SummaryWe have produced 22 090 primary transgenic rice plants that carry a T-DNA insertion, which has resulted in 18 358 fertile lines. Genomic DNA gel-blot and PCR analyses have shown that approximately 65% of the population contains more than one copy of the inserted T-DNA. Hygromycin resistance tests revealed that transgenic plants contain an average of 1.4 loci of T-DNA inserts. Therefore, it can be estimated that approximately 25 700 taggings have been generated. The binary vector used in the insertion contained the promoterless b-glucuronidase (GUS) reporter gene with an intron and multiple splicing donors and acceptors immediately next to the right border. Therefore, this gene trap vector is able to detect a gene fusion between GUS and an endogenous gene, which is tagged by T-DNA. Histochemical GUS assays were carried out in the leaves and roots from 5353 lines, mature¯owers from 7026 lines, and developing seeds from 1948 lines. The data revealed that 1.6±2.1% of tested organs were GUS-positive in the tested organs, and that their GUS expression patterns were organ-or tissue-speci®c or ubiquitous in all parts of the plant. The large population of T-DNA-tagged lines will be useful for identifying insertional mutants in various genes and for discovering new genes in rice.

Sucrose nonfermenting 1 (SNF1)-related protein kinase 2s (SnRK2s) are central components of abscisic acid (ABA) signaling pathways. The snrk2.2/2.3/2.6 triple-mutant plants are nearly completely insensitive to ABA, suggesting that most of the molecular actions of ABA are triggered by the SnRK2s-mediated phosphorylation of substrate proteins. Only a few substrate proteins of the SnRK2s are known. To identify additional substrate proteins of the SnRK2s and provide insight into the molecular actions of ABA, we used quantitative phosphoproteomics to compare the global changes in phosphopeptides in WT and snrk2.2/2.3/2.6 triple mutant seedlings in response to ABA treatment. Among the 5,386 unique phosphorylated peptides identified in this study, we found that ABA can increase the phosphorylation of 166 peptides and decrease the phosphorylation of 117 peptides in WT seedlings. In the snrk2.2/ 2.3/2.6 triple mutant, 84 of the 166 peptides, representing 58 proteins, could not be phosphorylated, or phosphorylation was not increased under ABA treatment. In vitro kinase assays suggest that most of the 58 proteins can serve as substrates of the SnRK2s. The SnRK2 substrates include proteins involved in flowering time regulation, RNA and DNA binding, miRNA and epigenetic regulation, signal transduction, chloroplast function, and many other cellular processes. Consistent with the SnRK2 phosphorylation of flowering time regulators, the snrk2.2/2.3/2.6 triple mutant flowered significantly earlier than WT. These results shed new light on the role of the SnRK2 protein kinases and on the downstream effectors of ABA action, and improve our understanding of plant responses to adverse environments. T he phytohormone abscisic acid (ABA) plays important roles in plant development and responses to stressful environments (1, 2). Recently, the discovery of the PYR1 (Pyrabactin Resistance 1)/ PYL (PYR1-Like)/RCAR (Regulatory Component of ABA Receptor) family of ABA receptors led to the elucidation of the core ABA signaling pathway. ABA binds to the PYLs, triggering the PYLs to interact with and inactivate clade A protein phosphatase 2Cs (PP2Cs). This releases Sucrose nonfermenting 1 (SNF1)-related protein kinase 2s (SnRK2s) from inhibition by the PP2Cs, allowing the kinases to phosphorylate downstream effectors of ABA responses (3-5).SnRK2s are a plant-specific protein kinase family related to the yeast SNF1 and animal AMP-dependent protein kinase (AMPK) (6), and the family has 10 members (SnRK2.1-2.10) in Arabidopsis. ABA treatment can quickly activate SnRK2.2, 2.3 and 2.6 (7), and the snrk2.2/2.3/2.6 triple-knockout mutant has a very strong ABAinsensitive phenotype and shows little response to even very high concentrations of ABA in seed germination, root growth, and stomatal movement (8). In contrast, mutations in the other seven SnRK2 family members do not cause significant ABA insensitivity (9). Notwithstanding the key role of SnRK2.2/2.3/2.6 in ABA signaling, some ABA responses are possibly independent of the SnRK2s, because the PYL r...

Plant MADS-box genes form a large gene family for transcription factors and are involved in various aspects of developmental processes, including flower development. They are known to be subject to birth-and-death evolution, but the detailed features of this mode of evolution remain unclear. To have a deeper insight into the evolutionary pattern of this gene family, we enumerated all available functional and nonfunctional (pseudogene) MADSbox genes from the Arabidopsis and rice genomes. Plant MADSbox genes can be classified into types I and II genes on the basis of phylogenetic analysis. Conducting extensive homology search and phylogenetic analysis, we found 64 presumed functional and 37 nonfunctional type I genes and 43 presumed functional and 4 nonfunctional type II genes in Arabidopsis. We also found 24 presumed functional and 6 nonfunctional type I genes and 47 presumed functional and 1 nonfunctional type II genes in rice. Our phylogenetic analysis indicated there were at least about four to eight type I genes and Ϸ15-20 type II genes in the most recent common ancestor of Arabidopsis and rice. It has also been suggested that type I genes have experienced a higher rate of birth-and-death evolution than type II genes in angiosperms. Furthermore, the higher rate of birth-and-death evolution in type I genes appeared partly due to a higher frequency of segmental gene duplication and weaker purifying selection in type I than in type II genes. M orphological͞physiological evolution of organisms has been driven mainly by the evolution of genetic toolkits for developmental͞physiological processes such as transcription factors and signaling pathways (1). A large proportion of genetic toolkits are highly conserved even between distantly related organisms. In flowering plants (angiosperms), MADS-box genes are among such toolkits that control various aspects of developmental processes. MADS-box genes are defined by the highly conserved 180-bp-long motif called the MADS-box and are found in animals, fungi, and plants (2). The protein region encoded by the MADS-box is called the MADS-domain (or Mdomain) and is part of the DNA-binding domain. It has been proposed that there are at least two evolutionary lineages (types I and II) of MADS-box genes in animals, fungi, and plants (3) (Fig. 1).There are Ϸ100 MADS-box genes in Arabidopsis thaliana (hereafter called Arabidopsis) and Ͼ70 MADS-box genes in Oryza sativa (hereafter called rice). There are Ϸ40 clearly identifiable type II MADS-box genes in each of Arabidopsis (4, 5) and rice (6). Most of the plant type II genes contain three additional plant-specific domains: intervening (I) domain (Ϸ30 codons), keratin-like coiled-coil (K) domain (Ϸ70 codons), and C-terminal (C) domain (variable length) (7) (Fig. 1). These genes are called MIKC-type genes. The MIKC-type genes can further be divided into two types based on the intron-exon structure: MIKC c -and MIKC*-type genes (8). The MIKC c -type genes have been identified in most major evolutionary lineages of green plants such as...

SummaryA late-¯owering mutant was isolated from rice T-DNA-tagging lines. T-DNA had been integrated into the K-box region of Oryza sativa MADS50 (OsMADS50), which shares 50.6% amino acid identity with the Arabidopsis MADS-box gene SUPPRESSOR OF OVEREXPRESSION OF CO 1/AGAMOUS-LIKE 20 (SOC1/ AGL20). While overexpression of OsMADS50 caused extremely early¯owering at the callus stage, OsMADS50 RNAi plants exhibited phenotypes of late¯owering and an increase in the number of elongated internodes. This con®rmed that the phenotypes observed in the knockout (KO) plants are because of the mutation in OsMADS50. RT-PCR analyses of the OsMADS50 KO and ubiquitin (ubi):OsMADS50 plants showed that OsMADS50 is an upstream regulator of OsMADS1, OsMADS14, OsMADS15, OsMADS18, and Hd (Heading date)3a, but works either parallel with or downstream of Hd1 and O. sativa GIGANTEA (OsGI). These results suggest that OsMADS50 is an important¯owering activator that controls various¯oral regulators in rice.