Marco Herde

Calcium-Dependent Protein Kinases: Hubs in Plant Stress Signaling and Development

These kinases are identified as integrators in plant signaling, with distinct as well as shared phosphorylation substrates mediating pathway specificity.

A bHLH-type transcription factor, ABA-INDUCIBLE BHLH-TYPE TRANSCRIPTION FACTOR/JA-ASSOCIATED MYC2-LIKE1, acts as a repressor to negatively regulate jasmonate signaling in arabidopsis.

This work identifies the bHLH transcription factor, JA-ASSOCIATED MYC2-LIKE1 (JAM1), which negatively regulates jasmonate responses independently from known regulators, namely, JASMONATE-ZIM DOMAIN proteins. JAM1 is involved in JA-mediated male fertility and the defense response to insect attack and represses the transcription of the target genes of MYC2 by competing for the binding sequences. Jasmonates (JAs) are plant hormones that regulate the balance between plant growth and responses to biotic and abiotic stresses. Although recent studies have uncovered the mechanisms for JA-induced responses in Arabidopsis thaliana, the mechanisms by which plants attenuate the JA-induced responses remain elusive. Here, we report that a basic helix-loop-helix–type transcription factor, ABA-INDUCIBLE BHLH-TYPE TRANSCRIPTION FACTOR/JA-ASSOCIATED MYC2-LIKE1 (JAM1), acts as a transcriptional repressor and negatively regulates JA signaling. Gain-of-function transgenic plants expressing the chimeric repressor for JAM1 exhibited substantial reduction of JA responses, including JA-induced inhibition of root growth, accumulation of anthocyanin, and male fertility. These plants were also compromised in resistance to attack by the insect herbivore Spodoptera exigua. Conversely, jam1 loss-of-function mutants showed enhanced JA responsiveness, including increased resistance to insect attack. JAM1 and MYC2 competitively bind to the target sequence of MYC2, which likely provides the mechanism for negative regulation of JA signaling and suppression of MYC2 functions by JAM1. These results indicate that JAM1 negatively regulates JA signaling, thereby playing a pivotal role in fine-tuning of JA-mediated stress responses and plant growth.

Identification and Regulation of TPS04/GES, an Arabidopsis Geranyllinalool Synthase Catalyzing the First Step in the Formation of the Insect-Induced Volatile C16-Homoterpene TMTT

Volatile secondary metabolites emitted by plants contribute to plant–plant, plant–fungus, and plant–insect interactions. The C16-homoterpene TMTT (for 4,8,12-trimethyltrideca-1,3,7,11-tetraene) is emitted after herbivore attack by a wide variety of plant species, including Arabidopsis thaliana, and is assumed to play a role in attracting predators or parasitoids of herbivores. TMTT has been suggested to be formed as a degradation product of the diterpene alcohol (E,E)-geranyllinalool. Here, we report the identification of Terpene Synthase 04 (TPS04; At1g61120) as a geranyllinalool synthase (GES). Recombinant TPS04/GES protein expressed in Escherichia coli catalyzes the formation of (E,E)-geranyllinalool from the substrate geranylgeranyl diphosphate. Transgenic Arabidopsis lines carrying T-DNA insertions in the TPS04 locus are deficient in (E,E)-geranyllinalool and TMTT synthesis, a phenotype that can be complemented by expressing the GES gene under the control of a heterologous promoter. GES transcription is upregulated under conditions that induce (E,E)-geranyllinalool and TMTT synthesis, including infestation of plants with larvae of the moth Plutella xylostella and treatment with the fungal peptide alamethicin or the octadecanoid mimic coronalon. Induction requires jasmonic acid but is independent from salicylic acid or ethylene. This study paves the ground to address the contribution of TMTT in ecological interactions and to elucidate the signaling network that regulates TMTT synthesis.

Herbivore-induced and floral homoterpene volatiles are biosynthesized by a single P450 enzyme (CYP82G1) in Arabidopsis

Terpene volatiles play important roles in plant-organism interactions as attractants of pollinators or as defense compounds against herbivores. Among the most common plant volatiles are homoterpenes, which are often emitted from night-scented flowers and from aerial tissues upon herbivore attack. Homoterpene volatiles released from herbivore-damaged tissue are thought to contribute to indirect plant defense by attracting natural enemies of pests. Moreover, homoterpenes have been demonstrated to induce defensive responses in plant–plant interaction. Although early steps in the biosynthesis of homoterpenes have been elucidated, the identity of the enzyme responsible for the direct formation of these volatiles has remained unknown. Here, we demonstrate that CYP82G1 (At3g25180), a cytochrome P450 monooxygenase of the Arabidopsis CYP82 family, is responsible for the breakdown of the C20-precursor (E,E)-geranyllinalool to the insect-induced C16-homoterpene (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT). Recombinant CYP82G1 shows narrow substrate specificity for (E,E)-geranyllinalool and its C15-analog (E)-nerolidol, which is converted to the respective C11-homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT). Homology-based modeling and substrate docking support an oxidative bond cleavage of the alcohol substrate via syn-elimination of the polar head, together with an allylic C-5 hydrogen atom. CYP82G1 is constitutively expressed in Arabidopsis stems and inflorescences and shows highly coordinated herbivore-induced expression with geranyllinalool synthase in leaves depending on the F-box protein COI-1. CYP82G1 represents a unique characterized enzyme in the plant CYP82 family with a function as a DMNT/TMTT homoterpene synthase.

From local to global: CDPKs in systemic defense signaling upon microbial and herbivore attack

Calcium-dependent protein kinases (CDPKs) are multifunctional proteins in which a calmodulin-like calcium-sensor and a protein kinase effector domain are combined in one molecule. Not surprisingly, CDPKs were primarily recognized as signaling mediators, which perceive rapid intracellular changes of Ca(2+) ion concentration, for example triggered by environmental stress cues, and relay them into specific phosphorylation events to induce further downstream stress responses. In the context of both, plant exposure to biotrophic pathogens-derived signals as well as plant attack by herbivores and wounding, CDPKs were shown to undergo rapid biochemical activation within seconds to minutes after stimulation and to induce local defence-responses including respective changes in gene expression patterns. In addition, CDPK function was correlated with the control of either salicylic acid-mediated or jasmonic acid-mediated phytohormone signaling pathways, mediating long term resistance to either biotrophic bacterial pathogens or herbivores also in distal parts of a plant. It has long been unclear how an individual enzyme can affect both rapid local as well as long-term distal immune responses. Here, we discuss recently raised topics from the field of CDPK research, in particular with a view on the identification of in vivo phosphorylation targets, which provide first mechanistic insights into the dual role of these enzymes: On the one hand as component of a self-activating circuit responsible for rapid plasma-membrane anchored cell-to-cell signal propagation from local to distal plant sites. On the other hand as nuclear-located regulators of transcription factor activity. Finally, we will highlight the dual function of calcium sensors in plasma-membrane/calcium-mediated signal propagation and in phytohormone signaling-dependent systemic resistance in immune responses to both, bacterial pathogens and herbivores.

The Calcium-Dependent Protein Kinase CPK28 Regulates Development by Inducing Growth Phase-Specific, Spatially Restricted Alterations in Jasmonic Acid Levels Independent of Defense Responses in Arabidopsis

A calcium-dependent protein kinase plays dual roles, maintaining tissue-specific phytohormone levels in adult plants without affecting defense responses and regulating defense signaling in seedlings. Phytohormones play an important role in development and stress adaptations in plants, and several interacting hormonal pathways have been suggested to accomplish fine-tuning of stress responses at the expense of growth. This work describes the role played by the CALCIUM-DEPENDENT PROTEIN KINASE CPK28 in balancing phytohormone-mediated development in Arabidopsis thaliana, specifically during generative growth. cpk28 mutants exhibit growth reduction solely as adult plants, coinciding with altered balance of the phytohormones jasmonic acid (JA) and gibberellic acid (GA). JA-dependent gene expression and the levels of several JA metabolites were elevated in a growth phase-dependent manner in cpk28, and accumulation of JA metabolites was confined locally to the central rosette tissue. No elevated resistance toward herbivores or necrotrophic pathogens was detected for cpk28 plants, either on the whole-plant level or specifically within the tissue displaying elevated JA levels. Abolishment of JA biosynthesis or JA signaling led to a full reversion of the cpk28 growth phenotype, while modification of GA signaling did not. Our data identify CPK28 as a growth phase-dependent key negative regulator of distinct processes: While in seedlings, CPK28 regulates reactive oxygen species-mediated defense signaling; in adult plants, CPK28 confers developmental processes by the tissue-specific balance of JA and GA without affecting JA-mediated defense responses.

Elicitation of Jasmonate-Mediated Defense Responses by Mechanical Wounding and Insect Herbivory

Many plant immune responses to biotic stress are mediated by the wound hormone jasmonate (JA). Functional and mechanistic studies of the JA signaling pathway often involve plant manipulations that elicit JA production and subsequent changes in gene expression in local and systemic tissues. Here, we describe a simple mechanical wounding procedure to effectively trigger JA responses in the Arabidopsis thaliana rosette. For comparison, we also present a plant-insect bioassay to elicit defense responses with the chewing insect Trichoplusia ni. This latter procedure can be used to determine the effect of JA-regulated defenses on growth and development of insect herbivores.

Jasmonic Acid Enhances Al-Induced Root Growth Inhibition

Jasmonate mediates aluminum-induced root growth inhibition through regulation of microtubule polymerization and ALMT1-regulated malate exudation in an auxin-independent manner in Arabidopsis. Phytohormones such as ethylene and auxin are involved in the regulation of the aluminum (Al)-induced root growth inhibition. Although jasmonate (JA) has been reported to play a crucial role in the regulation of root growth and development in response to environmental stresses through interplay with ethylene and auxin, its role in the regulation of root growth response to Al stress is not yet known. In an attempt to elucidate the role of JA, we found that exogenous application of JA enhanced the Al-induced root growth inhibition. Furthermore, phenotype analysis with mutants defective in either JA biosynthesis or signaling suggests that JA is involved in the regulation of Al-induced root growth inhibition. The expression of the JA receptor CORONATINE INSENSITIVE1 (COI1) and the key JA signaling regulator MYC2 was up-regulated in response to Al stress in the root tips. This process together with COI1-mediated Al-induced root growth inhibition under Al stress was controlled by ethylene but not auxin. Transcriptomic analysis revealed that many responsive genes under Al stress were regulated by JA signaling. The differential responsive of microtubule organization-related genes between the wild-type and coi1-2 mutant is consistent with the changed depolymerization of cortical microtubules in coi1 under Al stress. In addition, ALMT-mediated malate exudation and thus Al exclusion from roots in response to Al stress was also regulated by COI1-mediated JA signaling. Together, this study suggests that root growth inhibition is regulated by COI1-mediated JA signaling independent from auxin signaling and provides novel insights into the phytohormone-mediated root growth inhibition in response to Al stress.

Of the Nine Cytidine Deaminase-Like Genes in Arabidopsis, Eight Are Pseudogenes and Only One Is Required to Maintain Pyrimidine Homeostasis in Vivo

Bioinformatic, biochemical, and reverse genetics analyses suggest that only one of the nine annotated Arabidopsis cytidine deaminase genes encodes a fully functional enzyme involved in pyrimidine catabolism. CYTIDINE DEAMINASE (CDA) catalyzes the deamination of cytidine to uridine and ammonia in the catabolic route of C nucleotides. The Arabidopsis (Arabidopsis thaliana) CDA gene family comprises nine members, one of which (AtCDA) was shown previously in vitro to encode an active CDA. A possible role in C-to-U RNA editing or in antiviral defense has been discussed for other members. A comprehensive bioinformatic analysis of plant CDA sequences, combined with biochemical functionality tests, strongly suggests that all Arabidopsis CDA family members except AtCDA are pseudogenes and that most plants only require a single CDA gene. Soybean (Glycine max) possesses three CDA genes, but only two encode functional enzymes and just one has very high catalytic efficiency. AtCDA and soybean CDAs are located in the cytosol. The functionality of AtCDA in vivo was demonstrated with loss-of-function mutants accumulating high amounts of cytidine but also CMP, cytosine, and some uridine in seeds. Cytidine hydrolysis in cda mutants is likely caused by NUCLEOSIDE HYDROLASE1 (NSH1) because cytosine accumulation is strongly reduced in a cda nsh1 double mutant. Altered responses of the cda mutants to fluorocytidine and fluorouridine indicate that a dual specific nucleoside kinase is involved in cytidine as well as uridine salvage. CDA mutants display a reduction in rosette size and have fewer leaves compared with the wild type, which is probably not caused by defective pyrimidine catabolism but by the accumulation of pyrimidine catabolism intermediates reaching toxic concentrations.

Efficient generation of mutations mediated by CRISPR/Cas9 in the hairy root transformation system of Brassica carinata

A protocol for the induction of site-directed deletions and insertions in the genome of Brassica carinata with CRISPR is described. The construct containing the Cas9 nuclease and the guide RNA (gRNA) was delivered by the hairy root transformation technique, and a successful transformation was monitored by GFP fluorescence. PAGE analysis of an amplified region, presumably containing the deletions and insertions, demonstrated up to seven different indels in one transgenic root and in all analyzed roots a wildtype allele of the modified gene was not detectable. Interestingly, many of these mutations consisted of relatively large indels with up to 112 bp. The exact size of the deletions was determined to allow an estimation whether the targeted gene was not functional due to a considerable deletion or a frame shift within the open reading frame. This allowed a direct phenotypic assessment of the previously characterized roots and, in fact, deletions in FASCICLIN-LIKE ARABINOGALACTAN PROTEIN 1 (BcFLA1)–a gene with an expression pattern consistent with a role in root hair architecture–resulted in shorter root hairs compared to control roots ectopically expressing an allele of the gene that cannot be targeted by the gRNA in parallel to the CRISPR construct. As an additional line of evidence, we monitored BcFLA1 expression with qPCR and detected a significant reduction of the transcript in roots with an active CRISPR construct compared to the control, although residual amounts of the transcript were detected, possibly due to inefficient nonsense-mediated mRNA decay. Additionally, the presence of deletions and insertions were verified by Sanger sequencing of the respective amplicons. In summary we demonstrate the successful application of CRISPR/Cas9 in hairy roots of B. carinata, the proof of its effectiveness and its effect on the root hair phenotype. This study paves the way for experimental strategies involving the phenotypic assessment of gene lesions by CRISPR which do not require germline transmission.