Water is a crucial factor for plant growth. Drought stress can severely affect the normal growth and development of plants, leading to a decrease in water use efficiency. To adapt to arid environments, plants have evolved various mechanisms to reduce water loss and increase water absorption. Among these, abscisic acid (ABA) plays a central role in the plant’s response to drought stress. ABA accumulates in plant tissues, especially in the vascular bundles of leaves, and regulates stomatal closure and the expression of stress-responsive genes. The ABA signaling pathway involves three core components: PYR/PYL/RCAR receptors, PP2C phosphatases, and SnRK2 kinases, which jointly regulate the activity of downstream targets.
Plant transcription factors play a key role in responding to stress and hormone signals, and their activity is often regulated by post-translational modifications (PTMs). Ubiquitination is one of the most common PTMs. Ubiquitination marks target proteins for degradation by the 26S proteasome by attaching ubiquitin to the lysine residues of the target proteins, thus regulating protein stability. E3 ubiquitin ligases play a crucial role in this process. By interacting with substrate proteins, they determine the specificity of the ubiquitination process. Many E3 ligases have been found to interact with transcription factors related to ABA signaling and drought response, regulating their stability and thus having a positive or negative impact on ABA signaling and drought response. However, the mechanism by which drought stress affects the stability of the CaGRAS1 protein remains unclear.
A research paper titled “The Pepper E3 Ligase CaGIR1 Acts as a Negative Regulator of Drought Response via Controlling CaGRAS1 Stability” was published online in the journal Plant, Cell & Environment. This paper reveals how the pepper E3 ubiquitin ligase CaGIR1 affects the pepper’s response to drought stress by regulating the stability of the CaGRAS1 protein.
In the study, through yeast two-hybrid experiments, CaGIR1 that interacts with CaGRAS1 was screened from the pepper RING protein library. Using experiments such as co-immunoprecipitation (Co-IP), bimolecular fluorescence complementation (BiFC), and split luciferase complementation (SLC), the interaction between CaGIR1 and CaGRAS1 both in vitro and in vivo was verified, and this interaction mainly occurs in the nucleus and cytoplasm. Further in vitro and in vivo ubiquitination experiments showed that CaGIR1 has E3 ubiquitin ligase activity and can directly ubiquitinate CaGRAS1, leading to its degradation through the 26S proteasome pathway. This degradation is accelerated under drought stress, and the 26S proteasome inhibitor MG132 can partially inhibit this degradation.
In addition, by using the virus-induced gene silencing (VIGS) technique to silence the CaGIR1 gene in pepper, it was found that silencing CaGIR1 can enhance the drought tolerance of pepper, manifested as higher survival rate, lower water transpiration rate, and smaller stomatal aperture. Conversely, overexpressing the CaGIR1 gene in Arabidopsis reduces the plant’s drought tolerance, shown as lower survival rate, higher water transpiration rate, and larger stomatal aperture. Moreover, ABA sensitivity experiments indicated that CaGIR1 negatively regulates the ABA signaling pathway, and the analysis of the expression of drought-responsive genes further revealed that CaGIR1 affects the expression of the ABA signaling pathway and drought-responsive genes by regulating the stability of the CaGRAS1 protein.