Hormonal control of promoter activities of Cannabis sativa prenyltransferase 1 and 4 and salicylic acid mediated regulation of cannabinoid biosynthesis.

Cannabis sativa aromatic prenyltransferase 4 (CsPT4) and 1 (CsPT1) have been shown to catalyze cannabigerolic acid (CBGA) biosynthesis, a step that rate-limits the cannabinoid biosynthetic pathway; both genes are highly expressed in flowers. CsPT4 and CsPT1 promoter driven ß-glucuronidase (GUS) activities were detected in leaves of cannabis seedlings, and strong CsPT4 promoter activities were associated with glandular trichomes. Hormonal regulation of cannabinoid biosynthetic genes is poorly understood. An in silico analysis of the promoters identified putative hormone responsive elements. Our work examines hormone-responsive elements in the promoters of CsPT4 and CsPT1 in the context of physiological responses of the pathway to the hormone in planta. Dual luciferase assays confirmed the regulation of promoter activities by the hormones. Further studies with salicylic acid (SA) demonstrated that SA pretreatment increased the expression of genes located downstream of the cannabinoid biosynthetic pathway. The results from all aspects of this study demonstrated an interaction between certain hormones and cannabinoid synthesis. The work provides information relevant to plant biology, as we present evidence demonstrating correlations between molecular mechanisms that regulate gene expression and influence plant chemotypes.

Phenotypes of cyclic nucleotide-gated cation channel mutants: probing the nature of native channels.

Plant cyclic nucleotide gated channels (CNGCs) facilitate cytosolic Ca2+ influx as an early step in numerous signaling cascades. CNGC-mediated Ca2+ elevations are essential for plant immune defense and high temperature thermosensing. In the present study, we evaluated phenotypes of CNGC2, CNGC4, CNGC6, and CNGC12 null mutants in these two pathways. It is shown CNGC2, CNGC4, and CNGC6 physically interact in vivo, whereas CNGC12 does not. CNGC involvement in immune signaling was evaluated by monitoring mutant response to elicitor peptide Pep3. Pep3 response cascades involving CNGCs included mitogen-activated kinase activation mediated by Ca2+ -dependent protein kinase phosphorylation. Pep3-induced reactive oxygen species generation was impaired in cngc2, cngc4, and cngc6, but not in cngc12, suggesting that CNGC2, CNGC4, and CNGC6 (which physically interact) may be components of a multimeric CNGC channel complex for immune signaling. However, unlike cngc2 and cngc4, cngc6 is not sensitive to high Ca2+ and displays no pleiotropic dwarfism. All four cngc mutants showed thermotolerance compared to wild-type, although CNGC12 does not interact with the other three CNGCs. These results imply that physically interacting CNGCs may, in some cases, function in a signaling cascade as components of a heteromeric channel complex, although this may not be the case in other signaling pathways.

Delineating genetic regulation of cannabinoid biosynthesis during female flower development in Cannabis sativa.

Cannabinoids are predominantly produced in the glandular trichomes on cannabis female flowers. There is little known on how cannabinoid biosynthesis is regulated during female flower development. We aim to understand the rate-limiting step(s) in the cannabinoid biosynthetic pathway. We investigated the transcript levels of cannabinoid biosynthetic genes together with cannabinoid contents during 7 weeks of female flower development. We demonstrated that the enzymatic steps for producing cannabigerol (CBG), which involve genes GPPS, PT, TKS, and OAC, could rate limit cannabinoid biosynthesis. Our findings further suggest that upregulation of cannabinoid synthases, CBDAS and THCAS in a commercial hemp and medical marijuana variety, respectively, is not critical for cannabinoid biosynthesis. The cannabinoid biosynthetic genes are generally upregulated during flower maturation; increased expression occurs coincident with glandular trichome development and cannabinoid production in the maturing flower. The results also suggest that different cannabis varieties may experience discrete transcriptional regulation of cannabinoid biosynthetic genes. In addition, we showed that methyl jasmonate (MeJA) can potentially increase cannabinoid production. We propose that biweekly applications of 100 µM MeJA starting from flower initiation would be efficacious for promoting cannabinoid biosynthesis. Our findings provide important genetic information for cannabis breeding to generate new varieties with favorable traits.

Overexpression of CsMIXTA, a Transcription Factor from Cannabis sativa, Increases Glandular Trichome Density in Tobacco Leaves.

Cannabinoids are synthesized in glandular stalked trichomes on the female flowers of Cannabis sativa (cannabis). The regulation of glandular trichome development has not been characterized in cannabis. We recently identified an R2R3-MYB transcription factor, CsMIXTA, which could be involved in trichome morphogenesis in cannabis. Some homologous genes of CsMIXTA are known to function in glandular trichome initiation in other plant species. CsMIXTA is highly expressed in flower tissue compared to vegetative tissues. Interestingly, CsMIXTA is also highly expressed in trichomes isolated from female flower tissue. In addition, CsMIXTA is upregulated during the peak stages of female flower maturation in correlation with some cannabinoid biosynthetic genes. Transient expression in Nicotiana benthamiana showed that CsMIXTA is localized in the nucleus. Furthermore, yeast transcriptional activation assay demonstrated that CsMIXTA has transactivation activity. Overexpression of CsMIXTA in Nicotiana tabacum resulted in higher trichome density, larger trichome size, and more branching on stalked glandular trichomes. The results indicate that CsMIXTA not only promotes glandular trichome initiation in epidermal cells, but also regulates trichome development in tobacco leaves. In this report, we characterized the novel function of the first cannabis transcription factor that may be critical for glandular trichome morphogenesis.

Effects of Harpin and Flg22 on Growth Enhancement and Pathogen Defense in Cannabis sativa Seedlings.

Pathogen-associated molecular patterns, PAMPs, are a diverse group of molecules associated with pathogenic microbes and are known to activate immune response and in some cases enhance growth in plants. Two PAMPs, harpin and flg22, have shown these affects in various plant species. PAMPs are known to activate basal immunity, the ethylene signaling pathway, alter gene expression and change plant composition. Pretreatment with harpin enhanced hemp seedling resistance to Pythium aphanidermatum, while flg22 failed to induce the defense mechanism towards P. aphanidermatum. In the absence of the pathogen, both harpin and flg22 enhanced seedling growth when compared to the water control. Ethylene is a hormone involved in both plant defense signaling and growth. Both harpin and flg22 pretreatment induced certain ethylene responsive genes but not all the genes examined, indicating that harpin and flg22 act differently in ethylene and potentially defense signaling. In addition, both harpin and flg22 induced CsFRK1 and CsPR1, two marker genes for plant innate immunity. Both PAMPs can enhance growth but likely induce different defense signaling pathways.

The Receptor Kinases BAK1/SERK4 Regulate Ca2+ Channel-Mediated Cellular Homeostasis for Cell Death Containment.

Cell death is a vital and ubiquitous process that is tightly controlled in all organisms. However, the mechanisms underlying precise cell death control remain fragmented. As an important shared module in plant growth, development, and immunity, Arabidopsis thaliana BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 (BAK1) and somatic embryogenesis receptor kinase 4 (SERK4) redundantly and negatively regulate plant cell death. By deploying an RNAi-based genetic screen for bak1/serk4 cell death suppressors, we revealed that cyclic nucleotide-gated channel 20 (CNGC20) functions as a hyperpolarization-activated Ca2+-permeable channel specifically regulating bak1/serk4 cell death. BAK1 directly interacts with and phosphorylates CNGC20 at specific sites in the C-terminal cytosolic domain, which in turn regulates CNGC20 stability. CNGC19, the closest homolog of CNGC20 with a low abundance compared with CNGC20, makes a quantitative genetic contribution to bak1/serk4 cell death only in the absence of CNGC20, supporting the biochemical data showing homo- and heteromeric assembly of the CNGC20 and CNGC19 channel complexes. Transcripts of CNGC20 and CNGC19 are elevated in bak1/serk4 compared with wild-type plants, further substantiating a critical role of homeostasis of CNGC20 and CNGC19 in cell death control. Our studies not only uncover a unique regulation of ion channel stability by cell-surface-resident receptor kinase-mediated phosphorylation but also provide evidence for fine-tuning Ca2+ channel functions in maintaining cellular homeostasis by the formation of homo- and heterotetrameric complexes.

Intracellular Ca2+ is important for flagellin-triggered defense in Arabidopsis and involves inositol polyphosphate signaling.

Cytosolic Ca2+ increase is a crucial and early step of plant immunity evoked by pathogen-associated molecular patterns (PAMPs) such as flagellin (flg). Components responsible for this increase are still not uncovered, although current models of plant immune signaling portray extracellular Ca2+ influx as paramount to flg activation of defense pathways. Work presented here provides new insights into cytosolic Ca2+ increase associated with flg-induced defense responses. We show that extracellular Ca2+ contributes more to immune responses evoked by plant elicitor peptide (Pep3) than that evoked by flg, indicating an intracellular Ca2+ source responsible for immune responses evoked by flg. Genetic impairment of the inositol polyphosphate (InsP) and G-protein signal associated with flg perception reduced flg-dependent immune responses. Previous work indicates that prior exposure of Arabidopsis plants to flg leads to an immune response reflected by less vigorous growth of a pathogenic microbe. We found that this immune response to flg was compromised in mutants lacking the ability to generate an InsP or G-protein signal. We conclude that the recruitment of intracellular Ca2+ stores by flg may involve InsP and G-protein signaling. We also found a notable difference in contribution of intracellular stores of Ca2+ to the immune signaling evoked by another PAMP, elf18 peptide, which had a very different response profile to impairment of InsP signaling. Although Ca2+ signaling is at the core of the innate immune as well as hypersensitive response to plant pathogens, it appears that the molecular mechanisms generating the Ca2+ signal in response to different PAMPs are different.

The CLAVATA signaling pathway mediating stem cell fate in shoot meristems requires Ca(2+) as a secondary cytosolic messenger.

CLAVATA1 (CLV1) is a receptor protein expressed in the shoot apical meristem (SAM) that translates perception of a non-cell-autonomous CLAVATA3 (CLV3) peptide signal into altered stem cell fate. CLV3 reduces expression of WUSCHEL (WUS) and FANTASTIC FOUR 2 (FAF2) in the SAM. Expression of WUS and FAF2 leads to maintenance of undifferentiated stem cells in the SAM. CLV3 binding to CLV1 inhibits expression of these genes and controls stem cell fate in the SAM through an unidentified signaling pathway. Cytosolic Ca(2+) elevations, cyclic nucleotide (cGMP)-activated Ca(2+) channels, and cGMP have been linked to signaling downstream of receptors similar to CLV1. Hence, we hypothesized that cytosolic Ca(2+) elevation mediates the CLV3 ligand/CLV1 receptor signaling that controls meristem stem cell fate. CLV3 application to Arabidopsis seedlings results in elevation of cytosolic Ca(2+) and cGMP. CLV3 control of WUS was prevented in a genotype lacking a functional cGMP-activated Ca(2+) channel. In wild-type plants, CLV3 inhibition of WUS and FAF2 expression was impaired by treatment with either a Ca(2+) channel blocker or a guanylyl cyclase inhibitor. When CLV3-dependent repression of WUS is blocked, altered control of stem cell fate leads to an increase in SAM size; we observed a larger SAM size in seedlings treated with the Ca(2+) channel blocker. These results suggest that the CLV3 ligand/CLV1 receptor system initiates a signaling cascade that elevates cytosolic Ca(2+), and that this cytosolic secondary messenger is involved in the signal transduction cascade linking CLV3/CLV1 to control of gene expression and stem cell fate in the SAM.

Cloning and characterization of Na+/H+ antiporter (LfNHX1) gene from a halophyte grass Leptochloa fusca for drought and salt tolerance.

Abiotic stresses such as salinity and drought have adverse effects on plants. In the present study, a Na(+)/H(+) antiporter gene homologue (LfNHX1) has been cloned from a local halophyte grass (Leptochloa fusca). The LfNHX1 cDNA contains an open reading frame of 1,623 bp that encodes a polypeptide chain of 540 amino acid residues. LfNHX1 protein sequence showed high similarity with NHX1 homologs reported from other halophyte plants. Amino acid and nucleotide sequence similarity, protein topology modeling and the presence of conserved functional domains in the LfNHX1 protein sequence classified it as a vacuolar NHX1 homolog. The overexpression of LfNHX1 gene under CaMV35S promoter conferred salt and drought tolerance in tobacco plants. Under drought stress, transgenic plants showed higher relative water contents, photosynthetic rate, stomatal conductance and membrane stability index as compared to wild type plants. More negative value of leaf osmotic potential was also observed in transgenic plants when compared with wild type control plants. Transgenic plants showed better germination and root growth at 2 mg L(-1) Basta herbicide and three levels (100, 200 and 250 mM) of sodium chloride. These results showed that LfNHX1 is a potential candidate gene for enhancing drought and salt tolerance in crops.

Molecular steps in the immune signaling pathway evoked by plant elicitor peptides: Ca2+-dependent protein kinases, nitric oxide, and reactive oxygen species are downstream from the early Ca2+ signal.

Endogenous plant elicitor peptides (Peps) can act to facilitate immune signaling and pathogen defense responses. Binding of these peptides to the Arabidopsis (Arabidopsis thaliana) plasma membrane-localized Pep receptors (PEPRs) leads to cytosolic Ca(2+) elevation, an early event in a signaling cascade that activates immune responses. This immune response includes the amplification of signaling evoked by direct perception of pathogen-associated molecular patterns by plant cells under assault. Work included in this report further characterizes the Pep immune response and identifies new molecular steps in the signal transduction cascade. The PEPR coreceptor BRASSINOSTEROID-INSENSITIVE1 Associated Kinase1 contributes to generation of the Pep-activated Ca(2+) signal and leads to increased defense gene expression and resistance to a virulent bacterial pathogen. Ca(2+)-dependent protein kinases (CPKs) decode the Ca(2+) signal, also facilitating defense gene expression and enhanced resistance to the pathogen. Nitric oxide and reduced nicotinamide adenine dinucleotide phosphate oxidase-dependent reactive oxygen species generation (due to the function of Respiratory Burst Oxidase Homolog proteins D and F) are also involved downstream from the Ca(2+) signal in the Pep immune defense signal transduction cascade, as is the case with BRASSINOSTEROID-INSENSITIVE1 Associated Kinase1 and CPK5, CPK6, and CPK11. These steps of the pathogen defense response are required for maximal Pep immune activation that limits growth of a virulent bacterial pathogen in the plant. We find a synergism between function of the PEPR and Flagellin Sensing2 receptors in terms of both nitric oxide and reactive oxygen species generation. Presented results are also consistent with the involvement of the secondary messenger cyclic GMP and a cyclic GMP-activated Ca(2+)-conducting channel in the Pep immune signaling pathway.

Teaching an old hormone new tricks: cytosolic Ca2+ elevation involvement in plant brassinosteroid signal transduction cascades.

Brassinosteroids (BRs) are hormones that control many aspects of plant growth and development, acting at the cell level to promote division and expansion. BR regulation of plant and plant cell function occurs through altered expression of many genes. Transcriptional reprogramming downstream from cell perception of this hormone is currently known to be mediated by a phosphorylation/dephosphorylation ("phosphorelay") cascade that alters the stability of two master transcription regulators. Here, we provide evidence that BR perception by their receptor also causes an elevation in cytosolic Ca(2+), initiating a Ca(2+) signaling cascade in Arabidopsis (Arabidopsis thaliana) cell cytosol. BR-dependent increases in the expression of some genes (INDOLE-3-ACETIC ACID-INDUCIBLE1 and PHYTOCHROME B ACTIVATION-TAGGED SUPPRESSOR1) were impaired in wild-type plants by a Ca(2+) channel blocker and also in the defense-no-death (dnd1) mutant, which lacks a functional cyclic GMP-activated cell membrane Ca(2+)-conducting channel. Alternatively, mutations that impair the BR phosphorelay cascade did not much affect the BR-dependent expression of these genes. Similar effects of the Ca(2+) channel blocker and dnd1 mutation were observed on a BR plant growth phenotype, deetiolation of the seedling hypocotyl. Further evidence presented in this report suggests that a BR-dependent elevation in cyclic GMP may be involved in the Ca(2+) signaling cascade initiated by this hormone. The work presented here leads to a new model of the molecular steps that mediate some of the cell responses to this plant hormone.

There's more to the picture than meets the eye: nitric oxide cross talk with Ca2+ signaling.

Calcium and nitric oxide (NO) are two important biological messengers. Increasing evidence indicates that Ca(2+) and NO work together in mediating responses to pathogenic microorganisms and microbe-associated molecular patterns. Ca(2+) fluxes were recognized to account for NO production, whereas evidence gathered from a number of studies highlights that NO is one of the key messengers mediating Ca(2+) signaling. Here, we present a concise description of the current understanding of the molecular mechanisms underlying the cross talk between Ca(2+) and NO in plant cells exposed to biotic stress. Particular attention will be given to the involvement of cyclic nucleotide-gated ion channels and Ca(2+) sensors. Notably, we provide new evidence that calmodulin might be regulated at the posttranslational level by NO through S-nitrosylation. Furthermore, we report original transcriptomic data showing that NO produced in response to oligogalacturonide regulates the expression of genes related to Ca(2+) signaling. Deeper insight into the molecules involved in the interplay between Ca(2+) and NO not only permits a better characterization of the Ca(2+) signaling system but also allows us to further understand how plants respond to pathogen attack.

Identification of cyclic nucleotide gated channels using regular expressions.

Cyclic nucleotide-gated channels (CNGCs) are nonselective cation channels found in plants, animals, and some bacteria. They have a six-transmembrane/one-pore structure, a cytosolic cyclic nucleotide-binding domain, and a cytosolic calmodulin-binding domain. Despite their functional similarities, the plant CNGC family members appear to have different conserved amino acid motifs within corresponding functional domains than animal and bacterial CNGCs do. Here we describe the development and application of methods employing plant CNGC-specific sequence motifs as diagnostic tools to identify novel candidate channels in different plants. These methods are used to evaluate the validity of annotations of putative orthologs of CNGCs from plant genomes. The methods detail how to employ regular expressions of conserved amino acids in functional domains of annotated CNGCs and together with Web tools such as PHI-BLAST and ScanProsite to identify novel candidate CNGCs in species including Physcomitrella patens.

Detection of reactive oxygen species downstream of cyclic nucleotide signals in plants.

Cyclic nucleotides act in plant cell signal transduction cascades by activating cyclic nucleotide gated cation-conducting ion channels (CNGCs). Activation of CNGCs results in inward cation (including Ca(2+)) conductance across the plasma membrane. Elevation of cytosolic Ca(2+) is an early step in numerous plant cell signal transduction cascades, including plant immune responses to pathogens. CNGC involvement, along with cyclic nucleotides cAMP and cGMP, in pathogen defense programs is one relatively well-studied area of cyclic nucleotide signaling in plants. During plant immune responses, CNGC-dependent Ca(2+) elevations lead to a signaling cascade that results in the generation of defense molecules such as hydrogen peroxide and nitric oxide, and induction of defense gene expression. This pathogen defense response is discussed, and methods to detect some of the downstream signaling steps in the pathway are presented.

Linking ligand perception by PEPR pattern recognition receptors to cytosolic Ca2+ elevation and downstream immune signaling in plants.

Little is known about molecular steps linking perception of pathogen invasion by cell surface sentry proteins acting as pattern recognition receptors (PRRs) to downstream cytosolic Ca(2+) elevation, a critical step in plant immune signaling cascades. Some PRRs recognize molecules (such as flagellin) associated with microbial pathogens (pathogen-associated molecular patterns, PAMPs), whereas others bind endogenous plant compounds (damage-associated molecular patterns, DAMPs) such as peptides released from cells upon attack. This work focuses on the Arabidopsis DAMPs plant elicitor peptides (Peps) and their receptors, PEPR1 and PEPR2. Pep application causes in vivo cGMP generation and downstream signaling that is lost when the predicted PEPR receptor guanylyl cyclase (GC) active site is mutated. Pep-induced Ca(2+) elevation is attributable to cGMP activation of a Ca(2+) channel. Some differences were identified between Pep/PEPR signaling and the Ca(2+)-dependent immune signaling initiated by the flagellin peptide flg22 and its cognate receptor Flagellin-sensing 2 (FLS2). FLS2 signaling may have a greater requirement for intracellular Ca(2+) stores and inositol phosphate signaling, whereas Pep/PEPR signaling requires extracellular Ca(2+). Maximal FLS2 signaling requires a functional Pep/PEPR system. This dependence was evidenced as a requirement for functional PEPR receptors for maximal flg22-dependent Ca(2+) elevation, H(2)O(2) generation, defense gene [WRKY33 and Plant Defensin 1.2 (PDF1.2)] expression, and flg22/FLS2-dependent impairment of pathogen growth. In a corresponding fashion, FLS2 loss of function impaired Pep signaling. In addition, a role for PAMP and DAMP perception in bolstering effector-triggered immunity (ETI) is reported; loss of function of either FLS2 or PEPR receptors impaired the hypersensitive response (HR) to an avirulent pathogen.

Evolutionary and structural perspectives of plant cyclic nucleotide-gated cation channels.

Ligand-gated cation channels are a frequent component of signaling cascades in eukaryotes. Eukaryotes contain numerous diverse gene families encoding ion channels, some of which are shared and some of which are unique to particular kingdoms. Among the many different types are cyclic nucleotide-gated channels (CNGCs). CNGCs are cation channels with varying degrees of ion conduction selectivity. They are implicated in numerous signaling pathways and permit diffusion of divalent and monovalent cations, including Ca(2+) and K(+). CNGCs are present in both plant and animal cells, typically in the plasma membrane; recent studies have also documented their presence in prokaryotes. All eukaryote CNGC polypeptides have a cyclic nucleotide-binding domain and a calmodulin binding domain as well as a six transmembrane/one pore tertiary structure. This review summarizes existing knowledge about the functional domains present in these cation-conducting channels, and considers the evidence indicating that plant and animal CNGCs evolved separately. Additionally, an amino acid motif that is only found in the phosphate binding cassette and hinge regions of plant CNGCs, and is present in all experimentally confirmed CNGCs but no other channels was identified. This CNGC-specific amino acid motif provides an additional diagnostic tool to identify plant CNGCs, and can increase confidence in the annotation of open reading frames in newly sequenced genomes as putative CNGCs. Conversely, the absence of the motif in some plant sequences currently identified as probable CNGCs may suggest that they are misannotated or protein fragments.