Next Article in Journal
A Selective Melatonin 2 Receptor Agonist, IIK7, Relieves Blue Light-Induced Corneal Damage by Modulating the Process of Autophagy and Apoptosis
Next Article in Special Issue
Genome Analysis of BnCNGC Gene Family and Function Exploration of BnCNGC57 in Brassica napus L.
Previous Article in Journal
Evaluation of Blood–Brain Barrier Disruption Using Low- and High-Molecular-Weight Complexes in a Single Brain Sample in a Rat Traumatic Brain Injury Model: Comparison to an Established Magnetic Resonance Imaging Technique
Previous Article in Special Issue
Identification of Grape Laccase Genes and Their Potential Role in Secondary Metabolite Synthesis
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 25
Issue 20
10.3390/ijms252011242
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Downregulation of the GhROD1 Gene Improves Cotton Fiber Fineness by Decreasing Acyl Pool Saturation, Stimulating Small Heat Shock Proteins (sHSPs), and Reducing H2O2 Production

by
Bo Ding
1,2,
Bi Liu
1,2,
Xi Zhu
1,2,
Huiming Zhang
1,2,
Rongyu Hu
1,2,
Silu Li
1,2,
Liuqin Zhang
1,2,
Linzhu Jiang
1,2,
Yang Yang
1,2,
Mi Zhang
1,2,
Juan Zhao
1,2,
Yan Pei
1,2 and
Lei Hou
1,2,*
1
College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China
2
Chongqing Key Laboratory of Application and Safety Control of Genetically Modified Crops, Southwest University, Chongqing 400715, China
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2024, 25(20), 11242; https://doi.org/10.3390/ijms252011242
Submission received: 11 September 2024 / Revised: 16 October 2024 / Accepted: 17 October 2024 / Published: 19 October 2024
(This article belongs to the Special Issue Advances in Plant Genomics and Genetics: 2nd Edition)
Browse Figures
Review Reports Versions Notes

Abstract

:
Cotton fiber is one of the most important natural fiber sources in the world, and lipid metabolism plays a critical role in its development. However, the specific role of lipid molecules in fiber development and the impact of fatty acid alterations on fiber quality remain largely unknown. In this study, we demonstrate that the downregulation of GhROD1, a gene encoding phosphatidylcholine diacylglycerol cholinephosphotransferase (PDCT), results in an improvement of fiber fineness. We found that GhROD1 downregulation significantly increases the proportion of linoleic acid (18:2) in cotton fibers, which subsequently upregulates genes encoding small heat shock proteins (sHSPs). This, in turn, reduces H2O2 production, thus delaying secondary wall deposition and leading to finer fibers. Our findings reveal how alterations in linoleic acid influence cellulose synthesis and suggest a potential strategy to improve cotton fiber quality by regulating lipid metabolism pathways.

1. Introduction

Cotton fiber, the primary natural fiber source for the global textile industry, constitutes around 35% of the world fiber production [1]. The economic significance of the cotton textile industry is substantial, with a global market value exceeding USD 600 billion annually [2]. Cotton fibers develop as single cells, exhibiting remarkable uniformity and synchronicity during their growth. When fully matured, the fibers achieve a length-to-diameter ratio ranging from 1000 to 3000. Uniquely, mature cotton fibers consist of more than 95% cellulose by dry weight, with very low lignin content, distinguishing them from most other plant cell walls. These characteristics make cotton fibers an ideal model for investigating plant cell differentiation, elongation, and secondary cell wall development [3]. Fiber development can be broadly categorized into five overlapping stages: initiation, elongation, transition, secondary cell wall (SCW) biogenesis, and dehydration maturation. Among them, the elongation stage determines final fiber length, while the SCW biogenesis stage determines fiber fineness and strength. The transition stage, which marks the shift from primary to secondary cell wall synthesis, significantly influences both fiber length and cell wall thickness [4].
Lipids are vital for organisms, serving as key components of biological membranes that create a hydrophobic barrier to preserve the intracellular environment necessary for metabolic processes. They also function as energy storage compounds essential for cell growth and development as well as signaling molecules that regulate various cellular processes [5]. Accumulating investigations show that during fiber initiation and elongation, the lipid metabolism pathway is significantly enhanced [6,7,8,9]. However, the precise mechanisms of lipid metabolism in cotton fiber development remain poorly understood, and the specific roles of various lipid species in these processes await to be elucidated.
The rapid synthesis of cytoplasmic and vacuolar membranes during fiber elongation increases the cellular demand for glycerophospholipids [10,11]. Glycerophospholipids, essential components of cell membranes, are classified into six major types: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid (PA), and phosphatidylglycerol (PG) [12,13].
PC is a major component of cell membrane phospholipids, serving as a substrate for fatty acid desaturation [14]. In cotton fibers, PC is the most abundant lipid class during the elongation and secondary wall synthesis stages [10]. However, the specific function of PC in cotton fiber development remains largely unknown. Phosphatidylcholine cholinephosphotransferase (PDCT) catalyzes the transfer of the phosphocholine headgroup between PC and diacylglycerol (DAG), facilitating the exchange of fatty acyl groups between these lipid molecules [15]. In plants, the impact of PDCT, also known as ROD1, on fatty acid composition in seeds has been well-documented [16,17,18,19]. However, the influence of ROD1-mediated fatty acid alterations on cotton fiber development and fiber quality remains unexplored.
To investigate the function of PDCT in lipid metabolism in fiber cells and assess its role in fiber quality and yield traits, we cloned an AtROD1 homologous, GhROD1, from upland cotton (Gossypium hirsutum). We demonstrated that downregulation of GhROD1 resulted in an increase in C18:2 levels in fibers. Importantly, consecutive three-year field trails showed that the fineness of the GhROD1-downregulated fibers improved significantly. We further revealed that the elevated C18:2 level promoted the transcription of genes encoding small heat shock proteins (sHSPs), which in turn decreased the production of H2O2. This reduction in H2O2 inhibited secondary cell wall synthesis, ultimately resulting in finer fibers. A working model for the linoleic acid (C18:2)-induced inhibition of second cell wall deposition is proposed.

2. Results

2.1. Downregulation of GhROD1 Significantly Increases Linoleic Acid in Fibers

To investigate the role of fatty acids (FAs) in fiber development, we identified and cloned GhROD1, which encodes phosphatidylcholine diacylglycerol choline phosphotransferase (PDCT), from upland cotton. qRT-PCR results indicated that GhROD1 transcripts were abundant in cotton petals, boll shells, ovules, and fibers (Figure 1A). During fiber development, GhROD1 transcript levels steadily increased from 6 to 20 days post-anthesis (DPA), followed by a sharp decline after 20 DPA (Figure 1B).
To elucidate the role of GhROD1 in fiber development, we generated its overexpressing and RNA interference (RNAi) lines in upland cotton via Agrobacterium-mediated transformation (Figure S1). The fatty acid composition GC-FID assay showed that in the fibers at 20 DPA of GhROD1-RNAi lines Ri3 and Ri4, palmitic acid (C16:0) and linolenic acid (C18:3) levels were slightly lower than in the wild-type, while stearic acid (C18:0) and oleic acid (C18:1) levels were comparable. However, linoleic acid (C18:2) content was significantly higher in the transgenic lines Ri3 and Ri4 (17.21 ± 0.12% and 15.25 ± 0.12%, respectively) compared to the wild-type (12.91 ± 0.19%) (Figure 2A). In the fibers of overexpressing lines OE17 and OE25, C18:2 was slightly lower and C16:0 was slightly higher; while C18:0 (2.37 ± 0.16% and 2.67 ± 0.23%) was significantly higher and C18:1 (2.90 ± 0.02% and 2.71 ± 0.05%) was significantly lower compared to the wild-type (C18:0 = 1.33 ± 0.02% and C18:1 = 4.26 ± 0.16%, respectively).
To investigate whether alterations in fatty acid composition extended to the cell membrane, glycerophospholipid (GPL) species were extracted from fibers at 20 DPA and quantified. Analysis revealed significant changes in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species. PC34:3, the most abundant PC species, was significantly reduced in Ri3 and Ri4 fibers, while PC34:2 was increased significantly only in Ri4 fibers. Among PE species, the most abundant, PE34:3, was significantly reduced in Ri4 fibers. PE34:2, a less abundant PE species, displayed similar changes to PC34:2 in Ri4 fibers (Figure 2C). However, diacylglycerol (DAG) species exhibited more complex alterations. DAG34:2 (16:0–18:2) increased in the Ri3 and Ri4 fibers, while other components remained largely unchanged. In the OE17 and OE25 fibers, DAG34:3 (16:0–18:3) increased, while DAG36:2 (18:0–18:2), DAG36:3 (18:1–18:2), and DAG36:3 (18:0–18:3) decreased with no significant changes in other species (Figure 2D). Other GPLs, including phosphatidylglycerol (PG), phosphatidic acid (PA), and phosphatidylserine (PS), showed no significant differences between the transgenic and wild-type fibers (Figure S2).

2.2. Downregulating GhROD1 Improves Fiber Fineness

To assess the impact of altered fatty acids resulted from GhROD1 regulation on fiber quality, we tested fiber qualities of the transgenic cotton lines across three consecutive generations. Notably, the GhROD1-downregulated fibers exhibited significant improvements in fiber fineness, as indicated by reduced micronaire (MIC) values. Specifically, the Ri3 fibers displayed a 12% to 17% reduction in MIC values (Table 1). Additionally, the GhROD1-downregulated fibers, particularly the Ri3 fibers, showed increased fiber length and strength. However, no significant change in fiber quality was observed in GhROD1-upregulated fibers (Table 1).
The MIC value is a key indicator of both fiber fineness (linear density) and maturity (the degree of secondary cell wall development) [20]. To further support the fineness improvement in GhROD1-downregulated fibers, we examined cross-sections of mature fibers (Figure 3). The thicknesses of cell walls of fibers in the downregulated Ri3 and Ri4 lines were 3.71 ± 0.42 and 4.29 ± 0.30 μm, respectively, obviously thinner than that of the wild-type (4.86 ± 0.38 μm, Figure 3F), confirming that the GhROD1-downregulating fibers became thinner.

2.3. Downregulation of GhROD1 Leads to Upregulation of sHSPs and Downregulation of Secondary Wall Synthesis-Associated Genes

To investigate the role of GhROD1 in secondary wall synthesis, we performed a transcriptome analysis of fibers at 16 DPA from both the Ri3 line and the wild-type. A total of 81,931 transcripts were obtained and 70,478 hypothetical genes were annotated. A total of 540 differentially expressed genes (DEGs) were identified in the Ri3 fibers. Among them, 286 were upregulated and 254 were downregulated (Figure 4A).
To accurately determine the link between these DEGs and fiber fineness, we performed Gene Ontology (GO) term enrichment analysis. The analysis revealed that the DEGs were primarily associated with primary and secondary cell wall biogenesis and biosynthesis, metabolism of cell wall components, osmotic and heat stress response genes, and unfolded protein binding (Figure 4B). To validate the transcriptomic results, we further analyzed GO-enriched DEGs related to plant cell wall synthesis (Figure S3B). We selected GhCesA4B, GhCesA7B, and GhCesA8B, genes involved in fiber secondary wall synthesis, for qRT-PCR analysis. In the fibers at 16 DPA of the Ri3 and Ri4 lines, the expression levels of these genes were significantly reduced, while no significant changes were observed in the OE17 and OE25 lines compared to the wild-type (Figure 5B).
We also mapped the DEGs to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The DEGs were significantly enriched in two KEGG pathways: protein processing in the endoplasmic reticulum and phenylpropanoid biosynthesis (Figure 4C). The majority of the endoplasmic reticulum protein processing genes encode small heat shock proteins (sHSPs), such as GhHSP17.6B/GhHSP17.7C, GhHSP17.8A/B, GhHSP17.9A/C/F, GhHSP18.1B/C, GhHSP18.2B, GhHSP18.3B/D, GhHSP18.4, and GhHSP20.7A (Figure S3A). qRT-PCR results confirmed that the expression of these genes was significantly upregulated in the Ri3 and Ri4 fibers, while they remained unchanged in the OE17 and OE25 lines (Figure 5A). These data suggest a connection between GhROD1 downregulation, sHSPs-related protein processing, and cell wall biosynthesis.
To support this connection, we cultured wild-type ovules on the medium containing linoleic acid. The presence of linoleic acid resulted in a significant upregulation of several sHSP genes, including GhHSP17.6B/GhHSP17.7C, GhHSP17.9A/C/F, GhHSP18.1B/C, GhHSP18.3B/D, GhHSP18.4, and GhHSP20.7A, while there was a significant downregulation of the cellulose synthase genes GhCesA4B, GhCesA7B, and GhCesA8B (Figure 5D). Interestingly, adding dithiothreitol (DTT), an ER stress inducer, to the medium also resulted in the upregulation of sHSPs (Figure 5C).

2.4. Small HSPs Inhibit Secondary Wall Synthesis by Reducing ROS Levels in Fibers

Previous studies demonstrated that upregulation of sHSPs can reduce the accumulation of reactive oxygen species (ROS) [21,22,23]. This prompted us to investigate whether the overexpression of cotton sHSPs could similarly inhibit ROS production. Upon transiently expressing GhHSP17.6B, GhHSP17.9F, and GhHSP18.4 in tobacco, we observed a significant reduction in H2O2 levels in the treated leaves (Figure 6A). Moreover, in Ri3 and Ri4 fibers, where the sHSPs genes were upregulated, the H2O2 content was significantly lower than that of wild-type (Figure 6B). Examining the H2O2 content in the Arabidopsis rod1 siliques, we also observed a similar decrease in the mutant (Figure 6C). Importantly, culturing the wild-type ovules in the media containing linoleic acid (5 μM and 50 μM) resulted in a decrease in H2O2 content in the treated fibers (Figure 6D).
Previous research established that H2O2 acts as a signaling molecule that promotes cellulose deposition in plant cell wall [24,25,26]. When we treated wild-type ovules with diphenyleneiodonium (DPI) to block superoxide production, a significant reduction in H2O2 content was observed (Figure 6E). Concurrently, the expression of cellulose synthase genes involved in secondary wall deposition, GhCesA4B, GhCesA7B, and GhCesA8B, was significantly downregulated (Figure 6F). Taken together, these results indicate that downregulation of GhROD1 can upregulate sHSPs, which in turn inhibits the secondary wall synthesis by reducing ROS levels in fibers.

3. Discussion

3.1. GhROD1-Mediated DAG-to-PC Interconversion May Contribute to Fatty Acid Desaturation in Cotton Fibers

Following initiation, cotton fibers undergo rapid elongation to ultimately form lint. The period between 16 and 20 days post-anthesis (DPA) represents a critical transition from primary to secondary cell wall synthesis. Analysis of the fatty acid composition of fibers during this period revealed linolenic acid (18:3) and palmitic acid (16:0) as the two most abundant fatty acids (Figure 2A), consistent with previous findings [10,11] and highlighting the potential importance of 18:3 in fiber development. In fiber cells, as in most plant cells, 16:0 and 18:1 are the major fatty acids synthesized in plastids and exported to the endoplasmic reticulum (ER). These fatty acids undergo a series of enzymatic reactions to form phosphatidic acid (PA), which is then dephosphorylated to yield DAGs. DAGs are subsequently used to synthesize PC and PE. Finally, the 18:1 acyl chain is modified by FAD2 and FAD3 to produce 18:2 and 18:3, respectively [10,27]. Notably, DAGs can also be converted to PCs by PDCT, and the acyl chains of PCs are further desaturated by FADs in the ER to reduce overall fatty acid saturation and meet cellular demands [15,28]. Our study revealed high expression of GhROD1, encoding PDCT, in developing fibers from 14 to 20 DPA, in addition to its expression in other tissues. Furthermore, the encoded PDCT protein is primarily localized in the ER (Figure S4). These findings suggest that GhROD1 may facilitate the interconversion of DAG and PC to increase the unsaturation of the acyl pool during fiber development, potentially representing a complementary pathway to fatty acid desaturation. Consistent with this hypothesis, downregulation of GhROD1 led to an increase in PC 34:2 (16:0–18:2) and PE 34:2 (16:0–18:2) content, while PC 34:3 (16:0–18:3) and PE 34:3 (16:0–18:3) content decreased. Moreover, the 18:3 content in total fatty acids was reduced (Figure 2A). Conversely, upregulation of GhROD1 increased PC 36:6 (18:3–18:3) content in OE17 and OE25 fibers (Figure 2B), indicating that PDCT plays a key role in increasing polyunsaturated fatty acids (PUFAs) in fibers, similar to its function in Arabidopsis [16]. However, while GhROD1 overexpression increased 18:3 content in fatty acids by 2.4–8.4% and downregulation reduced it by 2.8–6.5% (Figure 2A), the magnitude of these changes was relatively modest. This suggests that while increased fatty acid unsaturation via the DAG-to-PC interconversion pathway mediated by PDCT may occur in fibers, it likely represents a complementary rather than primary route to achieving optimal fatty acid composition during fiber development.
Given the observed alterations in PC and PE fatty acid saturation in the fibers of Ri3 and Ri4 lines, a corresponding change in DAG saturation might be expected. However, our results revealed a less pronounced pattern of DAG variation (Figure 2D). DAG plays a central role in plant lipid metabolism, and its synthesis involves multiple reactions occurring in distinct subcellular compartments, including the ER, lipid droplets (LDs), and plasma membrane [29]. DAG serves as a precursor for triacylglycerol (TAG) synthesis, the important role of which is to storage energy, and serves as an intermediate in the synthesis of glycerophospholipids, such as PC and PE to participate in various lipid metabolic pathways [27,30,31]. Consequently, alterations in DAG saturation are likely influenced by other lipid metabolic pathways, providing a possible explanation for the complex changes in DAG saturation observed in our study.

3.2. Alterations in Phospholipid Fatty Acid of Fibers May Activate the ER Stress Response

The endoplasmic reticulum (ER) plays a crucial role in lipid and protein biosynthesis, serving as the primary site for phosphatidylcholine (PC) and phosphatidylethanolamine (PE) synthesis [32,33]. PC is the most abundant phospholipid in the ER membrane of eukaryotes [34,35]. Inhibition of PC biosynthesis is known to induce ER stress [36,37,38]. In this study, downregulation of GhROD1 not only increased fatty acid saturation but also altered PC and PE species in cotton fibers. Do these changes in PC and PE species induce ER stress in fiber cells? Our KEGG pathway enrichment analysis provides a potential clue for answering this question. The analysis identified protein processing in the ER as a significantly affected pathway, which is involved in the upregulation of genes from the small heat shock protein (sHSP) family (Figure 4), suggesting the activation of a stress response. In vitro supplementation with linoleic acid (18:2) resulted in a similar upregulation of several sHSP genes in fibers (Figure 5), as did the treatment with the ER stress inducer DTT. These results support a link between downregulation of GhROD1, encoding PDCT, and ER stress.

3.3. The Upregulation of sHSPs Can Reduce the Accumulation of ROS in Cells and Inhibit the Synthesis of Secondary Wall

Now the question is how does the downregulating PDCT-induced ER stress affect secondary cell wall biosynthesis in fibers? Heat shock proteins (HSPs) are a diverse group of proteins induced by various cellular stresses and are categorized into distinct families based on homology and molecular weight, including HSP100, HSP90, HSP70, HSP60, and small heat shock proteins (sHSPs) [39]. sHSPs, with molecular masses ranging from 12 to 42 kDa, are known for their protective roles in a variety of crops [40]. Previous studies showed that upregulation of some sHSPs could reduce H2O2 production. Overexpression of the maize HSP, ZmHSP16.9, has been shown to decrease H2O2 levels in tobacco under heat stress [41]. Similarly, overexpression of tomato SlHSP17.7 mitigated H2O2 accumulation under cold stress [42], and Arabidopsis plants overexpressing ClHSP20 from Camellia limonia exhibited reduced H2O2 levels [23]. Likely, our research demonstrated that transient expression of GhHSP17.6B, GhHSP17.9F, and GhHSP18.4 in tobacco leaves significantly reduced H2O2 accumulation (Figure 6A).
H2O2 is recognized as a signaling molecule that initiates secondary cell wall (SCW) formation in cotton fibers [43]. Transcriptome and metabolome analyses of upland cotton suggest that fiber cell wall thinning may be associated with enhanced antioxidant capacity, emerging as a strategy for improvement of fiber finesses by reducing reactive oxygen species (ROS) levels to delay SCW synthesis [44]. Mutations in GhMDHAR1AT/DT and GhMDHAR2AT/DT resulted in a reduction in H2O2 content, thus downregulating genes related to secondary wall synthesis and delaying the synthesis process [26]. In this study, downregulation of GhROD1 in cotton led to a marked decrease in H2O2 content (Figure 6B) and significant improvement in fiber finesses. Interestingly, the fiber strength of GhROD1-downregulated line Ri3 was simultaneously increased. Our findings reveal how the mechanism of downregulation of GhROD1 affects secondary cell wall synthesis through enhancing sHSP expression and H2O2 production. The study also suggests a strategy for improving cotton fiber quality by the regulation of the lipid metabolism pathway.
Our findings uncover the mechanism by which downregulation of GhROD1 affects secondary cell wall synthesis by inducing ER stress, which in turn enhances sHSP expression and H2O2 production (Figure 7). This study also suggests a potential strategy for improving cotton fiber quality through the regulation of lipid metabolism pathways.

4. Materials and Methods

4.1. Construction of Vectors

Construction of the pCB2012-GhROD1 overexpression vector: The coding sequence of GhROD1 (Gene ID: Gh_A06G1621/Gh_D06G1990) was amplified from upland cotton fiber cDNA. The purified PCR product was ligated into the pEASY-Blunt Cloning Vector (Transgen, China). The cloning vector containing GhROD1 was digested with BamHI and XbaI to release the gene fragment. The pCB2012 vector was digested with BamHI and SpeI to generate a compatible vector backbone. The GhROD1 fragment was ligated into the linearized pCB2012 vector using the Rapid DNA Ligation Kit (Roche, Basel, Switzerland), creating the 35S-GhROD1 overexpression construct.
Construction of the pCB2012-GhROD1-RNAi vector: Sense, intron, and antisense fragments of GhROD1 were sequentially fused via PCR to create a hairpin structure-forming sequence. The fusion fragment was cloned into the pUCm-T vector (Sangon, Shanghai, China). Both the pUCm-T containing the GhROD1-RNAi fragment and pCB2012 vectors were digested with KpnI and BamHI. The GhROD1-RNAi fragment was ligated into the linearized pCB2012 vector using the Rapid DNA Ligation Kit (Roche, Switzerland), resulting in the 35S-GhROD1-RNAi construct.
The coding sequences of GhHSP17.6B (Gene ID: Gh_D08G0057), GhHSP17.9F (Gene ID: Gh_D12G1364), and GhHSP18.4 (Gh_D03G1268) were amplified from wild-type fiber cDNA. Each amplified fragment was individually cloned into the pCB2016 vector, which was linearized with EcoRI and SpeI, using the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China). This process generated three separate 35S-GhHSP overexpression constructs: 35S-GhHSP17.6B, 35S-GhHSP17.9F, and 35S-GhHSP18.4.

4.2. Plant Materials and Growth Conditions

Upland cotton (Gossypium hirsutum L. cv. Jimian14) was used for transformation. The genetic transformation of cotton followed the protocol described by Luo et al. [45]. GhROD1 transgenic cotton plants were grown in experimental plots at Southwest University.

4.3. RNA Extraction and qRT-PCR

Total RNA was isolated from plant samples using the RN09-EASY Spin Plant RNA Rapid Kit (Aidlab Biotech, Beijing, China). Subsequently, complementary DNA (cDNA) synthesis was carried out using the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Beijing, China). qRT-PCR was performed in accordance with the methodology outlined by Yang et al. [46]. GhHis3 (Accession number: AF024716) served as reference. The specific primers employed are detailed in Supplementary Table S1.

4.4. Fatty Acid Analysis

Cotton fiber lipid extraction: Lipids were extracted from cotton fibers using a modified method based on Liu et al. [11]. All solvents used contained 0.01% butylated hydroxytoluene (BHT) to prevent lipid oxidation. Isopropanol was preheated to inactivate phospholipase D. Briefly, 100 mg of fresh cotton fiber was incubated with 3 mL of preheated isopropanol at 75 °C for 15 min. Heptadecanoic acid (C17:0) was added as an internal standard. Subsequently, 0.6 mL of water and 1.5 mL of chloroform were added, and the mixture was incubated at 4 °C with shaking at 200 rpm for 2 h. The lipid extracts were transferred to a new polytetrafluoroethylene tube. An additional 4 mL of a methanol/chloroform (1:2, v/v) mixture was added, and this extraction step was repeated twice. The combined lipid extracts were mixed with 1 mL of 1 M KCl, vortexed thoroughly, and centrifuged at 2000 rpm for 10 min. The resulting precipitate was transferred to a separate glass tube, dried, and stored at −80 °C.
Cotton fiber fatty acid (FA) extraction: Cotton fiber FAs were extracted with 2.5% H2SO4 in methanol (v/v), following the methodology outlined by Liu et al. [11].
Gas chromatography with flame ionization detection (GC-FID) analysis: Fatty acid methyl esters (FAMEs) in hexanes were analyzed by GC-FID using the following temperature program. Initial temperature: 100 °C for 1 min. Ramp 1: 20 °C/min to 170 °C, hold for 1 min. Ramp 2: 5 °C/min to 220 °C, hold for 12 min.

4.5. Lipidomic Analyses

The lipid was extracted by the same methods without internal standard. Lipidomic analyses were conducted by the National Key Laboratory of Crop Genetic Improvement at Huazhong Agricultural University, using direct infusion via LC-ESI-MS/MS, as described by Han and Gross [47].

4.6. Fiber Cell Wall Thickness and Quality Assessment

Cell wall thickness of mature fibers was evaluated using the method described by Han et al. [48]. Mature seed cotton was harvested from both transgenic and wild-type plants on the same day. Samples were dried and ginned before being submitted to the Supervision Inspection and Test Center of Cotton Quality, Ministry of Agriculture and Rural Affairs of China, for analysis. Fiber quality parameters were measured using a high-capacity integrated cotton fiber tester (model HVI-1000). The following properties were assessed: fiber length, uniformity index, fiber strength, micronaire value, and elongation ratio. Three independent samples were prepared for each material (transgenic and wild-type). Statistical analysis of the fiber quality data was performed using Student’s t-test to compare differences between transgenic and wild-type plants.

4.7. RNA Sequencing and Transcriptome Analysis

Total RNA was extracted from fibers 16 days post-anthesis (DPA) of both GhROD1-RNAi transgenic lines and wild-type plants using the MJZol total RNA extraction kit (TRIzol). Three biological replicates were included for each sample. RNA-seq libraries were constructed using the Illumina® Stranded mRNA Prep Kit (Illumina, San Diego, CA, USA) with 1 μg of total RNA per sample. Paired-end sequencing (150 bp reads) was performed on the Illumina Novaseq 6000 platform by Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China). Raw sequencing reads were deposited in the NCBI Sequence Read Archive (SRA) database under Accession Number SRP525959. Reads were aligned to the Gossypium hirsutum (AD1) TM-1 genome NAU-NBI_v1.1 as the reference genome. Data analysis was performed using Majorbio Cloud Platform (www.majorbio.com). Differentially expressed genes (DEGs) between GhROD1-RNAi lines and wild-type were identified based on an adjusted p-value threshold of <0.05 and a fold change criterion of ≥ 2.

4.8. Ovule Culture and Treatment

Cotton ovules at anthesis were harvested and incubated in BT medium at 32 °C for 12 days, as detailed in previous work [49]. Subsequently, the ovules were transferred to BT medium supplemented with various concentrations of linoleic acid (C18:2) or 1 μM diphenyleneiodonium (DPI) and further cultured for an additional 4 days. The BT medium was adjusted with the amount of DMSO (dimethyl sulfoxide) and 0.01% NP-40 equivalent to that used to dissolve linoleic acid (C18:2), or DMSO equivalent to that used to dissolve DPI as indicated, which was used as the mock control, respectively.
Dithiothreitol (DTT) treatment: Cotton ovules were collected at anthesis and cultured in BT medium at 32 °C for 4 h in the presence of 100 μM DTT. BT medium containing an equivalent amount of DMSO as used to dissolve DTT served as the mock control.

4.9. Statistical Data Analysis

The software used for statistical data analysis was GraphPad Prism 8.0 and SPSS 16.0. The heat map was built by Tbtools-II v2.119 [50].

5. Conclusions

This study identified GhROD1, a gene encoding phosphatidylcholine: diacylglycerol cholinephosphotransferase, in cotton. Downregulation of GhROD1 increased linoleic acid (C18:2) content in fiber cells, which was accompanied by an increase in C18:2-containing phosphatidylcholine (PC) and phosphatidylethanolamine (PE). This, in turn, promoted the expression of small heat shock protein (sHSP) genes. The upregulation of sHSP genes led to reduced H2O2 production in fiber cells, inhibiting secondary wall synthesis and resulting in thinner mature fibers. These findings reveal a novel genetic strategy for manipulating fiber quality in cotton by targeting GhROD1 and its downstream effects on lipid metabolism and sHSP gene expression.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ijms252011242/s1.

Author Contributions

Conceptualization, B.D. and L.H.; methodology, B.D., Y.Y., and X.Z.; software, B.D., S.L., and Y.Y.; validation, S.L. and H.Z.; formal analysis and investigation, X.Z., Y.Y., S.L., R.H., L.Z., M.Z., J.Z., L.J., and B.L.; resources, L.H.; data curation, B.D.; writing—original draft preparation, B.D. and L.H.; writing—review and editing, B.D., L.H., and Y.P.; visualization, B.D. and H.Z.; supervision, L.H. and Y.P.; project administration, L.H.; funding acquisition, L.H. All authors performed the experiments. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by National Natural Science Foundation of China, grant number 30971826, 3067132.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author due to privacy.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Jung, J.; Maeda, M.; Chang, A.; Landivar, J.; Yeom, J.; McGinty, J. Unmanned aerial system assisted framework for the selection of high yielding cotton genotypes. Comput. Electron. Agric. 2018, 152, 74–81. [Google Scholar] [CrossRef]
  2. Khan, M.A.; Wahid, A.; Ahmad, M.; Tahir, M.T.; Ahmed, M.; Ahmad, S.; Hasanuzzaman, M. World cotton production and consumption: An overview. In Cotton Production and Uses: Agronomy, Crop Protection, and Postharvest Technologies; Ahmad, S., Hasanuzzaman, M., Eds.; Springer: Singapore, 2020; pp. 1–7. [Google Scholar]
  3. Kim, H.J.; Triplett, B.A. Cotton fiber growth in planta and in vitro models for plant cell elongation and cell wall biogenesis. Plant Physiol. 2001, 127, 1361–1366. [Google Scholar] [CrossRef] [PubMed]
  4. Haigler, C.H.; Betancur, L.; Stiff, M.R.; Tuttle, J.R. Cotton fiber: A powerful single-cell model for cell wall and cellulose research. Front. Plant Sci. 2012, 3, 104. [Google Scholar] [CrossRef] [PubMed]
  5. Ohlrogge, J.; Browse, J.; Jaworski, J.; Somerville, C. Lipids. In Biochemistry and Molecular Biology of Plants; Buchanan, B.B., Gruissem, W., Jones, R.L., Eds.; Wiley: Oxford, UK, 2015; pp. 337–344. [Google Scholar]
  6. Shi, Y.H.; Zhu, S.W.; Mao, X.Z.; Feng, J.X.; Qin, Y.M.; Zhang, L.; Cheng, J.; Wei, L.P.; Wang, Z.Y.; Zhu, Y.X. Transcriptome profiling, molecular biological, and physiological studies reveal a major role for ethylene in cotton fiber cell elongation. Plant Cell 2006, 18, 651–664. [Google Scholar] [CrossRef] [PubMed]
  7. Gou, J.Y.; Wang, L.J.; Chen, S.P.; Hu, W.L.; Chen, X.Y. Gene expression and metabolite profiles of cotton fiber during cell elongation and secondary cell wall synthesis. Cell Res. 2007, 17, 422–434. [Google Scholar] [CrossRef]
  8. Wang, X.C.; Li, Q.; Jin, X.; Xiao, G.H.; Liu, G.J.; Liu, N.J.; Qin, Y.M. Quantitative proteomics and transcriptomics reveal key metabolic processes associated with cotton fiber initiation. J. Proteomics 2015, 114, 16–27. [Google Scholar] [CrossRef]
  9. Xu, F.; Chen, Q.; Huang, L.; Luo, M. Advances about the roles of membranes in cotton fiber development. Membranes 2021, 11, 471. [Google Scholar] [CrossRef]
  10. Wanjie, S.W.; Welti, R.; Moreau, R.A.; Chapman, K.D. Identification and quantification of glycerolipids in cotton fibers: Reconciliation with metabolic pathway predictions from DNA databases. Lipids 2005, 40, 773–785. [Google Scholar] [CrossRef]
  11. Liu, G.J.; Xiao, G.H.; Liu, N.J.; Liu, D.; Chen, P.S.; Qin, Y.M.; Zhu, Y.X. Targeted lipidomics studies reveal that linolenic acid promotes cotton fiber elongation by activating phosphatidylinositol and phosphatidylinositol monophosphate biosynthesis. Mol. Plant 2015, 8, 911–921. [Google Scholar] [CrossRef]
  12. Fahy, E.; Subramaniam, S.; Brown, H.A.; Glass, C.K.; Merrill, A.H., Jr.; Murphy, R.C.; Raetz, C.R.; Russell, D.W.; Seyama, Y.; Shaw, W.; et al. A comprehensive classification system for lipids. J. Lipid Res. 2005, 46, 839–861. [Google Scholar] [CrossRef]
  13. van Meer, G.; Voelker, D.R.; Feigenson, G.W. Membrane lipids: Where they are and how they behave. Nat. Rev. Mol. Cell Biol. 2008, 9, 112–124. [Google Scholar] [CrossRef] [PubMed]
  14. Stymne, S.; Appelqvist, L.A. The biosynthesis of linoleate from oleoyl-CoA via oleoyl-phosphatidylcholine in microsomes of developing safflower seeds. Eur. J. Biochem. 1978, 90, 223–229. [Google Scholar] [CrossRef] [PubMed]
  15. Bates, P.D. Understanding the control of acyl flux through the lipid metabolic network of plant oil biosynthesis. Biochim. Biophys. Acta 2016, 1861, 1214–1225. [Google Scholar] [CrossRef] [PubMed]
  16. Lu, C.; Xin, Z.; Ren, Z.; Miquel, M.; Browse, J. An enzyme regulating triacylglycerol composition is encoded by the ROD1 gene of Arabidopsis. Proc. Natl. Acad. Sci. USA 2009, 106, 18837–18842. [Google Scholar] [CrossRef] [PubMed]
  17. Jarvis, B.A.; Romsdahl, T.B.; McGinn, M.G.; Nazarenus, T.J.; Cahoon, E.B.; Chapman, K.D.; Sedbrook, J.C. CRISPR/Cas9-Induced fad2 and rod1 mutations stacked with fae1 confer high oleic acid seed oil in pennycress (Thlaspi arvense L.). Front. Plant Sci. 2021, 12, 652319. [Google Scholar] [CrossRef] [PubMed]
  18. Sandgrind, S.; Li, X.; Ivarson, E.; Wang, E.S.; Guan, R.; Kanagarajan, S.; Zhu, L.H. Improved fatty acid composition of field cress (Lepidium campestre) by CRISPR/Cas9-mediated genome editing. Front. Plant Sci. 2023, 14, 1076704. [Google Scholar] [CrossRef]
  19. Bai, S.; Wallis, J.G.; Denolf, P.; Engelen, S.; Bengtsson, J.D.; Van Thournout, M.; Dierickx, J.; Haesendonckx, B.; Browse, J. The biochemistry of headgroup exchange during triacylglycerol synthesis in canola. Plant J. 2020, 103, 83–94. [Google Scholar] [CrossRef]
  20. Joseph, G.; Montalvo, J. Relationships between micronaire, fineness, and maturity.part I. fundamentals. J. Cotton Sci. 2005, 9, 81–88. [Google Scholar]
  21. Kaur, H.; Petla, B.P.; Kamble, N.U.; Singh, A.; Rao, V.; Salvi, P.; Ghosh, S.; Majee, M. Differentially expressed seed aging responsive heat shock protein OsHSP18.2 implicates in seed vigor, longevity and improves germination and seedling establishment under abiotic stress. Front. Plant Sci. 2015, 6, 713. [Google Scholar] [CrossRef]
  22. Zhai, M.; Sun, Y.; Jia, C.; Peng, S.; Liu, Z.; Yang, G. Over-expression of JrsHSP17.3 gene from Juglans regia confer the tolerance to abnormal temperature and NaCl stresses. J. Plant Biol. 2016, 59, 549–558. [Google Scholar] [CrossRef]
  23. Zhong, L.; Shi, Y.; Xu, S.; Xie, S.; Huang, X.; Li, Y.; Qu, C.; Liu, J.; Liao, J.; Huang, Y.; et al. Heterologous overexpression of heat shock protein 20 genes of different species of yellow Camellia in Arabidopsis thaliana reveals their roles in high calcium resistance. BMC Plant Biol. 2024, 24, 5. [Google Scholar] [CrossRef] [PubMed]
  24. Potikha, T.S.; Collins, C.C.; Johnson, D.I.; Delmer, D.P.; Levine, A. The involvement of hydrogen peroxide in the differentiation of secondary walls in cotton fibers. Plant Physiol. 1999, 119, 849–858. [Google Scholar] [CrossRef] [PubMed]
  25. Ding, X.; Li, X.; Wang, L.; Zeng, J.; Huang, L.; Xiong, L.; Song, S.; Zhao, J.; Hou, L.; Wang, F.; et al. Sucrose enhanced reactive oxygen species generation promotes cotton fibre initiation and secondary cell wall deposition. Plant Biotechnol. J. 2021, 19, 1092–1094. [Google Scholar] [CrossRef] [PubMed]
  26. Tian, X.; Ji, M.; You, J.; Zhang, Y.; Lindsey, K.; Zhang, X.; Tu, L.; Wang, M. Synergistic interplay of redox homeostasis and polysaccharide synthesis promotes cotton fiber elongation. Plant J. 2024, 118, 405–422. [Google Scholar] [CrossRef]
  27. Nakamura, Y. Plant phospholipid diversity: Emerging functions in metabolism and protein-lipid interactions. Trends Plant Sci. 2017, 22, 1027–1040. [Google Scholar] [CrossRef]
  28. Bates, P.D.; Fatihi, A.; Snapp, A.R.; Carlsson, A.S.; Browse, J.; Lu, C. Acyl editing and headgroup exchange are the major mechanisms that direct polyunsaturated fatty acid flux into triacylglycerols. Plant Physiol. 2012, 160, 1530–1539. [Google Scholar] [CrossRef]
  29. Eichmann, T.O.; Lass, A. DAG tales: The multiple faces of diacylglycerol—Stereochemistry, metabolism, and signaling. Cell Mol. Life Sci. 2015, 72, 3931–3952. [Google Scholar] [CrossRef]
  30. Shindou, H.; Shimizu, T. Acyl-CoA:lysophospholipid acyltransferases. J. Biol. Chem. 2009, 284, 1–5. [Google Scholar] [CrossRef]
  31. Jennings, W.; Epand, R.M. CDP-diacylglycerol, a critical intermediate in lipid metabolism. Chem. Phys. Lipids 2020, 230, 104914. [Google Scholar] [CrossRef]
  32. McMaster, C.R.; Bell, R.M. Phosphatidylcholine biosynthesis via the CDP-choline pathway in Saccharomyces cerevisiae. Multiple mechanisms of regulation. J. Biol. Chem. 1994, 269, 14776–14783. [Google Scholar] [CrossRef]
  33. Kannan, M.; Sivaprakasam, C.; Prinz, W.A.; Nachiappan, V. Endoplasmic reticulum stress affects the transport of phosphatidylethanolamine from mitochondria to the endoplasmic reticulum in S. cerevisiae. Biochim. Biophys. Acta 2016, 1861, 1959–1967. [Google Scholar] [CrossRef] [PubMed]
  34. Fouillen, L.; Maneta-Peyret, L.; Moreau, P. ER membrane lipid composition and metabolism: Lipidomic analysis. Methods Mol. Biol. 2018, 1691, 125–137. [Google Scholar] [CrossRef] [PubMed]
  35. Wardhan, R.; Mudgal, P. Introduction to Biomembranes. In Textbook of Membrane Biology; Wardhan, R., Mudgal, P., Eds.; Springer: Singapore, 2017; pp. 1–28. [Google Scholar]
  36. Sanchez-Lopez, E.; Zimmerman, T.; Gomez del Pulgar, T.; Moyer, M.P.; Lacal Sanjuan, J.C.; Cebrian, A. Choline kinase inhibition induces exacerbated endoplasmic reticulum stress and triggers apoptosis via CHOP in cancer cells. Cell Death Dis. 2013, 4, e933. [Google Scholar] [CrossRef] [PubMed]
  37. Lin, Y.C.; Kanehara, K.; Nakamura, Y. Arabidopsis CHOLINE/ETHANOLAMINE KINASE 1 (CEK1) is a primary choline kinase localized at the endoplasmic reticulum (ER) and involved in ER stress tolerance. New Phytol. 2019, 223, 1904–1917. [Google Scholar] [CrossRef] [PubMed]
  38. Kanehara, K.; Cho, Y.; Yu, C.Y. A lipid viewpoint on the plant endoplasmic reticulum stress response. J. Exp. Bot. 2022, 73, 2835–2847. [Google Scholar] [CrossRef]
  39. Al-Whaibi, M.H. Plant heat-shock proteins: A mini review. J. King Saud. Univ. Sci. 2011, 23, 139–150. [Google Scholar] [CrossRef]
  40. Mishra, D.; Shekhar, S.; Singh, D.; Chakraborty, S.; Chakraborty, N. Heat Shock Proteins and Abiotic Stress Tolerance in Plants. In Regulation of Heat Shock Protein Responses; Asea, A., Kaur, P., Eds.; Springer: Cham, Switzerland, 2018; Volume 13, pp. 41–69. [Google Scholar]
  41. Sun, L.; Liu, Y.; Kong, X.; Zhang, D.; Pan, J.; Zhou, Y.; Wang, L.; Li, D.; Yang, X. ZmHSP16.9, a cytosolic class I small heat shock protein in maize (Zea mays), confers heat tolerance in transgenic tobacco. Plant Cell Rep. 2012, 31, 1473–1484. [Google Scholar] [CrossRef]
  42. Zhang, N.; Zhao, H.; Shi, J.; Wu, Y.; Jiang, J. Functional characterization of class I SlHSP17.7 gene responsible for tomato cold-stress tolerance. Plant Sci. 2020, 298, 110568. [Google Scholar] [CrossRef]
  43. Hovav, R.; Udall, J.A.; Chaudhary, B.; Hovav, E.; Flagel, L.; Hu, G.; Wendel, J.F. The evolution of spinnable cotton fiber entailed prolonged development and a novel metabolism. PLoS Genet. 2008, 4, e25. [Google Scholar] [CrossRef]
  44. Tuttle, J.R.; Nah, G.; Duke, M.V.; Alexander, D.C.; Guan, X.; Song, Q.; Chen, Z.J.; Scheffler, B.E.; Haigler, C.H. Metabolomic and transcriptomic insights into how cotton fiber transitions to secondary wall synthesis, represses lignification, and prolongs elongation. BMC Genom. 2015, 16, 477. [Google Scholar] [CrossRef]
  45. Luo, M.; Xiao, Y.; Li, X.; Lu, X.; Deng, W.; Li, D.; Hou, L.; Hu, M.; Li, Y.; Pei, Y. GhDET2, a steroid 5alpha-reductase, plays an important role in cotton fiber cell initiation and elongation. Plant J. 2007, 51, 419–430. [Google Scholar] [CrossRef] [PubMed]
  46. Yang, Y.; Zhou, X.; Zhu, X.; Ding, B.; Jiang, L.; Zhang, H.; Li, S.; Cao, S.; Zhang, M.; Pei, Y.; et al. GhMYB52 like: A key factor that enhances lint yield by negatively regulating the lignin biosynthesis pathway in fibers of upland cotton (Gossypium hirsutum L.). Int. J. Mol. Sci. 2024, 25, 4921. [Google Scholar] [CrossRef] [PubMed]
  47. Han, X.; Gross, R.W. Shotgun lipidomics: Electrospray ionization mass spectrometric analysis and quantitation of cellular lipidomes directly from crude extracts of biological samples. Mass. Spectrom. Rev. 2005, 24, 367–412. [Google Scholar] [CrossRef] [PubMed]
  48. Han, L.B.; Li, Y.B.; Wang, H.Y.; Wu, X.M.; Li, C.L.; Luo, M.; Wu, S.J.; Kong, Z.S.; Pei, Y.; Jiao, G.L.; et al. The dual functions of WLIM1a in cell elongation and secondary wall formation in developing cotton fibers. Plant Cell 2013, 25, 4421–4438. [Google Scholar] [CrossRef]
  49. Beasley, C.A.; Ting, I.P. The effects of plant growth substances on in vitro fiber development from fertilized cotton ovules. Am. J. Bot. 1973, 60, 130–139. [Google Scholar] [CrossRef]
  50. Chen, C.; Wu, Y.; Li, J.; Wang, X.; Zeng, Z.; Xu, J.; Liu, Y.; Feng, J.; Chen, H.; He, Y.; et al. TBtools-II: A “one for all, all for one“ bioinformatics platform for biological big-data mining. Mol. Plant 2023, 16, 1733–1742. [Google Scholar] [CrossRef]
Ijms 25 11242 g001
Figure 1. Expression of GhROD1 in cotton plants. (A) Expression levels of GhROD1 in various tissues. Total RNA was extracted from roots, stems, leaves, ovules at 20 DPA, and fibers, petals, sepals, bracts, and stamens at 20 DPA. GhHis3 was used as an internal control. The results are presented as mean ± SD (n = 3). (B) Expression levels of GhROD1 at different stages of fiber development. Total RNA was extracted from fibers at 6, 8, 10, 12, 14, 16, 18, 20, 22, and 25 DPA. The results are presented as mean ± SD (n = 3), with the x-axis indicating DPA.
Figure 1. Expression of GhROD1 in cotton plants. (A) Expression levels of GhROD1 in various tissues. Total RNA was extracted from roots, stems, leaves, ovules at 20 DPA, and fibers, petals, sepals, bracts, and stamens at 20 DPA. GhHis3 was used as an internal control. The results are presented as mean ± SD (n = 3). (B) Expression levels of GhROD1 at different stages of fiber development. Total RNA was extracted from fibers at 6, 8, 10, 12, 14, 16, 18, 20, 22, and 25 DPA. The results are presented as mean ± SD (n = 3), with the x-axis indicating DPA.
Ijms 25 11242 g001
Ijms 25 11242 g002
Figure 2. Targeted lipidomics analyses of PC, PE, and DAG in cotton fibers at the transition stage (20 DPA). (A) Total fatty acid composition in fibers of wild-type and transgenic cotton. C16:0: palmitic acid; C18:0: stearic acid; C18:1: oleic acid; C18:2: linoleic acid; and C18:3: linolenic acid. Data are presented as mean ± SD (n = 3). Data were analyzed by one-way ANOVA followed by Tukey’s test. Different lowercase letters indicate significant differences among groups. (B–D) Relative PC, PE, and DAG species content in the fibers at 20 DPA of wild-type and transgenic cotton. WT: wild-type (Gossypium hirsutum L. cv. Jimian14). Ri3 and Ri4: GhROD1-downregulated lines. OE17 and OE25: GhROD1-upregulated lines. PC: phosphatidylcholine. PE: phosphatidylethanolamine. DAG: diacylglycerol. Data for each molecular species represent the means and SDs of five independent extractions and are presented as mol% of total glycerophospholipids. Differences between transgenic lines and wild-type were analyzed by one-way ANOVA followed by Tukey’s test. Different lowercase letters indicate significant differences among groups. Components with lipid molecular species below 1% are not shown in the figure.
Figure 2. Targeted lipidomics analyses of PC, PE, and DAG in cotton fibers at the transition stage (20 DPA). (A) Total fatty acid composition in fibers of wild-type and transgenic cotton. C16:0: palmitic acid; C18:0: stearic acid; C18:1: oleic acid; C18:2: linoleic acid; and C18:3: linolenic acid. Data are presented as mean ± SD (n = 3). Data were analyzed by one-way ANOVA followed by Tukey’s test. Different lowercase letters indicate significant differences among groups. (B–D) Relative PC, PE, and DAG species content in the fibers at 20 DPA of wild-type and transgenic cotton. WT: wild-type (Gossypium hirsutum L. cv. Jimian14). Ri3 and Ri4: GhROD1-downregulated lines. OE17 and OE25: GhROD1-upregulated lines. PC: phosphatidylcholine. PE: phosphatidylethanolamine. DAG: diacylglycerol. Data for each molecular species represent the means and SDs of five independent extractions and are presented as mol% of total glycerophospholipids. Differences between transgenic lines and wild-type were analyzed by one-way ANOVA followed by Tukey’s test. Different lowercase letters indicate significant differences among groups. Components with lipid molecular species below 1% are not shown in the figure.
Ijms 25 11242 g002
Ijms 25 11242 g003
Figure 3. Cross-section of mature fibers in wild-type and GhROD1 transgenic cotton. (A–E) Cross-section of mature fibers in wild-type cv. Jimian14, Ri3, Ri4, OE17, and OE25. (F) Statistics of cell wall thickness of mature fibers in wild-type and transgenic cotton (n ≥ 150). Statistical data analysis was performed by one-way ANOVA followed by Tukey’s test. Different lowercase letters indicate significant differences among groups.
Figure 3. Cross-section of mature fibers in wild-type and GhROD1 transgenic cotton. (A–E) Cross-section of mature fibers in wild-type cv. Jimian14, Ri3, Ri4, OE17, and OE25. (F) Statistics of cell wall thickness of mature fibers in wild-type and transgenic cotton (n ≥ 150). Statistical data analysis was performed by one-way ANOVA followed by Tukey’s test. Different lowercase letters indicate significant differences among groups.
Ijms 25 11242 g003
Ijms 25 11242 g004
Figure 4. Transcriptome analysis of GhROD1-downregulated RNAi (Ri3) fibers at 16 DPA. (A) Volcano plot illustrating the differentially expressed genes (DEGs) between wild-type and Ri3 plants. In this plot, red and blue dots denote the DEGs between WT and Ri3. Red dots indicate genes that are significantly upregulated compared to WT, blue dots indicate genes that are significantly downregulated, and gray dots represent genes exhibiting no significant differential expression. (B,C) GO, KEGG enrichment analysis of DEGs between wild-type and Ri3 (bubble plot). The size of the dots represents the number of genes/transcripts associated with each GO term, showing the top 20 enrichment results. DEGs were defined by a fold change ≥ 2 and an adjusted p-value < 0.05.
Figure 4. Transcriptome analysis of GhROD1-downregulated RNAi (Ri3) fibers at 16 DPA. (A) Volcano plot illustrating the differentially expressed genes (DEGs) between wild-type and Ri3 plants. In this plot, red and blue dots denote the DEGs between WT and Ri3. Red dots indicate genes that are significantly upregulated compared to WT, blue dots indicate genes that are significantly downregulated, and gray dots represent genes exhibiting no significant differential expression. (B,C) GO, KEGG enrichment analysis of DEGs between wild-type and Ri3 (bubble plot). The size of the dots represents the number of genes/transcripts associated with each GO term, showing the top 20 enrichment results. DEGs were defined by a fold change ≥ 2 and an adjusted p-value < 0.05.
Ijms 25 11242 g004
Ijms 25 11242 g005
Figure 5. The expression profiles of sHSPs and GhCesAs. (A,B) Relative expression levels of sHSPs and GhCesAs in wild-type and transgenic cotton. Differences between transgenic lines and wild-type were analyzed by one-way ANOVA followed by Tukey’s test. Different lowercase letters indicate significant differences among groups. (C) Relative expression levels of sHSPs in ovules treated with 100 μM DTT. GhHis3 was used as an internal control. (D) Relative expression levels of sHSPs and GhCesAs in fibers treated with 5 μM linoleic acid (C18:2). CK: without linoleic acid (C18:2). Results are shown as mean ± SD (n = 3). Differences between the control and treatment were analyzed using Student’s t-test (* p < 0.05; ** p < 0.01) for (C,D).
Figure 5. The expression profiles of sHSPs and GhCesAs. (A,B) Relative expression levels of sHSPs and GhCesAs in wild-type and transgenic cotton. Differences between transgenic lines and wild-type were analyzed by one-way ANOVA followed by Tukey’s test. Different lowercase letters indicate significant differences among groups. (C) Relative expression levels of sHSPs in ovules treated with 100 μM DTT. GhHis3 was used as an internal control. (D) Relative expression levels of sHSPs and GhCesAs in fibers treated with 5 μM linoleic acid (C18:2). CK: without linoleic acid (C18:2). Results are shown as mean ± SD (n = 3). Differences between the control and treatment were analyzed using Student’s t-test (* p < 0.05; ** p < 0.01) for (C,D).
Ijms 25 11242 g005
Ijms 25 11242 g006
Figure 6. Analysis of H2O2 levels in various materials and treatments. (A) H2O2 content in tobacco leaves after transient expression of GhHSP17.6B, GhHSP17.9F, and GhHSP18.4. The empty vector (VC) served as a control. Values are the mean of three replicates, and vertical bars indicate ± SD (n = 3). (B) H2O2 content in wild-type and transgenic cotton fibers at 14 DPA and 16 DPA. Data were analyzed by one-way ANOVA followed by Tukey’s test. Different lowercase letters indicate significant differences among groups. (C) H2O2 content in siliques of wild-type Arabidopsis and rod1 mutant at 9 DPA. WT: wild-type Arabidopsis Col-0; rod1: loss-of-function rod1 mutant. Data are presented as mean ± SD (n = 3). (D,E) H2O2 content in cotton fibers treated with linoleic acid and DPI, respectively. CK: without linoleic acid or DPI as control; 0.5 μM, 5 μM, and 50 μM C18:2: supplemented with the corresponding concentration of linoleic acid in the medium. Data are presented as mean ± SD (n = 3). (F) Expression profiles of GhCesA4B, GhCesA7B, and GhCesA8B in fibers treated with DPI. CK: BT medium without DPI as control. Data are presented as mean ± SD (n = 3). Differences between the control and treatment were analyzed using Student’s t-test for (A,C–F). “*” indicates a significant difference (p < 0.05), and “**” indicates a highly significant difference (p < 0.01).
Figure 6. Analysis of H2O2 levels in various materials and treatments. (A) H2O2 content in tobacco leaves after transient expression of GhHSP17.6B, GhHSP17.9F, and GhHSP18.4. The empty vector (VC) served as a control. Values are the mean of three replicates, and vertical bars indicate ± SD (n = 3). (B) H2O2 content in wild-type and transgenic cotton fibers at 14 DPA and 16 DPA. Data were analyzed by one-way ANOVA followed by Tukey’s test. Different lowercase letters indicate significant differences among groups. (C) H2O2 content in siliques of wild-type Arabidopsis and rod1 mutant at 9 DPA. WT: wild-type Arabidopsis Col-0; rod1: loss-of-function rod1 mutant. Data are presented as mean ± SD (n = 3). (D,E) H2O2 content in cotton fibers treated with linoleic acid and DPI, respectively. CK: without linoleic acid or DPI as control; 0.5 μM, 5 μM, and 50 μM C18:2: supplemented with the corresponding concentration of linoleic acid in the medium. Data are presented as mean ± SD (n = 3). (F) Expression profiles of GhCesA4B, GhCesA7B, and GhCesA8B in fibers treated with DPI. CK: BT medium without DPI as control. Data are presented as mean ± SD (n = 3). Differences between the control and treatment were analyzed using Student’s t-test for (A,C–F). “*” indicates a significant difference (p < 0.05), and “**” indicates a highly significant difference (p < 0.01).
Ijms 25 11242 g006
Ijms 25 11242 g007
Figure 7. Proposed working model of GhROD1-mediated diacylglycerol (DAG)-to-phosphatidylcholine (PC) interconversion in cotton fiber. Downregulation of GhROD1 can reduce the rate of DAG-to-PC interconversion, leading to the accumulation of C18:2 in acyl pool of ER, which upregulates the expression of small heat shock proteins (sHSPs). The elevated sHSPs inhibit the production of H2O2, which reduces the secondary wall synthesis, resulting in finer fibers.
Figure 7. Proposed working model of GhROD1-mediated diacylglycerol (DAG)-to-phosphatidylcholine (PC) interconversion in cotton fiber. Downregulation of GhROD1 can reduce the rate of DAG-to-PC interconversion, leading to the accumulation of C18:2 in acyl pool of ER, which upregulates the expression of small heat shock proteins (sHSPs). The elevated sHSPs inhibit the production of H2O2, which reduces the secondary wall synthesis, resulting in finer fibers.
Ijms 25 11242 g007
Table 1. Comparison of fiber quality characteristics between GhROD1 transgenic cotton and wild-type.
Table 1. Comparison of fiber quality characteristics between GhROD1 transgenic cotton and wild-type.
Length (mm)Strength (cN/tex)MicronaireElongation
Ratio (%)		Uniformity
Line No. T3 generationWT30.2 ± 0.4 b28.8 ± 0.7 b5.3 ± 0.0 a6.8 ± 0.0 a86.1 ± 0.7 a
Ri331.3 ± 0.6 a30.0 ± 0.7 ab4.5 ± 0.1 c6.8 ± 0.0 a85.3 ± 0.7 ab
Ri429.7 ± 0.6 ab29.7 ± 0.4 ab5.0 ± 0.0 b6.8 ± 0.0 a85.8 ± 0.7 a
OE1728.8 ± 0.6 c29.4 ± 1.4 b5.3 ± 0.1 a6.8 ± 0.0 a85.0 ± 0.9 b
OE2529.1 ± 0.5 c31.1 ± 0.8 a5.4 ± 0.1 a6.8 ± 0.0 a86.4 ± 0.6 a
Line No. T4 generationWT30.1 ± 0.4 bc31.8 ± 1.2 b5.3 ± 0.1 b6.8 ± 0.0 a84.5 ± 0.6 b
Ri332.0 ± 0.5 a35.8 ± 1.2 a4.6 ± 0.1 d6.8 ± 0.0 a85.1 ± 0.4 b
Ri430.0 ± 0.4 bc32.4 ± 0.7 b5.0 ± 0.1 c6.8 ± 0.0 a84.8 ± 0.7 b
OE1730.6 ± 0.7 b31.5 ± 0.5 b5.3 ± 0.1 ab6.9 ± 0.1 a85.4 ± 0.4 a
OE2529.2 ± 0.9 c32.0 ± 0.6 b5.4 ± 0.1 a6.8 ± 0.0 a85.5 ± 0.5 a
Line No. T5 generationWT29.3 ± 0.4 ab31.7 ± 1.7 a5.3 ± 0.1 b6.9 ± 0.0 a86.1 ± 0.8 a
Ri330.8 ± 0.3 a33.9 ± 0.6 a4.6 ± 0.1 d6.9 ± 0.0 a84.6 ± 0.6 a
Ri429.8± 0.4 ab32.7 ± 1.1 a4.8 ± 0.0 c6.8 ± 0.0 a84.1 ± 0.8 a
OE1729.4 ± 0.8 ab31.7 ± 1.2 a5.2 ± 0.1 b6.9 ± 0.0 a86.2 ± 0.3 a
OE2528.7 ± 0.7 b31.7 ± 0.9 a5.6 ± 0.1 a6.8 ± 0.1 a86.1 ± 0.2 a
WT: wild-type; Ri3 and Ri4: transgenic GhROD1-RNAi lines; and OE17 and OE25: transgenic 35S-GhROD1 lines. Three independent samples were prepared for each material. Statistical data analysis was performed by one-way ANOVA followed by Tukey’s test. Different lowercase letters indicate significant differences among groups.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Ding, B.; Liu, B.; Zhu, X.; Zhang, H.; Hu, R.; Li, S.; Zhang, L.; Jiang, L.; Yang, Y.; Zhang, M.; et al. Downregulation of the GhROD1 Gene Improves Cotton Fiber Fineness by Decreasing Acyl Pool Saturation, Stimulating Small Heat Shock Proteins (sHSPs), and Reducing H2O2 Production. Int. J. Mol. Sci. 2024, 25, 11242. https://doi.org/10.3390/ijms252011242

AMA Style

Ding B, Liu B, Zhu X, Zhang H, Hu R, Li S, Zhang L, Jiang L, Yang Y, Zhang M, et al. Downregulation of the GhROD1 Gene Improves Cotton Fiber Fineness by Decreasing Acyl Pool Saturation, Stimulating Small Heat Shock Proteins (sHSPs), and Reducing H2O2 Production. International Journal of Molecular Sciences. 2024; 25(20):11242. https://doi.org/10.3390/ijms252011242

Chicago/Turabian Style

Ding, Bo, Bi Liu, Xi Zhu, Huiming Zhang, Rongyu Hu, Silu Li, Liuqin Zhang, Linzhu Jiang, Yang Yang, Mi Zhang, and et al. 2024. "Downregulation of the GhROD1 Gene Improves Cotton Fiber Fineness by Decreasing Acyl Pool Saturation, Stimulating Small Heat Shock Proteins (sHSPs), and Reducing H2O2 Production" International Journal of Molecular Sciences 25, no. 20: 11242. https://doi.org/10.3390/ijms252011242

APA Style

Ding, B., Liu, B., Zhu, X., Zhang, H., Hu, R., Li, S., Zhang, L., Jiang, L., Yang, Y., Zhang, M., Zhao, J., Pei, Y., & Hou, L. (2024). Downregulation of the GhROD1 Gene Improves Cotton Fiber Fineness by Decreasing Acyl Pool Saturation, Stimulating Small Heat Shock Proteins (sHSPs), and Reducing H2O2 Production. International Journal of Molecular Sciences, 25(20), 11242. https://doi.org/10.3390/ijms252011242

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop