Abstracts
A naturally occurring yellow tomato fruit mutant cv. Santa Clara was reciprocally crossed with the red wild type, after which F1 plants were self pollinated or backcrossed with both parents. Plants from F1 generations produced all fruits with a homogeneous deep red color when ripe. F2 plants showed a 3:1 red:yellow segregation of fruit color, and 100% red when backcrossed with red wild type or 1:1 red:yellow segregation in backcrosses with the yellow mutant; hence, yellow fruit color was determined by a recessive allele. Based on reciprocal crosses, fruit color is unlikely to be determined by maternal genes. Accumulation of lycopene dropped by 99.3% and<FONT FACE="Symbol"> b</font>-carotene by 77% in ripe yellow fruits, compared to the red wild type. Leaf and flower chlorophyll and total carotenoid concentrations were not affected by the yellow mutation. However, the mutant fruit had a higher rate of chlorophyll degradation during fruit ripening, whilst fruit from the F1 generation showed lower rates of degradation, similar to that observed in red wild type fruits.
Neste trabalho avaliou-se a herança da cor do fruto de um mutante natural da cv. Santa Clara, por meio da análise das gerações F1 e segregantes, obtidas mediante cruzamento entre plantas da cv. Santa Clara normal e o mutante amarelo. A caracterização das plantas normais, mutantes e F1 foi feita com base na análise quantitativa dos pigmentos carotenóides e clorofila em flores, folhas e frutos verdes e maduros. Plantas F1 e provenientes do retrocruzamento com o progenitor normal apresentaram 100% de frutos vermelhos. A semelhança entre os F1 recíprocos mostra que há ausência de herança materna para as características avaliadas. Em gerações segregantes, as freqüências observadas foram compatíveis com herança monogênica pelo teste qui-quadrado, com dominância completa para o gene que confere cor vermelha. Os frutos amarelos apresentaram teores reduzidos de <FONT FACE="Symbol">b</font>-caroteno e licopeno, enquanto o híbrido apresentou teores intermediários desses carotenóides quando comparados com o genótipo normal. Os níveis de clorofila em frutos verde-maduros e maduros mutantes foram menores que nos frutos normais, evidenciando o papel dos carotenóides sobre a fotoproteção da clorofila. A concentração de clorofila e carotenóides, em folhas e flores, não foi afetada pela mutação.
Inheritance of fruit color and pigment changes in a yellow tomato (Lycopersicon esculentum Mill.) mutant
Elizanilda R. do Rêgo1, Fernando L. Finger2, Vicente W.D. Casali2 and Antônio A. Cardoso2
1Escola Agrotécnica da Universidade Federal de Roraima, 69306-210 Boa Vista, RR, Brasil.
2Departamento de Fitotecnia, Universidade Federal de Viçosa, 36571-000 Viçosa, MG, Brasil. Send correspondence to F.L.F.
ABSTRACT
A naturally occurring yellow tomato fruit mutant cv. Santa Clara was reciprocally crossed with the red wild type, after which F1 plants were self pollinated or backcrossed with both parents. Plants from F1 generations produced all fruits with a homogeneous deep red color when ripe. F2 plants showed a 3:1 red:yellow segregation of fruit color, and 100% red when backcrossed with red wild type or 1:1 red:yellow segregation in backcrosses with the yellow mutant; hence, yellow fruit color was determined by a recessive allele. Based on reciprocal crosses, fruit color is unlikely to be determined by maternal genes. Accumulation of lycopene dropped by 99.3% and b-carotene by 77% in ripe yellow fruits, compared to the red wild type. Leaf and flower chlorophyll and total carotenoid concentrations were not affected by the yellow mutation. However, the mutant fruit had a higher rate of chlorophyll degradation during fruit ripening, whilst fruit from the F1 generation showed lower rates of degradation, similar to that observed in red wild type fruits.
INTRODUCTION
The tomato plant has been intensely used for fundamental genetic studies in the last three decades. In addition to their economical importance, tomato fruits are also used as model to explain important physiological and biochemical processes of ripening, for example ethylene and carotenoid synthesis, cell wall degradation and changes in starch, soluble sugars and acid metabolism (Kinet and Peet, 1997).
Several natural tomato mutants affecting fruit quality and ripening have been intensely studied. Pleiotropic fruit ripening mutants, such as ripening inhibitor (rin), never ripe (Nr), non-ripening (Nor) and alcobaça (alc), show a delay or inhibition of color development, softening, CO2 evolution and ethylene biosynthesis during ripening (Hobson and Grierson, 1993). An additional class of fruit pigment mutants is mentioned in the current literature, including apricot (at), beta carotene (B), greenflesh (gf), greenripe (gr), tangerine (t), high pigment (hp) and yellow flesh (r), among others (Jenkins and Mackinney, 1955; Rick and Butler, 1956). This latter class of tomato mutants shows a variety of changes in fruit color and quality when ripe, varying from the persistence of chlorophyll, development of yellow or orange color to a complete lack of lycopene synthesis (Gray et al., 1994). However, other aspects of fruit chemical composition may also be affected, including abscisic acid synthesis or content of b- or d-carotene.
Recently in Viçosa (MG), Brazil, a mutant fruit of tomato cv. Santa Clara was identified, which is yellow when the fruit is fully ripe. It is not possible to establish the nature of the mutated gene based only on fruit color development. Thus, the goals of the current work were to study the segregation of fruit color and pigment changes in segregating generations of crosses between the yellow mutant and the red wild type cultivated tomato.
MATERIAL AND METHODS
Self-pollinated yellow mutants and red wild type tomato plants of cv. Santa Clara were grown under greenhouse procedures to generate reciprocal crosses. F1 generations were then either self-pollinated or backcrossed with the original parents. Red wild type (P1), yellow mutant (P2), F1 (P2 ´ P1 or P1 ´ P2), F2 and backcrossed (F1´ P1 or F1´ P2) seeds were grown under field conditions. Fruits from first flower trusses were harvested at mature-green and ripe stages for color and pigment analyses.
Samples containing 20 g of outer pericarp from eight yellow or red ripe fruits were homogenized in 20 ml acetone and 65 ml hexane at 65oC using a Turrax homogenizer. The mixture was passed through filter paper and the filtrate transferred to a separation funnel, where it was sequentially washed with 90% methanol, 20% KOH, 90% methanol and finally, distilled water for 30 min each, keeping the upper phase after each wash. Total b-carotene and lycopene were estimated by spectrophotometry at 485.5 nm and 502 nm, as recommended by Zscheile and Porter (1947).
Total chlorophyll and carotenoids in leaves and fully opened flowers from the yellow mutant, red wild type and F1 generation were analyzed using the method described by Hendry and Price (1993): samples containing 1.0 g of mature leaves or 30 mg of flower petals were homogenized in 100 ml of fresh ammoniacal acetone (81.8 ml acetone: 18 ml distilled water:0.2 ml NH4OH). After the homogenate was filtered, absorbances at 470, 645, 663 and 710 nm were measured.
The experiment was performed in four random blocks for the pigment analysis and a completely random design for the color segregation study. Fruit color distribution frequency among plant generations was analyzed by chi-square (c2) test at P £ 0.01. Pigment concentration data were subjected to an analysis of variance, and the means were separated by Duncan's test at P £ 0.05.
RESULTS AND DISCUSSION
Tomato fruits harvested from the reciprocal F1 and backcrosses with the red wild type were all red when ripe (Table I). The F2 and backcrosses between the F1 andthe yellow mutant showed no significant differences from the expected result of a dominant gene with complete penetrance (Table I). The red color in the tomato fruit is the result of a single completely dominant gene for red color; yellow color in tomato fruit is the result of a homozygous recessive allele. Thompson (1955), working with a yellow cv. Snowball tomato mutant, found similar results for red and yellow color segregation. In addition, similarities in the color and shape of reciprocal F1 fruits indicate that it is unlikely that there are any maternal influences on tomato fruit color determination.
- Observed and expected frequencies of red (P1) and yellow mutant (P2) tomato fruits in F1 (P1´ P2), F1R reciprocal (P2 ´ P1), F2, and backcrosses BC1 (F1´ P1) and BC2 (F1´ P2).
ns = Nonsignificant at P = 0.01.
Fruit color mutations in some tomato plants may also determine changes in leaf or flower pigmentation (Giuliano et al., 1993). In the yellow mutant, total chlorophyll and carotenoid contents in leaves and flowers were similar to those observed in the red wild type and F1 plants (Table II). Carotenoid pigments are essential in preventing green tissue photobleaching (Goodwin and Mercer, 1982). As carotenoid contents in leaves and flowers were not affected by the yellow mutation, chlorophyll concentration in both tissues did not degrade faster in the mutant plants, compared to the red wild type (Table II). These data support the hypothesis that the recessive allele present in the 'Santa Clara' yellow tomato mutant differs from alleles r and ry, since they produce pale flowers and reddish fruit, respectively (Rick and Butler, 1956). Fray and Grierson (1993a), studying the mutated genes in yellow flesh tomato plants, also observed the presence of pale flowers. Thus, the mutant described in this work seems to be the result of a different recessive allele than the gene R studied by those authors.
- Chlorophyll and carotenoid contents of mature leaves and flowers from red wild type, yellow mutant and F1 tomatoes.
ns = Nonsignificant by F test at P > 0.05. FW = Fresh weight.
Lycopene and b-carotene are major pigments in ripe tomatoes, the former being responsible for the red color. In yellow ripe fruit, lycopene concentration was 99.3% less than that present in red fruits (Table III). Jenkins and Mackinney (1955), analyzing a similar yellow color tomato mutant, observed a 95% reduction in fruit lycopene content, when gene r was in the homozygous condition, compared to the genotype RR. Gene r encodes the enzyme phytoene synthase, which is responsible for transforming geranylgeranyl pyrophosphate into phytoene, a key intermediary in the synthesis of lycopene and b-carotene (Bartley and Scolnik, 1995). Fray and Grierson (1993a) cloned cDNA for the enzyme phytoene synthase (TOM 5), and transgenic plants expressing antisense mRNA for the enzyme decreased the lycopene content by 95% to 97% in ripe tomato fruits. The presence of only traces of lycopene in the 'Santa Clara' yellow mutant suggests that the carotenoid synthesis route is blocked in this fruit, similar to transgenic fruits expressing antisense mRNA for phytoene synthase. Contrary to lycopene, b-carotene concentration in yellow fruit was reduced by 77% (Table III), which indicates the presence of alternative routes for its synthesis, since b-carotene may be synthesized from the immediate precursors, such as lycopene, a-zeacarotene or b-zeacarotene (Bramley, 1997). The identity of the mutated gene in this yellow mutant cv. Santa Clara remains unknown. However, the presence of a single dominant gene R in F1 generation was able to restore the red wild type lycopene content in ripe fruits (Table III), therefore confirming its full dominance over yellow color.
- Content of lycopene and b-carotene in ripe red wild type, yellow mutant and F1 tomato fruits.
Different letters indicate significant differences within columns by
Duncan's test at P £ 0.05. FW = Fresh weight.
Chlorophyll concentration in the pericarp of mature-green and ripe yellow fruits was significantly reduced by the presence of the yellow mutated gene, but chlorophyll contents were fully restored when the yellow mutant was crossed with the red wild type plants (Table IV). Laval-Martin (1975), analyzing pigment changes in developing tomato fruit, pointed out that chlorophyll can be degraded faster if the carotenoid level is reduced throughout fruit growth. Fray and Grierson (1993b) observed that tomato plants transformed with constitutive antisense mRNA for phytoene synthase showed photobleaching, which was associated with reduced levels of carotenoids in foliage and unripe fruits. Once fruit ripening took place, the yellow tomato chlorophyll content dropped by a factor of 15.5, compared to the 8.3 and 8.8 reduction observed in red wild type and F1 fruits, respectively (Table IV). The sharper drop of chlorophyll degradation observed in the yellow mutant fruit could be attributed to the lower rate of b-carotene accumulation during ripening. However, a much less intense chlorophyll degradation was verified in F1 fruits, in which b-carotene formation was partially restored (Tables III and IV).
- Content of chlorophyll in mature-green and ripe red wild type, yellow mutant and F1 tomato fruit.
Different letters indicate significant differences within columns by
Duncan's test at P £ 0.05. FW = Fresh weight.
Transgenic plants expressing antisense cDNA for TOM 5 reduce carotenoid synthesis in fruits, without any apparent change in leaf carotenoid content (Bird et al., 1991; Fray and Grierson, 1993a). A similar trend was found in our study (Tables II and IV). On the other hand, carotenoid biosynthesis in variegated plant mutants is interrupted in albino leaf areas and a subsequent disruption of plastid development and chlorophyll accumulation takesplace (Bartley and Scolnik, 1995), as can be observed in the ghost tomato mutant, where an accumulation of phytoene and a lack of carotenoids in the white leaf areas occur (Giuliano et al., 1993).
This study showed that yellow 'Santa Clara' tomato mutant had reduced levels of carotenoid pigments in ripe fruits, but the mutation did not affect the accumulation of carotenoids and chlorophyll in leaves. The data suggest the existence of at least two different alleles controlling carotenoid biosynthesis in tomato, one expressed in leaves and flowers, and another in developing fruits. Furthermore, the allele mutated in this yellow tomato fruit does not correspond to the r and rymutations described elsewhere.
ACKNOWLEDGMENTS
CNPq is gratefully acknowledged for granting a fellowship to Elizanilda R. do Rêgo, and FAPEMIG (CAG 1610/95) for financial support during the execution of this experiment.
RESUMO
Neste trabalho avaliou-se a herança da cor do fruto de um mutante natural da cv. Santa Clara, por meio da análise das gerações F1 e segregantes, obtidas mediante cruzamento entre plantas da cv. Santa Clara normal e o mutante amarelo. A caracterização das plantas normais, mutantes e F1 foi feita com base na análise quantitativa dos pigmentos carotenóides e clorofila em flores, folhas e frutos verdes e maduros. Plantas F1 e provenientes do retrocruzamento com o progenitor normal apresentaram 100% de frutos vermelhos. A semelhança entre os F1 recíprocos mostra que há ausência de herança materna para as características avaliadas. Em gerações segregantes, as freqüências observadas foram compatíveis com herança monogênica pelo teste qui-quadrado, com dominância completa para o gene que confere cor vermelha. Os frutos amarelos apresentaram teores reduzidos de b-caroteno e licopeno, enquanto o híbrido apresentou teores intermediários desses carotenóides quando comparados com o genótipo normal. Os níveis de clorofila em frutos verde-maduros e maduros mutantes foram menores que nos frutos normais, evidenciando o papel dos carotenóides sobre a fotoproteção da clorofila. A concentração de clorofila e carotenóides, em folhas e flores, não foi afetada pela mutação.
(Received February 26, 1998)
- Bartley, G.E. and Scolnik, P. (1995). Plant carotenoids: pigments for photoprotection, visual attraction, and human health. Plant Cell 7: 1027-1038.
- Bird, C.R., Ray, J.A., Fletcher, J.D., Boniwell, J.M., Bird, S., Hughes, S. Morris, P.C. and Grierson, D. (1991). Using antisense RNA to study gene function: inhibition of carotenoid biosynthesis in transgenic tomatoes. Biotechnology 9: 635-639.
- Bramley, P.M. (1997). Isoprenoid metabolism. In: Plant Biochemistry (Dey, P.M. and Harborne, J.B., eds.). Academic Press, London, pp. 417-434.
- Fray, R.G. and Grierson, D. (1993a). Molecular genetics of tomato fruit ripening. Trends in Genet. 9: 438-443.
- Fray, R.G. and Grierson, D. (1993b). Identification and genetic analysis of normal and mutant phytoene synthase genes of tomato by sequencing, complementation and co-suppression. Plant Mol. Biol. 22: 589-602.
- Giuliano, G., Bartley, G.E. and Scolnik, P.A. (1993). Regulation of carotenoid biosynthesis during tomato development. Plant Cell 5: 379-387.
- Goodwin, T.W. and Mercer, E.I. (1982). Introduction to Plant Biochemistry 2nd edn. Pergamon Press, Oxford, pp. 677.
- Gray, J.E., Picton, S., Giovannoni, J.J. and Grierson, D. (1994). The use of transgenic and naturally occurring mutants to understand and manipulate tomato fruit ripening. Plant, Cell Environ. 17: 557-571.
- Hendry, G. and Price, A.H. (1993). Stress indicators: chlorophylls and carotenoids. In: Methods in Comparative Plant Ecology, a Laboratory Manual (Hendry, G.F. and Grime, J.P., eds.). Chapman and Hall, London, pp. 148-152.
- Hobson, G.E. and Grierson, D. (1993). Tomato. In: Biochemistry of Fruit Ripening (Seymour, G.B., Taylor, J.E. and Tucker, G., eds.). Chapman and Hall, London, pp. 405-442.
- Jenkins, J. and Mackinney, G. (1955). Carotenoids of the apricot tomato and its hybrids with yellow and tangerine. Genetics 40: 715-720.
- Kinet, J.M. and Peet, M.M. (1997). Tomato. In: The Physiology of Vegetable Crops (Wien, H.C., ed.). CAB International, Wallingford, pp. 207-258.
- Laval-Martin, D. (1975). Pigment evolution in Lycopersicon esculentum fruit growth and ripening. Phytochemistry 14: 2357-2362.
- Rick, C.M. and Butler, L. (1956). Cytogenetics of the tomato. Adv. Genet. 8: 267-382.
- Thompson, A.E. (1955). Inheritance of high total carotenoid pigments in tomato fruit. Science 121: 896-897.
- Zscheile, F.P. and Porter, J.W. (1947). Analytical methods for carotenes of Lycopersicon species and strains. Anal. Chem. 19: 47-51.
Publication Dates
-
Publication in this collection
02 June 1999 -
Date of issue
Mar 1999
History
-
Received
26 Feb 1998
