Saline Stress Alters the Temporal Patterns of Xylem Differentiation and Alternative Oxidase Expression in Developing Soybean Roots1
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Plant Physiol. (1998) 117: 695–701
Saline Stress Alters the Temporal Patterns of Xylem
Differentiation and Alternative Oxidase Expression in
Developing Soybean Roots1
Mirna Hilal, Ana M. Zenoff, Graciela Ponessa, Hortensia Moreno, and Eddy M. Massa*
Departamento Bioquı́mica de la Nutrición, Instituto Superior de Investigaciones Biológicas (Consejo Nacional de
Investigaciones Cientı́ficas y Tecnológicas–Universidad Nacional de Tucumán), and Instituto de Quı́mica
Biológica Dr. Bernabé Bloj, Chacabuco 461, San Miguel de Tucumán, 4000 Argentina (M.H., A.M.Z., H.M.,
E.M.M.); and Departamento de Morfologı́a Vegetal, Fundación Miguel Lillo, M. Lillo 251,
San Miguel de Tucumán, 4000 Argentina (G.P.)
velopmental stages. Plant roots provide an attractive ex-
We conducted a coordinated biochemical and morphometric perimental system for investigating salinity effects on
analysis of the effect of saline conditions on the differentiation zone growth and other parameters for the following reasons: (a)
of developing soybean (Glycine max L.) roots. Between d 3 and d 14 they have a definable growing region in the tip and a
for seedlings grown in control or NaCl-supplemented medium, we separate nongrowing region consisting of mature, elon-
studied (a) the temporal evolution of the respiratory alternative gated cells, some distance behind the tip (Ishikawa and
oxidase (AOX) capacity in correlation with the expression and
Evans, 1995); and (b) root cells can be directly exposed
localization of AOX protein analyzed by tissue-print immunoblot-
ting; (b) the temporal evolution and tissue localization of a perox-
to different NaCl concentrations by changing the root
idase activity involved in lignification; and (c) the structural medium.
changes, visualized by light microscopy and quantified by image Previously, it was reported that excess NaCl in the
digitization. The results revealed that saline stress retards primary growth medium induces structural changes in bean roots,
xylem differentiation. There is a corresponding delay in the tempo- as well as leakage of ions correlated with alterations of the
ral pattern of AOX expression, which is consistent with the xylem- cell membranes (Cachorro et al., 1995). It was also reported
specific localization of AOX protein and the idea that this enzyme that NaCl treatment leads to changes in the lipid compo-
is linked to xylem development. An NaCl-induced acceleration of sition of bean roots (Cachorro et al., 1993; Zenoff et al.,
the development of secondary xylem was also observed. However,
1994; Surjus and Durand, 1996) and affects the proton-
the temporal pattern of a peroxidase activity localized in the pri-
extrusion activity, which appears to be partially dependent
mary and secondary xylem was unaltered by NaCl treatment. Thus,
the NaCl-stressed root was specifically affected in the temporal on a H1-ATPase associated with the plasmalemma (Zenoff
patterns of AOX expression and xylem development. et al., 1994).
Knowledge about respiratory metabolism during saline
stress is scarce (Fernandes De Melo et al., 1994). In this
context, the role of the nonphosphorylating alternative
Salinity is an environmental stress that limits growth and pathway, which is a common feature of higher plant res-
development in plants. The response of plants to excess piration (Moore and Siedow, 1991; Siedow and Umbach,
NaCl is complex and involves changes in their morphol- 1995), has not been elucidated. This pathway can be in-
ogy, physiology, and metabolism. Most studies have been duced by a number of treatments generally described as
descriptive and have not elucidated mechanisms by which
stress conditions, and thus it was suggested that the AOX
salinity inhibits plant growth (Cheeseman, 1988; Munns,
pathway may be part of a stress response in plants (Purvis
1993). There are multiple genes that seem to act in concert
and Shewfelt, 1993; Day et al., 1995). The participation of
to increase NaCl tolerance, and certain proteins involved in
the AOX pathway in response to NaCl stress has been
salinity stress protection have been recognized (Bohnert
analyzed in barley leaves (Jolivet et al., 1990), but the
and Jensen, 1996; Hare et al., 1996).
reported data are difficult to interpret in part because they
Within any organ there exists a range of both cell types
were based on considerations, the validity of which has
and cell ages and, therefore, the metabolic functions and
been questioned (Millar et al., 1995; Day et al., 1996).
the responses to environmental stimuli may be expected to
An approach toward understanding the mechanisms of
vary with these different patterns of localization and de-
saline effects in young roots is to follow the time course of
a series of biochemical, physiological, and structural events
1
This work was partially supported by the Consejo de Investi- in the early stages of development. We studied the effect of
gaciones de la Universidad Nacional de Tucumán and by the
NaCl treatment on the differentiation zone of developing
Consejo Nacional de Investigaciones Cientı́ficas y Tecnológicas of
Argentina.
soybean roots by analyzing the temporal evolution of AOX
* Corresponding author; e-mail massa@insibio.unt.edu.ar; fax
54 – 81–24 – 8025. Abbreviation: AOX, alternative oxidase.
695
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.696 Hilal et al. Plant Physiol. Vol. 117, 1998
MATERIALS AND METHODS
Plant Growth and Saline Stress
Soybean (Glycine max L. var UFV-8) seeds were germi-
nated for 3 d at 28°C in sterile sand that was moistened
with tap water. Then the seedlings were transferred to
hydroponic culture in 25% Hoagland medium supple-
mented with 120 mm NaCl (saline stress) or without the
NaCl supplement (control). Plants were grown at 28°C
under greenhouse conditions and harvested when indi-
cated for each experiment during the period between d 0
(sowing) and d 14 of development. The nutrient medium
was renewed every 3 d. This standard protocol was fol-
lowed for all of the experiments, except for that experiment
whose results are shown in Figure 3.
In the experiment shown in Figure 3, the seedlings were
germinated and grown (at 28°C) in sand containing control
or NaCl-supplemented Hoagland medium (140 mL/kg
sand) over the whole period from 0 to 12 d of development.
The sand was periodically moistened with distilled water.
Selection of the Root Region Studied
Figure 1. Control (left) and NaCl-stressed (right) soybean seedlings at The differentiation zone of the primary root was studied.
d 8 of growth; magnification 30.4. To verify that the selected zone from both the control and
stressed roots was identical at the different developmental
capacity and peroxidase activity, in correlation with the stages, a segment about 4 mm long was marked gently
tissue localization of these enzymes and NaCl-induced with a pen in the differentiation zone of the primary root in
structural changes. These coordinated analyses during a 3-d-old seedlings grown in parallel with those used for the
defined growth period revealed that saline stress specifi- biochemical and morphological analyses. One-half of the
cally delays or advances the temporal evolution of deter- marked seedlings was transferred to the control medium
mined parameters and has no effect on the temporal pat- and the other half was transferred to the NaCl-
tern of others, leading to a plant that is not only smaller supplemented medium, and the localization of the selected
than the control but also with different biochemical and segment was observed during the following growth pe-
morphological characteristics. riod. This segment remained without substantial length
change and was localized almost in the middle of the
primary root in both the control and the stressed seedlings
over the period studied.
Figure 2. Temporal evolution of AOX capacity in the differentiation
zone of roots from control (E) and NaCl-stressed (F) seedlings. At d Figure 3. Temporal evolution of AOX capacity in the differentiation
6 of growth, a group of the seedlings was transferred from the control zone of roots from seedlings grown in sand containing control (E) or
to the saline medium (M) or from the saline to the control medium NaCl-supplemented (F) Hoagland medium from d 0 to 12. Each
(‚). Each value is the mean 6 SD of three separate measurements. Fw, value is the mean 6 SD of two separate measurements. Fw, Fresh
Fresh weight. weight.
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Assays of AOX Capacity and Peroxidase Activity phosphate buffer (pH 7.2). The samples were dehydrated
with a graded series of ethanol, ending with 100% acetone,
AOX capacity was measured as described previously
and then embedded in Spurr’s medium (Spurr, 1969) and
(Hilal et al., 1997) in slices of the selected segment from the
polymerized overnight in a 60°C oven. Cross-sections (0.5
primary root-differentiation zone of control and stressed
mm) were prepared with an ultramicrotome and stained
seedlings during the growth period between d 2 and 14.
with toluidine blue (Richardson et al., 1960) before visual-
Peroxidase activity was determined in extracts of the
ization with a light microscope.
selected root segments, as described by Peyrano et al.
The number of xylem vessels and the areas occupied by
(1997), using the substrate syringaldazine. The specific ac-
the xylem and phloem in the stele and the intercellular-to-
tivity was expressed as the increase in A530 per minute and
cellular-area ratios in the cortex were determined from
milligram of protein. Protein concentration was measured
images of the root cross-sections digitized with a charged-
by the procedure of Lowry et al. (1951).
coupled device 200E video camera (Videoscope Interna-
tional, Washington, DC) coupled to a Macintosh Quadra
Tissue Prints 700 computer. Image analysis and quantitation were per-
formed with NIH Image 1.45 software (Rasband W, Na-
Tissue printing of cross-sections from the differentiation
tional Institutes of Health, Bethesda, MD).
zone of primary roots (selected as indicated above) and
specific immunostaining with anti-AOX monoclonal
antibody were performed as described previously (Hilal et RESULTS
al., 1997) at d 8 of plant growth under control or saline
conditions. Figure 1 shows the appearance of control and NaCl-
Tissue prints of the same root zone were also made on d stressed seedlings at d 8 of growth. Roots of plants treated
3 and d 10 of control and stressed seedlings to detect with NaCl were shorter and had fewer secondary roots
activity of syringaldazine oxidase, a peroxidase associated than the controls. Saline stress decreased the growth rate of
with lignification (Goldberg et al., 1983). The assay condi- soybean seedlings, a well-known phenomenon.
tions were as described by Peyrano et al. (1997)
Temporal Evolution of AOX Capacity in Control and
Mophometric Analysis of the Root-Differentiation Zone NaCl-Stressed Roots
The selected segments from the root-differentiation zone AOX capacity in the differentiation zone of control roots
of control and stressed seedlings were fixed in filtered greatly decreased between d 3 and 8, as already reported
control or NaCl-supplemented Hoagland medium, respec- (Hilal et al., 1997), whereas in the stressed roots AOX
tively, with 3% glutaraldehyde for 6 h at 4°C and then capacity remained high at d 8 (Fig. 2) and declined several
postfixed overnight with 1% osmium tetroxide in 0.1 m days later than in the controls. At d 6 of development,
Figure 4. Localization of AOX protein. Tissue
prints of cross-sections from the differentiation
zone of control (A and B) and NaCl-stressed (C
and D) roots at d 8 of growth. A and C, Amido
black stains of total protein. B and D, Immuno-
stains specific for AOX. x, Xylem; p, phloem;
and c, cortex. Bars 5 250 mm.
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.698 Hilal et al. Plant Physiol. Vol. 117, 1998
when some of the stressed seedlings were transferred to the
control medium, their root AOX capacity decreased earlier
than that of the seedlings maintained in saline medium
(Fig. 2). When some of the control seedlings at d 6 of
development were transferred to the saline medium, they
retained their root AOX capacity for a longer period than
those remaining in the control medium. Results in Figure 2
show that saline stress delays the decline of AOX capacity
in developing roots, but it does not induce an increase of
this capacity.
In the experiment shown in Figure 2, saline stress was
initiated at d 3 of plant growth when AOX capacity in the
root-differentiation zone was highest (Hilal et al., 1997). A
different protocol was followed in the experiment pre-
sented in Figure 3. In this case, the seedlings were grown
on sand containing control or NaCl-supplemented Hoag-
land medium over the whole period from d 0 to 12 of
development. As shown in Figure 3, AOX capacity in the
differentiation zone of the control roots was maximal at d
3 to 5, whereas in the stressed roots the peak of AOX
capacity was shifted to 2 d later. Thus, the temporal pattern
of AOX capacity in developing roots is delayed by saline
stress.
Localization of AOX by Tissue-Print Immunoblots
Control roots at d 8 showed no specific immunostaining
in the differentiation zone using tissue-print immunoblots
(Fig. 4B) because, as already reported (Hilal et al., 1997),
AOX protein is no longer expressed at this developmental
stage. However, in roots of NaCl-stressed 8-d-old seed-
lings, the xylem strongly reacted with the anti-AOX mono-
clonal antibody (Fig. 4D), indicating that AOX protein was
still present in this tissue. This correlates with the delayed
decline of AOX capacity in stressed roots (Fig. 2) and
shows that the xylem-specific localization of AOX (Hilal et
al., 1997) is conserved under saline stress. Figure 4, A and
C, illustrates total protein, as evidenced by amido black
staining of tissue prints from control and NaCl-stressed
roots, respectively.
Figure 5. Anatomy of control and NaCl-stressed roots. Light pho-
tomicrograph of cross-sections from the differentiation zone of fixed
Morphometric Analysis of Developing Roots and embedded roots. A, At d 3 (after germination in sand). B, Control
at d 8. C, NaCl stressed at d 8. x, Xylem; p, phloem; and c, cortex.
To determine whether the NaCl-induced delay in AOX Bar 5 260 mm; all panels are shown at the same magnification.
expression was associated with retarded root differentia-
tion, root anatomy was examined by light microscopy of
cross-sections from the differentiation zone, which had
roots, whereas the number decreased significantly in con-
been previously fixed and embedded. As shown in Figure
trol roots between d 3 and 8 of growth. The changes in the
5, the most notable effect of the saline stress was to retard
xylem of control roots shown in Table I reflect the normal
primary xylem differentiation. The appearance of proto-
differentiation of protoxylem to metaxylem over the period
xylem and metaxylem in the stressed roots at d 8 of growth
was similar to that in the 3-d-old seedlings rather than to between d 3 and 8 of plant growth. These changes did not
that in the control roots at d 8. This effect was quantified occur in the NaCl-stressed roots, indicating delayed pri-
with an image analyzer and the data are summarized in mary xylem differentiation.
Table I. The total area of the xylem in the cross-sections of No appreciable effect of the saline stress was observed in
the root differentiation zone was significantly smaller in the phloem (Fig. 5; Table I). In the cortex the intercellular-
the control 8-d-old seedlings than at d 3 of growth, whereas to-cellular-area ratio was significantly decreased in the
in the NaCl-stressed 8-d-old seedlings, the xylem area re- NaCl-stressed roots (Table I), reflecting a reduction in the
mained similar to that at d 3 of development. Also, the apoplast in response to the increased NaCl concentration in
number of vessels remained constant in the NaCl-stressed the growth medium.
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Table I. Morphometric analysis of cross-sections from the differentiation zone of the primary root
Cross-sections similar to those shown in Figure 5 were analyzed as indicated in “Materials and
Methods.” Data are the means 6 SD of at least four seedlings from each group. Values in the same line
with different lowercase letters are significantly different (P , 0.05) by the Student’s t test.
d8
Tissue d3
Control Stressed
Conduction system
Phloem areaa 0.035 6 0.004a 0.026 6 0.003b 0.027 6 0.004b
Xylem areaa 0.017 6 0.003a 0.011 6 0.001b 0.015 6 0.002a
Xylem elementsb 49 6 1a 32 6 1b 49 6 7a
Cortex
Intercellular/cellular area 0.043 6 0.001a 0.049 6 0.002b 0.038 6 0.003c
a
Area (square millimeters) occupied by the phloem or the xylem in the cross-sections analyzed.
b
Number of vessels from the protoxylem and the metaxylem in the cross-sections analyzed.
Temporal Evolution and Tissue Localization of Peroxidase to that of the NaCl-stressed roots at d 10, as evidenced by
Activity in Developing Roots tissue-print analysis (not shown).
To determine whether the above results reflect a direct
effect of saline stress on the seedling growth rate leading to
a delayed evolution of every parameter linked to root DISCUSSION
development, we analyzed the effect of NaCl on a peroxi- The reduction in the apoplast of stressed roots relative to
dase involved in lignification (Goldberg et al., 1983). This the controls (Table I) is in agreement with previous data on
enzyme activity, measured with the substrate syringald- the effects of NaCl in bean roots (Cachorro et al., 1995) and
azine, was not affected in tomato roots under saline con- probably reflects an adaptive response to avoid NaCl load-
ditions (Peyrano et al., 1997). ing (Wegner and Raschke, 1994). Data in this paper re-
As shown in Figure 6, peroxidase activity in the differ- vealed that saline stress alters the temporal pattern of
entiation zone of control roots presented two maxima: one xylem differentiation, leading to the delayed development
at d 3 to 4, coincident with the peak of AOX capacity of the primary xylem (derived from the pro-cambium) and
reported by Hilal et al. (1997), and the other at d 9 to 10. precocious development of the secondary xylem (derived
The temporal pattern of peroxidase activity was unaffected from the cambium). Thus, saline stress had opposite effects
by the saline conditions. on the temporal evolution of primary and secondary xy-
The tissue localization of this peroxidase is shown in lem, two tissues with different ontogenic processes.
Figure 7. The enzyme was concentrated in the xylem at d 3 AOX protein, which has a xylem-specific localization
and 10 (Fig. 7, B, E and H). An unexpected result revealed (Hilal et al., 1997), exhibited a delayed pattern of expres-
by tissue prints in Figure 7 was the accelerated develop- sion that was apparently linked to primary xylem devel-
ment of secondary xylem in the NaCl-stressed roots (Fig. 7, opment. In this regard, it should be noted that depending
G and H) compared with the control roots (Fig. 7, D and E). on the developmental stage at which exposure to salinity is
The development of the secondary xylem in the control initiated, three different situations were observed: (a) when
roots was slower and its appearance at d 18 became similar NaCl treatment was initiated before the increase in AOX
capacity (Fig. 3), there was a shift in the peak and, thus,
values either lower or higher than the controls could be
obtained at different days of growth; (b) when NaCl treat-
ment was initiated when AOX capacity was high (Fig. 2),
there was a delay in the decline of AOX capacity and, thus,
values higher than the controls were obtained between d 4
and 12; and (c) when NaCl treatment was initiated after
AOX decline (Fig. 2), there was no NaCl-induced enhance-
ment of AOX capacity. Therefore, it is clear that salinity
delays developmental processes linked to AOX expression.
Once such events have occurred, NaCl is not able to modify
AOX capacity.
On the contrary, the temporal evolution of a peroxidase
activity localized in the xylem was not affected by saline
stress even though this enzyme presented a peak of activity
at d 3 to 4 of root development (Fig. 6), coincident with the
Figure 6. Temporal evolution of peroxidase activity in the differen- peak of AOX capacity. Therefore, saline stress does not
tiation zone of control (E) and NaCl-stressed (F) roots. Each value is alter the evolution of every parameter that has a temporal
the mean 6 SD of three separate measurements. prot, Protein. pattern linked to root development or seedling age.
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Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.700 Hilal et al. Plant Physiol. Vol. 117, 1998
Figure 7. Localization of peroxidase activity. Tissue prints of cross-sections from the root differentiation zone: at d 3, after
germination in sand (A, B, and C); at d 10, controls (D, E, and F); and at d 10, NaCl stressed (G, H, and I). A, D, and G,
Toluidine blue stain of total protein. B, E, and H, Stain for peroxidase activity. C, F, and I, Blanks for peroxidase activity,
omitting the substrate H2O2. PX, Primary xylem; SX, secondary xylem; P, phloem; and C, cortex. Bar 5 350 mm; all panels
are shown at the same magnification.
In conclusion, this work is the first demonstration, to our LITERATURE CITED
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