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.
SummaryResponse 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.
Transfer cells have specializations that facilitate the transport of solutes across plant exchange surfaces. ZmMRP-1 is a maize (Zea mays) endosperm transfer cellspeciWc transcriptional activator that plays a central role in the regulatory pathways controlling transfer cell diVerentiation 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. SigniWcantly, transfer cells also diVerentiated in these regions suggesting that, independent of species, location or morphological features, transfer cells might diVerentiate in a similar way under the inXuence of conserved induction signals. In planta and yeast experiments showed that the promoter activity is modulated by carbohydrates, glucose being the most eVective inducer. and milligram of total protein X-glucuronide 5-Bromo-4-chloro-3-indolyl-beta-D-glucuronic acid cyclohexylammonium salt ZmMRP-1 KeywordsZea mays Myb related protein 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.
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.
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