Plant Cell Tissue Organ Cult.2017 Mar;128(3):509-519

Transformation and characterization of transgenic rice and Cleome spinosa plants carrying the maize phosphoenolpyruvate carboxylase genomic DNA.

Full text

 

Supplement:

The classical photosynthetic mechanism discovered in the 1940s by Melvin Calvin and colleagues, where the first product of CO2 assimilation is the C3 acid phosphoglycerate via ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the mesophyll cells. Later in 1970s, it was discovered that certain plants use C4 photosynthetic pathway, which fixes atmospheric CO2 via phosphoenolpyruvate carboxylase (PEPC) in mesophyll cells and then the generated C4 dicarboxylic acids (oxaloacetate, malate, aspartate) are transported into the neighboring bundle sheath cells where conventional C3 cycle is used. Plants that possess the so-called C3 photosynthetic pathway tend to use CO2 much less efficiently than their C4 counterparts. Unfortunately for agriculture, many important crops (such as rice, wheat, soybean, potato) are of the C3 type. C4 crops (such as maize, sugarcane, sorghum) are considered more efficient assimilators of CO2 in warm environments, under intense light or drought conditions. Thus, most scientists in the field have been asked whether C3 plants can be “converted” into C4 plants.

 

Rice (Figure 1A), a C3 plant, is an important crop feeding nearly 50% of the world population. Due to the increase in population and reduction in land resources, it has been estimated that by 2050 it will necessitate a 60% increase in rice production to feed the growing population. In parallel, Cleome spinosa (Figure 1B) is a tough-stemmed shrub, also known as “spider flower”, which is widely used as a garden ornamental plant. Previous chemical studies have reported the isolation of glucosinolates, which are known as flavor compounds, cancer prevention agents and biopesticides, from seeds of C. spinosa. Moreover, a rare flavone flindulatin and five cembranes were isolated from aerial portions of C. spinosa; among them, cembranes are known to exhibit anti-HIV activity, neuroprotection, and cytotoxicity against several human cancer cell lines. In addition, five essential oil extracts, which displayed antimicrobial activities, were isolated from aerial parts of C. spinosa. The genus Cleome is closely to Arabidopsis; but more interestingly, contains C3 and C4 species. The genus Cleome was proposed as a model for understanding the differences between C3 and C4 photosynthesis over 10 years ago.

 

In this study, the vector PEPCgenome/pBIH2 carrying intact maize (a C4 plant) pepc genomic DNA fragment and two selective resistant genes (nptII and hptII) was used to transform the C3 monocot rice and C3 dicot C. spinosa plants by Agrobacterium tumefaciens-mediated method. For rice, both pepc-specific and hptII-specific fragments were detected in 28 transgenic plants we obtained. For C. spinosa, pepc-specific, hptII-specific and nptII-specific fragments were detected in 4 transgenic plants we obtained. More importantly, using the rigorous criteria during selection, all transformants we examined are independent lines as revealed by Southern blot analysis. Furthermore, high levels of maize PEPC protein and enzyme activity were detected in our transformants. Flowchart of Agrobacterium-mediated transformation in rice is shown in Figure 2.

 

Two T6 homozygous lines (Figure 3), each harboring a single insertion of maize pepc have been identified from transgenic japonica rice cultivar TNG67, which is an important cultivar in Taiwan. About 7- to 9.4-fold or 40-54% of PEPC enzyme activity were measured as compared to untransformed wild-type rice and wild-type maize, respectively. Thus, we believe that our transgenic rice lines with varying expression levels are valuable material for studying physiological responses or investigating the photosynthetic performance under various stress conditions. In addition, coexpression of one or more key genes from the C4 photosynthetic pathway into our transgenic lines carrying the maize pepc gene is also an interesting topic to be explored.

 

 

Figure 1. Rice (A) and Cleome spinosa (B) used in this study.

Figure 2. Flowchart of Agrobacterium-mediated transformation in rice.

Figure 3. Transgenic rice carrying maize pepc gene. (A) T6 generation of homozygous lines #3 and #17 carrying single insertion of maize pepc gene in rice genome were grown in a greenhouse. (B) Enlarged photo showed mature seeds.