Characterization of a pollen-expressed gene encoding a putative pectin esterase ofPetunia inflata

Plant Molecular Biology 25: 539-544, 1994. © 1994 Kluwer Academic Publishers. Printed in Belgium.

539

Short communication

Characterization of a pollen-expressed gene encoding a putative pectin esterase of Petunia inflata Jing-Hong Mu 1, Joseph P. Stains and Teh-hui Kao* Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA (* author for correspondence); lpresent address: Laboratory of Plant Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, N Y 10021, USA Received 7 January 1994; accepted in revised form 10 February 1994

Key words: pectin esterase, Petunia inflata, pollen, pollen tubes

Abstract From a pollen tube c D N A library of Petunia inflata, we isolated c D N A clones encoding a protein, PPE1, which exhibits sequence similarity with plant, bacterial, and fungal pectin esterases. Genomic clones containing the PPE1 gene were isolated using c D N A for PPE1 as a probe, and comparison of the c D N A and genomic sequences revealed the presence of a single intron in the PPEI gene. During pollen development, PPE1 m R N A was first detected in anthers containing uninucleate microspores; it reached the highest level in mature pollen and persisted at a high level in in vitro germinated pollen tubes. The observed expression pattern of the PPE1 gene suggests that its product may play a role in pollen germination and/or tube growth.

Pectinases are a family of enzymes involved in degradation of pectin, a structural component of the primary cell wall [5]. Pectin is demethylated by pectin esterase to yield pectate, which is subsequently depolymerized by polygalacturonase and pectate lyase [7]. Pectinase activity has been found in many plant tissues, indicating a general role in cell wall metabolism. In addition, pectinases have been implicated in specialized functions such as wall hydrolysis during fruit ripening [51. Pectinase activity has also been found in pathogenic bacteria and fungi, and this activity

has been implicated in pathogen invasion of host plants [4]. The process of pollen tube penetration of the transmitting tissue of the style is analogous to that of pathogen invasion [2]. This similarity would suggest that pectinases may also play a role in pollen germination and tube growth. In fact, pectinase activities have been found in mature pollen of many plant species [ 12]. Here we report the identification of a putative pectin esterase gene of Petunia inflata which is abundantly expressed in mature pollen and in vitro germinated pollen tubes. We constructed a pollen tube c D N A library

The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Sequence Databases under the accession number L27101 (PCPE22).

540 using poly(A) + R N A isolated from in vitro germinated pollen tubes, and screened the library for clones that hybridized more strongly to a c D N A probe derived from pollen tube poly(A) + R N A than to a c D N A probe derived from pollen poly(A) + RNA. One of the c D N A clones identified, PXC 1, contains a 1.1 kb c D N A which was found to exhibit sequence similarity with the pectin esterase genes ofBrassica [ 1] and tomato [ 13]. The c D N A library was rescreened with PXC1 c D N A as a probe to isolate clones which might contain longer c D N A inserts. A clone, PCPE22, containing 1.5 kb c D N A was isolated and sequenced. Screening of a P. inflata genomic library [3] using PCPE22 c D N A as a probe yielded seven positive clones. Four clones hybridized strongly to the probe, and, based on restriction mapping,

likely contain the same genomic D N A fragment. The other three clones hybridized weakly to the probe. One clone, PGPE2, from the former group was chosen for further analysis. A 4.5 kb Sac IB a m HI fragment which hybridized to the c D N A probe was subcloned, and its restriction map is shown in Fig. 1A. A total ca. 1.6 kb sequence was determined. Comparison of this sequence with the PCPE22 c D N A sequence confirmed that this genomic D N A contains the PPE1 gene, and revealed that the gene contains a single intron of 102 bp which interrupts the first and second bases of the codon for Gly-153. The deduced amino acid sequence of PPE1 contains an open reading frame of 374 amino acids, and has a calculated molecular mass of 41469 Da and a pI of 9.7. The putative translation initiation codon (nucleotides 175 to 177) was

Fig. 1. Structure of PPE1 gene (A) and genomic Southern blot hybridized with PCPE22 c D N A (B). Open boxes denote exons; straight line denotes 5'-flanking region or intron; broken lines (not drawn to scale) denote 3'-flanking region. Ten micrograms of Eco RI- or Hind llI-digested P. inflata DNA was used in Southern analysis.

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identified because the sequence surrounding it, TAAAATGGT, is similar to the consensus sequence A A C A A T G G C which is used for translation initiation of plant genes [8]. There is a putative signal peptide of 31 amino acids with the cleavage site located between Gly-31 and Leu-32. At -1 and - 3 position relative to the putative cleavage site are small, uncharged amino acids that conform to the ( - 3 , - 1 ) rule [16]. Four potential N-linked glycosylation sites (Asn-XSer/Thr) were found. Based on the restriction map shown in Fig. 1A, one Eco RI and one Hind III genomic fragment

P P E I (72) B p l 9 ( 273 ) PEI (62) P v V P E 2 (1 ) P v V P E 3 (1 ) E c P M E (30 ) A n P M E (28)

were expected to hybridize to PCPE22 cDNA. However, as shown in Fig. 1B, in addition to one strongly hybridizing Eco RI and Hind III fragment (7.6 kb and 4.4 kb, respectively), two Eco RI and two Hind III fragments hybridizing more weakly to the probe were also observed. These results suggest the presence of other PPEl-like genes in the P. inflata genome, and are consistent with the isolation of another group of genomic clones described above. A search of the E M B L and Genbank databases revealed four more pectin esterases, in addition to the tomato and Brassica pectin esterases,

A T V A L D G S . GQ. Y K T I K E A L D A V P K K N T E P F I I F I K A G V Y K E Y I D I P K 117 HVVAKDGS.GQ. FKTISEAVKACPEKNPGRCIIYIKAGVYKEQVTIPK 319 AWAKDGT.GK.YRTLAEAVAAAPDKSKTRYVIYVKRGTYKENVEVSS 107 A V V A K D G S .GK. F K T V A E A V .ASAPDNR. R Y V I Y V K K G T Y K E N V E I G K 44 A W A K D G S . GQ. F K T I A L K L V K K K S E . K. R F S V Y V K E G R Y V E N I D L D K 44 AVVSKSSSDGKTFKTIADAIASAPA. GSTPFVILIKNGVENERLTITR 75 I W A K S G . . GD. Y D T I S A A V D L S T T S T E T Q . TI F I E E G S Y D E Q V Y I P A 71

PPEI Bpl9 PE1 PvVPE2 PvVPE3 EcPME AnPME

SMTNVVL IGEGPTKTK ITGNKSVKDGPSTFHTTTVGVNG ............ K V N N V F M F G D G A T Q T I I T F D R S V G L S P G T T T S L S G T V Q V E S .......... R K M N L M I I G D G M Y A T I IT G S L N V V D G S T T F H S A T L A A V G . . . . . . . . . . . . K K K N V M L V G D G K D L T V I T G S L N Y ID G T G T F Q T A T V A A V G . . . . . . . . . . . . NTWNVMI YGDGKDKTFVLGSRNFMDGTPTFETATFAVKG ............ • .NNLLLKGESRNGAVIAAATAAGTLKSDGSKWGTAGSSTITISAKDFSAQ L S G K L I V Y G Q T E D T T T Y T ........ S N L V N I T I S T H A I A L A D V D N D D E T A

156 360 146 83 83 125 114

PPE 1 Bp19 PEI PvVPE2 PvVPE 3 EcPME AnPME

....... A N F V A K N I G F E N T A G P E K . E Q A V A L R V . . S A D K A I I Y N C Q I D G ....... E G F M A K W I G F Q N T A G P L G . H Q A V A F R V . . N G D R A V I F N C R F D G ....... K G F I L Q D I C I Q N T A G P A K . H Q A V A L R V . . G A D K S V I N R C R I D A ....... D G F IC-QD I W F Q N T A G P Q K .H Q A V A L R V . . G A D Q S V I N R C R V D A ....... K G F I A K D I G F V N N A G A S K . H Q A V A L R S . . G S D R S V F F R C S F D G S L T I R N D F D F P A N Q A K S D S D S SK I K D T Q A V A L Y V T K S G D R A Y F K D V S L V G TLRNYAEEGSAIYNLNIANTCGQAC.HQALAVSA. .YASEQGYYACQFTG I

196 400 186 123 123 175 161

PPE 1 Bpl9 PEI PvVPE 2 PvVPE 3 ECPME AnPME

Y Q D T L Y V H T Y R Q F Y R D C T I T G T V D F I F G N G E A V L Q N .C K V I V R K P A Q N Q YQDTLYVNNGRQFYRNIWSGTVDFIFGKSATVIQN .S L I L C R K G P S G Q Y Q D T L Y A H S Q R Q F Y Q S S Y V T G T IDF I F G N A A W F Q K .C Q L V A R K P G K Y Q Y Q D T L Y A H T N R Q F Y R D S F I T G T V D F I F G N A A V V F Q K .C Y L V A R K P M S N Q F ~ D T L Y A H S N R Q F Y R D C D I TGT ID F IF G N A A V V F Q S . C K IM P R Q P L P N Q Y Q D T L Y V S G G R S F F S D C R I S G T V D F I F G D G T A L F N N .C D L V S R Y R A D V K Y O D T L L A E T G Y Q V Y A G T Y I E G A V D F I F G Q H A R A W F H E C D . I R V L F G .P S II IlI

244 448 234 171 171 223 208

PPEI Bp19 PE1 PvVPE 2 PvVPE 3 ECPME AnPME

SCMVTAQGRTEP. IQKGAIVLQNCEI KPDTDYFSLSPPSKT¥. LGRPWK T N H V T A D G N E K G K A V K I G I V L H N C R I M A D K E L E A D R L T V K S Y. L G R P W K Q N M Y T A Q G R T D P •N Q A T G T S I Q F C D I I A S P D L K P V V K E F P T Y . L G R P W K K N M V T A O G R E D P .N Q S T G T S I Q Q C N I T P S L D L K P V A G S I K T Y .L G R P W K F N T I T A Q G K K D P •N Q N T G I I I Q K S T I T P .... F G N N L T A P T Y .L G R P W K S G N V S G Y L T A P S .T N I N Q K Y G L V I T N S R . V I R E S D S V P A K S Y G L G R P W H SAS ITANGRSSE. SDDSYYVIHKSTV... AAADGNDVSSGTYYLGRPWS IV

291 495 279 218 214 270 253

Fig. 2. An alignment of the deduced amino acid sequences of PPE1 and other pectin esterases. Only the regions of the proteins that share sequence similarity are included in the alignment. Gaps are introduced to maximize similarity. Conserved residues are in bold; the 4 conserved sequence motifs previously identified [ 1] are underlined and labeled I to IV. Bpl9 is from Brassica napus [1]; PE1 from tomato [13]; E c P M E from Erwinia chrysanthemi [11]; A n P M E from Aspergillus niger [6]; PvVPE2 and PvVPE3 from green beans [ 14].

542 to which PPE1 shares sequence similarity. They are a fungal enzyme from Aspergillus niger [6], a bacterial enzyme from Erwinia chrysanthemi [ 11 ], and two bean enzymes [14]. The amino acid sequences of the regions of PPE1 and the other 6 pectin esterases that share sequence similarity are aligned in Fig. 2. Four conserved sequence motifs previously identified [1] are perfectly conserved in PPE1, suggesting that PPE1 is likely to be a pectin esterase. In the region compared, PPE1 is 48~o identical to BP19 of Brassica pollen [ 1] and 47~o identical to PE1 of tomato fruits [13]. Northern analysis revealed that PCPE22 c D N A hybridized strongly to a 1.7 kb m R N A species of pollen, in vitro germinated pollen tubes, and pistils of open flowers, but did not hybridize to any m R N A species of petals, leaves, or roots (Fig. 3A). The finding of PPE1 m R N A in pollen is unexpected considering that the screening strategy was to isolate pollen tube-specific

cDNAs. Perhaps, radiolabeled cDNAs for PPE 1 were underrepresented in the pollen c D N A probe used for screening. The level of the 1.7 kb m R N A detected in pistils of open flowers was ca. 1/10 that detected in pollen, and this m R N A species was not detected in pistils of immature flower buds (Fig. 3B). Presently, it cannot be ruled out that the low level of m R N A detected in the pistils of open flowers is due to pollen contamination of pistils. The temporal expression of the PPE1 gene during pollen development was also examined (Fig. 3B). The development of the anther was divided into 5 stages based on the size of the flower buds. PPE1 m R N A was first detected at a very low level in stage 2 anthers, which contain uninucleate microspores. The level increased ca. 5-fold in stage 3 anthers, which contain binucleate microspores, and continued to increase until reaching a maximum (ca. 20-fold over the level in

Fig. 3. Northern blot analysis of the expression o f P P E 1 gene in various tissues (A) and during pollen and pistil development (B). Total RNA (30/~g) from each tissue were used. Both blots were first hybridized with PCPE22 cDNA, and after autoradiography, the probe was removed and the blots rehybridized with a D N A probe encoding 25S rRNA of P. inflata. Floral development was divided into 5 stages based on size of flower buds, and the corresponding development of pollen was determined microscopically. Stage 1 buds (0.5 cm or less) contain microspores in tetrad configuration; stage 2 buds (0.5-1.0 cm) contain free uninucleate microspores; stage 3 (1.0-1.5 cm) and stage 4 (1.5-2.0 cm) buds contain binucleate microspores; stage 5 buds (2.0-2.5 cm) and open flowers (o.f.) contain mature pollen. The pistils were collected from small (S) buds (1.5 cm or less), large (L) buds (1.5 cm-2.5 cm) and open flowers (o.f.). Arrows indicate PPE1 mRNA.

543 stage 2 anthers) in anthers of open flowers, which contain mature pollen. The level ofPPE1 mRNA decreased slightly in in vitro germinated pollen tubes (Fig. 3A). However, its level remained nearly constant over a 16-hr period after germination commenced (results not shown). The pattern of expression of the PPEI gene is different from that of Bpl9 gene of Brassiea, the only other pollen-expressed pectin esterase gene reported. The B p l 9 message is detected in small buds containing tetrads and uninucleate microspores, and peaks in buds containing mostly binucleate microspores, but declines considerably in open flowers containing mature pollen [ 1]. On the contrary, the PPE1 message is first detected only after completion of microspore meiosis, and reaches the highest level in mature pollen. In addition, the PPE1 message persists at a high level in in vitro germinated pollen tubes. Based on the expression pattern, Bpl9 gene belongs to the class of 'early' pollen genes, and PPEI gene belongs to the class of 'late' pollen genes [9]. Thus, Bpl9 and PPE1 may have different physiological roles, perhaps with Bp 19 involved in microspore development and PPE1 involved in pollen germination and/or tube growth. If PPE1 is indeed a pectin esterase as its sequence predicts, then a possible function may be to act along with polygalacturonase [10, 12] and/or pectate lyase [15, 17] to degrade part of pectin-containing pollen inner walls to facilitate pollen germination. PPE1, again along with the latter two enzymes, may also aid the growth of pollen tubes by degrading pectins in the transmitting tissue of the style and then using the degradation products as building blocks for the synthesis of new pollen tube walls. These enzymes may also be required for reorganization of pollen tube walls to allow continuous extension. Study of the function of PPE1 may lead to a better understanding of the mechanism of pollen germination and tube growth.

Acknowledgements J.-H.M. was supported by a Third Country Graduate Fellowship from Pioneer Hi-Bred In-

ternational, Inc. J.P.S. was supported by a Research Experience for Undergraduates award from NSF. This work was supported by an N S F grant (IBN-9220145) to T.-h. K.

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