Two Arabidopsis cyclin A3s possess G1 cyclin-like features.

A-type cyclins (CYCAs) are a type of mitotic cyclin and are closely related to cyclin B. Plant CYCAs are classified into three subtypes (CYCA1-CYCA3), among which CYCA3 has been suggested to show a biased expression during the G1-to-S phase. We characterised Arabidopsis CYCA3s (CYCA3;1-CYCA3;4) in terms of expression pattern and protein function. CYCA3;1 and CYCA3;2 transcripts were highly accumulated at the G1/S phase, whereas CYCA3;4 was constantly expressed during the cell cycle. Expressions of CYCA3;1 and CYCA3;2 were observed in actively dividing tissues, such as root and shoot apical meristems and lateral root primordia. Overexpression of CYCA3;1 or CYCA3;2 distorted apical dominance in Arabidopsis, indicating that they have critical functions in shoot meristems. In insect cells, CYCA3;1 formed an active kinase complex with CDKA;1, an orthologue of the yeast Cdc2/Cdc28p, and phosphorylated retinoblastoma-related protein, a key regulator in the transition from the G1 to the S phase. Our results suggest that Arabidopsis CYCA3;1 and CYCA3;2 are distinct members of the G1 cyclin family that play an important role in meristematic tissues.

Targeted degradation of the cyclin-dependent kinase inhibitor ICK4/KRP6 by RING-type E3 ligases is essential for mitotic cell cycle progression during Arabidopsis gametogenesis.

Following meiosis, plant gametophytes develop through two or three rounds of mitosis. Although the ontogeny of gametophyte development has been defined in Arabidopsis thaliana, the molecular mechanisms regulating mitotic cell cycle progression are not well understood. Here, we report that RING-H2 group F 1a (RHF1a) and RHF2a, two RING-finger E3 ligases, play an important role in Arabidopsis gametogenesis. The rhf1a rhf2a double mutants are defective in the formation of male and female gametophytes due to interphase arrest of the mitotic cell cycle at the microspore stage of pollen development and at female gametophyte stage 1 of embryo sac development. We demonstrate that RHF1a directly interacts with and targets a cyclin-dependent kinase inhibitor ICK4/KRP6 (for Interactors of Cdc2 Kinase 4/Kip-related protein 6) for proteasome-mediated degradation. Inactivation of the two redundant RHF genes leads to the accumulation of ICK4/KRP6, and reduction of ICK4/KRP6 expression largely rescues the gametophytic defects in rhf1a rhf2a double mutants, indicating that ICK4/KRP6 is a substrate of the RHF E3 ligases. Interestingly, in situ hybridization showed that ICK4/KRP6 was predominantly expressed in sporophytes during meiosis. Our findings indicate that RHF1a/2a-mediated degradation of the meiosis-accumulated ICK4/KRP6 is essential to ensure the progression of subsequent mitoses to form gametophytes in Arabidopsis.

Two OsGASR genes, rice GAST homologue genes that are abundant in proliferating tissues, show different expression patterns in developing panicles.

Two different types of genes for rice GA-stimulated transcript (GAST) homologue genes, Oryza sativa GA-stimulated transcript-related gene 1 (OsGASR1) and gene 2 (OsGASR2), were found. Both OsGASR proteins contain a cysteine-rich domain highly conserved among GAST family proteins in their C-terminal regions. Gibberellin A3 (GA3) stimulated expression of both OsGASRs in the wild-type Nipponbare and GA3 synthesis-deficient mutant. Expression of both OsGASRs apparently increased when cell proliferation entered the logarithmic phase, and rapidly reduced when cell proliferation was temporarily halted. RT-PCR analysis indicated different expression patterns of these genes in developing panicles. OsGASR1 was limitedly but strongly expressed in florets while OsGASR2 was expressed in both florets and branches. In situ hybridization showed that they were strongly expressed in the root apical meristem (RAM) and shoot apical meristem (SAM), but little signals were detected in mature leaves. Transient expression of OsGASR-GFP fusion proteins in onion epidermal cells revealed that both OsGASR proteins localized to the apoplasm or cell wall. These results suggest that OsGASR1 and OsGASR2 were involved in cell division and might play diverse roles in differentation of panicles.

Diverse phosphoregulatory mechanisms controlling cyclin-dependent kinase-activating kinases in Arabidopsis.

For the full activation of cyclin-dependent kinases (CDKs), not only cyclin binding but also phosphorylation of a threonine (Thr) residue within the T-loop is required. This phosphorylation is catalyzed by CDK-activating kinases (CAKs). In Arabidopsis three D-type CDK genes (CDKD;1-CDKD;3) encode vertebrate-type CAK orthologues, of which CDKD;2 exhibits high phosphorylation activity towards the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. Here, we show that CDKD;2 forms a stable complex with cyclin H and is downregulated by the phosphorylation of the ATP-binding site by WEE1 kinase. A knockout mutant of CDKD;3, which has a higher CDK kinase activity, displayed no defect in plant development. Instead, another type of CAK - CDKF;1 - exhibited significant activity towards CDKA;1 in Arabidopsis root protoplasts, and the activity was dependent on the T-loop phosphorylation of CDKF;1. We propose that two distinct types of CAK, namely CDKF;1 and CDKD;2, play a major role in CDK and CTD phosphorylation, respectively, in Arabidopsis.

Plastid DNA polymerases from higher plants, Arabidopsis thaliana.

Previously, we described a novel DNA polymerase, designated as OsPolI-like, from rice. The OsPolI-like showed a high degree of sequence homology with the DNA polymerase I of cyanobacteria and was localized in the plastid. Here, we describe two PolI-like polymerases, designated as AtPolI-like A and AtPolI-like B, from Arabidopsis thaliana. In situ hybridization analysis demonstrated expression of both mRNAs in proliferating tissues such as the shoot apical meristem. Analysis of the localizations of GFP fusion proteins showed that AtPolI-like A and AtPolI-like B were localized to plastids. AtPolI-like B expression could be induced by exposure to the mutagen H(2)O(2). These results suggested that AtPolI-like B has a role in the repair of oxidation-induced DNA damage. Our data indicate that higher plants possess two plastid DNA polymerases that are not found in animals and yeasts.

Characterization of the origin recognition complex (ORC) from a higher plant, rice (Oryza sativa L.).

The origin recognition complex (ORC) protein plays a critical role in DNA replication through binding to sites (origins) where replication commences. The protein is composed of six subunits (ORC1 to 6) in animals and yeasts. Our knowledge of the ORC protein in plants is, however, much less complete. We have performed cDNA cloning and characterization of ORC subunits in rice (Oryza sativa L. cv. Nipponbare) in order to facilitate study of plant DNA replication mechanisms. Our previous report provided a description of a gene, ORC1 (OsORC1), that encodes one of the protein subunits. The present report extends this initial analysis to include the genes that encode four other rice ORC subunits, OsORC2, 3, 4 and 5. Northern hybridization analyses demonstrated the presence of abundant transcripts for all OsORC subunits in shoot apical meristems (SAM) and cultured cells, but not in mature leaves. Interestingly, only OsORC5 showed high levels of expression in organs in which cell proliferation is not active, such as flag leaves, the ears and the non-tip roots. The pattern of expression of OsORC2 also differed from other OsORC subunits. When cell proliferation was temporarily halted for 6-10 days by removal of sucrose from the growth medium, expression of OsORC1, OsORC3, OsORC4 and OsORC5 was substantially reduced. However, the level of expression of OsORC2 remained constant. We suggest from these results that expression of OsORC1, 3, 4 and 5 are correlated with cell proliferation, but the expression of OsORC2 is not.

Characterization of Rad6 from a higher plant, rice (Oryza sativa L.) and its interaction with Sgt1, a subunit of the SCF ubiquitin ligase complex.

We report here the existence of interactions between a ubiquitin-conjugating enzyme, Rad6, from rice, Oryza sativa L. cv. Nipponbare (OsRad6), and Sgt1 (OsSgt1), a novel subunit of the SCF ubiquitin ligase complex. Rad6 is not only related to post-replicational repair but also to the proteasome system, while Sgt1 has a function in kinetochore assembly. The relationship between the two is unexpected, but of great interest. The open reading frames of OsRad6 and OsSgt1 encode predicted products of 152 and 367 amino acid residues, respectively, with molecular weights of 17.3 and 40.9kDa. Two-hybrid and pull-down analyses indicated that OsRad6 binds to OsSgt1, and transcripts of both OsRad6 and OsSgt1 were found to be strongly expressed only in the proliferating tissues such as the shoot apical meristem, suggesting that their expression is cell cycle-dependent. The amount of the Rad6 mRNA in cultured cells increased rapidly after division was halted, and mRNA levels of Rad6 and Sgt1 were induced by UV- and DNA-damaging agents such as MMS or H(2)O(2). The Rad6 pathway for repair or the proteasome system may thus require Sgt1 as ubiquitin-conjugating enzyme.