Analyses in transgenic tobacco of the promoter from a Brassica napus gene highly expressed in the stigma

A genomic clone, Pis G363, containing the Brassica napus stigma-expressed gene Pis 63-2 was isolated and sequenced. The coding region of Pis G363 does not possess introns and shows 82% identity to the nucleotide sequence of a gene from Arabidopsis BAC clone T01B08. A 2-kb promoter fragment from Pis G363 was fused to the coding sequence of the marker enzyme β-glucuronidase (GUS) and introduced into tobacco via Agrobacterium-mediated transformation. The promoter fragment directed expression of the GUS gene in the stigma of transgenic tobacco. Some transformants also showed relatively low GUS activity in the pollen. Received: 25 May 1998 / Revision received: 30 July 1998 / Accepted: 21 August 1998     

The 5′ untranslated region of Arabidopsis thaliana calmodulin cDNA is an independent cDNA containing an open reading frame

A cDNA clone, pAUK1, with an open reading frame (ORF) coding for a hypothetical 164-amino-acid protein was isolated from an Arabidopsis thaliana (L.) Heynh cDNA library. The clone was attached, tail to tail, to the 3′ end of A. thaliana hexokinase cDNA. An almost identical sequence had been previously described as the 5′ untranslated region (5′ UTR) of A. thaliana calmodulin cDNA (ACaM-2). Sequence comparison with three additional A. thaliana truncated cDNA clones which appear in a database (GenBank) supports the conclusion that pAUKl is identical to the 5′ UTR of ACaM-2 and that the 5′ UTR of ACaM-2 is an independent cDNA artificially linked to A. thaliana calmodulin cDNA.     

BcMF21 is important for pollen development and germination in Brassica campestris ssp. chinensis

Brassica campestris Male Fertility 21 (BcMF21) was previously isolated from the flower buds of Chinese cabbage (Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis) and expressed specifically in tapetum and microspores during the meiosis stage and the uninucleate stage of microspore development. Here, we used antisense RNA technology to knock down the expression level of BcMF21 in B. campestris and analyzed the phenotype of the transgenic plants. Alexander staining and scanning electron microscope revealed sterility and exine deformities in the mature pollen grains of BcMF21 antisense RNA transgenic plants. The germination furrow of the BcMF21 antisense RNA transgenic pollen was covered by lipid like materials. The pollen tubes burst and could not grow normally in vitro. Therefore, we presented here BcMF21 might be an important gene for pollen development and germination.     

BcMF9, a novel polygalacturonase gene, is required for both Brassica campestris intine and exine formation

Background and Aims

The polygalacturonase (PG) gene family has been found to be enriched in pollen of several species; however, little is currently known about the function of the PG gene in pollen development. To investigate the exact role that the PG gene has played in pollen development and about this family in general, one putative PG gene, Brassica campestris Male Fertility 9 (BcMF9), was isolated from Chinese cabbage (Brassica campestris ssp. chinensis, syn. B. rapa ssp. chinensis) and characterized.

Methods

RT-PCR, northern blotting and in situ hybridization were used to analyse the expression pattern of BcMF9, and antisense RNA technology was applied to study the function of this gene.

Key Results

BcMF9 is expressed in particular in the tapetum and microspore during the late stages of pollen development. Antisense RNA transgenic plants that displayed decreased expression of BcMF9 showed pollen morphological defects that resulted in reduced pollen germination efficiency. Transmission electron microscopy revealed that the homogeneous pectic exintine layer of pollen facing the exterior was over-developed and predominantly occupied the intine, reversing the normal proportional distribution of the internal endintine layer and the external exintine in transgenic pollen. Inhibition of BcMF9 also resulted in break-up of the previously formed tectum and baculae from the beginning of the binucleate stage, as a result of premature degradation of tapetum.

Conclusions

Several lines of evidence, including patterns of BcMF9 expression and phenotypic defects, suggest a sporophytic role in exine patterning, and a gametophytic mode of action of BcMF9 in intine formation. BcMF9 might act as a co-ordinator in the late stages of tapetum degeneration, and subsequently in the regulation of wall material secretion and, in turn, exine formation. BcMF9 might also play a role in intine formation, possibly via regulation of the dynamic metabolism of pectin.Key words: Brassica campestris, Chinese cabbage, exine, intine, PG, pollen wall, polygalacturonase, BcMF9     

Metallothioneins BcMT1 and BcMT2 from Brassica campestris enhance tolerance to cadmium and copper and decrease production of reactive oxygen species in Arabidopsis thaliana

Aims

Metallothioneins are cysteine-rich, metal-binding proteins, but their exact functions are not fully understood. In this study, we isolated two metallothionein genes, BcMT1 and BcMT2 from Brassica campestris to increase our understanding of metal tolerance mechanisms in Brassica plants.

Methods

Semi-quantitative RT-PCR was used to analyze expression of the two BcMTs genes. BcMT1 and BcMT2 were ectopically expressed in Arabidopsis thaliana. Quantitative real-time RT-PCR and GUS-staining method were used to select transgenic Arabidopsis plants. Cd and Cu concentrations were analyzed by flame atomic absorption spectrometry. Histochemical detection of H₂O₂ and O₂ ^?? were conducted by 3,3-diaminobenzidine and nitroblue tetrazoliu-staining methods.

Results

BcMT1 is expressed predominantly in roots, whereas BcMT2 is expressed mainly in leaves of B. campestris. Expression of BcMT1 was induced by both Cd and Cu, but expression of BcMT2 was enhanced only by Cd. Ectopic expression of BcMT1 and BcMT2 in Arabidopsis thaliana enhanced the tolerance to Cd and Cu and increased the Cu concentration in the shoots of the transgenic plants. Transgenic Arabidopsis accumulated less reactive oxygen species (ROS) than wild-type plants.

Conclusions

BcMT1 and BcMT2 increased Cd and Cu tolerance in transgenic Arabidopsis, and decreased production of Cd- and Cu-induced ROS, thereby protecting plants from oxidative damage.     

Induction of male sterile cabbage using a tapetum-specific promoter from<Emphasis Type="Italic"> Brassica campestris</Emphasis> L. ssp.<Emphasis Type="Italic"> pekinensis</Emphasis>

The anther (tapetum)-specific gene BcA9 was isolated from Chinese cabbage, Brassica campestris L. ssp. pekinensis cv. Jangwon, using the Arabidopsis tapetum-specific A9 gene as a probe. The DNA and amino acid sequences of the coding region of the BcA9 gene showed high homology with A9 genes from Arabidopsis and B. napus. However, the DNA sequences of the 5 noncoding (promoter) region were different, except for the sequence from –281 to –89. To test the specific activity of this promoter, a plant expression vector, pGR011, was constructed by fusing the BcA9 promoter and the cytotoxic diphtheria toxin A-chain (DTx-A) gene. Several transgenic plants from cabbage, B. oleracea ssp. capitata, were obtained by way of Agrobacterium-mediated transformation. Southern blot analysis indicated that the tapetum-specific BcA9 promoter and DTx-A gene were successfully integrated into the genome of the transgenic cabbage. Under the control of the BcA9 promoter, expression of the cytotoxic DTx-A gene in the tapetal cells of the transgenic plants resulted in male sterile cabbages. Microscopic examination revealed that pollen grains in anthers of the male sterile cabbages had not developed normally, but the vegetative growth and phenotype showed no difference compared to wild-type plants.Abbreviations At Arabidopsis thaliana - Bc Brassica camepstris - Bn Brassica napus - DTx-A Diphtheria toxin A-chain gene - hpt Hygromycin phosphotransferase - PCR Polymerase chain reaction - SDS Sodium dodecyl sulfate - SSC Sodium chloride-sodium citrate bufferThis revised version was published online September 2003 with corrections to Figure 6.Communicated by I.S. Chung     

Promoters of nuclear‐encoded respiratory chain Complex I genes from Arabidopsis thaliana contain a region essential for anther/pollen‐specific expression

Regulatory promoter regions responsible for the enhanced expression in anthers and pollen are defined in detail for three nuclear encoded mitochondrial Complex I (nCI) genes from Arabidopsis thaliana. Specific regulatory elements were found conserved in the 5′ upstream regions between three different genes encoding the 22 kDa (PSST), 55 kDa NADH binding (55 kDa) and 28 kDa (TYKY) subunits, respectively. Northern blot analysis and transgenic Arabidopsis plants carrying progressive deletions of the promoters fused to the β-glucuronidase (GUS) reporter gene by histochemical and fluorimetric methods showed that all three promoters drive enhanced expression of GUS specifically in anther tissues and in pollen grains. In at least two of these promoters the –200/–100 regions actively convey the pollen/anther-specific expression in gain of function experiments using CaMV 35S as a minimal promoter. These nCI promoters thus contain a specific regulatory region responding to the physiological demands on mitochondrial function during pollen maturation. Pollen-specific motifs located in these regions appear to consist of as little as seven nucleotides in the respective promoter context.     

In Silico and Deletion Analysis of Upstream Promoter Fragment of S-Adenosyl Homocysteine Hydrolase (SAHH1) Gene of Arabidopsis Leads to the Identification of a Fragment Capable of Driving Gene Expression in Developing Seeds and Anthers

S-adenosyl homocysteine hydrolase (SAHH) is a key enzyme in methylation metabolism of eukaryotes. A 1585 by fragment upstream to ATG of SAHH1 gene, was fused with a promoter-less β-Glucuronidase (GUS) gene and mobilized into Arabidopsis by Agrobacterium-mediated floral transformation to generate transgenic Arabidopsis. This fragment was found to drive constitutive expression of GUS in T₂ progeny of transgenic Arabidopsis. In silico analysis of the promoter region of SAHH1 suggested the presence of several cis-regulatory motifs including seed-specific motifs as well as anther-specific motifs in the 376 by (upstream to TSS of SAHH1) promoter fragment. Based on the partial deletion analysis carried out in the promoter region of SAHH1 (At4gl3940) this 376 by promoter fragment was found to be capable of driving GUS expression in developing seeds and in some anthers/micros pores.     

Pollen-Specific Expression of <Emphasis Type="Italic">Oryza sativa Indica</Emphasis> Pollen Allergen Gene (<Emphasis Type="Italic">OSIPA</Emphasis>) Promoter in Rice and <Emphasis Type="Italic">Arabidopsis</Emphasis> Transgenic Systems

Earlier, a pollen-specific Oryza sativa indica pollen allergen gene (OSIPA), coding for expansins/pollen allergens, was isolated from rice, and its promoter—upon expression in tobacco and Arabidopsis—was found active during the late stages of pollen development. In this investigation, to analyze the effects of different putative regulatory motifs of OSIPA promoter, a series of 5′ deletions were fused to β-glucuronidase gene (GUS) which were stably introduced into rice and Arabidopsis. Histochemical GUS analysis of the transgenic plants revealed that a 1631 bp promoter fragment mediates maximum GUS expression at different stages of anther/pollen development. Promoter deletions to −1272, −966, −617, and −199 bp did not change the expression profile of the pollen specificity. However, the activity of promoter was reduced as the length of promoter decreased. The region between −1567 and −199 bp was found adequate to confer pollen-specific expression in both rice and Arabidopsis systems. An approximate 4-fold increase in the GUS activity was observed in the pollen of rice when compared to that of Arabidopsis. As such, the OSIPA promoter seems promising for generation of stable male-sterile lines required for the production of hybrids in rice and other crop plants.     

Molecular and Histochemical Analysis of a T-DNA Tagged Mutant of Arabidopsis Exhibiting Anther — Specific GUS Expression

An Arabidopsis thaliana mutant, exhibiting anther specific GUS expression, identified from a mutant population of Arabidopsis tagged with a promoterless β-glucuronidase (GUS), carries the T-DNA insertions at two distinct loci. We have been able to segregate the two inserts from each other by backcrossing with wild type plants. The insertion responsible for anther specific GUS expression in segregating population has been identified and confirmed to be in the upstream region of a putative peroxidase gene, AT2G24800. Here we report detailed histochemical and molecular characterization of the mutant Anth85, carrying a single insertion of T-DNA in the peroxidase gene. In Anth85, the GUS expression was observed in the anthers and rosette of the young seedlings. The expression of GUS in the anthers was restricted to the tapetum and microspores. The mutant has no developmental defects and the gene appears to be redundant for normal plant growth. Cloning of upstream region and detailed deletion study of upstream region in transgenic plants is likely to lead to the identification of anther specific promoter elements.