Separable regulation of POW1 in grain size and leaf angle development in rice

Abstract

Leaf angle is one of the key factors that determines rice plant architecture. However, the improvement of leaf angle erectness is often accompanied by unfavourable changes in other traits, especially grain size reduction. In this study, we identified the pow1 (put on weight 1) mutant that leads to increased grain size and leaf angle, typical brassinosteroid (BR)-related phenotypes caused by excessive cell proliferation and cell expansion. We show that modulation of the BR biosynthesis genes OsDWARF4 (D4) and D11 and the BR signalling gene D61 could rescue the phenotype of leaf angle but not grain size in the pow1 mutant. We further demonstrated that POW1 functions in grain size regulation by repressing the transactivation activity of the interacting protein TAF2, a highly conserved member of the TFIID transcription initiation complex. Down-regulation of TAF2 rescued the enlarged grain size of pow1 but had little effect on the increased leaf angle phenotype of the mutant. The separable functions of the POW1-TAF2 and POW1-BR modules in grain size and leaf angle control provide a promising strategy for designing varieties with compact plant architecture and increased grain size, thus promoting high-yield breeding in rice.

Keywords: POW1; TAF2; brassinosteroid.; grain size; leaf angle; rice.

Figure 1

Phenotypic analysis of the pow1 mutant. (a–c) Grain size comparison between pow1 and WT. Bars = 5 mm. (d) Population morphology of pow1 and WT. Indicating randomly extended leaves of the mutant population. Bar = 10 cm. (e) Leaf angle phenotype of the main panicle of pow1 and WT. Bar = 3 cm. (f,g) Comparison of grain size (f) and leaf angle (g) between pow1 and WT. Data are means ± SE (n = 30). **: p < 0.01 and ***: p < 0.001 (Student’s t test). (h,i) Expression analysis of cell division markers MCM3 and CYCB2 and cell expansion makers EXPA2 and XTR2 in young inflorescences of about 2 mm long (h) and lamina joints of flag leaves of 2‐month‐old field grown plants (i) of pow1 and WT. The transcript levels were normalized against WT, which was set to 1. Data are means ± SE (n = 3). *: p < 0.05 and ***: p < 0.001 (Student’s t test).

Figure 2

Map‐based Cloning of POW1. (a) Identification of the POW1 candidate. Indicating one G–T point mutation occurred in LOC_Os07g07880 in the mutant, which results in an amino acid change from Arg to Ser. (b,c) Phenotypes of whole plant and grain size of pow1, WT, complemented T₀ line (pow1‐C), POW1 knockout (POW1‐KO), and POW1 overexpression (POW1‐OE). Bars = 20 cm (b) and 5 mm (c). (d) Expression analysis of POW1 in various plant tissues. Actin was used as the internal control. Data are means ± SD (n = 3). (e) Protein structure of POW1. (f) Subcellular localization of POW1. Bars = 10 μm.

Figure 3

POW1 controls leaf angle formation via BR biosynthesis pathway. (a) Endogenous BR quantification. Lamina joints of flag leaves of 2‐month‐old field grown plants were sampled. Data are means ± SE (n = 3). *: p < 0.05 (Student’s t test). (b) BRZ treatment assay. Sterilized seeds were sowed on half strength MS medium containing different concentrations of BRZ and grew for 1 week after germination. The white arrows indicate the second lamina joint. Bar = 1 cm. (c) Expression analysis of BR biosynthesis genes. Lamina joints of flag leaves of 2‐month‐old field grown plants was sampled. The transcript levels were normalized against WT, which was set to 1. Data are means ± SE (n = 3). ***: p < 0.001 (Student’s t‐test). (d) BR induction assay. The whole shoots of 2‐week‐old seedlings under 1 μM epiBL treatment were collected at the indicated time points after treatment. Actin was used as the internal control. Data are means ± SD (n = 3). *: p < 0.05 and ***: p < 0.001 (student’s t‐test). (e,f) Comparison of leaf angle and grain size among the D4‐RNAi, D11‐RNAi and D4‐D11‐RNAi plants under the background of pow1 and WT, respectively. Bars = 20 cm (e) and 5 mm (f).

Figure 4

POW1 controls leaf angle formation via BR signalling pathway. (a,b) Comparison of plant architecture (a) and grain appearance (b) among WT, pow1, d61‐2 and pow1‐d61‐2 double mutant. Bars == 20 cm (a) and 5 mm (b). (c–l) Stasistical analysis of leaf angle (c), grain length (d), grain width (e), plant height (f), tiller number (g), length of main panicle (h), spikelet number per main panicle (i), seed setting rate (j) and 1000‐grain weight (k) in WT, pow1, d61‐2 and pow1‐d61‐2 double mutant. Data are means ± SD (n = 20). Bars followed by the different letters represent significant difference at 5%.

Figure 5

Transactivation assay of TAF2 and its interaction with POW1. (a–c) Interaction assay between POW1/mPOW1 and TAF2. For pull‐down assay (c), the OsBZR1‐TAF2 combination was used as the negative control. Bars = 10 μm (b). (d) Expression analysis of TAF2 in various plant tissues. Actin was used as the internal control. Data are means ± SD (n = 3). (e,f) Transactivation assay. The transactivation activity of TAF2 was observed in yeast (e) and rice protoplast (f). ***: p < 0.001 (Student’s t‐test).

Figure 6

TAF2 controls grain size by affecting mainly cell division. (a‐c) Phenotypes of whole plant, grain size and leaf angle of the TAF2‐RNAi transgenic plants. Bars = 20 cm (a), 5 mm (b) and 1 cm (c). (d,e) Expression analysis of TAF2, MCM3, CYCB2, EXPA2, XTR2, D4 and D11 in the TAF2‐RNAi transgenic plants. Young inflorescences of about 2 mm long were sampled for detecting TAF2, MCM3, CYCB2, EXPA2 and XTR2, and lamina joints of flag leaves of 2‐month‐old field grown plants were sampled for detecting D4 and D11. The transcript levels were normalized against WT, which was set to 1. Data are means ± SE (n = 3). *: p < 0.05, **: p < 0.01, and ***: p < 0.001 (Student’s t‐test). Bars followed by different letters indicate significant difference at 5%. (f) Transcriptional activation of OsTAF2 on CYCB2 and EXPA2. Data are means ± SD (n = 3). *: p < 0.05 and ***: p < 0.001 (student’s t‐test). (g) Analysis of the effect of POW1 and mPOW1 on the transactivation activity of OsTAF2. Data are means ± SD (n = 3). Bars followed by different letters represent significant difference at 5%. (h,i) Inhibitory effects of POW1 on the transactivation activity of TAF2 in yeast (h) and rice protoplast (i). Bars followed by different letters indicate significant difference at 5%. Indicating that the inhibitory effect of mPOW1 on TAF2 was significantly weakened compared with that of the functional POW1.

Figure 7

Separable regulation of POW1 in grain size and leaf angle development. (a–c) Phenotypes of whole plant, grain size and leaf angle of TAF2‐RNAi and TAF2‐D4‐D11‐RNAi transgenic plants under the pow1 mutant background. Bars = 20 cm (a), 5 mm (b), and 2 cm (c). (d) Expression analysis of TAF2 in pow1, WT, TAF2‐RNAi and TAF2‐D4‐D11‐RNAi transgenic plants. Young inflorescences of about 2 mm long were sampled. The transcript levels were normalized against WT, which was set to 1. Data are means ± SE (n = 3). Bars followed by different letters represent significant difference at 5%. (e,f) Statistical analysis of grain size and leaf angle in pow1, WT, TAF2‐RNAi and TAF2‐D4‐D11‐RNAi transgenic plants. Data are means ± SE (n = 30). Bars followed by different letters represent significant difference at 5%. (g) Expression analysis of D4 and D11 in pow1, WT, TAF2‐RNAi and TAF2‐D4‐D11‐RNAi transgenic plants. Lamina joints of flag leaves of 2‐month‐old field grown plants were sampled. The transcript levels were normalized against WT, which was set to 1. Data are means ± SE (n = 3). Bars followed by different letters represent significant difference at 5%.

Figure 8

A working model of POW1 regulating leaf angle and grain size. POW1 and BR are feedback regulated. POW1 negatively regulates the expression of BR biosynthesis genes directly or by associating with TAF2. As a general transcription initiation factor, TAF2 functions not only in cell division and cell expansion but also in BR homeostasis. Because TAF2‐activated BR biosynthesis induces the expression of POW1, which in turn represses the transactivation activity of TAF2, genetic manipulation of TAF2 thus could not increase the endogenous BR content to a level high enough to affect leaf angle formation. Mutation of POW1 reduces the repression on D4/D11 expression and TAF2 transactivation activity, and the resultant increase of endogenous BR level enhances the accumulation of the mPOW1 protein. Because mPOW1 still has some repression activity, the basic cellular processes mediated by BR and TAF2 in the mutant are activated to a certain extent, resulting in the increase of leaf angle and grain size.