Zn nutrients-loaded chitosan nanocomposites and their efficacy as nanopriming agents for maize (Zea mays) seeds.

Recent breakthroughs in agro-inputs research have led to the development of nanomaterials that can promote precision agriculture and better environmental security. The agricultural sector is increasingly facing the negative impacts of changing climates due to various stress conditions. To curb this scenario, economical and low-risk practices such as decreasing fertilizer inputs and seed priming have been promoted. In the current study, the H. odoratissimum aqueous extract was used to nucleate the Zn ionic species and grow the zinc oxide nanoparticles (ZnO NPs). The developed nanocomposites and their ionic zinc precursor were then integrated into tripolyphosphate (TPP)-crosslinked chitosan (CS/TPP) nanostructures by ionic gelation. Advanced physicochemical characterization techniques (SEM, EDS, TEM, DLS, FTIR, TGA, and XPS) were exploited to report the morphology, hydrodynamic size, surface charge, and structural organization of the developed nanomaterials. These revealed positively charged particles with hydrodynamic size in the 149-257 nm range. The NPs were used as priming agents for Zea mays seeds. At 0.04%, the ZnO-loaded CS/TPP NPs achieved higher root and shoot elongation in 10-day old seedlings compared to other treatments. The pristine CS/TPP NPs, Zn(II)-laden CS/TPP NPs, and ZnO-loaded CS/TPP NPs at 0.01% significantly promoted the early seedling development of seeds under salt stress. This represents the first report showing ZnO integrated chitosan nanocomposites as an auspicious nanopriming agent for stimulating the seed germination of maize. The study envisages offering perspectives on utilizing green nanotechnology to improve the early seedling development of maize. Furthermore, it has the potential to contribute towards UN SDG 2, thus addressing the threats to global food insecurity and doubling agricultural productivity by 2030.

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 (<10%) 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.

An Arabidopsis Clathrin Assembly Protein with a Predicted Role in Plant Defense Can Function as an Adenylate Cyclase.

Adenylate cyclases (ACs), much like guanylate cyclases (GCs), are increasingly recognized as essential parts of many plant processes including biotic and abiotic stress responses. In order to identify novel ACs, we have applied a search motif derived from experimentally tested GCs and identified a number of Arabidopsis thaliana candidates including a clathrin assembly protein (AT1G68110; AtClAP). AtClAP contains a catalytic centre that can complement the AC-deficient mutant cyaA in E. coli, and a recombinant AtClAP fragment (AtClAP261-379) can produce cyclic adenosine 3',5' monophosphate (cAMP) from adenosine triphosphate (ATP) in vitro. Furthermore, an integrated analysis of gene expression and expression correlation implicate cAMP in pathogen defense and in actin cytoskeletal remodeling during endocytic internalization.

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.

Recombinant expression and functional testing of candidate adenylate cyclase domains.

Adenylate cyclases (ACs) are enzymes capable of converting adenosine-5'-triphosphate to cyclic 3', 5'--adenosine monophosphate (cAMP). In animals and lower eukaryotes, ACs and their product cAMP have firmly been established as important signalling molecules with important roles in several cellular signal transduction pathways. However, in higher plants, the only annotated and experimentally confirmed AC is a Zea mays pollen protein capable of generating cAMP. Recently a number of candidate AC-encoding genes in Arabidopsis thaliana have been proposed based on functionally assigned amino acids in the catalytic center of annotated and/or experimentally tested nucleotide cyclases in lower and higher eukaryotes. Here we detail the cloning and recombinant expression of functional candidate AC domains using, as an example, the A. thaliana pentatricopeptide repeat-containing protein (AtPPR-AC; At1g62590). Through a complementation test, in vivo adenylate cyclase activity of candidate recombinant molecules can be prescreened and promising candidates can subsequently be further evaluated in an in vitro AC immunoassay.

Infrared gas analysis technique for the study of the regulation of photosynthetic responses.

Homeostatic maintenance of physiological and biochemical processes is a key requirement for survival and adaptive responses of multicellular organisms such as plants. These important processes are in part mediated by various plant enzymes and hormones, many of which are in part, controlled by cyclic nucleotides and/or other signalling molecules. Infrared gas analysis (IRGA) technique is one of the modern methods which allows for rapid and accurate measurements of cyclic nucleotide mediated photosynthetic responses to plant hormones, and thus makes it a powerful and useful tool to study aspects of downstream cell signalling events in plants. In this chapter the basic protocols enabling the use of the IRGA technique to study signalling molecules, such as cyclic nucleotides on photosynthetic responses, are outlined.

Identification of a novel Arabidopsis thaliana nitric oxide-binding molecule with guanylate cyclase activity in vitro.

While there is evidence of nitric oxide (NO)-dependent signalling via the second messenger cyclic guanosine 3',5'-monophosphate (cGMP) in plants, guanylate cyclases (GCs), enzymes that catalyse the formation of cGMP from guanosine 5'-triphosphate (GTP) have until recently remained elusive and none of the candidates identified to-date are NO-dependent. Using both a GC and heme-binding domain specific (H-NOX) search motif, we have identified an Arabidopsis flavin monooxygenase (At1g62580) and shown electrochemically that it binds NO, has a higher affinity for NO than for O(2) and that this molecule can generate cGMP from GTP in vitro in an NO-dependent manner.

The Arabidopsis thaliana natriuretic peptide AtPNP-A is a systemic regulator of leaf dark respiration and signals via the phloem.

Plant natriuretic peptides (PNPs) belong to a novel class of peptidic signaling molecules that share some structural similarity to the N-terminal domain of expansins and affect physiological processes such as water and ion homeostasis at nano-molar concentrations. Here we show that a recombinant Arabidopsis thaliana PNP (AtPNP-A) rapidly increased the rate of dark respiration in treated leaves after 5 min. In addition, we observed increases in lower leaves, and with a lag time of 10 min, the effect spread to the upper leaves and subsequently (after 15 min) to the opposite leaves. This response signature is indicative of phloem mobility of the signal, a hypothesis that was further strengthened by the fact that cold girdling, which affects phloem but not xylem or apoplastic processes, delayed the long distance AtPNP-A effect. We conclude that locally applied AtPNP-A can induce a phloem-mobile signal that rapidly modifies plant homeostasis in distal parts.

The phytosulfokine (PSK) receptor is capable of guanylate cyclase activity and enabling cyclic GMP-dependent signaling in plants.

Phytosulfokines (PSKs) are sulfated pentapeptides that stimulate plant growth and differentiation mediated by the PSK receptor (PSKR1), which is a leucine-rich repeat receptor-like kinase. We identified a putative guanylate cyclase (GC) catalytic center in PSKR1 that is embedded within the kinase domain and hypothesized that the GC works in conjunction with the kinase in downstream PSK signaling. We expressed the recombinant complete kinase (cytoplasmic) domain of AtPSKR1 and show that it has serine/threonine kinase activity using the Ser/Thr peptide 1 as a substrate with an approximate K(m) of 7.5 µm and V(max) of 1800 nmol min(-1) mg(-1) of protein. This same recombinant protein also has GC activity in vitro that is dependent on the presence of either Mg(2+) or Mn(2+). Overexpression of the full-length AtPSKR1 receptor in Arabidopsis leaf protoplasts raised the endogenous basal cGMP levels over 20-fold, indicating that the receptor has GC activity in vivo. In addition, PSK-α itself, but not the non-sulfated backbone, induces rapid increases in cGMP levels in protoplasts. Together these results indicate that the PSKR1 contains dual GC and kinase catalytic activities that operate in vivo and that this receptor constitutes a novel class of enzymes with overlapping catalytic domains.

The Arabidopsis wall associated kinase-like 10 gene encodes a functional guanylyl cyclase and is co-expressed with pathogen defense related genes.

BACKGROUND: 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. PRINCIPAL FINDINGS: 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 AtWAKL10(431-700) 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. CONCLUSIONS: 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.