Identification And Functional Characterization Of Autophagy-related Zene ATG6 In Barley

Autophagy is a highly conserved degradation process among eukaryote cells in which an indispensable autophagy-related protein ATG6 could form the PI3K complex I, and then regulate autophagy and determine the autophagosome development. In Arabidopsis thaliana, Nicotiana benthamiana, Oryza sativa and Triticum aestivum, ATG6 has been reported to participate in autophagy process and function in response to various stress conditions, pathogen infection or other physiological processes such as pollen germination. Therefore, the study of ATG6 can help us to elucidate the molecular mechanism of autophagy and apply to regulate plant growth and development under stress conditions.Here, an ATG6 ortholog in Hordeum vulgare （HvATG6） had been identified. The conserved function of HvATG6 was investigated using sequence analysis, subcellular localization, yeast functional complementation, expression pattern analysis and knockdown experiment. The results are as follows:1 Screening and sequence analysis of ATG6 in barleyThe collections of ATG6 counterparts in Arabidopsis and rice were used as queries for the search in barley database and then one barley loci encoding the ATG6 protein tag had been found, which was designated as HvATG6 （Gene ID in Ensemble plants: MLOC₇1271）. HvATG6 shows a high sequence similarity to ATG6s in other plant species, especially in wheat and rice. Interestingly, it also has the corresponding autophagy domain structure of ATG6 homologs in which the repeated P-sheet-a-helix segments are included in, implying that the protein may play a role in autophagy.2 Morphological observation and functional analysis of HvATG62.1 Subcelluar localization of HvATG6 in barley protoplastsN-terminal GFP fusion constructs of barley ATG6 was transformed to barley protoplasts for the observation of subcelluar localization. Punctate florescence signals, considered as the PAS （Pre-autophagosomal structure） for autophagosome initiation, were observed only in the cytoplasm of cells.2.2 HvATG6 could rescue autophagic defect in yeast atg6 mutantThe expression of HvATG6 protein caused the reappearance of autophagosomes in yeast atg6 mutant in the absence of nitrogen source that could lead to an abundant autophagosomes in wild type yeast cells, which means that HvATG6 could replace the ATG6 protein and its function in yeast autophagy somehow.3 Expression patterns of HvATG6 under various abiotic stress conditionsKinds of treatment like nitrogen deficiency, high salinity, drought and aluminum toxicity were proved to result in an apparent upregulation of HvATG6 transcripts. Remarkably, in the GP seedlings, which is considered to be sensitive to aluminum stress, the expression of HvATG6 in root increased dramatically under 10μM or 30 μM Al3+ treatment for 72 h, which were over 4 times as that in the control. This correlates with the functional characteristics of autophagy in response to the stress conditions.4 Silencing of HvATG6 could make the seedlings more susceptible to stress conditionsHvATG6 konckdown plants were generated using BSMV-induced gene silencing. And the HvATG6 knockdown detached leaves could apparently exhibited an accelerated senescence under dark stress, and the same phenomenon was observed when the detached leaves were treated in 40 mM H2O2. These results show that silencing of HvATG6 could make the seedlings more susceptible to stress. This also correlates with the seedlings phenotype change resulted by autophagy defects in response to the stress conditions,In brief, kinds of evidence were provided that HvATG6 functions in autophagy and is implicated with abiotic stress. Further investigation of ATG6 and its interacting proteins will enable us to better understand the biological function of HvATG6 in autophagy and stress response.