Abstract

   A comprehensively analysis of the transcriptomics and metabolomics was
   conducted to investigate the mechanism of plant growth regulators on
   the quality of jujube fruit. After the application of plant growth
   regulators, a total of 3097 differentially expressed genes (DEGs) were
   identified, which were mainly annotated in 123 pathways such as
   flavonoid biosynthesis, metabolism of alanine, aspartate, and
   glutamate. In addition, 1091 differential expressed metabolites (DEMs),
   including 519 up-regulated and 572 down-regulated metabolites, were
   significantly altered after application of plant growth regulators.
   DEGs and DEMs simultaneously annotated 69 metabolic pathways, including
   biosynthesis of phenylpropane, flavonoid, starch and sucrose. The key
   genes in flavonoid biosynthesis pathway were revealed, which may play
   an important role in plant growth regulator regulation quality of
   jujube fruit. Besides, the application of plant growth regulator during
   the jujube flowering period increased the contents of gibberellin and
   indole-3-acetic acid in leaves, and decreased the contents of abscisic
   acid. The results may help to reveal the metabolic network and
   molecular mechanism of plant growth regulators in jujube fruit.

1. Introdcution

   Jujube, also known as red date (Ziziphus jujuba Mill.) is a small
   deciduous tree. Jujubes originated in Asia and have been cultivated in
   China for more than 4000 years [[45]1], leading to the selection of
   over 700 cultivars. China is the only exporter of jujube and accounts
   for over 90% of the world’s total production [[46]2]. In China, jujube
   cultivation is mainly distributed in five provinces in the middle and
   lower reaches of the Yellow River and the Aksu, Hotan, Kashgar and Hami
   of Xinjiang. According to the Chinese National Bureau of Statistics
   [[47]3] and Statistical Yearbook of Xinjiang [[48]4], the planting area
   of jujube trees in China in 2020 was approximately 3.3 million
   hectares, with a fruit yield of 8.8 million tons. In Xinjiang, the
   planting area was 414,000 hectares, with a yield of 3.8 million tons,
   accounting for 43.4% of the national production. The jujube industry is
   a characteristics and important pillar industry for agricultural
   development in southern Xinjiang, providing strong support for local
   growers to increase their income and playing a crucial role in
   promoting economic development [[49]5,[50]6].

   Jujube are rich in nutrients, including polysaccharides [[51]7],
   flavonoids [[52]8], polyphenols [[53]9–[54]11], cyclic adenosine
   monophosphate (cAMP) [[55]12], organic acids, and saponins [[56]13].
   They are also considered to assist in oxygen free radical scavenging,
   cardiovascular regulation, anti-aging, cancer prevention and to enhance
   immune function. Jujube trees display indefinite inflorescence with a
   long flowering period, during the growth and development of jujube
   fruit in most cultivars, this is accompanied by a low natural fruit
   setting rate due to abscisic acid [[57]1,[58]14,[59]15]. The main
   jujube cultivar in southern Xinjiang, jujube has a natural fruit
   setting rate of only about 1% of the total number of flowers [[60]14].
   Gibberellic acid (GA[3]) is often sprayed during the flowering period
   of jujube trees, which can improve fruit setting by up to 33% [[61]16].
   The effect of increasing yield is most significant when spraying a
   concentration of 40 mg/L GA[3] during the peak flowering period of
   jujube [[62]17]. The application of plant growth regulators can
   regulate the growth and quality of plant fruits [[63]18,[64]19]. GA[3]
   and other gibberellins (GAs) has been reported to promote seed
   germination, plant growth, flowering and fruit ripening
   [[65]20–[66]22]. During these processes, GAs play an important role in
   the regulation of other endogenous phytohormones, especially during
   flowering [[67]23].

   GAs promote the growth and development of flower organs [[68]24]. GA[3]
   is the most commonly used plant growth promoter in the commercial
   sector [[69]20,[70]21]. Exogenous GA[3] can differentially affect the
   content of other plant hormones in different plant species. In
   sugarcane and watermelon, the exogenous application of gibberellin
   caused significant increases in the endogenous hormones,
   indole-3-acetic acid (IAA) and cytokinin, while abscisic acid (ABA) was
   significantly decreased [[71]25]. In contrast, exogenous GA[3]
   treatment of jujube significantly increase the content of GA[3], IAA
   and ABA [[72]26]. In grapevine, the application of GA[3] can promote
   fruit setting, grape expansion, coloring, development and ripening,
   thereby improving grape yields [[73]27]. In addition, GA[3] has been
   reported to improve the hardness, total carotenoids, and polyphenol
   content of cashew nuts [[74]28]. GA[3] can also increase cell
   flexibility and peel extension, thus reducing the cracking of many
   cultivated fruits [[75]15,[76]29]. However, plant growth regulators are
   considered to be over-applied in fruit tree cultivation [[77]30,[78]31]
   and can lead to decreased product quality. For example, treatments of
   jujube with GA[3] resulted in a decrease in jujube soluble solids
   sugars [[79]32]. The brassinosteroids (BRs) is steroid plant hormones
   that play a major role in regulating fruit ripening in various species.
   In apples, BR was involved in the biosynthesis pathway of volatile
   compounds in the fruit [[80]33] and had also been applied in research
   on bananas [[81]34]. Recent studies have shown that green soybean seeds
   treated with BR can improve the quality and subsequent physiological
   development of soybean seedlings [[82]35], and BR also plays a role in
   regulating wheat flower degradation under low nitrogen stress [[83]36].

   Plant growth regulators regulate many plant metabolic processes
   [[84]37]. The thidiazuron (TDZ) affects plant growth and development in
   low concentrations [[85]38]. TDZ can promote grape berry enlargement
   and weight gain [[86]39]. Treating grape inflorescences with 2.5 mg/L
   TDZ and 25 mg/L GA[3] was also reported to improve the berry solid acid
   ratio [[87]40]. In recent studies, TDZ has effectively improved the
   quality and size of kiwifruit, as well as the content of soluble
   solids, and reduced fruit hardness [[88]41]. The TDZ significantly
   increased the size and weight of mango fruits, reduced the disease
   index, and maintained relatively high levels of hardness and
   antioxidant capacity during post-harvest storage [[89]42]. RNA-Seq
   analysis indicated GA[3] of grapes resulted mainly in the differential
   expression of genes involved in flavonoid and phenylpropanoid
   biosynthesis [[90]43]. GA[3] affects the regulation of phenylpropanoid
   synthesis and cell wall formation [[91]44]. RNA-Seq analysis showed
   that GA[3]-induced cherry parthenogenesis resulted in significant
   enrichment of differentially expressed genes (DEGs) in phenylalanine
   metabolism and phenylpropanoid biosynthesis [[92]45].

   The GA[3] of longan fruits was shown to up-regulate many tannins,
   phenolic acids and lignans during the on-tree preservation period
   [[93]46]. The IAA treatment of kiwifruit resulted in the activation of
   the phenylpropanoid synthesis pathway, including flavonoids, phenols,
   terpenoids, as well as carbohydrate metabolism and hormone signaling
   pathways [[94]47]. The present investigation utilized a comprehensive
   analysis of transcriptomics and metabolomics to comprehensively analyze
   the effect of spraying plant growth regulators on the quality of jujube
   fruit. The screening of candidate compounds and genes, as well as
   clarifying the metabolic regulatory network under the effects of GA[3],
   BR, and TDZ. We hypothesized that plant growth regulators increased the
   fruit setting rate of jujube and related to the biosynthesis of
   flavonoids. The study results are of great significance for
   understanding how plant growth regulators improve the fruit setting
   rate and quality of jujube.

2. Materials and methods

2.1. Experimental design

   The field trials of jujube were conducted in the 8^th regiment, Alar
   city, Xinjiang at May 30, 2022 during the jujube blooming period. The
   six experimental treatments were applied twice by spray with 7 days
   interval and consisted of J1 (18 g·hm^−2 GA[3]), J2 (18 g·hm^−2 GA[3]
   and 45 mg·hm^−2 BR), J3 (18 g·hm^−2 GA[3], 45 mg·hm^−2 BR and 1.8
   mg·hm^−2 TDZ), J4 (36 g·hm^−2 GA[3]), J5 (36 g·hm^−2 GA[3] and 45
   mg·hm^−2 BR), and J6 (36 g·hm^−2 GA[3], 45 mg·hm^−2 BR and 1.8 mg·hm^−2
   TDZ). Spraying water were as the control group (CK). The leaves were
   collected a 2 h, 1 d, 3 d, 5 d, 7 d and 14 d after the completion of
   each treatment. The mature fruits were collected in late September
   2022. In each case, three replicates were collected, immediately frozen
   in liquid nitrogen, placed in dry ice for transport and stored at −80°C
   until analysis.

2.2. The effects of plant growth regulator on quality of jujube fruit

2.2.1. Ultra performance liquid chromatography and tandem mass spectrometry
(UPLC-MS/MS) analysis of phytohormones, flavonoids and polyphenols

   The extraction of phytohormones was performed as described [[95]24].
   Flavonoids and polyphenols were extracted using a modification of a
   reported method [[96]48]. Briefly, 0.5 g of the sample was sonicated
   (40 kHz, 25°C) in 4 mL of acetonitrile for 30 minutes, then clarified
   by centrifugation at 5000 g and filtered through a 0.22 μm filter. The
   flavonoid and polyphenol contents were determined using a UPLC-MS/MS
   (Waters, Milford, MA, USA) with reference to standards ([97]S1 Table).
   The UPLC utilized an Acquity UPLC C18 column (2.1 × 100 mm, 1.7 μm;
   Waters, Milford, MA, USA). For the separation of phytohormones (5 μL
   injection volume), the column temperature was set at 40°C. The mobile
   phase consisted of acetonitrile (A) and water (B) with a flow rate of
   0.25 mL/min within a gradient of 10% A, 0–3.0 min; 3.0–7.0 min, 10%–50%
   A, 3.0–7.0 min; 50%–98% A, 7.0–18.0 min. For the separation of
   flavonoids and polyphenols (10 μL injection volume, column temperature
   30°C), the mobile phase consisted of methanol (A) and water (B) with a
   flow rate of 0.2 mL/min within a gradient consisting of 5% A, 0–3.0
   min; 5–45% A, 3.0–7.0 min; 45–50% A, 7.0–7.5 min; 50–75% A, 7.5–9.0
   min; 75–90% A, 9.0–13.0 min. The MS/MS conditions was set for multiple
   reaction monitoring mode (MRM) in the negative ion mode. The electron
   spray ionization gas temperature was set at 500°C, the ion spray
   voltage at −4500 V, the ion source temperature at 350°C and the
   de-solvent gas temperature at 650°C. The cone voltage, collision
   energy, quantitative and qualitative ion pair MS/MS parameters of each
   metabolite were shown in [98]S2 Table.

2.2.2. Determination of fructose, glucose and sucrose

   The contents of fructose, glucose, sucrose, organic acid and cyclic
   adenosine phosphate were determined using a Waters UPLC e2695
   separation module (Waters, Massachusetts, United States). The fruit
   contents of the soluble sugars fructose, glucose and sucrose were
   determined by UPLC according to GB 5009.8–2016 [[99]49]. The
   chromatographic column used was a NH[2]-RP column (250 × 4.6 mm, 5 μm)
   (Macherey Nagel, California, United States) with a column temperature
   of 40°C. The mobile phase consists of 70% acetonitrile in water with a
   flow rate of 1.0 mL/min. Detection employed a Waters 2414 refractive
   index detector (Waters, MA, USA) at 40°C.

2.2.3. Determination of organic acid

   Organic acids were determined by HPLC according to GB 5009.157–2016
   [[100]50]. The column used was a ODS column (250 mm × 4.6 mm, 5μm)
   (Dima Technology, Shanghai, China). Column temperature: 40°C. The
   mobile phase consists of (A) 0.1% phosphoric acid solution and (B)
   methanol (Thermo Field, Shanghai, China). The flow rate was 1.0 mL/min;
   injection volume: 20 μL; the mobile phase A: B ratio was 97.5%: 2.5%.
   Metabolite detection used a Waters 2489 UV/Vis Detector (Waters, MA,
   USA) set at 210 nm.

2.2.4. Determination of cyclic adenosine monophosphate (cAMP) and lignin

   The cAMP was extracted as described by Zhao [[101]51], and quantified
   by HPLC, using an Agilent Zorbax SB C18 column (4.6 mm × 250 mm, 5 μm)
   (Agilent, California, USA) with a column temperature of 35°C. The
   mobile phase was 15% methanol and 85% 50 mM potassium dihydrogen
   phosphate with a flow rate of 0.8 mL/min and an injection volume of 10
   μL. The detection wavelength was set at 254 nm. The lignin content in
   samples was determined using a commercial assay kit (Solarbio, Beijing,
   China) as the manufacturer’s instructions.

2.2.5. Determination of volatile compounds

   The extraction for volatile compounds of jujube fruit according to He
   [[102]52]. Take 10 g of powder sample, add 80, 50, and 50 mL of
   dichloromethane by extract three times, respectively. After shake for
   30 minutes each time, filter and merge the organic phase. The organic
   phase was evaporated to dryness in a rotating evaporator at 40°C, and
   finally fixed volume with 1 mL of ethyl acetate. After passing through
   a 0.22 μm filter membrane, it was loaded into an injection vial for
   GC-MS determination.

2.3 Transcriptomics (RNA-Seq)

   Three biological replicates of each treatment group were sequenced.
   Total RNA extraction and sequencing were undertaken by Metware Co.,
   Ltd. (Wuhan, China) on the Illumina Hiseq sequencer platform (Illumina,
   CA, USA). Raw reads were filtered to remove containing adapter
   sequences and Poly-N (> 10%) reads. Retained reads were mapped to
   predicted jujube mRNA using Hisat 2.2.1 software [[103]53] and the
   jujube reference genome obtained from the NCBI genome database
   ([104]https://www.ncbi.nlm.nih.gov/genome). The levels of gene
   expression were expressed in fragments per kilobase of transcript per
   million mapped reads (FPKM). The DESeq2 (1.22.1) was detected DEGs for
   each pair of experimental conditions [[105]54]. Genes displaying an
   adjusted p-value < 0.05 and a log[2] Fold Change (FC) > 1 were
   considered DEGs. Functional annotations of the DEGs were performed on 7
   different databases ([106]S3 Table). Gene Ontology (GO) [[107]55]
   enrichment analysis were performed using cluster profiler (V 4.6.0)
   software, while KEGG (Kyoto Encyclopedia of Genes and Genomes)
   signaling pathway enrichment analysis were performed using online KEGG
   resources ([108]https://www.genome.jp/kegg). The GO terms and KEGG
   pathways enriched with a false discovery rate (FDR) ≤ 0.05 were
   selected.

2.4. Quantitative real‑time polymerase chain reaction (qRT-PCR) analysis

   Total RNA was extracted using a total plant RNA extraction kit
   (Biospin, Hangzhou, China). The RNA quality was assayed using
   Nano-Photometer ®N60 instrument (IMPLE, Beijing, China). The total RNA
   was reverse-transcribed into cDNA using one-step gDNA removal and cDNA
   synthesis kit (Tran, Beijing, China) as per the manufacturer’s
   instructions. The qRT-PCR utilized the Light Cycler®96 instrument
   (Roche, Shanghai, China) using the primer sequences described in
   [109]S4 Table and ACT1 as the reference gene. The 2^−ΔΔCT method
   [[110]56] was used to determine the relative mRNA levels.

2.5. Detection and analysis of a wide range of targeted metabolomes

2.5.1. Metabolite extraction

   The metabolomics were carried out by Metware Biotechnology Co., Ltd.
   Jujube fruit were vacuum freeze-dried then ground to powder. The 50 mg
   of the powder was added to 1.2 mL of 70% precooled methanol and
   maintained at −20°C with vortexed for 30 s every 30 min for a total of
   6 times. The extract was then clarified by centrifugation at 13,400 g
   for 3 min and the supernatant was filtered with a 0.22 μm microporous
   filter membrane.

2.5.2. UPLC-MS/MS conditions

   The sample extracts were analyzed using an UPLC-MS/MS (UPLC, ExionLC™
   AD) and Tandem mass spectrometry system (Sciex, MA, USA). The
   analytical conditions were as follows: an Agilent SB-C18 (2.1 × 100 mm,
   1.8 μm) column with a column temperature of 40°C was used for
   metabolite separation. The mobile phase consisted of water (A) and
   acetonitrile (B), each containing 0.1% (v/v) formic acid. The elution
   gradient consisted of 5% B, 0–1 min; 5%−95% B, 1–9 min; 95% B, 9–10
   min, with a flow rate of 0.35 mL/min. The sample injection volume was 2
   μL. The MS/MS analysis of metabolites utilized MRM with optimization of
   the declustering potential and collision energy of each ion pair. The
   electrospray ionization (ESI) temperature used was 500°C and the ion
   spray voltages were set at 5500 V and -4500V in the positive and
   negative ion modes, respectively. The ion source gas I, II and curtain
   gas pressures were set at 50, 60 and 25 psi, respectively.

2.5.3. Metabolite quantification and the identification of differentially
expressed metabolites (DEMs)

   The DEMs were accepted a significantly different when variable
   interdependent parameter (VIP) scores > 1 [[111]57] and Log[2]FC ≥ 1.0
   were achieved. VIP values were extracted from OPLS-DA data, which also
   contain score plots and permutation plots, and was generated using R
   package Metabo Analyst R (1.0.1). The data was Log transform (Log[2])
   and mean centering before OPLS-DA. In order to avoid overfitting, a
   permutation test was performed.

2.6. Statistical analysis

   The least significant difference (LSD) of ANOVA was calculated using
   IBM SPSS statistics 26.0 and used at 5% probability level.

3. Results

3.1. Effects of different plant growth regulators on jujube leaf phytohormone
content

   To gain insight into the effects of plant growth regulator treatments
   on phytohormone levels, phytohormones which have been closely
   associated with jujube fruit setting [[112]58,[113]59] were determined
   during the blooming period of flowering, including four GA (GA[1],
   GA[3], GA[4] and GA[7]), IAA and ABA. The application of GA[3], BR and
   TDZ to leaves had a positive impact on the jujube phytohormone levels
   tested. The contents of the GA[1], GA[3] were increased with increasing
   GA[3] concentration, however, compared with CK, there was only a
   significant difference in the content of GA[4] after J1 and J4
   treatments at 5 d; the content of GA[7] in leaves showed no significant
   difference at 3 d, 14 d after treatment with JI and J4. The content of
   GA[1] and GA[3] were the main GA. The effects of plant growth
   regulators on jujube plant hormones were shown in [114]Table 1. After
   applying different plant growth regulators at 14 d, the content of
   GA[1], IAA, and ABA in the leaves showed significant changes under all
   treatment groups. The content of GA[3] did not change significantly at
   J3, while the content of GA[4] and GA[7] only showed significant
   differences at J2 and J5. The content of GA[7] and GA[4] at J4
   increased significantly by 344.8% and 471.2% compared with CK at 5 d,
   respectively. The content of IAA increased by 104.9%, 90.8%, 154.6%,
   270.2%, and 283.7% in J1, J2, J4, J5, and J6 treatment groups compared
   with the control group at 5 d, respectively. Conversely, the IAA
   content decreased by 26.0% at J3.

Table 1. Effects of different plant growth regulator formulations on jujube
leaf hormone levels (ng·g^-1).

   Time Item CK J1 J2 J3 J4 J5 J6
   2 h GA[1] 186.94±31.57^f 535.31±34.20^e 627.76±67.78^d 858.61±42.80^c
   974.91±51.01^b 1151.31±44.57^a 818.18±41.06^c
   GA[3] 75.23±14.41^f 1138.37±56.80^e 3148.98±69.07^b 2071.88±80.14^c
   3084.28±91.21^b 3604.38±93.25^a 1510.42±18.60^d
   GA[4] 0.16±0.12^e 3.07±0.41^d 5.41±0.20^c 6.57±0.28^b 2.82±0.36^d
   2.88±0.24^d 7.30±0.39^a
   GA[7] 0.43±0.04^f 6.03±0.04^b 7.96±0.19^a 5.31±0.25^c 3.60±0.26^d
   2.77±0.18^e 5.89±0.15^b
   IAA 1.16±0.05^c 1.66±0.29^abc 1.76±0.42^ab 1.22±0.19^bc 1.87±0.20^a
   1.20±0.49^bc 1.41±0.18^abc
   ABA 13.93±0.53^a 5.24±0.67^c 4.99±0.63^cd 7.49±0.54^b 3.61±1.37^cd
   4.17±1.87^cd 3.11±0.65^d
   1 d GA[1] 163.90±130.60^d 1166.85±92.94^a 949.40±59.38^bc
   1118.85±45.89^ab 820.87±50.98^c 951.38±158.18^bc 1260.74±62.04^a
   GA[3] 74.04±35.21^e 1876.77±29.36^d 2230.96±26.43^b 2396.84±39.59^a
   1837.50±36.95^d 2086.92±55.43^c 2106.68±46.01^c
   GA[4] 0.59±0.23^d 0.76±0.03^d 0.81±0.24^d 1.18±0.33^bc 0.89±0.04^cd
   1.40±0.23^b 1.84±0.09^a
   GA[7] 0.18±0.02^d 0.27±0.22^d 0.38±0.25^d 0.44±0.30^d 0.79±0.04^c
   1.70±0.04^b 2.31±0.22^a
   IAA 1.14±0.02^e 4.31±0.60^c 6.87±0.37^a 3.66±0.34^cd 3.87±0.12^c
   5.79±0.23^b 3.09±0.63^d
   ABA 10.41±0.41^a 10.23±0.55^a 3.38±0.33^cd 4.62±0.54^b 4.21±1.02^bc
   2.97±0.29^d 2.60±0.40^d
   3 d GA[1] 389.54±66.34^d 495.86±102.36^d 1253.34±39.23^a 841.82±36.05^c
   826.18±54.81^c 970.13±58.96^b 1264.44±95.66^a
   GA[3] 102.95±19.48^e 333.50±81.76^cd 937.70±66.83^a 384.16±26.10^c
   269.18±27.54^d 549.32±48.13^b 616.71±27.97^b
   GA[4] 0.20±0.05^c 0.09±0.05^c 0.76±0.06^a 0.35±0.21^bc 0.16±0.04^c
   0.51±0.27^ab 0.24±0.14^bc
   GA[7] 0.19±0.02^c 0.52±0.22^c 2.64±0.32^a 0.35±0.29^c 0.25±0.19^c
   0.93±0.05^b 0.32±0.29^c
   IAA 1.44±0.13^d 3.84±0.16^b 6.73±0.38^a 2.15±0.30^c 1.18±0.29^d
   2.60±0.45^c 2.43±0.49^c
   ABA 25.32±0.59^a 16.06±0.20^b 4.41±0.14^c 2.98±0.62^d 2.34±0.54^de
   1.60±0.33^e 2.11±0.46^e
   5 d GA[1] 483.21±46.95^f 935.80±47.99^e 1611.91±30.37^b 1071.52±80.46^d
   1189.71±56.05^c 2028.72±76.90^a 1277.99±76.43^c
   GA[3] 143.17±50.30^d 287.64±69.47^cd 910.41±85.28^a 220.28±99.21^d
   463.23±37.33^b 970.98±129.10^a 415.67±84.23^bc
   GA[4] 0.25±0.05^d 0.35±0.13^cd 0.68±0.28^bc 0.78±0.19^b 1.42±0.27^a
   0.60±0.03^bc 0.61±0.05^bc
   GA[7] 0.43±0.03^d 0.47±0.28^d 1.29±0.11^b 1.00±0.05^c 1.93±0.11^a
   0.76±0.23^c 0.82±0.15^c
   IAA 1.41±0.09^d 2.89±0.43^bc 2.69±0.26735^c 1.043±0.31^d 3.59±0.45^b
   5.22±0.43^a 5.41±0.63^a
   ABA 28.63±0.40^a 10.07±0.38^b 4.23±0.13^d 4.99±0.09^c 4.21±0.17^d
   3.08±0.12^e 3.33±0.35^e
   7 d GA[1] 575.64±16.97^c 937.95±126.29^b 894.36±92.57^b 635.10±56.60^c
   1157.22±48.81^a 718.41±89.08^c 641.66±62.41^c
   GA[3] 83.88±13.52^d 273.22±119.70^b 229.99±32.97^bc 223.76±43.41^bc
   463.10±70.25^a 325.91±90.05^b 118.52±33.83^cd
   GA[4] 0.26±0.04^a 0.36±0.20^a 0.40±0.14^a 0.66±0.10^a 0.37±0.27^a
   0.43±0.24^a 0.58±0.39^a
   GA[7] 0.63±0.06^c 0.80±0.08^bc 1.39±0.30^a 1.02±0.21^b 0.81±0.01^bc
   1.45±0.31^a 0.68±0.19^bc
   IAA 1.04±0.09^f 3.34±0.21^e 4.90±0.45^d 6.44±0.37^b 5.77±0.20^c
   6.14±0.13^bc 8.89±0.19^a
   ABA 19.21±0.46^a 17.08±0.51^b 5.36±1.10^c 3.91±0.19^d 2.86±0.80^e
   2.56±0.29^e 2.12±0.07^e
   14 d GA[1] 140.66±58.22^e 638.40±32.98^b 985.41±86.75^a 250.30±25.03^d
   379.95±97.16^c 423.09±52.79^c 621.08±23.34^b
   GA[3] 37.85±7.67^d 98.67±30.83^c 159.44±10.38^b 40.04±2.51^d
   213.57±70.01^ab 249.37±21.71^a 46.98±22.89^cd
   GA[4] 0.14±0.10^b 0.07±0.03^b 1.26±0.29^a 0.18±0.09^b 0.27±0.18^b
   0.99±0.19^a 0.04±0.02^b
   GA[7] 0.12±0.01^c 0.20±0.07^c 1.62±0.28^a 0.19±0.09^c 0.19±0.11^c
   1.20±0.22^b 0.13±0.10^c
   IAA 1.61±0.10^f 5.63±0.32^e 7.90±0.74^cd 10.72±0.62^b 7.02±0.35^d
   9.73±0.52^bc 13.76±0.52^a
   ABA 5.20±1.14^a 4.71±0.80^ab 3.59±0.49^bc 3.23±0.63^c 3.15±0.71^c
   1.39±0.33^d 1.10±0.42^d
   [115]Open in a new tab

   Note: Different lowercase letters in each row indicate significant
   differences (P<0.05) between the plant growth regulator treatments. CK:
   Control group; J1: 18 g·hm^-2 GA[3]; J2: 18 g·hm^-2 GA[3] and 45
   mg·hm^-2 BR; J3: 18 g·hm^-2 GA[3], 45 mg·hm^-2 BR and 1.8 mg·hm^-2 TDZ;
   J4: 36 g·hm^-2 GA[3]; J5: 36 g·hm^-2 GA[3] and 45 mg·hm^-2 BR; J6: 36
   g·hm^-2 GA[3], 45 mg·hm^-2 BR and 1.8 mg·hm^-2 TDZ.

   The ABA of all treatment groups were significantly lower than CK at
   7−14 d. The GA[3] content of J1 and J4 in the leaves increased and then
   rapidly decreased at 1 d. However, the GA[1] content decreased at 3 d,
   but showed an increasing by BR at 5 d. Within 0−14 days, the IAA
   content of J1, J2, J4, J5, and J6 significantly increased and more
   significant by TDZ at 7−14 d. The IAA content of J3 decreased at 5 d.
   All treatment groups showed inhibited ABA content. In summary, the
   content of IAA and GA were increased at J4 and J6, but resulted in a
   decreased ABA.

3.2. Effects of different plant growth regulators on indicators of jujube
fruit quality

   The effect of plant growth regulators on the transverse and
   longitudinal diameters of jujube are shown in [116]S5 Table. The
   transverse diameters followed the order of J6 (5.6 cm) > J1 | J3 | J4 |
   J5 (5.0 cm) > CK | J2 (4.2 cm), respectively. The significant effects
   on the longitudinal diameter were only observed at J4. Together, the
   results indicate that the largest significant effects on fruit size was
   obtained at J4 and J6. The effects of plant growth regulators on
   soluble sugars are shown in [117]Fig 1A–1C, which indicates that
   fructose levels were largely unaffected by the plant growth regulator.
   The minor effects on glucose levels were observed, which nevertheless
   achieved significance at J3 and J5. The larger relative changes were
   observed in sucrose levels, which follow the trend J6 > J4 > J1 | J3 >
   J2 | J5 > CK. The different plant hormone treatments had varying
   effects on jujube organic acids contents, except for tartaric acid,
   which was not significantly affected ([118]Fig 1D–1G). The effects on
   fumaric acid and citric acid were moderate. The citric acid
   significantly increased at J1, while it decreased significantly at J3,
   J4, and J5. The malic acid also showed significant decreases at J3, J4,
   J5, and J6.

Fig 1. The effect of different plant growth regulator formulations on sugars,
organic acid, cAMP and lignin of jujube fruit.

   [119]Fig 1
   [120]Open in a new tab

   (A-C) soluble sugars. (D-G) organic acids. (H) cyclic adenosine
   monophosphate (cAMP). (I) lignin. CK: Control; J1: 18 g·hm^-2 GA[3];
   J2: 18 g·hm^-2 GA[3] and 45 mg·hm^-2 BR; J3: 18 g·hm^-2 GA[3], 45
   mg·hm^-2 BR and 1.8 mg·hm^-2 TDZ; J4: 36 g·hm^-2 GA[3]; J5: 36 g·hm^-2
   GA[3] and 45 mg·hm^-2 BR; J6: 36 g·hm^-2 GA[3], 45 mg·hm^-2 BR and 1.8
   mg·hm^-2 TDZ. Different letters indicate a significant difference
   between treatments (p<0.05) and same letters indicate an insignificant
   difference between treatments (p≥0.05).

   The cAMP content was increased at J1, J4, and J5 ([121]Fig 1H).
   Similarly, higher lignin contents were observed after all treatment
   groups, with significant increases observed at J3, J4 and J6 ([122]Fig
   1I). The contents of hesperidin, catechin and rutin significantly
   increased, whereas the contents of naringenin, kaempferol and quercitin
   were not significantly affected ([123]Table 2). Conversely, the
   contents of caffeic acid and ferulic acid in fruit were reduced
   ([124]Table 3). The largest significant effects on the indicators could
   be seen at J4 and J6 relative to CK. The lignin, cAMP, and sucrose of
   J4 were significantly increased compared to CK, with decreases malic
   (26.0%), and citric acids (21.3%). The contents of lignin (94.2%), cAMP
   (44.1%), sucrose (97.4%) and fumaric acid (9.4%) of J6 significantly
   increased, with decreases in malic (22.7%), citric acids (11.3%).
   Therefore, the J4 and J6 were selected for further analysis. The effect
   of GA[3] on the types of volatile compounds in jujube fruit are shown
   in [125]S6 Table. The 6 categories and 50 volatile compounds were
   detected at CK, J1, and J4, with the highest number of acids, followed
   by esters, alcohols, and ketones. The number of ethers, aldehydes, and
   other volatile compounds was relatively small. The CK and treatment
   groups of jujube fruit contain 9 acids, 2 ketones, and 1 alcohol. The
   content of lauric acid and myristic acid is above 1%.

Table 2. UPLC analysis of plant growth regulator effects on flavonoid of
jujube fruit (μg·g^-1).

   Treatment Hesperidin Catechin Rutin Naringenin Kaempferol Quercitin
   CK 1.08±0.288^c 0.59±0.09^d 4.71±0.23e - - -
   J1 1.38±0.30b^c 0.75±0.07^d 5.81±0.50^d - 0.02±0.01^ab 0.03±0.01^a
   J2 2.91±1.90^ab 1.20±0.35^cd 7.73±0.19^c 0.09±0.01^a 0.04±0.01^ab
   0.06±0.03^a
   J3 3.26±1.05^a 1.73±0.16^bc 8.03±0.35^c 0.06±0.02^a 0.05±0.01^a
   0.06±0.05^a
   J4 2.24±0.48^abc 1.24±0.19^cd 10.39±0.28^b 0.05±0.01^ab 0.03±0.01^ab
   0.06±0.01^a
   J5 4.01±0.73^a 2.36±0.88^ab 11.03±0.67^b 0.06±0.05^a 0.04±0.02^a
   0.07±0.02^a
   J6 3.03±0.87^ab 2.48±0.24^a 12.29±0.49^a 0.08±0.02^a 0.04±0.01^a -
   [126]Open in a new tab

   Note: The lowercase letters, CK, J1, J2, J3, J4, J5 and J6 are as in
   [127]Table 1.

Table 3. UPLC analysis of plant growth regulator effects on phenolic acid of
jujube fruit (μg·g^-1).

   Treatment Quinic
   acid Chlorogenic acid Caffeic
   acid Ferulic
   acid [128]Trans-4-hydroxy-cinnamic acid
   CK 20.43±3.28^d - 0.06±0.01^a 0.05±0.01^a 0.18±0.02^ab
   J1 30.88±2.68^c - 0.03±0.01^a 0.03±0.01^a 0.18±0.04^ab
   J2 33.70±0.58^c 0.03±0.01^a 0.04±0.01^a 0.03±0.01^a 0.20±0.02^a
   J3 42.46±2.21^b 0.02±0.01^ab 0.05±0.01^a 0.04±0.02^a 0.17±0.01^ab
   J4 36.50±2.34^c 0.02±0.01^ab 0.04±0.01^a 0.03±0.01^a 0.14±0.02^b
   J5 46.30±5.23^b 0.02±0.01^ab 0.05±0.03^a 0.04±0.01^a 0.20±0.03^a
   J6 53.51±4.02^a 0.02±0.01^b 0.03±0.01^a 0.02±0.01^a 0.15±0.02^ab
   [129]Open in a new tab

   Note: The lowercase letters, CK, J1, J2, J3, J4, J5 and J6 are as in
   [130]Table 1.

3.3. RNA-Seq analysis of jujube fruit

   A PCA analysis of fruit RNA-Seq data from CK and treatment groups were
   conducted. The PC1 and PC2 explained 29.6% and 24.2% variance,
   respectively, with a clear distinction between CK and treatment groups
   ([131]Fig 2A). The correlation coefficient observed between samples of
   all treatments was greater than 0.89, and the correlation coefficient
   within treatment groups were greater than 0.93 ([132]S1 Fig). These 779
   DEGs (628 up-regulated and 151 down-regulated) were detected at J4,
   whereas 1527 (914 up-regulated and 613 down-regulated) were detected in
   fruit at J6. In addition, a comparison of J4 and J6 indicated 791 DEGs
   (92 up-regulated and 699 down-regulated) ([133]Fig 2B and 2C). The
   biosynthetic pathway of phenylpropanoids promotes the accumulation of
   secondary metabolites (flavonoids and phenolic acids) in fruits. These
   mRNA levels of acetaldehyde dehydrogenase (ALDH), phenylalanine ammonia
   lyase (PAL), cinnamic acid 4-hydroxylase (C4H), Cinnamoyl-CoA reductase
   1 (CCR1), 4-Coumarate Coenzyme A Ligase (4CL2, 4CL1) were significantly
   increased at J4 and J6 ([134]Fig 3). To confirm the validity of the
   RNA-Seq data, the relative expression levels of these phenylpropanoid
   pathway genes were measured by qRT-PCR. The relative expressions were
   approximately consistent with the FPKM value.

Fig 2. Transcriptomics and metabolomics analysis of jujube fruit treated with
different plant growth regulator.

   [135]Fig 2
   [136]Open in a new tab

   (A) PCA analyses of RNA-Seq samples. (B) Venn diagram of DEGs between
   the control and treatment groups. (C) The number of up-regulated and
   down-regulated DEGs between the control and treatment groups. (D) PCA
   analyses of metabolomics samples. (E) Venn diagram of DEMs between the
   control and the treatment groups, (F) The number of up-regulated and
   down-regulated DEMs between the control and treatment groups. The
   meaning of CK, J4 and J6 are same as [137]Fig 1.

Fig 3. Plant growth regulator effects on phenylpropanoid pathway gene
expression.

   [138]Fig 3
   [139]Open in a new tab

   (A-F) Relative expression levels of ALDH, PAL, C4H, CCR1, 4CL2, 4CL1
   from RNA-Seq and qRT-PCR, respectively. The meaning of CK, J4 and J6
   are same as [140]Fig 1.

3.4. GO term and KEGG pathway analysis of jujube DEGs

   An analysis of GO terms indicated that the DEGs affected at J4 and J6
   were mainly enriched at cellular processes, metabolic processes,
   cellular anatomical entities, binding and catalytic activity ([141]Fig
   4). The top 20 of which for each pair-wise comparison are presented in
   [142]Fig 5. Flavonoid biosynthetic and metabolic processes were
   enriched at J4 and J6 ([143]Fig 5A and 5B). An examination of DEGs
   pertaining at J4 indicated 16 pertained to the phenylpropanoid
   biosynthesis pathway (ko00940; 14 up-regulated and 2 down-regulated),
   whereas the J6 resulted in 21 DEGs (17 up-regulated and 4
   down-regulated). The six DEGs were selected for confirmation by qRT-PCR
   indicating that the RNA-Seq data was reliable ([144]Fig 3).

Fig 4. GO term analysis of DEGs in jujube fruit by application of plant
growth regulator.

   [145]Fig 4
   [146]Open in a new tab

   (A) CK vs. J4. (B) CK vs. J6. (C) J4 vs. J6. The meaning of CK, J4 and
   J6 are same as [147]Fig 1. The GO considered included cellular
   component (CC), biological process (BP) and molecular function (MF).

Fig 5. KEGG enrichment analysis of DEGs in jujube fruit by application of
plant growth regulator.

   [148]Fig 5
   [149]Open in a new tab

   (A) CK vs. J4. (B) CK vs. J6. (C) J4 vs. J6. The meaning of CK, J4 and
   J6 are same as [150]Fig 1.

3.5. Metabolomics analysis of different plant growth regulator effects in
jujube fruits

   The metabolomic analysis of jujube fruit revealed the presence of 1091
   DEMs between the control, J4 and J6. The PC1 and PC2 accounted for
   32.1% and 17.7% of the total variance, respectively. The control group
   was clearly distinguished from the treatment groups by PC2, whereas the
   J4 and J6 could be distinguished by PC1, although two samples from each
   were proximal ([151]Fig 2D). The orthogonal partial least
   squares-discriminant analysis (OPLS-DA) indicated 302 DEMs (229
   up-regulated and 73 down-regulated) at J4, 354 DEMs (162 up-regulated
   and 192 down-regulated) at J6 and 435 DEMs (128 up-regulated and 307
   down-regulated) between J4 vs. J6 ([152]Fig 2E and 2F). The volcano
   plot of these DEMs obtained through further screening under VIP, log[2]
   FC, P value screening conditions is shown in [153]Fig 6A–6C.

Fig 6. Volcano plots of DEMs in jujube fruit by application of plant growth
regulator.

   [154]Fig 6
   [155]Open in a new tab

   (A) CK vs. J4. (B) CK vs. J6. (C) J4 vs. J6. The meaning of CK, J4 and
   J6 are same as [156]Fig 1.

   To investigate the impact of plant growth regulator on different
   metabolic pathways, the DEMs identified were KEGG annotated ([157]S2
   Fig), followed by an enrichment analysis ([158]Fig 6). Pathways
   enriched at J4 and J6 included nucleotide metabolism, flavone and
   flavonol biosynthesis and phenylpropanoid biosynthesis, although with
   differing levels of significance ([159]Fig 7). KEGG pathway enrichment
   analysis of RNA-Seq data at J4 and J6 also indicated an enrichment of
   flavonoid biosynthesis and metabolism, with phenylpropanoid
   biosynthetic process at J4. Furthermore, fruit contents of lignin and
   flavonoids were increased at J4 and J6. Therefore, DEMs related with
   the phenylpropanoid pathway were examined further. The
   phenylpropanoid-related DEMs identified consisted of only cinnamic acid
   and sinapic acid at CK vs. J4; cinnamic acid, ferulic acid, syringin,
   caffeic aldehyde, 1-O-Sinapoyl-β-D-glucose and sinapyl alcohol at CK
   vs. J6.

Fig 7. KEGG enrichment analysis of DEMs in jujube fruit by application of
plant growth regulator.

   [160]Fig 7
   [161]Open in a new tab

   (A) CK vs. J4. (B) CK vs. J6. (C) J4 vs. J6. The meaning of CK, J4 and
   J6 are same as [162]Fig 1.

3.6. Joint analysis of plant growth regulator effects on DEGs and DEMs
related with the phenylpropanoid pathway in jujube fruit

   A joint analysis of DEGs and DEMs enrichments related with the
   phenylpropanoid pathway were conducted to identify potential
   correlations ([163]S3A–S3C Fig). These DEGs included the up-regulation
   of PAL, C4H, 4CL2, CCR, Caffeoyl-CoA 3-O-methyltransferase (CcoAoMT)
   and the down-regulation of cinnamyl-alcohol dehydrogenase (CAD) at J4.
   These alterations were accompanied by significant increases cinnamic
   and sinapic acids ([164]S3A Fig). These mRNA levels of PAL, C4H, 4CL,
   CCR and coniferyl-alcohol glucosyltransferase (CAGT) were up-regulated
   and CAD was down-regulated at J6, which occurred with the higher
   accumulation of cinnamic acid, syringin, 1-O-Sinapoyl-β-D-glucose and
   sinapyl alcohol, but a reduction of ferulic acid and caffeic aldehyde
   ([165]S3B Fig). These down-regulation of CCR, shikimate
   O-hydroxycinnamoyltransferase (HCT), CAD, peroxidase (POD) and CcoAoMT,
   and the up-regulation of CAGT occurred with increases in spermidine,
   syringin and sinapyl alcohol, with decreases in the levels of caffeic
   acid, sinapic acid, ferulic acid and caffeic aldehyde at J4 and J6 (C).

4. Discussion

   GAs participates in various biological processes during fruit
   development [[166]60]. The application of GA[3] to persimmon trees has
   a significant impact on persimmon fruit size [[167]61]. The treatment
   of green ripe bananas with cytokinin and GA[3] significantly inhibited
   fruit softening and delayed ripening-related increases in respiration,
   ethylene content and soluble sugar accumulation, with a reduced loss of
   ascorbic acid and increases in total phenolic content [[168]62]. In
   this study, all the plant growth regulator treatments utilized resulted
   in alterations to fruit phytohormone contents, including rapid, but
   short-term increases in GA[3] and GA[1] (2 h–1 d), and more prolonged
   changes, including relative increases in IAA and the suppression of ABA
   ([169]Table 1).

   The plant growth regulator treatments J4 and J6 significantly enhanced
   jujube fruit size. The fruit content of sucrose sugar in fruit was
   significantly increased by plant growth regulator treatments, which was
   consistent to that observed after similar treatment of grapevine, which
   was attributed to GA[3] effects on organic acid conversion, sugar
   transport and accumulation [[170]63]. Plant hormones play important
   roles in the regulation of phenylpropanoid biosynthesis
   [[171]64,[172]65]. GA treatment was reported to increase the phenolic
   content in wheat sprouts [[173]66]. GA[3] and cytokinin have a
   significant impact on the biosynthesis of phenylpropanoids and organic
   acids [[174]67,[175]68]. GA[3] was shown to affect the synthesis of
   sugars and the regulation phenylpropanoid biosynthetic genes, thereby
   affecting the coloring and quality of grapes [[176]68].

   The outputs from the phenylpropanoid pathway includes flavonoids,
   phenolic compounds and lignin [[177]69]. In this pathway, PAL is a key
   enzyme that catalyzes the deamination of phenylalanine to form cinnamic
   acid, which is then converted into p-coumaric acid by C4H, and
   p-coumaroyl-CoA by 4CL. The p-coumaroyl-CoA is a major precursor
   metabolite of the flavonoid biosynthetic pathway, where enzymes such as
   5’ hydroxylase (F3’5’H) and flavonol synthase (FLS1) are involved in
   conversion steps leading to the formation of flavonoids such as
   quercetin, kaempferol and the various anthocyanins. The GA[3] has been
   reported to increased PAL activity and the contents of phenols and
   flavonoids in long-on fruit [[178]46]. The GA[3] was also observed to
   increase flavonoid content in roselle [[179]70]. The pear fruit with
   GAs also reduced lignin biosynthesis by down-regulating the expression
   of PAL and 4CL [[180]71].

   In this study, we have shown that plant growth regulator treatments
   during jujube fruit development alter endogenous hormone levels,
   leading to alterations in the regulation of genes in aspects of the
   phenylpropanoid pathway (Figs [181]2B, 2C, [182]4 and [183]7), which is
   reflected in the contents of phenylpropanoid-related metabolites
   ([184]Fig 2E and 2F). The phenylpropanoid pathway was directed to an
   increase in lignin deposition at J4 and J6 ([185]Fig 1D). However, GO
   and KEGG analyses of RNA-Seq and metabolomics data also indicated an
   increased output from the flavonol/ flavonoid pathway (quercetin,
   epi-afzelechin, catechin, epicatechin, fustin) (Figs [186]4, [187]6 and
   [188]7). However, the specific regulatory mechanisms involved remain
   unclear.

5. Conclusions

   Application of plant growth regulators increased the contents of GAs
   and IAA, but reduced the content of ABA. The 18 g·hm^−2 GA[3] is
   beneficial for increasing the soluble sugar and cAMP in jujube fruit,
   and reducing the organic acid. Both GA[3] treatments increased lignin,
   but the lignin was lower in 18 g·hm^−2 GA[3]. Therefore, it is
   recommended that 18 g·hm^−2 GA[3] during the flowering period of
   jujube. The combined analysis of RNA-Seq and metabolomics treated with
   different plant growth regulators indicated an up-regulation of PAL,
   C4H, 4CL2, CCR, CcoAoMT, and the down-regulated CAD mRNA level of the
   phenylpropanoid pathway, which was accompanied by increased levels of
   cinnamic acid, eugenin, 1-O-sinapyl-D-glucose and sinapol, with
   decreases in ferulic and caffeic acids. These DEMs such as quercetin,
   epiafzelechin, catechin, epicatechin and fustin were increased at
   flavonoid pathway. Overall, our research findings have increased our
   understanding of how plant growth regulator affect quality of jujube
   fruit. In future, it would conduct in-depth research and validate the
   functional roles of key genes that control changes in flavonoids and
   polyphenols.

Supporting information

   S1 Fig. Heat map of correlation coefficients between RNA-Seq samples.

   CK: the control group; J1: 18 g·hm^-2 GA[3]; J2: 18 g·hm^-2 GA[3] and
   45 mg·hm^-2 BR; J3: 18 g·hm^-2 GA[3], 45 mg·hm^-2 BR and 1.8 mg·hm^-2
   TDZ; J4: 36 g·hm^-2 GA[3]; J5: 36 g·hm^-2 GA[3] and 45 mg·hm^-2 BR; J6:
   36 g·hm^-2 GA[3], 45 mg·hm^-2 BR and 1.8 mg·hm^-2 TDZ.

   (TIF)
   [189]pone.0305185.s001.tif^ (20MB, tif)
   S2 Fig. KEGG classification diagram of DEMs in jujube fruit.

   (A) CK vs. J4. (B) CK vs. J6. (C) J4 vs. J6. The meaning of CK, J4 and
   J6 are as in S1 Fig.

   (TIF)
   [190]pone.0305185.s002.tif^ (1.9MB, tif)
   S3 Fig. The plant growth regulator effects on the phenylpropanoid
   pathway from RNA-Seq and metabolomics.

   The red labels indicate the up-regulation of a gene/metabolite, while
   green labels indicate their down-regulation. (A) CK vs. J4. (B) CK vs.
   J6. (C) J4 vs. J6. The meaning of CK, J4 and J6 are as in S1 Fig.

   (TIF)
   [191]pone.0305185.s003.tif^ (1.6MB, tif)
   S4 Fig. TIC of volatile compounds in jujube fruit by GC-MS.

   (A) CK (B) J1 (C) J4. The meaning of CK, J1 and J4 are as in S1 Fig.

   (TIF)
   [192]pone.0305185.s004.tif^ (26.1MB, tif)
   S1 Table. Standard curves of plant hormone, sugar, organic acid,
   flavonoid and phenolic acid.

   (DOCX)
   [193]pone.0305185.s005.docx^ (19.4KB, docx)
   S2 Table. UPLC-MS/MS conditions for the detection of GA[1], GA[3],
   GA[4], GA[7], IAA, ABA, flavone and polyphenols.

   (DOCX)
   [194]pone.0305185.s006.docx^ (20.1KB, docx)
   S3 Table. Public databases used for the functional annotations of DEGs.

   (DOCX)
   [195]pone.0305185.s007.docx^ (16.1KB, docx)
   S4 Table. Primer sequences utilized in qRT-PCR.

   (DOCX)
   [196]pone.0305185.s008.docx^ (16.9KB, docx)
   S5 Table. Effects of different plant growth regulator formulations on
   the jujube fruit dimensions.

   (DOCX)
   [197]pone.0305185.s009.docx^ (17.3KB, docx)
   S6 Table. The effect of GA[3] on the types of volatile compounds in
   jujube.

   (DOCX)
   [198]pone.0305185.s010.docx^ (16.5KB, docx)
   S1 Graphical abstract

   (PNG)
   [199]pone.0305185.s011.png^ (1.3MB, png)

Data Availability

   All relevant data are within the paper and its [200]Supporting
   Information files.

Funding Statement

   The work was supported by the Key Cultivation Project of Scientific and
   Technological Innovation of Xinjiang Academy of Agricultural Sciences
   to LK (xjkcpy-2022002), the Key Laboratory of Forest Plant Ecology,
   Ministry of Education to LK (KLP2024B1), and the Xinjiang Production
   and Construction Crops Science and Technology Plan Project to LK
   (KY2024JBGS02). The funders had no role in study design, data
   collection and analysis, decision to publish, or preparation of the
   manuscript.

References