Planta (2009) 230:1185–1196
DOI 10.1007/s00425-009-1016-1
O R I G I N A L A R T I CL E
Brassinosteroids promote photosynthesis and growth
by enhancing activation of Rubisco and expression
of photosynthetic genes in Cucumis sativus
Xiao-Jian Xia · Li-Feng Huang · Yan-Hong Zhou ·
Wei-Hua Mao · Kai Shi · Jian-Xiang Wu ·
Tadao Asami · Zhixiang Chen · Jing-Quan Yu
Received: 29 May 2009 / Accepted: 4 September 2009 / Published online: 17 September 2009
© Springer-Verlag 2009
Abstract Brassinosteroids (BRs) are a new group of plant
growth substances that promote plant growth and produc-
tivity. We showed in this study that improved growth of
cucumber (Cucumis sativus) plants after treatment with 24-
epibrassinolide (EBR), an active BR, was associated with
increased CO2 assimilation and quantum yield of PSII
(
PSII). Treatment of brassinazole (Brz), a speciWc inhibitor
for BR biosynthesis, reduced plant growth and at the same
time decreased CO2 assimilation and
PSII. Thus, the
growth-promoting activity of BRs can be, at least partly,
attributed to enhanced plant photosynthesis. To understand
how BRs enhance photosynthesis, we have analyzed the
X.-J. Xia · L.-F. Huang · Y.-H. Zhou · W.-H. Mao · K. Shi ·
J.-Q. Yu (&)
Department of Horticulture, Huajiachi Campus,
Zhejiang University, Kaixuan Road 268, 310029 Hangzhou,
People’s Republic of China
e-mail: jqyu@zju.edu.cn
J.-X. Wu
Institute of Biotechnology, Zhejiang University,
Kaixuan Road 268, 310029 Hangzhou,
People’s Republic of China
T. Asami
Department of Applied Biological Chemistry,
University of Tokyo, Bunkyo Ku, Tokyo 1138657, Japan
Z. Chen
Department of Botany and Plant Pathology,
Purdue University, West Lafayette, IN 47907-2054, USA
J.-Q. Yu
Key Laboratory of Horticultural Plant Growth,
Development and Biotechnology,
Agricultural Ministry of China, Kaixuan Road 268,
310029 Hangzhou, People’s Republic of China
eVects of EBR and Brz on a number of photosynthetic
parameters and their aVecting factors, including the con-
tents and activity of ribulose-1,5-bisphosphate carboxylase/
oxygenase (Rubisco). Northern and Western blotting dem-
onstrated that EBR upregulated, while Brz downregulated,
the expressions of rbcL, rbcS and other photosynthetic
genes. In addition, EBR had a positive eVect on the activa-
tion of Rubisco based on increased maximum Rubisco car-
boxylation rates (Vc,max), total Rubisco activity and, to a
greater extent, initial Rubisco activity. The accumulation
patterns of Rubisco activase (RCA) based on immunogold-
labeling experiments suggested a role of RCA in BR-regu-
lated activation state of Rubisco. Enhanced expression of
genes encoding other Calvin cycle genes after EBR treat-
ment may also play a positive role in RuBP regeneration
(Jmax), thereby increasing maximum carboxylation rate of
Rubisco (Vc,max). Thus, BRs promote photosynthesis and
growth by positively regulating synthesis and activation of
a variety of photosynthetic enzymes including Rubisco in
cucumber.
Keywords Cucumber (Cucumis sativus) · Immunogold
labeling · Productivity · Rubisco activase · Sugar
metabolism
Abbreviations
Asat Light-saturated rate of CO2 assimilation
BRs Brassinosteroids
Brz Brassinazole
Chl Chlorophyll
Ci Intercellular CO2 concentration
EBR 24-Epibrassinolide
FBP Fructose-1,6-bisphosphate
FBPA Fructose-1,6-bisphosphate aldolase
FBPase Fructose-1,6-bisphosphatase
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1186 Planta (2009) 230:1185–1196

Fru6P Fructose 6-phosphate Yu et al. 2004). Moreover, BRs modulate plant response to
Fv/Fm Maximum quantum yield of PSII environmental stress and pathogen infection (Dhaubhadel
Fv⬘/Fm⬘ EYciency of energy capture by open PSII et al. 2002; Krishna 2003; Nakashita et al. 2003). Notably,
Gs Stomatal conductance application of BRs increases the yield of many crops
Jmax Maximum RuBP regeneration rates (Khripach et al. 2000). The yield-promoting activity of BRs
PRK Ribulose-5-phosphate kinase is consistent with their growth-promoting activity demon-
qP Photochemical quenching coeYcient strated in Arabidopsis. For example, in transgenic Arabid-
rbcL Rubisco large subunit gene opsis overexpressing DWF4, an overall increase in BR
rbcS Rubisco small subunit gene level results in increased per-plot seed yield (Choe et al.
rca Rubisco activase gene 2001), whereas an Arabidopsis mutant defective in sterol
Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase 7 reductase is deWcient in BR biosynthesis and produces
RuBP Ribulose-1,5-bisphosphate aberrantly shaped seeds (Choe et al. 2000). There is a
RuPE Ribulose phosphate epimerase promising prospect to increase crop yield and food produc-
SBP Sedoheptulose-1,7-bisphosphate tion through application of BR-derived growth-promoting
SBPase Sedoheptulose-1,7-bisphosphatase substances in modern agrosystem (Wu et al. 2008).
Sed7P Sedoheptulose 7-phosphate BRs promote plant growth likely through multiple mech-
TPI Triose-3-phosphate isomerase anisms. A number of studies have implicated BRs in the
Vc,max Maximum Rubisco carboxylation rates regulation of cell division and expansion (Hu et al. 2000;

PSII Quantum yield of PSII Catterou et al. 2001). Moreover, inhibition of BR biosyn-
thesis by the addition of brassinazole (Brz) inhibits the
growth of light-grown Chlorella vulgaris cells (Bajguz and
Introduction Asami 2004). It has been reported that BRs increase leaf
photosynthesis and this activity of BRs may contribute to
Since brassinolide (BL) was Wrst isolated from Brassicus increased crop yield after BR application (Yu et al. 2004).
napus pollen 30 years ago (Grove et al. 1979), approxi- It has been recently shown that tissue-speciWc overexpres-
mately 60 related compounds have been identiWed, which sion of sterol C-22 hydroxylases in stems, leaves and roots,
are collectively referred to as brassinosteroids (BRs). BRs but not in the embryos or endosperms, results in 15–44%
are essential for normal plant growth, reproduction and increase in grain yield per plant (Wu et al. 2008). Further
development. Plants impaired in biosynthesis, perception or analysis suggests that enhanced CO2 assimilation and
signaling of BRs exhibit the typical phenotypes of dark enlarged sugar pools in the Xag leaves may stimulate assim-
green dwarfs with epinastic leaves, reduced or abolished ilate Xow to the seeds and increase grain yields.
fertility and delayed development (Bishop and Koncz Increased rates of CO2 assimilation have been observed
2002). In Arabidopsis, the identiWcation of BR-deWcient in plants with increased BR levels through BR applications
mutants led to the cloning of several critical genes involved (Hayat et al. 2000; Yu et al. 2004). However, it is diYcult
in BR biosynthesis, such as constitutive photomorphogene- to determine whether reduced BR biosynthesis or signaling
sis and dwarWsm (CPD; Szekeres et al. 1996), dwarf4 has a corresponding negative eVect on CO2 assimilation
(DWF4; Choe et al. 1998) and deetiolated2 (DET2; Li et al. due to the strong and pleiotropic phenotypes of BR-deW-
1996). The CPD and DWF4 genes encode cytochrome cient or signaling mutants in Arabidopsis (Müssig et al.
P450 monooxygenases. Feeding experiments with BR bio- 2002). Alternatively, BR synthesis mutations can be pheno-
synthetic intermediates revealed that the C-22 and C-23 copied by exogenously applied Brz, a speciWc inhibitor of
positions of BRs are successively hydroxylated by DWF4 BR biosynthesis (Asami et al. 2000). Brz directly binds to
and CPD, respectively. The DET2 gene encodes a 5
-ste- DWF4 protein, inhibits the hydroxylation of the C-22 posi-
roid reductase, which catalyzes the Wrst step in the BR bio- tion of BR biosynthetic intermediates and eVectively
synthesis pathway (Noguchi et al. 1999). The BR-deWcient reduces the BR content in plants (Asami et al. 2001). By
Arabidopsis mutant, bul1, is defective in the 7-sterol-C5- using Brz, we can reduce the BR content within a short
desaturation step in the phytosterol pathway (Catterou et al. period of time without causing strong pleiotropic pheno-
2001). To date, the molecular genetics coupled with bio- types in cucumber. In the absence of secondary eVects of
chemical studies has established the BR biosynthetic path- strong pleiotropic phenotypes, we can determine whether
way (Yokota 1997; Bishop 2007). BR is directly involved in the regulation of photosynthesis.
Like other plant hormones, BRs aVect many physiologi- In addition, we have previously demonstrated that BR
cal processes, including cell expansion, cell division, xylem application induced an increase in ribulose-1,5-bisphos-
diVerentiation, proton-pump activity, ethylene biosynthesis phate carboxylase/oxygenase (Rubisco) activation state
and photosynthesis (Sasse 1997; Clouse and Sasse 1998; (Yu et al. 2004). The underlying mechanism for

123
Planta (2009) 230:1185–1196
BR-induced activation of Rubisco is unknown. To address
these issues, we analyzed the content of RCA, which is inti-
mately involved in regulation of Rubisco activity and the
expression of several genes involved in the Calvin cycle.
Our results indicate that BRs play an important role in regu-
lating synthesis and activation of a variety of enzymes in
the photosynthetic apparatus.
Materials and methods
Plant materials and experimental design
Cucumber (Cucumis sativus L. cv. Jinyan No. 4, Dandong
Shengyuan Agriculture Co., Ltd., Liaoning, China) seeds
were sown in a growth medium containing a mixture of soil
and perlite (1:1, v/v) in a growth chamber with a photope-
riod of 12 h, temperature of 25/17°C (day/night), and light
intensity of 600 mol m¡2 s¡1. When the cotyledons fully
expanded, groups of six plants were transplanted into a
container (40 cm £ 25 cm £ 15 cm) Wlled with Hoagland
nutrient solution. These plants were used for the following
two types of experiments.
To investigate the roles of BR in plant growth, cucumber
plants were divided into four groups and subjected to treat-
ment of 24-epibrassinolide (EBR), Brz, or both. Since the
EBR stock solution was made in ethanol, but the Brz stock
solution was made in DMSO, we included a very low level
of ethanol (0.01%, v/v) and DMSO (0.01%, v/v) in all our
working solutions including control water solution. Group I
control plants were treated with water. Group II plants were
treated with 0.1 M EBR every 5 days, from the cotyledon
stage to the four-leaf stage. Group III plants were treated
with 4 M Brz, a speciWc inhibitor of BR biosynthesis,
every 2 days from the cotyledon stage to the four-leaf stage.
Group IV plants were treated with 4 M Brz plus 0.1 M
EBR to test whether the eVects of Brz could be rescued by
EBR. Plants were harvested at 25 days for biomass and leaf
area analysis.
To examine the roles of BR in the regulation of photo-
synthesis, the third leaves of the plants at the four-leaf stage
were sprayed with water, 0.1 M EBR every 5 days, 4 M
Brz every 2 days or 4 M Brz plus 0.1 M EBR. Net CO2
assimilation rate and chlorophyll Xuorescence were mea-
sured at intervals. Leaf samples were harvested at 5 days
after EBR or Brz treatment, frozen immediately in liquid
nitrogen and stored at ¡80°C before biochemical and gene
expression analyses.
Gas exchange and chlorophyll Xuorescence measurements
Gas exchange analysis was conducted using an infrared gas
analyzer (CIRAS-1, PP System, Herts., UK) on the third
1187
leaf of each plant. Light-saturated rate of CO2 assimilation
(Asat) was measured at ambient CO2 concentration of
360 mol mol¡1 and saturating photosynthetic photo Xux
density (1,000 mol m¡2 s¡1) with a leaf temperature of
25 § 1.5°C and air relative humidity of 80–90%. Assimila-
tion versus intercellular CO2 concentration (A/Ci) curve
was measured according to von Caemmerer and Farquhar
(1981). Assimilation was Wrst measured at ambient CO2
concentration in which the plants had grown. Atmospheric
CO2 concentration was decreased in several steps to
50 mol mol¡1 and then returned to the growth concentra-
tion to check that the original rate could be regained and
was then Wnally increased stepwise to 2,000 mol mol¡1 to
complete the response curve. The maximum Rubisco car-
boxylation rates (Vc,max) and maximum ribulose-1,5-bis-
phosphate (RuBP) regeneration rates (Jmax) were estimated
from the A/Ci curves using the method of Ethier and
Livingston (2004).
A pulse-modulated Xuorometer (FMS-2, Hansatech
Instruments Ltd., Norfolk, UK) was used to determine the
chlorophyll Xuorescence parameters (Zhou et al. 2004).
Minimal Xuorescence (Fo) was measured under a weak
pulse of modulating light over a 0.8 s period, and maximal
Xuorescence (Fm) was induced by a saturating pulse of
light (4,000 mol m¡2 s¡1) applied over 0.8 s. The maxi-
mum quantum yield of PSII (
PSII) was determined as
Fv/Fm, where Fv is the diVerence between Fo and Fm. An
actinic light source (600 mol m¡2 s¡1) was then applied to
obtain Fs (steady-state Xuorescence yield), after which a
second saturation pulse was applied for 0.7 s to obtain Fm⬘
(light-adapted maximum Xuorescence). Minimal Xuores-
cence in the light (Fo⬘) was measured when the actinic light
was turned oV in the presence of far-red light. The quantum
yield of PSII (
PSII) and the eYciency of excitation capture
by open PSII centers at a steady state (Fv⬘/Fm⬘) was deW-
ned as (Fm⬘ ¡ Fs)/Fm⬘ and (Fm⬘ ¡ Fo⬘)/Fm⬘ (Genty et al.
1989). Photochemical quenching coeYcient (qP) was cal-
culated as (Fm⬘ ¡ Fs)/(Fm⬘ ¡ Fo⬘) (van Kooten and Snel
1990).
Measurement of total chlorophyll, soluble proteins
and carbohydrate content
Total chlorophyll content was determined by the method of
Arnon (1949). Total soluble protein content was measured
using Bradford reagent (Bradford 1976). Freeze-dried sam-
ples were used for the determination of carbohydrate con-
tent. Sucrose, starch and hexose contents were determined
using a modiWed phenol–sulfuric acid method (Buysse and
Merckx 1993). Soluble sugars were extracted from 200 mg
of dried material with 50 mL of 80% ethanol (v/v), using
Wve extraction steps. The supernatant was analyzed for hex-
ose, sucrose and total soluble sugars. The residue was
123
1188
boiled for 3 h in 10 mL 2% HCl (v/v) to hydrolyze starch.
The supernatant was analyzed for starch content.
Measurement of Rubisco activity
For measurements of Rubisco activity, frozen leaf samples
were ground to a Wne powder in liquid N2 and then
extracted in a solution containing 50 mM Tris–HCl (pH
7.5), 1 mM EDTA, 1 mM MgCl2, 12.5% (v/v) glycerin,
10% PVP and 10 mM -mercaptoethanol. The homogenate
was centrifuged at 15,000g for 15 min at 4°C. Total Rubi-
sco activity was assayed after the crude extract was acti-
vated in a 0.1 mL activation solution containing 33 mM
Tris–HCl (pH 7.5), 0.67 mM EDTA, 33 mM MgCl2 and
10 mM NaHCO3 for 15 min. Initial Rubisco activity was
measured by coupling the activity to NADH oxidation
using PGA kinase and GAP dehydrogenase according to
Lilley and Walker (1974) and Sharkey et al. (1991). The
oxidation of NADH was followed by changes in absor-
bance at 340 nm for 90 s.
Western-blot analysis
Leaf samples were ground in liquid N2 with mortar and pes-
tle. Total proteins were extracted with a buVer containing
50 mM Tris–HCl (pH6.8), 4.5% SDS, 7.5% -mercap-
toethanol and 9 M urea. For Western-blot analysis, proteins
(30 g from each sample) were separated by SDS-PAGE
using 12.5% (w/v) acrylamide gels according to the method
of Laemmli (1970) and electrophoretically transferred to
nitrocellulose membranes (Millipore, Saint-Quentin,
France). The proteins were detected with antibodies raised
against Rubisco activase (RCA), and Rubisco small and
large subunits.
Immunogold labeling of Rubisco activase
Subcellular RCA distribution was analyzed according to Jin
et al. (2006). Leaf segments were Wxed with 100 mM phos-
phate buVer (pH 7.2) containing 3% (v/v) paraformalde-
hyde and 1% (v/v) glutaraldehyde for 2 h at 4°C and
washed in the buVer. The segments were dehydrated in an
ethanol series and embedded in Lowicryl K4M according to
the following protocol: 100% ethanol/resin 1:1 (v/v) for
1 h, 100% ethanol/resin 1:2 (v/v) for 1 h, and pure resin for
12 h at ¡20°C. The embedded samples were polymerized
with UV radiation at ¡20°C for 72 h, followed by 24 h at
room temperature. Ultrathin sections (70–90 nm) were cut
with a diamond knife and placed on nickel grids. The sec-
tions were washed with distilled water for 15 min and then
incubated in blocking buVer containing 50 mM PBS, 1%
(w/v) BSA, 0.02% PEG20000, 0.1 M NaCl and 1% (w/v)
NaN3 for 1 h at room temperature. The sections were then
123
Planta (2009) 230:1185–1196
incubated for 1 h at room temperature with anti-RCA serum
applied at dilutions of 1:500 in blocking buVer. After wash-
ing with blocking buVer, the sections were incubated in
blocking buVer containing protein A conjugated with 15-nm
colloidal gold particles for 1 h, and were then washed in
PBS and in deionized distilled water. Finally, the sections
were observed and photographed with an electron micro-
scope (JEM-1200EX, JEOL Ltd., Tokyo, Japan) at 80 kV.
Total RNA extraction and gene expression analysis
Total RNA was extracted using Trizol according to the sup-
plier’s instruction. Contaminated DNA was removed with
purifying column. As much as 1 g of total RNA was
reverse-transcribed using 0.5 g of Oligo (dT) 12–18 and
200 units of Superscript II (Invitrogen, Hangzhou, China)
following the supplier’s recommendation. For Northern-
blot analysis, 10 g of total RNA was separated in a dena-
turing 1.2% (w/v) agarose gel containing 2% formaldehyde
and blotted onto a Hybond N+ membrane. Blots were
hybridized with 32P-labeled cDNA fragments prepared
from PCR ampliWcation using speciWc primers for Rubisco
activase gene (rca), Rubisco small subunit gene (rbcS) and
Rubisco large subunit gene (rbcL). The primers designed in
function of their conserved sequences are: 5⬘-GCTGAC
AACCCAACCAA-3⬘ and 5⬘-CATCCGACCATCACG
AA-3⬘ for rca; 5⬘-CGCTGTTGCCTCTGTGAA-3⬘ and
5⬘-AGAGTAGAACTTGGGGCTTGTA-3⬘ for rbcS; 5⬘-TA
TGGTCGCCCTCTATTG-3⬘ and 5⬘-GCGGCTTCGGTC
TTTTTC-3⬘ for rbcL. Following hybridization, membranes
were Wrst washed and then scanned and quantiWed by a
Typhoon 8600 (Amersham Pharmacia Biotech, New Jer-
sey, USA). For quantitative RT-PCR analysis, we ampliWed
PCR products in triplicate using iQ SYBR Green SuperMix
(Bio-Rad, Hercules, CA, USA) in 25-L qRT-PCR reac-
tions, an iCycler iQ 96-well real-time PCR detection system
(Bio-Rad), iCycler software to calculate threshold cycle
values and cucumber actin as an internal control. On the
basis of EST sequences, the following gene-speciWc primers
were designed and used for ampliWcation:
CPD, 5⬘-TCTCCGACGAGCAAATAGTG-3⬘ and 5⬘-C
GTCTCCGTCAGAAACTTGA-3⬘; DWF4, 5⬘-GGATG
GAAAGTGCTTCCTGT-3⬘ and 5⬘-GGGATACCCAAT
GATGAACC-3⬘; BR6ox (brassinosteroid-6-oxidase), 5⬘-A
TGAGAGGTGCTCTGCTTGC-3⬘ and 5⬘-TAGATGAG
CGGAGAGCCATC-3⬘; DIM (DIMINUTO), 5⬘-TTTGTA
TTGGGAGGGAAAGC-3⬘ and 5⬘-AAACTTTGGGAGG
CATCATC-3⬘; TPI (triose-3-phosphate isomerase), 5⬘-AC
TTCCTGTGCTTGCTCC-3⬘ and 5⬘-AACTTTTGATGTT
TGCTTAAC-3⬘; FBPA (fructose-1,6-bisphosphate aldol-
ase), 5⬘-CTCGTGCTGCTGCTTACT-3⬘ and 5⬘-CTGCCC
AAACTTTCTGTG-3⬘; FBPase (fructose-1,6-bisphospha-
tase), 5⬘-GGGAGAGGACCAGAAAAA-3⬘ and 5⬘-GGC
Planta (2009) 230:1185–1196
TGTAGATGCCAAAGA-3⬘; SBPase (sedoheptulose-1,7-
bisphosphatase), 5⬘-CAGGGTTATCAAATGTGG-3⬘ and
5⬘-GGAGTGAAGGGAAGCGAC-3⬘; RuPE (ribulose
phosphate epimerase), 5⬘-TGGGTTCACTCCCTTTTC-3⬘
and 5⬘-TTGGACCTCTTGTCGTTG-3⬘; PRK (ribulose-5-
phosphate kinase), 5⬘-ACAGGTTGCTTAGATGGC-3⬘
and 5⬘-TTGTTTGATGAAGGCTCG-3⬘; actin, 5⬘-TGGAC
TCTGGTGATGGTGTTA-3⬘ and 5⬘-CAATGAGGGATG
GCTGGAAAA-3⬘. The quantiWcation of mRNA levels is
based on the method of Livak and Schmittgen (2001). The
threshold cycle (Ct) value of actin was subtracted from that
of the gene of interest to obtain a Ct value. The Ct value
of untreated control sample was subtracted from the Ct
value to obtain a Ct value. The fold changes in
expression level relative to the control were expressed as
2¡Ct.
Statistical analysis
Tukey’s test was used for testing the mean diVerence
between treatments by using the SPSS statistical software
package.
Results
EVects of BR levels on plant growth and biomass
accumulation
To examine the eVects of BR on plant growth, we manipu-
lated BR levels in cucumber plants through application of
EBR, a synthetic BR, and Brz, a speciWc BR biosynthesis
inhibitor. EBR application resulted in improved plant
growth as indicated by increased biomass accumulation,
leaf area, and stem and root length (Table 1). However,
EBR had little eVect on total chlorophyll content. Inhibition
of BR biosynthesis by Brz, on the other hand, resulted in a
24% reduction in biomass accumulation. Reduced biomass
after Brz application was a result of reduced growth of all
plant organs as indicated by 46% reduction in leaf area,
22% reduction in stem length and 16% reduction in root
Table 1 EVects of EBR and Brz application on fresh weight, leaf area, leaf mass per unit leaf area, stem and root length and total chlorophyll
content in cucumber plants 25 days after treatments
Fresh weight Leaf area
(g plant¡1) (cm2 plant¡1)
Cont. 12.9 § 0.9b 121.7 § 19.0b
a a
EBR 17.3 § 0.4 170.1 § 6.6
c c
Brz 9.8 § 1.5 66.1 § 10.6
b b
Brz + EBR 13.4 § 2.5 129.3 § 15.7
Data are the means of four replicates with SDs. Means followed by the same letter are not signiWcantly diVerent according to Tukey’s test (P < 0.05)
1189
length relative to those of untreated plants. However, Brz
treatment caused signiWcant increases in leaf mass per
unit leaf area and total chlorophyll content, a characteris-
tic phenotype of BR-deWcient mutants. When EBR was
applied to the Brz-treated plants (Brz + EBR), all the
growth parameters and chlorophyll content were restored
to normal levels.
We also determined the eVects of EBR and Brz on the
expressions of CPD, DWF4, BR6ox and DIM, which are
involved in BR biosynthesis. CPD and DWF4 catalyze the
respective C-23 and C-22 hydroxylation of the steroid side
chain. BR6ox (brassinosteroid-6-oxidase) oxidizes 6-deox-
ocastasterone to castasterone via 6-hydroxycastasterone
and DIM (DIMINUTO) is responsible for the conversion
of 24-methylenecholesterol to campesterol. Figure 1
shows that EBR treatment reduced the amount of CPD,
DWF4, BR6ox and DIM transcripts by 30–50%, while Brz
treatment increased the transcript amount by 40–80%.
EBR reversed the eVect of Brz on gene expression. It is
well known that BR biosynthetic genes are subjected to
negative feedback regulation by BR (Bancos et al. 2002;
Tanaka et al. 2005). The observed opposite eVects of EBR
and Brz treatment on BR biosynthetic genes are consistent
with the expected changes of BR content in plants. These
results not only supported the important role for BR in
plant growth, but also indicated the feasibility of the chem-
ical genetic approach for manipulation of BR levels in
cucumber.
EVects on gas exchange and chlorophyll Xuorescence
parameters
To examine how BRs regulate photosynthetic capacity, we
determined the eVects of EBR and Brz application on gas
exchange and Chl Xuorescence quenching. Gas exchange
parameters of the third leaf were measured at ambient CO2
and saturating light level (1,000 mol m¡2 s¡1). As shown
in Fig. 2, the light-saturated rate of CO2 assimilation (Asat)
was not signiWcantly altered during the Wrst day, but sub-
stantially reduced at 3 days after Brz treatment and contin-
ued to decline during the remaining period of the
Leaf mass per unit Stem length Root length Chlorophyll
leaf area (g m¡2) (cm) (cm) content (g cm¡2)
21.2 § 0.2b 7.7 § 0.2b 30.6 § 1.5b 340.3 § 12.5b
b a a
21.9 § 0.4 9.8 § 0.3 50.8 § 7.3 347.4 § 15.5b
a c c
24.4 § 0.4 6.0 § 0.5 25.7 § 1.2 402.3 § 9.3a
b b b
21.7 § 0.3 8.0 § 0.5 33.2 § 2.4 357.2 § 10.5b
123
1190 Planta (2009) 230:1185–1196

CPD DWF4 Cont.

2.0 a a 2.0
20 EBR

Asat (µmol m-2 s-1)

1.5 b 1.5 Brz
b b b Brz+EBR
1.0 c 1.0 15
c
0.5 0.5
10
BR6ox a DIM a
1.5 1.5 5
b b b b
1.0 c 1.0
c
0.5 0.5
200

Gs (µmol m-2 s-1)

0.0 0.0
t

rz

rz

R
on

on

150
EB

EB

B
B

B
+E

+E
C

C
rz

rz
B

100
Fig. 1 EVects of EBR and Brz on the steady-state transcript level of
genes involved in BR biosynthesis. Leaf samples were harvested at
25 days after treatment. Data are the means of three replicates with SDs 50
shown by vertical bars. Means followed by the same letter are not sig-
niWcantly diVerent according to Tukey’s test (P < 0.05)

300

experiment. During this period of time, Asat was correlated 250

with the BR levels in the plants. Thus, the highest Asat was
Ci (µL L-1)

observed in the EBR-treated plants and the lowest Asat was
observed in the Brz-treated plants. Furthermore, decreased
Asat in Brz-treated plants could be recovered by EBR appli-
cation. An opposite eVect of BR levels was observed on the
intercellular CO2 concentration (Ci). Ci decreased in EBR-
treated plants, but increased in Brz-treated plants. Brz treat-
ment had no signiWcant eVect on stomatal conductance (Gs)
during the early stage, but had a negative eVect on Gs at
late stages after the treatment. The association of increased
Asat with decreased Ci in EBR-treated plants suggests that
the CO2 carboxylation capacity was a rate-limiting step in
photosynthesis.
Under our growth conditions, EBR and Brz treatments
did not cause any visible stress symptoms or changes in the
maximum quantum yield of PSII (Fv/Fm) (Table 2). Con-
sistent with Asat, EBR signiWcantly increased the quantum
yield of PSII (
PSII). Brz treatment, however, caused not
only reduced CO2 assimilation rate, but also reduced
PSII
and, again, this reduction of
PSII could be recovered by
EBR application. EBR signiWcantly increased, whereas Brz
slightly decreased photochemical quenching (qP), although
the eVect was not signiWcant. In contrast, EBR had no sig-
niWcant eVect on the eYciency of energy capture by open
PSII (Fv⬘/Fm⬘), while Brz signiWcantly reduced Fv⬘/Fm⬘.
EVects of EBR and Brz on carbohydrate contents
We subsequently tested the eVects of EBR and Brz applica-
tion on carbohydrate metabolism. Similar to the changes in
200
150
100
50
0 1 3 5 20
Time (d)
Fig. 2 EVects of EBR and Brz application on light-saturated rate of
CO2 assimilation (Asat), stomatal conductance (Gs) and intercellular
CO2 concentration (Ci) in cucumber leaves. Data are the means of four
replicates with SDs shown by vertical bars
Asat, contents of total soluble sugars, sucrose and starch
were all increased by EBR application and decreased by
Brz application (Table 3). The decrease in the contents of
total soluble sugars, sucrose and starch in Brz-treated plants
was signiWcantly restored by EBR treatment. However, the
content of hexose was not signiWcantly altered after EBR or
Brz treatment.
EVects on carboxylation and RuBP regeneration capacity
To further analyze the mechanism by which BRs regulate
CO2 Wxation, the Vc,max and Jmax were determined from the
steady-state measurements of the A/Ci response curves.
EBR-treated plants had higher and BR-deWcient plants had
lower Vc,max and Jmax than untreated control plants
(Table 4). When EBR was applied to Brz-treated plants, the

123
Planta (2009) 230:1185–1196 1191

Table 2 EVects of EBR and Brz on maximum quantum yield of PSII (Fv/Fm), quantum yield of PSII (
PSII), photochemical quenching coeYcient
(qP) and the eYciency of excitation capture by open PSII center (Fv⬘/Fm⬘) at 5 days after EBR or Brz treatment in cucumber leaves
Fv/Fm
PSII qP Fv⬘/Fm⬘

Cont. 0.85 § 0.006a 0.38 § 0.006b 0.61 § 0.033b 0.59 § 0.035a

a a a
EBR 0.86 § 0.006 0.44 § 0.022 0.72 § 0.032 0.64 § 0.021a
a c b
Brz 0.85 § 0.007 0.29 § 0.008 0.55 § 0.024 0.49 § 0.022b
Brz + EBR 0.85 § 0.008a 0.39 § 0.005b 0.63 § 0.023b 0.58 § 0.039a
Data are the means of four replicates with SDs. Means followed by the same letter are not signiWcantly diVerent according to Tukey’s test (P < 0.05)

Table 3 EVects of EBR and Brz on total soluble sugar, hexose, sucrose and starch content at 5 days after EBR or Brz treatment in cucumber leaves
Total soluble sugar Hexose Sucrose Starch
(mg g¡1 DW) (mg g¡1 DW) (mg g¡1 DW) (mg g¡1 DW)

Cont. 43.12 § 4.92b 23.76 § 4.52a 17.33 § 1.77b 27.68 § 1.66b

a a a
EBR 56.46 § 5.79 22.47 § 4.39 22.93 § 1.85 34.98 § 1.47a
c a c
Brz 27.56 § 1.36 26.6 § 1.69 12.1 § 1.5 19.2 § 1.11c
bc a bc
Brz + EBR 35.89 § 2.62 24.46 § 3.85 15.23 § 0.69 29.9 § 4.03b
Data are the means of four replicates with SDs. Means followed by the same letter are not signiWcantly diVerent according to Tukey’s test (P < 0.05)

Table 4 EVects of EBR and Brz on maximum Rubisco carboxylation rates (Vc,max), maximum RuBP regeneration rates (Jmax), total and initial
Rubisco activity and Rubisco activation state in cucumber leaves
Vc,max Jmax Total Rubisco Initial Rubisco Rubisco
(mol m¡2 s ¡1) (mol m¡2 s ¡1) activity (mol m¡2 s ¡1) activity (mol m¡2 s ¡1) activation state(%)

Cont. 70.3 § 5.9b 171.7 § 19.0b 40.3 § 3.3ab 21.3 § 1.4b 53b
a a a a
EBR 95.3 § 6.4 261.1 § 16.6 46.4 § 3.3 29.4 § 1.3 63a
c c b c
Brz 48.4 § 2.5 120.1 § 10.6 35.5 § 3.2 12.8 § 1.3 36c
Brz + EBR 66.3 § 6.2b 156.3 § 15.7b 37.4 § 2.1ab 20.1 § 1.5b 54b
Gas exchange measurements and sampling were carried out at 5 days after EBR or Brz treatment. Data are the means of four replicates with SDs.
Means followed by the same letter are not signiWcantly diVerent according to Tukey’s test (P < 0.05)

reduced Vc,max and Jmax were restored to those of control
plants.
EBR increased, while Brz decreased, total Rubisco
activity, although the eVects were not signiWcant. However,
the initial Rubisco activity was signiWcantly increased by
EBR treatment and decreased by Brz treatment and again
the reduced initial Rubisco activity in Brz-treated plants
was partially restored by EBR treatment. These results
strongly suggest that EBR treatment resulted in an
enhanced, and Brz treatment resulted in a reduced, Rubisco
activation state.
EVects on expression of rca, rbcS and rbcL
To further examine how BRs regulate photosynthesis, we
analyzed the eVects of EBR and Brz on the steady-state
transcriptional and translational products of rca, rbcS and
rbcL. As shown in Fig. 3a, the transcript levels of the rca,
rbcS and rbcL genes were elevated in EBR-treated plants,
but reduced in Brz-treated plants when compared with
those in control plants. Western blotting indicated that EBR
also increased the protein levels of RCA and RBCS, but not
RBCL (Fig. 3b). The protein levels of RCA, RBCS and
RBCL were all reduced in Brz-treated plants.
The subcellular localization of RCA was determined
using immunogold-labeling electron microscopy. When
sections of cucumber leaves were treated with the RCA-
speciWc antibody, large quantities of the immunogold
particles were observed within the chloroplast (Fig. 4).
Consistent with the increased protein levels of RCA from
Western blotting, there were signiWcantly increased gold
particle densities in EBR-treated leaves over those in the
control plants. By contrast, the gold particle density was
reduced in the chloroplasts of Brz-treated leaves com-
pared to the control. EBR application again could restore
the reduced gold particle density in the Brz-treated
plants. The observed eVects of EBR and Brz application
on the accumulation of RCA transcripts, proteins and

123
1192 Planta (2009) 230:1185–1196

subcellular distribution strongly suggest that RCA EVects on expression of Calvin cycle genes
played an important role in regulation of carboxylation
capacity by BR. To investigate how BR aVects Jmax, we examined the eVects
of EBR and Brz on the expression of six genes encoding
(a) enzymes involved in RuBP regeneration. TPI and FBPA

R
EB
catalyze the conversion of two triose-3-phosphates into
.

z+
nt

z
EB
Co

Br

Br
fructose-1,6-bisphosphate (FBP). FBPase and SBPase cata-
lyze the hydrolysis of FBP and sedoheptulose-1,7-bisphos-
rca phate (SBP) to Fru6P and sedoheptulose 7-phosphate
(Sed7P), respectively. RuPE and PRK catalyze the last two
rbcS steps in RuBP synthesis. Interestingly, expressions of all
the six genes increased by 1.5–2-fold in EBR-treated leaves
(Fig. 5). In contrast, the transcript levels of TPI, FBPase,
rbcL SBPase and PRK were reduced by more than 50% in Brz-
treated leaves. Brz had no signiWcant eVect on the expres-
sions of FBPA and RuPE. When Brz-treated plants were
rRNA
treated with EBR, the expression of TPI was completely
restored to control level, the expressions of SBPase and
(b)
R

PRK were partially restored, and the expression of FBPase

EB
.

z+
nt

was above the control level.

z
EB
Co

Br

Br

RCA
Discussion
RBCS
Brassinosteroids are plant steroidal hormones that regulate
RBCL
plant growth and development. Several studies have shown
that overexpression of genes involved in the BR biosyn-
Fig. 3 EVects of EBR and Brz on mRNA abundance (a) and protein
thetic pathway results in enhanced vegetative growth and
level (b) of RCA, RBCS and RBCL in cucumber leaves. Leaf samples increased seed yield, while reduced expression of these
were harvested at 5 days after EBR or Brz treatment genes leads to impaired plant growth (Choe et al. 2000,

Fig. 4 Immunogold labeling of

Rubisco activase (RCA) in Cont EBR
mesophyll cell chloroplasts of
cucumber leaves. The leaf seg-
ments were cut at 5 days after
EBR or Brz treatment. Bar 1 m

Brz Brz+EBR

123
Planta (2009) 230:1185–1196
Fig. 5 EVects of EBR and Brz
on the steady-state transcript Cont.
3 EBR
level of genes involved in regen-
eration of RuBP. Leaf samples Brz
Brz+EBR b
were harvested at 5 days after
EBR or Brz treatment. Data are 2 2
a a
the means of three replicates
with SDs shown by vertical
b c
bars. Means followed by the b c
1 1
same letter are not signiWcantly
c c
diVerent according to Tukey’s
test (P < 0.05)
0 0
TPI FBPA
2001). In the present study, we found that exogenous EBR
application resulted in enhanced growth, while inhibition of
BR biosynthesis by Brz resulted in reduced growth
(Table 1), supporting that BRs are actively involved in the
regulation of plant growth. Furthermore, these results
strongly suggest that the level of BR is a rate-limiting factor
for plant growth and, therefore, elevating BR levels in
plants through chemical or genetic means might be eVec-
tive in promoting plant growth and improving crop yield
(Wu et al. 2008).
We observed that Brz treatment caused a signiWcant
decrease in CO2 assimilation rate per unit leaf area at
3 days after treatment (Fig. 2), during which no signiWcant
growth aberration was observed. Brz speciWcally inhibits an
enzyme in the BR biosynthetic pathway (Asami et al. 2000,
2001). The inhibition of photosynthesis by Brz was an
eVect of reduced content of BR, because the eVect of Brz on
photosynthesis could be completely reversed by EBR.
Meanwhile, the reduction of photosynthesis was accompa-
nied by an increase in chlorophyll content in cucumber
plants (Table 1). Therefore, it appears that reduced photo-
synthesis as a result of reduced BR biosynthesis after Brz
treatment is not caused by reduced light-harvesting capac-
ity. We can also exclude reduced gas diVusion as a major
factor for reduced CO2 assimilation because BR deWciency
in Brz-treated plants did not reduce Gs (Fig. 2). Finally, we
observed that decreased Asat in Brz-treated plants was asso-
ciated with decreased contents of sucrose, soluble sugars
and starch (Table 3), making it less likely that reduced pho-
tosynthetic rates were caused by negative feedback regula-
tion by accumulated carbon metabolites (Paul and Pellny
2003). In our study, EBR treatment increased, but Brz treat-
ment decreased,
PSII (Table 2), which is a product of the
Fv⬘/Fm⬘ and qP (Genty et al. 1989). The change in
PSII
was mainly attributed to the increase in qP for EBR treat-
ment and to the decrease in Fv⬘/Fm⬘ for the Brz treatment,
respectively. The increase in qP was attributable to the
increase in the rate of consumption of reductants and ATP
produced from non-cyclic electron transport caused by
increased carboxylation rate, while the decrease in Fv⬘/Fm⬘
1193
a 3
a
a a
a a
b b b b
b
c
c
d d
FBPase SBPase RuPE PRK
after Brz treatment was associated with increase in excita-
tion energy quenching in the PSII antennae, which is con-
sidered as ‘downregulation’ of electron transport (Horton
et al. 1996). Consequently, inhibition of BR biosynthesis
may induce photo-protective mechanism, such as non-pho-
tochemical quenching as a result of its inhibitory eVect on
CO2 assimilation.
In the present study, we provide strong evidence that
BRs regulate photosynthetic capacity, to a large extent, by
aVecting the capacity of Rubisco carboxylation and RuBP
regeneration (Table 4). More speciWcally, EBR and Brz
treatments had discernible eVect on total Rubisco activity,
but had a substantial eVect on the maximum carboxylation
rate (Vc,max) and initial activity of Rubisco. It is apparent
that increase or decrease in Vc,max and initial Rubisco activ-
ity was associated with an increase or a decrease in mRNA
abundance and protein level of RBCS and RBCL (Fig. 3).
This is in agreement with that observed in rice leaves dur-
ing ontogeny (Suzuki et al. 2001; Irving and Robinson
2006). The consistent diVerence in transcript and protein
levels suggests that BRs play an important role in Rubisco
synthesis.
A survey of more than 100 plant species has shown a
strong correlation between Jmax and Vc,max (Wullschleger
1993). In agreement with this correlation, we have
observed that BR deWciency as a result of Brz treatment
caused a reduction of not only Vc,max, but also Jmax, while
increase in BR level after EBR treatment has an opposite
eVect on the two parameters (Table 4). Regeneration of
RuBP is dependent on both photosynthetic electron trans-
port chain and the enzymes downstream of Rubisco in the
Calvin cycle (Long et al. 2006). In the present study, we
observed that EBR promoted, while Brz inhibited, both
photosynthetic electron transport (Table 2) and expression
of genes encoding the Calvin cycle enzymes required for
RuBP regeneration (Fig. 5). Of the Calvin cycle enzymes
examined, SBPase is an important enzyme that determines
the rate of RuBP synthesis (Harrison et al. 2001), while
FBPase catalyzes a rate-limiting step in the Calvin cycle
and carbohydrate metabolism (Koßmann et al. 1994; Strand
123
1194
et al. 2000). Consistent with these previous studies, BR-
deWcient cucumber and Arabidopsis mutants have
decreased starch and sucrose contents (Table 3; Schlüter
et al. 2002), probably due to compromised balance of
source–sink relationship. The reduced activity of enzymes
involved in sucrose and starch metabolism in BR-deWcient
Arabidopsis mutants may reXect the decreased demand for
photo-assimilates (Schlüter et al. 2002).
We have previously shown that enhancement of photo-
synthetic capacity by BR is attributable to increase in the
Rubisco activation state (Yu et al. 2004). Now, we have
conWrmed this result and shown that BR is involved in the
regulation of Rubisco activation state (Table 4). Impor-
tantly, we have observed that changes in the Rubisco acti-
vation state were associated with the amounts of RCA
(Fig. 3). RCA plays an essential role in maintaining Rubi-
sco in an active conformation. Arabidopisis mutant deW-
cient in activase (Salvucci et al. 1985, 1986) and antisense
transgenic plants with reduced amounts of activase had
reduced rates of net photosynthesis (Mate et al. 1996;
Hammond et al. 1998). Immunogold labeling has shown
that RCA is mostly localized to the stroma of cucumber
chloroplast (Fig. 4), where it may interact directly with
Rubisco to alter the enzyme’s structure (Portis et al. 2008).
Enhanced activity of RCA would explain the observation
that BR can substantially enhance photosynthetic capacity.
Activation of Rubisco by RCA requires ATP (Streusand
and Portis 1987) and the reaction is inhibited by a high
ADP/ATP ratio. Increase in non-photochemical quench-
ing, as indicated by reduced Fv⬘/Fm⬘ (Table 2) in Brz-
treated plants suggests a large pH gradient across the
thylakoid membrane, which would result in an increase in
the ADP/ATP ratio. EBR treatment, however, had little
eVect on Fv⬘/Fm⬘ and, therefore, it is unclear how
increased BR level following EBR treatment leads to
enhanced activity of RCA.
Short-term treatment of BR induces CO2 assimilation
(Yu et al. 2004). In this study, we found that BR increased
photosynthesis, at least in part, through regulating the
expression of genes for the Rubisco subunits and other Cal-
vin cycle enzymes. However, the mechanisms by which BR
regulates these photosynthetic genes are still unclear. There
are lines of evidence showing that cellular redox status con-
trols photosynthetic gene expressions and enzyme activities
(Oswald et al. 2001; Pfannschmidt et al. 2001; Zhang et al.
2002). Interestingly, we discovered that BR treatment
induced a transient increase in leaf H2O2 content and
induced several genes related to redox status (Xia et al.
2009). Consistent with this, microarray analysis has shown
that BR regulates the expression of genes for monodehy-
droascorbate reductase and thioredoxin (Müssig et al.
2002). H2O2 can function as a second messenger to trigger
a signaling pathway via MAPK to regulate the cellular
123
Planta (2009) 230:1185–1196
redox status (Neill et al. 2002; Apel and Hirt 2004; Xia
et al. 2009). Therefore, it is possible that BR-induced H2O2
may alter cellular redox status and induce changes in
expression of photosynthetic genes.
Acknowledgments This work was supported by the National Basic
Research Program of China (2009CB119000), National Natural Sci-
ence Foundation of China (3050344; 30671428) and the Program for
Promotion of Basic Research Activities for Innovative Bioscience
(PROBRAIN). We thank Dr J Hong for help with the immunogold-
labeling experiment.
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