Three nuclear genes involved in plant mitochondrial recombination surveillance have been previously identified. Simultaneous disruption of two of these genes, MSH1 and RECA3, results in extensive rearrangement of the mitochondrial genome and dramatic changes in plant growth. We have capitalized on these changes in mitochondrial genome organization to understand the role mitochondria play in plant cellular and developmental processes. Transcript profiling of the double mutants grown under normal conditions revealed differential regulation of numerous nuclear genes involved in stress response together with increased levels of polyadenylated mitochondrial transcripts. We show that extensive rearrangement of the mitochondrial genome directly elicits physiological stress responses in plants, with msh1 recA3 double mutants exhibiting enhanced thermo-tolerance. Likewise, we show that mitochondrial transcriptional changes are associated with genome recombination, so that differential gene modulation is accomplished, at least in part, through altered gene copy number.

Rice seedlings are particularly sensitive to chilling in early spring in temperate and subtropical zones and in high elevation areas. Improvement of chilling tolerance in rice may significantly increase rice production. MYBS3 is a single DNA-binding repeat (1R) MYB transcription factor previously shown to mediate sugar signaling in rice. In the present study, we observed that MYBS3 also plays a critical role in cold adaptation in rice. Gain- and loss-of-function analyses indicated that MYBS3 was sufficient and necessary for enhancing cold tolerance in rice. Transgenic rice constitutively over-expressing MYBS3 tolerated 4°C for at least 1 week, and exhibited no yield penalty in normal field conditions. Transcription profiling of transgenic rice over- or under-expressing MYBS3 led to identification of many genes in the MYBS3-mediated cold signaling pathway. Several genes activated by MYBS3 as well as inducible by cold have previously been implicated in various abiotic stress response and/or tolerance in rice and other plant species. Surprisingly, MYBS3 repressed the well-known DREB1/CBF-dependent cold signaling pathway in rice, and the repression appears to act at the transcriptional level. DREB1 responded quickly and transiently while MYBS3 responded slowly to cold stress, which suggests distinct pathways act sequentially and complementarily for adapting short- and long-term cold stress in rice. Our studies thus reveal a hitherto undiscovered novel pathway which controls cold adaptation in rice.

The plant hormone abscisic acid (ABA) is involved in an array of plant processes, including the regulation of gene expression during adaptive responses to various environmental cues. Apart from its well-established role in abiotic stress adaptation, emerging evidence indicates that ABA is also prominently involved in the regulation and integration of pathogen defense responses. Here, we demonstrate that exogenously administered ABA enhances basal resistance of rice (Oryza sativa) against the brown spot-causing ascomycete Cochliobolus miyabeanus. Microscopic analysis of early infection events in control and ABA-treated plants revealed that this ABA-inducible resistance (ABA-IR) is based on restriction of fungal progression in the mesophyll. We also show that ABA-IR does not rely on boosted expression of SA-, JA-, or callose-dependent resistance mechanisms but, instead, requires a functional G-protein. In addition, several lines of evidence are presented suggesting that ABA steers its positive effect on brown spot resistance through antagonistic cross-talk with the ET-response pathway. Exogenous Ethephon application enhances susceptibility, whereas genetic disruption of ET signaling renders plants less vulnerable to C. miyabeanus attack, thereby inducing a level of resistance similar to that observed on ABA-treated wild-type plants. Moreover, ABA treatment alleviates C. miyabeanus-induced activation of the ET-reporter gene EPB89, while de-repression of pathogen-triggered EBP89 transcription via RNAi-mediated knockdown of OsMPK5, an ABA-primed MAP kinase gene, compromises ABA-IR. Collectively, these data favor a model whereby exogenous ABA enhances resistance against C. miyabeanus at least in part by suppressing pathogen-induced ET action in an OsMPK5-dependent manner.

Heterologous regulatory elements and flanking sequences have been used in chloroplast transformation of several crop species but their roles or mechanism has not yet been investigated. Nucleotide sequence identity in the psbA upstream region is 59% across all taxa; similar variation was consistent across all genes and taxa examined. Secondary structure and predicted G values of the psbA 5'UTR among different families reflected this variation. Therefore, chloroplast transformation vectors were made for tobacco (Nicotiana tabacum) and lettuce (Lactuca sativa), with endogenous (Nt-Nt, Ls-Ls) or heterologous (Nt-Ls, Ls-Nt) psbA promoter, 5'UTR and 3'UTR, regulating expression of the anthrax protective antigen (PA) or human proinsulin (Pins) fused with the cholera toxin B-subunit (CTB). Unique lettuce flanking sequences were completely eliminated during homologous recombination in the transplastomic tobacco genomes but not unique tobacco sequences. Nt-Ls or Ls-Nt transplastomic lines showed reduction of 80% PA and 97% CTB-Pins expression, when compared with endogenous psbA regulatory elements, which accumulated up to 29.6% TSP PA and 72.0% TLP CTB-Pins, two-fold higher than RuBisCO. Transgene transcripts were reduced by 84% in Ls-Nt-CTB-Pins and 72% in Nt-Ls-PA lines. Transcripts containing endogenous 5'UTR were stabilized in non-polysomal fractions. Stromal RNA binding proteins were preferentially associated with endogenous psbA 5'UTR. A rapid and reproducible regeneration system was developed for L. sativa commercial cultivars by optimizing plant growth regulators (PGRs). These findings underscore the need for sequencing complete crop chloroplast genomes, utilization of endogenous regulatory elements and flanking sequences as well as optimization of PGRs for efficient chloroplast transformation.

Complexation of arsenite (As(III)) with phytochelatins (PCs) is an important mechanism employed by plants to detoxify As; how this complexation affects As mobility was little known. We used high-resolution ICP-MS and accurate mass ESI-MS coupled to HPLC to identify and quantify As(III)-thiol complexes and free thiol compounds in Arabidopsis thaliana exposed to arsenate. Arsenate was efficiently reduced to As(III) in roots. In wild-type roots, 69% of As was complexed as As(III)-PC₄, As(III)-PC₃ and As(III)-(PC₂)₂. Both the glutathione (GSH)-deficient mutant cad2-1 and the PC-deficient mutant cad1-3 were approximately 20 times more sensitive to arsenate than wild-type. In cad1-3 roots only 8% of As was complexed with GSH as As(III)-(GS)₃ and no As(III)-PCs were detected, while in cad2-1 roots As(III)-PCs accounted for only 25% of the total As. The two mutants had a greater As mobility, with a significantly higher accumulation of As(III) in shoots and 4.5 - 12 times higher shoot-to-root As concentration ratio than wild-type. Roots also effluxed a substantial proportion of the arsenate taken up as As(III) to the external medium, and this efflux was larger in the two mutants. Furthermore, when wild-type plants were exposed to L-buthionine sulfoxime or deprived of sulfur, both As(III) efflux and root-to-shoot translocation were enhanced. The results indicate that complexation of As(III) with PCs in Arabidopsis roots decreases its mobility for both efflux to the external medium and for root-to-shoot translocation. Enhancing PC synthesis in roots may be an effective strategy to reduce As translocation to the edible organs of food crops.

Previous systems analyses in plants have focused on a single developmental stage or time point, although it is often important to additionally consider time-index changes. During seed development a cascade of events occurs within a relatively brief time-scale. We have collected protein and transcript expression data from five sequential stages of Arabidopsis thaliana seed development encompassing the period of reserve polymer accumulation. Protein expression profiling employed 2-dimensional gel electrophoresis coupled with tandem mass spectrometry, while transcript profiling used oligonucleotide microarrays. Analyses in biological triplicate yielded robust expression information for 523 proteins and 22,746 genes across the five developmental stages, and established 319 protein/transcript pairs for subsequent pattern analysis. General linear modeling was used to evaluate the protein/transcript expression patterns. Overall, application of this statistical assessment technique showed concurrence for a slight majority (56%) of expression pairs. Many specific examples of discordant protein/transcript expression patterns were detected, suggesting that this approach will be useful in revealing examples of post-transcriptional regulation.

Carotenoid turnover was investigated in mature leaves of Arabidopsis thaliana by ¹⁴CO₂ pulse-chase labeling under control-light (CL, 130) and high-light (HL, 1000 µmol photons m^-2 s^-1) conditions. Following 30-min ¹⁴CO₂ administration, photosynthetically fixed ¹⁴C was quickly incorporated in β-carotene (β-C) and chlorophyll a (Chl a) in all samples during the chase of up to 10 h. In contrast, ¹⁴C was not detected in Chl b and xanthophylls, even when steady-state amounts of the xanthophyll-cycle pigments and lutein increased markedly, presumably by de novo synthesis, in CL-grown plants under HL. Different light conditions during the chase did not affect the ¹⁴C fractions incorporated in β-C and Chl a, whereas long-term HL acclimation significantly enhanced ¹⁴C labeling of Chl a, but not β-C. Consequently, the maximal ¹⁴C signal ratio between β-C and Chl a was much lower in HL-grown plants (1:10) than in CL-grown plants (1:4). In lut5 mutants, containing -carotene (-C) together with reduced amounts of β-C, remarkably high ¹⁴C labeling was found for -C while the labeling efficiency of Chl a was similar to that of wild-type plants. The maximum ¹⁴C ratios between carotenes and Chl a were 1:2 for -C:Chl a and 1:5 for β-C:Chl a in CL-grown lut5 plants, suggesting high turnover of -C. The data demonstrate continuous synthesis and degradation of carotenes and Chl a in photosynthesizing leaves and indicate distinct acclimatory responses of their turnover to changing irradiance. In addition, the results are discussed in the context of photosystem II repair cycle and D1 protein turnover.

With the aim of determining the genetic basis of metabolic regulation in tomato fruit we constructed a detailed physical map of genomic regions, spanning previously described metabolic quantitative trait loci of a Solanum pennellii introgression line population. Two genomic libraries from S. pennellii were screened with 104 co-located markers from five selected genomic regions and a total of 614 BAC/cosmids were identified as seed clones. Integration of sequence data with the genetic and physical maps of S. lycopersicum facilitated the anchoring of 374 of these BAC/cosmid clones. The analyses of this information resulted in a genome-wide map of a non-domesticated plant species and covers 10 % of the physical distance of the selected regions corresponding to approximately 1 % of the wild tomato genome. Comparative analyses revealed that S. pennellii and domesticated tomato genomes can be considered as largely co-linear. A total of 1,238,705 bp from both BAC/cosmid ends and nine large insert clones were sequenced, annotated and functionally categorized. The sequence data allowed the evaluation of the level of polymorphism between the wild and cultivated tomato species. An exhaustive micro-synteny analysis allowed us to estimate the divergence date of S. pennellii and S. lycopersicum at 2.7 millon years ago. The combined results serve as a reference for comparative studies both at macro- and micro-syntenic levels. They also provide a valuable tool for fine mapping of quantitative trait loci in tomato. Furthermore, they will contribute to a deeper understanding of the regulatory factors underpinning metabolism and hence defining crop chemical composition.

The development of massively parallel sequencing technologies enables the sequencing of total cDNA (RNA-Seq) to derive accurate measure of individual gene expression, differential splicing activity and to discover novel regions of transcription, dramatically changing the way that the functional complexity of transcriptomes can be studied. Here we report on the first use of RNA-Seq to gain insight into the wide range of transcriptional responses that are associated with berry development in Vitis vinifera cv. Corvina.

More than 59 million sequence reads, 36–44 bp in length, were generated from three developmental stages: post-setting, véraison and ripening. The sequence reads were aligned onto the 8.4-fold draft sequence of the Pinot Noir 40024 genome and then analyzed to measure gene expression levels, to detect alternative splicing events and expressed single nucleotide polymorphisms (SNPs). We detected 17,324 genes expressed during berry development, 6,695 of which were expressed in a stage-specific manner, suggesting differences in expression for genes in numerous functional categories and a significant transcriptional complexity. This exhaustive overview of gene expression dynamics demonstrates the utility of RNA-Seq for identifying SNPs and splice variants and for describing how plant transcriptomes changes during development.

Transgenic tomato (Solanum lycopersicum cv. Moneymaker) plants independently expressing fragments of various genes encoding enzymes of the tricarboxylic acid cycle in antisense orientation have previously been characterised as exhibiting altered root growth. In the current study we evaluate the rates of respiration of roots from these lines in addition to determining their total dry weight accumulation. Given that these features were highly correlated we decided to carry out an evaluation of the cell wall composition in the transformants which revealed a substantial reduction in cellulose. Since the bulk of cellulose is associated with the secondary cell walls in roots we reasoned that the transformants most likely were deficient in secondary wall cellulose production. Consistent with these findings, cross sections of the root collar (approx. 15 mm from the junction between root and stem) displayed reduced lignified secondary cell walls for the transformants. In contrast, cell and cell wall patterning displayed no differences in elongating cells close to the root tip. To further characterise the modified cell wall metabolism, we performed feeding experiments in which we incubated excised root tips in [U¹⁴C]-glucose in the presence or absence of phosphonate inhibitors of the reaction catalysed by 2-oxoglutarate dehydrogenase. Taken together, the combined results suggest that restriction of root respiration leads to a deficit in secondary cell wall synthesis. These data are discussed in the context of current models of biomass partitioning and plant growth.

RsRGA4 (Ralstonia solanacearum-responsive gene A4) encodes a polypeptide similar to S-locus glycoprotein (SGP) from Brassica rapa, and SGP-like proteins from Ipomea trifida and Medicago truncatula. We therefore designated RsRGA4 as NtSGLP (Nicotiana tabacum S locus glycoprotein like protein) and NbSGLP (its N. benthamiana ortholog). NbSGLP is expressed in root, leaf, petal, gynoecium, and stamen. Expression of NbSGLP was strongly induced by inoculation with an avirulent strain of R. solanacearum (Rs8107) and slightly enhanced by inoculation with virulent R. solanacearum (RsOE1-1). Expression of NbSGLP was induced by inoculation with an hrp-deficient mutant of RsOE1-1 and Rs8107. Expression was also induced by aminocyclopropane carboxylic acid and salicylic acid. Virus-induced gene silencing of NbSGLP enhanced the growth of Rs8107. Growth of RsOE1-1 and appearance of wilt symptoms were also accelerated in silenced plants. Expression of PR-1a and EREBP was reduced, and markers for basal defense, such as callose deposition and reduced vascular flow were compromised in NbSGLP-silenced plants. Moreover, growth of Pseudomonas cichorii, P. syringae pv. tabaci, and P. syringae pv. mellea were also enhanced in the silenced plants. On the other hand, silencing of NbSGLP did not interfere with the appearance of the hypersensitive response (HR). NbSGLP was secreted in a signal peptide-dependent manner. Agrobacterium-mediated expression of NbSGLP induced PR-1a and EREBP expression, callose deposition, and reduced vascular flow. NbSGLP-induced callose deposition and reduced vascular flow were not observed in salicylic acid-deficient N. benthamiana NahG plant. Taken together SGLP might have a role in induction of basal defense in Nicotiana plants.

Autophagy is a catabolic membrane-trafficking process whereby cells recycle cytosolic proteins and organelles under stress conditions or during development. This degradative process is mediated by autophagy-related (ATG) proteins which have been described in yeasts, animals and more recently in plants. In this study we report the molecular characterization of autophagy in the unicellular green alga Chlamydomonas reinhardtii. We demonstrate that the ATG8 protein from Chlamydomonas (CrATG8) is functionally conserved and may be used as a molecular autophagy marker. Like yeast ATG8, CrATG8 is cleaved at the C-terminal conserved Glycine and is associated with membranes in Chlamydomonas. Cell aging or different stresses such as nutrient limitation, oxidative stress or the accumulation of misfolded proteins in the endoplasmic reticulum caused an increase in CrATG8 abundance as well as the detection of modified forms of this protein, both landmarks of autophagy activation. Furthermore, rapamycin-mediated inhibition of the TOR signaling pathway, a major regulator of autophagy in eukaryotes, results in identical effects on CrATG8 and a re-localization of this protein in Chlamydomonas cells similar to the one observed upon nutrient limitation. Thus, our findings indicate that Chlamydomonas cells may respond to stress conditions by inducing autophagy via TOR signaling modulation.

We previously characterized the rice (Oryza sativa L.) Sub1 locus encoding three Ethylene Responsive Factor (ERF) transcriptional regulators. Genotypes carrying the Sub1A-1 allele are tolerant of prolonged submergence. To elucidate the mechanism of Sub1A-1 mediated tolerance, we performed transcriptome analyses comparing the temporal submergence response of Sub1A-1 containing tolerant M202(Sub1) with the intolerant isoline M202 lacking this gene. We identified 898 genes displaying Sub1A-1-dependent regulation. Integration of the expression data with publicly available metabolic pathway data identified submergence tolerance-associated pathways governing anaerobic respiration, hormone responses, and antioxidant systems. Of particular interest were a set of AP2/ERF family transcriptional regulators that are associated with the Sub1A-1 mediated response upon submergence. Visualization of expression patterns of the AP2/ERF super family members in a phylogenetic context resolved 12 submergence-regulated AP2/ERFs into three putative functional groups: (I) anaerobic respiration and cytokinin-mediated delay in senescence via ethylene accumulation during submergence (3 ERFs); (II) negative regulation of ethylene-dependent gene expression (5 ERFs); and (III) negative regulation of GA-mediated shoot elongation (4 ERFs). These results confirm that the presence of Sub1A-1 impacts multiple pathways of response to submergence.

While malate and fumarate participate in a multiplicity of pathways in plant metabolism, the function of these organic acids as carbon stores in C₃ plants has not been deeply addressed. Here, Arabidopsis plants overexpressing a maize plastidic NADP-malic enzyme (MEm plants) were used to analyse the consequences of sustained low malate and fumarate levels on the physiology of this C₃ plant. When grown in short days (SD), MEm plants developed a pale green phenotype with decreased biomass and increased specific leaf area, with thin leaves having lower photosynthetic performance. These features were absent in plants growing in long days (LD). The analysis of metabolite levels of rosettes from transgenic plants indicated similar disturbances in both SD and LD, with very low levels of malate and fumarate. Determinations of the respiratory quotient by the end of the night indicated a shift from carbohydrates to organic acids as the main substrates for respiration in the wild type, while MEm plants use more reduced compounds, like fatty acids and proteins, to fuel respiration. It is concluded that the alterations observed in SD MEm plants are a consequence of impairment in the supply of carbon skeletons during a long dark period. This carbon starvation phenotype observed at the end of the night demonstrates a physiological role of the C₄ acids, which may be a constitutive function in plants.

The ability to non-destructively image and automatically phenotype complex root systems, like those of rice (Oryza sativa), is fundamental to identifying genes underlying root system architecture (RSA). Although root systems are central to plant fitness, identifying genes responsible for RSA remains an underexplored opportunity for crop improvement. Here we describe a non-destructive imaging and analysis system for automated phenotyping and trait ranking of RSA. Using this system we image rice roots from 12 genotypes. We automatically estimate RSA traits previously identified as important to plant function. In addition, we expand the suite of features examined for RSA to include traits that more comprehensively describe monocot RSA but that are difficult to measure with traditional methods. Using 16 automatically acquired phenotypic traits for 2297 images from 118 individuals, we observe (i) wide variation in phenotypes among the genotypes surveyed; (ii) greater inter-genotype variance of RSA features than variance within a genotype. RSA trait values are integrated into a computational pipeline which utilizes supervised learning methods to determine which traits best separate two genotypes, and then ranks the traits according to their contribution to each pair-wise comparison. This trait ranking step identifies candidate traits for subsequent QTL analysis and demonstrates that depth and average radius are key contributors to differences in rice RSA within our set of genotypes. Our results suggest a strong genetic component underlying rice RSA. This work enables the automatic phenotyping of RSA of individuals within mapping populations, providing an integrative framework for QTL analysis of RSA.

Plant metabolism is highly coordinated with development. However, an understanding of the whole picture of metabolism and its interactions with plant development is scarce. In this work we show that the deficiency in the plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPCp) leads to male sterility in Arabidopsis thaliana. Pollen from homozygous gapcp double mutant plants (gapcp1gapcp2) displayed shrunken and collapsed forms and were unable to germinate when cultured in vitro. The pollen alterations observed in gapcp1gapcp2 were attributed to a disorganized tapetum layer. Accordingly, the expression of several of the genes involved in tapetum development was down-regulated in gapcp1gapcp2. The fertility of gapcp1gapcp2 was rescued by transforming this mutant with a construct carrying the GAPCp1 cDNA under the control of its native promoter (pGAPCp1::GAPCp1c). However, the GAPCp1 or GAPCp2 cDNA under the control of the 35S promoter (p35S::GAPCp), which is poorly expressed in the tapetum, did not complement the mutant fertility. Mutant GAPCp isoforms deficient in the catalytic activity of the enzyme were unable to complement the sterile phenotype of gapcp1gapcp2, thus confirming that both the expression and catalytic activity of GAPCp in anthers are necessary for mature pollen development. A metabolomic study in flower buds indicated that the most important difference between the sterile (gapcp1gapcp2, gapcp1gapcp2-p35S::GAPCp) and the fertile (wild type plants, gapcp1gapcp2-pGAPCp1::GAPCp1c) lines was the increase in the signalling molecule trehalose. This work corroborates the importance of plastidial glycolysis in plant metabolism and provides evidence for the crucial role of GAPCps in pollen development. It additionally brings new insights into the complex interactions between metabolism and development.

Alkamides belong to a class of small lipid signals of wide distribution in plants, which are structurally related to the bacterial quorum-sensing (QS) signals N-acyl-L-homoserine lactones (AHLs). Arabidopsis (Arabidopsis thaliana) seedlings display a number of root developmental responses to alkamides, including primary root growth inhibition and greater formation of lateral roots. To gain insight into the regulatory mechanisms by which these compounds alter plant development, we performed a mutant screen for identifying Arabidopsis mutants that fail to inhibit primary root growth when grown under a high concentration of N-isobutyl decanamide. A recessive N-isobutyl decanamide-resistant mutant (decanamide resistant root-drr1) was isolated because of its continued primary root growth and reduced lateral root formation in response to this alkamide. Detailed characterization of lateral root primordia (LRP) development in wild-type and drr1 mutants revealed that DRR1 is required at an early stage of pericycle cell activation to form LRP in response to both N-isobutyl decanamide and N-decanoyl-L-homoserine lactone, a highly active bacterial QS signal. Exogenously supplied auxin similarly inhibited primary root growth and promoted lateral root formation in wild-type and drr1 seedlings, suggesting that alkamides and auxin act by different mechanisms to alter root system architecture. When grown both in vitro and in soil drr1 mutants showed dramatically increased longevity and reduced hormone and age dependent senescence, which were related to reduced lateral root formation when exposed to stimulatory concentrations of jasmonic acid (JA). Taken together, our results provide genetic evidence indicating that alkamides and AHLs can be perceived by plants to modulate root architecture and senescence-related processes possibly by interacting with JA signaling.

In epidermal and mesophyll cells of Arabidopsis thaliana leaves, nuclei become relocated in response to strong blue light. We previously reported that nuclear positions both in darkness and in strong blue light are regulated by the blue-light receptor phototropin2 in mesophyll cells. Here we investigate the involvement of phototropin as well as the actin cytoskeleton in nuclear positioning in epidermal cells. Analysis of geometrical parameters revealed that, in darkness, nuclei were distributed near the center of the cell, adjacent to the inner periclinal wall, independent of cell shape. Dividing the anticlinal wall into concave, convex, and intermediate regions indicated that, in strong blue light, nuclei became relocated preferably to a concave region of the anticlinal wall, nearest the center of the cell. Mutant analyses verified that light-dependent nuclear positioning was regulated by phototropin2, while dark positioning of nuclei was independent of phototropin. Nuclear movement was inhibited by an actin-depolymerizing reagent, latrunculin B, but not by a microtubule-disrupting reagent, propyzamide. Imaging actin organization by immunofluorescence microscopy revealed that thick actin bundles, periclinally arranged parallel to the longest axis of the epidermal cell, were associated with the nucleus in darkness; whereas under strong blue light, the actin bundles, especially in the vicinity of the nucleus, became arranged close to the anticlinal walls. Light-dependent changes in the actin organization were clear in phot1 mutant but not in phot2 and phot1phot2 mutants. We propose that, in A. thaliana, blue-light-dependent nuclear positioning is regulated by phototropin2-dependent reorganization of the actin cytoskeleton.

The pit membrane is a primary cell wall barrier that separates adjacent xylem water conduits, limiting the spread of xylem-localized pathogens and air embolisms from one conduit to the next. This paper provides a characterization of the size of the pores in the pit membranes (PMs) of grapevine (Vitis vinifera). The PM porosity (PMP) of stems infected with the bacterium Xylella fastidiosa was compared with the PMP of healthy stems. Stems were infused with pressurized water and flow rates were determined, gold particles of known size were introduced with the water to assist in determining the size of pit membrane pores. The effect of introducing CDTA, oligogalacturonides and polygalacturonic acid into stems on water flux via the xylem was also measured. The possibility that cell wall-degrading enzymes could alter the pore sizes, thus facilitating the ability of X. fastidiosa to cross the pit membranes was tested. Two cell wall-degrading enzymes likely to be produced by X. fastidiosa (polygalactuoronase and endo-1,4-β-glucanase), were infused into stems and particle passage tests were performed to check for changes in PMP. Scanning electron microscopy of control and enzyme-infused stem segments revealed that the combination of enzymes opened holes in PMs, probably explaining enzyme impacts on PMP and how a small X. fastidiosa population, introduced into grapevines by insect vectors, can multiply and spread throughout the vine and cause Pierce's disease.

In Arabidopsis, a family of six genes encodes acyl-CoA-binding proteins (ACBPs). A member of this family, ACBP1, contains an N-terminal transmembrane domain that targets it to the plasma membrane and the endoplasmic reticulum (ER). To investigate ACBP1 function, ACBP1-overexpressing transgenic Arabidopsis were characterized using lipid profiling analysis. ACBP1 overexpressors showed reduction in several species of di-unsaturated phosphatidycholine (PC), prompting us to investigate if they were altered in response to freezing stress. ACBP1 overexpressors demonstrated increased freezing sensitivity accompanied by a decrease in PC and an increase in phosphatidic acid (PA), while acbp1 mutant plants showed enhanced freezing tolerance associated with PC accumulation and PA reduction. We also showed binding of a recombinant eukaryotic ACBP (ACBP1) to PA, indicative of possibility in enhanced PA interaction in ACBP1 overexpressors. Since phospholipase D1 (PLD1) is a major enzyme promoting the hydrolysis of PC to PA, PLD1 expression was examined and was observed higher in ACBP1 overexpressors than acbp1 mutant plants. In contrast, the expression of phospholipase D (PLD), which plays a positive role in freezing tolerance, declined in the ACBP1 overexpressors but increased in acbp1 mutant plants. Given that ACBP1 is localized to the ER and plasma membrane, it may regulate the expression of PLD1 and PLD by maintaining a membrane-associated PA pool through its ability to bind PA. Moreover, both genotypes showed no alterations in proline and soluble sugar content or in cold-regulated (COR6.6 and COR47) gene expression, suggesting that the ACBP1-mediated response is phospholipase D-associated and is independent of osmolyte accumulation.

Abscisic acid (ABA) is postulated to be a ubiquitous hormone that plays a central role in seed development and responses to environmental stresses of vascular plants. However, in liverworts, which represent the oldest extant lineages of land plants, the role of ABA has been least emphasized and thus very little information is available on the molecular mechanisms underlying ABA responses. In this study, we isolated and characterized MpABI1, an ortholog of ABSCISIC ACID INSENSITIVE 1 (ABI1), from the liverwort Marchantia polymorpha L. The MpABI1 cDNA encoded a 568-amino-acid protein consisting of the C-terminal protein phosphatase 2C (PP2C) domain and a novel N-terminal regulatory domain. The MpABI1 transcript was detected in the gametophyte, and its expression level was increased by exogenous ABA treatment in the gemma, whose growth was strongly inhibited by ABA. Experiments using GFP fusion constructs indicated that MpABI1 was mainly localized in the nucleus and that its nuclear localization was directed by the N-terminal domain. Transient overexpression of MpABI1 in M. polymorpha and Physcomitrella patens cells resulted in suppression of ABA-induced expression of the Em-GUS gene. Transgenic P. patens expressing MpABI1 and its mutant construct, MpABI1-d2, lacking the N-terminal domain, had reduced freezing and osmotic-stress tolerance, associated with reduced accumulation of ABA-induced LEA-like boiling-soluble proteins. Furthermore, ABA-induced morphological changes leading to brood cells were not prominent in these transgenic plants. These results suggest that MpABI1 is a negative regulator of ABA signaling, providing unequivocal molecular evidence of PP2C-mediated ABA response mechanisms functioning in liverworts.

Chloroplasts in differentiated bundle sheath (BS) and mesophyll (M) cells of maize leaves are specialized to accommodate C4 photosynthesis. This study provides a reconstruction of how metabolic pathways, protein expression and homeostasis functions, are quantitatively distributed across BS and M chloroplasts. This yielded new insights in cellular specialization. The experimental analysis was based on high accuracy mass spectrometry, protein quantification by spectral counting and the first maize genome assembly. A bioinformatics workflow was developed to deal with gene models, protein families and gene duplications related to the polyploidy of maize; this avoided over-identification of proteins and resulted in more accurate protein quantification. 1105 proteins were assigned as potential chloroplast proteins, annotated for function, and quantified. Near complete coverage of primary carbon metabolism, starch and tetrapyrole metabolism, as well as excellent coverage for fatty acid synthesis, isoprenoid, sulfur, nitrogen and amino acid metabolism, was obtained. This showed e.g. quantitative and qualitative cell-type specific specialization in starch biosynthesis, Arg synthesis, N-assimilation, initial steps in S-assimilation. An extensive, overview of BS and M chloroplast protein expression and homeostasis machineries (>200 proteins) demonstrated qualitative and quantitative differences between M and BS chloroplasts, and BS-enhanced levels of the specialized chaperones ClpB3 and HSP90 that suggest active remodeling of the BS proteome. The reconstructed pathways are presented as detailed flow diagrams including annotation, relative protein abundances and cell-specific expression pattern. Protein annotation and identification data, and projection of matched peptides on the protein models, are available online through the Plant Proteome Database, PPDB.

Thylakoids are the chloroplast internal membrane systems that house light-harvesting and electron-transport reactions. Despite the important functions and well-studied constituents of thylakoids, the molecular mechanism of their development remains largely elusive. A recent genetic study has demonstrated that plastidic type I signal peptidase 1 (Plsp1) is vital for proper thylakoid development in Arabidopsis thaliana chloroplasts. Plsp1 was also shown to be necessary for processing of an envelope protein, Toc75, and a thylakoid lumenal protein, OE33; however, the relevance of the protein maturation in both of the two distinct subcompartments for proper chloroplast development remained unknown. Here we conducted an extensive analysis of the plsp1-null mutant to address the significance of lumenal protein maturation in thylakoid development. Plastids that lack Plsp1 were found to accumulate vesicles of variable sizes in the stroma. Analyses of the mutant plastids revealed that the lack of Plsp1 causes a reduction in accumulation of thylakoid proteins, and that Plsp1 is involved in maturation of two additional lumenal proteins, OE23 and plastocyanin. Further immunoblotting and electron microscopy immunolocalization studies showed that OE33 associates with the stromal vesicles of the mutant plastids. Finally, we used a genetic complementation system to demonstrate that accumulation of improperly-processed forms of Toc75 in the plastid envelope does not disrupt normal plant development. These results suggest that proper maturation of lumenal proteins may be a key process for correct assembly of thylakoids.

High-throughput technology has facilitated genome-scale analyses of transcriptomic adjustments in response to environmental perturbations with an oxygen deprivation component, such as transient hypoxia or anoxia, root waterlogging or complete submergence. We showed previously that Arabidopsis thaliana seedlings elevate the levels of hundreds of transcripts, including a core group of 49 genes that are prioritized for translation across cell types of both shoots and roots. To recognize low oxygen responses that are evolutionarily conserved versus species specific, we compared the transcriptomic reconfiguration in 21 organisms from four kingdoms (Plantae, Animalia, Fungi, Bacteria). Sorting of organism proteomes into clusters of putative orthologs identified broadly conserved responses associated with glycolysis, fermentation, alternative respiration, metabolite transport, reactive oxygen species amelioration, chaperone activity, and ribosome biogenesis. Differentially regulated genes involved in signaling and transcriptional regulation were poorly conserved across kingdoms. Strikingly, nearly half of the induced mRNAs of Arabidopsis seedlings encode proteins of unknown function (PUFs), of which over 40% had upregulated orthologs in poplar (Populus trichocarpa), rice (Oryza sativa) or Chlamydomonas (Chlamydomonas reinhardtii). Sixteen HYPOXIA-RESPONSIVE UNKNOWN PROTEIN (HUP) genes, including four that are Arabidopsis-specific, were ectopically overexpressed and evaluated for their effect on seedling tolerance to oxygen deprivation. This allowed identification of HUPs co-regulated with genes associated with anaerobic metabolism and other processes that significantly enhance or reduce stress survival when ectopically overexpressed. These findings illuminate both broadly conserved and plant-specific low oxygen stress responses and confirm that plant-specific HUPs with limited phylogenetic distribution influence low oxygen stress endurance.

The Ethylene responsive element binding factor-associated Amphiphilic Repression (EAR) motif is a transcriptional regulatory motif identified in members of the ERF, C2H2, and AUX/IAA families of transcriptional regulators. Sequence comparison of the core EAR motif sites from these proteins revealed two distinct conservation patterns: LxLxL and DLNxxP. Proteins containing these motifs play key roles in diverse biological functions by negatively regulating genes involved in developmental, hormonal and stress signaling pathways. Through a genome-wide bioinformatics analysis, we have identified the complete repertoire of the EAR repressome in Arabidopsis comprising 219 proteins belonging to 21 different transcriptional regulator families. Approximately 72% of these proteins contain a LxLxL type of EAR motif, whereas 22% contain a DLNxxP type of EAR motif and the remaining 6% have a motif where LxLxL and DLNxxP are overlapping. Published in vitro and in planta investigations support approximately 40% of these proteins functioning as negative regulators of gene expression. Comparative sequence analysis of EAR motif sites and adjoining regions has identified additional preferred residues and potential post-translational modification sites which may influence the functionality of the EAR motif. Homology searches against protein databases of poplar, grapevine, rice and sorghum revealed that the EAR motif is conserved across these diverse plant species. This genome-wide analysis represents the most extensive survey of EAR motif-containing proteins in Arabidopsis to-date, and provides a resource enabling investigations into their biological roles and the mechanism of EAR motif-mediated transcriptional regulation.

The alternative oxidase (AOX) is a cyanide-resistant oxidase that provides an alternative outlet for electrons from the respiratory electron transport chain embedded in the inner membrane of plant mitochondria. Examination of soybean (Glycine max L.) plants carrying a GmAOX2b antisense gene showed AOX to have a central role in reproductive development and fecundity. In three independently transformed antisense lines, seed set was reduced by 16 to 43%, whereas ovule abortion increased by 1.2- to 1.7-fold when compared to non-transgenic transformation control plants. Reduced fecundity was associated with reductions in whole-leaf cyanide-resistant, salicylhydroxamic acid-sensitive respiration and net photosynthesis, but there was no change in total respiration in the dark. The frequency of potential fertilisation events was reduced by at least one-third in the antisense plants as a likely consequence of pre-fertilisation defects. Pistils of the antisense plants contained a higher proportion of immature-sized, non-fertile embryo sacs compared to non-transgenic control plants. Increased rates of pollen abortion in vivo and reduced rates of pollen germination in vitro suggested that the antisense gene compromised pollen development and function. Reciprocal crosses between antisense and non-transgenic plants revealed that pollen produced by antisense plants was less active in fertilisation. Taken together, the results presented here indicate that AOX expression has an important role in determining normal gametophyte development and function.

Ca²⁺ is important for plant growth and development as a nutrient and a second messenger. However, the molecular nature and roles of Ca²⁺-permeable channels or transporters involved in Ca²⁺ uptake in roots are largely unknown. We recently identified a candidate for the Ca²⁺-permeable mechanosensitive channel in Arabidopsis thaliana, named MCA1. Here we investigated the only paralogue of MCA1 in Arabidopsis, MCA2. cDNA of MCA2 complemented a Ca²⁺-uptake deficiency in yeast cells lacking a Ca²⁺ channel composed of Mid1 and Cch1. RT-PCR analysis indicated that MCA2 was expressed in leaves, flowers, roots, siliques and stems and histochemical observation showed that an MCA2 promoter::β-glucuronidase (GUS) fusion reporter gene was universally expressed in 10-day old seedlings with some exceptions: it was relatively highly expressed in vascular tissues and undetectable in the cap and the elongation zone of the primary root. mca2-null plants were normal in growth and morphology. In addition, the primary root of mca2-null seedlings was able to normally sense the hardness of agar medium, unlike that of mca1-null or mca1-null mca2-null seedlings, as revealed by the two-phase-agar method. Ca²⁺ uptake activity was lower in the roots of mca2-null plants than those of wild-type plants. Finally, growth of mca1-null mca2-null plants was more retarded at a high concentration of Mg²⁺ added to medium, compared with that of mca1-null and mca2-null single mutants and wild-type plants. These results suggest that the MCA2 protein has a distinct role in Ca²⁺ uptake in roots and an overlapping role with MCA1 in plant growth.

Mitochondria play an essential role in NO signal transduction in plants. Using the biotin switch method in conjunction with nano liquid chromatography and mass spectrometry we identified eleven candidate proteins that were S-nitrosylated and/or glutathionylated in mitochondria of Arabidopsis leaves. These included glycine decarboxylase complex (GDC), a key enzyme of the photorespiratory C2 cycle in C3 plants. GDC activity was inhibited by S-nitrosoglutathione due to S-nitrosylation/S-glutathionylation of several Cys residues. Gas exchange measurements demonstrated that the bacterial elicitor harpin, a strong inducer of reactive oxygen species and NO, inhibits GDC activity. Furthermore, an inhibitor of GDC, aminoacetonitrile, was able to mimic mitochondrial depolarization, H₂O₂ production, and cell death in response to stress or harpin treatment of cultured Arabidopsis thaliana cells. These findings indicate that the mitochondrial photorespiratory system is involved in the regulation of NO signal transduction in Arabidopsis.

(1,3;1,4)-β-D-glucan (β-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum L.) and is an important source of dietary fibre for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CSLF6 gene as encoding a putative β-glucan synthase. RNAi constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high performance anion exchange chromatography (HPAEC) after digestion with lichenase showed decreases in total β-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable β-glucan was also reduced by about 50% in the transgenic lines, and the molecular weight distribution of the fraction was decreased from an average of 79-85 x 10⁴ g/mol in the controls to 36-57 x 10⁴ g/mol in the transgenics. Immunolocalisation of β-glucan in semi-thin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a β-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.

Although phenylpropanoid-polyamine conjugates (PPCs) occur ubiquitously in plants, their biological roles remain largely unexplored. The two major PPCs of Nicotiana attenuata plants, caffeoylputrescine (CP) and dicaffeoylspermidine (DCS), increase dramatically in local and systemic tissues after herbivore attack and simulations thereof. We identified NaMYB8, a homolog of NtMYBJS1, which in BY2 cells regulates PPCs biosynthesis, and silenced its expression by RNAi in N. attenuata (ir-MYB8), to understand the ecological role(s) of PPCs. The regulatory role of NaMYB8 in PPCs biosynthesis was validated by a microarray analysis, which revealed that transcripts of several key biosynthetic genes in shikimate and polyamine metabolism accumulated in a NaMYB8-dependent manner. Wild-type (WT) N. attenuata plants typically contain high levels of PPCs in their reproductive tissues; however, NaMYB8-silenced plants that completely lacked CP and DCS showed no changes in reproductive parameters of the plants. In contrast a defensive role for PPCs was clear; both specialist (Manduca sexta) and generalist (Spodoptera littoralis) caterpillars feeding on systemically pre-induced young stem leaves performed significantly better on ir-MYB8 plants lacking PPCs compared to WT plants expressing high levels of PPCs. Moreover, the growth of M. sexta caterpillars was significantly reduced when neonates were fed ir-MYB8 leaves sprayed with synthetic CP, corroborating the role of PPCs as direct plant defense. The spatial-temporal accumulation and function of PPCs in N. attenuata are consistent with the predictions of the Optimal Defense Theory: plants preferentially protect their most fitness enhancing and vulnerable parts, young tissues and reproductive organs, to maximize their fitness.

The role of nitrogen metabolism in the survival of prolonged periods of waterlogging was investigated in highly flood-tolerant, nodulated Lotus japonicus plants. Alanine production revealed to be a critical hypoxic pathway being the only amino acid whose biosynthesis is not inhibited by N-deficiency resulting from RNAi silencing of nodular leghemoglobin. The metabolic changes which were induced following waterlogging can be best explained by the activation of alanine metabolism in combination with the modular operation of a split tricarboxylic acid (TCA) pathway. The sum result of this metabolic scenario is the accumulation of alanine and succinate and the production of extra ATP under hypoxia. The importance of alanine metabolism is discussed with respect to its ability to regulate the level of pyruvate, and this and all other changes are discussed in the context of current models concerning the regulation of plant metabolism.

Light is the ultimate source of energy for photosynthesis; however, excessive light leads to photo-oxidative damage and hence reduced photosynthetic efficiency, especially when combined with other abiotic stresses. Although the photosystem II (PSII) reaction center D1 protein is the primary target of photo-oxidative damage, other PSII core proteins are also damaged and degraded. However, it is still largely unknown whether degradation of D1 and other PSII proteins involves previously uncharacterized proteases. Here we show that Deg7 is peripherally associated with the stromal side of the thylakoid membranes and that Deg7 interacts directly with PSII. Our results show that Deg7 is involved in the primary cleavage of photodamaged D1, D2, CP47, and CP43, and this activity is essential for its function in PSII repair. The double mutants of deg5 deg7 and deg8 deg7 showed no obvious phenotypic differences under normal growth conditions, but additive effects were observed under high light. These results suggest that Deg proteases on both the stromal and luminal sides of the thylakoid membranes are important for the efficient photosystem II repair in Arabidopsis.

Anoxia induces several heat shock proteins and a mild heat pre-treatment can acclimatize Arabidopsis seedlings to subsequent anoxic treatment. In this study we analyzed the response of Arabidopsis seedlings to anoxia, heat and combined heat+anoxia stress. A significant overlap between the anoxic and the heat responses was observed by whole-genome microarray analysis. Among the transcription factors induced by both heat and anoxia, the heat shock factor A2 (HsfA2), known to be involved in Arabidopsis acclimation to heat and to other abiotic stress, was strongly induced by anoxia. Heat-dependent acclimation to anoxia is lost in a HsfA2 knock-out mutant (hsfa2) as well as in a double mutant for the constitutively expressed HsfA1a / HsfA1b (hsfA1a/1b) indicating that these three heat-shock factors cooperate to confer anoxia tolerance. Arabidopsis seedlings that over-express HsfA2 showed an increased expression of several known targets of this transcription factor and were markedly more tolerant to anoxia as well as to submergence. Anoxia failed to induce HsfA2 target proteins in wild-type seedlings, while over-expression of HsfA2 resulted in the production of HsfA2 targets under anoxia, correlating well with the low anoxia tolerance experiments. The results indicate that there is a considerable overlap between the molecular mechanisms of heat and anoxia tolerance and that HsfA2 is a player in this mechanism.

Systemic acquired resistance is a widespread phenomenon in the plant kingdom that confers heightened and often enduring immunity to a range of diverse pathogens. Systemic immunity develops through activation of plant disease resistance protein signalling networks following local infection with an incompatible pathogen. The accumulation of the phytohormone salicylic acid in systemically responding tissues occurs within days after a local immunizing infection and is essential for systemic resistance. However our knowledge of the signalling components underpinning signal perception and establishment of systemic immunity are rudimentary. Previously we showed an early and transient increase in jasmonic acid in distal responding tissues was central to effective establishment of systemic immunity. Based upon predicted transcriptional networks induced in naive Arabidopsis thaliana leaves following avirulent Pseudomonas syringae challenge, we show that a variety of auxin mutants compromise the establishment of systemic immunity. Linking together transcriptional and targeted metabolite studies our data provide compelling evidence for a role of indole derived compounds, but not auxin itself, in establishment and maintenance of systemic immunity.

The actin cytoskeleton is a key target for signaling networks and plays a central role in translating signals into cellular responses in eukaryotic cells. Self-incompatibility (SI) is an important mechanism responsible for preventing self-fertilization. The SI system of Papaver rhoeas pollen involves a Ca²⁺-dependent signaling network, including massive actin depolymerization as one of the earliest cellular responses, followed by the formation of large actin foci. However, no analysis of these structures, which appear to be aggregates of filamentous (F-)actin based on phalloidin staining, has been carried out to date. Here, we characterize and quantify the formation of F-actin foci in incompatible Papaver pollen tubes over time. The F-actin foci increase in size over time and we provide evidence that their formation requires actin polymerization. Once formed, these SI-induced structures are unusually stable, being resistant to treatments with latrunculin B (LatB). Further, their formation is associated with changes in the intracellular localization of two actin-binding proteins, cyclase-associated protein (CAP) and actin-depolymerizing factor (ADF). Two other regulators of actin dynamics, profilin and fimbrin, do not associate with the F-actin foci. This study provides the first insights into the actin-binding proteins and mechanisms involved in the formation of these intriguing structures, which appear to be actively formed during the SI response.

Mature indeterminate Medicago truncatula nodules are zonated with an apical meristem, an infection zone, a fixation zone with nitrogen-fixing bacteroids, and a "developmental" senescence zone that follows nodule growth with a conical front originating in the center of the fixation zone. In nitrogen-fixing cells, senescence is initiated coincidently with the expression of a family of conserved cysteine proteases that might well be involved in the degradation of symbiotic structures. Environmental stress, such as prolonged dark treatment, interferes with nodule functioning and triggers a fast and global nodule senescence. Developmental and dark stress-induced senescence have several different structural and expression features, suggesting, at least, partly divergent underlying molecular mechanisms.

Phosphate homeostasis was studied in a monocotyledonous model plant through the characterization of the PHO1 gene family in rice (Oryza sativa). Bioinformatics and phylogenetic analysis showed that the rice genome has three PHO1 homologues, which cluster with the Arabidopsis AtPHO1 and AtPHO1;H1, the only two genes known to be involved in root-to-shoot transfer of phosphate. In contrast to the Arabidopsis PHO1 gene family, all three rice PHO1 genes have a cis-natural antisense transcript located at the 5' end of the genes. Strand-specific Q-PCR analyses revealed distinct patterns of expression for sense and antisense transcripts for all three genes, both at the level of tissue expression and in response to nutrient stress. The most abundantly expressed gene was OsPHO1;2 in the roots, for both sense and antisense transcripts. However, while OsPHO1;2 sense transcript was relatively stable under various nutrient deficiencies, the antisense transcript was highly induced by Pi deficiency. Characterization of Ospho1;1 and Ospho1;2 insertion mutants revealed that only Ospho1;2 mutants had defects in Pi homeostasis, namely strong reduction in Pi transfer from root to shoot, which was accompanied by low shoot and high root Pi. Our data identify OsPHO1;2 as playing a key role in the transfer of Pi from roots to shoots in rice, and indicate that this gene could be regulated by its cis-NAT. Furthermore, phylogenetic analysis of PHO1 homologues in monocotyledons and dicotyledons revealed the emergence of a distinct clade of PHO1 genes in dicotyledons, which include members having roles other than long-distance Pi transport.

Various fluorophore-based microscopic methods, comprising Forster Resonance Energy Transfer (FRET) and Bimolecular Fluorescence Complementation (BiFC), are suitable to study pairwise interactions of proteins in living cells. The analysis of interactions between more than two protein partners using these methods remains, however, difficult. In this study, we report the successful application of combined BiFC-FRET-FLIM (Fluorescence Lifetime Imaging Microscopy) and BiFC-FRET-APB (Acceptor Photobleaching) measurements to visualize the formation of ternary soluble N-ethylmaleimide sensitive factor attachment receptor (SNARE) complexes in leaf epidermal cells. This method expands the repertoire of techniques to study protein-protein interactions in living plant cells by a procedure capable of visualizing simultaneously interactions between three fluorophore-tagged polypeptide partners.

In Arabidopsis thaliana (L.), the blue light photoreceptors phototropins (phot1 and phot2) fine-tune the photosynthetic status of the plant by controlling several important adaptive processes in response to environmental light variations. These processes include stem and petiole phototropism (leaf positioning), leaf flattening, stomatal opening, and chloroplast movements. The PHYTOCHROME KINASE SUBSTRATE (PKS) protein family comprises four members in Arabidopsis (PKS1 to PKS4). PKS1 is a novel phot1 signaling element during phototropism as it interacts with phot1 and the important signaling element NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3), and is required for normal phot1-mediated phototropism. In this study, we have analyzed more globally the role of three PKS members (PKS1, 2 and 4). Systematic analysis of mutants reveals that PKS2 (and to a lesser extent PKS1) act in the same subset of phot-controlled responses as NPH3, namely leaf flattening and positioning. PKS1, PKS2 and NPH3 co-immunoprecipitate with both phot1-GFP and phot2-GFP in leaf extracts. Genetic experiments position PKS2 within phot1 and phot2 pathways controlling leaf positioning and leaf flattening, respectively. NPH3 can act in both phot1 and phot2 pathways, and synergistic interactions observed between pks2 and nph3 mutants suggest complementary roles of PKS2 and NPH3 during phot signaling. Finally, several observations further suggest that PKS2 may regulate leaf flattening and positioning by controlling auxin homeostasis. Together with previous findings, our results indicate that the PKS proteins represent an important family of phot-signaling proteins.

The classical effect of the plant hormone auxin is very rapid stimulation of cell expansion followed by sustained growth over a longer time period. However, auxins are also important in other responses such as cell division and differentiation. Recently, the TRANSPORT INHIBITOR RESPONSE1/AUXIN BINDING F-BOX PROTEIN (TIR1/AFB) family of auxin receptors regulating expression of auxin induced genes has garnered much attention regarding the precise role that TIR1/AFBs serve in auxin responses. Here, we show that major changes in gene expression mediated by these important receptors do not play the major role in triggering the very rapid phase of cell elongation.

Tension wood is widespread in the organs of woody plants. During its formation, it generates a large tensile mechanical stress, called maturation stress. Maturation stress performs essential biomechanical functions such as optimising the mechanical resistance of the stem, performing adaptive movements and ensuring long-term stability of growing plants. Although various hypotheses have recently been proposed, the mechanism generating maturation stress is not yet fully understood. In order to discriminate between these hypotheses, we investigated structural changes in cellulose microfibrils along sequences of xylem cell differentiation in tension and normal wood of poplar (Populus deltoides x P. trichocarpa cv. I45-51). Synchrotron radiation micro-diffraction was used to measure the evolution of the angle and lattice spacing of crystalline cellulose associated to the deposition of successive cell wall layers. Profiles of normal and tension wood were very similar in early development stages corresponding to the formation of the S1 and the outer part of the S2 layer. The microfibrils angle in the S2 layer was found lower in its inner part than in its outer part, especially in tension wood. In tension wood only, this decrease occurred together with an increase in cellulose lattice spacing, and this happened before the G-layer is visible. The relative increase in lattice spacing was found close to usual value of maturation strains, strongly suggesting that microfibrils of this layer are put into tension and contribute to the generation of maturation stress.

Bran from bread wheat (Triticum aestivum L. cv. Babbler) grain is composed of many outer layers of dead maternal tissues that overlie living aleurone cells. The dead cell layers function as a barrier resistant to degradation, whereas the aleurone layer is involved in mobilising organic substrates in the endosperm during germination. We microdissected three defined bran fractions – outer layers (epidermis and hypodermis), intermediate fraction (cross cells, tube cells, testa and nucellar tissue) and inner layer (aleurone cells) – and used proteomics to identify their individual protein complements. All proteins of the outer layers were enzymes whose function is to provide direct protection against pathogens or improve tissue strength. The more complex proteome of the intermediate layers suggests a greater diversity of function, including the inhibition of enzymes secreted by pathogens. The inner layer contains proteins involved in central metabolism, as would be expected from live aleurone cells, but this layer also included defense enzymes and inhibitors, as well as 7S globulin (specific to this layer). Using immunofluorescence microscopy, oxalate oxidase was localized predominantly to the outer layers, xylanase inhibitor protein-I to the xylan-rich nucellar layer of the intermediate fraction, and pathogenesis-related protein-4 mainly to the aleurone. Activities of the water-extractable enzymes oxalate oxidase, peroxidase and polyphenol oxidase were highest in the outer layers, whereas the chitinase activity was found only in the whole grain. We conclude that the differential protein complements of each bran layer in wheat provide distinct lines of defense in protecting the embryo and nutrient-rich endosperm.

In cyanobacteria fatty acids destined for lipid synthesis can be synthesized de novo, but also exogenous free fatty acids from the culture medium can be directly incorporated into lipids. Activation of exogenous fatty acids is likely required prior to their utilization. To identify the enzymatic activity responsible for activation we cloned candidate genes from Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 and identified the encoded proteins as acyl-acyl carrier protein synthetases (Aas). The enzymes catalyze the ATP-dependent esterification of fatty acids to the thiol of acyl carrier protein (ACP). The two protein sequences are only distantly related to known prokaryotic Aas proteins but they display strong similarity to sequences which can be found in almost all organisms that perform oxygenic photosynthesis. To investigate the biological role of Aas activity in cyanobacteria, aas knockout mutants were generated in the background of Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942. The mutant strains showed two phenotypes characterized by the inability to utilize exogenous fatty acids and by the secretion of endogenous fatty acids into the culture medium. The analyses of extracellular and intracellular fatty acid profiles of aas mutant strains as well as labeling experiments indicated that the detected free fatty acids are released from membrane lipids. The data suggest a considerable turnover of lipid molecules and a role for Aas activity in recycling the released fatty acids. In this model, lipid degradation represents a third supply of fatty acids for lipid synthesis in cyanobacteria.

Remobilization of phosphate (P_i) within a plant is critical for sustaining growth and seed production under external P_i fluctuation. The barley transporter, HvPHT1;6 has been implicated in P_i remobilization. In this report, we expressed HvPHT1;6 in Xenopus laevis oocytes allowing detailed characterization of voltage-dependent fluxes and currents induced by HvPHT1;6. HvPHT1;6 increased efflux of P_i near oocyte resting membrane potentials, dependent on external P_i concentration. Time-dependent inward currents were observed when membrane potentials were more negative than -160 mV, which was consistent with nH⁺:HPO₄^2-(n>2) co-transport, based on simultaneous radiotracer and oocyte voltage clamping, dependant upon P_i concentration gradient and pH. Time- and voltage-dependent inward currents through HvPHT1;6 were also observed for SO₄^2-, and to a lesser degree for NO₃^-, and Cl^-, but not for malate. Inward and outward currents showed linear dependence on the concentration of external HPO₄^2-, similar to low affinity P_i transport in plant studies. The electrophysiological properties of HvPHT1;6, which locates to the plasma membrane when expressed in onion epidermal cells, are consistent with its suggested role in the remobilization of P_i in barley plants.

Levels of the enzymes that produce wound response mediators have to be controlled tightly in unwounded tissues. The Arabidopsis fatty acid oxygenation up-regulated 8 (fou8) mutant catalyzes high rates of -linolenic acid (18:3) oxygenation and has higher-than-wild type (WT) levels of the 18:3-derived wound response mediator jasmonic acid (JA) in undamaged leaves. fou8 produces a null allele in the gene SAL1 (also known as FIERY1 or FRY1). Overexpression of the WT gene product had the opposite effect of the null allele suggesting a regulatory role of SAL1 acting in JA synthesis. The biochemical phenotypes in fou8 were complemented when the yeast sulfur metabolism 3‘(2’), 5‘-bisphosphate nucleotidase MET22 was targeted to chloroplasts in fou8. The data are consistent with a role of SAL1 in the chloroplast-localized dephosphorylation of 3’-phospho-5‘-adenosine phosphosulfate (PAPS) to 5’-adenosine phosphosulfate (APS) or in a closely related reaction (e.g. adenosine 3‘,5’-bisphospate (PAP) dephosphorylation). Furthermore, the fou8 phenotype was genetically suppressed in a triple mutant (fou8 apk1 apk2) affecting chloroplastic PAPS synthesis. These results show that a nucleotide component of the ‘sulfur futile-cycle’ regulates early steps of JA production and basal JA levels.

Fluorescence resonance energy transfer (FRET) sensitized emission of the yellow cameleon (YC) 3.60 was used to study the dynamics of cytoplasmic calcium ([Ca²⁺]_cyt) in different zones of living Arabidopsis (Arabidopsis thaliana) roots. Transient elevations of [Ca²⁺]_cyt were observed in response to glutamic acid (Glu), adenosine triphosphate (ATP) and aluminum (Al³⁺). Each chemical induced a [Ca²⁺]_cyt signature that differed among the three treatments in regard to the onset, duration and shape of the response. Glu and ATP triggered patterns of [Ca²⁺]_cyt increases that were similar among the different root zones, whereas Al³⁺ evoked [Ca²⁺]_cyt transients that had monophasic and biphasic shapes most notably in the root transition zone. The Al³⁺-induced [Ca²⁺]_cyt increases generally started in the maturation zone and propagated toward the cap while the earliest [Ca²⁺]_cyt response after Glu or ATP treatment occurred in an area that encompassed the meristem and elongation zone. The biphasic [Ca²⁺]_cyt signature resulting from Al³⁺ treatment originated mostly from cortical cells located at 300-500 µm from the root tip, which could be triggered in part through ligand-gated Glu receptors. Lanthanum (La³⁺) and gadolinium (Gd³⁺), cations commonly used as Ca²⁺channel blockers, elicited [Ca²⁺]_cyt responses similar to those induced by Al³⁺. The trivalent ion-induced [Ca²⁺]_cyt signatures in roots of an Al³⁺ resistant and Al³⁺ sensitive mutant were similar to those of wild-type indicating that the early [Ca²⁺]_cyt changes we report here may not be tightly linked to Al³⁺ toxicity but rather to a general response to trivalent cations.

As an overwhelming amount of functional genomics data has been generated, the retrieval, integration and interpretation of these data need to be facilitated to enable the advance of (systems) biological research. For example, gathering and processing microarray data that are related to a particular biological process is not straightforward, neither is the compilation of protein-protein interactions from numerous, partially overlapping databases, identified through diverse approaches. However, these tasks are inevitable to address the following questions: "Does a group of differentially expressed genes show similar expression in diverse microarray experiments?", "Was an identified protein-protein interaction previously detected by other approaches?" or "Are the interacting proteins encoded by genes with similar expression profiles and localization?". We developed CORNET (CORrelation NETworks) as an access point to transcriptome, protein interactome, localization data and functional information on Arabidopsis thaliana. It consists of two flexible and versatile tools, namely the co-expression tool and the PPI tool. The ability to browse and search microarray experiments using ontology terms and the incorporation of personal microarray data are distinctive features of the microarray repository. The co-expression tool enables either the alternate or simultaneous use of diverse expression compendia, whereas the PPI tool searches experimentally and computationally identified protein-protein interactions. Different search options are implemented to enable the construction of co-expression and/or protein-protein interaction networks centered around multiple input genes or proteins. Moreover, networks and associated evidence are visualized in Cytoscape. Localization is visualized in pie charts, thereby allowing multiple localizations per protein. CORNET is available at http://bioinformatics.psb.ugent.be/cornet.

The biosynthesis of the tetracyclic diterpene ent-kaurene is a critical step in the general (primary) metabolism of gibberellin hormones. ent-Kaurene is formed by a two-step cyclization of geranylgeranyl diphosphate via the intermediate ent-copalyl diphosphate. In a lower land plant, the moss Physcomitrella patens, a single bifunctional diterpene synthase (diTPS) catalyzes both steps. In contrast, in angiosperms, the two consecutive cyclizations are catalyzed by two distinct monofunctional enzymes, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). The enzyme, or enzymes, responsible for ent-kaurene biosynthesis in gymnosperms has been elusive. However, several bifunctional diTPS of specialized (secondary) metabolism have previously been characterized in gymnosperms, and all known diTPSs for resin acid biosynthesis in conifers are bifunctional. To further understand the evolution of ent-kaurene biosynthesis as well as the evolution of general and specialized diterpenoid metabolisms in gymnosperms, we set out to determine whether conifers use a single bifunctional diTPS or two monofunctional diTPSs in the ent-kaurene pathway. Using a combination of EST, full-length cDNA, gDNA, and targeted BAC sequencing, we identified two candidate CPS and KS genes from white spruce (Picea glauca) and their orthologues in Sitka spruce (P. sitchensis). Functional characterization of the recombinant enzymes established that ent-kaurene biosynthesis in white spruce is catalyzed by two monofunctional diTPSs, PgCPS and PgKS. Comparative analysis of gene structures and enzyme functions highlights the molecular evolution of these diTPSs as conserved between gymnosperms and angiosperms. In contrast, diTPSs for specialized metabolism have evolved differently in angiosperms and gymnosperms.

To study the influence of PIN1 mediated auxin transport during embryogenesis and endosperm development in monocots, the expression pattern of the three identified ZmPIN1 genes was determined at transcript level. Localization of the corresponding proteins was also analyzed during Zea mays kernel development. An anti-IAA monoclonal antibody was used to visualize IAA distribution and correlate the direction of auxin active transport, mediated by ZmPIN1 proteins, with the actual amount of auxin present in maize kernel at different developmental stages. ZmPIN1 genes are expressed in the endosperm soon after double fertilization occurs, however, differently to other tissues, the ZmPIN1 proteins were never polarly localized in the plasma-membrane of endosperm cells. ZmPIN1 transcripts and proteins also co-localize in developing embryos and the ZmPIN1 proteins are polarly localized in the embryo cell plasma-membrane from the first developmental stages, indicating the existence of ZmPIN1-mediated auxin fluxes. Auxin distribution visualization indicates that the aleurone, the basal endosperm transfer layer (BETL) and the embryo surrounding region (ESR) accumulate free auxin, which also has a maximum in the kernel maternal chalaza. During embryogenesis, polar auxin transport always correlates with the differentiation of embryo tissues and definition of the embryo organs. On the basis of these reports and of the observations on tissue differentiation and IAA distribution in defective endosperm-B18 mutant and in NPA treated kernels, a model for ZmPIN1-mediated transport of auxin and the related auxin fluxes during maize kernel development is proposed. Common features between this model and the model previously proposed for Arabidopsis are discussed.

Plant shoot organs arise from initial cells that are recruited from meristematic tissues. Previous studies have shown that members of the WUSCHEL-related homeob OX (WOX) gene family function to organize various initial cell populations during plant development. The function of the WOX4 gene is previously undescribed in any plant species. Comparative analyses of WOX4 transcription and function are presented in Arabidopsis thaliana, a simple-leafed plant with collateral vasculature, and in tomato (Solanum lycopersicum), a dissected-leafed species with bicollateral venation. WOX4 is transcribed in the developing vascular bundles of root and shoot lateral organs in both Arabidopsis and tomato. RNAi-induced down-regulation of WOX4 in Arabidopsis generated small plants whose vascular bundles accumulated undifferentiated ground tissue and exhibited severe reductions in differentiated xylem and phloem. In situ hybridization analyses of Atwox4-RNAi plants revealed delayed and reduced expression of both the phloem developmental marker ALTERED PHLOEM1 (APL1) and of HOMEOBOX GENE 8 (AtHB8), a marker of the vascular procambium. Over-expression of SlWOX4 correlated with over-proliferation of xylem and phloem in transgenic tomato seedlings. The cumulative data suggest that the conserved WOX4 function is to promote differentiation and/or maintenance of the vascular procambium, the initial cells of the developing vasculature.

To identify genes involved in vascular patterning, we screened for abnormal venation patterns in a large collection of leaf shape mutants isolated in our laboratory. The ron1-1 mutant, initially isolated because of its rounded leaves, exhibited an open venation pattern, which resulted from an increased number of free-ending veins. We positionally cloned the RON1 gene and found it to be identical to FRY1/SAL1, which encodes an enzyme with inositol polyphosphate 1-phosphatase and 3'(2'),5'-bisphosphate nucleotidase activities and has not previously been related to venation patterning. The ron1-1 mutant and mutants affected in auxin homeostasis share perturbations in venation patterning, lateral root formation, root hair length, shoot branching, and apical dominance. These similarities prompted us to monitor the auxin response using a DR5-GUS auxin-responsive reporter transgene, the expression levels of which were increased in roots and reduced in leaves in ron1-1 background. To gain insight into the function of RON1/FRY1/SAL1 during vascular development, we generated double mutants for genes involved in vein patterning, and found that ron1 synergistically interacts with axr1 and hve, but not with cvp1 and cvp2. These results suggest a role for inositol metabolism in the regulation of auxin responses. Microarray analysis of gene expression revealed that several hundred genes are misexpressed in ron1-1, which may explain the pleiotropic phenotype of this mutant. Metabolomic profiling of the ron1-1 mutant revealed changes in the levels of 38 metabolites, including myo-inositol and indole-3-acetonitrile, a precursor of auxin.

Aquaporins are channel proteins that facilitate the transport of water across plant cell membranes. In the present work, we used a combination of pharmacological and reverse genetic approaches to investigate the overall significance of aquaporins for tissue water conductivity in Arabidopsis. We addressed the function in roots and leaves of AtPIP1;2, one of most abundantly expressed isoforms of the Plasma membrane Intrinsic Protein family. At variance with the water transport phenotype previously described in AtPIP2;2 knockout mutants (Javot et al., 2003, Plant Cell, 15:509), disruption of AtPIP1;2 reduced by 20-30 % the root hydrostatic hydraulic conductivity but did not modify osmotic root water transport. These results document qualitatively distinct functions of different PIP isoforms in root water uptake. The hydraulic conductivity of excised rosettes (K_ros) was measured by a novel pressure chamber technique. Exposure of Arabidopsis plants to darkness increased K_ros by up to 90%. Mercury and azide, two aquaporin inhibitors with distinct modes of action, were able to induce similar inhibition of K_ros by ~13% and ~25% in rosettes from plant grown in the light or under prolonged (11-18 h) darkness, respectively. Prolonged darkness enhanced the transcript abundance of several PIP genes including AtPIP1;2. Mutant analysis showed that, under prolonged darkness conditions, AtPIP1;2 can contribute to up of ~ 20% of K_ros and to the osmotic water permeability of isolated mesophyll protoplasts. AtPIP1;2 therefore can account for a significant portion of aquaporin-mediated leaf water transport. The overall work shows that AtPIP1;2 represents a key component of whole plant hydraulics.

BET proteins are characterized by the presence of two types of domains, the bromodomain and the extra terminal (ET) domain. They bind to acetylated lysines present on histone tails and control gene transcription. They are also well known to play an important role in cell cycle regulation. In Arabidopsis there are 12 BET genes, however only two of them, IMBIBITION INDUCIBLE 1 (IMB1) and GENERAL TRANSCRIPTION FACTOR GROUP E6 (GTE6) were functionally analysed. We characterized GTE4 and show that gte4 mutant plants have some characteristic features of cell cycle mutants. Their size is reduced, they have jagged leaves and reduced number of cells in most organs. Moreover, cell size is considerably increased in the root, and, interestingly, the root quiescent center identity seems to be partially lost. Cell cycle analyses revealed that there is a delay in activation of the cell cycle during germination and a premature arrest of cell proliferation with a switch from mitosis to endocycling leading to a statistically significant increase in ploidy levels in the differentiated organs of gte4 plants. Our results point to a role of GTE4 in cell cycle regulation and specifically in the maintenance of the mitotic cell cycle.

Ribulose-1,5-bisphosphate carboxylase/oxygenase activase (RCA) catalyzes the activation of Rubisco in vivo, and plays a crucial role in photosynthesis. However, until now, little is known about the molecular genetics of RCA in soybean (Glycine max (L.) Merr.), one of the most important legume crops. Here, we cloned and characterized two genes encoding the longer isoform and the shorter β isoform of soybean RCA (GmRCA and GmRCAβ, respectively). The two corresponding cDNAs are divergent in both the translated and 3-untranslated regions. Analysis of genomic DNA sequences suggested that the corresponding mRNAs are transcripts of two different genes, and not the products of one single alternatively splicing pre-mRNA. Two additional possible -form RCA encoding genes, GmRCA03 and GmRCA14, and one additional β-form RCA encoding gene, GmRCA11, were also isolated. To examine the function and modulation of RCA genes in soybean, we determined the expression level of GmRCA and GmRCAβ, Rubisco initial activity, photosynthetic rate (P_N) and seed yield in 184 soybean recombinant inbred lines. Correlation of gene expression levels with three other traits indicates that RCA genes could play an important role in regulating soybean photosynthetic capacity and seed yield. Expression quantitative trait loci (eQTL) mapping revealed four trans eQTLs for GmRCA and GmRCAβ. These results could provide a new approach for the modulation of RCA genes to improve P_N and plant growth in soybean and other plants.

Using novel specially-designed instrumentation, fluorescence emission spectra were recorded from Arabidopsis thaliana leaves during the induction period of dark to high-light (HL) adaptation in order to follow the spectral changes associated with the formation of non-photochemical quenching. In addition to overall decrease of PSII fluorescence (quenching) across the entire spectrum, HL induced two specific relative changes in the spectra – i) a decrease of the main emission band at 682 nm relative to the far-red (FR, 750-760 nm) part of the spectrum (F₆₈₂) and ii) an increase at 720-730 nm (F₇₂₀) relative to 750-760 nm. The kinetics of the two relative spectral changes and their dependence on various mutants revealed that they do not originate from the same process but rather from at least two independent processes. The F₇₂₀ change is specifically associated with the rapidly-reversible energy-dependent quenching qE. Comparison of the wild type Arabidopsis with mutants unable to produce or overexpressing PsbS showed that PsbS was a necessary component for F₇₂₀. The spectral change F-₆₈₂ is induced both by qE and by PsbS-independent mechanism(s). A third novel quenching process, independent from both PsbS and zeaxanthin, is activated by a high turnover-rate of PSII. Its induction and relaxation occur on a time scale of a few minutes. Analysis of the spectral inhomogeneity of NPQ allows extracting mechanistically valuable information from the fluorescence induction kinetics when registered in a spectrally-resolved fashion.

Oxidative injury of the root elongation zone is a primary event in aluminum (Al) toxicity in plants, but the injurying species remain unidentified. We verified the hypothesis that lipid peroxide-derived aldehydes, especially highly electrophilic ,β-unsaturated aldehydes (2-alkenals), participate in Al toxicity. Transgenic tobaccos overexpressing Arabidopsis thaliana 2-alkenal reductase (AER-OE plants), wild-type SR1, and an empty vector-transformed control line (SR-Vec) were exposed to AlCl₃ on their roots. Compared with the two controls, AER-OE plants suffered less retardation of root elongation under AlCl₃ treatment and showed more rapid re-growth of roots upon Al removal. Under AlCl₃ treatment, the roots of AER-OE plants accumulated Al and H₂O₂ to the same levels as did the sensitive controls, while they accumulated lower levels of aldehydes and suffered less cell death than SR1 and SR-Vec roots. In SR1 roots, AlCl₃ treatment markedly increased the contents of the highly reactive 2-alkenals acrolein, 4-hydroxy-(E)-2-hexenal, and 4-hydroxy-(E)-2-nonenal and other aldehydes such as malondialdehyde and formaldehyde. In AER-OE roots, accumulation of these aldehydes was significantly less. Growth of the roots exposed to 4-hydroxy-(E)-2-nonenal and (E)-2-hexenal were retarded more in SR1 than in AER-OE plants. Thus, the lipid peroxide-derived aldehydes, formed downstream of reactive oxygen species, injured root cells directly. Their suppression by AER provides a new defense mechanism against Al toxicity.

Histidine (His) plays a critical role in plant growth and development, both as one of the standard amino acids in proteins, and as a metal binding ligand. While genes encoding seven of the eight enzymes in the pathway of His biosynthesis have been characterised from a number of plant species, the identity of the enzyme catalysing the dephosphorylation of histidinol-phosphate to histidinol has remained elusive. Recently, members of a novel family of histidinol-phosphate phosphatase (HPP) proteins, displaying significant sequence similarity to known myoinositol monophosphatases (IMP) have been identified from several Actinobacteria. Here we demonstrate that a member of the IMP family from Arabidopsis thaliana, myoinositol monophosphatase-like 2 (IMPL2, At4g39120) has HPP activity. Heterologous expression of IMPL2, but not the related IMPL1 protein, was sufficient to rescue the His auxotrophy of a Streptomyces coelicolor hisN mutant. Homozygous null impl2 Arabidopsis mutants displayed embryonic lethality, which could be rescued by supplying plants heterozygous for null impl2 alleles with His. In common with the previously characterised HISN genes from Arabidopsis, IMPL2 was expressed in all plant tissues and throughout development, and an IMPL2:GFP fusion protein was targeted to the plastid, where His biosynthesis occurs in plants. Our data demonstrate that IMPL2 is the HISN7 gene product, and suggest a lack of genetic redundancy at this metabolic step in Arabidopsis, which is characteristic of the His biosynthetic pathway.

Transporters move hydrophilic substrates across hydrophobic biological membranes and play key roles in plant nutrition, metabolism, signaling, and consequently in plant growth, development, and responses to the environment. To initiate and support systematic characterization of transporters in the model legume, Medicago truncatula, we identified 3,830 transporters and classified 2,673 of these into 113 families and 146 sub-families. Analysis of gene expression data for 2,611 of these transporters identified 129 that are expressed in an organ-specific manner, including 50 that are nodule-specific and 36 specific to mycorrhizal roots. Further analysis uncovered 196 transporters that are induced at least five fold during nodule development and 44 in roots during arbuscular mycorrhizal (AM) symbiosis. Amongst the nodule- and mycorrhizal-induced transporter genes are many candidates for known transport activities in these beneficial symbioses. The data presented here are a unique resource for selection and functional characterization of legume transporters.

In both applied and basic research, Agrobacterium-mediated transformation is commonly used to introduce genes into plants. We investigated the effect of three Agrobacterium tumefaciens strains and five T-DNA origins of replication on transformation frequency, transgene copy number, and the frequency of integration of non-T-DNA portions of the T-DNA-containing vector ("backbone") into the genome of Arabidopsis thaliana and Zea mays. Launching T-DNA from the picA locus of the Agrobacterium chromosome increases the frequency of single transgene integration events and almost eliminates the presence of vector backbone sequences in transgenic plants. Along with novel Agrobacterium strains we have developed, our findings are useful for improving the quality of T-DNA integration events.

Nectars are rich in primary metabolites and attract mutualistic animals, which serve as pollinators or as an indirect defence against herbivores. Their chemical composition makes nectars prone to microbial infestation. As protective strategy, floral nectar of ornamental tobacco contains ‘nectarins’, proteins producing reactive oxygen species such as H₂O₂. By contrast, pathogenesis-related (PR) proteins were detected in Acacia extrafloral nectar (EFN, which is secreted in the context of defensive ant-plant mutualisms). We investigated whether these PR-proteins protect EFN from phytopathogens. Five sympatric species (Acacia cornigera, A. hindsii, A. collinsii, A. farnesiana and Prosopis juliflora) were compared, which differ in their ant-plant mutualism. EFN of myrmecophytes, which are obligate ant-plants that secrete EFN constitutively to nourish specialised ant inhabitants, significantly inhibited the growth of four out of six tested phytopathogenic microorganisms. By contrast, EFN of non-myrmecophytes, which is secreted only transiently in response to herbivory, did not exhibit a detectable inhibitory activity. Combining two-dimensional SDS-PAGE with nanoLC-MS/MS analysis confirmed that PR-proteins represented over 90 % of all proteins in myrmecophyte EFN. The inhibition of microbial growth was exerted by the protein fraction, but not the small metabolites of this EFN and disappeared when nectar was heated. In-gel assays demonstrated the activity of acidic and basic chitinases in all EFNs, whereas glucanases were detected only in EFN of myrmecophytes. Our results demonstrate that PR-proteins causally underlie the protection of Acacia EFN from microorganisms and that acidic and basic glucanases likely represent the most important prerequisite in this defensive function.

Arabidopsis thaliana genes MLO2 (MILDEW RESISTANCE LOCUS O 2), MLO6 and MLO12 exhibit unequal genetic redundancy with respect to the modulation of defence responses against powdery mildew fungi and the control of developmental phenotypes such as premature leaf decay. We show that early chlorosis and necrosis of rosette leaves in mlo2 mlo6 mlo12 mutants reflects an authentic but untimely leaf senescence program. Comparative transcriptional profiling revealed that transcripts of several genes encoding tryptophan biosynthetic and metabolic enzymes hyper-accumulate during vegetative development in the mlo2 mlo6 mlo12 mutant. Elevated expression levels of these genes correlate with altered steady state levels of several indolic metabolites, including the phytoalexin camalexin and indolic glucosinolates, during development in the mlo2 single and the mlo2 mlo6 mlo12 triple mutant. Results of genetic epistasis analysis suggest a decisive role for indolic metabolites in mlo2-conditioned antifungal defence against both biotrophic powdery mildews and a camalexin-sensitive strain of the necrotrophic fungus, Botrytis cinerea. The wound- and pathogen-responsive callose synthase POWDERY MILDEW RESISTANCE 4/GLUCAN-SYNTHASE-LIKE 5 (PMR4/GSL5) was found to be responsible for the spontaneous callose deposits in mlo2 mutant plants but dispensable for mlo2-conditioned penetration resistance. Our data strengthen the notion that powdery mildew resistance of mlo2 genotypes is based on the same defence execution machinery as innate antifungal immune responses that restrict invasion of non-adapted fungal pathogens.

Besides abundant oleosin, three minor proteins, Sop 1, 2, and 3, are present in sesame (Sesamum indicum) oil bodies. The gene encoding Sop1, named caleosin for its calcium-binding capacity, has recently been cloned. In this study, Sop2 gene was obtained by immunoscreening, and it was subsequently confirmed by amino acid partial sequencing and immunological recognition of its overexpressed protein in Escherichia coli. Immunological cross recognition implies that Sop2 exists in seed oil bodies of diverse species. Along with oleosin and caleosin genes, Sop2 gene was transcribed in maturing seeds where oil bodies are actively assembled. Sequence analysis reveals that Sop2, tentatively named steroleosin, possesses a hydrophobic anchoring segment preceding a soluble domain homologous to sterol-binding dehydrogenases/reductases involved in signal transduction in diverse organisms. Three-dimensional structure of the soluble domain was predicted via homology modeling. The structure forms a seven-stranded parallel ß-sheet with the active site, S-(12X)-Y-(3X)-K, between an NADPH and a sterol-binding subdomain. Sterol-coupling dehydrogenase activity was demonstrated in the overexpressed soluble domain of steroleosin as well as in purified oil bodies. Southern hybridization suggests that one steroleosin gene and certain homologous genes may be present in the sesame genome. Comparably, eight hypothetical steroleosin-like proteins are present in the Arabidopsis genome with a conserved NADPH-binding subdomain, but a divergent sterol-binding subdomain. It is indicated that steroleosin-like proteins may represent a class of dehydrogenases/reductases that are involved in plant signal transduction regulated by various sterols.