Wounding and herbivore attack elicit the rapid (within minutes) accumulation of jasmonic acid (JA) which results from the activation of previously synthesized biosynthetic enzymes. Recently, several regulatory factors that affect JA production have been identified; however, how these regulators affect JA biosynthesis remains at present unknown. Here we demonstrate that Nicotiana attenuata salicylate-induced protein kinase (SIPK), wound-induced protein kinase (WIPK), nonexpressor of PR-1 (NPR1) and the insect elicitor N-linolenoyl-glutamate (18:3-Glu) participate in mechanisms affecting early enzymatic steps of the JA biosynthesis pathway. Plants silenced in the expression of SIPK and NPR1 were affected in the initial accumulation of 13-hydroperoxy-linolenic acid (13-OOH-18:3) after wounding and 18:3-Glu elicitation by mechanisms independent of changes in 13-lipoxygenase (13-LOX) activity. Moreover, 18:3-Glu elicited an enhanced and rapid accumulation of 13-OOH-18:3 that depended partially on SIPK and NPR1 but was independent of increased 13-LOX activity. Together, the results suggested that substrate supply for JA production was altered by 18:3-Glu elicitation and SIPK- and NPR1-mediated mechanisms. Consistent with a regulation at the level of substrate supply, we demonstrated by virus-induced gene silencing that a wound repressed plastidial glycerolipase (NaGLA1) plays an essential role in the induction of de novo JA biosynthesis. In contrast to SIPK and NPR1, mechanisms mediated by WIPK did not affect the production of 13-OOH-18:3 but were critical to control the conversion of this precursor into 12-oxo-phytodienoic acid. These differences could be partially accounted for by reduced allene oxide synthase activity in WIPK-silenced plants.

The Arabidopsis thaliana CLAVATA2 (CLV2) gene encodes a leucine-rich repeat receptor-like protein (LRR-RLP) that is involved in controlling the stem cell population size in the shoot apical meristem. Our previous genome-wide functional analysis of 57 Arabidopsis RLP (AtRLP) genes revealed only a few phenotypes for mutant alleles, despite screening a wide range of growth and developmental stages, and assaying sensitivity to various stress responses including susceptibility toward pathogens. To gain further insight into the biological role of AtRLPs, in particular of CLV2-related AtRLP genes, we tested their ability to complement the clv2 mutant phenotype. We found that out of four close CLV2 homologues tested, AtRLP2 and AtRLP12 could functionally complement the clv2 mutant when expressed under the control of the CLV2 promoter. This indicates that the functional specificity of these three genes is determined at the level of their transcriptional regulation. Single and double mutant combinations with impaired AtRLP2 and/or AtRLP12 did not show an aberrant phenotype, suggesting that other genes are redundant with these CLV2-like genes. To understand which protein domains are essential for CLV2 function and which parts are interchangeable between related CLV2-like proteins, we performed domain-deletion and domain-swap experiments. These experiments revealed that CLV2 remains functional without the island domain, whereas the C1 and C3 region of the LRR domain are essential for functionality. Analysis of domain-swap constructs showed that the C3-G region of CLV2 can be replaced by that of AtRLP38, although it could not complement the clv2 mutant under control of the CLV2 promoter. This suggests that the C3-G region is conserved among related AtRLP members, whereas the C1 domain may determine the functional specificity of CLV2.

Poly(ADP-ribosyl)ation is a post-translational protein modification in which ADP-ribose units derived from NAD⁺ are attached to proteins by poly(ADP-ribose) polymerase (PARP) enzymes. ADP-ribose groups are removed from these polymer chains by the enzyme poly(ADP-ribose) glycohydrolase (PARG). In animals, poly(ADP-ribosyl)ation is associated with DNA damage responses and programmed cell death. Previously, we hypothesized a role for poly(ADP-ribosyl)ation in plant defense responses when we detected defense-associated expression of the poly(ADP-ribosyl)ation-related genes PARG2 and NUDT7, and observed altered callose deposition in the presence of a chemical PARP inhibitor (Adams-Phillips et al., 2008). The role of poly(ADP-ribosyl)ation in plant defenses was more extensively investigated in the current study, using Arabidopsis thaliana. Pharmacological inhibition of PARP using 3-aminobenzamide (3AB) perturbs certain innate immune responses to MAMPs (flg22 and elf18), including callose deposition, lignin deposition, pigment accumulation, and phenylalanine ammonia lyase (PAL) activity, but does not disrupt other responses such as the initial oxidative burst and expression of some early defense-associated genes. Mutant parg1 seedlings exhibit an exaggerated seedling growth inhibition and pigment accumulation in response to elf18, and are hypersensitive to the DNA damaging agent mitomycin C. Both parg1 and parg2 knockout plants show accelerated onset of disease symptoms when infected with Botrytis cinerea. Cellular levels of ADP-ribose polymer increase after infection with avirulent Pseudomonas syringae pv. tomato DC3000 avrRpt2⁺, and pathogen-dependent changes in the poly(ADP-ribosyl)ation of discrete proteins were also observed. We conclude that poly(ADP-ribosyl)ation is a functional component in plant responses to biotic stress.

Processing bodies (P-bodies) are specialized cytoplasmic foci where mRNA turnover and translational repression can take place. Stress granules (SGs) are related cytoplasmic foci. The CCCH tandem zinc finger proteins (TZFs) play pivotal roles in gene expression, cell fate specification, and various developmental processes. Human TZF (hTTP) binds AU-rich elements at the 3'UTR, and recruit decapping, deadenylation, and exonucleolytic enzymes to P-bodies for RNA turnover. Recent genetic studies indicate that plant TZFs are involved in gene regulation and hormone-mediated environmental responses. It is unknown if plant TZFs can bind RNA and be localized to P-bodies or stress granules. The Arabidopsis AtTZF1/AtCTH/AtC3H23 was identified as a sugar sensitive gene in a previous microarray study. It is characterized by a TZF motif that is distinct from the human TZF. Higher plants such as Arabidopsis and rice each have a gene family containing this unique TZF motif. Here we show that AtTZF1 can traffic between the nucleus and cytoplasmic foci. AtTZF1 co-localizes with markers of P-bodies, and the morphology of these cytoplasmic foci resembles that of mammalian P-bodies and stress granules. AtTZF1-associated cytoplasmic foci are dynamic and tissue-specific. They can be induced by dark and wound stresses, and are preferentially present in actively growing tissues and stomatal precursor cells. Since AtTZF1 can bind both DNA and RNA in vitro, it raises the possibility that AtTZF1 might be involved in DNA and/or RNA regulation.

Phosphate (Pi) availability is a major constraint to plant growth. Consequently, plants have evolved complex adaptations to tolerate low Pi conditions. Numerous genes implicated in these adaptations have been identified, but their chromatin-level regulation has not been investigated. The nuclear actin-related protein ARP6 is conserved among all eukaryotes and is an essential component of the SWR1 chromatin remodeling complex, which regulates transcription via deposition of the H2A.Z histone variant into chromatin. Herein we demonstrate that ARP6 is required for proper H2A.Z deposition at a number of Pi-starvation response genes in Arabidopsis thaliana. The loss of H2A.Z at these target loci results in their de-repression in arp6 mutants, and correlates with the presence of multiple Pi-starvation-related phenotypes, including shortened primary roots and increases in the number and length of root hairs, as well as increased starch accumulation and phosphatase activity in shoots. Our data suggest a model for chromatin-level control of Pi-starvation responses in which ARP6-dependent H2A.Z deposition modulates the transcription of a suite of PSR genes.

Maize (Zea mays) endosperm ADP-glucose pyrophosphorylase (AGPase) is a highly regulated enzyme that catalyzes the rate-limiting step in starch biosynthesis. Although the structure of the heterotetrameric maize endosperm AGPase remains unsolved, structures of a non-native, low activity form of the potato tuber (Solanum tuberosum) AGPase (small subunit homotetramer) revealed that several sulfate ions bind to each enzyme [Jin, X., Ballicora, M.A., Preiss, J., Geiger, J.H. EMBO J. 2005, 24, 694-704]. These sites are also believed to interact with allosteric regulators such as inorganic phosphate (P_i) and 3-phosphoglycerate (3-PGA). Several arginine side chains contact the bound sulfate ions in the potato structure and likely play important roles in allosteric effector binding. Alanine scanning mutagenesis was applied to the corresponding Arg residues in both the small and large subunits of maize endosperm AGPase to determine their roles in allosteric regulation and thermal stability. Steady state kinetic and regulatory parameters were measured for each mutant. All of the arginine mutants examined – in both the small and large subunits – bound 3-PGA more weakly than the wild type (A₅₀ increased by 3.5 - 20 fold). By contrast, the binding of two other maize AGPase allosteric activators (fructose-6-phosphate and glucose-6-phosphate) did not always mimic the changes observed for 3-PGA . In fact, compared to 3-PGA, fructose-6-phosphate is a more efficient activator in two of the arginine mutants. Phosphate binding was also affected by arginine substitutions. The combined data support a model for the binding interactions associated with 3-PGA in which allosteric activators and P_i compete directly.

A role for differential glycoconjugation in the emission of phenylpropanoid volatiles from ripening tomato fruit (Solanum lycopersicum L.) upon fruit tissue disruption has been discovered in this study. Application of a multi-instrumental analytical platform for metabolic profiling of fruits from a diverse collection of tomato cultivars revealed that emission of three discriminatory phenylpropanoid volatiles, namely methyl salicylate, guaiacol and eugenol, took place upon disruption of fruit tissue through cleavage of the corresponding glycoconjugates, identified putatively as hexose-pentosides. However, in certain genotypes, phenylpropanoid volatile emission was arrested due to the corresponding hexose-pentoside precursors having been converted into glycoconjugate species of a higher complexity: dihexose-pentosides and malonyl-dihexose-pentosides. This glycoside conversion was established to occur in tomato fruit during the later phases of fruit ripening and has consequently led to the inability of red fruits of these genotypes to emit key phenylpropanoid volatiles upon fruit tissue disruption. This principle of volatile emission regulation can pave the way to new strategies for controlling tomato fruit flavor and taste.

The functional protein phosphatase type-2C (PP2C) FsPP2C1 from beechnut (Fagus sylvatica L.) was a negative regulator of ABA signaling in seeds. In this report, to get deeper insight on FsPP2C1 function, we aim to identify PP2C-interacting partners. Two close-related members (PYL8/RCAR3 and PYL7/RCAR2) of the Arabidopsis BetV I family were shown to bind FsPP2C1 in a yeast two-hybrid screening and in an ABA-independent manner. By transient expression of FsPP2C1 and PYL8/RCAR3 in epidermal onion cells and agroinfiltration in Nicotiana as GFP fusion proteins, we obtained evidence supporting the subcellular localization of both proteins mainly in the nucleus and in both the cytosol and the nucleus, respectively. The in planta interaction of both proteins in tobacco cells by BiFC assays resulted in a specific nuclear colocalization of this interaction. Constitutive overexpression of PYL8/RCAR3 confers ABA-hypersensitivity in Arabidopsis seeds and consequently, an enhanced degree of seed dormancy. Additionally, transgenic 35S:PYL8/RCAR3 plants are unable to germinate under low concentrations of mannitol, NaCl or paclobutrazol, which are not inhibiting conditions to the wild-type. In vegetative tissues, Arabidopsis PYL8/RCAR3 transgenic plants show ABA-resistant drought response and a strong inhibition of early root growth. These phenotypes are strengthened at the molecular level with the enhanced induction of several ABA-response genes. Both seed and vegetative phenotypes of Arabidopsis 35S:PYL8/RCAR3 plants are opposite to those of 35S:FsPP2C1 plants. Finally, double transgenic plants confirm the role of PYL8/RCAR3 by antagonizing FsPP2C1 function and demonstrating that PYL8/RCAR3 positively regulates ABA signaling during germination and abiotic stress responses.

Na⁺ and K⁺ homeostasis are crucial for plant growth and development. Two HKT transporter/channel classes have been characterized that mediate either Na⁺ transport or Na⁺ and K⁺ transport when expressed in Xenopus oocytes and yeast. However, the Na⁺/K⁺ selectivities of the K⁺ permeable HKT transporters have not yet been studied in plant cells. One study expressing 5' UTR-modified HKT constructs in yeast has questioned the relevance of cation selectivities found in heterologous systems for selectivity predictions in plant cells (Haro et al., 2005). Here we therefore analyze two highly homologous HKT transporters in plant cells, OsHKT2;1 and OsHKT2;2, that show differential K⁺ permeabilities in heterologous systems. Upon stable expression in cultured Nicotiana tabacum Bright-Yellow 2 (BY2) cells, OsHKT2;1 mediated Na⁺ uptake, but little Rb⁺ uptake, consistent with earlier studies and new findings presented here in oocytes. In contrast, OsHKT2;2 mediated Na⁺-K⁺ co-transport in plant cells such that extracellular K⁺ stimulates OsHKT2;2-mediated Na⁺ influx and vice versa. Furthermore, at millimolar Na⁺ concentrations OsHKT2;2 mediated Na⁺ influx into plant cells without adding extracellular K⁺. In addition, the presence of external K⁺ and Ca²⁺ down-regulated OsHKT2;1-mediated Na⁺ influx in two plant systems, BY2 cells and intact rice roots, and also in Xenopus oocytes. The present study shows that the Na⁺/K⁺ selectivities of these HKT transporters in plant cells coincide closely with the selectivities in oocytes and yeast and furthermore that OsHKT transporter selectivities in plant cells depend on the imposed cationic conditions, supporting the model that HKT transporters are multi-ion pores.

A vital quest in biology is comprehensible visualization and interpretation of correlation relationships on a genome scale. Such relationships may be represented in the form of networks, which usually require disassembly into smaller manageable units, or clusters, to facilitate interpretation. Several graph clustering algorithms that may be used to visualize biological networks are available. However, only some of these support weighted edges, and none provide good control of cluster sizes, which is crucial for comprehensible visualization of large networks. We constructed an interactive co-expression network for the Arabidopsis genome using a novel Heuristic Cluster Chiseling Algorithm (HCCA) that supports weighted edges, and that may control average cluster sizes. Comparative clustering analyses demonstrated that the HCCA performed as well as, or better than, both the commonly used Markov, MCODE, and k-means clustering algorithms. We mapped MapMan ontology terms onto co-expressed node vicinities of the network, which revealed transcriptional organization of previously unrelated cellular processes. We further explored the predictive power of this network through mutant analyses, and identified six new genes that are essential to plant growth. We show that the HCCA partitioned network constitutes an ideal "cartographic" platform for visualization of correlation networks. This approach rapidly provides network partitions with relative uniform cluster sizes on a genome-scale level, and may thus be used for correlation network lay-outs also for other species.

Many plants flower in response to seasonal changes in day length. This response often varies between accessions of a single species. We studied the variation in photoperiod response found in the model species Arabidopsis thaliana. Seventy-two accessions were grown under six day lengths varying in 2 h intervals from 6 h to 16 h. The typical response was sigmoidal so that plants flowered early under days longer than 14 h, late under days shorter than 10 h and at intermediate times under 12 h days. However, many accessions diverged from this pattern and were clustered into groups showing related phenotypes. Thirty-one mutants and transgenic lines were also scored under the same conditions. Statistical comparisons demonstrated that some accessions show stronger responses to different day lengths than are found among the mutants. Genetic analysis of two such accessions demonstrated that different quantitative trait loci conferred an enhanced response to shortening the day length from 16 h to 14 h. Our data illustrate the spectrum of day-length response phenotypes present in accessions of Arabidopsis and demonstrate that similar phenotypic variation in photoperiodic response can be conferred by different combinations of loci.

The viral genome-linked protein, VPg, of potyviruses is a multifunctional protein involved in viral genome translation and replication. Previous studies have shown that both eIF4E and eIF4G or their respective isoforms from the eIF4F complex, which modulates the initiation of protein translation, selectively interact with VPg and are required for potyvirus infection. Here, we report the identification of two DEAD-box RNA helicase-like proteins, PpDDXL and AtRH8 from Prunus persica and Arabidopsis thaliana, respectively, both interacting with VPg. We show that AtRH8 is dispensable for plant growth and development but necessary for potyvirus infection. In potyvirus-infected Nicotiana benthamiana leaf tissues, AtRH8 colocalizes with the chloroplast-bound virus accumulation vesicles, suggesting a possible role of AtRH8 in viral genome translation and replication. Deletion analyses of AtRH8 have identified the VPg-binding region. Comparison of this region and the corresponding region of PpDDXL suggests that they are highly conserved and share the same secondary structure. Moreover, overexpression of the VPg-binding region from either AtRH8 or PpDDXL suppresses potyvirus accumulation in infected N. benthamiana leaf tissues. Taken together these data demonstrate that AtRH8, interacting with VPg, is a host factor required for the potyvirus infection process and both AtRH8 and PpDDXL may be manipulated for the development of genetic resistance against potyvirus infections.

Rice is the staple food for over half the world's population yet may represent a significant dietary source of inorganic arsenic (As), a non-threshold, class 1 human carcinogen. Rice grain As is dominated by the inorganic species, and the organic species dimethylarsinic acid (DMA). To investigate how As species are unloaded into grain rice, panicles were excised during grain filling and hydroponically pulsed with arsenite, arsenate, glutathione complexed As (As(GS)₃), or DMA. Total As concentrations in flag leaf, grain and husk, were quantified by ICP-MS and As speciation in the fresh grain was determined by X-ray absorption near edge spectroscopy (XANES). The roles of phloem and xylem transport were investigated by applying a ± stem girdling treatment to a second set of panicles, limiting phloem transport to the grain in panicles pulsed with arsenite or DMA. The results demonstrate that DMA is translocated to the rice grain with over an order magnitude greater efficiency than inorganic species and is more mobile than arsenite in both the phloem and the xylem. Phloem transport accounted for 90% of arsenite, and 55% of DMA, transport to the grain. Synchrotron X-ray fluorescence (S-XRF) mapping and fluorescence microtomography revealed marked differences in the pattern of As unloading into the grain between DMA and arsenite challenged grain. Arsenite was retained in the ovular vascular trace and DMA dispersed throughout the external grain parts and into the endosperm. This study also demonstrates that DMA speciation is altered in planta, potentially through complexation with thiols.

While interspecific variation in the temperature response of photosynthesis is well documented, the underlying physiological mechanisms remain unknown. Moreover, mechanisms related to species-dependent differences in photosynthetic temperature acclimation are unclear. We compared photosynthetic temperature acclimation in 11 crop species differing in their cold tolerance, which were grown at 15°C or 30°C. Cold tolerant species exhibited a large decrease in optimum temperature for the photosynthetic rate at 360 µL L^-1 CO₂ concentrations (Opt (A₃₆₀ )) when growth temperature decreased from 30°C to 15°C, whereas cold sensitive species were less plastic in Opt (A₃₆₀ ). Analysis using the C₃ photosynthesis model shows that the limiting step of A₃₆₀ at the optimum temperature differed between cold tolerant and cold sensitive species; RuBP carboxylation rate was limiting in cold tolerant species, while RuBP regeneration rate was limiting in cold sensitive species. Alterations in parameters related to photosynthetic temperature acclimation, including the limiting step of A₃₆₀ , leaf nitrogen and Rubisco contents, were more plastic to growth temperature in cold tolerant species than cold sensitive species. These plastic alterations contributed to the noted growth temperature dependant changes in Opt (A₃₆₀ ) in cold tolerant species. Consequently, cold tolerant species were able to maintain high A₃₆₀ at their growth temperature at 15°C or 30°C, whereas cold sensitive species were not. We conclude that differences in the plasticity of photosynthetic parameters with respect to growth temperature were responsible for the noted interspecific differences in photosynthetic temperature acclimation between cold tolerant and cold sensitive species.

We identified an Arabidopsis thaliana EMS mutant, modified vacuole phenotype1-1 (mvp1-1), in a fluorescent confocal microscopy screen for plants with mislocalization of a GFP- tonoplast intrinsic protein fusion. The mvp1-1 mutant displayed static perinuclear aggregates of the reporter protein. mvp1 mutants also exhibited a number of vacuole-related phenotypes, as demonstrated by defects in growth, utilization of stored carbon, gravitropic response, salt sensitivity and specific susceptibility to the fungal necrotroph Alternaria brassicicola. Similarly, crosses with other endomembrane marker fusions identified mislocalization to aggregate structures, indicating a general defect in protein trafficking. Map-based cloning showed that the mvp1-1 mutation altered a gene encoding a putative myrosinase-associated protein, and GST-pulldown assays demonstrated that MVP1 interacted specifically with the Arabidopsis myrosinase protein, TGG2, but not TGG1. Moreover, the mvp1-1 mutant showed increased nitrile production during glucosinolate hydrolysis, suggesting that MVP1 may play a role in modulation of myrosinase activity. We propose that MVP1 is a myrosinase-associated protein that functions, in part, to correctly localize the myrosinase TGG2 and prevent inappropriate glucosinolate hydrolysis that could generate cytotoxic molecules.

Stomata arranged in crypts with trichomes are commonly considered to be adaptations to aridity due to the additional diffusion resistance associated with this arrangement; however, information on the effect of crypts on gas exchange, relative to stomata, is sparse. In this study, three-dimensional Finite-Element models of encrypted stomata were generated using commercial Computational Fluid Dynamics software. The models were based on crypt and stomatal architectural characteristics of the species Banksia ilicifolia, examined microscopically, and variations thereof. In leaves with open or partially closed stomata, crypts reduced transpiration by less than 15% compared to non-encrypted, superficially positioned stomata. A larger effect of crypts was found only in models with unrealistically high stomatal conductances. Trichomes inside the crypt had virtually no influence on transpiration. Crypt conductance varied with stomatal conductance, boundary layer conductance and ambient relative humidity, as these factors modified the three-dimensional diffusion patterns inside crypts. It was concluded that it is unlikely that the primary function of crypts and crypt trichomes is to reduce transpiration.

Root exudates influence the surrounding soil microbial community and recent evidence demonstrates the involvement of ABC transporters in root secretion of phytochemicals. In this study, we examined effects of seven Arabidopsis ABC transporter mutants on the microbial community in native soils. After two generations, only the Arabidopsis abcg30 (Atpdr2) mutant had significantly altered both the fungal and bacterial communities compared with the wild type using automated ribosomal intergenic spacer analysis (ARISA). Similarly, root exudate profiles differed between the mutants; however, the largest variance from the wild type (Col-0) was observed in abcg30, which showed increased phenolics and decreased sugars. In support of this biochemical observation, whole genome expression analyses of abcg30 roots revealed that some genes involved in biosynthesis and transport of secondary metabolites were up-regulated while some sugar transporters were down-regulated compared with genome expression in wild type roots. Microbial taxa associated with Col-0 and abcg30 cultured soils determined by pyrosequencing, revealed that exudates from abcg30 cultivated a microbial community with a relatively greater abundance of potentially beneficial bacteria (i.e. PGPRs, nitrogen fixers) and were specifically enriched in bacteria involved in heavy metal remediation. In summary, this is the first report of a single gene mutation from a functional plant mutant influencing the surrounding community of soil organisms, showing that genes are not only important for intrinsic plant physiology but for the interactions with the surrounding community of organisms.

Recent studies have demonstrated the important role of plant miRNAs under nutrient deficiencies. In this study, deep sequencing of Arabidopsis thaliana small RNAs was conducted to reveal microRNAs (miRNAs) and other small RNAs that were differentially expressed in response to phosphate (Pi) deficiency. About 3.5 million sequence reads corresponding to 0.6-1.2 million unique sequence tags from each Pi-sufficient or -deficient root or shoot sample were mapped to the Arabidopsis genome. We showed that upon Pi deprivation, the expression of miR156, miR399, miR778, miR827 and miR2111 was induced, whereas the expression of miR169, miR395 and miR398 was repressed. We found crosstalks coordinated by these miRNAs under different nutrient deficiencies. In addition to miRNAs, we identified one Pi starvation-induced DCL1-dependent small RNA derived from the long terminal repeat of a retrotransposon and a group of 19-nucleotide small RNAs corresponding to the 5‘ end of tRNA and expressed at a high level in Pi-starved roots. Importantly, we observed an increased abundance of TAS4-derived trans-acting siRNAs (ta-siRNAs) in Pi-deficient shoots and uncovered an autoregulatory mechanism of PAP1/MYB75 via miR828 and TAS4-siR81(-) that regulates the biosynthesis of anthocyanin. This finding sheds light on the regulatory network between miRNA/ta-siRNA and its target gene. Of note, a substantial amount of miR399* accumulated under Pi deficiency. Like miR399, miR399* can move across the graft junction, implying a potential biological role for miR399*. This study represents a comprehensive expression profiling of Pi-responsive small RNAs and advances our understanding of the regulation of Pi homeostasis mediated by small RNAs.

The early phase of the interaction between tree roots and ectomycorrhizal (ECM) fungi, prior to symbiosis establishment, is accompanied by a stimulation of lateral root (LR) development. We aimed to identify gene networks that regulate LR development during the early signal exchanges between Populus tremula x Populus alba and the ECM fungus Laccaria bicolor with a focus on auxin transport and signaling pathways. Our data demonstrated that increased LR development in poplar and Arabidopsis thaliana interacting with L. bicolor is not dependent on the ability of the plant to form ectomycorrhizae. LR stimulation paralleled an increase in auxin accumulation at root apices. Blocking plant polar auxin transport with 1-naphthylphthalamic acid (NPA) inhibited LR development and auxin accumulation. An oligoarray-based transcript profile of poplar roots exposed to molecules released by L. bicolor revealed the differential expression of 2945 genes, including several components of polar auxin transport (PtaPIN and PtaAUX genes), auxin conjugation (PtaGH3) and auxin signaling (PtaIAA). Transcripts of PtaPIN9, the homolog of Arabidopsis AtPIN2, and several PtaIAAs accumulated specifically during the early interaction phase. Expression of these rapidly induced genes was repressed by NPA. Accordingly, LR stimulation upon contact with L. bicolor in Arabidopsis transgenic plants defective in homologs of these genes was decreased or absent. Furthermore, in Arabidopsis pin2 the root apical auxin increase during contact with the fungus was modified. We propose a model in which fungus-induced auxin accumulation at the root apex stimulates LR formation through a mechanism involving PtaPIN9-dependent auxin redistribution together with PtaIAA-based auxin-signaling.

Zea mays (maize) leaves provide a useful system to study how proximal/distal patterning is established because of the distinct tissues found in the distal blade and the proximal sheath. Several mutants disrupt this pattern including the dominant knotted1-like homeobox (knox) mutants. knox genes encode homeodomain proteins of the TALE superclass of transcription factors. Class I knox genes are expressed in the meristem and down-regulated as leaves initiate. Gain-of-function phenotypes result from misexpression in leaves. We identified a new dominant allele of maize knotted1, Kn1-DL, which contains a transposon insertion in the promoter in addition to a tandem duplication of the kn1 locus. In situ hybridization shows that kn1 is misexpressed in two different parts of the blade that correlate with the different phenotypes observed. When kn1 is misexpressed along the margins, flaps of sheath-like tissue form along the margins. Expression in the distal tip leads to premature termination of the midrib into a knot and leaf bifurcation. The gain-of-function phenotypes suggest that kn1 establishes proximal/distal patterning when expressed in distal locations and lead to the hypothesis that kn1 normally participates in the establishment of proximal/distal polarity in the incipient leaf.

Widely conserved among eukaryotes, the microtubule-associated protein 215 (MAP215) family enhances microtubule dynamic instability. The family member studied most extensively, Xenopus laevis XMAP215, has been reported to enhance both assembly and disassembly parameters, although the mechanism whereby one protein can exert these apparently contradictory effects has not been clarified. Here, we analyze the activity of a plant MAP215 homolog, tobacco MAP200 on microtubule behavior in vitro. We show that, like XMAP215, MAP200 promotes both assembly and disassembly parameters, including microtubule growth rate and catastrophe frequency. When MAP200 is added to tubulin and taxol, strikingly long coiled structures form. When GDP partially replaces GTP, the increase of catastrophe frequency by MAP200 is strongly diminished, even though this replacement stimulates catastrophe in the absence of MAP200. This implies that MAP200 induces catastrophes by a specific, GTP-requiring pathway. We hypothesize that, in the presence of MAP200, a catastrophe-prone microtubule lattice forms occasionally when elongated but non-adjacent protofilaments make lateral contacts.

One of the key factors that defines plant form is the regulation of when and where branches develop. The diversity of form observed in nature results, in part, from variation in the regulation of branching between species. Two CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes CCD7 and CCD8 are required for the production of a branch suppressing plant hormone. Here we report that the dad3 mutant of Petunia hybrida results from the mutation of the PhCCD7 gene and has a less severe branching phenotype than mutation of PhCCD8 (dad1). An analysis of the expression of this gene in wild-type, mutant and grafted petunia suggests that in petunia CCD7 and CCD8 are co-ordinately regulated. In contrast to observations in Arabidopsis thaliana, ccd7ccd8 double mutants in petunia show an additive phenotype. An analysis using dad3 or dad1 mutant scions grafted to wild-type rootstocks showed that when these plants produce adventitious mutant roots branching is increased above that seen in plants where the mutant roots are removed. The results presented here indicate that mutation of either CCD7 or CCD8 in petunia results in both the loss of an inhibitor of branching and an increase in a promoter of branching.

Phosphatidylinositol 3,5 bisphosphate (PtdIns(3,5)P₂) is a phospholipid that has a role in controlling membrane trafficking events in yeast and animal cells. The function of this lipid in plants is unknown although its synthesis has been shown to be upregulated upon osmotic stress in plant cells. PtdIns(3,5)P₂ is synthesised by the PIKfyve/Fab1 family of proteins with two orthologues, FAB1A and FAB1B, being present in Arabidopsis thaliana. In this study we attempt to address the role of this lipid by analysing the phenotypes of plants mutated in FAB1A and FAB1B. It was not possible to generate plants homozygous for mutations in both genes though single mutants were isolated. Both homozygous single mutant plant lines exhibited a leaf curl phenotype which was more marked in FAB1B mutants. Genetic transmission analysis revealed that failure to generate double mutant lines was entirely due to inviability of pollen carrying mutant alleles of both FAB1A and FAB1B. This pollen displayed severe defects in vacuolar reorganisation following the first mitotic division of development. The presence of abnormally large vacuoles in pollen at the tricellular stage resulted in the collapse of the majority of grains carrying both mutant alleles. This demonstrates a crucial role for PtdIns(3,5)P₂ in modulating the dynamics of vacuolar rearrangement essential for successful pollen development. Taken together our results are consistent with PtdIns(3,5)P₂ production being central to cellular responses to changes in osmotic conditions.

Transcription factors of the DREB1/CBF family specifically interact with a cis-acting dehydration-responsive element/C-repeat (DRE/CRT) involved in low-temperature stress–responsive gene expression in Arabidopsis. Expression of DREB1s is induced by low temperatures and is regulated by the circadian clock under unstressed conditions. Promoter sequences of DREB1s contain six conserved motifs, Boxes I to VI. We analyzed the promoter region of DREB1C using transgenic plants and found that Box V with the G-box sequence negatively regulates DREB1C expression under circadian control. The region around Box VI contains positive regulatory elements for low-temperature-induced expression of DREB1C. Using yeast one-hybrid screens, we isolated cDNA encoding the transcriptional factor PIF7, which specifically binds to the G-box of the DREB1C promoter. The PIF7 gene was expressed in rosette leaves, and the PIF7 protein was localized in the nuclei of the cells. Transactivation experiments using Arabidopsis protoplasts indicated that PIF7 functions as a transcriptional repressor for DREB1C expression and that its activity is regulated by PIF7-interacting factors TOC1 and PhyB, which are components of the circadian oscillator and the red light photoreceptor, respectively. Moreover, in the pif7 mutant, expression of DREB1B and DREB1C was not repressed under light conditions, indicating that PIF7 functions as a transcriptional repressor for the expression of DREB1B and DREB1C under circadian control. This negative regulation of DREB1 expression may be important for avoiding plant growth retardation by the accumulation of DREB1 proteins under unstressed conditions.

The activation of Rubisco in vivo requires the presence of the regulatory protein Rubisco activase. To elucidate its role in maintaining CO₂ assimilation rate at high temperature, we examined the temperature response of CO₂ assimilation rate at 380 µL L^-1 CO₂ concentration (A₃₈₀) and Rubisco activation state in wild type and transgenic tobacco with reduced Rubisco activase content grown at either 20°C or 30°C. Analyses of gas-exchange and chlorophyll fluorescence showed that in wild type A₃₈₀ was limited by RuBP regeneration at lower temperatures, whereas at higher temperatures, by RuBP carboxylation irrespective of growth temperatures. Growth temperature induced modest differences on Rubisco activation state which declined with measuring temperature from mean values of 76% at 15°C to 63% at 40 °C in wild type plants. At measuring temperatures of 25°C and below, an 80% reduction in Rubisco activase content was required before Rubisco activation state was decreased. Above 35°C Rubisco activation state decreased slightly with more modest decreases in Rubisco activase content, but the extent of the reductions in Rubisco activation state were small such that a 55% reduction in Rubisco activase content did not alter the temperature sensitivity of Rubisco activation and had no effect on in vivo catalytic turnover rates of Rubisco. There was a strong correlation between Rubisco activase content and Rubisco activation state once Rubisco activase content was less that 20% of wild type at all measuring temperatures. We conclude that reduction in Rubisco activase content does not lead to an increase in the temperature sensitivity of Rubisco activation state in tobacco.

The consequences of altered ABA sensitivity in grey poplar development (Populus x canescens (Ait.) Sm.) were examined by ectopic expression of Arabidopsis mutant abi1 gene. The expression resulted in an ABA-insensitive phenotype revealed by a strong tendency of abi1 poplars to wilt, impaired responsiveness of their stomata to ABA, and an ABA-resistant bud outgrowth. These plants therefore required cultivation under very humid conditions to prevent drought stress symptoms. Morphological alterations became evident when comparing abi1 poplars with poplars expressing Arabidopsis non-mutant ABI1 or wild type (wt) plants. abi1 poplars showed increased stomatal size, enhanced shoot growth and retarded leaf and root development. The increased stomatal size and its reversion to the size of wt plants by exogenous ABA indicate a role for ABA in regulating stomatal development. Enhanced shoot growth and retarded leaf and root development support the hypothesis that ABA acts independently from drought stress as a negative regulator of growth in shoots and as a positive regulator of growth in leaves and roots. In shoots, we observed an interaction of ABA with ethylene: abi1 poplars exhibited elevated ethylene production, and the ethylene perception inhibitor Ag⁺ antagonised the enhanced shoot growth. Thus, we provide evidence that ABA acts as negative regulator of shoot growth in non-stressed poplars by restricting ethylene production. Furthermore, we show that ABA has a role in regulating shoot branching by inhibiting lateral bud outgrowth.

SIZ1 encodes the sole ortholog of mammalian PIAS and yeast SIZ SUMO E3 ligases in Arabidopsis (Arabidopsis thaliana). Four conserved motifs in SIZ1 include SAP (Scaffold attachment factor A/B//acinus/PIAS domain), PINIT, SP-RING (SIZ/PIAS-RING) and SXS motifs. SIZ1 contains, in addition, a PHD (Plant Homeodomain) typical of plant PIAS proteins. We determined phenotypes of siz1-2 knockout mutants transformed with SIZ1 alleles carrying point mutations in the predicted domains. Domain SP-RING is required for SUMO conjugation activity and nuclear localization of SIZ1. SA accumulation and SA-dependent phenotypes of siz1-2, such as diminished plant size, heightened innate immunity and ABA inhibition of cotyledon greening, as well as SA-independent basal thermotolerance were not complemented by the altered SP-RING allele of SIZ1. The SXS domain also controlled SA accumulation and was involved in greening and expansion of cotyledons of seedlings germinated in the presence of ABA. Mutations of the PHD zinc finger domain and the PINIT motif affected in vivo SUMOylation. Expression of the PHD and/or PINIT domain mutant alleles of SIZ1 in siz1-2 promoted hypocotyl elongation in response to sugar and light. The various domains of SIZ1 make unique contributions to the plant's ability to cope with its environment.

Storage protein activator (SPA) is a key regulator of the transcription of wheat (Triticum aestivum L.) grain storage protein (GSP) genes and belongs to the Opaque2 transcription factor subfamily. We analyzed the sequence polymorphism of the three homoeologous Spa genes in hexaploid wheat.). The level of polymorphism in these genes was high particularly in the promoter. The deduced protein sequences of each homoeolog and haplotype show >93% identity. Two major haplotypes were studied for each Spa gene. The three Spa homoeologs have similar patterns of expression during grain development with a peak in expression around 300 degree days after anthesis. On average, Spa-B is 10 and 7 times more strongly expressed than Spa-A and Spa-D, respectively. The haplotypes are associated with significant quantitative differences in Spa expression, especially for Spa-A and Spa-D. Significant differences were found in the quantity of total grain nitrogen allocated to the gliadin protein fractions for the Spa-A haplotypes, whereas the synthesis of glutenins is not modified. Genetic association analysis between Spa and dough viscoelasticity revealed that Spa polymorphisms are associated with dough tenacity, extensibility, and strength. Except for Spa-A, these associations can be explained by differences in grain hardness. No association was found between Spa markers and the average single grain dry mass or grain protein concentration. These results demonstrate in planta Spa is involved in the regulation of GSP synthesis. The associations between Spa and dough viscoelasticity and grain hardness strongly suggest Spa has complex pleiotropic functions during grain development.

The cuticle covering every plant aerial organ is largely made of cutin that consists of fatty acids, glycerol and aromatic monomers. Despite the huge importance of the cuticle to plant development and fitness our knowledge regarding the assembly of the cutin polymer and its integration in the complete cuticle structure is limited. Cutin composition implies the action of acyltransferase-type enzymes that mediate polymer construction through ester bond formation. Here we show that a member of the BAHD family of acyltransferases (DCR) is required for incorporation of the most abundant monomer into the polymeric structure of the Arabidopsis flower cutin. DCR deficient plants display phenotypes that are typically associated with a defective cuticle including altered epidermal cell differentiation and post-genital organ fusion. Moreover, levels of the major cutin monomer in flowers, 9(10), 16-dihydroxy-hexadecanoic acid, decreased to almost undetectable amount in the mutants. Interestingly, dcr mutants exhibit changes in the decoration of petal conical cells and mucilage extrusion in the seed coat, both phenotypes formerly not associated with cutin polymer assembly. Excessive root branching displayed by dcr mutants and DCR expression pattern in roots pointed to the function of DCR below ground, in shaping root architecture by influencing the lateral roots emergence and growth. In addition, the dcr mutants were more susceptible to salinity, osmotic and water deprivation stress conditions. Finally, the analysis of the DCR protein localization suggested that cutin polymerization, possibly the oligomerization step, is partially carried out in the cytoplasmic space. This study therefore extends our knowledge regarding the functionality of the cuticular layer and the formation of its major constituent the polymer cutin.

The information and resources generated from diverse ‘omics’ technologies provide opportunities for producing novel biological knowledge. It is essential to integrate various kinds of biological information and large-scale ‘omics’ datasets through systematic analysis in order to describe and understand complex biological phenomena. For this purpose, we have developed a web-based system, Plant MetGenMAP, which can comprehensively integrate and analyze large-scale gene expression and metabolite profile datasets along with diverse biological information. Using this system, significantly altered biochemical pathways and biological processes under given conditions can be retrieved rapidly and efficiently, and transcriptional events and/or metabolic changes in a pathway can be easily visualized. In addition, the system provides a unique function that can identify candidate promoter motifs associated with regulation of specific biochemical pathways. We demonstrate the functions and application of the system using datasets from Arabidopsis and tomato, respectively. The results obtained by Plant MetGenMAP can aid in a better understanding of the mechanisms that underlie interesting biological phenomena and provide novel insights into the biochemical changes associated with them at the gene and metabolite levels. Plant MetGenMAP is freely available at http://bioinfo.bti.cornell.edu/tool/MetGenMAP.

Arabidopsis plants subjected to water deficit, sodium chloride (NaCl), or abscisic acid (ABA) treatments were shown to exhibit a significant increase in the amount of leaf cuticular lipids. These stress treatments led to increases in cuticular wax amount per unit area of 32% to 80%, due primarily to 29% to 98% increases in wax alkanes. Of these treatments, only water deficit increased the total cutin monomer amount (by 65%), whereas both water deficit and NaCl altered the proportional amounts of cutin monomers. ABA had little effect on cutin composition. Water deficit, but not NaCl, increased leaf cuticle thickness (by 49%). Electron micrographs revealed that both water deprived and NaCl-treated plants had elevated osmium accumulation in their cuticles. The abundance of cuticle-associated gene transcripts in leaves was altered by all treatments; including those performed in both pot-grown and in vitro conditions. Notably, the abundance of the CER1 gene transcript, predicted to function in alkane synthesis, was highly induced by all treatments, results consistent with the elevated alkane amounts observed in all treatments. Further, this induction of cuticle lipids was associated with reduced cuticle permeability and may be important for plant acclimation to subsequent water-limited conditions. Taken together, these results show that Arabidopsis provides an excellent model system to study the role of the cuticle in plant response to drought and related stresses, and its associated genetic and cellular regulation.

Winter cress (Barbarea vulgaris) is resistant to a range of insect species. Some B. vulgaris genotypes are resistant whereas others are susceptible to herbivory by flea beetle larvae (Phyllotreta nemorum). Metabolites involved in resistance to herbivory by flea beetles were identified using an ecometabolomic approach.

An F2 population representing the whole range from full susceptibility to full resistance to flea beetle larvae was generated by a cross between a susceptible and a resistant B. vulgaris plant. This F2 offspring was evaluated with a bioassay measuring the ability of susceptible flea beetle larvae to survive on each plant. Metabolites that correlated negatively with larvae survival were identified through correlation, cluster and principal component analyses. Two main clusters of metabolites that correlate negatively with larvae survival were identified. Principal component analysis grouped resistant and susceptible plants as well as correlated metabolites. Known saponins, such as hederagenin cellobioside and oleanolic acid cellobioside, as well as two other saponins correlated significantly with plant resistance.

This study shows the potential of metabolomics to identify bioactive compounds involved in plant defense.

We had earlier reported that mutations to G and C at the 7^th and 8^th positions in the prototype TATA-box TCACTATATATAG inhibited light dependent activation of transcription from the promoter. In the present study we characterized mutations at the 9^th position of the prototype TATA-box. Substitution of T at the 9^th position with G or C enhanced transcription from the promoter in transgenic tobacco plants. The effect of T9G/C mutations was not light dependent though the 9G/C TATA-box showed synergy with the light responsive element (lre). However, the 9G/C mutants in the presence of lre failed to respond to phytochromes, sugar and calcium signaling in contrast to the prototype TATA-box with lre. The 9G/C mutation shifted the point of initiation of transcription and transcription activation was dependent upon the type of activating element present upstream. The synergy in activation was noticed with lre and legumin activators but not with rbcS, Pcec and PR-1a activators. The 9G mutation resulted in a micrococcal nuclease (MNase) sensitive region over TATA-box suggesting nucleosome free region in contrast to the prototype promoter which had a distinct nucleosome on the TATA-box. Thus, the transcriptional augmentation with mutation at the 9^th position might be because of the loss of a repressive nucleosomal structure on the TATA-box. In agreement with our findings, the promoters containing TATAGATA as identified by genome wide analysis of the Arabidopsis thaliana are not tightly repressed.

A cytoplasmically inherited chlorophyll-deficient mutant of barley (Hordeum vulgare L) termed CL3, displaying a viridis (homogeneously light-green colored) phenotype has been previously shown to be affected by elevated temperatures. In the present paper, biochemical, biophysical and molecular approaches were used to study the CL3 mutant under different temperature and light conditions. The results lead to the conclusion that an impaired assembly of photosystem I under higher temperatures and certain light conditions is the primary cause of the CL3 phenotype. Compromised splicing of ycf3 transcripts, particularly at elevated temperature, resulting from a mutation in a noncoding region (intron 1) in the mutant ycf3 gene results in a defective synthesis of Ycf3, which is a chaperone involved in PSI assembly. The defective PSI assembly causes severe photoinhibition and degradation of PSII.

As a genetic platform, tomato (Solanum lycopersicum) benefits from rich germplasm collection, ease of cultivation and transformation, and enables the analysis of biological processes impossible to investigate with other model species. To facilitate the assembly of an open genetic toolbox designed to study Solanaceae, we initiated a joint collection of publicly available gene manipulation tools. We focused on the characterization of promoters expressed at defined time windows during fruit development, for the regulated expression or silencing of genes of interest. Five promoter sequences were captured as entry clones compatible with the versatile MultiSite Gateway format: PPC2, PG, TPRP, and IMA from tomato, and CRC from Arabidopsis thaliana. Corresponding transcriptional fusions were made with the GUS gene, a nuclear localized GUS-GFP reporter, and the chimeric LhG4 transcription factor. The activity of the promoters during fruit development and in fruit tissues were confirmed in transgenic tomato lines. Novel Gateway destination vectors were generated for the transcription of artificial microRNA (amiRNA) precursors and hairpin RNAs under the control of these promoters, with schemes only involving Gateway BP and LR clonase reactions. Efficient silencing of the endogenous phytoene desaturase (PDS) gene was demonstrated in transgenic tomato lines producing a matching amiRNA under the CaMV 35S or PPC2 promoter. Lastly, taking advantage of the pOP/LhG4 two-component system, we found that well characterized flower-specific Arabidopsis promoters drive the expression of reporters in patterns generally compatible with heterologous expression. Tomato lines and plasmids will be distributed through a new NASC service unit dedicated to Solanaceae resources.

Herbivore-induced plant volatiles affect the systemic response of plants to local damage and hence represent potential plant hormones. These signals can also lead to ‘plant-plant communication’, a defense induction in yet undamaged plants growing close to damaged neighbors. We observed this phenomenon in the context of disease resistance. Lima bean (Phaseolus lunatus) plants in a natural population became more resistant against a bacterial pathogen, Pseudomonas syringae pv. syringae, when located close to conspecific neighbors in which systemic acquired resistance (SAR) to pathogens had been chemically induced with benzothiadiazole (BTH). Airborne disease resistance induction could also be triggered biologically by infection with avirulent P. syringae. Challenge inoculation after exposure to induced and non-induced plants revealed that the air coming from induced plants mainly primed resistance, since expression of pathogenesis-related protein 2 (PR-2) was significantly stronger in exposed than in non-exposed individuals when the plants were subsequently challenged by P. syringae. Among others, the plant-derived volatile, nonanal, was present in the headspace of BTH-treated plants and significantly enhanced PR-2 expression in the exposed plants, resulting in reduced symptom appearance. Negative effects on growth of BTH-treated plants, which usually occur as a consequence of high costs of direct resistance induction, were not observed in VOC-exposed plants. Volatile-mediated priming appears to be a highly attractive means for the tailoring of SAR against plant pathogens.

Ectomycorrhizas (EMs) alleviate stress tolerance of host plants, but the underlying molecular mechanisms are unknown. To elucidate the basis of EM-induced physiological changes and their involvement in stress adaptation, we investigated metabolic and transcriptional profiles in EM and non-EM roots of grey poplar (Populus x canescens) in the presence and abscence of osmotic stress imposed by excess salinity. Colonization with the ectomycorhizal fungus Paxillus involutus increased root cell volumes, a response associated with carbohydrate accumulation. Stress related hormones abscisic acid and salicylic acid (ABA, SA) were increased, whereas jasmonic acid and auxin were decreased in EM compared with non-EM roots. GH3::GUS-reporter plants showed that auxin decreased in the vascular system. The phytohormone changes in EMs are in contrast to those in arbuscular mycorrhizas (AMs) suggesting that EMs and AMs recruit different signaling pathways to influence plant stress responses. Transcriptome analyses on a whole genome poplar microarray revealed activation of genes related to abiotic and biotic stress responses as well as of genes involved in vesicle trafficking and suppression of auxin related pathways. Comparative transcriptome analysis indicated EM-related genes whose transcripts abundances were independent of salt stress and a set of salt-stress-related genes that were common to EM non-salt-stressed and non-EM salt-stressed plants. Salt-exposed EM roots showed stronger accumulation of myo-inositol, ABA and SA and higher K⁺ to Na⁺ ratio than stressed non-EM roots. In conclusion, EMs activated stress-related genes and signaling pathways apparently leading to priming of pathways conferring abiotic stress tolerance.

Replication protein A (RPA), a highly conserved single-stranded DNA-binding protein in eukaryotes, is a stable complex comprising three subunits termed RPA1, RPA2 and RPA3. RPA is required for multiple processes in DNA metabolism such as replication, repair and homologous recombination in yeast and human. Most eukaryotic organisms, including fungi, insects and vertebrates, have only a single RPA gene that encodes each RPA subunits. Arabidopsis and rice, however, possess multiple copies of an RPA gene. Rice has three paralogs each of RPA1 and RPA2, and one for RPA3. Previous studies have established their biochemical interactions in vitro and in vivo, but little is known about their exact function in rice. We examined the function of OsRPA1a in rice using a T-DNA insertional mutant. The osrpa1a mutants had a normal phenotype during vegetative growth but were sterile at the reproductive stage. Cytological examination confirmed that no embryo sac formed in female meiocytes and that abnormal chromosomal fragmentation occurred in male meiocytes after anaphase I. Compared with wild type, the osrpa1a mutant showed no visible defects in mitosis and chromosome pairing and synapsis during meiosis. In addition, the osrpa1a mutant was hypersensitive to UV-C irradiation and the DNA-damaging agents mitomycin C (MMC) and methyl methanesulfonate (MMS). Thus, our data suggest that OsRPA1a plays an essential role in DNA repair but may not participate in, or at least is dispensable for, DNA replication and homologous recombination in rice.

Plant roots and microorganisms interact and compete for nutrients within the rhizosphere, which is considered one of the most biologically complex systems on Earth. Unravelling the nitrogen (N) cycle is key to understanding and managing nutrient flows in terrestrial ecosystems, yet to date it has proved impossible to analyze and image N transfer in situ within such a complex system at a scale relevant to soil-microbe-plant interactions. Linking the physical heterogeneity of soil to biological processes marks a current frontier in plant and soil sciences. Here we present a new and widely applicable approach that allows imaging of the spatial and temporal dynamics of the stable isotope ¹⁵N assimilated within the rhizosphere. This approach allows visualization and measurement of nutrient resource capture between competing plant cells and microorganisms. For confirmation we show the correlative use of nanoscale secondary ion mass spectrometry (NanoSIMS), and transmission electron microscopy (TEM), to image differential partitioning of ¹⁵NH₄⁺ between plant roots and native soil microbial communities at the sub-micron scale. It is shown that ¹⁵N compounds can be detected and imaged in situ in individual microorganisms in the soil matrix and intracellularly within the root. NanoSIMS has potential to allow the study of assimilatory processes at the sub-micron level in a wide range of applications involving plants, microorganisms and animals.

Genetic analysis of interspecific populations derived from crosses between the wild tomato species Solanum habrochaites f glabratum, which synthesizes and accumulates insecticidal methylketones (MK), mostly 2-undecanone and 2-tridecanone, in glandular trichomes, and Solanum lycopersicum (cultivated tomato), which does not, demonstrated that several genetic loci contribute to MK metabolism in the wild species. A strong correlation was found between the shape of the glandular trichomes and their MK content, and significant associations were seen between allelic states of three genes and the amount of MK produced by the plant. Two genes belong to the fatty acid biosynthetic pathway and the third is the previously identified Methylketone Synthase 1 (MKS1) that mediates divergence to MK from β-ketoacyl intermediates. Comparative transcriptome analysis of the glandular trichomes of F2 progeny grouped into low- and high-MK- containing plants identified several additional genes whose transcripts were either more or less abundant in the high-MK bulk. In particular, a wild-species specific transcript for a gene which we named Methylketone Synthase 2 (MKS2), encoding a protein with some similarity to a well-characterized bacterial thioesterase, was approximately 300-fold more highly expressed in F2 plants with high MK content than in those with low MK content. Genetic analysis in the segregating population showed that MKS2's significant contribution to MK accumulation is mediated by an epistatic relationship with MKS1. Furthermore, heterologous expression of MKS2 in Escherichia coli resulted in the production of methylketones in this host.

In growing plant cells the combined activities of the cytoskeleton, endomembrane, and cell wall biosynthetic systems organize the cytoplasm and define the architecture and growth properties of the cell. These biosynthetic machineries efficiently synthesize, deliver, and recycle the raw materials that support cell expansion. The precise roles of the actin cytoskeleton in these processes are unclear. Certainly bundles of actin filaments position organelles and are a substrate for long distance intracellular transport, but the functional linkages between dynamic actin filament arrays and the cell growth machinery are poorly understood. The Arabidopsis "distorted group" mutants have defined protein complexes that appear to generate and convert small GTPase signals into an actin-related protein (ARP) 2/3-dependent actin filament nucleation response. However, direct biochemical knowledge about Arabidopsis ARP2/3 and its cellular distribution is lacking. In this paper we provide biochemical evidence for a plant ARP2/3. The plant complex utilizes a conserved assembly mechanism. ARPC4 is the most critical core subunit that controls the assembly and steady state levels of the complex. ARP2/3 in other systems is believed to be mostly a soluble complex that is locally recruited and activated. Unexpectedly, we find that Arabidopsis ARP2/3 interacts strongly with cell membranes. Membrane-binding is linked to complex assembly status and not to the extent to which it is activated. Mutant analyses implicate ARP2 as an important subunit for membrane association.

Unlike mammals whose development is limited to a short temporal window, plants produce organs de novo throughout their lifetime in order to adapt their architecture to the prevailing environmental conditions. The production of lateral roots represents one example of such post-embryonic organogenesis. An endogenous developmental program likely imposes an ordered arrangement on the position of new lateral roots. However, environmental stimuli such as nutrient levels also affect the patterning of lateral root production. In addition, we have found that mechanical forces can act as one of the triggers that entrain lateral root production to the environment of the plant. We observed that physical bending of the root recruited new lateral root formation to the convex side of the resultant bend. Transient bending of 20 s was sufficient to elicit this developmental program. Such bending triggered a Ca²⁺ transient within the pericycle, and blocking this change in Ca²⁺ also blocked recruitment of new lateral root production to the curved region of the root. The initial establishment of the mechanically induced LR primordium was independent of an auxin supply from the shoot and was not disrupted by mutants in a suite of auxin transporters and receptor/response elements. These results suggest that Ca²⁺ may be acting to translate the mechanical forces inherent in growth to a developmental response in roots.

Expansins are cell wall proteins associated with the process of plant growth. However, investigations in which expansin gene expression has been manipulated throughout the plant have often led to inconclusive results. In this paper, we report on a series of experiments in which overexpression of expansin was targeted to specific phases of leaf growth using an inducible promoter system. The data indicate that there is a restricted window of sensitivity when increased expansin gene expression leads to increased endogenous expansin activity and an increase in leaf growth. This phase of maximum expansin efficacy corresponds to the mid-phase of leaf growth. We propose that the effectiveness of expansin action depends on the presence of other modulating factors in the leaf and we suggest that it is the control of expression of these factors (in conjunction with expansin gene expression) which defines the extent of leaf growth. These data help to explain some of the previously observed variation in growth response following manipulation of expansin gene expression and highlight a potential linkage of the expression of modifiers of expansin activity with the process of exit from cell division.

Retroposition, as an important copy mechanism for generating new genes, was believed to play a negligible role in plants. As a representative dicot, the genomic sequences of Populus (poplar; Populus trichocarpa) provide an opportunity to investigate this issue. We identified 106 retrogenes and found the majority (89%) of them are associated with functional signatures in sequence evolution, transcription, and (or) translation. Remarkably, examination of gene structures revealed extensive structural renovation of these retrogenes: we identified 18 (17%) of them undergoing either chimerization to form new chimerical genes and (or) intronization (transformation into intron sequences of previously exonic sequences) to generate new intron-containing genes. Such a change might occur at a high speed, considering eight out of 18 such cases occurred recently after divergence between Arabidopsis (Arabidopsis thaliana) and Populus. This pattern also exists in Arabidopsis, with 15 intronized retrogenes occurring after the divergence between Arabidopsis and papaya (Carica papaya). Thus, the frequency of intronization in dicots revealed its importance as a mechanism in the evolution of exon-intron structure. In addition, we also examined the potential impact of the Populus nascent sex determination system on the chromosomal distribution of retrogenes and did not observe any significant effects of the extremely young sex chromosomes.

There currently exists a diverse array of molecular probes for the in situ localization of polysaccharides, nucleic acids, and proteins in plant cells, including reporter enzyme strategies (e.g., protein-glucuronidase fusions). In contrast, however, there is a paucity of methods for the direct analysis of endogenous glycoside hydrolases and transglycosidases responsible for cell wall remodeling. To exemplify the potential of fluorogenic resorufin glycosides to address this issue, a resorufin beta-glycoside of a xylogluco-oligosaccharide (XXXG-β-Res), was synthesized as a specific substrate for in planta analysis of xyloglucan endo-hydrolase (XEH) activity. The resorufin aglycone is particularly distinguished for high-sensitivity in muro assays due to a low pK_a (5.8) and large extinction coefficient ( 62 000 M^-1cm^-1), long-wavelength fluorescence (Ex 571 nm/Em 585 nm), and high quantum yield (0.74) of the corresponding anion. In vitro analyses demonstrated that XXXG-β-Res is hydrolyzed by the archetypal plant XEH, nasturtium (Tropaeolum majus L.) NXG1, with classical Michaelis-Menten substrate saturation kinetics and a linear dependence on both enzyme concentration and incubation time. Further, XEH activity could be visualized in real-time by observing the localized increase in fluorescence in germinating nasturtium seeds and Arabidopsis inflorescent stems by confocal microscopy. Importantly, this new in situ XEH assay provides an essential complement to the in situ xyloglucan endo-transglycosylase (XET) assay [Vissenberg et al. (2000) Plant Cell 12:1229-1237], thus allowing delineation of the disparate activities encoded by XTH genes directly in plant tissues. The observation that XXXG-β-Res is also hydrolyzed by diverse microbial XEHs indicates that this substrate, and resorufin glycosides in general, may find broad applicability for the analysis of wall restructuring by polysaccharide hydrolases during morphogenesis and plant-microbe interactions.

Proanthocyanidins (PAs) are secondary metabolites that contribute to the protection of the plant and also to the taste of the fruit, mainly through astringency. Persimmon (Diospyros kaki) is unique in being able to accumulate abundant PAs in the fruit flesh. Fruits of the non-astringent (NA) type mutants lose their ability to produce PA at an early stage of fruit development, while those of the normal astringent (A) type remain rich in PA until fully ripened. The expression of many PA pathway genes was coincidentally terminated in the NA-type at an early stage of fruit development. The five genes encoding the Myb transcription factor were isolated from an A-type cultivar (cv. Kuramitsu). One of them, DkMyb4 showed the expression pattern synchronous to that of the PA pathway genes in A- and NA-type fruit flesh. The ectopic expression of DkMyb4 in kiwifruit induced PA biosynthesis, but not anthocyanin biosynthesis. The suppression of DkMyb4 in persimmon calluses caused a substantial down-regulation of the PA pathway genes and PA biosynthesis. Furthermore, analysis of the DNA-binding ability of DkMyb4 showed that it directly binds to the MYBCORE cis-motif in the promoters of the some PA pathway genes. Our all results indicate that DkMyb4 acts as a regulator of PA biosynthesis in persimmon, and therefore, suggest that the reduction in the DkMyb4 expression causes the NA-type-specific down-regulation of PA biosynthesis and resultant non-astringent trait.

Peroxisomes are unique organelles involved in multiple cellular metabolic pathways. Nitric oxide (NO) is a free radical active in many physiological functions under normal and stress conditions. Using Arabidopsis thaliana wild-type and mutants expressing green fluorescent protein (GFP) through the addition of peroxisomal targeting signal 1 (PTS1), which enables peroxisomes to be visualized in vivo, this study analyzes the temporal and cell distribution of NO during the development of 3, 5, 8, and 11-day-old Arabidopsis seedlings and shows that Arabidopsis peroxisomes accumulate NO in vivo. Pharmacological analyses using nitric oxide synthase (NOS) inhibitors detected the presence of putative calcium-dependent NOS activity. Furthermore, peroxins PEX12 and PEX13 appear to be involved in transporting the putative NOS protein to peroxisomes, since pex12 and pex13 mutants, which are defective in PTS1- and PTS2-dependent protein transport to peroxisomes, registered lower NO content. Additionally, we show that under salinity stress (100 mM NaCl), peroxisomes are required for NO accumulation in the cytosol, thereby participating in the generation of peroxynitrite (ONOO^-) and in increasing protein tyrosine nitration which is a marker of nitrosative stress.

In order to elucidate transcriptional and metabolic networks associated with Lys metabolism, we utilized developing seeds as a system in which Lys synthesis could be stimulated developmentally without application of chemicals and coupled this to a T-DNA insertion knockout mutation impaired in Lys catabolism. This seed-specific metabolic perturbation stimulated Lys accumulation starting from the initiation of storage reserve accumulation. Our results revealed that the response of seed metabolism to the inducible alteration of Lys metabolism was relatively minor, however, that which was observable operated in a modular manner. They also demonstrated that Lys metabolism is strongly associated with the operation of the TCA cycle, whilst largely disconnected from other metabolic networks. In contrast, the inducible alteration of Lys metabolism was strongly associated with gene networks, stimulating the expression of hundreds of genes controlling anabolic processes that are associated with plant performance and vigor, whilst suppressing a small number of genes associated with plant stress interactions. The most pronounced effect of the developmentally-inducible alteration of Lys metabolism was an induction of expression of a large set of genes encoding ribosomal proteins as well as genes encoding translation initiation and elongation factors, all of which are associated with protein synthesis. With respect to metabolic regulation, the inducible alteration of Lys metabolism was primarily associated with altered expression of genes belonging to networks of amino acids and sugar metabolism. The combined data are discussed within the context of network interactions both between and within metabolic and transcriptional control systems.

BEL1-like transcription factors are ubiquitous in plants and interact with KNOTTED1-types to regulate numerous developmental processes. In potato, the BEL1-like transcription factor, StBEL5, and its Knox protein partner regulate tuber formation by targeting genes that control growth. RNA detection methods and heterografting experiments demonstrated that StBEL5 transcripts are present in phloem cells and move across a graft union to localize in stolon tips, the site of tuber induction. This movement of RNA originates in leaf veins and petioles and is induced by a short-day photoperiod, regulated by the untranslated regions, and correlated with enhanced tuber production. Assays for RNA mobility suggest that both 5' and 3' untranslated regions contribute to the preferential accumulation of the StBEL5 RNA but that the 3' untranslated region may contribute more to transport from the leaf to the stem, and into the stolons. Addition of the StBEL5 untranslated regions to another BEL1-like mRNA resulted in its preferential transport to stolon tips and enhanced tuber production. Transcript stability assays showed that the untranslated regions and a long-day photoperiod enhanced StBEL5 RNA stability in shoot tips. Upon fusion of the untranslated regions of StBEL5 to a GUS marker, translation in tobacco protoplasts was repressed by those constructs containing the 3' untranslated sequence. These results demonstrate that the untranslated regions of the mRNA of StBEL5 are involved in mediating its long-distance transport, in maintaining transcript stability, and in controlling translation.

The EMBRYONIC FLOWER (EMF) genes are required to maintain vegetative development in Arabidopsis. Loss-of-function emf mutants skip the vegetative phase, flower upon germination, and display pleiotropic phenotypes. EMF1 encodes a putative transcriptional regulator, while EMF2 encodes a Polycomb group (PcG) protein. PcG proteins form protein complexes that maintain gene silencing via histone modification. They are known to function as master regulators repressing multiple gene programs. Both EMF1 and EMF2 participate in PcG-mediated silencing of the flower homeotic genes, AGAMOUS (AG), PISTILLATA (PI) and APETALLA3 (AP3). Full-genome expression pattern analysis of emf mutants showed that both EMF proteins regulate additional gene programs including photosynthesis, seed development, hormone, stress and cold signaling. Chromatin Immunoprecipitation (ChIP) was carried out to investigate whether EMF regulates these genes directly. It was determined that EMF1 and EMF2 interact with genes encoding the transcription factors, ABSCISIC ACID 3 (ABI3), LONG VEGETATIVE PHASE 1 (LOV1) and FLOWERING LOCUS C (FLC), which control seed development, stress and cold signaling, and flowering, respectively. Our results suggest that the two EMFs repress the regulatory genes of individual gene programs to effectively silence the genetic pathways necessary for vegetative development and stress response. A model of the regulatory network mediated by EMF is proposed.

Plant architecture is determined by genetic and developmental programs, as well as by environmental factors. Sessile plants have evolved a subtle adaptive mechanism that allows them to alter their growth and development during periods of stress. Phytohormones play a central role in this process; however, the molecules responsible for integrating growth- and stress-related signals are unknown. Here, we report a gain-of-function rice mutant, tld1-D, characterized by an increased number of tillers, enlarged leaf angles, and dwarfism. TLD1 is a rice GH3.13 gene that encodes IAA-amido synthetase, which is suppressed in above-ground tissues under normal conditions, but which is dramatically induced by drought stress. The activation of TLD1 reduced the IAA maxima at the lamina joint, shoot base, and nodes, resulting in subsequent alterations in plant architecture and tissue patterning but enhancing drought tolerance. Accordingly, the decreased level of free IAA in tld1-D due to the conjugation of IAA with amino acids greatly facilitated the accumulation of late-embryogenesis abundant (LEA) mRNA compared with WT. The direct regulation of such drought-inducible genes by changes in the concentration of IAA provides a model for changes in plant architecture via the process of drought adaptation, which occurs frequently in nature.

A number of hypotheses have been suggested to explain why invasive exotic plants dramatically increase their abundance upon transport to a new range. The novel weapons hypothesis argues that phytotoxins secreted by roots of an exotic plant are more effective against naive resident competitors in the range being invaded. The common reed Phragmites australis has a diverse population structure including invasive populations that are noxious weeds in North America. P. australis exudes the common phenolic gallic acid, which restricts the growth of native plants. However, the pathway for free gallic acid production in soils colonized by P. australis requires further elucidation. Here, we show that exotic, invasive P. australis contain elevated levels of polymeric gallotannin relative to native, non-invasive P. australis. We hypothesized that polymeric gallotannin can be attacked by tannase, an enzymatic activity produced by native plant and microbial community members, to release gallic acid in the rhizosphere and exacerbate the noxiousness of P. australis. Native plants and microbes were found to produce high levels of tannase while invasive P. australis produced very little tannase. These results suggest that both invasive and native species participate in signaling events that initiate the execution of allelopathy potentially linking native plant and microbial biochemistry to the invasive traits of an exotic species.

The phytohormone abscisic acid (ABA) is known to be a negative regulator of legume root nodule formation. By screening Lotus japonicus seedlings for survival on an agar medium containing 70 µM ABA, we obtained mutants that not only showed increased root nodule number, but also enhanced nitrogen fixation. The mutant was designated enf1 (enhanced nitrogen fixation 1) and was confirmed to be monogenic and incompletely dominant. The low-sensitivity to ABA phenotype was thought to result from either a decrease in the concentration of the plant's endogenous ABA or from a disruption in ABA signaling. We determined that the endogenous ABA concentration of enf1 was lower than that of wild-type seedlings, and furthermore, when wild-type plants were treated with abamine, a specific inhibitor of 9-cis-epoxycarotenoid dioxygenase (NCED), which results in reduced ABA content, the N fixation activity of abamine-treated plants was elevated to the same levels as enf1. We also determined that production of nitric oxide (NO) in enf1 nodules was decreased. We conclude that endogenous ABA concentration not only regulates nodulation, but also nitrogen fixation activity by decreasing NO production in nodules.

In the photosynthetic apparatus, a major target of photodamage is the D1 reaction center protein of Photosystem II (PSII). Photosynthetic organisms have developed a PSII repair cycle in which photodamaged D1 is selectively degraded. A thylakoid membrane-bound metalloprotease FtsH was shown to play a critical role in this process. Here, the effect of FtsHs in D1 degradation was investigated in Arabidopsis (Arabidopsis thaliana) mutants lacking FtsH2 (var2) or FtsH5 (var1). Because these mutants are characterized by variegated leaves that sometimes complicate biochemical studies, we employed another mutation fug1 that suppresses leaf variegation in var1 and var2 to examine D1 degradation. Two-dimensional blue-native PAGE showed that var2 has less PSII supercomplex and more PSII intermediate lacking CP43 termed RC47 than wild type under normal growth light. Moreover, our histochemical and quantitative analyses revealed that chloroplasts in var2 accumulate significant levels of reactive oxygen species, such as superoxide radical and hydrogen peroxide. These results implicate that the lack of FtsH2 leads to impaired D1 degradation at the step of RC47 formation in the PSII repair, and to photooxidative stress even under non-photoinhibitory conditions. Our in vivo D1 degradation assays, carried out by non-variegated var2 fug1 and var1 fug1 leaves, demonstrated that D1 degradation was impaired in different light conditions. Taken together, our results suggest the important role of chloroplastic FtsHs, which was not precisely examined in vivo. Attenuated D1 degradation in the non-variegated mutants also suggests that leaf variegation seems to be independent of the PSII repair.