The Proline Dehydrogenase Gene CsProDH1 Regulates Homeostasis of the Pro-P5C Cycle Under Drought Stress in Tea Plants

Abstract

The homeostasis of the proline-Δ¹-pyrroline-5-carboxylate (Pro-P5C) cycle, mediated by proline dehydrogenase (ProDH), plays a critical role in plants in response to abiotic stresses. The biological function of gene CsProDH1 under drought stress and its effects on amino acid metabolism and photosynthesis through proline metabolism were investigated. Enzymatic characterization of the CsProDH1 protein was conducted in vitro. Overexpression of CsProDH1 aggravated plant stress, as evident by reduced photosynthetic efficiency and increased reactive oxygen species, which activated the Pro-P5C cycle. In contrast, silencing CsProDH1 enhanced plant drought resistance, increased proline accumulation, and protected photosynthesis. Studies indicate that exogenous amino acid application mitigates drought-induced physiological impairments in plants by maintaining cellular homeostasis, with particular efficacy observed in enhancing tea plant drought resilience through improved osmotic adjustment and antioxidant capacity. This study uncovers the significant role of CsProDH1 in plant drought resistance and its regulatory mechanism, offering potential gene targets and application strategies for enhancing crop drought resistance.

Keywords: Pro-P5C cycle; drought stress; proline dehydrogenase.

Figure 1

Expression patterns of tea plant CsProDH1 under different treatments. (A) Phylogenetic evolutionary tree of ProDH in different species; (B) expression of tea plant ProDH gene in different tissues; (C) expression of Pro metabolism-related genes in tea plant mature leaves after exogenous Pro treatment; (D) expression of Pro metabolism-related genes in tea plant mature leaves after exogenous Glu treatment; and (E) expression of proline metabolism-related genes in tea plant mature leaves after exogenous γ-aminobutyric acid treatment. The data are the mean ± SD (n = 3). Significance was verified by t-tests; ‘*’ represents significance at p ≤ 0.05.

Figure 2

Enzymatic characterization of tea plant CsProDH1 protein. (A) The purification effect of His-tagged recombinant protein under denaturing conditions. M, marker; CL, bacterial lysate (cell lysate); FT, flow-through; W1–W3, denaturing lysate washing 1–3; W4 and W5, denaturing washing solution washing; E1–E4, eluent 1–4; (B) results of the enzymatic characterization of CsProDH1 protein; (C) results of in vitro protein function validation of CsProDH1; (D) Lineweaver–Burke double inverse plots of CsProDH1 enzyme kinetics; (E) enzyme activity of CsProDH1 at different temperatures; (F) temperature stability of CsProDH1; (G) CsProDH1 enzyme activities at different pH; and (H) pH stability of CsProDH1.

Figure 3

Intracellular localization of the CsProDH1 protein. Scale bar = 50 μm.

Figure 4

Changes in parameters related to overexpression treatment of CsProDH1 in tea plants under drought conditions. (A) Phenotype and in vivo fluorescence imaging at 24 h processing. Scale bar = 10 cm; (B) leaf NBT staining at 24 h processing; (C) leaf O₂⁻ content at 24 h processing; (D) leaf MDA content at 24 h processing; (E) parameters related to in vivo fluorescence imaging at 24 h processing. Fv/Fm, maximum photosynthetic efficiency; Y(II), actual photosynthetic efficiency of PSII; Y(NPQ), quantum yield of regulated energy dissipation in PSII; and Y(NO), quantum yield of non-regulated energy dissipation in PSII. CK, green circle; OE, orange square. (F) OJIP curves; (G) fluorescence rise kinetics normalized by F₀ and F to W_OJ = (F_t − F₀)/(F − F₀); and (H) radar plot of fluorescence parameters. Tfm, the time required from dark adaptation to illumination to maximum fluorescence; Area, the OJIP curve, fluorescence intensity F = F_m, and the area between the Y-axis; F_v/F₀, the ability of the PSII reaction center to capture and convert light energy; ABS/RC, light energy absorbed per reaction center; (1 − Vj)/Vj, the efficiency/probability with which an electron from the intersystem electron carriers is transferred to reduce end electron acceptors at the PSI acceptor side; PI, performance index. CK, control group; OE, overexpression group. The data are the mean ± SD (n = 3). Significance was verified by t-tests; ‘*’ represents significance at p ≤ 0.05.

Figure 5

Changes in parameters related to transient silencing treatment of CsProDH1 in tea plants under drought conditions. (A) Phenotype and in vivo fluorescence imaging. Scale bar = 10 cm; CK, green circle; asODN, orange square (B) leaf NBT staining at 24 h processing; (C) leaf O₂⁻ content at 24 h processing; (D) radar plot of fluorescence parameters at 24 h processing; (E) parameters related to in vivo fluorescence imaging at 24 h processing; (F) OJIP curves at 24 h processing; and (G) fluorescence rise kinetics normalized by F₀ and F to W_OJ. CK, control group; asODN, silencing group. The data are the mean ± SD (n = 3). Significance was verified by t-tests; ‘*’ represents significance at p ≤ 0.05.

Figure 6

Exogenous application of amino acids for recovery treatment after 24 h of CsProDH1 overexpression under drought conditions. (A) Phenotype and in vivo fluorescence imaging. Scale bar = 1 cm; (B) leaf NBT staining 24 h after spraying with externally supported amino acids; and (C) leaf MDA content 24 h after spraying with externally supported amino acids. CK, samples of exogenous water; Pro, samples of 10 mM exogenous proline; GABA, samples of 1 mM exogenous γ-aminobutyric acid. The data are the mean ± SD (n = 3). Significance was verified by t-tests; ‘*’ represents significance at p ≤ 0.05.

Figure 7

A model of ProDH-mediated regulation of Pro-P5C cycle homeostasis under drought stress. ProDH, proline dehydrogenase; P5CDH, Δ¹-pyrroline-5-carboxylate dehydrogenase; P5CR: Δ¹-pyrroline-5-carboxylate reductase; P5CS: Δ¹-pyrroline-5-carboxylate synthase; Pro, proline; Glu, glutamate; P5C: Δ¹-pyrroline-5-carboxylate; ROS, reactive oxygen species.