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\begin{document}

\title{Research on soil enzymes and polysaccharides secreted by the roots of
Salvia miltiorrhiza Bunge under abiotic stress}



\author[1]{yong qin}%
\author[1]{Xiaoyu  Li}%
\author[1]{yanhong Wu}%
\author[1]{guiqi han}%
\author[1]{hai wang}%
\author[1]{Zhuyun Yan Yan}%
\affil[1]{Chengdu University of Traditional Chinese Medicine Wenjiang Campus}%


\vspace{-1em}



  
  \date{May 27, 2024}


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\selectlanguage{english}
\begin{abstract}
Root exudates serve as crucial mediators for the information exchange
between plants and soil, which is an important evolutionary mechanism
for plants to adapt to environmental changes. In this study, 15
different abiotic stress models were established using various stress
factors, including drought (D), high temperature (T), nitrogen
deficiency (N), and phosphorus deficiency (P) and their combinations. We
investigated their effects on the seedling growth of Salvia miltiorrhiza
Bunge and the activities of%
\end{abstract}%



\sloppy


\textbf{Research on soil enzymes and polysaccharides secreted by the
roots of \emph{Salvia miltiorrhiza}Bunge under abiotic stress}

\textbf{Yong Qin\textsuperscript{1} \textbar{} Xiaoyu
Li\textsuperscript{1} \textbar{} Yanhong Wu\textsuperscript{1}\textbar{}
Hai Wang\textsuperscript{1} \textbar{} Guiqi Han\textsuperscript{1}
\textbar{} Zhuyun Yan\textsuperscript{1}}

\textsuperscript{1} State Key Laboratory of Southwestern Chinese
Medicine Resources, Chengdu University of Traditional Chinese Medicine,
Chengdu 610075, China.

\textbf{Correspondence}

Prof. Dr. Zhu-Yun Yan, School of Pharmacy, Chengdu University of
Traditional Chinese Medicine, Chengdu, P.R. China. E-mail:
yanzhuyun@cdutcm.edu.cn Phone: +86 139 8196 4354 Fax: +86 61 80 02 31

\textbf{Abstract}

Root exudates serve as crucial mediators for the information exchange
between plants and soil, which is an important evolutionary mechanism
for plants to adapt to environmental changes. In this study, 15
different abiotic stress models were established using various stress
factors, including drought (D), high-temperature (T), nitrogen
deficiency (N), and phosphorus deficiency (P) and their combinations. We
investigated their effects on the seedling growth of \emph{Salvia
miltiorrhiza} Bunge and the activities of urease (S-UE), nitrite
reductase (S-NiR), nitrate reductase (S-NR), phosphotransferase (S-PT),
and catalase (S-CAT), as well as the contents of polysaccharides in the
culture medium. The results showed that the growth of \emph{S.
miltiorrhiza} was inhibited under 15 stress conditions, among which 13
stress conditions could increase the root-shoot ratio. These 15 stress
conditions significantly down-regulated the activity of S-NR,
synergistic stresses of drought and nitrogen deficiency (DN) and
synergistic stresses of high-temperature and nitrogen deficiency (TN)
significantly up-regulated the activity of S-NIR (\emph{p}
\textless{}0.05). The N, D, T, synergistic stresses of drought and
high-temperature (DT), DN, synergistic stresses of drought and
phosphorus deficiency (DP), and synergistic stresses of
high-temperature, nitrogen deficiency, and phosphorus deficiency (TNP)
stresses conditions significantly increased the activity of S-UE
(\emph{p} \textless{}0.05). The activity of soil enzyme S-PT could be
down-regulated under most stress conditions, but only D and T stresses
could significantly up-regulate S-PT activity (\emph{p}
\textless{}0.05). The N, DN, and TN stresses conditions significantly
reduced S-CAT activity. The content of total polysaccharides in soil was
decreased under most stress conditions, and P, DT, and synergistic
stresses of drought, high-temperature, and phosphorus deficiency (DTP)
stresses were significantly decreased (\emph{p} \textless{}0.05). These
results indicated that abiotic stress inhibited the growth of \emph{S.
miltiorrhiza} and altered the root secretion behavior. Plants respond to
different abiotic stresses by regulating root secretions, including
enzymes of the soil nitrogen cycle, phosphorus transport-related
enzymes, and antioxidant enzymes. In conclusion, plants regulate the
utilization of rhizosphere substances by regulating the intensity of
soil enzymes and polysaccharides secreted by roots to cope with abiotic
stress. At the same time, soil carbon sequestration is affected by the
adverse environment, which restricts the input of organic matter into
the soil.

\textbf{KEYWORDS}

\textbf{abiotic stress, \emph{Salvia miltiorrhiza} seedlings, soil
enzymes, total polysaccharides, soil carbon sequestration}

\textbf{1 \textbar{} INTRODUCTION}

Root exudates are a complex mixture of biochemical compounds that are
secreted actively or passively by plant roots. These compounds comprise
diverse small molecules, such as amino acids and organic acids, and
macromolecules, including polysaccharides, proteins, and biologically
active enzymes (Chai and Schachtman, 2022). Biological and abiotic
stress conditions alter the composition and quantity of plant root
exudates, thereby altering the rhizosphere ecosystem and assisting
plants in adapting to different stress environments (Chai and
Schachtman, 2022, Calvo et al., 2017). During the process of plant
growth, development and reproduction, about 21\% of the net
photosynthetic products of plants enter the soil as root exudates, and
when plants adapt to different stress environments (Wang et al., 2021,
Panchal et al., 2022, Pramanik and Phukan, 2020). Moreover, root
exudates provide nutrients and energy to the rhizosphere microbiome,
regulate the structure of microbial communities in soil, affect enzyme
activities produced by microorganisms, and ultimately influence the
decomposition, mineralization, and availability of organic compounds and
soil nutrients (Lu et al., 2021). It can be seen that root exudates
affect the action strength and direction of soil enzymes, and play a key
role in the soil material cycle, energy conversion and plant carbon
storage (Panchal et al., 2022). Especially, it plays an important role
in the soil nitrogen and phosphorus cycle (Malek et al., 2021).

Soil enzymes are a complex mixture of both plant and microbial sources.
Root secretion is not only an important source of soil enzymes but also
the main factor driving microorganisms to secrete soil enzymes. Many
enzymes play key roles in soil nitrogen cycling, such as Solid-Nitrate
Reductase (S-NR), Solid-Nitrite reductase (S-NiR) and Solid-Urease
(S-UE). Specifically, S-NR catalyzes the reduction of nitrate to
nitrite, S-NiR catalyzes the reduction of nitrite to nitric oxide
(Jian-guo and Wei-guo, 2018), and S-UE hydrolyzes urea to produce
ammonia and carbonic acid (Fisher et al., 2017). The
solid-phosphotransferase (S-PT) plays a crucial role in phosphorus
uptake and utilization by catalyzing the transfer of phosphate groups
from donors to acceptors (Wohlgemuth et al., 2017). Solid-Catalase
(S-CAT) prevents the accumulation of toxic substances by promoting the
degradation of hydrogen peroxide (Cusack et al., 2011). Additionally,
root-secreted polysaccharides can enhance the growth of
polysaccharide-utilizing microbial communities and stimulate the
production of extracellular enzymes, thereby facilitating the
decomposition of soil organic matter and nutrient cycling (Morcillo and
Manzanera, 2021). Furthermore, these polysaccharides also serve as a
significant source of organic matter in the soil (Pansu and Gautheyrou,
2006). Abiotic stresses such as drought (Staszel et al., 2022) and
nitrogen (Jia et al., 2020) can change soil enzyme activities, thereby
affecting soil-plant interactions. Nevertheless, root secretion plays an
important role in plant response to environmental stress, especially
soil enzymes secreted by roots and soil enzymes secreted by
microorganisms driven by root exudates. However, reports on soil enzymes
and polysaccharides in root exudates under sterile conditions less.

Global climate change is one of the important factors limiting crop
yields (Yuan et al., 2009), mainly causing the loss of nutrients such as
nitrogen and phosphorus in the soil, resulting in reduced crop yields
(Bojko and Kabala, 2016, Widdig et al., 2020, Twining et al., 2022).
High-temperature, drought, nitrogen deficiency, and phosphorus
deficiency are common factors that limit crop yield. In nature, plants
are often under combined stress of multiple environmental factors, but
how they affect plant growth and development is poorly understood
(Zandalinas et al., 2021). There is also a lack of direct evidence that
roots secrete soil enzymes and polysaccharides into the soil when plants
respond to these adverse environments. Based on this, under sterile
conditions, 15 stress models were designed with high-temperature (T),
drought (D), nitrogen deficiency (N), phosphorus deficiency (P) and
their combinations. After the stress culture of \emph{S.
miltiorrhiza}test-tube seedlings, the secretion of S-UE, S-NIR, S-NR,
S-PT, and S-CAT activities and total polysaccharide content changes were
measured. To understand the effects of \emph{S. miltiorrhiza} on soil
nitrogen, phosphorus cycling and soil polysaccharides in response to
abiotic stress, explore the ecological strategy and role of plant root
secretion response under abiotic stress.

\textbf{2 \textbar{} MATERIALS AND METHODS}

\textbf{2.1 \textbar{} Study site and test materials}

Tissue culture seedlings were selected from Chengdu University of
Traditional Chinese Medicine by excising young leaves from the stem apex
of the same plant and cultivating them into tissue culture seedlings
after surface sterilization, tissue healing, bud clustering, and root
induction in the laboratory (E: 103.81\selectlanguage{ngerman}°, N: 30.68°).

The culture medium utilized was shell shale sourced from Ximeishan
Village (E: 104.54°, N: 30.94°), Shiquan Township, Zhongjiang County,
Sichuan Province. The medium was air-dried, passed through a 1-5 mm
sieve, cleansed of impurities, sterilized at 180\selectlanguage{english} for 3 h in a dry
heating process, and cycled twice to eliminate microorganisms and their
exudates. After cooling, it was packed in kraft paper and then
sterilized again through dry heat at 120\selectlanguage{english} for 3-4 h before use. The pH
value of the shell shale was 7.78, with an organic matter content of
0.69 g/kg, alkaline nitrogen of 4.2 mg/kg, potassium of 3.79 mg/kg, and
phosphorus of 2.23 mg/kg.

The culture device used was a circular polypropylene box with a top
diameter of 17.2 cm, a bottom diameter of 11.8 cm, and a height of 9.7
cm. Each box was wrapped in kraft paper and sterilized at high-pressure
steam of 0.1MPa and 121\selectlanguage{english} for 30 min, and after cooling, filled with 600
g of culture medium.

\textbf{2.2 \textbar{} Experimental design}

Four stress factors, including high-temperature (T), drought (D),
nitrogen deficiency (N), and phosphorus deficiency (P), at 2 levels per
factor, for 16 treatments, of which CK was the control group the
treatment conditions for each group are shown in Table 1. As a full
nutrient solution, Hoagland's solution was used (table S1). In the
reference method (Torres-Rodriguez et al., 2021, Zhao et al., 2005), the
phosphorus content is set to 0 mM for the phosphorus stress treatment,
and the nitrogen content is set to 0 mM for the nitrogen deficiency
stress treatment. The corresponding concentrations of
KH\textsubscript{2}PO\textsubscript{4},
Ca(NO\textsubscript{3})\textsubscript{2}, KNO\textsubscript{3}, and
NH\textsubscript{4}NO\textsubscript{3} were adjusted to 0\%,
respectively. The lacking Ca and K in the deficit solutions were
CaCl\textsubscript{2} and KCl, respectively, while the remaining
components of Hoagland's solution remained unchanged. Based on ground
temperature data from the \emph{S. miltiorrhiza} cultivation base in
Zhongjiang, Sichuan, the temperature was chosen with 25\selectlanguage{english}/20\selectlanguage{english} (day/night)
for the normal conditions and 42\selectlanguage{english}/35\selectlanguage{english} (day/night) for the
high-temperature stress condition. The water holding capacity (WHC) was
75\% for normal moisture conditions and 30\% for drought stress
conditions (Liu et al., 2011).

\textbf{TABLE 1 Design of each condition for Salvia seedlings}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\textbf{Group} & \textbf{Abbreviations} & \textbf{Watering nutrients} &
\textbf{WHC} & \textbf{Incubation temperature (day/night
)}\tabularnewline
\midrule
\endhead
Control & CK & Full strength nutrient solution & 75\% &
25/20\tabularnewline
Drought & D & Full strength nutrient solution & 30\% &
25/20\tabularnewline
High-temperature & T & Full strength nutrient solution & 75\% &
42/35\tabularnewline
Nitrogen deficiency & N & Nitrogen-deficient nutrient solution & 75\% &
25/20\tabularnewline
Phosphorus deficiency & P & Phosphorus-deficient nutrient solution &
75\% & 25/20\tabularnewline
\bottomrule
\end{longtable}

Note: The experiments were carried out strictly by the above conditions.

\textbf{2.3 \textbar{} Seedling cultivation}

Under aseptic conditions, test tube seedlings with 3-5 uniform long
roots weighing approximately 0.5 g were carefully extracted from the
culture flasks, and any adhering agar was eliminated using sterile
filter paper. The seedlings were weighed and transferred to culture
boxes containing 600 g of shale pellets, 2 plants per box. A total of 96
boxes were planted. Each box was filled with 100 mL of sterile water and
5 mL of sterilized Hoagland's total nutrient solution at the seedlings'
roots. Each box's total weight was recorded as a reference value for
irrigation. Place on the culture rack in the tissue culture room for
cultivation at 25/20 (day/night) and a light intensity of 3000 lx.
Weigh and replace the water every 3 days to maintain 75\% WHC of the
culture medium, and water the roots of the seedlings with 5 mL of
Hoagland's full nutrient solution each week. After 4 weeks of
incubation, the plants were randomly divided into 16 groups of 6 boxes
each and watered with the nutrient solution required for the different
stress treatment groups. After 3 weeks of culture, observe the
morphological characteristics of the plant's nitrogen and phosphorus
deficiency stress and then perform other synergistic stress treatments.
The natural precipitation method was used for drought treatment in the
drought synergistic stress treatment group. After the WHC dropped to
30\%, the 30\% WHC was maintained until the end of the treatment. The
plants were transferred to an incubator at 42/35 (day/night) under the
same light conditions for the temperature stress treatment. All
experiments were completed after 5 days of heat stress.

\textbf{2.4 \textbar{} Sample collection and determination}

At the end of the culture experiments, the roots were washed with
deionized water and blotted dry with absorbent paper, and the weights of
the plants' above- and below-ground parts were measured separately. Two
boxes of culture substrates in each group were randomly combined into
one sample, mixed and sampled by quartering. Pass through a 40-mesh
sieve after grinding to determine soil enzymes and polysaccharides.
Enzymatic activities of S-UE, S-NiR, S-NR, and S-CAT were evaluated
using cominbio kits (Suzhou Keming Biotechnology Co., Ltd.) following
the manufacturer's guidelines, while mlbio kits (Shanghai Enzyme
Biotechnology Co., Ltd.) were employed to measure the activity of S-PT
enzymes. The determination of pentoses adopts the lichenol-hydrochloric
acid method (Haoli et al., 2014), and the determination of hexoses
adopts the anthrone-sulfuric acid method (wei-jie, 1999), and the sum of
the two is calculated as the total polysaccharide content.

S-NiR activity was assessed using the cominbio kit's method. The amount
of reducing 1 \selectlanguage{greek}μ\selectlanguage{english}mol NO\textsubscript{2}- \selectlanguage{english}per g soil sample per day was
regarded as an enzyme activity unit. In a 2 mL centrifuge tube, 0.02 g
of air-dried soil sample was mixed with 40 \selectlanguage{greek}µ\selectlanguage{english}L each of sodium nitrite and
glucose solutions. The mixture was thoroughly combined and reacted for
one hour at 25. Subsequently, 40 \selectlanguage{greek}µ\selectlanguage{english}L of aluminum potassium alum solution
was added, and the content was fully agitated for thirty seconds before
centrifugation was facilitated at 10,000 rpm, at 4, for ten minutes. At
this stage, 70\selectlanguage{greek}µ\selectlanguage{english}L of the supernatant was extracted, along with 1:1
p-amino benzene sulfonic acid-phosphoric acid solution and 140 \selectlanguage{greek}µ\selectlanguage{english}L of
N-1-naphthalene ethylenediamine hydrochloride solution. The components
were mixed well and evaluated for absorbance at 540 nm. Control tubes
were supplied with distilled water instead of sodium nitrite solution,
while blank tubes had no samples. S-NR activity was assessed using the
cominbio kit's method, and the amount of 1 \selectlanguage{greek}μ\selectlanguage{english}mol NO\textsubscript{2}-
\selectlanguage{english}produced per gram of soil sample per day was regarded as one S-NR
activity unit. Air-dried soil samples weighing 0.06 g were included in a
2 mL centrifuge tube containing 225 \selectlanguage{greek}µ\selectlanguage{english}L of KNO\textsubscript{3} solution
and 75 \selectlanguage{greek}µ\selectlanguage{english}L of Reduced Coenzyme I (NADH) solution. Samples were then
subjected to a 24-hour water bath at 37 before centrifugation at 8000g
and 25 for 10 min. At this stage, 130 \selectlanguage{greek}µ\selectlanguage{english}L of the supernatant was
collected and added to 85 \selectlanguage{greek}µ\selectlanguage{english}L each of p-amino benzene sulfonic acid
solution and \selectlanguage{greek}α-\selectlanguage{english}naphthylamine solution. The resulting mixture was
agitated and left for color development at 25 for 20 min before
centrifugation at 4000g for 10 min at 25. 200 \selectlanguage{greek}µ\selectlanguage{english}L of the supernatant was
transferred to a 96-well plate and monitored for absorbance at 540 nm.
Distilled water replaced the KNO\textsubscript{3} solution for the
control tube, while the blank tubes omitted soil samples. The standard
tube replaced the soil sample with 0.1 \selectlanguage{greek}μ\selectlanguage{english}mol/mL
NaNO\textsubscript{2}solution. S-UE activity was assessed using the
cominbio kit's method, and 1 \selectlanguage{greek}μ\selectlanguage{english}g of NH\textsubscript{3}-N produced per
gram of soil sample per day was defined as an enzyme activity unit.
Air-dried soil samples weighing 0.06 g were placed in 2 mL centrifuge
tubes after adding 20 \selectlanguage{greek}µ\selectlanguage{english}L of toluene and shaking, then placed at room
temperature for 15 min. Afterward, 90 \selectlanguage{greek}µ\selectlanguage{english}L of urea and 190 \selectlanguage{greek}µ\selectlanguage{english}L of citric
acid-potassium hydroxide solution were blended before a 24 h water bath
at 37. Following this, centrifugation was carried out at 10,000g and
25 for 10 min. The supernatant was then diluted ten-fold, with 80 \selectlanguage{greek}µ\selectlanguage{english}L
taken, and the mixture was coupled with 15 \selectlanguage{greek}µ\selectlanguage{english}L of phenol, methanol,
acetone, absolute ethanol, and NaOH solutions. Next, 15 \selectlanguage{greek}µ\selectlanguage{english}L of sodium
hypochlorite solution was added and mixed well before being placed at
room temperature for 20 min. The absorbance of the resultant mixture was
monitored at 578 nm after the inclusion of 90 \selectlanguage{greek}µ\selectlanguage{english}L of distilled water.
Distilled water substituted urea in the control tube. S-CAT activity was
assessed using the cominbio kit's method, and the degradation of 1 \selectlanguage{greek}μ\selectlanguage{english}mol
H\textsubscript{2}O\textsubscript{2} catalyzed per g of air-dried soil
sample per day was defined as an enzyme activity unit. Air-dried soil
samples weighing 0.03 g were included in a 2 mL centrifuge tube and
supplemented with 260 \selectlanguage{greek}µ\selectlanguage{english}L of hydrogen peroxide. Samples were subjected to
a 20 min incubation at 25 while agitated at 500r/min. Next, 10 \selectlanguage{greek}µ\selectlanguage{english}L of
aluminum potassium alum was added, followed by centrifugation at 8000g
and 25 for 5 min. 180 \selectlanguage{greek}µ\selectlanguage{english}L of the supernatant was collected and mixed
with 20 \selectlanguage{greek}µ\selectlanguage{english}L of sulfuric acid solution before monitoring absorptivity at
240 nm. The control tube contained distilled water instead of hydrogen
peroxide, while the blank tubes contained no soil samples. S-PT activity
was assessed using the mlbio kit's method. To prepare the soil samples
for testing, 0.1 g of soil was mixed with 0.9 mL of Phosphate buffer
saline (PBS) before centrifugation at 4000r for 15 min. The resultant
supernatant became the sample for examination. Next, 50 \selectlanguage{greek}µ\selectlanguage{english}L of the
diluted standard and 40 \selectlanguage{greek}µ\selectlanguage{english}L of the sample diluent were added to the
sample, which was then sealed with a sealing film and incubated at 37
for 30 min. Afterward, the liquid was discarded, followed by the
introduction of wash solution, left for 30 s, and discarded five times.
Subsequently, 50 \selectlanguage{greek}µ\selectlanguage{english}L of HRP enzyme-labeled reagent was included, and
incubation and washing processes resumed before adding 50 \selectlanguage{greek}µ\selectlanguage{english}L of
carbamide peroxide color reagent and 50 \selectlanguage{greek}µ\selectlanguage{english}L of color reagent. At this
stage, color development was initiated, with 10 min allotted for
completion in the dark at 37. Additionally, 50 \selectlanguage{greek}µ\selectlanguage{english}L of termination
solution was infused before absorbance levels were measured at 450 nm.
Blank wells that omitted samples and enzyme labeling reagents were also
featured. Phosphotransferase markers (Figure. S1). The content
determination method of the total polysaccharide in the soil adopts the
method of pansu et al. (Pansu and Gautheyrou, 2006). Preparation of the
test solution: weigh 2.5g of air-dried soil, put it in a 50mL Conical
flask, then add 20 mL of 2.5M H\textsubscript{2}SO\textsubscript{4},
heat and reflux in a water bath at 100 for 20 min, let cool, filter,
wash, and collect the filtrate. Put the residue in a 50mL Conical flask,
add 1mL 13M H\textsubscript{2}SO\textsubscript{4}, and place it at room
temperature for 16 h. Slowly add 25 mL of distilled water, let cool,
heat at 100 for 5 h after airtight, let cool, filter, combine the
filtrate with the above filtrate and dilute to 50mL to obtain the test
solution. The total polysaccharide content was determined using the
lichenol-hydrochloric acid method for pentose, while hexose was assessed
using the anthrone-sulfuric acid method. Pentose and hexose markers,
respectively (figure s3, figure s2). Finally, the sum of the two was
calculated as the polysaccharide content.

\textbf{2.5 \textbar{} Data statistics}

The data were analyzed using IBM SPSS 21.0 software for one-way analysis
of variance, and the least significant difference method and Duncan's
were used for multiple comparisons. Data presented here as a fully
randomized design with three replicates expressed as mean \selectlanguage{ngerman}± standard
deviation. Different lowercase letters in the table indicate a
significant difference from the blank. They were graphing with GraphPad
Prism 9 and Spearman correlation analysis with R (version 4.1.2).

\textbf{3. \textbar{} RESULTS}

\textbf{3.1 \textbar{} Changes in growing \emph{S.
miltiorrhiza}seedlings}

Abiotic stress is not conducive to the growth of \emph{S.
miltiorrhiza}seedlings, among which P, synergistic stresses of drought
and nitrogen deficiency (DN), synergistic stresses of drought, nitrogen
deficiency and phosphorus deficiency (DNP) and synergistic stresses of
drought, high-temperature, nitrogen deficiency and phosphorus deficiency
(DTNP) stresses reached a significant level (\emph{p} \textless{}0.05).
P, DN, synergistic stresses of drought, high-temperature and nitrogen
deficiency (DTN), synergistic stresses of drought high-temperature and
phosphorus deficiency (DTP), DNP, synergistic stresses of
high-temperature, nitrogen deficiency and phosphorus deficiency (TNP),
and DTNP stresses significantly inhibited the fresh weight of
aboveground parts of \emph{S. miltiorrhiza} (\emph{p} \textless{}0.05)
(Figure 1). Drought or synergistic stresses of drought and
high-temperature exacerbated the plant growth inhibition of nitrogen
deficiency, but not significantly. (e.g., DN and DTN stresses) (Figure
1-a). Drought or high-temperature or synergistic stresses of drought and
high-temperatures alleviated the plant growth inhibition of phosphorus
deficiency, but not significantly. (e.g., DP, TP, and DTP stresses)
(Figure 1-b). Synergistic stresses of drought and high-temperatures
aggravated the plant growth inhibition of nitrogen and phosphorus
deficiency (e.g., DTNP stresses) (\emph{p} \textless{}0.05) (Figure
1-c). The synergistic stresses of drought and high-temperatures
exacerbated the effects of single-factor inhibition, but not to a
significant extent (e.g., DT stress) (Figure 1-d).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{FIGURE 1} The growth changes of \emph{S. miltiorrhiza} seedlings
under abiotic stress. (a), (b), and (c) respectively represent the
changes in the growth effects of the superposition of various
environmental factors under the stress of nitrogen, phosphorus and
nitrogen and phosphorus deficiency, and (d) represents the changes in
the growth effects of the superposition of drought and high temperature.
* indicating a significant difference to the CK treatment,
\emph{p}\textless{} 0.05.

\textbf{3.2 \textbar{} The activity of enzymes related to nitrogen
metabolism in the medium}

Abiotic stress can significantly down-regulated S-NR activity in the
culture medium, with single factors N and T stresses having the
strongest inhibitory effect, while the combined effect was most severe
in synergistic stresses of high-temperature and nitrogen deficiency
(TN), synergistic stresses of nitrogen and phosphorus deficiency (NP),
and DTN stresses (\emph{p} \textless{}0.05). DN and TN stress
up-regulated S-NiR activity (\emph{p} \textless{}0.05), but the other
factors did not show statistical significance. Moreover, N, T, D,
synergistic stresses of high-temperature and drought (DT), DN,
synergistic stresses of drought and phosphorus deficiency (DP), and TNP
stresses all elevated S-UE activity in the culture medium, while
synergistic stresses of high-temperature and phosphorus deficiency (TP),
TN, NP, and DTN stresses significantly down-regulated S-UE activity in
the culture medium (\emph{p} \textless{}0.05) (Figure 2). These findings
indicate that under abiotic stress, plants reduce the secretion of S-NR
enzyme from their root system, which curtails denitrification and
prevents nitrate-nitrogen loss. In response to N, T, D, DT, DN, DP, and
TNP stresses, plants secrete S-UE to enhance organic nitrogen
utilization.

Drought or high-temperature stress can significantly up-regulate the
effect of nitrogen deficiency on the release of S-NiR from roots (e.g.,
DN and TN stresses) (\emph{p} \textless{}0.05). Drought stress can
significantly alleviate the effect of nitrogen deficiency on the release
of S-NR from roots and simultaneously significantly promote the release
of S-UE from roots (e.g., DN stress) (\emph{p} \textless{}0.05).
High-temperature or synergistic stresses of drought and high-temperature
inhibit the effect of nitrogen deficit on the induction of S-UE release
from roots (e.g., TN and DTN stresses) (\emph{p} \textless{}0.05)
(Figure 2-a). Moreover, drought stress can significantly enhance the
effect of phosphorus deficiency on the release of S-UE from roots, while
the high-temperature can alleviate the effect of phosphorus deficiency
on the release of S-NR from roots and inhibit the release of S-UE from
roots (e.g., DP and TP stresses) (\emph{p} \textless{}0.05) (Figure
2-b). Furthermore, drought or high-temperature or the synergistic
stresses of both can alleviate the effect of nitrogen and phosphorus
deficiency on the inhibition of root secretion of S-UE and the
synergistic stresses of high-temperature and drought can also
significantly promote the release of S-NR from roots (e.g., DNP, TNP,
and DTNP stresses) (\emph{p} \textless{}0.05) (Figure 2-c). Finally, the
synergistic stresses of drought and high-temperature can significantly
reduce the effect of drought on the release of S-UE from roots (e.g., DT
stress) (\emph{p} \textless{}0.05) (Figure 2-d).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{FIGURE 2} Roots in the culture medium secrete the activity of
enzymes related to nitrogen metabolism. (a), (b), and (c) respectively
represent the changes in nitrogen metabolism-related enzymes under the
superimposed environmental factors of nitrogen, phosphorus and
nitrogen-phosphorus deficit stresses. (d) indicates the changes in
nitrogen metabolism-related enzymes under the superimposed drought and
high temperature. * indicating a significant difference to the N, P, NP,
D, and CK treatment, \emph{p} \textless{} 0.05.

\textbf{3.3 \textbar{} The phosphotransferase activity in the medium}

D and T stresses can up-regulate the activity of S-PT in the culture
medium, whereas all stresses except N stress can reduce the activity of
S-PT in the culture medium (\emph{p} \textless{}0.05) (Figure 3).
Drought or high-temperature or the synergistic stresses of drought and
high-temperature can significantly reduce the effect of nitrogen
deficiency on up-regulating S-PT activity (e.g., DN, TN, and DTN
stresses) (\emph{p} \textless{}0.05) (Figure 3-a). Moreover, drought can
alleviate the effect of phosphorus deficiency on down-regulating S-PT
activity, while the synergistic stresses of drought and high-temperature
can aggravate the effect of phosphorus deficiency on down-regulating
S-PT activity (e.g., DP and DTP stresses) (\emph{p} \textless{}0.05)
(Figure 3-b). Furthermore, drought or high-temperature or synergistic
stresses of drought and high-temperature can alleviate the effect of
nitrogen and phosphorus deficiency on the inhibition of S-PT activity
(e.g., DNP, TNP, and DTNP stresses) (\emph{p} \textless{}0.05) (Figure
3-c). Finally, synergistic stresses of drought and high-temperature
inhibit the induction effect of a single factor on S-PT activity (e.g.,
DT stress) (\emph{p} \textless{}0.05) (Figure 3-d). It shows that under
D and T, plants secrete S-PT through the root system to improve the
transport and utilization of phosphorus, but most stresses are not
conducive to the transport and utilization of phosphorus.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{FIGURE 3} The root system secretes phosphotransferase activity
in the culture medium. (a), (b), and (c) respectively represent the
changes in phosphotransferases under the superimposed environmental
factors of nitrogen, phosphorus and nitrogen-phosphorus deficit stress.
(d) indicates the changes in phosphotransferase under the superposition
of drought and high temperature. * indicating a significant difference
to the N, P, NP, D, and CK treatment, \emph{p} \textless{} 0.05.

\textbf{3.4 \textbar{} The Catalase activity in the medium}

N, DN, and TN stress significantly down-regulated the activity of S-CAT
in the culture medium (\emph{p} \textless{}0.05), while the other groups
do not show statistical significance. In addition, synergistic stresses
of drought and high-temperature can significantly improve the effect of
nitrogen deficiency in down-regulating S-CAT activity (e.g., DTN stress)
(\emph{p} \textless{}0.05) (Figure 4-a), but the other groups are not
statistically different. It shows that N, DN, and TN stresses are not
conducive to removing root peroxide.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\par\null

\textbf{FIGURE 4} The root system secretes catalase activity in the
culture medium. (a), (b), and (c) respectively represent the changes in
catalase under the superimposed environmental factors of nitrogen,
phosphorus and nitrogen-phosphorus deficit stress. (d) indicates the
changes in catalase under the superimposed drought and high temperature.
* indicating a significant difference to the N, P, NP, D, and CK
treatment, \emph{p} \textless{} 0.05.

\textbf{3.5 \textbar{} The polysaccharide content in the medium}

P, DT, and DTP stresses can significantly reduce the polysaccharides
content in the culture medium (\emph{p} \textless{}0.05), and all groups
except DP, TP, and DN stresses tend to reduce the content of
polysaccharides in the culture medium (Figure 5). Additionally, drought
or high-temperature can significantly alleviate the effects of the
down-regulation of root polysaccharides secreted by phosphorus
deficiency (e.g., DP and TP stresses) (\emph{p} \textless{}0.05) (Figure
5-b). However, the synergistic stresses of drought and high-temperatures
can exacerbate the suppressive effects of drought (e.g., DT stress)
(\emph{p} \textless{}0.05) (Figure 5-d). These results suggest that
under abiotic stress, the root system of plants reduces the secretion of
polysaccharides, thereby reducing the consumption of carbon and energy,
but the effects of DP, TP, and DN stresses are not obvious.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\par\null

\textbf{FIGURE 5} Content of total polysaccharides secreted by roots in
culture media. (a), (b), and (c) respectively represent the changes in
polysaccharide content under the superimposed environmental factors of
nitrogen, phosphorus and nitrogen-phosphorus deficit stress. (d)
indicates the changes in polysaccharide content under superimposed
drought and high temperature. * indicating a significant difference to
the N, P, NP, D, and CK treatment, \emph{p} \textless{} 0.05.

\textbf{3.6 \textbar{} Correlation of soil enzyme activity and
polysaccharide content with the seedling weight}

The polysaccharide content in the culture medium showed a significant
positive correlation with the fresh weight of \emph{S.
miltiorrhiza}seedlings and the fresh weight of their aboveground or
underground parts (\emph{p} \textless{}0.05). Additionally, S-NR and
S-PT were found to have an extremely significant negative correlation
with the root-shoot ratio (\emph{p} \textless{}0.001), while S-NiR had a
significant positive correlation with the same ratio (\emph{p}
\textless{}0.05). Furthermore, S-UE had a significant positive
correlation with S-NR and S-PT, whereas S-NiR had a significant negative
correlation with S-NR (\emph{p} \textless{}0.05) (Figure 6). These
findings suggest that the release of polysaccharides from the root
system into the environment is closely related to the growth and
developmental status of the plant.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\par\null

\textbf{FIGURE 6} Correlation of soil enzyme activity and polysaccharide
content with the seedling weight. The top right corner of the graph
indicates correlation coefficients and the bottom left corner indicates
significant differences in correlation. * indicates a significant
correlation (\emph{p} \textless{}0.05), ** indicates a strong
significant correlation (\emph{p} \textless{}0.01).

\textbf{4 \textbar{} DISCUSSION}

\textbf{4.1 \textbar{} Root secretion and soil nitrate cycling and
utilization}

Plants require nitrogen to form new cells (Ke et al., 2020), and
nitrogen deficiency is a limiting factor in crop productivity (Zhao et
al., 2005). Soil nitrogen exists in organic and inorganic forms, with
nitrate and ammonium being the plant-available types (Xu et al., 2012).
Root secretion of soil nitrogen cycle enzymes influences nitrogen use
(Dong et al., 2021). This experiment revealed that S-NR activity
secreted by \emph{S. miltiorrhiza} roots was significantly
down-regulated under 15 different abiotic stress conditions. This
suggests that under stress, \emph{S. miltiorrhiza} can maintain the
supply of nitrate-nitrogen in the root environment by down-regulating
the activity of root-secreted S-NR and reducing its nitrate-nitrogen
reduction ability (Coskun et al., 2017). This is consistent with
nitrogen stress down-regulating NR secretion from maize roots (Qiang et
al., 2021) and phosphorus stress down-regulating NR activity from cowpea
roots (Qi bing-lin et al., 2010). The suggestion is that reducing
nitrogen loss is a strategy for plants to cope with abiotic stress.
However, the results of this study differ from the drought stress
induced upregulation of NR activity in wheat roots (Hosseini et al.,
2022) and the improvement of S-NR enzyme activity in soil by nutrient
stress treatment (Meng et al., 2021). The involvement of microorganisms
in root exudation activities, and the increased activity of denitrifying
bacteria, may contribute to the differences observed in nitrate
reductase activity in the soil.

This experiment shows that \emph{S. miltiorrhiza} often up-regulates the
activity of root-secreted S-NiR under most stress conditions, which is
consistent with the fact that drought stress significantly increases the
activity of S-NiR in the rhizosphere of maize (Lin et al., 2021) but
different from the fact that low nitrogen reduces the activity of NiR in
the rhizosphere of tobacco (Xihuan et al., 2020). Notably, \emph{S.
miltiorrhiza} significantly increased the activity of S-NiR in both DN
and TN stresses, indicating improved denitrification, which may be
related to the high-temperature causing soil oxygen overflow (Guan
hui-lin et al., 2010). It shows that after stress, plants can reduce the
damage of nitrite salt in the rhizosphere environment by up-regulating
the activity of S-NiR secreted from the root system.

S-UE plays a critical role in converting organic nitrogen in soil into
ammonium nitrogen available for plants. This study shows that \emph{S.
miltiorrhiza} can significantly up-regulate the activity of S-UE
secreted by roots under N, D, T, DT, DN, DP, and TNP stresses,
consistent with the results that increasing temperature increases the
activity of S-UE in soil (Bai et al., 2017). Moreover, nitrogen
deficiency and its synergistic stress with drought can significantly
up-regulate the activity of root-secreting S-UE, but the synergistic
stress of high-temperature or synergistic stresses of drought and
high-temperature can significantly down-regulate the activity of
root-secreting S-UE (Figure 2-a) (e.g., DN, TN, and DTN stresses). In
addition, nitrogen deficiency, phosphorus deficiency and their
synergistic stress with drought or high-temperature or drought and
high-temperature can significantly up-regulate the activity of
root-secreting S-UE (Figure 2-c) (e.g., DNP, TNP, and DTNP stresses).
These findings suggest that plants demonstrate unique responses to
different synergistic stress. Under abiotic environmental stress, plants
can reduce the reduction of nitrate nitrogen and accelerate the
transformation of organic nitrogen by altering the activities of enzymes
involved in root secretion and soil nitrogen cycling to maintain
nitrogen available to plants and improve the reduction of nitrite to
reduce its harmfulness, and then improve nitrogen conversion and use
efficiency.

\textbf{4.2 \textbar{} Root secretion and soil phosphorus utilization}

Phosphorus is one of the essential elements for plants (Arwenyo et al.,
2022), and the distribution and transport of soil phosphorus affect
plant growth and productivity (Xia et al., 2020, Yadav et al., 2020).
S-PT is an enzyme that catalyzes the transfer of phosphate groups from
donors to acceptors and is closely related to phosphorus uptake and
transport. This study found that under D, T, and N stresses, \emph{S.
miltiorrhiza} up-regulates the activity of root-secreted S-PT, while
other stresses down-regulate this activity significantly. In the context
of nitrogen stress, synergistic stress of drought or synergistic stress
of high-temperature, or synergistic stresses of drought and
high-temperature, can significantly down-regulate the activity of
root-secreted S-PT (Figure 3-a) (e.g., DN, TN, and DTN stresses).
However, under nitrogen and phosphorus stress, synergistic stress of
drought or synergistic stress of high-temperature or synergistic
stresses of drought and high-temperature synergistic stress can
significantly up-regulate the activity of root-secreted S-PT (Figure
3-c) (e.g., DNP, TNP, and DTNP stresses). This also supports the claim
that plants respond uniquely to different synergistic stress. It is also
suggested that in the absence of microbial participation, most stress
environments are not conducive to phosphorus uptake and transport. Most
stressful environments that promote phosphorus uptake and transport in
plant roots may be directly related to rhizosphere microorganisms
(Glaesner et al., 2016).

\textbf{4.3 \textbar{} Abiotic stress and inter-root antioxidant}

The accumulation of active oxygen free radicals in the rhizosphere of
plants causes oxidative damage to the cell membrane system of the root
tip and leads to a series of physiological and biochemical changes in
the root's absorptive function (Mittler et al., 2022). The results of
this study showed that \emph{S. miltiorrhiza} significantly
down-regulated the activity of S-CAT secreted by the root under N, DN,
and TN stresses. Under the synergistic stresses of three and four
factors, \emph{S. miltiorrhiza} has tended to up-regulate the activity
of root-secreted S-CAT. This is similar to the effect of nitrogen
inhibiting the secretion of enzymes in maize roots (Liang et al., 2016)
but different from the effect of drought stress stimulating the
secretion of antioxidant substances in maize roots (Song et al., 2012).
Overall, N, DN, and TN stresses can cause active oxygen hazards in the
rhizosphere, affecting the uptake and transport of rhizosphere
nutrients.

\textbf{4.4 \textbar{} Abiotic stress and soil polysaccharide
composition}

The saccharides secreted by plant roots are an important source of soil
organic matter, microbial carbon, and energy, mainly hexoses and a small
number of pentoses. This study shows that pentose content tends to
increase under most stress conditions, but it is not synchronized with
the change in hexose. Under N stress, the secretion of polysaccharides
from \emph{S. miltiorrhiza} roots was down-regulated, confirming that
nitrogen stress significantly reduced the secretion of saccharides from
maize roots (Carvalhais et al., 2010, Zhu et al., 2016). P, DTP, and DT
stress significantly down-regulated polysaccharide secretion in \emph{S.
miltiorrhiza} roots, unlike phosphorus deficiency, which promoted sugar
secretion in maize roots (Carvalhais et al., 2010), and drought stress,
which promoted polysaccharide secretion in plant roots (Ulrich et al.,
2022). The release of root exudates positively correlates with microbial
growth (Bengtson et al., 2012), suggesting that this result and the
literature results may be related to the lack of microbial involvement
in rhizosphere activities. Further analysis shows that the content of
polysaccharides secreted by roots is positively correlated with plant
yield. Abiotic stress is not conducive to the accumulation of plant
biomass and may also affect the distribution of plant polysaccharides,
resulting in most energy substances, such as carbon source synthetic
sugars, being supplied to plants, reducing the roots secrete
polysaccharides that are not conducive to carbon sequestration in the
soil. Thus, unfavorable environment affects plant growth and also
affects the carbon stored in the soil by the plants.

\textbf{5 \textbar{} CONCLUSIONS}

When \emph{S. miltiorrhiza} is subjected to different abiotic stress
conditions, changes occur in the soil enzyme activity that is secreted
by its root system, altering the soil nitrogen cycle, phosphorus
transport and utilization, and the antioxidant capacity of the
rhizosphere. Plants obtain nutrients and reduce oxidative damage to
roots by altering the levels of soil enzymes secreted by roots.
Unfavorable environments can directly affect plant growth and
development, affecting the amount of organic matter that plants put into
the soil, thereby affecting soil carbon storage.

\textbf{AUTHOR CONTRIBUTIONS}

\textbf{Yong Qin} : Conducting experiments, Formal analysis, Writing -
original draft. \textbf{Xiaoyu Li} : Data collection. \textbf{Yanhong
Wu} : Conducting experiments, Paper check. \textbf{Hai Wang} : Content
is critically revised. Paper check. \textbf{Guiqi Han} : Content is
critically revised. Paper check. \textbf{Zhuyun Yan} : Experimental
design, Methodology, Writing - review \& editing, Supervision.

\textbf{ACKNOWLEDGMENTS}

This research was funded by the National Natural Science Foundation of
China (grant number 81973416), and this research was funded by the
Science and Technology Department of Sichuan Province(2021YFS0045).
Special acknowledgments are given to the editors and the reviewers.

\textbf{CONFLICT OF INTEREST STATEMENT}

The authors declare no conflict of interest.

\textbf{DATA AVAILABILITY STATEMENT}

All data is available in the main text and the supplementary materials.

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