Gene expression of ascorbic acid biosynthesis related enzymes of the Smirnoff-Wheeler pathway in acerola (Malpighia glabra)

The Smirnoff-Wheeler (SW) pathway has been proven to be the only significant source of l-ascorbic acid (AsA; vitamin C) in the seedlings of the model plant Arabidopsis thaliana. It is yet uncertain whether the same pathway holds for all other plants and their various organs as AsA may also be synthesized through alternative pathways. In this study, we have cloned some of the genes involved in the SW-pathway from acerola (Malpighia glabra), a plant containing enormous amount of AsA, and examined the expression patterns of these genes in the plant. The AsA contents of acerola leaves were about 8-fold more than that of Arabidopsis with 5-700-fold higher mRNA abundance in AsA-biosynthesizing genes. The unripe fruits have the highest AsA content but the accumulation was substantially repressed as the fruit transitions to maturation. The mRNAs encoding these genes showed correlation in their expression with the AsA contents of the fruits. Although very little AsA was recorded in the seeds the mRNAs encoding all the genes, with the exception of the mitochondrially located L-galactono-1,4-lactone dehydrogenase, were clearly detected in the seeds of the unripe fruits. In young leaves of acerola, the expression of most genes were repressed by the dark and induced by light. However, the expression of GDP-D-mannose pyrophosphorylase similar to that encoded by A. thaliana VTC1 was induced in the dark. The expressions of all the genes surged after 24h following wounding stress on the young leaves. These findings will advance the investigation into the molecular factors regulating the biosynthesis of abundant AsA in acerola.     

Overexpression of the acerola (Malpighia glabra) monodehydroascorbate reductase gene in transgenic tobacco plants results in increased ascorbate levels and enhanced tolerance to salt stress

Monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) is a key enzyme of the ascorbate (AsA)-glutathione cycle that maintains reduced pools of AsA and serves as an important antioxidative enzyme. Previously, we have cloned MDHAR cDNA from acerola (Malpighia glabra), a plant that accumulates abundant amount of AsA. In this study, MDHAR cDNA from acerola was introduced into tobacco plants using an Agrobacterium-mediated gene delivery system. Transgenic tobacco plants accumulated greater amounts of AsA and showed higher MDHAR activity than the control plants. Lipid peroxidation and chlorophyll degradation, which were stimulated in control plants, were restrained in transgenic plants subjected to salt stress. These results indicate that overexpression of acerola MDHAR imparts greater tolerance to salt stress.     

High levels of expression of multiple enzymes in the Smirnoff-Wheeler pathway are important for high accumulation of ascorbic acid in acerola fruits

ABSTRACT

Acerola fruits contain abundant ascorbic acid (AsA). The gene expression levels of three upstream enzymes in the primary AsA biosynthesis pathway were correlated with AsA contents in the fruits of two acerola cultivars. Multiple overexpression of the enzymes increased AsA contents, suggesting their high expression is important for high AsA accumulation in acerola fruits and the breeding of AsA-rich plants.

Abbreviations: AsA: ascorbic acid; PMI: phosphomannose isomerase; PMM: phosphomannomutase; GMP: GDP-d-mannose pyrophosphorylase; GME: GDP-d-mannose 3?,5?-epimerase; GGP: GDP-l-galactose phosphorylase; GPP: l-galactose-1-phosphate phosphatase; GDH: l-galactose dehydrogenase; GLDH: l-galactono-1,4-lactone dehydrogenase     

A novel regulatory element responsible for high transcriptional expression of acerola GDP-d-mannose pyrophosphorylase gene

GDP-d-mannose pyrophosphorylase (GMP) is one of the enzymes that highly expressed in acerola plants. A promoter assay suggests the presence of a new cis-element in the ?1087 to ?1083 bp sequence of the MgGMP promoter. Moreover, cis-elements, present in the ?1080 to ?600 bp sequence of the MgGMP promoter, function as enhancers of MgGMP expression.     

Expression of the algal cytochrome c6 gene in Arabidopsis enhances photosynthesis and growth

Photosynthetic plants convert light energy into ATP and NADPH in photosynthetic electron transfer and photophosphorylation, and synthesize mainly carbohydrates in the Calvin-Benson cycle. Here we report the enhancement of photosynthesis and growth of plants by introducing the gene of an algal cytochrome c6, which has been evolutionarily eliminated from higher plant chloroplasts, into the model plant Arabidopsis thaliana. At 60 d after planting, the plant height, leaf length and root length of the transformants were 1.3-, 1.1- and 1.3-fold those in the wild-type plants, respectively. At the same time, in the transgenic plants, the amounts of chlorophyll, protein, ATP, NADPH and starch were 1.2-, 1.1-, 1.9-, 1.4- and 1.2-fold those in the wild-type plants, respectively. The CO2 assimilation capacity of the transgenic plants was 1.3-fold that of the wild type. Moreover, in transgenic Arabidopsis expressing algal cytochrome c6, the 1-qP, which reflects the reduced state of the plastoquinone pool, is 30% decreased compared with the wild type. These results show that the electron transfer of photosynthesis of Arabidopsis would be accelerated by the expression of algal cytochrome c6. Our results demonstrate that the growth and photosynthesis of Arabidopsis plants could be enhanced by the expression of the algal cytochrome c6 gene.     

Disruption of a gene encoding C4-dicarboxylate transporter-like protein increases ozone sensitivity through deregulation of the stomatal response in Arabidopsis thaliana

To understand better the plant response to ozone, we isolated and characterized an ozone-sensitive (ozs1) mutant strain from a set of T-DNA-tagged Arabidopsis thaliana ecotype Columbia. The mutant plants show enhanced sensitivity to ozone, desiccation and sulfur dioxide, but have normal sensitivity to hydrogen peroxide, low temperature and high light levels. The T-DNA was inserted at a single locus which is linked to ozone sensitivity. Identification of the genomic sequences flanking the T-DNA insertion revealed disruption of a gene encoding a transporter-like protein of the tellurite resistance/C(4)-dicarboxylate transporter family. Plants with either of two different T-DNA insertions in this gene were also sensitive to ozone, and these plants failed to complement ozs1. Transpiration levels, stomatal conductance levels and the size of stomatal apertures were greater in ozs1 mutant plants than in the wild type. The stomatal apertures of ozs1 mutant plants responded to light fluctuations but were always larger than those of the wild-type plants under the same conditions. The stomata of the mutant and wild-type plants responded similarly to stimuli such as light, abscisic acid, high concentrations of carbon dioxide and ozone. These results suggest that OZS1 helps to close stomata, being not involved in the responses to these signals.     

An ABC transporter gene of Arabidopsis thaliana, AtWBC11, is involved in cuticle development and prevention of organ fusion

Cuticle, including wax and cutin, is the barrier covering plant aerial organs and protecting the inner tissues. The Arabidopsis thaliana ATP-binding cassette (ABC) transporter CER5 (AtWBC12) has been identified as a wax exporter. In agreement with the latest report of another wax exporter, AtWBC11, here we show that atwbc11 mutants displayed organ fusions and stunted growth, and became vulnerable to chlorophyll leaching and toluidine blue staining. Chemical analysis showed that wax and cutin monomers were both reduced in the atwbc11 mutant. AtWBC11 was widely expressed in aerial organs. Interestingly, we found that the expression was light dependent, and the phytohormone ABA up-regulated AtWBC11 expression. We also found that while the AtWBC11 promoter had a broad pattern of activity, the expression was converted to epidermis specific when the reporter gene was fused to AtWBC11 cDNA. Furthermore, RNA blot analysis supported epidermis-specific expression of AtWBC11. Our results support that AtWBC11 is involved in cuticle development.     

Flowering in tobacco needs gibberellins but is not promoted by the levels of active GA1 and GA4 in the apical shoot

Flowering of Nicotiana tabacum cv Xhanti depends on gibberellins because gibberellin-deficient plants, due to overexpression of a gibberellin 2-oxidase gene (35S:NoGA2ox3) or to treatment with the gibberellin biosynthesis inhibitor paclobutrazol, flowered later than wild type. These plants also showed inhibition of the expression of molecular markers related to floral transition (NtMADS-4 and NtMADS-11). To investigate further the role of gibberellin in flowering, we quantified its content in tobacco plants during development. We found a progressive reduction in the levels of GA1 and GA4 in the apical shoot during vegetative growth, reaching very low levels at floral transition and beyond. This excludes these two gibberellins as flowering-promoting factors in the apex. The evolution of active gibberellin content in apical shoots agrees with the expression patterns of gibberellin metabolism genes: two encoding gibberellin 20-oxidases (NtGA20ox1 = Ntc12, NtGA20ox2 = Ntc16), one encoding a gibberellin 3-oxidase (NtGA3ox1 = Nty) and one encoding a gibberellin 2-oxidase (NtGA2ox1), suggesting that active gibberellins are locally synthesized. In young apical leaves, GA1 and GA4 content and the expression of gibberellin metabolism genes were rather constant. Our results support that floral transition in tobacco, in contrast to that in Arabidopsis, is not regulated by the levels of GA1 and GA4 in apical shoots, although reaching a threshold in gibberellin levels may be necessary to allow meristem competence for flowering.     

An Arabidopsis purple acid phosphatase with phytase activity increases foliar ascorbate

Ascorbate (AsA) is the most abundant antioxidant in plant cells and a cofactor for a large number of key enzymes. However, the mechanism of how AsA levels are regulated in plant cells remains unknown. The Arabidopsis (Arabidopsis thaliana) activation-tagged mutant AT23040 showed a pleiotropic phenotype, including ozone resistance, rapid growth, and leaves containing higher AsA than wild-type plants. The phenotype was caused by activation of a purple acid phosphatase (PAP) gene, AtPAP15, which contains a dinuclear metal center in the active site. AtPAP15 was universally expressed in all tested organs in wild-type plants. Overexpression of AtPAP15 with the 35S cauliflower mosaic virus promoter produced mutants with up to 2-fold increased foliar AsA, 20% to 30% decrease in foliar phytate, enhanced salt tolerance, and decreased abscisic acid sensitivity. Two independent SALK T-DNA insertion mutants in AtPAP15 had 30% less foliar AsA and 15% to 20% more phytate than wild-type plants and decreased tolerance to abiotic stresses. Enzyme activity of partially purified AtPAP15 from plant crude extract and recombinant AtPAP15 expressed in bacteria and yeast was highest when phytate was used as substrate, indicating that AtPAP15 is a phytase. Recombinant AtPAP15 also showed enzyme activity on the substrate myoinositol-1-phosphate, indicating that the AtPAP15 is a phytase that hydrolyzes myoinositol hexakisphosphate to yield myoinositol and free phosphate. Myoinositol is a known precursor for AsA biosynthesis in plants. Thus, AtPAP15 may modulate AsA levels by controlling the input of myoinositol into this branch of AsA biosynthesis in Arabidopsis.     

Bacterial lipopolysaccharides induce defense responses associated with programmed cell death in rice cells

PAMP (pathogen-associated molecular pattern) recognition plays an important role during the innate immune response in both plants and animals. Lipopolysaccharides (LPS) derived from Gram-negative bacteria are representative of typical PAMP molecules and have been reported to induce defense-related responses, including the suppression of the hypersensitive response, the expression of defense genes and systemic resistance in plants. However, the details regarding the precise molecular mechanisms underlying these cellular responses, such as the molecular machinery involved in the perception and transduction of LPS molecules, remain largely unknown. Furthermore, the biological activities of LPS on plants have so far been reported only in dicots and no information is thus available regarding their functions in monocots. In our current study, we report that LPS preparations for various becteria, including plant pathogens and non-pathogens, can induce defense responses in rice cells, including reactive oxygen generation and defense gene expression. In addition, global analysis of gene expression induced by two PAMPs, LPS and chitin oligosaccharide, also reveals a close correlation between the gene responses induced by these factors. This indicates that there is a convergence of signaling cascades downstream of their corresponding receptors. Furthermore, we show that the defense responses induced by LPS in the rice cells are associated with programmed cell death (PCD), which is a finding that has not been previously reported for the functional role of these molecules in plant cells. Interestingly, PCD induction by the LPS was not detected in cultured Arabidopsis thaliana cells.     

Molecular and functional analysis of phosphomannomutase (PMM) from higher plants and genetic evidence for the involvement of PMM in ascorbic acid biosynthesis in Arabidopsis and Nicotiana benthamiana

Phosphomannomutase (PMM) catalyzes the interconversion of mannose-6-phosphate and mannose-1-phosphate. However, systematic molecular and functional investigations on PMM from higher plants have hitherto not been reported. In this work, PMM cDNAs were isolated from Arabidopsis, Nicotiana benthamiana, soybean, tomato, rice and wheat. Amino acid sequence comparisons indicated that plant PMM proteins exhibited significant identity to their fungal and mammalian orthologs. In line with the similarity in primary structure, plant PMM complemented the sec53-6 temperature sensitive mutant of Saccharomyces cerevisiae. Histidine-tagged Arabidopsis PMM (AtPMM) purified from Escherichia coli converted mannose-1-phosphate into mannose-6-phosphate and glucose-1-phosphate into glucose-6-phosphate, with the former reaction being more efficient than the latter one. In Arabidopsis and N. benthamiana, PMM was constitutively expressed in both vegetative and reproductive organs. Reducing the PMM expression level through virus-induced gene silencing caused a substantial decrease in ascorbic acid (AsA) content in N. benthamiana leaves. Conversely, raising the PMM expression level in N. benthamiana using viral-vector-mediated ectopic expression led to a 20-50% increase in AsA content. Consistent with this finding, transgenic expression of an AtPMM-GFP fusion protein in Arabidopsis also increased AsA content by 25-33%. Collectively, this study improves our understanding on the molecular and functional properties of plant PMM and provides genetic evidence on the involvement of PMM in the biosynthesis of AsA in Arabidopsis and N. benthamiana plants.     

The plant growth-promoting fungus Penicillium simplicissimum GP17-2 induces resistance in Arabidopsis thaliana by activation of multiple defense signals

Arabidopsis thaliana grown in soil amended with barley grain inocula of Penicillium simplicissimum GP17-2 or receiving root treatment with its culture filtrate (CF) exhibited clear resistance to Pseudomonas syringae pv. tomato DC3000 (Pst). To assess the contribution of different defense pathways, Arabidopsis genotypes implicated in salicylic acid (SA) signaling expressing the NahG transgene or carrying disruption in NPR1 (npr1), jasmonic acid (JA) signaling (jar1) and ethylene (ET) signaling (ein2) were tested. All genotypes screened were protected by GP17-2 or its CF. However, the level of protection was significantly lower in NahG and npr1 plants than it was in similarly treated wild-type plants, indicating that the SA signaling pathway makes a minor contribution to the GP17-2-mediated resistance and is insufficient for a full response. Examination of local and systemic gene expression revealed that GP17-2 and its CF modulate the expression of genes involved in both the SA and JA/ET signaling pathways. Subsequent challenge of GP17-2-colonized plants with Pst was accompanied by direct activation of SA-inducible PR-2 and PR-5 genes as well as potentiated expression of the JA-inducible Vsp gene. In contrast, CF-treated plants infected with Pst exhibited elevated expression of most defense-related genes (PR-1, PR-2, PR-5, PDF1.2 and Hel) studied. Moreover, an initial elevation of SA responses was followed by late induction of JA responses during Pst infection of induced systemic resistance (ISR)-expressing plants. In conclusion, we hypothesize the involvement of multiple defense mechanisms leading to an ISR of Arabidopsis by GP17-2.