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Early response to salt ions in maize (Zea mays L.)
Christoph-Martin Geilfus1, Jutta Ludwig-Müller2, Gyöngyi Bárdos3, Christian Zörb3*
1Controlled Environment Horticulture, Faculty of Life Sciences, Albrecht Daniel Thaer-
Institute of Agricultural and Horticultural Sciences, Humboldt-University of Berlin,
Lentzeallee, 14195 Berlin, Germany. 2Institut für Botanik, Technische Universität Dresden,
Zellescher Weg 20b, D-01062 Dresden, Germany. 3Institute of Crop Science, Quality of Plant
Products, University of Hohenheim, Emil-Wolff-Straße 25, 70599 Stuttgart, Germany.
*Corresponding author: Christian Zörb, E-mail: [email protected]; Phone:
49(0)711 45922520; Fax 49(0)711 459-23960.
Key words: Chlorine, Sodium, Abscisic acid, Viviparous 14, NCED
Abbreviations: Abscisic acid (ABA); Dry weight (DW); Polyethylene glycol (PEG);
Relative turgidity (RT), Real-time quantitative reverse transcriptase-polymerase chain
reaction (qRT-PCR); 9-cis-epoxycarotenoid dioxygenase (NCED).
Key message The influence of chloride on the accumulation of ABA highlight the importance
of integrating chloride into models that elucidate early response during the establishment of
salt stress.
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Abstract Abscisic acid (ABA) regulates leaf growth and transpiration rate of plants exposed to
salt stress. Despite the known fact that cell dehydration is instrumental for the modulation of
ABA concentrations when NaCl is high in the external environment, it was never tested as to
whether sodium (Na) or chlorine (Cl) also modulate ABA concentrations. To answer this
question, a hydroponic study on maize (Zea mays) was established, by exposing plants to 50
mM of sodium glucosamide or glucosamine chloride. The effect of both ions on ABA was
investigated in an early stage before (i) the salt ions accumulated to toxic tissue
concentrations and before (ii) cells dehydrated. This allowed studying early responses to Na
and Cl separately, well before plants were stressed by these ions. Gas chromatography–mass
spectrometry analysis was used to quantify ABA concentrations in roots and in leaves after a
period of 2 hours after ion application. The transcript abundance of the key regulatory enzyme
of the biosynthesis of ABA in maize, the 9-cis-epoxycarotenoid dioxygenase gene viviparous
14, was quantified via real-time quantitative-reverse-transcriptase-polymerase-chain-reaction.
The results reveal that Cl and Na induce the increase of leaf tissue ABA concentrations at two
hours after plants were exposed to 50 mM of the ions. Surprisingly, this effect was more
pronounced in response to the Cl component. The increase in the guard-cell regulating ABA
concentration correlated with a reduced transpiration. Mainly because of this result we
suggest that the early accumulation of ABA is useful in maintaining cell turgor.
Introduction
Abscisic acid (ABA) is a plant hormone that is synthesized from carotenoids (Seo and
Koshiba, 2002). Together with other regulating factors, it controls leaf growth and
transpiration. In Arabidopsis thaliana, approximately 1 -10 % of the genome is regulated by 2
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ABA, being either induced or repressed by the 15-C weak organic acid (Finkelstein, 2013).
Many of those ABA-responsive genes are also related to the response to abiotic or biotic
stress (Choudhury and Lahiri, 2011; Finkelstein, 2013). It was shown for different plant
species, that a considerable amount of the stress-related genes that are under control of ABA
contribute to adaptive aspects of induced tolerance towards dehydration, i.e. by encoding
enzymes of the compatible solute synthesis, of the detoxification of reactive oxygen species,
or water channels (Ingram and Bartels, 1996; Yamaguchi-Shinozaki and Shinozaki, 2006;
Finkelstein, 2013). Moreover, the tissue concentration of ABA is relevant for the stress
response to salinity as well as for a possible tolerance mechanism. As a result of a salt-stress
induced reduction in the osmotic potential of the root solution, shoot ABA concentrations
increase in different plant species (Munns and Termaat, 1986; Munns, 2002; Zörb et al., 2013;
Geilfus et al., 2015). An increase in ABA inhibits leaf elongation rates in maize caryopses
(Cramer et al, 1997). Kutschera and Schopfer (1986) argued for maize coleoptiles that this
retardation of growth is based on an ABA-mediated inhibition of the capacity of the cell walls
to loosen, rather than by a reduction of turgor. Under salinity-induced water stress, this ABA-
mediated growth reduction is thought to counteract wilting which would otherwise be the
consequence of ongoing expansion growth under conditions of limited water availability
(Kutschera and Schopfer, 1986).
Another important mechanism to avoid wilting under conditions of salinity-induced water
stress is the regulation of the transpiration rate via ABA-based effects on stomatal aperture. A
change of the ABA concentration is well known to promote stomatal closure or inhibit
opening (McAinsh, et al., 1991; Leckie, et al., 1998; Hetherington, 2001; Kwak et al., 2008),
a process that is relevant for the water balances under salinity or drought stress (Iuchi et al.,
2001; Davies et al., 2002; Zhang et al., 2006; Bauer et al., 2013; Geilfus et al., 2015).
But what are the environmental stress conditions that activate ABA biosynthesis when plants
were exposed to salinity? Drought and salt (i.e. NaCl) are the two environmental stress events 3
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that result in the most pronounced accumulation of ABA, being a result of an activation of
ABA-biosynthesis genes (Xiong and Zhu, 2003). The common feature between both stress
events is the osmotic stress component. Water availability is reduced, which can result in a
reduced leaf turgor and reduced water content. Water stress is known to activate the
expression of the 9-cis-epoxycarotenoid dioxygenase (NCED) which is a key regulatory
enzyme for the production of ABA (Seo and Koshiba, 2002; Xiong and Zhu, 2003). Thus, a
reduction in the plant water content is a critical factor that activates ABA de novo synthesis
(Thompson et al., 1997; Wilkinson and Davies, 2002) that might also be instrumental for an
accumulation of ABA during NaCl-based salinity. However, irrespective of a reduction in
shoot water content, viz. fresh weight, it was never tested as to whether the salt ions Na+ or Cl-
represent environmental stress factor that are also instrumental in the modulation of ABA
tissue concentration. We studied maize that was subjected to a two-hour treatment with both
ions to clarify this, by analysing (i) ABA concentration and (ii) the transcript abundance of
the maize NCED ortholog viviparous 14 (vp14) in correlation to root and leaf sodium and
chloride concentration, transpiration and relative leaf turgidity. Our experimental design that
allowed to discriminate between early responses of the Na- and the Cl-component revealed
that Cl is besides Na an additional environmental factor that correlates with an increase of
ABA in leaves of corn that were exposed for two hours to salts.
Materials and Methods
Plant cultivation
Zea mays (cultivar STABIL, KWS Saat SE, Einbeck, Germany) was grown in hydroponic
culture in a controlled environment chamber. Seeds were imbibed in 2 mM CaSO4 for 1 d
with additional aeration followed by a germination period of 6 d in moistened quartz sand.
Afterwards, seedlings were transferred in 5-L plastic pots containing one-quarter-strength 4
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nutrient solution. After 2 d of cultivation, the nutrient concentration was increased to half-
strength and, after 4 d of cultivation to full-strength. This was done to adapt young plants
stepwise to nutrient concentration in the root medium. The solution was changed every 84
hours (3.5 days) to avoid nutrient depletion. The nutrient solution had the following
composition: 2.5 mM Ca(NO3)2, 1.0 mM K2SO4, 0.2 mM KH2PO4, 0.6 mM MgSO4, 2.5 mM
CaCl2, 0.5 mM NaCl, 1.0 µM H3BO4, 2.0 µM MnSO4, 0.5 µM ZnSO4, 0.3 µM CuSO4, 0.005
µM (NH4)6Mo7O24, 200 µM Fe-EDTA; pH, 6.8. Plants grew under a 14 h (22°C): 10 h (18°C)
dark: light cycle (photoperiod 07:00-21:00 h) with an atmospheric water vapour pressure
deficit of 0.58 kPa (75% RH) during photoperiod. Light intensity was 320 - 350 μmol s−1 m−2
above leaf canopy of the growing leaf number 4. Plants grew 10 days in full-strength nutrient
solution before being subjected to different short-term salt treatments over a period of 2 hours.
Experimental design
The separate effect of both ionic components of NaCl, viz. sodium and chloride, on abscisic
acid (ABA) abundance and viviparous 14 (vp14) mRNA abundance was investigated at 2
hours after salt ions were added into the nutrient solution of the plants. At this early phase,
plants were not yet stressed by the salt ions, but investigations during this early time point
may allow to elucidate early mechanisms of adaptations to increasing concentrations of salt
ions. The combined effect of Na+ and Cl- was tested by adding 50 mM of NaCl into the
nutrient solution (experimental group 1). In order to test for sodium-associated effects that are
conferred when chloride is absent, 50 mM Na+ were given together with the membrane
impermeable glucosamide- as counter anion (experimental group 2). Chloride-associated
effects were investigated by the substitution of Na+ by using the membrane impermeable
glucosamine+ as accompanying counter cation (experimental group 3). Osmotic effects that
are not related to ionic effects were investigated by treating the roots with 93 g PEG 6000 L-1
nutrient solution, a dose that has the same lowering effect of the osmotic potential as 50 mM 5
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NaCl (Sümer et al., 2004) (experimental group 4). Control plants were not treated with salts
or PEG (experimental group 5). At two hours after salt or PEG was added, respectively, the
growing leaf number four that emerged from the sheath two days ago was harvested, as were
the roots. Material was frozen in liquid nitrogen being stored at -80°C, either for ion analysis,
ABA quantification or quantification of the mRNA abundance of vp14. Five biological
replicates were taken for each experimental group.
Ion analysis
The analysis of Na+ and Cl− was performed with 15 mg of dried leaves that were boiled for
5 min in 1.6 ml of deionized water. After cooling, samples were centrifuged and proteins were
precipitated in the supernatant by washes in chloroform. Thereafter, samples were cleaned by
passage through C-18 column. Na+ and Cl− concentrations were analysed using ion
chromatography (Dionex ICS-5000+, Life Technologies GmbH, Darmstadt, Germany).
Before roots were subjected to ion analysis, roots were thoroughly washed for 30 sec with 1
mM Ca2SO4 to remove adhering salts from the surface.
Analysis of free ABA
At least 10 mg dry powder was used for one sample to determine the plant hormone ABA.
For each biological replicate two technical replicates were performed. The samples were
extracted with a mixture of iso-propanol and acetic acid (95:5, v/v) for 2 h under continuous
shaking at 4°C. Before the start of the extraction procedure 100 ng of heavy H labelled ABA
were added to each sample. [2H6]-ABA was from the Plant Biotechnology Institute, National
Research Council of Canada, Saskatoon, Canada. Further sample preparation was performed
according to Meixner et al., (2005). Briefly, the samples were centrifuged for 10 min at
10,000 g, the supernatant removed and evaporated to dryness under a stream of N2. The
residue was resuspended in methanol, centrifuged again for 10 min at 10,000 g, the 6
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supernatant was removed and transferred in a glass vial. The compounds in the methanolic
extract (20 µl) were methylated by addition of equal amounts of a 1:10 diluted solution (in
diethylether) of trimethylsilyldiazomethane (Sigma-Aldrich, Germany) for 30 min at room
temperature. The mixture was then evaporated and resuspended in 50 µl ethyl acetate for GC-
MS analysis. Gas chromatography–mass spectrometry analysis was carried out on a Varian
Saturn 2100 ion-trap mass spectrometer using electron impact ionization at 70 eV, connected
to a Varian CP-3900 gas chromatograph equipped with a CP-8400 autosampler (Varian,
Walnut Creek, CA, USA). For the analysis 1 µl of the methylated sample was injected in the
splitless mode (splitter opening 1:100 after 1 min) onto a Phenomenex (Aschaffenburg,
Germany) ZB-5 column (30 m x 0.25 mm x 0.25 µm) using Helium as carrier gas at 1 ml min -
1. Injector temperature was 250°C and the temperature program was 60°C for 1 min, followed
by an increase of 25°C min-1 to 180°C, 5°C min-1 to 250°C, 25°C min-1 to 280°C, then 5 min
isothermically at 280°C. For higher sensitivity, the µSIS mode (Varian Manual; Wells and
Huston, 1995) was used. The endogenous hormone concentrations were calculated by the
principles of isotope dilution (Cohen et al., 1986), using the ions at m/z 190/194 (endogenous
and labeled standard; while the molecular ion of ABA would have six deuterium
incorporated, during fragmentation of ABA two deuteriums are lost and the fragmentation ion
at m/z 194 has only four deuterium retained) for methylated ABA (Walker-Simmons et al.,
2000).
Vp14 primer design for polymerase chain reaction
Forward (f 5′–3′: TTCTCGGAGGAGGAACAGAGGA) and reverse (r 5′–3′:
CCAACTGTAACTCTGGTGTGCG) primer for amplifying Zea mays viviparous 14
(Zmvp14) mRNA were designed using the Primer-BLAST software
(https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and were purchased from ThermoFischer
Scientific (Darmstadt, Germany). For ensuring specificity and preventing false priming sites, 7
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primers were checked in silico by BLASTN against a Zea mays nucleotide collection (nr/nt)
and Zea mays reference mRNA sequences (refseq_rna). In order to avoid the formation of
base pairings within/between the primers, primer sequences were optimized with regard to the
formation of hairpins, self-dimer, or hetero-dimer by using the OligoAnalyzer (version 3.1;
Integrated DNA Technologies, Inc.; https://eu.idtdna.com/calc/analyzer). For demonstrating
the specificity of primer/template interactions, the real-time quantitative RT-PCR products
were sequenced (GATC Biotech, Konstanz, Germany) (supplementary material 1). Moreover,
agarose gels were run after real-time quantitative RT-PCR to ensure that only a single PCR
product was generated and to confirm the predicted PCR product size of 117 bp on the gel
(supplementary material 2).
RNA extraction and cDNA synthesis
100 mg ground lyophilized leaf or root material was used for RNA extraction. Total maize
leaf RNA was isolated by phenol-chloroform extraction according to the method by Cox and
Goldberg (1998). The quality of RNA was checked by OD260 and OD280. One µg of total RNA
was digested with PerfeCTa DNAseI (Quanta Biosciences, Beverly, USA) to eliminate
residual genomic DNA and reverse transcribed to single-stranded cDNA with SuperScript
VILO cDNA Synthesis Kit (Invitrogen by Life technologies, Karlsruhe, Germany) according
to the manufacturer’s instructions. Single-stranded cDNA was diluted to a concentration
depending on the level of expression of the gene studied, as assessed by measuring primer
efficiency; cDNA aliquots were used to avoid discrepancy in the data attributable to the
repetition of freezing-thawing cycles.
Real-time quantitative RT-PCR
The SYBR-Green-based real-time quantitative RT-PCR technique was performed on a
BioRad CFX96 real-time PCR system using SensiMix SYBR No-ROX Kit (Bioline, London, 8
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United Kingdom). For each reaction, 0.8 µL diluted single-stranded cDNA was used in a total
volume of 10 µL (0.5 µM forward and reverse primer, 5 µl SensiMix SYBR No-Rox 2x,
filled up with sterilized and autoclaved H2Obidest). After an initial denaturation step (95°C, 10
min), real-time quantitative RT-PCR was carried out over 45 cycles (denaturation: 95°C, 15 s;
amplification and quantification: 60°C, 15 s, elongation: 72°C, 15 s). To check the specificity
of the annealing of the oligonucleotides, dissociation kinetics was performed by the real-time
PCR system at the end of the experiment (65–95°C, continuous fluorescence measurement).
The comparative ∆∆Ct (threshold cycles) method for relative quantification was used to
analyse the real-time quantitative RT-PCR data according to Pfaffl (2001). With this method,
the Ct values were normalized by comparison with the two endogenous reference genes, actin
1 (Manoli et al., 2012) and ubiquitin-conjugating enzyme (Geilfus et al., 2011). The
normalized Ct values were then used to compare salt- or PEG-treated plants versus controls.
Data are shown as the relative change in transcript expression as fold-changes. Threshold
cycles were calculated by the internal software of the real-time PCR system and were the
means of three biological replicates of each run in triplicate. Negative controls without
templates were carried out concurrently.
Leaf transpiration rates and relative turgidity
A portable gas-exchange system (LI-COR 6400 XT, LI-COR; Bad Homburg, Germany) was
used to obtain transpiration rates (mmol H2O m-2 s-1) in leaves. Photon flux density was ~300
μmol m−2 s−1 as provided by the blue and red diodes of an integrated fluorescence chamber
head (6400-02B LED light source, LI-COR; Bad Homburg, Germany). CO2 at a flow rate of
300 μmol mol−1 CO2 was controlled by a CO2 injection system. Transpiration rate was
recorded between 10:00 h to 16:00 h (photoperiod 07:00-21:00 h) and was calculated by the
internal software. Relative turgidity (RT) was used as a measurement for the hydration status
of the leaf following the method by Barrs and Weatherley (1962). RT =((FW-DW)/(TW-9
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DW))*100, where FW is the fresh weight immediately taken after harvest, TW is turgid fresh
weight taken after leaf has been floated in chilled water over night to full turgidity, DW is dry
weight after oven drying.
Statistics
Five biological replicates (meaning five plants in five single pots) were generated for each
experimental group. Graphs show mean ± standard error (SE). Statistically significant mean
differences (P ≤ 0.05) are indicated by different letters. In order to maintain an experiment-
wise α of P < 0.05, multiple t-tests were adjusted according to Bonferroni–Holm.
Results
Chloride- and sodium increase shoot ABA concentration
The two-hour treatment with 50 mM of chloride, given as glucosamine-chloride to the roots,
increased the ABA concentration in the young growing leaf from 2.33 (±0.99 SE) to 5.68
(±1.05 SE) µg g-1 dry weight (dw) (Figure 1A). This increase was not that much pronounced
when plants were exposed to 50 mM sodium instead of chloride, given as sodium
glucosamide. Here ABA increased to 3.37 (±0.66 SE) µg g-1 dw. However, the highest
increase in leaf ABA concentration was detected after the combined sodium/chloride
treatment. Addition of 50 mM of NaCl resulted in 6.49 (±1.23 SE) µg ABA g -1 dw. When the
NaCl-induced decrease in the osmotic potential of the nutrient solution was mimicked by the
addition of PEG 6000 into nutrient solution (93 g PEG 6000 L-1 represents a dose that has
same lowering effect of the osmotic potential than 50 mM NaCl; Sümer et al., 2004), no
increase in leaf ABA was detected at two hours after addition (Figure 1A). Results from roots
contrast findings from the leaves. When compared to control plants, root ABA concentrations
decreased from 4.36 (±1.34 SE) to 2.38 (±0.51 SE), 1.93 (±0.57 SE), 2.63 (±1.1 SE), or 2.15
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(±0.96 SE) µg g-1 dw when being exposed for two hours to either PEG 6000, sodium
glucosamide, NaCl or glucosamine chloride, respectively.
Transpiration rate decreases when salts are added while relative turgidity remains
unchanged.
A two-hour treatment with either 50 mM sodium chloride, 50 sodium glucosamide or
glucosamine chloride resulted in a drop of the transpiration rate from 4.7 (±0.54 SE) mmol
H2O m-2 s-1 under control conditions to 2.3 (±0.52 SE), 2.9 (±0.34 SE) or 2.55 (±0.35 SE)
mmol H2O m-2 s-, respectively, while a two-hour treatment with PEG 6000 did not result in a
significate decrease of the transpiration rate (Figure 2; grey bars). Though, the PEG treatment
is known to lower the osmotic potential in the nutrient solution (Sümer et al., 2004), it was not
effective in inducing water deficiency in the maize leaves. It can be assumed that the two-
hour treatment did not lower leaf osmotic potential. Relative turgidity of the leaf remained
unchanged during the different two salt-treatments as well as the PEG treatment (Figure 2;
black squares).
Tissue salt ion concentration increases at two hours after addition of ions
A two-hour sodium treatment, established by adding 50 mM of sodium into the nutrient
solution, increased the root sodium concentration from 0.89 (±0.16 standard error; SE) mg g-1
dw under control conditions to 3.26 (±0.31 SE) or 2.93 (±0.20 SE) mg Na+ g-1 root dw when
given as NaCl or sodium glucosamide, respectively (Figure 3C). This increase was less
pronounced in the young growing leaf number four. Here, sodium concentration increased
from 0.84 (±0.16 SE) mg Na+ g-1 dw under control condition to 2.13 (±0.18 SE) or 1.78 (±0.14
SE) mg Na+ g-1 dw when given as NaCl or as sodium glucosamide, respectively (Figure 3A).
The two-hour treatment with chloride resulted in an increase of root chloride concentration
from 6.57 (±0.39 SE) mg g-1 dry weight under control conditions to 14.46 (±0.55 SE) or 13.36 11
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(±0.86 SE) mg Cl- g-1 root dw when given as NaCl or glucosamine chloride, respectively
(Figure 3D). This two fold increase was not observed in young leaf number four, although,
the chloride concentration increased from 5.98 (±0.11 SE) mg Cl- g-1 dw under control
condition to 9.25 (±0.46 SE) or 8.45 (±0.55 SE) mg Cl- g-1 dw when given as NaCl or
glucosamine chloride, respectively (Figure 3B).
Chloride- and sodium increase vp14 mRNA abundance
The two-hour treatment with 50 mM of chloride, given as glucosamine chloride to the roots,
resulted in a 14-fold enrichment of the mRNA abundance of the corn NCED gene vp14 in the
roots, relative to the control (Figure 4B). In leaf number four, vp14 transcript abundance was
only 1.6-fold enriched under same conditions (Figure 4A). This fold-enrichment in the root
was by far not that much pronounced when plants were exposed to 50 mM sodium instead of
chloride, given as sodium glucosamide. Here, a 9-fold enrichment was observed in the roots,
whereas vp14 transcript abundance was only 3.3-fold enriched in the leaf. The combined
sodium/chloride treatment, using 50 mM NaCl, led to a 31-fold enrichment of the mRNA
abundance of the vp14 gene in the roots (Figure 4B), whereas transcript abundance in the leaf
was only doubled (Figure 4A). The treatment with PEG 6000 resulted in a 10-fold enrichment
in the roots and a 4-fold enrichment in the leaves, relative to the control.
Discussion
This study increases knowledge regarding the environmental factor(s) that lead(s) to an
accumulation of ABA when plants were initially exposed to 50 mM NaCl, as measured at two
hour after salts were added into the nutrient solution. The load of a 50 mM concentration of
the salts is itself a low or not more than a moderate treatment. During this early phase, plants
were not yet stressed by the salt ions nor were cells dehydrated, which allowed studying early
mechanisms that may be linked to adaptation to changing environment.12
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It has previously been established for Arabidopsis that a five-hour treatment with 300 mM
NaCl leads to an enhanced expression of the biosynthetic ABA gene 9-cis-epoxycarotenoid
dioxygenase, viviparous 14 (Zmvp14; equals NCED3), which was shown to be an early rate
limiting step in controlling ABA biosynthesis (Barrero et al., 2006). While the study from
Barrero et al., (2006) has shown that the 9-cis-epoxycarotenoid cleavage reaction is the key
regulatory step for the biosynthesis of ABA under short-term NaCl stress, it remains an open
question as to whether sodium or chloride may lead to an activation of the enhanced
expression of ABA under short-term salt stress.
Chloride- and sodium are instrumental for increasing leaf ABA
Our results are clear: both ionic components of the NaCl, viz. chloride and sodium, lead to a
significant increase of the ABA concentration in leaves of maize that were exposed for two
hour to either 50 mM of sodium or chloride, or the combination thereof (NaCl) (Figure 1A).
The combined treatment of sodium and chloride, given as 50 mM of NaCl, resulted in the
highest leaf concentration of 6.49 µg ABA g-1 dw (with similar significance to the next
highest, the glucosamine chloride treatment). The osmotic treatment, with a dose of PEG
6000 that has the same lowering effect on the osmotic potential of the nutrient solution as the
treatment with 50 mM NaCl, did not result in increased leaf ABA concentration. However,
this does not mean that ABA synthesis is not activated by leaf water deficiency or by osmotic
stress, which was previously shown (Thompson et al., 1997; Wilkinson and Davies, 2002).
Instead, it appears that in in our experiment the two-hour treatment with PEG was not
effective in reducing transpiration and relative leaf turgidity (Figure 2). Thus, it can be
concluded that a two-hour treatment with 93 g PEG 6000 L-1 was not harsh enough to cause
osmotic stress in leaves, although PEG treatment lowers instantaneously the osmotic potential
of the nutrient solutions after addition. This implies that drought-induced leaf ABA de novo
synthesis is under control of the cell water status (i.e. turgidity) and not under control of the 13
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osmotic potential in the solution that harbours the roots. This assumption is in accordance to
other works that investigated the accumulation of ABA under water deficit (Thompson et al.,
1997; Wilkinson and Davies, 2002; Ren et al., 2006). It is well known that cell dehydration
induces the mRNA expression of the 9-cis-epoxycarotenoid dioxygenase (NCED) gene Vp14
in detached leaves of Arabidopsis seedlings that lost 15 % of the initial fresh weight by
transpiration over a period of 5 hours (Tan et al., 1997). The regulatory role of NCED during
the drought-induced ABA biosynthesis was also shown in detached leaves of Phaseolus
vulgaris seedlings that reduced 12–15 % of leaf fresh weight (Qin and Zeevaart, 1999).
The novelty of the presented results is that the increment in leaf ABA concentration can also –
irrespective of cell turgor – be a result of the ions, whereas the separate effect of chloride,
given without sodium, was more pronounced than that of sodium, given without chloride
(Figure 1A). However, the effects on ABA that we ascribe to chloride could also be
associated to pleiotropic effects of the chloride accompanying counter cation glucosamine.
That has to be ruled out because it was demonstrated in the yeast Rhodotorula gracilis
(Niemietz et al., 1981) and in the gibbous duckweed (Lemna gibba) (Sanz and Ullrich, 1989)
that the amino-sugar glucosamine can be taken up across the plasma membrane. Such an
uptake of glucosamine would invalidate the comparison performed between the sodium- and
the chloride-salt because it was shown for the hypocotyls of Arabidopsis seedlings that the
production of reactive oxygen species relies on glucosamine (Ju et al., 2009). However, we
measured that during the two-hour period during which the presented experiments were
performed, no glucosamine (and neither glucosamide) was taken up into roots or shoots
(suppl Tab. 1). Both ions, chloride and sodium, are instrumental for increasing leaf ABA
concentration, that is a novel fact that was not reported before. A major task for the future will
be to identify the genetic factors that regulate ABA biosynthesis genes in a chloride-specific
manner.
vp14 mRNA abundance was not effected in the leaves 14
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Although, chloride and sodium ion concentrations increased in the leaf (Figure 3), it cannot be
the direct effect of the ions in the leaf that lead to an increased leaf ABA concentration via the
induction of the ABA biosynthetic gene vp14. That is reasoned because qRT-PCR revealed
that the vp14 mRNA abundance was not affected in the leaves (Figure 4), although chloride
and sodium tissue concentration increased in the leaf. However, relative to the control plants,
the vp14 mRNA abundance significantly increased in the roots when exposed to 50 mM of
chloride, given as glucosamine chloride (14-fold), or when exposed to 50 mM of sodium,
given as sodium glucosamide (9-fold ) (Figure 4B). These, the increased vp14 mRNA
abundances in the roots coincidences with increased root chloride and sodium tissue
concentrations (Figure 3). Further research for the elucidation of the link between the
presence of chloride or sodium in the roots and the activation of the expression of the vp14
gene that codes for an enzyme that catalyses an early limiting step in controlling ABA
biosynthesis in the roots is needed. Which are the factors down-stream of the ions that link the
presence of the ions with ABA biosynthesis?
Root-to-shoot translocation of root-sourced ABA
The relative qRT-PCR-based quantification of the vp14 mRNA in roots and the leaf showed
that the accumulation of ABA in the leaf under conditions of either 50 mM NaCl, Na-
glucosamide, or glucosamine-Cl, cannot be ascribed to the expression of the 9-cis-
epoxycarotenoid dioxygenase gene in the leaves. More likely is an ABA de novo synthesis in
the roots and a subsequent acropetal transport towards the shoot, which is also known to occur
under salt stress (Jaschke et al., 1997; Hartung et al., 2002; Sauter et al., 2006; Jiang and
Hartung, 2008). The idea that ABA is synthesized in the roots before this root-sourced ABA
is translocated to the shoot is well established under the condition of salinity, however,
isotope tracers would be necessary to definitively prove the presence of root-shoot
translocation of ABA. Nevertheless, there is a very strong indication for root-to-shoot 15
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transport of root-sourced ABA. This is assumed for two reasons. First, the transcription of the
9-cis-epoxycarotenoid dioxygenase gene viviparous 14 (Zmvp14) is activated in the roots of
maize (Figure 4B), although a concomitant decrease in root ABA concentration was detected
(Figure 1B). Secondly, shoot ABA increases (Figure 1A), although vp14 transcription is not
affected in the shoot (Figure 4A). Overall, the novelty of our data is that under the conditions
studied here (two-hour of 50 mM salts) the activation of the ABA synthesizing gene vp14 in
the roots and the accumulation of ABA in the shoot can be a response to the chloride and
sodium component of NaCl.
Cl-- and Na+-related accumulation of ABA correlates with maintenance of turgor while
transpiration decreases
The plants that were exposed to 50 mM NaCl, Na- glucosamide, or glucosamine-Cl, showed a
higher leaf ABA concentration (Figure 1) and a reduced transpiration (Figure 2) at two hours
after initiation of the treatment. The increase in the guard cell regulating hormone ABA may
be functional in reducing the transpiration. This is important for avoiding transpiration-driven
water loss which would otherwise greatly augment wilting under conditions of salt stress.
Wilting is likely to occur if the transpiration rate is not reduced through ABA-signalling
networks, because the tissue accumulation of salts causes dehydration and cell death (Flowers
et al., 1991). Here, the salt-exposed plants that showed a reduction in the transpiration rate
(Figure 2; grey bars) together with an accumulation of leaf ABA (Figure 1) were able to
maintain relative turgidity (Figure 2; black line). This indicates that the accumulation of ABA
might be an effective means to lower transpiration in order to safe water for the maintenance
of turgor pressure during the onset of NaCl-salinity, well before salt ions accumulate to toxic
level. A reduction in transpiration rate is also useful for avoiding excess accumulation of salts
under conditions when salt concentration in the environment is high, because uptake of these
salts is driven by transpiration. 16
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Possible model indicating possible link between chloride-induced ABA increase and
reduced net xylem loading of chloride
In contrast to crops with halophytic ancestors such as sugar beet (Beta vulgaris), maize is a
glycophytic plant that is moderately sensitive to chloride (Parker et al., 1985). This means that
maize can only maintain growth and development when chloride is (i) included (stored) in the
vacuoles or in tissues far away from the active site of photosynthesis (e.g. epidermis), or (ii)
when chloride is excluded from being transferred from root to shoot. For the latter, Henderson
et al., (2014) explained in their working model for chloride exclusion in grapevine (Vitis
vinifera), that the passage of chloride from the root symplast into the root xylem is the critical
process that must be restricted in order to limit the accumulation of chloride in the shoot.
These authors explain two mechanisms that facilitate the exclusion of chloride from the root
xylem. First, a reduced abundance or inhibition of the activity of proteins that function in root
xylem loading of chloride. Secondly, an increase in the abundance of membrane proteins from
the cortical or epidermal root cells that facilitate chloride efflux into the soil solution. The
finding that the ABA receptor VvPYL1/RCAR11 was significantly more abundant in roots of
grapevine that were able to exclude chloride from being taken up into the laminae was
interpreted as indication that mechanisms of chloride exclusion might be induced by a
increases in ABA concentration (Henderson et al., 2014). Besides not investigated here, we
postulate that the chloride-induced increase in the transcript abundance of vp14 in the roots
might be related to such an ABA-related mechanism of chloride exclusion in maize. Since
maize is, just as grapevine, moderately sensitive to chloride, the avoidance of chloride uptake
and translocation into the shoots would favour growth. This is mode of action is suggestive
and awaits clarification.
Conclusion17
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It was investigated as to whether chloride and sodium induce the accumulation of ABA at
two-hour after exposure to NaCl; a phase that is well before (i) NaCl accumulates to toxic
tissue concentrations and (ii) cells dehydrate. A novelty is that the results clearly show that
ABA accumulates in response to both ions while transpiration decreases but turgor is
maintained stable. Surprisingly, ABA accumulation is more pronounced in response to
chloride. During this early time point, leaf cell turgor was not yet reduced, showing that an
osmotic stress-induced cell dehydration, which is well known to activate ABA biosynthetic
genes, has not yet occurred. The relative quantification of the transcript abundance of the 9-
cis-epoxycarotenoid dioxygenase gene viviparous 14 (Zmvp14), which catalyses the key
regulatory step of the biosynthesis of ABA, may indicate that the chloride- and sodium-
induced accumulation of leaf ABA might be caused by a de novo synthesis in the roots and a
subsequent and fast translocation towards the shoots, this has to be clarified by further
analyis. The finding that besides sodium chloride is also instrumental in the modulation of
tissue concentrations of ABA is relevant because aspects of chloride are largely neglected in
research efforts that aim to understand the stress response of maize to NaCl. The fact that
chloride adjusts tissue concentrations of a plant hormone that conveys a myriad of functions
reveals the need for more research into specific aspects of chloride.
The sodium- and chloride-induced accumulation of ABA, well before sodium and chloride
accumulate to toxic concentrations in the tissues, could be effective in lowering the root to
shoot transfer of those ions. This is assumed for two reasons: First, both ions travel via
transpiration and ABA will reduce transpiration during salt stress. Second, ABA might be
involved in the activation of an ion extrusion mechanisms as discussed in the preceding
section.
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Conflict of interest: We declare that there are no conflicts of interest.
Author contribution statement: CMG conceived the study, conducted some experiments
and analysed the data. JLM quantified ABA. GB and CZ did qRT-PCR measurements. CMG,
CZ, JLM interpreted the data and wrote the manuscript. GB modified the manuscript. All
authors reviewed and approved the manuscript.
Acknowledgement: The authors thank Xudong Zhang for helping with evaluation of qRT-
PCR data. Freia Benade, Technische Universität Dresden, is acknowledged for technical
assistance.
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Figure legends
Figure 1. Abscisic acid (ABA) tissue concentration. (A), growing leaf number 4; (B), root.
Mean ± SE of five independent replications (technically replicated in triplicate). Statistically
significant mean differences (P ≤ 0.05) are indicated by different letters. Multiple t-tests
adjusted according to Bonferroni–Holm; dw, dry weight.
Figure 2. Transpiration rate and relative turgidity in growing leaf number 4. Grey bar,
leaf transpiration rates (mmol H20 m-2 s-1, as shown on primary ordinate). Black squares,
relative turgidity (shown on secondary ordinate). Mean ± SE of five independent replications
(technically replicated in triplicate). Statistically significant mean differences (P ≤ 0.05) are
indicated by different letters. Multiple t-tests adjusted according to Bonferroni–Holm.
Figure 3. Sodium (Na+) or chloride (Cl-) tissue concentration. (A) & (B), growing leaf
number 4; (C) & (D), root. Mean ± SE of five independent replications (technically replicated
in triplicate). Statistically significant mean differences (P ≤ 0.05) are indicated by different
letters. Multiple t-tests adjusted according to Bonferroni–Holm; dw, dry weight.
Figure 4. mRNA abundance of the viviparous 14 (vp14) gene. (A), growing leaf number 4;
(B), root. Mean ± SE of five independent replications (technically replicated in triplicate).
Increases in transcript abundance shown as fold-changes, relative to the control. Statistically
significant mean differences (P ≤ 0.05) are indicated by different letters; ns, not significant.
Multiple t-tests adjusted according to Bonferroni–Holm.
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Figure files
Figure 1
0
2.5
5
7.5
10
Control PEG 6000 Sodiumglucosamide
NaCl Glucosaminechloride
ABA
(µg
g-1
DW
)
Shoot
0
2.5
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7.5
10
Control PEG 6000 Sodiumglucosamide
NaCl Glucosaminechloride
ABA
(µg
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DW
)
Root
aa
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B
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Figure 2Tr
ansp
iratio
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te (m
mol
H20
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s-1 )
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96
100
0
3
6
9
Control PEG 6000 Sodiumglucosamide
NaCl Glucosaminechloride
Rel
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ity
a ab b b b
nsns ns ns
Figure 3
0
2
4
6
8
Control PEG 6000 Sodiumglucosamide
NaCl Glucosaminechloride
Na+
(mg
g-1
DW
) Shoot
0
5
10
15
Control PEG 6000 Sodiumglucosamide
NaCl Glucosaminechloride
Cl- (
mg
g-1
DW
)
Shoot
0
2
4
6
8
Control PEG 6000 Sodiumglucosamide
NaCl Glucosaminechloride
Na+
(mg
g-1
DW
) Root
0
5
10
15
Control PEG 6000 Sodiumglucosamide
NaCl Glucosaminechloride
Cl- (
mg
g-1
DW
)
Root
a a ab b
a a a
bb
a a a
bb a a a
b b
A B
C D
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Figure 4
0
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ativ
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ized
vp14
Exp
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B
a a
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PEG 6000 Sodiumglucosamide
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ns ns ns ns
.
27
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Supplementary material
Supplementary material 1. Sequencing summary of Zmvp14 PCR product. The specificity of the Zea mays viviparous 14 (Zmvp14) primer pair
was demonstrated by sequencing the real-time quantitative RT-PCR product. The primer pair used for the amplification is shown in column 1. The
corresponding DNA sequencing result is presented in column 2. The sequences were alignment against NCBI’s reference mRNA sequences
(refseq_rna; blastn), columns 3-7. Search was limited to Zea mays L. (taxid:4577). Column 3, Genbank accession numbers. Column 4, description
of the amplificate. Parameters for statistical quality of the hit are presented in columns 5 - 7.
Zea mays viviparous 14 (Zmvp14) primer pair
Sequencing summary of real-time quantitative RT-PCR products(N, any base; primer sequences
highlighted grey)
Significant alignments of sequences(NCBI blastn on Zea mays L. [taxid:4577]; database: refseq_rna)
Genbank sequence ID Description Max score/
Total scoreQuery coverag. (%)/ Ident (%)
E value
f 5′–3′ TTCTCGGAGGAGGAACAGAGGA)
r 5′–3′ CCAACTGTAACTCTGGTGTGCG
TTTCTCGGAGGAGGAACAGAGGAGCCAGCCATGGATCAGGGGAGAAGTCACCAGAGGGAGCCCAGATCAGTTCCCCGGGGTCTTCNCTGTCNCCNNCNCACNGCACACNAGAGTTACAGTTGGA
NM_001112432.3
No other Blast hits found
Zea mays viviparous14
(vp14), mRNA187/187 99/ 94 4e-47
28
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650
Supplementary material 2. Specificity of the vp14 primer pair. After real-time quantitative qRT-PCR measurement, PCR products were
separated on agarose gels. Only one single DNA band was visualized. In-silico analysis using the Primer-BLAST software predicted a band size of
~ 117 bp. Gels contain 2% agarose, Tris-borate-EDTA, 5 µl SYBR Safe pro 100 ml Gel (SYBR® Safe DNA Gel Stain, Invitrogen).
1000 bp
500 bp
250 bp
200 bp
150 bp
100 bp
50 bp
Leaf Root
Glucosamine-Cl
Na-glucosamide
PEG 6000Control NaCl Glucosamine-Cl
Na-glucosamide
PEG 6000Control NaCl
29
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655656
657
Supplemental Material
Quantification of glucosamine and glucosamide
Glucosamine and its amide, glucosamide, in finely ground plant samples were extracted with chloroform:methanol (3:7, v ⁄ v) on ice for 30 min. Homogenates were then extracted in 2 mL ddH2O, evaporated in a SpeedVac, and dissolved in 2 mL of ddH2O. Glucosamine and glucosamide were analyzed as o-phthalaldehyde derivatives on a reversed-phase C18 column using a HPLC system as described by Ruan et al (2010). Standards were prepared from D-(+)-Glucosamine hydrochloride and its D(+)-glucosamid. (Ruan, J., Haerdter, R., Gerendás, J., 2010 Impact of nitrogen supply on carbon/nitrogen allocation: a case study on amino acids and catechins in green tea [Camellia sinensis (L.) O. Kuntze] plants. Plant Biol. 12:724–734.)
Supplemental Table 1. Glucosamine and glucosamide concentration in mg per 100 g dry weight. Multiple contrast tests reveal no mean differences for each factor group. SE, standard error of the mean.
Treatment Glucosamine mg/100 g DW Glucosamide mg/100 g DWLeaf Root Leaf Root
Control 12.78 ± 0.28 SE a 3.45 ± 0.08 SE a 2.37 ± 0.07 SE a 1.18± 0.08 SE a
Glucosamine-chloride 13.24 ± 0.53 SE a 2.78 ± 0.10 SE a 1.92 ± 0.12 SE a 0.95± 0.04 SE a
Sodium-glucosamide 11.16 ± 0.82 SE a 3.29 ± 0.07 SE a 2.11 ± 0.08 SE a 1.09± 0.07 SE a
30
658
659
660661662663664665
666
667
668669
670