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Isolation of dimorphic chloroplasts from the single-cell C4 species Bienertia sinuspersici.

ABSTRACT: Three terrestrial plants are known to perform C4 photosynthesis without the dual-cell system by partitioning two distinct types of chloroplasts in separate cytoplasmic compartments. We report herein a protocol for isolating the dimorphic chloroplasts from Bienertia sinuspersici. Hypo-osmotically lysed protoplasts under our defined conditions released intact compartments containing the central chloroplasts and intact vacuoles with adhering peripheral chloroplasts. Following Percoll step gradient purification both chloroplast preparations demonstrated high homogeneities as evaluated from the relative abundance of respective protein markers. This protocol will open novel research directions toward understanding the mechanism of single-cell C4 photosynthesis.

SUBMITTER: Lung SC

PROVIDER: S-EPMC3310801 | biostudies-other | 2012

REPOSITORIES: biostudies-other

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Isolation of dimorphic chloroplasts from the single-cell C4 species Bienertia sinuspersici.

Lung Shiu-Cheung SC Yanagisawa Makoto M Chuong Simon Dx SD

Plant methods 20120306 1

Three terrestrial plants are known to perform C4 photosynthesis without the dual-cell system by partitioning two distinct types of chloroplasts in separate cytoplasmic compartments. We report herein a protocol for isolating the dimorphic chloroplasts from Bienertia sinuspersici. Hypo-osmotically lysed protoplasts under our defined conditions released intact compartments containing the central chloroplasts and intact vacuoles with adhering peripheral chloroplasts. Following Percoll step gradient ...[more]

PMID: 22394490

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Project description:Background:Chloroplast genome information is critical to understanding forms of photosynthesis in the plant kingdom. During the evolutionary process, plants have developed different photosynthetic strategies that are accompanied by complementary biochemical and anatomical features. Members of family Chenopodiaceae have species with C3 photosynthesis, and variations of C4 photosynthesis in which photorespiration is reduced by concentrating CO2 around Rubisco through dual coordinated functioning of dimorphic chloroplasts. Among dicots, the family has the largest number of C4 species, and greatest structural and biochemical diversity in forms of C4 including the canonical dual-cell Kranz anatomy, and the recently identified single cell C4 with the presence of dimorphic chloroplasts separated by a vacuole. This is the first comparative analysis of chloroplast genomes in species representative of photosynthetic types in the family. Results:Methodology with high throughput sequencing complemented with Sanger sequencing of selected loci provided high quality and complete chloroplast genomes of seven species in the family and one species in the closely related Amaranthaceae family, representing C3, Kranz type C4 and single cell C4 (SSC4) photosynthesis six of the eight chloroplast genomes are new, while two are improved versions of previously published genomes. The depth of coverage obtained using high-throughput sequencing complemented with targeted resequencing of certain loci enabled superior resolution of the border junctions, directionality and repeat region sequences. Comparison of the chloroplast genomes with previously sequenced plastid genomes revealed similar genome organization, gene order and content with a few revisions. High-quality complete chloroplast genome sequences resulted in correcting the orientation the LSC region of the published Bienertia sinuspersici chloroplast genome, identification of stop codons in the rpl23 gene in B. sinuspersici and B. cycloptera, and identifying an instance of IR expansion in the Haloxylon ammodendron inverted repeat sequence. The rare observation of a mitochondria-to-chloroplast inter-organellar gene transfer event was identified in family Chenopodiaceae. Conclusions:This study reports complete chloroplast genomes from seven Chenopodiaceae and one Amaranthaceae species. The depth of coverage obtained using high-throughput sequencing complemented with targeted resequencing of certain loci enabled superior resolution of the border junctions, directionality, and repeat region sequences. Therefore, the use of high throughput and Sanger sequencing, in a hybrid method, reaffirms to be rapid, efficient, and reliable for chloroplast genome sequencing.

Project description:As the world population grows, our need for food increases drastically. Limited amounts of arable land lead to a competition between food and fuel crops, while changes in the global climate may impact future crop yields. Thus, a second "green revolution" will need a better understanding of the processes essential for plant growth and development. One approach toward the solution of this problem is to better understand regulatory and transport processes in C4 plants. C4 plants display an up to 10-fold higher apparent CO2 assimilation and higher yields while maintaining high water use efficiency. This requires differential regulation of mesophyll (M) and bundle sheath (BS) chloroplast development as well as higher metabolic fluxes of photosynthetic intermediates between cells and particularly across chloroplast envelopes. While previous analyses of overall chloroplast membranes have yielded significant insight, our comparative proteomics approach using enriched BS and M chloroplast envelopes of Zea mays allowed us to identify 37 proteins of unknown function that have not been seen in these earlier studies. We identified 280 proteins, 84% of which are known/predicted to be present in chloroplasts. Seventy-four percent have a known or predicted membrane association. Twenty-one membrane proteins were 2-15 times more abundant in BS cells, while 36 of the proteins were more abundant in M chloroplast envelopes. These proteins could represent additional candidates of proteins essential for development or metabolite transport processes in C4 plants. RT-PCR confirmed differential expression of 13 candidate genes. Chloroplast association for seven proteins was confirmed using YFP/GFP labeling. Gene expression of four putative transporters was examined throughout the leaf and during the greening of leaves. Genes for a PIC-like protein and an ER-AP-like protein show an early transient increase in gene expression during the transition to light. In addition, PIC gene expression is increased in the immature part of the leaf and was lower in the fully developed parts of the leaf, suggesting a need for/incorporation of the protein during chloroplast development.

Dataset Information

Isolation of dimorphic chloroplasts from the single-cell C4 species Bienertia sinuspersici.

Publications

Isolation of dimorphic chloroplasts from the single-cell C4 species Bienertia sinuspersici.

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