Backgroundβ-carotene, the most active provitamin A molecule produced by plants, plays important roles in human nutrition and health. β-carotene does not usually accumulate in the endosperm (i.e. flour) of mature wheat grains, which is a major food source of calories for humans. Therefore, enriching β-carotene accumulation in wheat grain endosperm will enable a sustainable dietary supplementation of provitamin A. Several metabolic genes affecting β-carotene accumulation have already been isolated from wheat, including phytoene synthase 1 (PSY1), lycopene ε-cyclase (LCYe) and carotenoid β-ring hydroxylase1/2 (HYD1/2).ResultsIn this work, we cloned and biochemically characterized two carotenoid cleavage dioxygenases (CCDs), CCD1 and CCD4, from wheat. While CCD1 homoeologs cleaved β-apo-8′-carotenal, β-carotene, lutein and zeaxanthin into apocarotenoid products, CCD4 homoeologs were inactive towards these substrates in in vitro assays. When analyzed by real-time qPCR, PSY1, LCYe, HYD1/2 and CCD1/4 homoeologs showed distinct expression patterns in vegetative tissues and sections of developing tetraploid and hexaploid wheat grains, suggesting that carotenoid metabolic genes and homoeologs are differentially regulated at the transcriptional level in wheat.ConclusionsThe CCD1/4 enzyme activity and the spatial-temporal gene expression data provide critical insights into the specific carotenoid metabolic gene homoeologs that control β-carotene accumulation in wheat grain endosperm, thus establishing the knowledge base for generation of wheat varieties with enhanced β-carotene in the endosperm through breeding and genome editing approaches.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0848-7) contains supplementary material, which is available to authorized users.
Griffithsin (Grft) is an antiviral lectin that has been shown to potently inhibit HIV-1 by binding high-mannose N-linked glycosylation sites on HIV-1 gp120. A key factor for Grft potency is glycosylation at N295 of gp120, which is directly adjacent to N332, a target glycan for an entire class of broadly neutralizing antibodies (bNAbs). Here, we unify previous work on the importance of other glycans to Grft potency against HIV-1 and Grft’s role in mediating the conformational change of gp120 by mutating nearly every glycosylation site in gp120. In addition to a significant loss of Grft activity by the removal of glycosylation at N295, glycan absence at N332 or N448 was found to have moderate effects on Grft potency. Interestingly, in the absence of N295, Grft effectiveness could be improved by a mutation that results in the glycan at N448 shifting to N446, indicating that the importance of individual glycans may be related to their effect on glycosylation density. Grft’s ability to alter the structure of gp120, exposing the CD4 binding site, correlated with the presence of glycosylation at N295 only in clade B strains, not clade C strains. We further demonstrate that Grft can rescue the activity of the bNAbs PGT121 and PGT126 in the event of a loss or a shift of glycosylation at N332, where the bNAbs suffer a drastic loss of potency. Despite targeting the same region, Grft in combination with PGT121 and PGT126 produced additive effects. This indicates that Grft could be an important combinational therapeutic.
Phytoene synthase (PSY) catalyzes a key reaction in the biosynthesis of carotenoids, which are major contributors of grain yellow pigment content (GYPC) in wheat (Triticum spp.). Although quantitative trait loci (QTL) for GYPC have been linked to PSY1 homoeologs in tetraploid and hexaploid wheat, the underlying molecular and biochemical mechanisms for such associations are not well understood. Functional characterization of wheat PSY1 homoeologs indicated that variations in PSY1 homoeolog enzyme activities did not correspond to different levels of GYPC in the wild‐type tetraploid and hexaploid wheat genotypes analyzed. However, a correlation between PSY1 homoeolog expression and carotenoid accumulation in grain endosperm was evident, suggesting that GYPC is at least partially modulated by transcriptional regulation of PSY1 homoeologs in wheat grains. Further promoter analysis of PSY‐A1 homoeologs with diverse expression profiles in three wheat genotypes revealed not only potential motifs for light and hormonal responses but also new candidate cis‐regulatory elements. Overall, the knowledge on the association of PSY1 homoeolog expression and GYPC can be directly applied to breeding tetraploid and hexaploid wheat varieties with desirable carotenoid levels.
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