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We cloned a gene encoding a member of the GRAS protein family; the latter show the highest identity to SCL7 in Arabidopsis, and we named it PeSCL7. The Populus euphratica SCL7 (PeSCL7) protein is predicted to be 64.7 kD in size and possesses a typical GRAS domain with two conserved LHR and VHIID motifs. A database search revealed that PeSCL7 is 49% and 46% identical to the Arabidopsis thaliana and Oryza sativa SCARECROW-LIKE 7 (SCL7) (Bolle, 2004; Tian et al., 2004) proteins, respectively. The genomic PCR products amplified by primers designed from the 5' and 3' untranslated region indicated that PeSCL7 had no intron interrupting its coding region. Our cDNA-AFLP data indicated that its transcript was induced to high levels after salt stress treatment, and this was later confirmed by RT- and qRT-PCR analysis (Fig. 1A). The cDNA-AFLP results suggested that PeSCL7 is involved in the stress response.
Expression profile of PeSCL7
To verify the cDNA-AFLP PeSCL7 expression result, the expression pattern of PeSCL7 under various stress treatments was investigated. PeSCL7 expression was up-regulated by salt and drought stress treatment and down-regulated by Gibberellin (GA) but not ABA application in the leaves (Fig. 1A). Under the salt treatment, PeSCL7 expression was induced quickly, and the level of the product peaked at 3 h. Under the drought treatment, the PeSCL7 transcript was not induced immediately, but then reached its maximum at 3 h. For GA treatment, the expression of PeSCL7 became weaker within 5 h. Further analysis by qRT-PCR showed similar results to that of semi-quantitative RT-PCR. We also determine the expression pattern by analyzing the expression of the PeSCL7 promoter-Î²-glucuronidase (GUS) fusion (Fig. 1B). Histochemical staining demonstrated that GUS activity increased throughout the plant in salt conditions and after drought treatment.
Targeting of PeSCL7 to the nucleus
To determine the subcellular localization of PeSCL7, p35S:PeSCL7-GFP and p35S:GFP were transiently expressed in onion epidermal cells. As shown in Fig. 2, the onion cells transformed with the p35S:GFP vector displayed fluorescence throughout the whole cells (Fig. 2, a-c). In contrast, fluorescence in the onion cell transformed with p35S:GFP-PeSCL7 was detected exclusively in the nucleus (Fig. 2B, d-f), indicating that PeSCL7 encoded a nuclear-localized protein.
Certain [Consider removing.] T-DNA Insertion Mutants of the AtSCL7 Gene
To elucidate the in vivo functions of PeSCL7, we applied reverse genetics, overexpression and complementation approaches. The loss-of-function mutant of the AtSCL7 gene and the two independent T-DNA insertion lines, scl7-1(Salk_106909) and scl7-2 (Salk_106426), were ordered from the ABRC seed stock center. The T-DNA insertion positions are illustrated in Fig. S1A; the homozygous mutants were verified by diagnostic PCR using AtSCL7 gene-specific and T-DNA border primers (Fig. S1B). Both the scl7-1 and scl7-2 null alleles were confirmed by RT-PCR (Fig. S1C).
Salt and Osmotic Responses of 35S-PeSCL7 and scl7-1 Plants
Salts inhibit germination and seedling growth in a concentration-dependent manner (Xiong et al., 2002). Since PeSCL7 is a salt-induced gene (Fig. 1), it is probable that PeSCL7 plays a role in plant responses to salt. Four different genotype seeds (WT, 35S-PeSCL7, scl7-1, scl7-1/PeSCL7) were germinated on MS medium containing 0, 50, 100 mM NaCl, and differences were observed at both germination and post-germinative growth stages. Under our experimental conditions, the germination ratio of the wild-type plants was reduced about 12% by 50 mM NaCl and 20% by 100 mM NaCl, while the scl7-1 mutant was more affected. In contrast, the germination efficiency of the 35S-PeSCL7 and scl7-1/PeSCL7 plants was less affected (below 10% reduction) by 100 mM NaCl (Fig. 3A). To further investigate PeSCL7 in plant salt or general osmotic effects, the WT, 35S-PeSCL7, scl7-1, and scl7-1/PeSCL7 plants were germinated and grown on MS medium with or without 100 mM NaCl and 200 mM mannitol (an osmotic agent) (Fig. 3C). The growth of the WT and scl7-1 plants was strongly inhibited compared with the transgenic plants upon NaCl and mannitol treatment. The transgenic plants were much healthier and had more green cotyledons than the WT and scl7-1 plants (Fig. 3C). To quantify the difference between the four plant genotypes upon salt or general osmotic stress, the root length of the plants were measured (Fig. 3D). The result showed that the WT and scl7-1 mutant were dramatically affected by a decrease of about 75% of the control plants under the stress treatments, whereas the transgenic plants were less influenced. Thus, both the germination (Fig. 3A) and post-germination (Fig. 3C and D) growth of the 35S-PeSCL7 transgenic plants are more tolerant to high salinity and osmotic stress.
Overexpression of PeSCL7 in transgenic Arabidopsis improved drought tolerance
To characterize the in vivo function of PeSCL7, 3-week-old 35S-PeSCL7, scl7-1/PeSCL7 and scl7-1 mutant plants, as well as wild-type control plants, were not watered for 15 d to induce drought stress. As shown in Fig. 4A, most of the wild-type plants and all scl7-1 mutant plants were withered, but the 35S-PeSCL7 plants exhibited continued survival and growth. Consistent with these results, the detached leaves of transgenic plants (35S-PeSCL7 and scl7-1/PeSCL7) lost water more slowly than did those of the wild-type and scl7-1 mutant plants (Fig. 3B). Thus, we concluded that the 35S-PeSCL7 transgenic plants were highly tolerant to drought stress.
To better understand the mechanisms of drought and salt tolerance conferred by overexpressing 35S-PeSCL7, we investigated the activity of several known enzymes related to abiotic stresses in transgenic Arabidopsis plants. As shown in Fig. 4B and C, the Î±-amylase and SOD activity assays showed that the transgenic plants exhibited the same level activity than the WT and mutants under normal growth conditions. However, after stresses, the elevation of the enzymatic activity in transgenic plants was much [Consider "significantly" if applicable.] larger than those of the WT and mutants (p<0.05). Since PeSCL7 is a transcription factor, the transcript levels of the AMY1 and Cu/Zn SOD enzymes were also investigated (Fig. 4D). The elevated transcript levels of AMY1 and Cu/Zn SOD were positively correlated with the Î±-amylase and SOD activities under stress conditions, as observed from the RT-PCR results. Under normal growth conditions, only the AMY1 transcript level was elevated in the transgenic plant, and no difference was observed for the Cu/Zn SOD transcript level.