I’ve
been wondering about orchid colors and color patterns for the past few weeks,
in response to an educational program at my local American Orchid Society judging
center. I had noticed on the California Sierra Nevada Judging Center’s website
(
http://www.csnjc.org/Sacto2012/masdzieglersloveAQ.html)
that they had recently awarded an AQ to Masdevalia Ziegler’s Love, whereby
siblings of this grex had different colors and/or color patterns. This range of
color diversity seems fairly uncommon to me, which prompted me into doing some
research about variation in Cymbidium colors and patterns.
The
topics more commonly discussed amongst Cymbidium growers presently seems to be
directed to peloric or feathered flowers, and their perceived increasing
appearance in the modern hybrids.
Cymbidium (Psychotic x Valley Zenith)
‘Barbara Uno’, exhibited at the 2012 Santa Barbara Int'l Orchid Show
Cymbidium Mimi ‘SanBar Feathered’, exhibited at the 2012 Santa Barbara Int'l Orchid Show
But
the discussion below will be directed to the picotee pattern, whereby the
margins of the sepals, petals and/or lip have different color than the body of
these segments.
The
picotee phenotype has long been recognized in the horticulture industry. For
example, Gustav Mehlquist co-authored a research paper sixty years ago
discussing the genetics of flower color variation, including the picotee, in
carnations (Geissman and Mehlquist, 1947). The
picotee can also be seen in Cymbidium species, e.g. Cymbidium bicolor and Cymbidium canaliculatum (check out
www.orchidspecies.com) and some hybrids.
Cymbidium (Golden Celebration 'Pink Tinges' x Kirby Lesh 'Fay', HCC/ACS) by Ezi-Gro Orchids (photo taken in greenhouse of Hatfield
Orchids)
Cymbidium (Mighty Sensation x Paul Robeson), exhibited at the 2011 Santa Barbara Int'l Orchid Show.
If
you ask any orchid hybridizer, they will likely tell you that flower color is
an important consideration in consumer choice for flowers and ornamental
plants. There is great interest in cultivars bearing flowers with altered
color, hues and patterns. Understanding the mechanisms regulating flower
coloration should aid in the breeding of new cultivars. Picotee and bicolor
phenotypes are extremely desirable, and are some of the main targets in ornamental
flower breeding (Nakatsuka et al, 2011).
However,
becoming familiar with all of the genes involved in pigment biochemistry and
genetics is almost like learning how to play chess. There are a multitude of
genes, both regulatory and structural, whose individual spatially- and temporally-controlled
activity give rise to the final flower color patterns that stand out from the
crowd and catch our eyes. Learning the names of these players, how each gene functions,
and then using this information to enhance one or more traits in a hybridizing
scheme borders on the arcane.
Flower
color biochemistry and genetics has been studied in established plant model
organisms such as petunia, snapdragon, morning glory, gentian, lisianthus and
lily. Within orchids, Phalaenopsis is the leader for the development of
molecular genetic tools applied to flower color variation. Only two of the
known pigment genes have been cloned from Cymbidium: the chalcone synthase
(CHS) gene (GenBank EU045579.1; Cymbidium floribundum) and the dihydroflavonol
4-reductase (DFR) gene (GenBank AF017451.1; unnamed Cymbidium hybrid).
Flower
colors are made up of a combination of flavonols, flavones, and anthocyanins.
Flavonols and flavones are two common co-pigments often associated with
anthocyanins. They play an important role in determining flower color since
they can form complexes with anthocyanins. Most flavones and flavonols are
colorless; they appear to provide ‘body’ to white, cream and ivory-colored
flowers. Since flavonols and flavones share common precursors with
anthocyanins, down-regulation of flavonols/flavones often reduce anthocyanin
levels (To and Wang, 2006). Anthocyanins accumulate in vacuoles or
anthocyanoplasts. The colored anthocyanins can be further chemically modified
by glycosylation, acylation or methylation in a plant species-specific manner.
Spatio-temporal inactivation of Myb
In
many plant species, many flavonoid structural genes (the genes that encode the
enzymes that synthesize flavonoids and anthocyanins) are co-regulated by
transcription factor genes belonging to three gene families: Myb, bHLH, and WDR. Each gene family can have dozens of
representatives, e.g. Myb-1 to Myb-15, each with its own purpose, but which may
or may not functionally overlap with another Myb family member. Importantly,
Myb-bHLH-WDR protein complexes regulate not only anthocyanin biosynthesis but
also generate plant epidermal cellular diversity, which includes the different
cell types of the flower (Nakatsuka et al, 2008).
Transgenic
technology is being used not only to better understand mechanism of
pigmentation, but also to increase the diversity of flower colors. Nakatsuka et
al (2011) introduced an engineered transgene to silence the Myb3 locus, which
regulates the pigmentation of gentian flowers. Starting with a blue-flowered
gentian cultivar, Nakatsuka et al obtained ten stable, transgenic lines that
showed no color changes, but two lines that produced white-centered flowers
with a blue picotee, indicating that the Myb3 locus was silenced in the central
part of the flower but not along the petal margins. Nakatsuka et al showed that
expression of some anthocyanin-producing genes, e.g. flavanone 3-hydroxylase
(F3H), are significantly reduced in the central flower portion, but not along
margins, which is consistent with the known Myb3 activity. However, Nakatsuka
et al did not explain why the silencing construct, although expressed in petal
margins, does not reduce anthocyanin production along petal margins.
Yamagishi
(2011) described the spatial and temporal expression of Myb12 in the
‘Sorbonne’ lily cultivar.
(Google image search;
Myb12 expression is lower during the earlier and later stages of tepal development,
and highest during the middle stages. Myb12 expression is also highest in the
central tepal regions, with decreasing levels in the intermediate regions, the
lowest expression in the margin tissue. This spatial and temporal expression
pattern correlates with the darker pink pigmentation in the central flower
portion and the white picotee margins. What is not yet known is the molecular
basis for these spatio-temporal differences in Myb12, as compared to lilies
that do not show the picotee phenotype.
Inactivation of Chalcone Synthase
Morita
et al (2012) teach that bicolor flower traits in Petunia are caused by the
spatial repression of the chalcone synthase A gene, which encodes an
anthocyanin biosynthetic enzyme. While other biosynthetic genes are expressed equally in both the
pigmented and white tissues, the suppression of CHS-A in the white tissue is
sufficient to result in the white color. The regulatory gene that specifically governs
the spatial regulation of CHS-A expression, but not the other pigmentation genes, giving rise to the picotee phenotype,
is unknown. The picotee phenotype appears to be a
recessive phenotype.
Change in Cell Fate
Broderick (2011)
described a new Petunia picotee, whereby the margin tissue is green.
In
this cultivar, a transposable element inserted into the floral binding protein 2 (fbp2) gene, which
promotes flower development over epidermal leaf tissue. Decreased expression of
fbp2 in petal margins causes the induction of green margin phenotype, including
the formation of epidermal tissue with stomata and tricomes. The trait is
inherited as a single-gene, homozygous recessive pattern.
Somatic ploidy conversion
De
Schepper et al (2001) described azalea sports having a broad, white picotee,
whereby the petal edges proved to be tetraploid (4N) tissue but the rest of the
flower tissue was diploid (2N). However, it is unclear why during petal
development there would be a switch from 2N to 4N cell development, nor why the
ploidy conversion would affect pigmentation. De Schepper et al indicate that
this somatic 2N to 4N picotee phenomenon is apparently unique to these azalea
sports (De Schepper, 2004).
Methods to select for picotee
Fukuta
et al (2009, 2010) describe methods of selecting for picotee traits in
Lisianthus, the methods comprising the steps of cultivating the plants at
temperatures at or below 20 deg C. Selection at the lower temperatures in which
the picotee phenotype became clear was effective for improving the stability of
the picotee formation in subsequent generations. Fukuta et al suggest that
selection of picotee formation stability in seed (pod) parents that show the
flavonoid-type picotee is more effective for improving stability than selection
of the pollen parent. Fertilizer application can also positively influence the
coloring rate, but the degree of influence depended upon the picotee stability
of each cultivar.
It
appears that pigmentation of the petal margin tissue is regulated differently
than in the central portions. One hypothesis to explain this difference is that
there may be a gradient of transcription factor expression along the petal
axis, resulting in spatial differences in pigmentation. A second hypothesis is
that changes in gene dosage, either through ploidy conversion or epigenetic
mechanisms, can result in the silencing of some pigmentation genes. At this
point in time, more molecular genetic tools need to be developed so that we can
improve the measurement of changes in gene expression during the many spatial
and temporal stages of flower development. Even in the model horticultural
plants, there are still more questions than answers to explain the mechanism of
picotee development.
What
I think would be really exciting to see Cymbidium flowers having a bold picotee
pattern such as Siskin Clock. Even though the picotee and bicolor
phenotypes are extremely desirable, and are some of the main targets in ornamental
flower breeding (Nakatsuka et al, 2011), it’s not clear to me that the leading Cymbidium
hybridizers have pursued a picotee breeding program. But if the buying public
shows some economic appreciation towards these color patterns, then perhaps we’ll
begin to see some real trait improvements in the near future Cymbidium hybrids.
References
Geissman
and Mehlquist, Inheritance in the
Carnation, Dianthus caryophyllus. IV. The Chemistry of Flower Color Variation,
I, Genetics 32:410-433, 1947.
To
and Wang, Molecular Breeding of Flower Color, in Floriculture, Ornamental and Plant Biotechnology Vol. 1, Chapter
35: 300-310, 2006.
Nakatsuka
et al, Production of picotee-type flowers
in Japanese gentian by CRES-T, Plant Biotechnol. 28: 173-180, 2011.
Nakatsuka
et al, Identification and
Characterization of R2R3-Myb and bHLH Transcription Factors Regulating Anthocyanin
Biosynthesis in Gentian Flowers, Plant Cell Physiol. 49(12):1818-1829,
2008.
Yamagishi,
Oriental Hybrid Lily Sorbonne Homologue
of LhMyb12 Regulates Anthocyanin Biosynthesis in Flower Tepals and Tepal Spots,
Mol. Breeding 28:381-389, 2011.
Morita
et al, Tandemly Arranged Chalcone
Synthase A Genes Contribute to the Spatially Regulated Expression of siRNA and
the Natural Bicolor Floral Phenotype in Petunia hybrida, The Plant Journal
70:739-749, 2012.
De
Schepper et al, Somatic Polyploid Petals:
Regeneration Offers New Roads for Breeding Belgian Pot Azaleas, Plant Cell,
Tissue and Organ Culture 76:183-188, 2004.
Fukuta
and Shibata, Influence of Fertilizer
Application on Coloring Area Rate in Picotee Petals of Eustoma grandiflorum
(Raf.) Shinn., Hort. Res. (Japan) 8(2):187-192, 2009.
Fukuta
et al, Environmental Variation and
Selection Efficiency of Picotee Colored Area Rate on Petals in Lisianthus
(Eustoma grandiflorum Raf. Shinn.), Hort. Res. (Japan) 9(3):255-261, 2010.
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