Abstract

   graphic file with name ao2c07258_0010.jpg

   Clematis ternifloraDC. (C. terniflora) has been used as an ancient
   Chinese traditional herbal medicine. The active substances in C.
   terniflora have been confirmed to be effective in treating diseases
   such as prostatitis. UV light radiation is a common environmental
   factor that damages plants and influences primary and secondary
   metabolism. Previous studies showed that ultraviolet B (UV-B) radiation
   followed by dark stress resulted in the accumulation of secondary
   metabolites in C. terniflora leaves. An in-depth understanding of how
   C. terniflora leaves respond to UV-B stress is crucial for improving C.
   terniflora value. Here, we conducted label-free proteomic and
   phosphoproteomic analyses to explore the protein changes under UV-B and
   UV-B combined with dark treatment. A total of 2839 proteins and 1638
   phosphorylated proteins were identified. Integrative omics revealed
   that the photosynthetic system and carbohydrate balance were modulated
   under both stresses. The phosphoproteomic data indicated that the
   mitogen-activated protein kinase signaling pathway was triggered, while
   the abundance of phosphorylated proteins related to osmotic stress was
   increased under UV-B stress. Differentially abundant phosphoproteins
   from UV-B followed by dark treatment were mainly enriched in response
   to stimulus including calcium-mediated proteins. This study provides
   new insight into the impact of UV-B stress on C. terniflora and plant
   molecular resistance mechanisms through proteomic and phosphoproteomic
   analyses.

1. Introduction

   Clematis ternifloraDC. (C. terniflora) is a kind of woody vine of
   Clematis L. that is mainly distributed in China, Japan, and Europe. It
   is widely used as an ornamental plant and has high application value in
   horticulture. In addition, C. terniflora has been famous as folk
   medicine for a long time in China, and its stems, leaves, and roots
   contain a variety of active ingredients such as coumarins, flavonoids,
   and alkaloids.^[36]1−[37]3 It has been used to treat rheumatoid
   arthritis, chronic pharyngitis, and inflammatory symptoms in the
   respiratory and urinary systems. Flavonoids extracted from the aerial
   part of C. terniflora are reported to be effective in treating
   prostatitis.^[38]4 The results of pharmacological experiments showed
   that tumor growth can be inhibited by the active ingredient isolated
   from C. terniflora.([39]5)

   For decades, researchers have been devoted to discovering the
   influential mechanism of ultraviolet B (UV-B) on plants. UV-B radiation
   usually causes damage to DNA, proteins, and plant morphological
   features. One of its major negative influences is triggering the
   accumulation of reactive oxygen species (ROS) in cells. UV-B radiation
   increased the abundance of antioxidases, such as superoxide dismutase
   in soybeans.^[40]6 In Catharanthus roseus, the calcium-related pathway
   and ROS scavenging system were activated under UV-B stress.^[41]7
   Moreover, UV-B stress also has the potential to increase the secondary
   metabolite contents, which can be used by plants for stress
   resistance.^[42]8 Berberine, jateorhizine, palmatine, and columbamine
   were increased when Mahonia bealei was exposed to UV-B radiation
   combined with dark treatment.^[43]9 It has been reported that the
   concentration of kaempferol increases after UV-B radiation in
   Arabidopsis thaliana.([44]10) UV-B followed by dark treatment also
   increased the contents of coumarins in C. terniflora according to a
   previous study,^[45]11 and the ATP content increased to provide energy
   for secondary metabolism.^[46]3 Therefore, exposure to UV-B stress
   might contribute to the increase in active ingredients, thereby
   elevating the quality of folk medicine.^[47]12 However, the induction
   and response mechanism of C. terniflora is not fully understood.

   Protein phosphorylation is catalyzed by protein kinases, which transfer
   the phosphate group of ATP/GTP to the protein amino acid
   residue.^[48]13 This posttranslational modification (PTM) is one of the
   most well-studied protein modifications. Protein phosphorylation is
   involved in many signal recognition and transduction pathways in
   plants. In Arabidopsis thaliana, the phosphorylation of tyrosine
   regulates the oxidative stress pathway in response to drought and salt
   stress.^[49]14 The mitogen-activated protein kinase (MAPK) signaling
   pathway transfers signals to downstream molecules through
   phosphorylation, and it can be activated by ROS, calcium (Ca^2+), and
   phytohormones. Proteomics combined with phosphoproteomics has become a
   major way to investigate plant resistance mechanisms under biotic and
   abiotic stress. For example, multiomics studies have explored the
   Spongospora subterranea resistance mechanisms of potato and identified
   that protein phosphorylation plays a role in the regulation of the
   immune response.^[50]15 Integrative proteomic and phosphoproteomic
   analyses showed that the early cold response in maize was associated
   with photosynthetic light reactions and the spliceosome.^[51]16
   Nevertheless, no research has combined proteomic and phosphoproteomic
   to study the molecular mechanism controlling changes in C. terniflora
   under UV-B stress.

   Phosphorylation may help C. terniflora respond to abiotic stress
   through a regulation mechanism. In this study, we integrated proteomic
   and phosphoproteomic analyses to further expand the understanding of C.
   terniflora leaves in response to UV-B treatment. The data of this study
   revealed the differentially abundant proteins and phosphoproteins
   between UV-B treated and untreated plants, which will help to infer the
   phosphorylation regulation mechanism. The results of this study provide
   new insights into the signal transduction of C. terniflora abiotic
   responses.

2. Materials and Methods

2.1. Plant Growth and Treatment

   C. terniflora plants were obtained from a greenhouse and grown at 25 °C
   under white light. Leaves of different groups, control (CK), 5 h
   ultraviolet B treatment (UVB), and 5 h ultraviolet B followed by 48 h
   dark treatment (UVD), were collected as reported previously.^[52]3 For
   proteomics analysis, three independent biological replicates were
   performed per treatment.

2.2. Analysis of Total Soluble Sugar Contents, Total Antioxidant Capacity
(T-AOC), and Chlorophyll Contents

   The total soluble sugar contents of CK, UVB, and UVD were measured with
   a plant soluble sugar content test kit (Solarbio, Beijing, China)
   according to the instructions. The absorbance of each sample was
   measured at 620 nm, and the content of soluble sugar was calculated on
   the basis of a standard curve. The T-AOC and chlorophyll content of
   each group were determined colorimetrically by a total antioxidant
   capacity assay kit (Nanjing Jiancheng Bioengineering Institute,
   Nanjing, China) and chlorophyll assay kit (Nanjing Jiancheng
   Bioengineering Institute). Chlorophyll content determination was
   performed with 0.1 g leaf samples. The absorbance values at OD[645] and
   OD[663] were measured. The calculation formula are as follows: (1)
   chlorophyll a = (12.7 × OD[663] – 2.69 × OD[645]) × volume × dilution
   rate/(sample weight × 1000). (2) Chlorophyll b = (22.9 × A645 – 4.68 ×
   A663) × volume×dilution rate/(sample weight × 1000). (3) Total
   chlorophyll content = chlorophyll a content + chlorophyll b content.
   T-AOC content measurement was performed after incubating the sample and
   mix buffer at 37 °C for 30 min. At least three independent biological
   replicates were performed per treatment.

2.3. Protein Sample Preparation and Phosphopeptide Enrichment

   Frozen leaves ground into powder were transferred to extraction
   solution (100 mM KCl, phosphatase inhibitor cocktails (Roche
   Diagnostics, NSW, Australia), 5 mM EDTA, 1% (w/v) DTT, 1 mM PMSF, 30%
   sucrose, and 50 mM Tris–HCl at pH 8.5). Tris-buffer phenol was added to
   the mixture, and the mixture was centrifuged at 6000 × g. Five volumes
   of 100 mM ammonium acetate were added to the protein supernatant and
   precipitated at −20 °C over 12 h. After centrifugation at 6000 × g for
   10 min, the precipitate was collected and then washed twice with
   acetone. The precipitate was dissolved in buffer containing 7 M urea, 2
   M thiourea, 4% CHAPS, 2 mM EDTA, 10 mM DTT, and 1 mM PMSF. The
   supernatant was collected after centrifugation as crude protein. The
   Bradford method was used for protein concentration determination with
   minor modifications. Protein purification, reduction, and alkylation
   were performed according to previous study.^[53]17 In brief, protein
   was purified by methanol and chloroform and then dissolved in
   NH[4]HCO[3]. Finally, trypsin was added at a 1:50 (trypsin-to-protein)
   ratio, and the sample was incubated at 37 °C for 16 h in the dark to
   digest protein and then desalted with a Strata X column (Phenomenex,
   United States) to obtain total peptide samples and phosphopeptide
   enrichment prior samples. The TiO[2] phosphopeptide enrichment kit (PTM
   BioLab, Hangzhou, China) was used to enrich the peptide according to
   the manufacturer’s instructions.

2.4. LC–MS/MS Analysis

   Peptides were redissolved in solvent A (0.1% formic acid) and analyzed
   by an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific
   Inc., San Jose, CA, USA), using an Acclaim PepMap 100 C18 column (25 cm
   × 75 μm, 2 μm-C18, Thermo Fisher Scientific Inc.). The peptides were
   separated by a Thermo Easy-nLC 1000 system. The parameters and gradient
   setting were in accordance to Zhong et al.’s^[54]18 protocol with minor
   modification. The gradient started at 3% solvent B (acetonitrile), 3% B
   to 8% B for 1 min, 8% B to 45% B for 101 min, and 45% B to 95% B for 4
   min and finally held at 95% for 9 min. The flow rate was 300 nL/min,
   and the column temperature was 40 °C.

2.5. Data Processing and Bioinformatics Analysis

   Raw data files were searched using the SEQUEST HT search engine
   embedded into Proteome Discoverer version 2.4 (Thermo Scientific).
   Spectral libraries were generated by searching the Uniprot database
   (Raninculales, 233,260 entries, 2022.6.15) and C. terniflora DC.
   transcriptome database.^[55]19 Trypsin/P was configured as a cleavage
   enzyme (up to 2 missing cleavages), and the false discovery rate (FDR)
   was set at a 1% cutoff. For the phosphoproteomic search, phosphoRS mode
   was enabled, phospho (S/T/Y) was specified as an additional variable
   modification, and default settings were used for other modifications.
   Relative quantitation of total proteins or phosphoproteins was
   performed according to the respective peptides or phosphopeptides.

   The proteins with an adjusted p value of <0.05 and a fold change of
   >1.2 or <0.83 were considered as differentially abundant proteins
   (DAPs) with statistical significance. Differentially phosphorylated
   proteins (DPPs) were identified using the same screening method, and
   overlapping proteins with the same change trend between DAPs and DPPs
   were deleted. Functional annotations of DAPs or DPPs were performed by
   an eggNOG-mapper,^[56]20 based on Gene Ontology (GO). GO term
   enrichment was performed by TBtools software.^[57]21 DAPs and DPPs were
   submitted to the online KOBAS 3.0^[58]22 software for Kyoto
   Encyclopedia of Genes and Genomes (KEGG) pathway enrichment.
   Subcellular prediction was performed by WoLF PSORT
   ([59]https://wolfpsort.hgc.jp/).

2.6. Statistical Analysis

   Statistical differences between three groups were tested using one-way
   ANOVA together with Tukey’s test in SPSS (version 25). Student’s t test
   was used to evaluate statistical significance between two groups. Data
   are expressed as the means ± SDs.

3. Results

   To explore the roles of proteins and phosphorylated proteins in C.
   terniflora under UV-B stress, we collected UVB-treated and UVD-treated
   leaf proteins, and the proteomic and phosphoproteomic were profiles by
   using an LC–MS/MS approach.

3.1. Overview of C. terniflora Proteomics under UV Stress

   In the global proteome, a total of 2839 proteins were identified in the
   CK, UVB, and UVD groups. In comparison with leaves from the CK group,
   based on a p value of <0.05 and a fold change ratio of >1.2 or <0.83,
   leaves in UVB/CK had 272 DAPs (increased/decreased: 86/186), and leaves
   in UVD/CK presented 267 DAPs (increased/decreased: 116/151) ([60]Figure
   [61]1
   A,B). Noticeably, 122 DAPs were identified under both UVB and UVD
   stresses, while over half of the DAPs changed specifically under the
   two different stresses ([62]Figure [63]1C).

Figure 1.

   [64]Figure 1
   [65]Open in a new tab

   Differentially abundant proteins (DAPs) identified in response to UVB
   and UVD stresses. Volcano maps of DAPs in (A) UVB/CK and (B) UVD/CK.
   Red dots refer to increased DAPs, and blue dots refer to decreased
   DAPs. (C) Venn diagram shows the overlap of DAPs under two different
   treatments. Bar chart of significantly enriched KEGG pathways of DAPs
   in (D) UVB/CK and (E) UVD/CK. The x axis of (D) and (E) represents
   −log10 (p value) of KEGG enrichment significance.

   GO classification of the DAPs according to molecular function (MF) and
   biological process (BP) ontological terms was performed. The top 20 GO
   enrichment terms in each category are shown in [66]Figure S1A,B. Tissue
   development and cellular metabolic compound salvage were the top two BP
   categories in which UVB DAPs were enriched. DAPs in UVD were associated
   with the transport of organophosphate esters and organic anions and
   lipid oxidation-related categories. Both groups of DAPs were enriched
   in the response to the light/abiotic stimulus. In MF, UVB DAPs were
   more related to DNA binding and sequence-specific DNA binding, while
   transporter activity and protein binding were the most common terms in
   UVD DAP enrichment.

   Based on the KEGG pathway enrichment analysis of UVB DAPs, the most
   significantly enriched pathways were photosynthesis and
   photosynthesis–antenna proteins. Other enriched pathways were
   carbohydrate metabolism pathways such as glyoxylate and dicarboxylate
   metabolism, starch and sucrose metabolism, and
   glycolysis/gluconeogenesis ([67]Figure [68]1
   D). Except for energy metabolism pathways and carbohydrate metabolism
   pathways, some lipid metabolism pathways (such as alpha-linolenic acid
   metabolism, fatty acid degradation, and fatty acid metabolism) also
   ranked highly among the enriched pathways in UVD ([69]Figure [70]1E).
   Furthermore, we found that DAPs were enriched in the phenylpropanoid
   biosynthesis pathway in both UVB and UVD, which meant that secondary
   metabolism was affected. Overall, the enriched pathways were mainly
   associated with metabolism, especially energy metabolism and
   carbohydrate metabolism.

   Photosynthesis and photosynthesis–antenna proteins were the top two
   enriched pathways in UVB/CK and UVD/CK. Therefore, we selected DAPs
   mapped to these two pathways in response to UVB and UVD. [71]Figure
   [72]2
   illustrated that, compared to the control group, the abundance of most
   proteins in the photosynthesis system were decreased under stress.

Figure 2.

   [73]Figure 2
   [74]Open in a new tab

   Clustering heatmap of DAPs related to photosynthesis and
   photosynthesis–antenna protein pathways. (A) DAPs in UVB/CK. (B) DAPs
   in UVD/CK.

3.2. Phosphoproteomic Analysis of C. terniflora Leaves in Response to UV
Stress

   In the phosphoproteomic analysis, we detected 1638 phosphorylated
   proteins, of which 238 (134 increased and 104 decreased) proteins in
   UVB and 218 (83 increased and 135 decreased) proteins in UVD
   significantly changed in abundance compared to those in the control
   group. Among the 1638 phosphoproteins, 3752 phosphorylation sites were
   identified. The distribution of phosphorylation sites was 88.1% Ser,
   11.5% Thr, and 0.4% Tyr ([75]Figure [76]3
   A). In this study, 49.8% of phosphoproteins were due to single
   phosphorylation, 22.4% were due to double phosphorylation, and the rest
   had three or more phosphorylation sites ([77]Figure [78]3B).

Figure 3.

   [79]Figure 3
   [80]Open in a new tab

   Statistical information of phosphorylation sites and subcellular
   localization of differentially phosphorylated proteins. Distribution of
   (A) phosphorylated sites (serine, Ser; threonine, Thr; tyrosine, Tyr)
   in identified phosphoproteins. (B) Proportion of phosphorylation site
   numbers per identified phosphoprotein. The subcellular localization of
   DPPs of (C) UVB/CK and (D) UVD/CK predicted by WoLF PSORT.

   Subcellular localization prediction analysis showed that nearly 50% of
   the significantly changed phosphoproteins were located in the nucleus,
   followed by the chloroplast, cytosol, and plasma membrane. The
   distributions of the subcellular localization of phosphoproteins in UVB
   ([81]Figure [82]3
   C) and UVD ([83]Figure [84]3D) were similar. The results showed that
   most of DPPs were nuclear-related proteins, which indicated that
   phosphorylation of nuclear proteins played an essential role in the
   response to UV light stress.

   GO enrichment revealed that the major effects of UV-B at the
   phosphoproteomic level involved the hyperosmotic response, plant organ
   development, and response to oxygen-containing compound in the BP
   category; plant-type vacuole in the CC category; and active
   transmembrane transporter activity in the MF category ([85]Figure S2A).
   In contrast, the majority of significantly changed phosphoproteins of
   UVD were related to the response to stimulus/water deprivation and
   DNA-binding transcription factor activity ([86]Figure S2B).

   KEGG enrichment pathways of DPPs in UVB were not only associated with
   photosynthesis-related energy metabolism pathways but also related to
   transcription and signal transduction. Specifically, these KEGG
   pathways were spliceosome, MAPK signaling pathway–plant, and plant
   hormone signal transduction ([87]Figure [88]4
   A). The KEGG pathway enrichment results revealed that ten pathways were
   significantly enriched, including photosynthesis, carbon fixation in
   photosynthetic organisms, photosynthesis–antenna proteins, plant
   hormone signal transduction, ABC transporters, and so on ([89]Figure
   [90]4B).

Figure 4.

   [91]Figure 4
   [92]Open in a new tab

   Bubble plot of significantly enriched KEGG pathways of DPPs in (A)
   UVB/CK and (B) UVD/CK. The color of the bubble represents the −log10 (p
   value) of KEGG enrichment significance. The size of the bubble
   represents the number of DPPs.

3.3. Effects of UV-B Radiation on Chlorophyll, Soluble Sugar, and T-AOC
Levels

   To investigate the photosynthetic response to the UV-B stress on C.
   terniflora leaves, we measured the chlorophyll content ([93]Figure
   [94]5
   A). The results showed that UV-B radiation reduced the total
   chlorophyll content in C. terniflora leaves. Furthermore, the total
   chlorophyll content was lower in the UVD group than in the UVB group.
   Consistent with the change in total chlorophyll, chlorophyll a and
   chlorophyll b ([95]Figure [96]5B,C) were decreased in the UVB group and
   UVD group, respectively.

Figure 5.

   [97]Figure 5
   [98]Open in a new tab

   Levels of (A) total chlorophyll, (B) chlorophyll a, and (C) chlorophyll
   b of C. terniflora leaves from CK, UVB, and UVD groups. The different
   letters indicate significant differences according to one-way ANOVA.

   The plant soluble sugar content and T-AOC were determined using test
   kits. [99]Figure [100]6
   A showed that the content of soluble sugar rose significantly after UVB
   stress. However, the soluble sugar content declined in the UVD group.
   For the T-AOC content, the UVB group showed a significantly higher
   value than the other two groups ([101]Figure [102]6B).

Figure 6.

   [103]Figure 6
   [104]Open in a new tab

   (A) Soluble sugar content and (B) T-AOC content change in different
   groups of C. terniflora (CK, UVB, and UVD). Letters a, b, and c
   indicated that the content changed significantly according to one-way
   ANOVA.

4. Discussion

   In this study, we used proteomic and phosphoproteomic analyses to
   investigate the response mechanisms of C. terniflora response to UV-B
   light stress. Leaves were treated with UV-B and UV-D separately. Taken
   together, we identified DAPs and DPPs between the treated and control
   groups, highlighting the significance of the C. terniflora defense
   regulation system. The results showed how UV-B affected the signal
   transduction and metabolism systems.

4.1. Photosynthetic Response Was Inhibited in Response to UV-B Stress

   [105]Figures [106]1
   D,E and [107]4 reveal that photosynthesis and photosynthesis–antenna
   proteins were the top two ranked pathways in almost all KEGG enrichment
   results for the proteomic and phosphoproteomic analyses. Regarding the
   DAPs and DPPs related to the above two pathways, most proteins in the
   UVB group and UVD group were downregulated ([108]Figure [109]2, Tables
   S1–S4). The antenna protein is also called light-harvesting chlorophyll
   (LHC) a and b binding protein and has the ability to absorb light
   energy.^[110]23 Zhang et al. reported that high NH[4]^+ stress
   decreased the expression of LHC genes and resulted in a decrease in
   chlorophyll concentration in Myriophyllum aquaticum.([111]24)
   Consistent with previous research, we noticed a decline in the
   chlorophyll content after UV-B stress, and the content was even lower
   in the UVD-treated samples ([112]Figure [113]5A). Ferredoxin is
   involved in chlorophyll metabolism^[114]25 and is an important protein
   that participates in electron transport in photosynthesis together with
   ferredoxin NADP reductase (FNR). FNR accepts electrons from ferredoxin
   and generates NADPH at the end of the photosynthetic electron transfer
   chain.^[115]26 Owing to the decrease in chlorophyll accumulation, rice
   mutants of the ferredoxin gene FdC2 were pale green^[116]27 according
   to previous research. In a barley mutant with C-type ferredoxin gene
   disruption, the plant presented a chlorophyll deficient
   phenotype.^[117]28 High light stress in Arabidopsis thaliana showed
   that FNR abundance was related to PSI inactivation.^[118]29 The
   overexpression of FNR increased the tolerance to high light and
   oxidative stress. According to total proteomics data, the abundance
   levels of ferredoxin and ferredoxin NADP reductase (FNR) were decreased
   in response to the stresses^[119]30 ([120]Tables S1 and S2). In line
   with the above analysis, the photosynthesis system may undergo massive
   damage under UV-B stress in C. terniflora.

4.2. Carbohydrate Balance Was Modulated in Resistance UV-B Stress

   Carbohydrates are the direct product of photosynthesis, which provides
   plants with energy.^[121]31 Soluble sugars are required for growth, as
   well as maintenance of the osmotic homeostasis of cells.^[122]32
   However, excess soluble sugars decrease the level of the photosynthesis
   in Arabidopsis.([123]33) According to the data on total protein levels,
   the abundance of sucrose synthase (SUS) and sucrose phosphate synthase
   (SPS) was induced by UVB stress, and SUS together with sucrose
   phosphatase (SPP) was increased under UVD treatment ([124]Figure [125]7
   ). SPS is the key rate-limiting enzyme of sucrose synthesis and is
   closely associated with starch/sucrose levels in leaves. High SPS
   activity represents an increase in the distribution of C in sucrose and
   a decrease in starch formation.^[126]34 When plants exposed to
   environmental pressure, improving the abundance of SPS can promote the
   synthesis of sucrose, which contributes to stabilizing protein and
   membrane structure and function.^[127]35 The abundance of glucan
   phosphorylase (GP) was increased in the UVB group ([128]Table S1), and
   another starch metabolism related enzyme starch synthase (SS) was
   upregulated in the UVD group ([129]Table S2). The presence of GP leads
   to the phosphorolytic degradation of leaf starch^[130]36 forming
   glucose 1 phosphate. SS is one of the major enzymes mediating starch
   biosynthesis. Studies on Oryza sativa revealed that the decrease in SS
   gene expression was correlated with the decreased starch content under
   drought stress.^[131]37 Under UVB single stress, C. terniflora may
   regulate osmotic homeostasis or generate energy substances to resist
   light stress by accumulating soluble sugars. When plants face long-term
   or multiple stresses, soluble sugar levels usually decrease after an
   initial rise. Therefore, C. terniflora leaves might adjust their
   carbohydrate balance to reduce the stress impact. The results indicated
   that the changes in carbohydrates and related proteins in leaves were
   differentially affected by UV-B and UV-D.

Figure 7.

   [132]Figure 7
   [133]Open in a new tab

   Identified DAPs from UVD/CK and UVB/CK mapped to carbohydrate
   metabolism. The red ellipses represent increased DAPs. The blocks with
   the letter “B” inside indicate that the DAP comes from UVB/CK, and
   those with the letter “D” mean that the DAP comes from UVD/CK. G3P,
   glyceraldehyde 3 phosphate; G1P, glucose 1 phosphate; F6P, fructose
   6-phosphate; S6P, sucrose 6 phosphate; GP, glycogen phosphorylase; SS,
   starch synthase; SUS, sucrose synthase; SPS, sucrose phosphate
   synthase; and SPP, sucrose phosphatase.

4.3. MAPK Signaling Pathway and Activated Hyperosmotic Response Were
Triggered to Resist UV-B Radiation

   Protein phosphorylation is known to alter the activity of proteins in
   response to environmental stimuli, signal transduction, and
   metabolism.^[134]15,[135]38 Here, phosphoproteomic analysis of C.
   terniflora leaves showed that phosphorylated proteins were involved in
   the MAPK signaling pathway and were significantly enriched in the
   UVB-treated group ([136]Figure [137]4
   A). The MAPK pathway is a conserved signaling pathway in eukaryotes.
   UV-B and other abiotic factors may cause wounding on plants under
   environmental conditions.^[138]39 When wounding does occur, plants
   respond to this damage by triggering the MAPK signaling
   pathway.^[139]40 MAPKs have four clades, among which MPK3 and MPK6
   belong to clade A.^[140]40 The plant MAPK cascade influences the
   expression of downstream genes by passing stress signals generated
   during in abiotic stress.^[141]41,[142]42 We mapped DPPs to the MAPK
   signaling pathway according to the KEGG database and found that MKK4/5
   and MPK3/6 cascade proteins were increased in the UVB group according
   to phosphoproteomic data ([143]Figure [144]8, [145]Table S3). The
   MKK4/5 and MPK3/6 cascades control ethylene biosynthesis to resist
   wounding in Arabidopsis thaliana.([146]43) Consistent with this result,
   another MAPK module, the type-2C protein-phosphatase (PP2C) and
   snf1-related protein kinase2 (SnRK2) cascade, was also affected.
   Abscisic acid (ABA) enables PP2C sequestration and then triggers SnRK2
   activation, thereby inhibiting plant growth.^[147]44 Nevertheless, the
   abundances of PP2C and SnRK2 were both reduced under UVB treatment, and
   the level of SnRK2 declined further, which might reduce the inhibition
   of growth. In addition, the hyperosmotic response could be activated by
   the MAPK pathway in response to a variety of stimuli. Heat stress
   triggers MAPK crosstalk to activate the hyperosmotic response by
   related protein phosphorylation.^[148]45 Hyperosmotic response was the
   most significantly enriched GO term in the BP category for UVB DPPs
   ([149]Figure S2A). Thus, increased hyperosmotic response proteins might
   contribute to the resistance of leaves against osmotic stress triggered
   by UV-B stress ([150]Table S3). Additionally, abiotic stress is usually
   accompanied by oxidative stress. MAPK is associated with the regulation
   of antioxidant capacity. In tomato, the overproduction of H[2]O[2] and
   O^2– was reduced after using a MAPK inhibitor under salt
   stress.^[151]46 The content of T-AOC increased in the UVB stress group
   and decreased in the UVD stress group. Improvement of antioxidant
   capacity may be one of the ways to resist UV light stress. Meanwhile,
   combined stress might contribute to the negative effects on antioxidant
   capacity in C. terniflora.

Figure 8.

   Figure 8
   [152]Open in a new tab

   DPPs of UVB/CK mapped to the MAPK signaling pathway. The red blocks
   represent increased phosphorylated proteins, while the green blocks
   represent decreased phosphorylated protein.

4.4. Ca^2+-Related Proteins Undergo Phosphorylation/Dephosphorylation in
Response to UV-B Radiation Followed by Dark Treatment

   Through the phosphorylation/dephosphorylation of kinases, the
   phosphorylation/dephosphorylation processes of stimuli
   resistance-related proteins are activated to initiate the protection
   response. DPPs of UVD were mainly enriched in response to stimulus,
   especially response to water deprivation. Water deprivation is a common
   reaction in various abiotic stresses. Among the proteins that respond
   to water deprivation, we noticed that Ca^2+ signaling related
   phosphoproteins had undergone changes. Previous analysis^[153]47
   indicated that Ca^2+ is a crucial macronutrient for plant defense
   responses, such as temperature shifts, light, salinity, and
   metalloids.^[154]48−[155]50 The phosphoproteomic data confirmed that
   Ca^2+-transporting ATPase was increased by UVD treatment, while
   Ca^2+-dependent protein kinase (CDPK) and calcineurin B-like protein
   (CBL) were decreased ([156]Table S4). Ca^2+-transporting ATPase pumps
   Ca^2+ ions out of the cell and binds to calmodulin in a Ca^2+-dependent
   manner.^[157]51 CDPK and CBL are sensor proteins that reflect Ca^2+
   concentrations.^[158]52 The CBL family is a family of plant-specific
   serine/threonine kinases^[159]53 and represents a key group of Ca^2+
   transients.^[160]54 Abiotic stress elicits a cytosolic Ca^2+
   concentration increase in a short time, and then, Ca^2+ signatures are
   relayed by CBLs and CDPK.^[161]55 CBL is involved in regulating
   freezing tolerance in Solanum tuberosum.([162]56) These results
   suggested that UV-D stress resistance in C. terniflora leaves might be
   achieved by Ca^2+ protein phosphorylation/dephosphorylation to maintain
   cell Ca^2+ homeostasis.

5. Conclusions

   In this work, UV light stress damaged the photosynthetic system of C.
   terniflora leaves. Proteomics revealed that C. terniflora leaves
   adjusted carbohydrate balance in response to UVB and UVD treatment.
   Moreover, UVB stress activated the MAPK pathway by phosphorylating or
   dephosphorylating pivotal proteins and triggered a hyperosmotic
   response and antioxidation capability. Phosphoproteins related to Ca^2+
   signal regulation were changed significantly to resist UVD stress. In
   conclusion, this research provided a new reference to study the
   mechanism of C. terniflora abiotic stress resistance from
   phosphoproteomic combined with proteomic data.

Data Availability

   The datasets of this study can be found in the ProteomeXchange
   Consortium^[163]57 via the PRIDE^[164]58 partner repository with
   identifier PXD038136 .

Acknowledgments