Photobiological hydrogen production and artificial photosynthesis for clean energy: from bio to nanotechnologies.

Global energy demand is increasing rapidly and due to intensive consumption of different forms of fuels, there are increasing concerns over the reduction in readily available conventional energy resources. Because of the deleterious atmospheric effects of fossil fuels and the uncertainties of future energy supplies, there is a surge of interest to find environmentally friendly alternative energy sources. Hydrogen (H2) has attracted worldwide attention as a secondary energy carrier, since it is the lightest carbon-neutral fuel rich in energy per unit mass and easy to store. Several methods and technologies have been developed for H2 production, but none of them are able to replace the traditional combustion fuel used in automobiles so far. Extensively modified and renovated methods and technologies are required to introduce H2 as an alternative efficient, clean, and cost-effective future fuel. Among several emerging renewable energy technologies, photobiological H2 production by oxygenic photosynthetic microbes such as green algae and cyanobacteria or by artificial photosynthesis has attracted significant interest. In this short review, we summarize the recent progress and challenges in H2-based energy production by means of biological and artificial photosynthesis routes.

Transgenic radish (Raphanus sativus L. longipinnatus Bailey) by floral-dip method--plant development and surfactant are important in optimizing transformation efficiency.

Transgenic radish (Raphanus sativus L. longipinnatus Bailey) plants were produced from the progeny of plants which were dipped into a suspension of Agrobacterium carrying both the beta-glucuronidase (gusA) gene and a gene for resistance to the herbicide Basta (bar) between T-DNA border sequences. The importance of development of the floral-dipped plant and presence of surfactant in the inoculation medium were evaluated in terms of transgenic plant production. Plants dipped at the primary bolt stage of growth, into a suspension of Agrobacterium containing 0.05% (v/v) Silwet L-77 resulted in optimum transformation efficiency, with 1.4% from 1110 seeds. The presence of Pluronic F-68 or Tween 20 in the inoculation medium was beneficial towards transgenic plant output compared to treatments without surfactant. Putative transformed T1 plants were efficiently selected by spraying with 0.03% (v/v) Basta and all herbicide-resistant plants tested positive for GUS activity when analysed both histochemically and fluorometrically. Southern analysis revealed that both the gusA and bar genes integrated into the genome of transformed plants and segregated as dominant Mendelian traits. These results demonstrate that radish can be genetically modified for the improvement of this important vegetable crop.

ORE9, an F-box protein that regulates leaf senescence in Arabidopsis.

Senescence is a sequence of biochemical and physiological events that constitute the final stage of development. The identification of genes that alter senescence has practical value and is helpful in revealing pathways that influence senescence. However, the genetic mechanisms of senescence are largely unknown. The leaf of the oresara9 (ore9) mutant of Arabidopsis exhibits increased longevity during age-dependent natural senescence by delaying the onset of various senescence symptoms. It also displays delayed senescence symptoms during hormone-modulated senescence. Map-based cloning of ORE9 identified a 693-amino acid polypeptide containing an F-box motif and 18 leucine-rich repeats. The F-box motif of ORE9 interacts with ASK1 (Arabidopsis Skp1-like 1), a component of the plant SCF complex. These results suggest that ORE9 functions to limit leaf longevity by removing, through ubiquitin-dependent proteolysis, target proteins that are required to delay the leaf senescence program in Arabidopsis.

Overexpression of the AtGluR2 gene encoding an Arabidopsis homolog of mammalian glutamate receptors impairs calcium utilization and sensitivity to ionic stress in transgenic plants.

We have identified a homolog of the mammalian ionotropic glutamate receptor genes in Arabidopsis thaliana (AtGluR2). This gene was found to alter Ca2+ utilization when overexpressed in A. thaliana. These transgenic plants displayed symptoms of Ca2+ deficiency, including browning and death of the shoot apex, necrosis of leaf tips, and deformation of leaves. Supplementation with Ca2+ alleviated these phenotypes. Overall levels of Ca2+ in tissues of control plants were not significantly different from those of transgenic plants, suggesting that overexpression of the AtGluR2 gene did not affect Ca2+ uptake. However, the relative growth yield as a function of Ca2+ levels revealed that the critical deficiency content of Ca2+ in transgenic plants was three times higher than that of control plants. The transgenic plants also exhibited hypersensitivity to Na+ and K+ ionic stresses. The ion hypersensitivity was ameliorated by supplementation with Ca2+. The results showed that overexpression of the AtGluR2 gene caused reduced efficiency of Ca2+ utilization in the transgenic plants. The promoter of the AtGluR2 gene was active in vascular tissues, particularly in cells adjacent to the conducting vessels. This suggests that AtGluR2 encodes a functional channel that unloads Ca2+ from the xylem vessels. The results together suggest that appropriate expression of the AtGluR2 protein may play critical roles in Ca2+ nutrition by controlling the ion allocation among different Ca2+ sinks both during normal development and during adaptation to ionic stresses.

Broad bean wilt virus Causes Necrotic Symptoms and Generates Defective RNAs in Capsicum annuum.

ABSTRACT A virus was isolated from hot pepper (Capsicum annuum cv. Hyang Chon) growing in Korea and displaying necrotic spots or streaks on leaves and stems followed by stunting and death of plants. Morphological and host range analyses of extracts from infected plants suggested that the causal agent of disease was a Broad bean wilt virus (BBWV), and the virus was tentatively named a Korean isolate of BBWV (BBWV-K). When the isolate was back-inoculated onto hot pepper plants, it induced symptoms similar to those of naturally infected hot pepper in the field. Two coat proteins (CPs) of 44 and 22 kDa, corresponding to a large CP and a small CP, respectively, were identified from the virus, and both reacted specifically with polyclonal antibody to BBWV 2. The complete nucleotide sequences of RNA 1 and RNA 2 of the isolate were determined from cDNA clones. The deduced amino acid sequence data from the putative proteins encoded by RNA 1 and 2 of the BBWV-K indicated a closer relationship with the isolates of BBWV 2 than BBWV 1. However, sequence comparison of the 5' noncoding regions of the viruses differentiates BBWV-K from other BBWV 2 isolates. Another distinctive feature of the BBWV-K is that it generates defective RNAs in hot pepper exhibiting necrotic symptoms, which is the first report of defective RNAs in the Fabavirus genera of BBWVs.

Control of circadian rhythms and photoperiodic flowering by the Arabidopsis GIGANTEA gene.

Photoperiodic responses in plants include flowering that is day-length-dependent. Mutations in the Arabidopsis thaliana GIGANTEA (GI) gene cause photoperiod-insensitive flowering and alteration of circadian rhythms. The GI gene encodes a protein containing six putative transmembrane domains. Circadian expression patterns of the GI gene and the clock-associated genes, LHY and CCA1, are altered in gi mutants, showing that GI is required for maintaining circadian amplitude and appropriate period length of these genes. The gi-1 mutation also affects light signaling to the clock, which suggests that GI participates in a feedback loop of the plant circadian system.

Photomorphogenic development of the Arabidopsis shy2-1D mutation and its interaction with phytochromes in darkness.

We previously reported a photomorphogenic mutation of Arabidopsis thaliana, shy2-1D, as a dominant suppressor of a hy2 mutation. Here, we report that shy2-1D confers various photo-responsive phenotypes in darkness and the dark phenotypes of the mutant are affected by phytochrome deficiency. Dark-grown seedlings of the mutant developed several photomorphogenic characteristics such as short hypocotyls, cotyledon expansion and opening, and partial differentiation of plastids. When grown further in darkness, the mutant plant underwent most of the developmental stages of a light-grown wild-type plant, including development of foliar leaves, an inflorescence stem with cauline leaves, and floral organs. In addition, two light-inducible genes, the nuclear-encoded CAB and the plastid-encoded PSBA genes, were highly expressed in the dark-grown mutant seedlings. Furthermore, reduced gravitropism, a phytochrome-modulated response, was observed in the mutant hypocotyl in darkness. Thus, shy2-1D is one of the most pleiotropic photomorphogenic mutations identified so far. The results indicate that SHY2 may be a key component regulating photomorphogenesis in Arabidopsis. Surprisingly, double mutants of the shy2-1D mutant with the phytochrome-deficient mutants hy2, hy3(phyB-1) and fre1-1(phyA-201) showed reduced photomorphogenic response in darkness with a longer hypocotyl, a longer inflorescence stem, and a lower level expression of the CAB gene than the shy2-1D single mutant. These results showed that phytochromes function in darkness in the shy2-1D mutant background. The implications of these results are discussed.

A Brassica cDNA clone encoding a bifunctional hydroxymethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase involved in thiamin biosynthesis.

We report the characterization of a Brassica napus cDNA clone (pBTHI) encoding a protein (BTHI) with two enzymatic activities in the thiamin biosynthetic pathway, thiamin-phosphate pyrophosphorylase (TMP-PPase) and 2-methyl-4-amino-5-hydroxymethylpyrimidine-monophosphate kinase (HMP-P kinase). The cDNA clone was isolated by a novel functional complementation strategy employing an Escherichia coli mutant deficient in the TMP-PPase activity. A biochemical assay showed the clone to confer recovery of TMP-PPase activity in the E. coli mutant strain. The cDNA clone is 1746 bp long and contains an open reading frame encoding a peptide of 524 amino acids. The C-terminal part of BTH1 showed 53% and 59% sequence similarity to the N-terminal TMP-PPase region of the bifunctional yeast proteins Saccharomyces THI6 and Schizosaccharomyces pombe THI4, respectively. The N-terminal part of BTH1 showed 58% sequence similarity to HMP-P kinase of Salmonella typhimurium. The cDNA clone functionally complemented the S. typhimurium and E. coli thiD mutants deficient in the HMP-P kinase activity. These results show that the clone encodes a bifunctional protein with TMP-PPase at the C-terminus and HMP-P kinase at the N-terminus. This is in contrast to the yeast bifunctional proteins that encode TMP-PPase at the N-terminus and 4-methyl-5-(2-hydroxyethyl)thiazole kinase at the C-terminus. Expression of the BTH1 gene is negatively regulated by thiamin, as in the cases for the thiamin biosynthetic genes of microorganisms. This is the first report of a plant thiamin biosynthetic gene on which a specific biochemical activity is assigned. The Brassica BTH1 gene may correspond to the Arabidopsis TH-1 gene.

Differential expression of senescence-associated mRNAs during leaf senescence induced by different senescence-inducing factors in Arabidopsis.

Four cDNA clones, named pSEN2, pSEN3, pSEN4, and pSEN5, for mRNAs induced during leaf senescence in Arabidopsis thaliana were characterized. The clones were isolated from a cDNA library of detached leaves incubated in darkness for 2 days to accelerate senescence, first by differential screening and then by examining expression of the primarily screened clones during age-dependent leaf senescence. Transcript levels detected by these cDNA clones, thus, were up-regulated in an age-dependent manner and during dark-induced leaf senescence. In contrast, when leaf senescence was induced by ethylene, ABA or methyljasmonate, the transcript level detected by the clones was differentially regulated depending on the senescence-inducing hormones. The transcript level for pSEN4 increased during senescence induced by all three hormones, while the transcript detected by the pSEN2 clone did not increase during senescence induced by ethylene. The transcript level for pSEN5 was increased upon ABA-induced senescence but decreased during ethylene-induced senescence. The pSEN3 clone detected multiple transcripts that are differentially regulated by these factors. The results show that, although the apparent senescence symptoms of Arabidopsis leaf appear similar regardless of the senescence-inducing factors, the detailed molecular state of leaf cells during senescence induced by different senescence-inducing factors is different. The pSEN3 clone encodes a polyubiquitin and the pSEN4 clone encodes a peptide related to endoxyloglucan transferase. This result is consistent with the expected roles of senescence-induced genes during leaf senescence.

Genetic identification of FIN2, a far red light-specific signaling component of Arabidopsis thaliana.

Phytochrome A (PhyA) mediates most, if not all, various plant responses to far-red (FR) light. Here, we report a novel genetic mutation that impairs a variety of responses in the PhyA-signaling pathway of Arabidopsis thaliana. The mutation was isolated by screening seedlings that show reduced sensitivity to continuous far-red (FRc) light irradiation, but not to continuous red (Rc) light irradiation. The mutation named fin2-1 is not allelic to a PHYA mutation. Furthermore, immunoblot analysis indicated that the amount of the phytochrome A apoprotein in the fin2-1 mutant was comparable to that in wild type. Seedling of the fin2-1 mutant showed defects in hypocotyl growth inhibition and apical hook and cotyledon opening in FRc light but not in Rc light. The results showed that the mutation occurred in a downstream signaling component potentially specific to PhyA. Other PhyA-mediated responses such as FR-preconditioned blocking of greening, anthocyanin accumulation, reduction of gravitropic response, and expression of the CAB and CHS genes were impaired by the fin2-1 mutation: the degree of the mutant effect on the responses was variable. However, FR light-mediated seed germination and photoperiodic flowering responses were not affected significantly in the mutant. These results showed that FIN2 defines an upstream branch point in the PhyA signaling pathway.

Large-scale analysis of expressed genes from the leaf of oilseed rape (Brassica napus L.).

While the number of leaf-specific expressed genes is estimated to be approximately 6,000, an overview of gene diversity and expression patterns in the leaf of oilseed rape (Brassica napus L.) has not yet been reported. In an effort to understand gene expression patterns and to identify new genes, we generated 754 expressed sequence tags (ESTs) from the leaf of B. napus. By comparing them to public databases, we showed that 204 of the ESTs (27.1%) have sequence homology to known genes, with 52 of them (6.9%) matching to genes not previously studied in B. napus. The most abundant transcripts were found to be involved in photosynthesis and energy metabolism. When compared with maize leaf ESTs and rice leaf ESTs, the pattern of gene expression was different depending on the developmental stages of the leaf.

Identification of three genetic loci controlling leaf senescence in Arabidopsis thaliana.

Four mutants that show the delayed leaf senescence phenotype were isolated from Arabidopsis thaliana. Genetic analyses revealed that they are all monogenic recessive mutations and fall into three complementation groups, identifying three genetic loci controlling leaf senescence in Arabidopsis. Mutations in these loci cause delay in all senescence parameters examined, including chlorophyll content, photochemical efficiency of photosystem II, relative amount of the large subunit of Rubisco, and RNase and peroxidase activity. Delay of the senescence symptoms was observed during both age-dependent in planta senescence and dark-induced artificial senescence in all of the mutant plants. The results indicate that the three genes defined by the mutations are key genetic elements controlling functional leaf senescence and provide decisive genetic evidence that leaf senescence is a genetically programmed phenomenon controlled by several monogenic loci in Arabidopsis. The results further suggest that the three genes function at a common step of age-dependent and dark-induced senescence processes. It is further shown that one of the mutations is allelic to ein2-1, an ethylene-insensitive mutation, confirming the role of ethylene signal transduction pathway in leaf senescence of Arabidopsis.

Resistance to tobamoviruses in transgenic tobacco plants expressing the coat protein gene of pepper mild mottle virus (Korean isolate).

Red pepper, one of the most important vegetable crops in Korea, is severely affected by viral diseases causing 20-50% reduction in product yield. A pepper strain of tobacco mosaic virus (TMV-p) is the most common virus in red pepper. To study the molecular structure of the TMV-p virus, we generated cDNA clones of the viral genome. Partial sequencing of a few cDNA clones revealed that TMV-p shares a 98% identity at the nucleotide level with the Spanish isolate of pepper mild mottle virus (PMMV-s). This suggests that TMV-p should be reclassified as the Korean isolate of PMMV (PMMV-k). The coat protein (CP) gene together with the 3' untranslated region of the PMMV-k virus was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) using oligomers deduced from the sequence of PMMV-s. The sequence analysis of the CP gene and the 3' untranslated region further confirmed that PMMV-k is highly related to PMMV-s. The CP gene and the 3' untranslated region of PMMV-k were cloned into a plant expression vector and the construct was introduced into tobacco plants. The transgenic plants expressing the PMMV-k CP gene were delayed in developing systemic disease or failed to develop symptoms at all after inoculation with PMMV-k. Delay of symptoms was also observed when the plants were inoculated with TMV-OM which shares a 74% homology with PMMV-k in the amino acid sequence of the CP region. In a local lesion host, the CP expressing plants exhibited a greatly reduced number of necrotic lesions as compared to control plants after inoculation with TMV-OM. Our results show that CP-mediated viral resistance is readily applicable in the case of PMMV-k and can provide resistance to other viruses in the tobamovirus group.

Identification of a receptor-like protein kinase gene rapidly induced by abscisic acid, dehydration, high salt, and cold treatments in Arabidopsis thaliana.

A cDNA clone for a receptor-like protein kinase gene (RPK1) was isolated from Arabidopsis thaliana. The clone is 1952 bp long with 1623 bp of an open reading frame encoding a peptide of 540 amino acids. The deduced peptide (RPK1) contains four distinctive domains characteristic of receptor kinases: (a) a putative amino-terminal signal sequence domain; (b) a domain with five extracellular leucine-rich repeat sequences; (c) a membrane-spanning domain; and (d) a cytoplasmic protein kinase domain that contains all of the 11 subdomains conserved among protein kinases. The RPK1 gene is expressed in flowers, stems, leaves, and roots. Expression of the RPK1 gene is induced within 1 h after treatment with abscisic acid (ABA). The gene is also rapidly induced by several environmental stresses such as dehydration, high salt, and low temperature, suggesting that the gene is involved in a general stress response. The dehydration-induced expression is not impaired in aba-1, abi1-1, abi2-1, and abi3-1 mutants, suggesting that the dehydration-induced expression of the RPK1 gene is ABA-independent. A possible role of this gene in the signal transduction pathway of ABA and the environmental stresses is discussed.

Evaluation of 515 expressed sequence tags obtained from guard cells of Brassica campestris.

As an attempt to examine the transcripts expressed in a single cell type and to unveil the physiology of guard cells at the molecular level, we generated 515 expressed sequence tags (ESTs) from a directional cDNA library constructed from guard-cell protoplasts of Brassica campestris L. ssp. pekinensis. A comparative analysis of the guard-cell ESTs against the National Center for Biotechnological Information non-redundant protein database revealed that 133 ESTs (26%) have significant similarity to protein coding sequences in the database. Among them were 35 clones related to genes that have not yet been identified in higher plants. Analysis of RNA gel blots of 14 database-matched clones revealed that five clones harbor the sequences for mRNAs expressed most abundantly in guard cells, one of them detecting an mRNA with highly preferential expression in guard cells. Functional categorization of the putatively identified guard-cell ESTs showed, when compared with maize leaf ESTs, that guard cells expressed a higher proportion of signal transduction components and a lower proportion of structural or photosynthetic genes, as is consistent with the roles of guard cells.

Insulin-induced maturation of Xenopus oocytes is inhibited by microinjection of a Brassica napus cDNA clone with high similarity to a mammalian receptor for activated protein kinase C.

A cDNA clone encoding a WD-40 repeat protein (BGB1) was characterized in Brassica napus L. The clone contained an open reading frame of 327 amino acid residues almost entirely composed of seven segments of WD-40 repeats. Among the WD-40 repeat proteins, BGB1 showed high similarity (63% identity) to a rat intracellular receptor for protein kinase C (RACK1) that functions in the translocation of activated protein kinase C (PKC) from the cytosolic fraction to the membrane fraction. BGB1 also had two sequence motifs involved in binding of RACK1 to PKC. The cDNA clone, when carried in a Xenopus oocyte expression vector and injected into Xenopus laevis oocytes, inhibited insulin-induced maturation of the oocytes, a PKC-mediated pathway, and this inhibition was accompanied by reduction of PKC in the membrane fraction, as in the case of mammalian RACKs. The data show that BGB1 shares some common functional characteristics with the mammalian RACK1 along with the structural similarity, suggesting that a mammalian RACK1-related cellular process might be operating in plants. Southern blot analyses of the genome of B. napus and Arabidopsis thaliana (L.) Heynh. revealed that BGB1-related genes constitute a small multigene family in both species. An approximately 1.4-kb transcript was constitutively expressed in all organs examined.

Pollen of a male-sterile mutant of Arabidopsis thaliana isolated from a T-DNA insertion pool is not effectively released from the anther locule.

We have isolated a male-sterile mutant from a pool of T-DNA insertional lines of Arabidopsis thaliana generated by an in planta transformation procedure [Chang et al. (1994) Plant J. 5: 551]. Pollen in this mutant is not effectively released from anther locules after cleavage of the stomium. Most mutant pollen grains are round, in contrast to the tricolpate wild-type pollen, and some pollen grains show an abnormal surface structure. Manually released mutant pollen grains are not fertile and show defects in pollen tube germination in vitro. Genetic analysis disclosed that this lesion is due to a single recessive nuclear mutation located on chromosome 3 closely linked to the gll locus. The mutation locus is tightly linked to the inserted T-DNA.

Two dominant photomorphogenic mutations of Arabidopsis thaliana identified as suppressor mutations of hy2.

By screening suppressor mutants of the hy2 mutation of Arabidopsis thaliana, two dominant photomorphogenic mutants, shy1-1D and shy2-1D, for two genetic loci designated as SHY1 and SHY2 (suppressor of hy2 mutation) have been isolated. Both of these non-allelic, extragenic suppressor mutations of hy2 are located on chromosome 1 of the Arabidopsis genome. Both mutations suppress the elongated hypocotyl phenotype of hy2 by light-independent inhibition of hypocotyl growth as well as by increasing the effectiveness of light inhibition of hypocotyl elongation. The shy1-1D mutation is partially photomorphogenic in darkness with apical hook opening and reduced hypocotyl elongation. The shy2-1D mutant displays highly photomorphogenic characteristics in darkness such as true leaf development, cotyledon expansion and extremely reduced hypocotyl growth. In regard to hypocotyl elongation, however, the shy2-1D mutation is still light sensitive. Examination of red-far-red light responses shows that the shy1-1D mutation suppresses the hypocotyl elongation of the hy2 mutation effectively in red light but not effectively in far-red light. The shy2-1D suppresses hypocotyl elongation of the hy2 mutation effectively in both red and far-red light. Both mutations can also suppress the early-flowering phenotype of hy2 and have a distinct pleiotropic effect on leaf development such as upward leaf rolling. The data obtained suggest that SHY1 and SHY2 represent a novel class of components involved in the photomorphogenic pathways of Arabidopsis. This is the first report on the identification of dominant mutations in the light signal transduction pathway of plants.

A senescence-associated gene of Arabidopsis thaliana is distinctively regulated during natural and artificially induced leaf senescence.

We have characterized the structure and expression of a senescence-associated gene (sen1) of Arabidopsis thaliana. The protein-coding region of the gene consists of 5 exons encoding 182 amino acids. The encoded peptide shows noticeable similarity to the bacterial sulfide dehydrogenase and 81% identity to the peptide encoded by the radish din1 gene. The 5'-upstream region contains sequence motifs resembling the heat-shock- and ABA-responsive elements and the TCA motif conserved among stress-inducible genes. Examination of the expression patterns of the sen1 gene under various senescing conditions along with measurements of photochemical efficiency and of chlorophyll content revealed that the sen1 gene expression is associated with Arabidopsis leaf senescence. During the normal growth phase, the gene is strongly induced in leaves at 25 days after germination when inflorescence stems are 2-3 cm high, and then the mRNA level is maintained at a comparable level in naturally senescing leaves. In addition, dark-induced senescence of detached leaves or of leaves in planta resulted in a high-level induction of the gene. Expression of the sen1 gene was also strongly induced in leaves subjected to senescence by 0.1mM abscisic acid or 1 mM ethephon treatment. The induced expression of the gene by dark treatment was not significantly repressed by treatment with 0.1 mM cytokinin or 50 mM CaCl2 which delayed loss of chlorophyll but not that of photochemical efficiency.