Immunodetection of retinoblastoma-related protein and its phosphorylated form in interphase and mitotic alfalfa cells

Plant retinoblastoma-related (RBR) proteins are primarily considered as key regulators of G(1)/S phase transition, with functional roles in a variety of cellular events during plant growth and organ development. Polyclonal antibody against the C-terminal region of the Arabidopsis RBR1 protein also specifically recognizes the alfalfa 115?kDa MsRBR protein, as shown by the antigen competition assay. The MsRBR protein was detected in all cell cycle phases, with a moderate increase in samples representing G(2)/M cells. Antibody against the human phospho-pRb peptide (Ser807/811) cross-reacted with the same 115?kDa MsRBR protein and with the in vitro phosphorylated MsRBR protein C-terminal fragment. Phospho-MsRBR protein was low in G(1) cells. Its amount increased upon entry into the S phase and remained high during the G(2)/M phases. Roscovitine treatment abolished the activity of alfalfa MsCDKA1;1 and MsCDKB2;1, and the phospho-MsRBR protein level was significantly decreased in the treated cells. Colchicine block increased the detected levels of both forms of MsRBR protein. Reduced levels of the MsRBR protein in cells at stationary phase or grown in hormone-free medium can be a sign of the division-dependent presence of plant RBR proteins. Immunolocalization of the phospho-MsRBR protein indicated spots of variable number and size in the labelled interphase nuclei and high signal intensity of nuclear granules in prophase. Structures similar to phospho-MsRBR proteins cannot be recognized in later mitotic phases. Based on the presented western blot and immunolocalization data, the possible involvement of RBR proteins in G(2)/M phase regulation in plant cells is discussed.     

The novel rice (Oryza sativa L.) gene OsSbf1 encodes a putative member of the Na+/bile acid symporter family

PCR-based differential screening was used to identify ethylene-induced genes in deep-water rice (Oryza sativa L.). One of the isolated cDNAs represented a novel protein, OsSBF1, with high homology to mammalian Na+/bile acid transporters and to sodium-dependent transporters from bacteria. One highly homologous protein and three less conserved homologues were identified in Arabidopsis thaliana indicating that Sbf proteins exist in monocot and dicot plant species. Expression of OsSbf1 in deep-water rice was shown to be elevated by growth-inducing treatments. Since bile acids have not been found in plants to date a possible function of SBF proteins may be in the transport of structurally related sulphonated brassinosteroids.     

Characterization of genes involved in cytokinin signaling and metabolism from rice

Two-component signaling elements play important roles in plants, including a central role in cytokinin signaling. We characterized two-component elements from the monocot rice (Oryza sativa) using several complementary approaches. Phylogenetic analysis reveals relatively simple orthologous relationships among the histidine kinases in rice and Arabidopsis (Arabidopsis thaliana). In contrast, the histidine-containing phosphotransfer proteins (OsHPs) and response regulators (OsRRs) display a higher degree of lineage-specific expansion. The intracellular localizations of several OsHPs and OsRRs were examined in rice and generally found to correspond to the localizations of their dicot counterparts. The functionality of rice type-B OsRRs was tested in Arabidopsis; one from a clade composed of both monocot and dicot type-B OsRRs complemented an Arabidopsis type-B response regulator mutant, but a type-B OsRR from a monocot-specific subfamily generally did not. The expression of genes encoding two-component elements and proteins involved in cytokinin biosynthesis and degradation was analyzed in rice roots and shoots and in response to phytohormones. Nearly all type-A OsRRs and OsHK4 were up-regulated in response to cytokinin, but other cytokinin signaling elements were not appreciably affected. Furthermore, multiple cytokinin oxidase (OsCKX) genes were up-regulated by cytokinin. Abscisic acid treatment decreased the expression of several genes involved in cytokinin biosynthesis and degradation. Auxin affected the expression of a few genes; brassinosteroid and gibberellin had only modest effects. Our results support a shared role for two-component elements in mediating cytokinin signaling in monocots and dicots and reveal how phytohormones can impact cytokinin function through modulating gene expression.     

Cloning and Characterization of AKR4C14, a Rice Aldo–Keto Reductase, from Thai Jasmine Rice

Aldo–keto reductase (AKR) is an enzyme superfamily whose members are involved in the metabolism of aldehydes/ketones. The AKR4 subfamily C (AKR4C) is a group of aldo–keto reductases that are found in plants. Some AKR4C(s) in dicot plants are capable of metabolizing reactive aldehydes whereas, such activities have not been reported for AKR4C(s) from monocot species. In this study, we have screened Indica rice genome for genes with significant homology to dicot AKR4C(s) and identified a cluster of putative AKR4C(s) located on the Indica rice chromosome I. The genes including OsI_04426, OsI_04428 and OsI_04429 were successfully cloned and sequenced by qRT-PCR from leaves of Thai Jasmine rice (KDML105). OsI_04428, later named AKR4C14, was chosen for further studies because it shares highest homology to the dicot AKR4C(s). The bacterially expressed recombinant protein of AKR4C14 was successfully produced as a MBP fusion protein and his-tagged protein. The recombinant AKR4C14 were capable of metabolizing sugars and reactive aldehydes i.e. methylglyoxal, a toxic by-product of the glycolysis pathway, glutaraldehyde, and trans-2-hexenal, a natural reactive 2-alkenal. AKR4C14 was highly expressed in green tissues, i.e. leaf sheets and stems, whereas flowers and roots had a significantly lower level of expression. These findings indicated that monocot AKR4C(s) can metabolize reactive aldehydes like the dicot AKR4C(s) and possibly play a role in detoxification mechanism of reactive aldehydes.     

Molecular analysis of the NAC gene family in rice

Genes that encode products containing a NAC domain, such as NO APICAL MERISTEM (NAM) in petunia, CUP-SHAPED COTYLEDON2 (CUC2) and NAP in Arabidopsis thaliana, have crucial functions in plant development. We describe here molecular aspects of the OsNAC genes that encode proteins with NAC domains in rice (Oryza sativa L.). Sequence analysis revealed that the NAC genes in plants can be divided into several subfamilies, such as the NAM, ATAF, and OsNAC3 subfamilies. In rice, OsNAC1 and OsNAC2 are classified in the NAM subfamily, which includes NAM and CUC2, while OsNAC5 and OsNAC6 fall into the ATAF subfamily. In addition to the members of these subfamilies, the rice genome contains the NAC genes OsNAC3, OsNAC4 (both in the OsNAC3 subfamily), OsNAC7, and OsNAC8. These results and Southern analysis indicate that the OsNAC genes constitute a large gene family in the rice genome. Each OsNAC gene is expressed in a specific pattern in different organs, suggesting that this family has diverse and important roles in rice development.     

Wound signaling-coordination of the octadecanoid and MAPK pathways

Plants have developed integrated signaling networks that play a pivotal role in mediating the perception of and response to environmental factors, including wounding, a common stress. Wound signaling is known to involve at least two major signaling pathways—the octadecanoid and the MAPK pathways. These two pathways have been investigated in model dicotyledoneous (dicot) and monocotyledoneous (monocot) plant species such as alfalfa, Arabidopsis, tobacco, tomato, barley, maize and rice. Our careful analysis of available findings, to date, on wound signaling point towards a remarkable difference in coordination between the octadecanoid and the MAPK pathways in dicots and rice (Oryza sativa L.), a monocot cereal crop model plant. In this review we discuss the current understanding on these two critical pathways and propose a view on their relationship in rice with respect to wound signaling in dicot plants.     

水稻U2snRNA基因的分离及结构分析

对水稻(Oryza sativa L.)基因文库中分离到的U2snRNA基因FDRGU2.3进行序列分析,其编码区与小麦(Triticum aestivum L、)、玉米(Zea mays L.)、豌豆(Pisum sativum L.)及拟南芥(Arabidopsis thaliana(L.)Heyhy.)等植物U2基因的同源性均大于80％,且5＇端70个碱基高度保守。在基因编码区上游-70及-30区分别包含有植物UsnRNA基因特有的上游顺序元件(USE)及类TATA元件。同其它植物一样,水稻U2.3snRNA的二级结构也有保守的4个茎环区。其中环Ⅱ的结构与单子叶植物中的小麦和玉米相同,但与双子叶植物的豌豆和拟南芥存在明显差异。环Ⅳ的结构在单子叶和双子叶植物中亦有不同的变化。这些差异可能意味着单子叶和双子叶植物的剪接机构有所区别。     

Is there evidence of optimisation for carbon efficiency in plant proteomes?

Flowering plants, angiosperms, can be divided into two major clades, monocots and dicots, and while differences in amino acid composition in different species from the two clades have been reported, a systematic analysis of amino acid content and distribution remains outstanding. Here, we show that monocot and dicot proteins have developed distinct amino acid content. In Arabidopsis thaliana and poplar, as in the ancestral moss Physcomitrella patens, the average mass per amino acid appears to be independent of protein length, while in the monocots rice, maize and sorghum, shorter proteins tend to be made of lighter amino acids. An examination of the elemental content of these proteomes reveals that the difference between monocot and dicot proteins can be largely attributed to their different carbon signatures. In monocots, the shorter proteins, which comprise the majority of all proteins, are made of amino acids with less carbon, while the nitrogen content is unchanged in both monocots and dicots. We hypothesise that this signature could be the result of carbon use and energy optimisation in fast-growing annual Poaceae (grasses).     

Large-scale, lineage-specific expansion of a bric-a-brac/tramtrack/broad complex ubiquitin-ligase gene family in rice

Selective ubiquitination of proteins is directed by diverse families of ubiquitin-protein ligases (or E3s) in plants. One important type uses Cullin-3 as a scaffold to assemble multisubunit E3 complexes containing one of a multitude of bric-a-brac/tramtrack/broad complex (BTB) proteins that function as substrate recognition factors. We previously described the 80-member BTB gene superfamily in Arabidopsis thaliana. Here, we describe the complete BTB superfamily in rice (Oryza sativa spp japonica cv Nipponbare) that contains 149 BTB domain-encoding genes and 43 putative pseudogenes. Amino acid sequence comparisons of the rice and Arabidopsis superfamilies revealed a near equal repertoire of putative substrate recognition module types. However, phylogenetic comparisons detected numerous gene duplication and/or loss events since the rice and Arabidopsis BTB lineages split, suggesting possible functional specialization within individual BTB families. In particular, a major expansion and diversification of a subset of BTB proteins containing Meprin and TRAF homology (MATH) substrate recognition sites was evident in rice and other monocots that likely occurred following the monocot/dicot split. The MATH domain of a subset appears to have evolved significantly faster than those in a smaller core subset that predates flowering plants, suggesting that the substrate recognition module in many monocot MATH-BTB E3s are diversifying to ubiquitinate a set of substrates that are themselves rapidly changing. Intriguing possibilities include pathogen proteins attempting to avoid inactivation by the monocot host.     

Grasses Like Mammals? Redundancy and Compensatory Regulation within the Retinoblastoma Protein Family

The retinoblastoma (RB) protein family plays a conserved and inhibitory role incell cycle progression in higher eukaryotes. In mammals, this family includes, in additionto RB, the related (RBR) proteins p107 and p130, which appear to have both specific andredundant functions compared to those of the prototypical RB protein. Whereas mostplant species seem to possess only one RBR gene, a recent study has shown that in maizethere are two types of distinctly regulated RBR proteins, RBR1 and RBR3. Expression ofRBR3 RNA is controlled by the RBR1-E2F pathway, and it is up-regulated uponinhibition of RBR1 activity by the wheat dwarf virus RepA protein in tissue culture,indicating the presence of a specific compensatory mechanism sustaining high pocketprotein activity. Database mining and phylogenetic analyses suggest the presence of twodistinct RBR genes to be a unique feature of grasses among plants, which might help toexplain their recalcitrance to genetic transformation.     

Comparative analysis of rice MADS-box genes expressed during flower development

MADS-box genes involved in flower development have been isolated and studied in a wide variety of plant species. However, most of these studies are related to dicot species like Antirrhinum majus, Arabidopsis thaliana and Petunia hybrida. Although the floral structures of typical monocot and dicot flowers differ substantially, previous studies indicate that MADS-box genes controlling floral organ identity in dicots can also be identified in monocot plants like rice and maize. To extend this study further to obtain a more global picture of monocot and dicot MADS-box gene evolution, we performed a phylogenetic study using MADS-box genes from A. thaliana and Oryza sativa. Furthermore, we investigated whether the identified orthologues of Arabidopsis and rice have a conserved expression profile that could indicate conservation of function.     

Codon usage in plant genes.

We have examined codon bias in 207 plant gene sequences collected from Genbank and the literature. When this sample was further divided into 53 monocot and 154 dicot genes, the pattern of relative use of synonymous codons was shown to differ between these taxonomic groups, primarily in the use of G + C in the degenerate third base. Maize and soybean codon bias were examined separately and followed the monocot and dicot codon usage patterns respectively. Codon preference in ribulose 1,5 bisphosphate and chlorophyll a/b binding protein, two of the most abundant proteins in leaves was investigated. These highly expressed are more restricted in their codon usage than plant genes in general.     

RBR Ubiquitin Ligases: Diversification and Streamlining in Animal Lineages

The patterns of emergence and disappearance in animal species of genes encoding RBR ubiquitin ligases are described. RBR genes can be classified into subfamilies (Parkin, Ariadne, Dorfin, ARA54, etc.) according to sequence and structural data. Here, I show that most animal-specific RBR subfamilies emerged early in animal evolution, and that ancient animals, before the cnidarian/bilaterian split, had a set of RBR genes, which was as complex as the one currently found in mammals. However, some lineages (nematodes, dipteran insects) have recently suffered multiple losses, leading to a highly simplified set of RBR genes. Genes of a particular RBR subfamily, characterized by containing a helicase domain and so far found only in plants, are present also in some animal species. The meaning of these patterns of diversification and streamlining are discussed at the light of functional data. Extreme evolutionary conservation may be related to gene products having housekeeping functions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Protein extraction/solubilization protocol for monocot and dicot plant gel-based proteomics

Sample preparation in plant proteomics is tedious, requiring modifications depending on the type of tissue involved. Here, we describe a protein extraction protocol for both monocotyledonous (monocot) and dicotyledonous (dicot) species, which significantly improves the solubilization of total proteins. For example, we used the primary leaf tissue and seeds from rice, a cereal crop and genome model system. Total protein was first precipitated with trichloroacetic acid/acetone extraction buffer (TCAAEB) and subsequently solubilized with a modified O’Farrell lysis buffer (LB) containing thiourea and tris (LB-TT). Separation of total leaf proteins by two-dimensional gel electrophoresis (2-DGE) revealed improved solubilization, as determined by an increased number of spots detected with Coomassie brilliant blue (CBB) staining. In addition, the resolution was better than when LB-TT was used alone for protein extraction. Seed proteins could be extracted in LB-TT itself without the need for TCAAEB, which resulted in a highly insoluble precipitate. Our CBB-stained 2-D gel protein profiles also demonstrated the efficacy of this protocol for total protein extraction/solubilization from the dicot genome model (Arabidopsis), a dicot disease model (cucumber), and two other important monocot cereal crop models (maize and wheat). Moreover, this is the first report on generating a 2-D gel proteome profile for wheat crown and cucumber leaf tissues. Finally, as examples of proteome reference maps, we obtained silver nitrate-stained, large-format 2-D gels for rice leaf and wheat crown LB-TT solubilized proteins.