Coping with inclement weather conditions due to high temperature and water deficit in rice: An insight from genetic and biochemical perspectives.

Climatic fluctuations, temperature extremes, and water scarcity are becoming increasingly unpredictable with the passage of time. Such environmental atrocities have been the scourge of agriculture over the ages, bringing with them poor harvests and threat of famine. Rice production, owing to its high-water requirement for cultivation, is highly vulnerable to the threat of changing climate, particularly prolonged drought and high temperature, individually or in combination. Amidst all the abiotic stresses, heat and drought are considered as the most important concurrent stressors, largely affecting rice yield and productivity under the current scenario. Such threats heighten the need for new breeding and cultivation strategies in generating abiotic stress-resilient rice varieties with better yield potential. Responses of rice to these stresses can be categorized at the morphological, physiological and biochemical levels. This review examines the physiological and molecular mechanism, in the form of up regulation of several defense machineries of rice varieties to cope with drought stress (DS), high temperature stress (HTS), and their combination (DS-HTS). Genotypic differences among rice varieties in their tolerance ability have also been addressed. The review also appraises research studies conducted in rice regarding various phenotypic traits, genetic loci and response mechanisms to stress conditions to help craft new breeding strategies for improved tolerance to DS and HTS, singly or in combination. The review also encompasses the gene regulatory networks and transcription factors, and their cross-talks in mediating tolerance to such stresses. Understanding the epigenetic regulation, involving DNA methylation and histone modification during such hostile situations, will also play a crucial role in our comprehensive understanding of combinatorial stress responses. Taken together, this review consolidates current research and available information on promising rice cultivars with desirable traits as well as advocates synergistic and complementary approaches in molecular and systems biology to develop new rice breeds that favorably respond to DS-HTS-induced abiotic stress.

Engineering PGPMOs through Gene Editing and Systems Biology: A Solution for Phytoremediation?

In light of extensive urbanization and deforestation, toxic wastes are being released into the atmosphere, causing increased air and soil pollution. Conventional methods of soil remediation are time consuming and labor and cost intensive, rendering them uneconomical to maintain sustainable agriculture. One solution is to use natural resources like plants and microbes for phytoremediation. A thorough systemic knowledge of plant-microbe interactions will allow the use of gene editing and gene manipulation techniques to increase the efficiency of plants in phytoremediation. This Opinion article focuses on gene editing techniques used in plants and microbes for phytoremediation and also emphasizes their effectiveness, advancement, and future implications for sustainable and environmentally friendly agriculture.

Effect of Spectral Quality of Monochromatic LED Lights on the Growth of Artichoke Seedlings.

Indoor farming is becoming a popular alternative approach in food production to meet the demand of a growing world population. Under this production system, artificial light provides the main source of illumination in sustaining plant growth and development. The use of light-emitting diodes (LEDs) is a popular source of artificial light for indoor farms due to its narrow light spectra, modular design and energy efficiency. This study purposely assessed the effect of monochromatic LED light quality on the growth of three varieties of artichoke seedlings compared to greenhouse condition. Spectral quality assessment showed that photosynthetic photon flux density (PPFD) was highest under red LED light, but only a third of the total PPFD under natural light. Seedlings grown under red light showed 60-100% more shoot dry weight and were 67-115% taller than seedlings grown in the greenhouse. However, seedlings under blue or white light conditions showed 67-76% less in biomass compared to greenhouse-grown seedlings. Overall, plant response of seedlings under red light condition was much better compared to greenhouse-grown seedlings emphasizing the importance of red light spectral quality in plant growth and development.

Comparative Metabolome Profile between Tobacco and Soybean Grown under Water-Stressed Conditions.

Understanding how plants respond to water deficit is important in order to develop crops tolerant to drought. In this study, we compare two large metabolomics datasets where we employed a nontargeted metabolomics approach to elucidate metabolic pathways perturbed by progressive dehydration in tobacco and soybean plants. The two datasets were created using the same strategy to create water deficit conditions and an identical metabolomics pipeline. Comparisons between the two datasets therefore reveal common responses between the two species, responses specific to one of the species, responses that occur in both root and leaf tissues, and responses that are specific to one tissue. Stomatal closure is the immediate response of the plant and this did not coincide with accumulation of abscisic acid. A total of 116 and 140 metabolites were observed in tobacco leaves and roots, respectively, while 241 and 207 were observed in soybean leaves and roots, respectively. Accumulation of metabolites is significantly correlated with the extent of dehydration in both species. Among the metabolites that show increases that are restricted to just one plant, 4-hydroxy-2-oxoglutaric acid (KHG) in tobacco roots and coumestrol in soybean roots show the highest tissue-specific accumulation. The comparisons of these two large nontargeted metabolomics datasets provide novel information and suggest that KHG will be a useful marker for drought stress for some members of Solanaceae and coumestrol for some legume species.

A toolbox of genes, proteins, metabolites and promoters for improving drought tolerance in soybean includes the metabolite coumestrol and stomatal development genes.

BACKGROUND: The purpose of this project was to identify metabolites, proteins, genes, and promoters associated with water stress responses in soybean. A number of these may serve as new targets for the biotechnological improvement of drought responses in soybean (Glycine max). RESULTS: We identified metabolites, proteins, and genes that are strongly up or down regulated during rapid water stress following removal from a hydroponics system. 163 metabolites showed significant changes during water stress in roots and 93 in leaves. The largest change was a root-specific 160-fold increase in the coumestan coumestrol making it a potential biomarker for drought and a promising target for improving drought responses. Previous reports suggest that coumestrol stimulates mycorrhizal colonization and under certain conditions mycorrhizal plants have improved drought tolerance. This suggests that coumestrol may be part of a call for help to the rhizobiome during stress. About 3,000 genes were strongly up-regulated by drought and we identified regulators such as ERF, MYB, NAC, bHLH, and WRKY transcription factors, receptor-like kinases, and calcium signaling components as potential targets for soybean improvement as well as the jasmonate and abscisic acid biosynthetic genes JMT, LOX1, and ABA1. Drought stressed soybean leaves show reduced mRNA levels of stomatal development genes including FAMA-like, MUTE-like and SPEECHLESS-like bHLH transcription factors and leaves formed after drought stress had a reduction in stomatal density of 22.34 % and stomatal index of 17.56 %. This suggests that reducing stomatal density may improve drought tolerance. MEME analyses suggest that ABRE (CACGT/CG), CRT/DRE (CCGAC) and a novel GTGCnTGC/G element play roles in transcriptional activation and these could form components of synthetic promoters to drive expression of transgenes. Using transformed hairy roots, we validated the increase in promoter activity of GmWRKY17 and GmWRKY67 during dehydration and after 20 µM ABA treatment. CONCLUSIONS: Our toolbox provides new targets and strategies for improving soybean drought tolerance and includes the coumestan coumestrol, transcription factors that regulate stomatal density, water stress-responsive WRKY gene promoters and a novel DNA element that appears to be enriched in water stress responsive promoters.

The WRKY transcription factor family and senescence in switchgrass.

BACKGROUND: Early 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. METHODS: All 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. RESULTS: We 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. CONCLUSIONS: We 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.

Transcriptome profiling of tobacco under water deficit conditions.

Drought is one of the limiting environmental factors that affect crop production. Understanding the molecular basis of how plants respond to this water deficit stress is key to developing drought tolerant crops. In this study we generated time course-based transcriptome profiles of tobacco plants under water deficit conditions using microarray technology. In this paper, we describe in detail the experimental procedures and analyses performed in our study. The data set we generated (available in the NCBI/GEO database under GSE67434) has been analysed to identify genes that are involved in the regulation of tobacco's responses to drought.

Transcriptomics analyses of soybean leaf and root samples during water-deficit.

Drought being a major challenge for crop productivity and yield affects multigenic and quantitative traits. It is also well documented that water stress shows a cross talk with other abiotic stresses such as high temperature and high light intensities (Tripathi et al., 2013) [1]. In this report, we documented the details of the methods and quality controls used and considered in our time course-based transcriptome profile of soybean plants under water deficit conditions using microarray technology. The findings of this study are recently published by the Rushton lab in BMC Genomics for a comparative study of tobacco and Soybean (Rabara et al., 2015) [2]. The raw microarray data set is deposited in GEO database with accession number GSE49537.

Tobacco drought stress responses reveal new targets for Solanaceae crop improvement.

BACKGROUND: The Solanaceae are an economically important family of plants that include tobacco (Nicotiana tabacum L.), tomato, and potato. Drought is a major cause of crop losses. RESULTS: We have identified major changes in physiology, metabolites, mRNA levels, and promoter activities during the tobacco response to drought. We have classified these as potential components of core responses that may be common to many plant species or responses that may be family/species-specific features of the drought stress response in tobacco or the Solanaceae. In tobacco the largest increase in any metabolite was a striking 70-fold increase in 4-hydroxy-2-oxoglutaric acid (KHG) in roots that appears to be tobacco/Solanaceae specific. KHG is poorly characterized in plants but is broken down to pyruvate and glyoxylate after the E. coli SOS response to facilitate the resumption of respiration. A similar process in tobacco would represent a mechanism to restart respiration upon water availability after drought. At the mRNA level, transcription factor gene induction by drought also showed both core and species/family specific responses. Many Group IX Subgroup 3 AP2/ERF transcription factors in tobacco appear to play roles in nicotine biosynthesis as a response to herbivory, whereas their counterparts in legume species appear to play roles in drought responses. We observed apparent Solanaceae-specific drought induction of several Group IId WRKY genes. One of these, NtWRKY69, showed ABA-independent drought stress-inducible promoter activity that moved into the leaf through the vascular tissue and then eventually into the surrounding leaf cells. CONCLUSIONS: We propose components of a core metabolic response to drought stress in plants and also show that some major responses to drought stress at the metabolome and transcriptome levels are family specific. We therefore propose that the observed family-specific changes in metabolism are regulated, at least in part, by family-specific changes in transcription factor activity. We also present a list of potential targets for the improvement of Solanaceae drought responses.

The interactome of soybean GmWRKY53 using yeast 2-hybrid library screening to saturation.

Soybean GmWRKY53 functions in both biotic and abiotic stress signaling. Using GmWRKY53 as a bait yeast 2-hybrid library screening to saturation isolated multiple independent fragments for many interacting proteins, enabling delineation of minimal interacting domains and computation of a confidence score. Multiple independent clones coding for the LATE ELONGATED HYPOCOTYL clock protein GmLCL2 (MYB114) were isolated and the binding site for GmWRKY53 was mapped to 90 amino acids separate from the MYB domain. This suggests a direct input from the clock on GmWRKY53 activity. The GmWRKY53-interacting proteins also included 3 water stress-inducible AP2/ERF transcription factors. One of these (Glyma03g26310) is one of the most strongly water stress induced genes in soybean roots, suggesting that GmWRKY53/ERF complexes regulate water stress responses.

The evolution of WRKY transcription factors.

BACKGROUND: The availability of increasing numbers of sequenced genomes has necessitated a re-evaluation of the evolution of the WRKY transcription factor family. Modern day plants descended from a charophyte green alga that colonized the land between 430 and 470 million years ago. The first charophyte genome sequence from Klebsormidium flaccidum filled a gap in the available genome sequences in the plant kingdom between unicellular green algae that typically have 1-3 WRKY genes and mosses that contain 30-40. WRKY genes have been previously found in non-plant species but their occurrence has been difficult to explain. RESULTS: Only two WRKY genes are present in the Klebsormidium flaccidum genome and the presence of a Group IIb gene was unexpected because it had previously been thought that Group IIb WRKY genes first appeared in mosses. We found WRKY transcription factor genes outside of the plant lineage in some diplomonads, social amoebae, fungi incertae sedis, and amoebozoa. This patchy distribution suggests that lateral gene transfer is responsible. These lateral gene transfer events appear to pre-date the formation of the WRKY groups in flowering plants. Flowering plants contain proteins with domains typical for both resistance (R) proteins and WRKY transcription factors. R protein-WRKY genes have evolved numerous times in flowering plants, each type being restricted to specific flowering plant lineages. These chimeric proteins contain not only novel combinations of protein domains but also novel combinations and numbers of WRKY domains. Once formed, R protein WRKY genes may combine different components of signalling pathways that may either create new diversity in signalling or accelerate signalling by short circuiting signalling pathways. CONCLUSIONS: We propose that the evolution of WRKY transcription factors includes early lateral gene transfers to non-plant organisms and the occurrence of algal WRKY genes that have no counterparts in flowering plants. We propose two alternative hypotheses of WRKY gene evolution: The "Group I Hypothesis" sees all WRKY genes evolving from Group I C-terminal WRKY domains. The alternative "IIa + b Separate Hypothesis" sees Groups IIa and IIb evolving directly from a single domain algal gene separate from the Group I-derived lineage.

Understanding Water-Stress Responses in Soybean Using Hydroponics System-A Systems Biology Perspective.

The deleterious changes in environmental conditions such as water stress bring physiological and biochemical changes in plants, which results in crop loss. Thus, combating water stress is important for crop improvement to manage the needs of growing population. Utilization of hydroponics system in growing plants is questionable to some researchers, as it does not represent an actual field condition. However, trying to address a complex problem like water stress we have to utilize a simpler growing condition like the hydroponics system wherein every input given to the plants can be controlled. With the advent of high-throughput technologies, it is still challenging to address all levels of the genetic machinery whether a gene, protein, metabolite, and promoter. Thus, using a system of reduced complexity like hydroponics can certainly direct us toward the right candidates, if not completely help us to resolve the issue.

The potential of transcription factor-based genetic engineering in improving crop tolerance to drought.

Drought is one of the major constraints in crop production and has an effect on a global scale. In order to improve crop production, it is necessary to understand how plants respond to stress. A good understanding of regulatory mechanisms involved in plant responses during drought will enable researchers to explore and manipulate key regulatory points in order to enhance stress tolerance in crops. Transcription factors (TFs) have played an important role in crop improvement from the dawn of agriculture. TFs are therefore good candidates for genetic engineering to improve crop tolerance to drought because of their role as master regulators of clusters of genes. Many families of TFs, such as CCAAT, homeodomain, bHLH, NAC, AP2/ERF, bZIP, and WRKY have members that may have the potential to be tools for improving crop tolerance to drought. In this review, the roles of TFs as tools to improve drought tolerance in crops are discussed. The review also focuses on current strategies in the use of TFs, with emphasis on several major TF families in improving drought tolerance of major crops. Finally, many promising transgenic lines that may have improved drought responses have been poorly characterized and consequently their usefulness in the field is uncertain. New advances in high-throughput phenotyping, both greenhouse and field based, should facilitate improved phenomics of transgenic lines. Systems biology approaches should then define the underlying changes that result in higher yields under water stress conditions. These new technologies should help show whether manipulating TFs can have effects on yield under field conditions.

A systems biology perspective on the role of WRKY transcription factors in drought responses in plants.

Drought is one of the major challenges affecting crop productivity and yield. However, water stress responses are notoriously multigenic and quantitative with strong environmental effects on phenotypes. It is also clear that water stress often does not occur alone under field conditions but rather in conjunction with other abiotic stresses such as high temperature and high light intensities. A multidisciplinary approach with successful integration of a whole range of -omics technologies will not only define the system, but also provide new gene targets for both transgenic approaches and marker-assisted selection. Transcription factors are major players in water stress signaling and some constitute major hubs in the signaling webs. The main transcription factors in this network include MYB, bHLH, bZIP, ERF, NAC, and WRKY transcription factors. The role of WRKY transcription factors in abiotic stress signaling networks is just becoming apparent and systems biology approaches are starting to define their places in the signaling network. Using systems biology approaches, there are now many transcriptomic analyses and promoter analyses that concern WRKY transcription factors. In addition, reports on nuclear proteomics have identified WRKY proteins that are up-regulated at the protein level by water stress. Interactomics has started to identify different classes of WRKY-interacting proteins. What are often lacking are connections between metabolomics, WRKY transcription factors, promoters, biosynthetic pathways, fluxes and downstream responses. As more levels of the system are characterized, a more detailed understanding of the roles of WRKY transcription factors in drought responses in crops will be obtained.

Extending MapMan Ontology to Tobacco for Visualization of Gene Expression.

Microarrays are a large-scale expression profiling method which has been used to study the transcriptome of plants under various environmental conditions. However, manual inspection of microarray data is difficult at the genome level because of the large number of genes (normally at least 30,000) and the many different processes that occur within any given plant. MapMan software, which was initially developed to visualize microarray data for Arabidopsis, has been adapted to other plant species by mapping other species onto MapMan ontology. This paper provides a detailed procedure and the relevant computing codes to generate a MapMan ontology mapping file for tobacco (Nicotiana tabacum L.) using potato and Arabidopsis as intermediates. The mapping file can be used directly with our custom made NimbleGen oligoarray, that contains gene sequences from both the tobacco gene space sequence and the tobacco gene index 4 (NTGI4) collection of ESTs. The generated data set will be informative for scientists working on tobacco as their model plant by providing a MapMan ontology mapping file to tobacco, homology between tobacco coding sequences and that of potato and Arabidopsis, as well as adapting our procedure and codes for other plant species where the complete genome is not yet available.

GmWRKY53, a water- and salt-inducible soybean gene for rapid dissection of regulatory elements in BY-2 cell culture.

Drought is the major cause of crop losses worldwide. Water stress-inducible promoters are important for understanding the mechanisms of water stress responses in crop plants. Here we utilized tobacco (Nicotiana tabacum L.) Bright Yellow 2 (BY-2) cell system in presence of polyethylene glycol, salt and phytohormones. Extension of the system to 85 mM NaCl led to inducibility of up to 10-fold with the water stress and salt responsive soybean GmWRKY53 promoter. Upon ABA and JA treatment fold inducibility was up to 5-fold and 14-fold, respectively. Thus, we hypothesize that GmWRKY53 could be used as potential model candidate for dissecting drought regulatory elements as well as understanding crosstalk utilizing a rapid heterologous system of BY-2 culture.

Dehydration-induced WRKY genes from tobacco and soybean respond to jasmonic acid treatments in BY-2 cell culture.

Drought is one of the important environmental factors affecting crop production worldwide and therefore understanding the molecular response of plant to stress is an important step in crop improvement. WRKY transcription factors are one of the 10 largest transcription factor families across the green lineage. In this study, highly upregulated dehydration-induced WRKY and enzyme-coding genes from tobacco and soybean were selected from microarray data for promoter analyses. Putative stress-related cis-regulatory elements such as TGACG motif, ABRE-like elements; W and G-like sequences were identified by an in silico analyses of promoter region of the selected genes. GFP quantification of transgenic BY-2 cell culture showed these promoters direct higher expression in-response to 100 µM JA treatment compared to 100 µM ABA, 10% PEG and 85 mM NaCl treatments. Thus promoter activity upon JA treatment and enrichment of MeJA-responsive elements in the promoter of the selected genes provides insights for these genes to be jasmonic acid responsive with potential of mediating cross-talk during dehydration responses.

The WRKY transcription factor family in Brachypodium distachyon.

BACKGROUND: A 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. RESULTS: We 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. CONCLUSIONS: The 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.

WRKY transcription factors: key components in abscisic acid signalling.

WRKY transcription factors (TFs) are key regulators of many plant processes, including the responses to biotic and abiotic stresses, senescence, seed dormancy and seed germination. For over 15 years, limited evidence has been available suggesting that WRKY TFs may play roles in regulating plant responses to the phytohormone abscisic acid (ABA), notably some WRKY TFs are ABA-inducible repressors of seed germination. However, the roles of WRKY TFs in other aspects of ABA signalling, and the mechanisms involved, have remained unclear. Recent significant progress in ABA research has now placed specific WRKY TFs firmly in ABA-responsive signalling pathways, where they act at multiple levels. In Arabidopsis, WRKY TFs appear to act downstream of at least two ABA receptors: the cytoplasmic PYR/PYL/RCAR-protein phosphatase 2C-ABA complex and the chloroplast envelope-located ABAR-ABA complex. In vivo and in vitro promoter-binding studies show that the target genes for WRKY TFs that are involved in ABA signalling include well-known ABA-responsive genes such as ABF2, ABF4, ABI4, ABI5, MYB2, DREB1a, DREB2a and RAB18. Additional well-characterized stress-inducible genes such as RD29A and COR47 are also found in signalling pathways downstream of WRKY TFs. These new insights also reveal that some WRKY TFs are positive regulators of ABA-mediated stomatal closure and hence drought responses. Conversely, many WRKY TFs are negative regulators of seed germination, and controlling seed germination appears a common function of a subset of WRKY TFs in flowering plants. Taken together, these new data demonstrate that WRKY TFs are key nodes in ABA-responsive signalling networks.