Karl H. Mühling

Proteomic changes in maize roots after short‐term adjustment to saline growth conditions

It is of fundamental importance to understand adaptation processes leading to salt resistance. The initial effects on maize roots in the first hour after the adjustment to saline conditions were monitored to elucidate initial responses. The subsequent proteome change was monitored using a 2‐D proteomic approach. We found several new salt‐inducible proteins, whose expression has not been previously reported to be modulated by salt. A set of phosphoproteins in maize was detected but only ten proteins were phosphorylated and six proteins were dephosphorylated after the application of 25 mM NaCl for 1 h. Some of the phosphorylated maize proteins such as fructokinase, UDP‐glucosyl transferase BX9, and 2‐Cys‐peroxyredoxine were enhanced, whereas an isocitrate‐dehydrogenase, calmodulin, maturase, and a 40‐S‐ribosomal protein were dephosphorylated after adjustment to saline conditions. The initial reaction of the proteome and phosphoproteome of maize after adjustment to saline conditions reveals members of sugar signalling and cell signalling pathways such as calmodulin, and gave hint to a transduction chain which is involved in NaCl‐induced signalling. An alteration of 14‐3‐3 proteins as detected may change plasma membrane ATPase activity and cell wall growth regulators such as xyloglucane endotransglycosylase were also found to be changed immediately after the adjustment to salt stress.

Contribution of nitrification and denitrification to nitrous oxide emissions from soils after application of biogas waste and other fertilizers

The attribution of nitrous oxide (N(2)O) emission to organic and inorganic N fertilizers requires understanding of how these inputs affect the two biological processes, i.e. denitrification and nitrification. Contradictory findings have been reported when the effects of organic and inorganic fertilizers on nitrous oxide emission were compared. Here we aimed to contribute to the understanding of such variation using (15)N-labelling techniques. We determined the processes producing N(2)O, and tested the effects of soil moisture, N rates, and the availability of organic matter. In a pot experiment, we compared soil treated with biogas waste (BGW) and mineral ammonium sulphate (Min-N) applied at four rates under two soil moisture regimes. We also tested biogas waste, conventional cattle slurry and mineral N fertilizer in a grassland field experiment. During the first 37 days after application we observed N(2)O emissions of 5.6 kg N(2)O-N ha(-1) from soils supplied with biogas waste at a rate of 360 kg N ha(-1). Fluxes were ca. 5-fold higher at 85% than at 65% water holding capacity (WHC). The effects of fertilizer types and N rates on N(2)O emission were significant only when the soil moisture was high. Organic fertilizer treated soils showed much higher N(2)O emissions than those receiving mineral fertilizer in both, pot and field experiment. Over all the treatments the percentage of the applied N emitted as N(2)O was 2.56% in BGW but only 0.68% in Min-N. In the pot experiment isotope labelling indicated that 65-95% of the N(2)O was derived from denitrification for all fertilizer types. However, the ratio of denitrification/nitrification derived N(2)O was lower at 65% than at 85% WHC. We speculate that the application of organic matter in conjunction with ammonium nitrogen first leads to a decrease in denitrification-derived N(2)O emission compared with soil receiving mineral fertilizer. However, at later stages when denitrification becomes C-limited, higher N(2)O emissions are induced when the soil moisture is high.

Apoplastic Ion Concentration of Intact Leaves of Field Bean (Vicia faba) as Influenced by Ammonium and Nitrate Nutrition

Summary In order to differentiate between free and exchangeable apoplastic cations, intact leaves of Vicia faba were vacuum infiltrated with solutions differing in cation exchange strength (water and 50 mol · m -3 BaCl 2 ). Apoplastic washing fluids were subsequently obtained by careful centrifugation. While for the bivalent cations Ca 2+ and Mg 2+ it was possible to differentiate between a free and bound fraction this was not the case with K + . Concentration of free apoplastic Ca 2+ and K + varied between 0.3–0.8 mol· m -3 and 1.3–4.5 mol · m -3 , respectively. Total apoplastic Ca 2+ accounted for up to 40 % of total leaf Ca 2+ content while just 1–8 % of total leaf Mg 2+ and K + content were found in this compartment. Surprisingly, the N-form revealed little influence on apoplastic ion concentration. Independently from the form of N-nutrition, NH 4 + was not found in the leaf apoplast while NO 3 - concentration was rather low (0.01–0.03 mol · m -3 ). The pH of the intercellular washing fluid (PH 6.1) remained unaffected by the N-form.

Membrane-Associated, Boron-Interacting Proteins Isolated by Boronate Affinity Chromatography

Boron deficiency symptoms point to a role for boron in plant membranes, but the molecular partners interacting with boron have not yet been identified. The objective of the present study was to isolate and identify membrane-associated proteins with an ability to interact with boron. Boron-interacting proteins were isolated from root microsomal preparations of arabidopsis (Arabidopsis thaliana) and maize (Zea mays) using phenylboronate affinity chromatography, subsequently separated by two-dimensional gel electrophoresis and identified using MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) peptide mass fingerprinting. Twenty-six boron-binding membrane-associated proteins were identified in A. thaliana, and nine in Z. mays roots. Additional unidentified proteins were also present. Common to both species were the beta-subunit of mitochondrial ATP synthase, several beta-glucosidases, a luminal-binding protein and fructose bisphosphate aldolase. In A. thaliana, binding of these proteins to boron was significantly reduced after 4 d of boron deprivation. The relatively high number of diverse proteins identified as boron interacting, many of which are usually enriched in membrane microdomains, supports the hypothesis that boron plays a role in plant membranes by cross-linking glycoproteins, and may be involved in their recruitment to membrane microdomains.

The influence of salt stress on ABA and auxin concentrations in two maize cultivars differing in salt resistance

The plant hormones abscisic acid (ABA) and auxin (IAA, IBA) play important roles in plant responses to environmental stresses such as salinity. Recent breeding improvements in terms of salt resistance of maize have lead to a genotype with improved growth under saline conditions. By comparing this salt-resistant hybrid with a sensitive hybrid, it was possible to show differences in hormone concentrations in expanding leaves and roots. In response to salinity, the salt-resistant maize significantly increased IBA concentrations in growing leaves and maintained IAA concentration in roots. These hormonal adaptations may help to establish favorable conditions for growth-promoting agents such as β-expansins and maintain growth of resistant maize hybrids under salt stress. Moreover, ABA concentrations significantly increased in resistant maize leaves under salt stress, which may contribute to acidifying the apoplast, which in turn is a prerequisite for growth.

Salt stress differentially affects growth-mediating β-expansins in resistant and sensitive maize (Zea mays L.).

Salinity mainly reduces shoot growth by the inhibition of cell division and elongation. Expansins loosen plant cell walls. Moreover, the expression of some isoforms is clearly correlated with growth. Effects of salinity on β-expansin transcripts protein abundance were recently reported for different crop species. This study provides a broad analysis of the impact of an 8-day 100mM NaCl stress treatment on the mRNA expression of different maize (Zea mays L.) β-Expansin isoforms using real-time quantitative RT-PCR. The composite β-expansin protein expression was analyzed by western blotting using an anti-peptide antibody raised against a conserved 15-amino-acid region shared by vegetatively expressed β-expansin isoforms. For the first time, changes in β-expansin transcript and protein abundance have been analyzed together with the salinity-induced inhibition of shoot growth. A salt-resistant and a salt-sensitive cultivar were compared in order to elucidate physiological changes. Genotypic differences in the relative concentration of six β-expansin transcripts together with differences in the abundance β-expansin protein are shown in response NaCl stress. In salt-sensitive Lector, reduced β-expansin protein expression was found to correlate positively with reduced shoot growth under stress. A down-regulation of ZmExpB2, ZmExpB6, and ZmExpB8 transcripts possibly contribute to this decrease in protein abundance. In contrast, the maintenance of shoot growth in salt-resistant SR03 might be related to an unaffected abundance of growth-mediating β-expansin proteins in the shoot. Our data suggest that the up-regulation of ZmExpB2, ZmExpB6, and ZmExpB8 may sustain the stable expression of β-expansin protein under conditions of salt stress.

Light-induced pH and K+ changes in the apoplast of intact leaves.

Abstract. The K+-sensitive fluorescent dye benzofuran isophthalate (PBFI) and the pH-sensitive fluorescein isothiocyanate dextran (FITC-Dextran) were used to investigate the influence of light/dark transitions on apoplastic pH and K+ concentration in intact leaves of Vicia faba L. with fluorescence ratio imaging microscopy. Illumination by red light led to an acidification in the leaf apoplast due to light-induced H+ extrusion. Similar apoplastic pH responses were found on adaxial and abaxial sides of leaves after light/dark transition. Stomatal opening resulted only in a slight pH decrease (0.2 units) in the leaf apoplast. Gradients of apoplastic pH exist in the leaf apoplast, being about 0.5–1.0 units lower in the center of the xylem veins as compared with surrounding cells. The apoplastic K+ concentration in intact leaves declined during the light period. A steeper light-induced decrease in apoplastic K+, possibly caused by higher apoplastic K+, was found on the abaxial side of leaves concentration. Simultaneous measurements of apoplastic pH and K+ demonstrated that a light-induced decline in apoplastic K+ concentration indicative of net K+ uptake into leaf cells occurs independent of apoplastic pH changes. It is suggested that the driving force that is generated by H+ extrusion into the leaf apoplast due to H+-ATPase activity is sufficient for passive K+ influx into the leaf cells.

Real-Time Imaging of Leaf Apoplastic pH Dynamics in Response to NaCl Stress.

Knowledge concerning apoplastic ion concentrations is important for the understanding of many processes in plant physiology. Ion-sensitive fluorescent probes in combination with quantitative imaging techniques offer opportunities to localize, visualize, and quantify apoplastic ion dynamics in situ. The application of this technique to the leaf apoplast is complicated because of problems associated with dye loading. We demonstrate a more sophisticated dye loading procedure that enables the mapping of spatial apoplastic ion gradients over a period of 3 h. The new technique has been used for the real-time monitoring of pH dynamics within the leaf apoplast in response to NaCl stress encountered by the roots.

Comparative evaluation of extraction methods for apoplastic proteins from maize leaves

Proteins in the plant apoplast are essential for many physiological processes. We have analysed and compared six different infiltration solutions for proteins contained in the apoplast to recognize the most suitable method for leaves and to establish proteome maps for each extraction. The efficiency of protocols was evaluated by comparing the protein patterns resolved by 1-DE and 2-DE, and revealed distinct characteristics for each infiltration solution. Nano-LC-ESI-Q-TOF MS analysis of all fractions was applied to cover all proteins differentially extracted by infiltration solutions and led to the identification of 328 proteins in total in apoplast preparations. The predicted subcellular protein localisation distinguished the examined infiltration solutions in those with high or low amounts of intracellular protein contaminations, and with high or low quantities of secreted proteins. All tested infiltration solution extracted different subsets of proteins, and those implications on apoplast-specific studies are discussed.

Differential Transcript Expression of Wall-loosening Candidates in Leaves of Maize Cultivars Differing in Salt Resistance

Salt-sensitive crop plants such as maize (Zea mays L.) exhibit a strong and rapid growth reduction in response to NaCl stress. The unique salt-resistant maize hybrid SR03 and the salt-sensitive maize hybrid Lector provide good tools to characterize various genotypic responses to salinity in terms of shoot growth, shoot extensibility, and the expression pattern of wall-loosening candidates. The mRNA transcript levels of wall-loosening candidates such as xyloglucan endotransglucosylase (XET), endo-1,4-β-D-endoglucanase (EGase), α-expansins (EXPA), and the plasma membrane proton pump (PM-H+-ATPase) are correlated with cell-wall extensibility and with shoot growth under NaCl stress. We have found for the salt-sensitive maize that a decrease in the relative transcript abundance of ZmXET1, ZmEXPA1, and the composite PM-H+-ATPase mRNAs correlates with a decrease in wall extensibility and shoot growth. We suggest that this downregulation of wall-loosening candidates contributes to a reduction in extensibility and consequently in growth. In contrast, the decrease in wall extensibility is less strong in the salt-sensitive hybrid SR03. In the salt-resistant maize genotype, an upregulation of ZmXET1, ZmEXPA1 and PM-H+-ATPase transcripts possibly mitigates the salinity-induced decrease in wall extensibility and thus in shoot growth.