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Ethylene induced cotton leaf abscission is associated with higher expression of cellulase (GhCel1) and increased activities of ethylene biosynthesis enzymes in abscission zone
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Plant Physiology and Biochemistry 46 (2008) 54e63 www.elsevier.com/locate/plaphy
Research article
Ethylene induced cotton leaf abscission is associated with higher expression of cellulase (GhCel1) and increased activities of ethylene biosynthesis enzymes in abscission zone Amita Mishra, Smriti Khare, Prabodh Kumar Trivedi, Pravendra Nath* Plant Gene Expression Laboratory, National Botanical Research Institute, Rana Pratap Marg, Lucknow, UP 226 001, India Received 14 February 2007 Available online 20 September 2007
Abstract Ethylene induced cotton (Gossypium hirusutum var RST-39) leaf abscission has been characterized by measuring the activities of ACC synthase (ACS, E.C. 4.4.1.14), ACC oxidase (ACO, E.C. 1.14.17.4) and cellulase (E.C. 3.2.1.4). In addition, a leaf abscission specific cDNA (GhCel1) has been cloned from cotton, which belongs to the a2 subgroup of cellulases that possess a C-terminus carbohydrate-binding domain. Measurement of enzyme activity in the abscission zones of cotton leaf explants exposed to ethylene for 48 h compared to non-treated controls indicated a more than 5-fold increase in the activity of ACS, 1.2-fold increase in the activity of ACO and about 2.7-fold increase in the activity of cellulase in the ethylene treated explants. This increase was accompanied by a substantial decrease in the force required to separate the petiole from the stem (break strength) and an increased accumulation of cellulase transcript in the abscission zone. Treatment of explants with 1-Methylcyclopropene (1-MCP) prior to ethylene resulted in significant inhibition of enzyme activities and transcript accumulation. It is concluded that ethylene response of cotton leaf abscission leads to higher cellulase expression and increased activities of ethylene biosynthesis enzymes in the abscission zone. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: 1-Methylcyclopropene; ACC oxidase; ACC synthase; Cellulase; Ethylene; Gossypium hirusutum; Leaf abscission
1. Introduction Abscission is the process by which plants shed organs from the parent body in response to developmental cues or to adapt to various environmental stresses including pathogen attack [26,31]. The signal that promotes abscission has been widely recognized as the gaseous plant hormone ethylene [1,37]. The role of ethylene in abscission was deduced by the use of chemical agents such as norbornadiene, silver ions or 1-MCP Abbreviations: 1-MCP, 1-Methylcyclopropene; ACC oxidase, 1-aminocyclopropane-1-carboxylate oxidase; ACC synthase, 1-aminocyclopropane1-carboxylate synthase; LAZ, leaf abscission zone. * Corresponding author: Plant Gene Expression Laboratory, National Botanical Research Institute, Rana Pratap Marg, P.O. Box 436, Lucknow, UP 226 001, India. Tel.: þ91 522 220 5841; fax: þ91 522 220 5836/5839. E-mail address: [email protected] (P. Nath). 0981-9428/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2007.09.002
that block ethylene action [10,40,41] or mutants, which are deficient in ethylene perception and response [7]. Organ separation is accomplished by degradation of the cell wall and middle lamella to form a fracture plane between the abscising organ and the parent plant. Although the precise sequence of events that bring about cell wall degradation is unclear, biochemical and molecular studies indicate that an increase in the activities of hydrolytic enzymes such as glucanases [5,38] and polygalacturanases [14,18] and proteins such as expansins [4] play a major role in the process of abscission. An increase in cellulase (endo-1,4-b-D-glucanase) activity has been noted in leaf abscission zones (LAZ) [20,40], fruit abscission zones [15,17], ripening fruits [24] senescing styles and anthers [34] and during adventitious root initiation [23]. The isoelectric points (pIs) of many of these cellulases differ from each other, which suggest that each may have distinct
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cellular function. Although cellulases have been identified and cloned from the abscission zones of several different sources, information relating to its role in leaf abscission is limited [31]. In the present study we have investigated petiole abscission in cotton using biochemical and molecular tools. The activities of cellulase and ethylene biosynthesis enzymes have been measured in LAZ and correlated with the progression of petiole separation. A leaf abscission specific cDNA has been cloned and characterized for the first time from cotton and accumulation of mRNA in abscission zones studied in the presence and absence of 1-MCP, an ethylene action inhibitor. The results of this study gain significance due to commercial importance of cotton leaf abscission. 2. Materials and methods 2.1. Plant material and treatments Seeds of cotton (Gossypium hirusutum var RST-39) were germinated in glasshouse under controlled temperature (32 3 C) for 12e15 days until the primary leaves were fully expanded and the secondary pinnate leaf was just beginning to open. At this time, the leaf blades for the primary leaves were removed and plants harvested by cutting 1 cm above the soil. Twenty-five explants, each of 8e10 cm long, were surface sterilized using bleach (10% v/v) for 5 min, washed 3e4 times with distilled water and kept in a beaker containing water and exposed to 10 mL/L ethylene in a 10 liter air tight desiccators for the time intervals indicated in the figure legends. Desiccators were opened every 4 h, flushed with air to remove CO2 accumulation and maintain O2 concentrations and 10 mL/L ethylene reintroduced fresh each time. For 1-MCP treatment, explants were exposed to 10 mL/L 1-MCP (EthylBlock, BioTechnologies for Horticulture, Inc., Waterboro, USA) for 1 h before ethylene exposure. For 1-MCP application, 1 ml water was added to 1-MCP powder kept in a 10 ml beaker and placed towards sidewall of the desiccators containing explants. Desiccators were closed immediately (within 2e3 s) to prevent diffusion of 1-MCP outside the desiccators. The concentration of 1-MCP was calculated as per manufacturers instructions. LAZs (1e2 mm on either side of fracture plane) were harvested and frozen in liquid nitrogen and stored at 70 C till further use. Other vegetative tissues were also collected after exposure to ethylene for 24e36 h. 2.2. Break strength measurement In order to determine the break strength, the petiolar stump of the explant was clamped using an alligator clip to which a string was attached for holding weight. Increasing weights were loaded on the string till the petiolar stump detached at the abscission zone. The distance of the explant from bench top as well as length and angle of the string were kept constant during each measurement to avoid any variations in the force applied. Total weight applied on the petiolar stump was determined and break strength of the abscission zone calculated as force in gram equivalents required to detach the petiole. Break
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strength values determined for each time point represented an average of at least 20 or more individual measurements. 2.3. Enzyme activity measurements For ACS activity measurements, LAZs (200 mg) were homogenized in extraction buffer consisting of 100 mM HepesKOH, pH 8.5, 4 mM DTT, 10 mM pyridoxal phosphate and 5% PVP 40,000 as described by Kato et al. [19]. The homogenate was centrifuged at 12,000 g for 20 min at 4 C. The supernatant was desalted using Sephadex G-25 and eluate used for enzyme assay as described by Pathak et al. [29]. ACS activity was expressed as nmoles ACC formed h1 g1 FW. ACO enzyme was extracted according to Moya-Leon and Jhon [27]. A total of 200 mg frozen tissue was homogenized in 1 ml of extraction buffer consisting of 0.1 M TriseHCl, pH 7.5, 10% glycerol, 2 mM DTT and 30 mM sodium acetate. The homogenate was centrifuged at 12,000 g for 20 min at 4 C. Further purification of supernatant and enzyme assay was carried out as described by Pathak et al. [29]. ACO activity was expressed as nmoles C2H4 produced h1 g1 FW. Cellulase activity was measured in 1 g of abscission zones homogenised in 3 ml of 0.1 M sod phosphate buffer (pH 7.8) containing 2 mM EDTA, 0.1 mM DTT and 0.2% Triton X100. The homogenate was transferred to eppendorf tubes on ice and 0.5 M EDTA (1/500 vol) and 5 M NaCl (1/10 vol) added, mixed thoroughly and kept on ice for 30 min. The homogenate was centrifuged at 15,000 g for 30 min at 4 C. The pellet was discarded and supernatant was used as enzyme preparation for the cellulase assay. The assay mixture contained 0.5 ml enzyme and 0.5 ml 1.3% (w/v) carboxy methyl cellulose (CMC) prepared in 0.02 M TriseHCl pH 8.0. Drainage time of assay mixture through a calibrated portion of 100 ml pipette was used as a measure of viscosity. Viscosity of the mixture was measured at an interval of 30 min from 0 to 2 h at room temperature. Viscosity data was converted to intrinsic viscosity and relative units of activity calculated as described by Durbin and Lewis [12]. Results were expressed as Relative activity h1 g1 FW. 2.4. Scanning electron microscopy For electron microscopy, abscission zones were fixed in 4% (v/v) gluteraldehyde in 0.5 M potassium phosphate buffer (pH 7.4) for 4 h at 25 C, rinsed four times in buffer, and then dehydrated in a graded ethanol series. Sputter coated sections were examined at different magnifications with Philips XL20 scanning electron microscope (Philips, Holond). 2.5. Cloning of cellulase gene and nucleotide sequence analysis Total RNA from abscission zones and other vegetative tissues was isolated according to Asif et al. [3]. In order to clone cellulase cDNA, RT-PCR was carried out using total RNA from 24 h ethylene treated abscission zones. Oligonucleotide primer pairs were designed from regions having maximum sequence
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identity amongst known cellulase nucleotide sequences. Partial cDNAs, from oligo(dT) primed RT reactions, of approximately 800 bp and 450 bp were amplified using oligonucleotide Cel1 (50 -GCAAATACGACAGCAGCATCACAGTTGCC-30 , forward primer), Cel2 (50 -GCCAAGAAGAGGAGCATTGT TGTAGGTAGC-30 , reverse primer) and Cel3 (50 -CTTCTTC GAAGCTCAGAGATCTGG-30 , forward primer), Cel4 (50 -AGCCCACAACAACTCGTCATTG-30 , reverse primer) respectively. The amplification consisted of denaturation at 94 C for 10 s, annealing at 53 C for 10 s and amplification at 72 C for 1 min for total of 35 cycles. The amplified products were cloned in pBluescriptII KSþ (Stratagene) at EcoRV site and sequenced from both strands using ThermoSequenase Dye Termination Kit and a 373 DNA sequencing system (Applied Biosystems). Sequence specific primers (Cel5, 50 -CGGGATGAGCTTTAATGAGGTAGTCAG-30 ; Cel6, 50 -C ATCGCATGGCCGAGCTCTCCAGTTGC-30 ; Cel7, 50 -CAAA CCGACCCAGGTACGTACAAC-30 and Cel8, 50 -ACAACAA TGCTCCTCTTCTTGGC-30 ) were designed to clone 50 and 30 regions of cDNA using 50 and 30 RACE system (Life Technologies, USA). PCR fragments were cloned and sequenced as described above. Sequence similarity searches were performed using the Basic Local Alignment Search Tool (BLAST, National Center for Biotechnology Information, Bethesda, MD). Deduced polypeptide alignment with hierarchical clustering [8] was carried out using the Florence Corpet method (http:// prodes.toulouse.inra.fr/multalin/multalin.html). Molecular weight and isoelectric point (pI ) was determined using ProtParam program in ExPASY server. A phylogenetic tree of full-length deduced amino acid sequences of cellulases was constructed using Phylip 3.5c package employing parsimony and bootstrap analysis (100 replicates).
kit (Life Technologies, USA) and the resulting single-stranded cDNA product was amplified by PCR. Equal amount of cDNA from each reaction was amplified using cotton cellulase specific oligonucleotides Cel1 and Cel2 primers. The amplification consisted of denaturation at 94 C for 10 s, annealing at 53 C for 10 s and amplification at 72 C for 1 min for total of 28 cycles. Actin forward (50 -GAGAGTTTTGATGTCCCT GCCATG-30 ) and reverse (50 -CAACGTCGCATTTCATGATG GAGT-30 ) primers were also included in the reaction as internal control. The amplified fragments were electrophoresed on 1% agarose gel in TAE buffer. 3. Results 3.1. Changes in break strength during leaf abscission In order to elucidate the effect of ethylene on the cell separation process the force (beak strength) required to pull the petiole away from the stem was measured in explants every 12 h after exposure to ethylene (Fig. 1). The break strength decreased gradually from 26.9 gr equivalent prior to ethylene exposure (0 h) to 1.2 gr equivalent after 48 h exposure to 10 ml/l ethylene. To establish whether ethylene plays a direct role in cotton leaf abscission, explants were treated with 1-MCP before exposing to ethylene and break strength measured. A 66% decrease in break strength was observed after 24 h ethylene exposure compared to only 34% decrease in explants pretreated with 1-MCP for the same period of time (Table 1). In control explants, which were neither exposed to ethylene nor 1-MCP, the decrease in break strength at 24 h could be due to generation of endogenous ethylene as a consequence of wounding.
2.6. RNA gel blot analysis 32
Break strength
24
Force (gr equivalent)
A total of 30 mg of total RNA was denatured at 65 C for 15 min in 10 mM MOPS, pH 7.0, containing 2.5 mM Naacetate, 2.2 M formaldehyde, and 50% (v/v) formamide. The denatured RNA was separated on 1.1% agarose gel containing 2.2 M formaldehyde and transferred to nylon membrane (Hybond Nþ, Amersham Pharmacia Biotech) by capillary transfer [33]. The DNA was fixed by baking at 80 C for 2 h. Blots were prehybrized at 42 C for 3 h in a solution containing 50% formamide, 5 SSC, 5 Denhardt’s solution, 0.1% SDS and 100 mg/ml denatured salmon sperm DNA followed by hybridization for 18 h in the same but fresh solution and containing 5 105 cpm of 32P-labelled cellulase cDNA from cotton LAZ. Membranes were washed twice in 2 SSC, 0.1% SDS for 15 min at room temperature followed by two washes of 0.1 SSC, 0.1% SDS at 60 C for 20 min. Hybridization signals were detected by exposing the blot to X-OMAT X-ray film (Kodak) for 3e5 days.
16
8
0
0
12
24
36
48
Time (h)
2.7. Semi-quantitative RT-PCR analysis RNase free DNase treated total RNA was reverse transcribed using oligo(dT) primer first strand cDNA synthesis
Fig. 1. Changes in break strength of cotton LAZ after exposure of explants to ethylene. The force in gr equivalent (N ) required to detach the petiole at the abscission zone was measured. Data are mean SD for at least 20 sets of observations.
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Table 1 Effect of 1-MCP on different parameters related to leaf abscission in cotton 0
BKS ACS ACO Cell
26.9 1.7 0.2 0.005 26.8 4.5 0.012 .005
Control
MCP þ Ethylene
Ethylene
24
48
24
48
24
48
18.9 1.2 0.6 0.01 109.2 7.7 11.9 0.3
10.9 0.8 0.67 0.04 289.2 8.5 34.8 7.4
8.9 1.9 2.30 0.05 165.8 9.0 39.5 1.0
1.2 0.3 3.45 0.05 356.6 8.5 93.5 4.2
17.7 1.9 0.17 0.06 23.4 3.5 1.4 0.15
6.3 1.4 0.44 0.05 36.37 5.0 1.3 0.14
Data are mean S.D. for at least three sets of observations. Units for the BKS, ACS, ACO and Cell are Force (gr equivalent), nmoles ACC g1 FW, nmoles C2H1 g1 FW and Relative Activity (103) h1 g1 respectively.
3.2. Activities of ACS, ACO and cellulase
4
n moles ACC h-1.g-1FW
ACS 3
2
1
0 500
n moles C2H4h-1. g-1FW
ACO 400
300
200
100
0 100
Cellulase Relative Activity (X103) h-1 g-1
The changes in the activities of enzymes involved in ethylene biosynthesis and cellulase during cotton leaf abscission are shown in Fig. 2. There was a continuous increase in the activities of all three enzymes with the length of exposure to ethylene, which suggests a role for these enzymes in cotton leaf abscission. Although a role for ethylene has already been documented by many groups for leaf abscission, changes in the activities of ethylene biosynthesis enzymes in the LAZ has not been cited extensively. A 12- and 17-fold increase in ACS activity was observed in the LAZs of explants exposed to ethylene for 24 and 48 h, respectively. In contrast, only 3.3 and 2.2-fold increase in ACS activity was observed after 48 h for control and 1-MCP treated explants, respectively. An approximate 5-fold increase in ACS activity was observed in the LAZ of ethylene treated explants compared to untreated control after 48 h, which suggests that exogenous ethylene is needed for the increase in ACS activity during abscission (Table 1). Similarly a continuous increase in ACO activity was observed in LAZs of ethylene treated explants, which was 6- and 13-fold higher for 24 and 48 h respectively in comparison to initial ACO activity at 0 h. Though 4- and 10-fold increase was also observed in control explants in 24 and 48 h respectively, no appreciable increase in ACO activity was observed in 1-MCP treated explants (Table 1). After 48 h, ACO activity in ethylene treated abscission zones was only 1.2 fold higher in comparison to respective control. There was a rapid increase in cellulase activity in LAZ of ethylene treated explants (Fig. 2C). In comparison to the initial activity at 0 h, there was more than a 3000- and 7700-fold increase in the activity observed after 24 and 48 h of ethylene treatment, respectively (Table 1). Control explants also showed increase in cellulase activity i.e., 1000- and 3000fold after 24 and 48 h respectively, but only 100-fold increase in activity was observed in 1-MCP treated explants. This suggests that, although control explants were not treated with ethylene, the ethylene produced by wounding of the tissue during explants generation promoted an increase in ACS, ACO and cellulase activities and a decrease in the break strength. 1-MCP treatment inhibited the increase in ACS, ACO and cellulase activity and decrease in the break strength at least till 24 h. Interestingly, there was a substantial decrease in break strength in the 1-MCP treated explants yet the increase in enzyme activities for ACS, ACO and cellulasts was not nearly so great after 48 h in comparison to ethylene treated explants.
80
60
40
20
0
0
4
8
12
24
36
48
Time (h) Fig. 2. Effect of ethylene on ACS, ACO and cellulase enzyme activities during cotton leaf abscission. Explants were exposed to ethylene and activities were measured at different time intervals. Data are mean SD for at least 3e4 sets of observations.
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3.3. Anatomical changes during abscission Abscission is accelerated if explants are exposed to ethylene. Disassembly of cell walls in the LAZ should lead to altered anatomical features in this separation layer. Scanning microscopy of LAZ was carried out in order to elucidate these differences. We examined changes in the surface and fracture plane of control, ethylene, and 1-MCP plus ethylene treated explants with scanning electron microscopy as shown in Fig. 3. Compact, well-organised and pentagonal cells were observed on the exterior surface of the petiole and stem junction, abscission zone, (Fig. 3A) and fracture plane (Fig. 3D) of explants prior to exposure to ethylene (0 h). After 24 h of ethylene treatment (Figs. 3B,E), the cells were elongated, disorganized and had a thin cell wall in comparison to the 0 h explants. Although changes were observed in the organization of cells in 1-MCP plus ethylene treated explants (Figs. 3C,F), the cells were less disorganized in comparison to 24 h ethylene treated explants. This suggests that the 1-MCP delayed changes at cellular level as was also evident from enzyme activity and break strength data (Table 1). 3.4. Molecular characterization of cotton cellulase Using a combination of RTPCR, 50 - and 30 -RACE, a cDNA of 2330 bp (GhCel1, Gene Bank Accession No. AF538680)
was cloned using total RNA from 24 h ethylene treated abscission zones. Nucleotide sequence analysis revealed a putative open reading frame starting with an ATG codon of 1863 bp encoding a polypeptide of 620 amino acid residues. The predicted polypeptide has a molecular weight of 68 KDa and theoretical pI of 9.17. Using SIGNALP, the signal sequence cleavage site of the polypeptide was predicted to be at amino acid residue 24 (AFA-GH). Amino acid residues from 27 to 480 showed high homology to glycosyl hydrolase family 9 sequences typical of cellulases. Sequence alignment of the deduced polypeptide with cotton fiber cellulase (EMBL Acc No. AY574906) showed only 39% identity with major differences in N-terminal and C-terminal regions. The LAZ cellulase lacked the membrane-anchored region present in the N-terminus of the fiber cellulase but had a long C-terminus region with sequence similarity to cellulose binding domains present in many other proteins. Three out of 4 amino acid residues at positions D163, D166 and H 519, which are essential for enzyme activity in bacterial cellulase celD [6], are conserved in GhCel1 as well as in all other plants cellulases (Fig. 4). A phylogenetic tree was generated from the alignment of the deduced full-length amino acid sequences for many of the plant cellulases in the public database. Sequences for the cellulose-synthesizing fungus, Phanerochaete chrysosporium, termite, Nasutitermes takasagoensis, and amoeba, Dictyostelium discoideum, were also included in the phylogenetic
Fig. 3. Scanning electron micrograph of the surface cells at the petiole and stem juncture, abscission zone (A, B, C), and fracture plane (D, E, F) of the cotton LAZ. A and D are micrographs for explants prior to exposure to ethylene (0 h); B and E are micrographs for 24 h ethylene treated explants; and C and F were explants treated with 1-MCP plus ethylene for 24 h.
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Fig. 4. Alignment of the deduced full-length amino acid sequence of GhCel1 with other plant cellulases. Gaps are shown as dashes. Deduced polypeptide alignment with hierarchical clustering was carried out using the Florence Corpet method [7]. Predicted processing site (:) for GhCel1 as identified by SIGNALP version 1.1 is shown in the figure. Conserved amino acid residues (C) necessary for the enzyme activity as shown by Ref. [5] are indicated overhead.
tree. The cellulases from fungus, amoeba and termite grouped separate from the plant cellulases (Fig. 5). The plant cellulase sequences aligned into 3 subgroups, similar to that previously documented by Libertini et al. [22]. Libertini et al. [22] designated these three clusters as a, b and g. In their analysis, most of the sequences were placed in a group. Only 2 sequences
from Arabidopsis were present in b group, which we have not included in our analysis. Nevertheless, we could still divide the a group in two subgroups i.e. a1 and a2 (Fig. 5). GhCel1 is included in subgroup a2, which includes the tomato Cel8 and three rice cellulases. All of these cellulases include a predicted cellulose-binding domain.
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83 100 100 85 100
100
99 100 66 99 96
2
100
77
99
83 100 64 43 91 54 100 76 96 99 85
100
56
100 84
99
85
100
1
100 100 57
96
100 100
42 100
81
100 88 47
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R
A
S
P
L
FI
Fi
36 GhCel1 rRNA
0
B
4
8
12
24
36
48 GhCel1 rRNA
E
C 0
4
8
12 24 36 48
M
C 24
48 24
48
Fig. 6. Analysis of GhCel1 mRNA abundance in different tissues (A), cotton LAZ after exposure to ethylene for various time (B) and in control, ethylene and 1-MCP plus ethylene treated (C) abscission zone. Northern blot analysis (A and B) was carried out using 30 mg each of total RNA electrophoresed and probed with radiolabelled GhCel1 probe. Ethidium bromide staining of gel is shown below the blot as loading control. In panel (A), different lanes are, R, root; S, stem; P, petiole; L, leaf; Fl, flower; Fi, fiber; and 36, abscission zone from 36 h ethylene treated explants. In panel (B), numbers above the lanes are time in h of ethylene exposure to explants. In panel C, amount of GhCel1 transcript is analyzed using semi-quantitative RTPCR in ethylene (E), control (C) and in 1-MCP plus ethylene (M) treated samples after different time points. Numbers above the lanes are time in h of ethylene exposure to explants. Actin primers were taken in RTPCR as a control for equal amount of RNA in the reaction.
3.5. Expression of GhCel1 The expression of GhCel1 was examined in LAZ of cotton explants exposed to ethylene (Fig. 6). The expression was also checked in other vegetative tissues exposed to ethylene for 24 h (Fig. 6). GHCel1 transcript was not detected in any other tissue except ethylene treated abscission zones and a smaller amount in the petioles (Fig. 6A). In abscission zones, though low level of transcript accumulation was detected at 0 h, increased transcript levels were apparent by 4 h after exposure to ethylene and continued to rise until 12 h after which it remained more or less constant till 36 h with a slight decrease at 48 h (Fig. 6B). The rise in GhCel1 transcript accumulation correlates with the decrease in the break strength and increase
61
in the cellulase activity during cotton leaf abscission. PCR amplification of GhCel1 in LAZ relative to actin was also performed in a separate experiment using RNA from ethylene treated, 1-MCP treated and non-treated LAZ (Fig. 6C). Similar to northern blot analysis, increased amplification was detected in ethylene treated LAZ from 0 to 48 h. Nil to very little amplification was detected in 24 h control and 1-MCP treated LAZ. However, amplification was visible at 48 h in both control as well as 1-MCP treated LAZ (Fig. 6C), though 3- to 4fold less in comparison to ethylene treated LAZ. This data confirms our northern analysis results and suggests that GhCel1 expression is ethylene regulated in LAZ of cotton. 4. Discussion Organ abscission is believed to be a highly coordinated process where the plant hormone ethylene plays an important role in regulating the breakdown of the cell wall of a layer of cells in the abscission zone. It is well documented that during leaf, flower and fruit abscission cell wall degradation is associated with an increase in the activity of several hydrolytic enzymes [14,16,38] and expansins [4]. These enzymes and proteins may either contribute to the cell separation process or play a role in protecting the exposed fracture surface from pathogen attack [9]. We have demonstrated that the process of leaf abscission in cotton is associated with higher biosynthesis of ethylene in abscission zones along with elevated levels of cellulase activity. Pretreatment of explants to 1-MCP, an ethylene action inhibitor, before ethylene exposure significantly delays the onset of abscission. Exposure of leaf explants to ethylene decreased break strength rapidly causing a 95% decrease in break strength after a 48 h treatment. The rapid decrease in break strength was not observed in explants pretreated with 1-MCP or in the first 24 h of explants not treated with ethyene. The decrease in break strength after 48 h in control explants not treated with ethylene and 1-MCP treated explants may be due to generation of endogenous ethylene as a consequence of wounding and loss of the affectivity of 1-MCP over time while explants were continuously exposed to ethylene. 1-MCP binds to the ethylene receptor [36]. The synthesis of new ethylene receptors, possibly in conjunction with the elimination of 1-MCP-bound receptor could be a possible explanation for loosing the affectivity of 1-MCP. However, the fact that the activities of ethylene synthesizing enzymes and cellulase are small
Fig. 5. Phylogenetic tree of the alignment of GhCel1 deduced amino acid sequence with other cellulases. Full-length protein sequences were aligned using CLUSTAL, and a phylogenetic tree was constructed using PHYLIP with the PROTPARS program. Numbers above the branches indicate bootstrap values. Sequences are GenBank accession numbers: Al (Atriplex lentiformis) Cel1 (BAB32662); At (Arabidopsis thaliana), Cel1 (At1g70710), Cel2 (At1g02800), KOR (NM_124350), Cel3 (NM_118487), KOR3 (At4g24260), Cel4 (NM_114254), Cel5 (NM_179018), Cel6 (AF074375) and Cel7 (U37702); Bn (Brassica napus) Cel16 (AJ242807); Ca (Capsicum annuum) Cel2 (CAA65828), Cl1 (X97188), Cl3 (X97189); Cs (Citrus sinensis) Cela1 (AAB65155); Dd (Dictyostelium discoideum) Cel (P22699); Fa (Fragaria ananassa) EG3 (AJ414708), Cel1 (AF051346), Cel2 (AF054615); Gh (Gossypium hirusutum) Cel1 (DQ060249, this study); Cel (AY574906); Le (Lycopersicon esculentum) Cel1 (U13054), Cel2 (U13055); Cel3 (U78526), Cel5 (AF077339), Cel7 (Y11268), Cel8 (AF098292); Ll (Lilium longiflorum) Cel1 (AAP38171); Md (Malus domestica) Cel1 (AAQ55294); Na (Nicotiana alata) Cel (AAD28258); Nt (Nicotiana tabacum) Cel (AAL30454); Ntter (Nasutitermes takasagoensis) Cel (AB019146); Os (Oryza sativa) Cel1 (NP_913380), Cel2 (NP_913378), Cel3 (XP_467689); Pa (Populus alba) Cel1 (BAB39482), Cel2 (BAB39483); Pam (Persea americana) Cel (CAA42569); PcFun (Phanerochaete chrysosporium) Cel (AAM22492); Pc (Pyrus communis) (BAC22691); Pr (Pinus radiata) Cel1 (U76725), Cel2 (U76756); Pp (Prunus persica) Cel (CAA65600); Ps (Pisum sativum) Cel1 (L41046), Cel2 (BAA85150); Pv (Phaseolus vulgaris) Cel (AAC78504); Sn (Sambucus nigra) Cel1 (CAA52343).
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compared to ethylene-treated and control explants may indicate that the reduction of break strength could be due to the contribution of additional cell wall degrading enzymes, or ethylene-independent induced genes [30]. Although the role of ethylene in initiation and progression of abscission is well documented, changes in the activity of ethylene synthesis enzymes, i.e. ACS and ACO, have not been documented for cotton abscission zones. The activities of ACS and ACO in LAZ increased many fold. 1-MCP treatment inhibited this increase. Jackson and Osborne [16] reported that an increase in endogenous level of ethylene correlates with the onset of abscission and proposed that ethylene might be a natural regulator. The endogenous synthesis of ethylene in abscission zone could play an important role in abscission by hastening the activities of various cell wall hydrolases, which have been shown to be regulated by ethylene. This observation is also supported by our results with control explants which were not exposed to ethylene. However, in these explants, wounding could have induced ACS and ACO activities to some extent and enough ethylene was generated to start abscission process. Though, it was much slower when compared to ethylene treated LAZ. Induction of ethylene biosynthesis has been reported in wounded mesocorp tissue of Cucurbita maxima where higher transcript levels of ACS and ACO and higher enzyme activities were observed in these cells [19]. Durbin et al. [13] observed a 40-fold increase in cellulase activity in bean LAZ and leaf abscission was complete in 48e60 h. We have also observed a several folds increase in the cellulase activity in cotton LAZs. It was demonstrated that progression of leaf abscission correlates positively with the de novo accumulation of a high salt extractable cellulase [21] in the abscission zones of bean with a pI of 9.5. Immunological localizations confirmed that the enzyme is indeed located in the separation layer and unexpectedly in vascular bundles up to several millimeters from the abscission zone [11,35]. Our scanning electron microscopy data is in accordance with observed changes in the cellular organization, which might be caused by the higher cellulase activity along with the other cell wall hydrolyzing enzymes. Similar changes in the cellular organization during abscission process have been reported in bean leaf [25,40], Arabidopsis [30] and tomato [32] flowers. Our sequence and phylogenetic analyses of cellulases show that subgroup a2 members LeCel8, GhCel1 and OsCel1 have 100e125 more amino acids at C-terminus, carbohydrate-binding domain, in comparison to a1 and g, whereas g group members possesses approximately 80 amino acid residues at N-terminus region, membrane anchored region. Although a role for the carbohydrate binding domain in cell wall extension is missing, a strawberry cellulase, FaEG3, containing this domain has been shown to involved in cell wall loosening [39]. As per our knowledge, GhCel1 is the first cellulase with the carbohydrate-binding domain involved in the leaf abscission. Cotton fiber-specific cellulase (GhCel ) is present in g group, which also includes Brassica napus cellulase (BnCel16), Arabidopsis KOR and KOR3 and tomato Cel3. They all have membrane-anchored region at N-terminus,
ubiquitously expressed, and linked to cell elongation, wall assembly and possibly to cellulose biosynthesis [28]. In addition to GhCel1, there is a cotton expressed sequence tag (EST) similar to GhCel1 that includes a cellulose-binding domain, which has been postulated to take part during cotton fiber elongation [2]. Our results indicates that GhCel1, characterized in this study, is not expressed in elongating cotton fibers (Fig. 6A), which supports the concept that different cellulases perform tissue-specific and specialized roles in cell wall extension in cotton. Abscission specific cellulases have been reported for many plant species [31]. The decrease in break strength of the cotton explants with an increase in expression of GhCel1 in the LAZ after ethylene treatment indicates that GhCel1 is ethylene-regulated and closely associated with abscission of cotton leaves. It is concluded that cotton leaf abscission is initiated and regulated by ethylene. Ethylene exposure increases the endogenous production of ethylene synthesis enzymes and increased synthesis of abscission specific cellulase in the leaf abscission zone, GhCel1, which includes a carbohydrate-binding domain at C-terminus.
Acknowledgements The authors are grateful to the Department of Science and Technology, New Delhi for financial support to carry out the work. Senior Research Fellowship to AM and Research Associate ship to SK from CSIR, India is acknowledged. Authors are thankful to Mr. V.K. Lal and Mr. Alok Saxena for scanning electron microscopy work.
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Plant Physiology and Biochemistry 46 (2008) 54e63 www.elsevier.com/locate/plaphy
Research article
Ethylene induced cotton leaf abscission is associated with higher expression of cellulase (GhCel1) and increased activities of ethylene biosynthesis enzymes in abscission zone Amita Mishra, Smriti Khare, Prabodh Kumar Trivedi, Pravendra Nath* Plant Gene Expression Laboratory, National Botanical Research Institute, Rana Pratap Marg, Lucknow, UP 226 001, India Received 14 February 2007 Available online 20 September 2007
Abstract Ethylene induced cotton (Gossypium hirusutum var RST-39) leaf abscission has been characterized by measuring the activities of ACC synthase (ACS, E.C. 4.4.1.14), ACC oxidase (ACO, E.C. 1.14.17.4) and cellulase (E.C. 3.2.1.4). In addition, a leaf abscission specific cDNA (GhCel1) has been cloned from cotton, which belongs to the a2 subgroup of cellulases that possess a C-terminus carbohydrate-binding domain. Measurement of enzyme activity in the abscission zones of cotton leaf explants exposed to ethylene for 48 h compared to non-treated controls indicated a more than 5-fold increase in the activity of ACS, 1.2-fold increase in the activity of ACO and about 2.7-fold increase in the activity of cellulase in the ethylene treated explants. This increase was accompanied by a substantial decrease in the force required to separate the petiole from the stem (break strength) and an increased accumulation of cellulase transcript in the abscission zone. Treatment of explants with 1-Methylcyclopropene (1-MCP) prior to ethylene resulted in significant inhibition of enzyme activities and transcript accumulation. It is concluded that ethylene response of cotton leaf abscission leads to higher cellulase expression and increased activities of ethylene biosynthesis enzymes in the abscission zone. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: 1-Methylcyclopropene; ACC oxidase; ACC synthase; Cellulase; Ethylene; Gossypium hirusutum; Leaf abscission
1. Introduction Abscission is the process by which plants shed organs from the parent body in response to developmental cues or to adapt to various environmental stresses including pathogen attack [26,31]. The signal that promotes abscission has been widely recognized as the gaseous plant hormone ethylene [1,37]. The role of ethylene in abscission was deduced by the use of chemical agents such as norbornadiene, silver ions or 1-MCP Abbreviations: 1-MCP, 1-Methylcyclopropene; ACC oxidase, 1-aminocyclopropane-1-carboxylate oxidase; ACC synthase, 1-aminocyclopropane1-carboxylate synthase; LAZ, leaf abscission zone. * Corresponding author: Plant Gene Expression Laboratory, National Botanical Research Institute, Rana Pratap Marg, P.O. Box 436, Lucknow, UP 226 001, India. Tel.: þ91 522 220 5841; fax: þ91 522 220 5836/5839. E-mail address: [email protected] (P. Nath). 0981-9428/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2007.09.002
that block ethylene action [10,40,41] or mutants, which are deficient in ethylene perception and response [7]. Organ separation is accomplished by degradation of the cell wall and middle lamella to form a fracture plane between the abscising organ and the parent plant. Although the precise sequence of events that bring about cell wall degradation is unclear, biochemical and molecular studies indicate that an increase in the activities of hydrolytic enzymes such as glucanases [5,38] and polygalacturanases [14,18] and proteins such as expansins [4] play a major role in the process of abscission. An increase in cellulase (endo-1,4-b-D-glucanase) activity has been noted in leaf abscission zones (LAZ) [20,40], fruit abscission zones [15,17], ripening fruits [24] senescing styles and anthers [34] and during adventitious root initiation [23]. The isoelectric points (pIs) of many of these cellulases differ from each other, which suggest that each may have distinct
A. Mishra et al. / Plant Physiology and Biochemistry 46 (2008) 54e63
cellular function. Although cellulases have been identified and cloned from the abscission zones of several different sources, information relating to its role in leaf abscission is limited [31]. In the present study we have investigated petiole abscission in cotton using biochemical and molecular tools. The activities of cellulase and ethylene biosynthesis enzymes have been measured in LAZ and correlated with the progression of petiole separation. A leaf abscission specific cDNA has been cloned and characterized for the first time from cotton and accumulation of mRNA in abscission zones studied in the presence and absence of 1-MCP, an ethylene action inhibitor. The results of this study gain significance due to commercial importance of cotton leaf abscission. 2. Materials and methods 2.1. Plant material and treatments Seeds of cotton (Gossypium hirusutum var RST-39) were germinated in glasshouse under controlled temperature (32 3 C) for 12e15 days until the primary leaves were fully expanded and the secondary pinnate leaf was just beginning to open. At this time, the leaf blades for the primary leaves were removed and plants harvested by cutting 1 cm above the soil. Twenty-five explants, each of 8e10 cm long, were surface sterilized using bleach (10% v/v) for 5 min, washed 3e4 times with distilled water and kept in a beaker containing water and exposed to 10 mL/L ethylene in a 10 liter air tight desiccators for the time intervals indicated in the figure legends. Desiccators were opened every 4 h, flushed with air to remove CO2 accumulation and maintain O2 concentrations and 10 mL/L ethylene reintroduced fresh each time. For 1-MCP treatment, explants were exposed to 10 mL/L 1-MCP (EthylBlock, BioTechnologies for Horticulture, Inc., Waterboro, USA) for 1 h before ethylene exposure. For 1-MCP application, 1 ml water was added to 1-MCP powder kept in a 10 ml beaker and placed towards sidewall of the desiccators containing explants. Desiccators were closed immediately (within 2e3 s) to prevent diffusion of 1-MCP outside the desiccators. The concentration of 1-MCP was calculated as per manufacturers instructions. LAZs (1e2 mm on either side of fracture plane) were harvested and frozen in liquid nitrogen and stored at 70 C till further use. Other vegetative tissues were also collected after exposure to ethylene for 24e36 h. 2.2. Break strength measurement In order to determine the break strength, the petiolar stump of the explant was clamped using an alligator clip to which a string was attached for holding weight. Increasing weights were loaded on the string till the petiolar stump detached at the abscission zone. The distance of the explant from bench top as well as length and angle of the string were kept constant during each measurement to avoid any variations in the force applied. Total weight applied on the petiolar stump was determined and break strength of the abscission zone calculated as force in gram equivalents required to detach the petiole. Break
55
strength values determined for each time point represented an average of at least 20 or more individual measurements. 2.3. Enzyme activity measurements For ACS activity measurements, LAZs (200 mg) were homogenized in extraction buffer consisting of 100 mM HepesKOH, pH 8.5, 4 mM DTT, 10 mM pyridoxal phosphate and 5% PVP 40,000 as described by Kato et al. [19]. The homogenate was centrifuged at 12,000 g for 20 min at 4 C. The supernatant was desalted using Sephadex G-25 and eluate used for enzyme assay as described by Pathak et al. [29]. ACS activity was expressed as nmoles ACC formed h1 g1 FW. ACO enzyme was extracted according to Moya-Leon and Jhon [27]. A total of 200 mg frozen tissue was homogenized in 1 ml of extraction buffer consisting of 0.1 M TriseHCl, pH 7.5, 10% glycerol, 2 mM DTT and 30 mM sodium acetate. The homogenate was centrifuged at 12,000 g for 20 min at 4 C. Further purification of supernatant and enzyme assay was carried out as described by Pathak et al. [29]. ACO activity was expressed as nmoles C2H4 produced h1 g1 FW. Cellulase activity was measured in 1 g of abscission zones homogenised in 3 ml of 0.1 M sod phosphate buffer (pH 7.8) containing 2 mM EDTA, 0.1 mM DTT and 0.2% Triton X100. The homogenate was transferred to eppendorf tubes on ice and 0.5 M EDTA (1/500 vol) and 5 M NaCl (1/10 vol) added, mixed thoroughly and kept on ice for 30 min. The homogenate was centrifuged at 15,000 g for 30 min at 4 C. The pellet was discarded and supernatant was used as enzyme preparation for the cellulase assay. The assay mixture contained 0.5 ml enzyme and 0.5 ml 1.3% (w/v) carboxy methyl cellulose (CMC) prepared in 0.02 M TriseHCl pH 8.0. Drainage time of assay mixture through a calibrated portion of 100 ml pipette was used as a measure of viscosity. Viscosity of the mixture was measured at an interval of 30 min from 0 to 2 h at room temperature. Viscosity data was converted to intrinsic viscosity and relative units of activity calculated as described by Durbin and Lewis [12]. Results were expressed as Relative activity h1 g1 FW. 2.4. Scanning electron microscopy For electron microscopy, abscission zones were fixed in 4% (v/v) gluteraldehyde in 0.5 M potassium phosphate buffer (pH 7.4) for 4 h at 25 C, rinsed four times in buffer, and then dehydrated in a graded ethanol series. Sputter coated sections were examined at different magnifications with Philips XL20 scanning electron microscope (Philips, Holond). 2.5. Cloning of cellulase gene and nucleotide sequence analysis Total RNA from abscission zones and other vegetative tissues was isolated according to Asif et al. [3]. In order to clone cellulase cDNA, RT-PCR was carried out using total RNA from 24 h ethylene treated abscission zones. Oligonucleotide primer pairs were designed from regions having maximum sequence
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A. Mishra et al. / Plant Physiology and Biochemistry 46 (2008) 54e63
identity amongst known cellulase nucleotide sequences. Partial cDNAs, from oligo(dT) primed RT reactions, of approximately 800 bp and 450 bp were amplified using oligonucleotide Cel1 (50 -GCAAATACGACAGCAGCATCACAGTTGCC-30 , forward primer), Cel2 (50 -GCCAAGAAGAGGAGCATTGT TGTAGGTAGC-30 , reverse primer) and Cel3 (50 -CTTCTTC GAAGCTCAGAGATCTGG-30 , forward primer), Cel4 (50 -AGCCCACAACAACTCGTCATTG-30 , reverse primer) respectively. The amplification consisted of denaturation at 94 C for 10 s, annealing at 53 C for 10 s and amplification at 72 C for 1 min for total of 35 cycles. The amplified products were cloned in pBluescriptII KSþ (Stratagene) at EcoRV site and sequenced from both strands using ThermoSequenase Dye Termination Kit and a 373 DNA sequencing system (Applied Biosystems). Sequence specific primers (Cel5, 50 -CGGGATGAGCTTTAATGAGGTAGTCAG-30 ; Cel6, 50 -C ATCGCATGGCCGAGCTCTCCAGTTGC-30 ; Cel7, 50 -CAAA CCGACCCAGGTACGTACAAC-30 and Cel8, 50 -ACAACAA TGCTCCTCTTCTTGGC-30 ) were designed to clone 50 and 30 regions of cDNA using 50 and 30 RACE system (Life Technologies, USA). PCR fragments were cloned and sequenced as described above. Sequence similarity searches were performed using the Basic Local Alignment Search Tool (BLAST, National Center for Biotechnology Information, Bethesda, MD). Deduced polypeptide alignment with hierarchical clustering [8] was carried out using the Florence Corpet method (http:// prodes.toulouse.inra.fr/multalin/multalin.html). Molecular weight and isoelectric point (pI ) was determined using ProtParam program in ExPASY server. A phylogenetic tree of full-length deduced amino acid sequences of cellulases was constructed using Phylip 3.5c package employing parsimony and bootstrap analysis (100 replicates).
kit (Life Technologies, USA) and the resulting single-stranded cDNA product was amplified by PCR. Equal amount of cDNA from each reaction was amplified using cotton cellulase specific oligonucleotides Cel1 and Cel2 primers. The amplification consisted of denaturation at 94 C for 10 s, annealing at 53 C for 10 s and amplification at 72 C for 1 min for total of 28 cycles. Actin forward (50 -GAGAGTTTTGATGTCCCT GCCATG-30 ) and reverse (50 -CAACGTCGCATTTCATGATG GAGT-30 ) primers were also included in the reaction as internal control. The amplified fragments were electrophoresed on 1% agarose gel in TAE buffer. 3. Results 3.1. Changes in break strength during leaf abscission In order to elucidate the effect of ethylene on the cell separation process the force (beak strength) required to pull the petiole away from the stem was measured in explants every 12 h after exposure to ethylene (Fig. 1). The break strength decreased gradually from 26.9 gr equivalent prior to ethylene exposure (0 h) to 1.2 gr equivalent after 48 h exposure to 10 ml/l ethylene. To establish whether ethylene plays a direct role in cotton leaf abscission, explants were treated with 1-MCP before exposing to ethylene and break strength measured. A 66% decrease in break strength was observed after 24 h ethylene exposure compared to only 34% decrease in explants pretreated with 1-MCP for the same period of time (Table 1). In control explants, which were neither exposed to ethylene nor 1-MCP, the decrease in break strength at 24 h could be due to generation of endogenous ethylene as a consequence of wounding.
2.6. RNA gel blot analysis 32
Break strength
24
Force (gr equivalent)
A total of 30 mg of total RNA was denatured at 65 C for 15 min in 10 mM MOPS, pH 7.0, containing 2.5 mM Naacetate, 2.2 M formaldehyde, and 50% (v/v) formamide. The denatured RNA was separated on 1.1% agarose gel containing 2.2 M formaldehyde and transferred to nylon membrane (Hybond Nþ, Amersham Pharmacia Biotech) by capillary transfer [33]. The DNA was fixed by baking at 80 C for 2 h. Blots were prehybrized at 42 C for 3 h in a solution containing 50% formamide, 5 SSC, 5 Denhardt’s solution, 0.1% SDS and 100 mg/ml denatured salmon sperm DNA followed by hybridization for 18 h in the same but fresh solution and containing 5 105 cpm of 32P-labelled cellulase cDNA from cotton LAZ. Membranes were washed twice in 2 SSC, 0.1% SDS for 15 min at room temperature followed by two washes of 0.1 SSC, 0.1% SDS at 60 C for 20 min. Hybridization signals were detected by exposing the blot to X-OMAT X-ray film (Kodak) for 3e5 days.
16
8
0
0
12
24
36
48
Time (h)
2.7. Semi-quantitative RT-PCR analysis RNase free DNase treated total RNA was reverse transcribed using oligo(dT) primer first strand cDNA synthesis
Fig. 1. Changes in break strength of cotton LAZ after exposure of explants to ethylene. The force in gr equivalent (N ) required to detach the petiole at the abscission zone was measured. Data are mean SD for at least 20 sets of observations.
A. Mishra et al. / Plant Physiology and Biochemistry 46 (2008) 54e63
57
Table 1 Effect of 1-MCP on different parameters related to leaf abscission in cotton 0
BKS ACS ACO Cell
26.9 1.7 0.2 0.005 26.8 4.5 0.012 .005
Control
MCP þ Ethylene
Ethylene
24
48
24
48
24
48
18.9 1.2 0.6 0.01 109.2 7.7 11.9 0.3
10.9 0.8 0.67 0.04 289.2 8.5 34.8 7.4
8.9 1.9 2.30 0.05 165.8 9.0 39.5 1.0
1.2 0.3 3.45 0.05 356.6 8.5 93.5 4.2
17.7 1.9 0.17 0.06 23.4 3.5 1.4 0.15
6.3 1.4 0.44 0.05 36.37 5.0 1.3 0.14
Data are mean S.D. for at least three sets of observations. Units for the BKS, ACS, ACO and Cell are Force (gr equivalent), nmoles ACC g1 FW, nmoles C2H1 g1 FW and Relative Activity (103) h1 g1 respectively.
3.2. Activities of ACS, ACO and cellulase
4
n moles ACC h-1.g-1FW
ACS 3
2
1
0 500
n moles C2H4h-1. g-1FW
ACO 400
300
200
100
0 100
Cellulase Relative Activity (X103) h-1 g-1
The changes in the activities of enzymes involved in ethylene biosynthesis and cellulase during cotton leaf abscission are shown in Fig. 2. There was a continuous increase in the activities of all three enzymes with the length of exposure to ethylene, which suggests a role for these enzymes in cotton leaf abscission. Although a role for ethylene has already been documented by many groups for leaf abscission, changes in the activities of ethylene biosynthesis enzymes in the LAZ has not been cited extensively. A 12- and 17-fold increase in ACS activity was observed in the LAZs of explants exposed to ethylene for 24 and 48 h, respectively. In contrast, only 3.3 and 2.2-fold increase in ACS activity was observed after 48 h for control and 1-MCP treated explants, respectively. An approximate 5-fold increase in ACS activity was observed in the LAZ of ethylene treated explants compared to untreated control after 48 h, which suggests that exogenous ethylene is needed for the increase in ACS activity during abscission (Table 1). Similarly a continuous increase in ACO activity was observed in LAZs of ethylene treated explants, which was 6- and 13-fold higher for 24 and 48 h respectively in comparison to initial ACO activity at 0 h. Though 4- and 10-fold increase was also observed in control explants in 24 and 48 h respectively, no appreciable increase in ACO activity was observed in 1-MCP treated explants (Table 1). After 48 h, ACO activity in ethylene treated abscission zones was only 1.2 fold higher in comparison to respective control. There was a rapid increase in cellulase activity in LAZ of ethylene treated explants (Fig. 2C). In comparison to the initial activity at 0 h, there was more than a 3000- and 7700-fold increase in the activity observed after 24 and 48 h of ethylene treatment, respectively (Table 1). Control explants also showed increase in cellulase activity i.e., 1000- and 3000fold after 24 and 48 h respectively, but only 100-fold increase in activity was observed in 1-MCP treated explants. This suggests that, although control explants were not treated with ethylene, the ethylene produced by wounding of the tissue during explants generation promoted an increase in ACS, ACO and cellulase activities and a decrease in the break strength. 1-MCP treatment inhibited the increase in ACS, ACO and cellulase activity and decrease in the break strength at least till 24 h. Interestingly, there was a substantial decrease in break strength in the 1-MCP treated explants yet the increase in enzyme activities for ACS, ACO and cellulasts was not nearly so great after 48 h in comparison to ethylene treated explants.
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Time (h) Fig. 2. Effect of ethylene on ACS, ACO and cellulase enzyme activities during cotton leaf abscission. Explants were exposed to ethylene and activities were measured at different time intervals. Data are mean SD for at least 3e4 sets of observations.
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3.3. Anatomical changes during abscission Abscission is accelerated if explants are exposed to ethylene. Disassembly of cell walls in the LAZ should lead to altered anatomical features in this separation layer. Scanning microscopy of LAZ was carried out in order to elucidate these differences. We examined changes in the surface and fracture plane of control, ethylene, and 1-MCP plus ethylene treated explants with scanning electron microscopy as shown in Fig. 3. Compact, well-organised and pentagonal cells were observed on the exterior surface of the petiole and stem junction, abscission zone, (Fig. 3A) and fracture plane (Fig. 3D) of explants prior to exposure to ethylene (0 h). After 24 h of ethylene treatment (Figs. 3B,E), the cells were elongated, disorganized and had a thin cell wall in comparison to the 0 h explants. Although changes were observed in the organization of cells in 1-MCP plus ethylene treated explants (Figs. 3C,F), the cells were less disorganized in comparison to 24 h ethylene treated explants. This suggests that the 1-MCP delayed changes at cellular level as was also evident from enzyme activity and break strength data (Table 1). 3.4. Molecular characterization of cotton cellulase Using a combination of RTPCR, 50 - and 30 -RACE, a cDNA of 2330 bp (GhCel1, Gene Bank Accession No. AF538680)
was cloned using total RNA from 24 h ethylene treated abscission zones. Nucleotide sequence analysis revealed a putative open reading frame starting with an ATG codon of 1863 bp encoding a polypeptide of 620 amino acid residues. The predicted polypeptide has a molecular weight of 68 KDa and theoretical pI of 9.17. Using SIGNALP, the signal sequence cleavage site of the polypeptide was predicted to be at amino acid residue 24 (AFA-GH). Amino acid residues from 27 to 480 showed high homology to glycosyl hydrolase family 9 sequences typical of cellulases. Sequence alignment of the deduced polypeptide with cotton fiber cellulase (EMBL Acc No. AY574906) showed only 39% identity with major differences in N-terminal and C-terminal regions. The LAZ cellulase lacked the membrane-anchored region present in the N-terminus of the fiber cellulase but had a long C-terminus region with sequence similarity to cellulose binding domains present in many other proteins. Three out of 4 amino acid residues at positions D163, D166 and H 519, which are essential for enzyme activity in bacterial cellulase celD [6], are conserved in GhCel1 as well as in all other plants cellulases (Fig. 4). A phylogenetic tree was generated from the alignment of the deduced full-length amino acid sequences for many of the plant cellulases in the public database. Sequences for the cellulose-synthesizing fungus, Phanerochaete chrysosporium, termite, Nasutitermes takasagoensis, and amoeba, Dictyostelium discoideum, were also included in the phylogenetic
Fig. 3. Scanning electron micrograph of the surface cells at the petiole and stem juncture, abscission zone (A, B, C), and fracture plane (D, E, F) of the cotton LAZ. A and D are micrographs for explants prior to exposure to ethylene (0 h); B and E are micrographs for 24 h ethylene treated explants; and C and F were explants treated with 1-MCP plus ethylene for 24 h.
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Fig. 4. Alignment of the deduced full-length amino acid sequence of GhCel1 with other plant cellulases. Gaps are shown as dashes. Deduced polypeptide alignment with hierarchical clustering was carried out using the Florence Corpet method [7]. Predicted processing site (:) for GhCel1 as identified by SIGNALP version 1.1 is shown in the figure. Conserved amino acid residues (C) necessary for the enzyme activity as shown by Ref. [5] are indicated overhead.
tree. The cellulases from fungus, amoeba and termite grouped separate from the plant cellulases (Fig. 5). The plant cellulase sequences aligned into 3 subgroups, similar to that previously documented by Libertini et al. [22]. Libertini et al. [22] designated these three clusters as a, b and g. In their analysis, most of the sequences were placed in a group. Only 2 sequences
from Arabidopsis were present in b group, which we have not included in our analysis. Nevertheless, we could still divide the a group in two subgroups i.e. a1 and a2 (Fig. 5). GhCel1 is included in subgroup a2, which includes the tomato Cel8 and three rice cellulases. All of these cellulases include a predicted cellulose-binding domain.
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Fig. 6. Analysis of GhCel1 mRNA abundance in different tissues (A), cotton LAZ after exposure to ethylene for various time (B) and in control, ethylene and 1-MCP plus ethylene treated (C) abscission zone. Northern blot analysis (A and B) was carried out using 30 mg each of total RNA electrophoresed and probed with radiolabelled GhCel1 probe. Ethidium bromide staining of gel is shown below the blot as loading control. In panel (A), different lanes are, R, root; S, stem; P, petiole; L, leaf; Fl, flower; Fi, fiber; and 36, abscission zone from 36 h ethylene treated explants. In panel (B), numbers above the lanes are time in h of ethylene exposure to explants. In panel C, amount of GhCel1 transcript is analyzed using semi-quantitative RTPCR in ethylene (E), control (C) and in 1-MCP plus ethylene (M) treated samples after different time points. Numbers above the lanes are time in h of ethylene exposure to explants. Actin primers were taken in RTPCR as a control for equal amount of RNA in the reaction.
3.5. Expression of GhCel1 The expression of GhCel1 was examined in LAZ of cotton explants exposed to ethylene (Fig. 6). The expression was also checked in other vegetative tissues exposed to ethylene for 24 h (Fig. 6). GHCel1 transcript was not detected in any other tissue except ethylene treated abscission zones and a smaller amount in the petioles (Fig. 6A). In abscission zones, though low level of transcript accumulation was detected at 0 h, increased transcript levels were apparent by 4 h after exposure to ethylene and continued to rise until 12 h after which it remained more or less constant till 36 h with a slight decrease at 48 h (Fig. 6B). The rise in GhCel1 transcript accumulation correlates with the decrease in the break strength and increase
61
in the cellulase activity during cotton leaf abscission. PCR amplification of GhCel1 in LAZ relative to actin was also performed in a separate experiment using RNA from ethylene treated, 1-MCP treated and non-treated LAZ (Fig. 6C). Similar to northern blot analysis, increased amplification was detected in ethylene treated LAZ from 0 to 48 h. Nil to very little amplification was detected in 24 h control and 1-MCP treated LAZ. However, amplification was visible at 48 h in both control as well as 1-MCP treated LAZ (Fig. 6C), though 3- to 4fold less in comparison to ethylene treated LAZ. This data confirms our northern analysis results and suggests that GhCel1 expression is ethylene regulated in LAZ of cotton. 4. Discussion Organ abscission is believed to be a highly coordinated process where the plant hormone ethylene plays an important role in regulating the breakdown of the cell wall of a layer of cells in the abscission zone. It is well documented that during leaf, flower and fruit abscission cell wall degradation is associated with an increase in the activity of several hydrolytic enzymes [14,16,38] and expansins [4]. These enzymes and proteins may either contribute to the cell separation process or play a role in protecting the exposed fracture surface from pathogen attack [9]. We have demonstrated that the process of leaf abscission in cotton is associated with higher biosynthesis of ethylene in abscission zones along with elevated levels of cellulase activity. Pretreatment of explants to 1-MCP, an ethylene action inhibitor, before ethylene exposure significantly delays the onset of abscission. Exposure of leaf explants to ethylene decreased break strength rapidly causing a 95% decrease in break strength after a 48 h treatment. The rapid decrease in break strength was not observed in explants pretreated with 1-MCP or in the first 24 h of explants not treated with ethyene. The decrease in break strength after 48 h in control explants not treated with ethylene and 1-MCP treated explants may be due to generation of endogenous ethylene as a consequence of wounding and loss of the affectivity of 1-MCP over time while explants were continuously exposed to ethylene. 1-MCP binds to the ethylene receptor [36]. The synthesis of new ethylene receptors, possibly in conjunction with the elimination of 1-MCP-bound receptor could be a possible explanation for loosing the affectivity of 1-MCP. However, the fact that the activities of ethylene synthesizing enzymes and cellulase are small
Fig. 5. Phylogenetic tree of the alignment of GhCel1 deduced amino acid sequence with other cellulases. Full-length protein sequences were aligned using CLUSTAL, and a phylogenetic tree was constructed using PHYLIP with the PROTPARS program. Numbers above the branches indicate bootstrap values. Sequences are GenBank accession numbers: Al (Atriplex lentiformis) Cel1 (BAB32662); At (Arabidopsis thaliana), Cel1 (At1g70710), Cel2 (At1g02800), KOR (NM_124350), Cel3 (NM_118487), KOR3 (At4g24260), Cel4 (NM_114254), Cel5 (NM_179018), Cel6 (AF074375) and Cel7 (U37702); Bn (Brassica napus) Cel16 (AJ242807); Ca (Capsicum annuum) Cel2 (CAA65828), Cl1 (X97188), Cl3 (X97189); Cs (Citrus sinensis) Cela1 (AAB65155); Dd (Dictyostelium discoideum) Cel (P22699); Fa (Fragaria ananassa) EG3 (AJ414708), Cel1 (AF051346), Cel2 (AF054615); Gh (Gossypium hirusutum) Cel1 (DQ060249, this study); Cel (AY574906); Le (Lycopersicon esculentum) Cel1 (U13054), Cel2 (U13055); Cel3 (U78526), Cel5 (AF077339), Cel7 (Y11268), Cel8 (AF098292); Ll (Lilium longiflorum) Cel1 (AAP38171); Md (Malus domestica) Cel1 (AAQ55294); Na (Nicotiana alata) Cel (AAD28258); Nt (Nicotiana tabacum) Cel (AAL30454); Ntter (Nasutitermes takasagoensis) Cel (AB019146); Os (Oryza sativa) Cel1 (NP_913380), Cel2 (NP_913378), Cel3 (XP_467689); Pa (Populus alba) Cel1 (BAB39482), Cel2 (BAB39483); Pam (Persea americana) Cel (CAA42569); PcFun (Phanerochaete chrysosporium) Cel (AAM22492); Pc (Pyrus communis) (BAC22691); Pr (Pinus radiata) Cel1 (U76725), Cel2 (U76756); Pp (Prunus persica) Cel (CAA65600); Ps (Pisum sativum) Cel1 (L41046), Cel2 (BAA85150); Pv (Phaseolus vulgaris) Cel (AAC78504); Sn (Sambucus nigra) Cel1 (CAA52343).
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compared to ethylene-treated and control explants may indicate that the reduction of break strength could be due to the contribution of additional cell wall degrading enzymes, or ethylene-independent induced genes [30]. Although the role of ethylene in initiation and progression of abscission is well documented, changes in the activity of ethylene synthesis enzymes, i.e. ACS and ACO, have not been documented for cotton abscission zones. The activities of ACS and ACO in LAZ increased many fold. 1-MCP treatment inhibited this increase. Jackson and Osborne [16] reported that an increase in endogenous level of ethylene correlates with the onset of abscission and proposed that ethylene might be a natural regulator. The endogenous synthesis of ethylene in abscission zone could play an important role in abscission by hastening the activities of various cell wall hydrolases, which have been shown to be regulated by ethylene. This observation is also supported by our results with control explants which were not exposed to ethylene. However, in these explants, wounding could have induced ACS and ACO activities to some extent and enough ethylene was generated to start abscission process. Though, it was much slower when compared to ethylene treated LAZ. Induction of ethylene biosynthesis has been reported in wounded mesocorp tissue of Cucurbita maxima where higher transcript levels of ACS and ACO and higher enzyme activities were observed in these cells [19]. Durbin et al. [13] observed a 40-fold increase in cellulase activity in bean LAZ and leaf abscission was complete in 48e60 h. We have also observed a several folds increase in the cellulase activity in cotton LAZs. It was demonstrated that progression of leaf abscission correlates positively with the de novo accumulation of a high salt extractable cellulase [21] in the abscission zones of bean with a pI of 9.5. Immunological localizations confirmed that the enzyme is indeed located in the separation layer and unexpectedly in vascular bundles up to several millimeters from the abscission zone [11,35]. Our scanning electron microscopy data is in accordance with observed changes in the cellular organization, which might be caused by the higher cellulase activity along with the other cell wall hydrolyzing enzymes. Similar changes in the cellular organization during abscission process have been reported in bean leaf [25,40], Arabidopsis [30] and tomato [32] flowers. Our sequence and phylogenetic analyses of cellulases show that subgroup a2 members LeCel8, GhCel1 and OsCel1 have 100e125 more amino acids at C-terminus, carbohydrate-binding domain, in comparison to a1 and g, whereas g group members possesses approximately 80 amino acid residues at N-terminus region, membrane anchored region. Although a role for the carbohydrate binding domain in cell wall extension is missing, a strawberry cellulase, FaEG3, containing this domain has been shown to involved in cell wall loosening [39]. As per our knowledge, GhCel1 is the first cellulase with the carbohydrate-binding domain involved in the leaf abscission. Cotton fiber-specific cellulase (GhCel ) is present in g group, which also includes Brassica napus cellulase (BnCel16), Arabidopsis KOR and KOR3 and tomato Cel3. They all have membrane-anchored region at N-terminus,
ubiquitously expressed, and linked to cell elongation, wall assembly and possibly to cellulose biosynthesis [28]. In addition to GhCel1, there is a cotton expressed sequence tag (EST) similar to GhCel1 that includes a cellulose-binding domain, which has been postulated to take part during cotton fiber elongation [2]. Our results indicates that GhCel1, characterized in this study, is not expressed in elongating cotton fibers (Fig. 6A), which supports the concept that different cellulases perform tissue-specific and specialized roles in cell wall extension in cotton. Abscission specific cellulases have been reported for many plant species [31]. The decrease in break strength of the cotton explants with an increase in expression of GhCel1 in the LAZ after ethylene treatment indicates that GhCel1 is ethylene-regulated and closely associated with abscission of cotton leaves. It is concluded that cotton leaf abscission is initiated and regulated by ethylene. Ethylene exposure increases the endogenous production of ethylene synthesis enzymes and increased synthesis of abscission specific cellulase in the leaf abscission zone, GhCel1, which includes a carbohydrate-binding domain at C-terminus.
Acknowledgements The authors are grateful to the Department of Science and Technology, New Delhi for financial support to carry out the work. Senior Research Fellowship to AM and Research Associate ship to SK from CSIR, India is acknowledged. Authors are thankful to Mr. V.K. Lal and Mr. Alok Saxena for scanning electron microscopy work.
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