Secondary Cell Wall Synthesis Research Articles

Expression profile analysis of cotton fiber secondary cell wall thickening stage.

To determine the genes associated with the fiber strength trait in cotton, three different cotton cultivars were selected: Sea Island cotton (Xinhai 32, with hyper-long fibers labeled as HL), and upland cotton (17-24, with long fibers labeled as L, and 62-33, with short fibers labeled as S). These cultivars were chosen to assess fiber samples with varying qualities. RNA-seq technology was used to analyze the expression profiles of cotton fibers at the secondary cell wall (SCW) thickening stage (20, 25, and 30 days post-anthesis (DPA)). The results showed that a large number of differentially expressed genes (DEGs) were obtained from the three assessed cotton cultivars at different stages of SCW development. For instance, at 20 DPA, Sea Island cotton (HL) had 6,215 and 5,364 DEGs compared to upland cotton 17-24 (L) and 62-33 (S), respectively. Meanwhile, there were 1,236 DEGs between two upland cotton cultivars, 17-24 (L) and 62-33 (S). Gene Ontology (GO) term enrichment identified 42 functions, including 20 biological processes, 11 cellular components, and 11 molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis identified several pathways involved in SCW synthesis and thickening, such as glycolysis/gluconeogenesis, galactose metabolism, propanoate metabolism, biosynthesis of unsaturated fatty acids pathway, valine, leucine and isoleucine degradation, fatty acid elongation pathways, and plant hormone signal transduction. Through the identification of shared DEGs, 46 DEGs were found to exhibit considerable expressional differences at different fiber stages from the three cotton cultivars. These shared DEGs have functions including REDOX enzymes, binding proteins, hydrolases (such as GDSL thioesterase), transferases, metalloproteins (cytochromatin-like genes), kinases, carbohydrates, and transcription factors (MYB and WRKY). Therefore, RT-qPCR was performed to verify the expression levels of nine of the 46 identified DEGs, an approach which demonstrated the reliability of RNA-seq data. Our results provided valuable molecular resources for clarifying the cell biology of SCW biosynthesis during fiber development in cotton.

CcNAC6 Acts as a Positive Regulator of Secondary Cell Wall Synthesis in Sudan Grass (Sorghum sudanense S.).

The degree of forage lignification is a key factor affecting its digestibility by ruminants such as cattle and sheep. Sudan grass (Sorghum sudanense S.) is a high-quality sorghum forage, and its lignocellulose is mostly stored in the secondary cell wall. However, the secondary cell wall synthesis mechanism of Sudan grass has not yet been studied in depth. To further study the secondary cell wall synthesis mechanism of Sudan grass using established transcriptome data, this study found that CcNAC6, a homologous gene of Arabidopsis AtSND2, is related to the secondary cell wall synthesis of Sudan grass. Accordingly, we constructed a CcNAC6-overexpressing line of Arabidopsis to investigate the function of the CcNAC6 gene in secondary cell wall synthesis. The results showed that the overexpression of the CcNAC6 gene could significantly increase the lignin content of Arabidopsis. Based on subcellular localization analysis, CcNAC6 is found in the nucleus. In addition, yeast two-hybridization screening showed that CcCP1, associated with secondary cell wall synthesis, can interact with CcNAC6. Therefore, the above results indicate that CcNAC6 has a positive regulatory effect on the secondary cell wall synthesis of Sudan grass, and it is speculated that CcNAC6 may be the main regulator of the secondary cell wall synthesis of Sudan grass through its interaction with another regulatory protein, CcCP1. This study provides a theoretical basis and new genetic resources for the creation of new Sudan grass germplasm with a low lignin content.

Comparative Transcriptomic Analysis of Gossypium hirsutum Fiber Development in Mutant Materials (xin w 139) Provides New Insights into Cotton Fiber Development.

Cotton is the most widely planted fiber crop in the world, and improving cotton fiber quality has long been a research hotspot. The development of cotton fibers is a complex process that includes four consecutive and overlapping stages, and although many studies on cotton fiber development have been reported, most of the studies have been based on cultivars that are promoted in production or based on lines that are used in breeding. Here, we report a phenotypic evaluation of Gossypium hirsutum based on immature fiber mutant (xin w 139) and wild-type (Xin W 139) lines and a comparative transcriptomic study at seven time points during fiber development. The results of the two-year study showed that the fiber length, fiber strength, single-boll weight and lint percentage of xin w 139 were significantly lower than those of Xin W 139, and there were no significant differences in the other traits. Principal component analysis (PCA) and cluster analysis of the RNA-sequencing (RNA-seq) data revealed that these seven time points could be clearly divided into three different groups corresponding to the initiation, elongation and secondary cell wall (SCW) synthesis stages of fiber development, and the differences in fiber development between the two lines were mainly due to developmental differences after twenty days post anthesis (DPA). Differential expression analysis revealed a total of 5131 unique differentially expressed genes (DEGs), including 290 transcription factors (TFs), between the 2 lines. These DEGs were divided into five clusters. Each cluster functional category was annotated based on the KEGG database, and different clusters could describe different stages of fiber development. In addition, we constructed a gene regulatory network by weighted correlation network analysis (WGCNA) and identified 15 key genes that determined the differences in fiber development between the 2 lines. We also screened seven candidate genes related to cotton fiber development through comparative sequence analysis and qRT-PCR; these genes included three TFs (GH_A08G1821 (bHLH), GH_D05G3074 (Dof), and GH_D13G0161 (C3H)). These results provide a theoretical basis for obtaining an in-depth understanding of the molecular mechanism of cotton fiber development and provide new genetic resources for cotton fiber research.

Deciphering the Molecular Mechanism of the Intermediate Secondary Growth and Internode Elongation of the Castor Bean (Ricinus communis L.) by the Combined Analysis of the Transcriptome and Metabolome.

The length of internodes plays a crucial role in determining the height of the castor plant (Ricinus communis L.). However, the specific mechanisms underlying internode elongation, particularly in the main stem of the castor plant, remain uncertain. To further investigate this, we conducted a study focusing on the internode tissue of the dwarf castor variety 071113, comparing it with the control high-stalk Zhuansihao. Our study included a cytological observation, physiological measurement, transcriptome sequencing, and metabolic determination. Our integrated findings reveal that the dwarf variety 071113 undergoes an earlier lignification development in the main stem and has a more active lignin synthesis pathway during internode intermediate development. In addition, the dwarf variety exhibited lower levels of the plant hormone indole-3-acetic acid (IAA), which had an impact on the development process. Furthermore, we identified specific enzymes and regulators that were enriched in the pathways of the cell cycle, auxin signal transduction, and secondary cell wall synthesis. Using these findings, we developed a model that explained the intermediate secondary growth observed in castor internode elongation and enhanced our comprehension of the dwarfing mechanism of the 071113 variety. This research provides a theoretical groundwork for the future breeding of dwarf castor varieties.

GhMYB30-GhMUR3 affects fiber elongation and secondary wall thickening in cotton.

Xyloglucan, an important hemicellulose, plays a crucial role in maintaining cell wall structure and cell elongation. However, the effects of xyloglucan on cotton fiber development are not well understood. GhMUR3 encodes a xyloglucan galactosyltransferase that is essential for xyloglucan synthesis and is highly expressed during fiber elongation. In this study, we report that GhMUR3 participates in cotton fiber development under the regulation of GhMYB30. Overexpression GhMUR3 affects the fiber elongation and cell wall thickening. Transcriptome showed that the expression of genes involved in secondary cell wall synthesis was prematurely activated in OE-MUR3 lines. In addition, GhMYB30 was identified as a key regulator of GhMUR3 by Y1H, Dual-Luc, and electrophoretic mobility shift assay (EMSA) assays. GhMYB30 directly bound the GhMUR3 promoter and activated GhMUR3 expression. Furthermore, DAP-seq of GhMYB30 was performed to identify its target genes in the whole genome. The results showed that many target genes were associated with fiber development, including cell wall synthesis-related genes, BR-related genes, reactive oxygen species pathway genes, and VLCFA synthesis genes. It was demonstrated that GhMYB30 may regulate fiber development through multiple pathways. Additionally, GhMYB46 was confirmed to be a target gene of GhMYB30 by EMSA, and GhMYB46 was significantly increased in GhMYB30-silenced lines, indicating that GhMYB30 inhibited GhMYB46 expression. Overall, these results revealed that GhMUR3 under the regulation of GhMYB30 and plays an essential role in cotton fiber elongation and secondary wall thickening. Additionally, GhMYB30 plays an important role in the regulation of fiber development and regulates fiber secondary wall synthesis by inhibiting the expression of GhMYB46.

An essential role for mannan degradation in both cell growth and secondary cell wall formation.

Coordination of secondary cell wall deposition and cell expansion during plant growth is required for cell development, particularly in vascular tissues. Yet the fundamental coordination process has received little attention. We observed that the Arabidopsis endo-1,4-mannanase gene, AtMAN6, is involved in the formation of cell walls in vascular tissues. In the inflorescence stem, the man6 mutant had smaller vessel cells with thicker secondary cell walls and shorter fiber cells. Elongation growth was reduced in the root, and secondary cell wall deposition in vessel cells occurred early. Overexpression of AtMAN6 resulted in the inverse phenotypes of the man6 mutant. AtMAN6 was discovered on the plasma membrane and was specifically expressed in vessel cells during its early development. The AtMAN6 protein degraded galactoglucomannan to produce oligosaccharides, which caused secondary cell wall deposition in vessel and fiber cells to be suppressed. Transcriptome analysis revealed that the expression of genes involved in the regulation of secondary cell wall synthesis was changed in both man6 mutant and AtMAN6 overexpression plants. AtMAN6's C-terminal cysteine repeat motif (CCRM) was found to facilitate homodimerization and is required for its activity. According to the findings, the oligosaccharides produced by AtMAN6 hydrolysis may act as a signal to mediate this coordination between cell growth and secondary cell wall deposition.

Enhancement of astaxanthin production, recovery, and bio-accessibility in Haematococcus pluvialis through taurine-mediated inhibition of secondary cell wall formation under high light conditions

This study explored the use of taurine in enhancing the production and bio-accessibility of astaxanthin in Haematococcus pluvialis, which typically forms a secondary cell wall hindering astaxanthin extraction. The biomass of taurine-treated group significantly increased by 18%, and astaxanthin yield surged by 34% in comparison to the control group. Without cell disruption, astaxanthin recovery from thin-walled cells in the taurine-treated group, using dimethyl sulfoxide and ethanol as extraction reagents, was 97% and 75%, respectively, which were 30-fold higher than those of thick-walled cells in the control group. Additionally, the cell fragmentation rate increased by 86% in taurine-treated group relative to the control group. Comparative transcriptome analysis identified taurine-induced upregulation of genes involved in the astaxanthin biosynthesis pathway and downregulation of those associated with secondary cell wall synthesis. This study thus offers an innovative taurine-based strategy to enhance astaxanthin production and bio-accessibility while shedding light on the mechanisms driving this process.

Role of Actin Dynamics and GhACTIN1 Gene in Cotton Fiber Development: A Prototypical Cell for Study.

Cotton crop is considered valuable for its fiber and seed oil. Cotton fiber is a single-celled outgrowth from the ovule epidermis, and it is a very dynamic cell for study. It has four distinct but overlapping developmental stages: initiation, elongation, secondary cell wall synthesis, and maturation. Among the various qualitative characteristics of cotton fiber, the important ones are the cotton fiber staple length, tensile strength, micronaire values, and fiber maturity. Actin dynamics are known to play an important role in fiber elongation and maturation. The current review gives an insight into the cotton fiber developmental stages, the qualitative traits associated with cotton fiber, and the set of genes involved in regulating these developmental stages and fiber traits. This review also highlights some prospects for how biotechnological approaches can improve cotton fiber quality.

A cell wall-localized β-1,3-glucanase promotes fiber cell elongation and secondary cell wall deposition.

β-1,3-glucanase functions in plant physiological and developmental processes. However, how β-1,3-glucanase participates in cell wall development remains largely unknown. Here, we answered this question by examining the role of GhGLU18, a β-1,3-glucanase, in cotton (Gossypium hirsutum) fibers, in which the content of β-1,3-glucan changes dynamically from 10% of the cell wall mass at the onset of secondary wall deposition to <1% at maturation. GhGLU18 was specifically expressed in cotton fiber with higher expression in late fiber elongation and secondary cell wall (SCW) synthesis stages. GhGLU18 largely localized to the cell wall and was able to hydrolyze β-1,3-glucan in vitro. Overexpression of GhGLU18 promoted polysaccharide accumulation, cell wall reconstruction, and cellulose synthesis, which led to increased fiber length and strength with thicker cell walls and shorter pitch of the fiber helix. However, GhGLU18-suppressed cotton resulted in opposite phenotypes. Additionally, GhGLU18 was directly activated by GhFSN1 (fiber SCW-related NAC1), a NAC transcription factor reported previously as the master regulator in SCW formation during fiber development. Our results demonstrate that cell wall-localized GhGLU18 promotes fiber elongation and SCW thickening by degrading callose and enhancing polysaccharide metabolism and cell wall synthesis.

Characterization of chromatin accessibility and gene expression reveal the key genes involved in cotton fiber elongation.

Cotton (Gossypium hirsutum L.) is an important economic crop, and cotton fiber is one of the longest plant cells, which provides an ideal model for the study of cell elongation and secondary cell wall synthesis. Cotton fiber length is regulated by a variety of transcription factors (TF) and their target genes; however, the mechanism of fiber elongation mediated by transcriptional regulatory networks is still unclear to a large extent. Here, we used a comparative assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) assay and RNA-seq analysis to identify fiber elongation transcription factors and genes using the short-fiber mutant ligon linless-2 (Li2 ) and wild type (WT). A total of 499 differential target genes were identified and GO analysis shows that differential genes are mainly involved in plant secondary wall synthesis and microtubule-binding processes. Analysis of the genomic regions preferentially accessible (Peak) has identified a number of overrepresented TF-binding motifs, highlighting sets of TFs that are important for cotton fiber development. Using ATAC-seq and RNA-seq data, we have constructed a functional regulatory network of each TF regulatory target gene and also the network pattern of TF regulating differential target genes. Further, to obtain the genes related to fiber length, the differential target genes were combined with FLGWAS data to identify the genes highly related to fiber length. Our work provides new insights into cotton fiber elongation.

The OsbHLH002/OsICE1-OSH1 module orchestrates secondary cell wall formation in rice

Transcriptional regulation of secondary cell wall (SCW) formation is strictly controlled by a complex network of transcription factors in vascular plants and has been shown to be mediated by a group of NAC master switches. In this study, we show that in a bHLH transcription factor, OsbHLH002/OsICE1, its loss-of-function mutant displays a lodging phenotype. Further results show that OsbHLH002 and Oryza sativa homeobox1 (OSH1) interact and share a set of common targets. In addition, the DELLA protein SLENDER RICE1, rice ortholog of KNOTTED ARABIDOPSIS THALIANA7, and OsNAC31 interact with OsbHLH002 and OSH1 and regulate their binding capacity on OsMYB61, a key regulatory factor in SCW development. Collectively, our results indicate OsbHLH002 and OSH1 as key regulators in SCW formation and shed light on molecular mechanisms of how active and repressive factors precisely orchestrate SCW synthesis in rice, which may provide a strategy for manipulating plant biomass production.

Bnt05G007257, a Novel NAC Transcription Factor, Predicts Developmental and Synthesis Capabilities of Fiber Cells in Ramie (Boehmeria nivea L.)

NAC transcription factors are one of the largest transcription factor families in plants, and they play a key role in the growth and development of a secondary cell wall. Despite the fact that ramie is well-known for its high fiber yield, the role of NAC transcription factors in ramie secondary cell wall synthesis and fiber development remains unknown. In this study, based on our previous study, we describe the characterization, physicochemical property analysis, protein structure and function prediction, subcellular localization, and functional validation of Bnt05G007257, which encodes an NAC transcription factor from ramie, in transgenic A. thaliana. Our findings show that the open reading frame of Bnt05G007257 was 1035 bp long and encodes for a protein comprising 344 amino acids, having a relative molecular mass of 39.0945 kDa and a theoretical isoelectric point of 6.55. The secondary structure of the encoded protein mainly consisted of random coiling, with a typical conserved structural domain of NAC. The phylogenetic tree revealed that Bnt05G007257 is a homolog of the NAC transcription factor SND2, which regulates secondary wall biosynthesis in A. thaliana. Subcellular localization showed that Bnt05G007257 was tentatively predicted to be localized in the cytoplasm. Furthermore, in stem sections, the secondary wall fiber cells’ thickness in Bnt05G007257 transgenic plants was 31.50% thicker than that in wild-type plants, and the radial width was significantly increased by approximately 21.75%. This indicates that the NAC family homolog Bnt05G007257 may have the potential function of promoting fiber cell development and secondary cell wall synthesis, providing a theoretical basis for the selection of high-fiber-yielding ramie varieties in the future.

Exploring the Biologically Active Metabolites Produced by Bacillus cereus for Plant Growth Promotion, Heat Stress Tolerance, and Resistance to Bacterial Soft Rot in Arabidopsis

Eight gene clusters responsible for synthesizing bioactive metabolites associated with plant growth promotion were identified in the Bacillus cereus strain D1 (BcD1) genome using the de novo whole-genome assembly method. The two largest gene clusters were responsible for synthesizing volatile organic compounds (VOCs) and encoding extracellular serine proteases. The treatment with BcD1 resulted in an increase in leaf chlorophyll content, plant size, and fresh weight in Arabidopsis seedlings. The BcD1-treated seedlings also accumulated higher levels of lignin and secondary metabolites including glucosinolates, triterpenoids, flavonoids, and phenolic compounds. Antioxidant enzyme activity and DPPH radical scavenging activity were also found to be higher in the treated seedlings as compared with the control. Seedlings pretreated with BcD1 exhibited increased tolerance to heat stress and reduced disease incidence of bacterial soft rot. RNA-seq analysis showed that BcD1 treatment activated Arabidopsis genes for diverse metabolite synthesis, including lignin and glucosinolates, and pathogenesis-related proteins such as serine protease inhibitors and defensin/PDF family proteins. The genes responsible for synthesizing indole acetic acid (IAA), abscisic acid (ABA), and jasmonic acid (JA) were expressed at higher levels, along with WRKY transcription factors involved in stress regulation and MYB54 for secondary cell wall synthesis. This study found that BcD1, a rhizobacterium producing VOCs and serine proteases, is capable of triggering the synthesis of diverse secondary metabolites and antioxidant enzymes in plants as a defense strategy against heat stress and pathogen attack.

Highly differentiated genomic properties underpin the different cell walls of Poaceae and eudicots.

Plant cell walls of Poaceae and eudicots differ substantially, both in the content and composition of their components. However, the genomic and genetic basis underlying these differences is not fully resolved. In this research, we analyzed multiple genomic properties of 150 cell wall gene families across 169 angiosperm genomes. The properties analyzed include gene presence/absence, copy number, synteny, occurrence of tandem gene clusters, and phylogenetic gene diversity. Results revealed a profound genomic differentiation of cell wall genes between Poaceae and eudicots, often associated with the cell wall diversity between these plant groups. For example, overall patterns of gene copy number variation and synteny were clearly divergent between Poaceae and eudicot species. Moreover, differential Poaceae-eudicot copy number and genomic contexts were observed for all the genes within the BEL1-like HOMEODOMAIN 6 regulatory pathway, which respectively induces and represses secondary cell wall synthesis in Poaceae and eudicots. Similarly, divergent synteny, copy number, and phylogenetic gene diversification were observed for the major biosynthetic genes of xyloglucans, mannans, and xylans, potentially contributing to the differences in content and types of hemicellulosic polysaccharides differences in Poaceae and eudicot cell walls. Additionally, the Poaceae-specific tandem clusters and/or higher copy number of PHENYLALANINE AMMONIA-LYASE, CAFFEIC ACID O-METHYLTRANSFERASE, or PEROXIDASE genes may underly the higher content and larger variety of phenylpropanoid compounds observed in Poaceae cell walls. All these patterns are discussed in detail in this study, along with their evolutionary and biological relevance for cell wall (genomic) diversification between Poaceae and eudicots.

CcNAC1 by Transcriptome Analysis Is Involved in Sudan Grass Secondary Cell Wall Formation as a Positive Regulator.

Sudan grass is a high-quality forage of sorghum. The degree of lignification of Sudan grass is the main factor affecting its digestibility in ruminants such as cattle and sheep. Almost all lignocellulose in Sudan grass is stored in the secondary cell wall, but the mechanism and synthesis of the secondary cell wall in Sudan grass is still unclear. In order to study the mechanism of secondary cell wall synthesis in Sudan grass, we used an in vitro induction system of Sudan grass secondary cell wall. Through transcriptome sequencing, it was found that the NAC transcription factor CcNAC1 gene was related to the synthesis of the Sudan grass secondary cell wall. This study further generated CcNAC1 overexpression lines of Arabidopsis to study CcNAC1 gene function in secondary cell wall synthesis. It was shown that the overexpression of the CcNAC1 gene can significantly increase lignin content in Arabidopsis lines. Through subcellular localization analysis, CcNAC1 genes could be expressed in the nucleus of a plant. In addition, we used yeast two-hybrid screening to find 26 proteins interacting with CcNAC1. GO and KEGG analysis showed that CcNAC1 relates to the metabolic pathways and biosynthesis of secondary metabolites. In summary, the synthesis of secondary cell wall of Sudan grass can be regulated by CcNAC1.

Open questions in plant cell wall synthesis.

Plant cells are surrounded by strong yet flexible polysaccharide-based cell walls that support the cell while also allowing growth by cell expansion. Plant cell wall research has advanced tremendously in recent years. Sequenced genomes of many model and crop plants have facilitated cataloging and characterization of many enzymes involved in cell wall synthesis. Structural information has been generated for several important cell wall synthesizing enzymes. Important tools have been developed including antibodies raised against a variety of cell wall polysaccharides and glycoproteins, collections of enzyme clones and synthetic glycan arrays for characterizing enzymes, herbicides that specifically affect cell wall synthesis, live-cell imaging probes to track cell wall synthesis, and an inducible secondary cell wall synthesis system. Despite these advances, and often because of the new information they provide, many open questions about plant cell wall polysaccharide synthesis persist. This article highlights some of the key questions that remain open, reviews the data supporting different hypotheses that address these questions, and discusses technological developments that may answer these questions in the future.

Cotton green fiber promotes suberin synthesis interfering cellulose deposition in the secondary cell wall

Naturally green cotton (NGC) represents one of two commercialized naturally colored cottons, but its application is limited in the textile industry for inferior productivity and fiber quality of NGC cultivars. NGC fiber deposits substantial suberin at maturation, possibly related to fiber green coloration. The effects of suberin deposition on fiber development and fiber yield and quality are still to be elucidated. In the present study, the green fiber gene (Lg) in upland cotton (Gossypium hirsutum L.) was cloned using map-based method. It was found that Lg encoded an R2R3-MYB transcription factor, promoting suberin synthesis and deposition in cotton fiber via regulating multiple suberin structural genes. Suberin accumulated in green fibers continuously from the beginning of the secondary cell wall synthesis, along with significantly inhibited cellulose deposition, leading to dramatically decreased fiber strength and lint percentage. Our data demonstrated that suberin accumulation significantly interfered with cellulose deposition in fiber secondary cell wall, which conferred low yield and inferior fiber quality to green-fiber cottons.

A receptor-like kinase controls the amplitude of secondary cell wall synthesis in rice

Cell wall expansion is a key element in determining plant morphology and growth, and cell wall integrity changes are relayed to the cell to fine-tune growth responses. Here, we show that variations in the ectodomain of a cell wall-associated receptor-like kinase, WAK10, in temperate Oryza japonica accessions differentially amplify fluctuations in cell wall integrity to control rice stem height. Mutation in the WAK10 gene exhibited increased cell wall thickening in stem sclerenchyma and reduced cell expansion in the stem. Two WAK10 ectodomain variants bound pectic oligosaccharides with different affinities. The pectic oligosaccharide binding regulated WAK10 phosphorylation activity, the amplitude of secondary wall deposition, and ultimately, stem height. Rice population analyses revealed active enrichment of the short-stem WAK10 ectodomain alleles in japonica subspecies during domestication. Our study outlines not only a mechanism for how variations in ligand affinities of a receptor kinase control cell wall biosynthesis and plant growth, but it also provides breeding targets for new semi-dwarf rice cultivars.

Cas9/gRNA-Mediated Mutations in PtrFLA40 and PtrFLA45 Reveal Redundant Roles in Modulating Wood Cell Size and SCW Synthesis in Poplar

Fasciclin-like arabinogalactan proteins (FLAs) play an important role in plant development and adaptation to the environment. However, the roles of FLAs in wood formation remain poorly understood. Here, we identified a total of 50 PtrFLA genes in poplar. They were classified into four groups: A to D, among which group A was the largest group with 28 members clustered into four branches. Most PtrFLAs of group A were dominantly expressed in developing xylem based on microarray and RT-qPCR data. The roles of PtrFLA40 and PtrFLA45 in group A were investigated via the Cas9/gRNA-induced mutation lines. Loss of PtrFLA40 and PtrFLA45 increased stem length and diameter in ptrfla40ptrfla45 double mutants, but not in ptrfla40 or ptrfla45 single mutants. Further, our findings indicated that the ptrfla40ptrfla45 mutants enlarged the cell size of xylem fibers and vessels, suggesting a negative modulation in stem xylem cell size. In addition, wood lignin content in the ptrfla40fla45 mutants was increased by nearly 9%, and the lignin biosynthesis-related genes were significantly up-regulated in the ptrfla40fla45 mutants, in agreement with the increase in wood lignin content. Overall, Cas9/gRNA-mediated mutations in PtrFLA40 and PtrFLA45 reveal redundant roles in modulating wood cell size and secondary cell wall (SCW) synthesis in poplar.

Tissue specific expression of bacterial cellulose synthase (Bcs) genes improves cotton fiber length and strength

Fiber growing inside the cotton bolls is a highly demandable product and its quality is key to the success of the textile industry. Despite the various efforts to improve cotton fiber staple length Pakistan has to import millions of bales to sustain its industrial needs. To improve cotton fiber quality Bacterial cellulose synthase (Bcs) genes (acsA, acsB) were expressed in a local cotton variety CEMB-00. In silico studies revealed a number of conserved domains both in the cotton-derived and bacterial cellulose synthases which are essential for the cellulose synthesis. Transformation efficiency of 1.27% was achieved by using Agrobacterium shoot apex cut method of transformation. The quantitative mRNA expression analysis of the Bcs genes in transgenic cotton fiber was found to be many folds higher during secondary cell wall synthesis stage (35 DPA) than the expression during elongation phase (10 DPA). Average fiber length of the transgenic cotton plant lines S-00–07, S-00–11, S-00–16 and S-00–23 was calculated to be 13.02% higher than that of the non-transgenic control plants. Likewise, the average fiber strength was found to be 20.92% higher with an enhanced cellulose content of 22.45%. The mutated indigenous cellulose synthase genes of cotton generated through application of CRISPR/Cas9 resulted in 6.03% and 12.10% decrease in fiber length and strength respectively. Furthermore, mature cotton fibers of transgenic cotton plants were found to have increased number of twists with smooth surface as compared to non-transgenic control when analyzed under scanning electron microscope. XRD analysis of cotton fibers revealed less cellulose crystallinity index in transgenic cotton fibers as compared to control fibers due to deposition of more amorphous cellulose in transgenic fibers as a result of Bcs gene expression. This study paved the way towards unraveling the fact that Bcs genes influence cellulose synthase activity and this enzyme helps in determining the fate of cotton fiber length and strength.