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This is to certify that the
thesis entitled
THE TOXIC ALUMINUM REACTION IN CORN AND
BARLEY ROOTS: AN ULTRASTRUCTURAL AND
MORPHOLOGICAL STUDY

presented by

Ian Bruce McLean

has been accepted towards fulfillment
of the requirements for

 

M. S 0 degree in Horticulture

 

Majo professor

Date October 16, 1979

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OVERDUE FINES:

25¢ per day per item

RETUMIM; LIBRARY MTERIALS:

Place in book return to remove
charge from circulation records

 

THE TOXIC ALUMINUM REACTION IN CORN AND
BARLEY ROOTS: AN ULTRASTRUCTURAL AND
MDRPHOLOGICAL STUDY

BY

Ian Bruce McLean

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Horticulture

1979

ABSTRACT

THE TOXIC ALUMINUM REACTION IN CORN AND
HARLEY ROOTS: AN ULTRASTRUCTURAL AND
MDRPHOLOGICAL STUDY

By
Ian’Bruce.McLean

The progressive gross morphological and ultrastructural changes in
the root tips of corn (£55 EELS. L., cv. Spring Gold) and barley (Hordeum
vulgare, cv. Coho) due to Al toxicity were studied. Roots from plants grown
in solution culture with 10-3M AlCl3 were examined by scanning and trans-
mission electron microscopy. Constriction of the corn root cortex followed
by transverse shearing, reduced root elongation, root cap cell sloughing,
cytoplasmic degeneration, and changes in the endoplasmic reticulum (ER) and
ribosomal frequency were observed. Barley roots exhibited cortical const—
riction, reduced elongation, and root cap cell sloughing and degeneration.
Longitudinal cortical fissures, abundant surface debris, and damaged root
hairs were observed. The frequency of ribosomes on cortical cell ER incr-

eased. A possible cause of cell elongation failure involving protein syn-

thesis is postulated.

ACKNOWLEDGEMENTS

IAppreciation and love are felt for my wife, Elly, and children,
James, Stephanie, and Heather, for their selfless sacrifice of material
comforts and time together during my studies.

The support and friendship of my adviser, Dr. H. Paul Rasmussen
and the other members of my committee, Drs. Gary Hooper and John Bukovac,
has been enjoyed and valued.

Gratitude is expressed to the United States Government and the
people of Michigan for the freedom and financial support received during
this degree.

The gracious assistance of Bonna Davis in the layout and typing

of this thesis was invaluable and most appreciated.

ii

Readers:

The paper-format utilized in this thesis meets the requirements
stipulated by the Horticulture Department and the University. The
thesis body was prepared for publication in the Journal of the American
Society for Horticultural Science and follows the manuscript style for

this journal.

iii

TABLE OF CONTENTS

LIST OF FIGURES O 0 O O O O O O O O O O O O O O O O 0 O O I O 0

JOURNAL PAPER -- The Toxic Aluminum Reaction in Corn and
Barley Roots: An Ultrastructural and Morphological Study.

Introduction

Materials and Methods. . . . . . . . . . . . . . . . . . .

Results and Discussion . . . . . . . . . . . . . . . . . .
1. Corn Root . . . . . . . . . . . . . . . . . . . . .

A. External Morphology. . . . . . . . . . . . . . .
B. Cytological Observations . . . . . . . . . . . .

2. Barley Root. . . . . . . . . . . . . . . . . . . . .

A. External Morphology. . . . . . . . . . . . . . .
B. Cytological Observations . . . . . . . . . . . .

3. Hypotheses of Al Toxicity. . . . . . . . . . . . . .

LITERATURE CITED 0 O O O O O O O O O O O I O I O O I O O O O O O

APPENDICES

APPENDIX I
General Literature Review. . . . . . . . . . . . . . . . .
A. Aluminum as an Element . . . . . . . . . . . . . . .

B. Aluminum and Plants. . . . . . . . . . . . . . . . .

Calcicoles and Calcifuges. . . . .

Differential Tolerance . . . . . . . . . . . . .
Aluminum Accumulators. . . . . . . . . . . . . .
Beneficial Effects . . . . . . . . . . . . . . .
Other Metal Toxicities . . . . . . . . . . . . .

U'I-l-‘wNH

C. Plant Reactions to Aluminum. . . . . . . . . . . . .
1. Symptoms of Toxicity . . . . . .

1A. Root System . . . . . .
1B. Leaves and Stems. . . . . . . . . . . . . .

iv

Page

. 46

. 46

. 48
. 49
. 49
. SO
. SO

. Sl

. 54

TABLE OF CONTENTS - continued
Page
2. Distribution of Al in the Plant. . . . . . . . . . . 54
3. Hypotheses of Al Toxicity. . . . . . . . . . . . . . 55
3A. Al-P Interaction. . . . . . . . . . . . . . . . 56
SB. Al-Ca Interaction . . . . . . . . . . . . . . . 57
3C. Al-DNA Interaction. . . . . . . . . . . . . . . 58

4. Mechanisms of Resistance . . . . . . . . . . . . . . 59

LIST OF REFERENCES 0 O O O O O O O O O O O O O O O O O O O O O O O O 61

APPENDIX II

Gel Electrophoresis of Corn and Barley Root
Water-Soluble Proteins . . . . . . . . . . . . . . . . . . . . 68

LIST OF REFEMNCES O O I O O 0 O O O O O O I O O O I O O O O O O O O 72

LIST OF FIGURES

FIGURE Page

1. Scanning electron micrographs of primary corn roots before
and after A1 treatment . . . . . . . . . . . . . . . . . . . . 11

2. Scanning electron micrographs of severe Al damage to corn
roots. 0 I O O O O O O O O O O I O O O I O I O I O O O O O O O 13

3. Transmission electron micrograph of a corn root cap cell . . . 15

4. Transmission electron micrograph of untreated corn root
cortical cells . . . . . . . . . . . . . . . . . . . . . . . . 17

5. Transmission electron micrographs of corn root tip after
12 hr A1 treatment 0 O O O O I O O O O O O O O O O I C O O O O 19

6. Transmission electron micrograph of a corn root cap cell
after 24 hr Al treatment . . . . . . . . . . . . . . . . . . . 21

7. Transmission electron micrograph of a corn cortical cell
after 12 hr Al treatment . . . . . . . . . . . . . . . . . . . 23

8. Transmission electron micrographs of endoplasmic reticulum
in corn root cap and epidermal cells . . . . . . . . . . . . . 25

9. Transmission electron micrographs of ribosomes and endo-
plasmic reticulum in corn root cells . . . . . . . . . . . . . 27

10. Simple linear regression of ribosome frequency on corn
and barley root endoplasmic reticulum. . . . . . . . . . . . . 29

ll. Scanning electron micrographs of barley root . . . . . . . . . 31

12. Scanning electron micrographs of a barley root after 30 hr
A1 treatmexlt o o o o o o o o o o o o o o o o o o o o o o o o o 33

13. Transmission electron micrograph of an untreated barley
root cap cell. . . . . . . . . . : . . . . . . . . . . . . . . 35

14. Transmission electron micrograph of untreated barley
root cortical cells. . . . . . . . . . . . . . . . . . . . . . 37

15. Transmission electron micrograph of a barley root cap
cell after 6 hr Al treatment . . . . . . . . . . . . . . . . . 39

vi

LIST OF FIGURES--Continued

FIGURE

l6.

17.

Page

Transmission electron micrograph of barley root
cortical cells after 6 hr Al treatment. . . . . . . . . . . . 41

Transmission electron micrographs of endoplasmic reticulum
of barley root cap cells. . . . . . . . . . . . . . . . . . . 43

APPENDICES

Diagram of gel after electrophoresis of corn root
water-SOIUble proteins 0 O O O O O O O O O O I O O O I O O O O 71

vii

THE TOXIC ALUMINUM REACTION IN CORN AND

BARLEY ROOTS: AN ULTRASTRUCTURAL

AND MORPHOLOGICAL STUDY

The Toxic Aluminum Reaction in Corn and
Barley Roots: An Ultrastructural
and Mbrphological Study

Ian B. McLean and H. Paul Rasmussen
Michigan State University, East Lansing

Abstract. The progressive gross morphological and ultra-
structural changes in the root tips of corn (Zea_mays L.,
cv. Spring Gold) and barley (Hordeum vulgare, cv. Coho)

due to Al toxicity were studied. Roots from plants grown

in solution culture with 10 M AlCl were examined by scan-
ning and transmission electron micr scopy. Constriction

of the corn root cortex followed by transverse shearing,
reduced root elongation, root cap cell sloughing, cyto-
plasmic degeneration, and changes in the endoplasmic reti-
culum (ER) and ribosomal frequency were observed. Barley
roots exhibited cortical constriction, reduced elongation,
and root cap cell sloughing and degeneration. Longitudinal
cortical fissures, abundant surface debris, and damaged root
hairs were observed. The frequency of ribosomes on cortical
cell ER increased. A possible cause of cell elongation
failure involving protein synthesis is postulated.

 

Losses in economic crops due to Al toxicity occur worldwide
where the soil pH is below 5.0. Rainfall leaching, crop harvesting, and
application of acid-forming fertilizers contribute to soil acidification
(6). Modification of surface soil pH by heavy applications of lime has
been attempted. However, the A2 and lower horizons frequently remain
untenable to roots. Water and nutrients located in those horizons are
unavailable to affected plants which are subsequently susceptible to
relatively short dry periods. The cultivation of a susceptible Species
under such conditions may be impracticable (14).

Aluminum toxicity causes stunted roots and tops in many crop
species. Common symptoms include severely stunted primary and lateral
roots which become stubby and discolored. Tops are dwarfed and may

appear deficient in P and be partially chlorotic (8).

2

Surveys have established wide inter- and intraspecific variability
in resistance to Al (7, 14, 16). The variability and the economic impact
of yield reduction in acid soils has motivated scientific interest in the
toxic reaction. Two hypotheses<fol toxicity predominate in the litera-
ture. First, a chronic internal P deficiency caused by symplastic precipi—
tation of Al(OH)2H2P04, and second, inhibition of mitosis by a nuclear
Al-DNA interaction (2, 3, 23).

This study investigated the progressive gross morphological and
ultrastructural changes in barley and corn roots subjected to toxic levels
of A1 in nutrient solution. Barley and corn are crop species respectively

sensitive and resistant to Al (16).

MATERIALS AND METHODS

Corn (Zea mays L., cv. Spring Gold) and barley (Hordeum vulgare,

 

cv. Coho) seeds were germinated between sheets of moist polyethylene-
backed paper in the dark at 24 C. Germinated seeds were transferred to a
nutrient culture as described by Rasmussen g£_al, (21), and 200 ppm fungi-
cide (Benlate) was added. The germinated seeds in nutrient culture were
placed in a growth chamber at 24 C under 40 ,uE incandescent and 210 pE
fluorescent illumination. After 5 days, either lO-BM CaCl2 or lO-BM AlCl3
was added to the nutrient cultures, resulting in pH 6.2 and 3.7 respectively.
The terminal 1 cm of primary roots was excised from the 5 day old plants at
hourly intervals from 0-12 hr, and at 12 and 18 hr, and prepared for both
transmission (TEM) and scanning electron microscope (SEM) observation.
Three roots in each of two replications were prepared for each instrument.

Root segments including the terminal 1.5 mm and a 1.5 mm section

from the zone of elongation were excised for TEM examination. They were

killed and fixed in 2% acrolein and 1Z‘K2Cr207 in .OSM Na cacodylate

buffer at pH 7.2 for 2 hr, rinsed in buffer, and post-fixed in 2% 0304
for 2 hr. Following fixation, tissues were dehydrated for 20 min at each
step in a 10, 25, 50, 75, 90, 95 and 100% ethanol series, transferred to
propylene oxide, and embedded in Epon 812 resin. Polymerization of the
resin was done at 70 C for 24 hr. Blocks were thin sectioned (Porter-
Blum MT-Z ultra microtome) and stained in uranyl acetate (saturated solution)
for 45 min and lead citrate (.42) for 7 min prior to TEM examination at 60 RV
accelerating voltage (Philips 201). The frequency of ribosomes per .4/mn
endoplasmic reticulum (ER) in root cap and epidermal cells at each sampling
time was determined from the TEM micrographs at 26,400X magnification and
a simple linear regression line was fitted to the data (19).

Preparation of roots for SEM studies was by dehydration through the
same ethanol series used for TEM for 30 min at each step. The samples were
transferred to a critical point dryer (Denton DCP-l) and dried in liquid

CO The dried roots were mounted on SEM stubs with double-sided sticky

2.
tape and sputter coated with 20-30 nm of Au (Film-Vac. Inc. mini-coater).
SEM observation was performed (181 S-III microscope) at 15 KV accelerating

voltage and 0 or 35'tilt.

RESULTS AND DISCUSSION
Examination of Al-treated corn and barley roots in the SEM and TEM
revealed rapid tissue degeneration in less than 24 hr.
1. CORN ROOT.
A. External Morphology.

The terminal 1 cm of the untreated corn primary root appeared smooth

and of uniform diameter to within 1-2 mm of the root cap. The root tip

tapered as it approached the root cap, which was compact and composed
of clearly defined cells (Fig. 1A). In Al treated roots a similar condi-
tion existed until 6 hr after treatment, when a constriction of the root
2-5 mm posterior to the root cap was observed (Fig. 1B). A shrinkage of
cortical and epidermal cell volume was evident. A similar constriction
in the zone of elongation of onion root was reported by Clarkson and
Sanderson (4). After 9 hr in Al root cap cells appeared disordered and
poorly adhered (Fig. 10). Transverse cracks appeared in the epidermis
and exterior ranks of cortical cells where the constriction had previ-
ously occurred (Fig. 1D). The separation of epidermal and deeper cortical
cells, and disorganization of the root cap increased through 12 hr
(Fig. 2A, B). After 30 hr the cortex was sheared transversely and appeared
as bands separated longitudinally over a 5 mm segment. The root cap was
an amorphous mass of irregularly shaped cells (Fig. 2C). Root elongation
was greatly reduced. The deformed root cap and bands of cortex contributed
to a stubby, spatulate appearance similar to that reported for other
species (11, 13, 22).

This progression of Al damage confirmed the observation of Hatch
(9) who showed that the stele continued to elongate at a reduced rate,
producing stress sufficient to crack and shear the non-elongating cortex
and epidermis. Differential growth on either side of the endodermis
reflected the presence of A1 in the respective tissues. Continued elon-
gation of the stele supported previous reports of the failure of Al to

penetrate the endodermis in large amounts (9, 20).

B. Cytological Observations.
Untreated corn root cap and epidermal cells contained many dictyo-

somes and parallel sheets of rough ER which were generally located near the

cell wall. Numerous mitochondria, ribosomes and small vacuoles were pre-
sent throughout the cytoplasm (Fig. 3). Nucleoli were present in nuclei
which contained areas of dark-staining material.

Cortical cells in the zone of elongation contained abundant cyto-
plasmic ribosomes but little rough ER and few dictyosomes. Mitochondria
were numerous and the nuclei appeared identical to those in the root cap
(Fig. 4).

Deterioration of the root ultrastructure after Al treatment was
observed. The cytoplasm of root cap cells contained remnants of membrane,
ribosomes and vacuoles after 12 hr (Fig. 5A, B). Organelle structure was
severely deformed compared to untreated cells (Fig. 3). Root cap cells
frequently contained large central areas of cytoplasmic debris and a band
of organelle remnants lying near the cell wall after 24 hr. The tonoplast
was not evident and no alteration of the cell wall was observed (Fig. 6).

Cells immediately interior to the epidermis and root cap retained
some organized structure after 12 hr (Fig. 5A, 7). Dictyosomes and mito-
chondria appeared intact even though the cyt0plasm appeared abnormal as
evidenced by distorted ER, irregular vacuoles, and a general reduction in
electron density (Fig. 7).

Changes in the ER were observed. Normal cells contained multiple
sheets of ER in which the two membranes were parallel (Fig. 8A). After
6 and 12 hr the two membranes were distended and irregular, and some
sections of smooth ER were present (Fig. 8B, C).

The frequency of ribosomes associated with the ER increased over
24 hr in the A1 treated cells. Groups of 3-5 ribosomes were common in
the untreated cells, with an average of 7 ribosomes per .4,um of rough

ER (Fig. 9a). The groups in Al-treated cells became larger and more

closely packed over time, reaching greater than 13 ribosomes per .4lum ER
by 24 hr (Fig. 9B). The slopes of simple linear regression lines fitted
to the ribosome count data revealed a progressive increase in the ribosome
frequency on the ER of the cells exposed to Al. There was a small
increase in the simple linear regression line for the control (Fig. 10).
This confirmed the suggestion by Hatch (9) that ribosomes appeared to
"jam" along corn root ER in toxic Al conditions. The implications of

the ER and ribosome changes are discussed below.

2 BARLEY ROOT.
A. External Mbrphology.

The terminal 1 cm of barley roots at time 0 tapered towards the
root cap. Root hairs were present to within 1.5 mm of the tip, and root
cap cells were clearly defined (Fig. 11A). A constriction of the root
beyond 2.5 mm from the root cap and a reduced number of root hairs was
observed after 4 hr in Al (Fig. 11B). The shrinkage was similar to that
observed in corn (Fig. 13). Sloughing of root cap cells was evident after
12 hr. Surface debris had accumulated on the root tip by this time and
some epidermal cells had shrunk radially (Fig. 11C).

The epidermis appeared severely damaged after 30 hr and root elon-
gation had virtually ceased (Fig. 12A). Longitudinal fissures separated
epidermal and cortical cells, some of which had plasmolysed. This was simi-
lar to a report of plasmolysed cells in the elongation zone of onion after
Al treatment (4). Some root hairs were fractured at the base (Fig. 123).
Large numbers of sloughed cells and other debris covered the length of the
root. Some of this material may have been dried cell contents since

transverse cracks in epidermal cell walls were observed (Fig. 123).

Bacterial cells were observed on this debris and their exudate may have

bound stray particulate matter in the nutrient culture to the root.

B. Cytological Observations.

The ultrastructure of untreated barley root cells was similar to
that of corn. The root cap cells contained numerous dictyosomes, rough ER,
mitochondria and small vacuoles (Fig. 13). Cortical cells contained some
dictyosomes and mitochondria, numerous ribosomes, but little rough ER
(Fig. 14).

The root cap ultrastructure showed evidence of damage 4 hr after
Al treatment and was deformed after 6 hr (Fig. 15). Fragments of ER and
mitochondria indicated their degeneration, and ribosomes were not evident.
Some cortical cells were also degenerated after 6 hr and no intact dictyo-
somes or mitochondria remained. Myelin-like figures symptomatic of mem-
brane breakdown were observed (Fig. 16).

Inflation and distortion of the rough ER was not observed. How-
ever, a 60% increase in the ribosome frequency from 13 to 21 ribosomes
per .qum ER was evident only 1 hr after Al treatment (Fig. 17 A, B, C).
The slope of simple linear regression lines fitted to the ribosome fre-
quency data of root cap and epidermal cells showed that the slope for the
Al treatment was 8.7 times greater than control (Fig. 10). Damage to root
cap and outer cortical cells by 6 hr was so severe that no ribosome counts
could be obtained. This result confirmed the evidence from corn of an
association between A1 treatment and ribosome frequency along the ER.

Several parallels existed in the reactions of corn and barley roots
to Al. The root cap and epidermis of both species were rapidly penetrated

and destroyed. Damage to the elongation zone took different forms but

cortical and epidermal cell elongation was effectively terminated in
both species.

The ribosome-ER association was altered more severely in barley.
Both the slope of the regression line and the increased ribosome fre-
quency after 1 hr were greater than in corn. This more severe reaction

confirmed previous reports of greater barley sensitivity to Al (7, l6).

3. HYPOTHESES 0F Al TOXICITY.

Aluminum exists as Al(HZO)2+ in acid solution and readily precipi-
tates P as Al6(OH)12(H2PO4)6. An Al-P reaction also occurs on the surface
of polymerized Al hydroxy aggregates at higher pH (10, 12).

Early research into A1 toxicity pointed to P deficiency as the
cause of the plant reaction. Plant symptoms of Al toxicity which could
be removed by addition of P to the growth medium were reported (1). In
acid soils, Al(H20)2+ enters the root intercellular space and adsorbs onto
the cell wall or middle lamella. Precipitation of P by the adsorbed Al
leads to a reduced supply of P for uptake to the shoots. Electron micro-
probe and elemental analysis has confirmed this model (15, 20). However,
the rapidity of the plant reaction and the absence of symptoms in solution
cultures devoid of both P and Al suggests an additional, more direct role
for Al.

The failure of mitosis in the root meristem of onion under toxic
Al conditions led Clarkson (3) to suggest an Al-DNA interaction. He
suggested that Al prevented DNA replication during the mitotic cycle.
This hypothesis has been supported by reports of Al binding to isolated
DNA of pea, and identification of Al in nuclei by staining techniques

(17, 18). However, the termination of cell division does not explain

the failure of cell elongation or the rapid degeneration of root cap
and epidermal cells.

The rapidity and breadth of disruption to cell processes by A1
suggests another causal factor of toxicity. Phosphorus is an integral
part of RNA nucleotides and therefore of ribosomes. Cell membranes
contain P in their phOSpholipid component. The changed configuration
of corn root ER membranes and the increased frequency of ribosomes
associated with ER in cells of Al-treated corn and barley roots suggests
interference in these structures and their activities. Aluminum may
directly disrupt protein synthesis at both the ER-ribosome and free
polysome sites. This would account for the prompt cessation of elon-
gation since enzymes are required for loosening of the cell wall fibers
(5). If the replacement of other enzymes which require constant renewal
was interrupted, then rapid cell degeneration would ensue.

The high r2 of .93 and .71 for corn and barley after A1 treatment
suggests constant interference of ribosome movement on the ER. Con-
versely, the lower r2 of .38 and .26 for Ca-treated corn and barley sug-
gest that movement of ribosomes on the ER is not inhibited, and normal
exit follows required protein synthesis.

Aluminum has been found to precipitate with P outside the cell
causing a P deficiency. A second Al-P interaction in the nuclear DNA
which would prevent mitosis has been suggested. This third Al-P inter-
action in the RNA and ER causing disruption to protein synthesis would
explain impeded cell elongation and rapid cell degeneration. The
extraction of RNA and confirmation of its association with A1 is indi-
cated. An understanding of how Al enters the cell and remains in solution
at cellular pH levels is also required before A1 toxicity may be fully

elucidated.

Figure l.

10

Scanning electron micrographs of primary corn roots before
and after Al treatment.

A) Normal control root with compact root cap (RC).

B) Constricted cortex and epidermis in zone of
elongation (arrow) after 6 hr Al treatment.

C) Shearing of cortex and epidermis (arrow) and
sloughing of root cap (RC) after 9 hr treatment.

D) Cracks several layers deep in cortex of elongation
zone (arrow) after 9 hr A1 treatment.
Arrows in (C) and (D) point to the same location
on the same root.

 

11

 

Figure 2.

12

Scanning electron micrographs of severe A1 damage to corn
roots.

A) Deepening cracks in cortex (arrow) and sloughing
root cap (RC) after 12 hr Al treatment.

B) Fissures (arrow) in cortex formed as cells were
pulled apart.

C) Final appearance of primary root after 30 hr Al
treatment. The cortex appeared as bands distributed
longitudinally along the root (arrow) and the root
cap (RC) was deformed and sloughing.

 

l3

 

Figure 3.

14

Transmission electron micrograph of a corn root cap cell.
Structures evident were cell wall (CW), dictyosomes (D),
rough endoplasmic reticulum (ER), mitochondria (M),
plasmalemma (P), small vacuoles (V) and ribosomes (R).

 

 

 

Figure 4.

16

Transmission electron micrograph of untreated corn root
cortical cells. Note the cell wall (CW), rough endo-
plasmic reticulum (ER), mitochondrion (M), nucleus (N),
nucleolus (Nu), ribosome (R) and vacuole (V).

 

 

l7

 

Figure 5.

18

Transmission electron micrographs of corn root tip after
12 hr Al treatment.

A) Root cap cell (arrow) with large vacuole (V) and
degenerated cytoplasm (C). Apical cell (Ap)
appeared intact.

B) Cytoplasm of root cap cell with remnants of mem-
brane (M), ribosome (R) and vacuole (V).

-
‘lr

 

 

19

 

Figure 6.

20

Transmission electron micrograph of a corn root cap cell
after 24 hr A1 treatment. Note large central area of
cytoplasmic debris (Cy), cell wall (CW), dictyosome (D),
distended rough endoplasmic reticulum (ER), mitochondrion
(M), plasmalemma (P1), polysome (P) and vacuole (V).

 

 

21

L11

 

M
.1

Figure 7.

22

Transmission electron micrograph of a corn cortical cell
after 12 hr Al treatment. Dictyosome (D), rough endo-
plasmic reticulum (ER), mitochondrion (M) and nucleus (N)
retain some structure.

 

 

Figure 8.

24

Transmission electron micrographs of endoplasmic reticulum
in corn root cap and epidermal cells.

A) Untreated root cap cell with multiple sheets of rough
endoplasmic reticulum (ER) in which the membranes are
parallel.

B) Rough endoplasmic reticulum (ER) of epidermal cell
with inflated distortions after 6 hr Al treatment.

C) Endoplasmic reticulum of epidermal cell showing
smooth (SER) and rough (RER) forms and disconnections
(arrow) after 12 hr Al treatment.

 

 

25

 

Figure 9.

26

Transmission electron micrographs of ribosomes and endo-
plasmic reticulum in corn root cells.

A) Untreated cell with infrequent groups of ribosomes
(R) associated with the endoplasmic reticulum (ER).

B) Numerous groups of ribosomes (R) associated with
endoplasmic reticulum (ER) after 24 hr A1 treatment.

 

 

27

 

Figure 10.

28

Simple linear regression of ribosome frequency on corn
and barley root endoplasmic reticulum.

l) Barley root following Al treatment:

A
Y = 19.5 + 1.38X
r2= .71

2) Barley root control:

A
Y = 12.7 + .158X
r2= .38

3) Corn root following Al treatment:

= 7.18 + .25X
2= .93
4) Corn root control:

A
Y = 7.24 + .053X

r2= .26

H r<>

29

26

 

24
22 ®BAI
20
C II
III
E to
§
' M
\3 @303
I2 (3)
g c..
: '
:2 @e—
c CCO
6
2
O

O 3 6 9 12 IS ‘8 2| 24

Time after Treatment (hr)

30

Figure 11. Scanning electron micrographs of barley root.

A) Untreated root with clearly defined root cap cells (C)
and root hairs (RH).

B) Root showing constriction (arrow) and reduced numbers
of root hairs (RH) after 4 hr Al treatment.

C) Sloughing root cap cells (RC), surface debris (arrow)
and shrunken epidermal cells (E) present after 12 hr
Al treatment.

 

 

31

 

Figure 12.

32

Scanning electron micrographs of a barley root after 30 hr
Al treatment.

A)

B)

Severely damaged epidermis and cortex showing
longitudinal fissures (F). Sloughed cells and
other debris adhered to the surface (arrow).
Some root hairs (RH) remained.

Fissures caused by epidermal and cortical cell
shrinkage (arrow). Transverse cracks (Cr)
appeared in some epidermal cells. Root hairs
(RH) were frequently broken off at the base.

 

 

33

 

Figure 13.

34

 

Transmission electron micrograph of an untreated barley root
cap cell. Cell wall (CW), dictyosomes (D), rough endoplasmic

reticulum (ER), mitochondrion (M), ribosome (R) and vacuole
were evident.

 

36

Figure 14. Transmission electron micrograph of untreated barley root
cortical cells. Dictyosome (D), mitochondrion (M) and
numerous ribosomes (R) were observed.

37

 

Figure 15.

38

Transmission electron micrograph of a barley root cap cell
after 6 hr Al treatment. Remnants of an amyloplast (Am),
endoplasmic reticulum (ER), mitochondrion (M), plasmalemma
(P1) and vacuole (V) were evident. The cell wall (CW)
appeared intact.

 

39

. 1b w.

. r

.
r‘, -

 

40

Transmission electron micrograph of barley root cortical
cells after 6 hr Al treatment. Myelin-like figure (My)
was evident in degenerated cytoplasm. Note cell wall
(CW) and remnants of ribosome (R) and vacuole (V).

Figure 16.

41

 

Figure 17.

42

Transmission electron micrographs of endoplasmic reticulum
of barley root cap cells.

A) Untreated cell showing groups of ribosomes (arrow)
along the endoplasmic reticulum (ER).

B) Increased frequency of ribosomes (R) on endoplasmic
reticulum (ER) after 1 hr Al treatment.

C) An almost continuous array of ribosomes (R) on the
endoplasmic reticulum (ER) after 4 hr treatment.

43

 

LITERATURE CITED

10.

11.

12.

13.

LITERATURE CITED

Blair, A. W. and A. L. Prince. 1923. Studies on the toxic properties
of soils. Soil Sci. 15:109-129.

Clarkson, D. T. 1967. Interactions between aluminum and phosphorus
on root surfaces and cell wall material. Plant and Soil 27:347-356.

 

. 1969. Metabolic aspects of aluminium toxicity and

 

some possible mechanisms for resistance. p. 381-397. In_I. W.
Rorison (ed.) British Ecological Society Symposium #9. Blackwell
Scientific Publications, Oxford.

and J. Sanderson. 1969. The uptake of a polyvalent

 

cation and its distribution in the root apices of Allium cepa:
tracer and auto-adiographic studies. Planta (Berlin) 89:136-154.

 

 

Cleland, R. 1971. Cell wall extension. Ann. Rev. P1. Physiol.
22:197-222.

 

Foy, C. D. 1975. Toxic factors in acid soil. Horticulture
53:38-43.

 

and J. C. Brown. 1964. Toxic factors in acid soils:

 

II. Differential aluminum tolerance of plant species. Soil Sci.
Soc. Amer. Proc. 28:27-32.

, L. R. Chaney, and M. C. White. 1978. The physiology

 

of metal toxicity in plants. Ann. Rev. P1. Physiol. 29:511-566.

Hatch, R. L. 1973. A morphological and anatomical study of the
toxic effect of aluminum on corn roots. M.S. Thesis, Michigan State
University.

Hem, J. D. 1968. Aluminum species in water. Advances in Chem.
73:98-114.

 

Hortenstine, C. C. and J. G. A. Fiskell. 1961. Effects of aluminum
on sunflower growth and uptake of boron and calcium from nutrient
solution. Soil Sci. Soc. Amer. Proc. 25:304-307.

Hsu, P. H. 1965. Fixation of phosphate by aluminum and iron in
acidic soils. Soil Sci. 99:398-402.

Ligon, W. S. and W. H. Pierre. 1932. Soluble aluminum studies:
II Minimum concentrations of aluminum found to be toxic to corn,
sorghum, and barley in culture solutions. Soil Sci. 34:307-319.

44

 

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

45

Long, F. L. and C. D. Foy. 1970. Plant varieties as indicators of
aluminum toxicity in the A2 horizon of a Norfolk soil. Agron. J.
62:679-681.

MacLean, A. A. and T. C. Chiasson. 1966. Differential performance
of two barley varieties to varying aluminum concentrations. Can. J.
Soil Sci. 46:147-153.

McLean, F. T. and B. E. Gilbert. 1927. The relative aluminum toler-
ance of crop plants. Soil Sci. 24:163-175.

Matsumoto, H., E. Hirasawa, H. Torikai, and E. Takahashi. 1976.
Localization of absorbed aluminium in pea root and its binding to
nucleic acids. Plant and Cell Physiol. 17:127-137.

 

Matsumoto, H., S. Morimura, and E. Takahashi. 1977. Binding of
aluminium to DNA of DNP in pea root nuclei. Plant and Cell Physiol.
18:987-993.

 

Neter, J. and W. Wasserman. 1974. Applied linear statistical
models. Richard D. Irwin, Inc., Homewood, Illinois.

Rasmussen, H. P. 1968. Entry and distribution of aluminum in Zea
mays. Planta (Berlin) 81:28-37.

 

, V. E. Shull, and H. T. Dryer. 1968. Determination
of element localization in plant tissue with the microprobe. Devel.
in Applied Spectroscopy 6:29-42.

 

Rios, M. A. and R. W. Pearson. 1964. The effect of some chemical
environmental factors on cotton root behavior. Soil Sci. Soc. Amer.

 

Wright, K. E. 1943. Internal precipitation of phosphorus in
relation to aluminum toxicity. Plant Physiol. 18:708-712.

 

APPENDICES

APPENDIX I

GENERAL LITERATURE REVIEW

GENERAL LITERATURE REVIEW

A. ALUMINUM AS AN ELEMENT

Aluminum is the third most abundant element in the outer litho-
sphere and exists generally in alumino-silicate rock-forming minerals.
Considerable quantities are found in all soils except certain sands and
peats (49, 68). The concentration in natural water is usually less than
100 ug per liter. Higher concentrations may occur at a pH less than 4.7
as the hydrated Al cation, and at pH higher than 8.5 as the aluminate
anion (41, 68).

The hydrated form, A1(H20)2+, predominates in acid solutions. As
pH increases deprotonation of one water molecule leads to the formation
of AlOH(H20)§+, which polymerizes via a double OH- bridge into A12(0H)2
(H20)g+. This process leads to gibbsite formation. Ionic Al in natural
water is generally complexed with F_ or OH-, or 804- at low pH. Polymer-
ized hydroxy aggregates of colloidal or sub-colloidal size predominate in
the range of pH 4.7 - 6.5 (41). Richburg and Adams (85) suggested that
A16(OH)i: was the major species at pH 5.5 - 6.0.

A complex relationship between soluble Al and pH exists in soils.
The actual value of the ionic product A1(OH)3 must be considered in order
to place their values on a consistent physicochemical basis (8). Evans
and Kamprath (16) found the A1 concentration in the soil solution of
mineral soils related to the percent Al saturation of the effective cation

exchange capacity (CEC). The concentration of soluble salts further

affected solubility.

46

47

In organic solution the Al concentration was related more closely
to exchangeable A1 than percent Al saturation. The Al concentration in
soil solution decreased with increasing organic matter at a given pH (8).

In 30 acid soils at pH of 4.0, the Al concentration ranged from
1.5 to 23 ppm, at pH 4.5 from O to 12 ppm with most less than 5 ppm, and
at pH 4.9 from 0 to 2 ppm with most less than 1 ppm (77). The A1 concen-
tration in soil solutions above pH 5.5 is less than 0.1 ppm (60). There-
fore, prediction of soil solution Al concentration is not possible at a
given pH, but toxic Al levels may exist in soils below pH 5.0.

The interaction between soil Al and P is critical to plant growth
due to possible Al toxicity_pg£-§e and P deficiencies from precipitation
in the soil matrix. In a moderately acidic solution (pH 4.0) with a
high P concentration, the precipitation reaction is:

3+

- +
6 Al + 6 HZPOA —>-A16(OH)12(H2PO + 12 H (1)

4)6
or

A1 (OH) 6+ + 6 n P0-——)-Al (on) (a PO ) (2)

6 12 2 4 6 12 2 4 6

From eq. (1) the reduction in pH upon addition of Al3+ to nutrient
solutions is evident. Equation (2) is the reaction to form the smallest
possible polymer unit. The phosphate ion tetrahedron reacts with two
different polymers to increase polymer size. In the first stage of pre-
cipitation phosphate reacts with both Al3+ and the polymer reducing pH
and turbidity. The second stage involves a continued drop in Al concen-
tration due to polymer formation (45, 46).

In slightly acidic to neutral solutions (pH 6-7), low in P,
phosphate is adsorbed on the surface of stable Al hydroxides. The surface-
reactive amorphous Al hydroxides are not limited in activity above pH 5

and are the real factors governing the P concentration in solution (45, 46).

48

B. ALUMINUM AND PLANTS

Soil acidity and high Al levels frequently limit plant growth in
the high rainfall areas of eastern and south-eastern United States and
other parts of the world. Basic elements such as Ca and Mg are removed
from the soil by leaching, erosion and harvest. Fertilizers such as
(NH4)ZSO4 intensify acidification (20).

Lime has been applied to acid soils to raise pH above 5.5 and
precipitate Al, but often the subsurface layers were not penetrated suffi—
ciently and the pH remained low (21, 22). For example, MacLeod and
Jackson (67) found that liming had a negligble effect on pH and therefore
upon water soluble and exchangeable Al below 23 cm, and that 9200 kg ha-1
of lime was required to influence soil pH at this depth and lower. Subse-
quent poor root development in the acidic subsurface layers effectively
prevented plant access to water and nutrients in the A2 and deeper hori-
zons. Plants with root systems largely confined to the limed layer were
susceptible to relatively short dry periods, particularly at critical

stages of plant growth (22, 62).

l. Calcicoles and Calcifuges.
Wide variability exists in the ability of plants to grow at low pH.
Two broad plant categories are evident. Calcicoles are species inhabiting

calcareous soils which are 100% base saturated due to the reaction:

CaCO3 + Hz-micelle -->-Ca-micelle + H20 + C02 (3)
Soil pH is controlled primarily by hydrolysis of CaCO3:
CaCO3 + 2 HzO--->-Ca(OH)2 + H2C03 (4)

The strong base, Ca(OH)2, exceeds the influence of the weak acid, H2C03,
and pH generally ranges from 7.0 - 8.3. Calcicoles grow only on soils

with high pH due to their sensitivity to A1. Calcifuges are rare or absent

49

on calcareous soils and may grow in conditions which cause Al toxicity in
calcicoles. On calcareous soils calcifuges suffer from lime chlorosis

(18, 90, 91).

2. Differential Tolerance.

Most important crop species are sensitive to A1, but a range of
reactions exists. Sensitive species include barley, beet, cabbage, cotton,
lettuce, mustard, oats, radish, rye, sorghum, timothy and turnip. Tolerant
or resistant species include buckwheat, corn, redtop and soybean (25, 62,
66). Surveys have revealed intraspecific variability in crop species
including barley, cotton, rye, snap bean, soybean, triticale and wheat
(l, 21, 22, 30, 48, 75, 83, 84).

Resistance is apparently genetically controlled. Natural and
breeding selection resulted in an association of geographic location and
the degree of tolerance. Generally, cultivars developed in high rainfall,
low soil pH areas of the world have greater Al tolerance (21, 23, 31, 52
84). Wide variability in barley and snap bean tolerance facilitated their

use as biological indicators of Al levels (62).

3. Aluminum Accumulators.

Aluminum accumulation is thought to be a primitive character and
has been reported for numerous woody genera (4, 7). Hutchinson (50)
reported an estimated mean content of .022 Al in herbaceous dry matter of
many species. However, accumulator species could take up prodigious
amounts. For example, young leaves of the tea bush contained approximately
100 ppm A1 with up to 5000-16000 ppm in leaves about to fall (6). Alumi-
num accumulated in the tea leaf epidermis (69). Foliar Al levels of

321-2173 ppm were reported in Pinus species which normally inhabit very

50

acid soils in eastern Australia (47). Aluminum influences flower color
of Hydrangea macrophylla. Chenery (5) found that a delphinidin flower
pigment which was pink in an acid cell-sap turned blue in the presence of

excess Al.

4. Beneficial Effects.

Beneficial effects of Al on plant growth have been reported.
Increased seed production and dry weight of oats and corn were observed
with small amounts of added Al (72, 89). Hackett (36) reported stimulated

root growth in 5 ppm Al in seedlings of the calcifuge Deschampsia flexuosa

 

(L.) Trin. Stimulated growth of Valencia orange and lemon in 2.5-5.0 ppm
Al was reported (58). However, reports of the beneficial effect of Al on

plant growth remain isolated.

5. Other Metal Toxicities.

Manganese and Fe toxicity in plants has been reported. Manganese
toxicity may occur in acid soils and mine spoils with pH below 5.5, and
also at high pH in soils under reducing conditions. Symptoms appear in
shoots and include marginal chlorosis and necrosis of leaves, leaf puck-
ering and necrotic leaf spots. Excess Mn caused "crinkle leaf" in cotton,
"stem streak necrosis" in potato and ”internal bark necrosis" in apple
trees. Plant roots may be injured and turn brown following severe injury
to the tops. Wide variability in species and cultivar tolerance to Mn
toxicity has been documented (27).

Iron toxicity was reported in some acid soils at pH below 6.0.
Symptoms included dark green foliage and overall stunted growth. Roots

were thickened and contained large deposits of inorganically bound

51

phosphates. Necrotic spots on leaves of rice and sugarcane were attributed
to Fe accumulation. Differential species and cultivar tolerance to Fe has
been reported (27).

Phytotoxicity has been observed for a few other heavy metals. Zinc,
Cu and Ni toxicities frequently occurred. Lead, Co, Be, As and Cd toxici-

ties under unusual conditions have also been reported (27).

C. PLANT REACTIONS TO ALUMINUM.
1. Symptoms of Toxicity.

Reactions to A1 differ qualitatively and quantitatively between
and within plant species. However, a coherent pattern has emerged from
data of numerous species and cultivars.

Typically plants are stunted above and below ground in toxic
levels of Al. Specifically, barley, clover, corn, flax, lettuce, potato
and sunflower developed both stunted tops and roots (39, 44, 56, 65, 66,

73, 87).

1A. Root System.

The primary injury from A1 is to the root system.where prolife-
ration is greatly reduced. de Waard and Sutton (93) found that the roots
of black pepper eventually died off centripetally. Less severe reactions
result in severely stunted primary and lateral roots. Clarkson and
Sanderson (13) found that elongation of onion roots in lO-BM Al slowed
progressively after 3-4 hr and ceased altogether after 8 hr. Reduction in
elongation has also been observed in corn, sunflower and sugar beet (40,
44, 48). Cessation of elongation was followed by brown discoloration and

thickening leading to a stubby and spatulate appearance of the tip in corn,

52

cotton and sunflower (44, 59, 86). Dissociation and sloughing of the
root cap were early symptoms in corn, while disorganization of the root
cap, root apex and vascular elements was observed in wheat (17, 40).
Hatch (40) found that the cortex and epidermis of corn roots sheared
perpendicular to the root axis after elongation had been arrested. Evi-
dently the stele continued to elongate, albeit at a reduced rate. A
related phenomenon was the collapse of cells in the cortex in the zone

of elongation of onion (13).

Lateral root development patterns are markedly altered by Al.
Root length and frequency of lateral root emergence were reduced in corn
(S9). Hutchinson §£_§l, (48) found that the emergence of lateral roots
of sugar beet seedlings grown at 4, 8 and 12 ppm Al extended down to the
region of the root apex. Lateral roots of flax grown at 5 ppm Al remained
as abortive stubs close to the primary root (73). Lateral roots of barley
in 20 ppm A1 either failed to penetrate the cortex or formed an irregular
mass of cells as a small tumor (82). Lateral roots of wheat penetrated
the cortex but became a shapeless mass of tissue with irregularly arranged
cells due to inhibition of differentiation of the root cap and vascular
elements (17).

Extended exposure to 10-3M A1 of Agrostis tenuis demonstrated the

 

cumulative effect of these abnormalities. Lateral roots were initiated
close to the apical meristem along with failure of the main axis to elon-
gate. These first order laterals in turn failed to develop and gave rise
to second order laterals. This process continued to higher orders. The
laterals of successive orders developed close to the apical meristems

of the preceding order and all the roots were of similar diameter.

Consequently the root system failed to effectively ramify in the soil

53

under natural conditions and predisposed the plant to dessication (12).

Cellular abnormalities were observed in roots subjected to toxic
levels of Al. Hatch (40) found that corn root epidermal and cortical
cells in the zone of elongation exhibited increased vacuole size after 8
hr and coalescence of most vacuoles occurred by 24 hr. He also observed
changes in the ribosome distribution. There appeared to be "jamming" of
ribosomes along the endoplasmic reticulum and an increase in the number of
polysomes in the cytoplasm. Nuclear abnormalities were observed in the
meristematic cells of cotton roots. Large numbers of binucleate cells were
present indicating inhibition of cell division (86). Clarkson (12) observed
no mitotic figures in onion roots which had ceased elongating, and con-
cluded that cell division was blocked. However, "sticky" chromosomes,
manifested mainly by the formation of anaphase bridges, have been reported
for onion (57).

Elemental analysis revealed changes in the root composition under
toxic A1 conditions. Rios and Pearson (86) found a higher concentration in
cotton roots grown at 2.5 ppm Al. Similarly, barley roots had increasing
P concentrations with increasing Al in the nutrient solution (65). Reduced
Ca levels in roots and/or tops were observed in alfalfa, barley, cotton,
rye, perennial ryegrass, soybean, sunflower, triticale and wheat (24, 28,
29, 34, 44, 55). Clarkson and Sanderson (14) found that 100 uM Al reduced
Ca uptake in barley to 31-37Z of control plants. However, inhibition of
root growth was not directly dependent on reduced Ca uptake, and in cotton
roots similarly treated the Al toxicity symptoms remained (55). Inhibition
of Cu uptake was reported for citrus, lettuce, potato and wheat (3, 39, 42,

56, 58). Absorption of Mg and Zn, and K at high Al levels, was inhibited

in potato (56).

54

The general debility and irregular tissue of roots damaged by Al
toxicity has been associated with increased root susceptibility to fungal
attack (32). For example, Hoffer and Trost (43) found that corn roots
were severely attacked by Fusarium moniliforme Sheldon. Older cotton
roots were subject to bacterial decomposition after growing in excess Al

(86).

1B. Leaves and Stems.

The leaves and stems of plants grown at toxic Al levels were
generally stunted and economic crop yield was reduced. Millikan (73)
found that shortened internodes and reduced leaf area caused the stunting
of flax.

Leaf symptoms reflected the root disorder. Petiole collapse
associated with decreased Ca was symptomatic of Al toxicity in soybean
(l, 28). Young barley leaves developed tip dieback while the older leaves
became yellow and brittle (65). Leaf margins of sunflower turned brown
and the cotyledons died (44). Leaf cupping, chlorosis, and in extreme
cases purpling has also been reported (82).

Elemental analysis of barley shoots revealed lower P with
increased Al concentration in the growth medium. This was contrary to
the root situation (65). Calcium was lower in the leaves of cotton and

soybean (24, 28).

2. Distribution of Al in the Plant.

The distribution of Al in roots has been investigated using
staining, autoradiographic and electron microprobe techniques. Aluminum
penetration into the root has been established. The initial phase of Al

adsorption in onion occurred in the mucigel (13). MbCormick and Borden

55

(64) found high levels of Al in the mucilaginous material at the barley
root surface. An adsorption intensity gradient in the vertical axis was
evident. Adsorption along the root surface decreased from a high at the
root cap to indiscernible amounts 3-4 mm back. The root cap was parti-
cularly susceptible to entry and passage of Al. Aluminum entered the
root cap of sugar beet and accumulated in the cells between the root and
the cortex. After 10 days in 4 ppm Al the root cap was separated from
the apex by a thick A1 phosphate layer and the cap cells had disinte-
grated (53).

Radial penetration behind the root cap diminishes centripetally.
Rasmussen (80) found that corn root epidermal cells largely excluded Al
from the cortex. Aluminum which had entered the root was generally found
in the cortex but most of it failed to penetrate the endodermis (13, 40,
53, 95). Clarkson and Sanderson (13) found that the A1 analog Sc failed
to penetrate further than 3 ranks of cortical cells at a point 3-4 mm
from the root apex of onion. However, a large accumulation of Al in or
on the endodermis, as well as slight penetration into the stele, has been
reported for sugar beet (54). Rasmussen (80) found that the rupture of
the endodermis by emerging lateral roots provided a pathway for Al move-
ment into the vascular tissue and on to the aerial parts of the corn
plant.

Cellular deposits of Al have been observed in the plasmalemma of
barley root cells, and the cell wall of root hair and root tip cells of

pea. Aluminum has been found in the nuclei of onion and pea (13, 64, 70).

3. Hypotheses of Al Toxicity.
Historically the cause of toxicity in acid soils remained contro-

versial for several decades. Hutchinson (49) reviewed the early debate

56

over the pre-eminence of Al or pH in the toxic reaction. Several workers

noted the decrease in pH of soils following repeated applications of
(NH4)ZSO4 fertilizer. In an effort to elucidate the causal factor plants
were grown in soil, soil extract, and nutrient solutions treated with Al.

In fact, soluble Al was the main cause of growth depression. Benefits
accrued from application of basic materials or acid phosphate to soil

came primarily from the precipitation of Al (2, 35, 37, 38). Other

workers concluded that native Al levels were too low to account for

plant injury and that toxicity was due to precipitation and subsequent
deficiency of P, low pH, or unfavorable percentage base saturation (61, 76).

The toxicity of A1 to plant growth is now accepted and several hypotheses

regarding its action have been proposed.

3A. Al-P Interaction.

Increased P concentrations were observed in roots of barley,
cotton and perennial ryegrass grown in toxic Al conditions (10, 79, 86).
However, Clarkson (10) observed that the increased P was not incorporated
into phosphorylated compounds but remained in an inorganic form in barley.
This was in marked contrast to a report regarding untreated barley roots
in which as much as 80% of the P was in an organic form 15 min after
adsorption (63).

Fixation of P by an adsorption-precipitation reaction in the
roots leading to P deficiency has been proposed (10, 78, 81, 94). Alumi-
num firmly adsorbs to isolated root cell wall material, perhaps adsorbing
to the free carboxyl groups of polygalacturonic acid chains in the middle
lamella. It has been suggested that Al may be precipitated on the root

or cell surface as hydroxide. Phosphorus could then be precipitated on

57

the hydroxide surface as A1(OH)2H2PO4. This process would reduce the P
available for active uptake from soils where Al and P arrive at the root
surface continuously (11). Electron microprobe analysis confirmed that
Al and P have similar distribution in corn roots (80).

Aluminum may affect root metabolism directly. Clarkson (10)
found that incorporation of 32P into sugar phosphates was markedly reduced
while the pool size of ATP and other nucleotide triphosphates was increased.
The rate of ATP synthesis was similar in treated and untreated roots. He

suggested that Al and P may react within the mitochondria resulting in a

decrease in the rate of sugar phosphorylation due to inhibition of hexo-

kinase,

3B. Al-Ca Interaction.

Calcium is involved in numerous plant processes. Calcium affected
the uptake of K, P and other ions, and influenced the activity of certain
enzymes. Its involvement in cell division, elongation, cell wall stability,
and membrane binding and/or stabilization has been reported (19, 51).
Therefore, any inhibition in Ca uptake leading to suboptimal plant Ca
levels could have multiple deleterious effects.

Clarkson and Sanderson (14) reported that Al inhibited the uptake
of Ca. They suggested that the control of Ca uptake and translocation
occurred in the epidermis and outer rank of cortical cells of barley roots.
The entry of A1 into the intercellular space could cause displacement of
cations of lower valence, thereby lowering the amount of exchangeable Ca
available for uptake. The superficial location of Al could therefore

restrict entry of Ca into the stele through the intercellular pathway.

58

3C. Al-DNA Interaction.

Several workers suggest that the precipitation of P does not
provide the whole answer to Al toxicity. Clymo (15) stated that the
kinetics of the toxic reaction, wherein deleterious change is almost im—
mediate and recovery follows a considerable lag, precludes a simple
blockage of exchange sites. Clarkson (9) reported that the cessation of
onion root elongation due to Al toxicity was closely correlated with the
disappearance of mitotic figures. Abnormal mitotic figures were not
observed. This inhibition of mitosis could not be duplicated by the with-
drawal of P in the absence of Al in the nutrient solution. Also,
inhibited roots required up to 7 days to restore cell division after
withdrawal of A1 and addition of P. He postulated that some Al-sensitive
mechanism of cell division had been permanently damaged by short exposures

to Al.

Aluminum was associated with nuclei of onion and pea by autoradio-
graphic and aluminum staining techniques respectively (13, 70). Clarkson
(12) suggested that the mitotic cycle of onion was blocked during the DNA
synthesis period. This point of blockage would explain the lack of
mitotic figures and fit the timing of the mitotic cycle. However, some
DNA synthesis was still possible (88).

An association between DNA and Al was observed in pea root nuclei.
Aluminum was recovered from the chromatin of isolated nuclei which had

3M AlCl for 1 day. Almost 90% of the Al was associated

3
with the DNA following separation of the DNA from chromatin proteins.

been treated in 10-

These workers suggested that partial displacement of histone ig_vivo
could increase accessibility of Al to DNA, thus stabilizing the double

helix and limiting template activity (71). Morimura and Matsumoto (74)

59

found that the absorption spectrum of DNA purified from pea seedlings did
not shift after treatment with Al, indicating that Al may bind to the DNA
phosphate. They suggested that the numerous binding sites on an Al poly-
mer may allow binding to phosphates of two strands of DNA causing stabi-
lization of the double strands. Such stabilization may inhibit mitosis

and hence cell division.

4. Mechanisms of Resistance.
Various mechanisms of resistance have been proposed. Clymo (15)

3+. Clarkson

found that Al EDTA was much less toxic and more mobile than Al
(13) suggested that an unidentified chelating agent may prevent Al from
being in a cationic form, thus avoiding injury-

Hydrolysis of A1 at the root cell surface removes Al from solution
due to its solubility product of 10-33 (33). The precipitation of free
Al by excess hydroxyl ions produced in the roots could provide a mechanism
for resistance (13).

Low CEC favors the uptake of monovalent and divalent cations, and
has been associated with Al resistance. The lower CEC of resistant plants
may increase monovalent cation uptake at the expense of the polyvalent Al
(92).

Differential tolerance of two wheat cultivars was associated with
plant-induced pH changes at the root surface. In nutrient culture the
sensitive cultivar lowered the pH while the resistant cultivar raised it,
resulting in a differential of pH 0.7 in the nutrient solution. Such a pH
change is sufficient to significantly alter the solubility of Al (26).

These hypotheses suggest that resistance to Al is based on its

elimination from the cell biochemical processes. This may be accomplished

60

by either blocking entry of Al to the cell or inactivating it chemically

after entry, or a combination of both factors.

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APPENDIX II
GEL ELECTROPHORESIS OF CORN AND

BARLEY ROOT WATER-SOLUBLE PROTEINS

GEL ELECTROPHORESIS 0F CORN AND
BARLEY ROOT WATER-SOLUBLE PROTEINS

Scanning and transmission electron micrographs of A1 treated corn
and barley roots showed severe structural damage, including retarded cell
elongation and changes in the ribosome/endoplasmic reticulum association.
It was postulated that the protein synthesizing process was altered.
Water-soluble proteins were extracted from the root and examined by gel
electrophoresis to detect any changes in the protein profile after Al
treatment .

Water-soluble proteins were extracted from 1 g corn or barley
primary root tips by the method of McCown £5 31, (3). Samples were
ground, strained and centrifuged at 20,000X g for 30 min at 4 C. The
supernatant was dialyzed at 4 C against .05M tris glycine buffer at pH

8.3, frozen in liquid N and lyophilized, and the protein estimated by

2
the Folin reaction (2).

Acrylamide gel electrophoresis was adapted from Davis (1). The
protein was dissolved in 20% sucrose in .05M tris glycine buffer at pH
8.3, and 100 ug samples were layered onto the sample gel. The running
gel was 13 cm long and electrophoresis was conducted at .4 watts/tube
for approximately 6 hr. Gels were stained for 1 hr in Coomassie Blue R
and destained for 24 hr in 7% acetic acid with 1 amp current across 12
gels.

Protein bands were identified in samples from Ca and Al treated

roots (Fig. 1). However, no change in the water-soluble protein profile

after Al treatment was observed for either corn or barley.

68

69

Two possible inferences may be drawn. First, no qualitative or
quantitative change in the water-soluble proteins had occurred. This
seems unlikely given the electron microscope data. Second, qualitative
changes were below the resolution of the gels. Refined protein extraction
and purification, and higher resolution gel electrophoresis, such as iso-
electric focusing, will be required to reveal apparently subtle protein

changes.

70

Figure 1. Diagram of gel after electrophoresis of corn root water-
soluble proteins. Note leading protein band (P).

71

\\\\\‘-CI

 

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72

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