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Calcium-dependent protein kinases (CPK) are key components of a wide array of signaling pathways, translating stress and nutrient signaling into the modulation of cellular processes such as ion transport and transcription. However, not much is known about CPKs in endomembrane trafficking. Here, we screened for CPKs that impact on root growth and gravitropism, by overexpressing constitutively active forms of CPKs under the control of an inducible promoter in Arabidopsis thaliana. We found that inducible overexpression of an constitutive active CPK30 (CA-CPK30) resulted in a loss of root gravitropism and ectopic auxin accumulation in the root tip. Immunolocalization revealed that CA-CPK30 roots have reduced PIN protein levels, PIN1 polarity defects and impaired Brefeldin A (BFA)-sensitive trafficking. Moreover, FM4-64 uptake was reduced, indicative of a defect in endocytosis. The effects on BFA-sensitive trafficking were not specific to PINs, as BFA could not induce aggregation of ARF1- and CHC-labeled endosomes in CA-CPK30. Interestingly, the interference with BFA-body formation, could be reverted by increasing the extracellular pH, indicating a pH-dependence of this CA-CPK30 effect. Altogether, our data reveal an important role for CPK30 in root growth regulation and endomembrane trafficking in Arabidopsis thaliana.

Genes encoding 45S ribosomal RNA (rDNA) are known for their abundance within eukaryotic genomes and for their unstable copy numbers in response to changes in various genetic and epigenetic factors. Commonly, we understand as epigenetic factors (affecting gene expression without a change in DNA sequence), namely DNA methylation, histone posttranslational modifications, histone variants, RNA interference, nucleosome remodeling and assembly, and chromosome position effect. All these were actually shown to affect activity and stability of rDNA. Here, we focus on another phenomenon – the potential of DNA containing shortly spaced oligo-guanine tracts to form quadruplex structures (G4). Interestingly, sites with a high propensity to form G4 were described in yeast, animal, and plant rDNAs, in addition to G4 at telomeres, some gene promoters, and transposons, suggesting the evolutionary ancient origin of G4 as a regulatory module. Here, we present examples of rDNA promoter regions with extremely high potential to form G4 in two model plants, Arabidopsis thaliana and Physcomitrella patens. The high G4 potential is balanced by the activity of G4-resolving enzymes. The ability of rDNA to undergo these “structural gymnastics” thus represents another layer of the rich repertoire of epigenetic regulations, which is pronounced in rDNA due to its highly repetitive character.

Thanks to recent innovative methodologies, key cellular processes such as replication or transcription can be visualized directly in situ in intact tissues. Many studies use so-called click iT chemistry where nascent DNA can be tracked by 5-ethynyl-2′-deoxyuridine (EdU), and nascent RNA by 5-ethynyl uridine (EU). While the labeling of replicating DNA by EdU has already been well established and further exploited in plants, the use of EU to reveal nascent RNA has not been developed to such an extent. In this article, we present a protocol for labeling of nucleolar RNA transcripts using EU and show that EU effectively highlights the nucleolus. The method is advantageous, because the need to prepare transgenic plants expressing fluorescently tagged nucleolar components when the nucleolus has to be visualized can be avoided.

Inter-individual variability of conspecific plants is governed by differences in their genetically determined growth and development traits, environmental conditions, and adaptive responses under epigenetic control involving histone post-translational modifications. The apparent variability in histone modifications among plants might be increased by technical variation introduced in sample processing during epigenetic analyses. Thus, to detect true variations in epigenetic histone patterns associated with given factors, the basal variability among samples that is not associated with them must be estimated. To improve knowledge of relative contribution of biological and technical variation, mass spectrometry was used to examine histone modification patterns (acetylation and methylation) among Arabidopsis thaliana plants of ecotypes Columbia 0 (Col-0) and Wassilewskija (Ws) homogenized by two techniques (grinding in a cryomill or with a mortar and pestle). We found little difference in histone modification profiles between the ecotypes. However, in comparison of the biological and technical components of variability, we found consistently higher inter-individual variability in histone mark levels among Ws plants than among Col-0 plants (grown from seeds collected either from single plants or sets of plants). Thus, more replicates of Ws would be needed for rigorous analysis of epigenetic marks. Regarding technical variability, the cryomill introduced detectably more heterogeneity in the data than the mortar and pestle treatment, but mass spectrometric analyses had minor apparent effects. Our study shows that it is essential to consider inter-sample variance and estimate suitable numbers of biological replicates for statistical analysis for each studied organism when investigating changes in epigenetic histone profiles.

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 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 chlorophyll fluorescence 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.