Cellulase (Endo β-1,4 Glucanase) and Cell Wall Breakdown during Anther Development in the Sweet Pea (Lathyrus odoratus L.): Isolation and Characterization of Partial cDNA Clones

J. Plant Physiol. Vol. 146. pp. 622-628 (1995)

Cellulase (Endo (3-1 ,4 Glucanase) and Cell Wall Breakdown during Anther Development in the Sweet Pea (Lathyrus odoratus L.): Isolation and Characterization of Partial cDNA Clones ANIL NEELAM1

and Roy SEXTON

Department of Biological and Molecular Sciences, University of Stirling, Stirling, FK9 4LA, UK 1 Present address: Department of Biology, Biomedical Sciences Building, University of Southampton, Bassett Crescent East, Southampton S016 7PX, UK Received February 22, 1995 . Accepted Apri14, 1995

Summary

The possible role of cellulase in cell wall breakdown during maturation of the anthers of Lathyrus odo· ratus 1. was studied. Cellulase (,6-1,4 glucanase) increased 1S0-fold from the premeiotic stage, reaching peak levels during the degradation of the primary walls around the tetrads and the tapetum. The enzyme activity declines as the pollen mature and anthers yellow but there is still large amounts of activity at the time of dehiscence. Four partial cDNA clones, which have a high degree of sequence identity with other plant cellulases, have been isolated by PCR from anther cDNA. Northern analysis showed that these clones differ dramatically in their mode of temporal expression.

Key words: Lathyrus odoratus L., anther, eDNA clones, cellulase (endo ,6·1,4 glucanase), cell wall break· down, pollen, tapetum. Abbreviations: AZ = Abscission zones; bp = base pairs; cDNA = DNA complementary to RNA; dNTP = Deoxynucleotide triphosphate; DTT ,., Dithiothreitol; kb = kilobase(s); PCR - Polymerase chain reaction; kD = kilodalton; SDS = Sodium dodecyl sulphate. Introduction

Endo ,6-1,4 glucanases are assayed by their ability to degrade carboxymethyl cellulose. Although commonly referred to as cellulase, they will not hydrolyse crystalline cellulose. These Cx cellulases, as they are sometimes called, are thought to act on the soluble ,6-1,4 glycosidic linkages found in the hemicelluloses of the cell wall (Reese et al., 19S0; Beguin, 1990) and have been implicated in many physiologically and functionally similar plant developmental processes such as fruit softening (Lashbrook et al., 1994), abscission (Sexton and Roberts, 1982), pod dehiscence (Meakin and Roberts, 1990), adventitious root formation (Kemmerer and Tucker, 1994), hormone mediated growth (Hayashi et al., 1984) and development of anthers (Sexton et al., 1990) and © 1995 by Gustav Fischer Verlag, Stuttgart

styles (del Campillo and Lewis, 1992), all of which involve cell wall degradation (Brummell et al., 1994). In a previous paper we described the purification from anthers of Lathyrus odoratus, two isoforms of cellulase. Both had very similar molecular weights but different isoelectric points (PI) (Sexton et al., 1990). The anther cellulases had very similar characteristics to the cellulase purified from bean abscission zones (AZ) (Durbin et al., 1981) but lacked some antigenic determinants found in the latter. More recently, del Campillo and Lewis (1992) have also demonstrated a similar cellulase present in the anthers and stigmal styles of bean (Phaseolus vulgaris). The aim of this study has been to investigate the involvement of cellulases in sweet pea anther development by seeking correlations between the accumulating cellulase activity

Anther cellulase gene family from Sweet pea

during anther development and cell wall hydrolysis which accompanies anther maturation. Partial anther cellulase cDNA clones have been isolated using peR with degenerate oligonucleotide primers for conserved plant cellulase sequences (Tucker et al., 1987; Tucker and Milligan, 1991; Lashbrook and Bennett, 1993). The pattern of expression of four cDNA clones at various stages of the development of anther were studied. Northern blot analysis revealed that the te~poral pattern of expression of each cellulase gene is umque.

Materials and Methods

Plant material Sweet pea seeds (Lathyrus odoratus L.) varieties Royal Wedding and Cream Southbourne were obtained from Unwins Seeds, Histon, Cambridgeshire. Plants were grown in unheated frost free greenhouses.

Cellulase assays Cellulase was extracted from 80-170 anthers at different stages of development by grinding them in 1.5 mL of extraction buffer (O.5M NaCI in 20mM Tris/HCI (PH 8.1) and 3mM EDTA) at 4 °C using a glass homogeniser. The mixture was spun at 1000 g for 10 mins to remove the cell debris and the resultant supernatant assayed for cellulase activity using the visco metric method described by Durbin and Lewis (1988).

Isolation of total RNA Total RNA was extracted from anthers at three different stages of development (green anthers, stages 1-2; yellow green anthers, stages 3-4; yellow anthers, stages 5-6), styles/stigmas, sepals, petals, roots, shoots (from seedlings) and bean leaf abscission zones (non abscising and after abscission) according to method described by Okayama et al. (1987). Bean leaf abscission zones were induced by ethylene treatment as described previously (Tucker et aI., 1988).

Cloning and sequencing ofanther cellulase cDNA sequences using Polymerase Chain Reaction (PCR) First strand cDNA was synthesised by reverse transcription of 5 Ilg of total RNA extracted.from anthers in 20 ilL reaction volume containing 50 mM Tris HCI (pH 8.3), 75 mM KCI, 3 mM MgCI 2, 10mM O.OlM DTT, ImM dNTPs, lunit/mL RNA guard (Pharmacia Biotech, St. Albans, UK) and 0.2 Ilg oligo (dT) 12-18 primer at 37°C for 1 hr with 200 units of Superscript reverse transcriptase (Gibco-BRL Lifetechnologies, Paisley, UK). The cDNA was denatured and reverse transcriptase was inactivated at 95°C for 5 min before using in the PCR reaction. Thirty cycles of PCR, each consisting of denaturing (94°C, for 1 min), annealing (50 °C for 2 min) and Amplitaq (Perkin Elmer, Warrington, UK) mediated primer extension (72 °C for 3 min), were used to amplify cellulase cDNA sequences from first strand cDNA. The PCR amplification was conducted in either 50 or 100 ilL reaction PCR buffer consisting of 10mM Tris HCI (PH 8.3), 50mM KCI, 2mM MgCb, 0.1 % Triton X-I00 with 0.2mM dNTPs and 20pmoles of each of the cellulasespecific degenerate primers (R & D Systems Europe Ltd., Abingdon, UK). The degenerate oligonucleotide primers used were 5' GGNTAYTAYGAYGCNGGNGA 3' as forward primer and 5' GCNGCCCANARNARYTCRTC 3' as reverse primer cor-

623

responding to amino acid regions GYYDAGD and DELL WAA respectively. The nucleotide bases in the primers are represented by IUB standard symbols, where G = Guanine, A = Adenosine, T = Tyrosine, C = Cytosine, Y = Cor T, R = G or A, and N = G,A, T or C. The PCR cDNA products were cloned into PCR II vector (Invitrogen, San Diego, USA) and amplified in E. coli strain INVaF. The nucleotide sequences of the positive clones were determined by double stranded dideoxy chain termination method using T7 DNA polymerase (Pharmacia Biotech, St. Albans, UK). The sequence data was analyzed and the predicted amino acid sequences determined using the computer programmes DNASIS and PROSIS (Pharmacia Biotech). Alignment of the amino acid sequences was performed using MultAlin programme (Cherwell Scientific, Oxford, UK) which uses the algorithm described by Lipman and Pearson (1985). DNA and protein database (GenBank, EMBL, NBRF-PIR and Swiss-Prot) searches for sequence homology with anther cDNA were made using DNASTAR software (DNASTAR Inc. Wisconsin, USA).

Northern analysis with partial cDNA clones The cDNA inserts used as probes were cleaved from the vectors with EcoRI and purified by gel elution using Qiaex gel suspension (Diagen, Germany). The DNA probes were labelled with 32p by random priming using an oligolabelling kit (Pharmacia Biotech, St. Albans, UK). Five micrograms of each of the total RNA samples were slot blotted onto Hybond N (Amersham, Little Chalfont, UK) using a BRL slot blotting apparatus (Gibco-BRL, Paisley, UK) as described in the manufacturer's protocol. Northern blots were prepared by electrophoresing (10 Ilg/lane) denatured, total RNA samples in 1 % agaroselformaldehyde gels. Subsequent transfer to Hybond N membranes was achieved using vacuum blotting apparatus (Pharmacia Biotech, St. Albans, UK). Both slot blots and Northern blots were prepared following the protocol described by Sam brook et al. (1989). The slot blots and Northern blots were hybridized at 68°C in Quick hyb solution (Stratagene, Cambridge, UK) containing denatured probe (1 x 106 cpm/ mL as described in the manufacturer's protocol. The blots were washed in wash buffer consisting of 2 x SSPE, 0.1 % SDS at room temperature (15 min x2), 2x SSPE, 0.1 % SDS at 50°C (15minx2) and 0.2x SSPE, 0.1 % SDS at 50°C (30 min). Final washes were carried out at approximately Tm -25°C, after which the blots were exposed to Kodak X-Omat AR film at -70°C.

Results

Cellulase activity increases as anthers mature Although some aspects of microsporogenesis in the Sweet pea anthers has been described (Latter, 1926; Wojciechowska, 1983) the details of wall breakdown during anther development is not well documented. A detailed light and electron microscope study was therefore undertaken, the results of which are beyond the scope of this paper. Six stages of anther maturity were examined. The morphological characteristics allowing each developmental stage (based on bud characteristics and pedicel length) to be recognized are summarized in Table 1. Green transluscent anthers of stage 1, found in the smallest recognizable buds in the apex, had polygonal pollen mother cells

624

ANIL NEELAM and Roy SEXTON

Table 1: Anatomical features and cellulase activity of anthers at six stages of development. Stage

Anatomical features

1 Transluscent

Pre and early meIOSIS Callose deposition around meiotic PMCs Post meiotic, callose degradation, separating tetrads Tapetal wall hydrolysis Microspore mitosis, tapetal cell degeneration, maturing anthers Imminent dehiscence

green 2 Opalescent green 3 Opalescent

green

4 Mainly green

some yellow 5 Mainly yellow some green 6 Bright yellow

Units of cellulase per gram fresh weight (Mean with standard deviation)

Units of cellulase per anther (Mean with standard deviation)

170±40

0.04±0.02

605±170

0.34±0.11

1610±380

1.01±0.17

5800±230

4.95±0.77

4810±1080

5.87±2.54

2070± 1090

2.76± 1.62

(PMCs) surrounded by a single layer of developing tapetal cells. In stage 2, the anthers were still green but more opaque. Meiosis had commenced in the PMCs and a callose wall was beginning to surround these cells. In these anthers, the tapetal cells were easily recognizable by their large lobed central nuclei and densely packed cytoplasm. Stage 3 anthers showed fully formed tetrads encased in a thick callosic wall. The primary tapetal cell wall was still intact. By stage 4 the anthers were beginning to turn yellow and were characterized by the dissociation of the tetrads brought about by degradation of the callose wall. Both the apical and lateral tapetal cell walls had degraded by this stage. The tapetal protoplasts persisted and most had retained their original peripheral positions around the locular wall. The anthers of stage 5 were completely yellow and contained maturing pollen grains. The tapetum was disorganized, degenerating completely by the end of this stage. At stage 6, the anthers had not dehisced but would do so under slight pressure. Internally, the cells of the septum between the two locules had separated from the locule wall apparently by cell separation along the line of the middle lamella leaving intact cells at the fracture faces. The septum then shrinks allowing the two 10cules to become confluent and the stomium eventually ruptures releasing the pollen. The levels of activity of cellulase per anther increased almost 150 times as the anther matured, reaching its maximum level when they had just completed yellowing (Table 1). When expressed on a per gram fresh weight basis the increase was not as great, the peak being reached during stage 4 when tapetal collapse was complete. The greater variation in assays of the different harvests of older anthers may be due to differences in climatically controlled drying rates. In an attempt to determine if the activity in the mature anthers was associated with the pollen or the anther wall,

pollen was washed out of the anthers and assayed. Both the pollen extract and the anther walls were quite rich in cellulase but the data is difficult to interpret since some pollen was left in the anther locules and the activity associated with the pollen could be due to the remains of the tapetum on the outside of the pollen grains.

Isolation and cloning ofanther cellulase eDNA sequences A number of conserved sequences present in the catalytic core of higher plant cellulase were identified using the sequence information available from two published sequences of bean abscission cellulase (Tucker et aI., 1987), avocado fruit cellulase (Tucker and Milligan, 1991) and also from tomato fruit cellulase (Lashbrook and Bennett, 1993). Two degenerate oligonucleotide primers designed for such conserved amino acid domains (GYYDAGD and DELLWAA) were used as PCR primers for the amplification of anther cellulase cDNA sequences. First strand cDNA was synthesised by reverse transcription of mRNA from total RNA derived from anthers at different stages of development, green (stages 1-2), yellow green (stages 3-4) and yellow (stages 5-6). Amplification of first strand cDNA with degenerate PCR primers for cellulase produced a 500 bp cDNA product at all three stages of anther development, but amplification was strongest at the predehiscing yellow anther stage. Since the size of the anther cDNA PCR product was consistent with those expected from the published sequences of bean abscission zone, avocado cellulases and tomato fruit cellulases, the cDNA sequences derived from predehiscing anthers were cloned into PCR II vector. A number of clones were obtained which were subsequently sequenced in both directions.

The four eDNA clones share considerable homology with other plant cellulases The nucleotide sequences were determined and the open reading frames for the clones were aligned with the sequences of bean abscission and avocado cellulases. The alignment of sequences to achieve the best fit required the introduction of gaps at certain residues. The results revealed four different cellulase sequences. An overall aminoacid sequence comparison of the four anther cellulase cDNA revealed that they shared considerable homology with one another (Table 2). DNA and protein database searches for sequence identity with anther cellulase cDNA sequences indicated that each of the anther sequences was most identical to either bean abscission (Tucker and Milligan, 1991) or to avocado fruit cellulase (Tucker et aI., 1987) (Table2). Three cysteine residues identified in bean and avocado cellulases, in the region corresponding to 500 bp were found in the same positions in three of the anther clones (Table 2). In clone pLAC5.2 only one of the cysteine residues matched. pLAC5.1 sequence contained an extra cysteine residue also found in avocado but at a different position. The amino acid and DNA sequence identities are shown in Table 3. Of all the four anther cellulases, pLAC5.9 shared maximum homology at the amino acid and nucleotide level (86.0 % and 82.6 % respectively) with bean abscission cellu-

Anther cellulase gene family from Sweet pea Table 2: Comparison of the deduced amino acid sequences of the four partial anther cellulase cDNA clones, pLAC5.1, 5.2, 5.5 and 5.9 with the corresponding sequences of avocado fruit (Tucker et al., 1987) and bean abscission (AZ) zone cellulases (Tucker and Milligan, 1991). Primer regions are underlined. Identically matched amino acid sequences are indicated in bold. Cysteine residues are indicated by asterisks. pLAC5.1 pLAC5.2 pLAC5.5 pLAC5.9 Bean AZ

Avocado

pLAC5.1 pLAC5.2 pLAC5.5 pLAC5.9 Bean AZ Avocado

pLAC5.1 pLAC5.2 pLAC5.5 pLAC5.9

Sean AZ

Avocado

G Y Y D A G D N V It Y G L P M AFT V T S L S If A GYYDAGDHMltFGFPMAFTASVLSlfA GYYDAGDNVltFGFPMAI'TTTMLSlfS GYYDAGDNVltFGlfPMSFTVSLLSlfA G Y Y D A G D N V It F G If P M A F S T S L L S If A G Y Y D A G D N L It F G L P M A l' T T T M L A If G

25

A I F Y K A E LEA T It E M G N I Q E A I R If G T ILEYGDQMDVVGQLEPAQDSLKlfIT VIEFGGLMKG • • ELPNAICEAVRIfAT AIEYESEISSAKQLDYLHSAIRIfGP AVEYESEISSVNQLGYLQSAIRIfGA I IEFGCLMPE • • QVENARAALRlfST

50

D Y F L K C • S S K K N R L Y V E V G E P H E D H DFLINA.HPSENVLYIQVGDPVADH DYLLKA. TAHPN I I YVQVGDAKKDH D F I L Q A • H T S PTA L F T Q V GOG NA D B DFMLRA. HTSPTTLYTQVGDGNADR DYLLKASTATSNSLYVQVGEPNADH

75

. .

pLAC5.1 pLAC5.2 pLAC5.5 pLAC5.9

Q C If A P P E K M K T K R S V K V lOT K T P G S 100 KClfNRPESITDARPLVRVNTTSPGS AClfERPEDMDTPRSVFKVDANAPGS NClfERPEDMDTPRTTYKIDANSPGT NClfERPEDMDTPRTVYKIDANSPGT RClfERPEDMDTPRNVYKVSTQNPGS

pLAC5.1 pLAC5.2 pLAC5.5 pLAC5.9

E I A A ETA A A M A ASS I V r R S T D R K Y A 125 DVAAETAAAMRSASLvrKKSDPTYS E V A A ETA A A L A A A S L V F R R S D P T Y A EVAAISAAAALSAASIVI'KKIDINYS EVAAEYAAALSAASIVFKKIDAKYS DVAAETAAALAAASIVFGDSDSSYS

Bean AZ Avocado

Bean AZ

Avocado

pLAC5.1 pLAC5.2 pLAC5.5 pLAC5.9 Bean AZ

Avocado

pLAC5.1 pLAC5.2 pLAC5.5 pLAC5.9

Bean AZ

Avocado

.

The expression of the cellulase cDNA clones was studied by slot blot and by Northern blot hybridization analysis of total RNA isolated from anthers at different stages of development and from different parts of the plant. All the four clones were found to be expressed specifically in anthers and not in other parts of the plant such as shoots and roots. Moreover, the four cDNA clones did not cross hybridize with each other even at low stringencies which is consistent with their low sequence homology. The unique pattern of expression of each of the four anther cellulase clones are indicated in figure 1. The gene corresponding to pLAC5.1 was found to be expressed exclusively in predehiscing anthers. Transcripts for pLAC5.2 was found to accumulate from the immature anther stage to the predehiscing stage and was also found to cross hybridize with the RNA in stigma and styles. Anther clone pLAC5.5 seemed to be expressed less abundantly in anthers but was found to cross hybridize very strongly to cellulase transcripts expressed in stigma and styles. pLAC5.9 was similar to pLAC5.5 in its pattern of expression at the various stages of anther development but was present more abundantly than pLAC5.5. It cross hybridized with bean abscission zone cellulase and also to stigma and style to some degree. The transcripts for both pLAC5.5 and 5.9 were found to accumulate to peak levels during tapetal wall breakdown and tetrad release.

R R L L N K A K L L .. T r A K S Y R G T FOG T • 150

KILVRRAIRVFQFADKHRGSYSNAL S K L L S Q S K S L F 0 FAD K Y R G T Y S T S • S T L L S H S K S L F 0 FAD K N R G S Y S G S • TKLLHTAVKVFEFADQYRGSYSDSL

172

• • • • CPFYCSYSGYNDELFlfAA PE • • VATYYNSTGFGDELLlfGA KPFVCPFYCSYSGYQDELFlfLA • • • • CPFYCSYSGYKDI!LLlfL. • • • • CPFYCSYSGYQDELLlfAA GSVV.CPFYCSYSGYNDELLlfGA

';' .... l

. .

pLAC5.1

pLACS.l

Expression pattern ofeach of the four clones is unique

G T L L K H A K Q L F T r A 0 N F K ElY S V S I

1,00

41:1 .'

pLAC5.9

Bean AZ.

Avocado

85.7

60.9

57.9

60.9

58.3

59.9

579

59.7

63.1

64.4

82.6

61.2

100

64.9

sU

100

pLAC5.2

pLAC5.5

58,2 100

pLAC5.5

~,.:." ."""

pLAC5.9 Bean AZ

1Il.b: ,::,,4a,~'>' :,-:.~~.,. 100 . , , ' :' . ,:aU .' !o&l'A •. :,;aiI,g

Avocado

11,5':.";,,,&

~:2>:'~',:

100

67.0

;,:

t f t. tj !

Table 3: Percentage homology between the amino acid and nucleotide sequences of the four anther cellulase cDNA clones and the corresponding regions of the avocado and bean abscission (AZ) cellulase sequences. Sequence comparions were performed using DNASTAR software. DNA sequences were compared by Wilbur and Lipman method and amino acid sequences by NeedlemanWunsch method.

pLAC52

625

!

Nucleotide

II sequence

'--______.........:_,'_ . _·Mdno~....·.'-':_.,;:;..~"" . ......:...i~'_.~_ . . .~. . .;.;".'-".;.~.-'-'-"""_________ J

lase, while pLAC5.5 shared 67.3 % identity at the amino acid level and 64.4 % at the nucleotide level with avocado fruit cellulase. The four anther cellulases shared an average identity of 50.1 % with one another at the amino acid level.

,

J1 J ! I'll

i ~

f fI ~

~

JJ pLACS.1

•••• ---- • •

" ,., . . . -.

-.~. ~

• .:

t

...

pLACS.2

I

...

.. --............;.,.. ............ .. .

:,

t

•

pLACS.S

pLACS.9

Fig. 1: RNA slot blot analysis showing the expression pattern of transcripts for anther cellulases (pLAC5.1, pLAC5.2, pLAC5.5 and pLAC5.9). Five micrograms of total RNA isolated from anthers at three different stages of development, various parts of the Sweet pea flower, roots, shoots, and abscising and non-abscising bean abscission zones were hybridized with 32p labelled cDNA probes.

626

ANn. NEELAM and Roy SEXTON

:! l

~

ttt

j J pLAC5.1

pLAC5.2

pLAC5.5

pLAC5.t .. Fig.2: Northern blot analysis showing the expression pattern of transcripts for anther cellulases. Ten micrograms of total RNA isolated from anthers at three different stages of development, stigmal styles, roots, shoots and abscising bean abscission zones were hybridized with 32p labelled cDNA probes (pLAC5.1, pLAC5.2, pLAC5.5 and pLAC5.9). The probes hybridized to 2.0kb transcripts.

Northern blot analysis of total RNA hybridized with the anther cellulase clones (Fig. 2) gave very similar results to those described for RNA slot blot analysis. All the four clones were found to hybridize to transcripts of approximately 2.0 kb in RNA samples from different stages of anther development, styles/stigma and bean abscission zones.

Discussion

The first important wall breakdown process during microsporogenesis is the degeneration of both the original pollen mother cell walls and the thick callosic wall which surrounds the tetrads. Our results showed cellulase levels increasing at this stage of anther development suggesting its involvement in the hydrolysis of the original pollen mother cell wall which appear to swell before disappearing. Callosic walls are predominately composed of a t3-1,3 linked glucan and the role of t3-1,3 glucanase or callase in its hydrolysis has been frequently proposed (Izhar and Frankel, 1971; Steiglitz and Stern, 1973; Steiglitz, 1977; Worrall et al., 1992). Cellulase may also be implicated in callose degradation since callose extracted from rye has been shown to contain t3-1,4 as well as t3-1,3 glycosidic linkages (Vithanage et al., 1980).

The highest levels of cellulase activity are associated with the stage of anther development when the degradation of the tapetal walls is in progress. Our EM observations confirmed that the tapetal walls adjacent to the developing microspores and down the lateral sides of the cells are almost entirely hydrolysed. Cytochemical staining studies in A vena suggests that not only the wall matrix but also cellulose is lost during this process (Steer, 1977). Presumably, cellulase together with other cell wall hydrolases such as polygalacturonase (enzyme activity was also detected in anthers) are involved in the process of the tapetal wall degradation. At the end of pollen maturation two adjacent anther 10cules become confluent as a result of the separation of two layers of cells located in the region where the septum joins the anther wall. This separation of apparently intact cells appears to be enzymatically mediated (Keijzer, 1987) and resembles very closely the similar process of abscission which involves the degradation of the central area of the primary wall (Sexton and Roberts, 1982). Since cellulase is implicated in cell separation during abscission (Sexton et al., 1980), it is quite possible that cellulase present at dehiscence has a similar role. Our results showed high levels of cellulase activity in pollen extracts of Sweet pea (data not shown). Besides cellulase, a number of cell wall hydro lases have been found to be associated with pollen including t3-1,3 glucanase (Roggen and Stanley, 1969), t3 galactosidase (Singh and Knox, 1985) and polygalacturonase (Pressey and Reger, 1989). Pollen specific genes for enzymes such as pectin esterase (Albani et al., 1991), polygalacturonase (Niogret et al., 1991) and pectate lyase (Wing et al., 1989) have also been isolated. Together with cellulase, these enzymes may participate in the wall modification associated with pollen germination, pollen tube growth and penetration of the style. Purification of the sweet pea anther cellulases revealed two antigenically related isoforms, differing in molecular weight (49 kD and 51 kD) and pI (Sexton et al., 1990). Such multiple isoforms of cellulase are common and have been reported in softening fruit (Kanellis and Kalaitzis, 1992; Maclachlan and Brady, 1992; Bonghi et al., 1992; Abeles et al., 1993) and in separating abscission zones (Sexton and Roberts, 1982). Our initial attempts to isolate anther cellulase cDNA clones from a sweet pea anther cDNA expression library employing both a heterologous bean abscission cellulase cDNA probe, pBACI0 (Tucker and Milligan, 1991) and bean abscission cellulase antibodies were unsuccessful. An alternative approach based on the use of the conserved cellulase sequences as degenerate oligonucleotide PCR primers proved successful. The PCR reaction amplified a 500 bp species, exactly the size anticipated from bean and avocado cellulase sequence data (Tucker and Milligan, 1991). The sequencing of partial cDNA clones derived from this 500 bp product revealed a family of four cellulases sharing significant homology to one another and to other plant cellulases. These results seem to be consistent with multiple cellulase sequences isolated recently from tomato fruits (Lashbrook et al., 1994) using cloning strategies based on conserved amino acid sequences. Cellulases have also been suggested to be a family of genes in avocado (Tucker et al., 1987; Cass et al., 1990) and bean (Tucker and Milligan, 1991).

Anther cellulase gene family from Sweet pea

Amino acid sequence comparison of bean and soybean abscission zone cellulases, avocado fruit and tomato fruit cellulases (Tucker and Milligan, 1991; Kemmerer and Tucker, 1994; Lashbrook et al., 1994) with anther cellulases, reveal the presence of multiple domains in which the sequence and its relative position in the cDNA are highly conserved. The functional significance of various conserved domains is not understood but as a result of this consistency it is thought that all plant and bacterial members of the E class family of cellulases have evolved from a common ancestor (Navarro et al., 1991). Our Northern analysis studies with the anther cellulase clones have shown that the temporal pattern of expression of different genes can be quite different, although their expression may overlap at a given stage. For instance, clone pLAC5.9 was found to be expressed at high levels during the degradation of the tapetal and pollen mother cell walls whereas transcripts which hybridized to pLAC5.1 were found exclusively towards dehiscence. It seems likely that these patterns reflect tissue specific changes rather than general changes in all the cells of the anther. The overlapping expressions of these genes also suggests the involvement of more than one cellulase gene at a given stage. It is likely that a combination of cellulase activities is required to carry out a particular cell wall degradation function (Lashbrook et al., 1994). A strong hybridization of three of the anther cellulase clones with cellulase transcripts in stigma and style correlated well with the very high levels of cellulase in stigma and style (data not shown). We have isolated and sequenced a partial cDNA clone for cellulase from stigma and styles. Although it shares considerable homology with all the anther cellulases reported here, it is not similar to any of them, suggesting the involvement of a number of different cellulases in the development of stigma and style. Cellulase activity accompanies cell wall breakdown in fruit softening, tapetal wall breakdown, anther dehiscence, stylar intercellular space formation and abscission, which have anatomically distinct features. This may be due to the fact that cellulases involved in modifying the cell wall in different ways in different tissues have different substrate specificities. Chromatographically separated tomato fruit 13-1,4 glucanases have been shown to be substrate specific (Maclachlan and Brady, 1992). Further work will involve in situ hybridization and chimeric gene constructs to investigate whether the anther cellulases we have identified have unique tissue specific expressions and functions during the development of the anther. Acknowledgements

This work was supported by an award from the UK Agricultural and Food Research Council's Plant Molecular Biology Initiative. We thank Dr. Mark L. Tucker and Prof. John L. Hall for critical reading of the manuscript.

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