File S1 - Overexpression of VrUBC1, a Mung Bean E2 Ubiquitin-Conjugating Enzyme, Enhances Osmotic Stress Tolerance in Arabidopsis

Table S1. Primer sequences used for genomic DNA PCR, RT-PCR and Northern blot experiments. Table S2. Primer sequences used for vector constructions. Figure S1. Multiple sequence alignment and phylogenetic analysis of mungbean VrUBC1 and its homologs. (A) Alignment of the deduced amino acid sequences of VrUBC1 with homologous UBCs. Proteins were aligned using CLUSTALW at the T-coffee website. The active Cys residue is denoted with an inverted delta symbol and the conserved E2 motif [HPN(I/V)(X)3-4GX(I/V/L)C(I/L)X(I/V)(I/L)] is over-lined [45]. Protein sequences are as follows: A. thaliana AtUBC10 (DQ027024), H. sapiens HsUBCH5D (NP_057067), and S. cerevisiae ScUBC5 (P15732). (B) Arabidopsis UBC domain-containing proteins and UBCs orthologous to VrUBC1 from other organisms were retrieved from databases. The protein sequences were used to construct the tree; P. trichocarpa (ABK94824), V. vinifera (CAO69355), Malus x domestica (ACB87920), P. patens subsp. patens (XP_001764055), S. tuberosum (P35135), S. lycopersicum (CAA51821), A. thaliana AtSCE1a (AEE79711.1), AtRCE1 (AAF19827.1), AtRCE2 (AAD12207.1), AtUBC1 (DQ027016), AtUBC2 (DQ027017), AtUBC3 (DQ027018), AtUBC4 (DQ027019), AtUBC5 (DQ027020), AtUBC6 (DQ027021), AtUBC7 (At5g59300), AtUBC8 (DQ027022), AtUBC9 (DQ027023), AtUBC10 (DQ027024), AtUBC11 (DQ027025), AtUBC12 (DQ027026), AtUBC13 (DQ027027), AtUBC14 (DQ027028), AtUBC15 (DQ027029), AtUBC16 (DQ027030), AtUBC17 (DQ027031), AtUBC18 (DQ027032), AtUBC19 (DQ027033), AtUBC20 (DQ027034), AtUBC21 (DQ027035), AtUBC22 (DQ027036), AtUBC23 (At2g16920), AtUBC24 (DQ027037), AtUBC25 (DQ027038), AtUBC26 (DQ027039), AtUBC27 (DQ027040), AtUBC28 (DQ027041), AtUBC29 (DQ027042), AtUBC30 (DQ027043), AtUBC31 (DQ027044), AtUBC32 (DQ027045), AtUBC33 (DQ027046), AtUBC34 (DQ027047), AtUBC35 (DQ027048), AtUBC36 (DQ027049), AtUBC37 (DQ027050), B. napus (ACC38297), C. annuum (AAR83891), P. sativum (AAA64427), O. sativa OsUBC5a (AB074411), OsUBC5b (AB074412), A. hypogaea (AAV34697), A. capillus-veneris (ABQ65169), G. max (AAN03469), G. thurberi (AAL99224), C. reinhardtii (EDO98738), P. resinosa (AAD00911), C. glabrata (CAG58813), S. pombe (CAA17917), G. cingulata (AAC39499), D. rerio (NP_001082922), H. sapiens HsUBCH5D (NP_057067), X. laevis (AAI42570), S. cerevisiae ScUBC4 (CAA35528), and ScUBC5 (P15732). Bootstrap values are shown for each node that had >50% support in a bootstrap analysis of 1,000 replicates. Figure S2. Expression analyses and growth phenotypes of the 35S:VrUBC1 transgenic plants. (A) RNA expression of VrUBC1 was examined by RT-PCR. Actin transcript level was used as a loading control. (B) VrUBC1 RNA expression in the wild-type and the 35S:VrUBC1 transgenic lines analyzed by qRT-PCR. Transcript levels of VrUBC1 were quantified by qRT-PCR against actin transcript level. Each value is the mean ± SD of three independent biological determinations. (C) Three-week-old seedlings of the wild-type and 35S:VrUBC1 Arabidopsis transgenic lines (L7, L9, L19 and L23) were grown in MS medium containing 2% (w/v) sucrose and 0.8% (w/v) phytoagar. (D) Root length was monitored after 3 weeks. The values are the means ± SD (n = 3). This experiment was carried out three times with consistent results. Figure S3. RNA expression of AtVBP1 and RAB18 in response to osmotic stress or ABA. Total RNA was extracted from the leaves of Arabidopsis treated with dehydration, NaCl (100 mM) or ABA (100 µM) for the indicated time period (0, 3, 6, 12, 24 h). Induction patterns of AtVBP1 were investigated by real-time qRT-PCR. RAB18 was used as a positive control for abiotic stress and ABA. Gene expression was normalized to Actin transcript levels as an internal control. Data represent means ± SD from three independent experiments. (DOCX)