We compare three Arabidopsis thaliana cgl1 alleles and report on genetic interaction with stt3a. STT3a encodes a subunit of oligo-saccharyl-trans-ferase that affects efficiency of N-glycan transfer to nascent secretory proteins in the ER; cgl1 mutants lack N-acetyl-gluco-saminyl-trans-ferase-I activity and are unable to form complex N-glycans in the Golgi apparatus. By studying CGL1-GFP fusions in transient assays we show that the extra N-glyco-sy-la-tion site created by a point mutation in cgl1 C5 is used in planta and interferes with folding of full-length membrane-anchored polypeptides in the ER. Tunicamycin-treatment or expression in the stt3a-2 mutant relieved the folding block and migration to Golgi stacks resumed. Comple-men-ta-tion tests with C5-GFP and other N-glycosylation variants of CGL1 demonstrated that suppression of aberrant N-glyco-sylation restores activity. Interestingly, CGL1 seems to be functional also as non-glyco-----sy-la-ted enzyme. Two other cgl1 alleles showed splicing defects of their transcripts: In cgl1 C6, a point mutation affects the 3'-splice site of intron 14, resulting in frame shifts; and in cgl1-T, intron 11 fails to splice due to insertion of a T-DNA copy. Introgression of stt3a-2 did not restore complex glycan formation in cgl1 C6 or cgl1-T but suppressed the GnTI defect in cgl1 C5. Root growth assays revealed synergistic effects in double mutants cgl1 C6 stt3a-2 and cgl1-T stt3a-2 only. Besides demonstrating the condi-tional nature of cgl1 C5 in planta, our observations with loss-of-function alleles cgl1 C6 and cgl1-T in the stt3a-2 underglycosylation background prove that correct N-glyco-sy-la-tion is important for normal root growth and morphology in Arabidopsis.

The degradation pathway of glutathione (GSH) in plants is not well understood. In mammals, GSH is predominately metabolized through the -glutamyl cycle, where GSH is degraded by the sequential reaction of -glutamyl transpeptidase (GGT), -glutamyl cyclotransferase (GGC) and 5-oxoprolinase (5OPase) to yield Glu and dipeptides that are subject to peptidase action. In this study we examined if GSH is degraded through the same pathway in Arabidopsis thaliana as occurs in mammals. In Arabidopsis the oxoprolinase knockout mutants (oxp1-1 and oxp1-2) accumulate more 5-oxoproline (5OP) and less Glu than wild-type plants, suggesting substantial metabolite flux though 5OP and that 5OP is a major contributor to Glu steady state levels. In the ggt1-1/ggt4-1/oxp1-1 triple mutant with no GGT activity in any organs except young siliques, the 5OP concentration in leaves was not different from that in oxp1-1 suggesting that GGTs are not major contributors to 5OP production in Arabidopsis. 5OP formation strongly tracked the level of GSH in Arabidopsis plants suggesting that GSH is the precursor of 5OP in a GGT-independent reaction. Kinetics analysis suggests that -glutamyl cyclotransferase is the major source of GSH degradation and 5OP formation in Arabidopsis. This discovery has led us to propose a new pathway for GSH turnover in plants where GSH is converted to 5OP and then glutamate by the combined action of -glutamyl cyclotransferase and 5-oxoprolinase in the cytoplasm.

Shade avoidance in plants involves rapid shoot elongation to grow towards the light. Cell wall modifying mechanisms are vital regulatory points for control of these elongation responses. Two protein families involved in cell wall modification are expansins and xyloglucan endotransglucosylase/hydrolases. We used an alpine and prairie ecotype of Stellaria longipes differing in their response to shade, to study regulation of cell wall extensibility in response to low red to far-red ratio (R/FR), an early neighbour detection signal and dense canopy shade (green shade: low R/FR, blue and total light intensity). Alpine plants were non-responsive to low R/FR, while prairie plants elongated rapidly. These responses reflect adaptation to the dense vegetation of the prairie habitat unlike the alpine plants that almost never encounter shade. Under green shade, both ecotypes rapidly elongate showing that alpine plants can react only to a deep shade treatment. Xyloglucan endotransglucosylase/hydrolase activity was strongly regulated by green shade and low blue light conditions, but not by low R/FR. Expansin activity expressed as acid induced extension, correlated with growth responses to all light changes. Expansin genes cloned from the internodes of the two ecotypes showed differential regulation in response to the light manipulations. This regulation was ecotype and light signal-specific and correlated with the growth responses. Our results imply that elongation responses to shade require the regulation of cell wall extensibility via the control of expansin gene expression. Ecotypic differences demonstrate how responses to environmental stimuli are differently regulated to survive a particular habitat.

Protein tyrosine phosphorylation plays a central role in many signaling pathways leading to cell growth and differentiation in animals. Tyrosine phosphorylated proteins have been detected in higher plants and the roles of protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) in some physiological responses have been shown. We investigated the involvement of tyrosine phosphorylation events in abscisic acid (ABA) signaling using a pharmacological approach. Phenylarsine oxide (PAO), a specific inhibitor of PTP activity, abolished the ABA-dependent accumulation of RAB18 transcripts. PTK inhibitors like genistein, tyrphostin A23 and erbstatin, blocked the RAB18 expression induced by ABA in Arabidopsis. Stomatal closure induced by ABA was also inhibited by PAO and genistein. We studied the changes in the tyrosine phosphorylation levels of proteins in Arabidopsis seeds after ABA treatment. Proteins were separated by two-dimensional gel electrophoresis and those phosphorylated on tyrosine residues were detected using an anti-phosphotyrosine antibody by Western blot. Changes were detected in the tyrosine phosphorylation levels of 19 proteins after ABA treatment. Genistein inhibited the ABA-dependent tyrosine phosphorylation of proteins. The 19 proteins were analysed by MALDI-TOF-TOF mass spectrometry. Among the proteins identified were storage proteins like cruciferins, enzymes involved in the mobilization of lipid reserves like aconitase, enolase, aldolase and a lipoprotein, and enzymes necessary for seedling development like the large subunit of Rubisco. Additionally, the identification of three putative signaling proteins: a peptidyl-prolyl isomerase, a RNA-binding protein and a Small Ubiquitin-like MOdifier (SUMO) conjugating enzyme, enlightens how tyrosine phosphorylation might regulate ABA transduction pathways in plants.

The SNF1/AMPK/SnRK1 kinases are evolutionary conserved kinases involved in yeast, mammals and plants in the control of energy balance. This heterotrimeric enzyme is composed of one -type catalytic subunit and two - and β-type regulatory subunits. In yeast it has been proposed that the β-type subunits regulate both the localization of the kinase complexes within the cell and the interaction of the kinase with its targets. In the present work, we demonstrate that the three β-type subunits of Arabidopsis thaliana (AKINβ1, β2 and β3) restore the growth phenotype of the yeast sip1sip2gal83 triple mutant thus suggesting the conservation of an ancestral function. Expression analyses, using AKINβ promoter::GUS transgenic lines, reveal different and specific patterns of expression for each subunit according to organs, developmental stages and environmental conditions. Finally our results show that the β-type subunits are involved in the specificity of interaction of the kinase with the cytosolic nitrate reductase (NR). Together with previous cell-free phosphorylation data, they strongly support the proposal that NR is a real target of SnRK1 in the physiological context. Altogether our data suggest the conservation of (an) ancestral basic function(s) together with specialized functions for each β-type subunit in plants.

A reverse genetic approach was used to investigate the functions of three members of the cellulose synthase super family in Arabidopsis thaliana, CSLD1, CSLD2 and CSLD4. CSLD2 is required for normal root hair growth but has a different role to that previously described for CSLD3 (KOJAK). CSLD2 is required during a later stage of hair development than CSLD3 and CSLD2 mutants produce root hairs with a range of abnormalities with many root hairs rupturing late in development. Remarkably though, it was often the case that in CSLD2 mutants, tip growth would resume after rupturing of root hairs. In silico, semi-quantitative RT-PCR and promoter-reporter construct analyses indicated that the expression of both CSLD2 and CSLD3 is elevated at reduced temperatures and the phenotypes of mutants homozygous for insertions in these genes were partially rescued by reduced temperature growth. However, this was not the case for a double mutant homozygous for insertions in both CSLD2 and CSLD3 suggesting that there may be partial redundancy in the functions of these genes. Mutants in CSLD1 and CSLD4 had a defect in male transmission and plants heterozygous for insertions in CSLD1 or CSLD4 were defective in their ability to produce pollen tubes - although the number and morphology of pollen grains was normal. We propose that the CSLD family of putative glycosyltransferases synthesise a polysaccharide that has a specialized structural role in the cell walls of tip growing cells.

Folates typically have -linked polyglutamyl tails that make them better enzyme substrates, and worse transport substrates, than the unglutamylated forms. The tail can be shortened or removed by the vacuolar enzyme -glutamyl hydrolase (GGH). It is known that GGH is active only as a dimer and that plants can have several GGH genes whose homodimeric products differ functionally. However, it is not known whether GGH dimers dissociate under in vivo conditions, whether heterodimers form, or how heterodimerization impacts enzyme activity. These issues were explored using the GGH system of tomato (Solanum lycopersicum). Tomato has three GGH genes that, like those in other eudicots, apparently diverged recently. LeGGH1 and LeGGH2 are expressed in fruit and all other organs whereas LeGGH3 is expressed mainly in flower buds. LeGGH1 and LeGGH2 homodimers differ in bond cleavage preference; the LeGGH3 homodimer is catalytically inactive. Homodimers did not dissociate in physiological conditions. When co-expressed in Escherichia coli, LeGGH1 and LeGGH2 formed heterodimers with an intermediate bond cleavage preference, while LeGGH3 formed heterodimers with LeGGH1 or LeGGH2 that had half the activity of the matching homodimer. E. coli cells expressing LeGGH2 showed ~95% reduction in folate polyglutamates but cells expressing LeGGH3 did not, confirming that LeGGH2 can function in vivo and LeGGH3 cannot. The formation of LeGGH1-LeGGH2 heterodimers was demonstrated in planta using bimolecular fluorescence complementation. Plant GGH heterodimers thus appear to form wherever different GGH genes are expressed simultaneously, and to have catalytic characteristics midway between those of the corresponding homodimers.

Plants have substantially higher gene duplication rates compared to most other eukaryotes. These plant gene duplicates are mostly derived from whole genome and/or tandem duplications. Earlier studies have shown that a large number of duplicate genes are retained over a long evolutionary time and there is a clear functional bias in retention. However, the influence of duplication mechanism, particularly tandem duplication, on duplicate retention has not been thoroughly investigated. We have defined orthologous groups (OGs) between Arabidopsis thaliana and three other land plants to examine the functional bias of retained duplicate genes during vascular plant evolution. Based on analysis of Gene Ontology categories, it is clear that genes in orthologous groups that expanded via tandem duplication tend to be involved in responses to environmental stimuli while those that expanded via non-tandem mechanisms tend to have intracellular regulatory roles. Using Arabidopsis stress expression data, we further demonstrate that tandem duplicates in expanded OGs are significantly enriched in genes that are up-regulated by biotic stress conditions. In addition, tandem duplication of genes in an OG tends to be highly asymmetric. That is, expansion of OGs with tandem genes in one organismal lineage tends to be coupled with losses in the other. This is consistent with the notion that these tandem genes have experienced lineage-specific selection. In contrast, OGs with genes duplicated via non-tandem mechanisms tend to experience convergent expansion where similar numbers of genes are gained in parallel. Our study demonstrates that the expansion of gene families and retention of duplicates in plants exhibits substantial functional biases that are strongly influenced by the mechanism of duplication. In particular, genes involved in stress response have an elevated probability of retention in a single-lineage fashion following tandem duplication, suggesting that these tandem duplicates are likely important for adaptive evolution to rapidly changing environments.

MicroRNAs (miRNAs) are ~21 nucleotide long RNAs processed from nuclear encoded transcripts, which include a characteristic hairpin-like structure. MiRNAs control the expression of target transcripts by binding to reverse complementary sequences directing cleavage or translational inhibition of the target RNA. Artificial miRNAs (amiRNAs) can be generated by exchanging the miRNA/miRNA* sequence within miRNA precursor genes, while maintaining the pattern of matches and mismatches in the foldback. Thus, for functional gene analysis, amiRNAs can be designed to target any gene of interest. The moss Physcomitrella patens exhibits the unique feature of a highly efficient homologous recombination mechanism, which allows for the generation of targeted gene knockout lines. However, the completion of the Physcomitrella genome necessitates the development of alternative techniques to speed up reverse genetics analyses, and to allow for more flexible inactivation of genes. To prove the adaptability of amiRNA expression in Physcomitrella we designed two amiRNAs, targeting the gene PpFtsZ2-1, which is indispensable for chloroplast division, and the gene PpGNT1 encoding an N-acetylglucosaminyltransferase. Both amiRNAs were expressed from the Arabidopsis thaliana miR319a precursor fused to a constitutive promoter. Transgenic Physcomitrella lines harboring the overexpression constructs showed precise processing of the amiRNAs and an efficient knock-down of the cognate target mRNAs. Furthermore, chloroplast division was impeded in PpFtsZ2-1-amiRNA lines which phenocopied PpFtsZ2-1 knockout mutants. We also provide evidence for the amplification of the initial amiRNA signal by secondary transitive siRNAs, although these siRNAs do not seem to have a major effect on sequence-related mRNAs, confirming specificity of the amiRNA approach.

Accumulation of reserve materials in filling grains involves coordination of different metabolic and cellular processes, and understanding the molecular mechanisms underlying the interconnections remains a major challenge for proteomics. Rice is an excellent model for studying grain filling because of its importance as a staple food and the available genome sequence database. Our observations showed that embryo differentiation and endosperm cellularization in developing rice seeds were completed approximately 6 days after flowering (DAF); and thereafter, the immature seeds mainly underwent cell enlargement and reached the size of mature seeds at 12 DAF. Grain filling began at 6 DAF and lasted until 20 DAF. Dynamic proteomic analyses revealed 396 protein spots differentially expressed throughout 8 sequential developmental stages from 6 to 20 DAF, and determined 345 identities. These proteins were involved in different cellular and metabolic processes with a prominently functional skew toward metabolism (45%) and protein synthesis/destination (20%). Expression analyses of protein groups associated with different functional categories/subcategories showed that substantially up-regulated proteins were involved in starch synthesis and alcoholic fermentation; whereas the proteins down-regulated in the process involved in central carbon metabolism and most of the other functional categories/subcategories such as cell growth/division, protein synthesis, proteolysis and signal transduction. The coordinated changes were consistent with the transition from cell growth and differentiation to starch synthesis and clearly indicated that a switch from central carbon metabolism to alcoholic fermentation may be important for starch synthesis and accumulation in the developmental process.

Lettuce (Lactuca sativa L. cv. Salinas) seeds fail to germinate when imbibed at temperatures above 25 to 30°C (termed thermoinhibition). However, seeds of an accession of L. serriola (UC96US23) do not exhibit thermoinhibition up to 37°C in the light. Comparative genetics, physiology, and gene expression were analyzed in these genotypes to determine the mechanisms governing the regulation of seed germination by temperature. Germination of the two genotypes was differentially sensitive to abscisic acid (ABA) and gibberellin (GA) at elevated temperatures. Quantitative trait loci (QTL) associated with these phenotypes collocated with a major QTL (Htg6.1) from UC96US23 conferring germination thermotolerance. ABA contents were elevated in Salinas seeds that exhibited thermoinhibition, consistent with the ability of fluridone (an ABA biosynthesis inhibitor) to improve germination at high temperatures. Expression of many genes involved in ABA, gibberellin (GA), and ethylene biosynthesis, metabolism and response was differentially affected by high temperature and light in the two genotypes. In general, ABA-related genes were more highly expressed when germination was inhibited and GA- and ethylene-related genes were more highly expressed when germination was permitted. In particular, LsNCED4, a gene encoding an enzyme in the ABA biosynthetic pathway, was up regulated by high temperature only in Salinas seeds and also collocated with Htg6.1. The temperature sensitivity of expression of LsNCED4 may determine the upper temperature limit for lettuce seed germination and indirectly influence other regulatory pathways via interconnected effects of increased ABA biosynthesis.

Cysteine synthesis in plants is carried out by two sequential reactions catalyzed by the rate-limiting enzyme serine acetyltransferase (SAT) and excess amounts of O-acetylserine(thiol)lyase (OAS-TL). Why these reactions occur in plastids, mitochondria and cytosol of plants remained unclear. Expression of artificial micro RNA (amiRNA) against Sat3 encoding mitochondrial SAT3 in transgenic Arabidopsis thaliana plants demonstrates that mitochondria are the most important compartment for synthesis of O-acetylserine (OAS), the precursor of cysteine. Reduction of RNA levels, protein contents, SAT enzymatic activity and phenotype strongly correlate in independent amiSAT3 lines and cause significantly retarded growth. The expression of the other four Sat genes in the Arabidopsis genome are not affected by amiRNA-SAT3 according to quantitative real time PCR and microarray analyses. Application of radiolabeled serine to leaf pieces reveales severely reduced incorporation rates into cysteine and even more so into glutathione. Accordingly steady-state levels of OAS are 4-fold reduced. Decrease of sulfate reduction related genes is accompanied by accumulation of sulfate in amiSAT3 lines. The results unequivocally show that mitochondria provide the bulk of OAS in the plant cell and are the likely site of flux regulation. Together with recent data [Heeg et al. (2008) Plant Cell 20: 168] the cytosol appears as major site of cysteine synthesis while plastids contribute reduced sulfur as sulfide. Thus, cysteine synthesis in plants is significantly different from that in non-photosynthetic eukaryotes at the cellular level.

During barley seed development PEPC activity increased and PEPC specific antibodies revealed housekeeping (103 kDa) and inducible (108 kDa) subunits. Bacterial-type PEPC fragments were immunologically detected in denatured protein extracts from dry and imbibed, however, in non-denaturing gels the activity of the recently reported octameric PEPC (in Castor oil seeds) was not detected. The phosphorylation state of the PEPC, as judged by L-malate IC₅₀ values, phosphoprotein chromatography and immunodetection of the phosphorylated N-terminus, was found to be high between 8 and 18 DPA and during imbibition. In contrast, the enzyme appeared to be in a low phosphorylation state from 20 DPA up to dry seed. The time course of 32/36 kDa, Ca²⁺-independent PEPC kinase activity exhibited a substantial increase after 30 DPA that did not coincide with the PEPC phosphorylation profile. This kinase was found to be inhibited by L-malate, and not by putative protein inhibitors, and the PEPC phosphorylation status correlated with high Glc-6P/malate ratios thereby suggesting an in vivo metabolic control of the kinase. PEPC phosphorylation was also regulated by photosynthate supply at 11 DPA. In addition, when fed exogenously to imbibing seeds, abscissic acid significantly increased PEPC kinase activity. This was further enhanced by the cytosolic protein synthesis inhibitor cycloheximide, but blocked by protease inhibitors, thereby suggesting that the phytohormone acts on the stability of the kinase. We propose that a similar ABA-dependent effect may contribute to produce the increase in PEPC kinase activity during desiccation stages.

Salicylic acid (SA) is a primary factor responsible for exerting diverse immune responses in plants, and is synthesized in response to attack by a wide range of pathogens. The Arabidopsis sid2 mutant is defective in a SA biosynthetic pathway involving isochorismate synthase 1 (ICS1) and consequently contains reduced levels of SA. However, the sid2 mutant as well as ICS-suppressed tobacco still accumulates a small but significant level of SA. These observations along with previous studies suggested that SA might also be synthesized by another pathway involving benzoic acid (BA). Here we isolated a benzoic acid hypersensitive 1-Dominant (bah1-D) mutant which excessively accumulated SA after application of BA from activation tagged lines. This mutant also accumulated higher levels of SA after inoculation with Pseudomonas syringae pv. tomato (Pst) DC3000. Analysis of the bah1-D sid2 double mutant suggested that the bah1-D mutation caused both ICS1-dependent and -independent accumulation. In addition, the bah1-D mutant showed SA-dependent localized cell death in response to Pst DC3000. The T-DNA insertional mutation which caused the bah1-D phenotypes resulted in the suppression of expression of the NLA gene, which encodes a RING-type ubiquitin E3 ligase. These results suggest that BAH1/NLA plays crucial roles in the ubiquitination-mediated regulation of immune responses, including BA- and pathogen-induced SA accumulation, and control of cell death.

Plant cells contain several thioredoxin isoforms which are characterized by subcellular localization and substrate specificity. Here we describe the functional characterization of an Oryza sativa thioredoxin m isoform (Ostrxm) using a reverse genetics technique. Ostrxm showed green tissue-specific and light-responsive mRNA expression. Ostrxm was localized in chloroplasts of rice mesophyll cells, and the recombinant protein showed DTT-dependent insulin β-chain reduction activity in vitro. RNA interference (RNAi) of Ostrxm resulted in rice plants with developmental defects including semi-dwarfism, pale green leaves, abnormal chloroplast structure, and reduced carotenoid and chlorophyll contents. Ostrxm RNAi plants showed remarkably decreased F_v/F_m values under high irradiance conditions (1,000 µmol m^-2 s^-1) with delayed recovery. Two-dimensional electrophoresis and MALDI-TOF analysis showed that the levels of several chloroplast proteins critical for photosynthesis and biogenesis were significantly decreased in Ostrxm RNAi plants. Furthermore, 2-Cys peroxiredoxin (2-Cys Prx), a known target of thioredoxin, was present in oxidized forms, and hydrogen peroxide levels were increased in Ostrxm RNAi plants. The pleiotropic effects of Ostrxm RNAi suggest that Ostrxm plays an important role in the redox regulation of chloroplast target proteins involved in diverse physiological functions.

Transporters for di- and tripeptides belong to the large and poorly characterized PTR/NRT1 (peptide transporter/nitrate transporter 1)-family. A new member of this gene family, AtPTR5, was isolated from Arabidopsis thaliana. Expression of AtPTR5 was analyzed and compared with tissue specificity of the closely related AtPTR1 to discern their roles in planta. Both transporters facilitate transport of dipeptides with high affinity and are localized at the plasma membrane. Mutants, double-mutants and overexpressing lines were exposed to several dipeptides, including toxic peptides, to analyse how the modified transporter expression affects pollen germination, growth of pollen tubes, root and shoot. Analysis of atptr5 mutants and AtPTR5 overexpressing lines showed that AtPTR5 facilitates peptide transport into germinating pollen and possibly into maturating pollen, ovules and seeds. In contrast, AtPTR1 plays a role in uptake of peptides by roots indicated by reduced nitrogen (N) levels and reduced growth of aptr1 mutants on medium with dipeptides as sole nitrogen source. Furthermore, overexpression of AtPTR5 resulted in enhanced shoot growth and increased N content. The function in peptide uptake was further confirmed with toxic peptides, which inhibited growth. The results show that closely related members of the PTR/NRT1 family have different functions in planta. This study also provides evidence that the use of organic nitrogen is not restricted to amino acids, but that dipeptides should be considered as nitrogen source and transport form in plants.

The sessile lifestyle of plants constrains their ability to acquire mobile nutrients such as nitrate. While proliferation of roots might help in the longer term, nitrate rich patches can shift rapidly with mass flow of water in the soil. A mechanism that allows roots to follow and capture this source of mobile nitrogen would be highly desirable. Here we report that variation in nitrate concentration around roots induces an immediate alteration of root hydraulic properties such that water is preferentially absorbed from the nitrate rich patch. Further, we show that this coupling between nitrate availability and water acquisition results from changes in cell membrane hydraulic properties and is directly related to intracellular nitrate concentrations. Split root experiments in which nitrate was applied to a portion of the root system showed that the response is both localized and reversible, resulting in rapid changes in water uptake to the portions of the roots exposed to the nitrate rich "patch." At the same time, water uptake by roots not supplied with nitrate was reduced. We believe that the increase in root hydraulic conductance in one part causes a decline of water uptake in the other part due to a collapse in the water potential gradient driving uptake. The translation of local information, in this case nitrate concentration, into a hydraulic signal that can be transmitted rapidly throughout the plant and thus coordinate responses at the whole plant level, represents an unexpected, higher level physiological interaction that precedes the level of gene expression.

Insect attack frequently down-regulates photosynthetic proteins. To understand how this influences the plant-insect interaction, we transformed Nicotiana attenuata to independently silence RuBPCase activase (RCA) and RuBPCase and selected lines whose photosynthetic capacity was similarly reduced. Decreases in plant growth mirrored the decreases in photosynthesis, but the effects on herbivore performance differed. Both generalist (Spodoptera littoralis) and specialist (Manduca sexta) larvae grew larger on RCA-silenced plants, which was consistent with decreased levels of trypsin protease inhibitors (TPIs) and diterpene glycosides (DTGs) and increased levels of RuBPCase, the larvae's main dietary protein. RCA-silenced plants were impaired in their attack-elicited jasmonate (JA)-Ile/Leu levels but RuBPCase-silenced plants were not, a deficiency which could not be restored by supplementation with Ile or attributed to lower transcript levels of JAR4/6, the key enzyme for JA-Ile conjugation. From these results, we infer that JA-Ile/Leu signaling and the herbivore resistance traits elicited by JA-Ile are influenced by adenylate charge, or more generally, carbon availability in RCA- but not RuBPCase-silenced plants. Growth of generalist larvae on RuBPCase-silenced plants did not differ from growth on EV controls, but the specialist larvae grew faster on RuBPCase-silenced plants, which suggests that the specialist can better tolerate the protein deficiency resulting from RuBPCase-silencing than the generalist can. We conclude that the plant-herbivore interaction is more influenced by the particular mechanisms that reduce photosynthetic capacity after herbivore attack than by the magnitude of the decrease, which highlights the value of understanding defense mechanisms in evaluating growth-defense tradeoffs.

The substrate supply system for respiration of the shoot and root of a perennial grass was characterized in terms of component pools, and pool's functional properties: size, half-life (t_1/2) and contribution to respiration of the root and shoot. The investigations were performed with Lolium perenne L. growing in constant conditions with continuous light. Plants were labeled with ¹³CO₂/¹²CO₂ for periods ranging from 1 h to 600 h, followed by measurements of the rates and ¹³C/¹²C ratios of CO₂ respired by shoots and roots in the dark. Label appearance in roots was delayed by approx. 1 h relative to shoots; otherwise the tracer time course was very similar in both organs. Compartmental analysis of respiratory tracer kinetics indicated that, in both organs, three pools supplied 95% of all respired carbon (a very slow pool whose kinetics could not be characterized provided the remaining 5%). Pool's half-lives and relative sizes were also near-identical in shoot and root (t_1/2 <15 min, ~3 h and 33 h). An important role of short-term storage in supplying respiration was apparent in both organs: only 43% of respiration was supplied by current photosynthate (fixed carbon transferred directly to centers of respiration via the two fastest pools). The residence time of carbon in the respiratory supply system was practically the same in shoot and root. From this and other evidence, we argue that both organs were supplied by the same pools, and that the residence time was controlled by the shoot via current photosynthate and storage deposition/mobilization fluxes.

ADP-glucose pyrophosphorylase (AGPase) catalyzes the rate limiting step in starch biosynthesis in plants and changes in its catalytic and/or allosteric properties can lead to increased starch production. Recently, a maize/potato small subunit mosaic, MP [Mos(1-198)], containing the first 198 amino acids of the small subunit of the maize endosperm enzyme and the last 277 amino acids from the potato tuber enzyme was expressed with the maize endosperm large subunit and was reported to have favorable kinetic and allosteric properties. Here we show that this mosaic, in the absence of activator, performs like a wild-type (wt) AGPase that is partially activated with 3-PGA. In the presence of 3-PGA, enzyme properties of Mos(1-198)/SH2 are quite similar to those of the wt maize enzyme. In the absence of 3-PGA, however, the mosaic enzyme exhibits greater activity, higher affinity for the substrates and partial inactivation by Pi. The Mos(1-198)/SH2 enzyme is also more stable to heat inactivation. The different properties of this protein were mapped using various mosaics containing smaller portions of the potato small subunit. Enhanced heat stability of Mos(1-198) was shown to originate from five potato-derived amino acids between 322 and 377. These amino acids were shown previously to be important in small subunit/large subunit interactions. These five potato-derived amino acids plus other potato-derived amino acids distributed throughout the carboxyl terminal portion of the protein are required for the enhanced catalytic and allosteric properties exhibited by Mos(1-198)/SH2.

Application of the widely used Farquhar et al. (1980) model of photosynthesis in interpretation of gas exchange data assumes that photosynthetic properties are homogeneous throughout the leaf. Previous studies showed that heterogeneity in stomatal conductance (g_s) across a leaf could affect the shape of the measured leaf photosynthetic CO₂ uptake rate (A) versus intercellular CO₂ concentration (C_i) response curve, and in turn estimation of the critical biochemical parameters of this model. These are the maximum rates of carboxylation (V_c,max), whole-chain electron transport (J_max), and triose phosphate utilization (V_TPU). The effects of spatial variation in V_c,max, J_max, and V_TPU on estimation of leaf averages of these parameters from A-C_i curves measured on a whole leaf have not been investigated. A mathematical model incorporating defined degrees of spatial variability in V_c,max and J_max was constructed. 110 theoretical leaves were simulated, each with the same average V_c,max and J_max, but different coefficients of variation of the mean (CV_VJ) and varying correlation between V_c,max and J_max (). Additionally, the interaction of variation in V_c,max and J_max with heterogeneity in V_TPU, g_s, and light gradients within the leaf was also investigated. Transition from V_c,max- to J_max-limited photosynthesis in the A-C_i curve was smooth in the most heterogeneous leaves, in contrast to a distinct inflection in the absence of heterogeneity. Spatial variability had little effect on the accuracy of estimation of V_c,max and J_max from A-C_i curves when the two varied in concert (=1.0), but resulted in underestimation of both parameters when they varied independently (up to 12.5% in V_c,max and 17.7% in J_max at CV_VJ=50% =0.3). Heterogeneity in V_TPU also significantly affected parameter estimates, but effects of heterogeneity in g_s or light gradients were comparatively small. If V_c,max and J_max derived from such heterogeneous leaves are used in models to project leaf photosynthesis, actual A is overestimated by up to 12% at the transition between V_c,max- and J_max-limited photosynthesis. This could have implications for both crop production and earth system models, including projections of the effects of atmospheric change.

The deduced amino acid sequence of an slr1923 gene of Synechocystis sp. PCC6803 is homologous to archaean F₄₂₀H₂ dehydrogenase, which acts as an soluble subcomplex of NADH dehydrogenase complex I. In the present study, the gene was inactivated and characteristics of the mutant were analyzed. The mutant grew slower than wild type under 100 µE m^-2 s^-1 but did not grow under high light intensity (300 µE m^-2s^-1). The cellular content of chlorophyll was lower in the mutant and absorption spectrum showed a shift in the absorption peak of the Soret band to longer wavelength by about 10 nm compared with wild type. It was found that retention time of chlorophyll of the mutant is shorter than wild type and peak wavelength of Soret band was also shifted to longer wavelength by 11 nm by HPLC analysis. ¹H-NMR analysis of the chlorophyll of the mutant revealed that the ethyl group of position 8 of ring B is replaced with vinyl group. The spectrum indicates that the chlorophyll of the mutant is not a normal (3-vinyl) chlorophyll a but a 3,8-divinylchlorophyll a. These results strongly suggest that Slr1923 protein is essential to convert from divinyl-chlorophyll(ide) to normal chlorophyll(ide). We thus designate this gene as cvrA (a gene indispensable for cyanobacterial vinyl reductase).

Chloroplasts change their intracellular distribution in response to light intensity. Previously, we isolated the chloroplast unusual positioning (chup1) mutant of Arabidopsis thaliana (Oikawa et al., 2003). This mutant is defective in normal chloroplast relocation movement and shows aggregation of chloroplasts at the bottom of palisade mesophyll cells. The isolated gene encodes a protein with an actin-binding motif. Here, we used biochemical analyses to determine the subcellular localization of full-length CHUP1 on the chloroplast outer envelope. A CHUP1-GFP fusion, which was detected at the outermost part of mesophyll cell chloroplasts, complemented the chup1 phenotype, but GFP-CHUP1, which localized mainly in the cytosol, did not. Overexpression of the N-terminal hydrophobic region (NtHR) of CHUP1 fused with GFP (NtHR-GFP) induced a chup1-like phenotype, indicating a dominant-negative effect on chloroplast relocation movement. A similar pattern was found in chloroplast outer envelope protein 7 (OEP7)-GFP transformants, and a protein containing OEP7 in place of NtHR complemented the mutant phenotype. Physiological analyses of transgenic Arabidopsis plants expressing truncated CHUP1 in a chup1-mutant background and cytoskeletal inhibitor experiments showed that the coiled-coil region of CHUP1 anchors chloroplasts firmly on the plasma membrane, consistent with the localization of coiled-coil-GFP on the plasma membrane. Thus, CHUP1 localization on chloroplasts, with the N-terminus inserted into the chloroplast outer envelope and the C-terminus facing the cytosol is essential for CHUP1 function, and the coiled-coil region of CHUP1 prevents chloroplast aggregation and participates in chloroplast relocation movement.

Chloroplasts are among the main targets of cytokinin action in the plant cell. We report here on the activation of transcription by cytokinin as detected by run-on assays with chloroplasts isolated from apical parts of first leaves detached from 9-day-old barley (Hordeum vulgare L.) seedlings and incubated for 3 h on a 2.2 x 10^-5M solution of benzyladenine (BA). Northern analysis detected also a BA-induced increase in the accumulation of chloroplast mRNAs. A prerequisite for BA activation of chloroplast transcription was pre-incubation of leaves for 24 h on water in the light resulting in a decreased chloroplast transcription and a drastic accumulation of abscisic acid. Cytokinin enhanced transcription of several chloroplast genes above the initial level measured before BA treatment, in case of rrn16 and petD even before pre-incubation. Cytokinin effects on basal (youngest), middle, and apical (oldest) segments of primary leaves detached from plants of different ages revealed an age dependence of chloroplast gene response to BA. BA-induced stimulation of transcription of rrn16, rrn23, rps4, rps16, rbcL, atpB, and ndhC required light during the period of pre-incubation and was further enhanced by light during the incubation on BA, whereas activation of transcription of trnEY, rps14, rpl16, matK, petD and petLG depended on light during both periods. Our data revealed positive and differential effects of cytokinin on the transcription of chloroplast genes that were dependent on light and on the age (developmental stage) of cells and leaves.

The anatomy of strawberry (Fragaria x ananassa ) fruit, in which the achene is found on the outer part of the fruit, makes it an excellent species for studying the regulation of fruit development. It can provide a model for the crosstalk between primary and secondary metabolism, whose role is of pivotal importance in the process. By combining GCMS and LC-MS with the aim of addressing the metabolic regulation underlying fruit-seed development, we simultaneously analysed the composition of primary and secondary metabolites, separately, in achene and receptacle during fruit ripening of strawberry cv. Herut. The results from these analyses suggest that changes in primary and secondary metabolism reflect organ and developmental specificities. For instance, the receptacle was characterised by increases in sugars and their direct derivatives, whilst the achene was characterised by a major decrease in the levels of carbon and nitrogen rich compounds with the exception of storage related metabolites, e.g. raffinose. Furthermore, the receptacle, and to a lower extent the achene, exhibited dynamic fluctuations in the levels and nature of secondary metabolites across the ripening process. In the receptacle, proanthocyanidins and flavonol derivatives characterized mainly early developmental stages, while anthocyanins were abundant in the mature red stage; in the achene, ellagitannin and flavonoids, were abundant during early and late development, respectively. Correlation-based network analysis suggested that metabolism is substantially coordinated during early development in either organ. Nonetheless, a higher degree of connectivity within and between metabolic pathways was measured in the achenes. The data are discussed within the context of current models both of the interaction of primary and secondary metabolism and of the metabolic interaction between the different plant organs.

Phytochrome A (phyA) is the primary photoreceptor for mediating various far-red light induced responses in higher plants. We recently showed that Arabidopsis FHY3 and FAR1, a pair of homologous proteins sharing significant sequence homology to Mutator-like transposases, act as novel transcription factors essential for activating the expression of FHY1 and FHL, whose products are required for light-induced phyA nuclear accumulation and subsequent light responses. FHY3, FAR1 and Mutator-like transposases also share a similar domain structure, including an N-terminal C2H2 zinc-finger domain, a central putative core transposase domain, and a C-terminal SWIM motif. In this study, we performed a promoter-swapping analysis of FHY3 and FAR1. Our results suggest that the partially overlapping function of FHY3 and FAR1 entails divergence of their promoter activities and protein sub-functionalization. To gain a better understanding of the molecular mode of FHY3 function, we performed a structure-function analysis, using site-directed mutagenesis and transgenic approaches. We show that the conserved N-terminal C2H2 zinc-finger domain is essential for direct DNA binding and biological function of FHY3 in mediating light signaling, whereas the central core transposase domain and C-terminal SWIM domain are essential for the transcriptional regulatory activity of FHY3 and its homodimerization or hetero-dimerization with FAR1. Further, the ability to form homo- or hetero-dimers largely correlates with the transcriptional regulatory activity of FHY3 in plant cells. Together, our results reveal discrete roles of the multiple domains of FHY3 and provide functional support for the proposition that FHY3 and FAR1 represent transcription factors derived from a Mutator-like transposase(s).

Aquaporins are water channel proteins which facilitate the passage of water through biological membranes and play a crucial role in plant growth. We showed that ethylene treatment significantly reduced petal size, inhibited expansion of petal abaxial sub-epidermal (AbsE) cells, and decreased petal water content in rose (Rosa hybrida) cv. Samantha. Here we report the isolation of a plasma membrane aquaporin (PIP) gene, Rh-PIP2;1 and characterized its potential role in ethylene inhibited petal expansion. Rh-PIP2;1 is mainly localized on the plasma membrane and belongs to the class 2 subfamily of PIP proteins. We have shown that Rh-PIP2;1 is an active water channel. The transcripts of Rh-PIP2;1 are highly abundant in petal epidermal cells, especially in the AbsE cells. The expression of Rh-PIP2;1 is highly correlated with petal expansion and tightly down-regulated by ethylene. Furthermore, we demonstrated that in Rh-PIP2;1-silenced flowers, petal expansion was greatly inhibited and anatomical features of the petals were similar to those of ethylene-treated flowers. We argue that Rh-PIP2;1 plays an important role in petal cell expansion and ethylene inhibits petal expansion of roses at least partially by suppressing Rh-PIP2;1 expression.

Root growth is mainly determined by cell division and subsequent elongation in the root apical area. Components regulating cell division in the root meristematic cells are largely unknown. Previous studies have identified OsRAA1 as a regulator in root development. Yet the function of OsRAA1 at the cellular and molecular level is unclear. Here, we show that OsRAA1 overexpressed transgenic rice showed reduced primary root growth, increased number of cells in metaphase, and reduced number of cells in anaphase, which suggests that OsRAA1 is responsible for limiting root growth by inhibiting the onset of anaphase. The expression of OsRAA1 in fission yeast also induced metaphase arrest, which is consistent with the fact that OsRAA1 functions through a conserved mechanism of cell cycle regulation. Moreover, co-localization assay has shown that OsRAA1 expresses predominantly at spindles during cell division. Yeast two-hybrid and pull-down, as well as bimolecular fluorescent complementation assays all have revealed that OsRAA1 interacts with a rice homolog of RPT4, a component that is involved in ubiquitin pathway. Treating transgenic rice with specific inhibitors of 26S proteasome blocked the degradation of OsRAA1 and increased the number of cells in metaphase. Mutation of a putative ubiquitination-targeting D-box (RGSLDLISL) in OsRAA1 interrupted the destruction of OsRAA1 in transgenic yeast. The results suggest that ubiquitination and proteasomic proteolysis are involved in the OsRAA1 degradation which is essential for the onset of anaphase and that OsRAA1 may modulate root development mediated by the ubiquitin-proteasome pathway as a novel regulatory factor of the cell cycle.

Pyrrolizidine alkaloids (PAs) are typical compounds of plant secondary metabolism and are believed to be part of the plant's chemical defense. Within the monocotyledonous plants, PAs have been described in only a few genera, mainly orchids, including Phalaenopsis. As phylogenetic analyses suggest an independent origin of PA biosynthesis within the monocot lineage, we have analyzed the developmentally regulated expression of homospermidine synthase (HSS), the first pathway-specific enzyme of PA biosynthesis, at the cell level. HSS is expressed in the tips of aerial roots exclusively in mitotically active cells. Raphide crystal idioblasts present within the root apical meristem do not show HSS expression. In addition, young flower buds but not mature flowers express HSS and have been shown by tracer feeding experiments to be able to catalyze PAs. This second site of PA biosynthesis ensures high concentrations of PAs in the reproductive structures of the Phalaenopsis flower, even after the flower opens. Thus, in spite of its identical function in PA biosynthesis, HSS shows in Phalaenopsis a completely different spacial and developmental expression pattern in comparison to other PA-producing species. These results show that the proverbial diversity of plant secondary metabolism is not just a matter of structural diversity but is also multifaceted in terms of pathway regulation and expression.

Both leaf production and leaf expansion are tightly linked to cell expansion and cell division but the functional relationships between all these variables are not clearly established. To get insight into these relationships, a quantitative genetic analysis was performed in 118 Recombinant Inbred Lines (RIL) derived from a cross between Ler and An-1 accessions and was combined with a structural equation modelling approach. Main effects and epistatic interactions at the QTL level were detected for rosette area, rosette leaf number, leaf 6 area, epidermal cell area and number. A QTL at ERECTA marker controlled cell expansion and cell division, in interaction with two other QTLs at SNP295 and SNP21 markers. Moreover, both the screening for marker association involved in the variation of the relationships between leaf growth variables and the test of alternative functional models by structural equation modelling revealed that the allelic value at ER controlled epidermal cell area and epidermal cell number in a leaf. These effects are driven both by a whole plant mechanism associated with leaf production and by a single leaf mechanism associated with leaf expansion. The complex effects of the QTL at ER were validated in selected Heterogeneous Inbred Families (HIF). The ERECTA gene, which is mutated in the Ler parental line, was found to be a putative candidate responsible for these mapped effects by phenotyping mutants of this gene at the cellular level. All together these results give insight into the complex determination of leaf epidermal cell number and area.

The levels of endogenous polyamines have been shown to increase in plant cells challenged with low temperature, however the functions of polyamines in the regulation of cold stress responses are unknown. Here we show that the accumulation of putrescine under cold stress is essential for proper cold-acclimation and survival at freezing temperatures, since Arabidopsis mutants defective in putrescine biosynthesis (adc1, adc2) display reduced freezing tolerance compared to wild type plants. Genes ADC1 and ADC2 show different transcriptional profile upon cold treatment, however they show similar and redundant contributions to cold responses in terms of putrescine accumulation kinetics and freezing sensitivity. Our data also demonstrate that detrimental consequences of putrescine depletion during cold stress are due, at least in part, to alterations in the levels of abscisic acid (ABA). Reduced expression of NCED3, a key gene involved in ABA biosynthesis, and downregulation of ABA-regulated genes are detected in both adc1 and adc2 mutant plants under cold stress. Complementation analysis of adc mutants with ABA, and reciprocal complementation tests of aba2-3 mutant with putrescine support the conclusion that putrescine controls the levels of ABA in response to low temperature by modulating ABA biosynthesis and gene expression.

Copper (Cu) is an essential element in plant nutrition, but it inhibits growth of roots at low concentrations. Accessions of Arabidopsis (Arabidopsis thaliana) vary in their tolerance to Cu. To understand the molecular mechanism of Cu tolerance in Arabidopsis, we performed QTL analysis and accession studies. One major QTL on chromosome 1 (QTL1) explained 52% of the phenotypic variation in Cu tolerance in roots in a Ler/Cvi recombinant inbred population. This QTL regulates Cu translocation capacity and involves a Cu-transporting P_1B-1-type ATPase, HMA5. The Cvi allele carries two amino acid substitutions in comparison with the Ler allele and is less functional than the Ler allele in Cu tolerance when judged by complementation assays using a T-DNA insertion mutant. Complementation assays of the ccc2 mutant of yeast using chimeric HMA5 proteins revealed that N923T of the Cvi allele, which was identified in the tightly conserved domain N(x)₆YN(x)₄P (where the former asparagine was substituted to threonine), is a cause of dysfunction of the Cvi HMA5 allele. Another dysfunctional HMA5 allele was identified in Chisdra-2 (Chi-2), which showed Cu sensitivity and low capacity of Cu translocation from roots to shoots. A unique amino acid substitution of Chi-2 was identified in another strictly conserved domain, CPC(x)₆P, where the latter proline was replaced with leucine. These results indicate that a portion of the variation in Cu tolerance of Arabidospsis is regulated by functional integrity of the Cu-translocating ATPase, HMA5, and in particular to the amino acid sequence in several strictly conserved motifs.

In plants, fatty acids are de novo synthesized predominantly in plastids from acetyl-CoA. Although fatty acid biosynthesis has been biochemically well-studied, little is known about the regulatory mechanisms of the pathway. Here, we show that overexpression of the Arabidopsis (Arabidopsis thaliana) LEAFY COTYLEDON1 (LEC1) gene causes globally increased expression of fatty acid biosynthetic genes, which are involved in key reactions of condensation, chain elongation and desaturation of fatty acid biosynthesis. In the plastidial fatty acid synthetic pathway, over 58% of known enzyme-coding genes are upregulated in LEC1-overexpressing transgenic plants, including those encoding three subunits of acetyl-CoA carboxylase, a key enzyme controlling the fatty acid biosynthesis flux. Moreover, genes involved in glycolysis and lipid accumulation are also upregulated. Consistent with these results, levels of major fatty acid species and lipids were substantially increased in the transgenic plants. Genetic analysis indicates that the LEC1 function is partially dependent on ABSCISIC ACID INSENSITIVE3, FUSCA3 and WRINKLED1 in the regulation of fatty acid biosynthesis. Moreover, a similar phenotype was observed in transgenic Arabidopsis plants overexpressing two LEC1-like genes of Brassica napus. These results suggest that LEC1 and LEC1-like genes act as key regulators to coordinate the expression of fatty acid biosynthetic genes, thereby representing a promising target for genetic improvement of oil-production plants.

IAA is an important regulator of the adventitious rooting via activation of complex signaling cascades. In animals, carbon monoxide (CO), mainly generated by heme oxygenases (HOs), is a significant modulator of inflammatory reactions, affecting cell proliferation and production of growth factors. In this report, we showed that the treatment with the auxin transport inhibitor naphthylphthalamic acid (NPA) besides prevented auxin-mediated induction of adventitious rooting, also decreased the activity of HO and its by-product CO content. The application of IAA, HO-1 activator/CO donor hematin or CO aqueous solution was able to alleviate the IAA depletion-induced inhibition of adventitious root formation. Meanwhile, IAA or hematin treatment rapidly activated HO activity or HO-1 protein expression, and CO content was also enhanced. The application of HO-1 specific inhibitor ZnPPIX could inhibit above IAA and hematin responses. While, CO aqueous solution treatment was able to ameliorate ZnPPIX-induced the inhibition of adventitious rooting. Molecular evidence further showed that ZnPPIX mimicked the effects of NPA on the inhibition of adventitious rooting, the down-regulation of one DnaJ-like gene CSDNAJ-1 and two calcium dependent protein kinases genes CSCDPK1/5. Application of CO aqueous solution could not only dose-dependently block IAA depletion-induced inhibition of adventitious rooting, but also enhance endogenous CO content and up-regulate CSDNAJ-1 and CSCDPK1/5 transcripts. Together, we provided pharmacological, physiological and molecular evidence that auxin rapidly activates HO activity and that the product of HO action, CO then triggers the signal transduction events that lead to the auxin responses of adventitious root formation in cucumber.

Ca²⁺ rise and nitric oxide (NO) generation are essential early steps in plant innate immunity, and initiate the hypersensitive response (HR) to avirulent pathogens. Previous work from this lab has demonstrated that a loss-of-function mutation of an Arabidopsis plasma membrane Ca²⁺ permeable inwardly conducting ion channel impairs HR and this phenotype could be rescued by the application of an NO donor. At present, the mechanism linking cytosolic Ca²⁺ rise to NO generation during pathogen response signaling in plants is still unclear. Animal NO synthase (NOS) activation is Ca²⁺/calmodulin (CaM) dependent. Here, we present biochemical and genetic evidence consistent with a similar regulatory mechanism in plants; a pathogen-induced Ca²⁺ signal leads to CaM and/or a CaM-like protein (CML) activation of NOS. In wild type Arabidopsis plants, the use of a CaM antagonist prevents NO generation and HR. Application of a CaM antagonist does not prevent pathogen-induced cytosolic Ca²⁺ elevation, excluding the possibility of CaM acting upstream from Ca²⁺. The CaM antagonist and Ca²⁺ chelation abolish NO generation in wild type Arabidopsis leaf protein extracts as well, suggesting that plant NOS activity is Ca²⁺/CaM dependent in vitro. The CaM like protein CML24 has been previously associated with NO-related phenotypes in Arabidopsis. Here, we find that innate immune response phenotypes (HR and (avirulent) pathogen-induced NO elevation in leaves) are inhibited in loss-of-function cml24-4 mutant plants. Pathogen associated molecular pattern mediated NO generation in cells of cml24-4 mutants is impaired as well. Our work suggests that the initial pathogen recognition signal of Ca²⁺ influx into the cytosol activates CaM and/or a CML which then acts to induce downstream NO synthesis as intermediary steps in a pathogen perception signaling cascade leading to innate immune responses including HR.

Arabidopsis thaliana overexpressing glycolate oxidase (GO) in chloroplasts accumulates both hydrogen peroxide (H₂O₂) and glyoxylate. GO overexpressing lines (GO plants) grown at 75 µmol quanta m^-2 s^-1 show retarded development, yellowish rosettes and impaired photosynthetic performance, while at 30 µmol quanta m^-2 s^-1 this phenotype virtually disappears. The GO plants develop oxidative stress lesions under photorespiratory conditions but grow like the wild-type under non-photorespiratory conditions. GO plants co-expressing enzymes which further metabolize glyoxylate but still accumulate H₂O₂ show all features of the GO phenotype indicating that H₂O₂ is responsible for the GO phenotype. The GO plants can complete their life cycle indicating that they are able to adapt to the stress conditions imposed by the accumulation of H₂O₂ during the light period. Moreover, the data indicate that a response to oxidative stress is installed, with increased expression and/or activity of known oxidative stress responsive components. Hence, the GO plants are an ideal non-invasive model system to study the effects of H₂O₂ directly in the chloroplasts because H₂O₂ accumulation is inducible and sustained perturbations can reproducibly be provoked by exposing the plants to different ambient conditions.

In our analysis of 5' and 3' end formation in plant mitochondria we compared the major transcript ends of all mitochondrial protein-coding genes between the three Arabidopsis thaliana accessions Columbia (Col), C24 and Landsberg erecta (Ler). Differences between transcript patterns were found for seven genes. For atp6-2 no transcripts at all were detected in Ler. This and further analyses suggest the atp6-2 gene arrangement to be absent from the mitochondrial DNA of this accession. All other transcript polymorphisms are attributed to variations at the 5' termini and were consistently observed in all tissues investigated. mRNA phenotyping of reciprocal Col/Ler, Col/C24 and Ler/C24 F₁ hybrids revealed the differing transcript pattern of ccmC to be inherited maternally suggesting these to arise from differences in the mitochondrial DNA. Bi-parental inheritance was observed for the polymorphic transcripts of nad4, nad9, ccmB and rpl5 indicating these differences to be caused by nuclear encoded trans-factors. Deviant transcript patterns were tested in further accessions and were found in at least three additional accessions. Detailed examination of the nad4 and the nad9 transcripts demonstrates that the respective polymorphisms affect the major mRNAs of these genes. This study shows that natural genetic variation in Arabidopsis thaliana can also affect mitochondrial mRNA end processing. These variations can now be used to identify the nuclear genes responsible as well as the mitochondrial cis-elements required for 5' end generation of mitochondrial transcripts.

Steady state labeling experiments with [1-¹³C]glucose were used to measure multiple metabolic fluxes through the pathways of central metabolism in a heterotrophic cell suspension culture of Arabidopsis thaliana. The protocol was based on in silico modeling to establish the optimal labeled precursor, validation of the isotopic and metabolic steady state, extensive NMR analysis of the redistribution of label into soluble metabolites, starch and protein, and a comprehensive set of biomass measurements. Following a simple modification of the cell culture procedure, cells were grown at two oxygen concentrations, and flux maps of central metabolism were constructed on the basis of replicated experiments and rigorous statistical analysis. Increased growth rate at the higher O₂ concentration was associated with an increase in fluxes throughout the network, and this was achieved without any significant change in relative fluxes, despite differences in the metabolite profile of organic acids, amino acids and carbohydrates. The balance between biosynthesis and respiration within the TCA cycle was unchanged with 38 ± 5% of carbon entering used for biosynthesis under standard O₂ conditions and 33% ± 2% under elevated O₂. These results add to the emerging picture of the stability of the central metabolic network and its capacity to respond to physiological perturbations with the minimum of rearrangement. The lack of correlation between the change in metabolite profile, which implied significant disruption of the metabolic network following the alteration in the oxygen supply, and the unchanging flux distribution highlights a potential difficulty in the interpretation of metabolomic data.

Peroxisomes are important for recycling carbon and nitrogen that would otherwise be lost during photorespiration. The reduction of hydroxypyruvate to glycerate catalyzed by hydroxypyruvate reductase (HPR) in the peroxisomes is thought to be facilitated by the production of NADH by peroxisomal malate dehydrogenases (PMDH). PMDH, which is encoded by two genes in Arabidopsis, reduces NAD⁺ to NADH via the oxidation of malate supplied from the cytoplasm to oxaloacetate. A double mutant lacking the expression of both PMDH genes was viable in air and had rates of photosynthesis only slightly lower than wild-type (WT). This is in contrast to other photorespiratory mutants which have severely reduced rates of photosynthesis and require high CO₂ to grow. The pmdh mutant had a higher O₂ dependent CO₂ compensation point than wild type (WT) implying that either Rubisco specificity had changed or that the rate of CO₂ released per Rubisco oxygenation was increased in the pmdh plants. Rates of gross O₂ evolution and uptake were similar in the pmdh and WT plants indicating chloroplast linear electron transport and photorespiratory O₂ uptake were similar between genotypes. The CO₂ post illumination burst and the rate of CO₂ released during photorespiration were both greater in the pmdh mutants compared to WT, suggesting the ratio of photorespiratory CO₂ release to Rubisco oxygenation was altered in the pmdh mutants. Without PMDH in the peroxisome the CO₂ released per Rubisco oxygenation reaction can be increased by over 50%. In summary, PMDH is essential for maintaining optimal rates of photorespiration at air; however, in its absence significant rates of photorespiration are still possible indicating that there are additional mechanisms for supplying reductant to the peroxisomal HPR reaction or the HPR reaction is altogether circumvented.

Initiation of leaves at the flanks of the shoot apical meristem occurs at sites of auxin accumulation and pronounced expression of auxin-inducible PIN genes, suggesting a feedback loop to progressively focus auxin in concrete spots. Since PIN expression is regulated by Auxin Response Factor (ARF) activity, including MONOPTEROS (MP), it appeared possible that MP affects leaf formation as a positive regulator of PIN genes and auxin transport. Here we analyze a novel, completely leafless phenotype arising from simultaneous interference with both auxin signaling and auxin transport. We show that mp pin1 double mutants, as well as mp mutants treated with auxin-efflux inhibitors, display synergistic abnormalities, not seen in wild type regardless of how strongly auxin transport was reduced. The synergism of abnormalities indicates that the role of MP in shoot meristem organization is not limited to auxin transport regulation. In mp mutant background, auxin transport inhibition completely abolishes leaf formation. Instead of forming leaves, the abnormal shoot meristems dramatically increase in size harboring correspondingly enlarged expression domains of CLAVATA3 and SHOOTMERISTEMLESS, molecular markers for the central stem cell zone and the complete meristem respectively. The observed synergism under conditions of auxin efflux inhibition was further supported by an unrestricted PIN1 expression in mp meristems, as compared to a partial restriction in wildtype meristems. Auxin transport-inhibited mp meristems also lacked detectable auxin maxima. We conclude that MP promotes the focusing of auxin and leaf initiation in part through pathways not affected by auxin efflux inhibitors.

Reactive oxygen species (ROS) act as signaling molecules but can also directly provoke cellular damage by rapidly oxidizing cellular components, including lipids. We developed an HPLC-electrospray ionization-MS/MS-based quantitative method that allowed to discriminate between free radical (type I) and singlet oxygen (type II) mediated lipid peroxidation (LPO) signatures by using hydroxy fatty acids as specific reporters. Using this method we observed that in non-photosynthesizing Arabidopsis thaliana tissues non-enzymatic LPO was almost exclusively catalyzed by free radicals both under normal and oxidative stress conditions. However in leaf tissues under optimal growth conditions, singlet oxygen was responsible for more than 80% of the non-enzymatic LPO. In Arabidopsis mutants favoring singlet oxygen production, photo-oxidative stress led to a dramatic increase of singlet oxygen (type II) LPO that preceded cell death. Furthermore, under all conditions and in mutants that favor the production of superoxide and hydrogen peroxide (two sources for type I LPO reactions), plant cell death was nevertheless always preceded by an increase in singlet oxygen dependent (type II) LPO. Thus, besides triggering a genetic cell death program, as demonstrated previously with the Arabidopsis fluorescent (flu) mutant, singlet oxygen plays a major destructive role during the execution of ROS-induced cell death in leaf tissues.

The "light" signal from the environment sets the circadian clock to regulate multiple physiological processes for optimal rhythmic growth and development. One such process is the control of flowering time by photoperiod perception in plants. In Arabidopsis, the flowering time is determined by the correct interconnection of light input and signal output by the circadian clock. The identification of additional clock proteins will help to better dissect the complex nature of the circadian clock in Arabidopsis. Here we show LWD1/LWD2 as new clock proteins involved in photoperiod control. The lwd1lwd2 double mutant has an early flowering phenotype, contributed by the significant phase shift of CO and, therefore, an increased expression of FT before dusk. Under entrainment conditions, the expression phase of oscillator (CCA1, LHY, TOC1 and ELF4) and output (GI, FKF1, CDF1, CO and FT) genes in the photoperiod pathway shifts ~3 hr forward in the lwd1lwd2 double mutant. Both the oscillator (CCA1, LHY, TOC1 and ELF4) and output (CCR2 and CAB2) genes have a short period length in the lwd1lwd2 double mutant. Our data imply that LWD1/LWD2 proteins function in close proximity to or within the circadian clock for photoperiodic flowering control.

Potassium homeostasis is essential for diverse cellular processes, though how various cation transporters collaborate to maintain a suitable K⁺ required for growth and development is poorly understood. The Arabidopsis thaliana genome contains numerous cation:proton antiporters (CHX) which may mediate K⁺ transport; however, the vast majority of these transporters remain uncharacterized. Here we show that AtCHX13 (At2g30240) has a role in K⁺ acquisition. AtCHX13 suppressed the sensitivity of yeast mutant cells defective in K⁺ uptake. Uptake experiments using ⁸⁶Rb⁺ as a tracer for K⁺ demonstrated that AtCHX13 mediated high-affinity K⁺ uptake in yeast and in plant cells with a Km of 136 µM and 196 µM, respectively. Functional GFP-tagged versions localized to the plasma membrane of both yeast and plant. Seedlings of null chx13 mutants were sensitive to K⁺ deficiency conditions, whereas over expression of AtCHX13 reduced the sensitivity to K⁺ deficiency. Collectively, these results suggest that AtCHX13 mediates relatively high-affinity K⁺ uptake though the mode of transport is unclear at present. AtCHX13 expression is induced in roots during K⁺ deficient conditions. These results indicate that one role of AtCHX13 is to promote K⁺ uptake into plants when K⁺ is limiting in the environment.

Pathogen-inducible antimicrobial defense-related proteins have emerged as key antibiotic peptides and enzymes involved in disease resistance in plants. A novel antimicrobial protein gene, CaAMP1, was isolated from pepper (Capsicum annuum) leaves infected with Xanthomonas campestris pv. vesicatoria (Xcv). Expression of the CaAMP1 gene was strongly induced in pepper leaves not only during pathogen infection but also after exposure to abiotic elicitors. The purified recombinant CaAMP1 protein possessed broad spectrum antimicrobial activity against phytopathogenic bacteria and fungi. CaAMP1:smGFP fusion protein was mainly localized in the external and intercellular regions of the onion epidermal cells. The virus-induced gene silencing technique and gain-of-function transgenic plants were used to determine the CaAMP1 gene function in plant defense. Silencing of CaAMP1 led to enhanced susceptibility to Xcv and Colletotrichum coccodes infection, accompanied by reduced PR gene expression. In contrast, overexpression of CaAMP1 in Arabidopsis thaliana conferred broad spectrum resistance to the hemi-biotrophic bacterial pathogen Pseudomonas syringae pv. tomato (Pst), the biotrophic oomycete Hyaloperonospora parasitica, and the fungal necrotrophic pathogens Fusarium oxysporum f.sp. matthiolae and Alternaria brassicicola. CaAMP1 overexpression induced the SA pathway-dependent genes PR1 and PR5, but not the jasmonic acid-dependent defense gene PDF1.2 during Pst infection. Together, these results suggest that the antimicrobial CaAMP1 protein is involved in broad spectrum resistance to bacterial and fungal pathogen infection.

Plant innate immunity to pathogenic microorganisms is activated in response to recognition of extracellular or intracellular pathogen molecules by transmembrane receptors or resistance proteins, respectively. The defense signaling pathways share components with those involved in plant responses to UV radiation, which can induce expression of plant genes important for pathogen resistance. Such intriguing links suggest that UV treatment might activate resistance to pathogens in normally susceptible host plants. Here we demonstrate that pre-inoculative UV (254 nm) irradiation of Arabidopsis thaliana susceptible to infection by the biotrophic oomycete Hyaloperonospora parasitica, the causative agent of Downy Mildew, induces dose- and time-dependent resistance to the pathogen detectable up to seven days after UV exposure. Limiting repair of UV photoproducts by post-irradiation incubation in the dark, or mutational inactivation of cyclobutane pyrimidine dimer (CPDs) photolyase, (6-4) photoproduct (6-4PP) photolyase or nucleotide excision repair (NER) increased the magnitude of UV-induced pathogen resistance. In the absence of treatment with 254-nm UV, plant NER mutants also defective for CPD or 6-4PP photolyase displayed resistance to H. parasitica partially attributable to short wavelength UV-B (280 to 320 nm) radiation emitted by incubator lights. These results indicate UV irradiation can initiate the development of resistance to H. parasitica in plants normally susceptible to the pathogen, and point to a key role for UV-induced DNA damage. They also suggest UV treatment can circumvent the requirement for recognition of H. parasitica molecules by A. thaliana proteins to activate an immune response.

In flowering plants the two sperm cells are embedded within the cytoplasm of the growing pollen tube and as such are passively transported to the embryo sac, wherein double fertilization occurs upon their release. Understanding the mechanisms and conditions by which male gametes mature and take part in fertilization are crucial goals in the study of plant reproduction. Studies of gene expression in male gametes of maize and Plumbago, and in lily generative cells already showed that the previously held view of transcriptionally inert male gametes was not true, but genome-wide studies were lacking. Analyses in the model plant Arabidopsis thaliana were hindered because no method to isolate sperm cells was available. Here we used Fluorescence-activated cell sorting (FACS) to isolate sperm cells from Arabidopsis, allowing GeneChip analysis of the transcriptome of sperm cells at a genome-wide level. Comparative analysis of the sperm cell transcriptome with those of representative sporophytic tissues and of pollen showed that sperm has a distinct and diverse transcriptional profile. Functional classifications of genes with enriched expression in sperm cells showed that DNA repair, ubiquitin-mediated proteolysis and cell cycle progression are over-represented Gene Ontology categories. Moreover, analysis of the small RNA and DNA methylation pathways suggests that distinct mechanisms might be involved in regulating the epigenetic state of the paternal genome. We identified numerous candidate genes whose involvement in sperm cell development and fertilization can now be directly tested in Arabidopsis. These results provide a roadmap to decipher the role of sperm-expressed proteins.

NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) A is the only thus far known example of a nucleus-encoded plastid protein that is imported to its final destination in a substrate-dependent, Pchlide-regulated manner. Previous work has shown that the cytosolic PORA precursor (pPORA) does not utilize the general import site but uses a distinct translocon designated the Pchlide-dependent translocon complex (PTC). Here we demonstrate that a pentapeptide motif, Thr-Thr-Ser-Pro-Gly (TTSPG) in pPORA's transit peptide (transA), is involved in Pchlide-dependent transport. Deletion of this motif from the COOH-terminal end of transA abolished both Pchlide binding and protein import. Incorporation of the TTSPG motif into normally non-Pchlide-responsive transit sequences conferred the pigment binding properties onto the engineered chimeric precursors but was insufficient to render protein import substrate-dependent. An additional motif was identified in the NH₂-terminal part of transA that was needed for binding of the precursor to the PTC complex. Point mutations of the TTSPG motif identified Gly as the Pchlide binding site. By analogy to the major light-harvesting chlorophyll a/b binding protein of photosystem II, we propose that the peptidyl carbonyl oxygen of Gly may bind directly or via a water molecule to the central Mg atom of the pigment.