An auxin signaling network translates low-sugar-state input into compensated cell enlargement in the fugu5 cotyledon.

In plants, the effective mobilization of seed nutrient reserves is crucial during germination and for seedling establishment. The Arabidopsis H+-PPase-loss-of-function fugu5 mutants exhibit a reduced number of cells in the cotyledons. This leads to enhanced post-mitotic cell expansion, also known as compensated cell enlargement (CCE). While decreased cell numbers have been ascribed to reduced gluconeogenesis from triacylglycerol, the molecular mechanisms underlying CCE remain ill-known. Given the role of indole 3-butyric acid (IBA) in cotyledon development, and because CCE in fugu5 is specifically and completely cancelled by ech2, which shows defective IBA-to-indoleacetic acid (IAA) conversion, IBA has emerged as a potential regulator of CCE. Here, to further illuminate the regulatory role of IBA in CCE, we used a series of high-order mutants that harbored a specific defect in IBA-to-IAA conversion, IBA efflux, IAA signaling, or vacuolar type H+-ATPase (V-ATPase) activity and analyzed the genetic interaction with fugu5-1. We found that while CCE in fugu5 was promoted by IBA, defects in IBA-to-IAA conversion, IAA response, or the V-ATPase activity alone cancelled CCE. Consistently, endogenous IAA in fugu5 reached a level 2.2-fold higher than the WT in 1-week-old seedlings. Finally, the above findings were validated in icl-2, mls-2, pck1-2 and ibr10 mutants, in which CCE was triggered by low sugar contents. This provides a scenario in which following seed germination, the low-sugar-state triggers IAA synthesis, leading to CCE through the activation of the V-ATPase. These findings illustrate how fine-tuning cell and organ size regulation depend on interplays between metabolism and IAA levels in plants.

AtMYB32 regulates the ABA response by targeting ABI3, ABI4 and ABI5 and the drought response by targeting CBF4 in Arabidopsis.

The Arabidopsis thaliana R2R3-MYB transcription factor AtMYB32 and its homologs AtMYB4 and AtMYB7 play crucial roles in the regulation of phenylpropanoid metabolism. In addition, AtMYB4 and AtMYB7 are involved in the response to abiotic stress. However, the function of AtMYB32 remains unclear. In this study, we found that AtMYB32 is induced by abscisic acid (ABA) and repressed by drought stress. AtMYB32 positively regulates ABA-mediated seed germination and early seedling development. The expression of ABSCISIC ACID-INSENSITIVE 3 (ABI3), ABI4 and ABI5, which encode key positive regulators of ABA signaling, was upregulated in response to ABA in AtMYB32-overexpressing plants and downregulated in the atmyb32-1 mutant. In addition, we found that the atmyb32-1 mutant was drought resistant. Consistent with the drought-resistant phenotype, the transcript levels of C-repeat binding factor 4 (CBF4) were higher in the atmyb32-1 mutant in response to drought stress. Electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP) assays revealed that AtMYB32 binds directly to the ABI3, ABI4, ABI5 and CBF4 promoters both in vitro and in vivo. Genetically, ABI4 was found to be epistatic to AtMYB32 for ABA-induced inhibition of seed germination and early seedling development. Taken together, our findings revealed that AtMYB32 regulates the ABA response by directly promoting ABI3, ABI4 and ABI5 expression and that the drought stress response likely occurs because of repression of CBF4 expression.

Synthesis of Profluorescent Strigolactone Probes for Biochemical Studies.

In this chapter, we will describe a method we set up to synthesize two profluorescent strigolactone (SL) mimic probes (GC240 and GC242) and the optimized protocols developed to study the enzymatic properties of various strigolactone receptors. The Arabidopsis AtD14 SL receptor is used here as a model for this purpose.

Coordinated cytokinin signaling and auxin biosynthesis mediates arsenate-induced root growth inhibition.

Interactions between plant hormones and environmental signals are important for the maintenance of root growth plasticity under ever-changing environmental conditions. Here, we demonstrate that arsenate (AsV), the most prevalent form of arsenic (As) in nature, restrains elongation of the primary root through transcriptional regulation of local auxin biosynthesis genes in the root tips of Arabidopsis (Arabidopsis thaliana) plants. The ANTHRANILATE SYNTHASE ALPHA SUBUNIT 1 (ASA1) and BETA SUBUNIT 1 (ASB1) genes encode enzymes that catalyze the conversion of chorismate to anthranilate (ANT) via the tryptophan-dependent auxin biosynthesis pathway. Our results showed that AsV upregulates ASA1 and ASB1 expression in root tips, and ASA1- and ASB1-mediated auxin biosynthesis is involved in AsV-induced root growth inhibition. Further investigation confirmed that AsV activates cytokinin signaling by stabilizing the type-B ARABIDOPSIS RESPONSE REGULATOR1 (ARR1) protein, which directly promotes the transcription of ASA1 and ASB1 genes by binding to their promoters. Genetic analysis revealed that ASA1 and ASB1 are epistatic to ARR1 in the AsV-induced inhibition of primary root elongation. Overall, the results of this study illustrate a molecular framework that explains AsV-induced root growth inhibition via crosstalk between two major plant growth regulators, auxin and cytokinin.

At3g53630 encodes a GUN1-interacting protein under norflurazon treatment.

Chloroplasts are semi-autonomous organelles, with more than 95% of their proteins encoded by the nuclear genome. The chloroplast-to-nucleus retrograde signals are critical for the nucleus to coordinate its gene expression for optimizing or repairing chloroplast functions in response to changing environments. In chloroplasts, the pentatricopeptide-repeat protein GENOMES UNCOUPLED 1 (GUN1) is a master switch that senses aberrant physiological states, such as the photooxidative stress induced by norflurazon (NF) treatment, and represses the expression of photosynthesis-associated nuclear genes (PhANGs). However, it is largely unknown how the retrograde signal is transmitted beyond GUN1. In this study, a protein GUN1-INTERACTING PROTEIN 1 (GIP1), encoded by At3g53630, was identified to interact with GUN1 by different approaches. We demonstrated that GIP1 has both cytosol and chloroplast localizations, and its abundance in chloroplasts is enhanced by NF treatment with the presence of GUN1. Our results suggest that GIP1 and GUN1 may function antagonistically in the retrograde signaling pathway.

Counteraction of ABA-Mediated Inhibition of Seed Germination and Seedling Establishment by ABA Signaling Terminator in Arabidopsis.

Seed germination and seedling establishment are important for the reproductive success of plants, but seeds and seedlings typically encounter constantly changing environmental conditions. By inhibiting seed germination and post-germinative growth through the PYR1/PYL/RCAR ABA receptors and PP2C co-receptors, the phytohormone abscisic acid (ABA) prevents premature germination and seedling growth under unfavorable conditions. However, little is known about how the ABA-mediated inhibition of seed germination and seedling establishment is thwarted. Here, we report that ABA Signaling Terminator (ABT), a WD40 protein, efficiently switches off ABA signaling and is critical for seed germination and seedling establishment. ABT is induced by ABA in a PYR1/PYL/RCAR-PP2C-dependent manner. Overexpression of ABT promotes seed germination and seedling greening in the presence of ABA, whereas knockout of ABT has the opposite effect. We found that ABT interacts with the PYR1/PYL/RCAR and PP2C proteins, interferes with the interaction between PYR1/PYL4 and ABI1/ABI2, and hampers the inhibition of ABI1/ABI2 by ABA-bound PYR1/PYL4, thereby terminating ABA signaling. Taken together, our results reveal a core mechanism of ABA signaling termination that is critical for seed germination and seedling establishment in Arabidopsis.

Quantitative Proteomic Analyses Identify STO/BBX24 -Related Proteins Induced by UV-B.

Plants use solar radiation for photosynthesis and are inevitably exposed to UV-B. To adapt to UV-B radiation, plants have evolved a sophisticated strategy, but the mechanism is not well understood. We have previously reported that STO (salt tolerance)/BBX24 is a negative regulator of UV-B-induced photomorphogenesis. However, there is limited knowledge of the regulatory network of STO in UV-B signaling. Here, we report the identification of proteins differentially expressed in the wild type (WT) and sto mutant after UV-B radiation by iTRAQ (isobaric tags for relative and absolute quantitation)-based proteomic analysis to explore differential proteins that depend on STO and UV-B signaling. A total of 8212 proteins were successfully identified, 221 of them were STO-dependent proteins in UV-B irradiated plants. The abundances of STO-dependent PSB and LHC (light-harvesting complex) proteins in sto mutants decreased under UV-B radiation, suggesting that STO is necessary to maintain the normal accumulation of photosynthetic system complex under UV-B radiation to facilitate photosynthesis photon capture. The abundance of phenylalanine lyase-1 (PAL1), chalcone synthetase (CHS), and flavonoid synthetase (FLS) increased significantly after UV-B irradiation, suggesting that the accumulation of flavonoids do not require STO, but UV-B is needed. Under UV-B radiation, STO stabilizes the structure of antenna protein complex by maintaining the accumulation of PSBs and LHCs, thereby enhancing the non-photochemical quenching (NPQ) ability, releasing extra energy, protecting photosynthesis, and ultimately promoting the elongation of hypocotyl. The accumulation of flavonoid synthesis key proteins is independent of STO under UV-B radiation. Overall, our results provide a comprehensive regulatory network of STO in UV-B signaling.

Salinity Stress-Mediated Suppression of Expression of Salt Overly Sensitive Signaling Pathway Genes Suggests Negative Regulation by AtbZIP62 Transcription Factor in Arabidopsis thaliana.

Salt stress is one of the most serious threats in plants, reducing crop yield and production. The salt overly sensitive (SOS) pathway in plants is a salt-responsive pathway that acts as a janitor of the cell to sweep out Na+ ions. Transcription factors (TFs) are key regulators of expression and/or repression of genes. The basic leucine zipper (bZIP) TF is a large family of TFs regulating various cellular processes in plants. In the current study, we investigated the role of the Arabidopsis thaliana bZIP62 TF in the regulation of SOS signaling pathway by measuring the transcript accumulation of its key genes such as SOS1, 2, and 3, in both wild-type (WT) and atbzip62 knock-out (KO) mutants under salinity stress. We further observed the activation of enzymatic and non-enzymatic antioxidant systems in the wild-type, atbzip62, atcat2 (lacking catalase activity), and atnced3 (lacking 9-cis-epoxycarotenoid dioxygenase involved in the ABA pathway) KO mutants. Our findings revealed that atbzip62 plants exhibited an enhanced salt-sensitive phenotypic response similar to atnced3 and atcat2 compared to WT, 10 days after 150 mM NaCl treatment. Interestingly, the transcriptional levels of SOS1, SOS2, and SOS3 increased significantly over time in the atbzip62 upon NaCl application, while they were downregulated in the wild type. We also measured chlorophyll a and b, pheophytin a and b, total pheophytin, and total carotenoids. We observed that the atbzip62 exhibited an increase in chlorophyll and total carotenoid contents, as well as proline contents, while it exhibited a non-significant increase in catalase activity. Our results suggest that AtbZIP62 negatively regulates the transcriptional events of SOS pathway genes, AtbZIP18 and AtbZIP69 while modulating the antioxidant response to salt tolerance in Arabidopsis.

TARGET OF RAPAMYCIN signaling plays a role in Arabidopsis growth promotion by Azospirillum brasilense Sp245.

Azospirillum brasilense colonizes plant roots and improves productivity, but the molecular mechanisms behind its phytostimulation properties remain mostly unknown. Here, we uncover an important role of TARGET OF RAPAMYCIN (TOR) signaling on the response of Arabidopsis thaliana to A. brasilense Sp245. The effect of the bacterium on TOR expression was analyzed in the transgenic line TOR/tor-1, which carries a translational fusion with the GUS reporter protein, and the activity of TOR was assayed thought the phosphorylation of its downstream signaling target S6K protein. Besides, the role of TOR on plant growth in inoculated plants was assessed using the ATP-competitive inhibitor AZD-8055. A decrease in growth of the primary root correlates with an improved branching and absorptive capacity via lateral root and root hair proliferation 6 days after transplant to different concentrations of the bacterium (103 or 105 CFU/mL). Bacterization increased the expression of TOR in shoot and root apexes and promoted phosphorylation of S6K 3 days after transplant. The TOR inhibitor AZD-8055 (1 µM) inhibited plant growth and cell division in root meristems and in lateral root primordia, interfering with the phytostimulation by A. brasilense. In addition, the role of auxin produced by the bacterium to stimulate TOR expression was explored. Noteworthy, the A. brasilense mutant FAJ009, impaired in auxin production, was unable to elicit TOR signaling to the level observed for the wild-type strain, showing the importance of this phyhormone to stimulate TOR signaling. Together, our findings establish an important role of TOR signaling for the probiotic traits elicited by A. brasilense in A. thaliana.

N-3-oxo-octanoyl-homoserine lactone-mediated priming of resistance to Pseudomonas syringae requires the salicylic acid signaling pathway in Arabidopsis thaliana.

BACKGROUD: Many Gram-negative bacteria use N-acyl-homoserine lactones (AHLs) to communicate each other and to coordinate their collective behaviors. Recently, accumulating evidence shows that host plants are able to sense and respond to bacterial AHLs. Once primed, plants are in an altered state that enables plant cells to more quickly and/or strongly respond to subsequent pathogen infection or abiotic stress. RESULTS: In this study, we report that pretreatment with N-3-oxo-octanoyl-homoserine lactone (3OC8-HSL) confers resistance against the pathogenic bacterium Pseudomonas syringae pv. tomato DC3000 (PstDC3000) in Arabidopsis. Pretreatment with 3OC8-HSL and subsequent pathogen invasion triggered an augmented burst of hydrogen peroxide, salicylic acid accumulation, and fortified expression of the pathogenesis-related genes PR1 and PR5. Upon PstDC3000 challenge, plants treated with 3OC8-HSL showed increased activities of defense-related enzymes including peroxidase, catalase, phenylalanine ammonialyase, and superoxide dismutase. In addition, the 3OC8-HSL-primed resistance to PstDC3000 in wild-type plants was impaired in plants expressing the bacterial NahG gene and in the npr1 mutant. Moreover, the expression levels of isochorismate synthases (ICS1), a critical salicylic acid biosynthesis enzyme, and two regulators of its expression, SARD1 and CBP60g, were potentiated by 3OC8-HSL pretreatment followed by pathogen inoculation. CONCLUSIONS: Our data indicate that 3OC8-HSL primes the Arabidopsis defense response upon hemibiotrophic bacterial infection and that 3OC8-HSL-primed resistance is dependent on the SA signaling pathway. These findings may help establish a novel strategy for the control of plant disease.

iTRAQ-Based Quantitative Proteomic Analysis of the Arabidopsis Mutant opr3-1 in Response to Exogenous MeJA.

Jasmonates (JAs) regulate the defense of biotic and abiotic stresses, growth, development, and many other important biological processes in plants. The comprehensive proteomic profiling of plants under JAs treatment provides insights into the regulation mechanism of JAs. Isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomic analysis was performed on the Arabidopsis wild type (Ws) and JA synthesis deficiency mutant opr3-1. The effects of exogenous MeJA treatment on the proteome of opr3-1, which lacks endogenous JAs, were investigated. A total of 3683 proteins were identified and 126 proteins were differentially regulated between different genotypes and treatment groups. The functional classification of these differentially regulated proteins showed that they were involved in metabolic processes, responses to abiotic stress or biotic stress, the defense against pathogens and wounds, photosynthesis, protein synthesis, and developmental processes. Exogenous MeJA treatment induced the up-regulation of a large number of defense-related proteins and photosynthesis-related proteins, it also induced the down-regulation of many ribosomal proteins in opr3-1. These results were further verified by a quantitative real-time PCR (qRT-PCR) analysis of 15 selected genes. Our research provides the basis for further understanding the molecular mechanism of JAs' regulation of plant defense, photosynthesis, protein synthesis, and development.

PIN FORMED 2 Modulates the Transport of Arsenite in Arabidopsis thaliana.

Arsenic contamination is a major environmental issue, as it may lead to serious health hazard. The reduced trivalent form of inorganic arsenic, arsenite, is in general more toxic to plants compared with the fully oxidized pentavalent arsenate. The uptake of arsenite in plants has been shown to be mediated through a large subfamily of plant aquaglyceroporins, nodulin 26-like intrinsic proteins (NIPs). However, the efflux mechanisms, as well as the mechanism of arsenite-induced root growth inhibition, remain poorly understood. Using molecular physiology, synchrotron imaging, and root transport assay approaches, we show that the cellular transport of trivalent arsenicals in Arabidopsis thaliana is strongly modulated by PIN FORMED 2 (PIN2) auxin efflux transporter. Root transport assay using radioactive arsenite, X-ray fluorescence imaging (XFI) coupled with X-ray absorption spectroscopy (XAS), and inductively coupled plasma mass spectrometry analysis revealed that pin2 plants accumulate higher concentrations of arsenite in roots compared with the wild-type. At the cellular level, arsenite specifically targets intracellular sorting of PIN2 and thereby alters the cellular auxin homeostasis. Consistently, loss of PIN2 function results in arsenite hypersensitivity in roots. XFI coupled with XAS further revealed that loss of PIN2 function results in specific accumulation of arsenical species, but not the other metals such as iron, zinc, or calcium in the root tip. Collectively, these results suggest that PIN2 likely functions as an arsenite efflux transporter for the distribution of arsenical species in planta.

BES1 Functions as the Co-regulator of D53-like SMXLs to Inhibit BRC1 Expression in Strigolactone-Regulated Shoot Branching in Arabidopsis.

Shoot branching, determining plant architecture and crop yield, is critically controlled by strigolactones (SLs). However, how SLs inhibit shoot branching after its perception by the receptor complex remains largely obscure. In this study, using the transcriptomic and genetic analyss as well as biochemical studies, we reveal the key role of BES1 in the SL-regulated shoot branching. We demonstrate that BES1 and D53-like SMXLs, the substrates of SL receptor complex D14-MAX2, interact with each other to inhibit BRC1 expression, which specifically triggers the SL-regulated transcriptional network in shoot branching. BES1 directly binds the BRC1 promoter and recruits SMXLs to inhibit BRC1 expression. Interestingly, despite being the shared component by SL and brassinosteroid (BR) signaling, BES1 gains signal specificity through different mechanisms in response to BR and SL signals.

The nitric oxide suppressed Arabidopsis mutants- Atnoa1 and Atnia1nia2noa1-2 produce nitric oxide in MS growth medium and on uranium exposure.

The mutants Atnoa1 and Atnia1nia2noa1-2 having a defective chloroplast developmental process, showed enhanced chlorophyll levels when they were grown on Murashige and Skoog (MS) medium and on exposure with uranium (U) on Hoagland medium. Thus we hypothesized that these mutants probably produced NO in MS medium and on exposure with U. Wild-type Col-0, Atnoa1, Atnia1nia2noa1-2 plants were cultured on modified Hoagland and 1/10 MS media and NO generation in the roots of these mutants was monitored using NO selective fluorescent dyes, DAF-2DA and Fl2E. Both Atnoa1 and Atnia1nia2noa1-2 triple mutants produced NO as observed by increases in DAF-2T and Fl2E fluorescence when these mutants were grown on MS medium but not on Hoagland medium. In presence of NO scavenger, methylene blue (MB, 200 µM), DAF-2T and Fl2E fluorescence was completely abolished. On the other hand treatment of the plants with 25 µM U triggered NO generation. U-treated Atnoa1 and Atnia1nia2noa1-2 plants upregulated genes (POR B, POR D, CHL D) involved in the chlorophyll biosynthesis. From these results it was concluded that Atnoa1 and Atnia1nia2noa1-2 are conditional NO producers and it appears that NO generation in plants substantially depends on growth medium and NIA1, NIA2 or NOA1 does not appear to be really involved in NO generation in MS medium or after U exposure.

The non-DNA binding bHLH transcription factor Paclobutrazol Resistances are involved in the regulation of ABA and salt responses in Arabidopsis.

Abscisic acid (ABA) is the key hormone that regulating plant responses to abiotic stresses. Several basic helix-loop-helix (bHLH) transcription factors have been reported to regulate ABA signaling in Arabidopsis. Paclobutrazol Resistances (PREs) are non-DNA binding bHLH transcription factors involved in the regulation of plant response to several different plant hormones including gibberellin, brassinosteroid and auxin. Here, we show that PREs are involved in the regulation of ABA and salt responses in Arabidopsis. Quantitative RT-PCR results showed that the expression levels of PRE6 as well as several other PRE genes were reduced in response to ABA treatment, but increased to salt treatment. Seed germination assays indicated that ABA sensitivity is reduced in the pre6 mutants, but increased in transgenic plants overexpressing PRE6. On the other hand, the 35S:PRE6 transgenic plants showed enhanced tolerance to salt, whereas little, if any changes were observed in the pre6 mutants. Similar responses to ABA and salt treatments were observed in the pre2 mutants and the transgenic plants overexpressing PRE2, and a slight increased resistance to ABA in seed germination was observed in the pre2 pre6 double mutants. Taken together, our results suggest that at least some of the PRE genes are ABA responsive genes, and PREs may function redundantly to regulate ABA and salt responses in Arabidopsis.

Unravelling calcium-alleviated aluminium toxicity in Arabidopsis thaliana: Insights into regulatory mechanisms using proteomics.

Aluminium (Al) toxicity is a major limiting factor for plant productivity in acidic soils. Calcium (Ca) is an essential element and participates in various physiological responses to environmental stress. Here, the aim of this work was to study the role of exogenous Ca in alleviating Al toxicity in Arabidopsis thaliana. For that we used the methods of physiology and proteomics. Results showed that Ca alleviated Al-induced growth inhibition and decreased Al accumulation. Proteomic analyses showed that 75 differentially expressed protein spots, including those related to organic acid metabolism, cell wall components, cellular transport, signal transduction and antioxidant activity, transcription and protein metabolism were identified during the response of Arabidopsis to Ca alleviated Al toxicity. Ca regulated tricarboxylic acid (TCA) cycle-related protein abundances and affected organic acid concentrations and related enzyme activities under Al stress. Vacuolar and mitochondrion adenosine triphosphate (ATP) synthase, and cell wall component-related proteins played important roles in Ca-alleviated Al toxicity. Ethylene-insensitive 3 (EIN3) participated in Ca-alleviated Al toxicity. Glutathione S-transferase (GST6) and glutathione S-transferase tau 19 (ATGSTU19) were associated with antioxidant activities induced by Ca under Al stress. Our results may contribute to an understanding of the functional mechanism by which Ca alleviates Al stress in plants. SIGNIFICANT: Our results elucidated how Ca alleviate the effects of Al toxicity on the inhibition of plant growth and Al accumulation in plants using the proteomics and physiological methods, which may contribute to a better understanding of the molecular mechanism of Ca alleviation Al stress in plants.

A B-box zinc finger protein, MdBBX10, enhanced salt and drought stresses tolerance in Arabidopsis.

KEY MESSAGE: The expression of MdBBX10 was significantly induced by different stresses and ABA treatments. Overexpression of MdBBX10 in Arabidopsis significantly enhanced abiotic stresses tolerance by ABA signalling. The roles of B-box domain(s) containing proteins (BBXs) in regulation of flowering and light morphogenesis of plants were intensively studied. However, the roles of plant BBXs in abiotic stresses are poorly understood. A B-box protein encoding gene from apple (MdBBX10) was found to be up-regulated from gene expression profile under salt stress. qRT-PCR analysis indicated that the expression of MdBBX10 was significantly induced by different stresses and exogenous abscisic acid (ABA) in apple roots and leaves. The ß-glucuronidase activity driven by the promoter of MdBBX10 was also strongly induced by NaCl, H2O2, polyethylene glycol and exogenous ABA, which was consistent to the existence of rich cis-acting elements related to the abiotic stresses in the promoter sequence. Over-expression of MdBBX10 in Arabidopsis significantly enhanced tolerance to abiotic stresses, with higher germination ratio and longer length of roots than the wild type plants. Transgenic plants of over-expressing MdBBX10 lines were more sensitive to exogenous ABA than the wild type plants. Under abiotic stress treatments, the transcript levels of ABA- and stress-related genes were higher in MdBBX10-overexpressing plants than wild type plants. Over-expression of MdBBX10 could enhance plant's ability to scavenge reactive oxygen species (ROS) under stresses, which is correlated with the expression of ROS-scavenging genes. These results provided the evidences that MdBBX10 plays an important role in enhanced plant tolerance to abiotic stresses, which were involved in ABA-mediated response and ROS response.

Metabolism of the anthelmintic drug fenbendazole in Arabidopsis thaliana and its effect on transcriptome and proteome.

Fenbendazole, a broad spectrum anthelmintic used especially in veterinary medicine, may impact non-target organisms in the environment. Nevertheless, information about the effects of fenbendazole in plants is limited. We investigated the biotransformation of fenbendazole and the effect of fenbendazole and its metabolites on gene expression in the model plant Arabidopsis thaliana. High-sensitive UHPLC coupled with tandem mass spectrometry, RNA-microarray analysis together with qPCR verification and nanoLC-MS proteome analysis were used in this study. Twelve fenbendazole metabolites were identified in the roots and leaves of A. thaliana plants. Hydroxylation, S-oxidation and glycosylation represent the main fenbendazole biotransformation pathways. Exposure of A. thaliana plants to 5 µM fenbendazole for 24 and 72 h significantly affected gene and protein expression. The changes in transcriptome were more pronounced in the leaves than in roots, protein expression was more greatly affected in the roots at a shorter period of exposure (24 h) and in leaf rosettes over a longer period (72 h). Up-regulated (>2-fold change, p < 0.1) proteins are involved in various biological processes (electron transport, energy generating pathways, signal transduction, transport), and in response to stresses (e.g. catalase, superoxide dismutase, cytochromes P450, UDP-glycosyltransferases). Some of the proteins which were up-regulated after fenbendazole-exposure probably participate in fenbendazole biotransformation (e.g. cytochromes P450, UDP-glucosyltransferases). Finally, fenbendazole in plants significantly affects many physiological and metabolic processes and thus the contamination of ecosystems by manure containing this anthelmintic should be restricted.

Arabidopsis γ-glutamylcyclotransferase affects glutathione content and root system architecture during sulfur starvation.

γ-Glutamylcyclotransferase initiates glutathione degradation to component amino acids l-glutamate, l-cysteine and l-glycine. The enzyme is encoded by three genes in Arabidopsis thaliana, one of which (GGCT2;1) is transcriptionally upregulated by starvation for the essential macronutrient sulfur (S). Regulation by S-starvation suggests that GGCT2;1 mobilizes l-cysteine from glutathione when there is insufficient sulfate for de novo l-cysteine synthesis. The response of wild-type seedlings to S-starvation was compared to ggct2;1 null mutants. S-starvation causes glutathione depletion in S-starved wild-type seedlings, but higher glutathione is maintained in the primary root tip than in other seedling tissues. Although GGCT2;1 is induced throughout seedlings, its expression is concentrated in the primary root tip where it activates the γ-glutamyl cycle. S-starved wild-type plants also produce longer primary roots, and lateral root growth is suppressed. While glutathione is also rapidly depleted in ggct2;1 null seedlings, much higher glutathione is maintained in the primary root tip compared to the wild-type. S-starved ggct2;1 primary roots grow longer than the wild-type, and lateral root growth is not suppressed. These results point to a role for GGCT2;1 in S-starvation-response changes to root system architecture through activity of the γ-glutamyl cycle in the primary root tip. l-Cysteine mobilization from glutathione is not solely a function of GGCT2;1.

Full-length transcriptome sequences of ephemeral plant Arabidopsis pumila provides insight into gene expression dynamics during continuous salt stress.

BACKGROUND: Arabidopsis pumila is native to the desert region of northwest China and it is extraordinarily well adapted to the local semi-desert saline soil, thus providing a candidate plant system for environmental adaptation and salt-tolerance gene mining. However, understanding of the salt-adaptation mechanism of this species is limited because of genomic sequences scarcity. In the present study, the transcriptome profiles of A. pumila leaf tissues treated with 250 mM NaCl for 0, 0.5, 3, 6, 12, 24 and 48 h were analyzed using a combination of second-generation sequencing (SGS) and third-generation single-molecule real-time (SMRT) sequencing. RESULTS: Correction of SMRT long reads by SGS short reads resulted in 59,328 transcripts. We found 8075 differentially expressed genes (DEGs) between salt-stressed tissues and controls, of which 483 were transcription factors and 1157 were transport proteins. Most DEGs were activated within 6 h of salt stress and their expression stabilized after 48 h; the number of DEGs was greatest within 12 h of salt stress. Gene annotation and functional analyses revealed that expression of genes associated with the osmotic and ionic phases rapidly and coordinately changed during the continuous salt stress in this species, and salt stress-related categories were highly enriched among these DEGs, including oxidation-reduction, transmembrane transport, transcription factor activity and ion channel activity. Orphan, MYB, HB, bHLH, C3H, PHD, bZIP, ARF and NAC TFs were most enriched in DEGs; ABCB1, CLC-A, CPK30, KEA2, KUP9, NHX1, SOS1, VHA-A and VP1 TPs were extensively up-regulated in salt-stressed samples, suggesting that they play important roles in slat tolerance. Importantly, further experimental studies identified a mitogen-activated protein kinase (MAPK) gene MAPKKK18 as continuously up-regulated throughout salt stress, suggesting its crucial role in salt tolerance. The expression patterns of the salt-responsive 24 genes resulted from quantitative real-time PCR were basically consistent with their transcript abundance changes identified by RNA-Seq. CONCLUSION: The full-length transcripts generated in this study provide a more accurate depiction of gene transcription of A. pumila. We identified potential genes involved in salt tolerance of A. pumila. These data present a genetic resource and facilitate better understanding of salt-adaptation mechanism for ephemeral plants.