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      Map-based cloning and functional analysis of YGL8, which controls leaf colour in rice ( Oryza sativa)

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          Abstract

          Background

          As the indispensable part of plant, leaf blade mainly functions as the production workshops where organic substance is produced by photosynthesis. Leaf colour mutation is a genetic phenomenon that has a high frequency and is easily identified. The mutations always exhibit negative impact on the development of plants in any of the different stages of growth. Up to now, numerous genes involved in leaf colour mutations have been cloned.

          Results

          In this study, a yellow-green leaf mutant, yellow-green leaf 8 ( ygl8), with stable genetic phenotype, has been screened out in the progeny of an excellent indica restorer line Jinhui 10 with seeds treated by EMS. The levels of Chl a, Chl b and total chlorophyll were significantly lower in ygl8 than those in the WT throughout the whole growth period, while no clear change was noted in the Chl a/ b ratio. Transmission electron microscopy demonstrated that the lamellae were clearly intumescent and intricately stacked in ygl8. Furthermore, compared with those of the WT, the stomatal conductance, intercellular CO 2 concentration, photosynthetic rate and transpiration rate of ylg8 were all significantly lower. Map-based cloning results showed that Loc_Os01g73450, encoding a chloroplast-targeted UMP kinase, corresponded to Ygl8 and played an important role in regulating leaf colour in rice ( Oryza sativa). Complementation of ygl8 with the WT DNA sequence of Loc_Os01g73450 led to restoration of the normal phenotype, and transgenic RNA interference plants showed a yellow-green colour. Analysis of the spatial and temporal expression of Ygl8 indicated that it was highly expressed in leaf blades and weakly expressed in other tissues. qRT-PCR also showed that the expression levels of the major Photosystem I core subunits plastome-encoded PsaA, PsaB and PsbC were significantly reduced in ygl8. The expression levels of nuclear-encoded gene involved in Chl biosynthesis HEMC, HEME, and PORA were also decreased when compared with the wild-type.

          Conclusions

          Independent of Chl biosynthesis and photosystem, YGL8 may affect the structure and function of chloroplasts grana lamellae by regulating plastid genome encoded thylakoid membrane constitutive gene expression and indirectly influences Chl biosynthesis.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s12870-016-0821-5) contains supplementary material, which is available to authorized users.

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          Most cited references20

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          GUN4, a regulator of chlorophyll synthesis and intracellular signaling.

          Nuclear genes control plastid differentiation in response to developmental signals, environmental signals, and retrograde signals from plastids themselves. In return, plastids emit signals that are essential for proper expression of many nuclear photosynthetic genes. Accumulation of magnesium-protoporphyrin IX (Mg-Proto), an intermediate in chlorophyll biosynthesis, is a plastid signal that represses nuclear transcription through a signaling pathway that, in Arabidopsis, requires the GUN4 gene. GUN4 binds the product and substrate of Mg- chelatase, an enzyme that produces Mg-Proto, and activates Mg-chelatase. Thus, GUN4 participates in plastid-to-nucleus signaling by regulating Mg-Proto synthesis or trafficking.
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            A chlorophyll-deficient rice mutant with impaired chlorophyllide esterification in chlorophyll biosynthesis.

            Chlorophyll (Chl) synthase catalyzes esterification of chlorophyllide to complete the last step of Chl biosynthesis. Although the Chl synthases and the corresponding genes from various organisms have been well characterized, Chl synthase mutants have not yet been reported in higher plants. In this study, a rice (Oryza Sativa) Chl-deficient mutant, yellow-green leaf1 (ygl1), was isolated, which showed yellow-green leaves in young plants with decreased Chl synthesis, increased level of tetrapyrrole intermediates, and delayed chloroplast development. Genetic analysis demonstrated that the phenotype of ygl1 was caused by a recessive mutation in a nuclear gene. The ygl1 locus was mapped to chromosome 5 and isolated by map-based cloning. Sequence analysis revealed that it encodes the Chl synthase and its identity was verified by transgenic complementation. A missense mutation was found in a highly conserved residue of YGL1 in the ygl1 mutant, resulting in reduction of the enzymatic activity. YGL1 is constitutively expressed in all tissues, and its expression is not significantly affected in the ygl1 mutant. Interestingly, the mRNA expression of the cab1R gene encoding the Chl a/b-binding protein was severely suppressed in the ygl1 mutant. Moreover, the expression of some nuclear genes associated with Chl biosynthesis or chloroplast development was also affected in ygl1 seedlings. These results indicate that the expression of nuclear genes encoding various chloroplast proteins might be feedback regulated by the level of Chl or Chl precursors.
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              Rice Chlorina-1 and Chlorina-9 encode ChlD and ChlI subunits of Mg-chelatase, a key enzyme for chlorophyll synthesis and chloroplast development.

              Photosynthetic organisms exhibit a green color due to the accumulation of chlorophyll pigments in chloroplasts. Mg-protoporphyrin IX chelatase (Mg-chelatase) comprises three subunits (ChlH, ChlD and ChlI) and catalyzes the insertion of Mg(2+) into protoporphyrin IX, the last common intermediate precursor in both chlorophyll and heme biosyntheses, to produce Mg-protoporphyrin IX (MgProto). Chlorophyll deficiency in higher plants results in chlorina (yellowish-green) phenotype. To date, 10 chlorina (chl) mutants have been isolated in rice, but the corresponding genes have not yet been identified. Rice Chl1 and Chl9 genes were mapped to chromosome 3 and isolated by map-based cloning. A missense mutation occurred in a highly conserved amino acid of ChlD in the chl1 mutant and ChlI in the chl9 mutant. Ultrastructural analyses have revealed that the grana are poorly stacked, resulting in the underdevelopment of chloroplasts. In the seedlings fed with aminolevulinate-dipyridyl in darkness, MgProto levels in the chl1 and chl9 mutants decreased up to 25% and 31% of that in wild-type, respectively, indicating that the Mg-chelatase activity is significantly reduced, causing the eventual decrease in chlorophyll synthesis. Furthermore, Northern blot analysis indicated that the nuclear genes encoding the three subunits of Mg-chelatase and LhcpII in chl1 mutant are expressed about 2-fold higher than those in WT, but are not altered in the chl9 mutant. This result indicates that the ChlD subunit participates in negative feedback regulation of plastid-to-nucleus in the expression of nuclear genes encoding chloroplast proteins, but not the ChlI subunit.
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                Author and article information

                Contributors
                hegh@swu.edu.cn
                Journal
                BMC Plant Biol
                BMC Plant Biol
                BMC Plant Biology
                BioMed Central (London )
                1471-2229
                13 June 2016
                13 June 2016
                2016
                : 16
                : 134
                Affiliations
                [ ]Key Laboratory of Application and Safety Control of Genetically Modified Crops, Rice Research Institute of Southwest University, Chongqing, 400716 China
                [ ]Institute of Rice, Chongqing Academy of Agricultural Sciences, Chongqing, 401329 China
                [ ]Forestry Bureau of Chuanshan District, Suining, Sichuan 629000 China
                Article
                821
                10.1186/s12870-016-0821-5
                4907030
                27297403
                d453677b-9584-4fcb-8ab4-96220a5373c6
                © 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
                : 13 December 2015
                : 25 May 2016
                Funding
                Funded by: Natural Science Foundation of China
                Award ID: 31171178
                Award Recipient :
                Funded by: The Application Development Key Project of Chongqing
                Award ID: cstc2014yykfB80009
                Funded by: the Ministry of Agriculture’s Public Welfare Industry Scientifci Research Special Project
                Award ID: 201303129
                Award Recipient :
                Funded by: the Ability Improvment Project of Chongqing
                Award ID: cstc2014pt-sy80001
                Award Recipient :
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2016

                Plant science & Botany
                rice (oryza sativa),yellow-green leaf 8 (ygl8),map-based cloning,functional analysis

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