The 'non-conventional' peptidome: A new layer in plant regulatory mechanisms.

A substantial but largely unexplored fraction of eukaryotic proteomes is composed of peptides and small proteins (the peptidome). In recent years, short open reading frames (sORFs) capable of encoding functional peptides have been identified within transcripts annotated as non-coding RNAs or in intergenic regions. These sORF-encoded peptides (SEPs) were previously overlooked due to their small size and difficulties in detection, both experimentally and computationally.

Chronology of transcriptome and proteome expression during early Arabidopsis flower development.

The complex gene regulatory landscape underlying early flower development in Arabidopsis has been extensively studied through transcriptome profiling, and gene networks controlling floral organ development have been derived from the analyses of genome-wide binding of key transcription factors. In contrast, the dynamic nature of the proteome during the flower development process is much less understood. In this study, we characterized the floral proteome at different stages during early flower development and correlated it with unbiased transcript expression data.

Peptidomics Methods Applied to the Study of Flower Development.

Understanding the global and dynamic nature of plant developmental processes requires not only the study of the transcriptome, but also of the proteome, including its largely uncharacterized peptidome fraction. Recent advances in proteomics and high-throughput analyses of translating RNAs (ribosome profiling) have begun to address this issue, evidencing the existence of novel, uncharacterized, and possibly functional peptides. To validate the accumulation in tissues of sORF-encoded polypeptides (SEPs), the basic setup of proteomic analyses (i.

Multi-Omics Methods Applied to Flower Development.

Developmental processes in multicellular organisms depend on the proficiency of cells to orchestrate different gene expression programs. Over the past years, several studies of reproductive organ development have considered genomic analyses of transcription factors and global gene expression changes, modeling complex gene regulatory networks. Nevertheless, the dynamic view of developmental processes requires, as well, the study of the proteome in its expression, complexity, and relationship with the transcriptome.

Gene Expression Analysis by Quantitative Real-Time PCR for Floral Tissues.

Real-time, or quantitative, reverse transcription polymerase chain reaction (qRT-PCR) is a powerful method for rapid and reliable quantification of mRNA abundance. Although it has not featured prominently in flower development research in the past, the availability of novel techniques for the synchronized induction of flower development, or for the isolation of cell-specific mRNA populations, suggests that detailed quantitative analyses of gene expression over time and in specific tissues and cell types by qRT-PCR will become more widely used. In this chapter, we discuss specific considerations for studying gene expression by using qRT-PCR, such as the identification of suitable reference genes for the experimental set-up used.

The floral repressors TEMPRANILLO1 and 2 modulate salt tolerance by regulating hormonal components and photo-protection in Arabidopsis.

Members of the plant specific RAV family of transcription factors regulate several developmental and physiological processes. In the model plant Arabidopsis thaliana, the RAV TEMPRANILLO 1 (TEM1) and TEM2 control important phase changes such as the juvenile to adult and the vegetative to reproductive transitions. Besides their known regulatory function in plant development, a transcriptomics analysis of transgenic plants overexpressing TEM1 also revealed overrepresentation of Gene Ontology (GO) categories related to abiotic stress responses.

Overexpression of the vascular brassinosteroid receptor BRL3 confers drought resistance without penalizing plant growth.

Drought represents a major threat to food security. Mechanistic data describing plant responses to drought have been studied extensively and genes conferring drought resistance have been introduced into crop plants. However, plants with enhanced drought resistance usually display lower growth, highlighting the need for strategies to uncouple drought resistance from growth.

Genome-wide analyses for dissecting gene regulatory networks in the shoot apical meristem.

Shoot apical meristem activity is controlled by complex regulatory networks in which components such as transcription factors, miRNAs, small peptides, hormones, enzymes and epigenetic marks all participate. Many key genes that determine the inherent characteristics of the shoot apical meristem have been identified through genetic approaches. Recent advances in genome-wide studies generating extensive transcriptomic and DNA-binding datasets have increased our understanding of the interactions within the regulatory networks that control the activity of the meristem, identifying new regulators and uncovering connections between previously unlinked network components.

Small RNA profiling reveals regulation of Arabidopsis miR168 and heterochromatic siRNA415 in response to fungal elicitors.

Background: Small RNAs (sRNAs), including small interfering RNAs (siRNAs) and microRNAs (miRNAs), have emerged as important regulators of eukaryotic gene expression. In plants, miRNAs play critical roles in development, nutrient homeostasis and abiotic stress responses. Accumulating evidence also reveals that sRNAs are involved in plant immunity.

Dynamics of chromatin accessibility and gene regulation by MADS-domain transcription factors in flower development.

Background: Development of eukaryotic organisms is controlled by transcription factors that trigger specific and global changes in gene expression programs. In plants, MADS-domain transcription factors act as master regulators of developmental switches and organ specification. However, the mechanisms by which these factors dynamically regulate the expression of their target genes at different developmental stages are still poorly understood.

Gene expression analysis by quantitative real-time PCR for floral tissues.

Real-time, or quantitative, reverse transcription polymerase chain reaction (qRT-PCR), is a powerful method for rapid and reliable quantification of mRNA abundance. Although it has not featured prominently in flower development research in the past, the availability of novel techniques for the synchronized induction of flower development, or for the isolation of cell-specific mRNA populations, suggests that detailed quantitative analyses of gene expression over time and in specific tissues and cell types by qRT-PCR will become more widely used. In this chapter, we discuss specific considerations for studying gene expression by using qRT-PCR, such as the identification of suitable reference genes for the experimental setup used.

Identification of Arabidopsis knockout lines for genes of interest.

Determining gene function through reverse genetics has been an important experimental approach in the field of flower development. The method largely relies on the availability of knockout lines for the gene of interest. Insertional mutagenesis can be performed using either T-DNA or transposable elements, but the former has been more frequently employed in Arabidopsis.

Flower development: open questions and future directions.

Almost three decades of genetic and molecular analyses have resulted in detailed insights into many of the processes that take place during flower development and in the identification of a large number of key regulatory genes that control these processes. Despite this impressive progress, many questions about how flower development is controlled in different angiosperm species remain unanswered. In this chapter, we discuss some of these open questions and the experimental strategies with which they could be addressed.

Specification of floral organs in Arabidopsis.

Floral organs are specified by the activities of a small group of transcriptional regulators, the floral organ identity factors. Extensive genetic and molecular analyses have shown that these proteins act as master regulators of flower development, and function not only in organ identity determination but also during organ morphogenesis. Although it is now well established that these transcription factors act in higher order protein complexes in the regulation of transcription, the gene expression programmes controlled by them have remained largely elusive.

Genome-wide profiling of uncapped mRNA.

Gene transcripts are under extensive posttranscriptional regulation, including the regulation of their stability. A major route for mRNA degradation produces uncapped mRNAs, which can be generated by decapping enzymes, endonucleases, and small RNAs. Profiling uncapped mRNA molecules is important for the understanding of the transcriptome, whose composition is determined by a balance between mRNA synthesis and degradation.

FRET-based real-time DNA microarrays.

We present a quantification method for affinity-based DNA microarrays which is based on the real-time measurements of hybridization kinetics. This method, i.e.

Arabidopsis paves the way: genomic and network analyses in crops.

Arabidopsis genomic and network analyses have facilitated crop research towards the understanding of many biological processes of fundamental importance for agriculture. Genes that were identified through genomic analyses in Arabidopsis have been used to manipulate crop traits such as pathogen resistance, yield, water-use efficiency, and drought tolerance, with the effects being tested in field conditions. The integration of diverse Arabidopsis genome-wide datasets in probabilistic functional networks has been demonstrated as a feasible strategy to associate novel genes with traits of interest, and novel genomic methods continue to be developed.

Orchestration of floral initiation by APETALA1.

The MADS-domain transcription factor APETALA1 (AP1) is a key regulator of Arabidopsis flower development. To understand the molecular mechanisms underlying AP1 function, we identified its target genes during floral initiation using a combination of gene expression profiling and genome-wide binding studies. Many of its targets encode transcriptional regulators, including known floral repressors.

Real-time DNA microarray analysis.

We present a quantification method for affinity-based DNA microarrays which is based on the real-time measurements of hybridization kinetics. This method, i.e.

Transcriptome-wide analysis of uncapped mRNAs in Arabidopsis reveals regulation of mRNA degradation.

The composition of the transcriptome is determined by a balance between mRNA synthesis and degradation. An important route for mRNA degradation produces uncapped mRNAs, and this decay process can be initiated by decapping enzymes, endonucleases, and small RNAs. Although uncapped mRNAs are an important intermediate for mRNA decay, their identity and abundance have never been studied on a large scale until recently.

Global expression profiling applied to the analysis of Arabidopsis stamen development.

To obtain detailed information about gene expression during stamen development in Arabidopsis (Arabidopsis thaliana), we compared, by microarray analysis, the gene expression profile of wild-type inflorescences to those of the floral mutants apetala3, sporocyteless/nozzle, and male sterile1 (ms1), in which different aspects of stamen formation are disrupted. These experiments led to the identification of groups of genes with predicted expression at early, intermediate, and late stages of stamen development. Validation experiments using in situ hybridization confirmed the predicted expression patterns.

The transcription factor WIN1/SHN1 regulates Cutin biosynthesis in Arabidopsis thaliana.

The composition and permeability of the cuticle has a large influence on its ability to protect the plant against various forms of biotic and abiotic stress. WAX INDUCER1 (WIN1) and related transcription factors have recently been shown to trigger wax production, enhance drought tolerance, and modulate cuticular permeability when overexpressed in Arabidopsis thaliana. We found that WIN1 influences the composition of cutin, a polyester that forms the backbone of the cuticle.

Redundancy and specialization among plant microRNAs: role of the MIR164 family in developmental robustness.

In plants, members of microRNA (miRNA) families are often predicted to target the same or overlapping sets of genes. It has thus been hypothesized that these miRNAs may act in a functionally redundant manner. This hypothesis is tested here by studying the effects of elimination of all three members of the MIR164 family from Arabidopsis.