by the late breaker stage of ripening PG is present in most. 17 .... Huber DJ (1983) The role of cell wall hydrolases in fruit soften- ing. Hortic Rev 5: 169-219. 14.
Received for publication December 5, 1988
Plant Physiol. (1989) 90, 17-20 0032-0889/89/90/001 7/04/$01 .00/0
Communication
Immunocytolocalization of Polygalacturonase in Ripening Tomato Fruit1 Denise M. Tieman and Avtar K. Handa* Center for Plant Environmental Stress Physiology, Department of Horticulture, Purdue University, West Lafayette, Indiana 47907 MATERIALS AND METHODS Tomato plants (Lycopersicon esculentum Mill. cv Rutgers) were grown in a greenhouse as described previously (2). Fruits were harvested according to developmental stage as determined by age, pigmentation, and ethylene production rates. Rates of ethylene production were measured by GC as described previously (3). Each fruit was divided into two halves with a sharp knife. Both halves were placed on Whatman 3MM paper saturated with 1 M NaCl for 5 min followed by brief blotting on dry Whatman 3MM paper. One-half of the tomato was then placed on a nitrocellulose membrane (BA, 85, 0.45 gm pore size, Schleicher and Schuell, Keene, NH) for 10 min. The nitrocellulose membrane was stained for total protein for 1 min with 0.1% amido black lOB in 25% isopropanol and 10% acetic acid and destained for 30 min in 25% isopropanol and 10% acetic acid with two changes of destain solution. To immunolocalize the PG protein using PG-specific polyclonal antibodies, the second half of the fruit was placed on an Immunodyne Immunoaffinity membrane (Pall Ultrafine Filteration Corp., East Hills, NY), which covalently binds protein. After blotting for 10 min, the tomato tissue prints were air dried and incubated for 15 min at 370C in Blotto (0.5% w/v nonfat dry milk in 130 mM NaCl, 10 mM sodium phosphate buffer [pH 7.4], 0.001% Antifoam A, and 0.01% thimersol) to saturate the remaining protein binding sites. Tissue blots were then incubated in plastic bags with 106 cpm of 1251-labeled PG-antibodies (17) in a gelatin solution (0.25% gelatin, 150 mM NaCl, 5 mm ethylene diaminetetraacetic acid, 0.05% Nonidet P-40, and 50 mm Tris-HCl [pH 7.4]) containing 0.2% SDS and 0.2% Triton X-100 overnight at 37°C with shaking. PG antibodies were immunopurified using a Sepharose-4B column to which purified PG was coupled (17). Blots were washed five times with 50 mL gelatin solution without SDS or Triton X- 100 for 15 min each. After air drying, membranes were covered with plastic wrap and autoradiogrammed using Kodak XAR-5 film at -80°C.
ABSTRACT Using tissue blotting and immunocytolocalization, we have investigated the appearance and accumulation of polygalacturonase (PG) during tomato (Lycopersicon esculentum Mill.) fruit ripening. Results show that PG first appears in the collumella region followed by sequential appearance in the exopericarp and endopericarp, respectively. Detectable levels of PG were not present in the jocular material containing seeds. This result indicates that PG synthesis initiates at the central collumella region of tomato fruit during ripening.
Tomato pericarp undergoes significant textural changes during fruit ripening (4, 13). Based on genetic and biochemical studies, PG2 has been implicated in playing an important role in fruit softening. It has been shown that the maximum loss of tomato fruit firmness and electron density in the middle lamellar region is coincidental with the maximum level of PG activity (6, 7). Also, nonripening mutants of tomato, which show little softening during aging, exhibit undetectable to extremely reduced levels of PG activity while showing normal levels of other hydrolases including cellulase(s) and pectinmethylesterase activities (22). Developmental regulation of PG expression during tomato fruit ripening has been demonstrated by several investigators (1, 8, 10, 19). We have recently shown (1) that overall accumulation of PG during tomato fruit ripening also is regulated at both posttranscriptional and posttranslational levels. PG accumulation in tomato pericarp begins at the onset of fruit ripening and accumulates to 3 to 5% of the total soluble protein present in the ripe pericarp tissue (1, 17). However, little is known about the sequential appearance of PG in other tissues of tomato fruit during ripening. Using techniques of tissue blotting and immunocytolocalization, we demonstrate here that PG protein appears first in the collumella region followed by a sequential appearance in the radial walls of pericarp, exocarp, and endocarp, respectively.
RESULTS AND DISCUSSION Immunocytolocalization of PG during tomato fruit ripening is shown in Figure 1. Detectable levels of PG were not present at the mature green stage of ripening which is in agreement with other published results (5, 12, 17, 23). Upon initiation of ripening, PG appears first in the collumella and by the late breaker stage of ripening PG is present in most
' Research supported by the Purdue University Agricultural Experimental Station. Journal paper No. 11,826 of the Purdue University Agricultural Experimental Station. 2 Abbreviations: PG, polygalacturonase; ACC, 1-aminocyclopropane- 1-carboxylic acid. 17
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Plant Physiol. Vol. 90, 1989
TIEMAN AND HANDA
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Figure 1. Tissue blotting and immunocytolocalization of PG protein with PG-specific antibodies in ripening tomato fruits. Stages of fruit ripening and respective rates of ethylene production (nL g1' h-1) were as follows: mature green, 0.15; breaker, 0.99; turning, 6.85; late turning, 9.83; ripe, 14.54; overripe, 11.88 and 6.76. Also shown are the total protein patterns and the immunolocalization of changes in PG protein during ripening of tomato fruits. Other details are described in "Materials and Methods."
TOMATO POLYGALACTURONASE IMMUNOCYTOLOCALIZATION parts of the collumella and in the exocarp at the blossom end of tomato fruit. As the fruit ripens, PG protein levels begin to
increase first in the exocarp followed by an increase in the endocarp tissue. In the fully ripe fruit, PG is present in all sections of pericarp, collumella, and the radial wall pericarp tissue. The overall levels of PG protein continue to increase even after fruit has attained fully red ripe stage of ripening (Fig. 1). PG was not present at detectable levels in the locular cavity with jelly-like parenchyma around the seeds. Absence of PG in the jelly-like parenchyma around the seeds has been suggested earlier based on enzyme activity assay ( 12). Immunoaffinity membrane was used for immunocytolocalization of PG in ripening fruit as this membrane binds protein covalently resulting in a higher signal to noise ratio (17). Only a faint signal was obtained when nitrocellulose membrane was used for immunolocalization of PG (data not shown). We have shown earlier that most PG elutes from nitrocellulose membrane under the conditions of incubation and washing of membranes required to remove the nonspecific binding of PG antibodies to other proteins, especially basic proteins (17). SDS was included in the incubation solution of gelatin since it helps reduce nonspecific binding of IgG to other basic proteins (9). Pretreatment of the cut surface of tomato fruit with 1 M NaCl before blotting onto the immunoaffinity membrane enhanced transfer of proteins from fruit to membrane resulting in increased sensitivity of immunocytolocalization and improved signal to noise ratio. This is probably a result of solubilization of PG protein bound to cell walls. Several other pretreatments also were tried to enhance the blotting of tissue proteins to nitrocellulose or immunoaffinity membranes. These treatments included buffers of different pHs and various concentrations of Triton X- 100 and Nonidet P-40 in the presence and absence of 1 M NaCl. In general, pH had little effect on sensitivity of immunocytolocalization after tissue blotting. However, the presence of either Triton X-100 or Nonidet P-40 in the pretreatment solution resulted in relatively diffuse patterns of PG localization (data not shown). Based on a correlation between the climacteric increase in the rate of ethylene production and PG levels, it has been suggested that ethylene plays an important role in PG expression during fruit ripening (5, 10, 11, 21). Also, ethylene stimulates the accumulation of PG mRNA in tomato fruits (18). However, the transcriptional regulation of PG gene expression by ethylene has not yet been demonstrated. Our data show that increases in PG levels in different sections of tomato fruit, including pericarp, are not uniform during tomato fruit ripening and that PG accumulation is differentially regulated in different sections of fruit. If one hypothesizes that ethylene, being a gaseous compound, is freely diffusible within the fruit resulting in similar concentrations of ethylene in various sections of fruit, then sensitivity of different parts of the fruit to ethylene may be responsible for the differential accumulation of PG in fruit tissues. Alternatively, it is possible that ethylene is not equally distributed in various sections of fruit or ethylene is not the primary regulator of PG gene expression and exerts its effect by activating a developmental cascade process which results in expression and accumulation of PG. Ethylene concentration dependent gene
19
expression has been demonstrated in the tomato fruit by Lincoln and Fisher (15). These authors have suggested that the ethylene concentration dependent response is most likely due to the differential sensitivity of different genes to ethylene within a tissue. Most studies in the past have utilized tomato pericarp to study biochemistry, physiology, and gene expression during tomato fruit ripening (1, 2, 5, 8, 10, 11, 15, 18, 23). Our results, however, show that PG first appears in the central collumella region of fruit during ripening. Due to wound induction of ethylene synthesis, it is not possible to determine the site of initiation of climacteric associated ethylene production. However, Kende and Boller (14) have shown that ACC and ACC synthase accumulate much more rapidly in the center portion of fruit than in the outer pericarp during the early stages of ripening. About 20-fold higher levels of ACC and 2-fold higher levels of ACC synthase were observed in the central portion of tomato as compared with the outer pericarp at the breaker stage ofripening (14). A similar pattern has been observed for the accumulation of lycopene ( 16, 20). Together, these data suggest that changes in various ripening parameters initiate in the collumella region of tomato fruit rather than the pericarp tissue and changes in collumella and connecting septa should be studied in order to understand the etiology of fruit ripening. LITERATURE CITED 1. Biggs MS, Handa AK (1989) Temporal regulation of polygalacturonase gene expression in fruits from normal mutant and heterozygous tomato genotypes. Plant Physiol 89: 117-125 2. Biggs MS, Harriman RW, Handa AK (1986) Changes in gene expression during tomato fruit ripening. Plant Physiol 81: 395-
403 3. Biggs MS, Woodson WR, Handa AK (1988) Biochemical basis of high temperature inhibition of ethylene biosynthesis in ripening tomato fruits. Physiol Plant 72: 572-578 4. Brady CJ (1987) Fruit ripening. Annu Rev Plant Physiol 38: 155-178 5. Brady CJ, MacAlpine G, McGlasson WB, Veda Y (1982) Polygalacturonase in tomato fruits and the induction of ripening. Aust J Plant Physiol 9: 171-178 6. Brady CJ, McGlasson WB, Pearson JA, Meldrum SK, Kopeliovitch E (1985) Interaction between the amount and molecular forms of polygalacturonase, calcium and firmness in tomato fruit. J Am Soc Hortic Sci 110: 254-258 7. Crookes PR, Grierson D (1983) Ultrastructure of tomato fruit ripening and the role of polygalacturonase isozymes in cell wall degradation. Plant Physiol 72: 1088-1093 8. DellaPenna D, Kates DS, Bennett AB (1987) Polygalacturonase gene expression in Rutgers, rin, nor and Nr tomato fruits. Plant Physiol 85: 502-507 9. Dimitriadis GJ (1979) Effect of detergents on antibody-antigen interaction. Anal Biochem 98: 445-451 10. Grierson D, Munders MJ, Slater A, Rag J, Bird CR, Schuch W, Holdworth MJ, Tucker GA, Knapp JE (1986) Gene expression during tomato ripening. Philos Trans R Soc Lond B Biol Sci 314: 399-410 11. Grierson D, Tucker GA (1983) Timing of ethylene and polygalacturonase synthesis in relation to the control of tomato fruit ripening. Planta 157: 174-179 12. Hobson GE (1964) Polygalacturonase in normal and abnormal tomato fruit. Biochem J 92: 324-332 13. Huber DJ (1983) The role of cell wall hydrolases in fruit softening. Hortic Rev 5: 169-219 14. Kende H, Boller T (1981) Wound ethylene and 1-aminocyclo-
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synthase in ripening tomato fruit. Planta 151: 476-481 Lincon JE, Fisher RL (1988) Diverse mechanisms for the regulation of ethylene-inducible gene expression. Mol Gen Genet 212: 71-75 Marlow SJ (1987) Effect of heat stress on ripening associated polygalacturonase synthesis of tomato fruit. MS thesis, Purdue University Marlow SJ, Handa AK (1987) Immuno slot-blot assay using a membrane which covalently binds protein. J Immunol Methods 101: 133-139 Munder MJ, Holdsworth MJ, Slater A, Knapp JE, Bird CR, Schuch W, Grierson D (1987) Ethylene stimulates the accumulation of ripening-related mRNAs in tomatoes. Plant Cell Environ 10: 177-184 Sheehy RE, Pearson J, Brady CJ, Hiatt WR (1987) Molecular propane- I -carboxylate
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characterization of tomato fruit polygalacturonase. Mol Gen Genet 208: 30-36 Simpson DJ, Baqar MR, McGlasson WB, Lee TH (1976) Changes in ultrastructure and pigment content during development and senescence of fruit of normal and rin and nor mutant tomatoes. Aust J Plant Physiol 3: 575-587 Su LY, McKeon T, Grierson D, Cantwell M, Yang SF (1984) Development of -amino-cyclopropane- -carboxylic acid synthase and polygalacturonase activities during maturation and ripening of tomato fruit. Relationship between internal ethylene concentration and ethylene production rate. HortScience 19: 576-578 Tigchelaar EC, McGlasson WB, Beuscher RW (1978) Genetic regulation of tomato fruit ripening. HortScience 13: 508-513 Tucker GA, Robertson NG, Grierson D (1980) Changes in polygalacturonase isozymes during the ripening of normal and mutant tomato fruit. Eur J Biochem 112: 119-124
