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\begin{document}

\title{Plant low-K responses are partly due to Ca and the low-K biomarker
putrescine does not protect from Ca side effects but acts as a metabolic
regulator}



\author[1]{Jing Cui}%
\author[2]{Marlene Davanture}%
\author[3]{Emmanuelle Lamade}%
\author[4]{Michel Zivy}%
\author[2]{Guillaume Tcherkez}%
\affil[1]{ANU}%
\affil[2]{Affiliation not available}%
\affil[3]{CIRAD}%
\affil[4]{INRAE Moulon}%


\vspace{-1em}



  
  \date{May 22, 2024}


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\selectlanguage{english}
\begin{abstract}
Effects of K deficiency have been investigated for several decades and
recently, progress has been made in identifying metabolomics signatures
thereby offering potential to monitor the K status of crops in the
field. However, effects of low K conditions could also be due to the
antagonism with other nutrients like calcium (Ca) and the well-known
biomarker of K deficiency, putrescine, could be a response to Ca/K
imbalance rather than K deficiency. We carried out experiments in
sunflower grown at either low or high K, at high or low Ca, with or
without putrescine added to the nutrient solution. Using metabolomics
and proteomics analysis, we show that a significant part of the low-K
response such as lower photosynthesis and N assimilation, is due to
calcium and can be suppressed by low Ca conditions. Putrescine addition
tends to restore photosynthesis and N assimilation but but aggravates
the impact of low-K conditions on catabolism. We conclude that (i)
effects of K deficiency can be partly alleviated by the use of low Ca
and not only by K fertilization, and (ii) in addition to its role as a
metabolite, putrescine participates in the regulation of the content in
enzymes involved in carbon primary metabolism.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Potassium (K) is an essential nutrient (up to 5\% of plant dry weight)
involved in crucial physiological processes such as electrochemical
homeostasis, stomatal aperture and enzyme catalysis (Wang \& Wu, 2013;
Ansch\selectlanguage{ngerman}ütz \emph{et al.} , 2014). K deficiency inhibits plant growth and
primary production and therefore, intense efforts are being devoted to
improve K acquisition by plants (Shin, 2014; Rawat \emph{et al.} ,
2016). K-deficient soils are relatively common in countries of the
intertropical region, such as Brazil, Equatorial Africa, China and
South-Eastern Asia (like Indonesia), and Australia (\emph{www.fao.org})
and thus important crops like oil palm, sunflower, cotton, or rice are
concerned. In addition, K-deficient areas have variable geological
substratum and soil total base content (in particular exchangeable
calcium), and as a result background nutrient (ionic) conditions that
accompany K deficiency are highly variable. For example, in Sumatra
(Indonesia) where oil palm is cultivated, both Ca-poor (histosol) and
Ca-rich (cambisol) soils can be found, and such a difference in Ca has
an important impact on K fertilization decisions.

Symptoms of K deficiency in plants have been studied for more than fifty
years and include metabolic effects such as an inhibition of glycolysis
due to a decrease in pyruvate kinase activity, as well as accumulation
of typical metabolites such as putrescine (Jones, 1961; Jones, 1966;
Okamoto, 1966; Freeman, 1967; Okamoto, 1967; Okamoto, 1968; Besford \&
Maw, 1976; Armengaud \emph{et al.} , 2009; Hussain \emph{et al.} , 2011;
Sung \emph{et al.} , 2015). Metabolomics analyses of K deficiency
in\emph{Arabidopsis} showed a reconfiguration of amino acid and organic
acid synthesis, consistent with a change in nitrogen assimilation (in
favour of neutral amino acids), and an inhibition of the most
K-sensitive enzyme, pyruvate kinase, thereby leading to altered
glycolytic metabolism (Armengaud \emph{et al.} , 2009). Recently,
metabolic changes caused by low K availability have been described in
details in sunflower, and it has been shown that in addition to the
well-known build-up of putrescine, low K induced a strong increase in
respiratory CO\textsubscript{2} efflux and modified the flux through the
C\textsubscript{5}-branched acid pathway; furthermore, the
natural\textsuperscript{15}N/\textsuperscript{14}N isotope composition
(\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{15}N) in leaf compounds showed that there was a
change in nitrate circulation, with less nitrate influx to leaves under
low K (Cui \emph{et al.} , 2019a).

Importantly, as pointed out recently (Cui \emph{et al.} , 2020), K
deficiency is not associated with a general decrease, but actually leads
to a significant increase in cation load, which comes from the
considerable increase in Ca\textsuperscript{2+} (and occasionally in
Mg\textsuperscript{2+}) in tissues. In fact, there is an antagonism
between K\textsuperscript{+} and Ca\textsuperscript{2+} (Dibb \&
Thompson, 1985; Jakobsen, 1993; Daliparthy \emph{et al.} , 1994), and
the K \emph{x} Ca interference in fertilization has been documented for
nearly 50 years in many crops (such as poplar, sugarcane, sunflower,
rapeseed, tobacco, oil palm, soybean, castor bean or wheat). Also, if
K-deficiency occurs under low Ca conditions, typical K symptoms such as
putrescine accumulation are partly suppressed (Richards \& Coleman,
1952; Coleman \& Richards, 1956). Therefore, despite the role of
Ca\textsuperscript{2+} in signaling to remobilize vacuolar
K\textsuperscript{+} ions under low K conditions (Amtmann \& Armengaud,
2007; Pandey \emph{et al.} , 2007; Tang \emph{et al.} , 2020), some of
the commonly observed K deficiency symptoms probably reflect the
response to a disequilibrium in external (soil) cation composition, in
which Ca\textsuperscript{2+} is over-represented. To our knowledge, this
question has never been tackled directly. In a recent meta-analysis of
papers dealing with the effect of nutrient antagonism on yield, the
antagonism between K and Ca appears to be understudied as compared to
K\emph{x} Mg antagonism (Rietra \emph{et al.} , 2017). Also, the
detailed metabolic responses to K availability when Ca is varied have
not been documented. In this context, putrescine is an interesting
biomolecule, since its role under K deficiency has been suggested to be
to both mitigate reactive oxygen species (ROS) production --and thus the
down-regulation of mitochondrial damage-- and participate in cellular
Ca\textsuperscript{2+} homeostasis (Cui \emph{et al.} , 2020).

To clarify these aspects, we cultivated sunflower plants under different
K availability conditions (low, 0.2 mM, and high, 4 mM), under low or
high Ca conditions, with or without addition of putrescine (experimental
design detailed in Fig. S1). We carried out physiological, ionomics,
metabolomics and proteomics analyses and also
used\textsuperscript{15}N-labelling to assess nitrate absorption. Our
objectives were to assess whether \emph{(i)} some of the typical
metabolic symptoms of K deficiency could actually be suppressed by the
use of low Ca conditions, and \emph{(ii)} putrescine addition alleviates
some metabolic effects of K deficiency or on the contrary, triggers a
low-K response. Our results show a considerable effect of Ca on
metabolism, low Ca conditions compensating for some of low-K symptoms.
Putrescine addition is found to improve N nutrition but exaggerates
glycolysis inhibition, suggesting that it plays a dual role under K
deficiency.

\textbf{Material and methods}

\emph{Plant material and photosynthesis}

Sunflower (\emph{Helianthus annuus} L.) var. XRQ was sown directly in
sand (washed with distilled water) in the greenhouse, using 7-L pots.
Growth conditions were: 12/12 h photoperiod, 25/18\selectlanguage{ngerman}°C air temperature,
70/60\% relative humidity day/night. Photosynthetic parameters were
measured using a portable open system Li-Cor 6400 XT. Net assimilation
(\emph{A} ) and conductance reported in Fig. 2 was obtained under
saturating light (1500 \selectlanguage{greek}µ\selectlanguage{english}mol m\textsuperscript{-2}
s\textsuperscript{-1}PAR) at 400 \selectlanguage{greek}µ\selectlanguage{english}mol mol\textsuperscript{-1}
CO\textsubscript{2} and 21\% O\textsubscript{2}. Night respiration
(\emph{R} \textsubscript{n}) was measured after photosynthesis
measurements on dark-adapted leaves (30 min) at 400 \selectlanguage{greek}µ\selectlanguage{english}mol
mol\textsuperscript{-1} CO\textsubscript{2}, 21\% O\textsubscript{2} and
25\selectlanguage{ngerman}°C.

\emph{Nutrient conditions and waterlogging}

The nutrient solution was after (Cui \emph{et al.} , 2019a), where the
amount of K\textsuperscript{+} was varied by changing the amount of KCl.
Two K availability conditions were used here: ``low K'' (0.2 mM) and
``high K'' (4 mM). The amount of nitrate and phosphate (in mM) in the
nutrient solution was kept constant throughout experiments (nutrient
solution composition in Table S1). Nitrate concentration of the nutrient
solution was 15 mM to cover plant N needs and avoid any N-limitation
effects. The amount of Ca\textsuperscript{2+} (low {[}0.4 mM{]}, and
high {[}4 mM{]}) was adjusted my changing the balance between Ca-nitrate
and Na-nitrate (Table S1). Putrescine was added at a concentration of
0.4 mM. Taken as a whole, there were six nutrient conditions: low K and
high Ca (LK-HCa), high K and high Ca (HK-HCa), low K and low Ca
(LK-LCa), high K and low Ca (HK-LCa), low K and high Ca with putrescine
addition (LK-HCa+P), and high K and high Ca with putrescine addition
(HK-HCa+P). It should be noted that here, the condition low Ca +
putrescine (under either low or high K) has not been used because the
objective was to assess whether putrescine addition can compensate for
the effects of high Ca. In figures, high Ca, low Ca and high Ca +
putrescine conditions are shown with blue, green and pink shades,
respectively.

\emph{Experimental design and sampling}

The experimental design is schematized in Fig. S1a. After emergence (1
week after sowing), plants were cultivated for 2 weeks under specific
nutrient conditions (described above). Three weeks after emergence,
plants had one true leaf pair, and after four weeks, there were two leaf
pairs. These two pairs are referred to as ``young'' and ``old'' (Fig.
S1b). There were two sampling campaigns: sampling 1 two weeks after
emergence (3 weeks old plants), and sampling 2 three weeks after
emergence (4 weeks old plants). Two weeks and four days after emergence,
half of the plants were labelled with \textsuperscript{15}N-nitrate
(nutrient solution of exactly the same composition, with just nitrate
replaced to its \textsuperscript{15}N form) while the other half was
kept at natural abundance. The day before sampling, photosynthesis and
dark respiration (reported in Fig. 2) were measured on the same plants.
Upon sampling, plants were measured for size, fresh weight, leaf
surface, and dissected and kept in liquid nitrogen for analyses. Roots
were sampled after having removed sand and washed with water. In
practice, roots were washed, rapidly dried with absorbing paper and
quenched in liquid nitrogen within 2-3 min. Extractions for metabolomics
and proteomics analyses were performed on fresh material. Isotope and
ionomics analyses were performed on freeze-dried material.

\emph{Omics}

Ionomics and metabolomics were performed ICP-OES and by GC-MS,
respectively, as in (Cui \emph{et al.} , 2019a). Proteomics analysis was
carried out as in (Cui \emph{et al.} , 2019b) on total protein obtained
by trichloroacetate-acetone extraction. Peptide identification and
quantitation was done using the same workflow, except that protein
abundance reported here are from extracted chromatograms (XIC). Details
are provided in Supplementary methods S1.

\emph{Isotope analysis and \%N}

Freeze-dried samples (powdered total organic matter) were weighed in tin
capsules, and \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{15}N values and \%N were measured using
an elemental analyser (Carlo-Erba) coupled to isotope ratio mass
spectrometer (Isochrom, Elementar) run in continuous flow. All sample
batches included standards (glycine, +0.66%0; cysteine, +7.78%0;
previously calibrated against IAEA standards glutamic acid USGS-40 and
caffeine IAEA-600) each ten samples.

\emph{Statistics}

Five replicates were done for all conditions for ionomics, isotopes,
metabolomics and four replicates were done for proteomics. Supervised
multivariate analysis of omics data was carried out by orthogonal
projection on latent structure (OPLS) (Bylesj\selectlanguage{ngerman}ö \emph{et al.} , 2007)
with Simca (Umetrics), using K, Ca and putrescine as predicted Y
variables and metabolites (or proteins) as predicting X variables. The
absence of statistical outliers was first checked using a principal
component analysis (PCA) to verify that no data point was outside the
99\% confidence Hotelling region. The goodness of the OPLS model was
appreciated using the determination coefficient R² and the predictive
power was quantified by the cross-validated determination coefficient,
Q². The significance of the statistical OPLS model was tested using a \selectlanguage{greek}χ\selectlanguage{ngerman}²
comparison with a random model (average~±~random error), and the
associated \emph{P} -value (\emph{P} \textsubscript{CV-ANOVA}) is
reported (Eriksson \emph{et al.} , 2008). Best discriminating features
were identified using volcano plots whereby the logarithm of the\emph{P}
-value obtained in univariate analysis (two-way ANOVA; factors used: K
\emph{x} Ca or K \emph{x} putrescine) was plotted against the rescaled
loading (p\textsubscript{corr}) obtained in the OPLS. In such a
representation, best biomarkers have both maximal --log(\emph{P} ) and
p\textsubscript{corr} values. Univariate analysis of statistical classes
in bar plots was performed using a two-way ANOVA (Fisher statistics),
with a threshold of \emph{P} as indicated in figure legends. When all
conditions were compared at once, a one-way ANOVA was conducted.

\textbf{Results}

\emph{Elemental composition}

The effect of nutrient conditions was first checked on leaf and root
elemental composition analyzed by ICP-OES (Fig. 1). As expected, there
was a clear and substantial effect of K conditions on tissue K content
in all organs. K conditions impacted on tissue Ca, Na, Mg demonstrating
the antagonism between major cations (Fig. 1a-c). But conversely, there
was no effect at all of Ca (and putrescine) on K tissue content. Low Ca
conditions were associated with lower Ca content in all tissues and this
was compensated for by higher Na content. There was little effect of K
on micronutrients, except for Fe which appeared to be more abundant at
low K in roots (other micronutrients are not shown). Taken as a whole,
when all cations contents were used to calculate the total positive
charge (cation charge balance), there was no effect of K and Ca
conditions in roots. By contrast, there was a very clear effect in
leaves which exhibited a much higher total cation charge balance at low
K (Fig. 1d).

\emph{Physiological parameters}

Total biomass, leaf photosynthesis (under standard conditions) and
respiration were also measured to appreciate the general impact of
nutrient conditions on carbon assimilation (Fig. 2). After 3 weeks under
high Ca, shoot and root biomass was significantly affected by K
conditions. Conversely, the effect of Ca and putrescine was modest under
high K. After 4 weeks, the effect of K conditions was very clear, with
no effect at all of Ca and putrescine in shoots. In roots, low Ca and
putrescine increased biomass very slightly (Fig. 2a). Net
CO\textsubscript{2} assimilation was significantly lower at low K under
high Ca, and this effect disappeared at low Ca in young leaves (Fig.
2b). Putrescine was also beneficial to photosynthesis and suppressed the
effect of low K conditions in young leaves. In old leaves, K
availability always had an effect on photosynthesis, and putrescine
increased photosynthesis only slightly under low K. In old leaves,
changes in photosynthesis were mostly caused by the effect of nutrient
conditions on conductance, the variations of which paralleled that of
photosynthesis (Fig. 2c). In young leaves, the effect of nutrient
conditions (K in particular) on conductance was not mirrored by
photosynthesis showing that photosynthetic capacity was also affected.
As expected, low K was associated with higher respiration rates (Fig.
2d). Surprisingly, low Ca conditions further increased respiration rates
in all leaves.

\emph{Nitrogen assimilation}

Nitrogen assimilation was examined using elemental N content analysis
and \textsuperscript{15}N-nitrate labelling to measure nitrate
absorption and distribution in shoots and roots (Fig. 3). In all
tissues, N elemental content was significantly lower at low K, showing
the general inhibition of N assimilation under low K availability (Fig.
3a). Low Ca conditions resulted in slightly lower N content in old
leaves under high K. Putrescine was beneficial to the N content in young
leaves under low K. N absorption measured
via\textsuperscript{15}N-labelling was much lower under low K, and this
was alleviated to some extent by putrescine. Interestingly, low Ca was
beneficial to N absorption at low K but detrimental at high K,
suggesting that the balance between cation species was essential for
nitrate absorption by root cells.

\emph{Metabolic signature of nutrient conditions}

The metabolic impact of nutrient conditions was analyzed using GC-MS
profiling. 178 (leaves) and 187 (roots) analytes were identified and
quantified. The multivariate statistical analysis by OPLS allowed facile
discrimination of samples in both leaves (Fig. 4a) and roots (Fig. 5a).
The multivariate statistical model was good and robust as shown by high
R² and Q² values (0.94 and 0.87, respectively, in both leaves and
roots), and highly significant (\emph{P}
\textsubscript{CV-ANOVA}\textless{}10\textsuperscript{-30} (K),
\textless{} 10\textsuperscript{-22} (Ca) and
\textless{}10\textsuperscript{-11}(putrescine)). Axis 1 was driven by K
conditions, while Ca and putrescine conditions were driven by both axes
2 and 3, showing some interaction in their metabolic effect. A specific
analysis of putrescine content is provided in Fig. S2. When all samples
were pooled together (old and new leaves), putrescine addition was not
found to cause an important increase in tissue putrescine pool in leaves
(on average, × 2) and roots (on average, × 1.25). In roots, putrescine
was 2-fold lower under low Ca.

In leaves, low K conditions were associated with many highly significant
changes, typically sugar (arabinose, ribose), putrescine, citrate and
ascorbate accumulation and a decrease in glucose 6-phosphate, inorganic
phosphate (Pi), threonate, amino acids (glutamate, aspartate) and their
derivatives (cystathionine, \selectlanguage{greek}β-\selectlanguage{english}alanine) (Fig. 4b). Ca conditions were not
associated with many significant metabolites and only one metabolite
increased by high Ca was above the Bonferroni threshold, tartarate
(reduced form of oxaloacetate) (Fig. 4c). Low Ca was associated with an
accumulation of organic acids of the tricarboxylic acid pathway (TCAP),
such as citrate, isocitrate, fumarate and aconitate. Under low Ca, there
was also more Pi and glutamine, suggesting a beneficial effect of low Ca
on N and P nutrition. Very few metabolites were affected above the
Bonferroni threshold by putrescine addition (Fig. 4d): putrescine
homoserine and serine (increased) and cellobiose and gulonate
(decreased). Interestingly, with a \emph{P} -value lower than 0.001,
tartarate appeared to be decreased by putrescine (thus counteracting the
effect of high Ca), while sucrose increased.

Like in leaves, many metabolites were significantly affected by low K
conditions in roots, including sugars (maltose, xylose, allose,
gentiobiose), putrescine and ascorbate (increased), and aspartate,
threonate, cystathionine, \selectlanguage{greek}β-\selectlanguage{english}alanine (decreased) (Fig. 5b). Also, low Ca
led to an accumulation in organic acids (glutarate, isocitrate,
succinate, aconitate, citrate, citramalate) (Fig. 5c). Interestingly,
spermidine (derivative of putrescine) was found to increase at high Ca.
Very few metabolites were affected by putrescine addition: putrescine,
glutamine, asparagine and methylaspartate (increased), and glutamine,
fumarate, xylulose, glucuronate (decreased) (Fig. 5d).

Despite the similarities in the response to K, Ca or putrescine,
metabolites associated with a significant K \emph{x} putrescine effect
were different in leaves and roots. Typically, under low K in leaves,
putrescine addition led to a decrease in several sugars, polyols and
shikimate, and an increase in glycolate (and, of course, putrescine)
(Fig. 4e). In roots, putrescine addition decreased isoprenoids (phytol,
tocopherol), sugars and sugar derivatives (2-oxogluconate, rhamnose,
xylulose) (Fig. 5e).

\emph{Proteome changes caused by nutrient conditions}

Proteomics analyses were carried out, and the full list of significant
proteins in tabulated in Table S2. A summary showing proteins involved
in primary CNS metabolism is provided in Table 1. As expected, in
leaves, many proteins involved in photosynthesis and photorespiration
(Rubisco, Rubisco activase, phosphoribulokinase, serine glyoxylate
aminotransferase, etc.) were significantly affected by K conditions,
showing the effect of K availability on the development of the
photosynthetic machinery. Importantly, key enzymes of catabolism were
more abundant under low K (isocitrate dehydrogenase, aconitase, malic
enzyme, pyruvate dehydrogenase, etc.) showing the up-regulation of
respiration. Also, low K was associated with an increase in carbamoyl
phosphate synthase (required for arginine and polyamine synthesis), and
a decrease in pyruvate kinase. The decrease in pyruvate kinase content
reflects the down-regulation of this K-dependent metabolic step when K
is limiting. In roots, a small number of proteins were significant, and
included also pyruvate kinase and proteins directly involved in N
assimilation (nitrate transporters, glutamine synthetase), all decreased
under low K.

High Ca impacted negatively on pyruvate kinase and several enzymes of
photosynthesis in leaves. In roots, only two enzymes had a\emph{P}
-value above the Bonferroni threshold and these included pyruvate kinase
(decreased at high Ca). Putrescine addition caused an increase in the
content of enzyme involved in photosynthesis and glucose oxidation
(glucose/ribitol dehydrogenase and glucose 6-phosphate dehydrogenase) in
leaves. In roots, putrescine addition caused a decline in proteins
involved in nitrogen assimilation: nitrate transporters, aspartate
aminotransferase and glutamine synthetase.

\emph{Effect of nutrient conditions on selected proteins}

A focus on enzymes involved in pyruvate metabolism is provided in Fig.
6. As mentioned above and shown in Table 1, pyruvate kinase was
down-regulated under low K and this effect could be alleviated by low Ca
conditions in both roots and leaves. Putrescine addition tended to
increase the content in pyruvate kinase under low K but decreased it at
high K. For other enzymes (NADP-dependent malic enzyme, aconitase,
phospho\emph{enol} pyruvate carboxylase), both low Ca and putrescine
tended to attenuate the increase in protein content observed under low K
in leaves. In roots, low Ca has little effect on these enzymes while
putrescine increased their content at high K.

Transporters and channels that were both detected and associated with
significant changes are shown in Figs. S3 and S4. As expected, a high
affinity K transporter was induced at low K in roots, and in both leaves
and roots, the subunit \selectlanguage{greek}β\selectlanguage{english}2 of the voltage dependent aldo/keto
reductase-potassium channel was downregulated at low K + high Ca and
upregulated at low K + low Ca. In leaves, low K caused a decline in two
ABC cassette containing proteins (TAP1 and cABCI6) and an ammonium/urea
transporter, suggesting changes in nitrogen/protein homeostasis. In
roots, nitrate transporters were upregulated at low K + high Ca, high K
+ low Ca and were clearly downregulated by putrescine.

The two-way ANOVAs carried out to look at significant proteins were used
to visualize proteins associated with specific interactions effects
(Fig. S5). In the group of roots proteins upregulated under high K + low
Ca, proteins involved in nitrogen assimilation appeared clearly (group
1, Fig. S5a). In leaves, some enzymes of catabolism (isocitrate
dehydrogenase, aconitase) were specifically enhanced at low K + high Ca
(group 1, Fig 5b). Interestingly, many proteins of the photosynthetic
machinery (including magnesium chelatase, Rubisco and proteins of the
chloroplastic electron transfer chain) were downregulated in this
specific condition, suggesting that the effect of low K was mostly
driven by high Ca (group 2, Fig. 5b). K \emph{x} putrescine interaction
analysis clearly showed that, as found above, the downregulation of
pyruvate kinase was strong under low K + high Ca, and that putrescine
addition minimized differences between low K and high K for key
catabolic enzymes (Figs. 5c-d).

\textbf{Discussion}

Response mechanisms to K deficiency have been extensively studied and
include changes in metabolism (such as putrescine build-up), gene
expression and physiological parameters such as photosynthesis,
chlorophyll synthesis and growth. Our study is broadly consistent with
documented effects of low K conditions on metabolism (Ashley \emph{et
al.} , 2006; Armengaud \emph{et al.} , 2009; Wang \& Wu, 2013).
Typically, there were \emph{(i)} a general decline in photosynthesis due
to lower stomatal conductance and a down-regulation of the biosynthesis
of the photosynthetic machinery; \emph{(ii)} an inhibition of nitrate
absorption and assimilation; and \emph{(iii)} changes in respiratory
metabolism, with higher respiration rates and higher content in several
catabolic enzymes (such as malic enzyme) despite an inhibition of
glycolysis due to lower pyruvate kinase activity. The typical inhibition
of pyruvate kinase by low K conditions comes from the fact that this
enzyme uses K\textsuperscript{+} as a cofactor (Evans, 1963; Nowak \&
Mildvan, 1972) but also, as found here, a lower enzyme abundance as also
found in tomato (Besford \& Maw, 1976). Here, we have carried out a
dissection of metabolic responses to K availability when it interacts
with calcium and putrescine to better understand specific pathways
affected by K itself, and effects of Ca/K balance or putrescine
accumulation.

\emph{The response to low K is partly driven by Ca}

A common drawback in manipulating nutrient composition is the potential
change in other cations due to electro-neutrality, making overall
mineral conditions not comparable (Cramer \emph{et al.} , 1986). Here,
we have used low and high Ca conditions that were compensated for by
sodium (Na) for the chemical form of nitrate. However, low Ca conditions
were not associated with a drastic augmentation of the Na content in
tissues and thus an onset of salt stress: in fact, (\emph{i} ) Na always
remained a minor component in leaves; and \emph{(ii)} in roots, Na was
increased by about 20\% only under low Ca. Therefore, the effects we
found here under low Ca conditions were not caused by salinity.

Our results clearly demonstrate that amongst effects observed under low
K conditions, the decline in photosynthesis was in reality partly due to
Ca. In fact, in young leaves, low Ca conditions could restore some
photosynthetic activity (while it was not so in old leaves) (Fig. 2). In
addition, the decline in the content of many proteins involved in
photosynthesis could be compensated for by low Ca conditions (Fig. S5b).
Surprisingly, low Ca aggravated the increase in leaf dark respiration
triggered by low K conditions (Fig. 2), in agreement with TCAP
intermediates being higher under both low K and low Ca (Fig. 4), whereas
some proteins of respiratory metabolism were more abundant under low K +
high Ca, not low K + low Ca (such as aconitase, Fig. 6). This
contradiction is perhaps explained by \emph{(i)} changes in
post-translational modifications, \emph{(ii)} the action of effectors on
enzymes and/or \emph{(iii)} a lower ATP/O\textsubscript{2} efficiency of
mitochondrial metabolism. In particular, we note that low Ca conditions
led to a decline in Mg leaf content, and in principle, this must have
affected mitochondrial-cytosolic ADP/ATP exchange and interconversion
(Bligny \& Gout, 2017).

It is worth noting that unlike photosynthesis, low Ca conditions did not
compensate for the alteration of N metabolism at low K. In fact, low Ca
aggravated the decline in the content of root proteins involved in N
metabolism (Fig. S5a) and increased the content in non-aminated
precursors (2-oxoglutarate) (Fig. 5c). The reverse was true at high K,
where low Ca upregulated the content in proteins involved in root N
metabolism, including nitrate transporters. However, nitrate
assimilation measured using \textsuperscript{15}N-nitrate was increased
by low Ca under low K conditions, and decreased by low Ca at high K
(Fig. 3). This surprising result likely reflects the fact that under
K-deficient conditions, the general effect of low K impacts not only on
root development and protein synthesis but also on root cation balance
(increase in Ca\textsuperscript{2+} but low content in unicharged
species) and this leads to a decline in nitrate capture by root cells.
In fact, Ca\textsuperscript{2+} addition has been found to be
detrimental to nitrate influx in root cells (Kafkafi \emph{et al.} ,
1992; Aslam \emph{et al.} , 1995). Accordingly,
here\textsuperscript{15}N-nitrate absorption was found to be low. When
low Ca conditions are used, Na\textsuperscript{+} can partly substitute
for K\textsuperscript{+} (Fig. 1) and this restores nitrate absorption,
thus downregulating the synthesis of high affinity nitrate transporters.

Under high K conditions, such a role for Na\textsuperscript{+} ions is
unnecessary and essentially, nitrate absorption is accompanied by
K\textsuperscript{+} absorption. In cotton grown at 4 mM
{[}K\textsuperscript{+}{]}, increasing {[}Ca\textsuperscript{2+}{]} from
2 to 10 mM has only a small effect on nitrate (and K) absorption
(Leidi\emph{et al.} , 1991). Also in cotton grown at 2.5 mM
{[}K\textsuperscript{+}{]} in the presence of NaCl, low
{[}Ca\textsuperscript{2+}{]} (\textless{}0.25 mM) inhibits nitrate
absorption (Gorham \& Bridges, 1995). Similarly, nitrate absorption by
wheat seedling is inhibited by Ca deficiency (Minotti \emph{et al.} ,
1968). K and nitrate absorption requires sufficient
Ca\textsuperscript{2+} to maintain transmembrane electrochemical
gradient and therefore, low Ca has a negative impact on nitrate
absorption. It is also interesting that low Ca conditions at high K
inhibited nitrate translocation to shoots (Fig. 3), probably due to
similar ion imbalance in xylem sap loading and electroneutrality.

\emph{Can putrescine alleviate effects of K deficiency?}

Putrescine addition partly alleviated the effects of low K on glycolysis
(regulation of pyruvate kinase content under low K) (Figs. 6, S5c-d),
photosynthesis (Fig. 2) and N assimilation (Fig. 3), and typically
increased root glutamine and asparagine content (Fig. 5). However,
putrescine did not suppress the increase in leaf respiration at low K
(Fig. 2) despite the down-regulation of several enzymes involved in
respiratory metabolism (Fig. S5d). Diverse mechanisms have been
suggested to explain why putrescine is accumulated under low K,
including the regulation of mitochondrial ion channels to avoid
excessive oxidative damage and mitochondrial permeability transition
(Cui \emph{et al.} , 2020). Here, the addition of putrescine did not
have a clear effect on mitochondrial proteins, except for alternative
NAD(P)H dehydrogenase in roots (Table 1, Fig. S5).

The fact that putrescine (\emph{i} ) had rather contrasted effects
(different effects in roots and leaves, e.g. Fig. 6), and
\emph{(ii)}caused a decline in pyruvate kinase content at high K,
suggests that putrescine is not a molecule that only compensates for
cellular K scarcity but rather, is involved (or one of its products) in
low-K signaling, perhaps including root-to-shoot signaling. It has been
proposed that putrescine regulates metabolism via an increase or a
decrease in reactive oxygen species (ROS) depending on its concentration
and the potential involvement of polyamine oxidase, which generates
H\textsubscript{2}O\textsubscript{2} (Verma \& Mishra, 2005;
Zepeda-Jazo\emph{et al.} , 2011; Shelp \emph{et al.} , 2012; Zhang
\emph{et al.} , 2014). It is worth noting that if putrescine is
effectively oxidized, this can interact directly with Ca and K
absorption since K efflux and Ca influx in root cells have been found to
be regulated by ROS (Demidchik \emph{et al.} , 2003). Here, we did not
find any protein annotated as polyamine oxidase but found two peptides
associated with copper amine oxidase (also referred to as diamine
oxidase; CAO). Interestingly, putrescine addition caused an increase in
CAO in roots but not in leaves (Fig. S6). It is plausible that
putrescine addition thus led to an increased production of
NH\textsubscript{4}\textsuperscript{+} and
H\textsubscript{2}O\textsubscript{2} in roots, thereby triggering ROS
signaling and improving N nutrition (higher glutamine and asparagine
content in roots). In leaves, the most significant metabolites under
putrescine addition were serine and homoserine. Putrescine oxidative
deamination produces \selectlanguage{greek}γ-\selectlanguage{english}aminobutyrate semialdehyde further oxidized to
GABA (Shelp \emph{et al.} , 2012), which can be in turn incorporated
into the TCAP via the GABA shunt and thus feed the synthesis of asparate
(precursor of homoserine). That said, the simultaneous increase in
serine and homoserine also likely reflects the regulation of sulfur (S)
metabolism by putrescine (serine and homoserine are precursors of
cysteine and methionine, respectively) (illustrated in Fig. S7).

In effect, low K conditions reconfigured S metabolism. Under low K
availability, the sulphur elemental content has been found to change
significantly in sunflower (increase in leaves, decline in roots)
(Cui\emph{et al.} , 2019a) and in Arabidopsis, the tissue content in
sulphate, cysteine and \emph{O} -acetylserine decrease strongly
(Forieri\emph{et al.} , 2017). Here, we observed at low K an increase in
cysteine metabolism (increased cysteine synthase, Table 1, lower
cystathionine content, Fig 4) at the expense of methionine metabolism
(decreased methionine synthase and SAM synthase, Table 1, lower
methionine-to-cysteine ratio, Fig. S7). That is, cysteine was consumed
to synthesize homocysteine (trans-sulfuration onto homoserine), which
was then recycled back to cystathionine by cystathionine \selectlanguage{greek}β-\selectlanguage{english}synthase
(CBS), thereby consuming serine and avoiding methionine synthesis. Such
an effect of low K on S metabolism is very likely a consequence of the
inhibition of SAM synthase catalysis, which has been shown to be
K\textsuperscript{+}-dependent (Takusagawa \emph{et al.} , 1996).
Parenthetically, the inhibition of SAM synthase further exaggerates
putrescine accumulation since the conversion of putrescine to other
polyamines requires SAM. It is also interesting to note that
homocysteine production liberates pyruvate (Fig. S7). Therefore,
homocysteine production and recycling to cystathionine represents an
alternative pathway for pyruvate production (from serine). This is
clearly advantageous under low K conditions where pyruvate kinase is
inhibited.

Quite critically, the effect of low K on cysteine metabolism appeared to
be modulated by Ca and putrescine: low Ca led to an increase in
cystathionine \selectlanguage{greek}β-\selectlanguage{english}synthase in leaves, and putrescine addition caused a
decline in cysteine synthase in roots (Table 1). This suppression effect
by putrescine was probably sufficiently strong to explain why both
serine and homoserine increased. Also, putrescine impacted modestly on
enzyme content in leaves (the effect of putrescine and K
\emph{x}putrescine on SAM synthase and CBS was associated with a
\emph{P} -value (3.64 10\textsuperscript{-5} and 4.5
10\textsuperscript{-5}, respectively) just above the Bonferroni
threshold (3.54 10\textsuperscript{-5}), Table S2).

\emph{Perspectives}

Taken as a whole, our results show that amongst typical symptoms of K
deficiency, the alteration of photosynthesis can be in part compensated
for by low Ca nutrition, via the upregulation of proteins involved in
the photosynthetic machinery. Similarly, the inhibition by low K of
pyruvate kinase could be partly alleviated by low Ca conditions. By
contrast, low Ca could not compensate for changes in N assimilation,
likely due to complicated interactions with other ions
(Na\textsuperscript{+}) and transmembrane potential. Low Ca
down-regulated the amount of putrescine in roots only. Putrescine itself
was able to alleviate some effects of low K such as the reconfiguration
of S metabolism, but exaggerated other effects (pyruvate kinase content)
suggesting a role of putrescine in signaling.

Other consequences of K and Ca availability have not been extensively
discussed here, such as the modulation of micronutrients: (\emph{i} )
iron (Fe) content and iron-dependent enzymes (2-oxoglutarate
Fe\textsuperscript{2+}-dependent, ferrochelatase, ferritin) are affected
by K and/or Ca level (Figs. 1, S5), and (\emph{ii} ) magnesium is
affected by both K and Ca (magnesium content, Fig. 1; magnesium
chelatase, Fig. S5). Of course, a detailed analysis of micronutrient
absorption and allocation would require further analyses, including the
use of isotopic tracers (\textsuperscript{54}Fe,\textsuperscript{25}Mg).
Similarly, more work would be required to better understand the specific
metabolic effects of putrescine, using isotopically labelled putrescine
(\textsuperscript{15}N or\textsuperscript{13}C) in particular to
identify key reactions and quantify putrescine catabolism to
NH\textsubscript{4}\textsuperscript{+}(via CAO) and CO\textsubscript{2}
(via the TCAP).

Our results also raise the question as to whether low Ca conditions or
putrescine addition are viable solutions to alleviate some effects on K
deficiency in crops. Although our results are limited to young sunflower
plants under controlled conditions, it seems that none of these two
possibilities is ideal. In fact, low Ca further increases respiration
and thus the overall impact on carbon mass balance might not be
beneficial. Putrescine addition is costly ([?] \$1 g\textsuperscript{-1})
and does not suppress the strong effect of low K on respiratory efflux
(Fig. 2). Therefore, monitoring plant K status using new technologies
such as metabolomics (Cui \emph{et al.} , 2021), and sustainable K
fertilization might be better options to ensure optimal K nutrition for
crops in the field.



\textbf{Acknowledgements}

The authors warmly thank members of the RSB Plant Service for their help
during sunflower cultivation. The support of the Joint Mass Spectrometry
Facility (ANU) is acknowledged. G.T. acknowledge the financial support
of the \selectlanguage{ngerman}\emph{Région Pays de la Loire} and \emph{Angers Loire
Métropole}via the grant Connect Talent Isoseed.

\textbf{Supplementary information}

Methods S1. Proteomics analyses.

Table S1. Summary of nutrient conditions.

Table S2. List of significant proteins.

Fig. S1. Overview of the experimental design.

Fig. S2. Summary of putrescine content in leaves and roots.

Fig. S3. Significant transporters and channels in leaves.

Fig. S4. Significant transporters and channels in roots.

Fig. S5. Proteins associated with a significant K \emph{x} Ca and
K\emph{x} putrescine interaction.

Fig. S6. Relative content in copper amine oxidase.

Fig. S7. S assimilation pathway, and methionine and cysteine content.

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