Os01g0104100

OsCOIN is expressed in all rice organs and strongly induced by low temperature, ABA, salt and drought.

Annotated Information

OsCOIN, a putative RING finger protein, is localized in both nuclear and cytoplasm membrane. OsCOIN is expressed in all rice organs and strongly induced by low temperature, ABA, salt and drought. Overexpression of OsCOIN in transgenic rice significantly enhances the proline content of the cells and tolerance to cold, salt and drought treatment. Our data supported a hypothesis that OsCOIN enhances ABA- dependent tolerance to cold, salt and drought by increasing the proline level in the cells.

Function

Cold (ranges 0–12°C) stress is one of the many obstacles for production of rice (Oryza sativa L.) cultivation in temperate zones and high-elevate environments. An important breeding objective in these regions is to develop cultivars tolerant to low temperatures at critical growth stages. Rice plants are injured at the seedling stage when they are grown in early spring in temperate or subtropical environments. The types of low-temperature effects on seedlings can be manifested as poor germination, slow growth, discoloration or yellowing, withering after transplanting, reduced tilling, and stunted growth. ABA as a phytohormone plays a critical role in response to various stress signals. The application of ABA to plant mimics the effect of a stress condition. Numerous studies have shown that ABA is essential for the normal adaptive response to water stress imposed by drought or high salinity. Many of the biochemical and physiological changes under stress result from ABA-induced changes in gene expression patterns. The importance of ABA in low-temperature adaptation is somewhat controversial. The endogenous ABA level increases under low-temperature conditions and application of exogenous ABA enhances cold tolerance of non-acclimatized plants. Gene expression induced by ABA often relies on the presence of cis acting element called ABRE. All three common abiotic stresses, drought, low temperature and high salinity, cause an accumulation of compatible solutes and antioxidants, such as sugars, proline.

Zinc finger proteins as members of transcriptional factors are further grouped into the subfamilies of TFIIIA, WRKY, Dof, LIM, and RING finger. In plants zinc finger proteins are involved in growth, development and responses to environmental stresses. bZIP proteins contain a region of basic amino acids followed by a region containing at least three to four repeats of Leu or another hydrophobic amino acid. The hydrophobic region mediates homodimer formation, whereas the basic region is involved in DNA binding. A notable exception is the cold-induced LIP19 in rice, which does not bind to DNA directly but rather binds to OsOBF1, another new bZIP protein, to form a heterodimer. ZFP245, a C2H2-type zinc finger protein, probably plays a role in cold and drought responses in monocots.

OsCOIN (Oryza sativa cold-inducible) is a novel cold-inducible gene in rice. It can increase tolerance to chilling, salt and drought, and enhance proline level in rice. Overexpression of OsCOIN in transgenic rice lines significantly enhances the proline content of the cells and improves tolerance to cold, salt and drought treatment.

Expression

The promoters of stress responsive genes have typical cis-regulatory elements like DRE/CRT, ABRE, MYCRS/MYBRS and are regulated by various upstream transcriptional factors. These upstream transcription factors fall in the category of early genes and are induced within minutes of stress. Our recent studies suggest that MYB3R-2 is involved in the gene control network in plant. The transcriptional activation of some of these genes including RD29A has been well worked out. The transcription factors CBF1, 2 and 3, are cold responsive and in turn bind DRE/CRT elements and activate the transcription of various stress responsive genes. The expression of the stress responsive genes leads to physiological responses.

Isolation and characterization of OsCOIN gene

To isolate the cold-inducible gene (GenBank AK104280) in rice (Oryzia sativa L.), the full-length OsCOIN cDNA was ampliWed by RT-PCR, cloned into pGEM-T easy and sequenced.

The cDNA of OsCOIN consists of 1,593 nucleotides, which include a 1,089-bp ORF (open reading frame) from 143 to 1,231 bp. The ORF encodes a putative protein of 363 amino acids with a predicted molecular mass of 42 kDa and a pI of 5.25. Analysis of the deduced amino acid sequence reveals that this protein may contain a conserved cysteine-rich domain of 40–60 residues (called“RING finger”) (Fig. 1a) that binds two atoms of zinc. The 3D structure of the zinc ligation system is referred to as“cross-brace” motif. This atypical conformation is also shared by the FYVE (PDOC50178) and PHD (PDOC50016) domains. Yeast hybrid assay determined that OsCOIN protein had no trans-activation activity (data not shown). Bioinformatic analysis revealed that the promoter of OsCOIN gene had the cold, ABA, salt and drought responsive elements (Fig. 1b). Multiple-alignment of OsCOIN with other related proteins showed that the deduced amino acid sequence of OsCOIN has more than 61% identity to gi21207099 (Zea mays), gi30694045 (Arabidopsis thaliana), gi47104584 (Lycopersicon esculentum) in the public database (Fig. 1c), indicating that these four plant proteins may belong to the same family.

OsCOIN protein is a nuclear and cytoplasma protein

The subcellular localization of OsCOIN had been studied in vivo using the transient transfection assay. Figure 2d showed that the OsCOIN–GFP fusion protein was located at the nuclear and the cytoplasm membrane under a confocal microscope. Under the same conditions, comparatively, GFP alone was distributed in the whole cell (Fig. 2b). The results suggested that OsCOIN was a protein located at nuclear and cytoplasm membrane.

OsCOIN expression in various rice organs and inductionby cold, ABA, salt and drought

The expression of the OsCOIN gene in various organs of the wild-type rice plants were analyzed by semi-quantitative RT-PCR using tubulin as an internal control. As shown in Fig. 3a, the OsCOIN gene is expressed in all organs of the wild-type rice plants indicating that this gene could be a house-keeping gene.

As shown in Fig. 3b, a huge increase in OsCOIN mRNA was already observed in rice seedlings (WT) after being exposed to cold (4°C) for only 30 min and this high level of expression was maintained for at least 48 h. At 72 h the OsCOIN expression declined to the level before the cold stress.

To examine the effect of exogenous ABA on OsCOIN expression, 2-week-old seedlings of wild type rice were immersed into the solutions with different concentrations of exogenous ABA. As shown in Fig. 3c, 50 μM of ABA induced the maximum OsCOIN expression. Analysis of the time course of OsCOIN induction by ABA showed that exogenous ABA (50 μM) resulted in a rapid induction of OsCOIN mRNA in the 2-week-old rice seedlings, reaching the peak around 1 h and maintaining the maximum level for up to 72 h, and then returned to the normal level after 96 h (Fig. 3d).

Figure 3e showed that 100 mM of NaCl induced the maximum OsCOIN expression in the 2-week-old rice seedlings. Analysis of the time course of OsCOIN induction by NaCl showed that 100 mM of NaCl treatment resulted in a rapid induction of OsCOIN mRNA in the 2-week-old rice seedlings, reaching the peak around 3 h and maintaining the maximum level for up to 60 h, and then returned to the normal level after 96 h (Fig. 3f).

To test the effect of the drought treatment on OsCOIN expression, 2-week-old seedlings of wild type rice were immersed into the solutions with different concentrations of PEG6000. Figure 3g showed that the maximum OsCOIN expression was induced by 10% of PEG6000. Analysis of the time course of OsCOIN induction by PEG6000 showed that 10% of PEG6000 treatment resulted in a very rapid induction of OsCOIN mRNA in the 2-week-old rice seedlings, reaching the peak around 1 h and maintaining the maximum level for up to 72 h, and then returned to the normal level after 96 h (Fig. 3h).

Identification of OsCOIN transgenic rice plants

To explore its function, the OsCOIN gene driven by an ubiquitin promoter together with a GUS gene as a marker was transformed into rice plants by the Agrobacterium transformation approach as described in experimental procedures. Southern blot with a probe of GUS gene showed that one hybridized band at different position (3, 4, and 6 kb for EcoRI digestion, and 2, 2.5, and 6 kb for HindIII) was presented in each of the three independent transgenic rice lines (Fig. 4a), indicating that the exogenous OsCOIN gene was integrated into the rice genome. The expression level of OsCOIN in T2 transgenic plants was analyzed by RTPCR. As shown in Fig. 4b, OsCOIN expression in the three OsCOIN transgenic rice lines was stronger than that in the wild type rice.

OsCOIN transgenic rice is more tolerant to cold, salt and drought

To examine the effect of overexpression of OsCOIN gene on cold tolerance, 2-week-old rice seedlings of both wild type and OsCOIN transgenic rice (T2 generation) were exposed to chilling temperature (4°C) for 60, 72, and 84 h after the MS agar had been washed off, and then returned to the normal growth conditions to allow their recovery. After 2 weeks in the greenhouse, the OsCOIN transgenic plants treated for 60, 72, and 84 h, re-grew 76.2, 71.4 and 50% respectively, while the wild type rice under the same conditions re-grew only 52.4, 22.2 and 14.8%, respectively (Fig. 5a, b). To analyze the effect of overexpression of OsCOIN gene on tolerance to drought, the 2-week-old seedlings of wild type and the OsCOIN transgenic rice plants were submerged in the solutions containing 20% of PEG6000 for 24 h after the MS agar had been washed off.

As shown in Fig. 6a, b, 92% of the leaves from the WT rice were rolled while only 8% of the leaves from the transgenic rice (T2 generation) were rolled. After 2 weeks culturing in the greenhouse for their recovery, the OsCOIN transgenic plants treated for 12 and 24 h, re-grew 85 and 60%, respectively, while the wild type rice under the same conditions re-grew only 40 and 7.5%, respectively (Fig. 5c, d). To examine the effect of overexpression of OsCOIN gene on salt tolerance, the seeds of both wild type and OsCOIN transgenic rice (T2 generation) were submerged in 1/2 MS medium with 250 mM NaCl and germinated for 20 days. As shown in Fig. 6c, d, the seeds from the OsCOIN transgenic rice germinated faster (35%) and grew taller than that from the wild type rice.

Expression of cold-responsive genes in OsCOIN transgenic rice

To investigate the possible gene regulation mechanisms of cold tolerance mediated by OsCOIN, we analyzed the expression of several known cold-induced genes by RTPCR. As demonstrated in Fig. 7a, the expression levels of OsNAC6, OsP5CS and OsLti6b were increased in OsCOIN transgenic rice compared with the levels in wild type rice after cold treatment (4°C) for 24 h. Interestingly, the expression of OsLti6a in transgenic rice remains the same as in wild type rice.

Evolution

Bioinformatic analysis showed that the OsCOIN protein had no classic mono-partite or bi-partite nuclear localization signal, but it is localized in both the nuclear and the cytoplasm membrane (Fig. 2). The reason is still unknown why the OsCOIN protein can enter the nucleus without an NLS. It is possible that the OsCOIN protein enters the nucleus by forming a heterodimer with another NLS-containing protein, just like OSISAP1, a zinc finger protein from rice. Another possibility is that a new kind of NLS is involved in the nuclear localization of the OsCOIN protein.

New experiments will be underway to determine this possibility. Some studies showed that the cold-response pathway was ABA-dependent. ABA level increased in response to low temperature and a set of cold-responsive genes such as OsLti6a/b were responsive to exogenous application of ABA. Thus, the expression of these cold-responsive genes was due, at least in part, to elevated levels of ABA. Nevertheless, recent effort has proved that cold-induced gene expression can also proceed through at least one ABA- independent pathway. The expression of OsCOIN was induced by exogenous ABA suggesting that the expression of the OsCOIN is ABA-dependent in rice(Fig. 3d). It is very interesting that the expression of OsP5CS was increased 2.2-fold in our OsCOIN transgenic rice compared with the wild type rice (Fig. 7a).

OsP5CS, an enzyme (delta1-pyrroline-5-carboxylate synthetase) induced by high salt, dehydration, ABA and cold treatments, was involved in the biosynthesis of proline. Proline has been reported to play roles in protecting enzymes from denaturation, stabilizing the machinery of protein synthesis, regulating the cytosolic acidity, increasing water-binding capacity, and acting as a reservoir of carbon and nitrogen source. Our results showed that the levels of proline in the OsCOIN transgenic rice were increased over threefold after 4°C cold treatment for 24 h compared with the levels of proline in the WT rice, although they remained at about the same level in both the OsCOIN transgenic rice and WT rice in the absence of any stress treatments (Fig. 7b).

Therefore, our OsCOIN transgenic rice increased the tolerance to cold, salt, and drought at least partially by increasing the concentration of proline in the cells. Previously the two closely related genes, OsLti6a and OsLti6b, have been shown to exhibit tissue-specific differential expression. OsLti6a showed high expression only in shoots in rice seedlings under cold stress and OsLti6b, in both shoots and roots. Both are involved in preserving the integrity of the plasma membrane. In the 2-week-old seedlings of our OsCOIN transgenic rice lines the expression of OsLti6b is increased during cold treatment while that of OsLti6a remains about the same (Fig. 7a).

Labs working on this gene

Plant materials and growth conditions

All rice materials used in the experiments were Oryza sativa L. cv Zhonghua 10. All rice plants (both wild type and trans-genic) were grown in Welds in natural conditions or in the greenhouse at 28°C/25°C (day/night) with a 16 h photoperiod under a relative humidity of 50%. For physiological experiments, the seeds of T2 transgenic and wild type rice were allowed to germinate in 30°C in darkness for 2 days, and then transferred to agar plates containing half of MS (Murashige and Skoog) medium and continued to culture for 12 days at 25–28°C with a 16 h photoperiod. To measure the OsCOIN expression in diVerent rice organs 2-week-old seedlings, young roots from trifoliate stage and other organs from adult rice were collected and used in RT-PCR.

Isolation of OsCOIN and construction of vectors

Total RNA from 2-week-old rice seedlings was isolated using Trizol Reagent (Invitrogen life technologies, Carlsbad CA 92008, USA), and then reverse transcribed as described. The first strand cDNAs were used as templates for PCR amplification with 5’-GGGGTACC ATG AGCTCT CTA TGC CCC TTT GCC A-3’ (KpnI site underlined) and 5’-GGGGATCC CTT GTC ATC CAA TTGTTT TTG TAG A-3’ (BamHI site underlined) as primers. PCR was performed with LA Taq DNA polymerase as follows: preheating at 94°C for 5 min, then 35 cycles of denaturation at 94°C for 1 min, annealing at 56°C for 30 s and extension at 72°C for 1 min, followed by a final extension at 72°C for 10 min. The PCR products were cloned into pGEM-T easy and sequenced. The OsCOIN gene fragment was then obtained by digestion with KpnI and BamHI and cloned downstream of a maize ubiquitin promoter in the pUN1301 vector (Ge et al. 2004). PCR and enzyme digestion were used to identify the positive clones.

Subcellular localization

The OsCOIN cDNA was amplified with the primers 5’-GCTCTAGA ATG AGC TCT CTA TGC CCC TTT GCCA-3’ (F) (XbaI site underlined) and 5’-GGGGTACC CTTGTC ATC CAA TTG TTT TTG TAG A-3’(R) (KpnI site underlined). The PCR products were digested with XbaI and KpnI, and ligated with XbaI and KpnI double-digested pGFP221 to created pGFP-OsCOIN, in which the coding region of the OsCOIN gene was fused to the N terminus of the GFP in frame, under the control of the cauliXower mosaic (CaMV) 35S promoter. The fusion construct and the GFP control vector were transformed into the onion epidermis cells by particle bombardment according to the protocol described. The transformed cells were cultured on MS medium for 24 h and observed under a confocal microscope.

Generation of OsCOIN transgenic rice lines

Rice calli were induced on scutella from germinated seeds and transformed with strain EHA 105 of Agrobacterium tumefaciens containing the desired binary vector, as described. Transgenic rice plants were selected in half-strength MS medium containing 75 mg L-1 hygromycin. Hygromycin-resistant plants from calli, designated as the T0 generation, were transplanted into soil and grown in a greenhouse at 28°C. To confirm the trans- genic rice plant further, seeds of the T0 generation were germinated in half-strength MS medium containing 75 mg L-1 hygromycin and analyzed by GUS staining. T2 generation of transgenic rice plants was used in subsequent experiments.

Southern blot analysis

Genomic DNA (20 μg), purified from transgenic or wild type rice plants (T2 generation), was digested with EcoRI or HindIII at 37°C for 20 h and then separated by 0.8% agarose gel. The genomic DNA was transferred and cross-linked onto a nylon membrane according to a protocol described. The GUS probe was synthesized by PCR with primers 5’-GCA GTG TAC GTC CTG TAG AAA CCC-3’ (F) and 5’-CAA AGC CAG TAA AGT AGA ACG GT-3’ (R) and labeled with [32P] dCTP. After pre-hybridization of about 6 h, the heat-denatured probe was added and then hybridized for another 20 h at 65°C. The membrane was washed twice with 2£ SSC plus 0.1% SDS at 65°C, and once with 1£ SSC plus 0.1% SDS at 65°C. The membrane was exposed to the X-ray film at -70°C for 1 day or more.

Four-degrees cold, ABA, NaCl and drought treatments

Rice seeds were sterilized and then germinated in half of MS agar medium before the treatments. To examine the time course of OsCOIN expression under cold stress, the cold treatment was accomplished by transferring the chamber with the 2-week-old seedlings of WT rice plants into a prechilled (4°C) Low Temperature Biochemical Incubator. At different time points samples were withdrawn from the Low Temperature Biochemical Incubator and frozen in liquid nitrogen immediately for subsequent RNA extraction. To examine the expression of OsCOIN under exogenous ABA, salt and drought stress, 2-week-old seedlings of the wild type rice (T2 generation) were treated in medium in the chamber with different concentrations of ABA, or NaCl, or PEG6000 for 6 h. Once the optimal concentrations for ABA, NaCl and PEG6000 have been determined, the time courses of OsCOIN induction by ABA, NaCl or PEG6000 have also been examined. To investigate the effect of overexpression of OsCOIN gene on salt tolerance, the seeds of both wild type and OsCOIN transgenic rice (T2 generation) were submerged into 1/2 MS medium with 250 mM NaCl and germinated for 20 days. To examine the effect of overexpression of OsCOIN gene on cold tolerance, 2-week-old rice seedlings of both wild type and OsCOIN transgenic rice (T2 generation) were exposed to chilling temperature (4°C) for 60, 72, and 84 h after the MS agar had been washed off, and then returned to the normal growth conditions to allow their recovery. To examine the effect of overexpression of OsCOIN gene on drought tolerance, 2-week-old seedlings of both wild type and OsCOIN transgenic rice (T2 generation) were submerged into the medium with 20% of PEG6000 for 24 h after the MS agar had been washed off.

RT-PCR analysis

Total RNA were extracted from the 2-week-old seedlings of the wild type or transgenic rice plants (T2 generation) describe above using Trizol Reagent (Invitrogen life technologies, Carlsbad CA 92008, USA), and then reverse transcribed as described. For the first-strand cDNA synthesis, 2 μg of total RNA was reversed-transcribed in a total volume of 20 μl reaction buffer that contained 10 ng of oligo(dT)-18 primer, 2.5 mM dNTP, and five units of AMV reverse transcriptase. PCR was performed in a 20 μl solution containing a 1 μl of the cDNA template, 0.1 μM of gene-special primers, 2 mM dNTPs, one unit of LA Taq DNA polymerase. The reaction included an initial 5 min denaturation at 94°C, followed by 20–40 cycles of PCR (94°C 1 min, 56°C 30 s, 72°C 1 min), and a final 10 min extension at 72°C. The PCR products were separated on a 0.8% agarose gel. The primers used for gene-specific PCR were listed as follows: (a) OsNAC6: 5’-CAT GGC CGG TGA ACT TTG AC-3’ (F), 5’-CTC GTCGTC GTT CAG TCC AG-3’ (R); (b) OsLti6a: 5’-AATACT GCG AGA GAA ATT AAT CA-3’(F), 5’-TAAGAG GGG AGC TTA TTC ACA C-3’(R); (c) OsLti6b: 5’-GCC TTA AAT TGG AGC TCA GTC-3’ (F), 5’-GTGCAG AAG ATA AAC TGG AGA A-3’(R); (d) OsP5CS:5’-AAG ATG GAA GAT TGG CTT TGG GCA G-3’ (F),5’-TCT CGT GTA GGT AGA GGA GGC ATG A-3’ (R);(e) OsTubulin: 5’-TCA GAT GCC CAG TGA CAG GA-3’(F), 5’-TTG GTG ATC TCG GCA ACA GA-3’(R); (f)OsCOIN: 5’-ATG AGC TCT CTA TGC CCC TTT GCCA-3’(F), 5’-CTT GTC ATC CAA TTG TTT TTG TAG A-3’(R).

Determination of cellular proline levels

Two-week-old seedlings (0.5 g) from wild type and OsCOIN transgenic rice (T2 generation) with or without cold treatment were homogenized in 2 ml of 3% aqueous sulfosalicylic acid and centrifuged. Free proline amount was measured by using spectrophotometer and reported as μmol g FW-1.

References

Kaimao Liu;Lei Wang;Yunyuan Xu;Na Chen;Qibin Ma;Fei Li;Kang Chong, Overexpression of OsCOIN, a putative cold inducible zinc finger protein, increased tolerance to chilling, salt and drought, and enhanced proline level in rice Planta, 2007, 226(4): 1007-1016

Aguan K, Sugawara K, Suzuki N, Kusano T (1993) Low-temperature-dependent expression of a rice gene encoding a protein with a leu-cine-zipper motif. Mol Gen Genet 240:1–8

Andaya VC, Mackill DJ (2003) Mapping of QTLs associated with coldtolerance during the vegetative stage in rice. J Exp Bot 54:2579–2585

Bates LS, Waldren RP, Teare ID (1973) Rapid determination of free proline for water-stress studies. Plant Soil 39:205–207