Suberin Biosynthesis Research Articles

Comparative anatomical and transcriptomic analyses of the color variation of leaves in Aquilaria sinensis.

Color variation in plant tissues is a common phenomenon accompanied with a series of biological changes. In this study, a special-phenotype Aquilaria sinensis (GS) with color variation of leaf was firstly reported, and DNA barcode sequences showed GS samples could not be discriminated clearly with the normal A. sinensis sample (NS), which suggested that the variety was not the cause of the GS formation. To reveal the characteristics of GS compared to NS, the anatomical and transcriptome sequencing studies were carried out. In microscopic observation, the leaves of golden-vein-leaf sample (LGS) and normal-vein-leaf sample (LNS) showed significant differences including the area of the included phloem in midrib and the thickness parameters of palisade and spongy tissues; the stems of golden-vein-leaf sample (SGS) and normal-vein-leaf sample (SNS) were also different in many aspects such as the area of vessels and included phloem. In addition, the structure of chloroplast was more complete in the midrib of LNS than that of LGS, and some particles suspected as virus were found through transmission electron microscope as well. Genes upregulated in LGS in contrast with LNS were mainly enriched in photosynthesis. As for stems, most of the genes upregulated in SGS compared to SNS were involved in translation and metabolism processes. The pathways about photosynthesis and chlorophyll metabolism as well as some important transcription factors may explain the molecular mechanism of the unique phenotypes of leaves and the genes related to suberin biosynthesis may result in the difference of stems. In addition, the genes about defense response especially biotic stress associated with numerous pathogenesis-related (PR) genes upregulated in LGS compared to LNS indicated that the pathogen may be the internal factor. Taken together, our results reveal the macro- and micro-phenotype variations as well as gene expression profiles between GS and NS, which could provide valuable clues for elucidating the mechanism of the color variation of Aquilaria.

Transcriptomic analysis of cork during seasonal growth highlights regulatory and developmental processes from phellogen to phellem formation

The phellogen or cork cambium stem cells that divide periclinally and outwardly specify phellem or cork. Despite the vital importance of phellem in protecting the radially-growing plant organs and wounded tissues, practically only the suberin biosynthetic process has been studied molecularly so far. Since cork oak (Quercus suber) phellogen is seasonally activated and its proliferation and specification to phellem cells is a continuous developmental process, the differentially expressed genes during the cork seasonal growth served us to identify molecular processes embracing from phellogen to mature differentiated phellem cell. At the beginning of cork growth (April), cell cycle regulation, meristem proliferation and maintenance and processes triggering cell differentiation were upregulated, showing an enrichment of phellogenic cells from which phellem cells are specified. Instead, at maximum (June) and advanced (July) cork growth, metabolic processes paralleling the phellem cell chemical composition, such as the biosynthesis of suberin, lignin, triterpenes and soluble aromatic compounds, were upregulated. Particularly in July, polysaccharides- and lignin-related secondary cell wall processes presented a maximal expression, indicating a cell wall reinforcement in the later stages of cork formation, presumably related with the initiation of latecork development. The putative function of relevant genes identified are discussed in the context of phellem ontogeny.

Leaf morphological and photosynthetic differences among four chemotypes of Cinnamomum camphora in different seasons

Linalool chemotype (LnL), eucalyptol chemotype (EuL), camphor chemotype (CmR) and borneol chemotype (BeL) are 4 main chemotypes of Cinnamomum camphora, an evergreen tree species with great economic and applied value. To facilitate their distinction, cultivation, and utilization, the differences of leaf morphology, photosynthetic abilities and related gene expression between the 4 chemotypes of C. camphora were investigated during spring (April and May), summer (July), and autumn (November). The 4 chemotypes of C. camphora exhibited different leaf morphology. Large leaves were detected in LnL and EuL, which might result from the high expression of the genes related to biosynthesis of brassinosteroids and cytokinins, as well as signal transduction and responses of brassinosteroids and auxins. Thick leaves were detected in EuL and BeL, as the genes related to biosynthesis of cutin, suberin and waxes were highly expressed. Among the 4 chemotypes, EuL and CmR showed strong photosynthetic abilities, which resulted from their high photosynthetic pigment content, photosystem II efficiency and photosynthetic rate, due to the high expression of the genes involving in photosynthetic pigment biosynthesis, photosynthetic electron transport, ATPase biogenesis, and CO2 fixation.

Transcriptional regulation of wound suberin deposition in potato cultivars with differential wound healing capacity

Wounding during mechanical harvesting and post-harvest handling results in tuber desiccation and provides an entry point for pathogens. Poor wound healing is a major cause of these losses. Wound tissue in potato (Solanum tuberosum) tubers, and all higher plants, is composed of a large proportion of suberin that is deposited in a specialized tissue called the wound periderm. However, the genetic regulatory pathway controlling wound-induced suberization remains unknown. Here, we implicate two potato transcription factors, StMYB102 (PGSC0003DMG400011250) and StMYB74 (PGSC0003DMG400022399), as regulators of wound suberin biosynthesis and deposition. Using targeted metabolomics and transcript profiling from the wound healing tissues of two commercial potato cultivars, as well as heterologous expression, we provide evidence for the molecular-genetic basis of the differential wound suberization capacities of different potato cultivars. Our results suggest that i) the export of suberin from the cytosol to the apoplast and ligno-suberin deposition may be limiting factors for wound suberization, ii) StMYB74 and StMYB102 are important regulators of the wound suberization process in tubers, and iii) polymorphisms in StMYB102 may influence cultivar-specific wound suberization capacity. These results represent an important step in understanding the regulated biosynthesis and deposition of wound suberin and provide a practical foundation for targeted breeding approaches aimed at improving potato tuber storage life.

Suberin Biopolymer in Rice Root Exodermis Reinforces Preformed Barrier Against Meloidogyne graminicola Infection

Exploration of novel genetic resources against root-knot nematode (RKN) is necessary to strengthen the resistance breeding program in cultivated rice, and investigations on the role of genotype-specific root anatomy in conferring a structural barrier against nematode invasion are largely underexplored. Here, we reported a highly-resistant rice germplasm Phule Radha that conferred remarkably lower RKN parasitic fitness in terms of reduced penetration and delayed development and reproduction when compared with susceptible cultivar PB1121. Using histological and biochemical analyses, we demonstrated that an enhanced suberin deposition in the exodermal root tip tissue of Phule Radha compared to PB1121 can effectively form a penetrative barrier against RKN infection, and this preformed barrier in the control tissue did not necessarily alter to a greater extent when challenged with RKN stress. Using qRT-PCR analysis, we showed that a number of suberin biosynthesis genes were greatly expressed in the exodermis of Phule Radha compared to PB1121. In sum, the present study established the role of rice exodermal barrier system in defense against an important soil-borne pathogen.

Cork cells in cork oak periderms undergo programmed cell death and proanthocyanidin deposition.

Vascular plants with secondary growth develop a periderm mostly composed of dead suberized cork cells to face environmental hostile conditions. Cork oak has a highly active and long-living phellogen forming a remarkably thick periderm that is periodically debarked for industrial purposes. This wounding originates the quick formation of a new traumatic periderm, making cork oak an exceptional model to study the first periderm differentiation during normal development in young sprigs and traumatic (wound) periderm formation after debarking. Here, we studied the poorly known first periderm differentiation steps that involve cell wall suberization, polyphenolic accumulation and programmed cell death (PCD) by combining transmission electron microscopy, histochemical and molecular methods in periderms from young sprigs. These processes were further compared with traumatic periderms formed after wounding using molecular and histochemical techniques, such as the polyphenolic accumulation. In the first periderms from young sprigs, four distinct differentiation stages were defined according to the presence of PCD morphological features. First young and traumatic periderms showed an upregulation of genes related to suberin biosynthesis, proanthocyanidins biosynthesis and transport, autophagy, and PCD. Traumatic periderms revealed an overall upregulation of these genes, likely resulting from ontogeny differences and distinct phellogen origin associated with a faster metabolism, highlighting the impact of wounding on phellogen activity after debarking. First periderms from young sprigs showed gradual accumulation of proanthocyanidins in the vacuoles throughout PCD stages until total filled lumens, whereas in traumatic periderms, these compounds were found cell wall linked in already empty cells. This work enabled a comprehensive overview of the cork cells differentiation processes contributing to deepening the knowledge of the fundamental ontogenic program of this protective tissue, which is also a unique forest product, constituting the basis of a sustainable and profitable industry.

AP2/ERF transcription factors orchestrate very long chain fatty acid biosynthesis during Arabidopsis lateral root development

AP2/ERF transcription factors orchestrate very long chain fatty acid biosynthesis during Arabidopsis lateral root development

Gene Regulatory Networks Shape Developmental Plasticity of Root Cell Types Under Water Extremes

Understanding how roots modulate development under varied irrigation or rainfall is crucial for development of climate resilient crops. We established a toolbox of tagged rice lines to profile translating mRNAs and chromatin accessibility within specific cell populations. We used these to study roots in a range of environments: plates in the lab, controlled greenhouse stress and recovery conditions, and outdoors in a paddy. Integration of chromatin and mRNA data resolves regulatory networks of: cycle genes in proliferating cells that attenuate DNA synthesis under submergence; genes involved in auxin signaling, the circadian clock, and small RNA regulation in ground tissue; and suberin biosynthesis, iron transporters, and nitrogen assimilation in endodermal/exodermal cells modulated with water availability. By applying a systems approach we identify known and candidate driver transcription factors of water deficit responses and xylem development plasticity. Collectively, this resource will facilitate genetic improvements in root systems for optimal climate resilience.

Expression analysis of genes for cytochrome P450 CYP86 and glycerol-3-phosphate acyltransferase related to suberin biosynthesis in rice roots under stagnant deoxygenated conditions

Expression analysis of genes for cytochrome P450 CYP86 and glycerol-3-phosphate acyltransferase related to suberin biosynthesis in rice roots under stagnant deoxygenated conditions

An integrated metabolic and transcriptomic analysis reveals the mechanism through which fruit bagging alleviates exocarp semi-russeting in pear fruit.

Fruit semi-russeting is an undesirable quality trait that occurs in fruit production. It is reported that preharvest fruit bagging could effectively alleviate fruit exocarp semi-russeting, but the physiological and molecular mechanisms remain unclear. In the present study, we performed an in-depth investigation into pear fruit semi-russeting from morphologic, metabolic and transcriptomic perspectives by comparing control (semi-russeted) and bagged (non-russeted) 'Cuiguan' pear fruits. The results showed that significant changes in cutin and suberin resulted in pear fruit semi-russeting. Compared with the skin of bagged fruits, the skin of the control fruits presented reduced cutin contents accompanied by an accumulation of suberin, which resulted in fruit semi-russeting; α, ω-dicarboxylic acids accounted for the largest proportion of typical suberin monomers. Moreover, combined transcriptomic and metabolic analysis revealed a series of genes involved in cutin and suberin biosynthesis, transport and polymerization differentially expressed between the two groups. Furthermore, the expression levels of genes involved in the stress response and in hormone biosynthesis and signaling were significantly altered in fruits with contrasting phenotypes. Finally, a number of transcription factors, including those of the MYB, NAC, bHLH and bZIP families, were differentially expressed. Taken together, the results suggest that the multilayered mechanism through which bagging alleviates pear fruit semi-russeting is complex, and the large number of candidate genes identified provides a good foundation for future functional studies.

Drought activates MYB41 orthologs and induces suberization of grapevine fine roots.

The permeability of roots to water and nutrients is controlled through a variety of mechanisms and one of the most conspicuous is the presence of the Casparian strips and suberin lamellae. Roots actively regulate the creation of these structures developmentally, along the length of the root, and in response to the environment, including drought. In the current study, we characterized the suberin composition along the length of grapevine fine roots during development and in response to water deficit, and in the same root systems we quantified changes in expression of suberin biosynthesis‐ and deposition‐related gene families (via RNAseq) allowing the identification of drought‐responsive suberin‐related genes. Grapevine suberin composition did not differ between primary and lateral roots, and was similar to that of other species. Under water deficit there was a global upregulation of suberin biosynthesis which resulted in an increase of suberin specific monomers, but without changes in their relative abundances, and this upregulation took place across all the developmental stages of fine roots. These changes corresponded to the upregulation of numerous suberin biosynthesis‐ and export‐related genes which included orthologs of the previously characterized AtMYB41 transcriptional factor. Functional validation of two grapevine MYB41 orthologs, VriMYB41 and VriMYB41‐like, confirmed their ability to globally upregulate suberin biosynthesis, export, and deposition. This study provides a detailed characterization of the developmental and water deficit induced suberization of grapevine fine roots and identifies important orthologs responsible for suberin biosynthesis, export, and its regulation in grape.

ABA stimulates wound suberization through antagonizing the MYB4-mediated transcriptional repression of CYP86A1 and FAR in postharvest kiwifruit

Abstract Suberin biosynthesis involves a large number of genes, and many of them are induced by abscisic acid (ABA). However, the regulation of transcription factor (TF) in response to ABA on suberin biosynthetic genes in kiwifruit has been unexplored. In this study, two genes, AchnCYP86A1 and AchnFAR respectively encoding a fatty acid ω-hydroxylase and fatty acyl-CoA reductase involved in suberin monomer biosynthesis were demonstrated in transient overexpressed tobacco (Nicotiana benthamiana). Notably, the negative regulation of AchnMYB4 on AchnCYP86A1 and AchnFAR was identified. AchnMYB4 could repress AchnCYP86A1 and AchnFAR transcript by directly binding to the gene promoter in yeast one-hybrid and dual-luciferase assays. These results were further confirmed in transient overexpressed tobacco leaves in that AchnMYB4 significantly down-regulated suberin biosynthetic genes including CYP86A1, FAR2 and FAR3, and reduced accumulation of ω-hydroxyacids and primary alcohols. Moreover, exogenous ABA could induce the expression of AchnCYP86A1 and AchnFAR, and the accumulation of corresponding suberin monomers by inhibition of AchnMYB4 in wound-tissue of kiwifruit. However, fluridone (an inhibitor of ABA biosynthesis) was found to counter the inductive effects of ABA. Taken together, the results suggest that ABA activates AchnCYP86A1 and AchnFAR to promote suberin monomers biosynthesis by inhibiting AchnMYB4.

Regulation of a Cytochrome P450 Gene CYP94B1 by WRKY33 Transcription Factor Controls Apoplastic Barrier Formation in Roots to Confer Salt Tolerance.

Salinity is an environmental stress that causes decline in crop yield. Avicennia officinalis and other mangroves have adaptations such as ultrafiltration at the roots aided by apoplastic cell wall barriers to thrive in saline conditions. We studied a cytochrome P450 gene from A. officinalis, AoCYP94B1, and its putative ortholog in Arabidopsis (Arabidopsis thaliana), AtCYP94B1, which are involved in apoplastic barrier formation. Both genes were induced by 30 min of salt treatment in the roots. Heterologous expression of AoCYP94B1 in the atcyp94b1 Arabidopsis mutant and wild-type rice (Oryza sativa) conferred increased NaCl tolerance to seedlings by enhancing root suberin deposition. Histochemical staining and gas chromatography-tandem mass spectrometry quantification of suberin precursors confirmed the role of CYP94B1 in suberin biosynthesis. Using chromatin immunoprecipitation and yeast one-hybrid and luciferase assays, we identified AtWRKY33 as the upstream regulator of AtCYP94B1 in Arabidopsis. In addition, atwrky33 mutants exhibited reduced suberin and salt-sensitive phenotypes, which were rescued by expressing 35S::AtCYP94B1 in the atwrky33 background. This further confirmed that AtWRKY33-mediated regulation of AtCYP94B1 is part of the salt tolerance mechanism. Our findings may help efforts aimed at generating salt-tolerant crops.

The sugarcane ShMYB78 transcription factor activates suberin biosynthesis in Nicotiana benthamiana.

A sugarcane MYB present in the culm induces suberin biosynthesis and is involved both with fatty acid and phenolics metabolism. Few transcription factors have been described as regulators of cell wall polymers deposition in C4 grasses. Particularly, regulation of suberin biosynthesis in this group of plants remains poorly understood. Here, we showed that the sugarcane MYB transcription factor ShMYB78 is an activator of suberin biosynthesis and deposition. ShMYB78 was identified upon screening genes whose expression was upregulated in sugarcane internodes undergoing suberization during culm development or triggered by wounding. Agrobacterium-mediated transient expression of ShMYB78 in Nicotiana benthamiana leaves induced the ectopic deposition of suberin and its aliphatic and aromatic monomers. Further, the expression of suberin-related genes was induced by ShMYB78 heterologous expression in Nicotiana benthamiana leaves. ShMYB78 was shown to be a nuclear protein based on its presence in sugarcane internode nuclear protein extracts, and protoplast transactivation assays demonstrated that ShMYB78 activates the promoters of the sugarcane suberin biosynthetic genes β-ketoacyl-CoA synthase (ShKCS20) and caffeic acid-O-methyltransferase (ShCOMT). Our results suggest that ShMYB78 may be involved in the transcriptional regulation of suberin deposition, from fatty acid metabolism to phenylpropanoid biosynthesis, in sugarcane internodes.

Crosstalk of phenylpropanoid biosynthesis with hormone signaling in Chinese cabbage is key to counteracting salt stress

Salt stress limits the regional distribution of Chinese cabbage, yet the molecular factors that confer tolerance to salt stress in Chinese cabbage remain poorly understood. To further investigate this issue, salt-tolerant and salt-sensitive Chinese cabbage cultivars were selected, and their responses to salt stress were tested. Then, comparative transcriptome profiling was performed on the roots of salt-tolerant and salt-sensitive Chinese cabbage cultivars under salt stress. The results showed that Qinghua and Biyu are considered to be salt-tolerant and salt-sensitive Chinese cabbage cultivars, respectively. Qinghua had higher water content and lower Na+/K+ in roots and leaves, better root development, higher lignin content and higher abscisic acid content in roots than Biyu. Comparative transcriptome profiling showed that the abscisic acid and ethylene signaling pathways of Qinghua roots were more actively expressed than those of Biyu. During root adaptation to salt stress, abscisic acid crosstalk with suberin biosynthesis and ethylene signaling crosstalk with lignin biosynthesis were observed. Suberin development and lignin accumulation in the Qinghua roots were found to be important strategies for resisting salt stress in this study. These strategies are not independent, but rather coordinate to promote salt tolerance in Chinese cabbage. The increased lignin content in the roots of Qinghua under salt stress may prevent embolism. Embolism prevention can maintain the flow of water transport, and thus compensate for the reduction in water permeability of these suberized cells in a process that occurs concurrently with greater sodium ion limitation. These results provide novel insight into the crosstalk of phenylpropanoid biosynthesis with hormone signaling under salt stress, and facilitate further dissection of molecular mechanism of adaptive response to salt stress in Chinese cabbage.

MYB41, MYB107, and MYC2 promote ABA-mediated primary fatty alcohol accumulation via activation of AchnFAR in wound suberization in kiwifruit

Wound damage triggers the accumulation of abscisic acid (ABA), which induces the expression of a large number of genes involved in wound suberization in plants. Fatty acyl-CoA reductase (FAR) catalyzes the generation of primary fatty alcohols by the reduction of fatty acids in suberin biosynthesis. However, the regulatory effects of transcription factors (TFs) on AchnFAR in response to ABA are unexplored. In this study, kiwifruit AchnFAR displayed a biological function analogous to that of FAR in transiently overexpressed tobacco (Nicotiana benthamiana) leaves. The positive role of TFs, including AchnMYB41, AchnMYB107, and AchnMYC2, in the regulation of AchnFAR was identified. The three TFs could individually bind to the AchnFAR promoter to activate gene transcription in yeast one-hybrid and dual-luciferase assays. Transient overexpression of TFs in tobacco leaves resulted in the upregulation of aliphatic synthesis genes (including FAR) and the increase in aliphatics, including primary alcohols, α,ω-diacids, ω-hydroxyacids, and fatty acids. Moreover, exogenous ABA treatment elevated TF-mediated AchnFAR expression and the accumulation of primary alcohols. Conversely, fluridone, an inhibitor of ABA biosynthesis, suppressed the expression of AchnFAR and TF genes and reduced the formation of primary alcohols. The results indicate that AchnMYB41, AchnMYB107, and AchnMYC2 activate AchnFAR transcription to promote ABA-mediated primary alcohol formation in wound suberization in kiwifruit.

Proteome and transcriptome profile analysis reveals regulatory and stress-responsive networks in the russet fruit skin of sand pear

The epidermal tissues of the cuticular membrane (CM) and periderm membrane (PM) confer first-line protection from environmental stresses in terrestrial plants. Although PM protection is essentially ubiquitous in plants, the protective mechanism, the function of many transcription factors and enzymes, and the genetic control of metabolic signaling pathways are poorly understood. Different microphenotypes and cellular components in russet (PM-covered) and green (CM-covered) fruit skins of pear were revealed by scanning and transmission electron microscopy. The two types of fruit skins showed distinct phytohormone accumulation, and different transcriptomic and proteomic profiles. The enriched pathways were detected by differentially expressed genes and proteins from the two omics analyses. A detailed analysis of the suberin biosynthesis pathways identified the regulatory signaling network, highlighting the general mechanisms required for periderm formation in russet fruit skin. The regulation of aquaporins at the protein level should play an important role in the specialized functions of russet fruit skin and PM-covered plant tissues.

Overexpression of ANAC046 Promotes Suberin Biosynthesis in Roots of Arabidopsis thaliana.

NAC (NAM (no apical meristem), ATAF1/2, and CUC2 (cup-shaped cotyledon)) proteins are one of the largest families of plant-specific transcription factors, and this family is present in a wide range of land plants. Here, we have investigated the role of ANAC046 in the regulation of suberin biosynthesis and deposition in Arabidopsis. Subcellular localization and transcriptional activity assays showed that ANAC046 localizes in the nucleus, where it functions as a transcription activator. Analysis of the PANAC046:GUS lines revealed that ANAC046 is mainly expressed in the root endodermis and periderm, and is also induced in leaves by wounding. The transgenic lines overexpressing ANAC046 exhibited defective surfaces on the aerial plant parts compared to the wild-type (WT) as characterized by increased permeability for Toluidine blue stain and greater chlorophyll leaching. Quantitative RT-PCR analysis showed that the expression of suberin biosynthesis genes was significantly higher in the roots and leaves of overexpression lines compared to the WT. The biochemical analysis of leaf cuticular waxes showed that the overexpression lines accumulated 30% more waxes than the WT. Concurrently, overexpression lines also deposited almost twice the amount of suberin content in their roots compared with the WT. Taken together, these results showed that ANAC046 is an important transcription factor that promotes suberin biosynthesis in Arabidopsis thaliana roots.

Seminal roots of wild and cultivated barley differentially respond to osmotic stress in gene expression, suberization, and hydraulic conductivity.

Wild barley, Hordeum vulgare spp. spontaneum, has a wider genetic diversity than its cultivated progeny, Hordeum vulgare spp. vulgare. Osmotic stress leads to a series of different responses in wild barley seminal roots, ranging from no changes in suberization to enhanced endodermal suberization of certain zones and the formation of a suberized exodermis, which was not observed in the modern cultivars studied so far. Further, as a response to osmotic stress, the hydraulic conductivity of roots was not affected in wild barley, but it was 2.5-fold reduced in cultivated barley. In both subspecies, osmotic adjustment by increasing proline concentration and decreasing osmotic potential in roots was observed. RNA-sequencing indicated that the regulation of suberin biosynthesis and water transport via aquaporins were different between wild and cultivated barley. These results indicate that wild barley uses different strategies to cope with osmotic stress compared with cultivated barley. Thus, it seems that wild barley is better adapted to cope with osmotic stress by maintaining a significantly higher hydraulic conductivity of roots during water deficit.

Transcriptome Sequencing and Metabolome Analysis Reveal Genes Involved in Pigmentation of Green-Colored Cotton Fibers.

Green-colored fiber (GCF) is the unique raw material for naturally colored cotton textile but we know little about the pigmentation process in GCF. Here we compared transcriptomes and metabolomes of 12, 18 and 24 days post-anthesis (DPA) fibers from a green fiber cotton accession and its white-colored fiber (WCF) near-isogenic line. We found a total of 2047 non-redundant metabolites in GCF and WCF that were enriched in 80 pathways, including those of biosynthesis of phenylpropanoid, cutin, suberin, and wax. Most metabolites, particularly sinapaldehyde, of the phenylpropanoid pathway had a higher level in GCF than in WCF, consistent with the significant up-regulation of the genes responsible for biosynthesis of those metabolites. Weighted gene co-expression network analysis (WGCNA) of genes differentially expressed between GCF and WCF was used to uncover gene-modules co-expressed or associated with the accumulation of green pigments. Of the 16 gene-modules co-expressed with fiber color or time points, the blue module associated with G24 (i.e., GCF at 24 DPA) was of particular importance because a large proportion of its genes were significantly up-regulated at 24 DPA when fiber color was visually distinguishable between GCF and WCF. A total of 56 hub genes, including the two homoeologous Gh4CL4 that could act in green pigment biosynthesis, were identified among the genes of the blue module that are mainly involved in lipid metabolism, phenylpropanoid biosynthesis, RNA transcription, signaling, and transport. Our results provide novel insights into the mechanisms underlying pigmentation of green fibers and clues for developing cottons with stable green colored fibers.