Preliminary analysis on the mechanism leading to difference in internode length between Venus Seedless grapevine and its shorter internode muant Zijinzaosheng

Abstract:【Objective】Grapeindustryislabor-intensive,especiallyinthemanagementofnewshoots.In practice, grape with shorter internode length has such unique advantages as labor saving and simplification in canopy management. Although the dwarfing mechanisms in many fruit trees have been studied, little has been studied in grapevine. The mechanism of short internode in grapevine remains unclear. Mutant is ideal materials for study the genetic mechanism of traits. However, short internode mutants of grapevine are scarce. A grape mutant named Zijinzaosheng with short internode was discovered from the progeny of Venus Seedless grapevines after treatment with colchicine. A previous study showed that the chromosome ploidy of Zijinzaosheng was the same as that of Venus Seedless. The DNA sequences had little difference between Zijinzaosheng and Venus Seedless, and the genetic background of the two materials is highly consistent. The short internode trait of Zijinzaosheng was stable in multi-plot trials for several years. The fruit of Zijinzaosheng was not sensitive to exogenous GA3. We speculated that the mutation mechanism of Zijinzaosheng was different from that of Point Meunier. In order to elucidate the mechanism and regulation of grapevine internode, comparative analyses on internode length, cell morphology of stem, endogenous hormone contents and small RNA sequencing were conducted in the study. Meanwhile, the influence of exogenous hormones on the internode length in Zijinzaosheng was also evaluated.【Methods】7-year-old trees of Venus Seedless and Zijinzaosheng were used as the experimental materials. They were planted in Lishui plant science station of Jiangsu Academy of Agricultural Sciences, a core demonstration vineyard of Nanjing Experimental Station of China Agriculture Research System for Grape Industry. All the plant materials were maintained under rain-shelter. The study was conducted in 2019, and shoot tips were collected for high throughput sequencing; the young leaves at the shoot tip were sampled to determine the endogenous hormones. Cell morphology were observed under a scanning electron microscopy, and endogenous hormone contents analyzed with an LC-MS/ MS. High throughput sequencing was used to analyze the different miRNA. The study of exogenous hormone treatments was conducted in 2020. Shoot tips and shoot internodes were treated with 0.25 mmol·L^-1 GA₃, 0.25 mmol·L-1 Jasmonic acid or 0.5 mmol·L-1 DIECA (biosynthesis inhibitor of Jasmonic acid), respectively. The treatment with ddH₂O was used as control. The internode length was mea- sured after exogenous hormone treatments.【Results】The results showed that the internode length of one-year hard shoots of Zijinzaosheng was lower (47%) than that of Venus Seedless. The cell number in the stem of Zijinzaosheng was smaller than that of Venus Seedless. There was no significant difference in cell size between the two cultivars. Jasmonic acid (JA), jasmonoyl-L-Isoleucine (JA-ILE) and salicyl- ic acid (SA) in Zijinzaoheng were significantly higher, while indole-3-carboxylic acid (ICA) and indole- 3-carboxaldehyde (ICAld) were significantly lower. The contents of gibberellins (GA15, GA19 and GA3), cytokinins (IP, cZ and tZ), auxin (IAA, ME-IAA) and ABA were not significant different between the two cultivars. Some gibberellins (GA1, GA4, GA7, GA9, GA20, GA24 and GA53), auxin (IBA), jasmonates (H2JA, MEJA) and cytokinin (DZ) were not detected in either of the two cultivars. Small RNA sequenc- ing results showed that 21 differentially expressed miRNA genes were detected, including 7 known miRNAs (miR156f, miR535a, miR535b, miR535c, miR3635-5p, miR319b and miR319f) and 14 novel miRNAs (miR48, miR28, miR33, miR68, miR55, miR81, miR70, miR78, miR165, miR162, miR152, miR58, miR108 and miR45). 556 target genes were predicted. GO annotation analysis revealed that these genes were involved in cell division, protein metabolism, environmental responses and organ development, etc. Two internode growth regulation miRNAs, miR319b and miR319f, were selected. The candidate target gene included TCP, which is involved in jasmonic acid metabolism and mitotic G2 phase. Compared with the control, the internode length of Zijinzaosheng was decreased by exogenous jasmonic acid while increased by DIECA. The difference in the internode length between the control and GA3 treatment was not significant.【Conclusion】The internode length of Zijinzaosheng was sensi- tive to exogenous jasmonic acid, and can be increased by exogenous DIECA treatment. The study sug- gests that jasmonic acid signaling pathway participates in the formation of short internode length in Zijinzaosheng. MiR319b/f may play a role in the grapevine internode length development. The study provides a new direction for further elucidation of the regulatory mechanism in grapevine internode growth and supplies material resource for breeding varieties with short internodes.