Functional Analysis of Light-harvesting-like Protein 3 (LIL3) and Its Light-harvesting Chlorophyll-binding Motif in Arabidopsis

The light-harvesting complex (LHC) constitutes the major light-harvesting antenna of photosynthetic eukaryotes. LHC contains a characteristic sequence motif, termed LHC motif, consisting of 25–30 mostly hydrophobic amino acids. This motif is shared by a number of transmembrane proteins from oxygenic photoautotrophs that are termed light-harvesting-like (LIL) proteins. To gain insights into the functions of LIL proteins and their LHC motifs, we functionally characterized a plant LIL protein, LIL3. This protein has been shown previously to stabilize geranylgeranyl reductase (GGR), a key enzyme in phytol biosynthesis. It is hypothesized that LIL3 functions to anchor GGR to membranes. First, we conjugated the transmembrane domain of LIL3 or that of ascorbate peroxidase to GGR and expressed these chimeric proteins in an Arabidopsis mutant lacking LIL3 protein. As a result, the transgenic plants restored phytol-synthesizing activity. These results indicate that GGR is active as long as it is anchored to membranes, even in the absence of LIL3. Subsequently, we addressed the question why the LHC motif is conserved in the LIL3 sequences. We modified the transmembrane domain of LIL3, which contains the LHC motif, by substituting its conserved amino acids (Glu-171, Asn-174, and Asp-189) with alanine. As a result, the Arabidopsis transgenic plants partly recovered the phytol-biosynthesizing activity. However, in these transgenic plants, the LIL3-GGR complexes were partially dissociated. Collectively, these results indicate that the LHC motif of LIL3 is involved in the complex formation of LIL3 and GGR, which might contribute to the GGR reaction.     

Comparison of thread-cutting behavior in three specialist predatory mites to cope with complex webs of <Emphasis Type="Italic">Tetranychus</Emphasis> spider mites

Anti-predator defenses provided by complex webs of Tetranychus mites can severely impede the performance of generalist predatory mites, whereas this may not be true for specialist predatory mites. Although some specialist predatory mites have developed morphological protection to reduce the adverse effects of complex webs, little is known about their behavioral abilities to cope with the webs. In this study, we compared thread-cutting behavior of three specialist predatory mites, Phytoseiulus persimilis, Neoseiulus womersleyi and N. californicus, exhibited inside the complex web of T. urticae. No major difference was observed among them in the basic pattern of this behavior, using chelicerae and palps, and in the number of silken threads severed while moving inside the web. These results and observations suggest that each predator species cut many sticky silken threads to move inside the complex web without suffering from serious obstruction. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evidence of the supercomplex organization of photosystem II and light-harvesting complexes in <Emphasis Type="Italic">Nannochloropsis granulata</Emphasis>

Diverse light-harvesting complexes (LHCs) have been found in photosynthetic microalgae that originated from secondary endosymbiosis involving primary red algae. However, the associations between LHCs and photosystem I (PSI) and photosystem II (PSII) in these microalgae are not fully understood. Eustigmatophyta is a red algal lineage that appears to have a unique organization in its photosynthetic machinery, consisting of only chlorophyll a and carotenoids that are atypical compared with other closely related groups. In this study, the supramolecular organization of pigment–protein complexes in the eustigmatophyte alga, Nannochloropsis granulata was investigated using Clear Native (CN) PAGE coupled with two-dimensional (2D) SDS-PAGE. Our results showed two slowly migrating green bands that corresponded to PSII supercomplexes, which consisted of reaction centers and LHCs. These green bands were also characterized as PSII complexes by their low temperature fluorescence emission spectra. The protein subunits of the PSII–LHC resolved by 2D CN/SDS-PAGE were analyzed by mass spectrometry, and four different LHC proteins were identified. Phylogenetic analysis of the identified LHC protein sequences revealed that they belonged to four different Lhc groups; (1) stress-related Lhcx proteins, (2) fucoxanthin chlorophyll a/c-binding Lhcf proteins, (3) red-shifted Chromera light-harvesting proteins (Red-CLH), and (4) Lhcr proteins, which are commonly found in organisms possessing red algal plastids. This is the first report showing evidence of a pigment–protein supercomplex consisting of PSII and LHCs, and to identify PSII-associated LHC proteins in Nannochloropsis.     

Constitutive and Inducible Proteins in the Root of Chinese Cabbage Infected with Plasmodiophora brassicae

A plant-pathogen system consisting of a Chinese cabbage cultivar and two isolates of Plasmodiophora brassicae was developed for analysing root proteins accumulated in susceptible and resistant responses to the fungus. Proteins extracted at pH 2.8 were analysed by two-dimensional gel electrophoresis. More than 150 protein spots were resolved. Spots indicating changes in the intensity by the infection of P. brassicae were classified into six types: class 1 contains proteins enhanced in susceptible response; class 2, proteins unique to susceptible response; class 3, proteins repressed in susceptible response: class 4, proteins enhanced in resistant response; class 5, proteins unique to resistant response; and class 6, proteins repressed in resistant response. Two proteins from class 1 and one protein from class 4 were subjected to an N-terminal amino acid sequencing. One of the class 1 protein (25 kDa, pl 7.0) revealed high homology with pathogenesis-related protein group 5.     

Identification and sequencing of cDNA clones for the rodent negative acute-phase protein alpha 1-inhibitor 3

Rat alpha 1-inhibitor 3 clones were isolated by immunological screening of a lambda gt11 cDNA library prepared from rat liver poly(A)-rich RNA. The recombinant cDNA clones were identified by the absence of their immunoprecipitable products following hybrid-arrested in vitro translation. The size of the cognate poly(A)-rich RNA was estimated to be roughly 5000 residues. Approximately 16 h after induction of inflammation the amount of alpha 1-inhibitor 3 poly(A)-rich RNA decreases as shown by dot-blot hybridization and Northern analyses. The response of this negative acute-phase plasma protein to inflammation may therefore be considered to be at the pretranslational level. The characterized DNA constitutes an open reading frame of 225 amino acids followed by a canonical eucaryotic polyadenylation signal and a poly(A) tail. Sequence microheterogeneity, particularly in the 3'-flanking region was observed. An amino acid homology of 70% for alpha 1-inhibitor 3 with human and rodent alpha 2-macroglobulin emphasizes the evolutionary relationship of the macroglobulins.     

Sister chromatid exchanges and chromosome aberrations in fibroblasts from patients with retinoblastoma

Summary The frequencies of sister chromatid exchanges (SCEs) and chromosome breaks were investigated in five diploid fibroblast strains derived from three patients with deletion 13[del(13)] retinoblastoma, one patient with a hereditary form of retinoblastoma, and one trisomy 13. The fibroblasts with del(13)(q14q22) showed slightly increased SCEs (at a P level of 5–10%), but the others, including del(13)(q12q14), the hereditary form of retinoblastoma, and trisomy 13, did not have increased SCEs as compared to normal controls. No increase in chromosome breaks was found in these fibroblasts. The results suggest that retinoblastoma is not associated with spontaneous increased chromosomal instability.