Plant Molecular Biology

Kawabe, Akira; Matsunaga, Sachihiro; Nakagawa, Katsuyuki; Kurihara, Daisuke; Yoneda, Arata; Hasezawa, Seiichiro; Uchiyama, Susumu; Fukui, Kiichi

doi: 10.1007/s11103-005-3454-xpmid: 16028112

The Aurora kinase family is a well-characterized serine/threonine protein kinase family that regulates different processes of mitotic events. Although functions of animal and yeast Aurora kinases have been analyzed, plant aurora kinases were not identified and characterized. We identified three Aurora kinase orthologs in Arabidopsis thaliana and designated these as AtAUR1, AtAUR2, and AtAUR3. These AtAURs could phosphorylate serine 10 in histone H3, in vitro. Dynamic analyses of GFP-fused AtAUR proteins revealed that AtAUR1 and AtAUR2 localized at the nuclear membrane in interphase and located in mitotic spindles during cell division. AtAUR1 also localized in the cell plates. AtAUR3 showed dot-like distribution on condensed chromosomes at prophase and then localized at the metaphase plate. At late anaphase, AtAUR3 is evenly localized on chromosomes. The localization of AtAUR3 during mitosis is very similar to that of phosphorylated histone H3. Interestingly, an overexpression of AtAUR3 induces disassembly of spindle microtubules and alteration of orientation of cell division. Our results indicate that plant Aurora kinases have different characters from that of Aurora kinases of other eukaryotes.

Peng, Hsiao-Ping; Lin, Ter-Yun; Wang, Ning-Ning; Shih, Ming-Che

doi: 10.1007/s11103-005-3573-4pmid: 16028113

Ethylene plays an essential role in response to hypoxic stress in plants. In most plant species, 1-aminocyclopropane-1-carboxylate synthase (ACS) is the key enzyme that regulates the production of ethylene. We examined the expression of ACS genes in Arabidopsis during hypoxia. Our data showed that the expression of 4 of the 12 Arabidopsis ACS genes, ACS2, ACS6, ACS7, and ACS9, is induced during hypoxia with three distinct patterns. The hypoxic induction of ACS9 is inhibited by aminooxy acetic acid, an inhibitor of ethylene biosynthesis. In addition, the hypoxic induction of ACS9 is also reduced in etr1-1 and ein2-1, two ethylene insensitive mutants in ethylene-signaling pathways, whereas the addition of 1-aminocyclopropane-1-carboxylic acid, a direct precursor of ethylene, does not induce ACS9 under normoxic conditions. These results indicate that ethylene is needed, but not sufficient, for the induction of ACS9 during hypoxia. This pattern of regulation is similar to that of ADH that encodes alcohol dehydrogenase, which we have reported previously. In contrast, the increased ethylene production during hypoxia has an inhibitory effect on ACS2 induction in roots, whereas ethylene has no effect on the hypoxic induction of ACS6 and ACS7. Based on these results, we propose that two signaling pathways are triggered during hypoxia. One pathway leads to the activation of ACS2, ACS6, and ACS7, whereas the other pathway leads to the activation of ADH and ACS9.

Khan, Md.; Jan, Asad; Karibe, Hideji; Komatsu, Setsuko

doi: 10.1007/s11103-005-4013-1pmid: 16028114

To identify the gibberellin (GA) signaling components involved in rice leaf sheath elongation process, protein phosphorylation changed by GA3 was analyzed. The protein kinase activities in rice leaf sheath were assessed in an in-gel kinase assay using SDS-polyacrylamide gel containing histone III-S as a substrate. The activity of a putative 54-kDa calcium dependent protein kinase (CDPK) in cytosolic fraction in rice leaf sheath increased significantly by GA3. Further, phosphorylation status of the proteins changed by GA3 in rice leaf sheath were detected by in vitro protein phosphorylation followed by two-dimensional polyacrylamide gel electrophoresis and the phosphoproteins were identified by mass spectrometry. Sixty phosphoproteins was detected after in vitro protein phosphorylation and the phosphorylation of 7 proteins was enhanced by GA3 treatment. The addition of GA3 treated cytosolic fraction of leaf sheath further increased the phosphorylation of 4 proteins, glyoxalase-I, cytoplasmic malate dehydrogenase, glyceraldehydes-3-phosphate dehydrogenase and another unknown protein. The protein kinase inhibitor, staurosporine inhibited the phosphorylation of these proteins in vitro. Other hormones, particularly, indole acetic acid, 6-benzylaminopurine and abscisic acid did not change the phosphorylation status of these proteins. The identified proteins did not show any change by GA3 treatment at transcription level. The abundance of glyoxalase-I and cytoplasmic malate dehydrogenase remained unchanged by GA3 treatment as detected on 2D-gel by silver staining, unlike for glyceraldehydes-3-phosphate dehydrogenase. Results suggest that the phosphoproteins, glyoxalase-I and cytoplasmic malate dehydrogenase in rice leaf sheath could be important signaling components of GA3, downstream to 54-kDa CDPK.

Casazza, Anna; Rossini, Silvia; Rosso, Mario; Soave, Carlo

doi: 10.1007/s11103-005-4090-1pmid: 16028115

Plants exposed to photoinhibitory conditions respond by accumulation of the early light-induced proteins (ELIPs) with a potential photoprotective function. In Arabidopsis thaliana two genes (Elip1 and Elip2) encode for two ELIP proteins: evidence exists that the two genes are differentially regulated but their precise function is unclear. Mutants null for one or the other Elip gene can help in elucidating ELIPs role and here we describe the expression profile of ELIP1 and ELIP2, and the phenotype of such null mutants. Both ELIPs accumulate during greening of etiolated seedlings and in mature plants the transcripts fluctuate diurnally without protein accumulation. Steady-state transcript level of both genes increases in response to high light with transcription of Elip1 much more sensitive than that of Elip2 to increasing irradiation at 22 °C. At 4 °C instead Elip2 is strongly transcribed even at growing light. Furthermore, only ELIP1 accumulates under high light at 22 °C while both proteins accumulate at 4 °C. These results indicate the existence of a differential regulation of ELIPs expression in response to light or chilling stress with mechanisms active either at transcriptional and post-transcriptional level. Phenotypically, the mutants behave as the wild type as far as sensitivity to light- or light and cold-induced short-term photoinhibition, while both ELIPs are necessary to ensure a high rate of chlorophyll accumulation during deetiolation in continuous high light.

Zhao, Xiang; Liu, Mao; Li, Jia; Guan, Chun; Zhang, Xian

doi: 10.1007/s11103-005-4162-2pmid: 16028116

Wheat (Triticum aestivum L.) is an important crop and requires long day and short night to flower. To study the molecular mechanism of photoperiodic regulation of flowering in this species, we isolated a wheat TaGI1 gene, an ortholog of GIGANTEA (GI) in Arabidopsis. RNA blot hybridization revealed that TaGI1 is expressed in leaves in a rhythmic manner under long day and short day conditions and its rhythmic expression is regulated by photoperiods and circadian clocks. Further study demonstrated that the TaGI1 rhythmic expression in the leaves of seedlings is initiated by photoperiods, implying that TaGI1 does not show circadian regulation until after being entrained in a light/dark cycle. Interestingly, TaGI1 mRNA was detected in adaxial epidermal cells right above the vascular bundles of leaves, suggesting that the localization of TaGI1 transcripts in leaves may function to regulate flowering in response to photoperiods. Since overexpression of TaGI1 altered flowering time in wild type and complemented the gi mutant in Arabidopsis, it confirmed that TaGI1 is an ortholog of GI in Arabidopsis.

Shigaki, Toshiro; Vyzasatya, Ravindranadha; Sivitz, Ali; Ward, John; Sze, Heven; Hirschi, Kendal

doi: 10.1007/s11103-005-4323-3pmid: 16028117

To facilitate the characterization of plant genes, the Cre-loxP site-specific recombination system was adapted to make reporter vectors for plant expression studies. This system allows promoter fragments to be cloned into a small vector (univector) and subsequently recombined in vitro with binary vectors containing different reporter genes precisely at near-perfect efficiency. We have constructed univector-adapted vectors with three reporters, β-glucuronidase, luciferase, and green fluorescent protein, and a BASTA-resistance gene for selection of plant transformants. Expression in plants using the new system was validated by comparison to conventional reporter vectors. These new vectors are efficient and economical alternatives to the other plant reporter vectors currently available. The royalty-free Cre-loxP system serves as a platform for the future expansion of recombination-based cloning vectors for plant research.

Ottow, Eric; Polle, Andrea; Brosché, Mikael; Kangasjärvi, Jaakko; Dibrov, Pavel; Zörb, Christian; Teichmann, Thomas

doi: 10.1007/s11103-005-4525-8pmid: 16028118

PeNhaD1 encodes a putative Na+/H+ antiporter from the salt-resistant tree Populus euphratica. It is the first characterization of a member of the NhaD type ion transporter family of plant origin. Homology searches revealed its close relation to functionally characterized microbial Na+/H+ antiporters VpNhaD and VcNhaD. Na+/H+ antiporters have proven to play a key role in salt resistance, both in plants and bacteria. Under salt stress transcript levels of PeNhaD1 were maintained only in the salt-resistant P. euphratica, but collapsed in Populus × canescens, a salt-sensitive species. To address the function of PeNhaD1, complementation studies with the salt-sensitive Escherichia coli EP432 mutant strain, lacking activity of the two Na+/H+ antiporters EcNhaA and EcNhaB were carried out. PeNhaD1 was able to restore growth of EP432 under stress imposed by up to 400 mM NaCl demonstrating its protective function. Growth rates of EP432 were always highest at pH 5.5 while growth was suppressed under salt stress at pH 7.0 and pH 8.0 suggesting that the antiporter activity is strongly pH dependent. Element analyses of EP432 cells complemented with PeNhaD1 growing under salt stress showed that salt resistance was correlated with a significant reduction in sodium accumulation. These results suggest that PeNhaD1 might play a role in the salt resistance of P. euphratica.

Lehti-Shiu, Melissa; Adamczyk, Benjamin; Fernandez, Donna

doi: 10.1007/s11103-005-4546-3pmid: 16028119

MADS domain factors play important roles as developmental regulators in plants. In Arabidopsis thaliana, MADS domain proteins have been shown to regulate various processes during the vegetative and reproductive phases. Relatively little is known, however, about family members expressed during the embryonic phase and their function. To determine which MADS-box genes are expressed during the embryonic phase in Arabidopsis, a family-wide survey involving gene-specific primers and RT-PCR was conducted. Transcripts corresponding to 64 (out of 109 total) family members could be detected in RNA samples isolated from embryonic culture tissue. Eight MADS-box genes that appear to be expressed at higher levels during the embryonic phase than in seedlings or in inflorescence apices were identified. The spatial pattern of expression in developing seeds was characterized for four MADS-box genes (FLOWERING LOCUS C, FLOWERING LOCUS M, AGAMOUS-LIKE 15, and AGAMOUS-LIKE 18) using reporter constructs encoding translational fusions to GUS. All four are expressed in cells throughout the endosperm and embryo. Finally, to test the hypothesis that AGAMOUS-LIKE15 (AGL15) and AGAMOUS-LIKE18 (AGL18) play essential roles during the embryonic phase, plants carrying T-DNA insertions that disrupt these genes were isolated. No embryo defects were observed in agl15 or agl18 single mutants or in agl15agl18 double mutants. These results indicate that multiple regulatory pathways that involve MADS domain factors are likely to operate in embryonic tissues, and that genetic and/or functional redundancy are likely to be as prevalent as in other phases of the life cycle.

Renna, Luciana; Hanton, Sally; Stefano, Giovanni; Bortolotti, Lauren; Misra, Vikram; Brandizzi, Federica

doi: 10.1007/s11103-005-4618-4pmid: 16028120

Golgins are a family of coiled-coil proteins that are associated with the Golgi apparatus. They are necessary for tethering events in membrane fusion and may act as structural support for Golgi cisternae. Here we report on the identification of an Arabidopsis golgin which is a homologue of CASP, a known transmembrane mammalian and yeast golgin. Similar to its homologues, the plant CASP contains a long N-terminal coiled-coil region protruding into the cytosol and a C-terminal transmembrane domain with amino acid residues which are highly conserved across species. Through fluorescent protein tagging experiments, we show that plant CASP localizes at the plant Golgi apparatus and that the C-terminus of this protein is sufficient for its localization, as has been shown for its mammalian counterpart. In addition, we demonstrate that the plant CASP is able to localize at the mammalian Golgi apparatus. However, mutagenesis of a conserved tyrosine in the transmembrane domain revealed that it is necessary for ER export and Golgi localization of the Arabidopsis CASP in mammalian cells, but is not required for its correct localization in plant cells. These data suggest that mammalian and plant cells have different mechanisms for concentrating CASP in the Golgi apparatus.

Khaled, Abdel-Sabour; Vernoud, Vanessa; Ingram, Gwyneth; Perez, Pascual; Sarda, Xavier; Rogowsky, Peter

doi: 10.1007/s11103-005-5219-ypmid: 16028121

ZmOCL1 is the founding member of the ZmOCL (Outer Cell Layer) family encoding putative transcription factors of the HD-ZIP IV class. It is expressed in the L1 cell layer of the embryo and several other tissues of maize. After determination of the intron/exon structure a mutator insertion was isolated in the upstream region. No notable phenotypes and wildtype levels of ZmOCL1 transcript were observed in homozygous mutant plants. In contrast transgenic plants carrying a fusion of the repressor domain of the Drosophila Engrailed gene with the DNA binding and dimerisation domains of ZmOCL1 showed a transient reduction of embryo, endosperm and kernel size that was most obvious around 15 DAP. An inverse relationship was observed between the degree of size reduction and the expression level of the transcript. In reciprocal crosses the size reduction was only observed when the transgenic plants were used as females and no expression of male transmitted transgenes was detected. Smaller kernels resembled younger kernels of wild-type siblings indicating that interference with ZmOCL1 function leads to an overall slow-down of early kernel development. Based on marker gene analysis ZmOCL1 may act via a modification of gibberellin levels. Phylogenetic analyses based on the intron/exon structure and sequence similarities of ZmOCL1 and other HD-ZIP IV proteins from maize, rice and Arabidopsis helped to identify orthologues and suggested an evolution in the function of individual genes after the divergence of monocots and dicots.