Jai S. Rohila

The WRKY transcription factor family in Brachypodium distachyon

BackgroundA complete assembled genome sequence of wheat is not yet available. Therefore, model plant systems for wheat are very valuable. Brachypodium distachyon (Brachypodium) is such a system. The WRKY family of transcription factors is one of the most important families of plant transcriptional regulators with members regulating important agronomic traits. Studies of WRKY transcription factors in Brachypodium and wheat therefore promise to lead to new strategies for wheat improvement.ResultsWe have identified and manually curated the WRKY transcription factor family from Brachypodium using a pipeline designed to identify all potential WRKY genes. 86 WRKY transcription factors were found, a total higher than all other current databases. We therefore propose that our numbering system (BdWRKY1-BdWRKY86) becomes the standard nomenclature. In the JGI v1.0 assembly of Brachypodium with the MIPS/JGI v1.0 annotation, nine of the transcription factors have no gene model and eleven gene models are probably incorrectly predicted. In total, twenty WRKY transcription factors (23.3%) do not appear to have accurate gene models. To facilitate use of our data, we have produced The Database of Brachypodium distachyon WRKY Transcription Factors. Each WRKY transcription factor has a gene page that includes predicted protein domains from MEME analyses. These conserved protein domains reflect possible input and output domains in signaling. The database also contains a BLAST search function where a large dataset of WRKY transcription factors, published genes, and an extensive set of wheat ESTs can be searched. We also produced a phylogram containing the WRKY transcription factor families from Brachypodium, rice, Arabidopsis, soybean, and Physcomitrella patens, together with published WRKY transcription factors from wheat. This phylogenetic tree provides evidence for orthologues, co-orthologues, and paralogues of Brachypodium WRKY transcription factors.ConclusionsThe description of the WRKY transcription factor family in Brachypodium that we report here provides a framework for functional genomics studies in an important model system. Our database is a resource for both Brachypodium and wheat studies and ultimately projects aimed at improving wheat through manipulation of WRKY transcription factors.

Leaf Proteome Analysis Reveals Prospective Drought and Heat Stress Response Mechanisms in Soybean

Drought and heat are among the major abiotic stresses that affect soybean crops worldwide. During the current investigation, the effect of drought, heat, and drought plus heat stresses was compared in the leaves of two soybean varieties, Surge and Davison, combining 2D-DIGE proteomic data with physiology and biochemical analyses. We demonstrated how 25 differentially expressed photosynthesis-related proteins affect RuBisCO regulation, electron transport, Calvin cycle, and carbon fixation during drought and heat stress. We also observed higher abundance of heat stress-induced EF-Tu protein in Surge. It is possible that EF-Tu might have activated heat tolerance mechanisms in the soybean. Higher level expressions of heat shock-related protein seem to be regulating the heat tolerance mechanisms. This study identifies the differential expression of various abiotic stress-responsive proteins that regulate various molecular processes and signaling cascades. One inevitable outcome from the biochemical and proteomics assays of this study is that increase of ROS levels during drought stress does not show significant changes at the phenotypic level in Davison and this seems to be due to a higher amount of carbonic anhydrase accumulation in the cell which aids the cell to become more resistant to cytotoxic concentrations of H2O2.

Proteomics: a biotechnology tool for crop improvement

A sharp decline in the availability of arable land and sufficient supply of irrigation water along with a continuous steep increase in food demands have exerted a pressure on farmers to produce more with fewer resources. A viable solution to release this pressure is to speed up the plant breeding process by employing biotechnology in breeding programs. The majority of biotechnological applications rely on information generated from various -omic technologies. The latest outstanding improvements in proteomic platforms and many other but related advances in plant biotechnology techniques offer various new ways to encourage the usage of these technologies by plant scientists for crop improvement programs. A combinatorial approach of accelerated gene discovery through genomics, proteomics, and other associated -omic branches of biotechnology, as an applied approach, is proving to be an effective way to speed up the crop improvement programs worldwide. In the near future, swift improvements in -omic databases are becoming critical and demand immediate attention for the effective utilization of these techniques to produce next-generation crops for the progressive farmers. Here, we have reviewed the recent advances in proteomics, as tools of biotechnology, which are offering great promise and leading the path toward crop improvement for sustainable agriculture.

The WRKY transcription factor family and senescence in switchgrass.

BackgroundEarly aerial senescence in switchgrass (Panicum virgatum) can significantly limit biomass yields. WRKY transcription factors that can regulate senescence could be used to reprogram senescence and enhance biomass yields.MethodsAll potential WRKY genes present in the version 1.0 of the switchgrass genome were identified and curated using manual and bioinformatic methods. Expression profiles of WRKY genes in switchgrass flag leaf RNA-Seq datasets were analyzed using clustering and network analyses tools to identify both WRKY and WRKY-associated gene co-expression networks during leaf development and senescence onset.ResultsWe identified 240 switchgrass WRKY genes including members of the RW5 and RW6 families of resistance proteins. Weighted gene co-expression network analysis of the flag leaf transcriptomes across development readily separated clusters of co-expressed genes into thirteen modules. A visualization highlighted separation of modules associated with the early and senescence-onset phases of flag leaf growth. The senescence-associated module contained 3000 genes including 23 WRKYs. Putative promoter regions of senescence-associated WRKY genes contained several cis-element-like sequences suggestive of responsiveness to both senescence and stress signaling pathways. A phylogenetic comparison of senescence-associated WRKY genes from switchgrass flag leaf with senescence-associated WRKY genes from other plants revealed notable hotspots in Group I, IIb, and IIe of the phylogenetic tree.ConclusionsWe have identified and named 240 WRKY genes in the switchgrass genome. Twenty three of these genes show elevated mRNA levels during the onset of flag leaf senescence. Eleven of the WRKY genes were found in hotspots of related senescence-associated genes from multiple species and thus represent promising targets for future switchgrass genetic improvement. Overall, individual WRKY gene expression profiles could be readily linked to developmental stages of flag leaves.

The Potential of Proteomics Technologies for Crop Improvement under Drought Conditions

Sustainable food production is a major challenge to our current agricultural systems. At the global level, water stress is one of the most critical abiotic factors that affect crop productivity. Currently, a vast amount of research efforts are being laid down to generate molecular information leading to an understanding of this abiotic stress factor. Although gene expression analysis at the transcription level has enhanced our understanding regarding the response of plants to drought stress, many questions remain unanswered at the protein level. In view of the fact that the plant proteome is highly dynamic in nature, proteomics is becoming essential for the study of many different aspects of plant functions. Further, proteomics is also a key part of the –omics suite of techniques and facilitates a systems biology approach to biological problems. Although proteomics studies of drought responses in crop plants are still in their infancy, recent significant progress illustrates the promise of proteomics projects for improving drought stress responses in crop plants. This review mainly focuses on the response of various plant proteomes under drought stress conditions. The results of previous comparative proteomics investigations are analyzed, which allow us to gather information on how different plant species alter their proteome to sustain themselves under the drought stress conditions. We predict that proteomics-based projects will continue the discovery of novel gene targets for crop improvement and that a systems biology approach, that includes proteomics, holds great promise for the future of agriculture.

Identification, Nomenclature, and Evolutionary Relationships of Mitogen-Activated Protein Kinase (MAPK) Genes in Soybean

Mitogen-activated protein kinase (MAPK) genes in eukaryotes regulate various developmental and physiological processes including those associated with biotic and abiotic stresses. Although MAPKs in some plant species including Arabidopsis have been identified, they are yet to be identified in soybean. Major objectives of this study were to identify GmMAPKs, assess their evolutionary relationships, and analyze their functional divergence. We identified a total of 38 MAPKs, eleven MAPKKs, and 150 MAPKKKs in soybean. Within the GmMAPK family, we also identified a new clade of six genes: four genes with TEY and two genes with TQY motifs requiring further investigation into possible legume-specific functions. The results indicated the expansion of the GmMAPK families attributable to the ancestral polyploidy events followed by chromosomal rearrangements. The GmMAPK and GmMAPKKK families were substantially larger than those in other plant species. The duplicated GmMAPK members presented complex evolutionary relationships and functional divergence when compared to their counterparts in Arabidopsis. We also highlighted existing nomenclatural issues, stressing the need for nomenclatural consistency. GmMAPK identification is vital to soybean crop improvement, and novel insights into the evolutionary relationships will enhance our understanding about plant genome evolution.

Comparative proteomics analysis by DIGE and iTRAQ provides insight into the regulation of phenylpropanoids in maize.

UNLABELLED The maize pericarp color1 (p1) gene encodes a Myb transcription factor that regulates the accumulation of 3-deoxyflavonoid pigments called phlobaphenes. The Unstable factor for orange1 (Ufo1) is a dominant epigenetic modifier of the p1 that results in ectopic pigmentation in pericarp. Presence of Ufo1-1 correlates with pleiotropic growth and developmental defects. To investigate the Ufo1-1-induced changes in the proteome, we conducted comparative proteomics analysis of P1-wr; Ufo1-1 pericarps using the 2-D DIGE and iTRAQ techniques. Most of the identified proteins were found to be involved in glycolysis, protein synthesis and modification, flavonoid and lignin biosynthesis and defense responses. Further, immunoblot analysis of internode protein extracts demonstrated that caffeoyl CoA O-methyltransferase (COMT) is post-transcriptionally down regulated in P1-wr; Ufo1-1 plants. Consistent with the down regulation of COMT, the concentrations of p-coumaric acid, syringaldehydes, and lignin are reduced in P1-wr; Ufo1-1 internodes. The reductions in these phenylpropanoids correlate with the bent stalk and stunted growth of P1-wr; Ufo1-1 plants. Finally, over-expression of the p1 in transgenic plants is also correlated with a lodging phenotype and reduced COMT expression. We conclude that ectopic expression of p1 can result in developmental defects that are correlated with altered regulation and synthesis of phenylpropanoid compounds including lignin. BIOLOGICAL SIGNIFICANCE Transcription factors have specific expression patterns that ensure that the biochemical pathways under their control are active in relevant tissues. Plant breeders can select for alleles of transcription factors that produce desirable expression patterns to improve a plants growth, development, and defense against insects and pathogens. The resulting de novo accumulation of metabolites in plant tissues in significant quantities could have beneficial and/or detrimental consequences. To understand this problem we investigated how the aberrant expression of a classically-studied transcription factor pericarp color1 (p1) which regulates phenylpropanoid metabolism, affects the maize proteome in pericarp tissue. We utilized a dominant mutant Unstable factor for orange 1-1 (Ufo1-1) which reduces the epigenetic suppression of p1 in various tissues throughout the maize plant. Our proteomic analysis shows how, in the presence of Ufo1-1, key enzymes of the glycolytic and shikimic acid pathways were modulated to produce substrates required for flavonoid synthesis. The finding that the presence of Ufo1-1 affected the expression levels of various enzymes in the lignin pathway was of particular interest. We show that lignin was reduced in Ufo1-1 plants expressing p1 and was associated with the post-transcriptional down regulation of CoA O-methyltransferase (COMT) enzyme. We further correlated the down-regulation of COMT with plant bending phenotype in Ufo1-1 plants expressing p1 and to a stalk lodging phenotype of transgenic p1 plants. This study demonstrates that although there can be adverse consequences to aberrantly overexpressing transcription factors, there might also be benefits such as being able to reduce lignin content for biofuel crops. However, more research will be required to understand the genetic and epigenetic regulation of transcription factors and how their expression can be optimized to obtain desired traits in preferred tissue types. This article is part of a Special Issue entitled: Translational Plant Proteomics.

Metabolomic Profiling of Soybeans (Glycine max L.) Reveals the Importance of Sugar and Nitrogen Metabolism under Drought and Heat Stress

Soybean is an important crop that is continually threatened by abiotic stresses, especially drought and heat stress. At molecular levels, reduced yields due to drought and heat stress can be seen as a result of alterations in metabolic homeostasis of vegetative tissues. At present an incomplete understanding of abiotic stress-associated metabolism and identification of associated metabolites remains a major gap in soybean stress research. A study with a goal to profile leaf metabolites under control conditions (28/24 °C), drought [28/24 °C, 10% volumetric water content (VWC)], and heat stress (43/35 °C) was conducted in a controlled environment. Analyses of non-targeted metabolomic data showed that in response to drought and heat stress, key metabolites (carbohydrates, amino acids, lipids, cofactors, nucleotides, peptides and secondary metabolites) were differentially accumulated in soybean leaves. The metabolites for various cellular processes, such as glycolysis, the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway, and starch biosynthesis, that regulate carbohydrate metabolism, amino acid metabolism, peptide metabolism, and purine and pyrimidine biosynthesis, were found to be affected by drought as well as heat stress. Computationally based regulatory networks predicted additional compounds that address the possibility of other metabolites and metabolic pathways that could also be important for soybean under drought and heat stress conditions. Metabolomic profiling demonstrated that in soybeans, keeping up with sugar and nitrogen metabolism is of prime significance, along with phytochemical metabolism under drought and heat stress conditions.

2D-DIGE-based proteome expression changes in leaves of rice seedlings exposed to low-level gamma radiation at Iitate village, Fukushima

The present study continues our previous research on investigating the biological effects of low-level gamma radiation in rice at the heavily contaminated Iitate village in Fukushima, by extending the experiments to unraveling the leaf proteome. 14-days-old plants of Japonica rice (Oryza sativa L. cv. Nipponbare) were subjected to gamma radiation level of upto 4 µSv/h, for 72 h. Following exposure, leaf samples were taken from the around 190 µSv/3 d exposed seedling and total proteins were extracted. The gamma irradiated leaf and control leaf (harvested at the start of the experiment) protein lysates were used in a 2-D differential gel electrophoresis (2D-DIGE) experiment using CyDye labeling in order to asses which spots were differentially represented, a novelty of the study. 2D-DIGE analysis revealed 91 spots with significantly different expression between samples (60 positive, 31 negative). MALDI-TOF and TOF/TOF mass spectrometry analyses revealed those as comprising of 59 different proteins (50 up-accumulated, 9 down-accumulated). The identified proteins were subdivided into 10 categories, according to their biological function, which indicated that the majority of the differentially expressed proteins consisted of the general (non-energy) metabolism and stress response categories. Proteome-wide data point to some effects of low-level gamma radiation exposure on the metabolism of rice leaves.

Proteomic Responses of Switchgrass and Prairie Cordgrass to Senescence

Senescence in biofuel grasses is a critical issue because early senescence decreases potential biomass production by limiting aerial growth and development. 2-Dimensional, differential in-gel electrophoresis (2D-DIGE) followed by mass spectrometry of selected protein spots was used to evaluate differences between leaf proteomes of early (ES)- and late- senescing (LS) genotypes of Prairie cordgrass (ES/LS PCG) and switchgrass (ES/LS SG), just before and after senescence was initiated. Analysis of the manually filtered and statistically evaluated data indicated that 69 proteins were significantly differentially abundant across all comparisons, and a majority (41%) were associated with photosynthetic processes as determined by gene ontology analysis. Ten proteins were found in common between PCG and SG, and nine and 18 proteins were unique to PCG and SG respectively. Five of the 10 differentially abundant spots common to both species were increased in abundance, and five were decreased in abundance. Leaf proteomes of the LS genotypes of both grasses analyzed before senescence contained significantly higher abundances of a 14-3-3 like protein and a glutathione-S-transferase protein when compared to the ES genotypes, suggesting differential cellular metabolism in the LS vs. the ES genotypes. The higher abundance of 14-3-3 like proteins may be one factor that impacts the senescence process in both LS PCG and LS SG. Aconitase dehydratase was found in greater abundance in all four genotypes after the onset of senescence, consistent with literature reports from genetic and transcriptomic studies. A Rab protein of the Ras family of G proteins and an s-adenosylmethionine synthase were more abundant in ES PCG when compared with the LS PCG. In contrast, several proteins associated with photosynthesis and carbon assimilation were detected in greater abundance in LS PCG when compared to ES PCG, suggesting that a loss of these proteins potentially contributed to the ES phenotype in PCG. Overall, this study provides important data that can be utilized toward delaying senescence in both PCG and SG, and sets a foundational base for future improvement of perennial grass germplasm for greater aerial biomass productivity.