Plant natriuretic peptides - elucidation of the mechanisms of action

Philosophiae Doctor - PhDSeveral lines of cellular and physiological evidence have suggested the presence of a novel class of systemically mobile plant molecules that are recognized by antibodies generated against vertebrate atrial natriuretic peptides (ANPs). Functional characterization of these immunoanalogues, referred to as immunoreactive plant natriuretic peptides (irPNPs) or plant natriuretic peptides (PNPs), has shown that they play important roles in a number of cellular processes crucial for plant growth and maintenance of cellular homeostasis. Although the various biological roles of PNPs in plants are known, their exact mode of action remains elusive. To elucidate the mechanisms of action for these immunoanalogues, we have prepared a biologically active recombinant PNP from Arabidopsis thaliana (AtPNP-A) and the biological activity was demonstrated by showing its ability to induce water uptake into Arabidopsis thaliana protoplasts. In addition, the molecule was shown to downregulate photosynthesis while at the same time up-regulating respiration, transpiration as well as net water uptake and retention capacities in the sage Plectranthus ecklonii. Further analysis of the recombinant AtPNP-A indicated that the peptide can induce systemic response signalling though the phloem. A recombinant Arabidopsis wall associated kinase-like protein (AtWAKL10) that has a domain organization resembling that of vertebrate natriuretic peptide (NP) receptors was also partially characterized as a possible receptor for the recombinant AtPNP-A. Vertebrate NP receptors contain an extracellular ligand-binding domain and an intracellular guanylate cyclase (GC)/kinase domain and signal through the activity of their GC domain that is capable of generating intracellular cGMP from GTP. The structural resemblance of AtWAKL10 to vertebrate NP receptors could suggest a functional homology with receptor molecules and it is conceivable that such a receptor may recognize PNPs as ligands. The characterization of the recombinant AtWAKL10 showed that the molecule functions as both a GC and a kinase in vitro. This strengthened the suggestion that AtWAKL10 could be a possible AtPNP-A receptor especially considering the fact that AtPNP-A applications to plant cells also trigger cGMP transients. Furthermore, a bioinformatic analysis of the functions of AtPNP-A and AtWAKL10 has inferred both molecules in plant pathogen responses and defense mechanisms, thus indirectly functionally linking the two proteins.South Afric

Elucidation of the morpho-physiological traits of maize (Zea mays L.) under salt stress

Agriculture is an essential sector for the increasing world population, hence the need for more food production.  However, the aim of increasing food crop production is mostly suppressed by abiotic stresses such as drought and salinity. Salinity is a major limiting factor that inhibits the potential of plant growth and productivity worldwide. Hence, understanding the mechanisms behind plant stress response is important for developing new biomarker approaches that will increase salt tolerance in crops.  To survive, plants exhibit various morphological, physiological, and biochemical processes when faced with saline conditions. This study was carried out to explore and evaluate the morphological and physiological effects of salinity on maize grown in the absence/presence of NaCl, followed by measurement of the various growth parameters at the end of a treatment cycle.  Results of the study revealed that salt stress significantly decreased growth parameters such as plant height, leaf number, leaf width, leaf area, leaf length, and shoot (weight and length).  On the other hand, salinity decreased physiological traits such as stomatal count, stomatal density, transpiration, and respiration rates.  This study has shown the negative effects of salt stress on the morphology and physiology of maize.  These findings can be used as a reference tool in stress response studies focusing on salt stress pathways in maize and other related crops

The arabidopsis wall associated kinase-like 10 gene encodes a functional guanylyl cyclase and is coexpressed with pathogen defense related genes

Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, guanosine 3′,5′-cyclic monophosphate (cGMP), has long been known to be an essential signaling molecule in many different physiological processes in higher plants, including biotic stress responses. To date, however, the guanylyl cyclase (GC) enzymes that catalyze the formation of cGMP from GTP have largely remained elusive in higher plants. We have identified an Arabidopsis receptor type wall associated kinase–like molecule (AtWAKL10) as a candidate GC and provide experimental evidence to show that the intracellular domain of AtWAKL10431–700 can generate cGMP in vitro. Further, we also demonstrate that the molecule has kinase activity indicating that AtWAKL10 is a twin-domain catalytic protein. A co-expression and stimulus-specific expression analysis revealed that AtWAKL10 is consistently co-expressed with well characterized pathogen defense related genes and along with these genes is induced early and sharply in response to a range of pathogens and their elicitors

The Arabidopsis Wall Associated Kinase-Like 10 Gene Encodes a Functional Guanylyl Cyclase and Is Co-Expressed with Pathogen Defense Related Genes

Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, guanosine 3′,5′-cyclic monophosphate (cGMP), has long been known to be an essential signaling molecule in many different physiological processes in higher plants, including biotic stress responses. To date, however, the guanylyl cyclase (GC) enzymes that catalyze the formation of cGMP from GTP have largely remained elusive in higher plants. is consistently co-expressed with well characterized pathogen defense related genes and along with these genes is induced early and sharply in response to a range of pathogens and their elicitors.We demonstrate that AtWAKL10 is a twin-domain, kinase-GC signaling molecule that may function in biotic stress responses that are critically dependent on the second messenger cGMP

The Arabidopsis thaliana Brassinosteroid Receptor (AtBRI1) Contains a Domain that Functions as a Guanylyl Cyclase In Vitro

BACKGROUND: Guanylyl cyclases (GCs) catalyze the formation of the second messenger guanosine 3′,5′-cyclic monophosphate (cGMP) from guanosine 5′-triphosphate (GTP). Cyclic GMP has been implicated in an increasing number of plant processes, including responses to abiotic stresses such as dehydration and salt, as well as hormones. PRINCIPLE FINDINGS: Here we used a rational search strategy based on conserved and functionally assigned residues in the catalytic centre of annotated GCs to identify candidate GCs in Arabidopsis thaliana and show that one of the candidates is the brassinosteroid receptor AtBR1, a leucine rich repeat receptor like kinase. We have cloned and expressed a 114 amino acid recombinant protein (AtBR1-GC) that harbours the putative catalytic domain, and demonstrate that this molecule can convert GTP to cGMP in vitro. CONCLUSIONS: Our results suggest that AtBR1 may belong to a novel class of GCs that contains both a cytosolic kinase and GC domain, and thus have a domain organisation that is not dissimilar to that of atrial natriuretic peptide receptors, NPR1 and NPR2. The findings also suggest that cGMP may have a role as a second messenger in brassinosteroid signalling. In addition, it is conceivable that other proteins containing the extended GC search motif may also have catalytic activity, thus implying that a significant number of GCs, both in plants and animals, remain to be discovered, and this is in keeping with the fact that the single cellular green alga Chlamydomonas reinhardtii contains over 90 annotated putative CGs

New Perspectives on Plant Adenylyl Cyclases

It is increasingly clear that plant genomes encode numerous complex multidomain proteins that harbor functional adenylyl cyclase (AC) centers. These AC containing proteins have well-documented roles in development and responses to the environment. However, it is only for a few of these proteins that we are beginning to understand the intramolecular mechanisms that govern their cellular and biological functions, as detailed characterizations are biochemically and structurally challenging given that these poorly conserved AC centers typically constitute only a small fraction (\u3c10%) of complex plant proteins. Here, we offer fresh perspectives on their seemingly cryptic activities specifically showing evidence for the presence of multiple functional AC centers in a single protein and linking their catalytic strengths to the Mg2+/Mn2+-binding amino acids. We used a previously described computational approach to identify candidate multidomain proteins from Arabidopsis thaliana that contain multiple AC centers and show, using an Arabidopsis leucine-rich repeat containing protein (TAIR ID: At3g14460; AtLRRAC1) as example, biochemical evidence for multienzymatic activities. Importantly, all AC-containing fragments of this protein can complement the AC-deficient mutant cyaA in Escherichia coli, while structural modeling coupled with molecular docking simulations supports catalytic feasibility albeit to varying degrees as determined by the frequency of suitable substrate binding poses predicted for the AC sites. This statistic correlates well with the enzymatic assays, which implied that the greatly reduced AC activities is due to the absence of the negatively charged [DE] amino acids previously assigned to cation-, in particular Mg2+/Mn2+-binding roles in ACs

Mopane Worm (<i>Gonimbrasia belina</i> Westwood) Meal as a Potential Protein Source for Sustainable Quail Production: A Review

Fast-growing and highly adaptable avian birds such as quail (Coturnix coturnix) possess great potential to meet the growing demand for animal protein by the rapidly increasing human population, and would contribute immensely to global food production and nutritional security. However, overreliance on conventional protein sources such as fish and soybean meals during the formulation of quail diets is economically and environmentally unsustainable. Alternatively, insect-based protein sources such as Gonimbrasia belina, commonly known as mopane worm (MW), can be used to increase quail production due to their high biological value and low feed-food competition. Indeed, MW is highly nutritious, with an average protein content of 55% and a well-balanced amino acid profile. Thus, its incorporation in quail diets could provide great potential to alleviate nutritional deficiencies in quail production and allow for their sustainable intensification. However, there are limited studies on the effect of partial or complete replacement of conventional protein sources with mopane worm meal (MWM) in quail diets. This paper reviews the nutritional profile and use of the MW as a protein source, as well as its potential future prospects in poultry diets. Finally, we postulate that mass production of this insect-based protein source and its sustainability would be an inventive strategy to develop a profitable quail business

Site-directed mutagenesis and functional testing of AtGC1(1–100).

<p>(A) Original 14 amino acid search motif for GCs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000449#pone.0000449-Ludidi1" target="_blank">[12]</a>; substitutions are in square brackets, X represents any amino acid and curly brackets define the number of amino acids. (B) AtGC1 (At5g05930): The position of the GC catalytic centre is marked in red; the underlined aspartic acid [D] is the amino acid that has been changed into a leucine [L] by site directed mutagenesis. The open arrow marks the conserved PPi-binding arginine [R] and the C-terminal putative metal binding site is highlighted in aquamarine. The green triangles point to exon borders and the solid arrow shows the border of the fragment that we have tested for GC activity. (C) SDS-PAGE of the 3 purification steps of the recombinant protein AtGC1(1–100); “M” is the molecular weight marker, “FT” is the protein in the flow-through, “W” is the wash and “E” is the eluted recombinant protein. (D) <i>In vitro</i> GC activity assay. The control (cont.; empty bar) was obtained by omitting protein in the reaction mix and the concentration of GTP was 1mM and that of Mn²⁺ or Mg²⁺ was 5 mM. The values for the wild-type protein (N-terminal fragment of 100 amino acids containing a [D] in position 7 of the catalytic centre) and the mutated protein (N-terminal fragment of 100 amino acids containing [L] in position 7 of the catalytic centre) are represented with solid bars. The bar values represent the mean cGMP (+/−SEM) generated in 15 minutes in three samples and the response pattern is representative of 3 independent experiments.</p

Calcium is the switch in the moonlighting dual function of the ligand–activated receptor kinase phytosulfokine receptor 1

Background: A number of receptor kinases contain guanylate cyclase (GC) catalytic centres encapsulated in the cytosolic kinase domain. A prototypical example is the phytosulfokine receptor 1 (PSKR1) that is involved in regulating growth responses in plants. PSKR1 contains both kinase and GC activities however the underlying mechanisms regulating the dual functions have remained elusive. Findings: Here, we confirm the dual activity of the cytoplasmic domain of the PSKR1 receptor. We show that mutations within the guanylate cyclase centre modulate the GC activity while not affecting the kinase catalytic activity. Using physiologically relevant Ca²⁺ levels, we demonstrate that its GC activity is enhanced over two-fold by Ca²⁺ in a concentration-dependent manner. Conversely, increasing Ca²⁺ levels inhibits kinase activity up to 500-fold at 100 nM Ca²⁺. Conclusions: Changes in calcium at physiological levels can regulate the kinase and GC activities of PSKR1. We therefore propose a functional model of how calcium acts as a bimodal switch between kinase and GC activity in PSKR1 that could be relevant to other members of this novel class of ligand-activated receptor kinases