Magdalena M. Julkowska

Halotropism is a response of plant roots to avoid a saline environment

Tropisms represent fascinating examples of how plants respond to environmental signals by adapting their growth and development. Here, a novel tropism is reported, halotropism, allowing plant seedlings to reduce their exposure to salinity by circumventing a saline environment. In response to a salt gradient, Arabidopsis, tomato, and sorghum roots were found to actively prioritize growth away from salinity above following the gravity axis. Directionality of this response is established by an active redistribution of the plant hormone auxin in the root tip, which is mediated by the PIN-FORMED 2 (PIN2) auxin efflux carrier. We show that salt-induced phospholipase D activity stimulates clathrin-mediated endocytosis of PIN2 at the side of the root facing the higher salt concentration. The intracellular relocalization of PIN2 allows for auxin redistribution and for the directional bending of the root away from the higher salt concentration. Our results thus identify a cellular pathway essential for the integration of environmental cues with auxin-regulated root growth that likely plays a key role in plant adaptative responses to salt stress.

Tuning plant signaling and growth to survive salt

Salinity is one of the major abiotic factors threatening food security worldwide. Recently, our understanding of early processes underlying salinity tolerance has expanded. In this review, early signaling events, such as phospholipid signaling, calcium ion (Ca(2+)) responses, and reactive oxygen species (ROS) production, together with salt stress-induced abscisic acid (ABA) accumulation, are brought into the context of long-term salt stress-specific responses and alteration of plant growth. Salt-induced quiescent and recovery growth phases rely on modification of cell cycle activity, cell expansion, and cell wall extensibility. The period of initial growth arrest varies among different organs, leading to altered plant morphology. Studying stress-induced changes in growth dynamics can be used for screening to discover novel genes contributing to salt stress tolerance in model species and crops.

The Snf1-related protein kinases SnRK2.4 and SnRK2.10 are involved in maintenance of root system architecture during salt stress

The sucrose non-fermenting-1-related protein kinase 2 (SnRK2) family represents a unique family of plant-specific protein kinases implicated in cellular signalling in response to osmotic stress. In our studies, we observed that two class 1 SnRK2 kinases, SnRK2.4 and SnRK2.10, are rapidly and transiently activated in Arabidopsis roots after exposure to salt. Under saline conditions, snrk2.4 knockout mutants had a reduced primary root length, while snrk2.10 mutants exhibited a reduction in the number of lateral roots. The reduced lateral root density was found to be a combinatory effect of a decrease in the number of lateral root primordia and an increase in the number of arrested lateral root primordia. The phenotypes were in agreement with the observed expression patterns of genomic yellow fluorescent protein (YFP) fusions of SnRK2.10 and -2.4, under control of their native promoter sequences. SnRK2.10 was found to be expressed in the vascular tissue at the base of a developing lateral root, whereas SnRK2.4 was expressed throughout the root, with higher expression in the vascular system. Salt stress triggered a rapid re-localization of SnRK2.4–YFP from the cytosol to punctate structures in root epidermal cells. Differential centrifugation experiments of isolated Arabidopsis root proteins confirmed recruitment of endogenous SnRK2.4/2.10 to membranes upon exposure to salt, supporting their observed binding affinity for the phospholipid phosphatidic acid. Together, our results reveal a role for SnRK2.4 and -2.10 in root growth and architecture in saline conditions.

Roles for blue light, jasmonate and nitric oxide in the regulation of dormancy and germination in wheat grain (Triticum aestivum L.)

Abscisic acid (ABA) plays a central role in seed dormancy and transcriptional regulation of genes coding for ABA biosynthetic and degradation enzymes is responsible for control of ABA content. However, little is known about signalling both before and after ABA regulation, in particular, how environmental signals are perceived and transduced. We are interested in these processes in cereal grains, particularly in relation to the development of strategies for controlling pre-harvest sprouting in barley and wheat. Our previous studies have indicated possible components of dormancy control and here we present evidence that blue light, nitric oxide (NO) and jasmonate are major controlling elements in wheat grain. Using microarray and pharmacological studies, we have found that blue light inhibits germination in dormant grain and that methyl jasmonate (MJ) and NO counteract this effect by reducing dormancy. We also present evidence that NO and jasmonate play roles in dormancy control in vivo. ABA was reduced by MJ and this was accompanied by reduced levels of expression of TaNCED1 and increased expression of TaABA8′OH-1 compared with dormant grain. Similar changes were caused by after-ripening. Analysis of global gene expression showed that although jasmonate and after-ripening caused important changes in gene expression, the changes were very different. While breaking dormancy, MJ had only a small number of target genes including gene(s) encoding beta-glucosidase. Our evidence indicates that NO and MJ act interdependently in controlling reduction of ABA and thus the demise of dormancy.

Capturing Arabidopsis root architecture dynamics with ROOT-FIT reveals diversity in responses to salinity

Natural variation in the dynamic response to salt stress of the Arabidopsis root system can be divided between four distinct strategies. The plant root is the first organ to encounter salinity stress, but the effect of salinity on root system architecture (RSA) remains elusive. Both the reduction in main root (MR) elongation and the redistribution of the root mass between MRs and lateral roots (LRs) are likely to play crucial roles in water extraction efficiency and ion exclusion. To establish which RSA parameters are responsive to salt stress, we performed a detailed time course experiment in which Arabidopsis (Arabidopsis thaliana) seedlings were grown on agar plates under different salt stress conditions. We captured RSA dynamics with quadratic growth functions (root-fit) and summarized the salt-induced differences in RSA dynamics in three growth parameters: MR elongation, average LR elongation, and increase in number of LRs. In the ecotype Columbia-0 accession of Arabidopsis, salt stress affected MR elongation more severely than LR elongation and an increase in LRs, leading to a significantly altered RSA. By quantifying RSA dynamics of 31 different Arabidopsis accessions in control and mild salt stress conditions, different strategies for regulation of MR and LR meristems and root branching were revealed. Different RSA strategies partially correlated with natural variation in abscisic acid sensitivity and different Na+/K+ ratios in shoots of seedlings grown under mild salt stress. Applying root-fit to describe the dynamics of RSA allowed us to uncover the natural diversity in root morphology and cluster it into four response types that otherwise would have been overlooked.

Genetic architecture of plant stress resistance: multi‐trait genome‐wide association mapping

Summary Plants are exposed to combinations of various biotic and abiotic stresses, but stress responses are usually investigated for single stresses only. Here, we investigated the genetic architecture underlying plant responses to 11 single stresses and several of their combinations by phenotyping 350 Arabidopsis thaliana accessions. A set of 214 000 single nucleotide polymorphisms (SNPs) was screened for marker‐trait associations in genome‐wide association (GWA) analyses using tailored multi‐trait mixed models. Stress responses that share phytohormonal signaling pathways also share genetic architecture underlying these responses. After removing the effects of general robustness, for the 30 most significant SNPs, average quantitative trait locus (QTL) effect sizes were larger for dual stresses than for single stresses. Plants appear to deploy broad‐spectrum defensive mechanisms influencing multiple traits in response to combined stresses. Association analyses identified QTLs with contrasting and with similar responses to biotic vs abiotic stresses, and below‐ground vs above‐ground stresses. Our approach allowed for an unprecedented comprehensive genetic analysis of how plants deal with a wide spectrum of stress conditions.

Phosphate-dependent root system architecture responses to salt stress

Arabidopsis accessions show different patterns of integrating root responses to phosphate starvation and salt stress. Nutrient availability and salinity of the soil affect the growth and development of plant roots. Here, we describe how inorganic phosphate (Pi) availability affects the root system architecture (RSA) of Arabidopsis (Arabidopsis thaliana) and how Pi levels modulate responses of the root to salt stress. Pi starvation reduced main root length and increased the number of lateral roots of Arabidopsis Columbia-0 seedlings. In combination with salt, low Pi dampened the inhibiting effect of mild salt stress (75 mm) on all measured RSA components. At higher salt concentrations, the Pi deprivation response prevailed over the salt stress only for lateral root elongation. The Pi deprivation response of lateral roots appeared to be oppositely affected by abscisic acid signaling compared with the salt stress response. Natural variation in the response to the combination treatment of salt and Pi starvation within 330 Arabidopsis accessions could be grouped into four response patterns. When exposed to double stress, in general, lateral roots prioritized responses to salt, while the effect on main root traits was additive. Interestingly, these patterns were not identical for all accessions studied, and multiple strategies to integrate the signals from Pi deprivation and salinity were identified. By genome-wide association mapping, 12 genomic loci were identified as putative factors integrating responses to salt stress and Pi starvation. From our experiments, we conclude that Pi starvation interferes with salt responses mainly at the level of lateral roots and that large natural variation exists in the available genetic repertoire of accessions to handle the combination of stresses.

High-Throughput Non-destructive Phenotyping of Traits that Contribute to Salinity Tolerance in Arabidopsis thaliana

Reproducible and efficient high-throughput phenotyping approaches, combined with advances in genome sequencing, are facilitating the discovery of genes affecting plant performance. Salinity tolerance is a desirable trait that can be achieved through breeding, where most have aimed at selecting for plants that perform effective ion exclusion from the shoots. To determine overall plant performance under salt stress, it is helpful to investigate several plant traits collectively in one experimental setup. Hence, we developed a quantitative phenotyping protocol using a high-throughput phenotyping system, with RGB and chlorophyll fluorescence (ChlF) imaging, which captures the growth, morphology, color and photosynthetic performance of Arabidopsis thaliana plants in response to salt stress. We optimized our salt treatment by controlling the soil-water content prior to introducing salt stress. We investigated these traits over time in two accessions in soil at 150, 100, or 50 mM NaCl to find that the plants subjected to 100 mM NaCl showed the most prominent responses in the absence of symptoms of severe stress. In these plants, salt stress induced significant changes in rosette area and morphology, but less prominent changes in rosette coloring and photosystem II efficiency. Clustering of ChlF traits with plant growth of nine accessions maintained at 100 mM NaCl revealed that in the early stage of salt stress, salinity tolerance correlated with non-photochemical quenching processes and during the later stage, plant performance correlated with quantum yield. This integrative approach allows the simultaneous analysis of several phenotypic traits. In combination with various genetic resources, the phenotyping protocol described here is expected to increase our understanding of plant performance and stress responses, ultimately identifying genes that improve plant performance in salt stress conditions.

Liposome-binding assays to assess specificity and affinity of phospholipid-protein interactions

Protein-lipid interactions play an important role in cellular protein relocation, activation and signal transduction. The liposome-binding assay is a simple and inexpensive method to examine protein-lipid binding in vitro. The phospholipids used for liposome production are dried and hydrated. Subsequent extrusion of the phospholipid mixture ensures the production of large unilamellar vesicles (LUV) filled with raffinose. Those LUVs can be easily separated from the aqueous solution by centrifugation. By incubating a protein of interest with the LUVs and subsequent centrifugation steps, the bound protein fraction can be determined using Western Blot or Coomassie staining. This technique enables analysis of protein-lipid binding affinity and specificity.

Identification and functional characterization of the Arabidopsis Snf1-related protein kinase SnRK2.4 phosphatidic acid-binding domain

Phosphatidic acid (PA) is an important signalling lipid involved in various stress-induced signalling cascades. Two SnRK2 protein kinases (SnRK2.4 and SnRK2.10), previously identified as PA-binding proteins, are shown here to prefer binding to PA over other anionic phospholipids and to associate with cellular membranes in response to salt stress in Arabidopsis roots. A 42 amino acid sequence was identified as the primary PA-binding domain (PABD) of SnRK2.4. Unlike the full-length SnRK2.4, neither the PABD-YFP fusion protein nor the SnRK2.10 re-localized into punctate structures upon salt stress treatment, showing that additional domains of the SnRK2.4 protein are required for its re-localization during salt stress. Within the PABD, five basic amino acids, conserved in class 1 SnRK2s, were found to be necessary for PA binding. Remarkably, plants overexpressing the PABD, but not a non-PA-binding mutant version, showed a severe reduction in root growth. Together, this study biochemically characterizes the PA-SnRK2.4 interaction and shows that functionality of the SnRK2.4 PABD affects root development.