GhFAD3-4 Promotes Fiber Cell Elongation and Cell Wall Thickness by Increasing PI and IP3 Accumulation in Cotton

The omega-3 fatty acid desaturase enzyme gene FAD3 is responsible for converting linoleic acid to linolenic acid in plant fatty acid synthesis. Despite limited knowledge of its role in cotton growth, our study focused on GhFAD3-4, a gene within the FAD3 family, which was found to promote fiber elongation and cell wall thickness in cotton. GhFAD3-4 was predominantly expressed in elongating fibers, and its suppression led to shorter fibers with reduced cell wall thickness and phosphoinositide (PI) and inositol triphosphate (IP3) levels. Transcriptome analysis of GhFAD3-4 knock-out mutants revealed significant impacts on genes involved in the phosphoinositol signaling pathway. Experimental evidence demonstrated that GhFAD3-4 positively regulated the expression of the GhBoGH3B and GhPIS genes, influencing cotton fiber development through the inositol signaling pathway. The application of PI and IP6 externally increased fiber length in GhFAD3-4 knock-out plants, while inhibiting PI led to a reduced fiber length in GhFAD3-4 overexpressing plants. These findings suggest that GhFAD3-4 plays a crucial role in enhancing fiber development by promoting PI and IP3 biosynthesis, offering the potential for breeding cotton varieties with superior fiber quality.


Introduction
Cotton, belonging to the Malvaceae family, can be an annual herb or a perennial shrub.Its leaves are broad and ovate, with an acuminate apex and a wide base.The petioles are sparsely pubescent, and the stipules are ovate and sickle-shaped.The flowers are solitary in the leaf axils, with pedicels larger than the petioles.The calyx is slightly short and cup-shaped, and the seeds are oval, covered with long white cotton hairs and gray-white short cotton hairs that are not easily removable.Cotton is used in the production of various textiles for clothing, furniture, and industrial purposes.It is considered one of the most important crops globally due to its high output and low production costs, resulting in relatively inexpensive cotton products.Transgenic cotton refers to cotton varieties that have been genetically modified by introducing beneficial genes from other species into the cotton genome.This genetic modification can lead to the development of high-yielding, high-quality, and insect-resistant cotton strains through scientific research.
Upland cotton (Gossypium hirsutum) is a significant fiber crop globally [1].Cotton fiber, a widely used natural material in textile industries, originates from a specialized single epidermal cell in the seed coat and serves as a valuable model for studying plant cell elongation [2].The development of cotton fiber involves four main stages: initiation, elongation, secondary cell wall (SCW) biosynthesis, and maturation [3].The majority of cell wall structures are synthesized in the Golgi apparatus and then transported to the apoplast for cell wall synthesis and remodeling [4,5].Fiber elongation determines the final length of the mature fiber, while the thickening of the SCW influences the strength and fineness Plants 2024, 13, 1510 2 of 13 of the mature fiber, which are crucial factors for assessing cotton fiber quality [6].Cotton fiber development initiates on the ovule surface on the day of anthesis and experiences rapid elongation, typically between 5 and 20 DPA (days post-anthesis), during which the ultimate fiber length is established [7].Therefore, an increase in fatty acid (FA) synthesis is essential for membrane expansion as fiber cells undergo rapid growth [8].
The biosynthesis of fatty acids occurs in the plastids of plant cells, where the continuous linking of 2-carbon units results in the formation of 16-or 18-carbon long fatty acids that are predominant in the cell membrane.Within the plastids, the soluble fatty acid desaturase can catalyze the conversion of 18:0 to 18:1, with the number 18 representing the carbon atoms in the molecule.The 18:1 fatty acids can then be further transformed into 18:2 either in the plastids or the endoplasmic reticulum (ER).The conversion of 18:1 to 18:2 is facilitated by FAD2 or FAD6 fatty acid desaturation enzymes.FAD2 and FAD6 exhibit similarities in their sequences, although FAD6 possesses a longer N-terminal.The production of 18:2 is triggered by FAD7 or FAD8 fatty acid dehydrogenase, and it can also be converted into 18:3 by the FAD3 enzyme before being exported to the endoplasmic reticulum.FAD7/FAD8 and FAD3 are recognized as omega-3 fatty acid desaturases due to their ability to introduce a double bond at the omega-3 position of the fatty acid structure.Consequently, the enzymes FAD6 and FAD2, responsible for producing 18:2, as well as FAD7/FAD8 and FAD3, which generate 18:3, play a pivotal role in the biosynthesis of polyunsaturated fatty acids (PUFAs) present in all plant species [9].
Fatty acids are crucial components of plant membrane phospholipids and seed storage triacylglycerols.The synthesis of fatty acids plays a significant role in regulating their content, composition, and fiber development [10].Fatty acid desaturase (FAD) is a key enzyme involved in the production of unsaturated fatty acids by introducing one or more carbon double bonds into hydrocarbon chains at various positions [9].Specifically, omega-3 fatty acid desaturase (FAD3) is an essential enzyme responsible for converting linoleic acid to linolenic acid (C18:3), which greatly enhances fiber development [10].
Phosphatidylinositol synthase catalyzes the conversion of the substrate C18:3 and CDP-DAG into PI, which is composed of 1,2-DAG phosphate and inositol.PI, a type of membrane lipid, plays a crucial role as a regulatory molecule in the secretion and assembly of cell-wall polymers [11].The phosphorylation of PI leads to the production of phosphatidylinositol 4-phosphate (PIP4), which can be further converted into PIP2 [12].In plants, PIPLC, an important lipid hydrolase, cleaves PIP2 to generate two significant secondary messengers, IP 3 and DAG [13].Upon phosphorylation, IP 3 forms inositol hexaphosphate (IP 6 ) and can promote fiber cell elongation by increasing ethylene biosynthesis [14,15].However, the role of FAD3 in cotton remains uncharacterized.
Targeted lipidomics studies reveal that linolenic acid (C18:3) promotes cotton fiber elongation by activating phosphatidylinositol and phosphatidylinositol ponophosphate biosynthesis [10].GhFAD3 has been found to promote cotton fiber development [10], but its specific mechanism for promoting cotton fiber development has not been confirmed.Secondly, there is no systematic comparison of GhFAD3 family genes.In this study, a GhFAD3 gene, GhFAD3-4, was identified as a promoter of both fiber cell elongation and cell wall thickness.Fibers of over-expressing GhFAD3-4 showed higher accumulation of PI and IP 3 compared to wild-type and GhFAD3-4 knock-out mutants.Silencing GhFAD3-4 resulted in reduced fiber length and cell wall thickness.Additionally, the application of exogenous PI and IP 6 significantly improved fiber development.These findings suggest that GhFAD3-4 plays a crucial role in promoting cotton fiber development by enhancing PI and IP 3 accumulation, leading to increased cell wall deposition and ethylene biosynthesis.

Genome-Wide Identification of GhFAD3 in Cotton
Previous studies have indicated that linolenic acid C18:3 can enhance cotton fiber development [10,15].The role of the key dehydrogenase fatty acid FAD3 in linolenic acid synthesis in cotton fiber development remains uncertain.By analyzing AtFAD3, we Plants 2024, 13, 1510 3 of 13 identified 11 GhFAD3 protein-encoding genes through homology searches in the Gossypium hirsutum genome (TM-1 v2.1).Phylogenetic analysis of these GhFAD3 proteins in cotton revealed that GhFAD3-4 and GhFAD3-5 have the closest phylogenetic relationship, while GhFAD3-1 and GhFAD3-2 show a closer phylogenetic connection (Figure S1a).Notably, based on database predictions, five GhFAD3 genes (GhFAD3-2A, GhFAD3-2D, GhFAD3-3A, GhFAD3-4A, and GhFAD3-4D) exhibit significantly higher expression levels during rapid fiber development stages (Figure 1a).Alignment of multiple sequences unveiled two conserved domains within FAD3 proteins (Figure S1b).Furthermore, we examined the expression levels of GhFAD3 homologous genes at different fiber developmental stages and observed high expression of GhFAD3-1A and GhFAD3-4A during rapid fiber elongation (Figure 1b-g).Importantly, GhFAD3-4A and GhFAD3-4D were notably highly expressed at both 10 DPA and 15 DPA during the rapid fiber elongation period, indicating their potential key role in this stage (Figure 1a,e).In this study, we selected GhFAD3-4D as the research object and abbreviated it as GhFAD3-4.

Genome-Wide Identification of GhFAD3 in Cotton
Previous studies have indicated that linolenic acid C18:3 can enhance cotton fiber development [10,15].The role of the key dehydrogenase fatty acid FAD3 in linolenic acid synthesis in cotton fiber development remains uncertain.By analyzing AtFAD3, we identified 11 GhFAD3 protein-encoding genes through homology searches in the Gossypium hirsutum genome (TM-1 v2.1).Phylogenetic analysis of these GhFAD3 proteins in cotton revealed that GhFAD3-4 and GhFAD3-5 have the closest phylogenetic relationship, while GhFAD3-1 and GhFAD3-2 show a closer phylogenetic connection (Figure S1a).Notably, based on database predictions, five GhFAD3 genes (GhFAD3-2A, GhFAD3-2D, GhFAD3-3A, GhFAD3-4A, and GhFAD3-4D) exhibit significantly higher expression levels during rapid fiber development stages (Figure 1a).Alignment of multiple sequences unveiled two conserved domains within FAD3 proteins (Figure S1b).Furthermore, we examined the expression levels of GhFAD3 homologous genes at different fiber developmental stages and observed high expression of GhFAD3-1A and GhFAD3-4A during rapid fiber elongation (Figure 1b-g).Importantly, GhFAD3-4A and GhFAD3-4D were notably highly expressed at both 10 DPA and 15 DPA during the rapid fiber elongation period, indicating their potential key role in this stage (Figure 1a,e).In this study, we selected GhFAD3-4D as the research object and abbreviated it as GhFAD3-4.
In the GhFAD3-4 transgenic materials analyzed, the expression levels of GhFAD3 homologous genes were investigated.GhFAD3-5A and GhFAD3-5D were notably upregulated in GhFAD3-4-OE and downregulated in GhFAD3-4-KO.Our study on the GhFAD3-4 gene's function may involve the influence of GhFAD3-5 (Figure S3).GhFAD3-4 exhibited widespread expression across all tested tissues, with particularly high levels in root and leaf tissues rich in membranous organelles, suggesting a potential key role in membrane expansion (Figure S2).GhFAD3-4-OE plants showed significant enhancement in fiber cell length compared to non-transgenic controls, while knocking out GhFAD3-4A/D resulted in reduced fiber cell length (Figure 2e,f).Consequently, GhFAD3-4-KO plants displayed Plants 2024, 13, 1510 4 of 13 a marked decrease in cell wall thickness compared to wild-type (WT) and GhFAD3-4-OE plants (Figure 2g,h).Fiber twist testing indicated that neither GhFAD3-4-OE nor GhFAD3-4-KO significantly affected fiber distortion (Figure S4).These findings suggest that GhFAD3-4 may play a role in promoting cotton fiber elongation and cell wall thickening.lated in GhFAD3-4-OE and downregulated in GhFAD3-4-KO.Our study on the GhFAD3-4 gene's function may involve the influence of GhFAD3-5 (Figure S3).GhFAD3-4 exhibited widespread expression across all tested tissues, with particularly high levels in root and leaf tissues rich in membranous organelles, suggesting a potential key role in membrane expansion (Figure S2).GhFAD3-4-OE plants showed significant enhancement in fiber cell length compared to non-transgenic controls, while knocking out GhFAD3-4A/D resulted in reduced fiber cell length (Figure 2e,f).Consequently, GhFAD3-4-KO plants displayed a marked decrease in cell wall thickness compared to wild-type (WT) and GhFAD3-4-OE plants (Figure 2g,h).Fiber twist testing indicated that neither GhFAD3-4-OE nor GhFAD3-4-KO significantly affected fiber distortion (Figure S4).These findings suggest that GhFAD3-4 may play a role in promoting cotton fiber elongation and cell wall thickening.

Transcriptome Identification and Characterization of GhFAD3-4 Downstream Genes
To investigate the role of GhFAD3-4 in cotton fiber development, we conducted transcriptome sequencing to identify differentially expressed genes in GhFAD3-4 transgenic lines.Our analysis revealed that 1265 and 3458 genes were upregulated in GhFAD3-4 overexpressing lines compared to control plants and GhFAD3-4A/D knock-out lines, respectively (Figure 3a-c).Interestingly, 763 genes were found to be commonly upregulated in both comparisons (Figure 3d).Furthermore, KEGG pathway analysis indicated enrichment of genes involved in inositol phosphate metabolism, with a particular focus on the phosphatidylinositol pathway in linolenic acid regulation (Figure 3e) [17].

Transcriptome Identification and Characterization of GhFAD3-4 Downstream Genes
To investigate the role of GhFAD3-4 in cotton fiber development, we conducted transcriptome sequencing to identify differentially expressed genes in GhFAD3-4 transgenic lines.Our analysis revealed that 1265 and 3458 genes were upregulated in GhFAD3-4 overexpressing lines compared to control plants and GhFAD3-4A/D knock-out lines, respectively (Figure 3a-c).Interestingly, 763 genes were found to be commonly upregulated in both comparisons (Figure 3d).Furthermore, KEGG pathway analysis indicated enrichment of genes involved in inositol phosphate metabolism, with a particular focus on the phosphatidylinositol pathway in linolenic acid regulation (Figure 3e) [17].

Expression Analysis of Genes Related to the Inositol Signaling Pathway in GhFAD3-4 Transgenic Materials
Transcriptome analysis of GhFAD3-4 transgenic material suggested that the phosphatidyl inositol metabolism pathway may be the downstream signaling pathway regulated by GhFAD3-4 in cotton fiber (Figure 3e).Genes enriched in this pathway include GhBoGH3B, GhMIOX4, GhPIS, and GhRNF144B.To investigate their roles in cotton fiber development, we assessed their expression levels at different development stages, including initiation, elongation, secondary cell wall synthesis, and maturation.During the development of cotton fibers from 0 to 20 days, the expression of GhBoGH3B gradually increased with time and reached its highest level at 20 days (Figure 4a).GhMIOX4 showed the highest expression at 0 DPA but was lower at other stages (Figure 4b).GhPIS has significantly higher expression during rapid elongation compared to initial and secondary cell wall synthesis (Figure 4c).The expression level of GhRNF144B gradually increased with the passage of time from 0 to 15 days of fiber development and then decreased after 15 days (Figure 4d).

PI and IP3 Can Promote the Development of Cotton Fiber
PI and IP3, key components of the inositol signaling pathway, play a crucial role in promoting cotton fiber development by enhancing ethylene biosynthesis [15].When phosphorylated, IP3 transforms into inositol hexaphosphate [18], which possesses diverse functions in plants.The catalytic action of PIPLC on PIP2 leads to the formation of IP3, which can subsequently undergo phosphorylation to generate IP6 [19].Our analysis of PI and IP3 levels across different developmental stages of cotton fibers revealed peak concentrations at 15 DPA, particularly during the rapid elongation phase of fiber growth (Figure 5a,b).Through an in vitro ovule culture system, we confirmed the positive impact of both PI and IP3 on cotton fiber development.Notably, the inhibition of PI resulted in a significant reduction in fiber development, aligning with previous findings (Figure 5b,c,e,f) [15].To investigate the regulatory role of GhFAD3-4 on four specific genes, we analyzed the expression levels of these genes in GhFAD3-4 transgenic materials (Figure 4e-h).Our results show that GhBoGH3B and GhPIS expression levels are significantly higher in GhFAD3-4-OE compared to the wild type, while GhFAD3-4-KO exhibits significantly lower expression levels for both genes (Figure 4e,g).The expression of GhMIOX4 in GhFAD3-4 transgenic materials did not show a significant difference (Figure 4f).Interestingly, GhRNF144B expression in GhFAD3-4-KO materials was significantly higher than in the wild type, with no significant difference observed between GhFAD3-4-OE and the wild type (Figure 4h).These findings suggest that GhFAD3-4 may regulate cotton fiber development by influ-encing the expression of GhBoGH3B and GhPIS, key components of the phosphoinositol signaling pathway.Further research is required to confirm the potential involvement of the phosphoinositol signaling pathway in the promotion of cotton fiber development by GhFAD3-4.

PI and IP 3 Can Promote the Development of Cotton Fiber
PI and IP 3 , key components of the inositol signaling pathway, play a crucial role in promoting cotton fiber development by enhancing ethylene biosynthesis [15].When phosphorylated, IP 3 transforms into inositol hexaphosphate [18], which possesses diverse functions in plants.The catalytic action of PIPLC on PIP2 leads to the formation of IP 3 , which can subsequently undergo phosphorylation to generate IP 6 [19].Our analysis of PI and IP 3 levels across different developmental stages of cotton fibers revealed peak concentrations at 15 DPA, particularly during the rapid elongation phase of fiber growth (Figure 5a,b).Through an in vitro ovule culture system, we confirmed the positive impact of both PI and IP 3 on cotton fiber development.Notably, the inhibition of PI resulted in a significant reduction in fiber development, aligning with previous findings (Figure 5b,c,e,f) [15].

GhFAD3-4 Promotes Cotton Fiber Development by Increasing PI and IP3 Accumulation
To investigate the mechanisms by which GhFAD3-4 regulates fiber development through phosphatidylinositol, we quantified phosphatidylinositol (PI) content and inositol 1,4,5-trisphosphate (IP3) accumulation in the GhFAD3-4 transgenic lines.Our results showed a significant increase in the levels of PI and IP3 in cotton fibers overexpressing GhFAD3-4 (GhFAD3-4-OE), while a decrease was observed in fibers with GhFAD3-4 knocked out (GhFAD3-4-KO) (Figure 6a,b).Subsequently, we utilized an in vitro cotton ovule culture system to validate the role of PI and IP3 in GhFAD3-4-mediated regulation of fiber development.The experiments demonstrated that GhFAD3-4-OE fibers exhibited increased length compared to the wild type, whereas GhFAD3-4-KO fibers were shorter

GhFAD3-4 Promotes Cotton Fiber Development by Increasing PI and IP 3 Accumulation
To investigate the mechanisms by which GhFAD3-4 regulates fiber development through phosphatidylinositol, we quantified phosphatidylinositol (PI) content and inositol 1,4,5-trisphosphate (IP 3 ) accumulation in the GhFAD3-4 transgenic lines.Our results showed a significant increase in the levels of PI and IP 3 in cotton fibers overexpressing GhFAD3-4 (GhFAD3-4-OE), while a decrease was observed in fibers with GhFAD3-4 knocked out (GhFAD3-4-KO) (Figure 6a,b).Subsequently, we utilized an in vitro cotton ovule culture system to validate the role of PI and IP 3 in GhFAD3-4-mediated regulation of fiber development.The experiments demonstrated that GhFAD3-4-OE fibers exhibited increased length compared to the wild type, whereas GhFAD3-4-KO fibers were shorter (Figure 6c,d).Furthermore, treatment with PI and IP 6 partially rescued the shortened fiber phenotype in GhFAD3-4-KO, while the application of a PI inhibitor significantly inhibited fiber elongation in GhFAD3-4-OE (Figure 6c,d).Overall, our findings indicate that the overexpression of GhFAD3-4 promotes fiber elongation, while the knockout of GhFAD3-4A/D hinders this process.Importantly, the in vitro supplementation of PI and IP 6 successfully reversed the short fiber phenotype induced by GhFAD3-4A/D knockout (Figure 6c).These results suggest that GhFAD3-4 may enhance cotton fiber elongation and thickening of the fiber cell wall by stimulating the accumulation of IP and IP 3 in cotton fibers.

Various Fatty Acid Synthases Are Involved in the Elongation of Cotton Fibers
Various fatty acids, such as very-long-chain fatty acids (VLCFAs), short-chain fatty acids, and unsaturated fatty acids, have been identified to regulate cotton fiber elongation [20][21][22].Specifically, two ketoacyl-CoA synthase genes (KCS) are involved in the biosynthesis of VLCFAs, which is crucial for high-grade cotton fiber development [22].Silencing experiments targeting fatty acid desaturases (GhFAD), PI synthase (PIS), and PI kinase (PIK) resulted in reduced mRNA levels of these genes in fibers, leading to a 10 mm decrease in fiber length compared to control plants [10].Furthermore, our study highlights the role of unsaturated fatty acid synthase GhFAD3-4 in promoting cotton fiber elongation through the production of C18:3 from C18:2, expanding our understanding of the regulatory mechanisms involved in cotton fiber development [15].

Various Fatty Acid Synthases Are Involved in the Elongation of Cotton Fibers
Various fatty acids, such as very-long-chain fatty acids (VLCFAs), short-chain fatty acids, and unsaturated fatty acids, have been identified to regulate cotton fiber elongation [20][21][22].Specifically, two ketoacyl-CoA synthase genes (KCS) are involved in the biosynthesis of VLCFAs, which is crucial for high-grade cotton fiber development [22].Silencing experiments targeting fatty acid desaturases (GhFAD), PI synthase (PIS), and Plants 2024, 13, 1510 9 of 13 PI kinase (PIK) resulted in reduced mRNA levels of these genes in fibers, leading to a 10 mm decrease in fiber length compared to control plants [10].Furthermore, our study highlights the role of unsaturated fatty acid synthase GhFAD3-4 in promoting cotton fiber elongation through the production of C18:3 from C18:2, expanding our understanding of the regulatory mechanisms involved in cotton fiber development [15].

Biological Function of FAD3 Homologs in Plants
Numerous studies have demonstrated the role of FAD3 in promoting the biosynthesis of linolenic acid [23][24][25].Physaria fendleri FAD3-1 overexpression increases A-linolenic acid content in camelina seeds [23].Some studies have even conducted molecular-level analyses to determine the specific mechanisms through which linolenic acid operates.Various plant species, including B. napus, Camelina sativa, Linum usitatissimum, Vernicia fordii, Gossypium hirsutum, S. hispanica, Cannabis sativa, and P. frutescens, exhibit a retention signal KXKXX/XKXX at their c-terminus, a key characteristic of FADs.Furthermore, research indicates that BnFAD3 acts as a transmembrane protein, converting omega-6 to omega-3 fatty acids while potentially functioning as a potassium ion channel in the endoplasmic reticulum [24].In addition, some studies on flaxseed (Linum usitatissimum) have identified the FAD3 protein as a potential source of peptides with angiotensin-converting enzyme (ACE) inhibitory and dipeptidyl peptidase-IV (DPP-IV) activities [25].This particular study delved into the downstream signaling pathway of GhFAD3-4 and confirmed its impact on cotton fiber development by influencing the accumulation of PI and IP3.The lack of a molecular-level explanation for this phenomenon could potentially pave the way for further in-depth exploration of the functions of FAD3-4 in future research.

The Role of the Phosphoinositol Signaling Pathway in Cotton Fiber Development
Inositol-1,4,5-trisphosphate (IP 3 ) is a crucial second messenger produced through the hydrolysis of phosphatidylinositol (4,5) bisphosphate (PIP2) by phosphoinositidespecific phospholipase C (PIPLC).GhPIPLC2D, predominantly expressed in elongating fibers, plays a key role in fiber elongation.A reduction in GhPIPLC2D transcription led to shorter fibers with decreased levels of IP 3 and ethylene production.Treatment with linolenic acid (C18:3) and phosphatidylinositol (PI), a precursor of IP 3 , enhanced the accumulation of IP 3 , myo-inositol-1,2,3,4,5,6-hexakisphosphate (IP 6 ), and ethylene biosynthesis.Silencing GhPIPLC2D resulted in reduced fiber length, which could be rescued by the exogenous application of IP 6 and ethylene.The positive regulation of fiber elongation by GhPIPLC2D and IP 3 is linked to enhanced ethylene biosynthesis [15].Additionally, external application of linolenic acid (C18:3), soybean L-alpha-PI, and specific PIPs containing PIP 34:3 significantly promoted fiber growth, while a liver PI lacking the C18:3 moiety, linoleic acid, and PIP 36:2 were found to be ineffective [10].
The study systematically analyzed the levels of PI and IP 3 in the inositol phosphate signaling pathway at various stages of cotton fiber development.The function of these molecules was assessed through in vitro ovule culture experiments (Figure 5).The research determined that GhFAD3-4 promotes cotton fiber development by increasing the accumulation of PI and IP 3 within the fibers (Figure 6).Furthermore, the study investigated the regulatory effects of GhFAD3-4 on the expression levels of four genes in the inositol phosphate signaling pathway, revealing significant differences in the expression levels of GhBoGH3B and GhPIS (Figure 4).These findings suggest that GhFAD3-4 may influence cotton fiber development by upregulating the expression of these two genes.However, additional experimental evidence is required to further support these conclusions.

Conclusions
Fiber quality and yield, determined by fiber length and strength, are crucial traits for the textile industry.Our study found that GhFAD3-4 plays a role in regulating fiber quality by increasing PI accumulation, which is essential for plant cell wall assembly and may enhance fiber cell wall thickness.Additionally, the results showed a significant increase in IP 3 content in GhFAD3-4-OE lines compared to control and GhFAD3-4 knockout lines, indicating a close relationship to PI decomposition.Therefore, PI and IP 3 are key regulators of GhFAD3-4 function.Overall, our study enhances the toolkit for improving agronomic traits (quality and yield) through genetic manipulation in the future.

Plant Material and Growth Conditions
The experimental wild-type upland cotton and transgenic cotton lines were cultivated in a greenhouse under controlled conditions of 28 • C with 14 h of light and 10 h of darkness.Ovules from the same-day flowering were collected for in vitro ovule culture experiments post-flowering, and fiber length was measured upon full maturation of cotton bolls.Cotton fibers were sampled at 5, 10, 15, 20, and 25 days after flowering to analyze the gene expression levels at various stages of fiber development.

Cotton Transformation
The vector construction primers for constructing GhFAD3-4 overexpression and gene knockout materials are shown in Table S1.For GhFAD3-4 genetic transformation, the constructed plasmids were introduced into the LB4404 Agrobacterium tumefaciens strain to be used on 'Jin668' cotton seedlings.5-7 mm hypocotyls from the seedlings were incubated with the A. tumefaciens suspension (OD600 = 0.1-0.5)for 20 min and then transferred onto a callus induction medium after being blotted dry on sterilized filter papers.After callus induction, proliferation, embryogenic callus induction, embryo differentiation, and plantlet regeneration, the putative transgenic plants were transferred to pots and grown in a greenhouse at 28 • C under a long-day (14 h light/10 h dark) cycle (Figure 7).

Cotton Transformation
The vector construction primers for constructing GhFAD3-4 overexpression and gene knockout materials are shown in Table S1.For GhFAD3-4 genetic transformation, the constructed plasmids were introduced into the LB4404 Agrobacterium tumefaciens strain to be used on 'Jin668' cotton seedlings.5-7 mm hypocotyls from the seedlings were incubated with the A. tumefaciens suspension (OD600 = 0.1-0.5)for 20 min and then transferred onto a callus induction medium after being blotted dry on sterilized filter papers.After callus induction, proliferation, embryogenic callus induction, embryo differentiation, and plantlet regeneration, the putative transgenic plants were transferred to pots and grown in a greenhouse at 28 °C under a long-day (14 h light/10 h dark) cycle (Figure 7).
Callus induction, proliferation medium: MS inorganic salts, B5 vitamins as the basic medium (hereinafter referred to as MSB), plus different types of hormone combinations.
Somatic embryo differentiation medium: MSB basic medium (KNO3 doubled, NH4NO3 removed) and supplemented with GIn 2.0 g/L and Asn 1.0 g/L.

Cotton Ovule Culture
The operation method of the cotton ovule culture experiment is as described in the previous literature [15].One day after fertilization, cotton bolls were collected, and the isolated ovule was removed for the ovule culture experiment.The ovules were first sterilized in a 10% sodium hypochlorite solution, peeled out in a sterile environment, and cultured separately on a fluid nutrient medium with 5 µM PI, 5 µM IP6, and 1 µM PI inhibitor (5-hydroxytryptamine) for 15 days.

RNA Extraction and RT-qPCR Analysis
Total RNA was extracted from cotton fibers, roots, stems, and leaves using an RNA isolation kit (Tiangen, Beijing, China) following the manufacturer's instructions.Firststrand cDNA was then reverse-transcribed from the total RNA using a kit from Takara (San Jose, CA, USA).RT-qPCR was conducted with gene-specific primers detailed in Supplemental Table S1, with GhUBQ7 (GenBank No. AY189972) serving as the internal control.The reactions were carried out using the Roche LightCycler ® 480 II instrument (Roche Sterile seedling germination medium: 1/2 MS macronutrients, supplemented with 15 g/L glucose and 2.5 g/L Phytagel (Sigma, St. Louis, MI, USA).
Callus induction, proliferation medium: MS inorganic salts, B5 vitamins as the basic medium (hereinafter referred to as MSB), plus different types of hormone combinations.
Somatic embryo differentiation medium: MSB basic medium (KNO 3 doubled, NH 4 NO 3 removed) and supplemented with GIn 2.0 g/L and Asn 1.0 g/L.
Embryo germination and rooting medium: 1/2 MS inorganic salts + B 5 organic matter, addition of 15 g/L glucose and 2.5 g/L phytagel.

Cotton Ovule Culture
The operation method of the cotton ovule culture experiment is as described in the previous literature [15].One day after fertilization, cotton bolls were collected, and the isolated ovule was removed for the ovule culture experiment.The ovules were first sterilized in a 10% sodium hypochlorite solution, peeled out in a sterile environment, and cultured separately on a fluid nutrient medium with 5 µM PI, 5 µM IP 6 , and 1 µM PI inhibitor (5-hydroxytryptamine) for 15 days.

Figure 1 .
Figure 1.Genome-wide identification of GhFAD3 in cotton.(a) Heatmap of GhFAD3 expression at five stages of fiber development.(b-g) Relative expression levels of GhFAD3-4 homologs in fibers at different developmental stages.DPA, day-post anthesis.

Figure 1 .
Figure 1.Genome-wide identification of GhFAD3 in cotton.(a) Heatmap of GhFAD3 expression at five stages of fiber development.(b-g) Relative expression levels of GhFAD3-4 homologs in fibers at different developmental stages.DPA, day-post anthesis.

Figure 3 .
Figure 3. Transcriptome identification and characterization of GhFAD3-4 downstream genes.(a) Volcanic maps of differentially expressing genes in GhFAD3-4-OE lines compared with the control.

Figure 3 .
Figure 3. Transcriptome identification and characterization of GhFAD3-4 downstream genes.(a) Volcanic maps of differentially expressing genes in GhFAD3-4-OE lines compared with the control.Blue dots: significantly upregulated genes; red dots: significantly downregulated genes; Grey dots, nondifferentially expressed genes.(b) Volcanic maps of differentially expressing genes in GhFAD3-4-OE lines compared with GhFAD3-4 knockout lines.Blue dots: significantly upregulated genes.Red dots: significantly downregulated genes.Grey dots: nondifferentially expressed genes.(c) The bar graph counts the number of differentially expressing genes in the GhFAD3-4-OE lines compared with control and GhFAD3-4 knockout lines.(d) Venn diagram showing the 763 overlapping genes in the DEGs that were upregulated in the GhFAD3-4-OE lines compared with control and GhFAD3-4 knockout lines, respectively.DEGs: differentially expressed genes.(e) KEGG enrichment analysis of the 763 overlapping upregulated DEGs.

Plants 2024 ,
13,  x FOR PEER REVIEW 11 of 14 experiments post-flowering, and fiber length was measured upon full maturation of cotton bolls.Cotton fibers were sampled at 5, 10, 15, 20, and 25 days after flowering to analyze the gene expression levels at various stages of fiber development.