Frontiers in Plant Science

Published by Frontiers
Online ISSN: 1664-462X
Publications
Article
Complex plant N-glycans containing β1,2-xylose and core α1,3-fucose are regarded as the major class of the so-called "carbohydrate cross-reactive determinants" reactive with IgE antibodies in sera of many allergic patients, but their clinical relevance is still under debate. Plant glycosyltransferases, β1,2-xylosyltransferase (XylT), and core α1,3-fucosyltransferase (FucT) are responsible for the transfer of β1,2-linked xylose and core α1,3-linked fucose residues to N-glycans of glycoproteins, respectively. To test the clinical relevance of β1,2-xylose-containing epitopes, expression of the tomato β1,2-xylosyltransferase was down-regulated by RNA interference (RNAi) in transgenic plants. Fruits harvested from these transgenic plants were analyzed for accumulation of XylT mRNA, abundance of β1,2-xylose epitopes and their allergenic potential. Based on quantitative real-time PCR analysis XylT mRNA levels were reduced up to 10-fold in independent transgenic lines as compared to untransformed control, whereas no xylosylated N-glycans could be revealed by MS analysis. Immunoblotting using anti-xylose-specific IgG antibodies revealed a strong reduction of β1,2-xylose-containing epitopes. Incubating protein extracts from untransformed controls and XylT_RNAi plants with sera from tomato allergic patients showed a patient-specific reduction in IgE-binding, indicating a reduced allergenic potential of XylT_RNAi tomato fruits, in vitro. To elucidate the clinical relevance of β1,2-xylose-containing complex N-glycans skin prick tests were performed demonstrating a reduced responsiveness of tomato allergic patients, in vivo. This study provides strong evidence for the clinical relevance of β1,2-xylose-containing epitopes in vivo.
 
Article
The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca(2+)-dependent protein kinases (CPKs). While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16, and 34). Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with K(M) ∼70 μM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites). Of these, 74 (27%) were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies.
 
Article
Carbon transport processes in plants can be followed non-invasively by repeated application of the short-lived positron-emitting radioisotope (11)C, a technique which has rarely been used with trees. Recently, positron emission tomography (PET) allowing 3D visualization has been adapted for use with plants. To investigate the effects of stem girdling on the flow of assimilates, leaves on first order branches of two-year-old oak (Quercus robur L.) trees were labeled with (11)C by supplying (11)CO2-gas to a leaf cuvette. Magnetic resonance imaging gave an indication of the plant structure, while PET registered the tracer flow in a stem region downstream from the labeled branches. After repeated pulse labeling, phloem translocation was shown to be sectorial in the stem: leaf orthostichy determined the position of the phloem sieve tubes containing labeled (11)C. The observed pathway remained unchanged for days. Tracer time-series derived from each pulse and analysed with a mechanistic model showed for two adjacent heights in the stem a similar velocity but different loss of recent assimilates. With either complete or partial girdling of bark within the monitored region, transport immediately stopped and then resumed in a new location in the stem cross-section, demonstrating the plasticity of sectoriality. One day after partial girdling, the loss of tracer along the interrupted transport pathway increased, while the velocity was enhanced in a non-girdled sector for several days. These findings suggest that lateral sugar transport was enhanced after wounding by a change in the lateral sugar transport path and the axial transport resumed with the development of new conductive tissue.
 
Illustration of procedures for reconstruction of bna572+ and integration of 13C-MFA data.
Loop topology of bna572+. Distribution of loop size (A) and loop membership (B).
Flux map for accession 3231 (13C-MFA). Flux distribution map showing glycolysis, pentosephosphate pathway, the TCA cycle and biosynthetic effluxes into protein, lipid and free soluble metabolites for accession 3231. Arrow thickness indicates carbon flux. Significant higher (lower) values in accession 3170 are marked in blue (red). Gray arrows: high statistical uncertainty (SD > 50% of flux value). Abbreviations: Metabolites (subscripts “p,” chloroplast; “c,” cytosol): AcCoA, acetyl-Coenzyme A; aKIV, 2-keto-isovalerate; C1, 5,10-methylene- or 5-formyl -tetrahydrofolate; Cit, citrate; GOX, glyoxylate; Hex, hexose, representing free hexoses and sucrose; HP, hexose phosphate; Icit, isocitrate; KG, ketoglutarate; OAA, lumped pool of subcellular pools of oxaloacetate and malate; PEP, phosphoenol pyruvate; PGA, 3-phospho glycerate; Pyr, pyruvate; RP, ribose 5-phosphate; RuP, Ribulose 5-phosphate; Succ, succinate; TP, triose phosphate (dihydroxyacetone phosphate, glyceraldehyde 3-phosphate); XP, xylulose 5-phosphate. Reactions: vACL, ATP:citrate lyase; vCS, citrate synthase; vFM, fumarase, malate dehydrogenase; vICDH, isocitrate dehydrogenase; vPDH_p, plastidic pyruvate dehydrogenase; vPK_p, plastidic pyruvate kinase; vRub_p, ribulose bisphosphate carboxylase.
Random sampling of loopless solutions showing the effect of flux- and flux ratio constraints. Genotype 3170. Euclidean distance between random sampled flux vectors, projected onto the 13C-MFA model, and 13C-MFA flux estimates.
Article
The use of large-scale or genome-scale metabolic reconstructions for modeling and simulation of plant metabolism and integration of those models with large-scale omics and experimental flux data is becoming increasingly important in plant metabolic research. Here we report an updated version of bna572, a bottom-up reconstruction of oilseed rape (Brassica napus L.; Brassicaceae) developing seeds with emphasis on representation of biomass-component biosynthesis. New features include additional seed-relevant pathways for isoprenoid, sterol, phenylpropanoid, flavonoid, and choline biosynthesis. Being now based on standardized data formats and procedures for model reconstruction, bna572+ is available as a COBRA-compliant Systems Biology Markup Language (SBML) model and conforms to the Minimum Information Requested in the Annotation of Biochemical Models (MIRIAM) standards for annotation of external data resources. Bna572+ contains 966 genes, 671 reactions, and 666 metabolites distributed among 11 subcellular compartments. It is referenced to the Arabidopsis thaliana genome, with gene-protein-reaction (GPR) associations resolving subcellular localization. Detailed mass and charge balancing and confidence scoring were applied to all reactions. Using B. napus seed specific transcriptome data, expression was verified for 78% of bna572+ genes and 97% of reactions. Alongside bna572+ we also present a revised carbon centric model for (13)C-Metabolic Flux Analysis ((13)C-MFA) with all its reactions being referenced to bna572+ based on linear projections. By integration of flux ratio constraints obtained from (13)C-MFA and by elimination of infinite flux bounds around thermodynamically infeasible loops based on COBRA loopless methods, we demonstrate improvements in predictive power of Flux Variability Analysis (FVA). Using this combined approach we characterize the difference in metabolic flux of developing seeds of two B. napus genotypes contrasting in starch and oil content.
 
Article
The 14-3-3 proteins have emerged as major phosphoprotein interaction proteins and thereby constitute a key node in the Arabidopsis Interactome Map, a node through which a large number of important signals pass. Throughout their history of discovery and description, the 14-3-3s have been described as protein families and there has been some evidence that the different 14-3-3 family members within any organism might carry isoform-specific functions. However, there has also been evidence for redundancy of 14-3-3 function, suggesting that the perceived 14-3-3 diversity may be the accumulation of neutral mutations over evolutionary time and as some 14-3-3 genes develop tissue or organ-specific expression. This situation has led to a currently unresolved question - does 14-3-3 isoform sequence diversity indicate functional diversity at the biochemical or cellular level? We discuss here some of the key observations on both sides of the resulting debate, and present a set of contrastable observations to address the theory functional diversity does exist among 14-3-3 isoforms. The resulting model suggests strongly that there are indeed functional specificities in the 14-3-3s of Arabidopsis. The model further suggests that 14-3-3 diversity and specificity should enter into the discussion of 14-3-3 roles in signal transduction and be directly approached in 14-3-3 experimentation. It is hoped that future studies involving 14-3-3s will continue to address specificity in experimental design and analysis.
 
| A summary of prominent membrane transporters recently identified as putative Arabidopsis 14-3-3 interactors.
Ribbon plot of a 14-3-3 dimer (gray) in complex with two phosphopeptides derived from the transcription factor OsFD1 (orange, the phosphorylated residue is indicated by an arrow, respectively) and two Hd3a proteins [color coded from the N-terminus (blue) to the C-terminus (red)] (Taoka et al., 2011). While binding of the OsFD1 phosphopeptides occurs in an extended conformation within the amphipathic groove of each 14-3-3 monomer, the “florigen” Hd3a attaches to the “outside” of the groove, respectively.
Article
14-3-3 Dimers are well known to interact with diverse target proteins throughout eukaryotes. Most notably, association of 14-3-3s commonly requires phosphorylation of a serine or threonine residue within a specific sequence motif of the client protein. Studies with a focus on individual target proteins have unequivocally demonstrated 14-3-3s to be the crucial factors modifying the client's activity state upon phosphorylation and, thus, finishing the job initiated by a kinase. In this respect, a recent in-depth analysis of the rice transcription factor FLOWERING LOCUS D1 (OsFD1) revealed 14-3-3s to be essential players in floral induction. Such fascinating discoveries, however, can often be ascribed to the random identification of 14-3-3 as an interaction partner of the favorite protein. In contrast, our understanding of 14-3-3 function in higher organisms is frustratingly limited, mainly due to an overwhelming spectrum of putative targets in combination with the existence of a multigene 14-3-3 family. In this review we will discuss our current understanding of the function of plant 14-3-3 proteins, taking into account recent surveys of the Arabidopsis 14-3-3 interactome.
 
Article
δ(15)N signals in plant and soil material integrate over a number of biogeochemical processes related to nitrogen (N) and therefore provide information on net effects of multiple processes on N dynamics. In general little is known in many grassland restoration projects on soil-plant N dynamics in relation to the restoration treatments. In particular, δ(15)N signals may be a useful tool to assess whether abiotic restoration treatments have produced the desired result. In this study we used the range of abiotic and biotic conditions provided by a restoration experiment to assess to whether the restoration treatments and/or plant functional identity and legume neighborhood affected plant δ(15)N signals. The restoration treatments consisted of hay transfer and topsoil removal, thus representing increasing restoration effort, from no restoration measures, through biotic manipulation to major abiotic manipulation. We measured δ(15)N and %N in six different plant species (two non-legumes and four legumes) across the restoration treatments. We found that restoration treatments were clearly reflected in δ(15)N of the non-legume species, with very depleted δ(15)N associated with low soil N, and our results suggest this may be linked to uptake of ammonium (rather than nitrate). The two non-legume species differed considerably in their δ(15)N signals, which may be related to the two species forming different kinds of mycorrhizal symbioses. Plant δ(15)N signals could clearly separate legumes from non-legumes, but our results did not allow for an assessment of legume neighborhood effects on non-legume δ(15)N signals. We discuss our results in the light of what the δ(15)N signals may be telling us about plant-soil N dynamics and their potential value as an indicator for N dynamics in restoration.
 
Article
Here we present the first study, in which a large number of different vascular epiphyte species were measured for their photosynthetic performance in the natural environment of their phorophyte in the lowland rainforest of French Guyana. More than 70 epiphyte species covered the host tree in a dense cover. Of these, the photosynthesis of 16 abundant species was analyzed intensely over several months. Moreover, the light environment was characterized with newly developed light sensors that recorded continuously and with high temporal resolution light intensity next to the epiphytes. Light intensity was highly fluctuating and showed great site specific spatio-temporal variations of photosynthetic photon flux. Using a novel computer routine we quantified the integrated light intensity the epiphytes were exposed to in a 3 h window and we related this light intensity to measurements of the actual photosynthetic status. It could be shown that the photosynthetic apparatus of the epiphytes was well adapted to the quickly changing light conditions. Some of the epiphytes were chronically photoinhibited at predawn and significant acute photoinhibition, expressed by a reduction of potential quantum efficiency (F(v)/F(m))(30'), was observed during the day. By correlating (F(v)/F(m))(30') to the integrated and weighted light intensity perceived during the previous 3 h, it became clear that acute photoinhibition was related to light environment prior to the measurements. Additionally photosynthetic performance was not determined by rain events, with the exception of an Aechmea species. This holds true for all the other 15 species of this study and we thus conclude that actual photosynthesis of these tropical epiphytes was determined by the specific and fluctuating light conditions of their microhabitat and cannot be simply attributed to light-adapted ancestors.
 
Article
Mutations in the Lotus japonicus nucleoporin genes, NUP85, NUP133, and NENA (SEH1), lead to defects in plant-microbe symbiotic signaling. The homologous proteins in yeast and vertebrates are part of the conserved NUP84/NUP107-160 subcomplex, which is an essential component of the nuclear pore scaffold and has a pivotal role in nuclear pore complex (NPC) assembly. Loss and down-regulation of NUP84/NUP107-160 members has previously been correlated with a variety of growth and molecular defects, however, in L. japonicus only surprisingly specific phenotypes have been reported. We investigated whether Lotus nup85, nup133, and nena mutants exhibit general defects in NPC composition and distribution. Whole mount immunolocalization confirmed a typical nucleoporin-like localization for NUP133, which was unchanged in the nup85-1 mutant. Severe NPC clustering and aberrations in the nuclear envelope have been reported for Saccharomyces cerevisiae nup85 and nup133 mutants. However, upon transmission electron microscopy analysis of L. japonicus nup85, nup133 and nena, we detected only a slight reduction in the average distances between neighboring NPCs in nup133. Using quantitative immunodetection on protein-blots we observed that loss of individual nucleoporins affected the protein levels of other NUP107-160 complex members. Unlike the single mutants, nup85/nup133 double mutants exhibited severe temperature dependent growth and developmental defects, suggesting that the loss of more than one NUP107-160 member affects basal functions of the NPC.
 
Article
Plant height is an important agronomic and horticultural trait that impacts plant productivity, durability and esthetic appeal. A number of the plant hormones such as gibberellic acid (GA), auxin and ethylene have been linked to control of plant architecture and size. Reduction in GA synthesis and auxin transport result in dwarfism while ethylene may have a permissive or repressive effect on tissue growth depending upon the age of plant tissues or the environmental conditions considered. We describe here an activation-tagged mutant of Populus tremula x P. alba clone 717-1B4 identified from 2000 independent transgenic lines due to its significantly reduced growth rate and smaller leaf size. Named dwarfy, the phenotype is due to increased expression of PtaACC SYNTHASE8, which codes for an enzyme in the first committed step in the biosynthesis of ethylene. Stems of dwarfy contain fiber and vessel elements that are reduced in length while leaves contain fewer cells. These morphological differences are linked to PtaACS8 inducing different transcriptomic programs in the stem and leaf, with genes related to auxin diffusion and sensing being repressed in the stem and genes related to cell division found to be repressed in the leaves. Altogether, our study gives mechanistic insight into the genetics underpinning ethylene-induced dwarfism in a perennial model organism.
 
Article
Over the past two decades revolutionary progress in plant biology became possible by focusing resources on a single plant reference system, Arabidopsis thaliana. After the completion of the Arabidopsis genome sequence in the year 2000, a coordinated multinational effort was launched to “determine the function of every gene in Arabidopsis” by the year 2010. Part of this effort was the German Arabidopsis Functional Genomics Network (AFGN). Established in 2001, AFGN was continuously supported for 9 years by the German Research Foundation (DFG). Over 85 German researchers contributed to AFGN, partially in close bilateral collaboration with scientists of the NSF-funded Arabidopsis 2010 initiative. While the ambitious goal of determining the function of every Arabidopsis gene has not yet been fully achieved, the Arabidopsis genome is now one of the best annotated and serves as the gold standard for plant and other genomes. A large and international community has established novel methods, toolkits and genomic resources, such as sequence-indexed mutant collections and comprehensive and easily accessible “omics-scale datasets,” ranging from transcriptome over proteome to the metabolome. One prominent example is the AtGenExpress data set, which was partially realized by AFGN scientists and serves as the gold standard in microarray-based transcriptomics. The Arabidopsis 2010 program evolved from studying the functions of single genes and gene families to comprehensive systems-wide analyses of functional networks, thereby paving the way from descriptive to predictive plant science. Progress does not stop here—in the near future, the genomes of 1000 Arabidopsis strains and accessions will become available, which will make it possible to exploit existing natural variation for addressing fundamental questions in ecology and evolutionary biology in an unprecedented manner. Further, due to ease of transformation and existing genetic and genomic resources, Arabidopsis will likely serve as a chassis for synthetic plant biology, an emerging field and challenge for the next decade of plant research (EU 2020 Vision of Plant Science, 2008; An International Model for the Future of Plant Science, 2010). This special issue of Frontiers in Plant Physiology will provides 20 examples from the ongoing research of AFGN and Arabidopsis 2010 members on how focusing on a single plant model system has impacted and revolutionized many fields of plant research and it will provide an outlook on the upcoming challenges and fields of research for the next decade of Arabidopsis research.
 
| Web presentation of Soybean Proteome Database (SPD). (A) Overview of 2-DE maps (http://proteome.dc.affrc.go.jp/cgi-bin/2d/ 2d_view_map.cgi). Particular gel maps can be selected and properties of proteins on the map can be retrieved from database. (B) The 'omics
| Clustering results for 36 proteins identified in more than one organ and/or subcellular compartment. Accession numbers for the proteins are indicated on the right-hand side. Colored boxes indicate an identified protein found in the organ (green) or a subcellular compartment (yellow). Samples from seven organs, Cot (cotyledon), EA (embryonic axis), RH (radicle plus hypocotyl), RT (root tip), Root, Hyp (hypocotyls), and Leaf, and four subcellular compartments, PM (plasma membrane), CW (cell wall), Mit (mitochondrion), and Chloro (chloroplast), were investigated. The samples of Cot, EA, RH, RT, Root, Hyp, and Leaf
Web presentation of Soybean Proteome Database (SPD). (A) Overview of 2-DE maps (http://proteome.dc.affrc.go.jp/cgi-bin/2d/2d_view_map.cgi). Particular gel maps can be selected and properties of proteins on the map can be retrieved from database. (B) The ‘omics table (http://proteome.dc.affrc.go.jp/Soybean/omics/). Resource information of transcripts, proteins, and metabolites can be accessed in a unified manner. Corresponding counterparts are shown in a row.
Clustering results for 36 proteins identified in more than one organ and/or subcellular compartment. Accession numbers for the proteins are indicated on the right-hand side. Colored boxes indicate an identified protein found in the organ (green) or a subcellular compartment (yellow). Samples from seven organs, Cot (cotyledon), EA (embryonic axis), RH (radicle plus hypocotyl), RT (root tip), Root, Hyp (hypocotyls), and Leaf, and four subcellular compartments, PM (plasma membrane), CW (cell wall), Mit (mitochondrion), and Chloro (chloroplast), were investigated. The samples of Cot, EA, RH, RT, Root, Hyp, and Leaf were extracted 0, 0, 2, 3, 7, 7, and 7 days after seedling emergence, respectively. The samples comprising PM, CW, Mit, and Chloro were extracted 3, 4, 4, and 7 days after seedling emergence, respectively. Clustering was conducted based on the identification of corresponding proteins, as identified (1) or not-identified (0), separately for organs and subcellular compartments. Hierarchical clustering was performed using Gene Cluster 3.0 (de Hoon et al., 2004) with Euclidean distance and centroid linkage method. The resulting clusters were visualized using JAVA TREEVIEW (Saldanha, 2004).
Article
The Soybean Proteome Database (SPD) was created to provide a data repository for functional analyses of soybean responses to flooding stress, thought to be a major constraint for establishment and production of this plant. Since the last publication of the SPD, we thoroughly enhanced the contents of database, particularly protein samples and their annotations from several organelles. The current release contains 23 reference maps of soybean (Glycine max cv. Enrei) proteins collected from several organs, tissues, and organelles including the maps for plasma membrane, cell wall, chloroplast, and mitochondrion, which were analyzed by two-dimensional polyacrylamide gels. Furthermore, the proteins analyzed with gel-free proteomics technique have been added and are available online. In addition to protein fluctuations under flooding, those of salt and drought stress have been included in the current release. A case analysis employing a portion of those newly released data was conducted, and the results will be shown. An 'omics table has also been provided to reveal relationships among mRNAs, proteins, and metabolites with a unified temporal-profile tag in order to facilitate retrieval of the data based on the temporal profiles. An intuitive user interface based on dynamic HTML enables users to browse the network as well as the profiles of the multiple "omes" in an integrated fashion. The SPD is available at: http://proteome.dc.affrc.go.jp/Soybean/
 
Function of E3 ligases in abiotic stress response. (A) Most common type of plant E3s. Ubiquitin ligases are categorized based on the presence of a RING, HECT, or U-box E2-binding domain. RING and U-box domain-containing E3s mediate transfer of ubiquitin (U) directly from the E2-Ub intermediate to the substrate protein. HECT domain-containing E3s form an E3-Ub intermediate prior to the transfer of ubiquitin to the substrate protein. RING domains are found in monomeric E3s and multisubunit CRLs. (B) Illustrations of the most common modes of action for E3 ligases in regulating abiotic stress responses. (1) E3 ligases may function as a negative response regulators required to supress stress response pathways by targeting positive regulators for degradation. (2) E3 ligases may promote stress signaling by functioning as positive response regulators that target negative regulators for degradation following stress perception. (3) E3 ligases may also function to attenuate stress signaling by targeting positive regulators for degradation.
Ubiquitin ligases that regulate ABA signaling. Illustration of E3 ligases that regulate ABA synthesis, signal transduction and response. Not all E3 ligases are shown, mainly those with identified substrates. Question marks and dashed lines denote instances where proteasomal-dependent degradation is reported but the E3 ligase involved is unknown.
Article
Ubiquitin is a small, highly conserved, ubiquitously expressed eukaryotic protein with immensely important and diverse regulatory functions. A well-studied function of ubiquitin is its role in selective proteolysis by the ubiquitin-proteasome system (UPS). The UPS has emerged as an integral player in plant response and adaptation to environmental stresses such as drought, salinity, cold and nutrient deprivation. The UPS has also been shown to influence the production and signal transduction of stress-related hormones such as abscisic acid. Understanding UPS function has centered mainly on defining the role of E3 ubiquitin ligases, which are the substrate-recruiting component of the ubiquitination pathway. The recent identification of stress signaling/regulatory proteins that are the subject of ubiquitin-dependent degradation has increased our knowledge of how the UPS facilitates responses to adverse environmental conditions. A brief overview is provided on role of the UPS in modulating protein stability during abiotic stress signaling. E3 ubiquitin ligases for which stress-related substrate proteins have been identified are discussed.
 
| Column names and information included in the "protein spreadsheet" file.
| Integration of the mitochondrial reference maps at GelMap into MASCP Gator (http://gator.masc-proteomics.org/). Upon submission of a protein accession (here: At2g47260), matching
Reference map of the mitochondrial proteome of Arabidopsis thaliana (Klodmann et al., 2011; http://www.gelmap.de/47). Proteins were separated by 2D Blue native/SDS PAGE. The two icons given in the header allow access to the “protein spreadsheet” and the “peptide spreadsheet.”
Integration of the mitochondrial reference maps at GelMap into MASCP Gator (http://gator.masc-proteomics.org/). Upon submission of a protein accession (here: At2g47260), matching peptides from GelMap are graphically displayed at the Gator site. Direct access to GelMap is provided by links in the left side menu of the Gator.
Article
In classical proteome analyses, final experimental data are (a) images of 2D protein separations obtained by gel electrophoresis and (b) corresponding lists of proteins which were identified by mass spectrometry (MS). For data annotation, software tools were developed which allow the linking of protein identity data directly to 2D gels ("clickable gels"). GelMap is a new online software tool to annotate 2D protein maps. It allows (i) functional annotation of all identified proteins according to biological categories defined by the user, e.g., subcellular localization, metabolic pathway, or assignment to a protein complex and (ii) annotation of several proteins per analyzed protein "spot" according to MS primary data. Options to differentially display proteins of functional categories offer new opportunities for data evaluation. For instance, if used for the annotation of 2D Blue native/SDS gels, GelMap allows the identification of protein complexes of low abundance. A web portal has been established for presentation and evaluation of protein identity data related to 2D gels and is freely accessible at http://www.gelmap.de/.
 
Article
The discovery of abundant plasmodesmata at the bundle sheath/phloem interface in Oleaceae (Gamalei, 1974) and Cucurbitaceae (Turgeon et al., 1975) raised the questions as to whether these plasmodesmata are functional in phloem loading and how widespread symplasmic loading would be. Analysis of over 800 dicot species allowed the definition of "open" and "closed" types of the minor vein phloem depending on the abundance of plasmodesmata between companion cells and bundle sheath (Gamalei, 1989, 1990). These types corresponded to potential symplasmic and apoplasmic phloem loaders, respectively; however, this definition covered a spectrum of diverse structures of phloem endings. Here, a review of detailed cytological analyses of minor veins in 320 species from the subclass Asteridae is presented, including data on companion cell types and their combinations which have not been reported previously. The percentage of Asteridae species with "open" minor vein cytology which also contain sieve-element-companion cell complexes with "closed" cytology, i.e., that show specialization for both symplasmic and apoplasmic phloem loading, was determined. Along with recent data confirming the dissimilar functional specialization of structurally different parts of minor vein phloem in the stachyose-translocating species Alonsoa meridionalis (Voitsekhovskaja et al., 2009), these findings suggest that apoplasmic loading is indispensable in a large group of species previously classified as putative symplasmic loaders. Altogether, this study provides formal classifications of companion cells and of minor veins, respectively, in 24 families of the Asteridae based on their structural features, opening the way to a close investigation of the relationship between structure and function in phloem loading.
 
Investigation of complex I subunits from different tissues of Arabidopsis thaliana by 3D BN/SDS/SDS-PAGE. Total mitochondrial protein from cell culture, leaves, and roots (1200 μg each) was resolved by BN-PAGE in a first dimension. Complex I was cut out from the BN gel and used for second gel dimensions [SDS-PAGE within a 10% polyacrylamide (PAA) gel in the presence of 6 M urea]. Lanes from the second dimension gels were again cut out and transferred horizontally onto third gel dimensions (SDS-PAGE within a 16% PAA gel in the absence of urea). Gels were stained with Coomassie colloidal. (A) Complex I of cell cultures, (B) of leaves, (C) of roots. Molecular masses (in kilodaltons) are given to the left and on the top of the gels.
| Complex I subunits in Arabidopsis thaliana.
3D map of complex I from Arabidopsis thaliana cell culture. Total mitochondrial protein (1200 μg each) was resolved by 3D BN/SDS/SDS-PAGE. (A) Coomassie-stained gel, (B) same gel as in (A) indicating protein spots which have been analyzed by mass spectrometry. Numbers correspond to those given in Table 1. Red arrows indicate the newly identified subunits B14.5a and B9. Molecular masses (in kilodaltons) are given to the left and on the top of the gels.
GelMap of complex I as resolved by 3D BN/SDS/SDS-PAGE (http://www.gelmap.de/arabidopsis-3d-complex-i/). Upon hovering with the cursor over a spot, a tooltip including information on all included proteins is opened. In the example given on the figure, the indicated spot includes the CAL2 protein and two isoforms of the TYKY subunit. Upon clicking into the spot the protein names are converted into stable links which can be used to obtain further information. Protein information also can be obtained by clicking into the menu given to the right or by entering protein names or accessions into the search field below the menu.
Article
Complex I has a unique structure in plants and includes extra subunits. Here, we present a novel study to define its protein constituents. Mitochondria were isolated from Arabidopsis thaliana cell cultures, leaves, and roots. Subunits of complex I were resolved by 3D blue-native (BN)/SDS/SDS-PAGE and identified by mass spectrometry. Overall, 55 distinct proteins were found, seven of which occur in pairs of isoforms. We present evidence that Arabidopsis complex I consists of 49 distinct types of subunits, 40 of which represent homologs of bovine complex I. The nine other subunits represent special proteins absent in the animal linage of eukaryotes, most prominently a group of subunits related to bacterial gamma-type carbonic anhydrases. A GelMap http://www.gelmap.de/arabidopsis-3d-complex-i/ is presented for promoting future complex I research in Arabidopsis thaliana.
 
The modeled 3D scene of the tomato crop: (A) A crop of 32 plants (wireframe with black leaves, green unripe trusses and red ripe trusses) on two double rows of slabs at 0.8 m above the floor, with LED (red squares) inside the double row at 2.4 m height and HPS lamps (brown squares) at 1.5 m above the crop, (B) cross-section of four plants with leaf angles increasing with height (LED position at brown square), (C) rendered image of a single leaf with its rachis at a specific angle (red arrow) with the main stem, and consisting of leaflets that hang down by 30-50° (see blue arrow) relative to the horizontal.
Diurnal pattern of light absorption (% of incoming radiation) by the crop on day 180 for three scenarios of leaf angles.
Projected light pattern of HPS lamps without plants, on a horizontal plane at 2.5 m distance from the lamps, with sensor trajectories (A – A and B – B) indicated by dashed lines, lamp positions are shown by star-like symbols, and the double plant rows by green lines.
Computed light intensity (W per sensor with 6 cm radius) at the trajectories of Figure 3, with and without light intercepting plants (plant positions indicated by arrows). For this simulation the HPS lamps had an arbitrary light output of 1000 W m-2.
Article
Reduction of energy use for assimilation lighting is one of the most urgent goals of current greenhouse horticulture in the Netherlands. In recent years numerous lighting systems have been tested in greenhouses, yet their efficiency has been very difficult to measure in practice. This simulation study evaluated a number of lighting strategies using a 3D light model for natural and artificial light in combination with a 3D model of tomato. The modeling platform GroIMP was used for the simulation study. The crop was represented by 3D virtual plants of tomato with fixed architecture. Detailed data on greenhouse architecture and lamp emission patterns of different light sources were incorporated in the model. A number of illumination strategies were modeled with the calibrated model. Results were compared to the standard configuration. Moreover, adaptation of leaf angles was incorporated for testing their effect on light use efficiency (LUE). A Farquhar photosynthesis model was used to translate the absorbed light for each leaf into a produced amount of carbohydrates. The carbohydrates produced by the crop per unit emitted light from sun or high pressure sodium lamps was the highest for horizontal leaf angles or slightly downward pointing leaves, and was less for more upward leaf orientations. The simulated leaf angles did not affect light absorption from inter-lighting LED modules, but the scenario with LEDs shining slightly upward (20(°)) increased light absorption and LUE relative to default horizontal beaming LEDs. Furthermore, the model showed that leaf orientation more perpendicular to the string of LEDs increased LED light interception. The combination of a ray tracer and a 3D crop model could compute optimal lighting of leaves by quantification of light fluxes and illustration by rendered lighting patterns. Results indicate that illumination efficiency increases when the lamp light is directed at most to leaves that have a high photosynthetic potential.
 
Article
The apical plasma membrane of pollen tubes contains different PI4P 5-kinases that all produce phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] but exert distinct cellular effects. In the present example, overexpression of Arabidopsis AtPIP5K5 or tobacco NtPIP5K6-1 caused growth defects previously attributed to increased pectin secretion. In contrast, overexpression of Arabidopsis AtPIP5K2 caused apical tip swelling implicated in altering actin fine structure in the pollen tube apex. AtPIP5K5, NtPIP5K6-1, and AtPIP5K2 share identical domain structures. Domains required for correct membrane association of the enzymes were identified by systematic deletion of N-terminal domains and subsequent expression of fluorescence-tagged enzyme truncations in tobacco pollen tubes. A variable linker region (Lin) contained in all PI4P 5-kinase isoforms of subfamily B, but not conserved in sequence, was recognized to be necessary for correct subcellular localization of AtPIP5K5, NtPIP5K6-1, and AtPIP5K2. Deletion of N-terminal domains including the Lin domain did not impair catalytic activity of recombinant AtPIP5K5, NtPIP5K6-1, or AtPIP5K2 in vitro; however, the presence of the Lin domain was necessary for in vivo effects on pollen tube growth upon overexpression of truncated enzymes. Overexpression of catalytically inactive variants of AtPIP5K5, NtPIP5K6-1, or AtPIP5K2 did not influence pollen tube growth, indicating that PtdIns(4,5)P2 production rather than structural properties of PI4P 5-kinases was relevant for the manifestation of growth phenotypes. When Lin domains were swapped between NtPIP5K6-1 and AtPIP5K2 and the chimeric enzymes overexpressed in pollen tubes, the chimeras reciprocally gained the capabilities to invoke tip swelling or secretion phenotypes, respectively. The data indicate that the Lin domain directed the enzymes into different regulatory contexts, possibly contributing to channeling of PtdIns(4,5)P2 at the interface of secretion and actin cytoskeleton.
 
| Overview of ubiquitin K63 chain dependent processes. The scheme is a composite of data from different organisms. The leftmost column, endocytosis of uracil permease Fur4 via ubiquitin ligase Rsp5, was investigated in S. cerevisiae. Ubiquitin transfer via Rsp5 occurs from a Cys
Structure of diubiquitin linked via different Lys residues. Linkage of donor ubiquitin (orange) to Lys63 of the acceptor ubiquitin (blue) results in positioning the donor opposite to the C-terminal Gly of the acceptor (cyan).This structure is similar, but not identical to ubiquitin linked via the amino-terminal Met, which is the co-translational peptide bond linkage of many ubiquitin precursors. In contrast, linkage of the donor to Lys48 gives a more compact structure, with the donor positioned on the side of the acceptor. Similarly, use of Lys11 for linkage positions the donor on the opposite side of the acceptor ubiquitin compared to Lys48 linkages. Longer chains of Lys48 or Lys11 linked ubiquitin are therefore more compact than Lys63 chains and are known as signals for proteasomal degradation, whereas Lys63 chains are believed to have distinct roles, independent of the proteasome. Amino acid Ile44 was colored in light green to highlight the orientation of ubiquitin moieties, the Lys residue of the acceptor and the C-terminal Gly of the donor used for linkage formation are colored in red. Images were withdrawn from RCSB protein data bank [2W9N (M1), 3H7P (K63), 2PE9 (K48), and 2MBO (K11)] and processed with program CCP4 Molecular Graphics.
Overview of ubiquitin K63 chain dependent processes. The scheme is a composite of data from different organisms. The leftmost column, endocytosis of uracil permease Fur4 via ubiquitin ligase Rsp5, was investigated in S. cerevisiae. Ubiquitin transfer via Rsp5 occurs from a Cys residue in the ligase HECT domain, Ubc13/Mms2 is not involved. RNF8 and RNF168 (next columns) were characterized in mammals, and no plant homologs are known to date. The other schemes refer directly to processes in plants. See text for further details.
Article
Covalent attachment of the small modifier ubiquitin to Lys ε-amino groups of proteins is surprisingly diverse. Once attached to a substrate, ubiquitin is itself frequently modified by ubiquitin, to form chains. All seven Lys residues of ubiquitin, as well as its N-terminal Met, can be ubiquitylated, implying cellular occurrence of different ubiquitin chain types. The available data suggest that the synthesis, recognition, and hydrolysis of different chain types are precisely regulated. This remarkable extent of control underlies a versatile cellular response to substrate ubiquitylation. In this review, we focus on roles of Lys63-linked ubiquitin chains in plants. Despite limited available knowledge, several recent findings illustrate the importance of these chains as signaling components in plants.
 
Article
Glutaredoxins are small redox proteins able to reduce disulfides and mixed disulfides between GSH and proteins. Synechocystis sp. PCC 6803 contains three genes coding for glutaredoxins: ssr2061 (grxA) and slr1562 (grxB) code for dithiolic glutaredoxins while slr1846 (grxC) codes for a monothiolic glutaredoxin. We have analyzed the expression of these glutaredoxins in response to different stresses, such as high light, H2O2 and heat shock. Analysis of the mRNA levels showed that grxA is only induced by heat while grxC is repressed by heat shock and is induced by high light and H2O2. In contrast, grxB expression was maintained almost constant under all conditions. Analysis of GrxA and GrxC protein levels by western blot showed that GrxA increases in response to high light, heat or H2O2 while GrxC is only induced by high light and H2O2, in accordance with its mRNA levels. In addition, we have also generated mutants that have interrupted one, two, or three glutaredoxin genes. These mutants were viable and did not show any different phenotype from the WT under standard growth conditions. Nevertheless, analysis of these mutants under several stress conditions revealed that single grxA mutants grow slower after H2O2, heat and high light treatments, while mutants in grxB are indistinguishable from WT. grxC mutants were hypersensitive to treatments with H2O2, heat, high light and metals. A double grxAgrxC mutant was found to be even more sensitive to H2O2 than each corresponding single mutants. Surprisingly a mutation in grxB suppressed totally or partially the phenotypes of grxA and grxC mutants except the H2O2 sensitivity of the grxC mutant. This suggests that grxA and grxC participate in independent pathways while grxA and grxB participate in a common pathway for H2O2 resistance. The data presented here show that glutaredoxins are essential for stress adaptation in cyanobacteria, although their targets and mechanism of action remain unidentified.
 
Article
Glutaredoxin from the cyanobacterium Synechocystis sp. PCC 6803 is a small protein, containing only 88 amino acids, that participates in a large number of redox reactions, serving both as an electron donor for enzyme-catalyzed reductions and as a regulator of diverse metabolic pathways. The crystal structures of glutaredoxins from several species have been solved, including the glutaredoxin A isoform from the cyanobacterium Synechocystis sp. PCC 6803. We have utilized the small size of Synechocystis glutaredoxin A and its propensity to form protein crystals that diffract to high resolution to explore a long-standing question in biochemistry; i.e., what are the effects of mutations on protein structure and function? Taking advantage of these properties, we have initiated a long-term educational project that would examine the structural and biochemical changes in glutaredoxin as a function of single-point mutational replacements. Here, we report some of the mutational effects that we have observed to date.
 
Morphological defects in the transgenic plants that express 6b protein. (A) Schematic domain organization of 6b protein. The acidic region (residues 164–184) is present in 6b. (B) Photographs showing clearly the reported phenotypes of transgenic tobacco plants. Transgenic plants transformed with the empty vector (a,c) or 6b gene (b,d,e). The 6b-transgenic seedling (b) and young plant (d) exhibiting mild defects and the 6b-plant (e) exhibiting severe defects. Arrowheads indicate protrusions on the abaxial side of leaves. Scale bars: 10 mm. (C) Photographs showing clearly the reported phenotypes of non-transgenic and transgenic Arabidopsis plants. The 6b-transgenic Arabidopsis plants (a,c); non-transgenic Col-0 plants (b,d). Scale bars: 1 mm (a,b), 10 mm (c,d). Some of pictures were published previously ( Terakura et al., 2006).
Working hypothesis of 6b action. See details in text. NtSIPs represent NtSIP1 or NtSIP2. They might independently bind to 6b and/or both might simultaneously bind to 6b.
Article
Crown gall tumors are formed mainly by actions of a group of genes in the T-DNA that is transferred from Agrobacterium tumefaciens and integrated into the nuclear DNA of host plants. These genes encode enzymes for biosynthesis of auxin and cytokinin in plant cells. Gene 6b in the T-DNA affects tumor morphology and this gene alone is able to induce small tumors on certain plant species. In addition, unorganized calli are induced from leaf disks of tobacco that are incubated on phytohormone-free media; shooty teratomas, and morphologically abnormal plants, which might be due to enhanced competence of cell division and meristematic states, are regenerated from the calli. Thus, the 6b gene appears to stimulate a reprogramming process in plants. To uncover mechanisms behind this process, various approaches including the yeast-two-hybrid system have been exploited and histone H3 was identified as one of the proteins that interact with 6b. It has been also demonstrated that 6b acts as a histone H3 chaperon in vitro and affects the expression of various genes related to cell division competence and the maintenance of meristematic states. We discuss current views on a role of 6b protein in tumorigenesis and reprogramming in plants.
 
Article
Heritable changes of phenotype arising in plant ontogenesis by the influence of environmental factors belong to the most intriguing genetic phenomena. An unusual inheritance pattern was detected during examination of male fertility restoration in the CMS-inducing "9E" type cytoplasm of sorghum: Rf-genes were functional in self-pollinated progeny of F(1) hybrids yet were either not expressed or poorly expressed in backcrosses of these hybrids to CMS-lines with the same cytoplasm type. In experiments on parallel growing of the same F(1) hybrid combinations in the "dry plot" and in the "irrigated plot," it was found that high level of plant water availability during panicle and pollen developmental stages significantly increased male fertility of F(1) and test-cross hybrid populations, in which fertility-restoring genes were in heterozygote state, whereas in F(2) populations the influences of water availability conditions cause less pronounce effects. Similarly, male-sterile F(1) plants, being transferred from the "dry plot" to greenhouse, produced male-fertile panicles. In addition, male-sterile plants from F(2) families, which segregated-out as recessives, being transferred to greenhouse also produced male-fertile panicles. In the progenies of these revertants that were grown in field conditions and in the "dry plot," stable inheritance of male fertility for three cycles of self-pollination was observed, and a number of stable fertile lines in the "9E" cytoplasm were obtained. However, in test-crosses of these fertile lines to CMS-lines with the "9E" cytoplasm restoration of male fertility was not observed, except the progeny of one revertant that behaved as fertility-restorer line. These data suggest that the functional state of fertility-restoring genes for the "9E" sorghum cytoplasm is epigenetically regulated trait established by the influence of environmental factors and is transmitted to sexual generations.
 
Article
Nitrogen is an essential mineral nutrient and it is often transported within living organisms in its reduced form, as amino acids. Transport of amino acids across cellular membranes requires proteins, and here we report the phylogenetic analysis across taxa of two amino acid transporter families, the amino acid permeases (AAPs) and the lysine-histidine-like transporters (LHTs). We found that the two transporter families form two distinct groups in plants supporting the concept that both are essential. AAP transporters seem to be restricted to land plants. They were found in Selaginella moellendorffii and Physcomitrella patens but not in Chlorophyte, Charophyte, or Rhodophyte algae. AAPs were strongly represented in vascular plants, consistent with their major function in phloem (vascular tissue) loading of amino acids for sink nitrogen supply. LHTs on the other hand appeared prior to land plants. LHTs were not found in chlorophyte algae Chlamydomonas reinhardtii and Volvox carterii. However, the characean alga Klebsormidium flaccidum encodes KfLHT13 and phylogenetic analysis indicates that it is basal to land plant LHTs. This is consistent with the hypothesis that characean algae are ancestral to land plants. LHTs were also found in both S. moellendorffii and P. patens as well as in monocots and eudicots. To date, AAPs and LHTs have mainly been characterized in Arabidopsis (eudicots) and these studies provide clues to the functions of the newly identified homologs.
 
ABA metabolism pathway. Zeaxanthin conversion into violaxanthin is catalyzed by zeaxanthin epoxidase (ZEP). ABA4 is involved in the synthesis of neoxanthin, which is then cis-isomerized, together with violaxanthin, by an unknown isomerase. Carotenoid cleavage is catalyzed by a family of 9-cis-epoxycarotenoid dioxygenases (NCED) to form xanthoxin. Xanthoxin moves to the cytosol by an unknown mechanism and is converted into abscisic aldehyde by a short-chain dehydrogenase reductase (SDR1), which is then oxidized into ABA by an abscisic aldehyde oxidase (AAO3). Sulfuration of AAO3 molybdenum co-factor by ABA3 is necessary for enzyme activity. The 8′-hydroxylation by CYP707A enzymes is thought to be the predominant pathway for ABA catabolism. Hydroxy-groups of ABA and its catabolites, phaseic acid (PA), neoPA, and dihydrophaseic acid (DPA) are targets for conjugation. ABA-glucose ester is formed by ABA glucosyltransferases (UGT) and hydrolyzed by glucosidases, including BG1 and BG2.
Interactions between ethylene, abscisic acid, and nitric oxide signaling pathways in the regulation of seed germination and dormancy. This scheme is based on genetic analyses, microarray data, and physiological studies on seed responsiveness to ABA, ethylene, or NO. ABA binding to PYR/PYL/RCAR receptor induces the formation of a protein complex with PP2C and the inhibition of phosphatase activity. In the absence of ABA, PP2C dephosphorylate SnRK2. When ABA is present, PP2C binding to the receptor releases inhibition of SnRK2 activity, which can phosphorylate downstream targets, including ABI5-related transcription factors. Interactions between ABI3 and ABI5 mediate transcriptional regulation of ABA-responsive genes. Ethylene positively regulates its own biosynthesis, by acting on ACC synthesis catalyzed by ACS and subsequent conversion to ethylene by ACO. This last step is also subject to ABA inhibition. Ethylene is perceived by receptors (among which ETR1) located in the endoplasmic reticulum; its binding leads to the deactivation of the receptors that become enable to recruit CTR1. Release of CTR1 inhibition allows EIN2 to act as a positive regulator of ethylene signaling pathway. EIN2 acts upstream of nuclear transcription factors, such as EIN3, EILs, and ERBPs/ERFs. Ethylene down-regulates ABA accumulation by both inhibiting its synthesis and promoting its inactivation, and also negatively regulates ABA signaling. In germinating seeds, NO enhances ABA catabolism and may also negatively regulate ABA synthesis and perception. Moreover, NO promotes both ethylene synthesis and signaling pathway. ABA, abscisic acid; ABI3, ABA insensitive3; ABI5, ABA insensitive5; ACC, 1-aminocyclopropane 1-carboxylic acid; ACO, ACC oxidase; ACS, ACC synthase; CTR1, constitutive triple response 1; CYP707A, ABA-8′-hydroxylase; EIL, EIN3-like; EIN, ethylene-insensitive; EREBP, ethylene-responsive element binding protein; ERF, ethylene response factor; Et, ethylene; ETR1, ethylene receptor1; NCED, 9-cis-epoxycarotenoid dioxygenase; NO, nitric oxide; PP2C, clade A type 2C protein phosphatases; PYR/PYL/RCAR, pyrabactin resistance1/PYR1-like/regulatory components of ABA receptor; SnRK2, group III sucrose non-fermenting-1-related protein kinase 2; a dashed line is used when regulatory targets are not precisely identified.
Ethylene biosynthesis pathway. S-adenosyl-methionine (S-AdoMet) is synthesized from the methionine by the S-adenosyl-methionine synthetase (SAM synthetase) with one ATP molecule expensed per S-AdoMet synthesized. S-AdoMet is then converted to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase, 5′-methylthioadenosine (MTA) being a by-product. MTA is recycled to methionine by successive enzymatic reactions involving various intermediates (MTR, 5-methylthioribose; KMB, 2-keto-4-methylthiobutyrate), which constitute the methionine (Yang) cycle. S-AdoMet is also the precursor of the spermidine/spermine biosynthesis pathway. Ethylene production is catalyzed by the ACC oxidase using ACC as substrate, and generates carbon dioxide and hydrogen cyanide. Malonylation of ACC to malonyl-ACC (MACC) reduces ACC content and consequently ethylene production.
Simplified overview of NO biosynthesis and homeostasis in plant cells. This scheme is inspired from Moreau et al. (2010). Nitrate (NO3− ) assimilation produces nitrite (NO2− ) in a reaction catalyzed by nitrate reductase (NR). The subsequent reduction of nitrite into NO can occur enzymatically, either through NR activity or mitochondrial electron transport chains, and via non-enzymatic reactions (reductive pathways). Alternatively, NO synthesis can result from oxidative reactions from hydroxylamine, polyamines or L-arginine (L-Arg; oxidative pathways). NO synthesis from L-Arg could account for the nitric oxide synthase-like (NOS-like) activity detected in plants. The pool of NO is then influenced by non-symbiotic hemoglobin 1 (nsHb1) dioxygenase activity, which converts NO into NO3− . NO can also react with reduced glutathione or thiol groups leading to the reversible formation of S-nitrosothiols (e.g., GSNO, S-nitrosoglutathione; S-nitrosylated proteins). Red arrows highlight the so-called nitrate-NO cycle that may take place under hypoxia. Green arrows correspond to biosynthesis reactions while blue arrows indicate reactions involved in NO homeostasis.
Article
Dormancy is an adaptive trait that enables seed germination to coincide with favorable environmental conditions. It has been clearly demonstrated that dormancy is induced by abscisic acid (ABA) during seed development on the mother plant. After seed dispersal, germination is preceded by a decline in ABA in imbibed seeds, which results from ABA catabolism through 8'-hydroxylation. The hormonal balance between ABA and gibberellins (GAs) has been shown to act as an integrator of environmental cues to maintain dormancy or activate germination. The interplay of ABA with other endogenous signals is however less documented. In numerous species, ethylene counteracts ABA signaling pathways and induces germination. In Brassicaceae seeds, ethylene prevents the inhibitory effects of ABA on endosperm cap weakening, thereby facilitating endosperm rupture and radicle emergence. Moreover, enhanced seed dormancy in Arabidopsis ethylene-insensitive mutants results from greater ABA sensitivity. Conversely, ABA limits ethylene action by down-regulating its biosynthesis. Nitric oxide (NO) has been proposed as a common actor in the ABA and ethylene crosstalk in seed. Indeed, convergent evidence indicates that NO is produced rapidly after seed imbibition and promotes germination by inducing the expression of the ABA 8'-hydroxylase gene, CYP707A2, and stimulating ethylene production. The role of NO and other nitrogen-containing compounds, such as nitrate, in seed dormancy breakage and germination stimulation has been reported in several species. This review will describe our current knowledge of ABA crosstalk with ethylene and NO, both volatile compounds that have been shown to counteract ABA action in seeds and to improve dormancy release and germination.
 
Article
Seed dormancy controls germination and plays a critical role in regulating the beginning of the life cycle of plants. Seed dormancy is established and maintained during seed maturation and is gradually broken during dry storage (after-ripening). The plant hormone abscisic acid (ABA) and DELAY OF GERMINATION1 (DOG1) protein are essential regulators of seed dormancy. Recent studies revealed that chromatin modifications are also involved in the transcription regulation of seed dormancy. Here, we showed that two Arabidopsis histone demethylases, LYSINESPECIFIC DEMETHYLASE LIKE 1 and 2 (LDL1 and LDL2) act redundantly in repressing of seed dormancy. LDL1 and LDL2 are highly expressed in the early silique developing stage. The ldl1 ldl2 double mutant displays increased seed dormancy, whereas overexpression of LDL1 or LDL2 in Arabidopsis causes reduced dormancy. Furthermore, we showed that LDL1 and LDL2 repress the expression of seed dormancy-related genes, including DOG1, ABA2 and ABI3 during seed dormancy establishment. Furthermore, genetic analysis revealed that the repression of seed dormancy by LDL1 and LDL2 requires DOG1, ABA2, and ABI3. Taken together, our findings revealed that LDL1 and LDL2 play an essential role in seed dormancy.
 
Article
Abscisic acid (ABA) and Gibberellic acid (GA) play key roles in regulating seed dormancy and germination. First, when examining germination of different rice cultivars, we found that their germination timing and dormancy status are rather distinct, coupled with different GA/ABA ratio. Second, we studied genomic sequences of ABA and GA dormancy- and germination-associated genes in rice and discovered single nucleotide polymorphisms and insertions/deletions (Indels) in both coding and regulatory sequences. We aligned all these variations to the genome assemblies of 9311 and PA64s and demonstrated their relevance to seed dormancy both quantitatively and qualitatively based on gene expression data. Third, we surveyed and compared differentially expressed genes in dry seeds between 9311 and PA64s to show that these differentially expressed genes may play roles in seed dormancy and germination.
 
Article
The unique physico-chemical properties of gold nanoparticles (AuNPs) find manifold applications in diagnostics, medicine and catalysis. Chemical synthesis produces reactive AuNPs and generates hazardous by-products. Alternatively, plants can be utilized to produce AuNPs in an eco-friendly manner. To better control the biosynthesis of AuNPs, we need to first understand the detailed molecular response induced by AuCl4- In this study, we carried out global transcriptome analysis in root tissue of Arabidopsis grown for 12- hours in presence of gold solution (HAuCl4) using the novel unbiased Affymetrix exon array. Transcriptomics analysis revealed differential regulation of a total of 704 genes and 4900 exons. Of these, 492 and 212 genes were up- and downregulated, respectively. The validation of the expressed key genes, such as glutathione-S-transferases, auxin responsive genes, cytochrome P450 82C2, methyl transferases, transducin (G protein beta subunit), ERF transcription factor, ABC, and MATE transporters, was carried out through quantitative RT-PCR. These key genes demonstrated specific induction under AuCl4- treatment relative to other heavy metals, suggesting a unique plant-gold interaction. GO enrichment analysis reveals the upregulation of processes like oxidative stress, glutathione binding, metal binding, transport, and plant hormonal responses. Changes predicted in biochemical pathways indicated major modulation in glutathione mediated detoxification, flavones and derivatives, and plant hormone biosynthesis. Motif search analysis identified a highly significant enriched motif, ACGT, which is an abscisic acid responsive core element (ABRE), suggesting the possibility of ABA- mediated signaling. Identification of abscisic acid response element (ABRE) points to the operation of a predominant signaling mechanism in response to AuCl4- exposure. Overall, this study presents a useful picture of plant-gold interaction with an identification of candidate genes invol
 
| Analysis of tissue-specific expression of PIN, AUX/LAX, and ABCB transcripts. Presence or absence of transcripts of genes coding for putative auxin transport proteins in the cortex, secondary phloem, cambial zone (i.e., initials and mother/daughter cells), and mature secondary xylem of Populus tremula × alba as determined by RT-PCR. Consensus of four biological replicates is shown, where GRAY = PRESENT, WHITE = ABSENT, and CROSS-HATCHED =VARIABLE among biological replicates. Samples were taken from the base of 6-month-old trees during active growth, approximately 100 internodes down from the top of the tree at a diameter of about 2 cm.
| Quantification of ABCB transcripts expression by qRT-PCR. Most notable among the ABCB family is PtaABCB1.1, which was highly expressed in internodes and developing xylem and whose ortholog in  
FIGURE A2 | Predicted model structures of putative auxin transport ABCBs from Populus trichocarpa. Tertiary protein structures have been generated using the python script Modeller 9v5. Predicted IAA docking sites are depicted in red.  
Article
Intercellular transport of the plant hormone auxin is mediated by three families of membrane-bound protein carriers, with the PIN and ABCB families coding primarily for efflux proteins and the AUX/LAX family coding for influx proteins. In the last decade our understanding of gene and protein function for these transporters in Arabidopsis has expanded rapidly but very little is known about their role in woody plant development. Here we present a comprehensive account of all three families in the model woody species Populus, including chromosome distribution, protein structure, quantitative gene expression, and evolutionary relationships. The PIN and AUX/LAX gene families in Populus comprise 16 and 8 members respectively and show evidence for the retention of paralogs following a relatively recent whole genome duplication. There is also differential expression across tissues within many gene pairs. The ABCB family is previously undescribed in Populus and includes 20 members, showing a much deeper evolutionary history, including both tandem and whole genome duplication as well as probable gene loss. A striking number of these transporters are expressed in developing Populus stems and we suggest that evolutionary and structural relationships with known auxin transporters in Arabidopsis can point toward candidate genes for further study in Populus. This is especially important for the ABCBs, which is a large family and includes members in Arabidopsis that are able to transport other substrates in addition to auxin. Protein modeling, sequence alignment and expression data all point to ABCB1.1 as a likely auxin transport protein in Populus. Given that basipetal auxin flow through the cambial zone shapes the development of woody stems, it is important that we identify the full complement of genes involved in this process. This work should lay the foundation for studies targeting specific proteins for functional characterization and in situ localization.
 
Article
The acquisition of plastids is a landmark event in plant evolution. The proper functionality of these organelles depends on strict and continuous communication between the plastids and the nucleus to precisely adjust gene expression in response to the organelle's requirements. Signals originating from the plastids impact the expression of a variety of nuclear genes, and this retrograde communication is essential to couple the nuclear expression of plastid-localized products with organelle gene expression and, ultimately, functionality. Major advances have been made in this field over the past few years with the characterization of independent retrograde signaling pathways and the identification of some of their components. One such factor is the nuclear transcriptional factor ABI4 (ABA-INSENTIVE 4). ABI4, together with the plastid PPR GUN1 protein, has been proposed to function as a node of convergence for multiple plastid retrograde signaling pathways. ABI4 is conserved among plants and also plays important roles in various critical developmental and metabolic processes. ABI4 is a versatile regulator that positively and negatively modulates the expression of many genes, including other transcriptional factors. However, its mode of action during plastid retrograde signaling is not fully understood. In this review, we describe the current evidence that supports the participation of ABI4 in different retrograde communication pathways. ABI4 is regulated at the transcriptional and post-transcriptional level. A known regulator of ABI4 includes the PTM transcription factor, which moves from the chloroplast to the nucleus. This transcription factor is a candidate for the transmission of retrograde signals between the plastid and ABI4.
 
Article
Morphological plasticity of ectomycorrhizal (EcM) short roots (known also as first and second order roots with primary development) allows trees to adjust their water and nutrient uptake to local environmental conditions. The morphological traits (MTs) of short-living EcM roots, such as specific root length (SRL) and area, root tip frequency per mass unit (RTF), root tissue density, as well as mean diameter, length, and mass of the root tips, are good indicators of acclimation. We investigated the role of EcM root morphological plasticity across the climate gradient (48-68°N) in Norway spruce (Picea abies (L.) Karst) and (53-66°N) birch (Betula pendula Roth., B. pubescens Ehrh.) forests, as well as in primary and secondary successional birch forests assuming higher plasticity of a respective root trait to reflect higher relevance of that characteristic in acclimation process. We hypothesized that although the morphological plasticity of EcM roots is subject to the abiotic and biotic environmental conditions in the changing climate; the tools to achieve the appropriate morphological acclimation are tree species-specific. Long-term (1994-2010) measurements of EcM roots morphology strongly imply that tree species have different acclimation-indicative root traits in response to changing environments. Birch EcM roots acclimated along latitude by changing mostly SRL [plasticity index (PI) = 0.60], while spruce EcM roots became adjusted by modifying RTF (PI = 0.68). Silver birch as a pioneer species must have a broader tolerance to environmental conditions across various environments; however, the mean PI of all MTs did not differ between early-successional birch and late-successional spruce. The differences between species in SRL, and RTF, diameter, and length decreased southward, toward temperate forests with more favorable growth conditions. EcM root traits reflected root-rhizosphere succession across forest succession stages.
 
Article
Rising levels of atmospheric CO(2) have often been reported to reduce plant water use. Such behavior is also predicted by standard equations relating photosynthesis, stomatal conductance, and atmospheric CO(2) concentration, which form the core of dynamic global vegetation models (DGVMs). Here, we provide first results from a free air CO(2) enrichment (FACE) experiment with naturally growing, mature (35 m) Picea abies (L.) (Norway spruce) and compare them to simulations by the DGVM LPJ-GUESS. We monitored sap flow, stem water deficit, stomatal conductance, leaf water potential, and soil moisture in five 35-40 m tall CO(2)-treated (550 ppm) trees over two seasons. Using LPJ-GUESS, we simulated this experiment using climate data from a nearby weather station. While the model predicted a stable reduction of transpiration of between 9% and 18% (at concentrations of 550-700 ppm atmospheric CO(2)), the combined evidence from various methods characterizing water use in our experimental trees suggest no changes in response to future CO(2) concentrations. The discrepancy between the modeled and the experimental results may be a scaling issue: while dynamic vegetation models correctly predict leaf-level responses, they may not sufficiently account for the processes involved at the canopy and ecosystem scale, which could offset the first-order stomatal response.
 
S-nitrosylated proteins under abiotic stress. Proteins that change their S-nitrosylation pattern and activity in response to abiotic stress are related to NO, ROS, and cellular metabolism. AHb1, haemoglobin 1; APX, ascorbate peroxidase; CAT, catalase; DHAR, dehydroascobate reductase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GOX, glycolate oxidase; Trx, thioredoxin. It is lacking: PrxII F, peroxiredoxin II F.
Article
Abiotic stress is one of the main problems affecting agricultural losses, and understanding the mechanisms behind plant tolerance and stress response will help us to develop new means of strengthening fruitful agronomy. The mechanisms of plant stress response are complex. Data obtained by experimental procedures are sometimes contradictory, depending on the species, strength, and timing applied. In recent years nitric oxide has been identified as a key signaling molecule involved in most plant responses to abiotic stress, either indirectly through gene activation or interaction with reactive oxygen species and hormones; or else directly, as a result of modifying enzyme activities mainly by nitration and S-nitrosylation. While the functional relevance of the S-nitrosylation of certain proteins has been assessed in response to biotic stress, it has yet to be characterized under abiotic stress. Here, we review initial works about S-nitrosylation in response to abiotic stress to conclude with a brief overview, and discuss further perspectives to obtain a clear outlook of the relevance of S-nitrosylation in plant response to abiotic stress.
 
| CK and crosstalks during abiotic stress responses. Under non-stress conditions, CK activates signaling mediated through AHK receptors, AHPs, and type-B response regulators ARRs. Type-B ARRs stimulate the expression of the early CK response genes, including type-A ARR genes that provide a negative feedback loop of the CK signaling. Besides this negative feedback loop, type-A ARRs also repress the expression of ABI5 and interfere with the ABA signaling, through the physical interaction with ABI5. In response to stress, ABA levels increase and, simultaneously, CK levels decrease. The recognition of ABA by the
CK and crosstalks during abiotic stress responses. Under non-stress conditions, CK activates signaling mediated through AHK receptors, AHPs, and type-B response regulators ARRs. Type-B ARRs stimulate the expression of the early CK response genes, including type-A ARR genes that provide a negative feedback loop of the CK signaling. Besides this negative feedback loop, type-A ARRs also repress the expression of ABI5 and interfere with the ABA signaling, through the physical interaction with ABI5. In response to stress, ABA levels increase and, simultaneously, CK levels decrease. The recognition of ABA by the receptors PYR/PYL/RCAR promotes the interaction with PP2C proteins that will activate downstream responses through signaling components including ABI5 and ABI4. At the same time, ABA interferes with the activity of CK and auxin and via ABI4 attenuates the expression of the PIN1 auxin efflux carrier and enhances the transcription of the CK signaling repressor ARR5. Interestingly, type-A ARRs, such as ARR5, are upregulated, despite the low CK levels, probably because of the indirect activation of the CK signaling pathway by alternative receptors of the histidine kinase family, such as AHK1.
CK and hormonal crosstalks during biotic stress responses. Pathogen attacks stimulated by PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) correlate with a dramatic production of SA and CK. The accumulation of CK will induce the production and accumulation of phytoalexins in a SA-independent manner and also enhance the SA-dependent immunity. In response to pathogens, NPR1 monomerizes and translocates to the nucleus where it interacts with TGA3. The NPR1-TGA3 activity is further regulated through interaction with the type-B ARR2 response regulator, a component of the CK signaling pathway. The TGA3-NPR1-ARR2 complex is required to induce the SA-mediated resistance and to trigger the expression of PR1 and PR2. High CK levels, induced after pathogen attacks, can activate the CRF5-mediated branch of the CK signaling pathway and contribute to the regulation of the PR1, PR3, PR4, and PR5 expression.
Article
As sessile organisms, plants have to be able to adapt to a continuously changing environment. Plants that perceive some of these changes as stress signals activate signaling pathways to modulate their development and to enable them to survive. The complex responses to environmental cues are to a large extent mediated by plant hormones that together orchestrate the final plant response. The phytohormone cytokinin is involved in many plant developmental processes. Recently, it has been established that cytokinin plays an important role in stress responses, but does not act alone. Indeed, the hormonal control of plant development and stress adaptation is the outcome of a complex network of multiple synergistic and antagonistic interactions between various hormones. Here, we review the recent findings on the cytokinin function as part of this hormonal network. We focus on the importance of the crosstalk between cytokinin and other hormones, such as abscisic acid, jasmonate, salicylic acid, ethylene, and auxin in the modulation of plant development and stress adaptation. Finally, the impact of the current research in the biotechnological industry will be discussed.
 
Article
Growth hormone auxin regulates various cellular processes by altering the expression of diverse genes in plants. Among various auxin-responsive genes, GH3 genes maintain endogenous auxin homeostasis by conjugating excess of auxin with amino acids. GH3 genes have been characterized in many plant species, but not in legumes. In the present work, we identified members of GH3 gene family and analyzed their chromosomal distribution, gene structure, gene duplication and phylogenetic analysis in different legumes, including chickpea, soybean, Medicago, and Lotus. A comprehensive expression analysis in different vegetative and reproductive tissues/stages revealed that many of GH3 genes were expressed in a tissue-specific manner. Notably, chickpea CaGH3-3, soybean GmGH3-8 and -25, and Lotus LjGH3-4, -5, -9 and -18 genes were up-regulated in root, indicating their putative role in root development. In addition, chickpea CaGH3-1 and -7, and Medicago MtGH3-7, -8, and -9 were found to be highly induced under drought and/or salt stresses, suggesting their role in abiotic stress responses. We also observed the examples of differential expression pattern of duplicated GH3 genes in soybean, indicating their functional diversification. Furthermore, analyses of three-dimensional structures, active site residues and ligand preferences provided molecular insights into function of GH3 genes in legumes. The analysis presented here would help in investigation of precise function of GH3 genes in legumes during development and stress conditions.
 
Subcellular localization of overexpressed protein and type of abiotic stress is classified. (A) Percentage of organelles in which overexpressed proteins were localized. (B) Distribution of abiotic stresses in which transgenic plants were tolerant.
Article
Environmental stresses are major factors limiting growth and development of crops. Plants respond to the stresses through a wide range of reactions from morphological changes to alterations in the patterns of protein expression. Understanding the mechanisms involved in the stress response is the first step to develop abiotic stress tolerant crops. Proteomics is a powerful tool in evaluating regulated proteins in the cell under stress and it is an efficient technique in studying stress tolerant plants. Because of the nature of abiotic stress, intracellular compartments play a main role in the stress response. Subcellular proteins such as ion and water transporters, reactive oxygen species (ROS) scavengers, and the proteins related to signaling and transcriptional regulation are frequently reported as being involved in stress tolerance. Overexpression of stress-responsive protein through generation of transgenic plants is one the main practical approaches in production of tolerant plants. In this article, recent studies on transgenic plants overexpressing subcellular proteins are reviewed and the role of organelles and over-expressed proteins is classified.
 
Transgenic plants in trehalose metabolism: toward an optimized approach for stress tolerant plants. (A)
OtsA OX plants contain high levels of T6P. They develop poor seed set, experience reduced apical dominance, flower early and display reduced rosette and darker, lancet shaped leaves. In tobacco OtsA OX, root development is disturbed, showing thicker roots. In Chinese cabbage plants OtsA OX, thicker roots, well developed lateral roots and extensive root hairs were noticed. In general, these plants are better stress tolerant (based on Goddijn et al., 1997; Park et al., 2003; Schluepmann et al., 2003). (B)
OtsB OX plants contain low levels of T6P. These plants show opposite phenotypes to (A). They develop plenty of seeds, they have distinct apical dominance, flower later, and they have a larger, paler-green rosette (based on Schluepmann et al., 2003). (C) Model for optimized transgenic trehalose metabolism plant. This plant has a controlled cellular level of T6P. This optimized model is the result of many factors; use of bifunctional constructs, use of condition specific promoters (for instance stress specific, depending on the destined region where the crop will be grown), use of cell-specific promoters (for instance in stomata, using TRE1 as target gene). In addition, this plant contains optimized constructs, yet unrevealed, connected to abiotic stress tolerance via trehalose metabolism. Ideally, this plant will be tolerant toward a wide range of abiotic stresses, generating high biomass, continuing growth in deteriorated circumstances, generating fertile seeds, and requiring little need for any other kind of maintenance (based on Karim et al., 2007; Van Houtte et al., 2013a).
Article
The impact of abiotic stress on plant growth and development has been and still is a major research topic. An important pathway that has been linked to abiotic stress tolerance is the trehalose biosynthetic pathway. Recent findings showed that trehalose metabolism is also important for normal plant growth and development. The intermediate compound - trehalose-6-phosphate (T6P) - is now confirmed to act as a sensor for available sucrose, hereby directly influencing the type of response to the changing environmental conditions. This is possible because T6P and/or trehalose or their biosynthetic enzymes are part of complex interaction networks with other crucial hormone and sugar-induced signaling pathways, which may function at different developmental stages. Because of its effect on plant growth and development, modification of trehalose biosynthesis, either at the level of T6P synthesis, T6P hydrolysis, or trehalose hydrolysis, has been utilized to try to improve crop yield and biomass. It was shown that alteration of the amounts of either T6P and/or trehalose did result in increased stress tolerance, but also resulted in many unexpected phenotypic alterations. A main challenge is to characterize the part of the signaling pathway resulting in improved stress tolerance, without affecting the pathways resulting in the unwanted phenotypes. One such specific pathway where modification of trehalose metabolism improved stress tolerance, without any side effects, was recently obtained by overexpression of trehalase, which results in a more sensitive reaction of the stomatal guard cells and closing of the stomata under drought stress conditions. We have used the data that have been obtained from different studies to generate the optimal plant that can be constructed based on modifications of trehalose metabolism.
 
Article
Understanding the global abiotic stress response is an important stepping stone for the development of universal stress tolerance in plants in the era of climate change. Although co-occurrence of several stress factors (abiotic and biotic) in nature is found to be frequent, current attempts are poor to understand the complex physiological processes impacting plant growth under combinatory factors. In this review article, we discuss the recent advances of reverse engineering approaches that led to seminal discoveries of key candidate regulatory genes involved in cross-talk of abiotic stress responses and summarized the available tools of reverse engineering and its relevant application. Among the universally induced regulators involved in various abiotic stress responses, we highlight the importance of (i) abscisic acid (ABA) and jasmonic acid (JA) hormonal cross-talks and (ii) the central role of WRKY transcription factors (TF), potentially mediating both abiotic and biotic stress responses. Such interactome networks help not only to derive hypotheses but also play a vital role in identifying key regulatory targets and interconnected hormonal responses. To explore the full potential of gene network inference in the area of abiotic stress tolerance, we need to validate hypotheses by implementing time-dependent gene expression data from genetically engineered plants with modulated expression of target genes. We further propose to combine information on gene-by-gene interactions with data from physical interaction platforms such as protein-protein or TF-gene networks.
 
The ascorbate–glutathione cycle in plants. Hydrogen peroxide (H2O2) within the plant cell can be detoxified by ascorbate peroxidase (APX). In this reaction, the reduced form of ascorbate (Asc) is oxidized to monodehydroascorbate (MDHA). MDHA is then either reduced by monodehydroascorbate reductase (MDHAR) to Asc or, since very unstable, reacts to dehydroascorbate (DHA). DHA is reduced by dehydroascorbate reductase (DHAR) to Asc. In this reaction, the reduced form of glutathione (GSH) is oxidized to glutathione disulfide (GSSG). GSSG is then reduced by glutathione reductase (GR) to GSH. The electron acceptor NADP is regenerated during the reduction of MDHA and GSSG by the respective enzymes. Asc and GSH are additional able to detoxify reactive oxygen species by direct chemical interaction. Thus, besides the total ascorbate and glutathione level their redox state (reduced vs. oxidized state) which depends on the activity of the described enzymes (gray boxes) is also very important for successful plant protection.
| Compartment-specific glutathione contents in leaves of Arabidopsis Col-0.
Compartment-specific production of reactive oxygen species (ROS) induced by different stress conditions and possible detoxification and signaling pathways involving ascorbate (Asc) and glutathione (GSH) and ROS. C, chloroplast; M, mitochondrium; N, nucleus; Px, peroxisome; V, vacuole.
Images show the typical distribution of glutathione. (A) Monochlorobimane staining in guard cells of the upper epidermis of tobacco cells in the light microscope. Fluorescence was observed in cytosol and nuclei (N) but not in vacuoles (V) and cell walls (arrowhead). Additionally, no fluorescence could be observed in chloroplasts (arrows in A and B) which can be best identified when comparing the autofluorescence of chloroplast (B) with monochlorobimane staining (A). Transmission electron micrographs show the subcellular distribution of glutathione (C,D) in mesophyll cells of leaves from Arabidopsis Col-0 plants. Glutathione-specific labeling could be observed in different concentrations in mitochondria (M), chloroplasts (C), peroxisomes (Px) but not in vacuoles (V) and cell walls (CW). Glutathione-specific labeling was observed in the stroma as well as inside the thylakoid lumen (arrowheads) when plants were exposed to high light intensities of 700 µmol m-2 s-1. Bars in (A,B) = 10 µm, (C,D) = 0.5 µm.
Article
The tripeptide thiol glutathione (γ-L-glutamyl-L-cysteinyl-glycine) is the most important sulfur containing antioxidant in plants and essential for plant defense against abiotic and biotic stress conditions. It is involved in the detoxification of reactive oxygen species (ROS), redox signaling, the modulation of defense gene expression, and the regulation of enzymatic activities. Even though changes in glutathione contents are well documented in plants and its roles in plant defense are well established, still too little is known about its compartment-specific importance during abiotic and biotic stress conditions. Due to technical advances in the visualization of glutathione and the redox state through microscopical methods some progress was made in the last few years in studying the importance of subcellular glutathione contents during stress conditions in plants. This review summarizes the data available on compartment-specific importance of glutathione in the protection against abiotic and biotic stress conditions such as high light stress, exposure to cadmium, drought, and pathogen attack (Pseudomonas, Botrytis, tobacco mosaic virus). The data will be discussed in connection with the subcellular accumulation of ROS during these conditions and glutathione synthesis which are both highly compartment specific (e.g., glutathione synthesis takes place in chloroplasts and the cytosol). Thus this review will reveal the compartment-specific importance of glutathione during abiotic and biotic stress conditions.
 
An overview of the central role of ROS in the responses towards abiotic stress factors. Different abiotic cues either directly or indirectly (through the action of other signals and hormones) lead to the production of ROS. In turn, ROS may influence a variety of signal transduction systems, thus providing positive or negative feedback control mechanisms (red arrow). The function of the antioxidant machinery is to prevent dangerous elevations of ROS levels. The outcome of ROS signaling depends mainly on the ROS concentration, but other factors like the site of ROS synthesis, previous stress exposure, developmental stage, and interaction with other signals like reactive nitrogen species (RNS) and Ca2+ are also integrated into the response. In general relatively weak stressors cause only a slight rise in ROS quantities which leads to adaptation. At more intensive abiotic stimuli the price for adaptation may be impaired growth and development of the plant. Severe stress usually causes massive accumulation of ROS and the initiation of PCD, or in extreme cases even necrosis of the tissue.
Article
During the course of their ontogenesis plants are continuously exposed to a large variety of abiotic stress factors which can damage tissues and jeopardize the survival of the organism unless properly countered. While animals can simply escape and thus evade stressors, plants as sessile organisms have developed complex strategies to withstand them. When the intensity of a detrimental factor is high, one of the defense programs employed by plants is the induction of programmed cell death (PCD). This is an active, genetically controlled process which is initiated to isolate and remove damaged tissues thereby ensuring the survival of the organism. The mechanism of PCD induction usually includes an increase in the levels of reactive oxygen species (ROS) which are utilized as mediators of the stress signal. Abiotic stress-induced PCD is not only a process of fundamental biological importance, but also of considerable interest to agricultural practice as it has the potential to significantly influence crop yield. Therefore, numerous scientific enterprises have focused on elucidating the mechanisms leading to and controlling PCD in response to adverse conditions in plants. This knowledge may help develop novel strategies to obtain more resilient crop varieties with improved tolerance and enhanced productivity. The aim of the present review is to summarize the recent advances in research on ROS-induced PCD related to abiotic stress and the role of the organelles in the process.
 
Venn map showing the proteins that were regulated by different abiotic stresses and ABA treatments. D, desiccation treatment: dehydrating to 90% fresh weight loss; S, salt treatment: >0.25 M NaCl for 3 days; C, cold treatment: 0°C incubation for >1 day; A, 50 µM ABA treat for 3 days. The raw data for this figure were from our previous studies ( Wang et al., 2008,  2009a,b, 2010).
Functional categorization of the differential displayed proteins under the treatment of salt, desiccation, and ABA . This figure was based on our previous data ( Wang et al., 2008, 2009a,b, 2010).
Article
Moss species Physcomitrella patens has been used as a model system in plant science for several years, because it has a short life cycle and is easy to be handled. With the completion of its genome sequencing, more and more proteomic analyses were conducted to study the mechanisms of P. patens abiotic stress resistance. It can be concluded from these studies that abiotic stresses could lead to the repression of photosynthesis and enhancement of respiration in P. patens, although different stresses could also result in specific responses. Comparative analysis showed that the responses to drought and salinity were very similar to that of abscisic acid, while the response to cold was quite different from these three. Based on previous studies, it is proposed that sub-proteomic studies on organelles or protein modifications, as well as functional characterization of those candidate proteins identified from proteomic studies will help us to further understand the mechanisms of abiotic stress resistance in P. patens.
 
Article
Reduced glutathione (GSH) is an abundant low molecular weight plant thiol. It fulfills multiple functions in plant biology, many of which remain poorly characterized. A phenomics approach was therefore used to investigate the effects of glutathione homeostasis on growth and stress tolerance in Arabidopsis thaliana. Rosette leaf area was compared in mutants that are either defective in GSH synthesis (cad2, pad2, and rax1) or the export of γ-glutamylcysteine and GSH from the chloroplast (clt) and in wild-type plants under standard growth conditions and following exposure to a range of abiotic stress treatments, including oxidative stress, water stress, and high salt. In the absence of stress, the GSH synthesis mutants had a significantly lower leaf area than the wild type. Conversely, the clt mutant has a greater leaf area and a significantly reduced lateral root density than the wild type. These findings demonstrate that cellular glutathione homeostasis exerts an influence on root architecture and on rosette area. An impaired capacity to synthesize GSH or a specific depletion of the cytosolic GSH pool did not adversely affect leaf area in plants exposed to short-term abiotic stress. However, the negative effects of long-term exposure to oxidative stress and high salt on leaf area were less marked in the GSH synthesis mutants than the wild type. These findings demonstrate the importance of cellular glutathione homeostasis in the regulation of plant growth under optimal and stress conditions.
 
Oxygen independent transcriptomic changes during rice germination. (A) Step-wise comparisons were carried out over the course of aerobic germination in Arabidopsis (1Narsai et al., 2011a) aerobic germination in rice (2Howell et al., 2009) and anaerobic germination in rice (3Narsai et al., 2009). (B) The differentially expressed genes (>2-fold, p < 0.05, PPDE > 0.96) were analyzed for common over-representation of functions in the up/down-regulated gene-sets. (C) Functional categories seen to be commonly up/down-regulated across all three germination studies are shown. Red font indicates that genes encoding these function(s) are over-represented in the up-regulated gene-sets and blue indicates over-representation of functions in the down-regulated gene-sets. Categories responsive at the same time in both aerobic and anaerobic germination in rice are denoted with an asterisk.
Transcriptomic changes specific to anaerobic germination in rice. (A) Core up/down-regulated genes exclusive to anaerobic germination were identified by isolating genes encoding functions that were over-represented in comparison to aerobic germination (Aerobic v Anaerobic), as well as over the course of anaerobic germination (step-wise), whilst not being over-represented over the course of aerobic germination (step-wise). (B) Differential expression of genes encoding core over-represented functions unique to anaerobic germination only [as defined in (A)]. Significant fold-changes (>2-fold, p < 0.05, PPDE > 0.96) are shown as a heatmap for genes both in comparison to aerobic germination (A v N—top rows) as well as over the course of anaerobic germination (step-wise—bottom rows). Categories showing similar responses during Arabidopsis germination are indicated with ∧ and categories containing genes that showed transient responses in Arabidopsis e.g., up then down or down then up, are denoted with #.
| Low oxygen marker genes in rice and Arabidopsis.
Defining the core low oxygen response. Pageman over-representation analysis was carried out for the significantly differentially expressed genesets (>2-fold, p < 0.05, PPDE > 0.96) in response to low oxygen in rice and Arabidopsis. The gene sets used included four different comparisons in rice and five different comparisons in Arabidopsis of control vs. anaerobic treatment (details in Table 1). Only the functional categories over-represented in rice only, Arabidopsis only or both species are shown (z-scores indicate over/under-representation, indicated by the green/yellow colors, respectively).
Overlapping responses between low oxygen and abiotic stress in rice and Arabidopsis. (A) Differentially expressed genes from each of the four low oxygen transcriptome datasets in rice (Lasanthi-Kudahettige et al., 2007; Narsai et al., 2009) were overlapped with the differentially expressed genes in response to cold, drought, salt, and heat stress (Jain et al., 2007; Hu et al., 2009). The numbers of genes showing overlapping responses are shown. The * indicates that the fold-changes were inversed for this comparison to show the response to anoxia. (B) Differentially expressed genes from each of the five low oxygen transcriptome datasets in Arabidopsis (Loreti et al., 2005; Branco-Price et al., 2008; Christianson et al., 2009, 2010; Banti et al., 2010) were overlapped with the differentially expressed genes in response to cold, drought, salt, and heat stress (Kilian et al., 2007). The number of genes showing overlapping responses is shown. (Ci) A heatmap showing differential expression of the 80 genes that were both significantly responsive to heat in Arabidopsis, as well as being previously identified as abiotic stress marker genes [by Gadjev et al. (2006), Lu et al. (2007), and Rasmussen et al. (2013) in Arabidopsis]. Expression is shown in response to low oxygen and abiotic stress. (Cii) A heatmap showing the expression of the 135 rice orthologs to the 80 Arabidopsis abiotic stress responsive genes (from Ci). (Di) The expression of three Arabidopsis genes representing examples of common transcriptomic responses to low oxygen and one or more abiotic stress in Arabidopsis. (Dii) Expression of the rice orthologs to the three Arabidopsis genes, showing that these differ in the transcriptomic responses to low oxygen and one or more abiotic stress in rice.
Article
Plants face a variety of environmental stresses and have evolved molecular mechanisms to survive these challenges. One of these stresses is low oxygen conditions, which can occur under flooding conditions. Rice (Oryza sativa) is somewhat unique for its ability to tolerate and even germinate under low to no oxygen conditions. In this study, we examined global transcriptomic responses over the course of germination and in response to low oxygen and other abiotic stress in rice and Arabidopsis (Arabidopsis thaliana). Over 150 microarray datasets were analyzed in parallel to determine just how unique the low oxygen response is in rice. Comparison of aerobic germination in rice and Arabidopsis, with anaerobic germination in rice revealed conserved transcriptomic responses that are not only conserved across both species but also occur in the absence of oxygen in rice. Thus, these genes may represent functions necessary for the developmental progression of germination, whether or not oxygen is present in rice. Analysis of genes that responded differently in rice compared to Arabidopsis revealed responses specific to anaerobic germination in rice, including the down-regulation of genes encoding redox functions and up-regulation of receptor kinases. Comparison of a range of hypoxia/anoxia studies within and across Arabidopsis and rice revealed both conserved and species specific changes in gene expression (e.g., Arabidopsis specific up-regulation of WRKYs and rice specific down-regulation of heme), unveiling unique transcriptomic signatures of the low oxygen response. Lastly, a comparison of the low oxygen response with cold, salt, drought and heat stress revealed some similarity with the response to heat stress in Arabidopsis, which was not seen in rice. Comparison of these heat-responsive, abiotic stress marker genes in Arabidopsis with their rice orthologs revealed that while low oxygen may be perceived as an abiotic stress in Arabidopsis, this is not the case in rice.
 
| A general workflow of comparative proteomic experiments in plants. Proteins are extracted and subjected to separation via gel (e.g., 2DE or 2 Dimensional gel electrophoresis) or non-gel (e.g., LC or liquid chromatography) based approaches. Reduction, alkylation and digestion are performed before or after the
A general workflow of comparative proteomic experiments in plants. Proteins are extracted and subjected to separation via gel (e.g., 2DE or 2 Dimensional gel electrophoresis) or non-gel (e.g., LC or liquid chromatography) based approaches. Reduction, alkylation and digestion are performed before or after the separation step as per the requirement to convert protein mixtures into peptides. Separated peptides are analyzed through mass spectrometer (MS) followed by tandem MS (MS/MS) for determining protein identity. The detected protein list is then used for data analysis using various bioinformatic tools.
A summary of proteome level changes detected under different abiotic stress conditions and current understanding of underlying molecular mechanisms. Color blocks inside all protein classes represent different types of abiotic stress condition as shown in the legend.
Article
Abiotic stress conditions adversely affect plant growth, resulting in significant decline in crop productivity. To mitigate and recover from the damaging effects of such adverse environmental conditions, plants have evolved various adaptive strategies at cellular and metabolic levels. Most of these strategies involve dynamic changes in protein abundance that can be best explored through proteomics. This review summarizes comparative proteomic studies conducted with roots of various plant species subjected to different abiotic stresses especially drought, salinity, flood, and cold. The main purpose of this article is to highlight and classify the protein level changes in abiotic stress response pathways specifically in plant roots. Shared as well as stressor-specific proteome signatures and adaptive mechanism(s) are simultaneously described. Such a comprehensive account will facilitate the design of genetic engineering strategies that enable the development of broad-spectrum abiotic stress-tolerant crops.
 
The dose-dependence of the vacuolar cation channels' block by intracellular polyamines and its implications for the salt stress resistance. Dose dependence for SV and FV channels at physiologically attainable (zero) tonoplast potential are drawn, using the values of blocking parameters from Brüggemann et al. (1998) and Dobrovinskaya et al., (1999a,b). Approximate ranges for intracellular PAs in plant cells are indicated by bars. At high salinity, efficient vacuolar Na+ sequestration is critical for the salt tolerance. This requires the increased Na+/H+ antiport activity and a decrease of Na+ leaks through non-selective FV and SV cation channels. The block by PAs would abolish the FV-mediated current, and strongly suppress the SV current. Continuous operation of the VK, weakly sensitive to PAs, acts as a shunt conductance for the electrogenic H+-pump, which fuels the active Na+ uptake, and contributes to the recuperation of the salt-induced cytosolic K+ loss. Salt stress stimulates expression of the cation-H+ antiporters, which may reduce the FV and SV activity via the increase of the luminal Ca2+. Over-expression of CAXs is also caused by the inhibition of the Spm4+ synthesis. Thus, PAs and vacuolar Ca2+ may act as alternative regulators of vacuolar cation channels.
Regulation of ion transport across the plasma membrane by polyamines and their catabolites. (1) PAs are exported from the cytosol to the apoplast against the electrochemical gradient. (2) PAs inhibit K+ (inward-rectifying, KIRC and outward rectifying, KORC) and non-selective voltage-independent cation (VI-NSCC) channels. (3) PAs oxidation by diamine (DAO) and/or polyamine (PAO) oxidases generates H2O2 in the apoplastic space. There H2O2 can be converted to •OH by the single electron reduction catalyzed by transient valency metal ions. (4) H2O2 and •OH activate a variety of non-selective Ca2+-permeable channels, including hyperpolarization-activated Ca2+ influx channel (HACC), annexin-formed channel (ANN), and non-selective voltage-independent conductance (ROSIC). (5) H2O2, released during PAs catabolization, causes a rapid NO generation. In its turn, NO inhibits KORC by a direct nitrosylation and in1duces the intracellular Ca2+ release via a pathway involving cGMP and cyclic adenosine ribose (cADPR). (6) Ca2+-sensitive network. Several PM channels, including slow anion channel (SLAC) and pumps, are regulated by cytosolic Ca2+. (7) PAs potentiate the ROSIC activation, activate the PM Ca2+-ATPase and alter the activity of the PM H+-ATPase.
| Summary of polyamine effects on plant ion channels and pumps.
Article
Polyamines are unique polycationic metabolites, controlling a variety of vital functions in plants, including growth and stress responses. Over the last two decades a bulk of data was accumulated providing explicit evidence that polyamines play an essential role in regulating plant membrane transport. The most straightforward example is a blockage of the two major vacuolar cation channels, namely slow (SV) and fast (FV) activating ones, by the micromolar concentrations of polyamines. This effect is direct and fully reversible, with a potency descending in a sequence Spm(4+) > Spd(3+) > Put(2+). On the contrary, effects of polyamines on the plasma membrane (PM) cation and K(+)-selective channels are hardly dependent on polyamine species, display a relatively low affinity, and are likely to be indirect. Polyamines also affect vacuolar and PM H(+) pumps and Ca(2+) pump of the PM. On the other hand, catabolization of polyamines generates H2O2 and other reactive oxygen species (ROS), including hydroxyl radicals. Export of polyamines to the apoplast and their oxidation there by available amine oxidases results in the induction of a novel ion conductance and confers Ca(2+) influx across the PM. This mechanism, initially established for plant responses to pathogen attack (including a hypersensitive response), has been recently shown to mediate plant responses to a variety of abiotic stresses. In this review we summarize the effects of polyamines and their catabolites on cation transport in plants and discuss the implications of these effects for ion homeostasis, signaling, and plant adaptive responses to environment.
 
Schematic illustration of organ-specific expression of proteins in response to drought, flooding, and salinity stresses. The scheme is based on the published organ-specific proteomic works on soybean (Glycine max L.) under the mentioned abiotic stresses. Up and down arrows indicate stress-induced increase and decrease protein abundance, respectively. Abbreviations: CS, Chalcone synthase; DHAR, dehydroascorbate reductase; GA3PDH, glyceraldehyde-3-phosphate dehydrogenase; LSU, large subunit; OEE, oxygen-evolving enhancer; SSU, small subunit; TK, Transketolase; TPI, Triosephosphate isomerase.
Article
Abiotic stresses, such as flooding, drought, salinity, and high/low temperatures, are the major constraints that global crop production faces at present. Plants respond to a stress by modulating abundance of candidate proteins, either by up-regulating expression or by the synthesizing novel proteins primarily associated with plant defense system. The cellular mechanisms of stress sensing and signal transduction into cellular organelles have been reported. Nevertheless, the responses of plant cells to abiotic stresses differ in each organ. As the correlation between the expression of mRNAs and the abundance of their corresponding proteins is difficult to assess in specific organs, proteomics techniques provide one of the best options for the functional analysis of translated regions of the genome. The present review summarizes the organ-specific proteome analyses for better understanding of the response mechanisms of crops to abiotic stresses, including flooding, drought, and salinity. The differential organ-specific responses against each of these stresses are discussed in detail to provide new insights into plant stress response mechanisms at protein level.
 
Proposed workflow that combines laboratory-based discovery proteomics followed by selected reaction monitoring mass spectrometry of field-grown plants to gain more relevant information to advance both gene discovery and gene evaluation for crop improvement programs.
Article
One major constraint upon the application of molecular crop breeding approaches is the small number of genes linked to agronomically desirable traits through defined biochemical mechanisms. Proteomic investigations of crop plants under abiotic stress treatments have identified many proteins that differ in control versus stress comparisons, however, this broad profiling of cell physiology is poorly suited to ranking the effects and identifying the specific proteins that are causative in agronomically relevant traits. Here we will reason that insights into a protein's function, its biochemical process and links to stress tolerance are more likely to arise through approaches that evaluate these differential abundances of proteins and include varietal comparisons, precise discrimination of protein isoforms, enrichment of functionally related proteins, and integration of proteomic datasets with physiological measurements of both lab and field-grown plants. We will briefly explain how applying the emerging proteomic technology of multiplexed selective reaction monitoring mass spectrometry with its accuracy and throughput can facilitate and enhance these approaches and provide a clear means to rank the growing cohort of stress responsive proteins. We will also highlight the benefit of integrating proteomic analyses with cultivar-specific genetic databases and physiological assessments of cultivar performance in relevant field environments for revealing deeper insights into molecular crop improvement.
 
Physiological responses to imposed environmental stress for all genotypes. Effect of environmental stress treatments on cob length and cob diameter are shown relative to the control treatment. Responses for inbred genotypes (open marker) or the hybrid genotype (solid marker) are depicted, with regression lines drawn in red.
Kernel iron concentrations by genotype-treatment combination. Iron concentration data are represented in μ g Fe g‒1 DW as an average of three technical replicates.
Multivariate partition analysis of inbred and environmental treatment factors important for kernel iron concentration. Combined response variables representing kernel Fe concentration and weight from each genotype were partitioned into subclasses defined by either inbred or environmental treatment conditions. Partitions were used to maximize differences in the combined Fe-weight response variable, leading to hierarchical splitting of genotype-treatment combinations until response variables were no longer considered significantly different among partition members. Factors used to distinguish subclasses are listed at the base of the node or data split; members of each partition are indicated within the box by levels of the splitting factor. Average values represent a combined kernel Fe concentration and weight variable, generated by partition analysis as indicated in methods section. The four inbred partitions representing genotype groupings with similar kernel Fe concentration and weight responses to environmental treatment are indicated with italic labels as partitions A, B, C, and D.
Genotype partition-specific response patterns of kernel weight and iron concentrations to stress environment. Kernel weight (100 KWT) and iron concentration for members of partition groups A, B, C and D in are plotted by environmental treatment. Relationship of kernel weight and kernel Fe concentration among all members of a partition group for a single treatment is demarcated by a linear regression. Regressions are flanked by a 90% confidence band. Average kernel iron concentrations and kernel weights from all members within a partition by environmental treatment are shown in the top right corner of each panel.
Stability of kernel weight and iron concentration under stress conditions across genotype partition groups. Fold change in kernel weight (X variable) or kernel iron concentration (Y variable) between environmental treatments are plotted as trait ratios (stress: control) for all genotypes in this study. Ratios are shown for: (A) drought vs. control, and; (B) combined stress vs. control. Genotypes from partitions A, C, and D are represented by open markers, and genotypes from partition B are represented by closed markers. Due to missing kernel weight data, trait ratios are not presented for all genotypes. (Genotypes with missing data points can be found in Supplementary File 1).
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Youssef Rouphael
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Rajeev K Varshney
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Shah Fahad
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