A new modification of silver staining is presented which utilizes two chemical properties of thiosulfate: image enhancement by pretreatment of fixed gels, and formation of soluble silver complexes which prevents unspecific background staining during image development. This procedure provides high sensitivity for proteins, RNA and DNA in the nanogram range on a colorless, transparent background. The performance of this method is documented by staining one-and two-dimensional patterns of plant leaf proteins. Moreover, we achieved, for the first time, the detection of the non-structural, tobacco mosaic virus-specific 126 kDa protein directly in the one-dimensional protein pattern of infected protoplasts by a staining procedure.

Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels

We have produced a draft sequence of the rice genome for the most widely cultivated subspecies in China, Oryza sativa L. ssp. indica, by whole-genome shotgun sequencing. The genome was 466 megabases in size, with an estimated 46,022 to 55,615 genes. Functional coverage in the assembled sequences was 92.0%. About 42.2% of the genome was in exact 20-nucleotide oligomer repeats, and most of the transposons were in the intergenic regions between genes. Although 80.6% of predicted Arabidopsis thaliana genes had a homolog in rice, only 49.4% of predicted rice genes had a homolog in A. thaliana. The large proportion of rice genes with no recognizable homologs is due to a gradient in the GC-content of rice coding sequences.

/pdf/a-draft-sequence-of-the-rice-genome-oryza-sativa-l-ssp-5212kgt1ff.pdf

A draft sequence of the rice genome (Oryza sativa L. ssp indica)

Rice, one of the world's most important food plants, has important syntenic relationships with the other cereal species and is a model plant for the grasses. Here we present a map-based, finished quality sequence that covers 95% of the 389 Mb genome, including virtually all of the euchromatin and two complete centromeres. A total of 37,544 non-transposable-element-related protein-coding genes were identified, of which 71% had a putative homologue in Arabidopsis. In a reciprocal analysis, 90% of the Arabidopsis proteins had a putative homologue in the predicted rice proteome. Twenty-nine per cent of the 37,544 predicted genes appear in clustered gene families. The number and classes of transposable elements found in the rice genome are consistent with the expansion of syntenic regions in the maize and sorghum genomes. We find evidence for widespread and recurrent gene transfer from the organelles to the nuclear chromosomes. The map-based sequence has proven useful for the identification of genes underlying agronomic traits. The additional single-nucleotide polymorphisms and simple sequence repeats identified in our study should accelerate improvements in rice production.

/pdf/the-map-based-sequence-of-the-rice-genome-2y5gce5ixc.pdf

The map-based sequence of the rice genome

Plant growth is greatly affected by drought and low temperature. Expression of a number of genes is induced by both drought and low temperature, although these stresses are quite different. Previous experiments have established that a cis-acting element named DRE (for dehydration-responsive element) plays an important role in both dehydration- and low-temperature-induced gene expression in Arabidopsis. Two cDNA clones that encode DRE binding proteins, DREB1A and DREB2A, were isolated by using the yeast one-hybrid screening technique. The two cDNA libraries were prepared from dehydrated and cold-treated rosette plants, respectively. The deduced amino acid sequences of DREB1A and DREB2A showed no significant sequence similarity, except in the conserved DNA binding domains found in the EREBP and APETALA2 proteins that function in ethylene-responsive expression and floral morphogenesis, respectively. Both the DREB1A and DREB2A proteins specifically bound to the DRE sequence in vitro and activated the transcription of the b-glucuronidase reporter gene driven by the DRE sequence in Arabidopsis leaf protoplasts. Expression of the DREB1A gene and its two homologs was induced by low-temperature stress, whereas expression of the DREB2A gene and its single homolog was induced by dehydration. Overexpression of the DREB1A cDNA in transgenic Arabidopsis plants not only induced strong expression of the target genes under unstressed conditions but also caused dwarfed phenotypes in the transgenic plants. These transgenic plants also revealed freezing and dehydration tolerance. In contrast, overexpression of the DREB2A cDNA induced weak expression of the target genes under unstressed conditions and caused growth retardation of the transgenic plants. These results indicate that two independent families of DREB proteins, DREB1 and DREB2, function as trans-acting factors in two separate signal transduction pathways under low-temperature and dehydration conditions, respectively.

/pdf/two-transcription-factors-dreb1-and-dreb2-with-an-erebp-ap2-3kypshugg1.pdf

Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis.

Plant productivity is greatly affected by environmental stresses such as drought, salt loading, and freezing. We reported previously that a cis-acting promoter element, the dehydration response element (DRE), plays an important role in regulating gene expression in response to these stresses. The transcription factor DREB1A specifically interacts with the DRE and induces expression of stress tolerance genes. We show here that overexpression of the cDNA encoding DREB1A in transgenic plants activated the expression of many of these stress tolerance genes under normal growing conditions and resulted in improved tolerance to drought, salt loading, and freezing. However, use of the strong constitutive 35S cauliflower mosaic virus (CaMV) promoter to drive expression of DREB1A also resulted in severe growth retardation under normal growing conditions. In contrast, expression of DREB1A from the stress inducible rd29A promoter gave rise to minimal effects on plant growth while providing an even greater tolerance to stress conditions than did expression of the gene from the CaMV promoter.

Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor

Five retrotransposon families of rice (Tos1-Tos5) have been reported previously Here we report 15 new retrotransposon families of rice (Tos6-Tos20) In contrast to yeast and Drosophila retrotransposons, all of the rice retrotransposons examined appear inactive (or almost inactive) under normal growth conditions Three of the rice retrotransposons (Tos10, Tos17, and Tos19) are activated under tissue culture conditions The most active one, Tos17, was studied in detail The copy number of Tos17 increased with prolonged culture period In all of the plants regenerated from tissue cultures, including transgenic plants, 5 to 30 transposed Tos17 copies were detected The transcript of Tos17 was only detected under tissue culture conditions, indicating that the transposition of Tos17 is mainly regulated at the transcriptional level To examine the target-site specificity of Tos17 transposition, sequences flanking transposed Tos17 copies were analyzed At least four out of eight target sites examined are coding regions Other target sites may also be in genes because two out of four were transcribed The regenerated plants with Tos17-insertions in the phytochrome A gene and the S-receptor kinase-related gene were identified These results indicate that activation of Tos17 is an important cause of tissue culture-induced mutations Tissue culture-induced activation of Tos17 may be a useful tool for insertional mutagenesis and functional analysis of genes

https://www.pnas.org/content/pnas/93/15/7783.full.pdf

Retrotransposons of rice involved in mutations induced by tissue culture

A series of chimeric promoters for higher-level expression of foreign genes in plants was constructed as fusions of a gene for beta-glucuronidase (GUS) with the terminator of a gene for nopaline synthase (nos) or of the cauliflower mosaic virus (CaMV) 35S transcript, and the strength of these promoters was assayed in transient and stable expression systems in tobacco and rice. As parts of these promoters, the CaMV 35S core promoter, three different 5'-upstream sequences of the 35S promoter, the first intron of a gene for phaseolin, and a 5'-untranslated sequence (omega sequence) of tobacco mosaic virus were used in various combinations. In tobacco and rice protoplasts, all three fragments of the 35S promoter (-419 to -90, -390 to -90 and -290 to -90, relative to the site of initiation of transcription), the intron, and the omega sequence effectively enhanced GUS activity. Some chimeric promoters allowed levels of GUS activity that were 20- to 70-fold higher than those obtained with the 35S promoter in pBI221. In tobacco protoplasts, the two longer fragments of the 35S promoter were more effective than the shortest fragment. In rice cells, by contrast, the shortest fragment was as effective as the two longer ones. The terminator of the 35S transcript was more effective than that of the nos gene for gene expression. In transgenic tobacco plants, a representative powerful promoter, as compared to the 35S promoter, allowed 10- and 50-fold higher levels of expression on average and at most, respectively, with no clear qualitative differences in tissue- and organ-specific patterns of expression. When the representative promoter was introduced into tobacco with a gene for luciferase, the autofluorescence of detached leaves after a supply of luciferin to petioles was great and was easily detectable by the naked eye in a dark room.

/pdf/efficient-promoter-cassettes-for-enhanced-expression-of-16htnafh1z.pdf

Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants.

It was proved that the protoplasts prepared from mesophyll of Nicotiana tabacum are infected by tobacco mosaic virus. The infection occurred when purified tobacco mosaic virus particles were added to a protoplast suspension in the presence of poly-L-ornithine. The virus multiplied in these protoplasts to a level of 106 virus particles per infected protoplast during 24 hours of incubation. The efficiency of infection was remarkably high, exceeding that by mechanical inoculation of tobacco leaves.

https://www.pnas.org/content/pnas/64/3/843.full.pdf

Infection of Tobacco Mesophyll Protoplasts by Tobacco Mosaic Virus

The complete nucleotide sequence of the tobacco retrotransposon Tto1, one of the few active retrotransposons of plants, was determined. The sequence analysis suggests that Tto1 carries all functions required for autonomous transposition through reverse transcription. Gene organization and the nature of the transcription product suggest that Tto1 uses a gene expression mechanism different from those employed by retroviruses and most retrotransposons to regulate Gag and Pol stoichiometry. Tto1 was introduced into rice to study its autonomous transposition in heterologous hosts. Transcription and transposition of Tto1 were observed in rice cells. To probe the autonomous transposition through reverse transcription, a modified Tto1 retrotransposon in which part of a reverse transcriptase gene was replaced with an intron-containing hygromycin resistance gene was constructed and introduced into rice cells. Loss of the intron was observed only when intact Tto1 was cotransfected. These results indicate that Tto1 can transpose autonomously through reverse transcription and that the host factors required for transposition are conserved among monocots (class Magnoliopsida; rice) and dicots (class Liliopsida; tobacco), which diverged approximately 200 million years ago. These findings are discussed in relation to the regulation and evolution of retrotransposons and the possible use of Tto1 as a molecular genetic tool.

Autonomous transposition of the tobacco retrotransposon Tto1 in rice.

Genomic libraries of rice,Oryza sativa L. cv. Nipponbare, in yeast artificial chromosomes were prepared for construction of a rice physical map. High-molecular-weight genomic DNA was extracted from cultured suspension cells embedded in agarose plugs. After size fractionation of theEco RI- andNot I-digested DNA fragments, they were ligated with pYAC4 and pYAC55, respectively, and used to transformSaccharomyces cerevisiae AB1380. A total of 6932 clones were obtained containing on average ca. 350 kb DNA. The YAC library was estimated to contain six haploid genome equivalents. The YACs were examined for their chimerism by mapping both ends on an RFLP linkage map. Most YACs withEco RI fragments below 400 kb were intact colinear clones. About 40% of clones were chimeric. Genetic mapping of end clones from large size YACs revealed that the physical distance corresponding to 1 cM genetic distance varies from 120 to 1000 kb, depending on the chromosome region. To select and order YAC clones for making contig maps, high-density colony hybridization using ECL was applied. With several probes, at least one and at most ten YAC clones could be selected in this library. The library size and clone insert size indicate that this YAC library is suitable for physical map construction and map-based cloning.

Yoshiaki Otsuki

Papers

Retrotransposons of rice involved in mutations induced by tissue culture

Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants.

Infection of Tobacco Mesophyll Protoplasts by Tobacco Mosaic Virus

Autonomous transposition of the tobacco retrotransposon Tto1 in rice.

Construction and characterization of a rice YAC library for physical mapping