Molecular Characterization of Pear 1-Aminocyclopropane-1-Carboxylate Synthase Gene Preferentially Expressed in Leaves

1-Aminocyclopropane-1-carboxylate (ACC) synthase catalyzes the conversion of S-adenosyl-L-methionine to ACC in the ethylene biosynthetic pathway. Lots of important ACC synthase genes have been isolated and characterized from the plant kingdom. In this study, a cDNA clone encoding putative ACC synthase was isolated from a cDNA library produced using mRNA from pear (Pyrus pyrifolia). The cDNA clone, designated PpACS1 (GenBank accession No. JQ284383), comprised an open reading frame of 1, 341 bp encoding a protein of 446 amino acids that shares high similarity with the known plant ACSs. Using PCR amplification techniques, a genomic clone corresponding to PpACS1 was isolated and shown to contain two introns with typical GT/AG boundaries defining the splice junctions. The PpACS1 gene product shared 97% identity with an ACC synthase from pear (Pyrus communis), and phylogenetic analyses clearly placed the gene product in the ACC synthase cluster of the plant ACS superfamily tree. RT-PCR analysis indicated that the PpACS1 gene was preferentially expressed in pear leaves. The transcript of PpACS1 gene was accumulated at relatively high levels in anthers. The expression signal was detected in shoot at relatively low levels, but none signal was detected in developing fruit of pear. These results suggested that the PpACS1 may participate in the regulation of ethylene production in pear leaves and anthers.


Introduction
Ethylene is a plant hormone regulating a wide range of physiological processes such as germination, growth, development, and senescence (Klee and Tieman, 2002).As a senescing hormone, it promotes leaf-yellowing, flower and leaf abscission, climacteric fruit ripening (Yang and Hoffman, 1984).The production of ethylene in higher plants is from S-adenosyl-L-methionine (SAM) via the intermediate 1-aminocyclopropane-1-carboxylic acid (ACC).The conversion of SAM to ACC is catalysed by ACC synthase, and the subsequent oxidation of ACC to ethylene is catalysed by ACC oxidase (Yang and Hoffman, 1984;Ververidis and John, 1991).
In watermelon, both CitACS1 and CitACS3 are expressed in floral tissue.CitACS1 is also expressed in vegetative tissue and it may be involved in cell growth processes.The CitACS3 gene is expressed in open flowers and in young staminate floral buds (male or hermaphrodite), but not in female flowers.CitACS3 is also up-regulated by ACC, and is likely to be involved in ethylene-regulated anther development.The expression of CitACS2 was not detected in vegetative or reproductive organs (Salman-Minkov et al., 2008).Silencing of ACACS2 causes delayed flowering in pineapple (Trusov et al., 2006).The expression of AtACS1 is under developmental control both in shoot and root.High expression was observed in young tissues and was switched off in mature tissues (Rodrigues-Pousada et al., 1993).However, little is known so far about the expression of pear ACS genes during early development.Here, isolation and characterization of one pear ACS gene and its expression pattern during early and fruit development are reported.

Collection of Plant Materials
Pear (Pyrus pyrifolia Nakai cv.Whangkeumbae) fruit was harvested at 120, 150, and 180 day-old after full bloom from a local orchard.The other tissues (such as shoots, leaves, petals, and anthers) were derived from the same pear trees from the local orchard.These samples were frozen immediately in liquid nitrogen, and then stored at -80℃ for RNA isolation.

Construction of Pear cDNA Library and Isolation of PpACS1 cDNA
Total RNA was extracted from pear tissues including shoots, leaves, petals, anthers, and fruit.Poly(A) + mRNA was prepared from a pool of total RNA by using an mRNA purification kit (Qiagen).Complementary DNA was synthesized and cloned into the EcoR I-Xho I sites of the ZAP express vector and packaged using a ZAPcDNA Gigapack Gold III cloning kit (Stratagene) according to the manufacturer's instruction.
More than 3,000 cDNA clones were randomly selected from the pear cDNA library for sequencing.One PpACS clone with complete sequences was identified.The corresponding PpACS gene was amplified from the genomic DNA of pear by PCR, using Pfu DNA polymerase and gene-specific primers that were designed according to the sequence of PpACS cDNA.In total, one PpACS gene was obtained.

RNA Isolation and RT-PCR Analysis
Total RNA was isolated from shoots, leaves, petals, anthers, and developing fruit (120, 150, and 180 day-old after full bloom) of pear by the method described previously (Shi et al., 2009).A 2-4 g aliquot of each pear tissue was randomly collected from 3-10 plants for RNA isolation.The concentration and purity of total RNA were identified by NanoDrop spectrophotometry and agarose gel electrophoresis.RNA samples were stored at -80℃ until use.
Expression profiling of the PpACS1 gene in different pear tissues (such as shoots, leaves, petals, and anthers) and during different stages of fruit development (including 120, 150, and 180 day-old after full bloom fruit) was carried out by semi-quantitative RT-PCR.A pear polyubiquitin gene (PpUBI, GenBank accession no.AF195224) was used as a standard pear in the RT-PCRs.A two-step RT-PCR procedure was performed in the experiments using a previously described method (Shi et al., 2009).First, 2 µg of purified total RNA using Qiagen RNeasy Mini kit was reversely transcribed into cDNAs by M-MLV reverse transcriptase according to the manufacturer's instructions.The cDNAs were used as templates for RT-PCR reactions using gene-specific primers (PpACS1 P1: 5'-CAAAGATCTTGGCCTTCCAGGCTT-3'; P2: 5'-CAGAAAGAGTAAATTGGGTTTTGTCC-3').

Homology Modeling
The 3D models of the whole and domains of the deduced PpACS1 protein have been built using the 3D structure 1IAX chain 'A' as template (Lambert et al., 2002) and SWISS-MODEL Workspace.Figures were generated using rw32b2a (http://www.37c.com.cn/topic/004/netguide/zip/RasMol26.zip).

Isolation and Characterization of PpACS1 cDNA
To isolate the genes that might be involved in the regulation of ethylene production, 3, 000 cDNA clones from the pear (Pyrus pyrifolia) cDNA library were randomly sequenced.One ACS cDNA clone containing full length ACS cDNA sequence was identified from these clones, designated as PpACS1 (GenBank accession no: JQ284383), encoding polypeptides consisting of 446 amino acids (50.00 kD, pI 5.55).The putative PpACS1 protein include 44 strongly basic(+) amino acids (K,R), 56 strongly acidic(-) amino acids (D,E), 147 hydrophobic amino acids (A,I,L,F,W,V), and 127 polar amino acids (N,C,Q,S,T,Y).Sequence alignment revealed that all of the predicted proteins encoded by these genes contain highly conserved Aminotransferase class I and II superfamily domain (Fig. 1).

Structural Analysis of the PpACS1 Protein
Analysis of protein structure revealed that the predicted PpACS1 polypeptide contain the typical Aminotransferase class I and II domain and other conserved motifs (i.e.Aminotransferases class-I pyridoxal-phosphate attachment site, N-glycosylation site, cAMP-and cGMP-dependent protein kinase phosphorylation site).As shown in Fig. 1, the typical Aminotransferase class I and II domains (V 51 -I 435 ) and Aminotransferases class-I pyridoxal-phosphate attachment sites (S 280 -G 293 ) are highly conserved among the plant ACSs.Interestingly, three substitutions at amino acid level occurred in PpACS1 compared with other pear ACS proteins (Fig. 1).At position 74, the polar Ser was substituted by a polar Cys.At position 430, the hydrophobic Ile was substituted by a hydrophobic Val.The strongly basic (+) Lys at residues 338 was substituted by a polar Asn, suggesting that PpACS1 may have a different structure and function compared with other pear ACS variants.Through PpACS1 protein, there are only one N-glycosylation site (PGSs; N in NxS/T motifs, positions 422) and cAMP-and cGMP-dependent protein kinase phosphorylation site (R 307 RMS 310 ).Sequence comparison revealed that PpACS1 shares the highest similarity (97% identity) with PcACS2b (El-Sharkawy et al., 2004) and pPPACS2 (Itai et al., 1999).It shares relatively high homology (96% identity) with MdACS3a-1 and MdACS3a-2, but relatively low homology (83-95% identities) with pPPACS1 (Itai et al., 1999) and other known plant ACSs (Fig. 1).This conserved structure may be related to the maintenance of ACS functions for plant development.
To understand the protein structure in detail, we predicted the 3D structure of PpACS1 from ESyPred3D (Lambert et al., 2002).3D homology model of PpACS1 has been built by using the 3D structure 1IAX chain 'A' as a template.We also used it to build the 3D structure of Aminotransferase class I and II domain.The whole modeling (Fig. 2A/B) is restricted to residues 1-446 of PpACS1, and shares 54.0% identity with the template sequence (using the ALIGN program).3D model of Aminotransferase class I and II domain (V 51 -I 435 ) (Fig. 2C/D) has been built using the 3D structure 1IAX chain 'A' as the template.It shares 50.9% identity with the template sequence.

Phylogenetic Relationship of PpACS Protein
To investigate the evolutionary relationships of the pear ACS proteins with apple and Arabidopsis ACS proteins, all of the known pear ACSs, apple ACSs, and Arabidopsis ACSs were selected from GenBank for phylogenetic analysis.The ACS proteins can be divided into two groups.As shown in Fig. 3, these proteins obviously split into two subgroups.PpACS1, PcACS2b (El-Sharkawy et al., 2004), PcACS2a (El-Sharkawy et al., 2004), pPPACS2 (Itai et al., 1999), MdACS3a-2, MdACS3a-1, MdACS3c (Wang et al., 2009), PcACS3a, MdACS3b (Wang et al., 2009) and AtACS7 (Mayer et al., 1999) form one subgroup, while PbACS1B, pPPACS1 (Itai et al., 1999) and PcACS1b (El-Sharkawy et al., 2004) together with the Arabidopsis ACS8 are located in the second branch of the tree.These results suggest that the divergence could have occurred before the differentiation of the plants.PpACS1 is located on the first subgroup of the tree, which displays that PpACS1 has the closest evolutional relationship with PcACS2b (El-Sharkawy et al., 2004), and relatively close relationship with PcACS2a (El-Sharkawy et al., 2004).Whereas the four ACSs, including AtACS8 (Mayer et al., 1999), PbACS1B, pPPACS1 (Itai et al., 1999) andPcACS1b (El-Sharkawy et al., 2004), occupy another branch of the tree, which suggests that PpACS1 might diverge earlier from these ACSs during evolution.

Isolation and Characterization of PpACS1 Gene
One PpACS gene was amplified from the pear (P.pyrifolia) genome by PCR, using gene-specific primers.Sequence comparison between the cDNA and its corresponding gene revealed that PpACS1 gene contains two introns splitting their ORF into three exons.The first intron is located within codons encoding valine, and the second intron is inserted within codons encoding phenylalanine in the gene (Fig. 4).

PpACS1 Gene is Preferentially Expressed in Leaves
To analyse the expression patterns of the isolated PpACS1 gene, mRNA levels of the gene in pear tissues were quantified by RT-PCR using gene-specific primers.As shown in Fig. 5, PpACS1 showed leaf-preferential expression pattern.PpACS1 was preferentially expressed in leaves, and moderate expression was found in anthers, but relatively weak signals were detected in shoots of pear.Little expression signal was detected in petals and developing fruit in pear.

Discussion
Pear and apple ACS genes are highly homologous throughout the protein coding regions but do show a degree of sequence divergence within the 3' untranslated regions.The data presented in Fig. 1 revealed that pear ACS proteins contain the conserved structure, Aminotransferase class I and II domain and Aminotransferases class-I pyridoxal-phosphate attachment site, like all the other ACS proteins.The structure of the Plant 1-aminocyclopropane-1-carboxylate (ACC) synthase genes has been studied.For example, the pear ACC synthase gene has three exons and two introns, with all of the boundaries between these introns and exons sharing a consensus dinucleotide sequence of GT-AG.
Previous studies revealed that ACS genes display their tissue-specific or preferential expression in plants.PnACS from Pharbitis nil was expressed in cotyledons, petioles, hypocotyls, root and shoot apexes both in light-and dark-grown seedlings.The highest expression level of PnACS was found in the roots.The expression of PnACS is up-regulated in all the organs examined under IAA applied to the cotyledons of P. nil seedlings.In this case, the most IAA-responsive were the hypocotyls (Frankowski et al., 2009).Likewise, our results also revealed that PpACS1 gene was predominantly expressed in leaves, moderate expression was found in anthers and shoots, but little signals were detected in developing fruit (Fig. 5), suggesting that it may be involved in early growth and development of pear.

Figure 1 .
Figure 1.Sequence alignment among the pear and apple ACS proteins

Figure 5 .
Figure 5. RT-PCR analysis of expression of PpACS1 gene in pear tissuesPpUBI was amplified as a control.120d, 150d, and 180d refer to 120, 150, and 180 day-old after full bloom fruit.