A Major Locus on Wheat Chromosome 7B Associated With Late-Maturity α-Amylase Encodes a Putative ent-Copalyl Diphosphate Synthase

Many wheat varieties have the potential to develop unacceptably high levels of α-amylase in the grains if exposed to a cool temperature shock or simply cool temperature during the early to middle stages of grain filling. This phenomenon is referred to as late maturity α-amylase (LMA). The enzyme persists in the grain until harvest and may result in wheat with a low Falling Number that does not meet receival and export specifications. Resistance to LMA is therefore a valuable target for wheat breeders and wheat industries in general. Genetic evidence implicating a locus on the long arm of chromosome 7B in variation in LMA phenotype was confirmed in this investigation. Through intensive fine-mapping an ent-copalyl diphosphate synthase (CPS), hitherto named LMA-1, was identified as the likely candidate gene associated with variation in LMA phenotype. Single Nucleotide Polymorphisms (SNPs) within the LMA-1 coding sequence of Chinese Spring, Maringa and Halberd result in either prematurely terminated or functionally altered proteins that are associated with useful levels of resistance to LMA. LMA-1 transcripts detected in de-embryonated grain tissue from around 15 days after anthesis, several days before the synthesis of α-amylase, were low in the resistant varieties Chinese Spring and Maringa compared with LMA susceptible genotype Spica. This was associated with a dramatic reduction in the concentrations of intermediates in the gibberellin biosynthesis pathway such as GA19, evidence that LMA-1 was functioning as CPS in the gibberellin biosynthesis pathway. A survey of a large collection of Australian and international wheat varieties distinguished 9 major haplotypes at the LMA-1 locus. Generally, within classes, there was notable variation for LMA phenotype and evidence for genotypes whose resistance is presumed to be due to genetic loci located elsewhere on the wheat genome. Further investigation is required to characterize the sequence of steps between LMA-1 and α-amylase synthesis as well as to gain a better understanding of the role and potential impact of other genetic loci. Diagnostic markers for sources of resistance and SNP variation reported in this study should assist breeders to deploy resistance associated with LMA-1 variants in breeding programs.

Expression of the aldehyde oxidase 3, ent-copalyl diphosphate synthase, and VIVIPAROUS 1 genes in wheat cultivars differing in their susceptibility to pre-harvest sprouting

The quality of wheat grains is often negatively affected by pre-harvest sprouting (PHS), a complex trait with a poorly understood genetic background. In this study two wheat cultivars differing in their susceptibility to PHS were used to investigate expression of three genes: AAO3, CPS3 and VP1. AAO3 is coding for aldehyde oxidase 3, an enzyme involved in the synthesis of abscisic acid. CPS3 codes for ent-copalyl diphosphate synthase which belongs to the pathway of gibberellic acid synthesis. The product of VP1 (VIVIPAROUS 1) is a transcription factor which controls expression of the former two genes. The study was carried out using both developing and sprouting-induced grains. In Piko, a wheat cultivar susceptible to PHS, accumulation of the AAO3 transcript was significantly decreased, during the last stages of grain development, in comparison to Sława, a cultivar tolerant to PHS. In case of the CPS3 and VP1 transcripts, the differences between cultivars were especially evident from 17th to 31st day after pollination. In turn, after induction of sprouting within spikes, accumulation of the AAO3 and VP1 mRNA in the Sława grains was lower in comparison to that observed in the Piko grains. Moreover, accumulation of the CPS3 transcript was significantly higher for Piko than for Sława, both in sprouting and non-sprouting grains. According to our knowledge this report provides the first description of the AAO3 and CPS3 expression in the context of PHS, and in the future it would be valuable to correlate this information with the data on the accumulation of ABA and GA3.

Isolation and characterization of a copalyl diphosphate synthase gene promoter from Salvia miltiorrhiza

<p>The promoter, 5' UTR, and 34-nt 5' fragments of protein encoding region of the <em>Salvia miltiorrhiza</em> copalyl diphosphate synthase gene were cloned and characterized. No tandem repeats, miRNA binding sites, or CpNpG islands were observed in the promoter, 5' UTR, or protein encoding fragments. The entire isolated promoter and 5' UTR is 2235 bp long and contains repetitions of many <em>cis</em>-active elements, recognized by homologous transcription factors, found in <em>Arabidopsis thaliana</em> and other plant species. A pyrimidine-rich fragment with only 6 non-pyrimidine bases was localized in the 33-nt stretch from nt 2185 to 2217 in the 5' UTR. The observed <em>cis</em>-active sequences are potential binding sites for <em>trans</em>-factors that could regulate spatio-temporal <em>CPS</em> gene expression in response to biotic and abiotic stress conditions. Obtained results are initially verified by in silico and co-expression studies based on <em>A. thaliana</em> microarray data.</p><p>The quantitative RT-PCR analysis confirmed that the entire 2269-bp copalyl diphosphate synthase gene fragment has the promoter activity.</p><p>Quantitative RT-PCR analysis was used to study changes in <em>CPS</em> promoter activity occurring in response to the application of four selected biotic and abiotic regulatory factors; auxin, gibberellin, salicylic acid, and high-salt concentration.</p>