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Subfunctionalization of the Ruby2–Ruby1 gene cluster during the domestication of citrus

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The evolution of fruit colour in plants is intriguing. Citrus fruit has repeatedly gained or lost the ability to synthesize anthocyanins. Chinese box orange, a primitive citrus, can accumulate anthocyanins both in its fruits and its leaves. Wild citrus can accumulate anthocyanins in its leaves. In contrast, most cultivated citrus have lost the ability to accumulate anthocyanins. We characterized a novel MYB regulatory gene, Ruby2, which is adjacent to Ruby1, a known anthocyanin activator of citrus. Different Ruby2 alleles can have opposite effects on the regulation of anthocyanin biosynthesis. AbRuby2Full encodes an anthocyanin activator that mainly functions in the pigmented leaves of Chinese box orange. CgRuby2Short was identified in purple pummelo and encodes an anthocyanin repressor. CgRuby2Short has lost the ability to activate anthocyanin biosynthesis. However, it retains the ability to interact with the same partner, CgbHLH1, as CgRuby1, thus acting as a passive competitor in the regulatory complex. Further investigation in different citrus species indicated that the Ruby2–Ruby1 cluster exhibits subfunctionalization among primitive, wild and cultivated citrus. Our study elucidates the regulatory mechanism and evolutionary history of the Ruby2–Ruby1 cluster in citrus, which are unique and different from that found in Arabidopsis, grape or petunia. © 2018, The Author(s), under exclusive licence to Springer Nature Limited.
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Articles
https://doi.org/10.1038/s41477-018-0287-6
1Key Laboratory of Horticultural Plant Biology of Ministry of Education, Huazhong Agricultural University, Wuhan, China. 2Robert W. Holley Center for
Agriculture and Health, Cornell University, Ithaca, NY, USA. 3John Innes Centre, Norwich, United Kingdom. *e-mail: xuqiang@mail.hzau.edu.cn
Citrus fruits exhibit vivid colour, ranging from yellow to pink,
red or purple. Citrus crops frequently gain or lose the ability
to synthesize anthocyanins in different species during evo-
lution, thus providing an interesting system for the analysis of the
evolution of fruit colour. Citrus fruit trees, belonging to the subtribe
Citrinae, consist of diverse species ranging from primitive, wild to
cultivated citrus based on botanical characteristics and domestica-
tion states1. Chinese box orange (Atalantia buxifolia)1, a species of
primitive citrus, accumulates high levels of anthocyanins in its fruits
and its young leaves. Although most wild and a few cultivated cit-
rus species, including trifoliate orange (Poncirus trifoliata), Ichang
papeda (Citrus ichangensis), citron (C. medica), lemon (C. limon)
and wild pummelo (C. grandis), do not accumulate anthocyanins
in their fruits but temporally synthesize anthocyanins in pigmented
leaves, some primitive citrus species and most cultivated citrus,
including mandarin (C. reticulata), sweet orange (C. sinensis) and
grapefruit (C. paradisi), do not accumulate anthocyanins in either
their fruits or leaves (Fig. 1).
Two unusual citrus accessions, blood orange and purple pum-
melo, do accumulate anthocyanins in fruit. Blood orange can accu-
mulate anthocyanins due to the insertion of a transposable element
and activation of the CsRuby1 gene, encoding a MYB transcription
factor that serves as the key positive regulator of anthocyanin bio-
synthesis2. Key polymorphisms, including nucleotide substitutions,
deletions and insertions in the coding region and in the promoter
of Ruby1, have been characterized in Citrus and related genera3.
Purple pummelo is a wild germplasm that was recently identified
from a mountain area in the Enshi region of Hubei Province, China.
Purple pummelo accumulates anthocyanins in the fruit peel and
pigmented leaves. The mechanism responsible for purple fruit in
pummelo is unknown.
Anthocyanins are synthesized as part of the flavonoid branch
within the phenylpropanoid pathway4. The regulation of anthocyanin
biosynthesis at the transcriptional level by the MYB-bHLH-WD40
(MBW) complex, formed by MYB, basic helix-loop-helix (bHLH)
and WD40 proteins, has been clearly elucidated57. The MBW tran-
scriptional regulators are conserved in eudicots8. The critical regu-
lators are MYB transcription factors, which have been identified
in various fruit crops, including grape9, apple10, pear11 and peach12.
Methylation of the key regulatory MYB gene may also be involved
in the regulation of anthocyanin biosynthesis. Hypermethylation in
the promoter of MdMYB10 causes striped pigmentation of ‘Honey
Crisp’ apple fruit13. In citrus, most cultivated varieties do not accu-
mulate anthocyanins and are associated with the presence of Ruby1
alleles that are predicted to be nonfunctional3.
Here, we demonstrate the function and evolutionary history of
a novel MYB regulatory gene, Ruby2, and a known transcriptional
activator, Ruby1, during the domestication of anthocyanin trait in
citrus. Two alleles, CgRuby2Short and AbRuby2Full, identified in pur-
ple pummelo and Chinese box orange, respectively, show opposite
effects in the regulation of anthocyanin biosynthesis. Sequence and
gene expression pattern of Ruby2Ruby1 cluster were investigated
in the primitive, wild and cultivated citrus.
Results
Identification of the natural ‘purple pummelo’ germplasm
and an active Ruby1 allele. Pummelo (C. grandis) is a true spe-
cies of Citrus. It has been cultivated for about 4,000 years in China,
Thailand and other Southeast Asian countries. Pummelo provided
genetic contributions to most citrus cultivars, including sweet and
sour orange, lemon and grapefruit. Several cultivated mandarins
also showed different patterns of pummelo introgression1416. Most
cultivated citrus do not synthesize anthocyanins except for lemon
and a few landraces that accumulate anthocyanins in pigmented
leaves (Fig. 1). None of the cultivated pummelo accumulate antho-
cyanins in fruit. A wild germplasm, called ‘purple pummelo’ in this
study, was discovered in a mountainous area in the Enshi region
of Hubei Province, China. The fruit peel of the purple pummelo
Subfunctionalization of the Ruby2Ruby1 gene
cluster during the domestication of citrus
DingHuang1, XiaWang1, ZhouzhouTang1, YueYuan1, YuantaoXu1, JiaxianHe1, XiaolinJiang1,
Shu-AngPeng1, LiLi2, EugenioButelli3, XiuxinDeng 1 and QiangXu 1*
The evolution of fruit colour in plants is intriguing. Citrus fruit has repeatedly gained or lost the ability to synthesize antho-
cyanins. Chinese box orange, a primitive citrus, can accumulate anthocyanins both in its fruits and its leaves. Wild citrus can
accumulate anthocyanins in its leaves. In contrast, most cultivated citrus have lost the ability to accumulate anthocyanins.
We characterized a novel MYB regulatory gene, Ruby2, which is adjacent to Ruby1, a known anthocyanin activator of citrus.
Different Ruby2 alleles can have opposite effects on the regulation of anthocyanin biosynthesis. AbRuby2Full encodes an antho-
cyanin activator that mainly functions in the pigmented leaves of Chinese box orange. CgRuby2Short was identified in purple pum-
melo and encodes an anthocyanin repressor. CgRuby2Short has lost the ability to activate anthocyanin biosynthesis. However, it
retains the ability to interact with the same partner, CgbHLH1, as CgRuby1, thus acting as a passive competitor in the regulatory
complex. Further investigation in different citrus species indicated that the Ruby2Ruby1 cluster exhibits subfunctionalization
among primitive, wild and cultivated citrus. Our study elucidates the regulatory mechanism and evolutionary history of the
Ruby2Ruby1 cluster in citrus, which are unique and different from that found in Arabidopsis, grape or petunia.
NATURE PLANTS | VOL 4 | NOVEMBER 2018 | 930–941 | www.nature.com/natureplants
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Transposable elements (TEs) are an important source for generating small interfering RNAs (siRNAs) in plants and animals. Although TE-siRNA-induced silencing of TEs by RNA-directed DNA methylation (RdDM) in the maintenance of genome integrity has been intensively studied, it is unknown whether this type of silencing occurs in suppressing endogenous non-TE genes during host–pathogen interactions. Here we show that a TE-siRNA, TE-siR815, causes opposite functions for the two alleles, WRKY45-1 and WRKY45-2, of the WRKY45 transcription factor in rice resistance to Xanthomonas oryzae pv. oryzae, which causes the most devastating bacterial disease in rice worldwide. Expression of WRKY45-1, but not WRKY45-2, generated TE-siR815, which in turn repressed ST1, an important component in WRKY45-mediated resistance, by RdDM. Suppression of ST1 abolished WRKY45-mediated resistance leading to pathogen susceptibility. These results suggest that TE-siR815 contributes to the natural variation of the WRKY45 locus and TE-siR815-induced suppression of ST1 results in the negative role of WRKY45-1 but positive role of WRKY45-2 in regulating disease resistance.
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Accumulation of anthocyanins and proanthocyanidins (PAs) is limited to specific cell types and developmental stages, but little is known about how antagonistically acting transcriptional regulators work together to determine temporal and spatial patterning of pigmentation at the cellular level, especially for PAs. Here, we characterize MYB2, a transcriptional repressor regulating both anthocyanin and PA biosynthesis in the model legume Medicago truncatula. MYB2 was strongly upregulated by MYB5, a major regulator of PA biosynthesis in M. truncatula and a component of MYB-basic helix loop helix-WD40 (MBW) activator complexes. Overexpression of MYB2 abolished anthocyanin and PA accumulation in M. truncatula hairy roots and Arabidopsis thaliana seeds, respectively. Anthocyanin deposition was expanded in myb2 mutant seedlings and flowers accompanied by increased anthocyanin content. PA mainly accumulated in the epidermal layer derived from the outer integument in the M. truncatula seed coat, starting from the hilum area. The area of PA accumulation and ANTHOCYANIDIN REDUCTASE expression was expanded into the seed body at the early stage of seed development in the myb2 mutant. Genetic, biochemical, and cell biological evidence suggests that MYB2 functions as part of a multidimensional regulatory network to define the temporal and spatial pattern of anthocyanin and PA accumulation linked to developmental processes.