全 文 ：第 49 卷 第 11 期
2 0 1 3 年 11 月
林 业 科 学
SCIENTIA SILVAE SINICAE
Vol. 49，No. 11
Nov．，2 0 1 3
doi:10．11707 / j．1001-7488．20131105
Received date: 2013 － 04 － 15; Revised date: 2013 － 08 － 15。
Foundation project: National science ＆ technology program in the twelfth five-year plan ( 2011BAD38B0305 ) ; Jiangsu agriculture science and
technology innovation fund［CX(11) 1041］．
* Corresponding author: Chen Qingsheng．
乌桕秋叶显色的生理生化与数字图像分析*
黄利斌1 陈庆生1 张 敏1 钱 猛2 窦全琴1
(1． 江苏省林业科学研究院 南京 211153; 2．南京农业大学生命科学学院生命科学实验中心 南京 210095)
摘 要: 研究不同颜色乌桕秋叶的色素含量、全氮及碳水化合物含量，定量分析叶片的 RGB 值，并观察叶绿素、
类胡萝卜素及花色素苷在叶片中的分布情况。结果表明:色素含量与单一的 RGB(红、绿、兰色)值无直接相关关
系，而 Chl a 与(G － B) /( R － B)呈正相关，Chl b 和总叶绿素均与 R /( R + G + B)呈显著负相关，花色素苷与
R /(G － B)呈显著正相关。绿叶全氮含量最高，而在转色后的叶片中，红色和紫色叶片的氮含量又高于橙色和黄色
叶片。全氮与总叶绿素含量呈显著正相关，蔗糖与花色素苷呈正相关，淀粉与叶绿素 a 和总叶绿素均呈极显著正
相关，非结构性碳水化合物总量与类胡萝卜素含量呈显著正相关。本研究期望从生理及结构等方面找到影响叶片
呈色的相关因素，丰富叶片呈色机制的基础资料，为选育秋季叶色纯正的乌桕优良观赏株系提供理论依据。
关键词: 乌桕; 叶色; RGB 值; 色素; 全氮; 碳水化合物
中图分类号: S718. 43 文献标识码: A 文章编号: 1001 － 7488(2013)11 － 0032 － 10
Analysis of Autumnal Leaf Colorization of Sapium sebiferum by
Using Physiological and Digital Imaging Methods
Huang Libin1 Chen Qingsheng1 Zhang Min1 Qian Meng2 Dou Quanqin1
(1 ． Jiangsu Academy of Forestry Nanjing 211153;
2 ． Life Science Laboratory Center School of Life Sciences，Nanjing Agricultural University Nanjing 210095)
Abstract: In the present study，foliar pigments，nitrogen (N) and carbohydrate concentrations in the autumn leaves of
Chinese tallow trees (Sapium sebiferum) with different colored leaves were measured． The R，G and B values of the leaves
were quantified by the digital imaging analysis． In addition，the distribution of chlorophyll，anthocyanins and carotenoids
in the leaves were observed in situ by their autofluorescence． The results showed that there was no correlation between
pigment contents and the single R，G and B value． Whereas，a significant positive correlation was observed between chl a
and (G － B) /(R － B) ． The chl b and total chlorophyll content were all negatively correlated with R /(R + G + B) ． There
was a positive correlation between anthocyanins contents and R / (G － B) ． Nitrogen concentration was highest in the green
leaves． After the leaves changed color，the red and purple leaves contained much more nitrogen than the orange and yellow
leaves did． The total nitrogen was negatively correlated with total chlorophyll． A linear regression analysis demonstrated a
positive correlation between sucrose and anthocyanins，and there were significant positive correlations between starch and
chl a with total chlorophyll． Moreover， the content of total non-structural carbohydrates was positively correlated to
carotenoid． The aim of this study was to find out the physiological and structural factors that influence autumn coloration of
S． sebiferum． This study would provide a basis for selective-breeding of Chinese tallow tree with attractive autumn colors．
Key words: Sapium sebiferum; leaf coloration; RGB values; pigment; total nitrogen; carbohydrate
The appearance of leaf color was linked to
differential expression of genes ( Nothnagel et al．，
2003)，changes in tissue and cell structures (Matile，
2000; Schaberg et al．， 2008 ) and physiological
conditions (Vitrac et al．，2000) in plant cells，as well
as the effect of external environment ( Chalker-Scott，
1999) ． The prevailing view among plant physiologists
is that foliage changes yellow and orange due to the
第 11 期 黄利斌等: 乌桕秋叶显色的生理生化与数字图像分析
breakdown of green chlorophyll molecules， which
resulting in unmasking of other pigments， like
carotenoids ( Lee et al．，2002)，and most red leaves
result from de novo synthesis of anthocyanins (Field et
al．，2001 ) ． Recent investigations have revealed that
many factors， including UV-B radiation
(Ranjbarfordoei et al．，2009)，light stress (Merzlyak
et al．，2000 )，abscisic acid ( Hung et al．，2008 )，
drought (Castellarin et al．，2007)，senescence (Hoch
et al．，2001) and ozone exposure ( de Rezende et al．，
2009) can influence biosynthesis of photosynthetic and
non-photosynthetic pigments such as chlorophyll a，
chlorophyll b， total chlorophyll， carotenoid and
anthocyanins． The physiological significance of autumn
leaf coloration through chlorophyll degradation and
anthocyanins accumulation has been well documented
(Field et al．，2001; Lee et al．，2002; Samuel et al．，
2003 ) ． However， the linkage among foliar color，
pigments contents and physiological conditions relating
the biochemical and physiological basis of autumn
coloration is not well understood ( Schaberg et al．，
2003) ． Furthermore， little is known concerning the
differences in cellular ultrastructure and distribution of
chloroplasts in leaves with different autumn hues．
Fig． 1 The scanogram of different color leaf of Sapium sebiferum
A． Green; B． Yellow; C． Orange; D． Red; E． Purple．
Chinese tallow tree ( Sapium sebiferum )
belonging to Euphorbiaceae， is widely used for
biomass-energy， timber and ornament． This tree
species enriches in seed oil，grows rapid，and abounds
in flowers． Chinese tallow trees show evidently
autumnal color change，and the senescing leaves show
various shades of yellow， orange， purple， or red．
Sapium sebiferum cultivars with brightly colored leaves
are valuable for ornament， which can enrich the
landscape level as well as make up the urban color
monotony．
In the present study， the differences in foliar
pigments， nitrogen ( N ) and carbohydrate
concentrations in various color leaves of Chinese tallow
trees were investigated． Leaf color was quantified using
digital imaging analysis． And the distribution and
ultrastructure of chloroplasts were examined using
transmission electron microscopy． Moreover，we took
advantage of the unique autouorescence of
chlorophyll，carotenoid and anthocyanins to study their
distribution in leaf tissues by laser confocal
microscopy． The purpose of this study was to find out
the physiological and structural factors that influence
autumn coloration of Sapium sebiferum． The results
may provide basis for selective-breeding of Chinese
tallow tree with better autumn colors．
1 Materials and methods
1. 1 Plant materials
The leaves were sampled from Sapium sebiferum
with different color ( green，yellow，orange，red and
purple) autumn leaves growing in Yongxing nursery at
Maoshan Town，Jurong County，Jiangsu，China． The
leaves were harvested separately from 5 seedlings (3-
year old and 2 m high) ． All collections were made in
the morning between 10: 00—11: 00 am on 13
November 2010．
1. 2 Foliar color analysis
Leaf color was measured by digital imaging
analysis using Image J software，which was downloaded
at http: ∥ imagej． nih． gov / ij / download． html． The
leaves were scanned as a color photo at a resolution of
300 dpi with an Epson Perfection 1 260 color scanner
and the images were saved in tag image file format
( TIFF) ( Fig． 1 ) ． Subsequently，the scanned color
images were manipulated in Image J software． The full
33
林 业 科 学 49 卷
color images consist of red (R)，green ( G)，and blue
(B) channels． And then the mean values of the red，
green and blue pixels for each leaf were calculated
using the Color histogram tool．
1. 3 Pigments determination
Fresh leaf tissues were pulverized with liquid
nitrogen and photosynthetic pigments were extracted
with 80% acetone for 24 h at － 5 ℃ ． Pigments were
determined spectrophotometrically with a Beckman DU
800 UV /VIS spectrophotometer． Contents of
chlorophyll a and b and total carotenoid were
determined according to Lichtenthaler ( 1987 )
formulae．
For the determination of anthocyanins， the
method of Pirie et al． ( 1976 ) was followed with
minor modification． Briefly， the leaf samples were
macerated initially in 10 mL of an aqueous methanol-
HCl solvent (methanol∶ water∶ HCl，79 ∶ 20 ∶ 1，V /V，
pH 3. 5 ) ． Total anthocyanins were assayed by
measuring absorbance at 530 nm using a Beckman DU
800 UV /VIS spectrophotometer and 10-mm quartz
cuvettes，and one absorbance unit was defined as the
amount of anthocyanins giving an absorbance of 0. 1 at
530 nm．
1. 4 Nitrogen analysis
The fresh leaves were dried at 105 ℃ for 30 min
to deactivate enzymes，and then the leaves were dried
at 75 ℃ until attaining a constant weight． Total
nitrogen ( N ) content in leaves was determined in
accordance with the Kjeldahl method with a 25 mL
aliquot of pure extract， followed by distillation and
titration as described by Bremmer et al． (1982) ．
1. 5 Carbohydrate determination
The fresh leaves were oven dried at 70 ℃ until
attaining a constant weight，and then the oven-dried
leaves were powdered． Subsequently，0. 15 g of the
powder was mixed with 30 mL distilled water and
stirred for 1 h． After then，50 mL distilled water was
added to the mixture，mixed thoroughly and filtered．
20 mL filtrate was transferred into a 50 mL flask and
2. 5 mL each of Carrez I and Carrez II was added and
mixed，and 5 mL 0. 1 mol·L － 1 NaOH was added．
After filtration， the filtrate was used for the
measurement of glucose，fructose and sucrose with the
Sucrose / D-Glucose / D-Fructose assay kit ( Roche，
Darmstadt，Germany) ．
For the measurement of starch，the Roche starch
assay kit ( Roche，Darmstadt，Germany ) was used
following the manufacturer’s instruction． In brief，
0. 15 g of the oven-dried leaf powder was added into 5
mL 40% ethanol and centrifuged at 3 000 rpm for 10
min． After centrifugation， the supernatant was
discarded，and the pellet was mixed with 5 mL of 40%
ethanol and centrifuged again under the same
condition． The pellet was then washed with 8 mL
DMSO for 4 times (2 mL each time)，and 2 mL 8
mol·L － 1 HCl was added into the eluent and bathed at
60 ℃ for 30 min． After being cooled at room
temperature for 30 min，10 mL distilled water was
added and then the pH value of mixture was adjusted to
4 － 5 with 5 mol·L － 1 NaOH． The final volume of the
mixture was made up to 50 mL with distilled water．
After filtration，the filtrate was used for the assessment
of starch in the leaves．
1. 6 Transmission electron microscopy
For transmission electron microscopy the leaves
were cut into slices (2 mm × 3 mm) and fixed with
2. 5% glutaraldehyde，postfixed with 1% OsO4，and
then embedded in EP 812 resin． Ultrathin sections
were cut on an ultramicrotome (LKB-V，Sweden) and
stained with 2% uranyl acetate followed by 6% lead
citrate． Chloroplast ultrastructure was observed with an
H-600 transmission electron microscope ( JEOL，
Japan) ．
1. 7 Laser confocal microscopy
The distribution of chlorophyll，anthocyanins and
carotenoid in the leaf mesophyll cells were visualized in
situ by their autofluorescence． Briefly，the fresh leaves
were embedded in OCT medium ( Sakura Finetek
USA，Inc，Torrance，CA，USA) and 7 μm continuous
sections were obtained with a Leica CM 1950 cryostat．
And then the sections were mounted onto the poly-
lysine coated slides． The 633 nm excitation ray line
was used to image chlorophyll and the emitted
uorescence was collected between 650 and 700 nm．
For the detection of carotenoid，the 488 nm excitation
ray line was used and the emitted uorescence was
collected between 500 and 600 nm ( Egea et al．，
2011 ) ． The autouorescence of anthocyanins was
excited using a helium-neon 543 nm laser ( long pass
43
第 11 期 黄利斌等: 乌桕秋叶显色的生理生化与数字图像分析
585 nm) (Gomez et al．，2011) ． And the sections were
counterstained with DAPI (4’，6’- diamidino-2-
phenylindole) ． The samples were then examined by a
Leica TCS SP5 confocal scanning microscope ( Leica
Microsystems， Heidelberg GmbH， Mannheim，
Germany) ．
1. 8 Statistical analysis
Statistical analysis was carried out by one-way
ANOVA using SPSS 13. 0 software． And data
presented were mean ± SEM of three experiments．
When the ANOVA was significant at P ＜ 0. 05，the
Duncan multiple range test was used for mean
comparison． Correlations between pigment contents and
various functions using R，G and B values or foliar
constituent concentrations were studied by multiple
linear regression． In the analysis， the regression
coefficients were showed in the equations．
2 Results
2. 1 Foliar pigments contents
All of the 5 individual trees of Sapium sebiferum
changed color from green to yellow，orange，red or
purple during senescence ( Fig． 1 ) ． The contents of
foliar pigments including chlorophyll，carotenoid and
anthocyanins in different color leaves were shown in
Tab． 1．
Tab． 1 Pigments，nitrogen and carbohydrates contents in different color leaves of S． sebiferum①
Constituent
Leaves
Green Yellow Orange Red Purple
Chlorophyll a + b /(mg·g － 1 FW) 1. 99 ± 0. 12a 0. 15 ± 0. 01c 0. 10 ± 0. 03c 0. 14 ± 0. 01c 0. 64 ± 0. 01b
Chlorophyll a / b 3. 13 ± 0. 43b 1. 22 ± 0. 21c 1. 30 ± 0. 06c 4. 82 ± 0. 97a 1. 87 ± 0. 76c
Carotenoid /(mg·g － 1 FW) 0. 35 ± 0. 03a 0. 29 ± 0. 01a 0. 31 ± 0. 01a 0. 30 ± 0. 01a 0. 32 ± 0. 02a
Anthocyanin /( units·g － 1 FW) 6. 04 ± 0. 15d 3. 68 ± 0. 10e 18. 05 ± 1. 35c 20. 95 ± 0. 60b 41. 95 ± 1. 16a
Nitrogen /(μg·g － 1 DW) 208. 02 ± 3. 27a 72. 29 ± 1. 21d 98. 49 ± 3. 39c 135. 79 ± 1. 46b 143. 35 ± 4. 04b
Glucose /(mg·g － 1 DW) 1. 78 ± 0. 31a 0. 60 ± 0. 11bc 1. 43 ± 0. 49ab 0. 53 ± 0. 11bc 0. 48 ± 0. 04c
Fructose /(mg·g － 1 DW) 4. 39 ± 0. 75a 1. 72 ± 0. 12b 4. 04 ± 0. 27a 3. 72 ± 0. 13a 3. 30 ± 0. 09a
Sucrose /(mg·g － 1 DW) 3. 22 ± 0. 18bc 2. 55 ± 0. 25c 3. 79 ± 0. 25ab 2. 90 ± 0. 28c 4. 13 ± 0. 16a
Starch /(mg·g － 1 DW) 1. 70 ± 0. 06a 0. 11 ± 0. 03b 0. 22 ± 0. 03b 0. 14 ± 0. 03b 0. 17 ± 0. 06b
TNC /(mg·g － 1 DW) 11. 09 ± 0. 66a 4. 98 ± 0. 30d 9. 48 ± 0. 49b 7. 28 ± 0. 22c 8. 08 ± 0. 15c
①TNC is the total non-structural carbohydrates，which comes from the sum of glucose，fructose，sucrose and starch． The significant levels of
difference among the samples were indicated by different letters for P ＜ 0. 05，respectively． Each value is the mean ± SEM of 3 replicates．
Chlorophyll a is the main green pigment，which
directly partakes in photosynthesis，while chlorophyll b
and carotenoid are the accessory pigments． In the
green leaves，chlorophyll a + b contents was 1. 99 mg·
g － 1 FW，which was much higher than those in other
colorized leaves． However， there was no significant
difference in carotenoid contents among the five
individual trees． Furthermore， the anthocyanins
concentration was determined in our studies to gain
better understand of the coloration mechanism of
autumn leaves of Sapium sebiferum． It showed that the
content of anthocyanins was significantly different in
the five individual trees ( P ＜ 0. 05 ) ． The highest
concentration of anthocyanins was 41. 95 units·g － 1 FW
in the purple leaves，and the orange and red leaves
were intermediate (18. 05 and 20. 95 units·g － 1 FW，
respectively) ． The anthocyanins contents in the green
and yellow leaves were significantly lower than those in
purple，orange and red leaves，especially in yellow
leaves，the concentration of anthocyanins was reduced
by 91. 23% as compared with purple leaves．
2. 2 Foliar nitrogen content
The content of nitrogen in different colored leaves
was presented in Tab． 1． It showed significant
variations in leaf nitrogen content in the five individual
trees． The total nitrogen content in green leaves was
the highest，which was 2. 88-fold higher than that in
the yellow leaves，and about twice higher than that in
the orange leaves． In contrast， there were no
significant differences in nitrogen concentrations
between the red and purple leaves．
2. 3 Foliar carbohydrates concentrations
To find out whether the changes of carbohydrates
influenced the development of autumn coloration in
leaves of Sapium sebiferum during senescence， the
contents of glucose，fructose，sucrose and starch were
determined in different color leaves． It indicated that
the concentrations of glucose were the highest in the
green leaves and lowest in the purple leaves and there
was significant difference between them ( Tab． 1 ) ．
53
林 业 科 学 49 卷
Moreover，the fructose content in the yellow leaves was
1. 72 mg·g － 1 DW，which was significantly lower than
those in other color leaves (P ＜ 0. 05) ． Additionally，
higher accumulation of sucrose in the purple leaves was
observed as compared with other color leaves． Starch，
another important carbohydrate，was also disparate in
different color leaves． The concentration of starch was
the highest in the green leaves，which was 15. 45-fold，
7. 73-fold，12. 14-fold and 10. 00-fold higher than that
in yellow， orange， red and purple leaves，
respectively． However， there were no significant
differences in starch contents among the yellow，
orange，red and purple leaves． As for the total non-
structural carbohydrate ( TNC )， the highest
concentration was found in the green leaves，while the
lowest content was present in the yellow leaves．
2. 4 Foliar color analysis by R，G and B values
Leaf color was quantified by digital imaging
analysis and the R ( red)，G ( green) and B ( blue)
values were presented in Tab． 2． The linear regression
analysis was conducted to investigate the relationship
between these data and various foliar pigments
contents． Correlations between various functions
derived from the R，G and B values and the parameters
of foliar pigments including Chl a，Chl b，Chl a / b，
Chl a + b，carotenoid and anthocyanins were examined
(Tab． 3 ) ． It revealed that there was a significant
negative relationship between R /( R + G + B ) and
chl b content with a correlation coefficient of － 0. 96．
It suggests that R /( R + G + B )， the red value
normalized by the sum of the three channels，may be
used as a good indicator to predict chlorophyll b
content in various color leaves of Sapium sebiferum． In
contrast，a significant positive relationship was found
between R /(G － B) and anthocyanins concentration in
the leaves ( r = 0. 92 ) ． Moreover，a strong positive
relationship was observed between (G － B) /(R － B)
and chl a content，G /( R － B ) and chl b content，
G /(R － B) and Chl a + b with the correlation
coefficients of 0. 90，0. 95 and 0. 90， respectively．
However， a significant negative relationship was
present in R /(G + B) and chl b，R /(R + G + B) and
Chl a + b，and the correlation coefficients were － 0. 95
and － 0. 90，respectively．
Tab． 2 Foliar color analysis of different color leaves in
S． sebiferum①
Color of
autumn leaves
Leaf color parameter
R G B
Green 195. 57 ± 1. 41c 211. 73 ± 1． 23a 181. 25 ± 2． 05a
Yellow 231. 88 ± 0. 59a 224. 69 ± 2． 64a 158. 54 ± 8． 58b
Orange 228. 26 ± 2. 16a 187. 31 ± 5． 43b 163. 01 ± 4． 82ab
Red 209. 10 ± 3. 90b 171. 70 ± 11． 06c 164. 75 ± 11． 53ab
Purple 197. 85 ± 0. 57c 186. 99 ± 2． 45b 182. 15 ± 2． 42a
① RGB image analysis of different color leaves harvested from 5
superior individual trees were done． The significant levels of difference
among the samples were indicated by different letters for P ＜ 0. 05，
respectively． Data were presented as mean ± SEM of 3 replicates．
Tab． 3 Correlation coefficients between various functions using R，G and B values and pigment contents
Chl a Chl b Chl a / b Chl a + b Carotenoid Anthocyanin
R － 0. 70 － 0. 75 － 0. 52 － 0. 72 － 0. 64 － 0. 42
G 0. 35 0. 36 － 0. 52 0. 35 0. 12 － 0. 68
B 0. 74 0. 85 0. 15 0. 77 0. 77 0. 46
G /R 0. 83 0. 87 － 0. 13 0. 84 0. 59 － 0. 35
G /B － 0. 09 － 0. 13 － 0. 52 － 0. 10 － 0. 31 － 0. 76
R /B － 0. 73 － 0. 80 － 0. 37 － 0. 75 － 0. 72 － 0. 45
G /(R + G + B) 0. 48 0. 49 － 0. 39 0. 49 0. 21 － 0. 66
R /(R + G + B) － 0. 88 － 0. 96 － 0. 10 － 0. 90 － 0. 72 － 0. 07
B /(R + G + B) 0. 47 0. 55 0. 46 0. 49 0. 56 0. 68
R － G － 0. 82 － 0. 87 0. 10 － 0. 83 － 0. 56 0. 31
R － B － 0. 73 － 0. 80 － 0. 38 － 0. 75 － 0. 70 － 0. 44
G － B － 0. 03 － 0. 06 － 0. 51 － 0. 04 － 0. 24 － 0. 79
(R － G) /(G + R + B) － 0. 80 － 0. 86 0. 15 － 0. 82 － 0. 56 0. 32
(R － B) /(G + R + B) － 0. 76 － 0. 84 － 0. 31 － 0. 78 － 0. 72 － 0. 41
(G － B) /(G + R + B) － 0. 02 － 0. 06 － 0. 61 － 0. 03 － 0. 23 － 0. 80
(G － B) /(R + B) － 0. 01 － 0. 05 － 0. 50 － 0. 02 － 0. 23 － 0. 80
(G － B) /(R － B) 0. 90 0. 84 0. 02 0. 89 0. 71 － 0. 61
(G － B) /(R + B) － 0. 01 － 0. 05 － 0. 50 － 0. 02 － 0. 23 － 0. 80
(G － B) /(R + G) － 0. 01 － 0. 03 － 0. 50 0. 004 － 0. 20 － 0. 81
R /(G + B) － 0. 86 － 0. 95 － 0. 10 － 0. 89 － 0. 71 － 0. 08
R /(G － B) － 0. 22 － 0. 09 0. 35 － 0. 19 － 0. 14 0. 92
G /(R + B) 0. 48 0. 49 － 0. 38 0. 48 0. 20 － 0. 67
G /(R － B) 0. 87 0. 95 0. 15 0. 90 0. 82 0. 21
B /(R + G) 0. 47 0. 60 0. 45 0. 49 0. 56 0. 69
B /(R － G) － 0. 61 － 0. 49 － 0. 53 － 0. 58 － 0. 75 － 0. 01
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第 11 期 黄利斌等: 乌桕秋叶显色的生理生化与数字图像分析
2. 5 Relationship between leaf constituents and
pigment contents
To gain better understanding of the relationship
between leaf constituents and foliar pigments，nitrogen
and carbohydrates concentrations were analyzed against
Chl a，Chl b，Chl a + b，carotenoid and anthocyanins．
As shown in Tab． 4，the results of correlation analysis
revealed that nitrogen and starch concentrations showed
significant positive correlation with Chl a，Chl b，Chl a
+ b and carotenoid ( r ＞ 0. 8) ． There was a less strong
correlation between fructose concentration and
carotenoid， and sucrose and anthocyanins． The
correlation coefficients were 0. 747 and 0. 77，
respectively． We also observed a significant positive
correlation between the TNC and carotenoid ( r =
0. 93) ． In contrast， weak negative correlation was
found between glucose concentration and anthocyanins
( r = － 0. 48 )， and starch concentration and
anthocyanins ( r = － 0. 42) ．
Tab． 4 Correlation coefficients between total nitrogen or
carbohydrates and pigment contents
Chl a Chl b Chl a + b Carotenoid Anthocyanin
Nitrogen 0. 89 0. 86 0. 90 0. 82 0. 07
Glucose 0. 67 0. 56 0. 64 0. 81 － 0. 48
Fructose 0. 51 0. 48 0. 50 0. 747 0. 17
Sucrose 0. 06 0. 22 0. 10 0. 46 0. 77
Starch 0. 97 0. 89 0. 96 0. 87 － 0. 42
TNC 0. 71 0. 67 0. 71 0. 93 0. 04
2. 6 Chloroplast ultrastructure
The ultrastructure of mesophyll cells of different
color leaves，especially the differences in chloroplasts，
was examined by transmission electron microscopy
(Fig. 2 ) ． The architecture of green leaf mesophyll
cells appeared to be intact with numerous chloroplasts
and other organelles such as mitochondria and vacuoles
( Fig. 2A， B ) ． Chloroplasts showed the typical
architecture with thylakoid membranes arranged in
granum and stroma lamellae ( Fig. 2C )， and large
starch grains and several osmiophilic granules were
visible within chloroplasts ( Fig. 2B ) ． In contrast，
almost no organelles could be observed in mesophyll
cells of yellow leaves，and some degrading chloroplasts
were found occasionally (Fig. 2D) ． The chloroplasts in
orange and red leaves were found to be abnormal，and
the microscopic data could not reveal obvious
ultrastructural differences between them (Fig. 2E，F) ．
The irregular or degraded chloroplasts were distributed
at the edge of the inside cell wall，and many of them
contained several osmiophilic granules． In the palisade
tissue cells of purple leaves，the number of chloroplasts
were reduced，whose membranes disintegrated， and
abundant fragments were found within the chloroplasts
( Fig. 2G ) ． Concurrently， considerable larger
osmiophilie globules，which might represent reserve
materials，were found in chloroplast and cytoplasm
(Fig. 2G) ． Rare chloroplasts could be found in the
spongy tissue cells，but enormous starch grains were
present in the cytoplasm (Fig. 2H) ．
2. 7 Distribution of pigments in different
color leaves
In order to determine the subcellular localization
of chlorophyll， carotenoid and anthocyanins， leaf
tissues were examined using confocal microscopy． As
seen in PlateⅡ，mesophyll chloroplasts showed red
autofluorescence． Confocal microscopy observations
revealed that in the green leaves both the palisade
tissue and spongy parenchyma contained large numbers
of chloroplasts exhibiting strong chlorophyll
fluorescence (Plate ⅠA) ． In contrast，the intensity of
the chlorophyll fluorescence was decreased in the
yellow (Plate ⅠB)，orange (Plate ⅠC)，red (Plate
ⅠD) and purple ( Plate ⅠE) leaves． Especially in
the yellow leaves， only very few chloroplasts were
found in the spongy parenchyma． Moreover， in the
orange leaves the palisade tissue contained more
chloroplasts than that in the spongy parenchyma (Plate
ⅠC) ． However，in the red and purple leaves，the
chloroplasts were mainly localized in the spongy
parenchyma (Plate ⅠD) ． In contrast to chlorophyll，
carotenoid was found to be distributed both in the
palisade tissue and spongy parenchyma in the five
different colored leaves ( Plate Ⅱ) ． Furthermore，the
fluorescence intensity in different colored leaves was
similar．
Using the autofluorescence of anthocyanins，we
observed numerous spherical vesicles in leaf mesophyll
cells，especially in the red and purple leaves ( Plate
Ⅲ) ． In the green leaves，only faint red fluorescence
was found in the palisade tissue， whereas， some
intensely red colored structures filled with anthocyanins
were present in the spongy parenchyma ( Plate ⅢA) ．
73
林 业 科 学 49 卷
Fig． 2 Transmission electron micrographs of Sapium sebiferum leaf mesophyll cells showing
chloroplast structure and arrangement
A. The cell of palisade tissue in green leaves; B. The spongy tissue cells of green leaves; C. Magnification of chloroplast; D. The ultrastructure of
mesophyll cells of yellow leaves; E. The deformation or degradation of chloroplasts in the orange leaf cells; F. The cellular structures in red leaves;
G. The cells of palisade tissue in purple leaves; H. The spongy tissue cells of the purple leaves． Chl． Chloroplasts; CW. Cell wall; GR. Thylakoid grana;
M. Mitochondria; OG. Osmiophilic granules; S. Starch grain; V. Vacuole; IS． Intercellular space．
In the yellow and orange leaves，only a small number
of vesicles were noticed in the spongy parenchyma
( Plate ⅢB， C ) ． These colored vesicles can be
classified into two groups according to the size as the
83
第 11 期 黄利斌等: 乌桕秋叶显色的生理生化与数字图像分析
previous report，which are Group I (GI) corresponds
to small particles，and Group II (GII) corresponds to
larger spherical vesicles． Unlike the yellow and orange
leaves，a great deal of vesicles was observed both in
the palisade tissue and spongy parenchyma in red and
purple leaves (Plate ⅢD － G) ． The GI structure was
found in the palisade tissue，while the GII structure
was localized in the spongy parenchyma．
3 Discussion
Pigments are the foundation of the variation of leaf
color． Autumn coloration was directly related to the
categories and amounts of pigments． Chlorophylls are
the most important pigments found in chloroplasts，and
chlorophyll contents were dramatically higher in green
leaves than other color leaves ( Tab． 1 ) ． Carotenoid，
as the“accessory”pigments of chlorophylls，was more
stable during senescence，and its contents were similar
in various leaves in our study． With declining autumn
temperatures， the leaves of Sapium sebiferum stop
producing chlorophyll，and in the yellow leaves the
chlorophyll breakdown unmasked the yellow carotenoid
pigments resulting in clear-yellow colored leaves (Field
et al．，2001 ) ． Unlike the yellow leaves，most red
leaves resulted from abundant synthesis of
anthocyanins． Anthocyanins contents varied in different
color leaves． There may be an admixture of
decomposed chlorophyll， yellow carotenoid and red
anthocyanins pigments to give the leaves various shades
of orange， red or purple． In addition， correlation
analyses showed a significant negative correlation
between chlorophyll a + b contents and R /(R + G + B)
(Tab. 3)， and there was a markedly positive
correlation between anthocyanins contents and
R / (G － B) ． These algorithms may be used as an
indicator to estimate the leaf color by digital imaging
analysis，which could provide quantitative expression
of color and be consistent with human color sensation
(Laliberte et al．，2007) ．
In this study，N level was the highest in the green
leaves，and there was a negative correlation between
Chl a + b and N concentrations ( r = 0. 90) ． Much of
the N in the leaves was translocated back to the spurs
in the early autumn before leaf falling ( Stephen et al．，
2007)， but after leaves turned color， the nitrogen
concentration of the red and purple leaves was
significantly higher than those of the yellow leaves，
which indicated that red leaves may have a greater
capacity for nutrient resorption than senescing yellow
leaves during autumn (Schaberg et al．，2003) ．
Analyses of leaf carbohydrates concentrations in
our study showed that: 1 ) the yellow leaves fixed a
little amount of TNC than other color leaves; 2 ) a
higher accumulation of sucrose was found in the purple
leaves， and the correlation analysis indicated that
sucrose contributed to the biosynthesis of anthocyanins;
3) there was a significant positive correlation between
chl a and starch ( r = 0. 97 ) ． As is known， the
anthocyanins pigments are related to the carbohydrates
and carbohydrate accumulation favors their formation
( Stephen et al．， 2007 ) ． The contents of stored
carbohydrates varied in different color leaves and
changed seasonally as well (Hughes et al．，2005) ． It
was suggested that trees of the same species growing
together showed much difference in color might be due
to variations in the amounts of soluble carbohydrates
among individual trees ( Stephen et al．， 2007 ) ．
Pigment contents，total N and TNC concentrations in
yellow leaves were lower than other color leaves． The
pigment contents were strongly correlated with
photosynthetic capacity，thus the lower chlorophyll and
anthocyanins contents in the yellow leaves may reflect
its lower nitrogen and TNC concentrations (Kytridis et
al．，2008) ． Among the soluble carbohydrates，sucrose
is the primary transportable and storage carbohydrate．
We assumed that the synthesis of carbohydrates or the
conversion of insoluble to soluble carbohydrates，
especially sucrose might favor anthocyanins formation
and bright autumn colors in Sapium sebiferum． Starch
is considered the most important reserve carbohydrate
and has often been used as the sole indicator of the
carbohydrate status of plants． Starch would convert to
sugars when sugar contents were low or at low
temperature (Kozlowski，1992)，which could explain
the correlativity of starch and chlorophyll．
Electron microscopic studies of different color
autumn leaves of Chinese tallow tree revealed various
pictures showing the differences in quantity，shape and
structure of chloroplasts，as well as some contents such
as starch grains and osmiophilic granules． Senescence
93
林 业 科 学 49 卷
of Chinese tallow tree in autumn progressed the
chloroplast disaggregated seriously， and the drastic
decrease in the number of chloroplasts led to reduction
in chlorophyll concentration． Swelling of chloroplast，
membrane reduction and accumulation of osmiophilic
granules in orange， red and purple leaves were
phenomena that were also observed in other plant
leaves under many other stresses，such as ultraviolet-B
( Peng et al．， 2009 )， cadmium pollution ( Baryla
et al．，2001)，freezing (Bourett et al．，1999) and low
temperature (Holzingerl et al．，2007 ) ． Furthermore，
we found starch grains were abundant in the green and
purple leaves，but the cells in the yellow，orange or
red leaves contained only negligible amounts of starch
grains． The synthesis of assimilation starch ceased may
in favor of the formation of various sugars ( Sensor et
al．，1975) ．
Consistent with the TEM results，the amount of
chloroplasts in yellow，orange，red and purple leaves
detected by laser confocal scanning microscopy was
apparently less than green leaves． Especially in the
yellow and orange leaves it displayed very feeble
fluorescence of chlorophyll， which may indicate
decreased photosynthetic capacity in autumn．
Moreover，the laser confocal microscopic observation
revealed an asymmetric distribution of chlorophyll in
the palisade tissue and spongy parenchyma in different
color leaves． These results suggested chlorophylls have
a specific cellular or subcellular location in different
individual trees (Modzińska，2009) ． In contrast，the
fluorescence intensity and subcellular location of
carotenoid was similar in different color leaves． The
confocal microscopy analysis of leaf tissues showed
different patterns of anthocyanins compartmentation． In
consistent with the observations of Gomez et al．
(2011)，we found that anthocyanins were present in
two types of structures，differing mainly by the size．
The authors suggested that GI and GII vesicles were
membrane surrounded structures，and the larger GII
vesicles were formed by the fusion of the small size GI
structures．
In summary，in the present study we utilized a
quantitative method，digital color analysis，to explore
autumnal leaf colorization of the woody plant S．
sebiferum． Based on the RGB image analysis， foliar
pigments of different color leaves can be quantitatively
determined by measuring image ( G － B ) /( R-B )，
R /(R + G + B) or R /( G － B ) value． Digital color
analysis provides a simple and fast tool to estimate leaf
colorization in comparison to traditional visual scale
method，and the former is more accurate than the
latter． Moreover， the physiological analysis also
provides some clues for autumnal leaf colorization of S．
sebiferum． However，autumn coloration may represent
an integration of genetic potential，structural support
and environmental stresses that influence leaf
senescence and anthocyanins biosynthesis． Further
study regarding the molecular mechanisms regulating
the induction and progression of autumn leaf coloration
as well as the biochemical and cell biological details
involved in the pathways of pigments catabolism should
be conducted． This could be useful to the selective-
breeding of improved cultivars with better ornamental
value．
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(责任编辑 王艳娜 郭广荣)
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