Cold acclimation is a well-known adaptive process through which plants can dramatically increase their tolerance to freezing temperature. Modifications of cell wall have been recognized as a key characteristic during plant acclimation to low temperature. However, the molecular mechanism responsible for such cellular adaptation still remains a mystery. Here, we report an unexpected regulatory role of TCF1 on lignin content during cold acclimation in Arabidopsis. TCF1 is specifically induced by cold and is required for chromatin based gene regulation of cold responsive genes such as BCB (a GAP) that regulates lignin genes. Further evidence shows that reduction in lignin dramatically increases plant freezing tolerance, while lignin maintenance required for cold acclimation is regulated by TCF-mediated signaling. Thus, our study has revealed, for the first time, lignin remodeling as a key function of cold acclimation and freezing tolerance. The findings provide the first direct molecular evidence that freezing tolerance is directly related to cell wall properties during cold acclimation and extra/intercellular freezing upon and freezing/thawing process.
Funding: This work was supported by the National Transgenic Key Project of MOA (2014ZX08002-002 to XL), National Science Foundation (31200962 and 31230050) and the Youth Innovation Promotion Association of the Chinese Academy of Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Freezing temperature is an important environmental factor that determines the natural geographical distribution of plants and limits crop productivity . Sudden exposure to freezing temperature causes intracellular freezing, membrane damage and cell death [1–3]. To better survive freezing low temperature, plants have evolved coping mechanisms through initiating cold acclimation when the temperature gradually drops lower in autumn in nature. Many signal transduction cascades are involved in this physiological adaptation process. In Arabidopsis, expression profiling of cold-treated plants revealed that up to 20% of genes in the genome are regulated by cold. Characterization of a group of cold-regulated (COR) genes which are highly induced by cold stress using forward and reverse genetics has led to identification of a key CBF (C-repeat binding factor, also known as dehydration-responsive element-binding protein 1 or DREB1) signaling pathway. CBF transcription factors (CBF1, CBF2, CBF3) can activate expression of the COR genes by binding to cis-elements in their promoters and induce cold acclimation and freezing tolerance [4–6]. Several regulators of CBF genes have been identified, such as Inducer of CBF expression 1 (ICE1), calmodulin binding transcription activator 3 (CAMTA3), MYB15 and Ethylene Insensitive 3 (EIN3) [7–10]. Most recently, it has been shown that OPEN STOMATA 1 (OST1), a central component in ABA signaling pathway, plays a crucial role in plant response to cold. OST1 is induced by cold and cold-activated OST1 can interact and phosphorylate ICE1 to enhance the stability of ICE1, resulting in increased plant tolerance to freezing . However, multiple studies have reported that the CBF signaling pathway is not the sole mechanism modulating plant cold acclimation and cold tolerance, because only 12% of the cold responsive genes are regulated by CBF transcription factors . The prominent example is HOS15, which regulates freezing tolerance through modification of histone acetylation [13,14]. In addition, SFR2 was found to modulate freezing tolerance through lipid remodeling of the outer chloroplast membrane . Most recently, it was found that AtHAP5A modulates freezing stress resistance in Arabidopsis independent of the CBF pathway . Through these signaling pathways, a wide variety of antifreeze/stress-related proteins and compounds are accumulated to minimize intracellular ice formation, to increase tolerance to dehydration caused by water outflow, and to maintain cell membrane stability and integrity that is considered central to the ability of plants to survive freezing [2,15,17–19].
The plant cell wall is the extracellular matrix consisting of cellulose, hemicellulose and lignin. It plays essential roles in plant growth and adaptive responses to adverse environmental conditions [20–22]. The cell wall integrity (CWI) and structures are dynamically regulated during plant development and are capable of being remodeled in response to various environmental stresses [23–26]. Fine-tuning regulation of the proportions and the amounts of each matrix component within the cell wall determines its nature and functions. Remarkably, deposition of lignin, phenylpropanoid polymer, which is highly hydrophobic in the cell wall, determines cell wall stiffness and permeability to water [27–29]. In yeast, the CWI signaling pathway plays a vital role in adjusting the cell wall thickness and composition to environmental cues, in particular freezing temperature and osmotic stress [30,31]. In plants, similar processes are employed for controlling cell wall integrity and performance during development, drought and defense , but the precise mechanisms remain unclear.
Previous studies have shown that the expression of genes related to cell wall biosynthesis and remodeling is dramatically altered under cold treatment . Using cryo-scanning electron microscopy (cryo-SEM), several studies have revealed that both cell membrane and cell wall properties play equally important roles in cold acclimation and freezing tolerance . Most strikingly, cell wall thickness and rigidity have been linked to dynamic water heterogeneities during cold acclimation and extra-/inter- or intracellular freezing upon freezing/thawing process. Variations in cell wall rigidity and composition in different types of plant tissues and cells (e.g. xylem, phloem, living fibers and mesophyll cells) showed altered intracellular freezing, tension-induced cavitation and cell viability during freezing/thawing [34–36]. Thus, resistance to freezing temperatures is dependent on the capacity for water outflow from the cells during cold acclimation and freezing and water reabsorption during thawing, on the capacity to accommodate growth of ice crystals in extra-/intercellular spaces, and on the ability of cell wall elasticity to respond to cellular shrinkage.
Investigation of the roles of the cell wall in cold acclimation and freezing/thawing has been limited to correlation of cellular changes in various plant species or different types of tissues and cells using cryo-SEM; mechanistic analysis of the regulatory genes and signaling cascades underlying cell wall mediated water movement and freezing tolerance is lacking. Lignin is a major component of the plant secondary cell wall, and the amounts of lignin are altered after cold treatment in various species [37,38]. In the past decades, some genes that regulate lignin biosynthesis have been identified [39–43]. Among them, Phenylalanine ammonia-lyase 1–4 (PAL; EC 4.3.1.5) encoding the enzymes that catalyze the first step in the phenylpropanoid pathway regulate biosynthesis of lignin and secondary metablites (e.g. flavonoids and salicylic acid) in Arabidopsis thaliana [39,44–48]. Arabidopsis thaliana blue copper binding gene (BCB) is another positive regulator of lignin synthesis, and AtBCB overexpression substantially increases lignin content in Arabidopsis roots . It has been shown that PAL1-PAL4 and BCB genes are responsive to a variety of environmental stimuli, including pathogen infection, wounding, nutrient depletion, UV irradiation, and extreme temperature, etc. [49,50], suggesting their roles in plant stress resistance. However, it remains unknown how these genes mediate plant responses to biotic and abiotic stresses.
Here we report that Tolerant to Chilling and Freezing 1 (TCF1), a gene encoding a RCC1 family protein, is required for chromatin based gene regulation of cold responsive genes in a CBF-independent pathway. Importantly, we reveal that lignin content in leaves is directly related to freezing tolerance, and that TCF1 plays a critical role in the adjustment of lignin accumulation through modulation of expression of BCB and downstream effectors PAL1/3/4 genes during cold acclimation and freezing tolerance in Arabidopsis.
To identify the genetic loci that regulate specifically plant cold acclimation and freezing tolerance through chromatin condensation and remodeling, we examined cold responses of the genes encoding Regulator of Chromatin Condensation 1 (RCC1) family proteins from AtGenExpress Data . The gene At3g55580 which is specifically responsive to cold was identified (S1A Fig), and designated Tolerant to Chilling and Freezing1 (TCF1) based on the phenotypes of its mutant. RT-PCR analysis and GUS assay of TCF1pro::GUS lines validated induction of TCF1 expression in response to cold but not to osmotic stress or ABA (Fig 1A–1D).
Fig 1. Analysis of TCF1 expression and nuclear localization of the TCF1 protein.
(A) Three-week-old Col-0 (WT) plants were subjected to low temperature (4°C) and the samples were harvested at the indicated time points for semi-quantitative RT-PCR analysis of TCF1 transcripts. ACT2 (At3g18780) was used as a loading control. (B) GUS staining of transgenic plants expressing TCF1pro::GUS under normal temperature or treated at 4°C for 7 days. (C) Semi-quantitative RT-PCR for TCF1 in different tissues with or without cold treatments for 7 days. R, root; Sh, shoot; L, leaves; F, flowers; Si, siliques. (D) Semi-quantitative RT-PCR analysis for TCF1 of three-week-old Col-0 plants treated with 100 μM ABA, 400 mM mannitol, 20% PEG6000, 300 mM NaCl for 3 h and 4°C Cold treatment for 24 h. (E) Localization of fluorescence in tcf1-1 plants expressing a TCF1pro::GFP-TCF1 fusion at 22°C (Upper) and 4°C for 7 days (Bottom). GFP: GFP-TCF1 fusion protein (tcf1-1TCF1-3), DAPI: DAPI staining, Merge: Merger of GFP and DAPI channels (Scale bars, 20 μm).
The TCF1 gene encodes a protein containing six predicted tandem RCC1 repeats that shows similarity to RCC1 in yeast and human [52,53] (S1B and S1C Fig). To determine whether TCF1 is localized in the nucleus like RCC1 [54,55], we made translational fusions with GFP and expressed them in tcf1-1 plants using the native promoters. Examination of independent transgenic lines revealed that GFP-TCF1 fluorescence was present in the nucleus (Fig 1E), and the level of the fusion protein GFP-TCF1 was also induced by cold (Fig 1E).
RCC1 is a guanine nucleotide exchange factor (GEF) for the small GTP-binding protein Ran. RCC1 is constitutively localized in the nucleus, binds to chromatin, and generates a Ran-GTP/Ran-GDP gradient across the nuclear envelope that is required both to drive nucleo-cytoplasmic transport and to regulate processes associated with progression of the cell cycle and mitosis [54,55]. We then ask whether TCF1 has the similar roles of RCC1. To address whether TCF1 interacts physically with chromatin, an in vitro assay was performed. The fusion protein GST-TCF1 expressed in E. coli bound strongly to a histone agarose column (Fig 2A). To further investigate which histone TCF1 interacts preferentially with, in vitro translated Myc-tagged TCF1 was pre-incubated with each kind of purified core histone in 20-fold excess followed by incubation with histone-agarose. As shown in Fig 2B, H3 and H4 were the histones that can compete effectively to diminish the binding of Myc-TCF1 to histone-agarose, suggesting high affinity binding between TCF1 and histones H3 and H4. Further yeast-two-hybrid results confirmed that TCF1 can indeed interact with the specific histone H4 (HFO2, at5g59690), but not other tested histone H3 and H4s (HTR9, At5g10400; HFO4, At1g07820) (Fig 2C). To exam whether TCF1 has GEF activity, an in vitro assay was performed. The results showed that TCF1 exhibited less than 1% of the GEF activity with human Ran as substrate of that measured for RCC1 (Fig 2D). Also expression of TCF1 did not complement the phenotype of the yeast prp20 mutant lacking yeast RCC1 (S2 Fig). The results indicate that TCF1 is not the ortholog of RCC1 in Arabidopsis, but it does associate with chromatin via its interaction with histones.
Fig 2. GEF activity assay of TCF1 and analysis of TCF1-histone interactions.
(A) Binding of E. coli-expressed GST-TCF1 to a calf thymus histone-agarose column. Western blots with anti-GST antibody showing GST-TCF1 (77 kDa) or control GST (26 kDa). Lane 1, protein applied to the columns; lane 2, unbound material that flowed through; lane 3, protein bound after a 5 min incubation, eluted with 0.3 M NaCl. (B) 0.3 μg of in vitro translated Myc-TCF1 was incubated with 20 μg of the appropriate histone (20-fold excess) for 30 min at room temperature. The samples were then incubated with histone-agarose overnight at 4°C. Myc-TCF1 binding to histone-agarose was analyzed by Western blot. The different histones used as competitors are indicated at the top of each lane. 'All' indicates the mixture of histones and 'None' indicates the control without competitors. (C) Yeast co-transformant strains carrying both TCF1 and vector control (TCF1/BD), histone H3/H4 and vector control (HFO2/AD, HFO4/AD and HTR9/AD), TCF1 and H3/H4 (TCF1/HFO2,TCF1/HFO4 and TCF1/HTR9), negative control (BD/AD) and positive control (BD-53/AD-T) were streaked onto selective media. Activation of the lacZ reporter gene is indicated by the formation of blue or blue-green colonies on plates containing X-Gal (left), the growth state of yeast co-transformant strains in YPD medium is shown on the right (right). (D) Ran-GEF activity of E. coli-expressed GST-RCC1 and GST-TCF1 is shown as the percentage of [3H]GDP remaining at the end of the GEF assay.
To verify the function of TCF1 in cold and/or freezing response, tcf1-1 and tcf1-2 were analyzed (Fig 3A and 3B). tcf1-1 with T-DNA insertion at the first exon of TCF1 showed no TCF1 transcript in response to cold treatment (Fig 3B), but tcf1-2 having a T-DNA insertion in the 3'-UTR region of TCF1 (Fig 3A) exhibited similar TCF1 expression to that of the wild-type under cold treatment (Fig 3B). Thus, the phenotypic analysis of tcf1-1 was shown thereafter. All of the F1 plants from tcf1-1×wild-type cross were resistant on MS medium containing 5 mg/L bialaphos. The F2 progeny of the selfed F1 plants segregated in a 3: 1 ratio (From 3130 plants, 2322 conferring resistant to bialaphos compared with 808 plants showing sensitive phenotype, x2 = 1.11 < 3.841; x2 test with one degree of freedom). Analysis of the bialaphos resistance revealed the presence of a single functional T-DNA that is inserted in the genome of the tcf1-1 mutant. The results indicated that the tcf1-1 mutation is recessive in a single nuclear gene.
Fig 3. tcf1-1 plants are tolerant to freezing treatment.
(A) Schematic presentation of the TCF1 gene structure and T-DNA insertions in the TCF1 gene (arrowheads). The closed rectangles represent exons and lines between the exons denote introns. (B) tcf1-1 is a null mutation of TCF1. The levels of TCF1 transcripts were determined by RT-PCR using 3-week-old tcf1-1 seedlings subjected to low temperature (4°C) for the indicated time periods (h); the ACT2 gene was used as loading control. (C) Tolerance of 3-week-old tcf1-1 and wild-type (WT) plants at the indicated temperatures below freezing under long-day photoperiod with cold acclimation for 7 days. The pictures were taken 7 days after treatments. (D) Quantification of survival rate of the treated plants in (C), (*, P < 0.05, t-test). (E) Leakage of electrolytes in tcf1-1 and WT plants treated at indicated temperatures below freezing. WT (cold) and tcf1-1 (cold): 3-week-old plants were cold-acclimated (4°C for 7 day), WT and tcf1-1: both plants were grown under normal conditions. Error bars are standard deviation (n = 8), (*, P < 0.05, t-test). (F) Tolerance of freezing treatments (-8°C for 2 h) of control and transgenic plants, which were cold-acclimated at 4°C for 7 days before the treatment. The plants included WT, tcf1-1, representative homozygous lines of tcf1-1 transformed with an empty vector pEZR(K)LC (tcf1-1Vector-2 (#2–3)) or TCF1 gene (tcf1-1TCF1-3 (#3–8), tcf1-1TCF1-12 (#12–4) and tcf1-1TCF1-13 (#13–2)).
To evaluate the effect of the tcf1-1 mutation on freezing tolerance, we performed the whole-plant freezing test. Without cold acclimation, tcf1-1 shows a slightly higher survival rate than wild-type, but there was no significant difference between wild-type and tcf1-1 (S3A Fig). When the plants were acclimated at 4°C for 7 days, 45.2% of the tcf1-1 plants survived freezing temperature as low as -10°C, but only 17.4% of the wild-type plants survived the treatment (Fig 3C and 3D). The electrolyte leakage assay confirmed the freezing tolerance of tcf1-1 plants under cold acclimation (Fig 3E). To verify the role of TCF1 in cold acclimation, we generated TCF1 RNA interference (TCF1-RNAi) lines and two TCF1-RNAi lines (TCF1-RNAi-2 and TCF1-RNAi-6) with reduced expression of TCF1 were used in freezing response assay (S3B Fig). Without cold acclimation, both TCF1-RNAi lines showed a similar survival rate to the wild-type (S3C Fig). When the plants were acclimated at 4°C for 7 days, these TCF1-RNAi lines also displayed significantly higher survival rates than the wild-type plants at -10°C (S3D and S3E Fig). The percentages of electrolyte leakage of TCF1-RNAi lines were also decreased under cold acclimation (S3F Fig), suggesting a role of TCF1 during cold acclimation and freezing tolerance in Arabidopsis.
To further determine that freezing tolerance of tcf1-1 was due to loss of function in TCF1, we expressed the GFP-CDS of TCF1 under the control of its native promoter in the tcf1-1 background (Fig 1E). Three independent transgenic lines (tcf1-1TCF1-3, tcf1-1TCF1-12 and tcf1-1TCF1-13) with increased levels of TCF1 showed a cold-sensitive phenotype compared with tcf1-1 mutant and had a similar response to the wild-type under freezing treatment with cold acclimation (Figs 3F and S4), thereby confirming that expression of TCF1 complemented the freezing tolerance phenotype of the mutant.
Because TCF1 associates with chromatin, we questioned whether TCF1 regulates freezing tolerance by modulating expression of CBF1-3 and the targeted genes. However, the expression levels and patterns of CBF1-3 under cold treatment in tcf1-1 did not show any significant difference from that in the wild-type (Fig 4A–4C and S5A). No significant changes in transcript levels of TCF1 in cbf2 (S5B Fig) were detected under cold (Fig 4D). In addition, the expression of the CBF regulon genes such as COR15A and COR47 was also not changed in tcf1-1 under cold (Fig 4E and 4F). Notably, TCF1 was not in the list of the CBF1, CBF2 and CBF3 coregulated genes in the previous study . These results indicate that TCF1 may influence cold/freezing tolerance through a mechanism different from the CBF-COR cascade.
Fig 4. Gene expression analyses of the CBFs and the CBF regulon in the tcf1-1 and wild-type plants.
(A) to (C) The levels of CBF1, CBF2 and CBF3 transcripts, respectively, in tcf1-1 and wild-type plants. The plants were treated with cold (4°C) at the indicated time points. (D) The level of TCF1 transcripts in the loss-of-function cbf2 mutant plants grown under normal conditions or given a 4°C treatment for 7 day. Transcript levels of the ACT2 gene were used as a loading control. (E) to (G) Transcript levels of COR47 (E), COR15A (F) and RD29A (G) in tcf1-1 and wild-type plants. The plants were treated with cold (4°C) for the indicated times. (H) Western blot analysis with anti-acetylated H4 antibody (Top) revealed that tcf1-1 and WT plants have similar levels of acetylated H4. Histone H4 antibody for core histone H4 was used as the immunoblot control (Bottom).
Because TCF1 reached the highest level of expression and loss of function in TCF1 enhanced freezing tolerance with a 7-day cold acclimation, the expression profiles of tcf1-1 and the wild-type grown under normal condition with a 7-day cold acclimation and without cold acclimation were compared by the GeneChip Array. Under normal conditions, only 20 genes up-regulated or down-regulated (> 1.5 fold) in tcf1-1 were identified and they are annotated to encode proteins with diverse cellular functions (S1 Table). Five genes that should be repressed during cold exposure were activated in tcf1-1, and 12 down-regulated genes are involved in diverse stress response processes. After cold acclimation, expression of 36 genes was varied (13 genes up-regulated and 23 down-regulated) in tcf1-1 (S2 Table). Among them, nineteen genes were cold responsive and are involved in different cell functions, but none of them appears to involve the CBF regulon. The highest and lowest two expression genes in microarray data under cold acclimation were validated by qRT-PCR (S6 Fig). The data supports that TCF1 functions in a novel pathway independent of the CBF-COR cascade.
HOS15 has been reported to function in a CBF-independent pathway to regulate cold acclimation and freezing tolerance . HOS15 interacts with H4 and represses RD29A expression by facilitating H4 deacetylation through association with the RD29A promoter. To determine whether TCF1 functions in the same way as HOS15, RD29A expression was analyzed in tcf1-1 plants under cold treatments. However, there were no changes in RD29A transcript levels detected in tcf1-1 plants under stress treatment (Fig 4G). Importantly, in contrast to hos15 the level of nuclear tetra-acetylated histone H4 in tcf1-1 was similar to that in wild-type plants (Fig 4H). Together with the results of expression of RD29A, which is elevated in hos15 plants in response to cold treatment  but not in tcf1-1 (Fig 4G), we conclude that TCF1 functions differently from HOS15.
To investigate whether TCF1 represses or activates the expression of target genes through direct interaction with chromatin, we performed chromatin immunoprecipitation (ChIP) assay using TCF1 complementational line (tcf1-1TCF1-3) that were cold-treated for 7 days. Seven genes (At1g23150, At1g69120, At1g75040, At5g50720, At5g10760, At5g20230 and At2g22500), which were responsive to cold in tcf1-1 in the microarray dataset were selected and three pairs of primers covering the entire genomic sequences of the candidate genes were designed. Using ChIP assay with an anti-GFP antibody, we found that among the tested genes, TCF1 was only associated with a chromatin fragment containing the coding region (BCBc1, CDS sequence +87 to +347) of the (BLUE-COPPER-BINDING PROTEIN) BCB gene (At5g20230), which is cold inducible (Fig 5A), whereas ACT2 whose expression level is not changed in the mutant was not immunoprecipitated (Fig 5B). The results suggest that the association of TCF1 with chromatin at the BCB locus mediates cold-induced and TCF1-regulated expression of BCB gene.
Fig 5. Chromatin immunoprecipitation assay of chromatin loci associated with TCF1.
(A) The levels of BCB transcripts in tcf1-1 (Grey) and wild-type (Black) plants. The plants were treated with cold at 4°C for 7 days. Data represent means of three replicates ± SD. Different Letters (P < 0.05; One Way ANOVA) show significant difference from WT. (B) ChIP assay for the association of TCF1 with BCB loci compared to the control ACT2. Lane 1, DNA immunoprecipitated by using anti-GFP antibody; lane 2, no antibody control; lane 3, input DNA before immunoprecipitation; lane 4, PCR without added DNA. TCF1pro::GFP-TCF1 (Complemental line tcf1-1TCF1-3-8, Left) and TCF1pro::GFP (tcf1-1Vector-2-3, Right) transgenic plants treated at 4°C for 4 day were used in the experiments. Below: schematic presentation of the gene structures and the positions of the PCR fragments amplified from the ChIP products. Three fragments covering the genomic sequences of BCB (BCBp1, -664bp-301bp; BCBc2, 87bp-347bp; and BCBc3, 552bp-680bp) were used in the ChIP assay. The experiments were repeated three times; each gave similar data and results from one experiment are shown. (C) Levels of H3K4me2 and H3K27me3 by ChIP-qPCR analysis with WT, tcf1-1 and two TCF1 complementary transgenic lines (tcf1-1TCF1-3 and tcf1-1TCF1-12) plants normalized to ACT2 or AGAMOUS in BCBc1 fragment with and without a 7-day cold treatment; data represent means of three biological replicates ± SD. Means with the same letter are not significantly different at P < 0.05 by One Way ANOVA analysis.
To get a better understanding of the relationships between histone modifications and BCB gene expression, we analyzed histone modifications across the BCB locus in non-stressed and stressed wild-type, tcf1-1 and TCF1 complementational lines (Figs 5C and S7A). As histone H3K4me2 has been demonstrated to play widespread roles in activation of gene expression, we first analyzed the H3K4me2 status of the BCB gene. In the wild-type and TCF1 complementation lines, BCB was induced by cold treatment. Accordingly, the level of H3K4me2 increased in the transcribed region of BCB (BCBc1 fragment) after cold treatment (Figs 5C and S7A). Under normal condition, tcf1-1 had comparable level of H3K4me2 to that of the wild-type. When exposed to cold for 7 days, tcf1-1 also exhibited an increase in H3K4me2, but the level of H3K4me2 at the BCB locus was lower than that of the wild-type and TCF1 complementation lines (Figs 5C and S7A).
Trimethylation of histone H3 at lysine 27 (H3K27me3) is a histone mark associated with gene silencing. We then analyzed the status of H3K27me3 at the BCB locus (BCBc1 fragment) in non-stressed and stressed wild-type, tcf1-1 and tcf1-1TCF1 seedlings. In the non-stressed wild-type and tcf1-1TCF1 lines, the level of H3K27me3 was relatively high. However, the level of H3K27me3 was significantly decreased when the seedlings were treated at low temperature for 7 days (Figs 5C and S7A). In sharp contrast, loss of TCF1 function caused an opposite trend in H3K27me3. Under normal condition, the tcf1-1 mutant displayed a similar level of H3K27me3 compared with that of the wild-type and TCF1 complementational lines (Figs 5C and S7A). As expected, the level of H3K27me3 at the BCBc1 region of BCB was higher in mutant compared with wild-type when exposed to low temperature for 7 days. We also tested H3K36me3 and H3K9me2 (for gene repression), H3K14ac and H3K9ac (for gene activation) and AcH4 (global histone H4 tetra-acetylation at K5/K8/K12/K16, which is also associated with gene activation) levels in the BCBc1 fragment and found there were no significant differences between WT and tcf1-1 plants (S7B–S7E Fig). The results indicate that TCF1 may regulate freezing tolerance of plants through modulation of histone modification and subsequent expression of target genes.
To test whether BCB is a functional target of TCF1 in plant response to cold, we used artificial microRNA (amiRNA) method to generate the BCB knock-down transgenic plants. Two transgenic lines with similar reduction in BCB expression to tcf1-1 mutant (named amiR-BCB4-9 and amiR-BCB9-2) (S8 Fig) were used for phenotypic analysis. Three week-old seedlings of the BCB knock-down lines and the wild type control with cold acclimation were treated at -8°C and -10°C for 2 h and were then grown under normal conditions for 7 days. The results showed that the survival rates of the amiR-BCB transgenic lines were markedly increased compared with that of the wild type (Figs 6A and 6B and S9A and S9B). The electrolyte leakage assay revealed that knock-down of BCB significantly reduced the electrolyte leakage from the treated plant cells (Fig 6C), confirming that BCB gene plays an important role in freezing tolerance of Arabidopsis plants during cold acclimation. However, we noticed that the average survival rate for the amRNAi-BCB lines (about 35%) is significantly lower than the tcf1-1 (45.5%) under -10°C treatment (Figs 3D and 6B), indicating that in addition to BCB, alteration of other genes may also contribute to the freezing tolerance of tcf1-1.
Fig 6. amiRNA-BCB plants are tolerant to freezing treatment.
(A) Freezing treatment of 3-week-old amiRNA-BCB transgenic plants (#4–9 and #9–2) and wild-type (WT) plants at the indicated temperature below freezing under long-day photoperiod with cold acclimation. The pictures were taken 7 days after treatments. (B) Quantification of survival of the plants in (A). Error bars are standard deviation (n = 80–100), (*, P < 0.05, t-test). (C) Leakage of electrolytes in amiRNA-BCB transgenic plants and WT plants treated at indicated temperature below freezing. Error bars are standard deviation (n = 8). (*, P < 0.05, t-test). (D) Quantitative determination of lignin content from whole rosettes of three-week-old WT and amiRNA-BCB transgenic plants grown in soil with (Grey) or without (Black) cold treatment (4°C for 7 days). Twelve independent experiments were performed and the data are expressed as mean ± S.E. Means with the same letter are not significantly different at P < 0.05 by One Way ANOVA analysis.
The immediate question is how BCB regulates plant freezing tolerance. Since a previous study has shown that overexpression of BCB results in increased lignin accumulation in Arabidopsis , we attempted to test whether BCB regulates plant freezing tolerance through modification of lignin content of plants. To this end, we analyzed the lignin levels of 3-week-old seedlings of the amiRBCB transgenic lines and the wild-type that were treated at 4°C for 7 days. As shown in Fig 6D, the lignin content of the wild type seedlings was not affected by cold treatment, by contrast, the lignin levels of the amiRBCB transgenic lines was significantly reduced compared with WT under cold treatment (Fig 6D). The results suggest that BCB is responsible for maintaining steady lignin content in Arabidopsis plants under cold stress conditions. Taken together, these results indicate that TCF1 may modulate freezing tolerance through a BCB-dependent mechanism that positively regulates lignin biosynthesis.
To investigated whether the genes responsible for lignin biosynthesis showed any differential expression in tcf1 in response to cold treatment. The transcript levels of PAL genes, which encode isoforms of a key enzyme Phe ammonia lyase involved in lignin biosynthesis were analyzed. In wild-type seedlings, the transcript levels of PAL1 and PAL3 remained not changed after cold treatment, but the PAL2 and PAL4 gene was induced by cold treatment (Fig 7A and 7B). In the tcf1-1 mutant, expression of PAL1-4 was similar to that of wild type under normal condition, but PAL1, PAL3 and PAL4 exhibited significantly decreased transcript accumulation after cold treatment. PAL2 showed a similar level of transcript in the non-stressed and stressed tcf1-1 mutant to that of the wild-type (Fig 7A and 7B).
Fig 7. Expression analysis of PAL genes in tcf1 and amiRNA-BCB mutants.
(A) to (D) Quantitative RT-PCR analysis of the expression of PALs in three-week-old WT, tcf1-1 and amiRNA-BCB plants grown in soil with or without cold treatment (4°C for 7 days). Three independent experiments were performed and the data are expressed as mean ±.SE. (*, t-test, P < 0.05).
We also checked the PALs expression in amiRNA-BCB transgenic lines, we found that PAL2 showed a similar level of transcript in the non-stressed and stressed amiRNB-BCB mutant to that of the wild-type, but PAL1, PAL3 and PAL4 exhibited significantly decreased transcript accumulation after cold treatment in the amiRNB-BCB mutant (Fig 7C and 7D).These results suggest that TCF1 and BCB positively regulates lignin content in rosettes under low temperature and that this may be due to regulation of genes involved in lignin biosynthesis.
To test whether TCF1 regulates plant freezing tolerance through modulating lignin biosynthesis, we measured the lignin contents in tcf1 and wild-type plants with cold treatments. As expected, at the end of a 7-day cold treatment, the tcf1 rosettes accumulated a significantly lower level of lignin compared with the wild-type (Fig 8A), suggesting a role of TCF1 in lignin biosynthesis.
Fig 8. Reduced lignin content increased freezing tolerance.
(A) Quantitative determination of lignin content from whole rosettes of three-week-old WT and tcf1-1 plants grown in soil with (Grey) or without (Black) cold treatment (4°C for 7 days). Twelve independent experiments were performed and the data are expressed as mean ±S.E. Means with the same letter are not significantly different at P < 0.05 by One Way ANOVA analysis. (B) Three week-old pal1pal2 and wild type (WT) plants with or without a 7-day cold treatment at 4°C were used for freezing treatments at indicated time point. The pictures were taken 7 days after treatments. (C) Quantification of survival of the wild type (Black) and pal1pal2 plants (Grey) in (B); Error bars represent standard deviation (n = 80–100). (*, t-test, P < 0.05). (D) Leakage of electrolytes in pal1pal2 and wild type plants treated (see experimental procedures) at indicated time point with and without cold acclimation. The experiments were repeated 3 times. Error bars are standard deviation (n = 6). (*, t-test, P < 0.05).
As the loss of function in TCF1 causes reduced lignin content and increased freezing tolerance, we proposed that under freezing temperature, reduced lignin may protect plant cells from freezing stress. To test this possibility, we analyzed the freezing tolerance of the pal1pal2 (pal1-2/pal2-2) double mutant with or without cold acclimation. Under normal condition, the pal1pal2 rosettes contained the lowest level of lignin reduction compared with the wild-type and tcf1-1 mutant (Fig 8A). The substantial low level of lignin in the pal1pal2 double mutant observed in this study is consistent with previous reports . Based on our hypothesis, the less lignin the plants accumulate, the higher freezing tolerance they have. Similar sized wild-type and pa11pal2 seedlings were subjected to freezing treatment with or without cold acclimation. As expected, the pal1pal2 mutant displayed the highest freezing tolerance (Fig 8B–8D and S10A and S10B). The stronger effect of pal1pal2 mutant on freezing tolerance than tcf1 and wild-type suggests that the reduced lignin level is correlated to plant freezing tolerance.
The present study reports isolation and functional characterization of a novel nuclear protein TCF1 as a determinant of cold acclimation and freezing tolerance. We show that TCF1 specifically activated by cold associates with chromatin and regulates a specific set of genes that are involved in cold acclimation and adaptation to freezing temperature. Importantly, TCF1 regulates lignin accumulation during acclimation/freezing and affects plant freezing tolerance via a CBF independent pathway. Thus, our study not only identifies a novel TCF1-mediated signaling cascade that plays a key role in cold acclimation and freezing tolerance, but also reveals a critical role of cell wall remodeling, in particular lignin homeostasis in cell wall in protecting cells from freezing damage.
TCF1 specifically responded to cold stress via both transcript and protein accumulation (Fig 1), and tcf1-1 plants displayed specific enhancement of freezing tolerance after cold acclimation (Fig 3C and 3D). These results suggest that TCF1 is functions as a negative regulator in cold acclimation and freezing tolerance in Arabidopsis. Recently, extensive attention has been paid to the CBF signaling pathway, however, several studies have shown that the CBF signaling pathway is not the sole mechanism modulating plant cold acclimation and cold tolerance, because at least 28% of the cold-responsive genes were not regulated by the CBFs . TCF1 may act independently of the CBF-COR signaling pathway, because expression of the CBF genes and CBF-regulated genes was unaltered in tcf1-1 (Fig 4A–4C, S1 and S2 Tables), and TCF1 expression remained unchanged in cbf2 (Fig 4D) and in the CBFs-overexpression plants . The TCF1-mediated pathway is also distinct from HOS15, because in tcf1-1, cold induction of RD29A and the level of nuclear tetra-acetylated histone H4 were not affected (Fig 4G and 4H). Thus, we conclude that TCF1 regulates plant cold acclimation and tolerance through at least one additional regulatory pathway. Further genetic analysis will be necessary to determine the genetic relationship between TCF1 and CBFs under cold acclimation and freezing tolerance.
TCF1 belongs to a family of RCC1-like proteins in Arabidopsis. RCC1 functions as the GEF for the small G-protein Ran and is critical for maintaining the RanGTP/RanGDP gradient across nuclear envelope . Our data show that unlike RCC1, TCF1 had very low Ran-GEF activity (Figs 2D and S2), indicating that TCF1 is not the ortholog of RCC1 in Arabidopsis. However, TCF1 shares several important features with UVR8, another RCC1 family protein: very low GEF activity, function in nucleus (Fig 1E) and histone/chromatin association (Fig 2A–2C), although UVR8 preferentially interacts with histone H2B [40,58]. UVR8 regulates plant responses specifically to UV-B through modifying expression of HY5 . Thus, we hypothesized that TCF1 may regulate cold acclimation and freezing tolerance through a similar regulatory mechanism. Indeed, we found that TCF1 directly interacted with the coding regions of BCB (Fig 5B). Further we show that activity of BCB correlates with the enrichment of the positive mark H3K4me2 and reduction of the repressive mark H3K27me3 as cold acclimation is initiated (Figs 5C and S7A). Most importantly, TCF1 is required to modulate levels of both H3K4me2 and H3K27me3 at the BCB locus and regulate BCB transcription (Figs 5C and S7A). Therefore, H3K4me2 and H3K27me3 appear to synergistically regulate transcription activation of BCB, pointing to a critical role of active and repressive marks in cold acclimation.
It is clear that cold activates TCF1 to induce or repress a set of target genes through a chromatin based mechanism. Interestingly, BCB encodes a Blue Copper Binding protein, which is a glycosylphosphatidylinositol-anchored protein (GAP) targeted to the cell surface [59,60] and seems to be responsible for lignin accumulation and cell wall-based resistance to aluminum and bacteria [49,61]. Therefore, our data support the hypothesis that TCF1 regulates cold acclimation and freezing tolerance through modulating BCB to adjust lignin accumulation and consequent cell wall remodeling (Fig 6). We observed a TCF1 dependent reduction in BCB expression and lignin content in rosette leaves of tcf1-1 during cold acclimation (Fig 8A). Importantly, we found that expression of PAL1, PAL3 and PAL4 was reduced under cold in tcf1-1 and BCB knock-down transgenic lines (Fig 7), suggesting that the transcriptional activity of the genes in leaves is also influenced by TCF1 and BCB. Our data reveals that TCF1 directly influences BCB activity and affects PAL1, PAL3 and PAL4 expression and lignification, although it is unclear whether BCB directly regulates PAL genes and lignification or whether TCF1 mediated alteration of chromatin state during cold acclimation indirectly affects PALs expression. Therefore, our data define TCF1 as a key factor able to bind to chromatin and epigenetically regulate the cold-specific GAP leading to establishment of cold specific transcriptional programs and consequently lignin and extracellular matrix remodeling. Although BCB level was almost the same in tcf1-1 and amiRNA-BCB lines, tcf1-1 showed higher survival rate than amRNAi-BCB lines under freezing treatments (Figs 3D and 6B), pointing out that BCB was not the only gene to involve in TCF1-mediated freezing tolerance. Further identification of the target gene(s) of TCF1 will help us to uncover the molecular mechanism underlying TCF1-mediated plant cold acclimation and freezing tolerance. Since UVR8 can also modulate plant response to UV-B through direct interaction with COP1 . We do not exclude the possibility that TCF1 regulates cold acclimation and freezing through association with the key regulator(s) in the cold signal transduction pathway.
It is well known that lignin fills the spaces in the cell wall to reduce water permeability and increase the stiffness of the cell wall [45,50]. Extensive cellular studies have shown that freezing tolerance is directly related to cell permeability and cell wall properties, in particular lignin content, so that water outflows and ice forms in the extracellular spaces without damaging cellular structures [18,35,63–65]. Although it is still technically difficult to measure the cell wall permeability of plants during cold acclimation, the important role of lignin in plant cold acclimation and freezing tolerance has been well documented. For example, the freezing tolerant Miscanthus contains lower lignin content and higher PAL gene expression than the freezing sensitive ecotype . Therefore, it is conceivable that lignin content is closely related to the cell wall permeability and freezing tolerance. However, the molecular mechanism by which lignin content is regulated during cellular adaptation to low temperature still remains a mystery. Here we demonstrate that during cold acclimation tcf1-1, amiRNA-BCB and pal1pal2 had increased freezing tolerance which correlates with their reduced lignin contents (Figs 6 and 8). Therefore, reduction of lignin deposition within the cell wall of the tcf1-1, amiRNA-BCB and pal1pal2 plants during acclimation and freezing may increase cell wall permeability and protect the cells from freezing damage. Reduction of lignin may also enhance elasticity of the cell wall to increase the capacity to accommodate growth of ice crystals with less damage to both the dehydrated cell and cell wall. Together, our data reveal a novel regulatory mechanism in cold acclimation and freezing tolerance in Arabidopsis that involves chromatin based regulation of lignification and cell wall remodeling.
The immediate question is what the biological significance of TCF1 induction is because absence of TCF1 confers freezing tolerance. It is known that cold hardy (freezing tolerant) plants frequently employ extracellular freezing to cope with the freezing temperature. Arabidopsis Col-0 is not hardy plant although it has moderate freezing tolerance compared with other ecotypes . It is possible that TCF1-mediated signaling is activated to maintain lignin content of cells that can enhance cell rigidity and reduce cell expansion, which is required for plant growth arrest under low temperature. Thus, it is conceivable that TCF1-mediated signaling modulates plastic development of the plants during cold acclimation, but not freezing tolerance. A mechanistic working model is presented in Fig 9. The fact that absence of TCF1 enhances freezing tolerance of Col-0 plants suggests that low expression or TCF1 absence may be related to plant freezing tolerance. Thus, our study also identifies a new gene that can be used for genetic improvement of plant freezing tolerance. It will be interesting to see whether TCF1 gene acts differently in cold hardy plants. Given that natural variation in TCF1 may contribute substantially to cold acclimation and freezing tolerance among Arabidopsis accessions, the role of TCF1 expression and its mediated signaling in cold acclimation and freezing tolerance is worthy of special focus. Furthermore, further study will also help to differentiate the molecular mechanisms in plant cold acclimation and freezing tolerance.
Fig 9. A proposed model for TCF1 in the Arabidopsis response to low temperature.
Under normal condition, PAL genes are modulated by the developmental signals to synthesize lignin that favor optimal plant growth and water transport. During cold acclimation, TCF1 is rapidly induced to activate BCB transcription and then stimulates expression of PAL1/3/4 genes to maintain lignin accumulation of the stressed cells. However, when the plants are exposed to freezing temperature, reduction of lignin deposition within the cell wall of the tcf1-1 plants may increase cell wall permeability and protect the cells from freezing damage. Reduction of lignin may also enhance elasticity of the cell wall to increase the capacity to accommodate growth of ice crystals with less damage to both the dehydrated cell and cell wall which is required for plant growth arrest. Lines with arrowheads denote direct regulation, and lines with blunt heads represent indirect regulation.
A. thaliana (L.) Heynh Col-0 and tcf1-1 (SAIL_97_D05) and tcf1-2 (SAIL_749_A09) from ABRC were germinated on MS medium containing 2% sucrose, pH 5.7. Three-week-old tcf1-1 and wild-type plants grown in soil with cold-acclimation (4°C for 7 days) and non-cold acclimation were used for whole plant freezing tolerance tests and an electrolyte leakage assay (ELA) as previously described . Briefly, the plants were grown in soil under a long-day photoperiod (16 h light/8 h dark) for 3 weeks in a growth chamber (22°C), and then placed in a temperature chamber (ZSP-A0160, ZHCHENG) at -6°C, -8°C or -10°C for 2 hours. Freezing tolerance was determined as the capacity of plants to resume growth after 7 days under control conditions. For ELA, the rosettes were placed in a temperature chamber starting at 0°C. The temperature was reduced by 2°C after 30 min and maintained for 1 h. Then an identical timing sequence was used for successive 2°C decreases until -10°C was reached. The percentage of EL was calculated as the percentage of the conductivity before autoclaving over that after autoclaving.
Total RNA was extracted from plants' leaves by using the TRizol Reagent. First-strand cDNA synthesis was performed according to standard procedures using reverse transcriptase (Promega, 18064–014) following the manufacturer's instructions. Semi-quantitative RT-PCR and qRT-PCR were done as described previously . A positive control was provided by a parallel analysis based on the ACT2 gene, and three independent replicates were performed per experiment. Gene-specific primers for CBF1, CBF2, CBF3 etc are shown in S3 Table.
For Microarray analysis, 3-week-old plants were treated with or without low temperature at 4°C for 7 day, 2 μg of total RNA was used to produce cyanine dye-tagged cRNA (cy5-WT, cy3-tcf1) and was hybridized to JingXin Array (CapitalBio Company) containing 29K Arabidopsis transcripts. Three biological replicates with 6 Microarray slides were used to check differential expression genes. Data from the GeneChip arrays were scanned on a GeneChip Scanner 3000 and analyzed using GeneChip Operating software (GCOS 1.4). The Significant Analysis of Microarray software (SAM) was used to identify significantly differentially expressed genes between tcf1 and WT groups. Different genes were determined to be significantly differentially expressed with a selection threshold of false discovery rate, FDR = 5% and fold change > 1.5 in the SAM output result. The Microarray data had submitted to GEO (Accession number: GSE70682) at NCBI website.
For histochemical analysis of TCF1 expression, a genomic fragment including 1,337 bp upstream of the translation initiation codon was amplified by PCR and cloned into the binary vector pCAMBIA1391 between the HindIII and BamHI sites. β-glucuronidase (GUS) activity was assayed as previously described . To make the TCF1pro::GFP-TCF1 fusion, the TCF1 CDS was first amplified by PCR and cloned in-frame into the binary vector pEZR(K)-LC  between the EcoRI and SalI sites, and then the 35S promoter was replaced by the 1,337 bp TCF1 promoter between the HindIII and SacI sites to generate TCF1pro::GFP-TCF1. The construct was introduced into wild-type and/or tcf1-1 plants through A. tumefaciens-mediated transformation. At least three independent homozygous T3 lines were tested for TCF1pro::GUS expression, protein subcellular localization, and gene expression analysis.
The TCF1 coding region was amplified by PCR and cloned in-frame between the EcoRI and BamHI sites of pGBKT7. HTA1 [histone H2A (AT5g54640)] and HTR9 [histone H3 (AT5g10400)] coding regions were amplified and cloned in-frame into pGADT7 between the NdeI and XhoI sites to generate prey constructs. pACT2-HTB1 (histone H2B) and pACT2-HFO1 (histone H4) were from Dr. R. A. Bressan at Purdue University. For analysis of specific histone H4 interaction with TCF1, TCF1 coding region was cloned in-frame between the EcoRI and BamHI sites of pGADT7, another two histone H4 variants (At5g59690 and At1g07820) with high expression in tcf1-1 were introduced into pGBKT7 at EcoRI and SalI sites. Plasmid DNA of bait and prey constructs was transformed into the S. cerevisiae strain Y190. Individual transformants were streaked on plates containing a synthetic, minimal (SD) medium lacking tryptophan and leucine and grown for 24 h. Yeast cells were transferred onto a filter paper, and β-galactosidase (β-gal) filter assays were performed .
Nuclei were isolated as described by . Twenty micrograms of nuclear protein and 1 μg of purified core histones from chicken (Upstate Biotechnology, 13–107) were separated by SDS/PAGE and blotted onto a PVDF membrane (Millipore, GVPPEAC12). Anti-tetra-acetylated-histone H4 (1:1,000) or anti-histone H4 (1:100,000) (Upstate Biotechnology) primary antibodies were used to detect acetylated and unacetylated histone H4, bands were visualized by using the BCIP/NBT Kit (Invitrogen). Data shown are representative of six independent experiments.
GEF activity assay was performed as described in . Briefly, RCC1, TCF1, and human Ran were expressed in Escherichia coli as fusions with GST. The Ran clone was provided by Dr. Murray Stewart (Medical Research Council Laboratory for Molecular Biology, Cambridge, U.K.). Assays of guanine nucleotide exchange activity were performed by using [3H]GDP to load 30 pmol GST-Ran and subsequent incubation with 0.5 nM recombinant RCC1 or TCF1 for 3 min. The exchange activity was calculated as ln (Ct/C0), where C0 and Ct are radioactive counts at the start and end of the reaction, respectively. The GEF assays were repeated four times. Analysis of TCF1-histone interaction was performed as previously described . Competition assays between TCF1 and histones were conducted as previously described by Cloix and Jenkins .
Chromatin was isolated and the chromatin immunoprecipitation assay was carried out [40,69] by using an anti-GFP antibody (Invitrogen A-11122). Before antibody treatment, the samples were precleared with protein A Dynabeads (Dynal Biotech, Great Neck, NY, 100.02). The immunoprecipitated DNA was used in PCR reactions to amplify fragments from the BCB and ACT2 genes, using primers shown in S3 Table. Data shown are representative of three independent experiments.
Cell wall fractions were isolated as described by Tokunaga et al . Briefly, three-week-old plants at rosette stage were ground in liquid nitrogen, and then washed with 95% ethanol and ethanol:hexane (1:2, v/v) in turn. The washed pellet was allowed to air-dry at 70°C overnight. Lignin content was measured according to the method of Fukuda and Komamine  with some modifications. Five mg of the air-dried samples suspended in a 1 ml aliquot of 25% acetyl bromide in acetic acid were treated at 70°C for 30 min. After cooling down, 0.9 ml of 2 M NaOH, 5 ml of acetic acid, 0.1 ml of 7.5 M hydroxylamine hydrochloride, and 3 ml of glacial acetic acid were added. The 10 ml samples were centrifuged and the absorbance of the supernatant was measured at 280 nm to determine the lignin content.
S1 Fig. TCF1 expression and computational analyses of TCF1 protein.
(A) Arabidopsis eFP Browser online software (http://bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb.cgi) shows the TCF1 gene expression was induced by cold stress. (B) Clustal W2 analysis of identity between RCC1 and TCF1 protein (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Arrows in different colors with letter ABCD stands marked seven RCC1 repeat domains. (C) Expasy online software (http://www.expasy.org/scanprosite) predicted that Homo sapiens RCC1 contains 7 RCC1 repeats. Using the same program, we found that the TCF1 protein contains six predicted RCC1 repeats that are located as follows: 83–135, 178–252, 253–329, 330–381, 382–435, and 436–486.
S2 Fig. TCF1 protein does not complement the temperature-sensitive phenotype of prp20 mutants.
Yeast prp20 mutant was transformed with the indicated vector. Individual colonies of transformants were streaked on SD plates lacking Ura with galactose as carbon source and put in 23°C /37°C for 24 h simultaneously. prp20+RCC1, prp20 transformed with human RCC1 gene. prp20+Vector, prp20 transformed with empty vector pMB150. prp20+TCF1, prp20 transformed with TCF1 gene.
S3 Fig. tcf1-1 plants show the same phenotype with WT without cold acclimation.
(A) Freezing analysis of 3-week-old tcf1-1 and wild-type (WT) plants at the indicated temperatures below freezing under long-day photoperiod without cold acclimation. Quantification of survival rate was taken at 7th days after treatments. (B) RT-PCR analysis of TCF1 expression in two TCF1-RNAi lines (TCF1-RNAi-2 and TCF1-RNAi-6) at 4°C for 7 day. (C) Freezing analysis of 3-week-old TCF1-RNAi lines and wild-type (WT) plants at the indicated temperatures below freezing under long-day photoperiod without cold acclimation. Quantification of survival rate was taken at 7th days after treatments. (D) Tolerance of 3-week-old TCF1-RNAi lines (TCF1-RNAi-2 and TCF1-RNAi-6) and wild-type (WT) plants at the indicated temperatures below freezing under long-day photoperiod with cold acclimation for 7 days. The pictures were taken 7 days after treatments. (E) Quantification of survival rate of the treated plants in (C), (*, P < 0.05, t-test). (F) Leakage of electrolytes in TCF1-RNAi lines and WT plants treated at indicated temperatures below freezing. Error bars are standard deviation (n = 8), (*, P < 0.05, t-test).
S4 Fig. TCF1 gene complements the tcf1-1 freezing tolerant phenotype.
(A) The three week-old non-acclimated plants were treated at -8°C for 2 h followed by a seven day recovery. These include tcf1-1, WT, a representative line of tcf1-1 transformed with an empty vector pEZR(K)LC (tcf1-1Vector-2), and three independent homozygous lines of tcf1-1 transformed with TCF1 gene (tcf1-1TCF1-3, tcf1-1TCF1-12 and tcf1-1TCF1-13) without cold acclimation. (B) Semi-quantitative RT-PCR for TCF1 expression in the wild type and transgenic plants. The three-week-old plants were treated with or without a seven-day cold acclimation at 4°C, respectively. Lane 1: WT, lane 2: tcf1-1, lane 3: tcf1-1Vector-2, lane 4: tcf1-1TCF1-3, lane 5: tcf1-1TCF1-12, lane 6: tcf1-1TCF1-13. (C) Quantification of survival rate of cold acclimated (black) and non-acclimated (grey) plants after freezing treatment at -8°C for 2 h and a seven day period of recovery. 1: WT, 2: tcf1-1, 3: tcf1-1Vector-2, 4: tcf1-1TCF1-3, 5: tcf1-1TCF1-12, 6: tcf1-1TCF1-13. Error bars are standard deviation (n = 80–100). Three biological experiments were performed and the data are expressed as mean ±S.E. Means with the same letter are not significantly different at P < 0.05 by One Way ANOVA analysis. (D) Leakage of electrolytes in WT, tcf1-1 and TCF1 complementary lines (tcf1-1TCF1-3, tcf1-1TCF1-12 and tcf1-1TCF1-13) treated at indicated temperatures below freezing. 3-week-old plants were cold-acclimated at 4°C for 7 day (Cold) or without treatment. Error bars are standard deviation (n = 10), (*, P < 0.05, t-test).
S5 Fig. Expression of CBFs in the wild-type and tcf1-1 plants under treatment.
(A) The three week-old tcf1-1 and wild type plants were treated with cold at 4°C and rosettes were harvested at the indicated time points for RNA extraction and semi-quantitative RT-PCR. Rosettes of the plants treated with 100 μM ABA, 400 mM mannitol or 300 mM NaCl for 3 h were also collected for gene expression analysis. Transcript levels were assayed for CBF genes and loading control ACTIN2. (B) Semi-quantitative RT-PCR for CBF2 expression in a T-DNA insertion line. Three-week-old SALK_025203 and Col-0 seedlings grown on soil were subjected to low temperature (4°C) for 3 h, and the shoots were collected for the expression analysis. ACT2 gene was used as a loading control. The result showed that the mutant is a null mutation for the CBF2 gene, and was named as cbf2.
S6 Fig. Quantitative RT-PCR analysis of the AT2G33810, AT1G23150, AT3G57260 and AT3G51330 genes expression.
Three-week-old WT and tcf1-1 grown on MS medium plates were treated with cold stress for 7 day, and the levels of four genes' transcripts were analyzed. The genes showed altered transcript levels in tcf1-1 in the microarray analysis were selected. ACT2 gene was used as a loading control. CA: Cold acclimation, NA: None cold acclimation.
S7 Fig. Level of histone markers in wild-type, tcf1-1 and two TCF1 complementary lines (tcf1-1TCF1-3 and tcf1-1TCF1-12) with or without cold acclimation.
(A) Levels of H3K4me2 and H3K27me3 by ChIP-PCR analysis with wild-type (WT), tcf1-1, tcf1-1TCF1-3 and tcf1-1TCF1-12 plants normalized to ACT2 or AGAMOUS in BCBc1 fragment with and without a 7-day cold treatment. (B) to (E) Levels of H3K9me2, H3K9ac, H4Ac (The level of global histone H4 tetra-acetylation at K5/K8/K12/K16), H3K14ac and H3K36me3 by ChIP-PCR analysis normalized to indicated control in BCBc1 fragment in wild type and tcf1-1 plants with a seven-day cold acclimation.
S8 Fig. Reduced BCB expression in amiRNA-BCB plants under cold acclimation.
The levels of BCB transcripts in wild-type (Black) and amiRNA-BCB (Grey) plants. The plants were treated with cold at 4°C for 7 days. Three biological experiments were performed and the data are expressed as mean ±S.E. Means with the same letter are not significantly different at P < 0.05 by One Way ANOVA analysis.
S9 Fig. amiRNA-BCB plants show similar freezing phenotype with WT without cold acclimation.
(A) Freezing treatment of three-week-old amiRNA-BCB transgenic plants (#4–9 and #9–2) and wild-type (WT) plants at the indicated temperature without cold acclimation. The pictures were taken 7-days after treatments. (B) Quantification of survival of the plants in (A). Error bars are standard deviation (n = 80–100).
S10 Fig. pal1pal2 is freezing tolerant compared with WT.
(A) Three week-old pal1pal2 and wild type (WT) plants without a 7-day cold treatment were used for freezing treatments at indicated time. The pictures were taken 7 days after treatments. (B) Quantification of survival of the wild type (Black) and pal1pal2 plants (Grey) in (A). Error bars represent standard deviation (n = 80–100).
S1 Table. Genes with increased and decreased expression levels by 1.5-fold in tcf1-1 without cold acclimation determined by Biocapital Jingxin microarray.
S2 Table. Genes with increased and decreased expression levels by 1.5-fold in tcf1-1 with cold acclimation determined by Biocapital Jingxin microarray.
We thank Dr. Abelson John, Division of Biology, California Institute of Technology, for kindly providing us the prp20 strains and the constructs. We also thank Prof. Zhixiang Chen, Department of Botany and Plant Pathology, Purdue University for pal1-2/pal2-2 double mutant.
Conceived and designed the experiments: XL. Performed the experiments: HJ YW CC KL SW YS. Analyzed the data: HJ YW CC KL. Contributed reagents/materials/analysis tools: YW KL GIJ SY ZS. Wrote the paper: HJ XL.
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In 1968, after 13 years in industry, Sol left the rat race to become a Professor of Electrical Engineering and Computer Programming at Union County College, Cranford New Jersey. He also taught in the Rutgers University Graduate School of Computer Science in the mid 1970s and was a visiting lecturer on topics related to microcomputer design at several other colleges and universities. He retired in 1993, after 25 years of teaching.
Sol built his first primative home digital computer in 1970. In 1973 he built his first microprocessor-based computer. Computer systems have been his hobby ever since.
In 1976 Sol co-founded the annual Trenton Computer Festival, the oldest and longest-running Personal Computer show in the world. TCF drew attendees from from across the U.S. and Canada.
During the 1970s and 1980s Sol spoke at many computer industry conventions and trade shows . He was a member of the IEEE (Institute of Electrical & Electronic Engineers) Computer Standards Committee which developed the interface standards for microcomputers. He also served as a consultant on microcomputer hardware and software design for several companies during the 1970s and 1980s.
He authored many articles in magazines and journals related to electronic and computer design over a thirty year period beginning in 1955. From 1975-1981 he authored a monthly column in Byte magazine, for many years the leading magazine devoted to microcomputer topics.
Sol began publishing and editing Microsystems magazine in 1979 and later sold it to Ziff-Davis Publishing, remaining as Editor. The magazine, written for designers of microcomputer-based hardware and software. It had a peak circulation of over 60,000. In 1984, after 28 issues, it ceased publication. At the urging of subscribers Sol restarted the magazine, as Micro/System Journal. The magazine had 22 issues and was published from 1985-1988. To view the covers and contents of all the issue go to Microsystems (1980-1984) and Microsystems Journal (1985-1988) magazines.
Sol also authored 16 books. His first book, was published in 1960, was "Repairing Transistor Radios". It was the first book on the subject and was translated in several languages. As Sol interests in digital logic and microcomputer hardware and software design developed, he wrote some of the first books on these subjects.
Sol is married to Lennie Libes, and they have a daughter, Susan, and a son, Don.


text:
ted ligety gs
Checking in with Ted Ligety to talk about uniting snow sports and SHRED.'s film, Art of Arc.Follow Ted Ligety and Antti Autti as they celebrate the beauty and simplicity of turning in their new film "Art of Arc".Ski legend John Moseley interviews alpine racer Ted Ligety about their shared experience handling FIS controversies and details from their famed careers.Ted Ligety seeks to repeat his win at this weekend's World Cup opener in Sölden, Austria.Forget the Kardashians, Victor Muffat-Jeandet is going to break the internet.A strong second run put Ligety on top of the podium at FIS World Championships in Garmisch-Partenkirchen, Germany.Follow Ted Ligety through a racecourse in Deer Valley.
FIS Alpine. Follow Ted Ligety through a racecourse in Deer Valley. I've spoken out on them before joking that Michael Von Gruenigen and Alberto Tomba were going to come out of retirement and use their old skis. Voir plus de contenu de Ted Ligety sur Facebook THE PERFECT TURN by TED LIGETY - Duration: 112 seconds. 273,205 views; 5 years ago; 3:23. Author: Emy Reznik Publish date: Mar 24, 2020. Ted has been a force on the World Cup circuit for over a decade. Photo courtesy of Shred. GoPro: From The Eyes of Ted Ligety - Duration: 3 minutes, 23 seconds.
In 2014 he won the first-ever giant slalom gold medal for the USA at the Olympics in Sochi, Russia. Ted Ligety is one of the fastest men on skis. With five World Cup Overall GS titles and five World Championship titles, including three Gold Medals at the 2013 World Championships, he is an accomplished skier that is still driven by his pure love of skiing.Head, Shred, Leki, Putnam, Deer Valley, Land Rover, GoProSkiing, backcountry skiing, mountain biking, basketball, family, business– At age 21, Ted became the youngest American man to win an Olympic Gold medal in Alpine Skiing.The rest of the World Pro Ski Tour (WPST) season has been cancelled due to growing concerns over health and safety issues resulting from the Coronavirus (COVID-19) pandemic. Thoughts on New GS ski rules. Both in 2013 and 2014, he was the racer with the most victories that season and among the top three with the most podiums. Perfect GS Turns with Ted Ligety. Although a GS specialist, Ligety is among the few alpine ski racers to have a World Cup podium finish in all five disciplines. GoPro. Hit the nailed on its head. Ted Ligety. 9,695 views; 5 months ago; 1:52. There's a lot of talk out there right now about the new FIS ski rules to be implemented in 2012, most specifically the GS radius rule.
NBC Sports. https://www.nytimes.com/.../on-giant-slalom-ted-ligety.html Ted Ligety . 990,721 views ; 5 years ago; 0:58. Ligety laying down a perfect arc in Deer Valley.
The World Pro Ski Tour™ is a nationwide tour of events featuring the popular "dual" format of alpine ski racing. Ted Ligety's superb GS run not enough to take down Kranjec | NBC Sports - Duration: 11 minutes, 22 seconds. Ted Ligety is one of the fastest men on skis. Hailing from Emy Reznik is an editorial intern at SKI Magazine.
In 2014 he won the first-ever giant slalom gold medal for the USA at the Olympics in Sochi, Russia. Ted has been a force on the World Cup circuit for over a decade.
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redpajama_set_name: RedPajamaCommonCrawl


text:
Created by Microsoft Corporation, 2015. Provided As-is without warranty. Trademarks mentioned here are the property of their owners.

### Intro
* [Universal Windows apps](http://blogs.windows.com/buildingapps/2014/04/02/extending-platform-commonality-through-universal-windows-apps/)
* This is a **newer and better** version of the previously released oneNote API [WinStore](https://github.com/OneNoteDev/OneNoteAPISampleWinStore) and [WinPhone](https://github.com/OneNoteDev/OneNoteAPISampleWinPhone) code samples. Use this code sample to build universal Windows 8.1 and above apps.
* As of August 2015, this code sample contains examples of all shipped features as well as most of the beta features released by the OneNote API team.

**If you wish to ignore the app UI/design and directly get to the part that interacts with the OneNote API, you can find the code under the [OneNoteServiceSamplesWinUniversal.Shared/OneNoteApi](https://github.com/OneNoteDev/OneNoteAPISampleWinUniversal/tree/master/OneNoteServiceSamplesWinUniversal.Shared/OneNoteApi) folder**.

### OneDrive based API

You can find additional documentation at the links below.

* [Authenticate the user using the OnlineIdAuthenticator API](http://msdn.microsoft.com/en-us/library/windows/apps/windows.security.authentication.onlineid.onlineidauthenticator.aspx)
    * In our previous [WinStore](https://github.com/OneNoteDev/OneNoteAPISampleWinStore) and [WinPhone](https://github.com/OneNoteDev/OneNoteAPISampleWinPhone) Code samples we demonstrated how to use the
[LiveSDK](http://msdn.microsoft.com/EN-US/library/office/dn575435.aspx) to do OAuth against the Microsoft Account service. This code sample, demonstrates an alternative way to do OAuth using the new Windows.Security.Authentication.OnlineId.OnlineIdAuthenticator class. Both the existing Live SDK approach and this alternative will work in Windows 8.1 universal apps
    * The usage of the OnlineIdAuthenticator class is based on the [Windows universal code sample](http://code.msdn.microsoft.com/windowsapps/Windows-account-authorizati-7c95e284)
* Create Pages:
    * [POST simple HTML to a new OneNote Quick Notes page](http://msdn.microsoft.com/EN-US/library/office/dn575428.aspx)
    * [POST multi-part message with image data included in the request](http://msdn.microsoft.com/EN-US/library/office/dn575432.aspx)
    * [POST page with a snapshot of an embedded web page HTML](http://msdn.microsoft.com/EN-US/library/office/dn575431.aspx)
    * [POST page with a URL rendered as an image](http://msdn.microsoft.com/EN-US/library/office/dn575431.aspx)
    * [POST page with a file attachment](http://msdn.microsoft.com/en-us/library/office/dn575436.aspx)
    * [POST page with a PDF file rendered and attached](http://msdn.microsoft.com/EN-US/library/office/dn655137.aspx)
    * [POST page with note tags](https://msdn.microsoft.com/EN-US/library/office/mt159148.aspx)
    * [POST page with a business card automatically extracted from an image](https://msdn.microsoft.com/EN-US/library/office/dn600338.aspx#ExtractBusinessCard)
    * [POST page with a recipe automatically extracted from a URL](https://msdn.microsoft.com/EN-US/library/office/dn600338.aspx#ExtractRecipe)
    * [POST page with a product info automatically extracted from a URL](https://msdn.microsoft.com/EN-US/library/office/dn600338.aspx#ExtractProduct)
    * [Extract the returned oneNoteClientURL and oneNoteWebURL links](http://msdn.microsoft.com/EN-US/library/office/dn575433.aspx)
    * [POST page in a specific named section](http://msdn.microsoft.com/EN-US/library/office/dn672416.aspx)
    * [POST page under a specific notebook and section](http://dev.onenote.com/docs#/reference/post-pages/v10menotessectionsidpages/post)
* Query and Search Pages:
    *  [GET a paginated list of all pages in OneNote](http://dev.onenote.com/docs#/reference/get-pages)
    *  [GET metadata for a specific page](http://dev.onenote.com/docs#/reference/get-pages/v10menotespagesid/get)
    *  [GET pages with title containing a specific substring using the $filter query parameter and contains method](http://dev.onenote.com/docs#/reference/get-pages/v10menotespagesfilterorderbyselecttopskipsearchcount/get)
    *  [GET pages using OData v4 query parameters like $skip and $top](http://dev.onenote.com/docs#/reference/get-pages/v10menotespagesfilterorderbyselecttopskipsearchcount/get)
        * [OData v4](http://docs.oasis-open.org/odata/odata/v4.0/os/part1-protocol/odata-v4.0-os-part1-protocol.html)
    *  [GET a sorted list of pages using the $orderby query parameter](http://dev.onenote.com/docs#/reference/get-pages/v10menotespagesfilterorderbyselecttopskipsearchcount/get)
    *  [GET selected metadata for pages using the $select query parameter](http://dev.onenote.com/docs#/reference/get-pages/v10menotespagesfilterorderbyselecttopskipsearchcount/get)
    *  [GET pages containing the matching search term using the search query parameter](http://dev.onenote.com/docs#/reference/get-pages/v10menotespagesfilterorderbyselecttopskipsearchcount/get)
    *  [GET back a specific page's content as HTML](http://dev.onenote.com/docs#/reference/get-pages/v10menotespagesidcontentincludeids/get)
* Manage Notebooks and Sections:
    * GET all notebooks and sections in one round trip using the $expand query parameter
    * [GET a list of all notebooks](http://dev.onenote.com/docs#/reference/get-notebooks)
    * [GET metadata for a specific notebook](http://dev.onenote.com/docs#/reference/get-notebooks/v10menotesnotebooksidselectexpand/get)
    * [GET a list of all sections](http://dev.onenote.com/docs#/reference/get-sections)
    * [GET metadata for a specific section](http://dev.onenote.com/docs#/reference/get-sections/v10menotessectionsidselectexpand/get)
    * [GET a list of all section groups](http://dev.onenote.com/docs#/reference/get-sectiongroups)
    * [GET notebooks and sections with a specific name using the $filter query parameter](http://dev.onenote.com/docs#/reference/get-notebooks/v10menotesnotebooksfilterorderbyselectexpandtopskipcount/get)
    * [GET notebooks shared by others using the $filter query parameter and userRole property](http://dev.onenote.com/docs#/reference/get-notebooks/v10menotesnotebooksfilterorderbyselectexpandtopskipcount/get)
    * [GET a sorted list of notebooks using the $orderby query parameter](http://dev.onenote.com/docs#/reference/get-notebooks/v10menotesnotebooksfilterorderbyselectexpandtopskipcount/get)
    * [GET selected metadata for notebooks using the $select query parameter](http://dev.onenote.com/docs#/reference/get-notebooks/v10menotesnotebooksfilterorderbyselectexpandtopskipcount/get)
    * [GET a list of all sections under a specific notebook](http://dev.onenote.com/docs#/reference/get-notebooks/v10menotesnotebooksfilterorderbyselectexpandtopskipcount/get)
    * [POST a new notebook](http://dev.onenote.com/docs#/reference/post-notebooks)
    * [POST a new section group under a specific notebook](http://dev.onenote.com/docs#/reference/post-sectiongroups/betamenotesnotebooksidsectiongroups/post)
    * [POST a new section group under a specific section group](http://dev.onenote.com/docs#/reference/post-sectiongroups/betamenotessectiongroupsidsectiongroups/post)
    * [POST a new section under a specific notebook](http://dev.onenote.com/docs#/reference/post-sections/v10menotesnotebooksidsections/post)
    * [POST a new section under a specific section group](http://dev.onenote.com/docs#/reference/post-sections/betamenotessectiongroupsidsections/post)
* Update Pages:
    * Examples of [PATCH Pages](http://dev.onenote.com/docs#/reference/patch-pages)
* Delete Pages:
    * [DELETE page](http://dev.onenote.com/docs#/reference/delete-pages)

### O365 based API (only available on beta)
All the APIs for Microsoft Account (LiveId) are supported in O365 as well except the following:
* $search query option is currently not supported.

### Prerequisites

**Tools and Libraries** you will need to download, install, and configure for your development environment.
* Operating system requirements for developing Windows universal apps:
    *   Client: Windows 8.1 OR
    *   Server: Windows Server 2012 R2
    *   Phone: Windows Phone 8.1 (developer unlocked phone for device testing. Alternatively emulators can be used)
* [Visual Studio 2013 Update 4 or later](http://www.visualstudio.com/en-us/downloads).

Be sure you enable the Windows Phone SDK when you install Visual Studio.
If you don't, you will need to un-install and re-install Visual Studio to get those features.

For using emulator (Windows Phone), make sure to enable hyper-v

* You can get a full list of tools needed to build Universal Windows app [here](http://dev.windows.com/en-us/develop/downloads)

** O365 specific Pre-requisites
* What you need to do is to go to Programs and Features, Turn on/off Windows features and then choose to add Windows Identity Foundation 3.5 to your system.

* **NuGet packages** used in the sample. These are handled using the package
manager, as described in the setup instructions. These should update
automatically at build time; if not, make sure your NuGet package manager
is up-to-date. You can learn more about the packages we used at the links below.
    * [Newtonsoft Json.NET package](http://newtonsoft.com/) provides Json parsing utilities.
    * [Active Directory Authentication Library package](https://www.nuget.org/packages/Microsoft.IdentityModel.Clients.ActiveDirectory/) provides AAD authentication utilities (for Office 365)

### Accounts
**To use this Code Sample in your own Microsoft Account based app, be sure to do the following:**
* As the developer, you'll need to [have a Microsoft account and get a client ID string](http://msdn.microsoft.com/EN-US/library/office/dn575426.aspx) so your app can authenticate. Get a client ID string and copy it into the file under [.../OneNoteServiceSamplesWinUniversal.Shared/OneNoteApi/LiveIdAuth.cs](https://github.com/OneNoteDev/OneNoteAPISampleWinUniversal/blob/master/OneNoteServiceSamplesWinUniversal.Shared/OneNoteApi/LiveIdAuth.cs#L54) (~line 54).
* Use the least permissible scopes by editing [.../OneNoteServiceSamplesWinUniversal.Shared/OneNoteApi/LiveIdAuth.cs](https://github.com/OneNoteDev/OneNoteAPISampleWinUniversal/blob/master/OneNoteServiceSamplesWinUniversal.Shared/OneNoteApi/LiveIdAuth.cs#L61) (~line 61).
* [Create an app package and use appropriate certificates](http://msdn.microsoft.com/en-us/library/windows/apps/xaml/hh975357.aspx)

**To use this Code Sample in your own Microsoft Office 365 based app, refer to the details in the blog [Support for work and school notebooks on Office 365](http://blogs.msdn.com/b/onenotedev/archive/2015/04/30/support-for-work-and-school-notebooks-on-office-365-in-preview.aspx)

### Using the sample

After you've set up your development tools, and installed the prerequisites listed above,...

1. Download the repository as a ZIP file to your local computer, and extract the files. Or, clone the repository into a local copy of Git.
2. Open the project (.sln file) in Visual Studio.
3. It is highly recommended that you get your own client ID string and copy it into the file under
	[.../OneNoteServiceSamplesWinUniversal.Shared/OneNoteApi/LiveIdAuth.cs](https://github.com/OneNoteDev/OneNoteAPISampleWinUniversal/blob/master/OneNoteServiceSamplesWinUniversal.Shared/OneNoteApi/LiveIdAuth.cs#L54) (~line 54)
	OR
	[.../OneNoteServiceSamplesWinUniversal.Shared/OneNoteApi/o365Auth.cs](https://github.com/OneNoteDev/OneNoteAPISampleWinUniversal/blob/master/OneNoteServiceSamplesWinUniversal.Shared/OneNoteApi/o365Auth.cs#L40) (~line 40)
4. For o365, additionally, change RedirectUri to one of your app under
	[.../OneNoteServiceSamplesWinUniversal.Shared/OneNoteApi/o365Auth.cs](https://github.com/OneNoteDev/OneNoteAPISampleWinUniversal/blob/master/OneNoteServiceSamplesWinUniversal.Shared/OneNoteApi/o365Auth.cs#L52) (~line 52)

5. Build and run the application (F5).

(If your copy of NuGet is up-to-date, it should automatically update the packages. If you get package-not-found errors, update NuGet and rebuild, and that should fix it.)

If you get any build or deployment errors related to the Publisher of the app (Microsoft Account), be sure to
[Create LiveId app package and use appropriate certificates](http://msdn.microsoft.com/en-us/library/windows/apps/xaml/hh975357.aspx)

If you get any build or deployment errors related to the Publisher of the app (Microsoft Office 365), be sure to
[Have your app appear in the Office 365 app launcher](https://msdn.microsoft.com/en-us/office/office365/howto/connect-your-app-to-o365-app-launcher)

### Version Info

| Date | Change |
|------|------|
| August 2015 | Added beta `POST /sectiongroups/{id}/sections`, `POST /notebooks/{id}/sectiongroups`, and  `POST /sectiongroups/{id}/sectiongroups` examples.
Changed `DELETE /pages/{id}` from beta to v1.0.
Support for `POST /pages` and `POST /pages?sectionName` in Office 365. |
| April 2015 | Added Office 365 with Azure AD auth support
Added beta `DELETE /pages/{id}` example. |
| March 2015 | Added `PATCH` example |
| January 2015 | Added `GET /sections/{id}/pages` examples. |
| December 2014 | Initial public release for this code sample. |

### Learning More

* Visit the [dev.onenote.com](http://dev.onenote.com) Dev Center
* Contact us on [StackOverflow (tagged OneNote)](http://go.microsoft.com/fwlink/?LinkID=390182)
* Follow us on [Twitter @onenotedev](http://www.twitter.com/onenotedev)
* Read our [OneNote Developer blog](http://go.microsoft.com/fwlink/?LinkID=390183)
* Explore the API using the [apigee.com interactive console](http://go.microsoft.com/fwlink/?LinkID=392871) OR the [apiary.io interactive console](http://dev.onenote.com/docs).
Also, see the [short overview/tutorial](http://go.microsoft.com/fwlink/?LinkID=390179).
* [API Reference](http://msdn.microsoft.com/en-us/library/office/dn575437.aspx) documentation
* [Debugging / Troubleshooting](http://msdn.microsoft.com/EN-US/library/office/dn575430.aspx)
* [Getting Started](http://go.microsoft.com/fwlink/?LinkID=331026) with the OneNote service API

This project has adopted the [Microsoft Open Source Code of Conduct](https://opensource.microsoft.com/codeofconduct/). For more information see the [Code of Conduct FAQ](https://opensource.microsoft.com/codeofconduct/faq/) or contact [opencode@microsoft.com](mailto:opencode@microsoft.com) with any additional questions or comments.

redpajama_set_name: RedPajamaGithub


Reflect on the social and cultural factors which have shaped your views of Aboriginal and Torres Strait Islander peoples.
Consider influences such as mass media and the literature that has shaped your views of Aboriginal and Torres Strait Islander people. Comparisons of indigenous peoples from other countries may inform your descriptions and discussion of similarity that might be experienced by Aboriginal and Torres Strait Islander people.
• review the marking rubric associated with this assessment item as your marks will be allocated based on rubric criteria.
Review the marking rubric associated with this assessment item as your marks will be allocated based on rubric criteria.


text:
package petablox.android.srcmap;

import soot.*;
import soot.jimple.*;
import soot.util.*;

import soot.jimple.internal.JDynamicInvokeExpr;
import soot.jimple.parser.node.*;
import soot.jimple.parser.analysis.*;

import java.util.*;
import java.io.*;

import static org.apache.commons.lang3.StringEscapeUtils.escapeXml;

public class JimpleAdapter extends DepthFirstAdapter
{
	private String className;
    private PrintWriter writer;

    public JimpleAdapter(File outputFile)
    {
        try{
            this.writer = new PrintWriter(outputFile);
        }catch(IOException e){
            throw new Error(e);
        }
        writer.println("<root>");
    }

    public void finish()
    {
        writer.println("</root>");
        writer.close();
    }

    /*
      file =
      modifier* file_type class_name extends_clause? implements_clause? file_body;
    */
    public void inAFile(AFile node)
    {
		Token tok = ((AFullIdentClassName) node.getClassName()).getFullIdentifier();
		className = tok.getText();

        writer.println("<class" +
					   " chordsig=\""+escapeXml(className)+"\""+
                       " line=\""+tok.getLine()+"\""+
                       ">");
    }

    public void outAFile(AFile node)
    {
		writer.println("</class>");
	}

    public void outAStaticInvokeExpr(AStaticInvokeExpr node)
	{
		AMethodSignature methSig = (AMethodSignature) node.getMethodSignature();
		String sig = getSignature(methSig);

		List<Token> args = getArgs(node.getArgList());

		StringBuilder expr = new StringBuilder();
		expr.append(sig).append('(');
		int count = args.size();
		for(int i=0; i < (count-1); i++)
			expr.append(args.get(i).getText()).append(", ");
		if(count > 0)
			expr.append(args.get(count-1).getText());
		expr.append(");");

		processInvkExpr(methSig, expr.toString(), chordSig(sig), args);
	}

	public void outANonstaticInvokeExpr(ANonstaticInvokeExpr node)
	{
		AMethodSignature methSig = (AMethodSignature) node.getMethodSignature();
		String sig = getSignature(methSig);
		Token base = ((AIdentName) ((ALocalName) node.getLocalName()).getName()).getIdentifier();
		List<Token> args = getArgs(node.getArgList());

		StringBuilder expr = new StringBuilder();
		expr.append(base.getText()).append('.').append(sig).append('(');
		int count = args.size();
		for(int i=0; i < (count-1); i++)
			expr.append(args.get(i).getText()).append(", ");
		if(count > 0)
			expr.append(args.get(count-1).getText());
		expr.append(");");

		args.add(0, base);

		processInvkExpr(methSig, expr.toString(), chordSig(sig), args);
	}

	private String chordSig(String sootSig)
	{
		return sootSig;
	}

	private void processInvkExpr(AMethodSignature methSig, String expr, String chordSig, List<Token> args)
    {
		Token cmpGt = methSig.getCmpgt();
		int lineNum = cmpGt.getLine();

		writer.println("\t<invkexpr"+
                       " line=\""+lineNum+"\""+
                       " chordsig=\""+escapeXml(chordSig)+"\""+
                       " start=\""+cmpGt.getPos()+"\""+
                       " length=\"1\""+
                       " type=\"invoke\""+
                       ">");
        writer.println("\t\t\t<expr><![CDATA["+expr+"]]></expr>");
		//for(Iterator it = args.iterator(); it.hasNext();){
		for(Token arg : args){
            writer.println("\t\t<param "+ getLocationStr(arg) + ">");
			writer.println("\t\t\t<expr><![CDATA["+arg.getText()+"]]></expr>");
            writer.println("\t\t</param>");
        }
        writer.println("\t</invkexpr>");
    }

	private List<Token> getArgs(PArgList aList)
	{
		List<Token> args = new ArrayList();
		if(aList == null)
			;//no args
		else if(aList instanceof AMultiArgList){
			do{
				PImmediate arg = (PImmediate) ((AMultiArgList) aList).getImmediate();
				Token tok = handleImmediate(arg);
				args.add(tok);
				aList = ((AMultiArgList) aList).getArgList();
			}while(aList instanceof AMultiArgList);
			PImmediate arg = (PImmediate) ((ASingleArgList) aList).getImmediate();
			Token tok = handleImmediate(arg);
			args.add(tok);
		}
		else if(aList instanceof ASingleArgList){
			PImmediate arg = (PImmediate) ((ASingleArgList) aList).getImmediate();
			Token tok = handleImmediate(arg);
			args.add(tok);
		}
		else
			throw new RuntimeException(aList.getClass().toString());
		return args;
	}

	private Token handleImmediate(PImmediate imm)
	{
		if(imm instanceof AConstantImmediate){
			PConstant c = ((AConstantImmediate) imm).getConstant();
			if(c instanceof AClzzConstant)
				return ((AIntegerConstant) c).getIntegerConstant();
			if(c instanceof AFloatConstant)
				return ((AFloatConstant) c).getFloatConstant();
			if(c instanceof AIntegerConstant)
				return ((AIntegerConstant) c).getIntegerConstant();
			if(c instanceof ANullConstant)
				return ((ANullConstant) c).getNull();
			if(c instanceof AStringConstant)
				return ((AStringConstant) c).getStringConstant();
		}
		if(imm instanceof ALocalImmediate){
			return ((AIdentName) ((ALocalName) ((ALocalImmediate) imm).getLocalName()).getName()).getIdentifier();
		}

		throw new RuntimeException(imm.getClass().toString());
	}

	private String getSignature(PMethodSignature signature)
	{
		String sig = signature.toString();
		//example sig: < com.example.android.snake.SnakeView : void loadTile ( int , android.graphics.drawable.Drawable ) >
		int colonIndex = sig.indexOf(':');
		String part1 = sig.substring(2, colonIndex-1);
		int lparenIndex = sig.indexOf('(');
		String part2 = sig.substring(colonIndex, lparenIndex-1);
		int rparenIndex = sig.indexOf(')');
		String part3 = sig.substring(lparenIndex, rparenIndex+1).replace(" ","");
		sig = "<"+part1+part2+part3+">";
		System.out.println(sig);
		return sig;
	}

    public void inAMethodMember(AMethodMember node)
    {
		Token nameToken = ((AIdentName) node.getName()).getIdentifier();
		String name = nameToken.getText();
		//System.out.println("++ "+node.toString());
		//buffer.append(" " + nameToken.getLine() + " "+nameToken.getPos());

		PType retType = node.getType();
		String retTypeStr;
		if(retType instanceof AVoidType)
			retTypeStr = "void";
		else if(retType instanceof ANovoidType)
			retTypeStr = toString(((ANovoidType) retType).getNonvoidType());
		else
			throw new RuntimeException(retType.getClass().toString());

		StringBuilder buffer = new StringBuilder();
		buffer.append('<').append(className).append(": ").append(retTypeStr).append(' ').append(name).append('(');
		PParameterList pList = node.getParameterList();
        if(pList instanceof AMultiParameterList){
			do{
				AParameter param = (AParameter) ((AMultiParameterList) pList).getParameter();
				//System.out.println("*"+param.getNonvoidType().getClass().toString());
				//System.out.println("*"+toString(param.getNonvoidType()));
				buffer.append(toString(param.getNonvoidType()).toString()+",");
				pList = ((AMultiParameterList) pList).getParameterList();
			}while(pList instanceof AMultiParameterList);
			AParameter param = (AParameter) ((ASingleParameterList) pList).getParameter();
			buffer.append(toString(param.getNonvoidType()));
			//System.out.println("*"+toString(param.getNonvoidType()));
			//System.out.println("*"+pList.toString());
        }
        else if(pList instanceof ASingleParameterList){
			AParameter param = (AParameter) ((ASingleParameterList) pList).getParameter();
			//System.out.println("*"+param.getNonvoidType().getClass().toString());
			buffer.append(toString(param.getNonvoidType()));
			//System.out.println("*"+toString(param.getNonvoidType()));
			//System.out.println("*"+pList.toString());
        }
		buffer.append(")>");

		String sig = buffer.toString();
		String chordSig = chordSig(sig);
        writer.println("<method");
		writer.println(" chordsig=\""+escapeXml(chordSig)+"\"");
        writer.println(" sig=\""+escapeXml(sig)+"\"");
        writer.println(" line=\""+nameToken.getLine()+"\"");
        writer.println(" startpos=\""+nameToken.getPos()+"\"");
        writer.println(" endpos=\""+(nameToken.getPos()+name.length())+"\"");
		writer.println(">");
    }

    public void outAMethodMember(AMethodMember node)
    {
		writer.println("</method>");
	}

	private String toString(PNonvoidType type)
	{
		if(type instanceof AFullIdentNonvoidType){
			AFullIdentNonvoidType baseType = (AFullIdentNonvoidType) type;
			String baseTypeStr = baseType.getFullIdentifier().getText();
			return toString(baseTypeStr, baseType.getArrayBrackets().size());
		}
		if(type instanceof ABaseNonvoidType){
			ABaseNonvoidType baseType = (ABaseNonvoidType) type;
			String baseTypeStr = toString(baseType.getBaseTypeNoName());
			return toString(baseTypeStr, baseType.getArrayBrackets().size());
		}
		throw new RuntimeException(type.getClass().toString());
	}

	private String toString(String baseTypeStr, int dim)
	{
		StringBuilder builder = new StringBuilder(baseTypeStr);
		for(int i = 0; i < dim; i++)
			builder.append("[]");
		return builder.toString();
	}

	private String toString(PBaseTypeNoName baseType)
	{
		if(baseType instanceof ABooleanBaseTypeNoName)
			return "boolean";
		if(baseType instanceof AByteBaseTypeNoName)
			return "byte";
		if(baseType instanceof ACharBaseTypeNoName)
			return "char";
		if(baseType instanceof ADoubleBaseTypeNoName)
			return "double";
		if(baseType instanceof AFloatBaseTypeNoName)
			return "float";
		if(baseType instanceof AIntBaseTypeNoName)
			return "int";
		if(baseType instanceof ALongBaseTypeNoName)
			return "long";
		if(baseType instanceof ANullBaseTypeNoName)
			return "nulltype";
		if(baseType instanceof AShortBaseTypeNoName)
			return "short";
		throw new RuntimeException(baseType.getClass().toString());
	}

	private String getLocationStr(Token node)
    {
        int start, length, line;
        if(node == null){
            start = length = line = -1;
        } else{
            start = node.getPos();
            length = node.getText().length();
            line = node.getLine();
        }
        return
            " start=\""+start+ "\""+
            " length=\""+length+ "\""+
            " line=\""+line+"\"";
    }
}