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. .

• Phosphate starvation could induce root elongation in Osglu3-1 (Figure 4A). Under starvation, both the root cell elongation and division of the primary root were almost restored to the WT level (Figure 4C(d), 4C(f), and 4D). The root cellulose content of Osglu3-1 was also increased (Figure 4F). This indicates that the phosphate-starvation-induced root elongation might be independent of OsGLU3. To further test this, the root growth response of the Osglu3-2 loss-of-function mutant under phosphate starvation was also examined. Similarly to the WT SSBM, the primary root growth of WT Dongjin is also induced by phosphate starvation, with an approximate 30% increase in primary root elongation and 15% increase in root cellulose content. However, the phosphate starvation- induced primary root elongation and cellulose-content increase are abolished in Osglu3-2, which suggests that phosphate starvation-induced primary root elongation depends on the activity of OsGLU3 (Figure 4B).

• To further determine whether the phosphate starvation condition induces the expression of OsGLU3, a quantitative RT–PCR analysis of OsGLU3 was made. It showed that the phosphate starvation could not induce OsGLU3 expression, which indicates that phosphate starvation might regulate OsGLU3 enzyme activity to induce cellulose synthesis for root elongation.

Figure 4. Phosphate Starvation Could Induce Root Elongation in Osglu3-1, But Not in the T-DNA Insert Mutant Osglu3-2. .

Expression

• Since the OsGLU3 is involved in root cell elongation and division, the researchers expected that it was expressed in the elongating root. To check that, we analyzed the relative OsGLU3 transcripts levels in root, stem-base, stem, leaf, and panicle by qRT–PCR. It showed that the OsGLU3 is highly expressed in root tissue and has relatively lower expression in the other tissues (Figure 5).

Figure 5. OsGLU3 Protein Is Strongly Presented in Elongation Region of Root and Lateral Root, Crown Root Primodia. GUS staining in various organs of proOsGLU3:OsGLU3:GUS transgenic plants. .

Subcellular localization

• To follow the subcellular distribution of OsGLU3, the researchers also generated transgenic plants with OsGLU3–GFP driven by the native OsGLU3 promoter in the Osglu3-1 mutant. The transgenic plants displayed normal root elongation, suggesting that the OsGLU3–GFP protein may reflect the native OsGLU3. In root elongation cells of T1 transgenic seedlings, the GFP fluorescent signals were detected in the plasma member (PM) and intracellular punctuate bodies (Figure 6A). However, the signals were only presented in plasma membrane (Figure 6A and 6B) and in cells within the mature region of the root.

• When the root was treated with FM4-64 for 30 min, all the compartments of the cell were labeled along the endocytic pathway

down to the vacuole. These compartments partially co-localized with OsGLU3–GFP in the root meristem, suggesting that the OsGLU3–GFP was located in the endosomal compartment and PM (Figure 6C). In meristem and elongating cells, a 90-min BFA treatment led to the disappearance of the OsGLU3–GFP-labeled punctate structures and a concomitant emergence of large aggregates in root cells (Figure 6E). OsGLU3–GFP largely co-localized with the FM4-64 label to those patches (Figure 6E). This suggest that OsGLU3 localizes in the PM and endosomes, and the export of OsGLU3 to the PM depends on vesicle transport.

Figure 6. OsGLU3 Is Present in PM and Endosomes. (A) Overlay (right) of PI staining (left) and OsGLU3–GFP (middle) in elongation region and maturation region. (B) Overlay (right) of PI staining (left) and OsGLU3–GFP (middle) in maturation region with 40% sucrose plasmolysis. (C) Overlay (right) of FM4-64 treated 30 min (left) and OsGLU3–GFP (middle) in meristem region. (D) Overlay (right) of FM4-64 treated 30 min (left) and OsGLU3–GFP (middle) in maturation region. (E) Overlay (down) of FM4-64 treated 30 min (up) and OsGLU3–GFP (middle) in meristem region after BFA treated for 1.5 h. (A, B) Bar = 100 lm; (C–E) Bar = 50 lm. .

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