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1

Over-expression of heat shock factor gene (AtHsfA1d) from Arabidopsis thaliana confers formaldehyde tolerance in tobacco

100%
Nian H.-J., Zhang D.-J., Zeng Z.-D., Yan J.-P., Li K.-Z., Chen L.-M.
Acta Physiologiae Plantarum
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2014
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tom 36
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nr 06
Formaldehyde (HCHO) is a major indoor air pollutant. Plants can be used to remove HCHO from polluted air because they can use HCHO as a carbon source to incorporate it into one-carbon (C1) metabolism. However, high concentrations of exogenous HCHO cause damages to plants. Therefore, genetic engineering is an effective measure to improve ability of plants to clear the HCHO pollution. Expression of AtHsfA1d encoding heat shock transcription factor of Arabidopsis was induced by HCHO stress. AtHsfA1d was cloned into the pYES3 vector and transformed into Saccharomyces cerevisiae. Yeast cells expression of AtHSFA1d showed higher tolerance to HCHO stress than wild-type (WT) cells. AtHsfA1d was introduced into tobacco and the expression of AtHSFA1d in transgenic lines was demonstrated using Western blot analysis. Transgenic tobacco showed higher uptake rate to aqueous HCHO, had the higher biomass and produced higher content of total proteins than WT plants. These results indicated that AtHsfA1d conferred HCHO tolerance to yeast and tobacco. AtHsfA1d is a good candidate to develop phytoremediation plants for HCHO pollution.
2

Overexpressions of dihydroxyacetone synthase and dihydroxyacetone kinase in chloroplasts install a novel photosynthetic HCHO-assimilation pathway in transgenic tobacco using modified Gateway entry vectors

100%
Xiao S.-Q., Sun Z., Wang S.-S., Zhang J., Li K.-Z., Chen L.-M.
Acta Physiologiae Plantarum
|
2012
|
tom 34
|
nr 5
Dihydroxyacetone synthase (DAS) and dihydroxyacetone kinase (DAK) are two key enzymes for formaldehyde assimilation in methylotrophic yeasts. In order to using a Gateway LR recombination reaction to construct a plant expression vector that contains the expression cassettes for the das and dak genes and allow the proteins encoded by the two target genes to be localized to the chloroplasts of transgenic plants, the entry vector pEN-L4*-PrbcS-*T-gfp-L3* contained the tomato rbcS 3C promoter (PrbcS) with its transit peptide sequence (*T) and a GFP reporter gene (gfp) was constructed in this study. To verify the applicability of pEN-L4*-PrbcS-*T-gfp-L3*, we generated an entry vector for the dak gene by replacing the gfp gene in this entry vector with the dak gene. We also generated an entry vector for the das gene by replacing the gus gene in another entry vector (pENTR*-PrbcS-*T-gus) with the das gene. Using these entry vectors and pK7m34GW2-8m21GW3, we successfully constructed the pKm-35S-PrbcS-*T-gfp-PROLD-PrbcS-*T-gus and the pKm-35S-PrbcS-*T-dak-PROLD-PrbcS-*T-das expression vectors. Our results showed that high expression of GUS was achieved in leaves, and the expressed GFP, DAS and DAK proteins could be targeted to the chloroplasts after the two expression vectors were used to transform tobacco. The overexpressions of DAS and DAK in the chloroplasts successfully created a novel photosynthetic HCHO-assimilation pathway in transgenic tobacco. By utilizing these expression vectors, we not only successfully expressed two target genes with one transformation but also localized the expressed proteins to chloroplasts via the transit peptide sequence (*T). Therefore, the construction of pEN-L4*-PrbcS-*T-gfp-L3* establishes a technique platform that provides a convenient means for chloroplast genetic engineering.
3

Deciphering the molecular responses to methanol-enhanced photosynthesis and stomatal conductance in broad bean

100%
Zhao Y., Zeng Z.-D., Qi C.-J., Yu X.-S., Guo C.-L., Chen Q., Chen L.-M.
Acta Physiologiae Plantarum
|
2014
|
tom 36
|
nr 11
This study showed that foliar spray of 2 % methanol significantly increased stomatal conductance, photosynthesis, and growth of broad bean (Vicia faba L.). To understand the molecular mechanisms behind the methanol-enhanced photosynthesis of broad bean, a forward suppression subtractive hybridization (SSH) cDNA library was constructed with 2 % methanol-sprayed leaves. A total of 145 methanol-induced genes were identified, and 63 of these had a known function. Clustering analysis indicated that the largest proportion (41.3 %) comprised genes associated with photosynthesis. RT-PCR analysis verified that the transcription of photosynthetic genes, including Rubisco and chlorophyll a/b-binding proteins of photosystem I and II, was upregulated in 2 % methanolsprayed leaves, which might contribute to the molecular mechanism of methanol-enhanced photosynthesis in broad bean. Further analysis suggested that the transcription and expression of 14-3-3 protein (present in the SSH library) and PM H⁺-ATPase encoding genes, PM H⁺-ATPase phosphorylation and its interactions with 14-3-3 proteins as well as stomatal aperture increased in leaves after 2 % methanol sprayed. Simultaneously, the leaf H₂O₂ content decreased in a time-dependent manner. Based on these results, we hypothesize that methanol spray induces the expression of 14-3-3 proteins and PM H⁺-ATPase and simultaneously reduces the H₂O₂ content in leaves. Consequently, the interaction between phosphorylated PM H⁺- ATPase and 14-3-3 proteins is enhanced, and PM H⁺- ATPase and H⁺ pump activities elevated; these functions may constitute the molecular mechanism through which methanol increases stomatal aperture and conductance in broad bean leaves.
4

Physiological and molecular responses of broad bean (Vicia faba L.) to aluminium stress

100%
Chen Q., Wu K.-H., Zhang Y.-N., Phan X.-H., Li K.-Z., Yu Y.-X., Chen L.-M.
Acta Physiologiae Plantarum
|
2012
|
tom 34
|
nr 6
In this study, the responses of broad bean cultivars resistant (YD) and sensitive (AD) to aluminum (Al) stress were investigated at physiological and molecular levels. The results showed that Al induced more citrate exudation in YD roots than that in AD roots, suggesting that citrate exudation is involved in broad bean Al resistance. The analyses for oxidative stress levels and antioxidant enzyme activities indicated that YD had a strong ability to cope with the oxidative stress induced by Al. To investigate the molecular responses of broad bean to Al stress further, a forward suppression subtractive hybridization cDNA library was constructed to identify Alresponsive genes in YD roots treated with 50-µM Al for a 24-h period. Of the obtained 162 high-quality ESTs, genes related to antioxidant enzymes including copper-zinc superoxide dismutase (SOD), class III peroxidase (POD) and germin-like protein (GEP) were up-regulated. Higher transcription levels of SOD and POD were observed in YD but not in AD roots, which is in agreement with the enhanced activities of antioxidant enzymes in YD roots under Al stress conditions. Furthermore, the up-regulated expression of vha2, encoding a plasma membrane (PM) H⁺-ATPase, and 14-3-3b in YD roots under Al stress were also detected and confirmed by RT-PCR analysis. Western and immunoprecipitation analyses indicated that Al-enhanced expressions and interactions of the PM H⁺-ATPase and 14-3-3 proteins might be involved in the regulation of citrate secretion in YD roots under Al stress.
5

Physiological and transcriptional analysis of the effects of formaldehyde exposure on Arabidopsis thaliana

84%
Wang S.-S., Song Z.-B., Sun Z., Zhang J., Mei Y., Nian H.-J., Li K.-Z., Chen L.-M.
Acta Physiologiae Plantarum
|
2012
|
tom 34
|
nr 3
Formaldehyde (HCHO) is highly toxic to all living organisms. In this study, the toxic effects of HCHO exposure on Arabidopsis thaliana were analyzed at the physiological and transcriptional levels. Exposure to 2 mM HCHO led to a significant decrease in plant growth and a massive increase in anthocyanin content. A remarkable increase in H₂O₂ content and elevation in the levels of protein carbonyl and DNA–protein crosslinks were detected in Arabidopsis plants exposed to 2 mM HCHO for a period of 17 h. In contrast, the malondialdehyde content decreased during this period. These results suggested that HCHO stress caused significant oxidative damage to proteins but not membrane lipids during this period. The Affymetrix ATH1 Genome Array was used to evaluate changes in the global gene expression in Arabidopsis plants exposed to 2 mM HCHO over the 17-h period. A total of 620 transcripts were shown to be regulated significantly (at least twofold). The number of down-regulated genes (467) was approximately threefold greater than the number of up-regulated genes (154). Down-regulation in a large number of genes encoding cell surface receptors, cell wall proteins, enzymes related to toxin metabolism, peroxidase, disease resistance protein, multidrug and toxin extrusion and ATP-binding cassette transporters might be an important part of the toxic effects of HCHO exposure on Arabidopsis at the transcriptional level. Up-regulation in many genes encoding heat shock proteins was suggested to be an important protective mechanism for Arabidopsis plants in response to the oxidative damage of proteins. Verification of microarray data by reverse transcription polymerase chain reaction analysis identified typical HCHO-induced and -repressed genes.
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