Fine-mapping of qRL6.1, a major QTL for root length of rice seedlings grown under a wide range of NH4 + concentrations in hydroponic conditions

Root system development is an important target for improving yield in cereal crops. Active root systems that can take up nutrients more efficiently are essential for enhancing grain yield. In this study, we attempted to identify quantitative trait loci (QTL) involved in root system development by measuring root length of rice seedlings grown in hydroponic culture. Reliable growth conditions for estimating the root length were first established to renew nutrient solutions daily and supply NH₄ ⁺ as a single nitrogen source. Thirty-eight chromosome segment substitution lines derived from a cross between ‘Koshihikari’, a japonica variety, and ‘Kasalath’, an indica variety, were used to detect QTL for seminal root length of seedlings grown in 5 or 500 μM NH₄ ⁺. Eight chromosomal regions were found to be involved in root elongation. Among them, the most effective QTL was detected on a ‘Kasalath’ segment of SL-218, which was localized to the long-arm of chromosome 6. The ‘Kasalath’ allele at this QTL, qRL6.1, greatly promoted root elongation under all NH₄ ⁺ concentrations tested. The genetic effect of this QTL was confirmed by analysis of the near-isogenic line (NIL) qRL6.1. The seminal root length of the NIL was 13.5–21.1% longer than that of ‘Koshihikari’ under different NH₄ ⁺ concentrations. Toward our goal of applying qRL6.1 in a molecular breeding program to enhance rice yield, a candidate genomic region of qRL6.1 was delimited within a 337 kb region in the ‘Nipponbare’ genome by means of progeny testing of F₂ plants/F₃ lines derived from a cross between SL-218 and ‘Koshihikari’.     

Genetic analysis of interspecific incompatibility in Brassica rapa

In interspecific pollination of Brassica rapa stigmas with Brassica oleracea pollen grains, pollen tubes cannot penetrate stigma tissues. This trait, called interspecific incompatibility, is similar to self-incompatibility in pollen tube behaviors of rejected pollen grains. Since some B. rapa lines have no interspecific incompatibility, genetic analysis of interspecific incompatibility was performed using two F₂ populations. Analysis with an F₂ population between an interspecific-incompatible line and a self-compatible cultivar ‘Yellow sarson’ having non-functional alleles of S-locus genes and MLPK, the stigmas of which are compatible with B. oleracea pollen grains, revealed no involvement of the S locus and MLPK in the difference of their interspecific incompatibility phenotypes. In QTL analysis of the strength of interspecific incompatibility, three peaks of LOD scores were found, but their LOD scores were as high as the threshold value, and the variance explained by each QTL was small. QTL analysis using another F₂ population derived from selected parents having the highest and lowest levels of interspecific incompatibility revealed five QTLs with high LOD scores, which did not correspond to those found in the former population. The QTL having the highest LOD score was found in linkage group A02. The effect of this QTL on interspecific incompatibility was confirmed by analyzing backcrossed progeny. Based on synteny of this QTL region with Arabidopsis thaliana chromosome 5, a possible candidate gene, which might be involved in interspecific incompatibility, is discussed.     

The REDUCED LEAFLET Genes Encode Key Components of the trans-Acting Small Interfering RNA Pathway and Regulate Compound Leaf and Flower Development in Lotus japonicus

The endogenous trans-acting small interfering RNA (ta-siRNA) pathway plays a conserved role in adaxial-abaxial patterning of lateral organs in simple-leafed plant species. However, its function in compound-leafed species is largely unknown. Using the compound-leafed species Lotus japonicus, we identified and characterized two independent mutants, reduced leaflet1 (rel1) and rel3, whose most conspicuous defects in compound leaves are abaxialized leaflets and reduction in leaflet number. Concurrent mutations in REL genes also compromise flower development and result in radial symmetric floral organs. Positional cloning revealed that REL1 and REL3 encode the homologs of Arabidopsis (Arabidopsis thaliana) SUPPRESSOR OF GENE SILENCING3 and ARGONAUTE7/ZIPPY, respectively, which are key components of the ta-siRNA pathway. These observations, together with the expression and functional data, demonstrated that the ta-siRNA pathway plays conserved yet distinct roles in the control of compound leaf and flower development in L. japonicus. Moreover, the phenotypic alterations of lateral organs in ta-siRNA-deficient mutants and the regulation of downstream targets by the ta-siRNA pathway in L. japonicus were similar to those in the monocots but different from Arabidopsis, indicating many parallels between L. japonicus and the monocots in the control of lateral organ development by the ta-siRNA pathway.Plant endogenous small RNAs can be categorized into microRNAs (miRNAs) and small interfering RNAs (siRNAs) according to their mechanism of biogenesis (Vaucheret, 2006). trans-Acting siRNAs (ta-siRNAs) are one type of siRNA, and their biogenesis requires several key components, such as SUPPRESSOR OF GENE SILENCING3 (SGS3), RNA-DEPENDENT RNA POLYMERASE6 (RDR6), DICER-LIKE4 (DCL4), ARGONAUTE7 (AGO7)/ZIPPY (ZIP), and dsRNA-BINDING4 (Peragine et al., 2004; Vazquez et al., 2004; Gasciolli et al., 2005; Xie et al., 2005; Yoshikawa et al., 2005; Adenot et al., 2006; Nakazawa et al., 2007). Recent studies revealed that the ta-siRNA pathway is integrated into different processes of plant development, such as vegetative phase transition in Arabidopsis (Arabidopsis thaliana; Hunter et al., 2003; Peragine et al., 2004; Xie et al., 2005; Nakazawa et al., 2007) and shoot apical meristem (SAM) initiation in rice (Oryza sativa; Satoh et al., 1999; Itoh et al., 2000; Nagasaki et al., 2007). Parallel studies of this pathway in simple-leafed species also showed that the ta-siRNA pathway plays critical roles in patterning of leaves and floral organs.In flowering plants, leaves and flowers are produced on the periphery of the apical meristem. These lateral organs are structurally asymmetric with regard to the apical meristem. The adaxial side is adjacent to the meristem, while the abaxial side is away from the meristem. The ta-siRNA pathway was found to play a conserved role in specifying the adaxial identity of lateral organs in both monocots and dicots, but defects in the ta-siRNA pathway caused more severe phenotypes in monocots than in dicot Arabidopsis. In Arabidopsis, no clear leaf polarity defects were detected in the ta-siRNA-defective mutants. However, blocking the ta-siRNA pathway in asymmetric1 (as1) or as2 background, which are regulators of leaf adaxial identity (Lin et al., 2003; Xu et al., 2003), results in enhanced adaxial-abaxial leaf defects (Li et al., 2005; Xu et al., 2006; Garcia et al., 2006). In addition, the as2rdr6 double mutants also display aberrant flowers with sepals failing to enwrap the inner whorl organs and some sepals and petals becoming needle-like structures (Li et al., 2005). In maize (Zea mays), mutations in LEAFBLADELESS1 (LBL1), which encodes the Arabidopsis SGS3 ortholog, give rise to abnormal leaves with partial or complete loss of adaxial cell identity (Timmermans et al., 1998; Nogueira et al., 2007). In severe lbl1 mutants, leaf-like lateral organs of inflorescences and flowers develop as symmetric, thread-like organs, and the immature ear is exposed and arrested in development (Timmermans et al., 1998). In rice, the osdcl4-1 mutants display an abaxialized epidermis in coleoptiles and in the first leaf, and knockdown of OsDCL4 can lead to the awn-like lemma with a radial abaxialized identity and the stamens and carpel not enwrapped by the lemma and pelea (Liu et al., 2007). Transgenic rice plants with ectopic expression of SHOOTLESS4 (SHL4), the homolog of Arabidopsis AGO7, exhibit partially adaxialized leaves (Nagasaki et al., 2007; Shi et al., 2007).In addition to the ta-siRNA pathway, other components have also been shown to be involved in the adaxial-abaxial patterning of lateral organs. The Antirrhinum majus PHANTASTICA (PHAN) gene (Waites et al., 1998; Byrne et al., 2000; Xu et al., 2003; Qi et al., 2004), which is the ortholog of Arabidopsis AS1, and CLASS III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) gene family members (McConnell et al., 2001; Emery et al., 2003) contribute to adaxial pattern formation of lateral organs, whereas members of YABBY (YAB; Sawa et al., 1999; Siegfried et al., 1999) and KANADI (Eshed et al., 2001; Kerstetter et al., 2001) gene families, AUXIN RESPONSE FACTOR3 (ARF3) and ARF4 (Pekker et al., 2005), and the miRNAs miR165/166 (Emery et al., 2003; Eshed et al., 2004; Mallory et al., 2004) are required for specifying abaxial identity. How the activities of these adaxial and abaxial determinants are coordinated has been extensively studied. It was found that ARF3 and ARF4 are regulated by the TAS3 ta-siRNA, and this regulation is conserved in both monocots and dicots (Allen et al., 2005; Williams et al., 2005). Recent studies in Arabidopsis suggest that ta-siRNAs act in a non-cell-autonomous manner to spatially restrict ARF activity (Chitwood et al., 2009; Schwab et al., 2009).In contrast to simple leaves with their single lamina, compound leaves are composed of one petiole and several leaflets. It is found that genes required for the adaxial-abaxial patterning of lateral organs in simple-leafed species also play critical roles in compound-leafed species, but these genes play multiple roles in compound leaf development. In tomato (Solanum lycopersicum), down-regulation of PHAN ortholog disturbs the leaf polarity as well as leaflet formation (Kim et al., 2003). Extensive studies of the PHAN expression in diverse compound-leafed species suggest that the function of PHAN in maintaining leaf adaxial identity is associated with leaflet formation in compound leaves and reduced adaxial identity of leaf primordia by down-regulation of PHAN could change pinnate compound leaves into palmate leaves (Kim et al., 2003). In pea (Pisum sativum), the role of PHAN in compound leaf development has also been elucidated by characterization of the phan mutant crispa (cri; Tattersall et al., 2005). However, unlike antisense PHAN transgenic tomato leaves, the cri mutant has the individual leaflet abaxialized, rather than the whole leaf. The number of lateral organs on the cri mutant compound leaves, including leaflets, is not altered, and the leaves remain pinnate. Apart from leaf development, the cri mutation also affects flower development. Although the floral organ identity and organ number are not altered, the laminar floral organ display abaxialized identity (Tattersall et al., 2005).The ta-siRNA pathway plays a critical role in simple-leafed species, but its role in compound-leafed species is not understood. Here, we address this question by analyzing loss-of-function reduced leaflet (rel1) and rel3 mutants in the compound-leafed species Lotus japonicus. Phenotypic characterization shows compound leaves of rel mutants exhibit a conspicuous disturbance in leaflet polarity as well as reduction in leaflet number. Besides the abnormal compound leaves, flower development is also severely affected in rel mutants, showing radial symmetric petals. REL1 and REL3 were identified by map-based cloning and were shown to be homologs of Arabidopsis SGS3 and AGO7, respectively. REL1 and REL3 act in the same genetic pathway and are both required for the biogenesis of TAS3 ta-siRNA. Further investigation reveals that the homolog of the Arabidopsis ARF3 is duplicated in the L. japonicus genome and that the duplicate ARF3 homologs and the ARF4 homolog are all negatively regulated by the ta-siRNA pathway. Furthermore, we found that the expression of LjYAB1, a homolog of Arabidopsis YAB1, was decreased in rel mutants, which may be associated with the reduced lamina.Taken together, our data reveal that the ta-siRNA pathway is integrated into the regulatory networks in the control of lateral organ development in L. japonicus and further emphasize the importance of the ta-siRNA pathway in compound leaf development. Moreover, our results also indicate many parallels between L. japonicus and monocots for the ta-siRNA pathway in the regulation of lateral organs.     

Classification of genus Pseudomonas by MALDI-TOF MS based on ribosomal protein coding in S10-spc-alpha operon at strain level

We have proposed a rapid phylogenetic classification at the strain level by MALDI-TOF MS using ribosomal protein matching profiling. In this study, the S10-spc-alpha operon, encoding half of the ribosomal subunit proteins and highly conserved in eubacterial genomes, was selected for construction of the ribosomal protein database as biomarkers for bacterial identification by MALDI-TOF MS analysis to establish a more reliable phylogenetic classification. Our method revealed that the 14 reliable and reproducible ribosomal subunit proteins with less than m/z 15,000, except for L14, coded in the S10-spc-alpha operon were significantly useful biomarkers for bacterial classification at species and strain levels by MALDI-TOF MS analysis of genus Pseudomonas strains. The obtained phylogenetic tree was consisted with that based on genetic sequence (gyrB). Since S10-spc-alpha operons of genus Pseudomonas strains were sequenced using specific primers designed based on nucleotide sequences of genome-sequenced strains, the ribosomal subunit proteins encoded in S10-spc-alpha operon were suitable biomarkers for construction and correction of the database. MALDI-TOF MS analysis using these 14 selected ribosomal proteins is a rapid, efficient, and versatile bacterial identification method with the validation procedure for the obtained results.     

Polygalacturonase mRNA of Tomato: Size and Content in Ripe Fruits

In ripe tomato fruits, polygalacturonase (PG) mRNA comprisedabout 1% of the translatable RNAs in the poly(A)(+)RNA fraction.Sucrose density gradient centrifugation showed that this PGmRNA is similar in size to 18S rRNA, which suggests the presenceof a non-coding region. (Received June 19, 1984; Accepted October 23, 1984)     

Novel Rana keratin genes and their expression during larval to adult epidermal conversion in bullfrog tadpoles

The conversion of the larval to adult epidermis during metamorphosis of tadpoles of bullfrog, Rana catesbeiana, was investigated utilizing newly cloned Rana keratin cDNAs as probes. Rana larval keratin (RLK) cDNA (rlk) was cloned using highly specific antisera against Xenopus larval keratin (XLK). Tail skin proteins of bullfrog tadpoles were separated by 2-dimensional gel electrophoresis and subjected to Western blot analysis with anti-XLK antisera. The Rana antigen detected by this method was sequenced and identified as a type II keratin. We cloned rlk from tadpole skin by PCR utilizing primers designed from these peptide sequences of RLK. RLK predicted by nucleotide sequences of rlk was a 549 amino acid -long type II keratin. Subtractive cloning between the body and the tail skin of bullfrog tadpole yielded a cDNA (rak) of Rana adult keratin (RAK). RAK was a 433 amino acid-long type I keratin. We also cloned a Rana keratin 8 (RK8) cDNA (rk8) from bullfrog tadpole epidermis. RK8 was 502 amino acid-long and homologous to cytokeratin 8. Northern blot analyses and in situ hybridization experiments showed that rlk was actively expressed through prometamorphosis in larva-specific epidermal cells called skein cells and became completely inactive at the climax stage of metamorphosis and in the adult skin. RAK mRNA was expressed in basal cells of the tadpole epidermis and germinative cells in the adult epidermis. The expression of rlk and rak was down- and up-regulated by thyroid hormone (TH), respectively. In contrast, there was no change in the expression of RK8 during spontaneous and TH-induced metamorphosis. RK8 mRNA was exclusively expressed in apical cells of the larval epidermis. These patterns of keratin gene expression indicated that the expression of keratin genes is differently regulated by TH depending on the type of larval epidermal cells. The present study demonstrated the usefulness of these genes for the study of molecular mechanism of postembryonic epidermal development and differentiation.