Progress towards the genetic engineering of tomato fruit softening

TIBTECH - JULY 1989 [Vol. 7]

Progress towards the genetic engineering of tomato fruit softening Matthew Kramer, Raymond E. Sheehy and William R. Hiatt Previous studies have correlated the softening of tomato fruit to changes in cell wall structure and the activities of cell wall degrading enzymes. In particular, the pectin-degrading enzyme polygalacturonase (PG) has been associated with softening. Studies of ripeningmutants deficient in PG activity have furthered this association. Recent advances in plant genetic engineering have enabled researchers to control PG gene expression, and to investigate further the role of this enzyme in fruit softening. The status and future directions of these experiments are discussed. This review focuses on the influence of cell wall composition on fruit texture during ripening and storage. Fruit texture is a major component of fruit quality and has commercial importance in both processing and fresh-market tomato varieties. In processing varieties, modifications of cell wall composition and fruit texture can affect the viscosity of the product and influence practices in the production facility. In fleshmarket varieties, textural changes dictate harvest and handling practices and affect firmness, flavor and storage of the fruit. The annual market values to US producers of flesh-market and processing tomatoes are estimated at $835 million and $472 million, respectively. The ripening of climacteric fruit (see Glossary) is characterized b y t h e production of ethylene, and involves coordinated biochemical and physiological changes which result in the final color, flavor and texture of ripe fruit. Tomato has been widely used as a model for the study of climacteric fruit ripening. Numerous studies have attempted to characterize the mechanisms and underlying biochemical processes which control tomato fruit ripening. These have focused on three major areas: (1) the

control of fruit ripening by growth regulators and, particularly, the induction of ripening by ethylene; (2) gene expression during ripening; and (3) the regulation of fruit softening.

M. Kramer, R. E. S h e e h y and W. R. Hiatt are at Calgene Inc., 1920 Fifth Street, Davis, CA 95616, USA. 1989, Elsevier Science Publishers Ltd (UK)

0167 - 9430/89/$02.00

Polygalacturonase and fruit softening A major feature of fruit cell walls (Fig. 1) is the interface between adjacent cells, the middle lamella, a fibrous layer composed primarily of pectin. One of the most characteristic changes in the cell wall associated with fruit softening is the solubilization of pectin, which is accompanied by dissolution of the middle lamella and eventual disruption of the primary cell wall 1. Enzymes involved in the metabolism of pectin include pectinmethylesterase (PE) and polygalacturonase (PG) (Fig. 2). Both PE and PG are physically associated with the cell wall fraction, and PG has been implicated as an important enzyme in fruit softening because: • its appearance during ripening corresponds to the increase in fruit softening; • in a number of cultivars, ihere is a correlation between levels of PG activity and the extent of fruit softening; e it degrades isolated fruit cell walls in vitro in a manner similar to that observed during ripening; • several ripening-mutants that have been described with delayed or decreased softening are deficient in PG activity. Hobson 2 demonstrated a correlation between fruit firmness and levels of PG activity. Additional studies in both fresh-market and processing tomato cultivars further characterized the molecular forms of PG and attempted to reconcile the activities of the isozymes to the textural changes observed in ripening fruit 3 7: these studies showed that extractable PG activity is first detected shortly after the onset of fruit ripening and becomes an abundant protein in ripe fruit. Measurements ofPG mRNA using isolated PG cDNAs (see Glossary) provided a similar resulta-11: expression of the PG gene seems to be developmentally regulated during fruit ripening in that PG mRNA is first detectable at the breaker stage (see Glossary) and accumulates rapidly to a final level of 1-2% of total mRNA as ripening progresses 11. Additional evidence for the involvement of PG in tomato fruit

TIBTECH- JULY 1989 [Vol. 7]

Fig. 1

rnic

softening comes from both ultrastructural and biochemical analyses of tomato fruit cell walls. These demonstrate first, that in vitro the middle lamella of cell walls of green tomato fruit is dissolved ~1 and second, that soluble pectin is released from isolated cell walls of green tomato fruit 12'13 when treated with purified PG.

Ripening mutants Studies of tomato fruit ripening and softening have been aided by the availability of numerous ripeningmutants. Three of the mutants, never ripe (Nr), ripening inhibitor (tin) and nonripening (nor), map to different chromosomes and exhibit pleiotropic (see Glossary) effects on fruit ripening 14,15. Nr, a dominant mutation, alters ripening in a number of ways: the onset of ripening is delayed, and less ethylene, lycopene and PG are produced; the rate of softening is substantially slower compared with normal fruit. The recessive tin mutation produces non-climacteric fruit with very low levels of ethylene, lycopene, and PG, relative to the wild type. Fruit with the rin mutation can be stored for over a year with almost no solubilization of the cell walls. The nor mutation is also recessive and produces non-climacteric fruit. As in rin, there is no rise in ethylene production or PG synthesis, while lycopene synthesis is both delayed and reduced. Fruit with the nor mutation can also be stored for much longer than normal fruit. Studies of these three ripening-mutants points to a correlation between PG activity and fruit softening.

Regulation of PG gene expression Recently, several groups have isolated and characterized tomato fruit PG cDNAs 8-1° and genomic clones 16-18 (see Glossary). The result of these efforts is that there now exists a tool with which to evaluate further the role of PG activity in tomato fruit softening. These studies are now under way and may be categorized as follows: • the use ofantisense RNA to cause a specific reduction in PG activity in a

polygalacturonic acid

The primary cell wall at the interface of two fruit cells. The cytoplasm of each cell is separated from the wall by the plasmalemma. The middle lamella is the interface between adjacent cells, and contains pectin as a major component. Pectin is primarily a polymer of galacturonic acid stabilized by calcium ions, and is thought to act as an intercellular adhesive. Tissue softening has generally been attributed to the degradation of pectin by polygalacturonase, leading to the release of the cellulose microfibrils and separation of cells. However, other cell wall polymers such as galactans and arabinogalactans 24 may act as a bridge between cellulose and pectin polysaccharides, and the enzymes which modify these polymers may be important in softening. (Reprinted from Ref. 25 with permission.)

cultivar which otherwise ripens normally; • the fruit-specific expression of functional PG activity in ripeningmutants; • the constitutive expression of a functional PG gene introduced into a normal cultivar.

Reduction in PG by antisense RNA Antisense RNA is complementary to a target mRNA. The formation of a duplex between antisense RNA and mRNA inactivates the mRNA by mechanisms which are as yet unclear. The introduction of stable antisense 'genes' (DNA which is transcribed into antisense RNA), has been used to inhibit the expression of specific genes in plants 19. Recently, the inhibition of PG activity in tomato fruit through the use of antisense RNA has been reported 2°,21. When an antisense PG gene was constitutively expressed in transformed tomato plants, PG mRNA and enzyme levels were lower than in controls. In many plants, both mRNA levels and PG activity were reduced

by as much as 90% compared with both non-transformed controls and transformed c o n t r o l s without the antisense PG gene. The reduction specifically of PG activity in these plants provides a phenocopy (see Glossary) of the PG-deficient mutants which will be useful in the study of fruit ripening and softening21. The antisense PG gene was co-transformed with a gene conferring antibiotic resistance. This allowed the rapid identification of transformed plants with segregation ratios consistent with integration at a single locus. In addition, following genetic crosses, second generation plants which are homozygous, heterozygous or negative for the antisense PG gene can be produced and identified. Thus, the effect of antisense PG gene dosage on PG levels and softening can be evaluated. The Nr, rin and nor mutants had an inhibited or retarded onset of ripening, and synthesized reduced levels of lycopene, and produced fruit which did not soften or softened very slowly. In contrast, in transgenic

TIBTECH - JULY 1989 [Vol. 7] -

-

Fig. 2

o¢.Ooo:o

pectinmethylesterase

COzH

CO~H

\ OH

The degradation of pectin in the middle lamella and primary cell wall can be viewed as a two-stage process. Pectinmethylesterase catalyses the demethylation of pectin, rendering it vulnerable to attack by polygalacturonase. Polygalacturonase can then catalyse endohydrolysis of the polygalacturonic acid polymers present in the middle lamella.

antisense-PG tomato plants, the onset of ripening is normal and the plants produce fruit with lycopene levels characteristic of red, ripe fruit 2°'21. The pectin composition and softening characteristics of these fruit are currently being investigated. However, Smith et al. 2° have already reported no difference in fruit softening (as measured by compressibility), between transformed and control fruit. Fruit-specific PG gen e expression in a r i p e n i n g - m u t a n t

Recently, a PG gene in the sense orientation has been used to try to complement the tin mutation in tomato. The gene was expressed from an ethylene-inducible promoter 18. When treated with the ethylene analogue propylene, the transgenic rin fruit expressed the PG gene: the polygalacturonase produced was biochemically identical to that in wild-type controls. In the propylenetreated transgenic rin mutant, extractable PG activity was equal to 60% of that in normally ripening fruit; however, this did not induce the synthesis of lycopene or the production of endogenous ethylene. The introduced PG activity increased the levels of soluble pectin, but did not induce fruit softening (as measured by compressibility). This supports the proposed role of PG in pectin solubilization, but brings into question the relationship between pectin solubilization and softening. However, this result must be qualified with regard to the genetic background into which the PG gene was introduced: since the rin mutation is pleiotropic, it may influence fruit softening by mechanisms not associated with PG activity.

OH

polygalacturonase 1

CO,H

OH +

OH

OH

Constitutive PG gene expression

We have also introduced a constitutively expressed functional PG gene into a normally ripening cultivar. This resulted in high level expression of PG mRNA and enzyme activity in all parts of the tomato plant at all developmental stages. The effect of PG activity on the pectin composition and softening of green fruit could then be evaluated without the influence of a ripening hormone or a pleiotropic ripening-mutant. Constitutive expression of a tomato fruit PG gene had, unexpectedly perhaps, no obvious effect on the growth or morphology of non-fruit tissue in transformed plants. Preliminary results indicate that PG activity in green fruit does not induce softening on the vine. Further shelf life experiments and an analysis of the pectin composition of the transformed fruit are in progress. Future directions

Genetic engineering has been used to manipulate the expression of the gene encoding polygalacturonase and has provided a further way of investigating the role of PG in pectin solubilization, and the role of pectin modifications in fruit softening. The results from genetic engineering studies suggest that PG is not the primary determinant of fruit softening although more work is required before this conclusion can be drawn with confidence. The antisense experiments resuited in a 90% reduction in PG activity. However, PG is an extremely abundant protein in ripe fruit, and the remaining 10% is easily detectable and potentially responsible for the ripening effects seen. In addition, previous studies have indicated that

the initial PG synthesized during ripening may be particularly important in pectin solubilization and softening. PG extracted early in ripening is a high molecular weight form, PG1 (Refs 3-7), and differences in firmness between cultivars are established when PG1 is the predominant form 7. PG in fruit with the Nr mutation is generally present at about 10-15% of normal levels and in the form of PG1 (Ref. 15). However, normal pectin solubilization occurs during ripening of Nr fruit 22. To clarify this picture, it will be important to reduce further the levels of PG. We will therefore manipulate antisense PG gene dosage, increase the expression of antisense RNA, and use antisense RNAs with enhanced ability to disrupt gene expression. Expression of functional PG activity in normally deficient (green wild type fruit) and fruit bearing the rin mutation, did not induce softening even though, in the case of rin, there was an increase in soluble pectins. However, further characterization of the peetin component in both situations (green, rin) is required to determine whether the modifications are similar to those observed during normal fruit ripening. The physical properties of pectin are influenced by a number of parameters including molecular weight, interactions with ions such as calcium, and the degree of methylation. The recent isolation of a cDNA clone encoding pectinmethylesterase (PE) 23 may provide a means of manipulating PE activity and pectin methylation. If so, then the role of methylation in altering the physical properties of pectin and its susceptibility to PG could be evaluated. While this would have undoubted value, it should be borne in mind that pectin modifications may represent only part of a complex process leading to fruit softening. Further characterization of the other enzymes involved would also then be necessary. References

1 Crookes, P. R. and Grierson, D. (1983) Plant Physiol. 72, 1088-1093 2 Hobson, G. E. (1965) J. Hort. Sci. 40, 66-72 3 Tucker, G. A., Robertson, N. G. and Grierson, D. (1980) Eur. J. Biochem. 112, 119-124

TIBTECH- JULY 1989 [Vol. 7]

4 Tucker, G. A., Robertson, N. G. and Grierson, D. (1981) Eur. J. Biochem. 115, 87-90 5 Pressey, R. (1986) in Chemistry and Functions of Pectins (Fishman, M. L. and Jen, J.J., eds), pp. 157-174, American Chemical Society 6 Brady, C. J., MacAlpine, G., McGlasson, W. B. and Ueda, Y. (1982) Aust. J. Plant Physiol. 9, 171-178 7 Brady, C. J., McGlasson, W. B., Pearson, J.A., Meldrum, S.K. and Kopeliovitch E. (1985) J. Am. Soc. Hort. Sci. 110, 254-258 8 Sheehy, R. E., Pearson, J., Brady, C. J. and Hiatt, W.R. (1987) Mol. Gen. Genet. 208, 30-36 9 Grierson, D., Tucker, G. A., Keen, J. et al. (1986) Nucleic Acids Res. 14, 8595-8603 10 DellaPenna, D., Alexander, D. C. and Bennett, A. B. (1986)Proc. NatlAcad. Sci. USA 83, 6420-6424 11 DellaPenna, D., Kates, D.S. and Bennett, A.B. (1987) Plant Physiol. 85,502-507 12 Wallner, S. J. and Bloom, H. L. (1977) Plant Physiol. 60, 207-210 13 Huber, D. J. (1983) J. Am. Soc. Hort. Sci. 108, 405-409 14 Tigchelaar, E. C., McGlasson, W.B. and Buescher, R.W. (1978) Hortscience 13,508-513 15 Grierson, D., Purton, M.E., Knapp, J.E. and Bathgate, B. (1987) in Developmental Mutants in Higher Plants (Thomas, H. and Grierson, D., eds), pp. 73-94, Cambridge University Press 16 Rose, R. E., Houck, C.M., Monson, E. K. et al. (1988) Nucleic Acids Res. 16, 7191 17 Bird, C. R., Smith, C. J. S., Ray, J. A. et al. (1988) Plant Mol. Biol. 11,651-662 18 Giovannoni, J. J., DellaPenna, D., Bennett, A.B. and Fischer, R.L. (1989) Plant Cell 1, 53-63 19 van der Krol, A. R., Mol, J. N. M. and Stuitje, A. R. (1988) Gene 72, 45-50 20 Smith, C. J. S., Watson, C. F., Ray, J. et al. (1988) Nature 334, 724-726 21 Sheehy, R. E., Kramer, M. and Hiatt, W.R. (1988) Proc. Natl Acad. Sci. USA 85, 8805-8809 22 Seymour, G. B., Harding, S.E., Taylor, A.J. et al. (1987) Phytochemistry 26, 1871-1875 23 Ray, J., Knapp, J., Grierson, D. et al. (1988) Eur. J. Biochem. 174, 119-124 24 Gross, K. C. and Wallner, S. J. (1979) Plant Physiol. 63,117-120 25 Nevins, D. J. (1987) in Models in Plant Physiology and Biochemistry Vol. 1 (Newman, D.W. and Wilson, K.C., eds), pp. 75-77, CRC Press

Practical Plants PLANT MOLECULAR BIOLOGY: A PRACTICAL APPROACH

edited by C. H. Shaw, IRL Press (Practical Approach series), 1988. (xx + 313 pages) ISBN 1 85221 057 5 (hardback) £29.00, 1 85221 056 7 (softback) £19.00 Molecular biology is a rapidly evolving discipline. It is also a discipline shrouded in mythology and made inaccessible by jargon. For anyone who is not a 'full-time' molecular biologist, but who needs, from time to time, to use molecular biological techniques, it is all too easy to find oneself 'off the pace' (as we say in athletics). It is also easy to be put off moving into the field because of the aura of difficulty which surrounds it. The editor of this volume, Charlie Shaw, is obviously aware of these problems and his stated aims in assembling the book are to abolish the mythology and 'to provide the newcomer and the practitioner with clear and sensible protocols to enable them to perform meaningful experiments in plant molecular biology'. In my view he has succeeded totally in fulfilling these aims. From the Preface and the moving dedication to the last entry in the Index this book is a winner. There are eleven chapters. There is also a brief Appendix describing the use of [5-glucuronidase as a reporter gene in transgenic plants: evidently the development of this reporter gene system occurred whilst the volume was in press. Every single chapter and the Appendix are mines of useful information. Protocols are described clearly and fully, with hints for troubleshooting. Theoretical background material is concise and relevant. The opening two chapters on analysis of plant gene structure and expression by Bob Goldberg, Kathleen Cox and Diane Jofuku are particularly impressive and beautifully fulfill the aims of the book. I also appreciated the inclusion of Chris Hawes' chapter on subcellular localization of macromolecules by microscopy; such material is not often included in molecular biology texts. In fact, all the authors have performed well and there is not one weak chapter in the book. Inevitably, there are some criticisms. The topic of plant molecular virology is

too large for one chapter- indeed it could almost have a volume of its o w n - and the authors necessarily omitted a lot of relevant material in order to keep the chapter a reasonable length. Why was one algal genus, Chlamydomonas, singled out when a more general chapter on algal molecular biology might have been even more useful? Within the chapter on Chlamydomonas, the use of lux as units of light intensity should have been avoided: this unit does not tell us anything about photosynthetically active radiation. I find it slightly odd that a chapter on cyanobacteria has been included (albeit a very useful chapter for anyone interested in cyanobacteria): cyanobacteria are not plants (even if they do photosynthesize!). Finally, many of the techniques described throughout the book have been shown to work with only a limited range of species. Some authors make this point whilst others claim that the techniques described are likely to be widely applicable. In my view, there are some very interesting plant species on which it is very difficult to carry out molecular biological studies. Nevertheless, even for those species, the techniques described in this volume should provide the reader with a place to start. However, my overriding and lasting impression is that this is a very good book maintaining and, in some places, even exceeding the high standards set by earlier volumes in the series. My review copy is hardback, but ring-bound. Full marks to the publishers for this format: the book will lie open and flat on the laboratory bench; indeed, now that I have finished this review, my copy will go straight into my laboratory. So, if you need to learn some plant molecular biology techniques, or want an update in such techniques, go out and buy this book. Do not allow it to gather dust in the library or in the office. Keep it in the laboratory where it belongs. JOHNA. BRYANT

Department of Biological Sciences, University of Exeter, Washington Singer Laboratories, Perry Road, Exeter EX4 4QG, UK.