Madana M. R. Ambavaram

Molecular and Physiological Analysis of Drought Stress in Arabidopsis Reveals Early Responses Leading to Acclimation in Plant Growth

Plant drought stress response and resistance are complex biological processes that need to be analyzed at a systems level using genomics and physiological approaches to dissect experimental models that address drought stresses encountered by crops in the field. Toward this goal, a controlled, sublethal, moderate drought (mDr) treatment system was developed in Arabidopsis (Arabidopsis thaliana) as a reproducible assay for the dissection of plant responses to drought. The drought assay was validated using Arabidopsis mutants in abscisic acid (ABA) biosynthesis and signaling displaying drought sensitivity and in jasmonate response mutants showing drought resistance, indicating the crucial role of ABA and jasmonate signaling in drought response and acclimation. A comparative transcriptome analysis of soil water deficit drought stress treatments revealed the similarity of early-stage mDr to progressive drought, identifying common and specific stress-responsive genes and their promoter cis-regulatory elements. The dissection of mDr stress responses using a time-course analysis of biochemical, physiological, and molecular processes revealed early accumulation of ABA and induction of associated signaling genes, coinciding with a decrease in stomatal conductance as an early avoidance response to drought stress. This is accompanied by a peak in the expression of expansin genes involved in cell wall expansion, as a preparatory step toward drought acclimation by the adjustment of the cell wall. The time-course analysis of mDr provides a model with three stages of plant responses: an early priming and preconditioning stage, followed by an intermediate stage preparatory for acclimation, and a late stage of new homeostasis with reduced growth.

Effects of Drought on Gene Expression in Maize Reproductive and Leaf Meristem Tissue Revealed by RNA-Seq

Drought stress affects cereals especially during the reproductive stage. The maize (Zea mays) drought transcriptome was studied using RNA-Seq analysis to compare drought-treated and well-watered fertilized ovary and basal leaf meristem tissue. More drought-responsive genes responded in the ovary compared with the leaf meristem. Gene Ontology enrichment analysis revealed a massive decrease in transcript abundance of cell division and cell cycle genes in the drought-stressed ovary only. Among Gene Ontology categories related to carbohydrate metabolism, changes in starch and Suc metabolism-related genes occurred in the ovary, consistent with a decrease in starch levels, and in Suc transporter function, with no comparable changes occurring in the leaf meristem. Abscisic acid (ABA)-related processes responded positively, but only in the ovaries. Related responses suggested the operation of low glucose sensing in drought-stressed ovaries. The data are discussed in the context of the susceptibility of maize kernel to drought stress leading to embryo abortion and the relative robustness of dividing vegetative tissue taken at the same time from the same plant subjected to the same conditions. Our working hypothesis involves signaling events associated with increased ABA levels, decreased glucose levels, disruption of ABA/sugar signaling, activation of programmed cell death/senescence through repression of a phospholipase C-mediated signaling pathway, and arrest of the cell cycle in the stressed ovary at 1 d after pollination. Increased invertase levels in the stressed leaf meristem, on the other hand, resulted in that tissue maintaining hexose levels at an “unstressed” level, and at lower ABA levels, which was correlated with successful resistance to drought stress.

Cyclophilin 20-3 relays a 12-oxo-phytodienoic acid signal during stress responsive regulation of cellular redox homeostasis

The jasmonate family of phytohormones plays central roles in plant development and stress acclimation. However, the architecture of their signaling circuits remains largely unknown. Here we describe a jasmonate family binding protein, cyclophilin 20-3 (CYP20-3), which regulates stress-responsive cellular redox homeostasis. (+)-12-oxo-phytodienoic acid (OPDA) binding promotes CYP20-3 to form a complex with serine acetyltransferase 1, which triggers the formation of a hetero-oligomeric cysteine synthase complex with O-acetylserine(thiol)lyase B in chloroplasts. The cysteine synthase complex formation then activates sulfur assimilation that leads to increased levels of thiol metabolites and the buildup of cellular reduction potential. The enhanced redox capacity in turn coordinates the expression of a subset of OPDA-responsive genes. Thus, we conclude that CYP20-3 is a key effector protein that links OPDA signaling to amino acid biosynthesis and cellular redox homeostasis in stress responses.

Coordinated Activation of Cellulose and Repression of Lignin Biosynthesis Pathways in Rice

Cellulose from plant biomass is the largest renewable energy resource of carbon fixed from the atmosphere, which can be converted into fermentable sugars for production into ethanol. However, the cellulose present as lignocellulosic biomass is embedded in a hemicellulose and lignin matrix from which it needs to be extracted for efficient processing. Here, we show that expression of an Arabidopsis (Arabidopsis thaliana) transcription factor, SHINE (SHN), in rice (Oryza sativa), a model for the grasses, causes a 34% increase in cellulose and a 45% reduction in lignin content. The rice AtSHN lines also exhibit an altered lignin composition correlated with improved digestibility, with no compromise in plant strength and performance. Using a detailed systems-level analysis of global gene expression in rice, we reveal the SHN regulatory network coordinating down-regulation of lignin biosynthesis and up-regulation of cellulose and other cell wall biosynthesis pathway genes. The results thus support the development of nonfood crops and crop wastes with increased cellulose and low lignin with good agronomic performance that could improve the economic viability of lignocellulosic crop utilization for biofuels.

Coordinated regulation of photosynthesis in rice increases yield and tolerance to environmental stress

Plants capture solar energy and atmospheric carbon dioxide (CO2) through photosynthesis, which is the primary component of crop yield, and needs to be increased considerably to meet the growing global demand for food. Environmental stresses, which are increasing with climate change, adversely affect photosynthetic carbon metabolism (PCM) and limit yield of cereals such as rice (Oryza sativa) that feeds half the world. To study the regulation of photosynthesis, we developed a rice gene regulatory network and identified a transcription factor HYR (HIGHER YIELD RICE) associated with PCM, which on expression in rice enhances photosynthesis under multiple environmental conditions, determining a morpho-physiological programme leading to higher grain yield under normal, drought and high-temperature stress conditions. We show HYR is a master regulator, directly activating photosynthesis genes, cascades of transcription factors and other downstream genes involved in PCM and yield stability under drought and high-temperature environmental stress conditions.

Phenotypic and physiological evaluation for drought and salinity stress responses in rice.

Drought and salinity stresses seriously affect rice plant growth and yield. The growing need to improve rice cultivars for drought and salt tolerance requires the development of reproducible screening methods that simulate field conditions, and which provide quantitative data for statistical testing and selection of genotypes with differential responses. In addition, the study of molecular responses to drought and salt stress requires controlled conditions for growth and treatments that are reportable and comparable between different laboratories. Drought, also known as soil water deficit, can result from insufficient moisture for a plant to grow adequately and complete its life cycle. Salinity due to excess sodium chloride affects rice at seedling and flowering stages, reducing root and leaf growth. Both these abiotic stresses can lead to major physiological and biochemical changes such as reduced photosynthesis and reprogramming of gene expression. The methods presented in this chapter can be applied for (a) examination of stress responses in rice vegetative and reproductive tissues to identify and characterize molecular and physiological responses; (b) testing of candidate genes by overexpression or knockout studies evaluated for altered stress response phenotypes; and (c) screening of different genotypes such as accessions or segregating populations for their quantitative responses to abiotic stress parameters.

RECoN: Rice Environment Coexpression Network for Systems Level Analysis of Abiotic-Stress Response

Transcriptional profiling is a prevalent and powerful approach for capturing the response of crop plants to environmental stresses, e.g., response of rice to drought. However, functionally interpreting the resulting genome-wide gene expression changes is severely hampered by the large gaps in our genomic knowledge about which genes work together in cellular pathways/processes in rice. Here, we present a new web resource – RECoN – that relies on a network-based approach to go beyond currently limited annotations in delineating functional and regulatory perturbations in new rice transcriptome datasets generated by a researcher. To build RECoN, we first enumerated 1,744 abiotic stress-specific gene modules covering 28,421 rice genes (>72% of the genes in the genome). Each module contains a group of genes tightly coexpressed across a large number of environmental conditions and, thus, is likely to be functionally coherent. When a user provides a new differential expression profile, RECoN identifies modules substantially perturbed in their experiment and further suggests deregulated functional and regulatory mechanisms based on the enrichment of current annotations within the predefined modules. We demonstrate the utility of this resource by analyzing new drought transcriptomes of rice in three developmental stages, which revealed large-scale insights into the cellular processes and regulatory mechanisms involved in common and stage-specific drought responses. RECoN enables biologists to functionally explore new data from all abiotic stresses on a genome-scale and to uncover gene candidates, including those that are currently functionally uncharacterized, for engineering stress tolerance.

Setting Up Reverse Transcription Quantitative-PCR Experiments

Quantitative real-time PCR (qRT-PCR), in conjunction with reverse transcriptase, has been used for the systematic measurement of plant physiological changes in gene expression. In the present paper, we describe a qRT-PCR protocol that illustrates the essential technical steps required to generate quantitative data that are reliable and reproducible. To demonstrate the methods used, we evaluated the expression stability of five [actin (ACT), actin1 (ACT1), β-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin (CYC), and elongation factor 1α (EF-1α)] frequently used housekeeping genes in rice. The expression stability of the five selected housekeeping genes varied considerably in different tissues (seedlings, vegetative and reproductive stages) in a given stress condition. The analysis allowed us to choose a set of two candidates (ACT1 and EF-1α) that showed more uniform expression and are also suitable for the validation of weakly expressed genes (≥0.5 fold), identified through microarray analysis.