A Tetratricopeptide Repeat Protein Regulates Carotenoid Biosynthesis and
Chromoplast Development in Monkeyflowers (Mimulus)
Lauren E. Stanley
1*
, Baoqing Ding
1
, Wei Sun
2
, Fengjuan Mou
1,3
, Connor Hill
1
,
Shilin Chen
2
, Yao-Wu Yuan
1,4*
1
Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, CT
06269, USA.
2
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences,
Beijing 100700, China.
3
Faculty of Forestry, Southwest Forestry University, Kunming, Yunnan 650224, China
4
Institute for Systems Genomics, University of Connecticut, Storrs, CT 06269, USA
*Corresponding authors: lauren.stanley@uconn.edu; yuan.colreeze@gmail.com
Short title: A TPR Protein Regulates Carotenoid Pigmentation
One-sentence summary: Mutant analyses and transgenic experiments in monkeyflowers
(Mimulus) identify a tetratricopeptide repeat protein required for chromoplast
development and carotenoid biosynthesis.
Keywords: Carotenoid pigmentation, chromoplast development, TPR Protein, retrograde
signaling, Mimulus
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ABSTRACT
The incredible diversity of floral color and pattern in nature is largely determined by the
transcriptional regulation of anthocyanin and carotenoid biosynthetic genes. While the
transcriptional control of anthocyanin biosynthesis is well understood, little is known
about the factors regulating the carotenoid biosynthetic pathway in flowers. Here, we
characterize the Reduced Carotenoid Pigmentation 2 (RCP2) locus from two
monkeyflower (Mimulus) species, the bumblebee-pollinated M. lewisii and hummingbird-
pollinated M. verbenaceus. We show that loss-of-function mutations of RCP2 cause
drastic down-regulation of the entire carotenoid biosynthetic pathway in these species.
Through bulk segregant analysis and transgenic experiments, we have identified the
causal gene underlying RCP2, encoding a tetratricopeptide repeat (TPR) protein that is
closely related to the Arabidopsis Reduced Chloroplast Coverage (REC) proteins. RCP2
appears to regulate carotenoid biosynthesis independently of RCP1, a previously
identified R2R3-MYB master regulator of carotenoid biosynthesis. We show that RCP2
is required for chromoplast development and suggest that it most likely regulates the
expression of carotenoid biosynthetic genes through chromoplast-to-nucleus retrograde
signaling. Furthermore, we demonstrate that M. verbenaceus is just as amenable to
chemical mutagenesis and in planta transformation as the more extensively studied M.
lewisii, making these two species an excellent platform for comparative developmental
genetics studies of two closely related species with dramatic phenotypic divergence.
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INTRODUCTION
Most flowers are colored by two classes of pigments: the red, pink, purple, or blue
anthocyanins, and the yellow, orange, or red carotenoids. The hydrophilic anthocyanins
are usually stored in the vacuoles of petal cells, whereas the hydrophobic carotenoids
accumulate in chromoplasts as various lipoprotein structures (e.g., plastoglobules,
crystals, fibrils). Frequently a plant can produce both pigment types in the same flower,
forming contrasting spatial patterns that serve as nectar guides for animal pollinators
(Glover, 2014). Common examples among horticultural plants include pansies,
primroses, lantanas, and hibiscus, to name but a few. As an example in nature, the vast
majority of the ~160 species of monkeyflowers (Mimulus) (Barker et al., 2012) produce
both anthocyanins and carotenoids in their petals with striking patterns. These
observations indicate that many plant genomes contain a full set of functional genes
encoding the anthocyanin and carotenoid biosynthetic pathways. The tremendous
diversity of floral pigmentation pattern, then, is largely due to when and where these
pathway genes are expressed. As such, identifying the transcriptional regulators of these
pigment biosynthetic pathways are critically important to understanding the
developmental mechanisms of pigment pattern formation and the molecular bases of
flower color variation.
The transcriptional control of anthocyanin biosynthesis is well understood. A
highly conserved MYB-bHLH-WD40 (MBW) protein complex has been shown to
coordinately activate all or some of the anthocyanin biosynthetic pathway genes in
multiple plant systems (Paz-Ares et al., 1987; Ludwig et al., 1989; Martin et al., 1991;
Goodrich et al., 1992; de Vetten et al., 1997; Quattrocchio et al., 1998; Borevitz et al.,
2000; Spelt et al., 2000; Schwinn et al., 2006; reviewed in Davies et al., 2012; Glover,
2014). Among the three components, the R2R3-MYB often displays tissue-specific
expression and causes spatial patterning of anthocyanin deposition in flower petals
(Shang et al., 2011; Albert et al., 2011; Yuan et al., 2014; Martins et al., 2016). In
contrast, little is known about the transcriptional regulators of the carotenoid biosynthetic
pathway (CBP) (Ruiz-Sola & Rodríguez-Concepción, 2012; Yuan et al. 2015),
particularly in flowers.
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The best characterized transcriptional regulators of carotenoid biosynthesis are the
phytochrome-interacting factors (PIFs) in Arabidopsis (Toledo-Ortiz et al., 2010). PIFs
directly bind the promoter of the phytotene synthase (PSY) gene and repress its
expression in dark-grown seedlings. During deetiolation, light-triggered degradation of
PIFs leads to rapid derepression of PSY and massive production of carotenoids in the
greening seedlings. However, the significance of PIFs in regulating carotenoid
pigmentation in flowers is unclear. In fact, there are good reasons to suspect that PIFs do
not play an important role in flower pigmentation: several studies have shown that
carotenoid production during petal development involves coordinated activation of
multiple CBP genes (Giuliano et al., 1993; Moehs et al., 2001; Zhu et al., 2002; Chiou et
al., 2010; Yamagishi et al., 2010; Yamamizo et al., 2010), whereas PIFs regulate only
PSY but none of the other CPB genes in Arabidopsis (Toledo-Ortiz et al., 2010).
To identify transcriptional regulators of floral carotenoid pigmentation, we
employed a new genetic model system, the monkeyflower species Mimulus lewisii. The
ventral (lower) petal of M. lewisii flowers has two yellow ridges that are pigmented by
carotenoids (Figure 1A and 1B), serving as nectar guides for bumblebee pollinators
(Owen and Bradshaw, 2011). We carried out an ethyl-methanesulfonate (EMS) mutant
screen using the inbred line LF10 for reduced carotenoid pigmentation in the nectar
guides, and recovered several recessive mutants with coordinated down-regulation of the
entire CBP compared to the wild-type (Sagawa et al., 2016). Complementation crosses
suggested that these mutants represent two different loci, RCP1 (Reduced Carotenoid
Pigmentation 1) and RCP2. RCP1 encodes a subgroup-21 R2R3-MYB that is clearly
distinguishable from the anthocyanin-activating R2R3-MYBs (subgroup-6) and is
specifically expressed in the yellow nectar guides (Sagawa et al., 2016) in M. lewisii.
The primary goal of this study is to characterize the RCP2 locus. Through bulk
segregant analysis and transgenic experiments, we have identified the causal gene of
RCP2, encoding a tetratricopeptide repeat (TPR) protein homologous to the Reduced
Chloroplast Coverage (REC) proteins in Arabidopsis. Loss-of-function REC mutants
have reduced chlorophyll content and smaller chloroplast compartment size compared to
wild-type (Larkin et al., 2016). Our analyses show that RCP2 is required for chromoplast
development and carotenoid biosynthesis in the flowers of both M. lewisii and a close
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relative, M. verbenaceus. We suggest that the coordinated down-regulation of the
nuclear-encoded CBP genes in the loss-of-function rcp2 mutant likely results from
chromoplast-to-nucleus retrograde signaling, which appears to be independent of RCP1
function.
RESULTS
rcp2-1 displays a distinct and stronger phenotype than rcp1-1
We recovered three independent rcp2 alleles from the previous mutant screen. rcp2-1 and
rcp2-2 are indistinguishable phenotypically, whereas rcp2-3 displays a slightly weaker
phenotype (Supplemental Figure 1). Like the rcp1-1 mutant, rcp2-1 has reduced
carotenoid content in the nectar guides compared to the wild-type (Figures 1A and 1B).
However, rcp2-1 can be readily distinguished from rcp1-1 in two aspects. First, the total
carotenoid content in the nectar guides of rcp2-1 is ~10-fold lower than the wild-type
(Figure 1C), whereas rcp1-1 is only ~4.4-fold lower (Sagawa et al., 2016). Second, the
residual carotenoid pigments in rcp1-1 and rcp2-1 show distinct spatial distributions. At
the base of the corolla tube (Figure 1B, white boxes), carotenoid pigments are completely
lacking in rcp2-1 but present in rcp1-1. In contrast, at the throat of the corolla tube
(Figure 1B, red boxes), carotenoid pigments are completely lacking in rcp1-1 but present
at low concentration in rcp2-1, giving a cream color. These spatial distributions of
residual pigments are consistent among allelic mutants within each complementation
group (Supplemental Figure 1).
To test whether RCP2 regulates CBP genes at the transcriptional level, we
performed qRT-PCR experiments on nectar guide tissue at the 15-mm corolla
developmental stage (the stage at which the CBP genes have their highest expression; this
is the same stage used previously for RCP1). Compared to wild-type, the rcp2-1 mutant
showed a coordinated down-regulation of the entire CBP (Figure 1D), with a 3- to 4-fold
decrease in expression of most CBP genes and ~10-fold decrease in BCH1 expression.
These results suggest that RCP2 is involved in the transcriptional regulation of CBP
genes in the nectar guides. Consistent with the more severe reduction in total carotenoid
content, the extent of CBP gene down-regulation is stronger in rcp2-1 than in rcp1-1
(Sagawa et al., 2016).
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was notthis version posted August 1, 2017. ; https://doi.org/10.1101/171249doi: bioRxiv preprint