Chemical induction of hypocotyl rooting reveals extensive conservation of auxin signalling controlling lateral and adventitious root formation.

Upon exposure to light, etiolated Arabidopsis seedlings form adventitious roots (AR) along the hypocotyl. While processes underlying lateral root formation are studied intensively, comparatively little is known about the molecular processes involved in the initiation of hypocotyl AR. AR and LR formation were studied using a small molecule named Hypocotyl Specific Adventitious Root INducer (HYSPARIN) that strongly induces AR but not LR formation. HYSPARIN does not trigger rapid DR5-reporter activation, DII-Venus degradation or Ca2+ signalling. Transcriptome analysis, auxin signalling reporter lines and mutants show that HYSPARIN AR induction involves nuclear TIR1/AFB and plasma membrane TMK auxin signalling, as well as multiple downstream LR development genes (SHY2/IAA3, PUCHI, MAKR4 and GATA23). Comparison of the AR and LR induction transcriptome identified SAURs, AGC kinases and OFP transcription factors as specifically upregulated by HYSPARIN. Members of the SAUR19 subfamily, OFP4 and AGC2 suppress HYS-induced AR formation. While SAUR19 and OFP subfamily members also mildly modulate LR formation, AGC2 regulates only AR induction. Analysis of HYSPARIN-induced AR formation uncovers an evolutionary conservation of auxin signalling controlling LR and AR induction in Arabidopsis seedlings and identifies SAUR19, OFP4 and AGC2 kinase as novel regulators of AR formation.

Asynapsis and meiotic restitution in tomato male meiosis induced by heat stress.

Susceptibility of the reproductive system to temperature fluctuations is a recurrent problem for crop production under a changing climate. The damage is complex as multiple processes in male and female gamete formation are affected, but in general, particularly pollen production is impaired. Here, the impact of short periods of elevated temperature on male meiosis of tomato (Solanum lycopersicon L.) is reported. Meiocytes in early stage flower buds exposed to heat stress (>35°C) exhibit impaired homolog synapsis resulting in partial to complete omission of chiasmata formation. In the absence of chiasmata, univalents segregate randomly developing unbalanced tetrads and polyads resulting in aneuploid spores. However, most heat-stressed meiotic buds primarily contain balanced dyads, indicating a propensity to execute meiotic restitution. With most meiocytes exhibiting a complete loss of chiasma formation and concomitantly showing a mitotic-like division, heat stress triggers first division restitution resulting in clonal spores. These findings corroborate with the plasticity of male meiosis under heat and establish a natural route for the induction of sexual polyploidization in plants and the engineering of clonal seed.

Heat stress promotes haploid formation during CENH3-mediated genome elimination in Arabidopsis.

Impaired activity of centromeric histone CENH3 causes inaccurate chromosome segregation and in crosses between the Arabidopsis recombinant CENH3 mutant GFP-tailswap and CENH3G83E with wild-type pollen it results in chromosome loss with the formation of haploids. This genome elimination in the zygote and embryo is not absolute as also aneuploid and diploid progeny is formed. Here, we report that a temporal and moderate heat stress during fertilization and early embryogenesis shifts the ratio in favour of haploid progeny in CENH3 mutant lines. Micronuclei formation, a proxy for genome elimination, was similar in control and heat-treated flowers, indicating that heat-induced seed abortion occurred at a late stage during the development of the seed. In the seeds derived from heat-treated crosses, the endosperm did not cellularize and many seeds aborted. Haploid seeds were formed, however, resulting in increased frequencies of haploids in CENH3-mediated genome elimination crosses performed under heat stress. Therefore, heat stress application is a selective force during genome elimination that promotes haploid formation and may be used to improve the development and efficacy of in vivo haploid induction systems.

Sunflower Bark Extract as a Biostimulant Suppresses Reactive Oxygen Species in Salt-Stressed Arabidopsis.

A survey of plant-based wastes identified sunflower (Helianthus annuus) bark extract (SBE), produced via twin-screw extrusion, as a potential biostimulant. The addition of SBE to Arabidopsis (Arabidopsis thaliana) seedlings cultured in vitro showed a dose-dependent response, with high concentrations causing severe growth inhibition. However, when priming seeds with SBE, a small but significant increase in leaf area was observed at a dose of 0.5 g of lyophilized powder per liter. This optimal concentration of SBE in the culturing medium alleviated the growth inhibition caused by 100 mM NaCl. The recovery in shoot growth was accompanied by a pronounced increase in photosynthetic pigment levels and a stabilization of osmotic homeostasis. SBE-primed leaf discs also showed a similar protective effect. SBE mitigated salt stress by reducing the production of reactive oxygen species (ROS) (e.g., hydrogen peroxide) by about 30% and developing more expanded true leaves. This reduction in ROS levels was due to the presence of antioxidative agents in SBE and by activating ROS-eliminating enzymes. Polyphenols, carbohydrates, proteins, and other bioactive compounds detected in SBE may have contributed to the cellular redox homeostasis in salt-stressed plants, thus promoting early leaf development by relieving shoot apical meristem arrest. Sunflower stalks from which SBE is prepared can therefore potentially be valorized as a source to produce biostimulants for improving salt stress tolerance in crops.

Belgian endive-derived biostimulants promote shoot and root growth in vitro.

Recovering biostimulant compounds from by-products of crops is a promising strategy to add value, enhance sustainability, and increase the environmental safety of the agricultural production chain. Here, we report consistent root and shoot growth-stimulating bioactivity present in water-based extracts from Belgian endive forced roots (Cichorium intybus var. foliosum) over two consecutive harvest years. The shoot and the primary root of in vitro cultivated Arabidopsis thaliana treated with Belgian endive extract were about 30% increased in size compared to plants grown under control conditions. The ornamental species Plectranthus esculentus also showed enhanced in vitro shoot and root growth, suggesting bioactivity on a broad range of species. Fractionation of the Belgian endive extracts into aqueous and organic subfractions coupled with bioactivity measurements showed that the principal root and shoot growth-promoting ingredients are primarily water-soluble. NMR-based characterization of the bioactive aqueous fractions revealed the presence of predominantly sugars and organic acids. Malate and sugars were abundant and common to all water fractions, suggesting these molecules contributed to the growth stimulation phenotype. The findings indicate that Belgian endive roots are a source for the development of organic waste-derived biostimulants with potential for application in tissue culture and putatively for soil-grown crop production.

Genetic Dissection of Light-Regulated Adventitious Root Induction in Arabidopsis thaliana Hypocotyls.

Photomorphogenic responses of etiolated seedlings include the inhibition of hypocotyl elongation and opening of the apical hook. In addition, dark-grown seedlings respond to light by the formation of adventitious roots (AR) on the hypocotyl. How light signaling controls adventitious rooting is less well understood. Hereto, we analyzed adventitious rooting under different light conditions in wild type and photomorphogenesis mutants in Arabidopsis thaliana. Etiolation was not essential for AR formation but raised the competence to form AR under white and blue light. The blue light receptors CRY1 and PHOT1/PHOT2 are key elements contributing to the induction of AR formation in response to light. Furthermore, etiolation-controlled competence for AR formation depended on the COP9 signalosome, E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC (COP1), the COP1 interacting SUPPRESSOR OF PHYA-105 (SPA) kinase family members (SPA1,2 and 3) and Phytochrome-Interacting Factors (PIF). In contrast, ELONGATED HYPOCOTYL5 (HY5), suppressed AR formation. These findings provide a genetic framework that explains the high and low AR competence of Arabidopsis thaliana hypocotyls that were treated with dark, and light, respectively. We propose that light-induced auxin signal dissipation generates a transient auxin maximum that explains AR induction by a dark to light switch.

Histidine kinase inhibitors impair shoot regeneration in Arabidopsis thaliana via cytokinin signaling and SAM patterning determinants.

Reversible protein phosphorylation is a post-translational modification involved in virtually all plant processes, as it mediates protein activity and signal transduction. Here, we probe dynamic protein phosphorylation during de novo shoot organogenesis in Arabidopsis thaliana. We find that application of three kinase inhibitors in various time intervals has different effects on root explants. Short exposures to the putative histidine (His) kinase inhibitor TCSA during the initial days on shoot induction medium (SIM) are detrimental for regeneration in seven natural accessions. Investigation of cytokinin signaling mutants, as well as reporter lines for hormone responses and shoot markers, suggests that TCSA impedes cytokinin signal transduction via AHK3, AHK4, AHP3, and AHP5. A mass spectrometry-based phosphoproteome analysis further reveals profound deregulation of Ser/Thr/Tyr phosphoproteins regulating protein modification, transcription, vesicle trafficking, organ morphogenesis, and cation transport. Among TCSA-responsive factors are prior candidates with a role in shoot apical meristem patterning, such as AGO1, BAM1, PLL5, FIP37, TOP1ALPHA, and RBR1, as well as proteins involved in polar auxin transport (e.g., PIN1) and brassinosteroid signaling (e.g., BIN2). Putative novel regeneration determinants regulated by TCSA include RD2, AT1G52780, PVA11, and AVT1C, while NAIP2, OPS, ARR1, QKY, and aquaporins exhibit differential phospholevels on control SIM. LC-MS/MS data are available via ProteomeXchange with identifier PXD030754.

Arabidopsis Hypocotyl Adventitious Root Formation Is Suppressed by ABA Signaling.

Roots are composed of different root types and, in the dicotyledonous Arabidopsis, typically consist of a primary root that branches into lateral roots. Adventitious roots emerge from non-root tissue and are formed upon wounding or other types of abiotic stress. Here, we investigated adventitious root (AR) formation in Arabidopsis hypocotyls under conditions of altered abscisic acid (ABA) signaling. Exogenously applied ABA suppressed AR formation at 0.25 µM or higher doses. AR formation was less sensitive to the synthetic ABA analog pyrabactin (PB). However, PB was a more potent inhibitor at concentrations above 1 µM, suggesting that it was more selective in triggering a root inhibition response. Analysis of a series of phosphonamide and phosphonate pyrabactin analogs suggested that adventitious root formation and lateral root branching are differentially regulated by ABA signaling. ABA biosynthesis and signaling mutants affirmed a general inhibitory role of ABA and point to PYL1 and PYL2 as candidate ABA receptors that regulate AR inhibition.

In Vitro Assay for Induction of Adventitious Rooting on Intact Arabidopsis Hypocotyls.

Adventitious roots (AR) are de novo formed roots that emerge from any part of the plant or from callus in tissue culture, except root tissue. The plant tissue origin and the method by which they are induced determine the physiological properties of emerged ARs. Hence, a standard method encompassing all types of AR does not exist. Here we describe a method for the induction and analysis of AR that emerge from the etiolated hypocotyl of dicot plants. The hypocotyl is formed during embryogenesis and shows a determined developmental pattern which usually does not involve AR formation. However, the hypocotyl shows propensity to form de novo roots under specific circumstances such as removal of the root system, high humidity or flooding, or during de-etiolation. The hypocotyl AR emerge from a pericycle-like cell layer surrounding the vascular tissue of the central cylinder, which is reminiscent to the developmental program of lateral roots. Here we propose an easy protocol for in vitro hypocotyl AR induction from etiolated Arabidopsis seedlings.