The plastid genome characteristics of a moth orchid (Phalaenopsis wilsoniii, Orchidaceae)

Phalaenopsis wilsonii is a typical deciduous species in the horticulturally well-known genus, Phalaenopsis. Tshi species is belonging to the section Aphyllae in moth orchid, and is endemic to South China. Although the Aphyllae section display the deciduous feature that is unique in this genus, their genetic information is still insufficient and limited them as breeding parent in moth orchid. Here, we reported and characterized the complete chloroplast genome for Phalaenopsis wilsonii. We found the total size of the chloroplast genome was 145,373 bp, constituting of a large single copy (LSC) region (84,996 bp), a small single-copy region (10,668 bp) and two inverted repeats (IRs) regions (24,855 bp). Based on homologous searching on database, we annotated 76 protein-coding genes, 38 tRNA, and 8 rRNA. The phylogenetic reconstruction revealed that P. wilsonii show the closest relationship with P. lowii within subgenus Parishianae.

Blue Light Acclimation Reduces the Photoinhibition of Phalaenopsis aphrodite (Moth Orchid)

The moth orchid is an important ornamental crop. It is very sensitive to high light irradiation due to photoinhibition. In this study, young orchid tissue culture seedlings and 2.5” potted plants pretreated under blue light (BL, λmax = 450 nm) at 100 µmol m−2 s−1 for 12 days (BL acclimation) were found to have an increased tolerance to high light irradiation. After BL acclimation, orchids had an increased anthocyanin accumulation, enhanced chloroplast avoidance, and increased chlorophyll fluorescence capacity whenever they were exposed to high light of 1000 μmol m−2 s−1 for two weeks (HL). They had higher Fv/Fm, electron transport rate (ETR), chlorophyll content, catalase activity and sucrose content when compared to the control without BL acclimation. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that transcript levels of phototropins, D1, RbcS, PEPCK, Catalase and SUT2 were upregulated in the BL-acclimated orchids. Consequently, BL acclimation orchids had better growth when compared to the control under long-term high light stress. In summary, this study provides a solution, i.e., BL acclimation, to reduce moth orchid photoinhibition and enhance growth before transplantation of the young tissue culture seedlings and potted plants into greenhouses, where they usually suffer from a high light fluctuation problem.

APPLICATION OF BENZYLADENINE (BA) IN THE FORM OF LANOLIN PASTE ON FLOWER-STALK BUDS SUCCESSFULLY INDUCED RE-BLOOMING OF HYBRID Phalaenopsis

Hybrid moth orchid (genus Phalaenopsis) is one of the most popular ornamentals in Indonesia. It has beautiful and long-lasting flowers, but cultivating this orchid is still become a challenging issues due to the need of specific condition to grow and long time period to re-blooming. Plant growth regulators (PGR) (i.e. Benzyladenine (BA)) has been widely documented as a flower-inducing substance in several orchids. However, the optimal concentration and its mechanism in inducing flower-stalk bud and re-blooming is still unclear. This research aimed to study the effects of BA application in the form of lanolin paste on hybrid Phalaenopsis flower-stalk buds. We conducted this study using completely randomized design with four replications at the greenhouse laboratory Faculty of Agriculture University of Lampung on August to December 2018. We divided the orchid into 5 group of BA concentration (0, 1000, 1500, 3000, or 6000 ppm). The sheath of the fourth or fifth buds of the flowers were carefully opened, then it smeared with BA. The percentage of bud break into flower spike or keiki, length of shoots or spike and number of open flowers were recorded until 10 weeks of observation. The results showed that, neither of the buds under the control treatment (without BA), 1000 ppm nor 1500 ppm BA broke and grew into keiki or spike. On the other hand, application of BA at 3000 ppm or 6000 ppm successfully induced 100% flower spikes on the buds treated. No keiki was formed in all buds treated. In addition, treatment of the buds with 6000 ppm BA produced longer flower spikes as well as more open flowers. We conclude that the application of BA (minimal 3000 ppm) successfully induced flower spike of hybrid Phalaenopsis.

Spike Activator 1, Encoding a bHLH, Mediates Axillary Bud Development and Spike Initiation in Phalaenopsis aphrodite

Double-spikes Phalaenopsis orchids have greater market value than those with single-spike. In this study, a gene designated as Spike Activator 1 (SPK1), which encodes a basic helix-loop-helix (bHLH) transcription factor, was isolated and characterized from Phalaenopsis aphrodite (moth orchid). SPK1 was highly expressed in the meristematic tissues. In the axillary bud, SPK1 was highly upregulated by a moderately low temperature of 20 °C but downregulated by a spike inhibition temperature of 30 °C. SPK1 protein is localized in the nucleus. Another bHLH, bHLH35, which is also highly expressed in young tissues in the same way as SPK1 was also identified. In contrast to SPK1, bHLH35 transcripts are downregulated at 20 °C but upregulated at 30 °C. Bimolecular florescence complementation assay and yeast two-hybrid assays indicated that SPK1 interacts with bHLH35 and forms a heterodimer. Virus-induced gene silencing (VIGS) showed that 7 out of 15 vector control plants produced double spikes but that only 1 out of 15 VIGS-spk1 plants produced double spikes. RT-qPCR results indicated that VIGS-spk1 downregulated gene expression levels of SPK1, FT, CYCB, and EXPA8. Overall, we propose that SPK1 plays an essential role in early axillary bud development and spike initiation of P. aphrodite.

Phototropins Mediate Chloroplast Movement in Phalaenopsis aphrodite (Moth Orchid)

Chloroplast movement is important for plants to avoid photodamage and to perform efficient photosynthesis. Phototropins are blue light receptors in plants that function in chloroplast movement, phototropism, stomatal opening, and they also affect plant growth and development. In this study, full-length cDNAs of two PHOTOTROPIN genes, PaPHOT1 and PaPHOT2, were cloned from a moth orchid Phalaenopsis aphrodite, and their functions in chloroplast movement were investigated. Phylogenetic analysis showed that PaPHOT1 and PaPHOT2 orthologs were highly similar to PHOT1 and PHOT2 of the close relative Phalaenopsis equestris, respectively, and clustered with monocots PHOT1 and PHOT2 orthologs, respectively. Phalaenopsis aphrodite expressed a moderate level of PaPHOT1 under low blue light of 5 μmol�m−2�s−1 (BL5) and a high levels of PaPHOT1 at >BL100. However, PaPHOT2 was expressed at low levels at <BL50 but expressed at high levels at > BL100. Analysis of light-induced chloroplast movements using the SPAD method indicated that orchid accumulated chloroplasts at <BL10. The chloroplast avoidance response was detectable at >BL25 and significant chloroplast avoidance movement was observed at >BL100. Virus-induced gene silencing of PaPHOTs in orchids showed decreased gene expression of PaPHOTs and reduced both chloroplast accumulation and avoidance responses. Heterologous expression of PaPHOT1 in Arabidopsis phot1phot2 double mutant recovered chloroplast accumulation response at BL5, but neither PaPHOT1 nor PaPHOT2 was able to restore mutant chloroplast avoidance at BL100. Overall, this study showed that phototropins mediate chloroplast movement in Phalaenopsis orchid is blue light-dependent but their function is slightly different from Arabidopsis which might be due to gene evolution.