Profiling of 1-aminocyclopropane-1-carboxylic acid and selected phytohormones in Arabidopsis using liquid chromatography-tandem mass spectrometry.

BACKGROUND: Gaseous phytohormone ethylene levels are directly influenced by the production of its immediate non-volatile precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Owing to the strongly acidic character of the ACC molecule, its quantification has been difficult to perform. Here, we present a simple and straightforward validated method for accurate quantification of not only ACC levels, but also major members of other important phytohormonal classes - auxins, cytokinins, jasmonic acid, abscisic acid and salicylic acid from the same biological sample. RESULTS: The presented technique facilitates the analysis of 15 compounds by liquid chromatography coupled with tandem mass spectrometry. It was optimized and validated for 10 mg of fresh weight plant material. The extraction procedure is composed of a minimal amount of necessary steps. Accuracy and precision were the basis for evaluating the method, together with process efficiency, recovery and matrix effects as validation parameters. The examined compounds comprise important groups of phytohormones, their active forms and some of their metabolites, including six cytokinins, four auxins, two jasmonates, abscisic acid, salicylic acid and 1-aminocyclopropane-1-carboxylic acid. The resulting method was used to examine their contents in selected Arabidopsis thaliana mutant lines. CONCLUSION: This profiling method enables a very straightforward approach for indirect ethylene study and explores how it interacts, based on content levels, with other phytohormonal groups in plants.

In situ seasonal patterns of root auxin concentrations and meristem length in an arctic sedge.

Seasonal dynamics of root growth play an important role in large-scale ecosystem processes; they are largely governed by growth regulatory compounds and influenced by environmental conditions. Yet, our knowledge about physiological drivers of root growth is mostly limited to laboratory-based studies on model plant species. We sampled root tips of Eriophorum vaginatum and analyzed their auxin concentrations and meristem lengths biweekly over a growing season in situ in a subarctic peatland, both in surface soil and at the permafrost thawfront. Auxin concentrations were almost five times higher in surface than in thawfront soils and increased over the season, especially at the thawfront. Surprisingly, meristem length showed an opposite pattern and was almost double in thawfront compared with surface soils. Meristem length increased from peak to late season in the surface soils but decreased at the thawfront. Our study of in situ seasonal dynamics in root physiological parameters illustrates the potential for physiological methods to be applied in ecological studies and emphasizes the importance of in situ measurements. The strong effect of root location and the unexpected opposite patterns of meristem length and auxin concentrations likely show that auxin actively governs root growth to ensure a high potential for nutrient uptake at the thawfront.

Unveiling Molecular Signatures in Light-Induced Seed Germination: Insights from PIN3, PIN7, and AUX1 in Arabidopsis thaliana.

Light provides seeds with information that is essential for the adjustment of their germination to the conditions that are most favorable for the successful establishment of the future seedling. The promotion of germination depends mainly on environmental factors, like temperature and light, as well as internal factors associated with the hormonal balance between gibberellins (GA) and abscisic acid (ABA), although other hormones such as auxins may act secondarily. While transcriptomic studies of light-germinating Arabidopsis thaliana seeds suggest that auxins and auxin transporters are necessary, there are still no functional studies connecting the activity of the auxin transporters in light-induced seed germination. In this study, we investigated the roles of two auxin efflux carrier (PIN3 and PIN7) proteins and one auxin influx (AUX1) carrier protein during Arabidopsis thaliana seed germination. By using next-generation sequencing (RNAseq), gene expression analyses, hormonal sensitivity assays, and the quantification of indole-3-acetic acid (IAA) levels, we assessed the functional roles of PIN3, PIN7, and AUX1 during light-induced seed germination. We showed that auxin levels are increased 24 h after a red-pulse (Rp). Additionally, we evaluated the germination responses of pin3, pin7, and aux1 mutant seeds and showed that PIN3, PIN7, and AUX1 auxin carriers are important players in the regulation of seed germination. By using gene expression analysis in water, fluridone (F), and ABA+F treated seeds, we confirmed that Rp-induced seed germination is associated with auxin transport, and ABA controls the function of PIN3, PIN7, and AUX1 during this process. Overall, our results highlight the relevant and positive role of auxin transporters in germinating the seeds of Arabidopsis thaliana.

Tree architecture: A strigolactone-deficient mutant reveals a connection between branching order and auxin gradient along the tree stem.

Due to their long lifespan, trees and bushes develop higher order of branches in a perennial manner. In contrast to a tall tree, with a clearly defined main stem and branching order, a bush is shorter and has a less apparent main stem and branching pattern. To address the developmental basis of these two forms, we studied several naturally occurring architectural variants in silver birch (Betula pendula). Using a candidate gene approach, we identified a bushy kanttarelli variant with a loss-of-function mutation in the BpMAX1 gene required for strigolactone (SL) biosynthesis. While kanttarelli is shorter than the wild type (WT), it has the same number of primary branches, whereas the number of secondary branches is increased, contributing to its bush-like phenotype. To confirm that the identified mutation was responsible for the phenotype, we phenocopied kanttarelli in transgenic BpMAX1::RNAi birch lines. SL profiling confirmed that both kanttarelli and the transgenic lines produced very limited amounts of SL. Interestingly, the auxin (IAA) distribution along the main stem differed between WT and BpMAX1::RNAi. In the WT, the auxin concentration formed a gradient, being higher in the uppermost internodes and decreasing toward the basal part of the stem, whereas in the transgenic line, this gradient was not observed. Through modeling, we showed that the different IAA distribution patterns may result from the difference in the number of higher-order branches and plant height. Future studies will determine whether the IAA gradient itself regulates aspects of plant architecture.

Flexure wood formation via growth reprogramming in hybrid aspen involves jasmonates and polyamines and transcriptional changes resembling tension wood development.

Stem bending in trees induces flexure wood but its properties and development are poorly understood. Here, we investigated the effects of low-intensity multidirectional stem flexing on growth and wood properties of hybrid aspen, and on its transcriptomic and hormonal responses. Glasshouse-grown trees were either kept stationary or subjected to several daily shakes for 5 wk, after which the transcriptomes and hormones were analyzed in the cambial region and developing wood tissues, and the wood properties were analyzed by physical, chemical and microscopy techniques. Shaking increased primary and secondary growth and altered wood differentiation by stimulating gelatinous-fiber formation, reducing secondary wall thickness, changing matrix polysaccharides and increasing cellulose, G- and H-lignin contents, cell wall porosity and saccharification yields. Wood-forming tissues exhibited elevated jasmonate, polyamine, ethylene and brassinosteroids and reduced abscisic acid and gibberellin signaling. Transcriptional responses resembled those during tension wood formation but not opposite wood formation and revealed several thigmomorphogenesis-related genes as well as novel gene networks including FLA and XTH genes encoding plasma membrane-bound proteins. Low-intensity stem flexing stimulates growth and induces wood having improved biorefinery properties through molecular and hormonal pathways similar to thigmomorphogenesis in herbaceous plants and largely overlapping with the tension wood program of hardwoods.

Fluorescence-activated multi-organelle mapping of subcellular plant hormone distribution.

Auxins and cytokinins are two major families of phytohormones that control most aspects of plant growth, development and plasticity. Their distribution in plants has been described, but the importance of cell- and subcellular-type specific phytohormone homeostasis remains undefined. Herein, we revealed auxin and cytokinin distribution maps showing their different organelle-specific allocations within the Arabidopsis plant cell. To do so, we have developed Fluorescence-Activated multi-Organelle Sorting (FAmOS), an innovative subcellular fractionation technique based on flow cytometric principles. FAmOS allows the simultaneous sorting of four differently labelled organelles based on their individual light scatter and fluorescence parameters while ensuring hormone metabolic stability. Our data showed different subcellular distribution of auxin and cytokinins, revealing the formation of phytohormone gradients that have been suggested by the subcellular localization of auxin and cytokinin transporters, receptors and metabolic enzymes. Both hormones showed enrichment in vacuoles, while cytokinins were also accumulated in the endoplasmic reticulum.

Changes in cell wall composition due to a pectin biosynthesis enzyme GAUT10 impact root growth.

Arabidopsis (Arabidopsis thaliana) root development is regulated by multiple dynamic growth cues that require central metabolism pathways such as ß-oxidation and auxin. Loss of the pectin biosynthesizing enzyme GALACTURONOSYLTRANSFERASE 10 (GAUT10) leads to a short-root phenotype under sucrose-limited conditions. The present study focused on determining the specific contributions of GAUT10 to pectin composition in primary roots and the underlying defects associated with gaut10 roots. Using live-cell microscopy, we determined reduced root growth in gaut10 is due to a reduction in both root apical meristem size and epidermal cell elongation. In addition, GAUT10 was required for normal pectin and hemicellulose composition in primary Arabidopsis roots. Specifically, loss of GAUT10 led to a reduction in galacturonic acid and xylose in root cell walls and altered the presence of rhamnogalacturonan-I (RG-I) and homogalacturonan (HG) polymers in the root. Transcriptomic analysis of gaut10 roots compared to wild type uncovered hundreds of genes differentially expressed in the mutant, including genes related to auxin metabolism and peroxisome function. Consistent with these results, both auxin signaling and metabolism were modified in gaut10 roots. The sucrose-dependent short-root phenotype in gaut10 was linked to ß-oxidation based on hypersensitivity to indole-3-butyric acid (IBA) and an epistatic interaction with TRANSPORTER OF IBA1 (TOB1). Altogether, these data support a growing body of evidence suggesting that pectin composition may influence auxin pathways and peroxisome activity.

Salicylic acid metabolism and signalling coordinate senescence initiation in aspen in nature.

Deciduous trees exhibit a spectacular phenomenon of autumn senescence driven by the seasonality of their growth environment, yet there is no consensus which external or internal cues trigger it. Senescence starts at different times in European aspen (Populus tremula L.) genotypes grown in same location. By integrating omics studies, we demonstrate that aspen genotypes utilize similar transcriptional cascades and metabolic cues to initiate senescence, but at different times during autumn. The timing of autumn senescence initiation appeared to be controlled by two consecutive "switches"; 1) first the environmental variation induced the rewiring of the transcriptional network, stress signalling pathways and metabolic perturbations and 2) the start of senescence process was defined by the ability of the genotype to activate and sustain stress tolerance mechanisms mediated by salicylic acid. We propose that salicylic acid represses the onset of leaf senescence in stressful natural conditions, rather than promoting it as often observed in annual plants.

GOLVEN peptides regulate lateral root spacing as part of a negative feedback loop on the establishment of auxin maxima.

Lateral root initiation requires the accumulation of auxin in lateral root founder cells, yielding a local auxin maximum. The positioning of auxin maxima along the primary root determines the density and spacing of lateral roots. The GOLVEN6 (GLV6) and GLV10 signaling peptides and their receptors have been established as regulators of lateral root spacing via their inhibitory effect on lateral root initiation in Arabidopsis. However, it was unclear how these GLV peptides interfere with auxin signaling or homeostasis. Here, we show that GLV6/10 signaling regulates the expression of a subset of auxin response genes, downstream of the canonical auxin signaling pathway, while simultaneously inhibiting the establishment of auxin maxima within xylem-pole pericycle cells that neighbor lateral root initiation sites. We present genetic evidence that this inhibitory effect relies on the activity of the PIN3 and PIN7 auxin export proteins. Furthermore, GLV6/10 peptide signaling was found to enhance PIN7 abundance in the plasma membranes of xylem-pole pericycle cells, which likely stimulates auxin efflux from these cells. Based on these findings, we propose a model in which the GLV6/10 signaling pathway serves as a negative feedback mechanism that contributes to the robust patterning of auxin maxima along the primary root.

Modulating auxin response stabilizes tomato fruit set.

Fruit formation depends on successful fertilization and is highly sensitive to weather fluctuations that affect pollination. Auxin promotes fruit initiation and growth following fertilization. Class A auxin response factors (Class A ARFs) repress transcription in the absence of auxin and activate transcription in its presence. Here, we explore how multiple members of the ARF family regulate fruit set and fruit growth in tomato (Solanum lycopersicum) and Arabidopsis thaliana, and test whether reduction of SlARF activity improves yield stability in fluctuating temperatures. We found that several tomato Slarf mutant combinations produced seedless parthenocarpic fruits, most notably mutants deficient in SlARF8A and SlARF8B genes. Arabidopsis Atarf8 mutants deficient in the orthologous gene had less complete parthenocarpy than did tomato Slarf8a Slarf8b mutants. Conversely, Atarf6 Atarf8 double mutants had reduced fruit growth after fertilization. AtARF6 and AtARF8 likely switch from repression to activation of fruit growth in response to a fertilization-induced auxin increase in gynoecia. Tomato plants with reduced SlARF8A and SlARF8B gene dosage had substantially higher yield than the wild type under controlled or ambient hot and cold growth conditions. In field trials, partial reduction in the SlARF8 dose increased yield under extreme temperature with minimal pleiotropic effects. The stable yield of the mutant plants resulted from a combination of early onset of fruit set, more fruit-bearing branches and more flowers setting fruits. Thus, ARF8 proteins mediate the control of fruit set, and relieving this control with Slarf8 mutations may be utilized in breeding to increase yield stability in tomato and other crops.

Flexible Organic Electronic Ion Pump for Flow-Free Phytohormone Delivery into Vasculature of Intact Plants.

Plant vasculature transports molecules that play a crucial role in plant signaling including systemic responses and acclimation to diverse environmental conditions. Targeted controlled delivery of molecules to the vascular tissue can be a biomimetic way to induce long distance responses, providing a new tool for the fundamental studies and engineering of stress-tolerant plants. Here, a flexible organic electronic ion pump, an electrophoretic delivery device, for controlled delivery of phytohormones directly in plant vascular tissue is developed. The c-OEIP is based on polyimide-coated glass capillaries that significantly enhance the mechanical robustness of these microscale devices while being minimally disruptive for the plant. The polyelectrolyte channel is based on low-cost and commercially available precursors that can be photocured with blue light, establishing much cheaper and safer system than the state-of-the-art. To trigger OEIP-induced plant response, the phytohormone abscisic acid (ABA) in the petiole of intact Arabidopsis plants is delivered. ABA is one of the main phytohormones involved in plant stress responses and induces stomata closure under drought conditions to reduce water loss and prevent wilting. The OEIP-mediated ABA delivery triggered fast and long-lasting stomata closure far away from the delivery point demonstrating systemic vascular transport of the delivered ABA, verified delivering deuterium-labeled ABA.

Long-distance turgor pressure changes induce local activation of plant glutamate receptor-like channels.

In Arabidopsis thaliana, local wounding and herbivore feeding provoke leaf-to-leaf propagating Ca2+ waves that are dependent on the activity of members of the glutamate receptor-like channels (GLRs). In systemic tissues, GLRs are needed to sustain the synthesis of jasmonic acid (JA) with the subsequent activation of JA-dependent signaling response required for the plant acclimation to the perceived stress. Even though the role of GLRs is well established, the mechanism through which they are activated remains unclear. Here, we report that in vivo, the amino-acid-dependent activation of the AtGLR3.3 channel and systemic responses require a functional ligand-binding domain. By combining imaging and genetics, we show that leaf mechanical injury, such as wounds and burns, as well as hypo-osmotic stress in root cells, induces the systemic apoplastic increase of L-glutamate (L-Glu), which is largely independent of AtGLR3.3 that is instead required for systemic cytosolic Ca2+ elevation. Moreover, by using a bioelectronic approach, we show that the local release of minute concentrations of L-Glu in the leaf lamina fails to induce any long-distance Ca2+ waves.

Physcomitrium patens PpRIC, an ancestral CRIB-domain ROP effector, inhibits auxin-induced differentiation of apical initial cells.

RHO guanosine triphosphatases are important eukaryotic regulators of cell differentiation and behavior. Plant ROP (RHO of plant) family members activate specific, incompletely characterized downstream signaling. The structurally simple land plant Physcomitrium patens is missing homologs of key animal and flowering plant RHO effectors but contains a single CRIB (CDC42/RAC interactive binding)-domain-containing RIC (ROP-interacting CRIB-containing) protein (PpRIC). Protonemal P. patens filaments elongate based on regular division and PpROP-dependent tip growth of apical initial cells, which upon stimulation by the hormone auxin differentiate caulonemal characteristics. PpRIC interacts with active PpROP1, co-localizes with this protein at the plasma membrane at the tip of apical initial cells, and accumulates in the nucleus. Remarkably, PpRIC is not required for tip growth but is targeted to the nucleus to block caulonema differentiation downstream of auxin-controlled gene expression. These observations establish functions of PpRIC in mediating crosstalk between ROP and auxin signaling, which contributes to the maintenance of apical initial cell identity.

Cytokinin signaling regulates two-stage inflorescence arrest in Arabidopsis.

To maximize reproductive success, flowering plants must correctly time entry and exit from the reproductive phase. While much is known about mechanisms that regulate initiation of flowering, end-of-flowering remains largely uncharacterized. End-of-flowering in Arabidopsis (Arabidopsis thaliana) consists of quasi-synchronous arrest of inflorescences, but it is unclear how arrest is correctly timed with respect to environmental stimuli and reproductive success. Here, we showed that Arabidopsis inflorescence arrest is a complex developmental phenomenon, which includes the arrest of the inflorescence meristem (IM), coupled with a separable "floral arrest" of all unopened floral primordia; these events occur well before visible inflorescence arrest. We showed that global inflorescence removal delays both IM and floral arrest, but that local fruit removal only delays floral arrest, emphasizing their separability. We tested whether cytokinin regulates inflorescence arrest, and found that cytokinin signaling dynamics mirror IM activity, while cytokinin treatment can delay both IM and floral arrest. We further showed that gain-of-function cytokinin receptor mutants can delay IM and floral arrest; conversely, loss-of-function mutants prevented the extension of flowering in response to inflorescence removal. Collectively, our data suggest that the dilution of cytokinin among an increasing number of sink organs leads to end-of-flowering in Arabidopsis by triggering IM and floral arrest.

The Arabidopsis ATP-Binding Cassette E protein ABCE2 is a conserved component of the translation machinery.

ATP-Binding Cassette E (ABCE) proteins dissociate cytoplasmic ribosomes after translation terminates, and contribute to ribosome recycling, thus linking translation termination to initiation. This function has been demonstrated to be essential in animals, fungi, and archaea, but remains unexplored in plants. In most species, ABCE is encoded by a single-copy gene; by contrast, Arabidopsis thaliana has two ABCE paralogs, of which ABCE2 seems to conserve the ancestral function. We isolated apiculata7-1 (api7-1), the first viable, hypomorphic allele of ABCE2, which has a pleiotropic morphological phenotype reminiscent of mutations affecting ribosome biogenesis factors and ribosomal proteins. We also studied api7-2, a null, recessive lethal allele of ABCE2. Co-immunoprecipitation experiments showed that ABCE2 physically interacts with components of the translation machinery. An RNA-seq study of the api7-1 mutant showed increased responses to iron and sulfur starvation. We also found increased transcript levels of genes related to auxin signaling and metabolism. Our results support for the first time a conserved role for ABCE proteins in translation in plants, as previously shown for the animal, fungal, and archaeal lineages. In Arabidopsis, the ABCE2 protein seems important for general growth and vascular development, likely due to an indirect effect through auxin metabolism.

IPT9, a cis-zeatin cytokinin biosynthesis gene, promotes root growth.

Cytokinin and auxin are plant hormones that coordinate many aspects of plant development. Their interactions in plant underground growth are well established, occurring at the levels of metabolism, signaling, and transport. Unlike many plant hormone classes, cytokinins are represented by more than one active molecule. Multiple mutant lines, blocking specific parts of cytokinin biosynthetic pathways, have enabled research in plants with deficiencies in specific cytokinin-types. While most of these mutants have confirmed the impeding effect of cytokinin on root growth, the ipt29 double mutant instead surprisingly exhibits reduced primary root length compared to the wild type. This mutant is impaired in cis-zeatin (cZ) production, a cytokinin-type that had been considered inactive in the past. Here we have further investigated the intriguing ipt29 root phenotype, opposite to known cytokinin functions, and the (bio)activity of cZ. Our data suggest that despite the ipt29 short-root phenotype, cZ application has a negative impact on primary root growth and can activate a cytokinin response in the stele. Grafting experiments revealed that the root phenotype of ipt29 depends mainly on local signaling which does not relate directly to cytokinin levels. Notably, ipt29 displayed increased auxin levels in the root tissue. Moreover, analyses of the differential contributions of ipt2 and ipt9 to the ipt29 short-root phenotype demonstrated that, despite its deficiency on cZ levels, ipt2 does not show any root phenotype or auxin homeostasis variation, while ipt9 mutants were indistinguishable from ipt29. We conclude that IPT9 functions may go beyond cZ biosynthesis, directly or indirectly, implicating effects on auxin homeostasis and therefore influencing plant growth.

The RPN12a proteasome subunit is essential for the multiple hormonal homeostasis controlling the progression of leaf senescence.

The 26S proteasome is a conserved multi-subunit machinery in eukaryotes. It selectively degrades ubiquitinated proteins, which in turn provides an efficient molecular mechanism to regulate numerous cellular functions and developmental processes. Here, we studied a new loss-of-function allele of RPN12a, a plant ortholog of the yeast and human structural component of the 19S proteasome RPN12. Combining a set of biochemical and molecular approaches, we confirmed that a rpn12a knock-out had exacerbated 20S and impaired 26S activities. The altered proteasomal activity led to a pleiotropic phenotype affecting both the vegetative growth and reproductive phase of the plant, including a striking repression of leaf senescence associate cell-death. Further investigation demonstrated that RPN12a is involved in the regulation of several conjugates associated with the auxin, cytokinin, ethylene and jasmonic acid homeostasis. Such enhanced aptitude of plant cells for survival in rpn12a contrasts with reports on animals, where 26S proteasome mutants generally show an accelerated cell death phenotype.

PIF7 is a master regulator of thermomorphogenesis in shade.

The size of plant organs is highly responsive to environmental conditions. The plant's embryonic stem, or hypocotyl, displays phenotypic plasticity, in response to light and temperature. The hypocotyl of shade avoiding species elongates to outcompete neighboring plants and secure access to sunlight. Similar elongation occurs in high temperature. However, it is poorly understood how environmental light and temperature cues interact to effect plant growth. We found that shade combined with warm temperature produces a synergistic hypocotyl growth response that dependent on PHYTOCHROME-INTERACTING FACTOR 7 (PIF7) and auxin. This unique but agriculturally relevant scenario was almost totally independent on PIF4 activity. We show that warm temperature is sufficient to promote PIF7 DNA binding but not transcriptional activation and we demonstrate that additional, unknown factor/s must be working downstream of the phyB-PIF-auxin module. Our findings will improve the predictions of how plants will respond to increased ambient temperatures when grown at high density.

Nitrogen represses haustoria formation through abscisic acid in the parasitic plant Phtheirospermum japonicum.

Parasitic plants are globally prevalent pathogens that withdraw nutrients from their host plants using an organ known as the haustorium. The external environment including nutrient availability affects the extent of parasitism and to understand this phenomenon, we investigated the role of nutrients and found that nitrogen is sufficient to repress haustoria formation in the root parasite Phtheirospermum japonicum. Nitrogen increases levels of abscisic acid (ABA) in P. japonicum and prevents the activation of hundreds of genes including cell cycle and xylem development genes. Blocking ABA signaling overcomes nitrogen's inhibitory effects indicating that nitrogen represses haustoria formation by increasing ABA. The effect of nitrogen appears more widespread since nitrogen also inhibits haustoria in the obligate root parasite Striga hermonthica. Together, our data show that nitrogen acts as a haustoria repressing factor and suggests a mechanism whereby parasitic plants use nitrogen availability in the external environment to regulate the extent of parasitism.