Characterization of Mitochondrial DNA in Citrus

Abstract

Characterization of mitochondrial DNA from leaves of four species of the genus Citrus by electron microscopy shows the presence of circular molecules with a great size dispersion and absence of discrete size classes. Restriction endonuclease patterns confirm the heterogeneity of the molecules.The mitochondrial genome ofhigher plants has been intensively studied in recent years, and most of its physicochemical characteristics are now well established. In the higher plant species so far studied, the mt DNA2 is quite distinguishable from both nuclear and chloroplastic DNAs, and represents nearly 1% of the total cell DNA.In animal cells, mt DNA appears as a sinile circular molecule of 5 to 6 ,im in contour length (9-12 x 10 mol wt). However, striking differences have been reported in the size of mt DNA in different plant species, ranging from 3 to 30,um. Plant species with a single or several discrete size classes have been described (2,4,8), but in other cases the occurrence of discrete classes is less obvious (6, 7).In addition, linear molecules of varying length are consistently observed in electron microscopic preparations of plant mt DNA. In fact, this form has been proposed as characteristic of the mt DNA of several species (5, 9, 11) with a wide range of variability in length. However, linear molecules appearing with closed ones in mt DNA samples from plants have been considered due to breakage during handling of molecules which were originally closed, and thus indirectly casting doubt on the in vivo existence of linear mt DNA.To characterize further the plant mitochondrial genome, we have studied the DNA of four species of the genus Citrus as representatives of a group of woody plants, not At the end of the incubation, 120 ml of buffer B (50 mm Tris, 0.3 M sucrose, 20 mi EDTA, pH 8.0) was added and the suspension was centrifuged at 20,000g for 20 min. The pellet was washed by suspending it in 120 ml of buffer B and the centrifugation repeated. The fmal pellet was carefully dispersed in 20 ml of buffer B using a Potter homogenizer and layered on top of a discontinuous sucrose density gradient formed by 12 ml 30% sucrose and 12 ml 60% sucrose in buffer B. After centrifugation at 22,000 rpm for 45 min in a SW-27 rotor, the mitochondrial band was recovered and washed two times with buffer B. The pellet was suspended in 8 ml of buffer C (50 aM Tris, 20 mM EDTA, pH 8.0) containing 0.1 mg/ml of Proteinase K (Merck), and sodium sarkosyl was added to give a final concentration of 2%.The mixture was allowed to stand at room temperature for 0.5 h. The lysates were brought to a refractive index of n = 1.3550 to 1.3600 with CsCl, chilled for 1 to 2 h in an ice bath, and centrifuged 30 min at 5,000g. The pellets were discarded. The supernatant was brought to n = 1.3900 with CsCl. Ethidium bromide (200 ,ug/ml, Sigma) was added and the solution was centrifuged in sodium sarkosyl-treated cellulose nitrate tubes at 175,000g, 20°C, for 48 to 60 h. After centrifugation, only one DNA band was observed in the centrifuge...

A review of the structure and organization of the mitochondrial genome of higher plants

“…This distribution of sizes could easily be interpreted as an apparent random size collection of circular DNA molecules with the 16.6 ~m class being the most abundant species (8). A similar, apparently random size distribution was observed in supercoiled DNA preparations from cultured cells of W38 tobacco (Nicotiani tabacum) (11) and in mtDNA preparations of Citrus (10).…”

“…Circular molecules were not seen in this earlier study, though some very long molecules were observed. DNA molecules of exceptional length have also been observed in Citrus mtDNA preparations (10). Such molecules may in fact represent the true mitochondrial genome but their susceptibility to breakage must significantly reduce their frequency.…”

“…In many studies, however, a low proportion of circular DNA molecules is observed (6)(7)(8)(9)(10). Electron microscopical studies on mtDNA preparations from the normal fertile and the S-and T-male sterile cytoplasms of maize showed that the circular DNA molecules fell into specific size classes and that these size classes were unique to each cytoplasm (9).…”

Phage T4-like intermediates of DNA replication and recombination in the mitochondria of the higher plant Chenopodium album (L.)

“…Huberman 1968;Powling and Knippers 1976;Masker et al 1978;Dannenberg and Mosig 1983) or vaccinia or herpes simplex viruses (Dahl and Kates 1970;Severini et al 1996) and highly supercoiled DNA as shown for protein-free phage P4 DNA (Liu et al 1981). Many of the extremely large rosette-like structures appeared to have distinct dense areas (hereafter called`cores'), as previously also observed in mtDNA preparations from other plants (Fontarnau and Herna ndez-Yago 1982;Dudareva et al 1988;Wang et al 1989) or petite yeast (Locker et al 1979). Usually, the rosettes had the appearance shown in gives the corresponding data on the total size of these molecules (measured in kb).…”

Identification and Screening of Citrus Vein Phloem Degeneration (CVPD) on Brastagi Citrus Variety Brastepu (Citrus nobilis Brastepu) in North Sumatra Indonesia

“…Molecular analysis with histochemical test was performed for molecular tissue by using a microscope. Extraction of the DNA, PCR, and electrophoresis are conducted followed the procedures explained in the references (Fontarnau & Hernandez-Yago, 1982;Weising et al, 2005;Amani et al, 2011). Confirmation test for infection of CVPD is conducted by using PCR.…”