No abstract

SummaryGeranyl 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.

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 alpha (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 microM; 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.

The harlequin/black flowers in Phalaenopsis 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 Phalaenopsis orchids. Most mesophyll cells of harlequin flowers showed extremely high accumulation of anthocyanins as well as a high expression of Phalaenopsis equestris MYB11 (PeMYB11) 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 MYB27-and MYBx-like repressors for their association with the spot pattern formation. To understand the high expression of PeMYB11 in harlequin flowers, we isolated the promoter sequences of PeMYB11 from red and harlequin flowers. A retrotransposon, named Harlequin Orchid RetroTransposon 1 (HORT1), was identified and inserted in the upstream regulatory region of PeMYB11. The insertion resulted in strong expression of PeMYB11 and thus extremely high accumulation of anthocyanins in the harlequin flowers of the Phalaenopsis Yushan Little Pearl variety. A dual luciferase assay showed that the insertion of HORT1 enhanced PeMYB11 expression by at least 2-fold compared with plants not carrying the insertion. Furthermore, the presence of HORT1 explains the high mutation rates resulting in many variations of pigmentation patterning in harlequin flowers of Phalaenopsis orchids.

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.