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.

Physiological and molecular adaptations to drought in Andean potato genotypes.

The drought stress tolerance of two Solanum tuberosum subsp. andigena landraces, one hybrid (adgxtbr) 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.

Validation and estimation of parameters for a general probabilistic model of the PCR process.

Earlier work rigorously derived a general probabilistic model for the PCR process that includes as a special case the Velikanov-Kapral model where all nucleotide reaction rates are the same. In this model, the probability of binding of deoxy-nucleoside triphosphate (dNTP) molecules with template strands is derived from the microscopic chemical kinetics. A recursive solution for the probability function of binding of dNTPs is developed for a single cycle and is used to calculate expected yield for a multicycle PCR. The model is able to reproduce important features of the PCR amplification process quantitatively. With a set of favorable reaction conditions, the amplification of the target sequence is fast enough to rapidly outnumber all side products. Furthermore, the final yield of the target sequence in a multicycle PCR run always approaches an asymptotic limit that is less than one. The amplification process itself is highly sensitive to initial concentrations and the reaction rates of addition to the template strand of each type of dNTP in the solution. This paper extends the earlier Saha model with a physics based model of the dependence of the reaction rates on temperature, and estimates parameters in this new model by nonlinear regression. The calibrated model is validated using RT-PCR data.

Early PLDalpha-mediated events in response to progressive drought stress in Arabidopsis: a transcriptome analysis.

Phospholipase D (PLD) has been implicated in a variety of stresses including osmotic stress and wounding. PLDalpha1-derived phosphatidic acid interacts with ABI1 phosphatase 2C and promotes abscisic acid signalling. It has also been shown to regulate proline biosynthesis negatively. Plants with abrogated PLDalpha show insensitivity to abscisic acid (ABA) and impaired stomatal conductance. The goal in the present study was to identify early PLDalpha-mediated events in response to progressive drought stress in Arabidopsis. Water was withheld from 7-week-old Arabidopsis thaliana (Col-0) and antisense-PLDalpha1 (anti-PLDalpha) in a controlled environment chamber. Diurnal leaf water potential (LWP) and photosynthesis measurements were recorded five and three times a day, respectively. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and microarray analyses were conducted using RNA from shoots collected at the fourth LWP time point on the ninth day after stress imposition. Anti-PLDalpha experienced severe water stress (-1.28 MPa) at the same time period that Col-0 experienced less water stress (-0.31 MPa). Diurnal LWP measurements showed that anti-PLDalpha had a lower LWP than Col-0 in both control and drought-stress conditions. Photosynthesis was also more affected in anti-PLDalpha than in Col-0. Anti-PLDalpha plants recovered fully following rehydration after 10 d of stress. qRT-PCR revealed up to 18-fold lower values for PLDalpha transcripts in stressed anti-PLDalpha plants when compared with stressed Col-0. Microarray expression profiles revealed distinct gene expression patterns in Col-0 and anti-PLDalpha. No differences in gene expression were detected between the two genotypes in the absence of drought stress. ROP8, PLDdelta, and lipid transfer proteins were among the differentially expressed genes between the two genotypes.

XcisClique: analysis of regulatory bicliques.

BACKGROUND: Modeling of cis-elements or regulatory motifs in promoter (upstream) regions of genes is a challenging computational problem. In this work, set of regulatory motifs simultaneously present in the promoters of a set of genes is modeled as a biclique in a suitably defined bipartite graph. A biologically meaningful co-occurrence of multiple cis-elements in a gene promoter is assessed by the combined analysis of genomic and gene expression data. Greater statistical significance is associated with a set of genes that shares a common set of regulatory motifs, while simultaneously exhibiting highly correlated gene expression under given experimental conditions. METHODS: XcisClique, the system developed in this work, is a comprehensive infrastructure that associates annotated genome and gene expression data, models known cis-elements as regular expressions, identifies maximal bicliques in a bipartite gene-motif graph; and ranks bicliques based on their computed statistical significance. Significance is a function of the probability of occurrence of those motifs in a biclique (a hypergeometric distribution), and on the new sum of absolute values statistic (SAV) that uses Spearman correlations of gene expression vectors. SAV is a statistic well-suited for this purpose as described in the discussion. RESULTS: XcisClique identifies new motif and gene combinations that might indicate as yet unidentified involvement of sets of genes in biological functions and processes. It currently supports Arabidopsis thaliana and can be adapted to other organisms, assuming the existence of annotated genomic sequences, suitable gene expression data, and identified regulatory motifs. A subset of Xcis Clique functionalities, including the motif visualization component MotifSee, source code, and supplementary material are available at https://bioinformatics.cs.vt.edu/xcisclique/.

A general probabilistic model of the PCR process.

This work describes a general probabilistic model for the PCR process; this model includes as a special case the Velikanov-Kapral model where all nucleotide reaction rates are the same. In this model the probability of binding of deoxynucleoside triphosphate (dNTP) molecules with template strands is derived from the microscopic chemical kinetics. A recursive solution for the probability distribution of binding of dNTPs is developed for a single cycle and is used to calculate expected yield for a multicycle PCR. The model is able to reproduce important features of the PCR amplification process quantitatively. This model also suggests that the amplification process itself is highly sensitive to initial concentrations and the reaction rates of addition to the template strand of each type of dNTP in the solution.

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 2173 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.