Distinct Hormone Signalling-Modulation Activities Characterize Two Maize Endosperm-Specific Type-A Response Regulators.

ZmTCRR1 and 2 are type-A response regulators expressed in the maize endosperm transfer cells (TC). While type-B response regulators transcriptionally control canonical type-A response regulators, as part of the cytokinin signal transduction mechanism, the ZmTCRRs are regulated by ZmMRP1, a master regulator of TC identity. In addition, the corresponding proteins are not detected in the TC, accumulating in the inner endosperm cells instead. These features suggest these molecules are not involved in classical, cell-autonomous, cytokinin signalling pathways. Using transgenic Arabidopsis plants ectopically expressing these genes, we have shown that ZmTCRR1 and 2 can modulate auxin and cytokinin signalling, respectively. In Arabidopsis, the ectopic expression of ZmTCRR2 blocked, almost completely, cytokinin perception. Given the conservation of these signalling pathways at the molecular level, our results suggest that the ZmTCRRs modulate cytokinin and auxin perception in the inner endosperm cells.

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

MAIN CONCLUSION: Studies 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.

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

MAIN CONCLUSION: Two 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.

A putative plant organelle RNA recognition protein gene is essential for maize kernel development.

Basal endosperm transfer layer (BETL) cells are responsible for transferring apoplastic solutes from the maternal pedicel into the endosperm, supplying the grain with compounds required for embryo development and storage reserve accumulation. Here, we analyze the maize (Zea mays L.) empty pericarp6 (emp6) mutant, which causes early arrest in grain development. The Emp6+gene function is required independently in both the embryo and endosperm. The emp6 mutant causes a notable effect on the differentiation of BETL cells; the extensive cell wall ingrowths that distinguish BETL cells are diminished and BETL marker gene expression is compromised in mutant kernels. Transposon tagging identified the emp6 locus as encoding a putative plant organelle RNA recognition (PORR) protein, 1 of 15 PORR family members in maize. The emp6 transcript is widely detected in plant tissues with highest levels in embryos and developing kernels. EMP6-green fluorescent protein (GFP) fusion proteins transiently expressed in Nicotiana benthamiana leaves were targeted specifically to mitochondria. These results suggest that BETL cell differentiation might be particularly energy intensive, or alternatively, that mitochondria might confer a developmental function.

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.

Two maize END-1 orthologs, BETL9 and BETL9like, are transcribed in a non-overlapping spatial pattern on the outer surface of the developing endosperm.

In the course of a project aimed to isolate transfer cells-specific genes in maize endosperm we have identified the BETL9 gene. BETL9 encodes for a small protein very similar in sequence to the product of the barley transfer cell-specific gene END-1. Both BETL9 and END-1 proteins are lipid transfer proteins, but their function is currently unknown. In situ hybridization analysis confirms that the BETL9 gene is exclusively transcribed in the basal endosperm transfer cell layer during seed development since 10 days after pollination. However, immunolocalization data indicates that the BETL9 protein accumulates in the maternal placento-chalaza cells located just beside the transfer cell layer. This suggests that the BETL9 protein should be transported to the maternal side to exert its, still unknown, function. In addition, we have identified a second maize gene very similar in sequence to BETL9 and we have named it BETL9like. In situ hybridization shows that BETL9like is also specifically transcribed in the developing maize endosperm within the same time frame that BETL9, but in this case it is exclusively expressed in the aleurone cell layer. Consequently, the BETL9 and BETL9like genes are transcribed in a non-overlapping pattern on the outer surface of the maize endosperm. The BETL9 and BETL9like promoter sequences, fused to the GUS reporter gene, accurately reflected the expression pattern observed for the genes in maize. Finally, we have identified in the Arabidopsis genome a set of four genes orthologous to BETL9 and BETL9like and analyzed the activity of their promoters in Arabidopsis transgenic plants carrying fusions of their promoter sequences to the GUS reporter. As in the case of the maize genes, the Arabidopsis orthologs showed highly complementary expression patterns.

Maize rough endosperm3 encodes an RNA splicing factor required for endosperm cell differentiation and has a nonautonomous effect on embryo development.

Endosperm and embryo development are coordinated via epigenetic regulation and signaling between these tissues. In maize (Zea mays), the endosperm-embryo signals are not known, but endosperm cellularization is a key event for embryos to form shoots and roots. We screened seed mutants for nonautonomous functions in endosperm and embryo development with genetically nonconcordant seeds and identified the recessive mutant rough endosperm3 (rgh3). The wild-type Rgh3 allele is required in the endosperm for embryos to develop and has an autonomous role in embryo and seedling development. Endosperm cell differentiation is defective in rgh3. Results from endosperm cell culture indicate that rgh3 mutants remain in a proliferative state through mid-seed development. Rgh3 encodes the maize U2AF(35) Related Protein (URP), an RNA splicing factor involved in both U2 and U12 splicing. The Rgh3 allele produces at least 19 alternative splice variants with only one isoform encoding a full-length ortholog to URP. The full-length RGH3α isoform localizes to the nucleolus and displays a speckled pattern within the nucleoplasm, and RGH3α colocalizes with U2AF(65). A survey of alternatively spliced transcripts found that, in the rgh3 mutant, a fraction of noncanonical splicing events are altered. Our findings suggest that differentiation of maize endosperm cell types is necessary for embryos to develop. The molecular cloning of Rgh3 suggests that alternative RNA splicing is needed for cell differentiation, development, and plant viability.

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.

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

BACKGROUND: Two 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. RESULTS: The 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. CONCLUSIONS: Our 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 C(2)H(2) 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.

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.

The promoter of ZmMRP-1, a maize transfer cell-specific transcriptional activator, is induced at solute exchange surfaces and responds to transport demands.

Transfer cells have specializations that facilitate the transport of solutes across plant exchange surfaces. ZmMRP-1 is a maize (Zea mays) endosperm transfer cell-specific transcriptional activator that plays a central role in the regulatory pathways controlling transfer cell differentiation and function. The present work investigates the signals controlling the expression of ZmMRP-1 through the production of transgenic lines of maize, Arabidopsis, tobacco and barley containing ZmMRP-1promoter:GUS reporter constructs. The GUS signal predominantly appeared in regions of active transport between source and sink tissues, including nematode-induced feeding structures and at sites of vascular connection between developing organs and the main plant vasculature. In those cases, promoter induction was associated with the initial developmental stages of transport structures. Significantly, transfer cells also differentiated in these regions suggesting that, independent of species, location or morphological features, transfer cells might differentiate in a similar way under the influence of conserved induction signals. In planta and yeast experiments showed that the promoter activity is modulated by carbohydrates, glucose being the most effective inducer.

Molecular dissection of the interaction between the transcriptional activator ZmMRP-1 and the promoter of BETL-1.

The interaction between the transfer cell specific transcriptional activator ZmMRP-1 and the promoter of the transfer cell specific gene BETL-1 constitutes an exceptionally robust system. Reporter constructs containing the BETL-1 promoter are virtually silent in a variety of cell types, from maize leaves to yeast. The introduction of ZmMRP-1 in co-transformation assays leads to the transactivation of the reporter construct by up to two orders of magnitude. In this work we have investigated the molecular basis of this interaction. We found that the BETL-1 promoter includes four potential targets for ZmMRP-1 binding, consisting of a 12 bp motif containing two repeats. Co-transformation assays and electrophoretic mobility shift experiments identified the sequence TATCTCTATCTC as the preferred one for the interaction with the transcription factor. Identification of similar sequences in other transfer cell specific promoters lead us to propose as a transfer cell box a sequence related to those identified in the BETL-1 promoter, positioned 50-100 bp upstream the TATA box.

The maize transfer cell-specific type-A response regulator ZmTCRR-1 appears to be involved in intercellular signalling.

Response regulators are signal-transduction molecules present in bacteria, yeast and plants, acting as relays for environmental challenges. This paper reports the characterization of a Zea mays gene, ZmTCRR-1, that codes for a member of the type-A response regulator class of proteins. The gene was found to be expressed exclusively in the endosperm transfer-cell layer 8-14 days after pollination, when transfer-cell differentiation is most active. The promoter of ZmTCRR-1 was strongly transactivated in heterologous systems by the transfer cell-specific transcription factor ZmMRP-1. The ZmTCRR-1 protein was detected not only in the transfer-cell layer, but also in the conductive tissue deep inside the endosperm, where there is no transcription of the gene. This suggests that two-component systems might be involved in intercellular signal transmission, in contrast to the generally held belief that these systems are involved only in cell-autonomous pathways.

ZmLrk-1, a receptor-like kinase induced by fungal infection in germinating seeds.

We report here on the identification and characterization of ZmLrk-1, a member of the Lrk class of receptor-like kinases in Zea mays. This gene was found to be located at the bin21.40 region on the short arm of maize chromosome 8, closely linked to the previously reported pseudogene of the same class psiZmLrk (originally called Zm2Lrk). Transient expression experiments in onion epithelium cells, using a ZmLrk-1:GFP fusion protein, indicate that ZmLrk-1 is a membrane protein. ZmLrk-1 is ubiquitously expressed in the maize plant, including roots and aerial parts. In seeds, ZmLrk-1 transcripts can be detected by in situ hybridization exclusively at the basal endosperm transfer cell layer during the first stages of development. However, from 14 days after pollination its transcripts are preferentially detected at the upper half of the kernel, including both the aleurone and the starchy endosperm. ZmLrk-1 expression is not induced after treatment with salicylic acid, jasmonic acid or wounding, but it clearly increases after infection of germinating seeds with Fusarium oxysporum. This suggests that ZmLrk-1 could be involved in a sensing system to activate plant defence mechanisms against fungal attacks during endosperm development and seed germination.

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.

Establishment of cereal endosperm expression domains: identification and properties of a maize transfer cell-specific transcription factor, ZmMRP-1.

In maize, cells at the base of the endosperm are transformed into transfer cells that facilitate nutrient uptake by the developing seed. ZmMRP-1 is the first transfer cell-specific transcriptional activator to be identified. The protein it encodes contains nuclear localization signals and a MYB-related DNA binding domain. A single gene copy is present in maize, mapping to a locus on chromosome 8. ZmMRP-1 is first expressed soon after fertilization, when the endosperm is still a multinuclear coenocyte. The transcript accumulates in the basal nucleocytoplasmic domain that gives rise to transfer cells after cellularization. The transcript can be detected throughout transfer cell development, but it is not found in mature cells. ZmMRP-1 strongly transactivates the promoters of two unrelated transfer cell-specific genes. The properties of ZmMRP-1 are consistent with it being a determinant of transfer cell-specific expression. Possible roles for ZmMRP-1 in the regulation of endosperm and transfer cell differentiation are discussed.