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Use of the atLEAF+ chlorophyll meter for a nondestructive estimate of

chlorophyll content

Article in Photosynthetica · August 2015

DOI: 10.1007/s11099-015-0172-8

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DOI: 10.1007/s11099-015-0172-8 PHOTOSYNTHETICA 54 (1): 130-137, 2016

Use of the atLEAF+ chlorophyll meter for a nondestructive estimate

of chlorophyll content

E.V. NOVICHONOK*,+, A.O. NOVICHONOK**, J.A. KURBATOVA***, and E.F. MARKOVSKAYA**

Laboratory of Physiology and Cytology of Woody Plants, Forest Research Institute, Karelian Research Centre
of the Russian Academy of Sciences, Pushkinskaya Street 11, Petrozavodsk, 185910, Russian Federation*
Petrozavodsk State University, Lenina Street, 33, Petrozavodsk, 185910, Russian Federation**
A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninsky Prospect, 33,
Moscow, 119071, Russian Federation***

Abstract
At present, сhlorophyll meters are widely used for a quick and nondestructive estimate of chlorophyll (Chl) contents in
plant leaves. Chl meters allow to estimate the Chl content in relative units – the Chl index (CI). However, using such
meters, one can face a problem of converting CI into absolute values of the pigment content and comparing data
acquired with different devices and for different plant species. Many Chl meters (SPAD-502, CL-01, CCM-200)
demonstrated a high degree of correlation between the CI and the absolute pigment content. A number of formulas have
been deduced for different plant species to convert the CI into the absolute value of the photosynthetic pigment content.
However, such data have not been yet acquired for the atLEAF+ Chl meter. The purpose of the present study was to
assess the applicability of the atLEAF+ Chl meter for estimating the Chl content. A significant species-specific
exponential relationships between the atLEAF value (corresponding to CI) and extractable Chl a, Chl b, Chl (a+b) for
Calamus dioicus and Cleistanthus sp. were shown. The correlations between the atLEAF values and the content of
Chl a, Chl b, and Chl (a+b) per unit of leaf area was stronger than that per unit of dry leaf mass. The atLEAF value–
Chl b correlation was weaker than that of atLEAF value–Chl a and atLEAF value–Chl (a+b) correlations. The influence
of light conditions (Chl a/b ratio) on the atLEAF value has been also shown. The obtained results indicated that the
atLEAF+ Chl meter is a cheap and convenient tool for a quick nondestructive estimate of the Chl content, if properly
calibrated, and can be used for this purpose along with other Chl meters.
Additional key words: absorption; adaptation; light; leaf water content; morphological trait; reflection; transmission.

Introduction
The photosynthetic pigment content and pigment ratio photosynthetic pigment content depends on abiotic
vary over a wide range in plants of different species and factors (light, soil moisture, soil fertility, salinity, etc.), as
growing in different latitudinal zones (Murchie and well as biological factors (competition, the presence or
Horton 1997, Shmakova and Markovskaya 2010). absence of herbivorous organisms) (Murchie and Horton
Estimating their content is of great practical and 1997, Wang and Nii 2000, Carter and Knapp 2001,
theoretical importance, since photosynthetic pigments Amujoyegbe et al. 2007, Dai et al. 2009, Guerfel et al.
participate in the absorption and transformation of light 2009, Nikolaeva et al. 2010). In this respect, monitoring
energy into chemical bound energy, and their content changes in the photosynthetic pigment content enables to
influences the photosynthetic rate and the plant estimate plant interactions with the environment and the
productivity (Šesták 1966, Buttery and Buzzell 1977, influence of stress factors.
Murchie and Horton 1997, Ghosh et al. 2004). The The spectrophotometric method is traditionally used

———
Received 7 April 2015, accepted 3 July 2015, published as online-first 15 August 2015.
+Corresponding author: e-mail: [email protected]

Abbreviations: atLEAF value – CI obtained with the atLEAF+ chlorophyll meter; Chl – chlorophyll; CI – chlorophyll index; LWC –
leaf water content; SLA – specific leaf area.
Acknowledgements: This research was carried out with the support of the Joint Russian–Vietnam Tropical Research and
Technological Centre. We thank the administration and staff of the Tropical Centre for the possibility of working in Vietnam, as well
as the administration of the Cát Tiên National Park (South Vietnam), where all the experimental studies were executed. The work of
J. Kurbatova was supported by Russian Science Foundation (Project № 14-27-00065). Also this study was partially supported by the
Ministry of Education and Science of Russian Federation (project № 6.724.2014/k).

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 atLEAF CHLOROPHYLL METER FOR ESTIMATE OF CHLOROPHYLL CONTENT

for the analysis of the photosynthetic pigment content in Chl meters estimate the Chl content in relative units –
plant leaves (Brougham 1960, Terborgh and Thimann the Chl index (CI), which is not directly comparable
1964, Einhellig and Rasmussen 1979, Baziramakenga et among different Chl meters. As a result, many problems
al. 1994, Zhao et al. 2001, Hallik et al. 2012). This arise in this connection with the conversion of CI into
method is based on the absorption spectrum of pigment absolute values of the pigment content and the
extracts (Mac Kinney 1941, Comar and Zscheile 1942, comparison of data acquired with different devices and
Vernon 1960, Wintermans and De Mots 1965, Hiscox for different plant species. For many Chl meters (SPAD-
and Israelstam 1979, Lichtenthaler 1987). The spectro- 502, CL-01, CCM-200), a high degree of correlation
photometric method allows us to acquire the data on the between the CI and the absolute values of the pigment
content of Chl a and Chl b, as well as carotenoids without content has been confirmed and various formulas for the
their prior separation. However, this method has some conversion of the CI into the absolute pigment content
limitations. It is labour- and time-consuming; it brings have been deduced (Van den Berg and Perkins 2004,
certain problems when a large number of samples is Pinkard et al. 2006, Cassol et al. 2008, Mielke et al.
involved. When using this method, minimal time lapses 2010). However, many researchers point out that the CI
between the sample collection and their analysis should may depend on a variety of factors, such as a leaf water
be ensured, because plant pigments break down very content, leaf thickness, anatomical traits, and peculiarities
quickly. Besides, the pigment extraction leads to of a Chl distribution in the leaf (Giunta et al. 2002,
destruction of the plant; it makes impossible to study a Marenco et al. 2009, Songsri et al. 2009, Wang et al.
dynamic pattern of the pigment content in the same 2009). In this respect for each particular plant species and
sample or to study rare and endangered plant species. environment, individual equations ought to be
All of those limitations can be overcome by using formulatted for dependence between the CI and the
nondestructive methods for the estimation of the photo- absolute photosynthetic pigment content.
synthetic pigment contents. Currently, various devices At present, the most popular Chl meter is the SPAD-
were used for a quick estimate of the relative pigment 502. It is widely used for estimation of the Chl content in
content without destructing plant samples: the SPAD-502 plant leaves (Ranganathan et al. 2006, Fotovat et al.
Chl meter (Spectrum Technologies, Inc., Plainfield, IL, 2007, Senger et al. 2014). However, similar instrument –
USA), the CL-01 Chl meter (Hansatech Instruments, the atLEAF+ Chl meter – is a cheaper and more
Ltd., United Kingdom), the CCM-200 Chl content meter accessible instrument than the SPAD-502. Although the
(Opti-Sciences, Inc., Hudson, NH, USA), the atLEAF+ SPAD-502 and the atLEAF+ Chl meters use the same
Chl meter (FT Green LLC, Wilmington, DE, USA). The principle, the data on the Chl content acquired with these
estimation of the photosynthetic pigment content with devices differ, because the radiation of different
these devices is based on the measurement of radiation wavelengths is used (Zhu et al. 2012). There are
absorption by Chl at different wavelengths. As a rule, one practically no information on the dependence between the
wavelength corresponds to the spectral range with the atLEAF value and the absolute Chl content in leaves as
maximum Chl activity, another wavelength – the infrared well as on the influence of leaf traits on this dependence.
region (where the Chl absorption is very low) – is used In the present study, we assessed the possibility of the
for the compensation of the leaf water content and leaf atLEAF+ Chl meter application for estimating the Chl
thickness (Hawkins et al. 2007). For example, the SPAD- content. The study objectives were to find out: (1) how
502, atLEAF+, and CL-01 Chl meters use the wavelengths effectively the atLEAF+ Chl meter allows to estimate the
of 650, 660, and 620 nm, respectively, as the region of content of Chl a, Chl b, and Chl (a+b); (2) whether the
maximum Chl absorption, while they use 940 nm in the accuracy of this estimation varies for different plant
infrared region (SPAD-502 Plus: product manual; species; (3) whether light conditions (which influence
atLEAF specification; CL-01 specification). The CCM- Chl a/b) and the leaf traits [specific leaf area (SLA) and
200 Chl meter uses the the wavelengths of 653 and 931 leaf water content (LWC)] influence the accuracy of the
nm (CCM-200 plus brochure). Chl content estimate with the atLEAF+ Chl meter.

Materials and methods

This study was carried out in the Cát Tiên National Park, For the study purposes, two plant species were
situated in South Vietnam (11о21'–11о48'N; 107о10'– chosen, Calamus dioicus (Arecaceae) and Cleistanthus
107о34'E). The climate of South Vietnam is tropical sp. (Phyllanthaceae). The choice of the species for the
monsoon (McKnight and Hess 2000). The average yearly study was determined by our concern about the fact that
air temperature is 26–27 оС, the annual precipitation is the data obtained for one species do not fully reflect the
about 2450 mm. There are two distinct seasons during the correlation between the atLEAF value and the absolute
year: the dry (November–April) and the wet season Chl content and thus they cannot be applied for other
(May–October) (Blanc et al. 2000, Deshcherevskaya et plant species. The selected plant species vary highly in
al. 2013). their morphological characteristics. For Calamus sp.,

131
E.V. NOVICHONOK et al.

xerophilous leaves are typical, and even in young leaves, calculated with regard to the SLA. In order to estimate
SLA quickly reaches low values. At the same time, in the SLA and LWC, the leaf parts (without the midribs) of
Cleistanthus sp., the young leaves with the low Chl the known area were weighed to determine fresh mass
content contain a larger amount of water and are thinner (FM). Then, these parts were dried until oven-dry mass at
(high SLA). These leaf features of the selected species 80°С and then weighed again to determine the dry mass
enabled us to explore the influence of the SLA and LWC (DM). The SLA was calculated as a ratio of leaf area to
on correlations between the atLEAF value and the dry leaf mass. The LWC was calculated using the
absolute Chl content. following formula:
For the analysis, 60 leaves of each species were
FM DM
collected. The leaves were taken from plants growing LWC ∗ 100
under different light conditions, such as unbroken forest FM
cover (under the canopy) and in gaps. The leaves differed The regression analysis was performed in order to
in their age (from young to old), and, consequently, in the determine relationships between the atLEAF values and
photosynthetic pigment content and SLA (Fig. 1). The the Chl content, SLA, and LWC for each species. Also a
leaves were collected at 11–12:00 h of local time general equation for the relationship between the atLEAF
(UTC+7), which corresponded to the local midday. The value and the Chl content was deduced for the whole data
leaves were placed into black plastic bags with ice and set (for both species). Student´s t-test for paired
then taken to the laboratory. For our study, the central observations was used to test differences between the
part of the leaf was used, avoiding the midribs. In the general and the species-specific equations. To estimate
laboratory, the atLEAF values were measured 5 times for the influence of the Chl a/b ratio on the relationship
each leaf, and then an average value for each leaf was between the Chl (a+b) and atLEAF values, two sets of
calculated. Immediately after this procedure, the absolute data were formed, differing in the Chl a/b ratio; in the
photosynthetic pigment content was measured in the first one, the Chl a/b ratio varied from 1.8 to 2.1, while in
same samples and SLA and LWC were estimated. The the second one, the Chl a/b ratio varied from 2.11 to 2.40.
absolute pigment content was determined spectrophoto- For each variant, the relationship between the Chl (a+b)
metrically. The pigments were extracted with 96% and atLEAF values was analysed separately with the aid
ethanol. The absorption was measured at wavelengths of of the regression analysis. For significance testing of the
665, 649, and 470 nm by a spectrophotometer (PD-303, regression equations and comparison of different models,
APEL, Japan). The content of Chl a and Chl b (per unit of the data were modified to linearity. The reliability of the
leaf dry mass) in the extract was calculated by equations regression equations was estimated by means of the
of Wintermans and De Mots (1965). The total Chl F-test. The STATISTICA, version 10 (StatSoft Inc.) was
content [Chl (a+b)] was calculated as the sum of Chl a used for the data analysis.
and Ch b. The Chl content per unit of leaf area was

Results

We estimated leaves with a wide range of photosynthetic tionship between the atLEAF values and Chl content was
pigment contents (Table 1). Consequently, the atLEAF stronger when calculated as the Chl content per unit
values varied over a wide range: 1.2–51.0 (Cleistanthus of leaf area than that per unit of DM. The atLEAF
sp.) and 12.5–51.2 (Calamus sp.) (Table 1). The relation- value–Chl b relationship was weaker than that of the
ships between the atLEAF values and the content of atLEAF–Chl a and atLEAF–Chl (a+b) relationships
Chl a, Chl b, Chl (a+b) were better expressed by means (Table 2, Fig. 1).
of an exponential function, not the linear one. We found The strength of the relationship between the atLEAF
out relationships between the atLEAF values and contents value and content of Chl (a+b) depended on the Chl a/b
of Chl a, Chl b, Chl (a+b) calculated both per unit of leaf ratio. Moreover, the significant relationship between
area and per unit of DM for Cleistanthus sp. and Calamus these values was observed when calculating the content
sp. (Table 2, Fig. 1). General equations differed from the of Chl (a+b) both per unit of leaf area (Fig. 2), and per
equations deduced separately for each species. The rela- unit of DM (data not shown). R2 for the relationship

Table 1. Range of the atLEAF value, the contents of chlorophyll (Chl) a, Chl b, and total Chl (a+b) (determined spectrophoto-
metrically), the specific leaf area (SLA), and leaf water content (LWC), estimated in Cleistanthus sp.and Calamus sp.

Species atLEAF Chl a Chl b Chl (a+b) SLA [cm2 g–1] LWC [%]
[mg g–1] [mg m–2] [mg g–1] [mg m–2] [mg g–1] [mg g–1]

Cleistanthus sp. 1.2–51.0 0.6–8.8 12.3–339.6 0.3–4.2 6.3–151.5 0.9–13.0 19.5–468.8 136.4–607.9 22.2–87.6
Calamus sp. 12.5–51.2 0.7–8.7 54.3–428.6 0.3–3.8 14.1–209.4 1.1–12.5 68.4–638.0 97.4–267.0 30.5–67.1

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 atLEAF CHLOROPHYLL METER FOR ESTIMATE OF CHLOROPHYLL CONTENT

Table 2. The relationships between the atLEAF value and the content of chlorophyll (Chl) a, Chl b, and total Chl (a+b). For all the
equation p<0.001. The regression equations marked with different letters differ significantly between species-specific equations and
general equation (p<0.05).

Variable Cleistanthus sp. Calamus sp. Both species (general)

Chl a [mg g–1] . . .

1.015 0.905 0.994
(R2 = 0.61)a (R2 = 0.52)b (R2 = 0.60)c
[mg m–2] . . .
22.03 47.65 27.31
(R2 = 0.84)a (R2 = 0.75)b (R2 = 0.82)с
Chl b [mg g–1] . . .
0.524 0.393 0.490
(R2 = 0.54)a (R2 = 0.50)b (R2 = 0.53)c
[mg m–2] . . .
11.36 20.57 13.46
(R2 = 0.81)a (R2 = 0.67)b (R2 = 0.79)c
Chl (a+b) [mg g–1] . . .
1.540 1.304 1.488
(R2 = 0.60)a (R2 = 0.52)b (R2 = 0.59)c
[mg m–2] . . .
33.40 68.71 40.88
(R2 = 0.84)a (R2 = 0.73)b (R2 = 0.82)с

Fig. 1. Relationships between the atLEAF value and the content of chlorophyll (Chl) a, Chl b, and total Chl (a+b) in the leaves of
Cleistanthus sp. (○, dotted line) and Calamus sp.(●, solid line).

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E.V. NOVICHONOK et al.

Fig. 2. Influence of chlorophyll (Chl) a and Chl a/b on the

relationship between total Chl and the atLEAF value for
Cleistanthus sp. (A) and Calamus sp. (B). There are two
variants: 1 – Chl a/b = 1.8–2.1 (○, dotted line); 2 – Chl a/b = Fig. 3. Relationships between the atLEAF value and A: the
2.11–2.4 (●, solid line). For all the equations p<0.001. The specific leaf area (SLA), B: the leaf water content (LWC) in
regression equations marked with different letters differ Cleistanthus sp. (○, dotted line) and Calamus sp. (●, solid line).
significantly (p<0.05). *** – p<0.001, ns – not significant.

between the atLEAF value and content of Chl (a+b) per over a wide range (Table 1). There were strong
unit of DM, when Chl (a+b) was equal to 1.8–2.1 for significant negative relationships between the atLEAF
Cleistanthus sp. amounted to 0.50, for Calamus sp. – value and SLA and LWC for Cleistanthus sp. For
0.82. When Chl (a+b) was equal to 2.11–2.4 R2 amounted Calamus sp., the relationships between the atLEAF
to 0.75 for Cleistanthus sp. and 0.30 Calamus sp. values and SLA, LWC were not significant (Fig. 3).
The SLA and LWC values in the studied leaves varied

Discussion

We found a significant relationships between the content Therefore, exponential functions (Marenco et al. 2009,
of Chl a, Chl b, and Chl (a+b) and the atLEAF values Mielke et al. 2010), logarithmic functions (Van den Berg
(Table 2, Fig. 1). The relationships between the CI values and Perkins 2004), and also more complex functions
obtained with different Chl meters (CCI 200, SPAD-502, (Netto et al. 2002) have been used. The loss of linearity
CL-01) and contents of Chl a, Chl b, and Chl (a+b) were can be related to several factors: the uneven distribution
noted by other authors as well (Van den Berg and Perkins of Chl and chloroplasts in the leaf, different values of
2004, Sheshshayee et al. 2006, Cassol et al. 2008, SLA (and the leaf thickness, dependent on them) and
Hawkins et al. 2009, Mielke et al. 2010, Zhu et al. 2012). different leaf water contents (Uddling et al. 2007,
The relationships between the atLEAF value and Marenco et al. 2009). These features of the leaf can
content of Chl a, Chl b, Chl (a+b) is better described by interfere with its properties for radiation absorption and
means of the exponential function. Some researchers reflection, which are used for determining the CI (Cassol
describe the relationship between the CI and Chl content et al. 2008).
by means of linear functions (Cassol et al. 2008, Wang et We observed strong relationships between the
al. 2009). However, the majority of scientists assume that atLEAF value and LWC and SLA in Cleistanthus sp. and
nonlinear functions are more suitable for expression of the absence of such relationships in Calamus sp., which
the relationship between the CI and Chl content. was connected to peculiarities of the leaf development in

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 atLEAF CHLOROPHYLL METER FOR ESTIMATE OF CHLOROPHYLL CONTENT

this species. For example, xerophilous leaves are typical atLEAF value–Chl a relationship, compared to the
for Calamus sp. and SLA quickly reaches low values atLEAF value–Chl b relationship, was most probably
even in the young leaves. At the same time, in connected with the radiation wavelength used in the
Cleistanthus sp., the young leaves with the low Chl atLEAF+ Chl meter for a Chl content determination. For
content contain a larger amount of water and are thinner such determination, the radiation with wavelengths of
(a high SLA), which can “strengthen” the relationship. 660 nm and 940 nm is used. The maximum light
For example, in Fig. 3, one can see that for leaves of absorption in the red region for Chl a is closer to 660 nm
Cleistanthus sp. with the higher atLEAF values the than that of Chl b. For example, the maximum light
relatioships of the atLEAF values–SLA and the atLEAF absorption in the red region for the extract of Chl a and
values–LWC was weaker. We separately analysed the Chl b in diethyl ether peaks at 661 and 642 nm,
relationship between the atLEAF value and LWC and respectively, while it is 665 and 649 nm, respectively, in
SLA for Cleistanthus sp. excluding too young leaves (the 96% ethanol (Wintermans and De Mots 1965,
atLEAF value lesser than 10). In this case, the atLEAF Lichtenthaler and Buschmann 2001). The variation
value–SLA relationship was weak (R2=0.24, p<0.05), and between the maximums of light absorption in the red
the atLEAF value–LWC relationship was not found region for Chl a and b can account for the observed
(R2=0.03, p>0.05) (data not shown). Thus, we could dependence of the atLEAF value–Chl (a+b) relationships
assume that the influence of LWC and SLA on the on the Chl a/b ratio (Fig. 2). The Chl a/b ratio depends,
atLEAF value was not strong when the very young leaves first of all, on the light conditions under which plants are
were excluded. But to confirm this hypothesis, more growing (Valladares and Niinemets 2008). Thus, it is
detailed studies of the atLEAF value in mature leaves necessary to take care when comparing the data acquired
with different leaf water contents and different SLA are with the atLEAF+ Chl meter for plants growing under
needed. It is expected that the compensation of the different light conditions. For a more accurate estimate of
influence of the leaf water content and leaf thickness on the Chl content, it is important to take into account the
the atLEAF values is made by using the radiation with a Chl a/b ratio when building calibration models; even the
wavelength of 940 nm (Hawkins et al. 2007). However, same plant species growing under different light
the researchers who were working with the SPAD-502 conditions require separate calibration equations.
Chl meter noted the dependence of CI on SLA and LWС The relationships between the atLEAF value and the
and attributed this dependence to the fact that these content of Chl a, b, and Chl (a+b) were species-specific
values strongly affect the leaf optical properties (Giunta for both calculations per unit of leaf area and per unit of
et al. 2002, Marenco et al. 2009, Songsri et al. 2009, DM (Table 2), which is consistent with the data acquired
Wang et al. 2009). by other authors with the SPAD-502 and CL-01 Chl
The relationships between the atLEAF values and the meters (Cassol et al. 2008, Marenco et al. 2009). In
content of Chl a, Chl b, and Chl (a+b) per unit of leaf summary, the acquired data indicated that despite high
area was stronger than that per unit of DM (Table 2). accuracy of the Chl content estimation in leaves with the
Similar results were achieved for the SPAD-502 and CL-1 atLEAF+ Chl meter, it is important to take into account
Chl meter (Cassol et al. 2008, Hawkins et al. 2009, some peculiarities. The maximum accuracy of the
Marenco et al. 2009). Marenco et al.(2009) attribute this estimate was achieved when the atLEAF+ Chl meter was
to the fact that when calculating the photosynthetic used for the determination of the Chl content per unit of
pigment content per unit of leaf area, one takes into leaf area. At the same time, it was necessary to use a
account SLA, which (as was proven by those scientists) species-specific equation when converting the atLEAF
affects Chl meter reading. However, in our study, the value into the absolute Chl content for each plant species.
SLA showed only a marginal effect on the atLEAF value When comparing the atLEAF value, one needs to take
for Cleistanthus sp. and did not affect this value for into account the growth conditions (particularly
Calamus sp. irradiance), which can affect the Chl a/b ratio. In case of
The atLEAF value–Chl b relationship was weaker large discrepancies in the Chl a/b ratio, the atLEAF value
than that of the atLEAF value–Chl a and atLEAF value– can vary even with the same absolute Chl content in the
Chl (a+b) relationships. The strength of the atLEAF leaf. Notwithstanding the above, the atLEAF+ Chl meter
value–Chl a relationship was similar to the strength of the could be a convenient tool for a quick, nondestructive
atLEAF value–Chl (a+b) relationship (Table 2). Similar estimate of the Chl content along with other сhlorophyll
results were achieved with the SPAD-502 Chl meter meters after the right calibration and taking into account
(Pinkard et al. 2006, Mielke et al. 2010). The stronger the leaf peculiarities and plant growth conditions.

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