Effects of supplementary light on the bud burst of ‘Red Globe’grape under protected cultivation based on transcriptome sequencing

Abstract: 【Objective】The early-maturing grape cultivation in greenhouse extends the market supply period of table grape, and improves the economic benefit of grape industry significantly.‘Red Globe’grape is the main cultivar of grape for the greenhouse culture in Ningxia. However, due to aging of the film and the dust on the surface of the film, the grapes are exposed to low density of light in the green-house, resulting in the reduction of fruit quality and economic income. Light can regulate the gene ex-pression, substance metabolism and morphological formation of plants. Artificial light is an efficient measure to improve the light conditions in the greenhouse. At present, researches on the effects of the light quality on grape mainly focus on plant growth, development and physiological metabolisms. In or-der to clarify the effect mechanism of different light quality supplementation on the bud burst of grape in the molecular level, transcriptome sequencing was used to analyze the light response related genes in the buds exposed to different light quality.【Methods】The experiment started in Helan Horticultural In-dustrial Park, experimental base of Ningxia University, on March 25, 2019. The experimental materials were eight-year-old‘Red Globe’grapes with a growing space of 0.8 m×1.5 m in the solar greenhouse.Four different light quality treatments were red light (H), blue light (L), white light (B), red blue light (red light: blue light = 2∶1, HL), the control was no supplementary light (CK). The intensity of supple-mentary light was 200 μmol·m^-2·s^-1, and the duration of supplementary light was 4h per day. On April 16, 2019, the buds in the leaf development stage were counted. The third and the fourth buds on the base of the annual branches with consistent growth in the leaf-developing stage were taken. 3 groups were randomly selected for each treatment, and 10 buds were taken for each group. The buds were im- mediately put in liquid nitrogen after stripping from the branches and stored in the refrigerator at -80 ℃. The soluble sugar, total protein, H2O2 content and CAT activity in the buds were measured. The RNA extraction and transcriptome sequencing of the samples were completed by Shanghai Meiji Biomedical Technology Co., Ltd. After passing the inventory inspection, Illumina Novaseq 6000 se- quencing platform sequencing. HISAT2 software was used for comparison with the reference genome.After obtaining the Read Counts of the gene, DESeq2 was used for differential expression analysis of the samples. Statistical analysis of gene differential expression was performed using FPKM value,|log2fold changes |≥1, and P-value ≤0.05 after correction as the screening standard. The differentially expressed genes in the gene set were compared with the COG, GO and KEGG databases in order to ob-tain the functional annotation and related metabolic pathway information of genes in different samples.In order to verify the accuracy of RNA-seq data results, 9 differentially expressed genes were randomly selected for real-time fluorescence quantitative expression verification analysis.【Results】Supplementa-ry light with different light quality accelerated the bud burst process of‘Red Globe’grapes. The leaf spreading rate of the buds under the HL treatment was the highest, followed by B, L and H, and the leaf spreading rate of the control was the lowest. The content of soluble sugar, total protein and H2O2 of the HL treatment was the highest, and CAT activity of the HL treatment was the lowest. Transcriptomic se-quencing was completed using Illumina platform. A total of 1423 differentially expressed genes were detected in all the samples, including 309 up-regulated genes and 1114 down-regulated genes. COG functional annotation was performed on the differentially expressed genes. It was found that the tran- scription and signal transduction pathways were significantly enriched in COG classification. GO analy-sis found that the differential genes related to bud burst were mainly concentrated in metabolic process-es, cell processes, cells, cell parts, membranes, binding and catalytic activities. KEGG analysis showed that the plant signal transduction was closely related to the bud burst. The SAUR gene was up-regulated in all the supplementary light treatments, and the effect of the HL treatment was the most significant in Auxin. The expression of b-type ARR related genes of Cytokinin growth inhibition factor was down-reg- ulated in the HL, the L and the H treatment, while the expression of B-type ARR of growth promotion factor was up-regulated in the H treatment. There was no significant difference in the DELLA genes in the GA pathway, and the positive regulatory factor GID1 was up-regulated in the HL, the L and the H treatment. It was found that PYR/PYL related genes were up-regulated inthe HL, the L and the H treat-ment in the ABA metabolic pathway, and the PP2C and the other ABA synthetic related genes were down-regulated. In this study, the MYC2 transcription factor, the ERF transcription factor, the JAZ and the TCH4 genes were down-regulated in all the supplementary light treatments. Nine differentially ex-pressed genes in the sequencing results were verified by qRT-PCR. The results showed that the expres-sion trend of the genes was consistent with the sequencing results, indicating that the transcriptomic se-quencing results were reliable.【Conclusion】The supplementary light treatment promoted the accumula-tion of nutrients and signal molecules in the grape buds, and accelerated the burst process of the buds. The bursting process of red blue light 2∶1 was faster than that of red, blue and white light, and it could be possibly used as the ideal light for the grape bud burst. The genes related to the plant hormone signal transduction pathways responded to the regulation of different light quality in the grape buds and might   play an important role in the bud burst. The expression differences of the SAUR, ARR, GID1, PYR/PYL, PP2C genes in the plant hormone signal transduction pathways would be the important reasons for the difference of the grape bud burst under different treatments.