Mitosis-specific promoter of the alfalfa cyclin-dependent kinase gene (Medsa;CDKB2;1) is activated by wounding and ethylene in a non-cell division-dependent manner

Abstract

Cyclin-dependent serine/threonine kinases (CDKs) have pivotal roles in regulating the eukaryotic cell cycle. Plants possess a unique class of CDKs (B-type CDKs) with preferential protein accumulation at G2/M-phases; however, their exact functions are still enigmatic. Here we describe the functional characterization of a 360-bp promoter region of the alfalfa (Medicago sativa) CDKB2;1 gene in transgenic plants and cell lines. It is shown that the activity of the analyzed promoter was characteristic for proliferating meristematic regions in planta and specific for cells in the G2/M-phases in synchronized cell cultures. Immunohistochemical analysis of transgenic root sections further confirmed the correlation of the expression of the CDKB2;1 promoter-linked reporter genes with the accumulation of the correspondent kinase. It was found that, in addition to auxin (2,4-dichlorophenoxyacetic acid) treatment, wounding could also induce both the reporter and endogenous genes in transgenic leaf explants. Furthermore, ethylene, known as a wound-response mediator, had a similar effect. The gene activation in response to wounding or ethephon was faster and occurred without the induction of cell cycle progression in contrast to the control auxin treatment. In silico analysis of this promoter indeed revealed the presence of a set of cis-elements, indicating not only cell cycle- but wound- and ethylene-dependent regulation of this CDK gene. Based on the presented data, we discuss the functional significance of the complex regulation of mitosis-specific CDK genes in plants.

Figure 1.

Alignment of cloned 5′ upstream regions of Medsa;CDKB2;1 gene. Corresponding alfalfa genomic DNA fragments were classified (fpr10, fpr13, and fpr15) and aligned with the homologous M. truncatula (AC144481) genomic sequence. Putative promoter motifs are underlined and indicated: W-box (WRKY-binding site), WUN, E2FB (E2FB transcription factor recognizable element), CCAAT-box, ERE, MSA, TATA-box, P-box (gibberellin-responsive element), and TCA (wound-responsive element). Numbers indicate the distance (bp) from the transcription start (0), and the sense or antisense orientation is shown as (+) or (−), respectively.

Figure 2.

In planta characterization of the activity of the Medsa;CDKB2;1 promoter. A, In vivo luc assay was carried out on transgenic alfalfa (fpr15:luc) calli. Normal light image (on the left) and luminescent light image produced after luciferin treatment (on the right) are shown. Red arrowheads point to the not transformed control calli treated with luciferin. B to H, Histochemical localization of GUS activity (blue) is shown in fpr15:GUS alfalfa plants: shoot apex (B), young leaf (C), lateral roots in different stages (D), root cross section with lateral root meristem (E), flower buds (F), young anthers (G), and seed primordia (H). Medsa;CDKB2;1 kinase and fpr15-driven luc reporter gene products were detected immunohistochemically on consecutive sections (I and J) with Medsa;CDKB2;1 and luc antibodies, respectively, displaying similar pattern of brown spots (highlighted with red arrowheads). LRM, Lateral root meristem; EP, epidermis; CX, cortex; S, stele.

Figure 3.

Expression analysis of fpr15:luc and Medsa;CDKB2;1 in a synchronously dividing alfalfa cell culture. Transcript level of Medsa;CDKB2;1, luc, and Medsa;CDKA;1 genes were monitored in an HU-synchronized cell culture initiated from fpr15:luc transgenic alfalfa plants. Samples were taken every 3 h after the release from HU block. BW, Before removing HU; AW, after removing HU. A, Frequency of cells in various cell cycle phases (G1, S, G2, mitosis) was determined by flow cytometry and by counting the mitotic index. B, Total RNA (bottom section) was hybridized with the [³²P]-labeled specific DNA probes: CDKB2;1 kinase, luc, and CDKA;1 kinase genes, respectively. The CDKB2;1 promoter regulated genes accumulated in the interval of 12 to 21 h; in contrast, the CDKA;1 expression was constitutive throughout the entire cell cycle.

Figure 4.

Induction of the Medsa;CDKB2;1 promoter by wounding (A and B), ethephon (C), and 2,4-D (D). Detached leaves were cultivated on B5II solid medium for 0.5, 1, 3, and 4 d following the indicated treatments. A, GUS expression appeared (blue) adjacent to the injury marked with red arrows after GUS assay in fpr15:GUS plant. Western blot confirmed the endogenous CDKB2;1 kinase accumulation in nontransgenic leaves after wounding. B, Cross section of wounded leaf after 4 d of cultivation demonstrates that GUS expression spread farther from the edge of the wound in the equatorial plane of the leaf. Arrowheads show the wounding sites. C, fpr15:luc leaves were placed on solid BII5 medium supplemented with 10 mg/L ethephon, and the luc assay-generated luminescent light was detected at given time points. In parallel experiments, immunoblot assays were executed with anti-Medsa;CDKB2;1 on protein isolated from ethephon-treated, nontransgenic alfalfa leaves, which revealed an increasing amount of CDKB2;1 kinase with elapsed time. D, fpr15:GUS leaves were incubated on BII5 medium, in the presence of 1 mg/L 2,4-D. The accumulation of the endogenous CDKB2;1 protein from the third day in nontransgenic leaves is shown by western blot. E, Cell cycle activity in wounded or hormone-treated leaves of alfalfa was characterized by flow-cytometric analysis after 0.5, 1, 3, and 4 d. The wounded or ethephon-treated samples had no change in relative DNA contents of cells, while 2,4-D-treated leaves exhibited an increased frequency of cells in the S and G2 cell cycle phases. Histograms represent average of three independent measurements. F, fpr15:luc leaves were wounded or 2,4-D treated, and placed onto solid BII5 medium in the presence or absence of HU. On the left side the leaves are shown in normal light, and on the right side the emitted luminescent light was captured upon luc assay carried out on the third day. In the absence of HU, the isolated promoter is active in the wounded or in the 2,4-D-treated leaves. In the presence of HU, the fpr15 remained active only in the case of wounding but was silent in the 2,4-D-treated leaves, indicating that the wound inducibility of the promoter is independent from the cell cycle.