Pepper pectin methylesterase inhibitor protein CaPMEI1 is required for antifungal activity, basal disease resistance and abiotic stress tolerance

Pectin is one of the main components of the plant cell wall that functions as the primary barrier against pathogens. Among the extracellular pectinolytic enzymes, pectin methylesterase (PME) demethylesterifies pectin, which is secreted into the cell wall in a highly methylesterified form. Here, we isolated and functionally characterized the pepper (Capsicum annuum L.) gene CaPMEI1, which encodes a pectin methylesterase inhibitor protein (PMEI), in pepper leaves infected by Xanthomonascampestris pv. vesicatoria (Xcv). CaPMEI1 transcripts are localized in the xylem of vascular bundles in leaf tissues, and pathogens and abiotic stresses can induce differential expression of this gene. Purified recombinant CaPMEI1 protein not only inhibits PME, but also exhibits antifungal activity against some plant pathogenic fungi. Virus-induced gene silencing of CaPMEI1 in pepper confers enhanced susceptibility to Xcv, accompanied by suppressed expression of some defense-related genes. Transgenic ArabidopsisCaPMEI1-overexpression lines exhibit enhanced resistance to Pseudomonas syringae pv. tomato, mannitol and methyl viologen, but not to the biotrophic pathogen Hyaloperonospora parasitica. Together, these results suggest that CaPMEI1, an antifungal protein, may be involved in basal disease resistance, as well as in drought and oxidative stress tolerance in plants.
Introduction
Plant cell wall, the first barrier of defense against invading pathogens, is composed of cellulose microfibrils cross-linked by hemicellulose, pectin and extensin. Pectin comprises a highly heterogeneous group of polymers that includes homogalacturonans and rhamnogalacturonans I and II (Willats et al.2001a, b). In pectin polymers, the galacturonic acid carboxyl groups can be methylesterified by a group of pectinases. These galacturonic acid methylesters are hydrolyzed by pectin methylesterase (PME) (Hagerman and Austin 1986; Pelloux et al. 2007). Many physiological processes, such as fruit maturation, microsporangenesis, seed germination and pollen growth, are affected by the degree to which PME changes the methlyesterification of galacturonic acid (Tieman and Handa 1994; Kagan-Zur et al. 1995; Ren and Kermode 2000; Bosch et al. 2005). The number and distribution of free and unesterified galacturonate carboxyl groups along the homogalacturonan chain has a great influence on the pectin properties and cell wall firmness (Willats et al. 2001a, b).
Plant pathogens hydrolyze the cell wall components of plants using extracellular pectinolytic enzymes (Collmer and Keen 1986), and PME is found in many plant pathogenic bacteria and fungi (Asoufi et al. 2007). The black filamentous fungus Aspergillus niger secretes a set of pectin-degrading enzymes that include PME, polygalacturonase and pectin lyase, and these decompose the plant cell wall to establish infection and absorb nutrients from the host (de Vries and Visser 2001). In soft rot disease caused by Erwinia chrysanthemi, bacterial PME activity is induced during infection of Saintpaulia plants; however, PME-deficient mutants are noninvasive to the host cells (Boccara and Chatain 1989). PMEs have also been found in higher plants; they play significant roles in physiological processes and interactions with pathogens (Micheli 2001). In tobacco plants, host cell pectin methylesterases are required for the tobacco mosaic virus movement protein to transfer the viruses between host cells (Chen et al. 2000). Furthermore, PME-degraded polygalacturonans are associated with recognition of fungal pathogens (Wietholter et al. 2003). PME is also involved in symbiosis-specific functions (Lievens et al. 2002). For example, plant PME isoenzymes may undergo organism-specific post-translational processing for structural and functional integrity during interactions with various microorganisms (Micheli 2001).
The PME enzyme activity is modulated specifically by inhibitor proteins such as the pectin methylesterase inhibitor (PMEI; Micheli 2001). Moreover, the PMEIs that inhibit demethylesterification of highly heterogeneous polymers (pectins) are the plant invertase inhibitor-related proteins, which are inhibitors of important metabolic enzymes (Koch 1996). Plant invertase inhibitor-related proteins play key roles in wounding, the plant defense reaction and developmental transitions (Raush and Greiner 2004), as well as during osmotic stress, senescence and seed development (Greiner et al. 1998, 1999). Investigation of gain- and loss-of-function mutants of tobacco cell wall invertase inhibitor (NtCIF) protein demonstrated that these inhibitor proteins play a role in seed development (Raush et al. 1998). Overexpression of the tobacco vacuolar invertase inhibitor protein (NtVIF) gene in transgenic potatoes is of potential use in the field of food technology (Greiner et al. 1998). However, there is little known about the in vivo functions of PMEI protein.
Proteinaceous inhibitors have been purified from kiwi (Actinidia deliciosa) (Giovane et al. 2004), Arabidopsis (Wolf et al. 2003; Raiola et al. 2004), rice (Han et al. 2005) and the jelly fig (Ficus awkeotsang cv. Makino) (Jiang et al. 2001, 2002). The kiwi PMEI is specific for PME (Balestrieri et al. 1990) and is active against PMEs from several plants, including kiwi, orange, apple, tomato, apricot, carrot, potato and banana (Ly-Nguyen et al. 2004). Four Cys residues conserved in several isoforms of PMEI are involved in the formation of disulfide bridges (Camardella et al. 2000). PME and PMEI form a stoichiometric 1:1 complex, in which the interaction between the PME and the inhibitor occurs in close proximity to the putative active site (Di Matteo et al. 2005). Since PME activity can be modulated by pH, the stability of the PME–PMEI complex is also affected by pH (Denès et al. 2000). Crystallographic work has revealed that an α-helical hairpin motif plays a structurally important role in PMEI activation (Hothorn et al. 2004). Many cDNAs encoding PMEIs have been isolated and functionally characterized from plants (Rausch and Greiner 2004); however, their role in plant defense remains relatively unknown.
To date, the functional analyses of genes associated with defense responses in plants have utilized reverse-genetics approaches based on loss-of-function via double-stranded RNA interference (Robertson 2004) or gain-of-function via transgenic gene expression (Clough and Bent 1998). Virus-induced gene silencing (VIGS) has been proven to be a useful method for assessing the function of target genes in Solanum species (Brigneti et al. 2004). In particular, VIGS studies have been used to investigate disease resistance signaling and defense-related genes such as SGT1 (Liu et al. 2002c; Peart et al. 2002b), EDS1 (Liu et al. 2002b; Peart et al. 2002a) and NPR1/NIM1 (Liu et al. 2002b) in Nicotiana benthamiana. AtPGIP1 is among the genes encoding pectic enzyme-related proteins; it encodes polygalacturonase (PG)-inhibiting protein (PGIP), and an antisense AtPGIP1 gene was used to silence its expression in transgenic Arabidopsis plants (Ferrari et al. 2006). Previous studies have revealed that overexpression of two closely related genes, AtPGIP1 and AtPGIP2, conferred resistance against Botrytis cinerea infection (Ferrari et al. 2003). Silencing of AtPGIP1 resulted in enhanced susceptibility to infection, as well as reduced activity of PGIP (Ferrari 2006). However, gene-silencing techniques such as VIGS and antisense RNA have not yet been used to investigate the role played in plant defense by other PMEI-encoding genes.
Here, we used a macroarray technique to isolate and functionally characterize a pectin methylesterase inhibitor gene, CaPMEI1, from a cDNA library of pepper (Capsicum annuum L.) leaves infected with Xanthomonas campestris pv. vesicatoria (Xcv; Jung and Hwang 2000). Local and systemic induction of CaPMEI1 was investigated in pepper plants following inoculation with pathogenic and non-pathogenic bacteria. We also examined the involvement of CaPMEI1 in defense-related signal transduction cascades via exogenous application of abiotic elicitors to pepper plants. Recombinant CaPMEI1 proteins were expressed in E. coli and exhibited antifungal activity against plant pathogenic fungi. Since it is difficult to transform pepper plants, we performed gene silencing and CaPMEI1 overexpression in pepper and Arabidopsis, respectively, to identify the cellular functions of the CaPMEI1 gene. The functional data obtained by VIGS and transgenic ectopic expression of CaPMEI1 suggest that this pepper pectin methylesterase inhibitor is involved in plant defense and abiotic stress responses.
Materials and methods
Plant materials and growth conditions
Pepper (Capsicum annuum L. cv. Nockwang) plants were grown at 28°C under a 16 h day at 70 μmol photons m−2 s−1. Plants were seeded into a plastic tray (55 × 35 × 15 cm3) containing steam-sterilized soil mix (peat moss, perlite and vermiculite; 5:3:2, v/v/v) and loam soil (1:1, v/v). At the two-leaf stage, seedlings were transplanted into plastic pots (5 × 15 × 10 cm3) containing the soil mix previously described.
Arabidopsisthaliana (ecotype Columbia) plants were grown in pots containing vermiculite, peat moss and perlite (1:1:0.5, v/v/v) in a growth chamber under a 12 h light/12 h dark photoperiod (130 μmol photons m−2 s−1) at 24°C and 60% relative humidity. Prior to sowing, the seeds were surface-sterilized using 1% sodium hypochlorite and vernalized at 4°C for 3 days to break dormancy.
Pathogens, inoculation procedures, disease rating and tissue staining
Xanthomonas campestris pv. vesicatoria (Xcv) strains Ds1 and Bv5-4a were used in this study. Bacteria were cultured overnight in yeast-nutrient (YN) broth (5 g L−1 yeast extract, 8 g L−1 nutrient broth) at 28°C. Prior to inoculation, bacteria were harvested by centrifugation and resuspended in sterile tap water (108 cfu mL−1). Pepper plants were inoculated at the six-leaf stage by infiltrating the bacterial suspension into the abaxial side of fully expanded leaves using an atomizer. Infected plants were then incubated for 16 h at 28°C in a moist chamber with 100% relative humidity. Bacteria-infected leaves were sampled at various time intervals after inoculation. To evaluate systemic induction in the upper leaves, bacterial suspensions (108 cfu ml−1) were infiltrated into the lower leaves of pepper plants at the two-leaf stage using a needless syringe. The bacterial strains used for this study included: virulent and avirulent strains of X. campestris pv. vesicatoria (Ds1 and Bv5-4a); a non-pathogenic strain (Pseudomonasfluorescens ATCC13525); and Escherichiacoli JM109.
Leaves of 6-week-old Arabidopsis plants were infiltrated with a suspension (OD600 = 0.001) of virulent Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000). The bacteria were cultured overnight at 28°C and suspended in 10 mM MgCl2. To determine bacterial growth, leaf discs were cut from infected leaves at different time intervals after inoculation. Bacterial growth was monitored by performing serial dilutions onto KB agar containing 100 μg mL−1 rifampicin. Each experiment was replicated three times.
Hyaloperonospora parasitica isolate Noco2 was propagated by weekly subculturing on 7- to 10-day-old Arabidopsis seedlings. The 7-day-old seedlings were inoculated with an H. parasitica asexual inoculum (5 × 104 conidiosporangia mL−1). The seedlings inoculated with H. parasitica were covered with a plastic dome to maintain a high relative humidity (80–100%) and grown in a growth chamber at 17°C. Seven days after inoculation, disease rating was scored for more than 50 plants per treatment. A visual disease rating was expressed as the number of sporangiophores on each cotyledon and was divided into five classes: 0–5, 6–10, 11–15, 16–20 and >20 sporangiophores per cotyledon. The cotyledons from inoculated plants were stained with lactophenol-trypan blue (10 mL lactic acid, 10 mL glycerol, 10 g phenol and 10 mg trypan blue, dissolved in 10 mL of distilled water) to assess H. parasitica growth. At 2–5 days after inoculation, the infected cotyledons were boiled for 5 min in the staining solution and de-stained overnight in chloral hydrate (2.5 g chloral hydrate dissolved in 1 mL distilled water). The destained cotyledons were subsequently mounted in 70% glycerol for microscopic observation.
Isolation and sequence analysis of pathogen-induced cDNAs
To construct a pathogen-induced cDNA library, pepper leaves were inoculated with the avirulent strain X. campestris pv. vesicatoria Bv5-4a. The pathogen-induced cDNA library was constructed using 5 μg poly(A)+ mRNA extracted from inoculated pepper leaves (Kim and Hwang 2000). To isolate pathogen-inducible cDNAs from the pepper cDNA library, we performed differential hybridization, as described previously by Jung and Hwang (2000). Digoxigenin (DIG)-labeled, single-stranded cDNA probes were generated from total RNA of healthy and Bv5-4a-infected leaves using RT-PCR. Nylon membranes were pre-hybridized at 65°C for 3 h in 5× SSC, 0.1% sodium lauroylsarcosine, 0.02% SDS and 1% blocking reagent (Boehringer Mannheim, Mannheim, Germany). Hybridization was then performed overnight at 65°C in the same buffer with single-stranded cDNA probes. Hybridized membranes were rinsed twice for 5 min with 2× SSC and 0.1% SDS at room temperature, and twice for 10 min with 0.1× SSC and 0.1% SDS at 65°C. The hybridization signals were detected according to the manufacturer’s protocol (Boehringer Mannheim).
We selected cDNA clones that were expressed strongly in pathogen-infected leaves, compared with those of healthy leaves. Clones were sequenced with an ABI 310 DNA sequencer (Applied Biosystems, Foster City, CA, USA) using the PRISM Big Dye™ Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems, Foster City, CA, USA). Sequencing results were analyzed using BLAST (National Center for Biotechnology Information; Altschul et al. 1997).
Treatment with abiotic elicitors and environmental stresses
The leaves of pepper plants at the six-leaf stage were sprayed with 5 mM salicylic acid (SA), 100 μM methyl jasmonate (MeJA) or 100 μM absicisic acid (ABA). Pepper plants treated with methyl jasmonate were sealed tightly in plastic bags. For ethylene treatment, whole plants were removed from soil, and then placed in a water-containing glass chamber, followed by injection of ethylene gas (5 μL L−1). For cold stress treatment, plants were placed at 4°C in a cold room. For wounding stress, the leaves were pricked with needles. To impose drought stress, the plants were removed from the soil and then incubated at room temperature without water. H2O2 treatment was performed by spraying leaves with 100 mM H2O2 solution. Leaves treated with various elicitors and abiotic stresses were removed from the plants, frozen in liquid nitrogen and stored at −70°C until used for RNA isolation.
RNA isolation and RNA gel blot analysis
Total RNA was extracted from pepper leaves, stems, roots, flowers and fruits using the guanidine isothiocyanate method (Chomczynski and Sacchi 1987). Frozen tissues (1 g) were ground to a powder and homogenized in 10 mL extraction buffer (4 M guanidine isothiocyanate, 25 mM sodium citrate [pH 7.0], 0.55% [w/v] N-laurylsarcosine and 0.1 M 2-mercaptoethanol). A mixture of 2 M sodium acetate (pH 4.0), water-saturated phenol and chloroform–isoamylalcohol (24:1) was added to the homogenate, followed by precipitation. Total RNA (20 μg) was separated by 1.2% formaldehyde-agarose gel electrophoresis and then blotted onto Hybond-N+ membranes (Amersham, Buckinghamshire, UK). Transferred RNA was fixed to the membrane using UV cross-linking.
The 3′ UTR region of CaPMEI1 was amplified for use in the generation of a gene-specific probe. The primers used for amplification of the CaPMEI1 gene-specific region were 5′-CATGGGTAAGTGCTGCCTTGACGGAC-3′ and 5′-GTTAACAAATGCATA TGGAACATTT-3′. The CaBPR1 coding region was amplified with the primers 5′-ATGGGACACTCTAATATTGCC-3′ and 5′-GACATCAGTTGGAAGTTCCAA-3′. The CaSAR82A coding region was amplified using the primers 5′-ATGGTTTCCAAAAGT AGTATTTTTATTT-3′ and 5′-TATGCTTAACAATTATTACTGAATA TAATC-3′. PCR-amplified products were 32P-labeled using a random priming kit (Boehringer, Mannheim). Hybridization was performed overnight at 65°C in 5% dextran sulfate, 0.25 M disodium phosphate (pH 7.2), 7% (w/v) sodium dodecyl sulfate (SDS) and 1 mM EDTA. Following hybridization, the membranes were washed twice for 10 min with 2× SSC and 0.1% SDS at room temperature, and then twice for 5 min with 0.1× SSC and 0.1% SDS at 65°C. Equal loading of RNA was confirmed by ethidium bromide-staining of ribosomal RNA.
In situ RNA localization
In situ RNA localization was performed as described previously (Lee et al. 2000). Leaf tissue was fixed for 2 h in a solution of 1× phosphate-buffered saline (PBS), 4% paraformaldehyde and 1 μL mL−1 Triton X-100 by vacuum infiltration for 10 min and shaking for 2 h at room temperature. The fixed samples were washed with 1× PBS, dehydrated through a graded ethanol and xylol series, and then embedded in liquid paraplast at 57°C (Sherwood Medical, St. Louis, MO, USA). Paraplast-embedded sections (10 μm in thickness) were placed on glass slides coated with poly-l-lysine (Sigma, St. Louis, MO, USA) and incubated at 42°C. Sections were deparafinated using xylene and 1-propanol, followed by rehydration with serial dilutions of ethanol. Tissue samples were treated with 0.01 M Tris–HCl (pH 8.0) and 1% bovine serum albumin (BSA) for 10 min, followed by incubation in 100 mM Tris–HCl solution (pH 8.0) containing proteinase K (5 mg mL−1) and 50 mM EDTA, for 30 min at 37°C. Sections were treated with 0.25% acetic anhydride in 100 mM triethanolamine (pH 8.0) for 10 min at room temperature to inhibit non-specific signals. Digoxigenin (DIG)-labeled probes were prepared using the Dig High Prime DNA Labeling and Detection kit, according to the manufacturer’s instructions (Boehringer Mannheim). Sections were prehybridized and hybridized at 42°C in 50% formamide, 4× SSC, 150 μg mL−1 tRNA and 0.5% blocking reagent (Boehringer Mannheim). After hybridization, the sections were washed twice with 50% formamide and 4× SSC at 42°C, twice with 4× SSC and then once with diethyl pyrocarbonate (DEPC)-treated water. The DIG signal was detected, according to the manufacturer’s instructions (Boehringer Mannheim). Color reactions were developed overnight at 37°C with nitro-blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP), and reactions were stopped with TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0). Sections were photographed with Kodak ISO 100 film under a bright field Olympus BH-2 microscope (Olympus, Tokyo, Japan). To demonstrate the specificity of in situ hybridization, control hybridizations were performed without DIG-labeled probes.
Purification of recombinant CaPMEI1 protein
The CaPMEI1 coding region, including the stop codon, was amplified by PCR using the forward and reverse primers 5′-GAATTCATGGAAGGTGGCAATTTTCT-3′ and 5′-CTCGAGTAGCCGTGAAGGGCAGCCAGACG-3′, respectively. Amplification products were cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA, USA), which was digested with EcoRI and XhoI and ligated into the similarly digested pET32a (Novagen, Madison, WI, USA).
Escherichia coli BL21 (DE3) pLysS (Novagen), which is defective for thioredoxin reductase, was used as a host for recombinant protein expression. Cultures were started from single colonies, grown in LB broth at 37°C, and then diluted 1:100 at OD600 = 0.6. After dilution, bacteria were grown to a density of OD600 = 0.6, induced with 10 mM IPTG and grown for a further 5 h at 37°C. Cells were harvested by centrifugation for 15 min at 5,000g, extracted with denaturation buffer (8 M urea, 20 mM sodium phosphate buffer [pH 7.8], 500 mM NaCl) and disrupted by sonication. Following centrifugation at 5,000g for 15 min at 4°C, the supernatant was loaded onto a 1.5 mL column of Ni-NTA resin (Qiagen, Hilden, Germany), which was washed with an initial denaturing wash buffer (8 M urea, 20 mM sodium phosphate buffer [pH 6.0], 500 mM NaCl), followed by a second denaturing wash buffer (8 M urea, 20 mM sodium phosphate buffer [pH 5.3], 500 mM NaCl). Bound fusion protein was eluted with a final denaturing elution buffer (8 M urea, 20 mM sodium phosphate buffer [pH 4.0], 500 mM NaCl). Recombinant proteins were dialyzed against a buffer containing 10 mM Tris (pH 8.0) and 0.1% Triton X-100, according to the manufacturer’s protocol. As a control, thioredoxin was purified using a native buffer that did not contain urea. The purified CaPMEI1 recombinant protein was digested for 16 h at room temperature with recombinant enterokinase (1 U/5 μg recombinant protein; Novagen).
SDS polyacrylamide gel electrophoresis
CaPMEI1 was dissolved in 1× SDS sample buffer (0.9 g glycerol, 5% SDS, 1% bromophenol blue, 0.1 mL mercaptoethanol and 1 L H2O), separated by 12% SDS-PAGE as described previously (Laemmli 1970), and stained with Coomassie Brilliant Blue R-250. Molecular weights were estimated using 6.5 to 200.5 kDa marker proteins (Bio-Rad, Hercules, CA, USA).
Pectin methylesterase enzyme inhibitor assay
The inhibitory effect of CaPMEI1 on the enzymatic activity of pectin methylesterase (PME) was assayed under standard conditions (Grsic-Rausch and Rausch 2004). The reaction mixture comprised 894 μL 0.4 mM NAD in 50 mM phosphate buffer (pH 7.5), 80 μL 5% (w/v) pectin (Sigma) in H2O, 8 μL formaldehyde dehydrogenase (0.35 U; Sigma) and 8 μL alcohol oxidase (1.0 U; Sigma). After mixing, the reaction was started with the addition of 10 μL (7.8 mU) PME from orange peel (Sigma). To analyze CaPMEI1 inhibition of PME, 1 μL inhibitor solution (0.5 mg CaPMEI1 fusion protein, 10 mM Tris buffer [pH 7.5], 0.1 M NaCl) was mixed with 10 μL PME (7.8 mU) and 0.5 μL 3 M K-acetate buffer (pH 5.3), followed by incubation for 15 min at room temperature. All reaction temperatures were maintained at 25°C. Reaction rates were recorded continuously at 340 nm using a DU650 UV-visible spectrophotometer (Beckman, Fullerton, CA, USA). PME–PMEI interactions were determined by measuring the rate of NADH formation per minute at pH 7.5 and 25°C.
In vitro antifungal activity
To determine the antifungal activity of CaPMEI1, we examined its effect on plant pathogenic fungi. Fusarium oxysporum f.sp. matthiole and Alternaria brassicicola were incubated on potato dextrose agar (PDA) at 28°C for 1–2 weeks; B. cinerea was incubated at 20°C for 1–2 weeks. Fungi were grown in 48-well plates (Cell Wells™, Corning Glass Works, Corning, NY, USA) containing sterile 4× potato dextrose broth (PDB, 100 μL) and a range of concentrations of purified CaPMEI1 protein (0–500 μg mL−1). Spore suspensions (104 spores mL−1) were prepared and 100 μL of inoculum was added to each microwell. Plates containing B. cinerea were incubated at 20°C; the other fungi were incubated at 28°C for 4–7 days.
To determine the inhibitory effect of CaPMEI1 on spore germination and hyphal growth, CaPMEI1 (0.1–500 μg mL−1) or the thioredoxin control were added to F. oxysporum f.sp matthiolae spore suspensions (105 spores mL−1), placed on glass slides and incubated for 12 h at 28°C. After 6 h incubation, 100 germinated spores were examined using a haemocytometer and the lengths of 50 individual hyphae were determined. The experiment was repeated three times.
Plasmid construction and plant transformation
The CaPMEI1 coding region was PCR-amplified without stop codon using the following primers to generate XbaI and BamHI sites: 5′-TCTAGAATGGAAGGTGGCA ATTTTCTCACA-3′ and 5′-GGATCCGCCGTGAAGGGCAGCCAGACGGT -3′. The fragment was inserted into pCR2.1-TOPO (Invitrogen) and digested with XbaI and BamHI. The construct was confirmed by sequencing. To generate the reporter construct p35S-CaPMEI1-GFP, the fragment was inserted into the XbaI–BamHI sites of the binary vector p35S-smGFP. p35S-smGFP was generated by fusing the gene encoding smGFP from the vector 326-GFP into pBIN35S digested with HindIII and EcoRI. The CaMV35S promoter-CaPMEI1-smGFP construct was introduced into the Agrobacterium tumefaciens strain EHA105 using electroporation.
Transgenic Arabidopsis plants were generated by floral-dipping wild-type (Col-0) plants into an A. tumefaciens culture containing the appropriate construct (Clough and Bent 1998). Agrobacterium was grown at 28°C and 250 rpm in YEP medium (10 g Bacto peptone, 10 g yeast extract, 5 g NaCl) supplemented with kanamycin (25 μg ml−1). Cells were harvested by centrifugation for 20 min at 5,500g and resuspended in inoculation media containing 5.0% sucrose and 0.05% Silwet L-77 (OSi Specialties, Inc., Danbury, CT, USA) to OD600 = 0.8. Arabidopsis plants with 2–10 cm long bolts were inoculated by dipping into an Agrobacterium suspension (OD600 = 0.8) and then left in the dark overnight, prior to return to a growth chamber. Seeds from the transformed Arabidopsis plants were collected and screened for kanamycin resistance.
Virus-induced gene silencing (VIGS)
The TRV-based VIGS system was used for silencing of CaPMEI1 in pepper plants, as described previously (Liu et al. 2002a; Chung et al. 2004). The pepper CaPMEI1 coding region was cloned into pTRV2 to generate the construct pTRV2:CaPMEI1. The fully extended cotyledons of 2-week-old pepper seedlings were co-infiltrated with A. tumefaciens GV3101 carrying pTRV1 or pTRV2:CaPMEI1 (OD600 = 0.2 for each construct). Plants were placed in a growth room at 25°C under a 16 h light/8 h dark photoperiod, to allow for growth and viral spread. Experiments were performed 5–6 weeks after the induction of silencing.
RT-PCR analysis
The RT reactions (20 μL) were performed at 42°C with total RNA (2 μg), oligo p(dT)15 primer (Roche, Mannheim, Germany) and AMV reverse transcriptase (Roche). Aliquots (1 μL) of the RT reaction products were used for RT-PCR analysis with the following gene-specific primers: 5′-CAGGATGCAACACTCTGGTGG-3′ and 5′-ATCAAAGGC CGGTTGGTC-3′ for CaBPR1 (accession no. AF053343); 5′-TGTCGAAGGTGGTCC AATAAA-3′ and 5′-TAGACAGAAGGATTGGCGAGG-3′ for CaPR10 (accession no. AF244121); 5′-ATCTGTACCAGCTTGCACGTGT-3′ and 5′-CCCTCACTGTGGCCT TGG-3′ for CaPOA1 (accession no. AF442387); and 5′-CAGGGAGATGAATTCTGA GGC-3′ and 5′-CATATGAACCTCTATGGATTTCTG-3′ for CaSAR82A (accession no. AF313766). RT-PCR conditions were 95°C for 10 min and 30 cycles of 95°C for 30 s, 52°C for 30 s, and 72°C for 1 min and 30 s. Single bands for PCR products were confirmed on an agarose gel.
Drought stress treatment and evaluation
For germination tests, seeds from Arabidopsis wild-type and CaPMEI1-OX transgenic lines were surface-sterilized and placed on MS media (Murashige and Skoog 1962) containing 200 or 600 mM mannitol. The seeds were maintained at 4°C for 48 h under dark conditions to synchronize germination, and then transferred to a growth chamber. Experiments were repeated at least three times, using approximately 100 seeds.
Surface-sterilized seeds were also used for examination of relative root length. Seedlings were grown on plates placed vertically in growth chambers and root length was estimated for 14 days. To assess mannitol tolerance, seedlings were grown for 7 days in 1× MS agar media supplemented with 1% sucrose, and then transferred to wells containing 1× MS liquid media supplemented with 100, 200, 300 or 400 mM mannitol. For drought treatment, 4-week-old soil-grown plants were deprived of water for 15 days and then re-watered on day 16. To minimize experimental variation, both the transgenic and control plants were grown in the same tray. Experiments were repeated at least three times.
For transpiration rate measurements, leaves of 4-week-old plants were detached and maintained at room temperature. Leaf weight was determined every 20 min for 2 h, and then every 1 h thereafter. Each measurement was performed using eight leaves. Experiments were repeated at least three times with similar results.
Oxidative stress treatment and evaluation
For analysis of oxidative stress tolerance, surface-sterilized seeds from Arabidopsis wild-type, vector control and CaPMEI1-OX transgenic lines were placed on MS medium containing different concentrations of methyl viologen (MV; Sigma). After synchronized germination, seeds were maintained in a growth chamber. To investigate whether or not MV treatment caused retardation of seedling development, 7-day-old seedlings were randomly transferred into well-plates containing 1× MS liquid medium supplemented with 0.05, 0.1 or 0.5 μM MV, and then grown for a further 2 weeks. To determine leaf senescence during MV treatment, fully expanded leaves were detached from 4-week-old plants and rinsed briefly with 70% ethanol. The detached leaves were floated on MS medium containing MV for 24 h following extraction with liquid nitrogen. Their chlorophyll content was measured spectrophotometrically according to the formula (Ca + b = 5.24 × A664 + 22.24 × A648), where C is the chlorophyll concentration (Ca + b) in micrograms per mL and A is absorption (Lichtenthaler 1987).
Results
Isolation and sequence analysis of CaPMEI1 cDNA in pepper
To identify the molecular mechanisms involved in plant defense against the invasion of microbial pathogens, we performed a differential hybridization screening of a cDNA library constructed from pepper leaves undergoing an incompatible interaction with Xcv (Jung and Hwang 2000). PR-1, PR-10, SAR8.2 and Myb transcription factor were among the pathogen-induced genes isolated by this screen. In addition, we isolated a full-length cDNA (834 bp) designated CaPMEI1 (Capsicum annuumpectin methylesterase inhibitor protein, accession no. AF477956), which contains a 54 bp 5′ untranslated sequence and a 174 bp 3′ untranslated region. The predicted open reading frame encodes a full-length protein of 200 amino acid residues, with an estimated molecular mass of 21.39 kDa and an isoelectric point (pI) of 6.51.
BLAST searches were performed between the predicted amino acid sequence of CaPMEI1S (accession no. AF477956) from C. annuum and the EMBL/GenBank database. These sequence analyses revealed that CaPMEI1 contains a conserved pectin methylesterase inhibitor (PMEI) domain of 156 amino acids (Fig. 1). Four cysteine residues were conserved in all the aligned proteins (Fig. 1), and these are predicted to be involved in the formation of disulfide bridges (Camardella et al. 2000). The predicted amino acid sequence of CaPMEI1 is 80% identical to that of Nicotiana tabacum DC1.2 protein (accession no. BAA95794) and has 50% identity with Arabidopsis thaliana ripening-related protein (accession no. BAA97200), 36% with Pinus radiata pectinesterase homolog (accession no. T08112) and 24% with A. thaliana invertase homolog (accession no. NP_201267).
Fig. 1Amino acid sequence alignments of pepper CaPMEI1 with Nicotiana tabacum DC1.2 protein (accession no. BAA95794), A. thaliana ripening-related protein (accession no. BAA97200), A. thaliana invertase homolog (accession no. NP201267) and Pinus radiata pectinesterase homolog (accession no. T08112). The boxed amino acid sequences represent the pectin methylesterase inhibitor (PMEI) domain. The conserved cysteine residues are marked by asterisks and the shaded regions represent conserved amino acid residues
Organ-specific expression of CaPMEI1 in pepper tissues
RNA gel blot analysis was performed to examine the organ-specific expression of CaPMEI1 in pepper plants. Although high levels of CaPMEI1 transcription were observed in stem tissues, only relatively low levels of expression were found in leaf, root, flower and green fruit (Fig. 2a). CaPMEI1 transcripts were not detected in red fruit.
Fig. 2RNA gel blot analysis of expression of CaPMEI1, CaSAR82A and CaBPR1 in pepper plants. Membranes were hybridized with probes from the 3′ UTR region of pepper CaPMEI1 cDNA or full-length CaBPR1 cDNA. Equal loading (20 μg) was verified by visualizing RNA on a gel stained with ethidium bromide. H healthy, M mock-inoculated or mock-treated. aCaPMEI1 expression in various organs of pepper plants. b Expression of CaPMEI1 and CaBPR1 in pepper leaves at various time intervals after inoculation with C. coccodes or virulent strain Ds1 (compatible interactions with pepper: susceptible response) and avirulent strain Bv5-4a (incompatible interactions with pepper: resistant response) of Xcv.cCaPMEI1 expression in lower (local) infected and upper (systemic) uninfected leaves at various time intervals after inoculation with the virulent and avirulent strains Ds1 and Bv5-4a of Xcv, P.fluorescence ATCC13525 and E.coli JM109. The lower leaves of pepper plants were inoculated at the 6-leaf stage. For the mock-inoculation, the lower leaves were infiltrated with 10 mM MgSO4. d Expression of CaPMEI1 and CABPR1 in pepper leaves at various time intervals after treatment with salicylic acid (SA, 5 mM), methyl jasmonate (MeJA, 100 μM), ethylene (5 μl L−1) and abscisic acid (100 μM). e Expression of CaPMEI1 and CaSAR82A in pepper leaves at various time intervals after treatment with drought, wounding, cold and H2O2
CaPMEI1 induction by pathogen infection
To assess the accumulation of CaPMEI1 transcripts in pepper leaves during the compatible and incompatible interactions with pathogens, leaves were inoculated with C. coccodes and Xcv Ds1 (virulent, compatible), as well as Xcv Bv5-4a (avirulent, incompatible). CaPMEI1 transcripts were not detected in leaves mock-inoculated with sterilized water (Fig. 2b, c). During fungal infection at the four- and eight-leaf stages, CaPMEI1 expression was induced at 48 and 12–48 h after inoculation, respectively, suggesting that the adult plant stage is more resistant to C. coccodes (Fig. 2b, upper panel). CaPMEI1 transcripts increased rapidly within 30 min after infection by both the virulent (Ds1) and avirulent (Bv5-4a) strains of Xcv (Fig. 2b, lower panel). High transcript levels accumulated by 18 h after infection with the virulent strain (Ds1) that causes a susceptible response; however, they started to decline rapidly by this time following infection with the avirulent strain (Bv5-4a) that causes a resistant response. CaBPR1 (Capsicum annuum basic PR1) expression was observed to confirm the success of individual inoculations. CaBPR1 transcripts were detected at 6 h after inoculation with the virulent and avirulent strains of Xcv and increased gradually during the 24 h following inoculation (Fig. 2b).
Local and systemic expression of CaPMEI1 in infected pepper plants
The local and systemic CaPMEI1 expression was analyzed in pepper leaves inoculated with Xcv and the non-pathogenic bacteria Pseudomonas fluorescens ATCC13525 and E. coli JM109 (Fig. 2c). In leaves infected with the virulent (Ds1) and avirulent (Bv5-4a) strains of Xcv, CaPMEI1 transcripts were detected 3 h after inoculation and levels decreased thereafter. Infection of lower (local) leaves by either Xcv strain induced systemic accumulation of CaPMEI1 transcripts in the upper leaves. The systemic induction by the avirulent strain (Bv5-4a) infection was stronger than that by the virulent strain (Ds1) infection. Local or systemic induction of CaPMEI1 transcripts was not observed 30 h after infection. In response to the non-pathogenic bacteria P. fluorescens and E. coli, CaPMEI1 transcripts were detected 3 h after inoculation and decreased subsequently in both local and systemic leaves.
CaPMEI1 induction following treatment with plant hormones
Plant hormones are involved in defense-related signal transduction pathways (Hammond-Kosack and Jones 1996; Chung et al. 2007). We examined whether or not CaPMEI1 expression is induced by plant hormones using pepper leaves treated with salicylic acid (SA), ethylene, methyl jasmonate (MeJA) or abscisic acid (ABA) (Fig. 2d). Salicylic acid strongly induced expression of CaPMEI1 at 1 and 2 h, as well as between 18 and 24 h after treatment. Following treatment with ethylene and MeJA, CaPMEI1 expression was strongly induced for 24 h. In addition, strong CaPMEI1 induction was observed 1 h after treatment with ABA.
In comparison, SA only induced CaBPR1 transcription 18–24 h after treatment (Fig. 2d), although transcripts were detected 6 h after treatment with ethylene and MeJA. In response to ethylene treatment, CaBPR1 transcripts increased rapidly between 18 and 24 h after treatment, but increased gradually between 6 and 24 h after MeJA treatment. CaBPR1 expression was induced between 2 and 12 h after ABA treatment.
CaPMEI1 induction by abiotic stresses
Abiotic stresses resulted in differential induction of CaPMEI1 in pepper leaves (Fig. 2e). The CaPMEI1 gene was responsive to mechanical injury, and expression was observed between 15 min and 6 h after wounding, although no transcription was detected thereafter. CaPMEI1 transcription also increased between 2 and 18 h after cold treatment. CaPMEI1 transcription was induced at 1 h of drought stress, but was undetectable thereafter. In response to H2O2 treatment, CaPMEI1 transcript levels increased for the first 6 h and declined thereafter. The marker genes CaBPR1 and CaSAR82A were used for comparison, as they also exhibit differential induction by these abiotic stresses.
In situ localization of CaPMEI1 transcripts
We performed an in situ hybridization using a CaPMEI1 probe to examine spatial expression of CaPMEI1 in pepper leaf tissues (Fig. 3). No hybridization signals were observed in non-treated leaves (Fig. 3a, b). Intense localization of CaPMEI1 transcripts was observed in the xylem area of the vascular bundle in non-treated stems (Fig. 3c). CaPMEI1 transcripts were detected in the vascular bundle of leaves inoculated with C. coccodes (Fig. 3e) and in the vascular bundles and upper epidermis of leaves treated with ethylene (Fig. 3g). No transcript signals were observed in any pepper tissues hybridized with the CaPMEI1 sense DIG-labeled RNA probe (Fig. 3b, d–f). These in situ hybridization results are supported by the temporal expression patterns observed in the RNA gel blot analysis of CaPMEI1 expression (Fig. 2).
Fig. 3In situ localization of CaPMEI1 transcripts in pepper leaf and stem tissues. Cross sections of leaf tissues were hybridized with CaPMEI1 antisense (a, c, e, g) and sense (b, d, f, h) DIG-labeled RNA probes, and then photographed under bright-field conditions. The transcript signal is purple. a, b Untreated leaf tissues. c, d Untreated stems. e, f Leaf tissues at 24 h after inoculation with C. coccodes. g, h Leaf tissues treated with 5 μl L−1 ethylene. P phloem, X xylem, UE upper epidermis, LE lower epidermis, Vs vascular bundle, C cortical cell
Inhibition of pectin methylesterase (PME) activity by CaPMEI1 protein
To prepare recombinant CaPMEI1 protein, the CaPMEI1 coding region was PCR-amplified and cloned into pET32a. E. coli strain BL21 (DE3) pLysS was used as a host for the recombinant expression of the CaPMEI1 construct and an empty vector control, yielding recombinant CaPMEI1 and thioredoxin, respectively. The purified CaPMEI1 (Fig. 4a, lane 5) and thioredoxin (Fig. 4a, lane 3) were examined by SDS-PAGE analysis. The purified thioredoxin–CaPMEI1 fusion protein formed a single band with an apparent molecular mass of 42 kDa (Fig. 4a, lane 5). Following cleavage of thioredoxin by enterokinase digestion, the purified CaPMEI1 protein (Fig. 4a, lane 6) was used for biological function determination. The cleaved recombinant CaPMEI1 protein was found exclusively in the insoluble fraction as inclusion bodies (data not shown). After attempting unsuccessfully to renature the cleaved protein using Triton X-100 as a detergent (Kim and Hwang 1994), we performed the PME inhibition assay using crude and purified CaPMEI1 fusion proteins.
Fig. 4Inhibition of pectin methylesterase (PME) activity by the pepper pectin methylesterase inhibitor (CaPMEI). a Recombinant CaPMEI1 expression in E. coli BL21 (DE3) pLysS. Cells were grown in LB media, and recombinant protein expression was induced with 1 mM IPTG. M, molecular marker (kDa); Lane 1, Uninduced E. coli BL21 cell extracts; Lane 2, Soluble fraction of E. coli BL21 cell extracts encoding thioredoxin protein after IPTG induction; Lane 3, Purified thioredoxin; Lane 4, Crude protein extracts of E. coli cells producing the thioredoxin–CaPMEI1 fusion protein after IPTG induction; Lane 5, Purified thioredoxin–CaPMEI1 fusion protein; Lane 6, Cleaved CaPMEI1 and thioredoxin proteins following enterokinase digestion. Protein staining was performed using Coomassie brilliant blue. b Inhibition of PME activity following treatment with CaPMEI1. For the inhibition assay, crude CaPMEI1 (0.5 μg) was mixed with 7.8 mU of orange peel PME in a total volume of 11 μL, and then preincubated at 25°C for 15 min, followed by addition to the reaction solution
CaPMEI1 contains a pectin methylesterase inhibitor (PMEI) domain (Fig. 1) and shares similarity with a plant pectin methylesterase inhibitor related to regulation of pectin degradation (Raiola et al. 2004). In addition, PMEIs have been found to play crucial roles in plant and microbial enzyme regulation of pectin degradation by de-esterification (Raiola et al. 2004). To determine whether or not orange peel PME activity is inhibited in the presence of crude CaPMEI1 fusion protein, we used the PME enzyme assay to measure the effect of PME inhibitor on the formation of NADH (Fig. 4b). Both crude and purified CaPMEI1 (ca. 0.5 μg) strongly inhibited PME enzyme activity, and a higher concentration of purified CaPMEI1 (ca. 1.0 μg) exhibited the strongest inhibition. However, thioredoxin alone did not exhibit any inhibitory effect. These results suggest that CaPMEI1 functions as an inhibitor of PME activity.
Antimicrobial activity of CaPMEI1
CaPMEI1 exhibited antifungal activity against the three plant pathogenic fungi examined: F. oxysporum f.sp. matthiole, A. brassicicola and B. cinerea (Fig. 5a). The thioredoxin–CaPMEI1 fusion protein (50 μg mL−1) suppressed mycelial growth of the three plant pathogenic fungi, whereas thioredoxin alone did not. Furthermore, the purified thioredoxin–CaPMEI1 (500 μg mL−1) fusion protein inhibited spore germination and hyphal growth of F.oxysporum f.sp. matthiole, whereas thioredoxin alone did not (Fig. 5b). Increasing concentrations of recombinant thioredoxin–CaPMEI1 fusion protein increasingly inhibited both spore germination and hyphal growth (Fig. 5c).
Fig. 5Assay of thioredoxin–CaPMEI1 fusion protein antimicrobial activity. a Inhibitory effects of the CaPMEI1–thioredoxin fusion protein on mycelial growth of the plant pathogenic fungi F. oxysporum f.sp. matthiolae, A. brassicicola and B. cinerea. In each plate, the upper wells (a) were treated with purified thioredoxin and the lower wells (b) were treated with the purified thioredoxin-CaPMEI1 fusion protein. b Inhibition of germination and hyphal growth of F.oxysporum f.sp. matthiolae. Fungal spores were allowed to germinate and grow in 100 μL potato dextrose broth medium alone (top), or with 500 μg mL−1 thioredoxin (middle) or 500 μg mL−1 thioredoxin–CaPMEI1 fusion protein (bottom). Photographs were taken after incubation for 10 h at 28°C. Bars 20 μm. c Inhibition of spore germination and hyphal growth of F.oxysporum f.sp. matthiolae by CaPMEI1. The percentage of germinated spores and the length of fungal hyphae were determined by light microscopy. Data represent means ± SD from three independent experiments
Enhanced susceptibility of CaPMEI1-silenced pepper plants to Xcv infection
Since inoculation with Xcv strongly induced CaPMEI1 expression in pepper plants, we performed the virus-induced gene silencing (VIGS) technique (Liu et al. 2002a; Chung et al. 2004) to examine its cellular function during pathogen infection. The full-length ORF of CaPMEI1 was used to construct pTRV2:CaPMEI1. Five to six weeks after induction of silencing, empty vector control (TRV:00) and CaPMEI1-silenced (TRV:CaPMEI1) pepper plants were inoculated with virulent and avirulent strains Ds1 and Bv5-4a of Xcv, respectively. To assess the efficiency of VIGS, CaPMEI1 transcript levels were examined by RT-PCR (Fig. 6). CaPMEI1 transcripts were nearly undetectable in both non-inoculated empty vector control and CaPMEI1-silenced plants. However, at 12 h after inoculation with virulent and avirulent Xcv, we observed strong induction of CaPMEI1 in the empty vector control plants, whereas only weak or undetectable transcript levels were found in CaPMEI1-silenced plants. These results confirm the effective silencing of the target gene in pepper plants.
Fig. 6RT-PCR analysis of expression of CaPMEI1 and several defense-related genes in empty vector control (TRV:00) and CaPMEI1 gene-silenced (TRV:CaPMEI1) pepper plants 12 h after inoculation with the virulent (Ds1; C, compatible) and avirulent (Bv5-4a; I, incompatible) strains of Xcv (5 × 106 cfu mL−1). 18S rRNA levels were visualized as a loading control. This experiment was repeated three times with similar results. H healthy leaves, CaBPR1 basic pathogenesis-related protein 1, CaPR10 putative ribonuclease-like protein, CaPOA1 ascorbate peroxidase 1, and CaSAR82A SAR8.2
To determine whether or not the expression of defense-related genes is affected by bacterial infection in the silenced plants, we used RT-PCR to analyze transcript levels of some defense-related genes (Fig. 6). In the empty vector control plants, both CaBPR1 (basic pathogenesis-related protein 1) and CaPR10 (putative ribonuclease-like protein) were strongly induced by virulent and avirulent Xcv infection. However, induction of these genes was slightly reduced in CaPMEI1-silenced plants infected with virulent Xcv, but was unaffected in plants infected with avirulent Xcv. Thus, CaPMEI1 gene silencing did not alter the induction of CaPOA1 (ascorbate peroxidase) or CaSAR82A (SAR 8.2) in response to Xcv infection.
To examine the function of the CaPMEI1 gene in basal defense or gene-for-gene resistance, empty vector control and CaPMEI1-silenced pepper plants were infected with virulent or avirulent Xcv (Fig. 7). CaPMEI1 gene silencing significantly increased susceptibility to virulent Xcv infection, but not to avirulent Xcv infection. The CaPMEI1-silenced pepper leaves exhibited more severe disease symptoms 5 days after virulent Xcv inoculation than did empty vector control plants, and these symptoms were accompanied by severe chlorosis and enlarged water-soaked lesions (Fig. 7a). However, we did not observe any phenotypical changes in the cell death response of CaPMEI1-silenced plants following avirulent Xcv inoculation (Fig. 7a). At 3 days after inoculation with virulent Xcv, bacterial growth was tenfold greater in CaPMEI1-silenced plants than in the empty vector controls (Fig. 7b). However, silencing of CaPMEI1 conferred a slightly enhanced susceptibility to avirulent Xcv. These findings suggest that CaPMEI1 may function in basal resistance of pepper plants against Xcv infection, rather than gene-for-gene resistance.
Fig. 7Enhanced disease susceptibility of CaPMEI1-silenced pepper plants to infection by the virulent Xcv strain Ds1, but not the avirulent Xcv strain Bv5-4a. a Disease symptoms developed on the leaves at different time points after inoculation with the virulent Xcv strain Ds1 (5 × 106 cfu mL−1) and the avirulent Xcv strain Bv5-4a (various bacterial concentrations). b Bacterial growth in leaves of empty vector control (TRV:00) or CaPMEI1-silenced (TRV:CaPMEI1) pepper plants at different time points after inoculation with the virulent Xcv strain Ds1 or the avirulent Xcv strain Bv5-4a (104 cfu mL−1). Data represent the mean ± SD from three independent experiments
Enhanced resistance of CaPMEI1-OX plants to Pst DC3000
The p35S-CaPMEI1-GFP and p35S-GFP overexpression (OX) plants were generated by transformation of A. thaliana using the floral dipping method (Clough and Bent 1998). Plants transformed with the empty vector p35S-GFP were used as a control. Seeds (T1) were collected from each transformed plant and screened for resistance to kanamycin. Northern blot analysis was performed on T1 plants exhibiting kanamycin resistance to determine the integrity of the inserted transgene. Three T2 plants showing strong CaPMEI1 expression were selected for analyses in planta (Fig. 8a).
Fig. 8Responses of wild-type (Col-0) Arabidopsis and CaPMEI1-OX transgenic plants to infection with P. syringae pv. tomato DC3000. a RNA gel blot analysis confirming CaPMEI1 overexpression (OX) in the transgenic Arabidopsis lines. Total RNA (10 μg) was loaded into each lane. The 3′ UTR region of pepper CaPMEI1 cDNA was used as a probe. b Growth of Pst DC3000 in the leaves of wild-type and transgenic plants. The mature leaves of the 6-week-old plants were infiltrated with a Pst Dc3000 suspension (105 cfu mL−1), and the degree of bacterial growth was rated at 0, 2 and 4 days after inoculation. c Disease symptoms on leaves of 6-week-old plants infiltrated with virulent Pst DC3000 (105 cfu mL−1). d Expression of pathogen-related (PR) genes in transgenic plants. Northern blot analyses were performed with 10 μg total RNA prepared from 5-week-old leaves of the wild-type (WT), vector control (smGFP) and transgenic (CaPMEI1::smGFP) plants. The samples were collected at 5, 15 and 25 h following pathogen infiltration with a suspension of the virulent strain Pst DC3000 (105 cfu mL−1)
To determine the contribution of CaPMEI1 to Arabidopsis resistance, wild-type and CaPMEI1-OX plants were inoculated with virulent P. syringae pv. tomato DC3000 (Pst; 105 cfu mL−1). Bacterial titers were determined 4 days after inoculation. CaPMEI1-OX plants exhibited much less growth of Pst bacteria than wild-type or empty vector control plants (Fig. 8b). In the transgenic plants, this reduced bacterial multiplication was most pronounced at 4 days after inoculation. At 6 days after inoculation, wild-type (Col-0) and empty vector control plants developed typical chlorotic symptoms in the infected leaves, whereas the transgenic plants displayed few disease symptoms (Fig. 8c).
Expression of defense-related genes in CaPMEI1-OX plants
To gain insight into the role played by CaPMEI1 in PR gene induction, we examined the expression patterns of the well-established marker genes AtPR1a, AtPR2 and AtPR5 in the defense responses of wild-type, vector control and the CaPMEI1-OX Arabidopsis plants infected with virulent Pst DC3000 (Fig. 8d). The expression of AtPR1a and AtPR2 was very similar in the CaPMEI1-OX, wild-type and vector control plants. AtPR5 expression was not induced in either un-inoculated or inoculated, wild-type or vector control plants. However, there was significant expression of AtPR5 in the transgenic CaPMEI1-OX plants at 15 and 25 h after Pst DC3000 infection. AtPR1a, AtPR2 and AtPR5 expression is known to be regulated via the SA-dependent pathway in Arabidopsis (Uknes et al. 1992), and in nahG plants, lack of SA reduces expression of these three PR genes during pathogenesis (Delany et al. 1994; Nawrath and Metraux 1999). In our experiments, AtPDF1.2 (defensin) transcripts were not detected in wild-type, vector control or CaPMEI1-OX plants (data not shown).
Responses of wild-type and CaPMEI1-OX plants to Hyaloperonospora parasitica
We examined whether or not ectopic CaPMEI1 expression in Arabidopsis plants affected resistance to the virulent biotrophic oomycte pathogen H. parasitica isolate Noco2 (Fig. 9). Over 100 seedlings of both wild-type (Col-0) and CaPMEI1-OX transgenic lines were inoculated with spores of H. parasitica isolate Noco2 (5 × 104 conidiosporangia mL−1). At 7 days after inoculation, over 50 plants in each line were sampled to estimate the disease and assess the degree of asexual sporulation, which was quantified and expressed as the mean number of sporangiophores per cotyledon.
Fig. 9Responses of wild-type (Col-0) Arabidopsis and CaPMEI1-OX transgenic plants to infection with H. parasitica isolate Noco2. a Disease symptoms and trypan blue-stained pathogen structures on 7-day-old cotyledons of wild-type and transgenic plants 7 days after inoculation; dpi days post-inoculation. Bars 0.5 mm. b Quantification of asexual sporangiophores per cotyledon for at least 50 cotyledons of wild-type and transgenic plants 7 days after inoculation. The average number of sporangiophores produced on the cotyledons of wild-type and transgenic lines are shown below each of the lines tested
As shown in Fig. 9a, the cotyledons of wild-type and transgenic seedlings responded to H. parasitica infection by stimulating similar levels of asexual parasite sporulation. Their trypan blue-stained cotyledons also exhibited abundant hyphae, conidiospores and oospores (Fig. 9a). Quantitative disease ratings are shown in Fig. 9b. Both the transgenic and the wild-type plants showed a high level of susceptibility to H. parasitica isolate Noco2, and similar levels of heavy asexual sporulation (>20 sporangiophores per cotyledon) were observed on both lines.
Drought tolerance of CaPMEI1-OX plants
To investigate a possible role for CaPMEI1 in the dehydration response, we tested seed germination and seedling growth under osmotic stress. The seeds of wild-type, vector control and CaPMEI1-OX plants were placed on MS media supplemented with various concentrations of mannitol. We observed no significant differences in seed germination between these lines (Fig. 10a). However, treatment with 200 and 600 mM mannitol strongly inhibited germination in the wild-type and vector control plants compared with the CaPMEI1-OX plants.
Fig. 10Transgenic ArabidopsisCaPMEI1-OX lines exhibit enhanced tolerance to drought stress. a Seed germination in wild-type, smGFP and transgenic plants on the MS media containing 0, 200 and 600 mM mannitol. The data represent the mean ± SD of 100 seeds for each line tested. b Relative root length of wild-type, smGFP and transgenic lines in MS agar medium containing different concentrations of mannitol. Three independent experiments were performed with 40 seedlings of both wild-type and transgenic lines. c Drought tolerance test of transgenic seedlings. Wild-type, smGFP and transgenic lines were germinated and grown in 1× MS agar medium. Each seedling was transferred to liquid medium containing 100 mM mannitol. d Wild-type, smGFP and the CaPMEI1 transgenic Arabidopsis plants after 15 days without water. e Water loss from the excised leaves of wild-type, smGFP and transgenic plants. Data represent the mean ± SD from three independent experiments
We also tested the sensitivity of root growth to osmotic stress (Fig. 10b). Drought tolerance was observed during post-germination growth. The root growth of wild-type and vector control seedlings was inhibited in the presence of 150 and 200 mM mannitol. However, root elongation of CaPMEI1-OX plants was less sensitive to mannitol-induced osmotic stress and in comparison with wild-type and vector control plants, mutant seedlings grew well in liquid medium supplemented with 100 mM mannitol (Fig. 10c).
In addition, we noticed that adult CaPMEI1-OX plants exhibited enhanced drought tolerance. Following 14 days without water, wild-type and vector plants had withered severely, whereas the CaPMEI1-OX plants remained healthy. To determine the effect of CaPMEI1 on survival, these plants were rewatered on day 16; wild-type and vector control plants died, whereas the transgenic lines survived (Fig. 10d). Next, we examined transpiration rates by measuring fresh weight loss in detached leaves. The leaves of CaPMEI1-OX lines exhibited slightly slower water loss than those of wild-type or vector control plants (Fig. 10e). Together, these results indicate that CaPMEI1 overexpression enhanced water stress resistance.
Oxidative tolerance of CaPMEI1-OX plants
To investigate the response of CaPMEI1-OX plants to oxidative stresses, seeds of wild-type, vector control and CaPMEI1-OX plants were exposed to MS medium containing methyl viologen (MV) (Fig. 11). Treatment with 5 or 10 μM MV significantly inhibited germination of wild-type and vector control seeds compared with CaPMEI1-OX seeds (Fig. 11a). Seven-day-old seedlings were transferred to a medium containing different MV concentrations (0–0.5 μM) and grown for 2 weeks. Wild-type and vector control seedlings turned white and started to die after 15 days of MV treatment, but CaPMEI1-OX lines were much less affected by the treatment (Fig. 11b). Furthermore, these differences in plant phenotype were also observed with respect to the higher fresh weight of transgenic seedlings compared to those of wild-type and vector control plants (Fig. 11c). Detached leaves of 4-week-old plants were treated with 10 μM MV for 24 h, after which chlorophyll content was measured (Fig. 11d). The leaves of transgenic lines treated with MV retained more chlorophyll than those of wild-type or vector control plants.
Fig. 11Transgenic ArabidopsisCaPMEI1-OX lines exhibit tolerance to oxidative stress. a Effects of methyl viologen on the seed germination of transgenic lines. Seeds from wild-type, vector control and transgenic lines were plated on media with or without methyl viologen (MV, 5 and 10 μM) and incubated for 3 days. The data represent mean ± SD of 100 seeds for each line tested. b Phenotypes of wild-type, vector control and transgenic line seedlings treated with different concentrations of MV. c Fresh weights of seedlings grown in the indicated concentrations of MV for 2 weeks. The results are presented as the average fresh weight per seedling. Data represent mean ± SD from three independent experiments. d Chlorophyll content of MV-treated leaves of wild-type, vector control and transgenic plants, which were floated on 0, 0.05, 0.1 and 0.5 μM MV in MS medium and then incubated for 24 h in a growth chamber
Discussion
Plants possess a diverse range of cell wall-modified enzymes, which are post-transcriptionally regulated by numerous inhibitor-related proteins (Tymowska-Lalanne and Kreis 1998; Raush and Greiner 2004). Several invertase inhibitor-related proteins have been isolated from higher plants such as kiwi, tobacco and Arabidopsis (Greiner et al. 1998; Wolf et al. 2003; Giovane et al. 2004; Raiola et al. 2004). Attempts to characterize the activity of invertase inhibitor proteins from plant species other than kiwi or Arabidopsis have been either unsuccessful or resulted in the isolation of invertase inhibitors that share structural similarities with pectin methylesterase inhibitor proteins (PMEI), but which represent completely different target enzymes (Greiner et al. 1998; Scognamiglio et al. 2003). In this study, we identified and functionally characterized a novel pepper CaPMEI1 gene encoding a PMEI. This CaPMEI1 protein contains the four cysteine residues that are conserved among other PMEI proteins (Camardella et al. 2000; Sato et al. 2000). These residues are expected to be engaged in two disulfide bridges, which constitute a common structural motif within the PMEI domain (Camardella et al. 2000).
At the molecular level, CaPMEI1 expression was induced in pepper leaves by infection with bacterial pathogens and treatment with plant hormones such as SA, ethylene, MeJA and ABA. In particular, these hormone treatments strongly induced CaPMEI1 transcription, suggesting that this gene may be involved in the early stages of the active defense responses to bacterial pathogen infection and exogenous treatment with plant hormones.
In enzymatic assays, purified CaPMEI1 proteins significantly inhibited activity of plant pectin methylesterase (PME). In addition, CaPMEI1 exhibited antifungal activity against a broad range of plant pathogenic fungi, including F. oxysporum f.sp. matthiolae, A. brassicicola and B. cinerea. To penetrate the cuticular layer, plant fungal pathogens produce plant cell wall-degrading enzymes such as polygalacturonase, pectin lyase and cellulase (Collmer and Keen 1986). An aggressive Phaeosphaeria nodorum isolate was shown to produce high amounts of xylanase, cellulase, polygalacturonase and butyrate esterase in vitro (Lalaoui et al. 2000). Recently, it was found that plant PMEIs do not inhibit the PMEs produced by plant pathogens (Giovane et al. 2004; Di Matteo et al. 2005). However, Arabidopsis plants expressing either AtPMEI-1 or AtPMEI-2 showed reduced infection by B. cinerea (Lionetti et al. 2007). This finding suggests that the increased level of pectin methylesterification caused by overexpression of AtPMEI-1 results in the inhibition of fungal endopolygalacturonase activity. Thus, the increase in PMEI activity resulted in reduced accessibility for fungal pectin degrading-enzymes and hence provided increased resistance to pathogens (Boudart et al. 1998; Lionetti et al. 2007). Consistent with these findings, our results suggest that CaPMEI1 may function as part of a new group of plant pectin methylesterase inhibitors, which restrict fungal pathogen infection in plants.
We used virus-induced gene silencing (VIGS) to investigate the effect of CaPMEI1 loss-of-function in pepper plants during Xcv infection. The CaPMEI1-silenced plants were susceptible to Xcv infection, and in particular to infection with the virulent strain, which resulted in enhanced bacterial growth and reduced PR1 and PR10 gene expression. Basal resistance is activated during the compatible bacterial interaction which restricts the spread of pathogens in the host plants to a certain extent (Glazebrook 2001). In addition, basal resistance is also effective in retarding proliferation of a wide range of microbial pathogens (Chisholm et al. 2006), but it is dependent upon SA accumulation (Cao et al. 1994, 1997; Kinkema et al. 2000). Therefore, we conclude that CaPMEI1 expression may be involved in basal resistance by triggering downstream PR gene induction in pepper plants.
To determine the effect of CaPMEI1 gain-of-function inplanta, we generated the CaPMEI1-OX Arabidopsis transgenic lines and investigated their response to P. syringae and H. parasitica infection, because these well-known model pathogens have been used extensively for the study of disease resistance mechanisms in Arabidopsis (Quirino and Bent 2003; Slusarenko and Schlaich 2003). The CaPMEI1-OX lines were resistant to Pst DC3000 infection, but not to infection by the biotrophic oomycete H. parasitica, which uses living cells as a nutrient source during the infection cycle (Alfano and Collmer 1996; Heath 2002). Moreover, as CaPMEI1 transcripts localize intensively in the xylem of vascular bundles in leaf tissues, they may not affect the accessibility of host plant cells to H.parasitica. In contrast, we hypothesize that intercellular growth of P. syringae may be restricted by the extracellular secretion of CaPMEI1 into host cells, resulting in triggering of the basal resistance response.
We found that fungal growth was inhibited by treatment with the recombinant CaPMEI1 protein in vitro, suggesting that CaPMEI1 may interfere directly with pathogen infection of host plants. Plant pathogenic microorganisms have been shown to produce a variety of pectinolytic enzymes that macerate and kill plant tissues (Collmer and Keen 1986). Cell wall fragments released by these pectinolytic enzymes may elicit the plant defense response (D’Ovidio et al. 2004). The polygalacturonase-inhibiting protein (PGIP) plays an important role in the recognition and inhibition of fungal polygalacturonase (PG). Overexpression of PGIPs in Arabidopsis not only significantly reduces disease symptoms, but also enhances defense gene activation during pathogen infection (Ferrari et al. 2003). Thus, we suggest that CaPMEI1 can disrupt invading pathogenic microorganisms by inhibiting pectin methylesterases produced by these pathogens.
The reduced bacterial growth observed in CaPMEI1-OX Arabidopsis lines may result from the expression of SA-inducible genes and CaPMEI1 overexpression. The SA-inducible genes such as PR1, PR2 and PR5 are activated in the SA defense pathway (Uknes et al. 1992). PR5 proteins are similar to thaumatin, which is a sweet-tasting protein from Thaumatococcus daniellii (Hu and Reddy 1997), and several pathogens can induce these proteins in a wide range of plant species (Ward et al. 1991; Hu and Reddy 1997; Reuber et al. 1998). Members of the PR5 group have been shown to exhibit antifungal activity against a broad spectrum of fungal pathogens (Coca et al. 2000) and to participate in the coordinated induction of systemic acquired resistance (SAR) against TMV (Ward et al. 1991). Therefore, the concomitant induction of PR genes may contribute to the enhanced resistance of CaPMEI1-OX Arabidopsis plants to bacterial pathogens.
The CaPMEI1-OX Arabidopsis lines showed a strong tolerance to drought stress, and CaPMEI1 overexpression resulted in reduced transpiration and enhanced root elongation. In contrast, transgene overexpression did not cause any obvious phenotypic differences under optimal growing conditions. Dehydration factors such as a mannitol and polyethyleneglycol (PEG) have been used to evaluate the effects of decreased water availability and simulate drought conditions in wild-type and CaPMEI1-OX Arabidopsis plants (Gupta and Kaur 2005; Verslues et al. 2006). Other plant invertase inhibitor-related protein genes such as NtCIF and NtVIF are also strongly induced by treatment with PEG or ABA (Rausch and Greiner 2004). However, the molecular and genetic roles played by PMEIs in drought and osmotic stress tolerance remain poorly understood.
Since plant responses to different abiotic stresses may be related to the accumulation of ROS and the mechanisms for their detoxification, the role played by ROS in stress signaling has been studied extensively (Apel and Hirt 2004). Methyl viologen binds to the thylakoid membranes of chloroplasts and in the presence of light, transfers electrons to O2 in a chain reaction causing continuous formation of superoxide radicals and oxidative stress (Asada 1996). The ArabidopsisCaPMEI1-OX lines exhibited tolerance to oxidative stress, both during seed germination and seedling growth. This tolerance to oxidative stress may reduce the damage caused by other stresses via the antioxidizing system, which suggests that CaPMEI1 overexpression results in detoxification of endogenous superoxide.
Here, we have determined that CaPMEI1 from pepper plays a role as an antifungal protein and has an inhibitory effect on PME. Furthermore, we have shown that Arabidopsis CaPMEI1-OX lines are resistant to bacterial pathogens. In addition, they exhibit tolerance to drought and oxidative stress. In conclusion, these multivariate functions of CaPMEI1 provide valuable insights into understanding the physiological significance of PMEIs in plant disease resistance and abiotic stress tolerance.
Electronic supplementary material
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pectin methylesterase inhibitor protein
antifungal activity
disease resistance
capsicum annuum
oxidative stress tolerance
drought tolerance


Bromodeoxyuridine Labeling Index as an Indicator of Early Tumor Response to Preoperative Radiotherapy in Patients with Rectal Cancer

Purpose Assessment of tumor proliferation rate using Bromodeoxyuridine labeling index (BrdUrdLI) as a possible predictor of rectal cancer response to preoperative radiotherapy (RT).
Introduction
In specialized centers, a refined surgical technique has resulted in high local control figures in rectal cancer. However, local recurrence rates after “standard” surgery are generally high, with figures ranging between 20 and 40%1,2, although after adopting the total mesorectal excision (TME) concept they fell down to 10–12%3,4. Radiotherapy in addition to surgery significantly diminishes the risk of local failure by more than half, from 8 to 2% after 2 years3. Therefore, combined treatment: radiotherapy (RT) and surgery in the treatment of patients with resectable rectal cancer has been proposed in many trials using either preoperative5,6 or postoperative irradiation7,8. Better results of preoperative RT for 5 days (25 Gy in five fractions) in comparison with postoperative 60 Gy in 30 fractions were achieved by a Swedish group4,9,10, with respect to the local recurrence rate11 and overall survival11,12.
A corresponding improvement in overall survival has not been demonstrated after postoperative radiotherapy alone13. Graf’s12 study provided a clinically significant biologic effect of a short preoperative course of radiotherapy on the tumor size and on the incidence of nodal metastases; however, this effect was minimized if surgery was performed immediately after radiotherapy. The effect is most likely caused by death of tumor cells in the primary tumor and in the involved nodes. A short treatment course of radiotherapy, i.e., 5 × 5 Gy is desirable, and this regimen is currently considered as the gold standard in many centers. However, using this schedule it is difficult to observe a down-staging and/or downsizing of the tumor, which is of importance for the selection of patients for sphincter-preserving surgery (anterior resection).
In clinical practice there are no certain methods able to predict tumor response to preoperative radiotherapy (RT). The optimal timing of surgery after preoperative radiotherapy in rectal cancer is unknown. However, it was shown that a long interval (6–8 weeks) between preoperative radiotherapy (39 Gy in 13 fractions) and surgery was associated with a significantly greater clinical tumor volume reduction than a short interval (2 weeks)14. On the other hand, it was shown that subclinical pelvic deposits of rectal cancer could grow rapidly during preoperative radiation therapy and during the radiotherapy–surgery interval, with an adverse influence on the rate of pelvic tumor control from protracting the overall treatment time15. Graf et al12 showed that low doses in short RT only offer clinically relevant reduction in the risk of pelvic relapses if the overall radiation treatment time is short. Thus, the rate of cancer cell proliferation seems to be a very important prognostic factor.
The aim of this study is to evaluate BrdUrd LI and S-phase fraction (SPF) as the possible indicators of tumor proliferation rate and predictors of the tumor response to neoadjuvant RT in patients with rectal cancer, and to suggest an optimal interval between short RT course and surgery.
Methods and Materials
Patients
Between November 2003 and January 2006 we recruited 92 patients with resectable rectal carcinoma for whom curative surgery was planned. Patients were eligible for the trial if they were less than 75 years old, had a histopathologically proved adenocarcinoma (T2/T3)16 situated less than 12 cm from the verge of the anus, and gave informed consent for their participation. The protocol was approved by the Ethical Committee of the Center of Oncology, and each patient gave written consent.
The criteria for exclusion were: locally nonresectable tumor; plan to perform only local tumor excision; known metastatic disease; previous radiotherapy of pelvis region; other malignant disease; and patient’s refusal.
Preoperative Radiotherapy
The patients assigned to preoperative radiotherapy received a total tumor dose of 25 Gy. The treatment was given in five fractions over 5 days, one posterior and two lateral wedged fields were irradiated with photons of maximum 6 MV energy. According to the random selection surgery was performed the following week (schedule I) or after longer interval of 4–5 weeks (schedule II).
Surgery
Anterior resection of rectum or abdominoperineal excision was performed within a week or a month after the completion of RT. Type of surgery was resection of the rectum and lower sigmoid with involved adjacent tissue and regional lymph nodes up to or above the origin of inferior mesenteric artery. A minimal touch technique was used with high tight ligation of the inferior mesenteric artery. The decision whether the patient should have an abdominoperineal resection or a sphincter-preserving surgery was made by the surgeon during the operation.
An abdominoperineal resection of rectum was performed in 41 (44.6%) of the patients, and sphincter preserving surgery was performed in 51 (55.4%).
Biological Assessment of Tumor Response
Tumor samples were taken twice: before radiotherapy (through a rectoscope) and during surgery from the same place, i.e., at the lowest edge of the tumor mass. Each biopsy was divided into two parts: one was used for BrdUrd LI assessment, and the second was used for immunohistochemical analysis (these results will be the subject of a separate study).
Bromodeoxyuridine Labeling Index
Incorporation of BrdUrd in tumor samples from a biopsy (0.3–0.5 cm3) was carried out in vitro according to the high-pressure oxygen method. The BrdUrd staining procedure and flow cytometry have been described in detail elsewhere17. The stained preparations were analyzed with a FACS Calibur flow cytometer (Becton Dickinson Immunocytometry Systems, Sunnyvale, CA, USA) by one coauthor (AG) and 20 × 103 events were collected in each histogram. The BrdUrdLI was calculated as a percentage of BrdUrd-labeled cells in a sample, which incorporated BrdUrd during 1 h of incubation at 37°C (with discrimination of diploid subpopulation in aneuploid tumors). The tumor ploidy and SPF were calculated from the DNA profile with ModFit software running on a MacIntosh computer. Apoptotic cells were identified as objects with a fractional DNA content not less than 20% of the 2n DNA content. Cell death was calculated as the sum of apoptosis and debris. The tumor ploidy was estimated by evaluating the DNA index, i.e., the ratio of the modal DNA fluorescence of abnormal to normal G1/0 cells. Aneuploidy was assessed in cases in which the normal and neoplastic cell populations gave two separate peaks. Human lymphocytes were used for the reference peak. Tumors with BrdUrdLI >8.5% (median value) were considered as fast, and those with BrdUrdLI ≤8.5% were considered as slowly proliferating tumors.
Clinical Assessment of Tumor Response
Tumor size before RT was assessed basing on measures taken during rectoscopy, and endorectal sonography. Tumor regression after RT was assessed at the time of operation by surgeons according to the following Response Evaluation Criteria in Solid Tumors (RECIST)18:
Complete response (CR): 100% disappearance; partial response (PR): 30–99% decrease; stable disease (SD): neither CR, PR or PD criteria met; progression of disease (PD): 20% increase in sums of tumor longest diameters.
Pathological Assessment of Tumor Response
Tumor regression after RT was evaluated by a pathologist on the excised tumor mass. The following criteria of tumor regression assessed by Dworak et al.19 were applied:
D0—no regression; D1—dominant tumor mass with obvious fibrosis and/or vasculopathy; D2—dominantly fibrotic changes with few tumor cells or groups; D3—very few (difficult to find microscopically) tumor cells in fibrotic tissue with or without mucous substance; D4—no tumor cells, only fibrotic mass (total regression or response).
Statistical Methods
Statistical analysis was performed with STATISTICA vs.5. Intergroup differences in the ordinal data were tested with ANOVA test or Student’s t test. P values of less than 0.05 were considered to indicate statistical significance. Linear regression was applied for assessing differences between fast and slowly proliferating tumors in relation with OTT, and its significance was determined by testing the difference between two correlation coefficients. Stratification by BrdUrd LI level was introduced and tested by the inclusion of dummy variable in the regression model.
Results
Patients
A total of 92 patients were included in the study. Twenty-eight (23.3%) out of 120 patients initially qualified for this study were excluded from the analysis because of discontinuation of treatment, metastatic tumor noticed at operation, or no tumor samples taken for biological assessment during surgery. Mean age for the entire group of patients was 61.6 years (range 30–75). There were 68 men and 24 women. There were no statistical differences between the two groups at the time of recruitment for prognostic factors such as: sex, age, histologic grade, or tumor stage (Table 1).
Table 1Selected Characteristics of Patients and Treatment ParametersCharacteristicsSchedule ISchedule IITotalAge mean (±SD) years(38)a 61.2 ± 12.0(54) 61.9± 9.5(92) 61.6 ± 10.6Sex Male303868 Female81624Histological grade G162026 G2293463 G3303Tumor stage T181927 T2253055 T35510PTNM 1162541 28614 3131730 4123Interval between RT and surgery Mean (range) days(38)a 8.8 (2–14)(54) 32.9 (17–45)(92) 22.9 (2–45) OTT mean (range) days(38) 13.8 (7–19)(54) 37.9 (22–50)(92) 27.9 (7–50)Surgery Sphincter-preserving20 (52.6 %)31(57.4 %)51 Abdominoperineal resection182341aNumber of patients
In our series of patients, there were 27 stage 1 (29.3%), 55 were T2 (59.8%), and 10 were T3 (10.9%). In 26 patients, tumor cells well differentiated (G1), 63 moderately differentiated (G2), and three poorly differentiated (G3) (Table 1). Thirty-eight patients were treated according to schedule I, in which time interval between end of irradiation and surgery averaged 8.8 days (range 2–14; Table 1). In 54 patients, schedule II was applied, in which mean break was 32.9 days (range 17–45). Because the interval between RT and surgery appeared to be longer than planned, overall treatment time (OTT), e.g., time from the beginning of RT to surgery, was calculated and it appeared to be 7–50 days (Table 1).
Biologic, Pathologic, and Clinical Assessment of Tumor Response
Mean BrdUrd LI before RT was 8.5% (range 1.0–24.2%) and SPF was 22.0% (range 3.8–49.9%) and the mean values did not differ between the two schedules (Table 2). Poorly differentiated tumors showed statistically significant higher BrdUrd LI than grades 1 and 2 tumors (P = 0.015; Table 3). After RT, tumors treated according to both schedules showed statistically significant growth inhibition (reduction of BrdUrd LI and percentage of SPF cells) in comparison with the values obtained before RT (Table 2). Radiation induced inhibition of tumor proliferation was expressed as a percentage of the after RT to before RT BrdUrd LI, and SPF as after/before RT percentage. This ratio ranged from 2.5 to 514% for BrdUrd LI (Fig. 1) and from 5.8 to 522.2% for SPF. When we stratified patients into two groups according to their biological RT response, those radioresponsive with reduction of pretreatment values after radiotherapy above 50% and those less responsive with reduction below 50%, it appeared that the mean values (of the after/before RT ratios of BrdUrd LI and SPF) for the more radioresponsive tumors were significantly higher than for the less responsive ones. Therefore, these ratios were presented separately for fast (BrdUrd LI >8.5%, SPF >22.0%) and slowly (BrdUrd LI ≤8.5%, SPF ≤22.0%) proliferating tumors. Mean BrdUrd LI value after RT for fast proliferating tumors (41 cases) showed statistically significant (P = 0.027) reduced pretreatment percentage (46.8%) in comparison with slowly proliferating tumors (85.3%, 51 cases). The same was true for SPF of fast (56.4%, 55 cases) and slowly (113.8%, 37) proliferating tumors (P = 0.006).
Table 2Status of Biological Parameters Before and After RTGroupBrdUrd LI (%) Mean (range)S-phase fraction (%) Mean (range)Apoptosis (%) Mean (range)All patientsBefore RT8.5 (1.0–24.2)22.0 (3.8–49.9)5.9 (0–52.8)After RT4.1* (0.4–18.3)16.8** (1.5–101.0)9.8*** (0–45.9)RT schedule IBefore RT8.4 (1.1–24.2)21.5 (6.1–49.2)6.6 (0–32.4)After RT3.8*(0.8–12.6)14.1**** (1.5–47.9)10.5 (0–43.3)RT schedule IIBefore RT8.6 (1.0–20.0)22.3 (3.8–49.9)5.4 (0–52.8)After RT4.5* (0.4–18.3)17.2 (2.6–101.0)9.5***** (0–45.9)*P = 0.000**P = 0.015***P = 0.010****P = 0.002*****P = 0.042Table 3The Relationship Between Tumor Biological Parameters and Histological GradeHistological gradeNBrdUrd LI (%) Mean (range)S-phase fraction (%) Mean (range)Apoptosis (%) Mean (range)G1268.5 (1.1–17.1)23.7 (5.8–49.9)3.4 (0–32.4)G2618.2 (1.0–20.0)21.0 (3.8–45.6)7.2 (0–52.8)G3316.2*, ** (9.5–24.2)27.7 (18.9–34.7)2.1 (0.4–4.8)*P = 0.015, difference between G1 and G3**P = 0.013, difference between G2 and G3Figure 1The association between biological tumor response for slowly (BrdUrd LI ≤ 8.5%; closed symbol) and faster proliferating tumors (BrdUrd LI > 8.5%; open symbol) and overall treatment time. Insert shows linear regression performed separately for each of the tumor subgroups for OTT > 30 days. P value shows difference between two correlation coefficients.
Next, the after/before RT ratios for BrdUrd LI and SPF were correlated with OTT. For SPF, statistical difference between linear regression coefficients for fast and slowly proliferating tumors was not obtained (P = 0.446), therefore the data for BrdUrd LI only are shown (Fig. 1). Insert on Fig. 1 shows a significant (P = 0.033) difference in proliferation rate between fast and slowly proliferating tumors treated within OTT >30 days. At that time slowly proliferating tumors, contrary to fast proliferating ones, show no inhibition but accelerated proliferation of tumor cells. This phenomenon was also confirmed by increased fraction of S-phase cells in tumors treated with longer RT schedule (Table 2). The influence of BrdUrdLI level has been also tested by the extended regression model between OTT and the percentage of after/before RT BrdUrd LI. BrdUrd LI level higher than 8.5% has been coded as dummy variable. It appeared to be significant (P = 0.025) in the relation between OTT and the percentage of after/before RT BrdUrd LI. The partial regression coefficient indicates that the average decrease of the percentage after/before RT BrdUrdLI for fast proliferating tumors (BrdUrd LI >8.5%) equals 39%.
All 92 irradiated rectal tumors were reviewed by the same pathologist (KN). The tumors were classified according to the World Health Organization classification of intestinal carcinoma16 and staged according to the TNM classification20. Of the 92 rectal tumors four had no pTNM classification, 41 were pT1 (46.6%), 14 were pT2 (15.9%), 30 were pT3 (34.1%), and three were pT4 (3.4%). Regional lymph node metastases were found in 27 (30.7%) patients, and 27 (30.7%) patients had their tumor down-staged.
Pathologic assessment of tumor regression after RT according to classification described by Dworak19 was performed in 90 out of 92 patients (for two patients the assessment was impossible). The analysis showed no tumor regression (D0) in 18 (20.0%) tumors, dominant tumor mass (D1) in 46 (51.1%) tumors, a few tumor cells in fibrotic mass (D2) in 18 (20.0%) tumors, single tumor cells (D3) in four (4.4%), and no tumor cells were observed in four (4.4%) of the examined tumors (Fig. 2a). In 25 (27.8%) out of 90 patients marked pathologic down-staging (no residual tumor confined to the rectal wall) was visible. Pretreatment BrdUrdLI and SPF were not correlated with early clinical and pathologic tumor response. However, patients having tumors with LI >8.5% were more radioresponsive (showed significant reduction in proliferative rate after radiotherapy) than patients with BrdUrdLI ≤8.5% tumors, although statistically significant difference between the two tumor subgroups was seen only for D0–D1 grade (Fig. 2b).
Figure 2Association between biological and pathological assessment (Dworak classification) of early tumor regression for (A) total group of patients and (B) for slowly (BrdUrdLI ≤ 8.5%) and fast proliferating (BrdUrdLI > 8.5%) tumors. Mean values ± SE are shown. For stages D0–D1, statistically significant lower inhibition of tumor cell proliferation after RT was observed for slowly than fast proliferating tumors.
In the clinical assessment of tumor mass resected during surgery, 34 (36.9%) tumors showed stable disease, 12 (13.0%) showed progressive disease, 41 (44.6%) showed partial response, and four (4.3%) showed complete response (Fig. 3a). And again, in fast proliferating tumors, greater inhibition in tumor proliferation rate (reduction of pretreatment BrdUrd LI value >50%) was observed in fast than in slowly proliferating tumors; however, this difference was not statistically significant (Fig. 3b). As the observed correlation between clinical assessment and SPF was weaker than for BrdUrd LI, the data were not shown. Partial and total tumor regression was observed in 45 (48.9%) tumors. However, tumor proliferation status was not in agreement with the kind of surgery. Sphincter-preserving surgery was performed in 51 out of 92 patients: in 22 (23.9%) fast proliferating and in 29 (31.5%) slowly proliferating tumors.
Figure 3Association between biological and clinical assessment of early tumor regression after RT for all tumors (A) and separately for slowly and fast proliferating tumors (B). Mean value ± SE are shown.
Discussion
This study provides evidence of a clinically significant biological effect of a short preoperative course of RT on tumor proliferation rate. The impact of irradiation on biological tumor response was assessed by BrdUrd LI, SPF, and the degree of subsequent pathologic and clinical down-staging of the tumors after surgery. The study showed differences in the pretreatment proliferation rate of the tumor. Mean BrdUrd LI before RT was equal to 8.5% and ranged from 1 to 24.2%. Mean SPF was 22.0% and ranged from 3.8 to 49.9%. The proportion of cells in S-phase as estimated by the DNA content overestimates the labeling index determined by the uptake of BrdUrd. This may be so because the exposure time is quite short and there may be subpopulations in the tumors that are synthesizing DNA at a very slow rate, or there may indeed be cells with an S-phase DNA content that are not synthesizing DNA (as a result of nutrient or oxygen supply, lack of growth factors, inadequate vascularity).
Mean value of the BrdUrd LI obtained in this study was lower than the one estimated by Bergstrom et al.21, Palmqvist et al.22, and Terry et al.23, and can be explained by a different method used by these authors: in vivo incorporation of iodouridine/bromodeoxyuridine, which can cause longer exposure of the tracers to S-phase cells. The differences in the LI value might be caused also by heterogeneity in proliferation within the tumor. It was shown by Bergstrom et al.21 that rectal tumors are polarized, having the superficial surface toward the lumen of the gut and the other toward deep structures facing totally different environments. Apart from Bergstrom et al., none of the above-mentioned authors gave account of site from where the tumor samples were taken. In each tumor analyzed by us, all the samples were taken from the same region, i.e., the bottom part of the mass.
In our study, pretreatment BrdUrd LI or SPF was not predictive for early clinical and pathologicl tumor response, probably because of different tumor microenvironment. However, BrdUrd LI after/before RT ratio gave information on the different significant biological processes that take place after irradiation, and have impact on cell death like redistribution, repopulation, and reoxygenation.
BrdUrd LI after RT decreased to mean 4.1% independently of the time interval between RT and surgery. Magnitude of LI reduction after RT was correlated with tumor proliferation rate. Greater reduction of BrdUrd LI value was observed in fast proliferating (LI >8.5%) tumors (to mean 46% of the pretreatment value) than in slowly (LI ≤8.5%) proliferating tumors (to mean 85.3% of pretreatment value). What then is the justification for better RT response of fast proliferating tumor cells? According to current knowledge on tumor proliferation, radiation therapy should preferentially inactivate rapidly dividing cells, leaving behind a population biased toward slow proliferation. However, recruitment is a known effect of cytotoxic treatment, and new cells from quiescent cell populations are recruited into active proliferation after irradiation. Probably, slowly proliferating tumors might have greater propensity to recruit cells into rapid cycle in response to treatment than fast proliferating tumors, which might have little reserve capacity for further accelerating their cell cycle24. That might be why we observed acceleration of proliferation rate in slowly proliferating tumors from 5 weeks after RT (basing on after/before RT BrdUrd LI ratio), which followed temporary reduction of the number of DNA-synthesizing cells, 4–5 weeks after the start of RT. Accelerated proliferation was confirmed by increased S-phase fraction. However, better biological tumor responsiveness of fast proliferating tumors on cellular level did not find confirmation on tissue level that is in surgery because a fewer number of sphincter saving resections were performed in patients with fast (22) than those with slowly proliferating tumors (29).
Regression of rectal carcinoma after preoperative irradiation varies, likely reflecting differences in the physical and biologic properties of these tumors. Apart from biological characteristics discussed here, tumor down-staging depends on the total irradiation dose, the fractionation, and the interval between irradiation and surgery25. We showed association of tumor proliferation rate after RT with tumor response basing on BrdUrd LI. SPF, considered as a less sensitive method of tumor proliferation, did not show such a correlation. The after/before radiotherapy BrdUrd LI ratios correlated, however nonsignificantly, with the degree of pathologic and clinical down-staging, which indicates that more radiation-induced cell death occurred in tumors that expressed high levels of BrUrd LI, or that an increased rate of tumor clearance occurred in more rapidly proliferating tumors. This effect was reflected by significantly higher incidence of apoptosis observed after RT only in fast proliferating tumors (4.1% vs 11.1%; P = 0.000). However, patients having tumors with LI >8.5% did not show higher rate (11.2%) of tumor pathological down-staging (D2–D4) than patients with BrdUrd LI ≤8.5% (16.8%) tumors, which may be suggestive of significant impact on tumor response also by biological processes other than proliferation. In the Spanish study26, high proliferative activity of rectal cancer, as determined by PCNA immunostaining, was predictive of response to preoperative chemoradiotherapy. Willett et al.27, in the same tumor type treated with higher RT dose (47–52 Gy) and 5 FU, showed that patients having tumors with extensive Ki-67 staining had also a higher rate of tumor down-staging (36%) 4–6 weeks after treatment than patients with minimal to moderate Ki-67 staining tumors (22–23%). These authors show that elevated postirradiation tumor proliferative activity correlated strongly with improved survival28. These authors, in contrast to our study, did not consider the proliferation profile of pre- and postirradiation for individual patients. The correlation of down-staging and higher survival rates was also found by other authors29,30.
In our study, even in totally regressed tumors (D4), the percentage of the after/before radiotherapy BrdUrd LI was about 50%, which may not indicate tumor but normal cell proliferation, mainly a fraction of activated fibroblasts or cycling endothelial cells in capillaries high in colorectal carcinoma31. Our study showed complete pathologic response (D4) similar to that in a Norwegian study (4.5%)32, where histological tumor slides were analyzed after treatment with a dose of 31.5 Gy in 18 fractions and 2–3 weeks interval between RT and surgery. However, it should be stressed that in this study, a high incidence (31.3%) of recurrences was observed at late follow-up. Our analysis showed that patients having fast proliferating tumors, as assessed by BrdUrdLI, experienced higher rates of regression than patients with slowly proliferating tumors, which could suggest a more frequent possibility of performing sphincter-preserving procedures in these tumors. However, this was not confirmed in surgical procedures. Therefore, we do not know yet if pretreatment BrdUrd LI assessment will be a good predictor for a locoregional failure. Berger et al.25, analyzing tumor sterilization after preoperative RT for rectal cancer, did not find a predictive factor for complete pathological response among such factors as age, sex, tumor stage, and pathologic grade. However, they found favorable influence of higher doses (>44 Gy) on pathologic stage.
There is no known optimal time for the interval between RT and surgery. The Swedish group keeps the interval at about a week; however, in other institutions, using longer RT treatments and higher total dose, longer intervals—4 to 6 weeks were adopted14,25. The main reason for a longer interval is tumor regression, which makes sphincter preservation possible. Similar to Francois et al14, we observed higher clinical and pathologic response rate after longer interval between RT and surgery. However, these authors14 showed nonsignificantly better overall survival for patients treated with shorter interval. Withers and Haustermans33 estimated the interval between long course of fractionated RT (40–54 Gy) and surgery and stated that the interval is not critical to either local recurrence or distant metastases. The authors offered the following arguments: the tumor cells do not disseminate until the primary tumor is large enough to be clinically detectable (probably 80% of patients whose rectal tumors have not metastasized to lymph nodes will be free of metastases). Irradiation with a dose of 40 Gy in 2 Gy fractions (equivalent to 25 Gy in five fractions) reduces tumor cell survival by about six decades, e.g., from 1010 to 104 cells. However, we have to remember that although the short overall treatment duration in the 25 Gy in five-fraction regimen provides a radiobiological advantage, this is a relatively low dose34, which causes about a 66% reduction in the rate of local recurrence11. A retrospective analysis of published results of preoperative radiation therapy for rectal cancer showed that local control probability curves were displaced toward higher doses as the overall duration of preoperative radiation therapy was increased15. Therefore, longer intervals between short RT schedule (25 Gy) and surgery may be inappropriate in case of patients with incomplete resection (cut-through) of primary tumor, in whom the average subclinical cancer cell burden increases during long interval. Also, subclinical disease beyond the future surgical margins, may be a potential target for future recurrences. Longer intervals after short RT can be dangerous because of potential subclinical tumor, which may grow more quickly than primary tumor15,33, the and risk of developing distant metastases. If we imply that moderately differentiated adenocarcinoma cells have different metastatic and proliferative activities from poorly differentiated cancer cells, which was shown by Taniyama et al35, then we could have an indication to adjuvant chemotherapy for patients with differentiated tumors. The authors35 indicated that moderately differentiated cancer cells are associated with hematogenous metastases to the liver, and the loss of tubular formation of cancer cells in poorly differentiated tumors may be fundamentally related to lymph node metastases and infiltrative growth. Therefore, particularly in patients with moderately differentiated and slowly proliferating tumors, adjuvant chemotherapy could be suggested after OTT shorter than 4 weeks, to prevent developing metastases to the liver.
In conclusion, our study shows that pretreatment BrdUrd LI or SPF were not predictive for early clinical and pathologic tumor response. After/before BrdUrd LI ratios showed inhibition of proliferation in responsive tumors, but this was not reflected in the number of sphincter preserving procedures performed. As 1 month after RT, accelerated proliferation of tumor cells is observed only in slowly proliferating tumors, we think that longer interval between RT and surgery is inadvisable.
If late tumor response confirms that patients having tumors with increased proliferative activity have statistically significantly less recurrences and improved survival rates compared with patients with less proliferative tumors, then we will be able to suggest a prognostic factor for individual rectal cancer patient, and a basis for selection to postoperative adjuvant chemotherapy.

early tumor response
radiotherapy
rectal cancer
proliferation rate
brdurdli