Received: 2 December 2023 | Revised: 28 March 2024 | Accepted: 30 April 2024

DOI: 10.1111/pce.14943

OR I G I NA L A R T I C L E

Salt tolerance in mungbean is associated with controlling
Na and Cl transport across roots, regulating Na and Cl
accumulation in chloroplasts and maintaining high K in root
and leaf mesophyll cells

Md Shahin Iqbal1,2,3 | Peta L. Clode4,5 | Al Imran Malik1,2,6 |

William Erskine1,2 | Lukasz Kotula2

1Center for Plant Genetics and Breeding, The UWA School of Agriculture and Environment, The University of Western Australia, Perth, Western Australia, Australia

2The UWA Institute of Agriculture, The University of Western Australia, Perth, Western Australia, Australia

3Pulses Research Center, Bangladesh Agricultural Research Institute, Ishurdi, Bangladesh

4Centre for Microscopy, Characterisation and Analysis, The University of Western Australia, Perth, Western Australia, Australia

5School of Biological Sciences, The University of Western Australia, Perth, Western Australia, Australia

6International Center for Tropical Agriculture (CIAT‐Asia), Lao People's Democratic Republic Office, Vientiane, Laos

Correspondence

Lukasz Kotula, The UWA Institute of

Agriculture, The University of Western

Australia, Perth, WA 6009, Australia.

Email: lukasz.kotula@uwa.edu.au

Present address

Lukasz Kotula, Department of Primary

Industries and Regional Development,

Perth, WA, Australia.

Funding information

Australian Centre for International Agricultural

Research (ACIAR) Project,

Grant/Award Number: CIM‐2014‐076;
John Allwright Fellowship Award (ACIAR)

Abstract

Salinity tolerance requires coordinated responses encompassing salt exclusion in

roots and tissue/cellular compartmentation of salt in leaves. We investigated the

possible control points for salt ions transport in roots and tissue tolerance to Na+

and Cl– in leaves of two contrasting mungbean genotypes, salt‐tolerant Jade AU and

salt‐sensitive BARI Mung‐6, grown in nonsaline and saline (75mM NaCl) soil. Cryo‐

SEM X‐ray microanalysis was used to determine concentrations of Na, Cl, K, Ca, Mg,

P, and S in various cell types in roots related to the development of apoplastic

barriers, and in leaves related to photosynthetic performance. Jade AU exhibited

superior salt exclusion by accumulating higher [Na] in the inner cortex, endodermis,

and pericycle with reduced [Na] in xylem vessels and accumulating [Cl] in cortical cell

vacuoles compared to BARI Mung‐6. Jade AU maintained higher [K] in root cells

than BARI Mung‐6. In leaves, Jade AU maintained lower [Na] and [Cl] in chloroplasts

and preferentially accumulated [K] in mesophyll cells than BARI Mung‐6, resulting in

higher photosynthetic efficiency. Salinity tolerance in Jade AU was associated with

shoot Na and Cl exclusion, effective regulation of Na and Cl accumulation in

chloroplasts, and maintenance of high K in root and leaf mesophyll cells.

K E YWORD S

cellular distribution, chloride, cryo‐SEM X‐ray microanalysis, endodermis, mungbean, salinity
tolerance, sodium, suberin lamellae

Plant Cell Environ. 2024;47:3638–3653.3638 | wileyonlinelibrary.com/journal/pce

This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any

medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

© 2024 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.

http://orcid.org/0000-0003-1687-0003
http://orcid.org/0000-0002-8688-2117
http://orcid.org/0000-0001-8760-7099
mailto:lukasz.kotula@uwa.edu.au
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1 | INTRODUCTION

Mungbean [Vigna radiata (L.) R. Wilczek var. radiata] is an important

short duration (65–90 days) grain legume rich in proteins, minerals,

and vitamins. It is an essential dietary component in highly populated

and economically challenged South and Southeast Asian countries

(Hou et al., 2019). Mungbean production needs to increase to meet

rising demand (Chauhan & Williams, 2018; Pataczek et al., 2018).

However, the continued expansion of salt‐affected land threatens

mungbean cultivation (Panta et al., 2014). Salinity adversely affects

mungbean germination (Breria et al., 2020), vegetative growth

(Alharby et al., 2019), and reproductive processes (Manasa

et al., 2017). However, variation in salinity tolerance among

mungbean genotypes has been reported (Breria et al., 2020; Liu

et al., 2022; Manasa et al., 2017).

Salinity tolerance is associated with (i) osmotic adjustment by

accumulating Na+ and Cl– in vacuoles and organic solutes in

cytoplasm to the more negative osmotic potential of NaCl in the

root zone; (ii) ion exclusion by restricting entry of salt ions into leaves

and avoiding ion toxicity; (iii) tissue tolerance by compartmentalizing

Na+ and Cl– in vacuoles and maintaining low Na+ and Cl–

concentrations in the cytoplasm and organelles (Munns &

Tester, 2008). In mungbean, an osmotic treatment of −0.43MPa

(equivalent to 100mM NaCl) and 100mM Na+ salts (without Cl–)

affected growth and yield less than the treatment of 100mM NaCl

and Cl– salts (without Na+), indicating that the adverse effects of

salinity are predominantly due to Cl– toxicity in leaf tissue (Le

et al., 2021). Moreover, mungbean maintained lower Na+ in leaves

than roots and accumulated more Cl– in leaves than roots (Le

et al., 2021). The lower leaf Na+ might be due to the interception of

Na+ in roots and its reduced transport to shoots, avoiding leaf Na+

accumulation and toxicity (Tester & Davenport, 2003). In contrast,

high leaf Cl– concentrations could exceed the storage capacity of

vacuoles resulting in Cl– entry and accumulation in cytoplasm and

organelles, such as chloroplasts, causing damage (Oi et al., 2022).

The present study assessed possible control points for salt ions

transport in roots and potential tissue tolerance to Na+ and Cl– in

mungbean leaves. Salt exclusion from leaves depends on several

transport processes in roots. Transport of solutes across roots can

occur through apoplastic or cell‐to‐cell (transmembrane and sym-

plastic) pathways arranged in parallel (Steudle, 2000). Sodium and

chloride can enter the root at the epidermis via channels/transporters

and move symplastically towards the xylem (Geilfus, 2018;

Kronzucker & Britto, 2011; Zhao et al., 2020). The amount of Na+

and Cl– reaching the xylem would depend on both the influx rate and

efflux back into the soil solution (Teakle & Tyerman, 2010; Tester &

Davenport, 2003). Additionally, Na+ or Cl– could be stored in

vacuoles of cortical cells (Wu, Shabala, et al., 2018; Zhang et al., 2021).

The amount of Na+ and Cl– reaching shoots would depend on Na+

and Cl– loading into the xylem and Na+ retrieval from the xylem

(Geilfus, 2018; Zhao et al., 2020). Several studies have reported a

high [Na] or [Cl] in pericycle or xylem parenchyma, suggesting that

these cells may play a role in controlling net Na+ or Cl– loading into

the xylem (Kotula et al., 2015a; Läuchli et al., 2008; Storey

et al., 2003).

The movement of Na+ and Cl– in the apoplast is regulated by the

development of apoplastic barriers in the endodermis, such as

Casparian bands and suberin lamellae (Cui et al., 2021). Casparian

bands located in the radial and transverse cell walls of endodermis

(stage I endodermis) restrict the bypass flow of water and ions into

the xylem (Ranathunge & Schreiber, 2011). Suberin lamellae

deposited between primary cell walls and the plasma membrane

cover the entire inner surface of endodermal cells (stage II

endodermis), isolating the endodermal protoplast from the apoplast

and thus preventing solute movement from the apoplastic space

(Ranathunge & Schreiber, 2011). Ions can only be transported to

xylem through the apoplast in young root zones, where Casparian

bands and suberin lamellae have not yet fully developed. Alterna-

tively, the emergence of lateral primordia that disrupts the continuity

of the endodermis can establish leakage sites between cortex and

stele (Krishnamurthy et al., 2009). Most studies of ion transport in

roots have been on plants grown hydroponically, however,

Krishnamurthy et al. (2009) reported less Na+ transport to shoots

in soil‐grown than hydroponically‐grown rice (Oryza sativa L.) plants.

Thus, a more detailed study is needed to explore how roots control

Na+ and Cl– transport to shoots in soil‐grown plants.

Tissue tolerance of salt ions is generally derived from the ability

of cells to compartmentalize Na+ and Cl– in vacuoles and to maintain

low Na+ and Cl– in the cytoplasm and organelles, like chloroplasts

(Bose et al., 2017; Munns et al., 2016; Oi et al., 2022). Both Na+ and

Cl– can also be partitioned between different cell types within leaves

(James et al., 2006; Kotula et al., 2019; Le et al., 2023; Oi et al., 2022).

Preferential accumulation of Na in epidermal cells and lower [Na] in

photosynthetically active mesophyll cells contributed to maintaining

photosynthesis and salt tolerance in chickpea (Cicer arietinum L.;

Kotula et al., 2019) and soybean (Glycine max L.; Le et al., 2023). In

both these species, Cl accumulation in mesophyll cells did not affect

photosynthesis. Similarly, Cl accumulation in mesophyll cells did not

affect photosynthesis in barley (Hordeum vulgare L.; Fricke et al., 1996;

James et al., 2006). Recently, Oi et al. (2022) showed that halophytic

C4 Rhodes grass (Chloris gayana L.) maintained lower [Na] and [Cl] in

chloroplasts of bundle sheath and mesophyll cells than their vacuoles

under 200mM NaCl. In contrast, salt‐sensitive common bean

(Phaseolus vulgaris L.) grown under 150mM NaCl accumulated

250–300mM Cl in cell vacuoles and chloroplasts, indicating

inefficient intracellular ion compartmentation (Seemann &

Critchley, 1985). Data on Na were not presented in that study due

to background noise. Thus, potential cellular and intracellular

compartmentation of salt ions related to photosynthesis warrants

further investigation, especially in agronomically important crop

species, such as mungbean.

The objectives of this mungbean study were to (i) investigate the

radial distribution and concentration of key elements in various root

cell types related to the development of apoplastic barriers in

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endodermis, and (ii) investigate the distribution and concentrations of

key elements in various cell types, vacuoles, and chloroplasts of

leaves and their relation to photosynthetic performance. We used

quantitative X‐ray microanalysis to determine cell‐specific concen-

trations of different elements across roots and leaves of two

contrasting mungbean genotypes grown in nonsaline control and

75mM NaCl treated soil. This is the first study to investigate

elemental distributions and concentrations in various cell types in

roots and leaves from the same plants and the first investigation of

radial elemental distribution in root cells related to apoplastic barriers

in salinized soil grown roots. This is also the first study directly

comparing elemental concentrations in chloroplasts and vacuoles and

their relation to leaf anatomical changes and photosynthetic

performances in grain legumes under salinity stress.

2 | MATERIALS AND METHODS

2.1 | Plant materials and growth conditions

Two contrasting mungbean varieties—salt‐sensitive BARI Mung‐6

(BM6) and salt‐tolerant Jade AU (Jade), and two contrasting

genotypes—VO2211 (putative salt‐tolerant) and VO1317 (putative

salt‐sensitive)—obtained from the Department of Agriculture and

Fisheries, Queensland were used based on a preliminary study with

salinized soil under glasshouse conditions (data not shown).

The experiment was conducted in a controlled temperature

glasshouse at The University of Western Australia, Perth, WA,

Australia (31°57'S, 115°47'E) at 30 ± 3/24 ± 2°C day/night tempera-

tures with 11–13 h day length and maximum PAR of

1400–1650 μmol m−2 s−1. Plants were grown in free‐draining plastic

pots (180mm in diameter, 180mm high) lined with waterproof

polybags and filled with 3.15 kg of 2 mm‐sieved oven‐dried red‐

brown sandy clay loam soil (pH = 8.79, electrical conductivity

0.28 dSm−1 in 1:5 soil:water extract) collected from Mukinbudin,

Western Australia (Kotula et al., 2015b). The water content (w/w) at

field capacity was 19.7%. The soil was fertilized, according to Kotula

et al. (2015b). Nutrients were added to the soil as (g kg–1 soil): 0.129

K2SO4, 0.225 CaSO4.2H2O, 0.191 KH2PO4, 0.025 MgSO4.7H2O, and

0.70mL kg–1 soil of half‐strength Hoagland solution micronutrients.

The nutrients were added with deionized water in a sufficient

solution volume to wet the soil to 80% field capacity before sowing.

Seeds were surface sterilized with 1% commercial bleach (active

ingredients NaOCl 40mg L–1) for 1 min, rinsed with deionized water

for 4 min and then pre‐germinated for 12 h in darkness in Petri dishes

containing filter paper moistened with deionized water. For each pot,

six seeds were sown at 30mm depth along with peat‐based Group I

mungbean Rhizobium (Bradyrhizobium spp.) strain CB 1015 (5.25 g

pot–1; Group N, New Edge Microbials Pty Ltd, Albury, New South

Wales, Australia). Pots were watered with deionized water to

maintain 80% field capacity every alternate day. Seedlings were

thinned to three per pot 7 days after sowing.

2.2 | Treatments and sampling procedure

Two treatments were applied: 0mM NaCl (nonsaline control) and

75mM NaCl. The 75mM NaCl treatment was selected based on a

preliminary experiment in the glasshouse with plants grown in soil

with five different NaCl levels (0, 25, 50, 75 and 100mM, data not

shown), with the 75mM NaCl treatment exhibited the largest

genotypic variation in salinity tolerance. The experiment was set up

in a completely randomized block design with two factors (4

genotypes × 2 treatments) and four replications. Pots were re‐

randomized weekly to reduce positional effects in the glasshouse.

Salinity treatments were imposed 15 days after sowing (DAS) when

most plants were approximately at the V1 stage (unifoliate leaves

attached to the first node are fully expanded and flat as the first

trifoliate leaf attached to the upper node starts to unroll). The NaCl

treatments were stepped up by the addition of 25mM NaCl (0.288 g

NaCl/kg soil) daily until the final concentration of 75mM had been

achieved. NaCl was applied to pots in a sufficient solution volume to

wet the soil to 80% field capacity and the equivalent volume of

deionized water was added to nonsaline control pots. The plants

were harvested at the vegetative stage (23 days after the first

addition of 25mM NaCl treatment) when distinct foliar injury

symptoms appeared. Leaf gas exchange measurements and leaf and

root samplings (for tissue ion analysis, morphology and anatomical

measurements and cell‐specific element analysis) were collected just

before harvesting (described below). Plant parts were separated into

green leaves (laminas), dead leaves, stems (with petioles), and roots.

Roots were washed carefully with flowing tap water on a sieve with a

2mm mesh size and blotted dry with a paper towel. Plant parts were

dried at 65°C for 72 h, and dry masses were recorded.

2.3 | Leaf gas exchange and chlorophyll
fluorescence measurements

Leaf gas exchange measurements were conducted on the youngest

fully expanded leaves (YFEL) of BM6 and Jade at 23 days after the

first 25mM NaCl application (see above) using a LI‐6400 gas

exchange system (LI‐COR Biosciences Inc.). Net photosynthetic rate

(Pn), transpiration rate (T), stomatal conductance (gs), and intercellular

CO2 concentration (Ci) were measured between 10:00 AM and

14:00 PM (data were collected in replications to minimize any

confounding influence from the timing of the measurements) at

photosynthetically active radiation of 1500 µmol photons m−2 s−1,

leaf chamber temperature of 28°C, 60%–70% relative humidity, and

CO2 concentrations of 400 μmol mol−1 (ambient) and 800 μmol mol−1

(elevated; to check the assimilation rate by removing any stomatal

limitation). Chlorophyll fluorescence or maximum quantum efficiency

of photosystem II (Fv/Fm) was determined on the same day using a

Handy PEA (Plant Efficiency Analyser) continuous excitation chloro-

phyll fluorimeter (Hansatech Instruments Ltd.) at a flash intensity of

3500 μmolm−2 s−1. Before measurements, leaflets were dark‐adapted

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for 30min using leaf clips. Leaf chlorophyll concentration was

measured on the same day using a SPAD meter (Minolta). All three

measurements were taken on the same YFEL.

2.4 | Total tissue ion analysis

Three leaflets from the YFEL (one sample per plant per pot) were

weighed and ground to a fine powder (2010 Geno/Grinder®, SPEX

SamplePrep) and extracted (100mg subsample) in 10mL of 0.5M HNO3

by shaking for 72 h in darkness at room temperature as described in

Munns et al. (2010). The extracts were diluted in Milli Q water as

required and analysed for Na+ and K+ using a flame photometer (PFP7;

Jenway) and for Cl– using a chloridometer (Model 50CL, SLAMED ING,

GmbH, Frankfurt, Germany). The reliability of the analyses was

confirmed by taking a reference tissue (broccoli, ASPAC no. 85) with

known ion concentrations through the same procedures.

2.5 | Morphology and anatomy of the YFEL

Leaf area was measured from the photographs of YFEL of BM6 and

Jade using Image J software (National Institutes of Health). Leaf dry

mass per area was calculated as the ratio of YFEL dry mass to leaf

area. Leaf water content was calculated using the fresh and dry mass

of the YFEL, and leaf succulence index (LS) was calculated as

LS = (fresh mass–dry mass)/area (Mantovani, 1999).

Leaflet lamina segments (5 × 5mm) were excised from the YFEL

of BM6 and Jade. Leaf segments were fixed with 2.5% glutar-

aldehyde in 0.1M phosphate buffer, pH 7, for 24 h at room

temperature and then stored at 4°C. After rinsing in deionized water,

fixed leaf segments were embedded in 5% agar in warm water. Cross‐

sections were prepared by cutting agarose blocks containing a leaf

segment using a vibrating microtome (Vibrotome 3000 Sectioning

System, The Vibrotome Company), removing adherent agar with a

clearing solution of 85%–92% (v/v) lactic acid saturated with chloral

hydrate for 1 h at 70°C and rinsing several times with deionised

water (Kotula et al., 2021). Leaf cross‐sections were stained for 1 min

in 0.05% (w/v) toluidine blue O in benzoate buffer at pH 4, washed

and viewed under white light using a microscope (Zeiss Axioscope2

plus; Carl Zeiss Microscopy GmbH). All images were photographed

with a Zeiss AxioCam Digital Camera (Carl Zeiss Microscopy GmbH,).

Leaf thickness and mesophyll thickness (without epidermis) were

measured in micrographs using Image J software with 13–19 cross‐

sections collected from three leaflets from three replicate plants. The

numbers of palisade mesophyll and spongy mesophyll cells per unit

area were counted. The area of mesophyll cells was also measured.

2.6 | Root anatomy

Lateral root segments (10mm long) were excised at 20 and 50mm

from the root apex of the BM6 and Jade. Root cross‐sections were

prepared, as described above for leaves. To visualize suberin lamellae,

root cross‐sections were stained for 1 h with 0.01% (w/v) fluorol

yellow 088 in polyethylene glycol‐glycerol (Brundrett et al., 1991).

The suberin lamellae in root cell walls were recognized by bright

yellow fluorescence under UV light (excitation G365, emission

LP397; Zeiss Axioscope2 plus; Carl Zeiss Microscopy GmbH). All

images were photographed with a Zeiss AxioCam Digital Camera

(Carl Zeiss Microscopy GmbH).

2.7 | Cell‐specific elemental analysis by cryo‐SEM
X‐ray microanalysis

Lateral root segments (10mm long) were excised at 20 and 50mm

from the root apex and leaflet lamina segments (2 × 3mm) were

excised from the middle part of the YFEL avoiding the central vein

from transpiring plants of BM6 and Jade (between 10.00 AM and

3.00 PM). Leaf and root segments were placed on an aluminium

grooved pin with optimal cutting temperature compound and

immediately plunge‐frozen into liquid N, immobilizing and preserving

cellular ions (Kotula et al., 2021; Oi et al., 2022). Samples were stored

in liquid N until required.

Perfectly flat transverse surfaces of frozen‐hydrated leaf and

root samples were prepared by stepwise cryo‐planing (1, 0.5, 0.25

and 0.1 micron) with a glass knife on a cryo‐microtome (FC7; Leica

Microsystems). Samples were transferred in the transfer shuttle

directly from the cryo‐microtome to the cryo‐preparation system

(ACE600; Leica Microsystems), where they were coated with 20 nm

Cr, without sublimation. Coated samples were transferred under

vacuum in the transfer shuttle to a field emission scanning electron

microscope (JSM‐IT800HR; JEOL) fitted with a cryo‐stage (VCT500,

Leica Microsystems) and an Ultim‐Max170 X‐ray detector interfaced

to AZtec software (Oxford Instruments). Samples were analysed at

–155°C, 15 kV, and a 1–2 nA beam current (measured using a probe

current detector in the column). Before each sample analysis, the

instrument was calibrated using a pure copper standard. Elemental

maps were acquired at 512 pixel resolution, for >1500 frames with a

dwell time of 10 μs per pixel. Drift correction and pulse pile‐up

correction (empirically determined to correct for O interference of Na

quantification was activated (Marshall, 2017).

Quantitative numerical data were extracted from regions of

interest (i.e. single cells) drawn on the element maps, with individual

spectra from each pixel summed and processed to yield concentra-

tion data using the AZtec software with inbuilt standards (Kotula

et al., 2021; Marshall, 2017; Oi et al., 2022). Quantitative elemental

analyses in root cells at 20 and 50mm from the root apex were

conducted on the area occupied by vacuole of the outer cortex,

middle cortex, endodermis, pericycle, and xylem vessels (epidermis

was excluded from the elemental analysis due to damage to the outer

layer of most soil‐growing roots), with 10–55 spectra collected from

each cell type from three different root segments from three

different replicate plants. Leaf cells analyzed were in the area

occupied by vacuole of the upper epidermis (UE), palisade mesophyll

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(PM), spongy mesophyll (SM), and lower epidermis (LE), with 10–50

spectra collected from each cell type from three different leaflets

from three different replicate plants. In addition, high magnification

analyses (1000 frames only) were conducted to undertake elemental

analyses of chloroplasts of PM and SM cells. Concentration data were

generated as weight % and converted to mmol kg−1 (mM) on a tissue

wet‐weight basis.

2.8 | Statistical analyses

Statistical analyses were performed using Genstat Software 21th

Edition (VSN International Ltd.) and R software (R‐4.0.3). Two‐way

ANOVA was used to assess the effects of genotypes, treatments, and

genotype × treatment interaction. Means were compared for signifi-

cant differences using LSD at 5% probability level. Differences in

elemental concentrations across cell types between two genotypes

were tested using general linear mixed‐effect models, with individual

plants as the random effects (Hayes et al., 2018; Pinheiro &

Bates, 2006). The residuals of each model were visually inspected

for heteroscedastically, and different variance structures and error

distributions were tested, and an appropriate model was considered.

3 | RESULTS

Growth differences between the genotypes and tissue ion (Na+, Cl–, K+

and K+/Na+) data for YFEL are presented in Supporting Information:

Supporting Results and Figures S1 and S2. Here, we focus on specific

traits of ion transport across roots and tissue tolerance, differentiating

the salt‐tolerant Jade from salt‐sensitive BM6.

3.1 | Radial Na, Cl and K concentrations in various
cell types of lateral roots

3.1.1 | Sodium – 20mm behind the apex

Sodium concentrations [Na] in the cells of nonsaline roots were

similar in BM6 and Jade (Figure 1a). In both genotypes, [Na] increased

across the cortex from 49mM in the outer cortex, reaching the

highest value in the inner pericycle (106mM) before declining to

44mM in the xylem vessels. The 75mM NaCl treatment increased

[Na] in all cell types for both genotypes (except for xylem vessels in

Jade). In BM6, [Na] increased from 119mM in the outer cortex (2.3‐

fold increase compared to nonsaline control) to 180mM in the inner

cortex (1.9‐fold increase compared to nonsaline control), then

declined towards the xylem vessels (85 mM). In Jade, [Na] increased

from 183mM in the outer and middle cortex, reaching the highest

values in the endodermis (283mM, 3.2‐fold increase compared to

nonsaline control), then decreased across the pericycle to the lowest

values in the xylem vessel (59 mM).

3.1.2 | Sodium – 50mm behind the apex

The [Na] in roots of nonsaline BM6 was similar in cells of the outer,

middle, and inner cortex (~127mM), decreasing in the endodermis

(84mM) before increasing to 135mM in the pericycle and declining to

85mM in the xylem vessels (Figure 1b). In nonsaline Jade, [Na] tended to

increase from 97mM in the outer cortex to 133mM in the endodermis,

decreasing across the pericycle to the lowest value in the xylem vessels

(27mM). The saline roots of BM6 had a similar Na profile to nonsaline

roots, but [Na] in all cell types, except xylem vessels (68mM), was 1.5‐

fold higher on average in the saline roots. In contrast, the saline roots of

Jade had 60% lower [Na] in the middle cortex, similar in the outer and

inner cortex, endodermis and xylem vessels, but 1.7‐fold higher in the

pericycle compared to nonsaline roots.

3.1.3 | Chloride – 20mm behind the apex

In nonsaline BM6, chloride concentration [Cl] increased from 88mM

in the outer cortex to 113mM in the middle cortex, then declined

towards the xylem vessels (38mM) (Figure 1c). In nonsaline Jade, [Cl]

was more uniform across roots ranging from 43mM in the inner

cortex to ~70mM in the outer cortex and endodermis, decreasing to

25mM in the xylem vessels. The 75mM NaCl treatment increased

[Cl] in all cell types in both genotypes. The saline roots of BM6 had a

similar Cl profile to nonsaline roots, but [Cl] in all cell types was 1.7‐

fold higher on average in saline roots. Similarly in Jade, the 75mM

NaCl treatment increased [Cl] in all cell types; however, Cl

distribution pattern was less uniform than in nonsaline roots, with

[Cl] decreasing markedly from 157mM in the outer and middle

cortex (2.4‐fold increase compared to nonsaline control) to 72mM in

the inner cortex, increasing to 129mM in the endodermis and

decreasing across the pericycle (71 mM) and xylem vessels (88mM,

3.5‐fold increase compared to nonsaline control).

3.1.4 | Chloride – 50mm behind the apex

In nonsaline controls, Jade had higher [Cl] in all cells than BM6

(Figure 1d). In roots of nonsaline BM6, the [Cl] was highest in the

outer and middle cortex (average 93mM), was lower in cells from the

inner cortex inwards and in the xylem vessels (average 37mM). In

Jade, [Cl] decreased from 117mM in the outer cortex to 81mM in

the inner cortex, then increased to the highest values in the

endodermis (142mm) before decreasing across the pericycle to

the lowest values in the xylem vessels (58mM). For BM6 and Jade,

the 75mM NaCl treatment produced similar Cl profiles to nonsaline

roots. In BM6, the salinity treatment increased [Cl] from 3.1‐fold in

the inner cortex to 1.4‐fold in the xylem vessels compared to

nonsaline controls. In Jade, [Cl] in saline roots was 1.6‐fold higher on

average in cortical cells and 1.2‐fold higher in the endodermis, but

[Cl] was similar to nonsaline controls in pericycle and xylem vessels.

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3.1.5 | Potassium– 20mm behind the apex

In nonsaline BM6, potassium concentration [K] decreased across

the cortex from 146 mM in the outer cortex to 64 mM in the

endodermis, it increased in the pericycle (102 mM) before

declining to the lowest values in the xylem vessels (49 mM)

(Figure 1e). In Jade, [K] was the highest in the outer cortex

(209 mM), was lower in cells from the middle cortex inwards and

in the xylem vessels (average 101 mM). The 75 mM NaCl

treatment decreased [K] in all cell types of BM6 to a similar

extent (average 47% of nonsaline control). In Jade, the 75 mM

NaCl treatment produced a different K profile across roots than

the nonsaline controls, with [K] decreasing from 73 mM (35% of

nonsaline control) in the outer cortex to 49 mM in the middle and

inner cortex (45% of nonsaline control), then markedly increasing

across the endodermis (101 mM, similar to nonsaline control) to

125 mM on average in the pericycle and xylem vessels (1.3‐fold

increase compared to nonsaline control).

F IGURE 1 Cellular concentrations of Na (a, b), Cl (c, d) and K (e, f) in various cell types across roots at 20mm (a, c, e) and 50mm (b, d, f) from
the apex of two mungbean genotypes at the vegetative stage grown in soil with 0 (nonsaline control) and 75mM NaCl treatments imposed on
15‐day‐old plants for 23 days. Elemental concentrations were measured by cryo‐scanning electron microscopy X‐ray microanalysis. The
concentrations in mM (mmol kg−1 water) are per unit fresh weight from fully hydrated, cryo‐fixed cells. Bars are means (n = 10–55 cells measured
for three different roots, each from a different replicate plant), and error bars represent 95% confidence intervals from generalized linear mixed‐
effect models. Three‐way ANOVA was used to compare genotype (G), salinity treatment (T), cell type (C) and genotype × treatment × cell type
(G × T × C) effects. Levels of significance for G × T, G × C, T × C and G × T × C effects are provided (* p < 0.05; ** p < 0.01; *** p < 0.001; n.s.,
nonsignificant). EN, endodermis; IC, inner cortex; MC, middle cortex; OC, outer cortex; PE, pericycle. The epidermis was excluded from the
elemental analysis due to damage to the outer layer of most soil‐grown roots.

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3.1.6 | Potassium – 50mm behind the apex

In nonsaline controls, both genotypes exhibited similar K distribution

profiles across roots but BM6 had higher [K] in the outer, middle, and

inner cortex, and the endodermis, and lower [K] in the xylem vessels

than Jade (Figure 1f). In both genotypes, [K] decreased from the

outer cortex (173mM in BM6 and 118mM in Jade) towards the

endodermis (53 mM in BM6 and 40mM in Jade), increased in

the pericycle (~100mM for both genotypes) before declining in the

xylem vessels (37 mM in BM6 and 84mM in Jade). The 75mM NaCl

treatment severely decreased [K] in all cell types of BM6 (average

15mM in the outer, middle and inner cortex, and endodermis, 36mM

in the pericycle and 18mM in the xylem vessels). Jade under 75mM

NaCl had similar [K] in the outer and middle cortex, 1.9‐fold higher on

average in the inner cortex, endodermis and pericycle, and 44% lower

in the xylem vessels compared to nonsaline roots.

3.2 | Development of suberin lamellae in lateral
roots

At 20mm, roots of BM6 grown in the 0 and 75mM NaCl treatments

showed yellow/green fluorescence in the endodermis, indicating the

presence of suberin in ~70% of all cells (Figure 2). In contrast, roots of

Jade laid down suberin lamellae in ~84% and 91% of endodermal

cells in the nonsaline control and 75mM NaCl treatments,

respectively. Deposition of suberin in the endodermal cells

proceeded along the roots towards the base. At 50mm, nonsaline

roots of the two genotypes had a few passage cells without suberin

located opposite the xylem poles. In the 75mM NaCl treatment, Jade

roots had a strongly suberized complete ring of endodermis, whereas

BM6 roots had 2–3 passage cells without suberin located opposite to

the xylem poles.

3.3 | Elemental concentration in various cell types
or organelles of the lamina of leaflets

3.3.1 | Sodium

For nonsaline control plants, vacuolar [Na] was similar in all cells

(average 8mM) in the two genotypes (Figures 3 and 4a). The 75mM

NaCl treatments increased [Na] in BM6 to similar degrees in all cell

types (average 22mM). In contrast, in Jade grown with 75mM NaCl

[Na] remained low in all cells (average 7mM). The nonsaline BM6 had

1.6‐fold higher [Na] in chloroplasts of mesophyll cells (PM and SM)

(average 13mM) compared to vacuoles (8 mM), whereas in Jade,

chloroplasts and vacuoles had similar [Na] (Figures 5 and 6a). The

75mM treatment increased [Na] in the chloroplasts of palisade and

spongy mesophyll cells to 33mM on average in BM6, while the

chloroplasts [Na] in PM and SM remained low in Jade (average

11mM). Compared to their respective vacuoles, BM6 had similar [Na]

in chloroplasts of PM and SM, but Jade had 1.9‐fold higher [Na] on

average in chloroplasts of mesophyll cells.

F IGURE 2 Cross‐sections of roots of BARI Mung‐6 and Jade AU showing deposition of suberin in the endodermis. Cross‐sections were
taken at 20 and 50mm from the apex of roots grown in 0 (nonsaline control) and 75mM NaCl treatments imposed on 15‐day‐old plants for
23 days. The presence of suberin was detected by yellow‐green fluorescence under UV illumination after staining of cross‐sections with Fluorol
Yellow 088 (indicated by white arrow). Yellow triangle indicates suberin‐free passage cells. Scale bars = 100 μm.

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3.3.2 | Chloride

For nonsaline controls, the two genotypes had similar [Cl] in the

vacuoles of different cells, with 357mM in UE and 316mM on

average in PM, SM and LE (Figures 3 and 4b). The 75mM NaCl

treatment increased vacuolar [Cl] in BM6 by 1.4‐fold on average in

epidermal cells (UE and LE) and 1.7‐fold in mesophyll cells (PM and

SM) compared to nonsaline controls. In contrast, the 75mM NaCl

treatment increased vacuolar [Cl] in Jade by 1.9‐fold in PM and 1.3‐

fold in SM but decreased vacuolar [Cl] to ~55% in UE and LE

compared to nonsaline controls. In nonsaline controls, [Cl] in

chloroplasts of mesophyll cells (PM and SM) was 88mM (32% of

vacuoles) in BM6 and 53mM (19% of vacuoles) in Jade (Figures 5

and 6b). The 75mM NaCl treatment increased chloroplastic [Cl] to

F IGURE 3 Typical quantitative element maps of Na, Cl, and K from cryo‐planed, frozen‐hydrated lamina of leaflets of the youngest fully
expanded leaves of BARI Mung‐6 and Jade AU. Qualitative maps of C are included to show cellular structure. Plants were grown in soil with 0
(nonsaline control) and 75mM NaCl treatments imposed on 15‐day‐old plants for 23 days. Elemental concentrations from different cell types
are summarized in Figure 4. For these maps, the concentrations (in mM) are scaled to best reveal element variations across cell layers and
treatments, with black = 0 (below detection, approximately <5mM) for all maps, and white >30mM for Na, >620mM for Cl, and >450mM for K.
Changes in concentration along the colour scale are linear. Typical cell types are labelled that were used to give cellular element profiles. LE,
lower epidermis; PM, palisade mesophyll; SM, spongy mesophyll; UE, upper epidermis. Scale bar for all images = 100 μm. [Color figure can be
viewed at wileyonlinelibrary.com]

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321mM in PM (3.6‐fold) and 246mM in SM (2.8‐fold) in BM6 and to

~150mM in both PM and SM (2.8‐fold) in Jade, compared to the

nonsaline control. Compared to their respective vacuoles, [Cl] in

chloroplasts of mesophyll cells was ~58% of [Cl] in vacuoles in

BM6 and 33% of [Cl] in vacuoles in Jade.

3.3.3 | Potassium and potassium/sodium ratio

In the nonsaline BM6, vacuolar [K] ranged from 60mM in PM to

165mM in LE. In nonsaline Jade, [K] ranged from 104mM in PM to

401mM in UE (Figures 3 and 4c). The 75mM NaCl treatment

increased vacuolar [K] in BM6 by 2.3‐fold in UE (206mM) and 1.6‐

fold in LE (265mM) but did not affect vacuolar [K] in PM and SM

(average 78mM), compared to the nonsaline control. Similarly, in

Jade, the 75mM NaCl treatment increased vacuolar [K] by 2.5‐fold in

PM (261mM) and 1.1‐fold in SM (172mM) but decreased vacuolar

[K] to 27% in UE (108mM) and 51% in LE (125mM), compared to the

nonsaline control. In nonsaline BM6, chloroplastic [K] ranged from

68mM in PM to 105mM in SM, similar to [K] in their respective

vacuoles (Figures 5 and 6c). In the nonsaline Jade, [K] in chloroplasts

and vacuoles of PM and SM was 123mM on average. The 75mM

NaCl treatment increased chloroplastic [K] by 1.5‐fold in PM and did

not affect [K] in chloroplasts of SM in BM6, whereas in Jade, [K]

remained relatively similar in chloroplasts of PM and SM, compared

to the nonsaline control. Compared to their respective vacuoles, [K]

in BM6 was 1.5‐fold higher in chloroplasts than vacuoles of PM but

similar in SM, whereas in Jade, [K] was 53% of vacuoles in PM but

similar in SM.

Cellular and vacuolar—chloroplastic potassium/sodium ratio (K/Na)

are presented in Figures 3 and 4d, and Figures 5 and 6d, respectively,

and described in Supporting Information: Supporting Results and

Figures S3–S8).

3.4 | Leaf gas exchange, morphology and anatomy

Significant genotype × salinity treatment interactions occurred for net

photosynthetic rate (Pn), transpiration rate (T), and intercellular CO2

concentration (Ci) of the YFEL at ambient CO2 level (400µmolmol−1),

with nonsignificant interactions for stomatal conductance (gs), maximum

quantum efficiency of photosystem II (Fv/Fm), and chlorophyll concen-

tration (Table 1). In the nonsaline control soil, the two genotypes had

similar Pn (average 12.2 µmol CO2 m−2 s−1), T (average 2.0mmol

F IGURE 4 Cellular concentrations of Na (a), Cl (b), K (c) and K/Na (d) in various cell types in the lamina of leaflets of the youngest fully
expanded leaves of BARI Mung‐6 and Jade AU. Plants were grown in soil with 0 (nonsaline control) and 75mM NaCl treatments imposed on
15‐day‐old plants for 23 days. Elemental concentrations were measured by cryo‐scanning electron microscopy X‐ray microanalysis. The
concentrations in mM (mmol kg−1 water) are per unit fresh weight from fully hydrated, cryo‐fixed cells. Bars are means (n = 10–50 cells measured
for three different leaflets from different replicate plants) and error bars represent 95% confidence intervals from generalized linear mixed‐
effect models. Three‐way ANOVA was used to compare genotype (G), salinity treatment (T), cell type (C) and genotype × treatment × cell type
(G × T × C) effects. Levels of significance for G × T, G × C, T × C and G × T × C effects are provided (* p < 0.05; ** p < 0.01; *** p < 0.001; n.s.,
nonsignificant). PM, palisade mesophyll; SM, spongy mesophyll; LE, lower epidermis; UE, upper epidermis.

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H2Om−2 s−1), gs (average 0.12mmol H2Om−2 s−1, Ci (average

194.7 µmol CO2 mol−1) and Fv/Fm (average 0.80) at ambient CO2 level.

For BM6, the 75mM NaCl treatment decreased Pn to 42%, T and gs to

~50% of controls but increased Ci 1.3‐fold and did not affect Fv/Fm. The

75mM NaCl did not significantly affect Pn, T, gs, Ci or Fv/Fm in Jade

relative to the nonsaline controls. Compared to the ambient CO2 level,

increasing the external CO2 level to 800µmolmol−1 doubled Pn and Ci

but did not affect T or gs in either genotype under nonsaline and 75mM

NaCl treatments. In the nonsaline soil, the genotypes did not differ in

chlorophyll concentration, with average SPAD values of 45.8. The

75mM NaCl treatment decreased SPAD values to 68% of controls in

BM6 and 89% of controls in Jade.

The salinity treatment significantly affected leaf area, leaf

succulence, leaf thickness, mesophyll thickness, PM and SM cell area

(Supporting Information: Table S1). BM6 and Jade had similar leaf

area (average 5.3 cm2), leaf dry mass/area ratio (average 9.8 mg

cm−2), and leaf succulence expressed as H2O content per leaf area

(average 23.8 mg H2O cm−2) under nonsaline conditions. Compared

to nonsaline controls, the 75mM NaCl treatment decreased leaf area

to 56% and increased leaf succulence by 1.8‐fold in BM6, whereas in

Jade, leaf area and leaf succulence were unaffected. Leaf dry mass/

area ratio decreased to 83% on average in both genotypes in the

75mM NaCl treatment, compared to the nonsaline control. In the

nonsaline controls, leaf thickness was 0.27mm in BM6 and 0.24mm

in Jade; however, mesophyll thickness (without epidermis) was similar

(average 0.20mm) in both genotypes. The 75mM NaCl treatment

increased leaf and mesophyll thickness in BM6 by 1.2‐fold compared

to the nonsaline control but did not affect leaf or mesophyll thickness

in Jade. The number of PM and SM cells per unit area (mm2) were

165 and 96 in BM6 and 188 and 113 in Jade, respectively, under

nonsaline control conditions. The 75mM NaCl treatment did not

significantly change the number of PM or SM cells per unit area in

either genotype. The two genotypes had similar PM cell area (average

527 μm2) and SM cell area (average 373 μm2) in nonsaline controls.

The 75mM NaCl treatment increased PM and SM cell areas in BM6

by 1.5‐fold compared to nonsaline controls, with no effect in Jade.

4 | DISCUSSION

4.1 | Controlling ion transport across roots

Ion transport across root cells is regulated by exo‐ or endodermal

apoplastic barriers, vacuolar sequestration or ion retrieval from the

F IGURE 5 Magnified views of quantitative element maps of Na, Cl, and K from cryo‐planed, frozen‐hydrated youngest fully expanded leaves
of BARI Mung‐6 and Jade AU in areas occupied by chloroplasts or vacuoles in palisade mesophyll (left panel) and spongy mesophyll (right panel).
Qualitative maps of C are included to show intracellular structure. Plants were grown in soil with 0 (nonsaline control) and 75mM NaCl
treatments imposed on 15‐day‐old plants for 23 days. Elemental concentrations from different cell types are summarized in Figure 6. For these
maps, the concentrations (in mM) are scaled to best reveal element variations across cell layers and treatments, with black = 0 (below detection,
approximately <5mM) for all maps, and white >50mM for Na, >620mM for Cl, and >325mM for K. The changes in concentration along the
colour scale are linear. Yellow triangles indicate chloroplasts, and white arrows indicate vacuoles used to give organelle element profiles. Scale
bar for all images = 25 μm. [Color figure can be viewed at wileyonlinelibrary.com]

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xylem, all of which contribute to ion exclusion from shoots

(Geilfus, 2018; Tester and Davenport, 2003). In this study, we

observed marked differences in [Na] at 20 and 50mm behind the

root apex of BM6 and Jade grown in saline soil. At 20mm behind

the root apex, salt‐tolerant Jade accumulated large amounts of

Na in the endodermis (283mM) with a decreasing concentration

gradient towards the outer cortex and xylem vessels. In salt‐sensitive

BM6, the highest [Na] was observed in the inner cortex with lower

[Na] in the middle and outer cortex, endodermis and pericycle. At

20mm from the apex, both genotypes developed suberin lamellae in

endodermal cells, but a few passage cells lacking suberin were

located in front of xylem poles in BM6. Mungbean, like other

Fabaceae, does not form suberized exodermis even under stressful

conditions (e.g. chickpea, soybean, common bean; Bramley

et al., 2009; Hartung et al., 2002; Perumalla et al., 1990; Ranathunge

et al., 2008); thus a suberized endodermis would provide the only

barrier for apoplastic bypass flow of ions in mungbean roots.

Casparian bands would divert the apoplastic flow of Na into the

plasma membrane of endodermal cells. Suberin lamellae that cover

the entire surface of endodermal cells would restrict Na import into

the symplast of endodermal cells (Barberon and Geldner, 2014). The

excess Na blocked by the endodermis would accumulate in vacuoles

of cortical cells exterior to the endodermis, and [Na] would decrease

towards the epidermis (Läuchli et al., 2008) and be low on the inner

side of endodermis. In contrast, Na accumulated to high concentra-

tions in endodermis and pericycle in both genotypes. These results

indicate that the role of suberized endodermis in preventing Na

uptake is minimal in mungbean roots.

Endodermal and pericycle cells contained high Na concentrations

at 20mm, as did the pericycle at 50mm from the apex in Jade. The

accumulation of ions such as Na, Cl or Ca has been reported in the

pericycle in roots of barley (Kotula et al., 2015a), durum wheat

(Triticum turgidum ssp. Durum; Läuchli et al., 2008), grapevine (Vitis

vinifera L.; Storey et al., 2003), and Australian native Proteacae

species (Kotula et al., 2021). Läuchli et al. (2008) suggested that the

pericycle may be an important control point limiting Na transport into

the xylem. However, given its low storage capacity, it is unclear how

this could be achieved. The pericycle is connected to the endodermis

F IGURE 6 Concentrations of Na (a), Cl (b), K (c) and K/Na (d) in vacuoles and chloroplasts of palisade mesophyll and spongy mesophyll cells
in the lamina of leaflets of the youngest fully expanded leaves of BARI Mung‐6 and Jade AU grown in soil with 0 (nonsaline control) and 75mM
NaCl treatments imposed on 15‐day‐old plants for 23 days. Elemental concentrations were measured by cryo‐scanning electron microscopy
X‐ray microanalysis. The concentrations in mM (mmol kg−1 water) are per unit fresh weight from fully hydrated, cryo‐fixed cells. Bars are means
(n = 10–50 cells measured for three different leaflets from different replicate plants) and error bars represent 95% confidence intervals from
generalized linear mixed‐effect models. Three‐way ANOVA was used to compare genotype (G), salinity treatment (T), cell type (C) and
genotype × treatment × cell type (G × T × C) effects. Levels of significance for G × T, G × C, T × C and G × T × C effects are provided (* p < 0.05;
** p < 0.01; *** p < 0.001; n.s., nonsignificant). PM Vac, palisade mesophyll vacuole; PM Chl, palisade mesophyll chloroplast; SM Vac, spongy
mesophyll vacuole; SM Chl, spongy mesophyll chloroplast.

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with plasmodesmata, and suberin lamellae do not block plasmodes-

matal connections (Clarkson, 1993) or affect the symplastic route

(Barberon et al., 2016). A suberized endodermis would resist

apoplastic ion movement towards the xylem and divert ions onto

the symplastic pathway (Geldner, 2013). The release of ions from the

symplast into the stele apoplast would be controlled by transport

proteins in the plasma membrane (Enstone et al., 2002; Maathuis

et al., 2014). However, how and where control mechanisms operate

remains unclear (Maathuis et al., 2014). In Arabidopsis, Na+/H+

antiporter CHX21 expressed in the endodermis is implicated in Na+

transport from the symplast into the stele apoplast and the absence

of this transporter reduced Na+ level in xylem sap (Hall et al., 2006).

However, the role of this transporter in endodermal cells covered

with suberin lamellae isolating protoplasm from the apoplast would

be questioned. In another study on Arabidopsis, Hunter et al. (2019)

showed that salt‐induced callose deposition in plasmodesmata

enhanced salt tolerance at the germination stage. Whether reduced

plasmodesmata permeability by callose deposition plays a role in

controlling Na+ or Cl– transport in crop roots remains to be seen. An

important mechanism controlling Na+ transport from roots to shoots

is Na+ retrieval from the xylem mediated by the HKT1 transporter

(Munns et al., 2012; Ren et al., 2005; Sunarpi et al., 2005). Sodium

retrieval from the xylem can stimulate K+ loading (Maathuis

et al., 2014). We found higher amounts of K and a higher K/Na

ratio in xylem vessels of Jade than BM6, which may result from the

net exchange of Na and K at the interface of the vessels and

neighbouring cells by HKT transporters (Läuchli et al., 2008). Møller

et al. (2009) suggested that Na+ retrieved in the stellar cells moves

back to cortical cells even under transpiring conditions. In our study,

we observed a decreasing gradient of Na from the pericycle to the

outer cortex at 50mm behind the root apex and a decreasing

gradient of Na from the endodermis to the outer cortex at 20mm

behind the root apex that would favour diffusion back into the

cortex. Similar patterns of low [Na] in outer cell layers and high [Na]

in the pericycle were reported in barley at 50mm behind the root

apex under 100mM NaCl (Kotula et al., 2015a) and wheat at 100mm

behind the root apex under 50mM NaCl (Läuchli et al., 2008). This

[Na] gradient might be due to a possible efflux of Na out of the root

via plasma membrane Na+/H+ antiporters encoded by the SOS1 gene

(Kotula et al., 2015a; Läuchli et al., 2008).

In addition to Na+ exclusion, retention of K+ in root tissue has

been established as one of the salt tolerance mechanisms (Shabala

and Cuin (2008); Wu, Zhang, et al., 2018). The K distribution profiles

at 20 and 50mm showed salt‐induced K loss in all cell types in salt‐

sensitive BM6. In contrast, salt‐tolerant Jade maintained high [K] in

stellar cells at 20mm and all cell types at 50mm behind the apex.

Thus, retention of K in root cells might contribute to higher salt

tolerance in Jade.

In contrast to [Na], a decreasing [Cl] gradient from the outer

cortex to xylem vessels was observed in both genotypes in the two

root regions. Apoplastic barriers in endodermis might not control Cl

transport to the xylem as chloride predominantly traverses the root

by a symplastic pathway (Abbaspour et al., 2014; Geilfus, 2018;

Pitman, 1982). At 20mm behind the root apex, both genotypes had

similar [Cl] across cells. However, at 50mm, Jade had higher [Cl] in

cortical cells and the endodermis than BM6. In lucerne (Medicago

TABLE 1 Net photosynthetic rate (Pn, µmol CO2 m−2 s−1), transpiration rate (T, mmol H2O m−2 s−1), stomatal conductance (gs, mol H2O
m−2 s−1), intercellular CO2 concentration (Ci, µmol CO2 mol−1), chlorophyll concentration (SPAD value) and maximum quantum efficiency of
photosystem II (Fv/Fm) measured in the youngest fully expanded leaf (YFEL) of BARI Mung‐6 and Jade AU at a CO2 concentration of 400
(ambient) and 800 µmol mol−1 (elevated).

CO2 level Traits
BARI Mung‐6 Jade AU p‐Value
Control 75mM Control 75mM G T G × T

400 Pn 13.5 ± 0.8 a 5.3 ± 0.5 c 10.9 ± 1.3 ab 9.2 ± 0.4 b p = 0.54 p < 0.001 p < 0.01

T 2.3 ± 0.2 a 1.1 ± 0.1 b 1.7 ± 0.2 ab 1.3 ± 0.2 b p = 0.19 p < 0.001 p < 0.01

gs 0.13 ± 0.01 0.06 ± 0.002 0.11 ± 0.01 0.08 ± 0.01 p = 0.64 p < 0.001 p = 0.09

Ci 197.5 ± 5.9 b 252.1 ± 7.1 a 191.9 ± 10.9 b 179.1 ± 14.7 b p < 0.001 p < 0.05 p < 0.01

800 Pn 26.0 ± 2.0 a 11.1 ± 1.7 c 19.3 ± 1.9 b 16.8 ± 0.9 ab p = 0.51 p < 0.001 p < 0.01

T 2.2 ± 0.3 0.9 ± 0.1 1.7 ± 0.1 0.9 ± 0.1 p = 0.09 p < 0.001 p = 0.16

gs 0.12 ± 0.02 0.05 ± 0.01 0.11 ± 0.02 0.06 ± 0.01 p = 0.78 p < 0.001 p = 0.64

Ci 373.6 ± 27.5 ab 428.9 ± 11.7 a 350.0 ± 31.7 ab 290.8 ± 34.6 b p < 0.01 p = 0.93 p < 0.05

SPAD 46.5 ± 3.5 31.4 ± 2.3 45.0 ± 2.4 40.0 ± 1.0 p = 0.17 p < 0.01 p = 0.06

Fv/Fm 0.81 ± 0.02 0.77 ± 0.01 0.79 ± 0.01 0.78 ± 0.01 p = 0.67 p = 0.08 p = 0.29

Note: Plants were grown in soil with 0 (nonsaline control) and 75mM NaCl treatments imposed on 15‐day‐old plants for 23 days. The measurements were

taken between 10:00 AM and 4:00 PM (data were taken in replications to minimize any confounding influence from the timing of the measurements;
Supporting Information: Table S2) at photosynthetically active radiation of 1500 µmol photons m−2 s−1, leaf chamber temperature of 28°C and 60%–70%
relative humidity. Data are mean ± SE of four replicates. Two‐way ANOVA was used to compare genotypes (G), salinity treatments (T) and
genotype × treatment (G × T) effects. Significant differences (treatment × genotype interaction at p = 0.05) are indicated by different letters for each mean.

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sativa L.), salinity tolerance was associated with the retention of Cl in

the vacuoles of the epidermis and outer cortex (Anderson & Van

Steveninck, 1987). Similarly, durum wheat grown in 50mM NaCl

accumulated the highest [Cl] in the epidermis (Läuchli et al., 2008). In

our study, Jade might accumulate higher [Cl] in the vacuoles of the

root cells, limiting Cl– transport to the shoots.

To summarise, Jade has better control of Na and Cl transport and

maintains higher K across roots than BM6. The control mechanisms

are possibly related to regulating Na loading into the stele apoplast

by the pericycle, and retrieving Na from the xylem and preferential

accumulation of higher Cl in the vacuoles of roots cells.

4.2 | Intra or intercellular sequestration of ions in
leaves

The capacity of cells to compartmentalize Na+ and Cl– in vacuoles

and restrict accumulation in cytoplasm and organelles, such as

chloroplasts, contributes to salt tissue tolerance (Bose et al., 2017;

Munns et al., 2016). In our study, BM6 had significantly higher [Na] in

all leaf cell types (~30mM) than Jade (~10mM) in saline conditions,

consistent with whole leaf [Na+]. In both genotypes, Na was

distributed similarly between epidermal and mesophyll cells. This is

in contrast to previous studies on other grain legumes, where Na was

preferentially accumulated in epidermal cells and maintained at lower

levels in photosynthetically active mesophyll cells (chickpea, Kotula

et al., 2019; soybean, Le et al., 2023). In chickpea, low [Na] in

mesophyll cells (<20mM) did not affect photosynthesis in the

tolerant genotype, but structural damage to chloroplasts that

impaired photosynthesis occurred at ~220mM mesophyll cell [Na]

in a sensitive genotype (Kotula et al., 2019). In cultivated soybean,

despite preferential Na partitioning to epidermal cells, ~30mM [Na]

in mesophyll cells contributed to decreased photosynthesis (Le

et al., 2023). In the present study, chloroplastic [Na] in salt‐tolerant

Jade was low (~11mM) and did not affect photosynthetic processes.

However, higher chloroplastic [Na] in salt‐sensitive BM6 (33mM)

could reduce the chloroplast function and decrease photosynthesis.

A slight increase of Na+ in chloroplasts of glycophytes (2.1–3.8 mM in

Arabidopsis) can decrease photosynthetic efficiency (Bose et al., 2017;

Müller et al., 2014).

Additionally, an increase in [K] in mesophyll cells along with a

concurrent decrease in epidermal cells, indicating K redistribution

from the epidermis to mesophyll cells (James et al., 2006; Kotula

et al., 2019), contributed to maintaining a favourable K/Na ratio in

the vacuoles (77) and chloroplasts (54) of mesophyll cells. In contrast

to Jade, higher vacuolar and chloroplastic [Na] (~30mM) and

preferential K accumulation in epidermal cells in BM6 resulted in a

lower K/Na ratio (~11) in the vacuoles and chloroplasts. Similar to Na,

Cl was distributed equally between epidermis and mesophyll cells in

BM6, but in Jade, Cl accumulated preferentially in mesophyll cells

compared to epidermal cells. These differences in the distribution

pattern of Cl between the two genotypes could be linked to charge

balance (higher [Cl] in the epidermis in BM6 could balance higher [K]).

Despite these differences in cellular Cl partitioning, both genotypes

had equally high vacuolar [Cl] in mesophyll cells (475mM), but

chloroplastic [Cl] was significantly lower in Jade (150mM) than in

BM6 (284mM). The concentration at which Cl becomes toxic is not

well defined (Munns & Tester, 2008). In barley and wheat,

170–200mM Cl in mesophyll cells did not affect photosynthetic

processes (Fricke et al., 1996; James et al., 2006). In chickpea, similar

Cl accumulation (353–403mM) in palisade mesophyll in salt‐tolerant

and salt‐sensitive genotypes was unlikely to reduce photosynthetic

rates in the salt‐sensitive genotype (Kotula et al., 2019). In contrast,

salt‐sensitive common bean grown in 150mM NaCl accumulated

250–300mM [Cl] in cell vacuoles and chloroplasts, which reduced

the efficiency of RuBP carboxylase and photosynthetic rate

(Seemann & Critchley, 1985). In spinach (Spinacia oleracea L.),

chloroplast [Cl–] remained below 100mM even at high leaf Cl– levels

(Robinson & Downton, 1984). In BM6, 284mM chloroplastic [Cl]

could be above the threshold level for Cl toxicity in mungbean,

contributing to reduced photosynthetic rates.

Maintenance of cellular ionic homeostasis is essential for the

optimal functioning of plant metabolic machinery including mainte-

nance of electro‐neutrality or osmotic adjustments to maintain cell

volume and turgor (Flowers & Colmer, 2015; Mulet et al., 2020;

Shabala & Pottosin, 2014). In the present study, leaf cellular [Na]

(both vacuolar and chloroplastic) was much lower than [Cl]. This

charge imbalance of Cl– >Na+ was overcome by the accumulation of

other cations such as K, Ca, and Mg (Figures 4, 6 and Supporting

Information S1: Figures S9–S11). Interestingly, Ca preferentially

accumulated in mesophyll cells, whereas Mg accumulated in

epidermal cells. Accumulation of P and S also appear to assist in

maintaining charge balance as anions in addition to Cl– in chloro-

plasts. In addition, Ca and P were selectively accumulated in different

cell types to avoid the precipitation of calcium phosphate, reducing

the availability of both nutrients and affecting multiple cellular

processes (Hayes et al., 2019; Oi et al., 2022).

These results illustrate that Jade restricted Na and Cl accumula-

tion in chloroplast of mesophyll cells better than BM6 and

preferentially accumulated K in mesophyll cells, which might

contribute to salt tolerance. Specific Ca, Mg, P and S accumulation

in different cell types or cellular organelles likely maintains electro‐

neutrality and/or osmotic balance.

4.3 | High leaf Na+ and Cl– alters leaf anatomy and
affects photosynthesis

High leaf Na+ and Cl– decreases leaf chlorophyll, induces reactive

oxygen species (ROS) production and inhibits photosynthetic CO2

enzymatic activities (Bose et al., 2017; Geilfus, 2018). In addition to

high Na+ and Cl– accumulation in leaf tissues (non‐stomatal

limitations of photosynthesis), osmotic component of salinity stress

can affect photosynthesis through stomatal closure leading to

decreased CO2 uptake (stomatal limitations) (Bose et al., 2017;

Chaves et al., 2009). In this study, salt treatment reduced Pn to 40%

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of nonsaline control in BM6 but did not affect Pn in Jade. We

evaluated the potential contribution of non‐stomatal and stomatal

limitation of photosynthesis by increasing external CO2 from

400 µmol mol−1 (ambient) to 800 µmol mol−1 (elevated) to over-

come any stomatal limitations. Elevated external CO2 concentration

(800 µmol mol−1) increased both photosynthesis and intercellular

CO2 by 2‐fold in both genotypes in the nonsaline and 75mM NaCl

treatments. However, genotypic differences in Pn were similar to

the ambient CO2, indicating that, in addition to stomatal limitation,

non‐stomatal factors predominantly limited photosynthesis in BM6.

Stomatal and non‐stomatal limitations of photosynthesis during

salinity stress have also been reported in faba bean (Vicia faba L;

Tavakkoli et al., 2010) and soybean (Le et al., 2023). High Cl– also

reduces leaf area, increases leaf succulence, thickness and cell size,

and alters leaf anatomy (Romero‐Aranda et al., 1998; Franco‐

Navarro et al., 2016; Rouphael et al., 2017). In our study, salinity

stress increased leaf succulence, leaf thickness and mesophyll

thickness and decreased leaf area in BM6. Salinity stress also

increased the palisade and spongy cell area in BM6 but did not

affect leaf morphology and anatomy in Jade. Larger mesophyll cells

tightly packed with reduced intercellular spaces in BM6 could

reduce the mesophyll surface area exposed to intercellular air space

per unit leaf area (Lundgren and Fleming, 2020). Salinity stress

significantly increased intercellular CO2 in BM6 but reduced

photosynthesis, whereas, in Jade, intercellular CO2 and photo-

synthesis were unaffected. These results indicate that, in addition to

stomatal limitations and Na and Cl toxicity, reduced photosynthesis

in BM6 could be correlated with changes in leaf anatomy, that is,

increased leaf thickness, leaf succulence and reduced intercellular

air spaces, which may increase resistance to CO2 diffusion to the

carboxylation site, known as mesophyll conductance (Lundgren and

Fleming, 2020).

5 | CONCLUSIONS

The [Na] profiles in roots indicate that salt‐tolerant Jade excludes

Na from shoots by regulating Na loading into the stele apoplast by

the pericycle, and retrieving Na from the xylem. The role of

suberized endodermis in preventing Na transport towards the

xylem is minimal in mungbean roots. The [Cl] profiles in roots

indicate that higher Cl accumulation in root cell vacuoles in Jade

contributed to lower [Cl] in leaves compared to BM6. Jade

maintained much lower [Na] and [Cl] in chloroplasts of mesophyll

cells compared to salt‐sensitive BM6, explaining the genotypic

variation in photosynthetic performance. In addition to regulating

[Na] and [Cl] accumulation in chloroplasts, retention of high [K] in

root cells and preferential accumulation of [K] in leaf mesophyll

cells contribute to salt tolerance in Jade. Thus, the superior ability

of Jade to exclude Na and Cl from shoots and regulate Na and Cl

accumulation in chloroplasts of mesophyll cells, as well as the

ability to maintain high K in root cells and leaf mesophyll cells,

increased salinity tolerance compared to BM6.

ACKNOWLEDGEMENTS

M. S. I. was supported by the John Allwright Fellowship Award from

the Australian Centre for International Agricultural Research (ACIAR).

The research was supported through project CIM‐2014‐076 funded

by ACIAR to W.E. The authors acknowledge the use of Microscopy

Australia facilities at the Centre for Microscopy, Characterisation and

Analysis, The University of Western Australia, a facility funded by the

University, State and Commonwealth Governments. We thank Joana

Kotula and Kosala Ranathunge for help in anatomy analysis, and

Robert Creasy and Bill Piasini for plant growth support in the

glasshouse. We also thank Col Douglas, Department of Agriculture

and Fisheries, Queensland for providing the seeds. Open access

publishing facilitated by The University of Western Australia, as part

of theWiley ‐ The University of Western Australia agreement via the

Council of Australian University Librarians.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the

corresponding author upon reasonable request.

ORCID

Md Shahin Iqbal http://orcid.org/0000-0003-1687-0003

Al Imran Malik http://orcid.org/0000-0002-8688-2117

Lukasz Kotula http://orcid.org/0000-0001-8760-7099

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SUPPORTING INFORMATION

Additional supporting information can be found online in the

Supporting Information section at the end of this article.

How to cite this article: Iqbal, M. S., Clode, P. L., Malik, A. I.,

Erskine, W. & Kotula, L. (2024) Salt tolerance in mungbean

is associated with controlling Na and Cl transport across

roots, regulating Na and Cl accumulation in chloroplasts

and maintaining high K in root and leaf mesophyll cells.

Plant, Cell & Environment, 47, 3638–3653.

https://doi.org/10.1111/pce.14943

MUNGBEAN SALTTOLERANCE: NA AND CL REGULATION AND K MAINTENANCE | 3653

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https://doi.org/10.1111/pce.14943

	Salt tolerance in mungbean is associated with controlling Na and Cl transport across roots, regulating Na and Cl accumulation in chloroplasts and maintaining high K in root and leaf mesophyll cells
	1 INTRODUCTION
	2 MATERIALS AND METHODS
	2.1 Plant materials and growth conditions
	2.2 Treatments and sampling procedure
	2.3 Leaf gas exchange and chlorophyll fluorescence measurements
	2.4 Total tissue ion analysis
	2.5 Morphology and anatomy of the YFEL
	2.6 Root anatomy
	2.7 Cell-specific elemental analysis by cryo-SEM X-ray microanalysis
	2.8 Statistical analyses

	3 RESULTS
	3.1 Radial Na, Cl and K concentrations in various cell types of lateral roots
	3.1.1 Sodium - 20mm behind the apex
	3.1.2 Sodium - 50mm behind the apex
	3.1.3 Chloride - 20mm behind the apex
	3.1.4 Chloride - 50mm behind the apex
	3.1.5 Potassium- 20mm behind the apex
	3.1.6 Potassium - 50mm behind the apex

	3.2 Development of suberin lamellae in lateral roots
	3.3 Elemental concentration in various cell types or organelles of the lamina of leaflets
	3.3.1 Sodium
	3.3.2 Chloride
	3.3.3 Potassium and potassium/sodium ratio

	3.4 Leaf gas exchange, morphology and anatomy

	4 DISCUSSION
	4.1 Controlling ion transport across roots
	4.2 Intra or intercellular sequestration of ions in leaves
	4.3 High leaf Na+ and Cl- alters leaf anatomy and affects photosynthesis

	5 CONCLUSIONS
	ACKNOWLEDGEMENTS
	DATA AVAILABILITY STATEMENT
	ORCID
	REFERENCES
	SUPPORTING INFORMATION