Isothiocyanates (ITCs), the biologically important glucosinolate breakdown products, can present health-promoting effects, play an important role in plant defense and affect plant cellular mechanisms. Here, we evaluated the biological effects of ITCs on Arabidopsis thaliana by assessing growth parameters after long-term exposure to low concentrations of aliphatic and aromatic ITCs, ranging from 1 to 1000 µM. Treatment with the aliphatic allylisothiocyanate (allyl-ITC) led to a significant reduction of root length and fresh weight in a dose-dependent manner and affected the formation of lateral roots. To assess the importance of a hormonal crosstalk in the allyl-ITC-mediated growth reduction, the response of auxin and ethylene mutants was investigated, but our results did not allow us to confirm a role for these hormones. Aromatic ITCs generally led to a more severe growth inhibition than the aliphatic allyl-ITC. Interestingly, we observed a correlation between the length of their side chain and the effect these aromatic ITCs caused on Arabidopsis thaliana, with the greatest inhibitory effect seen for 2-phenylethyl-ITC. Root growth recovered when seedlings were removed from exposure to ITCs.

Glucosinolates are defense-related secondary metabolites found in Brassicaceae. When Brassicaceae come under attack, glucosinolates are hydrolyzed into different forms of glucosinolate hydrolysis products (GHPs). Among the GHPs, isothiocyanates are the most comprehensively characterized defensive compounds, whereas the functional study of nitriles, another group of GHP, is still limited. Therefore, this study investigates whether 3-butenenitrile (3BN), a nitrile, can trigger the signaling pathways involved in the regulation of defense responses in Arabidopsis thaliana against biotic stresses. Briefly, the methodology is divided into three stages, (i) evaluate the physiological and biochemical effects of exogenous 3BN treatment on Arabidopsis, (ii) determine the metabolites involved in 3BN-mediated defense responses in Arabidopsis, and (iii) assess whether a 3BN treatment can enhance the disease tolerance of Arabidopsis against necrotrophic pathogens. As a result, a 2.5 mM 3BN treatment caused lesion formation in Arabidopsis Columbia (Col-0) plants, a process found to be modulated by nitric oxide (NO). Metabolite profiling revealed an increased production of soluble sugars, Krebs cycle associated carboxylic acids and amino acids in Arabidopsis upon a 2.5 mM 3BN treatment, presumably via NO action. Primary metabolites such as sugars and amino acids are known to be crucial components in modulating plant defense responses. Furthermore, exposure to 2.0 mM 3BN treatment began to increase the production of salicylic acid (SA) and jasmonic acid (JA) phytohormones in Arabidopsis Col-0 plants in the absence of lesion formation. The production of SA and JA in nitrate reductase loss-of function mutant (nia1nia2) plants was also induced by the 3BN treatments, with a greater induction for JA. The SA concentration in nia1nia2 plants was lower than in Col-0 plants, confirming the previously reported role of NO in controlling SA production in Arabidopsis. A 2.0 mM 3BN treatment prior to pathogen assays effectively alleviated the leaf lesion symptom of Arabidopsis Col-0 plants caused by Pectobacterium carotovorum ssp. carotovorum and Botrytis cinerea and reduced the pathogen growth on leaves. The findings of this study demonstrate that 3BN can elicit defense response pathways in Arabidopsis, which potentially involves a coordinated crosstalk between NO and phytohormone signaling.

Plants within the Brassicales order generate glucosinolate hydrolysis products that can exert different biological effects on several organisms. Here, we evaluated the physiological effects of one of these compounds, benzyl cyanide (phenylacetonitrile), when exogenously applied on Arabidopsis thaliana. Treatment with benzyl cyanide led to a dose-dependent reduction of primary root length and total biomass. Further morphological changes like elongated hypocotyls, epinastic cotyledons and increased formation of adventitious roots resembled a severe auxin-overproducer phenotype. The elevated auxin response was confirmed by histochemical staining and gene expression analysis of auxin-responsive genes. Nitriles are converted by specific enzymes, nitrilases (NIT1-3), to their corresponding carboxylic acids. The nitrilase mutants nit1 and nit2 tolerated benzyl cyanide treatments better than the wild type, with nit2 being less resistant than nit1. A NIT2RNAi line suppressing several nitrilases was resistant to all tested benzyl cyanide concentrations. When exposed to phenylacetic acid -the corresponding carboxylic acid of benzyl cyanide- wild type and mutant seedlings were however equally susceptible and showed a more severe auxin phenotype than upon cyanide treatment. Here, we demonstrate that the auxin-like effects triggered by benzyl cyanide on Arabidopsis are due to its nitrilase-mediated conversion to the natural auxin phenylacetic acid.

The glucosinolate-myrosinase system is a well-known plant chemical defence system. Two functional myrosinase-encoding genes, THIOGLUCOSIDASE 1 (TGG1) and THIOGLUCOSIDASE 2 (TGG2), express in aerial tissues of Arabidopsis. TGG1 expresses in guard cells (GCs) and is also a highly abundant protein in GCs. Recently, by studying wild type (WT), tgg single, and double mutants, we showed a novel association between the glucosinolate-myrosinase system defence system, and a physical barrier, the cuticle. In the current study, using imaging techniques, we further analysed stomata and ultrastructure of GCs of WT, tgg1, tgg2 single, and tgg1 tgg2 double mutants. The tgg mutants showed distinctive features of GCs. The GCs of tgg1 and tgg1 tgg2 mutants showed vacuoles that had less electron-dense granular material. Both tgg single mutants had bigger stomata complexes. The WT and tgg mutants also showed variations for cell wall, chloroplasts, and starch grains of GCs. Abscisic acid (ABA)-treated stomata showed that the stomatal aperture was reduced in tgg1 single and tgg1 tgg2 double mutants. The data provides a basis to perform comprehensive further studies to find physiological and molecular mechanisms associated with ultrastructure differences in tgg mutants. We speculate that the absence of myrosinase alters the endogenous chemical composition, hence affecting the physical structure of plants and the plants’ physical defence barriers.

The various applications of Ag(I) generated the necessity to obtain Ag(I)-accumulating organisms for the removal of surplus Ag(I) from contaminated sites or for the concentration of Ag(I) from Ag(I)-poor environments. In this study we obtained Ag(I)-accumulating cells by expressing plant metallothioneins (MTs) in the model Saccharomyces cerevisiae. The cDNAs of seven Arabidopsis thaliana MTs (AtMT1a, AtMT1c, AtMT2a, AtMT2b, AtMT3, AtMT4a and AtMT4b) and four Noccaea caerulescens MTs (NcMT1, NcMT2a, NcMT2b and NcMT3) fused to myrGFP displaying an N-terminal myristoylation sequence for plasma membrane targeting were expressed in S. cerevisiae and checked for Ag(I)-related phenotype. The transgenic yeast cells were grown in copper-deficient media to ensure the expression of the plasma membrane high-affinity Cu(I) transporter Ctr1, and also to elude the copper-related inhibition of Ag(I) transport into the cell. All plant MTs expressed in S. cerevisiae conferred Ag(I) tolerance to the yeast cells. Among them, myrGFP-NcMT3 afforded Ag(I) accumulation under high concentration (10–50 μM), while myrGFP-AtMT1a conferred increased accumulation capacity under low (1 μM) or even trace Ag(I) (0.02–0.05 μM). The ability to tolerate high concentrations of Ag(I) coupled with accumulative characteristics and robust growth showed by some of the transgenic yeasts highlighted the potential of these strains for biotechnology applications.

Diatoxanthin is a photoprotective carotenoid found in a few groups of microalgae displaying in vitro anti-inflammatory and anti-cancer properties, making it a promising candidate for nutraceutical, pharmaceutical, and cosmetic applications. However, large-scale production is currently nonexistent because of two major challenges: Instability during microalgae harvesting, where diatoxanthin is rapidly converted back to its inactive precursor diadinoxanthin under non-stressful light conditions, and dependence on prolonged exposure to high-intensity light, which is costly and technically challenging during indoor high-cell-density cultivation. The first limitation was previously addressed by knocking out zeaxanthin epoxidase 3 (ZEP3) in the marine diatom Phaeodactylum tricornutum, resulting in a mutant that stabilized diatoxanthin under non-stressful light conditions. Here, we report an improved diatoxanthin production line where both of the described challenges have been overcome. This was achieved by creating P. tricornutum mutants where the phenotype of the zep3 mutant was combined with the light-sensitive phenotype of the chloroplast signal recognition particle 54 (cpsrp54) mutant. Growth rates were maintained at wild-type levels at light intensities ≤ 150 µmol photons m⁻² s⁻¹ in the zep3cpsrp54 mutants, but prolonged medium light exposure resulted in a 1.5- and 7-fold increase in diatoxanthin concentration compared with zep3 and wild-type, respectively. When returned to low light, the zep3cpsrp54 cultures retained ~80% of their accumulated diatoxanthin. The improved production lines allow for diatoxanthin accumulation without the use of high-intensity light and with limited loss of diatoxanthin when returned to non-stressful light conditions.