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\begin{document}

\title{Why is phloem sap nitrate kept low?}



\author[1]{Jing Cui}%
\author[2]{Andreas D. Peuke}%
\author[3]{Anis Limami}%
\author[4]{Guillaume Tcherkez}%
\affil[1]{ANU}%
\affil[2]{ADP International Plant Science Consulting}%
\affil[3]{University of Angers, (IRHS 1345) Institute of Research of Horticulture and Seeds }%
\affil[4]{Affiliation not available}%


\vspace{-1em}



  
  \date{June 15, 2024}


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\selectlanguage{english}
\begin{abstract}
Since the first description of phloem sap composition nearly 60 years
ago, it is generally assumed that phloem sap does not contain nitrate
and that there is little or no backflow of nitrate from shoots to roots.
While it is true that nitrate can occasionally be absent from phloem
sap, there is now substantial evidence that phloem can carry nitrate and
furthermore, transporters involved in nitrate redistribution to shoot
sink organs and roots have been found. This raises the question of why
nitrate may or may not be present in phloem sap, why its concentration
is generally kept low, and whether plant shoot-root nutrient cycling
also involves nitrate. We propose here that phloem sap nitrate is not
only an essential component of plant nutritional signaling but also
contributes to physical properties of phloem sap and as such, its
concentration is controlled to ensure proper coordination of plant
development and nutrient transport.%
\end{abstract}%



\sloppy


\textbf{Introduction}

It has now been 61 years since phloem sap composition has first been
described, using sap extracted from willow (Peel \& Weatherley, 1959).
In that study, using colorimetric methods, nitrate was found to be
undetectable. Several subsequent studies also reported the absence of
nitrate in other species such as castor bean (\emph{Ricinus communis} )
(Hall \& Baker, 1972) and overviews of phloem sap composition
established the generally very low, or undetectable, concentration in
phloem sap nitrate (Ziegler, 1975). Furthermore, experiments
with\textsuperscript{15}N-nitrate labelling in legumes have shown that
nitrate could not be transported from xylem to phloem at detectable
levels (Pate, Sharkey, \& Lewis, 1975). There are more recent reports
where nitrate was found to be undetectable, for example in maize (Lohaus
et al., 2000). It is thus widely accepted that phloem sap nitrate is of
negligible importance and in particular, that nitrate circulation from
shoots to roots does not occur. For example, in a recent review, the
backflow of nitrate via the phloem to regulate root development is not
mentioned (Fig. 3 in (Tegeder \& Masclaux-Daubresse, 2018)). Also, in
textbooks and university lectures, it is often reported that nitrate is
absent from phloem sap and thus does not flow back from shoots to roots,
see for example (Taiz, Zeiger, Moller, \& Murphy, 2015). However, many
pieces of recent evidence suggest the contrary and provide possible
reasons explaining why phloem nitrate concentration is usually low. They
are presented in this Opinion.

\textbf{Phloem sap may contain nitrate}

In fact, there are noticeable exceptions where nitrate is not absent
from phloem sap composition (Fig. 1a-f). It is the case of cereals
(wheat, rice), with phloem sap nitrate of up to 8 mM (Hayashi \& Chino,
1985; Hayashi \& Chino, 1986). Also, in palm trees, nitrate has been
found in phloem exudates for either trunk and inflorescence peduncles
(van Die \& Tammes, 1975). Extensive analysis of castor bean phloem sap
has shown that average nitrate phloem concentration is 0.59 mM, ranging
from no detectability to 2.4 mM (Peuke, 2010). This is of course much
less than in xylem, which contains up to 25 mM nitrate. Interestingly,
nitrate has been found to be much more abundant in phloem (0.29 mM) than
xylem sap (3 \selectlanguage{greek}µ\selectlanguage{english}M) in Western candle tree (\emph{Banksia prionotes} )
(Jeschke \& Pate, 1995). These findings cannot be explained by the
technique of sap collection (i.e. potential adulteration by other
tissues upon sampling). The above-cited studies used very different
techniques (aphid-stylet and pure phloem sap exudation) and therefore a
systematic contamination is unlikely. In effect, nitrate can be found in
phloem sap using the aphid stylet method (i.e. involving stylectomy),
which gives access to very high purity phloem sap (van Helden,
Tjallingh, \& van Beek, 1994).

\textbf{Nitrate redistribution via phloem sap circulation}

Nitrate redistribution via phloem circulation has been recently
suggested to occur in three physiological contexts (Fig. 1g), supported
by the characterization of nitrate transporters in \emph{Arabidopsis} :
remobilization from leaves, development of reproductive structures and
nitrate backflow from shoots to roots.

\emph{Nitrate transfer and remobilization in leaves}

Three nitrate transporters have been shown to be involved in nitrate
transfer and/or redistribution to the phloem from xylem or source
nitrate-containing tissues: NPF2.13/NRT1.7, NPF1.1/NRT1.12, and
NRT1.2/NRT1.11 (Iqbal et al., 2020). In particular, it has been recently
shown that NPF2.13/NRT1.7 is essential for remobilization of nitrate
from old leaves to deliver nitrate into the phloem, making it available
for new, developing leaves and enhancing nitrogen use efficiency (Chen,
Chen, Tseng, \& Tsay, 2020). NPF1.1/NRT1.12 and NPF1.2/NRT1.11 have been
found to be involved in the transfer of xylem nitrate to phloem in the
petiole thereby facilitating nitrate remobilization from mature (old)
leaves to young leaves (Hsu \& Tsay, 2013).

\emph{Development of reproductive structures}

Up to now, two nitrate transporters have been shown to be of importance
in silique and seeds: NPF2.12/NRT1.6, and NPF5.5. NPF2.12/NRT1.6 is
capable of transporting both nitrate and gibberellin
GA\textsubscript{1/3}, and is expressed intensively in funiculus
vascular bundles, which are mostly made of phloem tissue
in\emph{Arabidopsis} (Almagro, Lin, \& Tsay, 2008).
In\emph{NPF2.12/NRT1.6} mutants, mature seeds contain effectively less
nitrate, suggesting that NPF2.12/NRT1.6 is involved in transporting
nitrate from maternal tissue to the developing embryo via the phloem
(Almagro et al., 2008). NPF5.5, which as been shown to transport
nitrate, is involved in controlling embryo N content: in knock-out
mutants, embryos at the bent cotyledon stage have a 7-8\% reduction in
the N content suggesting that the import of nitrate to the developing
seed has been impacted (L\selectlanguage{ngerman}éran et al., 2015).

\emph{Nitrate backflow from shoots to roots}

Two nitrate transporters are related to nitrate flow from shoots to
roots: NPF2.9/NRT1.9, and NPF1.1/NRT1.12. NPF2.9/NRT1.9 is expressed in
root phloem. In \emph{NPF2.9/NRT1.9} mutants, the nitrate content in
root phloem exudates is lower (by 20 to 30\%) (Wang \& Tsay, 2011);
furthermore, downwards nitrate transport to root tips, when assayed with
a compartmentalized root system, is reduced, suggesting that
NPF2.9/NRT1.9 effectively plays a role in the backflow of nitrate to
roots (Wang \& Tsay, 2011). Unlike NPF1.2/NRT1.11, NPF1.1/NRT1.12 is
expressed in roots (not only in shoots) and since it is involved in
xylem-to-phloem nitrate transfer (Hsu \& Tsay, 2013), it suggests that
it can be involved in nitrate cycling to roots.

Taken as a whole, there is substantial evidence that some nitrate
transporters allow nitrate transfer and circulation into the phloem
either downwards (to roots) or upwards (to seeds and young leaves). In
addition, although nitrate is not the major nitrogen form in the phloem,
the strong growth retardation phenotype observed in
the\emph{NPF1.2/NRT1.11 NPF1.1/NRT1.12} double mutant (Hsu \& Tsay,
2013) suggests that phloem-mediated nitrate redistribution is essential.

\textbf{Is phloem sap nitrate a signaling molecule?}

There is now considerable evidence that nitrate can play a signaling
role to control plant development (Kant, 2018) and nitrate redistributed
by the phloem seems to participate in this control (summarized in Fig.
2). Historically, an important step forward was the demonstration that
nitrate shoot content acts as a signal to regulate the shoot:root ratio.
In fact, manipulating nitrate reductase activity showed a correlation
between leaf nitrate concentration and shoot:root ratio, across
different levels of nitrate supply (Scheible, Lauerer, Schulze, Caboche,
\& Stitt, 1997). Furthermore, in split\selectlanguage{english}-root experiments, root growth is
inhibited by the accumulation of nitrate in the shoot, regardless of the
fact that one part of the root system was supplied with high nitrate and
the other part with low nitrate (Scheible et al., 1997). More recently,
split-root experiments have shown that there is a cytokinin-mediated
signaling from the root part under low N conditions, so as to promote
lateral root development in the root part under N-rich conditions
(Ruffel et al., 2011).

The signaling cascade associated with nitrate has been dissected
recently and shown to involve Ca\textsuperscript{2+}-sensor protein
kinases and NIN-like proteins (NLP) transcription factors to control
gene expression, including genes encoding enzymes and transporters
involved in nitrogen assimilation (Chu et al., 2020; Liu et al., 2017).
In addition, at the plant scale, nitrate signaling involves small
peptides transited via phloem sap. The signaling peptide C-TERMINALLY
ENCODED PEPTIDE 1 (CEP1) is produced by roots under N de\selectlanguage{english}ficiency, and
transported to the shoot via the xylem, leading to the induction of the
signaling peptides CEP DOWNSTREAM 1 (CEPD1) and CEPD2; these peptides
are then transported to roots via the phloem and upregulates the high
affinity nitrate transporter NRT2.1 (Gu et al., 2018; Ohkubo, Tanaka,
Tabata, Ogawa-Ohnishi, \& Matsubayashi, 2017).

In such a molecular context, could phloem nitrate itself have a
signaling role? It might be so, in particular in root tips where
longitudinal root growth (increase in root length) occurs. It is worth
noting that in root tips, protophloem develops first, that is, before
protoxylem (Bauby, Divol, Truernit, Grandjean, \& Palauqui, 2007;
Mahonen et al., 2000). Also, in root tips, nitrate absorption is very
small, compared to other root sections (Lazof, Rufty, \& Redinbaugh,
1992). Therefore, root tip metabolism depends on the import of organic
N, not nitrate. We can thus speculate that the influx of nitrate via
protophloem could contribute to determine root tip nitrate concentration
which has in turn a regulatory role. In effect, in Legumes, nitrate
inhibits primary root growth at the root tip via NPF6.8-mediated
abscisic acid signaling and reactive oxygen species (Limami \& Morere-Le
Paven, 2019; Pellizzaro et al., 2014; Zang et al., 2020).
In\emph{Arabidopsis} , nitrate stimulates meristematic activity (primary
root growth) at the root tip via cytokinin signaling (Naulin et al.,
2020).

If nitrate transported by phloem sap plays effectively a signaling role,
it raises two questions: (\emph{i} ) First, nitrate concentration in
phloem must be controlled to avoid both fluctuations and high
concentrations. That is, signal-carrying molecules have to be at low
concentration under steady-state conditions and their transient increase
can play the role of a signal. Unfortunately, relatively little is known
on possible fluctuations of nitrate concentration in phloem sap and
whether its appearance in sap is linked to specific circumstances
(although there is some correlation with xylem nitrate and other
nutrients, see below, \emph{Perspectives for N cycling in plants} ).
(\emph{ii} ) Second, unclear would be the reason to have both CEP- and
nitrate-based regulation of root growth. That said, while CEP-based
regulation seems to be associated with low N signaling, nitrate itself
could represent a more generic N status signal (not only low N
conditions). It is now well established that nitrate interacts with many
hormones such as cytokinins (Sakakibara, Takei, \& Hirose, 2006), and
this aspect has been reviewed elsewhere recently (Vega, O'Brien, \&
Gutierrez, 2019). Taken as a whole, nitrate concentration itself could
participate in defining the signaling milieu and thus the flow of
nitrate from shoots to roots via the phloem could be important to
control root growth.

\textbf{Low nitrate concentration may avoid protein aggregation}

Phloem sap contains very high content of sucrose (about 400 mM in castor
bean and palms, for example), amino acids (about 60 mM in most species,
up to 140 mM in some trees) and K\textsuperscript{+} (about 60 mM, up to
500 mM in \emph{Salix} , (Peel \& Weatherley, 1959)).
Mg\textsuperscript{2+} and Na\textsuperscript{+} are present in lower
abundance and Ca\textsuperscript{2+} is at low concentration (1 mM or
less) (Ziegler, 1975). Anions that counterbalance
K\textsuperscript{+}high content are mostly Cl\textsuperscript{-},
phosphate, sulphate and organic acids (malate). Nitrate is thus a minor
participant in phloem electroneutrality. Also, phloem sap contains
significant amounts of proteins, at about 1 g L\textsuperscript{-1}, and
an important proportion is made of the so-called P-proteins (SEOR
proteins in\emph{Arabidopsis} ) that are believed to play a role in
phloem occlusion by Ca\textsuperscript{2+}-dependent aggregation
(Anstead, Froelich, Knoblauch, \& Thompson, 2012; Jekat et al., 2013;
Knoblauch, Froelich, Pickard, \& Peters, 2014; van Bel et al., 2014). Of
course, phloem sap contains many other proteins, and recent proteomics
analyses have shown that this includes not only enzymes, but also
translation initiation or elongation factors, proteins involved in redox
homeostasis, chaperones, etc. (Rodriguez-Celma, Ceballos-Laita, Grusak,
Abadia, \& Lopez-Millan, 2016). It is worth mentioning that with such
high salt concentration (in particular K\textsuperscript{+}), there is a
risk of uncontrolled protein aggregation. In fact, both experiments and
theory have provided evidence that ion species have different propensity
to trigger protein aggregation (chaotropism), via denaturation and/or
instability (Kunz, 2010). Ca\textsuperscript{2+} is the most chaotropic
cation while tertiary amines are the least chaotropic,
K\textsuperscript{+} being intermediate. This provides a physical
justification for the role of Ca\textsuperscript{2+} in phloem
occlusion. Similarly for anions, phosphate, sulphate and organic acids
are the least chaotropic, but nitrate is much more chaotropic, just
behind perchlorate and iodide (Kunz, 2010). Accordingly, experiments
with lysozyme have demonstrated that the displacement of the
solubilization-aggregation equilibrium towards aggregation is larger
with nitrate than chloride forms of sodium salts (Kastelic, Kalyuzhnyi,
Hribar-Lee, Dill, \& Vlachy, 2015). As such, having high concentrations
of nitrate is not desirable for phloem protein stability when
K\textsuperscript{+} (and/or Mg\textsuperscript{2+}) is present at high
concentration. Conversely,\emph{in vitro} , in sucrose concentrated
solutions (at concentrations similar to that found in phloem sap),
ethylamine nitrate has been found to be beneficial to protein
renaturation and decreases viscosity due to the rescuing property of
ethylamine as a non-chaotropic cation (Byrne, Wang, Belieres, \& Angell,
2007).

\textbf{Nitrate cycling in plants}

While there might be good reasons to explain why nitrate concentration
must remain low in phloem sap (signaling and chaotropism), the
occasional presence of nitrate raises the question of its potential role
in nutrient cycling. Incubation with a nitrate-containing buffer (10 mM
nitrate) of castor bean cotyledons (the endosperm being removed) have
shown that nitrate can be found (at about 1 mM) in phloem sap exudated
from cut hypocotyls, suggesting that nitrate cycling via the phloem is
effectively possible (Schobert \& Komor, 1992). Furthermore, pioneering
N mass balance in castor bean proposed that nearly 50\% of nitrate
translocated to the shoot cycled back to roots (Marschner, Kirkby, \&
Engels, 1997). While this number is certainly overestimated, recent
isotopic data (\textsuperscript{15}N natural abundance,
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{15}N) also suggest that a small flux of a few percent
of xylem translocation to shoots can cycle back to roots, in both
sunflower and oil palm (Cui, Lamade, Fourel, \& Tcherkez, 2020).
Although quantitatively minor, this flux is important to explain the
natural, considerable \textsuperscript{15}N-enrichment in root nitrate:
in fact, nitrate reduction in shoots discriminates between N isotopes in
favor of \textsuperscript{14}N and thus \textsuperscript{15}N-nitrate
left behind is redistributed to roots via the phloem. Also, since
nitrate redistributed to the phloem is
naturally\textsuperscript{15}N-enriched, it is possible that variations
in phloem sap nitrate concentration along the day contribute to
explaining the diel pattern of \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{15}N of phloem in
castor bean (Peuke, Gessler, \& Tcherkez, 2013). Interestingly, the
\selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{15}N value of aphids (feeding on phloem sap) has been
shown to be lower (\textsuperscript{15}N-depleted) compared to host
plants and related to nitrate reduction capacity, suggesting that the
aphid-host isotopic difference is partly explained by the
natural\textsuperscript{15}N-enrichment in phloem nitrate, as opposed to
the\textsuperscript{15}N-depletion in phloem amino acids (Wilson,
Sternberg, \& Hurley, 2011).

The backflow of nitrate from shoots to roots depends on growth
conditions impacting on overall nutrition, since (Cui et al., 2020)
showed it depends on K nutrition and root hypoxia (waterlogging). The
interaction between K nutrition and nitrate absorption has been known
for decades and has been mostly explained by the key role of
K\textsuperscript{+} as a counteraction for nitrate (Casades\selectlanguage{ngerman}ús, Tapia,
\& Lambers, 1995; Coskun, Britto, \& Kronzucker, 2017; Wang \& Wu,
2013). Accordingly, the nitrate transporter NPF7.3/NRT1.5 has been shown
to act as an antiporter with
K\textsuperscript{+}/H\textsuperscript{+}(Li et al., 2017).
Nevertheless, there is no significant relationship between nitrate and
K\textsuperscript{+} concentrations in phloem (Peuke, 2010) and this
might not be surprising because K\textsuperscript{+} is present at very
high concentration in phloem. Interestingly, phloem sap nitrate
increases when nitrate availability increases and declines with salinity
(Peuke, Glaab, Kaiser, \& Jeschke, 1996). Meta-analyses have shown that
phloem nitrate concentration does not correlate significantly to other
cations and only correlates with xylem, leaf and root nitrate content
(Peuke, 2010). However, the nitrate flow in the phloem (expressed in
\selectlanguage{greek}µ\selectlanguage{english}mol nitrate g\textsuperscript{-1} FW d\textsuperscript{-1}) correlates
reasonably well with phloem carbon and Ca\textsuperscript{2+} flows
(Peuke, 2010). The nitrate backflow thus depends on other nutrients and
salinity and is maybe linked to metabolites (organic acids and amino
acids) present in phloem sap. The supply of amino acids to roots via the
phloem participates in the control of root N acquisition (for a specific
discussion, see (Tillard, Passama, \& Gojon, 1998)) and as discussed
above, nitrate also plays a regulatory role. Thus, more than individual
concentrations, the nitrate-to-amino acid ratio of phloem sap might be a
crucial component of plant development, root growth and nitrogen
assimilation. In the past years, there have been an increasing number of
publications on phloem (including proteomics data) but due to the
difficulty of phloem sap collection, there is limited information on
phloem composition under varying conditions, including metabolite
profiling (metabolomics), nitrate content and \selectlanguage{greek}δ\selectlanguage{english}\textsuperscript{15}N
value.

\textbf{Open questions for future research}

Taken as a whole, key questions that are still unanswered and deserve
more research can be summarized as follows:

\begin{enumerate}
\tightlist
\item
  What is the rationale of phloem sap nitrate variation between species?
  As stated above, the range of concentration is very large (from
  non-detectability to nearly 10 mM). Uncertainty remains as to whether
  this is related to N allocation, efficiency of nitrate remobilization
  from leaves to developing organs, or different strategies in N status
  signaling.
\item
  Does phloem nitrate contribute effectively to nitrate signaling and
  growth regulation? For example, is the response to nitrate modified
  when phloem nitrate transport is compromised? Is the xylem-to-phloem
  ratio of nitrate concentration or phloem nitrate-to-amino acid ratio
  indicators of N status?
\item
  Do the relationships between nutrients, xylem nitrate and phloem
  nitrate still hold in species other that castor bean? Up to now, there
  is no such extensive dataset like that obtained in this species.
\item
  Is low nitrate concentration an essential factor to avoid protein
  aggregation in phloem sap? Can experiments be designed where nitrate
  is trapped or added to manipulate protein aggregation propensity?
\end{enumerate}

Future studies are warranted to provide more insight on these aspects
and thus to clarify the roles of nitrate found in phloem sap and also
demonstrate why it is kept at a very low concentration in many cases.

\textbf{Acknowledgements}

The authors acknowledge the financial support of the R\selectlanguage{ngerman}égion Pays de la
Loire and Angers Loire Métropole via the grant Connect Talent Isoseed
awarded to G.T.

\textbf{Data availability}

There is no data associated with this manuscript.

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\textbf{Figure 1. Nitrate circulation via phloem sap} : (a-f) Typical
species where phloem nitrate has been directly measured or inferred from
isotopic mass-balance: palm trees (van Die \& Tammes, 1975) (a), cereals
(wheat and rice) (Hayashi \& Chino, 1985; Hayashi \& Chino, 1986) (b),
Western candle tree \emph{Banksia prionotes} (Jeschke \& Pate, 1995)
(c), castor bean (Peuke, 2010) (d), sunflower (Cui et al., 2020) (e),
and Arabidopsis (Wang \& Tsay, 2011) (f). (g) Summary of processes
involving nitrate circulation via phloem sap (see text for further
details).

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\textbf{Figure 2. Summary of potential roles played by phloem nitrate in
signaling for growth regulation} . This figure is a simplification and
does not mention transporters involved in nitrate transport and sensing
(see text for further details). Pathways are sub-divided into xylem
(green) and phloem (red) mediated signaling. Abbreviations: CaPK,
calcium-dependent protein kinases; CEP, C-terminally encoded peptide;
CEPD, CEP downstream; NLP, NIN-like proteins; ROS, reactive oxygen
species.\selectlanguage{english}
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