Joaquín Royo

Evidence for factors regulating transfer cell-specific expression in maize endosperm.

In maize, a layer of basal endosperm cells adjacent to the pedicel is modified for a function in solute transfer. Three genes specifically expressed in this region, termed the basal endosperm transfer layer (BETL-2 to -4), were isolated by differential hybridization. BETL-2 to -4 are coordinately expressed in early and mid-term endosperm development, but are absent at later stages. BETL-2 to -4 coding sequences all predict small (<100 amino acids), secreted, cysteine-rich polypeptides which lack close relatives in current database accessions. BETL-3 and BETL-1 display some sequence similarities with each other and to plant defensins. BETL-2 to -4 promoter regions were isolated and compared, revealing the presence of a promoter-proximal microsatellite repeat as the most highly conserved sequence element in each sequence. Electrophoretic mobility shift assays (EMSA) showed that specific BETL-2 to -4 promoter fragments competed for binding to the same DNA-binding activity in nuclear extracts prepared from maize endosperm. Although BETL-2 to -4 are only expressed in basal endosperm cells, the DNA-binding activities detected were of two types: distal endosperm-specific, or present in both basal and distal endosperm extracts. On the basis of these findings, a model to account for the coordinate regulation of BETL genes in endosperm cells is proposed.

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.

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.

A Maize Homologue of the Bacterial CMP-3-Deoxy-d-manno-2-octulosonate (KDO) Synthetases SIMILAR PATHWAYS OPERATE IN PLANTS AND BACTERIA FOR THE ACTIVATION OF KDO PRIOR TO ITS INCORPORATION INTO OUTER CELLULAR ENVELOPES

The eight-carbon acid sugar 3-deoxy-d-manno-2-octulosonate (KDO) is an essential component of Gram-negative bacterial cell walls and capsular polysaccharides. KDO is incorporated into these polymers as CMP-KDO, which is produced in an unusual activation step catalyzed by the enzyme CMP-KDO synthetase. CMP-KDO synthetase activity has traditionally been considered exclusive to Gram-negative bacteria. CMP-KDO synthetase inhibitors attract great interest owing to their potential as selective bactericides. The sugar KDO is also a component of the rhamnogalacturonan II pectin fraction of the primary cell walls of most higher plants and of the cell wall polysaccharides of some green algae. However, the metabolic pathway leading to its incorporation into the plant cell wall is unknown. This paper describes the isolation and characterization of a maize gene, which codes for a protein very similar in sequence and activity to prokaryotic CMP-KDO synthetases. Remarkably, the maize gene can complement a CMP-KDO synthetase (kdsB) Salmonella typhimurium mutant defective in cell wall synthesis. ZmCKS activity is novel in eukaryotes. The evolutionary origin of ZmCKS is discussed in relation to the high degree of conservation between the plant and bacterial genes and its atypical codon usage in maize.

Isolation and promoter characterization of barley geneItr1 encoding trypsin inhibitor BTI-CMe: differential activity in wild-type and mutantlys3a endosperm

The geneItr1, encoding trypsin inhibitor BTI-CMe, has been obtained from a genomic library ofHordeum vulgare L. The gene has no introns and presents in its 5′-upstream region 605 bp that are homologous to the long terminal repeats (LTR) of the ‘copia-like’ retro-transposon Bare-1. Functional analysis of theItr1 promoter by transient expression in protoplasts derived from different barley tissues, has shown that in this system theItr1 promoter retains its endosperm specifity and thetrans-regulation mediated by theLys3a gene. The proximal promoter extending 343 bp upstream of the translation initiation ATG codon is sufficient to confer fullGUS expression and for endosperm specifity. In protoplasts derived from thelys3a mutant, Risø 1508,GUS activity was less than 5% of that obtained with the same constructs in the protoplasts of wild-type Bomi from which it derives. Gel retardation experiments, after incubation with proteins obtained from both types of endosperm nuclei, also show differential patterns. Possible reasons for these differences are discussed.

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.

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.

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.