Effect of Benzyladenine on Diurnal Changes in DNA Content per Chloroplast and Chloroplast Replication in Intact Bean Leaves

The effect of benzyladenine (BA) on the diurnal changes in DNAand Chl contents per chloroplast and chloroplast replicationin primary leaves of bean plants (Phaseolus vulgaris L.) grownunder a 16 h light/8 h dark cycle was studied. Experiments weremade on primary leaves in the early expansion phase, where celldivision had been completed but chloroplasts were replicating.In untreated controls, chloroplast number, Chl content and freshweight per leaf showed daily periodic changes. Chl content perchloroplast increased in the light period every day, and freshweight per leaf increased most rapidly in the early dark period.Chloroplast number per leaf increased rapidly in the early darkperiod on day 9, though the increase began a little earlierand was less sharp on days 8 and 10. During these periods, DNAcontent per chloroplast was decreasing due to chloroplast divisionas chloroplast DNA (ctDNA) per leaf remained unchanged throughoutthe experimental period. BA induced increases in Chi contentper chloroplast, ctDNA content and fresh weight per leaf within6 h of its application, regardless of whether it was appliedat or 10 h after the beginning of the light period. Applicationof BA at 10 h in the light period shifted the start of chloroplastreplication by 6 h compared to that in untreated controls. However,when BA was applied at the beginning of illumination, the startof chloroplast replication showed the same relative change intime as above. 5-Fluorodeoxyuridine (5-FdU) promptly preventedBA-induced increase in Chl content and chloroplast number perleaf as well as ctDNA content per leaf.     

Benzyladenine-induced increase in DNA content in the nucleus of bean leaf cells

Primary leaves of bean plants (Phaseolus vulgaris L.) were treated with benzyladenine (BA) from 7 days after sowing. Nuclei were isolated and the DNA content per nucleus was determined. BA increased the amount of DNA per nucleus of untreated controls by about 50% without nuclear division.     

Benzyladenine-Induced Increase in DNA Content per Cell, Chloroplast Size, and Chloroplast Number per Cell in Intact Bean Leaves

Primary leaves of intact bean plants (Phaseolus vulgaris L.)were treated with benzyladenine (BA) at different stages oftheir growth. BA induced a marked increase in DNA content percell in growing leaves where no cell division occurred. BA stimulatedthe chloroplast replication compared with untreated leaves.After the replication period, chloroplast size continued toincrease in BA- treated leaves, but not in untreated controls.     

Effects of 6N-Benzyladenine on Primary Leaves of Intact Bean Plants and on their Sodium Absorption Capacity

The application of ⁶N-benzyladenine (BA) to primary bean-leaves attached to the intact plant resulted in increased leaf area. This was due to an extension of the duration of the period of leaf expansion. The dry-weight percentage of untreated leaves decreased continuously from emergence to abscission. In BA treated leaves this decrease stopped with the cessation of leaf expansion. Net chlorophyll synthesis occurs in untreated leaves as long as they continue to expand, shortly afterwards net chlorophyll decomposition starts. Benzyladenine treatments diminished the rate of chlorophyll synthesis immediately after its first application, but prevented the loss of chlorophyll afterwards. The abscission of primary leaves was also delayed by BA. During the period of rapid leaf expansion in slices cut from BA treated leaves, sodium absorption rates were lower than in those from untreated leaves. From the age of 14 days after sowing, till abscission time of untreated primary leaves, the BA treatment did not significantly affect sodium absorption rates by leaf slices.     

Increases in Nuclear DNA Content without Mitosis in Benzyladenine-treated Primary Leaves of Intact and Decapitated Bean Plants

Primary leaves of intact bean plants (Phaseolus vulgaris L.cv. Yamashiro-kurosando-saito) were treated with benzyladenine(BA) beginning on the seventh day after sowing when cell proliferationin the leaves had finished. Nuclear DNA contents were measuredby cytofluorometry combined with 4',6-diamidino-2-phenylindole(DAPI) staining. In the untreated controls, most mesophyll andabaxial epidermal cells contained a nucleus whose DNA contentwas 2C; whereas most adaxial epidermal cells contained a 4Cnucleus. Benzyladenine treatment induced 4C nuclec in mesophylland abaxial epidermal cells; but BA induced 8C nuclei in adaxialepidermal cells. To compare the effects of endogenous cytokininaccumulation, bean plants were decapitated above the primaryleaves on day 7 and continually disbudded thereafter. Changesin the nuclear DNA content in primary leaves attached to thedecapitated plants was similar to that for BA-treated primaryleaves. No multinucleate cells were formed and no mitotic figureswere present in the BA-treated leaves or in the primary leavesof decapitated plants. Our results indicate that both BA treatmentand decapitation induced one round of nuclear DNA synthesiswithout mitosis in a large number of mesophyll and epidermalcells.     

Amplification of Extrachromosomal Circular DNA in Intact Bean Leaves Treated with Benzyladenine

Bean (Phaseolus vulgaris L.) leaves were treated with benzyladenine(BA) beginning 7 days after sowing, and were harvested at 21days. There were less than 10 molecules per cell of small polydispersecircular (spc) DNA in mesophyll cells from untreated leaves,and more than 30 molecules in the BA-treated cells. The spcDNAranged in size from 0.2 to 7.5 µm, with a mean lengthof 1.7 µm. (Received May 29, 1985; Accepted December 24, 1985)     

Changes in mitochondrial DNA levels during development of pea (Pisum sativum L.)

The percentage of mitochondrial DNA (mtDNA) present in total DNA isolated from pea tissues was determined using labeled mtDNA in reassociation kinetics reactions. Embryos contained the highest level of mtDNA, 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 DNA contained only 0.3% mtDNA. This may be a reflection of increased nuclear ploidy levels without concomitant mtDNA synthesis. It was possible to compare the mtDNA values directly with previous estimates of the amount of chloroplast DNA (ctDNA) per cell because the same preparations of total DNA were used for both analyses. The embryo contained 1.5% of both mtDNA and ctDNA; this equals 410 copies of mtDNA and 1200 copies of ctDNA per diploid cell. Whereas mtDNA levels decreased to 260 copies in leaf cells of pea, the number of copies of ctDNA increased to 10300. In addition, the levels of ctDNA in first leaves of dark-grown and light-transferred pea were determined, and it was found that leaves of plants maintained in the dark had the same percentage of ctDNA as those transferred to the light.Abbreviations ctDNA chloroplast DNA - mtDNA mitochondrial DNA     

Differential effects of benzyladenine on the ultrastructure of chloroplasts in intact bean leaves according to their age

Summary Primary leaves of intact bean plants (Phaseolus vulgaris) were treated with benzyladenine (BA) at different stages of growth. Changes in the ultrastructure of chloroplasts and the contents of chlorophyll, carotenoid, and protein (soluble and insoluble) in leaves with different treatments were followed and compared. When BA was applied from an early stage, it increased the chloroplast size and the number of grana per chloroplast without any pronounced effect on the grana size. When BA treatment was stopped at the early stage, these effects remained for a while and then diminished. When BA treatment was begun at a late stage, such marked effects were not observed, suggesting that only young leaves could respond to BA in that manner. However, the late treatment efficiently prevented the process of the last stage of leaf senescence characterized by disintegration of thylakoids with concomitant increase in the plastoglobule size. Chlorophyll, carotenoid, and insoluble protein contents per leaf followed similar changes in chloroplast length and the number of grana per chloroplast section.     

Effects of Benzyladenine on Chlorophyll, DNA, RNA and Protein Content of Attached Young Bean (Phaseolus vulgaris L.) Leaves

N⁶-Benzyladenine (BA) was applied to intact bean (Phaseolusvulgaris L.) primary leaves at 2 and 6 days after imbibition,when they were in the cell division and post-cell division stages,respectively. BA treatment at day 2 temporarily inhibited an increase in chlorophyllcontent in the following day, but stimulated it in later days.No such inhibition by BA was observed for changes with timein DNA, RNA, and protein content and f. wt. On the other hand,BA treatment at day 6 enhanced RNA and protein content, withoutsignificant influence on DNA and chlorophyll content and f.wt. The mode of cytokinin action on greening in leaves during cell-divisiongrowth seems to be different from that in etiolated cotyledons. Phaseolus vulgaris L., bean, greening, benzyladenine, DNA, RNA, protein     

Chloroplast and Nuclear DNA Content of Cultured Spinach Leaf Discs

The amounts of chloroplast DNA and nuclear DNA in the cellsof spinach leaf discs cultutred under different light regimeshave been measured. The cellular level of ctDNA increased 10-foldin discs cultured in white light and was accompanied by a 2-foldincrease in the cellular level of nuclear DNA. In low intensitygreen light the cellular level of ctDNA increased 6-fold butwas not accompanied by an increase in the level of nuclear DNA.No net DNA synthesis on a per cell basis occurred in discs culturedin darkness. Chloroplasts of uncultured leaf discs containedan average of 83 plastome copies. The number of plastome copiesper chloroplast after 6 days culture decreased to 36 copiesin darkness, remained almost constant at 73 copies in whitelight and increased to 215 copies in low intensity green light. These results suggest that ctDNA replication can be independentof cellular levels of nuclear DNA and chloroplast division.     

Copy numbers of chloroplast and nuclear genomes are proportional in mature mesophyll cells of Triticum and Aegilops species

The possibility of estimating the proportion of chloroplast DNA (ctDNA) and nuclear DNA (nDNA) in nucleic-acid extracts by selective digestion with the methylation-sensitive restriction enzyme PstI, was tested using leaf extracts from Spinacia oleracea and Triticum aestivum. Values of ctDNA as percentage nDNA were estimated to be 14.58%±0.56 (SE) in S. oleracea leaves and 4.97%±0.36 (SE) in T. aestivum leaves. These estimates agree well with those already reported for the same type of leaf material. Selective digestion and quantitative dot-blot hybridisation were used to determine ctDNA as percentage nDNA in expanded leaf tissue from species of Triticum and Aegilops representing three levels of nuclear ploidy and six types of cytoplasm. No significant differences in leaf ctDNA content were detected: in the diploids the leaf ctDNA percentage ranged between 3.8% and 5.1%, and in the polyploids between 3.5% and 4.9%. Consequently, nuclear ploidy and nDNA amount were proportional to ctDNA amount (r₍₁₉₎=0.935, P>0.01) and hence to ctDNA copy number in the mature mesophyll cells of these species. There was a slight increase in ctDNA copy numbers per chloroplast at higher ploidy levels. The balance between numbers of nuclear and chloroplast genomes is discussed in relation to polyploidisation and to the nuclear control of ctDNA replication.Abbreviations ctDNA chloroplast DNA - nDNA nuclear DNA - RuBPCase ribulose-1,5-bisphosphate carboxylase - DAPI 4,6-diamidine-2-phenylindole     

Co-immunolocalization of topoisomerase II and chloroplast DNA in developing,dividing and mature wheat chloroplasts

This paper describes the first localization of immunofluorescence of topoisomerase II in developing chloroplasts. In order to investigate the relationship between topoisomerase II and chloroplast DNA (ctDNA) replication during chloroplast development the 7-day-old wheat leaf was used. Topoisomerase II was immunolabelled and fluorescein tagged and the ctDNA simultaneously stained with 4,6-diamidino-2-phenylindole (DAPI) in the same sections. Topoisomerase II was detected at every stage of chloroplast development and maximal levels of topoisomerase II were found in chloroplasts at the time of ctDNA replication. Topoisomerase II was localized around the plastid periphery, exactly mirroring the position of the ctDNA. After chloroplast division both topoisomerase II and ctDNA are seen to be restricted to small discrete areas within the plastid, but at different sites. These findings strongly suggest a role for topoisomerase II in ctDNA decatenation prior to chloroplast division.     

Increase in DNA Content of Primary Leaves of Phaseolus vulgaris upon Decapitation

Changes in DNA content of bean (Phaseolus vulgaris) primaryleaves after decapitation were investigated. When apical budswere removed at 11 d, DNA content per leaf increased by about20% at 15 d and then decreased in parallel with the controls.The RNA and chlorophyll contents, fresh weight, and leaf areaexpressed on a single leaf basis changed in the same manneras the DNA content in response to decapitation. But when bothapical and lateral buds were removed, all these values continuedincreasing during the test period. Thus, growing lateral budsand apical buds have the same effect on the DNA change in primaryleaves as that due to ageing of the leaves. Cell number perleaf was not increased by any treatment, indicating that theobserved increase in the DNA content of primary leaves is ascribableto an increase in DNA per cell. Next, the whole shoots above the nodes of primary leaves wereremoved at various ages. The response of primary leaves to decapitationvaried according to their age. With age, they lost the abilityto increase their fresh weight, area, and chlorophyll contentbut not their DNA and RNA contents in response to decapitation.Decapitation stimulated chloroplast replication only withinthe period in which chloroplasts were replicating in controlleaves, but it induced chloroplast enlargement at any age. Therefore,the increase in DNA content after decapitation may be partiallydue to an increase in the amount of chloroplast DNA. When stems were heat-girdled above the nodes of the primaryleaves, these leaves showed responses similar to but smallerthan those to decapitation. The senescence of primary leavesseems to be controlled by the distribution of substances whichare transported from the roots.     

Effect of Benzyladenine on DNA, RNA, Protein, and Chlorophyll Contents in Intact Bean Leaves: Differential Responses to Benzyladenine According to Leaf Age

Benzyladenine (BA) was applied to intact bean (Phaseolus vulgaris) leaves at different stages of their growth. Changes in the amounts of cellular constituents resulting from the different treatments were followed and compared. RNA, protein, and chlorophyll contents, dry weight, fresh weight, and leaf area per single leaf continued increasing when leaves were treated with BA from an early stage, whereas in untreated leaves all these values levelled off or declined with advancing age. Besides these changes, BA treatment induced an increase in the DNA content. Changes in RNA content was more remarkable in response to application or deprival of BA treatment than the corresponding ones in protein and chlorophyll contents. The pattern of response to BA varied greatly according to the age at which the leaf received the treatment. As leaves aged, they lost the ability to increase their area and fresh weight in response to BA. However, continuous treatment with BA from an early stage kept the leaves young and able to respond.     

Behavior of Chloroplast Nucleus during Chloroplast Development and Degeneration in Chlamydomonas reinhardii

Changes in morphology of chloroplast nuclei (cp-nuclei), totalcp-DNA content, number of cp-nuclei, oxygen-evolution activityand chlorophyll (a and b) content were examined during the degenerationand development of chloroplasts, using Chlamydomonas reinhardiicells which had been incubated on solid medium for various periods. Under 4'-6-diamidino-2-phenylindole (DAPI) epifluorescence microscopy,each cell that had been incubated for 7 days had one cell nucleus,one cup-shaped chloroplast and about 10 small, dispersed cp-nucleiin the chloroplast. One day after incubation of these cellson fresh medium, the cell volume and cp-nuclei increased insize 2-3 fold, but rapidly decreased in size after cell division.After about 7 days of incubation, cells ceased to divide andcp-nuclei began to associate with each other. At about 20 daysthey formed a ring-shaped structure surrounding the pyrenoid,followed by condensation into one cp-nuclear particle near thepyrenoid. When 41-day-old cells, having only one cp-nucleus,were reinoculated on fresh solid medium, the cp-nucleus increasedin size 2–3 fold, divided into several cp-nuclear particlesand then dispersed into the chloroplast, forming a bead-likestructure, before cell division. From microscopic fluorometry,a 4-fold increase in total cp-DNA content per chloroplast, withoutan increase in the number of cp-nuclear particles per chloroplast,occurred one day after the start of the experiment and one dayafter reinoculation of 41-day-old cells onto fresh medium. Theprocess of condensation of dispersed cp-nuclear particles intoone cp-nucleus during degeneration of the chloroplast was notaccompanied by any change in total cp-DNA content per chloroplast.A large peak of oxygen-evolution (0.6–0.9 pmoles/cell/hour)was seen one day after inoculation and reinoculation of thecells. The chlorophyll content (a+b) was high (1.2–2.2pg/cell) during the first week of incubation, after which itgradually decreased. (Received December 18, 1985; Accepted April 2, 1986)     

Age dependent changes in phospholipids and galactolipids in primary bean leaves (Phaseolus vulgaris)

Primary leaves of Phaseolus vulgaris show concomitant changes in phospholipid, galactolipid, chlorophyll and fresh weight during leaf development from 3 to 32 days after planting. Phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidyl inositol show only small changes on a mole per cent lipid phosphate basis during leaf development. The chloroplast lipids, phosphatidyl glycerol, monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) all show marked increases and decreases which are coincident with chloroplast development. The decline in the leaf content of chloroplast polar lipids and chlorophyll become evident upon reaching maximal leaf size. The molar ratio of galactolipids (MGDG/DGDG), reaches a maximum value of 2.3 in expanding leaves, but steadily declines during senescence to a minimum value of 1.5 at abscission. The declining ratio is caused by a preferential loss of MGDG in the senescing leaves.     

Response of soybean photosynthesis and chloroplast membrane function to canopy development and mutual shading

The effect of natural shading on photosynthetic capacity and chloroplast thylakoid membrane function was examined in soybean (Glycine max. cv Young) under field conditions using a randomized complete block design. Seedlings were thinned to 15 plants per square meter at 20 days after planting. Leaves destined to function in the shaded regions of the canopy were tagged during early expansion at 40 days after planting. To investigate the response of shaded leaves to an increase in available light, plants were removed from certain plots at 29 or 37 days after tagging to reduce the population from 15 to three plants per square meter and alter the irradiance and spectral quality of light. During the transition from a sun to a shade environment, maximum photosynthesis and chloroplast electron transport of control leaves decreased by two- to threefold over a period of 40 days followed by rapid senescence and abscission. Senescence and abscission of tagged leaves were delayed by more than 4 weeks in plots where plant populations were reduced to three plants per square meter. Maximum photosynthesis and chloroplast electron transport activity were stabilized or elevated in response to increased light when plant populations were reduced from 15 to three plants per square meter. Several chloroplast thylakoid membrane components were affected by light environment. Cytochrome f and coupling factor protein decreased by 40% and 80%, respectively, as control leaves became shaded and then increased when shaded leaves acclimated to high light. The concentrations of photosystem I (PSI) and photosystem II (PSII) reaction centers were not affected by light environment or leaf age in field grown plants, resulting in a constant PSII/PSI ratio of 1.6 ± 0.3. Analysis of the chlorophyll-protein composition revealed a shift in chlorophyll from PSI to PSII as leaves became shaded and a reversal of this process when shaded leaves were provided with increased light. These results were in contrast to those of soybeans grown in a growth chamber where the PSII/PSI ratio as well as cytochrome f and coupling factor protein levels were dependent on growth irradiance. To summarize, light environment regulated both the photosynthetic characteristics and the timing of senescence in soybean leaves grown under field conditions.     

Chemical suppression of the symptoms of two virus diseases

Carbendazim applied at the rate of 2 g per plant to the roots of tobacco (Nicotiana tabacum cv. White Burley) plants before infection with tobacco mosaic virus (TMV) caused very considerable reduction in the severity of disease symptoms in systemically infected leaves but did not affect their virus content. Leaves of untreated, infected plants had a greatly reduced chlorophyll content 100 days after infection whereas the chlorophyll content of leaves of infected plants treated with carbendazim was similar to that of normal uninfected leaves. Carbendazim had no effect on the infectivity of TMV in vitro or on the local lesion reaction of N. glutinosa plants when inoculated with TMV. Carbendazim was applied to lettuce cv. Cobham Green at a total rate of o-i g per plant before and after they were infected with beet western yellows virus and the plants were then grown on in the field. At harvest time (50 days after infection) almost all the treated virus-infected plants were of a normal green appearance, whereas the untreated controls were almost all very severely yellowed and unmarketable.     

Changes in chloroplast number per cell during leaf development in spinach

Summary The amounts of chlorophyll and nitrogen and the numbers of cells per unit area change as the green leaves of spinach plants grow and increase in size in the light. The changes in the numbers of chloroplasts per cell were measured by a new method. A 5-fold increase in the numbers of chloroplasts per cell took place in both palisade and mesophyll cells over a growing period of 10 days during which time the area of the leaves increased from 1 to 50 cm². Proplastids were not present in the young green leaves but electron-microscope and phase-contrast observations showed the presence of grana-containing chloroplasts, many of which appeared to be undergoing division by constriction. It is suggested that the large increase in chloroplast numbers as leaf cells grow and expand in the light is from the division of differentiated chloroplasts containing grana.