GhPIPLC2D promotes cotton fiber elongation by enhancing ethylene biosynthesis.

Inositol-1,4,5-trisphosphate (IP3) is an important second messenger and one of the products of phosphoinositide-specific phospholipase C (PIPLC)-mediated phosphatidylinositol (4,5) bisphosphate (PIP2) hydrolysis. However, the function of IP3 in cotton is unknown. Here, we characterized the function of GhPIPLC2D in cotton fiber elongation. GhPIPLC2D was preferentially expressed in elongating fibers. Suppression of GhPIPLC2D transcripts resulted in shorter fibers and decreased IP3 accumulation and ethylene biosynthesis. Exogenous application of linolenic acid (C18:3) and phosphatidylinositol (PI), the precursor of IP3, improved IP3 and myo-inositol-1,2,3,4,5,6-hexakisphosphate (IP6) accumulation, as well as ethylene biosynthesis. Moreover, fiber length in GhPIPLC2D-silenced plant was reduced after exogenous application of IP6 and ethylene. These results indicate that GhPIPLC2D positively regulates fiber elongation and IP3 promotes fiber elongation by enhancing ethylene biosynthesis. Our study broadens our understanding of the function of IP3 in cotton fiber elongation and highlights the possibility of cultivating better cotton varieties by manipulating GhPIPLC2D in the future.

Introduction

Inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) are two important second messengers that convert extracellular signals to intracellular signals in plants (Singh et al., 2015). Phosphoinositide-specific phospholipase C (PIPLC) catalysis of the substrate phosphatidylinositol (4,5) bisphosphate (PIP2) produces both messenger molecules (Abd-El-Haliem and Joosten, 2017). Reversible inactivation of guard cell K+ channels is controlled by cytoplasmic Ca2+ that rely on IP3 signal cascades (Blatt et al., 1990). In tomato plants, reduction of IP3 content modifies the inositol phosphate pathway and affects light signaling and secondary metabolism (Alimohammadi et al., 2012). IP3 suppresses protein degradation in plant vacuoles by regulating sorting nexin-mediated protein sorting (Chu et al., 2016). In post-harvest peach fruit, IP3 is also involved in nitric oxide-enhanced chilling tolerance and defense response (Jiao et al., 2019).

When phosphorylated, IP3 forms inositol hexaphosphate (Dong et al., 2019), which has many functions in plants. Also known as phytic acid, IP6 is the main form of storage of phosphorus in mature seeds (Gibson et al., 2018). Inositol hexaphosphate can stimulate Ca2+ release to participate in many signaling pathways (Lee et al., 2015). As an endomembrane-acting Ca2+ release signal, IP6 activates both fast and slow conductance of the guard cell vacuole (Lemtiri-Chlieh et al., 2003). In plant hormone perception, IP6 can bind to the auxin receptor complex TIR1/IAA (Tan et al., 2007). Gibberellic acid has been shown to affect the degradation of IP6 in soybean sprouts with the calcium transport (Hui et al., 2018).

Phosphatidylinositol (PI), the precursor of IP3, is composed of 1,2-DAG phosphate and inositol. As the major phospholipid in cell membranes, PI plays critical roles in various physiological processes in plants (Hänninen et al., 2017; Heilmann, 2016). Phosphorylation of PI produces phosphatidylinositol 4-phosphate (PIP4) of which can be further catalyzed to generate PIP2 (Munnik and Nielsen, 2011), a kind of membrane phospholipid involved in various developmental stages in plants (Shimada et al., 2019; Kusano et al., 2008). Mitogen-activated protein kinase 6 (MPK6)-mediated phosphorylation of PI 4-phosphate 5-kinase 6 limits the production of the pool of functional PIP2 in response to the pathogen-associated molecular pattern-triggered immunity in Arabidopsis thaliana (Menzel et al., 2019). Directional growth is regulated by MPK6 controlling PIP2 production and membrane trafficking in pollen tubes in Arabidopsis (Hempel et al., 2017). Arabidopsis plasma membrane-associated Ca2+-binding protein 2 regulates PIP2 in membranes to attenuate root hair elongation (Kato et al., 2019).

PIPLC, an important lipid hydrolase in plants, cleaves PIP2 into two important secondary messengers, IP3 and DAG (Mueller-Roeber and Pical, 2002; Kadamur and Ross, 2013). The four conserved domains of PIPLC are named EF-hand, PI-PLC-X, PI-PLC-Y, and C2 (Zhang et al., 2018a). The EF-hand domain consists of two helix-loop-helix folding motifs for calcium-binding. The catalytic activity of all PIPLCs is strictly dependent on the PI-PLC-X and PI-PLC-Y domains. The C2 domain has been identified in all plant PIPLCs and functions along with the participation of calcium, in binding phospholipids (Pokotylo et al., 2014). The PIPLC plays multiple roles in plant stress response and development.

There are nine AtPIPLC genes in Arabidopsis (Tasma et al., 2008). AtPIPLC2 is required for seedling growth (Di Fino et al., 2017) and AtPIPLC5 is involved in primary and secondary root growth (Zhang et al., 2018c). AtPIPLC3 and AtPIPLC9 play critical roles in thermo-tolerance response (Gao et al., 2014; Zheng et al., 2012). AtPIPLC4 is up-regulated after salt stimulation (Tasma et al., 2008). In addition, overexpression of AtPIPLC5 and AtPIPLC7 improves plant drought tolerance (Zhang et al., 2018c; Van Wijk et al., 2018). AtPIPLC2-silenced plants are more susceptible to bacterial and fungal infections, suggesting that AtPIPLC2 is involved in plant immune response (D'Ambrosio et al., 2017).

Cotton fiber is an important industrial textile material in the world (Li et al., 2015). Fuzz and lint are two types of cotton fibers. Fuzz fibers only grow to a maximum length of 5 mm after seed maturity which cannot be used in textile (Arpat et al., 2004). Lint fibers develop into sufficiently long fibers desired for textile products (Kim and Triplett, 2001). The fuzzless and lintness mutant (fl) has been widely used to investigate the developmental mechanism of cotton fibers (Wu et al., 2017; Hu et al., 2018). Multiple genes are reported to be involved in cotton fiber development, including genes related to phytohormones (Xiao et al., 2019; Zhang et al., 2011), plant growth and development (Zhang et al., 2018a), and biotic and abiotic stress responses (He et al., 2019). Linolenic acid (C18:3) enhanced cotton fiber elongation by improving PI and phosphatidylinositol monophosphate biosynthesis (Liu et al., 2015). The promoter of FLORAL BINDING PROTEIN 7 (FBP7) drives the iaaM gene expression in the cotton ovule epidermis at the fiber initiation stage, which increased IAA levels and enhanced the number of lint fibers (Zhang et al., 2011). Exogenous GA3 increases fiber length via regulating cellulose synthase (CesA) gene expression, because of the GA-responsive elements present in the promoters of several CesA genes (Xiao et al., 2016). A cotton NAC transcription factor (FSN) that acts a master switch in regulating secondary cell wall development, activates its downstream secondary cell wall-related genes to promote cotton fiber development (Zhang et al., 2018b). GhCFE1A plays a critical role in fiber cell initiation and elongation during cotton fiber development and likely functions as a dynamic link between the actin cytoskeleton and endoplasmic reticulum (ER) network (Lv et al., 2015).

Ethylene, one of the major hormones in plants, participates in cotton fiber development (Li et al., 2007; Qin et al., 2007; Shi et al., 2003, 2006). The transcripts of three ethylene biosynthesis genes 1-aminocyclopropane-1-carboxylic acid oxidases (GhACO1-3) were highly accumulated at the fiber elongation stage. Exogenous application of ethylene promotes fiber elongation, as evidenced by an in vitro application of an ethylene-synthesis inhibitor, L-(2-aminoethoxyvinyl)-glycine, that hindered cotton fiber elongation (Shi et al., 2003, 2006). Ethylene may also promote fiber elongation by enhancing H2O2 production, which in turn induces ascorbate peroxidase activity (GhAPX1) in cotton fibers. The high expression of GhAPX1 observed in wild-type (WT) cotton fibers and little to no expression of GhAPX1 observed in fuzzless-lintness (fl) mutant ovules suggest an important role of GhAPX1 in fiber development (Li et al., 2007). Lignoceric acid can also enhance fiber cell elongation by increasing ethylene biosynthesis. Moreover, ethylene can eliminate the inhibition of fiber cell elongation caused by application of 2-chloro-N-[ethoxymethyl]-N-[2-ethyl-6-methyl-phenyl]-acetamide, an inhibitor of the biosynthesis of very-long-chain fatty acids (Qin et al., 2007).

In this work, we found that the expression level of GhPIPLC2D was significantly upregulated in the cotton fiber elongation stage and IP3 accumulation was much higher in WT fibers compared to that in WT and fl ovules at 10 days post-anthesis (DPA). Furthermore, silencing GhPIPLC2D reduced fiber length, IP3 accumulation and ethylene content. Exogenous application of linolenic acid and PI, the precursor of PIP2, improved IP3 and IP6 contents as well as ethylene biosynthesis. Exogenous application of IP6, the phosphorylation product of IP3, also significantly enhanced ethylene biosynthesis. These results indicate that GhPIPLC2D promotes cotton fiber elongation by increasing IP3 accumulation, which in turn stimulates ethylene biosynthesis.

Results

Conserved domains and phylogenetic analysis of GhPIPLCs

In plants, PIPLCs are structurally composed of four conserved domains, the EF-hand-like, PI-PLC-X, PI-PLC-Y, and C2 domains (Abd-El-Haliem and Joosten, 2017). Amino acid sequences of 12 GhPIPLCs were obtained from a previous study (Zhang et al., 2018a). Here, we renamed the GhPIPLCs according to the phylogenetic relationships of GhPIPLCs and AtPIPLCs (Figure 1A); the names and corresponding genome IDs of GhPIPLCs are shown in Table S1. To investigate the sequence conservation of GhPIPLCs, all GhPIPLC members were submitted for analysis by the Pfam online tools (http://pfam.xfam.org/) to obtain more detailed conserved domain information. All GhPIPLCs possessed four domains (Figure 1B) with the exceptions of GhPIPLC1A, GhPIPLC1D, and GhPIPLC6D, which lacked the EF-hand-like domain (Figure S1), indicating that these three GhPIPLCs may be functionally more diverse than the GhPIPLCs that contain all four domains.

Figure 1

Phylogenetic analysis and conserved domains of GhPIPLCs

(A) phylogenetic analysis of 12 GhPIPLCs, 9 GaPIPLCs, 9 GrPIPLCs, 9 GhePIPLCs, 9 AtPIPLCs, and 4 OsPIPLCs. Numbers indicate bootstrap confidence percentages. Scale indicates evolutionary distance.

(B) conserved domains of GhPIPLCs. The gray line indicates protein sequence length. Boxes with different colors represent different conserved domains of the GhPIPLC proteins.

In order to explore the evolutionary relationships of PIPLCs, the protein sequences of PIPLCs from G. hirsutum, A. thaliana, G. arboreum, G. raimondii, G. herbaceum, and Oryza sativa were obtained to generate a rooted phylogenetic tree. As shown in Figure 1, GhPIPLC1A and GhPIPLC1D had the longest evolutionary distances compared with the distances of other GhPIPLCs. There were six GhPIPLCs (GhPIPLC4A-3, GhPIPLC4D-3, GhPIPLC4A-2, GhPIPLC4D-2, GhPIPLC4A-1, and GhPIPLC4D-1), four GaPIPLCs (GaPIPLC4-1, GaPIPLC4-2, GaPIPLC4-3, GaPIPLC4-4), three GhePIPLCs (GhePIPLC4-1, GhePIPLC4-2, GhePIPLC4-3), three GrPIPLCs (GrPIPLC4-1, GrPIPLC4-2, GrPIPLC4-3) and one AtPIPLC4 in the same branch, indicating that GhPIPLC4 may extensively expand in Gossypium. To explore the potential driving force of PIPLC4 expansion, we analyzed duplication events in the PIPLC genes and found that tandem duplication is the main contributor to the expansion of PIPLC4 genes in Gossypium (Table S2).

GhPIPLC and IP3 are associated with cotton fiber elongation

To investigate the potential functions of GhPIPLC genes in cotton, the expression profiles of individual members of GhPIPLCs in cotton fiber and ovules were obtained from CottonFDG (https://cottonfgd.org/) and examined over developmental time from 5 to 25 DPA. The results showed that six members of GhPIPLCs (GhPIPLC4A-2, GhPIPLC4D-2, GhPIPLC4A-3, GhPIPLC4D-3, GhPIPLC2A, and GhPIPLC2D) were predominantly expressed during cotton fiber development. Notably, GhPIPLC2A and GhPIPLC2D had the highest and similar expression patterns in the fiber elongation stage (Figure S2), suggesting these two genes might have similar contributions to cotton fiber development. GhPIPLC2A and GhPIPLC2D were likely homoeologous genes with 84.94% similarity in coding sequences. Therefore, we amplified GhPIPLC2D and checked the sequence specificity via clone sequencing for subsequent functional analyses. The results showed that the GhPIPLC2D coding sequence, but not the GhPIPLC2A coding sequence, was successfully amplified. The expression levels of GhPIPLC2D in different cotton fiber development stages were further confirmed using quantitative real-time polymerase chain reaction (qRT-PCR). As shown in Figure 2A, the expression level of GhPIPLC2D was significantly higher in the fiber elongation stage with peak levels occurring at 5 and 10 DPA than at prior sampling times (Figure 2A), implying the GhPIPLC2D gene may play a critical role in cotton fiber elongation. Furthermore, we detected content of IP3, one of the catalytic products of PIPLC, in fibers and ovules 10 DPA from Xuzhou-142 WT and mutant fl plants and found that IP3 content was higher in 10 DPA WT fibers than that in 10 DPA WT and fl ovules (Figure 2B). Taken together, these results suggest that GhPIPLC2D may promote cotton fiber cell development by regulating IP3 accumulation.

Figure 2

Expression of the GhPIPLC2D gene and IP3 accumulation in cotton fibers and ovules

(A) the expression levels of GhPIPLC2D during fiber development between 3 day prior to anthesis to 20 days post-anthesis. Gene expression data were obtained by quantitative real-time PCR with three independent replicates. Error bars represent the SE (n = 3 biological replicates). Statistical significance was determined using one-way ANOVA with Tukey's test.

(B) analysis of IP3 accumulation in fibers, WT ovules and fl ovules 10 DPA. Statistical significance was determined using one-way ANOVA with Tukey's test. Error bars represent the SE (n = 3 biological replicates). ∗p < 0.05, ∗∗∗p < 0.001. WT, wild-type; fl, fuzzless-lintless mutant; DPA, days post-anthesis.

Silencing GhPIPLC2D in cotton inhibits fiber elongation

To better understand the biological function of GhPIPLC2D in cotton fiber development, GhPIPLC2D was silenced in G. hirsutum using the virus-induced gene silencing (VIGS) strategy. Our results show that the expression level of GhPIPLC2D was clearly reduced in GhPIPLC2D-silenced cotton plants in contrast to the control plants (Figure 3A). We also analyzed the expression of GhPIPLC2A gene in GhPIPLC2D-silenced plants and the results show that GhPIPLC2A transcripts in GhPIPLC2D-silenced plants were similar to the control plants, indicating that GhPIPLC2A transcripts are not decreased in GhPIPLC2D-silenced plants (Figure S3). We further measured the lengths of mature fibers in GhPIPLC2D-silenced and control plants. GhPIPLC2D-silenced plants displayed shorter fiber length than that in control plants (Figures 3B and 3C). In addition, suppression of GhPIPLC2D expression significantly reduced IP3 accumulation in 10 DPA fiber cells (Figure 3D). These observations are additional evidence of GhPIPLC2D possibly regulating IP3 accumulation, which is essential for cotton fiber cell development.

Figure 3

GhPIPLC2D is involved in cotton fiber elongation

(A) the expression levels of GhPIPLC2D in fibers of WT and GhPIPLC2D-silenced plants 10 DPA. Gene expression data were obtained by quantitative real-time PCR with three independent replicates.

(B) comparison of fiber lengths in WT and GhPIPLC2D-silenced plants.

(C) representative seeds with attached fibers from the VIGS experiment. Scale bar = 1 cm.

(D) comparison analysis of IP3 contents in WT and GhPIPLC2D-silenced plants. Statistical significance was determined using one-way ANOVA with Tukey's test. Error bars represent the SE (n = 3 biological replicates). ∗∗p < 0.01, ∗∗∗p < 0.001. WT, wild-type.

GhPIPLC2D gene promotes cotton fiber growth by regulating ethylene biosynthesis

A previous study demonstrated that ethylene plays a key role in promoting cotton fiber elongation and the 1-aminocyclopropane-1-carboxylic acid oxidase1 (ACO1) and ACO3 genes, two key genes for ethylene biosynthesis, were highly expressed during the fiber growth stage (Shi et al., 2003, 2006). In order to explore the molecular mechanism of GhPIPLC2D in regulating fiber growth, we detected the expression of the GhACO1 and GhACO3 genes as well as ethylene accumulation in GhPIPLC2D-silenced and non-silenced plants. Our results show that the expression of GhACO1 and GhACO3 were significantly down-regulated in GhPIPLC2D-silenced cotton when compared with non-silenced cotton (Figures 4A and 4B).

Figure 4

Silencing the GhPIPLC2D gene reduced ethylene biosynthesis and production

Relative expression levels of GhAC O 1 (A) and GhAC O 3 (B) in 10 DPA fibers of WT and GhPIPLC2D-silenced plants. Gene expression data were obtained by quantitative real-time PCR with three independent replicates. The relative expression level of each gene was determined after normalizing to the expression level of the WT, which was set to 1.0.

(C) ethylene production in 10 DPA fibers of WT and GhPIPLC2D-silenced plants.

(D) ethylene production from ovules of WT and GhPIPLC2D-silenced plants cultured for six days. Statistical significance was determined using one-way ANOVA with Tukey's test. Error bars represent the SE (n = 3 biological replicates). ∗∗p < 0.01, ∗∗∗p < 0.001. WT, wild-type.

We also detected ethylene production in GhPIPLC2D-silenced and non-silenced plants. As expected, the accumulation of ethylene was significantly lower in GhPIPLC2D-silenced cotton (Figure 4C). Ethylene accumulation in GhPIPLC2D-silenced plants was reduced to half of that in the non-silenced plants after six days in culture (Figure 4D). These results suggest that the GhPIPLC2D gene may promote cotton fiber development by stimulating the expression of ethylene biosynthesis-related genes and ultimately enhance ethylene production.

Linolenic acid and PI increase IP3 and IP6 contents and ethylene biosynthesis

The synthetic precursor of PIP2 and the catalytic substrate of PIPLC is PI, which is composed of phosphoric acid 1,2-DAG and inositol (Mueller-Roeber and Pical, 2002). Linolenic acid (C18:3) and palmitic acid (C16:0) were the most abundant fatty acids (FA) in PI from the 10 DPA fiber samples. The structural formula of PI biosynthesis is shown in Figure S4.

Carbenoxolone and 5-hydroxytryptamine inhibit the biosynthesis of C18:3 and PI, respectively (Liu et al., 2015). To better understand the effects of C18:3 and PI on cotton fiber cell growth, we detected the amounts of IP3, IP6, ethylene and expression of ethylene biosynthesis-related genes after exogenous applications of C18:3, PI, carbenoxolone and 5-hydroxytryptamine to 1 DPA cotton ovules for six days. The results revealed that exogenous application of each C18:3 and PI markedly improved IP3 accumulation, whereas in vitro application of the corresponding inhibitors, carbenoxolone and 5-hydroxytryptamine, apparently reduced IP3 accumulation (Figure 5A). The qRT-PCR experiment showed that ethylene biosynthesis-related genes GhACO1 and GhACO3 were significantly upregulated after C18:3 or PI application (Figure 5B). Furthermore, in vitro applications of C18:3 or PI significantly promoted ethylene accumulation, whereas their corresponding inhibitors dramatically inhibited ethylene production (Figure 5C). After C18:3 or PI treatment for six days, ethylene accumulation nearly increased four times that of the control group. However, the corresponding inhibitor-treated samples decreased ethylene production to half of that of the control (Figure 5D). Meanwhile, although IP6 have higher content in ovules, the IP6 content was increased from 0 DPA and reached a peak at 20 DPA during fiber development (Figure S5), and IP6 accumulation was significantly improved after C18:3 and PI treatments (Figure 5E). These results imply that C18:3 and PI promote IP3 and IP6 accumulation as well as ethylene biosynthesis.

Figure 5

C18:3 and PI promote IP3 and IP6 production and ethylene biosynthesis

Accumulation of IP3 (A) and ethylene biosynthesis gene transcripts (B) in ovules treated in vitro with C18:3, the C18:3 inhibitor carbenoxolone, PI or the PI inhibitor 5-hydroxytryptamine. Gene transcripts in (B) were obtained by qRT-PCR with three replicates.

(C) ethylene production in the same treatments as in (A).

(D) ethylene production over six days of ovule cultivation with the same treatments as in (A).

(E) IP6 accumulation in ovules treated in vitro with C18:3, the C18:3 inhibitor carbenoxolone, PI or the PI inhibitor 5-hydroxytryptamine. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. Statistical significance was determined using one-way ANOVA with Tukey's test. Error bars represent the SE (n = 3 biological replicates). No chemicals were added the control.

The C18 fatty acid contains saturated fatty acid C18:0 and unsaturated fatty acids C18:1, C18:2, and C18:3 (Conte et al., 2018). To investigate whether other C18 fatty acids could stimulate IP3 accumulation, the levels of IP3 in ovules treated with C18:0, C18:1, C18:2, or C18:3 were measured. Exogenous application of C18:0, C18:1, and C18:2 did not increase IP3 contents compared with that of the control; only in vitro application of C18:3 improved IP3 accumulation (Figure S6). We further analyzed the total fatty acid signal intensities extracted from different tissues of cotton. The results showed that C16:0, C18:2, and C18:3 were the most abundant fatty acids in flowers, leaves, and ovules. Moreover, flowers, leaves, and ovules also contained higher amounts of total fatty acids than that from roots and stems (Figure S7).

IP6 improves fiber length and ethylene biosynthesis

Catalysis of PIP2 by PIPLC produces IP3, which can be further phosphorylated to form IP6 (Gibson et al., 2018). In order to determine whether IP6 potentially regulates cotton fiber elongation and ethylene biosynthesis, we measured cotton fiber length, ethylene biosynthesis-related gene expression and the amount of ethylene accumulation in response to different concentrations of IP6 treatment. We observed an increase of fiber length in a dose-dependent manner with the increase of IP6 concentrations from 1 to 10 μM (Figure 6A). Fiber length increased three-fold that of the control group after treatment with 10 μM IP6. Furthermore, exogenous application of IP6 increased the expression of GhACO1 (Figure 6B) and GhACO3 (Figure 6C). As expected, ethylene accumulation (Figure 6D) also increased after IP6 treatment in vitro. These results suggest that IP6 can promote fiber elongation and ethylene biosynthesis.

Figure 6

Fiber length and ethylene production increased after IP6 treatment in vitro

Analysis of fiber length (A) and GhAC O 1 (B) and GhAC O 3 (C) gene expression, as well as ethylene production (D) after treatment with different concentrations of IP6in vitro. Relative expression levels of each gene were determined after normalizing to the expression level in the control, which was set to 1.0. Statistical significance was determined using one-way ANOVA with Tukey's test. Error bars represent the SE (n = 3 biological replicates). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. No chemicals were added to the control.

Ethylene and IP6 significantly promoted fiber cell elongation in GhPIPLC2D-silenced cotton

To further understand the biological role of IP6 and ethylene on cotton fiber cell development, WT and GhPIPLC2D-silenced cotton ovules collected at 1 DPA were cultured with 5 μM IP6 and 0.01 μM ethylene for 6 days. Subsequently, the length of fiber cells was observed and measured in microscope. The result showed that exogenous application of ethylene and IP6 significantly enhanced the fiber length of GhPIPLC2D-silenced cotton and WT (Figure 7A). Furthermore, the fiber length of GhPIPLC2D-silenced plants treated with ethylene and IP6 was obviously longer than the samples without any treatment (Figure 7B). These results suggest that ethylene and IP6 can recover fiber length shortened by GhPIPLC2D gene silencing.

Figure 7

Exogenous application of ethylene and IP6 promoted fiber length of GhPIPLC2D-silenced cotton

(A) phenotype of fiber cells from WT, GhPIPLC2D-silenced plant and WT, GhPIPLC2D-silenced plant with ethylene and IP6 application, respectively. Scale bar = 2mm.

(B) comparison of fiber lengths in WT, GhPIPLC2D-silenced plants and WT, GhPIPLC2D-silenced plant with ethylene and IP6 application, respectively. Statistical significance was determined using one-way ANOVA with Tukey's test. Error bars represent the SE (n = 3 biological replicates). ∗∗p < 0.01, ∗∗∗p < 0.001. WT, wild-type.

Discussion

The PIPLC gene family contains nine members in Arabidopsis (Tasma et al., 2008), four members in rice (Singh et al., 2013) and twelve members in G. hirsutum (Zhang et al., 2018a). The PIPLC protein is usually composed of four conserved domains (Abd-El-Haliem and Joosten, 2017). In G. hirsutum, GhPIPLCs contained four conserved domains, except for GhPIPLC1A, GhPIPLC1D and GhPIPLC6D, which lacked the EF-hand-like domain (Figure S1). Interestingly, mutating an EF-hand-like domain of PIPLC did not affect Ca2+-dependent substrate hydrolysis in Dictyostelium discoideum (Drayer et al., 1995), suggesting the domain may not be a regulatory site of the Ca2+ dependence of the PIPLC reaction, although the EF-hand-like domain is required for enzyme activity.

Phylogenetic analysis showed that six GhPIPLC4s, four GaPIPLC4s, three GhePIPLC4s, three GrPIPLC4s and one AtPIPLC4 were in the same evolutionary branch (Figure 1), indicating that the PIPLC4 sequence might have expanded in Gossypium. In Arabidopsis, the expression of AtPIPLC4 is positively upregulated after salt stimulation (Tasma et al., 2008). OsPIPLC1 prefers to hydrolyze PIP2 and elicits stress-induced Ca2+ signals to regulate salt tolerance (Li et al., 2017). Meanwhile, cotton is a moderately salt-tolerant crop with a salinity threshold level of 7.7 dS m−1 and has a higher basal level of tolerance to NaCl compared to that of other major crops (Sharif et al., 2019), Li et al., 2015). The moderate level of salt tolerance implies that GhPIPLC4A-1, GhPIPLC4D-1, GhPIPLC4A-2, GhPIPLC4D-2, GhPIPLC4A-3, and GhPIPLC4D-3 may have an important role in salt stress response in cotton development, and the salt stress may be the driving force in the expansion of these six genes during the evolutionary process. To verify the role of these six GhPIPLCs in salt stress response, further investigations in the future, such as genetic verification experiments, are needed.

PI-specific phospholipase C is the key enzyme that catalyzes PIP2 to produce IP3 and DAG (Kadamur and Ross, 2013). IP3, the critical secondary messenger that mediates calcium release from the ER, serves as the precursor in inositol phosphate biosynthesis and can be phosphorylated to form IP6. Thus, IP3 affects the downstream regulatory pathway of phytic acid (Xia and Yang, 2005). In this study, we discovered that IP3 content in WT fibers was higher than that in WT and fl ovules at 10 DPA (Figure 2). Silencing GhPIPLC2D gene expression reduced IP3 content and fiber length (Figure 3). These results suggest that IP3 may contribute to cotton fiber elongation, which could be confirmed by observing the phenotypes resulting from stably transformed cotton plants. In addition, the GhPIPLC2D and GhPIPLC2A are allele and had similar expression patterns (Figure S2), suggesting both two genes might have similar functions. Silencing both GhPIPLC2D and GhPIPLC2A genes might have fiber length shorter than silencing only GhPIPLC2D, which needs to be further investigated.

A previous study revealed that linolenic acid promotes fiber elongation by activating PI and PIP biosynthesis (Liu et al., 2015). In eukaryotic cells, PI is the major phospholipid involved in a wide range of signaling pathways, such as hormone regulation, biotic and abiotic stress responses, and light response. PI is mainly phosphorylated to PIP2, and then PIP2 is cleaved to form IP3 and DAG, which are two important secondary messengers in cells (Abd-El-Haliem and Joosten, 2017). Our data showed that exogenous application of C18:3 and PI significantly increased IP3 and IP6 contents, while in vitro applications of their inhibitors expectedly reduced IP3 and IP6 accumulation (Figure 5). Exogenous application of IP6 significantly promoted cotton fiber length and the expression of ethylene biosynthesis genes (Figure 6). These results further indicate that IP3 and IP6 might play a critical role in cotton fiber elongation. The IP6 contant measurement also showed that the IP6 content was increased during fiber development (Figure S5). Meanwhile, exogenous applications of ethylene and IP6 significantly improve the fiber length in GhPIPLC2D-silenced plant (Figure 7). Our study revealed that the GhPIPLC2D gene acts as a positive regulator in cotton fiber elongation, which the enzyme it encodes catalyzes PIP2 to DAG and IP3. Furthermore, IP3 is phosphorylated to form IP6 to promote cotton fiber elongation (Figure 8). In addition, previous study showed that phytic acid is mainly accumulated in the embryo of seed in maize, and it mainly provide phosphate and minerals for use during seedling growth and germination (Shi et al., 2003). The phosphorus was translocated to seed from roots and leaves and for synthesizing phytic acid and stored in seeds and it breakdown during germination for early seedling growth (Taliman et al., 2019). The 10 DPA ovules have the highest IP6 content (Figure S5), indicating that IP6 may also play important roles in ovule development.

Figure 8

Proposed working model of GhPIPLC regulation of fiber elongation

Ethylene is a major phytohormone that participates in many developmental stages, such as cell division and root hair development (Song et al., 2019). In cotton, ethylene plays a major role in fiber cell elongation (Shi et al., 2003, 2006). One study showed that very-long-chain fatty acids promote fiber elongation by enhancing ethylene biosynthesis (Qin et al., 2007). In this study, we found that the expression of GhACO1 and GhACO3, as well as ethylene production, were significantly decreased in GhPIPLC2D-silenced cotton compared with those of WT cotton (Figure 4). In addition, exogenous application of linolenic acid (C18:3), PI and IP6 promoted ethylene biosynthesis (Figure 5). These results indicate that GhPIPLC2D and IP3 promoted cotton fiber cell development possibly by activating ethylene biosynthesis and enhancing ethylene accumulation (Figure 8). This study provides empirical evidence that IP3 regulates ethylene biosynthesis and promotes cotton fiber development, which is a branch in ethylene regulation of cotton fiber growth.

Calcium signals have been found to contribute to cotton fiber development (Guo et al., 2017). In Arabidopsis, PIPLC has been shown to be important in Ca2+ signaling, and piplc3 mutants showed decreased Ca2+ release (Gao et al., 2014). The reductions of IP3 and IP6 levels affect Ca2+ release from the cytosol and might contribute to flg22-dependent cytosolic Ca2+ bursts (Hilleary et al., 2020). Moreover, Hasenstein and Evans (1986) found that Ca2+ enhances the conversion of 1-aminocyclopropane carboxylic acid (ACC) to ethylene in primary roots of corn. Yu et al. (2019) showed that Ca2+ promotes root development in response to salt stress by regulating the biosynthesis of ethylene. As a secondary messenger, Ca2+ is central for plant signal transduction. Calcium is involved in most environmental responses and phytohormone signal pathways (Peiter, 2011; Guo et al., 2017). Therefore, we speculate that GhPIPLC2D may also affect Ca2+ release and thus participate in fiber development in cotton. In the future, molecular mechanisms and regulatory relationships between GhPIPLC2D-Ca2+-ethylene in regulating fiber cell elongation should be examined to deepen our understanding of the underlying processes in cotton fiber development.

Limitations of the study

In this study, we revealed a GhPIPLC2D gene serves as a positive regulator in cotton fiber elongation, which catalyzes PIP2 to produce IP3 and IP3 promotes fiber elongation by enhancing ethylene biosynthesis. However, as we have discussed in the article, the GhPIPLC2D expression impact IP3 content and IP3 accumulation promote fiber elongation through enhancing ethylene biosynthesis while the GhPIPLC2D-ethylene in regulating fiber cell elongation should be examined to deepen our understanding of the underlying processes in cotton fiber development. In addition, how IP3 promotes ethylene synthesis also needs to be further clarified in future studies.

Resource availability

Lead contact

Further information and requests for resources should be directed to and will be fulfilled by the lead contact, Guanghui Xiao (guanghuix@snnu.edu.cn).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate or analyze data sets and code.

Methods

All methods can be found in the accompanying Transparent methods supplemental file.