SUMMARY
Terrestrial plants emit volatiles into the atmosphere to attract both pollinators and the enemies of herbivores, for defense. Phalaenopsis bellina is a scented orchid species in which the ...main scent components are monoterpenes, including linalool and geraniol, and their derivatives. Here, we investigated whether ABC transporters are involved in floral scent emission. We carried out whole‐genome identification of ABC transporter‐related genes using four floral transcriptomics libraries of P. bellina. We identified 86 ABC subfamily G genes related to terpenoid transport. After comparing the gene expression patterns of P. bellina with that of Phalaenopsis aphrodite subsp. formosana, a scentless species, followed by gene‐to‐gene correlation analysis, PbABCG1 and PbABCG2 were selected. The temporal expression of both PbABCG1 and PbABCG2 was highly correlated with that of the key enzyme PbGDPS and the major transcription factor PbbHLH4 in monoterpene biosynthesis, with optimal expression on day 5 post‐anthesis. Spatial gene expression analysis showed that PbABCG1 was highly expressed in sepals, whereas PbABCG2 was expressed in the lip. Subcellular localization with a GFP fusion protein revealed that both PbABCG1 and PbABCG2 are cytoplasmic membrane proteins. Co‐downregulation of PbABCG1 and PbABCG2 using both double‐strand RNA interference and tobacco rattle virus‐based gene silencing led to a significant decrease in monoterpene emission, accompanied by an increase in the internal monoterpene pools. Furthermore, ectopic expression of PbABCG1 and PbABCG2 in an ABC16− mutant yeast strain rescued its tolerance to geraniol. Altogether, our results indicate that PbABCG1 and PbABCG2 play substantial roles in monoterpene transport/emission in P. bellina floral scent.
Significance Statement
The biosynthesis and regulation of volatile organic compounds are understood well, but the mechanism through which they are emitted to the atmosphere has remained unclear until now. We identified two PbABCG1 and PbABCG2 transporters and assessed them for involvement in the emission of monoterpene floral scent in Phalaenopsis bellina, the results suggest that PbABCG1 and PbABCG2 play a substantial role in the transport/emission of monoterpenes from flowers.
The harlequin/black flowers in
orchids contain dark purple spots and various pigmentation patterns, which appeared as a new color in 1996. We analyzed this phenotype by microscopy, HPLC, gene ...functional characterization, genome structure analysis, and transient overexpression system to obtain a better understanding of the black color formation in
orchids. Most mesophyll cells of harlequin flowers showed extremely high accumulation of anthocyanins as well as a high expression of
MYB11 (
) as the major regulatory R2R3-MYB transcription factor for regulating the production of the black color. In addition, we analyzed the expression of basic helix-loop-helix factors, WD40 repeat proteins, and
- and
-like repressors for their association with the spot pattern formation. To understand the high expression of
in harlequin flowers, we isolated the promoter sequences of
from red and harlequin flowers. A retrotransposon, named
(
), was identified and inserted in the upstream regulatory region of
The insertion resulted in strong expression of
and thus extremely high accumulation of anthocyanins in the harlequin flowers of the
Yushan Little Pearl variety. A dual luciferase assay showed that the insertion of
enhanced
expression by at least 2-fold compared with plants not carrying the insertion. Furthermore, the presence of
explains the high mutation rates resulting in many variations of pigmentation patterning in harlequin flowers of
orchids.
Phalaenopsis 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.
Cyanidin-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 Al
, Ca
and Fe
) to anthocyanin in violet-blue color Phalaenopsis was 190-, 49-, and 51-fold higher, respectively, than those in purple-color Phalaenopsis.
Cyanidin-based anthocyanin was detected in violet-blue color Phalaenopsis and was concomitant with a high pH value and high molar ratio of Al
, Ca
and Fe
to anthocyanin content. Enhanced expression of delphinidin is needed to produce true-blue Phalaenopsis.
Orchids are the most species-rich plants and highly interactive with pollinators via visual or olfactory cues. Biosynthesis and emission of volatile organic compounds (VOCs) to the atmosphere ...facilitate the olfactory cues and ensure successful pollination.
Phalaenopsis bellina
is a scented orchid with monoterpenes as major VOCs, comprising linalool, geraniol, and their derivatives. Comparative transcriptomics analysis identified four
terpene synthase-b
(
TPS-b
) genes and two
TPS-e/f
genes with differential gene expression between scented and scentless
Phalaenopsis
species. Here, we confirmed their differential expression between scented and scentless
Phalaenopsis
orchids and excluded one
TPS-b
candidate. We analyzed the temporal and spatial expression and functionally characterized these
TPSs
. Both
TPS-b
and
TPS-e/f
genes showed an increased expression on blooming day or 3 days post-anthesis (D + 3) before the optimal emission of floral scent on D + 5, with especially high expression of
PbTPS5
and
PbTPS10
. The
TPS-b
genes are expressed exclusively in reproductive organs, whereas the
TPS-e/f
genes are expressed in both reproductive and vegetative organs.
In planta
functional characterization of both
PbTPS5
and
PbTPS10
in tobacco and scentless
Phalaenopsis
plants did not produce terpenoids. Further ectopic expression in scented
Phalaenopsis
cultivar
P.
I-Hsin Venus showed that linalool was the main product, with
PbTPS10
displaying 3-fold higher activity than
PbTPS5.
On
in vitro
enzyme assay with purified recombinant TPS-b proteins ectopically expressed in
Escherichia coli
, geraniol was the product catalyzed by
PbTPS5
and
PbTPS9
.
PbTPS3
was a linalool/(β)-cis-ocimene synthase and
PbTPS4
a linalool synthase. In conclusion, both
TPS-b
and
TPS-e/f
enzymes orchestrated floral monoterpene biosynthesis in
P. bellina
.
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 Mg²⁺ 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.
In our previous studies, we identified four DEFICIENS (DEF)-like genes and one GLOBOSA (GLO)-like gene involved in floral organ development in Phalaenopsis equestris. Revealing the DNA binding ...properties and protein-protein interactions of these floral homeotic MADS-box protein complexes (PeMADS) in orchids is crucial for the elucidation of the unique orchid floral morphogenesis. In this study, the interactome of B-class PeMADS proteins was assayed by the yeast two-hybrid system (Y2H) and glutathione S-transferase (GST) pull-down assays. Furthermore, the DNA binding activities of these proteins were assessed by using electrophoretic mobility shift assay (EMSA). All four DEF-like PeMADS proteins interacted individually with the GLO-like PeMADS6 in Y2H assay, yet with different strengths of interaction. Generally, the PeMADS3/ PeMADS4 lineage interacted more strongly with PeMADS6 than the PeMADS2/PeMADS5 lineage did. In addition, independent homodimer formation for both PeMADS4 (DEF-like) and PeMADS6 (GLO-like) was detected. The protein-protein interactions between pairs of PeMADS proteins were further confirmed by using a GST pull-down assay. Furthermore, both the PeMADS4 homodimer and the PeMADS6 homodimer/homomultimer per se were able to bind to the MADS-box protein-binding motif CArG. The heterodimeric complexes PeMADS2-PeMADS6, PeMADS4-PeMADS6 and PeMADS5-PeMADS6 showed CArG binding activity. Taken together, these results suggest that various complexes formed among different combinations of the five B-class PeMADS proteins may increase the complexity of their regulatory functions and thus specify the molecular basis of whorl morphogenesis and combinatorial interactions of floral organ identity genes in orchids.
Kinetic and equilibrium isotope effects in peptide group hydrogen exchange reactions were evaluated. Unlike many other reactions, kinetic isotope effects in amide hydrogen exchange are small because ...exchange pathways are not limited by bond-breaking steps. Rate constants for the acid-catalyzed exchange of peptide group NH, ND, and NT in H2O are essentially identical, but a solvent isotope effect doubles the rate in D2O. Rate constants for base-catalyzed exchange in H2O decrease slowly in the order NH > ND > NT. The alkaline rate constant in D2O is very close to that in H2O when account is taken of the glass electrode pH artifact and the difference in solvent ionization constant. Small equilibrium isotope effects lead to an excess equilibrium accumulation of the heavier isotopes by the peptide group. Results obtained are expressed in terms of rate constants for the random coil polypeptide, poly-DL-alanine, to provide reference rates for protein hydrogen exchange studies as described in Bai et al. preceding paper in this issued.
In this study, we show how the red spotted grouper nervous necrosis virus (RGNNV) causes loss of mitochondrial membrane potential and promotes host secondary apoptotic necrosis. RGNNV viral proteins ...such as protein α (42 kDa) and protein A (110 kDa) were quickly expressed between 12 h and 24 h postinfection (p.i.) in GL-av cells. Annexin V staining revealed that the NNV infection of GL-av cells induced phosphatidylserine (PS) externalization and development of bulb-like vesicles (bleb formation) at 24 h p.i. NNV infection also induced DNA fragmentation detectable by TUNEL assay between 12 h (8%) and 72 h (32%) p.i. Bongkrekic acid (1.6 μM; BKA) blocked permeability of the mitochondrial permeability transition pore, but cyclosporine A (CsA) did not block secondary necrosis. Finally, secondary necrotic cells were not engulfed by neighboring cells. Our data suggest that RGNNV induces apoptotic death via opening the mitochondrial permeability transition pore thereby triggering secondary necrosis in the mid-apoptotic phase.
To investigate the role of Asp 26 and Lys 57, two conserved, buried residues, in the redox mechanism of Escherichia coli thioredoxin (Trx), three mutant proteins, Asp 26 → Ala (D26A), Lys 57 → Met ...(K57M), and the double mutant D26A/K57M, were prepared, replacing the charged amino acids with hydrophobic residues with similar sizes. Both the oxidized (Trx-S2) and reduced Trx-(SH)2 forms of the mutant thioredoxins are fully folded and similar in overall structure to the wild-type protein (wt). The structure of the active site hydrophobic surface is unchanged by the mutation of Asp 26 and Lys 57, since DNA polymerase activity in the 1:1 complex of the T7 gene 5 protein and mutant Trx-(SH)2 shows similar K d values (∼5 nM) for both mutants and wt. In contrast, redox reactions involving thioredoxin as a catalyst of the reduction of disulfides or oxidation of dithiols are strongly affected by the mutations. In the reaction of Trx-S2 with thioredoxin reductase at pH 8.0, the k cat/K m value for the D26A mutant is decreased by a factor of 10 from that of wt, while the value for the D26A/K57M mutant is reduced 40-fold. The activity of Trx-(SH)2 as a protein disulfide reductase was measured with insulin, using fluorescence to detect oxidation of thioredoxin. At 15 °C and pH 8.0, both the D26A and K57M mutants showed 5−10-fold decreases in rates of reaction compared to those of the wild type, and the pH−rate profiles for the mutants were shifted 1 (K57M) and 2 (D26A) units to higher pH compared with the wt curve. NMR measurements for the three mutant proteins indicate that the proteins have the same global fold as that of the wild type, although changes in the chemical shifts of a number of resonances indicate local structural changes in the active site region. The resonances of oxidized D26A and D26A/K57M are pH-independent between pH 6.0 and 10.0, confirming the identification of the active site group titrating with a pK a of 7.5 in wt Trx-S2 as Asp 26. A profound change in the pK a of Asp 26, from 7.5 in the wild type to 9.4 in the mutant, is observed for K57M Trx-S2. The pH-dependent behavior of the resonances is affected in all mutant Trx-(SH)2 proteins. A single pK a shifted to higher values is observed on both the Cys 32 and Cys 35 Cβ resonances. Ultraviolet absorbance measurements (A 240) as a function of pH for wt Trx-(SH)2 demonstrate that the cysteine thiols titrate with apparent pK as of about 7.1 and 9.9. The mutant proteins each show a single transition in the A 240 measurements, with a midpoint at pH 7.8−8.0, consistent with the NMR results. The change in absorbance at 240 nm with increasing pH indicates that the number of thiols titrating in each mutant is greater than one but less than two. It is clear that both thiol pK as have been significantly shifted by the mutations. The Cys 32 pK a is moved from 7.1 in wt to 7.8−8.0 in the mutants. The value of the Cys 35 pK a either is indistinguishable from that of Cys 32, thus accounting for more than one thiol titrating in the UV absorbance measurements or else is shifted to much higher pHs (>10) where its transition is masked in both UV and NMR measurements by the effects of ionization of the tyrosine residues and unfolding of the protein. Our results strongly suggest that the buried Asp 26 carboxyl and Lys 57 ε-amino groups significantly affect the pK as of the active site thiols, particularly that of the exposed low-pK a thiol Cys 32, thereby enhancing the rates of thiol−disulfide reactions at physiological pH.
It is known that the non-structural B2 protein of nervous necrosis virus (NNV) plays an important role in viral replication and can inhibit the RNA interference system of the host cell. Moreover, the ...mechanism of NNV B2 protein to inhibit RNAi is by sequestration and protection of double strand (ds) RNA. In the flock house virus (FHV), a model alphanodavirus, the structural and mutational analysis of B2 identified that the positively charged Arg54 of the α2 helix mediated the dsRNA-binding activity. According to the betanodavirus B2 protein alignment and modeling results, the amino acid sequences and the predicted structure of betanodavirus B2 are different from alphanodaviruses. It was suggested that the four Arg residues of α3 helix between amino residues 52–60 of B2 may be involved in dsRNA-binding activity. Thus, this study replaced these four Arg residues with Gln at position 52 (R52Q), 53 (R53Q), 59 (R59Q), and 60 (R60Q) by site-directed mutagenesis method. The dsRNA-binding assays of these B2 mutants demonstrated that mB2(R53Q) and mB2(R60Q) mutants are dsRNA-binding defective. Moreover, we have found mB2(R53Q) and mB2(R60Q) could not antagonize RNAi by using HeLa cell as an RNAi inhibition model. These results suggested that Arg53 and Arg60 of betanodavirus B2 protein may be similar to Arg54 of alphanodavirus FHV B2 protein and are critical for dsRNA binding and RNAi-inhibiting. This study may serve as an example where bioinformatic analysis of related viral genomes may lead to meaningful structural and functional clues for certain viral proteins.
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