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Monosaccharides and chitosan sensing in bud growth and petal pigmentation in Eustoma grandiflorum (Raf.) Shinn.
Scientia Horticulturae 100 (2004) 127–138
Monosaccharides and chitosan sensing in bud growth and petal pigmentation in Eustoma grandiflorum (Raf.) Shinn. A.F.M. Jamal Uddin, Fumio Hashimoto, Keiichi Shimizu, Yusuke Sakata∗ Ornamental Horticulture Laboratory, Faculty of Agriculture, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan Accepted 5 August 2003
Abstract This study focused on the application of chitosan and monosaccharide to lisianthus cultivars in vitro. Buds from three cultivars, ‘Asuka no Asa’, ‘Mickey Rose’, and ‘Royal Violet’, were kept in a holding solution containing different sugars, with or without chitosan. The cultivars showed distinct variations in flower bud development on treatment with chitosan. Following treatment with fructose and chitosan, a vivid petal color was produced in all the cultivars. Chitosan promoted various processes in developing flower buds, including the accumulation of anthocyanin in petals in vitro. An artificial neural network (ANN) model was used to simulate the measured and estimated values for the total amount of anthocyanins in lisianthus petals. The simulation could explain about 90% of the observed relationship between the input variations and relative anthocyanin synthesis in lisianthus flowers. © 2003 Elsevier B.V. All rights reserved. Keywords: Lisianthus; Chitosan; Sugars; Artificial neural network; Pigmentation
1. Introduction The use of sugars to promote flower development and pigmentation is common practice in pot harvest cut flower culture. The presence or an increase in the concentration of sucrose enhanced petal pigmentation in detached flowers of Eustoma grandiflorum (Kawabata et al., 1995; Uddin et al., 2001). Sugars are essential as general sources of carbohydrates for carbon metabolism, upon which the induction of pigmentation is dependent (Moalem-Beno et al., 1997). Tsukaya et al. (1991) reported that sugars induce the transcription of the ∗
Corresponding author. Tel./fax: +81-99-285-8560. E-mail address: [email protected] (Y. Sakata). 0304-4238/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2003.08.014
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petunia chalcone synthase (Chs) gene in transgenic Arabidopsis and suggested that the intercellulear level of sugars regulates Chs expression. It appears that high intracellular levels of sugars may promote the synthesis of anthocyanin. Sucrose alone does not induce Chs expression and anthocyanin synthesis. GA3 promotes sucrose uptake by the detached corollas and the sugars activate anthocyanin synthesis directly. Other metabolic sugars, such as glucose and fructose, had the same effect as sucrose (Weiss et al., 1992). Ohta et al. (2000) showed that chitosan (poly(1,4)--d-glucosamine) promotes plant growth in Eustoma in the presence of inorganic nitrogen. In addition, attention has been paid to chitosan as a source of polysaccharide (Nishimura et al., 1991; Majeti and Kumar, 2000). For commercial cut flowers, mature flower buds are harvested to ensure a long vase life, but flower color may not develop to the same extent as in intact flowers. The objective of this study was to investigate the use of monosaccharides and chitosan in the induction of flower coloration in lisianthus cut flowers.
2. Materials and methods 2.1. Cut flower culture Buds of three cultivars of lisianthus, ‘Asuka no Asa’, ‘Mickey Rose’ and ‘Royal Violet’ were detached from plants at the peduncle, at 7–10 days (approximately) before anthesis and placed in vials containing different monosaccharide (glucose, galactose, fructose, and rhamnose) solutions at 0.25 M with and without chitosan (poly(1,4)--d-glucosamine) (Sigma Co Ltd., USA; ash:moisture:protein:nitrogen:acetyl = 0.51:4.8:1.3:8.2:27.2) in a holding solution (Shepherd et al., 1997). 2.2. Phytotron conditions The treatment chambers were artificially illuminated by Toshiba lamps (TL 40W/12UV) with the same flux density (approximately 2100 lx) for 16 h per day. The temperature of the chambers was maintained via air conditioning units at 25 ◦ C/18 ◦ C (day/night) with 80% relative humidity (RH). 2.3. Bud growth and petal color intensity Bud length and width were measured daily before anthesis. To compare characteristics, intact flowers were observed in a greenhouse. The petal color of an individual was measured at three locations with a NR-3000 color analyzer (Nippon Denshoku, Japan). This measurement is based on the Commission Internationale de l’Eclairage (CIE D65/10◦ ) scale (McGuire, 1992) to obtain a correlation with petal anthocyanin content. CIE color data consist of a luminance or lightness component (L∗ ), corresponding to the vertical axis, and two chromatic components: a∗ (from green to red) and b∗ (from blue to yellow). At L∗ values from 0 to 100, the blackness gradually decreases, while the shade of green gradually decreases and the shade of red becomes more prominent from −a∗ to +a∗ . Moreover, from −b∗ to +b∗ , the tint of blue gradually diminishes and the yellow shade increases (CIE,
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1986). Chroma, C∗ , and hue (hue angle), hab , were calculated according to the following equations: C∗ = (a∗2 + b∗2 )0.5 and hab = tan−1 (b∗ /a∗ ) (Gonnet, 1998). C∗ , is the perpendicular distance from the lightness axis (more distance being more chroma). The hue angle is expressed in degrees. In the CIE color system, chromatic tonalities are spread across a continuous circle (0–360◦ , with 0◦ being on the +a∗ axis, red to purple; then continuing to 90◦ for the +b∗ axis, yellow, 180◦ for −a∗ bluish-green, 270◦ for −b∗ , blue, and back to 360◦ ). 2.4. Anthocyanin extraction Petal slices were macerated in 5 ml of an acidic methanol solution (1% HCl in MeOH) in a test tube and allowed to equilibrate overnight at 4 ◦ C. A 2 ml volume of sample solution with 4 ml of 2N HCl was heated to 100 ◦ C for around 2 h in a heating block, filtered with millipore disks (0.45 m), then collected for high-performance liquid chromatography (HPLC). 2.5. HPLC analyses The anthocyanidin composition was determined by HPLC (Gulliver CO-966 Intelligent Column Thermostat). For qualitative and quantitative analyses, the linear flow-gradient conditions were; 1.5% H3 PO4 as A eluent and H3 PO4 :HCOOH:CH3 CN:H2 O (1.5:20:25:53.5) as B eluent; A:B = 70:30 to 30:70 (v/v, %), 60 min; column, TSKgel ODS-80Ts QA, Toso Co., column, Cosmosil-5C18 (4.6 mm i.d. × 15 cm l); temperature, 40 ◦ C; flow rate, 0.8 ml min−1 ; wave length, 525 nm and injection volume, 10 l per sample. The following equipment was used: Intelligent Sampler AS950-10, three-line Degasser DG-980-02, Intelligent HPLC pump PU-980, Intelligent Column Thermostat CO-966, Ternary Gradient Unit LG980-02 and UV Spectrometer UVDEC-100-III FP-920 (JASCO Co. Ltd., Tokyo). Anthocyanidins were identified by matching their retention times to those of the anthocyanidins present in an authentic sample as described by Hong and Wrolstad (1990). The results are expressed as peak area percentages from the respective HPLC analyses. 2.6. Total anthocyanin (TA) concentrations in the petals Concentrations of anthocyanins in different samples were calculated semi-quantitatively from a simple linear regression using cyanin for anthocyanin as standards (Hashimoto et al., 2000, 2002). The optical density (OD) of individual samples was measured with a UV spectrophotometer (Toshiba, Spectra; Model SPM-60A) at 525 nm for anthocyanin, and calculated in mg per 100 mg fresh petal (mg, %). 2.7. Application of artificial neural networks Artificial neural networks (ANNs) are one of the most popular and successful neural network architectures. A range of different ANN architectures were trained and tested using the measured data, and the optimum design was a hybrid four-layer network (Fig. 1). Data were derived by ezVidCap.ocx for VB5/VB6 free software, Version 1.02. A hybrid network refers to a combination of different transfer functions in a single model. A linear
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Fig. 1. Principal structure of the 10–5–2–1 artificial neural network architecture (Qnet, 2000). Each neuron of the input layer is connected to each neuron of the hidden layers. Again, each neuron of the hidden layers is connected to a neuron of the output layer. The neurons are connected to neurons of the hidden layers with linear transfer functions. The neurons of the hidden layers (1 and 2) are connected to the neurons of the output layer via a sigmoid transfer function (1/(1 + e−x )).
transfer function was used for the input layers and a logistic function {sigmoid, 1/(1+e−x )} for the hidden layers 1 and 2, and output layer nodes. This type of model works by feeding function signals (inputs) in the input layer, propagates forward neuron by neuron through the network, and then emerges at the end of the network as an output signal. The number of iterations was set at 100,000 while monitoring the root mean square (RMS ≤ 0.001), which indicates the output precision. The momentum of training was set at α = 0.8 and training speed at η = 0.001 (Qnet, 2000). 3. Results 3.1. Bud growth in vitro and under field conditions In vitro, bud lengths of ‘Asuka no Kurenai’ were lowest under control (without sugar or chitosan) conditions (Fig. 2). Buds from sugar-added holding solutions had a longer
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Without chitosan Early Bud Stage Late Bud Stage 5.0
4.5
4.5
4.0
4.0
3.5
3.5
With chitosan Early Bud Stage Late Bud Stage
'Asuka no Kurenai' 3.0 0
1
-8 -7 -6 -5 -4 -3 -2 -1
5.0
5.0
4.5
4.5
4.0
4.0
3.5
3.5
0
1
Chitosan dependent
-8 -7 -6 -5 -4 -3 -2 -1
'Mickey Rose'
'Mickey Rose' 3.0
3.0 -8 -7 -6 -5 -4 -3 -2 -1
0
-8 -7 -6 -5 -4 -3 -2 -1
1
5.0
5.0
4.5
4.5
4.0
4.0
3.5
3.5
1
'Royal Violet'
'Royal Violet' 3.0
0
Sugar-sugar-chitosan dependent
Bud length (mm)
'Asuka no Kurenai' 3.0
Sugar-chitosan dependent
5.0
131
3.0 -8 -7 -6 -5 -4 -3 -2 -1
Glucose Rhamnose
0
1
-8 -7 -6 -5 -4 -3 -2 -1
Galactose Control
0
1
Fructose Field Conditions
Days before anthesis Fig. 2. Chitosan and sugar effects on length of detached flower buds of three lisianthus cultivars (Day 0 indicates anthesis).
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length but were smaller than those grown under field conditions. The different sugars did not produce any deviation in length. However, bud lengths of ‘Asuka no Kurenai’ in the presence of chitosan were the same as in control and sugar solutions. The buds were smaller than those grown in the field conditions. In ‘Mickey Rose’ grown without chitosan, control and sugar-added solutions produced similar length buds. They were smaller than those grown in the field conditions. But in the presence of chitosan, the lengths of the buds were the same as under field conditions. The holding solution with galactose produced larger buds in vitro. In the presence of chitosan and glucose, buds were larger than those obtained under field conditions (Fig. 2). Sugars in the holding solution without chitosan resulted in wider buds than the control. The width did not show any significant variation in the presence of chitosan in ‘Asuka no Kurenai’. In ‘Mickey Rose’, buds showed no variation with or without sugars but in the presence of chitosan, sugars increased the bud width. Maximum bud width was obtained in the presence of glucose in the holding solution. Significant variation in bud width was observed both with and without chitosan (Fig. 3). 3.2. Petal coloration and pigmentation The significant differences in CIE color coordinates show the distinct characteristics of the treatment variations (Table 1). The lowest L∗ (58.2) and highest C∗ (47.6) values were observed with a hue angle of 353.9◦ in ‘Asuka no Kurenai’, in fructose solution without chitosan. Flower petals became darker in the presence of chitosan in the holding solution with fructose. ‘Mickey Rose’ and ‘Royal Violet’ flower petals showed the lowest L∗ (76.7, 50.0) and highest C∗ (16.6, 37.5), in the fructose solution without chitosan at a hue angle of 354.6◦ and 335.2◦ , respectively. But with chitosan in the holding solution, fructose produced the lowest L∗ (68.0, 43.9) and highest C∗ (29.7, 50.2) values with a hue angle of 348.0◦ and 326.4◦ , respectively, and produced a more vivid petal color in vitro (Table 1). Some variation, 5–6%, was found in anthocyanidin composition among the treatments. The fructose solution with chitosan produced a 92:8 ratio of pelargonidin to cyanidin with the brightest flower color in ‘Asuka no Kurenai’ as well as the maximum amount of total anthocyanin (0.18 mg per 100 mg fresh petal). Similarly ‘Mickey Rose’ produced the largest amount of anthocyanin (0.12 mg per 100 mg fresh petal) in the fructose solution with chitosan along with the lowest L∗ and highest C∗ at a Cy to Pg ratio of 83:17. ‘Bridal Violet’ produced 0.31 mg per 100 mg fresh petal of anthocyanin with a ratio of Dp:Cy:Pn = 87:6:7 in the holding solution of fructose and chitosan. Results showed that the maximum TA was produced in the fructose solution with chitosan for all the cultivars. Though ‘Asuka no Kurenai’ in fructose produced more anthocyanin with chitosan, more anthocyanins were produced with fructose and chitosan than without chitosan (1.5 and 2.0 times more) in ‘Mickey Rose’ and ‘Royal Violet’, respectively. ANN was used to analyze the average contribution of treatments to the different output parameters. The greatest contribution of cultivars to all the outputs among the three treatments was more than 77% (Table 2). Cultivars contributed to up to 84% of the variation in TA, whereas chitosan and sugars contributed 9 and 7%, respectively, to the pigmentation.
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6
6
5
5
4
4
3
3
2
2 'Asuka no Kurenai'
0
1
'Asuka no Kurenai'
0 -8 -7 -6 -5 -4 -3 -2 -1
Bud width (mm)
Early Bud Stage Late Bud Stage
7
Sugar-chitosan dependent
Early Bud Stage Late Bud Stage
1
With chitosan
8
0
-8 -7 -6 -5 -4 -3 -2 -1
1
8
8
7
7
6
6
5
5
4
4
3
3
2
2
1
0
1
Chitosan dependent
7
Without chitosan
1 'Mickey Rose'
0
-8 -7 -6 -5 -4 -3 -2 -1
0
'Mickey Rose'
0 1
-8 -7 -6 -5 -4 -3 -2 -1
8
8
7
7
6
6
5
5
4
4
3
3
2
2
1
0
1
Sugar-sugar-chitosan dependent
8
133
1 'Royal Violet'
0
'Royal Violet'
0 -8 -7 -6 -5 -4 -3 -2 -1
Glucose Rhamnose
0
1
-8 -7 -6 -5 -4 -3 -2 -1
Galactose Control
0
1
Fructose Field Conditions
Days before anthesis Fig. 3. Chitosan and sugar effects on width of detached flower buds of three lisianthus cultivars (Day 0 indicates anthesis).
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Table 1 Petal color attributes and pigment constituents under different treatment conditions in lisianthusa Cultivars Asuka no Kurenai
Mickey Rose
Royal Violet
a
Treatments
Color coordinatesb,e
Anthocyanidins (%)c
Chitosan
Sugar
L∗
C∗
TAd,e
hab
Dp
Cy
Pt
Pg
Pn
Mv
−
–
69.2 a
31.7 a
356.2 a
−
9
+
91
−
−
0.08 a
− − − − + + + + + Field conditions
Glc Fru Gal Rha – Glc Fru Gal Rha –
60.5 b 58.2 c 72.5 d 71.6 e 69.0 a 58.7 f 56.1 c 71.1 e 66.9 g 38.0 h
45.1 b 47.6 c 27.4 d 28.0 e 33.0 f 47.9 g 51.8 c 30.7 h 37.6 i 68.0 j
353.6 b 353.9 b 353.0 c 355.0 d 351.2 e 352.3 f 351.9 f 352.2 f 350.7 g 355.2 h
− − − − − − − − − −
10 13 9 12 8 7 8 14 11 5
+ − − − − − − − − +
90 87 91 88 92 93 92 86 89 95
− − − − − − − − − −
− − − − − − − − − −
0.13 b 0.16 c 0.07 d 0.09 a 0.08 a 0.15 e 0.18 f 0.09 a 0.10 h 0.46 i
−
–
80.5 a
11.1 a
23.4 a
−
93
−
7
−
−
0.04 a
− − − − + + + + + Field conditions
Glc Fru Gal Rha – Glc Fru Gal Rha –
77.8 b 76.7 c 81.6 d 78.0 c 76.4 b 77.8 c 68.0 e 75.9 f 78.9 g 59.6 h
14.3 b 16.6 c 9.6 d 14.4 c 17.3 e 15.9 f 29.7 g 18.2 h 15.5 f 44.3 i
354.6 b 356.4 c 21.9 d 5.7 e 354.4 b 352.1 f 348.0 g 348.7 h 354.6 b 344.6 i
− − − − 1 − − − − 5
92 90 87 86 80 83 83 86 87 68
− − − − − − − − − −
8 10 13 14 19 17 17 14 13 26
− − − − − − − − − 1
− − − − − − − − − −
0.06 b 0.06 b 0.04 a 0.04 a 0.05 a 0.11 c 0.12 c 0.06 b 0.05 b 0.83 d
−
–
58.7 a
28.8 a
352.3 a
92
3
−
+
5
−
0.13 a
− − − − + + + + + Field conditions
Glc Fru Gal Rha – Glc Fru Gal Rha –
51.6 b 50.0 c 59.0 a 59.0 a 54.7 d 48.4 e 43.9 f 56.1 g 50.1 ab 27.6 h
32.0 b 37.5 c 29.8 d 28.7 a 37.3 b 44.4 e 50.2 f 33.5 g 45.2 h 74.2 i
335.2 b 326.2 c 329.7 d 333.4 e 328.1 f 327.6 g 326.4 c 327.8 b 325.0 f 333.3 i
86 90 89 87 90 89 87 89 88 95
5 4 5 6 5 5 6 5 6 4
− − − − − − − − − −
− − + + + − − − + −
9 6 6 7 5 6 7 6 6 −
− − − − − − − − − 1
0.18 b 0.24 c 0.11 a 0.13 ad 0.15 e 0.27 f 0.31 g 0.14 de 0.22 h 0.30 i
Fresh petals were observed and each value is expressed as a mean of five replicates. L∗ , lightness; C∗ , chroma (color saturation); hab , hue angle (◦ ; tan−1 (b∗ /a∗ )) (D65/10◦ CIE illuminant/observer condition, transmission, 1 cm optical pathlength). c Dp, delphinidin; Cy, cyanidin; Pg, pelargonidin; Pt, petunidin; Pn, peonidin; Mv, malvidin (−, not detected; +, detected, in which the respective anthocyanidin concentration is below 0.4% of total anthocyanidin in fresh petal). d TA, total anthocyanin in mg per 100 mg fresh petal. e Values in the column within each cultivar with different letters differ significantly (SAS, 1995) (at P = 0.05 by Duncan’s new multiple range test). b
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Table 2 Average contribution of treatments to output parameters Output parameter
Treatment
Percent contribution
Lightness (L∗ )
Cultivar Chitosan Sugars
83.1 9.5 7.4
Chroma (C∗ )
Cultivar Chitosan Sugars
77.1 11.6 11.3
Hue angle (hab )
Cultivar Chitosan Sugars
85.7 9.5 4.8
Delphinidin (Dp)
Cultivar Chitosan Sugars
91.4 5.9 2.7
Cyanidin (Cy)
Cultivar Chitosan Sugars
80.6 10.3 9.1
Pelargonidin (Pg)
Cultivar Chitosan Sugars
93.7 2.5 3.8
Peonidin (Pn)
Cultivar Chitosan Sugars
90.0 6.8 3.2
Total anthocyanin (TA)
Cultivar Chitosan Sugars
83.7 9.4 6.9
To compute the differences in estimated and measured amounts of anthocyanin in lisianthus flower petals, different input treatments were compared. The model was calibrated against a range of datasets of measured total amount of anthocyanin and anthocyanidin composition, chromatic values, and treatment components (cultivars, chitosan, and sugars). The good correlation between the estimated and measured amounts was surprising considering the heterogeneity involved and simplistic parameterization (RMS error = 0.02, correlation = 0.89). The creation of a model could explain 90% of the relationship of the treatments with the total amount of anthocyanin (Fig. 4).
4. Discussion Tsukaya et al. (1991) clearly showed that levels of sugars depend on the age of flower buds and the difference was about 20-fold between the early and late stages of bud development. The accumulation of a large amount of sugars in the later stage resulted in a rapid increase in bud growth. Consistent with the findings of Tsukaya et al. (1991), the presence of fructose produced a rapid increase in bud growth, in the late stage among lisianthus cultivars. This
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Total Anthocyanidin (mg/100mg fresh petal)
Measured and simulated outputs
'Asuka no Kurenai' 0.35
'Mickey Rose'
'Royal Violet'
Chitosan without with
Chitosan without with
Chitosan with without
ANN Correlation=0.89 ERS=0.02
0.3 0.25 0.2 0.15 0.1 0.05
Measured Simulated
0
5
10
15
20
25
30
control glucose fructose galactose rahmnose control glucose fructose galactose rahmnose control glucose fructose galactose rahmnose control glucose fructose galactose rahmnose control glucose fructose galactose rahmnose control glucose fructose galactose rahmnose
0
Input node patern sequence Fig. 4. Measured (open circle) and simulated (filled circle) variation in the total amount of anthocyanins in fresh flower petals against the input node pattern sequence (ANN, artificial neural network used to drive the model simulation).
suggests that fructose is the major substrate for the metabolic processes of bud growth, acting as an energy source and as a signaling molecule (Neta-Sharir et al., 2000). The role of sugars in flower development may be multifunctional: they can act as an energy source (Moalem-Beno et al., 1997), and as an osmotic regulator (Sacalis and Chin, 1976; Ho and Nichols, 1977; Beileski, 1993). This suggests that the presence of fructose and the increased bud osmoticum induced a rapid expansion. Fructose may serve as the best source of carbohydrates for the metabolism of carbon upon which the induction of specific signaling events is dependent for flower development. The presence of fructose in the holding solution causes a major increase in osmoticum in the flower petal (Schnyder and Nelson, 1987). Sugars play an essential role in the accumulation of pigment by signaling the activation of chalcone synthase gene (Chs) expression in the developing petals (Weiss, 2000). This study provided additional evidence that fructose is noticeably associated with pigment synthesis in lisianthus petals. It seems likely that a keto group at the C2 position in fructose boosts the Chs gene expression and is involved in the activation of pigment biosynthetic genes to produce a larger amount of anthocyanin. Chitosan is the universally accepted non-toxic N-de-acetylated derivative of chitin, a highly basic polysaccharide. It has been suggested that chitosan promotes tissue growth and differentiation in tissue culture. Chitosan is of commercial interest due to its high percentage of nitrogen (7.1%) compared to synthetically substituted celluloses. The presence of GA3
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is required for sucrose to induce chalcone synthase gene (Chs) expression and anthocyanin synthesis (Weiss, 2000). But the results obtained without using GA3 , revealed that chitosan along with fructose, contributes greatly to the increase in anthocyanin synthesis in petals of lisianthus cultivars. For determination of the extent of pigmentation, acceptable agreement was observed between the total amount of anthocyanin in fresh petals and the respective amount estimated using the artificial neural network model. Success in simulated modeling and numeric prediction on a limited scale, such as proposed here, would encourage commercial demand to increase the market value of cut flowers by maintaining vivid flower colors.
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Uddin, A.F.M.J., Hashimoto, F., Kaketani, M., Shimizu, K., Sakata, Y., 2001. Analysis of light and sucrose potencies on petal coloration and pigmentation of lisianthus cultivars (in vitro). Sci. Hort. 89, 73–82. Weiss, D., 2000. Regulation of flower pigmentation and growth: multiple signaling pathways control anthocyanin synthesis in expanding petals. Physiol. Plantarum 110, 152–157. Weiss, D., van Blokland, R., Kooter, J.M., Mol, J.N.M., van Tunen, A.J., 1992. Gibberellic acid regulates chalcone synthase gene transcription in the corolla of Petunia hybrida. Plant Physiol. 98, 191–197.
Monosaccharides and chitosan sensing in bud growth and petal pigmentation in Eustoma grandiflorum (Raf.) Shinn. A.F.M. Jamal Uddin, Fumio Hashimoto, Keiichi Shimizu, Yusuke Sakata∗ Ornamental Horticulture Laboratory, Faculty of Agriculture, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan Accepted 5 August 2003
Abstract This study focused on the application of chitosan and monosaccharide to lisianthus cultivars in vitro. Buds from three cultivars, ‘Asuka no Asa’, ‘Mickey Rose’, and ‘Royal Violet’, were kept in a holding solution containing different sugars, with or without chitosan. The cultivars showed distinct variations in flower bud development on treatment with chitosan. Following treatment with fructose and chitosan, a vivid petal color was produced in all the cultivars. Chitosan promoted various processes in developing flower buds, including the accumulation of anthocyanin in petals in vitro. An artificial neural network (ANN) model was used to simulate the measured and estimated values for the total amount of anthocyanins in lisianthus petals. The simulation could explain about 90% of the observed relationship between the input variations and relative anthocyanin synthesis in lisianthus flowers. © 2003 Elsevier B.V. All rights reserved. Keywords: Lisianthus; Chitosan; Sugars; Artificial neural network; Pigmentation
1. Introduction The use of sugars to promote flower development and pigmentation is common practice in pot harvest cut flower culture. The presence or an increase in the concentration of sucrose enhanced petal pigmentation in detached flowers of Eustoma grandiflorum (Kawabata et al., 1995; Uddin et al., 2001). Sugars are essential as general sources of carbohydrates for carbon metabolism, upon which the induction of pigmentation is dependent (Moalem-Beno et al., 1997). Tsukaya et al. (1991) reported that sugars induce the transcription of the ∗
Corresponding author. Tel./fax: +81-99-285-8560. E-mail address: [email protected] (Y. Sakata). 0304-4238/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2003.08.014
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petunia chalcone synthase (Chs) gene in transgenic Arabidopsis and suggested that the intercellulear level of sugars regulates Chs expression. It appears that high intracellular levels of sugars may promote the synthesis of anthocyanin. Sucrose alone does not induce Chs expression and anthocyanin synthesis. GA3 promotes sucrose uptake by the detached corollas and the sugars activate anthocyanin synthesis directly. Other metabolic sugars, such as glucose and fructose, had the same effect as sucrose (Weiss et al., 1992). Ohta et al. (2000) showed that chitosan (poly(1,4)--d-glucosamine) promotes plant growth in Eustoma in the presence of inorganic nitrogen. In addition, attention has been paid to chitosan as a source of polysaccharide (Nishimura et al., 1991; Majeti and Kumar, 2000). For commercial cut flowers, mature flower buds are harvested to ensure a long vase life, but flower color may not develop to the same extent as in intact flowers. The objective of this study was to investigate the use of monosaccharides and chitosan in the induction of flower coloration in lisianthus cut flowers.
2. Materials and methods 2.1. Cut flower culture Buds of three cultivars of lisianthus, ‘Asuka no Asa’, ‘Mickey Rose’ and ‘Royal Violet’ were detached from plants at the peduncle, at 7–10 days (approximately) before anthesis and placed in vials containing different monosaccharide (glucose, galactose, fructose, and rhamnose) solutions at 0.25 M with and without chitosan (poly(1,4)--d-glucosamine) (Sigma Co Ltd., USA; ash:moisture:protein:nitrogen:acetyl = 0.51:4.8:1.3:8.2:27.2) in a holding solution (Shepherd et al., 1997). 2.2. Phytotron conditions The treatment chambers were artificially illuminated by Toshiba lamps (TL 40W/12UV) with the same flux density (approximately 2100 lx) for 16 h per day. The temperature of the chambers was maintained via air conditioning units at 25 ◦ C/18 ◦ C (day/night) with 80% relative humidity (RH). 2.3. Bud growth and petal color intensity Bud length and width were measured daily before anthesis. To compare characteristics, intact flowers were observed in a greenhouse. The petal color of an individual was measured at three locations with a NR-3000 color analyzer (Nippon Denshoku, Japan). This measurement is based on the Commission Internationale de l’Eclairage (CIE D65/10◦ ) scale (McGuire, 1992) to obtain a correlation with petal anthocyanin content. CIE color data consist of a luminance or lightness component (L∗ ), corresponding to the vertical axis, and two chromatic components: a∗ (from green to red) and b∗ (from blue to yellow). At L∗ values from 0 to 100, the blackness gradually decreases, while the shade of green gradually decreases and the shade of red becomes more prominent from −a∗ to +a∗ . Moreover, from −b∗ to +b∗ , the tint of blue gradually diminishes and the yellow shade increases (CIE,
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1986). Chroma, C∗ , and hue (hue angle), hab , were calculated according to the following equations: C∗ = (a∗2 + b∗2 )0.5 and hab = tan−1 (b∗ /a∗ ) (Gonnet, 1998). C∗ , is the perpendicular distance from the lightness axis (more distance being more chroma). The hue angle is expressed in degrees. In the CIE color system, chromatic tonalities are spread across a continuous circle (0–360◦ , with 0◦ being on the +a∗ axis, red to purple; then continuing to 90◦ for the +b∗ axis, yellow, 180◦ for −a∗ bluish-green, 270◦ for −b∗ , blue, and back to 360◦ ). 2.4. Anthocyanin extraction Petal slices were macerated in 5 ml of an acidic methanol solution (1% HCl in MeOH) in a test tube and allowed to equilibrate overnight at 4 ◦ C. A 2 ml volume of sample solution with 4 ml of 2N HCl was heated to 100 ◦ C for around 2 h in a heating block, filtered with millipore disks (0.45 m), then collected for high-performance liquid chromatography (HPLC). 2.5. HPLC analyses The anthocyanidin composition was determined by HPLC (Gulliver CO-966 Intelligent Column Thermostat). For qualitative and quantitative analyses, the linear flow-gradient conditions were; 1.5% H3 PO4 as A eluent and H3 PO4 :HCOOH:CH3 CN:H2 O (1.5:20:25:53.5) as B eluent; A:B = 70:30 to 30:70 (v/v, %), 60 min; column, TSKgel ODS-80Ts QA, Toso Co., column, Cosmosil-5C18 (4.6 mm i.d. × 15 cm l); temperature, 40 ◦ C; flow rate, 0.8 ml min−1 ; wave length, 525 nm and injection volume, 10 l per sample. The following equipment was used: Intelligent Sampler AS950-10, three-line Degasser DG-980-02, Intelligent HPLC pump PU-980, Intelligent Column Thermostat CO-966, Ternary Gradient Unit LG980-02 and UV Spectrometer UVDEC-100-III FP-920 (JASCO Co. Ltd., Tokyo). Anthocyanidins were identified by matching their retention times to those of the anthocyanidins present in an authentic sample as described by Hong and Wrolstad (1990). The results are expressed as peak area percentages from the respective HPLC analyses. 2.6. Total anthocyanin (TA) concentrations in the petals Concentrations of anthocyanins in different samples were calculated semi-quantitatively from a simple linear regression using cyanin for anthocyanin as standards (Hashimoto et al., 2000, 2002). The optical density (OD) of individual samples was measured with a UV spectrophotometer (Toshiba, Spectra; Model SPM-60A) at 525 nm for anthocyanin, and calculated in mg per 100 mg fresh petal (mg, %). 2.7. Application of artificial neural networks Artificial neural networks (ANNs) are one of the most popular and successful neural network architectures. A range of different ANN architectures were trained and tested using the measured data, and the optimum design was a hybrid four-layer network (Fig. 1). Data were derived by ezVidCap.ocx for VB5/VB6 free software, Version 1.02. A hybrid network refers to a combination of different transfer functions in a single model. A linear
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Fig. 1. Principal structure of the 10–5–2–1 artificial neural network architecture (Qnet, 2000). Each neuron of the input layer is connected to each neuron of the hidden layers. Again, each neuron of the hidden layers is connected to a neuron of the output layer. The neurons are connected to neurons of the hidden layers with linear transfer functions. The neurons of the hidden layers (1 and 2) are connected to the neurons of the output layer via a sigmoid transfer function (1/(1 + e−x )).
transfer function was used for the input layers and a logistic function {sigmoid, 1/(1+e−x )} for the hidden layers 1 and 2, and output layer nodes. This type of model works by feeding function signals (inputs) in the input layer, propagates forward neuron by neuron through the network, and then emerges at the end of the network as an output signal. The number of iterations was set at 100,000 while monitoring the root mean square (RMS ≤ 0.001), which indicates the output precision. The momentum of training was set at α = 0.8 and training speed at η = 0.001 (Qnet, 2000). 3. Results 3.1. Bud growth in vitro and under field conditions In vitro, bud lengths of ‘Asuka no Kurenai’ were lowest under control (without sugar or chitosan) conditions (Fig. 2). Buds from sugar-added holding solutions had a longer
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Without chitosan Early Bud Stage Late Bud Stage 5.0
4.5
4.5
4.0
4.0
3.5
3.5
With chitosan Early Bud Stage Late Bud Stage
'Asuka no Kurenai' 3.0 0
1
-8 -7 -6 -5 -4 -3 -2 -1
5.0
5.0
4.5
4.5
4.0
4.0
3.5
3.5
0
1
Chitosan dependent
-8 -7 -6 -5 -4 -3 -2 -1
'Mickey Rose'
'Mickey Rose' 3.0
3.0 -8 -7 -6 -5 -4 -3 -2 -1
0
-8 -7 -6 -5 -4 -3 -2 -1
1
5.0
5.0
4.5
4.5
4.0
4.0
3.5
3.5
1
'Royal Violet'
'Royal Violet' 3.0
0
Sugar-sugar-chitosan dependent
Bud length (mm)
'Asuka no Kurenai' 3.0
Sugar-chitosan dependent
5.0
131
3.0 -8 -7 -6 -5 -4 -3 -2 -1
Glucose Rhamnose
0
1
-8 -7 -6 -5 -4 -3 -2 -1
Galactose Control
0
1
Fructose Field Conditions
Days before anthesis Fig. 2. Chitosan and sugar effects on length of detached flower buds of three lisianthus cultivars (Day 0 indicates anthesis).
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length but were smaller than those grown under field conditions. The different sugars did not produce any deviation in length. However, bud lengths of ‘Asuka no Kurenai’ in the presence of chitosan were the same as in control and sugar solutions. The buds were smaller than those grown in the field conditions. In ‘Mickey Rose’ grown without chitosan, control and sugar-added solutions produced similar length buds. They were smaller than those grown in the field conditions. But in the presence of chitosan, the lengths of the buds were the same as under field conditions. The holding solution with galactose produced larger buds in vitro. In the presence of chitosan and glucose, buds were larger than those obtained under field conditions (Fig. 2). Sugars in the holding solution without chitosan resulted in wider buds than the control. The width did not show any significant variation in the presence of chitosan in ‘Asuka no Kurenai’. In ‘Mickey Rose’, buds showed no variation with or without sugars but in the presence of chitosan, sugars increased the bud width. Maximum bud width was obtained in the presence of glucose in the holding solution. Significant variation in bud width was observed both with and without chitosan (Fig. 3). 3.2. Petal coloration and pigmentation The significant differences in CIE color coordinates show the distinct characteristics of the treatment variations (Table 1). The lowest L∗ (58.2) and highest C∗ (47.6) values were observed with a hue angle of 353.9◦ in ‘Asuka no Kurenai’, in fructose solution without chitosan. Flower petals became darker in the presence of chitosan in the holding solution with fructose. ‘Mickey Rose’ and ‘Royal Violet’ flower petals showed the lowest L∗ (76.7, 50.0) and highest C∗ (16.6, 37.5), in the fructose solution without chitosan at a hue angle of 354.6◦ and 335.2◦ , respectively. But with chitosan in the holding solution, fructose produced the lowest L∗ (68.0, 43.9) and highest C∗ (29.7, 50.2) values with a hue angle of 348.0◦ and 326.4◦ , respectively, and produced a more vivid petal color in vitro (Table 1). Some variation, 5–6%, was found in anthocyanidin composition among the treatments. The fructose solution with chitosan produced a 92:8 ratio of pelargonidin to cyanidin with the brightest flower color in ‘Asuka no Kurenai’ as well as the maximum amount of total anthocyanin (0.18 mg per 100 mg fresh petal). Similarly ‘Mickey Rose’ produced the largest amount of anthocyanin (0.12 mg per 100 mg fresh petal) in the fructose solution with chitosan along with the lowest L∗ and highest C∗ at a Cy to Pg ratio of 83:17. ‘Bridal Violet’ produced 0.31 mg per 100 mg fresh petal of anthocyanin with a ratio of Dp:Cy:Pn = 87:6:7 in the holding solution of fructose and chitosan. Results showed that the maximum TA was produced in the fructose solution with chitosan for all the cultivars. Though ‘Asuka no Kurenai’ in fructose produced more anthocyanin with chitosan, more anthocyanins were produced with fructose and chitosan than without chitosan (1.5 and 2.0 times more) in ‘Mickey Rose’ and ‘Royal Violet’, respectively. ANN was used to analyze the average contribution of treatments to the different output parameters. The greatest contribution of cultivars to all the outputs among the three treatments was more than 77% (Table 2). Cultivars contributed to up to 84% of the variation in TA, whereas chitosan and sugars contributed 9 and 7%, respectively, to the pigmentation.
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6
6
5
5
4
4
3
3
2
2 'Asuka no Kurenai'
0
1
'Asuka no Kurenai'
0 -8 -7 -6 -5 -4 -3 -2 -1
Bud width (mm)
Early Bud Stage Late Bud Stage
7
Sugar-chitosan dependent
Early Bud Stage Late Bud Stage
1
With chitosan
8
0
-8 -7 -6 -5 -4 -3 -2 -1
1
8
8
7
7
6
6
5
5
4
4
3
3
2
2
1
0
1
Chitosan dependent
7
Without chitosan
1 'Mickey Rose'
0
-8 -7 -6 -5 -4 -3 -2 -1
0
'Mickey Rose'
0 1
-8 -7 -6 -5 -4 -3 -2 -1
8
8
7
7
6
6
5
5
4
4
3
3
2
2
1
0
1
Sugar-sugar-chitosan dependent
8
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1 'Royal Violet'
0
'Royal Violet'
0 -8 -7 -6 -5 -4 -3 -2 -1
Glucose Rhamnose
0
1
-8 -7 -6 -5 -4 -3 -2 -1
Galactose Control
0
1
Fructose Field Conditions
Days before anthesis Fig. 3. Chitosan and sugar effects on width of detached flower buds of three lisianthus cultivars (Day 0 indicates anthesis).
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Table 1 Petal color attributes and pigment constituents under different treatment conditions in lisianthusa Cultivars Asuka no Kurenai
Mickey Rose
Royal Violet
a
Treatments
Color coordinatesb,e
Anthocyanidins (%)c
Chitosan
Sugar
L∗
C∗
TAd,e
hab
Dp
Cy
Pt
Pg
Pn
Mv
−
–
69.2 a
31.7 a
356.2 a
−
9
+
91
−
−
0.08 a
− − − − + + + + + Field conditions
Glc Fru Gal Rha – Glc Fru Gal Rha –
60.5 b 58.2 c 72.5 d 71.6 e 69.0 a 58.7 f 56.1 c 71.1 e 66.9 g 38.0 h
45.1 b 47.6 c 27.4 d 28.0 e 33.0 f 47.9 g 51.8 c 30.7 h 37.6 i 68.0 j
353.6 b 353.9 b 353.0 c 355.0 d 351.2 e 352.3 f 351.9 f 352.2 f 350.7 g 355.2 h
− − − − − − − − − −
10 13 9 12 8 7 8 14 11 5
+ − − − − − − − − +
90 87 91 88 92 93 92 86 89 95
− − − − − − − − − −
− − − − − − − − − −
0.13 b 0.16 c 0.07 d 0.09 a 0.08 a 0.15 e 0.18 f 0.09 a 0.10 h 0.46 i
−
–
80.5 a
11.1 a
23.4 a
−
93
−
7
−
−
0.04 a
− − − − + + + + + Field conditions
Glc Fru Gal Rha – Glc Fru Gal Rha –
77.8 b 76.7 c 81.6 d 78.0 c 76.4 b 77.8 c 68.0 e 75.9 f 78.9 g 59.6 h
14.3 b 16.6 c 9.6 d 14.4 c 17.3 e 15.9 f 29.7 g 18.2 h 15.5 f 44.3 i
354.6 b 356.4 c 21.9 d 5.7 e 354.4 b 352.1 f 348.0 g 348.7 h 354.6 b 344.6 i
− − − − 1 − − − − 5
92 90 87 86 80 83 83 86 87 68
− − − − − − − − − −
8 10 13 14 19 17 17 14 13 26
− − − − − − − − − 1
− − − − − − − − − −
0.06 b 0.06 b 0.04 a 0.04 a 0.05 a 0.11 c 0.12 c 0.06 b 0.05 b 0.83 d
−
–
58.7 a
28.8 a
352.3 a
92
3
−
+
5
−
0.13 a
− − − − + + + + + Field conditions
Glc Fru Gal Rha – Glc Fru Gal Rha –
51.6 b 50.0 c 59.0 a 59.0 a 54.7 d 48.4 e 43.9 f 56.1 g 50.1 ab 27.6 h
32.0 b 37.5 c 29.8 d 28.7 a 37.3 b 44.4 e 50.2 f 33.5 g 45.2 h 74.2 i
335.2 b 326.2 c 329.7 d 333.4 e 328.1 f 327.6 g 326.4 c 327.8 b 325.0 f 333.3 i
86 90 89 87 90 89 87 89 88 95
5 4 5 6 5 5 6 5 6 4
− − − − − − − − − −
− − + + + − − − + −
9 6 6 7 5 6 7 6 6 −
− − − − − − − − − 1
0.18 b 0.24 c 0.11 a 0.13 ad 0.15 e 0.27 f 0.31 g 0.14 de 0.22 h 0.30 i
Fresh petals were observed and each value is expressed as a mean of five replicates. L∗ , lightness; C∗ , chroma (color saturation); hab , hue angle (◦ ; tan−1 (b∗ /a∗ )) (D65/10◦ CIE illuminant/observer condition, transmission, 1 cm optical pathlength). c Dp, delphinidin; Cy, cyanidin; Pg, pelargonidin; Pt, petunidin; Pn, peonidin; Mv, malvidin (−, not detected; +, detected, in which the respective anthocyanidin concentration is below 0.4% of total anthocyanidin in fresh petal). d TA, total anthocyanin in mg per 100 mg fresh petal. e Values in the column within each cultivar with different letters differ significantly (SAS, 1995) (at P = 0.05 by Duncan’s new multiple range test). b
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Table 2 Average contribution of treatments to output parameters Output parameter
Treatment
Percent contribution
Lightness (L∗ )
Cultivar Chitosan Sugars
83.1 9.5 7.4
Chroma (C∗ )
Cultivar Chitosan Sugars
77.1 11.6 11.3
Hue angle (hab )
Cultivar Chitosan Sugars
85.7 9.5 4.8
Delphinidin (Dp)
Cultivar Chitosan Sugars
91.4 5.9 2.7
Cyanidin (Cy)
Cultivar Chitosan Sugars
80.6 10.3 9.1
Pelargonidin (Pg)
Cultivar Chitosan Sugars
93.7 2.5 3.8
Peonidin (Pn)
Cultivar Chitosan Sugars
90.0 6.8 3.2
Total anthocyanin (TA)
Cultivar Chitosan Sugars
83.7 9.4 6.9
To compute the differences in estimated and measured amounts of anthocyanin in lisianthus flower petals, different input treatments were compared. The model was calibrated against a range of datasets of measured total amount of anthocyanin and anthocyanidin composition, chromatic values, and treatment components (cultivars, chitosan, and sugars). The good correlation between the estimated and measured amounts was surprising considering the heterogeneity involved and simplistic parameterization (RMS error = 0.02, correlation = 0.89). The creation of a model could explain 90% of the relationship of the treatments with the total amount of anthocyanin (Fig. 4).
4. Discussion Tsukaya et al. (1991) clearly showed that levels of sugars depend on the age of flower buds and the difference was about 20-fold between the early and late stages of bud development. The accumulation of a large amount of sugars in the later stage resulted in a rapid increase in bud growth. Consistent with the findings of Tsukaya et al. (1991), the presence of fructose produced a rapid increase in bud growth, in the late stage among lisianthus cultivars. This
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Total Anthocyanidin (mg/100mg fresh petal)
Measured and simulated outputs
'Asuka no Kurenai' 0.35
'Mickey Rose'
'Royal Violet'
Chitosan without with
Chitosan without with
Chitosan with without
ANN Correlation=0.89 ERS=0.02
0.3 0.25 0.2 0.15 0.1 0.05
Measured Simulated
0
5
10
15
20
25
30
control glucose fructose galactose rahmnose control glucose fructose galactose rahmnose control glucose fructose galactose rahmnose control glucose fructose galactose rahmnose control glucose fructose galactose rahmnose control glucose fructose galactose rahmnose
0
Input node patern sequence Fig. 4. Measured (open circle) and simulated (filled circle) variation in the total amount of anthocyanins in fresh flower petals against the input node pattern sequence (ANN, artificial neural network used to drive the model simulation).
suggests that fructose is the major substrate for the metabolic processes of bud growth, acting as an energy source and as a signaling molecule (Neta-Sharir et al., 2000). The role of sugars in flower development may be multifunctional: they can act as an energy source (Moalem-Beno et al., 1997), and as an osmotic regulator (Sacalis and Chin, 1976; Ho and Nichols, 1977; Beileski, 1993). This suggests that the presence of fructose and the increased bud osmoticum induced a rapid expansion. Fructose may serve as the best source of carbohydrates for the metabolism of carbon upon which the induction of specific signaling events is dependent for flower development. The presence of fructose in the holding solution causes a major increase in osmoticum in the flower petal (Schnyder and Nelson, 1987). Sugars play an essential role in the accumulation of pigment by signaling the activation of chalcone synthase gene (Chs) expression in the developing petals (Weiss, 2000). This study provided additional evidence that fructose is noticeably associated with pigment synthesis in lisianthus petals. It seems likely that a keto group at the C2 position in fructose boosts the Chs gene expression and is involved in the activation of pigment biosynthetic genes to produce a larger amount of anthocyanin. Chitosan is the universally accepted non-toxic N-de-acetylated derivative of chitin, a highly basic polysaccharide. It has been suggested that chitosan promotes tissue growth and differentiation in tissue culture. Chitosan is of commercial interest due to its high percentage of nitrogen (7.1%) compared to synthetically substituted celluloses. The presence of GA3
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is required for sucrose to induce chalcone synthase gene (Chs) expression and anthocyanin synthesis (Weiss, 2000). But the results obtained without using GA3 , revealed that chitosan along with fructose, contributes greatly to the increase in anthocyanin synthesis in petals of lisianthus cultivars. For determination of the extent of pigmentation, acceptable agreement was observed between the total amount of anthocyanin in fresh petals and the respective amount estimated using the artificial neural network model. Success in simulated modeling and numeric prediction on a limited scale, such as proposed here, would encourage commercial demand to increase the market value of cut flowers by maintaining vivid flower colors.
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