Table_2_Transcriptomics‐based analysis of the causes of sugar receding in Feizixiao litchi (Litchi chinensis Sonn.) pulp.docx

To investigate the causes of the “sugar receding” in ‘Feizixiao’ litchi (Litchi chinensis Sonn.) pulp, the main sugar contents and sucrose metabolism enzyme activities were measured in pulp obtained in 2020 and 2021. Pulp RNA obtained in 2020 was extracted at 35, 63, and 69 days after anthesis (DAA) for transcriptome sequencing analysis. The differential expression of genes was verified by real-time PCR for both years. The results showed that after 63 DAA, the contents of soluble sugars and sucrose decreased, and the contents of fructose and glucose increased in both years. The dynamic changes in sucrose metabolism enzyme activities were similar in both years. After 63 DAA, except for acid invertase (AI) in 2021, the activities of other enzymes decreased significantly, and the net activity of sucrose metabolism enzymes showed a strong sucrose cleavage activity. There were 18061, 19575, and 985 differentially expressed genes in 35 d vs. 63 d, 35 d vs. 69 d, and 63 d vs. 69 d, respectively. Ninety-one sugar metabolism genes were screened out, including sucrose synthase (SS), sucrose phosphate synthase (SPS), AI, neutral invertase (NI), hexokinase (HK), glucose 6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), phosphofructokinase (PFK), and pyruvate kinase (PK) genes. In 63 d vs. 69 d, seventy-five percent of sucrose metabolism genes were downregulated, seventy-seven percent of genes in glycolysis (EMP) were upregulated and the PFK genes were significantly upregulated. There was a significant linear correlation between the expression of 15 genes detected by real-time PCR and the transcriptome sequencing results (r2020 = 0.9139, r2021 = 0.8912). These results suggest that the upregulated expression of PFK genes at maturity may enhance PFK activity and promote the degradation of soluble sugar in pulp through the EMP pathway, resulting in decreased soluble sugar and sucrose contents and “sugar receding” in pulp. Moreover, the downregulated expression of sucrose metabolism genes in pulp decreased the activities of these enzymes, but the net activity of these enzymes resulted in cleaved sucrose and replenished levels of reducing sugars, resulting in a stable reducing sugar content.

为探究‘妃子笑’荔枝（Litchi chinensis Sonn.）果肉‘退糖’现象的成因，本研究于2020年与2021年采集果肉样品，测定其主要糖分含量与蔗糖代谢相关酶活性。2020年的果肉RNA于花后35、63、69天（days after anthesis, DAA）提取后进行转录组测序分析；两年间的基因差异表达情况均通过实时PCR（real-time PCR）验证。结果显示，花后63天之后，两年间果肉的可溶性总糖与蔗糖含量均显著下降，而果糖与葡萄糖含量则呈上升趋势。两年间蔗糖代谢相关酶活性的动态变化趋势基本一致。花后63天之后，除2021年的酸性转化酶（acid invertase, AI）外，其余酶活性均显著降低，且蔗糖代谢酶的净活性表现出较强的蔗糖裂解能力。在花后35d与63d、35d与69d以及63d与69d的比较组中，分别筛选得到18061、19575和985个差异表达基因。本研究共筛选得到91个糖分代谢相关基因，涵盖蔗糖合酶（sucrose synthase, SS）、蔗糖磷酸合酶（sucrose phosphate synthase, SPS）、酸性转化酶（AI）、中性转化酶（neutral invertase, NI）、己糖激酶（hexokinase, HK）、葡萄糖-6-磷酸脱氢酶（glucose 6-phosphate dehydrogenase, G6PD）、6-磷酸葡糖酸脱氢酶（6-phosphogluconate dehydrogenase, 6PGD）、磷酸果糖激酶（phosphofructokinase, PFK）以及丙酮酸激酶（pyruvate kinase, PK）等基因。在63d与69d的比较组中，75%的蔗糖代谢相关基因呈下调表达，77%的糖酵解（EMP）通路基因呈上调表达，其中PFK基因的上调幅度尤为显著。经实时PCR检测的15个基因的表达量与转录组测序结果呈显著线性相关（2020年r²=0.9139，2021年r²=0.8912）。上述结果表明，果实成熟阶段PFK基因的上调表达可增强磷酸果糖激酶活性，并通过糖酵解通路促进果肉可溶性糖的降解，进而导致可溶性总糖与蔗糖含量下降，引发果肉‘退糖’现象。此外，果肉中蔗糖代谢相关基因的下调表达会降低此类酶的整体活性，但酶的净活性仍可裂解蔗糖并补充还原糖水平，最终维持还原糖含量的稳定。