Genome-Wide Identification of ATG Gene Family Members in Fagopyrum tataricum and Their Expression during Stress Responses

Abstract

Abiotic stresses such as drought and salinity are major environmental factors limiting plant productivity. Autophagy-related genes are extensively involved in plant growth, development, and adverse stress responses, which have not yet been characterized in Tartary buckwheat (Fagopyrum tataricum, TB). In this study, we verified that drought stress could induce autophagy in TB roots. Next, 49 FtATGs in the whole genome of TB were identified. All FtATGs were randomly distributed in 8 known chromosomes, while 11 FtATGs were predictably segmental repeats. As the core component of autophagy, there were 8 FtATG8s with similar gene structures in TB, while FtATG8s showed high expression at the transcription level under drought and salt stresses. The cis-acting element analysis identified that all FtATG8 promoters contain light-responsive and MYB-binding elements. FtATG8s showed a cell-wide protein interaction network and strongly correlated with distinct stress-associated transcription factors. Furthermore, overexpression of FtATG8a and FtATG8f enhanced the antioxidant enzyme activities of TB under adverse stresses. Remarkably, FtATG8a and FtATG8f may be vital candidates functioning in stress resistance in TB. This study prominently aids in understanding the biological role of FtATG genes in TB.

Keywords: FtATG8s; Tartary buckwheat; abiotic stress; autophagy; functional validation.

Figure 1

Accumulated autophagosomes in TB roots under drought stress. Autophagosomes were stained by MDC dye. Control indicates TB roots without drought treatment. Scale bars: 20 μm.

Figure 2

Phylogenetic analysis of ATG proteins from Fagopyrum tataricum and other plants. Neighbor-joining trees are constructed with 49 FtATGs of Fagopyrum tataricum (Ft), 39 OsATGs of Oryza sativa (Os), 29 NtATGs of Nicotiana tabacum (Nt), and 47 AtATGs of Arabidopsis thaliana (At) using the MEGAX software, with 2000 bootstrap replicates. The proteins of TB are marked in red. The phylogenetic tree shows bootstrap values above 50%.

Figure 3

Chromosomal distribution and gene duplication of FtATGs. FtATGs with fragment duplicates were marked in blue, and the red lines represent the collinearity between two genes.

Figure 4

Domains and gene structures of FtATGs. (A) The exon-intron structures. The colorful rectangles represent exons, except the gray rectangles indicate the untranslated regions (UTRs). Introns are indicated by black lines. (B,C) Domains of the corresponding genes. The 49 FtATG genes were described based on their functions in autophagy.

Figure 5

The expression heatmap of FtATGs under drought and salt stress. Each row represents one gene, and every three columns represent different replicates for each treatment.

Figure 6

Cis-acting elements in the promoter regions of FtATG8s. Cis-acting elements with analogous functions were presented in the same color as indicated. The colorful rectangles on the right column represent cis-acting elements with different functions.

Figure 7

Analysis of the functional interaction network of FtATG8 proteins. FtATG8s were highlighted in yellow. Proteins in red were predicted to encode ubiquitin-related proteins, and the pink indicated a peroxisomal protein. Other FtATGs were shown in blue, and undermined proteins were in green.

Figure 8

Correlations of expression patterns between FtATGs and other transcription factors. RStudio was applied for this visualization. Line thickness mapping absolute value of correlation, color mapping p value credibility (* p < 0.05, ** p < 0.01, *** p < 0.001). FtMYB7 (FtPinG0003734600.01), FtMYB9 (FtPinG0002001900.01), FtMYB10 (FtPinG0002706600.01), FtMYB11 (FtPinG0008533900.01), FtMYB13 (FtPinG0005410000.01), FtMYB17 (FtPinG0006925500.01), FtMYB21 (FtPinG0004929500.01), FtMYB22 (FtPinG0003119800.01), FtNAC2 (FtPinG0005692100.01), FtNAC3 (FtPinG0000381200.01), FtNAC4 (FtPinG0005791100.01), FtNAC5 (FtPinG0006190400.01), FtNAC6 (FtPinG0005624400.01), FtNAC7 (FtPinG0005167000.01), FtNAC8 (FtPinG0002252000.01), FtNAC9 (FtPinG0002967400.01), FtNAC31 (FtPinG0005167000.01), FtbHLH2 (GenBank:KU296218), FtbHLH4 (FtPinG0002267300.01), FtbHLH3 (GenBank:KU296217.1), FtbZIP83 (FtPinG0002143600.01), FtbZIP5 (FtPinG0003196200.01).

Figure 9

qRT-PCR analysis of FtATG8s in different tissues. R (root), S (stem), L (leaf), F (flower), FL (seed). The minimum expression levels of each gene in the five tissues were set to 1. Error bars represent SD calculated from three experiment repeats. Letters on the bars represent significant differences among (α = 0.05, Duncan) tissues.

Figure 10

Relative expression of FtATG8 genes under cold, NaCl, and drought treatments. The expression of FtATG8s was set to “1” after 3 and 12 h without stress. The R package was used to generate a heat map based on the mean log₂FC values of three biological replicates.

Figure 11

Effects of transient overexpression of FtATG8a and FtATG8f on antioxidant enzyme activity in TB. (A) Transient transformation of TB cotyledon by pCHF3-FtATG8a-YFP (termed FtATG8a-OE), pCHF3-FtATG8f-YFP (termed FtATG8a-OE), and pCHF3-YFP (termed Control) mediated by Agrobacterium tumefaciens in vacuum infiltration. (B) Relative expression of FtATG8a and FtATG8f after transient expression. (C,D) Determination of POD and SOD under abiotic stress. Error bars indicate the SD of three independent experiments. Asterisks indicate significant differences (* p < 0.05, ** p < 0.01, *** p < 0.001).