OsROXY2 Regulates Stamen Number Through Interaction with OsbZIP47 in Rice

Abstract

Precise regulation of floral primordia initiation is essential for normal flower development. However, the mechanisms regulating floral primordia initiation (PI) are complex and poorly understood. Herein, we identified a natural mutant in rice, stamen less (sl), which develops florets with reduced stamen number and no carpel due to defects in stamen and carpel PI. STAMENLESS (SL) encodes the CC-type glutaredoxin OsROXY2 and is involved in the regulation of stamen PI. OsROXY1, the closest homolog of OsROXY2, showed no function in stamen PI regulation. The osroxy1 single mutant showed normal reproductive development, while the floret phenotypes of osroxy1/2 double mutant were comparable to those of osroxy2 mutant. The TGA transcription factor OsbZIP47 showed a strong interaction with OsROXY2, and the two genes exhibited overlapping subcellular localizations and expression patterns during flower development. The number of stamens in the osbzip47 mutant was increased to seven (around 35%), indicating that OsbZIP47 is a negative regulator of stamen PI, in contrast to OsROXY2. Taken together, our results reveal that OsROXY2 regulates stamen number via interaction with OsbZIP47, indicating GRX-TGA-mediated floral organ number regulation mechanism is conserved in monocots and eudicots.

Keywords: OsROXY2; OsbZIP47; Floret development; Rice; Stamen number.

Fig. 1

Floret phenotypes of the wild type (C418) and sl mutant A Floret of C418. B1 sl floret showing five stamens and one carpel. B2 sl floret with four stamens and no carpel. B3 sl floret with three stamens and no carpel. Relative ratio of stamen number C and carpel number D in wild-type (C418) and sl mutant. E Panicle morphology of C418 and sl mutant at 35 days after pollination. F Seed setting rate of C418 and sl mutant. G– J SEM images of young floret. G C418 floret with six stamen primordia at the end of the stamen initiation stage. H Two stamen primordia formed in sl at the beginning of the stamen initiation stage. I Two stamen primordia formed in sl in the middle of the stamen initiation stage. J Three stamen primordia and one carpel primordium formed in sl at the end of the stamen initiation stage. Red, black, blue and white arrows indicate stamens, carpel, absent carpel, and stamen primordium, respectively. l, lemma; p, palea. Scale bars represent 2 mm in (A and B, and 25 μm in G to I)

Fig. 2

Map-based cloning of SL A Mapping of the SL gene. B Pin-shaped palea was not rescued in sl flowers complemented with empty vector pCAMBIA1301. C Reduced stamens and absent carpel were not rescued in sl flowers complemented with pCAMBIA1301; E)Palea was normal in sl florets transformed with the complementation vector pROXY2. F The sl floret transformed with the complementation vector pROXY2 had six stamens and one carpel. (D) and (G) are magnifications of (C) and (F), respectively. Relative ratios of stamen number (H) and carpel number (I) in sl flowers transformed with the empty vector pCAMBIA1301 and complementation vector pROXY2, respectively. Red arrows indicate stamens, white arrows indicate no carpels and blue arrows indicate carpels. Scale bar = 1 mm

Fig. 3

Expression and subcellular localization of OsROXY2. A Relative expression levels of OsROXY1 and OsROXY2 in different tissues of rice. B–E Subcellular localization of OsROXY2. Scale bar = 5 μm. (B) OsROXY2-GFP fusion protein. C mCherry (nuclear localization marker). D Image of bright field. E Merged image. F-I In situ hybridization of OsROXY2 during panicle development. Red arrows indicate detected signals. Scale bar = 100 μm. F Early stage of secondary branch development. GLate stage of secondary branch development and (H) spikelet development stage. I Negative control using a sense probe of OsROXY2

Fig. 4

Phenotypes of osroxy1, osroxy2 and osroxy1/2 double mutant A Gene structure of OsROXY1 and B OsROXY2. C The osroxy1/2 double mutant was generated by targeting the same sequence in both OsROXY1 and OsROXY2. Solid boxes represent the coding sequence (CDS), and white boxes represent UTR. Mutation sites in osroxy1, osroxy2 and osroxy1/2 double mutant are indicated by arrows, inserted bases are highlighted by red squares. D Phenotypes of osroxy1, osroxy2, and the osroxy1/2 double mutant. The upper panel displays complete florets. The lower panel shows the stamens and carpels in the open florets. Red, black and white arrows indicate stamens, carpels and absent carpels, respectively. Scale bar = 2 mm. E Relative ratio of stamen numbers in transgenic plants. (F) Seed-setting rates in transgenic plants

Fig. 5

OsROXY2 interacts with OsbZIP47 A Interaction and auto-activation of OsROXY2 with OsbZIP47, OsTGA2, and OsTGA6 in Y2H assays. T7-T + Lam and T7-T + 53 were negative and positive controls, respectively. B BiFC assays confirmed the interaction between OsROXY2 and OsbZIP47. Scale bar = 5 μm

Fig. 6

Subcellular location, expression, and function analysis of OsbZIP47 A–C Subcellular localization analysis of OsbZIP47. A OsbZIP47-eGFP fusion protein. B mCherry (nuclear marker). C Merged image. Scale bar is 5 µm. D Relative expression of OsbZIP47 in root, shoot, leaf, and young panicles of 1 cm (YP1), 3 cm (YP3), 5 cm (YP5), 10 cm (YP10), and 20 cm (YP20) in wild type (ZH) plants. Values are shown as means ± SD (n = 3). (E-H) In situ hybridization of OsbZIP47 during rice panicle development. Red arrows indicate detected signals. Scale bar = 100 µm. E Secondary branch stage. F Early stage of spikelet primordia. G Late stage of spikelet primordia. H Negative control with the sense probe of OsbZIP47. I Gene structure of OsbZIP47. Black boxes, white boxes and the lines represent exons, 5’ and 3’UTR, and introns. Mutation site is indicated by arrow, inserted base is highlighted by red square. J Florets of wild type ZH and osbzip47 mutant. Red arrows indicate stamens, and blue arrow indicates carpel. Scale bar = 1 mm. K Relative ratio of stamen number in osbzip47