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Metatranscriptomic study of nonmodel organisms requires strategies that retain the highly resolved genetic information generated from model organisms while allowing for identification of the unexpected. A real-world biological application of phytoremediation, the field growth of 10 Salix cultivars on polluted soils, was used as an exemplar nonmodel and multifaceted crop response well-disposed to the study of gene expression. Sequence reads were assembled de novo to create 10 independent transcriptomes, a global transcriptome, and were mapped against the Salix purpurea 94006 reference genome. Annotation of assembled contigs was performed without a priori assumption of the originating organism. Global transcriptome construction from 3.03 billion paired-end reads revealed 606,880 unique contigs annotated from 1588 species, often common in all 10 cultivars. Comparisons between transcriptomic and metatranscriptomic methodologies provide clear evidence that nonnative RNA can mistakenly map to reference genomes, especially to conserved regions of common housekeeping genes, such as actin, α/β-tubulin, and elongation factor 1-α. In Salix, Rubisco activase transcripts were down-regulated in contaminated trees across all 10 cultivars, whereas thiamine thizole synthase and CP12, a Calvin Cycle master regulator, were uniformly up-regulated. De novo assembly approaches, with unconstrained annotation, can improve data quality; care should be taken when exploring such plant genetics to reduce de facto data exclusion by mapping to a single reference genome alone. Salix gene expression patterns strongly suggest cultivar-wide alteration of specific photosynthetic apparatus and protection of the antenna complexes from oxidation damage in contaminated trees, providing an insight into common stress tolerance strategies in a real-world phytoremediation system. [ABSTRACT FROM AUTHOR]

Our knowledge of the factors mediating ethylene-dependent ripening of climacteric fruit remains limited. The transcription of ethylene-regulated genes is mediated by ethylene response factors (ERFs), but mutants providing information on the specific role of the ERFs in fruit ripening are still lacking, likely due to functional redundancy among this large multigene family of transcription factors. We present here a comprehensive expression profiling of tomato (Solanum lycopersicum) ERFs in wild-type and tomato ripening-impaired tomato mutants (Never-ripe [Nr], ripening-inhibitor [rin], and non-ripening [nor]), indicating that out of the 77 ERFs present in the tomato genome, 27 show enhanced expression at the onset of ripening while 28 display a ripening-associated decrease in expression, suggesting that different ERFs may have contrasting roles in fruit ripening. Among the 19 ERFs exhibiting the most consistent up-regulation during ripening, the expression of 11 ERFs is strongly down-regulated in rin, nor, and Nr tomato ripening mutants, while only three are consistently up-regulated. Members of subclass E, SlERF.E1, SlERF.E2, and SlERF.E4, show dramatic down-regulation in the ripening mutants, suggesting that their expression might be instrumental in fruit ripening. This study illustrates the high complexity of the regulatory network connecting RIN and ERFs and identifies subclass E members as the most active ERFs in ethylene- and RIN/NOR-dependent ripening. [ABSTRACT FROM AUTHOR]

Within the cereal grain, the endosperm and its nutrient reserves are critical for successful germination and in the context of grain utilization. The identification of molecular determinants of early endosperm development, particularly regulators of cell division and cell wall deposition, would help predict end-use properties such as yield, quality, and nutritional value. Custom microarray data have been generated using RNA isolated from developing barley grain endosperm 3 d to 8 d after pollination (DAP). Comparisons of transcript abundance over time revealed 47 gene expression modules that can be clustered into 10 broad groups. Superimposing these modules upon cytological data allowed patterns of transcript abundance to be linked with key stages of early grain development. Here, attention was focused on how the datasets could be mined to explore and define the processes of cell wall biosynthesis, remodeling, and degradation. Using a combination of spatial molecular network and gene ontology enrichment analyses, it is shown that genes involved in cell wall metabolism are found in multiple modules, but cluster into two main groups that exhibit peak expression at 3 DAP to 4 DAP and 5 DAP to 8 DAP. The presence of transcription factor genes in these modules allowed candidate genes for the control of wall metabolism during early barley grain development to be identified. The data are publicly available through a dedicated web interface (https://ics.hutton.ac.uk/barseed/), where they can be used to interrogate co- and differential expression for any other genes, groups of genes, or transcription factors expressed during early endosperm development. [ABSTRACT FROM AUTHOR]

The shapes of leaves are dynamic, changing over evolutionary time between species, within a single plant producing different shaped leaves at successive nodes, during the development of a single leaf as it allometrically expands, and in response to the environment. Notably, strong correlations between the dissection and size of leaves with temperature and precipitation exist in both the paleorecord and extant populations. Yet, a morphometric model integrating evolutionary, developmental, and environmental effects on leaf shape is lacking. Here, we continue a morphometric analysis of >5,500 leaves representing 270 grapevines of multiple Vitis species between two growing seasons. Leaves are paired one-to-one and vine-to-vine accounting for developmental context, between growing seasons. Linear discriminant analysis reveals shape features that specifically define growing season, regardless of species or developmental context. The shape feature, a more pronounced distal sinus, is associated with the colder, drier growing season, consistent with patterns observed in the paleorecord. We discuss the implications of such plasticity in a long-lived woody perennial, such as grapevine (Vitis spp.), with respect to the evolution and functionality of plant morphology and changes in climate. [ABSTRACT FROM AUTHOR]

Heterotrimeric G proteins composed of α β and γ subunits are central signal transducers mediating the cellular response to multiple stimuli in most eukaryotes. Gγ subunits provide proper cellular localization and functional specificity to the heterotrimer complex. Plant Gγ subunits, divided into three structurally distinct types, are more diverse than their animal counterparts. Type B Gγ subunits, lacking a carboxyl-terminal isoprenylation motif, are found only in flowering plants. We present the functional characterization of type B Gγ subunit (SlGGB1) in tomato (Solanum lycopersicum). We show that SlGGB1 is the most abundant Gγ subunit in tomato and strongly interacts with the Gβ subunit. Importantly, the green fluorescent protein- SlGGB1 fusion protein as well as the carboxyl-terminal yellow fluorescent protein-SlGGB1/amino-terminal yellow fluorescent protein-Gβ heterodimer were localized in the plasma membrane, nucleus, and cytoplasm. RNA interference-mediated silencing of SlGGB1 resulted in smaller seeds, higher number of lateral roots, and pointy fruits. The silenced lines were hypersensitive to exogenous auxin, while levels of endogenous auxins were lower or similar to those of the wild type. SlGGB1-silenced plants also showed strong hyposensitivity to abscisic acid (ABA) during seed germination but not in other related assays. Transcriptome analysis of the transgenic seeds revealed abnormal expression of genes involved in ABA sensing, signaling, and response. We conclude that the type B Gγ subunit SlGGB1 mediates auxin and ABA signaling in tomato. [ABSTRACT FROM AUTHOR]

In barley (Hordeum vulgare), PHOTOPERIOD1 (Ppd-H1) acts as a major positive regulator of flowering under long-day conditions, while VERNALIZATION2 (VRN-H2) is a strong repressor of flowering under long days before vernalization. By contrast, CONSTANS (CO) plays a key role in the photoperiodic regulation of flowering in Arabidopsis (Arabidopsis thaliana). Here, we study the role of the closest barley CO homologs, HvCO1 and HvCO2, in the long day-dependent control of flowering and their interactions with Ppd-H1 and VRN-H2. HvCO2 overexpression in spring barley, with a natural deletion of the VRN-H2 locus, caused a Ppd-H1-dependent induction of flowering and FLOWERING LOCUS T1 (HvFT1) expression. In winter barley, which carries the VRN-H2 locus, overexpression of HvCO1/CO2 caused an up-regulation of VRN-H2, resulting in a reduced expression of HvFT1 and delayed flowering under long- and short-day conditions. In addition, natural variation at Ppd-H1 altered the expression of VRN-H2 in wild-type plants under long days. VRN-H2, in turn, was involved in the down-regulation of Ppd-H1 and HvCO2, demonstrating strong reciprocal interactions between HvCO2, Ppd-H1, and VRN-H2. Consequently, this study showed that the induction of the floral repressor VRN-H2 and the floral activator HvFT1 was regulated by the same genes, Ppd-H1 and HvCO1/CO2. Our findings provide a novel insight into the photoperiodic regulation of the vernalization pathway in barley. [ABSTRACT FROM AUTHOR]

To maintain a stable cytosol phosphate (Pi) concentration, plant cells store Pi in their vacuoles. When the Pi concentration in the cytosol decreases, Pi is exported from the vacuole into the cytosol. This export is mediated by Pi transporters on the tonoplast. In this study, we demonstrate that SYG1, PHO81, and XPR1 (SPX)-Major Facility Superfamily (MFS) proteins have a similar structure with yeast (Saccharomyces cerevisiae) low-affinity Pi transporters Phosphatase87 (PHO87), PHO90, and PHO91. OsSPX-MFS1, OsSPX-MFS2, and OsSPX-MFS3 all localized on the tonoplast of rice (Oryza sativa) protoplasts, even in the absence of the SPX domain. At high external Pi concentration, OsSPX-MFS3 could partially complement the yeast mutant strain EY917 under pH 5.5, which lacks all five Pi transporters present in yeast. In oocytes, OsSPX-MFS3 was shown to facilitate Pi influx or efflux depending on the external pH and Pi concentrations. In contrast to tonoplast localization in plants cells, OsSPX-MFS3 was localized to the plasma membrane when expressed in both yeast and oocytes. Overexpression of OsSPX-MFS3 results in decreased Pi concentration in the vacuole of rice tissues. We conclude that OsSPX-MFS3 is a lowaffinity Pi transporter that mediates Pi efflux from the vacuole into cytosol and is coupled to proton movement. [ABSTRACT FROM AUTHOR]

Plant response mechanisms to deficiency of a single nutrient, such as sulfur (S) or iron (Fe), have been described at agronomic, physiological, biochemical, metabolomics, and transcriptomic levels. However, agroecosystems are often characterized by different scenarios, in which combined nutrient deficiencies are likely to occur. Soils are becoming depleted for S, whereas Fe, although highly abundant in the soil, is poorly available for uptake because of its insolubility in the soil matrix. To this end, earlier reports showed that a limited S availability reduces Fe uptake and that Fe deficiency results in the modulation of sulfate uptake and assimilation. However, the mechanistic basis of this interaction remains largely unknown. Metabolite profiling of tomato (Solanum lycopersicum) shoots and roots from plants exposed to Fe, S, and combined Fe and S deficiency was performed to improve the understanding of the S-Fe interaction through the identification of the main players in the considered pathways. Distinct changes were revealed under the different nutritional conditions. Furthermore, we investigated the development of the Fe deficiency response through the analysis of expression of ferric chelate reductase, iron-regulated transporter, and putative transcription factor genes and plant sulfate uptake and mobilization capacity by analyzing the expression of genes encoding sulfate transporters (STs) of groups 1, 2, and 4 (SlST1.1, SlST1.2, SlST2.1, SlST2.2, and SlST4.1). We identified a high degree of common and even synergistic response patterns as well as nutrient-specific responses. The results are discussed in the context of current models of nutrient deficiency responses in crop plants. [ABSTRACT FROM AUTHOR]

Linker (H1) histones play critical roles in chromatin compaction in higher eukaryotes. They are also the most variable of the histones, with numerous nonallelic variants cooccurring in the same cell. Plants contain a distinct subclass ofminor H1 variants that are induced by drought and abscisic acid and have been implicated in mediating adaptive responses to stress. However, how these variants facilitate adaptation remains poorly understood. Here, we show that the single Arabidopsis (Arabidopsis thaliana) stressinducible variant H1.3 occurs in plants in two separate and most likely autonomous pools: a constitutive guard cell-specific pool and a facultative environmentally controlled pool localized in other tissues. Physiological and transcriptomic analyses of h1.3 null mutants demonstrate that H1.3 is required for both proper stomatal functioning under normal growth conditions and adaptive developmental responses to combined light and water deficiency. Using fluorescence recovery after photobleaching analysis, we show that H1.3 has superfast chromatin dynamics, and in contrast to the main Arabidopsis H1 variants H1.1 and H1.2, it has no stable bound fraction. The results of global occupancy studies demonstrate that, while H1.3 has the same overall binding properties as the main H1 variants, including predominant heterochromatin localization, it differs from them in its preferences for chromatin regions with epigenetic signatures of active and repressed transcription. We also show that H1.3 is required for a substantial part of DNA methylation associated with environmental stress, suggesting that the likely mechanism underlying H1.3 function may be the facilitation of chromatin accessibility by direct competition with the main H1 variants. [ABSTRACT FROM AUTHOR]

Pollen tube growth is an essential aspect of plant reproduction because it is the mechanism through which nonmotile sperm cells are delivered to ovules, thus allowing fertilization to occur. A pollen tube is a single cell that only grows at the tip, and this tip growth has been shown to depend on actin filaments. It is generally assumed that myosin-driven movements along these actin filaments are required to sustain the high growth rates of pollen tubes. We tested this conjecture by examining seed set, pollen fitness, and pollen tube growth for knockout mutants of five of the six myosin XI genes expressed in pollen of Arabidopsis (Arabidopsis thaliana). Single mutants had little or no reduction in overall fertility, whereas double mutants of highly similar pollen myosins had greater defects in pollen tube growth. In particular, myo11c1 myo11c2 pollen tubes grew more slowly than wild-type pollen tubes, which resulted in reduced fitness compared with the wild type and a drastic reduction in seed set. Golgi stack and peroxisome movements were also significantly reduced, and actin filaments were less organized in myo11c1 myo11c2 pollen tubes. Interestingly, the movement of yellow fluorescent protein-RabA4d-labeled vesicles and their accumulation at pollen tube tips were not affected in the myo11c1 myo11c2 double mutant, demonstrating functional specialization among myosin isoforms. We conclude that class XI myosins are required for organelle motility, actin organization, and optimal growth of pollen tubes. [ABSTRACT FROM AUTHOR]

Plants sense the foliar shade of competitors and alter their developmental programs through the shade-avoidance response. Internode and petiole elongation, and changes in overall leaf area and leaf mass per area, are the stereotypical architectural responses to foliar shade in the shoot. However, changes in leaf shape and complexity in response to shade remain incompletely, and qualitatively, described. Using a meta-analysis of more than 18,000 previously published leaflet outlines, we demonstrate that shade avoidance alters leaf shape in domesticated tomato (Solanum lycopersicum) and wild relatives. The effects of shade avoidance on leaf shape are subtle with respect to individual traits but are combinatorially strong. We then seek to describe the developmental origins of shade-induced changes in leaf shape by swapping plants between light treatments. Leaf size is light responsive late into development, but patterning events, such as stomatal index, are irrevocably specified earlier. Observing that shade induces increases in shoot apical meristem size, we then describe gene expression changes in early leaf primordia and the meristem using laser microdissection. We find that in leaf primordia, shade avoidance is not mediated through canonical pathways described in mature organs but rather through the expression of KNOTTED1-LIKE HOMEOBOX and other indeterminacy genes, altering known developmental pathways responsible for patterning leaf shape. We also demonstrate that shade-induced changes in leaf primordium gene expression largely do not overlap with those found in successively initiated leaf primordia, providing evidence against classic hypotheses that shaded leaf morphology results from the prolonged production of juvenile leaf types. [ABSTRACT FROM AUTHOR]

Protein amino (N) termini are prone to modifications and are major determinants of protein stability in bacteria, eukaryotes, and perhaps also in chloroplasts. Most chloroplast proteins undergo N-terminal maturation, but this is poorly understood due to insufficient experimental information. Consequently, N termini of mature chloroplast proteins cannot be accurately predicted. This motivated an extensive characterization of chloroplast protein N termini in Arabidopsis (Arabidopsis thaliana) using terminal amine isotopic labeling of substrates and mass spectrometry, generating nearly 14,000 tandem mass spectrometry spectra matching to protein N termini. Many nucleus-encoded plastid proteins accumulated with two or three different N termini; we evaluated the significance of these different proteoforms. Alanine, valine, threonine (often in N-a-acetylated form), and serine were by far the most observed N-terminal residues, even after normalization for their frequency in the plastid proteome, while other residues were absent or highly underrepresented. Plastid-encoded proteins showed a comparable distribution of N-terminal residues, but with a higher frequency of methionine. Infrequent residues (e.g. isoleucine, arginine, cysteine, proline, aspartate, and glutamate) were observed for several abundant proteins (e.g. heat shock proteins 70 and 90, Rubisco large subunit, and ferredoxin-glutamate synthase), likely reflecting functional regulation through their N termini. In contrast, the thylakoid lumenal proteome showed a wide diversity of N-terminal residues, including those typically associated with instability (aspartate, glutamate, leucine, and phenylalanine). We propose that, after cleavage of the chloroplast transit peptide by stromal processing peptidase, additional processing by unidentified peptidases occurs to avoid unstable or otherwise unfavorable N-terminal residues. The possibility of a chloroplast N-end rule is discussed. [ABSTRACT FROM AUTHOR]

Anthocyanins are flavonoid compounds responsible for red/purple colors in the leaves, fruit, and flowers of many plant species. They are produced through a multistep pathway that is controlled by MYB transcription factors. VvMYBA1 and VvMYBA2 activate anthocyanin biosynthesis in grapevine (Vitis vinifera) and are nonfunctional in white grapevine cultivars. In this study, transgenic grapevines with altered VvMYBA gene expression were developed, and transcript analysis was carried out on berries using a microarray technique. The results showed that VvMYBA is a positive regulator of the later stages of anthocyanin synthesis, modification, and transport in cv Shiraz. One up-regulated gene, ANTHOCYANIN 3-O-GLUCOSIDE-699-O-ACYLTRANSFERASE (Vv3AT), encodes a BAHD acyltransferase protein (named after the first letter of the first four characterized proteins: BEAT [for acetyl CoA:benzylalcohol acetyltransferase], AHCT [for anthocyanin O-hydroxycinnamoyltransferase], HCBT [for anthranilate N-hydroxycinnamoyl/benzoyltransferase], and DAT [for deacetylvindoline 4-O-acetyltransferase]), belonging to a clade separate from most anthocyanin acyltransferases. Functional studies (in planta and in vitro) show that Vv3AT has a broad anthocyanin substrate specificity and can also utilize both aliphatic and aromatic acyl donors, a novel activity for this enzyme family found in nature. In cv Pinot Noir, a red-berried grapevine mutant lacking acylated anthocyanins, Vv3AT contains a nonsense mutation encoding a truncated protein that lacks two motifs required for BAHD protein activity. Promoter activation assays confirm that Vv3AT transcription is activated by VvMYBA1, which adds to the current understanding of the regulation of the BAHD gene family. The flexibility of Vv3AT to use both classes of acyl donors will be useful in the engineering of anthocyanins in planta or in vitro. [ABSTRACT FROM AUTHOR]

Autophagosomes are organelles that deliver cytosolic proteins for degradation in the vacuole of the cell. In contrast, exocystpositive organelles (EXPO) deliver cytosolic proteins to the cell surface and therefore represent a form of unconventional protein secretion. Because both structures have two boundary membranes, it has been suggested that they may have been falsely treated as separate entities. Using suspension culture cells and root tissue cells of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing either the EXPO marker Arabidopsis Exo70E2-GFP or the autophagosome marker yellow fluorescent protein (YFP)-autophagy-related gene 8e/f (ATG8e/f), and using specific antibodies against Exo70E2 and ATG8, we have now established that, in normally growing cells, EXPO and autophagosomes are distinct from one another. However, when cells/roots are subjected to autophagy induction, EXPO as well as autophagosomes fuse with the vacuole. In the presence of concanamycin A, the punctate fluorescent signals from both organelles inside the vacuole remain visible for hours and overlap to a significant degree. Tonoplast staining with FM4-64/YFP-Rab7-like GTPase/YFP-vesicleassociated membrane protein711 confirmed the internalization of tonoplast membrane concomitant with the sequestration of EXPO and autophagosomes. This suggests that EXPO and autophagosomes may be related to one another; however, whereas induction of autophagy led to an increase in the amount of ATG8 recruited to membranes, Exo70E2 did not respond in a similar manner. [ABSTRACT FROM AUTHOR]

Transcriptome resources for the medicinal plant Glaucium flavum were searched for orthologs showing identity with characterized O-methyltransferases (OMTs) involved in benzylisoquinoline alkaloid biosynthesis. Seven recombinant proteins were functionally tested using the signature alkaloid substrates for six OMTs: norlaudanosoline 6-OMT, 6-O-methyllaudanosoline 49-OMT, reticuline 7-OMT, norreticuline 7-OMT, scoulerine 9-OMT, and tetrahydrocolumbamine OMT. A notable alkaloid in yellow horned poppy (G. flavum [GFL]) is the aporphine alkaloid glaucine, which displays C8-C69 coupling and four O-methyl groups at C6, C7, C39, and C49 as numbered on the 1-benzylisoquinoline scaffold. Three recombinant enzymes accepted 1-benzylisoquinolines with differential substrate and regiospecificity. GFLOMT2 displayed the highest amino acid sequence identity with norlaudanosoline 6-OMT, showed a preference for the 6-O-methylation of norlaudanosoline, and O-methylated the 39 and 49 hydroxyl groups of certain alkaloids. GFLOMT1 showed the highest sequence identity with 6-O-methyllaudanosoline 49OMT and catalyzed the 6-O-methylation of norlaudanosoline, but more efficiently 49-O-methylated the GFLOMT2 reaction product 6-O-methylnorlaudanosoline and its N-methylated derivative 6-O-methyllaudanosoline. GFLOMT1 also effectively 39-O-methylated both reticuline and norreticuline. GFLOMT6 was most similar to scoulerine 9-OMT and efficiently catalyzed both 39- and 79-O-methylations of several 1-benzylisoquinolines, with a preference for N-methylated substrates. All active enzymes accepted scoulerine and tetrahydrocolumbamine. Exogenous norlaudanosoline was converted to tetra-O-methylated laudanosine using combinations of Escherichia coli producing (1) GFLOMT1, (2) either GFLOMT2 or GFLOMT6, and (3) coclaurine N-methyltransferase from Coptis japonica. Expression profiles of GFLOMT1, GFLOMT2, and GFLOMT6 in different plant organs were in agreement with the O-methylation patterns of alkaloids in G. flavum determined by high-resolution, Fourier-transform mass spectrometry. [ABSTRACT FROM AUTHOR]

The Arabidopsis (Arabidopsis thaliana) Early Light-Induced Protein (ELIP) is thought to act as a photoprotectant, reducing the damaging effects of high light (HL). Expression of ELIP2 is activated by multiple environmental stresses related to photoinhibition. We have identified putative regulatory elements in an ELIP2 promoter using an octamer-based frequency comparison method, analyzed the role of these elements using synthetic promoters, and revealed a key transcriptional regulatory unit for ultraviolet B (UV-B) radiation, HL, and cold stress responses. The unit is composed of two elements, designated as Elements A (TACACACC) and B (GGCCACGCCA), and shows functionality only when paired. Our genome-wide correlation analysis between possession of these elements in the promoter region and expression profiles in response to UV-B, HL, and cold suggests that Element B receives and integrates these multiple stress signals. In vitro protein-DNA binding assays revealed that LONG HYPOCOTYL5 (HY5), a basic domain-Leucine zipper transcription factor, directly binds to Element B. In addition, mutant analysis of HY5 showed partial involvement in the UV-B and HL responses but not in the cold stress response. These results suggest that signals for UV-B, HL, and cold stress join at Element B, which recognizes the signals of multiple transcription factors, including HY5. [ABSTRACT FROM AUTHOR]

The sensitivity of the circadian system to light allows entrainment of the clock, permitting coordination of plant metabolic function and flowering time across seasons. Light affects the circadian system via both photoreceptors, such as phytochromes and cryptochromes, and sugar production by photosynthesis. In the present study, we introduce a constitutively active version of phytochrome B-Y276H (YHB) into both wild-type and phytochrome null backgrounds of Arabidopsis (Arabidopsis thaliana) to distinguish the effects of photoreceptor signaling on clock function from those of photosynthesis. We find that the YHB mutation is sufficient to phenocopy red light input into the circadian mechanism and to sustain robust rhythms in steady-state mRNA levels even in plants grown without light or exogenous sugars. The pace of the clock is insensitive to light intensity in YHB plants, indicating that light input to the clock is constitutively activated by this allele. Mutation of YHB so that it is retained in the cytoplasm abrogates its effects on clock function, indicating that nuclear localization of phytochrome is necessary for its clock regulatory activity. We also demonstrate a role for phytochrome C as part of the red light sensing network that modulates phytochrome B signaling input into the circadian system. Our findings indicate that phytochrome signaling in the nucleus plays a critical role in sustaining robust clock function under red light, even in the absence of photosynthesis or exogenous sources of energy. [ABSTRACT FROM AUTHOR]

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin Blike proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes. [ABSTRACT FROM AUTHOR]

To improve seed iron (Fe) content and bioavailability, it is crucial to decipher the mechanisms that control Fe storage during seed development. In Arabidopsis (Arabidopsis thaliana) seeds, most Fe is concentrated in insoluble precipitates, with phytate in the vacuoles of cells surrounding the vasculature of the embryo. NATURAL RESISTANCE ASSOCIATED-MACROPHAGE PROTEIN3 (AtNRAMP3) and AtNRAMP4 function redundantly in Fe retrieval from vacuoles during germination. When germinated under Fe-deficient conditions, development of the nramp3nramp4 double mutant is arrested as a consequence of impaired Fe mobilization. To identify novel genes involved in seed Fe homeostasis, we screened an ethyl methanesulfonatemutagenized population of nramp3nramp4 seedlings for mutations suppressing their phenotypes on low Fe. Here, we report that, among the suppressors, two independent mutations in the VACUOLAR IRON TRANSPORTER1 (AtVIT1) gene caused the suppressor phenotype. The AtVIT1 transporter is involved in Fe influx into vacuoles of endodermal and bundle sheath cells. This result establishes a functional link between Fe loading in vacuoles by AtVIT1 and its remobilization by AtNRAMP3 and AtNRAMP4. Moreover, analysis of subcellular Fe localization indicates that simultaneous disruption of AtVIT1, AtNRAMP3, and AtNRAMP4 limits Fe accumulation in vacuolar globoids. [ABSTRACT FROM AUTHOR]

In plants, the copy number of the mitochondrial DNA (mtDNA) can be much lower than the number of mitochondria. The biological significance and regulatory mechanisms of this phenomenon remain poorly understood. Here, using the pollen vegetative cell, we examined the role of the Arabidopsis (Arabidopsis thaliana) mtDNA-binding protein WHIRLY2 (AtWHY2). AtWHY2 decreases during pollen development, in parallel with the rapid degradation of mtDNA; to examine the importance of this decrease, we used the pollen vegetative cell-specific promoter Lat52 to express AtWHY2. The transgenic plants (LWHY2) had very high mtDNA levels in pollen, more than 10 times more than in the wild type (ecotype Columbia-0). LWHY2 plants were fertile, morphologically normal, and set seeds; however, reciprocal crosses with heterozygous plants showed reduced transmission of LWHY2-1 through the male and slower growth of LWHY2-1 pollen tubes. We found that LWHY2-1 pollen had significantly more reactive oxygen species and less ATP compared with the wild type, indicating an effect on mitochondrial respiration. These findings reveal that AtWHY2 affects mtDNA copy number in pollen and suggest that low mtDNA copy numbers might be the normal means by which plant cells maintain mitochondrial genetic information. [ABSTRACT FROM AUTHOR]

In flowering plant reproduction, pollen tube reception is the signaling system that results in pollen tube discharge, synergid degeneration, and successful delivery of male gametes (two sperm cells) to the site where they can fuse with female gametes (egg cell and central cell). Some molecules required for this complex and essential signaling exchange have been identified; however, fundamental questions about the nature of the interactions between the pollen tube and the synergid cells remain to be clarified. Here, we monitor pollen tube arrival, pollen tube discharge, and synergid degeneration in Arabidopsis (Arabidopsis thaliana) wild type and in male and female gametophytic mutants that disrupt development and function of the gametophytes. By combining assays used previously to study these interactions and an assay that facilitates simultaneous analysis of pollen tube discharge and synergid degeneration, we find that synergid degeneration could be initiated without pollen tube discharge. Our data support the hypothesis that pollen tube-synergid contact, or signaling via secreted molecules, initiates receptive synergid degeneration. We also find that when pollen tubes successfully burst, they always discharge into a degenerated synergid. In addition to this pollen tube-dependent promotion of synergid degeneration, we also show that a basal developmental pathway mediates synergid degeneration in the absence of pollination. Our results are consistent with the model that a complex set of interactions between the pollen tube and synergid cells promote receptive synergid degeneration. [ABSTRACT FROM AUTHOR]

Grape (Vitis vinifera) accumulates various polyphenolic compounds, which protect against environmental stresses, including ultraviolet-C (UV-C) light and pathogens. In this study, we looked at the transcriptome and metabolome in grape berry skin after UV-C irradiation, which demonstrated the effectiveness of omics approaches to clarify important traits of grape. We performed transcriptome analysis using a genome-wide microarray, which revealed 238 genes up-regulated more than 5-fold by UV-C light. Enrichment analysis of Gene Ontology terms showed that genes encoding stilbene synthase, a key enzyme for resveratrol synthesis, were enriched in the up-regulated genes. We performed metabolome analysis using liquid chromatography-quadrupole time-of-flight mass spectrometry, and 2,012 metabolite peaks, including unidentified peaks, were detected. Principal component analysis using the peaks showed that only one metabolite peak, identified as resveratrol, was highly induced by UV-C light. We updated the metabolic pathway map of grape in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and in the KaPPA-View 4 KEGG system, then projected the transcriptome and metabolome data on a metabolic pathway map. The map showed specific induction of the resveratrol synthetic pathway by UV-C light. Our results showed that multiomics is a powerful tool to elucidate the accumulation mechanisms of secondary metabolites, and updated systems, such as KEGG and KaPPA-View 4 KEGG for grape, can support such studies. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H2S) is the third biological gasotransmitter, and in animals, it affects many physiological processes by modulating ion channels. H2S has been reported to protect plants from oxidative stress in diverse physiological responses. H2S closes stomata, but the underlying mechanism remains elusive. Here, we report the selective inactivation of current carried by inward-rectifying K+ channels of tobacco (Nicotiana tabacum) guard cells and show its close parallel with stomatal closure evoked by submicromolar concentrations of H2S. Experiments to scavenge H2S suggested an effect that is separable from that of abscisic acid, which is associated with water stress. Thus, H2S seems to define a unique and unresolved signaling pathway that selectively targets inward-rectifying K+ channels. [ABSTRACT FROM AUTHOR]

Glucose (Glc) plays a fundamental role in regulating lateral root (LR) development as well as LR emergence. In this study, we show that brassinosteroid (BR) signaling works downstream of Glc in controlling LR production/emergence in Arabidopsis (Arabidopsis thaliana) seedlings. Glc and BR can promote LR emergence at lower concentrations, while at higher concentrations, both have an inhibitory effect. The BR biosynthesis and perception mutants showed highly reduced numbers of emerged LRs at all the Glc concentrations tested. BR signaling works downstream of Glc signaling in regulating LR production, as in the glucose insensitive2- 1brassinosteroid insensitive1 double mutant, Glc-induced LR production/emergence was severely reduced. Differential auxin distribution via the in flux carriers AUXIN RESISTANT1/LIKE AUXIN RESISTANT1-3 and the efflux carrier PIN-FORMED2 plays a central role in controlling LR production in response to Glc and BR. Auxin signaling components AUXIN RESISTANT2,3 and SOLITARY ROOT act downstream of Glc and BR. AUXIN RESPONSE FACTOR7/19 work farther downstream and control LR production by regulating the expression of LATERAL ORGAN BOUNDARIES-DOMAIN29 and EXPANSIN17 genes. Increasing light flux could also mimic the Glc effect on LR production/emergence. However, increased light flux could not affect LR production in those BR and auxin signaling mutants that were defective for Glc-induced LR production. Altogether, our study suggests that, under natural environmental conditions, modulation of endogenous sugar levels can manipulate root architecture for optimized development by altering its nutrient/water uptake as well as its anchorage capacity. [ABSTRACT FROM AUTHOR]

The cuticle is a ubiquitous, predominantly waxy layer on the aerial parts of higher plants that fulfils a number of essential physiological roles, including regulating evapotranspiration, light reflection, and heat tolerance, control of development, and providing an essential barrier between the organism and environmental agents such as chemicals or some pathogens. The structure and composition of the cuticle are closely associated but are typically investigated separately using a combination of structural imaging and biochemical analysis of extracted waxes. Recently, techniques that combine stain-free imaging and biochemical analysis, including Fourier transform infrared spectroscopy microscopy and coherent anti-Stokes Raman spectroscopy microscopy, have been used to investigate the cuticle, but the detection sensitivity is severely limited by the background signals from plant pigments. We present a new method for label-free, in vivo structural and biochemical analysis of plant cuticles based on stimulated Raman scattering (SRS) microscopy. As a proof of principle, we used SRS microscopy to analyze the cuticles from a variety of plants at different times in development. We demonstrate that the SRS virtually eliminates the background interference compared with coherent anti-Stokes Raman spectroscopy imaging and results in label-free, chemically specific confocal images of cuticle architecture with simultaneous characterization of cuticle composition. This innovative use of the SRS spectroscopy may find applications in agrochemical research and development or in studies of wax deposition during leaf development and, as such, represents an important step in the study of higher plant cuticles. [ABSTRACT FROM AUTHOR]

Arabidopsis (Arabidopsis thaliana) leaf trichomes are single-cell structures with a well-studied development, but little is understood about their function. Developmental studies focused mainly on the early shaping stages, and little attention has been paid to the maturation stage. We focused on the EXO70H4 exocyst subunit, one of the most up-regulated genes in the mature trichome. We uncovered EXO70H4-dependent development of the secondary cell wall layer, highly auto fluorescent and callose rich, deposited only in the upper part of the trichome. The boundary is formed between the apical and the basal parts of mature trichome by a callose ring that is also deposited in an EXO70H4-dependent manner. We call this structure the Ortmannian ring (OR). Both the secondary cell wall layer and the OR are absent in the exo70H4 mutants. Ecophysiological aspects of the trichome cell wall thickening include interference with antiherbivore defense and heavy metal accumulation. Ultraviolet B light induces EXO70H4 transcription in a CONSTITUTIVE PHOTOMORPHOGENIC1-dependent way, resulting in stimulation of trichome cell wall thickening and the OR biogenesis. EXO70H4-dependent trichome cell wall hardening is a unique phenomenon, which may be conserved among a variety of the land plants. Our analyses support a concept that Arabidopsis trichome is an excellent model to study molecular mechanisms of secondary cell wall deposition. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) is highly toxic to plants. Modulation of Cd-responsive transcription is an important way for Cd detoxification in plants. Metal-responsive element (MRE) is originally described in animal metallothionein genes. Although functional MREs also exist in Cd-regulated plant genes, specific transcription factors that bind MRE to regulate Cd tolerance have not been identified. Previously, we showed that Cd-inducible bean (Phaseolus vulgaris) stress-related gene2 (PvSR2) produces a short (S) PvSR2 transcript (S-PvSR2) driven by an intronic promoter. Here, we demonstrate that S-PvSR2 encodes a bean MRE-binding transcription factor1 (PvMTF-1) that confers Cd tolerance in tobacco (Nicotiana tabacum). PvMTF-1 expression was up-regulated by Cd at the levels of RNA and protein. Importantly, expression of PvMTF-1 in tobacco enhanced Cd tolerance, indicating its role in regulating Cd resistance in planta. This was achieved through direct regulation of a feedback-insensitive Anthranilate Synthase α-2 chain gene (ASA2), which catalyzes the first step for tryptophan biosynthesis. In vitro and in vivo DNA-protein interaction studies further revealed that PvMTF-1 directly binds to the MRE in the ASA2 promoter, and this binding depends on the zinc finger-like motif of PvMTF-1. Through modulating ASA2 up-regulation by Cd, PvMTF-1 increased free tryptophan level and subsequently reduced Cd accumulation, thereby enhancing Cd tolerance of transgenic tobacco plants. Consistent with this observation, tobacco transiently overexpressing ASA2 also exhibited increased tolerance to Cd. We conclude that PvMTF-1 is a zinc finger-like transcription factor that links MRE to Cd resistance in transgenic tobacco through activation of tryptophan biosynthesis. [ABSTRACT FROM AUTHOR]

The hemibiotrophic fungus Zymoseptoria tritici causes Septoria tritici blotch disease of wheat (Triticum aestivum). Pathogen reproduction on wheat occurs without cell penetration, suggesting that dynamic and intimate intercellular communication occurs between fungus and plant throughout the disease cycle. We used deep RNA sequencing and metabolomics to investigate the physiology of plant and pathogen throughout an asexual reproductive cycle of Z. tritici on wheat leaves. Over 3,000 pathogen genes, more than 7,000 wheat genes, and more than 300 metabolites were differentially regulated. Intriguingly, individual fungal chromosomes contributed unequally to the overall gene expression changes. Early transcriptional down-regulation of putative host defense genes was detected in inoculated leaves. There was little evidence for fungal nutrient acquisition from the plant throughout symptomless colonization by Z. tritici, which may instead be utilizing lipid and fatty acid stores for growth. However, the fungus then subsequently manipulated specific plant carbohydrates, including fructan metabolites, during the switch to necrotrophic growth and reproduction. This switch coincided with increased expression of jasmonic acid biosynthesis genes and large-scale activation of other plant defense responses. Fungal genes encoding putative secondary metabolite clusters and secreted effector proteins were identified with distinct infection phase-specific expression patterns, although functional analysis suggested that many have overlapping/redundant functions in virulence. The pathogenic lifestyle of Z. tritici on wheat revealed through this study, involving initial defense suppression by a slow-growing extracellular and nutritionally limited pathogen followed by defense (hyper) activation during reproduction, reveals a subtle modification of the conceptual definition of hemibiotrophic plant infection. [ABSTRACT FROM AUTHOR]

Arabidopsis (Arabidopsis thaliana) leaf development relies on subsequent phases of cell proliferation and cell expansion. During the proliferation phase, chloroplasts need to divide extensively, and during the transition from cell proliferation to expansion, they differentiate into photosynthetically active chloroplasts, providing the plant with energy. The transcription factor GROWTH REGULATING FACTOR5 (GRF5) promotes the duration of the cell proliferation period during leaf development. Here, it is shown that GRF5 also stimulates chloroplast division, resulting in a higher chloroplast number per cell with a concomitant increase in chlorophyll levels in 35S:GRF5 leaves, which can sustain higher rates of photosynthesis. Moreover, 35S:GRF5 plants show delayed leaf senescence and are more tolerant for growth on nitrogen-depleted medium. Cytokinins also stimulate leaf growth in part by extending the cell proliferation phase, simultaneously delaying the onset of the cell expansion phase. In addition, cytokinins are known to be involved in chloroplast development, nitrogen signaling, and senescence. Evidence is provided that GRF5 and cytokinins synergistically enhance cell division and chlorophyll retention after dark-induced senescence, which suggests that they also cooperate to stimulate chloroplast division and nitrogen assimilation. Taken together with the increased leaf size, ectopic expression of GRF5 has great potential to improve plant productivity. [ABSTRACT FROM AUTHOR]

The mitochondrial electron transport chain includes an alternative oxidase (AOX) that is hypothesized to aid photosynthetic metabolism, perhaps by acting as an additional electron sink for photogenerated reductant or by dampening the generation of reactive oxygen species. Gas exchange, chlorophyll fluorescence, photosystem I (PSI) absorbance, and biochemical and protein analyses were used to compare respiration and photosynthesis of Nicotiana tabacum 'Petit Havana SRI' wild-type plants with that of transgenic AOX knockdown (RNA interference) and overexpression lines, under both well-watered and moderate drought- stressed conditions. During drought, AOX knockdown lines displayed a lower rate of respiration in the light than the wild type, as confirmed by two independent methods. Furthermore, CO2 and light response curves indicated a nonstomatal limitation of photosynthesis in the knockdowns during drought, relative to the wild type. Also relative to the wild type, the knockdowns under drought maintained PSI and PSII in a more reduced redox state, showed greater regulated nonphotochemical energy quenching by PSII, and displayed a higher relative rate of cyclic electron transport around PSI. The origin of these differences may lie in the chloroplast ATP synthase amount, which declined dramatically in the knockdowns in response to drought. None of these effects were seen in plants overexpressing AOX. The results show that AOX is necessary to maintain mitochondrial respiration during moderate drought. In its absence, respiration rate slows and the lack of this electron sink feeds back on the photosynthetic apparatus, resulting in a loss of chloroplast ATP synthase that then limits photosynthetic capacity. [ABSTRACT FROM AUTHOR]

The article discusses the effect of ultraviolet-B (UV-B) light on the transcription factor ELONGATED HYPOCOTYL5 (HY5) in Arabidopsis thaliana hypocotyls, based on the hypothesis that unilateral light occurs through unilateral photomorphegenic inhibition of growth as propose by Blaauw in 1919. Topics covered include the mechanism of the phototropin independent UV RESISTANCE LOCUS8 (UV8) function and the result of tests on the effect of UV-B on seedlings.

Fatty acids (FAs) and FA-derived complex lipids play important roles in plant growth and vegetative development and are a class of prominent metabolites stored in mature seeds. The factors and regulatory networks that control FA accumulation in plant seeds remain largely unknown. The role of TRANSPARENT TESTA8 (TT8) in the regulation of flavonoid biosynthesis and the formation of seed coat color is extensively studied; however, its fl2nction in affecting seed FA biosynthesis is poorly understood. In this article, we show that Arabidopsis (Arabidopsis thaliana) TT8 acts maternally to affect seed FA biosynthesis and inhibits seed FA accumulation by down-regulating a group of genes either critical to embryonic development or important in the FA biosynthesis pathway. Moreover, the tt8 mutation resulted in reduced deposition of protein in seeds during maturation. Posttranslational activation of a TT8-GLUCOCORTICOID RECEPTOR fusion protein and chromatin immmloprecipitation assays demonstrated that TT8 represses the activities of LEAFY COTYLEDON1, LEAFY COTYLEDON2, and FUSCA3, the critical transcriptional factors important for seed development, as well as CYTIDINEDIPHOSPHATE DIACYLGLYCEROL SYNTHASE2, which mediates glycerolipid biosynthesis. These results help us to understand the entire function of TT8 and increase our knowledge of the complicated networks regulating the formation of FA-derived complex lipids in plant seeds. [ABSTRACT FROM AUTHOR]

Plant water transport occurs through interconnected xylem conduits that are separated by partially digested regions in file cell wall known as pit membranes. These structures have a dual function. Their porous construction facilitates water movement between conduits while limiting fine spread of air that may enter the conduits and render them dysfunctional during a drought. Pit membranes have been well studied in woody plants, but very little is known about their function in more ancient lineages such as seedless vascular plants. Here, we examine the relationships between conduit air seeding, pit hydraulic resistance, and pit anatomy in 10 species of ferns (pteridophytes) and two lycophytes. Air seeding pressures ranged from 0.8 ± 0.15 MPa (mean ± SD) in the hydric fern Athyrium filix-femina to 4.9 ± 0.94 MPa in Psilotum nudum, an epiphytic species. Notably, a positive correlation was found between conduit pit area and vulnerability to air seeding, suggesting that the rare-pit hypothesis explains air seeding in early-diverging lineages much as it does in many angiosperms. Pit area resistance was variable but averaged 54.6 MPa s m-1 across all surveyed pteridophytes. End walls contributed 52% to the overall transport resistance, similar to the 56% in angiosperm vessels and 64% in conifer tracheids. Taken together, our data imply that, irrespective of phylogenetic placement, selection acted on transport efficiency in seedless vascular plants and woody plants in equal measure by compensating for shorter conduits in tracheid-bearing plants with more permeable pit membranes. [ABSTRACT FROM AUTHOR]

The signaling role of hydrogen gas (H2) has attracted increasing attention from animals to plants. However, the physiological significance and molecular mechanism of H2 in drought tolerance are still largely unexplored. In this article, we report that abscisic acid (ABA) induced stomatal closure in Arabidopsis (Arabidopsis thaliana) by triggering intracellular signaling events involving H2, reactive oxygen species (ROS), nitric oxide (NO), and the guard cell outward-rectifying K+ channel (GORK). AB elicited a rapid and sustained H2 release and production in Arabidopsis. Exogenous hydrogen-rich water (HRW) effectively led to an increase of intracellular H2 production, a reduction in the stomatal aperture, and enhanced drought tolerance. Subsequent results revealed that HRW stimulated significant inductions of NO and ROS synthesis associated with stomatal closure in the wild type, which were individually abolished in the nitric reductase mutant nitrate reductase1/2 (nia1/2) or the NADPH oxidase-deficient mutant rbohF (for respiratory burst oxidase homolog). Furthermore, we demonstrate that the HRW-promoted NO generation is dependent on ROS production. The rbohF mutant had impaired NO synthesis and stomatal closure in response to HRW, while these changes were rescued by exogenous application of NO. In addition, both HRW and hydrogen peroxide failed to induce NO production or stomatal closure in the nia1/2 mutant, while HRW-promoted ROS accumulation was not impair In the GORK-null mutant, stomatal closure induced by ABA, HRW, NO, or hydrogen peroxide was partially suppressed. Together, these results define a main branch of H2-regulated stomatal movement involved in the ABA signaling cascade in which RbohF-dependent ROS and nitric reductase-associated NO production, and subsequent GORK activation, were causally involved. [ABSTRACT FROM AUTHOR]

Root system architecture is a major determinant of water and nutrient acquisition as well as stress tolerance in plants. The Mediator complex is a conserved multiprotein complex that acts as a universal adaptor between transcription factors and the RNA polymerase II. In this article, we characterize possible roles of the MEDIATOR8 (MED8) and MED25 subunits of the plant Mediator complex in the regulation of root system architecture in Arabidopsis (Arabidopsis thaliana). We found that loss-of-function mutations in PHYTOCHROME AND FLOWERING TIME1 (PFT1)/MED25 increase primary and lateral root growth as well as lateral an adventitious root formation. In contrast, PFT1/MED25 overexpression reduces these responses, suggesting that PFT1/MED25 is an important element of meristematic cell proliferation and cell size control in both lateral and primary roots. PFT1/MED25 negatively regulates auxin transport and response gene expression in most parts of the plant, as evidenced by increased and decreased expression of the auxin-related reporters PIN-FORMED1 (PIN1)::PINI::GFP (for green fluorescent protein), DR5:GFP, DR5:uidA, and BA3:uidA in pftl-2 mutants and in 35S:PFT1 seedlings, respectively. No alterations in endogenous auxin levels could be found in pftl-2 mutants or in 35S:PFT1-overexpressing seedlings. However, detailed analyses of DR5:GFP and DR5:uidA activity in wild- type, pftl-2, and 35S:PFT1 seedlings in response to indole-3-acetic acid, naphthaleneacetic acid, and the polar auxin transport inhibitor 1-N-naphthylphthalamic acid indicated that PFT1/MED25 principally regulates auxin transport and response. These results provide compelling evidence for a new role for PFT1/MED25 as an important transcriptional regulator of root system architecture through auxin-related mechanisms in Arabidopsis. [ABSTRACT FROM AUTHOR]

The first step on the pathway of starch degradation in Arabidopsis (Arabidopsis thaliana) leaves at night is the phosphorylation of starch polymers, catalyzed by glucan, water dikinase (GWD). It has been suggested that GWD is important for the control of starch degradation, because its transcript levels undergo strong diel fluctuations, its activity is subject to redox regulation in vitro, and starch degradation is strongly decreased in gwd mutant plants. To test this suggestion, we analyzed changes in GWD protein abundance in relation to starch levels in wild-type plants, in transgenic plants in which GWD transcripts were strongly reduced by induction of RNA interference, and in transgenic plants overexpressing GWD. We found that GWD protein levels do not vary over the diel cycle and that the protein has a haft-life of 2 d. Overexpression of GWD does not accelerate starch degradation in leaves, and starch degradation is not inhibited until GWD levels are reduced by 70%. Surprisingly, this degree of reduction also inhibits starch synthesis in the light. To discover the importance of redox regulation, we generated transgenic plants expressing constitutively active GWD. These plants retained normal control of degradation. We conclude that GWD exerts only a low level of control over starch degradation in Arabidopsis leaves. [ABSTRACT FROM AUTHOR]

Indolic secondary metabolites play an important role in pathogen defense in cruciferous plants. In Arabidopsis (Arabidopsis thaliana), in addition to the characteristic phytoalexin camalexin, derivatives of indole-3-carbaldehyde (ICHO) and indole-3- carboxylic acid (ICOOH) are synthesized from tryptophan via the intermediates indole-3-acetaldoxime and indole-3-acetonitrile. Based on feeding experiments combined with nontargeted metabolite profiling, their composition in nontreated and silver nitrate (AgNOB)-treated leaf tissue was comprehensively analyzed. As major derivatives, glucose conjugates of 5-hydroxyindole- 3-carbaldehyde, ICOOH, and 6-hydroxyindole-3-carboxylic acid were identified. Quantification of ICHO and ICOOH derivative pools after glucosidase treatment revealed that, in response to AgNO treatment, their total accumulation level was similar to that of camalexin. ARABIDOPSIS ALDEHYDE OXIDASE1 (AAO1), initially discussed to be involved in the biosynthesis of indole-3- acetic acid, and Cytochrome P450 (CYP) 71B6 were found to be transcriptionally coexpressed with camalexin biosynthetic genes. CYP71B6 was expressed in Saccharomyces cerevisiae and shown to efficiently convert indole-3-acetonitrile into ICHO and ICOOH, thereby releasing cyanide. To evaluate the role of both enzymes in the biosynthesis of ICHO and ICOOH derivatives, knockout and overexpression lines for CYP71B6 and AA01 were established and analyzed for indolic metabolites. The observed metabolic phenotypes suggest that AAO1 functions in the oxidation of ICHO to ICOOH in both nontreated and AgNO3-treated leaves, whereas CYP71B6 is relevant for ICOOH derivative biosynthesis specifically after induction. In summary, a model for the biosynthesis of ICHO and ICOOH derivatives is presented. [ABSTRACT FROM AUTHOR]

The OsCPK4 gene is a member of the complex gene family of calcium-dependent protein kinases in rice (Oryza sativa). Here, we report that OsCPK4 expression is induced by high salinity, drought, and the phytohormone abscisic acid. Moreover, a plasma membrane localization of OsCPK4 was observed by transient expression assays of green fluorescent protein-tagged OsCPK4 in onion (Allium cepa) epidermal cells. Overexpression of OsCPK4 in rice plants significantly enhances tolerance to salt and drought stress. Knockdown rice plants, however, are severely impaired in growth and development. Compared with control plants, OsCPK4 overexpressor plants exhibit stronger water-holding capability and reduced levels of membrane lipid peroxidation and electrolyte leakage under drought or salt stress conditions. Also, salt-treated OsCPK4 seedlings accumulate less Na+ in their roots. We carried out microarray analysis of transgenic rice overexpressing OsCPK4 and found that overexpression of OsCPK4 has a low impact on the rice transcriptome. Moreover, no genes were found to be commonly regulated by OsCPK4 in roots and leaves of rice plants. A significant number of genes involved in lipid metabolism and protection against oxidative stress appear to be up-regulated by OsCPK4 in roots of overexpressor plants. Meanwhile, OsCPK4 overexpression has no effect on the expression of well-characterized abiotic stress-associated transcriptional regulatory networks (i.e. ORYZA SATIVA DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN1 and ORYZA SATIVA No Apical Meristem, Arabidopsis Transcription Activation Factor1-2, Cup-Shaped Cotyledon6 genes) and LATE EMBRYOGENESIS ABUNDANT genes in their roots. Taken together, our data show that OsCPK4 functions as a positive regulator of the salt and drought stress responses in rice via the protection of cellular membranes from stress-induced oxidative damage. [ABSTRACT FROM AUTHOR]

Systemic acquired resistance (SAR) is an inducible immune response that depends on ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1). Here, we show that Arabidopsis (Arabidopsis thaliana) EDS1 is required for both SAR signal generation in primary infected leaves and SAR signal perception in systemic uninfected tissues. In contrast to SAR signal generation, local resistance remains intact in eds1 mutant plants in response to Pseudomonas syringae delivering the effector protein AvrRpm1. We utilized the SAR-specific phenotype of the eds1 mutant to identify new SAR regulatory proteins in plants conditionally expressing AvrRpm1. Comparative proteomic analysis of apoplast-enriched extracts from AvrRpm1-expressing wild-type and eds1 mutant plants led to the identification of 12 APOPLASTIC, EDS1-DEPENDENT (AED) proteins. The genes encoding AED1, a predicted aspartyl protease, and another AED, LEGUME LECTIN-LIKE PROTEIN1 (LLP1), were induced locally and systemically during SAR signaling and locally by salicylic acid (SA) or its functional analog, benzo 1,2,3-thiadiazole-7-carbothioic acid S-methyl ester. Because conditional overaccumulation of AED1-hemagglutinin inhibited SA-induced resistance and SAR but not local resistance, the data suggest that AED1 is part of a homeostatic feedback mechanism regulating systemic immunity. In llp1 mutant plants, SAR was compromised, whereas the local resistance that is normally associated with EDS1 and SA as well as responses to exogenous SA appeared largely unaffected. Together, these data indicate that LLP1 promotes systemic rather than local immunity, possibly in parallel with SA. Our analysis reveals new positive and negative components of SAR and reinforces the notion that SAR represents a distinct phase of plant immunity beyond local resistance. [ABSTRACT FROM AUTHOR]

The development of the embryo in Arabidopsis (Arabidopsis thaliana) involves a carefully controlled set of cell divisions and cell fate decisions that lead to a mature embryo containing shoot and root meristems and all basic tissue types. Over the last 20 years, a number of transcriptional regulators of embryonic patterning have been described, but little is known about the role of posttranscriptional regulators such as microRNAs (miRNAs). Previous work has centered on the study of null or very weak alleles of miRNA biosynthetic genes, but these mutants either arrest early in embryogenesis or have wild-type-looking embryos. Here, we significantly extend those analyses by characterizing embryos mutant for a strong hypomorphic allele of DICER-LIKE1 (dcl1-15). Our data demonstrate that miRNAs are required for the patterning of most regions of the embryo, with the exception of the protoderm. In mutant embryos with the most severe morphological defects, the majority of tissue identities are lost. Different levels of miRNAs appear to be required to specify cell fates in various regions of the embryo. The suspensor needs the lowest levels, followed by the root apical meristem and hypocotyl, cotyledons, and shoot apical meristem. Furthermore, we show that erecta acts as a suppressor of dcl1-15, a novel role for this signaling pathway in embryos. Our results also indicate that the regulation of the messenger RNA levels of miRNA targets involves not just the action of miRNAs but has a significant transcriptional component as well. [ABSTRACT FROM AUTHOR]

Terpenoids represent one of file major classes of natural products and serve different biological ftmctions. In grape (Vitis vinifera), a large fraction of these compounds is present as nonvolatile terpene glycosides. We have extracted putative glycosyltransferase (GT) sequences from the grape genome database that show similarity to Arabidopsis (Arabidopsis thaliana) GTs whose encoded proteins glucosylate a diversity of terpenes. Spatial and temporal expression levels of the potential VvGT genes were determined in five different grapevine varieties. Heterologous expression and biochemical assays of candidate genes led to the identification of a UDP-glucose: monoterpenol β-D-glucosyltransferase (VvGT7). The VvGT7 gene was expressed in various tissues in accordance with monoterpenyl glucoside accumulation in grape cultivars. Twelve allelic VvGT7 genes were isolated from five cultivars, and their encoded proteins were biochemically analyzed. They varied in substrate preference and catalytic activity. Three amino acids, which corresponded to none of the determinants previously identified for other plant GTs, were found to be important for enzymatic catalysis. Site-specific mutagenesis along with the analysis of allelic proteins also revealed amino acids that impact catalytic activity and substrate tolerance. These results demonstrate that VvGT7 may contribute to the production of geranyl and neryl glucoside during grape ripening. [ABSTRACT FROM AUTHOR]

We previously reported that nitric oxide (NO) functions as a signal in thermotolerance. To illustrate its relationship with hydrogen peroxide (H2O2) in the tolerance of Arabidopsis (Arabidopsis thaliana) to heat shock (HS), we investigated the effects of heat on Arabidopsis seedlings of the following types: the wild type; three NADPH oxidase-defective mutants that exhibit reduced endogenous H202 levels (atrbohB, atrbohD, and atrbohB/D); and a mutant that is resistant to inhibition by fosmidomycin (noa1, for nitric oxide-associated proteinl). After HS, the NO levels in atrbohB, atrbohD, and atrbohB/D seedlings were lower than that in wild-type seedlings. Treatment of the seedlings with sodium nitroprusside or S-nitroso-N-acetylpenicillamine partially rescued their heat sensitivity, suggesting that NO is involved in H2O2 signaling as a downstream factor. This point was verified by phenotypic analyses and thermotolerance testing of transgenic seedlings that overexpressed Nitrate reductase! and NOA1, respectively, in an atrbohB/D background. Electrophoretic mobility shift assays, western blotting, and real-time reverse transcription-polymerase chain reaction demonstrated that NO stimulated the DNA-binding activity of HS factors and the accumulation of HS proteins through H2O2. These data indicate that H2O2 acts upstream of NO in thermotolerance, which requires increased HS factor DNA-binding activity and HS protein accumulation. [ABSTRACT FROM AUTHOR]

The effects of water deficit on carbon and nitrogen metabolism were investigated in flag leaves of wild-type and transgenic rice (Oryza sativa japonica 'Kitaake') plants expressing ISOPENTENYLTRANSFERASE (IPT; encoding the enzyme that mediates the rate-limiting step in cytokinin synthesis) under the control of PSARK, a maturation- and stress-induced promoter. While the wild-type plants displayed inhibition of photosynthesis and nitrogen assimilation during water stress, neither carbon nor nitrogen assimilation was affected by stress in the transgenic PSARK::IPT plants. In the transgenic plants, photosynthesis was maintained at control levels during stress and the flag leaf showed increased sucrose (Suc) phosphate synthase activity and reduced Suc synthase and invertase activities, leading to increased Suc contents. The sustained carbon assimilation in the transgenic PSARK::IPT plants was well correlated with enhanced nitrate content, higher nitrate reductase activity, and sustained ammonium contents, indicating that the stress-induced cytokinin synthesis in the transgenic plants played a role in maintaining nitrate acquisition. Protein contents decreased and free amino acids increased in wild-type plants during stress, while protein content was preserved in the transgenic plants. Our results indicate that the stress-induced cytokinin synthesis in the transgenic plants promoted sink strengthening through a cytokinin-dependent coordinated regulation of carbon and nitrogen metabolism that facilitates an enhanced tolerance of the transgenic plants to water deficit. [ABSTRACT FROM AUTHOR]

To better understand water uptake patterns in root systems of woody perennial crops, we detailed the developmental anatomy and hydraulic physiology along the length of grapevine (Vitis berlandieri × Vitis rupestris) fine roots from the tip to secondary growth zones. Our characterization included the localization of suberized structures and aquaporin gene expression and the determination of hydraulic conductivity (Lpr) and aquaporin protein activity (via chemical inhibition) in different root zones under both osmotic and hydrostatic pressure gradients. Tissue-specific messenger RNA levels of the plasma membrane aquaporin isogenes (VvPIPs) were quantified using laser-capture microdissection and quantitative polymerase chain reaction. Our results highlight dramatic changes in structure and function along the length of grapevine fine roots. Although the root tip lacked suberization altogether, a suberized exodermis and endodermis developed in the maturation zone, which gave way to the secondary growth zone containing a multilayer suberized periderm. Longitudinally, VvPIP isogenes exhibited strong peaks of expression in the root tip that decreased precipitously along the root length in a pattern similar to Arabidopsis (Arabidopsis thaliana) roots. In the radial orientation, expression was always greatest in interior tissues (i.e. stele, endodermis, and/or vascular tissues) for all root zones. High Lpr. and aquaporin protein activity were associated with peak VvPIP expression levels in the root tip. This suggests that aquaportns play a limited role in controlling water uptake in secondary growth zones, which contradicts existing theoretical predictions. Despite having significantly lower Lpr, woody roots can constitute the vast majority of the root system surface area in mature vines and thus provide for significant water uptake potential. [ABSTRACT FROM AUTHOR]

To better understand water uptake patterns in root systems of woody perennial crops, we detailed the developmental anatomy and hydraulic physiology along the length of grapevine (Vitis berlandieri × Vitis rupestris) fine roots from the tip to secondary growth zones. Our characterization included the localization of suberized structures and aquaporin gene expression and the determination of hydraulic conductivity (Lpr) and aquaporin protein activity (via chemical inhibition) in different root zones under both osmotic and hydrostatic pressure gradients. Tissue-specific messenger RNA levels of the plasma membrane aquaporin isogenes (VvPIPs) were quantified using laser-capture microdissection and quantitative polymerase chain reaction. Our results highlight dramatic changes in structure and function along the length of grapevine fine roots. Although the root tip lacked suberization altogether, a suberized exodermis and endodermis developed in the maturation zone, which gave way to the secondary growth zone containing a multilayer suberized periderm. Longitudinally, VvPIP isogenes exhibited strong peaks of expression in the root tip that decreased precipitously along the root length in a pattern similar to Arabidopsis (Arabidopsis thaliana) roots. In the radial orientation, expression was always greatest in interior tissues (i.e. stele, endodermis, and/or vascular tissues) for all root zones. High Lpr. and aquaporin protein activity were associated with peak VvPIP expression levels in the root tip. This suggests that aquaportns play a limited role in controlling water uptake in secondary growth zones, which contradicts existing theoretical predictions. Despite having significantly lower Lpr, woody roots can constitute the vast majority of the root system surface area in mature vines and thus provide for significant water uptake potential. [ABSTRACT FROM AUTHOR]

Plants have evolved a complex array of defensive responses against pathogenic microorganisms. Recognition of microbes initiates signaling cascades that activate plant defenses. The membrane lipid phosphatidic acid, produced by phospholipase D (PLD), has been shown to take part in both abiotic and biotic stress signaling. In this study, the involvement of PLD in the interaction between Arabidopsis (Arabidopsis thaliana) and the barley powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) was investigated. This nonadapted pathogen is normally resisted by a cell wall-based defense, which stops the fungal hyphae from penetrating the epidermal cell wall. Chemical inhibition of phosphatidic acid production by PLD increased the penetration rate of Bgh spores on wild-type leaves. The analysis of transfer DNA knockout lines for all Arabidopsis PLD genes revealed that PLDδ is involved in penetration resistance against Bgh, and chemical inhibition of PLDs in plants mutated in PLDδ indicated that this isoform alone is involved in Bgh resistance. In addition, we confirmed the involvement of PLDδ in penetration resistance against another nonadapted pea powdery mildew fungus, Erysiphe pisi. A green fluorescent protein fusion of PLDδ localized to the plasma membrane at the Bgh attack site, where it surrounded the cell wall reinforcement. Furthermore, in the pldδ mutant, transcriptional up-regulation of early microbe-associated molecular pattern response genes was delayed after chitin stimulation. In conclusion, we propose that PLD is involved in defense signaling in nonhost resistance against powdery mildew fungi and put PLDδ forward as the main isoform participating in this process. [ABSTRACT FROM AUTHOR]

Light and the phytohormone abscisic acid (ABA) regulate overlapping processes in plants, such as seed germination and seedling development. However, the molecular mechanism underlying the interaction between light and ABA signaling is largely unknown. Here, we show that FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and FAR-RED IMPAIRED RESPONSE1 (FAR1), two key positive transcription factors in the phytochrome A pathway, directly bind to the promoter of ABA-Insensitive5 and activate its expression in Arabidopsis (Arabidopsis thaliana). Disruption of FHY3 and/or FAR1 reduces the sensitivity to ABA-mediated inhibition of seed germination, seedling development, and primary root growth. The seed germination of the fhy3 mutant is also less sensitive to salt and osmotic stress than that of the wild type. Constitutive expression of ABA-Insensitive5 restores the seed germination response of fhy3. Furthermore, the expression of several ABA-responsive genes is decreased in the fhy3 and/or far1 mutants during seed imbibition. Consistently, FHY3 and FAR1 transcripts are up-regulated by ABA and abiotic stresses. Moreover, the fhy3 and far1 mutants have wider stomata, lose water faster, and are more sensitive to drought than the wild type. These findings demonstrate that FHY3 and FAR1 are positive regulators of ABA signaling and provide insight into the integration of light and ABA signaling, a process that may allow plants to better adapt to environmental stresses. [ABSTRACT FROM AUTHOR]

In the natural environment, days are generally warmer than the night, resulting in a positive day/night temperature difference (+DIF). Plants have adapted to these conditions, and when exposed to antiphase light and temperature cycles (cold photoperiod/warm night [-DIF]), most species exhibit reduced elongation growth. To study the physiological mechanism of how light and temperature cycles affect plant growth, we used infrared imaging to dissect growth dynamics under +DIF and -DIF in the model plant Arabidopsis (Arabidopsis thaliana). We found that -DIF altered leaf growth patterns, decreasing the amplitude and delaying the phase of leaf movement. Ethylene application restored leaf growth in -DIF conditions, and constitutive ethylene signaling mutants maintain robust leaf movement amplitudes under -DIF, indicating that ethylene signaling becomes limiting under these conditions. In response to -DIF, the phase of ethylene emission advanced 2 h, but total ethylene emission was not reduced. However, expression analysis on members of the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase ethylene biosynthesis gene family showed that ACS2 activity is specifically suppressed in the petiole region under -DIF conditions. Indeed, petioles of plants under -DIF had reduced ACC content, and application of ACC to the petiole restored leaf growth patterns. Moreover, acs2 mutants displayed reduced leaf movement under +DIF, similar to wild-type plants under -DIF. In addition, we demonstrate that the photoreceptor PHYTOCHROME B restricts ethylene biosynthesis and constrains the -DIF-induced phase shift in rhythmic growth. Our findings provide a mechanistic insight into how fluctuating temperature cycles regulate plant growth. [ABSTRACT FROM AUTHOR]

Seed endosperm development in Arabidopsis (Arabidopsis thaliana) is under control of the polycomb group complex, which includes Fertilization Independent Endosperm (FIE). The polycomb group complex regulates downstream factors, e.g. Pheres1 (PHE1), by genomic imprinting. In heterozygous fie mutants, an endosperm develops in ovules carrying a maternal fie allele without fertilization, finally leading to abortion. Another endosperm development pathway depends on MINISEED3 (a WRKY10 transcription factor) and HAIKU2 (a leucine-rich repeat kinase). While the role of seed development genes in the embryo and endosperm establishment has been studied in detail, their impact on metabolism and oil accumulation remained unclear. Analysis of oil, protein, and sucrose accumulation in mutants and overexpression plants of the four seed development genes revealed that (1) seeds carrying a maternal fie allele accumulate low oil with an altered composition of triacylglycerol molecular species; (2) homozygous mutant seeds of phe1, mini3, and iku2, which are smaller, accumulate less oil and slightly less protein, and starch, which accumulates early during seed development, remains elevated in mutant seeds; (3) embryo-specific overexpression of FIE, PHE1, and MINI3 has no influence on seed size and weight, nor on oil, protein, or sucrose content; and (4) overexpression of IKU2 results in seeds with increased size and weight, and oil content of overexpressed IKU2 seeds is increased by 35%. Thus, IKU2 overexpression represents a novel strategy for the genetic manipulation of the oil content in seeds. [ABSTRACT FROM AUTHOR]

The synthesis of cysteine (Cys) is a master control switch of plant primary metabolism that coordinates the flux of sulfur with carbon and nitrogen metabolism. In Arabidopsis (Arabidopsis thaliana), nine genes encode for O-acetylserine(thiol)lyase (OAS-TL)-like proteins, of which the major isoforms, OAS-TL A, OAS-TL B, and OAS-TL C, catalyze the formation of Cys by combining O-acetylserine and sulfide in the cytosol, the plastids, and the mitochondria, respectively. So far, the significance of individual OAS-TL-like enzymes is unresolved. Generation of all major OAS-TL double loss-of-function mutants in combination with radiolabeled tracer studies revealed that subcellular localization of OAS-TL proteins is more important for efficient Cys synthesis than total cellular OAS-TL activity in leaves. The absence of oastl triple embryos after targeted crosses indicated the exclusiveness of Cys synthesis by the three major OAS-TLs and ruled out alternative sulfur fixation by other OAS-TL-like proteins. Analyses of oastlABC pollen demonstrated that the presence of at least one functional OAS-TL isoform is essential for the proper function of the male gametophyte, although the synthesis of histidine, lysine, and tryptophan is dispensable in pollen. Comparisons of oastlABC pollen derived from genetically different parent plant combinations allowed us to separate distinct functions of Cys and glutathione in pollen and revealed an additional role of glutathione for pollen germination. In contrast, female gametogenesis was not affected by the absence of major OAS-TLs, indicating significant transport of Cys into the developing ovule from the mother plant. [ABSTRACT FROM AUTHOR]