To investigate the lignification process and its physiological significance under drought-stressed conditions, the changes in enzymes responsible for lignification and the related physiological parameters were determined in white clover (Trifolium repens L.) leaves during 28 d of water deficit treatment. Water deficit gradually decreased leaf water potential (Ψ_w) to −2.33 MPa at day 28. For the first 14 d of water deficit, ascorbate peroxidase and phenylalanine ammonia-lyase were highly activated. Neither a change in the parameters symptomatic of oxidative stress nor growth inhibition was observed. The reduction of leaf biomass occurred from 21 d of water deficit treatment when Ψ_w was −2.27 MPa or less, and was concomitant with the increase of lipid peroxidation and lignin content. As Ψ_w decreased below −1.67 MPa from 14 d of water deficit, the enhanced activation of guaiacol peroxidase, coniferyl alcohol peroxidase, syringaldazine peroxidase, and benzidine peroxidase was involved in lignification rather than in protection of plant tissues against the oxidative damage. The data indicate that a high activation of lignifying enzymes during terminal stress may be a drought stress-induced injurious symptom, which leads to reduced forage growth and digestibility.

Proline metabolism influences the metabolic and/or signaling pathway in regulating plant stress responses. This study aimed to characterize the physiological significance of glutamate (Glu)-mediated proline metabolism in the drought stress responses, focusing on the hormonal regulatory pathway. The responses of cytosolic Ca²⁺ signaling, proline metabolism, and redox components to the exogenous application of Glu in well-watered or drought-stressed plants were interpreted in relation to endogenous hormone status and their signaling genes. Drought-enhanced level of abscisic acid (ABA) was concomitant with the accumulation of ROS and proline, as well as loss of reducing potential, which was assessed by measuring NAD(P)H/NAD(P)⁺ and GSH/GSSG ratios. Glu application to drought-stressed plants increased both salicylic acid (SA) and cytosolic Ca²⁺ levels, with the highest expression of calcium-dependent protein kinase (CPK5) and salicylic acid synthesis-related ICS1. The SA-enhanced CPK5 expression was closely associated with further enhancement of proline synthesis-related genes (P5CS1, P5CS2, and P5CR) expression and a reset of reducing potential with enhanced expression of redox regulating genes (TRXh5 and GRXC9) in a SA-mediated NPR1- and/or PR1-dependent manner. These results clearly indicate that Glu-activated interplay between SA- and CPK5-signaling as well as Glu-enhanced proline synthesis are crucial in the amelioration of drought stress in Brassica napus.

The kinetics of protein incorporation from newly-absorbed nitrogen (N, de novo protein synthesis) was estimated by 15N tracing in 18-week-old white clover plants (Trifolium repens L. cv. Regal) during 7 d of water-deficit treatment. The physiological relationship between kinetics and accumulation of proline and ammonia in response to the change in leaf-water parameters was also assessed. All leaf-water parameters measured decreased gradually under water deficit. Leaf and root dry mass was not significantly affected during the first 3 d when decreases in leaf-water parameters were substantial. However, metabolic parameters such as total N, proline and ammonia were significantly affected within 1 d of commencement of water-deficit treatment. Water-deficit treatment significantly increased the proline and NH3–NH4+ concentrations in both leaves and roots. There was a marked reduction in the amount of N incorporated into the protein fraction from the newly absorbed N (NANP) in water-deficit stressed plants, particularly in leaf tissue. This reduction in NANP was strongly associated with an increased concentration of NH3–NH4+ in roots (P=0.05) and proline (P=0.01) in leaves and roots. These results suggest that proline accumulation may be a sensitive biochemical indicator of plant water status and of the dynamics of de novo protein synthesis in response to stress severity.