Arabidopsis PARC6 Is Critical for Plastid Morphogenesis in Pavement, Trichome, and Guard Cells in Leaf Epidermis.

Recently, a recessive Arabidopsis thaliana mutant with abundant stromules in leaf epidermal pavement cells was visually screened and isolated. The gene responsible for this mutant phenotype was identified as PARC6, a chloroplast division site regulator gene. The mutant allele parc6-5 carried two point mutations (G62R and W700stop) at the N- and C-terminal ends of the coding sequence, respectively. Here, we further characterized parc6-5 and other parc6 mutant alleles, and showed that PARC6 plays a critical role in plastid morphogenesis in all cell types of the leaf epidermis: pavement cells, trichome cells, and guard cells. Transient expression of PARC6 transit peptide (TP) fused to the green fluorescent protein (GFP) in plant cells showed that the G62R mutation has no or little effect on the TP activity of the PARC6 N-terminal region. Then, plastid morphology was microscopically analyzed in the leaf epidermis of wild-type (WT) and parc6 mutants (parc6-1, parc6-3, parc6-4 and parc6-5) with the aid of stroma-targeted fluorescent proteins. In parc6 pavement cells, plastids often assumed aberrant grape-like morphology, similar to those in severe plastid division mutants, atminE1, and arc6. In parc6 trichome cells, plastids exhibited extreme grape-like aggregations, without the production of giant plastids (>6 µm diameter), as a general phenotype. In parc6 guard cells, plastids exhibited a variety of abnormal phenotypes, including reduced number, enlarged size, and activated stromules, similar to those in atminE1 and arc6 guard cells. Nevertheless, unlike atminE1 and arc6, parc6 exhibited a low number of mini-chloroplasts (< 2 µm diameter) and rarely produced chloroplast-deficient guard cells. Importantly, unlike parc6, the chloroplast division site mutant arc11 exhibited WT-like plastid phenotypes in trichome and guard cells. Finally, observation of parc6 complementation lines expressing a functional PARC6-GFP protein indicated that PARC6-GFP formed a ring-like structure in both constricting and non-constricting chloroplasts, and that PARC6 dynamically changes its configuration during the process of chloroplast division.

Identification of novel C(20) and C (22) trienoic acids from arctiid and geometrid female moths that produce polyenyl Type II Sex pheromone components.

Gas chromatography-mass spectrometry (GC-MS) and GC-electroantennographic detection (EAD) analyses of the sex pheromone extract from a wasp moth, Syntomoides imaon (Lepidoptera: Arctiidae: Syntominae), showed that virgin females produced (Z,Z,Z)-3,6,9-henicosatriene and (Z,Z,Z)-1,3,6,9-henicosatetraene with a trace amount of their C(20) analogs. Identification of the chemical structures was facilitated by comparison with authentic standards and the double-bond positions were confirmed by dimethyl disulfide derivatization of monoenes produced by a diimide reduction. In a field test in the Yonaguni-jima Islands, males of the diurnal species were captured in traps baited with a 1:2 mixture of the above-described synthetic C(21) polyenes. Lipids were extracted from the abdominal integument and its associated oenocytes and peripheral fat bodies. Following derivatization, fatty acid methyl esters (FAMEs) were fractionated by HPLC equipped with an ODS column, and methyl (Z,Z,Z)-11,14,17-icosatrienoate and (Z,Z,Z)-13,16,19-docosatrienoate were identified by GC-MS. These novel C(20) and C(22) acid moieties are longer-chain analogs of linolenic acid, (Z,Z,Z)-9,12,15-octadecatrienoic acid. They are presumed to be biosynthetic precursors of the S. imaon pheromone because the C(21) trienyl component might be formed by decarboxylation of the C(22) acid. On the other hand, the C(20) acid, but not the C(22) acid, was found in FAMEs of Ascotis selenaria cretacea (Lepidoptera: Geometridae), which secretes C(19) pheromone components, (Z,Z,Z)-3,6,9-nonadecatriene and the monoepoxy derivative, indicating that different systems of the chain elongation might play an important role in developing species-specific communication systems mediated with polyunsaturated hydrocarbons and/or epoxy derivatives, components of Type II lepidopteran sex pheromones.

Identification of a unique pheromonotropic neuropeptide including double FXPRL motifs from a geometrid species, Ascotis selenaria cretacea, which produces an epoxyalkenyl sex pheromone.

Virgin females of the Japanese giant looper (Ascotis selenaria cretacea, Assc) in the family of Geometridae secrete an epoxyalkenyl sex pheromone to attract males. To regulate its biosynthesis in the pheromone gland, Assc females produce a pheromone biosynthesis-activating neuropeptide (PBAN) in the suboesophageal ganglion (SG), as do females in many lepidopteran species. We have isolated Assc-PBAN cDNA, which encodes 181 amino acids, including a PBAN homologue and four other putative peptides: a diapause hormone (DH) homologue, alpha-SG neuropeptide (SGNP), beta-SGNP, and gamma-SGNP, all of which shared an FXPR(K)L motif on their C-termini. Although PBANs with 30-35 amino acids have been characterized from 15 other species, the Assc-PBAN homologue consisted of 28 amino acids and showed low homology (<46%) compared with the others. Assc-beta-SGNP with eight amino acids was also shorter than the other beta-SGNPs (16-22 amino acids). Furthermore, all of the known PBAN cDNAs have a GRR sequence between beta-SGNP and PBAN as a cleavage site, but the Assc-PBAN cDNA showed an unusual GR sequence at the corresponding position, indicating the possibility of non-cleavage between the beta-SGNP and PBAN. When the GR sequence was a cleavage site, the question arose of whether or not the glutamine residue at the N-terminus of the Assc-PBAN homologue was cyclized. To identify the sequence of the Assc-PBAN, the brain-SG extract was fractionated by HPLC referring to three synthetic peptides with the predicted sequences. The chromatographic behavior of the natural pheromonotropic peptide revealed the unique structure of Assc-PBAN including beta-SGNP, i.e., SVDFTPRLGRQLVDDVPQRQQIEEDRLGSRTRFFSPRL-NH(2), as the first determination of PBAN from the insects producing an epoxyalkenyl sex pheromone.

Sex pheromones of two melittini species, Macroscelesia japona and m. Longipes: identification and field attraction.

Two Melittini species, Macroscelesia japona and M. longipes (Lepidoptera: Sesiidae), are native to Japan, but occupy different localities as their host plants seldom grow together. The contents of the sex pheromone gland of adult females of both species, obtained after rearing larvae collected from the field, were investigated by gas chromatograph-electroantennogram detection (GC-EAD) and gas chromatograph-mass spectrometry (GC-MS) analyses. Two GC-EAD-active components were found in a crude extract of M. japona female pheromone gland, and identified as (2E,13Z)-2,13-octadecadien-1-ol (E2,Z13-18:OH) and (2E,13Z)-2,13-octadecadienal (E2,Z13-18:Ald). The average ratio of these two components was about 1:10. In the field, M. japona males were attracted to traps baited with E2,Z13-18:Ald alone, but the strongest attraction was observed with a 1:100 mixture of E2,Z13-18:OH and E2,Z13-18:Ald. The same two components were found in extracts of M. longipes females, but in a markedly different ratio. Male M. longipes were attracted most strongly to lures containing a 20:1 mixture of E2,Z13-18:OH and E2,Z13-18:Ald, although some males were also attracted to lures with E2,Z13-18:OH alone. Although the two species do not generally occur in sympatry, our data indicate that, in the event of overlap, cross attraction of the two species is unlikely.

Transport of a hydrophobic biosynthetic precursor by lipophorin in the hemolymph of a geometrid female moth which secretes an epoxyalkenyl sex pheromone.

Previous experiments with a geometrid species, Ascotis selenaria cretacea, have suggested that a pheromonal C19 3,4-epoxy-6,9-diene is biosynthesized from the corresponding 3,6,9-triene produced outside a pheromone gland and transported to it via hemolymph after association with lipophorin. In order to clarify this transport, high-density lipophorin (HDLp) in the female moths showing two bands (apoLp I with ca. 250 kDa and apoLp II with ca. 80 kDa) on an SDS-PAGE was purified by KBr equilibrium density-gradient ultracentrifugation, and the association of the triene was confirmed by GC-MS analysis of a solvent extract from the isolated protein. Next, the role of HDLp was revealed by a topical application of the deuterated trienyl precursor to the abdomens of the females. The trienyl precursor was associated with HDLp. In their pheromone glands, the triene and the deuterated epoxy pheromone were detected, indicating movement of the triene via the hemolymph. Experiments with male moths of A. s. cretacea and female moths of Bombyx mori showed the same association of HDLp with the triene topically applied. This result suggested that the adult females of A. s. cretacea did not develop HDLp specialized in the triene transport. Furthermore, the topical application of a mixture including the trienyl precursor and two other related hydrocarbons showed equal amounts of association by HDLp but selective delivery of the precursor to pheromone glands in the A. s. cretacea females.