The bZIP transcription factor SPA Heterodimerizing Protein represses glutenin synthesis in Triticum aestivum.

The quality of wheat grain is mainly determined by the quantity and composition of its grain storage proteins (GSPs). Grain storage proteins consist of low- and high-molecular-weight glutenins (LMW-GS and HMW-GS, respectively) and gliadins. The synthesis of these proteins is essentially regulated at the transcriptional level and by the availability of nitrogen and sulfur. The regulation network has been extensively studied in barley where BLZ1 and BLZ2, members of the basic leucine zipper (bZIP) family, activate the synthesis of hordeins. To date, in wheat, only the ortholog of BLZ2, Storage Protein Activator (SPA), has been identified as playing a major role in the regulation of GSP synthesis. Here, the ortholog of BLZ1, named SPA Heterodimerizing Protein (SHP), was identified and its involvement in the transcriptional regulation of the genes coding for GSPs was analyzed. In gel mobility shift assays, SHP binds cis-motifs known to bind to bZIP family transcription factors in HMW-GS and LMW-GS promoters. Moreover, we showed by transient expression assays in wheat endosperm that SHP acts as a repressor of the activity of these gene promoters. This result was confirmed in transgenic lines overexpressing SHP, which were grown with low and high nitrogen supply. The phenotype of SHP-overexpressing lines showed a lower quantity of both LMW-GS and HMW-GS, while the quantity of gliadin was unchanged, whatever the nitrogen availability. Thus, the gliadin/glutenin ratio was increased, which suggests that gliadin and glutenin genes may be differently regulated.

Enhancement of grain number per spike by RNA interference of cytokinin oxidase 2 gene in bread wheat.

BACKGROUND: This study aimed to validate the function of CKX gene on grain numbers in wheat. METHODS: we constructed and transformed a RNA interference expression vector of TaCKX2.4 in bread wheat line NB1. Southern blotting analysis was used to select transgenic plants with single copy. The expression of TaCKX2.4 gene was estimated by Quantitative real-time PCR (qRT-PCR) analysis. Finally, the relation between expression of TaCKX2.4 gene and grain numbers was validated. RESULTS: Totally, 20 positive independent events were obtained. Homozygous lines from 5 events with a single copy of transformed gene each were selected to evaluate the expression of TaCKX2.4 and grain numbers per spike in T3 generation. Compared with the control NB1, the average grain numbers per spike significantly increased by 12.6%, 8.3%, 6.5% and 5.8% in the T3 lines JW39-3A, JW1-2B, JW1-1A and JW5-1A, respectively. CONCLUSION: Our study indicated that the expression level of TaCKX2.4 was negatively correlated with the grain number per spike, indicating that the reduced expression of TaCKX2.4 increased grain numbers per spike in wheat.

Storage protein activator controls grain protein accumulation in bread wheat in a nitrogen dependent manner.

The expression of cereal grain storage protein (GSP) genes is controlled by a complex network of transcription factors (TFs). Storage protein activator (SPA) is a major TF acting in this network but its specific function in wheat (Triticum aestivum L.) remains to be determined. Here we generated an RNAi line in which expression of the three SPA homoeologs was reduced. In this line and its null segregant we analyzed GSP accumulation and expression of GSP and regulatory TF genes under two regimes of nitrogen availability. We show that down regulation of SPA decreases grain protein concentration at maturity under low but not high nitrogen supply. Under low nitrogen supply, the decrease in SPA expression also caused a reduction in the total quantity of GSP per grain and in the ratio of GSP to albumin-globulins, without significantly affecting GSP composition. The slight reduction in GSP gene expression measured in the SPA RNAi line under low nitrogen supply did not entirely account for the more significant decrease in GSP accumulation, suggesting that SPA regulates additional levels of GSP synthesis. Our results demonstrate a clear role of SPA in the regulation of grain nitrogen metabolism when nitrogen is a limiting resource.

Highly efficient Agrobacterium-mediated transformation of wheat via in planta inoculation.

This chapter details a reproducible method for the transformation of spring wheat using Agrobacterium tumefaciens via the direct inoculation of bacteria into immature seeds in planta as described in patent WO 00/63398 (1). Transformation efficiencies from 1 to 30% have been obtained and average efficiencies of at least 5% are routinely achieved. Regenerated plants are phenotypically normal with 30-50% of transformation events carrying introduced genes at single insertion sites, a higher rate than is typically reported for transgenic plants produced using biolistic transformation methods.