Picotee

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;

http://www.novascotiagardening.com/products.php?offset=10&cat_id=59&search_query)

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. 

 http://www.springerimages.com/Images/LifeSciences/1-10.1007_s00425-008-0744-y-0

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.

 Dianthus ‘Siskin Clock’, Google Image search (http://www.pioneergardens.com/planthaven2.htm)

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. 

Broderick, Jewels in the Genome: Green Picotee Petunia, 2011. (http://www.extension.org/pages/61173/jewels-in-the-genome:-green-picotee-petunia) 

De Schepper et al, Somatic Polyploidy and its Consequences for Flower Coloration and Flower Morphology in Azalea, Plant Cell Rep. 20:583-590, 2001. (http://www.springerlink.com/content/vm377987q2wwpn7j/).

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.