Overexpression of soybean ubiquitin-conjugating enzyme gene GmUBC2 confers enhanced drought and salt tolerance through modulating abiotic stress-responsive gene expression in Arabidopsis

Abstract

Previous studies have shown that ubiquitination plays important roles in plant abiotic stress responses. In the present study, the ubiquitin-conjugating enzyme gene GmUBC2, a homologue of yeast RAD6, was cloned from soybean and functionally characterized. GmUBC2 was expressed in all tissues in soybean and was up-regulated by drought and salt stress. Arabidopsis plants overexpressing GmUBC2 were more tolerant to salinity and drought stresses compared with the control plants. Through expression analyses of putative downstream genes in the transgenic plants, we found that the expression levels of two ion antiporter genes AtNHX1 and AtCLCa, a key gene involved in the biosynthesis of proline, AtP5CS, and the copper chaperone for superoxide dismutase gene AtCCS, were all increased significantly in the transgenic plants. These results suggest that GmUBC2 is involved in the regulation of ion homeostasis, osmolyte synthesis, and oxidative stress responses. Our results also suggest that modulation of the ubiquitination pathway could be an effective means of improving salt and drought tolerance in plants through genetic engineering.

Fig. 1

Comparison of GmUBC2 and ubiquitin conjugation enzyme E2-related proteins. a Alignment of the deduced amino acid sequence of soybean GmUBC2 with Arabidopsis AtUBC2 (P42745), Mus musculus Ube2A (NP_062642), Saccharomyces cerevisiae RAD6 (AAA34952) and human HR6A (P49459). Fully and partially conserved amino acids were shaded in black and gray, respectively. The asterisk symbol above the alignment denotes the active-site cysteine of E2 enzymes. b Sequence comparison between consensus catalytic sites from UBC (PROSITE: PS000183) and soybean GmUBC2. The catalytic cysteine is highlighted. c Phylogenetic analyses of GmUBC2 and other representative E2-related enzymes. Multiple sequence alignment was performed using CluxtalW and the polygenetic tree was constructed via the Mega 4.1 program. Tree topology was calculated by the neighbor-joining method and 1,000 replicates were used for bootstrap test. The accession numbers for the proteins are given below in parentheses: A. thaliana AtUBC2 (P42745), AtUBC1 (P25865), AtUBC3 (P42746), AtUBC5 (P42749), AtUBC8 (P35131), AtUBC15 (AAC39324), and AtMMS2 (AAK68786); S. cerevisiae RAD6 (AAA34952), ScUBC9 (S52414), and ScMMS2 (AAC24241); M. musculus Ube2A (NP_062642); H. sapiens UbcM2 (AAD40197), HR6A (P49459), HR6B (P63146), HsUEV1 (AAB72016), HsMMS2 (CAA66717), and HsTSG101 (AAC52083); D. melanogaster DmTSG101 (AAG29564)

Fig. 2

Expression of GmUBC2 is regulated by salt, drought and ABA treatments. The seeds were first germinated in vermiculite irrigated with water. After opening of the first trifoliate, the seedlings were transferred into Hoagland’s solution supplemented with 200 mM NaCl, 20% polyethylene glycol (PEG), or 100 μM ABA for 6 h. Seedlings, roots, stems and leaves were collected for total RNA extraction and real time RT-PCR analysis. a GmUBC2 is response to NaCl, drought, and exogenous ABA treatments. b GmUBC2 is up-regulated in different organs under NaCl stress. The soybean actin gene was used as an internal control. Ck, untreated control. Error bars represent SD (n = 3)

Fig. 3

Subcellular localization of EGFP-GmUBC2 fusion protein in transgenic Arabidopsis root cells. a Localization of the EGFP protein. b Localization of EGFP-GmUBC2 protein. Scale bar, 20 μm

Fig. 4

Overexpression of the GmUBC2 in Arabidopsis confers improved NaCl tolerance. a The GmUBC2 transcript levels in WT and three transgenic lines (L1, L16, and L36) were assessed by quantitative RT-PCR. The Arabidopsis Actin gene was used as an internal control. b 5-day-old homozygous seedlings were transferred from normal MS media to MS media supplemented with 0 mM or 200 mM NaCl for 7 days. c Survival rates of WT plants and transgenic plants under 200 mM NaCl treatment for 7 days

Fig. 5

Seedling growth of GmUBC2 transgenic plants under different concentration levels of NaCl. 4-day-old WT and 35S-GmUBC2 seedlings grown on MS medium were transferred to MS medium supplemented with 1% sucrose containing 0, 100 and 150 mM NaCl for 5 days. The graph shows root length after 5 days under different levels of NaCl for WT and transgenic seedlings (n = 15)

Fig. 6

Drought tolerance of the 35S-GmUBC2 transgenic plants. a Three-week-old plants were grown in soil in the same container withheld from water for 15 days and then re-watered. The photographs were taken 1 day after re-watering. Percentages of surviving plants and numbers of surviving plants per total number of tested plants are indicated under the photographs. b Quantification of water loss in 3-week-old WT, L1 and L16 transgenic plants. Leaves of the same developmental stages were excised and weighed at various time points after detachment. Data represent average values ± SD (n = 8)

Fig. 7

Expression of stress-responsive genes in 35S-GmUBC2 lines. Relative RNA levels of stress-responsive genes were determined by Real time PCR using total RNAs isolated from the shoots of 3-week-old plants grown under normal conditions in soil. Two independent assays were performed and similar results were obtained (each n = 3). The Arabidopsis ACT3 (ATU39480) gene was used as a loading control

Fig. 8

Measurement of SOD activities, proline contents, and ion contents in wild type (WT) and two transgenic lines (L1 and L16) overexpressing GmUBC2. (A and B) SOD activity (a) and proline content (b) in WT and two transgenic lines (L1 and L16) overexpressing GmUBC2 grown at various salt concentrations. Na⁺ (C) and K⁺ (D) contents of WT and transgenic plants (n = 3). DW, dry weight. FW, fresh weight. Asterisks represent significant differences between the WT and transgenic plants (** and * represent P < 0.01 and P < 0.05 respectively, Student’s t test)