Imaging of plant calcium-sensor kinase conformation monitors real time calcium-dependent decoding in planta.

Changes in cytosolic calcium (Ca2+) concentration are among the earliest reactions to a multitude of stress cues. While a plethora of Ca2+-permeable channels may generate distinct Ca2+ signatures and contribute to response specificities, the mechanisms by which Ca2+ signatures are decoded are poorly understood. Here, we developed a genetically encoded Förster resonance energy transfer (FRET)-based reporter that visualizes the conformational changes in Ca2+-dependent protein kinases (CDPKs/CPKs). We focused on two CDPKs with distinct Ca2+-sensitivities, highly Ca2+-sensitive Arabidopsis (Arabidopsis thaliana) AtCPK21 and rather Ca2+-insensitive AtCPK23, to report conformational changes accompanying kinase activation. In tobacco (Nicotiana tabacum) pollen tubes, which naturally display coordinated spatial and temporal Ca2+ fluctuations, CPK21-FRET, but not CPK23-FRET, reported oscillatory emission ratio changes mirroring cytosolic Ca2+ changes, pointing to the isoform-specific Ca2+-sensitivity and reversibility of the conformational change. In Arabidopsis guard cells, CPK21-FRET-monitored conformational dynamics suggest that CPK21 serves as a decoder of signal-specific Ca2+ signatures in response to abscisic acid and the flagellin peptide flg22. Based on these data, CDPK-FRET is a powerful approach for tackling real-time live-cell Ca2+ decoding in a multitude of plant developmental and stress responses.

CAMTA3 repressor destabilization triggers TIR domain protein TN2-mediated autoimmunity in the Arabidopsis exo70B1 mutant.

Calcium-dependent protein kinases (CPKs) can decode and translate intracellular calcium signals to induce plant immunity. Mutation of the exocyst subunit gene EXO70B1 causes autoimmunity that depends on CPK5 and the Toll/interleukin-1 receptor (TIR) domain resistance protein TIR-NBS2 (TN2), where direct interaction with TN2 stabilizes CPK5 kinase activity. However, how the CPK5-TN2 interaction initiates downstream immune responses remains unclear. Here, we show that, besides CPK5 activity, the physical interaction between CPK5 and functional TN2 triggers immune activation in exo70B1 and may represent reciprocal regulation between CPK5 and the TIR domain functions of TN2 in Arabidopsis (Arabidopsis thaliana). Moreover, we detected differential phosphorylation of the calmodulin-binding transcription activator 3 (CAMTA3) in the cpk5 background. CPK5 directly phosphorylates CAMTA3 at S964, contributing to its destabilization. The gain-of-function CAMTA3A855V variant that resists CPK5-induced degradation rescues immunity activated through CPK5 overexpression or exo70B1 mutation. Thus, CPK5-mediated immunity is executed through CAMTA3 repressor degradation via phosphorylation-induced and/or calmodulin-regulated processes. Conversely, autoimmunity in camta3 also partially requires functional CPK5. While the TIR domain activity of TN2 remains to be tested, our study uncovers a TN2-CPK5-CAMTA3 signaling module for exo70B1-mediated autoimmunity, highlighting the direct embedding of a calcium-sensing decoder element within resistance signalosomes.

The terminal enzymatic step in piperine biosynthesis is co-localized with the product piperine in specialized cells of black pepper (Piper nigrum L.).

Piperine (1-piperoyl piperidine) is responsible for the pungent perception of dried black pepper (Piper nigrum) fruits and essentially contributes to the aromatic properties of this spice in combination with a blend of terpenoids. The final step in piperine biosynthesis involves piperine synthase (PS), which catalyzes the reaction of piperoyl CoA and piperidine to the biologically active and pungent amide. Nevertheless, experimental data on the cellular localization of piperine and the complete biosynthetic pathway are missing. Not only co-localization of enzymes and products, but also potential transport of piperamides to the sink organs is a possible alternative. This work, which includes purification of the native enzyme, immunolocalization, laser microdissection, fluorescence microscopy, and electron microscopy combined with liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), provides experimental evidence that piperine and PS are co-localized in specialized cells of the black pepper fruit perisperm. PS accumulates during early stages of fruit development and its level declines before the fruits are fully mature. The product piperine is co-localized to PS and can be monitored at the cellular level by its strong bluish fluorescence. Rising piperine levels during fruit maturation are consistent with the increasing numbers of fluorescent cells within the perisperm. Signal intensities of individual laser-dissected cells when monitored by LC-ESI-MS/MS indicate molar concentrations of this alkaloid. Significant levels of piperine and additional piperamides were also detected in cells distributed in the cortex of black pepper roots. In summary, the data provide comprehensive experimental evidence of and insights into cell-specific biosynthesis and storage of piperidine alkaloids, specific and characteristic for the Piperaceae. By a combination of fluorescence microscopy and LC-MS/MS analysis we localized the major piperidine alkaloids to specific cells of the fruit perisperm and the root cortex. Immunolocalization of native piperine and piperamide synthases shows that enzymes are co-localized with high concentrations of products in these idioblasts.

Integrating GWAS and TWAS to elucidate the genetic architecture of maize leaf cuticular conductance.

The cuticle, a hydrophobic layer of cutin and waxes synthesized by plant epidermal cells, is the major barrier to water loss when stomata are closed. Dissecting the genetic architecture of natural variation for maize (Zea mays L.) leaf cuticular conductance (gc) is important for identifying genes relevant to improving crop productivity in drought-prone environments. To this end, we performed an integrated genome- and transcriptome-wide association studies (GWAS and TWAS) to identify candidate genes putatively regulating variation in leaf gc. Of the 22 plausible candidate genes identified, 4 were predicted to be involved in cuticle precursor biosynthesis and export, 2 in cell wall modification, 9 in intracellular membrane trafficking, and 7 in the regulation of cuticle development. A gene encoding an INCREASED SALT TOLERANCE1-LIKE1 (ISTL1) protein putatively involved in intracellular protein and membrane trafficking was identified in GWAS and TWAS as the strongest candidate causal gene. A set of maize nested near-isogenic lines that harbor the ISTL1 genomic region from eight donor parents were evaluated for gc, confirming the association between gc and ISTL1 in a haplotype-based association analysis. The findings of this study provide insights into the role of regulatory variation in the development of the maize leaf cuticle and will ultimately assist breeders to develop drought-tolerant maize for target environments.

Transcriptomic network analyses shed light on the regulation of cuticle development in maize leaves.

Plant cuticles are composed of wax and cutin and evolved in the land plants as a hydrophobic boundary that reduces water loss from the plant epidermis. The expanding maize adult leaf displays a dynamic, proximodistal gradient of cuticle development, from the leaf base to the tip. Laser microdissection RNA Sequencing (LM-RNAseq) was performed along this proximodistal gradient, and complementary network analyses identified potential regulators of cuticle biosynthesis and deposition. A weighted gene coexpression network (WGCN) analysis suggested a previously undescribed function for PHYTOCHROME-mediated light signaling during the regulation of cuticular wax deposition. Genetic analyses reveal that phyB1 phyB2 double mutants of maize exhibit abnormal cuticle composition, supporting the predictions of our coexpression analysis. Reverse genetic analyses also show that phy mutants of the moss Physcomitrella patens exhibit abnormal cuticle composition, suggesting an ancestral role for PHYTOCHROME-mediated, light-stimulated regulation of cuticle development during plant evolution.

Constructing functional cuticles: analysis of relationships between cuticle lipid composition, ultrastructure and water barrier function in developing adult maize leaves.

BACKGROUND AND AIMS: Prior work has examined cuticle function, composition and ultrastructure in many plant species, but much remains to be learned about how these features are related. This study aims to elucidate relationships between these features via analysis of cuticle development in adult maize (Zea mays L.) leaves, while also providing the most comprehensive investigation to date of the composition and ultrastructure of adult leaf cuticles in this important crop plant. METHODS: We examined water permeability, wax and cutin composition via gas chromatography, and ultrastructure via transmission electron microscopy, along the developmental gradient of partially expanded adult maize leaves, and analysed the relationships between these features. KEY RESULTS: The water barrier property of the adult maize leaf cuticle is acquired at the cessation of cell expansion. Wax types and chain lengths accumulate asynchronously over the course of development, while overall wax load does not vary. Cutin begins to accumulate prior to establishment of the water barrier and continues thereafter. Ultrastructurally, pavement cell cuticles consist of an epicuticular layer, and a thin cuticle proper that acquires an inner, osmiophilic layer during development. CONCLUSIONS: Cuticular waxes of the adult maize leaf are dominated by alkanes and alkyl esters. Unexpectedly, these are localized mainly in the epicuticular layer. Establishment of the water barrier during development coincides with a switch from alkanes to esters as the major wax type, and the emergence of an osmiophilic (likely cutin-rich) layer of the cuticle proper. Thus, alkyl esters and the deposition of the cutin polyester are implicated as key components of the water barrier property of adult maize leaf cuticles.

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.

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.

Function of calcium-dependent protein kinase CPK28 of Arabidopsis thaliana in plant stem elongation and vascular development.

After a period of vegetative growth, plants undergo a developmental switch to the reproductive phase, inducing the transition to bolting, elongation of the inflorescence and flowering. We have identified calcium-dependent protein kinase CPK28 from Arabidopsis thaliana as a regulatory component that controls stem elongation and vascular development. In two independent mutant alleles of cpk28, a reduction of stem elongation, accompanied by shorter leaf petioles and enhanced anthocyanin levels, is observed upon the transition to the generative phase. Anatomical analysis revealed an altered vascular pattern characterised by fewer xylem tracheary elements but at the same time increased lignification and secondary growth. Coincident with these morphological changes, cpk28 mutants showed altered expression of NAC transcriptional regulators NST1 and NST3 as well as of GA3ox1, a key regulator of gibberellic acid homeostasis. In vitro protein kinase activity of CPK28 is strictly calcium-dependent. Furthermore, CPK28 is phosphorylated in vivo at several sites. Site-specific amino acid substitutions at these phosphorylation sites resulted in reduced in vitro activity. However, when introduced into a cpk28 mutant background, wild-type and phosphorylation site variants, but not kinase-inactive variants of CPK28 complemented the morphological and developmental defects. Our data identify CPK28 as a developmentally controlled regulator for coordinated stem elongation and secondary growth.

Activity of guard cell anion channel SLAC1 is controlled by drought-stress signaling kinase-phosphatase pair.

In response to drought stress the phytohormone ABA (abscisic acid) induces stomatal closure and, therein, activates guard cell anion channels in a calcium-dependent as well as-independent manner. Two key components of the ABA signaling pathway are the protein kinase OST1 (open stomata 1) and the protein phosphatase ABI1 (ABA insensitive 1). The recently identified guard cell anion channel SLAC1 appeared to be the key ion channel in this signaling pathway but remained electrically silent when expressed heterologously. Using split YFP assays, we identified OST1 as an interaction partner of SLAC1 and ABI1. Upon coexpression of SLAC1 with OST1 in Xenopus oocytes, SLAC1-related anion currents appeared similar to those observed in guard cells. Integration of ABI1 into the SLAC1/OST1 complex, however, prevented SLAC1 activation. Our studies demonstrate that SLAC1 represents the slow, deactivating, weak voltage-dependent anion channel of guard cells controlled by phosphorylation/dephosphorylation.

Endocytosis is a significant contributor to uranium(VI) uptake in tobacco (Nicotiana tabacum) BY-2 cells in phosphate-deficient culture.

Endocytosis of metals in plants is a growing field of study involving metal uptake from the rhizosphere. Uranium, which is naturally and artificially released into the rhizosphere, is known to be taken up by certain species of plant, such as Nicotiana tabacum, and we hypothesize that endocytosis contributes to the uptake of uranium in tobacco. The endocytic uptake of uranium was investigated in tobacco BY-2 cells using an optimized setup of culture in phosphate-deficient medium. A combination of methods in biochemistry, microscopy and spectroscopy, supplemented by proteomics, were used to study the interaction of uranium and the plant cell. We found that under environmentally relevant uranium concentrations, endocytosis remained active and contributed to 14% of the total uranium bioassociation. Proteomics analyses revealed that uranium induced a change in expression of the clathrin heavy chain variant, signifying a shift in the type of endocytosis taking place. However, the rate of endocytosis remained largely unaltered. Electron microscopy and energy-dispersive X-ray spectroscopy showed an adsorption of uranium to cell surfaces and deposition in vacuoles. Our results demonstrate that endocytosis constitutes a considerable proportion of uranium uptake in BY-2 cells, and that endocytosed uranium is likely targeted to the vacuole for sequestration, providing a physiologically safer route for the plant than uranium transported through the cytosol.

Structure-function analysis of the maize bulliform cell cuticle and its potential role in dehydration and leaf rolling.

The hydrophobic cuticle of plant shoots serves as an important interaction interface with the environment. It consists of the lipid polymer cutin, embedded with and covered by waxes, and provides protection against stresses including desiccation, UV radiation, and pathogen attack. Bulliform cells form in longitudinal strips on the adaxial leaf surface, and have been implicated in the leaf rolling response observed in drought-stressed grass leaves. In this study, we show that bulliform cells of the adult maize leaf epidermis have a specialized cuticle, and we investigate its function along with that of bulliform cells themselves. Bulliform cells displayed increased shrinkage compared to other epidermal cell types during dehydration of the leaf, providing a potential mechanism to facilitate leaf rolling. Analysis of natural variation was used to relate bulliform strip patterning to leaf rolling rate, providing further evidence of a role for bulliform cells in leaf rolling. Bulliform cell cuticles showed a distinct ultrastructure with increased cuticle thickness compared to other leaf epidermal cells. Comparisons of cuticular conductance between adaxial and abaxial leaf surfaces, and between bulliform-enriched mutants versus wild-type siblings, showed a correlation between elevated water loss rates and presence or increased density of bulliform cells, suggesting that bulliform cuticles are more water-permeable. Biochemical analysis revealed altered cutin composition and increased cutin monomer content in bulliform-enriched tissues. In particular, our findings suggest that an increase in 9,10-epoxy-18-hydroxyoctadecanoic acid content, and a lower proportion of ferulate, are characteristics of bulliform cuticles. We hypothesize that elevated water permeability of the bulliform cell cuticle contributes to the differential shrinkage of these cells during leaf dehydration, thereby facilitating the function of bulliform cells in stress-induced leaf rolling observed in grasses.

Genome-Wide Association Study for Maize Leaf Cuticular Conductance Identifies Candidate Genes Involved in the Regulation of Cuticle Development.

The cuticle, a hydrophobic layer of cutin and waxes synthesized by plant epidermal cells, is the major barrier to water loss when stomata are closed at night and under water-limited conditions. Elucidating the genetic architecture of natural variation for leaf cuticular conductance (gc) is important for identifying genes relevant to improving crop productivity in drought-prone environments. To this end, we conducted a genome-wide association study of gc of adult leaves in a maize inbred association panel that was evaluated in four environments (Maricopa, AZ, and San Diego, CA, in 2016 and 2017). Five genomic regions significantly associated with gc were resolved to seven plausible candidate genes (ISTL1, two SEC14 homologs, cyclase-associated protein, a CER7 homolog, GDSL lipase, and ß-D-XYLOSIDASE 4). These candidates are potentially involved in cuticle biosynthesis, trafficking and deposition of cuticle lipids, cutin polymerization, and cell wall modification. Laser microdissection RNA sequencing revealed that all these candidate genes, with the exception of the CER7 homolog, were expressed in the zone of the expanding adult maize leaf where cuticle maturation occurs. With direct application to genetic improvement, moderately high average predictive abilities were observed for whole-genome prediction of gc in locations (0.46 and 0.45) and across all environments (0.52). The findings of this study provide novel insights into the genetic control of gc and have the potential to help breeders more effectively develop drought-tolerant maize for target environments.

Machine Learning Enables High-Throughput Phenotyping for Analyses of the Genetic Architecture of Bulliform Cell Patterning in Maize.

Bulliform cells comprise specialized cell types that develop on the adaxial (upper) surface of grass leaves, and are patterned to form linear rows along the proximodistal axis of the adult leaf blade. Bulliform cell patterning affects leaf angle and is presumed to function during leaf rolling, thereby reducing water loss during temperature extremes and drought. In this study, epidermal leaf impressions were collected from a genetically and anatomically diverse population of maize inbred lines. Subsequently, convolutional neural networks were employed to measure microscopic, bulliform cell-patterning phenotypes in high-throughput. A genome-wide association study, combined with RNAseq analyses of the bulliform cell ontogenic zone, identified candidate regulatory genes affecting bulliform cell column number and cell width. This study is the first to combine machine learning approaches, transcriptomics, and genomics to study bulliform cell patterning, and the first to utilize natural variation to investigate the genetic architecture of this microscopic trait. In addition, this study provides insight toward the improvement of macroscopic traits such as drought resistance and plant architecture in an agronomically important crop plant.

The calcium-dependent protein kinase CPK28 negatively regulates the BIK1-mediated PAMP-induced calcium burst.

Plants are protected from microbial infection by a robust immune system. Two of the earliest responses mediated by surface-localized immune receptors include an increase in cytosolic calcium (Ca(2+)) and a burst of apoplastic reactive oxygen species (ROS). The Arabidopsis plasma membrane-associated cytoplasmic kinase BIK1 is an immediate convergent substrate of multiple surface-localized immune receptors that is genetically required for the PAMP-induced Ca(2+) burst and directly regulates ROS production catalyzed by the NADPH oxidase RBOHD. We recently demonstrated that Arabidopsis plants maintain an optimal level of BIK1 through a process of continuous degradation regulated by the Ca(2+)-dependent protein kinase CPK28. cpk28 mutants accumulate more BIK1 protein and display enhanced immune signaling, while plants over-expressing CPK28 accumulate less BIK1 protein and display impaired immune signaling. Here, we show that CPK28 additionally contributes to the PAMP-induced Ca(2+) burst, supporting its role as a negative regulator of BIK1.

The calcium-dependent protein kinase CPK28 buffers plant immunity and regulates BIK1 turnover.

Plant perception of pathogen-associated molecular patterns (PAMPs) triggers a phosphorylation relay leading to PAMP-triggered immunity (PTI). Despite increasing knowledge of PTI signaling, how immune homeostasis is maintained remains largely unknown. Here we describe a forward-genetic screen to identify loci involved in PTI and characterize the Arabidopsis calcium-dependent protein kinase CPK28 as a negative regulator of immune signaling. Genetic analyses demonstrate that CPK28 attenuates PAMP-triggered immune responses and antibacterial immunity. CPK28 interacts with and phosphorylates the plasma-membrane-associated cytoplasmic kinase BIK1, an important convergent substrate of multiple pattern recognition receptor (PRR) complexes. We find that BIK1 is rate limiting in PTI signaling and that it is continuously turned over to maintain cellular homeostasis. We further show that CPK28 contributes to BIK1 turnover. Our results suggest a negative regulatory mechanism that continually buffers immune signaling by controlling the turnover of this key signaling kinase.