Cytokinin activity of cis-zeatin and phenotypic alterations induced by overexpression of putative cis-Zeatin-O-glucosyltransferase in rice

Abstract

cis-Zeatin (cZ) is generally regarded as a cytokinin with little or no activity, compared with the highly active trans-zeatin (tZ). Although recent studies suggested possible roles for cZ, its physiological significance remains unclear. In our studies with rice (Oryza sativa), cZ inhibited seminal root elongation and up-regulated cytokinin-inducible genes, and its activities were comparable to those of tZ. Tracer experiments showed that exogenously supplied cZ-riboside was mainly converted into cZ derivatives but scarcely into tZ derivatives, indicating that isomerizations of cZ derivatives into tZ derivatives are a minor pathway in rice cytokinin metabolism. We identified three putative cZ-O-glucosyltransferases (cZOGT1, cZOGT2, and cZOGT3) in rice. The cZOGTs preferentially catalyzed O-glucosylation of cZ and cZ-riboside rather than tZ and tZ-riboside in vitro. Transgenic rice lines ectopically overexpressing the cZOGT1 and cZOGT2 genes exhibited short-shoot phenotypes, delay of leaf senescence, and decrease in crown root number, while cZOGT3 overexpressor lines did not show shortened shoots. These results propose that cZ activity has a physiological impact on the growth and development of rice.

Figure 1.

Comparison of the bioactivities of cZ and tZ in a root growth assay. Primary root length of Arabidopsis (A) and seminal root length of rice (B) were measured at 11 and 7 d, respectively, after sowing on agar medium containing various concentrations of tZ (white circles) and cZ (black circles). Asterisks indicate significant differences (Student’s t test; P < 0.01) between like concentrations of tZ and cZ. Mean values ± sd of at least 11 (Arabidopsis) and six (rice) plants are shown.

Figure 2.

Changes in mRNA levels of response regulator genes in response to zeatin isomers in roots of rice seedlings. Plants were grown in hydroponics for 2 weeks after sowing and then transferred to treatment solutions containing 100 nm tZ (white circles), cZ (black circles), or no cytokinin (gray circles). The roots were harvested before (0 min) and 15, 30, and 60 min after the transfer and submitted to qRT-PCR analysis for OsRR1 (top left), OsRR2 (top right), OsRR4 (middle left), OsRR6 (middle right), OsRR9 and/or OsRR10 (OsRR9/10; bottom left), and OsRR3 (bottom right). Because of their high degree of identity (Jain et al., 2006), mRNAs of OsRR9 and OsRR10 could not be separated. OsRR3, a gene not responsive to cytokinin, was used as an internal control (Jain et al., 2006). The mRNA contents were normalized to the value at 0 min. Mean values ± sd of at least three plants are shown. Different lowercase letters indicate statistically significant differences at a given time (Tukey-Kramer test following one-way ANOVA; P < 0.01). n.s., Not significant.

Figure 3.

Isomerization of tZ and cZ derivatives in rice seedlings. One micromolar [10¹³C,5¹⁵N]tZR (+tZR; gray bars) or [10¹³C,5¹⁵N]cZR (+cZR; black bars) was supplied to 2-week-old rice seedlings. After 1 h, roots (top panel) and shoots (bottom panel) were separately harvested. tZ and cZ derivatives containing isotope labels in the purine ring and the ribosyl group (total 15 D heavier than their authentic counterparts) and derivatives carrying label only in the purine ring (10 D heavier than their authentic counterparts) were quantified by UPLC-MS/MS. Means ± sd of five plants are shown. †, Not determined. FW, Fresh weight.

Figure 4.

Identification of rice genes encoding cZOGTs. A, Phylogenetic relations of P. lunatus ZOG1, maize cisZOG1 and cisZOG2, and putative rice cZ-O-glucosyltransferases. The deduced amino acid sequences of the proteins were obtained from GenBank (accession nos. AF101972 for P. lunatus ZOG1, AF318075 for maize cisZOG1, and AY082660 for cisZOG2 [http://www.ncbi.nlm.nih.gov/genbank/]) and the RAP-DB (locus identifiers Os04g0556500, Os04g0556600, Os04g0565200, Os04g0565400, and Os07g0660500 for putative rice cZ-O-glucosyltransferases [http://rapdb.dna.affrc.go.jp/]). The phylogenetic relations were analyzed with the MEGA 4 program (http://www.megasoftware.net/) using the neighbor-joining method. The bar represents 0.1 amino acid substitutions per site. B, SDS-PAGE analysis of the purified recombinant Os04g0556500 (55.1 kD), Os04g0556600 (55.4 kD), and Os04g0565400 (52.2 kD) proteins. The gel was stained with Coomassie Brilliant Blue; the arrowhead points to the recombinant proteins, and molecular mass markers (kD) are indicated at the left. C, O-Glucosylation activities of the recombinant cZOGT1 (black bars), cZOGT2 (gray bars), and cZOGT3 (white bars) for 50 μm tZ, cZ, cZR, and oT (top panel) and for 100 μm tZ, tZR, tZRMP, and cZRMP (bottom panel). ND, Not detected.

Figure 5.

Accumulation of cZOGT1 (A), cZOGT2 (B), cZOGT3 (C), and Gpc1 (D) mRNAs in various organs of rice. Total RNAs were extracted from shoots (Sht) and roots (Rt) of 2-week-old seedlings and from flowers (Flw), panicle branches (PBr), top and basal parts of internode I (InNt and InNb, respectively), node I (Nd), leaf blades of flag leaves (FLB), and blades of leaves 2 and 4 below the flag leaf (LB-2 and LB-4, respectively) of older plants. Total RNAs were subjected to qRT-PCR using gene-specific primers. The mRNA levels are indicated as amounts per total RNA without normalization by an internal control gene. The Gpc1 gene encoding glyceraldehyde-3-phosphate dehydrogenase is presented as an unrelated gene used to control for expression in the tissues. Mean values ± sd of three plants are shown. ND, Not detected.

Figure 6.

Distribution of GUS activity under the control of the 5′ upstream region of the cZOGT1 (A–C) and cZOGT2 (D–H) genes. A to F, Whole shoots of the transformants were stained for GUS activity. Overviews (A and D) and closeups of the laminar joint of the second leaves (B and E) and coleoptiles (C and F) are shown. Bars = 10 mm for A, B, D, and E and 1 mm for C and F. White and black arrowheads indicate laminar joints and vascular bundles of coleoptiles, respectively. G and H, Cross-sections of mature leaf blades; a large (G) and a small (H) vascular bundle are shown. Bars = 50 μm.

Figure 7.

The short-shoot phenotype of transgenic rice overexpressing cZOGT1 or cZOGT2. Transgenic rice lines that overexpressed cZOGT1 (1ox-7), cZOGT2 (2ox-2 and 2ox-3), and cZOGT3 (3ox-7 and 3ox-9) were grown on soil in parallel with VC plants (VC-1, VC-3, and VC-4). The generations of the transgenic plants analyzed are indicated underneath the names of the lines. A, Transgenic rice at 89 d after sowing. Bar = 200 mm. B, Shoot length of the transgenic rice lines at 22 d after sowing. Mean values ± sd of three plants are shown. Means were tested for significant differences by the Tukey-Kramer test (P < 0.05) following one-way ANOVA; different lowercase letters indicate statistically significant differences (P < 0.05). n.s., Not significant. C, Accumulation of cZOGT1 (black bar), cZOGT2 (gray bars), and cZOGT3 (white bars) mRNAs in the shoots of transgenic rice lines at 22 d after sowing. cZOGT mRNA contents were normalized to the content of Gpc1 mRNA. Values shown are means ± sd of three plants.

Figure 8.

Characterization of the short-shoot phenotype in transgenic rice overexpressing cZOGT1 and cZOGT2. A and B, Shoot growth in transgenic rice lines overexpressing cZOGT1 (1ox-1, gray circles; 1ox-7, gray triangles) and cZOGT2 (2ox-2, white circles; 2ox-7, white triangles) and the corresponding VCs (VC-4, black circles; VC-7, black triangles). Shoot length (A) and number of fully expanded leaves (B) on the main culms of transgenic plants (T2) grown in soil were determined from 21 to 91 d after seed imbibition every 7 d. Mean values ± sd of three plants are shown. C, Transgenic rice seedlings grown in hydroponics for 2 weeks after sowing. Arrowheads highlight the proximal and distal ends of the sheath of the third leaf. Bar = 50 mm. D, Length of the sheath of the third leaf (3LS; means ± sd of three plants). Different lowercase letters indicate statistically significant differences (P < 0.05) of mean values in the Tukey-Kramer test following one-way ANOVA. E, Epidermal cell lengths in the distal part of the sheath of the third leaf. Average lengths for each plant were determined from 185 individual values, and values from three plants were used to calculate the mean for each transgenic line. No significant differences (n.s.) were detected by one-way ANOVA (P < 0.05). [See online article for color version of this figure.]

Figure 9.

Chlorophyll retention in old leaf blades of cZOGT1- and cZOGT2-overexpressing rice lines. Relative chlorophyll contents in leaf blades at the ninth (9LB), 10th (10LB), 11th (11LB), 12th (12LB), and 13th (13LB) nodal positions of main culms were measured in transgenic lines that overexpressed cZOGT1 (1ox-1 and 1ox-7) or cZOGT2 (2ox-2 and 2ox-3) and in VC lines (VC-4 and VC-7); results are expressed in arbitrary units (AU). The plants were grown in soil for 77 d after seed imbibition. Mean values ± sd of three plants are given; no sd is shown where one or more leaves had started to wither.

Figure 10.

Contents of cZOG and tZOG and their precursors in cZOGT-overexpressing rice. Cytokinin derivatives were extracted from shoots of transgenic plants overexpressing cZOGT1 (1ox-7), cZOGT2 (2ox-2 and 2ox-3), and cZOGT3 (3ox-7 and 3ox-9) and from VC plants (VC-1, VC-3, and VC-4) grown in soil for 21 d after sowing (the same samples as in Fig. 7, B and C, were used) and were analyzed by UPLC-MS/MS. The contents of O-glucosides of cZ (cZOG; A), cZR (cZROG; B), cZRP (cZRPsOG; C), tZ (tZOG; D), and tZR (tZROG; E) are presented as means ± sd of three plants. The generations of transgenic plants are indicated underneath the names of the lines. Different lowercase letters indicate statistically significant differences in the same generation as detected by the Tukey-Kramer test (P < 0.05) following one-way ANOVA. FW, Fresh weight; n.s., not significant.