Summary Floral nectar composition beyond common sugars shows great diversity but contributing genetic factors are generally unknown. Mānuka (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 mānuka 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.

Paeonia delavayi is listed as an endangered species in the southwest of China. The flower colours of cultivated examples of this species are most commonly yellow and red, but a much wider range of flower colours can be found in plants growing in the natural habitat. To elucidate the basis of the colour range, pigments were extracted from flower petals of seven selected colours and profiled and quantified using High Performance Liquid Chromatography (HPLC) and Liquid Chromatography-Mass Spectrometry (LC-MS). All the petal colours were found to share the same base pigment types as the yellow flower - a composite colour based on a mix of flavonoids, carotenoids and chlorophyll. Anthocyanin profiling revealed the same anthocyanin compositions in all red coloured flowers, namely, cyanidin-3,5-diglucoside, cyanidin-3-glucoside, cyanidin-3-arabinoside, peonidin-3,5-diglucoside, peonidin-3-glucoside and peonidin 3-arabinoside. Quantitative analysis suggested the yellow colour was principally formed by the flavonoid naringenin chalcone 2'-glucoside (2',4',6',4-tetrahydroxychalcone 2'-glucoside). The various red/orange flower colours were the result of superimposing different amounts of anthocyanins onto the yellow flower background, with carotenoids and chlorophylls having only a minor contribution to the final colour. The results provide useful information for breeding programmes for ornamental varieties of P. delavayi.

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.

Orchids have a complex flower morphology with fascinating pigmentation patterns. As part of a study examining the development of anthocyanin pigmentation during bud development in Cymbidium orchids, we monitored expression patterns of a flavonoid biosynthetic gene dihydroflavanol reductase (DFR) and the transcription factor ChMYB1 in flower buds from a "white" cultivar ('Jungfrau dos Pueblos', JDP) and a "red" cultivar ('Clarisse Austin South Pacific', CASP). ChMYB1 was expressed during flower bud development in the two different cultivars but at much higher levels for the cultivar that accumulated anthocyanin pigments (CASP). Its expression was closely correlated with expression of the anthocyanin biosynthetic gene DFR and anthocyanin accumulation, suggesting it is associated with full petal/sepal coloration. The cultivar JDP appears to be an expression mutant for ChMYB1. ChMYB1 and DFR expression patterns vary across different floral organs too, with the JDP flower labelum showing some expression and some anthocyanin accumulation, compared with trace amounts in the white petals/sepals.

We assembled the genome of Leptospermum scoparium 'Crimson Glory' using a combination of Illumina paired-end sequencing, high-throughput chromosome conformation capture (Hi-C) and high density genetic mapping. As 'Crimson Glory' is a variety of manuka, this is the first genome assembly for a plant species culturally recognised as a treasure (taonga) by the indigenous Maori of Aotearoa New Zealand. The manuka genome spans a total of 297 Mbp organised in 11 pseudo-chromosomes that are syntenic with the Eucalyptus genome. A large proportion of the genome assembly corresponds to fungal and bacterial sequences, indicating the presence of an associated microbiome. A total of 31,220 protein-coding gene models were detected throughout the genome, including genes involved in biosynthesis of biologically active phenylpropanoids, triketones and terpenes, as well as genes involved in biotic resistance. The manuka genome sequence will help shed new light on the genetic control of unique characters such as nectar and foliage biochemical composition, flowering time and disease resistance.

Viral-induced gene silencing of selected biosynthetic genes decreased overall carotenoid accumulation in California poppy. Regulation of carotenogenesis was linked with pigment sequestration, not changes in biosynthetic gene expression.

MYB transcription factors (TFs) regulate diverse plant developmental processes and understanding their roles in controlling pigment accumulation in fruit is important for developing new cultivars. In this study, we characterised kiwifruit TFMYB7, which was found to activate the promoter of the kiwifruit lycopene beta-cyclase (AdLCY-beta) gene that plays a key role in the carotenoid biosynthetic pathway. To determine the role of MYB7, we analysed gene expression and metabolite profiles in Actinidia fruit which show different pigment profiles. The impact of MYB7 on metabolic biosynthetic pathways was then evaluated by overexpression in Nicotiana benthamiana followed by metabolite and gene expression analysis of the transformants. MYB7 was expressed in fruit that accumulated carotenoid and Chl pigments with high transcript levels associated with both pigments. Constitutive over-expression of MYB7, through transient or stable transformation of N. benthamiana, altered Chl and carotenoid pigment levels. MYB7 overexpression was associated with transcriptional activation of certain key genes involved in carotenoid biosynthesis, Chl biosynthesis, and other processes such as chloroplast and thylakoid membrane organization. Our results suggest that MYB7 plays a role in modulating carotenoid and Chl pigment accumulation in tissues through transcriptional activation of metabolic pathway genes.

A high-density linkage map was constructed for manuka (Leptospermum scoparium) genotyping by sequencing (GBS) and a segregating population developed using accessions from the East Cape region of New Zealand. Parental maps were constructed using 1140 and 1509 SNPs markers grouped into 11 linkage groups for both parents spanning a total of 1242.8 and 1616.2 cM, respectively. Quantitative trait loci (QTL) analysis was performed for tree height. In total, 10 QTLs were detected after the measured variable was adjusted for the block effect in the field. The significant QTLs were inherited from both parents (five each) and were located on LG2, 3, 6, 7, 9 and 10. This is the first high-density linkage map constructed using thousands of genetic markers obtained by GBS, the first QTL reported for a New Zealand native tree species, which represents a model for future genetic analysis of indigenous New Zealand species.

A multi-allelic genetic marker resource for future genetics studies in m (a) over bar nuka (Leptospermum scoparium) and its close relatives has been developed by mining simple sequence repeats (SSR) from a preliminary draft genome assembly of L. scoparium 'Crimson Glory'. In total, 469 and 169 trinucleotide and dinucleotide repeats, respectively, were detected in a set of 89,619 predicted gene models. Polymerase chain reaction primer pairs were designed for 32 loci exhibiting high sequence similarity to Eucalyptus grandis. These 32 primer pairs were screened over a population of m (a) over bar nuka and 3 accessions of sympatric, related k (a) over bar nuka (genus Kunzea). The 15 polymorphic SSR markers that were successfully developed are now available for genetic analysis in Myrtaceae.

The range of flower colours in Cymbidium hybrida orchid is extensive. All three main pigment groups have been detected, but our focus has been on the anthocyanins, which may be present in all sepals and petals, only in the labellum, or as a colour blush. Since anthocyanin pigments have an important influence on final flower colour, it is important to study how their biosynthesis is regulated. We report here the isolation of a MYB transcription factor (ChMYB1) that is shown to be an anthocyanin-related MYB and likely to be part of a regulatory complex controlling anthocyanin biosynthesis in Cymbidium flowers. There are at least two separate phases of anthocyanin development in Cymbidium flowers. One occurs during flower bud development, and the other occurs in open flowers after pollination. The relatively rapid induction of anthocyanin biosynthesis after anther cap removal was used to monitor expression of the MYB1 gene and the flavonoid biosynthetic gene dihydroflavonol 4-reductase (DFR). The involvement of ethylene in this anthocyanin induction was examined by applying the ethylene action inhibitor, 1-methyl-cyclopropene (1-MCP), and by studying the expression of ethylene biosynthesis genes. Expression of the MYB1 gene was first detected 12 h after pollinia removal, and it peaked after 24 h, while expression of the DFR gene was first detected at 24 h after pollinia removal and it peaked after 48 h. 1-MCP treatment abolished completely both anthocyanin accumulation and the expression of the ChMYB1 and ChDFR genes. The results suggest that ChMYB1 regulates in Cymbidium hybrida flowers the pollination-induced anthocyanin production mediated by ethylene signalling.

The mature pigmented spathe of Zantedeschia is characterized by a developmental process, wherein the spathe regreens after anthesis and prior to senescence of the inflorescence. Previous research has shown that spathe regreening involves redifferentiation of chloroplasts and re-accumulation of chlorophyll, but the detailed physiological changes associated with regreening are still largely unknown. Using Zantedeschia aethiopica and the Zantedeschia pentlandii variety 'Best Gold' as models, this study explores the physiological mechanism and possible roles of fructification, 6-benzylaminopurine (BAP) and gibberellin (GA3 ) in induction or progression of spathe regreening. Application of BAP stimulated regreening in spathe tissue of 'Best Gold' by enhancing accumulation of carotenoid and chlorophyll, and also increasing stacking of grana. In contrast, GA3 retarded formation of double-membrane lamella during chloroplast redifferentiation, thus delaying the onset of regreening. We suggest that these actions of BAP and GA3 have a synergistic effect in delaying the onset of regreening in 'Best Gold' so that when applied together retardation of chlorophyll accumulation, chloroplast redifferentiation and accumulation of carotenoids were enhanced. The elimination of fructification did not prevent the occurrence of regreening in either Zantedeschia model plants, indicating that fructification was not a prerequisite for the induction of regreening. It is still unclear how regreening in Zantedeschia is triggered. We propose that the onset of regreening in Zantedeschia is likely to be a genetically programmed event.

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.

BACKGROUND:Carotenoid compounds play essential roles in plants such as protecting the photosynthetic apparatus and in hormone signalling. Coloured carotenoids provide yellow, orange and red colour to plant tissues, as well as offering nutritional benefit to humans and animals. The enzyme phytoene synthase (PSY) catalyses the first committed step of the carotenoid biosynthetic pathway and has been associated with control of pathway flux. We characterised four PSY genes found in the apple genome to further understand their involvement in fruit carotenoid accumulation.RESULTS:The apple PSY gene family, containing six members, was predicted to have three functional members, PSY1, PSY2, and PSY4, based on translation of the predicted gene sequences and/or corresponding cDNAs. However, only PSY1 and PSY2 showed activity in a complementation assay. Protein localisation experiments revealed differential localization of the PSY proteins in chloroplasts; PSY1 and PSY2 localized to the thylakoid membranes, while PSY4 localized to plastoglobuli. Transcript levels in 'Granny Smith' and 'Royal Gala' apple cultivars showed PSY2 was most highly expressed in fruit and other vegetative tissues. We tested the transient activation of the apple PSY1 and PSY2 promoters and identified potential and differential regulation by AP2/ERF transcription factors, which suggested that the PSY genes are controlled by different transcriptional mechanisms.CONCLUSION:The first committed carotenoid pathway step in apple is controlled by MdPSY1 and MdPSY2, while MdPSY4 play little or no role in this respect. This has implications for apple breeding programmes where carotenoid enhancement is a target and would allow co-segregation with phenotypes to be tested during the development of new cultivars.

This study confirmed pigment profiles in different colour groups, isolated key anthocyanin biosynthetic genes and established a basis to examine the regulation of colour patterning in flowers of Cymbidium orchid.

Petunia line Mitchell [MP, Petunia axillaris × (P. axillaris × P. hybrida)] and Eustoma grandiflorum (lisianthus) plants were produced containing a transgene for over-expression of the R2R3-MYB transcription factor [TF; ROSEA1 (ROS1)] that up-regulates flavonoid biosynthesis in Antirrhinum majus. The petunia lines were also crossed with previously produced MP lines containing a Zea mays flavonoid-related basic helix-loop-helix TF transgene (LEAF COLOR, LC), which induces strong vegetative pigmentation when these 35S:LC plants are exposed to high-light levels. 35S:ROS1 lisianthus transgenics had limited changes in anthocyanin pigmentation, specifically, precocious pigmentation of flower petals and increased pigmentation of sepals. RNA transcript levels for two anthocyanin biosynthetic genes, chalcone synthase and anthocyanidin synthase, were increased in the 35S:ROS1 lisianthus petals compared to those of control lines. With MP, the 35S:ROS1 calli showed novel red pigmentation in culture, but this was generally not seen in tissue culture plantlets regenerated from the calli or young plants transferred to soil in the greenhouse. Anthocyanin pigmentation was enhanced in the stems of mature 35S:ROS1 MP plants, but the MP white-flower phenotype was not complemented. Progeny from a 35S:ROS1 × 35S:LC cross had novel pigmentation phenotypes that were not present in either parental line or MP. In particular, there was increased pigment in the petal throat region, and the anthers changed from yellow to purple pigmentation. An outdoor field trial was conducted with the 35S:ROS1, 35S:LC, 35S:ROS1 × 35S:LC and control MP lines. Field conditions rapidly induced intense foliage pigmentation in 35S:LC plants, a phenotype not observed in control MP or equivalent 35S:LC plants maintained in a greenhouse. No difference in plant stature, seed germination, or plant survival was observed between transgenic and control plants.

Plants require sophisticated regulatory mechanisms to ensure the degree of anthocyanin pigmentation is appropriate to myriad developmental and environmental signals. Central to this process are the activity of MYB-bHLH-WD repeat (MBW) complexes that regulate the transcription of anthocyanin genes. In this study, the gene regulatory network that regulates anthocyanin synthesis in petunia (Petunia hybrida) has been characterized. Genetic and molecular evidence show that the R2R3-MYB, MYB27, is an anthocyanin repressor that functions as part of the MBW complex and represses transcription through its C-terminal EAR motif. MYB27 targets both the anthocyanin pathway genes and basic-helix-loop-helix (bHLH) ANTHOCYANIN1 (AN1), itself an essential component of the MBW activation complex for pigmentation. Other features of the regulatory network identified include inhibition of AN1 activity by the competitive R3-MYB repressor MYBx and the activation of AN1, MYB27, and MYBx by the MBW activation complex, providing for both reinforcement and feedback regulation. We also demonstrate the intercellular movement of the WDR protein (AN11) and R3-repressor (MYBx), which may facilitate anthocyanin pigment pattern formation. The fundamental features of this regulatory network in the Asterid model of petunia are similar to those in the Rosid model of Arabidopsis thaliana and are thus likely to be widespread in the Eudicots.

Rhabdothamnus solandri A. Cunn. is the only species of the Gesneriaceae endemic to New Zealand. It forms monotypic genus and has flowers considered typical of bird-pollination, being bright orangered with strong visible striping. In addition, vegetative parts of R. solandri plants are also pigmented. Since anthocyanins contribute to significant traits in ornamental plants, elucidating the underlying patterns of pigmentation in flower and vegetative tissues in this species will advance the understanding of tissue-specific anthocyanin accumulation in ornamental plants. The floral and vegetative organs were examined microscopically to determine pigment distribution in different tissues. TLC, HPLC and LC-MS results showed that petals contained the anthocyanins pelargonidin-3-O-glucoside, pelargonidin3-O-(6"-(malony1)-glucoside), cyanidin-3-O-glucoside and cyanidin-3-O-(6"-(malonyl)-glucoside). The bright orange-red flower limb contained only two pelargonidin-based anthocyanins. The colour of the orange-red stripes in the tube was also predominately due to the same pelargonidin-based anthocyanins but with the addition of the two analogous cyanidin-based ones. The dark red stamens contained predominately the cyanidin-based anthocyanins. In addition, cyanidin-3-O-glucoside was found accumulating in leaves and stems. Flower colour patterning in R. solandri is due to the differential production of anthocyanins. The distribution of the anthocyanic vacuolar inclusions (AVIs) suggests that the pelargonidin- and cyanidin-based anthocyanins are capable of forming AVIs. The feature of differential anthocyanin accumulation in the plant makes it a valuable system for studying regulatory mechanisms of anthocyanins, providing new insights into ornamental breeding. (C) 2013 Elsevier B.V. All rights reserved.