OsJAZ11 regulates spikelet and seed development in rice

Abstract

Seed size is one of the major determinants of seed weight and eventually, crop yield. As the global population is increasing beyond the capacity of current food production, enhancing seed size is a key target for crop breeders. Despite the identification of several genes and QTLs, current understanding about the molecular regulation of seed size/weight remains fragmentary. In the present study, we report novel role of a jasmonic acid (JA) signaling repressor, OsJAZ11 controlling rice seed width and weight. Transgenic rice lines overexpressing OsJAZ11 exhibited up to a 14% increase in seed width and ~30% increase in seed weight compared to wild type (WT). Constitutive expression of OsJAZ11 dramatically influenced spikelet morphogenesis leading to extra glume-like structures, open hull, and abnormal numbers of floral organs. Furthermore, overexpression lines accumulated higher JA levels in spikelets and developing seeds. Expression studies uncovered altered expression of JA biosynthesis/signaling and MADS box genes in overexpression lines compared to WT. Yeast two-hybrid and pull-down assays revealed that OsJAZ11 interacts with OsMADS29 and OsMADS68. Remarkably, expression of OsGW7, a key negative regulator of grain size, was significantly reduced in overexpression lines. We propose that OsJAZ11 participates in the regulation of seed size and spikelet development by coordinating the expression of JA-related, OsGW7 and MADS genes.

Keywords: JASMONATE ZIM‐DOMAIN; Jasmonic acid; seed weight; seed width.

FIGURE 1

OsJAZ11 regulates seed width and weight. (a) Quantitation of GUS activity by MUG assay in pCAMV35S:OsJAZ11‐GUS (L14) and pCAMV35S:OsJAZ11ΔC‐GUS (L2). GUS activity was determined in different panicle (P0 to P6) and seed (S1 to S5) developmental stages (n = 6). P0 refers to panicle length up to 0.5 mm or shoot apical meristem. (b–d) Comparison of seed phenotype of WT and OsJAZ11 overexpression lines (pZmUbi1:OsJAZ11; OE3/6/8), RNAi lines (Ri1/2), OsJAZ11‐GUS lines (L12/L14), and OsJAZ11ΔC‐GUS lines (L1/2). Bar = 1 cm. (e–g) Quantitation of seed width in WT and OsJAZ11 transgenics. Each bar represents average of 100 seeds. (h) One‐hundred seed weight (in grams) of WT and OsJAZ11 transgenics (n = 4). The same WT control has been used in b–d and e–g for comparison between WT and transgenics. Error bar represents standard error. Significant differences between WT and transgenics were determined using Student's t test. *, **, and *** represent p values ≤.05, .01, and .001, respectively

FIGURE 2

Phenotypes of spikelet in OsJAZ11 overexpression transgenics. (a, b) Normal spikelet morphology of WT. (c) Abnormal spikelets of OsJAZ11 OE line showing extra glume‐like structure (indicated by arrow), (d) missing palea, (e, f) extra lemma, (g) fused anthers, (h) misplaced stamen protruding out of spikelet, (i) misplaced carpel protruding out of spikelet, (j) stamen and carpel outside spikelet, (k) supernumerary stamens and carpels, (l) curled anthers, (m) spikelet within spikelet, and (n) multiple stigma. (o–v) OsJAZ11 OE spikelets with abnormal number of stamens (N^s), carpels (N^c) and extra glume like structures (N^eg). Bar = 1 mm

FIGURE 3

Overexpression of OsJAZ11 suppresses JA signaling in rice. (a) Relative expression levels of OsMYC2, (b) OsJAZ1, (c) OsJAZ4, (d) OsJAZ5, (e) OsJAZ6, (f) OsJAZ8, (g) OsJAZ9, (h) OsJAZ12, and (i) OsJAZ15 in WT and OsJAZ11 OE lines. Expression levels were analyzed in spikelets at P4 stage of development (n = 3). Error bar represents standard error. Significant differences between WT and transgenics were determined using Student's t test. * and ** represent p values ≤.05 and .01, respectively

FIGURE 4

Overexpression of OsJA11 increased expression of JA biosynthesis genes. Expression profile of (a) OsAOS1, (b) OsAOS2, (c) OsJMT1, (d) OsLOX1, (e) OsOPR1, (f) OsOPR2, (g) OsOPR4, (h) OsOPR5, and (i) OsOPR6 in WT and OsJAZ11 OE lines. Expression levels were analyzed in spikelets at P4 stage of development (n = 3). Error bar represents standard error. Significant differences between WT and transgenics were determined using Student's t test. *, **, and *** represent p values ≤.05, .01, and .001, respectively

FIGURE 5

OsJAZ11 overexpressing lines showed increased JA biosynthesis. (a, b) JA content/g fresh weight of P4 spikelets of WT, OsJAZ11 OE, and Ri lines (n = 9). (c, d) JA content/g fresh weight of S2 seeds of WT, OsJAZ11 OE, and Ri lines (n = 6). Each white line in a bean represents data from single plant. Black line in each bean represents average for each line. p values were determined using Student's t test

FIGURE 6

OsJAZ11 interacts with OsMADS29 and OsMADS68. (a) Yeast two‐hybrid interaction assays between pBD‐OsJAZ11 and pAD‐OsMADS. Yeast cells co‐transformed with AD (prey) and BD (bait) plasmids were spotted on DDO medium (SD‐Leu/‐Trp) and QDO medium (SD‐Leu/‐Trp/‐His/‐Ade). pBD and pAD represent empty BD (pGBKT7) and AD (pGADT7) vector, respectively. Interaction between pAD‐T‐Antigen and pBD‐p53 was used as positive control, whereas interaction between pAD‐T‐Antigen and pBD‐Lam was used as a negative control. (b) GST pull‐down assay showing interaction of OsJAZ11‐GST and OsMADS68‐6XHis. (c) GST pull‐down assay showing interaction of OsJAZ11‐GST and OsMADS29‐6XHis

FIGURE 7

OsJAZ11 overexpression suppressed expression of OsGW7. Relative expression levels of (a) OsGW2, (b) OsGW7, (c) OsGW8, and (d) OsGS5 in P4 spikelets of WT, OsJAZ11 OE, and Ri lines (n = 3). Error bar represents standard error. Significant differences between WT and transgenics were determined using Student's t test. *, **, and *** represent p values ≤.05, .01, and .001, respectively. (e) Expression profile of OsGW7 in response to JA treatment. Expression pattern was retrieved from microarray database RiceXPro (Rice Expression Profile Database) version 3.0

FIGURE 8

OsJAZ11 regulates seed width and spikelet morphogenesis in rice. Schematic illustration of mode of action of OsJAZ11. Overexpression of OsJAZ11 blocks OsMYC2 regulated expression of other JAZ repressors and E‐class MADS. Suppressed JA signaling induces JA biosynthesis in spikelets (feedback loop) leading to abnormal spikelet morphogenesis. Increased hull size in overexpression lines allows wider seed growth. Overexpression of OsJAZ11 also indirectly downregulates OsGW7 leading to increased seed width