Boerner, T. | bioRxiv Science

bioRxiv2019

Chloroplast nucleoids are highly dynamic in ploidy, number, and structure during angiosperm leaf development

Chloroplast nucleoids are large, compact nucleoprotein structures containing multiple copies of the plastid genome. Studies on structural and quantitative changes of plastid DNA (ptDNA) during leaf development are scarce and have produced controversial data. We have systematically investigated nucleoid dynamics and ptDNA quantities in mesophyll of Arabidopsis, tobacco, sugar beet, and maize from the early post-meristematic stage until necrosis. DNA of individual nucleoids was quantified by DAPI-based supersensitive epifluorescence microscopy. Nucleoids occurred in scattered, stacked or ring-shaped arrangements and in recurring patterns during leaf development remarkably similar between the species studied. Nucleoids per organelle varied from few in meristematic plastids to >30 in mature chloroplasts (corresponding to about 20-750 nucleoids per cell). Nucleoid ploidies ranged from haploid to >20-fold even within individual organelles, with average values between 2.6- and 6.7-fold and little changes during leaf development. DNA quantities per organelle increased gradually from about a dozen plastome copies in tiny plastids of apex cells to 70-130 copies in chloroplasts of about 7 m diameter in mature mesophyll tissue, and from about 80 plastome copies in meristematic cells to 2,600-3,300 copies in mature diploid mesophyll cells without conspicuous decline during leaf development. Pulsed-field electrophoresis, restriction of high-molecular weight DNA from chloroplasts and gerontoplasts, and CsCl equilibrium centrifugation of single- and double-stranded ptDNA revealed no noticeable fragmentation of the organelle DNA during leaf development, implying that plastid genomes in mesophyll tissues are remarkably stable until senescence. Significance StatementPlastid DNA is organized in nucleoids that are highly dynamic in organization, structure and amount during leaf development. The present investigation fully resolves now this dynamic and is a precise cytogenetic characterization of nucleoids DNA spanning the entire life cycle of the leaf.

plant biology↗

bioRxiv2019

Leaf Variegation and Impaired Chloroplast Development Caused by a Truncated CCT Domain gene in albostrians Barley

Chloroplasts fuel plant development and growth by converting solar into chemical energy. They mature from proplastids through the concerted action of genes in both the organellar and the nuclear genome. Defects in such genes impair chloroplast development and may lead to pigment-deficient seedlings or seedlings with variegated leaves. Such mutants are instrumental as tools for dissecting genetic factors underlying the mechanisms involved in chloroplast biogenesis. Characterization of the green-white variegated albostrians mutant of barley has greatly broadened the field of chloroplast biology including the discovery of retrograde signaling. Here, we report the identification of the ALBOSTRIANS gene HvAST by positional cloning as well as its functional validation based on independently induced mutants by TILLING and RNA-guided Cas9 endonuclease mediated gene editing. The phenotypes of the independent HvAST mutants imply residual activity of HvAST in the original albostrians allele conferring an imperfect penetrance of the variegated phenotype even at homozygous state of the mutation. HvAST is a homolog of the Arabidopsis thaliana CCT Motif transcription factor gene AtCIA2, which was reported to be involved in the expression of nuclear genes essential for chloroplast biogenesis. Interestingly, in barley we localized HvAST to the chloroplast indicating novel without any clear evidence of nuclear localization. One-sentence summaryLeaf variegation in the barley mutant albostrians is caused by mutation of a single CCT-domain containing gene with residual activity, which is directed to the chloroplast.

genetics↗