The Landscape of Autophagy-Related (ATG) Genes and Functional Characterization of TaVAMP727 to Autophagy in Wheat

Autophagy is an indispensable biological process and plays crucial roles in plant growth and plant responses to both biotic and abiotic stresses. This study systematically identified autophagy-related proteins (ATGs) in wheat and its diploid and tetraploid progenitors and investigated their genomic organization, structure characteristics, expression patterns, genetic variation, and regulation network. We identified a total of 77, 51, 29, and 30 ATGs in wheat, wild emmer, T. urartu and A. tauschii, respectively, and grouped them into 19 subfamilies. We found that these autophagy-related genes (ATGs) suffered various degrees of selection during the wheat’s domestication and breeding processes. The genetic variations in the promoter region of Ta2A_ATG8a were associated with differences in seed size, which might be artificially selected for during the domestication process of tetraploid wheat. Overexpression of TaVAMP727 improved the cold, drought, and salt stresses resistance of the transgenic Arabidopsis and wheat. It also promoted wheat heading by regulating the expression of most ATGs. Our findings demonstrate how ATGs regulate wheat plant development and improve abiotic stress resistance. The results presented here provide the basis for wheat breeding programs for selecting varieties of higher yield which are capable of growing in colder, drier, and saltier areas.

Exploration of Autophagy Families in Legumes and Dissection of the ATG18 Family with a Special Focus on Phaseolus vulgaris

Macroautophagy/autophagy is a fundamental catabolic pathway that maintains cellular homeostasis in eukaryotic cells by forming double-membrane-bound vesicles named autophagosomes. The autophagy family genes remain largely unexplored except in some model organisms. Legumes are a large family of economically important crops, and knowledge of their important cellular processes is essential. Here, to first address the knowledge gaps, we identified 17 ATG families in Phaseolus vulgaris, Medicago truncatula and Glycine max based on Arabidopsis sequences and elucidated their phylogenetic relationships. Second, we dissected ATG18 in subfamilies from early plant lineages, chlorophytes to higher plants, legumes, which included a total of 27 photosynthetic organisms. Third, we focused on the ATG18 family in P. vulgaris to understand the protein structure and developed a 3D model for PvATG18b. Our results identified ATG homologs in the chosen legumes and differential expression data revealed the nitrate-responsive nature of ATG genes. A multidimensional scaling analysis of 280 protein sequences from 27 photosynthetic organisms classified ATG18 homologs into three subfamilies that were not based on the BCAS3 domain alone. The domain structure, protein motifs (FRRG) and the stable folding conformation structure of PvATG18b revealing the possible lipid-binding sites and transmembrane helices led us to propose PvATG18b as the functional homolog of AtATG18b. The findings of this study contribute to an in-depth understanding of the autophagy process in legumes and improve our knowledge of ATG18 subfamilies.

Genome-wide analysis of autophagy-related genes in Medicago truncatula highlights their roles in seed development and response to drought stress

Autophagy is a highly conserved process of degradation of cytoplasmic constituents in eukaryotes. It is involved in the growth and development of plants, as well as in biotic and abiotic stress response. Although autophagy-related (ATG) genes have been identified and characterized in many plant species, little is known about this process in Medicago truncatula. In this study, 39 ATGs were identified, and their gene structures and conserved domains were systematically characterized in M. truncatula. Many cis-elements, related to hormone and stress responsiveness, were identified in the promoters of MtATGs. Phylogenetic and interaction network analyses suggested that the function of MtATGs is evolutionarily conserved in Arabidopsis and M. truncatula. The expression of MtATGs, at varied levels, was detected in all examined tissues. In addition, most of the MtATGs were highly induced during seed development and drought stress, which indicates that autophagy plays an important role in seed development and responses to drought stress in M. truncatula. In conclusion, this study gives a comprehensive overview of MtATGs and provides important clues for further functional analysis of autophagy in M. truncatula.

Key Regulators of Autophagosome Closure

Autophagy is an evolutionarily conserved pathway, in which cytoplasmic components are sequestered within double-membrane vesicles called autophagosomes and then transported into lysosomes or vacuoles for degradation. Over 40 conserved autophagy-related (ATG) genes define the core machinery for the five processes of autophagy: initiation, nucleation, elongation, closure, and fusion. In this review, we focus on one of the least well-characterized events in autophagy, namely the closure of the isolation membrane/phagophore to form the sealed autophagosome. This process is tightly regulated by ESCRT machinery, ATG proteins, Rab GTPase and Rab-related proteins, SNAREs, sphingomyelin, and calcium. We summarize recent progress in the regulation of autophagosome closure and discuss the key questions remaining to be addressed.

The evolution of autophagy proteins – diversification in eukaryotes and potential ancestors in prokaryotes

ABSTRACT Autophagy is a degradative pathway for cytoplasmic constituents, and is conserved across eukaryotes. Autophagy-related (ATG) genes have undergone extensive multiplications and losses in different eukaryotic lineages, resulting in functional diversification and specialization. Notably, even though bacteria and archaea do not possess an autophagy pathway, they do harbor some remote homologs of Atg proteins, suggesting that preexisting proteins were recruited when the autophagy pathway developed during eukaryogenesis. In this Review, we summarize our current knowledge on the distribution of Atg proteins within eukaryotes and outline the major multiplication and loss events within the eukaryotic tree. We also discuss the potential prokaryotic homologs of Atg proteins identified to date, emphasizing the evolutionary relationships and functional differences between prokaryotic and eukaryotic proteins.

Autophagy gene-dependent intracellular immunity triggered by interferon-γ

Genes required for the lysosomal degradation pathway of autophagy play key roles in topologically distinct cellular processes with significant physiologic importance. One of the first-described of these ATG gene-dependent processes is the requirement for a subset of ATG genes in interferon-γ (IFNγ)-induced inhibition of Norovirus and Toxoplasma gondii replication. Herein we identified new genes that are required for or that negatively regulate this immune mechanism. Enzymes involved in the conjugation of UFM1 to target proteins including UFC1 and UBA5, negatively regulated IFNγ-induced inhibition of norovirus replication via effects of Ern1. IFNγ-induced inhibition of norovirus replication required Wipi2b and Atg9a, but not Becn1 (encoding Beclin1), Atg14, or Sqstm1. The phosphatidylinositol-3-phosphate and ATG16L1 binding domains of WIPI2B were required for IFNγ-induced inhibition of norovirus replication. Both WIPI2 and SQSTM1 were required for IFN?-induced inhibition of Toxoplasma gondii replication in HeLa cells. These studies further delineate the mechanisms of a programmable form of cytokine-induced intracellular immunity that relies on an expanding cassette of essential ATG genes to restrict the growth of phylogenetically diverse pathogens.

Genome-wide identification and expression analysis of autophagy genes in cucumber

Background Autophagy is an evolutionarily conserved physiological and developmental process in eukaryotes. In this process, damaged proteins in cells are degraded and cytoplasmic materials recycled. When plants are exposure to stress conditions or their growth and development are blocked, autophagy is induced to maintain the cell homeostasis by degrading long-lived proteins in the cells and organelles that function abnormally due to aging or damage. Cell autophagy has multiple functions in plants, it involved in growth and development, senescence, and responses to biotic and abiotic stress. So far, thirty three autophagy genes (ATG) have been found in rice, and more than 30 autophagy-related genes have been found in Arabidopsis, tobacco and corn, respectively. Four autophagy genes induced by salicylic acid were found in cucumber, but a little still unknown about all of autophagy genes in cucumber genome. Our experiment fully explored the ATG gene family of cucumber genome based on bioinformatics methods and identified 20 CsATG genes. We systematically analyzed the structure, conserved motifs, expression and phylogeny relationship of these ATG genes, which lays the foundation for exploring the function of the genes. Results A total of 20 putative ATG genes were identified in the cucumber genome. Gene duplication analysis showed that both fragmented and tandem duplication played vital roles in the amplification of cucumber ATG gene family. Gene expression analysis showed that 16 CsATG genes were induced by the salicylic acid (SA) treatment, and 16 CsATG genes were down-regulated by Methyl jasmonate (MeJA) treatment. Under high salinity stress, 10 CsATG genes were induced in roots. Under drought stress, 16 CsATG genes were induced in roots. Under carbon starvation stress, all of 20 CsATG genes were induced to express in leaves, suggesting that cell autophagy has a potential role in nutritional starvation tolerance. Conclusion Our results clearly have deepened our understanding of the characteristics and functions of cucumber ATG gene, and also found some new gene resources that can be used for the future development of cucumber and other crop varieties, which can resist stress.

ACSL3 is a novel GABARAPL2 interactor that links ufmylation and lipid droplet biogenesis

ABSTRACTWhile studies of the autophagy-related (ATG) genes in knockout models have led to an explosion of knowledge about the functions of autophagy components, the exact roles of LC3 and GABARAP family proteins (human ATG8 equivalents) are still poorly understood. A major drawback in understanding their roles is that the available interactome data has largely been acquired using overexpression systems. To overcome these limitations, we employed CRISPR/Cas9-based genome-editing to generate a panel of cells in which human ATG8 genes were tagged at their natural chromosomal locations with an N-terminal affinity epitope. This cellular resource was employed to map endogenous GABARAPL2 protein complexes using interaction proteomics. This approach identified the ER-associated protein and lipid droplet (LD) biogenesis factor ACSL3 as a stabilizing GABARAPL2-binding partner. GABARAPL2 bound ACSL3 in a manner dependent on its LC3-interacting regions, whose binding site in GABARAPL2 was required to recruit the latter to the ER. Through this interaction, the UFM1-activating enzyme UBA5 became anchored at the ER. Furthermore, ACSL3 depletion and LD induction affected the abundance of several ufmylation components and ER-phagy. Together these data allow us to define ACSL3 as a novel regulator of the enigmatic UFM1 conjugation pathway.

Overexpression of MdATG18a enhances alkaline tolerance and GABA shunt in apple through increased autophagy under alkaline conditions

Soil alkalization affects apple production in northwest China. Autophagy is a highly conserved degradative protein pathway in eukaryotes. Autophagy in plants can be activated by various abiotic factors. We previously identified the positive role of the autophagy-related gene MdATG18a in drought, nitrogen deficiency and resistance to Diplocarpon mali infection in apple. However, it is still unclear whether ATG18a is related to alkaline stress. In this study, we used hydroponic culture to simulate alkaline stress and found that the overexpression of MdATG18a significantly improved the tolerance of apple to alkaline stress. The overexpression of MdATG18a increased biomass, photosynthetic rate and antioxidant capacity of transgenic plants compared with wild-type plants under alkaline stress. The overexpression of MdATG18a promoted γ-aminobutyric acid (GABA) shunt via an increase in glutamate (GABA precursor) and GABA contents and upregulation of GABA shunt-related genes. In addition, the overexpression of MdATG18a significantly upregulated the expression of other core ATG genes and increased the formation of autophagosomes under alkaline stress. In conclusion, these results suggest that the overexpression of MdATG18a in apple enhances alkaline tolerance and the GABA shunt, which may be owing to the increase in autophagic activity.