Low expression of lipoxygenase 3 (LOX3) enhances the retention of kernel tocopherols in maize during storage.

BACKGROUND: Deficiency of vitamin E results in several neurological and age-related disorders in humans. Utilization of maize mutants with favourable vte4-allele led to the development of several α-tocopherol (vitamin E) rich (16-19 µg/g) maize hybrids worldwide. However, the degradation of tocopherols during post-harvest storage substantially affects the efficacy of these genotypes. METHODS AND RESULTS: We studied the role of lipoxygenase enzyme and Lipoxygenase 3 (LOX3) gene on the degradation of tocopherols at monthly intervals under traditional storage up to six months in two vte4-based contrasting-tocopherol retention maize inbreds viz. HKI323-PVE and HKI193-1-PVE. The analysis revealed significant degradation of tocopherols across storage intervals in both the inbreds. Lower retention of α-tocopherol was noticed in HKI193-1-PVE. HKI323-PVE with the higher retention of α-tocopherol showed lower lipoxygenase activity throughout the storage intervals. LOX3 gene expression was higher (~ 1.5-fold) in HKI193-1-PVE compared to HKI323-PVE across the storage intervals. Both lipoxygenase activity and LOX3 expression peaked at 120 days after storage (DAS) in both genotypes. Further, a similar trend was observed for LOX3 expression and lipoxygenase activity. The α-tocopherol exhibited a significantly negative correlation with lipoxygenase enzyme and expression of LOX3 across the storage intervals. CONCLUSIONS: HKI323-PVE with high tocopherol retention, low -lipoxygenase activity, and -LOX3 gene expression can act as a potential donor in the vitamin E biofortification program. Protein-protein association network analysis also indicated the independent effect of vte4 and LOX genes. This is the first comprehensive report analyzing the expression of the LOX3 gene and deciphering its vital role in the retention of α-tocopherol in biofortified maize varieties under traditional storage.

Development and validation of rapid and cost-effective protocol for estimation of amylose and amylopectin in maize kernels.

Maize possesses wide variation in amylose and amylopectin which assumes significance as a part of both food-chain and different industrial applications. Estimation of amylose and amylopectin in maize kernels is important for developing suitable hybrids. The existing protocols for estimation of amylose and amylopectin in maize are elaborate and lengthy, and involve high cost. Here, we developed a rapid and cost-effective method for estimation of amylose and amylopectin in maize kernels. 10% toluene and 80% ethanol were used for removal of proteins (~ 10%) and lipids (~ 4%) from maize flour. The over-estimation of amylose was minimized using NaOH with KI to stop free KI to bind with amylopectin. Standards were improved by mixing amylose and amylopectin in different concentrations (0-100%), rather than using amylose or amylopectin alone. Standard curve generated regression equation of y = 90.436x + 0.8535 with R 2 = 0.9989. Two types of samples viz., (1) protein, amylose and amylopectin (2) amylose and amylopectin, showed that starch fractions were highly comparable to expected values with correlation coefficient (r) of 0.9998 and mean standard deviation of 0.54. The protocol successfully estimated wide range of amylose (2.79-50.04%) and amylopectin (59.96-97.21%) among diverse maize inbreds including amylose extender1 (ae1) and waxy1 (wx1) mutants. Present protocol required 75% less time and 92.5% less cost compared to existing protocols. The newly developed method would be highly useful in developing maize hybrids high in amylose or amylopectin. This is the first report of rapid and cost-effective protocol for estimation of starch fractions in maize kernels.