Complex nested promoters control tissue-specific expression of acetyl-CoA carboxylase genes in wheat

Abstract

Cis-acting regulatory elements of the wheat acetyl-CoA carboxylase (ACC) gene family were identified by comparing the promoter activity of 5 end gene fragments fused to a reporter gene in two transient expression systems: wheat protoplasts and epidermal cells of mature embryos. Expression of the plastid and the cytosolic ACC genes is each driven by two nested promoters responsible for the synthesis of two transcript types. The internal promoter is located in an intron removed from transcripts originating at the first promoter. These complex promoters, which are different for the cytosolic and plastid ACC genes, control tissue-specific expression of the enzymatic activity supplying cytosolic, plastid, and mitochondrial pools of malonyl-CoA. The activity of one such complex promoter, driving expression of one of the cytosolic ACC genes, was studied throughout development of transgenic wheat plants carrying a full-length promoter-reporter gene fusion. High activity of the promoter was detected in the coleoptile, in the upper sheath section of the leaf, on the top surface of the ovary, in some sections of the main veins in the lemma and glume, and in abaxial epidermis hair cells of the lemma, glume, and rachis. The findings are consistent with the developmental and environmental requirements for very-long-chain fatty acids and flavonoids, whose synthesis begins with the ACC reaction in the cytosol of these specific cell types.M odern bread wheat has a large hexaploid genome, making molecular genetic studies difficult. We have been studying the structure, evolution, and function of the family of genes encoding the enzyme acetyl-CoA carboxylase (ACC) during wheat development (1-7).In plants, separate ACC isozymes supply the malonyl-CoA pools used for de novo fatty acid (FA) biosynthesis in plastids and mitochondria, and for FA elongation and flavonoid (FL) and stilbene biosynthesis in the cytosol (8, 9). FAs are essential in membrane biogenesis, lipoic acid synthesis, production of oil as storage material, cuticular wax (CW) synthesis, signaling, and pollen-stigma interaction. FLs play multiple roles in plants as pigments and UV protectants, defense compounds, and as signals (10). Malonyl-CoA is also used for protein and smallmolecule malonylation.Our previous study (5) showed that significant regulation of cytosolic and plastid ACC genes in young wheat plants is accomplished at the transcript level. The number and identity of transcribed genes were established by cDNA and genomic DNA sequence comparisons. Transcription start sites and splicing patterns of leader introns were identified for multiple genes, including homoeologs and paralogs. We found that in wheat seedlings, the plastid ACC mRNA level is high in the middle part of the plant and low in roots and leaf blades. The three plastid ACC-homoeologous genes are equivalent in their level of expression. Cytosolic ACC mRNA accumulates to a high level in the lower sheath section of the plant. For the cytosolic ACC genes, we found that transcripts of the three homoeo...

OPINIONS Acetyl-coenzyme A carboxylase – an attractive enzyme for biotechnology

“…The cytosolic (eukaryotic) type ACCase provides malonyl-CoA for the synthesis of very long fatty acids (20 or more carbon atoms), flavonoids and anthocyanins. Traditionally, these compounds are classified as secondary metabolites but a detailed analysis of plant cytosolic ACCases has proved their essential role in plant development (Zuther et al, 2004).…”

“…The cytosolic (eukaryotic) type ACCase provides malonyl-CoA for the synthesis of very long fatty acids (20 or more carbon atoms), flavonoids and anthocyanins. Traditionally, these compounds are classified as secondary metabolites but a detailed analysis of plant cytosolic ACCases has proved their essential role in plant development (Zuther et al, 2004).Animal and fungal ACCases are exclusively of eukaryotic type with one putative exception, the nematode Turbatrix aceti enzyme, which has not been applied for biotechnological purposes so far (Meyer and Meyer, 1978;Barber et al, 2005). Both animals and fungi have two isoforms of ACCases designated as ACC1 and ACC2.…”

Acc homoeoloci and the evolution of wheat genomes

“…The gene encoding plastid ACCase in grasses, which replaced the multisubunit plastid enzyme of bacterial origin still found in dicots, arose by duplication of a gene encoding the multidomain cytosolic ACCase (1). One copy of the ancestral gene acquired a plastid-targeting signal (1,2) and a new promoter and new regulatory elements (2,3). The wheat Acc-1 and Acc-2 genes show no sequence similarity outside the exons encoding the mature enzyme (2,3).…”

“…One copy of the ancestral gene acquired a plastid-targeting signal (1,2) and a new promoter and new regulatory elements (2,3). The wheat Acc-1 and Acc-2 genes show no sequence similarity outside the exons encoding the mature enzyme (2,3). For both Acc-1 and Acc-2, all three Triticum aestivum homoeologs are transcriptionally active, and each uses two nested promoters and alternative splicing of the first intron to produce transcripts with different organ specificity.…”

“…For both Acc-1 and Acc-2, all three Triticum aestivum homoeologs are transcriptionally active, and each uses two nested promoters and alternative splicing of the first intron to produce transcripts with different organ specificity. Each of the homoeologs contributes equally to the total mRNA level of each transcript type (2,3).…”