In order to further increase the plant ability of formaldehyde uptake and metabolism by synthetic biology technology, we choosed four enzyme-coding genes related to formaldehyde metabolic pathways from microorganism and plant: 3-hexulose-6-phosphate (HPS), 6-phospho-3-hexuloisomerase (PHI), formaldehyde dehydrogenase (FALDH) and formate-dehydrogenase (FDH). These genes are transformed into plants and will promote formaldehyde metabolism. For security reasons, we also induce AdCP gene into our plans because of its capability to lead to pollen abortion. At the same time, chloroplast transformation is taken into consideration to avoid gene flow and improve gene expression.

HPS-PHI

The ribulose monophosphate (RuMP) pathway is one of the formaldehyde-fixation pathways found in microorganisms called methylotrophs, which utilize one-carbon compounds as the sole carbon source. The key enzymes of this pathway are 3-hexulose-6-phosphate synthase (HPS), which fixes formaldehyde to D-ribulose-5-phosphate (Ru5P) to produce D-arabino-3-hexulose-6-phosphate (Hu6P), and 6-phospho-3-hexuloisomerase (PHI), which converts Hu6P to fructose 6-phosphate (F6P). The two key enzymes work in chloroplast both. We will use fusion expression method to conduct heterologous expression in tobacco (Chen et al., 2010).

Fig.1 Schematic Representation of the Bacterial RuMP Pathway and the Plant Calvin-Benson Cycle. HPS and PHI denote 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase respectively. The abbreviations for several sugar phosphates are as follows: Ru5P, ribulose 5-phosphate; Hu6P, hexulose 6-phosphate; F6P, fructose 6-phosphate; FBP, fructose 1,6-bisphosphate; RuBP, ribulose1, 5-bisphosphate; 3-PGA, 3-phosphoglyce-rate. The other metabolites in the pathway are symbolized merely by their carbon numbers for simplicity .

FALDH

The glutathione-dependent formaldehyde dehydrogenase (FALDH) plays a key role in formaldehyde metabolism (Fig.3). FALDH is identified as an enzyme expressed in the cytoplasm. If we make FALDH over-express in plants, we can enhance plants’ tolerance to formaldehyde and increase the ability of plants to absorb formaldehyde. In the process of metabolism of formaldehyde, the formaldehyde may first combined with glutathione (GSH) to form the product of S-hydroxymethyl glutathione (HM-GSH), then FALDH in cytoplasm will catalyzes the formation of a S-formyl glutathione (F-GSH). Next the F-GSH will be hydrolyzed to formate (HCOOH) and GSH by S-formyl glutathione hydrolase (FGH).

Fig.2 a. spectra from leaf extracts of transgenic tobacco plant treated with gaseous for 2 h. b. spectra from leaf extracts of WT treated with gaseous for 2 h. c. The extract from WT plant leaves without treatment was used to monitor the background signal levels (Nian et al., 2013).

FDH

Formate dehydrogenase is a mitochondrial-localized NAD-requiring enzyme while the HCOOH is getting into the mitochondrial, FDH will oxidize the formic acid into CO2, and reduce NAD+ to NADH with a high degree of specificity. In our project, the heterologous expression of Arabidopsis FDH gene in tobacco was completed.

Fig.3 Folate-independent pathway. The abbreviations are as follows: FALDH:glutathione-dependent formaldehyde dehydrogenase; FDH: Formate dehydrogenase; HM-GSH: S-Hydroxymethyl glutathione; Forml-GSH: Formyl glutathione; SMM cycle: Methionine cycle.

Stomatal opening

Stomata are microscopic pores surrounded by two guard cells and play an important role in the uptake of CO2 for photosynthesis. Recent researches revealed that light-induced stomatal opening is mediated by at least three key components: blue light receptor phototropin, plasma membrane H+-ATPase, and plasma membrane inward-rectifying K+ channels. However, Wang et al (2014) showed that only increasing the amount of H+-ATPase in guard cells had a significant effect on light-induced stomatal opening (Fig. 4). Transgenic Arabidopsis plants by overexpressing H+-ATPase in guard cells exhibited enhanced photosynthesis activity and plant growth. Therefore, in order to strengthen the ability of absorbing formaldehyde, we overexpressed H+-ATPase (AtAHA2) in transgenic tobacco guard cells, resulting in a significant effect on light-induced stomatal opening.

Fig.4 Typical stomata in the epidermis illuminated with light for 30 min (Wang et al., 2014).

Biosafty

In order to promise biology safety, we use male sterility systems which can be used as a biological safety containment to prevent horizontal transgene flow. Shukla et al (2014) has used a plant pathogen-induced gene, cysteine protease for inducing male sterility. This gene was identified in the wild peanut, Arachis diogoi differentially expressed when it was challenged with the late leaf spot pathogen, Phaeoisariopsis personata. Arachis diogoi cysteine protease (AdCP) was expressed under the strong tapetum-specific promoter (TA29) and tobacco transformants were generated. Morphological and histological analysis of AdCP transgenic plants showed ablated tapetum and complete pollen abortion.

Fig.5 Pollen germination of untransformed control plant and sterile transgenic plants in vitro. Pollen grains were germinated on sucrose-boric acid medium and over 500 pollen grains were observed. a. Untansformed control plant pollen, b. Sterile pollen. Scale bar 25 μm (Shukla et al., 2014).

Vectors

The product of HPS, PHI, and FDH are located in chloroplast, while the product of FALDH are located in cytoplasm. We used chloroplast transit peptides to locate these products of genes. So we constructed different vectors with and without transit peptide. We hope to compare the ability of metabolizing formaldehyde of transgenic tobacco between different transgenic lines. We planned to constructed 11 vectors (Fig. 6), including two backbones, six mono-gene expression vectors and three multi-gene expression vectors (Fig. 7). piGEM003, piGEM004 and piGEM005 are individual mono-gene expression vectors with transit peptides, while piGEM006, piGEM006, piGEM008 are individual multi-gene expression vectors without transit peptides. piGEM009 is a multi-gene expression vector without any transit peptides, while piGEM011 is a multi-gene expression vector with three peptides. piGEM010 is a multi-gene expression vector with two transit peptides.

Fig.6 The procedure of vectors construction.

Fig.7 Schematic of vectors construction.

References

Chen, L. M., H. Yurimoto, K. Z. Li, I. Orita, M. Akita, N. Kato, Y. Sakai and K. Izui (2010). "Assimilation of formaldehyde in transgenic plants due to the introduction of the bacterial ribulose monophosphate pathway genes." Biosci Biotechnol Biochem 74(3): 627-635.
Nian, H., Q. Meng, W. Zhang and L. Chen (2013). "Overexpression of the formaldehyde dehydrogenase gene from Brevibacillus brevis to enhance formaldehyde tolerance and detoxification of tobacco." Appl Biochem Biotechnol 169(1): 170-180.
Shukla, P., N. K. Singh, D. Kumar, S. Vijayan, I. Ahmed and P. B. Kirti (2014). "Expression of a pathogen-induced cysteine protease (AdCP) in tapetum results in male sterility in transgenic tobacco." Funct Integr Genomics 14(2): 307-317.
Wang, Y., K. Noguchi, N. Ono, S. Inoue, I. Terashima and T. Kinoshita (2014). "Overexpression of plasma membrane H+-ATPase in guard cells promotes light-induced stomatal opening and enhances plant growth." Proc Natl Acad Sci U S A 111(1): 533-538.