BACKGROUND
leaf senescence is the final stage of leaf development, ultimately leading to the death of the entire leaf.
although governed by the developmental age, it also can be stimulated by diverse environmental factors, including plant hormones, drought, salinity, extreme temperature, darkness, wounding and pathogenic infection  <cit> .
premature leaf senescence can eventually affect the yield of plants under adverse environmental conditions.
therefore, studying leaf senescence will not only strengthen our comprehension of a basic biological process, it also may provide methods to delay plant aging in order to improve agricultural traits of vegetable crops.
pepper  is an important vegetable crop that is extensive cultivated worldwide.
in recent years, premature senescence of pepper plants caused by various environmental stresses has become a universal phenomenon and considered an important field of research.
loss of green color, induced by degradation of chlorophyll, is the most visible symptom of leaf senescence.
pheophorbide a oxygenase  has been regarded as a crucial enzyme in chlorophyll degradation  <cit> .
it oxygenolytically cleaves the porphyrin macrocycle of pheophorbide  a and finally forms the primary fluorescent chlorophyll catabolite .
the pao gene was initially obtained from maize  and then discovered in other higher plants, such as rice, wheat, tomato, soybean and canola .
previous studies showed that the expression of pao is induced by natural senescence and environmental stresses in plants .
in arabidopsis, atpao is encoded by the accelerated cell death  <dig>  gene and is homologous to lethal leaf spot  <dig>  of maize.
it belongs to a small family of rieske-type iron-sulfur oxygenases  <cit> .
the absence of acd <dig> has been shown to result in the accumulation of pao and light-independent cell death when senescence is induced in permanent darkness  <cit> .
tobacco  is one of the most important model plants for transgenic research.
in previous studies, many new genes from other plants have been transferred into tobacco for further research on their functions  <cit> .
the rd29a promoter from arabidopsis thaliana is a stress-inducible promoter.
this promoter contains two dehydration- responsive elements , which are involved in response to salt, dehydration and low temperature  <cit> .
using the rd29a promoter instead of the constitutive 35s camv promoter for certain genes overexpression minimizes the negative effects on plant growth, which have been widely used in genetic transformation  <cit> .
in recent years, the research on pao focused on the functional analysis for inhibiting cell death  <cit> .
however, little is known about capao regulatory role after induction expression of inducible-promoter in pepper.
this study was conducted to clone this gene from the pepper plant and analyze its molecular characteristics.
patterns of capao gene expression in specific tissues and in response to various stresses were analyzed by quantitative real-time pcr.
furthermore, virus-induced gene silencing  and transgenic technologies were used to study capao gene function.
the results suggest that capao may play an important role during leaf senescence and chlorophyll degradation.
RESULTS
cloning and sequence analysis of capao
the full-length cdna designated capao was obtained using in silico cloning and race techniques.
the transcript consists of  <dig> nucleotides, including a 5′-untranslated region  of 32 bp, an orf of 1614 bp and a 3′-utr of 192 bp  .
capao was predicted to encode a  <dig> amino acid protein with a theoretical molecular weight  of  <dig>  kda and calculated isoelectric point  of  <dig> .
structural analysis revealed that capao belongs to a rieske-type iron-sulfur superfamily, containing a conserved rieske cluster, a mononuclear iron-binding site and a redox-active cxxc motif in the c-terminal end, which are necessary for oxygen activation .
subcellular analysis localized capao in the chloroplasts.
further sequence analysis indicated that the deduced capao protein contains a chloroplast transit peptide of  <dig> amino acid residues with a cleavage site located between r <dig> and v <dig>  but it does not contain a signal peptide region or transmembrane helix.fig.
<dig> multiple sequence alignment of the capao protein and pao proteins from other plants using dnaman software.
the arrow indicates the cleavage site of the chloroplast peptide, and the single line indicates the rieske iron-binding motif.
the dashed line indicates the mononuclear iron-binding motif, and a conserved cxxc motif is indicated by a double line.
besides capao, other amino acid sequences included in this alignment were s. lycopersicum lethal leaf spot 1-like protein , n. tabacum pao <dig> , n. tabacum pao <dig> , v. vinifera pao , r. communis pao , b. napus pao , and a. thaliana pao .
shaded regions show identical amino acid residues among all species
the deduced capao amino acid sequence showed high homology to other plant pao sequences via multiple alignments using dnaman software .
the percent identities of capao relative to solanum lycopersicum lethal leaf spot 1-like protein , n. tabacum pao <dig>  and pao <dig> , vitis vinifera pao , ricinus communis pao , brassica napus pao  and a. thaliana pao , were  <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and 71 %, respectively.
a phylogenetic tree, constructed using mega <dig>  software, was used to investigate the evolutionary relationship of the capao amino acid sequence with pao proteins of other plants.
two groups were formed using the  <dig> pao protein sequences from c. annuum l., n. tabacum, s. lycopersicum, r. communis, v. vinifera, b. napus, a. thaliana, medicago truncatula, aegilops tauschi, pisum sativum and brassica rapa var.
parachinensis .
capao clustered in the first group, which included sllls <dig>  ntpao, bopao, brpao and atpao.
capao was more closely related to sllls <dig>  ntpao than pao proteins of other plants.
all of the above-mentioned bioinformatic analyses demonstrated that capao should function as a pao.fig.
<dig> phylogenetic analysis of capao protein and pao proteins of other plant species.
the phylogenetic tree was constructed by the neighbor-joining method using mega <dig> .
branches were labeled with the names and genbank accession numbers of the different plant species
tissue-specific expression of capao
in order to investigate the expression levels of the capao gene in different tissues, total rna was extracted from the roots, stems, leaves and flowers, and quantitative real-time pcr  was performed .
capao transcripts were detected in all of these tissues and found to be higher in leaves than in other tissues.
for different leaf developmental stages, a low level of capao transcript was detected in young and fully mature leaves, but capao expression was increased in senescent leaves .fig.
<dig> tissue-specific expression of capao in pepper.
a tissue-specific expression of capao in pepper roots, leaves, stems and flowers.
relative expression levels of the capao transcript were determined in different tissues in comparison to that in roots; b the expression profiles of capao during pepper leaf developmental stage.
rna was extracted from young leaves, fully expanded leaves and senescent leaves, respectively.
error bars represent sd for three independent replicates
induction of signaling molecules, abiotic and biotic stresses
in order to study its function in pepper plants, the expression pattern of capao was first analyzed.
b <dig> cultivar seedlings at the six-leaf stage were treated with various stresses, including phytohormones, salt, osmosis and phytophthora capsici infection, and analyzed by qrt-pcr.
to analyze the response of the capao gene to abiotic stresses, pepper plants were exposed to 400 mm sodium chloride  and 400 mm mannitol, and then the abundance of capao transcripts was analyzed by quantitative rt-pcr.
as shown in fig.
4a, the capao expression level in plants treated with 400 mm nacl began to increase gradually at 4 h and peaked  at 24 h, compared to the control .
with osmotic treatment, capao was induced quickly in pepper plants  with a  <dig> -fold increase in expression at 2 h and a stronger  <dig> -fold elevation at 12 h. the highest transcript level was detected at 24 h .
these results indicate that the increased abundance of the capao gene transcript may be part of the response to abiotic stresses, including high salinity and osmotic stress.fig.
<dig>
capao expression patterns in pepper leaves treated with various stresses.
a salt stress; b osmotic stress; c phytohormone treatments ; d
p. capsici infection.
the relative transcriptional expression of capao was calculated in various treated leaves in comparison to that in the mock controls across time points.
error bars represent sd for three independent replicates
to examine whether stress-related signaling molecules can induce capao expression, three phytohormones were used to treat pepper leaves.
as shown in fig.
4c, the expression levels of capao in leaves sprayed with abscisic acid , methyl jasmonate  and salicylic acid  were elevated to different extents.
aba could induce quickly the expression of the capao gene within the first 4 h after treatment.
compared to the control, capao transcripts were detected at the highest level  at 4 h and then decreased gradually after 8 h. interestingly, there was a slight up-regulation at 24 h. spraying pepper plants with meja caused a gradual upregulation of capao within the first 4 h, but then it declined slowly.
however, the capao expression peaked by 24 h at a  <dig> -fold greater level than the control.
by contrast, the treatment of sa quickly induced the capao transcript abundance at 2 h to  <dig> -fold higher than the control and maintained a relatively steady level from 4 h to 12 h before a sharp downregulation at 24 h. at 48 h, the capao transcript decreased to nearly the same levels among all of the plants treated with the three phytohormones.
these results indicated that the capao gene could be induced and upregulated by all three stress-related signaling molecules tested .
capao expression was enhanced in pepper plants infected with p. capsici as shown in fig.
4d.
the capao transcriptional level was slightly decreased at 3 h and remained at a steady level from 6 h to 24 h, compared with the mock control.
this transcript then increased rapidly and peaked  at 48 h. subsequently, the capao expression level sharply downregulated at 72 h  and decreased to  <dig> -fold at 96 h, which was the lowest level after infection relative to the control.
these results revealed that capao may be involved in the pepper defense response against pathogens.
vigs assay of capao gene in pepper plants
silencing efficiency of capao gene
the results showed that capao participates in the chlorophyll degradation pathway and is involved in the response to various stresses in pepper plants.
to further examine the function of capao in pepper, a tobacco rattle virus -based vigs technique was used.
pepper plants inoculated with agrobacterium for 6– <dig> weeks were used for the following treatments.
an empty vector was applied to plants  as a negative control.
trv2: capds plants, in the endogenous phytoene desaturase  gene was silenced to cause photobleaching, were used as positive controls for testing vigs efficiency.
three weeks after the agrobacterium inoculation, we found that most of the plants clearly showed symptoms of viral infection.
furthermore, the trv2: capds plants began to exhibit the photobleached phenotype.
these results indicated that vigs was successfully applied in this experiment.
as shown in fig.
5a, no morphological distinction was observed between the capao-silenced plants  and the empty vector treated control plants   <dig> d after inoculation.
simultaneously, capao transcript levels in empty vector control plants  and capao-silenced plants  were examined by quantitative rt-pcr to screen the efficiency of capao gene silencing by vigs .
the results showed that the capao transcriptional level was reduced remarkably in capao-silenced plants compared to the empty vector control, which demonstrated that the gene silencing was successful.fig.
<dig> efficiency of capao gene-silencing in pepper plants.
a phenotypes of gene-silenced pepper plants  <dig> d after inoculation.
left: control plant ; middle: capds-silenced plant ; right: capao-silenced plant .
b quantitative real time-pcr analysis of capao expression levels in leaves of capao-silenced plants  and control plants   <dig> d after inoculation.
error bars represent sd for three independent replicates.
asterisks indicate a significant difference  compared to trv2: <dig> leaves
silencing of capao delay chlorophyll breakdown under high salt treatment
in order to analyse whether the capao response to salt stress, the leaf discs from empty vector control-treated  and capao-silenced  plants were exposed to various concentrations  of nacl solution with continuous lighting for  <dig> d. as shown in fig.
6a, the color of the leaf discs of trv2: <dig> plants turned yellow under 300 mm nacl treament and the leaf discs appeared to be a bleached phenotype under treatment with higher nacl concentrations .
however, the leaf discs of trv2: capao plants slightly shrinked and their color showed little change.
furthermore, chlorophyll breakdown in trv2: <dig> leaves was observed to occur faster than that in trv2: capao leaves  <dig> d after treatment with high nacl concentrations  .
these results suggested that silencing the capao gene can inhibit chlorophyll breakdown during salt stress-induced leaf senescence in pepper plants.fig.
<dig> chlorophyll degradation in leaves of gene-silenced pepper plants.
a phenotypes and b total chlorophyll contents of leaf discs of the gene-silenced plants in response to high salt stress.
leaf discs from the gene-silenced pepper leaves were floated in different concentrations of nacl solutions with continuous fluorescent lighting at 25 °c for 3 days.
error bars represent sd for three biological replicates.
asterisks indicate a significant difference  compared to trv2: <dig> leaves
effect of rd29a promoter-inducible overexpression of capao on salt tolerance of tobacco
identification of transgenic plants
to examine the relative contribution of capao to salt stress-induced leaf senescence, tobacco plants were transformed with pvbg2307-prd29a-capao vector.
a sequence of 491 bp, which contained a 284-bp fragment of the rd29a promoter and a 207-bp fragment of the capao gene, was produced from genomic dnas of the positive transgenic t <dig> lines.
nine plants were confirmed to be positive transgenic t <dig> lines.
t <dig> and t <dig> generation of transgenic tobacco plants were selected using 50 mg · l− <dig> kanamycin, and finally obtained transgenic homozygous t <dig> lines.
one t <dig> positive transgenic plants were used in subsequent studies.
semi-quantitative rt-pcr analysis of the transgenic tobacco plants after salt stress treatment
as shown in fig.
<dig>  capao showed no expression in the capao transgenic tobacco and wild type  plants before salt stress treatment.
capao was strongly induced in the leaves of the transgenic plants at 24 h after treatment with 150 mm nacl solution, while it was not expressed in wt plants at all.
this may suggest that the capao gene, controlled under the stress-inducible promoter rd29a, was induced to express under exogenous salt treatment in positive transgenic tobacco plants.fig.
<dig> rt-pcr analysis of capao in transgenic tobacco plants with and without salt treatment under 150 mm nacl.
t: t <dig> generation of transgenic tobacco plants, wt: wild type
phenotype of transgenic tobacco plants after salt stress treatment
before salt stress treatment, there were no phenotypic differences between capao transgenic and wt plants .
at early stage of the salt stress treatment , both leaves of transgenic and wt plants wilted with more serious wilting degree in the wt plants.
along with the time of treatment, the lower part of leaves became yellow.
after salt treatment for  <dig> d, the leaves of the transgenic plants became yellow and the edge of the lower leaves showed necrotic symptoms after salt stress treatment, while the upper young leaves were green.
however, leaves of wt plants appeared to shrink, with lower leaves turning yellow but upper leaves remaining green .fig.
<dig> phenotypes , chlorophyll contents  and mda contents  of transgenic tobacco plants after salt treatment under 150 mm nacl.
a phenotypes of wild type and transgenic plants before salt stress, b phenotypes of wild type and transgenic plants after salt treatment for  <dig> d, c phenotypes of leaves of wild type and transgenic plants after salt treatment for  <dig> d, d chlorophyll contents and e mda contents of transgenic and wild-type tobacco plants under salt stress for  <dig> and  <dig> d, respectively.
error bars represent sd for three biological replicates.
asterisks indicate a significant difference  compared to wild type leaves
chlorophyll and malondialdehyde  contents of transgenic plants after treatment with salt stress
leaf senescence is associated with chlorophyll breakdown and reactive oxygen species  accumulation  <cit> .
ros-generated lipid peroxidation  is an inherent feature of senescing cells and a source of ros  <cit> .
to further analyze the effect of capao overexpression on salt-induced leaf senescence in tobacco, chlorophyll and mda contents were measured after salt treatment for  <dig> and 14 days, respectively .
before the salt stress, there were few obvious differences in chlorophyll and mda content between wt and transgenic leaves.
chlorophyll content of wt and transgenic leaves decreased by  <dig>  and  <dig>  mg · g− <dig> after 7 days, and decreased by  <dig>  and  <dig>  mg · g− <dig> after 14 days of salt treatment, respectively.
the rate of chlorophyll breakdown in wt plants was higher than that in the transgenic plants.
correspondingly, mda content gradually increased with elongated treated time.
mda content of wt and transgenic leaves increased by  <dig>  and  <dig>  mmol · g - <dig> after 7 days, and increased by  <dig>  and  <dig>  mmol · g - <dig> after 14 days of salt treatment, respectively.
mda content in wt plants was significantly lower than that in transgenic plants.
these results indicated that overexpression of capao could accelerate the chlorophyll breakdown and lipid peroxidation accumulation of transgenic tobacco plants under salt treatment.
all these observations suggested that overexpression of capao could accelerate salt-induced leaf senescence in tobacco.
discussion
pao opens the porphyrin macrocycle of pheophorbide a and finally forms fccs  <cit> .
here, we identified the capao gene from pepper.
capao contained conserved domains that existed in pao homologues.
furthermore, capao shared high homology to other pao proteins, especially sllls <dig> and ntpao.
bioinformatics analyses demonstrated that capao should function as a pao.
tissue-specific analysis showed that capao was expressed in all tissues, and the expression level in leaves was higher than other tissues.
the findings were similar to the previous results in maize and rice  <cit> , it was reported that expression of the ospao gene was detected in all tissues and at the highest level in leaves.
however, the present results were different than those in a. thaliana  <cit> , in which atpao transcript was higher in flowers and siliques than other tissues.
these results revealed that pao is important to growth and development of all tissues, although its function may be distinct in different species.
various biotic and abiotic stresses can affect growth and development of pepper plants, causing chlorophyll breakdown, cell death and finally premature senescence  <cit> .
all of these consequences would impact the yield and quality of crop plants.
pao is an important intermediate in chlorophyll degradation, the expression and activity of which can be affected by various stresses.
wang et al.
reported that relative expression of and pao in drought-stressed leaves was greatly upregulated in apple  <cit> .
microarray analysis indicated that upregulation of pao in response to various stress conditions, coinciding with breakdown of chlorophyll under these conditions  <cit> .
results of the current study showed that transcript levels of the capao gene were upregulated in pepper plants after treatment with various stresses, including high salinity, osmosis and phytophthora capsici infection.
these results suggested that capao may be involved defense response to salt and osmotic stresses as well as phytophthora capsici in pepper plants.
previous studies have shown that phytohormone signaling pathways played an important role in mediating developmental processes and environmental responses in plants  <cit> .
these hormones could cause the expression of stress-related genes, which in turn affect senescence progress of leaf.
many studies have proved that capao gene expression or activity was regulated by hormones .
for instance, aba can enhance the expression of pao in rice leaves  <cit> .
rodoni et al.
suggested that the chlorophyll breakdown related to pao activity in barley detached leaves, a rapid loss of chlorophyll in conjunction with an increase of pao activity was occurred after treatment with aba and meja  <cit> .
in the current study, treatment with aba, meja or sa was found to increase the expression level of capao.
these findings suggested that the capao gene may be involved in aba-, ja- and sa-dependent signaling pathways.
these results were different from those of a study in wheat reporting that aba and meja enhanced the expression of tapao, while sa treatment did not cause significant changes in the level of this gene  <cit> .
sa is the hormone involved in pathogen response and abiotic stress tolerance in pepper plant  <cit> .
in perennials, sa may be involved in the regulation of drought-induced leaf senescence as sa accumulation preceded chlorophyll breakdown and nitrogen mobilization  <cit> .
here, high salinity, osmosis and phytophthora capsici infection induced capao expression in pepper leaves.
it is likely that salt, mannitol and phytophthora capsici-induced sa accumulation may trigger capao transcripts in pepper plants.
the function of the pao gene has been studied in many plants  <cit> .
tang et al.
reported that chlorophyll breakdown was delayed compared to controls in leaves detached from leaves of pao-silenced rice plants during dark-induced senescence  <cit> .
moreover, leaves with overexpression of pao in arabidopsis had lower chlorophyll content and earlier leaf yellowing than the control  <dig> d after dark treatment  <cit> .
leaf senescence was involved in chlorophyll degradation and accumulation of ros.
drought-induced senescence in the leaves of apple was reflected in chlorophyll loss and an increase in the levels of ros, with the induction of the pao gene  <cit> .
in this research, the delay of chlorophyll degradation was detectable in the detached leaves from capao silencing pepper plants 3 days after treatment with salt.
conversely, overexpression of capao, induced by the stress-inducible promoter rd29a, could accelerate the rate of chlorophyll breakdown and lipid peroxidation of the transgenic plants under salt stress.
the results of tang et al.
have shown that overexpressing tapao caused an accumulation of rccs in wheat leaves  <cit> .
rccs have been proved to be phototoxic and caused cell deach, resulting in senescence  <cit> .
all of these results demonstrated that cacp played crucial role in pepper plant defense response to salt stresses, thus delaying leaf senescence.
CONCLUSIONS
overall, we conclude that capao plays an important role in senescence and chlorophyll degradation, as well as defense responses to various stresses.
capao was induced in natural senescence and various stresses.
silence of capao resulted in a stay-green phenotype in pepper plants, and overexpression of capao accelerated the process of salt-induced leaf senescence.
in the future, the transgenic plants about overexpression or knockdown of capao gene in pepper would be used for studying detail function of capao.

 leaf senescence is the final stage of leaf development, ultimately leading to the death of the entire leaf. although governed by the developmental age, it also can be stimulated by diverse environmental factors, including plant hormones, drought, salinity, extreme temperature, darkness, wounding and pathogenic infection. premature leaf senescence can eventually affect the yield of plants under adverse environmental conditions. therefore, studying leaf senescence will not only strengthen our comprehension of a basic biological process, it also may provide methods to delay plant aging in order to improve agricultural traits of vegetable crops. pepper is an important vegetable crop that is extensive cultivated worldwide. in recent years, premature senescence of pepper plants caused by various environmental stresses has become a universal phenomenon and considered an important field of research. loss of green color, induced by degradation of chlorophyll, is the most visible symptom of leaf senescence. pheophorbide a oxygenase has been regarded as a crucial enzyme in chlorophyll degradation. it oxygenolytically cleaves the porphyrin macrocycle of pheophorbide a and finally forms the primary fluorescent chlorophyll catabolite. the pao gene was initially obtained from maize and then discovered in other higher plants, such as rice, wheat, tomato, soybean and canola. previous studies showed that the expression of pao is induced by natural senescence and environmental stresses in plants. in arabidopsis, atpao is encoded by the accelerated cell death gene and is homologous to lethal leaf spot of maize. it belongs to a small family of rieske-type iron-sulfur oxygenases. the absence of acd has been shown to result in the accumulation of pao and light-independent cell death when senescence is induced in permanent darkness. tobacco is one of the most important model plants for transgenic research. in previous studies, many new genes from other plants have been transferred into tobacco for further research on their functions. the rd29a promoter from arabidopsis thaliana is a stress-inducible promoter. this promoter contains two dehydration- responsive elements, which are involved in response to salt, dehydration and low temperature. using the rd29a promoter instead of the constitutive 35s camv promoter for certain genes overexpression minimizes the negative effects on plant growth, which have been widely used in genetic transformation. in recent years, the research on pao focused on the functional analysis for inhibiting cell death. however, little is known about capao regulatory role after induction expression of inducible-promoter in pepper. this study was conducted to clone this gene from the pepper plant and analyze its molecular characteristics. patterns of capao gene expression in specific tissues and in response to various stresses were analyzed by quantitative real-time pcr. furthermore, virus-induced gene silencing and transgenic technologies were used to study capao gene function. the results suggest that capao may play an important role during leaf senescence and chlorophyll degradation.  cloning and sequence analysis of capao the full-length cdna designated capao was obtained using in silico cloning and race techniques. the transcript consists of nucleotides, including a 5′-untranslated region of 32 bp, an orf of 1614 bp and a 3′-utr of 192 bp. capao was predicted to encode a amino acid protein with a theoretical molecular weight of  kda and calculated isoelectric point of. structural analysis revealed that capao belongs to a rieske-type iron-sulfur superfamily, containing a conserved rieske cluster, a mononuclear iron-binding site and a redox-active cxxc motif in the c-terminal end, which are necessary for oxygen activation. subcellular analysis localized capao in the chloroplasts. further sequence analysis indicated that the deduced capao protein contains a chloroplast transit peptide of amino acid residues with a cleavage site located between r and v but it does not contain a signal peptide region or transmembrane helix.fig. multiple sequence alignment of the capao protein and pao proteins from other plants using dnaman software. the arrow indicates the cleavage site of the chloroplast peptide, and the single line indicates the rieske iron-binding motif. the dashed line indicates the mononuclear iron-binding motif, and a conserved cxxc motif is indicated by a double line. besides capao, other amino acid sequences included in this alignment were s. lycopersicum lethal leaf spot 1-like protein, n. tabacum pao, n. tabacum pao, v. vinifera pao, r. communis pao, b. napus pao, and a. thaliana pao. shaded regions show identical amino acid residues among all species the deduced capao amino acid sequence showed high homology to other plant pao sequences via multiple alignments using dnaman software. the percent identities of capao relative to solanum lycopersicum lethal leaf spot 1-like protein, n. tabacum pao and pao, vitis vinifera pao, ricinus communis pao, brassica napus pao and a. thaliana pao, were and 71 %, respectively. a phylogenetic tree, constructed using mega software, was used to investigate the evolutionary relationship of the capao amino acid sequence with pao proteins of other plants. two groups were formed using the pao protein sequences from c. annuum l., n. tabacum, s. lycopersicum, r. communis, v. vinifera, b. napus, a. thaliana, medicago truncatula, aegilops tauschi, pisum sativum and brassica rapa var. parachinensis. capao clustered in the first group, which included sllls ntpao, bopao, brpao and atpao. capao was more closely related to sllls ntpao than pao proteins of other plants. all of the above-mentioned bioinformatic analyses demonstrated that capao should function as a pao.fig. phylogenetic analysis of capao protein and pao proteins of other plant species. the phylogenetic tree was constructed by the neighbor-joining method using mega. branches were labeled with the names and genbank accession numbers of the different plant species tissue-specific expression of capao in order to investigate the expression levels of the capao gene in different tissues, total rna was extracted from the roots, stems, leaves and flowers, and quantitative real-time pcr was performed. capao transcripts were detected in all of these tissues and found to be higher in leaves than in other tissues. for different leaf developmental stages, a low level of capao transcript was detected in young and fully mature leaves, but capao expression was increased in senescent leaves.fig. tissue-specific expression of capao in pepper. a tissue-specific expression of capao in pepper roots, leaves, stems and flowers. relative expression levels of the capao transcript were determined in different tissues in comparison to that in roots; b the expression profiles of capao during pepper leaf developmental stage. rna was extracted from young leaves, fully expanded leaves and senescent leaves, respectively. error bars represent sd for three independent replicates induction of signaling molecules, abiotic and biotic stresses in order to study its function in pepper plants, the expression pattern of capao was first analyzed. b cultivar seedlings at the six-leaf stage were treated with various stresses, including phytohormones, salt, osmosis and phytophthora capsici infection, and analyzed by qrt-pcr. to analyze the response of the capao gene to abiotic stresses, pepper plants were exposed to 400 mm sodium chloride and 400 mm mannitol, and then the abundance of capao transcripts was analyzed by quantitative rt-pcr. as shown in fig. 4a, the capao expression level in plants treated with 400 mm nacl began to increase gradually at 4 h and peaked at 24 h, compared to the control. with osmotic treatment, capao was induced quickly in pepper plants with a -fold increase in expression at 2 h and a stronger -fold elevation at 12 h. the highest transcript level was detected at 24 h. these results indicate that the increased abundance of the capao gene transcript may be part of the response to abiotic stresses, including high salinity and osmotic stress.fig. capao expression patterns in pepper leaves treated with various stresses. a salt stress; b osmotic stress; c phytohormone treatments; d p. capsici infection. the relative transcriptional expression of capao was calculated in various treated leaves in comparison to that in the mock controls across time points. error bars represent sd for three independent replicates to examine whether stress-related signaling molecules can induce capao expression, three phytohormones were used to treat pepper leaves. as shown in fig. 4c, the expression levels of capao in leaves sprayed with abscisic acid, methyl jasmonate and salicylic acid were elevated to different extents. aba could induce quickly the expression of the capao gene within the first 4 h after treatment. compared to the control, capao transcripts were detected at the highest level at 4 h and then decreased gradually after 8 h. interestingly, there was a slight up-regulation at 24 h. spraying pepper plants with meja caused a gradual upregulation of capao within the first 4 h, but then it declined slowly. however, the capao expression peaked by 24 h at a -fold greater level than the control. by contrast, the treatment of sa quickly induced the capao transcript abundance at 2 h to -fold higher than the control and maintained a relatively steady level from 4 h to 12 h before a sharp downregulation at 24 h. at 48 h, the capao transcript decreased to nearly the same levels among all of the plants treated with the three phytohormones. these results indicated that the capao gene could be induced and upregulated by all three stress-related signaling molecules tested. capao expression was enhanced in pepper plants infected with p. capsici as shown in fig. 4d. the capao transcriptional level was slightly decreased at 3 h and remained at a steady level from 6 h to 24 h, compared with the mock control. this transcript then increased rapidly and peaked at 48 h. subsequently, the capao expression level sharply downregulated at 72 h and decreased to -fold at 96 h, which was the lowest level after infection relative to the control. these results revealed that capao may be involved in the pepper defense response against pathogens. vigs assay of capao gene in pepper plants silencing efficiency of capao gene the results showed that capao participates in the chlorophyll degradation pathway and is involved in the response to various stresses in pepper plants. to further examine the function of capao in pepper, a tobacco rattle virus -based vigs technique was used. pepper plants inoculated with agrobacterium for 6– weeks were used for the following treatments. an empty vector was applied to plants as a negative control. trv2: capds plants, in the endogenous phytoene desaturase gene was silenced to cause photobleaching, were used as positive controls for testing vigs efficiency. three weeks after the agrobacterium inoculation, we found that most of the plants clearly showed symptoms of viral infection. furthermore, the trv2: capds plants began to exhibit the photobleached phenotype. these results indicated that vigs was successfully applied in this experiment. as shown in fig. 5a, no morphological distinction was observed between the capao-silenced plants and the empty vector treated control plants d after inoculation. simultaneously, capao transcript levels in empty vector control plants and capao-silenced plants were examined by quantitative rt-pcr to screen the efficiency of capao gene silencing by vigs. the results showed that the capao transcriptional level was reduced remarkably in capao-silenced plants compared to the empty vector control, which demonstrated that the gene silencing was successful.fig. efficiency of capao gene-silencing in pepper plants. a phenotypes of gene-silenced pepper plants d after inoculation. left: control plant; middle: capds-silenced plant; right: capao-silenced plant. b quantitative real time-pcr analysis of capao expression levels in leaves of capao-silenced plants and control plants d after inoculation. error bars represent sd for three independent replicates. asterisks indicate a significant difference compared to trv2: leaves silencing of capao delay chlorophyll breakdown under high salt treatment in order to analyse whether the capao response to salt stress, the leaf discs from empty vector control-treated and capao-silenced plants were exposed to various concentrations of nacl solution with continuous lighting for d. as shown in fig. 6a, the color of the leaf discs of trv2: plants turned yellow under 300 mm nacl treament and the leaf discs appeared to be a bleached phenotype under treatment with higher nacl concentrations. however, the leaf discs of trv2: capao plants slightly shrinked and their color showed little change. furthermore, chlorophyll breakdown in trv2: leaves was observed to occur faster than that in trv2: capao leaves d after treatment with high nacl concentrations. these results suggested that silencing the capao gene can inhibit chlorophyll breakdown during salt stress-induced leaf senescence in pepper plants.fig. chlorophyll degradation in leaves of gene-silenced pepper plants. a phenotypes and b total chlorophyll contents of leaf discs of the gene-silenced plants in response to high salt stress. leaf discs from the gene-silenced pepper leaves were floated in different concentrations of nacl solutions with continuous fluorescent lighting at 25 °c for 3 days. error bars represent sd for three biological replicates. asterisks indicate a significant difference compared to trv2: leaves effect of rd29a promoter-inducible overexpression of capao on salt tolerance of tobacco identification of transgenic plants to examine the relative contribution of capao to salt stress-induced leaf senescence, tobacco plants were transformed with pvbg2307-prd29a-capao vector. a sequence of 491 bp, which contained a 284-bp fragment of the rd29a promoter and a 207-bp fragment of the capao gene, was produced from genomic dnas of the positive transgenic t lines. nine plants were confirmed to be positive transgenic t lines. t and t generation of transgenic tobacco plants were selected using 50 mg · l− kanamycin, and finally obtained transgenic homozygous t lines. one t positive transgenic plants were used in subsequent studies. semi-quantitative rt-pcr analysis of the transgenic tobacco plants after salt stress treatment as shown in fig.  capao showed no expression in the capao transgenic tobacco and wild type plants before salt stress treatment. capao was strongly induced in the leaves of the transgenic plants at 24 h after treatment with 150 mm nacl solution, while it was not expressed in wt plants at all. this may suggest that the capao gene, controlled under the stress-inducible promoter rd29a, was induced to express under exogenous salt treatment in positive transgenic tobacco plants.fig. rt-pcr analysis of capao in transgenic tobacco plants with and without salt treatment under 150 mm nacl. t: t generation of transgenic tobacco plants, wt: wild type phenotype of transgenic tobacco plants after salt stress treatment before salt stress treatment, there were no phenotypic differences between capao transgenic and wt plants. at early stage of the salt stress treatment, both leaves of transgenic and wt plants wilted with more serious wilting degree in the wt plants. along with the time of treatment, the lower part of leaves became yellow. after salt treatment for d, the leaves of the transgenic plants became yellow and the edge of the lower leaves showed necrotic symptoms after salt stress treatment, while the upper young leaves were green. however, leaves of wt plants appeared to shrink, with lower leaves turning yellow but upper leaves remaining green.fig. phenotypes, chlorophyll contents and mda contents of transgenic tobacco plants after salt treatment under 150 mm nacl. a phenotypes of wild type and transgenic plants before salt stress, b phenotypes of wild type and transgenic plants after salt treatment for d, c phenotypes of leaves of wild type and transgenic plants after salt treatment for d, d chlorophyll contents and e mda contents of transgenic and wild-type tobacco plants under salt stress for and d, respectively. error bars represent sd for three biological replicates. asterisks indicate a significant difference compared to wild type leaves chlorophyll and malondialdehyde contents of transgenic plants after treatment with salt stress leaf senescence is associated with chlorophyll breakdown and reactive oxygen species accumulation. ros-generated lipid peroxidation is an inherent feature of senescing cells and a source of ros. to further analyze the effect of capao overexpression on salt-induced leaf senescence in tobacco, chlorophyll and mda contents were measured after salt treatment for and 14 days, respectively. before the salt stress, there were few obvious differences in chlorophyll and mda content between wt and transgenic leaves. chlorophyll content of wt and transgenic leaves decreased by and  mg · g− after 7 days, and decreased by and  mg · g− after 14 days of salt treatment, respectively. the rate of chlorophyll breakdown in wt plants was higher than that in the transgenic plants. correspondingly, mda content gradually increased with elongated treated time. mda content of wt and transgenic leaves increased by and  mmol · g - after 7 days, and increased by and  mmol · g - after 14 days of salt treatment, respectively. mda content in wt plants was significantly lower than that in transgenic plants. these results indicated that overexpression of capao could accelerate the chlorophyll breakdown and lipid peroxidation accumulation of transgenic tobacco plants under salt treatment. all these observations suggested that overexpression of capao could accelerate salt-induced leaf senescence in tobacco. discussion pao opens the porphyrin macrocycle of pheophorbide a and finally forms fccs. here, we identified the capao gene from pepper. capao contained conserved domains that existed in pao homologues. furthermore, capao shared high homology to other pao proteins, especially sllls and ntpao. bioinformatics analyses demonstrated that capao should function as a pao. tissue-specific analysis showed that capao was expressed in all tissues, and the expression level in leaves was higher than other tissues. the findings were similar to the previous results in maize and rice, it was reported that expression of the ospao gene was detected in all tissues and at the highest level in leaves. however, the present results were different than those in a. thaliana, in which atpao transcript was higher in flowers and siliques than other tissues. these results revealed that pao is important to growth and development of all tissues, although its function may be distinct in different species. various biotic and abiotic stresses can affect growth and development of pepper plants, causing chlorophyll breakdown, cell death and finally premature senescence. all of these consequences would impact the yield and quality of crop plants. pao is an important intermediate in chlorophyll degradation, the expression and activity of which can be affected by various stresses. wang et al. reported that relative expression of and pao in drought-stressed leaves was greatly upregulated in apple. microarray analysis indicated that upregulation of pao in response to various stress conditions, coinciding with breakdown of chlorophyll under these conditions. results of the current study showed that transcript levels of the capao gene were upregulated in pepper plants after treatment with various stresses, including high salinity, osmosis and phytophthora capsici infection. these results suggested that capao may be involved defense response to salt and osmotic stresses as well as phytophthora capsici in pepper plants. previous studies have shown that phytohormone signaling pathways played an important role in mediating developmental processes and environmental responses in plants. these hormones could cause the expression of stress-related genes, which in turn affect senescence progress of leaf. many studies have proved that capao gene expression or activity was regulated by hormones. for instance, aba can enhance the expression of pao in rice leaves. rodoni et al. suggested that the chlorophyll breakdown related to pao activity in barley detached leaves, a rapid loss of chlorophyll in conjunction with an increase of pao activity was occurred after treatment with aba and meja. in the current study, treatment with aba, meja or sa was found to increase the expression level of capao. these findings suggested that the capao gene may be involved in aba-, ja- and sa-dependent signaling pathways. these results were different from those of a study in wheat reporting that aba and meja enhanced the expression of tapao, while sa treatment did not cause significant changes in the level of this gene. sa is the hormone involved in pathogen response and abiotic stress tolerance in pepper plant. in perennials, sa may be involved in the regulation of drought-induced leaf senescence as sa accumulation preceded chlorophyll breakdown and nitrogen mobilization. here, high salinity, osmosis and phytophthora capsici infection induced capao expression in pepper leaves. it is likely that salt, mannitol and phytophthora capsici-induced sa accumulation may trigger capao transcripts in pepper plants. the function of the pao gene has been studied in many plants. tang et al. reported that chlorophyll breakdown was delayed compared to controls in leaves detached from leaves of pao-silenced rice plants during dark-induced senescence. moreover, leaves with overexpression of pao in arabidopsis had lower chlorophyll content and earlier leaf yellowing than the control d after dark treatment. leaf senescence was involved in chlorophyll degradation and accumulation of ros. drought-induced senescence in the leaves of apple was reflected in chlorophyll loss and an increase in the levels of ros, with the induction of the pao gene. in this research, the delay of chlorophyll degradation was detectable in the detached leaves from capao silencing pepper plants 3 days after treatment with salt. conversely, overexpression of capao, induced by the stress-inducible promoter rd29a, could accelerate the rate of chlorophyll breakdown and lipid peroxidation of the transgenic plants under salt stress. the results of tang et al. have shown that overexpressing tapao caused an accumulation of rccs in wheat leaves. rccs have been proved to be phototoxic and caused cell deach, resulting in senescence. all of these results demonstrated that cacp played crucial role in pepper plant defense response to salt stresses, thus delaying leaf senescence.  overall, we conclude that capao plays an important role in senescence and chlorophyll degradation, as well as defense responses to various stresses. capao was induced in natural senescence and various stresses. silence of capao resulted in a stay-green phenotype in pepper plants, and overexpression of capao accelerated the process of salt-induced leaf senescence. in the future, the transgenic plants about overexpression or knockdown of capao gene in pepper would be used for studying detail function of capao. 
the capao gene may be related to defense responses to various stresses and play an important role in salt-induced leaf senescence. the expression of capao gene was induced in natural senescence and various stresses. it opens the porphyrin macrocycle of pheophorbide a and finally forms the primary fluorescent chlorophyll catabolite. it was upregulated by abscisic acid, methyl jasmonate and salicylic acid. moreover, capao was significantly induced by high salinity and osmotic stress treatments and also was regulated by phytophthora capsici. pheophorbide a oxygenase is an important enzyme in the chlorophyll catabolism pathway and is involved in leaf senescence. however, the characterizaton of pao during leaf senescence induced by environmental factors is not well understood. after 3 days of high salt treatment, the chlorophyll breakdown of capao-silenced pepper plants was retarded. homology-based cloning and race techniques were used to obtain the full-length cdna of the capao gene. the virus-induced gene silencing technique was used to silence the capao gene in pepper plants. after 7 days of salt treatment, the leaves of transgenic plants were severely turned into yellow, the lower leaves showed necrotic symptom and chlorophyll content was significantly lower than that in the control plants. this deduced protein belongs to the rieske-type iron-sulfur superfamily, containing a conserved rieske cluster. previous studies have demonstrated the function of pao during cell death. capao expression was determined by quantitative real-time pcr. capao expression, as determined by quantitative real-time pcr, was higher in leaves than roots, stems and flowers. function of capao gene were studied using virus-induced gene silencing and transgenic techniques with tobacco plants. capsicum annuum lcapao geneexpression analysisfunctional analysistobaccoissue-copyright-statement© the author 2015
 pheophorbide a oxygenase is an important enzyme in the chlorophyll catabolism pathway and is involved in leaf senescence. it opens the porphyrin macrocycle of pheophorbide a and finally forms the primary fluorescent chlorophyll catabolite. previous studies have demonstrated the function of pao during cell death. however, the characterizaton of pao during leaf senescence induced by environmental factors is not well understood. methods homology-based cloning and race techniques were used to obtain the full-length cdna of the capao gene. capao expression was determined by quantitative real-time pcr. function of capao gene were studied using virus-induced gene silencing and transgenic techniques with tobacco plants.  a novel pao gene capao was isolated from pepper. the full-length capao cdna is comprised of 1838 bp, containing an open reading frame of 1614 bp, and encodes a amino acid protein. this deduced protein belongs to the rieske-type iron-sulfur superfamily, containing a conserved rieske cluster. capao expression, as determined by quantitative real-time pcr, was higher in leaves than roots, stems and flowers. it was upregulated by abscisic acid, methyl jasmonate and salicylic acid. moreover, capao was significantly induced by high salinity and osmotic stress treatments and also was regulated by phytophthora capsici. the virus-induced gene silencing technique was used to silence the capao gene in pepper plants. after 3 days of high salt treatment, the chlorophyll breakdown of capao-silenced pepper plants was retarded. rd29a promoter-inducible expression vector was constructed and transferred into tobacco plant. after 7 days of salt treatment, the leaves of transgenic plants were severely turned into yellow, the lower leaves showed necrotic symptom and chlorophyll content was significantly lower than that in the control plants.  the expression of capao gene was induced in natural senescence and various stresses. the capao gene may be related to defense responses to various stresses and play an important role in salt-induced leaf senescence. keywords capsicum annuum lcapao geneexpression analysisfunctional analysistobaccoissue-copyright-statement© the author 2015 