bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ratio‐imaging of Ca2+i in the self‐incompatibility response in pollen tubes of Papaver rhoeas
Authors: Franklin‐Tong Vernonica E. , Hackett Grant , Hepler Peter K. 
Published: March 2003

The data presented here describe ratio‐imaging of in intracellular free calcium (Ca2+i) during the self‐incompatibility (SI) response in pollen. Use of the ratiometric indicator, fura‐2 dextran, in pollen tubes of Papaver rhoeas has provided new, detailed information about the spatial‐temporal alterations in Ca2+i, and has permitted calibration of alterations in the concentration of intracellular free calcium ([Ca2+]i) in the SI response.Ratio images demonstrate that, like other pollen tubes, normally growing P. rhoeas pollen tubes exhibit a tip‐focused gradient of Ca2+bfi, with levels reaching 1–2 μM at the extreme apex of the pollen tube. Non‐growing pollen tubes did not exhibit this tip‐focused gradient. Basal levels of Ca2+i in the shank of the pollen tube were fairly consistent and had a mean value of 210 nM, with low‐level fluctuations +/− 50 nM observed. Challenge with incompatible S proteins resulted in S‐specific, rapid and dramatic alterations in [Ca2+]i within a few seconds of challenge. Increases in [Ca2+]i were visualized in the subapical/shank regions of the pollen tube and alterations in [Ca2+]i in this region subsequently increased for several minutes, reaching> 1.5 μM. At the pollen tube tip, a diminution of the tip‐focused gradient was observed, which following some fluctuation, was reduced to basal levels within ∼1 min. Our data suggest that some of these alterations in [Ca2+]i might be interpreted as a calcium wave, as the changes are not global. Although the increases in [Ca2+]i in the subapical/shank region are very rapid, because tip [Ca2+]i oscillates during normal growth, it is difficult to ascertain whether the increases in the shank of the pollen tube precede the decreases in [Ca2+]i at the pollen tube tip.
Gene identification with sequenced T‐DNA tags generated by transformation of Arabidopsis cell suspension
Authors: Mathur Jaideep , Szabados László , Schaefer Sabine , Grunenberg Britta , Lossow Andrea , Jonas‐Straube Esther , Schell Jeff , Koncz Csaba , Koncz‐Kálmán Zsuzsanna 
Published: March 2003

SummaryA protocol for establishment and high‐frequency Agrobacterium‐mediated transformation of morphogenic Arabidopsis cell suspensions was developed to facilitate saturation mutagenesis and identification of plant genes by sequenced T‐DNA tags. Thirty‐two self‐circularized T‐DNA tagged chromosomal loci were isolated from 21 transgenic plants by plasmid rescue and long‐range inverse polymerase chain reaction (LR‐iPCR). By bidirectional sequencing of the ends of T‐DNA‐linked plant DNA segments, nine T‐DNA inserts were thus localized in genes coding for the Arabidopsis ASK1 kinase, cyclin 3b, J‐domain protein, farnesyl diphosphate synthase, ORF02, an unknown EST, and homologues of a copper amine oxidase, a peripheral Golgi protein and a maize pollen‐specific transcript. In addition, 16 genes were identified in the vicinity of sequenced T‐DNA tags illustrating the efficiency of genome analysis by insertional mutagenesis.
Condensing enzymes from Cuphea wrightii associated with medium chain fatty acid biosynthesis
Authors: Slabaugh Mary B. , Leonard Jeffrey M. , Knapp Steven J. 
Published: March 2003

SummarySeed oils of most Cuphea species contain > 90% medium chain (C8–C14) fatty acids. Thioesterases with specificity for these substrates are important determinants of the medium chain phenotype. The role of condensing enzymes, however, has not been investigated. cDNA clones encoding β‐ketoacyl‐acyl carrier protein (ACP) synthase (KAS) were isolated from C. wrightii, a C10/C12‐producing species. Deduced amino acid sequences of four unique clones were ∼ 60% identical to plant KAS I sequences and ∼ 75% identical to a distinct class of KAS sequences recently identified in castor and barley. A 46 kDa protein that was observed only in developing and mature seed was detected using antiserum directed against recombinant Cuphea KAS protein. The 46 kDa protein was abundant in developing seeds of six medium chain‐producing Cuphea species but barely detected in one long chain‐producing species. A 48 kDa protein identified immunologically as KAS I was expressed in both medium and long chain‐producing Cuphea species and was detected in all tissues tested. In in vitro assays, extracts from C. wrightii and C. viscosissima developing embryos were unable to extend fatty acid chains beyond C10 following treatment with 10 μm cerulenin, a potent inhibitor of KAS I. However, a C. viscosissima mutant, cpr‐1, whose seed oils are deficient in caprate relative to wild type, was impaired in extension of C8 to C10 in this assay and Western analysis revealed a specific deficiency in 46 kDa KAS in cpr‐1 embryos. These results implicate cerulenin‐resistant condensing activity in production of medium chain fatty acids in Cuphea.
Characterization of acyl‐ACP thioesterases of mangosteen (Garcinia mangostana) seed and high levels of stearate production in transgenic canola
Authors: Hawkins Deborah J. , Kridl Jean C. 
Published: March 2003

SummaryAcyl‐acyl‐carrier protein (ACP) thioesterases are, at least in part, responsible for the fatty acyl chain length composition of seed storage oils. Acyl‐ACP thioesterases with specificity for each of the saturated acyl‐ACP substrates from 8:0 through 16:0 have been cloned, with the exception of 18:0, and are members of the FatB class of thioesterases. The authors have determined that the tropical tree species mangosteen (Garcinia mangostana) stores 18:0 (stearate) in its seed oil in amounts of up to 56% by weight. Acyl‐ACP thioesterase activity as measured in crude mangosteen seed extracts showed a preference for 18:1‐ACP substrates, but had significant activity with 18:0 relative to that with 16:0‐ACP, suggesting a thioesterase might be involved in the production of stearate. Three distinct acyl‐ACP thioesterases were cloned from mangosteen seed cDNA; two representative of the FatA class and one representative of the FatB class. When expressed in vitro, the enzyme encoded by one of the FatAs (Garm FatA1) while preferring 18:1‐ACP showed relatively low activity with 16:0‐ACP as compared to 18:0‐ACP, similar to the substrate preferences shown by the crude seed extract. Expression of Garm FatA1 in Brassica seeds led to the accumulation of stearate up to 22% in seed oil. These results suggest that Garm FatA1 is at least partially responsible for determining the high stearate composition of mangosteen seed oil and that FatA as well FatB thioesterases have evolved for specialized roles.
Requirement for the induced expression of a cell wall associated receptor kinase for survival during the pathogen response
Authors: He Zheng‐Hui , He Deze , Kohorn Bruce D. 
Published: March 2003

SummaryPathogen infection of angiosperms must rely on some interaction between the extracellular matrix (ECM) and the invading agent, and may be accompanied by signaling between the ECM and cytoplasm. AnArabidopsiscell wall associated receptor kinase (Wak1) has an amino‐terminal domain that is tightly associated with the ECM, spans the plasma membrane and has a cytoplasmic protein kinase domain.Wak1expression is induced whenArabidopsisplants are infected with pathogen, or when the pathogen response is stimulated either by exogenous salicylate (SA) or its analog 2,2‐dichloroisonicotinic acid (INA). ThisWak1induction requires the positive regulator NPR1/NIM1. Thus Wak1 is a pathogen‐related (PR) protein. Expression of an antisense and a dominant negative allele ofWak1shows that induced expression ofWak1is needed for a plant to survive if stimulated by INA. Ectopic expression of the entire Wak1, or the kinase domain alone, can provide resistance to otherwise lethal SA levels. These experiments suggest that Wak1 expression and other PR proteins are protecting plants from detrimental effects incurred during the pathogen response. These results provide a direct link between a protein kinase that could mediate signals from the ECM, to the events that are precipitated by a pathogen infection.
A gene encoding 1‐aminocyclopropane‐1‐carboxylate (ACC) synthase produces two transcripts: elucidation of a conserved response
Authors: Peck Scott C. , Kende Hans 
Published: March 2003

SummaryIndole‐3‐acetic acid (IAA) promotes ethylene biosynthesis in stems of etiolated pea (Pisum sativum L.) seedlings by rapidly increasing the expression of 1‐aminocyclopropane‐1‐carboxylate (ACC) synthase mRNA and by enhancing the activity of the enzyme. Two cDNA clones encoding ACC synthase, Ps‐ACS1 and Ps‐ACS2, were isolated from a cDNA library prepared from the apical hooks of etiolated pea seedlings that had been treated with 100 μM IAA for 4 h. While studying the expression pattern of IAA‐induced ACC synthase mRNA, we observed that the probe for Ps‐ACS1 hybridized to two transcripts of 1.6 and 1.9 kb on RNA gel blots. The shorter transcript accumulated before the longer one did, indicating that it is not a degradation product of the latter. Because a similar observation, namely hybridization of one ACC synthase probe to two transcripts, has also been reported in other species, we investigated the relationship between the 1.6‐ and 1.9‐kb transcripts. DNA gel blot analysis using the entire cDNA as probe and RNA gel blot analysis using the 3′‐untranslated region as probe indicated that both transcripts are encoded by the same gene. Oligonucleotide‐directed RNase H mapping showed that the transcripts differ in the sequence of their 5′‐ends. Using 5′‐RACE to obtain the DNA sequence of the shorter transcript, we determined that the 1.6‐kb transcript (Ps‐ACS1b) begins within the second exon of the 1.9‐kb transcript (Ps‐ACS1a) and lacks the first 383 bases. Thus, Ps‐ACS1b does not encode a full‐length ACC synthase protein. Because the Ps‐ACS1b sequence is identical to that of Ps‐ACS1a, including proper splicing of the second intron, Ps‐ACS1b appears to result from the use of an alternative, internal promoter.
Isolation of putative plant transcriptional coactivators using a modified two‐hybrid system incorporating a GFP reporter gene
Authors: Cormack Robert S. , Hahlbrock Klaus , Somssich Imre E. 
Published: March 2003

SummaryDual hybrid interacting screening in yeast led to the identification of two proteins from Arabidopsis both exhibiting sequence similarity to a family of transcriptional coactivators from a diverse range of organisms. Their discovery constitutes the first description of such plant proteins. A modified yeast two‐hybrid approach utilising the green fluorescent protein (GFP) of Aequora victoria was developed and used to clone one of the putative plant transcriptional coactivators from an Arabidopsis cDNA library. The two proteins, designated KIWI and KELP, can associate both hetero‐ and homomerically and their genes were cloned and mapped on the Arabidopsis genome. Both proteins are believed to play a role in gene activation during pathogen defence and plant development. The involvement of these proteins in general plant transcription as well as the advantages of using GFP as a reporter gene for detecting protein–protein interactions are discussed.
Cell‐to‐cell transport of proteins: requirement for unfolding and characterization of binding to a putative plasmodesmal receptor
Authors: Kragler Friedrich , Monzer Jan , Shash Khalid , Xoconostle‐Cázares Beatriz , Lucas William J. 
Published: March 2003

Summary Plasmodesmata and the nuclear pore complex (NPC) mediate the selective trafficking of proteins and protein‐nucleic acid complexes. The events underlying the translocation of endogenous and viral proteins through plasmodesmata were investigated to further explore the parallels between these cell‐to‐cell and intracellular communication systems. Studies performed with crosslinked KNOTTED1 (KN1) revealed that a conformational change is required for the cell‐to‐cell movement of this protein. Microinjection of gold‐conjugated KN1 established that, as with the NPC, a combination of protein unfolding and microchannel dilation appears to be involved in protein translocation. However, during this process the extent of microchannel dilation is much less than observed for the NPC, which may reflect a physical limitation imposed by the cell wall. Co‐injection of KN1‐gold with unbound KN1 or cucumber mosaic virus movement protein (CMV‐MP) established that the KN1‐gold probe is highly effective at blocking plasmodesmal transport of KN1 and CMV‐MP. This result provided the foundation for competition experiments which demonstrated that KN1 and the viral movement proteins of CMV and tobacco mosaic virus likely utilize a common receptor in the pathway for cell‐to‐cell transport of proteins. A combination of biochemical fractionation methods, an

in vitro

binding assay founded on the high affinity between KN1‐gold and the putative common plasmodesmal receptor, and microinjection techniques were used to isolate plasmodesmal constituents involved in cell‐to‐cell transport. A model describing the steps involved in protein transport through plasmodesmata is presented.
Cell marking inArabidopsis thaliana andits application to patch–clamp studies
Authors: Maathuis Frans J. M. , May Sean T. , Graham Neil S. , Bowen Helen C. , Jelitto Till C. , Trimmer Paul , Bennett Malcolm J. , Sanders Dale , White Philip J. 
Published: March 2003

Summary Ion transport processes at the plasma membrane of plant cells are frequently studied by applying membrane‐patch voltage‐clamp (patch–clamp) electrophysiological techniques to isolated protoplasts. As plants are composed of many tissues and cell types, and each tissue and cell type may be specialized to a particular function and possess a unique complement of transport proteins, it is important to certify the anatomical origin of the protoplasts used for patch–clamp studies. This paper describes a general molecular genetic approach to marking specific cell types for subsequent patch–clamp studies and presents a specific example: a comparison of the K+ currents in protoplasts from cortical and stelar cells of

Arabidopsis

roots. Transgenic

Arabidopsis

were generated in which the expression of green fluorescent protein (GFP) from

Aequoria victoria

was driven by the CaMV 35S promoter (line mGFP3). In roots of the transgenic mGFP3 line, visible fluorescence was restricted to the stele. Protoplasts were generated from roots of the mGFP3 line and K+ currents in non‐fluorescent (cortical/epidermal) and fluorescent (stelar) protoplasts were assayed using patch–clamp techniques. It was found that both the frequency of observing inward rectifying K+ channel (IRC) activity and the relative occurrence of IRC compared to outward rectifying K+ channels were significantly lower in protoplasts from cortical/epidermal cells compared to cells of the stele. The presence of GFP did not affect the occurrence or biophysical properties of K+ channels. It is concluded that the generation of transgenic

Arabidopsis

expressing GFP in a cell‐specific fashion is a convenient and reliable way to mark protoplasts derived from contrasting cell types for subsequent patch–clamp studies.
Generation of co‐dominant PCR‐based markers by duplex analysis on high resolution gels
Authors: Hauser Marie‐Theres , Adhami Farzaneh , Dorner Maria , Fuchs Esther , Glössl Josef 
Published: March 2003

Summary Rapid and efficient procedures for the detection of sequence polymorphisms are essential for chromosomal walking and mutation detection analyses. While DNA chip technology and denaturing high‐performance liquid chromatography (DHPLC) are the methods of choice for large scale facilities, small laboratories are dependent on simple ready‐to‐use techniques. We show that heteroduplex analysis on high resolution gel matrices efficiently detects sequence polymorphism differing as little as a single base pair (e.g. single‐nucleotide polymorphism, SNP) with standard laboratory equipment. Furthermore, the matrices also discerned differences between homoduplexes, a prerequisite for co‐dominant markers. The markers thus generated are referred to as duplex analysis markers. We designed PCR primers for 36

Arabidopsis thaliana

loci ranging in length from 230 bp to 1000 bp. Among three ecotypes, more than half (

n

= 19) of the loci examined were polymorphic; five of which contained three different alleles. This simple, high resolution technique can be used to rapidly convert sequence tagged sites into co‐dominant PCR‐based molecular markers for fine‐scale mapping studies and chromosomal walking strategies as well as for the detection of mutations in particular genes.
A bHLH transcription factor mediates organ, region and flower type specific signals on dihydroflavonol‐4‐reductase (dfr) gene expression in the inflorescence ofGerbera hybrida(Asteraceae)
Authors: Elomaa Paula , Mehto Merja , Kotilainen Mika , Helariutta Yrjö , Nevalainen Leena , Teeri Teemu H. 
Published: March 2003

Summary The angiosperm family Asteraceae is characterized by composite inflorescences, which are highly organized structures consisting of different types of flowers. In order to approach the control of floral organ differentiation in Asteraceae at molecular level, we are studying regulation of flavonoid biosynthesis in

Gerbera hybrida

. Dihydroflavonol‐4‐reductase (

dfr

) expression is regulated according to anthocyanin pigmentation patterns in all tested gerbera varieties at several anatomical levels. We have isolated a promoter for one of the

dfr

genes,

Pgdfr2

. Gerbera plants transgenic for a

Pgdfr2

‐

uidA

construct reveal that the activity of the

Pgdfr2

promoter from one variety follows the pigmentation in other varieties which have different color patterns. It is thus evident that the observed complex regulation of

dfr

expression occurs

in trans

. In order to identify the

trans

‐acting regulators, we isolated a cDNA (

gmyc1

) homologous to the previously characterized genes encoding bHLH‐type regulators of the anthocyanin pathway in plants. The expression of

gmyc1

in different varieties suggests that it has a major role in regulating

dfr

activity in corolla and carpel, but not in pappus and stamen. Specifically in gerbera, the identical patterns of

gmyc1

and

dfr

expression in corolla tissue suggest that GMYC1 also regulates

dfr

expression in a region and flower type specific manner. Our studies show that in gerbera GMYC1–

dfr

interaction is part of several developmental processes characteristic for Asteraceae (such as specification of flower types across the composite inflorescence), whereas in other processes (such as differentiation of sepal as pappus) other regulators control

dfr

expression to determine the spatial specificity.
Reciprocal regulation of distinct asparagine synthetase genes by light and metabolites inArabidopsis thaliana
Authors: Lam Hon‐Ming , Hsieh Ming‐Hsiun , Coruzzi Gloria 
Published: March 2003

Summary In plants, the amino acid asparagine serves as an important nitrogen transport compound whose levels are dramatically regulated by light in many plant species, including

Arabidopsis thaliana

. To elucidate the mechanisms regulating the flux of assimilated nitrogen into asparagine, we examined the regulation of the gene family for asparagine synthetase in Arabidopsis. In addition to the previously identified

ASN1

gene, we identified a novel class of asparagine synthetase genes in Arabidopsis (

ASN2

and

ASN3

) by functional complementation of a yeast asparagine auxotroph. The proteins encoded by the

ASN2/3

cDNAs contain a Pur‐F type glutamine‐binding triad suggesting that they, like

ASN1

, encode glutamine‐dependent asparagine synthetase isoenzymes. However, the ASN2/3 isoenyzmes form a novel dendritic group with monocot

AS

genes which is distinct from all other dicot

AS

genes including Arabidopsis

ASN1

. In addition to these distinctions in sequence, the

ASN1

and

ASN2

genes are reciprocally regulated by light and metabolites. Time‐course experiments reveal that light induces levels of

ASN2

mRNA while it represses levels of

ASN1

mRNA in a kinetically reciprocal fashion. Moreover, the levels of

ASN2

and

ASN1

mRNA are also reciprocally regulated by carbon and nitrogen metabolites. The distinct regulation of

ASN1

and

ASN2

genes combined with their distinct encoded isoenzymes suggest that they may play different roles in nitrogen metabolism, as discussed in this paper.
Reduced inositol content and altered morphology in transgenic potato plants inhibited for 1D‐myo‐inositol 3‐phosphate synthase
Authors: Keller Ruth , Brearley Charles A. , Trethewey Richard N. , Müller‐Röber Bernd 
Published: March 2003

Summary
Myo

‐inositol is a precursor of many plant metabolites, including polyols, cell wall components and phosphoinositides. The first committed step in the

de novo myo

‐inositol synthetic pathway is catalysed by the enzyme 1D‐

myo

‐inositol 3‐phosphate synthase (MIPS;
EC 5.5.1.4
), which converts D‐glucose 6‐phosphate to 1D‐

myo

‐inositol 3‐phosphate. Suppression of MIPS activity by an antisense RNA approach in transgenic potato (

Solanum tuberosum

L.) plants to below 20% of the wild‐type level in leaves resulted in strongly reduced levels of inositol, galactinol and raffinose (approximately 7%, 5% and 12%, respectively, of wild‐type values). In contrast, increases were observed for concentrations of hexose phosphates (up to 1.7‐fold), sucrose (twofold) and starch (two‐ to fourfold). Transgenic plants exhibited reduced apical dominance, altered leaf morphology, precocious leaf senescence and a decrease in overall tuber yield. These observations indicate a crucial role for

myo

‐inositol in plant physiology and development.
ATY3/GYPSYretrotransposon‐like sequence localizes to the centromeric regions of cereal chromosomes
Authors: Presting Gernot G. , Malysheva Ludmilla , Fuchs Jörg , Schubert Ingo 
Published: March 2003

SummaryA 745 bp sequence (pSau3A9) located at the centromeres of several cereal species was isolated from a sorghum BAC library byJianget al. (1996,Proc. Natl Acad. Sci. USA, 93, 14210–14213). We have amplified a partially homologous 809 bp sequence from barley genomic DNA by PCR and localized it to the centromeres of barley, wheat and rye chromosomes by fluorescentin situhybridization (FISH). Sequence analysis showed this barley homolog ofpSau3A9to have high similarity to the integrase region of the polyprotein gene ofTy3/gypsygroup retrotransposons. Using this integrase sequence as a probe, several clones were isolated from a lambda library constructed of genomic barley DNA. One of the lambda clones contained coding regions for all five catalytic sites characteristic of the retrotransposon polyprotein. Two direct repeats flanking the polyprotein gene are homologous to the cereal centromeric sequence described byAragón‐Alcaideet al. (1996,Chromosoma, 105, 261–268) and may represent all or part of the long‐terminal repeats (LTRs). Different plasmid subclones containing various regions of the lambda clone were used in FISH to show that the entire polyprotein gene and upstream flanking sequences, including the presumed LTR, are present at barley centromeres. The preferential (or exclusive) localization of an apparently complete retroelement within the centromeric regions of several cereal species raises interesting questions about its role in karyotype evolution and centromere function.