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The ability of a seed to germinate and establish a plant at the right time of year is of vital importance from an ecological and economical point of view. Due to the fragility of these early growth stages, their swiftness and robustness will impact later developmental stages and crop yield. These traits are modulated by a continuous interaction between the genetic makeup of the plant and the environment from seed production to germination stages. In this review, we have summarized the established knowledge on the control of seed germination from a molecular and a genetic perspective. This serves as a “backbone” to integrate the latest developments in the field. These include the link of germination to events occurring in the mother plant influenced by the environment, the impact of changes in the chromatin landscape, the discovery of new players and new insights related to well-known master regulators. Finally, results from recent studies on hormone transport, signaling, and biophysical and mechanical tissue properties are underscoring the relevance of tissue-specific regulation and the interplay of signals in this crucial developmental process.

Climate change is threatening crop productivity worldwide and new solutions to adapt crops to these environmental changes are urgently needed. Elevated temperatures driven by climate change affect developmental and physiological plant processes that, ultimately, impact on crop yield and quality. Plant roots are responsible for water and nutrients uptake, but changes in soil temperatures alters this process limiting crop growth. With the predicted variable climatic forecast, the development of an efficient root system better adapted to changing soil and environmental conditions is crucial for enhancing crop productivity. Root traits associated with improved adaptation to rising temperatures are increasingly being analyzed to obtain more suitable crop varieties. In this review, we will summarize the current knowledge about the effect of increasing temperatures on root growth and their impact on crop yield. First, we will describe the main alterations in root architecture that different crops undergo in response to warmer soils. Then, we will outline the main coordinated physiological and metabolic changes taking place in roots and aerial parts that modulate the global response of the plant to increased temperatures. We will discuss on some of the main regulatory mechanisms controlling root adaptation to warmer soils, including the activation of heat and oxidative pathways to prevent damage of root cells and disruption of root growth; the interplay between hormonal regulatory pathways and the global changes on gene expression and protein homeostasis. We will also consider that in the field, increasing temperatures are usually associated with other abiotic and biotic stresses such as drought, salinity, nutrient deficiencies, and pathogen infections. We will present recent advances on how the root system is able to integrate and respond to complex and different stimuli in order to adapt to an increasingly changing environment. Finally, we will discuss the new prospects and challenges in this field as well as the more promising pathways for future research.

A key component of seed germination is the interplay of mechanical forces governing embryo growth and the surrounding restraining endosperm tissue. Endosperm cell separation is therefore thought to play a critical role in the control of this developmental transition. Here we demonstrate that in Arabidopsis thaliana seeds, endosperm cell expansion is a key component of germination. Endosperm cells expand to accommodate embryo growth prior to germination. We show that this is an actively regulated process supported by spatiotemporal control of the cell expansion gene EXPANSIN 2 (EXPA2). The NAC transcription factors NAC25 and NAC1L were identified as upstream regulators of EXPA2 expression, gibberellin-mediated endosperm expansion, and seed germination. The DELLA protein RGL2 repressed activation of the EXPA2 promoter by NAC25/NAC1L. Taken together, our findings uncover a key role of the GA/DELLA-NAC25/NAC1L-EXPA2 network in regulating endosperm cell expansion to control the seed-to-seedling transition., This study demonstrates that endosperm cell expansion is crucial for germination and that two NAC transcription factors play pivotal roles in this process. NAC25 and NAC1L act downstream of GAs and the RGL2 DELLA protein to upregulate cell-wall remodeling enzyme gene expression.

Molecular interactions are an integral part of the regulatory mechanisms controlling gene expression. The yeast one- and two-hybrid systems (Y1H/Y2H) have been widely used by many laboratories to detect DNA-protein (Y1H) and protein-protein interactions (Y2H). The development of efficient cloning systems have promoted the generation of large open reading frame (ORF) clone collections (libraries) for several organisms. Functional analyses of such large collections require the establishment of adequate protocols. Here, we describe a simple straightforward procedure for high-throughput screenings of arrayed libraries with DNA or protein baits that can be carried out by one person with minimal labor and not requiring robotics. The protocol can also be scaled up or down and is compatible with several library formats. Procedures to make yeast stocks for long-term storage (tube and microplate formats) are also provided.

Since their original description more than 25 years ago, the yeast one- and two-hybrid systems (Y1H/Y2H) have been used by many laboratories to detect DNA-protein (Y1H) and protein-protein interactions (Y2H). These systems use yeast cells (Saccharomyces cerevisiae) as a eukaryotic "test tube" and are amenable for most labs in the world. The development of highly efficient cloning methods has fostered the generation of large collections of open reading frames (ORFs) for several organisms that have been used for yeast screenings. Here, we describe a simple mating based method for high-throughput screenings of arrayed ORF libraries with DNA (Y1H) or protein (Y2H) baits not requiring robotics. One person can easily carry out this protocol in approximately 10 h of labor spread over 5 days. It can also be scaled down to test one-to-one (few) interactions, scaled up (i.e., robotization) and is compatible with several library formats (i.e., 96, 384-well microtiter plates).

Seed dormancy prevents seeds from germinating under environmental conditions unfavourable for plant growth and development and constitutes an evolutionary advantage. Dry storage, also known as after-ripening, gradually decreases seed dormancy by mechanisms not well understood. An Arabidopsis thaliana DOF transcription factor gene (DOF6) affecting seed germination has been characterized. The transcript levels of this gene accumulate in dry seeds and decay gradually during after-ripening and also upon seed imbibition. While constitutive over-expression of DOF6 produced aberrant growth and sterility in the plant, its over-expression induced upon seed imbibition triggered delayed germination, abscisic acid (ABA)-hypersensitive phenotypes and increased expression of the ABA biosynthetic gene ABA1 and ABA-related stress genes. Wild-type germination and gene expression were gradually restored during seed after-ripening, despite of DOF6-induced over-expression. DOF6 was found to interact in a yeast two-hybrid system and in planta with TCP14, a previously described positive regulator of seed germination. The expression of ABA1 and ABA-related stress genes was also enhanced in tcp14 knock-out mutants. Taken together, these results indicate that DOF6 negatively affects seed germination and opposes TCP14 function in the regulation of a specific set of ABA-related genes.

Genomic approaches have generated large Arabidopsis open reading frame (ORF) collections. However, molecular tools are required to characterize this ORFeome functionally. A high-throughput microtiter plate-based protoplast transactivation (PTA) system has been established that can be used in a screening approach to define which transcription factor (TF) regulates a given promoter in planta. Using to this procedure, the transactivation properties of 96 TFs can be analyzed rapidly, making use of promoter:Luciferase (LUC)-reporters and luciferase imaging. Applying GATEWAY® technology, we have established a platform to assay more than 700 Arabidopsis TFs. As a proof-of-principle, the ethylene response factor (ERF) family has been studied to evaluate this system. Importantly, distinct subsets of related ERF factors were found to activate specifically the well described target promoters RD29A and PDF1.2 that are under control of DRE or GCC box cis-elements, respectively. Furthermore, several applications of the PTA system have been demonstrated, such as analysis of transcriptional repressors, salt-inducible gene expression or functional interaction of signaling molecules like kinases and TFs. This novel molecular tool will improve functional studies on transcriptional regulation in plants significantly.

Genomic approaches have generated large Arabidopsis thaliana open reading frame (ORF) collections. However, tools are required to functionally characterize this ORFeome. Here we describe a batch procedure to simultaneously recombine a population of GATEWAY-tagged full-length cDNAs into a plant expression vector. A pool of agrobacteria carrying these constructs has been used in flower-dip transformation experiments to obtain a collection of transgenic lines that over-express HA-tagged ORFs. This AtTORF-Ex library can be used in various screening approaches to identify particular gene family members involved in plant development or stress responses. The feasibility of the approach was studied using a near-complete collection of the Arabidopsis ethylene response factor (ERF) transcription factor (TF) family. Quality control performed at each step of the procedure revealed that the complexity of the population is maintained, and that almost all members of the ORF collection are covered by the plant library. The frequency of multiple transformation events has been determined as approximately 4%. Significant transgene expression was detected at the RNA and protein level in more than 60% and 30% of the transgenic plants, respectively. Striking phenotypic alterations were observed in approximately 4% of the plants. Many ERF TFs have been shown to participate in plant stress responses. As a proof of principle, the AtTORF-Ex library has been used in a selection procedure to isolate TFs involved in enhanced abiotic stress tolerance. The corresponding TF gene can be easily polymerase chain reaction-amplified using GATEWAY att site-specific primers. In summary, we describe here a method that can be generally applied for functional analysis of ORFeomes in planta.

Endo-β-mannanases (MAN; EC. 3.2.1.78) catalyze the cleavage of β1→4 bonds in mannan polymers and have been associated with the process of weakening the tissues surrounding the embryo during seed germination. In germinating Arabidopsis thaliana seeds, the most highly expressed MAN gene is AtMAN7 and its transcripts are restricted to the micropylar endosperm and to the radicle tip just before radicle emergence. Mutants with a T-DNA insertion in AtMAN7 have a slower germination than the wild type. To gain insight into the transcriptional regulation of the AtMAN7 gene, a bioinformatic search for conserved non-coding cis-elements (phylogenetic shadowing) within the Brassicaceae MAN7 gene promoters has been done, and these conserved motifs have been used as bait to look for their interacting transcription factors (TFs), using as a prey an arrayed yeast library from A. thaliana. The basic-leucine zipper TF AtbZIP44, but not the closely related AtbZIP11, has thus been identified and its transcriptional activation upon AtMAN7 has been validated at the molecular level. In the knock-out lines of AtbZIP44, not only is the expression of the AtMAN7 gene drastically reduced, but these mutants have a significantly slower germination than the wild type, being affected in the two phases of the germination process, both in the rupture of the seed coat and in the breakage of the micropylar endosperm cell walls. In the over-expression lines the opposite phenotype is observed.

International audience; Transcriptional regulation is an important mechanism underlying gene expression and has played a crucial role in evolution. The number, position and interactions between cis-elements and transcription factors (TFs) determine the expression pattern of a gene. To identify functionally relevant cis-elements in gene promoters, a phylogenetic shadowing approach with a lipase gene (LIP1) was used. As a proof of concept, in silico analyses of several Brassicaceae LIP1 promoters identified a highly conserved sequence (LIP1 element) that is sufficient to drive strong expression of a reporter gene in planta. A collection of ca. 1,200 Arabidopsis thaliana TF open reading frames (ORFs) was arrayed in a 96-well format (RR library) and a convenient mating based yeast one hybrid (Y1H) screening procedure was established. We constructed an episomal plasmid (pTUY1H) to clone the LIP1 element and used it as bait for Y1H screenings. A novel interaction with an HD-ZIP (AtML1) TF was identified and abolished by a 2 bp mutation in the LIP1 element. A role of this interaction in transcriptional regulation was confirmed in planta. In addition, we validated our strategy by reproducing the previously reported interaction between a MYB-CC (PHR1) TF, a central regulator of phosphate starvation responses, with a conserved promoter fragment (IPS1 element) containing its cognate binding sequence. Finally, we established that the LIP1 and IPS1 elements were differentially bound by HD-ZIP and MYB-CC family members in agreement with their genetic redundancy in planta. In conclusion, combining in silico analyses of orthologous gene promoters with Y1H screening of the RR library represents a powerful approach to decipher cis- and trans-regulatory codes.

Transcription of Arabidopsis thaliana seed maturation (MAT) genes is controlled by members of several transcription factor families, such as basic leucine zippers (bZIPs), B3s, MYBs, and DOFs. In this work, we identify Arabidopsis bZIP53 as a novel transcriptional regulator of MAT genes. bZIP53 expression in developing seeds precedes and overlaps that of its target genes. Gain- and loss-of-function approaches indicate a correlation between the amount of bZIP53 protein and MAT gene expression. Specific in vivo and in vitro binding of bZIP53 protein to a G-box element in the albumin 2S2 promoter is demonstrated. Importantly, heterodimerization with bZIP10 or bZIP25, previously described bZIP regulators of MAT gene expression, significantly enhances DNA binding activity and produces a synergistic increase in target gene activation. Full-level target gene activation is strongly correlated with the ratio of the correspondent bZIP heterodimerization partners. Whereas bZIP53 does not interact with ABI3, a crucial transcriptional regulator in Arabidopsis seeds, ternary complex formation between the bZIP heterodimers and ABI3 increases the expression of MAT genes in planta. We therefore propose that heterodimers containing bZIP53 participate in enhanceosome formation to produce a dramatic increase in MAT gene transcription.

The expression of many seed storage protein genes in cereals relies on transcription factors of the bZIP class, belonging to the maize OPAQUE2 family. Here, we describe a survey of such factors in the genome of Arabidopsis thaliana, and the characterization of two of them, AtbZIP10 and AtbZIP25. Expression analysis by in situ hybridization shows that the occurrence of their mRNAs in the seed starts from early stages of development, peaks at maturation, and declines later in seed development, matching temporally and spatially those of the seed storage protein genes encoding 2S albumins and cruciferins. Gel mobility shift assays showed that AtbZIP10 and AtbZIP25 bind the ACGT boxes present in At2S and CRU3 promoters. Moreover, using the yeast two-hybrid system we show that AtbZIP10 and AtbZIP25 can interact in vivo with ABI3, an important regulator of gene expression in the seed of Arabidopsis. Transient expression analyses of a reporter gene under the control of the At2S1 promoter in transgenic plants overexpressing ectopically AtbZIP10, AtbZIP25, and ABI3 reveal that none of these factors could activate significantly the reporter gene when expressed individually. However, co-expression of AtbZIP10/25 with ABI3 resulted in a remarkable increase in the activation capacity over the At2S1 promoter, suggesting that they are part of a regulatory complex involved in seed-specific expression. This study shows a common mechanism of ABI3 in regulating different seed-specific genes through combinatorial interactions with particular bZIP proteins and a conserved role of O2-like bZIPs in monocot and dicot species.

Background High throughput applications of the reverse transcriptase quantitative PCR (RT-qPCR) for quantification of gene expression demand straightforward procedures to isolate and analyze a considerable number of DNA-free RNA samples. Published protocols are labour intensive, use toxic organic chemicals and need a DNase digestion once pure RNAs have been isolated. In addition, for some tissues, the amount of starting material may be limiting. The convenience of commercial kits is often prohibitive when handling large number of samples. Findings We have established protocols to isolate DNA-free RNA from Arabidopsis thaliana tissues ready for RT-qPCR applications. Simple non-toxic buffers were used for RNA isolation from Arabidopsis tissues with the exception of seeds and siliques, which required the use of organic extractions. The protocols were designed to minimize the number of steps, labour time and the amount of starting tissue to as little as 10–20 mg without affecting RNA quality. In both protocols genomic DNA (gDNA) can be efficiently removed from RNA samples before the final alcohol precipitation step, saving extra purification steps before cDNA synthesis. The expression kinetics of previously characterized genes confirmed the robustness of the procedures. Conclusion Here, we present two protocols to isolate DNA-free RNA from Arabidopsis tissues ready for RT-qPCR applications that significantly improve existing ones by reducing labour time and the use of organic extractions. Accessibility to these protocols is ensured by its simplicity and the low cost of the materials used.