Characterization of a xyloglucan endotransglucosylase gene that is up-regulated by gibberellin in rice

Abstract

Xyloglucan endotransglucosylases/hydrolases (XTHs) that mediate cleavage and rejoining of the beta (1-4)-xyloglucans of the primary cell wall are considered to play an important role in the construction and restructuring of xyloglucan cross-links. A novel rice (Oryza sativa) XTH-related gene, OsXTH8, was cloned and characterized after being identified by cDNA microarray analysis of gibberellin-induced changes in gene expression in rice seedlings. OsXTH8 was a single copy gene; its full-length cDNA was 1,298 bp encoding a predicted protein of 290 amino acids. Phylogenetic analysis revealed that OsXTH8 falls outside of the three established subfamilies of XTH-related genes. OsXTH8 was preferentially expressed in rice leaf sheath in response to gibberellic acid. In situ hybridization and OsXTH8 promoter GUS fusion analysis revealed that OsXTH8 was highly expressed in vascular bundles of leaf sheath and young nodal roots where the cells are actively undergoing elongation and differentiation. OsXTH8 gene expression was up-regulated by gibberellic acid and there was very little effect of other hormones. In two genetic mutants of rice with abnormal height, the expression of OsXTH8 positively correlated with the height of the mutants. Transgenic rice expressing an RNAi construct of OsXTH8 exhibited repressed growth. These results indicate that OsXTH8 is differentially expressed in rice leaf sheath in relation to gibberellin and potentially involved in cell elongation processes.

Figure 1.

Genomic Southern-blot analysis of OsXTH8. Rice genomic DNA was digested completely with EcoRI, XbaI, and XhoI, respectively, separated by agarose gel electrophoresis, and then blotted onto positively charged nylon membrane. The blot was hybridized to 3′ UTR of OsXTH8. Molecular size markers are indicated on the left.

Figure 2.

Amino acid sequence alignment of OsXTH8 and other members of the rice XTH gene family. The deduced amino acid sequence of OsXTH8 is aligned with other members of rice XTH gene family by Genetyx-WIN. DEIDFEFLG motif (marked by line) indicates a possible conserved catalytic region shared with the Bacillus β-glucanase. The possible N-linked glycosylation residues are indicated by asterisks. Conserved Cys residues are marked by arrowheads.

Figure 3.

Phylogenic-alignment of the OsXTH8-deduced amino acid sequence with other plant XTHs. OsXTH8 was aligned with 54 full-length deduced amino acid sequences using the ClustalW and tree View software. Details and GenBank accession numbers are: AtXTH1, At4g13080; AtXTH2, At4g13090; AtXTH3, At3g25050; AtXTH4, At2g06850; AtXTH5, At5g13870; AtXTH6, At5g65730; AtXTH7, At4g37800; AtXTH8, At1g11545; AtXTH9, At4g03210; AtXTH10, At2g14620; AtXTH11, At3g48580; AtXTH12, At5g57530; AtXTH13, At5g57540; AtXTH14, At4g25820; AtXTH15, At4g14130; AtXTH16, At3g23730; AtXTH,17, At1g65310; AtXTH18, At4g30280; AtXTH19, At30290; AtXTH20, At5g48070; AtXTH21, At2g18800; AtXTH22, At5g57580; AtXTH23, At4g25810; AtXTH24, At4g30270; AtXTH25, At5g57550; AtXTH26, At4g28850; AtXTH27, At2g01850; AtXTH28, At1g14720; AtXTH29, At4g18990; AtXTH30, At1g32170; AtXTH31, At3g44990; AtXTH32, At2g36870; AtXTH33, At1g10550; TmNXG-1A, X68254; TmNXG-2A, X68255; TRUXET1G, L43094; VIREXT5, D16458; EXT2 (soybean), D16455; HVEXT, X91659; WHEXT4, D16457; TOMEXT3, D16456; XET (cotton), D88413; tXET-B1, X82685; tXET-B2, X82684; XEB X93175; XEA, X93174; XET (maize), U15781; HvPM2, X91660; HvPM5, X93173; and OsXTH8, AB110604. OsXTR1, OsXTR2, OsXTR3, and OsXTR4 sequences were noted down from Uozu et al. (2000).

Figure 4.

Tissue-specific expression of OsXTH8. A, Expression of OsXTH8 in different tissues. Two-week-old seedlings were treated with 5 μm GA₃ for 24 h. For tissue-specific expression, total RNAs were extracted from roots, leaf blades, leaf sheaths, and calli. B, Expression of OsXTH8 in five different sections of leaf sheaths using 1-month-old rice seedlings. The rice leaf sheath was divided in five sections: leafy section (I), second and third leaf section (II), first and second leaf section (III), coleoptile and first leaf section (IV), and coleoptile section (V) as shown in the figure drawing. Total RNA (20 μg each) transferred onto membrane was probed with PCR- amplified 3′ UTR of OsXTH8 cDNA clone. rRNA stained with ethidium bromide was used as a loading control.

Figure 5.

In situ hybridization of OsXTH8 and GUS expression driven by OsXTH8 promoter in different tissues of rice. A, In situ localization of OsXTH8 mRNA. Sections of rice leaf sheaths were hybridized with Dig-labeled antisense RNA prepared from OsXTH8 expressed sequence tag (A, right). Dig-labeled sense RNA was used as a negative control (A, left). B, GUS expression in different tissues of transgenic rice carrying OsXTH8∷GUS construct. Cross-section and longitudinal section of leaf sheath and intact roots were used for histochemical localization of GUS activity. VB, Vascular bundle; e, peripheral cylinder of vascular tissues; SAM, shoot apical meristem. Bar represents 500 μm.

Figure 6.

Hormonal regulation of OsXTH8 expression. A, Dose-dependent effect of GA₃. Rice leaf sheaths were treated with 0, 1, 5, 10, and 50 μm GA₃ for 24 h. B, Time-course changes in the expression of OsXTH8. Rice leaf sheaths were treated with 5 μm GA₃ for 1, 3, 6, 12, and 24 h for time-course experiment. C, Effects of different phytohormones on the expression of OsXTH8. Rice leaf sheaths were treated with 1 μm BL, 10 μm uniconazole, and 5 μm each GA₃, IAA, BA, and ABA for 24 h. D, OsXTH8 expression in different GA mutants exhibiting abnormal heights. Ginbozu and Nipponbare are the wild type of Tanginbozu and Slr1 mutant, respectively. Total RNA (20 μg each) was extracted from leaf sheaths of 2-week-old seedlings and probed with 3′ UTR of OsXTH8. Experiments were replicated three times.

Figure 7.

GUS activity in OsXTH8∷GUS transgenic rice seedlings in response to GA treatment. A, Binary vector pSMAHdN627 harboring GUS gene under the control of the 2,325-bp promoter region of OsXTH8. B, Histochemical localization of GUS activity in transgenic rice treated with or without GA₃. C, Ten-day-old seedlings were treated with 0, 0.1, 1, and 10 μm GA₃ for 24 h. GUS activity was measured by fluorescent method as described in “Methods and Materials.” Values are mean of triplicate experiment.

Figure 8.

Phenotypes of transgenic rice constitutively overexpressing RNAi OsXTH8. A, A binary vector pIG121-Hm harboring RNAi OsXTH8 under the control of CaMV 35S promoter. B, Transgenic rice 2 months after transferred to soil in the isolating green house. C, Elongation of the upper five internodes of the control (C) and RNAi OsXTH8 transgenic rice (T). D, Expression levels of OsXTR1, OsXTR2, OsXTR3, OsXTR4, and OsXTH8 mRNA in RNAi OsXTH8 transgenic lines. E, Growth curves of the three RNAi OsXTH8 transgenic plants and a vector control transgenic plant.