Kaoru Urano

Effects of abiotic stress on plants: a systems biology perspective.

The natural environment for plants is composed of a complex set of abiotic stresses and biotic stresses. Plant responses to these stresses are equally complex. Systems biology approaches facilitate a multi-targeted approach by allowing one to identify regulatory hubs in complex networks. Systems biology takes the molecular parts (transcripts, proteins and metabolites) of an organism and attempts to fit them into functional networks or models designed to describe and predict the dynamic activities of that organism in different environments. In this review, research progress in plant responses to abiotic stresses is summarized from the physiological level to the molecular level. New insights obtained from the integration of omics datasets are highlighted. Gaps in our knowledge are identified, providing additional focus areas for crop improvement research in the future.

Characterization of the ABA-regulated global responses to dehydration in Arabidopsis by metabolomics.

Drought is the major environmental threat to agricultural production and distribution worldwide. Adaptation by plants to dehydration stress is a complex biological process that involves global changes in gene expression and metabolite composition. Here, using one type of functional genomics analysis, metabolomics, we characterized the metabolic phenotypes of Arabidopsis wild-type and a knockout mutant of the NCED3 gene (nc3-2) under dehydration stress. NCED3 plays a role in the dehydration-inducible biosynthesis of abscisic acid (ABA), a phytohormone that is important in the dehydration-stress response in higher plants. Metabolite profiling performed using two types of mass spectrometry (MS) systems, gas chromatography/time-of-flight MS (GC/TOF-MS) and capillary electrophoresis MS (CE-MS), revealed that accumulation of amino acids depended on ABA production, but the level of the oligosaccharide raffinose was regulated by ABA independently under dehydration stress. Metabolic network analysis showed that global metabolite-metabolite correlations occurred in dehydration-increased amino acids in wild-type, and strong correlations with raffinose were reconstructed in nc3-2. An integrated metabolome and transcriptome analysis revealed ABA-dependent transcriptional regulation of the biosynthesis of the branched-chain amino acids, saccharopine, proline and polyamine. This metabolomics analysis revealed new molecular mechanisms of dynamic metabolic networks in response to dehydration stress.

‘Omics’ analyses of regulatory networks in plant abiotic stress responses

Plants must respond and adapt to abiotic stresses to survive in various environmental conditions. Plants have acquired various stress tolerance mechanisms, which are different processes involving physiological and biochemical changes that result in adaptive or morphological changes. Recent advances in genome-wide analyses have revealed complex regulatory networks that control global gene expression, protein modification, and metabolite composition. Genetic regulation and epigenetic regulation, including changes in nucleosome distribution, histone modification, DNA methylation, and npcRNAs (non-protein-coding RNA) play important roles in abiotic stress gene networks. Transcriptomics, metabolomics, bioinformatics, and high-through-put DNA sequencing have enabled active analyses of regulatory networks that control abiotic stress responses. Such analyses have markedly increased our understanding of global plant systems in responses and adaptation to stress conditions.

Metabolic Pathways Involved in Cold Acclimation Identified by Integrated Analysis of Metabolites and Transcripts Regulated by DREB1A and DREB2A

DREB1A/CBF3 and DREB2A are transcription factors that specifically interact with a cis-acting dehydration-responsive element (DRE), which is involved in cold- and dehydration-responsive gene expression in Arabidopsis (Arabidopsis thaliana). Overexpression of DREB1A improves stress tolerance to both freezing and dehydration in transgenic plants. In contrast, overexpression of an active form of DREB2A results in significant stress tolerance to dehydration but only slight tolerance to freezing in transgenic plants. The downstream gene products for DREB1A and DREB2A are reported to have similar putative functions, but downstream genes encoding enzymes for carbohydrate metabolism are very different between DREB1A and DREB2A. We demonstrate that under cold and dehydration conditions, the expression of many genes encoding starch-degrading enzymes, sucrose metabolism enzymes, and sugar alcohol synthases changes dynamically; consequently, many kinds of monosaccharides, disaccharides, trisaccharides, and sugar alcohols accumulate in Arabidopsis. We also show that DREB1A overexpression can cause almost the same changes in these metabolic processes and that these changes seem to improve freezing and dehydration stress tolerance in transgenic plants. In contrast, DREB2A overexpression did not increase the level of any of these metabolites in transgenic plants. Strong freezing stress tolerance of the transgenic plants overexpressing DREB1A may depend on accumulation of these metabolites.

Enhancement of oxidative and drought tolerance in Arabidopsis by overaccumulation of antioxidant flavonoids

The notion that plants use specialized metabolism to protect against environmental stresses needs to be experimentally proven by addressing the question of whether stress tolerance by specialized metabolism is directly due to metabolites such as flavonoids. We report that flavonoids with radical scavenging activity mitigate against oxidative and drought stress in Arabidopsis thaliana. Metabolome and transcriptome profiling and experiments with oxidative and drought stress in wild-type, single overexpressors of MYB12/PFG1 (PRODUCTION OF FLAVONOL GLYCOSIDES1) or MYB75/PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT1), double overexpressors of MYB12 and PAP1, transparent testa4 (tt4) as a flavonoid-deficient mutant, and flavonoid-deficient MYB12 or PAP1 overexpressing lines (obtained by crossing tt4 and the individual MYB overexpressor) demonstrated that flavonoid overaccumulation was key to enhanced tolerance to such stresses. Antioxidative activity assays using 2,2-diphenyl-1-picrylhydrazyl, methyl viologen, and 3,3′-diaminobenzidine clearly showed that anthocyanin overaccumulation with strong in vitro antioxidative activity mitigated the accumulation of reactive oxygen species in vivo under oxidative and drought stress. These data confirm the usefulness of flavonoids for enhancing both biotic and abiotic stress tolerance in crops.

Arabidopsis ADC genes involved in polyamine biosynthesis are essential for seed development

Arginine decarboxylase (ADC) is a rate‐limiting enzyme that catalyzes the first step of polyamine (PA) biosynthesis in Arabidopsis thaliana. We generated a double mutant deficient in Arabidopsis two ADC genes (ADC1 −/− ADC2 −/−) and examined their roles in seed development. None of the F2 seedlings from crosses of adc1‐1 and adc2‐2 had the ADC1 −/− ADC2 −/− genotype. In addition, some abnormal seeds were observed among the ADC1 +/− ADC2 −/− and ADC1 −/− ADC2 +/− siliques. Viable offspring with the ADC1 −/− ADC2 −/− genotype could not be obtained from the ADC1 +/− ADC2 −/− and ADC1 −/− ADC2 +/− plants. These results indicate that AtADC genes are required for production of polyamines that are essential for normal seed development in Arabidopsis.

Integrated Analysis of the Effects of Cold and Dehydration on Rice Metabolites, Phytohormones, and Gene Transcripts

In cold- or dehydration-stressed rice plants, up-regulation of genes related to starch degradation, sucrose metabolism, and the glyoxylate cycle results in the accumulation of sugars and with, abscisic acid signaling is inversely related to cytokinin signaling. Correlations between gene expression and metabolite/phytohormone levels under abiotic stress conditions have been reported for Arabidopsis (Arabidopsis thaliana). However, little is known about these correlations in rice (Oryza sativa ‘Nipponbare’), despite its importance as a model monocot. We performed an integrated analysis to clarify the relationships among cold- and dehydration-responsive metabolites, phytohormones, and gene transcription in rice. An integrated analysis of metabolites and gene expression indicated that several genes encoding enzymes involved in starch degradation, sucrose metabolism, and the glyoxylate cycle are up-regulated in rice plants exposed to cold or dehydration and that these changes are correlated with the accumulation of glucose (Glc), fructose, and sucrose. In particular, high expression levels of genes encoding isocitrate lyase and malate synthase in the glyoxylate cycle correlate with increased Glc levels in rice, but not in Arabidopsis, under dehydration conditions, indicating that the regulation of the glyoxylate cycle may be involved in Glc accumulation under dehydration conditions in rice but not Arabidopsis. An integrated analysis of phytohormones and gene transcripts revealed an inverse relationship between abscisic acid (ABA) signaling and cytokinin (CK) signaling under cold and dehydration stresses; these stresses increase ABA signaling and decrease CK signaling. High levels of Oryza sativa 9-cis-epoxycarotenoid dioxygenase transcripts correlate with ABA accumulation, and low levels of Cytochrome P450 (CYP) 735A transcripts correlate with decreased levels of a CK precursor in rice. This reduced expression of CYP735As occurs in rice but not Arabidopsis. Therefore, transcriptional regulation of CYP735As might be involved in regulating CK levels under cold and dehydration conditions in rice but not Arabidopsis.

Natural variation in a polyamine transporter determines paraquat tolerance in Arabidopsis

Polyamines (PAs) are ubiquitous, polycationic compounds that are essential for the growth and survival of all organisms. Although the PA-uptake system plays a key role in mammalian cancer and in plant survival, the underlying molecular mechanisms are not well understood. Here, we identified an Arabidopsis L-type amino acid transporter (LAT) family transporter, named RMV1 (resistant to methyl viologen 1), responsible for uptake of PA and its analog paraquat (PQ). The natural variation in PQ tolerance was determined in 22 Arabidopsis thaliana accessions based on the polymorphic variation of RMV1. An RMV1-GFP fusion protein localized to the plasma membrane in transformed cells. The Arabidopsis rmv1 mutant was highly resistant to PQ because of the reduction of PQ uptake activity. Uptake studies indicated that RMV1 mediates proton gradient-driven PQ transport. RMV1 overexpressing plants were hypersensitive to PA and PQ and showed elevated PA/PQ uptake activity, supporting the notion that PQ enters plant cells via a carrier system that inherently functions in PA transport. Furthermore, we demonstrated that polymorphic variation in RMV1 controls PA/PQ uptake activity. Our identification of a molecular entity for PA/PQ uptake and sensitivity provides an important clue for our understanding of the mechanism and biological significance of PA uptake.

Overexpression of AtABCG25 enhances the abscisic acid signal in guard cells and improves plant water use efficiency.

In addition to improving drought tolerance, improvement of water use efficiency is a major challenge in plant physiology. Due to their trade-off relationships, it is generally considered that achieving stress tolerance is incompatible with maintaining stable growth. Abscisic acid (ABA) is a key phytohormone that regulates the balance between intrinsic growth and environmental responses. Previously, we identified AtABCG25 as a cell-membrane ABA transporter that export ABA from the inside to the outside of cells. AtABCG25-overexpressing plants showed a lower transpiration phenotype without any growth retardation. Here, we dissected this useful trait using precise phenotyping approaches. AtABCG25 overexpression stimulated a local ABA response in guard cells. Furthermore, AtABCG25 overexpression enhanced drought tolerance, probably resulting from maintenance of water contents over the common threshold for survival after drought stress treatment. Finally, we observed enhanced water use efficiency by overexpression of AtABCG25, in addition to drought tolerance. These results were consistent with the function of AtABCG25 as an ABA efflux transporter. This unique trait may be generally useful for improving the water use efficiency and drought tolerance of plants.

Molecular, physiological, and agronomical characterization, in greenhouse and in field conditions, of soybean plants genetically modified with AtGolS2 gene for drought tolerance

Water deficit may occur at any stage of crop development, affecting productivity and causing economic losses. In response to drought, raffinose family oligosaccharides (RFOs) are accumulated in plant tissues stabilizing and protecting cell membranes and keeping the vital functions. The enzyme galactinol synthase (GolS, EC 2.4.1.123) catalyzes the first step in the biosynthesis of RFOs. In our study, soybean events overexpressing 35S:AtGolS2 were molecularly, physiological, and agronomical characterized, under drought simulated in greenhouse and in field conditions during the crop season 2014/2015. The conventional soybean cultivar BRS 184 was transformed and five positive events were obtained. Four events transmitted the transgene to further generations and in the events 2Ia1 and 2Ia4, two to four copies of AtGols2 gene were observed. Results in greenhouse showed that the overexpression of AtGolS2 in genetically modified (GM) plants led to increased galactinol transcripts, probably resulting in changes in carbohydrate metabolism. Accumulation of these transcripts that may have acted as osmoprotectors, lead to higher drought tolerance and survival rate of 2Ia4 plants. In addition, in field conditions, higher yield was observed for 2Ia4 plants under irrigated (IRR) and non-irrigated (NIRR) treatments. This result can be due to the increased synthesis of RFOs even under well-watered conditions. This field screening showed promising results for drought tolerance, suggesting that 2Ia4 plants may be useful in a breeding program for the development of drought-tolerant plants. However, additional studies are needed in further crop seasons and other sites to better characterize how these plants may outperform the WT plants under water deficit.