# Citrus Genomics

Authors: Manuel Talon; Fred G. Gmitter
Journal: International Journal of Plant Genomics
 (2008)

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## Abstract

Citrus is one of the most widespread fruit crops globally, with great economic and health value. It is among the most difficult plants to improve through traditional breeding approaches. Currently, there is risk of devastation by diseases threatening to limit production and future availability to the human population. As technologies rapidly advance in genomic science, they are quickly adapted to address the biological challenges of the citrus plant system and the world's industries. The historical developments of linkage mapping, markers and breeding, EST projects, physical mapping, an international citrus genome sequencing project, and critical functional analysis are described. Despite the challenges of working with citrus, there has been substantial progress. Citrus researchers engaged in international collaborations provide optimism about future productivity and contributions to the benefit of citrus industries worldwide and to the human population who can rely on future widespread availability of this health-promoting and aesthetically pleasing fruit crop.


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## Body

## 1. Introduction

Citrus is one of the most
important and widely grown of the fruit crops, with total global production
reported to be 105.4 million tons in 2004-2005 [1]. Citrus fruit is produced throughout the
tropical and subtropical regions of the world, where the winter temperatures
are adequate for tree survival and avoidance of freeze devastation, and where
there is sufficient water and suitable soils to support tree growth and fruit
production. The most significant
production areas are found in the Americas (led by Brazil,
the United States, Mexico, and Argentina),
the Mediterranean basin (led by Spain, Italy,
Egypt, and Turkey), and the south and east
Asian regions (led by China,
India, and Japan). Citrus production, whether for processed
or fresh fruit products, from the largest producing countries is an important
commodity for global trade and of tremendous economic value and impact. However, there is much citrus production of
great importance to local national and regional economies and of value to the
nutritional needs of people in less developed nations; this is born out, for
example, by the
fact that sweet oranges (Citrus sinensis L. Osb.) are reported to be grown
in 114 countries, grapefruit (Citrus paradisi Macf.) and pummelos (Citrus maxima Merr.) in 74 countries, and lemons/limes (Citrus limon [L.] Burm. F./Citrus
aurantifolia [Christm] Swing.) in 94 different countries [2].In addition to the use as a
food or beverage source, citrus products from some of the wild species not
grown commercially are also of value as agents of traditional medicinal and
sanitary utilization [3]. Several
closely related genera have varying degrees of sexual compatibility with Citrus, some of which produce edible
fruit for commerce (e.g., the kumquats, Fortunella [Swing.]) and
others that possess traits of economic value for rootstock and scion
improvement (e.g., the trifoliate orange or Poncirus trifoliata [L.] Raf.).
Despite
the diversity of fruit types, however, nearly 70% of the world’s citrus production
is sweet orange.Given the tremendous extent
and value of citrus production, it may be somewhat surprising on first
consideration that nearly all of the major scion and rootstock cultivars
utilized in much of the world have not arisen as a consequence of systematic
and targeted breeding programs. Rather, they
have arisen spontaneously as seedling and/or bud sport mutations or by
introduction and trials of materials from one location to another [4–6]. The reasons for the low level impact of
traditional breeding approaches to genetic improvement of this major fruit crop
are related to the peculiarities of citrus reproductive biology and the fairly
unique aspects of the taxonomic relationships of the major cultivar groups [7]. Citrus seedlings are subject to juvenile
periods ranging from one to as many as 20 years, though typically they will
flower and fruit within 3–7 years, depending on species. Even after first flowering, it is common for
fruit traits to be atypical of later characteristics as scion lines
mature. One consequence of juvenility is
the obvious delay between hybridization and selection for desired
characteristics; however, a secondary consequence is the requirement of large
unit areas of land to grow substantially large individual hybrids, thereby
increasing the cost of maintenance in the field and limiting the number of
families and individuals within families that can be grown. Further, many of the commercial citrus types
produce polyembryonic seeds through nucellar embryony, yielding seedlings that
are essentially clones of the maternal parent. These embryos arise autonomously prior to anthesis and their development
to maturity follows normal pollination and endosperm development [8]. These nucellar embryos most frequently grow
much more vigorously than any zygotic embryos and, consequently, the frequency
of true zygotics is extremely low. Finally, it is important to recognize that several of the so-called
“species” of economic significance (e.g., C. sinensis, C. paradisi, and C. limon) are not
biologically defined species; the cultivars in these groups represent
accumulated somatic mutations identified over centuries through on-tree or
nucellar seedling mutations [9]. Further, some cultivar groups within other species, such as the Clementine
and Satsuma mandarins, are likewise the result of somatic mutations and not a
consequence of hybridization. Market and
consumer expectations and demands for specific commodities (e.g., sweet
oranges, grapefruit, lemons, Clementines, and Satsumas) thereby limit the
possibilities for genetic improvement within these cultivar groups because the
commodities must meet the consumers’ expectations and concepts related to fruit
traits. These needs and the narrow
germplasm bases actually represented within these cultivar groups, along with
the reproductive factors, have precluded breeding as a strategy for cultivar
development and improvement. The exceptions
to this are pummelos (C. maxima), the development of new types of
mandarin hybrids (using selections that produce
monoembryonic seeds containing true zygotic embryos), and rootstock breeding, where
hybridization and selection are viable and productive approaches. In these cases, however, limited genetic
understanding of the inheritance and control of critical traits remains a
substantial issue.With globalization of
citrus production and increased human travel throughout the world, particularly
devastating citrus diseases have been rapidly spreading, thus threatening the
viability and the very future of citrus production globally. This is the case with much of agriculture; increasing human
populations and urban development have forced citrus production to less desirable regions where
environmental factors present greater challenges to sustained production. Increased demands for water resources that
follow increased populations and urbanization likewise limit the availability
of water resources with adequate quality to maintain tree growth and
production. There exist genetic
resources to address most of these challenges. However, the genetic challenges and the lack of understanding of the fundamental
mechanisms underlying these critical traits, as described above, present
tremendous impediments to the progress needed to incorporate needed genes and alleles
and to devise the appropriate strategies for the continued production and
economically feasible availability of citrus fruits to the future world
population. In this context, the advent
of genomic science and the powerful new tools that are being developed and
utilized for citrus improvement take on critical significance. This article will review the progress that
has taken place thus far in the development and application of genomic
information for citrus improvement and present the current status of research
and future directions envisioned.


## 2. Linkage Mapping

Citrus and the closely related genera are partially sexually compatible in
varying degrees; they are primarily diploid with a few known triploids and
occasional tetraploid forms (2n=2x=18),
and they possess fairly small genomes (e.g., sweet orange has been said to be
around 367 Mb, or approximately three times that of Arabidopsis [10]). As such, the citrus species should be
amenable to many of the commonly used techniques and approaches related to
genomic research, including genetic and physical mapping, full genome
sequencing, and functional genomics studies aimed at unraveling the complexities
of key traits of interest. Because of
the valuable
characteristics within some of the related genera that are absent from Citrus, particularly cold tolerance and
resistance to citrus canker (caused by Xanthomonas axonopodis pv. citri) from kumquat (Fortunella) and multiple stress-tolerant and
disease-resistance traits from P.
trifoliata (including freeze avoidance, and
resistance to citrus tristeza virus (CTV), Phytophthora, and citrus nematode [Tylenchulus semipenetrans]),
many of the genetic mapping projects and some of the physical mapping as well have focused on P. trifoliata through intergeneric
hybrids with Citrus.Citrus breeders and
geneticists have long desired to have linkage maps that empower selection
schemes based on easily scored, neutral molecular markers rather than relying
on frequently difficult, time-consuming, and inefficient approaches based on
phenotypic characterizations. Indeed,
genetic linkage maps have been produced across the past two decades with
increasing value and resolution, as the evolution of new marker systems has
taken place. The first published report
of linkage mapping in citrus (using a small intragenic
family ofCitrus, and a larger Citrus × Poncirus family) was based on leaf
isozymes [11]; five markers were found that defined two linkage groups, and
significantly this was also the first report of linkage distortion which has
been a common feature of the many intergeneric mapping efforts that followed
subsequently. As RFLP technology became
commonly applied in genetic studies, citrus scientists began to incorporate it
together with isozyme methods, and new maps resulted first with 35 markers in 8
linkage groups covering 314 cM within a citrus backcross family [12], followed
by maps with 52 and 35 markers each, defining 11 and 10 linkage groups and
533 and 351 cM, in an intergeneric backcross family and a population derived
from crossing two individual intergeneric F1 hybrids, respectively [13, 14]. As new marker systems were developed, the
maps produced from each of these families were further populated first by RAPDs
[15], thereby increasing the number of markers from Durham’s map from 52 to 189, decreasing the
number of linkage groups to 9, and more than doubling genomic coverage to 1192 cM. Sankar and Moore [16] increased
marker coverage in the same map to 310 markers through use of ISSRs. In similar fashion, Jarrell’s map was improved
through incorporation of SSR loci [17], and then further by ISSR marker
development to 156 markers defining 16 linkage groups across 701 cM [18]. AFLP markers were first reported to be used
for citrus genetic mapping in 1998 by de Simone et al. [19] and in 1999 by Ling
et al. [20] (also elaborating the Durham
map). Many other whole genome maps have been
produced as well as trait specific maps identifying single gene and QTL regions
of significance; these have been summarized by Chen et al. [21]. It is through the latter category of trait
specific mapping that some of the promise of genomic science for citrus genetic
improvement is being pursued and realized, including selection of disease
resistant and environmental stress tolerant hybrids in rootstock breeding
programs, and targeted gene cloning projects aimed at providing potential
solutions to serious disease problems.Although higher throughput
and increased marker density became possible through application of RAPD, AFLP,
and ISSR techniques, these systems were of limited value in comparative genomic
studies and in utilization for marker-assisted selection (MAS) methods because of the dominant nature of the markers and their low portability
among populations. RFLPs, SCARs, CAPS, SSRs,
and SNPs are obviously much more desirable for broad applications, and the citrus
research community has been developing these resources over time. SCAR markers for citrus were developed first
by Deng et al. [22], based on RAPD markers that were closely linked to Ctv, a gene for CTV resistance from P. trifoliata [23]. Polymorphism could not be revealed by some of
these SCAR markers without restriction of amplified products, thus they were
converted to CAPS. In 1999, García et
al. [24]  likewise used CAPS together with RFLPs, RAPDs, and isozymes to map
genes in Citrus and Poncirus associated with apomixis. Though codominant marker types such as RFLP,
SSR, SCAR, CAPS, and SNPs were being used earlier, the numbers of such loci available
were severely limited. Consequently,
there has been a limited ability to interrelate maps and/or markers developed
in different populations or targeting different QTLs, and there were very few
chromosome-specific anchor markers that could enable comparative mapping
efforts between different genetic resources of citrus. The processes for developing such markers
previously were very time and labor intensive, and the efficiencies were
extremely low. Further, some of these
markers were developed in the absence of genome or transcriptome sequences, and
as such they might be considered to be gene anonymous. However, the revolution in sequencing
technologies, including the sequencing of BAC clones and fairly extensive EST
libraries for citrus accessions under multiple conditions, has produced a very
substantial resource for high-throughput development, verification, and
utilization of molecular markers for citrus linkage mapping, which meets the
desired criteria. These markers
frequently are based on EST sequences and therefore represent specific genes,
which functions may be known or estimated in some cases. For example, Omura et al. [25, 26] first
reported development of 131 mapped CAPS markers derived from EST
sequences. Using a backcross citrus population,
these markers were assigned into nine linkage groups that accounted for 685 cM
coverage, and they were found to be portable to another population. With the rapid increase in publicly available
EST databases in the USA [27] and publicly available software programs, large-scale
searches for various types of SSR motifs and efficient design of appropriate
primers have made it possible to identify and map EST-SSRs in citrus [21, 28]. The first such map for sweet orange and P. trifoliata was published in 2007 [21],
and it is being expanded collaboratively [29]. New international, collaborative EST-SSR mapping efforts are currently
underway [30] using other citrus-based families as part of a plan intended to
lead to the full-length sequence of a haploid citrus genome, to be integrated
with physical and genetic maps based on BAC end sequencing, high-throughput
marker saturation, and mapping traits of economic importance to genetic
improvement of citrus. These extensive
international efforts are being promoted and coordinated by the International
Citrus Genome Consortium (ICGC),
currently chaired by F. G. Gmitter of the University of Florida, USA. New technology will continue to enhance
progress toward high-resolution and highly informative maps of citrus genomes
in the future. Currently, efforts are
underway in several labs around the world to utilize microarrays for mapping
SNPs in various families and genetic backgrounds, and, as a full genome sequence
comes forward for citrus, followed by additional resequencing of other genomes
of interest, the genomic identification and locations of thousands of
trait-relevant SNPs will become known and exploited for genetic improvement of
the crop.


## 3. Physical Mapping

The main challenge for a comprehensive and meaningful description of the
genomes is the integration of the DNA marker-based genetic maps with physical
maps, and eventually with DNA sequence of the whole genome, the ultimate
physical map. Large genomic DNA
insert-containing libraries are required for physical mapping, positional
cloning, and genome sequencing of complex genomes. The physical mapping of complex genomes is
based on the construction of a genomic library, and the determination of the
overlaps between the inserts of the mapping clones in order to generate an
ordered, cloned representation of nearly all the sequences present in the
target genome.For the generation of
high-resolution physical maps, the construction of bacterial artificial
chromosome (BAC) libraries containing
clones with large DNA fragments appears to be indispensable. The BAC cloning system has become a dominant
system over others to clone large genomic DNA inserts. BAC clone collections and BAC-based contig
maps are indeed powerful tools having multiple applications in genomics such as
supporting positional cloning or to aid large-scale assembly of whole genomes. In whole genome sequencing projects, BAC end
sequences (BES, paired-end reads) are
also of inestimable help for the integration of the physical map with the
genome sequence. Furthermore, in many
agricultural important species, BAC clones and physical maps are being rapidly
developed since they are essential components in linking phenotypic traits to
the responsible genetic variation, to integrate the genetic data, for the
comparative analysis of genomes, and to speed up and improve potential and
effectiveness of marker-assisted selection (MAS) for breeding.


### 3.1. BAC Libraries

In citrus, Yang et al. [31] and Deng et al. [32] independently
constructed two BAC libraries as part of a map-based, or positional, cloning
strategy with the idea of identifying BAC clones spanning the genetic region
identified as containing gene(s) for resistance
to CTV. CTV is the causal agent of
several diseases causing significant economic damage and losses to citrus
worldwide. Broad spectrum resistance to
CTV was previously associated with a single dominant gene, Ctv, characterized in P. trifoliata, a sexually compatible
relative of citrus [23]. In order to
clone this gene, Yang et al. [31] constructed a BAC library from an individual
plant homozygous for Ctv. The
library contained 45 000 clones with an average insert size of 80 kb. Ctv was initially mapped to a 282-kb region including a disease resistance gene
cluster with seven members and eight retrotransposons clustered [33]. Sequence
analysis of the Ctv surrounding genomic region located the locus into a
121-kb Poncirus region comprising 10 genes. All 10 genes were individually cloned in Agrobacterium-based
binary vector and used to transform susceptible varieties [34] to test their
resistance capability.In a parallel effort, a BAC
library was constructed from the genomic DNA of an intergenericCitrus and Poncirus hybrid
for molecular isolation of disease resistance genes, including Ctv [32]. The library of 24 000 clones with an average
insert size of 115 kb was screened with DNA markers linked to the Ctv gene and citrus disease resistance
gene candidate (RGC) sequences. A few clones were isolated with each of the CTV
resistance gene-linked markers, and several hundred others were identified
using previously cloned citrus RGC sequences as probes [35]. Further fingerprinting and assembly resulted
in the identification of 25 contigs of 120–250 kb. Additional libraries were developed from the
same intergeneric hybrid for the purpose of map-based cloning of Ctv. From these libraries, full contigs were constructed that spanned both
the resistance allele from Poncirus and the susceptibility allele from the Citrus chromosome. These clones were fully
sequenced and assembled. Comparisons of
the resistance and susceptibility allelic genomic sequences revealed that the
levels of similarity varied from region to region. Within the region where the most likely Ctv candidate genes were delimited,
based on fine genetic mapping and predicted by various sequence analysis
programs, there were 2 NBS-LRR candidate genes found in Ctv that were completely missing from the  Ctv sequence. Based on the
sequence analysis and the fine mapping results, it was concluded that either
one or both of these unique sequences should be considered the first priority
candidates for Ctv.A further exploitation of
this BAC library resulted in identification of other disease resistance
gene-like DNA sequences, using a PCR approach with degenerate primers designed
from conserved NBS (nucleotide-binding site) motifs [35]. In addition to the three
amplified DNA fragment markers associated with the citrus tristeza virus
resistance gene (Ctv),
another fragment (Pt8a) was found to be
associated with the major gene responsible for the citrus nematode resistance (Tyr1). In a similar approach, degenerate primers for
the conserved motifs in the kinase domains of the plant disease resistant genes
(R) of rice Xa21 and tomato Pto were
used in PCR amplification to identify resistance gene candidates. Twenty-nine sequences highly similar to the
kinase domain of Xa21 were cloned and
characterized [36]). Using the BAC
library, two full-length sequences, including upstream promoters and downstream
terminating sequences, were identified. Markers derived from these Xa21-like
sequences have been found linked to putative QTLs for citrus canker resistance
segregating among hybrids derived from Citrus
ichangensis Swing. with Citrus limettoides Tan. (Gmitter, unpublished data).


### 3.2. Physical Mapping

Two communications at the PAG XV meeting in San Diego,    2007, reported progress
on the construction of physical maps of
citrus [37, 38]. The Spanish Citrus
Genomic Consortium has constructed three BAC libraries from Clementine mandarin
(EcoR I, Hind III, and MboI) containing a total of 57 000 clones with an average insert size of 120 kb (19x coverage). Half of these BAC clones were end-sequenced (29 Mb), and these sequences
analyzed [37]. The sequence analysis
revealed that most abundant known retroelements were LTR elements, especially
Ty-1 Copia [39] and Gypsy [40] elements, while known DNA transposons were
scarce. Basic local alignment search
tool (BLAST) searches also identified about 14 000 clones with coding regions
in a least one end and therefore putative euchromatin regions. BAC end sequences were also searched for single
nucleotide polymorphisms (SNPs) and
simple sequence repeats (SSRs) in the Clementine
genome. These initial analyses
identified more than 2800 sequence repeats in coding regions out of 7700
putative SSRs. Some 1.7% of the reads had
high similarity with the sequence of the Citrus
sinensis chloroplast genome [41], suggesting that about 70% of the chloroplast
genome of Clementine was similarly recovered in this sequencing effort. In parallel, a physical map derived from the
same 28 000-clone set of the Clementine BAC libraries is being constructed by
restriction enzyme fragment fingerprinting, and work is in progress to place on
it a sufficient number of genetically mapped markers to anchor and orientate
the contigs (Ollitrault, personal communication). It is expected that the paired-end reads will
also aid integration of the genetic and physical maps.The Citrus Genome Analysis Team from Japan has also
communicated the construction of a physical map of
citrus by high-information-content fingerprinting (HICF) analysis of a BAC
library from Satsuma mandarin (Citrus unshiu Marc.) consisting of 37 000 clones, with 13.3x of citrus genome
[38]. More than 6000 BAC clones from the
library were fluorescent labeled using the SNaPshot kit after digestion with
BamHI, EcoRI, XbaI, and XhoI and assembly of BAC fingerprints by FPC resulted
in approximately 1000 contigs (1.6x coverage). Consistent assembly among contigs obtained by
fingerprint analysis and physical maps obtained by BAC walking with both
molecular markers and BAC end sequences was observed. Further evaluations by additional clones,
assignment of molecular markers for the contigs, and gap filling by BAC end
sequencing to complete the physical map were also reported to be in progress
[38].A BAC library of Ridge
Pineapple sweet orange was produced by Michael Bausher (USDA-ARS,
Ft. Pierce, FL, USA) containing 18 432 clones (BamHI/Mbo I) with an average insert size of 145 kb,
or an estimated 7x coverage. A total of 16 727 clones from this library have
been fingerprinted and assembled into 472 contigs, as of August 2006. Access is freely available to the public athttp://phymap.ucdavis.edu:8080/citrus. This resource was searched by EST-SSR overgo
probes to identify BAC clones in six known heterozygous genomic regions
containing polymorphic alleles of mandarin and/or pummelo ancestry (the putative ancestral genomic contributors to sweet orange). These BAC clones were then sequenced and
assembled “blind” to assess the difficulties in assembling sequences of the
heterozygous sweet orange.The goal of genome-wide
integrated physical and genetic maps is a priority in citrus genomics since it
will provide the essential and powerful tools for research into the citrus
genome, such as effective positional cloning, marker development, high-throughput
EST mapping, and large-scale genome sequencing and assembly.


## 3.1. BAC Libraries

In citrus, Yang et al. [31] and Deng et al. [32] independently
constructed two BAC libraries as part of a map-based, or positional, cloning
strategy with the idea of identifying BAC clones spanning the genetic region
identified as containing gene(s) for resistance
to CTV. CTV is the causal agent of
several diseases causing significant economic damage and losses to citrus
worldwide. Broad spectrum resistance to
CTV was previously associated with a single dominant gene, Ctv, characterized in P. trifoliata, a sexually compatible
relative of citrus [23]. In order to
clone this gene, Yang et al. [31] constructed a BAC library from an individual
plant homozygous for Ctv. The
library contained 45 000 clones with an average insert size of 80 kb. Ctv was initially mapped to a 282-kb region including a disease resistance gene
cluster with seven members and eight retrotransposons clustered [33]. Sequence
analysis of the Ctv surrounding genomic region located the locus into a
121-kb Poncirus region comprising 10 genes. All 10 genes were individually cloned in Agrobacterium-based
binary vector and used to transform susceptible varieties [34] to test their
resistance capability.In a parallel effort, a BAC
library was constructed from the genomic DNA of an intergenericCitrus and Poncirus hybrid
for molecular isolation of disease resistance genes, including Ctv [32]. The library of 24 000 clones with an average
insert size of 115 kb was screened with DNA markers linked to the Ctv gene and citrus disease resistance
gene candidate (RGC) sequences. A few clones were isolated with each of the CTV
resistance gene-linked markers, and several hundred others were identified
using previously cloned citrus RGC sequences as probes [35]. Further fingerprinting and assembly resulted
in the identification of 25 contigs of 120–250 kb. Additional libraries were developed from the
same intergeneric hybrid for the purpose of map-based cloning of Ctv. From these libraries, full contigs were constructed that spanned both
the resistance allele from Poncirus and the susceptibility allele from the Citrus chromosome. These clones were fully
sequenced and assembled. Comparisons of
the resistance and susceptibility allelic genomic sequences revealed that the
levels of similarity varied from region to region. Within the region where the most likely Ctv candidate genes were delimited,
based on fine genetic mapping and predicted by various sequence analysis
programs, there were 2 NBS-LRR candidate genes found in Ctv that were completely missing from the  Ctv sequence. Based on the
sequence analysis and the fine mapping results, it was concluded that either
one or both of these unique sequences should be considered the first priority
candidates for Ctv.A further exploitation of
this BAC library resulted in identification of other disease resistance
gene-like DNA sequences, using a PCR approach with degenerate primers designed
from conserved NBS (nucleotide-binding site) motifs [35]. In addition to the three
amplified DNA fragment markers associated with the citrus tristeza virus
resistance gene (Ctv),
another fragment (Pt8a) was found to be
associated with the major gene responsible for the citrus nematode resistance (Tyr1). In a similar approach, degenerate primers for
the conserved motifs in the kinase domains of the plant disease resistant genes
(R) of rice Xa21 and tomato Pto were
used in PCR amplification to identify resistance gene candidates. Twenty-nine sequences highly similar to the
kinase domain of Xa21 were cloned and
characterized [36]). Using the BAC
library, two full-length sequences, including upstream promoters and downstream
terminating sequences, were identified. Markers derived from these Xa21-like
sequences have been found linked to putative QTLs for citrus canker resistance
segregating among hybrids derived from Citrus
ichangensis Swing. with Citrus limettoides Tan. (Gmitter, unpublished data).


## 3.2. Physical Mapping

Two communications at the PAG XV meeting in San Diego,    2007, reported progress
on the construction of physical maps of
citrus [37, 38]. The Spanish Citrus
Genomic Consortium has constructed three BAC libraries from Clementine mandarin
(EcoR I, Hind III, and MboI) containing a total of 57 000 clones with an average insert size of 120 kb (19x coverage). Half of these BAC clones were end-sequenced (29 Mb), and these sequences
analyzed [37]. The sequence analysis
revealed that most abundant known retroelements were LTR elements, especially
Ty-1 Copia [39] and Gypsy [40] elements, while known DNA transposons were
scarce. Basic local alignment search
tool (BLAST) searches also identified about 14 000 clones with coding regions
in a least one end and therefore putative euchromatin regions. BAC end sequences were also searched for single
nucleotide polymorphisms (SNPs) and
simple sequence repeats (SSRs) in the Clementine
genome. These initial analyses
identified more than 2800 sequence repeats in coding regions out of 7700
putative SSRs. Some 1.7% of the reads had
high similarity with the sequence of the Citrus
sinensis chloroplast genome [41], suggesting that about 70% of the chloroplast
genome of Clementine was similarly recovered in this sequencing effort. In parallel, a physical map derived from the
same 28 000-clone set of the Clementine BAC libraries is being constructed by
restriction enzyme fragment fingerprinting, and work is in progress to place on
it a sufficient number of genetically mapped markers to anchor and orientate
the contigs (Ollitrault, personal communication). It is expected that the paired-end reads will
also aid integration of the genetic and physical maps.The Citrus Genome Analysis Team from Japan has also
communicated the construction of a physical map of
citrus by high-information-content fingerprinting (HICF) analysis of a BAC
library from Satsuma mandarin (Citrus unshiu Marc.) consisting of 37 000 clones, with 13.3x of citrus genome
[38]. More than 6000 BAC clones from the
library were fluorescent labeled using the SNaPshot kit after digestion with
BamHI, EcoRI, XbaI, and XhoI and assembly of BAC fingerprints by FPC resulted
in approximately 1000 contigs (1.6x coverage). Consistent assembly among contigs obtained by
fingerprint analysis and physical maps obtained by BAC walking with both
molecular markers and BAC end sequences was observed. Further evaluations by additional clones,
assignment of molecular markers for the contigs, and gap filling by BAC end
sequencing to complete the physical map were also reported to be in progress
[38].A BAC library of Ridge
Pineapple sweet orange was produced by Michael Bausher (USDA-ARS,
Ft. Pierce, FL, USA) containing 18 432 clones (BamHI/Mbo I) with an average insert size of 145 kb,
or an estimated 7x coverage. A total of 16 727 clones from this library have
been fingerprinted and assembled into 472 contigs, as of August 2006. Access is freely available to the public athttp://phymap.ucdavis.edu:8080/citrus. This resource was searched by EST-SSR overgo
probes to identify BAC clones in six known heterozygous genomic regions
containing polymorphic alleles of mandarin and/or pummelo ancestry (the putative ancestral genomic contributors to sweet orange). These BAC clones were then sequenced and
assembled “blind” to assess the difficulties in assembling sequences of the
heterozygous sweet orange.The goal of genome-wide
integrated physical and genetic maps is a priority in citrus genomics since it
will provide the essential and powerful tools for research into the citrus
genome, such as effective positional cloning, marker development, high-throughput
EST mapping, and large-scale genome sequencing and assembly.


## 4. Citrus Sequencing

One of the major goals of the International Citrus Genomics Consortium is
to provide a high quality sequence of a citrus genome. Sequencing
of the citrus genome will facilitate the comparison of herbaceous and woody
perennial genomes and provide a valuable resource for studying significant
biological questions of critical importance to genetic improvement of citrus. From a scientific perspective, citrus as a
fruit tree developing nonclimacteric fruits possesses a combination of interesting biological
characteristics such as apomixis,
gametophytic self- and cross-incompatibility, juvenility, deciduousness/evergreen
foliage, dormancy, seasonality, root/shoot interaction,
oil glands, nutraceutical compounds, and plant-pathogen interactions. Additionally, citrus is the most economically significant fruit crop produced in the
world, although citrus production is severely threatened by pest, disease, and
environmental problems to which current commercial rootstock and scion
cultivars are susceptible.In February 2004, a proposal prepared and supported by the National
Citrus Genomics Steering Committee (USA) and the International Citrus Genomics Consortium (ICGC,
composed of researchers from Australia,
Brazil, China, France,
Israel, Italy, Japan,
Spain, and USA) to sequence the genome of sweet orange was presented to the Joint Genome
Institute (JGI). This institution reported at the beginning of
2007 to have produced a low coverage (ca. 1.2x) whole-genome shotgun sequence ofCitrus sinensis (sweet
orange) by sequencing ends of about 126 000 fosmid clones
containing 40 kb inserts and 257 000 plasmid clones containing 8 kb inserts [42]. Total sequence coverage, available at http://harvest.ucr.edu/, was about 473 Mb, coverage
apparently insufficient to provide quality assembling of the high heterozygous
sweet orange.In January 2007, the Steering
Committee of the ICGC met at JGI in California,
USA
to reassess the present status of citrus genome research and to forge plans for
future collaborative efforts. The first
outcome of the meeting was a decision to shift focus to a haploid genome as the
target for sequencing, rather than the previously stated target of sweet
orange; this consideration was made to eliminate the difficulties associated
with quality assembly of highly heterozygous diploid sweet orange genome. A haploid derived genome sequence should
serve as the highest quality reference genome for all future genomic research
efforts. It was required that the
haploid (or di- or tri-haploid individual chosen) should be available for free distribution internationally, and that it should
be pathogen-free, exhibit robust vegetative growth (as
a partial guarantee against gross genome defects), and be
relatively easy to maintain. Teams were
established to verify chromosome number and to assess candidates for
homozygosity using SSR markers representing good genome coverage based on
linkage maps. Additionally, the
candidates will be assessed using at least two available citrus microarray
platforms in an effort to insure that there are no large deletions or other
cytogenetic defects. Currently there are
three candidates, all derived from Clementine mandarin, that are being
evaluated according to this plan. It is
envisaged that the international collaboration will be supported from various
agencies and sources in different partner nations, that the sequence
information will be quickly deposited and shared freely among the participating
laboratories, and that the goal will be achieved once there is 8 to 10x
coverage. Currently, there are funding
commitments from USA, Spain, France,
Italy, and  China.The complete chloroplast
genome sequence ofCitrus sinensis was recently provided by Bausher et al. [41].  It is 160,129 bp in length and contains 133 genes (89 protein-coding,
4 rRNAs, and 30 distinct tRNAs). The
genome included 29 direct and inverted repeats 30 bp or longer, and comparison
of protein-coding sequences with expressed sequence tags revealed six putative
RNA edits. Phylogenetic analyses provide
strong support for the monophyly of eurosids II and for the placement of Citrus
(Sapindales) sister to a clade including
the Malvales/Brassicales.


## 5. Functional Genomics

### 5.1. EST Sequencing

The first sets of ESTs (expressed sequence tags) from any citrus material came from the pioneering work of Omura and coworkers
who reported about 3000 partial sequences of cDNA clones from libraries derived
from seeds, and from developing and mature fruit and albedo tissue, during the second half of the 1990s
[43]. Later, a set of 6500 ESTs derived
from whole seedlings of sweet orange was developed by Bausher et al. [44], and
a new contribution of 600 sequences from Citrus
unshiu of the Japanese team was also reported [45]. Since then, various groups (including Roose and Close at University of California at Riverside
[UCR], Dandekar at University of California at Davis [UCD], and the Spanish Citrus
Genomics Consortium at Valencia) have contributed to EST
sequencing efforts using several species, mostly C. sinensis (sweet orange), C. clementina (Clementine
mandarin), C. paradisi (grapefruit), Poncirus trifoliata, and other hybrids (C. sinensis × Poncirus trifoliata, Carrizo citrange). The total resource has reached 232 808 citrus
sequences in the National Center
for Biotechnology Information
(NCBI) EST database as of May 2007. This EST collection includes a wide
representation of sequences from many cDNA libraries derived from multiple
reproductive (flowers, ovaries, fruits, seeds) and vegetative (roots, leaves, buds) organs and tissues (pulp flesh, flavedo, abscission
zones) at different developmental stages and challenged with
biotic (Phytophthora,
citrus tristeza virus, herbivory, Penicillium) and abiotic (salinity, iron deficiency, water deficit) agents, and elicitor and hormonal treatments.Although a compressive
analysis of all ESTs in public databases has not been performed, several
subsets of the data have been partially analyzed. Forment et al. [46], for example, generated
25 cDNA libraries covering different conditions and from 22 635 high-quality
ESTs identified 11 836 putative unigenes. A third of these unique sequences was reported not to have Arabidopsis
orthologues. From a deeper analysis of a
collection of 54 000 single-pass ESTs, derived mostly from a normalized full-length
cDNA library (41 000 ESTs) and nine additional standard libraries representing
particular treatments and tissues from several selected varieties and
rootstocks, Terol et al. [47] identified 13 000 putative unigenes with
significant BLAST hits. Further analyses
and comparisons with Arabidopsis suggested the occurrence of citrus paralogues, putative conserved orthologues,
single copy genes, duplication events, and increased number of genes for
specific pathways. Interestingly, the
sequences of the genes belonging to these different species were essentially
identical, suggesting that their differential behavior cannot be attributed to
major sequence divergences. Nearly 17%
(2250 total) of the predicted citrus unigenes had no detectable similarity to Arabidopsis genes, and of these, 647
unigenes produced significant hits only to Citrus species, suggesting
that these clusters might be putative Citrus exclusive genes [47]. This work also contributed over 8500 clones
carrying putative full-length cDNA sequences. Full-length sequences and clones that are valuable tools since they can facilitate
proper prediction of gene structures provide a useful resource for functional
analysis and may greatly facilitate annotation of the full genome sequence. BLAST searches against sequenced citrus ESTs
are possible through several open database projects (i.e.,
http://harvest.ucr.edu/; http://cgf.ucdavis.edu/;
http://bioinfo.ibmcp.upv.es/genomics/cfgpDB/) or data deposited in GenBank. Although
predictions from EST clustering tend to overestimate the total number of genes,
these citrus EST sequences are apparently derived from perhaps 40 to 50 000
genes, a number more similar to that reported in Populus than that in Arabidopsis.In addition, Machado et al. [48]
reported at the PAG-XV meeting that Brazilian researchers have developed a huge EST sequencing effort. According to this communication, the CitEST (http://citest.centrodecitricultura.br/) Brazilian database including more than 260 000 valid reads contained unigene
sets from several citrus species but mainly sweet orange, mandarin, and Poncirus trifoliata. These ESTs were generated from several
libraries under biotic (Xylella fastidiosa, CTV, Citrus Leprosis Virus, Phytophthora, mite) and abiotic
(drought) stresses, and during fruit
development.


### 5.2. Microarrays Platforms

The importance of microarray technology for transcript profiling
approaches in functional genomics is increasing exponentially in practically
all plant systems and, in particular, in many agricultural crops. In citrus, the first transcript profiling
data was reported by Shimada et al. [49] who constructed a cDNA microarray to
monitor expression of mRNA from 2213 genes during fruit development. Since then, several citrus DNA microarray
platforms were developed. The Spanish
Citrus Genomic Consortium developed a first generation cDNA microarray
containing 12 672 probes corresponding to 6875 putative unigenes of a 22 000-EST
collection [46]. Subsequently, a
second-generation microarray comprising 12 000 unigenes was released and,
shortly afterwards, the Consortium produced the current version, a higher
density citrus microarray composed of 24 000-element cDNA array containing 20 000
unigenes, based on nearly 90 000 high-quality sequences generated from 52
different cDNA libraries.Similarly, a citrus 22 K
oligoarray containing 21 495 independent ESTs from Citrus species has been
recently developed in Japan. The information regarding this platform is
available at the websitehttp://www.fruit.affrc.go.jp/index-e.html. This tool has already produced very useful
information [50]. In 2006, Affymetrix
developed and released a citrus GeneChip containing 960 444 total 25-mer oligos
in an 11 micron format
(http://www.affymetrix.com/analysis/index.affx), this product came through close collaboration with Close and Roose
(UCR) and was based on the NCBI citrus EST collection that was available at the
time of its design. About two-third of
the content was designed for gene expression analysis using 30 264 probe sets. Most of the remaining one-third was designed
to genotype 3219 genes using 5023 SNPs identified in ESTs from Citrus
sinensis and other citrus species. The
citrus chip also contained probe sets for detection of several pathogens and
commonly used transgenes and a representation of the region of the P. trifoliata genome containing Ctv, the CTV
resistance allele. In January 2007 at
PAG, it was communicated that the GeneChip Citrus Genome Array is being used in
Israel,
for example, to analyze transcription profiles of bud sprouting as related to
alternate bearing behavior [51]. Additional work is currently underway in various labs using the
Affymetrix product for high-throughput linkage mapping, and to assess gene
expression under various physiological and pathogen and/or pest challenges, it
is anticipated that there will be several reports published within the next
year on these research projects.Several other communications
presented at PAG XV, for instance, Deng et al. [52] and elsewhere reported
other research projects using cDNA citrus microarrays or smaller custom arrays
based on subtractive libraries. Some of
them include analysis of transcriptional responses of 1731 genes to herbivory [53],
of 312 subtracted genes in abscission zones (Tadeo and Talon, unpublished data),
and the investigation of citrus canker resistance in kumquat (Fortunella spp)
using an array with 2254 elements [54].


### 5.3. Gene Expression and Transcriptome Profiling

In a recent review, Jansson and Douglas [55] explained the usefulness of Populus as a new plant system model offering
new insights in many physiological processes that cannot be easily studied in Arabidopsis or rice, the two main models
for plant biology. The strength of this
proposition, sustained by the completion of the whole poplar genome sequence
and the development of several genetics and genomics tools, holds promise to
elucidate major tree-specific traits such as wood formation, long-term
perennial growth, biotic interactions, and others. The availability of a second tree model can
provide contrasting data on plant genome evolution, gene family structure, and
other pivotal tree traits. Citrus as a
fruit tree not only will promote achievement of these goals but more importantly
offers a suitable system to study “fruit growth and quality,” a fundamental
plant trait for which Arabidopsis,
rice, and poplar are not useful systems. Although these model plant systems, including tomato, are crucial to
understand plant growth and development, the dramatic developmental differences
found across species are channeling many efforts to genomic and post-genomic
studies of crop plant species, rather than retaining focus solely on these
model species.Citrus possesses an enormous
unexplored potential to reveal relevant plant growth processes and some
responses that probably cannot be studied in any other plant. Although functional genomics in citrus is currently
in its infancy, the particular citrus biology suggests that citrus may contain
a reservoir of genes with peculiar and unique functions. One of the first steps to assign functions to
unknown genes is the large-scale gene analysis of the transcriptome. In citrus, before microarray availability,
gene expression in developmental and environmentally regulated processes, as in
many other systems, was mostly studied through differential display techniques (i.e., DDRT-PCR). Genes involved in many processes were also identified after subtractive
hybridization of cDNA libraries constructed from two different conditions. The main targets in citrus research have been
those physiological processes that sustain major commercial traits. Below, we summarize the knowledge gained in
these several areas.


#### 5.3.1. Fruit Growth and Ripening

While in tomato (a climacteric fruit) great strides have been made in the areas of ethylene regulation, carotenoid
accumulation, and cell wall metabolism, in nonclimacteric citrus fruit the
general information is substantially less. Mature citrus fruits release low amounts of ethylene but respond to
exogenous ethylene by accelerating respiration, chlorophyll degradation, and
carotenoid deposition. In these fruits,
very low rates of ethylene production have been associated with constitutive
expression of the 1-aminocyclopropane-1-carboxylate synthase 2 (CsACS2) and ethylene receptor CsETR1 genes,
indicating that citrus possesses a system I machinery. However, it has been reported that a
climacteric-like rise in ethylene production, preceded by induction of the
genes for CsACS1, ACC oxidase1, and the ethylene receptor CsERS1,
characteristic of a system II-like, appears to be present in young fruitlets [56]. It is well known that ethylene accelerates
the molecular changes in the carotenoid biosynthesis naturally occurring during
maturation (see below), while
gibberellins and nitrates, two ripening retardants, reduce expression of early
carotenoid biosynthetic genes and repress pheophorbide a oxygenase (PaO) expression [57, 58], a gene involved in
chlorophyll disappearance. Other
characteristic genes induced by ethylene are ferredoxins or thi, a gene involved in thiamine biosynthesis. The large ABA
amounts found in the peel of citrus fruit
during maturation appear to be synthesized by two 9-cis-epoxycarotenoid
dioxygenases (NCED) with differential
spatial and environmental expression [59, 60].In citrus, an initial small-scale
EST sequencing project from mature fruit resulted in the identification of 20%
of the sequences as encoding for metallothionein [61]. The abundance of these kinds of genes was
confirmed later in more developed citrus arrays. Later, Shimada et al. [49] used a citrus cDNA
microarray containing 2213 independent genes to examine gene expression during
fruit development and reported that the expression profile in the different
tissues of the fruit, flesh, albedo, and flavedo was rather different. Recently,
a comprehensive transcriptome analysis using a citrus 22 K oligoarray was
performed to identify ethylene-responsive genes in mandarin fruit [50]. In the 72 hours after ethylene
treatment, 1493 genes were shown to be modulated by the hormone. Ethylene repressed the transcription of most
genes involved in photosynthesis, chloroplast biogenesis, and sugar metabolism,
while it induced the transcription of several genes related to resistance,
defense, stress, amino acid synthesis, protein degradation, and secondary
metabolism. The sensitivity and
responsive patterns to exogenous ethylene were significantly different among
carotenoid biosynthesis genes (see below). Furthermore, most of the ethylene
biosynthesis genes and its signal transduction components did not show any
significant expression change after ethylene treatment. Interestingly, a type II ethylene receptor (ETR2) showed higher sensitivity to exogenous ethylene
than two other type I ethylene receptors (CsETR1 and CsERS1), suggesting that ETR2 might be associated
with low ethylene sensitivity in mature fruit [50].During the last decades, research on citrus fruit flavor that depends
upon multiple compounds, mostly sugars, acids, and flavanones, has received
considerable attention because of both the uniqueness of the physiological
processes sustaining this trait and the potential importance of these
components to human health. To date, the
most comprehensive study on the transcriptome profiling of the citrus fruit
flesh was presented by Cercós et al. [62] who examined gene expression with the
first generation Spanish cDNA microarray during development and ripening of
self-incompatible Citrus clementina. They reported that as many as 2243 putative
unigenes showed significant expression changes while functional classification
revealed that genes encoding for regulatory proteins were most significantly
overrepresented approximately within the middle of the rapid fruit growth phase;
this suggested that fruits at this stage were reprogramming developmental
commands to face the complex cellular modifications during ripening. Most
pivotal changes were related to carbohydrate build up, acid reduction,
modifications in secondary metabolism, carotenoid accumulation, and chlorophyll
decreases. Alterations of the
transcriptome associated with carbon accumulation were expected since it was
known that expression of several homologues of pivotal genes implicated in
carbon metabolism (e.g., phosphoenolpyruvate
carboxykinase, ADP-glucose pyrophosporylase, sucrose synthase, and sucrose
phosphatase synthase) and transport (i.e.,
sucrose transporters) during fruit growth considerably changed [63, 64]. In general, these genes appear to
belong to small families including few members, showing differential spatial
and temporal expression.On the other hand, developing
citrus fruits accumulate
a considerable amount of citric acid in the vacuoles of the juice sac cells,
although before ripening this high concentration is considerably reduced. The rate of change and final acid levels are
perceived as major components for citrus fruit quality. Research in gene regulation of acid
metabolism, however, has not led to a full understanding of this essential
process. There is considerable evidence,
nevertheless, obtained comparing acidless and acidic varieties that activity
and expression of citrate synthase were not responsible for these differences [65]. Another gene characterized in citrus was NADP(+)-isocitrate dehydrogenase (NADP-IDH),
encoding for an enzyme involved in citrate metabolism. Recently, a citrate transporter gene has been
reported encoding a novel vacuolar citrate/symporter that is able to mediate
citrate vacuolar efflux through the electroneutral cotransport of H+ and citrate
ions [66].  Interestingly, the
transcriptomic study together with the analyses of selected metabolites
suggested the occurrence of specific metabolic alternatives during citric acid
catabolism [62]. Microarray data suggested
that citrate was sequentially metabolized to glutamate that was finally catabolized
through the gamma-aminobutyrate (GABA) shunt. This observation was of special
relevance since it linked an efficient major proton-consuming reaction with
high acid levels. This work provides a
convincing explanation for the strong reduction of both citrate and cytoplasmic
acidity that takes place in citrus fruit flesh during development and
ripening.Transcript profiling also
revealed down-regulation patterns of gene expression for anthocyanin and flavonoid
biosynthesis, confirming previous observations. Thus, it was known that in common oranges there was a differential
repression of some of the enzymes of anthocyanin biosynthetic pathway, namely
chalcone synthase (CHS), anthocyanidin
synthase (ANS), and
UDP-glucose-flavonoid 3-O-glucosyltransferase (UFGT) [67], in contrast to “blood” pigmented oranges. However, anthocyanin and gene expression associated with anthocyanin
synthesis increased at low temperature [68]. Flavanones, a flavonoid subgroup, that greatly contribute to the bitter flavor
of grapefruit and other citrus, have also been the subject of intensive work
and pivotal genes of this biosynthetic pathway such as CHS, chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H),
dihydroflavonol 4-reductase (DFR), and
flavonol synthase (FLS) have been isolated and
characterized [69, 70]. In an elegant
work, Frydman et al. [71] demonstrated that the key flavor-determining step of
citrus flavanone biosynthesis was catalyzed by rhamnosyltransferases. They demonstrated that 1,2
rhamnosyltransferases catalyzed biosynthesis of the bitter neohesperidosides,
while 1,6 rhamnosyltransferases catalyzed biosynthesis of the tasteless rutinosides. Bitter species, such as grapefruit and
pummelo, accumulated bitter flavanone-7-O-neohesperidosides (naringin, the major flavonoid glycoside in grapefruit) responsible, in part, for their characteristic juice flavor, while nonbitter
species, such as mandarin and orange, accumulated only tasteless flavanone-7-O-rutinosides.Bitterness in citrus also is
associated with the presence of limonoids, triterpene derivatives that confer
the scent to fresh lemon and oranges. Kita
et al. [72] isolated a cDNA clone encoding limonoid UDP-glucosyltransferase (limonoid GTase) that regulated the conversion of
limonoid aglycones such as limonin, a bitter compound, to their nonbitter
glucosides.In addition to limonoids,
citrus fruits possess unique aromas rarely found in other fruit species
produced by other terpenes. This is also
an area of high research interest. Monoterpenes
(d-limonene terpinene and pinene) and other low-abundance sesquiterpenes (valencene,
nootkatone, and α- and β-sinensal) stand out in citrus as
important aroma and also flavor compounds. Lücker et al. [73] and Shimada et al. [74] isolated various monoterpene
synthases (d-limonene, γ-terpinene, β-pinene
synthase, β-ocimene, and cineole synthase), and it has also
been shown that their metabolic engineering produced new aromas in tobacco [75].  Monoterpene synthesis takes place in
epithelial cells surrounding the secretory cavities that contain the oil glands
in the flavedo [76]. Regarding
sesquiterpene production, Sharon-Asa et al. [77] identified a sesquiterpene
synthase-encoding gene, regulating the conversion of farnesyl diphosphate to a
single sesquiterpene, valencene. They
reported the transcript that was responsive to ethylene naturally accumulated
only towards fruit maturation. Other
putative sesquiterpene synthases, such as β-farnesene synthase, have also been
cloned.Work is also in progress to
characterize induced mutants that exhibit altered fragrance (alf) and abnormal number
of oil glands in the flavedo [78]. Transcriptome
analysis of fruits from these mutants showed changes in expression profile of
genes encoding enzymes involved in the biosynthesis of volatile compounds
derived from isoprenoid and phenylpropanoid pathways. In fruits of alf, several genes with different biological functions were down-regulated
although genes coding for a new putative terpene synthase (TPS) and an O-methyltransferase (OMT), apparently involved in secondary metabolism of
volatile compounds, had the highest differences in expression. In the mutant with lower number of glands, transcript
profiling also revealed strong down-regulation of genes encoding enzymes from
the phenylpropanoid and isoprenoid biosynthetic pathways. In a similar approach, Ishikawa et al. [79] used
a 22 K citrus microarray to analyze gene expression in a mutant that shows
smoother rind and decreased numbers of oil glands. The authors reported that the genes of the
nonmevalonate pathway of isoprenoid synthesis and monoterpene synthases were
down-regulated in the mutant.Tetraterpenes are also
crucial components of citrus fruit that contains one of the greatest arrays of carotenoids
found in any plant. The simultaneous
carotenoid accumulation and chlorophyll reduction occurring during natural ripening
indeed determines the color of the fruit peel, a most valuable characteristic
of perceived fruit quality. Many pivotal
genes of the carotenoid pathway have been cloned in citrus (phytoene
synthase (CitPSY), phytoene desaturase (CitPDS), ζ-carotene (car) desaturase (CitZDS), carotenoid isomerase (CitCRTISO),
lycopeneβ-cyclase (CitLCYb), β-ring
hydroxylase (CitHYb), zeaxanthin (CitZEA) epoxidase (CitZEP),
lycopene β-cyclase (CitLCYb), and
lycopene ε-cyclase (CitLCYe), and their
expression has been correlated with the accumulation of carotenoids in fruit [80, 81]. It was reported that the transition
of peel color from green to orange, and the change from β,ε-carotenoid to
β,β-carotenoid accumulation was accompanied by the disappearance of CitLCYe and
the increase in CitLCYb transcripts. As
fruit maturation progressed, a concomitant increase in the expression of
CitPSY, CitPDS, CitZDS, CitLCYb, CitHYb, and CitZEP led to massive
β,β-xanthophyll accumulation. Cercós et
al. [62] showed that expression of carotenoid biosynthetic genes in fruit flesh
followed rather similar changes. Mutations
of flesh color are being investigated in China
using a citrus cDNA array
with 6000 unigenes [52].In contrast to carotenoid accumulation, there have been fewer studies of
the chlorophyll degradation processes in citrus. Previous work on the regulation of catabolism
showed that chlorophyllase (CLH) was constitutively expressed during natural fruit development [82]. Recent results suggest that CLH functions as a rate-limiting enzyme
in chlorophyllcatabolism controlled via post-translational
regulation [83]. It is also known that
pheophorbide a oxygenase (PaO) and geranylgeranyl reductase expression, correlated with chlorophyll
degradation [57]. Recent work upon “nan,”
a stay-green mutant of Navel orange that produces fruit with abnormal brown
flavedo, showed that typical ripening-related chlorophyll (Chl) degradation was impaired in this mutant. Transcript and proteomic profilings revealed that a citrus orthologue of
a number of SGR (stay green) genes was expressed at substantially
lower levels in ‘‘nan’’ both
prior to and during ripening [84]. The “nan” mutation also resulted in the suppressed expression of
numerous photosynthesis-related genes and in the induction of genes associated
with oxidative stress. The transcriptome
of other selected citrus mutants is also being investigated to identify gene
functions related to fruit quality that in citrus are barely accessible through
genetic approaches. To this end, three
collections of induced mutated lines (EMS,
gamma rays and fast neutrons) have been generated, comprising
10 000 potential [84] mutants.With
the exception of thermostable pectin methylesterase activity [85, 86] that
greatly reduces citrus juice quality, cell wall metabolism in citrus has been
studied as related to fruit abscission, a major component of final yield. One of the strategies for the identification
of abscission-related genes followed by Dr. Burns’ team (University of Florida) was based upon
the isolation of ethylene-induced genes in the calyx, the laminar, and the floral
abscission zones. The role of ethylene
on the regulation of abscission has been widely illustrated for decades, and
several works have shown that ethylene is the primary effector activating the abscission
pathway in citrus [87, 88]. Differential
display and subtractive cDNA library screening were also used to search for
abscission-related metabolism changes. Important
components of the citrus abscission process were thus associated with
expression and/or activity of pivotal enzymes of cell wall metabolism (glucanases, polygalacturonases, galactosidases, and other
hydrolases; [89, 90]), hormonal synthesis, and signal
transduction (i.e., ACC synthases and oxydases) and secondary metabolism/PR proteins (i.e.,
phenylalanine ammonia lyase, chitinases). Yuan et al. [91] also demonstrated that
differential expression of ACC synthase 1 and ACC oxidase genes was associated
with reduction of ethephon-enhanced leaf abscission by guanfacine, a G-protein-coupled
alpha-(2A)-adrenoreceptor selective antagonist,
and suggested a link between G-protein-related signalling and abscission. Interestingly, guanfacine had little effect
on ethephon-enhanced fruit loosening. In
spite of this information, major regulators of the abscission process in citrus
are still mostly unknown although both custom manufactured and large-scale
microarrays, in some instances, coupled to laser assisted microdissection (LAM) are currently being used in Florida and Spain
to gain new insight into this process. Part
of these transcriptomic profiling studies has been summarized in a recent Ph.D.
dissertation presenting a model of leaf abscission events occurring at the laminar
abscission zone [60].  The two-stage
model proposes a first phase of activation, mostly characterized by the
activation of signalling pathways (hormones,
phospholipids, calcium, and oxygen reactive species). In a second stage, the execution phase,
degradation of the cell wall by hydrolytic enzymes would be culminated and
sugar-nucleotide metabolism for cell elongation induced. The process would end with the promotion of a
double defensive program intended to protect the living zone remaining attached
to the plant including deposition of physical barriers (callose
and lignin) and induction of pathogen resistance.Several other microarray
studies on citrus growth and ripening are under development and have not been
published yet. For instance, Dr. Sadka
is investigating with the GeneChip Citrus Genome Array (Affymetrix) the transcriptome modifications occurring during the induction of flower bud
differentiation using “on” and “off” trees [51], taking advantage of the
alternate bearing behavior, a process regulating differentially
carbohydrate-related gene expression [92]. In Brazil, the
sequencing carried out at the Centro APTA Citros “Sylvio Moreira”—IAC (Brazil) that has generated one of the most important databases for this genus in the
world is being extensively used to produce “in
silico” analyses. This approach is yielding information not
only related to fruit growth and development (terpene
production, cell wall metabolism, etc.) but also in the biotic
stress field [48].


#### 5.3.2. Responses to Pathogenic and Environmental Stresses

In citrus, gene expression associated with the responses to biotic and
abiotic stresses has targeted a limited number of genes in spite of the
economical importance of the citrus diseases and environmental constrains. Multiple
pathogens provoke a range of citrus disorders, mostly fungal (leaf spot,Alternaria; mold, Penicillium; post-bloom
fruit drop, Colletotrichum acutatum;
root rot, Phytophthora), bacterial (canker, Xanthomonas axonopodis;
citrus variegated chlorosis, Xylella
fastidiosa; Huanglongbing or
greening, Candidatus Liberibacter),
and viral diseases (citrus tristeza virus, CTV; citrus
leprosis virus, CiLV). Environmental
stresses include cold temperatures, drought, flooding, salinity, and high and
low soil pH, among others.In response to the
inoculation with conidia ofAlternaria,
at least two cytosolic antifungal miraculins with protease inhibitor activity
were strongly up regulated. Actually,
induction of miraculin expression is one of the most prominent responses
observed in microarray experiments performed in open field experiments. Both miraculin genes responded to methyl
jasmonate and were antagonized by salicylate [93]. Several studies with the green mold pathogen, Penicillium digitatum, also indicated
that genes such as thioredoxins, the gnsl gene (beta-1,3-endoglucanase activity),
and chitinases are major components of the molecular mechanisms involved in
activation of pathogen defense in citrus. Other responsive genes reported in citrus were epoxide hydrolase and
hidroperoxide lyase. It has also been
shown that the fungus Colletotrichum acutatum altered hormonal homeostasis increasing both levels of ethylene,
indole-3-acetic acid, cis-jasmonic acid (JA) and salicylic acid (SA), and
associated gene expression [94].Gandía et al. [95] have
recently presented data on the transcriptional response of citrus to infection
with severe and mild isolates of citrus tristeza virus. These studies concluded that gene expression
was only significantly altered with the severe isolate. Changes detected in the citrus transcriptome
after infection with this isolate were predominantly associated with symptom
expression (chlorophyllases, SAM transferases, ACC oxidase,
and lipid transfer proteins), defense mechanism, and general
responses to stress (miraculins, superoxide dismutases,
glutation transferees, NBS-LRR resistance genes, thioredoxin, protease
inhibitors, ubiquitin ligases, etc.).To study the mechanisms of canker
resistance in kumquat, a custom microarray using 2254 ESTs from subtractive
libraries is being utilized to determine the response to infective bacteria in
an incompatible interaction [54]. The
macroscopic phenotype, a delayed hypersensitive response in the inoculated
leaves, was accompanied by altered expression of 1245 genes. This study identified major components of the
incompatible interaction, reactive oxygen species (ROS) production, and programmed cell death (PCD). In addition, a number of common defense
mechanisms besides a number of resistance genes and putative receptors were
also identified.Citrus plants are also very liable to infestation by
aphids, whitefly, and other insects as well as being susceptible to herbivory. Mozoruk et al. [53]
described how nylon filter cDNA arrays were used to analyze the transcriptional
changes of 1731 citrus unigenes that resulted from herbivory by a xylem-feeding
leafhopper, Homalodisca coagulata. Insect feeding led to a significant
expression change in 50 transcripts broadly functioning in direct defense,
defense signalling, ROS scavenging, transport, cell wall modification,
photosynthesis, and abiotic stress. The
authors also noted that the transcript profile recorded greatly resembled that
induced by wounding, likely through JA-independent pathways. In contrast to similar studies with aphids, SA-dependent
pathogenesis related genes were weakly induced.Although transcriptional
profiling using microarrays has developed into the most prominent tool for
functional genomics, none has yet reported on the effects and responses of
citrus to the major environmental constraints (salinity,
flooding, water deficit, chilling, and iron deficiency). High-throughput analyses of gene expression
in citrus challenged with major abiotic stresses, however, are currently
underway in several laboratories around the world and will soon produce
valuable information that might eventually lead to discovery of novel genes and
functions. For instance, it is known that in citrus, physiological
disturbances produced by salinity are associated with leaf chloride build up
rather than with sodium accumulation, as observed in many plants [96, 97]. Genes in principle associated with the
response of citrus to salinity were initially obtained
from a cDNA expression library of citrus salt-treated cell suspensions. These genes, homologues to phospholipid
hydroperoxides, glutathione peroxidases [98],  olesins, Lea5, or lypoxygenases,
were involved in the oxidative response rather than in the specific response to
salinity. Other genes involved in
oxidative stress well known in citrus are glutathione S-transferases [99] and
copper/zinc-superoxide dismutases. However,
recent microarray analyses are providing much-needed insights into chloride
tolerance mechanisms and short- and long-term adaptation of citrus to salinity. Several teams are engaged in a
Euro-Mediterranean MPC INCO project, started in 2006, between Spain, Morocco,
Tunisia, Turkey, and France
focused on tolerances to salinity
and iron deficiency associated with alkaline soils. One of the major components of this project
is the large-scale study and genome-wide acquisition of quantitative biological
information on gene expression from multiple tolerant and susceptible genotypes. Work on transcriptomic comparisons in this
area is confirming that Cl- is the most important ion involved in
the genetic response of citrus to salinity. In addition, major metabolic regulation changes are also apparent during
salinity acclimatization in tolerant rootstocks. In contrast, flooding is mostly characterized
by the rise of oxidative stress.Chilling
resistance in citrus is another area that has received much attention but lacks
current comprehensive gene expression analyses provided by microarrays. Since most commercially important citrus
varieties are cold-sensitive and therefore susceptible to freezing,Poncirus trifoliata (L.),
an interfertile Citrus relative that
can tolerate temperatures as low as −26°C after acclimation, is being used for
improving cold tolerance in citrus rootstocks and as a source for the
identification of cold-regulated genes. In
general, many studies have been performed through subtractive hybridization [100]
and DDRT-PCR [101] comparing expression in sensitive and resistant varieties. It has been shown, for example, that
expression of a C-repeat-binding factor (CBF) and one of its targets, COR19,
a cold-induced gene, accumulated both earlier and to higher levels in Poncirus. Moreover, COR19, COR11 [102], and COR15 were
found to belong to an unusual group 2 LEA gene family responsive to low
temperature. These dehydrins differ from
most other plant dehydrins in having an unusual K-segment similar to that of
gymnosperms and in having a serine cluster (S-segment) at an unusual position at the carboxy-terminus [103]. Citrus, however, also possesses the typical
plant angiosperm-type K-segment consensus sequence. Other up-regulated transcripts that may play
a role in cold sensitivity are a novel RING-H2 finger gene, AP2 domain
containing genes and CTL, and a homologue of a low-temperature-responsive gene
from Arabidopsis. During postharvest storage, chilling injury
in citrus fruit can be reduced by previous short heat treatments that activate
different molecular responses. Genes
differentially expressed in the chilling response have mostly been related to
lipid membrane and cell wall enzymes, to main regulators of secondary
metabolism and hormonal homeostasis, and to oxidative and general stress
responses [104, 105].Although the main
applications of microarrays to date are in transcriptome profiling analyses,
microarrays can also be used to study DNA variation. Oligonucleotide arrays are particularly
suited for the detection of single nucleotide mismatches during hybridization
and, hence, for the discovery of novel DNA variants or the determination of
known variants. The citrus GeneChip, for
example, was designed to genotype 3219 genes using 5023 SNPs. The 20 K Spanish Consortium microarrays have
been used to identify heterozygous deletions in fast neutron irradiated citrus
mutants through array-based comparative genomic hybridization (array-CGH) and
to study gene colinearity. Preliminary
CGH yielded several candidate genes that were in haploid gene dosage. After comparison with theArabidopsis and Populus genomes, it was observed that Populus orthologues of Citrus deleted genes grouped in two duplicated chromosomes in contrast to Arabidopsis orthologues that were
distributed in several chromosomes (Ríos and Talon, unpublished
data).


### 5.4. Genetic Transformation

Citrus transformation procedures, in general, followAg-  robacterium tumefaciens protocols, and subsequent regen- eration through organogenesis and somatic
embryogenesis are also rather typical and straightforward [106]. Trans-  formation efficiency of young material
is usually low, 15–20%, and a major achievement to overcome the juvenility
limitation was the direct transformation of adult material [107].In
citrus, genetic transformation is mostly being explored as an alternative to
classical genetic breeding and not many examples can be found in the literature
illustrating the use of genetic transformation for functional genomics. For example, there is interest in modulating the growth habit of rootstocks since this might eventually
affect the development of the scion and facilitate diverse cultural practices (e.g., pruning, pesticide applications, and harvesting). Thus, it
was known that the ectopic overexpression in tobacco of a citrus GA 20-oxidase,
a regulatory step of gibberellin biosynthesis in citrus, [108] enhanced gibberellin
content and shoot growth [109]. Later, Fagoaga
et al. [110] generated transgenic Carrizo rootstocks overexpressing this GA
20-oxidase and confirmed that the gene controls gibberellin flux through the
pathway since taller (sense) and shorter
(antisense) phenotypes correlating with
higher and lower levels of active GA1 were obtained. In these transgenic lines, however, cell
division was more affected than cell elongation, in contrast to the effects
observed in herbaceous plants [111]. In
another example, an antisense construction with a citrus ACC synthase gene
repressed ACC increase after a chilling treatment. A pectin methylesterase gene (Cs-PME4) isolated from sweet orange to prevent
juice cloud separation was also introduced via protoplasts and subsequent
regeneration through somatic embryogenesis [112].Generally,
characteristics related to commercial valuable traits are modified through the
use of transgenes. To accelerate
flowering time, Carrizo seedlings constitutively overexpressing theArabidopsis floral-regulatory genes LEAFY (LFY) or APETALA1 (AP1) were generated [113]. Both kind of
transgenic citrus produced fertile flowers in their first year considerably shortening
the juvenile phase. Consistently with
the role of LFY and AP1, juvenility
in citrus was positively correlated with CsTFL (homolog
to TERMINAL FLOWER) transcript accumulation and negatively
correlated with LEAFY and APETALA1 RNA levels [114].  In a similar approach but with a citrus gene,
it was showed that transgenic Poncirus carrying the CiFT gene (homolog to FLOWERING LOCUS T), another flowering time gene, also exhibited early
flowering although this phenotype was accompanied with several pleiotropic
effects [115]. It is possible that the early
flowering AP1 and CiFT transgenic
citrus could be used as rapid cycling genotypes for functional genomics studies. In a further example, Carrizo rootstock constitutively expressing a Δ1-pyrroline-5-carboxylate
synthetase mutant gene from Vigna,
showed higher water deficit tolerance [116]. Regarding tolerance to
stresses, however, a huge amount of work has been centered on resistance to
biotic stresses, a matter of major relevance in citrus industry. Thus, tolerance or resistance to Phytophthora
citrophthora, the most widely spread oomycete in citrus growing areas, was
generated by introducing the gene P23, that codes for a pathogenesis-related protein induced in
tomato. These results provided evidence
for the antifungal activity in vivo of the P23 pathogenesis-related protein
against P. citrophthora [117].A great
effort is also being developed to understand the basis of the tolerance to
citrus tristeza virus (CTV), the causal
agent of the most important virus disease in citrus. The strategy is generally supported by the
concept of pathogen-derived resistance (PDR),
based on expression of viral sequences interfering with the virus life cycle in
plants. CTV resistant transformants have
been obtained by genetically engineering thep25 and p23 genes
from CTV [118]. However, it still
remains to be elucidated if transgenic citrus plants expressing CTV-derived
sequences are a plausible alternative to cross protection to control CTV strains
in the field. In an alternative strategy,
heterologous expression of plant-derived resistance genes is promoted to confer
resistance against CTV. General
resistance to CTV has been found in Poncirus trifoliata, and a region
containing the resistance gene (Ctv) has been characterized. Furthermore,
work is under way for other pivotal diseases such as citrus mosaic virus (CiMV), citrus canker, and citrus blight.


### 5.5. Reverse Genetics

In addition to genetic transformation, the capability to perform reverse
genetic analyses is crucial to develop functional studies. The
creation of transgenic lines is a powerful and straightforward way to determine
gene function. However, in citrus, high-throughput
transgenic programs such as the generation of RNA interference knockouts,
activation tagging through enhancer elements, gene-trap T-DNA insertions, or
transposable tagging systems have not yet been developed. The capacity for the maintenance and
characterization of many transgenic lines of a perennial tree with both a long
juvenile phase, large individual plant size, and a complex reproductive biology
has probably hindered these developments. InPopulus, however, activation tagging and insertional mutagenesis approaches are been
explored despite logistical challenges in working with transgenic trees, a
direction that may well be followed by citrus researchers in the near future.


### 5.6. Tilling/Fast Neutrons

Since gene disruption is the most effective method to analyze gene
functions and no efficient tagging or insertional methods are available in
citrus, strategies based on genome-wide mutagenesis such as TILLING (targeted induced local lesions in genomes) and fast
neutron mutagenesis are being explored further [84]. These approaches are nontransgenic and have
particular interest for the industry where the debate on GMOs has restricted their
application in crop improvement. TILLING
identifies individuals carrying point mutations while the fast neutron
mutagenized population is searched for gene deletions using PCR amplification. Both approaches, at the moment, are of
limited usefulness as strategies for reverse genetics in citrus because of the
lack of genomic sequences and the large amounts of space required for mutated
populations of suitable size. ECOTILLING,
however, on natural citrus variants and microarray-based detection of deletions
on fast neutron citrus mutants in a more direct genetics strategy are very
straightforward approaches. However,
unless a high-throughput transformation protocol is developed for citrus,
functionally analyzing all genes with tagging approaches or genome-wide
mutagenesis and screening are not realistic strategies.


### 5.7. Viral-Induced Gene Silencing

Viral-induced
gene silencing (VIGS), on the other
hand, is an attractive and very promising alternative in citrus. Knocking out the expression of a gene by VIGS
does not require genetic transformation and has proven to be a very efficient
tool for function analysis of plant genes. VIGS is particularly suitable for woody plants like citrus with long
juvenile periods that require long periods between transformation and fruiting. In a hopeful work, Dr. Guerri and colleagues at IVIA have
recently showed that VIGS might be possible in citrus usingCitrus leaf
blotchvirus (CLBV) as a viral
vector (Dr. Guerri, personal communication). These workers cloned a full-length cDNA of
the CLBV genome [119] in a binary vector under the control of the 35S promoter
and demonstrated that tobacco and citrus plants Agro-infiltrated with this construct became infected and
replicated CLBV normally. Recently, they
have showed that tobacco plants
Agro-infiltrated with a CLBV chimeric construct carrying a fragment of the
phytoene desaturase gene developed photobleaching symptoms and reduced the
cognate transcripts. Parallel
experiments in citrus are planned. Availability
of the CLBV-based vector will certainly open new possibilities to study
functional genomics in citrus.


### 5.8. Proteomics/Metabolomics

Other powerful approaches for functional genomics studies such as
proteomics and metabolomics to comprehensively analyze proteomes and phenotypes
have just begun for citrus. For example,
Blumwald and coworkers (UCD) are using two main approaches, namely 2D
gel analyses coupled with MALDI-TOF-TOF from juice sac cell vacuoles and LC2-MS-MS
analyses of ER/Golgi, plasma membrane, tonoplast, mitochondria, and soluble
enriched fractions from citrus juice sac cells to define the “citrus fruit
proteome.” Current work is in progress
but they have already reported the identification of over 1500 proteins
involved in sugar metabolism, citrate cycle, signalling, transport, and
processing and have characterized changes in protein expression during
development [120]. In a further example,
proteome changes in the fruit albedo during postharvest ageing were studied
through 2D-PAGE, and relevant proteins were also identified through mass
spectrometry determinations [121]. This proteomic survey indicated that major
changes in protein content (ATP synthase beta subunit,
ascorbate peroxidase, translationally
controlled tumor protein, cysteine protease, etc.) were
apparently related to the activation of programmed cell death.Numerous analyses of citrus
metabolites, especially of ripening and matured fruits, have been reported in
the past. However, new methods to
characterize the metabolic phenotypes of representative lines from mutants and
natural varieties must be developed. Metabolic
profiling and metabolomic procedures using state-of-the-art gas chromatography-mass
spectrometry or fast gas chromatography-time-of-flight mass spectrometry need
to be setup.The final objective of citrus
functional genomics is to identify candidate genes, alleles, and genotypes
improving citrus fruit quality, correlating phenotypic analyses, metabolomic
profiling, and gene expression. At
completion, genes and alleles with major functions in nutritional quality could
be selected and genotypes with improved fruit composition searched among
existing collections or generated.


## 5.1. EST Sequencing

The first sets of ESTs (expressed sequence tags) from any citrus material came from the pioneering work of Omura and coworkers
who reported about 3000 partial sequences of cDNA clones from libraries derived
from seeds, and from developing and mature fruit and albedo tissue, during the second half of the 1990s
[43]. Later, a set of 6500 ESTs derived
from whole seedlings of sweet orange was developed by Bausher et al. [44], and
a new contribution of 600 sequences from Citrus
unshiu of the Japanese team was also reported [45]. Since then, various groups (including Roose and Close at University of California at Riverside
[UCR], Dandekar at University of California at Davis [UCD], and the Spanish Citrus
Genomics Consortium at Valencia) have contributed to EST
sequencing efforts using several species, mostly C. sinensis (sweet orange), C. clementina (Clementine
mandarin), C. paradisi (grapefruit), Poncirus trifoliata, and other hybrids (C. sinensis × Poncirus trifoliata, Carrizo citrange). The total resource has reached 232 808 citrus
sequences in the National Center
for Biotechnology Information
(NCBI) EST database as of May 2007. This EST collection includes a wide
representation of sequences from many cDNA libraries derived from multiple
reproductive (flowers, ovaries, fruits, seeds) and vegetative (roots, leaves, buds) organs and tissues (pulp flesh, flavedo, abscission
zones) at different developmental stages and challenged with
biotic (Phytophthora,
citrus tristeza virus, herbivory, Penicillium) and abiotic (salinity, iron deficiency, water deficit) agents, and elicitor and hormonal treatments.Although a compressive
analysis of all ESTs in public databases has not been performed, several
subsets of the data have been partially analyzed. Forment et al. [46], for example, generated
25 cDNA libraries covering different conditions and from 22 635 high-quality
ESTs identified 11 836 putative unigenes. A third of these unique sequences was reported not to have Arabidopsis
orthologues. From a deeper analysis of a
collection of 54 000 single-pass ESTs, derived mostly from a normalized full-length
cDNA library (41 000 ESTs) and nine additional standard libraries representing
particular treatments and tissues from several selected varieties and
rootstocks, Terol et al. [47] identified 13 000 putative unigenes with
significant BLAST hits. Further analyses
and comparisons with Arabidopsis suggested the occurrence of citrus paralogues, putative conserved orthologues,
single copy genes, duplication events, and increased number of genes for
specific pathways. Interestingly, the
sequences of the genes belonging to these different species were essentially
identical, suggesting that their differential behavior cannot be attributed to
major sequence divergences. Nearly 17%
(2250 total) of the predicted citrus unigenes had no detectable similarity to Arabidopsis genes, and of these, 647
unigenes produced significant hits only to Citrus species, suggesting
that these clusters might be putative Citrus exclusive genes [47]. This work also contributed over 8500 clones
carrying putative full-length cDNA sequences. Full-length sequences and clones that are valuable tools since they can facilitate
proper prediction of gene structures provide a useful resource for functional
analysis and may greatly facilitate annotation of the full genome sequence. BLAST searches against sequenced citrus ESTs
are possible through several open database projects (i.e.,
http://harvest.ucr.edu/; http://cgf.ucdavis.edu/;
http://bioinfo.ibmcp.upv.es/genomics/cfgpDB/) or data deposited in GenBank. Although
predictions from EST clustering tend to overestimate the total number of genes,
these citrus EST sequences are apparently derived from perhaps 40 to 50 000
genes, a number more similar to that reported in Populus than that in Arabidopsis.In addition, Machado et al. [48]
reported at the PAG-XV meeting that Brazilian researchers have developed a huge EST sequencing effort. According to this communication, the CitEST (http://citest.centrodecitricultura.br/) Brazilian database including more than 260 000 valid reads contained unigene
sets from several citrus species but mainly sweet orange, mandarin, and Poncirus trifoliata. These ESTs were generated from several
libraries under biotic (Xylella fastidiosa, CTV, Citrus Leprosis Virus, Phytophthora, mite) and abiotic
(drought) stresses, and during fruit
development.


## 5.2. Microarrays Platforms

The importance of microarray technology for transcript profiling
approaches in functional genomics is increasing exponentially in practically
all plant systems and, in particular, in many agricultural crops. In citrus, the first transcript profiling
data was reported by Shimada et al. [49] who constructed a cDNA microarray to
monitor expression of mRNA from 2213 genes during fruit development. Since then, several citrus DNA microarray
platforms were developed. The Spanish
Citrus Genomic Consortium developed a first generation cDNA microarray
containing 12 672 probes corresponding to 6875 putative unigenes of a 22 000-EST
collection [46]. Subsequently, a
second-generation microarray comprising 12 000 unigenes was released and,
shortly afterwards, the Consortium produced the current version, a higher
density citrus microarray composed of 24 000-element cDNA array containing 20 000
unigenes, based on nearly 90 000 high-quality sequences generated from 52
different cDNA libraries.Similarly, a citrus 22 K
oligoarray containing 21 495 independent ESTs from Citrus species has been
recently developed in Japan. The information regarding this platform is
available at the websitehttp://www.fruit.affrc.go.jp/index-e.html. This tool has already produced very useful
information [50]. In 2006, Affymetrix
developed and released a citrus GeneChip containing 960 444 total 25-mer oligos
in an 11 micron format
(http://www.affymetrix.com/analysis/index.affx), this product came through close collaboration with Close and Roose
(UCR) and was based on the NCBI citrus EST collection that was available at the
time of its design. About two-third of
the content was designed for gene expression analysis using 30 264 probe sets. Most of the remaining one-third was designed
to genotype 3219 genes using 5023 SNPs identified in ESTs from Citrus
sinensis and other citrus species. The
citrus chip also contained probe sets for detection of several pathogens and
commonly used transgenes and a representation of the region of the P. trifoliata genome containing Ctv, the CTV
resistance allele. In January 2007 at
PAG, it was communicated that the GeneChip Citrus Genome Array is being used in
Israel,
for example, to analyze transcription profiles of bud sprouting as related to
alternate bearing behavior [51]. Additional work is currently underway in various labs using the
Affymetrix product for high-throughput linkage mapping, and to assess gene
expression under various physiological and pathogen and/or pest challenges, it
is anticipated that there will be several reports published within the next
year on these research projects.Several other communications
presented at PAG XV, for instance, Deng et al. [52] and elsewhere reported
other research projects using cDNA citrus microarrays or smaller custom arrays
based on subtractive libraries. Some of
them include analysis of transcriptional responses of 1731 genes to herbivory [53],
of 312 subtracted genes in abscission zones (Tadeo and Talon, unpublished data),
and the investigation of citrus canker resistance in kumquat (Fortunella spp)
using an array with 2254 elements [54].


## 5.3. Gene Expression and Transcriptome Profiling

In a recent review, Jansson and Douglas [55] explained the usefulness of Populus as a new plant system model offering
new insights in many physiological processes that cannot be easily studied in Arabidopsis or rice, the two main models
for plant biology. The strength of this
proposition, sustained by the completion of the whole poplar genome sequence
and the development of several genetics and genomics tools, holds promise to
elucidate major tree-specific traits such as wood formation, long-term
perennial growth, biotic interactions, and others. The availability of a second tree model can
provide contrasting data on plant genome evolution, gene family structure, and
other pivotal tree traits. Citrus as a
fruit tree not only will promote achievement of these goals but more importantly
offers a suitable system to study “fruit growth and quality,” a fundamental
plant trait for which Arabidopsis,
rice, and poplar are not useful systems. Although these model plant systems, including tomato, are crucial to
understand plant growth and development, the dramatic developmental differences
found across species are channeling many efforts to genomic and post-genomic
studies of crop plant species, rather than retaining focus solely on these
model species.Citrus possesses an enormous
unexplored potential to reveal relevant plant growth processes and some
responses that probably cannot be studied in any other plant. Although functional genomics in citrus is currently
in its infancy, the particular citrus biology suggests that citrus may contain
a reservoir of genes with peculiar and unique functions. One of the first steps to assign functions to
unknown genes is the large-scale gene analysis of the transcriptome. In citrus, before microarray availability,
gene expression in developmental and environmentally regulated processes, as in
many other systems, was mostly studied through differential display techniques (i.e., DDRT-PCR). Genes involved in many processes were also identified after subtractive
hybridization of cDNA libraries constructed from two different conditions. The main targets in citrus research have been
those physiological processes that sustain major commercial traits. Below, we summarize the knowledge gained in
these several areas.


### 5.3.1. Fruit Growth and Ripening

While in tomato (a climacteric fruit) great strides have been made in the areas of ethylene regulation, carotenoid
accumulation, and cell wall metabolism, in nonclimacteric citrus fruit the
general information is substantially less. Mature citrus fruits release low amounts of ethylene but respond to
exogenous ethylene by accelerating respiration, chlorophyll degradation, and
carotenoid deposition. In these fruits,
very low rates of ethylene production have been associated with constitutive
expression of the 1-aminocyclopropane-1-carboxylate synthase 2 (CsACS2) and ethylene receptor CsETR1 genes,
indicating that citrus possesses a system I machinery. However, it has been reported that a
climacteric-like rise in ethylene production, preceded by induction of the
genes for CsACS1, ACC oxidase1, and the ethylene receptor CsERS1,
characteristic of a system II-like, appears to be present in young fruitlets [56]. It is well known that ethylene accelerates
the molecular changes in the carotenoid biosynthesis naturally occurring during
maturation (see below), while
gibberellins and nitrates, two ripening retardants, reduce expression of early
carotenoid biosynthetic genes and repress pheophorbide a oxygenase (PaO) expression [57, 58], a gene involved in
chlorophyll disappearance. Other
characteristic genes induced by ethylene are ferredoxins or thi, a gene involved in thiamine biosynthesis. The large ABA
amounts found in the peel of citrus fruit
during maturation appear to be synthesized by two 9-cis-epoxycarotenoid
dioxygenases (NCED) with differential
spatial and environmental expression [59, 60].In citrus, an initial small-scale
EST sequencing project from mature fruit resulted in the identification of 20%
of the sequences as encoding for metallothionein [61]. The abundance of these kinds of genes was
confirmed later in more developed citrus arrays. Later, Shimada et al. [49] used a citrus cDNA
microarray containing 2213 independent genes to examine gene expression during
fruit development and reported that the expression profile in the different
tissues of the fruit, flesh, albedo, and flavedo was rather different. Recently,
a comprehensive transcriptome analysis using a citrus 22 K oligoarray was
performed to identify ethylene-responsive genes in mandarin fruit [50]. In the 72 hours after ethylene
treatment, 1493 genes were shown to be modulated by the hormone. Ethylene repressed the transcription of most
genes involved in photosynthesis, chloroplast biogenesis, and sugar metabolism,
while it induced the transcription of several genes related to resistance,
defense, stress, amino acid synthesis, protein degradation, and secondary
metabolism. The sensitivity and
responsive patterns to exogenous ethylene were significantly different among
carotenoid biosynthesis genes (see below). Furthermore, most of the ethylene
biosynthesis genes and its signal transduction components did not show any
significant expression change after ethylene treatment. Interestingly, a type II ethylene receptor (ETR2) showed higher sensitivity to exogenous ethylene
than two other type I ethylene receptors (CsETR1 and CsERS1), suggesting that ETR2 might be associated
with low ethylene sensitivity in mature fruit [50].During the last decades, research on citrus fruit flavor that depends
upon multiple compounds, mostly sugars, acids, and flavanones, has received
considerable attention because of both the uniqueness of the physiological
processes sustaining this trait and the potential importance of these
components to human health. To date, the
most comprehensive study on the transcriptome profiling of the citrus fruit
flesh was presented by Cercós et al. [62] who examined gene expression with the
first generation Spanish cDNA microarray during development and ripening of
self-incompatible Citrus clementina. They reported that as many as 2243 putative
unigenes showed significant expression changes while functional classification
revealed that genes encoding for regulatory proteins were most significantly
overrepresented approximately within the middle of the rapid fruit growth phase;
this suggested that fruits at this stage were reprogramming developmental
commands to face the complex cellular modifications during ripening. Most
pivotal changes were related to carbohydrate build up, acid reduction,
modifications in secondary metabolism, carotenoid accumulation, and chlorophyll
decreases. Alterations of the
transcriptome associated with carbon accumulation were expected since it was
known that expression of several homologues of pivotal genes implicated in
carbon metabolism (e.g., phosphoenolpyruvate
carboxykinase, ADP-glucose pyrophosporylase, sucrose synthase, and sucrose
phosphatase synthase) and transport (i.e.,
sucrose transporters) during fruit growth considerably changed [63, 64]. In general, these genes appear to
belong to small families including few members, showing differential spatial
and temporal expression.On the other hand, developing
citrus fruits accumulate
a considerable amount of citric acid in the vacuoles of the juice sac cells,
although before ripening this high concentration is considerably reduced. The rate of change and final acid levels are
perceived as major components for citrus fruit quality. Research in gene regulation of acid
metabolism, however, has not led to a full understanding of this essential
process. There is considerable evidence,
nevertheless, obtained comparing acidless and acidic varieties that activity
and expression of citrate synthase were not responsible for these differences [65]. Another gene characterized in citrus was NADP(+)-isocitrate dehydrogenase (NADP-IDH),
encoding for an enzyme involved in citrate metabolism. Recently, a citrate transporter gene has been
reported encoding a novel vacuolar citrate/symporter that is able to mediate
citrate vacuolar efflux through the electroneutral cotransport of H+ and citrate
ions [66].  Interestingly, the
transcriptomic study together with the analyses of selected metabolites
suggested the occurrence of specific metabolic alternatives during citric acid
catabolism [62]. Microarray data suggested
that citrate was sequentially metabolized to glutamate that was finally catabolized
through the gamma-aminobutyrate (GABA) shunt. This observation was of special
relevance since it linked an efficient major proton-consuming reaction with
high acid levels. This work provides a
convincing explanation for the strong reduction of both citrate and cytoplasmic
acidity that takes place in citrus fruit flesh during development and
ripening.Transcript profiling also
revealed down-regulation patterns of gene expression for anthocyanin and flavonoid
biosynthesis, confirming previous observations. Thus, it was known that in common oranges there was a differential
repression of some of the enzymes of anthocyanin biosynthetic pathway, namely
chalcone synthase (CHS), anthocyanidin
synthase (ANS), and
UDP-glucose-flavonoid 3-O-glucosyltransferase (UFGT) [67], in contrast to “blood” pigmented oranges. However, anthocyanin and gene expression associated with anthocyanin
synthesis increased at low temperature [68]. Flavanones, a flavonoid subgroup, that greatly contribute to the bitter flavor
of grapefruit and other citrus, have also been the subject of intensive work
and pivotal genes of this biosynthetic pathway such as CHS, chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H),
dihydroflavonol 4-reductase (DFR), and
flavonol synthase (FLS) have been isolated and
characterized [69, 70]. In an elegant
work, Frydman et al. [71] demonstrated that the key flavor-determining step of
citrus flavanone biosynthesis was catalyzed by rhamnosyltransferases. They demonstrated that 1,2
rhamnosyltransferases catalyzed biosynthesis of the bitter neohesperidosides,
while 1,6 rhamnosyltransferases catalyzed biosynthesis of the tasteless rutinosides. Bitter species, such as grapefruit and
pummelo, accumulated bitter flavanone-7-O-neohesperidosides (naringin, the major flavonoid glycoside in grapefruit) responsible, in part, for their characteristic juice flavor, while nonbitter
species, such as mandarin and orange, accumulated only tasteless flavanone-7-O-rutinosides.Bitterness in citrus also is
associated with the presence of limonoids, triterpene derivatives that confer
the scent to fresh lemon and oranges. Kita
et al. [72] isolated a cDNA clone encoding limonoid UDP-glucosyltransferase (limonoid GTase) that regulated the conversion of
limonoid aglycones such as limonin, a bitter compound, to their nonbitter
glucosides.In addition to limonoids,
citrus fruits possess unique aromas rarely found in other fruit species
produced by other terpenes. This is also
an area of high research interest. Monoterpenes
(d-limonene terpinene and pinene) and other low-abundance sesquiterpenes (valencene,
nootkatone, and α- and β-sinensal) stand out in citrus as
important aroma and also flavor compounds. Lücker et al. [73] and Shimada et al. [74] isolated various monoterpene
synthases (d-limonene, γ-terpinene, β-pinene
synthase, β-ocimene, and cineole synthase), and it has also
been shown that their metabolic engineering produced new aromas in tobacco [75].  Monoterpene synthesis takes place in
epithelial cells surrounding the secretory cavities that contain the oil glands
in the flavedo [76]. Regarding
sesquiterpene production, Sharon-Asa et al. [77] identified a sesquiterpene
synthase-encoding gene, regulating the conversion of farnesyl diphosphate to a
single sesquiterpene, valencene. They
reported the transcript that was responsive to ethylene naturally accumulated
only towards fruit maturation. Other
putative sesquiterpene synthases, such as β-farnesene synthase, have also been
cloned.Work is also in progress to
characterize induced mutants that exhibit altered fragrance (alf) and abnormal number
of oil glands in the flavedo [78]. Transcriptome
analysis of fruits from these mutants showed changes in expression profile of
genes encoding enzymes involved in the biosynthesis of volatile compounds
derived from isoprenoid and phenylpropanoid pathways. In fruits of alf, several genes with different biological functions were down-regulated
although genes coding for a new putative terpene synthase (TPS) and an O-methyltransferase (OMT), apparently involved in secondary metabolism of
volatile compounds, had the highest differences in expression. In the mutant with lower number of glands, transcript
profiling also revealed strong down-regulation of genes encoding enzymes from
the phenylpropanoid and isoprenoid biosynthetic pathways. In a similar approach, Ishikawa et al. [79] used
a 22 K citrus microarray to analyze gene expression in a mutant that shows
smoother rind and decreased numbers of oil glands. The authors reported that the genes of the
nonmevalonate pathway of isoprenoid synthesis and monoterpene synthases were
down-regulated in the mutant.Tetraterpenes are also
crucial components of citrus fruit that contains one of the greatest arrays of carotenoids
found in any plant. The simultaneous
carotenoid accumulation and chlorophyll reduction occurring during natural ripening
indeed determines the color of the fruit peel, a most valuable characteristic
of perceived fruit quality. Many pivotal
genes of the carotenoid pathway have been cloned in citrus (phytoene
synthase (CitPSY), phytoene desaturase (CitPDS), ζ-carotene (car) desaturase (CitZDS), carotenoid isomerase (CitCRTISO),
lycopeneβ-cyclase (CitLCYb), β-ring
hydroxylase (CitHYb), zeaxanthin (CitZEA) epoxidase (CitZEP),
lycopene β-cyclase (CitLCYb), and
lycopene ε-cyclase (CitLCYe), and their
expression has been correlated with the accumulation of carotenoids in fruit [80, 81]. It was reported that the transition
of peel color from green to orange, and the change from β,ε-carotenoid to
β,β-carotenoid accumulation was accompanied by the disappearance of CitLCYe and
the increase in CitLCYb transcripts. As
fruit maturation progressed, a concomitant increase in the expression of
CitPSY, CitPDS, CitZDS, CitLCYb, CitHYb, and CitZEP led to massive
β,β-xanthophyll accumulation. Cercós et
al. [62] showed that expression of carotenoid biosynthetic genes in fruit flesh
followed rather similar changes. Mutations
of flesh color are being investigated in China
using a citrus cDNA array
with 6000 unigenes [52].In contrast to carotenoid accumulation, there have been fewer studies of
the chlorophyll degradation processes in citrus. Previous work on the regulation of catabolism
showed that chlorophyllase (CLH) was constitutively expressed during natural fruit development [82]. Recent results suggest that CLH functions as a rate-limiting enzyme
in chlorophyllcatabolism controlled via post-translational
regulation [83]. It is also known that
pheophorbide a oxygenase (PaO) and geranylgeranyl reductase expression, correlated with chlorophyll
degradation [57]. Recent work upon “nan,”
a stay-green mutant of Navel orange that produces fruit with abnormal brown
flavedo, showed that typical ripening-related chlorophyll (Chl) degradation was impaired in this mutant. Transcript and proteomic profilings revealed that a citrus orthologue of
a number of SGR (stay green) genes was expressed at substantially
lower levels in ‘‘nan’’ both
prior to and during ripening [84]. The “nan” mutation also resulted in the suppressed expression of
numerous photosynthesis-related genes and in the induction of genes associated
with oxidative stress. The transcriptome
of other selected citrus mutants is also being investigated to identify gene
functions related to fruit quality that in citrus are barely accessible through
genetic approaches. To this end, three
collections of induced mutated lines (EMS,
gamma rays and fast neutrons) have been generated, comprising
10 000 potential [84] mutants.With
the exception of thermostable pectin methylesterase activity [85, 86] that
greatly reduces citrus juice quality, cell wall metabolism in citrus has been
studied as related to fruit abscission, a major component of final yield. One of the strategies for the identification
of abscission-related genes followed by Dr. Burns’ team (University of Florida) was based upon
the isolation of ethylene-induced genes in the calyx, the laminar, and the floral
abscission zones. The role of ethylene
on the regulation of abscission has been widely illustrated for decades, and
several works have shown that ethylene is the primary effector activating the abscission
pathway in citrus [87, 88]. Differential
display and subtractive cDNA library screening were also used to search for
abscission-related metabolism changes. Important
components of the citrus abscission process were thus associated with
expression and/or activity of pivotal enzymes of cell wall metabolism (glucanases, polygalacturonases, galactosidases, and other
hydrolases; [89, 90]), hormonal synthesis, and signal
transduction (i.e., ACC synthases and oxydases) and secondary metabolism/PR proteins (i.e.,
phenylalanine ammonia lyase, chitinases). Yuan et al. [91] also demonstrated that
differential expression of ACC synthase 1 and ACC oxidase genes was associated
with reduction of ethephon-enhanced leaf abscission by guanfacine, a G-protein-coupled
alpha-(2A)-adrenoreceptor selective antagonist,
and suggested a link between G-protein-related signalling and abscission. Interestingly, guanfacine had little effect
on ethephon-enhanced fruit loosening. In
spite of this information, major regulators of the abscission process in citrus
are still mostly unknown although both custom manufactured and large-scale
microarrays, in some instances, coupled to laser assisted microdissection (LAM) are currently being used in Florida and Spain
to gain new insight into this process. Part
of these transcriptomic profiling studies has been summarized in a recent Ph.D.
dissertation presenting a model of leaf abscission events occurring at the laminar
abscission zone [60].  The two-stage
model proposes a first phase of activation, mostly characterized by the
activation of signalling pathways (hormones,
phospholipids, calcium, and oxygen reactive species). In a second stage, the execution phase,
degradation of the cell wall by hydrolytic enzymes would be culminated and
sugar-nucleotide metabolism for cell elongation induced. The process would end with the promotion of a
double defensive program intended to protect the living zone remaining attached
to the plant including deposition of physical barriers (callose
and lignin) and induction of pathogen resistance.Several other microarray
studies on citrus growth and ripening are under development and have not been
published yet. For instance, Dr. Sadka
is investigating with the GeneChip Citrus Genome Array (Affymetrix) the transcriptome modifications occurring during the induction of flower bud
differentiation using “on” and “off” trees [51], taking advantage of the
alternate bearing behavior, a process regulating differentially
carbohydrate-related gene expression [92]. In Brazil, the
sequencing carried out at the Centro APTA Citros “Sylvio Moreira”—IAC (Brazil) that has generated one of the most important databases for this genus in the
world is being extensively used to produce “in
silico” analyses. This approach is yielding information not
only related to fruit growth and development (terpene
production, cell wall metabolism, etc.) but also in the biotic
stress field [48].


### 5.3.2. Responses to Pathogenic and Environmental Stresses

In citrus, gene expression associated with the responses to biotic and
abiotic stresses has targeted a limited number of genes in spite of the
economical importance of the citrus diseases and environmental constrains. Multiple
pathogens provoke a range of citrus disorders, mostly fungal (leaf spot,Alternaria; mold, Penicillium; post-bloom
fruit drop, Colletotrichum acutatum;
root rot, Phytophthora), bacterial (canker, Xanthomonas axonopodis;
citrus variegated chlorosis, Xylella
fastidiosa; Huanglongbing or
greening, Candidatus Liberibacter),
and viral diseases (citrus tristeza virus, CTV; citrus
leprosis virus, CiLV). Environmental
stresses include cold temperatures, drought, flooding, salinity, and high and
low soil pH, among others.In response to the
inoculation with conidia ofAlternaria,
at least two cytosolic antifungal miraculins with protease inhibitor activity
were strongly up regulated. Actually,
induction of miraculin expression is one of the most prominent responses
observed in microarray experiments performed in open field experiments. Both miraculin genes responded to methyl
jasmonate and were antagonized by salicylate [93]. Several studies with the green mold pathogen, Penicillium digitatum, also indicated
that genes such as thioredoxins, the gnsl gene (beta-1,3-endoglucanase activity),
and chitinases are major components of the molecular mechanisms involved in
activation of pathogen defense in citrus. Other responsive genes reported in citrus were epoxide hydrolase and
hidroperoxide lyase. It has also been
shown that the fungus Colletotrichum acutatum altered hormonal homeostasis increasing both levels of ethylene,
indole-3-acetic acid, cis-jasmonic acid (JA) and salicylic acid (SA), and
associated gene expression [94].Gandía et al. [95] have
recently presented data on the transcriptional response of citrus to infection
with severe and mild isolates of citrus tristeza virus. These studies concluded that gene expression
was only significantly altered with the severe isolate. Changes detected in the citrus transcriptome
after infection with this isolate were predominantly associated with symptom
expression (chlorophyllases, SAM transferases, ACC oxidase,
and lipid transfer proteins), defense mechanism, and general
responses to stress (miraculins, superoxide dismutases,
glutation transferees, NBS-LRR resistance genes, thioredoxin, protease
inhibitors, ubiquitin ligases, etc.).To study the mechanisms of canker
resistance in kumquat, a custom microarray using 2254 ESTs from subtractive
libraries is being utilized to determine the response to infective bacteria in
an incompatible interaction [54]. The
macroscopic phenotype, a delayed hypersensitive response in the inoculated
leaves, was accompanied by altered expression of 1245 genes. This study identified major components of the
incompatible interaction, reactive oxygen species (ROS) production, and programmed cell death (PCD). In addition, a number of common defense
mechanisms besides a number of resistance genes and putative receptors were
also identified.Citrus plants are also very liable to infestation by
aphids, whitefly, and other insects as well as being susceptible to herbivory. Mozoruk et al. [53]
described how nylon filter cDNA arrays were used to analyze the transcriptional
changes of 1731 citrus unigenes that resulted from herbivory by a xylem-feeding
leafhopper, Homalodisca coagulata. Insect feeding led to a significant
expression change in 50 transcripts broadly functioning in direct defense,
defense signalling, ROS scavenging, transport, cell wall modification,
photosynthesis, and abiotic stress. The
authors also noted that the transcript profile recorded greatly resembled that
induced by wounding, likely through JA-independent pathways. In contrast to similar studies with aphids, SA-dependent
pathogenesis related genes were weakly induced.Although transcriptional
profiling using microarrays has developed into the most prominent tool for
functional genomics, none has yet reported on the effects and responses of
citrus to the major environmental constraints (salinity,
flooding, water deficit, chilling, and iron deficiency). High-throughput analyses of gene expression
in citrus challenged with major abiotic stresses, however, are currently
underway in several laboratories around the world and will soon produce
valuable information that might eventually lead to discovery of novel genes and
functions. For instance, it is known that in citrus, physiological
disturbances produced by salinity are associated with leaf chloride build up
rather than with sodium accumulation, as observed in many plants [96, 97]. Genes in principle associated with the
response of citrus to salinity were initially obtained
from a cDNA expression library of citrus salt-treated cell suspensions. These genes, homologues to phospholipid
hydroperoxides, glutathione peroxidases [98],  olesins, Lea5, or lypoxygenases,
were involved in the oxidative response rather than in the specific response to
salinity. Other genes involved in
oxidative stress well known in citrus are glutathione S-transferases [99] and
copper/zinc-superoxide dismutases. However,
recent microarray analyses are providing much-needed insights into chloride
tolerance mechanisms and short- and long-term adaptation of citrus to salinity. Several teams are engaged in a
Euro-Mediterranean MPC INCO project, started in 2006, between Spain, Morocco,
Tunisia, Turkey, and France
focused on tolerances to salinity
and iron deficiency associated with alkaline soils. One of the major components of this project
is the large-scale study and genome-wide acquisition of quantitative biological
information on gene expression from multiple tolerant and susceptible genotypes. Work on transcriptomic comparisons in this
area is confirming that Cl- is the most important ion involved in
the genetic response of citrus to salinity. In addition, major metabolic regulation changes are also apparent during
salinity acclimatization in tolerant rootstocks. In contrast, flooding is mostly characterized
by the rise of oxidative stress.Chilling
resistance in citrus is another area that has received much attention but lacks
current comprehensive gene expression analyses provided by microarrays. Since most commercially important citrus
varieties are cold-sensitive and therefore susceptible to freezing,Poncirus trifoliata (L.),
an interfertile Citrus relative that
can tolerate temperatures as low as −26°C after acclimation, is being used for
improving cold tolerance in citrus rootstocks and as a source for the
identification of cold-regulated genes. In
general, many studies have been performed through subtractive hybridization [100]
and DDRT-PCR [101] comparing expression in sensitive and resistant varieties. It has been shown, for example, that
expression of a C-repeat-binding factor (CBF) and one of its targets, COR19,
a cold-induced gene, accumulated both earlier and to higher levels in Poncirus. Moreover, COR19, COR11 [102], and COR15 were
found to belong to an unusual group 2 LEA gene family responsive to low
temperature. These dehydrins differ from
most other plant dehydrins in having an unusual K-segment similar to that of
gymnosperms and in having a serine cluster (S-segment) at an unusual position at the carboxy-terminus [103]. Citrus, however, also possesses the typical
plant angiosperm-type K-segment consensus sequence. Other up-regulated transcripts that may play
a role in cold sensitivity are a novel RING-H2 finger gene, AP2 domain
containing genes and CTL, and a homologue of a low-temperature-responsive gene
from Arabidopsis. During postharvest storage, chilling injury
in citrus fruit can be reduced by previous short heat treatments that activate
different molecular responses. Genes
differentially expressed in the chilling response have mostly been related to
lipid membrane and cell wall enzymes, to main regulators of secondary
metabolism and hormonal homeostasis, and to oxidative and general stress
responses [104, 105].Although the main
applications of microarrays to date are in transcriptome profiling analyses,
microarrays can also be used to study DNA variation. Oligonucleotide arrays are particularly
suited for the detection of single nucleotide mismatches during hybridization
and, hence, for the discovery of novel DNA variants or the determination of
known variants. The citrus GeneChip, for
example, was designed to genotype 3219 genes using 5023 SNPs. The 20 K Spanish Consortium microarrays have
been used to identify heterozygous deletions in fast neutron irradiated citrus
mutants through array-based comparative genomic hybridization (array-CGH) and
to study gene colinearity. Preliminary
CGH yielded several candidate genes that were in haploid gene dosage. After comparison with theArabidopsis and Populus genomes, it was observed that Populus orthologues of Citrus deleted genes grouped in two duplicated chromosomes in contrast to Arabidopsis orthologues that were
distributed in several chromosomes (Ríos and Talon, unpublished
data).


## 5.3.1. Fruit Growth and Ripening

While in tomato (a climacteric fruit) great strides have been made in the areas of ethylene regulation, carotenoid
accumulation, and cell wall metabolism, in nonclimacteric citrus fruit the
general information is substantially less. Mature citrus fruits release low amounts of ethylene but respond to
exogenous ethylene by accelerating respiration, chlorophyll degradation, and
carotenoid deposition. In these fruits,
very low rates of ethylene production have been associated with constitutive
expression of the 1-aminocyclopropane-1-carboxylate synthase 2 (CsACS2) and ethylene receptor CsETR1 genes,
indicating that citrus possesses a system I machinery. However, it has been reported that a
climacteric-like rise in ethylene production, preceded by induction of the
genes for CsACS1, ACC oxidase1, and the ethylene receptor CsERS1,
characteristic of a system II-like, appears to be present in young fruitlets [56]. It is well known that ethylene accelerates
the molecular changes in the carotenoid biosynthesis naturally occurring during
maturation (see below), while
gibberellins and nitrates, two ripening retardants, reduce expression of early
carotenoid biosynthetic genes and repress pheophorbide a oxygenase (PaO) expression [57, 58], a gene involved in
chlorophyll disappearance. Other
characteristic genes induced by ethylene are ferredoxins or thi, a gene involved in thiamine biosynthesis. The large ABA
amounts found in the peel of citrus fruit
during maturation appear to be synthesized by two 9-cis-epoxycarotenoid
dioxygenases (NCED) with differential
spatial and environmental expression [59, 60].In citrus, an initial small-scale
EST sequencing project from mature fruit resulted in the identification of 20%
of the sequences as encoding for metallothionein [61]. The abundance of these kinds of genes was
confirmed later in more developed citrus arrays. Later, Shimada et al. [49] used a citrus cDNA
microarray containing 2213 independent genes to examine gene expression during
fruit development and reported that the expression profile in the different
tissues of the fruit, flesh, albedo, and flavedo was rather different. Recently,
a comprehensive transcriptome analysis using a citrus 22 K oligoarray was
performed to identify ethylene-responsive genes in mandarin fruit [50]. In the 72 hours after ethylene
treatment, 1493 genes were shown to be modulated by the hormone. Ethylene repressed the transcription of most
genes involved in photosynthesis, chloroplast biogenesis, and sugar metabolism,
while it induced the transcription of several genes related to resistance,
defense, stress, amino acid synthesis, protein degradation, and secondary
metabolism. The sensitivity and
responsive patterns to exogenous ethylene were significantly different among
carotenoid biosynthesis genes (see below). Furthermore, most of the ethylene
biosynthesis genes and its signal transduction components did not show any
significant expression change after ethylene treatment. Interestingly, a type II ethylene receptor (ETR2) showed higher sensitivity to exogenous ethylene
than two other type I ethylene receptors (CsETR1 and CsERS1), suggesting that ETR2 might be associated
with low ethylene sensitivity in mature fruit [50].During the last decades, research on citrus fruit flavor that depends
upon multiple compounds, mostly sugars, acids, and flavanones, has received
considerable attention because of both the uniqueness of the physiological
processes sustaining this trait and the potential importance of these
components to human health. To date, the
most comprehensive study on the transcriptome profiling of the citrus fruit
flesh was presented by Cercós et al. [62] who examined gene expression with the
first generation Spanish cDNA microarray during development and ripening of
self-incompatible Citrus clementina. They reported that as many as 2243 putative
unigenes showed significant expression changes while functional classification
revealed that genes encoding for regulatory proteins were most significantly
overrepresented approximately within the middle of the rapid fruit growth phase;
this suggested that fruits at this stage were reprogramming developmental
commands to face the complex cellular modifications during ripening. Most
pivotal changes were related to carbohydrate build up, acid reduction,
modifications in secondary metabolism, carotenoid accumulation, and chlorophyll
decreases. Alterations of the
transcriptome associated with carbon accumulation were expected since it was
known that expression of several homologues of pivotal genes implicated in
carbon metabolism (e.g., phosphoenolpyruvate
carboxykinase, ADP-glucose pyrophosporylase, sucrose synthase, and sucrose
phosphatase synthase) and transport (i.e.,
sucrose transporters) during fruit growth considerably changed [63, 64]. In general, these genes appear to
belong to small families including few members, showing differential spatial
and temporal expression.On the other hand, developing
citrus fruits accumulate
a considerable amount of citric acid in the vacuoles of the juice sac cells,
although before ripening this high concentration is considerably reduced. The rate of change and final acid levels are
perceived as major components for citrus fruit quality. Research in gene regulation of acid
metabolism, however, has not led to a full understanding of this essential
process. There is considerable evidence,
nevertheless, obtained comparing acidless and acidic varieties that activity
and expression of citrate synthase were not responsible for these differences [65]. Another gene characterized in citrus was NADP(+)-isocitrate dehydrogenase (NADP-IDH),
encoding for an enzyme involved in citrate metabolism. Recently, a citrate transporter gene has been
reported encoding a novel vacuolar citrate/symporter that is able to mediate
citrate vacuolar efflux through the electroneutral cotransport of H+ and citrate
ions [66].  Interestingly, the
transcriptomic study together with the analyses of selected metabolites
suggested the occurrence of specific metabolic alternatives during citric acid
catabolism [62]. Microarray data suggested
that citrate was sequentially metabolized to glutamate that was finally catabolized
through the gamma-aminobutyrate (GABA) shunt. This observation was of special
relevance since it linked an efficient major proton-consuming reaction with
high acid levels. This work provides a
convincing explanation for the strong reduction of both citrate and cytoplasmic
acidity that takes place in citrus fruit flesh during development and
ripening.Transcript profiling also
revealed down-regulation patterns of gene expression for anthocyanin and flavonoid
biosynthesis, confirming previous observations. Thus, it was known that in common oranges there was a differential
repression of some of the enzymes of anthocyanin biosynthetic pathway, namely
chalcone synthase (CHS), anthocyanidin
synthase (ANS), and
UDP-glucose-flavonoid 3-O-glucosyltransferase (UFGT) [67], in contrast to “blood” pigmented oranges. However, anthocyanin and gene expression associated with anthocyanin
synthesis increased at low temperature [68]. Flavanones, a flavonoid subgroup, that greatly contribute to the bitter flavor
of grapefruit and other citrus, have also been the subject of intensive work
and pivotal genes of this biosynthetic pathway such as CHS, chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H),
dihydroflavonol 4-reductase (DFR), and
flavonol synthase (FLS) have been isolated and
characterized [69, 70]. In an elegant
work, Frydman et al. [71] demonstrated that the key flavor-determining step of
citrus flavanone biosynthesis was catalyzed by rhamnosyltransferases. They demonstrated that 1,2
rhamnosyltransferases catalyzed biosynthesis of the bitter neohesperidosides,
while 1,6 rhamnosyltransferases catalyzed biosynthesis of the tasteless rutinosides. Bitter species, such as grapefruit and
pummelo, accumulated bitter flavanone-7-O-neohesperidosides (naringin, the major flavonoid glycoside in grapefruit) responsible, in part, for their characteristic juice flavor, while nonbitter
species, such as mandarin and orange, accumulated only tasteless flavanone-7-O-rutinosides.Bitterness in citrus also is
associated with the presence of limonoids, triterpene derivatives that confer
the scent to fresh lemon and oranges. Kita
et al. [72] isolated a cDNA clone encoding limonoid UDP-glucosyltransferase (limonoid GTase) that regulated the conversion of
limonoid aglycones such as limonin, a bitter compound, to their nonbitter
glucosides.In addition to limonoids,
citrus fruits possess unique aromas rarely found in other fruit species
produced by other terpenes. This is also
an area of high research interest. Monoterpenes
(d-limonene terpinene and pinene) and other low-abundance sesquiterpenes (valencene,
nootkatone, and α- and β-sinensal) stand out in citrus as
important aroma and also flavor compounds. Lücker et al. [73] and Shimada et al. [74] isolated various monoterpene
synthases (d-limonene, γ-terpinene, β-pinene
synthase, β-ocimene, and cineole synthase), and it has also
been shown that their metabolic engineering produced new aromas in tobacco [75].  Monoterpene synthesis takes place in
epithelial cells surrounding the secretory cavities that contain the oil glands
in the flavedo [76]. Regarding
sesquiterpene production, Sharon-Asa et al. [77] identified a sesquiterpene
synthase-encoding gene, regulating the conversion of farnesyl diphosphate to a
single sesquiterpene, valencene. They
reported the transcript that was responsive to ethylene naturally accumulated
only towards fruit maturation. Other
putative sesquiterpene synthases, such as β-farnesene synthase, have also been
cloned.Work is also in progress to
characterize induced mutants that exhibit altered fragrance (alf) and abnormal number
of oil glands in the flavedo [78]. Transcriptome
analysis of fruits from these mutants showed changes in expression profile of
genes encoding enzymes involved in the biosynthesis of volatile compounds
derived from isoprenoid and phenylpropanoid pathways. In fruits of alf, several genes with different biological functions were down-regulated
although genes coding for a new putative terpene synthase (TPS) and an O-methyltransferase (OMT), apparently involved in secondary metabolism of
volatile compounds, had the highest differences in expression. In the mutant with lower number of glands, transcript
profiling also revealed strong down-regulation of genes encoding enzymes from
the phenylpropanoid and isoprenoid biosynthetic pathways. In a similar approach, Ishikawa et al. [79] used
a 22 K citrus microarray to analyze gene expression in a mutant that shows
smoother rind and decreased numbers of oil glands. The authors reported that the genes of the
nonmevalonate pathway of isoprenoid synthesis and monoterpene synthases were
down-regulated in the mutant.Tetraterpenes are also
crucial components of citrus fruit that contains one of the greatest arrays of carotenoids
found in any plant. The simultaneous
carotenoid accumulation and chlorophyll reduction occurring during natural ripening
indeed determines the color of the fruit peel, a most valuable characteristic
of perceived fruit quality. Many pivotal
genes of the carotenoid pathway have been cloned in citrus (phytoene
synthase (CitPSY), phytoene desaturase (CitPDS), ζ-carotene (car) desaturase (CitZDS), carotenoid isomerase (CitCRTISO),
lycopeneβ-cyclase (CitLCYb), β-ring
hydroxylase (CitHYb), zeaxanthin (CitZEA) epoxidase (CitZEP),
lycopene β-cyclase (CitLCYb), and
lycopene ε-cyclase (CitLCYe), and their
expression has been correlated with the accumulation of carotenoids in fruit [80, 81]. It was reported that the transition
of peel color from green to orange, and the change from β,ε-carotenoid to
β,β-carotenoid accumulation was accompanied by the disappearance of CitLCYe and
the increase in CitLCYb transcripts. As
fruit maturation progressed, a concomitant increase in the expression of
CitPSY, CitPDS, CitZDS, CitLCYb, CitHYb, and CitZEP led to massive
β,β-xanthophyll accumulation. Cercós et
al. [62] showed that expression of carotenoid biosynthetic genes in fruit flesh
followed rather similar changes. Mutations
of flesh color are being investigated in China
using a citrus cDNA array
with 6000 unigenes [52].In contrast to carotenoid accumulation, there have been fewer studies of
the chlorophyll degradation processes in citrus. Previous work on the regulation of catabolism
showed that chlorophyllase (CLH) was constitutively expressed during natural fruit development [82]. Recent results suggest that CLH functions as a rate-limiting enzyme
in chlorophyllcatabolism controlled via post-translational
regulation [83]. It is also known that
pheophorbide a oxygenase (PaO) and geranylgeranyl reductase expression, correlated with chlorophyll
degradation [57]. Recent work upon “nan,”
a stay-green mutant of Navel orange that produces fruit with abnormal brown
flavedo, showed that typical ripening-related chlorophyll (Chl) degradation was impaired in this mutant. Transcript and proteomic profilings revealed that a citrus orthologue of
a number of SGR (stay green) genes was expressed at substantially
lower levels in ‘‘nan’’ both
prior to and during ripening [84]. The “nan” mutation also resulted in the suppressed expression of
numerous photosynthesis-related genes and in the induction of genes associated
with oxidative stress. The transcriptome
of other selected citrus mutants is also being investigated to identify gene
functions related to fruit quality that in citrus are barely accessible through
genetic approaches. To this end, three
collections of induced mutated lines (EMS,
gamma rays and fast neutrons) have been generated, comprising
10 000 potential [84] mutants.With
the exception of thermostable pectin methylesterase activity [85, 86] that
greatly reduces citrus juice quality, cell wall metabolism in citrus has been
studied as related to fruit abscission, a major component of final yield. One of the strategies for the identification
of abscission-related genes followed by Dr. Burns’ team (University of Florida) was based upon
the isolation of ethylene-induced genes in the calyx, the laminar, and the floral
abscission zones. The role of ethylene
on the regulation of abscission has been widely illustrated for decades, and
several works have shown that ethylene is the primary effector activating the abscission
pathway in citrus [87, 88]. Differential
display and subtractive cDNA library screening were also used to search for
abscission-related metabolism changes. Important
components of the citrus abscission process were thus associated with
expression and/or activity of pivotal enzymes of cell wall metabolism (glucanases, polygalacturonases, galactosidases, and other
hydrolases; [89, 90]), hormonal synthesis, and signal
transduction (i.e., ACC synthases and oxydases) and secondary metabolism/PR proteins (i.e.,
phenylalanine ammonia lyase, chitinases). Yuan et al. [91] also demonstrated that
differential expression of ACC synthase 1 and ACC oxidase genes was associated
with reduction of ethephon-enhanced leaf abscission by guanfacine, a G-protein-coupled
alpha-(2A)-adrenoreceptor selective antagonist,
and suggested a link between G-protein-related signalling and abscission. Interestingly, guanfacine had little effect
on ethephon-enhanced fruit loosening. In
spite of this information, major regulators of the abscission process in citrus
are still mostly unknown although both custom manufactured and large-scale
microarrays, in some instances, coupled to laser assisted microdissection (LAM) are currently being used in Florida and Spain
to gain new insight into this process. Part
of these transcriptomic profiling studies has been summarized in a recent Ph.D.
dissertation presenting a model of leaf abscission events occurring at the laminar
abscission zone [60].  The two-stage
model proposes a first phase of activation, mostly characterized by the
activation of signalling pathways (hormones,
phospholipids, calcium, and oxygen reactive species). In a second stage, the execution phase,
degradation of the cell wall by hydrolytic enzymes would be culminated and
sugar-nucleotide metabolism for cell elongation induced. The process would end with the promotion of a
double defensive program intended to protect the living zone remaining attached
to the plant including deposition of physical barriers (callose
and lignin) and induction of pathogen resistance.Several other microarray
studies on citrus growth and ripening are under development and have not been
published yet. For instance, Dr. Sadka
is investigating with the GeneChip Citrus Genome Array (Affymetrix) the transcriptome modifications occurring during the induction of flower bud
differentiation using “on” and “off” trees [51], taking advantage of the
alternate bearing behavior, a process regulating differentially
carbohydrate-related gene expression [92]. In Brazil, the
sequencing carried out at the Centro APTA Citros “Sylvio Moreira”—IAC (Brazil) that has generated one of the most important databases for this genus in the
world is being extensively used to produce “in
silico” analyses. This approach is yielding information not
only related to fruit growth and development (terpene
production, cell wall metabolism, etc.) but also in the biotic
stress field [48].


## 5.3.2. Responses to Pathogenic and Environmental Stresses

In citrus, gene expression associated with the responses to biotic and
abiotic stresses has targeted a limited number of genes in spite of the
economical importance of the citrus diseases and environmental constrains. Multiple
pathogens provoke a range of citrus disorders, mostly fungal (leaf spot,Alternaria; mold, Penicillium; post-bloom
fruit drop, Colletotrichum acutatum;
root rot, Phytophthora), bacterial (canker, Xanthomonas axonopodis;
citrus variegated chlorosis, Xylella
fastidiosa; Huanglongbing or
greening, Candidatus Liberibacter),
and viral diseases (citrus tristeza virus, CTV; citrus
leprosis virus, CiLV). Environmental
stresses include cold temperatures, drought, flooding, salinity, and high and
low soil pH, among others.In response to the
inoculation with conidia ofAlternaria,
at least two cytosolic antifungal miraculins with protease inhibitor activity
were strongly up regulated. Actually,
induction of miraculin expression is one of the most prominent responses
observed in microarray experiments performed in open field experiments. Both miraculin genes responded to methyl
jasmonate and were antagonized by salicylate [93]. Several studies with the green mold pathogen, Penicillium digitatum, also indicated
that genes such as thioredoxins, the gnsl gene (beta-1,3-endoglucanase activity),
and chitinases are major components of the molecular mechanisms involved in
activation of pathogen defense in citrus. Other responsive genes reported in citrus were epoxide hydrolase and
hidroperoxide lyase. It has also been
shown that the fungus Colletotrichum acutatum altered hormonal homeostasis increasing both levels of ethylene,
indole-3-acetic acid, cis-jasmonic acid (JA) and salicylic acid (SA), and
associated gene expression [94].Gandía et al. [95] have
recently presented data on the transcriptional response of citrus to infection
with severe and mild isolates of citrus tristeza virus. These studies concluded that gene expression
was only significantly altered with the severe isolate. Changes detected in the citrus transcriptome
after infection with this isolate were predominantly associated with symptom
expression (chlorophyllases, SAM transferases, ACC oxidase,
and lipid transfer proteins), defense mechanism, and general
responses to stress (miraculins, superoxide dismutases,
glutation transferees, NBS-LRR resistance genes, thioredoxin, protease
inhibitors, ubiquitin ligases, etc.).To study the mechanisms of canker
resistance in kumquat, a custom microarray using 2254 ESTs from subtractive
libraries is being utilized to determine the response to infective bacteria in
an incompatible interaction [54]. The
macroscopic phenotype, a delayed hypersensitive response in the inoculated
leaves, was accompanied by altered expression of 1245 genes. This study identified major components of the
incompatible interaction, reactive oxygen species (ROS) production, and programmed cell death (PCD). In addition, a number of common defense
mechanisms besides a number of resistance genes and putative receptors were
also identified.Citrus plants are also very liable to infestation by
aphids, whitefly, and other insects as well as being susceptible to herbivory. Mozoruk et al. [53]
described how nylon filter cDNA arrays were used to analyze the transcriptional
changes of 1731 citrus unigenes that resulted from herbivory by a xylem-feeding
leafhopper, Homalodisca coagulata. Insect feeding led to a significant
expression change in 50 transcripts broadly functioning in direct defense,
defense signalling, ROS scavenging, transport, cell wall modification,
photosynthesis, and abiotic stress. The
authors also noted that the transcript profile recorded greatly resembled that
induced by wounding, likely through JA-independent pathways. In contrast to similar studies with aphids, SA-dependent
pathogenesis related genes were weakly induced.Although transcriptional
profiling using microarrays has developed into the most prominent tool for
functional genomics, none has yet reported on the effects and responses of
citrus to the major environmental constraints (salinity,
flooding, water deficit, chilling, and iron deficiency). High-throughput analyses of gene expression
in citrus challenged with major abiotic stresses, however, are currently
underway in several laboratories around the world and will soon produce
valuable information that might eventually lead to discovery of novel genes and
functions. For instance, it is known that in citrus, physiological
disturbances produced by salinity are associated with leaf chloride build up
rather than with sodium accumulation, as observed in many plants [96, 97]. Genes in principle associated with the
response of citrus to salinity were initially obtained
from a cDNA expression library of citrus salt-treated cell suspensions. These genes, homologues to phospholipid
hydroperoxides, glutathione peroxidases [98],  olesins, Lea5, or lypoxygenases,
were involved in the oxidative response rather than in the specific response to
salinity. Other genes involved in
oxidative stress well known in citrus are glutathione S-transferases [99] and
copper/zinc-superoxide dismutases. However,
recent microarray analyses are providing much-needed insights into chloride
tolerance mechanisms and short- and long-term adaptation of citrus to salinity. Several teams are engaged in a
Euro-Mediterranean MPC INCO project, started in 2006, between Spain, Morocco,
Tunisia, Turkey, and France
focused on tolerances to salinity
and iron deficiency associated with alkaline soils. One of the major components of this project
is the large-scale study and genome-wide acquisition of quantitative biological
information on gene expression from multiple tolerant and susceptible genotypes. Work on transcriptomic comparisons in this
area is confirming that Cl- is the most important ion involved in
the genetic response of citrus to salinity. In addition, major metabolic regulation changes are also apparent during
salinity acclimatization in tolerant rootstocks. In contrast, flooding is mostly characterized
by the rise of oxidative stress.Chilling
resistance in citrus is another area that has received much attention but lacks
current comprehensive gene expression analyses provided by microarrays. Since most commercially important citrus
varieties are cold-sensitive and therefore susceptible to freezing,Poncirus trifoliata (L.),
an interfertile Citrus relative that
can tolerate temperatures as low as −26°C after acclimation, is being used for
improving cold tolerance in citrus rootstocks and as a source for the
identification of cold-regulated genes. In
general, many studies have been performed through subtractive hybridization [100]
and DDRT-PCR [101] comparing expression in sensitive and resistant varieties. It has been shown, for example, that
expression of a C-repeat-binding factor (CBF) and one of its targets, COR19,
a cold-induced gene, accumulated both earlier and to higher levels in Poncirus. Moreover, COR19, COR11 [102], and COR15 were
found to belong to an unusual group 2 LEA gene family responsive to low
temperature. These dehydrins differ from
most other plant dehydrins in having an unusual K-segment similar to that of
gymnosperms and in having a serine cluster (S-segment) at an unusual position at the carboxy-terminus [103]. Citrus, however, also possesses the typical
plant angiosperm-type K-segment consensus sequence. Other up-regulated transcripts that may play
a role in cold sensitivity are a novel RING-H2 finger gene, AP2 domain
containing genes and CTL, and a homologue of a low-temperature-responsive gene
from Arabidopsis. During postharvest storage, chilling injury
in citrus fruit can be reduced by previous short heat treatments that activate
different molecular responses. Genes
differentially expressed in the chilling response have mostly been related to
lipid membrane and cell wall enzymes, to main regulators of secondary
metabolism and hormonal homeostasis, and to oxidative and general stress
responses [104, 105].Although the main
applications of microarrays to date are in transcriptome profiling analyses,
microarrays can also be used to study DNA variation. Oligonucleotide arrays are particularly
suited for the detection of single nucleotide mismatches during hybridization
and, hence, for the discovery of novel DNA variants or the determination of
known variants. The citrus GeneChip, for
example, was designed to genotype 3219 genes using 5023 SNPs. The 20 K Spanish Consortium microarrays have
been used to identify heterozygous deletions in fast neutron irradiated citrus
mutants through array-based comparative genomic hybridization (array-CGH) and
to study gene colinearity. Preliminary
CGH yielded several candidate genes that were in haploid gene dosage. After comparison with theArabidopsis and Populus genomes, it was observed that Populus orthologues of Citrus deleted genes grouped in two duplicated chromosomes in contrast to Arabidopsis orthologues that were
distributed in several chromosomes (Ríos and Talon, unpublished
data).


## 5.4. Genetic Transformation

Citrus transformation procedures, in general, followAg-  robacterium tumefaciens protocols, and subsequent regen- eration through organogenesis and somatic
embryogenesis are also rather typical and straightforward [106]. Trans-  formation efficiency of young material
is usually low, 15–20%, and a major achievement to overcome the juvenility
limitation was the direct transformation of adult material [107].In
citrus, genetic transformation is mostly being explored as an alternative to
classical genetic breeding and not many examples can be found in the literature
illustrating the use of genetic transformation for functional genomics. For example, there is interest in modulating the growth habit of rootstocks since this might eventually
affect the development of the scion and facilitate diverse cultural practices (e.g., pruning, pesticide applications, and harvesting). Thus, it
was known that the ectopic overexpression in tobacco of a citrus GA 20-oxidase,
a regulatory step of gibberellin biosynthesis in citrus, [108] enhanced gibberellin
content and shoot growth [109]. Later, Fagoaga
et al. [110] generated transgenic Carrizo rootstocks overexpressing this GA
20-oxidase and confirmed that the gene controls gibberellin flux through the
pathway since taller (sense) and shorter
(antisense) phenotypes correlating with
higher and lower levels of active GA1 were obtained. In these transgenic lines, however, cell
division was more affected than cell elongation, in contrast to the effects
observed in herbaceous plants [111]. In
another example, an antisense construction with a citrus ACC synthase gene
repressed ACC increase after a chilling treatment. A pectin methylesterase gene (Cs-PME4) isolated from sweet orange to prevent
juice cloud separation was also introduced via protoplasts and subsequent
regeneration through somatic embryogenesis [112].Generally,
characteristics related to commercial valuable traits are modified through the
use of transgenes. To accelerate
flowering time, Carrizo seedlings constitutively overexpressing theArabidopsis floral-regulatory genes LEAFY (LFY) or APETALA1 (AP1) were generated [113]. Both kind of
transgenic citrus produced fertile flowers in their first year considerably shortening
the juvenile phase. Consistently with
the role of LFY and AP1, juvenility
in citrus was positively correlated with CsTFL (homolog
to TERMINAL FLOWER) transcript accumulation and negatively
correlated with LEAFY and APETALA1 RNA levels [114].  In a similar approach but with a citrus gene,
it was showed that transgenic Poncirus carrying the CiFT gene (homolog to FLOWERING LOCUS T), another flowering time gene, also exhibited early
flowering although this phenotype was accompanied with several pleiotropic
effects [115]. It is possible that the early
flowering AP1 and CiFT transgenic
citrus could be used as rapid cycling genotypes for functional genomics studies. In a further example, Carrizo rootstock constitutively expressing a Δ1-pyrroline-5-carboxylate
synthetase mutant gene from Vigna,
showed higher water deficit tolerance [116]. Regarding tolerance to
stresses, however, a huge amount of work has been centered on resistance to
biotic stresses, a matter of major relevance in citrus industry. Thus, tolerance or resistance to Phytophthora
citrophthora, the most widely spread oomycete in citrus growing areas, was
generated by introducing the gene P23, that codes for a pathogenesis-related protein induced in
tomato. These results provided evidence
for the antifungal activity in vivo of the P23 pathogenesis-related protein
against P. citrophthora [117].A great
effort is also being developed to understand the basis of the tolerance to
citrus tristeza virus (CTV), the causal
agent of the most important virus disease in citrus. The strategy is generally supported by the
concept of pathogen-derived resistance (PDR),
based on expression of viral sequences interfering with the virus life cycle in
plants. CTV resistant transformants have
been obtained by genetically engineering thep25 and p23 genes
from CTV [118]. However, it still
remains to be elucidated if transgenic citrus plants expressing CTV-derived
sequences are a plausible alternative to cross protection to control CTV strains
in the field. In an alternative strategy,
heterologous expression of plant-derived resistance genes is promoted to confer
resistance against CTV. General
resistance to CTV has been found in Poncirus trifoliata, and a region
containing the resistance gene (Ctv) has been characterized. Furthermore,
work is under way for other pivotal diseases such as citrus mosaic virus (CiMV), citrus canker, and citrus blight.


## 5.5. Reverse Genetics

In addition to genetic transformation, the capability to perform reverse
genetic analyses is crucial to develop functional studies. The
creation of transgenic lines is a powerful and straightforward way to determine
gene function. However, in citrus, high-throughput
transgenic programs such as the generation of RNA interference knockouts,
activation tagging through enhancer elements, gene-trap T-DNA insertions, or
transposable tagging systems have not yet been developed. The capacity for the maintenance and
characterization of many transgenic lines of a perennial tree with both a long
juvenile phase, large individual plant size, and a complex reproductive biology
has probably hindered these developments. InPopulus, however, activation tagging and insertional mutagenesis approaches are been
explored despite logistical challenges in working with transgenic trees, a
direction that may well be followed by citrus researchers in the near future.


## 5.6. Tilling/Fast Neutrons

Since gene disruption is the most effective method to analyze gene
functions and no efficient tagging or insertional methods are available in
citrus, strategies based on genome-wide mutagenesis such as TILLING (targeted induced local lesions in genomes) and fast
neutron mutagenesis are being explored further [84]. These approaches are nontransgenic and have
particular interest for the industry where the debate on GMOs has restricted their
application in crop improvement. TILLING
identifies individuals carrying point mutations while the fast neutron
mutagenized population is searched for gene deletions using PCR amplification. Both approaches, at the moment, are of
limited usefulness as strategies for reverse genetics in citrus because of the
lack of genomic sequences and the large amounts of space required for mutated
populations of suitable size. ECOTILLING,
however, on natural citrus variants and microarray-based detection of deletions
on fast neutron citrus mutants in a more direct genetics strategy are very
straightforward approaches. However,
unless a high-throughput transformation protocol is developed for citrus,
functionally analyzing all genes with tagging approaches or genome-wide
mutagenesis and screening are not realistic strategies.


## 5.7. Viral-Induced Gene Silencing

Viral-induced
gene silencing (VIGS), on the other
hand, is an attractive and very promising alternative in citrus. Knocking out the expression of a gene by VIGS
does not require genetic transformation and has proven to be a very efficient
tool for function analysis of plant genes. VIGS is particularly suitable for woody plants like citrus with long
juvenile periods that require long periods between transformation and fruiting. In a hopeful work, Dr. Guerri and colleagues at IVIA have
recently showed that VIGS might be possible in citrus usingCitrus leaf
blotchvirus (CLBV) as a viral
vector (Dr. Guerri, personal communication). These workers cloned a full-length cDNA of
the CLBV genome [119] in a binary vector under the control of the 35S promoter
and demonstrated that tobacco and citrus plants Agro-infiltrated with this construct became infected and
replicated CLBV normally. Recently, they
have showed that tobacco plants
Agro-infiltrated with a CLBV chimeric construct carrying a fragment of the
phytoene desaturase gene developed photobleaching symptoms and reduced the
cognate transcripts. Parallel
experiments in citrus are planned. Availability
of the CLBV-based vector will certainly open new possibilities to study
functional genomics in citrus.


## 5.8. Proteomics/Metabolomics

Other powerful approaches for functional genomics studies such as
proteomics and metabolomics to comprehensively analyze proteomes and phenotypes
have just begun for citrus. For example,
Blumwald and coworkers (UCD) are using two main approaches, namely 2D
gel analyses coupled with MALDI-TOF-TOF from juice sac cell vacuoles and LC2-MS-MS
analyses of ER/Golgi, plasma membrane, tonoplast, mitochondria, and soluble
enriched fractions from citrus juice sac cells to define the “citrus fruit
proteome.” Current work is in progress
but they have already reported the identification of over 1500 proteins
involved in sugar metabolism, citrate cycle, signalling, transport, and
processing and have characterized changes in protein expression during
development [120]. In a further example,
proteome changes in the fruit albedo during postharvest ageing were studied
through 2D-PAGE, and relevant proteins were also identified through mass
spectrometry determinations [121]. This proteomic survey indicated that major
changes in protein content (ATP synthase beta subunit,
ascorbate peroxidase, translationally
controlled tumor protein, cysteine protease, etc.) were
apparently related to the activation of programmed cell death.Numerous analyses of citrus
metabolites, especially of ripening and matured fruits, have been reported in
the past. However, new methods to
characterize the metabolic phenotypes of representative lines from mutants and
natural varieties must be developed. Metabolic
profiling and metabolomic procedures using state-of-the-art gas chromatography-mass
spectrometry or fast gas chromatography-time-of-flight mass spectrometry need
to be setup.The final objective of citrus
functional genomics is to identify candidate genes, alleles, and genotypes
improving citrus fruit quality, correlating phenotypic analyses, metabolomic
profiling, and gene expression. At
completion, genes and alleles with major functions in nutritional quality could
be selected and genotypes with improved fruit composition searched among
existing collections or generated.


## 6. Conclusion and Future Prospects

This paper has reviewed
various aspects of the current status of citrus genome research, including the
development of fundamental tools, the applications currently under way and
envisaged leading to solutions to seemingly intractable problems facing the
citrus industries of the world, the
opportunities of improving further the perceived and real value of citrus fruit
and products, and the challenges that remain not only for genomic research but
for making progress in truly incorporating new knowledge into new plant
materials. The international citrus
research community has been growing closer together, and new international
alliances are making the achievement of truly great advances possible; this is
essential, as no one group or even nation has sufficient resources to address
all the needs for tool development and deployment, and many of the problems
faced are global in nature. It is clearly evident that by combining research resources and
by adopting the principle of depositing information in the public domain,
freely available to global research partners, the promise of genome research to
improve citrus plants, production, and protection from diseases, and enhanced
product quality and value, can be realized. The free availability of these tools and materials is truly the key to the success
in genomics research. Citrus is a very
important tree fruit crop throughout the world not only is it of great economic
significance but it is also of great value for human nutrition and well-being. In addition, it possesses many unique
characteristics of great biological interest. Consequently, the benefits of an expanded and focused effort into all
aspects of citrus genomics will be of great benefit to humanity in general as
well as to the realm of plant science. Citrus will have a first genome sequenced in the very near future; this
will not be the end of the process but the beginning of many more citrus genome
sequencing projects to add layers of valuable information to the already
developed and developing tools to understand the functions and
interrelationships of genes, their products, and their interactions with the
environment. Through the acquisition of
this knowledge and its application to the field, citrus will continue to be an
economically valuable fruit crop plant and a source of important health and
nutrition benefits to people throughout the world.



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*Source: 528361-2008-05-19.xml*