Effect of the Narrow Leaf Gene on Yield and Other Characters in a Soybean Cross

Agronom

Genome-Wide Atlas of Alternative Polyadenylation in the Forage Legume Red Clover

Studies on prevalence and significance of alternative polyadenylation (APA) in plants have been so far limited mostly to the model plants. Here, a genome-wide analysis of APA was carried out in different tissue types in the non-model forage legume red clover (Trifolium pratense L). A profile of poly(A) sites in different tissue types was generated using so-called ‘poly(A)-tag sequencing’ (PATseq) approach. Our analysis revealed tissue-wise dynamics of usage of poly(A) sites located at different genomic locations. We also identified poly(A) sites and underlying genes displaying APA in different tissues. Functional categories enriched in groups of genes manifesting APA between tissue types were determined. Analysis of spatial expression of genes encoding different poly(A) factors showed significant differential expression of genes encoding orthologs of FIP1(V) and PCFS4, suggesting that these two factors may play a role in regulating spatial APA in red clover. Our analysis also revealed a high degree of conservation in diverse plant species of APA events in mRNAs encoding two key polyadenylation factors, CPSF30 and FIP1(V). Together with our previously reported study of spatial gene expression in red clover, this study will provide a comprehensive account of transcriptome dynamics in this non-model forage legume

Distinctive interactions of the Arabidopsis homolog of the 30 kD subunit of the cleavage and polyadenylation specificity factor (AtCPSF30) with other polyadenylation factor subunits

BACKGROUND: The Arabidopsis ortholog of the 30 kD subunit of the mammalian Cleavage and Polyadenylation Specificity Factor (AtCPSF30) is an RNA-binding endonuclease that is associated with other Arabidopsis CPSF subunits (orthologs of the 160, 100, and 73 kD subunits of CPSF). In order to further explore the functions of AtCPSF30, the subcellular distribution of the protein was examined by over-expressing fusion proteins containing fluorescent reporters linked to different CPSF subunits. RESULTS: It was found that AtCPSF30 by itself localizes, not to the nucleus, but to the cytoplasm. AtCPSF30 could be found in the nucleus when co-expressed with AtCPSF160 or AtCPSF73(I), one of the two Arabidopsis orthologs of CPSF73. This re-directing of AtCPSF30 indicates that AtCPSF30 is retained in the nucleus via interactions with either or both of these other CPSF subunits. Co-expression of AtCSPF30 with AtCPSF100 altered the location, not of AtCPSF30, but rather of AtCPSF100, with these proteins residing in the cytoplasm. Deletion of plant-specific N- or C-terminal domains of AtCPSF30 abolished various of the interactions between AtCPSF30 and other CPSF subunits, suggesting that the plant CPSF complex assembles via novel protein-protein interactions. CONCLUSION: These results suggest that the nuclear CPSF complex in plants is a dynamic one, and that the interactions between AtCPSF30 and other CPSF subunits are different from those existing in other eukaryotes

Molecular Approaches in the Analysis of Red Clover Rhizobium Symbiosis

Red clover (Trifolium pratense L.) is an important forage and pasture legume grown throughout temperate regions. Because it can form a symbiotic relationship with rhizobium, it is able to fix atmospheric nitrogen. Due to the high cost of nitrogen fertilizers, pasture legumes have been increasingly important in forage production settings. Red clover has not been a model legume primarily due to selfincompatibility and the associated high level of genomic heterozygosity, therefore it has not been a significant contributor in molecular or genetic studies and basic information on red clover legume/rhizobium symbiosis is lacking. Using recently annotated genomic resources, RNA-seq expression analysis and CRISPR/Cas9 mutagenesis, we characterized a number of genes that are expressed only in nodule forming roots. These include genes that encode proteins with homology to nodule-specific cysteine rich proteins (NCRs) and nodule-specific polycystin-1, lipoxygenase, alpha toxic (PLAT) domain proteins (NPDs) that are postulated to be involved in plant rhizobium interactions. Our results indicate that red clover has one of the highest numbers of expressed NCR and ATS3-like/NPD peptides currently known in the inverted-repeat lacking clade (IRLC) of legumes. Knowledge of the expression of these genes and the continued analysis of the genetic variation in red clover should aid in breeding genotypes with increased rhizobium selection specificity and increased nitrogen fixation efficiency

Substrates of the \u3cem\u3eArabidopsis thaliana\u3c/em\u3e Protein Isoaspartyl Methyltransferase 1 Identified Using Phage Display and Biopanning

The role of protein isoaspartyl methyltransferase (PIMT) in repairing a wide assortment of damaged proteins in a host of organisms has been inferred from the affinity of the enzyme for isoaspartyl residues in a plethora of amino acid contexts. The identification of PIMT target proteins in plant seeds, where the enzyme is highly active and proteome long-lived, has been hindered by large amounts of isoaspartate-containing storage proteins. Mature seed phage display libraries circumvented this problem. Inclusion of the PIMT co-substrate, S-adenosylmethionine (AdoMet), during panning permitted PIMT to retain aged phage in greater numbers than controls lacking co-substrate or when PIMT protein binding was poisoned with S-adenosyl homocysteine. After four rounds, phage titer plateaued in AdoMet-containing pans, whereas titer declined in both controls. This strategy identified 17 in-frame PIMT target proteins, including a cupin-family protein similar to those identified previously using on-blot methylation. All recovered phage had at least one susceptible Asp or Asn residue. Five targets were recovered independently. Two in-frame targets were produced in Escherichia coli as recombinant proteins and shown by on-blot methylation to acquire isoAsp, becoming a PIMT target. Both gained isoAsp rapidly in solution upon thermal insult. Mutant analysis of plants deficient in any of three in-frame PIMT targets resulted in demonstrable phenotypes. An over-representation of clones encoding proteins involved in protein production suggests that the translational apparatus comprises a subgroup for which PIMT-mediated repair is vital for orthodox seed longevity. Impaired PIMT activity would hinder protein function in these targets, possibly resulting in poor seed performance

KELCH F-BOX Protein Positively Influences Arabidopsis Seed Germination by Targeting PHYTOCHROME-INTERACTING FACTOR1

Seeds employ sensory systems that assess various environmental cues over time to maximize the successful transition from embryo to seedling. Here we show that the Arabidopsis F-BOX protein COLD TEMPERATURE-GERMINATING (CTG)-10, identified by activation tagging, is a positive regulator of this process. When overexpressed (OE), CTG10 hastens aspects of seed germination. CTG10 is expressed predominantly in the hypocotyl, and the protein is localized to the nucleus. CTG10 interacts with PHYTOCHROME-INTERACTING FACTOR 1 (PIF1) and helps regulate its abundance in planta. CTG10-OE accelerates the loss of PIF1 in light, increasing germination efficiency, while PIF1-OE lines fail to complete germination in darkness, which is reversed by concurrent CTG10-OE. Double-mutant (pif1 ctg10) lines demonstrated that PIF1 is epistatic to CTG10. Both CTG10 and PIF1 amounts decline during seed germination in the light but reaccumulate in the dark. PIF1 in turn down-regulates CTG10 transcription, suggesting a feedback loop of CTG10/PIF1 control. The genetic, physiological, and biochemical evidence, when taken together, leads us to propose that PIF1 and CTG10 coexist, and even accumulate, in the nucleus in darkness, but that, following illumination, CTG10 assists in reducing PIF1 amounts, thus promoting the completion of seed germination and subsequent seedling development

Plant-Symbiotic Fungi as Chemical Engineers: Multi-Genome Analysis of the Clavicipitaceae Reveals Dynamics of Alkaloid Loci

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some—including the infamous ergot alkaloids—have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses

Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the Clavicipitaceae reveals dynamics of alkaloid Loci

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some—including the infamous ergot alkaloids—have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses