Changes of lignin content in poplar stem caused by high temperature
The biosynthesis of lignin can be altered by kinds of abiotic and biotic stresses [4, 36]. To investigate the effect of high temperature on lignin content of poplar, poplar “84k” of 40 days old tissue culture plantlets were treated with 35℃ high temperature and 25℃ normal temperature. Compared to the plantlets grown at normal temperature of 25°C, determination of lignin content showed a slight increase in lignin content when exposed to high temperatures (Fig. 1). The results showed that poplar tissue culture plantlets tended to accumulate more lignin to resist heat stress when exposed to high temperature.
Effect of high temperature treatment on metabolites in lignin synthesis pathway
In order to further explore the substance in lignin synthesis pathway, targeted metabolomics analysis of 14 metabolites in the lignin synthesis pathway was performed. Lignin metabolism intermediates such as sinapic acid, coniferyl alcohol, p-coumaric acid, sinapyl alcohol, p-coumaryl alcohol, caffeyl alcohol, caffeyl aldehyde were detected but could not be quantified and analyzed because of their low levels. Among the seven intermediates of lignin metabolism quantified,sinapinaldehyde, p-coumaraldehyde, l-phenylalanine, ferulic acid and cinnamic acide showed no significant changes at different time periods after high temperature treatment (Supplementary materials 1; Fig. 2). However, it was found that caffeate and coniferous aldehyde always showed an upward trend in different periods after high temperature treatment, which may be the main reason for the increase of lignin content in poplar under high temperature stress (Supplementary materials 1; Fig. 2).
Trait of transcriptional database libraries
Both transcription factors and structural genes are involved in lignin synthesis by affecting the relevant enzymes in the lignin pathway. In order to investigate which genes are involved in the regulation of lignin after high temperature stress, 18 cDNA libraries (6 samples, 3 replicates) from 35℃ treated poplar stems at different time periods were established. 892458006 high-quality reads were obtained and transcriptome data were stored in NCBI database with SRA accession number PRJNA742770. About 70.69–74.14% reads were matched to the genome. It was found that the percentage (about 68.25–71.80%) of unique reads and clean reads in the genome was not much different. Therefore, the data quality and quantity of the transcriptome were reliable (Supplementary materials 2).
Characterization of differential genes expression profiles of poplar stem in response to high temperature
Five two-by-two comparisons, including 0 hour versus 6, 12, 24, 48, 72 hour respectively, were used to analyze the expression profiles of differential genes in the response to high temperature treatment in poplar stems. By correlating FPKM values, the FPKM heat map was constructed. The correlation value of three repeat samples at the same time was 1 while the correlation values of heat maps at normal and cold temperature at different period were all less than 0.8, indicating a high degree of consistency in biological repetition with high temperature treatment (Fig. 3A).
The genes with log2 foldchange ≥ 1 and FDR ≤ 0.05 were significant DEGs. According to the analysis of transcriptome data, there were 6480, 2421, 1640, 1452, 778 genes changed at 6, 12, 24, 48, and 72 hours, respectively (Supplementary materials 3; Fig. 3B). In detail, compared with 0 hour, the transcription level of 3274, 1446, 939, 852, 534 genes increased respectively, while the transcription level of 3206, 975, 703, 600, 244 genes decreased after 6, 12, 24, 48, and 72 hours high temperature treatment (Supplementary materials 3; Fig. 3B). The studies showed that high temperature can stimulate the expression of related genes in poplar stem, and the gene change caused by high temperature reduced with time.
Functional analysis of differential expressed genes (DEG)
To further study the comprehensive annotation of DEGs in response to heat stress, the COG, KOG, GO and KEGG, protein databases were applied to annotate all the DEGs.
Twenty-six functional clusters were used for COG analysis. The COG analysis varies with the processing time. In general, both COG and KOG analysis revealed that the differential genes were mainly involved in inorganic ion transport and metabolism, carbohydrate transport and metabolism, energy production and conversion, signal transduction mechanisms, RNA processing and modification, posttranslational modification, protein turnover, chaperones, general function prediction only, cell wall/membrane/envelope biogenesis and other pathways (Supplementary materials 4, Supplementary materials 5). As can be seen from the results, the pathways involved in the protein data analysis of KOG and COG are almost identical. In addition, the changes of genes related to secondary metabolites biosynthesis, transport and catabolism suggest that some secondary metabolites may also participate in the response of poplar stems to high temperature. This indicated that poplar responds to the adverse effects of high temperature in a variety of ways, including energy conversion and signal transduction. For GO enrichment analysis, three terms including molecular function, biological process and cellular component were used to classify the DEGs function. Among all DEGs, 3319 genes were compared to the GO enrichment data. Of which, 1610, 665, 466, 423, and 228 genes changed after high temperature treatment of 6, 12, 24, 48, and 72 hours, respectively. The changed genes were mainly related to cell, organelle, cell part, cell junction, and membrane in cellular component; catalytic activity and binding in molecular function; and metabolic process, cellular process, single-organism process, response to stimulus, biological regulation in biological process (Supplementary materials 6). By analyzing the biological process, it was found that genes related toplant-type secondary cell wall biogenesis, the lignin catabolic process and lignin biosynthetic process which may influence lignin content changed significantly (Fig. 4).
Differential genes were selected for annotation analysis of KEGG database after high temperature treatment at 6, 12, 24, 48 and 72 hours. The results showed that the differential gene enrichment pathway decreased with the extension of treatment time (Supplementary materials 7). KEGG enrichment analysis at 0 h vs 6, 12, 24, 48, 72 h demonstrated that the enrichment pathways were mainly involved in starch and sucrose metabolism, biosynthesis of amino acids, plant hormone signal transduction, carbon metabolism, phenylpropanoid biosynthesis, etc (Supplementary materials 7). In general, the KEGG enrichment analysis of phenylpropanoid biosynthesis may be related with lignin synthesis.
Lignin synthesis related genes responding to high temperature stress
To investigate the genes involved in lignin synthesis in response to high temperatures, MYB, NAC and some structural genes in lignin synthesis pathway were analyzed. The results showed that a total of 843 genes may be involved in the process of lignin synthesis in poplar in response to high temperatures. In detail, there were 9 NACs, 558 MYBs, 6 PALs, 1 C4H, 2 C3'Hs, 16 4CLs, 35 HCTs, 32 CCRs, 1 COMT, 3 F5Hs, 4 CCoAOMTs, 42 CADs, 46 LACs, and 88 PODs (Supplementary materials 8).
The trends of the above lignin-related genes were analyzed by RPKM values (Fig. 5). Among the exchanged gene in lignin synthesis, PtrC3'H1 (Potri.006G033300), PtrCCR2 (Potri.003G181400), PtrMYB021 (Potri.009G053900) and PtrMYB074 (Potri.015G082700), PtrMYB103/46 (Potri.003G132000), PtrMYB090 (Potri.015G033600), PtrMYB161 (Potri.007G134500), PtrMYB3 (Potri.001G267300), PtrMYB125/85 (Potri.003G114100), PtrMYB093 (Potri.004G138000) may be involved in high temperature-induced synthesis of caffeate, coniferaldehyde and lignin. The other genes with more significant changes might also be involved in the process of lignin synthesis caused by high temperature.
RT-qPCR analysis of major lignin synthesis related genes
According to the network map of lignin synthesis pathway, some transcription factors such as MYBs, NACs and structural genes were found to be involved in the synthesis of caffeate, coniferaldehyde and lignin in response to high temperatures in poplar. By searching for related genes and designing primers, the transcript levels of several important genes, PtrC3'H1 (Potri.006G033300), PtrCCR2 (Potri.003G181400), PtrMYB021 (Potri.009G053900) and PtrMYB074 (Potri.015G082700), PtrMYB103/46 (Potri.003G132000), PtrMYB090 (Potri.015G033600), PtrMYB161 (Potri.007G134500), PtrMYB3 (Potri.001G267300), PtrMYB125/85 (Potri.003G114100), PtrMYB093 (Potri.004G138000),were examined by RT-qPCR analysis. The results showed that the transcript levels of these genes and the data from transcriptome analysis were consistent. It indicated that the transcriptome data were reliable and reproducible, and that these genes may play important roles in the regulation of lignin synthesis in response to high temperature in poplar seedlings.