Arabidopsis thaliana DOF6 negatively affects germination in non-after ripened seeds and interacts with TCP14

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 andin 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 gene

Lipid remodelling: unravelling the response to cold stress in Arabidopsis and its extremophile relative Eutrema salsugineum

This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License https://creativecommons.org/licenses/by/4.0/ Crown Copyright © 2017 Published by Elsevier Ireland Ltd.Environmental constraints limit the geographic distribution of many economically important crops. Cold stress is an important abiotic stress that affects plant growth and development, resulting in loss of vigour and surface lesions. These symptoms are caused by, among other metabolic processes, the altered physical and chemical composition of cell membranes. As a major component of cell membranes lipids have been recognized as having a significant role in cold stress, both as a mechanical defence through leaf surface protection and plasma membrane remodelling, and as signal transduction molecules. We present an overview integrating gene expression and lipidomic data published so far in Arabidopsis and its relative the extremophile Eutrema salsugineum. This data enables a better understanding of the contribution of the lipidome in determining the ability to tolerate suboptimal temperature conditions. Collectively this information will allow us to identify the key lipids and pathways responsible for resilience, enabling the development of new approaches for crop tolerance to stress.Peer reviewe

Caracterización molecular del factor de transcripción Dof6 de Arabidopsis thaliana

Los factores de transcripción (TFs) de tipo DOF (DNA binding with One Finger) son una familia de proteínas con papeles reguladores en procesos fisiológicos específicos de plantas, entre ellos el desarrollo y germinación de semillas. En semillas de cebada (Hordeum vulgare), nuestro grupo ha descrito cuatro DOFs: BPBF, SAD, HvDof19 y HvDof17, que ejercen un papel regulador en la expresión de genes que codifican hidrolasas en aleurona en germinación y en la de genes que codifican proteínas de reserva en endospermo en desarrollo (1-5). En los promotores de genes inducidos durante la germinación en Arabidopsis thaliana (p.e. Lipasas), se ha observado la presencia de motivos en cis reconocidos por TFs de tipo DOF (5’ T/A-AAAG 3’). Además, el análisis de expresión por RT-qPCR de distintos TFs de tipo DOF, nos ha permitido identificar el gen AtDof6 como un posible regulador negativo de la expresión génica durante la germinación de la semilla de Arabidopsis

bZIP transcription factors in the regulatory networks controlling seed maturation and germination of Arabidopsis thaliana

Se describe la expresión por RTqPCR de los genes que codifican los factores transcripcionales bZIP44 y bZIP9. Asimismo se establece la interacción entre ambas proteínas en el sistema de 2 híbridos de levadura y in planta por complementación bimolecular fluorescente

Regulación transcripcional en semillas de cebada cv. bomi y su mutante alto en lisina RISO-1508

En este trabajo se presenta un estudio de expresión global de los genes diferencialmente expresados (GDE) en Bomi y Riso 1508 en semillas en desarrollo a 15-18 dap (días después de la polinización), mediante hibridaciones de micromatrices de DNA (GeneChip Barley Genome Array, Affymetrix), donde están representados 25500 genes de cebada

The family of DOF transcription factors in Brachypodium distachyon: phylogenetic comparison with rice and barley DOFs and expression profiling

ABSTRACT: Transcription factors (TFs) are proteins that have played a central role both in evolution and in domestication, and are major regulators of development in living organisms. Plant genome sequences reveal that approximately 7% of all genes encode putative TFs. The DOF (DNA binding with One Finger) TF family has been associated with vital processes exclusive to higher plants and to their close ancestors (algae, mosses and ferns). These are seed maturation and germination, light-mediated regulation, phytohormone and plant responses to biotic and abiotic stresses, etc. In Hordeum vulgare and Oryza sativa, 26 and 30 different Dof genes, respectively, have been annotated. Brachypodium distachyon has been the first Pooideae grass to be sequenced and, due to its genomic, morphological and physiological characteristics, has emerged as the model system for temperate cereals, such as wheat and barley. RESULTS: Through searches in the B. distachyon genome, 27 Dof genes have been identified and a phylogenetic comparison with the Oryza sativa and the Hordeum vulgare DOFs has been performed. To explore the evolutionary relationship among these DOF proteins, a combined phylogenetic tree has been constructed with the Brachypodium DOFs and those from rice and barley. This phylogenetic analysis has classified the DOF proteins into four Major Cluster of Orthologous Groups (MCOGs). Using RT-qPCR analysis the expression profiles of the annotated BdDof genes across four organs (leaves, roots, spikes and seeds) has been investigated. These results have led to a classification of the BdDof genes into two groups, according to their expression levels. The genes highly or preferentially expressed in seeds have been subjected to a more detailed expression analysis (maturation, dry stage and germination). CONCLUSIONS: Comparison of the expression profiles of the Brachypodium Dof genes with the published functions of closely related DOF sequences from the cereal species considered here, deduced from the phylogenetic analysis, indicates that although the expression profile has been conserved in many of the putative orthologs, in some cases duplication followed by subsequent divergence may have occurred (neo-functionalization)

El gen DOFG4 de Arabidopsis participa en la regulación de la germinación de la semilla mediada por ABA

La semilla es un órgano clave en el ciclo de vida de las plantas y su capacidad para permanecer en estado de quiescencia y germinar sólo cuando las condiciones ambientales son favorables ha representado una ventaja evolutiva y permitido el desarrollo de la agricultura. La germinación comienza con una absorción de agua que permite la expansión celular y reiniciar el metabolismo. En sentido estricto, la germinación termina cuando la elongación del eje embrionario permite a la radícula penetrar la cubierta de la semilla. Durante la fase postgerminativa, las sustancias de reserva almacenadas en la semilla son utilizadas para el crecimiento de la plántula hasta que ésta es capaz de fotosintetizar. Los ácidos giberélico (GA) y abscísico (ABA) son las dos hormonas más importantes en el control de la germinación. Diferentes factores ambientales y genéticos son capaces de afectar al balance ABA/GA y el descenso de los niveles de ABA e incremento de los de GA durante la germinación reflejan su papel negativo y positivo, respectivamente, en este proceso. En cereales, los genes de hidrolasas que movilizan las sustancias de reserva responden al GA mediante un motivo en cis tripartito conservado llamado GARC que es reconocido por factores de transcripción (TFs) de las familias MYB (R2R3 y R1) y DOF. Sin embargo, el GARC no parece estar presente en los promotores inducibles por GA en dicotiledóneas (Ogawa et al., 2003) y las diferencias en la respuesta a ABA entre la capa de aleurona de cereales y el endospermo de las dicotiledóneas (Penfield et al., 2006), apunta a la existencia de mecanismos de regulación diferentes. Estamos abordando el estudio de estos mecanismos mediante dos aproximaciones diferentes para identificar y caracterizar elementos en cis y factores de transcripción implicados en la germinación de la dicotiledónea Arabidopsis thaliana

Arabidopsis thaliana bZIP44: a transcription factor affecting seed germination and expression of the mannanase-encoding gene AtMAN7.

Endo-β-mannanases (MAN; EC. 3.2.1.78) catalyze the cleavage of β1[RIGHTWARDS ARROW]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

The BdGAMYB protein from Brachypodium distachyon interacts with BdDOF24 and regulates transcription of the BdCathB gene upon seed germination

This document is the Accepted manuscript of the following article: González-Calle, V., Iglesias-Fernández, R., Carbonero, P. et al. Planta (2014) 240: 539. The final publication is available at Springer via: https://doi.org/10.1007/s00425-014-2105-3.Main conclusion: BdDOF24 interacting with BdGAMYB regulates the BdCathB gene upon germination. During barley seed germination, hydrolytic enzymes (α-amylases, proteases, etc.) synthesized in the aleurone layer in response to gibberellins (GA), catalyse the mobilization of storage reserves accumulated in the endosperm during seed maturation. In Brachypodium distachyon, the BdCathB gene that encodes a Cathepsin B-like thiol-protease, orthologous to the wheat Al21 and barley HvCathB, is highly induced in germinating seeds and its expression is regulated by transcription factors (TFs) encoded by genes BdGamyb and BdDof24, orthologous to the barley HvGamyb and BPBF-HvDof24, respectively. Transcripts of both TF genes increase during germination and treatments with abscisic acid (ABA) or paclobutrazol (PAC, an inhibitor of GA biosynthesis) decrease mRNA expression of BdGamyb but do not affect that of BdDof24. Besides, proteins BdDOF24 and BdGAMYB interact in yeast-2 hybrid systems and in plant nuclei, and in transient expression assays in aleurone layers BdDOF24 is a transcriptional repressor and BdGAMYB is an activator of the BdCathB promoter, as occurs with the putative orthologous in barley BPBF-HvDOF24 and HvGAMYB. However, when both TFs are co-bombarded, BdDOF24 enhances the activation driven by BdGAMYB while BPBF-HvDOF24 strongly decreases the HvGAMYB-mediated activation of the BdCathB promoter. The different results obtained when BdDOF24 and BPBF-HvDOF24 interact with BdGAMYB and HvGAMYB are discussed.Peer reviewedFinal Accepted Versio

Mannans and endo-β-mannanases (MAN) in Brachypodium distachyon : Expression profiling and possible role of the BdMAN genes during coleorhiza-limited seed germination

© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.Immunolocalization of mannans in the seeds of Brachypodium distachyon reveals the presence of these polysaccharides in the root embryo and in the coleorhiza in the early stages of germination (12h), decreasing thereafter to the point of being hardly detected at 27h. Concurrently, the activity of endo-β-mannanases (MANs; EC 3.2.1.78) that catalyse the hydrolysis of β-1,4 bonds in mannan polymers, increases as germination progresses. The MAN gene family is represented by six members in the Brachypodium genome, and their expression has been explored in different organs and especially in germinating seeds. Transcripts of BdMAN2, BdMAN4 and BdMAN6 accumulate in embryos, with a maximum at 24-30h, and are detected in the coleorhiza and in the root by in situ hybridization analyses, before root protrusion (germination sensu stricto). BdMAN4 is not only present in the embryo root and coleorhiza, but is abundant in the de-embryonated (endosperm) imbibed seeds, while BdMAN2 and BdMAN6 are faintly expressed in endosperm during post-germination (36-42h). BdMAN4 and BdMAN6 transcripts are detected in the aleurone layer. These data indicate that BdMAN2, BdMAN4 and BdMAN6 are important for germination sensu stricto and that BdMAN4 and BdMAN6 may also influence reserve mobilization. Whether the coleorhiza in monocots and the micropylar endosperm in eudicots have similar functions, is discussed.Peer reviewedFinal Published versio