Involvement of Both Subgroups A and B of Expansin Genes in Kiwifruit Fruit Ripening

in HortScience

Two complementary DNA fragments encoding expansin genes Ad-EXP1 and Ad-EXP2 were isolated from ripening kiwifruit (Actinidia deliciosa cv. Bruno) by reverse transcription–polymerase chain reaction amplification using a pair of degenerate primers. The homology between these two expansin family members was 50% in nucleotide sequence and 74% in amino acid sequence. It was revealed that Ad-EXP1 and Ad-EXP2 belong to subgroups A and B of an expansin gene family respectively. However, gene expression of these two members shared similar patterns. Both were upregulated by ethylene treatment and downregulated by acetylsalicylic acid treatment. The study suggests that members of both subgroups A and B of the expansin family are involved in kiwifruit fruit ripening.

Abstract

Two complementary DNA fragments encoding expansin genes Ad-EXP1 and Ad-EXP2 were isolated from ripening kiwifruit (Actinidia deliciosa cv. Bruno) by reverse transcription–polymerase chain reaction amplification using a pair of degenerate primers. The homology between these two expansin family members was 50% in nucleotide sequence and 74% in amino acid sequence. It was revealed that Ad-EXP1 and Ad-EXP2 belong to subgroups A and B of an expansin gene family respectively. However, gene expression of these two members shared similar patterns. Both were upregulated by ethylene treatment and downregulated by acetylsalicylic acid treatment. The study suggests that members of both subgroups A and B of the expansin family are involved in kiwifruit fruit ripening.

Fruit textural change is one of main processes that occur during fruit ripening and senescence. It affects the postharvest life of fruit as well as fruit quality and commercial value. Softening is a comprehensive result of complex primary cell wall disassembly and the disruption of adhesion between cells. Various enzymes, such as polygalacturonase (Bonghi et al., 1996), pectin esterase (Redgwell and Harker, 1995), cellulase (Gallego and Zarra, 1997), xyloglucan endotransglycosylase (Chen et al., 1999; Redgwell and Fry, 1993), and β-galactosidase (Redgwell and Harker, 1995; Ross et al., 1993) contributes to degradation of cell wall substance in kiwifruit as well as in other fruits (Bennett, 2002; Cosgrove, 2000; Rose and Bennett, 1999). However, these enzymes might not be the only important factors participating in the disassembly of the cell wall. Antisense inhibition of their expression in some fruit did not greatly retard fruit ripening (Giovannoni et al., 1989; Tieman and Handa, 1994; Tieman et al., 1992; Watson et al., 1994).

Recently, expansin (EXP), a nonenzymatic protein, was found to play an important role in cell wall loosening and extension. Expansin was first discovered while researching the “acid phenomenon” (McQueen-Mason et al., 1992). It is encoded by a large gene family with members that are extensively involved in plant growth, organ swelling, pollen tube elongation, and fruit ripening (Cosgrove, 2000). Some members express constitutively, whereas others each had individual tissue-specific or/and developmental-specific expression pattern (Cosgrove et al., 1997; Fleming et al., 1997; Im et al., 2000), although more than one EXP member had been reported to coexpress in a same tissue (Harrison et al., 2001).

In tomato, 18 EXP members have been identified. Le-EXP1 expressed specifically in the ripening fruit (Rose et al., 1997). Its important role in ripening of tomato fruit was noted by Brummell et al. (2002). Overexpression of Le-EXP1 accelerated fruit softening, whereas antisense inhibition of Le-EXP1 retarded it in transgenic tomato. Ripening-related members of the expansin family were also identified in peach (Hayama et al., 2000, 2003), strawberry (Harrison et al., 2001), pear (Hiwasa et al., 2003), litchi (Wang et al., 2006a), and banana (Wang et al., 2006b). Expression of ripening-related expansin was induced by ethylene and inhibited by 1-methylcyclopropene, a gaseous ethylene binding inhibitor (Rose et al., 1997, 2000). In addition, salicylic acid (SA), an endogenous plant growth substance regulating plant development and senescence, and its derivative acetylsalicylic acid (ASA) have been reported to inhibit ethylene production in fruit of kiwifruit, pear, banana, and apple (Fan et al., 1998; Leslie and Romani, 1988; Zhang et al., 2003). However, a relation between EXP expression and SA has not been reported. The objective of the current study was to isolate fruit ripening-related EXP members from kiwifruit and evaluate their response to ethylene and ASA.

Materials and Methods

Plant material.

Kiwifruit (Actinidia deliciosa cv. Bruno) fruit were harvested at commercial maturity with total soluble solids at around 7 ºBrix from an orchard at Wuyi, Zhejiang, China. They were transferred to the laboratory on the same day, where fruit of uniform size, maturity, and without disease and mechanical damage were selected and subjected to storage with or without treatments.

Treatments and sampling.

After precooling at 15 ºC for 2 h, the fruit were exposed to ethylene (100 μL·L−1, 12 h), stored at 20 ºC, and sampled at 0, 3, 6, 12, 24, 72, 120, and 156 h after treatment. For the last sampling, the fruit were sorted into four groups according to the rate of ethylene production as described later in the text. In another separate experiment, fruit were separated into three groups after precooling at 15 ºC for 2 h and exposed to ethylene (100 μL·L−1, 12 h), immersed in ASA (1 mm; pH, 3.5; 5 min), or not treated (control). These fruit were then stored at 20 ºC, sampled at –12 h (refers to the time point just before treatment), 0, 24, 48, 72, 96, 120, 144, 168, 216, and 264 h after treatment. Each sampling consisted of three replicates of 10 sample fruit. After measuring the rate of ethylene production, fruit firmness, and total soluble solids content, the pulp tissue without seeds from a core at an equatorial slice (≈2.5 cm thick) from each fruit was taken, combined, frozen in liquid nitrogen, and stored at –70 ºC until analysis.

Analysis methods.

Rates of ethylene production, fruit firmness, and total soluble solids were measured as described by Zhang et al. (2003).

Total RNA was extracted according to our previously published protocol (Xu et al., 2004). Degenerate sense primer EXPSP, 5′-ACAATGGGNGGDGCDTGTGG-3′ (D = A/G/T, N = A/C/G/T) and antisense primer EXPAP, 5′-TGCCARTTYTGNCCCCARTT-3′ (R = A/G, Y = T/C) were designed according to the conserved regions of plant expansin sequences deposited in GenBank with accession numbers AF038815, AF096776, AF297527, AB093029, and AB029083. Reverse transcription and polymerase chain reaction (PCR) amplification was performed according to Xu et al. (2004) with an annealing temperature of 50 ºC. The 3′ end of the fragment was amplified with a 3′ RACE kit (Takara, Dalian, China) according to the instructions of the supplier. The reaction products were analyzed on 1.0% agarose gel containing 0.5 μg·mL−1 ethidium bromide. The PCR products were cloned into pUCm-T (Shanghai Sangon, Shanghai, China) according to Sambrook and Russell (2001). Sequencing was completed by Shanghai Sangon. The sequences were aligned with Clustal X 1.81 (Institut de Genetiave et de Biologie Moleculaire et cellvlaire (CNRS/INSERM/VLP), Illkirch Cedex, France) and BioEdit 5.0.9, (Toln Hall, Ibis Therapeutics, Carlsbad, Calif.) and phylogenetic analysis was conducted with DNAMAN 5.1 (Lynnon Corp., Quebec).

The probe for Ad-EXP1 was generated using the Random Primed DNA Labeling Kit (Takara, Dalian, China), and Northern blot was performed according to Chen et al. (1999). The region of Ad-EXP1 for probe preparation was between EXPSP and EXPAP.

Two oligonucleotide primer sets used for real-time PCR analysis were designed according to the sequences of 3′-untranslated regions (UTR) of two individual members obtained in the current study using software Primer Premier 5.0 (Premier Biosoft Intl., Palo Alto, Calif.). The primers for member 1 (Ad-EXP1) and member 2 (Ad-EXP2) were 5′-TCGTTGGGGAATGTGAAA-3′ (ADEXP1SP, sense), 5′-TCGAAGAGCTGCGGGCTA-3′ (ADEXP1AP, antisense), 5′-TCATTCCAAGGCCCCATT-3′ (ADEXP2SP, sense), and 5′-AAGCACCTAAAACCAAAA-3′ (ADEXP2AP, antisense) respectively. Real-time PCR was performed using the iCycler iQ real-time PCR instrument (BioRad, Hercules, Calif.) in a total volume of 20 μL containing 2 μL complementary DNAs (cDNAs), 250 μm each primer, and 10 μL of 2× SYBR Green PCR Master Mix (BioRad). The predenaturation step was 10 min at 94 ºC, followed by 40 cycles of 94 ºC for 15 s and 60 ºC for 1 min. Negative controls without template for each primer pair were included in each run. Actin was selected as the internal control to normalize the differences in the amount of templates. The real-time PCR regime for actin was the same as described earlier, except that the primers were 5′-TGCATGAGCGATCAAGTTTCAAG-3′ (ACTSP, sense) and 5′-TGTCCCATGTCTGGTTGATGACT-3′ (ACTAP, antisense). Real-time PCR data analysis was performed according to Zhang et al. (2006). Quantification of gene expression was repeated three times from RNA extraction with three replicates in each repeat. The magnitude of expansin expression was expressed as x-fold multiples of 18 S recombinant RNA in Northern blot and of actin in real-time PCR.

Results and Discussion

Molecular characterization of kiwifruit expansin genes.

A fragment of expected size (≈500 bp) was amplified by PCR with cDNA reverse transcripted from pulp tissue of ripening kiwifruit as a template and EXPSP and EXPAP as primers. The fragment was cloned into a pUCm-T vector and sequenced. Two different sequences with 497 bp and 491 bp in length, respectively, were obtained, and both were highly homologous to plant EXP gene members. Sequences of 3′ ends of both members, 460 bp and 398 bp in length respectively, were obtained using the RACE kit. In combination, two cDNA fragments, designated as Ad-EXP1 (957 bp, GenBank accession number AY390358) and Ad-EXP2 (889 bp, accession number DQ915940), were obtained with full C-terminal but partial N-terminal protein sequences of 211 and 207 amino acids respectively. The homology between Ad-EXP1 and Ad-EXP2 was 50% in nucleotide sequence and 74% in amino acid sequence. Alignment of Ad-EXP1 and Ad-EXP2 amino acid sequences revealed common characteristics of plant EXP gene [e.g., conserved Cys (C) residues, Trp (W) residues, and His-Phe-Asp (HFD) domain (Cosgrove, 2000) as indicated in Fig. 1].

Fig. 1.
Fig. 1.

The deduced amino acid sequences of Ad-EXP1 and Ad-EXP2 fragments. Carboxy-terminal conserved amino acid domains, KNFRV unique to subgroup A and QF to B, are framed. Conserved cystein (C), tryptophan (W) residues, and a His-Phe-Asp (HFD) domain, characterized by plant expansins (EXPs), are in black boxes.

Citation: HortScience horts 42, 2; 10.21273/HORTSCI.42.2.315

Plant EXPs are encoded by a large superfamily that includes EXP (α-expansin), EXPB (β-expansin), EXPL (expansin liked), and EXPR (expansin related) (Cosgrove et al., 2002). Phylogenetic analysis of EXP members suggested that it includes at least four subgroups: A, B, C, and D (Gray-Mitsumune et al., 2004). In the current study, sequence alignment was done with 20 EXP amino acids sequences from seven plant species and a Hordeum vulgare β-expansin (Hv-EXPB1) as an outgroup. The results showed that Ad-EXP1 clustered with subgroup A expansin such as At-EXP6, whereas Ad-EXP2 clustered with subgroup B expansin such as At-EXP2 (Link and Cosgrove, 1998) (Fig. 2). Moreover, Fig. 1 showed that Ad-EXP1 and Ad-EXP2 had KNFRV and QF C-terminal amino acid sequences, which are unique to subgroups A and B of the plant EXP gene family (Gray-Mitsumune et al., 2004; Link and Cosgrove, 1998) respectively.

Fig. 2.
Fig. 2.

Phylogenetic tree based on deduced amino acid sequences of Ad-EXP1, Ad-EXP2 and other plant α-expansins with Hv-EXPB1 as an outgroup. Phylogenetic distances were indicated between branches. The plants applied are At (Arabidopsis thaliana), Le (Lycopersicon esculentum), Pa (Prunus armeniaca), Pc (Prunus cerasus), Pp (Prunus persica), Os (Oryza sativa), and Hv (Hordeum vulgare).

Citation: HortScience horts 42, 2; 10.21273/HORTSCI.42.2.315

Different EXP members were reported to be involved in various physiological processes such as seed germination, hypocotyl expansion, fruit development, and ripening (Chen et al., 2001). Members of subgroup A, such as Le-EXP1 in tomato, are mainly involved in fruit ripening (Gray-Mitsumune et al., 2004; Rose et al., 1997). Members of subgroup B were mostly related to rapid cell expansion (Gray-Mitsumune et al., 2004; Orford and Timmis, 1998). However, some members of this subgroup (e.g., Pa-EXP1 and Pa-EXP2 of apricot) were recently found to be expressed in ripening apricot fruit (Gray-Mitsumune et al., 2004; Mbéguié-A-Mbéguié et al., 2002). In addition, EXP members from different subgroups were also reported to participate in one specific process. For example, both Le-EXP18 of subgroup A and Le-EXP2 of subgroup B were involved in hypocotyl expansion (Chen et al., 2001). In the current study, both Ad-EXP1 and Ad-EXP2 were expressed in the ripening kiwifruit.

Expression patterns of Ad-EXP1 and Ad-EXP2 during fruit ripening.

Two techniques, Northern blot and real-time PCR, were applied to study the expression patterns of these two EXP members during fruit ripening. For Northern blot, a probe corresponding to the region of Ad-EXP1 between EXPSP and EXPAP was prepared. The probe sequence was only 70% homologous to Ad-EXP2, which successfully prevented a cross-hybridization signal from Ad-EXP2 (data not shown). For real-time PCR, the primer sets were designed according to the sequence of 3′-UTRs of two individual members (Fig. 3). Polymerase chain reaction amplification produced 142 bp and 198 bp fragments for Ad-EXP1 and Ad-EXP2 respectively. The authenticity of fragments was verified by cloning into the pUCm-T vector and then sequencing. Because there were significant sequence differences between two members where the primers were designed, no cross-PCR signal was observed (data not shown).

Fig. 3.
Fig. 3.

Comparison of partial 3′- untranslated region sequences of Ad-EXP1 and Ad-EXP2 showing framed regions for designed oligonucleotide primers for real-time polymerase chain reaction analysis.

Citation: HortScience horts 42, 2; 10.21273/HORTSCI.42.2.315

Fruit ripening and softening of kiwifruit were accompanied with rapid loss of fruit firmness and then climacteric ethylene production (Fig. 4A, B), which is consistent with previous studies (Chen et al., 1999; Wang et al., 2000; Zhang et al., 2003). Fruit at 156 h after ethylene treatment produced ethylene at a nonuniform rate with a range over 150-fold, although fruit firmness was similarly low (Fig. 4B). To evaluate the relationship between ethylene production and expression of Ad-EXP1, the fruit were separated, according to the ethylene production rate of individual fruit, into trace (T, <1.0 nL·g−1·h−1 ethylene), low (L, 1.0–5.0 nL·g−1·h−1 ethylene), medium (M, 5–20 nL·g−1·h−1 ethylene), and high (H, >20 nL·g−1·h−1 ethylene) groups. Expression of Ad-EXP1 increased by 6.5-fold within 6 h after treatment, which was much earlier than the marked dramatic increase in the rate of ethylene production (Fig. 4B, 4C). Ad-EXP1 in fruit of T, L, M, and H groups showed similar expression magnitudes (Fig. 4C). Ad-EXP1 responded to ethylene quickly, but the increased rate of ethylene production during the late softening stage, 72 h after treatment and later, did not result in corresponding increments in the expression of Ad-EXP1 (Fig. 4B, C).

Fig. 4.
Fig. 4.

(A–C) Changes in fruit firmness (A), ethylene production (B), and Ad-EXP1 expression (C) in ethylene-treated Actinidia deliciosa fruit. Ad-EXP1 expression was expressed as x-fold of 18 S recombinant RNA in Northern blot. T, trace ethylene production rates (<1.0 nL·g−1·h−1); L, low ethylene production rates (1.0–5.0 nL·g−1·h−1); M, medium ethylene production rates (5–20 nL·g−1·h−1); H, high ethylene production rates (>20 nL·g−1·h−1).

Citation: HortScience horts 42, 2; 10.21273/HORTSCI.42.2.315

Postharvest changes in firmness and ethylene production of ethylene-treated fruit were confirmed in a separate experiment (Fig. 5A, B). Similar to the preceding experiment (Fig. 4B), no significant increase in endogenous ethylene production rate within 24 h after exogenous ethylene treatment was observed (Fig. 5B). Ethylene production in ethylene-treated fruit was significantly higher than the control at 72 h and peaked at 216 h after treatment (Fig. 5B). The expression pattern of Ad-EXP1 during fruit softening in ethylene-treated fruit, revealed by real-time PCR analysis as shown in Fig. 5C, was consistent with that observed by Northern blot, as shown in Fig. 4C. Expression of Ad-EXP1 was enhanced several times by ethylene at 216 h after treatment. The expression pattern of Ad-EXP2 was similar to that of Ad-EXP1, although the expression magnitude was somewhat lower (Fig. 5C, D). Ethylene treatment enhanced expression of EXP members, either ripening or other related developmental process, is supported by previous work (Hayama et al., 2000; Rose et al., 1997).

Fig. 5.
Fig. 5.

(A–D) Changes in fruit firmness (A), ethylene production (B), expression of Ad-EXP1 (C), and expression of Ad-EXP2 (D) during fruit ripening of Actinidia deliciosa after ethylene or acetylsalicylic acid treatment. The expression magnitude of expansins was expressed as x-fold of actin in real-time polymerase chain reaction.

Citation: HortScience horts 42, 2; 10.21273/HORTSCI.42.2.315

Salicylic acid and ASA have been reported to delay fruit ripening and inhibit ethylene production in kiwifruit, pear, apple, and banana fruits (Fan et al., 1998; Leslie and Romani, 1988; Zhang et al., 2003). This observation for ASA has been confirmed in the current study. The strongest inhibitory effect of ASA on fruit softening was observed at 24 h after treatment (Fig. 5A). However, at this sampling time point, the rate of ethylene production was not obviously affected (Fig. 5B). Nonetheless, expression of both Ad-EXP1 and Ad-EXP2 were reduced by more than 50% (Fig. 5C–D). The data suggest that ASA might exert its effect on retarding fruit softening by inhibiting the expression of EXPs. The interactions between ASA, ethylene, and expression of EXPs merit further investigation.

In conclusion, two expansin cDNAs fragments, Ad-EXP1 and Ad-EXP2, were isolated from kiwifruit, and their expression patterns were characterized during fruit ripening and after either ethylene or ASA treatment. Phylogenetic analysis showed that these two expansin family members belong to subgroup A and subgroup B respectively. Expression patterns of both members were similar, although Ad-EXP1 showed higher messenger RNA (mRNA) abundance than Ad-EXP2. Both Ad-EXP1 and Ad-EXP2 mRNA accumulated during fruit ripening of kiwifruit, and were upregulated by ethylene and downregulated by ASA. The results suggest that both subgroup A and subgroup B expansins are involved in kiwifruit fruit ripening and softening.

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Contributor Notes

Supported by the National Natural Science Foundation of China (30571284), the University Doctoral Foundation of China (20040335022), and the 111 project (B06014).

To whom reprint requests should be addressed; e-mail akun@zju.edu.cn

  • View in gallery

    The deduced amino acid sequences of Ad-EXP1 and Ad-EXP2 fragments. Carboxy-terminal conserved amino acid domains, KNFRV unique to subgroup A and QF to B, are framed. Conserved cystein (C), tryptophan (W) residues, and a His-Phe-Asp (HFD) domain, characterized by plant expansins (EXPs), are in black boxes.

  • View in gallery

    Phylogenetic tree based on deduced amino acid sequences of Ad-EXP1, Ad-EXP2 and other plant α-expansins with Hv-EXPB1 as an outgroup. Phylogenetic distances were indicated between branches. The plants applied are At (Arabidopsis thaliana), Le (Lycopersicon esculentum), Pa (Prunus armeniaca), Pc (Prunus cerasus), Pp (Prunus persica), Os (Oryza sativa), and Hv (Hordeum vulgare).

  • View in gallery

    Comparison of partial 3′- untranslated region sequences of Ad-EXP1 and Ad-EXP2 showing framed regions for designed oligonucleotide primers for real-time polymerase chain reaction analysis.

  • View in gallery

    (A–C) Changes in fruit firmness (A), ethylene production (B), and Ad-EXP1 expression (C) in ethylene-treated Actinidia deliciosa fruit. Ad-EXP1 expression was expressed as x-fold of 18 S recombinant RNA in Northern blot. T, trace ethylene production rates (<1.0 nL·g−1·h−1); L, low ethylene production rates (1.0–5.0 nL·g−1·h−1); M, medium ethylene production rates (5–20 nL·g−1·h−1); H, high ethylene production rates (>20 nL·g−1·h−1).

  • View in gallery

    (A–D) Changes in fruit firmness (A), ethylene production (B), expression of Ad-EXP1 (C), and expression of Ad-EXP2 (D) during fruit ripening of Actinidia deliciosa after ethylene or acetylsalicylic acid treatment. The expression magnitude of expansins was expressed as x-fold of actin in real-time polymerase chain reaction.

  • BennettA.B.2002Biochemical and genetic determinants of cell wall disassembly in ripening fruit: A general modelHortScience37447450

  • BonghiC.PagniS.VidrihR.RaminaA.TonuttiP.1996Cell wall hydrolases and amylase in kiwifruit softeningPostharvest Biol. Technol.91929

  • BrummellD.A.HowieW.J.MaC.DunsmuirP.2002Postharvest fruit quality of transgenic tomatoes suppressed in expression of a ripening-related expansinPostharvest Biol. Technol.25209220

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