https://orcid.org/0000-0002-0058-9383
Abstract
The phytohormones ethylene and jasmonate play important roles in the adaptation of rice plants to salt stress. However, the molecular interactions between ethylene and jasmonate on rice seminal root growth under salt stress are unknown. In this study, the effects of NaCl on the homeostasis of ethylene and jasmonate, and on rice seminal root growth were investigated. Our results indicate that NaCl treatment promotes ethylene biosynthesis by up-regulating the expression of ethylene biosynthesis genes, whereas NaCl-induced ethylene does not inhibit rice seminal root growth directly, but rather indirectly, by promoting jasmonate biosynthesis. NaCl treatment also promotes jasmonate biosynthesis through an ethylene-independent pathway. Moreover, NaCl-induced jasmonate reduces meristem cell number and cell division activity via down-regulated expression of Oryza sativa PLETHORA (OsPLT) and cell division-related genes, respectively. Additionally, NaCl-induced jasmonate inhibits seminal root cell elongation by down-regulating the expression of cell elongation-related genes. Overall, salt stress promotes jasmonate biosynthesis through ethylene-dependent and -independent pathways in rice seminal roots, and jasmonate inhibits rice seminal root growth by inhibiting root meristem cell proliferation and root cell elongation.
Introduction
Soil salinity is one of the major abiotic stresses that limits food crop growth and productivity worldwide (Farooq et al., 2015). Rice (Oryza sativa L.) is one of the most important food crops and is a primary source of calories for more than half of the world’s population (Y. Wang et al., 2020). However, under saline stress conditions, the growth and development of rice is affected, resulting in reduced rice yield (Parihar et al., 2015; Negrão et al., 2017). Phytohormones, such as ethylene and jasmonate, play important roles in regulating the adaptation of rice plants to salt stress (Hazman et al., 2015; Qin et al., 2019).
Jasmonates, including jasmonic acid (JA) and the amino acid-conjugated jasmonate (jasmonoyl-isoleucine, JA-Ile), are lipid-derived plant hormones that play important roles in plant adaptation to stresses, including drought, wounding, and salt stress (Pedranzani et al., 2003; Tani et al., 2008; Seo et al., 2011; Wasternack and Hause, 2013; Zhao et al., 2014; Valenzuela et al., 2016; Hazman et al., 2019). Jasmonate can also regulate plant growth and development, such as leaf senescence (Liu et al., 2016), spikelet development (Liu et al., 2017), and root growth (Wang et al., 2002; Jiang et al., 2007). The roles of jasmonate in growth, development, and stress responses in plants are closely related to jasmonate homeostasis, which is mainly regulated by jasmonate biosynthesis. Usually, jasmonate-deficient mutants exhibit obvious phenotypes in growth (Hazman et al., 2015), development (You et al., 2019), or stress responses, compared with wild-type (WT) plants (Hazman et al., 2015). Jasmonate biosynthesis is catalyzed by a series of enzymes, including lipoxygenase (LOX), allene oxide synthase (AOS), allene oxide cyclase (AOC), cis-12-oxo-phytodienoic acid reductase (OPR), and acyl-CoA oxidase (ACX; Wasternack and Hause, 2013; Liu et al., 2017). It has been reported that over-expressing TaAOC1 led to increased jasmonate content in wheat (Zhao et al., 2014), and up-regulating OsOPR7 resulted in increased jasmonate content in rice (Tani et al., 2008). Moreover, previous results demonstrated that in rice leaves, NaCl treatment strongly up-regulated the expression of OsOPR genes such as OsOPR1, OsOPR3, and OsOPR7 (Jang et al., 2009), which in turn contribute to NaCl-induced jasmonate biosynthesis (Hazman et al., 2019). However, in rice seminal roots, the response pattern of jasmonate biosynthesis genes to salt stress is still unclear. Previous studies revealed that in Arabidopsis, a dicotyledonous model plant, jasmonate inhibits primary root growth mainly by inhibiting meristematic cell division under normal conditions, whereas jasmonate inhibits primary root growth by inhibiting cell elongation under salt stress conditions (Chen et al., 2011; Valenzuela et al., 2016). However, in rice, a monocotyledonous plant, although it has been reported that jasmonate plays an important role in adaptation of roots to salt stress (Toda et al., 2013), the underlying mechanism by which jasmonate regulates the growth of rice seminal roots under salt stress is poorly understood.
Ethylene, a gaseous phytohormone, is essential for various stress responses and developmental processes in plants (Lin et al., 2009; Alarcón et al., 2012; Wang et al., 2013; Dubois et al., 2018). There is increasing evidence to indicate that ethylene participates in salt-induced inhibition of root growth (Abiri et al., 2017; Qin et al., 2019), and that ethylene inhibits root growth in a dose-dependent manner (Qin et al., 2019). Ethylene homeostasis in plants is mainly determined by its biosynthesis, and the ethylene biosynthesis pathway has been well studied (Lin et al., 2009; Yamauchi et al., 2015). During ethylene biosynthesis, methionine is produced from S-adenosylmethionine (SAM) by SAM synthase (SAMS). SAM is then transformed to 1-aminocyclopropane-1-carboxylate (ACC) by ACC synthase (ACS; Yang and Hoffmang, 2003; Lin et al., 2009). Finally, ACC is converted to ethylene by ACC oxidase (ACO; Yang and Hoffman, 2003; Lin et al., 2009). It has been reported that salt stress can inhibit root growth by promoting ethylene biosynthesis via up-regulation of ethylene biosynthesis genes, such as ACS5 and ACS7 in Arabidopsis (Wang et al., 2005), ACS1 and ACO1-ACO3 in tobacco (Cao et al., 2006), and the ACS and ACO genes in rice (Qin et al., 2019). However, the effects of salt stress on ethylene biosynthesis genes, such as the OsSAMS genes in rice, remain unknown, and the long term (≥6 h) expression patterns of ethylene biosynthesis genes in response to salt stress have not been well studied in rice seminal roots.
The rice seminal root is the first root to emerge from the seed after germination, and is responsible for water uptake and nutrient absorption during seedling establishment (Wang et al., 2011). Rice seminal root growth is sustained by root meristem cell proliferation and root cell elongation (Ioio et al., 2008; Li et al., 2015; Xu et al., 2017). Meristem cell proliferation rate is positively correlated with meristem size and meristem cell division activity (Takatsuka and Umeda, 2014). It has been reported that PLETHORA (PLT) plays a critical role in regulating meristem size in Arabidopsis primary roots by increasing meristem cell number through maintenance of the root stem cell niche. Moreover, jasmonate can reduce root meristem size by inhibiting the transcription of PLT1 and PLT2 via the direct binding of MYC2 to the promoters of PLT1 and PLT2 (Chen et al., 2011). Jasmonate also reduces cell proliferation rate by decreasing cell division activity, by down-regulating the expression of cell division-related genes in Arabidopsis primary roots (Chen et al., 2011). In rice, OsPLT1 and OsPLT6, the homologs of Arabidopsis PLT1 and PLT2, are highly expressed in the seminal root (Li and Xue, 2011), and OsPLT genes, as well as cell division-related genes [such as Oryza sativa B-type cyclin1;1 (OsCycB1;1), Oryza sativa B-type cyclin-dependent kinase1;1 (OsCDKB1;1) and Oryza sativa B-type cyclin-dependent kinase2;1 (OsCDKB2;1)], can positively control meristem size and cell division activity, respectively (Zou et al., 2019; Q.Wang et al., 2020). In addition, rice root cell elongation is regulated by many proteins, including xyloglucan endotransglucosylase/hydrolase (XTH) and expansin (EXP; Yi and Kende, 2002; Wang et al., 2014; X.Zou et al., 2018). Although it has been reported that jasmonate can inhibit root growth, it is unclear whether jasmonate controls growth of the rice seminal root by mediating transcription of OsPLT genes and cell division-related genes, and/or cell elongation-related genes, under salt stress conditions.
Although there is increasing evidence to suggest that both ethylene and jasmonate may be involved in root responses to salt stress, the mechanism by which ethylene and jasmonate interact to affect rice seminal root growth under salt stress is unknown. In this study, we provide evidence that salt stress promotes ethylene biosynthesis in rice seminal roots. However, salt-induced ethylene does not directly inhibit rice seminal root growth, but rather inhibits growth of the seminal root growth by promoting jasmonate biosynthesis. Moreover, salt stress also promotes jasmonate biosynthesis through an ethylene-independent pathway. Additionally, our results indicate that salt-induced jasmonate inhibits rice seminal root growth by restricting root meristem cell proliferation via down-regulation of OsPLT genes and root cell division-related genes, and also by inhibiting root cell elongation via down-regulation of Oryza sativa xyloglucan endotransglucosylase/hydrolase (OsXTH) and Oryza sativa expansin (OsEXP) genes.
Materials and methods
Plant materials and growth conditions
The indica rice (Oryza sativa L.) ‘9311’, as well as the jasmonate-deficient mutant cpm2 and its wild-type (WT) rice (Oryza sativa L. ssp. japonica) ‘Nihonmasari’ were used in this study. The cpm2 mutant was isolated from a γ–ray-mutagenized M2 population of japonica rice (‘Nihonmasari’). cpm2 has a genomic deletion; within the deleted region, the gene OsAOC (Os03g0438100), which encodes a key enzyme for jasmonate biosynthesis, was identified (Riemann et al., 2013). The OsAOC of cpm2 was previously sequenced and a deletion of 11 bp in the first exon of this gene was detected. Therefore, OsAOC was identified as the most likely candidate gene causing the jasmonate deficiency in the cpm2 mutant (Riemann et al., 2013). In the cpm2 mutant the content of JA and JA-Ile is lower than that of WT (Riemann et al., 2013; Hazman et al., 2015).
Rice seeds were surface sterilized in 5% NaClO for 20 min, washed six times with distilled water, and then soaked in distilled water for 24 h at 28 °C Liu et al. (2016). The seeds were germinated for 24 h at 30 °C, and the germinated rice seedlings were then transferred to plastic screens floating in distilled water, with or without different treatments (Z. Zou et al., 2018). All of the germinated rice seedlings were incubated for 6, 12, 24, 48, or 96 h in an artificial climate incubator (HP 1500 GS, Ruhua Corporation, Wuhan, China) with a 12 h light (29 °C)/12 h dark (26 °C) photoperiod.
Chemicals and treatments
JA, JA-Ile, ACC, aminoethoxyvinylglycine (AVG), and ibuprofen (IBU) were purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). Each chemical was dissolved in dimethyl sulphoxide and diluted to the required concentration with distilled water. NaCl was directly dissolved in distilled water. ACC was used as an ethylene precursor, AVG, as an ethylene biosynthesis inhibitor (Swarup et al., 2007; Yin et al., 2011), and IBU, as a jasmonate biosynthesis inhibitor (Zhu et al., 2006; Liu et al., 2017). Germinated rice seedlings were incubated in distilled water with or without the following eight treatments: NaCl (30 mM, 60 mM, 90 mM, 120 mM), NaCl (120 mM) + AVG (0.2 μM), NaCl (120 mM) + IBU (5 μM), NaCl (120 mM) + AVG (0.2 μM) + IBU (5 μM), ACC (9 μM), ACC (9 μM) + IBU (5 μM), JA (3 μM), or JA (3 μM) + AVG (0.2 μM). The pH of each treatment solution was adjusted to 6.5. Each treatment solution contained the same concentration of dimethyl sulphoxide (0.01% v/v), and was refreshed every 2 d.
Measurement of seminal root length
Germinated rice seedlings were incubated in distilled water with or without the different treatments. After 96 h of treatment, the seminal root length was measured manually with a ruler. The data are presented as the means ±SD calculated from 10 biological replicates.
RNA isolation, cDNA synthesis, and quantitative real-time PCR (qRT–PCR) analysis
Germinated rice seedlings were incubated with different treatment solutions. After 6, 12, 24, 48, and 96 h of treatment, rice seminal roots were harvested and frozen in liquid nitrogen for RNA extraction. Total RNA was extracted from the seminal roots using an RNAprep Pure Plant Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1.5 μg of total RNA using the FastQuant RT kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. qRT–PCR was performed using 2× HSYBR qPCR mix (Zoman Biotech, Beijing, China) on a LightCycler 480 Real-Time System (Roche, Basel, Switzerland) according to the method of Liu et al. (2018). The PCR program included pre-denaturation at 95 °C for 3 min, followed by 40 cycles of 95 °C for 30 s, 57 °C for 20 s, and 72 °C for 30 s. Rice OsACTIN and OsUBQ5 have been widely used as reference genes for normalizing gene expression (Wang et al., 2016; El Mahi et al., 2019). In this study, the rice OsACTIN gene was used as the internal control for normalization of gene expression. Additionally, OsUBQ5 gene was used as another internal control for normalizing OsACTIN gene expression. The results demonstrated that OsACTIN gene expression was stable under all conditions described in this study (Supplementary Fig. S1). Relative gene expression data was analyzed using the 2−ΔΔCT method (Livak and Schmittgen, 2001). The primers used for qRT–PCR were designed using the primer design website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/), and the primers are provided in Supplementary Table S1.
Measurement of ethylene production
Ethylene production in rice seminal roots was measured as previously described (Lim et al., 2006; X. Zou et al., 2018). Germinated rice seedlings were incubated with different treatments. After 48 h and 96 h of incubation, rice seminal roots were harvested, weighed, and placed in 50 ml gas-tight glass vials containing 1 ml of distilled water. The samples were then incubated at 30 °C for 5 h in a controlled climate incubator (RDN-560C-4, Dongnan Instrument, Ningbo, China). Gas from the glass vessel headspace was withdrawn (3 ml) with a gas-tight syringe and injected into a gas chromatograph (Varian GC-3800,Varian Corporation, USA) fitted with a capillary column and flame ionization detector. The temperatures of the injector, column, and flame ionization detector were set to 100 °C, 60 °C, and 200 °C, respectively. The concentration of ethylene in the sample gas was determined against an authentic standard (Weichuang Corporation, Shanghai, China) also injected into the collection column. Peaks were identified by elution time and quantified relative to the local baseline. Data are presented as means ±SD calculated from three independent biological replicates.
Jasmonate measurement
Germinated rice seedlings were incubated with different treatments. Rice seminal roots were harvested after 48 h and 96 h of incubation and stored at –80 °C prior to JA and JA-Ile content analysis. Every treatment had three biological replicates. The concentrations of endogenous JA and JA-Ile were determined by LC-MS/MS according to the methods described by Liu et al. (2012). The stored rice seminal roots were ground to a fine powder in liquid nitrogen. The samples were then weighed (~0.1 g), transferred to 1.5 ml tubes, mixed with 750 μl pre-cooled extraction buffer (methanol:water:acetic acid, 80:19:1, v:v:v), vigorously shaken on a shaking bed for 16 h at 4 °C in the dark, and then centrifuged at 16 000 g for 15 min at 4 °C. The supernatant was transferred to a new 1.5 ml tube and the remaining residues were remixed with 400 μl extraction buffer, shaken for 4 h at 4 °C, and centrifuged as before. The supernatants were combined and filtered through 0.22 μm nylon filters. The filtrates were concentrated by evaporation with dry nitrogen at 25 °C and dissolved in 200 μl methanol. The resulting solutions were quantified using an Acquity UPLC H-Class Xevo G2-XS (Waters Corporation, USA). A Waters BEH C18 (Waters Corporation) column (2.1 × 30 mm, 1.7 μm) was used at 25 °C. The injected volume of each sample was 1 μl. The elution gradient was carried out with a binary solvent system consisting of 0.1% formic acid in H2O (solvent A) and 100% MeCN (solvent B) at a constant flow rate of 400 μl min-1. A linear gradient profile with the following proportions (v/v) of solvent B was applied: 20% of solvent B from 0 to 1 min; 20% to 100% of solvent B from 1 to 8 min; 100% of solvent B from 8 to 10 min; 100% to 20% of solvent B from 10 to 12 min. Data are presented as the means ±SD of three independent biological replicates.
Confocal microscopy analysis of root phenotypes
Germinated rice seedlings were incubated with different treatments. After 96 h of incubation, the rice seminal roots were selected for phenotypic analysis. For meristem size measurement, the seminal root tips were stained with 1 μg ml-1 4’,6-diamidino-2-phenylindole (DAPI) for 10 min in darkness and washed in distilled water for 5 min. For cell length measurement, the seminal root tips were stained with 5 μg ml-1 propidium iodide for 10 min in darkness and washed in distilled water for 5 min. The samples were mounted on a microscopic slide in 50% glycerol and then visualized using a laser fluorescence confocal microscope (Leica SP8, Leica Corporation, Solms, Germany). The root cell lengths and root meristem sizes were then measured using IMAGEJ software (Bethesda, MD, USA). The meristem cell number in rice seminal roots was expressed as the number of cortex cells from the quiescent center to the first elongated cell. Root meristem size was determined by measuring the length from the quiescent center to the first elongated epidermal cell. The cell proliferation rates were calculated as described previously (Li et al., 2015; Q. Wang et al., 2020). The data are presented as means ±SD calculated from ten biological replicates.
Statistical analyses
Statistical analyses were conducted using an independent sample t-test or one-way ANOVA followed by Duncan’s multiple range test at P<0.05. All data are presented as means ±SD from no fewer than three biological replicates.
Results
Salt inhibits rice seminal root growth in a dose-dependent manner
In order to investigate the effects of salt on rice seminal root growth, rice seedlings were treated with different concentrations of NaCl. As shown in Fig. 1, after 96 h of treatment, the average seminal root length in the control treatment was 9.01 cm. By contrast, rice seedlings treated with 30, 60, 90, and 120 mM NaCl exhibited a short-root phenotype, with average seminal root lengths of 7.47, 6.53, 5.61, and 4.67 cm, respectively (Fig. 1). This result shows that 30–120 mM NaCl significantly (P<0.05) inhibited growth of rice seminal roots in a dose-dependent manner.
Effects of different NaCl concentrations on rice (‘9311’) seminal root growth. (A) Seminal root phenotypes of rice seedlings. Scale bar =5 cm. (B) Statistical analysis of the seminal root lengths of NaCl-treated rice seedlings. The rice seedlings were treated with 0 (control), 30, 60, 90, and 120 mM NaCl for 96 h, after which the lengths of the seminal roots were measured and the seedlings were photographed. Data are the means ±SD calculated from 10 biological replicates. Significant differences (P<0.05; one-way ANOVA followed by Duncan’s multiple range test) are indicated by different lower-case letters.
Effects of salt on ethylene biosynthesis and seminal root growth
In order to test whether ethylene is involved in salt-induced growth inhibition of the seminal root, we investigated the effects of the ethylene biosynthesis inhibitor, AVG. The results demonstrated that the inhibition of rice seminal root growth by 120 mM NaCl was significantly (P<0.05) reversed by the application of AVG (Fig. 2A). Furthermore, we also analyzed the effects of NaCl and NaCl + AVG treatments on ethylene content. As shown in Fig. 2B, the ethylene concentration was increased by 2.3- and 2.1-fold in NaCl-treated seminal roots compared with the control, after 48 h and 96 h of treatment, respectively, whereas the increase in ethylene content was reversed by application of AVG. These results suggest that ethylene plays an important role in NaCl-induced growth inhibition of seminal roots in rice.
The effects of AVG on NaCl-induced growth inhibition and ethylene production in rice (‘9311’) seminal roots. (A) The alleviating effect of AVG on NaCl-induced growth inhibition of rice seminal roots. Data are presented as means ± SD calculated from 10 biological replicates, and significant differences (P <0.05; one-way ANOVA followed by Duncan’s multiple range test) are indicated by different lower-case letters. (B) AVG reduces NaCl-induced ethylene production in rice seminal roots. Data are presented as means ±SD calculated from three biological replicates, and significant differences (P<0.05; one-way ANOVA followed by Duncan’s multiple range test) are indicated by different lower-case letters at each sampling time. Rice seedlings were treated with solutions containing NaCl (120 mM) or NaCl + AVG (0.2 μM). The effects of the different treatments on seminal root lengths were investigated after 96 h, and the ethylene concentration was determined after 48 h and 96 h. AVG, aminoethoxyvinylglycine; CK, distilled water control; FW, fresh weight.
In order to investigate the mechanism by which salt treatment increases ethylene production in the rice seminal root, the effects of 120 mM NaCl on the expression of ethylene biosynthesis genes, including OsSAMS1/2/3, OsACS1/2/4/5, and OsACO1/2/4/5/7, were analyzed by qRT-PCR. After 6 h of NaCl treatment, only five ethylene biosynthesis genes, OsACS1, OsACS5, OsACO1, OsACO2, and OsACO5, responded to NaCl treatment, and the expression of these five genes was significantly (P<0.05) up-regulated compared with the control (Fig. 3). After 12 h of NaCl treatment, expression of all ethylene biosynthesis genes showed a strong response; the expression of seven ethylene biosynthesis genes (OsSAMS2, OsACS1, OsACS4, OsACO1, OsACO2, OsACO5, and OsACO7) was up-regulated following NaCl treatment by >2-fold (Fig. 3). Except for OsSAMS3 and OsACS1, the expression of other ethylene biosynthesis genes was significantly up-regulated by NaCl treatment at 24 h, compared with controls (Fig. 3). After 48 h of NaCl treatment, expression of the three OsSAMS genes had returned to control levels (Fig. 3). In contrast, the expression of two OsACS genes (OsACS2 and OsACS5) and three OsACO genes (OsACO1, OsACO2, and OsACO4) was up-regulated by NaCl treatment for 48 h, compared with the controls (Fig. 3). However, after 96 h of NaCl treatment, there was no significant difference (P>0.05) in the expression of most ethylene biosynthesis genes between the NaCl treatment and the control, although expression of both OsACS5 and OsACO2 was still up-regulated by NaCl treatment (Fig. 3). These results indicate that NaCl treatment promotes ethylene production by up-regulating the transcription of ethylene biosynthesis genes including the OsSAMS, OsACS, and OsACO genes.
Effects of NaCl on the expression of ethylene biosynthesis genes. Seminal roots of rice (‘9311’) seedlings were collected for qRT–PCR analysis after 6, 12, 24, 48, and 96 h of different treatments. The relative expression of each ethylene biosynthesis gene in control after 6 h of treatment was set as 1. Data are presented as the means ±SD, using three biological replicates with three technical replicates for the statistical analyses. Asterisks indicate significant differences (P<0.05; independent sample t-test) between the NaCl treatment and the CK at each sampling time. CK, distilled water control; NaCl, 120 mM NaCl.
Effect of salt on jasmonate biosynthesis and rice seminal root growth
To test whether jasmonate is involved in salt-induced inhibition of rice seminal root growth, we investigated the effect of the jasmonate biosynthesis inhibitor IBU on growth inhibition of the rice seminal root induced by 120 mM NaCl. The results demonstrated that NaCl-induced growth inhibition of rice seminal roots was rescued by the application of 5 μM IBU (Fig. 4A). Furthermore, the concentrations of JA and JA-Ile in seminal roots following treatment with NaCl and NaCl + IBU were measured, to ascertain whether JA was involved in salt-induced growth inhibition of the seminal roots. The JA and JA-Ile concentrations in rice seminal roots were significantly (P<0.05) increased after 48 h and 96 h of treatment with 120 mM NaCl, whereas the concentrations were reduced to control levels by application of IBU (Fig. 4B, C). These results suggest that JA plays a critical role in NaCl-induced growth inhibition of seminal roots in rice seedlings.
The effects of IBU on NaCl-induced jasmonate biosynthesis and growth inhibition in rice (‘9311’) seminal roots. (A) IBU alleviates the NaCl-induced growth inhibition of the rice seminal root. Data are presented as means ±SD calculated from 10 biological replicates and significant differences (P<0.05; one-way ANOVA followed by Duncan’s multiple range test) are indicated by different lower-case letters. IBU alleviates NaCl-induced biosynthesis of JA (B) and JA-Ile (C) in rice seminal roots. Data are presented as means ±SD calculated from three biological replicates and significant differences (P<0.05; one-way ANOVA followed by Duncan’s multiple range test) are indicated by different lower-case letters at each sampling time. Rice seedlings were treated with solutions containing 120 mM NaCl or 120 mM NaCl + 5 μM IBU. The seminal root lengths were measured after 96 h treatment, and the JA and JA-Ile concentrations were determined after 48 h and 96 h of different treatments. CK, distilled water control; FW, fresh weight; IBU, ibuprofen; JA, jasmonic acid; JA-Ile, jasmonoyl-isoleucine.
In order to investigate the mechanism by which salt increases the JA and JA-Ile content in rice seminal roots, the effect of NaCl on the expression of jasmonate biosynthesis genes was analyzed. As shown in Fig. 5, after 6 h of NaCl treatment, except for OsLOX2, OsLOX6, OsAOS1, OsACX1, and OsACX2, the expression of other jasmonate biosynthesis genes was up-regulated compared with the control treatment. Subsequently, except for OsAOS1, expression of all other jasmonate biosynthesis genes was up-regulated after 12 h of NaCl treatment (Fig. 5). After 24 h of NaCl treatment, with the exception of OsAOC, OsOPR1, and OsOPR2, expression of the jasmonate biosynthesis genes remained up-regulated by NaCl treatment (Fig. 5). After 48 h of NaCl treatment, 10 of the jasmonate biosynthesis genes were significantly (P<0.05) up-regulated by NaCl treatment, especially the expression of OsOPR10, which increased by 9.1-fold compared with the control (Fig. 5). After 96 h of NaCl treatment, there were no significant differences (P>0.05) in the expression of most jasmonate biosynthesis genes compared with the control treatment (Fig. 5). These results suggest that NaCl-induced growth inhibition of rice seminal roots might be due to increased JA and JA-Ile concentrations that result from the NaCl-induced transcriptional up-regulation of jasmonate biosynthesis genes.
Effects of NaCl on the transcription of jasmonate biosynthesis genes. Seminal roots of rice (‘9311’) seedlings were collected for qRT–PCR analysis after 6, 12, 24, 48, and 96 h of different treatments. Data are presented as the means ± SD, using three biological replicates with three technical replicates for the statistical and error range analyses. The relative expression of each jasmonate biosynthesis gene in control after 6 h of treatment was set as 1. Asterisks indicate significant differences (P<0.05; independent sample t-test) between the NaCl and the CK treatments at each sampling time. CK, distilled water control; NaCl, 120 mM NaCl.
The ethylene-jasmonate interaction in salt-induced growth inhibition of seminal roots
Because both ethylene and jasmonate are involved in the salt-induced growth inhibition of seminal roots, we investigated their interaction in this process. Although AVG treatment completely reversed NaCl-induced ethylene biosynthesis (Fig. 2B), AVG treatment did not fully rescue the NaCl-induced jasmonate biosynthesis and growth inhibition of the seminal root (Fig. 6; Supplementary Fig. S2); thus salt-induced growth inhibition of the seminal root cannot be due solely to salt-induced ethylene biosynthesis. In contrast, IBU treatment completely reversed NaCl-induced jasmonate biosynthesis and rescued the NaCl-induced growth inhibition of seminal roots (Figs 4; 6). However, there were no differences in seminal root lengths in seedlings treated with NaCl + IBU, NaCl + AVG + IBU, and the control (Fig. 6). This result demonstrated that there was no additive effect of AVG and IBU on NaCl-induced growth inhibition of the rice seminal root, suggesting an upstream or downstream relationship between ethylene and jasmonate in NaCl-induced growth inhibition of the seminal roots in rice. Moreover, application of the direct ethylene precursor ACC, or JA, also significantly (P<0.05) inhibited growth of seminal roots, and ACC-inhibition of rice seminal root growth was completely rescued by IBU treatment, whereas AVG enhanced the effect on the growth inhibition caused by JA (Fig. 6).
Ethylene and jasmonate interact in salt-induced growth inhibition of the rice (‘9311’) seminal roots. Rice seedlings were treated with solutions containing NaCl (120 mM), ACC (9 μM), AVG (0.2 μM), JA (3 μM), and IBU (5 μM) individually, and in combination. Seminal root lengths were measured after 96 h of treatment. Data are presented as means ± SD calculated from 10 biological replicates and significant differences (P<0.05; one-way ANOVA followed by Duncan’s multiple range test) are indicated by different lower-case letters. ACC, 1-aminocyclopropane-1-carboxylate; AVG, aminoethoxyvinylglycine; CK, distilled water control; IBU, ibuprofen; JA, jasmonic acid.
In addition, 9 μM ACC treatment significantly enhanced JA and JA-Ile content in roots, which could be reversed by IBU treatment (Fig. 7). As shown in Fig. 8, after 6 h of ACC treatment, except for OsLOX2, OsLOX4, OsLOX7, OsAOC, OsOPR1, OsOPR2, OsOPR7, OsOPR8, and OsACX3, the expression of other jasmonate biosynthesis genes was up-regulated compared with the control treatment. Subsequently, except for OsLOX1, OsLOX2, OsLOX4, OsLOX7, OsOPR1, OsOPR7, and OsOPR9, expression of all other jasmonate biosynthesis genes was up-regulated after 12 h of ACC treatment compared with the control treatment (Fig. 8). After 24 h of ACC treatment, 10 of the jasmonate biosynthesis genes were up-regulated by ACC treatment compared with the control treatment (Fig. 8). After 48 h of ACC treatment, expression of OsAOS2, OsOPR2, OsOPR5, OsOPR8, OsOPR10, OsACX2, and OsACX3, remained up-regulated by ACC treatment compared with the control treatment (Fig. 8). After 96 h of ACC treatment, only expression of OsOPR5, OsACX2, and OsACX3 was up-regulated by ACC treatment compared with the control treatment (Fig. 8).
The effect of IBU on ACC-induced jasmonate biosynthesis in the rice (‘9311’) seminal roots. (A) JA content. (B) JA-Ile content. Rice seedlings were incubated with ACC (9 μM) or ACC + IBU (5 μM) solutions. The JA and JA-Ile concentrations in rice seminal roots were determined after 48 h and 96 h of treatment. Data are presented as means ± SD calculated from three biological replicates and significant differences (P<0.05; one-way ANOVA followed by Duncan’s multiple range test) are indicated by different lower-case letters at each sampling time. ACC, 1-aminocyclopropane-1-carboxylate; CK, distilled water control; FW, fresh weight; IBU, ibuprofen; JA, jasmonic acid; JA-Ile, jasmonoyl-isoleucine.
Regulatory effects of ACC on the expression of jasmonate biosynthesis genes. Seminal roots of rice (‘9311’) seedling were collected for qRT–PCR analysis after 6, 12, 24, 48, and 96 h of different treatments. Data are presented as the means ± SD using three biological replicates with three technical replicates for the statistical and error range analyses. The relative expression of each jasmonate biosynthesis gene in control after 6 h of treatment was set as 1. Asterisks indicate significant differences (P<0.05; independent sample t-test) between different treatments at each sampling time. CK, distilled water control; ACC, 9 μM ACC. ACC, 1-aminocyclopropane-1-carboxylate.
Taken together, our results indicate that NaCl-induced ethylene biosynthesis does not directly inhibit rice seminal root growth, but indirectly, by promoting jasmonate biosynthesis. This evidence suggests that jasmonate, acting downstream of ethylene, inhibits seminal root growth in rice seedlings exposed to NaCl. Our results also indicate that NaCl treatment promotes jasmonate biosynthesis through an ethylene-dependent pathway. Additionally, NaCl treatment might also induce jasmonate biosynthesis through an ethylene-independent pathway because IBU treatment, rather than AVG treatment, completely rescued NaCl-induced growth inhibition of rice seminal roots. These findings strongly suggest that NaCl treatment promotes jasmonate biosynthesis through both ethylene-dependent and -independent pathways, and that jasmonate acts downstream of ethylene to inhibit rice seminal root growth under conditions of salt stress.
Salt-induced jasmonate restricts meristem cell proliferation in rice seminal roots
To clarify whether salt-induced jasmonate can inhibit seminal root length by restricting root meristem cell proliferation, we investigated the effects of 120 mM NaCl, 3 μM JA, and 120 mM NaCl + 5 μM IBU on the meristem cell proliferation rate, meristem size, meristem cell division activity, and the expression of OsPLT genes, and cell division-related genes.
Our results demonstrate that the cell proliferation rate was significantly (P<0.05) decreased by NaCl or JA treatment compared with the control, but the effect of NaCl was negated by the application of IBU (Fig. 9A). Additionally, the root meristem size and the meristem cell number were significantly (P<0.05) reduced by NaCl or JA treatment compared with the control (Fig. 9B–D). However, application of IBU rescued the NaCl-induced decreases in root meristem size and meristem cell number (Fig. 9B–D). Furthermore, compared with the control treatment, the expression of OsPLT1, OsPLT6, and cell division-related genes, such as OsCycB1;1, OsCDKB1;1, and OsCDKB2;1, was significantly (P<0.05) down-regulated by NaCl or JA treatment, and these effects were reversed by the application of IBU (Fig. 9E–I). All of these results indicate that NaCl-induced jasmonate production inhibits rice seminal root growth by restricting meristem cell proliferation.
Effects of NaCl-induced jasmonate production on root meristem cell proliferation in rice (‘9311’) seminal roots. (A) NaCl-induced jasmonate reduces the cell proliferation rate in the seminal root. Cell proliferation rate = root growth rate / mature cell length. (B–D) NaCl-induced jasmonate reduces meristem size and meristem cell number in rice seminal roots. Rice seminal root meristems were stained with 4’, 6-diamidino-2-phenylindole and visualized by confocal microscopy, and the meristem size and the meristem cell numbers were measured. Meristem cell number in the rice seminal root is expressed as the number of cortex cells from the quiescent center to the first elongated cell. Root meristem size was determined by measuring the length from the quiescent center to the first elongated epidermal cell. The distance between two arrows indicates the meristem size. Scale bar =100 μm. For (A–D), rice seedlings were incubated with different treatment solutions for 96 h, and the data are presented as means ±SD calculated from 10 biological replicates. (E–I) Comparison of the relative expression of OsPLT genes and meristem cell division-related genes between different treatments in rice seminal roots. The seminal roots were collected for qRT–PCR analysis after 6, 12, 24, 48, and 96 h of incubation. Data are presented as the means ±SD, using three biological replicates with three technical replicates for the statistical and error range analyses. The relative expression of OsCycB1;1, OsCDKB1;1, and OsCDKB2;1 was used as markers of the meristem cell division activities in the seminal roots. Different lower-case letters indicate significant differences (P<0.05; one-way ANOVA followed by Duncan’s multiple range test) between the different treatments at each sampling time. CK, distilled water control; JA, 3 μM JA; NaCl, 120 mM NaCl; NaCl + IBU, 120 mM NaCl + 5 μM IBU. IBU, ibuprofen; JA, jasmonic acid.
Salt-induced jasmonate inhibits cell elongation in rice seminal roots
To clarify whether salt-induced jasmonate inhibits seminal root length by inhibiting root cell elongation, we investigated the effects of NaCl, JA, and NaCl + IBU treatments on root cell length and the expression of cell elongation-related genes in rice seminal roots. Our results indicate that the average cell length was strongly reduced by NaCl or JA treatment compared with the control, while the NaCl-induced decrease in cell length could be rescued by IBU treatment (Fig. 10A, B). Furthermore, the expression of cell elongation-related genes, such as OsXTH1, OsXTH2, OsEXP12, OsEXP15, and OsEXPB7, was significantly (P<0.05) down-regulated by NaCl or JA treatment, and this effect was also reversed by IBU treatment (Fig. 10C–G). These results suggest that NaCl-induced jasmonate biosynthesis down-regulates the expression of cell-elongation related genes, which in turn contributes to the reduction in cell length in rice seminal roots under salt stress.
Effects of NaCl-induced jasmonate production on root cell elongation in rice (‘9311’) seminal roots. (A, B) NaCl-induced jasmonate reduces cell lengths in rice seminal roots. After 96 h of treatment, the mature zones of rice seminal roots were stained with propidium iodide and visualized using confocal microscopy, and the lengths of cells in the mature zones were measured. Data are presented as means ±SD calculated from 10 biological replicates. Scale bar =100 μm. (C–G) Comparison of the relative expression of root cell elongation-related genes between three different treatments in rice seminal roots. The seminal roots were collected for qRT–PCR analysis after 6, 12, 24, 48, and 96 h of incubation. Data are presented as the means ±SD, using three biological replicates with three technical replicates for the statistical and error range analyses. Different lower-case letters indicate significant differences (P<0.05; one-way ANOVA followed by Duncan’s multiple range test) between the treatments at each sampling time. CK, distilled water control; JA, 3 μM JA; NaCl, 120 mM NaCl; NaCl + IBU, 120 mM NaCl + 5 μM IBU. IBU, ibuprofen; JA, jasmonic acid.
Salt-induced growth inhibition of the rice seminal root is decreased in the jasmonate-deficient mutant
To further confirm the growth inhibition of salt-induced jasmonate on rice seminal root growth, the sensitivity of Nihonmasari (WT) and cpm2 (jasmonate-deficient mutant) to salt stress was compared in this study. The results reveal that the seminal root growth of WT, but not of cpm2, were significantly (P<0.05) inhibited after treatment with low concentrations (30 mM and 60 mM) of NaCl (Fig. 11A–C). Although treatment with relatively high concentrations (90 mM and 120 mM) of NaCl significantly (P<0.05) inhibited the seminal root growth of the WT and the cpm2 seedlings, the inhibition in cpm2 was obviously lower than that of WT (Fig. 11A–C). For example, after 96 h of NaCl (120 mM) treatment, the seminal root length of WT was decreased by 2.1-fold, whereas the seminal root length of cpm2 was only decreased by 1.2-fold (Fig. 11C). Moreover, the effects of NaCl on jasmonate biosynthesis, meristem cell proliferation rate, meristem size, meristem cell number, and the average cell length were weakened in cpm2 compared with WT (Fig. 11D–I; Supplementary Fig. S3). These results indicate that the jasmonate-deficient mutant cpm2 is hyposensitive to salt stress, and that salt-induced jasmonate biosynthesis plays a critical role in inhibiting rice seminal root growth.
Comparison of the sensitivity to NaCl between the jasmonate-deficient mutant (cpm2) and its WT (Nihonmasari). (A) Seminal root phenotypes of rice seedlings. Scale bar =5 cm. (B) Statistical analysis of the seminal root length of NaCl-treated rice seedlings. (C) Relative seminal root length of NaCl-treated rice seedlings. For (A–C), the rice seedlings were treated with 0 (control), 30, 60, 90, and 120 mM NaCl for 96 h, after which the lengths of the seminal roots were measured and the seedlings were photographed. The relative seminal root length is presented with the data from (B) of treatment/control. (D) Comparison of the cell proliferation rate in the seminal roots between the WT and the cpm2 seedlings under NaCl treatment. Cell proliferation rate = root growth rate / mature cell length. (E–G) Comparison of meristem size and meristem cell number in the seminal roots between the WT and the cpm2 seedlings under NaCl treatment. After 96 h of incubation, rice seminal root meristems were stained with 4’, 6-diamidino-2-phenylindole and visualized by confocal microscopy, and the meristem size and meristem cell number were measured. Meristem cell number in the rice seminal roots is expressed as the number of cortex cells from the quiescent center to the first elongated cell. Root meristem size was determined by measuring the length from the quiescent center to the first elongated epidermal cell. The distance between two arrows indicates the meristem size. Scale bar =100 μm. (H, I) Comparison of cell lengths in the seminal roots between the WT and the cpm2 seedlings under NaCl treatment. The mature zones of rice seminal roots were stained with propidium iodide and visualized using confocal microscopy, and the lengths of cells in the mature zones were measured. Scale bar =100 μm. For (D–I), rice seedlings were incubated with CK or NaCl (120 mM) for 96 h. Data are presented as means ±SD calculated from 10 biological replicates and significant differences (P<0.05; one-way ANOVA followed by Duncan’s multiple range test) are indicated by different lower-case letters. CK, distilled water control; WT, wild-type.
Discussion
Both ethylene and jasmonate are involved in salt-induced growth inhibition
In this study, our results indicate that NaCl inhibits rice seminal root growth in a dose-dependent manner, and thus the higher concentrations of NaCl resulted in shorter rice seminal roots (Fig. 1). Additionally, the results show that NaCl treatment not only increases ethylene content but also increases JA and JA-Ile content in the rice seminal root, and application of AVG and IBU inhibited ethylene and jasmonate biosynthesis, respectively, which in turn alleviated the NaCl-induced growth inhibition of rice seminal roots (Figs 2, 4). Moreover, the sensitivity to NaCl was decreased in the jasmonate-deficient mutant, cpm2 (Fig. 11). These results confirmed that both ethylene and jasmonate are involved in NaCl-induced growth inhibition of rice seminal roots.
Spatiotemporal specificity of ethylene and jasmonate biosynthesis genes in response to salt stress
Although the short term (≤3 h) response patterns of some ethylene biosynthesis genes, including the OsACS and OsACO genes, to salt stress have been investigated in rice roots (Qin et al., 2019), the long term expression patterns of these genes, as well as the OsSAMS genes, to salt stress are unknown in rice seminal roots. In this study, we investigated the long term (6–96 h) expression patterns of genes involved in ethylene biosynthesis, including the OsACS, OsACO and OsSAMS genes, in response to salt stress. Our results show that after 12 h of treatment, expression of all of the ethylene biosynthesis genes were up-regulated by NaCl (Fig. 3). In contrast, it has been previously shown that over the short term (≤3 h), some of the ethylene biosynthesis genes in rice seminal roots did not show changes in expression in response to NaCl treatment (Qin et al., 2019). However, only two of the ethylene biosynthesis genes, OsACS5 and OsACO2, were still up-regulated after 96 h of NaCl treatment (Fig. 3). Specifically, although short term (≤3 h) NaCl treatment had no effect on OsACO2 expression (Qin et al., 2019), we have shown here that long term (6–96 h) NaCl treatment significantly up-regulated the expression of OsACO2 (Fig. 3). These findings suggest that salt stress promotes ethylene biosynthesis in rice seminal roots by up-regulating the expression of ethylene biosynthesis genes, with different exposure times to salt accompanied by different expression patterns of ethylene biosynthesis genes.
It has been reported that NaCl treatment strongly up-regulates the expression of OsOPR genes such as OsOPR1, OsOPR3, and OsOPR7 in rice leaves (Jang et al., 2009), which in turn may contribute to salt-induced jasmonate biosynthesis (Hazman et al., 2019). In rice seminal roots, however, the response patterns of jasmonate biosynthesis genes to salt stress are still unclear. The results of our study show that the expression of jasmonate biosynthesis pathway genes, such as the OsLOX, OsAOS, OsAOC, OsOPR, and OsACX genes, was up-regulated by NaCl treatment, which in turn contributed to NaCl-induced jasmonate biosynthesis in the rice seminal root (Figs 4, 5). These results indicate that there are significant differences in the expression patterns of jasmonate biosynthesis genes between the different organs such as seminal roots and leaves, in response to salt stress in rice.
Salt stress promotes jasmonate biosynthesis to inhibit rice seminal root growth through both ethylene-dependent and -independent pathways
Plants have evolved sophisticated mechanisms to integrate exogenous and endogenous signals to adapt to the changing environment. Both ethylene and jasmonate regulate plant growth, development, and defense (Pedranzani et al., 2003; Tani et al., 2008; Lin et al., 2009; Seo et al., 2011; Wang et al., 2013; Wasternack and Hause, 2013; Song et al., 2014; Zhao et al., 2014; Valenzuela et al., 2016; Dubois et al., 2018; Hazman et al., 2019). In addition to antagonistic regulation of apical hook curvature, mesocotyl elongation, gene expression, and plant defense against insect attack (Song et al., 2014; Zhang et al., 2014; Xiong et al., 2017), ethylene and jasmonate act synergistically to regulate root hair development and resistance to necrotrophic fungi (Zhu et al., 2006; Pré et al., 2008). However, the molecular mechanisms connecting ethylene and jasmonate biosynthesis remain unclear in rice seminal roots under salt stress.
The results of our study have shown that although both AVG and IBU can alleviate the NaCl-induced growth inhibition of rice seminal roots, and had no additive effect on mitigating this growth inhibition (Fig. 6). Moreover, ACC-induced jasmonate production and growth inhibition in the rice seminal root were alleviated by IBU treatment (Figs 6; 7), whereas AVG treatment did not alleviate, but strengthened the JA-induced grow inhibition (Fig. 6). Previous studies revealed that a certain threshold level of ethylene is required to maintain rice seminal root growth, although supraoptimal concentrations of ethylene inhibited rice seminal root growth (Yin et al., 2011). Moreover, application of exogenous jasmonate could strongly inhibit ethylene biosynthesis (Kępczyński et al., 1999). Thus, we presume that application of exogenous JA may inhibit ethylene biosynthesis in the rice seminal root, and JA-induced growth inhibition of rice seminal roots may be strengthened by causing endogenous ethylene to drop below the certain threshold level through the application of AVG. These results demonstrate that, although NaCl treatment promoted ethylene biosynthesis in the seminal root, NaCl-induced ethylene production did not inhibit seminal root growth directly. Rather, seminal root growth was inhibited indirectly by ethylene by promoting jasmonate biosynthesis via up-regulation of the expression of jasmonate biosynthesis genes, including the OsLOX, OsAOS, OsAOC, OsOPR, and OsACX (Figs 6–8). Moreover, our results indicate that NaCl treatment can also promote jasmonate biosynthesis through an ethylene-independent pathway, because IBU rather than AVG completely rescued NaCl-induced jasmonate biosynthesis and growth inhibition of rice seminal roots (Fig. 6; Supplementary Fig. S2); however, the underlying mechanism of the ethylene-independent pathway is unclear and needs further investigation.
All of these results suggest that salt stress promotes jasmonate biosynthesis through ethylene-dependent and -independent pathways, and that jasmonate acts downstream of ethylene to inhibit the growth of rice seminal roots in response to salt stress. Our results are consistent with those of a previous report showing that ethylene may promote jasmonate biosynthesis in Arabidopsis, and thus applications of jasmonate biosynthesis inhibitors, including IBU and salicylhydroxamic acid, suppressed ethylene-induced root hair formation and reduced the number of root hairs in seedlings of the ethylene over-producing mutant eto1-1 (Zhu et al., 2006). Our results are also supported by the recent finding in maize that application of ethephon, an ethylene releasing compound, increased jasmonate content and inhibited mesocotyl elongation in the dark (Liu et al., 2020). In contrast, Xiong et al. (2017) reported that ethylene promoted mesocotyl elongation by inhibiting jasmonate biosynthesis in the dark. The variety of ethylene functions reveal that this hormone plays different roles in different organs, different species, and under different environmental conditions. This has been confirmed by previous findings showing that ethylene promotes the formation of adventitious roots and root hairs, but also inhibits root growth (Pitts et al., 1998; Zhu et al., 2006; Druege et al., 2014), and that in rice, ethylene significantly suppresses shoot growth under low relative humidity but promotes shoot growth under high relative humidity and under submergence stress (Azuma et al., 2007; Jackson, 2008).
Salt-induced jasmonate inhibits rice seminal root growth by restricting root meristem cell proliferation and root cell elongation
In this study, our objective was to explore the influence of salt-induced jasmonate production on meristem cell proliferation and cell elongation and their effects on growth of the rice seminal root. Our results indicate that salt-induced jasmonate reduced the root meristem cell number by down-regulating the transcription of OsPLT genes, which in turn reduced meristem cell proliferation rate and inhibited growth of the rice seminal root (Fig. 9). Moreover, our results also suggest that salt-induced jasmonate production can restrict meristem cell proliferation rate in the rice seminal root by inhibiting cell division activity via down-regulated expression of cell division-related genes (Fig. 9G–I). Additionally, compared with WT, the inhibitory effects of salt on meristem cell proliferation rate, the meristem size and the meristem cell number were weakened in the seminal roots of the jasmonate-deficient mutant cpm2 (Fig. 11). Our results are supported by previous findings in Arabidopsis that jasmonate inhibits primary root growth by inhibiting meristem cell division activity (Zhang and Turner, 2008; Chen et al., 2011). In addition, our results are consistent with those from a previous report in rice showing that application of exogenous JA reduced root meristem size and down-regulated the transcription of root cell division-related genes (Toda et al., 2013). Overall, our results suggest that salt-induced jasmonate production decreases root meristem cell number and cell division activity by down-regulating the transcription of OsPLT genes and cell division-related genes, respectively, which in turn inhibit seminal root growth by restricting root meristem cell proliferation.
Seminal root growth is positively controlled by cell elongation in the seminal root (Takatsuka and Umeda, 2014; X. Zou et al., 2018). It has been previously reported that the expression of cell-elongation-related genes, such as the OsEXP and OsXTH genes, is positively correlated with the growth rate of rice roots (Yi and Kende, 2002; X. Zou et al., 2018; Q. Wang et al., 2020). Here, we provide evidence that NaCl and JA treatment significantly down-regulate the expression of OsEXP and OsXTH and inhibit cell elongation in the rice seminal root, and that these these regulatory effects can be restored by IBU treatment (Fig. 10). Moreover, root cell elongation in the jasmonate-deficient rice mutant is hyposensitive to salt stress (Fig. 11). Our results are supported by previous studies showing that root cell elongation in jasmonate-deficient or jasmonate-insensitive Arabidopsis mutants was insensitive to salt stress (Valenzuela et al., 2016), and that application of exogenous JA inhibited root cell elongation and inhibited rice seminal root growth (Toda et al., 2013). These findings suggest that salt-induced jasmonate can inhibit rice seminal root growth by inhibiting cell elongation, by down-regulating the expression of OsEXP and OsXTH genes.
The results of our study suggest that salt stress promotes jasmonate biosynthesis through both ethylene-dependent and -independent pathways in the rice seminal root (Fig. 12). Salt-induced jasmonate production inhibits growth of the rice seminal root not only by restricting root meristem cell proliferation via the down-regulated expression of OsPLT and cell division-related genes, but also by inhibiting root cell elongation by down-regulating the expression of OsEXP and OsXTH genes. Therefore, we conclude that salt stress promotes jasmonate biosynthesis through ethylene-dependent and -independent pathways, and jasmonate acts downstream of ethylene to inhibit rice seminal root growth under salt stress conditions (Fig. 12).
Suggested model for the regulation of seminal root responses to salt stress in rice seedlings. Salt stress promotes ethylene biosynthesis by up-regulating expression of ethylene biosynthesis genes (OsSAMS1/2/3, OsACS1/2/4/5, and OsACO1/2/4/5/7), whereas salt-induced ethylene inhibits growth of the rice seminal root by promoting jasmonate biosynthesis via up-regulated expression of jasmonate biosynthesis genes (OsLOX1/5/6, OsAOS1/2, OsAOC, OsOPR2/3/4/5/6/8/9/10, and OsACX1/2/3). Salt stress also promotes jasmonate biosynthesis through an ethylene-independent pathway. Salt-induced jasmonate inhibits growth of the rice seminal root not only by restricting root meristem cell proliferation via the down-regulated expression of OsPLT (OsPLT1/2) and cell division-related genes (OsCycB1;1, OsCDKB1;1, and OsCDKB2;1), but also by inhibiting root cell elongation by down-regulating the expression of OsEXP (OsEXP12/15, and OsEXPB7) and OsXTH (OsXTH1/2) genes. The dashed line indicates the predicted ethylene-independent pathway. Arrows indicate positive regulation and bars denote negative effects.
Supplementary data
The following supplementary data are available at JXB online.
Table S1. Primers used in the quantitative real-time PCR (qRT-PCR) analysis.
Fig. S1. The relative expression of OsACTIN under all conditions described in this study.
Fig. S2. The effect of AVG on NaCl-induced jasmonate biosynthesis in rice (‘9311’) seminal roots.
Fig. S3. The effect of NaCl on jasmonate biosynthesis in rice seminal roots of a jasmonate-deficient mutant (cpm2) and its WT (‘Nihonmasari’).
Abbreviations:
- ACC
1-aminocyclopropane-1-carboxylate
- ACO
1-aminocyclopropane-1-carboxylate oxidase
- ACS
1-aminocyclopropane-1-carboxylate synthase
- ACX
acyl-CoA oxidase
- AOC
allene oxide cyclase
- AOS
allene oxide synthase
- AVG
aminoethoxyvinylglycine
- DAPI
4’,6-diamidino-2-phenylindole
- EXP
expansin
- FW
fresh weight
- IBU
ibuprofen
- JA
jasmonic acid
- JA-Ile
jasmonoyl-isoleucine
- LOX
lipoxygenase
- OPR
cis-12-oxo-phytodienoic acid reductase
- PLT
PLETHORA
- SAM
S-adenosylmethionine
- SAMS
S-adenosylmethionine synthase
- XTH
xyloglucan endotransglucosylase/hydrolase
Acknowledgements
We thank Dr. Michael Riemann (Botanical Institute, Karlsruhe Institute of Technology, Germany) for providing seeds of the jasmonate-deficient mutant cpm2 and its WT rice (Oryza sativa L. ssp. japonica) ‘Nihonmasari’. This research was supported by the National Key Research and Development Program of China (No. 2016YFD0300102), the Fundamental Research Funds for the Central Universities (No. 2662018PY076), the Innovative Scientific and Technological Talents in Henan Province (20HASTIT041 to Z.H.) and the 111 Project #D16014.
Author contributions
LL and XZ performed most of the experiments; YZ, XW, ZH, JZ, XL, and ZK took part in the experiments; XZ and LL analyzed the data; CY and YL designed the experiments; LL, XZ and CY wrote the manuscript; CY, YL, XW, and ZH revised the manuscript.
Conflict of interest
The authors declare that they have no conflicts of interest.
Data availability
All data supporting the findings of this study are available within the paper and within its supplementary data published online.
References
Author notes
X.Z and L.L contributed equally to this work.
Comments