SUMMARYLeaves of tobacco (Nicotiana tabacum) are covered with glandular trichomes that produce sucrose esters and diterpenoids in varying quantities, depending on cultivar type. The bicyclic diterpene Z-abienol is the major labdanoid present in some oriental tobacco cultivars, where it constitutes a precursor of important flavours and aromas. We describe here the identification and characterization of two genes governing the biosynthesis of Z-abienol in N. tabacum. As for other angiosperm labdanoid diterpenes, the biosynthesis of Z-abienol proceeds in two steps. NtCPS2 encodes a class-II terpene synthase that synthesizes 8-hydroxy-copalyl diphosphate, and NtABS encodes a kaurene synthase-like (KSL) protein that uses 8-hydroxy-copalyl diphosphate to produce Z-abienol. Phylogenetic analysis indicates that NtABS belongs to a distinct clade of KSL proteins that comprises the recently identified tomato (Solanum habrochaites) santalene and bergamotene synthase. RT-PCR results show that both genes are preferentially expressed in trichomes. Moreover, microscopy of NtCPS2 promoter-GUS fusion transgenics demonstrated a high specificity of expression to trichome glandular cells. Ectopic expression of both genes, but not of either one alone, driven by a trichomespecific promoter in transgenic Nicotiana sylvestris conferred Z-abienol formation to this species, which does not normally produce it. Furthermore, sequence analysis of over 100 tobacco cultivars revealed polymorphisms in NtCPS2 that lead to a prematurely truncated protein in cultivars lacking Z-abienol, thus establishing NtCPS2 as a major gene controlling Z-abienol biosynthesis in tobacco. These results offer new perspectives for tobacco breeding and the metabolic engineering of labdanoid diterpenes, as well as for structure-function relationship studies of terpene synthases.
Two types of gene encoding small subunits (SSU) of ADP-glucose pyrophosphorylase, a starch-biosynthetic enzyme, have been found in cereals and other grasses. One of these genes encodes two SSU proteins. These are targeted to different subcellular compartments and expressed in different organs of the plant: the endosperm cytosol and the leaf plastids. The SSU gene encoding two proteins evolved from an ancestral gene encoding a single protein by the acquisition of an alternative first exon. Prior to the work reported here, this type of SSU gene had been found in all grasses examined except maize. In maize, two separate genes, Bt2 and L2, were known to have the same roles as the alternatively spliced gene found in other grasses. The evolutionary origin of these maize genes and their relationship to the SSU genes in other grasses were unclear. Here we show that Bt2 and L2 are paralogous genes that arose as a result of the tetraploidization of the maize genome. Both genes derive from an ancestral alternatively spliced SSU gene orthologous to that found in other grasses. Following duplication, the Bt2 and L2 genes diverged in function. Each took a different one of the two functions of the ancestral gene. Now Bt2 encodes the endosperm cytosolic SSU but does not contribute significantly to leaf AGPase activity. Similarly, L2 has lost the use of one of its two alternative first exons. It can no longer contribute to the endosperm cytosolic SSU but is probably responsible for the bulk of the leaf AGPase SSU.
The barley (Hordeum vulgare) gene Hv.AGP.S.1 produces two different transcripts encoding small subunits (SSUs) of ADP-glucose pyrophosphorylase (AGPase). It was shown previously that one of these transcripts, Hv.1a, encodes the cytosolic SSU in the endosperm. It is shown here that the other transcript produced from Hv.AGP.S.1, Hv.1b, encodes a plastidial SSU that is required for >90% of the AGPase activity in the leaves. Thus, both of the alternative transcripts encoded by Hv.AGP.S.1 are physiologically relevant: One is important for starch synthesis in the endosperm and the other for starch synthesis in the leaves. Although the Hv.1b transcript is abundant in embryos and present in endosperm, there is no evidence that a protein is produced from this transcript in these organs. This suggests that some, as yet unidentified, post-transcriptional control mechanism prevents the accumulation of the protein encoded by Hv.1b in embryos and endosperm but not in leaves. There is one other known gene in barley, Hv.AGP.S.2, encoding a SSU of AGPase. This gene has been shown to be responsible for the plastidial SSU in the endosperm. It is shown here that Hv.AGP.S.2 probably also makes some contribution to the SSU of AGPase in the leaves and may be responsible for most or all of the plastidial SSU in a range of non-photosynthetic plant organs including the embryo.
A mutant of rice was identified with a Tos17 insertion in OsAPL1, a gene encoding a large subunit (LSU) of ADP-glucose pyrophosphorylase (AGPase). The insertion prevents production of a normal transcript from OsAPL1. Characterisation of the mutant (apl1) showed that the LSU encoded by OsAPL1 is required for AGPase activity in rice leaf blades. In mutant leaf blades, the AGPase small subunit protein is not detectable and the AGPase activity and starch content are reduced to <1 and <5% of that in wild type blades, respectively. The mutation also leads to a reduction in starch content in the leaf sheaths but does not significantly affect AGPase activity or starch synthesis in other parts of the plant. The sucrose, glucose and fructose contents of the leaves are not affected by the mutation. Despite the near absence of starch in the leaf blades, apl1 mutant rice plants grow and develop normally under controlled environmental conditions and show no reduction in productivity.
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