Cloning and characterization of the drought tolerance function of the autophagy-related gene PbATG5 in pear

【Objective】Drought is a major constraint on pear (Pyrus) production worldwide, and plants have evolved multiple adaptations to withstand water deficits. Autophagy, a conserved intracellular degradation and recycling pathway, plays a key role in plant responses to abiotic stress. However, the specific components underlying drought tolerance in pear have not yet been completely characterized. This study aimed to clarify the function of the key autophagy- related gene PbATG5 in regulating pear drought resistance, as well as the underlying mechanism, to aid genetic engineering of drought-resistant pear cultivars.【Methods】The PbATG5 was isolated from Pyrus betulifolia leaves and subjected to comprehensive bioinformatic analysis. Quantitative real-time PCR (qRT-PCR) was used to analyze the expression patterns of the PbATG5 under drought stress. The transgenic Arabidopsis thaliana plants overexpressing the PbATG5 were obtained via Agrobacterium-mediated transformation. Virus-induced gene silencing (VIGS) was used to generate the PbATG5-silenced pear plants. The transgenic Arabidopsis thaliana and the PbATG5-silenced pear plants were then subjected to drought treatment for two weeks, and the phenotypic performance was quantified via measurements of drought- relevant physio- logical and biochemical indices, including relative water content (RWC), total chlorophyll content, electrolyte leakage, malondialdehyde (MDA) content, antioxidant capacity, and autophagic activity, to clarify the function of the PbATG5 in regulating plant drought resistance.【Results】The PbATG5 was cloned from the leaves of P. betulifolia. The full-length open reading frame of the PbATG5 was 1110 bp, encoding 370 amino acids. The molecular weight of PbATG5 was 41.37 ku, and the isoelectric point was 4.83. PbATG5 contained the conserved APG5 domain. The multiple sequence alignment revealed that PbATG5 had high homology with ATG5 proteins from other plant species. The phylogenetic analysis demonstrated that pear ATG5 was closely related to homologous proteins in other Rosaceae species, including apple (Malus domestica), peach (Prunus persica), and strawberry (Fragaria vesca). The subcellular localization analysis showed that PbATG5 was distributed in the cell membrane and nucleus. The promoter prediction analysis demonstrated that the promoter region of the PbATG5 contained many cis- elements, which were related to plant hormones and abiotic stress. Several of these cis- elements were involved in drought stress responses, including ABRE (abscisic acid responsiveness element), DRE core (Dehydration- responsive element), MBS (MYB binding site involved in drought-inducibility), and MYC sites (MYC involved in drought-inducibility). Furthermore, the expression patterns of the PbATG5 in response to dehydration were characterized. After 3 h of dehydration treatment, the expression level of the PbATG5 was significantly up-regulated (7.0-fold relative to 0 h), which further indicated that the PbATG5 was involved in the physiological and biochemical processes of pear in response to drought stress. To further confirm the role of the PbATG5 in the drought response, the transgenic Arabidopsis thaliana overexpressing PbATG5 and PbATG5-silenced pear plants were generated and exposed to drought stress. After two weeks of drought treatment, the leaves of the transgenic Arabidopsis thaliana overexpressing PbATG5 exhibited less severe symptoms of dehydration than the leaves of the wildtype (WT) plants. The electrolyte leakage and the MDA content significantly increased in the two transgenic lines and WT plants under drought; however, increases in these two traits were less pronounced in transgenic lines compared with the WT plants. The RWC and total chlorophyll content were higher in the transgenic lines than in the WT plants. Drought stress could result in the excessive accumulation of reactive oxygen species (ROS), leading to oxidative damage to multiple cell components. Under drought stress, the ROS accumulation was reduced in the PbATG5-overexpressing lines compared with the WT plants, and the superoxide dismutase (SOD) activity was higher in the transgenic lines. Consistent with this, the drought tolerance of the PbATG5-silenced pear plants was significantly weaker than that of the control plants. Under drought stress, the RWC was lower in the leaves of the PbATG5-silenced pear plants than in the leaves of the control plants. After the drought treatment, increases in electrolyte leakage and the MDA content were greater in the PbATG5-silenced pear plants compared with the control plants. Compared with the control plants, the PbATG5-silenced pear plants showed greater ROS accumulation and reduced antioxidant enzyme activities under the drought conditions. The expression levels of the core autophagy-related genes were lower in the PbATG5-silenced pear plants than in the control plants. The transmission electron microscopy analysis was performed to further characterize autophagic activity. Under the drought stress, a large number of autophagic structures were present in the leaves of the control plants, and the number of autophagic structures was approximately 4.9 times higher in the control plants than those in the PbATG5-silenced pear plants. These findings indicated that the silencing of the PbATG5 could inhibit the activation of autophagy in pear under drought stress. 【Conclusion】The PbATG5 was cloned from the leaves of P. betulifolia and identified as a drought-responsive autophagy component that would enhance plant performance under water deficit. The promot-er region of the PbATG5 would harbor diverse drought- responsive cis- elements, and its expression could be induced by dehydration treatment. Under the drought stress, the PbATG5 mediated the activation of autophagy and could alleviate oxidative damage caused by drought stress to positively regulate the drought tolerance of the pear plants. Overall, this work would provide a theoretical and practical foundation for molecular breeding strategies to exploit PbATG5- mediated autophagy and generate drought-resistant pear plants.