Targeted inactivation of genes that encode plastid proteins

Abstract

Plant cell contains genetic material in three compartments; namely chloroplast, mitochondria and nucleus. Chloroplast genome encodes a small set of genes compared to nuclear genome. These genes are classified as housekeeping, regulatory and photosynthesis related genes. Most of these genes are subjected to various reverse genetics approaches that led them to classify as essential and nonessential genes based on their role in the plant growth, development and autotrophic status of plants. There are still a few plastid genes and open reading frames in the plastid genomes of higher plants that are yet to be analyzed for their functions. Of these genes, ycf4 and rpl16 were chosen to explore their functions. Plant knockouts were developed for both genes using standard chloroplast transformation approach coupled with reverse genetics strategy. Transgenic plants were confirmed for homoplasmic transplastomes using PCR and Southern blotting approaches. After complete deletion of ycf4 genetic material at genome level characterization at transcriptome level was carried out. It was observed through quantitative PCR approach that transcriptome of few genes were accumulated to low and of other genes to high levels compared to non-transformed wild type plant. It was observed that deletion of ycf4 does not affect the transcripts of PSI and PSII genes except psbC and psbE. Among the ribosomal protein genes and ATP synthase, rps16, rrn16 atpI were affected. Where other genes involved in photosynthesis and regulation of gene expression in Δycf4 plants ycf10, rpoA and rpoB remained unaffected. However, rbcL, and clpP were expressed in very low quantities. Transcripts of ATP synthase subunit (atpB) was accumulated to low levels whereas of clpP gene the transcripts were accumulated to very high levels compared to wild type plant. Photosynthetic rate of genes encoding photosynthesis subunits, contrary to their transcript levels, was greatly affected, and the measured rate was almost half of the wild type plants grown under in vitro controlled growth environment. Similarly, growth and developed of plants was severely affected. Therefore, plants were grown on growth medium supplemented with varied levels of carbon source. It was observed that levels of carbon source when decreased from normal (30g/L) to zero level the growth of plants was severely affected and plants became dead on zero sucrose levels. Similarly, peat moss in pots did not support plants’ autotrophic growth, confirming that ycf4 is an essential subunit of photosynthetic machinery of plants. Electron micrograph of Δplants revealed that structure of chloroplasts have been affected, became round in shape compared to chloroplasts of wild type plants, indicating that

ycf4 gene product has role in structural development of chloroplast. Rpl16 knock out were developed successfully however, to investigate its role in chloroplast functioning demands further experiments both transcriptome and proteome levels.