Expression of drought-stress-responsive genes in wild-type and <i>GpDSR7-</i>overexpressed <i>Arabidopsis</i>.

<p>Total RNA was extracted from control and drought-treated plants and the expression of several drought-responsive gene was analysed by qRT-PCR. The mean value of three technical replicates was normalised to the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Blank columns: control group; slash columns: #1 transgenic plants; black columns: #2 transgenic plants.</p

General summarisation of SSH cDNA libraries in <i>G</i>. <i>pilifera</i>.

<p>(A) Relative water contents were measured to determine the water status of dehydrated gametophytes. Arrows indicate 0.5- and 2.5-h treatments, which were selected for SSH cDNA library construction. Bars are means ± SD of five biological replicates. (B) Morphologies of <i>G</i>. <i>pilifera</i> gametophytes under fully hydrated and dehydrated conditions. Bar = 2 mm. (C) Percentage distribution of unigenes categorised into functional classes. (D) Expression patterns of several drought-responsive genes identified by SSH. Total RNA was extracted from <i>G</i>. <i>pilifera</i> at 0.00, 0.25, 0.50, 1.00, and 2.50 h after drought stress treatment and analysed by qRT-PCR using gene-specific primers. <i>Actin</i> was used as the internal control. *Numbers in parentheses are numbers of EST fragments of the <i>GpDSR</i> genes in the SSH library.</p

Sequence analysis of GpDSR7.

<p>(A) Schematic structure of the <i>GpDSR7</i> cDNA and predicted GpDSR7 protein. Hatched bar indicates the RING (<u>R</u>eally <u>I</u>nteresting <u>N</u>ew <u>G</u>ene) motif, and dark bar indicates the C-terminal putative TM domain. (B) Comparison of the derived amino acid sequence of GpDSR7 with its homologues from other species, including rice (Os06g0677300), <i>Musa acuminata</i> (ABF69983.1), <i>Arabidopsis</i> (At5g60580), maize (NP_001148132.1), <i>Medicago truncatula</i> (XP_003594026.1), and <i>Brassica rapa</i> (ADK63404.1). Solid line denotes the RING motif, and conserved metal ligand positions are indicated by numbered cysteine (C) and histidine (H) residues. Triangles indicate the sites of mutations (C224S, C242S, and H250Y).</p

Unigenes assembled with more than four ESTs annotated by sequence similarities.

<p>Unigenes assembled with more than four ESTs annotated by sequence similarities.</p

<i>In vitro</i> self-ubiquitination assay of GpDSR7.

<p>The C-terminal TM domain truncated form of GpDSR7 was defined as GpDSR7^Δ. (A) GST-tagged GpDSR7^Δ fusion protein was assayed for E3 ubiquitin ligase activity in the presence of E1, E2, and Ubi-Myc. Ubiquitinated proteins were detected by western blot analysis using an anti-GST antibody (left panel) and anti-Myc antibody (right panel). Triangles indicate non-ubiquitinated GST-GpDSR7^Δ. (B) Purified WT GST-GpDSR7^Δ and mutant proteins were used in the E3 Ub ligase enzyme assay.</p

Subcellular localisation of GpDSR7 in <i>Arabidopsis</i> mesophyll protoplasts.

<p>(A) Schematic of GpDSR7 and constructs used in the subcellular localisation assay. Lines indicate deleted regions. TM, transmembrane domain; RING, RING finger domain. (B) The fusion constructs GpDSR7-GFP, GpDSR7-TM-GFP and GpDSR7-ΔTM-GFP were transiently cotransformed with mCherry-labelled ER marker or cytoplasm (CT) marker into <i>Arabidopsis</i> mesophyll cells. All images were obtained from one optic section. Scale bars are equivalent to 10 μm.</p

GpDSR7, a Novel E3 Ubiquitin Ligase Gene in <i>Grimmia pilifera</i> Is Involved in Tolerance to Drought Stress in <i>Arabidopsis</i>

<div><p>The growth and development of plants under drought stress depends mainly on the expression levels of various genes and modification of proteins. To clarify the molecular mechanism of drought-tolerance of plants, suppression subtractive hybridisation cDNA libraries were screened to identify drought-stress-responsive unigenes in <i>Grimmia pilifera</i>, and a novel E3 ubiquitin ligase gene, <i>GpDSR7</i>, was identified among the 240 responsive unigenes. <i>GpDSR7</i> expression was induced by various abiotic stresses, particularly by drought. GpDSR7 displayed E3 ubiquitin ligase activity <i>in vitro</i> and was exclusively localised on the ER membrane in <i>Arabidopsis</i> mesophyll protoplasts. <i>GpDSR7-</i>overexpressing transgenic <i>Arabidopsis</i> plants showed a high water content and survival ratio under drought stress. Moreover, the expression levels of some marker genes involved in drought stress were higher in the transgenic plants than in wild-type plants. These results suggest that GpDSR7, an E3 ubiquitin ligase, is involved in tolerance to drought stress at the protein modification level.</p></div

Drought response of <i>35S</i>:<i>GpDSR7</i> over-expressed transgenic <i>Arabidospsis</i> plants.

<p>(A) Seven-day-old seedlings were transferred to soil for a further 2 weeks of normal growth (left panel), subjected to progressive drought by withholding water for 20 days (middle panel), and then re-watered for 7 days (right panel). (B) Survival rate of plants after re-watering (n > 40). Asterisks indicate significant differences determined by Student’s <i>t</i> test (* 0.01 < P < 0.05). (C) Leaves of 20-day-old plants were excised and weighed at various time points after detachment. Values are means ± SD of three individual plants. (D) Two independent T3 transgenic lines #1 and #2 were subjected to RT-PCR (upper) and immunoblotting (lower) for detecting the expression of GpDSR7. We have used the wildtype (WT) and the plants tranformed with empty vector (EV) as the control.</p

Additional file 5: Figure S1. of Genome-wide survey and expression analysis of the OSCA gene family in rice

Predicted conserved domains in OSCAs of Oryza sativa L. ssp. Japonica, Oryza sativa L. ssp. Indica, Oryza glaberrima, and Oryza brachyantha. (DOC 233 kb

Additional file 1: Table S1. of Genome-wide survey and expression analysis of the OSCA gene family in rice

General information and sequence characterisation of 45 OSCA genes from Oryza sativa L. ssp. Japonica, Oryza sativa L. ssp. Indica, Oryza glaberrima, and Oryza brachyantha. (DOC 89 kb