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\begin{document}

\title{ABA signaling in regulation of mesophyll conductance in tomato during
progressive soil drying}



\author[1]{Shuang Li}%
\author[1]{Junming Liu}%
\author[1]{Hao Liu}%
\author[1]{Yuan Lei}%
\author[2]{Yang Gao}%
\author[1]{Aiwang Duan}%
\affil[1]{Farmland Irrigation Research Institute}%
\affil[2]{Affiliation not available}%


\vspace{-1em}



  
  \date{May 05, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Objectives of this study were to clarify the involvement of root to
shoot ABA in regulation of leaf stomatal conductance (gs) and mesophyll
conductance (gm) during progressive soil drought, and to investigate its
impact on leaf photosynthesis and intrinsic water use efficiency (WUEi)
in pot-planted tomato. A fast-stomatal closure was related to decrease
of \selectlanguage{greek}Ψ\selectlanguage{english}leaf in the early stage of soil drought, whereas gm kept unchanged
until ABAxylem synchronously increased at threshold of relative soil
water content (RSWC) decreasing from 51.43 to 44.69\%. This out-of-step
response between gs and gm resulted in an increase of gm/gs ratio and
potentially improved WUEi during RSWC decreased from 51.43 to 40.16\%.
Meanwhile, gs and gm declined logarithmically with increasing xylem sap
ABA concentration (p\textless{}0.01). The sensitivity of gm response to
root to shoot ABA signaling, even though less than gs, played a key role
in regulation of CO2 diffusion into the chloroplast as soil drying
proceeded. gs mainly limited leaf photosynthesis with RSWC in range of
51.43 to 40.16\%, and relative contribution of gm limitation exceed that
of gs limitation as soil further dried. These results provided new
understanding of ABA in the regulation of gm and WUEi in response to
drought stress.%
\end{abstract}%



\sloppy


\textbf{ABA signaling in regulation ofmesophyll conductance in tomato
during progressive soil drying}

Shuang Li \textsuperscript{a,b,c}, Junming Liu \textsuperscript{a,b,c},
Hao Liu \textsuperscript{a,b}, Yuan Lei \textsuperscript{a,b,c}, Yang
Gao\textsuperscript{a,b*}, Aiwang Duan\textsuperscript{a,b*}

\textsuperscript{a} Key Laboratory of Crop Water Use and Regulation,
Ministry of Agriculture and Rural Affairs, Xinxiang, Henan 453003, PR
China

\textsuperscript{b} Institute of Farmland Irrigation, Chinese Academy of
Agricultural Sciences, Xinxiang, Henan 453003, PR China

\textsuperscript{c} Graduate School of Chinese Academy of Agricultural
Sciences (GSCAAS), Beijing 100081, PR China

*Corresponding author: gaoyang@caas.cn (Y. Gao); duanaiwang@caas.cn (A.
Duan)

\textbf{Abstract:} Objectives of this study were to clarify the
involvement of root to shoot ABA in regulation of leaf stomatal
conductance (g\textsubscript{s}) and mesophyll conductance
(g\textsubscript{m}) during progressive soil drought, and to investigate
its impact on leaf photosynthesis and intrinsic water use efficiency
(WUE\textsubscript{i}) in pot-planted tomato. A fast-stomatal closure
was related to decrease of \selectlanguage{greek}Ψ\selectlanguage{english}\textsubscript{leaf} in the early stage of
soil drought, whereas g\textsubscript{m} kept unchanged until
ABA\textsubscript{xylem}synchronously increased at threshold of relative
soil water content (RSWC) decreasing from 51.43 to 44.69\%. This
out-of-step response between g\textsubscript{s} and g\textsubscript{m}
resulted in an increase of g\textsubscript{m}/g\textsubscript{s} ratio
and potentially improved WUE\textsubscript{i} during RSWC decreased from
51.43 to 40.16\%. Meanwhile, g\textsubscript{s} and g\textsubscript{m}
declined logarithmically with increasing xylem sap ABA concentration
(\emph{p} \textless{}0.01). The sensitivity of
g\textsubscript{m}response to root to shoot ABA signaling, even though
less than g\textsubscript{s}, played a key role in regulation of
CO\textsubscript{2} diffusion into the chloroplast as soil drying
proceeded. g\textsubscript{s} mainly limited leaf photosynthesis with
RSWC in range of 51.43 to 40.16\%, and relative contribution of
g\textsubscript{m} limitation exceed that of
g\textsubscript{s}limitation as soil further dried. These results
provided new understanding of ABA in the regulation of
g\textsubscript{m} and WUE\textsubscript{i} in response to drought
stress.

\textbf{Key words} : stomatal conductance; mesophyll conductance; water
use efficiency; ABA signaling

\textbf{1. Introduction}

Water scarcity, as one of major environmental stress factors, could
strongly disturb the balance between H\textsubscript{2}O efflux via
stomata and CO\textsubscript{2} diffusion from air to the chloroplast
stroma, and simultaneously limit plant productivity. Stomatal closure
driven by hydraulic and chemical signals, mainly leaf water potential
and abscisic acid (ABA), has been recognized as an early response to
soil drought (Dodd et al. 2005; Jacobsen et al. 2008; Tombesi et al.
2015). Similar to stomatal conductance (g\textsubscript{s}), there was
increasing evidence that mesophyll conductance (g\textsubscript{m})
could be the important limiting factor in the regulation of plant
photosynthetic capacity response to environmental stresses, since
g\textsubscript{m} controlled the drawdown of CO\textsubscript{2} from
sub-stomatal cavities to chloroplasts (Flexasa et al. 2002; Niinemets et
al. 2009a; Qiu \& Katul 2019). Many studies have suggested that
g\textsubscript{m} decrease accompanied usually by a decline of
chloroplastic CO\textsubscript{2}concentration (C\textsubscript{c})
during progressive soil drying in many species, such as grapevines
(Flexasa et al. 2002; Ferrio et al. 2012), cotton (Han et al. 2016) and
rice (Wang et al. 2018), but the mechanisms for this decrease were still
unknown in tomato as a sensitive water-stressed vegetable in the world.
Besides the impact of leaf anatomical characteristics on
g\textsubscript{m}(Niinemets et al. 2009b; Muir et al. 2014), recent
study in leaf hydraulic signals suggested that the vulnerability of leaf
hydraulic conductance contributed to the decrease of g\textsubscript{m}
and g\textsubscript{s} in response to soil drought (Wang et al. 2018).
While Th\selectlanguage{ngerman}éroux-Rancourt et al (2014) considered that g\textsubscript{m}
was less sensitive to drought stress than g\textsubscript{s} or
hydraulic signals mediated by ABA, due to the unresponsive action of the
bulk of mesophyll cells to exogenous ABA application (Shatil-Cohen et
al. 2011). In line with this, Vrabl et al (2009) reported that 20 \selectlanguage{greek}μ\selectlanguage{english}M
application of ABA could not affect g\textsubscript{m} and the rate of
photosynthesis, while induced a reduction in g\textsubscript{s}. On the
contrary, several studies also showed exogenous ABA application could
result in a decrease in g\textsubscript{m} in soybean and tobacco
(Flexas et al., 2006) and in four woody species (Sorrentino et al.,
2016). Similarly, a recent study revealed that there was a negative
relationship between ABA concentration and g\textsubscript{m} under
drought condition, by means of ABA-deficient mutant (Mizokami et al.
2015). Nonetheless, the threshold of ABA signaling in regulation of
g\textsubscript{m} response to drought was still unclear.

Despite all of the negative response of leaf gas exchange to drought
stress, many studies have confirmed that leaf intrinsic water use
efficiency (WUE\textsubscript{i}), termed as ratio of net photosynthetic
rate to stomatal conductance, improved by decreasing g\textsubscript{s}
under mild drought stress (Liu et al. 2005; Xue et al. 2016). It was
also worth to be noted that drought--induced closure of stomatal
aperture could simultaneously in turn reduce assimilation rate and
yield, higher WUE\textsubscript{i}and A\textsubscript{n} seemed to be
only achieved with improving g\textsubscript{m}(Flexas et al. 2013,
2016). Correlations between WUE\textsubscript{i} and the ratio of
g\textsubscript{m}/g\textsubscript{s} was indeed observed based on the
Fick's law of diffusion, as higher g\textsubscript{m}/g\textsubscript{s}
contributed to higher A\textsubscript{n}and WUE\textsubscript{i} under
drought condition (Flexas et al. 2013; Cano et al. 2013; Han et al.
2016;). However, the relations between
g\textsubscript{m}/g\textsubscript{s} and ABA signaling during the
progressive soil drying were largely unknown. Therefore, revealing the
exact role of g\textsubscript{m} or
g\textsubscript{m}/g\textsubscript{s} in improving A\textsubscript{n}and
WUE\textsubscript{i} in response to progressive drought was necessary to
improve plant water productivity for solving food shortage problem all
over the world.

Moreover, it was important to address the impact of water stress on
photosynthetic capacity, which was commonly quantified as: stomatal
conductance (g\textsubscript{s}), mesophyll conductance
(g\textsubscript{m}) and photosynthetic biochemistry (including the
carboxylation capacity or the electron transport rate) (Cano, et al.
2013; Tosens et al. 2016; Wang et al. 2018). Since a quantitative
limitation analysis proposed by Grassi and Magnani (2005) has been
applied to estimate the contributions of each factor to photosynthesis,
several researches shown that g\textsubscript{s} and g\textsubscript{m}
were the main limitations to maximum photosynthesis under drought
condition (Tosens et al., 2016; Wang et al., 2018). Therefore, we
examined the applicability of this method to determine the main
constraint in tomato plants under progressive soil drought condition.

In this study, the photosynthetic diffusive components
(g\textsubscript{s} and g\textsubscript{m}) and possibly related to leaf
water potential and root to shoot ABA signaling were examined in tomato
seedlings during progressive soil drying. The objectives of this study
were (i) to reveal especially ABA signaling that might be involved in
the control of mesophyll conductance and leaf intrinsic water use
efficiency, as well as determining the threshold of mesophyll
conductance response to ABA signaling during progressive soil drying;
and (ii) to quantitatively analyze the relative contribution of each
limitation and determine the main constraint to photosynthesis during
progressive soil drying.

\textbf{2. Materials and methods}

\textbf{2.1 Plant material and water treatments}

Seeds of tomato (\emph{Solanum lycopersicum} L., cv. Helan108) were sown
on the trays with nursery substrate. When three true leaves emerged,
tomato seedlings were transplanted into 0.53 L pots filled with 6.5 kg
air-dried soil with sandy loam texture. The field water capacity
(\selectlanguage{greek}θ\selectlanguage{english}\textsubscript{FC}) was 22\% (g g\textsuperscript{-1}), the wilting
point was 6.8\% (g g\textsuperscript{-1}). After finishing
transplanting, all pots were irrigated to 85\% \selectlanguage{greek}θ\selectlanguage{english}\textsubscript{FC} with
Hoagland solution (5 mM KNO\textsubscript{3}, 5 mM
Ca(NO\textsubscript{3})\textsubscript{2} 4H\textsubscript{2}O, 1 mM
KH\textsubscript{2}PO\textsubscript{4}, and 1 mM
MgSO\textsubscript{4}7H\textsubscript{2}O, 1 ml l\textsuperscript{-1}
micronutrients, pH=6.0). Seedlings were cultivated in an
environment-controlled chamber (day/night air temperature 28/18 \selectlanguage{ngerman}°C, 50\%
relative humidity, 12 hours photoperiod at 600 \selectlanguage{greek}μ\selectlanguage{english}mol
m\textsuperscript{-2} s\textsuperscript{-1} photosynthetic photon flux
density (PPFD) supplied by LED lamps from 7:00 to 19:00). During 1-13
days after transplanting (DAT), water was applied sufficiently with same
volume of Hoagland solution and supplied distilled water to all pots.
All pots were weighted daily at 8:00 a.m. to calculate daily irrigation
quota according to water balance formula.

Soil water content in the pot was expressed as relative soil water
content (RSWC), the ratio between the current soil moisture
(\selectlanguage{greek}θ\selectlanguage{english}\textsubscript{C}) and the field water capacity. For well-watered
treatment, RSWC was maintained within the range of 70-82\% during the
experiment. For drought-stressed treatment, in the first circle of soil
progressive drying, the RSWC decreased from 85\% to 40\% from DAT 14 to
26. At DAT 27, both the well-watered and drought treatments were
irrigated to 82\% \selectlanguage{greek}θ\selectlanguage{english}\textsubscript{FC} with same volume of Hoagland
solution and additional distilled water. After that, the
drought-stressed tomato seedlings suffered second circle of drought
stress from DAT 28 to 33. During the second progressive soil drying, the
relevant experimental indexed were measured and collected for the two
treatments daily.

\textbf{2.2 Leaf gas exchange and chlorophyll fluorescence measurements}

The light and CO\textsubscript{2} response curves were measured both on
the latest and fully expanded leaves between 9:00-15:00 in a red-blue
chamber with a LI-6400 portable photosynthesis system (LiCor Inc.,
Lincoln, NE, USA). For the CO\textsubscript{2} response curve, the leaf
was consecutively exposed to different CO\textsubscript{2} levels: 400,
300, 200, 150, 100, 50, 400, 400, 600, 800, 1000, 1200, 1400, 1600 \selectlanguage{greek}µ\selectlanguage{english}mol
mol\textsuperscript{-1} under 1000 \selectlanguage{greek}µ\selectlanguage{english}mol
m\textsuperscript{-2}s\textsuperscript{-1} PPFD. For the light response
curve, the CO\textsubscript{2} was kept as constant of 400 \selectlanguage{greek}µ\selectlanguage{english}mol
mol\textsuperscript{-1} and PPFD decreased gradually in the order of
1800, 1600, 1400, 1200, 1000, 800, 600, 400, 200, 150, 100, 50, 20, 10,
0 \selectlanguage{greek}µ\selectlanguage{english}mol m\textsuperscript{-2} s\textsuperscript{-1}. Each sampled leaf
was acclimated for 30-minitue in environment with 1000 \selectlanguage{greek}μ\selectlanguage{english}mol
m\textsuperscript{-2} s\textsuperscript{-1} PPFD and 400 \selectlanguage{greek}μ\selectlanguage{english}mol
CO\textsubscript{2} mol\textsuperscript{-1}. Leaf gas-exchange data was
logged before starting A\textsubscript{n}/C\textsubscript{i} curve
measurement. Intrinsic water use efficiency (WUE\textsubscript{i}) was
calculated as the ratio of net photosynthetic rate divided by stomatal
conductance:

\(\text{WUE}_{i}=\frac{A_{n}}{g_{s}}\) (1)

Leaf chlorophyll fluorescence parameters were measured by the
fluorescence probe (MINI PAM-2000; Walz, Effeltrich, Germany) on the
same leaves as the measurement of response curves. The photochemical
efficiency of photosystem II (\selectlanguage{greek}\emph{Φ\textsubscript{PSII}}\selectlanguage{english} ) was
determined as follows:

\(\Phi_{\text{PSII}}=\frac{(\text{Fm}^{{}^{\prime}}-Fs)}{\text{Fm}^{{}^{\prime}}}\) (2)

where Fm' and Fs were maximal fluorescence and steady-state fluorescence
during a light-saturating pulse by the multiphase flash protocol (Genty
et al. 1989), respectively. The electron transport rate (\emph{J}
\textsubscript{f}) was then calculated as:

\(J_{f}=\Phi_{\text{PSII}}\times\text{PPFD}\times\alpha\times\beta\)(3)

where PPFD was maintained at 1000 \selectlanguage{greek}μ\selectlanguage{english}mol
m\textsuperscript{-2}s\textsuperscript{-1} on both the well-watered and
water-stressed leaves. \selectlanguage{greek}α\selectlanguage{ngerman} was the leaf absorptance
and\(\ \selectlanguage{greek}\text{β\ }\selectlanguage{english}\)reflected the partitioning of absorbed quanta
between photosystems II and I. \(\alpha\) and \(\beta\)
were considered to be 0.84 and 0.5, respectively (Laisk et al.
1996\textbf{;} Flexas et al. 2002).

Based on the variable J method described by Harley (1992),
g\textsubscript{m} was calculated as following:

\(g_{m}=\frac{A_{n}}{C_{i}-\frac{G^{*}\ (J_{f}+8\ (A_{n}+R_{d}))}{J_{f}-4(A_{n}+R_{d})}}\)(4)

where C\textsubscript{i} was intercellular
CO\textsubscript{2}concentration (\selectlanguage{greek}µ\selectlanguage{english}mol m\textsuperscript{-2}
s\textsuperscript{-1});\selectlanguage{greek}\emph{Γ\textsuperscript{*}}\selectlanguage{english} was chloroplast
CO\textsubscript{2}compensation point (\selectlanguage{greek}µ\selectlanguage{english}mol m\textsuperscript{-2}
s\textsuperscript{-1}); R\textsubscript{d} was day respiration (\selectlanguage{greek}µ\selectlanguage{english}mol
m\textsuperscript{-2}s\textsuperscript{-1}), calculated by the
Photosynthesis Assistant of Ye (2007) according to light curve and
changed as soil progressive drying from DAT 28 to 33.

\selectlanguage{greek}\emph{Γ\textsuperscript{*}}\selectlanguage{english}was a leaf temperature dependent parameter,
and could be estimated as:

\(\Gamma^{*}=exp\ (c-\frac{H_{a}}{RT_{K}})\)\begin{CJK}{UTF8}{gbsn}　　　　　　　　　　　　　\end{CJK}\selectlanguage{english}(\selectlanguage{english}5)

where c was the scaling constant
(dimensionless),\emph{H\textsubscript{a}} was the energies of activation
(KJ mol\textsuperscript{-1}), and R was the molar gas constant (KJ
K\textsuperscript{-1} mol\textsuperscript{-1}). According to the study
of Hermida-Carrera et al (2016), the value of these three parameters at
25 \selectlanguage{ngerman}°C was 12.7, 23.2, 8.314 in our experiment, respectively.
T\textsubscript{k} was the leaf absolute assay temperature (K), which
was recorded by the LI-6400 and corrected to Kelvin temperature.

\textbf{2.3 Photosynthetic limitation analysis}

Based on the Fickian diffusion of CO\textsubscript{2} from leaf surface
to the chloroplasts on steady-state conditions, photosynthesis
limitation to light-saturated photosynthesis could be defined as the
stomatal (l\textsubscript{s}), mesophyll (l\textsubscript{m}) and
biochemical characteristics (l\textsubscript{b}). And the relative
contribution of three components
(l\textsubscript{s}+l\textsubscript{m}+l\textsubscript{b}=1) was
calculated in terms of the method described by Grassi \& Magnani (2005).
Light-saturated photosynthesis model (Farquhar et al. 1980) was used as
follow:

\(A=\frac{V_{\text{cmax}}(C_{c}-\Gamma^{*})}{C_{c}+K_{c}(1+\frac{O}{K_{o}})}+R_{d}\)(6)

\(C_{c}=\frac{\Gamma^{*}(J_{f}+8(A+R_{d}))}{(J_{f}-4(A+R_{d})}\)(7)

\(l_{s}=\ \frac{\frac{g_{t}}{g_{\text{sc}}}\frac{\partial A}{\partial C_{C}}}{g_{t}+\frac{\partial A}{\partial C_{C}}}\)(8)

\(l_{m}=\ \frac{\frac{g_{t}}{g_{m}}\frac{\partial A}{\partial C_{C}}}{g_{t}+\frac{\partial A}{\partial C_{C}}}\)(9)

\(l_{b}=\ \frac{g_{t}}{g_{t}+\frac{\partial A}{\partial C_{C}}}\)(10)

where, \emph{K} \textsubscript{c} and \emph{K} \textsubscript{o} were
the Michaelis--Menten constants for CO\textsubscript{2} and
O\textsubscript{2}, and V\textsubscript{cmax} was the maximum
carboxylation capacity, all parameters were temperature dependent and
calculated with the equation 5. The specific value of these three
parameters at 25 °C was obtained from Sharkey et al., 2007. \emph{O} is
the O\textsubscript{2} concentration in air (=210 mmol
mol\textsuperscript{-1}). And g\textsubscript{t} was the total
conductance, which was calculated as:

\(g_{t}=\frac{1}{\frac{1}{g_{s}}+\frac{1}{g_{m}}}\) (11)

\textbf{2.4 Leaf water potential measurement and xylem sap collection}

Leaves measured for gas exchange and chlorophyll fluorescence were also
sampled for determination of leaf water potential by the WP4C Dewpoint
Potentiometer (Meter, USA). 5 repetitions were taken from each
treatment. Meanwhile, the de-topped shoot was put into the pressure
chamber (Model 3115, Soil Moisture Equipment, CA, USA), increased
gradually pressure until sap solution outflowed from the cut surface.
After the first 1-2 drops was discarded, nearly 2 ml of sap was
collected into centrifuge tube frozen in liquid nitrogen and then stored
at -80 \selectlanguage{english} for ABA analysis.

\textbf{2.5 ABA determination}

The concentration of ABA was determined following the procedure of You
et al (2006) with little modification. 2 ml of extracted xylem sap
sample was placed into a microcentrifuge tube containing 5 ml extraction
buffer composed of isopropanol/hydrochloric acid and 8 \selectlanguage{greek}μ\selectlanguage{english}l 1
\selectlanguage{greek}μ\selectlanguage{english}g\selectlanguage{ngerman}·ml\textsuperscript{-1} deuterated internal standard solution. After
10 ml dichloromethane was added, sap samples were shaken at 4 °C for 30
min and then centrifuged at 13,000 rpm for 5 min at 4 °C. The
supernatant was carefully removed and the lower organic phase was dried
by N\textsubscript{2} under shading condition and dissolved in 400 \selectlanguage{greek}μ\selectlanguage{english}l
methanol (containing 0.1\% methane acid) then filtered with a 0.22 mm
filter membrane. The purified sample was then injected into
high-performance liquid chromatography-tandem mass spectrometry
(HPLC-MS/MS), fitted with a POROSHELL120 SB-C18 (Agilent Technologies)
column (2.1 mm \selectlanguage{ngerman}× 150 mm; 2.7 mm), at 30 °C. The solvent gradient used
was 100\% A (99.9\% methanol: 0.1\% CHOOH) to 100\% B (99.9\%
H\textsubscript{2}O: 0.1\% CHOOH) over 15 min. The injection volume was
2 \selectlanguage{greek}μ\selectlanguage{english}l. MS conditions were as follows: the spray voltage was 4500 V; the
pressure of the air curtain, nebulizer, and aux gas were 15, 65, and 70
psi, respectively; and the atomizing temperature was
400\textsuperscript{*}C. The concentration of ABA was calculated with
reference to peak area of deuterated internal standard.

\textbf{2.6 Statistical analysis}

Statistically differences were analyzed by one-way variance (ANOVA)
using SPSS Statistic Software. The relationship between pairs of
variables was assessed by means of Pearson correlation (r) at\emph{p}
\textless{}0.05 and \emph{p} \textless{}0.01 level. All graphics and
regression with maximum likelihood (r\textsuperscript{2}) were performed
with OriginPro 2017 (USA).

\textbf{3. Results}

\textbf{3.1 Soil water statusat the day after transferring}

Dynamic change of RSWC in the pots during the two progressive soil
drying cycles was shown in Fig.1. RSWC of the well-watered pots were
maintained above 70\%-82\%, indicating no water stress occurred during
the experiment. By withholding irrigation from DAT 28 to 33 in second
drying cycle, RSWC in drought treatment gradually decreased from 80 to
37.27\%. There was a significant difference in water status between the
drought-stressed and well-watered seedlings from DAT 29 to 33.

\textbf{3.2 Leaf water potential (} \selectlanguage{greek}\textbf{Ψ\textsubscript{leaf}) and
xylem sap ABA concentration (ABA\textsubscript{xylem})}\selectlanguage{english}

In well-watered treatment, soil water content varied between 16\% and
19\% at DAT 28 to 33, and \selectlanguage{greek}Ψ\selectlanguage{english}\textsubscript{leaf} maintained with an
average of -0.82 MPa. \selectlanguage{greek}Ψ\selectlanguage{english}\textsubscript{leaf}of the drought-stressed
tomato decreased gradually as drought proceeded, and finally a
significant decline of \selectlanguage{greek}Ψ\selectlanguage{english}\textsubscript{leaf} in drought-stressed tomato
seedlings occurred at DAT 29 (Fig.2a). However, the ABA concentration of
xylem sap kept little changed until RSWC decreased from 51.43 to 44.69\%
during DAT from 30 to 31 (Fig.2b and Fig.1). As soil further dried, the
ABA\textsubscript{xylem} increased exponentially with increasing drought
intensity and resulted in a significant difference between the drought
and control treatment. In addition, it increased sharply to 97.86
ng\selectlanguage{ngerman}·ml\textsuperscript{-1} at the end of our experiment.

\selectlanguage{greek}\textbf{3.3 Drought effect on
T\textsubscript{leaf},\emph{Γ}\textsuperscript{*}, C\textsubscript{c}
and R\textsubscript{d}}\selectlanguage{english}

Parameters of T\textsubscript{leaf}, \selectlanguage{greek}\emph{Γ} \selectlanguage{english}\textsuperscript{*},
C\textsubscript{c} and R\textsubscript{d} were closely related to the
estimation of g\textsubscript{m}. To determine accurately
g\textsubscript{m}, we analyzed the effect of drought on four parameters
daily at the DAY from 28 to 33. The relative value of these four
parameters was calculated as ratio between drought and well-watered
treatment. The results showed that, compared to the well-watered
treatment, the relative R\textsubscript{d} decreased as RSWC decreased,
and a significant difference was observed at RSWC=44.68\% at the DAT 31
(Fig.3 and Fig.1). Drought stress had a more pronounced effect on
T\textsubscript{leaf}, \selectlanguage{greek}\emph{Γ} \selectlanguage{english}\textsuperscript{*} and
C\textsubscript{c}, and resulted in a significant increase in
T\textsubscript{leaf} and \selectlanguage{greek}\emph{Γ} \selectlanguage{english}\textsuperscript{*}, but decline in
C\textsubscript{c} at RSWC=51.43\%.

\textbf{3.4 Hydraulic and chemical signaling in regulation of leaf gas
exchange and mesophyll conductance (g\textsubscript{m})}

Sensitivity of g\textsubscript{s} and g\textsubscript{m} response to
leaf water potential and ABA signaling were present in Fig.4.
g\textsubscript{s}, g\textsubscript{m} and g\textsubscript{t} showed
remarkably hyperbolic regression response to
\selectlanguage{greek}Ψ\selectlanguage{english}\textsubscript{leaf}(r\textsuperscript{2}=0.55,
r\textsuperscript{2}=0.58, r\textsuperscript{2}=0.67, respectively),
while g\textsubscript{s}declined logarithmically with increasing ABA
concentration in root to shoot xylem sap with good correlation
(r\textsuperscript{2}=0.71), as well as same ways of g\textsubscript{m}
and g\textsubscript{t} response to ABA\textsubscript{xylem}
(r\textsuperscript{2}=0.68, r\textsuperscript{2}=0.81, respectively)
(Fig.4 e-l). The results of ANOVA indicated that g\textsubscript{s} and
Tr instantly decreased in parallel with decreasing \selectlanguage{greek}Ψ\selectlanguage{english}\textsubscript{leaf}
at onset of soil drought. Whereas, A\textsubscript{n},
g\textsubscript{m}, g\textsubscript{t} remained unchanged until
ABA\textsubscript{xylem}significantly increased from 2.03 to 9.708
ng\selectlanguage{ngerman}·ml\textsuperscript{-1}. The conductance of stomata and mesophyll
decreased markedly along with the further increase in
ABA\textsubscript{xylem} at DAT 31 to 33. The results indicated that
g\textsubscript{s} response quickly to hydraulic signal in mild drought,
whereas g\textsubscript{m} was more pronounced sensitive to root-shoot
ABA signaling as drought farther proceeded during RSWC decreased from
44.69 to 37.27\%.

\textbf{3.5 WUE\textsubscript{i} response to ratio of stomatal
conductance and mesophyll conductance}

WUE\textsubscript{i} increased with increase of
g\textsubscript{m}/g\textsubscript{s} during RSWC decreased from 62.55
to 40.16\%, accompanied by decrease of \selectlanguage{greek}Ψ\selectlanguage{english}\textsubscript{leaf} from -0.948
to -1.33 MPa, but decreased gradually with decrease of
g\textsubscript{m}/g\textsubscript{s} after \selectlanguage{greek}Ψ\selectlanguage{english}\textsubscript{leaf} was
less than -1.632 (Fig.5a). WUE\textsubscript{i} responded to
ABA\textsubscript{xylem} changed in the similar way as
WUE\textsubscript{i} response to \selectlanguage{greek}Ψ\selectlanguage{english}\textsubscript{leaf}:
WUE\textsubscript{i} increased in conjunction with increasing
g\textsubscript{m}/g\textsubscript{s} as ABA\textsubscript{xylem} rose
from 2.13 to 31.23 ng\selectlanguage{ngerman}·ml\textsuperscript{-1} at same soil water
condition, and then decreased due to the decline of
g\textsubscript{m}/g\textsubscript{s} as soil drought proceeded. In
addition, the relationship between WUE\textsubscript{i} and
g\textsubscript{m}/g\textsubscript{s} was tightly represented by a
logarithmic function (r\textsuperscript{2}=0.771).

\textbf{3.6 Quantitative analysis of photosynthetic limitation response
to soil drying}

Relative contribution of all limiting factors (stomatal conductance,
mesophyll conductance and photosynthetic biochemistry) to photosynthetic
capacity was shown in Fig.5. Photosynthetic biochemistry
(l\textsubscript{b}) was found to be the main factor in well-watered
plants with a good fitting (r\textsuperscript{2}=0.88). As soil drying,
the relative contribution of stomatal conductance (l\textsubscript{s})
limitation increased up to 50.43\% while seedlings were suffered mild
soil drought at \selectlanguage{greek}Ψ\selectlanguage{english}\textsubscript{leaf} =-1.05 MPa. As soil drought
proceeded, the relative contribution of mesophyll conductance limitation
(l\textsubscript{m}) increased up to 42.03\% when
\selectlanguage{greek}Ψ\selectlanguage{english}\textsubscript{leaf}decreased from -1.33 to -1.63 MPa. As a result, the
relative resistance of stomatal conductance and photosynthetic
biochemistry correspondingly decreased to 36.92\% and 21.07\% at RSWC
=37.23\% (Fig.2a).

\textbf{4. Discussion}

Drought-induced stomata movement was closely related to hydraulic
conductance, which has been extensively studied (Ripullone et al. 2007;
Rodriguez-Dominguez et al. 2016). We found that stomatal aperture
initially decreased concomitantly with decrease of leaf water potential
at early stage of drought-stress (Fig.3a), which was consistent with the
common consensus that stomatal closure acted as an early response to
soil drought (Martin-Stpaul et al. 2017). Here, the increase of ABA was
statistically insignificant when relative soil water decreased from
74.76 to 62.55\%, i.e., from 0.34 to 0.45 ng ml\textsuperscript{-1},
which implied that root to shoot ABA signaling might not play an
essential role in inducing stomatal closure in tomato under mild soil
drought condition (Fig.2a, Fig.4c and 4d). Consistent with this, Huber
et al. (2019) compared three signals of regulation in stomatal behavior
(chemical, hydraulic and electrical) and reported that hydraulic signals
were most passively linked to the stomata aperture due to the delayed
increase in leaf ABA occurred after the onset of close of stomata.
However, as soil drought proceeded, stomatal aperture decreased
concurrently with an exponential increase in ABA\textsubscript{xylem},
presenting a close relationship between g\textsubscript{s} and
ABA\textsubscript{xylem} (r=-0.548,\emph{p} \textless{}0.01). Therefore,
it could be concluded that stomatal closure was primarily regulated by
leaf hydraulic signal at the early stage of soil drought and maintained
by root to shoot ABA signaling as relative soil water dropped from 51.43
to 37.27\% (Tombesi et al. 2015). The ability of mesophyll conductance
(g\textsubscript{m}) decreasing the concentration of CO\textsubscript{2}
in the chloroplast stroma has been stressed in these years (Flexas et
al. 2002; Flexas et al. 2008; Cano et al. 2013). It should be firstly
mentioned that the four parameters involved in g\textsubscript{m}
estimation (T\textsubscript{leaf},\selectlanguage{greek}\emph{Γ} \selectlanguage{english}\textsuperscript{*},
C\textsubscript{c} and R\textsubscript{d}) were markedly affected by
soil drought and changed as soil drought proceeded (Fig.3). Here in my
study, to get precise estimation of g\textsubscript{m},
R\textsubscript{d} was calculated from the light response curve based on
the Photosynthesis Assistant of Ye (2007) and modified according to soil
water status during DAT from 28 to 33. Compared to the response of
g\textsubscript{s} , g\textsubscript{m} in the drought-stressed
seedlings kept little change in the beginning of soil drought until a
significant increase in ABA\textsubscript{xylem}occurred (Fig.4g and
4h), indicating that g\textsubscript{m} was less sensitive to mild
drought stress than g\textsubscript{s}. This delayed response of
g\textsubscript{m} to drought was in accordance with the results
reported by Theroux-Rancourt et al. (2014), who suggested that the
threshold of regulation in mesophyll aquaporins was expected to appear
under more severe soil drought stress, e.g. a 51.43-44.68\% decrease in
\selectlanguage{greek}θ\selectlanguage{english}\textsubscript{FC} in our study. It was important to note that the
concurrence between ABA\textsubscript{xylem} and g\textsubscript{m} was
not a mere coincidence. Moreover, it was found that the change of
g\textsubscript{m} was tightly related to
ABA\textsubscript{xylem}(r=-0.643, \emph{p} \textless{}0.01). In line
with this, Mizokami et al (2015) concluded that g\textsubscript{m} in
ABA-deficient mutant (\emph{abal} ) did not decrease under drought
condition as compared to the wild type of \emph{Nicotiana
plumbaginifolia} . Therefore, root to shoot ABA signaling seemed to be a
candidate for actively controlling the conductance of mesophyll as
relative soil water decreased from 51.43 to 37.27\%. There was no doubt
that, as reported in many studies (Ferrio et al. 2012; Theroux-Rancourt
et al. 2014; Wang et al., 2018), g\textsubscript{m} decreased as
\selectlanguage{greek}Ψ\selectlanguage{english}\textsubscript{leaf} decreased with a good relationship (r=0.760,
\emph{p} \textless{}0.01) at the later stage of soil drought, indicating
that the decrease in g\textsubscript{m}might be partly due to the impact
of changes in leaf hydraulics. The reason of g\textsubscript{m}
unaffected by decreasing \selectlanguage{greek}Ψ\selectlanguage{english}\textsubscript{leaf} at the early stage of
soil drought might resulted from the hydraulic compartmentalization of
mesophyll cells from the transpiration stream, meaning cells could be
buffered against short-term changes in leaf water status (Zwieniecki et
al. 2007; Theroux-Rancourt et al. 2014). We did not investigate the
specific interaction between ABA signaling and hydraulic conductance in
regulation of mesophyll conductance in this experiment, but dual ways of
ABA in controlling stomatal movement: a direct biochemical effect on
guard cells and an indirect effect on leaf hydraulic conductance have
been confirmed by Pantin et al (2013). Considering the coordinated role
between hydraulic and chemical signals (Pantin et al. 2013; Mizokami et
al. 2015; Tombesi et al. 2015), therefore, we concluded that ABA in
xylem could be one signaling responsible for regulation mesophyll
conductance at the severe soil drought, e.g. RASC \textless{}51.43\% in
the present study.

As a result of unaffected g\textsubscript{m} and g\textsubscript{t},
A\textsubscript{n} did not decrease until RSWC reached to 44.68\%.
Though A\textsubscript{n} significantly decreased during RSWC reduction
from 51.43\% to 40.16\%, WUE\textsubscript{i} increased with decrease of
soil moisture (Fig.5). Our data was in accordance with the report of Liu
et al. (2005), who thought that mild soil water shortage could improve
water use efficiency. In this study, this drought-induced
WUE\textsubscript{i}increase might be explained by the improving
g\textsubscript{m}/g\textsubscript{s} due to the more positive
correlation between WUE\textsubscript{i} and
g\textsubscript{m}/g\textsubscript{s} (r=0.771,\emph{p}
\textless{}0.01), compared to the correlation between
WUE\textsubscript{i} and g\textsubscript{s} or
g\textsubscript{m}(r=-0.759 and r=-0.439, respectively) (Fig.5).
Therefore, it was clear that, the less sensitivity to soil drought in
g\textsubscript{m} than that in g\textsubscript{s} improved
g\textsubscript{m}/g\textsubscript{s}, and thereby enhanced
WUE\textsubscript{i}. This explanation was supported by the results of
the important role of g\textsubscript{m} or
g\textsubscript{m}/g\textsubscript{s} in improving
WUE\textsubscript{i}and A\textsubscript{n} simultaneously (Flexas et
al.2013; Han et al. 2016). However, it was important to note that, such
improving behavior in g\textsubscript{m}/g\textsubscript{s} could be
beneficial for maintaining water status under short-term drought during
RSWC reduction from 51.43\% to 40.16\%, but difficult for a long-term
and under the further increasing intensity of soil drought. This was due
to the improved WUE\textsubscript{i} at leaf scale may not always result
in an improvement of WUE at the whole plant sacle resulted from
remarkable reduction of crop yield under serious soil drought (Flexas et
al. 2015). Nonetheless, drought-induced CO\textsubscript{2} drawdown
from leaf surface to the site of carboxylation could in turn prevented
water loss, which increased chances of survival under prolonged severe
drought stress (Yan et al. 2017).

In order to accurately understand the effects of progressive soil
drought on photosynthetic capacity, we quantitatively analyzed
contribution of each limiting factor according to the methods proposed
by Grassi and Magnani (2005). In our study, the relative contribution of
g\textsubscript{s} could account for about 41.99\% of the net
photosynthesis reduction (Fig.6), indicating that stomatal resistance
may be the main limiting factor during RSWC decrease from 62.55 to
40.16\% (Fig. 2a). Our result was closely coincident with Xue et al
(2016) who reported that stomatal conductance mainly dominated the rate
of assimilation in response to drought stress. However, it should be
noted that, with increasing drought intensity, the contribution of
g\textsubscript{m} increased rapidly and nearly equalled to that of
g\textsubscript{s} when RSWC reached to 37.27\%. In addition, although
small difference in the main limitation was observed as compared to the
study of Wang et al (2018) might due to the different plant species and
soil water status, generally speaking, diffusive limitations to
CO\textsubscript{2} were the major constraints to photosynthesis under
drought condition.

In conclusion, our results confirmed the important role of root to shoot
ABA in regulation of mesophyll conductance and identified the threshold
of ABA signaling, i.e. relative soil water content decreasing from 51.43
to 44.68\% in the present study. Compared to g\textsubscript{s},
g\textsubscript{m} was less sensitive to mild soil drought until a
concurrent marked increasing ABA in xylem occurred,which potentially
enhanced the ratio of g\textsubscript{m} and g\textsubscript{s}, and
thereby improved WUE\textsubscript{i} during RSWC reduced from 51.43 to
40.16\%. The decrease in g\textsubscript{s} and g\textsubscript{m} was
the main constraints to photosynthesis under progressively soil drought
condition.

\textbf{Acknowledgements}

This work was supported by National Natural Science Foundation of China
(51790534, 51879267), the Basic Scientific Research Project of Chinese
Academy of Agricultural Sciences (FIRI2016-05), and the Agricultural
Science and Technology Innovation Program (ASTIP), Chinese Academy of
Agricultural Sciences.

\textbf{Author contributions}

AD and YG planned and designed the experiments; SL and JL performed the
experiments; SL analyzed the data and wrote the manuscript. AD, YG and
HL revised the manuscript. We declare that we do not have any commercial
or associative interest that represents a conflict of interest in
connection with the work submitted.

\textbf{Declarations of interest: none.}

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\textbf{Figures}

Fig.1. Dynamic change of relative soil water content in well-watered and
drought-stressed treatments after transplanting.

Fig.2. Leaf water potential (n=6) and xylem sap ABA concentration (n=3)
in response to progressive soil drying. Bars indicated stand error. **
indicated significant difference at \emph{p} \textless{}0.01 level and
ns indicated no significant difference between drought and well-watered
treatment (one-way variance analysis).

Fig.3. Dynamic change of relative leaf temperature
(T\textsubscript{leaf}), chloroplast CO\textsubscript{2} compensation
point (\selectlanguage{greek}\emph{Γ\textsuperscript{*}}\selectlanguage{english} ), chloroplastic
CO\textsubscript{2}concentration (C\textsubscript{c}) and day
respiration (R\textsubscript{d}) as relative soil water content
decreased. The letter (ns and **) was shown in chronological order as
soil drying at the DAT from 28 to 33 (n=6).

Fig.4. Response of physiological parameters to leaf water potential
(\selectlanguage{greek}Ψ\selectlanguage{english}\textsubscript{leaf}) and ABA concentration (ABA\textsubscript{xylem})
was shown by bubble diagram: A\textsubscript{n}, Tr,
g\textsubscript{s}to \selectlanguage{greek}Ψ\selectlanguage{english}\textsubscript{leaf} (a, b) and to
ABA\textsubscript{xylem} (c, d); A\textsubscript{n}, Tr,
g\textsubscript{m} to \selectlanguage{greek}Ψ\selectlanguage{english}\textsubscript{leaf} (e, f) and to
ABA\textsubscript{xylem} (g, h); A\textsubscript{n}, Tr,
g\textsubscript{t} to \selectlanguage{greek}Ψ\selectlanguage{english}\textsubscript{leaf} (i, j) and to
ABA\textsubscript{xylem} (k, l). X coordinate axis indicated
\selectlanguage{greek}Ψ\selectlanguage{english}\textsubscript{leaf} and ABA, respectively.
Y\textsubscript{1}coordinate axis indicated the conductance of
CO\textsubscript{2}(g\textsubscript{s}, g\textsubscript{m},
g\textsubscript{t}). Y\textsubscript{2} coordinate axis indicated
A\textsubscript{n} and T\textsubscript{r}, respectively. The size of
bubble indicated the rate of A\textsubscript{n} and T\textsubscript{r}.
Correlations between X and Y\textsubscript{1}, Y\textsubscript{1} and
Y\textsubscript{2} were presented (** \emph{p} \textless{}0.01).

Fig.5. Relationship between WUE\textsubscript{i} and
g\textsubscript{m}/g\textsubscript{s} in response to (a) leaf water
potential and (b) xylem sap ABA concentration was shown by bubble
diagram. X coordinate axis indicated \selectlanguage{greek}Ψ\selectlanguage{english}\textsubscript{leaf} and ABA,
respectively. Y\textsubscript{1} coordinate axis indicated the
g\textsubscript{m}/g\textsubscript{s}. Y\textsubscript{2} coordinate
axis indicated WUE\textsubscript{i}. The size of bubble indicated the
value of WUE\textsubscript{i}. Correlations between X and
Y\textsubscript{1}, Y\textsubscript{1} and Y\textsubscript{2} were
presented (** \emph{p} \textless{}0.01).

Fig.6. Effect of leaf water potential (\selectlanguage{greek}Ψ\selectlanguage{english}\textsubscript{leaf}) on the
relative contribution of the photosynthesis capacity limiting factors:
stomatal conductance (l\textsubscript{s}), mesophyll conductance
(l\textsubscript{m}) and photosynthetic biochemistry
(l\textsubscript{b}).

\textbf{Hosted file}

\verb`2-Figure.docx`  available at \url{https://authorea.com/users/304366/articles/434777-aba-signaling-in-regulation-of-mesophyll-conductance-in-tomato-during-progressive-soil-drying}

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