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Plant Mol Biol (2010) 72:125–135 DOI 10.1007/s11103-009-9557-z OsPRP3, a flower specific proline-rich protein of rice, determines extracellular matrix structure of floral organs and its overexpression confers cold-tolerance Kodiveri Muthukalianan Gothandam • Easwaran Nalini • Sivashanmugam Karthikeyan Jeong Sheop Shin • Received: 8 August 2008 / Accepted: 2 October 2009 / Published online: 15 October 2009 Ó Springer Science+Business Media B.V. 2009 Abstract Proline-rich protein (PRP), a cell wall protein of plant, has been studied in many plant species. Yet, none of the PRPs has been functionally elucidated. Here we report a novel flower-specific PRP designated OsPRP3 from rice. Expression analysis showed that the OsPRP3 transcript was mainly present in rice flower and accumulated abundantly during the late stage of the flower development. To study the function of OsPRP3, we constructed and transformed a binary vector containing a full clone of OsPRP3 in sense orientation and also an RNAi vector to achieve overexpression and knockout of the gene, respectively. Our overexpression plants showed a significant increase in cold tolerance than the WT plants which is conferred by the accumulation of OsPRP3 protein during cold treatment. Further the microscopic analysis revealed that OsPRP3 enhances the cell wall integrity in the cold tolerant plant and confers cold-tolerance in rice. Microscopic analysis of the RNAi mutant flower revealed that blocking OsPRP3 function caused significant defects in floral organogenesis. Taken together, the results suggested that OsPRP3 is a cell wall protein, playing a crucial role in determining extracellular matrix structure of floral organs. Electronic supplementary material The online version of this article (doi:10.1007/s11103-009-9557-z) contains supplementary material, which is available to authorized users. K. M. Gothandam (&) E. Nalini S. Karthikeyan School of Bio Sciences & Technology, VIT University, Vellore 632 014, Tamil Nadu, India e-mail: [email protected] J. S. Shin School of Life Sciences & Biotechnology, Korea University, Seoul 136-701, Korea Keywords Cold tolerance Proline rich protein Rice Flower development Introduction Plant cell walls are complex structures that consist of carbohydrates, proteins, lignin, cellulose microfibrils, and also incrusting substances such as cutin and suberin (Showalter 1993). The composition and structure vary depending on different cell types due to their functional specializations and also can be modified as plants adapt to environmental signaling such as biotic and abiotic stresses (Showalter 1993). The cell walls are composed of about 10% proteins, including extensins, glycine-rich proteins, proline-rich proteins, the solanaceous lectins, and the arabinogalactan proteins (Showalter 1993; Lamport 1965). These proteins have their own unique distribution patterns among the various organs, tissues, and cell of plants, playing an integral role in the extracellular matrix structure of many plant cells (Vaner and Lin 1989). Proline-rich proteins (PRPs) are one of the structural cell wall proteins in plant (Chen and Varner 1985; Tierney et al. 1988) and these proteins have been shown to be expressed in many plant species. PRPs were first identified as the proteins in response to physical damage (Chen and Varner 1985; Tierney et al. 1988) and later the proteins were found during plant development. During the plant development, expression of PRP genes often appears to be regulated temporally and spatially. For instance, in soybean individual PRP genes were spatially expressed in different types of plant organs (Hong et al. 1989; Kleis-San Francisco and Tierney 1990; Lindstrom and Vodkin 1991; Wyatt et al. 1992). PRP gene expression was associated with root nodule formation, seedling growth (Sheng et al. 123 126 1991), and with fruit development (Santino et al. 1997). A potato gene, StGCPRP, was expressed in a highly differentiated cell type such as guard cells (Menke et al. 2000) and AtPRP3 in Arabidopsis was detected exclusively in root (Fowler et al. 1999). Expression of PRPs is also influenced by factors associated with biotic and abiotic stresses, suggesting that the synthesis of the proteins is sensitive to external stimuli (Tierney et al. 1988; Sheng et al. 1991; Marcus et al. 1991). To date, the precise functions of PRPs are unknown. However, there are increasing evidences that the proteins may have important roles in normal development, conferring the integrity of plant cell wall and the structure maintenance of organs (Sheng et al. 1991; Nicholas et al. 1993; Carpita and Gibeaut 1993). Localization studies suggest that PRPs may function in determining cell-type-specific wall structure during plant development (Menke et al. 2000). To our knowledge, two PRP genes have been studied in rice so far (Akiyama and Pillai 2003; Wu et al. 2003; Wang et al. 2006). Yet, none of these genes has been functionally elucidated. Here we report a novel rice PRP gene designated OsPRP3 (Oryza sativa Proline-Rich Protein 3) which accumulates during cold stress and confers cold-tolerance in the rice. We found that the OsPRP3 gene is regulated during flower development in rice. Also, our knockout mutant generated by RNAi demonstrated that the gene plays an important role in floral organ formation. Materials and methods Isolation and sequence analysis OsPRP3 clone was obtained from the rice anther cDNA library (Choi et al. 2000). Nucleotide and deduced amino acid sequence were analyzed with the Basic Local Alignment Search Tool (BLAST) at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov) and the Soft berry programme (http://www.softberry.com). The signal peptide cleavage site was predicted by the SignalP programme (Nielsen et al. 1997). Sequence comparisons were conducted using Clustal W (Altschul et al. 1990). Southern blot analysis Genomic DNA was isolated from rice seedlings as previously described (Gothandam et al. 2005). Fifteen micrograms of the genomic DNA were digested with EcoRI, HindIII and XbaI. The digested DNA was subjected to electrophoresis on a 0.8% agarose gel and transferred to Hybond-N membrane (Amersham). The blot was hybridized with 32P labeled C terminal region of OsPRP3 probe 123 Plant Mol Biol (2010) 72:125–135 at 65°C for 16 h. Following incubation, the blot was washed two times with 2 9 SSC, 0.5% of (w/v) of SDS for 5 min and two times with 2 9 SSC, 0.1% of (w/v) of SDS for 5 min at 65°C. Expression analysis Total RNA was isolated from flower, leaf, and different stages of flower using TRIzol reagent (Manufacturer’s method). Five microgram of total RNA was converted to single stranded cDNA. PCR was performed in a 20 ll total volume containing 1 ll (25 ng) of cDNA, 10 pm of each primer, 1 unit of taq polymerase, 500 mM KCl, 100 mM Tris–HCl (pH 9), 1% Triton X-100, 15 mM MgCl2 and 2.5 mM dNTPs. The PCR amplification was carried out in a 25 cycle setup (94°C, 5 min; followed by cyles of 94°C, 30 s; 62°C, 45 s; 72°C, 1 min; followed by 72°C, 5 min).The primers for OsPRP3 were 50 -CTT GCT GGT GAA CGT GCT CGC CGT TG-30 (forward) and 50 -CCA TTG CTT AAT TCG CCG GAG G-30 (reverse). The constitutively expressed OsActin gene was used as normalization control. Primers for cold inducible genes; Lip5 50 -GAA GAC GAG CAC AAG AAG GAG-30 (forward) and 50 -TAT TAC AAG GCA CCG TGC AG-30 (reverse); Lip9 50 -CTC CTG CTC CCG TGG TGA C-30 (forward) and 50 -GTA CCC CAC ACG AAA CAC AAA C-30 (reverse); COR413 50 -CTG GTG GGC TGT TCT CTC TG-30 (forward) and 50 -CAT CAG GAG GCA GGA GGT C-30 (reverse); Mapk2 50 -GAT GCT CAC CTT CAA CCC GCT G-30 (forward) and 50 -CAA TGT TCA GTC TAC CCG GCT CTC-30 (reverse); CDPK7 50 -CTG GAG CGA GAG GAA CAT CTT G-30 (forward) and 50 -CAT CCG CGG ACA TCT GAC AAC-30 (reverse). Construct and transformation analysis A binary vector, pGA1611, was used for over expression construct. The full cDNA clone of OsPRP3 was inserted in between the SacI and KpnI site of pGA1611 in the sense orientation. pANDA vector was used for RNAi mediated gene silencing. A 300-bp OsPRP3 fragment was used to make the RNAi construct following the procedure as previously described (Miki and Shimamoto 2004). A japonica rice variety, Dongjin, was used for transformation by the Agrobacterium co-cultivation method (Jeon et al. 1999). Agrobacterium tumefaciens LBA4404, containing Ach5 chromosomal background and a disarmed helper-Ti plasmid pAL4404 was used for rice transformation (Hoekema et al. 1983). Calli were induced from mature seeds on N6 medium. Agrobacterium-mediated transformation of rice callus was performed according to the method of Lee et al. (1999). Transformed calli were selected by hygromycin resistance, and the transgenic plants were regenerated from Plant Mol Biol (2010) 72:125–135 the transformed calli. Regenerated transgenic rice plants were grown in a greenhouse. Immuno-blot analysis Leaves and flowers were collected from wild type and transgenic plants. Total proteins were isolated and concentration of the protein samples were determined by the Bio-Rad DC protein assay. About 30 lg of each protein sample were separated on 10% SDS–polyacrylamide gel, and transferred to polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with OsPRP3 rabbit polyclonal antiserum (diluted 1:10,000) for 2 h in TBS buffer, containing 0.5% BSA [Primary antibody against OsPRP3 was produced from rabbit by injecting a synthetic oligopeptide derived from the OsPRP3 sequence (TPAYSHPTPVYKQPLPT)]. After two rinses in TBST (TBS ? 0.5% Tween 20), the membrane was incubated with anti-rabbit IgG-conjugated with horseradish peroxidase (diluted 1:10,000) for 1 h in TBS buffer containing 0.5% BSA. After two rinses in the TBST, OsPRP3 was visualized on the blot using the Alkaline Phosphate conjugate substrate kit (Bio-Rad). Cold tolerance, proline content and real time quantitative RT PCR analysis To assay cold tolerance, young and mature transgenic and wild type plants were grown in a cold room (set to 4°C) under 10 h day/14 h night cycle. Leaf samples were collected from cold treated and control plants and used for proline content and expression analysis. Proline content was determined by the method of Bates et al. (1973) using acid ninhydrin reagent and acetic acid. Samples were incubated in a boiling water bath for 1 h, cooled at room temperature and mixed with 1 ml toluene. After phase separation, the absorbance of the organic phase was measured at 520 nm and compared with standard curve generated with L-Pro. Amounts of proline were expressed as lmol g-1 initial fresh weight. Real-time quantitative RT-PCR analysis was performed on a Bio-Rad iCycler using SYBR green method. Thermal cycling conditions consisted of 10 min at 95°C and then 40 cycles of 30 s at 95°C, 45 s at 58°C and 45 s at 72°C. The primers for OsPRP3 were 50 -GAG GAG AAG AAG GTG GCG ATG-30 (forward) and 50 -CAG GCA GGC AAC AGA CCA AG-30 (reverse). OsPRP3 expression was normalized to Actin expression according to the following formula: 2DCt ; DCt ¼ CtOsPRP3 CtActin : Cytological analysis Rice samples from wild type, overexpression and RNAi plants were fixed in a solution containing 2.5% 127 glutaraldehyde, 2% paraformaldehyde, and 0.1 M Phosphate buffer (PBS) (pH 7.4) overnight at 4°C. The samples were then rinsed in 0.1 M PBS (pH 7.4) and further fixed in 1% (w/v) osmium tetroxide (OsO4) at 4°C overnight. After rinsing again in PBS buffer, the samples were dehydrated with an ethanol series and embedded in acrylic resin. The resin-embedded flower samples were sliced into 1-lm sections with an ultra-microtome (LKB, Bromma 2088) and stained with 0.5% toluidine blue containing 0.1% sodium carbonate. The tissue sections were then observed under a light microscope (Zeiss). For electron microscopy, thin sections (40–50 nm thickness) of rice leaves were prepared with an ultra-microtome (LKB, Bromma 2088) and were collected on nickel grids (1-GN, 150 mesh). These sections were then stained with uranyl acetate and lead citrate and examined under a transmission electron microscope (JEM—100CX-1). For immunolocalization, thin sections prepared as described above were etched with 10% hydrogen peroxide for 30 min, rinsed in a deionized water, and then incubated in 0.56 mM sodium meta periodate. The sections were incubated in a PBS buffer containing 1% BSA for 1 h, followed directly by incubation overnight in the PBS buffer containing the primary antibody diluted to 1:100 at 4°C. The sections were rinsed several times with PBS-BSA and continued to incubate in the PBS buffer containing an anti-rabbit IgG conjugated to 20 nm gold particle for 1 h. The sections were then stained and examined as described above. Preimmune serum was used as a negative control. Tetrazolium staining was done by using a solution containing a 1% (w/v) aqueous solution of 2,3,5-triphenyltetrazolium chloride in 50% sucrose at 28°C in darkness for 1 h. Results Molecular characterization of OsPRP3 OsPRP3 is a cDNA clone obtained from a rice anther cDNA library (Choi et al. 2000). Sequence analysis revealed that the clone contained an open reading frame of 271 amino acid residues with a predicted molecular mass of 28.8 kDa and encoded a protein homologous to a series of proline rich protein from plants. BLAST search indicated that OsPRP3 had 48% identity to both OsPRP1 and OsPRP2, 36% identity to NgGPP1 of Nicotiana glauca and 32% identity to AtPRP2 of Arabidopsis thaliana (Supplementary Fig. 1A). Signal P analysis revealed that OsPRP3 contained a signal peptide of 25 amino acid residues and a putative cleavage site at Ala26. We found that there were ten proline residues conserved with both monocot and dicot PRPs. Previously, among the two rice PRP genes, OsPRP1 and OsPRP2, OsPRP1 was analyzed in a detailed manner 123 128 (Wang et al. 2006). The report described that the OsPRP1 formed a gene family of four members and these four genes were tandemly organized within a 20 kb range in the chromosome 10. Our investigation in a rice genome database revealed that the OsPRP3 also lies in the same chromosome (Supplementary Fig. 2A). Phylogentic analysis, however, showed that the gene aligned apart from the two rice PRPs (Supplementary Fig. 1B). The results suggested that the gene contained an evolutionary divergence. DNA blot analysis was done to investigate organization of OsPRP3 in the rice genome and the blot hybridization using a gene-specific probe revealed that OsPRP3 exists as a single copy gene in the rice genome (Supplementary Fig. 2B). In our DNA blot hybridization probed with a full length of OsPRP3 cDNA clone at a high stringent condition, however, two or three hybridized bands were detected (data not shown), indicating that the gene belongs to a small gene family. Expression analysis was done by RTPCR (Fig. 1a, b). The result revealed that OsPRP3 transcript was accumulated in flower and not in leaf (Fig. 1a). RT-PCR analysis with the rice flowers at different developmental stages showed that OsPRP3 was highly expressed in mature flower (Fig. 1b). These results suggested that OsPRP3 expression was not only spatially but also temporally regulated. Generation of overexpression and knockout mutant plants To study function of OsPRP3 gene in rice, we constructed and transformed a binary vector containing a full clone of OsPRP3 in sense orientation and also an RNAi vector to achieve overexpression and knockout of the gene, respectively (Supplementary Fig. 3A). After transformation, we examined expression levels of OsPRP3 gene in a total of nineteen independent transgenic lines to check over- Fig. 1 Expression analysis of OsPRP3. a Expression of OsPRP3 in rice flower and leaf. The result indicated that the gene is expressed only in the flower, b expression of OsPRP3 at the different developmental stages of flower 1 young flower; 2 immature flower; 3 mature flower. OsActin, rice actin used as a positive control 123 Plant Mol Biol (2010) 72:125–135 expression and knockout of the gene by RT-PCR (Fig. 2a, b). The result indicated that leaves from the transgenic plants carrying overexpression of OsPRP3 appeared to accumulate the transcript at higher level (Fig. 2a) whereas flowers from RNAi plants showed either suppression or a complete knockout of the gene transcription (Fig. 2b). We also analysed the OsPRP1 and OsPRP2 mRNA levels in the RNAi flowers, the result showed that those mRNA levels were not reduced in the RNAi plants (data not shown). In addition, we also performed immuno-blotting with the total protein extracts. To do this, a polyclonal antibody was raised to recognize the OsPRP3 protein and we found that the antibody detected a protein band of approximately 28 kD, demonstrating its reliability. Our immunoblot assay confirmed that the OsPRP3 protein was expressed in the leaf of the overexpression plant whereas the protein was absent in the flower of the knockout plants (Fig. 2c). Taken together, the result indicated that the transformation strategy was successfully achieved. Also, the immunoblot study suggested that the OsPRP3 was flower-specific (Fig. 2c). Moreover, our immunolocalization showed that OsPRP3 proteins were localized in the cell wall of transgenic plant leaf (Fig. 3a). The result was consistent with our sequence analysis in which OsPRP3 protein showed common features with cell wall proteins (Supplementary Fig. 1A). This suggests that OsPRP3 is a cell wall protein of rice flower. Cold-tolerance conferred by overexpression of OsPRP3 In our observation with the overexpression plants grown under normal conditions, we found that their growth was perfectly normal as wild-type showed. However, when the transgenic plants were transferred to a growth chamber in which the growing temperature was set to 4°C, the transformants showed a significantly increased cold-tolerance than the wild-type plant (Fig. 4). In the first week of cold stress, the wild-type plant leaf became curling and then the leaf showed wilting symptoms in the second week (Fig. 4a). In 4 weeks of cold stress, the wild-type plant displayed a severe freeze-induced dehydration which eventually leaded to plant death, whereas the transgenic plants were consistently resistant to the cold-stress and survived at such low temperature (Fig. 4a). RNAi plants were exposed to cold treatment; the result was similar to that of wild type plants (Supplementary Fig. 4). To gain insight into the effect of OsPRP3 overexpression in conferring cold-tolerance, we measured expression levels of the cold-regulated genes such as CDPK7, MAPK2, COR413, LIP5 and LIP9 (Saijo et al. 2000; Xiong and Yang 2003; Lee et al. 2004; Aguan et al. 1991) in the overexpression plant (Fig. 4b). Our RT-PCR analysis, however, showed that increased OsPRP3 did not alter transcript levels of these cold inducible genes. The result suggests that the cold tolerance conferred by Plant Mol Biol (2010) 72:125–135 129 Fig. 2 OsPRP3 expression analysis in transgenic rice a accumulation of OsPRP3 mRNA in the leaves of overexpression transgenic plants. 1–9 Individual overexpressed transgenic plants; WT, wild type leaf. OsActin, rice actin gene used as a control, b transcript accumulation of OsPRP3 in the flowers of RNAi transgenic plants. The Gus linker indicates RT-PCR products of the gus linker region, indicative of the expression of the trigger dsRNA. 1–10, Individual RNAi transgenic plants; WT, wild type flower. OsActin, rice actin gene used as a control, c immunoblot analysis of OsPRP3. Top panel shows the immunoblot probed with OsPRP3 specific antibody. Bottom panel shows the corresponding SDS–PAGE gel (silver stained). L leaf; F flower overexpression of the OsPRP3 gene did not involve the pathway related to those cold-regulated genes. We further investigated the correlation between the cold-tolerance phenotype and the proline content of the transgenic plant (Fig. 5). Previously, proline was reported to be an effective cryoprotectant (Mahajan and Tuteja 2005). Furthermore, a correlation between freezing tolerance and increase in proline content during cold acclimation was also studied (Li et al. 2004; Wanner and Junttila 1999; Yelenosky 1979). In this experiment, thus, we used 6 weeks old T1 plants (after selection) in the cold-stress assay since young plants are more chilling sensitive than mature plants. To do this, the plants were treated continuously at 4°C growth condition and we measured expression of OsPRP3 gene and accumulation of proline in the leaf at a time course level. As we already observed in the mature plants, the T1 plants showed cold tolerance at the 4°C growth condition (Fig. 5c, e) whereas wild-type plants showed freeze induced symptoms such as wilting and dehydration within 9 days of cold treatment (Fig. 5d) and afterward in 15 days of cold treatment the plant was completely dehydrated (Fig. 5f). Real-time RT-PCR analysis and measurement of free proline content were effectively done to indicate the correlations with the coldtolerance of the transgenic plant. The real time RT-PCR result revealed that OsPRP3 mRNA level in the coldtreated transgenic plants was increased constantly (Fig. 5g). The transgenic plant also showed a continuous increase of proline content, reaching more than four fold within a week of cold treatment compared to the free proline accumulation in the control plant (data not shown). The result suggested that level of free proline accumulation of the transgenic plants was correlated to the OsPRP3 expression. To investigate structural injuries that could occur to the leaf tissue resulted from the chilling, we performed a cytological analysis (Fig. 6). Our microscopic observation showed that all mesophyll cells of wild type and RNAi plant leaves lost their cell wall integrity which allowed solute leakage and cell lysis (Fig. 6c, d, g, h), indicating that the cold-stress caused severe damage to the leaf tissue and thus the plant did not survive. Whereas the mesophyll cells of the overexpression plant well maintained their cell structure, retaining the cell wall integrity and thus the plant survived against the freeze induced injury (Fig. 6b, f). Taken together, we propose that the cell wall protein, OsPRP3, confers cold tolerance by stabilizing the cell wall integrity. Role of OsPRP3 Detailed function of the OsPRP3 gene during flower development was studied in the knockout mutants generated by the RNAi strategy. Looking into the overall structure of the RNAi plant during growth and development, we did not observed any abnormal phenotype in vegetative organs as we expected from the result of expression analysis; i.e., the OsPRP3 gene was flowerspecific (Fig. 1a). The knockout plants, however, showed defects in the spikelet on which floral buds appeared to be wilty and some were shrunken and white (Fig. 7a). The floral buds also lost their ability of opening. Staining with tetrazolium of the anther from the mutant flower revealed that the anther produced non-viable pollen (Fig. 7l). We also found that many flowers showed a complete loss of pollen production, resulting in male-sterility (Fig. 7m). Microscopic observation of anther locule of the knockout 123 130 Plant Mol Biol (2010) 72:125–135 cell layers (Fig. 7n–p) or a complete loss of the cell layers except for the outermost epidermal cell layer in many cases (Fig. 7o, q). Taken together, our data suggested that OsPRP3 plays a crucial role in determining the extracellular matrix structure of anther, palea and lemma. No significant alterations were observed in the overexpression transgenic plant (Fig. 7h–j). Discussion Fig. 3 Immunolocalization of OsPRP3. a, b Immunolocalization of OsPRP3 in the leaves from the overexpression transgenic and the wild-type plants, respectively. Arrowhead indicates the OsPRP3 localized on cell wall, indicating that the OsPRP3 is a cell wall protein. CH Chloroplast; CW cell wall, scale bar = 1 lm mutant showed that the anther did not contain tapetum, middle layer, and endothecium (Fig. 7q) whereas the anthers at the same developmental stage of wild-type and overexpression plants showed normal development of those tissue layers (Fig. 7f, k). Our microscopic observation revealed that blocking of the OsPRP3 function affected not only the anther development but also other floral organs, such as palea and lemma (Fig. 7n–p). Palea and lemma of the wild-type flower contained an outermost epidermal cell layer, three to six layers of fibrous sclerenchyma and spongy parenchyma cells, and then an innermost cell layer (Fig. 7c–e). The knockout florets, however, showed a severe reduction in formation of those 123 PRPs are cell wall proteins in plant (Chen and Varner 1985; Tierney et al. 1988). These proteins can be classified into five groups on the basis of motifs, domains and biochemical characters (Wang et al. 2006). The first group is characterized by PRPs which contain tandem copies of the pentapeptide PPVXK/T (X is often H, Y or E) as appeared in MtPRP2, SbPRP2, (Wilson and Cooper 1994; Hong et al. 1987, 1989). The second group is characterized by two domain proteins, which contain a Proline-rich N-terminal domain with tandem repeats of PPYV motifs, and a C-terminal domain that lacks Proline-rich sequences, such as the sequence of AtPRP1/3 (Fowler et al. 1999). The third group also consists of two domains but its N-terminal domain is non-repetitive and its C-terminal domain contains proline-rich repeats which are often to be either PPV or PV/IY. This group of PRPs contains the Cys-rich motif, KKPCPP, as found in AtPRP2 and AtPRP4 (Fowler et al. 1999). The fourth group is characterized by the tandem repeats of PEPK motifs in whole protein sequence, as appeared in OsPRP (Akiyama and Pillai 2003) and TaPRP (Raines et al. 1991). The fifth group of PRP characterized by the tandem repeats of PKPE, P(V/E)PPK in the C terminal of the protein sequence, as found in OsPRP1.1-4 (Wang et al. 2006). OsPRP3 contains three PPXY (X = S, V, I) and 2 P(V/I)YK motifs in the C terminal region indicating that OsPRP3 belongs to the third group of PRP (Supplementary Table 1 and Supplementary Fig 1A). Our phylogenetic analysis also confirmed that OsPRP3 aligned under the third group of PRPs (Supplementary Fig. 1B). Wang et al. (2006) described that OsPRP1 formed a gene family with tandem duplication and the duplication showed a expression divergence in spatial specificity. Alignment of OsPRP3 with the rice PRPs and also with PRPs from other plant species showed a low sequence identity (Supplementary Fig. 1A). Taken together, the results suggest that plant PRP genes are evolved in diversity. Expression analysis of OsPRP3 gene revealed that the gene was regulated during the plant development (Fig. 1). This regulation was not surprising because PRP genes from plants were often appeared to be regulated spatially and also temporally. For instance, transcripts of wheat WPRP and maize ZmPRP were detected in the growing tissues like Plant Mol Biol (2010) 72:125–135 131 Fig. 4 Phenotype and coldregulated gene expression in transgenic and wild type plants under 4°C cold stress. a Phenotype of the Ubi:OsPRP3 overexpression transgenic and wild type plants grown at 4°C. Overexpression transgenic plant was more tolerant to the cold stress than the wild type plant, b expression analysis of cold regulated genes in rice leaves after 0, 1, 2, 3, 7 and 14 days under 4°C cold-stress. WT wildtype; OsPRP3, OsPRP3 overexpression transgenic plant. A constitutive expression of OsPRP3 in the transgenic rice leaf was shown whereas the gene transcript was hardly detected in wild-type leaf, indicating that the transgene was over-expressed in the transgenic plant Fig. 5 Cold-stress assay. a, c, e, Overexpression plant (T1), b, d, f, wild-type plant as control. 9 and 15 days after cold treatment were shown in the pictures, g real-time quantitative RT-PCR analysis of OsPRP3 in 4°C cold treated leaves 123 132 Plant Mol Biol (2010) 72:125–135 Fig. 6 Cytological analysis of cold-treated transgenic and wild-type leaves. a–d Cross section of control (untreated), cold-treated transgenic (overexpression), cold treated wild type and cold treated (RNAi) leaves, respectively, e–h magnification of mesophyll cells from the cross sections (a–d) of the leaves, respectively. Arrows indicates the mesophyll cells that lost their cell wall in the wild-type plant with cold-induced injury. UE upper epidermis; LE lower epidermis; VB vascular bundle; MC mesophyll cells; CP chloroplast. Scale bar = 30 lm root and meristem (Raines et al. 1991) and in the xylem and epidermis (Vignols et al. 1999), respectively. This suggests that PRPs are encoded by a number of different genes whose mRNAs are accumulated preferentially in 123 distinct plant tissues and thereby the proteins play a crucial role in the tissues (Tierney et al. 1988). Arabidopsis contains four PRPs (designated AtPRP1-4) and also shows different expression patterns (Fowler et al. 1999). Plant Mol Biol (2010) 72:125–135 133 Fig. 7 Characterization of OsPRP3 RNAi transgenic plants. a Spikelet of RNAi plant. Inset box shows magnification of an abnormal flower appeared in the spikelet. Microscopic observation of wild type (b–f), OsPRP3 overexpression (h–k), and knockout (m–q) flowers. le lemma; pa palea; ep epidermis; sl sclerenchyma layer; ie inner epidermis; ms microspore; t tapetum; a anther; vb vascular bundle. c, h, p Magnification of the interlocking of lemma and palea. d, j ,o Palea histology showing different cell layers. The knockout plant showed a severe reduction in sclerenchyma layer and inner epidermis. f, k, q anther locule at micropsore stage of anther development. The knockout plant showed a severe abnormality in anther development. Tetrazolium staining of anther from wild type (g) and knockout (l) plants. Scale bar = 20 lm Expression divergence in spatial specificity of PRPs has been studied in rice (Wang et al. 2006). Our study showed that transcript of OsPRP3 is accumulated most abundantly during the late stage of flower development, suggesting that it may contribute to the cell wall integrity during the flower maturation. In order to elucidate function of the OsPRP3, we transformed the binary constructs to bring either overexpression or knockout of the gene in rice (Supplementary Fig. 3A). After transformation, we confirmed that the protein was successively synthesized in the leaf of the overexpression plant whereas the protein was absent in the flower of the knockout plant (Fig. 2c). From our cold treatment study, we found that the overexpression plant tolerated to cold stress whereas the wild-type plant showed chilling sensitivity (Figs. 4, 5, 6). It has been reported that chilling sensitive plants characteristically exhibit structural injuries and may suffer from metabolic dysfunction when chilled (Kacperska 1999). Chilling ultimately results in loss of membrane integrity, which leads to solute leakage. Integrity of intracellular organelles is also disrupted leading to the loss of compartmentalization, reduction and impairing of photosynthesis, protein assembly and general metabolic processes. The primary function of cold acclimation is to stabilize the integrity of cellular membranes against freezing induced injury (Mahajan and Tuteja 2005). Cold acclimation also results in enhancement of the antioxidative mechanisms and increased cellular sugar levels as well as accumulation of cryoprotectants (Xin and Browse 2000). All these modifications help the plant to withstand and surpass the severe dehydration associated with cold stress. In our primary observation using T0 mature plants shown in the Fig. 5, we found that the leaf of the transgenic plants expresses the OsPRP3 in the leaves 123 134 and the expression was increased during the cold treatment (Fig. 4b) and also the transgenic plants accumulated proline at a high level (data not shown). Based on this, we measured the amount of OsPRP3 and free proline content in the T1 plants during cold treatment. Expression of OsPRP3 in the overexpression plant is by Ubiquitin promoter. Whereas increased expression of OsPRP3 under cold stress is also because of ubiquitin promoter. Recently Perales et al. (2008) elucidated that ubiquitin promoter responds to various environmental stresses by enhancing the expression of any gene under its control. Proline rich protein/glycoproteins are thought to play an integral role in extracellular matrix structure of many plant cells that adds mechanical strength to the cell wall and assists in proper wall assembly (Vaner and Lin 1989). Free proline is known to be one of the compatible osmolytes preventing dehydration in response to freezing and drought stress (Delauney and Verma 1993). Increase in proline content occurs in many plant species during cold acclimation (Koster and Lynch 1992). Proline is also known to protect membranes and proteins against the adverse effects of temperature extremes (Paleg et al. 1984; Rudolph et al. 1986; Santarius 1992). Rapid catabolism of proline upon relief of stress may provide reducing equivalents that support mitochondrial oxidative phosphorylation and the generation of ATP for recovery from stress and repair of stress-induced damage (Hare and Cress 1997; Hare et al. 1998). All of those reports support that proline plays an important role in plant cold-tolerance. Furthermore, our cytological analysis of the leaf tissue from the transgenic plant revealed that the leaf cells maintained their cell wall integrity against the chilling induced injury (Fig. 6e). It is well reported that PRP proteins accumulated in response to wounding, infection and other stresses (Sheng et al. 1991; Showalter 1993; Cassab 1998; Bernhardt and Tierney 2000). Their accumulation during theses processes has been associated with a possible role as structural proteins within the extracellular matrix to add mechanical strength to the wall and assist in proper wall assembly. It is also proposed that these kinds of proteins are secreted into the wall, where eventually they become insolublised in response to a hydrogen peroxide burst elicited by environmental signals (Marshall et al. 1999; Somerville et al. 2004). Further the insolubilization of the PRPs may lead to the formation of protein/protein or protein/carbohydrate linkages within the cell wall and contributes to the stability of the extracellular matrix (Fowler et al. 1999). Therefore, it is possible that the overexpression of OsPRP3 is related to remodeling of plant cell wall components during cold stress and indirectly confers cold tolerance in the rice plant. Knockout of OsPRP3 gene showed a severe disruption of the cell walls in the floral organs (Fig. 7m–q). The extracellular matrix has a central role both in plant 123 Plant Mol Biol (2010) 72:125–135 development and in the interactions with pathogenic microorganisms. Although the matrix is mainly composed of polysaccharide, the less abundant proteins, plays a crucial role in differentiation of different tissues (Knox 1995; Davis et al. 1997). Moreover proline rich protein/glycoproteins are thought to play an integral role in extracellular matrix structure of many plant cells and the structural cell wall proteins form an independent structure-determining network within the extracellular matrix that adds to the mechanical strength of the wall and assists in proper wall assembly (Vaner and Lin 1989). Our microscopic observation revealed that the knockout mutant displayed loss of cell layers in the palea, lemma, and anther due to the cell wall collapse (Fig. 7n, q). Taken together, our study suggested that loss of OsPRP3 function resulted in the failure of determining cellular structure during the floral organ formation in rice. In summary, we report a novel PRP gene from rice. 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