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Abstract In order to understand the mechanism of spathe color variation in Anthurium andraeanum at the protein level， the leaves， inflorescences and spathes of the wild type and two mutants of A. andraeanum ‘Madural’ were used as research objects in which the differential expression of proteins related to flower color mutants was analyzed by onedimensional electrophoresis and mass spectrometry （1DE/MS）. The 1DE patterns showed that the protein components expressed highly in spathes were mainly concentrated in the molecular weight range of 20-42 kD， and differential bands were detected between the wild type and the mutant， while no significant differences were detected in the leaf and inflorescence proteins. According to the results of mass spectrometry analysis of the differential bands， 21 known functional proteins involved in life processes such as glucose metabolism， resistance， cytoskeleton， gene regulation and signal transduction were identified. It showed that in addition to the influences from anthocyanidins， the spathe color variation of A. andraeanum ‘Madural’ is also regulated by a variety of metabolic pathwayrelated proteins.
Key words Anthurium andraeanum; Flower color mutant; Differential protein; Gel electrophoresis; Mass spectrometry
Flower color is an important characteristic and economic trait of ornamental plants， which is affected by a variety of internal and external factors. Researches on the flower color of ornamental plants at home and abroad mainly focus on pigment types and contents[1]， pigment distribution[2]， pigment anabolism[3-6]and gene expression regulation[7]. In recent years， people have also studied the molecular mechanism of flower color variation in ornamental plants at the protein level. Su[8]isolated and identified differentially expressed proteins from the yellow and purple petals of flower color chimera in Chrysanthemum， and discussed the formation of flower color differences. Wu[9]studied the molecular mechanism of flower color performance in the white and red flower buds of Japanese apricot ‘Fuban Tiaozhi’ using the twodimensional electrophoresis combined with mass spectrometry. In this study， somatic mutants with rose red and white spathes were found in the Anthurium andraeanum variety ‘Madural’ and used to explore the molecular mechanism of flower color variation， during which protein gel electrophoresis and mass spectrometry （1DE/MS） were applied to analyze the expression differences of proteins related to flower color variation in ‘Madural’ mutants， providing a new experimental basis for elucidating the mechanism of A. andraeanum flower color variation. Materials and Methods
Plant materials
The test materials were the wild type of A. andraeanum ‘Madural’ （with red spathes， marked as MR） and its rose red mutant （marked as MM） and white mutant （marked as MW）. The leaves， inflorescences and spathes of similar development were cut from potted A. andraeanum. After washing with double distilled water， the materials were dried with filter paper. After quick freezing with liquid nitrogen， they were stored in an ultralow temperature refrigerator for storage.
Comparison of flower color
The color types of the wild type and its mutants were analyzed using the Royal Horticultural Society Color Card （RHSCC）.
Extraction of A. andraeanum protein
A certain amount of leaves， inflorescences and spathes of A. andraeanum （0.2 g） were weighed and ground into powder with the addition of liquid nitrogen， respectively. Each powder was added with 3 ml of protein extracting solution （10% glycerol， 0.1 mol/L TrisHCl （pH 6.8）， 5% ┑mercaptoethanol， 2% SDS）， and ground to homogenate. The homogenate was transferred to a centrifuge tube and centrifuged at 4 → and 14 000 r/min for 30 min. The precipitate was discarded， and the supernatant was transferred to a new centrifuge tube and centrifuged at 4 → and 14 000 r/min for 20 min. The supernatant was transferred to a new centrifuge tube， added with three volumes of precooled 80% acetone and shaken well， followed by storage in a freezer at -20 → overnight. The solution was then centrifuged at 4 → and 14 000 r/min for 3 min. The supernatant was discarded， and the precipitate was added with 30-100 l of protein extract to resuspend and dissolve it， obtaining the protein sample which was quantified and stored in the refrigerator at -20 →.
SDSPAGE of A. andraeanum protein
The polyacrylamide gels were 12% separation gel and 5% spacer gel. The loading quantity of the sample was adjusted according to the protein concentration of the sample. And 35 g of sample was added and mixed with 5 l of bromophenol blue indicator， and then loaded. SDSPAGE （1DE） was performed at a fixed current of 35 mA. After the electrophoresis， the gel was removed and stained with CBBR250 for 1 h， and finally decolored to a clear gel background.
MS identification of protein differential bands
From the protein 1DE gel， the differential bands were taken and sent to Shanghai Applied Protein Technology Co.， Ltd. for mass spectrometry analysis. Results and Analysis
Phenotypic characteristics of spathe color mutants of A. andraeanum ‘Madural’
According to the description specification of A. andraeanum resources[10]， the phenotypic characteristics of the three tested plant materials were analyzed. From the perspective of the shape of the spathe （Fig. 1）， the mutants and the wild type all have heartshaped， leathery， smooth， and shiny spathes. However， the wild type （Fig. 1， MR） belongs to the red group （RHSCC code： 42A）， and the rose red mutant （MM） also belongs to the red group though the color is slightly pink （RHSCC code： 51A）， while the white mutant （MW） falls into the white group （RHSCC code： NN155B）. In contrast， the morphology and color of the inflorescences are not significantly different from the wild type. In addition， in terms of the leaf shape， the two mutants are the same as the wild type， both of which show heartshaped leaves， with pointed leaf tip， heartshaped leaf base and green leaf color.
SDSPAGE analysis of different tissue proteins in A. andraeanum
From the SDSPAGE patterns of the tested materials， many bands were detected， and the bands of the spathe proteins were separated clearly. The highabundance protein components were mainly concentrated in the range of 20-42 kD; and compared with the white mutants， a differential band with a molecular weight of approximately 130 kD was detected in the wild type and the rose red mutant （Fig. 2， a and b）， and the expression level of the wild type was higher than that of the rose red mutant. Secondly， the leaf protein bands were also clearly separated， and the number of bands detected was also relatively high， while the inflorescence proteins were poorly separated. However， there were no significant differences in the types and contents of the leaf and inflorescence proteins between the wild type and the mutants.
MS Identification and analysis of differential proteins
From the 1DE gels of the wild type and the rose red mutant， two differential bands （Fig. 2， a and b） were selected for mass spectrometry. The results showed that 17 proteins with known functions were identified from the spathes of the wild type， and 12 proteins with known functions were identified from the rose red mutant. It could be seen from the comparison of the two that a total of 21 proteins were identified. Nine proteins were specifically expressed in the wild type， four proteins were specifically expressed in the rose red mutant， and eight proteins were detected in both materials （Table 1）. According to the function of the identified proteins， they can be divided into 6 types： proteins involved in metabolic regulation， such as mannosespecific lectin， subtilisinlike protease， ATP synthase subunit beta， mannosidase， xylose isomerase， glyeerophosphodiester phosphodiesterase and laccase， cytoskeletonassociated proteins， such as tubulinalpha chain and actin， resistancerelated proteins， such as polyubiquitin， early nodulinlike proteins and mitochondrial peroxiredoxin， gene regulationrelated proteins， such as histones， transportrelated proteins， such as aquaporins， ADP/ATP carrier proteins， polyol transporters， pleiotropic drug resistance protein， and ? signalingrelated proteins， such as auxinbinding proteins. It was speculated that these proteins are involved in the regulation of the spathe color variation from a variety of metabolic pathways. Agricultural Biotechnology2019
Discussion
A. andraeanum is a perennial evergreen plant with beautiful flower and leaves， as well as an important ornamental plant in the form of potted flower or fresh cut flower. Its spathes are diverse in color， and the red variety is the most popular among Chinese people. In this study， the red variety ‘Madural’ was used as a test material， which was investigated for the differential expression of proteins related to flower color variation by protein gel electrophoresis combined with mass spectrometry. The results showed that there were no significant differences in the 1DE bands of the leaf and inflorescence proteins between the wild type and the mutants of ‘Madural’， but significant differences were detected during the comparison of the 1DE patterns of the spathes， indicating the proteins related to flower color variation were mainly expressed in the spathe tissue.
From the 1DE gels of spathes of the test materials， a differential band with a molecular weight of about 130 kD was detected， which was most abundant in the wild type， followed by the rose red mutant， while no expression was detected in the white mutant. Based on the results of mass spectrometry analysis of the two differential bands， 21 proteins were identified， nine of which were specifically expressed in the wild type， four were specifically expressed in the rose red mutant， and eight presented in both the two materials. Their functions involved physiological processes such as glucose metabolism， cytoskeleton formation， resistancerelated metabolism and gene regulation.
Interestingly， the differential proteins identified by the above mass spectrometry， whose functional characteristics indicated that they were not directly involved in the anabolism of flavonoids and anthocyanins， suggested that they might have some regulatory functions on the anthocyanidin metabolic pathway. For instance， the identified laccase （A0A1D1Y305） is a glycoprotein oxidase that affects plant growth and development in cell wall lignin synthesis， promotion of wound healing， disease resistance， and stress resistance， and is also involved in the pathway of pigment synthesis[11-12]. In Arabidopsis thaliana， the laccase gene AtLAC15 is involved in the oxidative polymerization of the flavonoids of the seed coat. The browning of the seed coat of the AtLAC15deletion mutants might be delayed or not happen， and the seed coat is pale yellow or milky white[13]. In the present study， laccase expression was detected in the red spathes of the wild type of ‘Madural’， but was not detected in the rose red mutant， suggesting that A. andraeanum laccase might be involved in the regulation process of anthocyanin anabolism in spathes. In addition， among the identified differential proteins， some were detected only in the wild type， such as mannosidase， three histones （H1， H2， H4）， early nodulinlike proteins， actin， mitochondrial peroxiredoxin PRX， ATP synthase subunit beta and ADP/ATP carrier protein. Conversely， some of them were only detected in the rose red mutant， such as tubulin， auxinbinding protein， aquaporin PIP22 and xylose isomerase. Although their biological functions are relatively clear， the relationships with A. andraeanum flower color variation still need further study.
References
[1]ZHI YJ， WANG WH， LENG PS， et al. Components and content of flower pigments in the petals of different color Lilium species[J]. Northern Horticulture， 2017（9）： 62-69. （in Chinese）
[2]ZHANG YANGQINGHUI， WANG YG， FANG WM， et al. Changes of colors and pigment compositions during the senescence process of Chrysanthemum morifolium[J]. Acta Horticulturae Sinica， 2018， 45（3）： 519-529. （in Chinese）
[3]ZHANG N， HU ZL， CHEN SQ， et al. Analysis of metabolic pathway and establishment of regulating model of anthocyanin synthesis[J]. Journal of Chinese Biotechnology， 2008， （1）： 97-105. （in Chinese）
[4]ALBERT NW， LEWIS DH， ZHANG H， et al. Members of an R2R3MYB transcription factor family in petunia are developmentally and environmentally regulated to control complex floral and vegetative pigmentation patterning[J]. Plant Journal， 2011， 65（5）： 771-784.
[5]ALI MB， HOWARD S， CHEN S， et al. Berry skin development in Norton grape： Distinct patterns of transcriptional regulation and flavonoid biosynthesis[J]. Bmc Plant Biology， 2011， 11（1）： 7.
[6]HOU FY， WANG QM， LI ZX， et al. Study progress on anthocyanidin synthase of plants[J]. Chinese Agricultural Science Bulletin， 2009， 25（21）： 188-190. （in Chinese）
[7]LI J， GAO GC， LI B. Cloning and expression analysis of anthocyanin biosynthesis genes from redyellow flower chimera in Canna generalis[J]. Molecular Plant Breeding， 2015， 13（3）： 634- 640. （in Chinese）
[8]SU M. Study on biological characteristics and formation mechanism of flower color chimera in Chrysanthemum[D]. Suzhou： Suzhou University， 2011. （in Chinese）
[9]WU XX. Plum petal colorrelated proteome and transcriptome analysis of ‘Armeniaca mume f. versicolor T. Y. Chen et H. H. Lu’[D]. Nanjing： Nanjing Agricultural University， 2014. （in Chinese）
[10]YIN JM. Tropical flower germplasm resource description specification[M]. Beijing： China agriculture Press， 2005： 85-102. （in Chinese）
[11]XU XP， CHEN XH， LYU KL， et al. Genomewide identification and function analysis of the laccase gene family in Dimocarpus longan Lour.[J]. Chinese Journal of Applied and Environmental Biology， 2018， 24（4）： 833-844. （in Chinese）
[12]GAO MX， FENG X， LAI RL， et al. Cloning and expression analysis of laccase gene of "Miliang No. 1" kiwi fruit[J]. Chinese journal of agricultural biotechnology， 2018， 26（1）： 64-76. （in Chinese）
[13]LUCILLE P， JEANMARC R， LUCIEN K， et al. TRANSPARENT TESTA10 encodes a laccaselike enzyme involved in oxidative polymerization of flavonoids in Arabidopsis seed coat[J].The Plant Cell， 2005， 17（11）： 2966-2980.
Key words Anthurium andraeanum; Flower color mutant; Differential protein; Gel electrophoresis; Mass spectrometry
Flower color is an important characteristic and economic trait of ornamental plants， which is affected by a variety of internal and external factors. Researches on the flower color of ornamental plants at home and abroad mainly focus on pigment types and contents[1]， pigment distribution[2]， pigment anabolism[3-6]and gene expression regulation[7]. In recent years， people have also studied the molecular mechanism of flower color variation in ornamental plants at the protein level. Su[8]isolated and identified differentially expressed proteins from the yellow and purple petals of flower color chimera in Chrysanthemum， and discussed the formation of flower color differences. Wu[9]studied the molecular mechanism of flower color performance in the white and red flower buds of Japanese apricot ‘Fuban Tiaozhi’ using the twodimensional electrophoresis combined with mass spectrometry. In this study， somatic mutants with rose red and white spathes were found in the Anthurium andraeanum variety ‘Madural’ and used to explore the molecular mechanism of flower color variation， during which protein gel electrophoresis and mass spectrometry （1DE/MS） were applied to analyze the expression differences of proteins related to flower color variation in ‘Madural’ mutants， providing a new experimental basis for elucidating the mechanism of A. andraeanum flower color variation. Materials and Methods
Plant materials
The test materials were the wild type of A. andraeanum ‘Madural’ （with red spathes， marked as MR） and its rose red mutant （marked as MM） and white mutant （marked as MW）. The leaves， inflorescences and spathes of similar development were cut from potted A. andraeanum. After washing with double distilled water， the materials were dried with filter paper. After quick freezing with liquid nitrogen， they were stored in an ultralow temperature refrigerator for storage.
Comparison of flower color
The color types of the wild type and its mutants were analyzed using the Royal Horticultural Society Color Card （RHSCC）.
Extraction of A. andraeanum protein
A certain amount of leaves， inflorescences and spathes of A. andraeanum （0.2 g） were weighed and ground into powder with the addition of liquid nitrogen， respectively. Each powder was added with 3 ml of protein extracting solution （10% glycerol， 0.1 mol/L TrisHCl （pH 6.8）， 5% ┑mercaptoethanol， 2% SDS）， and ground to homogenate. The homogenate was transferred to a centrifuge tube and centrifuged at 4 → and 14 000 r/min for 30 min. The precipitate was discarded， and the supernatant was transferred to a new centrifuge tube and centrifuged at 4 → and 14 000 r/min for 20 min. The supernatant was transferred to a new centrifuge tube， added with three volumes of precooled 80% acetone and shaken well， followed by storage in a freezer at -20 → overnight. The solution was then centrifuged at 4 → and 14 000 r/min for 3 min. The supernatant was discarded， and the precipitate was added with 30-100 l of protein extract to resuspend and dissolve it， obtaining the protein sample which was quantified and stored in the refrigerator at -20 →.
SDSPAGE of A. andraeanum protein
The polyacrylamide gels were 12% separation gel and 5% spacer gel. The loading quantity of the sample was adjusted according to the protein concentration of the sample. And 35 g of sample was added and mixed with 5 l of bromophenol blue indicator， and then loaded. SDSPAGE （1DE） was performed at a fixed current of 35 mA. After the electrophoresis， the gel was removed and stained with CBBR250 for 1 h， and finally decolored to a clear gel background.
MS identification of protein differential bands
From the protein 1DE gel， the differential bands were taken and sent to Shanghai Applied Protein Technology Co.， Ltd. for mass spectrometry analysis. Results and Analysis
Phenotypic characteristics of spathe color mutants of A. andraeanum ‘Madural’
According to the description specification of A. andraeanum resources[10]， the phenotypic characteristics of the three tested plant materials were analyzed. From the perspective of the shape of the spathe （Fig. 1）， the mutants and the wild type all have heartshaped， leathery， smooth， and shiny spathes. However， the wild type （Fig. 1， MR） belongs to the red group （RHSCC code： 42A）， and the rose red mutant （MM） also belongs to the red group though the color is slightly pink （RHSCC code： 51A）， while the white mutant （MW） falls into the white group （RHSCC code： NN155B）. In contrast， the morphology and color of the inflorescences are not significantly different from the wild type. In addition， in terms of the leaf shape， the two mutants are the same as the wild type， both of which show heartshaped leaves， with pointed leaf tip， heartshaped leaf base and green leaf color.
SDSPAGE analysis of different tissue proteins in A. andraeanum
From the SDSPAGE patterns of the tested materials， many bands were detected， and the bands of the spathe proteins were separated clearly. The highabundance protein components were mainly concentrated in the range of 20-42 kD; and compared with the white mutants， a differential band with a molecular weight of approximately 130 kD was detected in the wild type and the rose red mutant （Fig. 2， a and b）， and the expression level of the wild type was higher than that of the rose red mutant. Secondly， the leaf protein bands were also clearly separated， and the number of bands detected was also relatively high， while the inflorescence proteins were poorly separated. However， there were no significant differences in the types and contents of the leaf and inflorescence proteins between the wild type and the mutants.
MS Identification and analysis of differential proteins
From the 1DE gels of the wild type and the rose red mutant， two differential bands （Fig. 2， a and b） were selected for mass spectrometry. The results showed that 17 proteins with known functions were identified from the spathes of the wild type， and 12 proteins with known functions were identified from the rose red mutant. It could be seen from the comparison of the two that a total of 21 proteins were identified. Nine proteins were specifically expressed in the wild type， four proteins were specifically expressed in the rose red mutant， and eight proteins were detected in both materials （Table 1）. According to the function of the identified proteins， they can be divided into 6 types： proteins involved in metabolic regulation， such as mannosespecific lectin， subtilisinlike protease， ATP synthase subunit beta， mannosidase， xylose isomerase， glyeerophosphodiester phosphodiesterase and laccase， cytoskeletonassociated proteins， such as tubulinalpha chain and actin， resistancerelated proteins， such as polyubiquitin， early nodulinlike proteins and mitochondrial peroxiredoxin， gene regulationrelated proteins， such as histones， transportrelated proteins， such as aquaporins， ADP/ATP carrier proteins， polyol transporters， pleiotropic drug resistance protein， and ? signalingrelated proteins， such as auxinbinding proteins. It was speculated that these proteins are involved in the regulation of the spathe color variation from a variety of metabolic pathways. Agricultural Biotechnology2019
Discussion
A. andraeanum is a perennial evergreen plant with beautiful flower and leaves， as well as an important ornamental plant in the form of potted flower or fresh cut flower. Its spathes are diverse in color， and the red variety is the most popular among Chinese people. In this study， the red variety ‘Madural’ was used as a test material， which was investigated for the differential expression of proteins related to flower color variation by protein gel electrophoresis combined with mass spectrometry. The results showed that there were no significant differences in the 1DE bands of the leaf and inflorescence proteins between the wild type and the mutants of ‘Madural’， but significant differences were detected during the comparison of the 1DE patterns of the spathes， indicating the proteins related to flower color variation were mainly expressed in the spathe tissue.
From the 1DE gels of spathes of the test materials， a differential band with a molecular weight of about 130 kD was detected， which was most abundant in the wild type， followed by the rose red mutant， while no expression was detected in the white mutant. Based on the results of mass spectrometry analysis of the two differential bands， 21 proteins were identified， nine of which were specifically expressed in the wild type， four were specifically expressed in the rose red mutant， and eight presented in both the two materials. Their functions involved physiological processes such as glucose metabolism， cytoskeleton formation， resistancerelated metabolism and gene regulation.
Interestingly， the differential proteins identified by the above mass spectrometry， whose functional characteristics indicated that they were not directly involved in the anabolism of flavonoids and anthocyanins， suggested that they might have some regulatory functions on the anthocyanidin metabolic pathway. For instance， the identified laccase （A0A1D1Y305） is a glycoprotein oxidase that affects plant growth and development in cell wall lignin synthesis， promotion of wound healing， disease resistance， and stress resistance， and is also involved in the pathway of pigment synthesis[11-12]. In Arabidopsis thaliana， the laccase gene AtLAC15 is involved in the oxidative polymerization of the flavonoids of the seed coat. The browning of the seed coat of the AtLAC15deletion mutants might be delayed or not happen， and the seed coat is pale yellow or milky white[13]. In the present study， laccase expression was detected in the red spathes of the wild type of ‘Madural’， but was not detected in the rose red mutant， suggesting that A. andraeanum laccase might be involved in the regulation process of anthocyanin anabolism in spathes. In addition， among the identified differential proteins， some were detected only in the wild type， such as mannosidase， three histones （H1， H2， H4）， early nodulinlike proteins， actin， mitochondrial peroxiredoxin PRX， ATP synthase subunit beta and ADP/ATP carrier protein. Conversely， some of them were only detected in the rose red mutant， such as tubulin， auxinbinding protein， aquaporin PIP22 and xylose isomerase. Although their biological functions are relatively clear， the relationships with A. andraeanum flower color variation still need further study.
References
[1]ZHI YJ， WANG WH， LENG PS， et al. Components and content of flower pigments in the petals of different color Lilium species[J]. Northern Horticulture， 2017（9）： 62-69. （in Chinese）
[2]ZHANG YANGQINGHUI， WANG YG， FANG WM， et al. Changes of colors and pigment compositions during the senescence process of Chrysanthemum morifolium[J]. Acta Horticulturae Sinica， 2018， 45（3）： 519-529. （in Chinese）
[3]ZHANG N， HU ZL， CHEN SQ， et al. Analysis of metabolic pathway and establishment of regulating model of anthocyanin synthesis[J]. Journal of Chinese Biotechnology， 2008， （1）： 97-105. （in Chinese）
[4]ALBERT NW， LEWIS DH， ZHANG H， et al. Members of an R2R3MYB transcription factor family in petunia are developmentally and environmentally regulated to control complex floral and vegetative pigmentation patterning[J]. Plant Journal， 2011， 65（5）： 771-784.
[5]ALI MB， HOWARD S， CHEN S， et al. Berry skin development in Norton grape： Distinct patterns of transcriptional regulation and flavonoid biosynthesis[J]. Bmc Plant Biology， 2011， 11（1）： 7.
[6]HOU FY， WANG QM， LI ZX， et al. Study progress on anthocyanidin synthase of plants[J]. Chinese Agricultural Science Bulletin， 2009， 25（21）： 188-190. （in Chinese）
[7]LI J， GAO GC， LI B. Cloning and expression analysis of anthocyanin biosynthesis genes from redyellow flower chimera in Canna generalis[J]. Molecular Plant Breeding， 2015， 13（3）： 634- 640. （in Chinese）
[8]SU M. Study on biological characteristics and formation mechanism of flower color chimera in Chrysanthemum[D]. Suzhou： Suzhou University， 2011. （in Chinese）
[9]WU XX. Plum petal colorrelated proteome and transcriptome analysis of ‘Armeniaca mume f. versicolor T. Y. Chen et H. H. Lu’[D]. Nanjing： Nanjing Agricultural University， 2014. （in Chinese）
[10]YIN JM. Tropical flower germplasm resource description specification[M]. Beijing： China agriculture Press， 2005： 85-102. （in Chinese）
[11]XU XP， CHEN XH， LYU KL， et al. Genomewide identification and function analysis of the laccase gene family in Dimocarpus longan Lour.[J]. Chinese Journal of Applied and Environmental Biology， 2018， 24（4）： 833-844. （in Chinese）
[12]GAO MX， FENG X， LAI RL， et al. Cloning and expression analysis of laccase gene of "Miliang No. 1" kiwi fruit[J]. Chinese journal of agricultural biotechnology， 2018， 26（1）： 64-76. （in Chinese）
[13]LUCILLE P， JEANMARC R， LUCIEN K， et al. TRANSPARENT TESTA10 encodes a laccaselike enzyme involved in oxidative polymerization of flavonoids in Arabidopsis seed coat[J].The Plant Cell， 2005， 17（11）： 2966-2980.