The endophytic fungus Serendipita indica affects auxin distribution in Arabidopsis thaliana roots through alteration of auxin transport and conjugation to promote plant growth.

Plants share their habitats with a multitude of different microbes. This close vicinity promoted the evolution of interorganismic interactions between plants and many different microorganisms that provide mutual growth benefits both to the plant and the microbial partner. The symbiosis of Arabidopsis thaliana with the beneficial root colonizing endophyte Serendipita indica represents a well-studied system. Colonization of Arabidopsis roots with S. indica promotes plant growth and stress tolerance of the host plant. However, until now, the molecular mechanism by which S. indica reprograms plant growth remains largely unknown. This study used comprehensive transcriptomics, metabolomics, reverse genetics, and life cell imaging to reveal the intricacies of auxin-related processes that affect root growth in the symbiosis between A. thaliana and S. indica. Our experiments revealed the sustained stimulation of auxin signalling in fungus infected Arabidopsis roots and disclosed the essential role of tightly controlled auxin conjugation in the plant-fungus interaction. It particularly highlighted the importance of two GRETCHEN HAGEN 3 (GH3) genes, GH3.5 and GH3.17, for the fungus infection-triggered stimulation of biomass production, thus broadening our knowledge about the function of GH3s in plants. Furthermore, we provide evidence for the transcriptional alteration of the PIN2 auxin transporter gene in roots of Arabidopsis seedlings infected with S. indica and demonstrate that this transcriptional adjustment affects auxin signalling in roots, which results in increased plant growth.

Growth and Molecular Responses of Tomato to Prolonged and Short-Term Heat Exposure.

Tomatoes are one of the most important vegetables for human consumption. In the Mediterranean's semi-arid and arid regions, where tomatoes are grown in the field, global average surface temperatures are predicted to increase. We investigated tomato seed germination at elevated temperatures and the impact of two different heat regimes on seedlings and adult plants. Selected exposures to 37 °C and heat waves at 45 °C mirrored frequent summer conditions in areas with a continental climate. Exposure to 37 °C or 45 °C differently affected seedlings' root development. Both heat stresses inhibited primary root length, while lateral root number was significantly suppressed only after exposure to 37 °C. Heat stress treatments induced significant accumulation of indole-3-acetic acid (IAA) and reduced abscisic acid (ABA) levels in seedlings. As opposed to the heat wave treatment, exposure to 37 °C increased the accumulation of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), which may have been involved in the root architecture modification of seedlings. Generally, more drastic phenotypic changes (chlorosis and wilting of leaves and bending of stems) were found in both seedlings and adult plants after the heat wave-like treatment. This was also reflected by proline, malondialdehyde and heat shock protein HSP90 accumulation. The gene expression of heat stress-related transcription factors was perturbed and DREB1 was shown to be the most consistent heat stress marker.

BPM1 regulates RdDM-mediated DNA methylation via a cullin 3 independent mechanism.

KEY MESSAGE: BPM1 interacts with components of the DDR complex and stimulates DNA methylation at CHH sites, suggesting its involvement in the RdDM methylation pathway. The best-known function of MATH-BTB proteins, including Arabidopsis BPM proteins, is their role as substrate-specific adaptors of CUL3-based E3 ligases in the ubiquitin-proteasome pathway. This paper reports a new CUL3-independent role of BPM1 in RNA-directed DNA methylation (RdDM). Using quantitative and qualitative Y2H, pull down, microscale thermophoresis and FRET-FLIM, we demonstrate that BPM1 interacts with DMS3 and RDM1, components of the chromatin remodeling DDR complex involved in the recruitment of the RdDM methylation machinery. All three proteins colocalized predominantly in the nucleus. The MATH domain, which specifically binds proteins destined for degradation, was not essential for interactions with DMS3 and RDM1. In plants overexpressing BPM1, endogenous DMS3 protein levels were stable, indicating that BPM1 does not induce proteasomal degradation. In RDM1-overexpressing plants, RDM1 was not ubiquitinated. Together, these results suggest that BPM1 does not mediate the degradation of DMS3 and RDM1. Additionally, overexpression of BPM1 caused increased global methylation levels as well as CHH methylation in promoters of two RdDM-regulated genes, FWA and CML41. Overall, BPM1 seems to have a stimulating effect on RdDM activity, and this role appears to be unrelated to its known function as a Cul3-based E3 ligase adaptor.

Mechanisms of Kale (Brassica oleracea var. acephala) Tolerance to Individual and Combined Stresses of Drought and Elevated Temperature.

Rising temperatures and pronounced drought are significantly affecting biodiversity worldwide and reducing yields and quality of Brassica crops. To elucidate the mechanisms of tolerance, 33 kale accessions (B. oleracea var. acephala) were evaluated for individual (osmotic and elevated temperature stress) and combined stress (osmotic + temperature). Using root growth, biomass and proline content as reliable markers, accessions were evaluated for stress responses. Four representatives were selected for further investigation (photosynthetic performance, biochemical markers, sugar content, specialized metabolites, transcription level of transcription factors NAC, HSF, DREB and expression of heat shock proteins HSP70 and HSP90): very sensitive (392), moderately sensitive (395), tolerant (404) and most tolerant (411). Accessions more tolerant to stress conditions were characterized by higher basal content of proline, total sugars, glucosinolates and higher transcription of NAC and DREB. Under all stress conditions, 392 was characterized by a significant decrease in biomass, root growth, photosynthesis performance, fructan content, especially under osmotic and combined stress, a significant increase in HSF transcription and HSP accumulation under temperature stress and a significant decrease in NAC transcription under all stresses. The most tolerant accession under all applied stresses, 411 showed the least changes in all analyzed parameters compared with the other accessions.

Altered Root Growth, Auxin Metabolism and Distribution in Arabidopsis thaliana Exposed to Salt and Osmotic Stress.

Salt and osmotic stress are the main abiotic stress factors affecting plant root growth and architecture. We investigated the effect of salt (100 mM NaCl) and osmotic (200 mM mannitol) stress on the auxin metabolome by UHPLC-MS/MS, auxin distribution by confocal microscopy, and transcript levels of selected genes by qRT-PCR in Arabidopsis thaliana ecotype Columbia-0 (Col-0) and DR5rev::GFP (DR5) line. During long-term stress (13 days), a stability of the auxin metabolome and a tendency to increase indole-3-acetic acid (IAA) were observed, especially during salt stress. Short-term stress (3 h) caused significant changes in the auxin metabolome, especially NaCl treatment resulted in a significant reduction of IAA. The data derived from auxin profiling were consistent with gene expressions showing the most striking changes in the transcripts of YUC, GH3, and UGT transcripts, suggesting disruption of auxin biosynthesis, but especially in the processes of amide and ester conjugation. These data were consistent with the auxin distribution observed in the DR5 line. Moreover, NaCl treatment caused a redistribution of auxin signals from the quiescent center and the inner layers of the root cap to the epidermal and cortical cells of the root elongation zone. The distribution of PIN proteins was also disrupted by salt stress; in particular, PIN2 was suppressed, even after 5 min of treatment. Based on our results, the DR5 line was more sensitive to the applied stresses than Col-0, although both lines showed similar trends in root morphology, as well as transcriptome and metabolome parameters under stress conditions.

The protein turnover of Arabidopsis BPM1 is involved in regulation of flowering time and abiotic stress response.

KEY MESSAGE: Protein degradation is essential in plant growth and development. The stability of Cullin3 substrate adaptor protein BPM1 is regulated by multiple environmental cues pointing on manifold control of targeted protein degradation. A small family of six MATH-BTB genes (BPM1-6) is described in Arabidopsis thaliana. BPM proteins are part of the Cullin E3 ubiquitin ligase complexes and are known to bind at least three families of transcription factors: ERF/AP2 class I, homeobox-leucine zipper and R2R3 MYB. By targeting these transcription factors for ubiquitination and subsequent proteasomal degradation, BPMs play an important role in plant flowering, seed development and abiotic stress response. In this study, we generated BPM1-overexpressing plants that showed an early flowering phenotype, resistance to abscisic acid and tolerance to osmotic stress. We analyzed BPM1-GFP protein stability and found that the protein has a high turnover rate and is degraded by the proteasome 26S in a Cullin-dependent manner. Finally, we found that BPM1 protein stability is environmentally conditioned. Darkness and salt stress triggered BPM1 degradation, whereas elevated temperature enhanced BPM1 stability and accumulation in planta.

Potential of Different Coleus blumei Tissues for Rosmarinic Acid Production.

Rosmarinic acid is one of the main active components of Coleus blumei and is known to have numerous health benefits. The pharmacological significance of rosmarinic acid and its production through in vitro culture has been the subject of numerous studies. Here, the ability of different tissues to accumulate rosmarinic acid and sustainability in production over long cultivation have been tested. Calli, tumours, normal roots and hairy roots were established routinely by application of plant growth regulators or by transformation with agrobacteria. The differences among the established tumour lines were highly heterogeneous. Hairy root lines showed the highest mean growth rate and consistency in rosmarinic acid production. Although some tumour lines produced more rosmarinic acid than the hairy root lines, over a long cultivation period their productivity was unstable and decreased. Further, the effects of plant growth regulators on growth and rosmarinic acid accumulation were tested. 2,4-Dichlorophenoxyacetic acid significantly reduced tumour growth and rosmarinic acid production. 1-Naphthaleneacetic acid strongly stimulated hairy root growth whilst abscisic acid strongly enhanced rosmarinic acid production. Hairy roots cultured in an airlift bioreactor exhibited the highest potential for mass production of rosmarinic acid.

Taking the Wheel - de novo DNA Methylation as a Driving Force of Plant Embryonic Development.

During plant embryogenesis, regardless of whether it begins with a fertilized egg cell (zygotic embryogenesis) or an induced somatic cell (somatic embryogenesis), significant epigenetic reprogramming occurs with the purpose of parental or vegetative transcript silencing and establishment of a next-generation epigenetic patterning. To ensure genome stability of a developing embryo, large-scale transposon silencing occurs by an RNA-directed DNA methylation (RdDM) pathway, which introduces methylation patterns de novo and as such potentially serves as a global mechanism of transcription control during developmental transitions. RdDM is controlled by a two-armed mechanism based around the activity of two RNA polymerases. While PolIV produces siRNAs accompanied by protein complexes comprising the methylation machinery, PolV produces lncRNA which guides the methylation machinery toward specific genomic locations. Recently, RdDM has been proposed as a dominant methylation mechanism during gamete formation and early embryo development in Arabidopsis thaliana, overshadowing all other methylation mechanisms. Here, we bring an overview of current knowledge about different roles of DNA methylation with emphasis on RdDM during plant zygotic and somatic embryogenesis. Based on published chromatin immunoprecipitation data on PolV binding sites within the A. thaliana genome, we uncover groups of auxin metabolism, reproductive development and embryogenesis-related genes, and discuss possible roles of RdDM at the onset of early embryonic development via targeted methylation at sites involved in different embryogenesis-related developmental mechanisms.

The MATH-BTB Protein TaMAB2 Accumulates in Ubiquitin-Containing Foci and Interacts With the Translation Initiation Machinery in Arabidopsis.

MATH-BTB proteins are known to act as substrate-specific adaptors of CUL3-based E3 ligases in the ubiquitin proteasome pathway. Their BTB domain binds to CUL3 scaffold proteins and the less conserved MATH domain targets a highly diverse collection of substrate proteins to promote their ubiquitination and subsequent degradation. In plants, a significant expansion of the MATH-BTB family occurred in the grasses. Here, we report analysis of TaMAB2, a MATH-BTB protein transiently expressed at the onset of embryogenesis in wheat. Due to difficulties in studying its role in zygotes and early embryos, we have overexpressed TaMAB2 in Arabidopsis to generate gain-of-function mutants and to elucidate interaction partners and substrates. Overexpression plants showed severe growth defects as well as disorganization of microtubule bundles indicating that TaMAB2 interacts with substrates in Arabidopsis. In tobacco BY-2 cells, TaMAB2 showed a microtubule and ubiquitin-associated cytoplasmic localization pattern in form of foci. Its direct interaction with CUL3 suggests functions in targeting specific substrates for ubiquitin-dependent degradation. Although direct interactions with tubulin could not be confimed, tandem affinity purification of TaMAB2 interactors point towards cytoskeletal proteins including tubulin and actin as well as the translation initiation machinery. The idenification of various subunits of eucaryotic translation initiation factors eIF3 and eIF4 as TaMAB2 interactors indicate regulation of translation initiation as a major function during onset of embryogenesis in plants.

Somatic embryogenesis in pumpkin (Cucurbita pepo L.): control of somatic embryo development by nitrogen compounds.

Embryogenic cultures of pumpkin (Cucurbita pepo L.) were initiated from mechanically wounded mature zygotic embryos on 2,4-D-containing MS medium, and on hormone-free, semisolid modified MS medium containing NH4Cl as the sole source of nitrogen. The habituated line was derived from the embryogenic tissue induced with 2,4-D and maintained on medium without growth regulators. Sustained subculturing of the three embryogenic lines on a medium with NH4Cl as the sole source of nitrogen enabled the establishment of highly uniform cultures in which no further development into mature embryo stages occurred. The tissue consisting of proembryogenic globules or globular stage embryos was maintained, without decline, for over six years. Globular embryos proceeded to maturity when a combination of reduced (NH4) and unreduced (NO3) forms of nitrogen was provided in the medium. Different nitrogen sources in the medium caused changes of medium pH during subculture in the pH range of 4.0-6.5. The tissue growth and embryo development were blocked on medium with pH adjusted and stabilized at 4.0 or at 3.2.