Genome wide expression profiling of two accession of G. herbaceum L. in response to drought

<p>Abstract</p> <p>Background</p> <p>Genome-wide gene expression profiling and detailed physiological investigation were used for understanding the molecular mechanism and physiological response of <it>Gossypium herbaceum</it>, which governs the adaptability of plants in drought conditions. Recently, microarray-based gene expression analysis is commonly used to decipher genes and genetic networks controlling the traits of interest. However, the results of such an analysis are often plagued due to a limited number of genes (probe sets) on microarrays. On the other hand, pyrosequencing of a transcriptome has the potential to detect rare as well as a large number of transcripts in the samples quantitatively. We used Affymetrix microarray as well as Roche's GS-FLX transcriptome sequencing for a comparative analysis of cotton transcriptome in leaf tissues under drought conditions.</p> <p>Results</p> <p>Fourteen accessions of <it>Gossypium herbaceum </it>were subjected to mannitol stress for preliminary screening; two accessions, namely Vagad and RAHS-14, were selected as being the most tolerant and most sensitive to osmotic stress, respectively. Affymetrix cotton arrays containing 24,045 probe sets and Roche's GS-FLX transcriptome sequencing of leaf tissue were used to analyze the gene expression profiling of Vagad and RAHS-14 under drought conditions. The analysis of physiological measurements and gene expression profiling showed that Vagad has the inherent ability to sense drought at a much earlier stage and to respond to it in a much more efficient manner than does RAHS-14. Gene Ontology (GO) studies showed that the phenyl propanoid pathway, pigment biosynthesis, polyketide biosynthesis, and other secondary metabolite pathways were enriched in Vagad under control and drought conditions as compared with RAHS-14. Similarly, GO analysis of transcriptome sequencing showed that the GO terms <it>responses to various abiotic stresses </it>were significantly higher in Vagad. Among the classes of transcription factors (TFs) uniquely expressed in both accessions, RAHS-14 showed the expression of ERF and WRKY families. The unique expression of ERFs in response to drought conditions reveals that RAHS-14 responds to drought by inducing senescence. This was further supported by transcriptome analysis which revealed that RAHS-14 responds to drought by inducing many transcripts related to senescence and cell death.</p> <p>Conclusion</p> <p>The comparative genome-wide gene expression profiling study of two accessions of <it>G.herbaceum </it>under drought stress deciphers the differential patterns of gene expression, including TFs and physiologically relevant processes. Our results indicate that drought tolerance observed in Vagad is not because of a single molecular reason but is rather due to several unique mechanisms which Vagad has developed as an adaptation strategy.</p

Purification and properties of leucine aminotransferase from soybean seedlings

Two isoenzymes of leucine aminotransferase (LAT I and LAT II) were extracted and partially purified from etiolated soybean seedlings. LAT I accounted for about 87% and LAT II about 13% of the total LAT activity. LAT I was eluted from a DEAE-cellulose column with a buffer having lower ionic strength than LAT II. Both isoenzymes gave pH optima of 8.9. Kinetic data for the forward reaction were consistent with the accepted ping-pong bi-bi mechanism for aminotransferases. Isoenzymes were inhibited by excess of substrate and product. Inhibition by the substrate analogue maleate suggested that both substrates utilized the same catalytic site of the enzyme. Hydroxylamine inhibited the aldehyde form of the LAT while the amino form was found to be inert

Comparative transcriptomic analysis of roots of contrasting <it>Gossypium herbaceum</it> genotypes revealing adaptation to drought

<p>Abstract</p> <p>Background</p> <p>Root length and its architecture govern the adaptability of plants to various stress conditions, including drought stress. Genetic variations in root growth, length, and architecture are genotypes dependent. In this study, we compared the drought-induced transcriptome of four genotypes of <it>Gossypium herbaceum</it> that differed in their drought tolerance adaptability. Three different methodologies, namely, microarray, pyrosequencing, and qRT–PCR, were used for transcriptome analysis and validation.</p> <p>Results</p> <p>The variations in root length and growth were found among four genotypes of <it>G.herbaceum</it> when exposed to mannitol-induced osmotic stress. Under osmotic stress, the drought tolerant genotypes Vagad and GujCot-21 showed a longer root length than did by drought sensitive RAHS-14 and RAHS-IPS-187. Further, the gene expression patterns in the root tissue of all genotypes were analyzed. We obtained a total of 794 differentially expressed genes by microarray and 104928 high-quality reads representing 53195 unigenes from the root transcriptome. The Vagad and GujCot-21 respond to water stress by inducing various genes and pathways such as response to stresses, response to water deprivation, and flavonoid pathways. Some key regulatory genes involved in abiotic stress such as AP2 EREBP, MYB, WRKY, ERF, ERD9, and LEA were highly expressed in Vagad and GujCot-21. The genes RHD3, NAP1, LBD, and transcription factor WRKY75, known for root development under various stress conditions, were expressed specifically in Vagad and GujCot-21. The genes related to peroxidases, transporters, cell wall-modifying enzymes, and compatible solutes (amino acids, amino sugars, betaine, sugars, or sugar alcohols) were also highly expressed in Vagad and Gujcot-21.</p> <p>Conclusion</p> <p>Our analysis highlights changes in the expression pattern of genes and depicts a small but highly specific set of drought responsive genes induced in response to drought stress. Some of these genes were very likely to be involved in drought stress signaling and adaptation, such as transmembrane nitrate transporter, alcohol dehydrogenase, pyruvate decarboxylase, sucrose synthase, and LEA. These results might serve as the basis for an in-depth genomics study of <it>Gossypium herbaceum</it>, including a comparative transcriptome analysis and the selection of genes for root traits and drought tolerance.</p

The EAR motif controls the early flowering and senescence phenotype mediated by over-expression of SlERF36 and is partly responsible for changes in stomatal density and photosynthesis.

The EAR motif is a small seven amino acid motif associated with active repression of several target genes. We had previously identified SlERF36 as an EAR motif containing gene from tomato and shown that its over-expression results in early flowering and senescence and a 25-35% reduction of stomatal density, photosynthesis and stomatal conductance in transgenic tobacco. In order to understand the role of the EAR motif in governing the phenotypes, we have expressed the full-length SlERF36 and a truncated form, lacking the EAR motif under the CaMV35S promoter, in transgenic Arabidopsis. Plants over-expressing the full-length SlERF36 show prominent early flowering under long day as well as short day conditions. The early flowering leads to an earlier onset of senescence in these transgenic plants which in turn reduces vegetative growth, affecting rosette, flower and silique sizes. Stomatal number is reduced by 38-39% while photosynthesis and stomatal conductance decrease by about 30-40%. Transgenic plants over-expressing the truncated version of SlERF36 (lacking the C-terminal EAR motif), show phenotypes largely matching the control with normal flowering and senescence indicating that the early flowering and senescence is governed by the EAR motif. On the other hand, photosynthetic rates and stomatal number were also reduced in plants expressing SlERF36ΔEAR although to a lesser degree compared to the full- length version indicating that these are partly controlled by the EAR motif. These studies show that the major phenotypic changes in plant growth caused by over-expression of SlERF36 are actually mediated by the EAR motif

Reduction in organ size and plant height of transgenic <i>SlERF36</i> over-expressing plants grown under long day conditions.

<p>A. Graphical representation of rosette diameter of 28-day-old transgenic plants over-expressing <i>SlERF36</i> and <i>SlERF36ΔEAR</i>. Values represent the average ± SD of 3–5 homozygous plants of each independent transformant.** P<0.01; ***P<0.001. B. Flower (top) and silique (bottom) size variation in transgenic <i>SlERF36</i> and <i>SlERF36</i>ΔEAR <i>Arabidopsis</i> plants. C. Graphical representation of the variation in silique sizes in transgenic <i>SlERF36</i> and <i>SlERF36</i>ΔEAR <i>Arabidopsis</i> plants. Values represent the average ± SD of 10 siliques each from 3–5 homozygous plants of each independent transformant.** P<0.01; ***P<0.001. D. Comparison of transgenic <i>SlERF36</i> and <i>SlERF36</i>ΔEAR over-expressing plants showing differences in height. E. Graphical representation of plant height of transgenic <i>SlERF36</i> and <i>SlERF36</i>ΔEAR plants. Values represent the average ± SD of 3–5 homozygous plants of each independent transformant. ** P<0.01; ***P<0.001.</p

Early flowering in transgenic <i>SlERF36</i> over-expressing plants.

<p><b>A.</b> Early flowering in transgenic <i>SlERF36</i> over-expressing plants grown under long day (16 h light/8 h dark) conditions. Col-0 wild type; Lines 1-8 and 2-1 – <i>SlERF36</i> over-expressing lines; Lines 6-1 and 9-1 – <i>SlERF36ΔEAR</i> over-expressing lines. <b>B.</b> Graphical representation of days to flowering in transgenic <i>SlERF36</i> and <i>SlERF36ΔEAR</i> over-expressing lines. Values represent the average ± SD of 3–5 homozygous plants of each independent transformant.** P<0.01; ***P<0.001. <b>C.</b> Graphical representation of number of leaves at the time of flowering in transgenic <i>SlERF36</i> and <i>SlERF36ΔEAR</i> over-expressing lines. Values represent the average ± SD of 3–5 homozygous plants of each independent transformant.** P<0.01; ***P<0.001. <b>D.</b> Transcript accumulation of the <i>FT</i> gene in control and transgenic lines in 12-day-old plants by semi-quantitative RT-PCR. Actin was used as internal control. <b>E.</b> Early flowering in transgenic <i>SlERF36</i> over-expressing plants grown under short day (10 h light/14 h dark) conditions. Note the delay in flowering and the larger rosettes compared to plants grown in long days in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101995#pone-0101995-g003" target="_blank">Fig. 3A</a>.</p

Reduction in stomatal density in transgenic <i>SlERF36</i> and <i>SlERF36ΔEAR</i> plants. A.

<p>Stomatal density on the leaf abaxial surface in control (C) and transgenic <i>Arabidopsis</i> plants from two independent lines (lines 1-8 and 2-1 over-expressing <i>SlERF36</i> and lines 6-1 and 9-1 over-expressing <i>SlERF36ΔEAR</i>). Stomatal density from leaf epidermal peels was estimated in the leaf sections in three different regions of three different leaves (7^th leaf from bottom from 30-day-old plants) under a light microscope (Nikon Eclipse TE300 Inverted microscope). The small black bar at the base of each picture on the left hand side represents a length of 10 µm. <b>B.</b> Graphical estimation of the stomatal density of the lower leaf epidermis of control (Col-0) and transgenic <i>SlERF36</i> and <i>SlERF36ΔEAR</i> over-expressing lines from Fig. 6A. Values represent the average stomatal density ± SD in an area of 240 µm² of three independent leaves (from the same position). ** P<0.01; ***P<0.001, ****P<0.0001. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101995#pone-0101995-g006" target="_blank">Figure 6C</a>. Graphical estimation of the non-stomatal cell number of the lower leaf epidermis of control (Col-0) and transgenic <i>SlERF36</i> and <i>SlERF36ΔEAR</i> over-expressing lines from Fig. 6A. Values represent the average cell number± SD in an area of 240 µm² of three independent leaves (from the same position). * P<0.05.</p

Early senescence in transgenic <i>SlERF36</i> over-expressing plants.

<p>One and half month old plants (grown under long day conditions from Fig. 3A) showing early leaf senescence and death. Graphical representation of differences in chlorophyll content between control, <i>SlERF36</i> over-expressing (1-8 and 2-1) and <i>SlERF36ΔEAR</i> over-expressing (6-1 and 9-1) plants. Values represent the average ± SD of 5 leaves of each independent transformant (4^th leaf from bottom from 12-day-old plants). Transcript accumulation of the senescence associated <i>SEN4</i> gene in control and transgenic lines in 12-day-old plants. Actin was used for normalization.</p

Net photosynthesis (A), stomatal conductance (gs) and transpiration (E) rates of transgenic <i>Arabidopsis</i> plants over-expressing <i>SlERF36</i> and <i>SlERF36ΔEAR</i>.

<p>Five plants of homozygous progeny of two independent lines for each gene (lines 1-8 and 2-1 over-expressing <i>SlERF36</i> and lines 6-1 and 9-1 over-expressing <i>SlERF36ΔEAR</i>) were grown as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101995#s2" target="_blank">methods</a> section. Measurements were carried out using a GFS-3000 system under a light intensity of 400 µmole photons m⁻²s⁻¹ and a CO₂ concentration of 400 µmol mol⁻¹. Values are average ± SEs of five replicates. *P<0.05, ** P<0.01; ***P<0.001, ****P<0.0001.</p