Kunitz Proteinase Inhibitors Limit Water Stress Responses in White Clover (Trifolium repens L.) Plants

Abstract

The response of plants to water deficiency or drought is a complex process, the perception of which is triggered at the molecular level before any visible morphological responses are detected. It was found that different groups of plant proteinase inhibitors (PIs) are induced and play an active role during abiotic stress conditions such as drought. Our previous work with the white clover (Trifolium repens L.) Kunitz Proteinase Inhibitor (Tr-KPI) gene family showed that Tr-KPIs are differentially regulated to ontogenetic and biotic stress associated cues and that, at least some members of this gene family may be required to maintain cellular homeostasis. Altered cellular homeostasis may also affect abiotic stress responses and therefore, we aimed to understand if distinct Tr-PKI members function during drought stress. First, the expression level of three Tr-KPI genes, Tr-KPI1, Tr-KPI2, and Tr-KPI5, was measured in two cultivars and one white clover ecotype with differing capacity to tolerate drought. The expression of Tr-KPI1 and Tr-KPI5 increased in response to water deficiency and this was exaggerated when the plants were treated with a previous period of water deficiency. In contrast, proline accumulation and increased expression of Tr-NCED1, a gene encoding a protein involved in ABA biosynthesis, was delayed in plants that experienced a previous drought period. RNAi knock-down of Tr-KPI1 and Tr-KPI5 resulted in increased proline accumulation in leaf tissue of plants grown under both well-watered and water-deficit conditions. In addition, increased expression of genes involved in ethylene biosynthesis was found. The data suggests that Tr-KPIs, particularly Tr-KPI5, have an explicit function during water limitation. The results also imply that the Tr-KPI family has different in planta proteinase targets and that the functions of this protein family are not solely restricted to one of storage proteins or in response to biotic stress.

The roles of plant proteases and protease inhibitors in drought response: a review

“…The authors, however, concluded that HvPap1 and HvPap19 are drought-inducible genes and may cause differential effects on barley plants subjected to drought (Gomez-sanchez et al, 2019). Islam et al (2017) investigated the functions of Kunitz proteinase inhibitors (KPIs) in white clover (Trifolium repens L.) plants subjected to water deficits using several knockdown lines of different Tr-KPIs. The study utilised two distinct water withholding regimes, a Non-PreStress treatment with direct exposure of plants to water deficits and a PreStress treatment in which plants were initially exposed to drought stress and rehydration, followed by a final drought stress treatment.…”

“…For example, Gomez-sanchez et al (2019) suggested that the observed unexpected increase in cuticle thickness of KDPap1, an HvPap1 knockdown mutant line, could have been due to pleiotropic effects associated with the knocked-down cysteine protease gene. In addition, knockdown mutants exhibit some residual mRNA expression or protein accumulation of the knocked-down gene to varying extents (Islam et al, 2017;Gomezsanchez et al, 2019;Mishra et al, 2021), hence the knockdown character (Gomez-sanchez et al, 2019). For example, while Mishra et al (2021) reported a 99% reduction in the transcript expression of the target AtFtsHi3 gene in the generated knockdown mutant, ftshi3-I (kd), relative to the wild-type, other studies reported much lower reduction rates of the target genes (Islam et al, 2017).…”

Physiological and Molecular Responses of ‘Dusa’ Avocado Rootstock to Water Stress: Insights for Drought Adaptation

“…Cluster 1 was the largest group with 427 DEGs induced under both water shortage treatments. The most representative biological processes identified with GO term enriched analysis (p < 0.05) in this cluster comprised, among others, "response to water deprivation" (GO0009414), "negative regulation of endopeptidase activity" (GO0010951), "glutathione metabolic process" (GO0006749), "response to wounding" (GO0009611), "sodium ion transport" (GO0006814), and "serine-type endopeptidase inhibitor activity" (GO0004867), which have been extensively associated with both biotic and abiotic stress [59,60]. In particular, serine protease inhibitors play an important role in cell survival, development, and host defense, i.e., transgenic Arabidopsis thaliana plants overexpressing serine protease inhibitors showed higher RWC, reduced lipid peroxidation, and enhanced activity of antioxidant glutathione-S-transferase [61].…”

Crystal structure of a novel Kunitz type inhibitor, alocasin with anti‐Aedes aegypti activity targeting midgut proteases

“…The KNIs belong to a β‐trefoil fold superfamily with high loop plasticity and an approximate molecular mass of 21 kDa. The β‐trefoil fold also has two disulfide bridges to inhibit a wide range of proteinases . This suggests that KNIs could act as proteinase regulators in plant germination and in a defense response during insect attack .…”

“…The -trefoil fold also has two disulfide bridges to inhibit a wide range of proteinases. 22 This suggests that KNIs could act as proteinase regulators in plant germination and in a defense response during insect attack. [23][24][25][26] Recent evidence also suggests that KNIs are involved in programmed cell death, growth, and development in plants.…”