Isolation, phylogenetic analysis, and expression of a Somatic Embryogenesis Receptor like Kinase (SERK) gene in Cattleya maxima Lindl

Orchidaceae is a central family of vascular plants in Ecuador because of its huge diversity and endemism. A prominent commercial native species well known for its richness of color is Cattleya maxima Lindl. Somatic embryogenesis is recognized as an important process for mass propagation of many ornamental crops including orchids. Somatic Embryogenesis Receptor like Kinases (SERK) genes, in particular, are highly expressed during the early phases of somatic embryogenesis and therefore, the study of their involvement in this process is of paramount importance to improve the commercial propagation of orchids. For this reason, we decided to isolate and characterize a SERK orthologue from Cattleya maxima L. The deduced amino acid sequence of a partial CmSERK cDNA shows the presence of all SERK typical domains, suggesting that it may be functionally active in C. maxima. Its role in somatic embryogenesis is further supported by its high expression level during embryogenesis and in protocorm-like bodies. Moreover, the basal expression of CmSERK in roots, leaves, and ovaries points to a broader developmental role of this gene. Keywords: CmSERK, Somatic embryogenesis, Orchids, Leucine Rich Repeat, Molecular characterizatio

Arabidopsis Topless-related 1 mitigates physiological damage and growth penalties of induced immunity

Transcriptional corepressors of the Topless (TPL) family regulate plant hormone and immunity signaling. The lack of a genome-wide profile of their chromatin associations limits understanding of the TPL family roles in transcriptional regulation. Chromatin immunoprecipitation with sequencing (ChIP-Seq) was performed on Arabidopsis thaliana lines expressing GFP-tagged Topless-related 1 (TPR1-GFP) with and without constitutive immunity via Enhanced Disease Susceptibility 1 (EDS1). RNA-Seq profiling of the TPR1-GFP lines and pathogen-infected tpl/tpr mutants, combined with measuring immunity, growth, and physiological parameters was employed to investigate TPL/TPR roles in immunity and defense homeostasis. TPR1 was enriched at promoter regions of c. 1400 genes and c. 10% of the detected binding required EDS1 immunity signaling. In a tpr1 tpl tpr4 (t3) mutant, resistance to bacteria was slightly compromised, and defense-related transcriptional reprogramming was weakly reduced or enhanced, respectively, at early (< 1 h) and late 24 h stages of bacterial infection. The t3 plants challenged with bacteria or pathogen-associated molecular pattern nlp24 displayed photosystem II dysfunctions. Also, t3 plants were hypersensitive to phytocytokine pep1 at the level of root growth inhibition. Transgenic expression of TPR1 rescued these t3 physiological defects. We propose that TPR1 and TPL family proteins function in Arabidopsis to reduce detrimental effects associated with activated transcriptional immunity

Arabidopsis Topless-related 1 mitigates physiological damage and growth penalties of induced immunity

Transcriptional corepressors of the Topless (TPL) family regulate plant hormone and immunity signaling. The lack of a genome-wide profile of their chromatin associations limits understanding of the TPL family roles in transcriptional regulation. Chromatin immunoprecipitation with sequencing (ChIP-Seq) was performed on Arabidopsis thaliana lines expressing GFP-tagged Topless-related 1 (TPR1-GFP) with and without constitutive immunity via Enhanced Disease Susceptibility 1 (EDS1). RNA-Seq profiling of the TPR1-GFP lines and pathogen-infected tpl/tpr mutants, combined with measuring immunity, growth, and physiological parameters was employed to investigate TPL/TPR roles in immunity and defense homeostasis. TPR1 was enriched at promoter regions of c. 1400 genes and c. 10% of the detected binding required EDS1 immunity signaling. In a tpr1 tpl tpr4 (t3) mutant, resistance to bacteria was slightly compromised, and defense-related transcriptional reprogramming was weakly reduced or enhanced, respectively, at early (< 1 h) and late 24 h stages of bacterial infection. The t3 plants challenged with bacteria or pathogen-associated molecular pattern nlp24 displayed photosystem II dysfunctions. Also, t3 plants were hypersensitive to phytocytokine pep1 at the level of root growth inhibition. Transgenic expression of TPR1 rescued these t3 physiological defects. We propose that TPR1 and TPL family proteins function in Arabidopsis to reduce detrimental effects associated with activated transcriptional immunity

Immunity onset alters plant chromatin and utilizes EDA16 to regulate oxidative homeostasis

Perception of microbes by plants leads to dynamic reprogramming of the transcriptome, which is essential for plant health. The appropriate amplitude of this transcriptional response can be regulated at multiple levels, including chromatin. However, the mechanisms underlying the interplay between chromatin remodeling and transcription dynamics upon activation of plant immunity remain poorly understood. Here, we present evidence that activation of plant immunity by bacteria leads to nucleosome repositioning, which correlates with altered transcription. Nucleosome remodeling follows distinct patterns of nucleosome repositioning at different loci. Using a reverse genetic screen, we identify multiple chromatin remodeling ATPases with previously undescribed roles in immunity, including EMBRYO SAC DEVELOPMENT ARREST 16, EDA16. Functional characterization of the immune-inducible chromatin remodeling ATPase EDA16 revealed a mechanism to negatively regulate immunity activation and limit changes in redox homeostasis. Our transcriptomic data combined with MNase-seq data for EDA16 functional knock-out and over-expressor mutants show that EDA16 selectively regulates a defined subset of genes involved in redox signaling through nucleosome repositioning. Thus, collectively, chromatin remodeling ATPases fine-tune immune responses and provide a previously uncharacterized mechanism of immune regulation

GCN5 modulates salicylic acid homeostasis by regulating H3K14ac levels at the 5ʹ and 3ʹ ends of its target genes

The modification of histones by acetyl groups has a key role in the regulation of chromatin structure and transcription. The Arabidopsis thaliana histone acetyltransferase GCN5 regulates histone modifications as part of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) transcriptional coactivator complex. GCN5 was previously shown to acetylate lysine 14 of histone 3 (H3K14ac) in the promoter regions of its target genes even though GCN5 binding did not systematically correlate with gene activation. Here, we explored the mechanism through which GCN5 controls transcription. First, we fine-mapped its GCN5 binding sites genome-wide and then used several global methodologies (ATAC-seq, ChIP-seq and RNA-seq) to assess the effect of GCN5 loss-of-function on the expression and epigenetic regulation of its target genes. These analyses provided evidence that GCN5 has a dual role in the regulation of H3K14ac levels in their 5′ and 3′ ends of its target genes. While the gcn5 mutation led to a genome-wide decrease of H3K14ac in the 5′ end of the GCN5 down-regulated targets, it also led to an increase of H3K14ac in the 3′ ends of GCN5 up-regulated targets. Furthermore, genome-wide changes in H3K14ac levels in the gcn5 mutant correlated with changes in H3K9ac at both 5′ and 3′ ends, providing evidence for a molecular link between the depositions of these two histone modifications. To understand the biological relevance of these regulations, we showed that GCN5 participates in the responses to biotic stress by repressing salicylic acid (SA) accumulation and SA-mediated immunity, highlighting the role of this protein in the regulation of the crosstalk between diverse developmental and stress-responsive physiological programs. Hence, our results demonstrate that GCN5, through the modulation of H3K14ac levels on its targets, controls the balance between biotic and abiotic stress responses and is a master regulator of plant-environmental interactions

Shifting the limits in wheat research and breeding using a fully annotated reference genome

Introduction: Wheat (Triticum aestivum L.) is the most widely cultivated crop on Earth, contributing about a fifth of the total calories consumed by humans. Consequently, wheat yields and production affect the global economy, and failed harvests can lead to social unrest. Breeders continuously strive to develop improved varieties by fine-tuning genetically complex yield and end-use quality parameters while maintaining stable yields and adapting the crop to regionally specific biotic and abiotic stresses. Rationale: Breeding efforts are limited by insufficient knowledge and understanding of wheat biology and the molecular basis of central agronomic traits. To meet the demands of human population growth, there is an urgent need for wheat research and breeding to accelerate genetic gain as well as to increase and protect wheat yield and quality traits. In other plant and animal species, access to a fully annotated and ordered genome sequence, including regulatory sequences and genome-diversity information, has promoted the development of systematic and more time-efficient approaches for the selection and understanding of important traits. Wheat has lagged behind, primarily owing to the challenges of assembling a genome that is more than five times as large as the human genome, polyploid, and complex, containing more than 85% repetitive DNA. To provide a foundation for improvement through molecular breeding, in 2005, the International Wheat Genome Sequencing Consortium set out to deliver a high-quality annotated reference genome sequence of bread wheat. Results: An annotated reference sequence representing the hexaploid bread wheat genome in the form of 21 chromosome-like sequence assemblies has now been delivered, giving access to 107,891 high-confidence genes, including their genomic context of regulatory sequences. This assembly enabled the discovery of tissue- and developmental stage–related gene coexpression networks using a transcriptome atlas representing all stages of wheat development. The dynamics of change in complex gene families involved in environmental adaptation and end-use quality were revealed at subgenome resolution and contextualized to known agronomic single-gene or quantitative trait loci. Aspects of the future value of the annotated assembly for molecular breeding and research were exemplarily illustrated by resolving the genetic basis of a quantitative trait locus conferring resistance to abiotic stress and insect damage as well as by serving as the basis for genome editing of the flowering-time trait. Conclusion: This annotated reference sequence of wheat is a resource that can now drive disruptive innovation in wheat improvement, as this community resource establishes the foundation for accelerating wheat research and application through improved understanding of wheat biology and genomics-assisted breeding. Importantly, the bioinformatics capacity developed for model-organism genomes will facilitate a better understanding of the wheat genome as a result of the high-quality chromosome-based genome assembly. By necessity, breeders work with the genome at the whole chromosome level, as each new cross involves the modification of genome-wide gene networks that control the expression of complex traits such as yield. With the annotated and ordered reference genome sequence in place, researchers and breeders can now easily access sequence-level information to precisely define the necessary changes in the genomes for breeding programs. This will be realized through the implementation of new DNA marker platforms and targeted breeding technologies, including genome editing

Biochemical characterization of Arabidopsis thaliana DNA Polymerase λ: Role in the oxidative DNA Damage bypass

As obliged phototropic organisms, plants are continuously exposed to high levels of reactive oxygen species (ROS), generated within the cell as a result of the exposure to biotic stresses, sunlight and metabolic activity. The most frequent DNA oxidation damage generated by ROS is the 7,8-dihydro-8-oxo-guanine (8oxoG ) adduct. When present in the replicating DNA strand, this lesion is highly mutagenic, leading to the insertion by the replicative DNA polymerases (pols) of an adenine in place of the correct cytosine. Here we present the first characterization of an error-free mechanism for 8-oxo-G bypass operating in crude extracts of Arabidopsis thaliana. For the first time, our results show that, similarly to what observed in mammalian cells, plant cells possess an efficient pathway for faithful translesion synthesis of oxidative DNA lesions

Functional Analysis of the Degradation of Cellulosic Substrates by a Chaetomium globosum Endophytic Isolate

Most photosynthetically fixed carbon is contained in cell wall polymers present in plant biomasses, the largest organic carbon source in the biosphere. The degradation of these polymers for biotechnological purposes requires the combined action of several enzymes. To identify new activities, we examined which enzymes are activated by an endophytic strain of Chaetomium globosum to degrade cellulose-containing substrates. After biochemical analyses of the secretome of the fungus grown on cellulose or woody substrates, we took advantage of the available genomic data to identify potentially involved genes. After in silico identification of putative genes encoding either proteins able to bind to cellulose or glycohydrolases (GHs) of family 7, we investigated their transcript levels by reverse transcription-quantitative PCR (RT-qPCR). Our data suggest that eight genes compose the core of the cellulose-degrading system of C. globosum. Notably, the related enzymes belong structurally to the well-described GH families 5, 6, 7, 16, and 45, which are known to be the core of the cellulose degradation systems of several ascomycetes. The high expression levels of cellobiose dehydrogenase and two GH 61 enzymes suggest the involvement of this oxidoreductive synergic system in C. globosum. Transcript analysis along with relevant coding sequence (CDS) isolation and expression of recombinant proteins proved to be a key strategy for the determination of the features of two endoglucanases used by C. globosum for the first attack of crystalline cellulose. Finally, the possible involvement of transcriptional regulators described for other ascomycetes is discussed

Interplay between typical and atypical Arabidopsis E2Fs in cell cycle progression and development

Arabidopsis contains six E2F genes: E2Fa-E2Fc possess all the features of typical animal and plant E2Fs while E2Fd-E2Ff are atypical insofar as they are characterized by the presence of a duplicated DNA-binding domain allowing binding to E2F sites without interactions with a DP partner. E2Fa and E2Fb are activators of E2F-dependent genes while E2Fc is a repressor. Arabidopsis plants overexpressing E2Fa (E2FaOE) have shown increased expression of E2Fb while we have recently demonstrated that in E2FbOE plants the content of E2Fa protein was reduced. This suggested an interplay between activator E2Fs in cell cycle progression. We are now analyzing the consequences of downregulating E2Fb. To gain insight into the role of E2Fd/DEL2, during cell division cycle and development, we have overexpressed and silenced E2Fd in transgenic plants. We found that E2Fd exerts a positive influence on the proliferative activity of dividing cells. This unexpected result derives from repression of E2Fc and RBR gene expression and activation of E2Fa. Thus, our data support the notion of a complex interplay between E2F members forming a regulatory network that involves activator as well as atypical E2Fs

CHARACTERIZATION OF THE RESCUED “VOGHERA” SWEET PEPPER LANDRACE GROWN IN NORTHERN ITALY

A traditional Italian sweet pepper landrace, ‘Peperone di Voghera’, which faced the risk of extinction, was analyzed for its genetic, phenological, morphological, agronomic and biochemical traits. An extant population was compared with cultivars ‘Quadrato d’Asti’, ‘Cuneo’, and ‘Giallo d’Asti’, cultivated in the same area, in order to evaluate the chance of the landrace recovery. Amplified fragment length polymorphism (AFLP) analysis demonstrated that Voghera landrace is a distinct population with respect to reference cultivars, thus excluding extensive genetic contamination and providing a molecular basis of both phenological and biochemical differences. Leaf chlorophyll content is lower and fruits start ripening earlier than controls; and yield does not significantly differ. Sensitivity to root pathogens, the main factor that led to the decline in the past, does not seem to compromise the future chance of recovery. Vitamin C concentration is high and preserved by cold storage; a good amount of vitamin C is also kept in pickled fruits. ‘Voghera’ landrace has nutritional and gastronomic properties that can be appreciated by consumers. The high internal genetic variability shown by AFLP analysis can allow future selection work necessary to fully maintain the original traits of the landrace and to improve it