Genome-wide analysis of cellulose synthase (CesA) and cellulose synthase-like (Csl) proteins in Cannabis sativa L

Introduction

Cannabis sativa L., an annual herbaceous plant, has versatile usage features as raw material in paper, textile, biofuel, automotive, and construction industries, mainly due to its cell wall content and other strains are useful as food and pharmaceuticals. Hemp is a preferable fiber source to cotton and other petroleum-derived synthetic fibers because it can be grown even in areas where water, fertilizer, and pesticide use is limited, and it yields a large amount of biomass in a short time (Manaia, Manaia & Rodriges, 2019). The hemp bast fiber, the outer part of the hemp stem known as phloem fiber, consists of cellulose microfibrils, which are contained in a matrix of hemicellulose and lignin (Behr et al., 2016).

The production and processing of hemp fiber in textiles depend on the amount and distribution of cellulose, hemicellulose, and lignin components in the fiber, which affects the mechanical properties of the fiber. Hemp fibers consist of 53–91% cellulose, 4–18% hemicellulose, 1–17% pectin, and 1–21% lignin, depending on the growing conditions, harvest year and location (Liu et al., 2017). 

Cellulose is synthesized in the plasma membrane and located in the cell wall as microfibrils, which are the long chain structure of 1,4-D-glucose units connected by glycosidic bonds (Mujtaba et al., 2017). These microfibrils constitute the scaffold of the cell wall. The stiffness and organization of cellulose microfibrils in the cell wall affect cell growth and increase the cell’s resistance to osmotic pressure (Cosgrove, 2005; Hu et al., 2018). The second major structural component of the cell wall is hemicellulose. Hemicellulose, which has branched polymers consisting of 500–3000 sugar units, is a heteropolymer composed of different types of hemicellulose such as xylan, glucuronoxylan, glucomannan, and xyloglucan. After cellulose fibers are synthesized in the cell wall, they are cross-linked with pectin and hemicelluloses.

Exposing the biosynthesis, assembly, organization, and networking of the cell wall load-bearing cellulosic fibrils is complex and essential (Zhang et al., 2021). Cellulose and hemicellulose polysaccharides are synthesized by the enzymes of the cellulose synthase A (CESA) family and the cellulose synthase-like (CSL) family, respectively. These families belong to the glycosyltransferase 2 (GT2) superfamily (Richmond & Somerville, 2000). In Arabidopsis, ten members of the cellulose synthase (CESA) family have been identified, designated as CESA1 to CESA10. Among these, CESA1, CESA3, and one member from the group consisting of CESA2, CESA5, CESA6, or CESA9, are assembled into the Cellulose Synthase Complex responsible for cellulose synthesis in the primary cell wall (Zhang et al., 2021). Another distinct Cellulose Synthase Complex, composed of CESA4, CESA7, and CESA8, is involved in cellulose synthesis in the secondary cell wall (Taylor et al., 2003).

The genes of the cellulose synthase-like (CSL) family, grouping in from CSLA to CSLG, encode enzymes that generate hemicellulose, including xylans, xyloglucans, mannans, glucomannans and β-(1,3;1,4) glucan (Holland et al., 2000). A total of 30 genes belonging to the Cellulose synthase-like (CSL) family have been identified in Arabidopsis. CSLA, CSLC, and CSLF  are responsible for the biosynthesis of mannan, xyloglucan, and (1 → 3; 1 → 4)- β-D-glucan, respectively (Arioli et al., 1998; Richmond & Somerville, 2000; Richmond & Somerville, 2001; Lerouxel et al., 2006; Cocuron et al., 2007; Dwivany et al., 2009; Doblin, Pettolino & Bacic, 2010).  CSLD  genes are involved in synthesizing xylan, homogalacturonan, and mannan (Bernal et al., 2007). CSLJ are responsible for (1, 3; 1, 4)- β-glucan biosynthesis (Little et al., 2018). 

Because CESA and CSL  genes play a crucial role in plant growth, immunity responses to pathogens, and plant biomass increase, genome-wide characterization of cellulose synthase and cellulose synthase-like gene families have been studied in a variety of plants, such as rice (Hazen, Scott-Craig & Walton, 2002), flax (Guo et al., 2022), pineapple (Cao et al., 2019), maize (Appenzeller et al., 2004), tomato (Song et al., 2019), tea (Li et al., 2022) and strawberry (Huang et al., 2022). Subsequently, over-expression and silencing experiments of certain CESA and CSL genes were performed to identify their roles in plant development and growth, plant phenotype and defense, cell wall integrity, and osmotic stress (Held et al., 2008; Zhu et al., 2010; Chowdhury et al., 2016; Douchkov et al., 2016; Mabuchi et al., 2016; Mazarei et al., 2018; Huang et al., 2022; Li et al., 2022; Zhao et al., 2022). For example, rice OsCSLD4 may regulate the function of cell wall localized proteins through its effect on cell wall polysaccharide composition, which in turn modulates intracellular signaling and regulates ABA synthesis, which has a critical role in regulating plant responses to salt stress, growth and development (Zhao et al., 2022). In a study on the role of CSLs in plant defence, down-regulation of glucan synthase-like 6 gene (HvGsl6) in barley led to lower papillary and wound callose deposition and increased cell wall penetration of Blumeria graminis f (Bgf) (Chowdhury et al., 2016). Thus, it has been proven that the HvGsl6 plays a role in the deposition of callose, which contributes positively to the penetration resistance mechanism of Bgf. Likewise, the papillae of HvCslD2-silenced barley were more successfully penetrated by host-adapted, virulent, and avirulent non-host isolates of the powdery mildew fungus, and this papillary penetration was associated with lower cellulose content in the epidermal cell walls and increased digestion by fungal cell wall-degrading enzymes (Douchkov et al., 2016). Thus, barley CslDs have been identified to exhibit functional diversity, such as cell wall biosynthesis in dividing cells or local cell wall reinforcement during pathogen invasion attempts (Douchkov et al., 2016).

Although cellulose/hemicellulose is important in increasing the economic value of cannabis in terms of protection from environmental effects such as disease resistance and providing raw materials for industrial applications such as textiles and biodiesel, there is no report on the genome-wide identification and functional analysis of cellulose/hemicellulose synthesis genes in cannabis. Therefore, this study aimed to identify CESA/CSL gene family members in the cannabis genome and reveal their structure and evolution relationships. In this study, detailed identification of CESA/CSL genes and analysis of their gene expression in tissues will create a foundation for understanding their structural, functional, and evolutionary properties.

Materials & Methods

Results

A total of 29, 30 and 39 CESA/CSL genes were identified in ASM2916894v1 (cultivar pink pepper), JL_Mother and JL_Father (cultivar Jamaican Lion $\stackrel{ˆ}{}$4) genomes of C. sativa, respectively (Tables S2 and S3). Two conserved domains, namely RING/U-box type zinc-binding domain (PF14569) and cellulose synthase (PF03552), were identified in the cellulose synthase A protein (CESA). The structural characteristics of the cellulose synthase-like proteins showed that CSLA proteins (glucomannan 4-beta-mannosyltransferase 9) and CSLC (xyloglucan glycosyltransferase) contained glycosyltransferase-like family 2 (PFAM 13641) and glycosyl transferase family group 2 (PF13632) domains, respectively. CSLD (cellulose-synthase-like D1) proteins included RING/Ubox like zinc-binding (PF14570) and/or cellulose synthase (PF03552) domains. Finally, the CSLB, CSLE, CSLG cellulose synthase-like proteins contained the cellulose synthase domain (PF03552). Also, all proteins included transmembrane and signal domains.

A total of eight CsCESA and 21 CsCSL genes (3 CsCSLA, 2 CsCSLB, 4 CsCSLC, 5 CsCSLD, 5 CsCSLE, 2 CsCSLG) from ASM2916894v1 genome were numbered according to their chromosomal locations and named species names (Table S2), These 29 genes were analyzed bioinformatically.

Based on the phylogenetic classification, eight CsCESA proteins were grouped in the CESA clade, which is the largest clade. The cellulose synthase-like proteins were classified into six CsCSL subfamilies (CSLA, CSLB, CSLC, CSLD, CSLE, CSLG) (Fig. 1). Eexceptionally, while no any CsCSL proteins from the pink pepper genome were clustered in the CSLM subgroup, only one protein from the JL_Mother genome (Fig. S1) and two from the and JL_Father genome were clustered in this subgroup (Fig. S2).

Many cellulose synthases were predicted to be localized mainly in the plasma membrane and endoplasmic reticulum, followed by the nucleus, vacuole, mitochondria and Golgi body (Fig. 2). Only CsCSLB2 was present in the chloroplast and CsCESA1 and CSLA2 in the extracellular membrane.

All CsCESA/CsCSL genes detected in cannabis genome (ASM2916894v1) were mapped and unequally distributed on all chromosomes of C. sativa except chromosome 7 (Fig. 3). The highest number of genes were found on chromosome 1 with six, while the lowest number was on chromosome 2 and 9. Also, a total of two duplication events were observed.

Cis-acting elements are conserved nucleotide sequences in the gene’s promoter to which transcription factors can bind, and they regulate the transcription of the gene of interest. Variations in the Cis-acting elements in the promoter regions of these genes may lead to variations in the phenotypic characteristics of the organism, such as its development and response to biotic and abiotic factors. A two kilo base upstream sequence of the CsCESA/CsCSL genes was searched to identify cis-acting elements. These cis-elements were categorized into four main groups: light-responsive elements, environmental stress-responsive elements, hormone-responsive elements, and development-related elements. Twenty-five light-responsive elements were found (Fig. 4A). All but one CsCESA/CsCSL contained the Box 4 motif, making it the most abundant light response element. Thirteen cis-acting elements were involved in environmental stress response (Fig. 4B). MYB (drought and ABA signaling), MYC (drought, salt, and stress response), STRE (multiple signaling including heat stress), LTR (low-temperature response), ARE (anaerobic induction) motifs were most abundant in CsCESA/CsCSL gene promotors. Thirteen cis-acting hormone response elements were detected (Fig. 4C). ABRE (abscisic-acid-responsive element), ERE (the ethylene-responsive element), CGTCA motifs and TGACG motifs (MeJA-responsive elements) were the most common. CsCESA/CsCSL genes carrying hormone response cis-regulatory elements might be upregulated by these hormone treatments. TGA-box (auxin-responsive element) was found only in one CsCSLE4. Finally, fourteen development response cis-acting elements were found (Fig. 4D). Among them, the AAGAA-motif (the endosperm-specific negative expression), O2-site (zein metabolism regulation), and as-1 (the root-specific expression) were found to be the most abundant elements.

A total of 15 conserved protein motifs were searched (Fig. 5B). Consistent with conserved domain analysis, CsCSLA shared similar motif composition with CsCSLC, CsCSLG with CsCSLE and CsCSLB, and CsCESA with class CsCSLD. CsCESA and CSLDs have the highest number of motifs, with 15 motifs.

Considerable diversity of the exon-intron structures of the CsCESA/CsCSL genes was noted (Fig. 5C). CsCESA genes usually have 14 exons, with the exciting exceptions that CsCESA1 has only one exon, while the longest gene, CsCESA3 has 26 exons. Although the exon-intron numbers of the CsCSLA and CsCSLB genes were the same within the members of their subfamilies, diversities were observed in the lengths of the introns. CsCSLC and CsCSLD genes have between four to five exons. The highest intron length variation was in the CsCSLE subfamily, with eight exons.

Syntenic maps were constructed between CESA/CSL genes of C. sativa and A. thaliana (Fig. 6, Table S4). Nine C. sativa CESA/CSL genes were found orthologous to A. thaliana genes.

The nonsynonymous (Ka)/synonymous (Ks) ratios were calculated to reveal the evolutionary status of the two linked CsCESA/CsCSL gene pairs (Table S6). The Ka/Ks ratios of two linked CsCESA gene pairs (CsCESA1/CsCESA6 and CsCESA7/CsCESA8) were found to be less than one, indicating the presence of purifying selection and, in this case, the maintenance of the number of members in this gene family.

The members of the cellulose synthase family exhibit tissue-specific expression (Hamann et al., 2004). To investigate the expression patterns of CESA/CSL genes in different tissues (flower, leaf, root, stem) of cannabis, the expression of 14 CESA/CSL genes, selected based on phylogenetic groups, gene structures, and cis-elements was evaluated by RT-qPCR (Fig. 7, Table S7). All examined CsCESA genes were expressed in four different tissues with variable levels, suggesting that all these CESA genes are necessary for primary or secondary cell wall formation. One of the CsCESA genes (Fig. 7A) was expressed relatively higher in flower and the other two (b, c) in leaf, but the difference in the expression levels of these genes between the tissues was not statistically significant. In addition, the expression level of one CsCESA gene (Fig. 7D) was highest in stem and lowest in flower. CsCSLA (Fig. 7F), CsCSLB (Fig. 7G), CsCSLG (Fig. 7M) showed good leaf-specific expression. CsCSLC (Fig. 7H) exhibited the highest expression in the flower, whereas CsCSLD, CsCSLD, CsCSLG genes (Figs. 7J, 7K and 7N) were expressed only in root and stem.

Discussion

The structure, composition, and organization of cell wall components have critical effects in terms of affecting the growth and development of the plant and its response to environmental stimuli by determining the cell’s shape, strength, structural integrity and response to abiotic and biotic environmental stresses. Furthermore, the substances of the cell wall affect the quality and processing of the plant for use as paper, textiles, bioethanol, feed and food.

The plant cell wall matrix mainly comprises polysaccharides such as cellulose, hemicellulose and pectin. The cellulose is embedded in the cell wall matrix as microfibrils. Microfibrils are a bundle of 40 cellulose molecules that run parallel to each other and are attached by hydrogen bonds. Each cellulose molecule is an unbranched polymer composed of glucose units linked by an β-1,4-bond. It is estimated that hundreds of enzymes are responsible for cell wall biosynthesis (Keegstra & Raikhel, 2001; Scheible & Pauly, 2004; Liepman & Cavalier, 2012). Cellulose Synthase (CESA) and Cellulose Synthase-Like (CSL) family proteins synthesize various β-glycan polymers. In this study, CESA and CSL family proteins were identified and characterized, and their phylogenetic relationships were revealed by using genome sequence and bioinformatics analysis tools in cannabis, which is a fiber and biofuel source and has a crucial phytochemical content.

To better understand the CsCESA/CsCSL characteristics in cannabis, genome-wide analyses were carried out via phylogenetic relationships with existing gene structures and expression in different tissues. 8 CsCESA and 21 CsCSL (3 CsCSLA, 2 CsCSLB, 4 CsCSLC, 5 CsCSLD, 5 CsCSLE, 2 CsCSLG) genes in cannabis ASM2916894v1 genome resemble the CESA/CSL phylogenetic classification in other species. As expected, the monocot-specific CSLF, CSLH and CSLJ protein subfamilies were not found in the cannabis genome.

Syntenic relationships are shaped by conservation or DNA substitution rates among taxa and genes in the genomes of different species. Gene orthology relationship determined by synteny data analysis can support the phylogenetic relationships of multiple gene families (Gabaldón & Koonin, 2013). The orthologous relationships of CsCSL genes with A. thaliana, with the exception of CsCESA4 gene, supported their close relationship in the phylogenetic tree (Table S4, Fig. 1).

The existence of closely clustered CESA/CSL subfamily members between cannabis and other eight species used in phylogenetic tree resulted from evolutionary conservation and closer homology (Fig. 1). According to phylogenetic tree analysis (Fig. 1), CsCESA1/7 and CsCESA2 were closely clustered with AtCESA1 and AtCESA3, respectively. In addition, AtCESA5/6/2/9 are grouped with CsCESA5/8/6. AtCESA1, AtCESA3, and any of AtCESA2/ 5/ 6, or /9 complex cause primary wall cellulose accumulation with unequal enzymatic activity in cells undergoing cell division and elongation (Persson et al., 2007; Hu et al., 2018). The results inferred that these orthologous CsCESA1/7/2, or 5/8/6 genes may have similar functions.

The expression analysis showed that four CsCESA genes were expressed in four different tissues with varying levels. Only one CESA gene (Fig. 7D) were expressed at highest level in the stem. These results may support that CsCESA genes play roles in the growth of different tissues. In addition, Arabidopsis CESA4/7/8 are involved in secondary wall thickness by increasing the amount of cellulose (Tanaka et al., 2003; Taylor et al., 2003; Zhong, Cui & Ye, 2019) and phylogenetically clustered together with CsCESA4/6/3 gene homologous, respectively (Fig. 1). These results suggest that CsCESA4/6/3 might participate in establishing secondary cell walls.

Revealing gene structures and protein motif patterns of gene family members may elucidate the evolution and diversity of their structure and function. Most CsCESA and CsCSL subfamily members, closely grouped in the phylogenetic tree according to their amino acid sequences (Fig. 5A), share the same or similar motif distributions.

The motif components of CsCESA and CsCSLD were highly similar. However, the CsCSLA and CsCSLC genes had different motif patterns. Although the members of the CsCESA/CsCSL subfamily were generally similar in terms of exon and intron number and length, they also showed some differences (Fig. 5C). A similar situation has been noted for members of the DcCSLD subfamily in Dendrobium catenatum, and it has been reported that intron gain/loss may occur in the evolution of DcCSLD genes (Xi et al., 2021).

Several members of the CESA/CSL gene family have been reported to have varying expression levels depending on tissue type, growth stage, and environmental factors (Cao et al., 2019; Li et al., 2020; Li et al., 2022; Song et al., 2019; Kaur et al., 2017; Yuan et al., 2021; Liu et al., 2022; Nawaz et al., 2017; Marcotuli et al., 2018; Hou et al., 2023). Likewise, the results of this study identified different expression levels of several CsCESA/CsCSL genes in four tissues. CSLA (Fig. 7F), CSLB (Fig. 7G) and CSLG (Fig. 7M) were expressed mainly in the leaf. In contrast, CsCSLD (Figs. 7J and 7K) and CsCSLG (Fig. 7N) were expressed only in root and stem, whereas CSLC (Fig. 7H) was mainly expressed in the flower. In the previous study conducted to determine transcriptomic changes associated with bast fiber development stage in textile hemp, certain CES/CSL family members showed differential expression patterns in the stem’s upper, middle, and lower internodes (Guerriero et al., 2017). For example, the annotation against the Arabidopsis database showed that some contigs annotated with CSLC5 were more highly expressed in the upper internode, with CSLC04 in the middle internode, with CSLE1, CSLG1 and CSLB04 in the lower internode. The progressive decrease in expression from the top to the bottom of the stem was detected in CSLC04 and CSLC5 genes, while progressive increase in expression along the stem axis for CSLE1, CSLG1, IRX1, CSLG3, CSLE1, CSLB04 genes.

In another important fiber plant, flax, expression differences were also found in different growth stages and stem parts, confirming the role of CESA/CSL genes in cell wall thickening (Guo et al., 2022). The flax CESA3/8 (Lus10007538, Lus10007296) and CSLD4 (Lus10008225) genes have been shown to be active during early fiber development. These genes are specifically expressed at the stage of fiber development when there is an increased amount of secondary cell wall deposition during the period of rapid growth. The flax CESA6 genes (Lus10006161.g and Lus10041063.g) were found to be specifically expressed in the stem during fiber maturation. The expression levels of LusCSLE1 (Lus10016625) and the two LusCSLG3 (Lus10023056.g and Lus10023057.g) genes were higher in 30 cm plants than in 50 cm plants. The opposite trend occurred for CESA6 (Lus10006161.g and Lus10041063.g) genes. The phylogenetic tree of flax and cannabis CESA/CSL genes showed that the flax CESA/CSL genes were closely clustered with those of cannabis (Fig. 1). This may indicate that flax and cannabis CESA/CSL genes may have similar functions during the fiber development stages.

In addition to improving fiber quality, a mutant allele of CESA3 gene was developed using CRISPR/Cas9 base editing to improve herbicide resistance in Arabidopsis plants, and this mutant allele was found to confer plant resistance to the herbicide C17 (Hu et al., 2019). Shortly, editing CESA/CSL genes could lead to advances in areas such as enhancing plant growth and development, and improving disease and pest resistance. Also, the silencing or overexpression of genes associated with cell wall synthesis may indicate changes in plant-pathogen interactions in the cell wall, which may shed light on whether cannabinoid production is affected. In this study, the genome-wide identification of CsCESA/CsCSL genes will provide the basis for future investigation of their role.

Conclusions

In this study, eight CsCESA and 21 CsCSL genes in C. sativa genome (ASM2916894v1) were identified according to their conserved domains and motifs. Their features were characterized, including gene structure, chromosome location, phylogenetic analysis and syntenic relationships. The cis-element analysis identified the multifunctional role of CsCESA/CsCSL genes in growth, hormone responsiveness, and biotic and abiotic stress responsiveness. CsCESA/CsCSL genes exhibited diverse expression patterns in flower, leaf, root, and stem tissues. The detailed characterization of CESA/CSL in cannabis may aid in designing experiments for future genetic manipulation of cellulose and hemicellulose synthesis genes to breed cultivars with high fiber quality and bioethanol yield.

Supplemental Information