GmNFYA13 Improves Salt and Drought Tolerance in Transgenic Soybean Plants

Abstract

NF-YA transcription factors function in modulating tolerance to abiotic stresses that are serious threats to crop yields. In this study, GmNFYA13, an NF-YA gene in soybean, was strongly induced by salt, drought, ABA, and H₂O₂, and suppressed by tungstate, an ABA synthesis inhibitor. The GmNFYA13 transcripts were detected in different tissues in seedling and flowering stages, and the expression levels in roots were highest. GmNFYA13 is a nuclear localization protein with self-activating activity. Transgenic Arabidopsis plants overexpressing GmNFYA13 with higher transcript levels of stress-related genes showed ABA hypersensitivity and enhanced tolerance to salt and drought stresses compared with WT plants. Moreover, overexpression of GmNFYA13 resulted in higher salt and drought tolerance in OE soybean plants, while suppressing it produced the opposite results. In addition, GmNFYA13 could bind to the promoters of GmSALT3, GmMYB84, GmNCED3, and GmRbohB to regulate their expression abundance in vivo. The data in this study suggested that GmNFYA13 enhanced salt and drought tolerance in soybean plants.

Keywords: ABA hypersensitivity; crop yield; nuclear factor YA; salt and drought tolerance; soybean.

FIGURE 1

GmNFYA13 transcripts were induced by treatment of salt, H₂O₂, and ABA. (A) The expression levels of 21 NF-YA members in Glycine max were evaluated with qRT-PCR under salt treatment. (B–D) H₂O, H₂O₂, and ABA induced GmNFYA13 transcripts. (E) Tungstate suppressed salt- and drought-induced GmNFYA13 transcripts. Control, salt, salt + tungstate, drought, and drought + tungstate are indicated by CTR, ST, ST + T, DH, and DH + T, respectively. GmCYP2 was used as the internal control and transcripts of all genes used in the assay were normalized to the non-treated expression level, which was set as 1.0. Data indicate three biological replicates ± SD. Different letters above the columns represent significant differences at P < 0.05.

FIGURE 2

Analysis of tissue-specific expression, self-activating activity, and subcellular localization of GmNFYA13. (A,B) GmNFYA13 transcripts were assessed in various tissues at two growth periods. The expression level of GmNFYA13 in roots was set as 1.0, and GmCYP2 was the internal control. Data indicate three biological replicates ± SD. Different letters above the columns represent significant differences at P < 0.05. (C) GUS staining in each tissue in P_GmNFYA13:GUS transgenic Arabidopsis plants. a, 5-day-old seedling; b, rosette leaf (RL); c, cauline leaf (CL); d, root (R); e, flower (F); f, silique (S); g, transcripts of GUS were measured in various tissues of transgenic Arabidopsis by qRT-PCR. Scale bar = 4 mm. The transcripts of GUS in roots were set as 1.0, and AtTub8 was the internal control. Data indicate three biological replicates ± SD. Different letters above the columns represent significant differences at P < 0.05. (D) Self-activating activity analysis of GmNFYA13. (E) GFP fluorescence was detected in roots of WT and 35S:GmNFYA13 Arabidopsis plants. Scale bar = 200 μm. Each experiment had three biological replicates.

FIGURE 3

Root growth of WT and 35S:GmNFYA13 transgenic Arabidopsis seedlings under treatments of salt, drought, and ABA. (A) The root elongation of WT and 35S:GmNFYA13 with/without treatments. Scale bar = 2 cm. (B) The transcript levels of GmNFYA13 in WT and 35S:GmNFYA13 lines were evaluated with RT-PCR. AtTub8 was the internal control. (C–E) The root lengths of WT and 35S:GmNFYA13 lines subjected to various concentrations of NaCl, PEG, and ABA. Data indicate three biological replicates (n = 18) ± SD. Different letters above the columns represent significant differences at P < 0.05. Three biological replicates were used in each experiment.

FIGURE 4

35S:GmNFYA13 Arabidopsis seedlings showed enhanced salt tolerance compared to WT lines. (A) Assessment of salt tolerance in WT and 35S:GmNFYA13 transgenic Arabidopsis plants. Three-week-old seedlings were treated with 350 mM NaCl solution for 7 days. (B–E) The survival rate, ion leakage, MDA, and chlorophyll content of WT and 35S:GmNFYA13 lines were measured after salt treatment. (F) The ABA concentration of WT and 35S:GmNFYA13 lines were measured following salt treatment for 4 days. (G–J) The expression levels of SOS1, SOS2, SOS3, and NCED3 in WT and 35S:GmNFYA13 lines were evaluated under control and salt treatments. Transcript levels of these genes were normalized to those in WT plants under control conditions, and AtTub8 was used as the internal control. Data indicate three biological replicates ± SD. Different letters above the columns represent significant differences at P < 0.05. Each experiment had three biological replicates.

FIGURE 5

35S:GmNFYA13 Arabidopsis seedlings showed enhanced drought tolerance compared to WT lines at the seedling stage. (A) Evaluation of drought tolerance in WT and 35S:GmNFYA13 transgenic Arabidopsis plants. Three-week-old seedlings were subjected to drought treatment for 9 days and recovered for 7 days. (B) The SWP of the soil where WT and 35S:GmNFYA13 were grown. (C) The water loss of detached leaves from WT and 35S:GmNFYA13 Arabidopsis seedlings. (D) The survival rate of WT and 35S:GmNFYA13 Arabidopsis seedlings after recovery from drought treatment. (E,F) The relative water content and proline content of WT and 35S:GmNFYA13 Arabidopsis seedlings after drought treatment. (G) The ABA concentration of WT and 35S:GmNFYA13 Arabidopsis seedlings following drought treatment for 4 days. (H–K) Transcript levels of DREB2A, ABI3, NCED3, and RD29A under control and drought treatment. Transcript levels of these genes were normalized to those in WT plants under control conditions, AtTub8 was used as the internal control. Data indicate three biological replicates ± SD. Different letters above the columns represent significant differences at P < 0.05. Three biological replicates were used in each experiment.

FIGURE 6

OE and RNAi transgenic soybean plants, respectively, displayed improved and decreased salt tolerance compared with EV plants. (A) Assessment of salt tolerance in RNAi, EV, and OE transgenic soybean plants. Soybean plants with positive hairy roots were subjected to 500 mM NaCl solution for 7 days. Scale bar = 6 cm. (B) GmNFYA13 transcript was evaluated in RNAi, EV and OE transgenic soybean plants. GmCYP2 was used as the internal control and transcripts of GmNFYA13 were normalized to the expression level in RNAi transgenic soybean plants, which was set as 1.0. Data indicate three biological replicates ± SD. Different letters above the columns represent significant differences at P < 0.05. (C–E) The survival rate, ion leakage, and MDA content in RNAi, EV, and OE transgenic soybean plants. Data indicate three biological replicates (n = 18) ± SD. Different letters above the columns represent significant differences at P < 0.05.

FIGURE 7

OE and RNAi transgenic soybean plants, respectively, displayed increased and decreased tolerance to drought stress compared with EV plants. (A) Drought tolerances were evaluated in RNAi, EV, and OE transgenic soybean plants. Soybean plants were subjected to drought treatment for 15 days and recovery for 5 days. Scale bar = 6 cm. (B) The SWP of the soil where RNAi, EV, and OE transgenic plants grew were assessed. Asterisks represent significant differences at P < 0.05 in comparison to the corresponding controls. (C) The survival rate was measured in RNAi, EV, and OE transgenic plants after recovery from drought treatment. (D–F) The relative water content, proline, and MDA content were measured in RNAi, EV, and OE transgenic plants after drought treatment. Data indicate three biological replicates (n = 18) ± SD. Different letters above the columns represent significant differences at P < 0.05. Each experiment had three biological replicates.

FIGURE 8

The expression levels of GmSALT3, GmNHX1, GmSOS1, GmNCED3, GmMYB84, GmDREB2, GmWRKY46, and GmRbohB in RNAi, EV, and OE transgenic soybean plants under control, salt, and drought conditions. (A) Transcripts of GmSALT3, GmNHX1, GmSOS1, GmNCED3, GmMYB84, GmDREB2, GmWRKY46, and GmRbohB in EV and OE transgenic lines under control, salt, and drought conditions. (B) Transcript levels of these genes in RNAi and EV transgenic lines under control, salt, and drought conditions. GmCYP2 was used as the internal control and transcripts of GmNFYA13 were normalized to the expression level in EV transgenic soybean plants under control conditions, which was set as 1.0. CTR represents the control condition, and salt and DH represent salt and drought treatment, respectively. For salt treatment, roots were subjected to 500 mM NaCl solution for 1 h; for drought treatment, roots were placed on a plastic plate for 2 h; roots under normal condition were used as the control sample. Data indicate three biological replicates ± SD. Different letters above the columns represent significant differences at P < 0.05.

FIGURE 9

LUC activity was enhanced due to the regulation of GmNFYA13 on promoters of GmSALT3, GmNCED3, GmMYB84, and GmRbohB. (A–D) The LUC activity was evaluated in tobacco leaves with LB985 NightSHADE. (E–H) The LUC expression levels of the protein solution extracted from the leaves, indicated by ratio of LUC/REN, were measured with GloMax Multi Jr. The ratio of LUC/REN of GFP-GmNFYA13 + P_{GmSALT3/GmNCED3/GmMYB84/GmRbohB} was normalized to that of GFP + P_{GmSALT3/GmNCED3/GmMYB84/GmRbohB}, which was set as 1.0. Data indicate three biological replicates ± SD. Different letters above the columns represent significant differences at P < 0.05.