MsTHI1 overexpression improves drought tolerance in transgenic alfalfa (Medicago sativa L.)

Abstract

In recent years, drought stress caused by global warming has become a major constraint on agriculture. The thiamine thiazole synthase (THI1) is responsible for controlling thiamine production in plants displaying a response to various abiotic stresses. Nonetheless, most of the THI1 activities in plants remain largely unknown. In this study, we extracted MsTHI1 from alfalfa and demonstrated its beneficial impact on improving the resistance of plants to stress conditions. The highest levels of MsTHI1 expression were identified in alfalfa leaves, triggered by exposure to cold, drought, salt, or alkaline conditions. The upregulation of MsTHI1 in drought-stressed transgenic plants resulted in enhanced accumulation of vitamin B1 (VB1), chlorophyll a (Chl a), chlorophyll b (Chl b), soluble protein, higher soil and plant analyzer development (SPAD) value, and the activity of peroxidase (POD), maintained Fv/Fm, and decreased lipid peroxidation. Moreover, overexpression of MsTHI1 upregulated the transcription of THI4, TPK1, RbcX2, Cu/Zn-SOD, CPK13, and CPK32 and downregulated the transcription of TH1 and CPK17 in transgenic alfalfa under drought stress. These results suggested that MsTHI1 enhances drought tolerance by strengthening photosynthesis, regulating the antioxidant defense system, maintaining osmotic homeostasis, and mediating plant signal transduction.

Keywords: MsTHI1; alfalfa; antioxidant defense; drought tolerance; osmotic homeostasis; photosynthesis.

Figure 1

Subcellular localization of MsTHI1. (A) fusion GFP fluorescence. (B) bright field under infected leaves, (C) fusion picture. Scale bar, 20 μm.

Figure 2

Expression pattern of MsTHI1. (A) Tissue-specific expression of MsTHI in the alfalfa root, stem, and leaf. (B) Expression analysis of MsTHI1 in leaves of alfalfa subjected to cold (4°C), drought (15% PEG), salt (150 mM of NaCl), and alkaline (150 mM of NaHCO₃) stresses. The expression levels were normalized in the alfalfa GAPDH gene. Mean ± standard deviations from three biological replicates are shown with error bars. These letters denote variations that are statistically significant (α = 0.05).

Figure 3

Identification of transformation with MsTHI1 via qPCR and phenotype of transformation with MsTHI1 under control conditions and drought stress. (A) Identifying transgenic tobacco via quantitative real-time PCR (qPCR). (B) Identifying transgenic alfalfa via qPCR. (C) Phenotype of three-week-old transgenic tobacco plants with overexpressed MsTHI1 under control and drought stress for seven days. (D) Phenotype of six-week-old transgenic alfalfa plants with overexpressed MsTHI1 under control and drought stress for seven days. NtActin and alfalfa GAPDH gene were used as the internal reference control for identifying transgenic tobacco and alfalfa, respectively, via qPCR. Mean ± standard deviations from three biological replicates are shown with error bars. “**”: extremely significant difference between WT and transformation line (P < 0.01). WT: wild type; OV#Nt2, OV#Nt3, and OV#Nt7: transgenic tobacco lines 2, 3, and 7, respectively. OV#Ms7 and OV#Ms9: transgenic alfalfa lines 7 and 9, respectively.

Figure 4

Modifications in the physiology of transgenic tobacco plants that overexpress MsTHI1 during control or under drought stress. Mean ± standard deviations from three biological replicates are displayed with error bars. MDA, malondialdehyde. SPAD, soil and plant analyzer development. Fv/Fm, the PS II primary light energy conversion efficiency. ${\text{O}}_2^{.-}$, superoxide anion radical. SOD, superoxide dismutase. POD, peroxidase. Pro, proline. WT, wild type. OV#Nt2, OV#Nt3, and OV#Nt7, transgenic tobacco lines 2, 3, and 7, respectively. “*”, significant difference between WT and transgenic tobacco lines (P < 0.05). “**”, extremely significant difference between WT and transgenic tobacco lines (P < 0.01).

Figure 5

Physiological changes of transgenic alfalfa plants with overexpressing MsTHI1 under control and drought stress. Mean ± standard deviations from three biological replicates are depicted with error bars. MDA, malondialdehyde. ${\text{O}}_2^{.-}$ superoxide anion radical. SOD, superoxide dismutase. POD, peroxidase. GSH, reduced glutathione. Pro, proline. WT, wild type. OV#Ms7 and OV#Ms9, transgenic alfalfa lines 7 and 9, respectively. “*”, significant difference between WT and transgenic tobacco lines (P < 0.05). “**”, extremely significant difference between WT and transgenic tobacco lines (P < 0.01).

Figure 6

Expression of eight associated genes in transgenic alfalfa plants with overexpressing MsTHI1 under drought stress. WT, wild-type alfalfa. OV, overexpressing MsTHI1 alfalfa. THI4, thiazole biosynthetic enzyme THIAMIN4. TH1, HMP-P kinase/TMP pyrophosphorylase. TPK1, thiamine pyrophosphokinase. RbcX2, chaperonin-like RbcX protein 2. Cu/Zn-SOD, superoxide dismutase [Cu-Zn]. CPK13, CPK17, and CPK32: calcium-dependent protein kinases. Mean ± standard deviations from three biological replicates are shown with error bars. These letters denote statistically significant variations (α = 0.05).

Figure 7

Possible function model of MsTHI1 response to drought stress. The red arrows represent significant increases in physiological signs or genes being overexpressed. Green arrows represent decreased expressions of genes or declines in physiological markers. Dotted lines represent potential regulating pathways. CPK13, CPK 17, and CPK32: calcium-dependent protein kinases. Cu/Zn-SOD, superoxide dismutase [Cu-Zn]. ${\text{O}}_2^{.-}$, superoxide anion radical. POD, peroxidase. RbcX2, chaperonin-like RbcX protein 2. TH1, HMP-P kinase/TMP pyrophosphorylase. THI4, thiazole biosynthetic enzyme THIAMIN4. TPK1, thiamine pyrophosphokinase. WT, wild type.