Download Effects of excess and deficient boron and niacin

Turkish Journal of Botany
http://journals.tubitak.gov.tr/botany/
Research Article
Turk J Bot
(2013) 37: 160-166
© TÜBİTAK
doi:10.3906/bot-1202-22
Effects of excess and deficient boron and niacin on the ultrastructure of root cells in
Daucus carota cv. Nantes
Hatice DEMİRAY*, Aylin EŞİZ DEREBOYLU
Section of Biology, Department of Botany, Faculty of Science, Ege University, 35100 Bornova, İzmir, Turkey
Received: 14.02.2012
Accepted: 17.09.2012
Published Online: 26.12.2012
Printed: 22.01.2013
Abstract: The effects of excess and deficient boron and niacin on vascular tissues of carrot roots (Daucus carota L. cv. Nantes) were
investigated in plants grown in medium both rich and poor in boron and also boron with niacin. Five media were investigated: control
(MS medium), boron-deficient MS medium, MS medium with excess boron, niacin-deficient MS medium, MS medium with niacin
excess, and MS medium with excess boron and niacin. In anatomical cross sections, lignification was seen in middle lamellar pectins
in the tracheary cells of boron deficit grown carrot roots, while in the other applications including excess boron lignification was in the
secondary walls. Number of xylem arches and tracheary lengths of root cells were different, but not significantly so. Scanning electron
microscopic (SEM) sections of vessels from roots grown in media with excess boron and deficient boron revealed paramural bodies in
the tracheary walls. Paramural bodies were found in the tracheary cell walls of both boron deficient and boron excess grown carrot roots.
In root cells grown in media with excess and deficient boron, tracheary cells had amyloplasts. While the boron deficient medium grown
carrot roots had amyloplasts scarcely, in boron excess grown root cells these amyloplasts filled the vessels densely.
Key words: Daucus carota, amyloplasts, excess boron
1. Introduction
Boron (B) is an essential microelement for higher plants
and has important physiological functions in plant
growth and development (Goldbach et al., 2007). During
the past decade, B has been shown to be essential to the
structure and function of plant cell walls and membranes,
in addition to causing different effects on root elongation,
carbohydrate metabolism, and pollen tube growth (O’Neill
et al., 2004; Goldbach & Wimmer, 2007; CamachoCristóbal et al., 2008). B deficiency is frequently observed
because the boric acid in soil is easily leached under
high rainfall conditions, thus reducing the yield of many
agricultural products, such as wheat (Triticum aestivum
L.), barley (Hordeum sativum L.), and Citrus L. species
(Jamjod et al., 2004; Yan et al., 2006; Tanaka & Fujiwara,
2007). In addition, considerable genotypic variations in
response to B deficiency exist among many agricultural
crops, or even cultivars within a species, as reported by
Jamjod et al. (2004).
Due to the importance of B as an essential element for
higher plants, B deficiency and B toxicity are a worldwide
problem in food production because of their reducing crop
quality and yields in soils, especially in arid areas (Nable et
al., 1977). The primary effect of B deficiency is reduction
*Correspondence: [email protected]
160
of cell enlargement in growing tissues, which has been
explained mainly by the structural role of B in the cell
wall. Borate cross-linked (RG-II) rhamnogalacturonan
II, a pectic polysaccharide, was shown to be essential for
plant growth and B was found to be located predominantly
in cell walls in association with rhamnogalacturonan II
(O’Neill et al., 2004). In addition to B cross-linked RG-II
pectins (Höfte, 2001), the pectin network of calcium crosslinked de-esterified homogalacturonan pectins (Matoh
& Kobayashi, 1998; Jarvis, 1984) is also important for
the regulation of the mechanical properties of cell walls
(Li et al., 1997). Ricardo et al. (2004) suggested that the
formation of di-pentose-borate complexes might have
stabilised ribose/ribulose (besides other cyclic pentoses
such as arabinose, xylose, and lyxose) in pre-biotic phases
in interstellar dust or during the earth’s early history.
As a consequence of its essential role in growing tissues
and inherent phloem mobility of B in most plant species,
many species are also sensitive to high levels of B in soil and
water and growth inhibition has been retarded as a result
of excess B uptake in many agricultural regions. B uptake
under conditions of adequate or excessive B concentration
is the result of passive diffusion of undissociated boric
acid (H3BO3) (Brown & Shelp, 1997; Hu & Brown, 1997).
DEMİRAY and EŞİZ DEREBOYLU / Turk J Bot
In fact, at high external B levels, significant B transport
occurs through the plasma membrane aquaporins
(Dordas et al., 2000; Dordas & Brown, 2001). Moreover,
B deficiency caused a weakening of the interaction
among pectic polysaccharides due to a decrease in boronrhamnogalacturonan II cross-linkage (Kakegawa et al.,
2005). Occasionally, the disorganisation of middle lamellae
occurred and the accumulation of numerous vesicles was
observed in the disorganised area of the cortical cells of
B deficient grown tomato (Kouchi and Kumazawa, 1976).
B toxicity causes significant changes in the physiology
and activity of numerous enzymes and, consequently, the
metabolism during the life cycle of plants. Three main
candidate sites have been suggested in view of the ability
of B to bind compounds with 2 hydroxyl groups in the
cis-configuration: (i) alteration of cell wall structure; (ii)
metabolic disruption by binding to the ribose moieties
of molecules such as adenosine triphosphate (ATP),
nicotinamide adenine dinucleotide (reduced form,
NADH), or nicotinamide adenine dinucleotide phosphate
(reduced form, NADPH); and (iii) disruption of cell
division and development by binding to ribose, either
as free sugar or within RNA (Reid et al., 2004). In the
present study, we used niacin (nicotinic acid) to restrict B
accumulation in root cells, and we examined the effects of
excess B and excess B/excess niacin on the ultrastructure
of the vascular system in carrot roots.
2. Materials and methods
2.1. Seed germination and culture conditions
In our experiments we used Nantes carrot (Daucus carota
L.) seeds. The seeds were germinated in Murashige-Skoog
(MS) (Murashige & Skoog, 1962) nutrient medium and in
vitro conditions. Media were prepared as follows: without
boron (0), with 6.2 mg/L boron and 0.5 mg/L niacin
(control = MS), with 31 mg/L (5-fold) boron (5B), with 2.5
mg/L niacin (5-fold) (5N), without niacin (0N), and with
31 mg/L boron and 2.5 mg/L niacin (5B+5N) added.
Seeds were sterilised with hypochlorite solution (7.5%)
by shaking for 20 min before sowing to the media. After
sterilisation, the seeds were washed with sterile distilled
water and sowed to culture jars of 200 mL, with 5 seeds in
each. The jars were placed in a Fitotron® regulated to 16
h/8 h photoperiod and 25 ± 2 °C temperature conditioned.
The experiments were performed 3 times and 10 seeds
were used in every application. After 8 weeks the seedlings
were harvested.
2.2. Anatomical investigations
Transverse hand sections, approximately 0.2 to 0.5 mm
thick, were made with a razor blade from roots. Replicate
sections were stained with Mirande’s reagent (a mixture of
carmin alum and iodine green) (Deysson, 1954), iodinegreen and Wiesner (characterises aldehyde groups of the
cinnamic type) and Mäule solutions (to observe syringyl
groups) (Monties, 1989), and 0.2% (w/v) iodine in 5% (w/v)
KI (10 min) (Johannes et al., 2001) to reveal and confirm the
location of lignification and starch containing amyloplasts.
In the anatomical longitudinal sections the lengths of
tracheary cells were measured in all application groups.
In the cross-sections of root cells the numbers of xylem
arches in the central cylinder were counted.
Elongation zones of the roots of the seedlings in
different applications were fixed in FAA [ethyl alcohol
(50 cc): formaldehyde (10 cc): acetic acid (5 cc): distilled
water (35 cc)], embedded, and sectioned (15 μm) with a
rotary microtome. Anatomical cross and longitudinal
sections were taken from them and photographed using
an Olympus 50 Jena microscope and Leica DM 4000 B.
In addition, specimens were taken for scanning electron
microscopy (SEM) using a JSM-5200; they were covered
with gold after being fixed with gluteraldehyde, cacodylate
tampons, and osmium tetraoxide and dehydrated with
different alcohol and xylol series. These samples were
processed using 2% glutaraldehyde, in a 0.2 M cacodylate
buffer. After 2 h at 4 °C, these samples were washed in 0.1
M cacodylate buffer and dehydrated with sequences of
acetone series (25%, 50%, 75%, 90%, and 100%). They were
then dried to critical point, and covered with gold. The
coated specimens were examined in a scanning electron
microscope operating at 20 kV.
2.3. Statistical analysis
Data obtained for the lengths of vessel members and the
number of xylem arches were analysed by applying the
analysis of variance and the means were compared by the
LSD test (Steel & Torrie, 1980).
3. Results
In our application groups the lengths of trachearies
increased in MS (85.200 μm) < 5B (87.700 μm) < 5N
(90.900 μm) < 0B (96.000 μm) < 5B/5N (110.500 μm), and
finally in niacin deficit (0N) (113.000 μm) media, while
the numbers of xylem arches in vascular cylinders were
simultaneously reduced in 5N (12.83) > MS (9.83) > 0N
(9) > 5B/5N (8.83) > 0B (8) > 5B (6.83) media (Tables 1, 2;
Figure 1). There were no statistically significant differences
between the groups.
Transverse sections from the roots of carrot (Daucus
carota) in B-deficient medium stained with Wiesner and
Mäule reaction showed bright red coloration in the middle
lamellae of the cell walls of the trachearies. The colour was
dark red in the secondary walls of tracheary cells of carrot
roots grown in the other media except for the B-deficient
medium in our experiment (Figure 1).
Carrot tracheary cell walls were stained by iodine
green to determine the lignification in the walls (Mandolot
et al., 2001). The red coloration of the middle lamellae of
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DEMİRAY and EŞİZ DEREBOYLU / Turk J Bot
Table 1. Number of xylem arches in Daucus carota.
Application groups
Number of xylem arches
Table 2. Length of vessel members in Daucus carota.
Application groups
Tracheary lengths (μm)
MS
9.83
MS
85.200 ± 1.564df
5B
6.83
5B
87.700 ± 2.336df
5N
12.83
5N
90.900 ± 2.039df
5B/5N
8.83
5B/5N
B deficient
8
B deficient
96.000 ± 2.501df
N deficient
9
N deficient
113.000 ± 1.956abce
There are no significant differences between the groups (P <
0.05).
110.500 ± 2.912abce
The difference between “a” and control (MS) group, “b” and 5B
group, “c” and 5N group, “d” and 5B/5N group, “e” and boron
deficient group, “f ” and niacin deficient application group is
statistically significant (P < 0.05).
a
b
c
d
e
f
g
h
i
Figure 1. Xylem arch numbers in the cross sections of carrot roots. a- MS, b- excess boron (5B), c- deficient boron, d- excess niacin (5N),
e- deficient niacin (1 cm equals 66 μm). Cross sections of tracheary cells. f- MS, g- excess boron (secondary walls of tracheary cells were
red in staining with Mäule reagent). h- deficient boron (primary walls were red because of lignin, secondary walls were green in staining
with iodine green) (1 cm equals 66 μm). Longitudinal section of tracheary cells. i- MS (1 cm equals 66 μm).
the cell walls increased in the presence of lignified tissues
in carrot tracheary cells grown in B-deficient medium,
while the coloration was blue in excess B grown cell walls
162
of trachearies because of the absence of lignified tissues
(Figure 1). Our control group was the root cells of carrots
grown in MS standard growth medium (Figure 1).
DEMİRAY and EŞİZ DEREBOYLU / Turk J Bot
a
b
c
d
e
f
g
h
i
j
Figure 2. Amyloplasts were seen as granules in the centre of the tracheary cells in the cross sections taken from carrot root cells grown
in media with excess boron by SEM. a, b, c- Longitudinally fractured central cells with adhering globules, d, e, f, g, h, and i- Longitudinal
sections of carrot root cells grown in boron deficient medium with microporate wall layer, f and g- Paramural bodies were seen in boron
deficient root cells. j- No amyloplasts were seen in excess boron and excess niacin together.
In the root cells of carrots grown in the medium with
excess B, the rod-shaped globular amyloplasts were layered in
clusters in the middle of the tracheary cells (Figure 2), while
no globular amyloplasts were seen in the vascular tissues of
the root cells grown in the other media, except those with
excess and deficient B. The formation of paramural bodies
was observed in the cell walls of trachearies of root cells grown
in deficient and excess B, besides the observation of different
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DEMİRAY and EŞİZ DEREBOYLU / Turk J Bot
accumulation types of amyloplast globules in tracheary cells.
In the tracheary cells of carrot roots grown in B-deficient
medium, amyloplasts were scarcely observed (Figure 2).
Anatomical investigations using SEM of the tracheary cells
of carrot roots grown in media with both excess and deficient
B also revealed the presence of paramural bodies (Figure 2).
While B deficiency reduced the amyloplast accumulations
(Figure 2), amyloplast accumulations were never observed
in carrot root cells grown in excess B with excess niacin
together, only in medium with excess niacin and deficient
niacin (Figure 2), in contrast with the B-deficient carrot cells
(Figure 2).
4. Discussion
Cell walls of vascular tissues demonstrated different
responses to B deficiency and toxicity. Middle lamellae
tracheary cells were especially lignified because of the B
deficiency. B deficiency affects the structure of middle
lamellae by attending phenols that were precursors of
lignin synthesis (Lewis, 1980; Pilbeam & Kirkby, 1983;
Shkolnik, 1984; Downes et al., 1991). In addition, the
formation of dimeric B-rhamnogalacturonan-2(BRGII)borate-ester crosslinking (Ishii & Matsunaga, 1996;
Kobayashi et al., 1996; O’Neill et al., 1996) cannot occur.
This crosslink forms a macromolecular complex that
controls cellular growth (Fleischer et al., 1999) and the
mechanical properties of primary cell walls (Ishii et al.,
2001). Consequently, B deficiency leads to an accumulation
of soluble carbohydrates, leaky membranes, and DNA,
RNA, and lignin synthesis are inhibited, causing an
accumulation of low molecular weight phenolics (Çakmak
& Römheld, 1977).
B promotes sugar transport and stabilises cell walls.
Therefore, in B excess grown roots lignification was
observed in the primary and secondary walls of tracheary
cells, whereas the middle lamellae were not lignified.
An investigation on the ultrastructural effects of B
deficiency and toxicity in castor bean (Ricinus communis)
plants revealed thickening of the middle lamellae in
B deficiency and a low level of starch granules in leaves
(Herisson da Silva et al., 2008). It is known that vessel
length distribution varies in different branching levels
and under different environmental conditions (Makbul
et al., 2011). More distal branches mostly have shorter
vessels, and many trees produce longer vessels in spring
than in summer (Zimmermann & Potter, 1982). Auxins
and cytokinins play roles in vessel element differentiation
and vascular patterning (Ye, 2002). The vessel termination
chance might be the result of a hormone flux or gradient.
The high-concentration IAA streams induce protoxylem
vessels. Cytokinin (CK), synthesised in the root cap,
promotes cytokinesis, vascular cambium sensitivity,
vascular differentiation, and root apical dominance.
Auxin (indole-3-acetic acid (IAA)), produced in young
164
shoot organs, promotes root development and induces
vascular differentiation. Both IAA and CK regulate root
gravitropism (Aloni et al., 2006). B deficiency symptoms
may be the result of supraoptimal endogenous levels of
IAA. These high levels of IAA may inhibit cell division
and lead to an induction of the IAA oxidase enzyme
(Charles et al., 1977). For the reasons mentioned above,
B excess induced fewer xylem arches compared with the
other application groups while the niacin (nicotinic acid)
excess induced the most, although the results were not
statistically significant.
Of the water-soluble vitamins, nicotinic acid, precursor
of NAD and NADP, participates in cellular redox reactions.
A concentration of 32.4 μM nicotinic acid induced 76%
embryogenesis (Barwale et al., 1986). It was concluded
that reducing the concentration to half or quarter of
the Murashige et al. (1974) medium, or removing the
vitamins, did not have any significant effect on growth
over 3 passages (each 4 weeks), except in the case of 1
cultivar requiring nicotinic acid (Soczek & Hempel, 1988).
It has previously been shown that B deficiency inhibits
growth of the plant apex, which consequently results in
a relatively weak apical dominance, and a subsequent
sprouting of lateral buds. Auxin and CKs are the 2 most
important phytohormones involved in the regulation of
apical dominance. In B-deficient plants, the levels of both
auxin and CKs were reduced, and the export of auxin
from the shoot apex was considerably decreased relative to
plants well supplied with B (Wang et al., 2006).
Nicotinic acid seems to be related to the evolution
process by participating in cellular redox reactions.
Deficiency of this vitamin increased the flow of auxin
to the apex, resulting in the highest length of tracheal
cells in carrot roots. Recent studies have suggested that
Palaeozoic hyperoxia enabled insect gigantism, and the
subsequent hypoxia drove a reduction in animal size. This
evolutionary hypothesis depends on the gas exchange
in insects being almost exclusively determined by the
tracheal system, providing a particularly suitable model to
investigate possible limitations of oxygen delivery on size
(Kaiser et al., 2007). A number of highly successful insect
groups were found to be evolved in conjunction with
flowering plants, a powerful illustration of coevolution
(Carter, 2005).
Paramural bodies (lomasomes, plasmalemmasomes)
originate when exocytotic plasma membrane invaginations
contain additional internal membranous structures.
These membrane-bound vesicles are usually involved
in the deposition of cell wall components, especially
calluses, often leading to the synthesis of irregularly
thickened and folded cell walls or papillae (Siegfried,
1999). They may become more obvious in cases where
the plasma membrane has slightly retracted from the cell
wall, thus leaving a space where the normal discharge of
DEMİRAY and EŞİZ DEREBOYLU / Turk J Bot
Golgi vesicles is somehow interrupted. In root cells of
sunflower (Helianthuus annuus), paramural bodies have
been observed in response to B deficiency, adding new
cell material (Hirsch & Torrey, 1980). Similar structures
may also occur during the process of frost-hardening
(Wisniewski & Ashworth, 1986).
Paramural bodies and amyloplast presence improved
the severe effects of both B deficiency and excess in carrot
root cells’ vascular system. Higher plant organs sense
gravity primarily through the sedimentation of starchfilled amyloplasts in specialised cells called statocytes
(Caspar & Pickard, 1989; Kiss et al., 1989; Kuznetsov &
Hasenstein, 1996; Blancaflor et al., 1998; Prabhu et al.,
2011). These cells constitute the columella of the root
cap and the endodermal layer of the shoot. Amyloplast
displacement and sedimentation are thought to activate
signal transduction pathways that lead to the asymmetric
redistribution of the plant hormone auxin across the
stimulated organ (Masson et al., 2002). A greater amount
of auxin accumulates in the bottom flank of the organ,
where it inhibits cell elongation in the root and promotes it
in the shoot. As a result of this growth differential, the root
curves downward and the shoot curves upward (Masson
et al., 2002). Therefore, amyloplast flow was more densely
observed in the tracheal cells of excess B grown root cells
than in B deficient ones. Moreover, paramural bodies and
amyloplast movement were absent in the tracheary cells of
carrot roots grown in excess B/excess N media and only
excess niacin media since niacin eliminated the toxic effects
of B with a metabolic disruption by binding to the ribose
moieties of molecules such as adenosine triphosphate
(ATP), nicotinamide adenine dinucleotide (reduced form)
(NADH), or nicotinamide adenine dinucleotide phosphate
(reduced form) (NADPH) (Reid et al., 2004).
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