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

Abstract

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.

Figure 1

Comparison of ZmTCRRs to plant type-A response regulators. a, phylogenetic tree based on the protein sequences of type-A response regulators from Arabidopsis thaliana, Oryza sativa and Zea mays. Note that OsRR13 and OsRR13H refer to different sequences named similarly in Ito et al. 2006 and Hirose et al. 2007, respectively. NCBI GI numbers for all sequences are supplied in Table 1. The alignment and distance calculations were performed using the MEGA 3.1 software [64] and the Neighbour-Joining algorithm with 1000 bootstrap iterations. The circle indicates the cluster where both ZmTCRRs are located together with four putative flower specific molecules from rice. b, alignment of ZmTCRRs and rice related proteins. The arrowheads indicate conserved residues forming the active site of response regulators, as indicated in the Conserved Domain Database (CDD) [65]. Conservative changes are shown on a dark background, identical residues on a light grey background. The common c-terminal motif specific for this cluster of proteins is underlined.

Figure 2

Expression analysis of ZmTCRR-2. Upper panel, the graph represents the accumulation of the transcript along early development, relative to its level in the first time point tested (3 DAP). Numbers in the X-axis indicate days after pollination, EMB indicates embryo at 20 DAP, T and B indicate top (upper) and bottom (lower) half of the seed, respectively. Lower panel, Comparison of transcript accumulation sites of ZmTCRR-1(a to e) and ZmTCRR-2 (f to J) at 6 (c, h), 10 (a, b, d, f, g, i) and 14 (e, j) days after development, covering the transfer cells differentiation period. Images in a, b, f and g were taken in bright field using 1.25× (a, f) and 2.5 × (b, g) objectives. The condenser diaphragm was closed to increase contrast and visualize the unstained tissue. The hybridisation signal appears as a layer of black spots in the transfer cells. Images in c, d, e, h, i and j are dark field images, the signal is shown as white dots. Sections were counterstained with Calcofluor White to show the tissue structure. En, inner endosperm; Em, embryo; TC, transfer cells; Pd, pedicel. Scale bars represent 1 mm in a, b, f and g and 100 μm in c, d, e, h, i and j.

Figure 3

Expression of TCS components along early seed development. Results are the average and standard deviation of three replicates. Numbers in the X-axis indicate days after pollination. All values are relative to the expression of the housekeeping gene ZmFKBP66. Results are shown as lines and divided into upper (left panels) and lower (right panels) seed halves, except at 3 and 6 DAP, for which the sample represents the whole kernel. The expression in the embryo for each transcript at 20 DAP is represented as a single time point in a bar graphic (g).

Figure 4

Characterization of protein profiles along development. Detection of native peptides in soluble extracts of 3 to 30 DAP kernels. T and B indicate upper (Top) and lower (Bottom) half of the kernel, respectively. a ZmTCRR-2; b ZmHP1 and 3; c ZmHP2; d ZmFKBP66, used as a loading control. The polypeptide with the predicted size is marked by an arrow in each case.

Figure 5

Immunolocation of ZmTCRR-2 and ZmHPs on 10 DAP seeds. Areas squared in the low magnification image are shown on the left after detection with anti-ZmTCRR-2, ZmHP1/3 and ZmHP2 antisera and preimmune sera as indicated below the images. The signal appears as dark/light grey precipitates depending on its intensity. a upper endosperm and aleurone show detection of ZmTCRR-2 and a strong signal from the ZmHP1/3 serum, while ZmHP2 is only weakly detectable. No unspecific signal is visible in the presera treated sections. b transfer cells and transmitting tissue. ZmTCRR-2 is present in the transfer cells (black arrowheads) and in the transmitting tissue located above them, as is the case for ZmHP1/3. Anti-ZmHP2, on the other hand, produces a weaker signal in this area, still clearly distinguishable from the preserum. c placento-chalaza and pedicel. A dark precipitate appears in the crushed placento-chalaza in all sections shown (yellow arrowheads), including presera, which indicates its unspecific nature. Apart from this, no signal from ZmTCRR-2 is detected in the area. Anti-ZmHP1/3, on the other hand, broadly decorates the pedicel specially at the vascular bundles (blue circle), while anti-ZmHP2 labels (apart from the unspecific signal mentioned above) two distinctive patches at the edges of the placento-chalaza. This area at the adgerminal end is shown in the panels for immune serum and preserum (yellow circles). En, inner endosperm; Em, embryo; TCL, transfer cell layer; Pd, pedicel. The scale bars represent 1 mm in the low magnification image and 100 μm in the other images.

Figure 6

Molecular interaction between ZmTCRRs and ZmHPs. a, growth of AH109 yeast cells carrying ZmTCRRs-binding domain and ZmHPs-activation domain fusions on histidine-lacking medium. Upper left image, growth control on non selective medium after 10 days. Upper right image, distribution of ZmHPs-activation domain clones on the interaction plates. Lower left image, interaction plate for ZmTCRR-1 after 5 days; lower right image, interaction plate for ZmTCRR-2 after 10 days. b, quantification of interaction based on galactosidase activity in a 2-hybrid assay in YRG-2 cells. White bars, interaction of the ZmTCRR-1-activation domain fusion protein with ZmHP-binding domain fusions. Grey bars, interaction of the ZmTCRR-2-activation domain peptide with ZmHP-binding domain fusions. A yeast clone carrying only ZmTCRR-1-activation domain was used as to establish the background level. Each bar represents the mean and standard deviation of 3 independent experiments.

Figure 7

Localization of GUS activity driven by the ZmTCRR-1 promoter in Arabidopsis. a, young developing seeds inside the siliques. The signal is concentrated on the chalazal endosperm (Ch-e) in contact with the funiculus, with a fade blue colour visible in the rest of the endosperm (peripheral endosperm, P-e). b, discontinuous staining of vessel bundles in the cotyledon. c, staining at the branching points of the vasculature below the apical meristem in 14 days old seedlings. d, longitudinal section showing one of the stained cells along the cotyledon vasculature and its association to the xylem tracheary elements (Te). e, f, g, transversal sections of vascular bundles at the intercotyledonary node (position indicated by a dashed line in d); sections were stained with toluidine blue (e), fuchsine (f) and ruthenium red (g). The primary cell wall appears pink in e and g, secondary cell wall appears dark blue in e and remains unstained in g. Cytoplasm is stained blue in e and red in f. GUS staining appears in all cases as a light blue signal. Ph, phloem; Pc, procambium; Xy, xylem. The scale bar in d, e, f and g is 20 μm.