AtCIPK 19 aggrandizes polyamines-involved cold stress tolerance in plant cells

The CALCINEURIN B-LIKE PROTEIN-INTERACTING PROTEIN KINASEs (CIPKs) play important roles in regulating ion homeostasis and stress responses. However, molecular mechanism of the Arabidopsis (Arabidopsis thaliana) CALCINEURIN B-LIKE PROTEININTERACTING PROTEIN KINASE19 (CIPK19) enhanced cold stress tolerance is not fully understood. Here, we report that overexpression of the Arabidopsis CIPK19 gene (AtCIPK19) results in increasing cell viability and growth rate under cold stress in O. sativa, G. hirsutum, and P. strobus. AtCIPK19 increases cold stress tolerance by decreases the thiobarbituric acid reactive substances (TBARS) and increases the levels of putrescine (Put), spermidine (Spd), and spermine (Spm). In AtCIPK19 transgenic rice cell lines, the transcript levels of genes associated with biosynthesis of Put, Spd, and Spm include OsADC1, OsADC2, OsADC3, OsODC1, OsODC2, OsODC3, OsCPA1, OsCPA2, OsCPA3, OsAIH, OsSAMDC1, OsSAMDC2, OsSAMDC3, OsSAMDC4, OsSAMDC5, OsSAMDC6, OsSPD/SPM1, OsSPD/SPM2, OsSPD/SPM3, and OsSPD/SPM4 are increased under cold stress. These results will increase our understanding of AtCIPK19-related cold stress tolerance in different plant species and are valuable in plant molecular breeding application.


Introduction
Cold stress reduces plant growth and crop yield.To counteract the damage caused by cold stress, plants have developed capabilities to adapt to this extreme environment, including activation of hormone signaling and alterations of expression of cold stress-related genes.In order to fully characterize the adaptive responses of plants to cold stress, it is important to determine the final levels of mRNAs and proteins in order to reveal the precise novel regulatory mechanisms which specifically function in the response to cold stress [1].Transcription factors participate in biological processes of cold stress responses.In Prunus mume, increased expression of 15 putative PmNACs transcription factors was observed to tolerate freezing-stress [2].In rice, OsmiR156 enhances cold stress tolerance by regulating the expression of transcription factor genes [3].Membrane-to-nucleus signals modulate plant cold tolerance [4].A yeast one-hybrid assay demonstrated that AaDREB1 encodes a transcription activator and specifically binds to DRE/CRT to enhance tolerance to low temperature [5].In cucumber, CsWRKY46 was up-regulated in response to cold stress and its overexpression increased the expression of stress-inducible genes, including RD29A and COR47 [5].In K. obovata, mRNA expression analysis indicated that the KoHSP70 was increased significantly after 48 h cold stress, and reached the highest level at 168 h after cold treatment [6].In wheat and barley, high transcription level of dehydrin was observed after plants exposed to cold stress [7].In Glyycine soja, over-expression of GsZFP1 increased the expression of stress-response marker genes, including CBF1, CBF2, CBF3, NCED3, COR47, and RD29A in response to cold stress [8].
Polyamines including putrescine, spermidine, and spermine might help plants to deal with cold stress by interacting with negatively charged macromolecules and regulate their functions [9].In apple (Malus domestica), genome-wide investigation showed that 18 Sadenosylmethionine decarboxylase and spermidine synthase-related sequences were involved in polyamines biosynthesis in response to cold stressed condition [10].In rubber tree, S-Adenosylmethionine decarboxylase (SAMDC), a key rate-limiting enzyme involved in polyamines biosynthesis, plays important roles in cold stress response [11].In bamboo, the endogenous polyamines significantly increased to avoid injury during cold stress [12].In trifoliate orange [Poncirus trifoliata (L.) Raf.], miRNA396b (ptr-miR396b) positively regulates cold tolerance through reducing ACO transcript levels and simultaneously promoting polyamine synthesis [13].In Banana, the chilling tolerance induced by NO treatment might be ascribed to the enhanced catabolism of polyamine [14].In tea (Camellia sinensis) subjected to lowtemperature stress, the CsSPMS gene is quickly induced by cold stress [15].In rice, SamDC, a key enzyme in the polyamine biosynthesis pathway, functions in response to the abiotic stress treatments of cold [16,17].In Siberian spruce (Picea obovata), the accumulation of polyamines may act as compatible solutes or cryoprotectants to act on freezing tolerance development [18,19].
It has been reported that the Arabidopsis (Arabidopsis thaliana) CALCINEURIN B-LIKE PROTEIN-INTERACTING PROTEIN KINASE19 (AtCIPK19) acts as an important element to pollen tube growth [27].Overexpression of AtCIPK19 was associated with elevated cytosolic Ca 2+ throughout the bulging tip, indicating that CIPK19 may be involved in maintaining Ca 2+ homeostasis through its potential function in the modulation of Ca 2+ influx [28].Although CIPKs play important role in cold stress tolerance, how AtCIPK19 regulates cell response to cold stress is not fully understood.In this investigation, we demonstrated that AtCIPK19 increases cold stress tolerance in O. sativa, G. hirsutum, and P. strobus.In rice, AtCIPK19 enhanced cold stress tolerance by expression of OsADC1, OsADC2, OsADC3, OsODC1, OsODC2, OsODC3, OsCPA1, OsCPA2, OsCPA3, OsAIH, OsSAMDC1, OsSAMDC2, OsSAMDC3, OsSAMDC4, OsSAMDC5, OsSAMDC6, OsSPD/SPM1, OsSPD/SPM2, OsSPD/SPM3, and OsSPD/SPM4.To our best knowledge, this is the first report that describes a detailed interaction between AtCIPK19 and genes associated with biosynthesis of Put, Spd, and Spm in improving cold stress tolerance in different plant species.

Plasmid constructs
The cDNA sequence of AtCIPK19 was amplified from the Arabidopsis genome and cloned into expression vector pCAMBIA1301 as previously described [27].DNA of pCAMBIA1301 and AtCIPK19 were digested by KpnI and BamHI (Promega, Madison, WI, USA) at 37 o C. DNA was purified using QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA, USA) and ligated to generate the expression vector pCAMBIA1301-AtCIPK19. Expression vectors were introduced into Agrobacterium tumefaciens strain GV3101 by electroporation.
The neomycin phosphotransferase II (NPT II) gene was used as a select marker in the transformation.

Transformation of O. sativa, G. hirsutum, and P. strobus cells
AtCIPK19 transgenic cell lines of O. sativa, G. hirsutum, and P. strobus were generated as described before [28], using Agrobacterium tumefaciens strain GV3101 carrying pCAMBIA1301-AtCIPK19 to transform cultured cells.After transformation, cell cultures of different cell lines were growing for 4-5 week, and then these cell cultures were used for further analysis including cold stress and gene expression.

Southern blot analysis of transgenic cells
Southern blotting analysis of transgenic cells of O. sativa, G. hirsutum, and P. strobus was conducted as previously described [28].Five grams of control cells and transgenic cells were used to isolate genomic DNA, using a Genomic DNA Isolation Kit (Sigma).Twenty-five micrograms of DNA were digested 16 hours with the enzymes Xba I (Boehringer Mannheim) at 37 o C. The molecular probes (1452 pb fragment of AtCIPK19) were labeled by Digoxigenin (DIG) (Roche Diagnostics, Indianapolis, IN, USA).

RNA isolation and Northern blot analysis
Five grams of fresh cultures of transgenic and control cells of O. sativa, G. hirsutum, and P. strobus were used to isolate total RNA, using a RNeasy Mini Plant Kit (Germantown, MD, USA) by following the manual.Six g of total RNA was separated by agarose-gel electrophoresis.Electrophoresis of RNAs and northern blotting were performed as described before [28].The hybridization probe is the Digoxigenin (DIG)-labelling AtCIPk19 DNA (1452 pb) (Roche Diagnostics).The rRNA was used as the loading control of RNA samples.

Cold treatment and determination of cell viability and cell growth rate
Cold treatment was performed by incubation of AtCIPK19 transgenic cells (at the age of 4-5 weeks) and control cells of O. sativa, G. hirsutum, and P. strobus at -10, 4, 12, and 24 o C in the dark for 24 hours in growth chambers (Beijing ZNYT, China).Control seedlings were grown under the same conditions.After 24 h of chilling treatment, cells were moved to the normal growth environment.The influences of cold stress on cell growth and cell viability of O. sativa, G. hirsutum, and P. strobus were determined, as previously described [29].The average growth was expressed as mg/g FW/day.The rate of cell growth and the cell viability were measured 7 days after treatment.Samples from both chilled and control samples were collected with 3 biological replicates.

Measurement of thiobarbituric acid reactive substances (TBARS)
Lipid peroxidation was determined as the amount of thiobarbituric acid reactive substances (TBARS) measured by the thiobarbituric acid (TBA) reaction as described previously (Tang and Page 2013).Transgenic and control cells (1 g) were homogenized in 3 ml of 20 % (w/v) trichloroacetic acid (TCA), then centrifuged at 5,000 rpm for 20 min, following by mixing with 20 % TCA containing 0.5 % (w/v) TBA and100 l 4 % BHT in ethanol at 1:1.The absorbance of the extracts of different cell lines was measured at 532 nm.The value of TBARS was calculated using the method described previously [28,29].

Determination of polyamines
Determination of polyamines putrescine (Put), spermidine (Spd), and spermine (Spm) from tissues of P. strobus was carried out as described previously [30].Samples were examined using a HPLC and Spector Monitor 3200 Detector by following the manual of the facility.The measured polyamines are total PAs.Page: 7 www.raftpubs.com

Statistical analyses
Statistical analysis was performed using the General Linear Model procedure of SAS (Cary, NC, USA), employing ANOVA models.The significant differences between mean values of different cell lines derived from O. sativa, G. hirsutum, and P. strobus were made at 5 % level of probability, using the T-test.Each value of different cell lines derived from O. sativa, G. hirsutum, and P. strobus was presented as means standard errors of the mean.

Generation of transgenic cell lines in O. sativa, G. hirsutum, and P. strobus
AtCIPK19 transgenic cell lines of O. sativa, G. hirsutum, and P. strobus were generated using Agrobacterium tumefaciens strain GV3101 carrying pCAMBIA1301-AtCIPK19 (Figure 1A) to transform cultured cells.After transformation, a total of 29 transgenic cell lines of O. sativa, a total of 32 transgenic cell lines of G. hirsutum, and a total of 36 transgenic cell lines of P. strobus were produced.After transgenic cell lines of O. sativa, G. hirsutum, and P. strobus were confirmed by PCR (Figure 1B,C), Southern blotting (Figure 1D), and northern blotting analyses (Figure 1E), nine cell lines (Os1, Os2, and Os3 from O. sativa, Gh1, Gh2, and Gh3 from G. hirsutum, and Ps1, Ps2, and Ps3 from P. strobus) from each of O. sativa, G. hirsutum, and P. strobus were selected and analyzed in this study.

Overexpression of AtCIPK19 increases cell viability, cell growth rate, and decreases TBARS
To determine if overexpression of the AtCIPK19 gene increases cold stress tolerance, the cell growth rate, the cell viability, and the TBARS in transgenic cell lines of O. sativa, G. hirsutum, and P. strobus were examined.The cell growth rate and the cell viability were significantly increased in transgenic cell lines of O. sativa (Figure 2A

Overexpression of AtCIPK19 increases contents of Put, Spd, and Spm
To examine if overexpression of the AtCIPK19 gene-enhanced cold stress tolerance is related to the biosynthesis of putrescine (Put), spermidine (Spd), and spermine (Spm), the contents of Put, Spd, and Spm were analyzed in transgenic cell lines of O. sativa, G. hirsutum, and P. strobus.Overexpression of the AtCIPK19 gene significantly increased the levels of putrescine (Put), spermidine (Spd), and spermine (Spm) in transgenic cells of O. sativa (Figure 3A,B and  C), G. hirsutum (Figure 3D,E, and F), and P. strobus (Fig. 3G, H, and I), respectively, under stress at -10 o C and -4 o C. Overexpression of the AtCIPK19 gene did not significantly increase the levels of putrescine (Put), spermidine (Spd), and spermine (Spm) in transgenic cells of O. sativa (Fig. 3A, B, and C), G. hirsutum (Figure 3D,E, and F), and P. strobus (Figure 3G,H

Morphological changes of transgenic cell lines.
To examine if overexpression of the AtCIPK19 gene-increased contents of Put, Spd, and Spm caused morphological changes of transgenic cell lines, cell morphology was analyzed in transgenic cells in O. sativa (Figure 7A-D), G. hirsutum (Figure 6 7E-H

Discussion
Cold stress, which causes dehydration damage to the plant cell, is one of the most common abiotic stresses that adversely affect plant growth and crop productivity.Cold stress response is mediated by multiple signaling pathways and is regulated by many factors, among which CIPKs and polyamines may play a role.In Scots pine (Pinus sylvestris L.), temperature was a more effective signal than day-length for dehardening [36].Phosphorylation-mediated signaling transduction plays a crucial role in the regulation of plant defense mechanisms against cold stress.In tomato, about 5500 phosphoproteins were identified to be involved in cold tolerant signaling [37].In Carpobrotus edulis, cold stress reduced melatonin contents and increased salicylic acid contents [38].GhHyPRP4 gene was isolated from cotton genome.Overexpression of GhHyPRP4 in yeast (Schizosaccharomyces pombe) significantly enhanced the cell survival rate upon treatment under -20 degrees C for 60 h, indicating that GhHyPRP4 may be involved in plant response to cold stress [39].Expression of dehydrins is upregulated under cold stress, suggesting their involvement in membrane stabilization [40].In Brassica napus, BnCOR25 was expressed at high levels in hypocotyls, cotyledons, stems, and flowers, and BnCOR25 transcripts were significantly induced by cold stress treatment [41].
The signal transduction of the plant hormone abscisic acid (ABA) has been studied extensively.ABA plays an important role in improving plant tolerance to cold via integrating sugars and reactive oxygen species signaling pathways [42][43].Plants counteract cold stress through signal transduction and molecular genetic regulation.In our study, genetic and biochemical assays were performed to explore the effect of AtCIPK19 in response Page: 15 www.raftpubs.com

Conclusion
In this investigation, we identified a mechanism of AtCIPK19 enhanced cold stress tolerance.We found that overexpression of AtCIPK19 increased cell viability and growth rate under cold stress.AtCIPK19 increased cold stress tolerance by regulating expression of genes associated with putrescine biosynthesis including OsADC1, OsADC2, OsADC3, OsODC1, OsODC2, OsODC3, OsCPA1, OsCPA2, OsCPA3, OsAIH.In AtCIPK19 transgenic rice cell lines, the transcript levels of OsSAMDC1, OsSAMDC2, OsSAMDC3, OsSAMDC4, OsSAMDC5, OsSAMDC6, OsSPD/SPM1, OsSPD/SPM2, OsSPD/SPM3, and OsSPD/SPM4 were increased, indicating that AtCIPK19 enhances cold stress tolerance by regulating expression of these genes in plant cells.These results will increase our understanding of AtCIPK19-related cold stress tolerance in different plant species including monocotyledonous, dicotyledonous, and gymnosperm plants.

Figure 1 :
Figure 1: Expression vector and generation of transgenic cells.
,D), G. hirsutum (Figure 2B,E), and P. strobus (Figure 2C,F), respectively, under stress at -10 o C and -4 o C. The cell growth rate and the cell viability were not significantly increased in transgenic cell lines of O. sativa (Figure 2A,D), G. hirsutum (Figure 2B,E), and P. strobus (Figure 2C,F), respectively, under stress at 10 o C and 24 o C. The amount of TBARS was significantly increased in transgenic cell lines of O. sativa (Fig. 2G), G. hirsutum (Figure 2H), and P. strobus (Figure 2I), respectively, under stress at -10 o C and -4 o C. The amount of TBARS was not Page: 9 www.raftpubs.comsignificantly increased in transgenic cell lines of O. sativa (Figure 2G), G. hirsutum (Fig. 2H), and P. strobus (Figure 2I), respectively, under stress at 10 o C and 24 o C.

Figure 2 :
Figure 2: Overexpression of AtCIPK19 increases cell viability, cell growth rate, and decreases TBARS.
, and I), respectively, under stress at 10 o C and 24 o C.

Figure 3 :
Figure 3: Overexpression of AtCIPK19 increases contents of Put, Spd, and Spm.
) under stress at -10 o C and -4 o C. Overexpression of the AtCIPK19 gene does not significantly increases expression of putrescine biosynthesis enzymes genes (Figure 4) under stress at 10 o C and 24 o C. Page: 11 www.raftpubs.com

Figure 4 :
Figure 4: Expression of genes associated with putrescine biosynthesis in transgenic rice cells.

Figure 5 :
Figure 5: Expression of genes associated with decarboxylated Sadenosylmethionine biosynthesis in transgenic rice cells.

Figure 6 :
Figure 6: Expression of genes associated with spermidine and spermine biosynthesis in transgenic rice cells.
), and P. strobus (Figure 7I-L), respectively, under treatment of -10 o C -4 o C, 10 o C, and 24 o C. Cold stress causes cell morphological change 3 days after treatment of different temperature at -10 o C -4 o C, 10 o C, and 24 o C. Page: 14 www.raftpubs.com

Figure 7 :
Figure 7: Morphological changes of transgenic cell lines.
to cold stress in different plant species.Overexpression of AtCIPK19 increased the cell viability, the cell growth rate, and decreases the TBARS in cells of O. sativa, G. hirsutum, and P. strobus after treatment at -10, and 4 o C, respectively.These results demonstrated that overexpression of AtCIPK19 enhanced cold stress tolerance could be achieved in different plant species[27,49,50].Polyamines (PAs) are vital signals in modulating plant response to abiotic stress.Polyamines have been globally associated to plant responses to abiotic stress.Particularly, putrescine has been related to a better response to cold and dehydration stresses [51,52].It is known that this polyamine is involved in cold tolerance.Cold temperature inhibits stomatal opening and causes stomatal closure.Coldacclimated plants often exhibit marked changes in their lipid composition, particularly of the membranes.Cold stress often leads to the accumulation of ABA, glycine betaine, polyamines, and proline [51].In Leymus chinensis, the S-adenosylmethionine decarboxylase gene, LcSAMDC1, was upregulated by cold stress.Overexpression of LcSAMDC1 in transgenic Arabidopsis promoted increased tolerance to cold stress, indicating that LcSAMDC1 could be used to improve the abiotic resistance of crops [52].Studies on the relationship between NO and PAs in response to cold stress in tomato showed that NO induced by Spd plays a crucial role in tomato's response to chilling stress [53].In Arabidopsis, the level of putrescine increased substantially under cold stress [54].In Arabidopsis thaliana, the increment in putrescine upon cold treatment correlated with the induction of known stress-responsive genes, and putrescine may be directly or indirectly involved in ABA metabolism and gene expression [55].The levels of endogenous polyamines have been shown to increase in plant cells challenged with low temperature.The accumulation of putrescine under cold stress is essential for proper cold acclimation and survival at freezing temperatures [56-58].Cold stress has been the subject of intense investigation to unravel the complex mechanisms responsible for cold tolerance.In this study, we foud that AtCIPK19 increases cold stress tolerance by regulating expression of OsADC1, OsADC2, OsADC3, OsODC1, OsODC2, OsODC3, OsCPA1, OsCPA2, OsCPA3, OsAIH, OsSAMDC1, OsSAMDC2, OsSAMDC3, OsSAMDC4, OsSAMDC5, OsSAMDC6, OsSPD/SPM1, OsSPD/SPM2, OsSPD/SPM3, and OsSPD/SPM4.Calcineurin B-like proteins interacting protein kinases (CIPKs) play important roles in diverse plant stress responses.In Arabidopsis thaliana, CIPK21 ubiquitously expressed in tissues and up-regulated under multiple abiotic stress conditions.CIPK21 mediated responses to cold stress is associated with ion and water homeostasis [49].In maize, ZmCIPK21 was primarily localized in the cytoplasm and the nucleus of cells and displayed enhanced expression under stress.Over-expression of ZmCIPK21 in wild-type Arabidopsis plants increased their tolerance to salt stress [59].In Arabidopsis, overexpressing of CIPK9 resulted in a low-K(+)-sensitive phenotype [60].In wheat, TaCIPK14 was upregulated under cold conditions.Transgenic tobaccos overexpressing TaCIPK14 exhibited higher contents of chlorophyll and sugar under cold stress [61].In rice (Oryza sativa), OsCIPK14/15 play a crucial role in the microbe-associated molecular functions of stress tolerance [62].Three CIPK genes (OsCIPK03, OsCIPK12, and OsCIPK15) were overexpressed in japonica rice and transgenic plants overexpressing the transgenes OsCIPK03, OsCIPK12, and OsCIPK15 showed significantly improved tolerance to cold stress [63].In this investigation, our results demonstrated that AtCIPK19 increased cold stress tolerance by regulating expression of genes associated with putrescine biosynthesis including OsADC1, OsADC2, OsADC3, Page: 16 www.raftpubs.comOsODC1, OsODC2, OsODC3, OsCPA1, OsCPA2, OsCPA3, OsAIH.