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 (Psiw) 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 Psiw was -2.27 MPa or less, and was concomitant with the increase of lipid peroxidation and lignin content. As Psiw 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.

Splicing of plant organellar transcripts is facilitated by members of a large protein family, the pentatricopeptide repeat proteins. We have identified a pentatricopeptide repeat protein in a genetic screen for mutants resistant to inhibition of root growth by buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis and consequently named BIR6 (BSO-insensitive roots 6). BIR6 is involved in splicing of intron 1 of the mitochondrial nad7 transcript. Loss-of-function mutations in BIR6 result in a strongly reduced accumulation of fully processed nad7 transcript. This affects assembly of Complex I and results in moderate growth retardation. In agreement with disruption of Complex I function, the genes encoding alternative NADH oxidizing enzymes are induced in the mutant, and the mutant plants are less sensitive to mannitol and salt stress. Mutation in the BIR6 gene allowed normal root growth in presence of BSO and strongly attenuated depletion of glutathione content at these conditions. The same phenotype was observed with other mutants affected in function of Complex I, thus reinforcing the importance of Complex I function for cellular redox homeostasis.

Glucosinolates are a major class of sulphur-containing secondary metabolites involved in plant defence against pathogens. Recently many regulatory links between glucosinolate biosynthesis and sulphate assimilation were established. Since sulphate assimilation undergoes diurnal rhythm and is light regulated, this study analysed whether the same is true for glucosinolate biosynthesis. The levels of glucosinolates and glutathione were found to be higher during the day than during the night. This agreed with variation in sulphate uptake as well as activity of the key enzyme of the sulphate assimilation pathway, adenosine 5’-phosphosulphate reductase. Correspondingly, the flux through sulphate assimilation was higher during the day than during the night, with the maximum flux through primary assimilation preceding maximal incorporation into glucosinolates. Prolonged darkness resulted in a strong reduction in glucosinolate content. Re-illumination of such dark-adapted plants induced accumulation of mRNA for many genes of glucosinolate biosynthesis, leading to increased glucosinolate biosynthesis. The light regulation of the glucosinolate synthesis genes as well as many genes of primary sulphate assimilation was controlled at least partly by the LONG HYPOCOTYL5 (HY5) transcription regulator. Thus, glucosinolate biosynthesis is highly co-regulated with sulphate assimilation.