A phosphatase gene is linked to nectar dihydroxyacetone accumulation in manuka (Leptospermum scoparium).

Floral nectar composition beyond common sugars shows great diversity but contributing genetic factors are generally unknown. Manuka (Leptospermum scoparium) is renowned for the antimicrobial compound methylglyoxal in its derived honey, which originates from the precursor, dihydroxyacetone (DHA), accumulating in the nectar. Although this nectar trait is highly variable, genetic contribution to the trait is unclear. Therefore, we investigated key gene(s) and genomic regions underpinning this trait. We used RNAseq analysis to identify nectary-associated genes differentially expressed between high and low nectar DHA genotypes. We also used a manuka high-density linkage map and quantitative trait loci (QTL) mapping population, supported by an improved genome assembly, to reveal genetic regions associated with nectar DHA content. Expression and QTL analyses both pointed to the involvement of a phosphatase gene, LsSgpp2. The expression pattern of LsSgpp2 correlated with nectar DHA accumulation, and it co-located with a QTL on chromosome 4. The identification of three QTLs, some of the first reported for a plant nectar trait, indicates polygenic control of DHA content. We have established plant genetics as a key influence on DHA accumulation. The data suggest the hypothesis of LsSGPP2 releasing DHA from DHA-phosphate and variability in LsSgpp2 gene expression contributing to the trait variability.

The fate of sex chromosomes during the evolution of monoicy from dioicy in liverworts.

Liverworts comprise one of six primary land plant lineages, with the predicted origin of extant liverwort diversity dating to the Silurian. The ancestral liverwort has been inferred to have been dioicous (unisexual) with chromosomal sex determination in which the U chromosome of females and the V chromosome of males were dimorphic with an extensive non-recombining region. In liverworts, sex is determined by a U chromosomal "feminizer" gene that promotes female development, and in its absence, male development ensues. Monoicy (bisexuality) has independently evolved multiple times within liverworts. Here, we explore the evolution of monoicy, focusing on the monoicous species Ricciocarpos natans, and propose that the evolution of monoicy in R. natans involved the appearance of an aneuploid spore that possessed both U and V chromosomes. Chromosomal rearrangements involving the U chromosome resulted in distribution of essential U chromosome genes, including the feminizer, to several autosomal locations. By contrast, we infer that the ancestral V chromosome was inherited largely intact, probably because it carries numerous dispersed "motility" genes distributed across the chromosome. The genetic networks for sex differentiation in R. natans appear largely unchanged except that the feminizer is developmentally regulated, allowing for temporally separated differentiation of female and male reproductive organs on a single plant. A survey of other monoicous liverworts suggests that similar genomic rearrangements may have occurred repeatedly in lineages transitioning to monoicy from dioicy. These data provide a foundation for understanding how genetic networks controlling sex determination can be subtly rewired to produce profound changes in sexual systems.

Evolution and function of red pigmentation in land plants.

BACKGROUND: Land plants commonly produce red pigmentation as a response to environmental stressors, both abiotic and biotic. The type of pigment produced varies among different land plant lineages. In the majority of species they are flavonoids, a large branch of the phenylpropanoid pathway. Flavonoids that can confer red colours include 3-hydroxyanthocyanins, 3-deoxyanthocyanins, sphagnorubins and auronidins, which are the predominant red pigments in flowering plants, ferns, mosses and liverworts, respectively. However, some flowering plants have lost the capacity for anthocyanin biosynthesis and produce nitrogen-containing betalain pigments instead. Some terrestrial algal species also produce red pigmentation as an abiotic stress response, and these include both carotenoid and phenolic pigments. SCOPE: In this review, we examine: which environmental triggers induce red pigmentation in non-reproductive tissues; theories on the functions of stress-induced pigmentation; the evolution of the biosynthetic pathways; and structure-function aspects of different pigment types. We also compare data on stress-induced pigmentation in land plants with those for terrestrial algae, and discuss possible explanations for the lack of red pigmentation in the hornwort lineage of land plants. CONCLUSIONS: The evidence suggests that pigment biosynthetic pathways have evolved numerous times in land plants to provide compounds that have red colour to screen damaging photosynthetically active radiation but that also have secondary functions that provide specific benefits to the particular land plant lineage.

The renaissance and enlightenment of Marchantia as a model system.

The liverwort Marchantia polymorpha has been utilized as a model for biological studies since the 18th century. In the past few decades, there has been a Renaissance in its utilization in genomic and genetic approaches to investigating physiological, developmental, and evolutionary aspects of land plant biology. The reasons for its adoption are similar to those of other genetic models, e.g. simple cultivation, ready access via its worldwide distribution, ease of crossing, facile genetics, and more recently, efficient transformation, genome editing, and genomic resources. The haploid gametophyte dominant life cycle of M. polymorpha is conducive to forward genetic approaches. The lack of ancient whole-genome duplications within liverworts facilitates reverse genetic approaches, and possibly related to this genomic stability, liverworts possess sex chromosomes that evolved in the ancestral liverwort. As a representative of one of the three bryophyte lineages, its phylogenetic position allows comparative approaches to provide insights into ancestral land plants. Given the karyotype and genome stability within liverworts, the resources developed for M. polymorpha have facilitated the development of related species as models for biological processes lacking in M. polymorpha.

Stress, senescence, and specialized metabolites in bryophytes.

Life on land exposes plants to varied abiotic and biotic environmental stresses. These environmental drivers contributed to a large expansion of metabolic capabilities during land plant evolution and species diversification. In this review we summarize knowledge on how the specialized metabolite pathways of bryophytes may contribute to stress tolerance capabilities. Bryophytes are the non-tracheophyte land plant group (comprising the hornworts, liverworts, and mosses) and rapidly diversified following the colonization of land. Mosses and liverworts have as wide a distribution as flowering plants with regard to available environments, able to grow in polar regions through to hot desert landscapes. Yet in contrast to flowering plants, for which the biosynthetic pathways, transcriptional regulation, and compound function of stress tolerance-related metabolite pathways have been extensively characterized, it is only recently that similar data have become available for bryophytes. The bryophyte data are compared with those available for angiosperms, including examining how the differing plant forms of bryophytes and angiosperms may influence specialized metabolite diversity and function. The involvement of stress-induced specialized metabolites in senescence and nutrient response pathways is also discussed.

Production of Betacyanins in Transgenic Nicotiana tabacum Increases Tolerance to Salinity.

Although red betalain pigments (betacyanins) have been associated with salinity tolerance in some halophytes like Disphyma australe, efforts to determine whether they have a causal role and the underlying mechanisms have been hampered by a lack of a model system. To address this, we engineered betalain-producing Nicotiana tabacum, by the introduction of three betalain biosynthetic genes. The plants were violet-red due to the accumulation of three betacyanins: betanin, isobetanin, and betanidin. Under salt stress, betacyanic seedlings had increased survivability and leaves of mature plants had higher photochemical quantum yields of photosystem II (F v /F m ) and faster photosynthetic recovery after saturating light treatment. Under salt stress, compared to controls betacyanic leaf disks had no loss of carotenoids, a slower rate of chlorophyll degradation, and higher F v /F m values. Furthermore, simulation of betacyanin pigmentation by using a red filter cover improved F v /F m value of green tissue under salt stress. Our results confirm a direct causal role of betacyanins in plant salinity tolerance and indicate a key mechanism is photoprotection. A role in delaying leaf senescence was also indicated, and the enhanced antioxidant capability of the betacyanic leaves suggested a potential contribution to scavenging reactive oxygen species. The study can inform the development of novel biotechnological approaches to improving agricultural productivity in saline-affected areas.

Discrete bHLH transcription factors play functionally overlapping roles in pigmentation patterning in flowers of Antirrhinum majus.

Floral pigmentation patterning is important for pollinator attraction as well as aesthetic appeal. Patterning of anthocyanin accumulation is frequently associated with variation in activity of the Myb, bHLH and WDR transcription factor complex (MBW) that regulates anthocyanin biosynthesis. Investigation of two classic mutants in Antirrhinum majus, mutabilis and incolorata I, showed they affect a gene encoding a bHLH protein belonging to subclade bHLH-2. The previously characterised gene, Delila, which encodes a bHLH-1 protein, has a bicoloured mutant phenotype, with residual lobe-specific pigmentation conferred by Incolorata I. Both Incolorata I and Delila induce expression of the anthocyanin biosynthetic gene DFR. Rosea 1 (Myb) and WDR1 proteins compete for interaction with Delila, but interact positively to promote Incolorata I activity. Delila positively regulates Incolorata I and WDR1 expression. Hierarchical regulation can explain the bicoloured patterning of delila mutants, through effects on both regulatory gene expression and the activity of promoters of biosynthetic genes like DFR that mediate MBW regulation. bHLH-1 and bHLH-2 proteins contribute to establishing patterns of pigment distribution in A. majus flowers in two ways: through functional redundancy in regulating anthocyanin biosynthetic gene expression, and through differences between the proteins in their ability to regulate genes encoding transcription factors.

The Evolution of Flavonoid Biosynthesis: A Bryophyte Perspective.

The flavonoid pathway is one of the best characterized specialized metabolite pathways of plants. In angiosperms, the flavonoids have varied roles in assisting with tolerance to abiotic stress and are also key for signaling to pollinators and seed dispersal agents. The pathway is thought to be specific to land plants and to have arisen during the period of land colonization around 550-470 million years ago. In this review we consider current knowledge of the flavonoid pathway in the bryophytes, consisting of the liverworts, hornworts, and mosses. The pathway is less characterized for bryophytes than angiosperms, and the first genetic and molecular studies on bryophytes are finding both commonalities and significant differences in flavonoid biosynthesis and pathway regulation between angiosperms and bryophytes. This includes biosynthetic pathway branches specific to each plant group and the apparent complete absence of flavonoids from the hornworts.

Auronidins are a previously unreported class of flavonoid pigments that challenges when anthocyanin biosynthesis evolved in plants.

Anthocyanins are key pigments of plants, providing color to flowers, fruit, and foliage and helping to counter the harmful effects of environmental stresses. It is generally assumed that anthocyanin biosynthesis arose during the evolutionary transition of plants from aquatic to land environments. Liverworts, which may be the closest living relatives to the first land plants, have been reported to produce red cell wall-bound riccionidin pigments in response to stresses such as UV-B light, drought, and nutrient deprivation, and these have been proposed to correspond to the first anthocyanidins present in early land plant ancestors. Taking advantage of the liverwort model species Marchantia polymorpha, we show that the red pigments of Marchantia are formed by a phenylpropanoid biosynthetic branch distinct from that leading to anthocyanins. They constitute a previously unreported flavonoid class, for which we propose the name "auronidin," with similar colors as anthocyanin but different chemistry, including strong fluorescence. Auronidins might contribute to the remarkable ability of liverworts to survive in extreme environments on land, and their discovery calls into question the possible pigment status of the first land plants.

MYBA From Blueberry (Vaccinium Section Cyanococcus) Is a Subgroup 6 Type R2R3MYB Transcription Factor That Activates Anthocyanin Production.

The Vaccinium genus in the family Ericaceae comprises many species, including the fruit-bearing blueberry, bilberry, cranberry, huckleberry, and lingonberry. Commercially, the most important are the blueberries (Vaccinium section Cyanococcus), such as Vaccinium corymbosum (northern highbush blueberry), Vaccinium virgatum (rabbiteye blueberry), and Vaccinium angustifolium (lowbush blueberry). The rising popularity of blueberries can partly be attributed to their "superfood" status, with an increasing body of evidence around human health benefits resulting from the fruit metabolites, particularly products of the phenylpropanoid pathway such as anthocyanins. Activation of anthocyanin production by R2R3-MYB transcription factors (TFs) has been characterized in many species, but despite recent studies on blueberry, cranberry, and bilberry, no MYB anthocyanin regulators have been reported for Vaccinium. Indeed, there has been conjecture that at least in bilberry, MYB TFs divergent to the usual type are involved. We report identification of MYBA from blueberry, and show through sequence analysis and functional studies that it is homologous to known anthocyanin-promoting R2R3-MYBs of subgroup 6 of the MYB superfamily. In transient assays, MYBA complemented an anthocyanin MYB mutant of Antirrhinum majus and, together with a heterologous bHLH anthocyanin regulator, activated anthocyanin production in Nicotiana benthamiana. Furthermore anthocyanin accumulation and anthocyanin structural gene expression (assayed by qPCR and RNA-seq analyses) correlated with MYBA expression, and MYBA was able to transactivate the DFR promoter from blueberry and other species. The RNA-seq data also revealed a range of other candidate genes involved in the regulation of anthocyanin production in blueberry fruit. The identification of MYBA will help to resolve the regulatory mechanism for anthocyanin pigmentation in the Vaccinium genus. The sequence information should also prove useful in developing tools for the accelerated breeding of new Vaccinium cultivars.

UVR8-mediated induction of flavonoid biosynthesis for UVB tolerance is conserved between the liverwort Marchantia polymorpha and flowering plants.

Damaging UVB radiation is a major abiotic stress facing land plants. In angiosperms the UV RESISTANCE LOCUS8 (UVR8) photoreceptor coordinates UVB responses, including inducing biosynthesis of protective flavonoids. We characterised the UVB responses of Marchantia polymorpha (marchantia), the model species for the liverwort group of basal plants. Physiological, chemical and transcriptomic analyses were conducted on wild-type marchantia exposed to three different UVB regimes. CRISPR/Cas9 was used to obtain plant lines with mutations for components of the UVB signal pathway or the flavonoid biosynthetic pathway, and transgenics overexpressing the marchantia UVR8 sequence were generated. The mutant and transgenic lines were analysed for changes in flavonoid content, their response to UVB exposure, and transcript abundance of a set of 48 genes that included components of the UVB response pathway characterised for angiosperms. The marchantia UVB response included many components in common with Arabidopsis, including production of UVB-absorbing flavonoids, the central activator role of ELONGATED HYPOCOTYL5 (HY5), and negative feedback regulation by REPRESSOR OF UV-B PHOTOMORPHOGENESIS1 (RUP1). Notable differences included the greater importance of CHALCONE ISOMERASE-LIKE (CHIL). Mutants disrupted in the response pathway (hy5) or flavonoid production (chalcone isomerase, chil) were more easily damaged by UVB. Mutants (rup1) or transgenics (35S:MpMYB14) with increased flavonoid content had increased UVB tolerance. The results suggest that UVR8-mediated flavonoid induction is a UVB tolerance character conserved across land plants and may have been an early adaptation to life on land.

A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants.

BACKGROUND: Most published genome sequences are drafts, and most are dominated by computational gene prediction. Draft genomes typically incorporate considerable sequence data that are not assigned to chromosomes, and predicted genes without quality confidence measures. The current Actinidia chinensis (kiwifruit) 'Hongyang' draft genome has 164 Mb of sequences unassigned to pseudo-chromosomes, and omissions have been identified in the gene models. RESULTS: A second genome of an A. chinensis (genotype Red5) was fully sequenced. This new sequence resulted in a 554.0 Mb assembly with all but 6 Mb assigned to pseudo-chromosomes. Pseudo-chromosomal comparisons showed a considerable number of translocation events have occurred following a whole genome duplication (WGD) event some consistent with centromeric Robertsonian-like translocations. RNA sequencing data from 12 tissues and ab initio analysis informed a genome-wide manual annotation, using the WebApollo tool. In total, 33,044 gene loci represented by 33,123 isoforms were identified, named and tagged for quality of evidential support. Of these 3114 (9.4%) were identical to a protein within 'Hongyang' The Kiwifruit Information Resource (KIR v2). Some proportion of the differences will be varietal polymorphisms. However, as most computationally predicted Red5 models required manual re-annotation this proportion is expected to be small. The quality of the new gene models was tested by fully sequencing 550 cloned 'Hort16A' cDNAs and comparing with the predicted protein models for Red5 and both the original 'Hongyang' assembly and the revised annotation from KIR v2. Only 48.9% and 63.5% of the cDNAs had a match with 90% identity or better to the original and revised 'Hongyang' annotation, respectively, compared with 90.9% to the Red5 models. CONCLUSIONS: Our study highlights the need to take a cautious approach to draft genomes and computationally predicted genes. Our use of the manual annotation tool WebApollo facilitated manual checking and correction of gene models enabling improvement of computational prediction. This utility was especially relevant for certain types of gene families such as the EXPANSIN like genes. Finally, this high quality gene set will supply the kiwifruit and general plant community with a new tool for genomics and other comparative analysis.

Chromosome-scale scaffolding of the black raspberry (Rubus occidentalis L.) genome based on chromatin interaction data.

Black raspberry (Rubus occidentalis L.) is a niche fruit crop valued for its flavor and potential health benefits. The improvement of fruit and cane characteristics via molecular breeding technologies has been hindered by the lack of a high-quality reference genome. The recently released draft genome for black raspberry (ORUS 4115-3) lacks assembly of scaffolds to chromosome scale. We used high-throughput chromatin conformation capture (Hi-C) and Proximity-Guided Assembly (PGA) to cluster and order 9650 out of 11,936 contigs of this draft genome assembly into seven pseudo-chromosomes. The seven pseudo-chromosomes cover ~97.2% of the total contig length (~223.8 Mb). Locating existing genetic markers on the physical map resolved multiple discrepancies in marker order on the genetic map. Centromeric regions were inferred from recombination frequencies of genetic markers, alignment of 303 bp centromeric sequence with the PGA, and heat map showing the physical contact matrix over the entire genome. We demonstrate a high degree of synteny between each of the seven chromosomes of black raspberry and a high-quality reference genome for strawberry (Fragaria vesca L.) assembled using only PacBio long-read sequences. We conclude that PGA is a cost-effective and rapid method of generating chromosome-scale assemblies from Illumina short-read sequencing data.

Genetic analysis of the liverwort Marchantia polymorpha reveals that R2R3MYB activation of flavonoid production in response to abiotic stress is an ancient character in land plants.

The flavonoid pathway is hypothesized to have evolved during land colonization by plants c. 450 Myr ago for protection against abiotic stresses. In angiosperms, R2R3MYB transcription factors are key for environmental regulation of flavonoid production. However, angiosperm R2R3MYB gene families are larger than those of basal plants, and it is not known whether the regulatory system is conserved across land plants. We examined whether R2R3MYBs regulate the flavonoid pathway in liverworts, one of the earliest diverging land plant lineages. We characterized MpMyb14 from the liverwort Marchantia polymorpha using genetic mutagenesis, transgenic overexpression, gene promoter analysis, and transcriptomic and chemical analysis. MpMyb14 is phylogenetically basal to characterized angiosperm R2R3MYB flavonoid regulators. Mpmyb14 knockout lines lost all red pigmentation from the flavonoid riccionidin A, whereas overexpression conferred production of large amounts of flavones and riccionidin A, activation of associated biosynthetic genes, and constitutive red pigmentation. MpMyb14 expression and flavonoid pigmentation were induced by light- and nutrient-deprivation stress in M. polymorpha as for anthocyanins in angiosperms. MpMyb14 regulates stress-induced flavonoid production in M. polymorpha, and is essential for red pigmentation. This suggests that R2R3MYB regulated flavonoid production is a conserved character across land plants which arose early during land colonization.

Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome.

The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant. PAPERCLIP.

The Onion (Allium cepa L.) R2R3-MYB Gene MYB1 Regulates Anthocyanin Biosynthesis.

Bulb color is an important consumer trait for onion (Allium cepa L., Allioideae, Asparagales). The bulbs accumulate a range of flavonoid compounds, including anthocyanins (red), flavonols (pale yellow), and chalcones (bright yellow). Flavonoid regulation is poorly characterized in onion and in other plants belonging to the Asparagales, despite being a major plant order containing many important crop and ornamental species. R2R3-MYB transcription factors associated with the regulation of distinct branches of the flavonoid pathway were isolated from onion. These belonged to sub-groups (SGs) that commonly activate anthocyanin (SG6, MYB1) or flavonol (SG7, MYB29) production, or repress phenylpropanoid/flavonoid synthesis (SG4, MYB4, MYB5). MYB1 was demonstrated to be a positive regulator of anthocyanin biosynthesis by the induction of anthocyanin production in onion tissue when transiently overexpressed and by reduction of pigmentation when transiently repressed via RNAi. Furthermore, ectopic red pigmentation was observed in garlic (Allium sativum L.) plants stably transformed with a construct for co-overexpression of MYB1 and a bHLH partner. MYB1 also was able to complement the acyanic petal phenotype of a defined R2R3-MYB anthocyanin mutant in Antirrhinum majus of the asterid clade of eudicots. The availability of sequence information for flavonoid-related MYBs from onion enabled phylogenetic groupings to be determined across monocotyledonous and dicotyledonous species, including the identification of characteristic amino acid motifs. This analysis suggests that divergent evolution of the R2R3-MYB family has occurred between Poaceae/Orchidaceae and Allioideae species. The DNA sequences identified will be valuable for future analysis of classical flavonoid genetic loci in Allium crops and will assist the breeding of these important crop species.

High concentrations of aromatic acylated anthocyanins found in cauline hairs in Plectranthus ciliatus.

Vegetative shoots of a naturalized population of purple-leaved plectranthus (Plectranthus ciliatus, Lamiaceae) were found to contain four main anthocyanins: peonidin 3-(6″-caffeoyl-ß-glucopyranoside)-5-ß-glucopyranoside, peonidin 3-(6″-caffeoyl-ß-glucopyranoside)-5-(6‴-malonyl-ß-glucopyranoside), peonidin 3-(6″-E-p-coumaroyl-ß-glucopyranoside)-5-(6‴-malonyl-ß-glucopyranoside), and peonidin 3-(6″-E-p-coumaroyl-ß-glucopyranoside)-5-ß-glucopyranoside. The first three of these pigments have not been reported previously from any plant. They all follow the typical anthocyanin pattern of Lamiaceae, with universal occurrence of anthocyanidin 3,5-diglucosides and aromatic acylation with p-coumaric and sometimes caffeic acids; however, they differ by being based on peonidin. The four anthocyanins were present in the leaves (22.2 mg g(-1) DW), and in the xylem and interfascicular parenchyma of the stem. They were exceptionally abundant, among the highest reported for any plant organ, in epidermal hairs on some of the stem internodes (101 mg g(-1) DW). Anthocyanin content in these hairs increased more than three-fold from the youngest to the fourth-youngest internodes. In situ absorbances (λmax ≈ 545 nm) were bathochromic in comparison to absorbances of the isolated anthocyanins in their flavylium form in acidified aqueous solutions (λmax = 525 nm), suggesting that the anthocyanins occur both in quinoidal and flavylium forms in constant proportions in the anthocyanic hair cells. The most distinctive observation with respect to relative proportions of individual anthocyanins was found in de-haired internodes, for which anthocyanin caffeoyl-derivatives decreased, and anthocyanin coumaroyl-derivatives increased, from the youngest to the fourth-youngest internode.

Characterisation of betalain biosynthesis in Parakeelya flowers identifies the key biosynthetic gene DOD as belonging to an expanded LigB gene family that is conserved in betalain-producing species.

Plant betalain pigments are intriguing because they are restricted to the Caryophyllales and are mutually exclusive with the more common anthocyanins. However, betalain biosynthesis is poorly understood compared to that of anthocyanins. In this study, betalain production and betalain-related genes were characterized in Parakeelya mirabilis (Montiaceae). RT-PCR and transcriptomics identified three sequences related to the key biosynthetic enzyme Dopa 4,5-dioxgenase (DOD). In addition to a LigB gene similar to that of non-Caryophyllales species (Class I genes), two other P. mirabilis LigB genes were found (DOD and DOD-like, termed Class II). PmDOD and PmDOD-like had 70% amino acid identity. Only PmDOD was implicated in betalain synthesis based on transient assays of enzyme activity and correlation of transcript abundance to spatio-temporal betalain accumulation. The role of PmDOD-like remains unknown. The striking pigment patterning of the flowers was due to distinct zones of red betacyanin and yellow betaxanthin production. The major betacyanin was the unglycosylated betanidin rather than the commonly found glycosides, an occurrence for which there are a few previous reports. The white petal zones lacked pigment but had DOD activity suggesting alternate regulation of the pathway in this tissue. DOD and DOD-like sequences were also identified in other betalain-producing species but not in examples of anthocyanin-producing Caryophyllales or non-Caryophyllales species. A Class I LigB sequence from the anthocyanin-producing Caryophyllaceae species Dianthus superbus and two DOD-like sequences from the Amaranthaceae species Beta vulgaris and Ptilotus spp. did not show DOD activity in the transient assay. The additional sequences suggests that DOD is part of a larger LigB gene family in betalain-producing Caryophyllales taxa, and the tandem genomic arrangement of two of the three B. vulgaris LigB genes suggests the involvement of duplication in the gene family evolution.

Failure to launch: the self-regulating Md-MYB10 R6 gene from apple is active in flowers but not leaves of Petunia.

KEY MESSAGE: The Md - MYB10 R6 gene from apple is capable of self-regulating in heterologous host species and enhancing anthocyanin pigmentation, but the activity of MYB10 is dependent on endogenous protein partners. Coloured foliage due to anthocyanin pigments (bronze/red/black) is an attractive trait that is often lacking in many bedding, ornamental and horticultural plants. Apples (Malus × domestica) containing an allelic variant of the anthocyanin regulator, Md-MYB10 R6 , are highly pigmented throughout the plant, due to autoregulation by MYB10 upon its own promoter. We investigated whether Md-MYB10 R6 from apple is capable of functioning within the heterologous host Petunia hybrida to generate plants with novel pigmentation patterns. The Md-MYB10 R6 transgene (MYB10-R6 pro :MYB10:MYB10 term ) activated anthocyanin synthesis when transiently expressed in Antirrhinum rosea (dorsea) petals and petunia leaf discs. Stable transgenic petunias containing Md-MYB10 R6 lacked foliar pigmentation but had coloured flowers, complementing the an2 phenotype of 'Mitchell' petunia. The absence of foliar pigmentation was due to the failure of the Md-MYB10 R6 gene to self-activate in vegetative tissues, suggesting that additional protein partners are required for Md-MYB10 to activate target genes in this heterologous system. In petunia flowers, where endogenous components including MYB-bHLH-WDR (MBW) proteins were present, expression of the Md-MYB10 R6 promoter was initiated, allowing auto-regulation to occur and activating anthocyanin production. Md-MYB10 is capable of operating within the petunia MBW gene regulation network that controls the expression of the anthocyanin biosynthesis genes, AN1 (bHLH) and MYBx (R3-MYB repressor) in petals.