Transcriptomic, proteomic, and physiological comparative analyses of ooding mitigating low temperature stress on direct-seeded early indica rice at seedling stage

In study, treatments were established for 3d to aim to determine the response mechanisms on physiological, transcriptomic, and proteomic of direct-seeded rice seedlings at seedling stage. The results showed that the chloroplasts was severely degraded, thylakoid lamellae were seriously damaged and osmiophilic body increased gradually in LT contrast to CK, but LTF could alleviate the damage of low temperature on chloroplast structure. Compared with LT, LTF signicantly increased the contents of Rubisco, chlorophyll, PEPCK, ATP and GA 3 of rice seedlings whereas signicantly decreased soluble protein, MDA and ABA content, suggesting the higher photosynthetic traits, antioxidant ability and better growth characteristic, although it could also affect the physiological activity contrast to CK. The identied differentially expressed genes and differentially expressed proteins indicated that photosynthesis metabolism pathway, reactive oxygen species and metabolic regulation had signicant differences between LT and LTF stress, which were the main reasons that reduced the LT damage of rice seedlings for LTF.


Background
Rice (Oryza sativa L.) is the staple food for more than half of the world's population (Khush. 2000). Given that the development of green revolution since the 1960s, rice yield has increased largely. As known as a typical thermophilic crop, the growth and development of rice are susceptibly affected by temperature, especially for low temperature (Sun et al. 2016). With the rise of global temperature and frequent occurrence of extreme weather, direct-seeded early indica rice often suffers from "Cold Spell in Later Spring" disaster at sowing and seedling stage. During the 30 years from 1951 to 1980, japonica and indica rice cultivars in the middle and lower reaches of the Yangtze River had encountered this disaster event for 7 times and 9 times, respectively, which resulted in a large area of rotten seeds and rotten seedlings of direct seeded rice, and seriously affected the production of direct seeded rice. In August 2009, the Yanbian region of China was affected by continuous low temperature, resulting in the total crop failure for an area of 2310 hm 2 (Zhang et al. 2015). At the same time, although rice is a water-loving crop, the growth and development were also affected by long-term ooding stress, resulting in anaerobic respiration of roots and leaves, producing alcohol toxicity and reducing leaf photosynthesis. Therefore, it is particularly urgent to carry out indepth research on the response mechanism to low temperature stress of direct seeded early rice and its prevention measures to improve the high e ciency and stable yield of direct seeded rice.
In direct-seeded rice production, when the low temperature comes, farmers often reduce the damage of low temperature to seedlings by irrigating a certain amount of deep water layer, so as to achieve the role of protection seedling with deep water. Through this measure, the survival rate of seedlings emergence at seedling stage can be effectively increased and the production risk of direct-seeded rice can be reduced.
To date, many studies have payed close attention to the points on rice cultivation, physiological traits, genetics mechanisms and other aspects injuries induced by low temperature stress (Zhang et al. 2012;Confalonieri et al. 2005;Zia et al. 2008) and ooding stress (Mishra et al. 2010;Lal et al. 2015). Previous studies on ooding mitigating low temperature stress mainly focus on agronomic traits or related physiological characteristics (Farrell et al. 2006). However, the molecular mechanism response on the seedlings damage of direct-seeded early indica rice to low temperature stress mitigating by ooding irrigation at seedling stage was rarely reported.
With the rapid development of biotechnology, an increasing number of studies about rice in response to different stress were profoundly analyzed by transcriptomic technologies (Cohen et al. 2017;Jithesh et al. 2018). The transcriptome analysis is fast and comprehensive, which has been constructed and annotated to assist in identi cation of differentially expressed genes (DEGs) in different plant populations (Vera et al. 2008). However, a comparison of mRNA expression levels is de ned as indirect and temporarily messages in transmit information. On the contrary, proteins play a direct role in biological processes, and are the basis of organism (Marino et al. 2016). Protein is the embodiment and executor of plant functions, which not only regulates plant stress tolerance by changing the catalytic activity of enzymes, but also acts as a transcription factor to regulate the expression of other genes Tse et al. 2013;Liang et al. 2013). Through the combination of transcriptome and proteome, many differentially expressed proteins (DEPs) have been identi ed and metabolic pathways have been found. On this basis, many differentially expressed genes related to metabolic pathways have been excavated, providing a molecular mechanism for nding responses to environmental stress.
At present, many studies have elucidated the in uence mechanism of low temperature stress or ooding stress on rice seedlings from the aspects of proteomics and transcriptomics (Yang et al. 2015;Hussain et al. 2016). However, the mechanism of transcriptomics and proteomics based on low temperature ooding is still unclear. The molecular mechanism of the mitigative effect rather than superposition effect of the hypoxic treatment caused by ooding under low temperature is a scienti c problem worthy of further study. This study combined transcriptomics and 4D-label free quantitative proteomics analysis to explore the molecular mechanism of ooding mitigating low temperature stress on direct-seeded early indica rice at seedling stage. Here, in this study, we identi ed genes and proteins that were obtained from Illumina-Hiseq and 4D-label free searching for likely protein identi cation in Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Klustersof eukaryotic Orthologous Groups (KOG), Swissport and Uniprot database, respectively, and focused on the DEGs and DEPs in ooding mitigating low temperature stress. This study has guiding signi cance to breeding and cultivation of low temperature tolerant crops cultivars. It also provides the evidence for the meteorological disaster mitigation and low temperature prevention.

Result
Transmission electron microscopic observation of chloroplast structure In this study, transmission electron microscopy was used to analyze leaf phenotypic characteristics and compare the differences on chloroplasts structural of early indica rice seedlings after 3 days between LT and LTF. The results showed that chloroplasts were regular boat shaped or spindle shaped and thylakoid lamellae clear arranged close to the inner wall of cells in CK (Fig. 1C). Compared with CK, chloroplasts began to degrade, shaped distorted and loosely structured in LTF (Fig. 1B), however, chloroplasts had been severely degraded, thylakoid lamellae were seriously damaged and osmiophilic body increased gradually in LT (Fig. 1A). The damage to chloroplast structure in LTF was less than that in LT. These results showed that chloroplast were affected to some extent after low temperature stress, and ooding could alleviate low temperature damage on chloroplast structure.

Analysis of photosynthesis activity andendogenous hormone content
Rubisco is the key enzyme of photosynthetic carbon metabolism in C 3 plants, which is positively correlated with the photosynthetic rate (Yu et al. 2012). Under LT stress, the balance of plant cell free radical production and elimination system is damaged, chlorophyll synthesis and energy metabolism are affected (Wang et al. 2006). This study showed that the rubisco content of LT was signi cantly decreased by 26.97% (P < 0.05) compared to CK, but there was no signi cant difference between LTF and CK ( Fig. 2A). The PEPCK activity, chlorophyll content and ATP content of LT and LTF were signi cantly decreased (P < 0.05) compared with CK, and those indexs were lower in LT than in LTF ( Fig. 2B; Fig. 2C; 2D). Endogenous hormones are signal molecules produced in the process of plant metabolism, which play an important role in plant growth and environmental response (Sakamoto T. 2006). Compared with CK, the GA 3 content of pro-growth hormones in LT and LTF decreased signi cantly (Fig. 2E). In contrast, the ABA content of anti-growth hormones in LT and LTF signi cantly increased by 35.71% and 16.67% (P < 0.05), respectively (Fig. 2F). There were the same trends on GA 3 and ABA between LT and LTF, which reached a signi cant level. These results indicated that ooding could improve photosynthesis activity and endogenous hormone content under low temperature stress of direct-seeded early indica rice at seedling stage.
Analysis of soluble protein content, antioxidase and osmotic regulatory substances Soluble protein is an important osmotic regulator that affects the osmotic potential of cells (Zhu et al. 2015). After plants were stressed, plant cell membrane would be damaged by free radicals and reactive oxygen species, resulting in membrane lipid peroxidation and protein activity affected (Vighi et al. 2017;Hu et al. 2017). This study showed that the content of soluble protein and MDA in LT and LTF increased signi cantly (P < 0.05) compared with CK, and LT also signi cantly increased soluble protein and MDA contrast to LTF ( Fig. 3A; Fig. 3B). In addition, LT and LTF signi cantly increased the activities of SOD and POD (P < 0.05) compared with CK, and there were no signi cant differences between LT and LTF, although SOD and POD were higher in LT than in LTF ( Fig. 3C; Fig. 3D).

Identi cation of DEPs
To evaluate the reliability of the data generated by proteomic analysis, the Pearson correlation coe cient was calculated for each of three samples, which indicated good reproducibility of the three biological replicates in each treatment (Fig. 4A). In addition, a total of 412489 spectrums were detected, 236880 of which could be matched to peptides in the database and 28934 were unique peptides. In total, 5639 proteins could be identi ed and 4518 proteins were experimentally quanti ed (Table 1).
Proteins with a fold change (FC) > 1.5 or (FC) < 0.67 (P < 0.05) between the treatment (LT, LTF) and control groups (CK) were regarded as DEPs, and DEPs were hence considered as low temperature and low temperature ooding responsive proteins at the seedling stage. There were 567 DEPs between LT and CK, 239 DEPs between LTF and CK, and 235 DEPs between LTF and LT. The number of up-regulated and down-regulated DEPs was shown in Fig. 4B and the three groups had 16 DEPs in common (Fig. 4C).
In this study, 72818 transcripts and 5639 proteins were identi ed by quantitative transcriptome and proteome studies. 4983 genes were identi ed at both transcriptome and proteome levels (Fig. 4D). The correlation coe cient between transcripts and proteins in LT and CK treatment groups was 0.19, that in LTF and CK treatment groups was 0.25, and that in LT and LTF treatment groups was 0.22. It indicates that the correlation degree of samples in each treatment group is low, and they are basically consistent with the expectation (Fig. 5).

Protein-protein interaction
The functional DEPs of all annotated were used to analyze protein interactions. This revealed that most enzymatic proteins and proteins related to biosynthesis of secondary metabolites, monobactam biosynthesis, metabolic pathways, pentose phosphate pathway, fructose and mannose metabolism, glycolysis / gluconeogenesis, glycine, serine and threonine metabolism, arachidonic acid metabolism, biosynthesis of amino acids, phenylalanine, tyrosine and tryptophan biosynthesis and proteasome related proteins interactions were affected by LT and CK (Additional le 7: Figure S4). Most enzymatic proteins and metabolic pathways, biosynthesis of secondary metabolites, carotenoid biosynthesis, ribosome biogenesis in eukaryotes, glycolysis/gluconeogenesis, glycine, serine and threonine metabolism photosynthesis and thiamine metabolism were observed for the interaction between LTF and CK (Additional le 8: Figure S5). Most enzymatic proteins and photosynthesis-antenna proteins, photosynthesis related proteins interactions were affected by LTF and LT (Additional le 9: Figure S6). In this study, the photosynthesis pathway and energy metabolism pathway were observed for being highly enriched under low temperature and low temperature ooding. This result showed that low temperature ooding played an important role in regulating the photosynthetic capacity of rice leaves. Consistent with our GO analysis ndings, the majority of proteins were involved in photosynthesis and metabolic processes. We could focus on proteins related to photosynthesis and metabolism at the proteomic level.

KEGG pathway analysis
All of the DEGs and DEPs were analyzed for the KEGG over-representation of pathways to obtain functional insights into the difference between LT, LTF and CK treatment. The signi cantly (P < 0.01) enriched KEGG pathways are shown in Table 2. The KEGG pathways (ordered by rank) are monobactam biosynthesis, glycine, serine and threonine metabolism, biosynthesis of secondary metabolites, pentose phosphate pathway, biosynthesis of amino acids, metabolic pathways, arachidonic acid metabolism, glycolysis / gluconeogenesis, proteasome, phenylalanine, tyrosine and tryptophan biosynthesis, fructose and mannose metabolism between LT and CK. The KEGG pathways (ordered by rank) are thiamine metabolism, ribosome biogenesis in eukaryotes, carotenoid biosynthesis, biosynthesis of secondary metabolites, metabolic pathways, glycine, serine and threonine metabolism, glycolysis / gluconeogenesis between LTF and CK. The KEGG pathways (ordered by rank) are photosynthesis, photosynthesisantenna proteins between LTF and LT.

Analysis of DEGs and DEPs by qRT-PCR
To verify the proteomes and transcriptomes results, eighteen related genes including ve up-regulated and thirteen down-regulated were detected. As shown that the mRNA expression of A2XLW5, B8AYU2, A2X822, B8ASV8 and A2XYC2 were down-regulated between LT and CK (Fig. 7A). The mRNA expression levels of A2YM28, A2XKN7, A2WRR8 and A2X8P7 were down-regulated between LTF and CK, B8AZB8, B8BJP8, B8AS16 were signi cantly up-regulated between LTF and CK (Fig. 7B). In LTF and LT, A2YMZ1, A2YCB9, A2YHC5 and A2YLE6 were down-regulated, and B8B7M5 and A2YP23 were up-regulated (Fig.   7C). Those as mentioned indicated that transcriptomes and proteomes results indeed re ected the relative expression of each gene, in which up-regulated or down-regulated genes in qRT-PCR were completely consistent with transcriptome and proteome trends. Therefore, the transcriptome results were reliable.

Discussion
Photosynthesis response to ooding under low temperature stress Photosynthesis is one of the sensitive physiological and biochemical processes under low temperature (Winkel et al. 2014). After LT stress, the photosynthetic mechanism was damaged, electron transfer chain was interrupted and the stomatal conductance decreased (Qiu et al. 2017). In addition, LT not only affects the synthesis of chlorophyll in leaves, but also rapidly degrades chlorophyll and damages chloroplast cells (Yamori et al. 2014). In this study, transmission electron microscope observation showed that under LT stress, chloroplast and membrane lipids were seriously degraded, and osmiophilic granule were also numerous and large. LTF reduces the damage to chloroplast structure and protects seedlings through shallow ooding. LT affects the metabolism and physiology of crops, and has an indirect effect on leaf photosynthesis (Ayub et al. 2011;Nelson et al. 2006).
Rubisco is the key enzyme in photosynthesis that determines the carbon assimilation rate (Bowes et al. 2006). In this study, LTF could signi cantly increase the contents of rubisco and chlorophyll of rice seedlings compared with LT ( Fig. 2A; Fig. 2B), and photosynthesis and glycine, serine and threonine metabolism were also signi cant enrichment between LT, LTF and CK ( Table 2). The results of DEPs analysis indicated that phytochrome B (protein ID: A2XFW2) and phytochrome C (protein ID: A2XM23) were signi cantly down-regulated (P < 0.05) between LT and CK (Dataset 1), and phytochrome C (protein ID: A2XM23), regulation of photosynthesis (protein ID: B8AWA3) were also signi cantly down-regulated (P < 0.05) between LTF and CK (Dataset 2). However, phytochrome B (protein ID: A2XFW2) and phytochrome C (protein ID: A2XM23) were signi cantly up-regulated (P < 0.05) between LTF and LT (Dataset 3), which supported the above results that LT could signi cantly inhibit the photosynthesis of rice leaf, and ooding could alleviate the effects. This was consistent with the results of previous studies (Makino et al., 2007;Liu et al., 2018). In addition, the DEPs also showed that chlorophyllide oxygenase activity (protein ID: A2XAH0, A2XCH9, A2XN43), and chloroplastic (protein ID: B8AD72) were signi cantly down-regulated (P < 0.05) between LT and CK (Additional le 1: Dataset S1), and chlorophyll proteins (protein ID: A2XIZ1, B8AM17) were signi cantly down-regulated (P < 0.05) between LTF and CK (Additional le 2: Dataset S2). On the contrary, chlorophyllide oxygenase activity (protein ID: A2XCH9) were signi cantly up-regulated (P < 0.05) between LTF and LT (Additional le 3: Dataset S3). The results showed that LT would lead to the degradation of pigment proteins in leaves, while LTF would improve a large amount of synthesis of pigment proteins in leaves, which was bene cial to the absorption of more solar energy in rice leaves and remission the effects of LT stress on photosynthesis.
ROS response to ooding under low temperature stress When rice was subjected to LT stress, the balance of intracellular oxygen metabolism was disturbed, resulting in reactive oxygen species (ROS) production, aggravating membrane lipid peroxidation, producing MDA and damaging the cell membrane system (Sanghera et al. 2011 2018). In this study, the activities of SOD and POD in LT and LTF were signi cantly increased contrast to CK ( Fig. 3C; Fig. 3D). The results of DEPs analysis indicated that the antioxidant activity, response to stimulus, defense response and metabolic process were signi cantly enriched between LT and CK (Fig.  6). Here, the scavenging of the ROS related protein oxidase (protein ID: A2XX54, B8AKJ8), oxidoreductase (protein ID: A2YAP5) and glutathione peroxidase (protein ID: A2X822, B8ASV8) were signi cantly upregulated (P < 0.05) between LT and CK (Additional le 1: Dataset S1). Meanwhile, superoxide dismutase activity (protein ID: A2XAA0), oxidoreductase (protein ID: A2YAP5) and glutathione peroxidase (protein ID: B8ASV8) were signi cantly up-regulated (P < 0.05) between LTF and CK (Additional le 2: Dataset S2), although POD and SOD did not differ between LTF and LT. However, peroxidase (protein ID: B8ARU3) were signi cantly down-regulated between LTF and LT (Additional le 3: Dataset S3). These could show that the results of physiological and proteomic data were mutually veri ed. To survive under LT stress, rice plants activated their reactive oxygen scavenging system and reduced the damage caused by low temperature to rice seedling. However, because the temperature was too low and the damage of rice seedlings were serious, excessive ROS attacked rice, resulting in cell structure damage and metabolic disorder, which might be one of the crucial reasons for the damage of chloroplast structure. In this study, ooding plays a role in protecting seedlings from the direct stress of LT due to shallow ooding, which could improve the antioxidant enzyme protection system of direct seeded rice seedlings under LT.

Metabolic regulation response to ooding under low temperature stress
Under LT stress, the growth and development of rice involves the comprehensive regulation of many kinds of plant endogenous hormones, such as ABA, GA 3 , etc., in which low temperature stress will cause a large amount of ABA accumulation (Nishiuchi et al. 2012). This study showed that compared with LTF, LT signi cantly increased the contents of ABA of rice seedlings and signi cantly decreased GA 3 Fig. 2C;  Fig. 2D), and KEGG pathway analysis indicated that biosynthesis of secondary metabolites, metabolic pathways, glycolysis / gluconeogenesis, glycine, serine and threonine metabolism were signi cantly enriched between LT, LTF and CK ( Table 2). The results of DEPs analysis indicated that the glycolysis protein 6-phosphofructokinase (protein ID: B8BF81) and phosphoenolpyruvate carboxykinase (protein ID:A2XEP1) were signi cantly down-regulated (P < 0.05) between LT and CK (Additional le 1: Dataset S1), tricarboxylic acid cycle (protein ID: A2X2W9) and fructose 1,6-bisphosphate 1-phosphatase (protein ID: B8AYU2) were also signi cantly down-regulated between LTF and CK (Additional le 2: Dataset S2), which might be the key position to limit the glycolysis pathway, resulting in insu cient energy supply under LT at the seedling stage. However, galactosidase (protein ID: B8A982), pyruvate kinase (protein ID: B8ACJ0), glycoprotein glucosyltransferase (protein ID: B8AGC9), glucan branching enzyme (protein ID: B8ATS0) were signi cantly up-regulated (P < 0.05) between LTF and LT (Additional le 3: Dataset S3), and that could improve energy supply for the seedlings damage through ooding under LT stress. As a consequence, for low temperature stress, due to the disorder of energy metabolism, rice seedlings cannot be provided with the normal energy needed for growth and development, resulting in plant wilting.

Conclusions
The study provides novel insight into the physiological and molecular mechanisms of rice response to LT stress and LTF at seedling stage. The damage of chloroplast structure in rice leaves was alleviated by ooding under low temperature. LTF signi cantly increased the contents of rubisco, chlorophyll, PEPCK, ATP and GA 3 , and also enhanced photosynthesis and energy metabolism compared with LT. GO and KEGG enrichment analysis revealed that the DEPs were signi cantly associated with the photosynthesis pathway, metabolic regulation and reactive oxygen species. 4D-label-free quantitative proteomic and transcriptomic conjoint analysis showed that biosynthesis of secondary metabolites, metabolic pathways, glycolysis / gluconeogenesis, glycine, serine and threonine metabolism were signi cantly enriched under LT and LTF. Our results could provide comprehensive interpretation of physiological characteristics, genes and proteins expression changes in low temperature and low temperature ooding.

Plant materials and growth conditions
In this study, early indica rice cultivar Zhongjiazao 17 (ZJZ17) was selected for pot experiment, which is mainly generalized for rice production in Jiangxi Province mg·kg -1 , and pH 6.1. The soil was naturally blown-dried, crushed by a soil grinder (FT-1000A, Changzhou WIK Instrument Manufacturing Co. Ltd., China), and then sifted by a 100-mm mesh. Each pot lled approximately six kilograms of dry soil that was soaked in water for two weeks before direct seeding. 3 g compound fertilizer (N-P-K = 15-15-15%) was applied as base fertilizer 1 day before direct seeding. Other management measures were in accordance with local recommendations.

Experimental design
Germinated seeds were selected for direct seeding, then all pots were placed in arti cial climate chamber with a diurnal temperature of 27/23°C (day/night). After 20 days of direct seeded, one-third pots of rice seedlings with three leaves continued to be kept in suitable temperature arti cial climate chamber 27/23°C (day/night) as control (CK), and the two-thirds pots of rice seedlings were equally moved to another arti cial climate chamber for low temperature (LT) and low temperature ooding (LTF) treatment. For low temperature (LT), the diurnal temperature was set 10/6°C (day/night), and the period of low temperature treatment was 3 days that the light intensity was 100mmol·m -2 s -1 and the relative humidity (RH) of the arti cial climate chamber was 75%, and potting soil remained moist. During the treatment, the positions of each pot were rotated from time to time to avoid the in uence of light on seedling growth. For low temperature ooding treatment (LTF), temperature and treatment period were both the same as LT treatment, and the rice plants were maintained in a 5-6 cm ood water layer. For control (CK), rice seedlings were grown in arti cial climate chamber that the diurnal temperature was 27/23°C (day/night), keep the soil moist. Each treatment was consisted of three replicates, and 20 pots constituted each replication.
Transmission electron microscopic observation After 3 days of low temperature and low temperature ooding, the middle and upper parts of the leaves were cut into 1×1cm long with a blade, and immediately put into the 2.5% glutaric dialdehyde xative, vacuumized until the sample was completely submerged, and then they were xed overnight at 4°C, taken about 10 pieces for each sample. The sample processing methods were according to the method of Huang et al (2018).

Physiological and biochemical characteristic
Enzymatic activity and endogenous hormones contents were measured on the third days of low temperature and low temperature ooding treatment. In each replicate experiment, three pots of plants were selected for each treatment group, 3 g leaf sample was collected and immediately frozen in liquid N 2 and stored at -80°C until extraction. PEPCK activity and Rubisco, chlorophyll, ATP, ABA, GA 3 content

RNA isolation, library preparation, and sequencing
According to the RNA extraction scheme, the total RNA was separated by Trizol reagent (Invitrogen, Waltham, MA, USA). RNA purity was checked by the Nano Photometer® spectrophotometer (IMPLEN, CA, USA). And the integrity of the RNA was assessed by the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The quality and quantity of the RNA samples were determined by Nanodrop 2000 spectrophotometer (Thermo, USA).
The total amount of 3µg RNA per sample was used as the input material for RNA sample preparation.
Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Brie y, mRNA was puri ed from total RNA using poly-Toligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3′ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were puri ed with AMPure  . DESeq provide statistical routines for determining differential expression in digital gene expression data using a model based on the beta negative binomial distribution. The resulting Pvalues were corrected by the Benjamini-Hochberg method. Genes with an adjusted P-value < 0.01and fold change > 2 found by DESeq were assigned as differentially expressed.
Protein extraction, trypsin digestion and TMT labeling The sample was rst grinded by liquid nitrogen, and then the powder was transferred to a 5 mL centrifuge tube. After that, four volumes of lysis buffer (8 M urea, 1% Protease Inhibitor Cocktail) were added to the cell powder, followed by sonication three times on ice using a high intensity ultrasonic processor (Scientz, China). The samples were centrifuged at 12,000×g at 4°C for 10min and the debris was removed. Finally, the protein concentration was determined with BCA kit (Beyotime, China) according to the manufacturer's instructions.
For digestion, the protein solution was reduced with 5mM dithiothreitol for 30min at 56°C and alkylated with 11mM iodoacetamide for15 min at room temperature in darkness. The protein sample was then diluted by adding 100 mM TEAB to urea concentration less than 2M. Finally, trypsin was added at 1: 50 trypsin-to-protein mass ratio for the rst digestion overnight and 1: 100 trypsin-to-protein mass ratio for a second 4 h-digestion.
After trypsin digestion, peptide was desalted by Strata X C18 SPE column (Phenomenex, USA) and vacuum-dried. Peptide was reconstituted in 0.5M TEAB and processed according to the manufacturer's protocol for TMT kit. The peptide mixtures were then incubated for 2h at room temperature and pooled, desalted and dried by vacuum centrifugation.

LC-MS/MS analysis and database search
The tryptic peptides were dissolved in 0.1% formic acid (solvent A), directly loaded onto a home-made reversed-phase analytical column (15 cm length, 75 μm i.d.). The gradient was comprised of an increase from 6% to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, 23%-35% in 8 min and climbing to 80% in 3 min then holding at 80% for the last 3 min, all at a constant ow rate of 400 nL/min on an EASY-nLC 1000 UPLC system (Thermo, Waltham, USA).
The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1.800 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE setting as 28 and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. Fixed rst mass was set as 100 m/z.
The resulting MS/MS data were processed using Maxquant search engine (v.1.5.2.8). Tandem mass spectra were searched against human uniprot database concatenated with reverse decoy database.
Trypsin/P was speci ed as cleavage enzyme allowing up to 4 missing cleavages. The mass tolerance for precursor ions was set as 20 ppm in First search and 5 ppm in main search, and the mass tolerance for fragment ions was set as 0.02 Da. Carbamidomethyl on Cys was speci ed as xed modi cation and acetylation modi cation and oxidation on Met were speci ed as variable modi cations. FDR was adjusted to < 1% and minimum score for modi ed peptides was set > 40.

Con rmation using qRT-PCR
To verify the accuracy of transcriptome and proteome results, eighteen DEGs involved in LT, LTF and CK responses were selected for veri cation using qRT-PCR. The RNA used for qRT-PCR was the same with those used to construct cDNA library. The primers were designed using Primer 5.0 (Additional le 10: Table S1) and synthesized by Xiamen Life-Int Technology Co., Ltd and 18s (GenBank Accession NO. AK059783) acted as the reference gene.

Statistical Analysis
The data were analyzed by analysis of variance (SAS Institute Inc., Cary, NC), and the means of different treatments were examined by Tukey's honest signi cant difference (HSD) test to compare the differences at the probability level of 0.05. reagents/materials/analysis tools. Wang Wenxia wrote the manuscript. All authors read and approved the manuscript.  The pathway ranking in this table is in order from high to low between LT, LTF and CK. The "mapping" number represents the number of annotated DEGs and DEPs in the pathway, while the "all" number represents the total number of proteins in the pathway. CK: control; LT: low temperature; LTF: low temperature ooding. Figure 1 Transmission electron microscope analysis of the top leaves at rice seedlings stage after low temperature and low temperature ooding stress. CP: chloroplast, Thy: thylakoid lamellae, OB: osmiophilic body. LT: low temperature, LTF: low temperature ooding, CK: control.    The transcript and its corresponding protein expression scatter diagram. LT: low temperature, LTF: low temperature ooding, CK: control.