OsJAZ11 regulates phosphate starvation responses in rice

Abstract

OsJAZ11 regulates phosphate homeostasis by suppressing jasmonic acid signaling and biosynthesis in rice roots. Jasmonic Acid (JA) is a key plant signaling molecule which negatively regulates growth processes including root elongation. JAZ (JASMONATE ZIM-DOMAIN) proteins function as transcriptional repressors of JA signaling. Therefore, targeting JA signaling by deploying JAZ repressors may enhance root length in crops. In this study, we overexpressed JAZ repressor OsJAZ11 in rice to alleviate the root growth inhibitory action of JA. OsJAZ11 is a low phosphate (Pi) responsive gene which is transcriptionally regulated by OsPHR2. We report that OsJAZ11 overexpression promoted primary and seminal root elongation which enhanced Pi foraging. Expression studies revealed that overexpression of OsJAZ11 also reduced Pi starvation response (PSR) under Pi limiting conditions. Moreover, OsJAZ11 overexpression also suppressed JA signaling and biosynthesis as compared to wild type (WT). We further demonstrated that the C-terminal region of OsJAZ11 was crucial for stimulating root elongation in overexpression lines. Rice transgenics overexpressing truncated OsJAZ11ΔC transgene (i.e., missing C-terminal region) exhibited reduced root length and Pi uptake. Interestingly, OsJAZ11 also regulates Pi homeostasis via physical interaction with a key Pi sensing protein, OsSPX1. Our study highlights the functional connections between JA and Pi signaling and reveals JAZ repressors as a promising candidate for improving low Pi tolerance of elite rice genotypes.

Keywords: JASMONATE ZIM-DOMAIN; Jasmonic acid; OsSPX1; Phosphate deficiency; Root length.

Fig. 1

OsJAZ11 is induced by Pi deficiency. a Expression pattern of OsJAZ11 in shoot (PB1.S) and root (PB1.R) of low-Pi-sensitive rice genotype PB1 under +P and −P conditions. Signal intensities were retrieved from previous microarray dataset (Mehra et al. 2016) submitted in Gene Expression Omnibus (GEO) database (accession no. GSE74795). b RT-qPCR of OsJAZ11 in roots and shoots of 30-day-old PB1 plants under +P (320 μM NaH₂PO₄) and −P (1 μM NaH₂PO₄) conditions. Each bar is the mean of three independent replicates with standard error. Significant differences between +P vs −P treatments were evaluated by Student’s t test. Asterisks * and *** indicate P values, ≤ 0.05 and 0.001, respectively (n = 3). c EMSA assays showing physical interaction of OsJAZ11 promoter with OsPHR2-6XHIS at P1BS cis-element. Interactions were performed using 50 ng of DIG-labeled OsJAZ11 promoter probe (− 1057 to − 1028 bp) flanking P1BS (lanes 1–6). Interactions were carried out with different concentrations of OsPHR2-6XHIS (100 ng, 200 ng, 300 ng and 400 ng in lane 2, 3, 4 and 5, respectively). In lane 6, for competition assays, 200 ng of OsPHR2-6XHIS and 200-fold excess of unlabeled OsJAZ11 promoter was used. In lanes 7–10, 50 ng of OsJAZ11 promoter probe was used with mutations in P1BS element (mP1BS). 100 ng, 200 ng, 300 ng and 400 ng of OsPHR2-6XHIS was used in lanes 7, 8, 9 and 10, respectively

Fig. 2

Total phosphorus and JA content of WT and OsJAZ11 transgenics. a Total phosphorus content in shoots of WT and OsJAZ11 transgenics. Each bar represents mean of ten independent replicates with standard error (n = 10). b JA content/g root fresh weight of WT and OsJAZ11 transgenics. Plants were grown under –P conditions for 30 days. Each bar represents mean of six independent replicates with standard error (n = 6). Significant differences between WT and transgenics were evaluated by Student’s t test. Asterisks; * and ** indicate P values, ≤ 0.05 and 0.01, respectively

Fig. 3

Overexpression transgenics of OsJAZ11 developed longer roots than WT. Root phenotype of 30-day-old OsJAZ11 overexpression (OE) lines (a, d) and silencing RNAi (Ri) lines (b, e) compared to WT under +P (a, b) and −P (d, e) conditions. For imaging, three representative plants of each line were stacked together. Scale bar = 10 cm. Quantitation of root lengths of WT, OE and Ri lines under +P conditions (c) and −P (f) conditions. Each bar represents mean of ten biological replicates with standard error. Significant differences between WT and transgenics were evaluated by Student’s t test. Asterisks; *, ** and *** indicate P values, ≤ 0.05, 0.01 and 0.001, respectively (n = 10)

Fig. 4

Effect of OsJAZ11 on root traits. a–d Average lateral root length, total number of lateral roots per plant, seminal root length and total seminal root length per plant in 15-day-old WT and OsJAZ11 transgenics under +P and −P conditions. e Root phenotypes of 15-day-old WT and OsJAZ11 transgenics under +P and −P conditions. White line at bottom of each image represents scale of 1 cm. Each bar represents mean from four replicates with standard error. Significant differences between WT and transgenics were evaluated by Student’s t test. Asterisks; * and ** indicate P values, ≤ 0.05 and 0.01, respectively (n = 4)

Fig. 5

C-terminal domain of OsJAZ11 regulates root growth. Plant phenotype of 30-day-old OsJAZ11-GUS (a, d), OsJAZ11ΔC-GUS (b, e) translational reporter lines and WT under +P (a, b) and −P (d, e) conditions. For imaging three representative plants of each line were stacked together. Scale bar = 10 cm. c Quantitation of root lengths of WT, OsJAZ11-GUS and OsJAZ11ΔC-GUS lines under +P conditions. f Quantitation of root lengths of WT, OsJAZ11-GUS and OsJAZ11ΔC-GUS lines under −P conditions. Each bar represents mean of ten biological replicates with standard error. Significant differences between WT and transgenics were evaluated by Student’s t test. Asterisks; *, ** and *** indicate P values, ≤ 0.05, 0.01 and 0.001, respectively (n = 10). g-h Fluorometric quantitation of GUS activity in 30-day-old OsJAZ11-GUS (L3, L12, L14) and OsJAZ11ΔC-GUS (L1, L2) transgenics under +P and –P conditions. Each bar represents mean of six biological replicates with standard error. Different letters on top of each bar denotes significant differences determined by one-way ANOVA followed by Duncan’s multiple comparison test (α < 0.05) (n = 6)

Fig. 6

OsJAZ11 interacts with OsSPX1 to regulate Pi homeostasis. a Yeast two-hybrid assay showing interaction of OsJAZ11 (pBD-OsJAZ11) with OsSPX1 (pAD-OsSPX1). Serial dilutions of Y2H Gold strain co-transformed with AD (prey) and BD (bait) clones were spotted on DDO (SD-Leu/-Trp), TDO (SD- Leu/-Trp/-His), QDO (SD- Leu/-Trp/-His/-Ade) and QDO/A/X (QDO + Aureobasidin A + X-α-Gal). b GST pull-down assay showing interaction of OsJAZ11-GST and OsSPX1-6XHis. GST (negative control) and OsJAZ11-GST were immobilized on glutathione-agarose beads and incubated with OsSPX1-6XHIS (input protein). Pulled-down complexes were detected by immunoblotting using anti-GST and anti-HIS antibodies. c EMSA assay showing no effect of OsJAZ11 on OsSPX1-mediated inhibition of OsPHR2 binding to P1BS. OsPHR2-6XHIS (0.7 μg) and 50 ng DIG-labeled P1BS probe was incubated with either 7.5 μg SPX-6HIS (lane 3), SPX-6XHIS + 7.5 μg OsJAZ11-GST (lane 4) or SPX-6XHIS + 7.5 μg GST (lane 5). d–g Relative expression levels of OsSPX1, OsSPX2, OsSPX3 and OsSPX5 in roots of 30-day-old WT and OsJAZ11 transgenics. Each bar is the mean of three independent replicates with standard error. Different letters on top of each bar denote significant differences determined by one-way ANOVA followed by Duncan’s multiple comparison test (α < 0.05) (n = 3)

Fig. 7

Schematic model illustrating mechanisms of action of OsJAZ11 in rice. OsJAZ11 interacts with NINJA and TOPLESS (TPL) co-repressors. Upon its overexpression, OsJAZ11 blocks downstream transcription factors such as OsMYC2. Repressor activity of OsJAZ11 suppresses the expression of OsMYC2-regulated JA-responsive genes. This consequently increases root growth in overexpression lines. Enhanced root growth favors higher Pi acquisition and suppression of phosphate starvation response (PSR) in overexpression lines. OsJAZ11 also regulates Pi homeostasis through OsSPXs. OsJAZ11 interacts with OsSPX1 and also alters expression of OsSPXs in overexpression lines under Pi deficiency. Other downstream component of OsJAZ11 signaling pathway such as OsMYC2 interacts with key Pi homeostasis proteins such as OsSPX3 to regulate Pi homeostasis. All these mechanisms together support relatively enhanced plant growth in OsJAZ11 overexpression lines under Pi deficiency