ABSTRACT
Alfalfa is widely recognized as an important forage crop. To understand the morphological characteristics and genetic basis of seed morphology in alfalfa, we screened 318 Medicago spp., including 244 Medicago sativa subsp. sativa (alfalfa) and 23 other Medicago spp., for seed area size, length, width, length-to-width ratio, perimeter, circularity, the distance between the intersection of length & width (IS) and center of gravity (CG), and seed darkness & red-green-blue (RGB) intensities. The results revealed phenotypic diversity and correlations among the tested accessions. Based on the phenotypic data of M. sativa subsp. sativa, a genome-wide association study (GWAS) was conducted using single nucleotide polymorphisms (SNPs) called against the Medicago truncatula genome. Genes in proximity to associated markers were detected, including CPR1, MON1, a PPR protein, and Wun1(threshold of 1E-04). Machine learning models were utilized to validate GWAS, and identify additional marker-trait associations for potentially complex traits. Marker S7_33375673, upstream of Wun1, was the most important predictor variable for red color intensity and highly important for brightness. Fifty-two markers were identified in coding regions. Along with strong correlations observed between seed morphology traits, these genes will facilitate the process of understanding the genetic basis of seed morphology in Medicago spp.
Subject(s)
Genome-Wide Association Study , Machine Learning , Medicago , Polymorphism, Single Nucleotide , Seeds , Seeds/genetics , Medicago/genetics , Phenotype , Quantitative Trait Loci , Medicago sativa/genetics , Medicago truncatula/genetics , Genome, PlantABSTRACT
KEY MESSAGE: Sustainable winter production in lettuce requires freezing tolerant varieties. This study identified a wild-type allele of LsCBF7 that could contribute to freezing tolerance improvement in lettuce. Lettuce is one of the most consumed vegetables globally. While ideally grown in 13-21 °C, its cultivation extends into winter in milder climates. However, occasional freezing temperatures can significantly reduce yields. Therefore, the development of freezing-tolerant lettuce varieties has become a long-term goal of lettuce breeding programs. Despite its significance, our understanding of freezing tolerance in lettuce remains limited. Plants have evolved a coping mechanism against freezing, known as cold acclimation, whereby they can increase freezing tolerance when pre-exposed to low nonfreezing temperatures. The CBF pathway is well-known for its central role in cold acclimation. Previously, we identified 14 CBF genes in lettuce and discovered that one of them, LsCBF7, had a loss-of-function mutation. In this study, we uncovered that accessions from colder regions carried the wild-type allele of LsCBF7 and this allele likely contributed to increased freezing tolerance, with 14% of the lettuce population carrying this allele. Interestingly, in wild lettuce (L. serriola) that is considered a progenitor of cultivated lettuce, this wild-type allele was much more common, with a frequency of 90%. This finding suggests that this wild-type allele may have undergone negative selection during the domestication or breeding of lettuce. Our data strongly indicate that this allele could be linked to early bolting, an undesirable trait in lettuce, which may have driven the negative selection. While this wild-type allele shows promise for improving freezing tolerance in lettuce, it is crucial to decouple it from the early bolting trait to fully harness its potential in lettuce breeding.
Subject(s)
Acclimatization , Alleles , Domestication , Freezing , Lactuca , Plant Breeding , Lactuca/genetics , Lactuca/growth & development , Lactuca/physiology , Acclimatization/genetics , Selection, Genetic , Plant Proteins/genetics , PhenotypeABSTRACT
A newly documented pathotype 5 of the soil-borne fungus Sporisorium reilianum, causing head smut in sorghum, was tested against 153 unexplored Senegalese sorghum accessions. Among the 153 sorghum accessions tested, 63 (41%) exhibited complete resistance, showing no signs of infection by the fungus. The remaining 90 accessions (59%) displayed varying degrees of susceptibility. Sorghum responses against S. reilianum were explored to analyze the potential link with previously known seed morphology-related traits and new phenotype data from 59 lines for seed weight. A genome-wide association study (GWAS) screened 297,876 SNPs and identified highly significant associations (p < 1 × 10-5) with head smut resistance in sorghum. By mapping these significant SNPs to the reference genome, this study revealed 35 novel candidate defense genes potentially involved in disease resistance.
ABSTRACT
The APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) gene family plays vital roles in plants, serving as a key regulator in responses to abiotic stresses. Despite its significance, a comprehensive understanding of this family in lettuce remains incomplete. In this study, we performed a genome-wide search for the AP2/ERF family in lettuce and identified a total of 224 members. The duplication patterns provided evidence that both tandem and segmental duplications contributed to the expansion of this family. Ka/Ks ratio analysis demonstrated that, following duplication events, the genes have been subjected to purifying selection pressure, leading to selective constraints on their protein sequence. This selective pressure provides a dosage benefit against stresses in plants. Additionally, a transcriptome analysis indicated that some duplicated genes gained novel functions, emphasizing the contribution of both dosage effect and functional divergence to the family functionalities. Furthermore, an orthologous relationship study showed that 60% of genes descended from a common ancestor of Rosid and Asterid lineages, 28% from the Asterid ancestor, and 12% evolved in the lettuce lineage, suggesting lineage-specific roles in adaptive evolution. These results provide valuable insights into the evolutionary mechanisms of the AP2/ERF gene family in lettuce, with implications for enhancing abiotic stress tolerance, ultimately contributing to the genetic improvement of lettuce crop production.
Subject(s)
Lactuca , Plant Proteins , Ethylenes , Gene Expression Regulation, Plant , Genome, Plant/genetics , Lactuca/genetics , Multigene Family , Phylogeny , Plant Proteins/genetics , Plant Proteins/metabolismABSTRACT
Since the early 19th century, a substantial amount of jujube (Ziziphus spp.) germplasm has been introduced from China and Europe into the United States. However, due to a lack of passport data, cultivar mislabeling is common and the genetic background of the introduced germplasm remains unknown. In the present study, a low-density SNP array was employed to genotype 204 jujube trees sampled from multiple locations in New Mexico, Texas, Missouri, and Kentucky. Multilocus matching of SNP profiles revealed a significant rate of genetic redundancy among these jujube samples. A total of 14 synonymous groups were detected, comprising 48 accessions. Bayesian clustering analysis and neighbor-joining tree partitioned the US jujube germplasm into two major clusters. The first cluster included cultivated genotypes (Ziziphus jujuba Mill.), whereas the other major cluster comprised the wild/sour jujube (Ziziphus spinosa Hu.). The results also revealed a unique jujube population at Fabens/Tornillo, Texas, and a semi-naturalized population at Tucumcari, NM. These findings will provide valuable guidance to jujube growers and researchers on the effective utilization of jujube germplasm in the horticultural industry.
ABSTRACT
The World Collection of Sugarcane and Related Grasses, maintained at the USDA-ARS in Miami, FL, is one of the largest sugarcane germplasm repositories in the world. However, the genetic integrity of the Saccharum spp. germplasm in this collection has not been fully analyzed. In this study, we employed a single-dose SNP panel to genotype 901 sugarcane accessions, representing six Saccharum species and various hybrids. Our analysis uncovered a high rate of clone mislabeling in the collection. Specifically, we identified 86 groups of duplicates, characterized by identical SNP genotypes, which encompassed 211 accessions (23% of the total clones), while 135 groups, constituting 471 clones (52% of the total), exhibited near-identical genotypes. In addition, twenty-seven homonymous groups were detected, which shared the same clone name but differed in SNP genotypes. Hierarchical analysis of population structure partitioned the Saccharum germplasm into five clusters, corresponding to S. barberi, S. sinense, S. officinarum, S. spontaneum and S. robustum/S. edule. An assignment test, based on the five Saccharum species, enabled correcting 141 instances of mislabeled species memberships and inaccuracies. Moreover, we clarified the species membership and parentage of 298 clones that had ambiguous passport records (e.g., 'Saccharum spp', 'unknown', and 'hybrid'). Population structure and genetic diversity in these five species were further supported by Principal Coordinate Analysis and neighbor-joining clustering analysis. Analysis of Molecular Variance revealed that within-species genetic variations accounted for 85% of the total molecular variance, with the remaining 15% attributed to among-species genetic variations. The single-dose SNP markers developed in this study offer a robust tool for characterizing sugarcane germplasm worldwide. These findings have important implications for sugarcane genebank management, germplasm exchange, and crop genetic improvement.
ABSTRACT
Genetic diversity is an important resource in crop breeding to improve cultivars with desirable traits. Selective breeding can lead to a reduction of genetic diversity. However, our understanding on this subject remains limited in lettuce (Lactuca sativa L.). Genotyping-by-sequencing (GBS) can provide a reduced version of the genome as a cost-effective method to identify genetic variants across the genome. We genotyped a diverse set of 441 lettuce accessions using the GBS method. Phylogenetic and population genetic analyses indicated substantial genetic divergence among four horticultural types of lettuce: butterhead, crisphead, leaf, and romaine. Genetic-diversity estimates between and within the four types indicated that the crisphead type was the most differentiated from other types, whereas its population was the most homogenous with the slowest linkage disequilibrium (LD) decay among the four types. These results suggested that crisphead lettuces had relatively less genetic variation across the genome as well as low gene flow from other types. We identified putative selective sweep regions that showed low genetic variation in the crisphead type. Genome-wide association study (GWAS) and quantitative trait loci (QTL) analyses provided evidence that these genomic regions were, in part, associated with delayed bolting, implicating the positive selection of delayed bolting in reducing variation. Our findings enhance the current understanding of genetic diversity and the impacts of selective breeding on patterning genetic variation in lettuce.
Subject(s)
Genome-Wide Association Study , Lactuca , Genetics, Population , Lactuca/genetics , Phylogeny , Plant Breeding , Polymorphism, Single Nucleotide , Selective BreedingABSTRACT
The C-repeat binding factor (CBF)/dehydration-responsive element binding (DREB1) proteins play a prominent role in freezing tolerance and are highly conserved in higher plants. Here we performed a genome-wide search of the CBF/DREB1 gene family in lettuce (Lactuca sativa L.) and identified 14 members of the family with one member gene containing a non-sense mutation within the AP2 DNA-binding domain. A comprehensive phylogenetic analysis of the CBF/DREB1 family members in 20 plant species from the Asterid or Rosid clade provided evidence that tandem duplication played an important role in the expansion of the CBF/DREB1 family. Expression analysis showed that twelve of the lettuce CBF genes were responsive to low temperature (4 °C), and that three and six of them could also be responsive to salt and heat stresses, respectively. Unlike Arabidopsis thaliana whose members of the CBF/DREB1 family respond only to a particular stress, lettuce CBFs provide wider protection from combinations of abiotic stresses. A global transcriptome analysis revealed distinctive temporal expression patterns among the cold-regulated genes in lettuce plants exposed to low temperature. Genes induced throughout the cold treatment are enriched in functions associated with protection from UV and high-light intensity and the genes suppressed after 7 days of cold exposure are enriched in photosynthesis-associated functions. These results provide insight into the molecular evolutionary properties of the CBF/DREB1 gene family in lettuce and a reference for genetic improvement of the lettuce response to cold acclimation.
Subject(s)
Acclimatization/genetics , Gene Expression Regulation, Plant , Lactuca/genetics , Plant Proteins/genetics , Transcription Factors/genetics , Gene ExpressionABSTRACT
PREMISE: Despite myriad examples of local adaptation, the phenotypes and genetic variants underlying such adaptive differentiation are seldom known. Recent work on freezing tolerance and local adaptation in ecotypes of Arabidopsis thaliana from Italy and Sweden provides an essential foundation for uncovering the genotype-phenotype-fitness map for an adaptive response to a key environmental stress. METHODS: We examined the consequences of a naturally occurring loss-of-function (LOF) mutation in an Italian allele of the gene that encodes the transcription factor CBF2, which underlies a major freezing-tolerance locus. We used four lines with a Swedish genetic background, each containing a LOF CBF2 allele. Two lines had introgression segments containing the Italian CBF2 allele, and two contained deletions created using CRISPR-Cas9. We used a growth chamber experiment to quantify freezing tolerance and gene expression before and after cold acclimation. RESULTS: Freezing tolerance was lower in the Italian (11%) compared to the Swedish (72%) ecotype, and all four experimental CBF2 LOF lines had reduced freezing tolerance compared to the Swedish ecotype. Differential expression analyses identified 10 genes for which all CBF2 LOF lines, and the IT ecotype had similar patterns of reduced cold responsive expression compared to the SW ecotype. CONCLUSIONS: We identified 10 genes that are at least partially regulated by CBF2 that may contribute to the differences in cold-acclimated freezing tolerance between the Italian and Swedish ecotypes. These results provide novel insight into the molecular and physiological mechanisms connecting a naturally occurring sequence polymorphism to an adaptive response to freezing conditions.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Acclimatization , Cold Temperature , Freezing , Gene Expression Regulation, Plant , SwedenABSTRACT
The Arabidopsis thaliana Calmodulin-binding Transcription Activator (CAMTA) transcription factors CAMTA1, CAMTA2, and CAMTA3 (CAMTA123) serve as master regulators of salicylic acid (SA)-mediated immunity, repressing the biosynthesis of SA in healthy plants. Here, we show that CAMTA123 also repress the biosynthesis of pipecolic acid (Pip) in healthy plants. Loss of CAMTA123 function resulted in the induction of AGD2-like defense response protein 1 (ALD1), which encodes an enzyme involved in Pip biosynthesis. Induction of ALD1 resulted in the accumulation of high levels of Pip, which brought about increased levels of the SA receptor protein NPR1 without induction of NPR1 expression or requirement for an increase in SA levels. Pip-mediated induction of ALD1 and genes regulating the biosynthesis of SA-CBP60g, SARD1, PAD4, and EDS1-was largely dependent on NPR1. Furthermore, Pip-mediated increase in NPR1 protein levels was associated with priming of Pip and SA biosynthesis genes to induction by low levels of SA. Taken together, our findings expand the role for CAMTA123 in regulating key immunity genes and suggest a working model whereby loss of CAMTA123 repression leads to the induction of plant defense genes and initiation of SAR.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/physiology , Calcium-Binding Proteins/metabolism , Pipecolic Acids/metabolism , Trans-Activators/metabolism , Transaminases/metabolism , Arabidopsis Proteins/genetics , Calcium-Binding Proteins/genetics , Calmodulin-Binding Proteins/genetics , Calmodulin-Binding Proteins/metabolism , Gene Expression Regulation, Plant , Genes, Plant , Plant Immunity , Salicylic Acid/metabolism , Trans-Activators/genetics , Transaminases/geneticsABSTRACT
Arabidopsis thaliana (Arabidopsis) increases in freezing tolerance in response to low nonfreezing temperatures, a phenomenon known as cold acclimation. The CBF regulatory pathway, which contributes to cold acclimation, includes three genes-CBF1, CBF2 and CBF3-encoding closely-related transcription factors that regulate the expression of more than 100 genes-the CBF regulon-that impart freezing tolerance. Here we compare the CBF pathways of two Arabidopsis ecotypes collected from sites in Sweden (SW) and Italy (IT). Previous studies showed that the SW ecotype was more freezing tolerant than the IT ecotype and that the IT ecotype had a nonfunctional CBF2 gene. Here we present results establishing that the difference in CBF2 alleles contributes to the difference in freezing tolerance between the two ecotypes. However, other differences in the CBF pathway as well as CBF-independent pathways contribute the large majority of the difference in freezing tolerance between the two ecotypes. The results also provided evidence that most cold-induced CBF regulon genes in both the SW and IT ecotypes are coregulated by CBF-independent pathways. Additional analysis comparing our results with those published by others examining the Col-0 accession resulted in the identification of 44 CBF regulon genes that were conserved among the three accessions suggesting that they likely have important functions in life at low temperature. The comparison further supported the conclusion that the CBF pathway can account for a large portion of the increase in freezing tolerance that occurs with cold acclimation in a given accession, but that CBF-independent pathways can also make a major contribution.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis Proteins/physiology , Arabidopsis/genetics , Arabidopsis/physiology , Trans-Activators/genetics , Trans-Activators/physiology , Acclimatization/genetics , Acclimatization/physiology , Alleles , Arabidopsis/growth & development , CRISPR-Cas Systems , Ecotype , Flowers/genetics , Flowers/growth & development , Freezing , Gene Expression Regulation, Plant , Gene Regulatory Networks , Genes, Plant , Italy , Mutagenesis , Plants, Genetically Modified , Regulon , Sweden , Transcription Factors/genetics , Transcription Factors/physiologyABSTRACT
Arabidopsis thaliana calmodulin binding transcription activator (CAMTA) factors repress the expression of genes involved in salicylic acid (SA) biosynthesis and SA-mediated immunity in healthy plants grown at warm temperature (22°C). This repression is overcome in plants exposed to low temperature (4°C) for more than a week and in plants infected by biotrophic and hemibiotrophic pathogens. Here, we present evidence that CAMTA3-mediated repression of SA pathway genes in nonstressed plants involves the action of an N-terminal repression module (NRM) that acts independently of calmodulin (CaM) binding to the IQ and CaM binding (CaMB) domains, a finding that is contrary to current thinking that CAMTA3 repression activity requires binding of CaM to the CaMB domain. Induction of SA pathway genes in response to low temperature did not occur in plants expressing only the CAMTA3-NRM region of the protein. Mutational analysis provided evidence that the repression activity of the NRM was suppressed by action of the IQ and CaMB domains responding to signals generated in response to low temperature. Plants expressing the CAMTA3-NRM region were also impaired in defense against the bacterial hemibiotrophic pathogen Pseudomonas syringae pv tomato DC3000. Our results indicate that the regulation of CAMTA3 repression activity by low temperature and pathogen infection involves related mechanisms, but with distinct differences.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Arabidopsis/microbiology , Salicylic Acid/metabolism , Arabidopsis Proteins/genetics , Calmodulin/genetics , Calmodulin/metabolism , Cold Temperature , Gene Expression Regulation, Plant/physiology , Pseudomonas syringae/pathogenicity , Temperature , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
The natural range of Arabidopsis thaliana (Arabidopsis) encompasses geographical regions that have greatly differing local climates, including harshness of winter temperatures. A question thus raised is whether differences in freezing tolerance might contribute to local adaptation in Arabidopsis. Consistent with this possibility is that Arabidopsis accessions differ in freezing tolerance and that those collected from colder northern latitudes are generally more tolerant to freezing than those collected from warmer southern latitudes. Moreover, recent studies with Arabidopsis genotypes collected from sites in Sweden (SW) and Italy (IT) have established that the two accessions are locally adapted, that the SW ecotype is more tolerant of freezing than the IT ecotype, and that genetic differences between the two ecotypes that condition local adaptation and freezing tolerance map to a region that includes the C-repeat binding factor (CBF) locus. The CBF locus includes three genes - CBF1, CBF2 and CBF3 - that are induced by low temperature and encode transcription factors that regulate a group of more than 100 genes, the CBF regulon, which impart freezing tolerance. Here we show that cold induction of most CBF regulon genes is lower in IT plants compared with SW plants, and that this is due to the IT CBF2 gene encoding a non-functional CBF2 protein. The non-functional IT CBF2 protein also contributes to the lower freezing tolerance of the IT plants compared with the SW plants. Taken together, studies on the SW and IT ecotypes provide evidence that natural variation in the CBF pathway has contributed to adaptive evolution in these Arabidopsis populations.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Cold Temperature , Genetic Variation , Trans-Activators/genetics , Transcription Factors/genetics , Adaptation, Physiological/genetics , Amino Acid Sequence , Arabidopsis/classification , Ecotype , Gene Expression Profiling , Gene Expression Regulation, Plant , Geography , Italy , Molecular Sequence Data , Plants, Genetically Modified , Quantitative Trait Loci/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Signal Transduction/genetics , Species Specificity , SwedenABSTRACT
Exposure of Arabidopsis thaliana plants to low non-freezing temperatures results in an increase in freezing tolerance that involves action of the C-repeat binding factor (CBF) regulatory pathway. CBF1, CBF2 and CBF3, which are rapidly induced in response to low temperature, encode closely related AP2/ERF DNA-binding proteins that recognize the C-repeat (CRT)/dehydration-responsive element (DRE) DNA regulatory element present in the promoters of CBF-regulated genes. The CBF transcription factors alter the expression of more than 100 genes, known as the CBF regulon, which contribute to an increase in freezing tolerance. In this study, we investigated the extent to which cold induction of the CBF regulon is regulated by transcription factors other than CBF1, CBF2 and CBF3, and whether freezing tolerance is dependent on a functional CBF-CRT/DRE regulatory module. To address these issues we generated transgenic lines that constitutively overexpressed a truncated version of CBF2 that had dominant negative effects on the function of the CBF-CRT/DRE regulatory module, and 11 transcription factors encoded by genes that were rapidly cold-induced in parallel with the 'first-wave' CBF genes, and determined the effects that overexpressing these proteins had on global gene expression and freezing tolerance. Our results indicate that cold regulation of the CBF regulon involves extensive co-regulation by other first-wave transcription factors; that the low-temperature regulatory network beyond the CBF pathway is complex and highly interconnected; and that the increase in freezing tolerance that occurs with cold acclimation is only partially dependent on the CBF-CRT/DRE regulatory module.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Cold Temperature , Gene Expression Regulation, Plant , Regulon/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/metabolismABSTRACT
Mannans are hemicellulosic polysaccharides that have a structural role and serve as storage reserves during plant growth and development. Previous studies led to the conclusion that mannan synthase enzymes in several plant species are encoded by members of the cellulose synthase-like A (CSLA) gene family. Arabidopsis has nine members of the CSLA gene family. Earlier work has shown that CSLA9 is responsible for the majority of glucomannan synthesis in both primary and secondary cell walls of Arabidopsis inflorescence stems. Little is known about how expression of the CLSA9 gene is regulated. Sequence analysis of the CSLA9 promoter region revealed the presence of multiple copies of a cis-regulatory motif (M46RE) recognized by transcription factor MYB46, leading to the hypothesis that MYB46 (At5g12870) is a direct regulator of the mannan synthase CLSA9. We obtained several lines of experimental evidence in support of this hypothesis. First, the expression of CSLA9 was substantially upregulated by MYB46 overexpression. Second, electrophoretic mobility shift assay (EMSA) was used to demonstrate the direct binding of MYB46 to the promoter of CSLA9 in vitro. This interaction was further confirmed in vivo by a chromatin immunoprecipitation assay. Finally, over-expression of MYB46 resulted in a significant increase in mannan content. Considering the multifaceted nature of MYB46-mediated transcriptional regulation of secondary wall biosynthesis, we reasoned that additional transcription factors are involved in the CSLA9 regulation. This hypothesis was tested by carrying out yeast-one hybrid screening, which identified ANAC041 and bZIP1 as direct regulators of CSLA9. Transcriptional activation assays and EMSA were used to confirm the yeast-one hybrid results. Taken together, we report that transcription factors ANAC041, bZIP1 and MYB46 directly regulate the expression of CSLA9.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Gene Expression Regulation, Plant , Glucosyltransferases/metabolism , Mannosyltransferases/metabolism , Transcription Factors/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Basic-Leucine Zipper Transcription Factors/genetics , Basic-Leucine Zipper Transcription Factors/metabolism , Chromatin Immunoprecipitation , Electrophoretic Mobility Shift Assay , Glucosyltransferases/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Mannosyltransferases/genetics , Microscopy, Confocal , Plants, Genetically Modified , Promoter Regions, Genetic/genetics , Protein Binding , Regulatory Sequences, Nucleic Acid/genetics , Transcription Factors/genetics , Two-Hybrid System TechniquesABSTRACT
Previous studies in Arabidopsis thaliana established roles for CALMODULIN BINDING TRANSCRIPTION ACTIVATOR 3 (CAMTA3) in the rapid cold induction of CRT/DRE BINDING FACTOR (CBF) genes CBF1 and CBF2, and the repression of salicylic acid (SA) biosynthesis at warm temperature. Here we show that CAMTA1 and CAMTA2 work in concert with CAMTA3 at low temperature (4°C) to induce peak transcript levels of CBF1, CBF2 and CBF3 at 2 h, contribute to up-regulation of approximately 15% of the genes induced at 24 h, most of which fall outside the CBF pathway, and increase plant freezing tolerance. In addition, CAMTA1, CAMTA2 and CAMTA3 function together to inhibit SA biosynthesis at warm temperature (22°C). However, SA levels increase in Arabidopsis plants that are exposed to low temperature for more than 1 week. We show that this chilling-induced SA biosynthesis proceeds through the isochorismate synthase (ICS) pathway, with cold induction of ICS1 (which encodes ICS), and two genes encoding transcription factors that positively regulate ICS1 - CBP60g and SARD1 -, paralleling SA accumulation. The three CAMTA proteins effectively repress the accumulation of ICS1, CBP60g and SARD1 transcripts at warm temperature but not at low temperature. This impairment of CAMTA function may involve post-transcriptional regulation, as CAMTA transcript levels did not decrease at low temperature. Salicylic acid biosynthesis at low temperature did not contribute to freezing tolerance, but had a major role in configuring the transcriptome, including the induction of 'defense response' genes, suggesting the possible existence of a pre-emptive defense strategy programmed by prolonged chilling temperatures.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/physiology , Calcium-Binding Proteins/metabolism , Salicylic Acid/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Adaptation, Physiological , Arabidopsis Proteins/genetics , Calcium-Binding Proteins/genetics , Calmodulin-Binding Proteins/genetics , Calmodulin-Binding Proteins/metabolism , Cold Temperature , Freezing , Gene Expression Regulation, Plant , Intramolecular Transferases/genetics , Intramolecular Transferases/metabolism , Plants, Genetically Modified , Temperature , Trans-Activators/genetics , Transcription Factors/genetics , TranscriptomeABSTRACT
Histone lysine methylation patterns underlie much of the functional diversity of nucleosomes in eukaryotes, and an interesting aspect of histone methylation is the potential functional specificity for different methylation states on a given lysine. Trimethylation of histone H3 (H3K27me3) is intimately related to developmental gene silencing through the so-called Polycomb Group (PcG) mechanism. How this modification becomes established at PcG-repressed loci is generally not known, but it has been suggested that it may be facilitated by prior occupancy by H3K27me2. In this study we mapped the genomic and gene-level distribution of H3K27me2 in Arabidopsis thaliana using ChIP and a high-density tiling microarray, and integrated this with previous maps of other chromatin features and gene expression data. At the genome level, H3K27me2 enrichment sites were sparsely distributed across chromosomes, within an average size expected for a single nucleosome, and contrasted with the longer domains seen for H3K27me3. In both heterochromatic and euchromatic segments of the genome, H3K27me2 enrichment was often localized within transposon-related genes, with the longest genomic stretches of this modification corresponding to retroelements. However, H3K27me2 was more frequently found within protein-coding genes. These genes generally also showed moderate enrichment for H3K27me3, but H3K27me2 was strongly depleted within those genes most enriched in H3K27me3. H3K27me2 within highly transcribed genes was at highest levels at transcriptional starts and was strongly depleted throughout the transcribed regions, and reached higher levels at active than at silent promoters.
Subject(s)
Arabidopsis/genetics , Histones/metabolism , 3' Flanking Region , 5' Flanking Region , Arabidopsis/metabolism , Chromosome Mapping , Cluster Analysis , DNA Transposable Elements , Euchromatin/genetics , Euchromatin/metabolism , Genes, Plant , Genomics , Lysine/metabolism , Open Reading FramesABSTRACT
Paf1C is a transcriptional cofactor that has been implicated in various transcription-associated mechanisms spanning initiation, elongation and RNA processing, and is important for multiple aspects of development in Arabidopsis. Our recent studies suggest Arabidopsis Paf1C is crucial for proper regulation of genes within H3K27me3-enriched chromatin, and that a protein named PHP may act as an accessory subunit of Paf1C that promotes this function.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Histones/metabolism , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Chromatin/metabolism , Gene Expression Regulation, PlantABSTRACT
The human Paf1 complex (Paf1C) subunit Parafibromin assists in mediating output from the Wingless/Int signaling pathway, and dysfunction of the encoding gene HRPT2 conditions specific cancer-related disease phenotypes. Here, we characterize the organismal and molecular roles of PLANT HOMOLOGOUS TO PARAFIBROMIN (PHP), the Arabidopsis (Arabidopsis thaliana) homolog of Parafibromin. PHP resides in an approximately 670-kD protein complex in nuclear extracts, and physically interacts with other known Paf1C-related proteins in vivo. In striking contrast to the developmental pleiotropy conferred by mutation in other plant Paf1C component genes in Arabidopsis, loss of PHP specifically conditioned accelerated phase transition from vegetative growth to flowering and resulted in misregulation of a very limited subset of genes that included the flowering repressor FLOWERING LOCUS C. Those genes targeted by PHP were distinguished from the bulk of Arabidopsis genes and other plant Paf1C targets by strong enrichment for trimethylation of lysine-27 on histone H3 (H3K27me3) within chromatin. These findings suggest that PHP is a component of a plant Paf1C protein in Arabidopsis, but has a more specialized role in modulating expression of a subset of Paf1C targets.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Chromatin/metabolism , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Flowers/growth & development , Gene Expression Profiling , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Genes, Plant , Histones/metabolism , Methylation , MutationABSTRACT
In budding yeast, intragenic histone modification is linked with transcriptional elongation through the conserved regulator Paf1C. To investigate Paf1C-related function in higher eukaryotes, we analyzed the effects of loss of Paf1C on histone H3 density and patterns of H3 methylated at K4, K27, and K36 in Arabidopsis genes, and integrated this with existing gene expression data. Loss of Paf1C did not change global abundance of H3K4me3 or H3K36me2 within chromatin, but instead led to a 3' shift in the distribution of H3K4me3 and a 5' shift in the distribution of H3K36me2 within genes. We found that genes regulated by plant Paf1C showed strong enrichment for both H3K4me3 and H3K27me3 and also showed a high degree of tissue-specific expression. At the Paf1C- and PcG-regulated gene FLC, transcriptional silencing and loss of H3K4me3 and H3K36me2 were accompanied by expansion of H3K27me3 into the promoter and transcriptional start regions and further enrichment of H3K27me3 within the transcribed region. These results highlight both genic and global functions for plant Paf1C in histone modification and gene expression, and link transcriptional activity with cellular memory.