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      Distinct expression and function of carotenoid metabolic genes and homoeologs in developing wheat grains

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          Abstract

          Background

          β-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).

          Results

          In 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.

          Conclusions

          The 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 material

          The online version of this article (doi:10.1186/s12870-016-0848-7) contains supplementary material, which is available to authorized users.

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          Most cited references50

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          Genome plasticity a key factor in the success of polyploid wheat under domestication.

          Wheat was domesticated about 10,000 years ago and has since spread worldwide to become one of the major crops. Its adaptability to diverse environments and end uses is surprising given the diversity bottlenecks expected from recent domestication and polyploid speciation events. Wheat compensates for these bottlenecks by capturing part of the genetic diversity of its progenitors and by generating new diversity at a relatively fast pace. Frequent gene deletions and disruptions generated by a fast replacement rate of repetitive sequences are buffered by the polyploid nature of wheat, resulting in subtle dosage effects on which selection can operate.
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            The path from β-carotene to carlactone, a strigolactone-like plant hormone.

            Strigolactones, phytohormones with diverse signaling activities, have a common structure consisting of two lactones connected by an enol-ether bridge. Strigolactones derive from carotenoids via a pathway involving the carotenoid cleavage dioxygenases 7 and 8 (CCD7 and CCD8) and the iron-binding protein D27. We show that D27 is a β-carotene isomerase that converts all-trans-β-carotene into 9-cis-β-carotene, which is cleaved by CCD7 into a 9-cis-configured aldehyde. CCD8 incorporates three oxygens into 9-cis-β-apo-10'-carotenal and performs molecular rearrangement, linking carotenoids with strigolactones and producing carlactone, a compound with strigolactone-like biological activities. Knowledge of the structure of carlactone will be crucial for understanding the biology of strigolactones and may have applications in combating parasitic weeds.
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              Identification and validation of reference genes for quantitative RT-PCR normalization in wheat

              Background Usually the reference genes used in gene expression analysis have been chosen for their known or suspected housekeeping roles, however the variation observed in most of them hinders their effective use. The assessed lack of validated reference genes emphasizes the importance of a systematic study for their identification. For selecting candidate reference genes we have developed a simple in silico method based on the data publicly available in the wheat databases Unigene and TIGR. Results The expression stability of 32 genes was assessed by qRT-PCR using a set of cDNAs from 24 different plant samples, which included different tissues, developmental stages and temperature stresses. The selected sequences included 12 well-known HKGs representing different functional classes and 20 genes novel with reference to the normalization issue. The expression stability of the 32 candidate genes was tested by the computer programs geNorm and NormFinder using five different data-sets. Some discrepancies were detected in the ranking of the candidate reference genes, but there was substantial agreement between the groups of genes with the most and least stable expression. Three new identified reference genes appear more effective than the well-known and frequently used HKGs to normalize gene expression in wheat. Finally, the expression study of a gene encoding a PDI-like protein showed that its correct evaluation relies on the adoption of suitable normalization genes and can be negatively affected by the use of traditional HKGs with unstable expression, such as actin and α-tubulin. Conclusion The present research represents the first wide screening aimed to the identification of reference genes and of the corresponding primer pairs specifically designed for gene expression studies in wheat, in particular for qRT-PCR analyses. Several of the new identified reference genes outperformed the traditional HKGs in terms of expression stability under all the tested conditions. The new reference genes will enable more accurate normalization and quantification of gene expression in wheat and will be helpful for designing primer pairs targeting orthologous genes in other plant species.
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                Author and article information

                Contributors
                qin@ucdavis.edu
                kfischer4@ucmerced.edu
                shuyu@ucdavis.edu
                jdubcovsky@ucdavis.edu
                (530) 752-0940 , ltian@ucdavis.edu
                Journal
                BMC Plant Biol
                BMC Plant Biol
                BMC Plant Biology
                BioMed Central (London )
                1471-2229
                12 July 2016
                12 July 2016
                2016
                : 16
                : 155
                Affiliations
                [ ]Department of Plant Sciences, Mail Stop 3, University of California, Davis, CA 95616 USA
                [ ]Quantitative and Systems Biology Program, University of California, Merced, CA 95343 USA
                [ ]Howard Hughes Medical Institute, Chevy Chase, MD 20815 USA
                [ ]Shanghai Key Laboratory of Plant Functional Genomics and Resources, Shanghai Chenshan Botanical Garden, Shanghai, 201602 China
                [ ]Shanghai Chenshan Plant Science Research Center, Chinese Academy of Sciences, Shanghai, 201602 China
                Author information
                http://orcid.org/0000-0001-6461-6072
                Article
                848
                10.1186/s12870-016-0848-7
                4943016
                27405473
                af7d3982-0d7e-47c3-916a-f484c49e379b
                © The Author(s). 2016

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 23 April 2016
                : 7 July 2016
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2016

                Plant science & Botany
                β-carotene,carotenoid,carotenoid cleavage dioxygenase,endosperm,grain,provitamin a,spatial expression,wheat

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