Comprehensive comparative analysis and expression profiles and effects on physiological response of DEAD-box RNA helicase genes in Lumnitzera littorea (Jack) Voigt under cold stress

ABSTRACT The DEAD-box family has been shown to play an important role in a variety of abiotic stresses, but little is known in studies of mangrove plants. Here, the effects of cold stress on various physiological changes and the role of the DEAD-box RNA helicase family in response to cold stress were determined. First, we identified 73 DEAD-box RNA helicase family members in L. littorea. Second, the evolutionary relationships between the DEAD family in L. littorea and the model species Arabidopsis thaliana were investigated by evolutionary phylogenetic analysis. Finally, qRT-PCR study of representative DEAD-box genes showed that DEAD-box genes played a major role in the low-temperature stress response of L. littorea. Furthermore, we found that LlDEAD48, LlDEAD36, and LlDEAD47 might be involved in the maintenance of chlorophyll function, and LlDEAD43 might play a role in the maintenance of mitochondrial function in L. littorea under cold stress.


Introduction
Lumnitzera littorea (Jack) Voigt is a typical mangrove plant found in tropical and subtropical coastal areas (Su et al. 2019) with various roles (Siikamäki et al. 2012;Martin et al. 2019;Wilda et al. 2020). L. littorea is listed as a national class I key protected plant, with only nine wild individuals existing in Sanya Tielu harbor ). There are many reasons for the low number of L. littorea, including low pollen viability, low seed fertility, and low seedling survival rate (Zhang et al. 2016;Zhang et al. 2021). The natural range of L. littorea is in the southernmost part of the country, which is considered as the least cold-tolerant actual mangrove plant. L. littorea is highly sensitive to low temperature, and shows heavy damage under low temperature and is difficult to recover (Chen et al. 2010). In addition, due to the increased frequency of extreme cold weather, improving the cold resistance of L. littorea is an urgent task to protect this species safely through the winter.
Among abiotic stresses, low temperature is one of the most critical environmental factors, which plants need to address during their evolution and development since it limits the species, growth, and distribution of plants (Stuart et al. 2007;Osland et al. 2017). Cold stress may cause structural rupture of plant cell membranes (Wolfe 1978;Palta 1990;Mahajan and Tuteja 2005;Vigh et al. 2007), reduce the activity of various enzymes involved in plant metabolic synthesis, water loss in plant cells, plant wilting (Ashworth and Pearce 2002;Jin et al. 2022), and impede photosynthesis (Allen and Ort 2001;Asada 2006). Low-temperature stress leads to increase in reactive oxygen species (ROS), which causes damage to organelles and macromolecules, including DNA, proteins, lipids, and ultimately leads to cell death (Foyer and Noctor 2005;Mahajan and Tuteja 2005;Das and Roychoudhury 2014). At the same time, under low temperature stress, plants altered a series of biological processes such as accelerating ROS scavenging, enhancing antioxidant mechanisms and protective protein synthesis (Guy et al. 1987; Barrero-Gil et al. 2016;Chang et al. 2021), increasing osmotic substances (Sun et al. 2021), and altering endogenous cellular phytohormones (Karimi et al. 2016).
The mechanisms of photosynthesis involve numerous components including photosynthetic pigments and photosystems, electron transport systems, and CO 2 reduction pathways, damage to each part caused by low-temperature stress may decrease the overall photosynthetic capacity of mangrove plants (Ashraf and Harris 2013). Photosynthesis is considered as one of the criteria for healthy plant growth (Sharma et al. 2020), and several studies have reported the mechanism of low-temperature stress damage to mangrove plants. Lowtemperature stress inhibits photosynthesis in mangrove plants by blocking photosynthetic pigment synthesis, inhibiting antioxidant metabolism (e.g. CAT, POD, and APX), suppressing sucrose transport, reducing the maximum photochemical efficiency of PSII, and accelerating nucleic acid endonuclease senescence. It is complicated to recover from impaired photosynthesis due to constant low temperature (Peng et al. 2015;Zheng et al. 2016). There were several cases of widespread mangrove plant mortality due to cold stress caused by extremely cold weather in North America and southern China (Li et al. 2013;Chen et al. 2017).
The DEAD-box family is named DEAD because motif II, which makes up the majority of the SF2 helicase subfamily, contains the amino acid sequence Asp-Glu-Ala-Asp (D-E-A-D) feature (Ali 2021). DEAD-box RNA helicases contain nine conserved motifs (Q, I, Ia, Ib, and II-VI) (Ali 2021), each of which plays its role (Pause et al. 1993;Tanner and Linder 2001;Shen et al. 2007). Although their sequence and structure are similar, each DEAD-box gene has a different function. For instance, AtRH47/ISE1 is associated with embryonic development (Stonebloom et al. 2009). AtCAF plays a vital role in meristematic tissue determination in Arabidopsis thaliana (Jacobsen et al. 1999), PRH75 is involved in accelerated cell growth and division (Lorković et al. 1997), OsRH34 and OsRH2 are involved in the regulation of rice plant height and seed development (Huang et al. 2016). With the first confirmation of the function of FL25A4, an abiotic stress-related gene, it was found to limit the plant growth and development under low-temperature stress (Seki et al. 2001). PMH1 and PMH2, which localized on mitochondria, also played essential roles during low-temperature stress in Arabidopsis thaliana (Matthes et al. 2007). LOS4 participates in the regulation of plant cold response by mediating the expression of CBF family transcription factors (Gong et al. 2002;Gong et al. 2005;Braud et al. 2012). AtRH3 localized on chloroplasts and involved in low-temperature stress and salt stress in Arabidopsis thaliana (Asakura et al. 2012;Gu et al. 2014), and OsRH58 in rice improved tolerance to drought and salt in Arabidopsis thaliana by regulating the translation level of chloroplasts (Asakura et al. 2012). The continuous exploration of the functions of these genes in the DEAD box provided the new insights into the mechanism of cold resistance in L. littorea.
In this study, we identified the DEAD-box gene family and predicted its physicochemical properties, performed a phylogenetic analysis of DEAD protein sequences in Arabidopsis thaliana and L. littorea, and then analysed the conserved structural domains of DEAD protein sequences in L. littorea. In addition, we investigated the physiological characteristics of L. littorea and the response of DEAD family genes under low temperature to lay the preliminary foundation for improving the cold resistance of L. littorea.

Materials and methods
2.1 Identification of the DEAD-box RNA helicase family in L. littorea The transcriptome data of L. littorea was obtained from the college of life science and technology, Lingnan Normal College, and submitted to NCBI with the accession number (PRJNA791944). The DEAD-box sequence of the model plant Arabidopsis thaliana was downloaded from the TAIR (https://www.arabidopsis.org/) and used as a probe to perform the blast search in L. littorea transcriptome database. Meanwhile, a direct search was performed by using the keyword 'DEAD-box'. The conserved sequences were identified by InterPro (https://www.ebi.ac.uk/interpro/), SMAR (https://smart.embl-heidelberg.de/smart/set_mode.cgi?) and CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd. shtml) software. The genes and repetitive sequences without no complete DEAD-box structural domain were excluded, 73 DEAD-box gene sequences were obtained.

Characteristics, structure and motif analysis of DEAD genes
The protein characteristics, including isoelectric point, lengths, and molecular weight of DEAD-box proteins, were analyzed by ExPASy (https://web.expasy.org/compute_pi/). The conserved motifs were detected by using the online MEME tool (https://meme-suite.org/meme/doc/meme.html). Parameters were set as follows: number of repetitions; optimum motif width set to ≥ 6 and ≤ 200; the maximum number of motifs set to 8. The conserved structural domains were predicted by the Batch CD-Search function on the NCBI website (https:// www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The conserved motifs and their structural domains were visualized against the evolutionary tree of the L. littorea DEAD-box gene family using by TBtools software. Subcellular localization analysis was done by using PSORT (https://www.genscript. com/psort.html?src=leftbar) software.

Multiple sequence alignment and phylogenetic analysis
Multiple sequence alignments of homologous proteins in L. littorea were performed by using the ClustalW program with the default parameters together with 54 Arabidopsis thaliana DEAD-box protein sequences. The DEAD-box protein sequences of Arabidopsis thaliana and L. littorea were downloaded from UniProt (https://www.uniprot.org/) and Arabidopsis Information Resource (https://www.arabidopsis.org/), respectively. The phylogenetic trees for DEAD-box RNA helicase genes were constructed by using the Nearest-Neighbor-Interchange (NNI) method in MEGA7 software and assessed by bootstrap analysis with 1000 resampling replicates. Trees were visualized by TBtools and drawn by using the Interactive Tree of Life (iTOL; https://itol.embl.de/).

Plant materials and growth conditions
One-year-old L. littorea seedlings with 5-6 true leaves and a height of 30 ± 2 cm were chosen as experimental materials, and then divided into four groups: (1) Seedlings were treated at a temperature of 8°C day/5°C night (SLS); (2) Seedlings treated under temperature 15°C day/12°C night (MLS); (3) Seedlings treated under temperature 25°C day/23°C night (LLS); (4) Seedlings handled in temperature 34°C day/30°C night (HLS). All plants were cultured for 48 h in a cold light plant incubator (LRG-450-LED, LV BO, China) under controlled climatic conditions: day/night photoperiod, 12/12 h; 50% relative humidity and 80% relative light intensity during the day; and 70% relative humidity and zero relative light intensity during the night. All harvested leaves, stems, and roots were quickly washed and wrapped in aluminum foil and immediately placed in liquid nitrogen, and stored at -80°C until use. Three biological replicates were analysed for each treatment.

Determination of physiological indicators at different temperatures
2.5.1 Measurements of antioxidant enzymes, osmoregulatory substances, hydrogen peroxide (H 2 O 2 ) and chlorophyll content After chilling stress, leaves from each treatment were collected for physiological analysis. The contents of antioxidant enzymes, osmoregulatory substances, hydrogen peroxide (H 2 O 2 ) and Chlorophyll were determined with commercial assay kits (Jiancheng, China).
One unit of CAT enzyme activity is defined as 1 umol of H 2 O 2 being decomposed by 1 mg histone per second. One unit of POD activity is defined as the amount of enzyme per mg of tissue catalyzing 1ug of substrate per minute at 37°C. The enzyme activity was expressed as U/g fresh weight.
Proline content was determined by the Proline Assay Kit (Jiancheng, China) according to the operating instructions. Soluble sugars were determined with plant soluble sugar content Assay Kit (Jiancheng, China) by adding ten times the volume of distilled water and grinding into a homogeneous solution, heating in a boiling water bath for 10 min, and then centrifuging at 4000 rpm for 10 min, followed by determination of their content by anthrone colorimetry. The concentration of total soluble phenols was determined by using commercial assay kits (Nanjing, China).
The content of H 2 O 2 was determined by using a commercially available kit (Nanjing, China). Chlorophyll a, and chlorophyll b were determined by the Chlorophyll assay kit (Jiancheng, China) according to the operating instructions.
Chlorophyll fluorescence was measured simultaneously with CO 2 gas exchange on the third leaves using an OS5p + Portable Pulse Modulated Chlorophyll Fluorometer. The seedling leaves were dark-adapted for 20 min before measurements. The fluorescence parameters for non-photochemical (NPQ) and photochemical (qP) quenching, the maximum fluorescence (Fm), the maximal efficiency of PSII photochemistry (Fv/Fm), and the actual quantum yield of PSII photochemistry (ΦPSII) were obtained by the method described in the previous study (Livak and Schmittgen 2001;Leckie and Neal Stewart 2011).

RNA isolation and quantitative real-time RT-PCR analysis
Total RNA was extracted from L. littorea using TRI reagent according to the manufacturer's protocol. RNA quantity and integrity were estimated at 260 nm using a Nano-Drop ND-8000 UV-Vis spectrophotometer, and gel electrophoresis was performed in a 1% agarose gel stained with ethidium bromide in 1x TAE (Tris-Acetate-EDTA) buffer. Then, firststrand cDNAs were synthesized using the HiScript QRT SuperMix for qPCR (+gDNA wiper) with Oligo (dT) primers. Twenty DEAD-box genes were chosen for quantitative real-time RT-PCR analysis including those localized to different organelles and had high similarity to the DEADbox amino acid sequences of the other plant species. The primers are listed in Table S1. The Real-time PCR experiments were performed three times.
The expression of selected L. littorea DEAD-box family genes in five tissues (root, stem, leaf, flower and fruit) and at different temperatures was researched. The average threshold cycle (Ct) was calculated for each sample, AthU6 was used as the internal control. The relative expression levels of individual genes were calculated using the 2 −ΔΔCt method described by Livak and Schmittgen (Livak and Schmittgen 2001). The average expression folds of three biological replicates were calculated. The heatmap of DEAD gene expression in five tissues of L. littorea was plotted by TBtools software.

Identification of the DEAD-box family proteins in L. littorea
We identified 73 non-redundant DEAD-box genes (LlDEAD1 to LlDEAD73) from the transcriptome analysis databases of L. littorea. Bioinformatic analysis of the DEAD-box gene family of L. littorea revealed 73 newly identified LlDEAD proteins ranging from 406 to 2084 aa in length, with LlDEAD18 having the longest amino acids sequences. Molecular weight analysis showed that the molecular weight of this family member was between 9546.8 and 198608.51 Da, which positively correlated with the number of amino acids. Isoelectric point analysis shows that LlDEAD52 has the lowest isoelectric point, with strong sedimentation ability and weak solubility. LlDEAD72 has the highest isoelectric point with more vital intermolecular forces. The lipid coefficient ranged from 41.25 to 99.07. According to the predicted analysis of subcellular localization, a total of 50.68% of 73 DEAD-box genes localized in the nucleus, 17.81% in the mitochondria, 27.40% in the cytoplasm and 4.11% in the endoplasmic reticulum (Table S3).

Conserved structural domain and motif analysis
A total of 9 conserved motifs were identified among 73 LlDEAD proteins (Figure 1(A)). Figure 1(B) shows the details of those conserved amino acid sequences and lengths of the nine motifs. Most of the closely related members have the same motif compositions, indicating functional similarity between the evolutionary closely related LlDEAD proteins.

Multiple Sequence Alignment and Phylogenetic Analysis
To investigate the phylogenetic relationship of DEAD-box proteins between L. littorea and Arabidopsis thaliana, we constructed an unrooted phylogenetic tree (Figure 2). The L. littorea DEAD-box members exhibit interspersed distribution among different subgroups.

Physiological responses caused by chilling stress in L. littorea
As shown in Figure 3, the phenology of L. littorea changed significantly under different culture temperature conditions. No significant changes were observed in LLS and HLS culture conditions. Under MLS conditions, the leaves of L. littorea appeared to wilt. Under SLS culture conditions, the leaves of L. littorea appeared to dry up and turn yellow. As shown in Figure 4(A-G), after treatment at different temperatures, the activities of CAT and POD as well as the contents of soluble sugars, total phenols, proline, soluble protein and hydrogen peroxide were measured in L. littorea seedlings. In the process of low-temperature stress, the activity of POD, soluble sugar, total phenol, proline, and soluble protein content increased significantly compared with the control. Still, the range of CAT and H 2 O 2 decreased. As shown in Figure 5, there was no significant difference in chlorophyll content under LLS and HLS treatments. Under MLS treatment, the content of chlorophyll increased, while chlorophyll a/b decreased, indicating that chlorophyll b increased more than chlorophyll a. Under SLS treatment, the content of chlorophyll decreased, while the amount of chlorophyll a/b increased, indicating that chlorophyll b decreased more than chlorophyll a and it shows that chlorophyll b is more susceptible to the influence of environmental temperature.
Furthermore, as shown in Figure 6(A-I), photosynthetic characteristics and chlorophyll fluorescence characteristics of L. littorea treated at different temperatures revealed that the photosynthesis of L. littorea seedings was severely hindered under low-temperature stress. After 48 h of treatment, all measured indicators in SLS changed significantly, with the net photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration (Tr), the fluorescence parameters for nonphotochemical (NPQ) and photochemical (qP) quenching, the maximum fluorescence (Fm), the maximal efficiency of PSII photochemistry (Fv/Fm), and the actual quantum yield of PSII photochemistry (ΦPSII) decreased to 114.45%, 85.79%, 94.83%, 73.6%, 30.17%, 62.92%, 66.28% and 87.54% in the HLS group, respectively. And the intercellular CO 2 concentration (Ci) increased to 29.34% in the HLS group.

Expression profiling of DEAD genes
To study the expression characteristics of the DEAD-box gene in the growth and development of L. littorea, the expression characteristics of 20 representative DEAD-box genes were studied in different tissues and organs ( Figure   7). LlDEAD13 are highly expressed in roots; LlDEAD68 expressed explicitly in stems; a total of 15 genes are highly expressed in leaves; LlDEAD46 highly expressed in flowers; LlDEAD1 showed highly in fruits. A total of 19 genes of those LlDEAD genes were not defined in the roots of L. littorea and most genes were highly expressed in leaves, followed by stems and flowers.

Quantitative real-time PCR analysis under cold stress
As shown in Figure 8, quantitative real-time PCR analysis was performed on 20 representative genes of the DEADbox family. These 20 genes were all significantly down-regulated under low-temperature stress.

Discussion
Based on previous studies of DEAD-box genes in rice (Tuteja et al. 2013;, wheat (Zhang et al. 2014), Arabidopsis thaliana (Gong et al. 2002;Western et al. 2002;Matthes et al. 2007;Nishimura et al. 2010;Guan et al. 2013), kale (Nawaz, Sai, et al. 2018), maize (Li et al. 2001), and tomato (Zhu et al. 2015;Cai et al. 2018; Pandey et al. 2019), DEAD-box genes are found to be structurally similar, high functional similarity and versatility so that they can be used as candidate genes to study cold resistance in plants. 73 LlDEAD genes obtained in L. littorea   In this paper, the Pn, Gs, Tr, Fm, Fv/Fm, ΦPSII of the leaves in L. littorea showed a trend of reduction under low-temperature stress, which was same with the studies of Sonneratia apetala and Kandelia candel Wang et al. 2018). The hindrance of photosynthesis in plants includes two reasons: stomatal limitation and non-stomatal limitation (Farquhar and Sharkey 1982). Under MLS, Pn, Gs and Tr of L. littorea decreased while Ci increased, indicating that stomatal limitation was the main factor in the decrease of photosynthesis. Moreover, at SLS, L. littorea was severely stressed by low temperature, and the Pn of the plant is less than 0, indicating that only respiration, without photosynthesis is present in the seedings and non-stomatal limitation plays a major role in the reduction of photosynthesis (Allen and Ort 2001;Ploschuk et al. 2014). Chlorophyll fluorescence parameters, which can reflect the state of the PSII reaction center, can reveal the intrinsic characteristics of the plant photosynthetic system better than photosynthetic parameters (Murchie and Lawson 2013). Fv/Fm and ΦPSII showed a decreasing trend with decreasing temperature, reflecting that the activity of PSII decreased (Jiang et al. 2002;Krause et al. 2010;Krause et al. 2013). Under SLS conditions, NPQ decreased, indicating a deepening of plant exposure to low temperature stress, demonstrating that non-stomatal limitation is the main cause of reduced photosynthesis.
Moreover, chlorophyll a, chlorophyll b, and total chlorophyll of L. littorea showed a trend of increasing and then decreasing with the enhanced intensity of low-temperature stress, indicating that the chlorophyll synthesis system was disturbed, which was also a reason for the decrease of photosynthesis. The osmoregulatory substances such as soluble protein, proline, and total phenols increased significantly under low-temperature stress and were used to maintain osmotic pressure homeostasis, which was similar to the results of the study in Kandelia candel (Yong et al. 2011), but a further drop in temperature, proline, and total phenols Data is expressed as the mean ± standard error of three independent biological replicates. SLS indicates seedlings treated at a temperature of 8°Cday/5°C night for 48 h; MLS indicates seedlings treated at a temperature of 15°Cday/12°C night for 48 h; LLS indicates seedlings treated at a temperature of 25°Cday/23°C night for 48 h; and HLS indicates seedlings treated at a temperature of 34°Cday/30°C night for 48 h. The experiments were repeated for three times. Different letters above the bars indicate a significant difference determined by one-way ANOVA with Tukey's test (P < 0.05).
showed a decrease, indicating that their synthesis mechanism was blocked and the defense function of the organism was weakened. The results of CAT were similar to the results of the study on Cerops tagal, both with the drop in temperature and the inhibition of enzyme activity, probably due to the impairment of the physiological metabolic sites of CAT by low temperature (Zhong et al. 2012). The activity of POD showed an increasing trend with decreasing temperature, the same as in the study of Kandelia candel (Yong et al. 2011), probably due to the dual role of POD in both scavenging H 2 O 2 and mediating the conversion of H 2 O 2 to hydroxyl radicals, so the decrease in H 2 O 2 content may due to the scavenging effect of POD.
As the primary site of photosynthesis, the maintenance of chloroplast function is vital for plant growth and development under abiotic stresses (Zhao et al. 2020). The ability of plants to survive in adversity is positively correlated with the expression of genes regulating the chloroplast stress response (Nawaz and Kang 2019). DEAD-box gene is the majority of the second subfamily of RNA helicases whose mechanism of function involves every step of RNA metabolism, including nuclear transcription, precursor mRNA splicing, ribosome biogenesis, nucleoplasmic translocation, translation, RNA decay, and organelle gene expression (de la Cruz et al. 1999;Lorsch 2002).
AtRH3 is a chloroplast-localized RNA helicase, which plays a role in the splicing of introns and the assembly of 50S ribosomal particles (Asakura et al. 2012). And AtRH3 plays an essential role in the processing of rRNA precursors during the conversion of plastids to chloroplasts, which is vital for the maintaining normal chloroplast function (Lee et al. 2013). Furthermore, the correct splicing of introns controlled by the RNA chaperone activity of AtRH3 has an essential role in the low-temperature response of plants, as proven by the severely inhibited growth of AtRH3 knockout mutant plants under low-temperature stress (Gu et al. 2014). By evolutionary analysis, LlDEAD44 and LlDEAD45 in L. littorea have a close evolutionary relationship with AtRH3, and it can be guessed that LlDEAD44 and LlDEAD45 may play a similar function. Validation by realtime fluorescence quantitative PCR revealed that these two genes were significantly down-regulated under low-temperature stress. RH42 is an essential gene in the low-temperature response of plants. It is required for the precise regulation of cold-induced precursor mRNA splicing in rice during mRNA maturation at low temperatures (Guan et al. 2013). Combined with evolutionary analysis and real-time fluorescence quantitative PCR verification, it is hypothesized that LlDEAD36 has a possible role in mRNA splicing under low temperature stress. It has been shown that AtRH22 can play a role in the assembly of 50S ribosomal subunits in chloroplasts and the maintenance of plastid mRNA levels through its RNA chaperone activity (Chi et al. 2012;Chi et al. 2012;, and play a positive role in abiotic stresses such as low-temperature stress by affecting the translation of chloroplast genes (Tripurani et al. 2011). AtRH38/LOS4 is a cold stress response gene that plays a positive role in plant response to cold stress by regulating the export of RNA molecules from the nucleus to the cytoplasm (Gong et al. 2002;Gong et al. 2005). In addition, AtRH7 governs plant growth and development and responds to cold temperatures by participating in ribosome assembly (Lorković et al. 1997). It can interact with a molecular chaperone, Arabidopsis thaliana cold-excited structural domain protein 3 (AtCSP3) improved plant tolerance to low temperatures . Through evolutionary analysis, it can be inferred that LlDEAD48 and LlDEAD47, which are more closely related to the evolution of AtRH38 and AtRH7, may have the function of maintaining chloroplast function under low temperature stress, and the specific functions need to be verified later. Under low-temperature stress, reactive oxygen species accumulate rapidly in plants, resulting in oxidative damage to the organism. Existing studies have shown that OsBIRH1 is a DEAD-box gene and transgenic plants with overexpression of OsBIRH1 exhibit increased tolerance to oxidative stress. They increased expression levels of oxidative defense genes AtApx1, AtApx2, and AtFSD1, which are essential for scavenging the accumulation of reactive oxygen species under low-temperature stress (Li et al. 2008). LlDEAD34 had a close evolutionary relationship with RH1. Combined with the decrease in H 2 O 2 content and the increase in the activities of antioxidant enzymes such as CAT and POD, it can indirectly prove that LlDEAD34 may reduce the reactive oxygen species damage due to low temperature by increasing the activities of antioxidant enzymes.
Mitochondrial energy metabolism is an essential cellular process for plant growth and survival under low-temperature stress and normal growth conditions (Zsigmond et al. 2008). DEAD-box RNA helicases (RHS) can aid in the formation of mature RNA function in mitochondria (Cordin et al. 2006). For instance, PMH1 (AtRH9) is a mitochondrial localization protein with the potential to function as an RNA chaperone required for the formation or maintenance of intron complex RNA secondary structures, aiding the formation of functionally mature RNAs in mitochondria (Cordin et al. 2006). Both AtRH9 and AtRH25 were up-regulated under cold stress with different nucleic acid binding properties between them (Kim et al. 2008). The evolutionary relationship analysis showed that LlDEAD43 have a high similarity in structure to AtRH25 that has been demonstrated to be Figure 8. The expression of 20 representative LlDEAD genes under different temperature treatments was analyzed by qRT-PCR. The relative expression of each gene relative to SLS was calculated. The treatment temperature of treatment SLS was 8/5°C (day/night), the treatment temperature of treatment MLS was 15/12°C (day/night), the treatment temperature of treatment LLS was 25/23°C (day/night), and the treatment temperature of treatment HLS was 34/30°C (day/night). The treatment time was 48 h and the bars are standard deviations (SD) of three biological replicates. Different letters above the bars indicate a significant difference determined by one-way ANOVA with Tukey's test (P < 0.05). functional in cold stress in Arabidopsis thaliana. These predicted genes are the priority genes for our subsequent studies to inprove cold resistance in L. littorea.

Conclusions
In this study, we investigated the response mechanism of L. littorea under cold stress from two aspects: physiological changes and DEAD-box gene family analysis. Our physiological data showed that cold stress severely impaired photosynthesis in L. littorea, which radically reduced net photosynthetic rate, stomatal conductance, and transpiration rate. We identified 73 DEAD-box genes in L. littorea by transcriptome data analysis. After evolutionary relationship analysis, we found that LlDEAD44, LlDEAD45, LlDEAD36, LlDEAD47, and LlDEAD48 might play a role in the maintenance of chloroplast function under low-temperature stress, LlDEAD34 might be associated with the scavenging of excess ROS caused by low-temperature stress, and LlDEAD43 might be involved in the maintenance of mitochondrial function. These potential cold-responsive genes provide the basis for our subsequent studies to improve cold resistance in L. littorea.

Notes on contributors
Lulu Hao, a master student, focuses on resistance research of mangrove plants.
Ying Zhang, a professor of phytoecology, researches the endangered mechanisms of mangrove species. She recently published Comparative Transcriptome Reveals the Genes' Adaption to Herkogamy of Lumnitzera littorea (Jack) Voigt.
Yin Li, an undergraduate student.
Chunfang Zheng, a professor of physiology, researches the chilling resistance mechanism of mangrove plants. She has recently published academic papers such as Transcriptome analysis of Sonneratia caseolaris seedlings under chilling stress.
Danfei Yue, a master's student, studied the cold resistance of mangrove plants.
Huiyu Zhang, a master's student, studied the cold resistance of mangrove plants.