Molecular characterization and function analysis of the rice OsDUF1664 family

Abstract The functions of a large number of genes, including gene families with domains of unknown functions (DUF), still remain unclear. In this study, we analyzed four members of OsDUF1664 (OsDUF1664.1-OsDUF1664.4) in rice Nipponbare. By phylogenetic analysis, DUF1664 members in rice and Arabidopsis were classified into three major groups (I, II, III). Under drought conditions, the expression level of OsDUF1664.3 in rice was significantly elevated. Overexpression of OsDUF1664.3 in Escherichia coli led to a significant enhancement of catalase (CAT) and peroxidase (POD) activities, and improved bacterial resistance to drought. The results of this study will provide important information for further study of the function of the OsDUF1664 family in rice.


Introduction
Due to the variability of external environmental factors, plants often face the stresses of one or more adverse environmental factors, which ultimately affect the normal growth and development of plants [1]. Abiotic stresses in environmental stress mainly include drought stress, salt stress, high and low temperature stress, nutrient stress and heavy metal stress [2]. Affected by abiotic stress, the average yield of crops can even be reduced by more than 50%, and by 2050, world food production needs to increase at least twice to meet the needs of population growth [3]. Drought is an important limiting factor for food production. In general, drought affects the normal growth and development of plants by affecting the plasma membrane, the photosynthetic rate and disrupting the levels of plant hormones [4,5]. In order to avoid the harmful effects of the changing environment, plants have developed a number of physiological and biochemical strategies [6], but the specific regulatory mechanisms for drought are not fully understood.
The domain of unknown function (DUF) is a protein domain with unknown function. When a protein family with no functional annotation is added to the Pfam database (http://pfam.xfam.org/family), this family is named DUF plus a number. Currently, there are approximately 3000 unknown functional domain protein families that are collected in the protein family database, accounting for 26.5% of the protein families (Pfam) [7,8]. At present, the functions of only a few DUF proteins have been elucidated recently. Some DUF-containing proteins are involved in plant growth and development, such as members of DUF231, DUF724 and DUF1218 families [9][10][11], while some other DUF-containing proteins [12][13][14], play important roles in plant stress responses. TaSRG, a wheat DUF662 domain-containing transcription factor, significantly affects salt tolerance in transgenic rice and Arabidopsis [12]. Arabidopsis RING-DUF1117 E3 ubiquitin ligases genes AtRDUF1 and AtRDUF2 are induced by ABA and drought stress, and suppression of Arabidopsis AtRDUF1 and AtRDUF2 reduced tolerance to ABA-mediated drought stress [13]. OsDSR2, encoding a protein with a DUF966 domain, negatively regulated rice response to salt and simulated drought stresses as well as ABA signaling [14]. The transcriptional levels of some DUF1618 gene family members varied under stress and hormone treatment conditions, so it was suggested that some DUF1618 members were involved in stress responses [15]. OsSIDP366, a DUF1644 gene, positively regulates salt and drought resistance in rice [16].
The OsDUF1664 family consists of plant proteins of unknown specific function with an average length of approximately 100 residues. There are five DUF1664 members in Arabidopsis and the function of all members is also not reported. Currently, the OsDUF1664 family in rice has not been functionally identified. In this study, we focused on the identification and functional analysis of the OsDUF1664 family. We also studied the expression profiles of four members of OsDUF1664 and their expression patterns under various stresses. We found that overexpression of OsDUF1664.3 in Escherichia coli could improve the tolerance to drought.

Database searches
The sequence of assumed OsDUF1664 family proteins in rice was obtained by retrieving the rice genome annotation project(RGAP version 7)database (http:// rice.plantbiology.msu.edu/) with domain number PF07889. Then we downloaded all the related protein sequences of the OsDUF1664 family from RGAP and confirmed with the SMART database (http://smart. embl-heidelberg.de/smart/batch.pl) [17]. Five DUF1664 members' sequences in Arabidopsis were also downloaded from the Arabidopsis information resource (https://www.arabidopsis.org). If there were several gene models at one locus, only the complete gene model was selected for further sequence analysis.

Sequence analysis
The information about chromosomal localization, amino acid length, full-length cDNA accessions for each OsDUF1664 family member was obtained from the rice genome annotation project. The protein localization of OsDUF1664 members was predicted by online protein location prediction software TargetP 2.0 (http://www.cbs.dtu.dk/services/TargetP/) [18]. Sequence alignment was done using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo). In order to understand the evolutionary relationship among members of DUF1664, we constructed a neighbor-joining (NJ) phylogenetic tree with 1000 bootstrap replicates using MEGA4 [19].

Plant growth and various stress and ABA treatment
Rice (Oryza sativa L. subsp. japonica cv. Nipponbare) seeds were germinated for 3 days before they were transferred to a 30-L nutrient solution container for hydroponic growth [20]. In order to further understand the expression patterns of OsDUF1664 family members, 15 different tissues of rice Nipponbare under normal conditions were sampled, as follows: Lb1, leaf blade at the four-leaf stage; Lb2, leaf blade from plants with four tillers; Lb3, leaf blade at the ripening stage; Ls1, leaf sheath at the four-leaf stage; Ls2, leaf sheath from plants with four tillers; Rt1, root at the four-leaf stage; Rt2, root from plants with four tillers; St1, stem from plants with four tillers; St2, stem at the ripening stage; An, 1.2-1.5 mm anther; Pi, pistil from 10-14 cm inflorescence; Em1, embryo at 7 days after flowering; Em2, embryo at 28 days after flowering; En1, endosperm at 7 days after flowering; En2, endosperm at 28 days after flowering.
When the fourth leaf of rice seedlings appeared, they were subjected to various stresses and ABA treatment. For the salt treatment, high salinity stress was achieved by adding NaCl to the solution to a final concentration of about 200 mmol/L. For the drought tests, intact seedlings were put in the air without water supply. For the cold tests, the seedlings were transferred to a chamber at 4 C (12-h-light/12-h-dark cycle). For ABA treatment, 100 lmol/L ABA was sprayed onto the leaves of seedlings. For heat treatment, the seedlings were exposed to 42 C. Stress treatment continued until the seedlings were sampled. For salt stress and drought stress, seedlings were sampled at 0, 4, 8 and 16 h. For ABA, heat and cold stress, seedlings were sampled at 0, 1, 3, 8 h.

RNA extraction and real-time polymerase chain reaction
The total RNA was extracted using Trizol reagent (GIBCO, Burlington, ON, USA) according to the manufacturer's instructions. In order to degrade any possible DNA contamination in total RNA, it was treated with RNase-free DNase for 15 min. Real-time PCR was performed as described before [21]. The rice Actin1 gene (LOC_Os03g50885) was used as an internal control with primers 5 0 -TGGCATCTCTCAGCACATTC-3 0 and 5 0 -TGCACAATGGATGGGCCAGA-3 0 . The primers used in the expression analysis of the OsDUF1664 gene family members are listed in Table S1 in the Supplementary Appendix. The 2 -᭝᭝Ct method was used to analyze the relative expression data as mentioned previously [22].

Assay for drought stress tolerance of E. coli transformants
In order to construct the expression vector pET32a-OsDUF1664.3, specific primers were designed as follows: sense, 5 0 -GGATCCATGGTGCTTGGCAACGTCGC-3 0 (BamHI site underlined) and antisense, 5 0 -AAGCTT AACAAAGGCAACCAACCAGT-3 0 (HindIII site underlined). The amplified products were cloned into a pET32a vector at the BamHI-HindIII site to express the fusion protein, which has a TrxTag TM thioredoxin at the Nterminus [23]. The transformed E. coli Rosetta cells (Sanborn, MN, USA) were grown in Luria-Bertani (LB) liquid medium containing 100 mg/mL of ampicillin at 37 C overnight, then they were inoculated into fresh LB medium (1:100 dilution) supplemented with ampicillin (100 mg/mL) to be incubated until the exponential growth phase (optical density at 600 nm of 0.5-0.6). Isopropylthio-b-D-galactoside (IPTG) was added into the cultures to induce the expression of the transformed gene. For the drought (mimic) tolerance assay, the growth curve of E. coli transformants was plotted. The OD values of pET-32a, pET32a-

Measurements of antioxidant enzyme activity
The bacteria were collected by centrifugation. After ultrasonic cell-breakage at 4 C, the supernatant (as a crude enzyme solution) was collected by spinning at 12,000 rpm (13,200 xg rcf) for 20 min. Catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) activities were determined as described previously [24].

Data analysis
The experiment was arranged as a completely randomized design and all the experiments were repeated three times. Data are mean values from three independent experiments, with standard error of the means. Statistical analysis was performed using the Student's t-test by EXCEL version 2003.

Results and discussion
Identification and sequence analysis of OsDUF1664 family members in rice We searched the rice genome annotation project (RGAP) using domain number PF07889 and obtained four members of the OsDUF1664 family. And then these OsDUF1664 family members were confirmed by the SMART database ( Figure S1 in  The rice genome includes 10 duplicated blocks accounting for 45% of its total size [25]. Both OsDUF1664.1 and OsDUF1664.2 are located on chromosome 1, and both OsDUF1664.3 and OsDUF1664.4 are located on chromosome 5. It is possible that these genes of OsDUF1664 family members were the results of a segmental duplication event in the evolution of the genome, similar to that previously suggested for OsDUF866, OsDUF946, OsDUF936, OsDUF829 and OsDUF1191 family members [21,[26][27][28][29].

Expression patterns of OsDUF1664 family members
So far, few studies have been done about the gene expression patterns of OsDUF1664 genes. Therefore, we used real-time PCR to investigate the expression patterns of OsDUF1664 family genes in 15 samples from different tissues or stages (Figure 2). The results showed that the expression profiles of OsDUF1664.   Real-time PCR was performed to examine the expression of OsDUF1664 family genes under various stress conditions and ABA treatment (Figure 3). The ABA-signaling pathway is central to drought-and saltstress responses in plants. In addition, ABA is required for freezing tolerance, which also involves the induction of dehydration tolerance genes [30]. So ABA treatment was added when rice seedlings were subjected to various stresses. The expression of OsDUF1664.1 was significantly (P < 0.01) downregulated at the mRNA level under drought conditions. For OsDUF1664.2, the expression remained unchanged under drought, salt, cold, heat stress conditions and ABA treatment. Under drought and salt conditions, the expression of OsDUF1664.3 was significantly (P < 0.01) upregulated. For OsDUF1664.4, the expression was significantly (P < 0.01) upregulated under drought treatment, but significantly (P < 0.05) downregulated under salt conditions.
In general, gene family members display similar increase or decrease under various stresses conditions [31][32][33]. In this study, the expression of OsDUF1664 family under abiotic stresses conditions and ABA treatment was examined, and surprisingly, we found the four members of OsDUF1164 family in rice displayed greatly distinct expression patterns under various stresses conditions and ABA treatment (Figure 3). For OsDUF1164.2 and OsDUF1164.3 especially, although they had similar sequence and expression profiles in 15 samples from different tissues or stages, their coping strategies were completely different when dealing with abiotic stresses. We thus suggest that OsDUF1664 family members may play different roles in response to various stresses.

OsDUF1664.3 improved drought stress resistance in transgenic E. coli
Since the expression levels of OsDUF1664.3 were significantly elevated under drought conditions, we hypothesized that this gene may be involved in response to drought stress. We overexpressed the gene in E. coli and then examined the resistance of recombinant bacteria to drought stress. Under normal conditions, there was no evident difference in the number of colonies between transgenic lines (E. coli transformed with pET32a-OsDUF1664.3) and the control (E. coli transformed with pET-32a). Under drought conditions, the growth rate in E. coli transformed with pET32a-OsDUF1664.3 was much higher than that of the control from 4 to 18 h (Figure 4). This result indicated that the overexpression of OsDUF1664.3 in E. coli may significantly improve the resistance to drought.

OsDUF1664.3 improves the antioxidant defenses of E. coli transformants grown under drought stress conditions
In order to investigate the effects of OsDUF1664.3 on the intracellular antioxidant ability, we determined catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) activities of E. coli transformants and control bacteria. Under normal conditions, there was no evident difference in CAT, SOD and POD activities between the transgenic line and the control. Under drought conditions, CAT and POD activities of E. coli transformed with pET32a-OsDUF1664.3 were significantly (P < 0.01) higher than those of the control from 4 to 8 h, while there was no significant difference in SOD activity ( Figure 5). These results indicated that the improvement in tolerance to stress in E. coli OsDUF1664.3 transformants may be related to the increase in the activities of CAT and POD under drought stress conditions. Production of reactive oxygen species (ROS) is a frequent event in organisms when they suffer from abiotic stresses, including drought [34][35][36]. ROS production can cause oxidative damage on membrane lipids and perturbation of cell membrane function [37]. It is therefore of high importance to maintain the balance between ROS generation and ROS scavenging in cells subjected to abiotic stresses. In this study, we  After E. coli transformants were exposed to drought conditions (300 mmol/L mannitol treatment) for 0, 4, and 8 h, respectively, catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) activities were determined. Error bars indicate standard error of the means based on three biological replicates. ÃÃ P < 0.01 (Student's t test).
observed an increase in the activities of CAT and POD in transgenic E. coli. This increase possibly indicates that the extent of damage to the E. coli transformed with pET32a-OsDUF1664.3 exposed to drought stress triggered a more pronounced antioxidant defense response than that in the control bacteria exposed to the same stress.
At present, the specific mechanism by which drought tolerance is enhanced in E. coli recombinants transformed with OsDUF1664.3 remains unclear. Under drought conditions, it is possible that the drought stress signal is enhanced in E. coli recombinants transformed with OsDUF1664.3, which, in turn, promotes the expression of downstream stress-related genes, including genes encoding ROS scavenging enzymes, leading to better improvement in drought tolerance in transgenic E. coli.

Conclusions
In summary, this study describes sequence information regarding the OsDUF1664 family members and the expression patterns of OsDUF1664 family members in different tissues, and under various abiotic stresses and ABA. OsDUF1664.3 could enhance the activity of ROS scavenging enzymes and improve the resistance to drought of transgenic E. coli. These data can provide important reference for further studies of the functions of the OsDUF1664 family.

Disclosure statement
No potential conflict of interest was reported by the authors.