Enhanced vitamin E content in an Indica rice cultivar harbouring two transgenes from Arabidopsis thaliana involved in tocopherol biosynthesis pathway

Micronutrient deficiency results in malnutrition, which is prevalent all over the world and may lead to premature death in women and children (White and Broadley, 2009). Strategies formulated earlier including supplementation and fortified foods were not successful, owing to socio-economic and technical hurdles (Mayer et al. 2008). The subsequently evolved strategy of bio-fortification is a viable biotechnological tool to achieve desired results without compromising the agronomical values of crops. Conferring the genetic trait to improve vital nutrient accumulation in the edible parts of staple food crops, such as rice, through metabolic engineering is considered a fast, sustainable, and cost-effective alternative to conventional breeding (Maestre et al. 2017). In earlier reports from our lab, enhanced α-tocopherol levels in the stable transformants of Nicotiana tabacum (Harish et al. 2013a, b) and in Nicotiana benthamiana adopting a transient expression system using A. thaliana tocopherol cyclase (TC) and homogentisate phytyl transferase (HPT) (Sathish et al. 2018) were shown. In the present study, Agrobacterium-mediated transformation of Indica rice ASD16 with two genes involved in tocopherol biosynthesis, viz., TC and HPT were carried out and the transgenic plants were analyzed for the vitamin E (α- tocopherol) content.

Micronutrient deficiency results in malnutrition, which is prevalent all over the world and may lead to premature death in women and children (White and Broadley, 2009). Strategies formulated earlier including supplementation and fortified foods were not successful, owing to socio-economic and technical hurdles (Mayer et al., 2008). The subsequently evolved strategy of biofortification is a viable biotechnological tool to achieve desired results without compromising the agronomical values of crops. Conferring the genetic trait to improve vital nutrient accumulation in the edible parts of staple food crops, such as rice, through metabolic engineering is considered a fast, sustainable and costeffective alternative to conventional breeding (Maestre et al., 2017). In earlier reports from our laboratory, enhanced atocopherol levels in the stable transformants of Nicotiana tabacum (Harish et al., 2013a(Harish et al., , 2013b and in Nicotiana benthamiana adopting a transient expression system using A. thaliana tocopherol cyclase (TC) and homogentisate phytyl transferase (HPT) (Sathish et al., 2018) were shown. In the present study, Agrobacterium-mediated transformation of Indica rice ASD16 with two genes involved in tocopherol biosynthesis, viz., TC and HPT, was carried out and the transgenic plants were analysed for the vitamin E (a-tocopherol) content.
Mature seed-derived embryogenic calli were subjected to genetic transformation (Sundararajan et al., 2020) by employing two gene constructs, viz., pCAMBIA 1305.1 harbouring TC and pNutKan harbouring HPT (Fig 1a), individually, and in a cotransformation system, the calli were infected with both gene constructs. Putative transgenic plants recovered from all three independent experiments were established in a containment facility and no detectable morphological variations were found between the acclimatized NT and the transgenic plants (Fig 1b). Ten plants from all three experiments were taken for initial PCR analysis with the marker genes respective to each gene construct, that is hptII in pCAMBIA 1305.1 TC and nptII in pNutKan HPT. Nine plants harbouring TC, 6 plants harbouring HPT and 7 TC + HPT plants showed positive PCR amplification of the marker genes (data not shown).
A preliminary GUS histochemical assay for the transgenic lines generated with TC and TC + HPT revealed the formation of blue colour in the leaves and roots of transgenic plants confirming the presence of the GUS gene, whereas no blue coloration was detected in the NT control. PCR analysis of the transgenic plants harbouring individual TC and HPT showed the expected band sizes (1.2 Kb for HPT and 1.4 Kb for TC). With the TC + HPT plants, amplification of both selectable marker genes during the initial PCR analysis was observed. However, in the PCR using gene-specific primers, among the seven plants, only five showed the presence of both the transgenes (plants subsequently renamed as TH1 to TH5). Southern blot analysis revealed the expected hybridization signals corresponding to HPT (1.2 kb) in all the six plants digested with PstI and NotI, and TC (1.4 kb) in all the nine plants digested with KpnI and BamHI. For the TC + HPT lines, two individual blots were prepared using genomic DNA digested with KpnI and BamHI for TC and PstI and NotI for HPT. The HPT-probed blot showed a corresponding hybridization signal of 1.2 kb, and a hybridization signal corresponding to 1.4 kb of the TC gene was observed in the TC-probed blot in all the five transgenic lines demonstrating the stable integration of the transgenes in the plants (data not shown).
Transcript analysis indicated that among the TC plants, TC3, TC4 and TC7 showed significantly higher levels of transgene expression (19.7-fold, 21-fold and 20.2-fold, respectively) as compared to the control. The relative gene expression of HPT lines was found to be higher as compared to TC lines. The quantum increase ranged from 9.5 to 17.0 among HPT lines with HPT4 showing the highest gene expression (26.3-fold) followed by HPT5 (23.6-fold increase). Among the TC + HPT lines, TH4 showed maximum gene expression to the tune of 26.7-fold (HPT) and 32.5-fold (TC) (data not shown).
Two plants each harbouring TC, HPT and TC + HPT were analysed for a-tocopherol content in the transgenic leaves by HPLC. The determination of a-tocopherol in the samples was based on peaks observed at 295 nm with a UV detection lamp. The retention time of the metabolite was confirmed by injecting the authentic standard (Sigma-Aldrich, St. Louis, MO), and the quantification in transgenic lines was performed accordingly. Results showed that among the TC lines, TC1 showed a 3.26-fold and TC4 showed a 2.2-fold increase in a-tocopherol. The HPT lines showed relatively higher a-tocopherol content as compared to that of the TC lines, wherein HPT3 showed a 3.5-fold and HPT2 showed a 2.8-fold increase in a-tocopherol. The TC + HPT plants exhibited the highest a-tocopherol content among all the transgenic lines. Accordingly, the line TH4 showed a 5.3-fold increase in a-tocopherol followed by TH3 (4.3-fold). Subsequently, three lines (TC1, HPT3 and TH4) were chosen for T 1 analysis where transgenic plants germinated from T 0 seeds showed a segregation ratio of 3:1 confirming the Mendelian pattern of inheritance. Ten samples for each progeny were checked by PCR, and the progenies showed respective positive amplicons for the TC (1.4 kb) and HPT (1.2 kb) and presence of both the genes in the TC + HPT progenies (data not shown).
Subsequently, Southern blot analysis revealed the stable integration of the transgenes in the T 1 progenies. Accordingly, the hybridization signals corresponding to the expected sizes of transgenes were detected in all the samples and no hybridization signal was detected in the NT control. qRT-PCR analysis of the two randomly selected PCR-positive plants from each gene construct revealed comparable results as that of the T 0 plants. The TC + HPT lines showed higher relative gene expression as compared to that of the lines that harbour only one gene. Both TC1 progenies showed a comparable increase to the tune of 8.36-fold (TC 1-1) and 11.6-fold (TC 1-2), and HPT4 progenies showed a significant increase of 11.26-fold (HPT 4-1) and 8.95fold (HPT 4-2). The TC + HPT progeny TH4 showed the highest relative gene expression about 15.35 (TH 4-1) and 14.80 (TH 4-2) folds (data not shown). A sample each from T 1 progeny that carried TC, HPT and TC + HPT were analysed for their a-tocopherol content. Results revealed that TC1-1 showed a 2.7-fold increase, HPT3-1 showed an increase of 3.3-fold and the TC + HPT progeny TH4-1 showed a 4.1-fold increase in a-tocopherol content in the transgenic leaves similar to that of the T 0 progenies where the TC + HPT line showed the highest a-tocopherol as compared to the lines harbouring either TC or HPT (Fig. 1c, d). The transgenic T 1 seeds from the selected progenies were assessed for their a-tocopherol content and a 0.29-fold increase in the TC + HPT line (TH4-1) was observed as compared to TC1-1 (0.12-fold) and HPT3-1 (0.18-fold). Enhanced level of vitamin E in numerous plant species including A. thaliana leaves (4.4-fold) and seeds (40%), tobacco leaves (5.5-fold), lettuce (2-fold), Brassica napus (2.7-fold) and potato tuber (106%) has been reported utilizing various enzymes of the vitamin E biosynthesis pathway (reviewed by M ene-Saffran e and Pellaud, 2017). Harish et al., (2013a) proposed that in higher plants, co-expression of TC and HPT might increase the overall rate of total pathway flux resulting in a higher content of the end products. In summary, successful enhancement of vitamin E in rice was achieved using overexpression of both TC and HPT. Though the accumulation is not significant in the seeds, improvement with a multi-gene strategy with appropriate endosperm-specific promoters might offer an attractive model for vitamin E biofortification in rice, which would immensely benefit human nutrition and health care.