A Cryophyte Transcription Factor, CbABF1, Confers Freezing, and Drought Tolerance in Tobacco

Abscisic acid responsive element binding factors (ABFs) play crucial roles in plant responses to abiotic stress. However, little is known about the roles of ABFs in alpine subnival plants, which can survive under extreme environmental conditions. Here, we cloned and characterized an ABF1 homolog, CbABF1, from the alpine subnival plant Chorispora bungeana. Expression of CbABF1 was induced by cold, drought, and abscisic acid. Subcellular localization analysis revealed that CbABF1 was located in the nucleus. Further, CbABF1 had transactivation activity, which was dependent on the N-terminal region containing 89 residues. A Snf1-related protein kinase, CbSnRK2.6, interacted with CbABF1 in yeast two-hybrid analysis and bimolecular fluorescence complementation assays. Transient expression assay revealed that CbSnRK2.6 enhanced the transactivation of CbABF1 on ABRE cis-element. We further found that heterologous expression of CbABF1 in tobacco improved plant tolerance to freezing and drought stress, in which the survival rates of the transgenic plants increased around 40 and 60%, respectively, compared with wild-type plants. Moreover, the transgenic plants accumulated less reactive oxygen species, accompanied by high activities of antioxidant enzymes and elevated expression of stress-responsive genes. Our results thus suggest that CbABF1 is a transcription factor that plays an important role in cold and drought tolerance and is a candidate gene in molecular breeding of stress-tolerant crops

A Nuclear Calcium-Sensing Pathway Is Critical for Gene Regulation and Salt Stress Tolerance in <i>Arabidopsis</i>

<div><p>Salt stress is an important environmental factor that significantly limits crop productivity worldwide. Studies on responses of plants to salt stress in recent years have identified novel signaling pathways and have been at the forefront of plant stress biology and plant biology in general. Thus far, research on salt stress in plants has been focused on cytoplasmic signaling pathways. In this study, we discovered a nuclear calcium-sensing and signaling pathway that is critical for salt stress tolerance in the reference plant <i>Arabidopsis</i>. Through a forward genetic screen, we found a nuclear-localized calcium-binding protein, RSA1 (SHORT <u>R</u>OOT IN <u>SA</u>LT MEDIUM 1), which is required for salt tolerance, and identified its interacting partner, RITF1, a bHLH transcription factor. We show that RSA1 and RITF1 regulate the transcription of several genes involved in the detoxification of reactive oxygen species generated by salt stress and that they also regulate the <i>SOS1</i> gene that encodes a plasma membrane Na⁺/H⁺ antiporter essential for salt tolerance. Together, our results suggest the existence of a novel nuclear calcium-sensing and -signaling pathway that is important for gene regulation and salt stress tolerance.</p></div

RSA1 interacts with RITF1.

<p>(A) RSA1 interacts with RITF1 as determined by yeast two-hybrid assays. Yeast strain AH109 co-transformed with RSA1-pDEST32 (bait) and RITF1-pDEST22 (prey) was subjected to x-gal assay. AH109 cells co-transformed with RSA1-pDEST32/pDEST22 (empty vector) or RITF1-pDEST22/pDEST32 (empty vector) were used as negative controls. Yeast cells grown on SD medium-L-W or SD medium-L-W-H+3-AT are shown. 3-AT, 3-amino-1,2,4-triazole. L, W, H, symbols for amino acids leucine, tryptophan, and histidine, respectively. SD, yeast minimal media. (B) Localization of RITF1-GFP in <i>Arabidopsis</i> protoplasts. Bar = 25 µm. (C) RSA1 interacts with RITF1 <i>in vivo</i> as determined by BiFC assays in tobacco leaf epidermal cells. Bars = 25 µm in (a), and 50 µm in (b) and (c). YFP images were detected at an approximate frequency of 4.04% (101 out of 2,501 tobacco leaf epidermal cells analyzed exhibited BiFC events). (D) RSA1 interacts with RITF1 <i>in vivo</i> as determined by Split-LUC assays. (E) RSA1 interacts with RITF1 <i>in vivo</i> as determined by Co-IP assays. (F) <i>RITF1</i> expression under salt stress. The qRT-PCR analysis was carried out with 14-d-old wild-type seedlings grown for 6 h on MS medium containing 0, 100, or 150 mM NaCl. Error bars represent the standard deviation (n = 20 in [D], 4 in [F]). The experiments in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003755#pgen-1003755-g004" target="_blank">Figure 4</a> were performed at least three times with similar results, and data from one representative experiment are presented.</p

A working model for RSA1 and RITF1 function under salt stress.

<p>The calcium-binding protein RSA1 senses salt-induced changes in nuclear free calcium and interacts with a bHLH transcription factor, RITF1. RITF1 may be phosphorylated by nuclear-localized MAPKs. The RSA1-RITF1 complex controls expression of genes important for detoxification of salt-induced ROS and for Na⁺ homeostasis under salt stress. Some RITF1 target genes may play a role in salt tolerance with so far unknown mechanisms. The calcium-binding protein SOS3 senses salt-induced cytosolic calcium increases and interacts with SOS2, a protein kinase. The SOS3-SOS2 protein kinase complex then phosphorylates and thereby activates the plasma membrane-localized Na⁺/H⁺ antiporter SOS1.</p

<i>ritf1</i> mutant plants are sensitive to salt and oxidative stresses, and overexpression of <i>RITF1</i> increases plant tolerance to salt and oxidative stresses.

<p>(A) Seed germination of wild type and <i>ritf1</i> in response to various NaCl levels. There were 80–150 seeds per genotype per biological replicate. (B) Fresh weight of wild-type and <i>ritf1</i> seedlings under salt stress. Five-d-old seedlings grown on MS medium were transferred to MS medium containing 0 or 100 mM NaCl and allowed to grow for an additional 7 d. (C) Growth responses of wild-type and <i>ritf1</i> seedlings to oxidative stress-inducing reagents H₂O₂ and methyl viologen (MV). (D) and (E) Fresh weight of seedlings grown on MS medium containing various levels of H₂O₂ (D) or MV (E) as shown in (C). (F) Salt tolerance of <i>RITF1</i> overexpression plants. Five-d-old seedlings grown on MS medium were transferred to MS medium containing 0 or 100 mM NaCl and allowed to grow for an additional 10 d. (G) and (H) Fresh weight of wild-type and <i>RITF1</i> overexpression plants grown on MS medium containing various levels of H₂O₂ (G) or MV (H). In (C)–(E), (G), and (H), seeds were sown directly on MS medium supplemented with various levels of H₂O₂ or MV and allowed to grow for an additional 10 d. Error bars represent the standard deviation (n = 8 in [A], 40 in [B], [D]–[H]). One-way ANOVA (Tukey-Kramer test) was performed, and statistically significant differences are indicated by different lowercase letters (p<0.05). The experiments in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003755#pgen-1003755-g005" target="_blank">Figure 5</a> were repeated at least four times with similar results, and data from one representative experiment are presented.</p

<i>rsa1-1</i> plants are hypersensitive to NaCl, and RSA1 is involved in Na⁺ homeostasis under salt stress.

<p>(A)–(C) Five-d-old wild-type and <i>rsa1-1</i> seedlings grown on MS medium were transferred to MS medium supplemented with different levels of NaCl and allowed to grow for an additional 8 d. Root elongation or shoot fresh weight was measured and is shown as a percentage relative to growth on normal MS medium. (D) Two-week-old wild-type and <i>rsa1-1</i> plants grown in soil were irrigated with 300 mM NaCl for 0 or 14 d. (E) Survival rate of wild-type and <i>rsa1-1</i> plants as shown in (D). (F) Seed germination of wild type and <i>rsa1-1</i> in response to various NaCl levels. There were 80–150 seeds per genotype per biological replicate. Seeds in which the radical had emerged through the seed coat were considered germinated. (G) Na⁺ content in soil-grown wild-type and <i>rsa1-1</i> plants. DW, dry weight. (H) K⁺ content in soil-grown wild-type and <i>rsa1-1</i> plants. (I) Ratio of Na⁺ to K⁺ accumulation in soil-grown wild-type and <i>rsa1-1</i> plants. WT, wild type. Error bars indicate the standard deviation (n = 30–40). The experiments in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003755#pgen-1003755-g001" target="_blank">Figure 1</a> were repeated at least five times with similar results, and data from one representative experiment are presented.</p

Chilling- and Freezing- Induced Alterations in Cytosine Methylation and Its Association with the Cold Tolerance of an Alpine Subnival Plant, <i>Chorispora bungeana</i>

<div><p>Chilling (0–18°C) and freezing (<0°C) are two distinct types of cold stresses. Epigenetic regulation can play an important role in plant adaptation to abiotic stresses. However, it is not yet clear whether and how epigenetic modification (i.e., DNA methylation) mediates the adaptation to cold stresses in nature (e.g., in alpine regions). Especially, whether the adaptation to chilling and freezing is involved in differential epigenetic regulations in plants is largely unknown. <i>Chorispora bungeana</i> is an alpine subnival plant that is distributed in the freeze-thaw tundra in Asia, where chilling and freezing frequently fluctuate daily (24 h). To disentangle how <i>C</i>. <i>bungeana</i> copes with these intricate cold stresses through epigenetic modifications, plants of <i>C</i>. <i>bungeana</i> were treated at 4°C (chilling) and -4°C (freezing) over five periods of time (0–24 h). Methylation-sensitive amplified fragment-length polymorphism markers were used to investigate the variation in DNA methylation of <i>C</i>. <i>bungeana</i> in response to chilling and freezing. It was found that the alterations in DNA methylation of <i>C</i>. <i>bungeana</i> largely occurred over the period of chilling and freezing. Moreover, chilling and freezing appeared to gradually induce distinct DNA methylation variations, as the treatment went on (e.g., after 12 h). Forty-three cold-induced polymorphic fragments were randomly selected and further analyzed, and three of the cloned fragments were homologous to genes encoding alcohol dehydrogenase, UDP-glucosyltransferase and polygalacturonase-inhibiting protein. These candidate genes verified the existence of different expressive patterns between chilling and freezing. Our results showed that <i>C</i>. <i>bungeana</i> responded to cold stresses rapidly through the alterations of DNA methylation, and that chilling and freezing induced different DNA methylation changes. Therefore, we conclude that epigenetic modifications can potentially serve as a rapid and flexible mechanism for <i>C</i>. <i>bungeana</i> to adapt to the intricate cold stresses in the alpine areas.</p></div

Non-metric multidimensional scaling (NMDS) analysis for variation in methylation epigenotypes.

<p>Non-metric multidimensional scaling representing the variation in methylation epigenotypes between samples. The dynamic pattern of epigenetic divergence among the 0, 0.5, 3, 12 and 24 h in 23°C, 4°C and -4°C treatments based on presence (1) ⁄ absence (0) scores of 16 polymorphic methylation-sensitive amplified fragment-length polymorphism (MS-AFLP) markers. The first two components of the NMDS analysis are extracted and plotted against each other; the small symbols are individual plants (n = 5), while the large symbols with the cross indicate the mean ± SD of the treatment group. (A) At 4°C for 0–24 h. (B) At -4°C for 0–24 h. <i>R</i>² values represent the proportion of the variance that is explained by the first two components. Axis 1 explains the majority of the total variation in both (A) and (B).</p