Arabidopsis Rho-related GTPases: differential gene expression in pollen and polar localization in fission yeast

Abstract

The Rho small GTP-binding proteins are versatile, conserved molecular switches in eukaryotic signal transduction. Plants contain a unique subfamily of Rho-GTPases called Rop (Rho-related GTPases from plants). Our previous studies involving injection of antibodies indicated that the pea Rop GTPase Rop1Ps is critical for pollen tube growth. In this study we show that overexpression of an apparent Arabidopsis ortholog of Rop1Ps, Rop1At, induces isotropic cell growth in fission yeast (Schizosaccharomyces pombe) and that green fluorescence protein-tagged Rop1At displays polar localization to the site of growth in yeast. We found that Rop1At and two other Arabidopsis Rops, Rop3At and Rop5At, are all expressed in mature pollen. All three pollen Rops fall into the same subgroup as Rop1Ps and diverge from those Rops that are not expressed in mature pollen, suggesting a coupling of the structural conservation of Rop GTPases to their gene expression in pollen. However, pollen-specific transcript accumulation for Rop1At is much higher than that for Rop3At and Rop5At. Furthermore, Rop1At is specifically expressed in anthers, whereas Rop3At and Rop5At are also expressed in vegetative tissues. In transgenic plants containing the Rop1At promoter:GUS fusion gene, GUS is specifically expressed in mature pollen and pollen tubes. We propose that Rop1At may play a predominant role in the regulation of polarized cell growth in pollen, whereas its close relatives Rop3At and Rop5At may be functionally redundant to Rop1At in pollen.

Figure 4

Construction of Rop1At promoter:GUS fusion gene. Rop1At genomic clone containing the putative promoter region was cloned into the binary vector as described in the text. Shown is the region joining Rop1At and the GUS gene. The restriction sites used for translational fusion with the GUS gene are shown in bold; underlined sequences are the partial coding region for Rop1At. The last ATG codon shown is the translation-initiation codon for the GUS gene.

Figure 1

Phylogenetic relationship between different Rho-GTPases. Unrooted trees were constructed using PAUP (Swofford, 1993). Amino acid sequences for various plant, animal, and yeast Rho-GTPases were obtained from GenBank using the Blast program. At, Arabidopsis thaliana; Bv, Beta vulgaris; Ce, Caenorhabditis elegans; Dd, Dictyostelium discoideum; Dm, Drosophila melanogaster; Gg, Gallus gallus; Gh, Goosypium hirsutum; Hs, Homo sapiens; Lj, Lotus japonicus; Mm, Mus musculus; Ps, Pisum sativum; Sc, Saccharomyces cerevisiae; Sp, S. pombe. A, Rho family tree showing phylogenetic relationships among major subfamilies from different eukaryotic kingdoms. This tree does not include all known members of Rho-GTPases, since several novel Rho-GTPases that do not fall within any of the major subfamilies are not included. B, Arabidopsis Rop family tree. Members shown include those described in this paper and those whose complete coding sequences are available in the Arabidopsis database.

Figure 2

Alignment of the C-terminal divergent sequences of Arabidopsis Rop proteins. Predicted amino acid sequences including the C-terminal variable region for Arabidopsis Rop members were aligned using the MegAlign program. Residues identical to Rop1Ps sequences are indicated by dots; dashes represent gaps introduced into the alignment.

Figure 3

RT-PCR analyses of Rop gene expression in various Arabidopsis tissues. A, Demonstration of Rop isogene-specific PCR amplification. Two sets of PCR reactions for each Rop isogene were performed. Lane M, DNA marker; lanes N, negative control, which involves the same primers and template DNAs containing a mixture of equal amounts of cDNA or genomic DNA for each of the other five Rop genes; lanes P, positive control, which involves a specific cDNA (for Rop1At, Rop2At, Rop3At, and Rop6At) or genomic DNA (for Rop4At and Rop5At) as templates and corresponding gene-specific primers. Expected cDNA lengths of amplified fragments are shown in Table I. The PCR reaction conditions are described in text. B, Accumulation of various Arabidopsis Rop transcripts in mature pollen. Total pollen RNA was isolated and the cDNA derived was amplified for 40 cycles using Rop gene-specific primers as described in the text. C, Organ distribution of various Arabidopsis Rop transcripts. RT-PCR was performed using Rop gene-specific primers described in A and total RNAs from different tissues as indicated. Act2 RT-PCR was included as a constitutive control. The number of PCR cycles was: 25 for Rop1At, Rop2At, Rop3At Rop4At, and act2, and 45 for Rop5At and Rop6At. D, Analyses of Rop1At mRNA accumulation during floral development. Total RNAs isolated from Arabidopsis floral buds and flowers at different stages were used for RT-PCR using the reaction conditions described in B. Flower stages were estimated as described previously (Smyth et al., 1990). Stages 1 to 9, Initiation and formation of floral primordia and organ differentiation; stages 10 to 13, organs fully developed, anthesis; stage 14, anthers extended above stigma, pollination; stage 15, stigma extends above anthers; stage 16, petals and sepals wither; siliques, developing siliques before seed maturation.

Figure 5

Histochemical localization of GUS expression in transgenic Arabidopsis plants carrying the Rop1At promoter:GUS fusion gene. Various parts of transgenic T₂ plants were stained with 5-bromo-4-chloro-3-indolyl-β-glucuronide cyclohexylamine salt, as described in the text. Typical staining patterns are shown. Anthers were costained with DAPI to determine the developmental stages of pollen. an, Anther; ms, microspore; p, pollen; tp, tapetum; tt, transmitting tissue; 2n, pollen at bicellular stage; 3n, pollen at tricellular stage. A, Early stages of floral buds; B, anthers at the stage of microspore development; C, anthers just prior to anthesis; D, anthers just after anthesis; E, stigma and anthers at anthesis; F, stigma and anthers after anthesis; G, stigma from stage-16 flowers; H, pollen at various developmental stages; I, pollen from H costained with DAPI.

Figure 6

Overexpression of Rop1At and polar localization of GFP:Rop1At fusion in fission yeast. The Rop1At or GFP:Rop1At fusion genes were cloned into pREP3X under the control of a thiamine-repressible promoter and introduced into fission yeast, as described in the text. A, Yeast cells with pREP3X-Rop1At grown in a repressive medium containing 5 mm thiamine. Cells have a normal morphology. B, Yeast cells containing pREP3X-Rop1At grown in a nonrepressive medium lacking thiamine. Greater than 90% were abnormal in shape. C and D, Yeast cells containing pREP3X-GFP:Rop1At grown in a partially repressive medium containing 2 mm thiamine and examined under an epifluorescence microscope. Typical GFP localization patterns are indicated: long arrow, unipolar localization; thick arrow, bipolar localization; long arrowhead, localization to the septum; short arrowhead, nonpolar localization in GFP-Rop1At-overexpressing cells.