A reference-grade genome of the xerophyte Ammopiptanthus mongolicus sheds light on its evolution history in legumes and drought-tolerance mechanisms.

Plants that grow in extreme environments represent unique sources of stress-resistance genes and mechanisms. Ammopiptanthus mongolicus (Leguminosae) is a xerophytic evergreen broadleaf shrub native to semi-arid and desert regions; however, its drought-tolerance mechanisms remain poorly understood. Here, we report the assembly of a reference-grade genome for A. mongolicus, describe its evolutionary history within the legume family, and examine its drought-tolerance mechanisms. The assembled genome is 843.07 Mb in length, with 98.7% of the sequences successfully anchored to the nine chromosomes of A. mongolicus. The genome is predicted to contain 47 611 protein-coding genes, and 70.71% of the genome is composed of repetitive sequences; these are dominated by transposable elements, particularly long-terminal-repeat retrotransposons. Evolutionary analyses revealed two whole-genome duplication (WGD) events at 130 and 58 million years ago (mya) that are shared by the genus Ammopiptanthus and other legumes, but no species-specific WGDs were found within this genus. Ancestral genome reconstruction revealed that the A. mongolicus genome has undergone fewer rearrangements than other genomes in the legume family, confirming its status as a "relict plant". Transcriptomic analyses demonstrated that genes involved in cuticular wax biosynthesis and transport are highly expressed, both under normal conditions and in response to polyethylene glycol-induced dehydration. Significant induction of genes related to ethylene biosynthesis and signaling was also observed in leaves under dehydration stress, suggesting that enhanced ethylene response and formation of thick waxy cuticles are two major mechanisms of drought tolerance in A. mongolicus. Ectopic expression of AmERF2, an ethylene response factor unique to A. mongolicus, can markedly increase the drought tolerance of transgenic Arabidopsis thaliana plants, demonstrating the potential for application of A. mongolicus genes in crop improvement.

Over-activation of cold tolerance in arabidopsis causes carbohydrate shortage compared with Chorispora bungeana.

Many plants cope with cold stress by developing acquired freezing tolerance (AFT) through cold acclimation (CA), and some species have strong basal freezing tolerance (BFT) independent of CA. Although CA has been extensively studied, its potential in agricultural applications is still unclear. Here, carbohydrate metabolism and transcriptome in AFT plant Arabidopsis and BFT plant Chorispora bungeana were compared with each other. The results showed that, although both species were able to accumulate soluble sugars during CA, leaf starch accumulation in the daytime was almost blocked in Arabidopsis while it was greatly enhanced in C. bungeana, revealing that Arabidopsis experienced carbohydrate shortage during CA. Transcriptome and pathway enrichment analysis found that genes for photosynthesis antenna proteins were generally repressed by cold stress in both species. However, cold-up-regulated genes were enriched in protein translation in Arabidopsis, whilst they were enriched in carotenoid biosynthesis, flavonoid biosynthesis, and beta-amylases in C. bungeana. Furthermore, weighted gene co-expression network analysis (WGCNA) showed that the inhibition of starch accumulation was associated with down-regulation of genes for photosynthesis antenna proteins and up-regulation of genes for protein translation, DNA repair, and proteasome in Arabidopsis but not in C. bungeana. Taken together, our results revealed that over-activation of common tolerant mechanisms resulted in insufficient carbohydrate supplies in Arabidopsis during CA, and photoprotective mechanisms played important roles in cold adaptation of C. bungeana. These findings uncovered the drawback of CA in improving freezing tolerance and highlighted photoprotection as a possible solution for agricultural applications.

Genome-Wide Identification and Functional Analysis of the TIFY Family Genes in Response to Abiotic Stresses and Hormone Treatments in Tartary Buckwheat (Fagopyrum tataricum).

TIFY is a plant-specific gene family with four subfamilies: ZML, TIFY, PPD, and JAZ. Recently, this family was found to have regulatory functions in hormone stimulation, environmental response, and development. However, little is known about the roles of the TIFY family in Tartary buckwheat (Fagopyrum tataricum), a significant crop for both food and medicine. In this study, 18 TIFY family genes (FtTIFYs) in Tartary buckwheat were identified. The characteristics, motif compositions, and evolutionary relationships of the TIFY proteins, as well as the gene structures, cis-acting elements, and synteny of the TIFY genes, are discussed in detail. Moreover, we found that most FtTIFYs responded to various abiotic stresses (cold, heat, salt, or drought) and hormone treatments (ABA, MeJA, or SA). Through yeast two-hybrid assays, we revealed that two FtTIFYs, FtTIFY1 and FtJAZ7, interacted with FtABI5, a homolog protein of AtABI5 involved in ABA-mediated germination and stress responses, implying crosstalk between ABA and JA signaling in Tartary buckwheat. Furthermore, the overexpression of FtJAZ10 and FtJAZ12 enhanced the heat stress tolerance of tobacco. Consequently, our study suggests that the FtTIFY family plays important roles in responses to abiotic stress and provides two candidate genes (FtJAZ10 and FtJAZ12) for the cultivation of stress-resistant crops.

BYPASS1-LIKE regulates lateral root initiation via exocytic vesicular trafficking-mediated PIN recycling in Arabidopsis.

Auxin and auxin-mediated signaling pathways are known to regulate lateral root development. Although exocytic vesicle trafficking plays an important role in recycling the PIN-FORMED (PIN) auxin efflux carriers and in polar auxin transport during lateral root formation, the mechanistic details of these processes are not well understood. Here, we demonstrate that BYPASS1-LIKE (B1L) regulates lateral root initiation via exocytic vesicular trafficking-mediated PIN recycling in Arabidopsis thaliana. b1l mutants contained significantly more lateral roots than the wild type, primarily due to increased lateral root primordium initiation. Furthermore, the auxin signal was stronger in stage I lateral root primordia of b1l than in those of the wild type. Treatment with exogenous auxin and an auxin transport inhibitor indicated that the lateral root phenotype of b1l could be attributed to higher auxin levels and that B1L regulates auxin efflux. Indeed, compared to the wild type, C-terminally green fluorescent protein-tagged PIN1 and PIN3 accumulated at higher levels in b1l lateral root primordia. B1L interacted with the exocyst, and b1l showed defective PIN exocytosis. These observations indicate that B1L interacts with the exocyst to regulate PIN-mediated polar auxin transport and lateral root initiation in Arabidopsis.

Comparison with Arabidopsis reveals optimal nitrogen allocation strategy and mechanism in Chorispora bungeana, a cryophyte with strong freezing tolerance.

The stress responses of plant compete for resources with growth and development. Resource allocations among these processes may have been optimized in plants adapted to natural habitats. Here, nitrogen (N) allocations were compared in leaves of Arabidopsis and Chorispora bungeana, a cryophyte with strong freezing tolerance. The results showed that the two species differed not only in N partitions among N forms and allocations among leaves, but also in their responses to cold stress. Interestingly, leaf protein contents were enhanced in C. bungeana while reduced in Arabidopsis, though the N allocations to leaves were reduced in both plants upon cold stress. Profoundly, when grown at warm temperature, contents of total free amino acids (FAAs) in leaves of C. bungeana were 6-11 times higher than those in Arabidopsis. In contrast, cold treatment induced FAAs accumulation in leaves of Arabidopsis without having significant effect in any leaf of C. bungeana. Considerable discrepancy was also found between the two species in the expressions of nitrate transporter genes and the activities of nitrate assimilation enzymes. Correlation and network analysis showed that most NPFs were clustered in a single network module and had loose relations with protein synthesis in Arabidopsis, while they were distributed in different modules in a decentralized network in C. bungeana. Therefore, our results reveal that C. bungeana may have optimized its N allocation strategy by producing and storing amino acids as efficient N reserve and adopting a decentralized network for N utilization, which may equip the plant with powerful capabilities for environmental adaptions.

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.

Integrated regulation triggered by a cryophyte ω-3 desaturase gene confers multiple-stress tolerance in tobacco.

ω-3 fatty acid desaturases (FADs) are thought to contribute to plant stress tolerance mainly through linolenic acid (C18:3)-induced membrane stabilization, but a comprehensive analysis of their roles in stress adaptation is lacking. Here, we isolated a microsomal ω-3 FAD gene (CbFAD3) from a cryophyte (Chorispora bungeana) and elucidated its functions in stress tolerance. CbFAD3, exhibiting a high identity to Arabidopsis AtFAD3, was up-regulated by abiotic stresses. Its functionality was verified by heterogonous expression in yeast. Overexpression of CbFAD3 in tobacco constitutively increased C18:3 in both leaves and roots, which maintained the membrane fluidity, and enhanced plant tolerance to cold, drought, and salt stresses. Notably, the constitutively increased C18:3 induced a sustained activation of plasma membrane Ca2+-ATPase, thereby, changing the stress-induced Ca2+ signaling. The reactive oxygen species (ROS) scavenging system, which was positively correlated with the level of C18:3, was also activated in the transgenic lines. Microarray analysis showed that CbFAD3-overexpressing plants increased the expression of stress-responsive genes, most of which are affected by C18:3, Ca2+, or ROS. Together, CbFAD3 confers tolerance to multiple stresses in tobacco through the C18:3-induced integrated regulation of membrane, Ca2+, ROS, and stress-responsive genes. This is in contrast with previous observations that simply attribute stress tolerance to membrane stabilization.

Metabolite Profiling of adh1 Mutant Response to Cold Stress in Arabidopsis.

As a result of global warming, vegetation suffers from repeated freeze-thaw cycles caused by more frequent short-term low temperatures induced by hail, snow, or night frost. Therefore, short-term freezing stress of plants should be investigated particularly in light of the current climatic conditions. Alcohol dehydrogenase (ADH) plays a central role in the metabolism of alcohols and aldehydes and it is a key enzyme in anaerobic fermentation. ADH1 responds to plant growth and environmental stress; however, the function of ADH1 in the response to short-term freezing stress remains unknown. Using real-time quantitative fluorescence PCR, the expression level of ADH1 was analyzed at low temperature (4°C). The lethal temperature was calculated based on the electrolyte leakage tests for both ADH1 deletion mutants (adh1) and wild type (WT) plants. To further investigate the relationship between ADH1 and cold tolerance in plants, low-Mr polar metabolite analyses of Arabidopsis adh1 and WT were performed at cold temperatures using gas chromatography-mass spectrometry. This investigation focused on freezing treatments (cold acclimation group: -6°C for 2 h with prior 4°C for 7 d, cold shock group: -6°C for 2 h without cold acclimation) and recovery (23°C for 24 h) with respect to seedling growth at optimum temperature. The experimental results revealed a significant increase in ADH1 expression during low temperature treatment (4°C) and at a higher lethal temperature in adh1 compared to that in the WT. Retention time indices and specific mass fragments were used to monitor 263 variables and annotate 78 identified metabolites. From these analyses, differences in the degree of metabolite accumulation between adh1 and WT were detected, including soluble sugars (e.g., sucrose) and amino acids (e.g., asparagine). In addition, the correlation-based network analysis highlighted some metabolites, e.g., melibiose, fumaric acid, succinic acid, glycolic acid, and xylose, which enhanced connectedness in adh1 network under cold chock. When considered collectively, the results showed that adh1 possessed a metabolic response to freezing stress and ADH1 played an important role in the cold stress response of a plant. These results expands our understanding of the short-term freeze response of ADH1 in plants.

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

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. Chorispora bungeana 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 C. bungeana copes with these intricate cold stresses through epigenetic modifications, plants of C. bungeana 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 C. bungeana in response to chilling and freezing. It was found that the alterations in DNA methylation of C. bungeana 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 C. bungeana 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 C. bungeana to adapt to the intricate cold stresses in the alpine areas.

Molecular cloning and characterization of an S-adenosylmethionine synthetase gene from Chorispora bungeana.

S-adenosylmethionine synthetase (SAMS) catalyzes the formation of S-adenosylmethionine (SAM) which is a molecule essential for polyamines and ethylene biosynthesis, methylation modifications of protein, DNA and lipids. SAMS also plays an important role in abiotic stress response. Chorispora bungeana (C. bungeana) is an alpine subnival plant species which possesses strong tolerance to cold stress. Here, we cloned and characterized an S-adenosylmethionine synthetase gene, CbSAMS (C. bungeana S-adenosylmethionine synthetase), from C. bungeana, which encodes a protein of 393 amino acids containing a methionine binding motif GHPDK, an ATP binding motif GAGDQG and a phosphate binding motif GGGAFSGDK. Furthermore, an NES (nuclear export signal) peptide was identified through bioinformatics analysis. To explore the CbSAMS gene expression regulation, we isolated the promoter region of CbSAMS gene 1919bp upstream the ATG start codon, CbSAMSp, and analyzed its cis-acting elements by bioinformatics method. It was revealed that a transcription start site located at 320 bp upstream the ATG start codon and cis-acting elements related to light, ABA, auxin, ethylene, MeJA, low temperature and drought had been found in the CbSAMSp sequence. The gene expression pattern of CbSAMS was then analyzed by TR-qPCR and GUS assay method. The result showed that CbSAMS is expressed in all examined tissues including callus, roots, petioles, leaves, and flowers with a significant higher expression level in roots and flowers. Furthermore, the expression level of CbSAMS was induced by low temperature, ethylene and NaCl. Subcellular localization revealed that CbSAMS was located in the cytoplasm and nucleus but has a significant higher level in the nucleus. These results indicated a potential role of CbSAMS in abiotic stresses and plant growth in C. bungeana.

The protein phosphatase RCF2 and its interacting partner NAC019 are critical for heat stress-responsive gene regulation and thermotolerance in Arabidopsis.

Heat stress is a major environmental constraint for crop production worldwide. To respond to and cope with heat stress, plants synthesize heat shock proteins (HSPs), which are often molecular chaperones and are under the control of heat stress transcription factors (HSFs). Very little is known about the upstream regulators of HSFs. In a forward genetic screen for regulators of C-REPEAT BINDING FACTOR (CBF) gene expression (RCFs), we identified RCF2 and found that it is allelic to CPL1/FIERY2, which encodes a homolog of C-terminal domain phosphatase. Our results also showed that, in addition to being critical for cold stress tolerance, RCF2 is required for heat stress-responsive gene regulation and thermotolerance, because, compared with the wild type, the rcf2-1 mutant is hypersensitive to heat stress and because the reduced thermotolerance is correlated with lower expression of most of the 21 HSFs and some of the HSPs in the mutant plants. We found that RCF2 interacts with the NAC transcription factor NAC019 and that RCF2 dephosphorylates NAC019 in vivo. The nac019 mutant is more sensitive to heat stress than the wild type, and chromatin immunoprecipitation followed by quantitative PCR analysis revealed that NAC019 binds to the promoters of HSFA1b, HSFA6b, HSFA7a, and HSFC1. Overexpression of RCF2 or NAC019 in Arabidopsis thaliana increases thermotolerance. Together, our results suggest that, through dephosphorylation of NAC019, RCF2 is an integrator of high-temperature signal transduction and a mechanism for HSF and HSP activation.

A nuclear calcium-sensing pathway is critical for gene regulation and salt stress tolerance in Arabidopsis.

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 Arabidopsis. Through a forward genetic screen, we found a nuclear-localized calcium-binding protein, RSA1 (SHORT ROOT IN SALT 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 SOS1 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.