Luis M. Muñiz

ACAULIS5 controls Arabidopsis xylem specification through the prevention of premature cell death.

Cell size and secondary cell wall patterning are crucial for the proper functioning of xylem vessel elements in the vascular tissues of plants. Through detailed anatomical characterization of Arabidopsis thaliana hypocotyls, we observed that mutations in the putative spermine biosynthetic gene ACL5 severely affected xylem specification: the xylem vessel elements of the acl5 mutant were small and mainly of the spiral type, and the normally predominant pitted vessels as well as the xylem fibers were completely missing. The cell-specific expression of ACL5 in the early developing vessel elements, as detected by in situ hybridization and reporter gene analyses, suggested that the observed xylem vessel defects were caused directly by the acl5 mutation. Exogenous spermine prolonged xylem element differentiation and stimulated cell expansion and cell wall elaboration in xylogenic cell cultures of Zinnia elegans, suggesting that ACL5 prevents premature death of the developing vessel elements to allow complete expansion and secondary cell wall patterning. This was further supported by our observations that the vessel elements of acl5 seemed to initiate the cell death program too early and that the xylem defects associated with acl5 could be largely phenocopied by induction of premature, diphtheria toxin-mediated cell death in the ACL5-expressing vessel elements. We therefore provide, for the first time, mechanistic evidence for the function of ACL5 in xylem specification through its action on the duration of xylem element differentiation.

The Maize Transcription Factor Myb-Related Protein-1 Is a Key Regulator of the Differentiation of Transfer Cells

Transfer cells are highly modified plant cells specialized in the transport of solutes. They differentiate at many plant exchange surfaces, including phloem loading and unloading zones such as those present in the sink organs and seeds. In maize (Zea mays) seeds, transfer cells are located at the base of the endosperm. It is currently unknown how apical-basal polarity is established or why the peripheral cells at the base of the endosperm differentiate into transfer instead of aleurone cells. Here, we show that in epidermal cells committed to develop into aleurone cells, the ectopic expression of the transfer cell-specific transcriptional activator Myb-Related Protein-1 (MRP-1) is sufficient to temporarily transform them into transfer cells. These transformed cells acquire distinct transfer cell features, such as cell wall ingrowths and an elongated shape. In addition, they express a number of MRP-1 target genes presumably involved in defense. We also show that the expression of MRP-1 is needed to maintain the transfer cell phenotype. Later in development, an observed reduction in the ectopic expression of MRP-1 was followed by the reversion of the transformed cells, which then acquire aleurone cell features.

A protective role for the embryo surrounding region of the maize endosperm, as evidenced by the characterisation of ZmESR-6, a defensin gene specifically expressed in this region

A Zea mays cDNA clone, ZmESR-6, was isolated as a gene specifically expressed at the basal region of immature kernels. ZmESR-6 cDNA encoded for a small (11.1 kDa) protein homologous to plant defensins. As for other defensins, the protein contained an N-terminal signal peptide signature and a C-terminal acidic peptide, the mature peptide has a molecular mass of 5.5 kDa. ZmESR-6 was highly expressed in developing kernels but the transcript could not be detected in any other maize tissue. The recombinant ZmESR-6 protein, purified from E. coli, showed strong in vitro inhibitory activity against bacterial and fungal plant pathogens, suggesting a role for ZmESR-6 in plant defence. The distribution of the transcripts was restricted to the embryo surrounding region (ESR) of the kernel. Immunolocalisation experiments revealed, however, that at the grain filling phase ZmESR-6 was accumulated in the placentochalaza-cells, rather than in the ESR cells that produce it. Our results suggest that the ESR has a role in protecting the embryo at the very early stages of seed development, whilst contributes to the general defence mechanism of the kernel at later developmental stages.

Atypical response regulators expressed in the maize endosperm transfer cells link canonical two component systems and seed biology

BackgroundTwo component systems (TCS) are phosphotransfer-based signal transduction pathways first discovered in bacteria, where they perform most of the sensing tasks. They present a highly modular structure, comprising a receptor with histidine kinase activity and a response regulator which regulates gene expression or interacts with other cell components. A more complex framework is usually found in plants and fungi, in which a third component transfers the phosphate group from the receptor to the response regulator. They play a central role in cytokinin mediated functions in plants, affecting processes such as meristem growth, phyllotaxy, seed development, leaf senescence or tissue differentiation. We have previously reported the expression and cellular localization of a type A response regulator, ZmTCRR-1, in the transfer cells of the maize seed, a tissue critical for seed filling and development, and described its regulation by a tissue specific transcription factor. In this work we investigate the expression and localization of other components of the TCS signalling routes in the maize seed and initiate the characterization of their interactions.ResultsThe discovery of a new type A response regulator, ZmTCRR-2, specifically expressed in the transfer cells and controlled by a tissue specific transcription factor suggests a previously unknown role for TCS in the biology of transfer cells. We have characterized other canonical TCS molecules, including 6 histidine kinases and 3 phosphotransfer proteins, potentially involved in the atypical transduction pathway defined by ZmTCRR-1 and 2. We have identified potential upstream interactors for both proteins and shown that they both move into the developing endosperm. Furthermore, ZmTCRR-1 expression in an heterologous system (Arabidopsis thaliana) is directed to xylem parenchyma cells, probably involved in transport processes, one of the major roles attributed to the transfer cell layer.ConclusionsOur data prove the expression of the effector elements of a TCS route operating in the transfer cells under developmental control. Its possible role in integrating external signals with seed developmental processes is discussed.

Transcriptional activation of the maize endosperm transfer cell-specific gene BETL1 by ZmMRP-1 is enhanced by two C2H2 zinc finger-containing proteins

ZmMRP-1 is a single MYB-domain transcription factor specifically expressed in the transfer cell layer of the maize endosperm, where it directly regulates the expression of a number of transfer cell specific genes and very likely contributes to the regulation of the transfer cell differentiation process. It is still a matter of debate, however, how this type of transcription factors interact with the promoter sequences they regulate. In this work we have investigated the existence of proteins interacting with ZmMRP-1 in the transfer cell nuclei. In a yeast double-hybrid screen we identified two related maize proteins, ZmMRPI-1 and ZmMRPI-2 belonging to the C2H2 zinc finger protein family, which interact with ZmMRP-1 and modulate its activity on transfer cell specific promoters. Two ZmMRPI orthologous genes were also identified in the rice and Arabidopsis genomes. The expression pattern in maize and Arabidopsis suggest a role for these proteins in gene regulation at the exchange surfaces where ZmMRP-1 is expressed providing the first indication of their function. We show that this previously uncharacterized family of proteins encodes nuclear proteins that interact with MYB-related transcription factors through their C-terminal conserved domain.

A PCR-based forward genetics screening, using expression domain-specific markers, identifies mutants in endosperm transfer cell development.

Mutant collections are an invaluable source of material on which forward genetic approaches allow the identification of genes affecting a wide variety of biological processes. However, some particular developmental stages and morphological structures may resist analysis due to their physical inaccessibility or to deleterious effects associated to their modification. Furthermore, lethal mutations acting early in development may escape detection. We have approached the characterization of 101 maize seed mutants, selected from a collection of 27,500 visually screened Mu-insertion lines, using a molecular marker approach based on a set of genes previously ascribed to different tissue compartments within the early developing kernel. A streamlined combination of qRT-PCR assays has allowed us to preliminary pinpoint the affected compartment, establish developmental comparisons to WT siblings and select mutant lines with alterations in the different compartments. Furthermore, clusters of markers co-affected by the underlying mutation were identified. We have analyzed more extensively a set of lines presenting significant variation in transfer cell-associated expression markers, and have performed morphological observations, and immunolocalization experiments to confirm the results, validating this approach as an efficient mutant description tool.

Cell wall invertase activity regulates the expression of the transfer cell-specific transcription factor ZmMRP-1

AbstractMain conclusionStudies in cell wall bound invertase mutants indicate that the promoter of the transfer cell-specific transcription factor,ZmMRP-1, is modulated by the carbohydrate balance. Transfer cells are highly specialized plant cells located at the surfaces that need to support an intensive exchange of nutrients, such as the entrance of fruits, seeds and nodules or the young branching points along the stem. ZmMRP-1 is a one-domain MYB-related transcription factor specifically expressed at the transfer cell layer of the maize endosperm. Previous studies demonstrated that this factor regulates the expression of a large number of transfer cell-specific genes, and suggested that ZmMRP-1 is a key regulator of the differentiation of this tissue. The expression of this gene is largely dominated by positional cues, but within the ZmMRP-1 expressing cells the promoter appears to be modulated by sugars. Here we have investigated in vivo this modulation. Using maize and Arabidopsis mutants for cell wall invertase genes, we found that the absence of cell wall invertase activity is a major inductive signal of the ZmMRP-1 expression.

Robust and efficient genotyping of factor V and prothrombin alleles using amplicon high-resolution melting and synthetic DNA controls: Validation of the test with three different commercial chemistries

OBJECTIVES Developing robust HRM (amplicon High Resolution Melting) analysis valid for different commercial reaction mixes, using synthetic control DNA samples and the RotorGeneQ (Qiagen) instrument. DESIGN AND METHODS 126 samples were analyzed for the presence of the factor Leiden and the 20210G>A prothrombin alleles. The four alleles were cloned and used to prepare synthetic controls. RESULTS All mutant alleles present in the sample were successfully detected. Genotyping confidence mean was higher than 95%. CONCLUSIONS Cost effective HRM genotyping is very reliable using synthetic control DNAs and the RotorGenQ instrument.

zmsbt1 and zmsbt2, two new subtilisin-like serine proteases genes expressed in early maize kernel development

AbstractMain conclusionTwo subtilisin-like proteases show highly specific and complementary expression patterns in developing grains. These genes label the complete surface of the filial–maternal interface, suggesting a role in filial epithelial differentiation. The cereal endosperm is the most important source of nutrition and raw materials for mankind, as well as the storage compartment enabling initial growth of the germinating plantlets. The development of the different cell types in this tissue is regulated environmentally, genetically and epigenetically, resulting in the formation of top–bottom, adaxial–abaxial and surface–central axes. However, the mechanisms governing the interactions among the different inputs are mostly unknown. We have screened a kernel cDNA library for tissue-specific transcripts as initial step to identify genes relevant in cell differentiation. We report here on the isolation of two maize subtilisin-related genes that show grain-specific, surficial expression. zmsbt1 (Zea mays Subtilisin1) is expressed at the developing aleurone in a time-regulated manner, while zmsbt2 concentrates at the pedicel in front of the endosperm basal transfer layer. We have shown that their presence, early in the maize caryopsis development, is dependent on proper initial tissue determination, and have isolated their promoters to produce transgenic reporter lines that assist in the study of their regulation.