Hypoxia-Responsive Class III Peroxidases in Maize Roots: Soluble and Membrane-Bound Isoenzymes.

Flooding induces low-oxygen environments (hypoxia or anoxia) that lead to energy disruption and an imbalance of reactive oxygen species (ROS) production and scavenging enzymes in plants. The influence of hypoxia on roots of hydroponically grown maize (Zea mays L.) plants was investigated. Gene expression (RNA Seq and RT-qPCR) and proteome (LC-MS/MS and 2D-PAGE) analyses were used to determine the alterations in soluble and membrane-bound class III peroxidases under hypoxia. Gel-free peroxidase analyses of plasma membrane-bound proteins showed an increased abundance of ZmPrx03, ZmPrx24, ZmPrx81, and ZmPr85 in stressed samples. Furthermore, RT-qPCR analyses of the corresponding peroxidase genes revealed an increased expression. These peroxidases could be separated with 2D-PAGE and identified by mass spectrometry. An increased abundance of ZmPrx03 and ZmPrx85 was determined. Further peroxidases were identified in detergent-insoluble membranes. Co-regulation with a respiratory burst oxidase homolog (Rboh) and key enzymes of the phenylpropanoid pathway indicates a function of the peroxidases in membrane protection, aerenchyma formation, and cell wall remodeling under hypoxia. This hypothesis was supported by the following: (i) an elevated level of hydrogen peroxide and aerenchyma formation; (ii) an increased guaiacol peroxidase activity in membrane fractions of stressed samples, whereas a decrease was observed in soluble fractions; and (iii) alterations in lignified cells, cellulose, and suberin in root cross-sections.

A Rhodamine B-based fluorescent probe for imaging Cu2+ in maize roots.

A new Rhodamine B-based fluorescent probe (RBO) is successfully designed and synthesized, which is a higher selective and sensitive chemosensor for Cu2+ than other ions. Under physiological conditions (pH = 7.0), the non emission RBO displays a rapid fluorescence increase together with a color change after addition of Cu2+ and the detection limit is down to 28nM, which can clearly illustrate the distribution of Cu2+ with the help of laser scanning confocal microscope in plant tissues. Eventually, it confirmed that the Cu2+ accumulates mostly in the vascular cylinder and very less in the epidermal cells of maize roots, which is important to understand how the plants take up, transport and store in the Cu2+.

Some properties of the walls of metaxylem vessels of maize roots, including tests of the wettability of their lumenal wall surfaces.

BACKGROUND AND AIMS: Since the proposal of the cohesion theory there has been a paradox that the lumenal surface of vessels is rich in hydrophobic lignin, while tension in the rising sap requires adhesion to a hydrophilic surface. This study sought to characterize the strength of that adhesion in maize (Zea mays), the wettability of the vessel surface, and to reconcile this with its histochemical and physical nature. METHODS: Wettability was assessed by emptying the maize root vessels of sap, perfusing them with either water or oil, and examining the adhesion (as revealed by contact angles) of the two liquids to vessel walls by cryo-scanning electron microscopy. The phobicity of the lumenal surface was also assessed histochemically with hydrophilic and hydrophobic probes. KEY RESULTS: Pit borders in the lumen-facing vessel wall surface were wetted by both sap/water and oil. The attraction for oil was weaker: water could replace oil but not vice versa. Pit apertures repelled oil and were strongly stained by hydrophilic probes. Pit chambers were probably hydrophilic. Oil never entered the pits. When vessels were emptied and cryo-fixed immediately, pit chambers facing away from the vessels were always sap-filled. Pit chambers facing vessel lumens were either sap- or gas-filled. Sap from adjoining tracheary elements entering empty vessels accumulated on the lumenal surface in hemispherical drops, which spread out with decreasing contact angles to fill the lumen. CONCLUSIONS: The vessel lumenal surface has a dual nature, namely a mosaic of hydrophilic and hydrophobic patches at the micrometre scale, with hydrophilic predominating. A key role is shown, for the first time, of overarching borders of pits in determining the dual nature of the surface. In gas-filled (embolized) vessels they are hydrophobic. When wetted by sap (vessels refilling or full) they are hydrophilic. A hypothesis is proposed to explain the switch between the two states.

Characterization of AMT-mediated high-affinity ammonium uptake in roots of maize (Zea mays L.).

High-affinity ammonium uptake in plant roots is mainly mediated by AMT1-type ammonium transporters, and their regulation varies depending on the plant species. In this study we aimed at characterizing AMT-mediated ammonium transport in maize, for which ammonium-based fertilizer is an important nitrogen (N) source. Two ammonium transporter genes, ZmAMT1;1a and ZmAMT1;3, were isolated from a maize root-specific cDNA library by functional complementation of an ammonium uptake-defective yeast mutant. Ectopic expression of both genes in an ammonium uptake-defective Arabidopsis mutant conferred high-affinity ammonium uptake capacities in roots with substrate affinities of 48 and 33 µM for ZmAMT1;1a and ZmAMT1;3, respectively. In situ hybridization revealed co-localization of both ZmAMT genes on the rhizodermis, suggesting an involvement in capturing ammonium from the rhizosphere. In N-deficient maize roots, influx increased significantly while ZmAMT expression did not. Ammonium resupply to N-deficient or nitrate-pre-cultured roots, however, rapidly enhanced both influx and ZmAMT transcript levels, revealing a substrate-inducible regulation of ammonium uptake. In conclusion, the two rhizodermis-localized transporters ZmAMT1;1a and ZmAMT1;3 are most probably the major components in the high-affinity transport system in maize roots. A particular regulatory feature is their persistent induction by ammonium rather than an up-regulation under N deficiency.

Examining the Effects of the Nitrogen Environment on Growth and N2-Fixation of Endophytic Herbaspirillum seropedicae in Maize Seedlings by Applying 11C Radiotracing.

Herbaspirillum seropedicae, as an endophyte and prolific root colonizer of numerous cereal crops, occupies an important ecological niche in agriculture because of its ability to promote plant growth and potentially improve crop yield. More importantly, there exists the untapped potential to harness its ability, as a diazotroph, to fix atmospheric N2 as an alternative nitrogen resource to synthetic fertilizers. While mechanisms for plant growth promotion remain controversial, especially in cereal crops, one irrefutable fact is these microorganisms rely heavily on plant-borne carbon as their main energy source in support of their own growth and biological functions. Biological nitrogen fixation (BNF), a microbial function that is reliant on nitrogenase enzyme activity, is extremely sensitive to the localized nitrogen environment of the microorganism. However, whether internal root colonization can serve to shield the microorganisms and de-sensitize nitrogenase activity to changes in the soil nitrogen status remains unanswered. We used RAM10, a GFP-reporting strain of H. seropedicae, and administered radioactive 11CO2 tracer to intact 3-week-old maize leaves and followed 11C-photosynthates to sites within intact roots where actively fluorescing microbial colonies assimilated the tracer. We examined the influence of administering either 1 mM or 10 mM nitrate during plant growth on microbial demands for plant-borne 11C. Nitrogenase activity was also examined under the same growth conditions using the acetylene reduction assay. We found that plant growth under low nitrate resulted in higher nitrogenase activity as well as higher microbial demands for plant-borne carbon than plant growth under high nitrate. However, carbon availability was significantly diminished under low nitrate growth due to reduced host CO2 fixation and reduced allocation of carbon resources to the roots. This response of the host caused significant inhibition of microbial growth. In summary, internal root colonization did little to shield these endophytic microorganisms from the nitrogen environment.

Quantitative proteomics suggests changes in the carbohydrate metabolism of maize in response to larvae of the belowground herbivore Holotrichia parallela.

The larvae of Holotrichia parallela, a destructive belowground herbivore, may cause yield losses of up to 20% in maize in a typical year. To understand the protein-level mechanisms governing the response of maize to this herbivore, tandem mass tag (TMT) quantitative proteomics was used for the comparative analysis of protein abundance in the maize roots after H. parallela larval attack. A total of 351 upregulated proteins and 303 downregulated proteins were identified. Pathway enrichment analysis revealed that the differentially abundant proteins (DAPs) were most strongly associated with carbohydrate and energy metabolism pathways, such as glycolysis, pentose phosphate pathway and fructose and mannose metabolism. Most glycolysis-related proteins were significantly induced. In addition, H. parallela larval attack decreased the glucose concentrations in the roots. This study demonstrates that maize can manipulate carbohydrate metabolism by modifying glycolysis and pentose phosphate pathway response to root-feeding herbivorous attackers. The results of this study may help to establish a foundation for further functional studies of key protein-mediated responses to H. parallela larvae in maize.

Quantifying Plant-Borne Carbon Assimilation by Root-Associating Bacteria.

Herbaspirillum seropedicae is a rhizobacteria that occupies a specialized ecological niche in agriculture. As an endophyte and prolific grass root colonizer it has the potential to promote plant growth, enhancing crop yield in many cereal crops. While the mechanisms for plant growth promotion are controversial, the one irrefutable fact is these microorganisms rely heavily on plant-borne carbon as their main energy source in support of their biological functions. Unfortunately, the tools and technology enabling researchers to trace carbon exchange between plants and the microorganisms associating with them has been limiting. Here, we demonstrate that radioactive 11CO2 administered to intact maize leaves with translocation of 11C-photosynthates to roots can provide a 'traceable' source of carbon whose assimilation by microbial organisms can be quantified with enormous sensitivity. Fluorescence root imaging of RAM10, a green fluorescent protein (GFP) reporting strain of H. seropedicae, was used to identify regions of high microbial colonization. Microbes were mechanically removed from these regions via sonication in saline solution and extracts were subjected to fluorescence measurement and gamma counting to correlate carbon-11 atoms with numbers of colony forming units. The method has potential to translate to other microorganisms provided they possess an optical reporting trait.

A Pseudomonas strain isolated from date-palm rhizospheres improves root growth and promotes root formation in maize exposed to salt and aluminum stress.

The aim of this study was to evaluate the effectiveness of Pseudomonas fluorescens 002 (P.f.002.), isolated from the rhizosphere of date palms from the Ghardaia region in the Algerian Sahara, to promote root growth of two varieties of maize under conditions of salt and aluminum stress. Primary roots of 5-day-old seedlings were inoculated with P.f.002., and seedlings were then grown under both control and stressed conditions. Primary, lateral, and seminal root lengths and numbers, as well as root dry mass, were evaluated. P.f.002 increased all parameters measured under both salt and aluminum stress. Hence, the use of P.f.002 may represent an important biotechnological approach to decrease the impact of salinity and acidity in crops.

The epidermal surface of the maize root tip: III. Isolation of the surface and characterization of some of its structural and mechanical properties.

The surface of the young epidermal cells of maize roots is composed o) three layers; the inner layer (LI), which is the outer epidermal Wall, overlaid by a pellicle consisting of a thick, coherent inner layer (L2) and a very thin, loosely organized outer layer (L3). The entire surface can be removed intact to produce either narrow, circumferential strips or apical halftones, by gently prying it loose in the circumferential direction by hand with insect pins. Usually only short remnants of anticlinical walls of the epidermal cells remain attached. These isolated surface pieces always curl outward at the free circumferential edges in the longitudinal direction of the original intact root. When the strips are deliberated stretched alone their long axis (i.e., the original circumferential direction) they elongate irreversibly by us much us two thirds of their length, before showing some elastic deformation and breaking. Some plastic deformation may occur in the original longitudinal direction of the root during removal of the strips. The plastic deformation opens the helicoidal array of microfibrils in the L1 layer. Deformation also produces structural changes over the original radial walls and those transverse anticlinal walls that form boundaries of cell packets derived from single cells. In these positions the L1 layers over adjacent cells separate in the direction of the applied stress. This occurs by the separation of the L I layers of adjacent cells and the stretching of the inward projection of the amorphous L2 layer of the pellicle which lies; over these original anticlinal walls. There is much less or no separation of the L1 layers over anticlinal walls Of adjoining daughter cells in the epidermis. The pellicle always remains firmly attached to the outer epidermal wall during removal of the surface strips. On removal, these strips shorten in their original longitudinal direction in situ, indicating a release of tension imposed by underlying cells. Their outward curling suggests stress between the wall and pellicle of the outer epidermal surface in the intact root. These findings focus attention on structural differences between sites where anticlinal walls of different origin join the outer surface, and on possible differences in surface extensibility at each sites.