Identification of autophagy-related genes ATG18 subfamily genes in potato (Solanum tuberosum L.) and the role of StATG18a gene in heat stress

Abstract

Autophagy is a highly conserved process in eukaryotes that is used to recycle the cellular components from the cytoplasm. It plays a crucial function in responding to both biotic and abiotic stress, as well as in the growth and development of plants. Autophagy-related genes (ATG) and their functions have been identified in numerous crop species. However, their specific tasks in potatoes (Solanum tuberosum L.), are still not well understood. This work is the first to identify and characterize the potato StATG18 subfamily gene at the whole-genome level, resulting in a total of 6 potential StATG18 subfamily genes. We analyzed the phylogenetic relationships, chromosome distribution and gene replication, conserved motifs and gene structure, interspecific collinearity relationship, and cis-regulatory elements of the ATG18 subfamily members using bioinformatics approaches. Furthermore, the quantitative real-time polymerase chain reaction (qRT-PCR) analysis suggested that StATG18 subfamily genes exhibit differential expression in various tissues and organs of potato plants. When exposed to heat stress, their expression pattern was observed in the root, stem, and leaf. Based on a higher expression profile, the StATG18a gene was further analyzed under heat stress in potatoes. The subcellular localization analysis of StATG18a revealed its presence in both the cytoplasm and nucleus. In addition, StATG18a altered the growth indicators, physiological characteristics, and photosynthesis of potato plants under heat stresses. In conclusion, this work offers a thorough assessment of StATG18 subfamily genes and provides essential recommendations for additional functional investigation of autophagy-associated genes in potato plants. Moreover, these results also contribute to our understanding of the potential mechanism and functional validation of the StATG18a gene's persistent tolerance to heat stress in potato plants.

Keywords: StATG18a; autophagy; heat stress; photosynthesis; physiological; potato.

Figure 1

Expression profiles of StATG18 subfamily genes in different tissues of potato. Transcript levels of (A) StATG18a, (B) StATG18b, (C) StATG18c, (D) StATG18d, (E) StATG18f, and (F) StATG18h at mRNA levels, in the tuber, flower, root, petiole, stem, leaf and root. Data were the means ± standard deviation. Different letters indicated significant differences between the two groups (p-value less than 0.05, calculated by one-way ANOVA, followed by LSD and Duncan or Dunnett’s T3).

Figure 2

Expression profiles of StATG18 subfamily genes in potato leaves, stem and roots in response to heat stress. Transcript levels of (A–C) StATG18a, (D–F) StATG18b, (G–I) StATG18c, (J–L) StATG18d, (M–O) StATG18f and (P–R) StATG18h in leaves, stems, and roots under heat stress treatment at different time intervals (0, 1, 2, 4, 8, 16, 24, and 48h). Data were the means ± standard deviation. Different letters indicated significant difference between two groups (p-value less than 0.05, calculated by one-way ANOVA, followed by LSD and Duncan or Dunnett’s T3).

Figure 3

Subcellular localization of StATG18a-GFP fusion protein in Nicotiana benthamiana leaves. (A) Confocal scanning laser microscopy analysis of tobacco transformed with pBI121-EGFP-StATG18a. (B) The empty vector (GFP) served as a control. Bar = 50 μm.

Figure 4

Effects of StATG18a on potato plant growth phenotypes. Transcript levels of StATG18a in (A) overexpressing StATG18a and (B) knock down potato plants (*p < 0.05, **p < 0.01, ***p< 0.001). One-Way ANOVA corrected by Dunnett ( Figures 5A, B , n = 9). (C1) The phenotypes of transgenic and non-transgenic potato plants were grown normally for 6 weeks, (C2) The phenotypes of transgenic and non-transgenic potato plants treated with 35°C heat stress for 48 hours after 6 weeks of normal growth, (C3) The phenotypes of transgenic and non-transgenic potato plants that recovered 7 d after heat stress treatment, (C4) The phenotypes of transgenic and non-transgenic potato plants that recovered 14 d after heat stress treatment, and (D) plant height, (E) total fresh weights and (F) dry weights, as well as (G) root fresh weights and (H) dry weights were measured after the heat treatment at different time intervals (0 d, 7 d and 14 d), Bar = 10 cm. NT, non-transgenic plants; OE, pBI121-EGFP-StATG18a-transgenic plants (OE-2 and OE-5); Ri, pART-StATG18a-RNAi-transgenic plants (Ri-1 and Ri-4); Two-Way ANOVA amended by Tukey ( Figures 4D–H , n = 9; *p < 0.05, **p< 0.01).

Figure 5

Effects of StATG18a on (A) SOD activity, (B) CAT activity, (C) POD activity, (D) H₂O₂ content, (E) MDA content, and (F) proline content. Potato plants (transgenic and non-transgenic) were treated with heat stress at different time intervals (0, 4, 8, 16, 24, and 48h), and examined the SOD, CAT, as well as POD activities, and H₂O₂ MDA as well as proline content. NT, non-transgenic plants; NT, non-transgenic plants; OE, pBI121-EGFP-StATG18a-transgenic plants (OE-2 and OE-5); Ri, pART-StATG18a-RNAi-transgenic plants (Ri-1 and Ri-4); Data were computed by means ± standard deviation; p-values (*p<0.05, **p<0.01) were calculated by ordinary two-way ANOVA, followed by Tukey’s multiple comparisons tests (n = 9).

Figure 6

Effects of StATG18a on (A) net photosynthetic rate, (B) transpiration rate and (C) stomatal conductance under heat conditions.Potato tubers stimulated by transgenic and non-transgenic lines were monitored by cultivation with heat stress treatment at different time intervals (0h, 4h, 8h, 12h, 24h, and 48h). Analyses for heat stress (A) net photosynthetic rate, (B) transpiration rate, and (C) stomatal conductance. NT, non-transgenic plants; OE, pBI121-EGFP-StATG18a-transgenic plants (OE-2 and OE-5); Ri, pART-StATG18a-RNAi-transgenic plants (Ri-1 and Ri-4); Data were computed by means ± standard deviation; p-values (*p<0.05, **p<0.01) were estimated by ordinary two-way ANOVA, followed by Tukey’s multiple comparisons tests (n = 9).