Association between starch branching enzymes and modification of opaque-2 (o2) in quality protein maize

Maize is the most important cereal in Uganda and many other countries of Sub-Saharan Africa. It is the main source of basal energy for humans, livestock and poultry. Maize is however deficient in two essential amino acids, lysine and tryptophan. Being a major food source implies that such populations are prone to protein allied malnutrition especially among poor households. High quality proteins maize (QPM) derived from a natural mutant called opaque-2 (o2) that increases the level of lysine and tryptophan in maize grain could provide cheap sources of proteins. Unfortunately, the rate of QPM development has been slow due to frequently poor kernel texture associated with opaque-2. Identification of opaque-2 modifier genes with ability to overcome negative effects of the mutation, while maintaining high nutritional value paved way for development of QPM. Screening for opaque modifiers by conventional means has been difficult and poses a big challenge to large scale adoption of the technology. This is mainly because of the complex nature of opaque-2 modifier genes which is poorly understood. They have however been shown to be closely associated with the starch synthesis genes like starch branching enzymes (sbe’s). Arising from this, study aimed at finding out the nature of the association between the opaque-2 modifiers and the sbe’s encoding starch branching enzymes in maize. The field experiment was conducted at the National Crops Resources Research Institute (NaCRRI), central Uganda, about 27 Km north of Kampala. The plant materials included QPM derived materials with varying levels of modification, normal maize (wild type) and opaque-2 lines. The laboratory work was conducted in the biotechnology laboratory at the Department of Crop Science, Makerere University. Plants were grown in adjacent plots and hand pollinated. Ears were harvested from each maize line starting from 6 days after pollination (DAP) and at 14, 18, 22, 26, 28 and 30 DAP. The harvested materials were immediately frozen in liquid nitrogen and stored at -800 C until use. Total RNA was isolated and reverse transcribes into first strand cDNA which was used in a semi quantitative PCR to study the expression levels and patterns in test lines. Proteins were extracted, resolved on 10% SDS-PAGE and visualized by coomassie staining. Band intensity analysis and quantification was performed using the Image Quant TLTM 1-D analysis software and the corresponding expression levels generated for the same software as band volumes. Sbel transcripts were detectable as early as 6DAP, reached a peak between 18 and 28 DAP, but declined at 30 DAP similarly, sbella transcripts were low at 6 DAP but highest starting from 18 DAP until 30 DAP. sbe proteins levels matched the observed transcript expression patterns with a peak around 22 dap. The observed peak for SBE at 22 DAP corresponded to that of the depending on the background of the QPM test line. Higher expression of the sbe genes associated with better or g=highly modified endosperms. Moreover, higher sbs gene expression in modified backgrounds correspond to high SBE protein levels. Most notable however, sbel expression was more affected by the QPM background than sbella. This observation was fortified by the slight or no difference between the QPM parent or a fully modified background (level1) and a non QPM (normal) parent with respect to sbe/SBE expression. It is concluded that the suppressed expression of sbe and SBE and probably the activity of the enzymes in backgrounds having low percentage of the modifier genes implied that branching enzymes condition modification in QPM. Or, they are an important component of the complex gene pleiotropy involved in reversing the soft chalky endosperm of opaque-2 materials to the hard vitreous one of QPM. Modification of QPM is also likely more due to the SBEI and SBEIIb isoforms rather than the SBEIIa isoform as was depicted by a greater suppression of expression of SBEI in the poorly modified backgrounds compared to the later. This study suggested that starch branching enzymes can only be appropriately used as a diagnostic in identifying potential good modifier backgrounds between 6 and 18DAP when gene/protein expression is detectably variable. In practice, the application of immunoblot detection to visualize the proteins separated by SDS-PAGE and quantitative PCR to directly quantitate changes in gene expression is likely to significantly contribute to fat selection for high protein maize. In order to develop a full picture on the roles of starch synthesis genes in protein accumulation in maize, it is very important to understand the function to starch debranching enzymes (DBE) and SBEIIb.