Os03g0646900 - Revision history

Chenxi Sang: /* References */

2016-10-27T13:20:41Z

‎References

Zhennan: /* Knoledge Extension */

2016-06-24T07:53:07Z

‎Knoledge Extension

Zhennan: /* Influences protein phosphorylation */

2016-06-24T07:48:24Z

‎Influences protein phosphorylation

Zhennan: /* Regulate grain length */

2016-06-24T07:36:43Z

‎Regulate grain length

Zhennan: /* Ser/Thr phosphatase */

2016-06-24T07:34:15Z

‎Ser/Thr phosphatase

Zhennan: /* Ser/Thr phosphatase */

2016-06-24T07:33:50Z

‎Ser/Thr phosphatase

Zhennan: /* Regulates spikelet hull cell division */

2016-06-24T07:32:55Z

‎Regulates spikelet hull cell division

Zhennan: /* Ser/Thr phosphatase */

2016-06-24T07:29:51Z

‎Ser/Thr phosphatase

Zhennan: /* Ser/Thr phosphatase */

2016-06-24T07:29:08Z

‎Ser/Thr phosphatase

Zhennan: /* Regulates rice grain yield */

2016-06-24T07:26:26Z

‎Regulates rice grain yield

← Older revision		Revision as of 13:20, 27 October 2016
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	<ref name="ref4">Hirofumi Nakagami, Naoyuki Sugiyama, Keiichi Mochida, Arsalan Daudi, Yuko Yoshida, Tetsuro Toyoda, Masaru Tomita, Yasushi Ishihama, Ken Shirasu .Large-Scale Comparative Phosphoproteomics Identifies Conserved Phosphorylation Sites in Plant.Plant Physiol(2010) 153(3): 1161–1174</ref>		<ref name="ref4">Hirofumi Nakagami, Naoyuki Sugiyama, Keiichi Mochida, Arsalan Daudi, Yuko Yoshida, Tetsuro Toyoda, Masaru Tomita, Yasushi Ishihama, Ken Shirasu .Large-Scale Comparative Phosphoproteomics Identifies Conserved Phosphorylation Sites in Plant.Plant Physiol(2010) 153(3): 1161–1174</ref>
		+	</references>
		+	== Structured Information==

← Older revision		Revision as of 07:53, 24 June 2016
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	== Knoledge Extension ==		== Knoledge Extension ==
		+
		+	* GL3.1-WY3 differs from GL3.1-FAZ1 by two amino acid substitutions, induces a longer grain length as a result of an increased number of cells in the spikelet hull, and accelerates milk filling, which leads to increased grain weight and yield. GL3.1 influenced grain length through an increased rate of cell division during spikelet hull development, as evidenced by the upregulation of cell cycle-related genes and an increase in the proportion of cells with 4C DNA content; however, there was no effect on cell size. Protein phosphorylation and dephosphorylation critically regulate the cell cycle. The dephosphorylation activity of GL3.1-FAZ1 was higher than that of GL3.1-WY3, GL3.1-M1 and GL3.1-M2; therefore, we hypothesize that the ability of GL3.1 to dephosphorylate substrates, such as Cyclin-T1;3, may regulate cell proliferation during spikelet development.
		+	* We propose that the high activity of GL3.1-FAZ1 dephosphorylates substrates, such as Cyclin-T1;3, to promote cell proliferation, which leads to normal cell proliferation during spikelet hull development ('''Figure 6C'''). For the WY3 allele, the weaker dephosphorylation activity of GL3.1-WY3 resulted in an accumulation of phosphorylated substrates that promoted cell proliferation during spikelet development and increased the grain length ('''Figure 6D'''). Only transgenic lines expressing high levels of GL3.1-WY3 had an increased grain size, which indicates that a significant increase in the expression of the low-activity GL3.1-WY3 allele would result in competition for substrates with the abundant endogenous GL3.1-FAZ1, which has increased dephosphorylation activity. As a result, higher quantities of phosphorylated substrates accumulate in GL3.1-WY3-overexpressing transgenic plants, which leads to accelerated cell division and eventually results in an elongated grain. Thus, we propose that Cyclin-T1;3 is one of the substrates in this pathway. In summary, we hypothesize that the larger grain size in lines overexpressing GL3.1-WY3, GL3.1-M1 and GL3.1-M2 that contain high levels of exogenous GL3.1 may result from dominant negative effects.
		+	* Efficient breeding requires a thorough understanding of the molecular and genetic factors that regulate seed development. Although considerable research has been performed to this end, an investigation of the relationship among cloned QTLs for grain size is needed. According to a previous study, GW2 and GW5 both function in the ubiquitin-proteasome pathway and control grain size through the regulation of cell division; thus, GW2 and GW5 may be involved in the same pathway for grain size control. However, GL3.1 and GS3 encode different types of proteins [10], and whether they function in the same pathway remains unknown. Determining the relationship between these QTLs would be useful for understanding grain development. However, GL3.1 is similar to BSU1, and whether GL3.1 plays a role in BR responses needs to be further investigated. In this study, we cloned a new QTL, GL3.1, characterized its function, and further identified the GL3.1-mediated pathway that regulates grain size and yield. This results obtained in this study contribute to the understanding of the molecular and genetic factors involved in seed development and provide a new approach for improving crop grain yield.

	== Labs working on this gene ==		== Labs working on this gene ==

@@ Line 46: / Line 46: @@
 ===Influences protein phosphorylation===
-[[File:fig2.6.jpg|right|thumb|150px|''Figure 6 GL3.1 influences protein phosphorylation status as a potential model for grain size control. (from reference <ref name="ref1" /><ref name="ref2" /><ref name="ref3" /><ref name="ref4" />).'']]
+[[File:fig2.6.jpg|right|thumb|500px|'''Figure 6''' ''GL3.1 influences protein phosphorylation status as a potential model for grain size control. (from reference <ref name="ref1" /><ref name="ref2" /><ref name="ref3" /><ref name="ref4" />).'']]
-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.
+* 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. 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.
 == Knoledge Extension ==

@@ Line 38: / Line 38: @@
 ===Regulate grain length===
-[[File:fig2.5.jpg|right|thumb|150px|''Figure5 GL3.1 and Cyclin-T13 interact(from reference <ref name="ref1" /><ref name="ref2" /><ref name="ref3" />).'']]
+[[File:fig2.5.jpg|right|thumb|500px|'''Figure5''' ''GL3.1 and Cyclin-T13 interact(from reference <ref name="ref1" /><ref name="ref2" /><ref name="ref3" />).'']]
-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.
+* 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''').
-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.
+* 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, 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. These results demonstrate that Cyclin-T1;3 interacts with GL3.1 and is dephosphorylated through GL3.1.
 ===Influences protein phosphorylation===

@@ Line 22: / Line 22: @@
 ===Ser/Thr phosphatase===
-[[File:fig2.3.jpg|left|thumb|500px|'''Figure 3'''  ''Expression pattern and molecular function of GL3.1 (from reference <ref name="ref1" />).'']]
+[[File:fig2.3.jpg|right|thumb|500px|'''Figure 3'''  ''Expression pattern and molecular function of GL3.1 (from reference <ref name="ref1" />).'']]
 * Although some nucleotides were different between the promoters of FAZ1 and WY3, the GL3.1 expression pattern remained similar. GL3.1 was expressed in all organs and developmental stages tested in FAZ1 and NIL. 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.

@@ Line 30: / Line 30: @@
 ===Regulates spikelet hull cell division===
-[[File:fig2.4.jpg|right|thumb|150px|''Figure 4 GL3.1 alters spikelet hull cell division to regulate grain length. <ref name="ref1" />).'']]
+[[File:fig2.4.jpg|right|thumb|500px|'''Figure 4'''  ''GL3.1 alters spikelet hull cell division to regulate grain length. <ref name="ref1" />).'']]
-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.
+* 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,''') and the distance between tubercles at the outer spikelet hull surface ('''Figure 4C-4D'''), 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,
 ===Regulate grain length===

@@ Line 25: / Line 25: @@
 * Although some nucleotides were different between the promoters of FAZ1 and WY3, the GL3.1 expression pattern remained similar. GL3.1 was expressed in all organs and developmental stages tested in FAZ1 and NIL. 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.
-* 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, whereas the PPKL family member BSU1 is sensitive to OA. Furthermore, we observed that the phosphatase domain of GL3.1 was also not sensitive to these two inhibitors. * A comparative analysis of phosphatases that are typically sensitive to OA revealed that 929G in GL3.1 conferred resistance to OA. According to a previous study 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.
+* 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, whereas the PPKL family member BSU1 is sensitive to OA. Furthermore, we observed that the phosphatase domain of GL3.1 was also not sensitive to these two inhibitors.
 ===Regulates spikelet hull cell division===

@@ Line 15: / Line 15: @@
 ===Regulates rice grain yield===
-[[File:fig2.1.jpg|right|thumb|550px|''fig2.1 Map-based cloning of GL3.1 (from reference <ref name="ref1" />).'']]
+[[File:fig2.1.jpg|right|thumb|550px|'''Figure 2''' ''Map-based cloning of GL3.1 (from reference <ref name="ref1" />).'']]
 ===Ser/Thr phosphatase===