Abstract

   Small fructans improve plant tolerance for cold stress. However, the
   underlying molecular mechanisms are poorly understood. Here, we have
   demonstrated that the small fructan tetrasaccharide nystose improves
   the cold stress tolerance of primary rice roots. Roots developed from
   seeds soaked in nystose showed lower browning rate, higher root
   activity, and faster growth compared to seeds soaked in water under
   chilling stress. Comparative proteomics analysis of nystose-treated and
   control roots identified a total of 497 differentially expressed
   proteins. GO classification and KEGG pathway analysis documented that
   some of the upregulated differentially expressed proteins were
   implicated in the regulation of serine/threonine protein phosphatase
   activity, abscisic acid-activated signaling, removal of superoxide
   radicals, and the response to oxidative stress and defense responses.
   Western blot analysis indicated that nystose promotes the growth of
   primary rice roots by increasing the level of RSOsPR10, and the cold
   stress-induced change in RSOsPR10levelis regulated by jasmonate,
   salicylic acid, and abscisic acid signaling pathways in rice roots.
   Furthermore, OsMKK4-dependentmitogen-activated protein kinase signaling
   cascades may be involved in the nystose-induced cold tolerance of
   primary rice roots. Together, these results indicate that nystose acts
   as an immunostimulator of the response to cold stress by multiple
   signaling pathways.

Introduction

   Fructans are acid-labile, water-soluble, polydisperse fructose polymers
   with a sucrose starter unit. They represent the major reserve
   carbohydrates in approximately 15% of flowering plants [[46]1].
   Fructans can be linear or branched, and their degree of polymerization
   (DP) ranges from 3 to 300, depending on the species and developmental
   stage of the plant as well as environmental conditions [[47]2].
   Fructans are classified according to their glycosidic linkages: β(2,1),
   β(2,6), or both. There are two forms of trisaccharide fructans,
   1-kestotriose, 6-kestotriose, and 6G-kestotriose (neokestose); they
   belong to inulin- and levan-type fructans, respectively.
   Tetrasaccharide nystose is a fructooligosaccharide with two fructose
   molecules linked via β(2,1) bonds to the fructosyl moiety of sucrose.
   Nystose typically occurs in the eudicot family Asteraceae and the
   monocot families Asparagaceae, Liliaceae, and Poaceae; however, it is
   absent in rice [[48]3].

   It has been suggested that fructans function as reserve carbohydrates
   deposited in the underground storage organs of dicots and above-ground
   parts of monocots [[49]2, [50]4, [51]5]. Additionally, fructans
   regulate flower opening. Closed petals of Campanula rapunculoides and
   Hemerocallis have a high fructan content, while no fructan is present
   in the petals of opened flowers [[52]6, [53]7]. Fructanexohydrolases
   quickly release a significant amount of fructose, facilitating water
   inflow and flower opening by lowering the osmotic pressure. In
   addition, fructans protect plants from cold stress and water deficit
   [[54]8–[55]11] and maintain the sucrose concentration necessary for
   photosynthesis and transport [[56]12]. These properties of fructans led
   to the hypothesis that they act as membrane stabilizers. Indeed, by
   interacting with membrane lipids, fructans increase membrane stability
   during freezing and cellular dehydration [[57]10, [58]13–[59]17].
   Fructans also scavenge hydroxyl radicals (·OH) generated by the
   activity of tonoplast-associated class III peroxidase [[60]18, [61]19].
   In contrast to glucose-, fructose-, and sucrose-specific signaling
   pathways, which are well characterized in plants [[62]20–[63]22], the
   cold stress-related fructan signaling pathways are poorly understood.

   Cold stress, including chilling (0–15°C) and freezing (< 0°C), is a
   major environmental factor affecting plant growth and development, and
   influencing crop yield [[64]23–[65]27]. Cold stress triggers numerous
   physiological responses in plants, including membrane rigidification,
   suppression of nutrient absorption, reduced photosynthetic rate, growth
   inhibition [[66]28], transient increases in the levels of hormones such
   as abscisic acid (ABA) and jasmonic acid (JA) [[67]29], changes in
   membrane lipid composition [[68]30], accumulation of compatible
   osmolytes such as soluble sugars, betaine, and proline [[69]31–[70]33],
   and increased formation of antioxidants [[71]34]. Cold stress signaling
   pathways involve calcium signaling and mitogen-activated protein kinase
   (MAPK) cascade pathways, hormone-mediated signaling (e.g., JA, ABA),
   and reactive oxygen species (ROS) signaling [[72]24].

   The development of molecular technologies and proteomics enables a
   comprehensive analysis of changes in plant protein expression in
   response to cold stress. Kawamura and Uemura (2003) were the first to
   apply mass spectrometry to elucidate the relationship between plasma
   membrane proteins and cold acclimation. They successfully identified 38
   relevant proteins in Arabidopsis [[73]35], including early response to
   dehydration proteins ERD10 and ERD14, members of the dehydrins family
   that protect against cold-induced dehydration [[74]36, [75]37]. In rice
   seedlings exposed to the stress of a progressively decreasing
   temperature, from normal t to 15, 10, and 5°C, 41 cold-responsive
   proteins were identified using 2-D electrophoresis combined with
   MALDI-TOF MS or ESI/MS/MS. These proteins were involved in protein
   quality control mediated by chaperones and proteases, and in the
   enhancement of cell wall components [[76]38]. Neilson and coworkers
   identified 236 cold-responsive proteins compared using label-free
   quantification to 85 using iTRAQ, with only 24 common proteins
   identified. Functional analysis revealed cold stress-related
   differential expression of proteins involved in transport,
   photosynthesis, generation of precursor metabolites, histones, and
   vitamin B biosynthesis [[77]39]. These findings can guide molecular
   breeding strategies aiming at the improvement of tolerance to cold in
   crops [[78]40].

   Microarray technology provides a powerful tool for genome-wide
   identification of genes involved in the response of rice to cold stress
   and dissecting the mechanisms responsible for their effects.
   Comparative transcriptome analysis of two rice genotypes with
   contrasting responses to cold stress revealed that the major effect of
   low temperature was the upregulation of gene expression in the
   cold-tolerant genotype, and downregulation in the cold-sensitive
   genotype [[79]41]. Genome-wide profiling of gene expression in a
   cold-tolerant japonica variety identified 5557 differentially expressed
   genes (DEGs) at four distinct time points during moderate cold stress
   (8°C). Further analysis revealed that the glutathione system and the
   ABA signaling pathway played a dominant role in the cold stress
   response [[80]42]. In the present study, integrated analysis of
   morphology, physiology, and comparative proteomics demonstrated that
   nystose regulates the response of rice roots to cold stress via
   multiple signaling pathways, including jasmonate, SA and ABA signaling
   pathways, and MAPK signaling cascades. These findings provide novel
   insights into the role of nystose-specific signaling pathways in the
   response of rice roots to cold stress.

Materials and methods

Plant materials and growth conditions

Rice growth conditions

   Seeds of rice variety ‘Zhongzheyou 1’ were supplied by the WuWangNong
   Seed Group (Hangzhou, Zhejiang province, China). The seeds were soaked
   in 50 ml of water containing 75 mg/L nystose (Seebio Biotechnology Co.,
   Ltd., Shanghai, China) for 1 d at 30°C, and rinsed with distilled water
   three times. Subsequently, 20 rice seeds were transferred to 50 ml of
   agar containing 75 mg/L nystose and incubated at 15°C for 7 d.

Rice cold treatment and recovery protocol

   Rice seeds were soaked in 50ml water containing 75 mg/L nystose, 2.5
   mg/L ABA, 2.5 mg/L JA, and 10 mg/L SA, respectively. After germinating
   for 40h at 30°C, 50 rice seedlings were transferred to germination
   boxes containing two layers of filter papers moistened with distilled
   water and incubated at 4°C for 24h and 48h. For the recovery, seedlings
   subjected to cold stress for 48 h were transferred to normal culture
   conditions.

Measurement of primary root length

   The length of primary roots in 20 control and treated plants was
   measured after 7 d of growth at 15°C.

Determination of browning

   The number of rice roots that turned from white to brown after 5d of
   recovering in normal culture conditions was counted. The percentage of
   browning was calculated as the number of browned roots/50*100%.

TTC reduction assay

   For the TTC assay, 50 root samples were cut into 1 cm segments and
   placed in 10 ml of TTC solution (5 ml 0.4% TTC and 5 ml 0.05 M sodium
   phosphate buffer, pH 7.4) for 2 h in the dark at 37°C. TTC solution was
   then drained, and the specimens were washed in sterile water. TTC was
   extracted in 10 ml of methanol for 1 d at 37°C, and its absorbance was
   measured at 485 nm. The amount of reduced TTC was calculated according
   to the standard curve prepared using 20, 40, 80, 120, 160, and 200 μg
   of TTC.

Protein preparation

   Rice roots (1 g) were ground in liquid nitrogen, suspended in 5 ml
   acetone with 10% (w/v) trichloroacetic acid and 0.07% (w/v)
   β-mercaptoethanol at -20°C for 1 h, and centrifuged for 30 min at
   14,000 g. Pellets were resuspended in 0.07% (w/v) β-mercaptoethanol in
   acetone, incubated at -20°C for 1 h, and centrifuged14,000 g for 15 min
   at 4°C. This step was repeated three times. The pellets containing
   crude protein were dried and solubilized in lysis buffer (8 M urea, 100
   mMTris-HCl (pH 8.5), and 1 mM PMSF) for 1 h at room temperature. The
   samples were then centrifuged15 min at 14,000g, and the supernatants
   were collected in 1.5 ml tubes. A 4 μl aliquot was used to measure
   protein concentration by the Bradford assay, using bovine serum albumin
   as the standard.

Protein digestion

   Protein samples (100 μg) were mixed with 50 mM NH[4]HCO[3] to reach the
   final volume of 150 μl. Subsequently, 100mM stock solution of DTT was
   added for the final concentration of 10 mM, mixed at 600 rpm for 1 min,
   and incubated at 37°C for 1h. IAA (500 mM) was added to obtain the
   final concentration of 50 mM, mixed at 600 rpm for 1 min, and stored at
   room temperature in the dark for 30 min. The samples were then passed
   through a 10 kDa filter and centrifuged at 14,000 g for 15 min at 4°C.
   Next, 100 μl of trypsin buffer (2 μg trypsin in 100 μl NH[4]HCO[3]
   buffer) was added, and the tryptic digestion was performed for 16–18 h
   at 37°C. The products of digestion were collected after centrifugation
   at 14,000g for 10 min and subsequent addition of 100 μl
   NH[4]HCO[3,]followed by centrifugation at 14,000g for 10 min. Peptides
   were desalted using the C18 tip (Thermo Fisher Scientific, Waltham, MA,
   USA).

LC-MS/MS analysis

   RP-HPLC separation was performed using a nanoflow HPLC instrument
   (ProxeonBiosystems, now Thermo Fisher Scientific) equipped with a
   self-packed tip column (75 μm × 150 mm; C18, 3.0 μm). A 120 min
   gradient was applied at a flow rate of 300 nl/min. For mass
   spectrometry, a Q-Exactive mass spectrometer (Thermo Fisher Scientific)
   equipped with a nanoelectrospray ion source (ProxeonBiosystems, now
   Thermo Fisher Scientific) was used. Data were acquired in the
   data-dependent “top10” mode, in which the ten precursor ions of the
   highest abundance were selected with at a high resolution (70,000 at
   m/z 200) from the full scan (300–2000 m/z) for HCD fragmentation.
   Precursor ions with single or unassigned charges information were
   excluded. The resolution for the MS/MS spectra was set to 17500 at m/z
   200, with a target value of 2E5 at enabled AGC control. The isolation
   window was set to 2.0 m/z, with a lock mass option enabled for the
   445.120025 ion. The normalized collision energy was 29%.

Database search and bioinformatics

   MS data were analyzed using MaxQuant software version 1.6.0.1 to obtain
   matched protein datasets for label-free quantification (LFQ) ([81]S4
   File). The data were searched against the Oryza sativa subsp. japonica
   database (122,587 total entries). The main search parameters were as
   follows: main search ppm: 6, missed cleavage: 2, MS/MS tolerance ppm:
   20, enzyme: trypsin, variable modification: oxidation (M) and acetyl
   (protein N-term), decoy database pattern: reverse, LFQ: true, LFQ min.
   ratio count: 1, match between runs: 2 min, peptide FDR: 0.01, and
   protein FDR: 0.01 [[82]43]. DEP analysis was performed using the
   Perseus software (version 1.6.5.0). KEGG and GO analyses of the
   differentially expressed proteins (DEPs)were performed in DAVID 6.8
   ([83]https://david.ncifcrf.gov/). Figures were constructed in the R
   environment using the ggplot2 package.

Preparation of antibodies and Western blot analysis

   Peptide fragments were examined as protein-surface antigens (for the
   sequences, see [84]S5 File). Peptides containing an additional
   N-terminal cysteine were synthesized, purified on resin, and used to
   raise polyclonal antibodies in rabbits (HuaAn Biotechnology Co., Ltd.,
   Hangzhou, China, or Beijing Protein Innovation Co.,Ltd., Beijing,
   China). For Western blotting, rice roots (0.5 g) were ground into a
   fine powder in liquid nitrogen and suspended in 2 ml of protein
   extraction buffer containing 50 mM Tris-HCl, pH8.0, 1 mM EDTA, 10 mM
   NaCl, 1% SDS, 0.5% (v/v) 2-mercaptoethanol, 0.1 mM PMSF, 0.1 mM DTT and
   0.1% (v/v) Triton X-100. The suspension was ground until the powder was
   fully homogenized. Mixtures were then centrifuged at 14,000 g for 15
   min at 4°C, and supernatants were transferred into 5 ml centrifuge
   tubes. Protein concentration was determined using the RC DC protein
   assay kit II (Bio-Rad, Hercules, CA, USA). Samples containing 20 μg of
   total protein were subjected to electrophoresis on 15%
   SDS-polyacrylamide gel. Separated proteins were transferred to PVDF
   membranes, which were blocked with 5% (w/v) skimmed milk. Blots were
   incubated with rabbit antibodies diluted 1:1000 in TBST (25 mM Tris
   base pH 8.0, 140 mM NaCl, 3 mM KCl, 0.05% (v/v) Tween 20) for 1 h and
   washed three times for 5 min in TBST. Subsequently, the blots were
   probed with HRP-labeled goat anti-rabbit IgG (H+L) diluted 1:5000.
   Reactive bands were visualized using ECL (Multiscience Biotech Co.,
   Ltd., Hangzhou, China).

Statistical analysis

   Each treatment was performed in three independent experimental
   replicates, and the data are presented as the mean ± SE. The analysis
   of variance (ANOVA) was performed using Duncan’s multiple range test.
   Percentages were transformed according to y = arcsin [SQR(x/100)]. All
   data were analyzed according to a factorial model and replicates as
   random effects. The treatments were compared using the least
   significant difference at the 0.05 confidence level.

Results

Effect of nystose on the phenotypes and physiological parameters of rice
primary roots under normal conditions and cold stress

   Rice seeds were soaked in nystose solution (SSN, treatment group) or
   water (SSW, control group) at 4°C for 2 d and allowed to recover at
   25°C for 1 d, 3 d, and 5 d. After 1 day of the recovery, the apical
   parts of the primary roots in the SSW group showed browning ([85]Fig
   1A), and the degree of root tip browning gradually increased with
   recovery time ([86]Fig 1C and 1E). The majority of primary root tips in
   the control group exhibited a typical white phenotype ([87]Fig 1B, 1D
   and 1F). Under normal growth conditions, no significant differences in
   the phenotypes of primary root tips were observed between the two
   groups, and the tips had a normal white phenotype ([88]S1 Fig). Thus,
   the percentage of browning primary roots in the SSW group was higher
   than in the SSN group ([89]Fig 1H). The ability of roots to reduce TTC
   was then used as an indicator of their tolerance to cold. The results
   of TTC staining assays documented the metabolic activity of primary
   roots in the treatment group was higher in the control group under cold
   stress, while no significant differences were observed between SSN and
   SSW ([90]Fig 1I). In addition, the length of the primary roots in the
   SSN group was greater than in the control group after growing at 15°C
   for 10 days after sowing ([91]Fig 1G). Together, these data suggest
   that nystose improves the cold tolerance of primary roots of rice.

Fig 1. Phenotypes and physiological indices of rice roots under normal growth
and cold stress.

   [92]Fig 1
   [93]Open in a new tab

   (A, C, E) Phenotypes of primary rice roots from seeds soaked in water,
   grown at 4°C for 2 d, and allowed to recover at 25°C for 1 d (A), 3 d
   (C), and 5 d (E). Red arrows in the boxes point to the brown
   discoloration of primary rice roots in the control group. Selected
   areas indicated by small red boxes are shown at higher magnification
   large red boxes. (B, D, F). Phenotypes of primary rice roots from seeds
   soaked in 75 mg/L nystose, grown at 4°C for 2 d, and allowed to recover
   at 25°C for 1 d (B), 3 d (D) and 5 d (F). (G) Length of primary rice
   roots after growing at 15°C for 10 days after sowing. Twenty
   independent experimental replicates were analyzed for each treatment,
   and data are presented as the mean ± SE. An independent t-test was
   performed to determine the differences between the SSN and SSW groups
   (*P<0.05). (H) The fraction of browning primary roots. Each experiment
   was performed in triplicate, and data are presented as the mean ± SE.
   An independent t-test was performed to determine the differences
   between groups (**P<0.01). (I) Metabolic activity of the roots. Each
   experiment was performed in triplicate, and data are indicated as the
   mean ± SE. An independent t-test was performed to determine the
   difference between groups (*P<0.05).

Protein expression profiles of nystose-treated and control primary roots
under normal growth conditions and cold stress

   To determine the molecular mechanisms underlying the ability of nystose
   to improve the cold tolerance of primary roots of rice, comparative
   proteomics analysis was performed. The objective was to identify DEPs
   in the primary roots in the treatment and control groups under normal
   growth conditions (25°C for 24 h, 48 h, and 7 d), cold stress (4°C for
   24 h and 48 h) and recovery from cold stress (25°C for 5d after growth
   at 4°C for 48 h). As shown in [94]S2 Fig, a total of 916 DEPs were
   identified, with similar numbers of upregulated and downregulated
   proteins. Among them, 497 DEPs, including 248 upregulated DEPs and 249
   downregulated DEPs, were characterized by fold-change ≥ 1.5 ([95]Fig
   2). In addition, the number of upregulated proteins was greater than
   the number of downregulated proteins under normal growth conditions and
   cold stress, while the number of upregulated proteins was lower than
   the number of downregulated proteins under normal growth conditions and
   recovery from cold stress ([96]Fig 2, [97]S2 Fig).

Fig 2. Protein expression profiles in control and nystose-treated rice roots
under normal growth conditions and cold stress.

   [98]Fig 2
   [99]Open in a new tab

   A: Venn diagram of the number of DEPs with fold change >1.5 (P<0.05).
   B: Venn diagram of the number of DEPs with fold change<2/3 (P<0.05).
   NW-vs-NN (24 h): rice roots that grew at 25°C for 24 h after treatment
   with water and nystose. CW-vs-CN (24 h): rice roots that grew at 4°C
   for 24 h after treatment with water and nystose. NW-vs-NN (48h): rice
   roots that grew at 25°C for 48 h after treatment with water and
   nystose. CW-vs-CN (48 h): rice roots that grew at 4°C for 48 h after
   treatment with water and nystose. NW-vs-NN (R): rice roots that grew at
   25°C for 7 d after treatment with water and nystose. CW-vs-CN (R): rice
   roots that grew at 4°C for 2 d and then at 25°C for 5 d after treatment
   with water and nystose.

GO classification of differentially expressed proteins

   To explore the biological functions of the 497 identified DEPs, GO
   classification was conducted. The DEPs were assigned to 18 cell
   components, classified into 22 functional categories, and involved in
   27 biological processes ([100]S3 Fig). Specifically, the 248
   upregulated DEPs were assigned to 13 cell components, classified into
   14 functional categories, and involved in 18 biological processes
   ([101]S5 Fig), while the 249 downregulated DEPs were assigned to 10
   cell components, classified into 18 functional categories, and involved
   in 16 biological processes ([102]S4 Fig). Some DEPs were classified
   into 6 functional categories, which may be closely related to stress
   responses. These categories included abscisic acid binding, receptor
   activity, protein phosphatase inhibitor activity, FK506 binding, signal
   recognition particle binding, and 12-oxophytodienoate reductase
   activity. The DEPs were also involved in the regulation of protein
   serine/threonine phosphatase activity, chaperone-mediated protein
   folding, and oxylipin biosynthesis ([103]S3A and S3F Fig). Some of the
   upregulated DEPs were within functional categories of abscisic acid
   binding, receptor activity, protein phosphatase inhibitor activity,
   superoxide dismutase activity, peroxidase activity, and kinase
   activity. Upregulated DEPS were also implicated in the regulation of
   serine/threonine protein phosphatase activity, abscisic acid-activated
   signaling, removal of superoxide radicals, response to oxidative
   stress, and defense responses to bacteria ([104]S5A, S5C and S5D Fig).
   The downregulated DEPs belonged to 3 functional categories: glutathione
   transferase activity, signal recognition particle binding, and
   12-oxophytodienoate reductase activity. Additionally, downregulated
   DEPs were involved in glutathione metabolism, type I hypersensitivity,
   and defense responses to fungi ([105]S6A and S6F Fig). It is generally
   accepted that root function is closely related to dehydrogenase
   activity [[106]44, [107]45]. In this regard, we have identified DEPs
   with dehydrogenase activity ([108]S3E and S3F Fig), which are likely to
   be responsible for the higher activity of the root after the treatment
   with nystose.

Pathway analysis of the differentially expressed proteins

   To identify potential target genes of the 497 DEPs detected in primary
   roots, pathway analysis was performed for all 248 upregulated and 249
   downregulated proteins. The 497 DEPs are involved in 16 distinct
   metabolic pathways: glutathione metabolism, ribosome, carbon
   metabolism, valine, leucine, and isoleucine degradation, citrate cycle
   (TCA cycle), synthesis of antibiotics, pyruvate metabolism, RNA
   transport, glycolysis/gluconeogenesis, galactose metabolism, starch and
   sucrose metabolism, pentose and glucuronate interconversions, ascorbate
   and aldarate metabolism, synthesis of amino acids, metabolic pathways,
   and the biosynthesis of secondary metabolites ([109]S4 Fig).
   Upregulated and downregulated DEPs were involved in 11 and 14 distinct
   metabolic pathways ([110]S7 & [111]S8 Figs), respectively. Of these
   pathways, 7 were shared by up- and downregulated DEPs: synthesis of
   antibiotics, pyruvate metabolism, carbon metabolism,
   glycolysis/gluconeogenesis, ribosome, carbon fixation in photosynthetic
   organisms, the biosynthesis of secondary metabolites. Importantly, the
   glutathione and galactose metabolism associated with upregulated DEPs
   ([112]S7A and S7C Fig) and the ascorbate, aldarate, and alpha-linolenic
   acid metabolism associated with downregulated DEPs ([113]S8E Fig) is
   closely related to stress responses.

Validation of differentially expressed candidate proteins

   To validate the proteome datasets and the patterns of the DEPs revealed
   by label-free quantification ([114]S1–[115]S3 Files), Western blot
   analysis was performed to document the changes in expression of 7 DEPs,
   including 4 upregulated and 3 downregulated DEPs ([116]Fig 3, [117]S2 &
   [118]S3 Files). This experiment demonstrated that 4 proteins in the
   roots of the nystose-treated group, [119]Q7FAE1 (momilactone A
   synthase), [120]Q75T45 (RSOsPR10), [121]P17654 (alpha-amylase), and
   [122]Q6K9Q5 (serine/threonine-protein phosphatase), had a higher level
   of expression than in the untreated control group after 24 h and 48 h
   of cold stress and growth recovery, respectively ([123]Fig 3A and 3B).
   Three DEPs downregulated in the roots of the nystose-treated group,
   [124]Q69RL1 (GST), [125]Q6YUR8 (CSP1), and [126]Q6ESR4 (DHN1), had a
   lower level of expression than in the untreated control group at 24 h
   of cold stress ([127]Fig 3B). These results are consistent with the
   quantification data of label-free proteomics in primary rice roots from
   both groups.

Fig 3. Validation of DEPs.

   [128]Fig 3
   [129]Open in a new tab

   (A) Comparison of fold-change of DEPs obtained by label-free
   quantification and Western blot analysis. The values on the y-axis
   indicate fold-change. (B) Western blot analysis of DEPs showing four
   upregulated proteins, including [130]Q7FAE1 (momilactone A synthase),
   [131]Q75T45 (RSOsPR10),[132]P17654 (alpha-amylase) and [133]Q6K9Q5
   (serine/threonine-protein phosphatase), and three downregulated
   proteins, including [134]Q69RL1 (GST), [135]Q6YUR8 (CSP1) and
   [136]Q6ESR4 (DHN1). Four DEPs, including [137]Q7FAE1, [138]Q75T45,
   [139]Q69RL1, and [140]Q6YUR8, of rice roots grown at 25°C for 24 h and
   48 h after the treatment with water and nystose. Three DEPs, including
   [141]P17654, [142]Q6K9Q5, and [143]Q6ESR4, of rice roots grown at 25°C
   for 7 d and 4°C for 2 d and then at 25°C for 5 d after the treatment
   with water and nystose. Normal (24 h) represents rice roots grown at
   25°C for 24 h after the treatment with water and nystose. Normal (48 h)
   represents rice roots grown at 25°C for 24 h after the treatment with
   water and nystose. Normal (R) represents rice roots grown at 25°C for 7
   d after the treatment with water and nystose. Cold (R) represents rice
   roots grown at 4°C for 2 d and then at 25°C for 5 d after the treatment
   with water and nystose. “+” indicates soaking of the seeds in 75 mg/L
   nystose, “-” indicates soaking of the seeds in water.

Discussion

   Rice is a cold-sensitive cereal crop. When exposed to low temperatures,
   rice plants suffer from chilling injury, which leads to growth
   inhibition and the browning of leaves and roots, decreasing grain yield
   and quality [[144]46–[145]48]. The present study identified the
   browning phenotype in primary roots of untreated rice grown at 4°C for
   2 d and allowed to recover at 25°C for 1 d, 3 d, and 5 d. The browning
   indicated chilling injury, growth inhibition, and decreased root
   activity. The majority of primary root tips in the nystose-treated
   group exhibited normal white phenotype and higher activity, indicating
   that nystose protects against chilling injury. Furthermore, a primary
   root-based stress tolerance-induced system was developed. The system
   has the advantages of simple operation, clear phenotypic
   identification, and the lack of interference from housekeeping proteins
   in isolation and identification, providing an effective tool for future
   proteome-level research on the mechanisms underlying the impact of
   nystose on the regulation of the responses of rice roots to cold
   stress.

   The possibility has been raised that fructans act as important
   immunostimulators [[146]49, [147]50]. In the current investigation, 4
   antioxidant enzymes, including one Cu-Zn superoxide dismutase
   ([148]P93407) and three peroxidases (A0A0P0X9R7,[149]Q5U1P7,
   [150]Q5U1Q2) showed higher levels in nystose-treated primary roots
   grown at 25°C and 4°C for 48h than in untreated plants ([151]S1 File).
   It is generally accepted that these antioxidant enzymes are directly
   involved in the scavenging of excess free radicals in primary rice
   roots, and may contribute to their protection against browning under
   cold stress. Fructans enhance membrane stability at low temperatures by
   interacting with membrane lipids [[152]10, [153]13–[154]17] and
   scavenging hydroxyl radicals (·OH) generated by the activity of
   tonoplast-associated class III peroxidase [[155]18, [156]19].
   Therefore, nystose is likely to participate in antioxidant mechanisms
   in primary rice roots, protecting them from browning under cold stress.

   Interestingly, we have identified 5 upregulated DEPs, including
   [157]Q10MP7 (PR10c), [158]Q2QNS7 (PR10a), [159]Q75T45 (RSOsPR10),
   [160]Q941F5 (PR4), and [161]Q84J76 (putative pathogenesis-related
   protein) ([162]S1 File). These DEPs are likely to be involved in immune
   and stress responses. The expression of RSOsPR10, a root-specific
   pathogenesis-related (PR) protein, is rapidly induced in roots by salt,
   drought stress, and blast fungus infection, possibly by the activation
   of the JA signaling [[163]51]. Also, RSOsPR10 is strongly induced by JA
   and the ethylene (ET) precursor 1-aminocyclopropane-1-carboxylic acid
   (ACC), while salicylic acid (SA) almost completely suppresses these
   effects [[164]52]. Similarly, OsERF1, a transcription factor in the
   JA/ET pathway, is regulated by JA, ACC, and SA [[165]50], but is
   induced before RSOsPR10 upon the treatment with JA and ACC [[166]52].
   Therefore, it was proposed that the environmental stress-induced
   changes in RSOsPR10 expression in rice roots are antagonistically
   regulated by JA/ET and SA [[167]52]. The present work documented that
   under normal conditions or cold stress, the expression of RSOsPR10 was
   strongly upregulated by nystose and JA, but was significantly inhibited
   by SA ([168]Fig 4). Under normal conditions, no significant differences
   in RSOsPR10 expression were observed between rice roots treated with
   water or ABA, while under cold stress, RSOsPR10 level was significantly
   lower in ABA-treated roots than in the controls ([169]Fig 4). Indeed,
   the integration of our datasets was in agreement with previous findings
   that fructans act as essential immunostimulators involved in universal
   antioxidant mechanisms in plants. Recently, RSOsPR10 was shown to
   mediate environmental stress tolerance by increasing root growth and
   development [[170]53]. Here, we have demonstrated that nystose
   increases RSOsPR10 level and promotes the growth of primary rice roots
   under cold stress. Together, these data suggest that nystose improves
   the tolerance of primary rice roots to low temperatures by upregulating
   the expression of RSOsPR10. In addition, RSOsPR10 expression is induced
   by cold stress, contrasting the previous report that RSOsPR10mRNA does
   not accumulate after the exposure to low temperature [[171]51].
   However, inconsistencies in the correlation between gene transcription
   and protein translation can occur, and that report [[172]51] did not
   evaluate the expression pattern of RSOsPR10 protein under cold stress.

Fig 4. Western blot analysis of RSOsPR10 expression in response to nystose,
JA, SA, and ABA.

   [173]Fig 4
   [174]Open in a new tab

   CK: rice roots grown at 25°C for 2 d after the treatment. Cold: rice
   roots grown at 4°C for 2 d after the treatment. “-“: seeds treated with
   water; “+”: seeds treated with 2.5 mg/L JA, 5 mg/L SA, 2.5 mg/L ABA, or
   75mg/L nystose.

   Two upregulated DEPs involved in hormonal signaling, [175]Q5Z8S0
   (abscisic acid receptor PYL9) and [176]Q10AI2 (SnRK1-interacting
   protein 1) were identified ([177]S1 File). These proteins are likely
   functioning as components of ABA signaling pathways involved in stress
   responses. Interestingly, nystose-treated primary rice roots had a
   higher ABA level under cold stress than untreated roots ([178]S9 Fig),
   indicating that nystose promotes ABA biosynthesis. Therefore, it can be
   concluded that nystose regulates the response of rice primary roots to
   cold stress by ABA-dependent signaling ([179]Fig 5).

Fig 5. Flow chart of the mechanisms underlying the improvement in cold
tolerance of primary rice roots by nystose.

   [180]Fig 5
   [181]Open in a new tab

   In addition to hormonal signaling, the MAPK cascade may be involved in
   mediating the effects of nystose. MAPK cascade comprises modules that
   transduce extracellular signals into a variety of cellular responses.
   Plant MAPK cascades have been implicated in the development and stress
   responses. We have identified an upregulated protein [182]Q6K4Q0 that
   was confirmed as the mitogen-activated protein kinase 4 (OsMKK4).
   OsMKK4 is strongly regulated by cold and salt stress [[183]54]. Thus,
   OsMKK4-dependent MAPK signaling cascades may be implicated in the
   nystose-induced improvement of cold tolerance by primary rice roots
   ([184]Fig 5).

Conclusions

   This study integrates the results of morphologic, physiologic, and
   comparative proteomics analyses of primary rice roots to address the
   molecular mechanism by which nystose improves plant tolerance to cold
   stress. Nystose promotes the growth of primary rice roots, possibly by
   upregulating the expression of RSOsPR10. In turn, the changes in
   RSOsPR10 level associated with the response of rice roots to cold
   stress are probably regulated by the JA, SA, and ABA signaling
   pathways. Furthermore, OsMKK4-dependent MAPK signaling cascades may be
   involved in the nystose-induced tolerance of rice roots to the cold.
   Therefore, our findings indicate that nystose acts as an
   immunostimulator of the response to cold stress by multiple signaling
   pathways.

Supporting information

   S1 Fig. Phenotypes of rice roots under the condition of normal growth.

   Phenotypes of rice primary roots from soaking seeds in water after
   growing at 25°C for 3 d (a), 5 d (c) and 7 d (e). Phenotypes of rice
   primary roots from soaking seeds in 75 mg/L nystose after growing at
   25°C for 3 d (b), 5 d (d) and 7 d (f).

   (TIF)
   [185]Click here for additional data file.^ (9.1MB, tif)
   S2 Fig. Protein expression profiles of rice roots treated with water
   and nystose under normal growth and cold stress conditions.

   Number of DEPs (P<0.05).

   (TIF)
   [186]Click here for additional data file.^ (320.9KB, tif)
   S3 Fig. GO classification of the DEPs of rice roots treated with water
   and nystose grown under normal and cold stress conditions.

   (A) NW-vs-NN (24h), rice roots grown at 25°C for 24 h after water and
   nystose treatments. (B) CW-vs-CN (24h), rice roots grown at 4°C for 24
   h after water and nystose treatments. (C) NW-vs-NN (48 h), rice roots
   grown at 25°C for 48 h after water and nystose treatments. (D) CW-vs-CN
   (48 h), rice roots grown at 4°C for 48 h after water and nystose
   treatments. (E) NW-vs-NN (recovery), rice roots grown at 25°C for 7 d
   after water and nystose treatments. (F) CW-vs-CN (recovery), rice roots
   grown at 4°C for 2 d and then at 25°C for 5 d after water and nystose
   treatments.

   (TIF)
   [187]Click here for additional data file.^ (2.5MB, tif)
   S4 Fig. KEGG pathway enrichment analysis based on DEPs of control and
   nystose-treated rice roots grown under normal and cold stress
   conditions.

   (A) NW-vs-NN (24h), water- or nystose-treated rice roots grown at 25°C
   for 24 h. (B) CW-vs-CN (24h), water- or nystose-treated rice roots
   grown at 4°C for 24 h. (C) NW-vs-NN (48h), water- or nystose-treated
   rice roots grown at 25°C for 48 h. (D) CW-vs-CN (48h), water- or
   nystose-treated rice roots grown at 4°C for 48 h. (E) NW-vs-NN
   (recovery), water- or nystose-treated rice roots grown at 25°C for 7 d.
   (F) CW-vs-CN (recovery), water- or nystose-treated rice roots grown at
   4°C for 2 d and then at 25°C for 5 d.

   (TIF)
   [188]Click here for additional data file.^ (2.7MB, tif)
   S5 Fig. GO classification of up-regulated DEPs of rice roots treated
   with water and nystose under normal and cold stress conditions.

   (A) NW-vs-NN (24 h), rice roots that grew at 25°C for 24 h after
   treatment with water and nystose. (B) NW-vs-NN (48 h), rice roots that
   grew at 25°C for 48 h after treatment with water and nystose. (C)
   CW-vs-CN (48h), rice roots that grew at 4°C for 48 h after treatment
   with water and nystose. (D) NW-vs-NN (recovery), rice roots that grew
   at 25°C for 7 d after treatment with water and nystose. (E) CW-vs-CN
   (recovery), rice roots that grew at 4°C for 2 d and then at 25°C for 5
   d after treatment with water and nystose.

   (TIF)
   [189]Click here for additional data file.^ (2.2MB, tif)
   S6 Fig. GO classification of down-regulated DEPs in rice roots treated
   with water and nystose under normal and cold stress conditions.

   (A) NW-vs-NN (24 h), rice roots that grew at 25°C for 24 h after
   treatment with water and nystose. (B) CW-vs-CN (24 h), rice roots that
   grew at 4°C for 24 h after treatment with water and nystose. (C)
   NW-vs-NN (48 h), rice roots that grew at 25°C for 48 h after treatment
   with water and nystose. (D) CW-vs-CN (48 h), rice roots that grew at
   4°C for 48 h after treatment with water and nystose. (E) NW-vs-NN
   (recovery), rice roots that grew at 25°C for 7 d after treatment with
   water and nystose. (F) CW-vs-CN (recovery), rice roots that grew at 4°C
   for 2 d and then at 25°C for 5 d after treatment with water and
   nystose.

   (TIF)
   [190]Click here for additional data file.^ (2.3MB, tif)
   S7 Fig. Pathway enrichment analysis based on the upregulated DEPs of
   rice roots treated with water and nystose under the condition of normal
   growth and cold stress.

   (A) NW-vs-NN (24h), rice roots that grew at 25°C for 24 h after
   treatment with water and nystose. (B) NW-vs-NN (48h), rice roots that
   grew at 25°C for 48 h after treatment with water and nystose. (C)
   CW-vs-CN (48h), rice roots that grew at 4°C for 48 h after treatment
   with water and nystose.

   (TIF)
   [191]Click here for additional data file.^ (1.2MB, tif)
   S8 Fig. Pathway enrichment analysis based on the downregulated DEPs of
   rice roots treated with water and nystose under normal and cold stress
   conditions.

   (A) NW-vs-NN (24h), rice roots that grew at 25°C for 24 h after
   treatment with water and nystose. (B) NW-vs-NN (48h), rice roots that
   grew at 25°C for 48 h after treatment with water and nystose. (C)
   CW-vs-CN (48h), rice roots that grew at 4°C for 48 h after treatment
   with water and nystose. (D) NW-vs-NN (recovery), rice roots that grew
   at 25°C for 7 d after treatment with water and nystose. (E) CW-vs-CN
   (recovery), rice roots that grew at 4°C for 2 d and then at 25°C for 5
   d after treatment with water and nystose.

   (TIF)
   [192]Click here for additional data file.^ (2.2MB, tif)
   S9 Fig. ABA content in rice primary roots grown at 25°C and 4°C for 48
   h after treatment with water and nystose.

   HPLC-MS/MS was used for the analysis of ABA in rice roots. Experiments
   were repeated on at least three occasions. Data are the mean ± SE (n =
   3). Different lower case letters on the top of each of the bars
   indicate significant differences (P<0.05, two-way ANOVA followed by the
   Tukey test).

   (TIF)
   [193]Click here for additional data file.^ (8.8MB, tif)
   S1 File. DEPs following the analysis of Perseus software.

   NW-vs-NN (24 h): rice roots that grew at 25°C for 24 h after treatment
   with water and nystose. CW-vs-CN (24h): rice roots that grew at 4°C for
   24 h after treatment with water and nystose. NW-vs-NN (48h): rice roots
   that grew at 25°C for 48 h after treatment with water and nystose.
   CW-vs-CN (48h): rice roots that grew at 4°C for 48 h after treatment
   with water and nystose. NW-vs-NN (recovery): rice roots that grew at
   25°C for 7 d after treatment with water and nystose. CW-vs-CN
   (recovery): rice roots that grew at 4°C for 2 d and then at 25°C for 5
   d after treatment with water and nystose.

   (XLSX)
   [194]Click here for additional data file.^ (3.3MB, xlsx)
   S2 File. List of upregulated proteins (P<0.05, fold change>1.5).

   NW-vs-NN (24h): rice roots that grew at 25°C for 24 h after treatment
   with water and nystose. CW-vs-CN (24h): rice roots that grew at 4°C for
   24 h after treatment with water and nystose. NW-vs-NN (48h): rice roots
   that grew at 25°C for 48 h after treatment with water and nystose.
   CW-vs-CN (48h): rice roots that grew at 4°C for 48 h after treatment
   with water and nystose. NW-vs-NN (recovery): rice roots that grew at
   25°C for 7 d after treatment with water and nystose. CW-vs-CN
   (recovery): rice roots that grew at 4°C for 2 d and then at 25°C for 5
   d after treatment with water and nystose.

   (XLSX)
   [195]Click here for additional data file.^ (32.2KB, xlsx)
   S3 File. Lists of down-regulated proteins (P<0.05, fold change<2/3).

   NW-vs-NN (24h): rice roots that grew at 25°C for 24 h after treatment
   with water and nystose. CW-vs-CN (24h): rice roots that grew at 4°C for
   24 h after treatment with water and nystose. NW-vs-NN (48h): rice roots
   that grew at 25°C for 48 h after treatment with water and nystose.
   CW-vs-CN (48h): rice roots that grew at 4°C for 48 h after treatment
   with water and nystose. NW-vs-NN (recovery): rice roots that grew at
   25°C for 7 d after treatment with water and nystose. CW-vs-CN
   (recovery): rice roots that grew at 4°C for 2 d and then at 25°C for 5
   d after treatment with water and nystose.

   (XLSX)
   [196]Click here for additional data file.^ (31.4KB, xlsx)
   S4 File. Matched protein datasets following the analysis of MaxQuant
   software.

   NW-NN-CW-CN (24 h): rice roots that grew at 25°C and 4°C for 24 h after
   treatment with water and nystose. NW-NN-CW-CN (48 h): rice roots that
   grew at 25°C and 4°C for 48 h after treatment with water and nystose.
   NW-NN-CW-CN (recovery): rice roots that grew at 25°C for 7 d and 4°C
   for 2 d and then at 25°C for 5 d after treatment with water and
   nystose.

   (XLSX)
   [197]Click here for additional data file.^ (9.9MB, xlsx)
   S5 File. List of peptides for antibody preparation.

   (XLSX)
   [198]Click here for additional data file.^ (12.5KB, xlsx)

Data Availability

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

Funding Statement

   This work was sponsored by National Key R&D Program of China
   (No.2017YFD0200900) grant to DQ and SR, Active Design Project of
   Hangzhou Agricultural and Social Development Scientific Research (No.
   20172015A02) grant to WX, Project 454 of Hangzhou Great Science and
   Technology innovation (No. 20131812A02) grant to SR, Science and
   Technology Innovation and Demonstration extension Fund of Hangzhou
   Academy of Agricultural Sciences (No.2019HNCT-28) grant to SR. We also
   received funding from the Ministry of Science and Technology, Hangzhou
   Science and Technology Bureau and Hangzhou Academy of Agricultural
   Sciences.

References