Dehydrin ERD14 activates glutathione transferase Phi9 in Arabidopsis thaliana under osmotic stress.

BACKGROUND: Fully intrinsically disordered plant dehydrin ERD14 can protect enzymes via its chaperone-like activity, but it was not formally linked with enzymes of the plant redox system yet. This is of particular interest, as the level of H2O2 in Arabidopsis plants increases during osmotic stress, which can be counteracted by overexpression of ERD14. METHODS: The proteomic mass-spectrometry analysis of stressed plants was performed to find the candidates affected by ERD14. With cross-linking, microscale thermophoresis, and active-site titration kinetics, the interaction and influence of ERD14 on the function of two target proteins: glutathione transferase Phi9 and catalase was examined. RESULTS: Under osmotic stress, redox enzymes, specifically the glutathione transferase Phi enzymes, are upregulated. Using microscale thermophoresis, we showed that ERD14 directly interacts with GSTF9 with a KD of ~25 µM. ERD14 activates the inactive GSTF9 molecules, protects GSTF9 from oxidation, and can also increases the activity of the enzyme. Aside from GSTF9, we found that ERD14 can also interact with catalase, an important cellular H2O2 scavenging enzyme, with a KD of ~0.13 µM, and protects it from dehydration-induced loss of activity. CONCLUSIONS: We propose that fully intrinsically disordered dehydrin ERD14 might protect and even activate redox enzymes, helping plants to survive oxidative stress under dehydration conditions. GENERAL SIGNIFICANCE: ERD14 has a direct effect on the activity of redox enzymes.

Medicago truncatula dihydrodipicolinate synthase (DHDPS) enzymes display novel regulatory properties.

Lysine biosynthesis in plants is tightly regulated by feedback inhibition of the end product on the first enzyme of the lysine-specific branch, dihydrodipicolinate synthase (DHDPS). Three complete DHDPS coding sequences and one partial sequence were obtained in Medicago truncatula via inverse PCR. Analysis of the MtDHDPS sequences indicated the presence of isozymes (MtDHDPS2 and MtDHDPS3) with multiple amino acid substitutions on positions previously shown to be involved in feedback inhibition and of residues important for catalytic activity, possibly affecting the enzymatic properties of these isoforms. Sequences similar to MtDHDPS2 and 3 are present in Lotus japonicus and Glycine max, suggesting the existence of a specific conserved class of DHDPS genes within the Fabaceae family. The MtDHDPS genes were found by quantitative RT-PCR analysis to be expressed in an organ-specific manner in M. truncatula. All four MtDHDPS enzymes were expressed separately in Escherichia coli, revealing a strongly reduced sensitivity of the MtDHDPS2 protein to lysine feedback inhibition and a severely reduced activity of the MtDHDPS3 protein. Remarkably, MtDHDPS3 expression in Arabidopsis thaliana produced transgenic plants with a significantly increased threonine level, suggesting a dominant DHDPS inhibiting role of this isoform. This is supported by co-expression experiments in E. coli which indicate that AtDHDPS and MtDHDPS3 interact and may form hetero-oligomers with strongly reduced enzymatic activity. In conclusion, analysis of DHDPS in M. truncatula revealed the presence of unique isozymes displaying novel regulatory properties.