This study explored the potential of 120 wild yeasts obtained from eight different fruits, with particular emphasis on Saccharomyces cerevisiae S-1. This strain demonstrated remarkable attributes, notably its superior alcohol tolerance when compared to the conventional yeast, US-05. Significantly, the S-1 yeast unveiled its ability to produce three distinct aroma compounds—isoamyl acetate, β-phenylethyl acetate, and ethyl decanoate— not typically found in beer brewed with US-05 stain. These compounds are essential for enriching the sensory experience of beer drinkers. Further investigation revealed that the diminsihed expression of Leu2 in US-05 hindered metabolite synthesis, resulting in the absence of these aroma compounds. In contrast, S-1 exhibited approximately three times higher Leu2 expression, which may explain the observed differences in aroma compounds production. This sheds light on the potential mechanism behind the disparity in aroma profiles between the two strains. Overall, the study highlighted S-1’s capacity to enhance beer flavor and aroma. It suggested that wild yeast strains, such as S-1 yeast isolated from grapes, present innovative opportunities for the brewing industry. The hightened Leu2 expression and synthesis of unique aroma compounds in S-1 yeast underscore the latent capabilities of wild yeast strains in elevating beer characteristics.

Phalaenopsis orchids have a spectacular floral morphology with a highly evolved lip that offers a landing platform for pollinators. The typical morphological orchid lip features are essential for the special pollination mechanism of Phalaenopsis flowers. Previously, we found that in the lip, a member of the AP2/EREBP protein family was highly expressed. Here, we further confirmed its high expression and characterized its function during lip development. Phylogenetic analysis showed that AP2/EREBP belongs to the Va2 subgroup of ERF transcription factors. We named it PeERF1. We found that PeERF1 was only expressed at stage 5, as flowers opened. This coincided with both thickening of the cuticle and development of nanoridges. We performed knockdown expression of PeERF1 using CymMV-based virus-induced gene silencing in either the AP2 conserved domain, producing PeERF1_AP2-silenced plants, or the SHN specific domain, producing PeERF1_SHN-silenced plants. Using cryo-SEM, we found that the number of nanoridges was reduced only in the PeERF1_AP2-silenced group. This change was found on both the abaxial and adaxial surfaces of the central lip lobe. Expression of PeERF1 was reduced significantly in PeERF1_AP2-silenced plants. In cutin biosynthesis genes, expression of both PeCYP86A2 and PeDCR was significantly decreased in both groups. The expression of PeCYP77A4 was reduced significantly only in the PeERF1_AP2-silenced plants. Although PeGPAT expression was reduced in both silenced plants, but to a lesser degree. The expression of PeERF1 was significantly reduced in the petal-like lip of a big-lip variant. PeCYP77A4 and PeGPAT in the lip were also reduced, but PeDCR was not. Furthermore, heterologous overexpression of PeERF1 in the genus Arabidopsis produced leaves that were shiny on the adaxial surface. Taken together, our results show that in Phalaenopsis orchids PeERF1 plays an important role in formation of nanoridges during lip epidermis development.

BackgroundPhalaenopsis represents an important cash crop worldwide. Abundant flower colors observed in Phalaenopsis orchids range from red-purple, purple, purple-violet, violet, and violet-blue. However, violet-blue orchids are less bred than are those of other colors. Anthocyanin, vacuolar pH and metal ions are three major factors influencing flower color. This study aimed to identify the factors causing the violet-blue color in Phalaenopsis flowers and to analyze whether delphinidin accumulation and blue pigmentation formation can be achieved by transient overexpression of heterologous F3'5'H in Phalaenopsis.ResultsCyanidin-based anthocyanin was highly accumulated in Phalaenopsis flowers with red-purple, purple, purple-violet, and violet to violet-blue color, but no true-blue color and no delphinidin was detected. Concomitantly, the expression of PeF3'H (Phalaenopsis equestrsis) was high, but that of PhF3'5'H (Phalaenopsis hybrid) was low or absent in various-colored Phalaenopsis flowers. Transient overexpression of DgF3'5'H (Delphinium grandiflorum) and PeMYB2 in a white Phalaenopsis cultivar resulted a 53.6% delphinidin accumulation and a novel blue color formation. In contrast, transient overexpression of both PhF3'5'H and PeMYB2 did not lead to delphinidin accumulation. Sequence analysis showed that the substrate recognition site 6 (SRS6) of PhF3'5'H was consistently different from DgF3'5'Hs at positions 5, 8 and 10. Prediction of molecular docking of the substrates showed a contrary binding direction of aromatic rings (B-ring) with the SRS6 domain of DgF3'5'H and PhF3'5'H. In addition, the pH values of violet-blue and purple Phalaenopsis flowers ranged from 5.33 to 5.54 and 4.77 to 5.04, respectively. Furthermore, the molar ratio of metal ions (including Al3+, Ca2+ and Fe3+) to anthocyanin in violet-blue color Phalaenopsis was 190-, 49-, and 51-fold higher, respectively, than those in purple-color Phalaenopsis.ConclusionCyanidin-based anthocyanin was detected in violet-blue color Phalaenopsis and was concomitant with a high pH value and high molar ratio of Al3+, Ca2+ and Fe3+ to anthocyanin content. Enhanced expression of delphinidin is needed to produce true-blue Phalaenopsis.

Polyploidy plays an important role in the breeding of many crops as well as horticultural plants of the world. It is also important for the variety improvement of Phalaenopsis orchids. Most of the commercial orchids are tetraploids. However, most of the wild species of Phalaenopsis are diploid. Thus, a barrier to hybridization between these two groups of varieties limits the source of germplasm available for the breeding programs. Hence, a simple technique to scale up the ploidy level of diploid species is needed. Traditionally, chemical induction using anti-microtubule agents such as colchicine or oryzalin is used for polyploid induction. However, there are several disadvantages to these methods, such as complicated procedures, problems of toxicity to plants, and the occurrence of unfavorable chimeras. Search for alternate procedures might give better result to achieve the same objective. Due to the occurrence of endopolyploid cells in the tissues of protocorms and PLBs of the orchid, there is a chance to regenerate polyploid plants from these cells. A protocol for dissecting protocorms or PLBs in tissue culture without using anti-microtubule agents and the use of flow cytometry with 4,6-diamidino-2-phenylindole (DAPI) staining is described in details in this chapter. This is a simple, effective, and reliable technique to produce large numbers of polyploid plants in Phalaenopsis orchids. It might have a great impact on new variety development of the orchid in the future.

Phalaenopsis bellina is a scented orchid emitting large amount of monoterpenes. GERANYL DIPHOSPHATE SYNTHASE (PbGDPS) is the key enzyme for monoterpene biosynthesis, and shows concomitant expression with the emission of monoterpenes during flower development in P. bellina. Here, we identified a dual repeat cis-element in the GDPS promoter that is critical for monoterpene biosynthesis in Phalaenopsis orchids. A strong correlation between the dual repeat and the monoterpene production was revealed by examination of the GDPS promoter fragments over 12 Phalaenopsis species. Serial-deletion of the 2-kb GDPS promoter fragments demonstrated that the integrity of the dual repeat was crucial for its promoter activities. By screening the Arabidopsis transcription factors (TFs) cDNA library using yeast one-hybrid assay, AtbZIP18, a member of group I of bZIP TFs, was identified to be able to bind the dual repeat. We then identified PbbZIP4 in the transcriptome of P. bellina, showing 83% identity in the DNA binding region with that of AtbZIP18, and the expression level of PbbZIP4 was higher in the scented orchids. In addition, PbbZIP4 transactivated the GDPS promoter fragment containing the dual repeat in dual luciferase assay. Furthermore, transient ectopic expression of PbbZIP4 induced a 10-fold production of monoterpenoids in the scentless orchid. In conclusion, these results indicate that the dual repeat is a real TF-bound cis-element significant for GDPS gene expression, and thus subsequent monoterpene biosynthesis in the scented Phalaenopsis orchids.

Orchid Biotechnology III, pp. 1-21 (2017) No AccessCHAPTER 1: Genome Size Variation in Species of the Genus Phalaenopsis Blume (Orchidaceae) and Its Application in Variety ImprovementWen-Huei Chen and Ching-Yan TangWen-Huei ChenOrchid Research & Development Center, National Cheng Kung University, No. 1, University Road, Tainan 701, Taiwan and Ching-Yan Tanghttps://doi.org/10.1142/9789813109223_0001Cited by:1 PreviousNext AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsRecommend to Library ShareShare onFacebookTwitterLinked InRedditEmail Abstract: The genome size of Phalaenopsis species of four subgenera with eight sections was estimated by flow cytometry using propidium iodide (PI) and 4,6-diamidino-2-phenylindol (DAPI). Young ovary tissues of the flowers were used for this study. 2C values of 50 species obtained by using the PI fluorochrome were highly variable. These values varied to different degrees within each subgenus/section. The Nuclear DNA contents of species within the sections of Phalaenopsis and Fuscatae were homogenous without significant difference, while those in the sections of Zibrinae and Amboinenses were highly variable. The subgenera Parishianae and Aphyllae and two sections of the subgenus Phalaenopsis (Esmeralda and Deliciosae) showed higher mean 2C values than those of other sections. Intraspecific variations in 2C values were observed in some species. Comparison of the fluorescence ratios of DAPI and PI of the same species showed highly positive correlation between these two sets of data. Furthermore, there was a tendency for the increase in DAPI fluorescence ratios to be higher when the nuclear DNA content of a species increased. Nuclear DNA measurements obtained from PI and DAPI gave further insight into the genomic structure of the Phalaenopsis species. Considering the taxonomic classification and studies on the molecular phylogeny and biogeographic distribution, our data indicate the possibility of different patterns of phylogenetic relationship among different taxonomic groups of Phalaenopsis. The information provided by this study could also be useful for orchid breeders to enhance the efficiency of their improvement programs. This chapter is dedicated to Dr. Chi-Chu Tsai (1968–2015), who made an excellent contribution to the research on germplasm conservation, molecular phylogeny and biogeography in Phalaenopsis orchids. FiguresReferencesRelatedDetailsCited By 1A Target Capture-Based Method to Estimate Ploidy From Herbarium SpecimensJuan Viruel, María Conejero, Oriane Hidalgo, Lisa Pokorny and Robyn F. Powell et al.24 July 2019 | Frontiers in Plant Science, Vol. 10 Orchid Biotechnology IIIMetrics History PDF download

Malayan Orchid Review, pp. 41-47 (2016) No AccessThe breeding achievements from Phalaenopsis equestrisChia-Chi Hsu and Wen-Huei ChenChia-Chi HsuDepartment of Life Sciences, National Cheng Kung University, Tainan 701, Taiwan and Wen-Huei ChenOrchid Research and Development Center, National Cheng Kung University, Tainan 701, Taiwanhttps://doi.org/10.1142/9789814749954_0007Cited by:1 PreviousNext AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsRecommend to Library ShareShare onFacebookTwitterLinked InRedditEmail Abstract: Phalaenopsis equestris (Schauer) Rchb.f. is native to Philippines and Taiwan. It is a small plant and has short floral stalks with many flowers in various colours. Three varieties are distinguishable according to the flower colours, including var. rosea for the rose-colored sepals and petals and purple-red lip; var. alba for the pure white sepals, petals, and lip; and var. aurea for the white sepals and petals with a highly contrasting solid yellow lip. These varieties have been used as breeding parents with other species and hybrids to generate many distinguished cultivars with flowers of various types and colours. There are 541 first generation (G1) hybrids and a total of 20,906 hybrids from Phal. equestris in 13 generations registered with the Royal Horticultural Society (RHS, 2015). Several G1 and G2 hybrids of Phal. equestris have been popularly used as breeding parents, such as G1-generation Phal. Cassandra and Phal. Zuma's Pixie, and G2-generation Phal. Timothy Christopher, Phal. Carmela's Pixie, Phal. Sogo Vivien, Phal. Be Glad, and Phal. Little Gem Stripes. The other generations from Phal. equestris varieties as breeding parents were preferred for the multi-flowered and colorful features, but not for the small flowers, such as the G5-generation Phal. Golden Buddha, G6-generation Phal. Happy Valentine, and G7-generation Phal. Golden Peoker which have medium flower size. Recently, the breeding of cultivars with multi-flowered short stems has been widely accepted in the market and the use of Phal. equestris' progeny as breeding parents will continue and last for a long time. FiguresReferencesRelatedDetailsCited By 1Transcriptome atlas of Phalaenopsis equestrisAnna V. Klepikova, Artem S. Kasianov, Margarita A. Ezhova, Aleksey A. Penin and Maria D. Logacheva10 December 2021 | PeerJ, Vol. 9 Malayan Orchid ReviewMetrics History PDF download

Different night temperature treatments have been adopted for spike emergence in Phalaenopsis. The aim of this research was to study the dawn-dusk carbohydrate and organic acid levels responding to warm-(28 degrees C day/28 degrees C night, spike inhibitory) and cool-night (28 degrees C day/20 degrees C night, spike inducing) temperatures for six weeks with respect to different ages of leaves, including the new and not fully expanded 1st leaf (L1), the new and fully expanded 2nd leaf (L2), the older 5th leaf (L5) and the stem, the locus of spiking, and the dawn-dusk oscillations of metabolites associated with spiking. The results showed that L2 exhibited the maximum amplitude of nocturnal malate accumulation and daytime starch deposition on both treatments. Compared with the control, the warm-night treatment dramatically reduced the soluble sugar concentration, particularly the hexose levels in L2 and L5. However, in the stem, the sucrose concentration after warm-night treatment was significantly higher than that of the control at dawn and dusk. We deduce that the potential role of sucrose in the stem is to sustain the viability of the dormant spike bud. The occurrence of a large amount of soluble sugars in the L5 of the control implies that the breaking of bud dormancy may rely on the supply of sugars from the older leaves. The warm-night treatment significantly increased the citrate concentration in all leaves at dawn, implying that citrate might be playing a crucial role in protecting the leaves from warm-night stress. (C) 2013 Elsevier B.V. All rights reserved.

Endopolyploidy is frequently observed during development in plant species. Patterns of endopolyploidy are diverse in the various organs of different plant species. However, little is known about the role of endopolyploidization and its significance in orchids. This study was undertaken to determine the extent of endopolyploidy in different tissues of the diploid and tetraploid genotypes of Phalaenopsis aphrodite subsp. formosana and to examine the factors that contribute to increased ploidy levels. Endopolyploidy occurs in various tissues of diploid and tetraploid orchids, at different developmental stages and under different culture conditions, as determined by flow cytometry. In this study, different patterns of endopolyploidy were observed in parts of the protocorms, leaves, roots and flowers. Endopolyploidy was found in all tissues studied except the pollinia and the tetraploid ovaries. A higher degree of endopolyploidy was observed in mature tissues compared to young tissues, greenhouse-grown plants compared to in vitro plants and diploid plants compared to tetraploid plants. We discuss the relationships between endopolyploidization and several factors related to plant growth, as well as some practical considerations of these findings.

Distribution of nuclei of different ploidy levels was studied at different developmental stages in the embryonic tissue of the ovule, seed and protocorm of Phalaenopsis aphrodite subsp. formosana (Miwa) E.A. Christ. by a combination of flow cytometry and fluorescence microscopy with Apo Tome Slider. Three stages of ploidy patterns were identified in the ovular tissue at different days after pollination (DAP). Firstly, between pollination and fertilization (0 to 50 DAP), 2C nuclei were dominant over 4C nuclei and resulted in low level of cycle value. Secondly, between fertilization and seed maturation (50 to 110 DAP), amount of 4C nuclei increased rapidly, maintained at a high level and then decreased gradually to a low level. Small amount of 8C nuclei was also detected at this stage. Thirdly, at seed maturation (110 to 130 DAP), 2C nuclei became dominant over 4C nuclei again and the cycle value remained at a low but significant level at this stage. After seed sowing, nuclei with ploidy levels of 2C, 4C and 8C were observed in the developing protocorms as early as at 4 DAS (days after sowing). Nuclei with high ploidy levels (8C and 16C) increased gradually until 40 DAS in this study. Significant level of cycle value at this stage of protocorm development indicated the presence of endopolyploidy. 4,6-diamido-2-phenylindol (DAPI) staining showed large and prominent nuclei in the basal portions of the mature seeds before sowing and in the developing protocorms at 20 DAS. These findings clearly demonstrate the occurrence of different distribution patterns of nuclei with different ploidy levels during ovule, seed and protocorm de-velopment in Phalaenopsis aphrodite. These observations will provide fundamental information for further studies in Phalaenopsis orchids.

Orchid Biotechnology II, pp. 25-48 (2011) No AccessEndopolyploidy in Phalaenopsis Orchids and Its Application in Polyploid BreedingWen-Huei Chen, Yu-Lin Kao, Ching-Yan Tang, and Goamg-Tyng JeanWen-Huei ChenOrchid Research Center, National Cheng Kung University, Tainan 701, TaiwanCorresponding author., Yu-Lin KaoDepartment of Life Sciences and Institute of Biotechnology, National University of Kaohsiung, Kaohsiung, Taiwan, Ching-Yan TangDepartment of Life Sciences and Institute of Biotechnology, National University of Kaohsiung, Kaohsiung, Taiwan, and Goamg-Tyng JeanDepartment of Life Sciences and Institute of Biotechnology, National University of Kaohsiung, Kaohsiung, Taiwanhttps://doi.org/10.1142/9789814327930_0002Cited by:4 PreviousNext AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsRecommend to Library ShareShare onFacebookTwitterLinked InRedditEmail Abstract: Polyploidy plays an important role in the development of different varieties of Phalaenopsis orchids and other orchids. However, evidence indicates that the genetic base of commercial orchid hybrids is quite narrow. On the other hand, sterility in progenies derived from hybridization between species with different chromosome sizes or ploidy levels is one of the problems encountered by Phalaenopsis breeders. For these reasons, a research project was initiated to study the occurrence of endopolyploidy in Phalaenopsis species and to develop simple and effective techniques to determine nuclear DNA content and to double chromosome numbers. By employing flow cytometry, a database was set up for the nuclear DNA content of the wild species and some related hybrids of Phalaenopsis collected from several germplasm banks and orchid nurseries in Taiwan. Information in the database will be helpful to orchid breeders to select parental varieties for hybridization to increase their breeding efficiency. In addition, endopolyploidy in different tissues of Phalaenopsis species was thoroughly studied using flow cytometry. It was found that different patterns of endopolyploidy occurred in different tissues of Phalaenopsis species at various stages of development. Based on these findings, a simple and effective protocol was developed for the production of polyploid plants by sectioning of protocorms or protocorm-like bodies (PLBs) without using anti-microtubule agents. Through this technique, a conversion program to develop a series of tetraploid species of Phalaenopsis is in progress. This project will provide a wide range of tetraploid germplasm which will match the ploidy level of the commercial hybrids to accelerate the breeding of novel varieties of Phalaenopsis for the market. With the full-scale upgrading of the breeding program to the polyploid level, the production of a wide range of new hybrids of Phalaenopsis is expected to create a significant impact on the orchid industry in the coming years. FiguresReferencesRelatedDetailsCited By 4Polyploidization in Orchids: From Cellular Changes to Breeding ApplicationsJoe Abdul Vilcherrez-Atoche, Carla Midori Iiyama and Jean Carlos Cardoso9 February 2022 | Plants, Vol. 11, No. 4Evaluation of endopolyploidy patterns in selected Capsicum and Nicotiana species (Solanaceae)Viera Fráková, Lukáš Koprivý, Marianna Paľová, Vladislav Kolarčik and Pavol Mártonfi2 March 2021 | Biologia, Vol. 76, No. 7Floral Induction and Flower Development of OrchidsShan-Li Wang, Kotapati Kasi Viswanath, Chii-Gong Tong, Hye Ryun An and Seonghoe Jang et al.10 October 2019 | Frontiers in Plant Science, Vol. 10Understanding Seed and Protocorm Development in OrchidsEdward C. Yeung, Yuan-Yuan Li and Yung-I Lee9 May 2018 Orchid Biotechnology IIMetrics History PDF download

Orchid Biotechnology II, pp. 181-194 (2011) No AccessWarm- and Cool-Night Temperatures Alter Carbon Dioxide Fixation Pattern and Biochemical Metabolism in Phalaenopsis aphroditeYa-Chen Tseng, Yo-Ching Liu, Kai-Meng Tseng, Wen-Huei Chen and Heng-Long WangYa-Chen TsengInstitute of Biotechnology, National University of Kaohsiung, Kaohsiung 811, Taiwan, Yo-Ching LiuDepartment of Life Sciences, National University of Kaohsiung, Kaohsiung 811, Taiwan, Kai-Meng TsengDepartment of Life Sciences, National University of Kaohsiung, Kaohsiung 811, Taiwan, Wen-Huei ChenInstitute of Biotechnology, National University of Kaohsiung, Kaohsiung 811, TaiwanDepartment of Life Sciences, National University of Kaohsiung, Kaohsiung 811, Taiwan and Heng-Long WangInstitute of Biotechnology, National University of Kaohsiung, Kaohsiung 811, TaiwanDepartment of Life Sciences, National University of Kaohsiung, Kaohsiung 811, TaiwanCorresponding author.https://doi.org/10.1142/9789814327930_0010Cited by:0 PreviousNext AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsRecommend to Library ShareShare onFacebookTwitterLinked InRedditEmail Abstract: After being acclimated to constant warm- (28°C day/28°C night) and coolnight (28°C day/20°C night) temperature regimes in growth chambers for two weeks, two groups of mature Phalaenopsis aphrodite subsp. formosana plants both clearly exhibited a diurnal oscillation in stomatal conductance, net CO2 uptake rate, malate and starch levels, and phosphoenolpyruvate carboxylase (EC 4.1.1.31) and NAD+-malic enzyme (EC 1.1.1.39) activities. Hence, P. aphrodite is an obligate crassulacean acid metabolism plant. Nevertheless, a different night temperature greatly affected both the stomatal conductance and the contribution of ambient and respiratory CO2 to the nocturnal accumulation of malate. However, there appeared to be no significant difference between the two groups in the amounts of nocturnal accumulated malate and daily deposited starch. These results demonstrate that P. aphrodite is congruent with the characteristics of CAM plants in having great flexibility and plasticity in response to changes in environmental conditions. In addition, the formation of reproductive stem, viz. spike, was noticeably inhibited by a constant warm temperature, but induced by fluctuating warm-day and cool-night conditions. The relationship between the metabolic pool variation and spike induction in Phalaenopsis is also discussed. FiguresReferencesRelatedDetails Orchid Biotechnology IIMetrics History PDF download

Endopolyploidy was observed in the protocorms of diploid Phalaenopsis aphrodite subsp. formosana with ploidy doubling achieved by in vitro regeneration of excised protocorms, or protocorm-like bodies (PLBs). Thirty-four per cent of the PLBs regenerated from the first cycle of sectioned protocorms were found to be polyploids with ploidy doubled once or twice as determined by flow-cytometry. The frequency of ploidy doubling increased as the sectioning cycles increased and was highest in diploid followed by the triploid and tetraploid. Regeneration of the endopolyploid cells in the tissue of the protocorms or PLBs is proposed as the source of the development of ploidy doubled plantlets. The frequency of ploidy doubling was similar in seven other Phalaenopsis species, although the rate of increase within cycles was genotype specific. In two species, a comparison of five parameters between 5-month-old diploid and tetraploid potted plants showed only the stomata density differed significantly. The flowers of the tetraploid plant were larger and heavier than those of the diploids. This ploidy doubling method is a simple and effective means to produce large number of polyploid Phalaenopsis species plants as well as their hybrids. The method will be beneficial to orchid breeding programs especially for the interspecific hybridization between varieties having different chromosome sizes and ploidy levels.

Orchidaceae constitutes one of the largest families in angiosperms. The versatility and specialization in orchid floral morphology, scent and colour patterns endear orchidologists and plant biologists to orchid plants. Moreover, the co-evolution of the sophisticated orchid floral presentation and pollinators leads to the ingenious device of the orchid flower. Because of market needs and the current level of breeding technologies, the industry for biotech seedling products of Phalaenopsis spp. in Taiwan is currently focussed on the development of orchid species. Research into the molecular regulatory mechanism of floral development, scent production and colour presentation can undoubtedly enhance the understanding of orchid floral biology. Owing to advances in genomics and functional genomics, such as karyotypes, expressed sequence tags, bacterial artificial chromosomes and molecular markers, the isolation and identification of orchid floral genes has been increased rapidly. Orchid floral development was revealed to involve MADS-box-containing transcriptional regulators. The modified ‘ABCDE model’ of duplication and diversification of MADS-box genes has been proposed as a major driving force behind orchid floral organ identities. The scent biosynthesis pathway in the Phalaenopsis bellina flower was unravelled and found to be controlled by geraniol and linalool metabolism. Further interest has been promoted by the recent expansion of studies of orchid floral molecular biology. This information will provide broad scope for study of orchid floral development and serves as a starting point for uncovering the mystery of orchid evolution.

SUMMARY:Geranyl diphosphate (GDP) is the precursor of monoterpenes, which are the major floral scent compounds in Phalaenopsis bellina. The cDNA of P. bellina GDP synthase (PbGDPS) was cloned, and its sequence corresponds to the second Asp-rich motif (SARM), but not to any aspartate-rich (Asp-rich) motif. The recombinant PbGDPS enzyme exhibits dual prenyltransferase activity, producing both GDP and farnesyl diphosphate (FDP), and a yeast two-hybrid assay and gel filtration revealed that PbGDPS was able to form a homodimer. Spatial and temporal expression analyses showed that the expression of PbGDPS was flower specific, and that maximal PbGDPS expression was concomitant with maximal emission of monoterpenes on day 5 post-anthesis. Homology modelling of PbGDPS indicated that the Glu-rich motif might provide a binding site for Mg(2+) and catalyze the formation of prenyl products in a similar way to SARM. Replacement of the key Glu residues with alanine totally abolished enzyme activity, whereas their mutation to Asp resulted in a mutant with two-thirds of the activity of the wild-type protein. Phylogenetic analysis indicated that plant GDPS proteins formed four clades: members of both GDPS-a and GDPS-b clades contain Asp-rich motifs, and function as homodimers. In contrast, proteins in the GDPS-c and GDPS-d clades do not contain Asp-rich motifs, but although members of the GDPS-c clade function as heterodimers, PbGDPS, which is more closely related to the GDPS-c clade proteins than to GDPS-a and GDPS-b proteins, and is currently the sole member of the GDPS-d clade, functions as a homodimer.

After being acclimated to constant warm (28°C day/28°C night) and cool-night temperature (28°C day/20°C night) regimes in growth chambers for 2 weeks, the two groups of mature Phalaenopsis aphrodite subsp. formosana plants both clearly exhibited a diurnal oscillation of stomatal conductance, net CO2 uptake rate, malate and starch levels, and the phosphoenolpyruvate carboxylase (EC 4.1.1.31) and NAD+-malic enzyme (EC 1.1.1.39) activities. Hence, P. aphrodite is an obligate crassulacean acid metabolism plant. Nevertheless, different night temperature greatly affected both the stomatal conductance and the contribution of ambient and respiratory CO2 to the nocturnal accumulation of malate. However, the amounts of nocturnal accumulated malate and daily deposited starch appeared to have no significant difference between the two groups. These results demonstrate that P. ahrodite is congruent with the characteristics of CAM plants having great flexibility and plasticity in response to changes in environmental conditions. In addition, the formation of reproductive stem, viz. spike, was noticeably inhibited by a constant warm temperature, but induced by a fluctuating warm day and cool night condition. The relationship between the metabolic pool variation and spike induction of Phalaenopsis is also discussed.