Os04g0497200

The rice gene Os04g0497200 was reported as OsGLU3 in 2012. OsGLU3 encodes a putative membrane-bound endo-1,4-b-glucanase.

Annotated Information

Function

• OsGLU3 modulates root cell wall cellulose synthesis and affects root cell elongation and division.

• The researchers also found that phosphate starvation, an environmental stress, altered cell wall cellulose content to modulate root elongation in a OsGLU3-dependant way.

Mutation

• A rice mutant with short root and short lateral root was obtained at 10 d after germination (DAG) (Figure 1A). The mutant was named Osglu3-1 after the mutation, which will be described later. To determine whether the short root phenotype of Osglu3-1 is due to defects in root cell elongation or division, root longitudinal sections for theWT and Osglu3-1 were made. These showed that the cell length of mature epidermal cells was only one-third of those in the WT, while the root hair length of the mutant is similar to that of WT (Figure 1B and 1C and Supplemental Figure 1). However, the root radical pattern organization and meristem of the mutant seems normal. In order to check the root mitotic activity, a mitotic activity reporter, the OsCYCB1,1:GUS, was incorporated into the mutant. The region expressing the OsCYCB1,1:GUS in the root meristem of mutant is about 90% of that in WT (Figure 1D and 1E). Together, these data suggest that the short root phenotype of Osglu3-1 results from defects in both root cell elongation and division, particularly defect in cell elongation.

Figure 1. Osglu3-1 Is Defective in Root Cell Elongation and Division. (A) Seedlings of wild-type (left), Osglu3-1 (middle), and Osglu3-1 transformed with whole OsGLU3 gene (right). Bar = 2 cm. Bottom figures are enlargements of the lateral roots in rectangles. Bar = 200 lm. (B) Longitudinal sections of root tip and maturation zone of wild-type (left) and Osglu3-1 (right). Bar = 50 lm. (C) Cell length ofwild-type (left) and Osglu3-1 (right). Two asterisks indicate statistical significance relative to control (**P , 0.01, Student’s t-test). (D) OsCYCB1,1–GUS activity in primary root tip of 5-day-old wild-type (left) and Osglu3-1 (right) by histochemical GUS-staining. Bar = 200 lm. (E) The width of OsCYCB1;1:GUS expression region in Osglu3-1 and WT. .

• To determine whether the OsGLU3 participates in cell wall synthesis, the content of the root cell wall components of Osglu3 mutants was measured. It was found that Osglu3-1 has 20% less crystalline cellulose in its roots than that in WT (Figure 3C). Then the WT and the Osglu3-1 mutants were grown on a solid media with 3% glucose. The addition of 3% glucose to the culture medium almost rescued the short lateral root defects of Osglu3-1, although the treatment inhibited the primary root elongation in the WT plants, SSBM (Figure 3A). To determine whether the complementation is due to the restoration of cellulose content in the Osglu3-1, the level of crystalline cellulose in the seedlings grownwith glucose and inWTseedlings was measured. These results showed that Osglu3-1 had almost the same concentration of cell wall cellulose asWT grown onmedium with 3% exogenous glucose. Moreover, the cellulose content of WT and Osglu3-1 had statistically increased after the application of 3% glucose (Figure 3C). The root growth response of Osglu3-2 to glucose treatment was also tested. As with the Osglu3-1 mutant, the application of 3% glucose suppressed the short root and short lateral root defect of Osglu3-2 (Figure 3B).

Figure 3. Three Percent Exogenous Glucose Rescued the Defects of Osglu3-1 and Partially Rescued T-DNA Insert Mutant Osglu3-2. .

• After being grown for 10 d in media without phosphate, the SSBM showed 20% increase of primary root elongation as compared to WT. To determine whether root elongation under phosphate starvation is due to the induction of cell elongation or enhanced mitotic activity, longitudinal sections of the primary root maturation zone were taken and the expression patterns of OsCYCB1;1:GUS under the treatment were analyzed. The longitudinal sections showed that phosphate starvation can lead to an increase of approximately 10% in rice root cell elongation (Figure 4A, 4C(c), and 4D). Also, the starvation can activate root mitotic activity as visualized by a broader GUS staining in the root tip (Figure 4C(e) and 4E). Together, these indicate that phosphate starvation stimulates primary root elongation by inducing root cell elongation and activating root mitotic activity.

Figure 4. Phosphate Starvation Could Induce Root Elongation in Osglu3-1, But Not in the T-DNA Insert Mutant Osglu3-2. (A) The phenotype of Osglu3-1 under phosphate starvation. Bar = 2 cm. CK, control; –P, phosphate starvation. (B) The phenotype of T-DNA insert mutant Osglu3-2 under phosphate starvation. Bar = 2 cm. CK, control; –P, phosphate starvation. (C) Phosphate starvation induced the cell elongation and mitotic activity in Osglu3-1. (a, b) The root tip of WT (a) and Osglu3-1 (b) under phosphate starvation. Bar = 1 mm. Left panel, CK; right panel, phosphate starvation. (c, d) Longitudinal sections of maturation zone ofWT (c) and Osglu3-1 (d) under phosphate starvation. Bar = 50 lm. Left panel, CK; right panel, phosphate starvation. (e, f) Expression patterns of OsCYCB1,1:GUS of 10-day-old WT (e) (bar = 100 lm) and Osglu3-1 (f) under phosphate starvation (bar = 50 lm). Left panel, CK; right panel, phosphate starvation. (D) Phosphate starvation induced root cell elongation in Osglu3-1. (E) Phosphate starvation increased the width of OsCYCB1,1:GUS expression region. (F) The crystalline cellulose (TFA-insoluble) content of Osglu3-1 was partly rescued in phosphate starvation. (G) The crystalline cellulose (TFA-insoluble) content of Osglu3-2 was not rescued in phosphate starvation. CWM, cell wall material. Data are mean values from three independent experiments, each performed in triplicate. Error bars represent standard error (SE). Different letters are used to indicate means that differ significantly (P , 0.05). CWM, cell wall material. .

Expression

内切-1,4-β左旋葡聚糖酶在水稻中组织特异性表达，在根尖、侧根和冠状根原基强表达，但在其他组织表达量低，如茎、叶和穗。

Evolution

在突变体中，Osglu3-1突变体的根细胞壁中晶体纤维素含量减少，根细胞长度变短，突变体根的分生组织略微变小。外施葡萄糖能够抑制突变体根细胞壁纤维素含量的降低和短根的表型。OsGLU3在不同组织遍在表达，但是在根尖、侧根和冠状根原基强表达。OsGLU3-GFP融合蛋白在根分生组织和伸长区的质膜和FM4-64标记的细胞器表达。 rt突变体根的伸长区的细胞伸长有严重缺陷，而且由于根冠细胞不能顺利剥落，导致根尖表皮细胞和皮层细胞的崩溃。rt突变体根中纤维素含量略有增加而纤维素结晶度明显降低，此外，半纤维素的组分在野生型和突变体根中也有差异，rt根的外植体的延展性明显降低。

Labs working on this gene

• The State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, China
• College of Science and Technology, Ningbo University, Ningbo, Zhejiang 315211, China
• State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China