Chloroplast DNA in Mature and Senescing Leaves: A Reappraisal  

Abstract

The fate of plastid DNA (ptDNA) during leaf development has become a matter of contention. Reports on little change in ptDNA copy number per cell contrast with claims of complete or nearly complete DNA loss already in mature leaves. We employed highresolution fluorescence microscopy, transmission electron microscopy, semithin sectioning of leaf tissue, and real-time quantitative PCR to study structural and quantitative aspects of ptDNA during leaf development in four higher plant species (Arabidopsis thaliana, sugar beet [Beta vulgaris], tobacco [Nicotiana tabacum], and maize [Zea mays]) for which controversial findings have been reported. Our data demonstrate the retention of substantial amounts of ptDNA in mesophyll cells until leaf necrosis. In ageing and senescent leaves of Arabidopsis, tobacco, and maize, ptDNA amounts remain largely unchanged and nucleoids visible, in spite of marked structural changes during chloroplast-to-gerontoplast transition. This excludes the possibility that ptDNA degradation triggers senescence. In senescent sugar beet leaves, reduction of ptDNA per cell to ;30% was observed reflecting primarily a decrease in plastid number per cell rather than a decline in DNA per organelle, as reported previously. Our findings are at variance with reports claiming loss of ptDNA at or after leaf maturation.

On the Fate of Plastid DNA Molecules during Leaf Development: Response to the Golczyk et al. Commentary

“…In their commentary, Golczyk et al (2014) state that the level of plastid DNA (ptDNA) remains approximately constant during mature, aging, and senescent stages of leaf development, in apparent contrast with our conclusion of ptDNA decline from previous studies. However, the ptDNA decline that we reported was found during proplastid-to-chloroplast development, before the mature-to-senescent leaf stages.…”

“…Inspection of the images ( Figure 2 and Supplemental Figures 1 to 4) presented by Golczyk et al (2014) can be interpreted to indicate this progression. Highly fragmented ptDNA, either still aggregated to form a nucleoid or dissociated from the nucleoid, could also affect the interpretation of results from the qPCR assay.…”

“…Although Golczyk et al (2014) used four methods for assessing the presence of ptDNA during the development of green leaves, three of these methods involved microscopic examination that did not provide quantitative information for either ptDNA per plastid, ptDNA per cell, or ptDNA per nuclear DNA (nucDNA) amount. DAPI-DNA fluorescence revealed DNA within chloroplast nucleoids that was characterized by "visual inspection.indicating unaltered DNA contents per organelle" in young and mature leaves.…”

“…The only quantitative assay of ptDNA reported by Golczyk et al (2014) was real-time qPCR. Using standard qPCR procedures, our group and Thomas Bö rner and colleagues (Zoschke et al, 2007) reported constant values for plastid genome equivalents per nuclear genome equivalent (ptDNA/nucDNA) during Arabidopsis leaf development.…”

“…Our conclusions for three plant species with respect to changes in ptDNA content during leaf development are summarized in Figure 1, based on four methods that provide quantitative data on ptDNA mass per plastid and per cell and on molecular structure. The only quantitative data provided by Golczyk et al (2014) are based on amounts of DNA between two closely spaced quantitative PCR (qPCR) primers and thus depend on the unvalidated assumption that such tiny DNA fragments represent genome-sized ptDNA molecules. We conclude that an accurate assessment of ptDNA must consider not only ptDNA quantity but also molecular quality, a parameter not addressed by Golczyk et al In our opinion, the manner of data presentation is the principal difference leading to our opposing conclusions.…”