Conversion of perianth into reproductive organs by ectopic expression of the tobacco floral homeotic gene NAG1.

Mutations in the AGAMOUS (AG) gene of Arabidopsis thaliana result in the conversion of reproductive organs, stamens and carpels, into perianth organs, sepals and petals. We have isolated and characterized the putative AG gene from Nicotiana tabacum, NAG1, whose deduced protein product shares 73% identical amino acid residues with the Arabidopsis AG gene product. RNA tissue in situ hybridizations show that NAG1 RNA accumulates early in tobacco flower development in the region of the floral meristem that will later give rise to stamens and carpels. Ectopic expression of NAG1 in transgenic tobacco plants results in a conversion of sepals and petals into carpels and stamens, respectively, indicating that NAG1 is sufficient to convert perianth into reproductive floral organs.     

Tissue-specific expression of esterase isoenzymes inLinum usitatissimum L.

Esterase isoenzyme spectra of different organs of seedlings and field-grown plants of fiber flax (Linum usitatissimum L., cv. Belinka) were studied by electrophoresis in polyacrylamide gel for estimating ontogenetic variability of gene expression. Formation of individual isoesterases depended on the type of tissue and the stage of its development. Isoesterases characteristic of exclusively one or some tissues of the same developmental stage were revealed simultaneously with basic esterase isoforms active in all analysed parts of seeds, seedlings and field-grown plants. The revealed changes of esterase isoenzyme spectrum during germination show tissue and time specificity of the endogenous regulation of genes controlling their formation.     

The small subunit ADP-glucose pyrophosphorylase (<Emphasis Type="Italic">ApS</Emphasis>) promoter mediates okadaic acid-sensitive<Emphasis Type="Italic"> uidA</Emphasis> expression in starch-synthesizing tissues and cells in<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Transgenic plants of Arabidopsis thaliana Heynh., transformed with a bacterial beta-glucuronidase (GUS) gene under the control of the promoter of the small subunit (ApS) of ADP-glucose pyrophosphorylase (AGPase), exhibited GUS staining in leaves (including stomata), stems, roots and flowers. Cross-sections of stems revealed GUS staining in protoxylem parenchyma, primary phloem and cortex. In young roots, the staining was found in the root tips, including the root cap, and in vascular tissue, while the older root-hypocotyl axis showed prominent staining in the secondary phloem and paratracheary parenchyma of secondary xylem. The GUS staining co-localized with ApS protein, as found by tissue printing using antibodies against ApS. Starch was found only in cell and tissue types exhibiting GUS staining and ApS labelling, but not in all of them. For example, starch was lacking in the xylem parenchyma and secondary phloem of the root-hypocotyl axis. Sucrose potently activated ApS gene expression in leaves of wild-type (wt) plants, and in transgenic seedlings grown on sucrose medium where GUS activity was quantified with 4-methylumbelliferyl-beta-glucuronide as substrate. Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, completely blocked expression of ApS in mature leaves of wt plants and prevented GUS staining in root tips and flowers of the transgenic plants, suggesting a similar signal transduction mechanism for ApS expression in various tissues. The data support the key role of AGPase in starch synthesis, but they also underlie the ubiquitous importance of the ApS gene for AGPase function in all organs/tissues of Arabidopsis.     

Subtissue-specific evaluation of promoter efficiency by quantitative fluorometric assay in laser microdissected tissues of rapeseed

β-glucuronidase (GUS) is a useful reporter for the evaluation of promoter characteristics in transgenic plants. Here, we introduce an original technique to quantify the strength of promoters at subtissue resolution of cell clusters. The method combines cryotomy, laser microdissection, and improved fluorometric analysis of GUS activity using 6-chloro-4-methylumbelliferyl-β-D-glucuronide as an efficient fluorogenic substrate for kinetic studies in plants. The laser microdissection/6-chloro-4-methylumbelliferyl-β-D-glucuronide method is robust and reliable in a wide range of GUS expression levels and requires extremely low (few cells) tissue amounts. Suitability of the assay was demonstrated on rapeseed (Brassica napus) plants transformed with a P35S2::GUS construct. GUS expression patterns were visualized and quantified in approximately 30 tissues of vegetative and generative organs. Considerable differences in promoter activity within the tissues are discussed in relation to the cell type and developmental state.     

Duplication and paralog sorting of RPB2 and RPB1 genes in core eudicots

RPB1 and RPB2, which encode the largest and second largest subunits of RNA polymerase II, respectively, are essential single copy genes in fungi, animals and most plants. Two paralogs of the RPB2 gene have been found in some groups of angioperms [Oxelman, B., Yoshikawa, N., McConaughy, B.L., Luo, J., Denton, A.L., Hall, B.D., 2004. RPB2 gene phylogeny in flowering plants, with particular emphasis on asterids. Mol. Phylogenet. Evol. 32, 462-479]. Here, we report the results of experiments designed to identify the evolutionary origin of the RPB2 duplicate copies. Through careful sampling and phylogenetic analysis, we were able to construct the RPB2 gene tree in angiosperms and infer the phylogenetic positions of the gene duplication and gene loss events that occurred. Our study shows that an RPB2 gene duplication occurred early in core eudicot evolution, at or near the time of the Buxaceae/Trochodendraceae divergence. Subsequently, multiple gene duplication and paralog sorting events happened independently in different core eudicot taxa. Differential expression of the two RPB2 gene paralogs may explain the preservation of both paralogs in the asterids. One gene (RPB2-i) accounts for most of the RPB2 mRNA made in the flower organs while the other gene (RPB2-d) is predominantly used in the vegetative tissues. We also found two paralogs of the RPB1 gene in some core eudicot species. The RPB1 gene duplication occurred before core eudicot divergence, around the time of RPB2 gene duplication. Several independent RPB1 paralog sorting events happened in different core eudicot taxa; their occurrence was independent of the RPB2 paralog sorting events. Our results suggest that a polyploidization event happened at or near the time of the Buxaceae/Trochodendraceae divergence. We propose that this polyploidization and the partial diploidization processes thereafter may have been the driving force of core eudicot radiation.     

利用Ⅱ型启动子转录的U6 RNA提高植物病毒表达载体在植物中表达外源基因的水平

Ⅱ型启动子转录的外源短链RNA可以竞争性抑制细胞内源mRNA的核质转运,因而可能会提高植物RNA 病毒载体表达的外源基因在植物中的积累. 为了验证这一假说,利用OE-PCR技术合成拟南芥U6-1核内小RNA序列,并构建其Ⅱ型启动子转录的植物表达载体. 以农杆菌渗滤技术,与烟草花叶病毒（Tobacco mosaic virus, TMV）表达载体共接种寄主植物本氏烟,通过对报告基因绿色荧光蛋白（green fluorescence protein, GFP）的荧光观察,并以Western印迹和ELISA测定GFP在烟草中的表达情况,分析共表达Ⅱ型启动子转录的U6 RNA对外源基因在植物中表达的作用效果. 结果表明,共接种Ⅱ型启动子转录的U6 RNA对TMV病毒表达载体表达外源基因的水平有明显的增效作用,推测RNA核质转运干扰是提高外源基因表达的可能机制.     

Axillary meristem development in the branchless Zu-0 ecotype of Arabidopsis thaliana

Axillary meristems form in the leaf axils during post-embryonic development. In order to initiate the genetic dissection of axillary meristem development, we have characterized the late-flowering branchless ecotype of Arabidopsis thaliana (L.) Heynh., Zu-0. The first-formed rosette leaves of Zu-0 plants all initiate axillary meristems, but later-formed leaves of the rosette remain branchless. Alteration in the meristem development is axillary meristem-specific because the shoot apical and floral meristems develop normally. Scanning electron microscopy, histology and RNA in situ analysis with SHOOTMERISTEMLESS ( STM), a marker for meristematic tissues, show that a mound of cells form and STM mRNA accumulates in barren leaf axils, indicating that axillary meristems initiate but arrest in their development prior to organizing a meristem proper. Expression and retention of the STM RNA in barren leaf axils further suggests that STM expression is not sufficient for the establishment of the axillary meristem proper.     

The sugarcane bacilliform badnavirus promoter is active in both monocots and dicots

Regions of the sugarcane bacilliform badnavirus genome were tested for promoter activity. The genomic region spanning nucleotides 5999–7420 was shown to possess promoter activity as exemplified by its ability to drive the expression of the coding region of the uidA gene of Escherichia coli, in both Avena sativa and Arabidopsis thaliana. In A. sativa, the promoter was active in all organs examined and, with the exception of the anthers where the expression was localized, this activity was constitutive. In A. thaliana, the promoter activity was constitutive in the rosette leaf, stem, stamen, and root and limited primarily to vascular tissue in the sepal and the silique. The transgene was inherited and active in progeny plants of both A. sativa and A. thaliana.     

Induction of trehalase in Arabidopsis plants infected with the trehalose-producing pathogen Plasmodiophora brassicae

Various microorganisms produce the disaccharide trehalose during their symbiotic and pathogenic interactions with plants. Trehalose has strong effects on plant metabolism and growth; therefore, we became interested to study its possible role in the interaction of Arabidopsis thaliana with Plasmodiophora brassicae, the causal agent of clubroot disease. We found that trehalose accumulated strongly in the infected organs (i.e., the roots and hypocotyls) and, to a lesser extent, in the leaves and stems of infected plants. This accumulation pattern of trehalose correlated with the expression of a putative trehalose-6-phosphate synthase (EC 2.4.1.15) gene from P. brassicae, PbTPS1. Clubroot formation also resulted in an induction of the Arabidopsis trehalase gene, ATTRE1, and in a concomitant increase in trehalase (EC 3.2.1.28) activity in the roots and hypocotyls, but not in the leaves and stems of infected plants. Thus, induction of ATTRE1 expression was probably responsible for the increased trehalase activity. Trehalase activity increased before trehalose accumulated; therefore, it is unlikely that trehalase was induced by its substrate. The induction of trehalase may be part of the plant's defense response and may prevent excess accumulation of trehalose in the plant cells, where it could interfere with the regulation of carbon metabolism.