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\begin{document}

\title{Amelioration of saline-alkali land by cultivating Melia azedarach and
characterization of underlying mechanisms via metabolome analysis}



\author[1]{Xiaohua Long}%
\author[1]{Na Li}%
\author[1]{Tianyun Shao}%
\author[2]{Tianyun  Shao}%
\author[1]{Bin  Li}%
\author[1]{Xinglan Wang}%
\author[1]{Chenyunzhu Tao}%
\author[1]{Tianyun  Shao}%
\author[1]{Xiumei  Gao}%
\author[3]{ Zed  Rengel}%
\affil[1]{Nanjing Agricultural University College of Resources and Environmental Sciences}%
\affil[2]{Jiangsu Fangyang Group co ltd}%
\affil[3]{UWA School of Agriculture and Environment}%


\vspace{-1em}



  
  \date{May 17, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Soil salinization is a serious problem leading to ecological
degradation. \emph{Melia azedarach} is highly salt-tolerant, and its
application to saline-alkali land is a promising strategy for restoring
degraded lands. In this study, we analyzed the soil properties and
metabolome of \emph{M. azedarach} roots grown in low- (\textless{} 3
g/kg; L), medium- (5\textasciitilde{}8 g/kg; M), and high-
(\textgreater{} 10 g/kg; H) salinity soils to explore the amelioration
effect and adaptation mechanism of \emph{M. azedarach} to soils with
differential salinity. Cultivation of \emph{M. azedarach} was associated
with a decrease in the concentration of Na \textsuperscript{+} and
increases in organic matter content and alkaline phosphatase and urease
activities in the rhizosphere soil. The metabolome analysis revealed
that a total of 382 (ESI+) and 277 (ESI-) differential metabolites
(DEMs) were detected. The number of DEMs in roots rose with increased
soil salinity, such as sugars and flavonoids in H vs. L, and amino acids
in M vs. L. The most up-regulated DEMs were 13-S-hydroxyoctadecadienoic
acid, 2'-Deoxyuridine and 20-hydroxyleukotriene B4. Combined analysis of
soil properties and \emph{M. azedarach} DEMs indicated that alkaline
phosphatase activity was positively correlated with traumatic acid
concentration. Taken together, these results indicate that \emph{M.
azedarach} has the potential to reduce soil salinity and enhance soil
enzyme activity, and it can adapt to salt stress by regulating
metabolites like sugars, amino acids, and flavonoids \emph{.} This study
provided a basis for understanding the mechanism underlying the
adaptation of \emph{M. azedarach} to saline-alkali soil and its
amelioration.%
\end{abstract}%



\sloppy


Amelioration of saline-alkali land by cultivating \emph{Melia azedarach}
and characterization of underlying mechanisms via metabolome analysis

Na Li\textsuperscript{1}, Tianyun Shao\textsuperscript{1}*, Bingzhi
Jia\textsuperscript{2}, Xiao Yan\textsuperscript{1}, Xinglan
Wang\textsuperscript{1}, Chenyunzhu Tao\textsuperscript{1}, Xiaohua
Long\textsuperscript{1}*, Manqiang, Liu\textsuperscript{1}, Xiumei
Gao\textsuperscript{1}, Zed Rengel\textsuperscript{3,4}

\emph{\textsuperscript{1} College of Resources and Environmental
Sciences/Jiangsu Province Key Laboratory of Marine Biology, Nanjing
Agricultural University, Nanjing 210095, China}

\emph{\textsuperscript{2}Jiangsu Fangyang Group co., ltd} ,

\emph{Lianyungang 222065, China}

\emph{\textsuperscript{3} Soil Science and Plant Nutrition, UWA School
of Agriculture and Environment, The University of Western Australia, 35
Stirling Highway, Perth WA 6009, Australia}

\emph{\textsuperscript{4} Institute for Adriatic Crops and Karst
Reclamation, 21000 Split, Croatia}

* Corresponding author.

E-mail address: t2022065@njau.edu.cn (T.Y. Shao),
longxiaohua@njau.edu.cn (X.H. Long).

\textbf{ABSTRACT}

Soil salinization is a serious problem leading to ecological
degradation.\emph{Melia azedarach} is highly salt-tolerant, and its
application to saline-alkali land is a promising strategy for restoring
degraded lands. In this study, we analyzed the soil properties and
metabolome of \emph{M. azedarach} roots grown in low- (\textless{} 3
g/kg; L), medium- (5\textasciitilde{}8 g/kg; M), and high-
(\textgreater{} 10 g/kg; H) salinity soils to explore the amelioration
effect and adaptation mechanism of \emph{M. azedarach} to soils with
differential salinity. Cultivation of \emph{M. azedarach} was associated
with a decrease in the concentration of Na\textsuperscript{+}and
increases in organic matter content and alkaline phosphatase and urease
activities in the rhizosphere soil. The metabolome analysis revealed
that a total of 382 (ESI+) and 277 (ESI-) differential metabolites
(DEMs) were detected. The number of DEMs in roots rose with increased
soil salinity, such as sugars and flavonoids in H vs. L, and amino acids
in M vs. L. The most up-regulated DEMs were 13-S-hydroxyoctadecadienoic
acid, 2'-Deoxyuridine and 20-hydroxyleukotriene B4. Combined analysis of
soil properties and\emph{M. azedarach} DEMs indicated that alkaline
phosphatase activity was positively correlated with traumatic acid
concentration. Taken together, these results indicate that \emph{M.
azedarach} has the potential to reduce soil salinity and enhance soil
enzyme activity, and it can adapt to salt stress by regulating
metabolites like sugars, amino acids, and flavonoids\emph{.} This study
provided a basis for understanding the mechanism underlying the
adaptation of \emph{M. azedarach} to saline-alkali soil and its
amelioration.

\textbf{Key words:} Adaptation; In-situ restoration; \emph{Melia
azedarach} ; Root metabolome; Salt stress; Soil salinization

\section*{1. Introduction}

{\label{introduction}}

Soil salinization, as one of the major factors compromising crop growth
and leading to soil degeneration in natural ecological environment,
which have become a global concern (Haj-Amor et al., 2022; Singh, 2016).
Currently, 20\% of cultivated land and 50\% of irrigated land globally
are under the threat of salinization (Hopmans et al., 2021; Rozentsvet
et al., 2017). With climate change, population increase, intensive food
production and irrigation practices, soil salinization is expected to be
exacerbated (Singh, 2021). The area of saline-alkali land in China is
approximately 36 million ha, distributed mainly in the north-eastern and
north-western parts and along the coast (Jiang et al., 2022; Wang et
al., 2020).

Excessive salt in saline-alkali soils have always been regarded to be
the greatest hazard to crop production (Z\selectlanguage{ngerman}örb et al., 2019). Salt stress
have profound influences on the regulation of plant morphology,
physiology and metabolic process (Yang \& Guo, 2018), which can lead to
plant dehydration, ion toxicity, and even plant death (McDowell et al.,
2022).

Plants possess a wide range of metabolic strategies to promote osmotic
adaptation and antioxidant mechanisms under salt stress (de Freitas et
al., 2019). Roots are the main organ for sensing salt stress signals and
responding to excess salt (Redwan et al., 2017). The analysis of root
metabolite differences under different salinity conditions is important
in elucidating the salt tolerance mechanism of plants (Barding et al.,
2013; Ben Hsouna et al., 2022).

\emph{M. azedarach} has high adaptability and strong salt tolerance;
hence, growing \emph{M. azedarach} on saline-alkali land has been
considered as a promising strategy for degraded land restoration (Dias
et al., 2014; Husain \& Anis, 2009). \emph{M. azedarach} can effectively
increase bacterial communities diversity and enhance environmental
carrying capacity (Li et al., 2021b; Shahbaz \& Ashraf, 2013).

Understanding of how \emph{M. azedarach} metabolome responds to
different soil salinities is poor. In this paper, soil properties
and\emph{M. azedarach} root metabolome were characterized under high,
medium and low salinity to explore the ameliorative effects of \emph{M.
azedarach} on soils with different salt content and the metabolome
changes of \emph{M. azedarach} in response to different severities of
salt stress.

\section*{2. Materials and methods}

{\label{materials-and-methods}}

\subsection*{\texorpdfstring{\emph{2.1. Study
area}}{2.1. Study area}}

{\label{study-area}}

The field experiment was conducted in Xuwei New District (34°37\selectlanguage{english}'N,
119deg29\selectlanguage{english}'E), Lianyungang City, Jiangsu Province, where the annual average
rainfall and evaporation were 901 mm and 855 mm, respectively. The
experimental site was divided into three parts: low salinity
(\textless{} 3 g/kg), medium salinity (5\textasciitilde{}8 g/kg), and
high salinity (\textgreater{} 10 g/kg). Before\emph{M. azedarach} was
cultivated, the experimental site was plowed to a depth of 40 cm, and
the row spacing was 2.0 x 3.0 m.

\subsection*{\texorpdfstring{\emph{2.2. Sample
collection}}{2.2. Sample collection}}

{\label{sample-collection}}

In August 2020 (after 40 months of \emph{M. azedarach} growth), the
samples of soil and roots of\emph{M. azedarach} were collected from each
plot {[}high (H), medium (M) and low (L) salinity{]}. Samples of
rhizosphere soil (soil attached to \emph{M. azedarach} roots) and bulk
soil (soil away from \emph{M. azedarach} roots) were collected in each
plot. Three replicates were collected in each plot, with each replicate
consisting of six sampling points. All soil samples were air-dried and
then sieved through a 0.147 mm sieve for soil chemical and enzymatic
activities analysis.

Six replicates of roots were sampled in each plot, and each replicate
comprised three sampling points. The samples were stored at -80 \selectlanguage{english} for
analysis of root metabolome. Root samples (R) collected in different
salinities were labelled: RH, RM and RL for high, medium and low
salinity, respectively.

\subsection*{\texorpdfstring{\emph{2.3. Measurement of soil
properties}}{2.3. Measurement of soil properties}}

{\label{measurement-of-soil-properties}}

Soil salinity was determined by the conductivity method (Yue et al.,
2020). Soil pH was assessed by a calibrated pH meter (FE28-Standard,
Mettler Toledo, Greifensee, Switzerland) (Zhang \& Pang, 1999). Soil
water-soluble Na\textsuperscript{+}, K\textsuperscript{+},
Ca\textsuperscript{2+}, and Mg\textsuperscript{2+}were measured by
ICP-OES (Optima 2100DV, Perkin-Elmer, Waltham, Massachusetts, USA) (Yang
et al., 2016). Soil water-soluble Cl\textsuperscript{-} content was
determined by the silver nitrate titration method (Asakai, 2018).

Soil organic matter was determined using the potassium dichromate method
(Osman et al., 2013), available phosphorus by the molybdenum-antimony
colorimetric method (Wang et al., 2011) and available potassium by flame
photometry (Bilias \& Barbayiannis, 2019).

Soil enzyme activities were measured using aprpriate kits (Solarbio,
Beijing, China). Activity of soil alkaline phosphatase (S-AP) was
measured by the determination of phenol produced during the hydrolysis
of the substrate (Li et al., 2021b). Activity of soil urease activity
(S-UE) was obtained by measuring NH\textsubscript{3}-N produced by the
urease hydrolysis of urea based on the indophenol blue colorimetric
method (Huang et al., 2014).

\subsection*{\texorpdfstring{\emph{2.4. Characterizations of} \emph{M.
azedarach
metabolome}}{2.4. Characterizations of M. azedarach metabolome}}

{\label{characterizations-of-m.-azedarach-metabolome}}

\subsubsection*{\texorpdfstring{\emph{2.4.1. Metabolite
extraction}}{2.4.1. Metabolite extraction}}

{\label{metabolite-extraction}}

An amount of 50 mg of fresh \emph{M. azedarach} roots was weighed
accurately ground in 1000 \selectlanguage{greek}μ\selectlanguage{english}L of extractant (methanol-acetonitrile-water,
2:2:1, v/v) containing internal standard (l,2-chlorophenylalanine, 2 mg)
at 45 Hz for 10 min in a Tissuelyser-32; the suspension was allowed to
stand at -20  for 1 hour, followed by centrifugation at 16,114 \emph{g}
for 15 min (Sun et al., 2022). The supernatant was collected and dried
in a vacuum concentrator. The dried metabolites were reconstructed in
the extractant (acetonitrile-water, 1:1, v/v), sonicated in an ice-water
bath for 10 min, and centrifuged at 16,114 \emph{g} for 15 minutes; the
supernatant was used for metabolomic analysis (Li et al., 2022).

\subsubsection*{\texorpdfstring{\emph{2.4.2. UPLC-Q-TOF-MS for metabolite
analysis}}{2.4.2. UPLC-Q-TOF-MS for metabolite analysis}}

{\label{uplc-q-tof-ms-for-metabolite-analysis}}

Extracts were analyzed by UPLC (Waters UPLC Acquity I-Class PLUS) and
QTOF system (Waters UPLC Xevo G2-XS QTof) with a chromatographic column
Acquity UPLC HSS T3, mobile phase A (0.1\% formic acid aqueous
solution), and mobile phase B (0.1\% formic acid acetonitrile) (Fan et
al., 2022). Metabolites need to be ionized before they can be detected
by mass spectrometry. The ESI source operation parameters were as
follows: capillary voltage at 2000 V (positive ion mode) or -1500 V
(negative ion mode), a cone voltage at 30 V, a source temperature of
150 \selectlanguage{ngerman}°C, desolvation to 500\selectlanguage{english} degC and a desolvation gas flow to 800
L*h\textsuperscript{-1} (Liu et al., 2022).

\subsubsection*{\texorpdfstring{\emph{2.4.3. Metabolome data
processing}}{2.4.3. Metabolome data processing}}

{\label{metabolome-data-processing}}

The raw data were collected by MassLynx (v4.2) and processed by
Progenesis QI software (Huang et al., 2022), before identified based on
Progenesis QI software, the online METLIN database, and Bemec's
proprietary library. The criteria for differential metabolites (DEM)
were FC\selectlanguage{english}[?]2.0 or FC\selectlanguage{english}[?]0.5,\emph{p} -value\textless{}0.05, and
VIP\textgreater{}1.0.

\subsection*{\texorpdfstring{\emph{2.5. Statistical
analyses}}{2.5. Statistical analyses}}

{\label{statistical-analyses}}

An independent sample \emph{t} -test was done by IBM SPSS Statistics 20
(IBM, Armonk, NY, USA), with \emph{p} \textless{} 0.05 considered
statistically significant. Graphs were drawn by Graphpad prism v8.0.1
and R package ggplot 2 (v3.2.0). The Venn diagrams were constructed by
the R package Venn diagram (v1.6.20). The network diagram was produced
by Cytoscape (v3.6.1).

\section*{3. Results}

{\label{results}}

\subsection*{\texorpdfstring{\emph{3.1. Soil chemical properties and
enzyme
activities}}{3.1. Soil chemical properties and enzyme activities}}

{\label{soil-chemical-properties-and-enzyme-activities}}

The soil salinity (Figure 1a) and soluble ions (Na\textsuperscript{+},
Mg\textsuperscript{2+}, K\textsuperscript{+}, Ca\textsuperscript{2+},
and Cl\textsuperscript{-}) (Figure 1c-g) in rhizosphere soil were
significantly lower than those in bulk soil under medium- and high-
salinity. The salinity of rhizosphere soil decreased by 57\% and 62\%
compared with that of bulk soil under medium and high salinity
conditions, respectively.

Regardless of soil salinity, the rhizosphere soil had higher organic
matter content than the bulk soil (Figure 1h). The same changes were
found in the activities of S-AP and S-UE (Figure 1k, 1l). Under medium
salinity, the contents of available phosphorus and available potassium
in the rhizosphere soil were 2.13- and 1.09-fold greater than in the
bulk soil, respectively (Figure 1j-k).

\subsection*{\texorpdfstring{\emph{3.2. Metabolomic analysis of M.
azedarach under salt
stress}}{3.2. Metabolomic analysis of M. azedarach under salt stress}}

{\label{metabolomic-analysis-of-m.-azedarach-under-salt-stress}}

\subsection*{\texorpdfstring{\emph{3.2.1. Venn diagram and volcanic plot
analysis of
DEMs}}{3.2.1. Venn diagram and volcanic plot analysis of DEMs}}

{\label{venn-diagram-and-volcanic-plot-analysis-of-dems}}

There were 382 (ESI+) (Figure 2a) and 277 (ESI-) DEMs (Figure 2b). Venn
diagram analysis of DEMs showed there were more common DEMs between RH
vs. RL and RM vs. RL than in other combinations. \emph{M. azedarach}had
most of unique DEMs in RH vs. RL, including 72 DEMs in ESI+ and 90 DEMs
in ESI-.

The analysis of the volcano plots showed there were more up-regulated
than down-regulated DEMs under salt stress. The DEMs significantly
up-regulated in RM vs. RL were homoveratric acid, 2'-deoxyuridine and
7,8-dihydroneopterin (Figure 3a-b). The significantly up-regulated DEMs
in RH vs. RL were 13-S-hydroxyoctadecadienoic acid,
20-hydroxyleukotriene B4 and 7,8-dihydroneopterin (Figure 3c-d). The
significantly up-regulated DEMs in RH vs. RM were anhwiedelphinine,
hyperoside and histidinyl-lysine (Figure 3e-f).

\subsubsection*{\texorpdfstring{\emph{3.2.2. Sugars, amino acids, and
flavonoids}}{3.2.2. Sugars, amino acids, and flavonoids}}

{\label{sugars-amino-acids-and-flavonoids}}

Nine sugars (D-mannose, maltotetraose, maltohexaose, stachyose,
1-kestose, raffinose, 3-alpha-mannobiose, GDP-glucose, and D-(-)-lyxose)
were identified in DEMs, most sugars were up-regulated in RM vs. RL and
RH vs. RL (Table 1).

Six amino acids were detected in DEMs, including L-theanine, L-proline,
L-lysine, L-isoleucine, DL-leucine and aspartic acid. Five out of six
amino acids in DEMs (except L-Theanine) were up-regulated in RM vs. RL
(Table 2).

Flavonoids have antioxidant activity. Twenty-seven types of flavonoids
were detected in DEMs. Among them, 19 were up-regulated in RM vs. RL and
23 were up-regulated in RH vs. RL (Table 3).

\emph{3.2.3. KEGG pathway analysis}

The number of DEMs enriched in the biosynthesis pathway of flavonoids
and amino acids ranked the top five among KEGG pathways (Figure 4). The
expressions of L-proline, L-lysine, L-isoleucine, S-adenosylmethionine,
and L-saccharopine in the biosynthesis of amino acids were higher in RM
than in RL (Figure 5a), while citrate and L-saccharopine were higher in
RH than in RM (Figure 5c). With soil salinity increased (RH vs. RL),
three of the four DEMs in the biosynthesis of amino acids (L-proline,
aspartic acid, and citrate) were up-regulated (Figure 5b). Among the
DEMs involved in flavonoid biosynthesis, the expressions of
(-)-naringenin, vitexin, (+)-gallocatechin, pinocembrin, (-)-epicatechin
and epigallocatechin were higher in RM vs. RL (Figure 5a) and RH vs. RL
(Figure 5b).

\subsection*{\texorpdfstring{\emph{3.3.} \emph{Combined analysis of soil
properties and M. azedarach
DEMs}}{3.3. Combined analysis of soil properties and M. azedarach DEMs}}

{\label{combined-analysis-of-soil-properties-and-m.-azedarach-dems}}

Six soil chemical properties (salinity, Na\textsuperscript{+},
Cl\textsuperscript{-}, K\textsuperscript{+}, available phosphorus and
available potassium) were correlated negatively with four DEMs
of\emph{M. azedarach} : prunasin, androstendione,\emph{o}
-succinyl-L-homoserine and traumatic acid (Figure 6). By contrast,
alkaline phosphatase activity was positively correlated with these four
DEMs. Soil pH was negatively correlated with prunasin, and
Ca\textsuperscript{2+} was positively correlated with prunasin and
corticosterone.

\section*{4. Discussion}

{\label{discussion}}

Coastal saline soil contains excess salinity, with Na\textsuperscript{+}
and Cl\textsuperscript{-} being the main base ions (Zhang et al., 2022).
Soil organic matter (SOM) is dominant to global carbon cycle
(Bhattacharyya, 2022), S-AP activity is an indicator of soil phosphorus
cycle (Liberti et al., 2013), and S-UE plays a crucial role in
influencing the content of soil nitrogen (Albiach et al., 2000). In this
paper, salinity, contents of Na\textsuperscript{+} and
Cl\textsuperscript{-} in the rhizosphere soils were significantly lower
than bulk soils, whereas SOM and activities of S-AP and S-UE showed the
opposite trend (Figure 1). These results indicated that \emph{M.
azedarach} played a critical role in reducing soil salinity and
promoting soil nutrient cycling.

Excess salinity in saline-alkali land can cause osmotic stress, ion
toxicity, oxidative damage, nutrient deficiency and even cell death in
plants (Elkelish et al., 2019; Ghazali, 2020). Plants can cope with salt
stress by regulating biosynthesis of metabolites such as sugars, amino
acids and flavonoids (Batista-Silva et al., 2019).

Sugars can be bound to inorganic solutes (e.g. K\textsuperscript{+}) to
maintain cell expansion and are active in osmotic regulation under salt
stress (Arbelet-Bonnin et al., 2018). Similar results were reported in
other studies, with sugars acting not only as osmoprotectants and cell
membrane stabilizer, but also as scavengers of reactive oxygen species
(Nishizawa et al., 2008). The contents of various sugars were
up-regulated under medium and high salinity (Table 1), indicated that
\emph{M. azedarach} could produce more sugars to regulate osmotic
pressure when exposed to a gradient of salt stress.

Besieds, amino acids are important osmoprotectants and antioxidants, and
their accumulation can enhance the stability of cell membrane and
improve salt tolerance of plants (Slama et al., 2015; Widodo et al.,
2009). Hosseinifard et al. (2022) discovered that proline could inhibit
the accumulation of toxic ions under salt stress. With the increase in
the severity of salt stress, the amino acid DEMs were up-regulated, and
more amino acids were synthesized under medium compared with low
salinity (Figure 5a-b, Table 2). The result showed that \emph{M.
azedarach} increased its salt tolerance by up-regulating amino acid as
the severity of salinity progressed from low to medium.

Flavonoids have antioxidant activity, protecting cell structures from
oxidative damage and enhance the stress resistance of plants (Saeed et
al., 2012; Sumner et al., 2003). More than 70\% of the flavonoids were
up-regulated in \emph{M. azedarach} roots with soil salinity increased
(Table 3). And DEMs involved in flavonoid biosynthesis were largely
up-regulated with salinity increased (Figure 5). These results indicated
that\emph{M. azedarach} could enhance the antioxidant activity and salt
tolerance of plants by up-regulating the expression of flavonoids.

Among \emph{M. azedarach}metabolites, traumatic acid was shown to
participate in the plant adaptation and tolerance to salinity by
diminishing the negative effects of sodium and chloride ions and
increasing the monosaccharide content (Pietryczuk et al., 2014).
Alkaline phosphatase can release inorganic phosphorus for plants growing
on phosphorus-deficient saline soils (Li et al., 2021a). In the present
study, alkaline phosphatase activity was positively correlated with
traumatic acid concentration (Figure 6), indicating that there should be
a synergistic effect between soil alkaline phosphatase activity and
traumatic acid in \emph{M. azedarach} , facilitating\emph{M. azedarach}
tolerance to salt stress.

\section*{5. Conclusion}

{\label{conclusion}}

In conclusion, the cultivation of\emph{M. azedarach} could reduce soil
salinity and promote soil nutrient cycling. \emph{M.
azedarach}alleviated salt stress by up-regulating the expression of
metabolites such as sugars, amino acids and flavonoids. There was a
synergistic effect between alkaline phosphatase and traumatic acid in
improving the salt tolerance of \emph{M. azedarach} . This research
provided a solid basis for clarifying the adaptation and amelioration
mechanism of\emph{M. azedarach} to salt stress on saline-alkali land.

\section*{Aacknowledgments}

{\label{aacknowledgments}}

This work was supported by funding from the following projects: National
Key Project of Scientific and Technical Supporting Programs funded by
the Ministry of Science \& Technology of Jiangsu Province (BE2022304),
Forestry Science and Technology Innovation and Extension Project in
Jiangsu Province (LYKJ{[}2019{]}07), and the Programs funded by the
Ministry of Science \& Technology of Inner Mongolia (2020CG0057).

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{\label{references}}

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\section*{Figure and Table captions}

{\label{figure-and-table-captions}}

FIGURE 1 Chemical properties and enzyme activities in the rhizosphere
and bulk soils with different salinities. (a) Salinity, (b) pH, (c)
Na\textsuperscript{+}, (d) Mg\textsuperscript{2+}, (e)
K\textsuperscript{+}, (f) Ca\textsuperscript{2+}, (g)
Cl\textsuperscript{-}, (h) organic matter, (i) available phosphorus, (j)
available potassium, (k) alkaline phosphatase, and (l) urease. Note:
Asterisks denote significant differences (\emph{p} \selectlanguage{english}[?]0.05). Means +- SE (n
= 3). L, low-salinity soil; M, medium-salinity soil; H, high-salinity
soil.

FIGURE 2 Venn diagrams of DEMs in \emph{Melia azedarach} roots: (a) ESI+
mode, (b) ESI- mode. Note: RL- roots in low salinity soil, RM- roots in
medium salinity soil, RH- roots in high salinity soil.

FIGURE 3 Volcano plots of DEMs in \emph{Melia azedarach} roots. Note:
RL- roots in low salinity soil, RM- roots in medium salinity soil, RH-
roots in high salinity soil. Red represents up-regulated and blue
represents down-regulated DEMs.

FIGURE 4 Enrichment diagram of \emph{Melia azedarach} root DEMs in KEGG
pathways. Note: RL- roots in low salinity soil, RM- roots in medium
salinity soil, RH- roots in high salinity soil.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

FIGURE 5 DEMs enrichment network diagram of biosynthesis of amino acids
and flavonoid biosynthesis. (a) RM vs. RL, (b) RH vs. RL, (c) RH vs. RM.
Note: RL- roots in low salinity soil, RM- roots in medium salinity soil,
RH- roots in high salinity soil. - upregulated DEMs, - downregulated
DEMs. The small nodes connected to the large node (KEGG pathway) are
DEMs annotated to the pathway. The thickness of the line indicates the
fold change.

FIGURE 6 Combined analysis of soil properties and DEMs in \emph{Melia
azedarach} roots. Green circles represent soil properties, and orange
circles represent DEMs. The color of connecting lines indicates the
correlation between nodes (red- positive correlation, blue- negative
correlation).

TABLE 1 Regulation of sugars in DEMs

TABLE 2 Regulation of amino acids in DEMs

TABLE 3 Regulation of flavonoids in DEMs

TABLE 1 Regulation of sugars in DEMs\selectlanguage{english}
\begin{longtable}[]{@{}llllllllll@{}}
\toprule
Flavonoids & RM vs. RL & RM vs. RL & RM vs. RL & RH vs. RL & RH vs. RL &
RH vs. RL & RH vs. RM & RH vs. RM & RH vs. RM\tabularnewline
& log\textsubscript{2}FC & \emph{p}-value & regulated &
log\textsubscript{2}FC & \emph{p}-value & regulated &
log\textsubscript{2}FC & \emph{p-}value & regulated\tabularnewline
D-Mannose & 2.49 & 0.005 & up & 4.00 & 0.000 & up & 1.50 & 0.000 &
up\tabularnewline
Maltotetraose & 1.79 & 0.000 & up & 2.88 & 0.000 & up & 1.09 & 0.002 &
up\tabularnewline
Maltohexaose & 0.96 & 0.000 & unchanged & 1.32 & 0.000 & up & 0.35 &
0.038 & unchanged\tabularnewline
Stachyose & 2.34 & 0.000 & up & 2.15 & 0.000 & up & -0.19 & 0.316 &
unchanged\tabularnewline
1-Kestose & 1.44 & 0.001 & up & 2.97 & 0.000 & up & 1.52 & 0.001 &
up\tabularnewline
Raffinose & 1.55 & 0.005 & up & 1.08 & 0.039 & up & -0.47 & 0.190 &
unchanged\tabularnewline
3-alpha-Mannobiose & 1.27 & 0.000 & up & 1.27 & 0.001 & up & 0.01 &
0.973 & unchanged\tabularnewline
GDP-glucose & 0.85 & 0.018 & unchanged & 1.21 & 0.002 & up & 0.36 &
0.042 & unchanged\tabularnewline
D-(-)-Lyxose & 2.35 & 0.025 & up & 4.08 & 0.030 & up & 1.73 & 0.077 &
unchanged\tabularnewline
\bottomrule
\end{longtable}

TABLE 2 Regulation of amino acids in DEMs\selectlanguage{english}
\begin{longtable}[]{@{}llllllllll@{}}
\toprule
Flavonoids & RM vs. RL & RM vs. RL & RM vs. RL & RH vs. RL & RH vs. RL &
RH vs. RL & RH vs. RM & RH vs. RM & RH vs. RM\tabularnewline
& log\textsubscript{2}FC & \emph{p}-value & regulated &
log\textsubscript{2}FC & \emph{p}-value & regulated &
log\textsubscript{2}FC & \emph{p}-value & regulated\tabularnewline
L-Theanine & 0.92 & 2.3E-05 & unchanged & -1.24 & 4E-04 & down & -2.15 &
2E-07 & down\tabularnewline
L-Proline & 3.31 & 1E-06 & up & 2.93 & 1E-07 & up & -0.38 & 0.0004 &
unchanged\tabularnewline
L-Lysine & 1.16 & 8.1E-05 & up & 1.00 & 4E-05 & unchanged & -0.16 &
0.1736 & unchanged\tabularnewline
L-Isoleucine & 1.72 & 2.1E-05 & up & 0.96 & 1E-04 & unchanged & -0.76 &
6E-05 & unchanged\tabularnewline
DL-Leucine & 1.95 & 3.8E-05 & up & 0.62 & 0.019 & unchanged & -1.33 &
4E-05 & down\tabularnewline
Aspartic acid & 1.93 & 8.5E-05 & up & -0.03 & 0.958 & unchanged & -1.96
& 8E-05 & down\tabularnewline
\bottomrule
\end{longtable}

TABLE 3 Regulation of flavonoids in DEMs\selectlanguage{english}
\begin{longtable}[]{@{}llllllllll@{}}
\toprule
Flavonoids & RM vs. RL & RM vs. RL & RM vs. RL & RH vs. RL & RH vs. RL &
RH vs. RL & RH vs. RM & RH vs. RM & RH vs. RM\tabularnewline
& log\textsubscript{2}FC & \emph{p}-value & regulated &
log\textsubscript{2}FC & \emph{p}-value & regulated &
log\textsubscript{2}FC & \emph{p}-value & regulated\tabularnewline
5,7,3',4',5'-Pentahydroxyflavanone & 1.37 & 0.002 & up & 1.43 & 6E-09 &
up & 0.07 & 0.71 & unchanged\tabularnewline
Prodelphinidin A1 & 1.64 & 2E-04 & up & 1.75 & 2E-08 & up & 0.11 & 0.42
& unchanged\tabularnewline
(+)-Gallocatechin & 2.61 & 0.001 & up & 2.39 & 2E-06 & up & -0.22 & 0.33
& unchanged\tabularnewline
5,7-Dimethoxy-6-C-methylflavone & 0.94 & 4E-05 & unchanged & 1.04 &
5E-04 & up & 0.10 & 0.44 & unchanged\tabularnewline
Phellodensin F & 1.70 & 2E-05 & up & 1.61 & 3E-06 & up & -0.09 & 0.4 &
unchanged\tabularnewline
Licoagrodin & 1.04 & 8E-06 & up & 0.85 & 5E-05 & unchanged & -0.19 &
0.04 & unchanged\tabularnewline
(-)-Catechin gallate & 1.58 & 0.002 & up & 1.54 & 1E-06 & up & -0.03 &
0.86 & unchanged\tabularnewline
Taxifolin 3-arabinoside & 1.67 & 3E-04 & up & 1.37 & 7E-05 & up & -0.30
& 0.12 & unchanged\tabularnewline
Nympholide A & 1.22 & 2E-05 & up & 1.62 & 1E-06 & up & 0.40 & 0 &
unchanged\tabularnewline
Munsericin & 0.97 & 2E-05 & unchanged & 1.21 & 2E-04 & up & 0.24 & 0.09
& unchanged\tabularnewline
(-)-Epicatechin & 1.82 & 2E-04 & up & 2.15 & 9E-04 & up & 0.33 & 0.16 &
unchanged\tabularnewline
Vitexin & 3.03 & 0.012 & up & 3.23 & 1E-03 & up & 0.20 & 0.60 &
unchanged\tabularnewline
3-Hydroxyflavone & 1.75 & 0.003 & up & 2.03 & 4E-04 & up & 0.28 & 0.29 &
unchanged\tabularnewline
Pinocembrin & 1.18 & 0.001 & up & 1.45 & 8E-05 & up & 0.27 & 0.02 &
unchanged\tabularnewline
Rutin & 0.77 & 0.001 & unchanged & 1.03 & 2E-07 & up & 0.26 & 0.04 &
unchanged\tabularnewline
6-O-Malonyldaidzin & 1.33 & 2E-04 & up & 1.32 & 4E-06 & up & -0.01 &
0.96 & unchanged\tabularnewline
(-)-Naringenin & 1.14 & 0.003 & up & 1.05 & 5E-06 & up & -0.09 & 0.63 &
unchanged\tabularnewline
Catechin & -4.01 & 0.049 & down & -5.06 & 0.044 & down & -1.05 & 0.30 &
unchanged\tabularnewline
Epigallocatechin & 3.03 & 0.003 & up & 2.63 & 0.008 & up & -0.40 & 0.32
& unchanged\tabularnewline
Theaflavin & 1.82 & 3E-04 & up & 1.60 & 0.011 & up & -0.22 & 0.46 &
unchanged\tabularnewline
Taxifolin & 3.10 & 0.001 & up & 2.79 & 0.008 & up & -0.30 & 0.40 &
unchanged\tabularnewline
Biochanin A & 0.66 & 0.077 & unchanged & 1.03 & 0.017 & up & 0.37 & 0.27
& unchanged\tabularnewline
Xanthohumol & -1.64 & 0.060 & unchanged & -2.09 & 0.041 & down & -0.45 &
0.19 & unchanged\tabularnewline
Baicalin & 0.19 & 0.677 & unchanged & 2.29 & 0.004 & up & 2.10 & 0 &
up\tabularnewline
Apigenin 7-glucoside & 4.39 & 0.009 & up & 4.74 & 0.008 & up & 0.35 &
0.47 & unchanged\tabularnewline
Hyperoside & -0.59 & 0.224 & unchanged & 0.92 & 0.011 & unchanged & 1.50
& 0 & up\tabularnewline
Deguelin & 1.80 & 0.024 & up & 2.06 & 0.039 & up & 0.26 & 0.64 &
unchanged\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-3/Figure-3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
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\includegraphics[width=0.70\columnwidth]{figures/Figure-5/Figure-5}

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\includegraphics[width=0.70\columnwidth]{figures/Figure-6/Figure-6}

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