Molecular integrity of chloroplast DNA and mitochondrial DNA in mesophyll and bundle sheath cells of maize.

MAIN CONCLUSION: When compared to maize mesophyll cells, the plastid and mitochondrial DNAs in bundle sheath cells are less fragmented, less damaged, and contain fewer DNA polymerase-blocking impediments. Plants that conduct C4 photosynthesis differ from those that employ C3 photosynthesis with respect to leaf anatomy, biochemical pathways, and the proteins and RNA transcripts present in the leaf mesophyll (M) and bundle sheath (BS) cells. Here, we investigate the organellar DNA (orgDNA) from plastids and mitochondria in these two cell types. We use standard qPCR, long PCR, and DNA damage analysis to quantify the amount and quality of orgDNA in isolated M and BS cells of maize. When compared to M cells, BS cells have less orgDNA damage and a higher percentage of unimpeded orgDNA. In addition, the orgDNA is more fragmented in M than BS cells, although orgDNA in BS is subject to more in vitro repair. We suggest that the differences in molecular integrity of orgDNA in these two cells are due to higher levels of reactive oxygen species in M than BS cells.

Changes in DNA damage, molecular integrity, and copy number for plastid DNA and mitochondrial DNA during maize development.

The amount and structural integrity of organellar DNAs change during plant development, although the mechanisms of change are poorly understood. Using PCR-based methods, we quantified DNA damage, molecular integrity, and genome copy number for plastid and mitochondrial DNAs of maize seedlings. A DNA repair assay was also used to assess DNA impediments. During development, DNA damage increased and molecules with impediments that prevented amplification by Taq DNA polymerase increased, with light causing the greatest change. DNA copy number values depended on the assay method, with standard real-time quantitative PCR (qPCR) values exceeding those determined by long-PCR by 100- to 1000-fold. As the organelles develop, their DNAs may be damaged in oxidative environments created by photo-oxidative reactions and photosynthetic/respiratory electron transfer. Some molecules may be repaired, while molecules with unrepaired damage may be degraded to non-functional fragments measured by standard qPCR but not by long-PCR.

The amount and integrity of mtDNA in maize decline with development.

In maize and other grasses there is a developmental gradient from the meristematic cells at the base of the stalk to the differentiated cells at the leaf tip. This gradient presents an opportunity to investigate changes in mitochondrial DNA (mtDNA) that accompany growth under light and dark conditions, as done previously for plastid DNA. Maize mtDNA was analyzed by DAPI-DNA staining of individual mitochondria, gel electrophoresis/blot hybridization, and real-time qPCR. Both the amount and integrity of the mtDNA were found to decline with development. There was a 20-fold decline in mtDNA copy number per cell from the embryo to the light-grown leaf blade. The amount of DNA per mitochondrial particle was greater in dark-grown leaf blade (24 copies, on average) than in the light (2 copies), with some mitochondria lacking any detectable DNA. Three factors that influence the demise of mtDNA during development are considered: (1) the decision to either repair or degrade mtDNA molecules that are damaged by the reactive oxygen species produced as byproducts of respiration; (2) the generation of ATP by photophosphorylation in chloroplasts, reducing the need for respiratory-competent mitochondria; and (3) the shift in mitochondrial function from energy-generating respiration to photorespiration during the transition from non-green to green tissue.

Distinguishing authentic mitochondrial and plastid DNAs from similar DNA sequences in the nucleus using the polymerase chain reaction.

DNA sequences similar to those in the organellar genomes are also found in the nucleus. These non-coding sequences may be co-amplified by PCR with the authentic organellar DNA sequences, leading to erroneous conclusions. To avoid this problem, we describe an experimental procedure to prevent amplification of this "promiscuous" DNA when total tissue DNA is used with PCR. First, primers are designed for organelle-specific sequences using a bioinformatics method. These primers are then tested using methylation-sensitive PCR. The method is demonstrated for both end-point and real-time PCR with Zea mays, where most of the DNA sequences in the organellar genomes are also present in the nucleus. We use this procedure to quantify those nuclear DNA sequences that are near-perfect replicas of organellar DNA. This method should be useful for applications including phylogenetic analysis, organellar DNA quantification and clinical testing.

Virus-induced gene silencing as a tool for comparative functional studies in Thalictrum.

Perennial woodland herbs in the genus Thalictrum exhibit high diversity of floral morphology, including four breeding and two pollination systems. Their phylogenetic position, in the early-diverging eudicots, makes them especially suitable for exploring the evolution of floral traits and the fate of gene paralogs that may have shaped the radiation of the eudicots. A current limitation in evolution of plant development studies is the lack of genetic tools for conducting functional assays in key taxa spanning the angiosperm phylogeny. We first show that virus-induced gene silencing (VIGS) of a PHYTOENE DESATURASE ortholog (TdPDS) can be achieved in Thalictrum dioicum with an efficiency of 42% and a survival rate of 97%, using tobacco rattle virus (TRV) vectors. The photobleached leaf phenotype of silenced plants significantly correlates with the down-regulation of endogenous TdPDS (P<0.05), as compared to controls. Floral silencing of PDS was achieved in the faster flowering spring ephemeral T. thalictroides. In its close relative, T. clavatum, silencing of the floral MADS box gene AGAMOUS (AG) resulted in strong homeotic conversions of floral organs. In conclusion, we set forth our optimized protocol for VIGS by vacuum-infiltration of Thalictrum seedlings or dormant tubers as a reference for the research community. The three species reported here span the range of floral morphologies and pollination syndromes present in Thalictrum. The evidence presented on floral silencing of orthologs of the marker gene PDS and the floral homeotic gene AG will enable a comparative approach to the study of the evolution of flower development in this group.