A Novel Image-Based Screening Method to Study Water-Deficit Response and Recovery of Barley Populations Using Canopy Dynamics Phenotyping and Simple Metabolite Profiling.

Plant phenotyping platforms offer automated, fast scoring of traits that simplify the selection of varieties that are more competitive under stress conditions. However, indoor phenotyping methods are frequently based on the analysis of plant growth in individual pots. We present a reproducible indoor phenotyping method for screening young barley populations under water stress conditions and after subsequent rewatering. The method is based on a simple read-out of data using RGB imaging, projected canopy height, as a useful feature for indirectly following the kinetics of growth and water loss in a population of barley. A total of 47 variables including 15 traits and 32 biochemical metabolites measured (morphometric parameters, chlorophyll fluorescence imaging, quantification of stress-related metabolites; amino acids and polyamines, and enzymatic activities) were used to validate the method. The study allowed the identification of metabolites related to water stress response and recovery. Specifically, we found that cadaverine (Cad), 1,3-aminopropane (DAP), tryptamine (Tryp), and tyramine (Tyra) were the major contributors to the water stress response, whereas Cad, DAP, and Tyra, but not Tryp, remained at higher levels in the stressed plants even after rewatering. In this work, we designed, optimized and validated a non-invasive image-based method for automated screening of potential water stress tolerance genotypes in barley populations. We demonstrated the applicability of the method using transgenic barley lines with different sensitivity to drought stress showing that combining canopy height and the metabolite profile we can discriminate tolerant from sensitive genotypes. We showed that the projected canopy height a sensitive trait that truly reflects other invasively studied morphological, physiological, and metabolic traits and that our presented methodological setup can be easily applicable for large-scale screenings in low-cost systems equipped with a simple RGB camera. We believe that our approach will contribute to accelerate the study and understanding of the plant water stress response and recovery capacity in crops, such as barley.

Occurrence and biosynthesis of cytokinins in poplar.

MAIN CONCLUSION: Isoprenoid and aromatic cytokinins occur in poplar as free compounds and constituents of tRNA, poplar isopentenyltransferases are involved in the production of isoprenoid cytokinins, while biosynthesis of their aromatic counterparts remains unsolved. Cytokinins are phytohormones with a fundamental role in the regulation of plant growth and development. They occur naturally either as isoprenoid or aromatic derivatives, but the latter are quite rare and less studied. Here, the spatial expression of all nine isopentenyl transferase genes of Populus × canadensis cv. Robusta (PcIPTs) as analyzed by RT-qPCR revealed a tissue preference and strong differences in expression levels for the different adenylate and tRNA PcIPTs. Together with their phylogeny, this result suggests a functional diversification for the different PcIPT proteins. Additionally, the majority of PcIPT genes were cloned and expressed in Arabidopsis thaliana under an inducible promoter. The cytokinin levels measured in the Arabidopsis-overexpressing lines as well as their phenotype indicate that the studied adenylate and tRNA PcIPT proteins are functional in vivo and thus will contribute to the cytokinin pool in poplar. We screened the cytokinin content in leaves of 12 Populus species by ultra-high performance-tandem mass spectrometry (UHPLC-MS/MS) and discovered that the capacity to produce not only isoprenoid, but also aromatic cytokinins is widespread amongst the Populus accessions studied. Important for future studies is that the levels of aromatic cytokinins transiently increase after daybreak and are much higher in older plants.

Phytohormones and polyamines regulate plant stress responses by altering GABA pathway.

In plants, γ-aminobutyric acid (GABA) accumulates rapidly in response to environmental stress and variations in its endogenous concentration have been shown to affect plant growth. Exogenous application of GABA has also conferred higher stress tolerance by modulating the expression of genes involved in plant signalling, transcriptional regulation, hormone biosynthesis, reactive oxygen species production and polyamine metabolism. Plant hormones play critical roles in adaptation of plants to adverse environmental conditions through a sophisticated crosstalk among them. Several studies have provided evidence for the relationships between GABA, polyamines and hormones such as abscisic acid, cytokinins, auxins, gibberellins and ethylene, among others, focussing on the effect that one specific group of compounds exerts over the metabolic and signalling pathways of others. In this review, we bring together information obtained from plants exposed to several stress conditions and discuss the possible links among these different groups of molecules. The analysis supports the view that highly conserved pathways connect primary and secondary metabolism, with an overlap of regulatory functions related to stress responses and tolerance among phytohormones, amino acids and polyamines.

Characterization of Biostimulant Mode of Action Using Novel Multi-Trait High-Throughput Screening of Arabidopsis Germination and Rosette Growth.

Environmental stresses have a significant effect on agricultural crop productivity worldwide. Exposure of seeds to abiotic stresses, such as salinity among others, results in lower seed viability, reduced germination, and poor seedling establishment. Alternative agronomic practices, e.g., the use of plant biostimulants, have attracted considerable interest from the scientific community and commercial enterprises. Biostimulants, i.e., products of biological origin (including bacteria, fungi, seaweeds, higher plants, or animals) have significant potential for (i) improving physiological processes in plants and (ii) stimulating germination, growth and stress tolerance. However, biostimulants are diverse, and can range from single compounds to complex matrices with different groups of bioactive components that have only been partly characterized. Due to the complex mixtures of biologically active compounds present in biostimulants, efficient methods for characterizing their potential mode of action are needed. In this study, we report the development of a novel complex approach to biological activity testing, based on multi-trait high-throughput screening (MTHTS) of Arabidopsis characteristics. These include the in vitro germination rate, early seedling establishment capacity, growth capacity under stress and stress response. The method is suitable for identifying new biostimulants and characterizing their mode of action. Representatives of compatible solutes such as amino acids and polyamines known to be present in many of the biostimulant irrespective of their origin, i.e., well-established biostimulants that enhance stress tolerance and crop productivity, were used for the assay optimization and validation. The selected compounds were applied through seed priming over a broad concentration range and the effect was investigated simultaneously under control, moderate stress and severe salt stress conditions. The new MTHTS approach represents a powerful tool in the field of biostimulant research and development and offers direct classification of the biostimulants mode of action into three categories: (1) plant growth promotors/inhibitors, (2) stress alleviators, and (3) combined action.

An Automated Method for High-Throughput Screening of Arabidopsis Rosette Growth in Multi-Well Plates and Its Validation in Stress Conditions.

High-throughput plant phenotyping platforms provide new possibilities for automated, fast scoring of several plant growth and development traits, followed over time using non-invasive sensors. Using Arabidopsis as a model offers important advantages for high-throughput screening with the opportunity to extrapolate the results obtained to other crops of commercial interest. In this study we describe the development of a highly reproducible high-throughput Arabidopsis in vitro bioassay established using our OloPhen platform, suitable for analysis of rosette growth in multi-well plates. This method was successfully validated on example of multivariate analysis of Arabidopsis rosette growth in different salt concentrations and the interaction with varying nutritional composition of the growth medium. Several traits such as changes in the rosette area, relative growth rate, survival rate and homogeneity of the population are scored using fully automated RGB imaging and subsequent image analysis. The assay can be used for fast screening of the biological activity of chemical libraries, phenotypes of transgenic or recombinant inbred lines, or to search for potential quantitative trait loci. It is especially valuable for selecting genotypes or growth conditions that improve plant stress tolerance.

Isopentenyltransferase-1 (IPT1) knockout in Physcomitrella together with phylogenetic analyses of IPTs provide insights into evolution of plant cytokinin biosynthesis.

The moss Physcomitrella patens is part of an early divergent clade of land plants utilizing the plant hormone cytokinin for growth control. The rate-limiting step of cytokinin biosynthesis is mediated by isopentenyltransferases (IPTs), found in land plants either as adenylate-IPTs or as tRNA-IPTs. Although a dominant part of cytokinins in flowering plants are synthesized by adenylate-IPTs, the Physcomitrella genome only encodes homologues of tRNA-IPTs. This study therefore looked into the question of whether cytokinins in moss derive from tRNA exclusively. Targeted gene knockout of ipt1 (d|ipt1) along with localization studies revealed that the chloroplast-bound IPT1 was almost exclusively responsible for the A37 prenylation of tRNA in Physcomitrella. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)-based cytokinin profiling demonstrated that the total amount of all free cytokinins in tissue was almost unaffected. However, the knockout plants showed increased levels of the N (6) -isopentenyladenine (iP)- and trans-zeatin (tZ)-type cytokinins, considered to provide active forms, while cis-zeatin (cZ)-type cytokinins were reduced. The data provide evidence for an additional and unexpected tRNA-independent cytokinin biosynthetic pathway in moss. Comprehensive phylogenetic analysis indicates a diversification of tRNA-IPT-like genes in bryophytes probably related to additional functions.

Biochemical characterization of the maize cytokinin dehydrogenase family and cytokinin profiling in developing maize plantlets in relation to the expression of cytokinin dehydrogenase genes.

The cytokinin dehydrogenases (CKX; EC 1.5.99.12) are a protein family that maintains the endogenous levels of cytokinins in plants by catalyzing their oxidative degradation. The CKX family in maize (Zea mays L.) has thirteen members, only two of which--ZmCKX1 and ZmCKX10--have previously been characterized in detail. In this study, nine further maize CKX isoforms were heterologously expressed in Escherichia coli, purified by affinity and ion-exchange chromatography and biochemically characterized. ZmCKX6 and ZmCKX9 could only be expressed successfully after the removal of putative sequence-specific vacuolar sorting signals (LLPT and LPTS, respectively), suggesting that these proteins are localized to the vacuole. Substrate specificity analyses revealed that the CKX isoforms can be grouped into two subfamilies: members of the first strongly prefer cytokinin free bases while members of the second degrade a broad range of substrates. The most active isoform was found to be ZmCKX1. One of the studied isoforms, ZmCKX6, seemed to encode a nonfunctional enzyme due to a mutation in a conserved HFG protein domain at the C-terminus. Site-directed mutagenesis experiments revealed that this domain is essential for CKX activity. The roles of the maize CKX enzymes in the development of maize seedlings during the two weeks immediately after radicle emergence were also investigated. It appears that ZmCKX1 is a key regulator of active cytokinin levels in developing maize roots. However, the expression of individual CKX isoforms in the shoots varied and none of them seemed to have strong effects on the cytokinin pool.

Rhizobial synthesized cytokinins contribute to but are not essential for the symbiotic interaction between photosynthetic Bradyrhizobia and Aeschynomene legumes.

Cytokinins (CK) play an important role in the formation of nitrogen-fixing root nodules. It has been known for years that rhizobia secrete CK in the extracellular medium but whether they play a role in nodule formation is not known. We have examined this question using the photosynthetic Bradyrhizobium sp. strain ORS285 which is able to nodulate Aeschynomene afraspera and A. indica using a Nod-dependent or Nod-independent symbiotic process, respectively. CK profiling showed that the most abundant CK secreted by Bradyrhizobium sp. strain ORS285 are the 2MeS (2-methylthiol) derivatives of trans-zeatin and isopentenyladenine. In their pure form, these CK can activate legume CK receptors in vitro, and their exogenous addition induced nodule-like structures on host plants. Deletion of the miaA gene showed that transfer RNA degradation is the source of CK production in Bradyrhizobium sp. strain ORS285. In nodulation studies performed with A. indica and A. afraspera, the miaA mutant had a 1-day delay in nodulation and nitrogen fixation. Moreover, A. indica plants formed considerably smaller but more abundant nodules when inoculated with the miaA mutant. These data show that CK produced by Bradyrhizobium sp. strain ORS285 are not the key signal triggering nodule formation during the Nod-independent symbiosis but they contribute positively to nodule development in Aeschynomene plants.

Spatiotemporal regulation of lateral root organogenesis in Arabidopsis by cytokinin.

The architecture of a plant's root system, established postembryonically, results from both coordinated root growth and lateral root branching. The plant hormones auxin and cytokinin are central endogenous signaling molecules that regulate lateral root organogenesis positively and negatively, respectively. Tight control and mutual balance of their antagonistic activities are particularly important during the early phases of lateral root organogenesis to ensure continuous lateral root initiation (LRI) and proper development of lateral root primordia (LRP). Here, we show that the early phases of lateral root organogenesis, including priming and initiation, take place in root zones with a repressed cytokinin response. Accordingly, ectopic overproduction of cytokinin in the root basal meristem most efficiently inhibits LRI. Enhanced cytokinin responses in pericycle cells between existing LRP might restrict LRI near existing LRP and, when compromised, ectopic LRI occurs. Furthermore, our results demonstrate that young LRP are more sensitive to perturbations in the cytokinin activity than are developmentally more advanced primordia. We hypothesize that the effect of cytokinin on the development of primordia possibly depends on the robustness and stability of the auxin gradient.

Novel cytokinin derivatives do not show negative effects on root growth and proliferation in submicromolar range.

BACKGROUND: When applied to a nutrition solution or agar media, the non-substituted aromatic cytokinins caused thickening and shortening of the primary root, had an inhibitory effect on lateral root branching, and even showed some negative effects on development of the aerial part at as low as a 10 nanomolar concentration. Novel analogues of aromatic cytokinins ranking among topolins substituted on N9-atom of adenine by tetrahydropyranyl or 4-chlorobutyl group have been prepared and tested in standardized cytokinin bioassays [1]. Those showing comparable activities with N(6)-benzylaminopurine were further tested in planta. METHODOLOGY/PRINCIPAL FINDINGS: The main aim of the study was to explain molecular mechanism of function of novel cytokinin derivatives on plant development. Precise quantification of cytokinin content and profiling of genes involved in cytokinin metabolism and perception in treated plants revealed several aspects of different action of m-methoxytopolin base and its substituted derivative on plant development. In contrast to standard cytokinins, N9- tetrahydropyranyl derivative of m-topolin and its methoxy-counterpart showed the negative effects on root development only at three orders of magnitude higher concentrations. Moreover, the methoxy-derivative demonstrates a positive effect on lateral root branching and leaf emerging in a nanomolar range of concentrations, in comparison with untreated plants. CONCLUSIONS/SIGNIFICANCE: Tetrahydropyranyl substitution at N9-position of cytokinin purine ring significantly enhances acropetal transport of a given cytokinins. Together with the methoxy-substitution, impedes accumulation of non-active cytokinin glucoside forms in roots, allows gradual release of the active base, and has a significant effect on the distribution and amount of endogenous isoprenoid cytokinins in different plant tissues. The utilization of novel aromatic cytokinin derivatives can distinctively improve expected hormonal effects in plant propagation techniques in the future.