Differential expression of carotenoid-related genes determines diversified carotenoid coloration in floral tissues of Oncidium cultivars

Three cultivars of Oncidium orchid with varied coloration, such as Oncidium Gower Ramsey (yellow), Sunkist (orange), and White Jade (white), were analyzed for carotenoid metabolites and gene expression of carotenoid-biosynthetic genes. The HPLC analysis revealed that yellow Gower Ramsey accumulates violaxanthin, 9-cis-violaxanthin and neoxanthin, orange Sunkist accumulates an additional β-carotene, and White Jade is devoid of carotenoid compounds. Molecular characterization indicated that the three Oncidium cultivars exhibited varied expression pattern and level in carotenoid-biosynthetic pathway. Among them, high expression level of β-hydroxylase (OgHYB) and zeaxanthin epoxidase (OgZEP) was displayed in yellow Gower Ramsey, relative to the down-regulation of OgHYB and OgZEP exhibited in orange Sunkist, which results in the accumulation of β-carotene and orange coloration in floral tissues. However, White Jade is caused by the up-regulation of OgCCD1 (Carotenoid Cleavage Dioxygenase 1), which catabolizes carotenoid metabolites. Methylation assay of OgCCD1 promoter in White Jade and Gower Ramsey revealed that a high level of DNA methylation was present in OgCCD1 promoter region of Gower Ramsey. Transient expression of OgCCD1 in yellow lip tissues of Gower Ramsey by bombardment confirmed its function of disintegrating carotenoid compounds. Our results suggest an evolutionary significance that genetic variation of carotenoid-related genes in Oncidium generates the complexity of floral pigmentation and consequently provides the profound varieties in Oncidium population.     

Color genes in the orchid Oncidium Gower Ramsey: identification, expression, and potential genetic instability in an interspecific cross

Orchids are one of the most unique and evolved of flowering plants, with many being valuable floricultural crops. Spatial localization of pigments within the flower of the commercially important bi-color Oncidium Gower Ramsey demonstrated a mixture of carotenoids and anthocyanins concentrated in the adaxial epidermis. Chromatography identified the predominant yellow pigment to be an equal mixture of all-trans and 9-cis isomers of violaxanthin, with esterification specific to the 9-cis isomer. Red ornamentation was comprised of the anthocyanins cyanidin and its methylated derivate, peonidin. Five key pigment biosynthesis genes encoding dihydroflavonol 4-reductase (DFR), phytoene synthase (PSY), phytoene desaturase, carotenoid isomerase, and the downstream 9-cis epoxycarotenoid dioxygenase were isolated and their expression profiles determined. Northern analyses showed both phytoene desaturase and carotenoid isomerase expression to be up-regulated in floral tissue relative to leaves whereas PSY was not. Three closely related DFR genes were isolated, including one with an insertion in the 3′ coding region. DFR expression occurred throughout flower development in Oncidium, unlike in Dendrobium and Bromheadia orchids. A number of the isolated anthocyanin and carotenoid genes showed variations due to insertion events. These findings raise questions about the genetic stability in interspecific crosses in orchids, such as the tri-specific Oncidium Gower Ramsey.     

Modification of flower colour by suppressing β‐ring carotene hydroxylase genes in Oncidium

Oncidium ‘Gower Ramsey’ (Onc. GR) is a popular cut flower, but its colour is limited to bright yellow. The β‐ring carotene hydroxylase (BCH2) gene is involved in carotenoid biogenesis for pigment formation. However, the role of BCH2 in Onc. GR is poorly understood. Here, we investigated the functions of three BCH2 genes, BCH‐A2, BCH‐B2 and BCH‐C2 isolated from Onc. GR, to analyse their roles in flower colour. RT‐PCR expression profiling suggested that BCH2 was mainly expressed in flowers. The expression of BCH‐B2 remained constant while that of BCH‐A2 gradually decreased during flower development. Using Agrobacterium tumefaciens to introduce BCH2 RNA interference (RNAi), we created transgenic Oncidium plants with down‐regulated BCH expression. In the transgenic plants, flower colour changed from the bright yellow of the wild type to light and white‐yellow. BCH‐A2 and BCH‐B2 expression levels were significantly reduced in the transgenic flower lips, which make up the major portion of the Oncidium flower. Sectional magnification of the flower lip showed that the amount of pigmentation in the papillate cells of the adaxial epidermis was proportional to the intensity of yellow colouration. HPLC analyses of the carotenoid composition of the transgenic flowers suggested major reductions in neoxanthin and violaxanthin. In conclusion, BCH2 expression regulated the accumulation of yellow pigments in the Oncidium flower, and the down‐regulation of BCH‐A2 and BCH‐B2 changed the flower colour from bright yellow to light and white‐yellow.     

Overexpression of Oncidium MADS box (OMADS1) gene promotes early flowering in transgenic orchid (Oncidium Gower Ramsey)

Oncidium is a popular ornamental orchid and is produced as a high value cash crop for cut flower sold worldwide. Genetically transformed plants of Oncidium were regenerated after cocultivating protocorm-like bodies (PLBs) with Agrobacterium tumefaciens strain LBA4404 harboring pBI121 with OMADS1. The chopped PLBs pre-cultured for 3?days in darkness produced more kanamycin-resistant PLBs. G10 medium containing 200?mg?l^?1 kanamycin was effective for the selection of transformed lines at a frequency of 9%. The rooted plantlets were transferred to pots, acclimated for 3?weeks in the culture room and then moved to the greenhouse. OMADS1 transgene was detected in transgenic lines by PCR, Southern blot analysis and RT-PCR were performed, and the results confirmed that OMADS1 was expressed in these 35S::OMADS1 transgenic plants. CaMV35S::OMADS1 transgenic Oncidium orchid plants flowered significantly earlier, produced more flowers and pseudobulbs than non-transgenic plants. The flower organ conversions were not observed in 35S::OMADS1 transgenic flowers of Oncidium. This is the first report on the ectopic expression of MADS box gene in O. Gower Ramsey using a simple and efficient gene transfer protocol.     

Characterization of promoter activity of the ethylene receptor gene OgERS1 from Oncidesa in transgenic Arabidopsis

Physiological changes associated with senescence of flowers and abscission of floral parts in Oncidesa (formerly Oncidium) cv. Gower Ramsey are caused by a plant hormone ethylene which is produced by pollinia cap dislodgment during postharvest handling and transportation. The ethylene receptor gene OgERS1 of Oncidesa has been previously cloned and characterized. To analyze promoter activity of OgERS1, transgenic Arabidopsis thaliana plants were generated to express the ß-glucuronidase (GUS) reporter gene under the control of 5’-upstream sequence of OgERS1 from Oncidesa. The expression pattern of the OgERS1 promoter at the cellular level was investigated by analysis of GUS activity. This promoter can activate gene expression in both actively dividing young tissues and abscission-related aging tissues. Expression of GUS was detected in the shoot meristem uniquely in 10 to 30 d-old-plants and was found in flower buds, axillary buds, flower stems, and abscission layers during later development. In 2- to 3-week-old transgenic Arabidopsis, exogenous ethylene, glucose, lactose, and maltose enhanced promoter activity implying that crosstalk between sugar and an ethylene receptor may exist. However, indole-3-acetic acid, benzylaminopurine, abscisic acid, heat, wounding, salinity, drought, and flooding slightly suppressed promoter activity. These results demonstrate that the promoter of OgERS1 was developmentally and environmentally regulated, and imply a potential for application of this bi-functional promoter to increase branching or enhanced dwarfing.     

Molecular characterization of the Oncidium orchid HDR gene encoding 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase, the last step of the methylerythritol phosphate pathway

Two pathways are used by higher plants for the biosynthesis of isoprenoid precursors: the mevalonate pathway in the cytosol and a 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in the plastids, with 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) catalyzing the last step in the MEP pathway. In order to understand the contribution of MEP pathway in isoprenoid biosynthesis of Oncidium orchid, a full-length cDNA corresponding to HDR from the flower tissues of Oncidium Gower Ramsey was cloned. The deduced OncHDR amino acid sequence contains a plastid signal peptide at the N-terminus and four conserved cysteine residues. RT-PCR analysis of HDR in Oncidium flowering plants revealed ubiquitous expression in organs and tissues, with preferential expression in the floral organs. Phylogenetic analysis revealed evolutionary conservation of the encoding HDR protein sequence. The genomic sequence of the HDR in Oncidium is similar to that in Arabidopsis, grape, and rice in structure. Successful complementation by OncHDR of an E. coli hdr ⁻ mutant confirmed its function. Transgenic tobacco carrying the OncHDR promoter-GUS gene fusion showed expression in most tissues, as well as in reproductive organs, as revealed by histochemical staining. Light induced strong GUS expression driven by the OncHDR promoter in transgenic tobacco seedlings. Taken together, our data suggest a role for OncHDR as a light-activated gene.     

Carbohydrate and Amino Acid Composition of α-Glucosidase from Saccharomyces logos

A water-soluble and neutral polysaccharide was extracted from the current pseudobulbs of Oncidium “Gower Ramsey” during the early inflorescence stage (flower stalk less than 4 cm) by hot water, precipitated with ethanol, and purified with an anion exchanger. From the data of monosaccharide composition and linkage and anomeric configuration analyses, the polysaccharide was identified as a linear β-1→4 linked mannan.     

Carotenoid Accumulation and Carotenogenic Genes Expression During Two Types of Persimmon Fruit (<Emphasis Type="Italic">Diospyros kaki</Emphasis> L.) Development

Persimmon (Diospyros kaki L.), belonging to the Ebenaceae family, has been used not only as a fresh fruit, but also for many medicinal uses. Carotenoids are the main pigment in persimmon fruit, which contribute significantly to fruit color and nutritional quality due to their composition and content. In this study, fruit quality indices, carotenoid contents and expression of carotenogenic genes were analyzed in two types of persimmon fruit. The results demonstrated that there was a positive correlation between fruit color and the contents of main composition and total carotenoids. Carotenoid accumulation in persimmon fruit resulted from the interaction of carotenogenic genes, but the molecular mechanisms responsible for accumulation of carotenoids in two types of persimmon fruit had a few differences. As a complete unit, the relatively low expression level of phytoene synthase gene (DkPSY) in “Niuxinshi” resulted in low carotenoid contents or even under the detection limit at the early fruit developmental stages; but low carotenoid contents in “Nishimurawase” were due to the relatively low expression level of carotenogenic genes other than DkPSY. At the late fruit developmental stages, increased expression levels of DkPSY, phytoene desaturase gene and beta-carotene hydroxylase gene (DkBCH) induced elevated carotenoid contents; because all carotenogenic genes strongly expressed in “Nishimurawase”, a large amount of carotenoids were accumulated. In addition, β-cryptoxanthin was the main composition whose content increased with the fruit maturity changes, which was mainly because of DkBCH which might lead more conversion of β-carotene to β-cryptoxanthin.     

Alteration of flower colour in <Emphasis Type="Italic">Ipomoea nil</Emphasis> through CRISPR/Cas9-mediated mutagenesis of <Emphasis Type="Italic">carotenoid cleavage dioxygenase 4</Emphasis>

Japanese morning glory, Ipomoea nil, exhibits a variety of flower colours, except yellow, reflecting the accumulation of only trace amounts of carotenoids in the petals. In a previous study, we attributed this effect to the low expression levels of carotenogenic genes in the petals, but there may be other contributing factors. In the present study, we investigated the possible involvement of carotenoid cleavage dioxygenase (CCD), which cleaves specific double bonds of the polyene chains of carotenoids, in the regulation of carotenoid accumulation in the petals of I. nil. Using bioinformatics analysis, seven InCCD genes were identified in the I. nil genome. Sequencing and expression analyses indicated potential involvement of InCCD4 in carotenoid degradation in the petals. Successful knockout of InCCD4 using the CRISPR/Cas9 system in the white-flowered cultivar I. nil cv. AK77 caused the white petals to turn pale yellow. The total amount of carotenoids in the petals of ccd4 plants was increased 20-fold relative to non-transgenic plants. This result indicates that in the petals of I. nil, not only low carotenogenic gene expression but also carotenoid degradation leads to extremely low levels of carotenoids.     

Heterologous Carotenoid-Biosynthetic Enzymes: Functional Complementation and Effects on Carotenoid Profiles in Escherichia coli

A limited number of carotenoid pathway genes from microbial sources have been studied for analyzing the pathway complementation in the heterologous host Escherichia coli. In order to systematically investigate the functionality of carotenoid pathway enzymes in E. coli, the pathway genes of carotenogenic microorganisms (Brevibacterium linens, Corynebacterium glutamicum, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodopirellula baltica, and Pantoea ananatis) were modified to form synthetic expression modules and then were complemented with Pantoea agglomerans pathway enzymes (CrtE, CrtB, CrtI, CrtY, and CrtZ). The carotenogenic pathway enzymes in the synthetic modules showed unusual activities when complemented with E. coli. For example, the expression of heterologous CrtEs of B. linens, C. glutamicum, and R. baltica influenced P. agglomerans CrtI to convert its substrate phytoene into a rare product—3,4,3′,4′-tetradehydrolycopene—along with lycopene, which was an expected product, indicating that CrtE, the first enzyme in the carotenoid biosynthesis pathway, can influence carotenoid profiles. In addition, CrtIs of R. sphaeroides and R. capsulatus converted phytoene into an unusual lycopene as well as into neurosporene. Thus, this study shows that the functional complementation of pathway enzymes from different sources is a useful methodology for diversifying biosynthesis as nature does.     

Isolation and characterization of the carotenoid biosynthesis genes of Flavobacterium sp. strain R1534

The Gram-negative bacterium Flavobacterium sp. strain R1534 is a natural producer of zeaxanthin. A 14 kb genomic DNA fragment of this organism has been cloned and a 5.1 kb piece containing the carotenoid biosynthesis genes sequenced. The carotenoid biosynthesis cluster consists of five genes arranged in at least two operons. The five genes are necessary and sufficient for the synthesis of zeaxanthin. The encoded proteins have significant homology to the crtE, crtB, crtY, crtI and crtZ gene products of other carotenogenic organisms. Biochemical assignment of the individual gene products was done by HPLC analysis of the carotenoid accumulation in Escherichia coli host strains transformed with plasmids carrying deletions of the Flavobacterium sp. strain R1534 carotenoid biosynthesis cluster.     

Phosphomannose-isomerase as a selectable marker to recover transgenic orchid plants (<Emphasis Type="Italic">Oncidium</Emphasis> Gower Ramsey)

A transformation method using the phosphomannose-isomerase (pmi) gene as a selectable marker was developed for orchid Oncidium Gower Ramsey. The pmi-gene, which converts mannose-6-phosphate to fructose-6-phosphate allowing for selection of transgenic plants on mannose selective medium. Genetically transformed plants of Oncidium were regenerated after cocultivating protocorm-like bodies with Agrobacterium tumefaciens strain GV3101 containing the vectors pEPYON-42P and pEPYON-42H with 35S::PMI and 35S::HPTII genes respectively. We observed that 35S::PMI (pEPYON-42P) produced high rate (27 plants) of mannose resistant transgenic plants compared to 35S::HPTII (pEPYON-42H) in which only fourteen hygromycin resistant transgenic plants were obtained. Mannose resistant transgenic plants were confirmed by PCR and Southern blot. The pmi gene expression in 35S::PMI (pEPYON-42P) transgenic plants was confirmed by RT-PCR. Furthermore, the duration of regeneration time of transgenic plants was significantly shorter in mannose selected system (4 months) than in hygromycin selected system (8 months). The pmi/mannose selection system is shown to be highly efficient for producing transgenic O. Gower Ramsey without using antibiotics or herbicides. For the first time, the pmi/mannose-based “positive” selection system has been used to obtain genetically engineered O. Gower Ramsey.     

An orchid (Oncidium Gower Ramsey) AP3-like MADS gene regulates floral formation and initiation

cDNA for a B group MADS box gene OMADS3 was isolated and characterized from Oncidium Gower Ramsey, an important species of orchid. OMADS3 encoding a 204 amino acid protein showed high sequence homology to both paleoAP3 and TM6 lineage of B group MADS box gene such as monocots AP3 homologue LMADS1 in lily and GDEF1 in Gerbera hybrida. Despite the sequence homology, consensus motifs identified in the C-terminal region of B group genes were absent in OMADS3. Southern analysis indicated that OMADS3 was present in O. Gower Ramsey genome in low copy numbers. Different from most B group genes, OMADS3 mRNA was detected in all four floral organs as well as in vegetative leaves. This is similar to the expression pattern of GDEF1. 35S::OMADS3 transgenic plants showed novel phenotypes by producing terminal flowers similar to those observed in transgenic plants ectopically expressed A functional genes such as AP1. Ectopic expression of OMADS3 cDNA truncated with the MADS box or C terminal region in Arabidopsis generated novel ap2-like flowers in which sepals and petals were converted into carpel-like and stamen-like structures. Yeast two-hybrid analysis indicated that OMADS3 is able to strongly form homodimers. Our results suggested that OMADS3 might represent an ancestral form of TM6-like gene which was conserved in monocots with a function similar to A functional gene in regulating flower formation as well as floral initiation.     

Isolation and Characterization of Three New Promoters from Gossypium hirsutum that Show High Activity in Reproductive Tissues

Engineering of plant protection requires well-characterized tissue-specific promoters for the targeted expression of insecticidal resistance genes. Herein, we describe the isolation of five different fragments of promoters of three distinct flower-specific cotton (Gossypium hirsutum) genes. Expression analyses of the three genes GhPME-like1, GhβGal-like1 and GhPL-like1 revealed that they are expressed highly in flowers buds ranging from 4 to 12 mm in size. Several putative regulatory cis-elements were identified in the promoter regions, including elements involved in the control of tissue-specific gene expression in pollen grains and fruits. In vivo analyses of these promoters were performed using the heterologous plant system Arabidopsis thaliana by fusing them with the gene uidA (GUS). GUS staining in Arabidopsis tissues revealed that their expression was restricted to anthers, with the majority of expression in pollen grains and in the upper portion of the carpels and siliques. A comparison between a CaMV35S::GUS constitutive promoter and the promoters isolated in this study revealed that the cotton promoters were more active and were specific to flowers and fruits, which are organs that are preferentially attacked by important pest insects such as the boll weevil (Anthonomus grandis). The activity of the promoters was also confirmed using transient expression assays in flower buds of G. hirsutum. The promoters of GhPME-like1, GhβGal-like1 and GhPL-like1 are specific to reproductive tissues and could represent important biotechnological tools for controlling insect pests, in particular the cotton boll weevil, which attacks floral and fruit tissues.