The Ionic Environment and

Plant Ionic Relations

The Ionic Environment and Plant Ionic Relations

Ionic Environment
Ionic Atmosphere is a concept employed in Debye-Hückel theory which explains the
conductivity behaviour of solutions.

“It can be generally defined as the area at which a charged entity is capable of attracting an entity
of the opposite charge”

The role of salts and of their ions in the metabolism of the cell still remains one of the
most important problems in the biological sciences. It was believed that enzyme activity would
be modified in plants when growing in solutions lacking certain salts or ions, and to test this idea
out a number of experiments was undertaken with sugar beets as the test crop. At certain
arbitrary periods during the growth of the plants, leaf material was gathered and the enzymes
extracted. Certain tests were then made on these enzymes to determine their activity under
various conditions. Method. 1. Sand Cultures.

The plants used in the experiments were grown in pure quartz sand cultures in enamelled
earthenware crocks of 2 gallon capacity. There were seventeen of these cultures, nine being
reserved to be watered with a complete nutrient solution (containing all the essential salts in
proper balance) and the other eight to be watered with a solution like the above with the
exception of a deficiency of potassium. In this solution another salt, sodium, was substituted for
the potassium. Care had to be exercised also to adjust the solution to the same osmotic
concentration as that of the complete solution. The gravel layer at the top of the crock, as shown
in the sketch acted as a mulch to check a too rapid evaporation of water and to restrain the
growth of alga. The sugar beet plants’ were grown in the field from pure line seed. When about 2
months old they were carefully transplanted to pots and brought into the greenhouse. Here they
were left for a while to become accustomed to the new environmental conditions before being
again transplanted into the culture crocks. Transplanting was done carefully and the roots freed
of soil particles by a jet of water. Three plants of uniform size were put in each crock. The
complete nutrient solution developed by Stoklasa and Matousek (11) and reported as supporting
luxuriant growth was slightly modified for our purpose and also found to give excellent growth.
A glance at Table I will show that the modification consisted in eliminating certain ions that
occurred frequently and in using magnesium sulfate in the place of calcium sulfate. The two sets
of sand cultures thus prepared were saturated with their respective solutions, which in 10 minutes
were partially withdrawn, by means of a suction pump, to a final water content of 16 per cent.
The total weight was then recorded on the jar. At weekly intervals the solutions were renewed in
the same way and then sucked out to original weight. By this means the water content from week
to week throughout the growing season was approximately maintained. The cultures were set on
a rotating table in the greenhouse. Additional light (five 200 watt nitrogen-filled Mazda lamps)
was used during the winter months. The plants were carefully watched and kept free from insect

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and fungous pests. The tests on these plants were made at two different periods during their
growth, February 10 to 14 and April 7 to 13. On the first date, about 2$ months after being
transplanted, the plants were in good vigorous condition, those in the complete nutrient being
from one-third to one-fourth larger than the plants in the potassium-deficient nutrient. At the
beginning of the second period the plants in the potassium-deficient nutrient had gained on an
average of 46 per cent on their previous measurements, while those in the complete nutrient had
made a gain of 75 per cent. Since all other conditions for growth were the same, it is evident that
potassium has much to do with the vigor and growth of plants. What then can be the influence of
a potassium-deficient nutrient on enzyme activity? How would the enzymes extracted from the
leaves of plants in these two types of nutrient differ in amount, character, and salt activation?

Plant ionic relation and whole-plant physiological responses to

waterlogging, salinity and their combination in barley

The world’s population is expected to reach over 9.3 billion by 2050, prompting the need
to double current agricultural food production. At the same time, agricultural lands globally are
suffering from human-induced and natural environmental stresses such as salinity and
waterlogging. Soil salinisation is affecting more than 800 million ha (~6%) of the land.

Although some of this land is naturally saline, other parts have become saline as a result
of secondary salinisation caused by irrigation. According to the Food and Agriculture
Organisation (FAO), 11% of irrigated lands (~34 million ha) are suffering from the secondary
(human-induced) salinity. As the growth of most agricultural crops is greatly reduced by high
concentrations of salt in the soil, economic penalties are high, and so is the threat to the global
food security. About 60–80 million ha of land are affected to some extent by combined
waterlogging (WL) and salinity stress (FAO 2011).Waterlogging reduces the available oxygen in
the soil and has a profound effect on plant growth. These two stresses are often interrelated, as
waterlogging can lead to land salinisation by transporting the salts to the surface. In many parts
of the world (e.g. Australia, USA, Pakistan, India, Iran, Thailand and Egypt) these two
environmental stresses coexist. Barley (Hordeum vulgare L.) is the fourth major crop plant
amongst the cereals that is used as a forage grain, staple food and to supply malt industries
(Meng et al. 2016). Barley plants are known for their moderate tolerance to salinity (Munns et al.
1995) and sensitivity to waterlogging (Garthwaite et al. 2003).

Waterlogging plays the major role in exacerbating the effects of secondary salinity in
barley (John et al. 1977). Thus, the combination of waterlogging and salinity could seriously
affect the salinity tolerance of barley. Combined WL/NaCl stress significantly decreases most
agronomical and physiological characteristics (e.g. seed germination rate, plant biomass,
chlorophyll content, leaf photochemistry) in many plant species (including barley) compared
with saline and hypoxic saline conditions alone (Kırmızı and Bell 2012; Zeng et al. 2013). The

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physiological and molecular mechanisms of plant responses to each of these separate
environmental constraints have been studied in detail, but the mechanisms underlying plant
tolerance to combined stress have not been well studied. This gap in knowledge is jeopardising
the success of breeding programs so studies addressing this area are needed.

The present study focussed on plant physiological traits under combined stress compared
with separate stresses. The current knowledge of the physiological mechanisms behind the
observed metabolic perturbations and reduction in plant growth under combined waterlogging
and salinity stress were comprehensively reviewed by Barrett-Lennard, and until now were
thought to be associated primarily with increased Na+ and Cl– concentrations in the plant shoot
(Barrett-Lennard 1986, 2003). The rate of Na+ and Cl– transfer from root to shoot was increased
whereas the rate of K+ transport was decreased under combined WL/NaCl conditions compared
with salinity alone (Barrett-Lennard 2003; Malik et al. 2009).

Experiments using different accessions of Hordeum marinum Huds. revealed that their
tolerance to combined WL/NaCl stress was conferred by several complementary mechanisms.
First, tolerant accessions kept Na+ and Cl– concentrations relatively low in the leaves by
restricting the entry of these ions to roots and subsequently the shoots. Second, Na+ and Cl–
were preferentially accumulated in older leaves, to avoid ion toxicity in photosynthetically active
young leaves (Alamri et al. 2013). These results were consistent with observations on wheat,
reporting that Na+ retrieval from the xylem and its storage in the basal shoot tissues reduced Na+
accumulation and mature leaves death, in order to continue the growth under WL/NaCl
conditions (James et al. 2011). Under saline conditions, xylem Na+ concentration is controlled
by several active transport processes in specific root cells, which limit the amount of Na+
transfer to the shoot (Tester and Davenport 2003).

However, these energy-dependent mechanisms can be inhibited when salinity is

combined with waterlogging due to O2 deficiency in the roots (Drew and Läuchli 1985; Shabala
et al. 2014). This notion was further confirmed by comparing the relative tolerance to WL/NaCl
stress of some plants contrasting in their patterns for Na+ accumulation and extrusion (e.g. H.
marinum and H. vulgare) (Malik et al. 2009). It was concluded that any explanation of the
mechanisms by which hypoxia increases the fluxes of Na+ and Cl into roots at the cellular level
needs to be reconciled with our understanding of the regulatory roles of ion channels, H+ -
gradients across cell membranes and H+ -pumping ATPases (Barrett-Lennard 2003). In this
context, the increased Na+ accumulation under combined WL/NaCl stress could be a result of
either an increase in n Na+ uptake or a decrease in the roots ability to extrude Na+ back to the
external media. From the thermodynamics point of view, the former mechanism is unlikely, as
the electrochemical gradient for Na+ entry through non-selective cation channels (NSCC; the
main Na+ uptake pathway) is expected to decrease due to root plasma membrane-depolarisation
caused by hypoxic conditions (Demidchik and Maathuis 2007; Kronzucker and Britto 2011). The
decreased ability of the roots to extrude Na+ back to external media is more likely (Barrett-

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Lennard and Shabala 2013). This active extrusion is mediated by plasma membrane Na+ /H+
antiporters that are powered by the plasma membrane H+ - ATPase (Shi et al. 2000; Qiu et al.
2003). Another aspect that should be considered is a cytosol acidification under hypoxic
conditions (Felle 2005). The intracellular pH regulation under oxygen deprivation stress is a
complex interaction of biophysical (active H+ transport from the cytoplasm) and biochemical
(H+ buffering and H+ producing or consuming reactions) mechanisms (Kulichikhin et al. 2008;
Couldwell et al. 2009). Nuclear magnetic resonance (NMR) studies have revealed a significant
difference in the hypoxia stress-induced pHcyt kinetics between contrasting species. Although in
the hypoxia-tolerant rice cytosolic acidification was only transient, apical root cells failed to
regain optimal pH levels in the hypoxia-sensitive wheat species (Kulichikhin et al. 2007). As
many plasma- and organelle-based membrane transporters show strong pH dependence, hypoxia-
induced changes in cytosolic pH may significantly affect ionic homeostasis. This is specifically
true for Na+ and its distribution in intracellular compartments. The SOS1 Na+ /H+ antiporter
shows a clear pH-dependency (Oh et al. 2010), and cytosolic acidosis under hypoxic conditions
is expected to reduce efficiency of Na+ removal from the cytosol, as a consequence of reduced
chemical gradient for H+ . A deletion of SOS1 affects proton-flux in the Arabidopsis root
(Shabala et al. 2005), suggesting an involvement in pH homeostasis. The effect of hypoxia on
barley plant ionic relations has clearly pronounced tissue specificity (Zeng et al. 2014). Barley
plants exposed to WL conditions showed severe O2 deficiency in the stele and apical regions of
the roots, and the most pronounced reduction in concentration of K, Na and Cl was observed in
the xylem parenchyma and pericycle cells at the subapical region, due to the stele oxygen
deficiency. Near the root apex of plants under NaCl conditions, Na+ concentration had declined
across the cortex although it was relatively high in all cell types in roots under WL/NaCl stress
(Kotula et al. 2015). At the same time, Na+ accumulation in the outer cell layers helped in Na+
reduction in the tip inner cells. In order to prevent oxygen deficiency under combined WL/ NaCl
stress, H. marinum shows a larger percentage of root porosity than wheat. Moreover, high rates
of radial O2 loss (ROL) close to the adventitious roots tips and low ROL rates near the shoot
base indicated that H. marinum generated a barrier to ROL (Alamri et al. 2013). Aerenchyma
development in the root of wheat varieties under combined WL/NaCl conditions, and was higher
close to the root–shoot interface than near the root tip. However, the rate of aerenchyma
development in both areas was significantly less than in WL alone, and aerenechyma formation
was not found to be responsible for varietal difference in tolerance to WL/NaCl conditions
(Akhtar et al. 1998).

The essential nature of Na+ exclusion from the cytosol for plant adaptive responses to
saline environment is well recognised and widely accepted (Munns and Gilliham 2015; Munns et
al. 2016), but the essential nature of potassium homeostasis for plant adaptive responses to
adverse soil conditions has emerged over the last decade (reviewed by Shabala and Pottosin
2014; Shabala et al. 2016). Another comprehensive literature analysis by Barrett-Lennard and
coauthors comparing halophytes and glycophytes species suggests that this may be indeed the
case for plant responses to combined WL/NaCl stress (Barrett-Lennard and Shabala 2013). What

5
about glycophytes per se? Does their tolerance to combined salinity and hypoxia stress correlate
with K+ retention? If yes, which trait is more essential: Na+ exclusion or K+ retention? Finally,
are detrimental effects of combined WL/NaCl stress synergistic or additive, compared with
separate stresses? The aim of the present study was to answer all these questions.

Plant Nutrient Ions and Enzymes

The two enzymes for study were those chiefly responsible for carbohydrate changes,
amylase and saccharase. Both of these were extracted from the leaves at the same time by the
following method: 20 to 30 gm. of fresh leaf material were shredded up after the petioles had
been thrown out. A definite weighed sample was then mixed with clean quartz sand and ground
in a porcelain mortar until it had become a thin paste. The juice was then pressed out by hand
through cheese-cloth, diluted with an equal quantity of distilled water, and centrifuged for 5
minutes at 2000 R.P.M. The clear liquid was pipetted off and used immediately. Toluene was
added as a preservative where necessary. This method of making the enzyme preparation is
preferable to that in which the leaf material is first dried and then extracted. The enzyme appears
to be obtained in unmodified form by this method. The presence of amylase in the sugar beet was
demonstrated long ago by Gonnermann (8) and later by Brasse (2). No quantitative experiments
were reported.

Later Palladin and Popoff (9) isolated amylase from sugar beet leaves and the leaves of
many other kinds of plants. These men reported in addition more amylase in young leaves than
in older ones. Saccharase was shown by Gonnermann (8) to be present in the sugar beet plant.
Later Stoklasa, Jelinek and Vftex (10) found this enzyme in roots undergoing anaerobic
respiration. Cohn (4) made a study of the roots and leaves separately and found it always in the
leaf, but absent in the roots that were growing normally. At the same time Bodnar (1) showed
that it was absent in healthy roots but present in roots suffering from a disease, commonly known
as “root-rot” (Schwanzfaule).. Several preliminary experiments, which will be briefly noted, had
to be conducted in order to become acquainted with the activity of the enzyme preparation and
the various experimental conditions. For these studies good vigorous plants were selected from
the general supply brought into the greenhouse from the field and potted in their own soil. The
enzyme preparations were so active that considerable dilution (10: 1000) was necessary at the
start. No buffers were used as it was thought that natural ones in sufficient quantities were
present in plant juices.

Furthermore, under the conditions arbitrarily set, it was necessary to study the influence
of alkali salts without the disturbance of other buffers. The salts used in the study of enzyme
activation were potassium nitrate, sodium nitrate, and potassium chloride. Studies were also
made to determine the proper salt concentrations and to find out which salt and which ion
exerted the greatest influence. In testing for amylase and saccharase in young and older leaves it
was found that young leaves showed more amylase than older ones. It did not appear to be a
matter of the size, but just a matter of age. In regard to saccharase activity the young leaves
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contained more enzyme than the older ones. Great care then had to be exercised in the collection
of material for comparative tests., All leaf collections were made at noon. The method of
procedure for determining the diastatic power or the sucroclastic activity of the leaf juice was
that used by the senior author in his studies on the enzymes of the potato leaf (5, 6). For the
amylase studies the method consisted in incubating a 5 cc. sample for 24 hours at 38”C., and
then determining by Wohlgemuth’s (13) calorimetric iodine method the diastatic power. In the
tables this is represented as Di$, (diastatic power at 38°C. for 24 hours). The 5 cc. sample
contains a definite amount of a 1 per cent starch solution, a definite concentration of enzymatic
juice, and 0.1 cc. of toluene as a preservative. The details of the method as used in the studies on
saccharase are as follows: 2 cc. of enzymatic preparation are put into a total of 50 cc. of a
solution which is made up of saccharase at 5 per cent, toluene (0.1 cc.) as a preservative, and the
necessary amount of activating salt to reach the concentration called for in the test. This solution
is then put into the incubator at 38°C. At certain time intervals, previously determined upon, 5
cc. quantities are withdrawn and treated with 1.5 cc. of lead subacetate (saturated solution diluted
2:5). After shaking, 0.5 cc. of a 12.8 per cent NazSOI solution is introduced and the solution
shaken again. It is now centrifuged and 4 cc. of the clear liquid are mixed with 1 cc. of a 5 per
cent solution of Na2C03 (made from crystals). This is again centrifuged and the clear solution is
polarized in a 20 cm. tube at 20°C. The constant (Ic) of the reaction is calculated according to
von Euler and Svanberg (7), R-FL k = f + log d+L where t is time in hours from the beginning of
the reaction, R the initial rotation, L the final rotation, and d the observed rotation at t time.

Ionic Homeostasis
Ionic homeostasis is a fundamental cellular phenomenon. All living cells maintain an
intracellular ionic composition compatible with their constituent molecules, and this requires the
regulation of multiple membrane transporters and signal transduction pathways. Other
biophysical parameters such as turgor and electrical potential are also part of this essential
regulation. How ionic homeostasis is achieved, however, is not completely understood. Although
most transporters have already been identified, their physiological function is only starting to be
demonstrated and the receptors and most components of the regulatory pathways that effect ionic
homeostasis remain unknown. In the case of plants, this problem is related to mineral nutrition
and salinity tolerance, both of which have great relevance for agriculture. In fact, as
demonstrated by this meeting, salinity stress has been one of the keys to opening the black box of
ionic homeostasis in general. Another has been the novel molecular genetics of the
plant Arabidopsis thaliana. Of course, other approaches have also contributed to our present
understanding of ion homeostasis in plants and were represented at the meeting.

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Some physiology of salt tolerance
Salt stress is an important threat to the future of agriculture in many productive areas of
the planet. In countries such as Australia and Pakistan, salinity is already a national concern, as it
was in the past in ancient Mesopotamia. Areas of California and the Mediterranean region are
also threatened.
The greatest challenges faced by plants cultivated in the presence of excess salt are
osmotic regulation, ion transport and toxicity and oxidative stress. The signal transduction
pathways that are involved are not completely understood but include calcium-activated protein
kinases, stress-activated MAP (mitogen-activated protein) kinases and the hormone abscisic
acid. Different speakers discussed these physiological aspects. Salinity stress results in increased
production of reactive oxygen species, and detoxifying enzymes such as ascorbate peroxidase
and glutathione peroxidase (G. Ben-Hayyim, Bet Dagan, Israel) are important components of the
cellular response to salt stress. Plants rarely experience stress from a single environmental
source, and multi-stress interactions were demonstrated by salt-stressed plants whose state was
exacerbated by the resulting increase in their uptake of toxic boron and cadmium (A. Laüchli,
Davis, CA). Genetic studies in wheat have demonstrated the important role of Na+ exclusion
from the shoot in salt tolerance. This Na+ exclusion trait may be explained by
K+/Na+ discrimination during xylem loading and the genes responsible are being approached by
molecular markers (R. Munns, Canberra, Australia). It is probable that these will encode
components of cation uptake and efflux systems. The synthesis of organic molecules known as
‘osmolytes’ is an important stress response for osmotic adjustment and the stabilization of
cellular structures. A.D. Hanson (Gainesville, FL) described a very ambitious, long-term project
on the metabolic engineering of glycine betaine, one of the most effective osmoprotectants and
naturally found in salt- or drought-tolerant plants like corn or sugarbeet. Manipulating the
synthetic pathway in plants, such as tobacco, that are unable to accumulate this osmolyte is
proving difficult, however, because successive bottlenecks need to be overcome. For example,
once the conversion of choline into glycine betaine was increased by overexpression of
chloroplast choline monooxygenase and betaine aldehyde dehydrogenase, the limiting role of
choline synthesis and uptake by the chloroplast became apparent and only a modest
accumulation of glycine betaine was obtained.

Sodium and potassium transporters

In order to survive extreme saline conditions, plants must maintain a high cytoplasmic
K+/Na+ ratio and therefore must be efficient at K+ uptake in a high Na+ background and be able
to exclude or remove Na+from the cytoplasm. H. Sentenac (Montpellier, France) presented a
systematic study of the different isoforms of the K+ channels of Arabidopsis. The approaches
used included the characterization of knock-out mutants, determination of the expression patterns
in plant tissues and expression of recombinant proteins in yeast and insect cells. The inward
rectifier AKT1 is a major route of K+ uptake from soil by root epidermis. The outward rectifier
SKOR present at the root stele largely mediates xylem loading of K+. In guard cells the inward
rectifiers KAT1 and KAT2 participate in stomata opening, whereas the outward rectifier GORK
is required for stomata closing. The pollen-specific AKT5 (inward rectifier) is required for pollen
tube development. Other isoforms that are less well characterized include the phloem-specific

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AKT2 and AKT3, the flower-specific AKT6 and KC1. These channels are highly selective for
K+ over Na+ and therefore are unlikely to mediate Na+ transport during salt stress.
One major pathway for Na+ uptake is blocked by external calcium and occurs via non-
selective cation channels. D. Sanders (York, UK) presented evidence for members of the cyclic-
nucleotide-gated channel (CNGC) family, specifically Arabidopsis CNGC1 and CNGC3, being
mediators of such activity. Expression of CNGC3 in yeast increased Na+ uptake, whereas an
Arabidopsis knock-out mutant in CNGC1 was more tolerant to moderate Na+ concentrations.
The other, calcium-insensitive, pathway for Na+ uptake was clarified by the poster of A.
Rus (West Lafayette, IN), winner of the special poster award of the workshop, and also discussed
in the presentation by R.A. Bressan (West Lafayette, IN). His laboratory is collaborating with
those of P.M. Hasegawa (West Lafayette, IN), J.-K. Zhu (Tucson, AZ) and M. Reddy
(Bhavanagar, India) to perform a screen for suppressor mutations of the salt-sensitive
Arabidopsis sos3 mutant, which hyperaccumulates Na+. From >65 000 T-DNA-tagged lines
derived from the sos3 mutant, two null alleles of the HKT1 transporter were identified as
suppressors of both the Na+ sensitivity and Na+ hyperaccumulation of the sos3 mutant. These
results constitute the in vivo demonstration that HKT1 is an entry system for Na+. Whether SOS3
simply modulates the SOS1 Na+ efflux system or also the HKT1 influx system is still unclear.
E. Blumwald (Davis, CA) presented data on transgenic tomato and rapeseed (Canola)
plants overexpressing the Arabidopsis NHX1 vacuolar Na+–H+ antiporter, and the resulting salt
tolerance was impressive (Zhang and Blumwald, 2001; Zhang et al., 2001). However, it was
argued by some participants that the resistance observed, compared with the control plants, may
have been dependent on the salinization regime, as a gradual increase in salinization resulted in
greater tolerance of the transgenic plants, which had not always been observed in other
laboratories.
A. Rodriguez-Navarro (Madrid, Spain) discussed the ENA-like cation extrusion ATPase,
which is a major determinant of salt tolerance in fungi but is absent from plants. Members of the
family exhibit different relative activities with Na+ and K+ as the pumped cations. The natural
history of the enzyme suggests that it was originally a K+-extrusion pump of fungi associated
with plants and therefore exposed to high external K+ concentrations. The most Na+-specific
members, such as the Neurospora crassa ENA-like ATPase, could be useful tools to improve
salt tolerance in plants when specifically expressed in root epithelia.