PAT (Periderm Assessment Toolkit): A Quantitative and Large-Scale Screening Method for Periderm Measurements.

The periderm is a vital protective tissue found in the roots, stems, and woody elements of diverse plant species. It plays an important function in these plants by assuming the role of the epidermis as the outermost layer. Despite its critical role for protecting plants from environmental stresses and pathogens, research on root periderm development has been limited due to its late formation during root development, its presence only in mature root regions, and its impermeability. One of the most straightforward measurements for comparing periderm formation between different genotypes and treatments is periderm (phellem) length. We have developed PAT (Periderm Assessment Toolkit), a high-throughput user-friendly pipeline that integrates an efficient staining protocol, automated imaging, and a deep-learning-based image analysis approach to accurately detect and measure periderm length in the roots of Arabidopsis thaliana. The reliability and reproducibility of our method was evaluated using a diverse set of 20 Arabidopsis natural accessions. Our automated measurements exhibited a strong correlation with human-expert-generated measurements, achieving a 94% efficiency in periderm length quantification. This robust PAT pipeline streamlines large-scale periderm measurements, thereby being able to facilitate comprehensive genetic studies and screens. Although PAT proves highly effective with automated digital microscopes in Arabidopsis roots, its application may pose challenges with nonautomated microscopy. Although the workflow and principles could be adapted for other plant species, additional optimization would be necessary. While we show that periderm length can be used to distinguish a mutant impaired in periderm development from wild type, we also find it is a plastic trait. Therefore, care must be taken to include sufficient repeats and controls, to minimize variation, and to ensure comparability of periderm length measurements between different genotypes and growth conditions.

HISTONE DEACETYLASE19 Controls Ovule Number Determination and Transmitting Tract Differentiation.

The gynoecium is critical for the reproduction of flowering plants as it contains the ovules and the tissues that foster pollen germination, growth, and guidance. These tissues, known as the reproductive tract (ReT), comprise the stigma, style, and transmitting tract (TT). The ReT and ovules originate from the carpel margin meristem (CMM) within the pistil. SHOOT MERISTEMLESS (STM) is a key transcription factor for meristem formation and maintenance. In all above-ground meristems, including the CMM, local STM downregulation is required for organ formation. However, how this downregulation is achieved in the CMM is unknown. Here, we have studied the role of HISTONE DEACETYLASE 19 (HDA19) in Arabidopsis (Arabidopsis thaliana) during ovule and ReT differentiation based on the observation that the hda19-3 mutant displays a reduced ovule number and fails to differentiate the TT properly. Fluorescence-activated cell sorting coupled with RNA-sequencing revealed that in the CMM of hda19-3 mutants, genes promoting organ development are downregulated while meristematic markers, including STM, are upregulated. HDA19 was essential to downregulate STM in the CMM, thereby allowing ovule formation and TT differentiation. STM is ectopically expressed in hda19-3 at intermediate stages of pistil development, and its downregulation by RNA interference alleviated the hda19-3 phenotype. Chromatin immunoprecipitation assays indicated that STM is a direct target of HDA19 during pistil development and that the transcription factor SEEDSTICK is also required to regulate STM via histone acetylation. Thus, we identified factors required for the downregulation of STM in the CMM, which is necessary for organogenesis and tissue differentiation.

The histone H3.1 variant regulates TONSOKU-mediated DNA repair during replication.

The tail of replication-dependent histone H3.1 varies from that of replication-independent H3.3 at the amino acid located at position 31 in plants and animals, but no function has been assigned to this residue to demonstrate a unique and conserved role for H3.1 during replication. We found that TONSOKU (TSK/TONSL), which rescues broken replication forks, specifically interacts with H3.1 via recognition of alanine 31 by its tetratricopeptide repeat domain. Our results indicate that genomic instability in the absence of ATXR5/ATXR6-catalyzed histone H3 lysine 27 monomethylation in plants depends on H3.1, TSK, and DNA polymerase theta (Pol θ). This work reveals an H3.1-specific function during replication and a common strategy used in multicellular eukaryotes for regulating post-replicative chromatin maturation and TSK, which relies on histone monomethyltransferases and reading of the H3.1 variant.

H3.1K27me1 maintains transcriptional silencing and genome stability by preventing GCN5-mediated histone acetylation.

Epigenetic mechanisms play diverse roles in the regulation of genome stability in eukaryotes. In Arabidopsis thaliana, genome stability is maintained during DNA replication by the H3.1K27 methyltransferases ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) and ATXR6, which catalyze the deposition of K27me1 on replication-dependent H3.1 variants. The loss of H3.1K27me1 in atxr5 atxr6 double mutants leads to heterochromatin defects, including transcriptional de-repression and genomic instability, but the molecular mechanisms involved remain largely unknown. In this study, we identified the transcriptional co-activator and conserved histone acetyltransferase GCN5 as a mediator of transcriptional de-repression and genomic instability in the absence of H3.1K27me1. GCN5 is part of a SAGA-like complex in plants that requires the GCN5-interacting protein ADA2b and the chromatin remodeler CHR6 to mediate the heterochromatic defects in atxr5 atxr6 mutants. Our results also indicate that Arabidopsis GCN5 acetylates multiple lysine residues on H3.1 variants, but H3.1K27 and H3.1K36 play essential functions in inducing genomic instability in the absence of H3.1K27me1. Finally, we show that H3.1K36 acetylation by GCN5 is negatively regulated by H3.1K27me1 in vitro. Overall, this work reveals a key molecular role for H3.1K27me1 in maintaining transcriptional silencing and genome stability in heterochromatin by restricting GCN5-mediated histone acetylation in plants.

Developmental Analysis of Mimulus Seed Transcriptomes Reveals Functional Gene Expression Clusters and Four Imprinted, Endosperm-Expressed Genes.

The double fertilization of the female gametophyte initiates embryogenesis and endosperm development in seeds via the activation of genes involved in cell differentiation, organ patterning, and growth. A subset of genes expressed in endosperm exhibit imprinted expression, and the correct balance of gene expression between parental alleles is critical for proper endosperm and seed development. We use a transcriptional time series analysis to identify genes that are associated with key shifts in seed development, including genes associated with secondary cell wall synthesis, mitotic cell cycle, chromatin organization, auxin synthesis, fatty acid metabolism, and seed maturation. We relate these genes to morphological changes in Mimulus seeds. We also identify four endosperm-expressed transcripts that display imprinted (paternal) expression bias. The imprinted status of these four genes is conserved in other flowering plants, suggesting that they are functionally important in endosperm development. Our study explores gene regulatory dynamics in a species with ab initio cellular endosperm development, broadening the taxonomic focus of the literature on gene expression in seeds. Moreover, it is the first to validate genes with imprinted endosperm expression in Mimulus guttatus, and will inform future studies on the genetic causes of seed failure in this model system.

miR167 limits anther growth to potentiate anther dehiscence.

In flowering plants, anther dehiscence and pollen release are essential for sexual reproduction. Anthers dehisce after cell wall degradation weakens stomium cell junctions in each anther locule, and desiccation creates mechanical forces that open the locules. Either effect or both together may break stomium cell junctions. The microRNA miR167 negatively regulates ARF6 and ARF8, which encode auxin response transcription factors. Arabidopsis mARF6 or mARF8 plants with mutated miR167 target sites have defective anther dehiscence and ovule development. Null mir167a mutations recapitulated mARF6 and mARF8 anther and ovule phenotypes, indicating that MIR167a is the main miR167 precursor gene that delimits ARF6 and ARF8 expression in these organs. Anthers of mir167a or mARF6/8 plants overexpressed genes encoding cell wall loosening functions associated with cell expansion, and grew larger than wild-type anthers did starting at flower stage 11. Experimental desiccation enabled dehiscence of miR167-deficient anthers, indicating competence to dehisce. Conversely, high humidity conditions delayed anther dehiscence in wild-type flowers. These results support a model in which miR167-mediated anther growth arrest permits anther dehiscence. Without miR167 regulation, excess anther growth delays dehiscence by prolonging desiccation.

Dissecting Tissue-Specific Transcriptomic Responses from Leaf and Roots under Salt Stress in Petunia hybrida Mitchell.

One of the primary objectives of plant biotechnology is to increase resistance to abiotic stresses, such as salinity. Salinity is a major abiotic stress and increasing crop resistant to salt continues to the present day as a major challenge. Salt stress disturbs cellular environment leading to protein misfolding, affecting normal plant growth and causing agricultural losses worldwide. The advent of state-of-the-art technologies such as high throughput mRNA sequencing (RNA-seq) has revolutionized whole-transcriptome analysis by allowing, with high precision, to measure changes in gene expression. In this work, we used tissue-specific RNA-seq to gain insight into the Petunia hybrida transcriptional responses under NaCl stress using a controlled hydroponic system. Roots and leaves samples were taken from a continuum of 48 h of acute 150 mM NaCl. This analysis revealed a set of tissue and time point specific differentially expressed genes, such as genes related to transport, signal transduction, ion homeostasis as well as novel and undescribed genes, such as Peaxi162Scf00003g04130 and Peaxi162Scf00589g00323 expressed only in roots under salt stress. In this work, we identified early and late expressed genes in response to salt stress while providing a core of differentially express genes across all time points and tissues, including the trehalose-6-phosphate synthase 1 (TPS1), a glycosyltransferase reported in salt tolerance in other species. To test the function of the novel petunia TPS1 allele, we cloned and showed that TPS1 is a functional plant gene capable of complementing the trehalose biosynthesis pathway in a yeast tps1 mutant. The list of candidate genes to enhance salt tolerance provided in this work constitutes a major effort to better understand the detrimental effects of salinity in petunia with direct implications for other economically important Solanaceous species.

Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida.

Petunia hybrida is a popular bedding plant that has a long history as a genetic model system. We report the whole-genome sequencing and assembly of inbred derivatives of its two wild parents, P. axillaris N and P. inflata S6. The assemblies include 91.3% and 90.2% coverage of their diploid genomes (1.4 Gb; 2n = 14) containing 32,928 and 36,697 protein-coding genes, respectively. The genomes reveal that the Petunia lineage has experienced at least two rounds of hexaploidization: the older gamma event, which is shared with most Eudicots, and a more recent Solanaceae event that is shared with tomato and other solanaceous species. Transcription factors involved in the shift from bee to moth pollination reside in particularly dynamic regions of the genome, which may have been key to the remarkable diversity of floral colour patterns and pollination systems. The high-quality genome sequences will enhance the value of Petunia as a model system for research on unique biological phenomena such as small RNAs, symbiosis, self-incompatibility and circadian rhythms.

Transcriptomic Signature of the SHATTERPROOF2 Expression Domain Reveals the Meristematic Nature of Arabidopsis Gynoecial Medial Domain.

Plant meristems, like animal stem cell niches, maintain a pool of multipotent, undifferentiated cells that divide and differentiate to give rise to organs. In Arabidopsis (Arabidopsis thaliana), the carpel margin meristem is a vital meristematic structure that generates ovules from the medial domain of the gynoecium, the female floral reproductive structure. The molecular mechanisms that specify this meristematic region and regulate its organogenic potential are poorly understood. Here, we present a novel approach to analyze the transcriptional signature of the medial domain of the Arabidopsis gynoecium, highlighting the developmental stages that immediately proceed ovule initiation, the earliest stages of seed development. Using a floral synchronization system and a SHATTERPROOF2 (SHP2) domain-specific reporter, paired with FACS and RNA sequencing, we assayed the transcriptome of the gynoecial medial domain with temporal and spatial precision. This analysis reveals a set of genes that are differentially expressed within the SHP2 expression domain, including genes that have been shown previously to function during the development of medial domain-derived structures, including the ovules, thus validating our approach. Global analyses of the transcriptomic data set indicate a similarity of the pSHP2-expressing cell population to previously characterized meristematic domains, further supporting the meristematic nature of this gynoecial tissue. Our method identifies additional genes including novel isoforms, cis-natural antisense transcripts, and a previously unrecognized member of the REPRODUCTIVE MERISTEM family of transcriptional regulators that are potential novel regulators of medial domain development. This data set provides genome-wide transcriptional insight into the development of the carpel margin meristem in Arabidopsis.

Transcriptomic analysis of Petunia hybrida in response to salt stress using high throughput RNA sequencing.

Salinity and drought stress are the primary cause of crop losses worldwide. In sodic saline soils sodium chloride (NaCl) disrupts normal plant growth and development. The complex interactions of plant systems with abiotic stress have made RNA sequencing a more holistic and appealing approach to study transcriptome level responses in a single cell and/or tissue. In this work, we determined the Petunia transcriptome response to NaCl stress by sequencing leaf samples and assembling 196 million Illumina reads with Trinity software. Using our reference transcriptome we identified more than 7,000 genes that were differentially expressed within 24 h of acute NaCl stress. The proposed transcriptome can also be used as an excellent tool for biological and bioinformatics in the absence of an available Petunia genome and it is available at the SOL Genomics Network (SGN) http://solgenomics.net. Genes related to regulation of reactive oxygen species, transport, and signal transductions as well as novel and undescribed transcripts were among those differentially expressed in response to salt stress. The candidate genes identified in this study can be applied as markers for breeding or to genetically engineer plants to enhance salt tolerance. Gene Ontology analyses indicated that most of the NaCl damage happened at 24 h inducing genotoxicity, affecting transport and organelles due to the high concentration of Na+ ions. Finally, we report a modification to the library preparation protocol whereby cDNA samples were bar-coded with non-HPLC purified primers, without affecting the quality and quantity of the RNA-seq data. The methodological improvement presented here could substantially reduce the cost of sample preparation for future high-throughput RNA sequencing experiments.