An anther-specific dihydroflavonol 4-reductase-like gene (DRL1) is essential for male fertility in Arabidopsis.

Arabidopsis contains only one functional dihydroflavonol 4-reductase (DFR) gene, but several DFR-like genes encoding proteins with the conserved NAD(P)H binding domain. At4g35420, named DRL1 (Dihydroflavonol 4-reductase-like1), is a closely related homolog of the rice anther-specific gene OsDFR2 reported previously. Two T-DNA mutants (drl1-1 and drl1-2) were found to have impaired pollen formation and seed production. Histological analysis revealed defective microspore development after tetrad release in both mutants. Microspore walls were found to rupture, releasing the protoplasts which eventually degenerated. The DRL1 promoter is anther-specific in closed flower buds. Promoter-GUS analysis in transgenic Arabidopsis revealed expression in tapetum, tetrads, and developing microspores, but not in mature anthers. Enhanced yellow fluorescent protein (EYFP)-localization analysis demonstrated that DRL1 is a soluble cytosolic protein that may also be localized in the nucleus. Restoration of male fertility and seed formation was only achieved by a native promoter-DRL1 construct, but not by a 35S-DRL1 construct, demonstrating the importance of spatial and temporal specificities of DRL1 expression. DRL1 may be involved in a novel metabolic pathway essential for pollen wall development. DRL1 homologs were identified as anther- and floral-specific expressed sequence tags from different species, suggesting that DRL1 may have a conserved functional role in male fertility in flowering plants.

Heterologous expression analyses of rice OsCAS in Arabidopsis and in yeast provide evidence for its roles in cyanide detoxification rather than in cysteine synthesis in vivo.

While most dicot plants produce little ethylene in their vegetative stage, many monocots such as rice liberate a relatively large amount of ethylene with cyanide as a co-product in their seedling stage when etiolated. One of the known functions of beta-cyanoalanine synthase (CAS) is to detoxify the co-product cyanide during ethylene biosynthesis in higher plants. Based on a tryptic peptide sequence obtained from a partially purified CAS activity protein preparation in etiolated rice seedlings, the full-length putative rice CAS-encoding cDNA sequence (OsCAS), which is homologous to those O-acetylserine sulphydrylase (OASS) genes, was cloned. Unlike most of the CAS genes reported from dicots, the transcription of OsCAS is promoted by auxins but suppressed by ethylene. To address the function and the subcellular localization of this gene product in planta, a binary vector construct consisting of this gene appended with a yellow fluorescent protein-encoding sequence was employed to transform Arabidopsis. Specific activities on CAS and OASS of the purified recombinant protein from transgenic Arabidopsis were 181.04 micromol H(2)S mg(-1) protein min(-1) and 0.92 micromol Cys mg(-1) protein min(-1), respectively, indicating that OsCAS favours CAS activity. The subcellular localization of OsCAS was found mostly in the mitochondria by immunogold electron-microscopy. Chemical cross-linking and in-gel assay on a heterodimer composed of functional and non-functional mutants in a yeast expression system on OsCAS suggested that OsCAS functions as a homodimer, similar to that of OASS. Despite the structural similarity of OsCAS with OASS, it has also been confirmed that OsCAS could not interact with serine-acetyltransferase, indicating that OsCAS mainly functions in cyanide detoxification.

Soft contact lens cleaning: rub or no-rub?

PURPOSE: To compare effectiveness of cleaning with and without rubbing of soft contact lenses. METHODS: Three-hundred new biweekly disposable hydrogel lenses (Ocufilcon D, FDA Group IV; 55% water content) were artificially deposited with serum albumin, hand cream (semi-transparent deposits) and mascara (black deposits). The treated lenses were randomly divided into three groups, each group cleaned by one of three methods of cleaning--Rubbing (R), No-Rub following the manufacturer's instruction on duration of rinsing (NR1) and No-Rub with a shorter duration of rinsing (NR2). Four commercially-available multipurpose solutions (MPS) and a saline were used. The cleaning effectiveness was determined by the amount of deposits remaining on the contact lenses after cleaning, assessed with the aid of a slit-lamp. The level of deposits remaining (in terms of coverage of lens surface) were determined using a five-point scale [0 (no observable deposits)--4 (>80% deposits remained)] for semi-transparent deposits (protein and hand cream) and black deposits (mascara). The investigators were masked as to the solutions used (except for one MPS which has a different rinsing time than the other MPS), and the investigator who assessed the deposits left on the lenses did not know which solution or cleaning method was used to clean each lens. RESULTS: Lenses cleaned by the R method were significantly cleaner than those cleaned by methods NR1 and NR2. No significant difference was found between lenses cleaned by NR1 and NR2 methods. The median grade of deposits for lenses cleaned by R method was 0.5 for both semi-transparent and black deposits. For lenses cleaned by NR1 and NR2 methods, the median grade of deposits left on lens surfaces was 4.0 for both types of deposits. Different solutions used did not affect the level of deposits left on lens surfaces for all three cleaning methods. CONCLUSIONS: Not rubbing the soft lens when cleaning is ineffective in removing loosely-bound deposits. A longer rinse, as recommended by the manufacturers, does not remove significantly more deposits than a shorter rinse with the MPS. This work supports the view that contact lens wearers should be encouraged to rub their lenses when cleaning.

Differentially localized rice ethylene receptors OsERS1 and OsETR2 and their potential role during submergence.

Ethylene is gaseous plant hormone that controls a variety of physiologic activities. OsERS1 and OsETR2 are major ethylene receptors in rice that have been reported to have different regulatory functions. The GFP fused N-terminus of OsERS1 and OsETR2 showed differentially localization patterns when transiently expressed in onion epidermal cells. Base on these results, we suggested that OsERS1 could be localized to plasma membranes, whereas OsETR2 could be localized to the endoplasmic reticulum. Furthermore, instead of the constitutive expression profile of OsERS1, OsETR2 is differentially expressed in seedlings of light/dark-grown conditions, submergence or exogenous ethylene treatments. Our results and others support the notion that OsERS1 and OsETR2 could have different roles during rice plant submergence.

A truncated hepatitis E virus ORF2 protein expressed in tobacco plastids is immunogenic in mice.

AIM: To cost-effectively express the 23-ku pE2, the most promising subunit vaccine encoded by the E2 fragment comprising of the 3'-portion of hepatitis E virus (HEV) open reading frame 2 (ORF2) in plastids of tobacco (Nicotiana tabacum cv. SR1), to investigate the transgene expression and pE2 accumulation in plastids, and to evaluate the antigenic effect of the plastid-derived pE2 in mice. METHODS: Plastid-targeting vector pRB94-E2 containing the E2 fragment driven by rice psbA promoter was constructed. Upon delivery into tobacco plastids, this construct could initiate homologous recombination in psaB-trnfM and trnG-psbC fragments in plastid genome, and result in transgene inserted between the two fragments. The pRB94-E2 was delivered with a biolistic particle bombardment method, and the plastid-transformed plants were obtained following the regeneration of the bombarded leaf tissues on a spectinomycin-supplemented medium. Transplastomic status of the regenerated plants was confirmed by PCR and Southern blot analysis, transgene expression was investigated by Northern blot analysis, and accumulation of pE2 was measured by ELISA. Furthermore, protein extracts were used to immunize mice, and the presence of the pE2-reactive antibodies in serum samples of the immunized mice was studied by ELISA. RESULTS: Transplastomic lines confirmed by PCR and Southern blot analysis could actively transcribe the E2 mRNA. The pE2 polypeptide was accumulated to a level as high as 13.27 microg/g fresh leaves. The pE2 could stimulate the immunized mice to generate pE2-specific antibodies. CONCLUSION: HEV-E2 fragment can be inserted into the plastid genome and the recombinant pE2 antigen derived is antigenic in mice. Hence, plastids may be a novel source for cost-effective production of HEV vaccines.

Differential expression of two flavonoid 3'-hydroxylase cDNAs involved in biosynthesis of anthocyanin pigments and 3-deoxyanthocyanidin phytoalexins in sorghum.

Three unique sorghum flavonoid 3'-hydroxylase (F3'H) cDNAs (SbF3'H1, SbF3'H2 and SbF3'H3) were discovered through bioinformatics analysis. Their encoded proteins showed >60% identity to the Arabidopsis TT7 (F3'H) protein. Overexpression of SbF3'H1 or SbF3'H2 restored the ability of tt7 mutants to produce 3'-hydroxylated flavonoids, establishing their roles as functional F3'H enzymes. In sorghum mesocotyls, SbF3'H1 expression was involved in light-specific anthocyanin accumulation while SbF3'H2 expression was involved in pathogen-specific 3-deoxyanthocyanidin synthesis. No SbF3'H3 expression was detected in all tissues examined. The sorghum mesocotyls represent a good system for investigation of differential regulation of F3'H genes/alleles responding to different external stimuli.

Expression of viral capsid protein antigen against Epstein-Barr virus in plastids of Nicotiana tabacum cv. SR1.

Epstein-Barr virus (EBV) infects nearly 90% of adults worldwide and is the pathogenic source of a broad spectrum of malignancies originating from lymphoid and epithelial cells. Currently, no vaccine has been developed to immunologically inactivate this virus. In infected patients, anti-EBV viral capsid antigen (VCA) immunoglobins represent some of the useful diagnostic markers for carcinoma development. To demonstrate that the EBV VCA antigen can be produced in plants, the plastid genome of tobacco (Nicotiana tabacum cv. SR1) was transformed with a VCA-expressing cassette. The EBV VCA mRNA was actively transcribed in the transplastomic plants and antigen production was detected. This study indicates that plastid transformation could be a promising strategy in EBV VCA antigen production.

A stilbene synthase gene (SbSTS1) is involved in host and nonhost defense responses in sorghum.

A chalcone synthase (CHS)-like gene, SbCHS8, with high expressed sequence tag abundance in a pathogen-induced cDNA library, was identified previously in sorghum (Sorghum bicolor). Genomic Southern analysis revealed that SbCHS8 represents a single-copy gene. SbCHS8 expression was induced in sorghum mesocotyls following inoculation with Cochliobolus heterotrophus and Colletotrichum sublineolum, corresponding to nonhost and host defense responses, respectively. However, the induction was delayed by approximately 24 h when compared to the expression of at least one of the other SbCHS genes. In addition, SbCHS8 expression was not induced by light and did not occur in a tissue-specific manner. SbCHS8, together with SbCHS2, was overexpressed in transgenic Arabidopsis (Arabidopsis thaliana) tt4 (transparent testa) mutants defective in CHS activities. SbCHS2 rescued the ability of these mutants to accumulate flavonoids in seed coats and seedlings. In contrast, SbCHS8 failed to complement the mutation, suggesting that the encoded enzyme does not function as a CHS. To elucidate their biochemical functions, recombinant proteins were assayed with different phenylpropanoid-Coenzyme A esters. Flavanones and stilbenes were detected in the reaction products of SbCHS2 and SbCHS8, respectively. Taken together, our data demonstrated that SbCHS2 encodes a typical CHS that synthesizes naringenin chalcone, which is necessary for the formation of different flavonoid metabolites. On the other hand, SbCHS8, now retermed SbSTS1, encodes an enzyme with stilbene synthase activity, suggesting that sorghum accumulates stilbene-derived defense metabolites in addition to the well-characterized 3-deoxyanthocyanidin phytoalexins.

Differential expression of three genes encoding an ethylene receptor in rice during development, and in response to indole-3-acetic acid and silver ions.

Five ethylene receptor genes, OS-ERS1, OS-ERS2, OS-ETR2, OS-ETR3, and OS-ETR4 were isolated and characterized from rice. The genomic structure of OS-ERS1 and OS-ERS2 revealed that the introns within the coding sequences occurred in conserved positions to those of At-ETR1 and At-ERS1, whereas each of the OS-ETR2, OS-ETR3, and OS-ETR4 genes contained 1 intron within its coding region located at a position equivalent to those of At-ERS2, At-ETR2, and At-EIN4. Deduced amino acid sequences of OS-ERS1, OS-ERS2, OS-ETR2, OS-ETR3, and OS-ETR4 showed that they exhibited significant homology to the prokaryotic two-component signal transducer and a wide range of ethylene receptors in a variety of plant species. Northern analysis revealed that the level of OS-ETR2 mRNA was markedly elevated either by the exogenous application of IAA or by ethylene treatment in young etiolated rice seedlings, whereas the OS-ERS1 transcript level was only slightly induced under the same experimental conditions. Pretreatment with silver prevented IAA-induced and ethylene-induced accumulation of both mRNAs (OS-ERS1 and OS-ETR2). However, the abundance of OS-ERS2 mRNA was shown to be down-regulated by both IAA and ethylene treatments, indicating that it was not positively regulated by ethylene. Analysis of the expression of the three ethylene receptor genes in different tissues of rice has unravelled their corresponding tissue-specificity in which OS-ERS1 was constitutively expressed in considerable amounts in all tissues studied, while OS-ERS2 and OS-ETR2 exhibited differential expression patterns in different tissues of rice. Moreover, higher levels of these three mRNAs were commonly observed in anthers when compared with their corresponding levels in other tissues, suggesting the important role played by ethylene involved in the regulation of pollen development in rice. Among the five ethylene receptor genes, the expression levels of both OS-ETR3 and OS-ETR4 were too low to be detected by the northern blot analysis. Results from RT-PCR illustrated that both mRNAs were present in young green rice seedlings and anthers.