Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (22): 4732-4745.doi: 10.3864/j.issn.0578-1752.2025.22.013

• HORTICULTURE • Previous Articles     Next Articles

Functional Analysis of CaIAA8, An Interacting Protein of the Autophagy-Related Protein CaATG8c, in the Heat Tolerance of Pepper

LI NiFei(), YANG QiaoMin, YANG KeCheng, XING YuTeng, WANG MengYuan, ZANG TianBao, LU MingHui()   

  1. College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi
  • Received:2025-05-13 Accepted:2025-07-07 Online:2025-11-16 Published:2025-11-21
  • Contact: LU MingHui

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Abstract:

【Objective】Autophagy is a conserved protein degradation system in eukaryotic cells and plays an important role in plant stress tolerance. Previous studies showed that silencing the autophagy-related gene CaATG8c reduces heat tolerance in pepper. A candidate interacting protein of CaATG8c, the auxin responsive protein CaIAA8, was identified via yeast two-hybrid screening. This study aimed to analyze the sequence characteristics of CaIAA8, confirm its interaction with CaATG8c, and investigate its role in regulating heat tolerance in pepper, thereby providing insights into the molecular mechanisms of heat tolerance and supporting the breeding of heat-tolerant cultivars. 【Method】 Protein sequence alignment, phylogenetic analysis, and conserved motif identification of CaIAA8 were conducted using BLASTP (NCBI), IQ-TREE, and MEME, respectively. Protein physicochemical properties, signal peptide, transmembrane domain, and phosphorylation sites were predicted using ExPASy, SignalP-6.0, TMHMM-2.0, and NetPhos, respectively. Protein secondary and tertiary structures were predicted using SOPMA and SWISS-MODEL respectively. The interaction between CaATG8c and CaIAA8 was validated by yeast two-hybrid and luciferase complementation assays. Subcellular localization of CaIAA8 was examined by transient expression in tobacco leaves. The expression pattern of CaIAA8 gene in pepper was analyzed using real-time quantitative PCR (RT-qPCR), and its function in heat tolerance was further evaluated by virus-induced gene silencing (VIGS) and transient overexpression in pepper. 【Result】CaIAA8 encodes a 349-amino-acid protein with a molecular weight of 37.48 kDa and an isoelectric point of 7.52. It contains 65 predicted phosphorylation sites and is a basic, hydrophilic, and structurally unstable protein. CaIAA8 localizes primarily to the cytoplasm and co-localizes with chloroplasts. Its expression is highest in stems, followed by fruits and flowers, and lowest in leaves. Under 45 ℃ heat stress, CaIAA8 transcript level decreases. Silencing CaIAA8 enhances pepper heat tolerance, as evidenced by reduced leaf wilting, lower relative electrolyte leakage, decreased cell death and H2O2 accumulation, and increased expression levels of heat shock protein and heat shock transcription factor genes. In contrast, transient overexpression of CaIAA8 reduces pepper heat tolerance, leading to more severe heat-induced damage and elevated levels of cell death and H2O2. 【Conclusion】CaIAA8 physically interacts with CaATG8c and functions as a negative regulator of heat tolerance in pepper.

Key words: pepper, heat tolerance, autophagy, CaATG8c, auxin responsive protein, CaIAA8

Table 1

Sequence of PCR primers"

用途Purpose 名称Name 序列Sequence (5′-3′)
酵母双杂交
Y2H		 T7 TAATACGACTCACTATAGG
3AD GAGATGGTGCACGATGCACAGT
AD-CaIAA8-F GCCATGGAGGCCAGTGAATTC
AD-CaIAA8-R CAGCTCGAGCTCGATGGATCC
BD-CaATG8c-F ATGGCCATGGAGGCCGAATTCATGGCGAAGAGTTCATTC
BD-CaATG8c-R CCGCTGCAGGTCGACGGATCCTTACACAAAGTTAAGGTCCC
荧光素酶互补成像
LCI		 CLuc-F GTGTTTGTGGACGAAGTACC
CLuc-R CCATTTCACAGTTCGATAGCG
NLuc-F TGATGTGATATCTCCACTGACG
NLuc-R GGGCGTATCTCTTCATAGCC
Cluc-CaIAA8-F TACGCGTCCCGGGGCGGTACC
Cluc-CaIAA8-R ACGAAAGCTCTGCAGGTCGAC
NLuc-CaATG8c-F ACGAAAGCTCTGCAGGTCGACCTACTGATGCACGGAGGAATC
NLuc-CaATG8c-R CGCGTACGAGATCTGGTCGACCACAAAGTTAAGGTCCCCAAAT
亚细胞定位及基因过表达
Subcellular localization and
gene overexpression		 pART27-CaIAA8-F GATGAACTATACAAAGAATTCA
pART27-CaIAA8-R CAGGACTCTAGATTAGGTACCC
基因表达分析
Analysis of gene expression		 CaUbi3-F TGTCCATCTGCTCTCTGTTG
CaUbi3-R CACCCCAAGCACAATAAGAC
qCaIAA8-F AAAGGCTCAGGTTGTGGGTT
qCaIAA8-R AACATTTCCTTCCCAGGAGC
qCaHsfA2-F GTAGCATCAGTAGCCACAGC
qCaHsfA2-R CAAGCAACTCTTCCCAAATA
qCaHSP16.4-F ATGTCAAAGATGATCAGCTTATTG
qCaHSP16.4-R GGGTACTCAACACGGGACAC
qCaHSP25.9-F AGGAGACGACGCCTTAGTAG
qCaHSP25.9-R TCTGACCCTTCTTTCTCTTCC
qCaHSP70.1-F CAGGTGTGCTAGTTCAGGTGT
qCaHSP70.1-R TGACCTGAGGCACTCCTCTT
基因沉默
Gene-silencing		 TRV2:CaIAA8-F GCTCTAGAATGTCTCCACCACT
TRV2:CaIAA8-R CGGGATCCCTCTTCAACCTTTGT

Fig. 1

Phylogenetic analysis of CaIAA8 A: Conserved motif analysis; B: Conserved domain analysis; C: Phylogenetic evolutionary tree"

Fig. 2

Sequence alignment within conserved domains of CaIAA8"

Fig. 3

Physicochemical properties of CaIAA8 protein A: Prediction of signal peptide; B: Prediction of structure prediction; C: Prediction of phosphorylation sites; D: Prediction of hydrophilicity and hydrophobicity; E: Prediction of secondary structure; F: Prediction of tertiary structure"

Fig. 4

Verification of the interaction between CaIAA8 and CaATG8c A: Yeast two-hybrid assay; B: Luciferase complementation imaging assay"

Fig. 5

Subcellular localization of CaIAA8"

Fig. 6

Expression pattern analysis of CaIAA8 gene A: Expression in different tissues and organs of pepper; B: Expression at different time points of heat stress. Different lowercase letters indicate the significant differences at P<0.05 level"

Fig. 7

Phenotype and silencing efficiency of CaIAA8 gene-silenced pepper plants A: Phenotype of pepper seedlings with CaIAA8 gene-silenced; B: Relative expression level of CaIAA8. *: The significant difference at P<0.05 levels. The same as below"

Fig. 8

Effects of CaIAA8-silencing on the heat tolerance of pepper A: Heat stress phenotypes; B: Relative conductivity determination; C: Tissue chemical staining, with DAB staining on the left and trypan blue staining on the right. ns: There is no significant difference; ****: The significant difference at P<0.0001 levels. The same as below"

Fig. 9

Effects of CaIAA8-silencing on the expression levels of marker genes for heat tolerance in pepper leaves under heat stress A: CaHsfA2; B: CaHSP16.4; C: CaHSP25.9; D: CaHSP70.1. **: The significant difference at P<0.01 levels; ***: The significant difference at P<0.001 levels. The same as below"

Fig. 10

Effects of transient overexpression of CaIAA8 gene on the heat tolerance of pepper A: Relative expression level of CaIAA8 ; B: Heat damage phenotypes; C: Tissue chemical staining, with trypan blue staining on the left and DAB staining on the right"

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