Plant Gene Engineering Center

The Plant Gene Engineering Center was formed in 2010 upon the arrival of David Ow. In 2012, the PGEC was merged into the newly formed Molecular Analysis & Genetic Improvement Center(MAGIC).

Research Direction

Site-specific gene stacking

In many crop species, transgenic traits are introduced into transformable varieties before introgressing them out to field cultivars. For diploids or polyploids that behave as diploids, the ‘n’ number of unlinked transgenic loci can be assorted as homozygous into a single genome at a probability of (1/4)ⁿ. However, along with the ‘x’ number of other nontransgenic traits that breeders need to assemble into the same genome, the (1/4)^n+x probability for a ‘breeding stack’ makes line conversion difficult. To minimize the number of segregating transgenic loci, the option of in vitro stacking prior to its introduction into the plant genome would mean the re-engineering and re-deregulating of previously introduced traits each time a new trait is introduced. The option of bypassing introgression by directly transforming field cultivars is also not practical as most field cultivars are difficult to transform. Moreover, each locale-specific cultivar would harbor an independent integration event that requires individual de-regulation.

PhD student HOU Lili and colleagues demonstrated recombinase-mediated gene stacking in tobacco. Through two rounds of integration, followed by deletion of unneeded DNA, precise structure and reproducible expression of the sequentially added traits were obtained.

Hou, L., Yau, Y-Y, Wei, J., Han, Z., Dong, Z., Ow, D.W. 2014. An open source system for in planta gene stacking by Bxb1 and Cre recombinases. Molecular Plant 7:1756-1765.

Associate Researcher LI Ruyu and colleagues have developed a biolistic mediated method for site-specific integration and have demonstrated gene insertions into those target sites work efficiently. To implementing this system in rice, a number of precise target sites in the rice genome were screened.

Li, R., Han, Z., Hou, L., Kaur, G., Qian, Y., Ow, D.W. 2016. Method for biolistic site-specific integration in rice and tobacco catalyzed by Bxb1 integrase. In: Methods Mol Biol. 1469: 15-30 (Chromosome and Genomic Engineering in Plants, Ed. M. Murata), Humana Press, (Book Chapter).

New features were being developed adding flexibility to the gene stacking system. PhD student CHEN Weiqiang tested an in vitro gene stacking method that can integrate into the in vivo stacking strategy. Ph.D. student Maryam RAJAEE found a new location to split the Cre recombinase into two parts such that each part is stably produced by separate plants, but activity is reconstituted in the progeny of the hybrid, and PhD student Gurmindar KAUR demonstrated replacing previously inserted transgenes with new DNA.

Chen, W., Ow, D.W. 2016. Protocol for in vitro stacked molecules compatible with in vivo recombinase mediated gene stacking. In: Methods Mol Biol. 1469: 31-47 (Chromosome and Genomic Engineering in Plants, Ed. M. Murata), Humana Press, (Book Chapter).

Rajaee, M., Ow, D.W. 2017. A new location to split Cre recombinase for protein fragment complementation. Plant Biotechnology Journal 15: 1420-1428.

Chen*, W., Kaur*, G., Hou, L., Li, R., Ow, D.W. 2019. Replacement of stacked transgenes in planta. Plant Biotechnology Journal, https://doi.org/10.1111/pbi.13172 (*co-first authors).

Recent reviews on recombinase mediated gene stacking:

Ow, D.W. 2016. The long road to recombinase-mediated plant transformation. Plant Biotechnology Journal 14:441-117.

Chen, W., Ow, D.W. 2017. Precise, flexible and affordable gene stacking for crop improvement. Bioengineered 8:451-456.

Metal and oxidative stress tolerance

Environmental stresses reduce plant productivity. Both abiotic and biotic stresses disrupt normal cellular homeostasis leading to elevated levels of reactive oxygen species that in turn leads to cellular damage and cell death. We have been interested in the molecular mechanisms of plant tolerance to heavy metals, which also leads to oxidative stress. In particular, we have been studying three genes involved in oxidative and metal stress tolerance.

OXS1

Using a fission yeast model system to probe into the molecular mechanisms of OXS1 (Oxidative Stress 1), postdoctoral fellow HE Yumei and colleagues elucidated a new cadmium induced disulfide stress pathway in the fission yeast. This new Oxs1-Pap1 regulatory pathway appears evolutionarily conserved, as heterologous (human, mouse and Arabidopsis) Oxs1 and Pap1-homologues can bind interchangeably with each other in vitro, and at least in the fission yeast, heterologous Oxs1 and Pap1-homologues can substitute for S. pombe Oxs1 and Pap1 to enhance stress tolerance.

He, Y., Chen, Y., Song, W., Zhu, L., Dong, Z., Ow, D.W. 2017. A Pap1-Oxs1 signaling pathway for disulfide stress in Schizosaccharomyces pombe. Nucleic Acids Research 45: 106-114.

PhD student CHEN Yan and colleagues found that the Oxs1 nuclear export signal can protect fission yeast against oxidative stress by serving as a competitive substrate for Crm1-mediated export of Pap1. Higher Pap1 concentration in the nucleus primes the expression of stress tolerance genes. This method of blocking Crm1 mediated export of nuclear proteins may find medical relevance as an alternative to chemical drugs currently being tested to block the nuclear export of tumor suppressor p53 in cancer therapy, or proteins necessary for maturation of HIV-1 and influenza viruses.

Chen, Y., Zhang, Y., Dong, Z.,Ow, D.W. 2018. Protection from disulfide stress by inhibition of Pap1 nuclear export in Schizosaccharomyces pombe. Genetics 210: 857-868.

OXS2

Previously, we described Arabidopsis Oxidative Stress 2 (OXS2) as a zinc-finger transcription factor that resides predominantly in the cytoplasm in the absence of stress, but upon encountering stress it relocates to the nucleus until stress is abated. Since loss of OXS2 function causes stress sensitivity, nuclear OXS2 was proposed to play a positive role in alleviating the stress challenge. In other words, in response to tolerable stress, plants respond by delaying reproduction in hopes for better times and OXS2 activates tolerance pathways to facilitate this response. However, when encountering intolerable stress, plants accelerate early flowering to insure survival of the species, by directly binding to the promoter of floral integrator SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1) to enhance SOC1 transcription that leads to flowering. An observation that was also noted when OXS2 remains in the cytoplasm, it causes the opposite effect of retaining vegetative growth. At the time, we were unable to offer an explanation on how OXS2 could suppress flowering while in the cytoplasm. Now, Postdoctoral Fellow LIANG Minting has shown that OXS2 can interact with another key component of the flowering pathway. Through a linker protein, OXS2 can indirectly bind the florigen FT (FLOWERING LOCUS T) and its associated transcription factor FD (FLOWERING LOCUS D). Whereas FD is always found in the nucleus, FT can be either cytoplasmic or nuclear. The OXS2 binding to FT in the cytoplasm could account for how cytoplasmic OXS2 maintains vegetable growth, by preventing FT entry to the nucleus to activate the flowering pathway.

Liang, M., Ow, D.W. 2019. Nucleocytoplasmic OXIDATIVE STRESS 2 can relocate FLOWERING LOCUS T. Biochemical and Biophysical Research Communications 517: 735-740.

In extending the research of OXS2 in major crops, Ph.D. student HE Lilong and colleagues have since extended this research to crop species and reported that members of the maize OXS2 family can activate transcription of a gene encoding a putative SAM-dependent methyltransferase, a new factor for enhanced Cd tolerance.

He, L., M., X., Li, Z., Jiao, Z., Li, Y., Ow, D.W. 2016. Maize OXIDATIVE STRESS 2 homologs enhance cadmium tolerance in Arabidopsis through activation of a putative SAM-dependent methyltransferase gene. Plant Physiology 171:1675-1685.

OXS3

We had reported previously that Arabidopsis Oxidative Stress 3 (OXS3) was a putative histone modification factor in response to oxidative and metal stress. Postdoctoral fellow LIANG Minting also found that loss of Arabidopsis OXS3 causes early flowering under drought stress, and this led to finding that OXS3 can bind SOC1 to cause AP1 gene repression. Since AP1 is needed for floral development, repressing AP1 expression may serve as a “safety check” to attenuate stress-induced precocious flowering.

Liang, M., Xiao, S., Cai, J., Ow, D.W. 2019. OXIDATIVE STRESS 3 regulates drought-induced flowering through APETALA 1. Biochemical and Biophysical Research Communications 519: 585-590.

Most interesting of all, Assistant Researcher Wang Changhu and colleagues have shown that overproducing certain rice OXS3 family member proteins can lower the cadmium content in rice grain. Due to soil pollution problems in China, rice with high Cd content has been found in recent years. As this problem cannot be solved easily through soil remediation, the engineering of low cadmium rice may provide a solution to minimize dietary intake of cadmium.

Wang, C., Guo, W., Ye, S., Wei, P., Ow, D.W. 2016. Reduction of Cd in rice through expression of OXS3-like gene fragments. Molecular Plant, 9:301-304.

Wang, C., Guo, W., Cai, X., Li, R., Ow, D.W. 2018. Engineering low-cadmium rice through stress-inducible expression of OXS3-family member genes. New Biotechnology https://doi.org/10.1016/j.nbt.2018.04.004.

Urban farming

By 2040, Chinese cities will house a billion residents, and the need to provide fresh food for this large population will become a greater challenge. Urban development reduces arable land for traditional agriculture while urban industrialization pushes clean growing areas further away from urban centers. Hence, not only is China facing the prospect of less capacity for food production, but also escalating costs (including environmental costs) for food transport and packaging. Roof top agriculture can reclaim some of the land displaced by urban sprawl. Locally grown vegetables would save otherwise costly handling, packaging and transport that depend on fossil fuel. Water and fertilizers are more efficient in closed hydroponics systems without causing eutrophication of scarce drinking water supplies. Herbicides are not necessary, and pesticides can be substantially reduced. Hydroponically grown vegetables can be healthier than soil-grown counterparts given the high soil pollution near most urban surrounds. PhD student LIU Ting and colleagues experimentally tested roof top farming, and the data showed that rooftop grown leafy vegetables can be produced more cost effectively and with higher quality than market equivalents.

Liu, T., Yang, M, Han, Z., Ow, D.W. 2016. Rooftop production of leafy vegetables can be profitable and less contaminated than farm grown vegetables. Agronomy for Sustainable Development 36:41 DOI 10.1007/s13593-016-0378-6.