The virescent3 (v3) and stripe1 (st1) mutants in rice (Oryza sativa) produce chlorotic leaves in a growth stage-dependent manner under field conditions. They are temperature-conditional mutants that produce bleached leaves at a constant 20°C or 30°C but almost green leaves under diurnal 30°C/20°C conditions. Here, we show V3 and St1, which encode the large and small subunits of ribonucleotide reductase (RNR), RNRL1, and RNRS1, respectively. RNR regulates the rate of deoxyribonucleotide production for DNA synthesis and repair. RNRL1 and RNRS1 are highly expressed in the shoot base and in young leaves, and the expression of the genes that function in plastid transcription/translation and in photosynthesis is altered in v3 and st1 mutants, indicating that a threshold activity of RNR is required for chloroplast biogenesis in developing leaves. There are additional RNR homologs in rice, RNRL2 and RNRS2, and eukaryotic RNRs comprise a 2 b 2 heterodimers. In yeast, RNRL1 interacts with RNRS1 (RNRL1:RNRS1) and RNRL2:RNRS2, but no interaction occurs between other combinations of the large and small subunits. The interacting activities are RNRL1:RNRS1 . RNRL1:rnrs1(st1) . rnrl1(v3):RNRS1 . rnrl1(v3):rnrs1(st1), which correlate with the degree of chlorosis for each genotype. This suggests that missense mutations in rnrl1(v3) and rnrs1 (st1) attenuate the first ab dimerization. Moreover, wild-type plants exposed to a low concentration of an RNR inhibitor, hydroxyurea, produce chlorotic leaves without growth retardation, reminiscent of v3 and st1 mutants. We thus propose that upon insufficient activity of RNR, plastid DNA synthesis is preferentially arrested to allow nuclear genome replication in developing leaves, leading to continuous plant growth.Plastid development from proplastids to photosynthetically active chloroplasts is one of the most important metabolic processes during plant growth and is coordinately regulated by both plastid and nuclear genes. Chloroplast development is largely under nuclear control, because the coding capacity of plastids is very limited and nuclear genes encode more than 95% of the chloroplast proteins. Thus, the precise coordination of gene expression through two-way signaling between plastids and the nucleus is essential for chloroplast biogenesis in plant cells (Mandel et al., 1996;Koussevitzky et al., 2007).A number of chlorophyll (Chl)-and chloroplastassociated mutations that affect leaf coloration and/or seedling viability have been identified and are referred to as virescent (v), stripe (st), albino, chlorina, zebra, and yellow variegated depending on their diverse phenotypes. Among these mutants, v plants suffer from Chl deficiency in the leaves that develop during the early growth stages and produce mostly green leaves during the late growth stages (Archer and Bonnett, 1987). This developmental phenotype suggests that some of the key factors required for Chl synthesis and/or chloroplast development are absent or insufficient at the earlier developmental stages but are present at ade...

SummaryGuanylate kinase (GK) is a critical enzyme in guanine nucleotide metabolism pathways, catalyzing the phosphorylation of (d)GMP to (d)GDP. Here we show that a novel gene, VIRESCENT 2 (V2), encodes a new type of GK (designated pt/mtGK) that is localized in plastids and mitochondria. We initially identified the V2 gene by positional cloning of the rice v2 mutant. The v2 mutant is temperature-sensitive and develops chlorotic leaves at restrictive temperatures. The v2 mutation causes inhibition of chloroplast differentiation; in particular, it disrupts the chloroplast translation machinery during early leaf development [Sugimoto et al. (2004) Plant Cell Physiol. 45, 985]. In the bacterial and animal species studied to date, GK is localized in the cytoplasm and participates in maintenance of the guanine nucleotide pools required for many fundamental cellular processes. Phenotypic analysis of rice seedlings with RNAi knockdown of cytosolic GK (designated cGK) showed that cGK is indispensable for the growth and development of plants, but not for chloroplast development. Thus, rice has two types of GK, as does Arabidopsis, suggesting that higher plants have two types of GK. Our results suggest that, of the two types of GK, only pt/mtGK is essential for chloroplast differentiation.

The rice virescent-2 mutant (v(2)) is temperature conditional and develops chlorotic, chloroplast-deficient leaves at the restrictive temperature. In the v(2) mutant, plastid-encoded proteins involved in photosynthesis and plastid transcriptional regulation were not detectable at any time during chloroplast differentiation. However, the plastid transcripts for these two classes of proteins behaved differently in the mutant, with those for the plastid transcription/translation apparatus accumulating to wild-type levels and those for photosynthetic apparatus being suppressed. Polysome analysis showed that translation of the plastid transcripts encoding the plastid transcription/translation apparatus was blocked at an early stage of chloroplast differentiation. Accumulation of transcripts of nuclear-encoded photosynthetic genes, such as cab and rbcS, was strongly suppressed in the mutant at later stages of chloroplast differentiation, whereas transcripts of genes for the plastid transcription apparatus, such as OsRpoTp and OsSIG2A, accumulated to abnormally high levels at these stages. These results suggest that activation of the plastid translation machinery at an early stage of chloroplast differentiation is important for triggering the transmission of information about plastid developmental state to the nucleus, which in turn is required for the induction of nuclear-encoded chloroplast proteins at later stages of chloroplast differentiation.

DNA double-strand break (DSB)-mediated genome rearrangements are assumed to provide diverse raw genetic materials enabling accelerated adaptive evolution; however, it remains unclear about the consequences of massive simultaneous DSB formation in cells and their resulting phenotypic impact. Here, we establish an artificial genome-restructuring technology by conditionally introducing multiple genomic DSBs in vivo using a temperature-dependent endonuclease TaqI. Application in yeast and Arabidopsis thaliana generates strains with phenotypes, including improved ethanol production from xylose at higher temperature and increased plant biomass, that are stably inherited to offspring after multiple passages. High-throughput genome resequencing revealed that these strains harbor diverse rearrangements, including copy number variations, translocations in retrotransposons, and direct end-joinings at TaqI-cleavage sites. Furthermore, large-scale rearrangements occur frequently in diploid yeasts (28.1%) and tetraploid plants (46.3%), whereas haploid yeasts and diploid plants undergo minimal rearrangement. This genome-restructuring system (TAQing system) will enable rapid genome breeding and aid genome-evolution studies.

In contrast to mammals, higher plants have evolved to express diverse protein phosphatase 2Cs (PP2Cs). Of all Arabidopsis thaliana PP2Cs, members of PP2C subfamily A, including ABI1, have been shown to be key negative regulators of abscisic acid (ABA) signalling pathways, which regulate plant growth and development as well as tolerance to adverse environmental conditions. However, little is known about the enzymatic and signalling roles of other PP2C subfamilies. Here, we report a novel Arabidopsis subfamily E PP2C gene, At3g05640, designated AtPP2CF1. AtPP2CF1 was dramatically expressed in response to exogenous ABA and was expressed in vascular tissues and guard cells, similar to most subfamily A PP2C genes. In vitro enzymatic activity assays showed that AtPP2CF1 possessed functional PP2C activity. However, yeast two-hybrid analysis revealed that AtPP2CF1 did not interact with PYR/PYL/RCAR receptors or three SnRK2 kinases, which are ABI1-interacting proteins. This was supported by homology-based structural modelling demonstrating that the putative active- and substrate-binding site of AtPP2CF1 differed from that of ABI1. Furthermore, while overexpression of ABI1 in plants induced an ABA-insensitive phenotype, Arabidopsis plants overexpressing AtPP2CF1 (AtPP2CF1oe) were weakly hypersensitive to ABA during seed germination and drought stress. Unexpectedly, AtPP2CF1oe plants also exhibited increased biomass yield, mainly due to accelerated growth of inflorescence stems through the activation of cell proliferation and expansion. Our results provide new insights into the physiological significance of AtPP2CF1 as a candidate gene for plant growth production and for potential application in the sustainable supply of plant biomass.

The fruiting body-forming ciliate Sorogena stoianovitchae is a protist that is multicellular in one stage of its life cycle. When nutrient levels are depleted, a number of Sorogena cells aggregate beneath the water surface to form an aerial fruiting body. Based on morphologies and the inhibition of protein synthesis, fruiting body development is divided into five distinct stages: (1) aggregation before sunrise, (2) compact aggregation after sunrise, (3) secretion of mucous matrix, (4) stalk-elongation, and (5) completion of the fruiting-body. In the aggregation stage, the cells were trapped in a matrix material that stained orange with 4',6-diamino-2-phenylindole (DAPI), but differed from the mucous matrix in the later stage. A short interruption of the dark period, at 6-8 h after the onset of dark, inhibited fruiting body development. Irrespective of the length of the dark period (10-16 h), the cells remained in the aggregation stage until the beginning of the light period. Therefore, an uninterrupted dark period of more than 8 h is critical for the initial aggregation of cells, but subsequent development is triggered by light.

SUMMARY We previously developed a large‐scale genome restructuring technology called the TAQing system. It can induce genomic rearrangements by introducing transient and conditional formation of DNA double‐strand breaks (DSBs) via heat activation of a restriction enzyme TaqI, which can cleave DNA at 5′‐TCGA‐3′ sequences in the genome at higher temperatures (37–42°C). Such heat treatment sometimes confers lethal damage in certain plant species and TaqI cannot induce rearrangements in AT‐rich regions. To overcome such problems we developed an extended TAQing (Ex‐TAQing) system, which enables the use of a wider range of restriction enzymes active at standard plant‐growing temperatures. We established the Ex‐TAQing system using MseI that can efficiently cleave DNA at room temperature (at temperatures ranging from 22 to 25°C) and the 5′‐TTAA‐3′ sequence which is highly abundant in the Arabidopsis genome. A synthetic intron‐spanning MseI gene, which was placed downstream of a heat‐shock‐inducible promoter, was conditionally expressed upon milder heat treatment (33°C) to generate DSBs in Arabidopsis chromosomes. Genome resequencing revealed various types of genomic rearrangements, including copy number variations, translocation and direct end‐joining at MseI cleavage sites. The Ex‐TAQing system could induce large‐scale rearrangements in diploids more frequently (17.4%, n = 23) than the standard TAQing system. The application of this system to tetraploids generated several strains with chromosomal rearrangements associated with beneficial phenotypes, such as high salinity stress tolerance and hypersensitivity to abscisic acid. We have developed the Ex‐TAQing system, allowing more diverse patterns of genomic rearrangements, by employing various types of endonucleases and have opened a way to expand the capacity for artificial genome reorganization.

Plant cells have acquired chloroplasts (plastids) with a unique genome (ptDNA), which developed during the evolution of endosymbiosis. The gene content and genome structure of ptDNAs in land plants are considerably stable, although those of algal ptDNAs are highly varied. Plant cells seem, therefore, to be intolerant of any structural or organizational changes in the ptDNA. Genome rearrangement functions as a driver of genomic evolutionary divergence. Here, we aimed to create various types of rearrangements in the ptDNA of Arabidopsis genomes using plastid-targeted forms of restriction endonucleases (pREs). Arabidopsis plants expressing each of the three specific pREs, i.e., pTaqI, pHinP1I, and pMseI, were generated; they showed the leaf variegation phenotypes associated with impaired chloroplast development. We confirmed that these pREs caused double-stranded breaks (DSB) at their recognition sites in ptDNAs. Genome-wide analysis of ptDNAs revealed that the transgenic lines exhibited a large number of rearrangements such as inversions and deletions/duplications, which were dominantly repaired by microhomology-mediated recombination and microhomology-mediated end-joining, and less by non-homologous end-joining. Notably, pHinP1I, which recognized a small number of sites in ptDNA, induced drastic structural changes, including regional copy number variations throughout ptDNAs. In contrast, the transient expression of either pTaqI or pMseI, whose recognition site numbers were relatively larger, resulted in small-scale changes at the whole genome level. These results indicated that DSB frequencies and their distribution are major determinants in shaping ptDNAs.