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.

Danger-Associated Peptides Close Stomata by OST1-Independent Activation of Anion Channels in Guard Cells.

The plant elicitor peptides (Peps), a family of damage/danger-associated molecular patterns (DAMPs), are perceived by two receptors, PEPR1 and PEPR2, and contribute to plant defense against pathogen attack and abiotic stress. Here, we show that the Peps-PEPR signaling pathway functions in stomatal immunity by activating guard cell anion channels in Arabidopsis thaliana The mutant plants lacking both PEPR1 and PEPR2 (pepr1 pepr2) displayed enhanced bacterial growth after being sprayed with Pseudomonas syringae pv tomato (Pst) DC3000, but not after pathogen infiltration into leaves, implicating PEPR function in stomatal immunity. Indeed, synthetic Arabidopsis Peps (AtPeps) effectively induced stomatal closure in wild-type but not pepr1 pepr2 mutant leaves, suggesting that the AtPeps-PEPR signaling pathway triggers stomatal closure. Consistent with this finding, patch-clamp recording revealed AtPep1-induced activation of anion channels in the guard cells of wild-type but not pepr1 pepr2 mutant plants. We further identified two guard cell-expressed anion channels, SLOW ANION CHANNEL1 (SLAC1) and its homolog SLAH3, as functionally overlapping components responsible for AtPep1-induced stomatal closure. The slac1 slah3 double mutant, but not slac1 or slah3 single mutants, failed to respond to AtPep1 in stomatal closure assays. Interestingly, disruption of OPEN STOMATA1 (OST1), an essential gene for abscisic acid-triggered stomatal closure, did not affect the AtPep1-induced anion channel activity and stomatal response. Together, these results illustrate a DAMP-triggered signaling pathway that, unlike the flagellin22-FLAGELLIN-SENSITIVE2 pathway, triggers stomata immunity through an OST1-independent mechanism.

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.

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.

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.

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.

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.

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.

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.

Ca2+ signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca2+ channels.

A family of peptide signaling molecules (AtPeps) and their plasma membrane receptor AtPepR1 are known to act in pathogen-defense signaling cascades in plants. Little is currently known about the molecular mechanisms that link these signaling peptides and their receptor, a leucine-rich repeat receptor-like kinase, to downstream pathogen-defense responses. We identify some cellular activities of these molecules that provide the context for a model for their action in signaling cascades. AtPeps activate plasma membrane inwardly conducting Ca(2+) permeable channels in mesophyll cells, resulting in cytosolic Ca(2+) elevation. This activity is dependent on their receptor as well as a cyclic nucleotide-gated channel (CNGC2). We also show that the leucine-rich repeat receptor-like kinase receptor AtPepR1 has guanylyl cyclase activity, generating cGMP from GTP, and that cGMP can activate CNGC2-dependent cytosolic Ca(2+) elevation. AtPep-dependent expression of pathogen-defense genes (PDF1.2, MPK3, and WRKY33) is mediated by the Ca(2+) signaling pathway associated with AtPep peptides and their receptor. The work presented here indicates that extracellular AtPeps, which can act as danger-associated molecular patterns, signal by interaction with their receptor, AtPepR1, a plasma membrane protein that can generate cGMP. Downstream from AtPep and AtPepR1 in a signaling cascade, the cGMP-activated channel CNGC2 is involved in AtPep- and AtPepR1-dependent inward Ca(2+) conductance and resulting cytosolic Ca(2+) elevation. The signaling cascade initiated by AtPeps leads to expression of pathogen-defense genes in a Ca(2+)-dependent manner.

Plant Elicitor Peptide (Pep) Signaling and Pathogen Defense in Tomato.

Endogenous signaling compounds are intermediaries in signaling pathways that plants use to respond to the perception of harmful and beneficial organisms. The plant elicitor peptides (Peps) of plants are important endogenous signaling molecules that induce elements of defense responses such as hormone production, increased expression of defensive genes, the activation of phosphorelays, and the induction of cell secondary messenger synthesis. The processes by which Peps confer resistance to pathogenic microorganisms have been extensively studied in Arabidopsis but are less known in crop plants. Tomato and many other solanaceous plants have an endogenous signaling polypeptide, systemin, that is involved in the defense against herbivorous insects and necrotrophic pathogens. This paper explores the similarity of the effects and chemical properties of Pep and systemin in tomato. Additionally, the relationship of the Pep receptor and systemin receptors is explored, and the identification of a second tomato Pep receptor in the literature is called into question. We suggest future directions for research on Pep signaling in solanaceous crops during interactions with microbes.

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.

Ca2+, cAMP, and transduction of non-self perception during plant immune responses.

Ca(2+) influx is an early signal initiating cytosolic immune responses to pathogen perception in plant cells; molecular components linking pathogen recognition to Ca(2+) influx are not delineated. Work presented here provides insights into this biological system of non-self recognition and response activation. We have recently identified a cyclic nucleotide-activated ion channel as facilitating the Ca(2+) flux that initiates immune signaling in the plant cell cytosol. Work in this report shows that elevation of cAMP is a key player in this signaling cascade. We show that cytosolic Ca(2+) elevation, nitric oxide (NO) and reactive oxygen species generation, as well as immune signaling, lead to a hypersensitive response upon application of pathogens and/or conserved molecules that are components of microbes and are all dependent on cAMP generation. Exogenous cAMP leads to Ca(2+) channel-dependent cytosolic Ca(2+) elevation, NO generation, and defense response gene expression in the absence of the non-self pathogen signal. Inoculation of leaves with a bacterial pathogen leads to cAMP elevation coordinated with Ca(2+) rise. cAMP acts as a secondary messenger in plants; however, no specific protein has been heretofore identified as activated by cAMP in a manner associated with a signaling cascade in plants, as we report here. Our linkage of cAMP elevation in pathogen-inoculated plant leaves to Ca(2+) channels and immune signaling downstream from cytosolic Ca(2+) elevation provides a model for how non-self detection can be transduced to initiate the cascade of events in the cell cytosol that orchestrate pathogen defense responses.

Genome-Wide Identification and Expression Analysis of Wall-Associated Kinase (WAK) Gene Family in Cannabis sativa L.

Wall-associated kinases (WAKs) are receptors that bind pectin or small pectic fragments in the cell wall and play roles in cell elongation and pathogen response. In the Cannabis sativa (Cs) genome, 53 CsWAK/CsWAKL (WAK-like) protein family members were identified and characterized; their amino acid lengths and molecular weights varied from 582 to 983, and from 65.6 to 108.8 kDa, respectively. They were classified into four main groups by a phylogenetic tree. Out of the 53 identified CsWAK/CsWAKL genes, 23 CsWAK/CsWAKL genes were unevenly distributed among six chromosomes. Two pairs of genes on chromosomes 4 and 7 have undergone duplication. The number of introns and exons among CsWAK/CsWAKL genes ranged from 1 to 6 and from 2 to 7, respectively. The promoter regions of 23 CsWAKs/CsWAKLs possessed diverse cis-regulatory elements that are involved in light, development, environmental stress, and hormone responsiveness. The expression profiles indicated that our candidate genes (CsWAK1, CsWAK4, CsWAK7, CsWAKL1, and CsWAKL7) are expressed in leaf tissue. These genes exhibit different expression patterns than their homologs in other plant species. These initial findings are useful resources for further research work on the potential roles of CsWAK/CsWAKL in cellular signalling during development, environmental stress conditions, and hormone treatments.

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.

Genome-Wide Identification and Expression Analysis of Homeodomain Leucine Zipper Subfamily IV (HD-ZIP IV) Gene Family in Cannabis sativa L.

The plant-specific homeodomain zipper family (HD-ZIP) of transcription factors plays central roles in regulating plant development and environmental resistance. HD-ZIP transcription factors IV (HDZ IV) have been involved primarily in the regulation of epidermal structure development, such as stomata and trichomes. In our study, we identified nine HDZ IV-encoding genes in Cannabis sativa L. by conducting a computational analysis of cannabis genome resources. Our analysis suggests that these genes putatively encode proteins that have all the conserved domains of HDZ IV transcription factors. The phylogenetic analysis of HDZ IV gene family members of cannabis, rice (Oryza sativa), and Arabidopsis further implies that they might have followed distinct evolutionary paths after divergence from a common ancestor. All the identified cannabis HDZ IV gene promoter sequences have multiple regulation motifs, such as light- and hormone-responsive elements. Furthermore, experimental evidence shows that different HDZ IV genes have different expression patterns in root, stem, leaf, and flower tissues. Four genes were primarily expressed in flowers, and the expression of CsHDG5 (XP_030501222.1) was also correlated with flower maturity. Fifty-nine genes were predicted as targets of HDZ IV transcription factors. Some of these genes play central roles in pathogen response, flower development, and brassinosteroid signaling. A subcellular localization assay indicated that one gene of this family is localized in the Arabidopsis protoplast nucleus. Taken together, our work lays fundamental groundwork to illuminate the function of cannabis HDZ IV genes and their possible future uses in increasing cannabis trichome morphogenesis and secondary metabolite production.

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.

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.

Ca2+ conduction by plant cyclic nucleotide gated channels and associated signaling components in pathogen defense signal transduction cascades.

Ca(2+) elevation in the cytosol is an essential early event during pathogen response signaling cascades. However, the specific ion channels involved in Ca(2+) influx into plant cells, and how Ca(2+) signals are initiated and regulate downstream events during pathogen defense responses, are at present unclear. Plant cyclic nucleotide gated ion channels (CNGCs) provide a pathway for Ca(2+) conductance across the plasma membrane (PM) and facilitate cytosolic Ca(2+) elevation in response to pathogen signals. Recent studies indicate that the recognition of pathogens results in cyclic nucleotide production and the activation of CNGCs, which leads to downstream generation of pivotal signaling molecules (such as nitric oxide (NO)). Calmodulins (CaMs) and CaM-like proteins (CMLs) are also involved in this signaling, functioning as Ca(2+) sensors and mediating the synthesis of NO during the plant pathogen response signaling cascade. In this article, these and other pivotal signaling components downstream from the Ca(2+) signal, such as Ca(2+)-dependent protein kinases (CDPKs) and CaM-binding transcription activators (CAMTAs), are discussed in terms of their involvement in the pathogen response signal transduction cascade.