9 Springer-Verlag 1984
Changes in mitoehondrial DNA levels during development of pea (Pisum sativum L.) Gayle K. Lamppa* and Arnold J. Bendich** Departments of Botany and Genetics, University of Washington, Seattle, WA 98195, USA
Abstract. The percentage of mitochondrial D N A (mtDNA) present in total D N A isolated from pea tissues was determined using labeled m t D N A in reassociation kinetics reactions. Embryos contained the highest level of m t D N A , equal to 1.5% of total DNA. This value decreased in light- and dark-grown shoots and leaves, and roots. The lowest value found was in dark-grown shoots; their total D N A contained only 0.3% mtDNA. This may be a reflection of increased nuclear ploidy levels without concomitant m t D N A synthesis. It was possible to compare the m t D N A values directly with previous estimates of the amount of chloroplast D N A (ctDNA) per cell because the same preparations of total D N A were used for both analyses. The embryo contained 1.5% of both m t D N A and ctDNA; this equals 410 copies of m t D N A and 1200 copies o f c t D N A per diploid cell. Whereas m t D N A levels decreased to 260 copies in leaf cells of pea, the number of copies of ctDNA increased to 10300. In addition, the levels of c t D N A in first leaves of dark-grown and lighttransferred pea were determined, and it was found that leaves of plants maintained in the dark had the same percentage of c t D N A as those transferred to the light.
Key words: Chlorolast D N A - D N A (chloroplast, mitochondrial) - Mitochondrial D N A - Pisum (mitochondrial DNA).
Two extra-nuclear genomes contribute to the development of a plant cell: the mitochondrial and * Present address. Laboratory of Plant Molecular Biology, Rockefeller University, New York, NY 10021, USA ** To whom correspondence should be addressed Abbreviations: ctDNA = chloroplast DNA; m t D N A - m i t o chondrial DNA
chloroplast DNAs. For pea, we have previously demonstrated that the percentage of chloroplast D N A (ctDNA) in total cellular D N A increases as much as eightfold from the embryonic level to the amount found in light-grown leaves, i.e. from 1.5% to 12% of total D N A (Lamppa and Bendich 1979, 1980). This represents an increase from about 1 200 copies per diploid embryonic cell to 10000 copies of c t D N A per diploid leaf cell (or 20 000 per tetraploid cell). The maximum number of chloroplast genome copies per leaf cell has been estimated at 1 900 for beet (Tymms et al. 1983), 12000 for spinach (Tymms etal. 1982), and 50000 for wheat (Boffey and Leech 1982). We know very little of the number of copies of the mitochondrial genome in plant cells, and nothing at all of the changes in number during development. Is there a corresponding change in the number of mitochondrial and chloroplast gehomes, or is the replication of these DNAs regulated independently? In the present study we measure the percentage of mitochondrial D N A (mtDNA) in various pea tissues by reassociationkinetics analysis, using the very same total DNAs as "drivers" that we used previously to estimate the levels of c t D N A per cell and per plastid (Lamppa and Bendich 1979, 1980). Our objectives were to 1) establish the amount of m t D N A in a plant cell, and 2) compare the levels of the two extra-nuclear DNAs during pea development. The results show that both the m t D N A and c t D N A levels decrease in roots with respect to those in the embryo. However, in shoots, m t D N A levels decrease (or remain nearly constant) when c t D N A levels increase.
Material and methods Growth of plants and preparation of total DNA. The conditions of plant growth and the procedure for isolating total DNA from peas (Pisum sativum L. cv. Alaska wilt-resistant, from L.L. Olds Seed Co., Madison, Wis., USA) were the same as
G.K. Lamppa and A.J. Bendich: Mitochondrial D N A during development of pea
described in Lamppa and Bendich (1979, 1980). The same total D N A preparations were employed in this study, permitting the direct comparison of percentages of m t D N A and ctDNA in total DNA. For the experiment described in the last two columns of Table 1, plants were grown in the dark for 4 d; then half of them were transferred to light. At the end of 24 h the first leaves were excised from both the dark-maintained plants and those introduced to the light, and total D N A was prepared.
Preparation of mitochondrial DNA. The procedure used for isolating m t D N A was described in Ward et al. (1981). The D N A extracted from mitochondria was examined for purity by restriction-enzyme digestion and density-gradient centrifugation (Ward et al. 1981). The patterns of fragments obtained with Kpn I and Sal I were the same as found previously for pea m t D N A (Ward et al. 1981). No other bands were visible, indicating that the D N A was essentially free of another D N A component, such as chloroplast DNA. Analytical CsC1 densitygradient analysis showed the presence of a single D N A component, as we had obtained previously for pea m t D N A (Ward et al. 1981). Thus, the m t D N A preparation is not detectably contaminated by any other DNA. Reassociation-kineties analysis. The same protocol was used to establish the percentage of m t D N A in total D N A as we used in our earlier report on the percentages of ctDNA in total pea D N A (Lamppa and Bendich 1979). Briefly, purified m t D N A was labeled in vitro by the nick-translation method (Maniatis et al. 1975) with 3ZP-labeled ATP, and sonicated in 0.2 M sodium-phosphate buffer (pH 6.8) to about 300 bases in the presence of unlabeled calf thymus DNA. The labeled m t D N A or ctDNA (minimum specific activity of 10 6 cpm/gg) was reassociated with total pea D N A in 1 M NaC104, 30 mM 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris) pH 8, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1% sarkosyl at 25 ~ C below its thermal denaturation temperature. Hydroxyapatite chromatography was used to separate reassociated from single-stranded DNA. We present the data in terms of Cot (M.s for total DNA) in contrast to Po t (M.s for labeled probe DNA), to compare data using different probes for each series of curves. This obviates the necessity of including a control driver D N A for reactions when different probes with different specific activities were employed.
Percentage of mtDNA in total DNA estimated by reassociation kinetics. The m t D N A was labeled in vitro and reassociated following the procedure used previously to determine the percentage of c t D N A in total cellular D N A from pea (Lamppa and Bendich 1979). Figure 1 shows the reassociation kinetics for labeled m t D N A in the presence of calf thymus DNA, or in the presence of unlabeled m t D N A mixed with calf thymus D N A as a model reaction. Nearly all of the reassociation of labeled m t D N A in the model reaction occurred as a single kinetic component, with less than 10% reassociating rapidly as would be expected for repetitive D N A sequences. A repetitive component of about 5% was detected previously in our spec-
Fig. 1. Levels of mitochondrial D N A in total D N A from pea shoots and roots. Labeled m t D N A (about 10 ngm1-1) was reassociated with 150 ggm1-1 of unlabeled total D N A from shoots (,), roots (,,), calf thymus (A), or calf thymus containing 2% m t D N A as a model reaction (o). Since the reaction rates for shoots and roots were fourfold slower than for the model, total D N A for these tissues contained 0.5% mtDNA. Singlecomponent kinetic curves, equal to 80%, are drawn through the data points. Zero-time binding (quick-cool, QC) was about 10% in each case and is indicated by the arrow
trophotometric analysis of pea m t D N A reassociation (Ward et al. 1981). Figure 1 also shows the reassociation of labeled m t D N A with unlabeled total pea D N A isolated from shoots and roots. The rightward displacement of the curves indicates that the shoot and root DNAs contained less m t D N A than was added to the model reaction. The model reaction contained 3.0 gg m t D N A per 150 Ixg of calf thymus DNA, or 2% mtDNA. The percentages of m t D N A in total pea D N A from the different tissues were estimated from the relative positions of the reassociation curves; the values are given in Table 1, line 1. Similar reactions were performed using total D N A extracted from embryos (plumule, epicotyl and radicle), shoots grown in the dark, and leaves from different stages of devel9opment. There is about 9" 106 dalton of m t D N A homologous to c t D N A in corn which includes sequences representing the genes for 16S ribosomal R N A (Stern and Lonsdale 1982) and the large subunit of ribulose-bisphosphate carboxylase (Lonsdale et al. 1983). If found for pea, such sequences would constitute only 4% of the mitochondrial genome (see below) and would not appreciably affect our reassociation-kinetic curves. Additional sequences on pea m t D N A restriction-enzyme fragments cross-react with cloned hybridization probes from the mung-bean and spinach chloroplast genomes (Stern and Palmer 1984). However, since we found no large repetitive component in any of our reassociation-kinetic curves, even with total leaf D N A
G.K. Lamppa and A.J. Bendich: Mitochondrial DNA during development of pea
Table 1. The mitochondrial D N A and chloroplast D N A contents in pea tissues Embryo
2. ctDNA (%)c
3. Nuclear D N A e ploidy (C value)
m t D N A (%)
4. Copies/cell ~ mtDNA ctDNA
First leaf b Mature
0.9 12 2-4
All of the first leaves from young (6 d after planting) or mature (9 d after planting) plants were pooled for D N A extraction b The first leaf set was obtained from plants maintained in the dark or transferred to the light 24 h before harvest (see Material and methods). N D means not determined c First-leaf data are from this work; all other ctDNA data were obtained previously (Lamppa and Bendich 1979) a This is an average of a range of 1.3-7.3 Ploidy values are from Van Oostveldt and Van Parijs (1975, 1976), Jones (1977) for embryonic axis, Evans and Van't Hof (1975) for root and leaves, and Evans and Van't H o f (1975), Van Oostveldt and Van Parijs (1975, 1976) for shoots f Values are calculated as described (Lamppa and Bendich 1979) using the lowest nuclear ploidy for each tissue and the Feulgenstaining-based value of 6.32. ]012 dalton (10.5 pg) of D N A per diploid nucleus (Bennett and Smith 1976). For light-grown shoots the range was 990-5900 for 1.3-7.3% c t D N A ; the 140 value for m t D N A was obtained with the D N A containing 7.3% ctDNA. Recently the diploid D N A content for pea was reported as 7.7 pg based on flow cytometry of mithramycin fluorescence from leaf nuclei (Galbraith et at. 1983). To use 7.7 pg for calculation, the copy numbers should be multiplied by 7.7/10.5 (for example, embryo would contain 300 copies of mtDNA)
that contains 12% ctDNA (Table 1), we conclude that the fraction of our labeled mtDNA that is homologous to ctDNA is less than 10% and would not appreciably influence our estimates of mtDNA as a fraction of total cellular DNA. Total cellular DNA from pea embryos contains 1.5% mtDNA. This is the highest percentage of mtDNA in the total DNAs isolated from the different pea tissues (Table 1, line I). Shoot and root DNAs contain 0.5% or less mtDNA, and lightgrown leaves contain 0.9%. In contrast, we have found previously that the percentage of ctDNA in the same DNA preparations increased above the embryonic level in light-grown tissues. Mature pea leaves had eight times more ctDNA than embryonic tissue (Lamppa and Bendich 1979). The last two columns of Table 1 show that the first leaf contained 4.5% ctDNA whether etiolated or grown in the light for 1 d. The level of mtDNA in plants kept in the dark was 0.8%, the same as the average value (0.9%) of all the leaves of plants grown for 9 d in either the dark or light. The histogram in Fig. 2 compares the relative levels of mtDNA and ctDNA for the different developmental stages. The amount of organellar DNA in the embryo is set at unity to illustrate the decrease in the level of mtDNA in total DNA during development, and the increase in ctDNA. Nuclear DNA content in pea usually increases during tissue differentiation (Evans and Van't Hof 1975; Van
Oostveldt and Van Parijs 1975, 1976; Jones 1977). Cells in roots and dark-grown shoots may attain nuclear ploidy values of 8C and 16C, respectively (Evans and Van't Hof 1975; Van Oostveldt and Van Parijs 1975). We argued previously that the decrease in the contribution of ctDNA to total root DNA reflects nuclear DNA endoreduplication without concomitant ctDNA synthesis (Lamppa and Bendich 1979). We find a parallel decrease
7~ 6_ 5_ 4_ 3. 2.
FIRST LEAF LIGHT
Fig. 2. Relative levels of mitochondrial and chloroplast D N A s in pea tissues. Embryo D N A contains 1.5% of both m t D N A (spotted bars) and ctDNA (open bars) and is given the value of 1, The levels of organellar D N A in total D N A from Table 1 are given relative to the embryo level. Total D N A was extracted from entire shoots grown in the light or dark, from young (at the onset of greening) and mature first leaves, and from first leaves either maintained in the dark or transferred to the light for 24 h (see Material and methods)
G.K. Lamppa and A.J. Bendich: Mitochondrial D N A during development of pea
in the level of mtDNA during root differentiation that could be explained in the same way. Since we find that the percentage of mtDNA in shoots and leaves decreases as well, we conclude that mtDNA synthesis does not keep pace with nuclear ploidy increases, and mtDNA is diluted during cell division and differentiation.
Estimation of the number of copies of the mitochondriaI genome per cell. We have estimated the number of mitochondrial genomes per cell based on the following information: 1) The size of the pea mitochondrial genome is 240.106 dalton, as determined by reassociationkinetics analysis (restriction-enzyme analysis gave a similar value) (Ward et al. 1981). We equate this value with the genome of the mitochondrion, although the relationship between the genome and the variously sized circular mtDNA molecules is still not clear (Bendich 1982; Dale et al. 1983). 2) The cells within a tissue have the same amount of nuclear DNA. The range of nuclear DNA content that has been reported is listed in Table 1, line 3. We have used the minimum ploidy values (2C) to estimate the copies of the mitochondrial genome. Most cells in the pea embryo have a 2C amount of DNA (Van Oostveldt and Van Parijs 1975, 1976; Jones 1977). At least 90% of the cells of mature leaves are diploid (Evans and Van't Hof 1975). The increases in nuclear DNA ploidy during seedling development are greater in roots (Evans and Van't Hof 1975) and most dramatic in dark-grown epicotyl tissue (Van Oostveldt and Van Parijs 1975, 1976). 3) The contribution of the chloroplast DNA varies as previously established (Lamppa and Bendich 1979). We calculate that a diploid embryonic cell from pea contains an average of 410 copies of mtDNA with a complexity of 240 x 10 6 dalton. Light-grown leaves have 260 copies, and light-grown shoots have 140 copies. Dark-grown shoots, which contain the lowest percentage of mtDNA, have only 80 copies of mtDNA per cell, assuming a diploid nuclear genome throughout the tissue. It is probable that half of the cells from dark-grown shoots are octoploid; the cells in pea stems have been shown to have a high ploidy level (Van Oostveldt and Van Parijs 1975, 1976). If one takes this into account, the average number of copies of the mitochondrial genome increases 2.5-fold (0.5 [80 copies]+0.5 [320 copies]=200 copies). Specialized cells may replicate their mtDNA (or nuclear or chloroplast DNAs) above the average values given in Table 1. In any case, the number of copies of
the mitochondrial genome do not increase in parallel with the number of copies of the chloroplast genome during tissue development. Discussion
The major finding of this work is that during the development of shoots, leaves and roots from embryonic tissue in pea, the number of mitochondrial genomes per cell either decreases or remains the same. It appears that there is little net synthesis of mtDNA during the time when the cells of the plant are expanding and greening. As the nuclear DNA ploidy increases from the embryonic 2C-4C level to reach 4C-8C in shoots, there may be, in fact, no change in the number of copies of mtDNA per cell so that a dilution of mtDNA occurs. In sharp contrast, we have found (Lamppa and Bendich 1979), using the same total DNA preparations, that the level of ctDNA increases almost threefold in shoots and as much as eightfold (or 16-fold for a 4C cell) above the embryonic level during light-controlled development of leaves. In roots a threefold decrease from the embryonic level is found for both mtDNA and ctDNA, and is likely a consequence of dilution by increasing nuclear ploidy. We conclude that the level of each of the three genomes in a pea cell can change independently during development: nuclear DNA increases slightly in leaves and more so in roots, plastid DNA increases greatly in leaves but not in roots, and mtDNA does not increase in either tissue. The percentage of mtDNA decreases in both dark- and light-grown leaves (Table 1). On the other hand, we find an increase in ctDNA content above the embryonic level whether the first leaf is grown in the dark (etiolated) or greened for one day before harvest. The fact that the increase in ctDNA occurs in the dark suggests that leaf development requires the amplification of the chloroplast genome, and an intrinsic program is followed regardless of the light conditions. Certain transcripts may be required in large amounts early in development to produce the photosynthetically competent cell. The only previous measurement of which we are aware for the number of mitochondrial gehomes in plants was 110-140 per diploid DNA amount for etiolated hypocotyls of watermelon, zucchini and muskmelon, species with a sevenfold range in the size of the mitochondrial genome (Ward etal. 1981). After considering nuclear ploidy, the estimate was 200-300 copies per cell (Bendich and Gauriloff 1984). This is about the
G.K. Lamppa and A.J. Bendich: Mitochondrial DNA during development of pea
same number we find for the pea tissues of comparable ploidy (leaves and embryo) and it will be interesting to learn whether other plants have similar numbers of genomes per cell. The copy number, however, does increase in at least one case. For muskmelon the fraction of D N A that is m t D N A in dry seeds is four- to fivefold greater than in hypocotyls or cotyledons of seedlings (Bendich and Ward 1980). The m t D N A s from dry seeds and seedlings were indistinguishable by comparing SalI, SmaI and EcoRI restriction-fragment patterns, indicating that selective changes in the structure of the genome did not occur (Bendich 1982). Most of the D N A in oocytes of Xenopus laevis and about one third in mouse eggs is m t D N A (Clayton 1982). We are aware of no copy-number measurements for somatic cells in animals, but in cultured mammalian cells the copy number is several thousand (Clayton 1982; Schmookler Reis and Goldstein 1983). For yeasts there are usually some 50-100 mitochondrial genomes per cell, but in some strains the variation can be greater depending on carbon source and growth phase (Dujon 1981). The thousands of copies of the chloroplast gehome in a plant cell are distributed among a variable number of plastids. Chloroplast multiplication during leaf development results in a dilution of the number of copies of the c t D N A molecule per plastid in pea (Lamppa and Bendich 1980), spinach (Scott and Possingham 1980), wheat (Boffey and Leech 1982) and beet (Tymms et al. 1983). A similar dilution occurs during the cell-cycle of the alga Olisthodiscus (Cattolico 1978), and can be inferred to occur during development of the single-celled green algae Acetabularia (Coleman 1979; Lfittke and Bonotto 1981) and Batophora (Coleman 1979) until many plastids contain no D N A detectable by 4'-6-diamidino-2-phenylindole (DAPI) fluorescence. What is the relationship between the number of mitochondrial genomes and the number of mitochondria per plant cell? Using serial sectioning and morphometric analysis of electron micrographs to count organelles, we calculated that there is an average of one or fewer mitochondrial genomes per mitochondrion in hypocotyls, leaves and cotyledons of watermelon, zucchini and muskmelon (Bendich and Gauriloff 1984). There were 400-1 100 ovoid mitochondria per cell in these differentiated tissues, whereas apical meristem cells of watermelon and muskmelon contained only about ten large, branching mitochondria. We do not known the number of mitochondria present in pea cells. If it is necessary for each mitochon-
drion to have at least one copy of its genome, then a pea leaf cell would have at most 260 mitochondria, since this is the number of copies of m t D N A per cell that we find (Table 1). The possibility exists, however, that not every mitochondrion contains one complete copy of its genome. In yeast the number of genomes per mitochondrion appears to have little functional importance since the organelles undergo continued fusion and fragmentation (Dujon 1981; Stevens 1981); a similar inference was drawn for plants (Bendich and Gauriloff 1984). Mitochondrial number, plasticity and ploidy may each affect the way in which these organelles participate in energy metabolism in plant tissues. There is, unfortunately, very little information currently available concerning the biogenesis of plant mitochondria. This work was supported by a grant from the National Science Foundation. We thank Shawna Gandy for assisting in some of the experiments.
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G.K. Lamppa and A.J. Bendich: Mitochondrial DNA during development of pea
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Received 15 March; accepted 16 July 1984