GUN4-porphyrin complexes bind the ChlH/GUN5 subunit of Mg-Chelatase and promote chlorophyll biosynthesis in Arabidopsis

The GENOMES UNCOUPLED4 (GUN4) protein stimulates chlorophyll biosynthesis by activating Mg-chelatase, the enzyme that commits protoporphyrin IX to chlorophyll biosynthesis. This stimulation depends on GUN4 binding the ChlH subunit of Mg-chelatase and the porphyrin substrate and product of Mg-chelatase. After binding porphyrins, GUN4 associates more stably with chloroplast membranes and was proposed to promote interactions between ChlH and chloroplast membranes—the site of Mg-chelatase activity. GUN4 was also proposed to attenuate the production of reactive oxygen species (ROS) by binding and shielding light-exposed porphyrins from collisions with O₂. To test these proposals, we first engineered Arabidopsis thaliana plants that express only porphyrin binding–deficient forms of GUN4. Using these transgenic plants and particular mutants, we found that the porphyrin binding activity of GUN4 and Mg-chelatase contribute to the accumulation of chlorophyll, GUN4, and Mg-chelatase subunits. Also, we found that the porphyrin binding activity of GUN4 and Mg-chelatase affect the associations of GUN4 and ChlH with chloroplast membranes and have various effects on the expression of ROS-inducible genes. Based on our findings, we conclude that ChlH and GUN4 use distinct mechanisms to associate with chloroplast membranes and that mutant alleles of GUN4 and Mg-chelatase genes cause sensitivity to intense light by a mechanism that is potentially complex.     

A J-like protein influences fatty acid composition of chloroplast lipids in Arabidopsis

A comprehensive understanding of the lipid and fatty acid metabolic machinery is needed for optimizing production of oils and fatty acids for fuel, industrial feedstocks and nutritional improvement in plants. T-DNA mutants in the poorly annotated Arabidopsis thaliana gene At1g08640 were identified as containing moderately high levels (50-100%) of 16∶1Δ7 and 18∶1Δ9 leaf fatty acids and subtle decreases (5-30%) of 16∶3 and 18∶3 (http://www.plastid.msu.edu/). TLC separation of fatty acids in the leaf polar lipids revealed that the chloroplastic galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were the main lipid types affected by this mutation. Analysis of the inferred amino acid sequence of At1g08640 predicted the presence of a transit peptide, three transmembrane domains and an N-terminal J-like domain, and the gene was named CJD1 for Chloroplast J-like Domain 1. GFP reporter experiments and in vitro chloroplast import assays demonstrated CJD1 is a chloroplast membrane protein. Screening of an Arabidopsis cDNA library by yeast-2-hybrid (Y2H) using the J-like domain of CJD1 as bait identified a plastidial inner envelope protein (Accumulation and Replication of Chloroplasts 6, ARC6) as the primary interacting partner in the Y2H assay. ARC6 plays a central role in chloroplast division and binds CJD1 via its own J-like domain along with an adjacent conserved region whose function is not fully known. These results provide a starting point for future investigations of how mutations in CJD1 affect lipid composition.     

PDV1 and PDV2 mediate recruitment of the dynamin-related protein ARC5 to the plastid division site

During plastid division, the dynamin-related protein ACCUMULATION AND REPLICATION OF CHLOROPLASTS5 (ARC5) is recruited from the cytosol to the surface of the outer chloroplast envelope membrane. In Arabidopsis thaliana arc5 mutants, chloroplasts arrest during division site constriction. Analysis of mutants similar to arc5 along with map-based cloning identified PLASTID DIVISION1 (PDV1), an integral outer envelope membrane protein, and its homolog PDV2 as components of the plastid division machinery. Similar to ARC5, PDV1 localized to a discontinuous ring at the division site in wild-type plants. The midplastid PDV1 ring formed in arc5 mutants and the ARC5 ring formed in pdv1 and pdv2 mutants, but not in pdv1 pdv2. Stromal FtsZ ring assembly occurred in pdv1, pdv2, and pdv1 pdv2, as it does in arc5. Topological analysis showed that the large N-terminal region of PDV1 upstream of the transmembrane helix bearing a putative coiled-coil domain is exposed to the cytosol. Mutation of the conserved PDV1 C-terminal Gly residue did not block PDV1 insertion into the outer envelope membrane but did abolish its localization to the division site. Our results indicate that plastid division involves the stepwise localization of FtsZ, PDV1, and ARC5 at the division site and that PDV1 and PDV2 together mediate the recruitment of ARC5 to the midplastid constriction at a late stage of division.     

Disc Volume Reduction with Percutaneous Nucleoplasty in an Animal Model

Study Design

We assessed volume following nucleoplasty disc decompression in lower lumbar spines from cadaveric pigs using 7.1Tesla magnetic resonance imaging (MRI).

Purpose

To investigate coblation-induced volume reductions as a possible mechanism underlying nucleoplasty.

Methods

We assessed volume following nucleoplastic disc decompression in pig spines using 7.1-Tesla MRI. Volumetry was performed in lumbar discs of 21 postmortem pigs. A preoperative image data set was obtained, volume was determined, and either disc decompression or placebo therapy was performed in a randomized manner. Group 1 (nucleoplasty group) was treated according to the usual nucleoplasty protocol with coblation current applied to 6 channels for 10 seconds each in an application field of 360°; in group 2 (placebo group) the same procedure was performed but without coblation current. After the procedure, a second data set was generated and volumes calculated and matched with the preoperative measurements in a blinded manner. To analyze the effectiveness of nucleoplasty, volumes between treatment and placebo groups were compared.

Results

The average preoperative nucleus volume was 0.994 ml (SD: 0.298 ml). In the nucleoplasty group (n = 21) volume was reduced by an average of 0.087 ml (SD: 0.110 ml) or 7.14%. In the placebo group (n = 21) volume was increased by an average of 0.075 ml (SD: 0.075 ml) or 8.94%. The average nucleoplasty-induced volume reduction was 0.162 ml (SD: 0.124 ml) or 16.08%. Volume reduction in lumbar discs was significant in favor of the nucleoplasty group (p<0.0001).

Conclusions

Our study demonstrates that nucleoplasty has a volume-reducing effect on the lumbar nucleus pulposus in an animal model. Furthermore, we show the volume reduction to be a coblation effect of nucleoplasty in porcine discs.     

Karyotype description of two species of Hypostomus (Siluriformes, Loricariidae) of the Planalto da Bodoquena, Brazil

Hypostomus sp 3-Córrego Salobrinha NUP 4247 and Hypostomus sp 2-Rio Perdido NUP 4249, collected in the Planalto da Bodoquena, Paraguay River basin, Brazil, were characterized cytogenetically. Hypostomus sp 3-Córrego Salobrinha showed two modal numbers. This polymorphism consists of the presence of two extrachromosomes. It was not possible to define the diploid number in four specimens, where cell lineages had 2n = 83 and 2n = 84 chromosomes in one individual, and 2n = 82, 2n = 83 and 2n = 84 chromosomes in the others. These results reveal the existence of a genetic mosaic due to the occurrence of one or two extrachromosomes in this species. Hypostomus sp 2-Rio Perdido NUP 4249 showed a 2n = 84, FN = 106 with size heteromorphism in one pair of chromosomes stained with AgNO3. In both species, C banding showed a pattern of heterochromatin distribution with a few small bands in the centromeric and pericentromeric regions coinciding with chromomycin A3 staining. Until now, the major diploid number for the genus Hypostomus was 2n = 80, but the species studied here had chromosomes that in creased this number and the variation for this genus. Our results are also the first cytogenetic data on Hypostomus from the Paraguay River basin.     

Low-coverage massively parallel pyrosequencing of cDNAs enables proteomics in non-model species: comparison of a species-specific database generated by pyrosequencing with databases from related species for proteome analysis of pea chloroplast envelopes

Proteomics is a valuable tool for establishing and comparing the protein content of defined tissues, cell types, or subcellular structures. Its use in non-model species is currently limited because the identification of peptides critically depends on sequence databases. In this study, we explored the potential of a preliminary cDNA database for the non-model species Pisum sativum created by a small number of massively parallel pyrosequencing (MPSS) runs for its use in proteomics and compared it to comprehensive cDNA databases from Medicago truncatula and Arabidopsis thaliana created by Sanger sequencing. Each database was used to identify proteins from a pea leaf chloroplast envelope preparation. It is shown that the pea database identified more proteins with higher accuracy, although the sequence quality was low and the sequence contigs were short compared to databases from model species. Although the number of identified proteins in non-species-specific databases could potentially be increased by lowering the threshold for successful protein identifications, this strategy markedly increases the number of wrongly identified proteins. The identification rate with non-species-specific databases correlated with spectral abundance but not with the predicted membrane helix content, and strong conservation is necessary but not sufficient for protein identification with a non-species-specific database. It is concluded that massively parallel sequencing of cDNAs substantially increases the power of proteomics in non-model species.     

Colocalization of Plastid Division Proteins in the Chloroplast Stromal Compartment Establishes a New Functional Relationship between FtsZ1 and FtsZ2 in Higher Plants

Chloroplast division is driven by a macromolecular complex containing components that are positioned on the cytosolic surface of the outer envelope, the stromal surface of the inner envelope, and in the intermembrane space. The only constituents of the division apparatus identified thus far are the tubulin-like proteins FtsZ1 and FtsZ2, which colocalize to rings at the plastid division site. However, the precise positioning of these rings relative to the envelope membranes and to each other has not been previously defined. Using newly isolated cDNAs with open reading frames longer than those reported previously, we demonstrate here that both FtsZ2 proteins in Arabidopsis, like FtsZ1 proteins, contain cleavable transit peptides that target them across the outer envelope membrane. To determine their topological arrangement, protease protection experiments designed to distinguish between stromal and intermembrane space localization were performed on both in vitro imported and endogenous forms of FtsZ1 and FtsZ2. Both proteins were shown to reside in the stromal compartment of the chloroplast, indicating that the FtsZ1- and FtsZ2-containing rings have similar topologies and may physically interact. Consistent with this hypothesis, double immunofluorescence labeling of various plastid division mutants revealed precise colocalization of FtsZ1 and FtsZ2, even when their levels and assembly patterns were perturbed. Overexpression of FtsZ2 in transgenic Arabidopsis inhibited plastid division in a dose-dependent manner, suggesting that the stoichiometry between FtsZ1 and FtsZ2 is an important aspect of their function. These studies raise new questions concerning the functional and evolutionary significance of two distinct but colocalized forms of FtsZ in plants and establish a revised framework within which to understand the molecular architecture of the plastid division apparatus in higher plants.     

Evaluation of methods for estimating macroinvertebrate species richness using individual stones in tropical streams

1. The most straightforward way to assess diversity in a site is the species count. However, a relatively large sample is needed for a reliable result because of the presence of many rare species in rich assemblages. The use of richness estimation methods is suggested by many authors as a solution for this problem in many cases.
2. We examined the performance of 13 methods for estimating richness of stream macroinvertebrates inhabiting riffles both at local (stream) and regional (catchment) scales. The evaluation was based on (1) the smallest sub-sample size needed to estimate total richness in the sample, (2) constancy of this size, (3) lack of erratic behaviour in curve shape and (4) similarity in curve shape through different data sets. Samples were from three single stream sites (local) and three from several streams within the same catchment basin (regional). All collections were made from protected forest areas in south-east Brazil.
3. All estimation methods were dependent on sub-sample size, producing higher estimates when using larger sub-sample sizes. The Stout and Vandermeer method estimated total richness in the samples with the smallest sub-sample size, but showed some erratic behaviour at small sub-sample sizes, and the estimated curves were not similar among the six samples. The Bootstrap method was the best estimator in relation to constancy of sub-sample sizes, but needed an unacceptably large sub-sample to estimate total richness in the samples. The second order Jackknife method was the second best estimator both for minimum sub-sample size and constancy of this size and we suggest its use in future studies of diversity in tropical streams. Despite the inferior performance of several other methods, some produced acceptable results. Comments are made on the utility of using these estimators for predicting species richness in an area and for comparative purposes in diversity studies.     

Second-site revertants of the P1 copN22 copy mutant.

Miniplasmids with the P1 copN22 mutation have a copy number about seven times that of the wild type. Selection for reduced copy number from this plasmid led to the isolation of second-site pseudorevertants, called poc mutants. DNA sequence analysis showed that all six independent poc mutants have a single base change in the same codon of the repA gene. This implicates the amino acid at this location, either directly or indirectly, in interactions important for copy number control.