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Identification of autophagy-related genes ATG4 and ATG8 from wheat (Triticum aestivum L.) and profiling of their expression patterns responding to biotic and abiotic stresses

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Abstract

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The genes coding for wheat ATG4 and ATG8 were cloned and their roles in autophagy were verified. Implications of ATG4/ATG8 in wheat responses to stresses were suggested by expression profiling.

Abstract

Autophagy-related proteins ATG4 and ATG8 are crucial for autophagy biogenesis. ATG4 processes ATG8 precursor to expose its C-terminal glycine for phosphatidyl ethanolamine (PE) lipidation. ATG8, in the form of ATG8-PE adduct, functions in the organization dynamics of autophagic membranes. Here, we report the identification of two/nine members of the ATG4/ATG8 family from common wheat (Triticum aestivum L.). Expression of each wheat ATG4/ATG8 could complement the autophagy activity of yeast atg4/atg8 mutant cells. GFP fusion proteins of ATG8s, especially of ATG8s with innate C-terminal-exposed glycines, localized to punctate autophagic membranes. Both of purified ATG4s could cleave ATG8s in vitro, but they had different activities and different preferences for ATG8 substrates. Two times of transcript accumulation, an early one and a late one, of ATG4s/ATG8s were detected in the early phases of the Pm21- and Pm3f-triggered wheat incompatible reactions to the powdery mildew causal fungus Blumeria graminis f. sp. tritici (Bgt), and fluorescence microscopy also revealed a Bgt-induced enhanced wheat autophagy level in the Pm21-triggered incompatible reaction. Only one time of Bgt-induced transcript accumulation of ATG4s/ATG8s, corresponding to but much higher than the late one in incompatible reactions, was detected in a susceptible line isogenic to the Pm21 resistance line. These results suggested positive roles of ATG4/ATG8-associated autophagy process in the early stage and possible negative roles in the late stage of wheat immunity response to Bgt. In addition, expression of wheat ATG4s/ATG8s was also found to be upregulated by abiotic stress factors and distinctively regulated by different phytohormones.

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Abbreviations

ATG:

Autophagy-related gene

PE:

Phosphatidyl ethanolamine

EST:

Expressed sequence tag

ORF:

Open reading frame

GFP:

Green fluorescent protein

ET:

Ethylene

SA:

Salicylic acid

MeJA:

Methyl jasmonate

ABA:

Abscisic acid

PEG:

Polyethylene glycol

qRT-PCR:

Quantitative real-time PCR

TA:

Transcript accumulation

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Acknowledgments

This work was supported by the Natural Science Foundation of Tianjin, China (Grant number 12JCZDJC23000); the Fok Ying-Tong Education Foundation for Young Teachers in the Higher Education Institutions of China (Grant number 131026); and the Open Fund of Tianjin Key Laboratory of Animal and Plant Resistance, Tianjin Normal University (Grant number 52LX12).

Conflict of interest

The authors do not have any conflict of interest.

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Authors and Affiliations

  1. School of Life Sciences, Tianjin Normal University, Tianjin Key Laboratory of Animal and Plant Resistance, Tianjin, 300387, China

    Dan Pei, Wei Zhang, Hong Sun, Xiaojing Wei, Jieyu Yue & Huazhong Wang

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  1. Dan Pei
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  2. Wei Zhang
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  3. Hong Sun
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  4. Xiaojing Wei
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  5. Jieyu Yue
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  6. Huazhong Wang
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Correspondence to Huazhong Wang.

Additional information

Communicated by Jeong Sheop Shin.

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Table S1. List of primers used in this paper.

Figure S1. Multiple alignments of wheat ATG8s and homologues from other species. The alignment was produced in ClustalX 2.1 software. Sequences in the alignment include the nine wheat ATG8s here identified, AER27507 (TdATG8) from Triticum dicoccoides, XP_003581707 (BdATG8a) and XP_003571383 (BdATG8b) from Brachypodium distachyon, Os07g0512200 (OsATG8a), Os04g0624000 (OsATG8b), Os08g0191600 (OsATG8c) and Os02g0529150 (OsATG8d) from Oryza sativa, ACJ73920 (ZmATG8a), ACJ73921 (ZmATG8b), ACJ73922 (ZmATG8c), ACJ73923 (ZmATG8d) and ACJ73925 (ZmATG8e) from Zea mays, At4g21980 (AtATG8a), At4g04620 (AtATG8b), At1g62040 (AtATG8c), At2g05630 (AtATG8d), At2G45170 (AtATG8e), At4g16520 (AtATG8f), At3g60640 (AtATG8 g), At3g06420 (AtATG8h) and At3g15580 (AtATG8i) from Arabidopsis thaliana, ACU13796 (GmATG8a), ACU14633 (GmATG8b), ACU17086 (GmATG8c), BAH22449 (GmATG8d), ACU15101 (GmATG8e), ACU19559 (GmATG8f), ACU13862 (GmATG8g), ACU16419 (GmATG8h), BAH22448 (GmATG8i) and ACU19611 (GmATG8k) from Glycine max, NP_009216 (HsATG8/GATE16/GABARAPL2) from Human and NP_009475 (ScATG8) from Yeast. Names of wheat ATG8s are boxed. One hundred percent similarity, black; 80–100 % similarity, dark gray; 60–80 % similarity, light gray; <60 % similarity, white. Hashes and asterisks, respectively, indicate conserved essential residues at the N-terminal microtubule binding site and at the ATG7 binding site. The C-terminal conservative glycine for PE-conjugation is indicated by an arrowhead.

Figure S2. Multiple alignments of wheat ATG4s and homologues from other species. The alignment was produced in ClustalX 2.1 software. Sequences in the alignment include the two wheat ATG4s here identified, Os03g0391000 (OsATG4a) and Os04g0682000 (OsATG4b) from Oryza sativa, ACJ73912 (ZmATG4a) and ACJ73914 (ZmATG4b) from Zea mays, At2g44140 (AtATG4a) and At3g59950 (AtATG4b) from Arabidopsis thaliana, NP_443168 (HsATG4a) from Human and NP_014176 (ScATG4) from Yeast. One hundred percent similarity, black; 80 to 100 % similarity, dark gray; 60 to 80 % similarity, light gray; <60 % similarity, white. The conserved Peptidase_C54 domain is under thick line;Arrowheads mask the canonical catalytic triad of cysteine protease: Cys, Asp and His.

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Pei, D., Zhang, W., Sun, H. et al. Identification of autophagy-related genes ATG4 and ATG8 from wheat (Triticum aestivum L.) and profiling of their expression patterns responding to biotic and abiotic stresses. Plant Cell Rep 33, 1697–1710 (2014). https://doi.org/10.1007/s00299-014-1648-x

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