Consequences of induced brassinosteroid deficiency in Arabidopsis leaves
BMC Plant Biology
Consequences of induced brassinosteroid deficiency in Arabidopsis leaves
Florian Schrder 0 2
Janina Lisso 0 2
Toshihiro Obata 1
Alexander Erban 1
Eugenia Maximova 1
Patrick Giavalisco 1
Joachim Kopka 1
Alisdair R Fernie 1
Lothar Willmitzer 1
Carsten Mssig 0 2
0 University of Potsdam, c/o Max Planck Institute of Molecular Plant Physiology , Am Muhlenberg 1, 14476 Potsdam-Golm , Germany
1 Max Planck Institute of Molecular Plant Physiology , Am Muhlenberg 1, 14476 Potsdam-Golm , Germany
2 University of Potsdam, c/o Max Planck Institute of Molecular Plant Physiology , Am Muhlenberg 1, 14476 Potsdam-Golm , Germany
Background: The identification of brassinosteroid (BR) deficient and BR insensitive mutants provided conclusive evidence that BR is a potent growth-promoting phytohormone. Arabidopsis mutants are characterized by a compact rosette structure, decreased plant height and reduced root system, delayed development, and reduced fertility. Cell expansion, cell division, and multiple developmental processes depend on BR. The molecular and physiological basis of BR action is diverse. The BR signalling pathway controls the activity of transcription factors, and numerous BR responsive genes have been identified. The analysis of dwarf mutants, however, may to some extent reveal phenotypic changes that are an effect of the altered morphology and physiology. This restriction holds particularly true for the analysis of established organs such as rosette leaves. Results: In this study, the mode of BR action was analysed in established leaves by means of two approaches. First, an inhibitor of BR biosynthesis (brassinazole) was applied to 21-day-old wild-type plants. Secondly, BR complementation of BR deficient plants, namely CPD (constitutive photomorphogenic dwarf)-antisense and cbb1 (cabbage1) mutant plants was stopped after 21 days. BR action in established leaves is associated with stimulated cell expansion, an increase in leaf index, starch accumulation, enhanced CO2 release by the tricarboxylic acid cycle, and increased biomass production. Cell number and protein content were barely affected. Conclusion: Previous analysis of BR promoted growth focused on genomic effects. However, the link between growth and changes in gene expression patterns barely provided clues to the physiological and metabolic basis of growth. Our study analysed comprehensive metabolic data sets of leaves with altered BR levels. The data suggest that BR promoted growth may depend on the increased provision and use of carbohydrates and energy. BR may stimulate both anabolic and catabolic pathways.
Brassinosteroids; Arabidopsis; Tricarboxylic acid cycle; Biomass; Cell expansion; Growth
Brassinolide (BL) was identified in 1979 through its
ability to promote internode growth . Since then, the
growth-promoting effect has been studied in hundreds
of articles. Excised tissues (e.g. hypocotyl, epicotyl,
cotyledons, internodes, leaves, and roots), protoplasts, cell
suspension cultures, intact seedlings, and whole plants
were subjected to brassinosteroid (BR) treatments, and
in all cases BR had the potential to stimulate growth .
A large number of BR deficient and BR insensitive
mutants in Arabidopsis thaliana and in crops such as rice,
tomato, and pea were identified . These mutants are
generally dwarfed and exhibit rounded, dark green leaves,
delayed flowering, reduced male fertility and seed set, and
delayed senescence. Further proven roles of BR include
the control of xylem formation [4,5], stomata
development [6-8], and further developmental processes [9-12].
Numerous studies analysed gene expression patterns upon
BR treatment and BR deficiency. However, these studies
barely clarified the metabolic and physiological basis of
BR dependent growth because the precise functions of
isoenzymes, cell wall proteins, and other factors often
BR plays non redundant roles since it is not possible
to complement BR mutants with other phytohormones or
their antagonists . Overexpression of the major BR
receptor, BRI1 (BRASSINOSTEROID-INSENSITIVE 1),
stimulated growth. However, the underlying changes at
transcript and metabolite level are largely different from
other growth-stimulating pathways . The most
prominent direct BR effect is the modification of gene
expression patterns . Transcription factors such as BES1
(bri1-EMS-suppressor 1) and BZR1 (Brassinazole-resistant
1) regulate BR responsive genes [16-19]. The physiological
mechanisms underlying BR promoted growth appear to
be manifold, and depend on the tissue and developmental
stage . They include the control of aquaporin activity
and water movement across membranes , cytoskeleton
organisation [21-23], and the modification of mechanical
cell wall properties [24,25]. A few studies in Arabidopsis
and crops addressed the influence of BR on primary
metabolism. The focus of these studies was on
photosynthesis and sink strength, and specific enzyme activities or
metabolites were measured. An early article showed that
BR stimulates CO2 assimilation in wheat . This finding
was confirmed in other plants such as cucumber  and
rice . In Arabidopsis, expression of a mutated BRI1
receptor (Y831F) enhanced shoot growth and conferred
elevated photosynthetic rates and starch levels at the end of
the light period . Rubisco activity and regeneration of
Ribulose-1,5-bisphosphate are the most important limiting
factors of photosynthesis under natural conditions.
Positive effects of BR on Rubisco activity have been shown
[26,27]. Yu et al.  also postulated a positive effect on
photosynthesis correlated with an increase of soluble sugar and
starch content and parallel enhancement of fresh and dry
weights. In line with these data, BR deficient Arabidopsis
mutants showed drastically reduced CO2 assimilation
rates, reduced starch levels, a tendency to reduced
sucrose levels, and reduced biomass accumulation .
In addition to source efficiency, BR also increases sink
strength. The tomato dx mutant produces bioactive BR
in fruits but not in the shoot, and provides an option to
dissect BR dependent processes in fruits and shoots. Dry
weight and starch levels of dx fruits were significantly
reduced . BR application to leaves partly normalized
metabolic changes in dx fruits suggesting that
shootderived BR dependent factors are required for proper
fruit metabolism. Previous research emphasized the
relevance of BR for invertase activity in the growing zone of
tomato hypocotyls . Thus, several reports
demonstrated the requirement of BR for source efficiency and
In this study, induced BR deficiency was analysed in
Arabidopsis rosette leaves by means of complementary
time-series experiments. First, BR deficient plants were
complemented by exogenous BR. Subsequent omission
of BR treatment caused BR deficiency. Secondly,
brassinazole (BRZ) was applied to wild-type plants. BRZ binds
to the DWF4 enzyme and specifically blocks BR
biosynthesis at the C-22 hydroxylation step . BR dependent
growth of established leaves is associated with elevated
starch levels, higher metabolic flux through the
tricarboxylic acid (TCA) cycle, and increased cell expansion
and biomass production.
Time course experiments for the analysis of morphological
and biochemical consequences of BR deficiency
The analysis of BR deficient or BR insensitive mutants is
complicated by the severe dwarfism and major
morphological changes . The use of mutants with mild
phenotypic changes such as cbb1/dwf1 (cabbage1/dwarf1) [13,34]
can alleviate that difficulty. The known alleles result in
milder phenotypic changes in comparison to det2
(de-etiolated2), cpd (constitutive photomorphogenic dwarf), dwf4
(dwarf4), and other biosynthetic mutants. The cbb1/dwf1
mutants are presumably able to produce unusual bioactive
BR as a consequence of the accumulation of precursors
and display altered BR responses [34,35].
In order to start the analysis of BR deficiency symptoms
with morphologically intact plants, two complementary
sets of time course experiments were performed (Figure 1).
During the time course experiments, plants were grown
in parallel in a randomized manner in a controlled growth
chamber (see Methods for details). The time course
experiments were performed three times each, resulting in a
total of six independent experiments.
The first approach used BR deficient mutants.
CPDantisense (aCPD) plants and the BR-deficient cbb1/dwf1-6
mutant [13,30] were treated with 200 nM brassinolide
(BL) for three weeks. Wild-type plants were grown in
parallel and were simultaneously treated with a control
solution. The control solution was identical to the BR solution
apart from the addition of BR (for details see Methods).
BR supplementation fully normalized the morphology and
biomass production of CPD-antisense plants. The
CPDantisense plants were nearly indistinguishable from the
Figure 1 Design of time series experiments. Plants were grown
on soil for 5 weeks in controlled growth chambers under the
specified conditions. Black bars indicate a treatment of the plants
with brassinolide (BL) or brassinazole (BRZ). Samples were taken at
day 0 (21 days after sowing), 1, 3, 6, 9 and 14. LD, long day; SD,
short day; CN, cold night.
wild type. The growth defect of cbb1 plants was partly
complemented by exogenous BR (Figure 2, day 0). Fresh
weight of 21-day-old cbb1 shoots was identical to the wild
type. However, leaf length and width were diminished in
comparison to the wild type, leaves were more erect and
had a slightly crinkled surface, and rosettes appeared
compact. Thus, exogenous BR could not fully substitute for
endogenous BR. After three weeks, BR treatment was stopped
(day 0). The CPD-antisense and cbb1 plants started to run
into BR deficiency or pronounced BR deficiency,
respectively. At this point the sampling began. Samples for
biochemical analysis were taken at day 0, 1, 3, 6, 9 and 14 after
the respective treatment was stopped. Under the applied
conditions, plants were in the vegetative phase during the
complete experiments and did not start bolting.
The second approach used wild-type plants (C24) that
were grown for three weeks without any treatment
(Figure 3, day 0). Subsequently, plants were treated with
10 M BRZ, 20 M BRZ, or control solution. Lower
concentrations such as 1 to 5 M BRZ have previously
been applied in synthetic growth medium (e.g. [36,37])
but were inapplicable in our time course experiments
since they induced only minor growth effects in
soilgrown plants. The necessity for higher BRZ
concentrations may reflect weaker uptake by leaves through a
functional epidermis. After the onset of BRZ application
(day 0), samples for biochemical analysis were taken at
the same points in time as described above (i.e. day 1, 3,
6, 9, and 14).
The parallel analysis of CPD-antisense, cbb1, and BRZ
treated wild-type plants allows avoiding genotype- or
treatment-specific limitations during the evaluation of
Elevated CPD and DWF4 transcript levels indicate
emerging BR deficiency
The CPD  and DWF4  genes encode enzymes
involved in BR biosynthesis. The expression of these genes
is negatively associated with endogenous BR levels. High
transcript levels indicate low BR levels and vice versa .
CPD and DWF4 transcript levels were analysed by means
of quantitative RT-PCR (Figure 4).
Unchanged transcript levels one day after the BR
supplementation was stopped or after the BRZ treatment
was started may indicate the presence of remaining BR
or a time lag in the induction of BR biosynthetic genes.
Stronger differences were observed from day 3 onwards.
CPD transcript levels in CPD-antisense plants were
previously described . Due to incomplete CPD gene
repression, the phenotypic changes of CPD-antisense plants are
considerably milder in comparison to the cpd/cbb3
knockout mutant and other BR deficient mutants such as cbb1/
Figure 2 Growth parameters of CPD-antisense and cbb1 plants in comparison to the wild type. Wild-type (C24), CPD-antisense, and cbb1
plants were grown as described in Figure 1. A, Shoot fresh weight. B, Representative plants at day 0, 1, 3, 6, 9, and 14. C, Length of rosette leaves
three and four. D, Width of rosette leaves three and four. Data are given as mean SE (n =10 plants). Values denoted with an asterisk are
significantly different from the wild type (t test, P <0.05).
Figure 3 Growth parameters of BRZ treated plants in comparison to mock treated plants. Wild-type plants were grown and treated as
described in Figure 1. A, Shoot fresh weight. B, Representative plants at day 0, 1, 3, 6, 9, and 14. C, Length of rosette leaves three and four.
D, Width of rosette leaves three and four. Data are given as mean SE (n =10 plants). Values denoted with an asterisk are significantly different
from the wild type (t test, P <0.05).
dwf1-6 [13,38]. Stronger DWF4 expression in the cbb1
mutant in comparison to the CPD-antisense plants
corresponds to the observed growth defect (Figure 4A and B).
Only minor differences were detected between plants
treated with different concentrations of BRZ. Application
of 10 M BRZ induced CPD and DWF4 expression nearly
as effectively as 20 M BRZ (Figure 4C and D).
Induced BR deficiency impairs leaf expansion
Significant differences in shoot fresh weight and length
of rosette leaves three and four developed after one day
in cbb1 plants (Figure 2A and C). Both growth parameters
of CPD-antisense plants became significantly different
from the wild type after six days and one day, respectively
(Figure 2A and C). At the end of the analysed period (day
14), cbb1 and CPD-antisense plants had 36% and 68% of
the wild-type fresh weight, respectively (Figure 2A). BR
deficiency caused a less pronounced effect on leaf width
(Figure 2D). The resulting decrease in the leaf index (i.e.
more roundish leaves; ) is a well described feature
of BR deficient plants (e.g. ). BRZ treated plants
exhibited a small reduction in the shoot fresh weight at
day 1 (Figure 3A). The biomass difference to control
plants increased over time. Leaf length and leaf width
were reduced. Similarly to the CPD-antisense and cbb1
plants, leaf width was less affected than leaf length
(Figure 3C and D).
Leaf thickness depends on the BR level and genotype.
For example, BR deficient mutants such as det2
exhibited an increased leaf thickness. Low concentrations of
exogenous BR decreased leaf thickness of det2 and
wildtype plants. Higher concentrations caused an increase of
leaf thickness in the wild type . In this study, leaf
thickness of CPD-antisense plants and BRZ treated plants
displayed diminished enhancement from day 0 to day 6.
In contrast, leaf thickness of cbb1 plants increased more
during that period (Figure 5).
Leaves five and six were analysed in parallel at day 3
and day 6. Similar effects on leaf length, leaf width, and
leaf thickness were observed (Additional file 1: Figures S1
and S2, A-C).
Reduced growth is mainly due to reduced cell size
Smaller leaf size of BR deficient plants could be based
on impaired cell expansion, cell proliferation, or a
combination of both. Previous analyses of BR mutants revealed
effects on both cell proliferation and cell expansion. BR
deficient mutants such as dwf1, det2  and cpd  are
characterized by reduced cell division rates and reduced
Figure 4 Quantitative RT-PCR analysis of CPD and DWF4 transcript levels. Plants were grown and harvested as described in Figure 1. A,
Relative CPD transcript levels in wild-type (C24) and cbb1 plants. B, Relative DWF4 transcript levels in wild-type, CPD-antisense, and cbb1 plants.
C, Relative CPD transcript levels in BRZ treated plants. D, Relative DWF4 transcript levels in BRZ treated plants. eIF1a CT values were subtracted
from respective CT values of the gene of interest resulting in dCT. Subsequently, differences were subtracted from an arbitrary value (i.e. 40).
Higher numbers indicate higher transcript levels. A difference of one unit indicates a fold change of two. Data are given as mean SE of gene of
interest in three technical replicates. The data shown are from one experiment representative of three independent biological replicates.
In this study, similar size of palisade and spongy
parenchyma cells in CPD-antisense, cbb1, and wild-type plants
were observed at day 0 (Figure 6A and B), suggesting that
the previous BR application normalized cell expansion.
Later on, palisade and spongy parenchyma cells of
CPDantisense and cbb1 plants became smaller in comparison
to the wild type (Figure 6A and B). Similar results were
obtained for younger leaves (Additional file 1: Figure S1,
D and E). Effects on cell number were less evident. The
cbb1 leaves exhibited a tendency towards lower cell
numbers, indicating an incomplete normalization of cell
division rates by the previous BR treatment. In contrast,
Figure 5 Leaf thickness. Plants were grown and harvested as described in Figure 1. Thickness of the leaves three and four was measured using
transversal sections. Values are given as mean SE. 20 leaves were analysed per point in time. Values denoted with an asterisk are significantly
different from the wild type (C24) or control (t test, P <0.05). A, Leaf thickness of wild-type (C24), CPD-antisense, and cbb1 plants. B, Leaf thickness
of BRZ treated plants.
Figure 6 Cell sizes and cell numbers of rosette leaves of CPD-antisense and cbb1 plants. Wild-type (C24), CPD-antisense, and cbb1 plants
were grown and harvested as described in Figure 1. Cell sizes were measured using transversal sections of rosette leaves three and four. Cell
numbers were calculated from the respective other half of the same leaf. Data are given as mean SE. 20 leaves were analysed per point in time.
Values denoted with an asterisk are significantly different from the wild type (t test, P <0.05). A, Area of palisade cells. B, Area of spongy
parenchyma cells. C, Palisade cells per leaf. D, Spongy parenchyma cells per leaf.
CPD-antisense plants were identical to the wild type at
day 0 and later (Figure 6C and D; Additional file 1:
Figure S1, F-H; Additional file 1: Figure S3A). Application
of BRZ to the wild type reduced cell sizes (Figure 7A
and B; Additional file 1: Figure S2, D and E), but did not
significantly reduce cell numbers (Figure 7C and D;
Additional file 1: Figure S2, F-H).
Reduced starch and unchanged protein levels in BR
Synthetic BR stimulates CO2 assimilation [26-29], and
cbb1 and CPD-antisense plants exhibit reduced
photosynthetic rates . Both biochemical and morphological
factors could account for reduced photosynthesis. The
consequences of reduced carbon supply include reduced
starch levels, impaired energy balance, reduced provision
of biosynthetic precursors, and decreased growth .
In line with previous reports, starch levels were
diminished in CPD-antisense, cbb1, and BRZ treated plants
from day 3 onwards (Figure 8A and B). The reduction of
starch levels in CPD-antisense and cbb1 plants is relatively
small in comparison to previously determined levels .
This may reflect the lack of severe cellular abnormalities
that were avoided by the initial BR supplementation.
Hexose and sucrose levels were not significantly altered
(Additional file 1: Table S1). Examination of plastid
ultrastructure revealed intact chloroplasts in BR deficient
plants. BRZ treated, CPD-antisense, and cbb1 chloroplasts
tended to develop a thylakoid network with reduced
grana stacking at day 3 and to a more minor extent at
day 6 (Figures 9 and 10).
Early studies on BR demonstrated that inhibitors of
protein synthesis (e.g. cycloheximide and puromycin)
interfere with BR dependent growth . It was suggested that
BR induces the synthesis of a large number of specific
proteins, but does not indiscriminately increase overall
protein synthesis. In agreement with that view, the overall
protein content was not significantly altered in leaves of
BR deficient plants (Figure 8C and D).
Reduced TCA cycle activity in BRZ treated plants
Mitochondrial respiratory metabolism is the major source
of ATP and associated with proper maintenance of
cellular metabolism as a whole [47,48]. The tricarboxylic acid
(TCA) cycle is a crucial component of respiratory
metabolism. It links the oxidation of the acetyl group of
acetyl-CoA to CO2 with the generation of NADH for
the oxidation by the mitochondrial respiratory chain. In
Figure 7 Cell sizes and cell numbers of rosette leaves of BRZ treated plants. BRZ treated plants were grown and harvested as described
in Figure 1. Cell sizes were measured using transversal sections of rosette leaves three and four, and cell numbers were calculated from the
respective other half of the same leaf. Data are given as mean SE. 20 leaves were analysed per point in time. Values denoted with an asterisk
are significantly different from the wild type (t test, P <0.05). A, Area of palisade cells. B, Area of spongy parenchyma cells. C, Palisade cells per
leaf. D, Spongy parenchyma cells per leaf.
plants, acetyl-CoA is derived from the products of
glycolysis through oxidative decarboxylation of pyruvate by
the pyruvate dehydrogenase [49,50].
Leaf discs were incubated in [3:4-14C]-glucose or
[1-14C]glucose. CO2 from the C3 and C4 positions is preferentially
released by the actions of pyruvate dehydrogenase or malic
enzyme [51,52]. Feeding with [3:4-14C]-glucose to BRZ
treated leaves resulted in a lower evolution of 14CO2 in
comparison to the control (Figure 11A). C1 of glucose is
released either by an enzyme of the oxidative pentose
phosphate pathway (OPPP), namely 6-phosphogluconate
dehydrogenase, or an enzyme of the TCA cycle, isocitrate
dehydrogenase [51,52]. Feeding of BRZ treated plants with
[1-14C]-glucose tended to result in a lower 14CO2
evolution in comparison to mock-treated plants (Figure 11B).
The relative content of TCA cycle intermediates was
determined by mass spectrometry. Aspartate is
synthesized by transamination of oxaloacetate and can be used
to estimate oxaloacetate levels. Levels of several TCA
cycle intermediates were increased in BRZ treated plants
(Figure 12). Levels of citrate, malate, and aspartate were
significantly different from the control (Additional file 1:
Table S2). A tendency to higher levels of TCA cycle
intermediates was also observed in the cbb1 mutant (Additional
file 1: Figure S5). The ketoglutarate level was significantly
increased at day 6 (Additional file 1: Table S3).
Lower CO2 release from [3:4-14C]-glucose by the
pyruvate decarboxylase and/or malic enzyme and increased
levels of TCA cycle intermediates suggest a weaker TCA
cycle activity in BR deficient plants. The release of CO2
from [1-14C]-glucose is furthermore consistent with a
reduced flux through the TCA cycle, but could also suggest
a reduced activity of the oxidative pentose phosphate
Experimental approaches to study BR deficiency
BR deficient plants display dwarfism and multiple
defects in cell elongation, cell division, cell differentiation,
reproduction and senescence, and light control of
development . Reduced fertility and male sterility are common
features of BR deficient mutants. BR appears to be largely
dispensable for embryogenesis . Seedling development,
however, critically depends on BR. Hypocotyl length,
cotyledon growth, and responses to environmental stimuli were
impaired in Arabidopsis mutants [2,3,12,13]. BR deficiency
impairs plant growth at early stages, and later phenotypic
changes are inevitably modified by the early growth
Figure 8 Starch and protein levels. Plants were harvested at the middle of the light period. Data are given as mean SE (n =3 pools of 10
plants). Values denoted with an asterisk are significantly different from the wild type or control (t test, P <0.05). A, Starch levels of wild-type (C24),
CPD-antisense, and cbb1 plants. B, Starch levels of BRZ treated plants. C, Protein levels of C24, CPD-antisense, and cbb1 plants. D, Protein levels of
BRZ treated plants.
defects. Thus, mutant analyses can properly address early
phases in plant development, but conclusions about BR
function at later stages are fraught with uncertainty.
One approach to study the mode of action of BR at
later developmental stages is the application of inhibitors
of BR biosynthesis . Previous approaches usually
supplemented BRZ [33,37,54] and other azole
derivatives (e.g. propiconazole, ; voriconazole, ; YCZ,
) to synthetic growth medium, implying that
seedlings or small plantlets were analysed. An alternative
Figure 9 Transmission electron microscopy of plastids in leaf three of CPD-antisense and cbb1 plants. Plants were grown and harvested
as described in Figure 1 at the middle of the light period. A, Wild type (C24) at day 3. B, CPD-antisense at day 3. C, cbb1 at day 3. D, C24 at day 6.
E, CPD-antisense at day 6. F, cbb1 at day 6. Subcellular structures are exemplarily indicated in A; cw, cell wall; g, granum (stack of thylakoids);
s, starch granule; bar: 1 m.
Figure 10 Transmission electron microscopy of plastids in leaf three of BRZ treated plants. Plants were grown and harvested as described
in Figure 1 at the middle of the light period. A, control (0 M BRZ) at day 3. B, 10 M BRZ at day 3. C, 20 M BRZ at day 3. D, control at day 6.
E, 10 M BRZ at day 6. F, 20 M BRZ at day 6. Subcellular structures are exemplarily indicated in A; cw, cell wall; g, granum (stack of thylakoids);
s, starch granule; bar: 1 m.
approach is the complementation of BR deficient
mutants for a limited period, and the subsequent deprivation
of synthetic BR. Both approaches have pros and cons.
For example, BRZ is seen as a highly specific
inhibitor, but it presumably also affects other P450s.
Although BR deficient mutants respond to synthetic BR,
BR feeding cannot fully mimic the endogenous
distribution of BR.
For those reasons, both approaches were followed in
the current study and analysed in parallel (Figure 1).
The first three weeks of the experiments presumed the
presence of wild-type BR levels or continuous supply of
synthetic BR (mutant complementation for three weeks).
At this point (day 0), transcript levels of BR biosynthesis
genes and growth were similar in the mutants and the
control (Figures 2, 3, 4). Increased CPD and DWF4
transcript levels suggest that plants became impoverished for
BR within one to three days (Figure 4).
BR deficiency in established leaves impairs cell expansion
Leaves grow initially mainly by cell proliferation. Cells
divide and grow simultaneously. A proliferation
gradient develops between cell division and expansion at the
transition zone. The transition from cell proliferation to
expansion (cell growth without cell division) is
controlled by a network of factors. Cell division first ceases
at the tip of the leaf, and progressively ceases along the
longitudinal axis . The final size of the organ is
achieved by elongation growth.
Mutant analyses indicated that both cell division
and cell elongation are affected by BR, because leaves
of BR deficient mutant such as det2 and cpd exhibit
both decreased cell size and cell numbers [42,44]. BR
controls the transition between cell division and
expansion . In addition, BR controls organ boundary
formation [59,60], xylem formation , and stomata
Figure 11 TCA cycle flux. Evolution of 14CO2 of BRZ treated plants at day 6 when incubated with labelled glucose. Plants were grown and
harvested as described in Figure 1. A, Leaf discs were incubated with [3:4-14C]-glucose. B, Leaf discs were incubated with [1-14C] -glucose. Data
are given as mean SE (n =5 pools of leaf discs from 10 plants).
Figure 12 Levels of TCA cycle intermediates in BRZ treated plants. Plants were grown and harvested as described in Figure 1. Relative
metabolite levels are given as mean SE of three biological replicates. Fold change values are given in Additional file 1: Table S2.
We focused our analysis on established leaves at later
developmental stages. At this stage (21 days after sowing
and later), cell expansion and cell proliferation took
place in leaves three to six. Palisade and spongy
parenchyma were significantly smaller in CPD-antisense, cbb1,
and BRZ treated plants (Figures 6, 7, Additional file 1:
Figures S1, S2). The cell number was slightly reduced in
the cbb1 mutant (Figure 6, Additional file 1: Figures S1, S3).
However, cell proliferation in CPD-antisense and BRZ
treated plants was similar to the wild type (Figures 6, 7,
Additional file 1: Figures S1-3). Thus, cell expansion in
established leaves depends on BR, but cell division is
Leaf thickness is determined by the mesophyll
anatomy and cell size. The cellular organization of mesophyll
tissues modifies the interception of light and CO2
diffusion to the sites of photosynthesis. The effects of BR or
other phytohormones and stimuli on leaf thickness are
not well documented, because changes in leaf growth
have usually been assessed in two dimensions. In this
study, induced BR deficiency in BRZ treated and
CPDantisense plants was associated with reduced leaf
thickness (Figure 5, Additional file 1: Figures S1, S2, S4). In
contrast, leaf thickness in cbb1 plants increased more
until day 6 (Figure 5, Additional file 1: Figures S1, S4).
The reason for the difference between the genotypes
could be the incomplete normalization of cbb1 plants.
Alternatively, cbb1 plants could synthesize alternative
BR and respond in a different manner to BR as has been
reported for the rice brd2 (BR-deficient dwarf2) mutant
(Hong et al. ).
Reduced starch accumulation may cause reduced growth
Reduced growth of BR deficient plants may be a
consequence of reduced carbon availability. Starch levels in cbb1,
CPD-antisense, and BRZ treated leaves were lower in
comparison to the wild type (Figure 8). This presumably is a
consequence of drastically reduced CO2 assimilation rates
. Optimal starch metabolism is pivotal for the diurnal
carbon balance and growth . Mutants impaired in
starch synthesis such as phosphoglucomutase (pgm) or
mutants impaired in starch degradation such as starch excess
1 (sex1) show dwarfism . Both too rapid and too slow
mobilization of starch during the night can result in
diminished growth rates . Thus, carbon undersupply may
cause impaired growth in BR deficient mutants.
Electron micrographs of BRZ treated plants and the
cbb1 mutant revealed a tendency to less grana thylakoids
(Figures 9, 10). The molecular basis of reduced thylakoid
stacking is unknown. It could reflect a delay in plastid
development, an altered adaptation of the thylakoid
architecture to the light conditions, an altered protein
composition, or changes in other regulatory mechanisms. Grana
confer functional advantages such as enhancement of light
capture and fine-tuning of energy distribution between the
photosystems [65,66]. Conceivably, the observed changes
in chloroplast structure contribute to the reduced
photosynthetic rate and starch accumulation. Plastid structure
and function were previously analysed in BR mutants.
One reason for that interest is the link between BR action
and photomorphogenesis . Light-grown det2 plants
developed structurally altered chloroplasts. For example,
eight-day-old det2 chloroplasts had a smaller, rounder
shape, reduced grana stacking, and an abnormally high
ratio of chlorophyll a/b in comparison to the wild type,
indicating an immature status . However, Azpiroz and
coworkers  did not describe an altered chloroplast
structure of light-grown dwf4 plants. Given the multiple
reports that describe altered properties of plastids of BR
treated plants or BR mutants ( and references therein),
a more detailed analysis of the underlying structural and
molecular changes may be worthwhile.
Reduced growth is associated with reduced TCA cycle
The TCA cycle links the oxidation of pyruvate and
malate with the generation of NADH. NADH is used by the
mitochondrial respiratory chain for ATP production. The
reduced release of 14CO2 from labelled glucose in BRZ
treated plants (Figure 11) and elevated levels of TCA cycle
intermediates in BRZ treated (Figure 12) and cbb1 plants
(Additional file 1: Figure S5) suggest a reduced carbon flux
through the TCA cycle in BR deficient plants. Reduced
TCA cycle activity may compromise efficient use of
carbohydrates and impair growth especially during the dark
period. Furthermore, the TCA cycle provides precursors
for various biosynthetic pathways [48,50].
Reduced production of ATP for sucrose synthesis and
carbon precursors for anabolism could be consequence
or cause of reduced growth. BR deficiency and reduced
growth presumably goes along with reduced demand
for carbohydrates, amino acids, and other biosynthetic
precursors. On the other hand, reduced photosynthesis
in BR deficient plants could diminish the supply of
substrates for mitochondrial reactions and reduce the flux
through the TCA cycle. The situation becomes even
more complex in view of the multifaceted links between
photosynthesis and TCA cycle. Altered TCA cycle enzyme
activities can result in increased, decreased, or unvaried
photosynthesis [70,71]. Thus, identification of cause and
effect of metabolic changes is complicated. Labelling
studies and application of network models will be necessary to
precisely determine the flux of metabolites and interplay
of metabolic pathways in BR deficient plants.
The morphology of BR deficient mutants was described in
detail. Numerous studies addressed the consequences of
BR deficiency at the molecular and cellular level. In that
way, the current understanding of BR was developed.
However, the analysis of BR deficient mutants is complicated by
the dwarfism and multiple morphological changes. The
mode of action of BR at later developmental stages cannot
be faultlessly determined. In this study, we used two
approaches for the analysis of BR action in established leaves.
An inhibitor of BR biosynthesis was applied and BR
complementation of BR deficient plants was stopped after three
weeks. For the first time the metabolic changes upon BR
deficiency were analysed comprehensively by means of
metabolic profiling. Our analyses revealed that induced BR
deficiency impairs starch accumulation, TCA cycle activity,
cell expansion, and biomass production. Further studies are
needed to determine alterations in metabolic fluxes and the
precise link between genomic BR effects and catabolic and
anabolic pathways. Transgenic approaches such as the
inducible expression of RNAi hairpins represent another
approach that would enable tissue-specific repression of BR
biosynthesis. This could particularly help to separate the
role of BR in sink and source tissues.
C24 wild type was obtained from the Nottingham
Arabidopsis Stock Centre (NASC) - NASC ID: N906.
The CPD-antisense line and the cbb1 mutant were
described before [13,30]. Seeds for growth experiments
were derived from plants grown in parallel in a
greenhouse. The cbb1 mutant was repeatedly treated with BR
before and during seed set. Seeds were allowed to
germinate and seedlings grew for two weeks in controlled
growth chambers (7 days: 16 h light [140 mol m2 s1,
20C, 75% relative humidity]/8 h night [6C, 75% relative
humidity]; thereafter 7 days: 8 h light [140 mol m2 s1,
20C, 60% relative humidity]/16 h night [16C, 75%
relative humidity]). Subsequently, plants were transferred to
long-day conditions in a controlled growth chamber (16 h
light [140 mol m2 s1, 20C, 60% relative humidity]/8 h
night [16C, 75% relative humidity]). All genotypes were
grown in the same chamber at the same time in a
randomized manner, each replicate one after another. All
necessary measures were taken in order to avoid biotic and
Plants were sprayed at midday for three (BL) or five
(BRZ) times a week with an aqueous solution containing
BL or BRZ, respectively, and 0.01% Tween 20. Methanol
was used as solvent for stock solutions. The same
volume of methanol was added to the control solution. BL
and BRZ experiments were performed in the same growth
chamber. BRZ was sprayed more often to ensure lowered
Gene expression analysis
Gene expression analyses were performed as described
before . Primer sequences for quantitative RT-PCR
were as follows: CPD_fw 5 GGA AAC ACT CTC TGC
TTC TTA TGA AAG GT 3, CPD_rev 5 AAG TAA
AGC CAC CAA GAA GTC AAC AAT CT 3, DWF4_fw
5 AAT CCT TGG AGA TGG CAA CAG C 3, DWF4_rev
5 TCT GAA CCA GCA CAT AGC CTT GG 3, eIF1_fw
5 TTG ACA GGC GTT CTG GTA AGG 3 and
eIF1_rev 5CAG CGT CAC CAT TCT TCA AAA A 3
Light microscopy was performed as described before .
Cell size and number determination covered all parts of
the leaf sparing cells surrounding the primary vein and at
the edge of the leaves. For transmission electron
microscopy, leaf samples were fixed in 2.5% glutaraldehyde,
0.1 M cacodylate buffer (pH 7.4), 5 mM calcium chloride
for 4 h at 4C, and post-fixed with 1% Os04 and 0.8% K3Fe
(CN)6 for 2 h at 4C. The samples were washed with water
and post-stained with 2% aqueous uranyl acetate for 2 h.
Subsequently, the tissue was dehydrated in a series of
ethanol and propylene oxide and embedded in Spurrs low
viscosity epoxy resin. Ultrathin sections (6070 nm) were
cut with a Leica UC6 ultramicrotome using a diamond
knife, stained with uranyl acetate and lead citrate and
examined on an energy-filtering transmission electron
microscope (EFTEM, Zeiss) at 120 kV.
Protein and starch levels
Protein and starch levels were determined in leaves one to
four. The Quick Start Bradford Protein Assay (BioRad)
was used as described in the manufacturers description.
Starch levels were determined as described before .
50 mg of powdered plant material from leaves one to
four was used per extraction. Extraction of metabolites,
LC-MS measurements and data analysis was performed
as described by Giavalisco and coworkers . For
GCMS analysis, the polar phase of the same extraction was
used and carried out as described before .
Availability of supporting data
The data sets supporting the results of this article are
included within the article and its additional file.
Additional file 1: Figure S1. Growth parameters of rosette leaves five
and six of CPD-antisense and cbb1 plants. Figure S2. Growth parameters
of rosette leaves five and six of BRZ treated plants. Figure S3. Epidermis
cell number of leaves three and four. Figure S4. Transversal sections of
rosette leaves three and four. Figure S5. Relative levels of TCA cycle
intermediates in the wild type and cbb1 mutant. Table S1. Relative
hexose and sucrose levels. Table S2. Relative levels of TCA cycle
intermediates in BRZ treated plants. Table S3. Relative -ketoglutarate
levels in the cbb1 mutant.
FS carried out the plant experiments, gene expression, protein and
metabolite analysis and helped to draft the manuscript. JL carried out the
light microscopy. TO contributed to the flux analysis. AE and PG did the
pre-processing of the metabolite data. EM performed the electron microscopy.
JK, ARF and LW participated in the design of this study. CM conceived the
study, participated in its design and coordination and drafted the manuscript.
All authors read and approved the final manuscript.
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