Proteomic response of hybrid wild rice to cold stress at the seedling stage

Low temperature at the seedling stage is a major damaging factor for rice production in southern China. To better understand the cold response of cultivated and wild rice, cold-sensitive cultivar 93–11 (Oryza sativa L. ssp. Indica) and cold-resistant hybrid wild rice DC907 with a 93–11 genetic background were used for a quantitative proteomic analysis with tandem mass tags (TMT) in parallel. Rice seedlings grown for four weeks at a normal temperature (25°C) were treated at 8–10°C for 24, 72 and 120 h. The number of differentially expressed proteins increased gradually over time in the cold-exposed rice in comparison with the untreated rice. A total of 366 unique proteins involved in ATP synthesis, photosystem, reactive oxygen species, stress response, cell growth and integrity were identified as responding to cold stress in DC907. While both DC907 and 93–11 underwent similar alterations in proteomic profiles in response to cold stress, DC907 responded in a prompter manner in terms of expressing cold-responding proteins, maintained a higher level of photosynthesis to power the cells, and possessed a stable and higher level of DIR proteins to prevent the plant from obtaining irreversible cell structure damage. The observations made in this study may lay a new foundation for further investigation of cold sensitivity or tolerance mechanisms in rice.


Introduction
Low temperature is a major environmental stress affecting plant growth. Chilling stress causes water reduction and osmotic changes in the cellular milieu and suppresses the activities of cellular macromolecules, resulting in reduced growth and extensive losses in agricultural production [1]. Rice (Oryza sativa), a monocot plant and widely grown as food crop in tropical and subtropical areas, is particularly sensitive to cold stress at the seedling and flowering stages [2,3]. Molecular genetic studies have already identified components of cold tolerance, such as CTB4a, which confers cold resistance by mediating ATP supply [4], and the WRKY gene superfamily in rice [5]. COLD1, as one of the best-characterized rice genes, is considered as a regulator of G-protein signaling (RGS) that regulates Ca 2+ signaling in cells and confers chilling tolerance in rice [2,6]. The dehydrin gene OsDhn1 has been identified as being highly expressed in developing seeds under low temperatures and protects rice floral organs against abiotic stress [7]. qCTS-9, found in hybrid rice under different cold environments, was confirmed to be a functional gene associated with cold tolerance at rice seedling stage [8]. In a genome-wide association mapping of cold tolerance in cultivated rice from rice diversity panel 1 (RDP1), 87 cold tolerance-related quantitative trait loci (QTLs) with significant enrichment for genes related to lipid metabolism, response to stress and oxygen binding were identified [9,10].
Recently, proteomic technologies have been used to monitor and characterize protein profiles in rice [11]. For example, two-dimensional gel electrophoresis  and isobaric tags labelling approaches were used to monitor the proteomic response of rice to the cold treatment and proteins involved in energy metabolism, transport, photosynthesis, precursor metabolites generation, histones and vitamin B biosynthesis, which were found to be differentially expressed by cold stress [12][13][14]. However, to date, only a limited number of proteins in the cold-response pathway have been identified.
Early season rice in South China generally suffers from cold weather characterized by a temperature drop to approximately 10˚C or lower that results in seedling rot one in every three years in mid-to late March [15]. However, wild rice (Oryza rufipogon Griff.) in the same region survives the cold stress. Efforts to introduce the cold tolerance trait of wild rice into cultivar rice have been carried out by crossing cultivar rice with wild rice. To better understand the cold resistance mechanism of wild rice, a cold-tolerant hybrid wild rice DC907 with cultivar 93-11 genetic background and cold-sensitive 93-11 were investigated in parallel in this study by a comparative proteomics approach. Our results show that cold-tolerant DC907 was different from cold-sensitive 93-11 in its protein expression pattern. While a small portion of the differentially expressed proteins match those previously reported, a large proportion of cold stress-induced proteins were reported for the first time.

Rice growth and cold treatment conditions
Seeds of the indica rice cultivar 93-11 and hybrid wild rice DC907, derived from crossing of Guangxi wild rice (Oryza rufipogon Griff.) with 93-11 and sequential back cross with 93-11 for 4 rounds, were germinated in soil and grown in a phytotron with a 12-h day/night cycle, at 25˚C in the day and 18˚C at night. Seedlings at the four-leaf stage were subject to cold treatment at 10˚C in the day and 8˚C at night to simulate natural cold conditions for varied time durations. Seedlings were separated into groups for varied cold treatment conditions. Each group contained a total of 100 individual plants. After cold treatment for a fixed amount of time, plants were then transferred to an environment of 25˚C in the day and 18˚C at night for 5 days for survival rate determination, defined as the ratio of surviving plants to total plants. The light intensity was set at 30000 lux.

Preparation of protein samples for TMT analysis
Whole rice seedlings were ground into powder with liquid nitrogen and then five volumes of pre-cold acetone containing 10% trichloroacetic acid (TCA) and 0.07% β-mercaptoethanol was added. The mixture was kept at -20˚C overnight and centrifuged at 18,000 g for 30 min. The crude protein pellet was washed with pre-cold acetone containing 0.07% β-mercaptoethanol three times by centrifugation at 18,000 g. After vacuum drying, lysis buffer (7 M urea, 2% 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS), 40 mM Tris, 1 mM phenylmethanesulfonyl fluoride (PMSF)) was added to dissolve the protein pellet that was then centrifuged at 18,000 g for 30 min to remove debris. Five volumes of pre-chilled acetone was added to the supernatant and kept at -20˚C overnight. The mixture was centrifuged at 18,000 g for 30 min, and the pellet was dissolved in triethylamine borane (TEAB, 100 mM). The protein concentration was determined using the Bradford method [16].
An amount of 100 μg of protein was mixed with 5 μl of 200 mM tris (2-carboxyethyl) phosphine (TCEP) and incubated at 55˚C for 60 min. Then, 5 μl of 375 mM iodoacetamide was added and incubated for another 30 min in the dark. Six volumes of pre-cold acetone was added to precipitate proteins overnight. The protein mixture was centrifuged at 18,000 g for 30 min, and the pellet was dried at room temperature and dissolved in 100 μl of triethylamonium bicarbonat (TEAB). Trypsin solution (2.5 μg/100 μg protein) was used to digest protein samples at 37˚C overnight. The peptides were labeled with 41 μl of the TMTsixplex label reagent set (Thermo Fisher Scientific, CAT 90061) and incubated for 60 min at room temperature. Then, 8 μl of 5% hydroxylamine was used to quench the labeling reaction for 15 min, and the labeled peptides were stored at -80˚C.

Strong cation exchange chromatography
A PolyLC polysulfoethyl aspartamide column (100 mm X 2.1 mm, 5 μm, 300Å pore size) was used on a Waters high-performance liquid chromatography system (HPLC, Waters, series 2695) for off-line strong-cation exchange (SCX) chromatography fractionation. A 40 min gradient elution of 100% solvent A (10 mM monopotassium phosphate, 15% acetonitrile) to 100% solvent B (500 mM potassium chloride in solvent A) at 200 μl/min flow rate was performed. The SCX elution was monitored under a Waters 2998 PDA detector module (220 nm) and collected into 25 fractions for further mass spectrometry (MS) analysis.

Nanoflow LC-MS/MS analysis and data processing
The SCX fractions were loaded onto the trap column (nanoViper C18, 75 μm X 2 cm) and then eluted using capillary analytical column (nanoViper C18, 50 μm X 15 cm) at a 300 nl/ min flow rate using Easy-nLC 1000 nanoflow liquid chromatography system (Thermo Fisher Scientific). The linear gradient for peptide elution was from 95% solvent A (0.1% formic acid) and 5% solvent B (0.1% formic acid, 98% acetonitrile) to 40% solvent B for a 60 min program. The peptides from the untreated control and cold treated samples were labeled, mixed and fractionated by SCX chromatography and then analyzed using LTQ-Orbitrap Elite hybrid mass spectrometer system. Three biological replicates for each sample were performed.
The scan range of mass spectrometric analysis was set at 350-1800 m/z in a data-dependent mode. The survey scan was set at 400 m/z with a mass resolution of 60,000. Tandem mass spectrometry (MS/MS or MS2) was preceded with ten of the most intense precursor ions in the collision-induced dissociation (CID) mode with 35% normalized collision energy. MS2 spectrum was acquired in the ion trap analyzer at normal speed. The software Proteome Discoverer 1.3 was used to search the mass spectrometric data against rice genome database v7.0 (http://rice. plantbiology.msu.edu/). Search parameters were set as a standard method: 2 missed cleavages using trypsin as endoprotease, lysine residues as fixed modification, peptide N-termini as variable modification, 10 ppm precursor ion mass tolerance, 0.8 Da fragment mass tolerance, and 1% maximum false discovery rate (FDR). The identified proteins were filtered with high peptide confidence. Proteins with a 1.5-fold change (p<0.05) were considered to be differentially expressed.

Western blot analysis
A Western blot analysis was carried out according to a previous study [17]. Samples of 40 μg of total protein were loaded on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to polyvinylidene fluoride (PVDF) membranes using a semidry transfer unit after electrophoretic separation. Specific antibodies against ribulose bisphosphate carboxylase oxygenase (Rubisco) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Abcam (California, USA) and used to detect and verify the protein expression level in different samples by enhanced chemiluminescence (SuperSignal West Pico Substrate, Thermo Scientific).

Bioinformatics
Identified differentially expressed proteins were classified according to Gene Ontology (GO) and KEGG. The protein information from the Database of Rice Genome Annotation Project [18] was converted to corresponding access numbers of the UniProt protein database and classified using QuickGo online annotation tool (http://www.ebi.ac.uk/QuickGO/GMultiTerm) [19]. The protein network of differentially expressed proteins was performed using agriGO v2.0 [20].

Cold-response proteins and their time course
Cold stress that is harmful to the seedlings of early season rice in South China (mid-to late March) is typically at approximately 10˚C [15]. Thus, temperatures of 8-10˚C were selected for cold treatment in this study. Based on our previous observations that 93-11 primarily survived cold stress for 24 h but died completely after 120 h (survival rate was counted at day 5 after the stress was relieved) and wild rice survived at both conditions, we opted to use 24, 72, and 120 h as the times for proteomic analysis. As seen in Fig 1, the survival rates of 93-11 after cold stress for 24 h, 72 h, and 120 h were 90%, 20%, and 0%, respectively, while the survival rates of DC907 were 100% at all time points. A total of 1781 unique proteins were identified by TMT labeling from cold-stressed DC907 and 93-11 (S1 Table). By comparing protein abundance between the two accessions, 99 proteins (75 up-and 24 down-regulated) with changes greater than 1.5-fold were found after the first 24 h of cold treatment ( Table 1). As the cold treatment time was extended, the number of differentially expressed proteins slightly increased: 120 (37 up-and 83 down-regulated) at 72 h ( Table 2) and 105 (46 up-and 59 downregulated) at 120 h (Table 3). These proteins fall into different functional groups with obvious  time course characteristics; ATP synthesis, photosystems and other functional groups increased while DNA binding and transcription, cell growth and integrity, and structural protein decreased as the cold treatment time was extended (Fig 2A). The structural proteins, DNA binding proteins, and transcriptional factors were among the most differentially expressed at the time point of 24 h, and the most significant changes found at 72 h were the proteins involved in stress response. Accumulations of protein related to photosynthesis and ATP synthesis were affected more severely in 93-11 as the cold treatment proceeded. A total of 366 unique proteins from DC907 were found to significantly change in terms of accumulation under cold treatment for all three time points when using the untreated sample as a reference (S2 Table).
More interestingly, we found that most of the differentially expressed proteins at 24 h returned to the level at time zero at 72 h, but changed to an even higher level as the cold treatment continued to 120 h in DC907 ( Fig 2B). However, no such change was found in 93-11. According to these observations, we assume that self-regulation in the hybrid wild rice is important for cold resistance to ensure that the expression level of key proteins would not overload and threaten plant survival. Compared with previous studies of cold-response proteomics of cultivar rice, a proportion of proteins were identified that included rubisco and GAPDH whose expression level were both decreased under cold stress [12,13]. As shown in Fig 3, the expression level of rubisco and GAPDH in DC907 detected by Western blotting also decreased, in good accordance with TMT quantification. In terms of time frames, the differentially expressed proteins in DC907 contained several functional groups: ATP synthesis, photosystem, reactive oxygen species (ROS), stress response, transcription factors, structural proteins, and cell growth and integrity (S2 Table). Under cold stress, the proteome pattern of hybrid wild rice showed a more sensitive and faster change than cultivar rice, e.g., the vigorous change in protein functional classification was seen at 24 h (Fig 4A), but a similar change occurred in cultivar rice at 72 h (Fig 4B). The delay of cold response in cultivar rice could be a crucial reason for low survival rates. The protein networks of differentially expressed proteins in DC907 show that the centrality of protein change is mainly related to cell structure, stress response, ATP synthesis and photosynthesis (S1 Fig). These networks of functional proteins were generally consistent with those from cultivated  rice previously reported using the 2-DE method [13,14], suggesting that the timely response to cold stress and self-regulation of wild rice is more important than a change in single proteins during cold stress.

Functional grouping of cold-responding proteins
Differentially expressed proteins between DC907 and 93-11 with assigned functions at all three cold treatment times are summarized in Fig 5. Some of these proteins have been shown or implicated to have functions against cold stress.  Non-specific lipid-transfer proteins. Non-specific lipid-transfer proteins (nsLTPs) are a group of small lipophilic proteins that accumulate between the plant epidermis and cell wall [21]. Previous reports have revealed that nsLTPs participate in the process of plant biotic and abiotic stress resistance [22][23][24]. As seen from the comparative proteomic results between 93-11 and DC907, approximately 10% of the cold-responsive proteins were related to stress response. Although the nsLTPs were both up-regulated in DC907 and 93-11 after cold stress, the comparative expression level of nsLTPs was higher in hybrid wild rice DC907 in the first 24 h and 120 h (Tables 1 and 2), implying that nsLTPs may play an important part in cold tolerance in DC907.
Heat shock proteins. Heat shock proteins (HSPs) have been shown to facilitate plant adaptation to environmental changes [25]. A higher level of HSPs, acting as molecular chaperones, may help plants adapt to abnormal temperature, light, drought and salt [26]. As shown in S2 Table, many small HSPs were found to be up-regulated by cold stress in DC907, similar to those found for the cold-tolerance response in japonica rice [13].
LEA proteins. Up-regulation of late embryogenesis abundant (LEA) proteins was also identified in DC907 (S2 Table). These proteins have been implicated as enhancing plant cold stress tolerance [27-29].

ROS-related proteins
Reactive oxygen species (ROS) function to oxidize the harmful substrates that may produce and accumulate in the cell. It is now known that ROS-derived signals regulate plant growth, development and stress adaption [30,31] and are crucial for removing harmful substances from cells and avoiding plant frostbite. Under stress conditions, the ROS level could be increased by 3-10 folds to help the plant adapt to the harsh environment [32]. The ROS related proteins were mainly down-regulated under cold stress in 93-11. The death of 93-11 indicated that harmful substrates may not have been efficiently scavenged in a timely manner and damage of the cells may occur eventually, whereas the expression level of this kind of proteins was relatively stable in DC907 (S1 Table). Thus, insufficiency in ROS may result in plant cell dysfunction and cell death [33,34].

Cell structure proteins
A total of 60 differentially expressed proteins related to cell growth, integrity and structure were found (S2 Table). A large amount of ribosomal proteins was found to increase in expression during cold stress both in hybrid DC907 and cultivar rice 93-11, but the time points were different; for 93-11, the highest peak was at 72 h and returned to a normal level at 120 h, and for hybrid DC907, the highest peak was at 24 h and returned to a normal level at 72 h, thus a much faster response in the cold-tolerant hybrid DC907. In Escherichia coli, a 70 kDa ribosomal-associated protein (CsdA) was induced when the temperature was shifted from 37 to 15˚C, and this protein was further demonstrated to be involved in derepression of HSPs and cell growth at low temperatures [35]. In soybean, three low-temperature inducible ribosomal proteins were found to be increasingly expressed after cold treatment [36]. Dirigent (DIR) proteins were identified as being induced at cold conditions in cultivated rice for the first time in this study. This kind of protein was reported to be involved in lignification and to respond to pathogen infection and abiotic stress in plants [37,38]. In response to a temperature shift, the expression level of DIRs showed a relatively stable pattern in DC907, similar to hardy plants [39]. In contrast, three DIR proteins were all down-regulated and decreased substantially following the prolonged cold treatment in cultivar rice 93-11. It was reported that a decrease in DIR proteins weakened the process of lignification, which is crucial for the structural integrity of the plant cell wall and cell wall apposition (CWA)-mediated defense [40]. Thus, it is speculated that the decreased expression of DIR proteins in 93-11 results in the vulnerability of indica rice to the cold stress since lignification may prevent cells from collapsing and responding to abiotic stress [41].

Proteins related to photosynthesis and energy metabolism
In the current study, most of the differentially expressed proteins involved in photosynthesis are from photosystem II (PSII), indicating that PSII is more sensitive than photosystem I (PSI) to cold stress [42]. When the cold treatment started, the expression level of chlorophyll a-b binding proteins from the light-harvesting complex (LHC) as a light receptor decreased very fast and was significantly lower in DC907 than in 93-11 (Tables 1-3). This phenomenon is comparable to the previous observation that photoinhibition was a protection mechanism for cold tolerant plants but more significant and protective for cultivar rice in low temperature after long time exposure [43,44].
Stress-induced inhibition of plant photosynthesis is always coupled with a loss of ATP and ATP synthases [45,46]. Thus, most of the proteins related to ATP synthesis were found to decrease gradually in DC907 (S2 Table). The decreased ATP synthase resulted in a shortage of ATP, implying a weakened biological activity. The reduction in ATP and ATP synthases Gene Ontology Cellular Component. 93-11, C0 represents the control group without cold treatment; C1 represents cold treatment for 24 h; C2 represents cold treatment for 72 h; C3 represents cold treatment for 120 h. DC907, T0 represents the control group without cold treatment; T1 represents cold treatment for 24 h; T2 represents cold treatment for 72 h; T3 represents cold treatment for 120 h.
https://doi.org/10.1371/journal.pone.0198675.g004 directly relates to the reduction in photosynthetic energy captured at low temperatures, and a shortage of energy supply would result in the restriction of normal metabolic processes of plant cells. It is believed that this is a protective mechanism for plant cells under abnormal cold stress [47,48].

Interaction networks among cold induced proteins
As show in S1 Fig, protein interaction networks constructed with differentially expressed proteins from hybrid wild rice contain biological processes, molecular functions, and cellular components. In biological processes, proteins that function in photosynthesis, metabolite and energy generation, protein translation, and stress response are the highest positively regulated in DC907; in terms of molecular function, proteins that function in structural activities are most positively regulated in DC907. However, the largest group of regulated proteins at the highest level was in the cellular component domain, including membranes, macromolecule complexes, vacuoles, ribosomes, mitochondria and other organelles. An important observation is that hybrid wild rice responded to cold stress in a more timely manner by mobilizing its signal transduction and self-regulation mechanisms, similar to other cold tolerant plants studied [49].

Conclusions
In this work, the proteomes of cold-resistant DC907 with 93-11 genetic background and the cold-sensitive 93-11 were compared. The protein expression level of several important functional categories, including photosynthesis, energy generation, ROS, cell growth and development, were found to be changed under cold stress. While both DC907 and 93-11 underwent similar alterations in proteomic profiles to cold stress, DC907 responded in a prompter manner in expressing cold-response proteins, maintained a higher level of photosynthesis to power the cells, and possessed a stable and higher level of DIR proteins to prevent the plant from obtaining irreversible cell structure damage induced by ROS activity (Fig 6). Since DC907 carries chromosome fragments of wild rice, future studies should focus on the genetic elements of wild rice that confer the cold tolerant trait in DC907.