# Sugarcane Functional Genomics: Gene Discovery for Agronomic Trait Development

Authors: M. Menossi; M. C. Silva-Filho; M. Vincentz; M.-A. Van-Sluys; G. M. Souza
Journal: International Journal of Plant Genomics
 (2008)

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## Abstract

Sugarcane is a highly productive crop used for centuries as the main source of sugar and recently to produce ethanol, a renewable bio-fuel energy source. There is increased interest in this crop due to the impending need to decrease fossil fuel usage. Sugarcane has a highly polyploid genome. Expressed sequence tag (EST) sequencing has significantly contributed to gene discovery and expression studies used to associate function with sugarcane genes. A significant amount of data exists on regulatory events controlling responses to herbivory, drought, and phosphate deficiency, which cause important constraints on yield and on endophytic bacteria, which are highly beneficial. The means to reduce drought, phosphate deficiency, and herbivory by the sugarcane borer have a negative impact on the environment. Improved tolerance for these constraints is being sought. Sugarcane's ability to accumulate sucrose up to 16% of its culm dry weight is a challenge for genetic manipulation. Genome-based technology such as cDNA microarray data indicates genes associated with sugar content that may be used to develop new varieties improved for sucrose content or for traits that restrict the expansion of the cultivated land. The genes can also be used as molecular markers of agronomic traits in traditional breeding programs.


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## Body

## 1. Sugarcane: A Highly Successful Crop  with a Challenging Genome

Sugarcane is an important tropical crop and has served as a source of sugar for hundreds of years. With
an originally soft, watery culm sugarcane acquired through human selection a
distinctive feature of partitioning carbon into sucrose in the stem. The
striking ability of accumulating levels of sucrose that can reach around 0.7 M in mature internodes [1] is an almost unique feature in cultivated plants.Sugarcane is cultivated in
more than 20 million hectares in tropical and subtropical regions of the world,
producing up to 1.3 billion metric tons of crushable stems. It is generally
used to produce sugar, accounting for almost two thirds of the world’s
production and has recently gained increased attention because ethanol derived
from cane sugar represents an important renewable biofuel source, which could
turn it into a global commodity and important energy source. Sugarcane bagasse
(the major waste product generated by sugar mills after extraction of the
sucrose from cane juice) is largely used for energy cogeneration at the mill or
for the production of animal feed increasing the overall efficiency of the crop
system. Recently, there has been increased interest in using bagasse for
processes such as paper production, as a dietary fiber in bread, as a wood
substitute in the production of wood composite, and in the synthesis of carbon
fibres [2–6]. It is expected that enzymatic and hydrolytic processes
that allow the bagasse carbon units from cellulose and hemicellulose to be
fermented, will soon be scaled up for ethanol production, turning sugarcane
into an efficient crop for energy production.Commercial sugarcane relies on vegetative propagation
through stem cuttings to generate a new clonal plant, resulting from lateral
bud growth, and subsequently stools, with a large number of tillers. In 12
months the plant will reach 4-5
meters, with extractable culms measuring 2-3 meters and a sugar content
of 13–16%. After harvest, underground buds will sprout starting a new crop
season. In most situations 4–6 harvests are possible before the field is
renewed. After each harvest, leaves and plant toppings removed from the stems
are left in the fields allowing for nutrient recycling, soil protection and
growth without crop rotation.Sugarcane belongs to the genusSaccharum L composed of hybrids [7, 8] derived from Saccharum
officinarum (Noble clones), S.
sinense (Chinese clones), S. barberi (North Indian clones), and S. spontaneum [9]. The hybrids are highly polyploid and aneuploid and
on average contain 100–120 chromosomes with an estimated somatic cell size of
10,000 Mbp  [10]. The number of chromosomes can vary in commercial
cultivars. The basic genome size ranges from 760 to 926 Mbp, which is twice the
size of the rice genome (389 Mbp) and similar to sorghum's (760 Mbp) [11]. Even in the face of the economic importance, it
represents to many countries, the complexity of the sugarcane genome inhibited
large efforts and investments in the development of biotechnology and genetic
tools for this crop. Cultivar improvement has been achieved over the years
using traditional breeding, which can take up to 15 years of selections.
Nevertheless sugarcane transgenics are still lagging behind. Herbicide-,
herbivory-, and viral-resistant transgenic plants have been reported but so far
there has been no commercial release. This is probably due to intellectual
property and regulatory issues, but may also be related to the fact that for
complex traits, such as sucrose content, the genes to be used have not yet been
proved ideal for improving agronomic performance. Gene discovery and
identification is essential for breeding programs, either for transgenic plant
development or for marker-assisted breeding.The complete genome
sequence of a sugarcane cultivar is not yet available. Significant progress has
been noted recently with the development of tools such as expressed sequence tags
(ESTs). Large collections have become available to explore the large polyploid
sugarcane genome and consequently renewed the interest in sugarcane genetics
[12–14]. This review will focus on describing EST development
and subsequent progress that led to the identification of genes associated with
agronomic traits of interest in sugarcane. It will also highlight some of the
possible functions of genes associated with sucrose content, including biotic and abiotic stress and the role that
phytohormones may play in the adaptive responses of this plant.


## 2. EST Profiling for Gene Discovery

ESTs represent tags of the
expressed portion of a genome and therefore potentially identify genes encoding
proteins, natural antisense transcripts [15–18], miRNA, transacting siRNA precursors [19, 20],  and more
generally noncoding RNA [21]. The information carried by an EST collection is a
significant starting point to determine an organism's genome content but more
pragmatically, when considering important crops, it can directly point to genes
which may contribute to agronomical trait development (e.g., tolerance to
abiotic and biotic stresses, mineral nutrition, and sugar content amongst others).Several sugarcane ESTs
collections have been developed [22–28]. The publicly available sugarcane ESTs were
assembled into tentative consensus sequences (virtual transcripts), singletons,
and mature transcripts, referred to as the Sugarcane Gene Index (SGI; http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=s_officinarum).
The Brazilian sugarcane EST project collection (SUCEST, http://sucest-fun.org, [26]) generated 237,954 ESTs, which were organized into
43,141 putative unique sugarcane transcripts (26,803 contigs and 16,338
singletons) referred to as sugarcane assembled sequences (SASs). An internal
redundancy analysis suggested that this collection of SASs represented 33,000
sugarcane genes [13, 26, 29] but this estimation was likely to have been an
overestimation, since a two-fold redundancy among SASs that presented
significant similarity with Arabidopsis proteins (60% of the SASs) was detected (M. Vincentz, unpublished data). A
detailed organization of sugarcane genes into functional categories (i.e., signal
transduction components, regulation of gene expression, development, biotic and
abiotic stresses, transposable elements, metabolism, etc., [26]) was completed and represents the basis to develop
functional genomic approaches.The contribution of this
large set of SASs to our understanding of the processes underlying angiosperm
evolution was also of significance. A comparison of the SASs with the DNA and
protein sequences from other angiosperms confirmed that lineage-specific gene
loss, high evolutionary rate of specific sequences, and exon shuffling were
important processes involved in the divergence among angiosperms [30]. Of particular interest are the monocot-specific
sequences that evolve at high rates and are found in members of conserved
angiosperm gene families, because they may lead to functional diversification
and may therefore be related to the differentiation of specific lineages.
Interestingly, two SASs (SCEZSD2038A10.g and SCSFRT2070F09.g), only detected in
sugarcane, sorghum and maize, point to the existence of recent innovations in
the Andropogoneae tribe (M. Vincentz, unpublished) and raise the question of
what kind of adaptive traits are associated with these sequences.Finally, it is important
to note the contribution that the EST collections have made to our
understanding of the sugarcane genome structure, number of alleles and the
complex relationship of specific alleles and allele dosage to phenotypes. Single
sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) have been
annotated in a number of genotypes [31, 32]; and with the advent of pyrosequencing, their
identification has been increasingly adding to our knowledge of large genomes [33]. A comprehensive functional map of the sugarcane
genome has recently been described with an enhanced resolution, creating the means for developing “perfect markers” associated with
key QTL [34].


## 3. Gene Expression Blueprint of  Sugarcane Tissues

The availability of ESTs allows for
large-scale gene expression analysis using a variety of tools. Several studies
have reported an in silicoanalysis of transcript enrichment when
different cDNA libraries were compared [26, 35]. Of the 43,141 SUCEST SASs, 1234 were considered to
be tissue-enriched. The maximum number of ESTs in a tissue-enriched SAS (i.e.,
with higher transcript amounts in one or more tissues) was found to be in
prolamin, which contained 360 ESTs. Developing seeds contained 1902 specific
ESTs (33% of the total), with almost half of these (919) encoding prolamins,
the major seed storage protein found in cereals. These ESTs included six
putative new genes with a high level of expression in seeds (up to 32
ESTs/SAS). The most frequent protein domains found in tissue-enriched SASs were
the protein kinase domain, followed by the trypsin-amylase inhibitor, seed
storage protein, and lipid transfer protein domains. Overall, 13 transcription
factor families were found to be specific for flowers, five for roots, three
for Herbaspirillum-inoculated
plantlets, and two for developing seeds and other tissues.Following
sequence identification, a functional genomics project, the SUCEST-FUN Project
(http:sucest-fun.org), was implemented to associate putative roles with the
sugarcane genes. cDNA microarrays containing sugarcane ESTs were used to
determine temporal and spatial gene expression. Determining the distribution of
gene transcripts in sugarcane tissues has helped define tissue-specific
activities and ubiquitous genes, and point out genes in which promoter
sequences could be searched for. This is of particular interest if one is
interested in directing the expression of transgenes to particular plant
tissues to avoid pleiotropic effects. Individual gene expression variation was
investigated using cDNA microarrays containing 1280 distinct elements, on
plants grown in the field. Transcript abundance in six plant organs (flowers,
roots, leaves, lateral buds, 1st (immature), and 4th (mature) internodes) was analyzed  [36], resulting in the identification of 217 genes with a
tissue-enriched expression patterns, while 153 genes showed highly similar
expression levels in all the tissues analyzed. A virtual profile matrix was
constructed where tissue expression was compared amongst 24 tissue samples.
Amongst the tissue-enriched genes,a
caffeic acid 3-O-methyltransferase
(COMT) gene expressed primarily in the mature internode was identified. This
enzyme is involved in lignin biosynthesis and, in association with other
enzymes like the CCOMT (caffeoyl CoA 3-O-methyltransferase),
keeps the cell lignin content and composition in check. The identification of
this culm-enriched enzyme may lead to improved sugarcane varieties with an altered
lignin content: a trait highly valuable for the paper industry and for those
interested in increasing hydrolysis of the sugarcane bagasse for fermentation
purposes. The tissue specificity data was also evaluated against data from
plants submitted to biotic and abiotic stresses, which can shed light on the
putative roles of newly identified genes, such as genes for which no similarity
has been found with genes in the public databases [37].Active
transcription of transposable elements (TEs) was also detected in the SUCEST
database [38] and enabled the identification of a previously
unknown set of genetic mobile elements in sugarcane. Further studies confirmed
the expression profile of 68 individual TE clones [39]. Four actively dividing tissues were examined
(callus, apical meristem, leaf roll, and flower), and callus was determined to
be the tissue expressing the most diverse group of TEs. Both transposons and
retrotransposons are expressed, which suggest that some of these mobile
elements may have an important role in genome metabolism, as previously
described for other elements in several biological systems [40, 41]. Further analysis of these transcribed TEs revealed
that some of the families were constituted by both bona fide transposable
elements and domesticated variants that had been captured by the plant genome
to perform a yet unknown function [42]. Mutator-like elements were the most expressed
transposons in sugarcane, and four groups were identified that showed
similarity with the MURA transposase protein [43], of which two represented domesticated elements
related to the Mustang-like genes described in rice [42, 44]. Amongst the retrotransposons, Hopscotch-like
sequences, the most prevalent in the SUCEST database, showed a highly diverse
expression profile. Retrotransposons carry their promoter region along the
length of their transcribed mRNA and GUS-fusion expression analyses for three
out of four TEs, this is being confirmed in transient assays [44], leading to the possibility of using these sequences
as promoters for the expression of genes of interest in sugarcane.


## 4. Insights in the Sugarcane Responses  to Biotic Stress

Plants are constantly challenged by a
wide array of biotic stresses, such as herbivorous insects, nematodes, and by
fungal, bacterial, and viral infestations. Phytohormones largely mediate plant
responses to attacks by triggering conserved defence mechanisms, each with an
intricate signalling pathway leading to plant protection. Cross-talk signalling
pathways leading to plant defence have been reported, with synergistic and
antagonistic outcomes [45]. Specific and general responses are mediated by
distinct signals, mainly jasmonic acid, ethylene, and salicylic acid. It has
been shown that both the ethylene and jasmonic acid signalling pathways act
synergistically in plant defence. For example, ethylene synthesis increases the
response to severaltypes of biotic challenges (e.g., bacteria, fungi [46], and insects [47]).
In sugarcane, a putative ethylene receptor and two putative transcription
factors, which are members of the ethylene signalling pathway, have been shown
to be regulated during the association with nitrogen-fixing endophytic bacteria
[48]. In addition, other signals such as green leaf volatiles
(GLVs) may be involved in the orchestration of plant defences since their
production is drastically enhanced when they are under biotic stress [49].Biotic stress is responsible for significant sugarcane
losses, posing a demand for the development of new stress-tolerant cultivars.
In order to reduce insect and pathogen damage, plants have developed complex
and varied defence mechanisms, including chemical and physical barriers. In the
last few years, an extensive amount of work has been undertaken in order to
decipher the sugarcane response to biotic stress, mainly related to some insect
herbivores and pathogens. Amongst the SUCEST sequences, dozens of orthologous
genes involved in the sugarcane response to insect herbivores [50]  and Diazothophic endophytes [51, 52] were identified. Although the sugarcane-endophytic
bacteria interaction is an advantageous association for both organisms, it is
thought that sugarcane plants activate defence responses before the
establishment of such symbiosis [53]. A study based on a wide gene expression analysis of
1,545 genes in sugarcane revealed that Gluconacetobacter
diazotrophicus and Herbaspirillum
seropedicae endophytic bacteria activated distinct classes of defence
proteins, including four plant disease-resistant genes (R-genes), salicylic
acid biosynthesis genes, five transcription factors, and so on. On the other
hand, Diatraea saccharalis herbivory
specifically upregulated the expression of a pathogenesis-related protein
similar to thaumatin [37]. Transcript profiling of sugarcane-resistant plants
to either Ustilago scitaminea or Bipolaris sacchari (also known as Helminthosporium sacchari or Drechslera sacchari), causal agents of
smut and eyespot, respectively, identified 62 differentially regulated genes,
of which 10 were downregulated and 52 were induced. Nineteen out of 52
transcript-derived fragments showed homology to known plant gene sequences,
most being related to defense or signaling [54].A considerable amount of data was
obtained on how the plant hormone methyl jasmonate (MeJa) could be regulating
plant defence reactions [28, 37, 55]. cDNA
microarrays containing 829 ESTs from roots treated with MeJA, and 4793 ESTs
from immature and mature stem tissues were used to evaluate gene expression
changes produced by MeJA [28]. An MeJA solution was applied to the soil containing
the plants, and the roots were harvested after 1, 3, and 10 days. The highest
induction was observed for genes encoding the dirigentprotein, which is involved in lignin assembly and
can protect plants against fungal attack [56]. Gene categories with increased transcript levels
included signal transduction, the phenylpropanoid pathway, oxidative stress,
and MeJA synthesis, indicating that several processes were altered by MeJA. In
agreement with several studies involving transcription profiling, most of the
up- or downregulated genes had unknown functions, reinforcing the great
challenge of understanding plant gene function. Responses of sugarcane leaves
sprayed with MeJA for 0.5, 1, 3, 6, and 12 hours, were investigated using nylon
cDNA arrays containing 1536 ESTs from several cDNA libraries [55]. A total of 15 genes were upregulated, while 11 were
downregulated. As observed in sugarcane roots [28], MeJA changed the expression of genes involved in
several biological processes including transcription (a zinc finger protein),
signalling (a protein kinase), and abiotic stress responses (a
carboxy-peptidase, a peroxidase, and a heat shock factor). The authors
complemented their analysis using a digital mRNA expression profiling of the
differentially expressed genes, providing an overview of their expression
patterns in different sugarcane tissues. These results support the idea that
different in silicostrategies can be used to enrich
functional genomics analyses.Changes in gene expression in leaves
exposed to MeJA were also evaluated using cDNA
microarrays containing 1545 genes [37], mostly corresponding to signal transduction
components [57]. The upregulation of transcription factors (MYB, NAC,
and Aux/IAA) and histone homologues (H4 and H2B) strongly suggested chromatin
remodelling followed by the activation of a cascade of signalling genes.
Several protein kinases were up- and downregulated, indicating a complex network
of sugarcane responses to MeJA.Several
strategies have been used to improve plant defence against insects and
pathogens. The activation of stress-response transcription factors was found to
enhance plant tolerance to fungal and bacterial pathogens in transgenic plants
[58]. However, little is known about the function of other
components of the plant transcription machinery during stress. The
identification and characterization of agronomically-interesting genes related
to herbivores and pathogens is a major challenge for sugarcane functional
genomics.  Several candidates have been
tested in the last few years and incorporated into elite genotypes [59–66]. The heterologous expression of defence-related proteins in
sugarcane, such as the soybean proteinase inhibitors encoding genes [67] or cry proteins from Bacillus thuringiensis [68], has led to increased resistance against the
sugarcane borer D. saccharalis, the
major sugarcane pest in Brazil. In addition, the molecular and functional characterization of cysteine
proteinase inhibitors opened up new perspectives on pathogen control, since
sugarcane cystatins inhibited the growth of the filamentous fungus Trichoderma reesei, suggesting that it
can also be employed to inhibit the growth of pathogenic sugarcane fungi [69]. The use of inducible promoters will have a
significant impact on the effectiveness and management of transgenic plants.
One such promoter has been cloned in sugarcane that responds to the sugarcane
borer (Silva-Filho, unpublished results). Taken together, the combination of
new genes with appropriate regulatory sequences will be a major outcome of the
sugar cane OMICS in breeding programs.


## 5. Assessing Sugarcane Genes Related  to Abiotic Stress

Plants face several restrictions in
their environment and have developed a wide array of strategies to either avoid
or cope with the stress condition. Most of the studies using high throughput
assays, such as cDNA microarrays have been conducted with model plants, such asArabidopsis or species not considered
as tropical crops. Recently, the first insights into the responses of sugarcane
to environmental stress have been provided.Amongst abiotic stress,
water deficit plays a major role, and increasing water scarcity has been
observed throughout the world. Plant irrigation currently accounts for approximately
65% of global freshwater use, indicating that the development of drought-resistant
plant varieties will be a necessity in the near future [70, 71]. Agricultural irrigation is one of the most water
demanding human activities. In the case of sugarcane, agricultural frontiers
are expanding, in part, in areas where irrigation is needed [72, 73].To increase the knowledge on the sugarcane responses to drought, cDNA microarrays
were used to evaluate gene expression in plants submitted to 24, 72, and 120 hours
of water deprivation [37]. Drought stress caused dramatic changes in the gene
expression profile of sugarcane plants, with 93 genes being up- or downregulated. Among the genes differentially expressed,
transcription factor orthologs of the Myb, WRKY, NAC, and DREB proteins, which are
known as role players in the drought responses of other systems [74–77], were upregulated. Sugarcane plants also selectively
activated proteases in response to hydric stress, since a homologue to the
cysteine proteinase RD19A precursor was induced. This gene is also induced by
water stress in Arabidopsis [78].Although it may sound surprising,
another important stress in the case of sugarcane is cold stress, caused by
temperatures below 0°C (freezing) or by low temperatures above 0°C (chilling). Cold stress is unusual in
tropical areas, where most of the world's sugarcane is grown, but occasionally
cold can severely affect crops in these regions. This is because most plants in
the tropics have not developed strategies to avoid the devastating consequences
of cold to the cells [79]. There is evidence that sugarcane varieties differ in
their sensitivity to cold [80], suggesting the presence of alleles that might help
this tropical crop to cope with this stress. These genes would have a great
potential in breeding programs and also in the engineering of sugarcane plants
with higher cold tolerance, a highly valuable trait that would allow the
cultivation of this plant in temperate climates.The first report of the use of cDNA arrays to discover sugarcane genes modulated by
cold stress was conducted by Nogueira et al. [81]. The exposure of sugarcane plantlets to 4°C
repressed the expression of 25 genes, while a further 34 genes were upregulated.
Sugarcane homologues to several genes known to be induced by cold stress were
found together with genes induced by drought in other species. This is probably
because the cold induces the formation of ice, dehydrating the cell.
Interestingly, 20 genes that had not previously been associated with cold or
drought stress were identified, suggesting that sugarcane might activate novel
cold response pathways. One example is the gene encoding a putative
NAD-dependent dehydrogenase that might be involved in the protection against
oxidative stress due to cold exposure. One of the genes, SsNAC23, is a member of the NAC family of transcriptional factors
that are involved in biotic and abiotic stress and development [82]. In a further characterization of SsNAC23, Nogueira
et al. [81] showed that the protein is targeted to the nucleus.
In addition, SsNAC23 transcripts also
increased in response to herbivory and water stress. This data further
reinforces the view that different kinds of stress may have common signalling
pathways. Based on this expression profiling experiment, the authors proposed a
hypothetical model integrating the several components activated by sugarcane in
response to low temperature. The same data analyzed  using PmmA [83] revealed a new set of 30 genes as differentially
expressed. Among the genes upregulated was a putative endonuclease involved in
nucleic acid repair, indicating that low temperature stress might cause DNA damage.
Most genes in this new set were repressed by cold stress, such as those
encoding a myo-inositol 1-phosphate synthase and an MAP Kinase.Several plant responses to
environmental stress are mediated by phytohormones, with a well-known
cross-talk between them [84, 85]. To assess the role of ABA in sugarcane, Rocha et al. [37] sprayed ABA
on sugarcane leaves and evaluated the gene expression profile using the cDNA
arrays described above. Two genes encoding orthologs to receptor Ser/Thr
kinases were upregulated. A phosphatase and a small GTPase were also induced,
while a protein kinase was repressed. These findings help to depict an overview
of the network of ABA
signal transduction in sugarcane. The cDNA array data pinpointed several
aspects of the sugarcane metabolism that seem to have been changed in response
to ABA. For example, changes in the fatty acid composition probably take place, since a
fatty acid desaturase was induced, while transpiration would be decreased due
to the action of a PP2C-like protein homologous to ABI1 and ABI2. Moreover, the
work of Rocha et al. [37] also showed drought responses similar to those
elicited by ABA. For example, two delta-12 oleate desaturases, an S-adenosylmethionine
decarboxylase, and a PP2C-like protein phosphatase were induced by both ABA and
drought.The cross-talk between ABA and MeJA also become
evident from the activation of two genes involved in salicylic acid and MeJA
biosynthesis in the ABA-treated plants [37]. ABA treatment
elicited an antagonistic response between the ABA
and auxin pathways. A gene coding for a
protein similar to the auxin responsive protein GH3 [86] was found to be repressed by ABA. Furthermore, a gene
coding for a protein with a predicted auxin-repressed domain found in
dormancy-associated and auxin-repressed proteins [87] was upregulated by this hormone. The cross-talk of
other hormone signalling pathways during water stress was further highlighted
by the differential expression of several genes encoding proteins involved in
ethylene, gibberellin, salicylic acid biosynthesis, as well as other proteins
involved in hormone perception and action. In the same line, several genes
induced by drought stress were also observed in sugarcane plants exposed to
MeJA, suggesting that this hormone might play a role in gene expression changes
during water deficit in this crop. These genes are interesting tools in the
engineering of plants aimed at increasing drought tolerance. In fact,
transgenic tobacco and rice plants over expressing a DREB protein and an NAC
protein, respectively, had improved performance in response to water scarcity [88, 89].Last, but not least, a study on the
evaluation of sugarcane responses to low P availability was also reported. Most
of the world’s agriculture takes place in soils with low availability of P and
other nutrients [90]. Phosphorus, a key nutrient for plant growth and
development, is taken up as inorganic phosphate (Pi), and most soils have very
low Pi concentrations (around 2
mM) compared to the range 5–20 mM found inside the plant
cells [91]. Soil supplementation with rock phosphate is widely
used to increase P availability, increasing the productivity of several crops,
including sugarcane [92]. However, since the P fertilizers may be exhausted
within the next 60–90 years, and the P released into watercourses increases
eutrophication of the water sources, there is a need to minimize P
fertilization.Phosphorus starvation experiments were used to access the changes in the gene expression profiles and
gain information on the strategies used by sugarcane to overcome this nutrient
deficiency stress [37]. The effect of P deficiency on the gene expression
was evaluated in the roots of sugarcane plantlets. Fourteen genes were found to
be repressed after 6 hours and 48 hours due to the absence of P in the nutrient
solution. Surprisingly, no upregulated genes were identified. This was probably
because of the highly stringent statistical test used, based on the outliers
searching method [93]. When an alternative approach was used, based on the
SOM algorithm [94], 146 genes were found, of which several were upregulated
due to P stress [37]. This is an example of how the use of multiple
statistical tests might improve the reach of large-scale gene expression
profiling. Based on this larger set of genes, it was clear that P starvation
triggered oxidative stress, since genes involved in the detoxification of
reactive oxygen species, such as those encoding a gluthatione S-transferase and
a superoxide dismutase, were induced. The role of GTPases in sugarcane
responses to low P was pointed out by the differential expression of several
small GTPases and their regulators, one Ran GTPase activator, one Rho GTPase
activator, and one Rho GDP dissociation inhibitor. P starvation repressed one
homologue of an auxin-repressed protein. The authors [37] found an interesting link between this protein and
the fact that P-starvation in Arabidopsis caused an increase in the number of lateral roots, which is linked to increased
auxin sensitivity. Interestingly, MeJA treatment also repressed the expression
of this sugarcane gene, again showing a complex cross-talk between the
hormones. Another indication of the hormonal regulation of the root
architecture in response to low P levels was the repression of an EIL
transcription factor, which was involved in root development in rice. The wide array of genes induced by P stress in
sugarcane were in line with the complex responses observed in other species,
such as tomato [95], Arabidopsis [96], white lupin [97], and rice [98].


## 6. The Search for Regulators of Sugar  Synthesis, Transport, and Accumulation

CO2 fixed during photosynthesis is used to synthesize carbohydrates [1, 99, 100]. Several adaptations were developed by some grass
species, such as sugarcane and maize, aiming at optimizing CO2 fixation for carbohydrate biosynthesis. They developed a distinct carbon cycle,
which defines them as the “C4 plants” [101]. The compound transported in bundle sheath cells
(malate or aspartate), or the compound returned to the mesophyll cells (alanine
or pyruvate), varies between the species. Also, different enzymes are involved
in the decarboxylation reactions: phosphoenolpyruvate carboxykinase, NAD malic
enzyme, and NADP malic enzyme (which is the case with sugarcane) [102].  In sugarcane
leaves, CO2 fixation starts in the mesophyll cells, where CO2 is combined with phosphoenol pyruvate acid in a reaction catalysed by
phosphenolpyruvate carboxylase. The resulting C4 compound, oxaloacetate, is
converted to malate by NADP-malate dehydrogenase. Malate is transported to the
bundle sheath cells and decarboxylated by the NADP-malic enzyme, releasing (and
concentrating) CO2 for the RuBisCo action, which catalyzes the
carboxylation of CO2 with ribulose-1,5-bisphosphate, the first step
in the Benson-Calvin cycle. Glyceraldehyde 3-phosphate is formed and after
several steps during which fructose-1,6-bisphosphatase (FBPase) and
sucrose-phosphate synthase (SPS) play a major control role, sucrose is
synthesized. Sucrose transfer to the phloem cells allows for its transport to
the parenchyma cells located in the stem, the major sink tissue in sugarcane [100]. All these steps raise the possibility that the
sugarcane sucrose content in the stem could be even higher, considering that it
is possible, at least theoretically, to have higher rates of phloem loading
transport to the stalks, to its parenchyma cells, and finally to the vacuoles
of these cells, as well as the control of the use of sucrose for vegetative
growth [100].The interest in sugar
transporters is obvious in sugarcane, and recent findings have indicated that
sink strength is a driver for photosynthesis [103], highlighting their potential for sugarcane improvement.
The SUCEST database contains nine monosaccharide and four disaccharide
transporters (M. Menossi, unpublished results), and this diversity of transporters is in line with the findings that
sugar transport involves either symplastic or apoplastic steps [104, 105]. An EST survey comparing transcripts from immature
and mature internodes revealed transcripts encoding proteins homologous with
known sugar transporters more abundant in the mature internodes [24]. The only sugarcane transporter showing high
selectivity for sucrose, ShSUT1, was characterized in [106, 107]. This protein is supposed to act in the loading of
sucrose from the vascular tissue into the parenchyma cells from the stem.The large-scale analysis
of gene expression in a population segregated for brix was used to identify
genes associated with sucrose content [108]. The plants analyzed derived from multiple crossings among S. officinarum and S. spontaneum genotypes, and from commercial varieties selected
for sugar content over 12–15 years. Sucrose accumulating internodes from field
grown plants were assayed using cDNA microarrays containing 1545 elements.
Transcriptome comparisons aimed at identifying differentially expressed genes
were made by comparing high-sugar and low-sugar plants directly, and also by
comparing high-sugar and low-sugar internodes. A total of 125 genes were found
to have expression patterns correlated with sugar content. Genes encoding
SNF-related kinases and involved in auxin signalling were found, providing
insights into the regulatory network that might control sucrose accumulation.
Intriguingly, several proteins related to stress responses, such as cytochrome
P450 monoxygenases, were also found. Approximately half of the sucrose
content-associated genes were found to be developmentally regulated during culm
maturation, and many were related to stress responses. A comparison of this
differential expression dataset with the results obtained when the plants were
submitted to drought [37] revealed that approximately half of the genes
identified as associated  with the
sucrose content were responsive to drought. They belonged to several functional
categories including calcium signalling, stress responses, and protein
phosphorylation. The data indicated that the
sucrose accumulating tissues activate pathways during culm development, which
overlap with drought and other stress responses such as cold and injury. This
is corroborated by the observation that several SnRKs associated with the
sucrose content and with drought belonged to the SnRK2 and SnRK3 family of
kinases involved in osmotic stress responses [109].The usefulness of
evaluating progenies for gene expression studies has been reviewed by Casu et
al. [110]. From their studies of a progeny contrasting for
sucrose content, they showed that few of the differentially expressed genes
were involved in carbohydrate metabolism. Additionally, a collection of 7409
ESTs from maturing sugarcane stems in combination with a smaller collection
(1089) of ESTs from immature stems [23, 24, 111] were
analyzed  by bioinformatic techniques and
by cDNA microarray methods, allowing for the identification of genes that are
differentially regulated by stem maturity. The low level of sucrose metabolism
gene expression observed indicated that when the culm matured and the sugar
content increased, so sucrose synthesis and catalysis decreased. GeneChips from
Affymetrix containing approximately 6,024 distinct S. officinarum genes were also used to study culm maturation,
leading to the identification of the developmentally regulated genes involved
in cellulose synthesis, cell wall metabolism, and lignification [112].Source tissues might
affect the efficiency and control of carbon fixation and allocation [113]. Gene expression in source tissues has been
investigated in sugarcane using the EST analysis conducted by Ma et al. [27], and more recently using SAGE [114]. As pointed out before, the sugarcane photosynthetic
carbon cycle is suspected to rely on the NADP malic enzyme pathway [115], but a high expression of a phosphoenolpyruvate carboxykinase
was found in the bundle sheath cells from maize leaves, a species  considered to employ the same mechanism as
sugarcane [116, 117]. Recent evidence [114] indicated that the phosphoenolpyruvate carboxykinase
might even be more active than the NADP malic enzyme in sugarcane leaves. This
data highlighted how large-scale gene expression profiling can help in
understanding complex traits such as sucrose content. In another study of
sugarcane leaves, 24 genes were found to be differentially expressed in plants
with high- and low-sugar content, selected from an F1 segregating population
derived from a cross between two commercial sugarcane varieties [118]. Evidence that hormone signalling is related to the
sucrose content was also found. One of the upregulated genes encoded an omega-3
fatty acid desaturase that might be involved in methyl jasmonate signalling.
Other genes associated with high brix include a receptor-like serine/threonine
kinase and a transcription factor containing an Myb domain. Surprisingly, from
the 24 differentially expressed genes, 19 were more expressed in plants
containing low-sugar content. Three of these genes encoded 14-3-3 like
proteins, which have been found to reduce SPS activity [119, 120].  Another
encoded an SNF1-related protein similar to a protein quinase that
phosphorylates SPS in vitro [121] making it a target for the interaction with 14-3-3
proteins, which in turn reduces SPS activity. This data reinforced the
usefulness of genomic approaches to uncover how sucrose metabolism can be
regulated in sugarcane.


## 7. Concluding Remarks

Sugarcane cultivars tolerant to
drought, cold, and poor soils are increasingly important in countries that are
aiming to expand their plantations. The impending need to decrease fossil fuel
usage together with the fact that ethanol is a less-pollutant renewable source
of energy has renewed interest in the cultivation of sugarcane around the
world, and many countries are developing an ethanol/biofuel industry. Cultivars
adapted to grow in colder climates and high altitudes would be a highly
attractive option. In Brazil, the largest sugarcane producer, sugarcane
cultivation increased 11.20% in 2007 and the area planted increased by 7.4% [73]. This expansion occurred mostly in pastures and was
possible due to the increased usage of irrigation and of new varieties adapted
to the climate and to the soil of the regions.
Small increases in sucrose content also contributed to the increased
productivity.Knowledge of the plant
responses to drought, cold, and low levels of P help to provide a framework for
improving sugarcane production using biotechnological tools. The possibility of
using these genes as markers for breeding purposes or by genetic engineering of
the sugarcane will certainly reduce the impact of the sugarcane crop on the
environment. For example, the use of plants capable of growing in low-P soil
would lead to reduced liming in thecerrados (savannas), which is a new agricultural frontier, and is characterized by low P
availability. Additionally, as stated before, a large amount of the water used
by men goes into agriculture, and improvements in sugarcane drought tolerance
would reduce the impact on the water supply. A better understanding of how
sugarcane plants cope with cold and drought stresses could aid in the
development of cultivars better suited to particular areas.The classical breeding of
sugarcane takes 15 years of crosses and agronomical evaluation before a new
cultivar is released for commercialisation. Gene discovery through the SUCEST
sequencing program has been a major breakthrough for the breeding programs
throughout the world, and functional studies based on cDNA arrays are
uncovering pathways of plant adaptation and responses to the environment. EST-simple sequence
repeats (SSRs) have been successfully used for genetic relationship analysis,
extending the knowledge of the genetic diversity of sugarcane to a functional
level. Development of new markers based on ESTs and their integration in
genetic maps will renew breeding programs and help MAB technology speed up the
breeding programs.For over ten years now [122, 123] the directed genetic modification of sugarcane has
been a reality in laboratories, and field trials have been conducted [124–127]. Genes can be silenced or overexpressed to study
their function and to produce new phenotypes not possible through conventional
breeding. Metabolic profiling associated with gene expression
studies are certainly the future tools of the sugarcane industry. Also, the
analysis of the transcriptome in transgenic plants altered for genes of
interest would certainly prove to be an excellent tool to unravel sugarcane
regulatory networks associated with important traits.



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*Source: 458732-2007-12-16.xml*