Spinach Gene Research Articles

Genome-wide analysis of MADS-box genes and their expression patterns in unisexual flower development in dioecious spinach

Evolution of unisexual flowers involves extreme changes in floral development. Spinach is one of the species to discern the formation and evolution of dioecy. MADS-box gene family is involved in regulation of floral organ identity and development and in many other plant developmental processes. However, there is no systematic analysis of MADS-box family genes in spinach. A comprehensive genome-wide analysis and transcriptome profiling of MADS-box genes were undertaken to understand their involvement in unisexual flower development at different stages in spinach. In total, 54 MADS-box genes found to be unevenly located across 6 chromosomes and can be divided into type I and type II genes. Twenty type I MADS-box genes are subdivided into Mα, Mβ and Mγ subgroups. While thirty-four type II SoMADSs consist of 3 MIKC*, and 31 MIKCC -type genes including sixteen floral homeotic MADS-box genes that are orthologous to the proposed Arabidopsis ABCDE model of floral organ identity determination, were identified in spinach. Gene structure, motif distribution, physiochemical properties, gene duplication and collinearity analyses for these genes are performed in detail. Promoters of both types of SoMADS genes contain mainly MeJA and ABA response elements. Expression profiling indicated that MIKCc genes exhibited more dynamic and intricate expression patterns compared to M-type genes and the majority of type-II genes AP1, SVP, and SOC1 sub-groups showed female flower-biased expression profiles, suggesting their role in carpel development, while PI showed male-biased expression throughout flower developmental stages, suggesting their role in stamen development. These results provide genomic resources and insights into spinach dioecious flower development and expedite spinach improvement.

Genome-wide characterization, functional analysis, and expression profiling of the Aux/IAA gene family in spinach

BackgroundThe auxin/indole-3-acetic acid (Aux/IAA) gene family is a crucial element of the auxin signaling pathway, significantly influencing plant growth and development. Hence, we conducted a comprehensive investigation of Aux/IAAs gene family using the Sp75 and Monoe-Viroflay genomes in spinach.ResultsA total of 24 definitive Aux/IAA genes were identified, exhibiting diverse attributes in terms of amino acid length, molecular weight, and isoelectric points. This diversity underscores potential specific roles within the family, such as growth regulation and stress response. Structural analysis revealed significant variations in gene length and molecular weight. These variations indicate distinct roles within the Aux/IAA gene family. Chromosomal distribution analysis exhibited a dispersed pattern, with chromosomes 4 and 1 hosting the highest and lowest numbers of Aux/IAA genes, respectively. Phylogenetic analysis grouped the identified genes into distinct clades, revealing potential evolutionary relationships. Notably, the phylogenetic tree highlighted specific gene clusters suggesting shared genetic ancestry and potential functional synergies within spinach. Expression analysis under NAA treatment unveiled gene-specific and time-dependent responses, with certain genes exhibiting distinct temporal expression patterns. Specifically, SpoIAA5 displayed a substantial increase at 2 h post-NAA treatment, while SpoIAA7 and SpoIAA9 demonstrated continuous rises, peaking at the 4-hour time point.ConclusionsThese observations indicate a complex interplay of gene-specific and temporal regulation in response to auxin. Moreover, the comparison with other plant species emphasized both shared characteristics and unique features in Aux/IAA gene numbers, providing insights into the evolutionary dynamics of this gene family. This comprehensive characterization of Aux/IAA genes in spinach not only establishes the foundation for understanding their specific functions in spinach development but also provides a valuable resource for experimental validation and further exploration of their roles in the intricate network of auxin signaling pathways.

Genome-Wide Identification and Characterization of MYB Gene Family and Analysis of Its Sex-Biased Expression Pattern in Spinacia oleracea L.

The members of the myeloblastosis (MYB) family of transcription factors (TFs) participate in a variety of biological regulatory processes in plants, such as circadian rhythm, metabolism, and flower development. However, the characterization of MYB genes across the genomes of spinach Spinacia oleracea L. has not been reported. Here, we identified 140 MYB genes in spinach and described their characteristics using bioinformatics approaches. Among the MYB genes, 54 were 1R-MYB, 80 were 2R-MYB, 5 were 3R-MYB, and 1 was 4R-MYB. Almost all MYB genes were located in the 0-30 Mb region of autosomes; however, the 20 MYB genes were enriched at both ends of the sex chromosome (chromosome 4). Based on phylogeny, conserved motifs, and the structure of genes, 2R-MYB exhibited higher conservation relative to 1R-MYB genes. Tandem duplication and collinearity of spinach MYB genes drive their evolution, enabling the functional diversification of spinach genes. Subcellular localization prediction indicated that spinach MYB genes were mainly located in the nucleus. Cis-acting element analysis confirmed that MYB genes were involved in various processes of spinach growth and development, such as circadian rhythm, cell differentiation, and reproduction through hormone synthesis. Furthermore, through the transcriptome data analysis of male and female flower organs at five different periods, ten candidate genes showed biased expression in spinach males, suggesting that these genes might be related to the development of spinach anthers. Collectively, this study provides useful information for further investigating the function of MYB TFs and novel insights into the regulation of sex determination in spinach.

Expression profiling of the Dof gene family under abiotic stresses in spinach

DNA-binding with one finger (Dof) are plant-specific transcription factors involved in numerous pathways of plant development, such as abiotic stresses responses. Although genome-wide analysis of Dof genes has been performed in many species, but these genes in spinach have not been analyzed yet. We performed a genome-wide analysis and characterization of Dof gene family in spinach (Spinacia oleracea L.). Twenty-two Dof genes were identified and classified into four groups with nine subgroups, which was further corroborated by gene structure and motif analyses. Ka/Ks analysis revealed that SoDofs were subjected to purifying selection. Using cis-acting elements analysis, SoDofs were involved in plant growth and development, plant hormones, and stress responses. Expression profiling demonstrated that SoDofs expressed in leaf and inflorescence, and responded to cold, heat, and drought stresses. SoDof22 expressed the highest level in male flowers and under cold stress. These results provided a genome-wide analysis of SoDof genes, their gender- and tissue-specific expression, and response to abiotic stresses. The knowledge and resources gained from these analyses will benefit spinach improvement.

Selection and Validation of Reference Genes for RT-qPCR Analysis in Spinacia oleracea under Abiotic Stress

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is an accurate and convenient method for mRNA quantification. Selection of optimal reference gene(s) is an important step in RT-qPCR experiments. However, the stability of housekeeping genes in spinach (Spinacia oleracea) under various abiotic stresses is unclear. Evaluating the stability of candidate genes and determining the optimal gene(s) for normalization of gene expression in spinach are necessary to investigate the gene expression patterns during development and stress response. In this study, ten housekeeping genes, 18S ribosomal RNA (18S rRNA), actin, ADP ribosylation factor (ARF), cytochrome c oxidase subunit 5C (COX), cyclophilin (CYP), elongation factor 1-alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H3 (H3), 50S ribosomal protein L2 (RPL2), and tubulin alpha chain (TUBα) from spinach, were selected as candidates in roots, stems, leaves, flowers, and seedlings in response to high temperature, CdCl2, NaCl, NaHCO3, and Na2CO3 stresses. The expression of these genes was quantified by RT-qPCR and evaluated by NormFinder, BestKeeper, and geNorm. 18S rRNA, actin, ARF, COX, CYP, EF1α, GAPDH, H3, and RPL2 were detected as optimal reference genes for gene expression analysis of different organs and stress responses. The results were further confirmed by the expression pattern normalized with different reference genes of two heat-responsive genes. Here, we optimized the detection method of the gene expression pattern in spinach. Our results provide the optimal candidate reference genes which were crucial for RT-qPCR analysis.

Identification and characterization of the NPF, NRT2 and NRT3 in spinach

Nitrate transporters (NRTs) participate in nitrate uptake, transport and allocation within the plant. However, this gene family has not been studied thoroughly in spinach. This study provided the general information about spinach SoNRTs and their transcriptional and functional responses to different levels of nitrate supplies. Resultes showed that fifty-seven NPF (also known as NRT1), nine NRT2 and one NRT3 were identified in spinach homologous to characterized Arabidopsis NRT genes. Phylogenetic analysis organized the SoNRT family into three clades: NPF with three subclades, NRT2, and NRT3. The tested SoNRT genes showed the various expression profiles in relation to tissue specificity and nitrate supply, indicating their functional diversity in response to external nitrate supply. Among them, transgenic Arabidopsis plants overexpressing SoNPF30 showed improved biomass, decreased shoot nitrate contents but no significant difference of 15NO3− uptake rates when compared with those of the wild type in response to high N treatment. Under low N treatment, overexpressing of SoNRT3 significantly increased whole plant biomass, root nitrate contents and 15NO3− uptake rates. These demonstrated that SoNPF30 and SoNRT3 confer greater capacity for nitrate translocation or nitrate uptake, and could help to improve the ability of plant nitrogen utilization under those conditions. Our findings provide a valuable basis for future research on this family of genes in spinach.

Construction of high-density genetic maps defined sex determination region of the Y chromosome in spinach

Spinach (Spinacia olracea L.) is a dioecious leafy vegetable with a highly repetitive genome of around 990Mb, which is challenging for de-novo genome assembly. In our study, a segregating F1 (double pseudo-testcross) population from 'Viroflay' × 'Cornell-NO. 9' was used for genetic mapping by resequencing genotyping. In the paternal 'Cornell-NO. 9' map, 212,414 SNPs were mapped, and the total linkage distance was 476.83cM; the maternal 'Viroflay' map included 29,282 SNPs with 401.28cM total genetic distance. Both paternal and maternal maps have the expected number of six linkage groups (LGs). A non-recombining region with 5678 SNPs (39 bin markers) co-segregates with sex type which located at 45.2cM of LG1 in the 'Cornell-NO. 9' map while indicates the sex determination region (SDR). Integration of two maps into a consensus map guided us to anchor additional 1242 contigs to six pseudomolecules from the published reference genome, which improved additional 233Mb (23.4%) assembly based on spinach estimated genome size. Particularly, the X counterpart of SDR in our assembly is estimated around 18.4Mb which locates at the largest chromosome, as consensus with sex-biased FISH signals from previous cytogenetics studies. The region is featured by reduced gene density, higher percentage of repetitive sequences, and no recombination. Our linkage maps provide the resource for improving spinach genome de-novo assembly and identification of sex-determining genes in spinach.

Isolation and characterization of Salt Overly Sensitive family genes in spinach

The Salt Overly Sensitive (SOS) pathway regulates intracellular sodium ion homeostasis as a salt-stress response in plants. This pathway involves three main genes designated as SOS1, SOS2 and SOS3, which are members of the Na+ /H+ exchanger (NHX), CBL-interacting protein kinase (CIPK) and Calcineurin B-like (CBL) gene families, respectively. To identify and characterize SOS genes in spinach (Spinacia oleracea), a species of the Amaranthaceae family, we conducted genome-wide identification and phylogenetic analyses of NHX, CIPK and CBL genes from four Amaranthaceae species, Arabidopsis and rice. Most Amaranthaceae genes exhibited orthologous relationships with Arabidopsis and/or rice, except a clade of Vac-type Amaranthaceae NHX genes. Phylogenetic analyses also revealed gene gain/loss events in Amaranthaceae species and the intron-less to intron-rich evolution of CIPK genes. A bacterial protein-rooted CIPK tree allowed naming most of the phylogenetic clades based on their evolutionary history. Single S. oleracea (So) SOS1, SOS2 and SOS3 proteins were identified. Direct protein-protein interaction was observed between SoSOS2 and SoSOS3 but not between SoSOS2 and SoSOS1 based on yeast two-hybrid assay. This may suggest distinct modes of action of spinach SOS proteins compared to Arabidopsis SOS proteins. Unlike SoSOS1 and SoSOS2, which were expressed at similar or higher levels in leaves than roots, SoSOS3 expression was significantly higher in roots than leaves, suggesting its greater importance in roots. The expression of SoSOS3 was upregulated in both roots and leaves under salinity compared to the control; however, SoSOS1 was only upregulated in roots. Thus, this study demonstrated the conservation of SOS pathway genes in spinach and also highlighted the complexity of SOS signaling in Amaranthaceae species.

Application of cucumber mosaic virus to efficient induction and long-term maintenance of virus-induced gene silencing in spinach.

Virus-induced gene silencing (VIGS) is a useful tool for functional genomics in plants. In this study, we tried to apply cucumber mosaic virus (CMV) to efficient induction of VIGS in spinach. Although VIGS for spinach had been previously developed based on two viruses (beet curly top virus and tobacco rattle virus), they still have some problems with systemic movement and long-term maintenance of VIGS in spinach. Although ordinary CMV strains infect spinach inducing distinct mosaic symptoms, using a CMV pseudorecombinant, we can modify the viral pathogenicity to attenuate viral symptoms that may mask the silencing phenotype. We here successfully demonstrated the viral ability to silence the phytoene desaturase (PDS) and the dihydroflavonol 4-reductase (DFR) genes in spinach. Because CMV could quickly induce VIGS even at 7-10 days postinoculation and the virus did not disappear even at the flowering stage, this CMV-based VIGS system would contribute to functional genomics in spinach and especially to the elucidation of molecular mechanisms for some properties unique to spinach such as plasticity of sex expression; the CMV-induced VIGS can last until the flowering stage after the virus was inoculated onto the seedling.

DNA Extraction for Sensitive Detection of Shiga Toxin-Producing Escherichia coli in Food by Real-Time PCR Assays

Alkali-heat DNA extraction, a rapid and economical method, was evaluated for use in the detection of Shiga toxin-producing Escherichia coli in food using real-time PCR assays. Alkali-heat DNA extracts led to highly sensitive detection (102-104 CFU/mL) of stx and O-antigen genes in beef liver, ground beef, sliced pork, cheese, lettuce, radish sprouts, tomato, and spinach, equivalent to the sensitivity obtained using a commercial DNA extraction kit that utilizes proteinase K lysis, and silica membrane purification. Although there were differences in DNA concentration and purity between DNA extraction methods, the sensitivity of real-time PCR assays was similar. These results indicate that alkali-heat DNA extraction is a viable method when testing food products with real-time PCR assays for the presence of stx and O-antigen genes.

Higher anthocyanin accumulation associated with higher transcription levels of anthocyanin biosynthesis genes in spinach.

Spinach (Spinacia oleracea L.) is widely cultivated as an economically important green leafy vegetable crop for fresh and processing consumption. The red-purple spinach shows abundant anthocyanin accumulation in the leaf and leaf petiole. However, the molecular mechanisms of anthocyanin synthesis in this species are still undetermined. In the present study, we investigated pigment formation and identified anthocyanin biosynthetic genes in spinach. We also analyzed the expression of these genes in purple and green cultivars by quantitative PCR. The accumulation of anthocyanin showed that it was the dominant pigment resulting in the red coloration in spinach. In total, 22 biosynthesis genes and 25 regulatory genes were identified in spinach, based on the spinach genomic and transcriptomic database. Furthermore, the expression patterns of genes encoding enzymes indicated that SoPAL, SoUFGT3, and SoUFGT4 were possible candidate genes for anthocyanin biosynthesis in red-purple spinach. The expression patterns of transcription factors indicated that two SoMYB genes, three SobHLH genes, and one SoWD40 gene were drastically up-regulated and co-expression in red-purple spinach, suggesting an essential role of regulatory genes in the anthocyanin biosynthesis of spinach. These results will enhance our understanding of the molecular mechanisms of anthocyanin biosynthesis in purple spinach.

Optimization of Agrobacterium-mediated transient expression of heterologous genes in spinach

The Agrobacterium-mediated transient assay is a relatively rapid technique and a promising approach for assessing the expression of a gene of interest. Despite the successful application of this transient expression system in several plant species, it is not well understood in spinach. In this study, we analyzed various factors, including infiltration method, Agrobacterium strain and density, and co-infiltration of an RNA silencing suppressor (p19), that affect transient expression following agroinfiltration in spinach. To evaluate the effects of these factors on the transient expression system, we used the β-glucuronidase (GUS) reporter gene construct pB7WG2D as a positive control. The vacuum-based infiltration method was much more effective at GUS gene expression than was the syringe-based infiltration method. Among the three Agrobacterium strains examined (EHA105, LBA4404, and GV2260), infiltration with the GV2260 strain suspension at a final optical cell density (OD600) of 1.0 resulted in the highest gene expression. Furthermore, co-expression of suppressor p19 also increased the efficiency and duration of gene expression and protein accumulation. The results indicate that the use of optimized conditions for transient gene expression could be a simple, rapid, and effective tool for functional genomics in spinach.

Development of a virus-induced gene silencing (VIGS) system for Spinacia oleracea L.

Virus-induced gene silencing (VIGS) is known as a rapid and efficient system for studying functions of interesting genes in plants. Tobacco rattle virus (TRV) is widely applied for the gene silencing of many plants. Although spinach is a TRV-susceptible plant, a TRV-based VIGS system has not yet been developed for spinach. In this study, we established a TRV-based VIGS system for spinach. To evaluate the functionality of the TRV-based VIGS system, the phytoene desaturase gene (SoPDS) was first isolated from spinach as a marker gene. Then, the VIGS vector pTRV2 was combined with the partial fragment of SoPDS gene in sense or antisense orientation. Using the Agrobacterium infiltration method, we introduced the pTRV2-SoPDS clone to silence the SoPDS gene in spinach. SoPDS was efficiently silenced, and consequently, greater than 90% of newly emerging leaves exhibited severe chlorosis symptoms in the treated plants. Levels of chlorosis symptoms were similar in both plants infected with pTRV2 vectors harboring sense (SoPDS_S) or antisense (SoPDS_A) gene fragments. Quantitative analysis of SoPDS gene expression by qRT-PCR revealed that gene expression was reduced by greater than 90% in both SoPDS_S and SoPDS_A VIGS plants. Chlorosis on leaves was prolonged up to 4~5 wk after Agrobacterium infiltration. The TRV-based VIGS system was effective in silencing the SoPDS gene in spinach, suggesting that it can be a useful reverse genetics tool for the functional study of spinach genes.

SpinachDB: A Well-Characterized Genomic Database for Gene Family Classification and SNP Information of Spinach.

Spinach (Spinacia oleracea L.), which originated in central and western Asia, belongs to the family Amaranthaceae. Spinach is one of most important leafy vegetables with a high nutritional value as well as being a perfect research material for plant sex chromosome models. As the completion of genome assembly and gene prediction of spinach, we developed SpinachDB (http://222.73.98.124/spinachdb) to store, annotate, mine and analyze genomics and genetics datasets efficiently. In this study, all of 21702 spinach genes were annotated. A total of 15741 spinach genes were catalogued into 4351 families, including identification of a substantial number of transcription factors. To construct a high-density genetic map, a total of 131592 SSRs and 1125743 potential SNPs located in 548801 loci of spinach genome were identified in 11 cultivated and wild spinach cultivars. The expression profiles were also performed with RNA-seq data using the FPKM method, which could be used to compare the genes. Paralogs in spinach and the orthologous genes in Arabidopsis, grape, sugar beet and rice were identified for comparative genome analysis. Finally, the SpinachDB website contains seven main sections, including the homepage; the GBrowse map that integrates genome, genes, SSR and SNP marker information; the Blast alignment service; the gene family classification search tool; the orthologous and paralogous gene pairs search tool; and the download and useful contact information. SpinachDB will be continually expanded to include newly generated robust genomics and genetics data sets along with the associated data mining and analysis tools.

Functional analysis of B and C class floral organ genes in spinach demonstrates their role in sexual dimorphism

BackgroundEvolution of unisexual flowers entails one of the most extreme changes in plant development. Cultivated spinach, Spinacia oleracea L., is uniquely suited for the study of unisexual flower development as it is dioecious and it achieves unisexually by the absence of organ development, rather than by organ abortion or suppression. Male staminate flowers lack fourth whorl primordia and female pistillate flowers lack third whorl primordia. Based on theoretical considerations, early inflorescence or floral organ identity genes would likely be directly involved in sex-determination in those species in which organ initiation rather than organ maturation is regulated. In this study, we tested the hypothesis that sexual dimorphism occurs through the regulation of B class floral organ gene expression by experimentally knocking down gene expression by viral induced gene silencing.ResultsSuppression of B class genes in spinach resulted in the expected homeotic transformation of stamens into carpels but also affected the number of perianth parts and the presence of fourth whorl. Phenotypically normal female flowers developed on SpPI-silenced male plants. Suppression of the spinach C class floral organ identity gene, SpAG, resulted in loss of reproductive organ identity, and indeterminate flowers, but did not result in additional sex-specific characteristics or structures. Analysis of the genomic sequences of both SpAP3 and SpPI did not reveal any allelic differences between males and females.ConclusionSexual dimorphism in spinach is not the result of homeotic transformation of established organs, but rather is the result of differential initiation and development of the third and fourth whorl primordia. SpAG is inferred to have organ identity and meristem termination functions similar to other angiosperm C class genes. In contrast, while SpPI and SpAP3 resemble other angiosperms in their essential functions in establishing stamen identity, they also appear to have an additional function in regulating organ number and identity outside of the third whorl. We present a model for the evolution of dioecy in spinach based on the regulation of B class expression.

A genetic linkage map of Spinacia oleracea and localization of a sex determination locus

A genetic map of Spinach (Spinacia oleracea) was constructed in a classical back cross population using 101 AFLP and 9 microsatellite markers. The map was divided into seven linkage groups with a total length of 585 cM and an average distance between the markers of 5.18 cM. The linkage map was constructed with LOD 3.5, but was quite stable with seven linkage groups remaining until LOD 7.0. Gender segregated 1 male to 1 female in the mapping population and was mapped to a small area of one linkage group with a distance of 1.9 cM to a microsatellite marker termed SO4. This small chromosomal region co-segregating with sex determination in the species is in contrast to previous reports on a heterologous XY chromosome system in spinach. Microsatellite markers used as anchors in the map construction were isolated from sequences of known nuclear encoded genes in spinach. This enabled simultaneous positioning on the map of these genes: Rubisco activase (Rca), Photosytem 1 subunit V (PsaG), Protein Kinase (Pk), Nitrate reductase (Nir), ferrodoxin:thioredoxin reductase (Ftr), Ribosomal protein L1 (Rps22), Choline monooxygenase (Cmo), Pseudogene for BZIP protein (Bzip), Glycerol-3-phosphate acyltransferase (Act1) and stromal ascorbate peroxidase, thylakoid-bound ascorbate peroxidase (Apx2). Spinach has a small genome, which makes it suitable for basic genomic studies and many physiologically important genes have been cloned from the species. The present map anchored with user friendly microsatellite markers will be useful for future studies of physiology and genetics of the species as well as studies of the nature of gender determination.

Sequence evolution and sex-specific expression patterns of the C class floral identity gene, SpAGAMOUS, in dioecious Spinacia oleracea L

Development in dioecious cultivated spinach, Spinacia oleracea, is distinguished by the absence of alternative reproductive organ primordia in male and female flowers. Given the highly derived floral developmental program in spinach, we wished to characterize a spinach C class floral identity gene and to determine the patterns of sequence evolution as well as compare the spatial and temporal expression patterns with those of AGAMOUS. The isolated cDNA sequence clusters phylogenetically within the AGAMOUS/FARINELLI C class clade. In comparison with the SLM1 sequence from the related Silene latifolia, amino acid replacements are highly conservative and non-randomly distributed, being predominantly found in hinge regions or on exposed surfaces of helices. The spinach gene (SpAGAMOUS) appears to be exclusively expressed in reproductive tissues and not in vegetative organs. Initial expression of SpAGAMOUS is similar in male and female floral primordia. However, upon initiation of the first whorl organs, SpAGAMOUS becomes restricted to meristemic regions from which the reproductive primordia will develop. This results in an early gender-specific pattern. Thus, the spinach C class gene is differentially expressed prior to reproductive organ development and is, at least, correlated with, if not directly involved in, the sexual dimorphism in spinach.

In Brief

A Japanese group claims to be the first to make animals genetically modified with a gene from a plant, according to a report on newscientist.com/news. Described by a spokesperson from the Royal Society for the Prevention of Cruelty to Animals as ‘totally disgraceful’, the work has come as a surprise to many experts who had no idea that the work was going on. In order to produce pork with reduced saturated fat content, the group introduced a spinach gene encoding the enzyme FAD2 into pigs. FAD2 converts saturated fats to the unsaturated fat linoleic acid. The data are not yet published, but the scientist in charge of the experiments, Akira Iritani, based at Kinki University in Western Japan, is reported to have said that the FAD2 transgene has been passed successfully through three generations of pigs and seven generations of mice, and that the level of saturated fats in the animals has been reduced by 20%. Iritani, also known for his attempts to clone mammoth DNA, hopes that tests will be conducted to show that the meat from the modified pigs is safe for consumption, and must now be under strong pressure to make his data public. S.L.

Molecular characterization of nucleus-localized RNA-binding proteins from higher plants.

We report the isolation and characterization of genes from the higher plants Arabidopsis, spinach and tobacco which code for nucleus-localized RNA-binding proteins. Common features of these polypeptides are glycine/arginine-rich regions with several RGG repeats at their N- and C-termini, which are sufficient for RNA binding in northwestern assays. All polypeptides analysed contain two basic bipartite nuclear localization signals and translational fusions harbouring these regions with the beta-glucuronidase gene direct the fusion proteins into the nucleus. Nuclear localization was confirmed by cellular fractionation with a polyclonal antiserum raised against the over-expressed tobacco protein NtRGG1p. Two or three copies of related RGG genes appear to be present in the analysed organisms and the expression of some of them is regulated: a tobacco gene is light-regulated and a spinach gene is preferentially expressed in roots. Possible biological functions of this class of RNA-binding proteins as well as structure/function relationships related to the modular structure are discussed.

Identification of a Catalytic Aspartyl Residue ofd-Ribulose 5-Phosphate 3-Epimerase by Site-directed Mutagenesis

Guided by comparative sequence considerations, we have examined the possibility of a catalytic role of Asp186 of D-ribulose 5-phosphate epimerase by site-directed mutagenesis of the recombinant spinach enzyme. Accordingly, D186A, D186N, and D186E mutants of the epimerase were constructed, purified, and characterized; as judged by their electrophoretic mobilities the mutants are properly assembled into octamers like the wild-type enzyme. Based on the extent of internal quenching of Trp fluorescence, the conformational integrity of the wild-type enzyme is preserved in the mutants. The wild-type kcat of 7.1 x 10(3) s-1 is lowered to 3.3 x 10(-4) s-1 in D186A, 0.13 s-1 in D186N, and 1.1 s-1 in D186E; as gauged by D186A, altogether lacking a functional side chain at position 186, the beta-carboxyl of Asp186 facilitates catalysis by >10(7)-fold. Relative to the wild-type enzyme, the Km for D-ribulose 5-phosphate is essentially unaltered with D186N and D186E but increased 10-fold with D186A. Apart from their impairments in epimerase activity, the mutants are unable to catalyze exchange between solvent protons and the C3 proton of substrates. This deficiency and the differential alterations of kinetic parameters among the mutants are consistent with Asp186 serving as an electrophile to facilitate alpha-proton abstraction. This study is the first to identify a catalytic group of the epimerase.