PLANT SIGNALING & BEHAVIOR

2018, VOL. 13, NO. 6, e1482175 (7 pages)

https://doi.org/10.1080/15592324.2018.1482175

RESEARCH PAPER

Development of sugar beet leaves: contents of hormones, localization of abscisic

acid, and the level of products of photosynthesis
G. R. Kudoyarovaa, A. K. Romanovab, N. S. Novichkovab, L. B. Vysotskayaa, Z. Akhtyamovaa, G. R. Akhiyarovaa,
S. Y. Veselovc, and B. N. Ivanovb
a
Ufa Institute of Biology, Ufa Research Centre, Russian Academy of Sciences, Ufa, Russia; bInstitute of Basic Biological Problems, Russian Academy of
Science, Pushchino, Russia; cBashkir State University, Biological department, Ufa, Russia

ABSTRACT ARTICLE HISTORY

The level of hormones in the tissues of sugar beet leaves of different age in parallel with their growth Received 17 April 2018
and metabolic activity was assayed; the latter was analyzed, measuring the contents of sugars and Accepted 23 May 2018
N-containing compounds, and the activities of Rubisco and proteases. The highest auxin and ABA KEYWORDS
concentration was detected in the actively growing upper leaf, while high level of cytokinins was Beta vulgaris; abscisic acid;
maintained in the middle and upper leaves characterized by intensive photosynthesis. Leaf senescence carbohydrates; chlorophyll;
being manifested in decline of chlorophyll content, decrease of photosynthesis and activation of glucose; Rubisco;
proteolysis was accompanied by a decline in concentration of cytokinins. Glucose level gradually senescence
increased from upper (younger) to a lower (elder) leaves; this was accompanied with the signs of
senescence on the background of decreased cytokinins level. Immuno-histochemical technique revealed
increased level of abscisic acid in phloem parenchyma of the lowest leaf. The results suggest a possible
involvement of auxins in maintaining leaf growth, an implication of decreased cytokinins level in the
hypothesized induction of senescence by glucose, and a participation of abscisic acid in the active
loading of metabolites into the phloem of senescing leaf.

Introduction was in some cases accompanied by accumulation of glucose,-

10,11
the increase in glucose concentration might stimulate
Leaf growth and development are of vital importance for
ABA accumulation during leaf aging and senescence.
plants, since these processes result in the formation of CO2
Sugar beet is a suitable model organism for the study of
assimilating organ. Leaf capacity to supply sink organs with
interaction of sugars and hormones in the control of senes-
assimilates increases with the development of the source
cence. In the zones with temperate climate, sugar beet vege-
leaves. Source function of the leaves reaches its maximum
tates as a biennial plant with pronounced rosette
during their senescence, when degradation and remobilization
morphology capable of accumulating record store of sucrose
of polysaccharides and proteins take place1,2. The process of
in the roots that are the main sink organ. During the first
senescence is manifested in destruction of chloroplasts; these
season of sugar beet development, when only vegetative
organoids containing most part of leaf proteins, whose degra-
growth takes place and hormonal regulation of flowering
dation is a rich source for reutilization of nitrogen.3
does not interfere with the processes, gradual transition
Importance of leaf development and senescence explains per-
from sink properties of young leaves to the source properties
sistent interest of researchers to the mechanisms regulating
of fully developed leaves takes place. Mesophyll cells of fully
these processes. Hormone abscisic acid (ABA) is one of endo-
developed leaves are both utilizing and exporting sucrose,
genous factors implicated in the process of senescence4.
while with further aging and senescent leaf acquirers predo-
Acceleration of senescence detected under stress conditions
minantly source properties.2 As far as we know ABA content
was related to accumulation of ABA5-7 frequently character-
has been previously reported for this species only in infected
ized as stress hormone. Meanwhile the role of ABA in reg-
plants12 and during seed formation,13 and not in the leaves
ulating leaf senescence was studied to a lesser extent in the
of different position and age.
absence of stress, while, contrary to expectations, some experi-
The goal of the present research was to reveal peculiarities
ments with the mutant plants showed delayed senescence in
of the changes in the levels of ABA, cytokinins (zeatin deri-
plants with elevated ABA content8.
vatives) and auxin (indoleactetic acid, IAA) and in distribu-
Interaction between ABA and sugars is one of important
tion of ABA in the leaves of sugar beet, and to collate them
aspects of the control of growth and development. Glucose-
with the level of metabolites during transition from sink
induced growth inhibition detected at the early stages of plant
properties of the young emerged leaf to the predominately
development is suggested to be due to up-regulation of the gin
source properties of the eldest leaf. Study of ABA distribution
(glucose insensible) gene coding for dehydrogenase/reductase
seems to be important in the light of the data showing that
involved in ABA synthesis.9 Since the process of senescence

CONTACT Boris Ivanov ivboni@rambler.ru Institute of Basic Biological Problems, Russian Academy of Science, Pushchino, Russia
© 2018 Taylor & Francis Group, LLC
e1482175-2 G. R. KUDOYAROVA ET AL.
estimation of signal molecules content in the whole organ
may be not sufficient for detecting its role in the control of
senescence, and that the analysis on the level of separate cells
may be necessary14. It was important to reveal possible impact
on leaf growth and senescence in sugar beet of both cytoki-
nins, since the hormones has been previously shown to reg-
ulate these processes in other species15, and auxins due to the
previously reported their role in the regulation of wheat leaf
growth16. Not only the content of sugars, but also that of
chlorophyll and proteins served as indicators of leaf matura-
tion and senescence as well as of an increase in their source
properties.17
Results
Out of 15 leaves that had appeared, cotyledons and the first
pair of true leaves were already absent by the time of sam-
pling. The upper leaves including 18d leaf had only partially
emerged and were still growing, while the middle 40d leaves
were already mature. The fresh weight and the area of leaves
increased with their age from 0.54 g in 18d leaf to 1.78 and
2.22 g in 40d and 58d leaves, correspondingly, but specific leaf
mass (leaf mass per unit leaf area) was only about 1.2 times
higher in 40d and 58d leaves than in 18d leaves (Table 1). At
the same time, the dry mass expressed as percentage per unit
of fresh mass was highest in the 18d leaves and decreased in
the 40d leaves and 58d leaves.
The details of mesostructure of leaves of different age can
be seen under light microscope on the transverse leaf sections
treated with immune serum after fixation (Figure 1).
Parenchyma cells of 18d leaf are rather small, tightly pressed
together, while intercellular space is almost absent
(Figure 1A). Structure of the 40d leaf (described below as
“mature” leaf) is less dense, vacuoles are increased, and cyto-
plasm stretches as a thin layer along the cell walls (Figure 1C).
The eldest leaf of the sampled leaves (58d) was characterized
by loose parenchyma and great intercellular space (Figure 1E).
These peculiarities of structure correlated with the decline in
the dry mass percentage with the leaf age as seen in the
Table 1.
In the sections of 18d leaves, the most intensive immunos-
taining for ABA was detected in the cell periphery and chlor-
oplasts (Figure 1A). Staining decreased visibly with the leaf
age (Figure 1C and E). Study of the leaf section with greater
resolution at the site of small vessels showed weak staining for
ABA in the 18d leaves (Figure 2B). However, distinct staining
appeared in the cells accompanying the vessels of the eldest
Table 1. Morphological characteristics of sugar beet leaves of different age.
Leaf age (days)
Characteristics 18 40
Fresh leaf weight, g 0.54 ± 0.06a 1.78 ± 0.18b
Leaf area, см2 17.2 ± 1.3a 46.3 ± 4.0b
Specific leaf weight (fresh leaf 314 ± 37a 384 ± 49b
weight per unit leaf area),
g/m2
Dry mass, % of the fresh 18.5 ± 1.9 a
15.1 ± 1.8
weight
Similar letters mean the absence of difference, different letters – significant
difference.
58d leaves (Figure 2A). Staining of the sections with non-
immune serum was very weak and cell structures were hardly
visible (Figure 2C), supporting specificity of staining.
Table 2 shows that the content of the sum of soluble
carbohydrates without glucose in the 18d leaves was lower
than in the fully emerged leaves. The difference between the
40d and 58d leaves was not great and statistically insignificant.
The content of glucose was lowest in the 18d leaves, visibly
increased in the 40d leaves and was even greater in the 58d
leaves; the difference in glucose content between the eldest
and intermediate leaves being statistically significant.
Meanwhile, comparison of the content of different
N-containing substances in the leaves draws attention to the
similar tendencies in their changes with the leaf age. The
content of soluble proteins was equally high in the 18d and
40d leaves and then declined in the 58d leaves. Chlorophyll
content (a + b) was similar in the 18d and 40d leaves, but was
about 2.6 times lower in the 58d leaves. The decline in
Rubisco activity in the 40d vs. 18d leaves was negligible, but
the enzyme was about 2 times less active in the 58d leaves.
Protease activity was about 1.5 times higher in the mature
leaves than in the upper growing 18d leaves and more than 6
times higher in the eldest leaves in comparison with the 18d
leaves (Table 2).
Determination of the bulk content of hormones in the
leaves of different age showed high level of the three hor-
mones (IAA, ABA and cytokinins) in the youngest upper leaf
(at the age of 18 days since its appearance) (Table 3). In the
next mature (middle 40d leaf), total cytokinin content (sum of
zeatin, its riboside and nucleotide) was maintained at the
similar high level as in the young 18-d leaves, but was more
than 2 times lower in the ageing 58d leaves, than in the upper
leaves. The content of IAA decreased from the upper to the
lowest leaves: 1.5 -fold decline was detected when the young-
est 18d leaves and mature 40d leaves were compared, and in
the eldest 58-d leaves IAA content was about 6 times lower
than in the upper leaves. The content of ABA was lower in the
middle leaf as compared to the youngest upper leaf, while no
difference was detected in its content in between the middle
(40-d) and lowest (58d) leaves.
Discussion
The upper sampled leaves were smaller than the lower leaves,
indicating that 18d leaves were still actively growing. The
growth of upper leaves of sugar beet may be related to ele-
vated concentration of IAA detected in these leaves (Table 3).
It was shown previously that the increase in the content of
this hormone contributes to activation of leaf cell
elongation.16
The middle-age leaf was distinguished by high level of chlor-
58 ophyll, soluble proteins and Rubisco, indicating high activity of
2.22 ± 0.24c metabolic processes during the period between 18d and 40d
58.5 ± 4.9c
380 ± 48b leaves. These leaves were enriched with most of studied meta-
bolites. In the oldest leaves, photosynthesis decreased as shown
b
14.2 ± 1.7 b in the previous reports.17,18 The indicated decrease in the photo-
synthesis of the oldest leaves is in line with the drop of Rubisco
activity found in the present study (Table 2). These leaves were
characterized by the decline in protein content and Rubisco
 PLANT SIGNALING & BEHAVIOR e1482175-3

Figure 1. Immunolocalization of ABA in the sections of sugar beet leaves of different age (A, B – 18-days old, C, D – 40-days-old; E, F – 58-days-old), treated with
serum against ABA (A, C, E) and normal non-immune serum (B, D, F). 1 – epidermis; 2 – palisade mesophyll; 3 – spongy mesophyll; 4 – vascular tissue; 5 – cells
containing inclusions. Scale bars – 100 µm.

activity, while the content of soluble sugars was maintained at
comparatively high level (Table 2). The maintenance of soluble
sugar levels might be due to hydrolysis of starch accumulated
earlier. The data obtained showed that the most important
processes related to senescence were likely to occur during the
period of development in between 40-days- and 58-days-old
leaves. The drop in the potential activity of Rubisco was the
most demonstrative. This effect was likely to be due to hydrolysis
of the enzyme suggested by increased protease activity in the
aging leaf. Thus, the estimations of the leaf size and the content
of metabolites in them served as criteria for characterizing leaf
growth, development and senescence that was important for
interpretation of the data on hormone content presented below.
The fact that highest ABA content was detected in the
upper still growing leaf of sugar beet (Figure 1 and Table 3)
is in accordance with some reports on high level of this
hormone in young leaves of Ricinus, Xanthium, and
Tradescantia virginiana19,20. These data contradicts with the
notion that ABA is a growth inhibitor that has been dominat-
ing until recently. Meanwhile this assumption has been refuted
by the data showing that ABA deficient Arabidopsis mutants
e1482175-4 G. R. KUDOYAROVA ET AL.

Figure 2. Immunolocalization of ABA in the cells of vascular bundles of leaves of different age (A – 58-days-old; B, C – 18 days old). C – section treated with non-immune
serum. 1 – phloem, 2 – xylem. Scale bars – 50 µm.

Table 2. Biochemical characteristics of sugar beet leaves of different age. Values

of substances were calculated per 1 g of leaf dry mass. No accumulation of ABA was detected in the aging senes-
Leaf age, days cent leaf of sugar beet (Table 3). It is not excluded that at the
Characteristics 18 40 58
later stages of senescence such an accumulation could have
С-substances
been detected, since elevated level of this hormone was regis-
Soluble carbohydrates 62.22 ± 3.14a 73.51 ± 3.0b 79.65 ± 2.4b tered at the stage close to leaf death,10 while leaves at this stage
without glucose, mg were not sampled in our experiments. Nevertheless, increased
Glucose, mg 3.73 ± 0.11a 4.90 ± 0.13b 6.79 ± 0.14c
N-substances content of ABA was detected in the leaves of phloem par-
Soluble protein, mg 130.59 ± 3.51a 136.23 ± 5.8a 76.90 ± 11.0b enchyma of the lowest senescent leaf of sugar beet (Figure 2A)
Chlorophyll a plus b, mg 9.51 ± 0.59a 9.60 ± 0.66a 3.73 ± 0.21b
Activities of enzymes
suggesting implication of ABA in increased export of meta-
Rubisco, *µmol min−1 24.54 ± 0.65a 22.19 ± 0.60a 9.01 ± 0.28b bolites from the senescent leaf. This assumption is in accor-
Protease activity, µmol 99.5 ± 1.6a 185.4 ± 24.5b 696.0 ± 233.1c dance with the data, showing correlation between ABA
NNH2 h−1
content in the flag leaf of the wheat plants and remobilization
*Changes in potential activities are equivalent to the changes in enzyme protein
content. of carbohydrates under moderate drought in accordance with
Similar letters mean the absence of difference, different letters – significant decreased chlorophyll content in the leaf treated with exogen-
difference. ous ABA.26
Increased content of ABA in the phloem parenchyma of
the lowest senescent leaf of sugar beet was likely to be due to
Table 3. Content of hormones (ng g−1 fresh weight) in sugar beet leaves of
different age.
redistribution of the hormone between the leaf cells. These
Leaf age, days
results suggest direct participation of this hormone in the
increased phloem loading in the senescent leaves, which is
Hormones 18 40 58
in accordance with the data showing capacity of ABA to
Cytokinins 4.1 ± 0.3a 4.9 ± 0.5a 2.3 ± 0.2b
IAA 33 ± 3a 22 ± 1b 5,3 ± 0.6c activate sugar transporters. This is achieved either at the
ABA 6.1 ± 0.5a 3.7 ± 0.4b 3.1 ± 0.2b level of up-regulation by ABA of the genes coding for sugar
Similar letters mean the absence of difference, different letters – significant transporters,27 or at the expense of ABA-induced activation of
difference
ATPases,28 providing the energy for secondary active trans-
port. There is at least one more possible mechanism connect-
ing ABA with the export of metabolites from senescent leaf
had smaller sizes as compared to the wild type plants even through phloem. Thus it was shown that callose lining of sieve
under conditions of high humidity excluding the effects of plate pores is essential for normal phloem transport because it
ABA at the level of transpiration.21 ABA has been shown to confers favorable flow characteristics on the pores,29 while
accelerate leaf growth by inhibiting production of ethylene in exogenous ABA has been shown to promote callose
tomato.22 This hormone is suggested to induce different set of deposition.30
genes in mature and aging leaves.23 It is stated in the review by The role of sugars in leaf senescence is actively discussed.
Schippers et al.,1 that ABA has a dual role. It down-regulates Nevertheless, both increased concentration of sugars and their
expression of the genes coding for the proteins of chloroplasts, deficit may serve as stimulus for senescence, while accumula-
and thereby induces the processes of senescence24 and, on the tion of sugars in the senescent leaf may be not the cause, but
contrary, demonstrates the positive effect on chloroplast devel- the consequence of senescence.14 Previously no significant
opment in younger plants25 (possibly in cooperation with increase in the bulk soluble sugars was detected in the course
other factors). Thus, high level of ABA registered by us in of leaf senescence in sugar beet,18 reproduced in the present
young leaves and their chloroplasts of sugar beet (Figure 1A) is experiments. Nevertheless, in the present experiments we
not likely to prevent subsequent growth and development of detected accumulation of glucose in the eldest leaf, while
the leaves and accumulation of chlorophyll in them. previous experiments showed a decline in the content of
chlorophyll in glucose-treated leaves of sugar beet.18 Glucose

is believed to serve as a signal regulating many processes in
the plants linked to hexokinases that executes not only
enzyme, but also glucose sensor functions.31 Addition of
glucose to the nutrient solution of young plants activated
ABA synthesis, while sensitivity to glucose (manifested in its
ability to inhibit growth and photosynthesis in young plants)
decreased in ABA deficient plants.9 However, interaction of
ABA and glucose signaling was not detected in senescent
plants.4 These data are in accordance with the results of the
present experiments, where no bulk accumulation of ABA was
detected in senescent leaves, where concentration of glucose
increased.
It is of interest that increased concentration of glucose
registered in the mature leaves as compared to the younger
leaves was not accompanied by a decline in chlorophyll con-
centration, which remained high in this mature leaf in the
present experiments. The content cytokinins remained high in
the mature leaf of sugar beet. These hormones have been
previously shown to eliminate the inhibiting effect of glucose
on plants.32 Furthermore, capacity of cytokinins to stimulate
photosynthesis and to prevent senescence is well-known.33
These data suggest that high level of cytokinins detected in
the mature leaf of sugar beet were capable of preventing the
induction of the processes of senescence by high concentra-
tion of glucose in the leaves. As a continuation of this
assumption we may suggest that, only on the background of
decreased concentration of cytokinins, accumulation of glu-
cose was accompanied by senescence and was likely to stimu-
late this process. This assumption needs further experimental
confirmation.
Thus, estimation of the contents of hormones, sugars,
chlorophyll and proteins in the leaves of different age of
sugar beet allowed revealing the relationship between
increased concentration of auxins in the young leaves and
their active growth, as well as participation of cytokinins in
the regulation of leaf development by preventing senescence
in actively functioning leaf. Accumulation of glucose in the
leaves was likely to initiate the processes of senescence only on
the background of decreased cytokinin concentration. Bulk
ABA concentration was high in young leaves and decreased
with their aging. Nevertheless, we were first to show increased
content of ABA in the cells of phloem parenchyma of old
leaves with the help of immunohistochemical technique,
which is accordance with the assumed role of this hormone
in the export of metabolites from the senescent leaves.
Materials and methods
Plant growth
Sugar beet plants (Beta vulgaris L., subspecies saccharifera
(“white beet”)) were grown at 16-h photoperiod and irradi-
ance of 400 µmol m–2 s–1 in one-liter pots filled with sand
(one plant in a pot) as described.2 In the present work nitro-
gen and water deficit were excluded due to regular watering
and supply of plants with Knop’s nutrient solution. Chemical
analysis was performed for 70-days-old plants. The age of
individual leaves was determined visually from the time of
their appearance till the date of sampling. The following leaves
PLANT SIGNALING & BEHAVIOR e1482175-5
have been chosen for analysis: young still growing leaves (18-
days-old – 18d), mature fully developed leaves that reached
their maximum size (40-days-old – 40d) and the eldest leaves
(58-days-old – 58d). Dry weight of leaves fixed at 105°C was
determined after further drying at 80º С. Discs excised from
leaf parts without great vessels sampled at the same time after
3 h of illumination following dark night period were used for
all analysis. The content of soluble carbohydrates (sCH) in
water-alcohol extract was determined with the help of phenol-
sulfuric acid reagent using sucrose for calibration;34 glucose
was measured with the help of glucose-oxidase using Clark
oxygen electrode;18 sum of chlorophyll a plus b was deter-
mined spectrophotometrically in 96% ethanol extract;35 the
content of soluble proteins (sProt) was measured with thehelp
of Folin reagent36 after sedimentation of proteins in the buf-
fered extract with 4% thrichloroacetic acid. Extracts for deter-
mination of Rubisco activity were obtained by leaf
homogenization in cooled 0.05 M Tris-HCl buffer in the
presence of stabilizing additives. Potential Rubisco activity
was measured under conditions of full saturation of carbox-
ylase function with bicarbonate as described.2,37 Preparations
for determination of protease activity were obtained according
to Wittenbach.38 Intensity of biochemical processes was cal-
culated per mg of leaf dry.
The obtained results were processes with the help of one-
way ANOVA and Bonferroni’s method for confirmation of
data variability and significance of the differences.
Immunoassay of plant hormones
Plant hormones were extracted from leaves with the help of
80% ethanol. Further purification of IAA and ABA was per-
formed according to modified scheme as described39. The
hormones were partitioned twice with diethyl ether from
acidified aliquots of aqueous residue (ratio of organic to aqu-
eous phase 1:3) and then returned to 1% solution of sodium
hydrocarbonate (ratio of aqueous to organic phase 1:3), acid-
ified, partitioned twice with diethyl ether and methylated.
Dried samples were diluted in a small amount of ethanol
and enzyme immunoassay was performed as described with
specific antibodies to IAA and ABA.40
Cytokinins from aliquots of aqueous residue were concen-
trated on C18 cartridge [Bond-Elut, RP-C18] and after eva-
poration of ethanol eluates; hormones were dissolved in 70%
ethanol and placed on thin layer plates for chromatography as
described.41,42, Zeatin, its riboside and nucleotide, whose posi-
tion was detected with the help of standards under ultraviolet
(250 nm), were eluted with phosphate buffer and immunoas-
sayed. Previously, reliability of the data obtained by means of
immunoassay was confirmed by their comparison with the
results of physicochemical methods based on chromatography
and mass-spectrometry.43
Immunohistochemical localization of hormones in the leaf
cells was performed with the same specific antibodies to ABA
as described.44 In short, hormones present in the leaf cuts
were fixed with 4% 1-ethyl-3-(3-dimethylaminopropyl) carbo-
diimide (Sigma, St Louis, MO, USA) dehydrated in ethanol
series and embedded in methacrylate resin JB-4 (Electron
Microscopy Sciences l, USA). Hormones on the sections
e1482175-6 G. R. KUDOYAROVA ET AL.
were revealed with the help of specific antibodies against
ABA. First antibodies bound to cell structures were revealed
with the secondary antibodies labeled with colloidal gold and
developed with silver enhancer as described.44 Development
of staining was visualized with the help of light microscope
(Carl Zeiss, Jena, Germany). Specificity of antibody binding in
the present experiments was confirmed with by the absence of
staining, when specific serum was substituted with non-
immune serum. Previously specificity was also confirmed in
experiments, where uptake of exogenous ABA increased
immunostaining, while decreased staining was detected in
the case of sections obtained from ABA deficient mutant.44
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed
Funding
This work was supported by Russian Science Foundation (project No.
17-14-01371) in the part of the hormone studies. The study was partly
supported by funding the theme No. АААА-А17-117030110135-1 by
FASO of Russia
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