Abstract: INTRODUCTION: Zizania latifolia is an important aquatic vegetable in China with both economic and nutritional values, but it is currently facing industrial challenges such as lack of crop rotation, declining quality, and insufficient supply, and is in urgent need of efficient means to increase production. As a novel small peptide hormone, phytosulfokine (PSK) has significant potential in plant growth regulation and agricultural applications, but its mechanism of regulating tillering in Z. latifolia has not yet been clarified. In this study, we used PSK-treated Z. latifolia as the material, combined with transcriptome sequencing (accession No.PRJNA1377146), fluorescence quantitative PCR validation and endogenous hormone determination, to investigate the molecular and physiological mechanisms of PSK in regulating the tillering of Z. latifolia, and to provide a theoretical basis and technological support for the high-yield and high-quality cultivation of Z. latifolia.  RATIONALE: Z. latifolia is the second largest aquatic vegetable after lotus root in China, with remarkable economic and nutritional value, but the current cultivation is facing problems such as lack of crop rotation, strong seasonality, and declining soil fertility, which leads to quality degradation and insufficient supply, and there is an urgent need for efficient means of yield increase; PSK, as a small peptide hormone in plants, can regulate the physiological processes such as cell elongation and development of meristematic tissues, and it has demonstrated its value of promoting crop growth and delaying senescence in agriculture, but the molecular mechanism of PSK regulation has not been clearly defined. In agriculture, PSK has demonstrated its value in promoting crop growth, delaying aging and other applications. However, there are fewer studies on the regulation of plant growth and development, and the molecular mechanism has not yet been clarified. Therefore, it is of great significance to systematically analyze the changes in gene expression of Z. latifolia after PSK treatment by means of RNA-seq sequencing, combined with the determination of endogenous hormones and observation of the tillering phenotypes, to excavate key metabolic pathways and regulatory genes, and to elucidate the molecular and physiological mechanisms of its regulation of water bamboo tillering.  RESULTS: After transcriptome sequencing, a total of 806 differentially expressed genes (365 up-regulated and 441 down-regulated) were screened out, and GO enrichment analysis showed that the differentially expressed genes were mainly involved in biological processes such as response to water deficit, cell wall organization, etc., and KEGG analysis showed that the differentially expressed genes were significantly enriched in the pathways of phenylpropane biosynthesis and tryptophan metabolism, etc. The results of fluorescence quantitative PCR of four differentially expressed genes were in line with the trend of expression of transcriptome, PSK treatment significantly promoted tillering in Z. latifolia, and the number of tillers at 14 days of treatment was 122.22% higher than that of the control group, which was the most significant effect. Two-way ANOVA confirmed that the treatment, time and the interaction effect were all highly significant, and there was a time-dependent “window period”. As for endogenous hormones, the content of growth hormone, gibberellin and jasmonic acid increased significantly after PSK treatment (GA3 increased by 188.59%), and the content of abscisic acid and cytokinin decreased significantly, and the hormone ratio was significantly reconfigured.  CONCLUSION: Exogenous PSK regulates the tillering and growth of Z. latifolia through multiple pathways: it significantly alters the endogenous hormone levels, promotes the increase of growth-related hormones and inhibits the decrease of hormone-like hormones; it regulates the key metabolic pathways such as phenylpropane biosynthesis and fatty acid degradation, and influences the synthesis of the cell wall and energy metabolism; and it promotes the synthesis of indoleacetic acid through the expression of the key genes in the tryptophan metabolic pathway, which provides the material and signal basis for tillering. The optimal concentration of PSK treatment is 1 μmol·L^–1, and the optimal application “window” is about 14 days after treatment. This study reveals the molecular mechanism of PSK regulation tillering of Z. latifolia, and provides feasible technical solutions and theoretical support for large-scale high-yield and high-quality cultivation of Z. latifolia.

Key words: Phytosulfokine, Zizania latifolia, tillering, endogenous hormone, transcriptome