Doctoral Thesis

Physiological Responses of Rice to SalineAlkaline Stress

Rowland Maganizo Kamanga

Graduate School of Integrated Sciences for Life
Hiroshima University

September 2021

Doctoral Thesis

Physiological Responses of Rice to SalineAlkaline Stress

Rowland Maganizo Kamanga

Program of Bioresource Science
Graduate School of Integrated Sciences for Life
Hiroshima University

September 2021

List of Abbreviations
APX

Ascorbate peroxidase

CAT

Catalase

FAO

Food and Agriculture Organization

GPX

Guaiacol peroxidase

GR

Glutathione reductase

HAs

H+-ATPases (Adenosine triphosphate)

IRT

Iron (Fe) regulated transporter

MA

Moderate alkalinity

MDA

MalondialdehydemM

Millimolar

MSA

Moderate saline alkalinity

MSI

Membrane stability index

NA

Non-acclimating

NaCl

Sodium chloride

NPT

No pretreatment

NSA

Na+ content under saline alkalinity

PT

Pre-treatment

RDW

Root dry weight

ROS

Reactive oxygen species

SA

Severe alkalinity

SDW

Shoot dry weight

SEL

Shoot electrolyte leakage

SOD

Superoxide dismutase

SPAD

Soil – plant analyses development

SSA

Severe saline alkalinity

UN

United Nations

UNESCO

United Nations Educational, Scientific and Cultural Organization

μM

Micromolar

Table of Contents
Chapter 1 General Introduction ............................................................................................. 1
Chapter 2 Time – Course Study on Differential Responses of Rice Seedlings to Saline,
Saline – Alkaline and Alkaline Stress................................................................................... 13
Introduction .......................................................................................................................... 14
Materials and methods ......................................................................................................... 16
Results.. ................................................................................................................................ 20
Discussion ............................................................................................................................ 39
Conclusion ........................................................................................................................... 44
Chapter 3 Study of Acclimation and Induced Cross – Tolerance of Rice Seedlings toSaline – Alkaline Stress ......................................................................................................... 45
Introduction .......................................................................................................................... 46
Study objectives ................................................................................................................... 47
Materials and methods ......................................................................................................... 48
Results. ................................................................................................................................. 56
Discussion ............................................................................................................................ 70
Conclusion ........................................................................................................................... 74
Chapter 4 General Discussion............................................................................................... 76
Chapter 5 Summary .............................................................................................................. 81
References ............................................................................................................................... 86
Acknowledgements ................................................................................................................ 99

Chapter 1

General Introduction

11.1. Background information
Global changes in the climate scenario undeniably pose insurmountable challenge on global
food supply system. Formerly agriculturally productive lands are insidiously mutating to
irreparably unarable. Yet, global population is steadily increasing, projected to reach an insane
9.8 billion by 2050 (UN 2017). Therefore finding ways to sustain agricultural productivity inlight of the prevailing and worsening climate to support the growing population is one of thecurrent’s and future’s major global hurdles. Persistent droughts, extreme temperatures, soil
salinization, and heat stresses have gradually become abiotic norms in the agricultural setting.
Soil salinization for one has victimized agriculture since time immemorial, and has remained
an important factor constraining worldwide crop productivity (Negrão et al. 2011). As a result,remarkable strides have been made in unmasking plants’ responses and tolerance mechanisms
to salinity stress. In the same way, soil pH has long been recognized as an important factor
influencing plant growth, as such optimal pH ranges for plant growth have been accordingly
established. For plant growth purposes, most plant species grow better in slightly acidic toneutral pH range (5.5 to 7.0), and growth is considerably downregulated at alkaline pH, this is
generally referred to as “alkaline” stress.

Meanwhile, there is a growing awareness and recognition that salinity stress often co-occurs
with alkaline stress (Kawanabe & Zhu 1991). Simply put, this is as a result of soil salts that areboth saline (impose Na+ toxicity) and alkaline (raise soil pH), this is referred to as salinealkaline stress or mixed saline-alkaline stress, and its effects on crop growth and productivity
are cruelly devastating (Islam et al. 2011; Zhang & Mu 2009) than neutral salinity stress. Prior,soil scientists had endeavoured to classify soils based on salt composition. While there has
hardly been a consensus in nomenclature, distinctions have been made on what are considered“saline” from “sodic” soils (FAO 2019; Szabolcs & Fink 1974). Based on these, it is generally2agreed that “saline” soils are those containing a substantial amount of “neutral” soluble sodium
salts with a capacity to negatively alter plant growth and productivity, in principal, these salts
are sodium chlorides (NaCl) and sodium sulphates (Na2SO4). Conversely, soils are considered“sodic” when they contain sodium salts that are capable of undergoing “alkaline hydrolysis”,
principally, these salts include sodium carbonates (Na2CO3) and sodium bicarbonates(NaHCO3). Accordingly, salt stress arising from the former has been termed neutral salinity
stress, or simply salinity stress whereas the latter has been termed “alkali” stress (Peng et al.
2008; Szabolcs & Fink 1974). Therefore, these terms will be used interchangeably in thepresent context. Meanwhile, the need for development of crop cultivars bearing resistance toalkali stress has never been more exigent. Attaining this therefore calls for a comprehensive
understanding of plants’ responses governing resistance and adaptability to alkali stress.

1.2. Comparative outlook of salinity, alkalinity and saline-alkalinity
Salinity stress has comprehensively been studied, hence knowledge regarding its effects onplant growth is well documented. Majorly, salinity stress (1) imposes toxicity of Na + in plantorgans at the expense of beneficial minerals particularly K+, (2) results in osmotic stress(practically synonymous to “physiological drought”) and (3) elicits hyperaccumulation ofreactive oxygen species (ROS) (Munns & Tester 2008; Shabala et al. 2010). Saline-alkaline
stress exerts all the above effects, in addition to an inordinately high pH resulting fromcarbonates which primarily affects root properties (Peng et al. 2008; Yang et al. 2009; Zhang& Mu 2009; Zhang et al. 2019) and absorption of metal cations particularly Fe and P(Kobayashi & Nishizawa 2012; Takahashi et al. 2001). Fe deficiency manifests itself inchlorosis of younger leaves. These latter effects are also imposed by “alkaline” stress arisingfrom non-sodic alkaline salts such as K2CO3, KHCO3, CaCO3 and Ca(HCO3)2.

3

Na+ and K+ have similar physicochemical properties, resulting into their competitive uptake
systems and failure to discriminate against their identities under higher salt concentrations.
This results into higher preferential Na+ uptake at the expense of K+. Na+ toxicity in leaf blades
is typified with premature senescence of older leaves (Munns & Tester 2008). Osmotic effectof salt stress manifests itself in failure to produce new leaves and reduced expansion of young
leaves. Plants are surfeited with various strategies and mechanisms to tolerate to salinity,
saline-alkaline and alkaline stress, these may be shared across the stress types while others may
be distinct. A comparative outlook of salinity, alkalinity and saline-alkaline stress is shown in
Table 1.1.

1.3. Growth of rice under saline-alkaline stress
Rice (Oryza sativa L.) is one of the most important cereal crops in the world. However, despite
its worldwide agronomical prominence, it is considered the most sensitive cereal crop to
salinity stress (Munns & Tester 2008). Coupled with its poor Fe uptake capacities (Masuda etal. 2008 2009; Ishimaru et al. 2007) and leaky nature of its roots (Munns & Tester 2008), it isconceivable that its sensitivity to alkali stress is all the more extreme. Meanwhile, growing
evidence ascertain that its response to alkali stress is poorer (Chuamnakthong et al. 2019;
Zhang et al. 2017). What remains a fundamental question is whether plants bearing increased
tolerance to salinity stress would potentially be better adapted to alkali stress. Lv et al. (2015)
reported that a majority of japonica rice cultivars showed tolerance to salinity stress but weresensitive to saline-alkaline stress. This demonstrates either severity of alkali stress over salinity
stress or involvement of different mechanisms in tolerance to the two. Earlier, similar findings
were also reported (Lv et al. 2013). However, some cultivars demonstrated tolerance to bothsalinity and saline-alkaline stress. Chuamnakthong et al. (2019) showed that a salinity tolerant4rice cultivar exhibited higher tolerance to saline-alkaline stress by effectively excluding
accumulation of Na+ in the leaf blades.

Recently, Guo et al. (2014) developed an alkali tolerant mutant alt1 in rice that grows wellunder saline-alkaline stress with less chlorosis, higher survival rate and better growth througha stable acquisition of metal ions particularly Fe. Increasing number of reports indicate
considerable damage elicited by saline-alkaline stress in roots. Being directly exposed to highNa+ and high pH, roots are particularly at higher risk of damage and injury. Marked increasein root cell injury and expression of cell death related genes have been repeatedly reported (Lvet al. 2013; Zhang et al. 2017). Consequently, preserving better root properties in rice underalkali stress has been shown to correlate with resistance to alkali stress (Guo et al. 2014; Lv et
al. 2015). Zhang et al. (2017) showed that a saline-alkaline stress tolerant rice cultivar had
better root properties and suffered less lipid peroxidation and cellular damage by efficiently
scavenging ROS. In summary, present knowledge permits the conclusion that rice is moresensitive to saline-alkaline stress, or at least that saline-alkaline stress is more severe thanneutral salinity stress. Furthermore, it is imperative to learn from plants that are naturally
adapted to grow on sodic soils. Meanwhile, a number of such plants have been studied and may
therefore present better prototypes for saline-alkaline stress tolerance improvement programs
for agriculturally important crops such as rice.

1.4. Growth and ionic responses of other crops to saline-alkaline stress
A number of plant species, halophytes and glycophytes alike are known to have remarkable
growth characteristics under salt stress. Among the cereal crops, barley (Garthwaite et al. 2005;
Munns & Tester 2008; Shabala et al. 2010) and quinoa (Hariadi et al. 2011) offer excellent
prototypes for studying comparative responses to salt stress.

5Na+, CO32-, HCO3High pH induced nutrient deficiency
K+, Ca2+, Fe, P

Na+, Cl+, SO42-

Osmotic, ion toxicity, oxidative

stressK+, Ca2+

Dominant ions

Main stresscomponents

Major nutrient

Key tolerant strategies

Symptoms

deficiencies

Na2CO3, NaHCO3

NaCl, Na2SO4

Dominant salts

in younger leaves, darker roots
Na+ exclusion, Osmotic tolerance, Tissue
tolerance, ROS scavenging, Ion balance,

leavesNa+ exclusion, Osmotic tolerance,

Tissue tolerance, ROS scavenging

6

Induce Fe-uptake systems

darker roots

new leaves, death of older leaves, chlorosis

develop new leaves, death of older

Induce Fe-uptake systems

ROS scavenging,chlorosis in younger leaves,

Limited leaf expansion, failure to develop

Fe, P

induced nutrient deficiency

Oxidative stress, High pH

K+, Ca+, CO32-, HCO3-

Ca(HCO3)2

K2CO3, KHCO3, CaCO3,> 7.5

Alkaline

Limited leaf expansion, failure to

Osmotic, ion toxicity, oxidative stress,> 7.5

5.0 – 6.0

pH

AlkaliSalinity

Characteristics

Salt stress

Table 1.1. Comparative outlook of neutral salinity stress, saline-alkaline (alkali) and alkaline stressYang et al. (2009) showed that barley plants were also more sensitive to saline-alkaline stress,
but were able to tolerate up to 150 mM Na+ at a pH above 9.0. Similar observations were sharedby other studies (Shi & Yin 1993; Yang et al. 2007; Zhang et al. 2019). Consistent withobservations in rice Yang et al. (2009) observed nearly ten-folds reduction in root activity
under saline-alkaline stress relative to salinity stress. Similar findings were also reported inalfalfa (Peng et al. 2008), providing independent support for the fundamental role roots playunder saline-alkaline stress.

On ionic relations, barley accumulates larger amounts of Na+ in leaf blades that it effectively
sequesters in vacuoles, attaining both turgor and avoidance of cytosolic Na+ toxicity (Shabalaet al. 2010). Under saline-alkaline stress, barley accumulates even more Na+ (Yang et al. 2009)in leaf blades than that under salinity stress, consistent with observations in rice and wheat(Yang et al. 2008). Halophyte Kochia sieversiana is naturally adapted to saline-alkaline soils.
Yang et al. (2007) studied its growth and tolerance to saline-alkaline soils, and reported thatthe halophyte can grow at up to 400 mM soil Na+.

Strikingly, in K. sieversiana uptake and transport of Na+ to the shoots were similar between
salinity stress and saline-alkaline stress. Not only that, increases in soil Na+ concentration underboth salinity and alkali stress were associated with concomitant increases in both Na + and K+
in shoots, a unique and striking feature. This was also earlier observed in another halophyte
Sea buckthorn (Chen et al. 2009). These observations signal lack of competitive inhibition
between Na+ and K+, highlighting a distinctive pathway for K+ absorption in halophytes. While
this demands further in-depth inquiry, it undeniably provides for a splendid learning andstarting point for the improvement of alkali tolerance in glycophytes.

7

1.5. Na+ homeostasis under saline-alkaline stress
Saline-alkaline stress is characterized by toxicity of Na+ in plant organs, hence plants’ ability
to minimize uptake and transport of Na+ to shoots is an important feature of tolerance.
Comparative studies have shown that rice plants accumulate significantly higher contents of
Na+ in leaf blades under saline-alkaline stress than neutral salinity (Cao et al. 2020; Yang et al.
2009; Yang et al. 2008). These observations instigate the thinking that high pH under alkali
stress either reduces plant roots’ ability to control Na+ uptake or compromises plant roots’ability for Na+ extrusion.

In rice, extrusion of Na+ from roots into the rhizosphere is mediated by the plasma membrane
Na+/H+ antiporter OsSOS1 that exchanges cytoplasmic Na+ for external H+ (Munns & Tester
2008; Zhu 2003). This banks on transmembrane proton gradient between cytosol andrhizosphere, generated by H+ pumps (H+-ATPases) (Duby & Boutry 2009; Moriyama 2015)
which is otherwise deficient under high pH conditions, consequently impairing extrusion of
Na+ into the external environment. Cao et al. (2020) has identified Zea mays L. Na+ content
under Saline Alkaline 1 (ZmNSA1), a Ca2+ dependent gene that mediates lower shoot Na+
accumulation by promoting H+-ATPase (HAs) activity and consequent root Na+ efflux. This is
an important revelation for saline-alkaline studies, revealing important insights into high pH
induced hyperaccumulation of Na+ in leaf blades.
1.6. Fe uptake under saline-alkaline stress
Plants growing under high pH conditions have long been characterized by Fe deficiency
chlorosis (Pushnik et al. 1984), hence dominating efforts aiming to increase tolerance of plants
to alkaline soils have entailed improvement in Fe uptake (Gómez-Galera et al. 2012; Kobayashi
et al. 2008 2007; Ogo et al. 2011; Takahashi et al. 2001; Ishimaru et al. 2007). Despite being

8

naturally abundant in the soil, Fe forms highly insoluble ferric compounds under high pH
resulting into decreased bioavailability for plant uptake (Nakanishi et al. 2004).
Chuamnakthong et al. (2019) reported higher differential expression of Fe deficiency
responsive genes (OsNAS1, OsNAS2, OsIRT1 and OsIRO2) under saline-alkaline stress in atolerant rice cultivar. Li et al. (2016) also attributed the tolerance of a saline-alkaline tolerant
rice cultivar to efficient Fe acquisition. These findings emphasize the crucial role enhanced Fe
uptake plays under saline-alkaline stress hence its utilization in interventions for increasing
resistance.

Roots of graminaceous plants such as rice secrete mugineic acids (MAs), a family ofphytosiderophores that solubilize ferric iron (Fe3+) forming Fe(III)-MA complexes that aresubsequently taken up by plant roots (Ishimaru et al. 2006; Takagi 1976) using Fe(III)-MA
transporter, YS1/ YSL, this is referred to as strategy II. Transgenic approaches have been
explored by utilizing genes involved in synthesis of mugineic acids from barley into rice suchas HvNAAT-A and HvNAAT-B (Takahashi et al. 2001) and YS1/YSL that codes for transporterof Fe(III)-MA complexes into the root cells (Gómez-Galera et al. 2012).

Other transgenic strategies to introduce genes involved in strategy I of Fe uptake have also
been explored following revelations that rice plant roots can also take up Fe2+ directly using
the Iron regulated transporter OsIRT1 (Bughio et al. 2002; Ishimaru et al. 2006) especially
under anaerobic conditions. These include introduction of a yeast chelate reductase gene,refre1/372 (Ishimaru et al. 2007). Put together, these studies highlight the essentiality enhanced
Fe uptake offers under alkaline conditions, and should thus be a fundamental attribute efforts
aiming to improve rice plants’ resistance to saline-alkaline stress should explore.

91.7. Abiotic stress, reactive oxygen species generation and oxidative stress in plants
An inevitable consequence of aerobic metabolism is the accumulation of ROS, extremely
harmful radicals that are overproduced under environmental stresses (Apel & Hirt 2004;
Kamanga et al. 2018; Mekawy et al. 2018; Sharma et al. 2012). ROS are kept in check by thesynthesis of enzymatic and non-enzymatic antioxidants that detoxify them, keeping theirconcentrations optimal for beneficial purposes (Sharma et al. 2012). However, under stressed
environments, synthesis of ROS is uncontrollably increased (Apel & Hirt 2004) past plants’
detoxification capacity thereby putting the plant in a state of oxidative stress. Under such astate, ROS can potentially cause peroxidation of lipids, protein damage and programmed cell
death (Apel & Hirt 2004; Sharma et al. 2012).

Under saline-alkaline stress, most studies have associated the sensitivity of plants to root
damage caused by high pH environment in combination with Na+. Being directly exposed tothe saline-alkaline environment, roots are particularly at a higher risk of damage as shown by
the remarkable reductions in root activity in a range of plant species (Peng et al. 2008; Yang etal. 2009; Zhang & Mu 2009; Zhang et al. 2019). Zhang et al. (2017) showed a significant
accumulation of H2O2 and O2•- in roots exposed to saline-alkaline stress. Furthermore, Guo etal. (2014) found that in an alkaline tolerant rice mutant (alt1) 50 out of 78 abiotic stress relateddifferentially expressed genes were involved in generation and detoxification of ROS andrepair of ROS mediated DNA damage. It is reasonable therefore, that alkali stress tolerance in
breeding programs should consider designing plants that are able to avoid ROS production,
detoxify ROS and or repair ROS mediated damage, these three processes may translate into anoxidative stress tolerance strategy.

10

1.8. Study rationale
Global population is estimated to reach 9.8 billion by 2050 (UN 2017), posing a striking socioeconomic challenge; to sustainably support the ever growing population. Rice is a prominentcereal crop serving as a top staple food crop for more than half of the world’s population.
Notwithstanding its agronomical and food value, it is also regarded as the most sensitive cereal
crop to salt stress (Munns & Tester 2008). Moreover, its growth is considerably impeded byhigh pH, in part owing to its poor Fe uptake capacities despite being equipped with a dual Fe
uptake system (Ishimaru et al. 2006; Masuda et al. 2008; Takahashi et al. 2001).

Saline-alkaline stress is supposedly more devastating, exhibiting effects of both salinity (Na +toxicity, osmotic stress and oxidative stress) and alkalinity (high pH). However, unlike solesalinity and alkalinity stress, rice plants responses to saline-alkaline stress have not been clearly
elucidated. In order to develop rice crop cultivars tolerant to saline-alkalinity, an understandingof how saline-alkalinity stress affects survival, growth and physiology of rice plants isimperative. Present knowledge on rice plants’ responses to sole salinity and sole alkaline stressis vast, hence comparative studies with saline-alkalinity may offer an optimal baseline forstudying effects of saline-alkalinity.

Furthermore, it is imperative to explore quicker and cost effective options of enhancing rice
plants’ tolerance to saline-alkaline stress. Plants are known to develop tolerance to abiotic
stress following pre-exposure to a mild level of similar abiotic stress, a process known as
acclimation or acquired resistance. Furthermore, plants pre-exposed to one abiotic stress also
reportedly develop tolerance to another type of stress, this is referred to as induced crosstolerance. Hence, this study further attempts to explore an option for inducing cross-tolerance
to saline-alkaline stress in rice plants by pre-exposure to salinity stress, oxidative stress andsaline-alkalinity.
11

1.9. Study objectives
The study was conducted to achieve the following overarching objectives;

1.

To investigate comparative physiology of neutral salinity, alkalinity and saline-alkaline
stresses in rice.

2.

To study acclimation induced cross-tolerance of rice seedlings to saline-alkaline stresspre-exposed to mild salinity, oxidative and saline-alkaline stress.

3.

To investigate the role of pre-treatments in post saline-alkaline stress recovery of rice.

12

Chapter 2

Time-Course Study on Differential Responsesof Rice Seedlings to Saline, Saline-Alkaline, andAlkaline Stress

13

2.1.

Introduction

Land salinization and alkalinisation are widespread agricultural problems worldwide and havethus haunted mankind for centuries. Relative to neutral salinity stress, saline-alkaline stress is
considerably detrimental. Meanwhile, effects of salinity stress on crop growth and productivity
have been comprehensively studied in nearly all major crop plants, rice inclusive.
Notwithstanding recent increases in land saline-alkalinisation and its associated aggravatedeffects on crop growth compared to those of neutral salinity stress, plants’ responses to salinealkaline stress are poorly understood. A large number of reports indicate that saline-alkaline
stress is much more harmful than either sole salinity stress or sole alkaline stress, insinuatinga cooperative effect of their co-occurrence on plants (Chan et al. 2003). As a result, plants haveover the years developed sophisticated mechanisms to withstand both high Na + toxicity andhigh pH. Unlike most halophytes, glycophytes prioritize prevention of Na+ accumulation insensitive organs such as leaf blades and opt to partition inevitably accumulated Na + in thevacuoles albeit less adept. Na+ is a toxic element in plant cells, considerably interfering withcytosolic metabolic activities, hence sequestration in the vacuole not only averts its toxicity inthe cytosol but also serves as a cheap osmoticum aiding in turgor maintenance. Meanwhile,
rice (Oryza sativa L.) has a well-known reputation for its tremendous sensitivity to salt stress(Munns & Tester 2008), hence, coupled with the leaky nature of its roots, high pH may further
weaken its root properties and consequent growth and development under saline-alkaline stress.

Decreases in root activity, compromised root cell vigour, deteriorated root cell viability andaggravated cell death have often been reported under saline-alkaline stress (Guo et al. 2014;
Lv et al. 2015; Lv et al. 2013; Zhang et al. 2017). Reactive oxygen species (ROS) accumulation
is invariably implicated in decreased root cell viability and vigour, and altered cell membrane
properties (Guo et al. 2014; Zhang et al. 2017), hence have remained central to investigations
14

of plants’ responses to saline-alkaline stress. Moreover, high pH substantially constrains
bioavailability of essential micronutrients particularly Fe that forms insoluble ferric complexes(Nakanishi et al. 2004), making it non-bioavailable for plant uptake. This is despite riceuniquely having two Fe uptake pathways; the strategy I and strategy II (Ishimaru et al. 2006;
Masuda et al. 2008; Takahashi et al. 2001). Therefore, it is hypothesized that its sensitivity tomixed saline-alkaline stress would all the more be extreme. Presently, information regarding
comparative performance of rice to neutral salinity, alkaline (high pH) and saline-alkaline
stress is quite sketchy. This study therefore attempts to highlight key growth and physiologicaldifferences governing responses of rice to salinity stress, alkaline stress and saline-alkaline
stress to inform formulation of approaches to enhance tolerance of rice to saline-alkaline stressby targeting key physiological and molecular signatures principally affected by saline-alkaline
stress.

2.2.

Study objectives

The study was carried out to achieve the following specific objectives;

1. To study comparative time-course changes in growth of rice seedlings to neutral salinity
stress, alkalinity stress and saline-alkalinity stress.
2. To elucidate differences in Na+ toxicity and high pH induced chlorosis under salinity stress,
alkalinity stress and saline-alkalinity.
3. To elucidate comparative physiological mechanisms of damage of salinity, alkalinity andsaline-alkalinity in rice plants.

15

2.3. Materials and methods
2.3.1. Plant material and growth conditions
Rice (Oryza sativa L.) seeds for the cultivar Hinohikari were incubated in tap water at 60 oC
for 10 minutes and then surface sterilized with 0.1 % (v/v) Benlate solution for 30 minutes andrinsed thoroughly with distilled water. Thereafter, seeds were incubated in tap water for 24 h
at 30 oC. The seeds were then transferred onto a nylon mesh wire floated in a tap water filled
bucket for one week, at a water temperature of 28 oC. After one week, slightly modified Kimura
B nutrient solution was added to water with the following nutrient composition in μM; 365(NH4)2SO4, 547 MgSO4 7H2O, 183 KNO3, 365 Ca(NO3)2 4H2O, 185 KH2PO4, 28 FeSO4 7H2O
was used in place of Fe-EDTA, 48.7 H3BO3, 9.0 MnSO4 5H2O, 0.3 CuSO4 7H2O, 0.7 ZnSO4
7H2O, 0.1 Na2MoO4. The experiment was conducted in the greenhouse at Hiroshima
University’s faculty of Applied Biological Sciences experimental field under hydroponic
conditions. Hydroponic culture temperature was maintained at 28 oC using water heaters,
average day air temperature was 30 oC. After 18 days, seedlings were transplanted from nylon
mesh wire to six buckets. Treatment conditions were induced 8 days later as follows; Controls(0 mM Na+, pH 5.5), salinity (50 mM Na+, pH 5.5), moderate saline-alkaline (50 mM Na+, pH7.5), severe saline-alkaline (50 mM Na+, pH 8.5), moderate alkalinity (0 mM Na+, pH 7.5), andsevere alkalinity (0 mM Na+, pH 8.5). Chemical composition for the treatment conditions isshown in Table 2.1. below. pH changes were monitored on daily basis using a pH meter (AS700,
As One Corp., Osaka, Japan) and were adjusted using 2 N HCl and 2 N KOH throughout thegrowth period. Nutrient solution was changed every 8 days, water lost throughevapotranspiration was replaced by addition of tap water.

16

Table 2.1. Treatment conditions and chemical compositions
No.

Treatment

Supplements

pH

1

Control

0 mM Na, pH 5.5

0 mM NaCl

5.5

2

Salinity

50 mM Na, pH 5.5 50 mM NaCl

5.5

3

Moderate saline-alkaline 50 mM Na, pH 7.5 3 mM NaHCO3 + 47 mM 7.5
(MSA)

4

SevereNaClsaline-alkaline 50 mM Na, pH 8.5 48 mM NaHCO3 + 1.0 mM 8.5(SSA)

Na2CO35Moderate alkaline (MA)5 mM K, pH 7.5

4 mM KHCO3 + 1 mM KCl

7.5

6

Severe alkaline (SA)5 mM K, pH 8.5

0.6 mM K2CO3 + 3.8 mM KCl 8.5

2.3.2. Determination of fresh weight, dry weight and water content
Seedlings were harvested in replicates of four prior to imposition of stress treatments (Day 0),
24 hours after stress imposition (Day 1) and subsequently every four days up to the 16 th day.
Plant height and root length were measured using a measuring ruler. Plants were then dissected
into leaf blades, leaf sheaths and roots and immediately measured for fresh weight (FW). Dry
weights (DW) were obtained after oven drying the samples for 72 hours at 70 oC. Water content
in leaf blades and roots was obtained as followsܹܽ‫ݐ݊݁ݐ݊݋ܿݎ݁ݐ‬ሺΨሻ ൌ ‫ ܹܨ‬െ ‫ܹܦ‬‫ͲͲͳݔ‬‫ܹܦ‬

2.3.3. Chlorophyll concentration (SPAD)
Leaf blade chlorophyll concentration was determined using a chlorophyll meter (SPAD – 502,
Minolta Camera Co. Ltd., Osaka, Japan) on three fresh fully expanded leaf blades of the same
positions in all plants according to Kamanga et al. (2020).
17

2.3.4. Determination of macro and micronutrient elements
Elemental analysis was performed on dried leaf blades and root samples after 16 days of stress
imposition using inductively coupled plasma-optical emission spectrometry (ICP-OES). For
this purpose, 100 mg of dried leaf blades and roots were crushed using a freeze crusher (μT –48, Taitec, Saitama, Japan). Acid digestion was conducted using 2 mL of H 2SO4 followed byrepetitive cycles of 1 mL of H2O2 addition and heating in a heat block at 150–200oC until thesample turned colourless. An extra hour of heating was performed to remove the remaining
H2O2 after which the sample was filled to 25 mL using Milli-Q water. ICP analysis was
performed using iCAP – 6300 (Thermo-Scientific, Massachusetts, USA) at wavelengths 769.8,
818.3, 422.6, 285.2, 257.6, 213.8, 324.7 and 259.9 nm for K, Na, Ca, Mg, Mn, Zn, Cu and Fe
respectively. Quantification was performed using various elemental standard solutions preparedin 1 % HNO3.

2.3.5. K+ leakage and Na+ efflux experiment
In order to determine plant roots’ ability to retain K+, leakage of K+ was assessed by a method
previously described in Pandolfi et al. (2016) with slight modifications. For this purpose, plants
were pretreated with various pre-treatments for 7 days, at the conclusion of which, four uniform
seedlings of pretreated and non-pretreated plants were transferred into 50 mL falcon tubes
containing 30 mL of 50 mM Na+, pH 8.5 (48 mM NaHCO3 + 1 mM Na2CO3). Four more
seedlings were transferred into 30 mL deionized water as control plants. After 24 hours,
solution was sampled for K+ analysis.

Efflux of Na+ from roots was assessed by a previously outlined method (Pandolfi et al. 2016;
Shabala et al. 2010), for this purpose, 7 day pretreated and non-pretreated plants were subjected
to 30 mL of 50 mM Na+, pH 8.5 for 24 hours, after which plants were transferred into bathing
medium containing 0.1 mM CaCl2, 0.5 mM KCl and 50 mM Na+, pH 8.5. After 1 hour, the18

solution was poured off, roots were rinsed with 10 mM CaCl2 three times. Then, the roots weretransferred into 10 mL bathing medium containing 0.1 mM CaCl2 and 0.5 mM KCl for 4 hours.
The solution was then sampled and Na+ concentration was measured. Both K+ and Na+ analysis
were done using flame photometry (ANA – 135, Tokyo Photoelectric, Tokyo, Japan).

2.3.6. Determination of hydrogen peroxide concentration
H2O2 concentration was determined using ground frozen samples based on ferrous oxidation of
xylenol orange (FOX) method (Kaur et al. 2016) with slight modifications (Mekawy et al. 2018).
For this purpose, 100 mg of frozen leaf blade and root samples were ground using mortar andpestle in liquid nitrogen, and homogenized with 1 mL cold acetone. The homogenate wascentrifuged at 8000g for 15 minutes at 4°C, and 200 μL of the supernatant was added to 2 mL
FOX reagent comprising 0.25 mM FeSO4, 0.25 mM (NH4)2SO4, 25 mM H2SO4, 125 μM
xylenol orange, and 10 mM sorbitol. The reaction mixture was left at room temperature for 1
hour, afterwards, H2O2 levels were determined spectrophotometrically at 560 nm and quantified
using H2O2 standard solutions.

2.3.7. Membrane integrity measurements
Membrane integrity was measured using electrolyte leakage method at the end of 16 days ofmain stress. Freshly harvested leaf blade disks and roots (200 mg) were cut into 2 cm pieces
and fully immersed into 25 mL deionised water in 50 mL falcon tubes and kept at room
temperature for 24 hours. Electrical conductivity (EC) was then obtained (EC1) using an EC
meter (CM-31P, Toa DKK Co., Tokyo, Japan). Immediately, the samples were killed byincubating at 100 oC for 15 minutes, cooled and EC2 was obtained. Electrolyte leakage rate
was calculated according to Farooq & Azam (2006).

19

2.3.8. Principal component analysis (PCA).
PCA was analysed using R studio (RStudio Team 2020) to identify traits associated with plants
growing under salinity stress, alkalinity and saline-alkalinity.

2.3.9. Statistical analyses
The experiment was arranged in a Completely Randomized Design (CRD). Each measurement
was done in biological replicates of 4. Data collected was subjected to analysis of variance(ANOVA) at 0.05 level of significance. Multiple comparison tests were performed using Tukey
Test. All data was analysed using R studio (RStudio Team 2020).

2.4. Results
2.4.1. pH changes in hydroponic cultures
Prior to imposition of stress treatments, various salt supplement proportions (50 mM Na +, 5mM K+) were tested by addition to 1x Kimura B nutrient solution and measuring their resultant
pH for alkaline and saline-alkaline treatments. Salt supplements were considered ideal for theparticular treatment if their pH just after addition to 1x Kimura B was within ± 0.10 of therequired pH. Required and the obtained pH values were shown in Table 2.2. pH changes weremonitored every 24 hours. The average pH values were 5.0, 5.1, 7.8, 8.7, 7.8 and 8.5 forcontrols, salinity stress, moderate saline-alkaline (MSA) stress, severe saline-alkaline (SSA)
stress, moderate alkaline (MA) stress and severe alkaline (SA) stress, respectively (Table 2.2).
The pH evolution during the growth period indicate that for MSA, SSA, and MA treatments
pH increased to much severer levels than required.

2.4.2. Growth
After four days of stress treatments, growth nearly stopped under SSA stress, however
differences were insignificant from all other treatments until day 8 when differences became20apparent on which MA, SA, MSA, and SSA had significantly shorter plants compared with
controls and salinity stressed plants. However control plants were comparable to salinity
stressed plants until day 16 (Fig. 2.1A – D and Fig. 2.2). Shoot (SFW) and root fresh weights(RFW) became significantly (P < 0.05) different on day 12 and day 16. On day 12, SFW and
RFW were significantly lower under SSA treatment whereas no differences were shown among
MA, SA and MSA, but were significantly lower than controls and salinity treatment. At day
16, SFW for both saline-alkaline treatments decreased as older leaves begun to die off. Root
length for salinity stressed plants was comparable to controls throughout the treatment period
whereas plant height diverged at 12th day of stress imposition, decreasing significantly undersalinity stress (Fig. 2.1E and F). On overall, biomass results show that the cultivar Hinohikari
displayed some level of tolerance to salinity stress for up to 12 days, but was considerably
sensitive to high pH treatments, particularly when combined with salinity (saline-alkaline).
Typical features of all high pH treatments were darkening of roots and yellowing of younger
leaves (Fig 2.2), however both of which were severer under moderate than severe treatment
conditions.

2.4.3. Chlorophyll concentration
In order to distinguish chlorosis resulting from high pH – induced essential element
deficiencies from that emanating from leaf Na+ toxicity, chlorophyll concentration wasmeasured in both younger and older leaves representing younger and older leaves respectively.
From stress imposition to day 4, no visual differences were observed among treatments, andso was chlorophyll concentration in both younger and older leaves (Fig. 2.3A and B). At day
8, younger leaves of high pH treatments (MSA, SSA, MA, SA stress) became chlorotic, levelof chlorosis was significantly higher in sole alkaline (MA and SA) treatments (Fig. 2.3A)
indicating essential element deficiency. On day 16, chlorophyll concentration in salinity stress,
MSA and SSA stress also drastically dropped. The lowest values were in MA, SA & MSA
21

stress (Fig. 2.3A). In SSA stress, new leaf formation had completely stopped hence their upper
leaves were relatively older with less chlorosis (Fig 2.3A). In lower (older) leaves, chlorophyll
concentration remained relatively stable from stress imposition until day 16, on which SSA
stress had drastically lower chlorophyll concentration as the older leaves were nearly dying(Figs. 2.2 and 2.3B). The distinct patterns in chlorosis between lower and upper leaves among
treatments suggest that the underlying causes was different.

2.4.4. Water content
In leaves, after stress imposition for 12 days, no significant differences were shown in watercontent among the treatments. Leaf blade water content became significantly lower in bothsaline-alkaline treatments (MSA and SSA) at day 16 dropping to 64 and 58% respectively,
indicative that leaf blades were drying up and dying (Fig. 2.3C). Conversely, leaves of plants
grown under high alkalinity (MA and SA) and salinity stress remained turgid and freshcomparable to controls (Figs. 2.2 and 2.3C). In roots, differences also became more apparent
at day 16, however, root water content was lowest in alkaline treatments, particularly inmoderate alkaline treatment (Fig. 2.3D). No differences were shown in root water content
between controls and salinity stressed plants, and between MSA and SSA stress (Fig. 2.3D).

2.4.5. Macronutrient concentrations
K+ concentration in leaf blades was significantly reduced by all Na treatments (Salinity, MSA
and SSA) but was unaffected by MA stress and increased by severe alkalinity (SA) (Fig. 2.4A).
However, in leaf sheaths, K concentrations were reduced regardless of treatment, but thedecrease was less pronounced under salinity stress and alkaline stress, and extreme undersaline-alkaline stress (Fig. 2.4B). In roots, saline-alkaline treatments significantly decreased K
concentration followed by salinity stress (Fig. 4C). Na concentration was extremely high inleaf blades and sheaths under saline-alkaline treatments, whereas under sole salinity stress,22

concentrations were statistically comparable to controls (Fig. 2.4D and E). In roots however,no differences were shown between salinity stress and saline-alkaline treatments (Fig. 2.4F).
Ca concentration was not affected by salinity stress and SSA stress, but was increased by MSA
stress and both alkaline treatments in leaf blades (Fig. 2.5A). In leaf sheaths, all treatments
except salinity and severe alkaline stress reduced Ca concentration (Fig. 2.5B). In roots, Caconcentration was considerably increased by all high pH treatments, increases were more
pronounced with increases in growth medium pH, accumulating to about 9 folds higher under
severe saline-alkaline compared to controls (Fig. 2.5C). In leaf blades and sheaths, Mg
concentration followed a similar trend as Ca (Fig. 2.5D and E), however, in roots, Mg
concentration was significantly reduced by all high pH treatment by the same magnitude,
whereas salinity stressed plants accumulated similar amount of Mg as did control plants (Fig.
2.5F).

2.4.6. K+ leakage and Na+ efflux from the roots
It was endeavoured to investigate whether the lower leaf K concentration shown in Fig 2.4A
was as a result of K leakage from the roots. It was shown that both salinity and saline-alkaline
stress elicited much higher leakage of K from the roots, however the leak was extreme undersaline-alkaline stress affirming the K deficiency observed in leaf blades (Fig. 2.6A). Secondly,
root Na+ efflux was measured to check whether efflux of Na is perturbed by high pH undersaline-alkaline stress as hypothesized by Cao et al. (2020). Results showed that while bothstresses triggered significant Na efflux from the roots, the efflux was actually much higher insaline-alkaline stressed plants compared to salinity stress (Fig. 2.6B), invalidating the
hypothesis that higher leaf Na was as a result of reduced root Na efflux.

23

2.4.7. Micronutrient concentrations
All high pH treatments significantly reduced leaf blade Fe concentrations. Under salinity stress,
Fe concentration was also significantly reduced (Fig. 2.7A). In leaf sheaths, only high pH
treatments suffered remarkable reductions in Fe concentration (Fig. 2.7B). In roots, all high pH
treatments experienced remarkable reductions, but Fe concentrations were over 10 times higher
than in leaf blades. Mn concentration in leaf blades was significantly higher in salinity,
moderate alkaline and moderate saline-alkaline stressed plants than control plants. However,under severe saline-alkaline treatment, Mn concentration was significantly reduced, whereas
alkaline stress was comparable to controls (Fig. 2.7D). In leaf sheaths, Mn concentration wasconsiderably lowered by all stress treatments, and the decreases were more pronounced withincreases in pH (Fig. 2.7E). However, in roots, control and salinity stressed plants had
significantly lower Mn concentration, whereas concentrations were considerably increased byhigh pH treatments (Fig 2.7F). In leaf blades effect of the stress factors on Zn concentration
depended on the severity of the medium pH. Under pH 5.5, Zn concentration was similar
between control and salinity stressed plants, but was considerably increased by moderately
high pH (pH 7.5) by 2.6 and 2.4, respectively, whereas under severely high pH (pH 8.5), Zn
concentration was unaffected (Fig. 2.8A). In leaf sheaths, no significant changes in Zn
concentration were observed except under severely high pH 8.5 (SA and SSA stress) (Fig.
2.8B). In roots, Zn concentration was unaffected by salinity but was considerably increased inall high pH treatments (Fig. 2.8C). Cu concentration was increased by salinity stress in leaf
blades whereas all high pH treatments drastically decreased it, decreases were more
pronounced under severe saline-alkaline stress (Fig. 2.8D). In leaf sheaths, Cu concentration
was reduced by MSA, SA and SSA stress by 58.0, 66.0 and 76.8, % respectively (Fig. 2.8E).
In roots, all high pH treatments considerably decreased Cu concentration, with decreases being
more pronounced under alkaline treatments, whereas salinity stress did not affect it (Fig. 2.8F).

24

2.4.8. Correlation of SPAD values with Fe and Na concentration
In order to clarify the causes of leaf blade chlorophyll concentration loss in plants subjected todifferent stresses, Pearson correlation analysis was performed. It was shown that chlorophyll
concentration (R = 0.73, P < 0.00084) in younger leaf blades significantly correlated with leaf
blade Fe concentration (Fig. 2.9A) signifying that in younger leaf blades, Fe deficiency was
the prime cause of chlorosis. In older leaf blades, there was a significant negative correlation(R = -0.72, P < 0.00072) between chlorophyll concentration and Na concentration (Fig. 2.9B).
Therefore, in older leaf blades, Na accumulation in leaf blades contributed significantly tochlorosis.

2.4.9. Membrane integrity parameters
Saline-alkaline treatments (MSA and SSA) caused remarkable leakage of electrolytes in leaf
blades, significantly higher than alkaline treatments (MA and SA) and salinity stress (Fig.
2.10A). Electrolyte leakage in salinity stressed plants was comparable to controls. In roots,leakage of electrolytes was generally higher than in leaves, except for saline-alkaline
treatments (Fig. 2.10B). Nonetheless, root electrolyte leakage was highest in SSA, however
differences were not as pronounced. Membrane injury in leaves showed significantly higher
injury in saline-alkaline treatments but were significantly lower under salinity stress andalkalinity stress (Fig. 2.10C). In roots, severe saline-alkalinity also caused considerable
membrane damage whereas the rest of the treatments showed insignificant differences (Fig.
2.10D).

2.4.10. H2O2 concentration
H2O2 concentration was much higher in leaves than in roots (Fig. 2.11A and B). In leaves,severe saline-alkaline treatment caused significantly higher H2O2 accumulation followed bymoderate saline-alkaline and salinity treatments. Alkaline treatments did not induce any
25significant H2O2 accumulation in leaf blades wherein concentration was similar to controls (Fig.
2.11A). In roots however, moderate alkalinity significantly induced H 2O2 accumulation
followed by severe saline-alkaline stress (Fig. 2.11B).

2.4.11. Principal component analysis
Principal component analysis was performed using several traits of plants growing under all
treatment conditions. It was shown that plants growing under saline-alkaline conditions (MSAand SSA stress) were associated with higher values of Na+ concentration in both leaf blades
and roots, H2O2 concentration in leaf blades and membrane damage in shoots and roots (Fig.
2.12) and lower values of SDW, K concentration and chlorophyll concentration.

Table 2.2. Chemical composition of various treatment conditions and their respective actual
pH, targeted pH and average daily pH values.
pH
pH†

pH‡

-

5.5

5.0

50 mM NaCl

5.5

5.1

7.32

7.5

7.8

saline-alkaline 48 mM NaHCO3 + 1.0 mM 8.50

8.5

8.7

No.

Treatment

Chemical composition

1

Control

2

Saline3Moderate saline-alkaline 3 mM NaHCO3 + 47 mM NaCl

pH*

(MSA)
4

Severe(SSA)

Na2CO35Moderate alkaline (MA)4 mM KHCO3 + 1 mM KCl

7.51

7.5

7.8

6

Severe alkaline (SA)

0.6 mM K2CO3 + 3.8 mM KCl

8.55

8.5

8.5

*

The actual pH after adding chemicals to 1x Kimura B.

†

The required/ target pH.

‡

Average pH at 24 hours after adjusting to required range (before adjusting)26

Fig. 2.1. Time course changes in growth of rice plants to salinity, saline-alkalinity andalkalinity showing shoot fresh weight (A), root fresh weight (B), shoot dry weight (C), root dry
weight (D), plant height (E) and root length (F). Plants were exposed to salinity stress, moderate
alkalinity (MA), severe alkalinity (SA), moderate saline-alkalinity (MSA) and severe salinealkalinity (SSA) for 16 days. Data shown was taken every four days and represents means of
6 biological replicates.

27

Fig. 2.2. Comparative shoot and root growth of rice plants showing photograph of plants after
16 days of being exposed to neutral salinity stress, moderate saline-alkalinity (MSA), severe
saline-alkalinity (SSA), moderate alkalinity (MA) and severe alkalinity (SA).

28

Fig. 2.3. Time-course physiological responses of rice plants to salinity, saline-alkalinity andalkalinity showing leaf chlorophyll concentration measured as SPAD units in younger upper
leaves (A) and in older lower leaves (B); leaf water content (C) and root water content (D).
Plants were exposed to salinity stress, moderate alkalinity (MA), severe alkalinity (SA),moderate saline-alkalinity (MSA) and severe saline-alkalinity (SSA) for 16 days. Data shown
was taken every four days and represents means of 6 biological replicates.

29

30

7.5) and severe saline-alkalinity (50 mM Na+, pH 8.5) for 16 days. Data shown represents means and standard errors of 6 biological replicates.

mM Na+, pH 5.5), moderate alkalinity (0 mM Na+, pH 7.5), severe alkalinity (0 mM Na+, pH 8.5), moderate saline-alkalinity (50 mM Na+, pH

concentration in leaf blades (D), leaf sheaths (E), and roots (F). Plants were exposed to control conditions (0 mM Na+, pH 5.5), salinity stress (50Fig. 2.4. Macronutrient concentration in rice plants showing K+ concentration in leaf blades (A), leaf sheaths (B), and roots (C); and Na+317.5) and severe saline-alkalinity (50 mM Na+, pH 8.5) for 16 days. Data shown represents means and standard errors of 6 biological replicates.

mM Na+, pH 5.5), moderate alkalinity (0 mM Na+, pH 7.5), severe alkalinity (0 mM Na+, pH 8.5), moderate saline-alkalinity (50 mM Na+, pH

concentration in leaf blades (D), leaf sheaths (E), and roots (F). Plants were exposed to control conditions (0 mM Na+, pH 5.5), salinity stress (50Fig. 2.5. Macronutrient concentration in rice plants showing Ca2+ concentration in leaf blades (A), leaf sheaths (B), and roots (C); and Mg2+

Fig. 2.6. Na+ and K+ concentrations in rice plants after 16 days of exposure to different
treatments showing leakage of K+ in plant roots subjected to 50 mM Na+, pH 5.5 (Salinity stress)
and 50 mM Na+ pH 8.50 (SSA stress) for 24 hours (A) and efflux of Na+ from roots of riceseedlings subjected to 50 mM Na+, pH 5.5 (Salinity stress) and 50 mM Na+ pH 8.50 (SSA stress)for 24 hours, and deionized water for 2 hours. Data shown represents means and standard errorsof 6 biological replicates.

32

33

7.5) and severe saline-alkalinity (50 mM Na+, pH 8.5) for 16 days. Data shown represents means and standard errors of 6 biological replicates.

mM Na+, pH 5.5), moderate alkalinity (0 mM Na+, pH 7.5), severe alkalinity (0 mM Na+, pH 8.5), moderate saline-alkalinity (50 mM Na+, pH

concentration in leaf blades (D), leaf sheaths (E), and roots (F). Plants were exposed to control conditions (0 mM Na+, pH 5.5), salinity stress (0Fig. 2.7. Micronutrient concentration in rice plants showing Fe concentration in leaf blades (A), leaf sheaths (B), and roots (C); and Mn

347.5) and severe saline-alkalinity (50 mM Na+, pH 8.5) for 16 days. Data shown represents means and standard errors of 6 biological replicates.

mM Na+, pH 5.5), moderate alkalinity (0 mM Na+, pH 7.5), severe alkalinity (0 mM Na+, pH 8.5), moderate saline-alkalinity (50 mM Na+, pH

concentration in leaf blades (D), leaf sheaths (E), and roots (F). Plants were exposed to control conditions (0 mM Na+, pH 5.5), salinity stress (0Fig. 2.8. Micronutrient concentration rice plants showing Zn2+ concentration in leaf blades (A), leaf sheaths (B), and roots (C); and Cu2+

Fig. 2.9. Correlation plot showing Pearson correlation between chlorophyll concentration(SPAD values) in younger leaf blades with leaf blade Fe concentration (A) and correlation ofchlorophyll concentration in older leaf blades (LB) with leaf blade Na concentration (B). Plants
were exposed to salinity stress (0 mM Na+, pH 5.5), moderate alkalinity (0 mM Na+, pH 7.5),
severe alkalinity (0 mM Na+, pH 8.5), moderate saline-alkalinity (50 mM Na+, pH 7.5) andsevere saline-alkalinity (50 mM Na+, pH 8.5) for 16 days.

Fig. 2.10. Membrane damage parameters in rice plants after 16 days of exposure to different
treatments showing electrolyte leakage ratio in shoots (A) and roots (B); and membrane injury
in shoots (C) and roots (D) concentration in leaf blades (A) and roots (B). Plants were exposed
to salinity stress (0 mM Na+, pH 5.5), moderate alkalinity (0 mM Na+, pH 7.5), severe alkalinity(0 mM Na+, pH 8.5), moderate saline-alkalinity (50 mM Na+, pH 7.5) and severe salinealkalinity (50 mM Na+, pH 8.5) for 16 days. Data shown represents means and standard errorsof 6 biological replicates.

36

Fig. 2.11. Reactive oxygen species accumulation in rice plants after 16 days of exposure todifferent treatments showing hydrogen peroxide (H2O2) concentration in leaf blades (A) androots (B). Plants were exposed to salinity stress (0 mM Na+, pH 5.5), moderate alkalinity (0mM Na+, pH 7.5), severe alkalinity (0 mM Na+, pH 8.5), moderate saline-alkalinity (50 mM
Na+, pH 7.5) and severe saline-alkalinity (50 mM Na+, pH 8.5) for 16 days. Data shown
represents means and standard errors of 6 biological replicates.

37

Fig. 2.12. A summary of key traits useful for studying comparative responses to saline-alkaline
stress, salinity and alkalinity stress using Principal Component Analysis (PCA). Traits usedwere relative water content (RWC), SPAD units in young and old leaves (Chl_Y and Chl_O,
respectively), Fe, K, Na in leaf blades (Fe_L, K_L, Na_L) and roots (Fe_R, K_R, and Na_R,
respectively), shoot dry weight (SDW), root dry weight (RDW), leaf water content (LWC),
shoot electrolyte leakage (SEL), root electrolyte leakage (REL) and leaf blade hydrogen
peroxide concentration (H2O2_L). All values were measured after exposure to salinity stress(0 mM Na+, pH 5.5), moderate alkalinity (0 mM Na+, pH 7.5, MA), severe alkalinity (0 mM
Na+, pH 8.5, SA), moderate saline-alkalinity (50 mM Na+, pH 7.5, MSA) and severe salinealkalinity (50 mM Na+, pH 8.5, SSA) for 16 days.

38

2.5. Discussion
2.5.1. Interactive effects of high pH and Na+ toxicity are more detrimental than theirsole effects
Saline-alkaline stress is a major detriment to plant growth. Rice is a glycophytic cereal crop
characterized as extremely sensitive to a range of abiotic stresses including salinity stress andhigh pH. This study finds that the interactive effects of high pH and salinity stress are far much
more devastating than sole salinity stress and sole alkaline stress. Here, rice cultivar Hinohikari
was able to grow normally under salinity stress for up to 12 days of stress imposition,
considerable growth reductions were only noticeable on 16th day of stress imposition, whereas
under high pH treatments, growth reductions were apparent even four days after stresstreatment. Munns & Tester (2008) distinguished between the osmotic and ionic effects ofsalinity stress based on speed of onset and primary visible site of effect. Based on that and thepresent results, it is inferable that under salinity stress treatment, the slower nature of visible
effect on plant growth (16 days) and sustained production of younger leaves present more of
an ionic response than osmotic, albeit not severe enough to cause remarkable leaf senescence.
Root growth was the most sensitive to severe saline-alkaline stress (Fig. 2.1B; Fig. 2.2D andF), which might have severely constrained water uptake ability of plants. Previous studies haveshown that saline-alkaline stress severely affects root growth (Guo et al. 2014; Lv et al. 2013;
Zhang et al. 2017), hence plants with intact root structure and morphology are well capacitatedfor absorptive functions such as water uptake and metal ion acquisition under saline-alkaline
stress. The dramatically reduced leaf water content in this study (Fig. 2.3C) in part reflects
incapacitated roots as a result of both Na+ toxic and a high pH rhizosphere, generating
hyperosmotic stress and causing premature leaf senescence due to hyperaccumulation of Na+
in the leaf blades (Fig. 2.4A). In total, it is inferable that at the present experimental conditions,high rhizosphere pH interferes with growth and some essential physiological processes more39

than sole salinity, and furthermore, the toxicity of Na+ is inordinately aggravated in an alkaline
rhizosphere than a neutral one. The physiological rationale for these observations will be
detailed in the subsequent sections.

2.5.2. High pH boosts shoot Na accumulation at the expense of root K
Plants growing under saline-alkaline stress are not only confronted with a high pH rhizospherebut also toxicity of Na+, an element that plants do not need in excess. In this study, it wasobserved that under the same molar concentration of Na+, saline-alkaline treated plants
accumulated 4.8 and 8.2 times more Na in the leaf blades than sole salinity stressed plants
under moderate and severe saline-alkaline stress respectively. This high pH induced Na+
hyperaccumulation in leaf blades has been repeatedly reported (Cao et al. 2020; Chen et al.
2009; Paz et al. 2012; Yang et al. 2009; Yang et al. 2008), yet no consensus is reached on the
proposed mechanisms for the high pH induced hyperaccumulation of Na+ in the shoots. A
recent study in maize identified and characterized a Ca2+ dependent Zea mays Na+ Content
under Saline-Alkaline condition (ZmNSA1), a gene responsible for natural variation in shootNa+ contents and saline-alkaline tolerance (Cao et al. 2020). Plants lacking ZmNSA1 promote
ZmSOS1 mediated root Na+ efflux by increasing plasma membrane H+-ATPases (MHA2 andMHA4). SOS1 mediated root Na+ efflux exchanges cytoplasmic Na+ for external H+. However,high rhizospheric and cytosolic pH weakens function of H+ gradient dependent transporters(Haruta & Sussman 2012; Yang et al. 2019) such as SOS1 (Duby & Boutry 2009; Moriyama
2015). Contrarily, measurements of root Na+ efflux showed considerably higher root Na+ efflux
under saline-alkaline stress relative to plants exposed to salinity stress (Fig. 2.6B), annullingthe hypothesis that root Na+ efflux was upset by high pH. Therefore, in this study, themechanism of high shoot Na+ accumulation may likely relate to processes regarding uptake of
Na+ by roots and its subsequent transport to the shoots. Strikingly, this ionic burst was40

associated with considerable reductions in K+ accumulation particularly in roots under salinealkaline stress (MA and SA, Fig. 2.4A). Measurement of K+ leakage revealed that while bothsalinity stress and saline-alkalinity induced significant leakage of K+ in the roots, leakage wasintensified by saline-alkalinity (Fig. 2.6A). Therefore, the observed K deficiency under salinealkalinity was primarily as a result of root K+ leakage. The present study did not record any
decreases in leaf blade Ca2+ concentration under all stress treatments but was enhanced bysome high pH treatments, particularly in roots, recording a 9-fold increase under SSA. That
notwithstanding, the failure for the root increase in Ca2+ uptake to translate similarly in shoots
reveal that it is Ca2+ transport to the shoot other than uptake by the roots that is perturbed byhigh pH under saline-alkaline stress. Also, this observed increase in root Ca2+ uptake reflects
more of a high pH specific response, than a salinity one, but is tremendously provoked by theirinteraction. The physiological rationale behind this observation remains a mystery.

2.5.3. Chlorosis is distinct among salinity, saline-alkalinity and alkalinity
Chlorosis is an apparent feature of a high pH rhizosphere (Gómez-Galera et al. 2012; Guo et
al. 2014), a chlorophyll deficiency symptom that impacts on plants’ photosynthetic
competency. It is known that high pH reduces plant roots’ ability for Fe uptake (Ishimaru et al.
2006; Takahashi et al. 2001). Fe is crucial for chlorophyll biosynthesis, the deficiency of which
manifests itself through consequent chlorosis particularly in younger leaves due to Fe’s
immobile nature (Taiz et al. 2015). Under salinity stress, Na+ toxicity in leaf blades coupledwith failure to compartmentalize it in the vacuoles degrades chlorophyll, resulting into
somewhat similar symptoms. According to Munns & Tester (2008), Na+ toxicity manifests
itself through premature senescence of older leaves that are no longer expanding hence areincapable of diluting incoming Na+. Therefore, under saline-alkaline stress, toxic presence of
Na+ in the rhizosphere and inordinately high pH may produce somewhat physiologically41

confusing symptoms of chlorosis and senescence, both of which entail lack of chlorophyll. The
observation that in younger leaves, all high pH treatments induced severe chlorosis (Fig. 2.3A),
coupled with a significant positive correlation (R = 0.73, P < 0.00073) between Fe
concentration and SPAD units in young leaf blades (Fig. 2.9A) suggests that Fe deficiency was
the prime cause of chlorosis. It is crucial to note that following onset of severe saline-alkaline
stress, literally no new leaves were formed, hence the supposedly newer leaves did notessentially represent younger leaves, with Fe already partitioned to them, consequently having
relatively higher chlorophyll. Conversely, in older leaves, sole alkaline and salinity treatments
had much turgid and greener leaves (Figs. 2.2, 2.3B), whilst severe saline-alkaline plants
experienced 71.4 % reduction in leaf chlorophyll concentration. These leaves represent older
leaves undergoing senescence due to Na+ toxicity, this is justifiable noting that leaf blades
accumulated 4.8 and 8.1 times more Na+ than salinity stress (Fig. 2.4D). This was further
supported by a significant and high negative correlation (R = -0.72, P < 0.00072) between
chlorophyll concentration in older leaf blades and Na concentration (Fig. 2.9B). This implies
that in older leaf blades under saline-alkaline stress, lower chlorophyll concentration was
primarily due to leaf blade Na toxicity. This result not only reflects leaf Na+ toxicity but alsoinability to sequester Na+ away from the cytosol, indicative that these concentrations wereclearly above its vacuolar sequestration capacity (Pandolfi et al. 2012; Shabala et al. 2010). It
is therefore unsurprising that severe saline-alkaline treatments experienced such an agonizing
chlorosis, representing an overwhelming photosynthetic incompetency that in part validates its
poor growth. Therefore, while both Na+ toxicity and high pH stress reduce chlorophyll
concentration, the former does so by damaging chlorophyll whereas the latter reduces its
biosynthesis, hence under saline-alkaline stress, the co-existence of these dual factors and theirresultant chlorosis is distinguishable by leaf age.

42

2.5.4. Saline-alkaline stress elicits oxidative stress in leaves
Generation of reactive oxygen species (ROS) is an inevitable fate of organisms undergoing
aerobic metabolism (Apel & Hirt 2004; Sharma et al. 2012). Abiotic stresses such as salinity
and high pH upset plants’ inherent abilities to tightly regulate ROS generation, throwing plants
into a state of “oxidative stress” that may potentially cause lipid peroxidation, protein andnucleic acid damage, and programmed cell death (Kamanga et al. 2018). In the present study,saline-alkaline stress caused considerably higher electrolyte leakage and membrane injury (Fig.
2.10A – D), indices indicative of membrane damage (Elsawy et al. 2018; Nyoka et al. 2018;
Ueda et al. 2013). This was particularly more pronounced in leaves than roots. In roots, all highpH treatments considerably elicited root membrane injury (Fig. 2.10D), producing visuallydarker roots with conspicuous symptoms of damage (Fig. 2.2). A number of reports indicatethat roots are the primary physiological sites damaged by saline-alkaline stress (Peng et al.
2008; Yang et al. 2009; Yang et al. 2008; Zhang & Mu 2009; Zhang et al. 2019). This study
was consistent with previous reports, but further report high membrane injury in leaves, thismay be primarily attributed to high pH, whether through accelerated Na+ uptake andaccumulation of ROS. However, measurements of H 2O2 concentration in leaves showed that
only saline-alkaline treatments considerably increased it, whereas in roots it was elicited bymoderate alkalinity. Therefore, while growth media pH alone could be implicated in root
oxidative stress and resultant membrane damage, it certainly wasn’t an important cause in leaf
blades, neither was the sole growth media Na+, but the interactive effects of both and theirresultant ionic burst in the leaves. It has been suggested that oxidative stress is the prime causeof lethality under saline-alkaline stress (Zhang et al. 2017) such that its amelioration improvestolerance to saline-alkalinity. Guo et al. (2014) showed that an alkaline tolerance mutant (alt1)
rice had lower H2O2 production, with 64 % of abiotic stress differentially expressed genes
under saline-alkaline stress were involved in generation and detoxification of ROS and repair43

of ROS mediated damage, highlighting the role that ROS homeostasis may play under salinealkaline stress.

2.6.

Conclusion

From this study, it is derivable that the rice cultivar Hinohikari is extremely sensitive to highpH, and that its sensitivity is aggravated under saline-alkalinity than sole salinity stress.
Essentially, saline-alkalinity promotes Fe deficiency chlorosis in younger leaf blades and Na+
toxicity induced senescence of older leaf blades. Furthermore, high pH rhizosphere boosts
shoot Na+ accumulation at the expense of K+ that leaks excessively in the roots. A secondary
consequence of Na+ toxicity and high pH under saline-alkalinity is compromised membrane
integrity particularly in plant leaves as a result of excessive accumulation of ROS. Therefore,
efforts attempting to enhance tolerance of rice plants to saline-alkalinity should consider
incorporating these priority traits in breeding programs.

44

Chapter 3

Study of Acclimation and Induced CrossTolerance of Rice Seedlings to Saline-Alkaline
Stress45

3.1. Introduction
Plants are known to have remarkable abilities to acclimate to lethal stress events whensubjected to a prior mild stress. This is referred to as acclimation, describing plants’physiological ability to adapt to a stress event (Djanaguiraman et al. 2006; Pandolfi et al. 2012).
Acclimation has been demonstrated as an effective and quicker way to improve plants’tolerance to abiotic stresses such as drought (Bruce et al. 2007; Conrath et al. 2006; Crisp et al.
2016; Ding et al. 2012, 2013; Walter et al. 2011) and salinity stress (Kamanga et al. 2020;
Pandolfi et al. 2012, 2016; Sriskantharajah et al. 2020). In a primed state, plants are typified
with faster and stronger responses triggered by systemic defence systems that confer enhanced
resistance. Under alkali stress, Na+ toxicity, osmotic stress, oxidative stress, and high pH are
key known stress factors responsible for growth inhibition and plant death (Yan et al. 2005;
Yang et al. 2008, 2009; Zhang et al. 2019), hence choice of acclimating agents should aim at
those that may enhance defence responses that counteract these stress factors. It is speculated
that high pH reduces plant roots’ SOS1 mediated Na+ efflux as a result of deficiency intransmembrane proton gradient required for SOS1 activity (Haruta & Sussman 2012; Yang etal. 2019). Therefore, in order to address the latter, ability to maintain a lower pH through H +ATPases mediated proton secretion is a prerequisite for regulation of leaf Na+ toxicity throughroot Na+ extrusion. Moreover, rhizospheric acidification is also necessary for Fe uptake inplants utilizing strategy I, solubilizing ferric iron (Fe3+) to utilizable ferrous form (Fe2+) for
uptake by plant roots. Rice employs both strategy I and strategy II of Fe uptake, hence
maintenance of a lower rhizospheric pH cannot be overlooked.

Reactive oxygen species (ROS) are unstable molecules inevitably produced by plants both asa result of normal aerobic metabolism and in response to abiotic stresses (Kamanga et al. 2018;
Sharma et al. 2012), and also participate in cellular signalling. It is alleged that an important46

overlap exists in signalling and response pathways to different abiotic stresses, involving ROS,
hormones, protein kinase cascades and calcium gradients as common elements (Atkinson &Urwin 2012). This crosstalk may promote tolerance to a range of abiotic stresses, illustrating a
concept of cross-tolerance, in which exposure to one stress type enhances tolerance to anothertype (Foyer et al. 2016; Mittler 2006; Perez & Brown 2014), offering a prospect for breeding
crop plants resistant to multiple forms of stress. The recognition that a crosstalk to multiple
stressors in part involves ROS (Chamnongpol et al. 1998; Pastori & Foyer 2002; Perez &Brown 2014) has lured agronomists to pursue chemical means to activate ROS induced
systemic resistance. This has included direct application of ROS particularly H 2O2 (Fedina et
al. 2009; Gondim et al. 2013; He et al. 2009; Wahid et al. 2007) and application of salts suchas NaCl (Kamanga et al. 2020; Sriskantharajah et al. 2020) and NaHCO3 (Liu & Saneoka 2019)
which are hypothesized to activate antioxidant defence systems. Nevertheless, little is known
regarding the physiological and molecular mechanisms governing cross-tolerance between
oxidative stress and saline-alkaline stress, and whether acclimation to salinity stress would
increase resistance to alkali stress. It is hypothesized here, that acclimation to either or a
combination of the four key stress factors under alkali stress i.e. Na+ toxicity, osmotic stress,
oxidative stress and high pH may trigger an inter-stress crosstalk that may upsurge resistanceof rice seedlings to alkali stress.

3.2. Study objectives
The study was conducted to achieve the following objectives;
1. To determine optimal pretreatment concentrations that promote acclimation to salinealkaline stress in rice
2. To elucidate the physiological processes underpinning acclimation and cross-tolerance ofrice to saline-alkaline stress47

3. To investigate the effect of pretreatments in post saline-alkaline stress recovery of rice

3.3. Materials and methods
3.3.1.

Plant materials and growth conditions

Seeds of rice (Oryza sativa L.) cultivar Hinohikari were incubated, surface-sterilized, andgerminated according to Sriskantharajah et al. (2020). After one week, a slightly modified
Kimura B nutrient solution was added to the hydroponic culture following Chuamnakthong etal. (2019). The nutrient solution was provided by immersing the plant roots directly in thehydroponic solution. The experiment was conducted in a greenhouse at Hiroshima University’s
Faculty of Biological Sciences Experimental Field. The hydroponic culture temperature wasmaintained at 28 oC, and the average day air temperature was 30 oC. After 18 days, seedlings
were transplanted from a nylon mesh wire to 20 L buckets.

3.3.2.

Treatment conditions

Twenty-six (26) day-old seedlings were subjected to three concentrations of each of thefollowing three sets (N = 20) of pre-treatments for 7 days: (1) NaCl (S) (2.5, 5.0, and 10 mM),
(2) H2O2 (OX) (1.0, 5.0, and 10 μM), and (3) NaHCO 3 (SA) (0.5, 1.0, and 2.5 mM). A fourth(4) set of plants was non-pretreated and maintained as controls (N = 40). Out of these, after 7
days acclimation period, half were subjected to saline-alkalinity (NPT) while the remaining
were maintained as control plants (no pre-treatment and no saline-alkaline stress). Except for
set 3, all sets were maintained at a pH of 5.0–5.5 using 2.0 N HCl and 2.0 N KOH. After sevendays, pretreated (PT) and non-pretreated (NPT) plants were subjected to main SAS treatment(50 mM Na, pH 8.25) using a mixture of 0.75 mM Na2CO3, 25 mM NaHCO3, and 23.5 mM
NaCl. Growth medium pH was monitored every 24 hours but was left unadjusted except forcontrol conditions adjusted to 5.0–5.5 daily. To evaluate whether pre-treatments facilitate a48

quicker recovery after the disappearance of SAS conditions, all pretreated plants were subjected
to 50 mM Na, pH 8.25 for 14 days, at the conclusion of that all stressed plants were recovered
when exposed to 0 mM Na (1x Kimura B), pH 5.0–5.5 for 10 days. Shoot and root dry weight,
chlorophyll concentration, and membrane integrity were measured.

Table 3.1. Pre-treatment conditions used. pH for PT No. 1 – 8 was being adjusted daily to theindicated range. pH for PT No. 9 – 11 was left unadjusted, indicated values are round averagevalues during the 7 day PT period.

No.

Pre-treatment

Treatment

Designation

(PT)
1

None

2
3

Control-

5.0 – 5.5

None

NPT

-

5.0 – 5.5

SalinityS2.5

2.5 mM NaCl

5.0 – 5.5S5.0

5.0 mM NaCl

S10

10.0 mM NaCl

Ox1

1 μM H2O2

Ox5

5 μM H2O2

Ox10

10 μM H2O2

SA0.5

0.5 mM NaHCO3

~ 6.5

SA1.0

1.0 mM NaHCO3

~ 7.0

SA2.5

2.5 mM NaHCO3~ 7.5

Control

7
8
9

Saline-10alkalinity

Saline-alkaline stress (SAS,Oxidative

50 mM Na+, pH 8.25)

5

11

pH

composition

4

6

Chemical49

5.0 – 5.5

3.3.3.

Determination of plant fresh and dry weight

Seedlings were harvested in replicates of 4 – 5 after 7 days of pre-treatment period, after 14days of main stress and after 10 days of recovery period. Plant height and root length were
measured using a measuring ruler. Plants were then dissected into leaf blades, leaf sheaths androots and immediately measured for fresh weight (FW). Dry weights (DW) were obtained afteroven drying the samples for at least 72 hours at 70 oC.

3.3.4.

Chlorophyll concentration (SPAD)

Leaf blade chlorophyll concentration was determined using a chlorophyll meter (SPAD – 502,
Minolta Camera Co. Ltd., Osaka, Japan) on three fresh fully expanded leaf blades of the same
positions in all plants according to Kamanga et al. (2020).

3.3.5.

Determination of macro and micronutrient elements

Elemental analysis was performed in dried leaf blades and root samples after 14 days of stress
imposition using Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES).
iCAP – 6300 (Thermo-Scientific, Massachusetts, USA). Plant samples were digested using
H2SO4 and H2O2 following a procedure outlined in section 2.2.4. Quantification was done using
various elemental standard solutions prepared in 1 % HNO3.

Table 3.2. Wavelengths used for ICP analysis
Element

Wavelength (nm)Element

Wavelength (nm)Potassium (K)766.4

Iron (Fe)259.9

Sodium (Na)818.3

Boron (B)249.7

Phosphorus (P)

177.4

Zinc (Zn)

213.8

50

3.3.6.

K+ leakage and Na+ efflux experiment

In order to determine plant roots’ ability to retain K+, leakage of K+ was assessed by a method
previously described in Pandolfi et al. (2016) with slight modifications. For this purpose, plants
were pretreated with various pre-treatments for 7 days, at the conclusion of which, four uniform
seedlings of pretreated and non-pretreated plants were transferred into falcon tubes containing
30 mL of 50 mM Na+, pH 8.5 (48 mM NaHCO3 + 1 mM Na2CO3). Four more seedlings weretransferred into 30 mL deionized water as control plants. After 24 hours, solution was sampled
for K+ analysis.

Efflux of Na+ from roots was assessed by a previously outlined method (Pandolfi et al. 2016;
Shabala et al. 2010), for this purpose, 7 day pretreated and non-pretreated plants were subjected
to 30 mL of 50 mM Na+, pH 8.5 for 24 hours, after which plants were transferred into bathing
medium containing 0.1 mM CaCl2, 0.5 mM KCl and 50 mM Na+, pH 8.5. After 1 hour, thesolution was poured off, roots were rinsed with 10 mM CaCl2 three times. Then, the roots weretransferred into 10 mL bathing medium containing 0.1 mM CaCl2 and 0.5 mM KCl for 4 hours.
The solution was then sampled and Na+ concentration was measured. Both K+ and Na+ analysis
were done using flame photometry (ANA – 135, Tokyo Photoelectric, Tokyo, Japan).

3.3.7.

Na+ uptake in excised leaves

In order to exclude contribution of Na+ exclusion from uptake by roots, rice seedlings werepretreated with various pre-treatments for 7 days, thereafter uniform leaf blades were excisedunder running water, and immersed into 30 mM Na+, pH 8.0 at about the same depth (30 mm)
for 3 days following a method described in Shabala et al. (2010). Thereafter, immersed cut
ends of leaf blades were thoroughly washed using deionized water, and dried at 65 oC for 72

51

hours. Then, 50 mg of leaf blades was agitated on a shaker in 1.0 N HCl for 24 hours andmeasured for Na+ concentration using flame photometry according to Kamanga et al. (2020).

3.3.8.

Determination of membrane integrity

Membrane integrity was measured using electrolyte leakage method and lipid peroxidation at
the end of 14 days of main stress and 7 days of recovery. Freshly harvested leaf blade disks
and roots (200 mg) were cut into 2 cm pieces and fully immersed into 25 mL deionised waterin 50 mL falcon tubes and kept at room temperature for 24 hours. Electrical conductivity (EC)
was then obtained (EC1) using an EC meter (CM-31P, Toa DKK Co., Tokyo, Japan).
Immediately, the samples were killed by incubating at 100 oC for 15 minutes, cooled and EC2
was obtained. Electrolyte leakage rate was calculated according to Farooq & Azam (2006).

Lipid peroxidation was determined by obtaining malondialdehyde (MDA) concentration using
the improved thiorbarbituric acid (TBA) reaction previously described in Hodges et al. (1999).
For this purpose, 100 mg of fresh leaf and root samples were ground in presence of liquid
nitrogen using mortar and pestle and extracted with 3 mL 80 % ethanol after incubating underroom temperature for 20 minutes and centrifuging at 3,000 x g for 10 minutes. Thereafter, twosets of 1 mL of the supernatant were transferred into two glass tubes, respectively, one
containing 1 mL TBA (-) solution (20 % trichloro acetic acid and 0.01 % Butyl Hydroxy
Toluene, BHT) and another containing 1 mL TBA (+) solution (20 % trichloro acetic acid,0.01 % BHT and 0.65 % TBA). MDA concentration was calculated from the absorbances ofthe samples, which were measured spectrophotometrically (U-3310, Hitachi, Tokyo, Japan) at
440, 532, and 600 nm after incubating the samples at 95 oC for 30 minutes, cooled at room
temperature for 5 minutes followed by ice for 5 minutes, and centrifuging the samples at 3,000

52

x g for 10 minutes. MDA concentration was calculated using a 155 mM-1 cm-1 extinction
coefficient.

3.3.9.

Measurement of root and leaf cell viability

Root and leaf cell viability were assessed using Evan’s blue staining procedure as previously
described (Rodriguez-Serrano et al. 2006; Romero-Puertas et al. 2004; Zhang et al. 2017). In
brief, fresh root tips and leaf blades were cut into 2 cm long pieces, 100 mg of which were
infiltrated with 1.5 mL of 0.25 % aqueous solution of Evan’s blue for 30 min under room
temperature. Upon absorption of the stain, leaf and root samples were rinsed with deionizedwater until no further blue dye was eluted from the samples. To quantify the Evan’s blue stain
absorbed, the stain was solubilized by incubating in 4 mL of 1 % (w/v) sodium dodecyl sulfate(SDS) prepared in 50 % (v/v) methanol at 50 °C for 30 min, and measure absorbance at 600
nm using SDS as a blank. Stain absorption was then quantified using Evan’s blue standard
solutions.

3.3.10. Determination of hydrogen peroxide concentration
H2O2 concentration was determined using ground frozen samples based on ferrous oxidation of
xylenol orange (FOX) method (Kaur et al. 2016) with slight modifications (Mekawy et al. 2018)
similar to section 2.2.6. The reaction mixture was left at room temperature for 1 hour and H2O2
concentrations were determined spectrophotometrically at 560 nm and quantified using H 2O2
standard solutions.

3.3.11. Antioxidant enzyme activity essays
In order to analyze antioxidant enzyme activities, 300 mg frozen leaf blade and root samples
were ground in presence of liquid nitrogen using mortar and pestle and extracted using 3 mL

53

potassium phosphate (KPB) buffer (pH 7.0) and centrifuged at 10,000 x g for 15 min at 4 °C.
Catalase activity was obtained as previously described (Mekawy et al. 2018; Takagi and
Yamada 2013). The 700 μL reaction mixture for catalase (CAT, EC 1.11.1.6) activity
comprised of 665 μL of 10 mM H2O2 in 50 mM KPB, pH 7.0, and 35 μL crude extract. Catalase
activity was measured by following decrease in absorbance at 240 nm for 1 minute. CAT
activity was calculated using H2O2 extinction coefficient of 0.0436 mM-1 cm-1. One unit of CAT
activity was determined as mmol of H2O2 consumed min-1 mg-1 protein.

Ascorbate peroxidase (APX, EC 1.11.1.11) was essayed according to Mekawy et al. (2018).
For this purpose, 700 μL reaction mixture contained 630 μL APX reaction mixture containing
0.2 mM H2O2, 0.15 mM EDTA, 50 mM ascorbic acid and 70 μL crude extract in 50 mM KPB,
pH 7.0. APX activity was obtained by recording decrease in absorbance at 290 nm for 1 minute,and calculated using extinction coefficient of 2.8 mM-1 cm-1. One unit of APX activity wasdetermined as μmol of ascorbate oxidized min-1 mg-1 protein.

Guaiacol peroxidase (GPX, EC 1.11.1.7) activity was investigated as described previously(Jebara et al. 2005; Uarrota et al. 2016). In brief, the 700 μL GPX reaction mixture contained
665 μL reaction buffer containing 20 mM H2O2, 10 mM guaiacol, and 35 μL crude extract in
50 mM KPB, pH 7.0. GPX activity was obtained by recording increase in absorbance at 470
nm for 1 minute, and calculated using extinction coefficient of 26.6 mM-1 cm-1. One unit of
GPX activity was determined as μmol tetraguaiacol formed min-1 mg-1 protein.

Glutathione reductase (GR, EC 1.8.1.7) was essayed according to outlined methods in Liu &Saneoka (2019) with slight modifications. For this purpose 200 μL crude extract was added to
1,800 μL GR reaction mixture, containing 1 mM oxidized glutathione (GSSG), 0.5 mM EDTA,

54

0.04 mM reduced nicotinamide adenine dinucleotide phosphate (NADPH). GR activity wascalculated by following decrease in absorbance at 340 nm using extinction coefficient of 6.22
mM-1 cm-1. One unit of GR was determined as μmol NADPH oxidized min-1 mg-1 protein.

Activity of superoxide dismutase (SOD, EC.1.15.1.1) was essayed based on its ability to inhibit
photochemical reduction of nitro blue tetrazolium (NBT) as outlined in Beauchamp &Fridovich (1971) and Dhindsa et al. (1980). Here, SOD activity was estimated by recording thedecrease in absorbance of superoxide-nitro blue tetrazolium complex by the enzyme. The
complete 3,200 μL SOD reaction mixture contained 100 μL of 1.5 M sodium carbonate 200
μL of 200 mM methionine, 100 μL of 2.25 mM NBT, 100 μL of 3 mM EDTA, 1,500 μL of
100 mM potassium phosphate buffer, 1,000 μL distilled water and 100 μL of enzyme. Fourtubes without enzyme extract were taken as control. The reaction was started by adding 100
μL of 1 mM riboflavin and placing the tubes below a light source of two 15 W florescent lamps
for 15 min. Reaction was stopped by switching off the light and covering the tubes withaluminium foil. Tubes without enzyme developed maximal colour. Another set with complete
reaction mixture but without the enzyme was nonirradiated, did not develop colour hence
served as blank. Absorbance was recorded at 560 nm and one unit of enzyme activity was taken
as the quantity of enzyme which caused 50 % inhibition of NBT photoreduction. Protein
concentration in the enzyme extract was measured using Bradford reagent (Bradford 1976)
using bovine serum albumin as standards.

3.3.12. Statistical analyses
The experiment was arranged in a completely randomized design (CRD). Each measurement
was performed using four to five biological replicates. Inferential statistics were performed
using analysis of variance (ANOVA) at a significance level of 0.05. Multiple comparison tests

55

were performed using Tukey’s test. All data were analyzed using R Studio (RStudio team2020).

3.4. Results
3.4.1.

Effectiveness of PTs on growth

After 7 days of PTs, no significant reductions in growth were recorded by PTs, instead, somePTs, notably 1 μM H2O2 slightly increased shoot and root growth (Fig. 3.1A). At the end of 14days of main stress, shoot and root dry weight were considerably decreased in non-pretreatedplants (Fig. 3.1B). However, pre-treatment with 5 mM and 10 mM NaCl, and all concentrationsof H2O2 (1, 5, 10 μM) significantly increased shoot dry weight. Acclimation to saline-alkaline
stress (SAS) using mild SA PTs was not as effective (Fig. 3.1B), despite no significant
reductions in growth during the acclimation period (Fig. 3.1A). Nonetheless, 0.5 mM NaHCO3
proved relatively effective, slightly increasing both shoot and root growth compared with nonpretreated (NPT) plants. Plant height and root length were not significantly affected undersaline-alkaline pre-treatments (data not shown), however, the tendencies for 10 mM NaCl PT
favoured shoot growth whereas H2O2 PTs favoured root growth. Overall, 10 mM NaCl, 10 μM
H2O2 and 0.5 mM NaHCO3 PTs were the most effective in their respective groups, with theformer two being the best, hence were chosen for subsequent experiments, and will be the main
focus subsequently.

3.4.2.

Chlorophyll concentration

At the conclusion of the pre-treatment period, chlorophyll concentration was significantly
reduced by NaHCO3 PTs whereas no differences were shown between controls and NaCl andH2O2 PTs (Fig. 3.1C). After 14 days of main stress, chlorophyll concentration was significantly56

reduced in non-pretreated and NaHCO3 pretreated seedlings, whereas NaCl and H2O2 PTs
maintained it (Fig. 3.1C).

3.4.3.

Changes in growth medium pH

Growth medium pH was monitored on daily basis and showed that during the onset of stress,
pH generally progressively increased. On the first day of stress for example, medium pH wasset at 8.0 for all plants, but increased to around 8.45 by the end of 14 days of stress for nonpretreated plants (Fig. 3.2). In comparison to non-pretreated plants, PTs with NaCl were themost effective in reducing growth medium pH (Fig. 3.2A) followed by H2O2 PTs (Fig. 3.2B).
It was hypothesized that acclimating rice with saline-alkaline PT (NaHCO3) would reduce
medium pH under high saline-alkaline stress, however, it was striking to observe that 1.0 and
2.5 mM NaHCO3 considerably increased growth medium pH to about 8.7 by the end of 14 day
stress period, whereas 0.5 mM did not significantly change it (Fig. 3.2C).

3.4.4.

Accumulation of K, Na and P in roots and leaf blades

Significant reductions in K concentration were observed in both leaf blades and roots,
particularly pronounced in roots (Fig. 3.3A). However, in leaf blades, PT with 10 μM H 2O2
and 0.5 mM NaHCO3 significantly increased K concentration while in roots all PTs
significantly increased it (Fig. 3.3A). Content of K per plant was significantly reduced bysaline-alkaline treatment, however, all PTs significantly increased K content per plant (Fig.
3.3B). This was coincidental with significant increases in Na concentration especially in theroots. In leaf blades, PT with 10 mM NaCl significantly reduced Na accumulation, and so did
NaHCO3, but H2O2 PT accumulated the highest Na concentration (Fig. 3.3C). In roots however,
10 mM NaCl and 10 μM H2O2 PTs significantly reduced Na concentration. However, content
of Na+ was significantly higher in all pretreated plants compared to NPT under saline-alkaline57

conditions (Fig. 3.3D). P concentration was significantly reduced in both leaves and roots,
whereas PTs with 0.5 mM NaHCO3 and 10 μM H2O2 significantly increased it especially inroots (Fig. 3.3E).

3.4.5.

Effectiveness of PTs on micronutrient acquisition

All three PTs significantly increased Fe concentration in both organs, this was however mostpronounced in roots (Fig. 3.4A). Likewise, content of Fe was considerably reduced undersaline-alkaline stress especially in NPT plants (Fig. 3.4B), however, all PTs were able tosignificantly increase Fe content per plant. The same was observed for boron (B) in both leaf
blades and roots (Fig. 3.4C). In leaf blades, Zn concentration was significantly reduced bysaline-alkaline stress, except for plants acclimated using 0.5 mM NaHCO3 that accumulated
nearly two-folds higher Zn than NPT, NaCl and H2O2 pretreated plants (Fig. 3.4D), whereas inroots, Zn concentration was significantly increased by saline-alkaline stress in all PTs (Fig.
3D) and so was Mn (data not shown). Overall, 0.5 mM NaHCO 3 was the most effective atincreasing micronutrient concentration, implying that acclimation to saline-alkaline stressincreases the efficiency for uptake of micronutrients in rice.

3.4.6.

Effect of PTs on root K+ retention and root Na+ efflux

In the previous result (Fig. 3.3A and B), rice plants had significantly lower concentrations andcontent of K+, but this was somehow ameliorated by PTs. Plant roots’ K+ leakage showed
extremely high leakage of K+ regardless of PT (> 60 folds higher), implying that lower root K
concentrations under saline-alkaline stress were chiefly as a result of leakage of K+ from roots(Fig. 3.5A), and that PTs were not effective enough to reduce it.

58

Then, efflux of Na+ was studied in a 4 hour period, and showed that saline-alkaline stressed
plants exhibited significantly higher Na+ efflux (Fig. 3.5B), however this was more pronounced
in pretreated plants, particularly by 10 mM NaCl, in part explaining its lower leaf blade androot Na+ concentrations.

3.4.7.

Na+ in excised leaves

In order to eliminate contribution of plant roots’ Na+ exclusion and efflux, Na+ uptake wasmeasured in excised leaves and showed that devoid of these factors, 10 mM NaCl and 10 μM
H2O2 significantly reduced Na+ concentration accumulating in the leaf blades, whereas nonpretreated and 0.5 mM NaHCO3 pretreated plants accumulated significantly higher amounts(Fig. 3.5C). Also, there was significantly lower chlorophyll concentration in non-pretreated
and 0.5 mM NaHCO3 pretreated leaf blades (Fig. 3.5D), which could be due to the higher Na+
accumulation and inefficient sequestration of Na+ away from the cytosol.

3.4.8.

Effectives of PTs on membrane integrity and cell viability

Leaves of non-pretreated plants exhibited considerably higher leakage of electrolytes (Fig.
3.6A), signifying loss of membrane integrity, whereas in roots, despite having much higher
leakage of electrolytes than leaf blades, no significant differences were detected among all
treatments (Fig. 3.6A). This was further verified by measurement of MDA, a marker of lipid
peroxidation that showed significantly lower concentration in leaf blades of NaCl and H2O2
pretreated plants, whereas in roots, only NaCl reduced MDA accumulation (Fig. 3.6B).

Cell viability using Evan’s blue staining method was significantly enhanced in leaf blades by
all PTs (Fig. 3.6C). In roots, cell viability was only significantly increased by NaCl and H 2O2
PT (Fig. 3.6C). Finally, it was checked whether changes in membrane integrity and cell59

viability among various PTs could be attributed to oxidative stress. For this purpose, H 2O2
concentration was analyzed in both leaf and root organs. It was shown that in leaves, 10 μM
H2O2 and 0.5 mM NaHCO2 PTs significantly reduced H2O2 accumulation whereas in roots all
PTs significantly reduced oxidative stress (Fig. 3.6D).

3.4.9.

Oxidative stress and antioxidant enzyme activity

In order to elucidate the biochemical basis in which pre-treatments ameliorate oxidative stress,
antioxidant enzyme activities were essayed in leaf blades and roots. Catalase activity wassignificantly increased in leaf blades of NaCl and H2O2 PTs, whereas in roots, only NaCl and
NaHCO3 PTs significantly increased its activity (Fig. 3.7A). Guaiacol peroxidase (GPX) was
not enhanced by any of the PTs in both leaf blades and roots (data not shown). However,ascorbate peroxidase (APX) was considerably increased in both leaves and roots of NaCl andH2O2 PTs (Fig. 3.7B). Next, antioxidant activity of glutathione reductase (GR) was also
essayed, and showed that its activity was significantly induced in leaf blades, however, only
pretreated plants were able to significantly induce GR activity (Fig. 3.7C). In roots, no GR
activity was detected in all samples. Finally, superoxide dismutase (SOD), an important
enzyme for detoxification of superoxide radicles was essayed. No any significant differenceswere shown in leaf blades among PTs, except for H2O2 pre-treatment that considerably
decreased its activity (Fig. 3.7D). On the other hand, in roots, SOD activity was significantly
increased by all PTs whereas in non-pretreated plants it was considerably supressed (Fig. 3.7D).
Protein concentration was measured for purposes of expressing specific enzyme activity, andwas highest in leaf blades than roots (Fig. 3.7E).

60

3.4.10. Effect of PTs on post – stress recovery
In order to investigate if acclimation and cross-tolerance would influence recovery of rice
plants after conclusion of stress, saline-alkaline stressed plants were recovered by cultivating
under normal growth conditions (0 mM Na+, pH 5.5) for 10 days. It was observed that during
recovery, plants under control conditions grew about 3 times faster than during the stress period(Fig. 3.8A and B). However, in previously stressed plants, despite subjecting them to similar
recovery conditions, growth rates did not significantly change, except for H 2O2 pretreatedplants that considerably increased both shoot and root mean growth rates (Fig. 3.8A and B).
Therefore 10 μM H2O2 PT facilitates a quicker recovery and resumption of growth when stressis concluded. Strikingly, in roots, NaHCO3 PT significantly reduced root mean growth rateduring recovery (Fig. 3.8B), suggesting that here and in NPT plants, irreparable damage was
inflicted. Despite the 10 day recovery, non pretreated plants were unable to recover chlorophyll
concentration, but other PTs were able to accumulate chlorophyll concentration comparable tocontrol plants (Fig. 3.8C). All plants suffered considerably less membrane damage as measured
by electrolyte leakage, and no differences were detected among the pretreated seedlings (Fig.
3.8D). These observations imply that during the recovery period, some plants were able tophysiologically recover, despite the inability of some to resume rapid growth.

61

Fig. 3.1. Vegetative growth and physiological data showing dry weight after 7 days acclimation
period (A) and dry weight (B) and leaf chlorophyll concentration shown as SPAD units (C)after 14 days main saline-alkaline treatment. Plants were pretreated with various concentrationsof NaCl (S) (2.5, 5.0, 10 mM), H2O2 (OX) (1.0, 5.0, 10.0 μM) and NaHCO3 (SA) (0.5, 1.0, 2.5mM) for 7 days and subjected to 50 mM Na, pH 8.25 for 14 days. The data represents means± standard errors from five biological replicates. Different letters indicate significant
differences by Tukey multiple comparison tests at 0.05 level of significance, same/ no letters
indicate no significant differences.
62

Fig. 3.2. Rhizosphere acidification data showing daily growth medium pH changes during a
14 day period of main saline-alkaline treatment in plants pretreated with various concentrationsof NaCl (A), H2O2 (B) and NaHCO3 (C) versus non-pretreated seedlings. Plants were pretreatedwith various concentrations of NaCl (S) (2.5, 5.0, 10 mM), H2O2 (OX) (1.0, 5.0, 10.0 μM) and
NaHCO3 (SA) (0.5, 1.0, 2.5 mM) for 7 days and subjected to 50 mM Na, pH 8.25 for 14 days.

63Fig. 3.3. Macronutrient analysis showing K concentration (A) and content (B), Na
concentration (C) and content (D), and P concentration (E) in leaf blades, roots and whole plant
after 14 days of main saline-alkaline treatment. Plants were pretreated with 0 (NPT), 10 mM
NaCl (S10), 10 μM H2O2 (OX10), and 0.5 mM NaHCO3 (SA0.5), for 7 days and subjected to
50 mM Na, pH 8.25 for 14 days. The data represents means ± standard errors from fourbiological replicates. Different letters indicate significant differences by Tukey multiple
comparison tests at 0.05 level of significance.

64Fig. 3.4. Micronutrient analysis showing Fe concentration in leaf blades and roots (A) andcontent in the whole plant (B), and concentrations of B (C) and Zn (D) in leaf blades and roots
after 14 days of main saline-alkaline treatment. Plants were pretreated with 0 (NPT), 10 mM
NaCl (S10), 10 μM H2O2 (OX10), and 0.5 mM NaHCO3 (SA0.5), for 7 days and subjected to
50 mM Na, pH 8.25 for 14 days. The data represents means ± standard errors from fourbiological replicates. Different letters indicate significant differences by Tukey multiple
comparison tests at 0.05 level of significance.

65Fig. 3.5. Physiological experiments for K+ and Na+ homeostasis showing K+ leakage in rootsof 7 day pretreated and non-pretreated seedlings subjected to 50 mM Na, pH 8.50 for 24 hours(A), efflux of Na+ from roots of 7 day pretreated and non-pretreated seedlings, subjected to 50
mM Na, pH 8.50 for 24 hours, and deionized water for 2 hours (B), and concentrations of Na+(C) and chlorophyll (D) in excised leaves from 7 day pretreated and non-pretreated plants
subjected to 30 mM Na, pH 8.0 for 3 days. Plants were pretreated with 0 (NPT), 10 mM NaCl
(S10), 10 μM H2O2 (OX10), and 0.5 mM NaHCO3 (SA0.5), for 7 days and subjected to theabove experiments. The data represents means ± standard errors from four biological replicates.
Different letters indicate significant differences by Tukey multiple comparison tests at 0.05
level of significance.

66Fig. 3.6. Oxidative stress, membrane integrity and cell viability parameters showing leakageof electrolytes (A), malondialdehyde concentrations (B), Evans Blue staining (C) andconcentrations of hydrogen peroxide (D), in leaf blades and roots after 14 days of main salinealkaline treatment. Plants were pretreated with 0 (NPT), 10 mM NaCl (S10), 10 μM H 2O2(OX10), and 0.5 mM NaHCO3 (SA0.5), for 7 days and subjected to 50 mM Na, pH 8.25 for 14
days. The data represents means ± standard errors from four biological replicates. Different
letters indicate significant differences by Tukey multiple comparison tests at 0.05 level ofsignificance.

67

from four biological replicates. Different letters indicate significant differences by Tukey multiple comparison tests at 0.05 level of significance(OX10), and 0.5 mM NaHCO3 (SA0.5), for 7 days and subjected to 50 mM Na, pH 8.25 for 14 days. The data represents means ± standard errors

protein concentration (E) after 14 days of main saline-alkaline treatment. Plants were pretreated with 0 (NPT), 10 mM NaCl (S10), 10 μM H 2O2

Fig. 3.7. Antioxidative enzyme activities of catalase (A), ascorbate peroxidase (B), glutathione reductase (C), and superoxide dismutase (D) andFig. 3.8. Effect of pre-treatments on recovery of plants after exposure to saline-alkaline stress.
Comparative mean growth rates during and after stress in shoots (A) and roots (B). Chlorophyll
concentration (C) and membrane integrity assessed by the electrolyte leakage method in shoots(D) at the end of the 10-day recovery period. “Stress” group represents mean growth rate ofplants during the 14 day growth period under main saline-alkaline stress (SAS) (50 mM Na+,pH 8.25) after pre-treatment with 0 (NPT), 10 mM NaCl (S10), 10 μM H2O2 (OX10), and 0.5
mM NaHCO3 (SA0.5) for 7 days. “Recovery” group represents the growth rate of the sameplants upon completion of main saline-alkaline stress (SAS) and subjecting them to recovery
under normal conditions (0 mM Na+, pH 5.5) for 10 days. The data represents means ± standard
errors from five biological replicates. Different letters indicate significant differences by
Tukey’s multiple comparison tests at P < 0.05.

69

3.5. Discussion
Rice is extremely sensitive to salt stress, although it is one of the prominent staples in the world.
Hence, it remains imperative to identify quicker, yet effective means to increase rice growthand yield in such environments. Plants are adept at acclimating to salt stress, characterized byquicker and stronger responses to subsequent lethal exposure to the same stress (Kamanga et
al. 2020; Liu & Saneoka 2019; Sriskantharajah et al. 2020). While acclimation studies arewidespread, the physiological processes governing responses of plants to multiple exposuresof different stresses are unclear. Here, we have shown that plants pre-exposed to lower
concentrations of NaCl and H2O2 show improved growth and physiological characteristics
under SAS conditions. Contrary to the hypothesis, the failure of NaHCO 3 pre-treatment toenhance growth suggests that the concentrations used were too high for pre-treatment in rice,
likely causing physiological injuries. However, a similar concentration of 1 mM NaHCO3 in
Rye (Secale cereale) effectively improved tolerance to SAS conditions (Liu & Saneoka 2019).
Overall, 10 mM NaCl and 10 μM H2O2 were the most effective (Fig. 3.1B). These latter results
illustrate the concept of cross-tolerance, wherein pre-exposure to one stress, such as salinity oroxidative stress, promotes tolerance to another stress, such as saline-alkaline stress, as
previously described {Formatting Citation}. In light of a myriad of abiotic stresses as currently
faced, this critical attribute offers a prospect for generating plants tolerant to multiple abiotic
stresses.

Under SAS conditions, the first line of defence entails acidification of the rhizosphere through
H+-ATPase-mediated proton efflux by plant roots (Li et al. 2016). In the present study, thefailure of NaHCO3 pretreated plants to suppress rise in growth medium pH (Fig. 3.2C) may
partly elucidate their inability to effectively promote tolerance to SAS conditions (Fig. 3.1B).
In contrast, plants pretreated with NaCl and H2O2 maintained a much lower growth medium70

pH throughout the growth period compared to the NPT plants, indicative of higher root activity.
Lower rhizospheric pH is also advantageous for maintaining the transmembrane proton
gradient required for SOS-mediated efflux of Na+ by roots SOS1 (Cao et al. 2020; Moriyama
2015). Experiments on Na+ efflux showed that all pretreated plants had higher root Na+ efflux
than non-pretreated (Fig. 3.5B) including NaHCO3. Therefore, whether higher root Na+ efflux
and resultant lower leaf blade Na accumulation (Fig. 3.3C) were as a result of lower growth
medium pH are difficult to untangle. Furthermore, when the contribution of Na + uptake byroots was eliminated using experiments on Na+ accumulation in excised leaf blades, both 10
mM NaCl and 10 μM H2O2 pretreated plants accumulated significantly lower Na in the leaf
blades (Fig. 3.5C). In H2O2 pretreated plants, notwithstanding higher leaf blade Na+
accumulation, the deleterious effects of Na+ were ameliorated and leaf photosynthetic
competency (Fig. 3.1C) was preserved. Moreover, despite the “statistical” significance of leaf
blade Na+ accumulation among PTs, this result suggests that the observed differences may nothave been “biologically significant”.

Na and K have similar physicochemical properties; hence, they exhibit competitive uptake
systems (Marschner & Marschner 2012). Moreover, rice plant roots are leaky (Munns & Tester2008), potentially losing significant amounts of K+ under SAS conditions. Retention of root K+
has been cited as a principal trait for salt tolerance (Chen et al. 2007). In the present study, pretreatment with NaCl and H2O2 maintained a higher root K concentration (Fig. 3.3A) and K
content (Fig. 3.3B). Separate experiments revealed extremely high root K+ leakage under SAS(Fig. 3.5A), which could however not be alleviated by pre-treatments. Therefore, while root
K+ leakage was the primary cause of K deficiency under SAS, its contribution to the observed
differences among various pre-treatments could not be verified. Nonetheless, considering theextreme loss of K+ in roots under SAS conditions, these results suggest that even the slightest

71improvements in root K+ retention may be beneficial under saline-alkaline stress, primarily forosmotic adjustments and maintenance of root growth as also suggested by Chen et al. (2007).
Additional benefits of acclimation included increased P uptake and accumulation especially inroots (Fig. 3.3E and D). Uptake of P is driven by H + gradient across the plasma membrane,such that when the rhizospheric pH is lower than root cytosolic pH, P uptake is promoted (Caoet al. 2020). Hence, under saline-alkaline stress, maintenance of P uptake is an extremely
important attribute for plant growth and development, and also dependant on plant roots ability
to lower rhizospheric pH through H+-ATPases mediated proton efflux. However, further
studies are required to elucidate the physiological mechanisms underlying this enhanced P
uptake.

Fe deficiency, which is a typical feature of plants growing under high pH conditions, ischaracterized by chlorosis. Although Fe is a naturally abundant element, it forms insoluble
ferric compounds that are non-bioavailable (Marschner 1995; Nakanishi et al. 2004) for uptake
under high pH conditions. As shown in Fig. 3.1C, NPT plant leaves were chlorotic, which is atypical sign of Fe deficiency (Marschner & Marschner 2012). However, in NaCl and H2O2
pretreated plants, leaves were healthier (Fig. 3.1C) and accumulated higher Fe concentrationand content (Fig. 3.4A and B). Therefore, it is tempting to attribute the high Fe concentration
to the ability of plant roots to suppress rise in growth medium pH, as observed in Fig. 3.2A and
B. Lower growth medium pH is thought to increase the solubility of ferric ions or support the
reducing capacity of ferric Fe on the root surface (Kobayashi & Nishizawa 2012). However,similar increases in Fe concentration and content were also observed in NaHCO3 pretreatedplants (Fig. 3.4A and B, respectively) which otherwise did not show different growth medium
pH from NPT plants (Fig. 3.2C), raising doubts on the causality between Fe concentration andlower rhizospheric pH. Alternative causes for the variations in Fe uptake could relate to72differences in key enzyme activities involved in Fe uptake (Nakanishi et al. 2004; Kobayashi& Nishizawa 2012), however, further studies to elucidate the physiological and molecularbases of this acclimation induced increase in accumulation are imperative. In NaHCO3
pretreated plants (SA0.5), despite having higher leaf blade and root Fe concentration (Fig.
3.4A), leaf blades were relatively chlorotic (Fig. 3.1C), similar to non-pretreated plants,
inconsistent with our expectations. This might have been as a result of considerably reduced
chlorophyll concentration during the 7-day acclimation period (Fig. 3.1C), such that enhanced
Fe uptake and accumulation during the main stress could not fully recover chlorophyll
concentration by the sampling time.

Several reports have implicated the lethality of SAS to ROS-mediated damage. In an alkaline
tolerant (Alt1) rice mutant, Guo et al. (2014) credited tolerance to enhanced defence against
oxidative stress. Oxidative damage to membrane lipids causes peroxidation, which isanalytically deduced from the concentration of malondialdehyde (MDA), an aldehyde byproduct of lipid peroxidation (Ayala et al. 2014). In the present study, NaCl and H2O2 pretreatments considerably alleviated SAS-induced membrane damage and cell death (Fig. 3.6A
– C). These results, in part, indicate mitigated oxidative stress, owing to the enhanced ROS
scavenging capacity in pretreated plants. ROS generation in plants is an inevitable consequenceof aerobic metabolism. Until recently, the traditional paradigm that considers ROS asdeleterious to plant cells is gradually fading owing to recent revelations of their critical role assecondary messengers (Apel and Hirt 2004; Mittler 2017). We hypothesized that pretreatments would spark a mild generation of ROS, which would eventually activate systemic
antioxidant defence responses. In agreement, H2O2 generation was significantly reduced inroots in all pretreated plants, whereas only H2O2 and NaHCO3 pre-treatments were effective inleaf blades. In NaCl pretreated plants, despite having exceptional growth and lower Na

73

concentration in leaf blades, the higher H2O2 in tandem cautions against the inevitable
expectation that ROS results in poor growth, harmonizing with notions earlier expressed inMittler (2017). Perhaps the crucial aspect is maintaining a delicate balance between ROS
generation and detoxification. Given that NaCl pretreated plants had higher antioxidant
activities of CAT, APX, and GR (Fig. 3.7A – C), this balance might have been attained,cushioning against vicious functions of ROS (Apel and Hirt 2004). Better antioxidant enzyme
activities were also observed in H2O2 pretreated plants, in agreement with earlier observations(Fedina et al. 2009; Wahid et al. 2007). Moreover, all pre-treatments considerably increased
the SOD activity (Fig. 3.7D). Since H2O2 and hydroxyl radicals are superoxide radical
derivatives, SOD offers a first line of defence against oxidative stress; hence, acclimation
provided nearly complete protection against oxidative stress. Besides, H 2O2 concentration (Fig.
3.6A) did not essentially correspond to the trends of electrolyte leakage, MDA concentration,and cell viability (Fig. 3.6B – C) in some PTs, in part cementing the above notions. Apparently,
this may be ascribed to different antioxidant enzyme capacities (Fig. 7A – C) or capacities torepair of ROS mediated damage. Strategies adopted by plants to confront oxidative stressmediated damage engage three lines of steps; avoidance of ROS generation, detoxification of
ROS (scavenging) and repair of ROS mediated damage (Guo et al. 2014; Mittler 2017). Hence,measurement of ROS generation alone may not sufficiently predict resultant ROS mediateddamage. Furthermore, this result may point to involvement of other ROS, such as superoxideand hydroxyl radicles that were otherwise not assayed in the present study.

3.6. Conclusion
The present study strengthens the role of ROS in stress tolerance, and reports that acclimation
to salinity stress, or oxidative stress promotes cross-tolerance to saline-alkaline stress byfostering stable Fe acquisition and ROS homeostasis. These processes are interlinked to plant74

roots’ ability to resist excessive changes in growth media pH, such that maintaining a lower
pH is the first line of defence against saline-alkaline stress. These findings further suggest acrosstalk between salinity stress, oxidative stress and saline-alkaline stress, likely engaging
common signals and pathways that develop cross resistance. While these signalling elements
need to be fully investigated, results suggest involvement of ROS such as hydrogen peroxide,
but calls for exploration of molecular signatures underlying these responses. These results
demonstrate that acclimation to salinity stress and oxidative stress triggers systemic defence inrice plants, that aids stronger and quicker responses to saline-alkaline stress. Moreover, except
for NaHCO2 pretreated plants, during recovery period, plants quickly physiologically recoverand resume rapid growth. This may be related to lower accumulation of H2O2 in the leaf blades(Fig. 3.6D), as a result of higher antioxidant enzyme activities specifically CAT, APX and GR(Fig. 3.7A – C) that detoxify cellular H2O2.This is a fundamental attribute for recurrent stresses
with prolonged recovery periods, therefore, pretreatment with optimal NaCl and H2O2 may be
a recommended option for quick improvements in tolerance to saline-alkaline stress in rice.
This trait offers a prospect for developing plants tolerant to multiple stressors.

75

Chapter 4

General Discussion

76Notwithstanding the worldwide agronomic prominence of rice, its growth and productivity
remains considerably affected by a range of abiotic stresses. This study (1) comparatively
investigated the effects of salinity stress, saline-alkalinity and alkaline stress on growth andphysiology of rice seedlings; and (2) elucidated physiological mechanisms of acclimationinduced cross-tolerance to saline-alkalinity. This study finds that rice plants are far muchsensitive to saline-alkaline stress than to either sole salinity or sole alkalinity. Furthermore,
relative to salinity stress, at the concentrations used in the study, rice plants are more sensitive
to high growth media pH than salinity stress. These results highlight the gravity of salinealkaline stress to rice plants. Similar conclusions have been derived at in a number of studies(Lv et al. 2013; Shi & Wang 2005; Yang et al. 2007; Yang et al. 2008; Zhang et al. 2019).

Plants have a unique ability to tolerate to one stress following previous exposure to anotherstress, this is referred to as cross-tolerance (Foyer et al. 2016; Pastori & Foyer 2002; Perez &Brown 2014). However, this phenomenon has never been reported under saline-alkaline stress.
In order to explore this option in rice, this study endeavoured to investigate the physiologicalmechanisms in which saline-alkaline stress confronts growth of rice to understand key stresscomponents of saline-alkaline stress that can be harnessed as pre-treatments to elicit systemic
defence responses. Consistent with previous reports, plants grown under high pH conditions,both saline-alkalinity and sole alkalinity exhibited clear signs of chlorosis (Fig 2.3) in bothyounger and older leaf blades as a result of Fe deficiency and Na toxicity, respectively (Li etal. 2016; Munns & Tester 2008). Saline-alkaline stressed plants accumulated multiple folds
more Na in leaf blades than salinity stressed plants (Fig. 2.4D). This finding harmonized withpre-existing reports and further revealed chronic K deficiency in plant roots grown undersaline-alkalinity (Fig. 2.4C), as a result of excessive K+ leakage in the roots (Fig. 2.6A).
Furthermore, this study observed excessive accumulation of H2O2 in leaf blades, that may have77

exceeded plants’ inherent antioxidative capacity causing membrane damage. Oxidative stresshas been implicated in lethality of saline-alkalinity, particularly in plant roots of many species(Fu et al. 2017; Peng et al. 2008; Zhang et al. 2017; Zhang & Mu 2009).

Premised on the above results, the study acclimated rice seedlings to mild stress components;
Na+ toxicity (NaCl), oxidative stress (H2O2) and high pH (NaHCO3). Direct application of ROS
by oxidative agents such as H2O2 has been investigated as a potential means of inducing crosstolerance in plants (Perez & Brown 2014). H2O2 is a recognized inducer of antioxidant
responses, hence has been utilized as a seed pretreatment for plants growing under abiotic
stresses (He et al. 2009; Wahid et al. 2007). Besides, we had previously shown that plants preexposed to mild NaCl develops tolerance upon subsequent exposure to a higher NaCl stress(Kamanga et al. 2020; Sriskantharajah et al. 2020). Hence, this study has shown that riceseedlings pre-exposed to 10 mM NaCl and 10 μM H2O2 showed greater tolerance to and better
photosynthetic properties under saline-alkalinity (Fig. 3.1A – C). Moreover, upon conclusionof the stress treatment, H2O2 pretreated plants showed better recovery compared to nonpretreated plants, in part attributed to lower H2O2 accumulation, an essential attribute forrecurrent stresses with prolonged recovery periods. In total, these results reveal that riceseedlings pre-exposed to salinity stress and oxidative stress develop cross-tolerance to salinealkalinity, pointing to a likelihood for involvement of common pathways and signalling
elements among these stresses.

In this study, it was shown that NaCl and H 2O2 pretreatments (PTs) were able to supress
excessive rise in growth medium pH (Fig. 3.2A and B), a rhizosphere acidification strategy
that is dubbed a first line of defence under alkaline conditions (Li et al. 2016). This crucial
attribute also plays a critical role in increasing Fe solubility, facilitating rice plants’ Fe uptake

78

(Ishimaru et al. 2006; Kobayashi & Nishizawa 2012). As a consequence, plants pretreated with
10 mM NaCl and 10 μM H2O2 exhibited higher leaf blade Fe concentration and content in theplant (Fig. 3.4 A and B) and accordingly averted leaf Fe deficiency chlorosis (Fig. 3.1C). Lower
rhizospheric pH is also crucial for SOS root Na+ efflux, to avoid excessive accumulation of
Na+ in shoots. Consequently, plants pretreated with NaCl and H2O2 showed considerably
higher efflux of Na+ (Fig. 3.5B) and lower leaf blade Na+ concentration (Fig. 3.3C) but it did
not translate into any decreases in leaf blade Na+ content (Fig 3.3D). That notwithstanding,deleterious effects of Na+ were ameliorated and leaf photosynthetic competency was preserved
by both H2O2 and NaCl PTs. Besides, the PTs enhanced K and P uptake capacities, elements
critical for plant growth under saline-alkaline conditions, however mechanisms behind theenhanced uptake need to be further investigated.

Considering the high oxidative stress mediated membrane damage observed in chapter 2 (Fig.
2.10 A – D, Fig. 2.11 A and B), I investigated whether acclimation to saline-alkaline stresswould ameliorate oxidative stress induced damage as previously reported (He et al. 2009;
Wahid et al. 2007; Kamanga et al. 2020; Liu & Saneoka 2019). Accordingly, leaf and root
membrane integrity (Fig. 3.6A and B) and cell viability (Fig. 3.6C) were considerablyenhanced in H2O2 pretreated plants by tremendously reducing leaf and root H2O2 accumulation(Fig. 3.6D). While NaCl PT also enhanced membrane properties and cell viability, oxidative
stress was only significantly reduced in roots (Fig. 3.6A – D). Plants are surfeited with a batteryof antioxidants to detoxify generated ROS under abiotic stresses. Here, NaCl and H2O2
pretreated plants exhibited higher CAT and APX activities in both leaf blades and roots, andGR activity in leaves (Fig. 3.7A – C), these enzymes are crucial for detoxification of H2O2.
Superoxide dismutase (SOD) offers the first line of defence against oxidative stress, since H2O2
and hydroxyl radicles are derivatives of superoxide radicles. SOD antioxidant activity was also79

considerably increased in root tissues (Fig. 3.7D) of all pretreated plants. Being directly
exposed to Na+ toxic and alkaline rhizosphere, efficient antioxidant capacity in roots is crucial.
These results suggest that a nearly complete shield against oxidative stress was instituted in
H2O2 and NaCl pretreated plants. Albeit having higher H2O2 concentration in the leaf bladesin NaCl pretreated plants, efficient antioxidant capacities cushioned against their harmful
effects attaining an optimal equilibrium between their generation and scavenging.

In conclusion, this study reports that saline-alkaline stress is a highly lethal abiotic stressrelative to sole salinity and sole alkaline stress. Plants exposed to saline-alkaline stressexperience severe growth reductions due to leaf blade Na+ toxicity, chronic Fe deficiencies
particularly in younger leaves, aggravated root K+ deficiency as a result of tremendous root K+
leakage and oxidative stress mediated damage in leaf and root tissues. However, these effects
were ameliorated by pre-exposure to mild ionic (NaCl) and oxidative (H2O2) stress componentsthat developed cross-tolerance to saline-alkalinity. While mechanisms of acquired resistancediffered across PTs, an aggregated consensus suggests the crucial role played by the following
physiological mechanisms; (1) ability to maintain a lower growth medium pH (2) maintenanceof a stable acquisition of Fe, P and K and (3) regulated detoxification of reaction oxygen species(ROS) via enhanced antioxidant enzyme activities. These results suggest that mechanisms
which protect against salinity stress or oxidative stress may also provide protection against
saline-alkalinity. Furthermore, they also suggest and strengthen the possibility that a crosstalk
exists between salinity stress, oxidative stress and saline-alkalinity, likely engaging someoverlapping signalling elements. This is an important finding, offering a prospect to develop
plants tolerant to multiple forms of stress, an important attribute and a major breeding goal inlight of a myriad of abiotic stresses.

80

Chapter 5

Summary81

Salt stress is a major abiotic stress globally, affecting over 800 million ha of land. Depending
on soil pH, salt stress can be categorized into neutral salt stress, popularly known as “salinity
stress” and saline-alkaline stress, also known as “alkali” stress. Over the years, salinity stresshas been extensively studied, hence physiological and molecular mechanisms underpinningplants’ responses to salinity stress have been thoroughly elucidated. Contrarily, relatively lessis known regarding how saline-alkalinity confronts plant growth. Meanwhile, physiologicalmechanisms regulating plants’ resistance under saline-alkaline stress remain hugely
conjectural. Rice (Oryza sativa L.) represents a crop of prominent worldwide food value in
addition to being a key model research crop, yet known for its reputation of extreme sensitivity
to salt stress. This study was aimed at unravelling comparative physiological mechanisms inwhich saline-alkalinity confronts growth and physiology of rice plants. Furthermore, the study
explored the potential of acclimation to different stress components in developing crosstolerance to saline-alkaline stress.

In order to evaluate comparative physiological responses of rice to saline-alkalinity, 21 day oldseedlings of rice cultivar Hinohikari were subjected to control conditions (0 mM Na+, pH 5.5)
neutral salinity stress (50 mM Na+, pH 5.5), moderate and severe alkalinity (0 mM Na+, pH 7.5
and 8.5 respectively) and moderate and severe saline-alkalinity (50 mM Na+, pH 7.5 and 8.5
respectively) for 16 days. Time course changes in growth were monitored every 4 days. The
study revealed that saline-alkaline stress was the most growth limiting, with growth nearlyceasing on the fourth day of stress imposition, whereas under salinity stress growth decreases
were only apparent from the 12th day. Furthermore, at the same growth media pH, solealkalinity was less detrimental relative to saline-alkalinity, but more lethal than salinity stress.
Therefore, effects of rhizospheric Na+ are aggravated by growth media pH, rendering salinealkalinity more harmful on growth than sole salinity and sole alkalinity.

82

Next, comparative physiology underlying saline-alkaline damage on rice seedlings wasinvestigated. Plant grown under both alkaline and saline-alkaline conditions was characterized
by chlorosis, which was confirmed to have resulted from Fe deficiency, particularly in younger
leaves owing to immobile nature of Fe. In older leaves, alkaline stressed plants exhibited
healthier leaves, whereas saline-alkaline stressed plants were severely chlorotic and nearlydying off. This chlorosis was ascribed to leaf blade Na+ toxicity, which was inordinately highin saline-alkaline stressed plants, resulting into premature senescence of older leaves.
Additionally, saline-alkaline stress incited K+ deficiency predominantly in roots, due totremendous root K+ leakage, thus, while resistance to saline-alkaline stress may be complex
and multigenetic, ability to regulate root K+ loss may be a valuable trait. Furthermore, it wasobserved that under saline-alkaline stress, leaf blade membrane integrity was substantiallycompromised, due to toxic accumulation of Na+ in leaf blades, and resultant oxidative stress asshown by high accumulation of H2O2 in leaf blades. Hence, it was concluded that at the used
experimental conditions, rice plants are more sensitive to high growth media pH, and thissensitivity is aggravated under mixed saline-alkaline conditions. Primarily, hyperaccumulationof Na+ in leaf blades, accelerated root K+ deficiency due to root K+ leakage, Fe deficiency inyounger leaves, and oxidative stress mediated membrane damage were crucial causes ofseverity under saline-alkalinity.

Premised on the these findings, the study further explored the potential of acclimation indeveloping cross-tolerance to saline-alkalinity. Rice plants were pretreated (PT) with three
concentrations of each of NaCl, NaHCO2, and H2O2 for one week, then subjected to 50 mM
Na+ at pH 8.25 for 14 days and investigated growth and physiological responses relative to
non-pretreated (NPT) plants. Acclimation to 10 mM NaCl (salinity) and 10 μM H 2O2

83

(oxidative stress) were the most effective, hence were the focus for further investigation. ThesePTs developed cross-tolerance to saline-alkaline stress, in addition to promoting quicker
recovery and resumption of growth upon conclusion of stress. Physiologically, both PTs
noticeably maintained a lower growth medium pH, employing a growth medium acidification
strategy which was not further investigated. The lower growth medium pH also facilitated Fe
acquisition, whose uptake is else constrained under high pH. On ionic relations, NaCl
pretreated plants accumulated much lower Na+ in leaf blades by enhancing root Na+ efflux. In
H2O2 pretreated plants, despite higher root Na+ efflux, leaf blade Na+ accumulation was not
reduced. However, experiments on Na+ uptake in excised leaf blades showed lower
accumulation of Na+. These results may suggest that the high leaf blade Na+ accumulation in
H2O2 pretreated plants emanated from processes relating to either uptake by roots and/ or
resultant transport to the shoots. That notwithstanding, deleterious effects of Na+ were ameliorated
and leaf photosynthetic competency was preserved by both H2O2 and NaCl PTs. Additionally, bothPTs were unable to lower saline-alkaline – triggered root K+ leakage, but efficiently reduced
oxidative stress – triggered membrane damage and lipid peroxidation, and improved cell
viability by constituting a complete shield of antioxidant enzymes SOD, CAT, APX and GR,
establishing an ultimate equilibrium between ROS generation and scavenging.

These findings propose a possible crosstalk between salinity stress, oxidative stress and salinealkaline stress, such that exposure to salinity stress or oxidative stress develops cross-tolerance
to saline-alkaline stress. This is an important finding particularly in light of a myriad of abiotic
stresses, offering a prospect for breeding plants resistant to a range of abiotic stresses. Besides,
upon elimination of stress and resumption of normal growth conditions, pretreated plants,
particularly those pretreated with H2O2 quickly resumed growth and physiologically recovered,a crucial attribute especially under recurrent abiotic stresses with some prolonged inter stress84

recovery periods. In order to provide a fairly broad picture of the mechanism of cross-tolerance,
this study recommends a further exploration of molecular and perhaps epigenetic marks
underpinning cross-tolerance, and an investigation of overlapping signalling elements between
salinity, oxidative and saline-alkaline stress.

Box 5.1. Key summary points of the study
Key summary points of the studyx

The rice cultivar Hinohikari is extremely sensitive to high pH, and its sensitivity is aggravatedunder saline-alkalinity than sole salinity stress.

x

Saline-alkalinity promotes Fe deficiency chlorosis in younger leaf blades and Na + toxicity
induced senescence in older leaf blades.

xHigh pH rhizosphere accelerates above ground organ Na+ accumulation relative to same molar
growth medium Na+ under neutral rhizospheric pH.

xSaline-alkaline stress incites root K+ deficiency through excessive root K+ leakagexMembrane damage in plant leaves through excessive accumulation of ROS was also an important
effect of saline-alkaline stress in rice plants.

x

Acclimation to 10 mM NaCl and 10 μM H2O2 develops cross-tolerance to saline-alkalinity by;Supressing rise in growth medium pH
Sustaining a stable acquisition of Fe and other micronutrients
Enhancing uptake of K and P
Regulating generation of ROS via enhanced antioxidant enzyme activities of SOD, CAT,APX and GR

85

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Acknowledgements
I thank the Almighty God for the gift of healthy life, wisdom and talents. To my academic
supervisor, Associate Prof. Dr. Akihiro Ueda of Plant Nutritional Physiology Laboratory, thankyou so much for offering me with the opportunity to study under your guidance. Your
mentoring, advices, teaching and guidance have shaped me into a better scientist. Further
gratitude goes to my co-advisors Prof. Rumi Tominaga, Prof. Jun Wasaki, Associate Prof.
Toshinori Nagaoka, and Prof. Sakae Agarie for always being there to offer their academic
guidance towards my doctoral thesis. To Prof. Hirofumi Saneoka, thank you for your academic
mentoring and guidance and always making my stay in the laboratory more comfortable. Also,
I sincerely thank Ms. Himiko Koi of the student support office of graduate school of Integrated
Sciences for Life, and all members of the Laboratory of Plant Nutritional Physiology for theirsupport in various day to day laboratory and personal activities. Further gratitude goes to myfamily, my late father Mr. Maganizo Phillip Kamanga, to whom this thesis is dedicated, mymother Mrs. Gertrude Thabu Kamanga, my wife Ethel N. Kamanga, and all the family andfriends. Finally, to the People of Japan, through the Ministry of Education, Culture, Sports,
Science and Technology (MEXT) for funding my doctoral studies.

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