Effect of exogenous IAA on the growth and development of walnut endocarp

Abstract：【Objective】The accumulation of lignin is the main events of walnut endocarp development, and plant hormones regulate the formation of thick-walled tissues and the synthesis of lignin, which involves in xylem development, through the expression of related genes, thus affecting the development of the secondary wall. The study was carried out in order to understand the effect of the growth hormone IAA on the development of endocarp in walnuts.【Methods】In the present study, we selected the 16 year old trees with robust and consistent growth and consistent levels of water and fertilizer management. At about 50 d after flowering, bearing spurs were wrapped and treated with different concentrations of growth regulators. Changes in lignin deposition in the endocarp of walnuts after the treatments with different concentrations of IAA was observed by resorcinol staining, and the contents of lignin, cellulose, hemicellulose, and IAA in the endocarp were determined. At the same time, samples at 12 d after IAA treatment were subjected to high- throughput sequencing and analysis.【Results】The resultsshowed that at the early stage of treatment there was a significant difference in the accumulation of endocarp lignin between the treatments with differential IAA concentrations. Lignin deposition started from tissues near the vascular bundles, and the color shifted from light red to dark red and from localized staining to overall staining. At the late stage, the endocarp of the late walnuts was partially hardened and significantly thickened, but the development of the endocarp was incomplete, and the dewy kernel was still present. Lignin content in the endocarp increased significantly after the treatments with 50 mg ·L^-1 IAA and 100 mg ·L^-1 IAA at the 12th d after treatment, and the difference in lignin content was not significant between the control and the treatment with 200 mg ·L^-1. In the late stages, low concentration IAA treatment promoted lignin accumulation and lignin content increased slowly, but lignin content in treatments high concentrations was lower than that in the control. The overall endocarp cellulose content showed an increasing trend, compared with the control. The pre- cellulose content in the treatments was higher than in the control. Compared with the control, the overall endocarp hemicellulose content showed an increasing trend. The endogenous IAA content in walnut endocarp after different concentrations of IAA treatments showed an overall increasing trend. On the 12th d after treatment, the endogenous IAA content in the treatments with 50 mg·L^-1 IAA and 100 mg·L^-1 IAA was significantly higher than that in the control. It was 37.77 ng·g^-1 in the treatment with 50 mg ·L^-1 IAA, which was 24.2% higher than that of the control, and 100 mg ·L^-1 IAA treatment had a IAA content 21.5% higher than that in the control. Treatments with 150 mg·L^-1 IAA and 200 mg·L^-1 IAA were lower than the control. Therefore, with the increase in concentration exogenous IAA, the endogenous IAA content showed a decreasing trend. The number of differentially expressed genes between 50 mg·L^-1 IAA treatment and the control was the largest, with 3292 genes, of which 1922 were up-regulated and 1370 down-regulated, and the number of common differential genes among the treatments with different concentrations were 591 based on the Veen diagram. GO enrichment and KEGG enrichment analyses revealed that the phytohormone signaling pathway was the common metabolic pathway, and the phenylpropane biosynthesis pathway was significantly altered with the increase in IAA concentration, but it was not enriched in the treatments with 150 mg·L^-1 IAA and 200 mg·L^-1 IAA. It was initially hypothesized that there were significant differences in the phytohormone signaling and phenylpropane biosynthesis metabolic pathways in the endocarp of walnuts after exogenous application of IAA. The genes related to the two pathways, which were differentially expressed, were gradually down- regulated or non-expressed with the increase in IAA concentration.【Conclusion】The results showed that IAA affected the accumulation of lignin in the endocarp as well as the expression of genes in the lignin synthesis pathway. The results provided reference for the later study to explore the mechanism of the effect.