Characteristics of a Virescent Cotton Mutant - NCBI

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Plant Physiologist and Geneticist, Crops Research Division, Agricultural Research Service,. United States ..... Recovery. Green. Recovery. Ether soluble. H20. Soluble. Basic (amino acids) ..... aggregatioll of the lamelflar disc,s in the (chloro-.
Plant Physiol. (1968)

43,

1611-1616

Characteristics of a Virescent Cotton Mutant' C. R. Benedict and R. J. Kohel Plant Physiologist and Geneticist, Crops Research Division, Agricultural Research Service, United States Department of Agriculture and Texas A&M University, College Station, Texas 77843

Received April 2 1968. Abstract. The virescent cotton (Gossypiumn kirsuttum) mutant described here differs from normal cultivated cotton by a single mutation in the nucleus. The mutant exhibits nuclear control of chlorophyll and carotenoid development. Young leaves are distinctly yellow and become green with age. There is no unusual photometabolism of 14CO., or 14C-acetate in this mutant. It is probable that the nuclear virescent mutation is in a locus ooncerned with making structural units. The yellow leaves do show a high photosynthetic capacity on a chlorophyll basis. At saturating light intensity the rate of C02 fixation is 8 fold higher than the green control leaves. Thus, impaired pigment synthesis which could be lethal is offset by a high photosynthetic capacitv in the virescent leaves.

Voni Wettstein (13. 14, 15) hlas examined the formation of lamellar systenms in plastids of mutant barley. In this tissue there is evidence that the development of the fine structure of the plastids is controlled by nuclear genes. Products of the nuclear genes affect the genes of the chloroplast (3) although it is uncertain how this affect is brought about. Nuclear genes could affect the development of the chloroplast by limiting the supply of metabolites. or bv producing activators or inhibitors that affect certain key genes of the chloroplast. Rhyne (5) has described the genetics of a virescent cotton mutant that differs from normal cultivated cottons by a recessive allele at a single locus. This mutant exhibits nuclear control of chlorophyll synthesis and possibly plastid development. The yellow color of these leaves can be maintained for relatively long periods under conditions of low temperature, i.e., 250. This is typical of most virescent plants ( 10). We have examined these vireseent leaves to determ,in,e if the development of metabolic reactions (as well as chlorophyll development) are arrested in this mutant.

Materials and Methods Materials. Glycine, serine, fructose, glucose, sucrose, ATP, GSH, phosphoglvceric acid tricyclohexylammonium salt, and ribulose- 1,5-diP tetrasodium salt were purchased from Sigma Chemical Company. 14C-Am.no acid mixture, sodium 'IC-4-

1 This work was supported in part by the Cotton Producers Institite and the Cotton Genetics Regional Project S-I.

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acetate, anid sodium 14IC-bicarbonate were purchased from New England Nuclear Corporation. Plants. Plants of virescent and normal green Gossypihin hirsutum were grown in the greenhouse and in the field. In all experiments an attempt was made to use green and virescent leaves of comparable development by harvesting leaves from the same node. CO, Fixation Experimtents. Leaves were harvested, rinsed with distilled water, blotted on papeltowels and weighed. Each leaf was suspended (froml a thread attached to the petiole) in a 500 ml separatory funnel through a serum stopper. The funnel was mounted on a ring stand between 2 flat, rectangular chromatographv jars of water. Light of about 4000 ft-c was provided by two 150 watt flood bulbs through the tanks of wAater. The temiperatuire in the flask was about 30 to 320. The leaves were equilibrated 2 min in the light. -1CO.2 was released fromii 20.0 mg of Bal4CO,. containing 500 1Ac of radioactiv,ity by adding 1.5 ml of lactic acid through the serum stopper with a syringe. The leaves were allowed to fix 14jC09 for 8 miii, then were removed from the funnel and plunged into boiling 95 % ethyl alcohol. The leaves were simmered for 20 min and extracted 4 times with boiling 95 % ethyl alcohol. The extracts were combined and evaporated to dryness on a flash evaporator in the presence of formic acid. The residue was dissolved in water and ether, and the water soluble and ether soluble phases were separated. Acetate 3MetabolismII. Leaves were harvested. rinsed with distilled water and blotted. Twentv half-inch leaf punches were taken at random from the leaves and weighed. The punches were floated on 10.0 nil of 0.1 M tris buffer pH 7.5 containing 100 ,Lmoles of potassium acetate in a 125 ml Erlen-

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meyer flask. The leaf punches in the Erlenmeyer flask were equilibrated for 5.0 minutes in a beaker of water at 370 which was illuminated from the bottom with one 150 watt flood bulb. The light intensity was approximately 4000 ft-c. After equilibration, l100 uc of 14C-1-acetate was added to the flask. The leaves had metabolized the 14C-acetate for 15 min when the reactioni was stopped with boilinig 95 % ethyl alcohol. The alcohol solulble comnipounds were extracted in a maniner similar to that used for CO. fixation experimiienlts. Preparation of Solutble Enzyme Extracts. Leaves were harvested, rinsed with distilled water, blotted anid weighed, then ground in a chilled mortar with sand and 0.1 M tris buffer plI 7.5 containing 0.1 mM GSH. The homogenate was squeezed through 2 layers of cheesecloth. The filtrate was centrifuged for 30 ;min'at 20,000g in a refrigerated Sorval12 centrifuge. The supernatant fraction was used to assay ribtulose diphosphate carboxylase. Ribulose Diphosphate Carboxylase. This enzyme was assayed by t-he procedure described by Fuller and Gibbs (2) The reaction mixture contained in Fmoles: 100, tris buffer pH 7.5; 10, MgCI,; 5, GSH, 2.5, ribulose-1,5-diP; 50, potassium bicarbonate containing 10 j,c of radioactivTity; and 0.5 ml of supernatant protein and water to 1.0 ml. The reactio,n was incubated 15 min at 31". The reaction was stopped by adding 1.0 ml concentrated HCI to each tube. The precipitated protein was removed by centrifugation and washed 3 times with water. The original supernatant and washings were conmbined and evaporated to dryness on a flash evaporator. The residue was dissolved in water and evaporated to dryness in the presence of formic acid. This process was repeated 2 or 3 tirnes to rid the vessel of unreacted '1C-bicarbonate. The residue was dissolved in water and assayed for radioactivity in a Beckman liquid scintillation system. Pignewt Analysis. For chlorophyll and carotenoid analysis the leaves were harvested, washed with distilled H0O, blotted oni paper toweling and weighed. The leaves were ground in a mortar with sand and cold acetone. The mixture was transferred to cups and centrifuged. The supernatant fraction was removed and the residue washed with cold acetone until it was colorless. The combined acetone extracts were evaporated to dryness in vacuo. The residue was dissolved in methyl alcohol and the visible absorption spectra determined with a Beckman DK-2 Recording Spectrophotometer. The methanol extract was saponified bv treatment with 30 % methanolic KOH at room temperature for 8 hr in the dark. Following saponification the carotenoids were transferred to hexane. The visible absorption spectrum of the carotenoid fraction was determined.

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Chlorophyll Determzinationi. Leaf and chloroplast chlorophvll was extracted bv acetone and transferred to methyl alcohol or the chlorophyll was extractedl by methyl alcohol alone. The visible absorption spectrum of the methaiiolic extract was determined. The anmounit of chlorophyll a+b in the extract was determined from the absorption at 650 nimu anid 665 m,u by the miietlhod of MacKinneey ( 12). Separationl of Radioactive Comiipounds in Water Soluble Ei-tracts. The water soluble extracts fronm the 14CO., or l C-acetate experimenits were passed through Do\vex-;() (H+) resini columins, lO) X 15.0 cm. The effluenit from these columns was evaporated to dryniess. The ' iC-mino acids (basic fractioni)

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FIG. 1. Visible absorption spectra of the methanolic extract of virescent and normal green cotton leaves. The visible absorption spectra are: 1) immature virescent leaf; 2) immature normal green leaf; 3) mature virescent leaf and 4) mature normal green leaf. The immature

virescent leaf and the immature normal green leaf contained 0.512 and 1.31 mg chlorophyll a+b/G FWT, respectively. The mature virescent leaf and mat:ure normlal greeni leaf containerl 1.54 atnd 1.68 m11g chlorophyll t+bl/G FWT, respectively.

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were eluted from the Dowex-50 columns with 50.0 ml of 1.0 N NHOH. These acids were evaporated to dryness, dissolved in water and assayed for radioactivity. The effluent residue was dissolved in water and passed through a Dowex-1 (formate) resin columin. 1.0 X 15.0 cnm. The effluent containing the 1*Csugars (neutral Fractions) was evaporated to dryness, dissolved in water anid assayed for radioactivity. The 14C-organic acids (acidic fraction) were eluited from the Dowex-1 columns with 50.0t ml of 8 N formic acid. The acid fraction was evaporated to dryness, dissolved in water and assayed for radioactivity. The radioactive amiino acid mixture was separated by paper chromatography by the procedure of Rinne et al. (6). The 14C-sugars were separated by paper chromatography by the procedure of Benedict and Beevers (1). Mleasurement of Radioactivzity. The amount of radioactivity in the aqueous samples was assaved in a Beckman liquid scintillation system. Radioactive sample of 0.2 ml each was added to 15.0 ml of scintillation fluid containing 5 g of PPO (diphenyloxazole), 100 g of napthalene, 10 ml of water and dioxane to 1 liter. Tihe scintillation vials were dark-adapted for several hours anid the counts were assayed with + 2 % error.

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Results The visible spectrum of a methanol extract of green and virescent leaves is shown in figure 1. Absorbance at 665 m,u and 650 mni indicates the presence of chlorophyll a k b in all leaves. The mature virescent leaf contains nearly the same amount of chlorophyll as the mature green leaf. The immature virescent leaf, which is a prominent yellow color, contains a much reduced concentration of chlorophyll. There is no major qualitative difference in the pigments extracted by methyl alcohol from the virescent anXd green leaves. There is no

FIG. 2. Visible absorption slpectra of the hexane extract of virescent and normal green cotton leaves. The immature leaves used for the determination of chlorophyll in figure 1 were used for the determination of the carotenoids. The carotenoids of all of these leaves were partitioned into 100 ml of hexane. The visible absorption spectra are: 1) immature virescent leaf and 2) immature normal green leaf.

substantial accumulation of protochlorophyll in these leaves as indicated by the absence of any peak at 630 m,u.

Table I. Distribution of Radioactivity From 14CO2 Fixation in Virescent and Green Icaves

Radioactivity Fraction Ether

soluble

H2 0 Soluble Basic (amino acids) Neutral (sugars) Acidic (acids)

Virescent cpm/g fr wt 1,558,000 52,192,000 15,155,000 35,669,000 1,385,000 cpm/mg chlor

Ether

soluble

H,O Soluble

Basic (amino acids) Neutral (sugars) Acidic (acids)

2,265,000 75,883,000 22,034.000

51,860,00

2,014,000

Recovery

Green

Recovery

%

cpm/g fr wt 804,000 26,846,000 6,480,000

% 100 24

2

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%

100 29 68

100 29 68

3

433,000 14,472,000 3,491.000 10,105 000 647,000

100 24

70 5

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Table II. Distribution of Radioactivity? From 14C-acetatc Incorporation Into Green and Virescent Lcaves

Fraction Ether soluble H20 Soluble Basic (amino acids) Neutral (sugars) Acidic (alcids')

Radioactivitx Recovery Green % cpniz/gq fr wet 804,000 100 1,412,000

Virescent cpr/nj fr zt

742.000

1,559,000 660,000 47,000 852,000

The absorption spectra of the carotenioids from immiiature virescent and green leaves is showni ill figure 2. The carotenoids were l)artitioned into hexane from the methanolic extracts of the imiimature leaves in figure 1. Compared to normal green immature leaves the immature virescent leaves contain a reduced amount of carotenoids. Therefore, virescent leaves show a reduced content of the major photosynthetic pigments, chlorophyll, and carotenoids. There is anl equal distribtution of radioactivity from 14CO. fixation (table I) into sugars, amino acids and organic acids in virescent and green leaves. In addition, the chromatograms of the sugar and amino acid fractions show no unusual accumulation of radioactivity in these fractions in virescent and green leaves. Apparently the metabolic distribution of CO.. fixation products is tlle same in both these leaves. These leaves do not show any accumulation of 1,C-sucrose from "CO.. fixation. The major difference of virescent and green leaves is in the rate of 14CO., fixation. On a fresh weight basis, the rate of '4CO., fixation is twice as rapid in virescent leaves as in green. On a milligram of chlorophyll basis. the virescent leaves fix CO2 5 times as fast as the green leaves. As noted above, this faster rate of 14CO., fixation does not lead to an unusual accumulation of radioactivitv in any detectable fraction of compound. The result of the metabolisnm of 14C-acetate is shown in table II. There is an equal distribution of radioactivity from "GC-acetate in organic acid. amino acid and sugar fractions of virescent and green leaves. This distribution indicates that there is no unusual route of acetate respiration in virescent leaves. The fact that virescent leaves accumulate the same amount of radioactivity from 14C-acetate as green leaves, indicates no unusual rate of respiration in the yellow leaves. Because the rate of 14CO0 fixation was more rapid in virescent leaves this photosynthetic rate was studied as a function of light intensity. Gaffron and his associates (8, 9) have shown that a dominant gene mutant of tobacco has a high rate of CO, fixation. The tobacco mutant did not show a saturating level of CO. uptake in blue or red light. This is probablv due to impairment of the lamellar system (7).

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virescent and greeni thle lphotosynthetic rate per iimg of chlorophyll is 5 to 8 timiies ral)i(l in the virescent lea.ves. .\t the low liglht initelnsities, the rate of CO., fixationi is greater in the greeni leaves. as

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BENEDICT AND KOH EL-V IRESCENT COTTON AMUTANT

Table III shows the amount of ribulose diphosphate carboxylase activity in extracts of normnal green and virescent leaves. The amount of carboxylase activity per mg of chlorophyll in the virescent extract greatly exceeds the enzymlie activity in the green extract. These enzyme extracts were prepared for leaves which wvere about 50 % exl)andedl. Younger leaves (harvested fromii the first or seconId( node) did not always show this increased carboxylase activity in the virescent plants. On a freslh weight bas,is the amounit of ribulose diphosphate carboxyl7ase is nearly the saame in virescent and green leaves.

Discussion The concentrationi of chlorophyll a+b and carotenoid pigments in the yellow leaves of this virescent cotton mutant is less than the concentration in comparable green leaves. The mature virescent leaves contain ani amounit of chlorophyll comparable to green leaves. There is no unusual photometabolism of CO., or acetate in this mutanit nor does this mutant accumulate any unusual amounts of radioactivity in metabolic intermediates. This mutant differs from the normal by a single gene mutation (5)). These facts make it unilikely that the mutation involved is in a locus which determines the structure of an enzyme in the pigment synthesizing pathways for both carotenoids and chlorophyll. Rather, it is probable that the mutationi is in a locus concerned with making structural units. 'Gibor and Granick (3) have proposed a schenme wherebyinuclear genes control chloroplast development. In accord with this hypothesis nuclear virescent genes may prodclue inihibitors which affect the genes in the chloroplast concerned with making pigmient structuires. In virescent lcaves, showing poor pigment development, the labeling of sugars from 14CO., is evidence that the photosynthetic enzymes are operating as well as in the control leaves. The mietabolism of radioactive acetate in mutant anid greeni leaves is similar. It appears likely that metabolic enzymes involving acetate metabolism are o)peratingo normally in the virescent leaves. This muititanit slowly matures and completes its life cycle. The y\ello\ mutant shows the remarkable ability of iapidlv fixilln CO., into sugars anl amiinlo aci(s. Thus, impaire(l pigment synthesis, wlhich couil(d he lethal, is offset by high photosynthetic rates. Trown (11) has suggested that Fraction T protein which exhibits ribulose (liphosphate carhoxylase activity may be analogous to the proteill conistituient of the protochlorophyll holocchromie. This work su1ggests a close relation between thle synthesis of chlorophyll and ribulose diphosphate carboxylase. Huffaker et al. (5) have indeed showln that there is a close relationship of carboxylase activitv and chlorophyll synthesis. The fact that this virescent mutant has a decreased amount of chlorophyll and carotenoids but a normal level of ribulose diphos-

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phate carboxylase may relate to this problem. The high photosynthetic capacity per molecule of chlorophyll in virescent cotton leaves appears very simiilar to the high photosynthetic capacity of goldeni tobacco mutanits (9). The 2 mutants differ. however, itl responses to increases in light intensity. Further the tobacco mutant remainis yellow whereas the virescent muitant greens with age. The tobacco muttants cointain an ab'berant laimiellar svstem (8) anid the mi-ajor factor limiting growth and photosviytlhetic rate is light intensity. Good growth and hiigh photosynthetic rates requtire high light intensity. The cottoin mutant does not require high light intensity for growtlh or high photosynthetic rates. The nuclear mutation in tobacco leads to a chlorophyll deficienicy whereas the nuclear mutation in cotton causes a lag in chlorophyll development.

Literature Cited 1. BENEDICT, C. R. AND H. BEEVERS. 1962. Formation of sucrose from malate in germinating castor beans. Reactioni sequence from phosphoenol-pyruvate to sucrose. Plant Physiol. 37: 176-78. 2. FULLER, R. C. AND M. GIBBS. 1959. Intracellular andfl phylogenetic distribution of ribulose-1,5-diphosphate carboxylase and D-glyceraldehyde dehydrogenases. Plaint Physiol. 34: 324-28. 3. GIBOR, A. AND S. GRANICK. 1964. Plastids and mitochondria: Inheritable systems. Science 145: 89(-97. 4. HUFFAKER. R. C., R. L. OBENDORF, C. J. KELLER, .AN D G. E. KLEINKOPF. 1966. Effects of light intensity on photosynthetic carboxylative phase enzymies and( chloroplhyll synthesis in greening leaves of Hordeumt? 'uldare L. Planit Physiol. 41: 913-18. 5. RiHYNE, C. L. 1955. The inheritanice of yellowgreenl, a possible multation in cottoil. Genetics 40: 235-45. 6. RINNF, R. W., R. W. BU CKiNAN, AND C. R. BENEDICT. 1965. Acetate imietabolism in plhotosnlthetic bacteria. Plant Physiol. 40: 1066-73. 7. SCHMITD, G., J. M. PRICE, AND H. GAFFRON. 1966. Lainellar structure in chlorophyll deficient but normally active chloroplasts. J. Microscopie 5: 205-12. 8. SCHMII, G. ANI) H. GAFFRON. 1967. Light meta)ol isin and chloroplast structure in chlorophyll (leficiellt tobacco miutanits. J. Gen. Phvsiol. 50: 563-82. 9. SC(HMI1), (G. A\ND H. GAFERON. 1967. Quaintum re(juitremneiits for photosynthesis in chlorophyll (leficienlt p)lants with unlusual laiellar strtnctinres. J. Geln. Physiol. 50: 2131-44 10. SRB, A. M. A\ND R. D. OWENS. 1955. General Ge-netics. Chapt. 5. The impact of environment. W. H. Freeman and Company, San Francisco, California. 11. TROWN, P. W. 1965. An improved method for the isolation of carboxydismutase. Probable identity with Fraction I protein and the protein moiety of protochlorophyll holochrome. Biochemistry 4: 908-18.

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12. V1SHNIAC, W. 1957. Metlhods for study of the Hill Reaction. Methods in Enzymology. Vol. IV. S. P. Colowick and N. 0. Kaplan, eds. Academic Press Inc., New York. p 342-55.

13.

WETTSTEIN, D. 1958. The formation of plastid structures. Brookhaven Symposia in Biology 11: 138-59.

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WVETT.STEIN, D. 1959. The effect of genietic factors oni the submicroscopic structures of chloropilasts. J. Ultrastruct. Res. 3: 235-36. WVETTSTEIN, D. 19O. Multiple allelisim in iin(luce(I chlorophy ll mutants. 11. Error in the

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