Down-regulation of ZmEXPB6 (Zea mays β-expansin 6) protein is correlated with salt-mediated growth reduction in the leaves of Z. mays L

Abstract

The salt-sensitive crop Zea mays L. shows a rapid leaf growth reduction upon NaCl stress. There is increasing evidence that salinity impairs the ability of the cell walls to expand, ultimately inhibiting growth. Wall-loosening is a prerequisite for cell wall expansion, a process that is under the control of cell wall-located expansin proteins. In this study the abundance of those proteins was analyzed against salt stress using gel-based two-dimensional proteomics and two-dimensional Western blotting. Results show that ZmEXPB6 (Z. mays β-expansin 6) protein is lacking in growth-inhibited leaves of salt-stressed maize. Of note, the exogenous application of heterologously expressed and metal-chelate-affinity chromatography-purified ZmEXPB6 on growth-reduced leaves that lack native ZmEXPB6 under NaCl stress partially restored leaf growth. In vitro assays on frozen-thawed leaf sections revealed that recombinant ZmEXPB6 acts on the capacity of the walls to extend. Our results identify expansins as a factor that partially restores leaf growth of maize in saline environments.

Keywords: Cell Growth; Growth Inhibition; Linear Variable Differential Transducer; Plant; Plant Biochemistry; Plant Cell Wall; Real-time Fluorescence Ratio Imaging; Salinity; Stress; Zea mays L.

FIGURE 1.

Two-dimensional SDS-PAGE gel of Z. mays L leaf. Separation was achieved by isoelectric focusing (pH 3–11) in the horizontal dimension and by SDS-PAGE in the vertical dimension. The spots were visualized by staining with Coomassie Blue R250.

FIGURE 2.

His tag-labeled metal-chelate-affinity chromatography-purified ZmEXPB6 expressed in E. coli. Cell lysate, cells induced with IPTG; wash, third washing step shown; eluate, second elution step shown. Expansin identity in eluate was confirmed by WB (data not shown).

FIGURE 3.

In situ apoplastic pH calibration curve for Oregon Green 488 dextran fluorescence excitation ratio R (490 excitation/440 excitation; 525 emission) in maize leaves. The Boltzmann fit was chosen for fitting sigmoidal curves to in situ calibration ratio data. Fitting resulted in an optimal dynamic range for pH measurements between 3.2 and 6.6 (corresponds to the ratios 0.95 and 2.15). In situ calibration was conducted on 12 different plants.

FIGURE 4.

Salinity-induced leaf growth inhibition. Leaf growth and biomass formation with 8-day 100 mm NaCl treatment. Shown are the daily increase in leaf length as averaged over the 8-day salt treatment (A), leaf thickness (B), leaf area (C), and leaf fresh weight (D), each at the last day of the 8-day 100-mm NaCl treatment (A). Light gray, control; dark gray, 100 mm NaCl. All measurements are the means of four biological replicates ± S.E., each replicated in triplicate. Asterisks indicate mean differences betweens salt treatment and control (*, p ≤ 0.05; **, p ≤ 0.01; ns = not significant; t test).

FIGURE 5.

NaCl decreases abundance of one specific β-expansin protein isoform in size-reduced leaves. For labeling the β-expansin isoforms, two-dimensional WB was performed on whole leaf protein extracts. Gel fragments that contained proteins of <40 kDa were electroblotted onto a PVDF membrane that was subsequently used for immunodetection. Gel fragments that contained proteins of ≥40 kDa were stained to guarantee equal loading of the gels. A, two-dimensional WB showing β-expansin isoforms in control-treated (no NaCl) and salt-stressed (100 mm NaCl) leaves. Arrows indicate the isoform that could not be detected under saline conditions. A negative control with preimmune serum detected no signals (data not shown). B, Coomassie-stained SDS-gels served as loading controls confirming equal loading of proteins in all gels. Numbers 1–3 indicate biological replicates.

FIGURE 6.

Clustal O (1.2.1) multiple amino acid sequence alignment and fragmentation spectrum of ZmEXPB6 peptide. A, alignment between Z. mays (Zm) β-expansin (EXPB) 2 (AF332175), ZmEXPB3 (AF332176), ZmEXPB4 (AF332177), ZmEXPB5 (AF332178), ZmEXPB6 (AF332179), ZmEXPB7 (AF332180), and ZmEXPB8 (AF332181). ZmEXPB6 was identified by the underlined peptide sequence TGAGPLDNG by mass spectrometry analysis of proteins extracted from a PVDF blotting membrane. B, 19 fragmentation spectra of peptide 730.29 (2+) were recorded in positive ion modus with an ion score of 5.12E3, and spectra were combined for peptide sequence analysis.

FIGURE 7.

Growth-mediating effects of exogenously applied ZmEXPB6. A, daily length growth of salt-stressed (100 mm NaCl) and osmotic-stressed (22.5 mm PEG 6000) leaves in response to exogenous application of recombinant ZmEXPB6. Stressed plants were divided into the following groups: NaCl- or PEG-treated plants plus exogenous application of heterologous ZmEXPB6; NaCl- or PEG-treated plants plus exogenous application of heat-inactivated heterologous ZmEXPB6; NaCl- or PEG-treated plants without exogenous application of heterologous ZmEXPB6. Control-treated plants were cultivated without NaCl or PEG. The exogenously application was carried out by brushing the ZmEXPB6-containing solution onto the adaxial- and abaxial leaf sides. To keep conditions identical, plants that were not treated with exogenously added expansin were wetted with the identical quantity of brushing solution. Leaf application treatment was repeated on four consecutive days and averaged. Statistical significance (p ≤ 0.01) is indicated by letters (all-pair comparison according to Tukey (45)). Confocal images in b–d show the adaxial view on the leaf apoplast. B, exogenously applied Alexa Fluor 488 goat anti-chicken IgG antibody at 3 h after leaf surface application appears as a green ring labeled by white arrows. C, identical details show autofluorescence of the palisade cell chloroplasts in red, #. D, overlay of B and C. The overlay demonstrates that the fluorescently labeled and exogenously applied protein can enter the apoplast and appears as a ring surrounding the palisade cell walls. *, apoplastic space.

FIGURE 8.

Leaf apoplastic pH. In planta quantitation of apoplastic pH in intact maize leaves under control and NaCl-stress conditions is shown. Leaf apoplastic pH as quantified in the stomatal cavities and in the apoplast that is surrounded by the mesophyll cells. Light gray, control; dark gray, 100 mm NaCl. Measurements were conducted on 45 different plants, with 5 regions of interest quantified for each apoplastic subcompartment. Thus, each mean is based on 225 single data points. Error bars indicate S.D. The t test was calculated based on de-logarithmized pH values.

FIGURE 9.

Assaying activity of recombinant ZmEXPB6 proteins in vitro. Extension of frozen-thawed basal leaf sections (5-cm length/1.5-cm width) in response to a force applied to leaf sections treated with recombinant ZmEXPB6 (light gray) or heat-inactivated recombinant ZmEXPB6 protein (dark gray). Extension is in mm, as measured with a linear variable differential transducer until point of breakage of leaf sections, as plotted over time. Extension is in response to application of 0.2 μg of ZmEXPB6 protein per cm² ad- and abaxial leaf surface. Representative kinetics of equivalent recordings are from independent experiments (n = 14). Inset, comparability between leaf segments was achieved by using a segment left of the mid rib and the congruent segment right of the mid rib.