TY - CHAP
T1 - Reverse Genetics in Flowering Plant Plastids
T2 - 10.1007/978-94-007-2920-9
AU - Day, A
A2 - Bock, R
A2 - Knoop, V
N1 - BBSRC
PY - 2012/6
Y1 - 2012/6
N2 - Plastid reverse genetics exploits the predominance of homologous DNA recombination inthis organelle, which allows targeted mutations to be introduced into plastid genes. Most studies have used tobacco and involve replacement of wild-type plastid genes with mutant alleles. Mutant alleles are either disrupted by the marker gene or lie adjacent to the marker gene. Marker selection with antibiotics is required to remove wild-type plastid genomes and reveal the phenotype of homoplasmic mutant plants. Targeted knock-outs have shown that tobacco plastid genes are either dispensable or essential. Dispensable plastid genes include those encoding photosynthesis-related proteins, subunits of the plastid-encoded RNApolymerase, ribosomal proteins rps15, rpl33 and rpl36, valyl transfer RNA(GAC), glycyltransfer RNA(GCC) and origins of DNA replication. Loss-of-photosynthesis is dispensable if mutants are propagated on sucrose medium. Knock-outs were particularly useful for elucidating the roles of conserved but dispensable hypothetical reading frames in photosynthesis. Site-directed mutations allow structure-function studies on the products of plastid genes. Marker-free plants containing deletions of dispensableplastid genes, facilitate the rapid isolation of plants containing site-directed mutant alleles. Knock-outs of essential tobacco plastid genes (accD, clpP, ycf1, ycf2, rps2, rps3, rps4, rps16, rps18, rpl20, rpl22, rpl23, rpl32, trnC-GCA,trnN-GUU, trnG-UCC) persist as heteroplasmic mixtures with the wild-type allele under antibiotic selection. Homoplasmic cells containing knock out alleles of essential genes would not be viable and this explains the leaf-lamina-loss phenotype of mutant plants. Strong selection for the wild-type gene may hinder the isolation of partial-function alleles of essential plastid genes containing site directed mutations. Progress may require the use of angiosperm species, in which homologues of essential tobacco plastid genes are dispensable.
AB - Plastid reverse genetics exploits the predominance of homologous DNA recombination inthis organelle, which allows targeted mutations to be introduced into plastid genes. Most studies have used tobacco and involve replacement of wild-type plastid genes with mutant alleles. Mutant alleles are either disrupted by the marker gene or lie adjacent to the marker gene. Marker selection with antibiotics is required to remove wild-type plastid genomes and reveal the phenotype of homoplasmic mutant plants. Targeted knock-outs have shown that tobacco plastid genes are either dispensable or essential. Dispensable plastid genes include those encoding photosynthesis-related proteins, subunits of the plastid-encoded RNApolymerase, ribosomal proteins rps15, rpl33 and rpl36, valyl transfer RNA(GAC), glycyltransfer RNA(GCC) and origins of DNA replication. Loss-of-photosynthesis is dispensable if mutants are propagated on sucrose medium. Knock-outs were particularly useful for elucidating the roles of conserved but dispensable hypothetical reading frames in photosynthesis. Site-directed mutations allow structure-function studies on the products of plastid genes. Marker-free plants containing deletions of dispensableplastid genes, facilitate the rapid isolation of plants containing site-directed mutant alleles. Knock-outs of essential tobacco plastid genes (accD, clpP, ycf1, ycf2, rps2, rps3, rps4, rps16, rps18, rpl20, rpl22, rpl23, rpl32, trnC-GCA,trnN-GUU, trnG-UCC) persist as heteroplasmic mixtures with the wild-type allele under antibiotic selection. Homoplasmic cells containing knock out alleles of essential genes would not be viable and this explains the leaf-lamina-loss phenotype of mutant plants. Strong selection for the wild-type gene may hinder the isolation of partial-function alleles of essential plastid genes containing site directed mutations. Progress may require the use of angiosperm species, in which homologues of essential tobacco plastid genes are dispensable.
KW - reverse genetics, chloroplasts, plastid, targeted knockouts
U2 - 10.1007/978-94-007-2920-9
DO - 10.1007/978-94-007-2920-9
M3 - Chapter
SN - 978-94-007-2920-9
T3 - Advances in Photosynthesis and Respiration
SP - 415
EP - 441
BT - Genomics of Chloroplasts and Mitochondria
PB - Springer Nature
ER -