Overexpression of BrSAC1 encoding a phosphoinositide phosphatase isolated from Chinese cabbage (Brassica rapa L) improved tolerance to cold, dehydration, and salt stresses in transgenic tobacco

1 Department of Horticulture, Hankyong National University, 67 Seokjeong-dong, Ansung 456-749, South Korea. 2 Institute of Genetic Engineering, Hankyong National University, Ansung 456-749, Korea. 3 Clone Company, 67 Seokjeong-dong, Ansung 456-749, South Korea. 4 Department of Horticulture, Sunchon National University, Suncheon 540-742, Korea. 5 Department of Crop Science, Chungbuk National University, Cheongju, Korea. 6 National Institute of Horticultural and Herbal Science, Suwon, 440-746, Korea.


INTRODUCTION
Plant growth is strongly affected by a variety of major *Corresponding author.#These authors contributed equally to this work.obstacles affecting plant growth and crop environmental stresses.Cold, salt, and drought are the productivity.In response to these stresses, plants initiate signaling cascades leading to the development of defense responses.Phosphoinositides (PIs) are signaling molecules that regulate cellular events, including vesicle targeting and interactions between membrane and cytoskeleton.To date, various enzymes involved in PI signaling and biosynthesis have been identified and characterized in animals (Martin, 1998;De Matteis et al., 2002).Compared to animals, in plants the role of PIs is still poorly understood.During the last decade, however, new information has emerged showing involvement of PIs in various plant processes, such as calcium mobilization, signal transduction, cytoskeletal rearrangements and membrane trafficking (Wang, 2004) in addition to the defense against attack by pathogens (Laxalt and Munnik, 2002).Recent research indicates that PIs play important roles in development, including stress signaling (Meijer et al., 2001;Xiong et al., 2001), growth (Williams et al., 2005), and vascular patterning (Carland and Nelson, 2004).PI phosphatases (PIP) are classified into three main groups, 3-, 4-, or 5-phosphatases on the basis of the position of the phosphate-inositol head group being hydrolyzed (Takenawa and Itoh, 2001).The group of phosphatases called suppressor of actin (SAC) domain phosphatases have been identified in yeast and animal; these phosphatases were shown to hydrolyze the phosphates on multiple positions on the inositol head group of PIs (Hughes et al., 2000).As in other eukaryotic systems, PI metabolism in higher plants is considered important in signal transduction.The involvement of phospholipid signaling in the cell responses to stress stimuli, including salt, osmotic, temperature, and pathogen stresses, has been shown, and multiple members of the PI pathway are thought to mediate stress responses through different mechanisms.
In this study, we isolated the BrSAC1 gene encoding a PIP from the cDNA of Brassica rapa seedling and characterized its modulation of PI signals during abiotic stress response.Also, introgression of BrSAC1 into tobacco led to stress tolerance when assessed at the seedling stage.

Plant materials and stress treatments
Chinese cabbage (B.rapa var.OSOME) was aseptically grown on Murashige Skoog (MS) agar medium (Murashige and Skoog, 1962) in a culture room under a 16:8 h light:dark period at 25°C.Salt and hormone treatments were conducted, after nine days of growth, on seedlings that were transferred to fresh liquid half-strength MS medium without sucrose (MSH medium) containing either 250 mM NaCl or 100 µM abscisic acid (ABA) for 24 h.Induction of cold stress was examined on seedlings grown in the above conditions for 24 h after transferring to 4°C.Membrane fluidity treatments using chemical modulators were conducted on cut leaves of seedlings exposed to 5, 10, and 15 mM benzyl alcohol at 25°C for 3 h followed by growth at 8°C for 24 h.Cut leaves of seedlings were kept at 25°C for 6 h in the presence of 2, 4 and 6% dimethyl sulfoxide (DMSO), while the control leaves were kept in water at 25°C.

Isolation of full-length cDNA of BrSAC1
A partial BrSAC1 sequence was identified in the dbEST division of GenBank and searches were conducted using basic local alignment Han et al. 1783 search tool (BLAST) with a specific SAC domain motif as the query sequence.The full-length cDNA was isolated by RACE technology using the SMART RACE-PCR kit (Clontech, Palo Alto, CA, USA) (Lee et al., 2009).

RNA isolation and real-time PCR
Total RNA from leaf tissues was extracted using Trizol reagent (Invitrogen, USA) according to the manufacturer's protocol.The specific primers for the analysis of BrSAC1 expression in germinating seeds were: and BrSAC1R3 5′-ATGATGGCTGGAATGCCCT-3′. Real-time polymerase chain reaction (qRT-PCR) was performed using a Bio-RAD I Cycler IQ5 machine as previously described (Ali-Benali et al., 2005) using RT pre-mix (TOYOBO Co., Japan).The mean threshold cycle (Ct) values of triplicate PCR reactions were computed and relative quantification of expression levels was performed using the comparative Ct method (Livak and Schmittgen, 2001).The fold change in total RNA of the target gene relative to the reference gene (actin) was determined by the following formula: fold change = 2 -∆∆Ct , where ∆∆Ct = (Ct target gene -Ct actin gene ) transgenic plants -(Ct target gene -Ct actin gene ) wild-type plants.
First-strand cDNA was generated using Superscript TM III reverse transcriptase according to manufacturer's instructions (Invitrogen, USA).BrSAC1 was amplified using 1 ul cDNA template added with the primer pairs BrSAC1F2 5′-ATGGAGGCTCCTAAGCA-3′ and BrSAC1R2 5′-CGCAAAGAGTTGCATTCAGC-3′.The PCR amplification program consisted of an initial step at 94°C for 5 min followed by 30 cycles of 94°C for 1 min, 58°C for 1 min, 72°C for 2 min, and a final step at 72°C for 10 min.

Generation of BrSAC1 transgenic tobacco plants
Full-length sequence of BrSAC1 amplified from a pGEM-T easy vector with the primers BrSAC1F1 (5′-ATGGAGGCTCCTCCTAAGCACA-3′) and BrSAC1R1 (5′-TCAAGGTCTGGGCTTGTGCAG-3′) was confirmed by sequencing and inserted into the SfiI site of a pBig plant binary vector system for overexpression; the vector also contained a modified cauliflower mosaic virus (CaMV) 35S promoter.The pBig plant binary vector containing complete coding sequence of BrSAC1 was introduced into tobacco seedlings via Agrobacterium tumefaciens-mediated transformation according to the procedure of Horsch et al. (1985).
Four-week-old T2 generation tobacco seedlings were analysed for the presence of the transgene.DNA was extracted from the leaves of the tobacco seedlings by using cetyltrimethyl ammonium bromide (CTAB) method (Rogers and Bendich, 1994).The primers used for PCR amplification were BrSAC1F2 and BrSAC1R2, with reaction conditions as described above.

Analysis of transgenic plants for abiotic stress tolerance
Four homozygous T2 generation transgenic tobacco (Nicotiana tabacum cv.Samson) plants were used in all subsequent abiotic stress assays.The seeds of transgenic and non-transgenic (wild type, WT) plants were germinated and grown on MS medium for 16 days in a culture room under a 16:8 h light:dark period at 24°C.
For salt, cold, and dehydration stress treatments, (Figure 4), the seedlings were germinated and grown according to the above procedure for 14 days and then transferred to ½ MS media containing 250 mM NaCl, 4°C cold, and 0.3 M and 0.4 M mannitol.After 14 days, the fresh weight (FW) and shoot and root lengths were measured.Statistical analyses were performed using a Student's t test in Microsoft Excel.P-values below 0.05 were consi-dered significant.To determine the effects of salt, cold, and dehydration stress on transgenic tobacco plants (Figures 5 to 7), seeds of WT and transgenic plants were germinated and grown on ½ MS medium for various times (noted below) in a culture room according to the procedure described above.The effect of salt stress resulting in chlorosis was examined in WT and transgenic seedlings (21 days) that had been transferred to sterile tissue paper soaked with a 250 mM NaCl MSH solution for four days.After treatment, seedlings were washed briefly in sterile Milli-Q water and allowed to grow and recover for eight days on MSH in a culture room, and then the number of green seedlings was determined.Four leaf discs of transgenic (lines 2 and 4) and WT plants (21 days) were floated in 250 mM NaCl MSH solution treatment versus no salt control MSH solution for four days to examine status and chlorophyll content.The leaf disk chlorophyll content (mg/g FW) was measured using the method described by Aono et al. (1993) and was shown as mean ± standard deviation.To examine the effects of cold stress treatment, transgenic and WT plants (21 days) were transferred to a cold chamber (4°C) for 15 days.After treatment, the plants were transferred to culture room conditions and allowed to grow and recover for 15 days.Individual plant FW (mg) was measured and is shown as the mean ± standard deviation.To examine the effects of dehydration stress on germinating seeds of transgenic and WT plants, the seeds were germinated on 0.3 and 0.4 M mannitol and observations (percent germination, FW) of representative plants were recorded over a period of eight days according to the method of Mukhopadhyay et al. (2004).

Multiple sequence alignment and phylogenetic analysis
Multiple sequence alignment of the BrSAC1 gene was performed using Clustal X version 1.83 (Thompson et al., 1997).A phylogenetic tree was constructed using DNA Data Bank of Japan (DDBJ; http://clustalw.ddbj.nig.ac.jp/top-e.html),and bootstrap analysis was performed with 1,000 samplings.
Further, both BrSAC1 and AtSac1B contain N-terminal KXKXX motifs that serve as binding sites for the coatomer (COPI) complex.This COPI interaction motif is considered a unique feature of mammalian Sac1s, as it is not conserved in other plants, yeast, or Drosophila Sac1 proteins (Figure 1B).The phylogenetic relationship of the deduced amino acid sequence of BrSAC1 to the SAC domains from other organisms was determined using Clustal W within the BioEdit program (Figure 1C).

Expression of the BrSAC1 gene in various organs and during seed germination
Expression analysis of the BrSAC1 gene transcript was performed in various vegetative tissues (leaf, stem and root), reproductive tissues (stamen, pistil, petal, flower bud, calyx of flower, and flower stalk), and during seed germination.Semi-quantitative reverse transcription (RT)-PCR analysis showed varying expression of BrSAC1 gene across tissues, highest in the stamen among reproductive tissues and in leaf among vegetative tissues (Figure 2A).An examination of BrSAC1 expression during the first 72 h of seed germination revealed an increase in mRNA levels of BrSAC1 that peaked at 36 h, about 20 fold higher than that at 0 h, with a fast decrease at subsequent times examined (Figure 2B).

Expression pattern of BrSAC1 under different abiotic stresses
The results of qRT-PCR analysis showed that BrSAC1 was induced under various abiotic stresses.BrSAC1 transcripts showed rapid induction (9.0 fold) peaking at 1 to 2 h after exposure to cold (4°C) conditions, followed by a gradual decrease in expression levels to pre-treatment levels over the next several hours of exposure.In the presence of salt stress (250 mM NaCl), transcript levels were rapidly induced several fold by 1 h, maintaining a high level of induction after 3 to 8 h, then returning to pretreatment levels (Figure 3).Expression of BrSAC1 in response to the stress hormone ABA was also determined and exogenous ABA treatment (100 µM) resulted in strong BrSAC1 induction within 1 h, gradually decreasing to pre-treatment levels by 8 h.We also wanted to examine the role of membrane fluidity and the cold induction of BrSAC1 transcript.Membrane rigidification at low temperatures can be prevented by benzyl alcohol (BA) that acts as a membrane fluidizer.Similar examinations can also be simulated at room temperature by treatment with a membrane rigidifier DMSO (Mukhopadhyay et al., 2004).In order to examine     In this analysis, significant P values of 0.015 to 0.02 (FW), 0.015 to 0.02 (shoot lengths), and 0.02 to 0.025 (root lengths) were obtained between WT and transgenic lines (TG1 and TG2).
trations of the membrane rigidifier, DMSO, greater than 4% (Figure 3).The decline was thought to be caused by the toxic influence of this compound.

BrSAC1 overexpression increases stress tolerance
To study the physiological functions of BrSAC1, we generated several transgenic tobacco plant lines that overexpressed BrSAC1 by fusing its full-length cDNA sequence to the CaMV 35S promoter in the pBig vector (data not shown).Four positive transgenic plant lines were confirmed by genomic PCR, Southern blot, and RT-PCR (data not shown).No significant morphological or growth differences were observed between the transgenic and non-transgenic plants under standard growth conditions.Each of the transgenic lines was examined for responses to salt stress in comparison to WT. Seedlings were stressed for 18 days with salt (250 mM NaCl), cold (4°C), or dehydration (0.3 M and 0.4 M mannitol) stresses, and FW, and shoot and root lengths were examined (Figure 4).After salt stress, all seedlings (WT and TG) showed significant reductions in each of the physiological categories measured.Overexpressing  Tobacco seedlings from transgenic lines (1 to 4) overexpressing BrSAC1 were examined for effects of cold stress: A. Fresh weight of 21-day-old seedlings (WT and transgenic lines) that were cold-stressed (4°C) for 15 days, followed by a 15 day recovery, was measured for comparison with that of unstressed controls.B. Pictures of representative seedlings from (A) at the end of the recovery period.BrSAC1-carrying lines showed greater tolerance to salt stress than WT having significant increases in FW and shoot and root length of almost twice those of WT-levels.Plants were also examined after salt stress followed by a recovery period.Seedlings were subjected to salt stress in 250 mM NaCl for 4 days and then returned to nonstressed conditions for eight days after which the number of plants without any chlorosis (green seedlings) was counted (Figure 5) while about 30% of WT seedlings remained green; at the end of this period several overexpressing lines showed significant increases in the number of green seedlings, some with more than 70% (Figure 5A).Representative examples of surviving plants from this experiment are shown (Figure 5B) (P values of 0.015 and 0.02, respectively, by student's t-test).
Overexpressing lines were also examined to determine the role of BrSAC1 during cold stress by growing seedlings at 4°C for 15 days followed by a recovery period of the same time (Figure 6).An increase in FW was noted in some transgenic lines with advantage levels accounted to 50 to 100% of WT FW (Figure 6A) (P < 0.02, by student's t-test).In addition, transgenic seedlings appeared to be larger and healthier than WT seedlings after cold stress treatment showing recovery marked by emergence and expansion of third and fourth leaves compared to WT seedlings that only produced undeveloped first two leaves (Figure 6B).
In order to examine the role of BrSAC1 in dehydration stress tolerance, WT and transgenic lines were grown and analyzed in the presence of stressing levels of mannitol over an eight-day period (Figure 7).Germination was dramatically effected in WT plants, with levels reduced to less than 20% by day 4.At the same time point, transgenic BrSAC1 lines showed almost three fold increases in percent germination in both the lower (0.3 M) and higher (0.4 M) concentrations of mannitol examined (Figures 7A and B).Similar increased levels of percent germination were observed in almost every BrSAC1 transgenic line over the period examined, often at levels two-fold or greater compared to WT, suggesting an important role for this gene in dehydration stress tolerance.In addition, the FW of transgenic lines stressed by mannitol was also increased in several cases to twofold above that of WT plants (Figures 7A and B) (P values of 0.01 and 0.015, respectively, by student's t test).
Together these results suggest that BrSAC1 plays an important role in several stress related processes, and overexpression of BrSAC1 may lead to transgenic plants becoming tolerant to salt, cold, and dehydration stresses.

DISCUSSION
The SAC domain was first identified in the yeast (S. cerevisiae) Sac1p PIP protein and subsequently in a number of proteins from yeast and animals.It is approximately 400 amino acids in length and characterized by seven conserved motifs.The Sac1 phosphatases represent a major class of PIP phosphatases and have a signature feature of being integral membrane proteins (Cleves et al., 1989;Whitters et al., 1993).
PIPs are phosphorylated derivatives of phosphatidylinositol (PtdIns) that serve a variety of primary intracellular roles in the specification of dedicated membrane microdomains that organize signal transduction processes, as co-factors for the regulated activities of proteins, and as precursors for second messengers such as diacylglycerol and soluble inositol phosphates (Fruman et al., 1998;Di Paolo and De Camilli, 2006).In plant cells, all PI except PI (3, 4 and 5) P3 have been identified.Several lines of evidence suggest that as in yeast, PIs in plants may regulate many cellular activities such as vesicle trafficking (Matsuoka et al., 1995;Kim et al., 2001), pollen tube growth (Kost et al., 1999), and responses to stress and hormonal treatments (Meijer et al., 1999(Meijer et al., , 2001;;DeWald et al., 2001).A number of kinases and phospholipase Cs involved in the metabolism of PIs have been characterized in plants (Cleves et al., 1991;Stevenson et al., 2000).
Homologs of the Sac1 PIP phosphatase are disseminated throughout the eukaryotic kingdom as evidenced by inspection of insect, plant, and mammalian genomes (Figure 1).The intracellular distribution of Sac1 PIP phosphatases in the endoplasmic reticulum (ER) and golgi of mammalian cells recapitulates aspects of what is observed in yeast (Xie et al., 1998;Nemoto et al., 2000), but with some unanticipated twists.Of interest, hSac1 also contains a C-terminal KXKXX motif that serves as binding site for the coatomer (COPI) complex.This COPI interaction motif is a unique feature of mammalian Sac1s as it is not conserved in yeast, plant or Drosophila Sac1 proteins (Figure 1).
Our examinations reveal that BrSAC1 plays a role in salt, cold and drought responses, as each of these processes regulated BrSAC1 expression.Rapid induction of BrSAC1 protein was seen within 1 h of both salt (NaCl) and cold (4°C) treatments.Although BrSAC1 is rapidly induced by both of these stresses, the pattern of transcript induction over 24 h between them is quite different.BrSAC1 induction by cold was rapid and peaked at 17-fold in the first 2 h, then declined rapidly until it was close to pre-induced levels by 24 h (Figure 3).The difference in transcript induction pattern may suggest different modes of action for BrSAC1 in response to stress.Stress responses, such as salt and cold, have been proposed to be activated through different signaling pathways: ABA dependent or ABA independent (Xiong et al., 2002;Shinozaki et al., 2003;Bartels and Sunkar, 2005;Tuteja, 2007).The influence of BrSAC1 on responses to abiotic stresses of salt, cold and dehydra- Han et al. 1791 tion were further examined through the creation of several overexpressing transgenic lines in tobacco.
In conclusion, we cloned, characterized, and investigated the function of Sac1; PIP phosphatase encoded by the BrSAC1 gene of B. rapa L. Overexpression of the BrSAC1 gene in tobacco plants enhanced their tolerance to salt, cold and dehydration stresses.These results suggest that BrSAC1 may be important to the understanding, and potential breeding, of plants towards improved abiotic stress response.

Figure 1 .
Figure 1.Sequence alignment, domain structure and phylogenetic relationship of BrSAC1. A. Comparison and deduced amino acid sequence of BrSAC1 and other Sac1 proteins, AtSac1B (Arabidopsis thaliana), NP201403; AtRHD4 (A. thaliana), NP190714; SAC1 (Saccharomyces cerevisiae), CAA82057; Sac1 (Drosophila melanogaster), NM138243; rat Sac1 (Rattus norvegicus), NP446250; mouse Sac1 (Mus musculus), NM030692; human Sac1 (Homo sapiens), NM014016; rice Sac1 (Oryza sativa), NP001067755; and Maize Sac1 (Zea mays), BT086272.Sequence alignment of the core catalytic domains and the KXKXX COPI binding motifs of the SAC1 protein family.The CX 5 R(T/S) core catalytic domain of Sac1 is highly conserved in yeast, higher plants, Drosophila, and mammals.Identical and conserved residues in all aligned sequences are indicated by the rectangle box.B. (a) Diagrams of the domain organization of Sac1 proteins.Domains and motifs of interest are highlighted by colored boxes.Blue boxes show the Sac1-catalytic domain, yellow boxes show the CX5R(T/S) core catalytic motif, and orange boxes show the KXKXX COPI binding motif.Three key amino acids within the Sac1 domain known to affect catalytic activity (Rivas et al., 1999; Liu et al., 2008) are indicated in red.(b) Sequence alignment of the core catalytic domains and the KXKXX COPI binding motifs of the Sac1 protein families.Identical amino acids are highlighted by black boxes.C. The phylogenetic tree of the Sac1 protein family.BrSAC1 isolated in this study.A phylogenetic tree was constructed using CLC Main workbench 5 with CLUSTAL W alignments as input using the neighbor-joining method (Saitou and Nei 1987) to construct the tree.

Figure 2 .
Figure 2. BrSAC1 showing the broad expression across plant tissues using real-time PCR analysis.A. Expression levels of BrSAC1 were measured in various tissues collected from Brassica rapa.B. Real-time PCR analysis of BrSAC1 in germinating seedlings (0-72 h after germination).Amounts of template were calibrated using the mRNA as a reference.Data are presented as means ± standard deviation of three independently replicated experiments.

Figure 3 .
Figure 3. BrSAC1 expression is induced by abiotic stress treatments in Brassica seedlings.Expression levels were examined by RT-PCR at various times after treatment with cold (4°C), salt (250 mM NaCl), or 100 µM abscisic acid (ABA).Expression profiles were also examined in the presence of various concentrations of chemical modulators of membrane fluidity benzyl alcohol (BA) at 4°C and DMSO at room temperature.Actin was used as a positive control and to determine relative expression for each treatment.

Figure 4 .
Figure 4. Plants overexpressing BrSAC1 showing enhanced tolerance to stress.Tobacco plants overexpressing BrSAC1(transgenic lines: TG) and WT plants were examined under various stress conditions.The 10-day-old seedlings grown on ½ MS medium were transferred to new media containing 250 mM NaCl for salt stress, 0.3 M or 0.4 M mannitol for dehydration stress, or placed at 4℃ for cold stress.After 18 days of stress treatment, plants were photographed (A) and examined for relative fresh weight and shoot and root lengths.Data are represented as average ± standard deviation for 5 plants from 1 representative experiment (B).In this analysis, significant P values of 0.015 to 0.02 (FW), 0.015 to 0.02 (shoot lengths), and 0.02 to 0.025 (root lengths) were obtained between WT and transgenic lines (TG1 and TG2).

Figure 5 .
Figure 5. Plants overexpressing BrSAC1 showed increased salt tolerance.Seedlings of tobacco seedlings from transgenic lines (1 to 4) overexpressing BrSAC1 were examined for effects of salt treatment.A. The number of non-chlorotic (Green) 21 day old seedlings (WT and transgenic lines) were counted following a 4 day 250 mM NaCl salt-stress treatment and eight day recovery period.B. Pictures of representative seedlings from (A) are shown at the end of the recovery period.

Figure 6 .
Figure 6.Plants overexpressing BrSAC1 showing increased cold tolerance.Tobacco seedlings from transgenic lines (1 to 4) overexpressing BrSAC1 were examined for effects of cold stress: A. Fresh weight of 21-day-old seedlings (WT and transgenic lines) that were cold-stressed (4°C) for 15 days, followed by a 15 day recovery, was measured for comparison with that of unstressed controls.B. Pictures of representative seedlings from (A) at the end of the recovery period.

Figure 7 .
Figure 7. Plants overexpressing BrSAC1 showing increased dehydration tolerance.Tobacco seedlings overexpressing BrSAC1 (transgenic lines 1 to 4) were examined for effects of dehydration stress.Percent germination of seeds (WT and transgenic lines) sown on 0.3 M (A) and 0.4 M (B) mannitol is shown for four to eight days after the start of germination.(C) Percent increase in relative fresh weight (FW) of 8-day-old seedlings germinated on 0.3 M or 0.4 M mannitol is shown relative to the FW of unstressed seedlings.(D) Pictures of representative seedlings of WT and transgenic lines taken 8 days after germination on 0.3 M (upper) and 0.4 M (lower) mannitol.