Possible involvement of leaf gibberellins in the clock-controlled expression of XSP30, a gene encoding a xylem sap lectin, in cucumber roots

Abstract

Root-produced organic compounds in xylem sap, such as hormones and amino acids, are known to be important in plant development. Recently, biochemical approaches have revealed the identities of several xylem sap proteins, but the biological functions and the regulation of the production of these proteins are not fully understood. XYLEM SAP PROTEIN 30 kD (XSP30), which is specifically expressed in the roots of cucumber (Cucumis sativus), encodes a lectin and is hypothesized as affecting the development of above-ground organs. In this report, we demonstrate that XSP30 gene expression and the level of XSP30 protein fluctuate in a diurnal rhythm in cucumber roots. The rhythmic gene expression continues for at least two or three cycles, even under continuous light or dark conditions, demonstrating that the expression of this gene is controlled by a circadian clock. Removal of mature leaves or treatment of shoots with uniconazole-P, an inhibitor of gibberellic acid (GA) biosynthesis, dampens the amplitude of the rhythmic expression; the application of GA negates these effects. These results suggest that light signals perceived by above-ground organs, as well as GA that is produced, possibly, in mature leaves, are important for the rhythmic expression of XSP30 in roots. This is the first demonstration of the regulation of the expression of a clock-controlled gene by GA.

Figure 1.

Root-vascular tissue-specific XSP30 expression. A, Root-specific expression of XSP30. Total RNA (10 μg lane^-1) extracted from aboveground organs or roots of 30-d-old cucumber plants was subjected to RNA gel-blot analysis. The transcripts were probed using a full-length XSP30 cDNA and ubiquitin fragments as probes. The ubiquitin cDNA was isolated from cucumber roots, where it is expressed constitutively. B through D, β-Glucuronidase (GUS) activity in P_XSP30::GUS-transgenic hairy roots. GUS activity was observed in the central cylinder of mature roots, but not in the root tip shown in B. GUS-stained transgenic roots were embedded in Technovit 7100 (Kulzer and Co., Werheim, Germany), and thin serial sections were stained with toluidine blue (C) or left un-stained (D). The central cylinder is shown. Scale bars correspond to 100 μm. The arrow and arrowhead indicate GUS staining in the xylem parenchyma and pericycle cells, respectively. E, Seedling development-dependent expression of XSP30 in roots. Total RNA (10 μg lane^-1) extracted at dusk from roots of seedlings at 4, 6, 8, 10, 12, 14, 16, and 18 d after sowing were subjected to RNA gel-blot analysis. The transcripts were probed using a full-length XSP30 cDNA and a ubiquitin fragment as probes.

Figure 2.

Diurnal regulation of XSP30 gene expression and XSP30 content of xylem sap. A and B, Time course of XSP30 expression. Cucumber plants were grown under a 16-h light/8-h dark photoperiod. Total RNA (10 μg lane^-1) was extracted from the roots of seedlings every 4 h, beginning on the 11th d after sowing. RNA samples were subjected to RNA gel-blot analysis with XSP30 cDNA and rRNA probes. Shown are the original autoradiograph in A and the ratio of the intensity of hybridization of XSP30 and rRNA in B. C through E, Detection of XSP30 protein in xylem sap. Xylem sap was collected at 4-h intervals from stems of cucumber plants 30 d after sowing. The proteins in equal volumes of sap (5 μL lane^-1) were separated by SDS-PAGE and detected with anti-XSP30 serum on nylon membranes (C) or silver stained (D). The ratio of the intensity of the XSP30 signal and staining of the total proteins is shown in E. The XSP30 gene expression in cucumber roots, 30 d after sowing, is shown in F. Total RNA (10 μg lane^-1) was extracted from the roots of seedlings every 8 h, beginning on d 30 after sowing. RNA samples were subjected to RNA gel-blot analysis with an XSP30 cDNA and a ubiquitin fragment as probes. The original autoradiograph and the ratio of the intensity of hybridization to the XSP30 and ubiquitin transcripts are shown. Dawn was defined as zeitgeber time 0. The periods of light and dark are indicated as shaded and black bars, respectively.

Figure 3.

Expression of XSP30 under DD or LL. Thirteen-day-old seedlings grown under a 16-h light/8-h dark photoperiod were transferred to DD (A-C) or LL (D-F). Total RNA was extracted every 8 h from roots of treated seedlings. RNA samples were subjected to RNA gel-blot analysis with an XSP30 cDNA and rRNA as probes. The original autoradiograph is shown (A and D), as well as the ratio of the intensity of hybridization to XSP30 and rRNA (B and E). The data were Fourier transformed and the period were estimated using Fast Fourier Transform-Non-Linear Least Squares program as described (Plautz et al., 1997; C and F). The shaded and black bars indicate the original periods of light and dark, respectively.

Figure 4.

Effect of decapitation of aboveground organs on XSP30 expression in roots. Total RNA (10 μg lane^-1) was extracted every 4 h from the roots of 15-d-old cucumber plants grown under a 16-h light/8-h dark photoperiod from intact (A) or decapitated plants (B). Root RNA samples were subjected to RNA gel-blot analysis with an XSP30 cDNA and rRNA as probes. The original autoradiograph is shown (A and B), as well as the ratio of the intensity of hybridization to XSP30 and rRNA (C). The periods of light and dark are indicated as shaded and black bars, respectively.

Figure 5.

Effect of leaf removal on XSP30 expression in roots. The cotyledons plus the shoot apex (A), or the mature first leaf (B), were removed from 13-d-old seedlings, and the roots were collected every 8 h for RNA extraction. Root RNA samples were subjected to RNA gel-blot analysis with an XSP30 cDNA and rRNA as probes. The original autoradiograph is shown (A and B), as well as the ratio of the intensity of hybridization to XSP30 and rRNA (C). The periods of light and dark are indicated as shaded and black bars, respectively.

Figure 6.

Effects of gibberellin on XSP30 expression in roots. Mature first leaves were removed from 13-d-old seedlings, and 2 × 10^-4 m GA₃ (A) or water (B) was applied every 2 d to the cotyledons and shoot apex. Roots were collected every 8 h for RNA extraction. Root RNA samples were subjected to RNA gel-blot analysis with an XSP30 cDNA and rRNA as probes. The original autoradiograph is shown (A and B), as well as the ratio of the intensity of hybridization to XSP30 and rRNA (C). The periods of light and dark are indicated as shaded and black bars, respectively.

Figure 7.

Effect of uniconazole-P and gibberellin on XSP30 expression in roots. Uniconazole-P (10^-4 m; A) or 10^-4 m uniconazole-P plus 2 × 10^-4 m GA₃ (B) was applied to the shoot, including the cotyledons, first leaf, and shoot apex, of 13-d-old seedlings. Roots were collected every 8 h for RNA extraction. Root RNA samples were subjected to RNA gel-blot analysis with an XSP30 cDNA and rRNA as probes. The original autoradiograph is shown (A and B), as well as the ratio of the intensity of hybridization to XSP30 and rRNA (C). The periods of light and dark are indicated as shaded and black bars, respectively.