Arabidopsis WRKY71 regulates ethylene-mediated leaf senescence by directly activating EIN2, ORE1 and ACS2 genes.

Leaf senescence is a pivotal step in the last stage of the plant life cycle and is influenced by various external and endogenous cues. A series of reports have indicated the involvement of the WRKY transcription factors in regulating leaf senescence, but the molecular mechanisms and signaling pathways remain largely unclear. Here we provide evidence demonstrating that WRKY71 acts as a positive regulator of leaf senescence in Arabidopsis. WRKY71-1D, an overexpressor of WRKY71, exhibited early leaf senescence, while wrky71-1, the WRKY71 loss-of-function mutant, displayed delayed leaf senescence. Accordingly, a set of senescence-associated genes (SAGs) were substantially elevated in WRKY71-1D but markedly decreased in wrky71-1. Chromatin immunoprecipitation assays indicated that WRKY71 can bind directly to the promoters of SAG13 and SAG201. Transcriptome analysis suggested that WRKY71 might mediate multiple cues to accelerate leaf senescence, such as abiotic stresses, dark and ethylene. WRKY71 was ethylene inducible, and treatment with the ethylene precursor 1-amino-cyclopropane-1-carboxylic acid enhanced leaf senescence in WRKY71-1D but caused only a marginal delay in leaf senescence in wrky71-1. In vitro and in vivo assays demonstrated that WRKY71 can directly regulate ETHYLENE INSENSITIVE2 (EIN2) and ORESARA1 (ORE1), genes of the ethylene signaling pathway. Consistently, leaf senescence of WRKY71-1D was obviously retarded in the ein2-5 and nac2-1 mutants. Moreover, WRKY71 was also proved to interact with ACS2 in vitro and in vivo. Treatment with AgNO3 and aminoethoxyvinylglycine and acs2-1 could greatly arrest the leaf senescence of WRKY71-1D. In conclusion, our data revealed that WRKY71 mediates ethylene signaling and synthesis to hasten leaf senescence in Arabidopsis.

Distinct Functions of Ethylene and ACC in the Basal Land Plant Marchantia polymorpha.

Ethylene is a gaseous phytohormone involved in various physiological processes, including fruit ripening, senescence, root hair development and stress responses. Recent genomics studies have suggested that most homologous genes of ethylene biosynthesis and signaling are conserved from algae to angiosperms, whereas the function and biosynthesis of ethylene remain unknown in basal plants. Here, we examined the physiological effects of ethylene, an ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC) and an inhibitor of ethylene perception, silver thiosulfate (STS), in a basal land plant, Marchantia polymorpha. M. polymorpha plants biosynthesized ethylene, and treatment with high concentrations of ACC slightly promoted ethylene production. ACC remarkably suppressed the growth of thalli (vegetative organs) and rhizoids (root-hair-like cells), whereas exogenous ethylene slightly promoted thallus growth. STS suppressed thallus growth and induced ectopic rhizoid formation on the dorsal surface of thalli. Thus, ACC and ethylene have different effects on the vegetative growth of M. polymorpha. We generated single and double mutants of ACC synthase-like (ACSL) genes, MpACSL1 and MpACSL2. The mutants did not show obvious defects in thallus growth, ACC content and ethylene production, indicating that MpACSL genes are not essential for the vegetative growth and biosynthesis of ACC and ethylene. Gene expression analysis suggested the involvement of MpACSL1 and MpACSL2 in stress responses. Collectively, our results imply ethylene-independent function of ACC and the absence of ACC-mediated ethylene biosynthesis in M. polymorpha.

1-Aminocyclopropane-1-carboxylic acid stimulates tomato pollen tube growth independently of ethylene receptors.

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.

Zinc finger protein 5 (ZFP5) associates with ethylene signaling to regulate the phosphate and potassium deficiency-induced root hair development in Arabidopsis.

KEY MESSAGE: Zinc finger protein transcription factor ZFP5 positively regulates root hair elongation in response to Pi and potassium deficiency by mainly activating the expression of EIN2 in Arabidopsis. Phosphate (Pi) and potassium (K+) are major plant nutrients required for plant growth and development, and plants respond to low-nutrient conditions via metabolic and morphology changes. The C2H2 transcription factor ZFP5 is a key regulator of trichome and root hair development in Arabidopsis. However, its role in regulating root hair development under nutrient deprivations remains unknown. Here, we show that Pi and potassium deficiency could not restore the short root hair phenotype of zfp5 mutant and ZFP5 RNAi lines to wild type level. The deprivation of either of these nutrients also induced the expression of ZFP5 and the activity of an ethylene reporter, pEBS:GUS. The significant reduction of root hair length in ein2-1 and ein3-1 as compared to wild-type under Pi and potassium deficiency supports the involvement of ethylene in root hair elongation. Furthermore, the application of 1-aminocyclopropane-1-carboxylic acid (ACC) significantly enhanced the expression level of ZFP5 while the application of 2-aminoethoxyvinyl glycine (AVG) had the opposite effect when either Pi or potassium was deprived. Further experiments reveal that ZFP5 mainly regulates transcription of ETHYLENE INSENSITIVE 2 (EIN2) to control deficiency-mediated root hair development through ethylene signaling. Generally, these results suggest that ZFP5 regulates root hair elongation by interacting with ethylene signaling mainly through regulates the expression of EIN2 in response to Pi and potassium deficiency in Arabidopsis.

Identification of Transcriptional and Receptor Networks That Control Root Responses to Ethylene.

Transcriptomic analyses with high temporal resolution provide substantial new insight into hormonal response networks. This study identified the kinetics of genome-wide transcript abundance changes in response to elevated levels of the plant hormone ethylene in roots from light-grown Arabidopsis (Arabidopsis thaliana) seedlings, which were overlaid on time-matched developmental changes. Functional annotation of clusters of transcripts with similar temporal patterns revealed rapidly induced clusters with known ethylene function and more slowly regulated clusters with novel predicted functions linked to root development. In contrast to studies with dark-grown seedlings, where the canonical ethylene response transcription factor, EIN3, is central to ethylene-mediated development, the roots of ein3 and eil1 single and double mutants still respond to ethylene in light-grown seedlings. Additionally, a subset of these clusters of ethylene-responsive transcripts were enriched in targets of EIN3 and ERFs. These results are consistent with EIN3-independent developmental and transcriptional changes in light-grown roots. Examination of single and multiple gain-of-function and loss-of-function receptor mutants revealed that, of the five ethylene receptors, ETR1 controls lateral root and root hair initiation and elongation and the synthesis of other receptors. These results provide new insight into the transcriptional and developmental responses to ethylene in light-grown seedlings.

Isolation and identification of Ammodendron bifolium endophytic bacteria and the action mechanism of selected isolates-induced seed germination and their effects on host osmotic-stress tolerance.

This study aimed to identify Ammodendron bifolium endophytic bacteria, and to evaluate promoting mechanism of selected isolates on seed germination and their effects on host osmotic-stress tolerance. Forty-five strains were isolated from A. bifolium and were classified into 13 different genera by 16S rDNA gene sequence analysis. AY3, AY9 and AG18, which were identified as Staphylococcus, Kocuria, Bacillus sp., promoted host seed ethylene release during germination. Ethrel and 1-aminocyclopropane-1-carboxylic acid (ACC) imitated the effect of AY3, AY9 and AG18 on seed germination. The data suggest that ethylene mediates AY3-, AY9-, AG18-induced A. bifolium seed germination. In addition, osmotic stress prevented seed germination and radicle elongation. However, the inhibitory effect of osmotic stress on seed germination and radicle elongation were rescued by AY3, AY9 and AG18. The results show that AY3, AY9 and AG18 increased osmotic-stress tolerance in A. bifolium. AY3, AY9, AG18 induced A. bifolium seed germination through promoting ethylene production during endophytic bacteria-plant interaction, and increase osmotic-stress tolerance in A. bifolium. AY3, AY9 and AG18 are potential candidates for the protection of A. bifolium.

On the origin of the 2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate scaffold's unique group II selectivity for the mGlu receptors.

Analogs based on the 2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate scaffold showed high potency and selectivity as both group II mGlu receptors orthosteric agonists and antagonists. This scaffold was initially designed to mimic the fully extended glutamate backbone conformation that was hypothesized to be the active conformation for the group II mGlu receptors. With the availability of crystal structures of l-Glu-bound amino terminal domain proteins from multiple mGlu receptor subtypes spanning all three subgroups, a new steric hindrance hypothesis was proposed to account for the scaffold's unique group II selectivity that explores the subtle distance differences between the α-carbon of l-Glu and the center of the tyrosine phenyl ring from the bottom lobe (e.g. Y216 of mGlu2).

Ethylene signaling induces gelatinous layers with typical features of tension wood in hybrid aspen.

The phytohormone ethylene impacts secondary stem growth in plants by stimulating cambial activity, xylem development and fiber over vessel formation. We report the effect of ethylene on secondary cell wall formation and the molecular connection between ethylene signaling and wood formation. We applied exogenous ethylene or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) to wild-type and ethylene-insensitive hybrid aspen trees (Populus tremula × tremuloides) and studied secondary cell wall anatomy, chemistry and ultrastructure. We furthermore analyzed the transcriptome (RNA Seq) after ACC application to wild-type and ethylene-insensitive trees. We demonstrate that ACC and ethylene induce gelatinous layers (G-layers) and alter the fiber cell wall cellulose microfibril angle. G-layers are tertiary wall layers rich in cellulose, typically found in tension wood of aspen trees. A vast majority of transcripts affected by ACC are downstream of ethylene perception and include a large number of transcription factors (TFs). Motif-analyses reveal potential connections between ethylene TFs (Ethylene Response Factors (ERFs), ETHYLENE INSENSITIVE 3/ETHYLENE INSENSITIVE3-LIKE1 (EIN3/EIL1)) and wood formation. G-layer formation upon ethylene application suggests that the increase in ethylene biosynthesis observed during tension wood formation is important for its formation. Ethylene-regulated TFs of the ERF and EIN3/EIL1 type could transmit the ethylene signal.

Defense Responses in Aspen with Altered Pectin Methylesterase Activity Reveal the Hormonal Inducers of Tyloses.

Tyloses are ingrowths of parenchyma cells into the lumen of embolized xylem vessels, thereby protecting the remaining xylem from pathogens. They are found in heartwood, sapwood, and in abscission zones and can be induced by various stresses, but their molecular triggers are unknown. Here, we report that down-regulation of PECTIN METHYLESTERASE1 (PtxtPME1) in aspen (Populus tremula × tremuloides) triggers the formation of tyloses and activation of oxidative stress. We tested whether any of the oxidative stress-related hormones could induce tyloses in intact plantlets grown in sterile culture. Jasmonates, including jasmonic acid (JA) and methyl jasmonate, induced the formation of tyloses, whereas treatments with salicylic acid (SA) and 1-aminocyclopropane-1-carboxylic acid (ACC) were ineffective. SA abolished the induction of tyloses by JA, whereas ACC was synergistic with JA. The ability of ACC to stimulate tyloses formation when combined with JA depended on ethylene (ET) signaling, as shown by a decrease in the response in ET-insensitive plants. Measurements of internal ACC and JA concentrations in wild-type and ET-insensitive plants treated simultaneously with these two compounds indicated that ACC and JA regulate each other's concentration in an ET-dependent manner. The findings indicate that jasmonates acting synergistically with ethylene are the key molecular triggers of tyloses.

Fibroblast-like synoviocyte migration is enhanced by IL-17-mediated overexpression of L-type amino acid transporter 1 (LAT1) via the mTOR/4E-BP1 pathway.

In rheumatoid arthritis (RA), activated synovial fibroblasts have the ability to invade joint cartilage, actively contributing to joint destruction in RA. The mechanisms underlying this cell migration and invasion remain unclear. Our previous results and data from the GEO profile indicate that the L-type amino acid transporter gene, LAT1, is overexpressed in the synovium of RA. To identify its potential role in RA, fibroblast-like synoviocytes (FLS) from patients with RA were used to determine the effects of suppressing the LAT1 genes using RNA interference and the LAT inhibitor, BCH. We found that BCH exposure reduced the phosphorylation of mTOR and its downstream target 4EBP1, radiolabeled leucine uptake, and migration of RA FLS. LAT1 silencing by siRNA presented effects similar to BCH inhibition. Treatment of cells with IL-17 stimulated the expression of LAT1. In contrast, applying an inhibitor of mTOR pathway, temsirolimus, or silencing eIF4E neutralized the stimulation of IL-17 on LAT1. BCH and siLAT1 also resulted in lower IL-17-stimulated leucine uptake and cell migration. These results suggest that the migration of RA FLS is aggravated by IL-17-mediated overexpression of LAT1 via mTOR/4E-BP1 pathway. In conclusion, further investigation is warranted into LAT1 as a potential target for drug therapies aimed at attenuating migration of transformed-appearing fibroblasts and subsequently preventing further erosion of bone and cartilage.

Design of cyclic and d-amino acids containing peptidomimetics for inhibition of protein-protein interactions of HER2-HER3.

HER2 receptors are surface proteins belonging to the epidermal growth factor family of receptors. Their numbers are elevated in breast, lung, and ovarian cancers. HER2-positive cancers are aggressive, have higher mortality rate, and have a poor prognosis. We have designed peptidomimetics that bind to HER2 and block the HER2-mediated dimerization of epidermal growth factor family of receptors. Among these, a symmetrical cyclic peptidomimetic (compound 18) exhibited antiproliferative activity in HER2-overexpressing lung cancer cell lines with IC50 values in the nanomolar concentration range. To improve the stability of the peptidomimetic, d-amino acids were introduced into the peptidomimetic, and several analogs of compound 18 were designed. Among the analogs of compound 18, compound 32, a cyclic, d-amino acid-containing peptidomimetic, was found to have an IC50 value in the nanomolar range in HER2-overexpressing cancer cell lines. The antiproliferative activity of compound 32 was also measured by using a 3D cell culture model that mimics the in vivo conditions. The binding of compound 32 to the HER2 protein was studied by surface plasmon resonance. In vitro stability studies indicated that compound 32 was stable in serum for 48 hours and intact peptide was detectable in vivo for 12 hours. Results from our studies indicated that 1 of the d-amino acid analogs of 18, compound 32, binds to the HER2 extracellular domain, inhibiting the phosphorylation of kinase of HER2.

Effects of glucose and ethylene on root hair initiation and elongation in lettuce (Lactuca sativa L.) seedlings.

Root hair formation occurs in lettuce seedlings after transfer to an acidic medium (pH 4.0). This process requires cortical microtubule (CMT) randomization in root epidermal cells and the plant hormone ethylene. We investigated the interaction between ethylene and glucose, a new signaling molecule in plants, in lettuce root development, with an emphasis on root hair formation. Dark-grown seedlings were used to exclude the effect of photosynthetically produced glucose. In the dark, neither root hair formation nor the CMT randomization preceding it occurred, even after transfer to the acidic medium (pH 4.0). Adding 1-aminocyclopropane-1-carboxylic-acid (ACC) to the medium rescued the induction, while adding glucose did not. Although CMT randomization occurred when glucose was applied together with ACC, it was somewhat suppressed compared to that in ACC-treated seedlings. This was not due to a decrease in the speed of randomization, but due to lowering of the maximum degree of randomization. Despite the negative effect of glucose on ACC-induced CMT randomization, the density and length of ACC-induced root hairs increased when glucose was also added. The hair-cell length of the ACC-treated seedlings was comparable to that in the combined-treatment seedlings, indicating that the increase in hair density caused by glucose results from an increase in the root hair number. Furthermore, quantitative RT-PCR revealed that glucose suppressed ethylene signaling. These results suggest that glucose has a negative and positive effect on the earlier and later stages of root hair formation, respectively, and that the promotion of the initiation and elongation of root hairs by glucose may be mediated in an ethylene-independent manner.

The ARM Domain of ARMADILLO-REPEAT KINESIN 1 is Not Required for Microtubule Catastrophe But Can Negatively Regulate NIMA-RELATED KINASE 6 in Arabidopsis thaliana.

Microtubules are dynamic filaments, the assembly and disassembly of which are under precise control of various associated proteins, including motor proteins and regulatory enzymes. In Arabidopsis thaliana, two such proteins are the ARMADILLO-REPEAT KINESIN 1 (ARK1), which promotes microtubule disassembly, and the NIMA-RELATED KINASE 6 (NEK6), which has a role in organizing microtubule arrays. Previous yeast two-hybrid and in vitro pull-down assays determined that NEK6 can interact with ARK1 through the latter protein's Armadillo-repeat (ARM) cargo domain. To explore the function of the ARM domain, we generated fluorescent reporter fusion proteins to ARK1 lacking the ARM domain (ARK1ΔARM-GFP) and to the ARM domain alone (ARM-GFP). Both of these constructs strongly associated with the growing plus ends of microtubules, but only ARK1ΔARM-GFP was capable of inducing microtubule catastrophe and rescuing the ark1-1 root hair phenotype. These results indicate that neither the ARM domain nor NEK6's putative interaction with it is required for ARK1 to induce microtubule catastrophe. In further exploration of the ARK1-NEK6 relationship, we demonstrated that, despite evidence that NEK6 can phosphorylate ARK1 in vitro, the in vivo distribution and function of ARK1 were not affected by the loss of NEK6, and vice versa. Moreover, NEK6 and ARK1 were found to have overlapping but non-identical distribution on microtubules, and hormone treatments known to affect NEK6 activity did not stimulate interaction. These findings suggest that ARK1 and NEK6 function independently in microtubule dynamics and cell morphogenesis. Despite the results of this functional analysis, we found that overexpression of the ARM domain led to complete loss of NEK6 transcription, suggesting that the ARM domain might have a regulatory role in NEK6 expression.

Ethylene sensitivity and relative air humidity regulate root hydraulic properties in tomato plants.

MAIN CONCLUSION: The effect of ethylene and its precursor ACC on root hydraulic properties, including aquaporin expression and abundance, is modulated by relative air humidity and plant sensitivity to ethylene. Relative air humidity (RH) is a main factor contributing to water balance in plants. Ethylene (ET) is known to be involved in the regulation of root water uptake and stomatal opening although its role on plant water balance under different RH is not very well understood. We studied, at the physiological, hormonal and molecular levels (aquaporins expression, abundance and phosphorylation state), the plant responses to exogenous 1-aminocyclopropane-1-carboxylic acid (ACC; precursor of ET) and 2-aminoisobutyric acid (AIB; inhibitor of ET biosynthesis), after 24 h of application to the roots of tomato wild type (WT) plants and its ET-insensitive never ripe (nr) mutant, at two RH levels: regular (50%) and close to saturation RH. Highest RH induced an increase of root hydraulic conductivity (Lpo) of non-treated WT plants, and the opposite effect in nr mutants. The treatment with ACC reduced Lpo in WT plants at low RH and in nr plants at high RH. The application of AIB increased Lpo only in nr plants at high RH. In untreated plants, the RH treatment changed the abundance and phosphorylation of aquaporins that affected differently both genotypes according to their ET sensitivity. We show that RH is critical in regulating root hydraulic properties, and that Lpo is affected by the plant sensitivity to ET, and possibly to ACC, by regulating aquaporins expression and their phosphorylation status. These results incorporate the relationship between RH and ET in the response of Lpo to environmental changes.

Amino acids regulate mTOR pathway and milk protein synthesis in a mouse mammary epithelial cell line is partly mediated by T1R1/T1R3.

PURPOSE: The mechanism of dietary amino acids in regulating milk protein synthesis at the translational level is not well understood. Numerous studies have shown that the amino acid signal is transferred through the mammalian target of rapamycin (mTOR) pathway; however, the extracellular amino acid-sensing mechanism that activates mTOR complex 1 is unknown. We tested the hypotheses that the T1R1/T1R3 heterodimer functions as a direct sensor of the fed state and amino acid availability preceding the mTOR pathway and affects milk protein synthesis in mammary epithelial cells. METHODS: The expression of T1R1 was repressed by T1R1 siRNA in mouse mammary epithelial cells model (HC11). Western blot was used to analyze activity of the mTOR pathway and ß-casein expression, and quantitative real-time RT-PCR was used to analyze the change in mRNA abundance of amino acid transporters. RESULTS: The transcripts and proteins of T1R1 and T1R3 were detected in HC11 cells and mouse mammary gland tissue. siRNA silencing of T1R1 repressed ß-casein synthesis in HC11 cells both with and without essential amino acids present in the culture medium. The phosphorylation of mTOR, S6K, and 4EBP1 in T1R1 knockdown HC11 cells declined to 25, 50, and 30 %, indicating T1R1 knockdown repressed the activity of the mTOR pathway. T1R1 knockdown increased the mRNAs coding three important amino acid transporters (SLC1A5 and SLC3A2/SLC7A5). Activation of the mTOR pathway was partially repressed by T1R1 siRNA or SLC7A5/SLC3A2 inhibitor (BCH, 10 mM), and the combination of these two treatments further repressed the activity of this pathway. CONCLUSION: T1R1/T1R3 serves as sensor of extracellular amino acids in mouse mammary epithelial cells and involved in milk protein synthesis regulation.

Identification of a Novel Activator of Mammalian Glutamate Dehydrogenase.

Glutamate dehydrogenase (GDH) catalyzes the oxidative deamination of l-glutamate and in animals is highly regulated. GDH in hyperinsulinism/hyperammonemia syndrome patients lacks GTP inhibition, resulting in hypersecretion of insulin upon protein consumption. This suggests insulin secretion could be stimulated with GDH activators. A high-throughput screen yielded one potent activator, N1-[4-(2-aminopyrimidin-4-yl)phenyl]-3-(trifluoromethyl)benzene-1-sulfonamide (75-E10). 75-E10 is ∼1000-fold more efficacious than the synthetic activator, BCH, and is at least as effective as ADP. 75-E10 compound is highly effective at alleviating GTP inhibition and may be binding to the ADP site. Unlike ADP, 75-E10 is activated over a broad range of conditions.

LAT1 is a putative therapeutic target in endometrioid endometrial carcinoma.

l-type amino acid transporters (LAT1-4) are expressed in various cancer types and are involved in the uptake of essential amino acids such as leucine. Here we investigated the expression of LAT1-4 in endometrial adenocarcinoma and evaluated the contribution of LATs to endometrial cancer cell growth. Analysis of human gene expression data showed that all four LAT family members are expressed in endometrial adenocarcinomas. LAT1 was the most highly expressed, and showed a significant increase in both serous and endometrioid subtypes compared to normal endometrium. Endometrioid patients with the highest LAT1 levels exhibited the lowest disease-free survival. The pan-LAT inhibitor BCH led to a significant decrease in cell growth and spheroid area in four endometrial cancer cell lines tested in vitro. Knockdown of LAT1 by shRNA inhibited cell growth in HEC1A and Ishikawa cells, as well as inhibiting spheroid area in HEC1A cells. These data show that LAT1 plays an important role in regulating the uptake of essential amino acids such as leucine into endometrial cancer cells. Increased ability of BCH compared to LAT1 shRNA at inhibiting Ishikawa spheroid area suggests that other LAT family members may also contribute to cell growth. LAT1 inhibition may offer an effective therapeutic strategy in endometrial cancer patients whose tumours exhibit high LAT1 expression.

Ethylene Biosynthesis Is Promoted by Very-Long-Chain Fatty Acids during Lysigenous Aerenchyma Formation in Rice Roots.

In rice (Oryza sativa) roots, lysigenous aerenchyma, which is created by programmed cell death and lysis of cortical cells, is constitutively formed under aerobic conditions, and its formation is further induced under oxygen-deficient conditions. Ethylene is involved in the induction of aerenchyma formation. reduced culm number1 (rcn1) is a rice mutant in which the gene encoding the ATP-binding cassette transporter RCN1/OsABCG5 is defective. Here, we report that the induction of aerenchyma formation was reduced in roots of rcn1 grown in stagnant deoxygenated nutrient solution (i.e. under stagnant conditions, which mimic oxygen-deficient conditions in waterlogged soils). 1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is a key enzyme in ethylene biosynthesis. Stagnant conditions hardly induced the expression of ACS1 in rcn1 roots, resulting in low ethylene production in the roots. Accumulation of saturated very-long-chain fatty acids (VLCFAs) of 24, 26, and 28 carbons was reduced in rcn1 roots. Exogenously supplied VLCFA (26 carbons) increased the expression level of ACS1 and induced aerenchyma formation in rcn1 roots. Moreover, in rice lines in which the gene encoding a fatty acid elongase, CUT1-LIKE (CUT1L; a homolog of the gene encoding Arabidopsis CUT1, which is required for cuticular wax production), was silenced, both ACS1 expression and aerenchyma formation were reduced. Interestingly, the expression of ACS1, CUT1L, and RCN1/OsABCG5 was induced predominantly in the outer part of roots under stagnant conditions. These results suggest that, in rice under oxygen-deficient conditions, VLCFAs increase ethylene production by promoting 1-aminocyclopropane-1-carboxylic acid biosynthesis in the outer part of roots, which, in turn, induces aerenchyma formation in the root cortex.

Thermoperiodic control of hypocotyl elongation depends on auxin-induced ethylene signaling that controls downstream PHYTOCHROME INTERACTING FACTOR3 activity.

We show that antiphase light-temperature cycles (negative day-night temperature difference [-DIF]) inhibit hypocotyl growth in Arabidopsis (Arabidopsis thaliana). This is caused by reduced cell elongation during the cold photoperiod. Cell elongation in the basal part of the hypocotyl under -DIF was restored by both 1-aminocyclopropane-1-carboxylic acid (ACC; ethylene precursor) and auxin, indicating limited auxin and ethylene signaling under -DIF. Both auxin biosynthesis and auxin signaling were reduced during -DIF. In addition, expression of several ACC Synthase was reduced under -DIF but could be restored by auxin application. In contrast, the reduced hypocotyl elongation of ethylene biosynthesis and signaling mutants could not be complemented by auxin, indicating that auxin functions upstream of ethylene. The PHYTOCHROME INTERACTING FACTORS (PIFs) PIF3, PIF4, and PIF5 were previously shown to be important regulators of hypocotyl elongation. We now show that, in contrast to pif4 and pif5 mutants, the reduced hypocotyl length in pif3 cannot be rescued by either ACC or auxin. In line with this, treatment with ethylene or auxin inhibitors reduced hypocotyl elongation in PIF4 overexpressor (PIF4ox) and PIF5ox but not PIF3ox plants. PIF3 promoter activity was strongly reduced under -DIF but could be restored by auxin application in an ACC Synthase-dependent manner. Combined, these results show that PIF3 regulates hypocotyl length downstream, whereas PIF4 and PIF5 regulate hypocotyl length upstream of an auxin and ethylene cascade. We show that, under -DIF, lower auxin biosynthesis activity limits the signaling in this pathway, resulting in low activity of PIF3 and short hypocotyls.

The ETHYLENE RESPONSE FACTORs ERF6 and ERF11 Antagonistically Regulate Mannitol-Induced Growth Inhibition in Arabidopsis.

Leaf growth is a tightly regulated and complex process, which responds in a dynamic manner to changing environmental conditions, but the mechanisms that reduce growth under adverse conditions are rather poorly understood. We previously identified a growth inhibitory pathway regulating leaf growth upon exposure to a low concentration of mannitol and characterized the ETHYLENE RESPONSE FACTOR (ERF)/APETALA2 transcription factor ERF6 as a central activator of both leaf growth inhibition and induction of stress tolerance genes. Here, we describe the role of the transcriptional repressor ERF11 in relation to the ERF6-mediated stress response in Arabidopsis (Arabidopsis thaliana). Using inducible overexpression lines, we show that ERF6 induces the expression of ERF11. ERF11 in turn molecularly counteracts the action of ERF6 and represses at least some of the ERF6-induced genes by directly competing for the target gene promoters. As a phenotypical consequence of the ERF6-ERF11 antagonism, the extreme dwarfism caused by ERF6 overexpression is suppressed by overexpression of ERF11. Together, our data demonstrate that dynamic mechanisms exist to fine-tune the stress response and that ERF11 counteracts ERF6 to maintain a balance between plant growth and stress defense.