Ruth Grene

The iPlant Collaborative: Cyberinfrastructure for Plant Biology

The iPlant Collaborative (iPlant) is a United States National Science Foundation (NSF) funded project that aims to create an innovative, comprehensive, and foundational cyberinfrastructure in support of plant biology research (PSCIC, 2006). iPlant is developing cyberinfrastructure that uniquely enables scientists throughout the diverse fields that comprise plant biology to address Grand Challenges in new ways, to stimulate and facilitate cross-disciplinary research, to promote biology and computer science research interactions, and to train the next generation of scientists on the use of cyberinfrastructure in research and education. Meeting humanitys projected demands for agricultural and forest products and the expectation that natural ecosystems be managed sustainably will require synergies from the application of information technologies. The iPlant cyberinfrastructure design is based on an unprecedented period of research community input, and leverages developments in high-performance computing, data storage, and cyberinfrastructure for the physical sciences. iPlant is an open-source project with application programming interfaces that allow the community to extend the infrastructure to meet its needs. iPlant is sponsoring community-driven workshops addressing specific scientific questions via analysis tool integration and hypothesis testing. These workshops teach researchers how to add bioinformatics tools and/or datasets into the iPlant cyberinfrastructure enabling plant scientists to perform complex analyses on large datasets without the need to master the command-line or high-performance computational services.

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.

Photosynthetic Acclimation Is Reflected in Specific Patterns of Gene Expression in Drought-Stressed Loblolly Pine

Because the product of a single gene can influence many aspects of plant growth and development, it is necessary to understand how gene products act in concert and upon each other to effect adaptive changes to stressful conditions. We conducted experiments to improve our understanding of the responses of loblolly pine (Pinus taeda) to drought stress. Water was withheld from rooted plantlets of to a measured water potential of -1 MPa for mild stress and -1.5 MPa for severe stress. Net photosynthesis was measured for each level of stress. RNA was isolated from needles and used in hybridizations against a microarray consisting of 2,173 cDNA clones from five pine expressed sequence tag libraries. Gene expression was estimated using a two-stage mixed linear model. Subsequently, data mining via inductive logic programming identified rules (relationships) among gene expression, treatments, and functional categories. Changes in RNA transcript profiles of loblolly pine due to drought stress were correlated with physiological data reflecting photosynthetic acclimation to mild stress or photosynthetic failure during severe stress. Analysis of transcript profiles indicated that there are distinct patterns of expression related to the two levels of stress. Genes encoding heat shock proteins, late embryogenic-abundant proteins, enzymes from the aromatic acid and flavonoid biosynthetic pathways, and from carbon metabolism showed distinctive responses associated with acclimation. Five genes shown to have different transcript levels in response to either mild or severe stress were chosen for further analysis using real-time polymerase chain reaction. The real-time polymerase chain reaction results were in good agreement with those obtained on microarrays.

Physiological and molecular adaptations to drought in Andean potato genotypes

The drought stress tolerance of two Solanum tuberosum subsp. andigena landraces, one hybrid (adg×tbr) and Atlantic (S. tuberosum subsp. tuberosum) has been evaluated. Photosynthesis in the Andigena landraces during prolonged drought was maintained significantly longer than in the Tuberosum (Atlantic) line. Among the Andigena landraces, ‘Sullu’ (SUL) was more drought resistant than ‘Negra Ojosa’ (NOJ). Microarray analysis and metabolite data from leaf samples taken at the point of maximum stress suggested higher mitochondrial metabolic activity in SUL than in NOJ. A greater induction of chloroplast-localized antioxidant and chaperone genes in SUL compared with NOJ was evident. ABA-responsive TFs were more induced in NOJ compared with SUL, including WRKY1, mediating a response in SA signalling that may give rise to increased ROS. NOJ may be experiencing higher ROS levels than SUL. Metabolite profiles of NOJ were characterized by compounds indicative of stress, for example, proline, trehalose, and GABA, which accumulated to a higher degree than in SUL. The differences between the Andigena lines were not explained by protective roles of compatible solutes; hexoses and complex sugars were similar in both landraces. Instead, lower levels of ROS accumulation, greater mitochondrial activity and active chloroplast defences contributed to a lower stress load in SUL than in NOJ during drought.

Oxidative Stress and Acclimation Mechanisms in Plants

One of the most crucial functions of plant cells is their ability to respond to fluctuations in their environment. Understanding the connections between a plants initial responses and the downstream events that constitute successful adjustment to its altered environment is one of the next grand challenges of plant biology. Oxidative stress from environmental sources and developmental transitions such as seed maturation involves the formation of reactive oxygen species (ROS) in plant cells. The redox-modulated changes that follow are central events in cellular responses. Thiol redox regulation (Figure 1) partially mediated through the redox state of the glutathione pool (GSH/GSSG), regulation of the glutathione biosynthetic pathway, and ROS themselves are each thought to have important roles as environmental sensors and/or modulators of global patterns of gene expression in development and defense. Exposure of green tissue to potentially damaging light intensities involves redox sensing molecular events throughout the plant, originating at the plastoquinone (PQ) pool in the thylakoid membrane. Major defense genes whose expression is affected by the redox state of the PQ pool include both cytosolic and chloroplast ascorbate peroxidases (APX) ( Karpinska et al. 2000 ). The superoxide dismutase (SOD) gene families appear to be specialized in function with respect to subcellular location and other as yet unknown factors (Alscher et al. 2002). In the case of peroxisomes, the imposition of oxidative stress gives rise to organelle proliferation, thus adding another layer of complexity to stress responses (Lopez-Huertas et al. 2000). Defense mechanisms involving molecular chaperones and methionine sulfoxide reductase are becoming recognized as important players in resistance to oxidative stress throughout the cell. Figure 1. Thiol redox control and stress defense Intracellular origins of ROS and their multiple damaging effects Any circumstance in which cellular redox homeostasis is disrupted can lead to oxidative stress or the generation of ROS (Asada 1994). Production of ROS during environmental stress is one of the main causes for decreases in productivity, injury, and death that accompany these stresses in plants. ROS are produced in both unstressed and stressed cells, and in various locations (Halliwell and Gutteridge 1989) (Figure 2). They are generated endogenously during certain developmental transitions such as seed maturation and as a result of normal, unstressed, photosynthetic and respiratory metabolism. An initial oxyradical product, the superoxide radical (O2-.), upon further reaction within the cell, can form more ROS such as hydroxyl radicals and singlet oxygen. Superoxide is a charged molecule and cannot cross biological membranes. Subcellular compartmentation of defense mechanisms is, therefore, crucial for efficient removal of superoxide anions at their sites of generation throughout the cell. Hydrogen peroxide, on the other hand, which is formed as a result of SOD action, is capable of diffusing across membranes and is thought to fulfil a signaling function in defense responses (Mullineaux et al. 2000). Figure 2. Reactive oxygen species (ROS) arise throughout the cell. ROS play an important role in endonuclease activation and consequent DNA damage (Hagar et al. 1996). In the presence of metal ions such as Fe or Cu(II), hydroxyl radicals are formed very rapidly. Hydroxyl radicals can cause damage to all classes of biologically important macromolecules, especially nucleic acids. Hydroxyl radicals can also modify proteins so as to make them more susceptible to proteolytic attack. There is evidently considerable specificity associated with this degradative process since proteins have widely differing susceptibilities to attack by ROS (Davies 1987). Once damaged, proteins can be broken down further by specific endopeptidases such as the one found bound to the thylakoid membrane (Casano et al. 1994). A multicatalytic proteinase complex has been demonstrated in plant systems, with the capacity to selectively break down oxidatively damaged proteins (Van Nocker et al. 1996)

Studying the functional genomics of stress responses in loblolly pine with the Expresso microarray experiment management system

Conception, design, and implementation of cDNA microarray experiments present a variety of bioinformatics challenges for biologists and computational scientists. The multiple stages of data acquisition and analysis have motivated the design of Expresso, a system for microarray experiment management. Salient aspects of Expresso include support for clone replication and randomized placement; automatic gridding, extraction of expression data from each spot, and quality monitoring; flexible methods of combining data from individual spots into information about clones and functional categories; and the use of inductive logic programming for higher-level data analysis and mining. The development of Expresso is occurring in parallel with several generations of microarray experiments aimed at elucidating genomic responses to drought stress in loblolly pine seedlings. The current experimental design incorporates 384 pine cDNAs replicated and randomly placed in two specific microarray layouts. We describe the design of Expresso as well as results of analysis with Expresso that suggest the importance of molecular chaperones and membrane transport proteins in mechanisms conferring successful adaptation to long-term drought stress.

Molecular and physiological adaptation to prolonged drought stress in the leaves of two Andean potato genotypes

Responses to prolonged drought and recovery from drought of two South American potato (Solanum tuberosum L. ssp. andigena (Juz & Buk) Hawkes) landraces, Sullu and Ccompis were compared under field conditions. Physiological and biomass measurements, yield analysis, the results of hybridisation to a potato microarray platform (44 000 probes) and metabolite profiling were used to characterise responses to water deficit. Drought affected shoot and root biomass negatively in Ccompis but not in Sullu, whereas both genotypes maintained tuber yield under water stress. Ccompis showed stronger reduction in maximum quantum yield under stress than Sullu, and less decrease in stomatal resistance. Genes associated with PSII functions were activated during recovery in Sullu only. Evidence for sucrose accumulation in Sullu only during maximum stress and recovery was observed, in addition to increases in cell wall biosynthesis. A depression in the abundance of plastid superoxide dismutase transcripts was observed under maximum stress in Ccompis. Both sucrose and the regulatory molecule trehalose accumulated in the leaves of Sullu only. In contrast, in Ccompis, the raffinose oligosaccharide family pathway was activated, whereas low levels of sucrose and minor stress-mediated changes in trehalose were observed. Proline, and expression of the associated genes, rose in both genotypes under drought, with a 3-fold higher increase in Sullu than in Ccompis. The results demonstrate the presence of distinct molecular and biochemical drought responses in the two potato landraces leading to yield maintenance but differential biomass accumulation in vegetative tissues.

Metabolic and Transcriptional Reprogramming in Developing Soybean (Glycine max) Embryos

Soybean (Glycine max) seeds are an important source of seed storage compounds, including protein, oil, and sugar used for food, feed, chemical, and biofuel production. We assessed detailed temporal transcriptional and metabolic changes in developing soybean embryos to gain a systems biology view of developmental and metabolic changes and to identify potential targets for metabolic engineering. Two major developmental and metabolic transitions were captured enabling identification of potential metabolic engineering targets specific to seed filling and to desiccation. The first transition involved a switch between different types of metabolism in dividing and elongating cells. The second transition involved the onset of maturation and desiccation tolerance during seed filling and a switch from photoheterotrophic to heterotrophic metabolism. Clustering analyses of metabolite and transcript data revealed clusters of functionally related metabolites and transcripts active in these different developmental and metabolic programs. The gene clusters provide a resource to generate predictions about the associations and interactions of unknown regulators with their targets based on “guilt-by-association” relationships. The inferred regulators also represent potential targets for future metabolic engineering of relevant pathways and steps in central carbon and nitrogen metabolism in soybean embryos and drought and desiccation tolerance in plants.

Differential expression of heat shock protein genes in preconditioning for photosynthetic acclimation in water-stressed loblolly pine

Heat shock proteins (HSPs) are induced not only under heat stress conditions but also under other environmental stresses such as water stress. In plants, HSPs families are larger than those of other eukaryotes. In order to elucidate a possible connection between HSP expression and photosynthetic acclimation or conditioning, we conducted a water stress experiment in loblolly pine (Pinus taeda L.) seedlings involving progressive treatment consisting of one cycle of mild stress (-1 MPa) followed by two cycles of severe stress (-1.7 MPa). Net photosynthesis was measured at each stress level. Photosynthetic acclimation occurred in the progressive treatment after the first cycle, but not in the severe treatment, suggesting that a cycle of mild stress conditioned the trees to adapt to a more severe stress. Real time results indicated specific patterns in needles in the expression of HSP70, HSP90 and sHSP genes for each treatment, both at maximum stress and at recovery. We identified a pine homolog to GRP94 (ER resident HSP90) that was induced after rehydration coincident with acclimation. Further analysis of the promoter region of the pine GRP94 showed putative cis-elements associated with water stress and rehydration, corresponding to the expression pattern observed in our experiment.

Multilevel Regulation of Abiotic Stress Responses in Plants

The sessile lifestyle of plants requires them to cope with stresses in situ. Plants overcome abiotic stresses by altering structure/morphology, and in some extreme conditions, by compressing the life cycle to survive the stresses in the form of seeds. Genetic and molecular studies have uncovered complex regulatory processes that coordinate stress adaptation and tolerance in plants, which are integrated at various levels. Investigating natural variation in stress responses has provided important insights into the evolutionary processes that shape the integrated regulation of adaptation and tolerance. This review primarily focuses on the current understanding of how transcriptional, post-transcriptional, post-translational, and epigenetic processes along with genetic variation orchestrate stress responses in plants. We also discuss the current and future development of computational tools to identify biologically meaningful factors from high dimensional, genome-scale data and construct the signaling networks consisting of these components.