Plant Growth Regul (2009) 58:73–84 DOI 10.1007/s10725-008-9353-9
ORIGINAL PAPER
Spectral reflectance indices and pigment functions during leaf ontogenesis in six subtropical landscape plants Nan Liu Æ Zhi-Fang Lin Æ Anna Van Devender Æ Gui-Zhu Lin Æ Chang-Lian Peng Æ Xiao-Ping Pan Æ Shao-Wei Chen Æ Qun Gu
Received: 9 July 2007 / Accepted: 3 December 2008 / Published online: 24 December 2008 Ó Springer Science+Business Media B.V. 2008
Abstract Pigment combinations are regulated during leaf ontogenesis. To better understand pigment function, alterations in chlorophyll, carotenoid and anthocyanin concentrations were investigated during different leaf development stages in six subtropical landscape plants, namely Ixora chinensis Lam, Camellia japonica Linn, Eugenia oleina Wight, Mangifera indica L., Osmanthus fragrans Lowr and Saraca dives Pierre. High concentrations of anthocyanin were associated with reduced chlorophyll in juvenile leaves. As leaves developed, the photosynthetic pigments (chlorophyll and carotenoid) of all six species increased while anthocyanin concentration declined.
N. Liu Z.-F. Lin (&) G.-Z. Lin C.-L. Peng X.-P. Pan S.-W. Chen Guangdong Key Laboratory of Digital Botanical Garden, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China e-mail:
[email protected] N. Liu C.-L. Peng College of Life Sciences, MOE Key Laboratory of Laser Life Science, South China Normal University, Guangzhou 510631, China A. Van Devender School of Natural Resources, College of Agriculture and Life Sciences, University of Arizona, Tucson, AZ 85721, USA Q. Gu Zeal Quest Scientific Technology Co. Ltd., Shanghai 200333, China
Chlorophyll fluorescence imaging of UPSII (effective quantum yield of PSII) and of NPQ (non-photochemical fluorescence quenching) and determination of electron transport rate-rapid light curve (RLC) showed that maximum ETR (leaf electron transport rate), UPSII and the saturation point in RLC increased during leaf development but declined as they aged. Juvenile leaves displayed higher values of NPQ and Car/Chl ratios than leaves at other developmental stages. Leaf reflectance spectra (400–800 nm) were measured to provide an in vivo non-destructive assessment of pigments in leaves during ontogenesis. Four reflectance indices, related to pigment characters, were compared with data obtained quantitatively from biochemical analysis. The results showed that the ARI (anthocyanin reflectance index) was linearly correlated to anthocyanin concentration in juvenile leaves, while a positive correlation of Chl NDI (chlorophyll normalized difference vegetation index) to chlorophyll a concentration was species dependent. Photosynthetic reflectance index was not closely related to Car/Chl ratio, while a structural-independent pigment index was not greatly altered by leaf development or species. Accordingly, it is suggested that the high concentration of anthocyanin, higher NPQ and Car/Chl ratio in juvenile leaves are important functional responses to cope with high radiation when the photosynthetic apparatus is not fully developed. Another two leaf reflectance indices, ARI and Chl NDI, are valuable for in vivo pigment evaluation during leaf development.
123
74
Keywords Anthocyanin Carotenoid Chlorophyll Fluorescence Reflectance spectrum Leaf development Abbreviations ARI Anthocyanin reflectance index Chl NDI Chlorophyll normalized difference vegetation index PRI Photosynthetic reflectance index SIPI Structural-independent pigment index UPSII Effective quantum yield of PSII NPQ Non-photochemical fluorescence quenching ETR Electron transport rate RLC Rapid light curve
Introduction Two classes of pigments, photosynthetic and nonphotosynthetic, exist in different organelles of plant leaf cells. Within the photosynthetic class of pigments, chlorophylls in the chloroplasts participate in absorbing and transforming light energy into storable chemical energy (Curran et al. 1990; Filella et al. 1995). Carotenoids, as antenna pigments, are closely attached to the chlorophyll molecule and act to funnel light energy to the photosynthetic reaction center. Besides the function of light harvesting, carotenoids also play an important protective role guarding against pigment damage induced by intensive radiation and/or oxidative stresses (Miki 1991; Marschner 1995; Tracewell et al. 2001). One of the typical nonphotosynthetic pigments, anthocyanins are watersoluble pigments primarily within the cell vacuole, which function as antioxidants particularly in young and aging leaves (Moyer et al. 2002; Peng et al. 2006). During leaf ontogenesis, pigment concentrations and their ratios change significantly. Juvenile leaves contain low concentration of photosynthetic pigments and a greater concentration of non-photosynthetic pigments. During leaf expansion, photosynthetic pigments concentration increases which coincides with a concomitant up regulation in gene expression, an increase in activities of ribulose bisphosphate carboxylase and electron transport and increase in chlorophyll concentration (Taiz and Zeiger 1998; Yoo et al. 2003). Mature leaves contain relatively constant pigment
123
Plant Growth Regul (2009) 58:73–84
ratios which are associated with a decrease in photosynthetic capacity and reduced gene expression relative to juvenile leaves. Chlorophyll degradation and changes in the ratio between individual pigments are the most obvious visible symptoms of the aging process, because plant tissues undergo changes in colour from the edges to the center of the leaf (Biswall 1995; Buchanan-Wollastin 1998). Leaves approaching death are characterized by a reduction in chlorophyll concentration and photosynthetic activities. Simultaneously, the decline in chlorophyll concentration and photosynthetic rate accelerates leaf senescence (Smart 1994; Bleecker and Patterson 1997). It is widely accepted that, as a result of changes in anthocyanin, chlorophyll and carotenoid pigment concentrations, the physiological age of leaves directly influences coloration, energy production and plant photoprotection capacity. For instance, the increased coloration in plant tissues attributed to maturity is often indicative of increases in carotenoid concentrations (Gross 1991). Also, colour produced by anthocyanin has been commonly observed to precede and accompany chlorophyll formation in juvenile leaves of plants with delayed greening in tropical areas (Lee and Collins 2001; Cai et al. 2005). However, it still remains unclear at which stage the potential changes in both photosynthetic and non-photosynthetic pigment concentrations and their ratios occur. Despite widespread phylogenetic distribution, we assume that the abundance of anthocyanin in juvenile leaves, especially in our six test species, must be functionally significant in responding to environmental light condition. The goal of this study was to describe anthocyanin, chlorophyll and carotenoid pigment accumulations linked to photosynthetic activity of PSII within leaves of six subtropical woody plants at different developing stages. Furthermore, leaf reflectance indices measured in situ were compared to pigment concentrations measured using biochemical methods in order to validate the feasibility of earlier established nondestructive formulae to analyze leaf pigments.
Materials and methods Experimental site and plant materials The experimental site was located in South China Botanical Garden, Chinese Academy of Sciences,
Plant Growth Regul (2009) 58:73–84
Guangzhou, China. Six subtropical heliophyte woody species including Ixora chinensis Lam, Camellia japonica Linn, Eugenia oleina Wight, Mangifera indica L., Osmanthus fragrans Lowr and Saraca dives Pierre were selected in our experiments. Leaves of the six plants at the juvenile stage all exhibit a reddish colour, and then turn green during maturation. The reflectance spectra, chlorophyll fluorescence emission and pigment concentrations were measured for juvenile (J, crimson or red colour), young (Y, lawngreen or greenyellow colour), mature (M, green or dark green colour) and senescent (S, olivegreen or yellowgreen colour) leaves, respectively. Spectral reflectance of leaf upper surface Leaf disks (78.5 mm2 in size) excised from the central part of newly detached leaves were washed with distilled water and fixed adaxial side up on filter paper, using the same filter paper as a control. Spectral reflectance at wavelengths from 400 to 800 nm was measured using a UV-spectrophotometer equipped with an integrating sphere (Lambda 650, Perkin-Elmer Inst. USA). For all species, three to five reflectance spectra were determined for each leaf stage, and expressed as a mean. To express pigment concentration, the mathematical formulae derived from reflectance curves were calculated as reflectance indices. In the present research, four indices were used for each species. Chlorophyll normalized difference vegetation index (Chl NDI) is calculated as (R750 - R705)/(R750 ? R705) (Gitelson and Merzlyak 1994); Structuralindependent pigment index (SIPI) as (R800 - R445)/ (R800 - R680) (Pen˜uelas and Filella 1998); Anthocyanin reflectance index (ARI) as 1/R550 - 1/R700 (Gitelson et al. 2003); and Photochemical reflectance index (PRI) as (R530 - R570)/(R530 ? R570) (Winkel et al. 2002). Where R is reflectance, the subscript number of R represents a specific wavelength. Total chlorophyll and carotenoid determinations Leaf sections (0.2 g) were submerged in 80% acetone (4°C, dark) for 3 days then removed. Absorptions of the remaining solution were measured at 663, 645 and 440 nm, respectively, with a UV-spectrophotometer (Lambda 650, Perkin-Elmer Inst. USA). Total
75
chlorophyll and carotenoid concentrations were calculated as lg g-1 FW (Lin and Ehleringer 1982). Anthocyanin determination Anthocyanin was extracted with a mixture of methanol and HCl (99:1, V/V) at 4°C in the dark. Absorption was measured at 530 nm by an UVspectrophotometer (Lambda 650, Perkin-Elmer Inst. USA). Anthocyanin concentration (lg g-1 FW) was calculated according to the formula of (A530 - 0.24 9 A650) 9 V/(W 9 E) (Murray and Hackett 1991; Reddy et al. 1995). A is the absorption at the specific wavelengths, V is the volume of extracted solution, W is the fresh weight of leaf samples and E is the extinction coefficient [(31.6 mmol/L)-1 cm-1]. Rapid light curve and fluorescence imaging Measurement of rapid light curve (RLC) of whole leaves from different plant individuals at each developmental stage and fluorescence imaging at saturation pulse mode of the same leaves were conducted by a chlorophyll fluorescence system (Imaging-PAM, MAXI-Version, for imaging areas up to 10 9 13 cm; Walz, Efeltrich, Germany). According to Ralph and Gademann (2005), when measuring RLC, data was recorded from 16 areas of interest (AOI, diameter 0.5–1 cm) on each plant leaf. The apparent absorptivity of the leaf surface was automatically calculated pixel by pixel from the R-(red) and NI-(near infrared) images using the formula: Abs = 1 - (R/NI). The definition of the ETR (electron transport rate) assumes a uniform absorption of incident light over the whole sample area: rETR = Yield 9 PAR 9 0.5 9 Absorptivity, where, Yield = DF/Fm0 and PAR is the actinic light intensity. To generate RLCs, leaves were irradiated with a series of actinic light intensities (0, 1, 21, 56, 111, 186, 281, 336, 396, 461, 531, 611, 701, 801, 926, 1076 lmol m-2 s-1) for 10 s which always finished with a saturating pulse after each level of illumination. Simultaneously, ETR values were determined and plotted against irradiances automatically by the inherent software. From the surface of the tested leaves, images of the chlorophyll fluorescence indices, UPSII (effective quantum yield of PSII), and NPQ/4 (non-photochemical quenching) were taken under saturation pulse mode.
123
76
Plant Growth Regul (2009) 58:73–84
The measured light intensity was 0.5 lmol m-2 s-1, the saturation pulse light was 2,700 lmol m-2 s-1 (duration 0.8 s; interval 20 s) and the actinic light intensity was 185 lmol m-2 s-1. Fluorescence results and the corresponding images were recorded simultaneously.
667 nm, after which the values increased and remained high for all species. Compared to other developing stages, reflectance curves of mature leaves were lowest in S. dives, I. chinensis, M. indica and C. japonica and were higher only than senescent leaves in E. oleina and O. fragrans. With regard to the whole curve from 400 to 800 nm, it was clear that juvenile leaves reflected the incident light much more than young, mature and senescent leaves.
Results Spectral reflectance
Spectral reflectance indices Spectral reflectance of leaf upper surface from the incident light is the combined responses of leaf photochemical and morphological properties. As shown in Fig. 1, no significant peak was found in the reflectance curves within 400–500 nm. However, peaks appeared at about 554 nm for young, mature and senescent leaves and at about 635 nm for red juvenile leaves. Blue shifts were apparent to different extents for juvenile leaves of I. chinensis and C. japonica, with peak values at 604 and 630 nm, respectively. Most of the leaves measured except juvenile ones showed the lowest reflectance value at Fig. 1 Leaf reflectance spectra of six species (I. chinensis, C. japonica, E. oleina, M. indica, O. fragrans and S dives). J, juvenile; Y, young; M, mature; S, senescent. Each curve is the mean of three or four individual experiments
Table 1 shows four indices calculated from data in Fig. 1 for each individual plant at different developmental stages. Chl NDI has previously been correlated to chlorophyll a concentration (Gitelson et al. 2003). For these six plant species, Chl NDI initially increased and then declined in most of the senescent leaves. The mature leaves of I. chinensis showed the highest Chl NDI value among the six species. This value of senescent leaves of E. oleina and O. fragrans did not decline but were also not significantly lower.
100 Ixora chinensis
Cmellia japonica
80 Y
60 S 40
Y J
J
S M
M
20 0 100
Reflectance (%)
Eugenia oleina 80
Mangifera indica
J
60 Y
Y S
40
M
J
S
M
20 0 100 Osmanthus fragans 80
Saraca dives
J
60 S
40
J
Y
Y
S
M
M 20 0 400
500
600
700
800
400
Wavelength (nm)
123
500
600
700
800
Senescent (S)
Mature (M) 0.01 ± \0.01 c
ND
0.06 ± 0.02 a 0.02 ± 0.01 b
0.02 ± \0.01 d
Senescent (S)
Juvenile (J) Young (Y)
0.30 ± \0.01 c
0.05 ± 0.01 c \0.01 ± 0.02 d
Mature (M)
-0.03 ± 0.01 a
0.01 ± \0.01 a
\0.01 ± \0.01 c
ND
0.03 ± 0.01 a \0.01 ± \0.01 b
-0.02 ± 0.01 b
-0.06 ± 0.02 b
-0.03 ± 0.02 a
-0.15 ± 0.03 a
0.99 ± \0.01 a
0.99 ± 0.01 a
Senescent (S)
Young (Y)
0.99 ± \0.01 a
0.99 ± \0.01 a
0.989 ± \0.01 a
Mature (M)
Juvenile (J)
0.99 ± 0.02 b
0.99 ± \0.01 a
0.99 ± \0.01 a
0.99 ± \0.01 a
ND
\0.01 ± \0.01 c
ND
0.04 ± \0.01 a 0.01 ± \0.01 b
0.03 ± \0.01 a ND 0.01 ± \0.01 b
-0.98 ± \0.01 d
0.02 ± \0.01 c
0.01 ± \0.01 b
0.99 ± \0.01 b
1.00 ± 0.01 b
-0.98 ± \0.01 c
-0.01 ± \0.01 b
0.02 ± \0.01 a
0.99 ± \0.01 a
0.41 ± 0.03 c 1.29 ± 0.08 a
Young (Y)
0.49 ± 0.03 b 1.04 ± 0.01 a
0.47 ± 0.02 b 1.02 ± 0.01 a
0.31 ± 0.04 ab 1.02 ± \0.01 a
Senescent (S)
0.36 ± 0.02 c
0.23 ± 0.03 b
0.07 ± 0.01 a
Eugenia oleina
Juvenile (J)
0.50 ± 0.01 b
0.42 ± 0.02 b
0.15 ± 0.01 a
Mangifera indica
Different small letters in each index column of leaf developing stages are significantly different at P \ 0.05. ND, not detectable
ARI
PRI
SIPI
0.50 ± 0.01 b
0.40 ± 0.03 b
0.39 ± 0.02 b 0.56 ± 0.03 c
0.23 ± 0.02 a
Camellia japonica
0.21 ± 0.02 a
Young (Y)
Juvenile (J)
ChlNDI
Ixora chinensis
Mature (M)
Developing phases
Indices
Table 1 Changes in leaf reflectance indices in leaves of six species at various developmental stages (n = 5)
ND
ND
0.04 ± 0.01 a 0.01 ± 0.01 b
-1.95 ± \0.01 d
0.24 ± 0.02 c
-0.17 ± 0.02 b
-0.02 ± 0.01 a
1.00 ± 0.01 a
0.99 ± 0.01 a
1.02 ± 0.01 a
1.05 ± 0.03 a
0.36 ± 0.06 b
0.34 ± 0.02 b
0.19 ± 0.03 a
0.16 ± 0.03 a
Osmanthus fragrans
0.01 ± \0.01 b
0.01 ± \0.01 b
0.03 ± \0.01 a 0.01 ± \0.01 b
0.02 ± 0.01 a
0.04 ± \0.01 c
\0.01 ± \0.01 b
0.02 ± \0.01 a
0.99 ± \0.01 a
0.99 ± \0.01 a
1.01 ± \0.01 a
1.04 ± \0.01 a
0.37 ± 0.04 d
0.51 ± \0.01 c
0.24 ± 0.01 b
0.11 ± \0.01 a
Saraca dives
Plant Growth Regul (2009) 58:73–84 77
123
78
Plant Growth Regul (2009) 58:73–84
SIPI is used to quantify the ratio of carotenoid and chlorophyll a (Pen˜uelas and Filella 1998). In all six species, the ratio did not significantly vary from 1.00 among the development stage. Similar SIPI values in different plant species showed that the ratios of photosynthetic pigments were mainly regulated in response to leaf development stages rather than to species-specific leaf morphology. Photochemical reflectance index, the PRI, has been demonstrated to be related to light use efficiency (Winkel et al. 2002). For all species but M. indica, PRI values were highest in mature leaves but lowest in juvenile or senescent ones. Therefore, light use efficiency initially increased significantly and subsequently decreased during the leaf life. Anthocyanin reflectance index, the commonly used index in estimating anthocyanin concentration (Gitelson et al. 2003), showed the highest values in the red coloured juvenile leaves, sharply declining values in young leaves, and the lowest values in senescent leaves. The ARI values were lower than the detectable limit in some of the mature and senescent leaves. These values are coincident with changes in leaf colour.
Chlorophyll, carotenoid and anthocyanin concentrations Chlorophyll concentrations in the six species at different developmental stages are shown in Table 2. Among the six species, the juvenile and young leaves of M. indica had the highest chlorophyll concentrations and S. dives the lowest. Chlorophyll concentrations increased initially then decreased subsequently with leaf development, with leaves contains more chlorophyll than juvenile ones, except in E. oleina. In E. oleina, the highest chlorophyll concentrations were in senescent leaves. Carotenoid concentrations and the ratios of carotenoid to chlorophyll (Car/Chl) are also shown in Table 2. Carotenoid concentration in M. indica and E. oleina, showed the mean values of 444 and 326 lg g-1 FW across all four developmental stages. I. chinensis and C. japonica contained the lowest carotenoid concentrations of 154 and 142 lg g-1 FW. Mean values of Car/Chl in leaves varied among the six species from 0.143 to 0.399. Carotenoid concentrations were correlated with developmental tendencies differently than Car/Chl ratios were. Carotenoid
Table 2 Changes in concentrations of chlorophyll and carotenoid in plant leaves at different developmental stages (n = 5) Plant species
Pigment (lg g-1 FW)
Junivenile (J)
Ixora chinensis
Chl
273 ± 12 a
Camellia japonica
Car/Chl Chl
Car
Car Eugenia oleina
Mangifera indica
Osmanthus fragrans
Saraca dives
83 ± 9.2 a
Young (Y)
Mature (M)
464 ± 9.1 b
790 ± 6.3 c
548 ± 19 b
123 ± 0.2 ab
240 ± 6.2 c
172 ± 13 bc
0.30 285 ± 36 a
0.27 386 ± 23 b
0.30 607 ± 3.5 c
0.31 544 ± 20 c
94 ± 13 a
119 ± 26 ab
150 ± 2.5 b
207 ± 0.6 c
0.31
0.25
0.38
Car/Chl
0.33
Chl
137 ± 1.5 a
451 ± 38 b
1314 ± 5.7 c
1804 ± 81 d
Car
54 ± 0.5 a
143 ± 14 b
423 ± 3.2 c
683 ± 9.8 d
0.32
0.32
Car/Chl
0.40
Chl
503 ± 20 a
1333 ± 3.6 b
2687 ± 159 c
2377 ± 35 c
Car
143 ± 8.1 a
336 ± 3.5 b
657 ± 45.8 c
641 ± 5.5 c
0.24
0.38
Car/Chl
0.28
0.25
Chl
458 ± 6.0 a
496 ± 12 a
2080 ± 64 b
1770 ± 65 c
Car
168 ± 3.0 a
171 ± 2.7 a
424 ± 26 b
282 ± 20 c
Car/Chl
0.37
0.35
0.20
0.27
0.16
Chl
31 ± 0.8 a
54 ± 2.9 b
3591 ± 34 c
1776 ± 31 d
Car
63 ± 3.9 a
84 ± 11 a
515 ± 39 b
337 ± 15 c
0.14
0.19
Car/Chl
0.20
0.16
Different small letters in each row of leaf developing stages are significantly different at P \ 0.05
123
Senescent (S)
Plant Growth Regul (2009) 58:73–84
79
Table 3 Changes in concentration of anthocyanin in plant leaves at different developmental stages (n = 5) Plant species
Anthocyanin (lmol g-1 FW) Juvenile (J)
Young (Y)
Mature (M)
Senescent (S)
Ixora chinensis
0.71 ± 0.09 a
0.24 ± 0.02 b
0.29 ± 0.08b
0.57 ± 0.07 c
Cmellia japonia
1.34 ± 0.04 a
0.34 ± 0.04 b
0.29 ± 0.04 b
0.69 ± 0.09 c
Eugenia oleina
1.46 ± 0.11 a
0.50 ± 0.04 b
0.42 ± 0.18 b
1.22 ± 0.08 c
Mangifera indica
1.66 ± 0.04 a
0.21 ± 0.01 b
0.57 ± 0.08 c
0.44 ± 0.07 c
Osmanthus fragrans
0.89 ± 0.02 a
0.17 ± \0.01 b
0.11 ± 0.01 b
0.07 ± 0.01 c
Saraca dives
0.85 ± \0.01 a
0.14 ± 0.01 b
0.10 ± 0.01 b
0.14 ± 0.02 b
Different small letters in each row of leaf developing stages are significantly different at P \ 0.05
Relationships between spectral reflectance indices and pigment concentrations Figure 2 shows the regression relation between ARI values and anthocyanin concentrations in six tested species at different developmental stages, wherein positive correlation was found linearly with a slope of 0.0191. Similarly, two positive regressions were found between Chl NDI and chlorophyll a concentration (Fig. 3). However, based on specimen together with chlorophyll a concentrations the positive relationships were distinctly described by two formulae with slopes of 0.1817 and 0.9105, respectively.
Rapid light curve (RLC) In the present study, 16 AOIs were chosen on plant leaves for measuring rETR and plotting RLC. In leaves of most tested species, RLCs were elevated from the juvenile to the mature stages, but largely decreased in senescent leaves (Fig. 4). Light saturation points in RLCs and maximum rETR were different among species, being much higher in
0.05
0.04
0.03
ARI
concentration was lowest in mature leaves but higher in all other stages, in all species except O. fragrans. Anthocyanin concentrations, in red coloured juvenile leaves, ranged between 0.7 and 1.6 lg g-1 FW (Table 3). Leaves of E. oleina and M. indica had the highest anthocyanin concentrations compared with the other species. As the leaves turned green, anthocyanin concentrations declined but increased significantly to some extent in senescent leaves.
0.02
0.01
0
y = 0.0191x + 0.0067 R2 = 0.8456 0
0.5
1
1.5
2
Anthocyanin (µmol g-1FW) Fig. 2 Linear relationships between anthocyanin concentration and anthocyanin reflectance index in juvenile leaves of six species
I. chinensis, M. indica and O. fragrans compared to S. dives and E. oleina. Chlorophyll fluorescence imaging Values of two chlorophyll fluorescence indices were taken as photographic images (Fig. 5) for five species. From the resultant images of UPSII, effective quantum yield of photosystem II, most juvenile and senescent leaves showed ‘‘warmer colours’’ than young and mature leaves. Therefore most juvenile and senescent leaves produced lower UPSII values than young and mature leaves which were closely related to their difference in chlorophyll concentration. Non-photochemical quenching chlorophyll fluorescence (NPQ) showed that juvenile leaves revealed mostly ‘‘colder colours’’ corresponding to higher fluorescent values in
123
80 0.8
0.8
A
B
0.6
0.6
Chl NDI
Chl NDI
Fig. 3 Linear relationships between chlorophyll a concentration and chlorophyll normalized difference index during leaf development. a Four species: E. oleina, M. indica, O. fragrans and S. dives. b Two species: I. chinensis and C. japonica
Plant Growth Regul (2009) 58:73–84
0.4
y = 0.1817x + 0.12
0.2
0.2
y = 0.9105x + 0.0804
2
2
R = 0.8312 0
R = 0.7419
0 0
1
2 -1
Chla (mg g FW)
juvenile leaves compared to leaves at other developmental stages, especially for O. fragrans (Fig. 5).
Discussion Emerging leaves of many woody and herbaceous species in subtropical areas conspicuously appear initially red, pink or purple and become greener with leaf development. Immature leaves are vulnerable to stresses from high solar radiation, caused by several internal factors which include underdeveloped chloroplasts (Pettigrew and Vaughn 1998; Choinski et al. 2003) and low photosynthetic enzyme activities (Miranda et al. 1981; Pettigrew and Vaughn 1998). Therefore, numerous strategies, including elevated xanthophyll cycle pigments (Krause et al. 1995; Barker et al. 1997), reduced chlorophyll concentrations (Choinski et al. 2003; Cai et al. 2005) and elevated anthocyanin as a light dissipation pigment (Manetas et al. 2002; Karageorgou and Manetas 2006; Liakopoulos et al. 2006) are apparent in juvenile leaves to avoid photoinhibition. Dominy et al. (2002) reported that the number of species with young anthocyanic leaves ranged from 7 to 62% in four forests sampled. Murray and Hackett (1991) compared the enzyme activities of dihydroflavonol reductase (DFR) and phenylalanine aminotransferase (PAT) linked to anthocyanin synthesis in juvenile and mature leaves of Hedera helix L. and concluded that juvenile-phase leaves accumulate anthocyanin pigment in the hypodermis of stems and petioles. In the present study, juvenile leaves of the six landscape species were more abundant in anthocyanin compared with older leaves (Table 3). Anthocyanin concentrations became lower with leaf coloration changes from red to green in young and mature leaves, except for M. indica. This type of non-photosynthetic pigment exists in epidermal or
123
0.4
3
0
0.2
0.4
0.6
-1
Chla (mg g FW)
mesophyll vacuoles and disappeared within hours to days with leaf expansion and maturation (Choinski et al. 2003; Cai et al. 2005). It is suggested that anthocyanin-containing juvenile leaves are able to act as light attenuators to protect developing cells from high irradiance (Chalker-Scott 1999; Gould 2004). Anthocyanin as an antioxidant is capable of scavenging organic free radical DPPH and active oxygen; therefore, it can restrain peroxidation of membrane lipids and photoinhibition (Peng et al. 2006) in the early stage of leaf development (Yamasaki et al. 1996; Tsuda et al. 1996). In comparison with mature leaves, juvenile leaves of six species had 450% of the mean anthocyanin concentration that mature leaves had but only 27.6% of the mean carotenoid concentration accompanied with higher NPQ in juvenile leaves of six species (Tables 2, 3; Fig. 5). This correlation provides support for anthocyanin playing a main function to alleviate photoinhibition in juvenile leaves when the photosynthetic apparatus is not well established. With relatively abundant chlorophyll concentration, mature leaves are commonly matched with the higher photosynthetic activity. Among different developmental stages, leaves with higher total chlorophyll concentration at a mature stage showed higher rETR values and saturation point of RLCs than that at the other stages (Fig. 4). In green mature leaves with high chlorophyll, high UPSII, high electron transport rate and low anthocyanin, the obviously elevated carotenoid became the main protective pigment against photoinhibition. Therefore, the richness of anthocyanin in the earliest stage and its significant reduction while the leaves grew from young to mature stages, together with changes in chlorophyll and carotenoid combinations (Tables 2, 3), well demonstrated that the adjustment in ratios of pigments align with the enhancement of light use efficiency of foliar cells. This would
Plant Growth Regul (2009) 58:73–84
81
Ixora chinensis
60
40
20
0
Mangifera indica 60
40
0
-2
-1
Electron Transport Rate (µmol m s )
20
Eugenia oleina
60
40
20
0
Osmanthus fragans
60
40
20
0
Saraca dives
60
40
20
0 0
250
500
750
1000
1250
Light intensity (µmol m-2 s-1) Fig. 4 Electron transport rate-rapid light curve of five landscape species (n = 16) -r- juvenile leaf; -j- young leaf; -m- mature leaf; -9- senescent leaf
effectively reduce oxidative damage and potentially improve photosynthetic performance at different development stages of the leaf. Leaf extraction with organic solvents and spectrophotometric determination in solution is generally required for pigment analysis with wet chemical methods. However, alternative optical methods of leaf pigment analysis (i.e., chlorophylls, carotenoids and anthocyanins) have been developed. These newer methods are non-destructive, inexpensive, quick and now possible in the field (Gamon and Surfus 1999; Gitelson et al. 2003). Leaf spectral reflectance indices have been used to evaluate Chl a/b ratio, chlorophyll, carotenoid, anthocyanin and xanthophyll cycle concentrations due to different pigment reflectance characters (Lin and Ehleringer 1982; Sims and Gamon 2002). Leaf consisting of cuticula, epidermis, palisade and spongy parenchyma etc., with numerous boundaries and containing high amounts of pigments is very complicated from an optical point of view. Relationships between reflectance in the visible range and leaf chlorophyll concentration are essentially nonlinear (Buschmann and Nagel 1993; Gitelson and Merzlyak 1994). However, it was found that reciprocal reflectance (R-1) alone at certain wavelengths could be used for chlorophyll quantification (Gitelson et al. 2003). Although leaf spectral reflectance indices have been testified alone or in combination by other researchers, their validity was still limited by research scales and species. To our knowledge, few reports had simultaneously analyzed the relationships among the spectral reflectance indices, pigment concentrations, different plant species and developmental stages. In this study, the four indices of leaf reflectance chosen were widely used and tested in earlier works. Figure 2 showed that ARI, the anthocyanin reflectance index, was significantly related to the anthocyanin concentration (r = 0.920) in plant leaves measured by biochemical methods. The reflectance index Chl NDI was also directly related to chlorophyll a concentration, but the linear slopes of chlorophyll a concentration versus Chl NDI value were divided into two categories due to different chlorophyll a concentrations and leaf development stages in the six species. The species rich in chlorophyll a, including E. oleina, M. indica, O. fragrans and S. dives, had a correlation coefficient r = 0.912 and linear slope 0.182 (Fig. 3a), while the other two species, I. chinensis and C. japonica containing less chlorophyll a, had a correlation
123
82 Fig. 5 Series of chlorophyll fluorescence images (UPSII and NPQ/4) measured by a saturation pulse kinetic process in leaves belong to different developmental stages (juvenile, young, mature and senescent leaves from left to right) of five species. Bar along the bottom of images indicates relative value of each index as a percentage
Plant Growth Regul (2009) 58:73–84
Species
ΦPSII
NPQ/4
Ixora chinensis
Mangifera indica
Eugenia oleina
Osmanthus fragrans
Saraca dives
coefficient r = 0.861 and linear slope 0.091 (Fig. 3b). However, PRI showed no direct relationship with total chlorophyll concentrations. Our results showed that within these indices, ARI was linearly correlated to anthocyanin concentrations determined in juvenile
123
leaves of six species (Fig. 2). Hence, ARI is likely as a nondestructive index which can detect anthocyanin concentration effectively in red colour leaves only. The feasibility of Chl NDI in estimating photosynthetic pigment concentration and capacity was
Plant Growth Regul (2009) 58:73–84
demonstrated by the direct correlation between Chl NDI and chlorophyll concentration in different developmental stages of leaves (Fig. 3). Nevertheless, this linear correlation depends on plant species and developmental stages, and is therefore ubiquitous. PRI as the index of xanthophyll cycle pigments was not closely related to Car/Chl in our six species, which was not accordant with former studies using remote sensing technique (Sims and Gamon 2002). For plant foliage, reflectance spectra are the product of complex patterns of scattering and absorption by numerous structural and biochemical components (Andrew et al. 2003). Besides, other factors (i.e., individual or species differences, water or other environmental stresses) may affect changes of leaf structure and reflectance characters. In this context, we believe that when using the nondestructive technique of spectral reflectance, some indices show greater promise as estimators of pigment concentrations than others. Our newly results also agree the point of view that the mechanisms responsible for a close relationship between reciprocal reflectance and pigment concentration still need more detailed investigations (Gitelson et al. 2003). Therefore, further research is requested in a systematic and deep study to solve this problem. Acknowledgments This research is financially supported by ‘‘National Natural Science Foundation of China (30770173, 30870385)’’, ‘‘the Opening Project of MOE Key Laboratory of Laser Life Science, South China Normal University’’, ‘‘Arizona Science Teacher Advancement and Research Training Program, USA’’, ‘‘Scientific Start-up Foundation of South China Botanical Garden, Chinese Academy of Sciences (200748)’’, National Basic Research Program of China (973 Program) (2009CB118504) and ‘‘Zealquest Scientific Foundation’’.
References Andrew DR, James BR, Timothy GG (2003) Multivariate analyses of visible/near infrared (VIS/NIR) absorbance spectra reveal underlying spectral differences among dried, ground conifer needle samples form different growth environments. New Phytol 161:291–301 Barker DH, Seaton GGR, Robinson SA (1997) Internal and external photoprotection in developing leaves of the CAM plant Cotyledon orbiculata. Plant Cell Environ 20:617– 624. doi:10.1111/j.1365-3040.1997.00078.x Biswall B (1995) Carotenoid catabolism during leaf senescence and its control by light. Photochem Photobiol B Biol 30:3–14. doi:10.1016/1011-1344(95)07197-A Bleecker A, Patterson S (1997) Last exit: senescence abscission and meristem arrest in Arabidopsis. Plant Cell 9: 1169–1179. doi:10.1105/tpc.9.7.1169
83 Buchanan-Wollastin V (1998) The molecular biology of leaf senescence. J Exp Bot 49:181–199. doi:10.1093/jexbot/ 49.319.181 Buschmann C, Nagel E (1993) In vivo spectroscopy and internal optics of leaves as basis for remote sensing of vegetation. Int J Remote Sens 14:711–722. doi:10.1080/ 01431169308904370 Cai ZQ, Slot M, Fan ZX (2005) Leaf development and photosynthetic properties of three tropical tree species with delayed greening. Photosynthetica 43:91–98. doi:10.1007/ s11099-005-1098-3 Chalker-Scott L (1999) Environmental significance of anthocyanins in plant stress responses. Photochem Photobiol 70:1–9. doi:10.1111/j.1751-1097.1999.tb01944.x Choinski JS, Ralph P, Eamus D (2003) Changes in photosynthesis during leaf expansion in Corymbia gummifera. Aust J Bot 51:111–118. doi:10.1071/BT02008 Curran PJ, Dungan JL, Gholz HL (1990) Exploring the relation-ship between reflectance red edge and chlorophyll concentration in slash pine. Tree Physiol 7:33–48 Dominy NJ, Lucas PW, Ramsden LW, Riba-Hernandez P, Stoner KE, Turner IM (2002) Why are young leaves red? Oikos 98:163–176. doi:10.1034/j.1600-0706.2002. 980117.x Filella I, Serrano L, Serra J, Penuelas J (1995) Evaluating wheat nitrogen status with canopy reflectance indices and discriminant analysis. Crop Sci 35:1400–1405 Gamon JA, Surfus JS (1999) Assessing leaf pigment concentration and activity with a reflectometer. New Phytol 143:105–117. doi:10.1046/j.1469-8137.1999. 00424.x Gitelson AA, Merzlyak MN (1994) Spectral reflectance changes associated with autumn senescence of Aesculus hippocastanum L. leaves: spectral features and relation to chlorophyll estimation. J Plant Physiol 143:286–292 Gitelson AA, Gritz U, Merzlyak MN (2003) Relationships between leaf chlorophyll concentration and spectral reflectance and algorithms for non-destructive chlorophyll assessment in higher plant leaves. J Plant Physiol 160:271–282. doi:10.1078/0176-1617-00887 Gould KS (2004) Nature’s swiss army knife: the diverse protective roles of anthocyanins in leaves. J Biomed Biotechnol 5:314–320. doi:10.1155/S1110724304406147 Gross J (1991) Pigments in vegetables: chlorophylls and carotenoids. AVI/Van Nostrand Reinhold, New York Karageorgou P, Manetas Y (2006) The importance of being red when young: anthocyanins and the protection of young leaves of Quercus coccifera from insect herbivory and excess light. Tree Physiol 26:613–621 Krause GH, Virgo A, Winter K (1995) High susceptibility to photoinhibition of young leaves of tropical forest trees. Planta 197:583–591. doi:10.1007/BF00191564 Lee DW, Collins TM (2001) Phylogenetic and ontogenetic influences on the distribution of anthocyanins in betacyanins in leaves of tropical plants. Int J Plant Sci 162:1141– 1153. doi:10.1086/321926 Liakopoulos G, Nikolopoulos D, Klouvatou A, Vekkos K-A, Manetas Y, Karabourniotis G (2006) The photoprotective role of epidermal anthocyanins and surface pubescence in young leaves of grapevine (Vitis vinifera). Ann Bot (Lond) 98:257–265. doi:10.1093/aob/mcl097
123
84 Lin ZF, Ehleringer J (1982) Changes in spectral properties of leaves as related to chlorophyll and age of Papaya. Photosynthetica 16:520–525 Manetas Y, Drinia A, Petropoulou Y (2002) High concentrations of anthocyanins in young leaves are correlated with low pools of xanthophyll cycle components and low risk of photoinhibition. Photosynthetica 40:349–354. doi: 10.1023/A:1022614722629 Marschner H (1995) Mineral nutrition of higher plants, 2nd edn. Academic Press, New York Miki W (1991) Biological functions and activities of animal carotenoids. Pure Appl Chem 63:141–146. doi:10.1351/ pac199163010141 Miranda V, Baker NR, Long SP (1981) Limitation of photosynthesis in different regions of the Zea mays leaf. New Phytol 89:179–190. doi:10.1111/j.1469-8137.1981.tb07481.x Moyer RA, Hummer KE, Finn CE, Frei B, Wrolstad RE (2002) Anthocyanins, phenolics, and antioxidant capacity in diverse small fruits: vaccinium, rubus, and ribes. J Agric Food Chem 50:519–525. doi:10.1021/jf011062r Murray JR, Hackett WP (1991) Dihydroflavonol reductase activity in relation to differential anthocyanin accumulation in juvenile and mature phase Hedera helix L. Plant Physiol 97:343–351 Peng CL, Lin ZF, Lin GZ, Chen SW (2006) Antioxidant functions of purple rice with abundant anthocyanin. Sci China ser C 36:209–216 Pen˜uelas J, Filella I (1998) Visible and near-infrared reflectance techniques for diagnosing plant physiological status. Trends Plant Sci 3:151–156. doi:10.1016/S1360-1385(98) 01213-8 Pettigrew WT, Vaughn KC (1998) Physiological, structural, and immunological characterization of leaf and chloroplast development in cotton. Protoplasma 202:23–37. doi: 10.1007/BF01280872 Ralph PJ, Gademann R (2005) Rapid light curves: a powerful tool to assess photosynthetic activity. Aquat Bot 82:222– 237. doi:10.1016/j.aquabot.2005.02.006
123
Plant Growth Regul (2009) 58:73–84 Reddy VS, Dash S, Reddy AR (1995) Anthocyanin pathway in rice (Oryza sativa L.): identification of a mutant showing dominant inhibition of anthocyanins in leaf and accumulation of proanthocyanins in pericarp. Theor Appl Genet 91:301–312. doi:10.1007/BF00220892 Sims DA, Gamon JA (2002) Relationships between leaf pigment concentration and spectral reflectance across a wide range of species, leaf structures and developmental stages. Remote Sens Environ 81:337–354. doi:10.1016/S00344257(02)00010-X Smart CM (1994) Gene expression during leaf senescence. New Phytol 126:419–448. doi:10.1111/j.1469-8137.1994. tb04243.x Taiz L, Zeiger E (1998) Plant physiology, 2nd edn. Sinauer Associates, Inc., Sunderland Tracewell CA, Vrettos JS, Bautista JA, Frank HA, Brudvig GW (2001) Carotenoid photooxidation in photosystem II. Arch Biochem Biophys 385:61–69. doi:10.1006/abbi. 2000.2150 Tsuda T, Shiga K, Ohshima K, Kawakishi S, Osawa T (1996) Inhibition of lipid peroxidation and the active oxygen radical scavenging effect of anthocyanin pigments isolated from Phaseolus vulgaris L. Biochem Pharmacol 52:1033–1039. doi:10.1016/0006-2952(96)00421-2 Winkel T, Me´thy M, The´not F (2002) Radiation use efficiency, chlorophyll fluorescence, and reflectance indices associated with ontogenic changes in water-limited Chenopodium quinoa leaves. Photosynthetica 40:227–232. doi:10.1023/ A:1021345724248 Yamasaki H, Uefuji H, Sakihama Y (1996) Bleaching of the red anthocyanin induced by superoxide radical. Arch Biochem Biophys 332:183–186. doi:10.1006/abbi.1996. 0331 Yoo SD, Greer DH, Laing WA, McManus MT (2003) Changes in photosynthetic efficiency and carotenoid compostion in leaves of white clover at different developmental stages. Plant Physiol Biochem 41:887–893. doi:10.1016/S09819428(03)00138-4
