Role of callose synthases in transfer cell wall development in tocopherol deficient Arabidopsis mutants

Tocopherols (vitamin E) are lipid-soluble antioxidants produced by all plants and algae, and many cyanobacteria, yet their functions in these photosynthetic organisms are still not fully understood. We have previously reported that the vitamin E deficient 2 (vte2) mutant of Arabidopsis thaliana is sensitive to low temperature (LT) due to impaired transfer cell wall (TCW) development and photoassimilate export associated with massive callose deposition in transfer cells of the phloem. To further understand the roles of tocopherols in LT induced TCW development we compared the global transcript profiles of vte2 and wild-type leaves during LT treatment. Tocopherol deficiency had no significant impact on global gene expression in permissive conditions, but significantly affected expression of 77 genes after 48 h of LT treatment. In vte2 relative to wild type, genes associated with solute transport were repressed, while those involved in various pathogen responses and cell wall modifications, including two members of callose synthase gene family, GLUCAN SYNTHASE LIKE 4 (GSL4) and GSL11, were induced. However, introduction of gsl4 or gsl11 mutations individually into the vte2 background did not suppress callose deposition or the overall LT-induced phenotypes of vte2. Intriguingly, introduction of a mutation disrupting GSL5, the major GSL responsible for pathogen-induced callose deposition, into vte2 substantially reduced vascular callose deposition at LT, but again had no effect on the photoassimilate export phenotype of LT-treated vte2. These results suggest that GSL5 plays a major role in TCW callose deposition in LT-treated vte2 but that this GSL5-dependent callose deposition is not the primary cause of the impaired photoassimilate export phenotype

A foundation for provitamin A biofortification of maize: genome-wide association and genomic prediction models of carotenoid levels.

Efforts are underway for development of crops with improved levels of provitamin A carotenoids to help combat dietary vitamin A deficiency. As a global staple crop with considerable variation in kernel carotenoid composition, maize (Zea mays L.) could have a widespread impact. We performed a genome-wide association study (GWAS) of quantified seed carotenoids across a panel of maize inbreds ranging from light yellow to dark orange in grain color to identify some of the key genes controlling maize grain carotenoid composition. Significant associations at the genome-wide level were detected within the coding regions of zep1 and lut1, carotenoid biosynthetic genes not previously shown to impact grain carotenoid composition in association studies, as well as within previously associated lcyE and crtRB1 genes. We leveraged existing biochemical and genomic information to identify 58 a priori candidate genes relevant to the biosynthesis and retention of carotenoids in maize to test in a pathway-level analysis. This revealed dxs2 and lut5, genes not previously associated with kernel carotenoids. In genomic prediction models, use of markers that targeted a small set of quantitative trait loci associated with carotenoid levels in prior linkage studies were as effective as genome-wide markers for predicting carotenoid traits. Based on GWAS, pathway-level analysis, and genomic prediction studies, we outline a flexible strategy involving use of a small number of genes that can be selected for rapid conversion of elite white grain germplasm, with minimal amounts of carotenoids, to orange grain versions containing high levels of provitamin A

Rare genetic variation at Zea mays crtRB1 increases β-carotene in maize grain

Breeding to increase β-carotene levels in cereal grains, termed provitamin A biofortification, is an economical approach to address dietary vitamin A deficiency in the developing world. Experimental evidence from association and linkage populations in maize (Zea maysL.) demonstrate that the gene encoding β-carotene hydroxylase 1 (crtRB1) underlies a principal quantitative trait locus associated with β-carotene concentration and conversion in maize kernels. crtRB1 alleles associated with reduced transcript expression correlate with higher β-carotene concentrations. Genetic variation at crtRB1 also affects hydroxylation efficiency among encoded allozymes, as observed by resultant carotenoid profiles in recombinant expression assays. The most favorable crtRB1 alleles, rare in frequency and unique to temperate germplasm, are being introgressed via inexpensive PCR marker-assisted selection into tropical maize germplasm adapted to developing countries, where it is most needed for human health

Functional Identification of Valerena-1,10-diene Synthase, a Terpene Synthase Catalyzing a Unique Chemical Cascade in the Biosynthesis of Biologically Active Sesquiterpenes in Valeriana officinalis

Valerian is an herbal preparation from the roots of Valeriana officinalis used as an anxiolytic and sedative and in the treatment of insomnia. The biological activities of valerian are attributed to valerenic acid and its putative biosynthetic precursor valerenadiene, sesquiterpenes, found in V. officinalis roots. These sesquiterpenes retain an isobutenyl side chain whose origin has been long recognized as enigmatic because a chemical rationalization for their biosynthesis has not been obvious. Using recently developed metabolomic and transcriptomic resources, we identified seven V. officinalis terpene synthase genes (VoTPSs), two that were functionally characterized as monoterpene synthases and three that preferred farnesyl diphosphate, the substrate for sesquiterpene synthases. The reaction products for two of the sesquiterpene synthases exhibiting root-specific expression were characterized by a combination of GC-MS and NMR in comparison to the terpenes accumulating in planta. VoTPS7 encodes for a synthase that biosynthesizes predominately germacrene C, whereas VoTPS1 catalyzes the conversion of farnesyl diphosphate to valerena-1,10-diene. Using a yeast expression system, specific labeled [13C]acetate, and NMR, we investigated the catalytic mechanism for VoTPS1 and provide evidence for the involvement of a caryophyllenyl carbocation, a cyclobutyl intermediate, in the biosynthesis of valerena-1,10-diene. We suggest a similar mechanism for the biosynthesis of several other biologically related isobutenyl-containing sesquiterpenes

Characterization of three members of the Arabidopsis carotenoid cleavage dioxygenase family demonstrates the divergent roles of this multifunctional enzyme family

Arabidopsis thaliana has nine genes that constitute a family of putative carotenoid cleavage dioxygenases (CCDs). While five members of the family are believed to be involved in synthesis of the phytohormone abscisic acid, the functions of the other four enzymes are less clear. Recently two of the enzymes, CCD7/MAX3 and CCD8/MAX4, have been implicated in synthesis of a novel apocarotenoid hormone that controls lateral shoot growth. Here, we report on the molecular and genetic interactions between CCD1, CCD7/MAX3 and CCD8/ MAX4. CCD1 distinguishes itself from other reported CCDs as being the only member not targeted to the plastid. Unlike ccd7/max3 and ccd8/max4, both characterized as having highly branched phenotypes, ccd1 loss-of-function mutants are indistinguishable from wild-type plants. Thus, even though CCD1 has similar enzymatic activity to CCD7/MAX3, it does not have a role in synthesis of the lateral shoot growth inhibitor. Rather, it may have a role in synthesis of apocarotenoid flavor and aroma volatiles, especially in maturing seeds where loss of function leads to significantly higher carotenoid levels

Homogentisate Phytyltransferase Activity Is Limiting for Tocopherol Biosynthesis in Arabidopsis

Tocopherols are essential components of the human diet and are synthesized exclusively by photosynthetic organisms. These lipophilic antioxidants consist of a chromanol ring and a 15-carbon tail derived from homogentisate (HGA) and phytyl diphosphate, respectively. Condensation of HGA and phytyl diphosphate, the committed step in tocopherol biosynthesis, is catalyzed by HGA phytyltransferase (HPT). To investigate whether HPT activity is limiting for tocopherol synthesis in plants, the gene encoding Arabidopsis HPT, HPT1, was constitutively overexpressed in Arabidopsis. In leaves, HPT1 overexpression resulted in a 10-fold increase in HPT specific activity and a 4.4-fold increase in total tocopherol content relative to wild type. In seeds, HPT1 overexpression resulted in a 4-fold increase in HPT specific activity and a total seed tocopherol content that was 40% higher than wild type, primarily because of an increase in γ-tocopherol content. This enlarged pool of γ-tocopherol was almost entirely converted to α-tocopherol by crossing HPT1 overexpressing plants with lines constitutively overexpressing γ-tocopherol methyltransferase. Seed of the resulting double overexpressing lines had a 12-fold increase in vitamin E activity relative to wild type. These results indicate that HPT activity is limiting in various Arabidopsis tissues and that total tocopherol levels and vitamin E activity can be elevated in leaves and seeds by combined overexpression of the HPT1 and γ-tocopherol methyltransferase genes