Monogalactosyldiacylglycerol (MGDG) is the major lipid constituent of chloroplast membranes, and has been proposed to act directly in several important plastidic processes, particularly during photosynthesis. In this study, the effect of MGDG deficiency, as observed in the monogalactosyldiacylglycerol synthase 1 (mgd1-1) mutant, on chloroplast protein targeting, phototransformation of pigments, and photosynthetic light reactions was analyzed. The targeting of plastid proteins into or across the envelope, or into the thylakoid membrane, was not different from wild-type in the mgd1 mutant, suggesting that the residual amount of MGDG in mgd1 was sufficient to maintain functional targeting mechanisms. In dark-grown plants, the ratio of bound protochlorophyllide (Pchlide, F656) versus free Pchlide (F631) was increased in mgd1 compared to the wild type. Increased levels of the photoconvertible pigment-protein complex (F656), which is photoprotective and suppresses photooxidative damage caused by an excess of free Pchlide, may be an adaptive response to the mgd1 mutation. Leaves of mgd1 suffered from a massively impaired capacity for thermal dissipation of excess light (qE), due to an inefficient operation of the xanthophyll cycle: the mutant contained less zeaxanthin and more violaxanthin than wild type after 60 min of high-light exposure, and suffered from increased PSII photoinhibition. This is attributable to an increased conductivity of the thylakoid membrane at high light intensities, so that the proton motive force is reduced and the thylakoid lumen is less acidic than in wild type. Thus, the pH-dependent activation of the violaxanthin de-epoxidase and of the PsbS protein is impaired.

Arbuscular mycorrhizal (AM) fungi are obligate biotrophs that participate in a highly beneficial root symbiosis with 80 % of land plants. Strigolactones are trace molecules in plant root exudates that are perceived by AM fungi at sub nanomolar concentrations. Within just a few hours, they were shown to stimulate fungal mitochondria, spore germination and branching of germinating hyphae. In the present study we show that treatment of Gigaspora rosea with a strigolactone analogue (GR24) causes a rapid increase in the NADH concentration, the NADH dehydrogenase activity and the ATP content of the fungal cell. This fully and rapidly (within minutes) activated oxidative metabolism does not require new gene expression. Up-regulation of the genes involved in mitochondrial metabolism and hyphal growth, and stimulation of the fungal mitotic activity, take place several days after this initial boost to the cellular energy of the fungus. Such a rapid and powerful action of GR24 on G. rosea cells suggests that strigolactones are important plant signals involved in switching arbuscular mycorrhizal fungi towards full germination and a pre-symbiotic state.

Prenylation is a posttranslational protein modification essential for developmental processes and response to abscicic acid. Following prenylation, the three C-terminal residues are proteoliticaly removed and in turn the free carboxyl group of the isoprenyl cysteine is methylated. The proteolysis and methylation, collectively referred to as CaaX processing, are catalyzed by Ste24 or Rce1 endoproteases and by an isoprenyl cysteine methyltrasnferase, ICMT. Arabidopsis contains single STE24 and RCE1 and two ICMT homologues. Here we show that in yeast AtRCE1 promoted a-mating factor secretion and membrane localization of a ROP GTPase. Furthermore, GFP fusion proteins of AtSTE24, AtRCE1, AtICMTA and AtICMTB are co-localized in the ER, indicating that prenylated proteins reach this compartment and that CaaX processing is likely required for subcellular targeting. AtICMTB can process yeast a-factor more efficiently than AtICMTA. Sequence and mutational analyses revealed that the higher activity AtICMTB is conferred by five residues, which are conserved between yeast Ste14p, human ICMT and AtICMTB but not in AtICMTA. Quantitative real-time RT-PCR and microarray data show that AtICMTA expression is significantly lower compared to AtICMTB. AtICMTA null mutants have a wild type phenotype indicating that its function is redundant. However, AtICMT RNAi lines had fasciated inflorescence stems, altered phylotaxis and developed multiple buds without stem elongation. The phenotype of the ICMT RNAi lines is similar to farnesyltrasnferase β-subunit mutant era1 but is more subtle. Collectively, the data suggest that AtICMTB is likely the major isoprenyl carboxy methyltansferase and that methylation modulates activity of prenylated proteins.

Fruit color-break is the visual manifestation of the developmentally-regulated transition of chloroplasts to chromoplasts during fruit ripening and often involves biosynthesis of copious amounts of carotenoids concomitant with massive breakdown of chlorophyll. Regulation of chlorophyll breakdown at different physiological and developmental stages of the plant life cycle, particularly at fruit color-break, is still not well understood. Here we present the dynamics of native chlorophyllase and chlorophyll breakdown in Citrus limon fruit during ethylene induced color-break. We show, using in-situ immunofluorescence on ethylene treated fruit peel (flavedo) tissue, that citrus chlorophyllase is located in the plastid, in contrast to recent reports suggesting cytoplasmic localization of Arabidopsis chlorophyllases. At the intra-organellar level, chlorophyllase signal was found to overlap mostly with chlorophyll fluorescence, suggesting association of most of the chlorophyllase protein with the photosynthetic membranes. Confocal microscopy analysis showed that the kinetics of chlorophyll breakdown was not uniform in the flavedo cells. Chlorophyll quantity at the cellular level was negatively correlated with plastid chlorophyllase accumulation; plastids with reduced chlorophyll content were found by in-situ immunofluorescence to contain significant levels of chlorophyllase, while plastids containing still intact chlorophyll lacked any chlorophyllase signal. Immunoblot and protein-MS analyses were used to demonstrate that citrus chlorophyllase initially accumulates as a ~35-kDa precursor, which is subsequently N-terminally processed to ~33-kDa mature forms by cleavage at either of three consecutive amino acid positions. Chlorophyllase plastid localization, expression kinetics and the negative correlation with chlorophyll levels support the central role of the enzyme in chlorophyll breakdown during citrus fruit color-break.

cpSRP54 is involved in co- and post-translational sorting of thylakoid proteins. The Arabidopsis cpSRP54 null mutant, ffc1-2, is pale green with delayed development. Western blot analysis of individual leaves showed that the SRP sorting pathway, but not the SecYE translocon, was strongly down-regulated with progressive leaf development in both wild-type and ffc1-2. To further understand the impact of cpSRP54 deletion, a quantitative comparison between ffc2-1 was carried out for total leaf proteomes of young seedlings and for chloroplast proteomes of fully developed leaves, using stable isotope labeling (iTRAQ and ICAT) and 2-dimensional gels. This showed that cpSRP54 deletion led to a change in LHC composition, increase of PsbS, and a decreased Photosystem I/II ratio. Moreover, the cpSRP54 deletion led in young leaves to up-regulation of thylakoid proteases and stromal chaperones, including ClpC. In contrast, the stromal protein homeostasis machinery returned to wild-type levels in mature leaves, consistent with the developmental down-regulation of the SRP pathway. A differential response between young and mature leaves was also found in carbon metabolism, with an up-regulation of the Calvin cycle and the photorespiratory pathway in peroxisomes and mitochondria in young leaves but not in old leaves. In contrast, the Calvin cycle was down-regulated in mature leaves, likely to adjust to the reduced capacity of the light reaction, while reactive oxygen species defense proteins were up-regulated. The significance of ClpC up-regulation was confirmed through the generation of an ffc2-1 x clpc1 double mutant. This mutant was seedling-lethal under autotrophic conditions, but could be partially rescued under heterotrophic conditions.

Oilseeds are the main source of lipids used in both food and bio-fuels. The growing demand for vegetable oil has focused research toward increasing the amount of this valuable component in oilseed crops. Globally, soybean is one of the most important oilseed crops grown contributing about 30% of the vegetable oil used for food, feed and industrial applications. Breeding efforts in soy have shown that multiple loci contribute to the final content of oil and protein stored in seeds. Genetically, the levels of these two storage products appear to be inversely correlated with an increase in oil coming at the expense of protein and vice versa. One way to overcome the linkage between oil and protein is to introduce a transgene that can specifically modulate one pathway without disrupting the other. We describe the first transgenic soy crop with increased oil that shows no major impact on protein content or yield. This was achieved by expressing a codon-optimized version of a diacylglycerol acyltransferase (DGAT2A) from the soil fungus Umbelopsis (formerly Mortierella) ramanniana in soybean seed during development resulting in an absolute increase in oil of 1.5% (by weight) in the mature seed.

During the establishment of symbiosis in legume roots, the rhizobial Nod factor signal is perceived by the host cells via receptor-like kinases including SymRK. The Nodule Inception (NIN) gene in Lotus japonicus is required for rhizobial entry into root cells and for nodule organogenesis. We describe here a novel DNA-binding protein from Lotus japonicus, referred to as SIP1, because it was identified as a SymRK-interacting protein. SIP1 contains a conserved AT-rich interaction domain (ARID) and represents a unique member of the ARID-containing proteins in plants. The C-terminus of SIP1 was found to be responsible for its interaction with the kinase domain of SymRK, and for homo-dimerization in the absence of DNA. SIP1 specifically binds to the promoter of LjNIN, but not to that of LjCBP1 (a calcium-binding protein gene), both of which are known to be inducible by Nod factors. SIP1 recognizes two of the three AT-rich domains present in the NIN gene promoter. Deletion of one of the AT-rich domain at the NIN promoter diminishes the binding of SIP1 to the NIN promoter. The protein is localized to the nuclei when expressed as a red fluorescence fusion protein in the onion epidermal cells. The SIP1 gene is expressed constitutively in the uninfected roots, and its expression levels are elevated after infection by Mesorhizobium loti. It is proposed that SIP1 may be required for the expression of NIN and involved in the initial communications between the rhizobia and the host root cells.

Eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein found in all eukaryotic kingdoms. The present study demonstrates that plant eIF5A is involved in the development of disease symptoms induced by a common necrotrophic bacterial phytopathogen. Specifically, AteIF5A-2, one of the three eIF5A genes in Arabidopsis thaliana, is shown to regulate programmed cell death caused by infection with virulent Pseudomonas syringae pv tomato DC3000 (Pst DC3000). Transgenic Arabidopsis plants with constitutively suppressed AteIF5A-2 exhibited marked resistance to programmed cell death induced by virulent Pst DC3000, and there was a corresponding reduction in pathogen growth and development of disease symptoms in the plant tissue. Constitutive over-expression of AteIF5A-2 circumvented the apparent post-transcriptional regulation of AteIF5A-2 protein expression characteristic of wild-type plants, but did not increase susceptibility to virulent Pst DC3000 ingression. The transgenic plants with constitutive AteIF5A-2 over-expression did, however, display phenotypes consistent with precocious cell death. The results indicate that AteIF5A-2 is a key element of the signal transduction pathway resulting in plant programmed cell death.

Forster resonance energy transfer (FRET) measurements based on fluorescence lifetime imaging microscopy (FLIM) are increasingly being used to assess molecular conformations and associations in living systems. Reduction in the excited state lifetime of the donor fluorophore in the presence of an appropriately positioned acceptor is taken as a strong evidence of FRET. Traditionally CFP has been widely used as a donor fluorophore in FRET experiments. However given its photolabile nature, low quantum yield and multiexponential lifetime, CFP is far from an ideal donor in FRET imaging. Here we report the application and use of TSapphire mutant of GFP as an efficient donor to mOrange in FLIM based-FRET imaging in intact plant cells. Using time correlated single photon counting (TSCPC)-FLIM we show that TSapphire expressed in living plant cells decays with lifetime of 2.93 ± 0.09 ns. Chimerically linked TSapphire and mOrange (with 16 aa linker in-between) exhibit substantial energy transfer based on the reduction in the lifetime of TSapphire in the presence of the acceptor mOrange. Experiments performed with various genetically and/or biochemically-known interacting plant proteins demonstrate the versatility of the FRET-FLIM system presented here in different sub-cellular compartments tested (cytosol, nucleus and at plasma-membrane). The better spectral overlap with red monomers, higher photostability and monoexponential lifetime of TSapphire makes it an ideal FRET-FLIM donor to study protein-protein interactions in diverse eukaryotic systems overcoming in particular many technical challenges encountered (like autofluorescence of cell walls and fluorescence of pigments associated with photosynthetic apparatus) while studying plant protein dynamics and interactions.

Coenzyme A (CoA) is an essential cofactor in the metabolism of both prokaryotic and eukaryotic organisms and a universal five-step pathway is utilized to synthesize CoA from pantothenate. Null mutations in two of the five steps of this pathway led to embryo lethality, and therefore viable reduction-of-function mutations are required to further study its role in plant biology. In this work, we have characterized a viable Arabidopsis thaliana T-DNA mutant affected in the penultimate step of the CoA biosynthesis pathway, which is catalyzed by the enzyme phosphopantetheine adenylyltransferase (PPAT). This ppat-1 knockdown mutation showed a ~90 % reduction in PPAT transcript levels and was severely impaired in plant growth and seed production. The sum of CoA and AcCoA levels was severely reduced (60-80%) in ppat-1 seedlings compared to wild type, and catabolism of storage lipids during seedling establishment was delayed. Conversely, PPAT over-expressing (OE) lines showed on average ~1.6-fold higher levels of CoA + AcCoA levels, as well as enhanced vegetative and reproductive growth and salt/osmotic stress resistance. Interestingly, dry seeds of OE lines contained between 35-50% more FAs than wild type, which suggests that CoA biosynthesis plays a crucial role in storage oil accumulation. Finally, biochemical analysis of the recombinant PPAT enzyme revealed an inhibitory effect of CoA on PPAT activity. Taken together, these results suggest that the reaction catalyzed by PPAT is a regulatory step in the CoA biosynthetic pathway that plays a key role for plant growth, stress resistance and seed lipid storage.

A silencing vector for cotton was developed from the geminivirus Cotton leaf crumple virus (CLCrV). The CLCrV coat protein gene was replaced by up to 500 bp of DNA homologous to one of two endogenous genes, ChlI or PDS. Cotyledons of Gossypium hirsutum cv. DeltaPine 5415 bombarded with the modified viral vectors manifested chlorosis due to silencing of either ChlI or PDS in ~70% of inoculated plants after 2-3 weeks. Use of the green fluorescence protein gene showed that replication of viral DNA was restricted to vascular tissue and that the viral vector could transmit to leaves, roots, and the ovule integument from which fibers originate. Temperature had profound effects on vector DNA accumulation and the spread of endogenous gene silencing. Consistent with reports that silencing against viruses increases at higher temperatures, plants grown at a 30/26°C day/night cycle had a greater than 10-fold reduction in viral DNA accumulation compared to plants grown at 22/18°C. However, endogenous gene silencing decreased at 30/26°C. There was a ~7 day delay in the onset of gene silencing at 22/18°C, but silencing was extensive and persisted throughout the life of the plant. The extent of silencing in new growth could be increased or decreased by changing temperature regimes at various times following the onset of silencing. Our experiments establish the use of the CLCrV silencing vector to study gene function in cotton and show that temperature can have a major impact on the extent of geminivirus-induced gene silencing.

Gibberellins (GAs) regulate many aspects of plant development such as germination, growth, and flowering. The barley Amy32b -amylase promoter contains at least five cis-acting elements that govern its GA-induced expression. Our previous studies indicates that a barley WRKY gene, HvWRKY38 and its rice ortholog, OsWRKY71, blocks GA induced expression of Amy32b-GUS. In this work, we investigated the functional and physical interactions of HvWRKY38 with another repressor and two activators in barley. HvWRKY38 blocks the inductive activities of SAD (a DOF protein) and HvGAMYB (a R2R3 MYB protein) when either of these proteins is present individually. However, SAD and HvGAMYB together overcome the inhibitory effect of HvWRKY38. Yet combination of HvWRKY38 and BPBF (another DOF protein) almost diminishes the synergistic effect of SAD and HvGAMYB transcriptional activators. Electrophoretic mobility shift assays indicate that HvWRKY38 blocks GA-induced expression of Amy32b by interfering with the binding of HvGAMYB to the cis-acting elements in the -amylase promoter. The physical interaction of HvWRKY38 and BPBF repressors is demonstrated via Bimolecular Fluorescence Complementation (BiFC) assays. These data suggest that the expression of Amy32b is modulated by protein complexes that contain either activators (e.g. HvGAMYB and SAD) or repressors (e.g. HvWRKY38 and BPBF). The relative amounts of the repressor or activator complexes binding to the Amy32b promoter regulate its expression level in barley aleurone cells.

Wax esters are neutral lipids composed of aliphatic alcohols and acids, with both moieties usually long-chain (C₁₆ and C₁₈) or very-long-chain (C₂₀ and longer) carbon structures. They have diverse biological functions in bacteria, insects, mammals and terrestrial plants, and are also important substrates for a variety of industrial applications. In plants, wax esters are mostly found in the cuticles coating the primary shoot surfaces, but they also accumulate to high concentrations in the seed oils of a few plant species, including jojoba, a desert shrub which is the major commercial source of these compounds. Here we report the identification and characterization of WSD1, a member of the bifunctional wax ester synthase/diacylglycerol acyltransferase gene family, which plays a key role in wax ester synthesis in Arabidopsis stems, as first evidenced by severely reduced wax ester levels of in the stem wax of wsd1 mutants. In vitro assays using protein extracts from E. coli expressing WSD1 showed that this enzyme has a high level of WS activity, and approximately tenfold lower level of DGAT activity. Expression of the WSD1 gene in Saccharomyces cerevisiae resulted in the accumulation of wax esters, but not triacylglycerol, indicating that WSD1 predominantly functions as a wax synthase. Analyses of WSD1 expression revealed that this gene is transcribed in flowers, top parts of stems and leaves. Fully functional yellow fluorescent protein-tagged WSD1 protein was localized to the endoplasmic reticulum, demonstrating that biosynthesis of wax esters, the final products of the alcohol-forming pathway, occurs in this subcellular compartment.

Small 10-kD acyl-CoA-binding proteins (ACBPs) are highly-conserved proteins that are prevalent in eukaryotes. In Arabidopsis thaliana, other than the 10-kD ACBP homologue (designated as Arabidopsis ACBP6), there are 5 larger forms of ACBPs ranging from 37.5 to 73.1 kD. In this study, the cytosolic subcellular localization of Arabidopsis ACBP6 was confirmed by analyses on transgenic Arabidopsis expressing autofluorescence-tagged ACBP6 and western blot analysis of subcellular fractions using ACBP6-specific antibodies. The expression of Arabidopsis ACBP6 was noticeably induced at 48 h after 4 ^oC treatment in northern blot analysis and western blot analysis. Further, an acbp6 T-DNA insertional mutant that lacked ACBP6 mRNA and protein, displayed increased sensitivity to freezing temperature (-8 ^oC), while ACBP6-overexpressing transgenic Arabidopsis were conferred enhanced freezing tolerance. Northern blot analyses indicated that ACBP6-associated freezing tolerance was not dependent on induction of cold-regulated COR gene expression. Instead, ACBP6-overexpressors showed increased expression of mRNA encoding phospholipase D (PLD). Lipid profiling analyses on cold-acclimated freezing-treated (-8 ^oC) transgenic Arabidopsis plants overexpressing ACBP6 showed a decline in phosphatidylcholine (-36% and -46%) and elevation of phosphatidic acid (73% and 67%) in comparison to wild type. From our comparison, the gain in freezing tolerance in ACBP6-overexpressors that were accompanied by decreases in phosphatidylcholine and accumulation in phosphatidic acid is consistent with previous findings on PLD-overexpressing transgenic Arabidopsis. In vitro filter-binding assays indicating His-tagged ACBP6 binds phosphatidylcholine, but not phosphatidic acid or lysophosphatidylcholine, further implicates a role for ACBP6 in phospholipid metabolism in Arabidopsis.

We report the molecular characterization and functional analysis of a gene (PsOAT) from Scots pine (Pinus sylvestris L.) encoding ornithine--aminotransferase (-OAT, EC 2.6.1.13), an enzyme of arginine metabolism. The deduced amino acid sequence contains a putative N-terminal signal peptide for mitochondrial targeting. The polypeptide is similar to other -OATs from plants, yeast and mammals and encoded by a single copy gene in pine. PsOAT encodes a functional -OAT as determined by expression of the recombinant protein in Escherichia coli and analysis of the active enzyme. The expression of PsOAT was undetectable in the embryo, but highly induced at early stages of germination and seedling development in all different organs. Transcript levels decreased in later developmental stages, although an increase was observed in lignified stems of 90-day-old plants. An increase of -OAT activity was observed in germinating embryos and seedlings and appears to mirror the observed alterations in PsOAT transcript levels. Similar expression patterns were also observed for genes encoding arginase and isocitrate dehydrogenase. Transcripts of PsOAT and the arginase gene were found widely distributed in different cell types of pine organs. Consistent with these results a metabolic pathway is proposed for the nitrogen flow from the megagametophyte to the developing seedling, which is also supported by the relative abundance of free amino acids in embryos and seedlings. Taken together, our data support that -OAT plays an important role in this process providing glutamate for glutamine biosynthesis during early pine growth.

Beta-barrel proteins of the Omp85 (Outer membrane protein, 85 kD) superfamily exist in the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts. Prominent Omp85 proteins in bacteria and mitochondria mediate biogenesis of other β-barrel proteins, and are indispensable for viability. In Arabidopsis (Arabidopsis thaliana) chloroplasts, there are two distinct types of Omp85-related protein: Toc75 (Translocon at the outer envelope membrane of chloroplasts, 75 kD) and OEP80 (Outer Envelope Protein, 80 kD). Toc75 functions as a preprotein translocation channel during chloroplast import, but the role of OEP80 remains elusive. We characterized three T-DNA mutants of the Arabidopsis OEP80 (AtOEP80) gene. Selectable markers associated with the oep80-1 and oep80-2 insertions segregated abnormally, suggesting embryo-lethality of the homozygous genotypes. Indeed, no homozygotes were identified amongst >100 individuals, and heterozygotes of both mutants produced ~25% aborted seeds upon self-pollination. Embryo arrest occurred at a relatively late stage (globular embryo-proper), as revealed by analysis using Nomarski optics microscopy. This is substantially later than arrest caused by loss of the principal Toc75 isoform, atToc75-III (two-cell stage), suggesting a more specialized role for AtOEP80. Surprisingly, the oep80-3 T-DNA (located in exon 1, between the first and second ATG codons of the open reading frame) did not cause any detectable developmental defects, or affect the size of the AtOEP80 protein in chloroplasts. This indicates that the N-terminal region of AtOEP80 is not essential for the targeting, biogenesis or functionality of the protein, in contrast with atToc75-III which requires a bipartite targeting sequence.

Building a cell plate during cytokinesis in plant cells requires the participation of a number of proteins in a multistep process. We previously identified phragmoplastin as a cell plate-specific protein involved in creating a tubulo-vesicular network at the cell plate. We report here the identification and characterization of a phragmoplastin interacting protein, PHIP1, in Arabidopsis. It contains multiple functional motifs including a lysine (K)-rich domain (KRD), two RNA recognition motifs (RRMs) and three CCHC-type zinc-fingers (ZnFs). Polypeptides with similar motif structures were found only in plant protein databases, but not in the sequenced prokaryotic, fungal and animal genomes, suggesting that PHIP1 represents a plant-specific RNA-binding protein. In addition to phragmoplastin, two Arabidopsis small GTP-binding proteins, Rop1 and Ran2, are also found to interact with PHIP1. The zinc-fingers of PHIP1 were not required for its interaction with Rop1 and phragmoplastin, but may participate in its binding with the Ran2 mRNA. Immunofluorescence, in situ RNA hybridization and green fluorescence protein (GFP)-tagging experiments showed the association of PHIP1 with the forming cell plate during cytokinesis. Taken together, our data suggest that PHIP1 is a novel RNA-binding protein and may play a unique role in the polarized mRNA transport to the vicinity of the cell plate.

Over a quarter of all plant genes encode proteins of unknown function which can be further classified as Proteins with Obscure Features (POFs), that lack currently defined motifs or domains, or Proteins with Define Features (PDFs), that contain at least one previously defined domain or motif. Although empirical data in the form of transcriptome and proteome profiling suggest that many of these proteins play important roles in plants, their functional characterization remains one of the main challenges in modern biology. To begin the functional annotation of proteins with unknown function, which are involved in the oxidative stress response of Arabidopsis thaliana, we generated transgenic Arabidopsis plants that constitutively expressed 23 different POFs (of which 4 were specific to Arabidopsis), and 18 different PDFs. All previously found to be expressed in response to oxidative stress in Arabidopsis. Transgenic plants were tested for their tolerance to oxidative stress imposed by paraquat or t-butyl hydroperoxide, or subjected to osmotic, salinity, cold and heat stresses. More than 70% of all expressed proteins conferred tolerance to oxidative stress. In contrast, over 90% of the expressed proteins did not confer enhanced tolerance to the other abiotic stresses tested, and about 50% rendered plants more susceptible to osmotic or salinity stress. Two Arabidopsis-specific POFs, and an Arabidopsis and Brassica-specific protein of unknown function, conferred enhanced tolerance to oxidative stress. Our findings suggest that tolerance to oxidative stress involves mechanisms and pathways that are unknown at present, including some that are specific to Arabidopsis or the Brassicaceae.

The sensitive to freezing 6 (sfr6) mutant of Arabidopsis thaliana is late flowering in long days due to reduced expression of components in the photoperiodic flowering pathway in long day photoperiods. Microarray analysis of gene expression showed that a circadian clock-associated motif, the evening element, was over-represented in promoters of genes down-regulated in sfr6 plants. Analysis of leaf movement rhythms found sfr6 plants showed a sucrose-dependent long period phenotype; unlike wild type Arabidopsis, the clock in sfr6 plants did not have a shorter rhythm in the presence of sucrose. Other developmental responses to sucrose were unaltered in sfr6 plants, suggesting insensitivity to sucrose is restricted to the clock. We investigated the effect of sfr6 and sucrose upon clock gene expression over twenty-four hours. The sfr6 mutation resulted in reduced expression of the clock components CIRCADIAN CLOCK ASSOCIATED 1, GIGANTEA and TIMING OF CAB1. These changes occurred independently of sucrose-supplementation. Wild type plants showed small increases clock gene expression in the presence of sucrose; this response to sucrose was reduced in sfr6 plants. This study shows that large changes in level and timing of clock gene expression may have little effect upon clock outputs. Moreover, although sucrose influences the period and accuracy of the Arabidopsis clock it results in relatively minor changes in clock gene expression.

Thioredoxins (Trxs) constitute a family of small proteins in plants. This family has been extensively characterized in Arabidopsis thaliana, which contains six different Trx types: f, m, x and y in chloroplasts, o in mitochondria and h mainly in cytosol. A detailed study of this family in the model legume Medicago truncatula, realized here, has established the existence of two isoforms that do not belong to any of the types previously described. As no possible orthologs were further found in either rice or poplar, these novel isoforms may be specific for legumes. Nevertheless, on the basis of protein sequence and gene structure, they are both related to Trxs m, and probably have evolved from Trxs m after the divergence of the higher plant families. They have redox potential values similar to those of the classical Trxs and one of them can act as a substrate for the M. truncatula NADP-thioredoxin reductase A. However, they differ from classical Trxs in that they possess an atypical putative catalytic site and lack disulfide reductase activity with insulin. Another important feature is the presence in both proteins of an N-terminal extension containing a putative signal peptide that targets them to the endoplasmic reticulum, as demonstrated by their transient expression in fusion with the green fluorescent protein in M. truncatula or Nicotiana benthamiana leaves. According to their pattern of expression, these novel isoforms function specifically in symbiotic interactions in legumes. They were therefore given the name of Trxs s, s for symbiosis.

ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the first committed step in starch biosynthesis. Higher plant ADP-Glc PPase is a heterotetramer (₂β₂) consisting of two small and two large subunits. There is increasing evidence that suggests that catalytic and regulatory properties of the enzyme from higher plants results from the synergy of both types of subunits. In Arabidopsis thaliana two genes encode small subunits (APS1 and APS2) and four large subunits (APL1 to 4). Here we show that in Arabidopsis thaliana APL1 and APL2, besides their regulatory role, have catalytic activity. Heterotetramers formed by combinations of a non-catalytic APS1 and the four large subunits showed that APL1 and APL2 exhibited ADP-Glc PPase activity with distinctive sensitivities to the allosteric activator (3-PGA). Mutation of the glucose-1-phosphate binding site of Arabidopsis and potato isoforms confirmed these observations. To determine the relevance of these activities in planta a T-DNA mutant of APS1 (aps1) was characterized. aps1 is starchless, lacked ADP-Glc PPase activity, APS1 mRNA, APS1 protein and is late flowering in long days. Transgenic lines of the aps1 mutant, expressing an inactivated form of APS1, recovered the wild type phenotype indicating that APL1 and APL2 have catalytic activity and may contribute to ADP-Glucose synthesis in planta.

During senescence and at times of stress, plants can mobilize needed nitrogen from chloroplasts from leaves to other organs. Much of the total leaf nitrogen is allocated to the most abundant plant protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). While bulk degradation of the cytosol and organelles in plants occurs by autophagy, the role of autophagy in the degradation of chloroplast proteins is still unclear. We have visualized the fate of Rubisco, stromal-targeted green fluorescent protein (GFP) and DsRed, and GFP-labeled Rubisco in order to investigate the involvement of autophagy in the mobilization of stromal proteins to the vacuole. Using immuno-electron microscopy (IEM), we previously demonstrated that Rubisco is released from the chloroplast into Rubisco-containing bodies (RCBs) in naturally senescent leaves (Chiba et al., 2003, Plant Cell Physiol 44: 914-921). When leaves of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing stromal-targeted fluorescent proteins were incubated with concanamycin A to inhibit vacuolar H⁺-ATPase activity, spherical bodies exhibiting GFP or DsRed fluorescence without chlorophyll fluorescence were observed in the vacuolar lumen. Double-labeled IEM with anti-Rubisco and anti-GFP antibodies confirmed that the fluorescent bodies correspond to RCBs. RCBs could also be visualized using GFP-labeled Rubisco directly. RCBs were not observed in leaves of a T-DNA insertion mutant in ATG5, one of the essential genes for autophagy. Stromal-targeted DsRed and GFP-ATG8 fusion proteins were observed together in autophagic bodies in the vacuole. We conclude that Rubisco and stromal-targeted fluorescent proteins can be mobilized to the vacuole through an ATG gene-dependent autophagic process without prior chloroplast destruction.

The cellular import of the hormone auxin is a fundamental requirement for the generation of auxin gradients which control a multitude of plant developmental processes. The AUX/LAX family of auxin importers, exemplified by AUX1 from Arabidopsis thaliana, has been shown to mediate auxin import when expressed heterologously. The quantitative nature of the interaction between AUX1 and its transport substrate indole-3-acetic acid (IAA) is incompletely understood and we sought to address this in the present investigation. We expressed AUX1 to high levels in a baculovirus expression system and prepared membrane fragments from baculovirus-infected insect cells. These membranes proved suitable for determination of the binding of IAA to AUX1 and enabled us to determine a Kd of 2.6 µM, comparable with estimates for the Km for IAA transport. The efficacy of a number of auxin analogues and auxin transport inhibitors to displace IAA binding from AUX1 has also been determined and can be rationalized in terms of their physiological effects. Determination of the parameters describing the initial interaction between a plant transporter and its hormone ligand provides novel quantitative data for modelling auxin fluxes.

In plants and many other organisms, phytochelatin synthase (PCS) catalyzes the synthesis of phytochelatins (PCs) from glutathione in the presence of certain metals and metalloids. We have used the budding yeast (Saccharomyces cerevisiae) as an heterologous system to characterize two PCS proteins, LjPCS1 and LjPCS3, of the model legume Lotus japonicus. Initial experiments revealed that the metal tolerance of yeast cells in vivo depends on the concentrations of divalent cations in the growth medium. Detailed in vivo (intact cells) and in vitro (broken cells) assays of PCS activity were performed with yeast expressing the plant enzymes, and values of PC production for each metal tested were normalized to those of cadmium to correct for the lower expression level of LjPCS3. Our results showed that lead was the best activator of LjPCS1 in the in vitro assay, whereas, for both assays, arsenic, iron, and aluminum were better activators of LjPCS3 and mercury was similarly active with the two enzymes. Most interestingly, zinc was a powerful activator, especially of LjPCS3, when assayed in vivo, whereas copper and silver were the strongest activators in the in vitro assay. We conclude that the in vivo and in vitro assays are useful and complementary to assess the response of LjPCS1 and LjPCS3 to a wide range of metals and that the differences in the C-terminal domains of the two proteins are responsible for their distinct expression levels or stabilities in heterologous systems and patterns of metal activation.

Leaf sucrose transporters are essential for phloem loading and long-distance partitioning of assimilates in plants that load their phloem from the apoplast. Sucrose loading into the phloem is indispensable for the generation of the osmotic potential difference that drives phloem bulk flow and is central for the long-distance movement of phloem sap compounds, including hormones and signaling molecules. In previous analyses, solanaceous SUT1 sucrose transporters from tobacco, potato and tomato were immunolocalized in plasma membranes of enucleate SEs. Here we present data that identify solanaceous SUT1 proteins with high specificity in phloem CCs. Moreover, comparisons of SUT1 localization in the abaxial and adaxial phloem revealed higher levels of SUT1 protein in the abaxial phloem of all three solanaceous species, suggesting different physiological roles for these two types of phloem. Finally, SUT1 proteins were identified in files of xylem parenchyma cells, mainly in the bicollateral veins. Together, our data provide new insight into the role of SUT1 proteins in solanaceous species.

Manganese (Mn) deficiency is an important plant nutritional disorder in many parts of the world. Barley (Hordeum vulgare) genotypes differ considerably in their ability to grow in soils with low Mn²⁺ availability. Differential genotypic Mn efficiency can be attributed to differences in Mn²⁺ uptake kinetics in the low nM concentration range. However, the molecular basis for these differences has not yet been clarified. We here present the identification and characterization of the first barley gene encoding a plasma membrane localized metal transport protein being able to transport Mn²⁺. The gene is designated HvIRT1 because it belongs to the ZIP gene family and has a high similarity to OsIRT1. A novel yeast uptake assay based on ICP-MS analysis of 31 different metal and metalloid ions showed that the HvIRT1 protein, in addition to Mn²⁺, also transported Fe²⁺/Fe³⁺, Zn²⁺ and Cd²⁺. Both Mn and Fe deficiency induced an up-regulation of HvIRT1 in two barley genotypes differing in Mn efficiency but the expression levels were in all cases highest (up to 40%) in the Mn-efficient genotype. The higher expression of HvIRT1 correlated with an increased Mn²⁺ uptake rate. It is concluded that HvIRT1 is an important component controlling Mn²⁺ uptake in barley roots and HvIRT1 contributes to genotypic differences in Mn²⁺ uptake kinetics.

The aim of this study was to clone and characterize the SUGAR-DEPENDENT6 gene, which is essential for post-germinative growth in Arabidopsis (Arabidopsis thaliana). Mutant alleles of sdp6 were able to breakdown triacylglycerol following seed germination but failed to accumulate soluble sugars suggesting that they had a defect in gluconeogenesis. Map-based cloning of SDP6 revealed that it encodes a mitochondrial flavin adenine dinucleotide–dependent glycerol-3-phosphate dehydrogenase:ubiquinone oxidoreductase called FAD-GPDH. This gene has previously been proposed to play a role both in the breakdown of glycerol (derived from triacylglycerol) and in NAD⁺/NADH homeostasis. Germinated seeds of sdp6 were severely impaired in the metabolism of [U-¹⁴C]glycerol to CO₂ and accumulated high levels of glycerol-3-phosphate (G3P). These data suggest that SDP6 is essential for glycerol catabolism. The activity of the glycolytic enzyme phosphoglucose isomerase (PGI) is competitively inhibited by G3P in vitro. We show that PGI is likely to be inhibited in vivo, since there is a 6-fold reduction in the transfer of ¹⁴C-label into the opposing hexosyl moiety of sucrose when [U-¹⁴C]glucose or [U-¹⁴C]fructose is fed to sdp6 seedlings. A block in gluconeogenesis, at the level of hexose phosphate isomerization, would account for the arrested seedling growth phenotype of sdp6 and explain its rescue by sucrose and glucose but not by fructose. Measurements of NAD⁺ and NADH levels in sdp6 seedlings also suggest that NAD⁺/NADH homeostasis is altered, and this observation is consistent with the hypothesis that SDP6 participates in a mitochondrial G3P shuttle by cooperating with the cytosolic NAD-dependent GPDH protein GPDHC1.

Seedlings of several species of gymnosperm trees, angiosperm trees, and angiosperm lianas were grown under tropical field conditions in the Republic of Panama; physiological processes controlling plant C and water fluxes were assessed across this functionally diverse range of species. Relative growth rate, r, was primarily controlled by the ratio of leaf area to plant mass, of which specific leaf area was a key component. Instantaneous photosynthesis, when expressed on a leaf-mass basis, explained 69% of variation in r (P<0.0001, n=94). Mean r of angiosperms was significantly higher than that of the gymnosperms; within angiosperms, mean r of lianas was higher than that of trees. Whole-plant N use efficiency was also significantly higher in angiosperm than in gymnosperm species, and was primarily controlled by the rate of photosynthesis for a given amount of leaf N. Whole-plant water use efficiency, TE_c, varied significantly among species, and was primarily controlled by c_i/c_a, the ratio of intercellular to ambient CO₂ partial pressures during photosynthesis. Instantaneous measurements of c_i/c_a explained 51% of variation in TE_c (P<0.0001, n=94). Whole-plant ¹³C discrimination also varied significantly as a function of c_i/c_a (R²=0.57, P<0.0001, n=94), and was, accordingly, a good predictor of TE_c. The ¹⁸O enrichment of stem dry matter was primarily controlled by the predicted ¹⁸O enrichment of evaporative sites within leaves (R²=0.61, P<0.0001, n=94), with some residual variation explained by mean transpiration rate. Measurements of C and O stable isotope ratios could provide a useful means of parameterizing physiological models of tropical forest trees.

Light drives the production of chemical energy and reducing equivalents in photosynthetic organisms required for the assimilation of essential nutrients. This process also generates strong oxidants and reductants that can be damaging to the cellular processes especially during absorption of excess excitation energy. Cyanobacteria, like other oxygenic photosynthetic organisms, respond to increase in the excitation energy such as during exposure of cells to high light by the reduction of antenna size and photosystem content. However, the mechanism of how Synechocystis sp. PCC 6803, a cyanobacterium, maintains redox homeostasis and coordinates various metabolic processes under high light stress remains poorly understood. In this study, we have utilized time series transcriptome data to elucidate the global responses of Synechocystis to high light. Identification of differentially regulated genes involved in the regulation, protection and maintenance of redox homeostasis has offered important insights into the optimized response of Synechocystis to high light. Our results indicate a comprehensive integrated homeostatic interaction between energy production (photosynthesis) and energy consumption (assimilation of carbon and nitrogen). In addition, measurements of physiological parameters under different growth conditions showed that integration between the two processes is not a consequence of limitations in the external carbon and nitrogen levels available to the cells. We have also discovered the existence of a novel glycosylation pathway, to date known as an important nutrient sensor only in eukaryotes. Upregulation of a gene encoding the rate-limiting enzyme in the hexosamine pathway suggests a regulatory role for protein glycosylation in Synechocystis under high light.

A variety of mechanisms has been proposed to account for the extension of life span in seeds (seed longevity). In the present work, we have used Arabidopsis thaliana seeds as a model and carried out differential proteomics to investigate this trait, which is of both ecological and agricultural importance. In our system based on a controlled deterioration treatment (CDT), we compared seed samples treated for different periods of time, up to seven days. Germination tests showed a progressive decrease of germination vigor depending on the duration of CDT. Proteomic analyses revealed that this loss in seed vigor can be accounted for by protein changes in the dry seeds and by an inability of the low-vigor seeds to display a normal proteome during germination. Furthermore, the CDT strongly increased the extent of protein oxidation (carbonylation), which might induce a loss of functional properties of seed proteins and enzymes and/or enhance their susceptibility toward proteolysis. These results unveiled essential mechanisms for seed vigor as translational capacity, mobilization of seed storage reserves and detoxification efficiency. Finally, the present work shows that similar molecular events accompany artificial and natural seed aging.

C₄ plants have up to ten-fold higher apparent CO₂ assimilation rates than the most productive C₃ plants. This requires higher fluxes of metabolic intermediates across the chloroplast envelope membranes of C₄ plants in comparison to that of C₃ plants. In particular, the fluxes of metabolites involved in the biochemical inorganic carbon pump of C₄ plants, such as malate, pyruvate, oxaloacetate, and phosphoenolpyruvate must be considerably higher in C₄ plants because they exceed the apparent rate of photosynthetic CO₂ assimilation whereas they represent relatively minor fluxes in C₃ plants. While the enzymatic steps involved in the C₄ biochemical inorganic carbon pump have been studied in much detail, little is known about the metabolite transporters in the envelope membranes of C₄ chloroplasts. In this study, we have used comparative proteomics of chloroplast envelope membranes from the C₃ plant Pisum sativum and mesophyll cell chloroplast envelopes from the C₄ plant Zea mays to analyze the adaptation of the mesophyll cell chloroplast envelope proteome to the requirements of C₄ photosynthesis. We show that C₃ and C₄-type chloroplasts have qualitatively similar but quantitatively very different chloroplast envelope membrane proteomes. In particular, translocators involved in the transport of triose phosphate and phosphoenolpyruvate as well as two outer envelope porins are much more abundant in C₄ plants. Several putative transport proteins have been identified that are highly abundant in C₄ plants, but relatively minor in C₃ envelopes. These represent prime candidates for the transport of C₄ photosynthetic intermediates, such as pyruvate, oxaloacetate, and malate.

Viroids are small self-replicating RNAs that infect plants. How these non-coding pathogenic RNAs interact with hosts to induce disease symptoms is a long-standing unanswered question. Recent experimental data has led to the suggestive proposal of a pathogenic model based on the RNA silencing mechanism. However, evidences of a direct relation between key components of the RNA silencing pathway and the symptom expression in infected plants remains elusive. To address this issue, we used a symptomatic transgenic line of Nicotiana benthamiana that expresses and processes dimeric forms of Hop stunt viroid (HSVd). These plants were analyzed under different growing temperature conditions, and were used as stocks in grafting assays with the rdr6i-Nb line, in which the RNA-dependent RNA polymerase 6 (NbRDR6) is constitutively silenced. Here we show that the symptom expression in N. benthamiana plants is independent of HSVd accumulation levels, but dependent on an active state of the viroid-specific RNA silencing pathway. The scion of rdr6i-Nb plants remained asymptomatic when grafted onto symptomatic plants, despite an accumulation of a high level of mature forms of HSVd indicating the requirement of RDR6 for the viroid-induced symptom production. In addition, the RDR6 requirement for symptom expression was also observed in wild type N. benthamiana plants mechanically infected with HSVd. These results provide biological evidence of the involvement of the viroid-specific RNA silencing pathway in the symptoms expression associated with viroid pathogenesis.

The Arabidopsis thaliana MKK1 and MKK2 MAP kinase kinases have been implicated in biotic and abiotic stress responses as part of a signalling cascade including MEKK1 and MPK4. Here, the double loss-of-function mutant (mkk1/2) of MKK1 and MKK2 is shown to have marked phenotypes in development and disease resistance similar to those of the single mekk1 and mpk4 mutants. Since mkk1 or mkk2 single mutants appear wild type, basal levels of MPK4 activity are not impaired in them, and MKK1 and MKK2 are in part functionally redundant in unchallenged plants. These findings are confirmed and extended by biochemical and molecular analyses implicating the kinases in jasmonate- and salicylate-dependent defense responses, mediated in part via the MPK4 substrate MKS1. In addition, transcriptome analyses delineate overlapping and specific effects of the kinases on global gene expression patterns demonstrating both redundant and unique functions for MKK1 and MKK2.

Plants elaborate a vast array of enzymes that synthesize defensive secondary metabolites in response to pathogen attack. Here, we isolated the pathogen responsive-CaMNR1 (menthone: (+)-(3S)-neomenthol reductase) gene, a member of the short-chain dehydrogenase/reductase (SDR) superfamily, from pepper (Capsicum annuum) plants. Gas chromatography-mass spectrometry analysis revealed that purified CaMNR1 and its ortholog AtSDR1 catalyze a menthone reduction with NADPH as a cofactor to produce neomenthol with antimicrobial activity. CaMNR1 and AtSDR1 also possess a significant catalytic activity for neomenthol oxidation. We examined the cellular function of the CaMNR1 gene by virus-induced gene silencing and ectopic overexpression in pepper and Arabidopsis plants, respectively. CaMNR1-silenced pepper plants were significantly more susceptible to Xanthomonas campestris pv. vesicatoria and Colletotrichum coccodes infection, and expressed lower levels of salicylic acid (SA)-responsive CaBPR1 and CaPR10 and jasmonic acid (JA)-responsive CaDEF1. CaMNR1-overexpression (OX) Arabidopsis plants exhibited enhanced resistance to the hemi-biotrophic pathogen Pseudomonas syringae pv. tomato DC3000 and the biotrophic pathogen Hyaloperonospora parasitica isolate Noco2, accompanied by induction of AtPR1 and AtPDF1.2. In contrast, mutation in the CaMNR1 ortholog Arabidopsis SDR1 (AtSDR1) significantly enhanced susceptibility to both pathogens. Together, these results indicate that the novel menthone reductase gene CaMNR1 and its ortholog AtSDR1 positively regulate plant defenses against a broad spectrum of pathogens.

Protein ubiquitination is a posttranslational regulatory process essential for plant growth and interaction with the environment. E3 ligases, to which the seven in absentia (SINA) proteins belong, determine the specificity by selecting the target proteins for ubiquitination. SINA proteins are found in animals as well as in plants and a small gene family with highly related members has been identified in the genome of rice (Oryza sativa), Arabidopsis thaliana, Medicago truncatula, and poplar (Populus trichocarpa). To acquire insight into the function of SINA proteins in nodulation, a dominant negative form of the Arabidopsis SINAT5 was ectopically expressed in the model legume M. truncatula. After rhizobial inoculation of the 35S:SINAT5DN transgenic plants, fewer nodules were formed than in control plants and most nodules remained small and white, a sign of impaired symbiosis. Defects in rhizobial infection and symbiosome formation were observed by extensive microscopic analysis. Besides the nodulation phenotype, transgenic plants were affected in shoot growth, leaf size, and lateral root number. This work illustrates a function for SINA E3 ligases in a broad spectrum of plant developmental processes, including nodulation.

The central cell of the female gametophyte plays a role in pollen tube guidance and in regulating the initiation of endosperm development. Following fertilization, the central cell gives rise to the seed's endosperm, which nourishes the developing embryo within the seed. The molecular mechanisms controlling specification and differentiation of the central cell are poorly understood. We identified AGL61 in a screen for transcription factor genes expressed in the female gametophyte. AGL61 encodes a Type I MADS domain protein, which likely functions as a transcription factor. Consistent with this, an AGL61-GFP fusion protein is localized to the nucleus. In the context of the ovule and seed, AGL61 is expressed exclusively in the central cell and early endosperm. agl61 female gametophytes are affected in the central cell specifically. The morphological defects include an overall reduction in size of the central cell and a reduced or absent central cell vacuole. When fertilized with wild-type pollen, agl61 central cells fail to give rise to endosperm. In addition, synergid- and antipodal-expressed genes are ectopically expressed in agl61 central cells. The expression pattern and mutant phenotype of AGL61 are similar to those of AGL80, suggesting that AGL61 may function as a heterodimer with AGL80 within the central cell; consistent with this, AGL61 and AGL80 interact in yeast. Together, these data suggest that AGL61 functions as a transcription factor and controls the expression of downstream genes during central cell development.

To better understand the metabolic processes of seed filling in soybean, two complementary proteomic approaches, two-dimensional gel electrophoresis (2-DGE) and semi-continuous multi-dimensional protein identification technology (Sec-MudPIT) coupled with liquid chromatography-mass spectrometry, were employed to analyze whole seed proteins at five developmental stages. 2-DGE and Sec-MudPIT analyses collectively identified 478 non-redundant proteins with only 70 proteins common to both datasets. 2-DGE data revealed that 38% of identified proteins were represented by multiple 2-DGE species. Identified proteins belonged to 13 (2-DGE) and 15 (Sec-MudPIT) functional classes. Proteins involved in metabolism, protein destination and storage, and energy were highly represented, collectively accounting for 61.1% (2-DGE) and 42.2% (Sec-MudPIT) of total identified proteins. Membrane proteins, based upon transmembrane predictions, were 3-fold more prominent in Sec-MudPIT than 2-DGE. Data were integrated into an existing soybean proteome database (www.oilseedproteomics.missouri.edu). The integrated quantitative soybean database was compared to a parallel study of rapeseed to further understand the regulation of intermediary metabolism in protein- versus oil-rich seeds. Comparative analyses revealed: (i) up to 3-fold higher expression of fatty acid biosynthetic proteins during seed filling in rapeseed compared to soybean and (ii) approximately 48% higher number of protein species and a net 80% higher protein abundance for carbon assimilatory and glycolytic pathways leading to fatty acid synthesis in rapeseed versus soybean. Increased expression of glycolytic and fatty acid biosynthetic proteins in rapeseed compared to soybean suggests a possible mechanistic basis for higher oil in rapeseed involves the concerted commitment of hexoses to glycolysis and eventual de novo fatty acid synthesis pathways.

During the interaction between sedentary plant-parasitic nematodes and their host, complex morphological and physiological changes occur in the infected plant tissue finally resulting in the establishment of a nematode feeding site. This cellular transformation is the result of altered plant gene expression most likely induced by proteins injected in the plant cell by the nematode. Here we report on the identification of a WRKY transcription factor expressed during nematode infection. Using both promoter-reporter gene fusions and in situ RT-PCR we could show that AtWRKY23 is expressed during the early stages of feeding site establishment. Knocking-down the expression of WRKY23 resulted in lower infection of the cyst nematode H. schachtii. WRKY23 is an auxin-inducible gene and in uninfected plants WRKY23 acts downstream of the Aux/IAA protein SLR/IAA14. Although auxin is known to be involved in feeding site formation, our results suggest that during early stages auxin-independent signals might be at play to activate the initial expression of WRKY23.

Plants utilize tightly regulated mechanisms to defend themselves against pathogens. Initial recognition results in activation of specific Resistance (R) proteins that trigger downstream immune responses, in which the signalling networks remain largely unknown. A point mutation in SNC1, an RPP4 R gene homolog, renders plants constitutively resistant to virulent pathogens. Genetic suppressors of snc1 may carry mutations in genes encoding novel signalling components downstream of activated R proteins. One such suppressor was identified as a novel loss-of-function allele of ENHANCED RESPONSE TO ABSCISIC ACID 1 (ERA1), which encodes the beta subunit of protein farnesyltransferase. Protein farnesylation involves attachment of C15-prenyl residues to the carboxyl termini of specific target proteins. Mutant era1 plants display enhanced susceptibility to virulent bacterial and oomycete pathogens, implying a role for farnesylation in basal defence. In addition to its role in snc1-mediated resistance, era1 affects several other R-protein-mediated resistance responses against bacteria and oomycetes. ERA1 acts partly independent of abscisic acid and additively with the resistance regulator NON-EXPRESSOR OF PR-GENES 1 (NPR1) in the signalling network. Defects in geranylgeranyl transferase I, a protein modification similar to farnesylation, do not affect resistance responses, indicating that farnesylation is most likely specifically required in plant defence signalling. Taken together, we present a novel role for farnesyltransferase in plant-pathogen interactions, suggesting the importance of protein farnesylation, which contributes to the specificity and efficacy of signal transduction events.

The switch from vegetative to reproductive growth is marked by the termination of vegetative development and the adoption of floral identity by the shoot apical meristem (SAM). This process is called the floral transition. To elucidate the molecular determinants involved in this process, we performed genome-wide RNA expression profiling on Zea mays (maize) shoot apices at vegetative and early reproductive stages using Massively Parallel Signature Sequencing^TM (MPSS^TM) technology. Profiling revealed significant up-regulation of two Z. mays MADS-box (ZMM) genes, ZMM4 and ZMM15, after the floral transition. ZMM4 and ZMM15 map to duplicated regions on chromosomes 1 and 5 and are linked to neighboring MADS-box genes ZMM24 and ZMM31, respectively. This gene order is syntenic with the vernalization1 (VRN1) locus responsible for floral induction in winter wheat (Triticum monococcum) and similar loci in other cereals. Analyses of temporal and spatial expression patterns indicated that the duplicated pairs ZMM4-ZMM24 and ZMM15-ZMM31 are coordinately activated after the floral transition in early developing inflorescences. More detailed analyses revealed ZMM4 expression initiates in leaf primordia of vegetative shoot apices and later increases within elongating meristems acquiring inflorescence identity. Expression analysis in late flowering mutants positioned all four genes downstream of the floral activators indeterminate1 (id1) and delayed flowering1 (dlf1). Over expression of ZMM4 leads to early flowering in transgenic maize and suppresses the late flowering phenotype of both the id1 and dlf1 mutations. Our results suggest ZMM4 may play roles in both floral induction and inflorescence development.

In contrast to animals, where polyamine (PA) catabolism efficiently converts Spermine (Spm) to Putrescine (Put), plants have been considered to possess a polyamine catabolic pathway producing 1,3-diaminopropane (1,3-Dap), ¹-pyrroline, the corresponding aldehyde and H₂O₂, but unable to back-convert Spm to Put. Arabidopsis thaliana genome contains at least five putative polyamine oxidase (PAO) members, with yet unknown localization and physiological role(s). Recently, Tavladoraki et al., (2006) identified AtPAO1 as an enzyme similar to the mammalian Spm oxidase (SMO), which converts Spm to Spermidine (Spd). In this work we have performed in silico analysis of the five Arabidopsis thaliana genes and have identified Polyamine Oxidase 3 (AtPAO3) as a non-typical Polyamine Oxidase (PAO) in terms of homology, compared to other known PAOs. We have expressed the gene AtPAO3 and have purified a protein corresponding to it, using the inducible heterologous expression system of Escherichia coli. AtPAO3 catalyzed the sequential conversion/oxidation of Spm to Spd, and of Spd to Put, thus exhibiting functional homology to the mammalian PAOs. The best substrate for this pathway was Spd, whereas the N¹-acetyl-derivatives of Spm and Spd were oxidized less efficiently. On the other hand, no activity was detected when diamines (Agmatine, Cadaverine and Put) were used as substrates. Moreover, although AtPAO3 does not exhibit significant similarity to the other known PAOs, it is efficiently inhibited by guazatine, a potent PAO inhibitor. AtPAO3 contains a peroxisomal targeting motif at the C-terminus, and it targets Green Fluorescence Protein (GFP) to peroxisomes when fused at the N-terminus but not at the C-terminus. These results reveal that AtPAO3 is a peroxisomal protein and that the C-terminus of the protein contains the sorting information. The overall data reinforce the view that plants and mammals possess a similar PA oxidation system, concerning both the subcellular localization and the mode of its action.

In tip growing cells, the tip-high Ca²⁺ gradient is thought to regulate the activity of components of the growth machinery including the cytoskeleton, Ca²⁺-dependent regulatory proteins and the secretory apparatus. In pollen tubes, both the Ca²⁺ gradient and cell elongation show oscillatory behavior reinforcing the link between the two. We report that in growing root hairs of Arabidopsis thaliana, an oscillating tip-focused Ca²⁺ gradient can be resolved through imaging of a cytosolically expressed Yellow Cameleon (YC) 3.6 FRET-based Ca²⁺ sensor. Both elongation of the root hairs and the associated tip-focused Ca²⁺ gradient show a similar dynamic character, oscillating with a frequency of 2-4 min^-1. Cross-correlation analysis indicates that the Ca²⁺ oscillations lag the growth oscillations by 5.3 ± 0.3 s. However, growth never completely stops, even during the slow cycle of an oscillation, and the concomitant tip Ca²⁺ level is always slightly elevated compared to the resting Ca²⁺ concentration along the distal shaft, behind the growing tip. Artificially increasing Ca²⁺ using the Ca²⁺ ionophore A23187 leads to immediate cessation of elongation and thickening of the apical cell wall. In contrast, dissipating the Ca²⁺ gradient using either the Ca²⁺ channel blocker La³⁺ or the Ca²⁺ chelator EGTA is accompanied by an increase in the rate of cell expansion and eventual bursting of the root hair tip. These observations are consistent with a model where the maximal oscillatory increase in cytosolic Ca²⁺ is triggered by cell expansion associated with tip growth and plays a role in the subsequent restriction of growth.

Through a single genetic transformation in onion, a crop recalcitrant to genetic transformation, we suppressed the lachrymatory factor synthase gene (lfs) using RNAi silencing in six plants. This reduced lachrymatory synthase activity by up to 1544-fold, so that when wounded the onions produced significantly reduced levels of tear-inducing lachrymatory factor. We then confirmed, through a novel colorimetric assay, that this silencing had shifted the trans-S-1-propenyl-L-cysteine sulfoxide (1-PRENCSO) breakdown pathway so that more 1-propenyl sulfenic acid was converted into di-1-propenyl thiosulfinate. A consequence of this raised thiosulfinate level was a marked increase in the downstream production of a non-enzymatically produced zwiebelane isomer and other volatile sulfur compounds, di-1-propenyl disulfide and 2-mercapto-3,4-dimethyl-2,3-dihydrothiophene, which had previously been reported either in trace amounts or had not been detected in onion. The consequences of this dramatic simultaneous down- and up-regulation of secondary sulfur products on the health and flavour attributes of the onion are discussed.

The role of polyamine (PA) metabolism in tobacco (Nicotiana tabacum) defense against pathogens with contrasting pathogenic strategies was evaluated. Infection by the necrotrophic fungus Sclerotinia sclerotiorum resulted in increased arginine decarboxylase expression and activity in host tissues, as well as putrescine and spermine accumulation in leaf apoplast. Enhancement of leaf PA levels, either by using transgenic plants or infiltration with exogenous PAs, led to increased necrosis due to infection by S. sclerotiorum. Specific inhibition of diamine and polyamine oxidases (DAO and PAO) attenuated the PA-induced enhancement of leaf necrosis during fungal infection. When tobacco responses to infection by the biotrophic bacterium Pseudomonas viridiflava were investigated, an increase of apoplastic spermine levels was detected. Enhancement of host PA levels by the above-described experimental approaches strongly decreased in planta bacterial growth, an effect that was blocked by a PAO inhibitor.

It can be concluded that accumulation and further oxidation of free PAs in the leaf apoplast of tobacco plants occurs in a similar, although not identical way during tobacco defense against infection by microorganisms with contrasting pathogenesis strategies. This response affects pathogen's ability to colonize host tissues and results detrimental for plant defense against necrotrophic pathogens that feed on necrotic tissue and, on the contrary, plays a beneficial role in defense against biotrophic pathogens that depend on living tissue for successful host colonization. Thus, apoplastic PAs play important roles in plant-pathogen interactions, and modulation of host PA levels, particularly in the leaf apoplast, may lead to significant changes in host susceptibility to different kinds of pathogens.

Prenylated Rab acceptor 1 (PRA1) domain proteins are small transmembrane proteins that regulate vesicle trafficking as receptors of Rab GTPases and the v-SNARE protein VAMP2. In plants, however, little is known about the PRA1 family members in plants. Sequence analysis revealed that higher plants, compared to animals and primitive plants, possess an expanded family of PRA1 domain-containing proteins. The Arabidopsis thaliana PRA1 (AtPRA1) proteins were found to homo- and heterodimerize in a manner corresponding to their phylogenetical distribution. Different AtPRA1 family members displayed distinct expression patterns, with a preference for vascular cells and expanding or developing tissues. AtPRA1 genes were significantly co-expressed with Rab GTPases and genes encoding vesicle transport proteins, suggesting an involvement in the vesicle trafficking process, similar to that of their animal counterparts. Correspondingly, AtPRA1 proteins were localized in the endoplasmic reticulum, Golgi apparatus, and endosomes/prevacuolar compartments, hinting at a function in both secretory and endocytic intracellular trafficking pathways. Taken together, our data reveal a high functional diversity of AtPRA1 proteins probably dealing with the various demands of the complex trafficking system.

Mutation of either arginase structural gene (ARGAH1 or ARGAH2) resulted in increased formation of lateral and adventitious roots in Arabidopsis seedlings and increased nitric oxide (NO) accumulation and efflux, detected by the fluorogenic traps, DAF FM-DA and DAR-4M, respectively. Upon seedling exposure to the synthetic auxin naphthalene acetic acid, NO accumulation was differentially enhanced in argah1-1 and argah2-1 compared to wild type. In all genotypes much DAF FM-DA fluorescence originated from mitochondria. The arginases are both localized to the mitochondrial matrix and are closely related. However, their expression levels and patterns differ: ARGAH1 encoded the minor activity, and ARGAH1-driven GUS was expressed throughout the seedling; the ARGAH2::GUS expression pattern was more localized. Naphthalene acetic acid increased seedling lateral root numbers (total lateral roots per primary root) in the mutants to twice the number in the wild type- patterns consistent with increased internal NO leading to enhanced auxin signaling in roots In agreement, argah1-1 and argah2-1 showed increased expression of the auxin-responsive reporter, DR5::GUS, in root tips, emerging lateral roots and hypocotyls. We propose that arginine, or an arginine derivative, is a potential NO source, and that reduced arginase activity in the mutants results in greater conversion of Arg to NO, thereby potentiating auxin