Pectins and pectolytic enzymes in relation to development and processing of green beans (Phaseolus vulgaris L.)

J. Visserand A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996Elsevier ScienceB.V.All rights reserved.

399

Pectins and pectolytic enzymes in relation to development and processing of green beans (Phaseolus vulgaris L.). K. Recourt a, T. Stolle-Smits ~, J.M. Laats ~, J.G. Beekhuizen ~, C.E.M. Ebbelaa#, A.G.J. Voragen b, H.J. Wichers a and C. van DijkL

aAgrotechnological Research Institute (ATO-DLO), Department of Biochemistry and Food Processing, P.O. Box 17, 6700 AA Wageningen, The Netherlands. bWageningen Agricultural University, Department of Food Sciences, P.O. Box 8129, 6700 EV Wageningen, The Netherlands.

Abstract Processing of green beans involves major changes within the composition of cell wall pectins. About 20% of homogalacturonan is degraded while 65% of rhamnogalacturonan is solubilized. Since B-eliminative breakdown, which is dependant on the degree of pectin methylesterification, is probably the main mechanism explaining this phenomenon, a biochemical and molecular biological study was initiated on the cell wall enzyme pectin methylesterase [PE]. Two groups of isoenzymes with molecular weights of 33 kDa and 42 kDa and different thermostabilities were partly purified. In addition, it appeared that two PE genes, most likely encoding precursor proteins of 63 kDa, are expressed during pod development.

1. INTRODUCTION The texture of processed vegetables is an important quality attribute and is determined by (i) the composition of the cell walls from the fresh product and (ii) cell wall changes occurring during processing. Cell walls of fruit and vegetables consist of about 40% of pectic polymers which can be distinguished in linear homogalacturonan (smooth) and branched rhamnogalacturonan (hairy) regions [1]. Plant enzymes capable to modify cell wall pectins include the de-esterifying pectin methylesterase (PE, EC 3.1.1.11) and the depolymerizing polygalacturonase (PG), existing as either endo-acting (EC 3.2.1.15) or exo-acting (EC 3.2.1.67) enzymes. Both PE and endoPG are considered to play a role during fruit softening and have been studied in detail during tomato maturation [2]. Processing of vegetables at 100 ~ to 120 ~ results in a major decrease of firmness. Several studies suggest that the major cleavage reaction leading to softening is the B-eliminative depolymerization of intercellular pectin. Since this chemical breakdown of pectins only occurs at methylated smooth pectic regions, the

400 demethylating properties of PE might be a tool to control the processing quality of vegetables. Moreover, for potatoes it has been shown that PE is activated during moderate heating procedures ranging between 50~ and 80~ resulting in an increased firmness of the end product [3]. In this paper, we report on changes of cell wall pectins during processing of green beans. Furthermore, biochemical and molecular biological research is presented on the role of PE and PG during pod development.

2. C E L L WALL CHANGES D U R I N G P R O C E S S I N G OF G R E E N BEANS To gain insight in textural changes during processing, cell walls were analyzed from fresh, blanched (5 min 90 ~ and sterilized (30 min 118 ~ green beans. This was accomplished by preparing AIR (Alcohol Insoluble Residue), WIR (Water Insoluble Residue) and WSP (Water Soluble Polymers). Sugar analysis showed that due to the sterilization procedure about 20% of uronic acids was degraded and could not be retrieved from any of the cell wall fractions. Since the overall degree of pectin methylation decreased during processing, this fraction was most likely highly methylated and poorly branched and might originate from the middle lamellae [4]. In addition, sterilization resulted in a significant shift of uronic acid, galactose and arabinose from the WIR to the WSP fraction suggesting a solubilization of branched pectic regions. To study the cell wall alterations during processing in detail, total pectic fractions were separated by extraction of AIR from fresh, blanched and sterilized beans with acetate buffer, CDTA and Na2CO 3. Analysis showed that sterilization caused a large increase in the amount of buffer soluble pectins (Figure 1).

mg/g AIR in: I I Buffer 171CDTA EEl Carbonate 4 "C I;~ Carbonate 20 "C

Fresh

Blanched

Sterilized

Figure 1. Distribution of pectins over different fractions during industrial processing of green beans.

401 Additionally, FPLC analysis showed that sterilization caused a significant reduction of the molecular weight of the solubilized pectins. Interestingly, blanching (5 min 90 ~ also affected the distribution of cell wall pectins (Figure 1). Moreover, processing of green beans at reduced blanching temperatures of 60 ~ to 70 ~ yielded relatively firm products which was accompanied by less pectin degradation (not shown). 3. P E C T I N METHYLESTERASE (PE) AND P O L Y G A I ~ C T U R O N A S E (PG) D U R I N G POD D E V E L O P M E N T To investigate the role of pectic enzymes during green bean development, plants were grown in a green house and pods were harvested at different 'developmental stages. Analysis showed that PG activities were only detectable during early developmental stages and activities ranged between 50 to 100 p k a t / m g protein. Due to the low activities, the endo or exo nature of the enzyme could not be determined. PE activities were measurable during all developmental stages and ranged between 100 and 150 n k a t / m g protein. Seeds contained significantly higher levels of enzyme activity which ranged between 200 and 250 n k a t / m g protein. To purify PE from green beans, mature pods and seeds were extracted with water, NaC1 (1.25 M) and total protein was precipitated with ammoniumsulphate (35%-90%). For seeds, PE activities were only detectable in the hull fraction which was used for further purification studies. After dialysis, PE activities were further purified using weak cation exchange chromatography and heparin affinity chromatography. The active fractions were characterized using gelfiltration chromatography, SDS-PAGE and isoelectric focusing. The identity of the isoforms was confirmed with polyclonal antibodies directed towards a pectin methylesterase from tomato fruit [5]. The results show that both pods and seed hulls contain at least two PE isoenzymes of 42 kDa and 33 kDa respectively. Isoelectric focusing of purified fractions showed the occurrence of one or more isoforms with relative alkaline pI values (Table 1).

Table 1 Molecular weights (MW) and isoelectric points (pIs) of PE fractions from green beans

Seed (hull)

Pod

Fraction

MW (kDa)

pI

I

42

9.8

II

33

>11.5, 10.5

I

42

9.8, 8.4

II

33

>11.5, 10.5

402 Both pod fractions were analyzed for thermostability by incubation at different time-temperature combinations. At a temperature of 70 ~ 50 % of fraction 1 activity was lost after 1 minute of incubation. Fraction 2 was more stabile and contained 50% of PE activity at 11 minutes of incubation. Total pods contained 50% of PE activity after 10 minutes of incubation at this temperature.

4. CHARACTERIZATION OF G R E E N B E A N PE (C)DNA CLONES 4.1 I s o l a t i o n o f (c)DNA c l o n e s Based on the homology between previously published PE sequences [6], three oligonucleotides were constructed and used to isolate green bean-specific PE clones. By using genomic DNA and mRNA from young beans of cv. Verona as templates for the PCR reaction, one putative genomic clone of 210 bp (PE1V) and two cDNA clones of 660 bp (PE2V&PE3V) were isolated. The identities of the respective cv. Verona clones ranged between 50% and 60% at the deduced amino acid level. In addition, a cDNA library was constructed using poly(A)+ RNA from young developing pods of cv. Masai. By screening this library with the cv. Verona PCR clones, a cDNA clone (PE3M) of 1990 bp with 99 % identity as compared to PE3V was isolated [7]. Figure 2 shows an alignment of part of the deduced amino acid sequences. The identity of the partial and full length cDNA clones was confirmed by producing the corresponding polypeptides in E.coli using the pGEX4T expression system and Western blot analysis with antibodies directed towards tomato fruit PE [5]. I PE3M PE3V PE2V PEIV

FIAKDIGFVN ********** **GQ**W*Q*

II

NAGASKHQA ********* T**PQ****

VALRSGSDRS ********** ********Q*

VFFRCRFDGFQ *********** ******V****

DTLYAHSNRQ ********** ******T***

FYRDCDITGT ********** ****SF**A* .........

420

VFQSCKIMP ********* ***K*YLVA *L*E*N**S

RQPLPNQFNT ********** *KP*S**K*M *K**HG*ATV

ITAQGKKDPNQ *********** V****RE**** ****S**DP**

NTGIIIQKST ********** S**TS**QCN *TGIV**GCN

ITPFGNNLTA ******~*** ***SLDLKPV *KASFD*SSV

480

PWKDFSTTV IMQSDIGALL NPVGWMSWVPN VEPPTTIFYA EYQNSGPGAD *** ...... ......................................... *** ............................................... *** ...............................................

536

III PE3M PE3V PE2V PEIV

IDFXFGNAAV ********** V********* ********** IV

PE3M PE3V PE2V PEIV

.... PTYLGR .... ****** AGSIK***** .... KS****

Figure 2. Comparison of Phaseolus vulgaris pectin methylesterase cDNA clones PE3M (cv. Masai), PE3V & PE2V (cv. Verona) and genomic clone PEIV (cv. Verona). Homology boxes are printed in bold [6]. Numbers refer to the aa sequence of the full length cDNA clone PE3M.

403 The 2 kb clone PE3M contains an open reading frame of 582 amino acids (nucleotides 51 to 1796) encoding a polypeptide of 63.5 kDa. Following the rule of von Heijne, a putative signal peptide of 39 amino acids with the cleavage site at position Ala 45 can be predicted [8]. In addition, the N-terminal segment of the protein contains a putative transmembrane segment of 23 residues within the first 55 amino acids. This region is also identified for other (putative) plant PEs and forms part of the more than 200 N-terminal residues which precede the mature protein [5,9].

4.2 PE gene expression during pod development Southern blot experiments showed that the respective PE clones crosshybridized less than 10% using stringent conditions. Northern blot analysis was performed to determine the pattern of expression of the different PE genes. By using the genomic DNA fragment PE1 as a probe, no significant transcripts could be detected in any of the tissues analyzed. PE2 and PE3 cDNAs hybridized with transcripts of about 1.8 kb in length (Figure 3). Interestingly, PE2 expression levels were high during early stages of pod development while PE3 expression increased during pod maturation. Both bean PEs were also expressed in other plant tissues while tomato PE1 mRNA was only detectable during tomato fruit development (Figure 3, [5]).

A

Y.pods Seeds ,---=1 :=;~ ~:~: ~ , ;

2

! ~ ,; ~i

: ::ii=~i~::i~!;~

PE2

'~'"

D.pods

3 4 5 3 45 "~ . . . .

~"~=~~': ~~~, ~'~176 ~"~ ; ~ : :~" ~i: : , , ; ~ o~=o~.......; :~:~::?~:~:=:

9

iii

(1.8 kB) PE3

11.8 kB)

MG

11.9 kB)

Figure 3. Developmental pattern of expression of PE2 and PE3 in pods and other tissues of green bean cultivar Verona. Numbered samples concur with the following developmental stages in Days Post Flowering (DPF). 1" 1-5 DPF, 2:6-7 DPF, 3: 811 DPF, 4:12-22 DPF, 5:23-35 DPF. Abbreviations: Y.pods = Young pods, D. pods = Deseeded pods. rt, If and fl represent root, leaf and flower tissue respectively. Mature green (MG) tomato RNA was probed with tomato pPE1 [5].

404 5. S U M M A R Y

Processing of green beans involves modifications of the cell wall composition. Based on the analysis of the cell wall fractions from fresh and processed beans, we propose that due to the sterilization procedure demethylated smooth pectic regions (homogalacturonans ?) are degraded by means of B-elimination [10]. Analysis of the pectic fractions showed that (i) uronic acids are released into the brine and (ii) hairy regions are solubilized (rhamnogalacturonans). At decreased blanching temperatures of 60 ~ to 70 ~ a significant reduction of the release from uronic acids was observed. Further studies will indicate to which extent this effect is due to a temperature-dependant activation of PE. During early stages of pod development, low levels of endo- or exoPG were detected. In contrast, PE activities were measurable during all developmental stages. Purification studies showed that both pods and seeds contained two groups of PE isoenzymes with molecular weights of 42 kDa and 33 kDa respectively. Most likely, the relative thermostable isoenzymes of 33 kDa account for the firming effect at reduced blanching temperatures. At a molecular level, three PE clones were isolated from which the two cDNA clones (PE2V and PE3V), corresponding with mRNAs of 1.8 kb, were expressed during pod development and other plant tissues. Based on the deduced amino acid sequence of the full length cDNA clone (PE3M) we postulate that, similar to other plant PEs [5,9], green bean PEs are synthesised as precursor proteins of about 63 kDa. It is not known yet to which extent the proteins encoded by the PE2 and PE3 cDNAs correspond with the biochemically characterized isoenzymes. To investigate the function of PE3M in detail, this cDNA clone will be expressed in potato tubers using constitutive and tuber-specific promoter constructs.

6. R E F E R E N C E S

1.

J.A. De Vries, F.M. Rombouts, A.G.J. Voragen and W. Pilnik. Carbohydr. Polym., 2 (1982), 25-33. 2. G.A. Tucker and J. Mitchell. In: D. Grierson (ed), Biosynthesis and manipulation of plant products, London (1993), 55-103. 3. L.G. Bartolome and J.E. Hoff. J. Agric. Food Chem. 20 (1972), 266-270. 4. P. Ryden and R.R. Selvendran. Carbohydr. Res. 195 (1989), 257-272. 5. L.N. Hall, C.R. Bird, S. Picton, G.A. Tucker, G.B. Seymour and D. Grierson. Plant Mol. Biol. 25 (1994), 313-318. 6. D. Albani, I. Altosaar, P.G. Arnison and S.F. Fabijanski. Plant Mol. Biol. 16 (1991), 501-513. 7. K. Recourt, J.M. Laats, T. Stolle-Smits, H.J. Wichers, C. van Dijk and C.E.M. Ebbelaar. GenBank Data Base (1995) Accession no. X85216. 8. G. von Heijne. J. Mol. Biol. 189 (1986), 239-242. 9. L. Richard, L-X Qin and R. Goldberg. FEBS Letters 355 (1994), 135-139. 10. T. Stolle-Smits, J.G. Beekhuizen, C. van Dijk, A.G.J. Voragen and K. Recourt. J. Agric. and Food Chem. 43 (1995), 2480-2486.