Os03g0646900

one sentence summary

  GL3.1 encodes a protein phosphatase kelch (PPKL) family — Ser/Thr phosphatase and GL3.1 is a member of the large grain WY3 variety, which is associated with weaker dephosphorylation activity than the small grain FAZ1 variety GL3.1

Annotated Information

2.1 Fine-mapping of a new QTL, GL3.1, which regulates rice grain yield

fig2.1 Map-based cloning of GL3.1 (from reference ).

• Fengaizhan-1 (FAZ1) and Waiyin-3 (WY3) rice varieties were selected as parents to map the QTLs that affect grain length. FAZ1 is a small grain indica variety (1 000-grain weight: 20.18 ± 0.89 g), whereas WY3 is a larger grain japonica variety (1 000-grain weight: 43.40 ± 0.92 g; Figure 1A). We fine-mapped a new major QTL (GL3.1) for grain length to a 20-kb region between the L012 and L008 markers on chromosome 3 (25 036 192 bp to 25 060 567 bp at chromosome 3) (Figure 1B), which is distinct from other previously reported QTLs [35-38]. This region contains two genes: Os03g44510, which is a predicted transposon that was excluded from further analysis because the transcript was not detected in both parents, and the predicted phosphatase Os03g44500, which was expressed in both parents and considered as the GL3.1 candidate. Based on the mapping results, we developed a nearisogenic line (NIL) from BC4F2 generations that contained a 30-kb WY3 chromosomal region at the GL3.1 locus in a FAZ1 genetic background (Figure 1C; Supplementary information, Figure S1A-S1C).
• NIL had longer grains (+16.1%) than FAZ1 (10.71 ± 0.13 mm vs 9.22 ± 0.09 mm), but there were no significant differences in grain width or thickness (Figure 1D-1F), plant height or tiller number (Supplementary information, Figure S1DS1E). NIL had a significantly greater 1 000-grain weight than FAZ1 (+43.5%; Figure 1G) and reduced grain number per main panicle (21.3%, Supplementary information, Figure S1F). NIL exhibited an increase in the milk filling rate (Figure 1H-1I) and higher expression of milk filling-related genes (Supplementary information, Figure S1G). The plot grain yield was significantly increased in NIL (+ 11.1%; Figure 1J); however, the grain quality was not affected, as the packing density of starch granules was similar in the mature seeds of NIL and FAZ1 (Supplementary information, Figure S1H-S1I), and the chalky grain percentage and protein and amylose contents were similar between the NIL and FAZ1 grains (Supplementary information, Figure S1J-S1L).
• We crossed NIL with Huanghuazhan, which is a relatively high-yield elite indica variety that is widely cultivated in Southern China, and subsequently backcrossed the F1 generation with Huanghuazhan to obtain a Huanghuazhan (GL3.1) variety that exhibited a longer and heavier grain (Supplementary information, Figure S2A-S2E) and a higher grain yield than Huanghuazhan under field conditions (Supplementary information, Figure S2F). These findings confirmed that GL3.1 potentially increases grain yield.

2.2 Confirmation of GL3.1 function

Figure 2.2 Transgenic analysis of GL3.1.(from reference ).

GL3.1 contains a 3 012-bp open reading frame (ORF) that encodes 21 exons and 20 introns. The FAZ1 GL3.1 allele contains 4-bp differences when compared with the WY3 GL3.1 allele (FAZ1 to WY3: 1092C-A, 1495CT, 2643A-G, 2838T-C), which results in two amino acid substitutions (364 aspartic acid — glutamic acid (364DE), 499 histidine — tyrosine (499H-Y); Figure 2A). We sequenced GL3.1 in several large grain varieties and detected the japonica variety Nanyangzhan with a truncated GL3.1 allele as well as Jizi1560 and Jizi1581, which contained 15 additional amino acids at the C-terminus compared with FAZ1 and WY3. At the positions 364 and 499, Jizi1560 and Jizi1581 exhibited the same amino acid substitutions as WY3 (Supplementary information, Figure S3). Transgenic rice plants were generated to determine whether GL3.1 controls grain length. FAZ1 and WY3 failed to regenerate shoots from the callus, and therefore we used the small-grain japonica variety Zhonghua 11, which was easily regenerated [39]. We generated constructs containing the full-length GL3.1 ORFs from FAZ1 or WY3 under the CaMV 35S promoter. Some of the obtained transgenic lines that overexpressed the WY3 GL3.1 allele showed an increased grain length (GL3.1-WY3), whereas the grain length was not changed in all lines overexpressing the FAZ1 GL3.1 allele (GL3.1-FAZ1; data not shown). Only the GL3.1- WY3 line, which expressed relatively high levels of GL3.1-WY3, exhibited increases in grain length (Figure 2B-2D, Supplementary information, Figure S4A-S4B), which confirms that GL3.1 controls grain length. GL3.1- FAZ1 RNA interference (RNAi) and antisense transgenic plants were generated; however, no phenotypic changes in grain length were observed (data not shown). We also observed that GL3.1 was downregulated but not completely suppressed in these lines; therefore, we hypothesized that these lines retained adequate GL3.1 function, as GL3.1 was abundantly expressed. GL3.1 is predicted to encode a Ser/Thr phosphatase of unknown function and with two predicted domains: a Kelch_1 protein interaction domain and a Ser/Thr phosphatase domain (Figure 2A). Transgenic plants were generated to investigate the effect of the GL3.1 point substitutions GL3.1-M1 (364E, 499H) and GL3.1-M2 (364D, 499Y) in FAZ1 and WY3. Both transgenic lines exhibited significant increases in grain length (Supplementary information, Figure S4C-S4H). Similar to the GL3.1-WY3 transgenic lines, only high levels of GL3.1- M1 or GL3.1-M2 overexpression led to enhanced grain length. These results suggest that the 364D-E and 499HY substitutions both influence the function of GL3.1.

2.3 GL3.1 functions as a Ser/Thr phosphatase

Figure 3 Expression pattern and molecular function of GL3.1 (from reference ).

Although some nucleotides were different between the promoters of FAZ1 and WY3, the GL3.1 expression pattern remained similar (Supplementary information, Figure S5). GL3.1 was expressed in all organs and developmental stages tested in FAZ1 and NIL (Supplementary information, Figure S6A-S6C). Notably, expression of GL3.1 was higher in the panicle of NIL at the heading stage than in the panicle of FAZ1 (Figure 3A) and lower in the calluses from FAZ1 and NIL, which primarily con- sist of dividing cells (Supplementary information, Figure S6D). GL3.1 in both parents was detected throughout the entire cell (Figure 3B). Purified GL3.1-FAZ1 and GL3.1- WY3 dephosphorylated myelin basic protein (MyBP; a standard substrate) in vitro, which demonstrates that GL3.1 is a functional Ser/Thr phosphatase (Figure 3C); however, GL3.1-FAZ1 exhibited higher activity than GL3.1-WY3. In addition, GL3.1 from Nanyangzhan (GL3.1-NYZ) did not show dephosphorylation ability (Figure 3C). GL3.1 was insensitive to both okadaic acid (OA) and Inhibitor 2 (Figure 3D), which suggests that GL3.1 may encode a novel type of PPKL, as the PPKL and PP1 enzymes are generally sensitive to Inhibitor 2 [27], whereas the PPKL family member BSU1 is sensitive to OA [32]. Furthermore, we observed that the phosphatase domain of GL3.1 was also not sensitive to these two inhibitors (Supplementary information, Figure S6E). A comparative analysis of phosphatases that are typically sensitive to OA revealed that 929G in GL3.1 conferred resistance to OA (Supplementary information, Figure S7). According to a previous study [40] in rats, PP2Aα is sensitive to OA, but the Y267G mutant of this protein is resistant to OA, which indicates that Y267G is a key mutation for OA resistance. Alignment analysis revealed that 267Y in rat PP2Aα corresponds to 929G in GL3.1, which suggests that GL3.1 harbors a Y to G mutation at this key site that may be responsible for the OA insensitivity of GL3.1.

2.4 GL3.1 regulates spikelet hull cell division

Figure 4 GL3.1 alters spikelet hull cell division to regulate grain length. ).

We measured the lemma cell length to determine whether GL3.1 regulates grain length. There were no significant differences in cell length at the central point of the lemma in the vertical orientation (Figure 4A-4B, Supplementary information, Figure S6F) and the distance between tubercles at the outer spikelet hull surface (Figure 4C-4D, Supplementary information, Figure S6G), which indicates that cell length is not responsible for the difference in grain length between FAZ1 and NIL. As the FAZ1 and NIL life cycles and heading days are similar, we hypothesized that an increased rate of cell division may be responsible for the longer spikelet hull in NIL. Therefore, we assessed the cell division rate during different developmental stages of the spikelet hull in the two parents. The detection points were set at the stages when the spikelet hull length reached 25%, 50%, 65%, 80% and 100% of the full spikelet hull length in FAZ1 and NIL, and the percentage of cells with 4C DNA content in the spikelet hull as well as the cell lengths at the central zone of the spikelet hull at these points were also recorded (Figure 4E, Supplementary information, Figure S6F). At the five detection points, the cell length in the vertical orientation was not different between FAZ1 and NIL. Notably, at 50% of full spikelet hull length, the percentage of cells with a 4C DNA content was significantly higher in NIL than in FAZ1 (Figure 4E). Consistent with this, the expression of cell cycle-related genes was significantly higher in the NIL than in the FAZ1 spikelet (Figure 4F). Therefore, we propose that rapid cell division occurs in NIL during spikelet hull development. Furthermore, we synchronized cells from FAZ1 and NIL using hydroxycarbamide, which blocks cell division at the G1/S boundary. 8 h after release from hydroxycarbamide, the expression of Histone H4 was maximal in FAZ1 and NIL (Supplementary information, Figure S6H), which suggests that the cells from FAZ1 and NIL had entered the S phase. In addition, a higher percentage of cells with 4C DNA content and a lower percentage of cells in S phase were observed in NIL when compared with FAZ1 (Figure 4G-4I), which implies that more cells from NIL completed DNA duplication. We also observed that the maximal expression of CYCD4;1, which was expressed from early G2 phase to M phase, was earlier in NIL (28 h after release) than in FAZ1 (32 h after release) (Supplementary information, Figure S6I), which implies faster entry into the G2 phase in NIL cells. Furthermore, we synchronized cells from FAZ1 and NIL using nocodazole, which blocks cell division at the G2/M boundary. The expression of CYCD3;1, which is specifically expressed at the G1/S stage, remained the same between FAZ1 and NIL (Supplementary information, Figure S6J), which implies that the transformation from the G2 phase to G1 phase was not different between the two parents. These results suggest that the transformation from G1 to G2 may be accelerated in NIL. Thus, our results collectively demonstrate that the GL3.1-WY3 allele increases the rate of cell division during spikelet hull development compared with the GL3.1-FAZ1 allele, which results in a longer spikelet hull.

2.5 GL3.1 interacts with Cyclin-T1;3 to regulate grain length

Figure5 GL3.1 and Cyclin-T13 interact(from reference ).

To determine the mechanism underlying the GL3.1- mediated regulation of grain size, a yeast two-hybrid system was used to screen a cDNA library constructed from Zhonghua 11 spikelets using GL3.1-FAZ1 as bait. We identified 23 interacting proteins, of which Os11g05850, annotated as Cyclin-T1;3, was selected for further analysis (Figure 5A). The expression of Cyclin-T1;3 was localized to the nucleus of Arabidopsis protoplasts (Figure 5B), which was consistent with the expression pattern observed in humans. When GL3.1 was co-expressed with Cyclin-T1;3, increased accumulation of GL3.1 was observed in the nuclei from both parents when compared with expression without Cyclin-T1;3 (Figures 5C and 3B). We confirmed that GL3.1 dephosphorylated Cyclin-T1;3 in vitro. GL3.1-FAZ1 exhibited stronger Cyclin-T1;3 dephosphorylation activity than GL3.1- WY3, GL3.1-M1 and GL3.1-M2 (Figure 5D), which was consistent with the effects observed for the common substrate MyBP (Figure 3C). As the kelch-repeat domain has demonstrated potential for protein interactions [41], we used a bimolecular fluorescence complementation (BiFC) assay to identify such interactions. GL3.1ΔP, which contains a kelch-repeat domain, interacted with Cyclin-T1;3 in the absence of the GL3.1 Ser/Thr phosphatase domain (Figure 5E). In addition, Cyclin-T1;3 was constitutively expressed in various tissues and organs in a pattern similar to that of GL3.1 (Supplementary information, Figure S8A-S8D). These results demonstrate that Cyclin-T1;3 interacts with GL3.1 and is dephosphorylated through GL3.1. Real-time PCR was performed to analyze the expression of Cyclin-T1;3 after cell synchronization. In contrast to the high expression levels observed at 16 h and 32 h in FAZ1 cells, Cyclin-T1;3 showed increased expression at 8 h and 28 h in NIL cells (Figure 5F). At these two specific points, the cells were entering the S and G2 phases, respectively, which indicates that Cyclin-T1;3 may be involved in cell cycle control. Moreover, we used transgenic rice plants to determine whether Cyclin-T1;3 influences grain size. No obvious phenotype was observed when we overexpressed Cyclin-T1;3 in Zhonghua 11. However, antisense strands of Cyclin-T1;3 resulted in smaller grain sizes in the transgenic plants as well as reduced expression of CyclinT1;3 (Figure 5G-5I). Thus, our data indicate that Cyclin-T1;3 is involved in the GL3.1-mediated regulation of grain length.

2.6 GL3.1 is a widespread gene that influences protein phosphorylation in vivo

Figure 6 GL3.1 influences protein phosphorylation status as a potential model for grain size control. (from reference ).

A quantitative proteomic analysis using two-dimensional difference gel electrophoresis (2-D DIGE) indicated that 21 proteins were differentially expressed in FAZ1 and NIL, of which 18 were upregulated in NIL (Supplementary information, Table S1). Mass spectrometry revealed that the 21 proteins were associated with cellular metabolic processes. Interestingly, actin was upregulated in NIL, which is consistent with the observation that GL3.1 influences the rate of cell proliferation. The phosphopeptides from the young spikelets of FAZ1 and NIL were enriched on the TiO2 beads and quantified using iTRAQ, which confirmed that GL3.1 is a phosphatase. 556 phosphopeptides were detected, and 464 of these molecules were quantified. Proteins showing a 1.5- fold difference between FAZ1 and NIL and demonstrating the same trend when quantified using two different labelling systems were chosen for further analysis. At least 130 proteins demonstrated a different phosphorylation status between FAZ1 and NIL during spikelet development (Figure 6A and Supplementary information, Table S2). Gene ontology analysis revealed that these proteins are primarily involved in processes related to nucleic acid metabolism and protein complex assembly (Figure 6B and Supplementary information, Table S3). The molecular functions of these proteins include nucleotide binding and the activities of phosphotransferases, helicases and the RNA polymerase II transcription factor. These results strongly suggest that GL3.1 could influence DNA duplication. Thus, we propose that GL3.1 regulates the expression and phosphorylation of a variety of genes involved in metabolism and cell division. Further phylogenetic analyzes based on genomic BLAST searches demonstrated the widespread existence of GL3.1 in plants (Supplementary information, Figure S9), which suggests that GL3.1 has an important conserved function in plants.

Labs working on this gene

• Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China
• State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China
• Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi-110021, India
• RIKEN Plant Science Center (H.N., K.M., A.D., K.S.) and RIKEN Bioinformatics and Systems Engineering Division (Y.Y., T.T.), Tsurumi-ku, Yokohama 230–0045, Japan
• Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997–0017, Japan (N.S., M.T., Y.I.)

References

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