Changes of cellulose, hemicellulose and lignin content in elephant grass stem at different development stages
The content of cellulose, hemicellulose and lignin in different stem segments ofT1, T2, T3 stage weremeasured. It was found that the content of cellulose and hemicellulose increased first and then decreased, for example,T1-S2 was higher than T1-S1, but T1-S3 was lower than T1-S2, whereas the content of lignin decreased gradually, that is, T1-S1 is the highestwhile T1-S3 is the lowest.The similar changes also appeared in different stem nodes at T2 and T3 stages. Meanwhile, by analyzing the content change of the same stem node in different development time, it was also found that with the increase of growth time of elephant grass, the content of cellulose and hemicellulose decreased, while the content of lignin increased (Fig.1b).
The results indicated that with the development of elephant grass, the cell gradually matures and ages, the main macromolecular cellulose and hemicellulose content of cell wall structure gradually decreases, while the lignin content gradually increases to maintain and support the strength of elephant grass stems.
Characteristics of cell wall in different developmental stages of elephant grass stem
The changes of cell wall morphology at different development stages of elephant grass, especially the primary and secondary cell wall were observed (Fig. 2a).In T1-S1 and T1-S2, the ratio of secondary cell wall (sw) thickness to primary cell wall (pw) thickness was 1.18 and 0.85, the ratio of sw/pw thickness in T2-S1 and T2-S2 was 1.67 and 0.92, and the ratio of sw/pw thickness of T3-S1 and T3-S3 was 2.15 and 1.25, respectively (Fig.2b).The above data showed that the change trendof sw/pw thickness of T1, T2 and T3 was consistent, that is, with the increase of development time, the development speed of secondary cell wall (sw) was faster than that of primary cell wall (pw). The ratio of sw/pw in S1 node of three different development stages was alsoanalyzed. It was found that with the increase of development time,the thickness ratio of sw/pw in T1-S1T2-S1, T3-S1 increasedsubsequently.The change of sw/pw thickness ratio in S2 and S3 stages of three different development stages showed same trends.
In order to further verify the morphological changes of cell wall during the development of elephant grass, we selected S1 and S5 stem segments in T3 period with a longer development time span as samples for micro-CT observation.The stem of elephant grass consists of epidermis, parenchyma cells and vascular bundles, in which vascular bundles are composed of phloem and xylem without cambium. Vascular bundles are scattered in parenchyma cells and cannot be thickened. With the development of stem tissue, S1 vascular bundle showed regular and compact arrangement(Fig. S1), and the content of cellulose and hemicellulose in vascular bundle decreases gradually, while the content of lignin increases gradually (Fig.1b), so as to meet the mechanical support need in the process of stem maturity of elephant grass.
Differential gene expression during the development of elephant grass stems
12 deepest samples (Table S1)were selected to reassemble the elephant grass transcriptome(Fig. S2) using Trinity software.The Pearson correlation coefficient based on the expression valueofeach library indicated that there was a high correlation between sample replicates (Fig. S3).Cluster analysis among samples showed that the development time of elephant grass was the main factor affecting the clustering. The DEGs in three developmental stages of elephant grass stems were analyzed, a total of 15611, 10235and27389 DEGs were identified in T1, T2, and T3, respectively(Fig.3).In addition, we also analyzed the DEGs of different stem segments of three development stages, and found that 147 of which were co-expressed in three segments of T1, 54 in the four segments of T2,and 91 in the five segments of T3 (Fig.3). The intersection of all DEGs at three different developmental stages was compared to determine the shared core set. It was found that 3852 genes were differentially expressed in three developmental stages (Fig.3d).
GO and KEGG analysis were performed on 3852 DEGs which were co-expressed in the three developmental stages.Four of the top 10 enriched GO annotation functions are related to cell composition such as apoplast, cell wall, extracellularregion, plant-type cell wall, 4 are related to molecular functions such as peroxidase activity,xyloglucan and xyloglucosyl transferase activity,heme-binding,xyloglucan−specific endo−beta−1,4−glucanase activity, and 2 were related to biological processes such as cell wall macromolecule catabolic process, hydrogenperoxidecatabolicprocess (Fig.4a).According to KEGG analysis, all DEGs were enriched to 9 pathways, of which the two most significant were phenylpropane metabolism（23 DEGs），and starch and sucrose metabolism（23 DEGs）(Fig.4b).
Geneshighly correlated with the synthesis of cellulose, hemicellulose and lignin by WGCNA analysis
Weighted gene co-expression network analysis (WGCNA) was performed on 3852 DEGs in three stages, and the network was divided into 3 modules. The analysis of module-trait relationship showed that the ‘blue’ module was highly correlated with the synthesis of cellulose (r=0.67, P=6.0×10-6) and hemicellulose (r=0.51, P=0.001), whereas the ‘turquoise’ module was related to lignin synthesis (r=0.68, P=5.0×10–6)(Fig. S4).
The ‘blue’ module was filtered according to Module membership > 0.9, the absolute value of the correlation coefficient between the ‘turquoise’ module and lignin was greater than 0.75, and the 20 and 23 genes remained in the ‘blue’ and‘turquoise’module respectively. The WGCNA gene significance (GS) (i.e. related to traits) showed that the genes with highest GS in the ‘blue’ and ‘turquoise’ modules were Cluster-55067.0 (0.659) and Cluster-17353.3 (0.798), respectively. Six of the 20 genes in the module ‘blue’ are known to be functional, such asGTL1 transcription factor, O-methyltransferase (OMT), expansin-likeA2, alpha-humulenesynthase, probable galactinolsucrose, GhGalT1. At the same time, 14 genes with unknown functions are also covered by the ‘blue’ module, which needs further study.The GO function annotations of these 20 genes in the module ‘blue’ included the genes which were related toextracellular region, C-4 methylsterol oxidase activity, terpene biosynthesis, etc.
Among the 23 genes in the module ‘turquoise’, there are eight coding genes such as GTL1, MYB2 transcription factors, threonine-protein kinase ERECTA, probable methionine-tRNA ligase, alcohol holding hydrogenase-like2, zinc finger proteinGIS3, protein slr0074, proline-rich receptor-like protein kinase PERK8. The GO function annotations of these 23 genes in the module ‘turquoise’included genes which were related tocell growth regulation, secondary cell wall formation regulation, cell wall composition regulation, etc. 15 genes remained with unknown functions (Table S2).
Lignin and cellulose synthesis pathway during stem development of elephant grass
Cellulose synthase (CesA) is the most important enzyme in the cellulose synthesis pathway.It can directly use UDPG produced in starch and sucrose metabolism to synthesize cellulose.In elephantgrass, we identified 27 CesA genes. In T1 stage, the expression level of CesA1-CesA6was higher in the whole stem, but decreased in T2 and T3 stages, while the expression level of CesA7-CesA27 gene in tender stems was much higher than that in mature stems in T1, T2, and T3 stages. This indicated that CesA gene mainly synthesizes cellulose in the tender stem tissue, when the cellulose accumulated to a certain level, its expression level gradually decreased (Fig.6a).
Lignin synthesis is one of the most important pathways of phenylpropane metabolism. In the lignin metabolism pathway, CAD (21 Unigenes), 4CL9 (21 Unigenes), C4H (8Unigenes), PAL (16Unigenes), CCR (25Unigenes), F5H (6Unigenes),CCoAOMT (4Unigenes) were identified to be related. Expression analysis found that most members of these gene families showed higher expression levels inmature stem, while a few gene members were continuously expressed in the whole tissue (Fig.6b).
The results of qRT-PCR demonstratedthat the expression trends of these genes were consistent with that of RNA-seq data. Overall, with the development of stem, the expression level of cellulose synthesis genes and the expression level of lignin synthesis related genes have the opposite trend, while the changes of stem cellulose, hemicellulose and lignin content, as well as the changes of stem primary wall and secondary cell wall thickness, are positively related to the expression of these two types of genes(Fig. S5).