4.1 Metabolic composition of phloem exudates during new shoot development
To explore the metabolic composition of phloem exudates in mature leaves, we performed widely-targeted metabolomics using the UPLC-MS/MS detection platform, The results showed that a total of 326 metabolites were detected, mainly including 93 amino acids and their derivatives (28.53%), 61 sugars (18.71%), 60 organic acids (18.40%), 51 lipids (15.64%), 50 nucleotides and their derivatives (15.34%), and 11 vitamins (3.37%) (Fig. 2A). To analyze the proportion of each metabolite, the relative peak areas of metabolites were compared. It was found that amino acids and their derivatives were the highest relative content metabolites in the phloem exudates (39.13%), followed by organic acids (30.25%), lipids (16.04%), and sugars (12.02%) (Fig. 2B). In the phloem exudates, the highest relative content of amino acids were Glu, Gln, Thea, followed by Ile, Phe (Fig. 2C).
4.2 Quantitative analysis of amino acid components in phloem exudates
In order to further investigate the transport characteristics of amino acids during the development of tea shoots, we quantitatively analyzed amino acids and their derivatives in the phloem exudates using UPLC-MS/MS. Cluster analysis based on 20 major amino acids showed that there was a significant difference between different leaf positions (Fig. 3A). The content of most amino acids in the phloem exudates collected from upper leaves was significantly higher than that in lower leaves. The content of various amino acids in the phloem exudates of the lower leaves was stable. The 20 amino acids could be roughly clustered into 3 branches, with Thea, Glu, and Gln clustered into a group. As the new shoots develop, the content in the exudates of the upper mature leaves gradually increases, reaching its highest level in the C3 stage. Ala (Alanine), Arg (Arginine), and His (Histidine) were clustered into a group, with their content reaching its highest in the C2 stage. It showed a trend of first increasing and then decreasing with the development of new shoots; Ser (Serine), Lys (Lysine), Gly (Glycine), Cys (Cysteine), Asp (Aspartic acid), Asn (Asparagine), Thr (Threonine), Ile (Isoleucine), Leu (Leucine), Val (Valine), Trp (Tryptophan), Pro (Proline), Phe (Phenylalanine) were clustered into a group, and their content remained at high levels during the C2 and C3 stages as new shoots developed.
Quantitative analysis of amino acids showed that Thea, Gln, and Glu had the highest content among the amino acid components in the phloem exudates (Fig. 3B). At the same stage, the content of three amino acids in the phloem exudates of the upper mature leaves of the plant was significantly higher than that of the lower. The content of Glu and Gln in the phloem exudates of the upper mature leaves significantly increased with the development of new shoots. Interestingly, the content of Thea did not show significant differences in the early stages of shoot development, but increased significantly in the later stages.
4.3 Identification of key genes in the regulation of amino acid transport from source to sink
In order to reveal the molecular mechanism of amino acid transport in the phloem, transcriptome sequencing was performed on the corresponding mature leaves at different development stages of new shoots (Table S3). The transcriptome sequencing analysis of 18 mature leaf samples obtained 120.47 Gb Clean Data after filtering. The Clean Data of each sample reached 6 GB. More than 80% of the sequences can be mapped to the reference genome, including a large number of genes involved in amino acid transport and metabolism (1260) and carbohydrate transport and metabolism (1846) (Fig. 4A).
A total of 12169 differential genes were identified, which were significantly enriched in metabolic pathways such as "starch and sugar metabolism", "plant hormone signaling", "flavonoid metabolism", "fatty acid metabolism", and "nitrogen metabolism" (Fig. 4B). Among them, 25 amino acid transporters were identified. Cluster expression analysis found that the expression of these amino acid transporter genes showed diversity in different parts and developmental stages (Fig. 5). Overall, the expression level in the upper mature leaves was significantly higher than that in the lower. It can be divided into four branches according to different expression tendencies. In the first branch, including AVT1A (CSS0026804), AAP3 (CSS0000586) and AAP3 (CSS0016393), the gene expression levels were significantly up-regulated in the mature leaves at the bottom of the one bud three leaf (C3) stage. The second branch including AVT1C (CSS00022138), AVT6C (CSS0029068), AAP7 (CSS0035834), AVT6A (CSS0043484), LHTL8 (CSS0033052), AVT1J (CSS0010301), AVT6A (CSS0014663), AVT1C (CSS0006346), AAP7 (CSS0034324), which were significantly up-regulated in the upper leaves that formed a source-sink relationship with new shoots. and the third branch including CAAT1 (CSS0020714), AAP6 (CSS0035405), LHTL8 (CSS0010852) and AVT3CA (CSS0027657), which were gradually up-regulated in upper mature leaves as new shoots developed; The fourth branch, including GAT1 (CSS0038477), GAT1 (CSS0006735), AVT1H (CSS0009001), AVT3C (CSS0002567), AAP3 (CSS0024604), CAAT1 (CSS0029843), AAP7 (CSS0035046), GAT1 (CSS0006897), AAP7 (CSS0013258), and the expression levels in upper mature leaves decreased with the development of new shoots.
4.4 WGCNA analysis
In order to further identify the key genes that regulate the amino acid transport in the phloem during the development of tea shoots, a weighted gene co-expression network analysis was performed. Based on similar expression patterns, a total of 12 co-expression modules were identified. The MEterqoise module exhibited a high positive correlation with most amino acids such as Thea, Glu, Gln, Asp, Trp, Leu, Phe, Val, Thr, and Met (Fig. 6A), indicating that the genes in this module might play an important role in the transport of amino acids from source to sink during shoot development.
Five DEGs with higher correlation with amino acid transport were selected and verified by the qRT-PCR method. The five genes related to amino acid transport were identified in the MEterqoise module, including LHTL8 (CSS0010852), AVT1J (CSS0010301), AVT1C (CSS0002138), AVT1C (CSS0006346), and AAP6 (CSS0035405). To verify the transcriptome data, qRT-PCR was performed. The results showed that the expression levels of five functional genes in the upper leaves were significantly higher than those in the lower leaves, and gradually up-regulated with the development of new shoots (Fig. 6B). The gene expression level reached its highest at the C3 stage. These results are consistent with the RNA-Seq data, indicating that the data can be used to evaluate the up-regulation and down-regulation of gene expression.
4.5 Tissue specific expression analysis and transient suppression of CsAAP6 in mature leaf
Among the 5 amino acid transporter genes, the CsAAP6 was the highest up-regulated gene. And our previous study showed expression of CsAAP6 had a significant positive correlation with total nitrogen amount in new shoots (Fan et al. 2020). So, the CsAAP6 was selected for the further research. The tissue specific expression analysis showed that expression level of CsAAP6 was highest in major veins of mature leaves (Fig. 7A).
To verify the function of CsAAP6 in loading amino acids in the phloem of mature leaves, the asOND was injected into mature leaves for 2 h, 6 h, 12 h, and 24 h. The qRT-PCR results showed that significant reductions in transcript levels were detected at 12 h (Fig. 7C,D). Among them, the best effect of gene silencing occurred at 12 h. The amino acid content of phloem exudates collected from silencing leaves at 12 h was measured.
Interestingly, the results showed that silencing the CsAAP6 gene reduced the content of various amino acid components in the phloem exudates (Fig. 7C). The contents of Glu, Asp, Gln, Asn, Ala in phloem exudates decreased (p < 0.05), and the Glu and Asp decreased significantly (p < 0.01).