SALINITY ACCELERATES ENDODERMAL DEVELOPMENT AND …

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Enllronmenla[andF.xperzmentalBotqt~y, Vol. 35, No. 4, pp. 563 574. 1995 Copyright @ 1995 Elsevier Sciente Ltd

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SALINITY A C C E L E R A T E S E N D O D E R M A L D E V E L O P M E N T AND I N D U C E S AN E X O D E R M I S IN C O T T O N SEEDLING R O O T S

D. H. R E I N ~ T * and T. L. ROST Section of Plant Biology, University of California, Davis, CA 95616-8537, U.S.A.

(Received 19 Seplember 1994; accepted in revised form 27 january 1995)

Reinhardt D. H. and Rost T. L. Salinity accelerates endodermal development and induces an exodermis in cotton seedling roots. Environmental and Experimental Botany 35, 563 574, 1995. The development of the endodermis was studied in 5- to 48-day-old cotton (Gossypium hirsutum L., cv. Acala SJ-2) seedling roots g o w n in vermiculite at different NaC1 salinity levels. Sensitive fluorochromes (berberine aniline blue for Casparian bands, and fluorol yellow aniline blue for suberin lamellae) were used to detect cell wall modifications. Endodermal cells progressed through several developmental stages. In the primary stage, endodermal Casparian bands appeared synchronously in the radial walls. Then, suberin lamellae were laid down asynchronously in all cell walls, but always first in endodermal cells in the phloem sectors. Passage cells without suberin lamellae were usually present in the endodermal layer opposite protoxylem poles. The positions relative to the root tip at which Casparian bands and suberin lamellae developed were dependent on plant age and primary root len~h for control and salt-stressed seedlings. Salinity induced those structures to mature closer to the root tip for plants up to 10 days old, whereas in older plants no differences between treatments were observed. Exposure to high salinity (200 mM NaC1) induced the formation of an exodermis with Casparian bands and suberin lamellae close to the root base and in the transition zone to the hypocotyl. The exoderrnis, which never developed in control roots, may play a role in protecting the root from water loss and/or leakage of solutes important for osmotic adjustment. An exodermis was detected in 5- to 28-day-old seedlings. Older plants (48 days), with advanced secondary growth in the root stele, did not dif- ferentiate an exodermis.

Key words: Gossypium hirsutum, salt stress, root development, endodermis, exodermis.

INTRODUCTION

Roots o f all vascular plants possess an endodermis . This layer, essential tbr root functioning, seems to have evolved together with the conduct ive system (xylem) in land p l a n t s 9 ) T h e endodermis surrounds the stele and is considered to be the ma jo r constraint

for the m o v e m e n t o f solutes f rom the cor tex to the vascular tissues, due to the impregna t ion o f its walls with hydrophobic substances, i<

T h e endodermis originates f rom the g round mer- istem, and its deve lopmen t is usually divided into four stages, based upon the occur rence o f cell wall modifications. T h e system devised by K r o e m e r 05i

*Current address: EMBRAPA/CNPMF (National Research Center for Cassava and Tropical Fruit Crops), Caixa Postal 7, 44380 Cruz das Almas, Bahia, Brazil.

563

564 D.H. REINHARDT and T. L. ROST

and followed by Robards and Jackson, (:36/ has the following stages: (1) the proendodermis; (2) the primary stage endodermis with the Casparian band; (3) the secondary stage endodermis with the deposition of suberin lamellae on the internal wall surface; and (4) the tertiary stage endodermis with additional internal cellulosic wall material. The endodermal cells of many plant roots do not pro- gress beyond the primary stage, whereas others typi- cally show the complete range, as it often occurs for monocots, especially Poaceae (e.g. corn, barley). No reports have been published on cotton endodermis development.

Wall modifications ofendodermal cells have been shown and correlated with physiological functions of roots. (9/All roots possess a Casparian band, which consists of deposition of suberin and/or lignin between cellulose microfibrils in the anticlinal walls of the endodermis. Those substances are a major barrier for ions and possibly water. (31/The presence of the Casparian band insures that ions on their radial pathway towards the stele are subject to a selection process at the plasma membrane of an endodermal, cortical or epidermal cell. The additional deposition of the suberin lamellae in endodermal cells interposes a new layer of material which may not affect the radial transport of ions moving mostly through the symplast (K +, PO4:~-), but may inhibit the movement of ions flowing mainly along the apoplast, like Ca2+. (37'3~/

In addition to an endodermis, many plants develop an exodermis (defined as "a hypodermis with a Casparian band" by Peterson and Perumalla)J ~2/The exodermis is the outermost layer(s) of the cortex and undergoes cell wall modifications with the deposition of Casparian bands and suberin lamellae. In some monocots, yet another cellulosic wall is formed inside the suberin layer. (8/ The exodermis may function similarly to the endodermis, controlling the uptake and radial transport of ions. (2~) The endodermis and exodermis may also play an important role as a barrier to soil pathogens.(33,4~)

Many plants develop an exodermis. Brundrett and Kendrick ~~'6/have found a root exodermis in all vascular plants of a deciduous forest, except for a few conifer species. In an extensive survey of 200 angiosperm species, Perumalla et al. ('24'251 and Peter- son and Perumalla (~2} observed the existence of uni-, bi- or multiseriate exodermis in over 90%

of the species examined, including monocots and dicots, and members of primitive and advanced plant families. However, no hypodermis or exodermis was found in cotton plants, as well as in other species of the Malvaceae and Fabaceae.

Several factors may influence the development of the endodermis and exodermis. In general, Casp- arian bands in the exodermis mature further from the root tip than their counterpart in the endodermis, and the position of both layers depends on the plant species, and the age and growth rate of the individual root. (23/ Endodermal Casparian bands mature mostly from 3 to 16 mm behind the root tip. (~2'4°i In a study of the chemical composition of root cell walls in 27 plant species, Wilson and Peterson/~vr' could, in most cases, detect wall-mod- ifying components within 5 mm of the root apex.

Stress effects on endodermal and/or exodermal differentiation have been the issue of very few studies. Walker et al. (421 observed an intensified suberization in endo- and exodermis in citrus root- stocks grown under salinity stress. Root and cell hydraulic conductivities were reduced by salt stress in corn seedlings, (1'2/ but the relationship of that inhibition with structural changes was not studied. Accelerated suberization of barley root endodermis resulted from mechanical impedance. (45i In water-stressed corn and broad bean roots, endodermal Casparian bands appeared closer to the tip than in controlsJ '2:~'29/In sorghum grown at low soil water potential, an accelerated deposition of suberin and lignin occurred in both endodermis and hypodermis.(l 1!

Although cotton is an important model system for physiological studies on salt stress and salt tol- erance in plants, and the endo- and exodermis play crucial roles in root functioning, salinity effects on cotton root endodermis have not been reported yet. In this study, we observed the endodermal development in cotton seedlings of different ages, grown under non-saline and saline conditions. We expected to see a typical primary stage endodermis with Casparian bands, which should appear closer to the root tip for seedlings grown under salinity, independently from plant age.

MATERIAL AND M E T H O D S

Cotton seeds (Gossypium hirsutum L. cv. Acala SJ-2, California Planting Cotton Seed Distr., Bakersfield,

SALINITY ACCELERATES ENDODERMAL DEVELOPMENT 565

CA) were selected by weight (120 135 mg per seed) and surface sterilized with a solution of sodium hypochlorite (0.5% w/v) and 0.75% (w/v) detergent (Alconox Inc., NY) for 10 min. After brief rinsing with distilled water, the seeds were imbibed over- night in aerated distilled water at room temperature in the dark. Imbibed seeds were planted into pots filled with vermiculite saturated with treatment solutions, and placed in a growth chamber with a 16 hr photoper iod at 200 /2mol s ~m -~ photon fluence rate, and 26 27°C day and 21 22°C night temperatures. Vermiculite and solutions were pre- viously sterilized in an autoclave. The control con- sisted of a basic solution (1/10 strength modified Hoagland 's solution, (HI with 1 m M NaC1 and 1 m M CaC12). Salt treatments were 100 m M NaC1 (low salinity) and 200 m M NaC1 (high salinity), added to the basic solution. Trea tment solutions were sup- plied to each pot every other day to keep the vermiculite moist.

Plants were harvested 5, 7, 10 and 14 days after the start of imbibition. At each harvest, pr imary root lengths were measured and five roots were sampled per treatment. The experiment was repeated three times. In an additional study, 28- and 48-day-old seedlings grown at 1 m M NaCI (control) and 200 m M NaC1 salinity from the time of planting, were harvested. Growth conditions and all other procedures before and after harvest were the same as described above. Sections were obtained only from pr imary root zones close to the root base (at 10, 40 and 100 m m from the root base/hypocotyl transition).

Freehand sections were made from roots immediately after harvesting plants and taking root growth measurements. Folded Parafihn was used to immobilize the root pieces during sectioning with a razor blade. !1:~i Sections were obtained for the following pr imary root zones: 4 6, 6-8, 8 10, 10- 12, 18 20 m m from the tip, then for every 10 mm segment up to 100 m m from the tip, and for every 50 mm segment from 100 mm up to the root base (immediately above the root hair zone, at the transition to the hypocotyl). The numerous sections generated for each sampled root zone were examined under a dissecting microscope, and the thin- nest were selected for staining. Sections were placed into small polyethylene tubes with mesh bottoms (Balzers Union, Liechtenstein) and handled in this way throughout the staining procedures.

Several techniques for the staining of lignin and suberin in plant tissues were studied, including fluorescent procedures using berberine aniline blue, (4: neutral red toluidine blue O, (~G~ and fluorol yellow 088 from BASF. i7:' Fluorol yellow was also tested together with aniline blue as a counterstain to quench autofluorescence. These procedures were compared with the perfbrmance of non-fluorescent stains like Sudan IV and Sudan Red 7B (Sigma), which are reported to be the best non-fluorescent stains for lipids in plant material by Brundrett et al.( 7',

For detection of Casparian bands and suberin lamellae at early stages o fendodermal development in cotton roots, the fluorescent stains berberine for Casparian bands, and fluorol yellow for suberin lamellae, both counterstained with aniline blue, were the most efficient. The former was mostly used for sections from root zones up to 20 mm from the tip, and the latter for sections from all other root zones. The berberine aniline blue staining procedure was as described by Brundrett el al. ~4~ For the fluorol yellow aniline blue staining method sections were immersed in fluorol yellow 0.01% solution in a polyethylene glycoNglycerol solvent system (as described by Brundrett et a/.) '7 for 1 h, rinsed with distilled water fi~r about 1 rain and counterstained in 0.5% (w/v) aniline blue solution in water for 30 min. All sections stained by either one of the two methods, were briefly rinsed, and immersed for a few minutes in 0.1% (w/v) FeCI3 in 50% (w/v) glycerin (glycerin added to filtered aqueous FeCI,~). Cover glasses were mounted in that same FeCI:~ solution on microscope slides.

Sections were examined within 24 h after harvest and photographed with an Olympus Vanox AH-2 microscope using epifluorescence optics and UV- light (Dichroic mirror D M 400; excitation filter U G 1; and barr ier filter L 435). Images were r ecorded on 50 ASA Kodak Techpan 2415 black and white film.

R E S U L T S

Endodermis Conlrolplants . Plant age, and hence pr imary root

length, affected the position of the endodermal Casparian band deposition in control plants (Table 1). The location for its differentiation decreased, relative to the root tip, with increasing plant age.

566 D . H . R E I N H A R D T and T. L. R O S T

Table 1. Effects of salinity and plant age on prima~ root length and the position of endodermal and exodermal wall modifications in prima~ roots of cotton seedlings

Plant age Primary root Treatment (days) length (mm)

Distance from root tip (mm) Endodermal Endodermal Exodermal

Caspafian band suberin lamellae* suberin lamellae*

Control 5 127 18 20+ 90 i10 No 7 201 l0 20 60 150(100--120)§ No

10 256 6-12 30 150(80--100)§ No 14 344 4 6 38 40 No

1 0 0 m M 5 113 10 12 58 60 No NaC1 7 203 8 12 58 60 No{

10 252 4 8 38 40 No 14 332 4 6 38 40 No

2 0 0 m M 5 82 4 10 38-40 68 70 NaCI 7 139 8-10 38 40 108 110

10 208 4-8 38 40 180 200 14 290 4 6 38 40 248-250

* Detected in at least one endodermal or exodermal cell. "~ Not detected at 12 mm; no sections observed at 13 17 mm. { Suberin lamellae present in a few samples only. § Range for most of the roots observed.

T h e C a s p a r i a n b a n d was la id d o w n in a s y n c h r o n - ous m a n n e r in the rad ia l walls of all e n d o d e r m a l cells (Fig. 1).* In 5-day-o ld seedlings, the C a s p a r i a n

b a n d s cou ld no t be obse rved in sect ions up to 12 m m f rom the root tip (Tab le l), whe rea s in 14-day- old cont ro l p lan t s all sect ions s h o w e d C a s p a r i a n

Fig. 1. Stele and part of the cortex of a 10-day old control plant, at about 8 mm from the root tip. Note the Casparian bands (arrow) in the radial walls of all endodermal cells. B+A staining. Scale bar = 47/~m.

Fig. 9. Full cross-section of a 14-day old control plant at about 5 mm from the root tip. Note the Casparian bands in the endodermal cells (arrowhead) and the fluorescence in the epidermis (arrow). B + A staining. Scale bar = 32/~m. Fig. 3. Suberin lamellae (arrow) in some phloem sector endodermal cells in a control plant. Section taken 150 mm from the root tip. Casparian bands are Faintly visible in other endodermal cells (arrowhead). F + A staining. Scale

bar = 72 pm. Fig. 4. Higher ma~lification of the stele of a 10-day old control plant. Section taken 200 mm from the root tip. Suberin lamellae are present in the endodermal cells in the phloem sectors only (long arrow). Note the staining of phloem fibers (short arrow), which usually appeared at this stage of initial secondary growth. F + A staining. Scale

bar = 94 pro. Fig. 5. Section at 150 mm from the root tip of a 7-day old control plant. Note the two positions of lateral root emergence (arrowheads) and the hear T suberization in all endodermal cells situated right above these laterals (arrow-); in this r e , o n , suberin lamellae are present in the endodermal cells overlying both the phloem and the xylem sectors.

Casparian bands are still visible in all other endodermal cells. F + A staining. Scale bar = 120 pm. Fig. 6. Lateral root emergence is visible in this cross-section of a 7-day old control plant due to the fluorescence of the endodermal suberin lamellae (arrow'). Note the continuity' of this structure from the main root to the lateral root.

Section taken at 180 mm from the root tip. F + A staining. Scale bar = 177 pm.

*All figures are micrographs of fresh, freehand cross-sections of pr ima W roots from vermiculitc-grown cotton seedlings, stained with berberine aniline blue (B+A) or fluorol yellow" aniline blue (F+A). Figures 1 6 are from control roots and Figs 7 12 from salt-treated roots.


568 D . H . R E I N H A R D T and T. L. R O S T


bands within 6 m m from the root tip (Fig. 2). From previous experiments in our laboratory it was known that older cotton seedling roots grow at a lower rates than younger plants. Therefore, a reduction of the distances from the root tip for Caspar ian band differentiation was expected in the pr imary roots of older control plants. (43i Flu- orescence was not only detected in endodermal Casparian bands, but also in epidermal cell walls near the root tip (Fig. 2).

Cotton roots developed a stage I | endodermis characterized by the formation of suberin lamellae in endodermal cell walls. The deposition ofsuber in lamellae occurred asynchronously in endodermal cells. Suberin lamellae always appeared first in a few cells of the endodermis located in the phloem sectors (Fig. 3). With increasing distance from the root tip, more and more endodermal cells developed suberin lamellae. However, even in root regions with fully mature pr imary structure, one or more endodermal cells opposite the protoxylem poles usually did not develop a suberin lamella (Fig. 4). None of these so-called 'passage cells' could be observed at sites immediately adjacent to lateral root emergences; at these locations, usually all endodermal cells, opposite to both phloem and xylem sectors, on the side of the lateral root, deposited suberin lamellae (Fig. 5). In older, emerged, lateral roots, the endodermal suberin lamellae formed a continuous cylinder connected to the main root axis (Fig. 6).

Plant age affected the position ofsuberin lamellae

formation in the endodermis. This event started much closer to the root tip in 14-day-old seedlings when compared to its position in pr imary roots of younger plants (Table 1).

Salt-stressedplants. Fluorescing epidermal cell walls occurred farther from the root tip in salt-stressed plants, but salinity stress did not change the general pat tern of cell wall modifications in the endodermis. Casparian bands appeared in all radial walls sim- ultaneously, whereas suberin lamellae were deposited asynchronously and predominant ly in endodermal cells opposite the phloem sectors. In these plants, especially those exposed to 200 m M NaC1, the number of passage cells in the endodermis was usually fewer than in control seedlings. In some cases, an almost full ring of endodermal cells with suberin lamellae could be observed in roots grown under high salinity, with fully mature pr imary structure (Fig. 7).

Increasing salinity level caused both Casparian band and suberin lamellae formations to occur close to the root tip (Table 1). The differences between treatments were the largest for 5-day-old seedlings, whereas by 14 days no differences could be detected. At this plant age, Casparian bands were differentiated within 4 6 mm from the root tip, and the first endodermal cells with suberin lamellae appeared within 40 mm from the tip, for plants grown at all levels of NaC1 studied (Table 1). In addition, the position for suberin lamellae differentiation was more consistent for salt-stressed plants than for control seedlings, which showed

Fig. 7. Higher magnification of the stele of a 14-day old plant grown at 100 mM NaC1 salinity. Section obtained 250 mm fi'om the root tip. Note the almost full ring of suberin lamellae in the endodermis (arrow). F+A staining. Scale

bar = 119/~m. Fig. 8. Exodermis in 5-day old seedling exposed to 200 mM NaCI salinity. Note suberin lamellae in outermost cortex

cell layer (arrow). F + A staining. Scale bar = 228/~m. Fig. 9. Higher magnification of the exodermis in Fig. 8 (arrow) The arrowhead indicates the epidermis. F + A staining.

Scale bar = 49/~m. Fig. 10. Exodermis (arrow) in 10-day old seedling grown at 200 mM NaC1 salinity. Section 150 mm from the root tip. Note the well developed endodermal suberin lamellae (large arrowhead), except for the cells opposite the protoxylem poles (small arrowhead). A pith is present in the center of the stele, indicating the transition to the

hypocotyl. F + A staining. Scale bar = 264/ml. Fig. 11. Similar to Fig. 10 but exodermis is less developed in this 14-day old plant exposed to 200 mM NaCI. Suberin lamellae are present in some of the exodermal cells (arrow). Section taken at 250 mm from the tip. F + A staining.

Scale bar = 268 #m. Fig. 12. Higher magnification of an exodermis of a 28-day old plant grown at 200 mM NaC1 salinity. Note the brighter fluorescence in the radial walls of exodermal cells due to Casparian bands which are often mostly obscured

by the fluorescence of the suberin lamellae (arrowheads). B + A staining. Scale bar = 40 m.


major variations until 10 days after planting (Table 1).

Plant age had a less pronounced effect on the distances for both Casparian band and suberin lamellae maturation in the endodermis of salt- stressed plants than that reported for the control treatment (Table 1). This was especially the case for seedlings exposed to high salinity (200 mM NaC1), for which the variations with age were smaller than in control plants. There was, for example, no change with increasing age for the distance from the root tip at which suberin lamellae appeared in the first endodermal cells (Table 1). Consistent with these observations, primary root lengths (and elongation rates) were smaller for plants exposed to high salinity.

Exodermis. Control plants of all ages studied failed to develop an exodermis. No exodermis was formed at any developmental stage of the primary root, neither with primary nor with secondary growth (for 5- to 14-day-old plants, see Table 1).

Similarly, in most plants exposed to low salinity (100 mM NaC1), no exodermis differentiated. In this treatment, some exodermal suberin lamellae could be detected in a few primary roots of 7-day- old cotton seedlings (Table 1). When grown at high salinity, however, cotton seedlings did develop a typical exodermis characterized by the deposition of suberin lamellae in cells of the outermost cortex layer. This occurred in primary roots of 200 mM NaC1 treated plants, 5 28 days old, with primary growth (Figs 8-11) or with some secondary growth.

The formation of an exodermis in cotton seedlings exposed to high salinity was mostly restricted to the region rather close to the base of the primary root (Figs 8 and 9) or to the root base/hypocotyl transition zone characterized by the appearance of a pith in the central region of the stele (Figs 10 and 1 1). Exodermal suberin lamellae could only be detected within about 50 mm from the root base (Table 1). Increasing plant age and correspondingly longer primary root length, did correlate with the maturation of the exodermis at larger distances from the root tip (Table 1).

The development of Casparian bands could not be separated from the deposition of the exodermal suberin lamellae. No root section was obtained in which only Casparian bands could be visualized in exodermal cells, without the masking presence of

the very bright yellow fluorescent suberin lamellae in sections stained with fluorol yellow, even when counterstained with aniline blue. There was, however, no doubt about the deposition of Casp- arian bands in exodermal cells, which could be better distinguished when the berberine aniline blue staining procedure was used (Fig. 12). The observations suggested that both Casparian bands and suberin lamellae were laid down almost con- comitantly in exodermal cells.

D I S C U S S I O N

Root growth inhibition is part of an overall plant growth reduction in response to salinity. Associated with it are changes in root function which are often reflected by modifications in root structure. The most pronounced structural responses to salinity, and other environmental stresses, are often related to cell wall modifications. (44}

Our data and observations agreed only with part of our hypothesis. As expected, salt stress induced endodermal differentiation events to occur mostly at shorter distances from the root tip. However, this was not observed for older roots. In addition, endodermal development in cotton roots reached the secondary stage characterized by the formation of suberin lamellae. Another unexpected observation was the formation of an exodermis close to the root base in response to high salinity.

Endodermis. An increase in suberization, closer to the root tip, has been detected in endodermal cells of some crop species in response to salt stress (42) and to other environmental stressors like mechanical impedance, (45! osmotic stress/29/and water deficit. (~ ~/ The current study showed that cotton root endodermis does develop to stage II, which is characterized by the differentiation of suberin lamellae in its cell walls. It also was observed that Casparian bands and suberin lamellae mature closer to the root tip due to salinity.

That response was, however, root age- and root length-dependent (Table 1). For 14-day old roots, no differences could be detected between control and salt-stressed plants. Salinity changed the position ofendodermis maturation only in plants up to a certain age (10 days). This salt-stress effect was similar to that observed for protoxylem tracheary element differentiation in other studies on cotton


roots at this same laboratory, which was also restricted to roots of a certain range of lengths, cor- responding to a range of plant ages. (35} In the current study, some positive relationship between differentiation and root elongation rates (as indicated by root length increases between harvests - - T a b l e 1) was suggested for both the endodermal Casp- arian band, and to a lesser extent, the suberin lamellae. In older plants with smaller growth rates, the deposition of Casparian bands and suberin lamellae occurred closer to the root tip.

Very few studies have related root age to the position of suberization in the endodermis. In most reports, stress effects have been determined for plants of only one age. Perumalla and Peterson, i23/ however, studied the differentiation of the endodermis in 2- to 6-day-old onion and corn plants. Similar to our results, they observed that the position for endodermal cell wall modifications from the root tip was dependent on plant age and root growth rates.

The pattern of suberin lamellae deposition in the root endodermis of cotton seedlings was not different from that known tbr other plants. (8! The presence of passage cells, with Casparian bands but without suberin lamellae in their walls, here observed for cotton, has also been mentioned for some other plant species (e.g. onion, (3'19) Iridaceae, cited by Clarkson) (8i. Although these cells have not been examined in more detail, it is frequently assumed that in roots, where they are charac- teristically present, they represent the major entry points of water and solutes into the stele. (8/

The emergence of lateral roots disrupts the endodermis of the main root. At early stages of development, rapidly growing lateral root pr imordia may emerge without the formation of any mature Casp- arian bands, thereby establishing an apoplastic continuity, at least temporarily, between cortex and stele, i~'28'2~''m) At later stages, a continuous Casparian band is laid down in all endodermal cells from the main root to the lateral root, closing off the apoplastic pathway. '28'>'3°i These latter observations made on several other plant species (e.g. broad bean, corn), are similar to our circumstancial observations on cotton roots in this study. At early stages of lateral root formation (at initiation and emergence), usually no suberin lamellae and apparently no Casparian bands as well, could be seen in the root primordia. In more developed lateral roots,

however, the suberin lamellae were present (Fig. 6). In addition, a continuous half-ring of cells with suberin lamellae in the endodermis of the main root, could be detected at sites immediately adjacent to lateral root emergence (Fig. 5). At these locations, no passage cells interrupted the continuity of the suberin depositions in the tangential walls of the endodermis. This may contribute to the control of the radial solute flow towards the stele. However, the apoplastic pathway through the newly formed root primordia, presumably remains open for a short period during the initial stages of lateral root development of cotton, similar to that reported for broad bean. i29)

Suberized walls in the endodermis are known as the major barriers to radial ion and water movement in plant roots, based on evidence from old and modern literature. <9'~7'~~'2°'22) Environmental stressors like drought, have been reported to reduce radial hydraulic conductivities in roots of sorghum 'l ~! and Agave deserti, i21i which has been at least partially attributed to the accelerated formation of suberin lamellae. In cotton roots, growth-limiting deficiencies of nitrogen and phosphorous decreased considerably the hydraulic conductivity of cortical cells. ':~4) In that study, it was also shown that sub- stantial water flow follows the apoplastic pathway. Therefore, radial hydraulic conductivity in cotton is most likely affected by the earlier deposition of suberin lamellae in the endodermis, in spite of the presence of passage cells. This assumption remains, however, to be confirmed.

The radial movement of ions that move mostly via the apoplastic pathway to the stele, should be similarly affected by the accelerated suberization in the endodermis of salt-stressed roots. This could be especially critical for the uptake of calcium, which seems to be greatly restricted to unsuberized root zones.'.lS. ~8)

Exodermis. Cotton has been classified among plant species that do not possess an exodermis, as reported by Perumalla et al. (24~ for the species (;os- sypium herbaceum L. This was confirmed in this work for unstressed control plants of Gossypium hirsutum L., cv. Acala SJ-2. For all ages studied (5- to 48- day-old plants), no exodermis could be observed. However, exposure to high salinity (200 m M NaC1) induced the deposition of suberin in the cell walls of the outermost cortical layer, forming a typical exodermis. This new structure was detected in


plants of all ages, except for those with advanced secondary growth in the root (48-day-old plants).

No similar induction of an exodermis differentiation due to salinity or other environmental stresses, has been published. Stressors have, however, been reported to cause an intensified suberization in exodermal cell walls of several other crop species, which already develop an exodermis under unstressful conditions. This has been observed in response to salt stress (citrus) (4~) and to other environmental stressors like water deficit (sorghum)(11/and osmotic stress (maize) (24).

As discussed above for the endodermis, suberization of cell walls affects the main root function of water and ion uptake and transport. An exodermis represents an apoplastic barrier similar to the endodermis and thus must have some physiological significance. In salt-stressed cotton roots, the presence of the exodermis was restricted to the root zone close to its base or even to the transition zone to the hypocotyl. These sites are presumably no longer critical for the entry of mineral nutrients and water into the root, as most of these functions are executed by younger root tissues closer to the tip, and also by lateral roots in older seedlings. There- fore the presence of an exodermis at these sites should not prevent the exposure of the cortex to high ion concentrations in the external solution.

There are, however, some other functions to be considered. Casparian bands are known to prevent the growth of ectomycorrhizal fungi, and suberin lamellae to block the development of hyphae of endomycorrhizal and pathogenic fungi .(27'33) In some species, the exodermis may inhibit the loss of the root cortex during periods of severe drought. 'y':~~i This latter observation was made in onion roots, where long periods of water deficits resulted in epidermal death; the exodermis, however, survived and presumably prevented critical water loss from the cortex to the dry external medium. Similarly, the exodermis formed in roots of cotton seedlings subjected to high salinity, could play a role in preventing the leakage of water and/or solutes needed for osmotic adjustment. It is known that salinity affects the integrity of cell membranes which become leaky resulting, for example, in the loss o f K + to the external medium. '1°) The presence of Casparian bands and suberin lamellae in the exodermis could reduce that leakage. Some improved protection against soil pathogens could

also be of adaptive value, since stressed and thus physiologically weakened plants are usually more susceptible to diseases.

In conclusion, this study showed that in cotton roots, the endodermis develops not only Casparian bands but also suberin lamellae (stage II endodermis). The position of both Casparian bands and suberin lamellae in relation to the root tip was dependent on seedling age and pr imary root length for both control plants and salt-stressed seedlings. NaC1 salinity induced those endodermal wall modifications to occur closer to the root tips for up to 10-day-old plants, but not in the pr imary roots of older plants. High salinity induced the formation of an exodermis in all seedlings except for those with pr imary roots at advanced stages of secondary growth.

Acknowledgements--We are ~ateful to S.B. Reinhardt for her help in the preparation of the micrographic plates. The author D.H. Reinhardt was supported by a schol- arship from EMBRAPA (Brazilian Corporation for Agri- cultural Research). We also acknowledge D. Gladish, J. Jernstedt, S. Baum and P. Lu for their assistance at times during this study.

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