The toxic metalloid arsenic is prevalent in the environment and poses a threat to nearly all organisms. However, the mechanism by which phytohormones modulate arsenic resistance is not well-understood. Therefore, we analyzed multiple phytohormones based on the results of transcriptome sequencing, content changes, and related mutant growth under arsenic stress. We found that ethylene was the key phytohormone in Arabidopsis thaliana response to arsenic. Further investigation showed the ethylene-overproducing mutant eto1-1 generated less malondialdehyde (MDA), H2O2, and O2•- under arsenic stress compared to wild-type, while the ethylene-insensitive mutant ein2-5 displayed opposite patterns. Compared to wild-type, eto1-1 accumulated a smaller amount of arsenic and a larger amount of non-protein thiols. Additionally, the immediate ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), enhanced resistance to arsenic in wide-type, but not in mutants with impaired detoxification capability (i.e., cad1-3, pad2-1, abcc1abcc2), which confirmed that ethylene regulated arsenic detoxification by enhancing arsenic chelation. ACC also upregulated the expression of gene(s) involved in arsenic detoxification, among which ABCC2 was directly transcriptionally activated by the ethylene master transcription factor ethylene-insensitive 3 (EIN3). Overall, our study shows that ethylene is the key phytohormone to enhance arsenic resistance by reducing arsenic accumulation and promoting arsenic detoxification at both physiological and molecular levels.

The plant hormone ethylene is of vital importance in the regulation of plant development and stress responses. Recent studies revealed that 1-aminocyclopropane-1-carboxylic acid (ACC) plays a role beyond its function as an ethylene precursor. However, the absence of reliable methods to quantify ACC and its conjugates malonyl-ACC (MACC), glutamyl-ACC (GACC), and jasmonyl-ACC (JA-ACC) hinders related research. Combining synthetic and analytical chemistry, we present the first, validated methodology to rapidly extract and quantify ACC and its conjugates using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Its relevance was confirmed by application to Arabidopsis mutants with altered ACC metabolism and wild-type plants under stress. Pharmacological and genetic suppression of ACC synthesis resulted in decreased ACC and MACC content, whereas induction led to elevated levels. Salt, wounding, and submergence stress enhanced ACC and MACC production. GACC and JA-ACC were undetectable in vivo; however, GACC was identified in vitro, underscoring the broad applicability of the method. This method provides an efficient tool to study individual functions of ACC and its conjugates, paving the road toward exploration of novel avenues in ACC and ethylene metabolism, and revisiting ethylene literature in view of the recent discovery of an ethylene-independent role of ACC.

The urea cycle has been found to be closely associated with certain types of cancers and other diseases such as cardiovascular disease and chronic kidney disease. An analytical method for the precise quantification of urea cycle amino acids (arginine, ornithine, citrulline, and argininosuccinate) by off-line two-dimensional liquid chromatography (2D-LC) combined with fluorescence-based detection was developed. Before analysis, the amino acids were derivatised with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to obtain NBD-amino acids. The first dimension involved the reversed-phase separation, in which NBD derivatives of urea cycle amino acids were completely separated from each other and mostly separated from the 18 NBD-proteinogenic amino acids. The samples were eluted with stepwise gradient using 0.02% trifluoroacetic acid in water-acetonitrile as the mobile phase. In the second dimension, an amino column was used for the separation of NBD-ornithine, -citrulline, and -argininosuccinate, while a sulfonic acid column was used to separate NBD-arginine. The developed 2D-LC system was used to analyse human plasma samples. The fractions of NBD-urea cycle amino acids obtained in the first dimension were collected manually and introduced into the second dimension. By choosing appropriate mobile phases for the second dimension, each NBD-urea cycle amino acid eluted in the first dimension was well separated from the other proteinogenic amino acids and interference from endogenous substance. This could not be achieved in the first dimension. The urea cycle amino acids in human plasma sample were quantified, and the method was well validated. The calibration curves for each NBD-urea cycle amino acid showed good linearity from 3 (ASA) or 15 (Orn, Cit, and Arg) to 600 nM, with correlation coefficients higher than 0.9969. The intraday and interday precisions were less than 7.9% and 15%, respectively. The 2D-LC system is expected to be useful for understanding the involvement of the urea cycle in disease progression.

A stoma forms by a series of asymmetric divisions of stomatal lineage precursor cell and the terminal division of a guard mother cell (GMC). GMC division is restricted to once through genetic regulation mechanisms. Here, we show that nitric oxide (NO) is involved in the regulation of the GMC division. NO donor treatment results in the formation of single guard cells (SGCs). SGCs are also produced in plants that accumulate high NO, whereas clustered guard cells (GCs) appear in plants with low NO accumulation. NO treatment promotes the formation of SGCs in the stomatal signalling mutants sdd1, epf1 epf2, tmm1, erl1 erl2 and er erl1 erl2, reduces the cell number per stomatal cluster in the fama-1 and flp1 myb88, but has no effect on stomatal of cdkb1;1 cyca2;234. Aminocyclopropane-1-carboxylic acid (ACC), a positive regulator of GMC division, reduces the NO-induced SGC formation. Further investigation found NO inhibits ACC synthesis by repressing the expression of several ACC SYNTHASE (ACS) genes, and in turn ACC represses NO accumulation by promoting the expression of HEMOGLOBIN 1 (HB1) encoding a NO scavenger. This work shows NO plays a role in the regulation of GMC division by modulating ACC accumulation in the Arabidopsis cotyledon.

The photoionisation and photofragmentation of the two cyclic dipetides cyclo(alanyl-glycine) cGA and cyclo(glycyl-glycine) cGG, have been studied combining experiments and simulations. State selected fragments from the ionized molecules are detected using photo-electron photo-ion coincidence (PEPICO) measurements and specific fragmentation paths are identified and characterized via the use of ion-neutral coincidence maps. The simulations, performed using Quantum Chemistry methods, allow us to infer the fragmentation mechanisms of the ionized and excited molecules. We show that ring opening is followed by emission of the neutral fragments CO and HNCO. In the case of cGG the emission of neutral CO leads to a metastable structure that breaks producing small cationic fragments. The studied cyclic dipeptides evolve under ionizing radiation generating different small aziridin moieties and oxazolidinones. These two species are key reactants to elongate producing peptide chains. The corresponding mechanisms have been computed and show that the reaction requires very low energy and may occur in the presence of ionizing radiation.

In seed plants, 1-aminocyclopropane-1-carboxylic acid (ACC) is the precursor of the plant hormone ethylene but also has ethylene-independent signaling roles. Nonseed plants produce ACC but do not efficiently convert it to ethylene. In Arabidopsis thaliana, ACC is transported by amino acid transporters, LYSINE HISTIDINE TRANSPORTER 1 (LHT1) and LHT2. In nonseed plants, LHT homologs have been uncharacterized. Here, we isolated an ACC-insensitive mutant (Mpain) that is defective in ACC uptake in the liverwort Marchantia polymorpha. Mpain contained a frameshift mutation (1 bp deletion) in the MpLHT1 coding sequence, and was complemented by expression of a wild-type MpLHT1 transgene. Additionally, ACC insensitivity was re-created in CRISPR/Cas9-Mplht1 knockout mutants. We found that MpLHT1 can also transport l-hydroxyproline and l-histidine. We examined the physiological functions of MpLHT1 in vegetative growth and reproduction based on mutant phenotypes. Mpain and Mplht1 plants were smaller and developed fewer gemmae cups compared to wild-type plants. Mplht1 mutants also had reduced fertility, and archegoniophores displayed early senescence. These findings reveal that MpLHT1 serves as an ACC and amino acid transporter in M. polymorpha and has diverse physiological functions. We propose that MpLHT1 contributes to homeostasis of ACC and other amino acids in M. polymorpha growth and reproduction.

BACKGROUND: Verticillium wilt of cotton is a serious disease caused by the infection of soil borne fungus Verticillium dahliae Kleb, and the infection mechanisms may involve the regulation of phytohormone ethylene. The precursor of ethylene biosynthesis is 1-aminocyclopropane-1-carboxylic acid (ACC), whose biosynthesis in vivo depends on activation of ACC synthase (ACS). Here, we investigated how ACS activation and ACC accumulation affected the infection of V. dahliae strain Vd991 on cotton (Gossypium hirsutum L.) cultivar YZ1.
RESULTS: Preliminary observations indicated that ACC applications reduced the disease incidence, disease index and stem vascular browning by impeding fungal biomass accumulation. Transcriptome and qRT-PCR data disclosed that Vd991 induced GhACS2 and GhACS6 expression. GhACS2- or GhACS6-overexpressing transgenic YZ1 lines were generated, respectively. In a Verticillium disease nursery with about 50 microsclerotia per gram of soil, these ACC-accumulated plants showed decreased disease indexes, stem fungal biomasses and vascular browning. More importantly, these transgenic plants decreased the green fluorescent protein-marked Vd991 colonization and diffusion in root tissues. Further, either ACC treatment or ACC-accumulating cotton plants activated salicylic acid (SA)-dependent resistance responses.
CONCLUSIONS: The GhACS2- and GhACS6-dependent ACC accumulations enhanced the resistance of cotton to V. dahliae in a SA-dependent manner, and this lays a foundation for cotton resistance breeding.

The use of halotolerant beneficial plant-growth-promoting (PGP) bacteria is considered as a promising eco-friendly approach to improve the salt tolerance of cash crops. One strategy to enhance the possibility of obtaining stress-alleviating bacteria is to screen salt impacted soils. In this study, amongst the 40 endophytic bacteria isolated from the roots of Sahara-inhabiting halophytes Atriplex halimus L. and Lygeum spartum L., 8 showed interesting NaCl tolerance in vitro. Their evaluation, through different tomato plant trials, permitted the isolate IS26 to be distinguished as the most effective seed inoculum for both plant growth promotion and mitigation of salt stress. On the basis of 16S rRNA gene sequence, the isolate was closely related to Stenotrophomonas rhizophila. It was then screened in vitro for multiple PGP traits and the strain-complete genome was sequenced and analysed to further decipher the genomic basis of the putative mechanisms underlying its osmoprotective and plant growth abilities. A remarkable number of genes putatively involved in mechanisms responsible for rhizosphere colonization, plant association, strong competition for nutrients, and the production of important plant growth regulator compounds, such as AIA and spermidine, were highlighted, as were substances protecting against stress, including different osmolytes like trehalose, glucosylglycerol, proline, and glycine betaine. By having genes related to complementary mechanisms of osmosensing, osmoregulation and osmoprotection, the strain confirmed its great capacity to adapt to highly saline environments. Moreover, the presence of various genes potentially related to multiple enzymatic antioxidant processes, able to reduce salt-induced overproduction of ROS, was also detected.

In seed plants, 1-amino-cyclopropane-1-carboxylic acid (ACC) is the well-known precursor of the plant hormone ethylene. In nonseed plants, the current view is that ACC is produced but is inefficiently converted to ethylene. Distinct responses to ACC that are uncoupled from ethylene biosynthesis have been discovered in diverse aspects of growth and development in liverworts and angiosperms, indicating that ACC itself can function as a signal. Evolutionarily, ACC may have served as a signal before acquiring its role as the ethylene precursor in seed plants. These findings pave the way for unraveling a potentially conserved ACC signaling pathway in plants and have ramifications for the use of ACC as a substitute for ethylene treatment in seed plants.

The plant hormone ethylene plays vital roles in plant development, including pollen tube (PT) growth. Many studies have used the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), as a tool to trigger ethylene signaling. Several studies have suggested that ACC can act as a signal molecule independently of ethylene, inducing responses that are distinct from those induced by ethylene. In this study, we confirmed that ethylene receptor function is essential for promoting PT growth in tomato, but interestingly, we discovered that ACC itself can act as a signal that also promotes PT growth. Exogenous ACC stimulated PT growth even when ethylene perception was inhibited either chemically by treating with 1-methylcyclopropene (1-MCP) or genetically by using the ethylene-insensitive Never Ripe (NR) mutant. Treatment with aminoethoxyvinylglycine, which reduces endogenous ACC levels, led to a reduction of PT growth, even in the NR mutants. Furthermore, GUS activity driven by an EIN3 Binding Site promoter (EBS:GUS transgene) was triggered by ACC in the presence of 1-MCP. Taken together, these results suggest that ACC signaling can bypass the ethylene receptor step to stimulate PT growth and EBS driven gene expression.