Variation in Plastid Number

Photosynthetically-active protoplasts isolated from isogenic sets of diploid-tetraploid and tetraplold-octoplold alfalfa (Medicago sativa L.) leaves were used to investigate the consequences of polyploidization on several aspects related to photosynthesis at the cellular level. Protoplasts from the tetraploid population contained twice the amount of DNA, ribulose-1,5-bisphosphate carboxylase (RuBPCase), chlorophyll (Chi), and chloroplasts per cell compared to protoplasts from the diploid population. Although protoplasts from the octoploid population contained nearly twice the number ofchloroplasts and amount of ChW per cell as tetraploid protoplasts, the amount of DNA and RuBPCase per octoploid cell was only 50% higher than in protoplasts from the tetraploWd population. The rate of C02-dependent 02 evolution in protoplasts nearly doubled with an increase in ploidy from the diploid to tetraploid level, but increased only 67% with an increase in ploidy from the tetraplold to octoploid level. Whereas leaves and-protoplasts had simflar increases in RuBPCase, DNA, and Chi with increase in ploidy level, it was concluded that increased cell volume rather than increased cell number per leaf is responsible for the increase in leaf size with ploidy.A better understanding of the physiological consequences of polyploidization should facilitate the development of agronomically useful genotypes from experimental polyploids. A principal difficulty inherent in physiological comparisons of polyploids is that cell volume (18), chloroplast number per cell (2), and the amount of DNA per nucleus (5) have been shown, in some cases, not to increase in exact proportion to an increase in genome size. In these instances, data expressed on a fresh weight, leaf area, or Chl basis may be misleading if the increase in these characteristics in relation to ploidy level has not been determined. The preferred method of comparing polyploids is to isolate protoplasts so that the amount of DNA can be correlated with genome size, and physiological data can be expressed on a per cell basis. Protoplast isolation also eliminates variability resulting from differences such as stomatal density (14) or anatomical characteristics (3).Allopolyploids (3,7,12) and colchicine-induced polyploids (14, 18) have been used to study the effects of polyploidization on biochemical and physiological processes. Results from these studies are confounded by genetic differences in the case of allopolyploids and problems associated with inbreeding depression in colchicine-induced and spontaneously appearing polyploids. 'To whom correspondence should be addressed. topolyploids of alfalfa (11) avoid these influences and allow a more direct study of polyploidization. These polyploids consist of an isogenic diploid-tetraploid (DDC 2X-4X) population of alfalfa to which maximum heterozygosity was restored and an isogenic tetraploid-octoploid (IC 4X-8X) population that was passed through a single sexual cycle, thus restoring some heterozygosity to the plants. These isogeni...

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Ploidy Effects in Isogenic Populations of Alfalfa

Molin¹,

Meyers²,

Baer³

et al. 1982

“…It has been found that ct DNA comprises about 5% of total DNA in the primitive alga Olisthodiscus throughout the growth cycle (2), and as much as 11 to 15% in Chlamydomonas (22,26). Although 9% ct DNA has been estimated for tobacco leaves by the density method (23), reassociation kinetics analysis has indicated that tobacco leaves contain 4% ct DNA and roots less than 1% (21).…”

mentioning

confidence: 99%

Changes in Chloroplast DNA Levels during Development of Pea (Pisum sativum)

Lamppa¹,

Bendich²

1979

Determinations were made of the percentage of chloroplast DNA (ct DNA) in total cell DNA isolated from shoots of pea at different stages of development. Labeled pea ct DNA was reassociated with a high concentration of total DNA; the percentage of ct DNA was estimated by comparing the rate of reassociation of this reaction with that of a model reaction containing a known concentration of unlabeled ct DNA. The maximum change in ct DNA content was from 1.3% of total DNA in young shoots to 7.3% in fully greened shoots. Analyses were also performed on DNA from embryos, etiolated tissue, roots, and leaves. The first leaf set to develop in pea was excised over a growth period of 8 days during which leaf length increased from 4 to 12 millimeters. Young leaves contained about 8% ct DNA; in fuly greened leaves the level of ct DNA approached 12%, equivalent to as many as 9,575 copies of ct DNA per cell. Root tissue contained only 0.4% ct DNA.Chloroplast development is essentially a light-induced phenomenon. The process includes reorganization of the prolamellar bodies to form the thylakoids and grana, reduction of Pchl to Chl, and plastid enlargement (11). In higher plants the number of chloroplasts per cell can increase by as much as a factor of 20 during greening and leaf expansion (19).There is some evidence that chloroplast development is accompanied by ct DNA2 synthesis (6, 19, 20). However, the contribution of ct DNA to the total cellular DNA complement at any stage of higher plant development has not been accurately determined. A major difficulty is the similar base composition of chloroplast and nuclear DNAs (10), which has prevented resolution of these components by CsCl density gradient analysis of total cell DNA.In the algae the buoyant densities of the two DNAs may differ considerably. It has been found that ct DNA comprises about 5% of total DNA in the primitive alga Olisthodiscus throughout the growth cycle (2), and as much as 11 to 15% in Chlamydomonas (22,26). Although 9% ct DNA has been estimated for tobacco leaves by the density method (23), reassociation kinetics analysis has indicated that tobacco leaves contain 4% ct DNA and roots less than 1% (21).The purpose of this study was to determine the amount of ct DNA in total DNA extracted from shoots throughout development of peas using reassociation kinetics analysis. In an earlier report we noted that ct DNA could increase by a factor of 2.5

“…Upon lysis completion, which was monitored by light microscropy, solid CsCl was added and Hoechst 33258 eye-density centrifugation followed. Alternatively, chloroplasts prepared by Protocol I were lysed as described above and the DNA was purified by the phenol extraction technique of Cattolico (9). Occasionally, in this phenol extraction technique, a pronase digestion step was included before centrifugation.…”

mentioning

confidence: 99%

Isolation and Characterization of Chloroplast DNA from the Marine Chromophyte, Olisthodiscus luteus: Electron Microscopic Visualization of Isomeric Molecular Forms

Aldrich¹,

Cattolico²

1981

Self Cite

Chloroplast DNA (ctDNA) from the marine chromophytic alga, Olisthodiscus luteus, has been isolated using a whole cel lysis method folowed by CsCI-Hoechst 33258 dye gradient centrifugation. This DNA, which has a buoyant density of 1.691 grams per cubic centimeter was identified as plastidic in origin by enrichment experiments. Inclusion of the nuclease inhibitor aurintricarboxylic acid in all lysis buffers was mandatory for isolation of high molecular weight DNA. Long linear molecules (40 to 48 micrometers) with considerable internal organization comprised the majority of the ctDNA isolated, whereas supertwisted ctDNA and open circular molecules averaging 46 micrometers were occasionally present. Also observed in this study were folded ctDNA molecules with electron dense centers ("rosettes") and plastid DNA molecules which have a tightly wound "key-ring" center. The ctDNA of Olisthodiscus has a contour length that is median to the size range reported for chiorophytic plants.A minor component of the total celular DNA, which originates from a DNase insensitive cellular structure, has a buoyant density of 1.694 grams per cubic centimeter. This DNA consists predominantly oflinear molecules, but open circles 11.5 micrometers in length and rare 22-micrometer dimers were also present.This study represents the first analysis of the extranuclear DNA of a chromophytic alga.