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.

AIM: METHODS: RESULTS: CONCLUSION:

Vegetation has been well recognized for its ability in improving air quality and alleviating global warming. Tree planting has been used to mitigate urban heat island phenomena, sequester carbon dioxide, and help to trap air pollutants on leaves. Because some plant species emit biogenic volatile organic compounds (BVOCs), planting of some cultivars increase ozone and particulate matter ambient concentration and hence deteriorate air quality. Moreover, pollen grains and fungal spores from plants are health hazards for allergic or other sensitive members of the population. This paper reviews benefits and limited hazards of urban vegetation on air quality, highlighting useful computer simulations for predicting some of the interaction between urban forestry and the ambient atmosphere. To maximize the beneficial environmental effects of urban forestry, careful design, planning, and cost-benefit analysis would be required.

Wild type (WT) tomato seedlings responded to a low red to far-red (R/FR) ratio with increased stem elongation, similar leaflet area expansion and lower shoot ethylene levels. The levels of endogenous growth-active GA1 and its immediate precursor GA20 were decreased by low R/FR ratio, whereas the levels of GA1 catabolite, GA8, increased. To examine the interaction of ethylene with GAs in regulating tomato shoot growth under low R/FR ratio, transgenic (T) seedlings bearing Le-ACS2 and Le-ACS4 antisense mRNA were utilized. Low R/FR ratio increased stem elongation and decreased ethylene levels in T tomato shoots, as it did in WT shoots. However, T stems were significantly taller than the WT stems under low R/FR ratio. Leaflet areas were significantly larger for T, than WT seedlings under both R/FR ratios. Low R/FR ratio did not decrease endogenous levels of GA1 and GA20 in T shoots, but did increase GA8 levels, which were higher than in WT shoots. These results, and hormone/inhibitor application studies, showed that in tomato shoots subjected to low R/FR ratio, GAs play a growth-promotive role in stem elongation, whereas ethylene is growth-inhibitory. Further, these results may imply that decreasing ethylene production under low R/FR ratio causes increases in stem elongation and GA levels.

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 β-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 μmol H2S mg−1 protein min−1 and 0.92 μmol 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.

Biogenic volatile organic compounds (BVOCs) in the atmosphere react to form ozone and secondary organic aerosols, which deteriorate air quality, affect human health, and indirectly influence global climate changes. The present study aims to provide a preliminary assessment of BVOC emissions in Hong Kong (HKSAR). Thriteen local tree species were measured for their isoprene emission potential. Tree distribution was estimated for country park areas based on field survey data. Plant emission data obtained from measurements and the literature, tree distribution estimation data, land use information, and meteorological data were combined to estimate annual BVOC emissions of 8.6×109 g C for Hong Kong. Isoprene, monoterpenes, and other VOCs contributed about 30%, 40%, and 30% of the estimated total annual emissions, respectively. Although hundreds of plant species are found in Hong Kong country parks, the model results indicate that only 10 tree species contribute about 76% of total annual VOC emissions. Prominent seasonal and diurnal variations in emissions were also predicted by the model. The present study lays a solid foundation for future local research, and results can be applied for studying BVOC emissions in nearby southern China and Asian regions that share similar climate and plant distributions.

UDP-glucose pyrophosphorylase (UGPase) is predominantly present and plays significant role in carbohydrate metabolism in plants. Two homologous UGPase genes, OsUgp1 and OsUgp2, exist in rice genome. OsUgp1 has recently been reported to be essential for callose deposition during pollen mother cell and meiosis stages as well as for seed carbohydrate metabolism. In this study, a full-length cDNA of OsUgp2 was isolated from rice anther. Northern blot and RNA in situ hybridization indicated that the expression of OsUgp2 was preferentially in pollen and developmentally regulated. No transcripts were found in leaf, stem, lemma/palea, ripening grain and florets before the uninucleate microspore developmental stage, but a large quantity of OsUgp2 mRNA was found in pollen at the binucleate and mature stages. The immunolocalization of OsUgp2 showed a similar expression pattern to that by RNA in situ hybridization. The function of OsUgp2 was investigated by dsRNA-mediated transcriptional gene silencing. The pollen fertility of 16 independent transgenic rice plants was found between 25% and 90%, which was correlated with the amount of OsUgp2 mRNA. The results of morphological changes and starch variation during pollen development in transgenic rice showed that the abnormal feature of pollen development appeared after the uninucleate microspore stage. Starch failed to accumulate in pollen and thus led to sterile pollens. These results demonstrated that OsUgp2 is a pollen-preferential “late gene” and plays a key role during pollen maturation, especially for starch accumulation. OsUgp2 complements OsUgp1 to fulfill the UGPase’s functions necessary for the full process of pollen development.

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.

Plants make use of two-component systems for signal transduction, and these are involved in vital cellular processes such as the responses to cytokinins, ethylene, red/far-red light, and osmosensing ([Schaller et al., 2002][1]). Two-component systems were originally identified in bacteria, and in

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.

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.

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.

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.

A large proportion of soil phosphorus (P) exists as organic compounds, of which phytic acid (IHP) is the dominant form. To generate transgenic plants capable of utilizing exogenous IHP, β-propeller phytase from Bacillus subtilis (168phyA) was constitutively expressed in tobacco and Arabidopsis, and was shown to be secreted from their roots. In tobacco, phytase activities in transgenic leaf and root extracts were seven to nine times higher than those in wild-type extracts; whereas, the extracellular phytase activities of transgenic plants were enhanced by four to six times. In sterile hydroponic culture using 1mM Na-IHP as the sole P source, the transgenic tobacco lines accumulated 1.7–2.2 times more shoot biomass than the wild-type plants after 30 days of growth, concomitant with a 27–36% increase in shoot P concentration. Similar results were observed from the transgenic Arabidopsis. Our work on the exudation of recombinant Bacillus phytase from plant roots may offer a new perspective on mobilizing soil phytate into inorganic phosphate for plant uptake.

In an attempt to understand more about the molecular mechanism by which pollen development is regulated and coordinated in rice, we employed a cDNA subtraction strategy to identify genes that differentially expressed between the wild-type, AN-N and its thermosensitive genic male-sterile mutant (TGMS), AN S-1 during microsporogenesis. Eleven clones exhibiting differential expression patterns were isolated and identified. We report here in detail the molecular cloning and characterization of one of the clones, OS-DFR2 which encodes a dihydroflavonol 4-reductase-like protein. RNA gel blot analysis revealed that OS-DFR2 expressed strongly in anthers but no expression was detected in roots, leaves, stems and pistils, indicating that OS-DFR2 was a tissue-specific gene. Furthermore, the expression of OS-DFR2 was developmentally regulated during early microsporogenesis. It accumulated at high levels in anthers undergoing meiosis and reached a maximal value during tetrad formation stage in the wild-type, AN-N. In AN S-1, the expression of OS-DFR2 resembled that of AN-N except its mRNA level is approximately one-fourth of that found in AN-N at the tetrad formation stage. In situ analysis indicated that OS-DFR2 transcript was specifically expressed in microsporocyte of AN-N; whereas OS-DFR2 was present both in microsporocyte and tapetal cells in AN S-1. The significance and possible role played by OS-DFR2 during male gametophyte development in rice is discussed.

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.

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.