Induction Of Ethylene Biosynthesis Research Articles

Assessing the Role of Gaseous Chlorine Dioxide in Modulating the Postharvest Ripening of Keitt Mangoes through the Induction of Ethylene Biosynthesis.

Consumer acceptance of Keitt mangoes (Mangifera indica L.) is significantly affected by their slow postharvest ripening. This work used gaseous chlorine dioxide (ClO2(g)) to prepare the ready-to-eat Keitt mango and explored the potential mechanisms for the mango ripening. Harvested mangoes were treated with 20 mg·L-1 of ClO2(g) or ethephon for 3 h (25 °C) and left in a climatic chamber with a temperature of 25 ± 1 °C and a relative humidity of 85 ± 5% for 4 d. The results showed that ClO2(g) treatment significantly promoted the orange coloration of mango flesh compared to the untreated control group. Moreover, ClO2(g) treatment significantly elevated the total soluble solids, total soluble sugar, and total carotenoids content of mangoes, whereas the firmness and titratable acidity were reduced. ClO2(g)-treated mangoes reached the edible window on day 2, as did mangoes treated with ethephon at the same concentration, except that the sweetness was prominent. The residual ClO2 level of the mangoes was <0.3 mg/kg during the whole storage time, which is a safe level for fruit. In addition, ClO2(g) significantly advanced the onset of ethylene peaks by 0.5 days and increased its production between days 0.5 and 2 compared to the control group. Consistently, the genes involved in ethylene biosynthesis including miACS6, miACO1, and miACO were upregulated. In sum, ClO2(g) can be a potential technique to reduce the time for harvested mango to reach the edible window, and it functions in modulating postharvest ripening by inducing ethylene biosynthesis.

MaSPL16 positively regulates fruit ripening in bananas via the direct transcriptional induction of MaNAC029

Fruit ripening (FR) is attributed to the selective expression of several genes precisely governed by various specific transcription factors (TFs). The NAC (NAM, ATAF, and CUC) TF, MaNAC029, positively regulated banana ripening by directly inducing ethylene biosynthesis and transcription of fruit quality-related genes. However, its upstream regulatory mechanism still needs to be clarified. Herein, yeast one-hybrid screening revealed that a SQUAMOSA promoter binding protein-like (SPL) TF, MaSPL16, was a potentially upstream regulator of Musa acuminata NAC (NAM, ATAF, CUC) 029 (MaNAC029). Furthermore, gel mobility shift assay revealed that MaSPL16 can directly bound with the “GTAC” element of the MaNAC029 promoter. The gene expression and promoter activity assays demonstrated that Musa acuminata SPL (SQUAMOSA promoter binding protein-like) 16 (MaSPL16) expression was inducible by ethylene and ripening. MaSPL16 was localized to the nucleus, displayed a potenial capacity for transcriptional activation of MaNAC029. More critically, the transient expression of MaSPL16 in bananas accelerated FR via the upregulation of MaNAC029 and its downstream genes. Collectively, the mechanistic basis of a regulatory cascade involving MaSPL16-MaNAC029 that governed ethylene biosynthesis and fruit quality throughout the entire process of banana fruit ripening was unveiled. These outcomes increase the understanding of the gene-transcriptional regulatory mechanisms in FR. They are envisaged to help devise molecular techniques to regulate maturation and improve future fruit quality.

Nitric oxide (NO) modulates low temperature-stress signaling via S-nitrosation, a NO PTM, inducing ethylene biosynthesis inhibition leading to enhanced post-harvest shelf-life of agricultural produce.

The online version contains supplementary material available at 10.1007/s12298-023-01371-z.

PpARF6 acts as an integrator of auxin and ethylene signaling to promote fruit ripening in peach.

Although auxin is known to induce ethylene biosynthesis in some Rosaceae fruit crops, the mechanisms underlying the auxin-ethylene interaction during fruit ripening remain largely unknown. Here, the regulatory role of an auxin response factor, PpARF6, in fruit ripening was investigated in peach. Peach fruits showed accelerated ripening after treatment with auxin and PpARF6 was found to be significantly induced. PpARF6 not only could induce ethylene synthesis by directly activating the transcription of ethylene biosynthetic genes, but also competed with EIN3-binding F-box proteins PpEBF1/2 for binding to ethylene-insensitive3-like proteins PpEIL2/3, thereby keeping PpEIL2/3 active. Moreover, PpARF6 showed an interaction with PpEIL2/3 to enhance the PpEIL2/3-activated transcription of ethylene biosynthetic genes. Additionally, ectopic overexpression of PpARF6 in tomato accelerated fruit ripening by promoting the expression of genes involved in ethylene synthesis and fruit texture. In summary, our results revealed a positive regulatory role of PpARF6 in peach fruit ripening via integrating auxin and ethylene signaling.

Pathotypes of Fusarium oxysporum f. sp. fragariae express discrete repertoires of accessory genes and induce distinct host transcriptional responses during root infection.

Convergent evolution of phytopathogenicity is poorly described, especially among multiple strains of a single microbial species. We investigated this phenomenon with genetically diverse isolates of Fusarium oxysporum f. sp. fragariae (Fof) that cause one of two syndromes: chlorosis and wilting (the 'yellows-fragariae' pathotype), or only wilting (the 'wilt-fragariae' pathotype). We challenged strawberry (Fragaria × ananassa) plants to root infection by five fungal isolates: three yellows-fragariae, one wilt-fragariae and one that is not pathogenic to strawberry. All Fof isolates had chromosome-level assemblies; three were newly generated. The two pathotypes triggered distinct host responses, especially among phytohormone-associated genes; yellows-fragariae isolates strongly induced jasmonic acid-associated genes, whereas the wilt-fragariae isolate primarily induced ethylene biosynthesis and signalling. The differentially expressed genes on fungal accessory chromosomes were almost entirely distinct between pathotypes. We identified an ~150 kbp 'pathogenicity island' that was horizontally transferred between wilt-fragariae strains. This predicted pathogenicity island was enriched with differentially expressed genes whose predicted functions were related to plant infection, and only one of these genes was also upregulated in planta by yellows-fragariae isolates. These results support the conclusion that wilt- and yellows-fragariae cause physiologically distinct syndromes by the expression of discrete repertoires of genes on accessory chromosomes.

Feedback loop promotes sucrose accumulation in cotyledons to facilitate sugar-ethylene signaling-mediated, etiolated-seedling greening.

De-etiolation is indispensable for seedling survival and development. However, how sugars regulate de-etiolation and how sugars induce ethylene (ET) for seedlings to grow out of soil remain elusive. Here, we reveal how a sucrose (Suc) feedback loop promotes de-etiolation by inducing ET biosynthesis. Under darkness, Suc in germinating seeds preferentially induces 1-amino-cyclopropane-1-carboxylate synthase (ACS7; encoding a key ET biosynthesis enzyme) and associated ET biosynthesis, thereby activating ET core component ETHYLENE-INSENSITIVE3 (EIN3). Activated EIN3 directly inhibits the function of Suc transporter 2 (SUC2; a major Suc transporter) to block Suc export from cotyledons and thereby elevate Suc accumulation of cotyledons to induce ET. Under light, ET-activated EIN3 directly inhibits the function of phytochrome A (phyA; a de-etiolation inhibitor) to promote de-etiolation. We therefore propose that under darkness, the Suc feedback loop (Suc-ACS7-EIN3-|SUC2-Suc) promotes Suc accumulation in cotyledons to guarantee ET biosynthesis, facilitate de-etiolation, and enable seedlings to grow out of soil.

Cytokinin regulates apical hook development via the coordinated actions of EIN3/EIL1 and PIF transcription factors in Arabidopsis.

The apical hook is indispensable for protecting the delicate shoot apical meristem while dicot seedlings emerge from soil after germination in darkness. The development of the apical hook is co-ordinately regulated by multiple phytohormones and environmental factors. Yet, a holistic understanding of the spatial-temporal interactions between different phytohormones and environmental factors remains to be achieved. Using a chemical genetic approach, we identified kinetin riboside, as a proxy of kinetin, which promotes apical hook development of Arabidopsis thaliana in a partially ethylene-signaling-independent pathway. Further genetic and biochemical analysis revealed that cytokinin is able to regulate apical hook development via post-transcriptional regulation of the PHYTOCHROME INTERACTING FACTORs (PIFs), together with its canonical roles in inducing ethylene biosynthesis. Dynamic observations of apical hook development processes showed that ETHYLENE INSENSITVE3 (EIN3) and EIN3-LIKE1 (EIL1) are necessary for the exaggeration of hook curvature in response to cytokinin, while PIFs are crucial for the cytokinin-induced maintenance of hook curvature in darkness. Furthermore, these two families of transcription factors display divergent roles in light-triggered hook opening. Our findings reveal that cytokinin integrates ethylene signaling and light signaling via EIN3/EIL1 and PIFs, respectively, to dynamically regulate apical hook development during early seedling development.

Interaction between reactive oxygen species and hormones during the breaking of embryo dormancy in Sorbus pohuashanensis by exogenous nitric oxide

The breaking of dormancy mediated by reactive nitrogen species (RNS) is related to the accumulation of reactive oxygen species (ROS) in germinating embryos but the underlying mechanism is unclear. The objectives of this study were: (1) to explore the relationship between RNS-mediated dormancy release and ROS accumulation in germinating embryos of Sorbus pohuashanensis; and, (2) to investigate the relationships among germination time, ROS metabolism, and endogenous hormone synthesis. We studied the effects of exogenous nitric oxide (NO) donor sodium nitroprusside (SNP), the NO scavenger (4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), abscisic acid (ABA), the exogenous ethylene donor ethrel, and the ethylene receptor inhibitor 2,5-norbornadien (NBD) on embryo germination and seedling growth. Embryos were released from dormancy by pretreatment with NO or ethylene, which was related to increased ethylene biosynthesis and decreased ABA levels. Breaking of dormancy by SNP was related to increased levels of ethylene, hydrogen peroxide, and glutathione, increased activities of superoxide dismutase and glutathione peroxidase, and decreased levels of ABA, superoxide anions, and malondialdehyde. These effects of nitric oxide were especially significant in seedling hypocotyls and radicles. These results demonstrate that NO can break S. pohuashanensis embryo dormancy by inducing ethylene biosynthesis, and that this signalling pathway is closely related to ROS accumulation and the antioxidant defence response.

Dichromate-induced ethylene biosynthesis, perception, and signaling regulate the variance in root growth inhibition among Shaheen basmati and basmati-385 rice varieties.

Heavy metals, including a hexavalent form of chromium (Cr(VI)) increasing accumulation in agricultural soil, cause a significant reduction in quality, yield, and growth of rice varieties worldwide. Screening for the selection of tolerant varieties is essential for conventional and molecular breeding. Shaheen basmati (SB) and basmati-385 (B-385) rice varieties, a subspecies of indica, show different sensitivity to Cr(VI), but the underlying mechanisms of this different sensitivity remain elusive. In the current study, we examine the sensitivity of SB and B-385 based on the root, which is the primary organ that encounters water and soil containing Cr(VI), elongation assay, and ethylene's possible role (a stress-responsive phytohormone) in the process. Our results show that SB's seedlings exhibit hypersensitivity as a higher root elongation inhibition than B-385 under different Cr(VI) concentrations. Hypersensitive SB consistently expresses a higher level of ethylene biosynthesis and signaling-related genes than B-385. Moreover, ethylene signaling antagonist (silver, Ag) and biosynthesis inhibitor (aminoethoxy vinyl glycine, AVG) alleviate the difference in Cr(VI)-induced root growth inhibition between SB and B-385, respectively. Taken together, we conclude that ethylene mediates difference in sensitivity based on the difference in root growth inhibition in different rice varieties. The difference in Cr(VI)-induced root growth inhibition in SB and B-385. (A) Root growth of SB is slightly more as compared to B-385 in control conditions in the Hoagland solutions. (B) Seedlings of SB showed hypersensitivity to 200 μM Cr(VI) compared to B-385 in terms of primary root growth inhibition, which was higher in SB than B-385. Interestingly, Cr(VI)-induced relative transcript level of ethylene biosynthesis, perception, and signaling-related genes was significantly higher in hypersensitive SB than B-385. Current results in association with previous literature show that Cr(VI)-induced ethylene biosynthesis is regulating Cr(VI)-induced ethylene perception, signaling, and associated Cr(VI)-induced ethylene-mediated primary root growth inhibition. Conclusively, the difference in ethylene quantities in both varieties mediates the difference in root growth inhibition between SB and B-385 (C and E). The difference in Cr(VI)-induce root growth inhibition between SB and B-385 was significantly alleviated by ethylene signaling inhibitor (10 μM Ag, as AgNO3) and ethylene biosynthesis inhibitor (10 μM AVG) treatment in the presence of 200 μM Cr(VI), respectively. (D) Ethylene biosynthesis precursor (10 μM ACC) treatment-mediated induced root growth inhibition difference between SB and B-385 was not significant, which may be because of enough quantity of the Cr(VI)-mediated ethylene accumulation or unknown limiting factor. Arrows mean addition and an increase in expression, and T-line means suppression or inhibition. The width of the pointers (arrows) is proportional to the gene expression level.

Modifying the effect of stressed spring wheat seeds on intact ones

Spring wheat seeds react to the effect of stress factors of various natures with a nonspecific adaptive reaction, accompanied by intense emission of stress ethylene in trace amounts during the entire time, while. On the one hand, they remain viable, and on the other hand, have mechanical, radiation-chemical and temperature damage. Damaged air-dry seeds are not able to regenerate damaged organs and tissues and, being in a state of chronic stress, permanently induce ethylene biosynthesis. The effect of stressed seeds on intact ones is achieved remotely when they are stored together in the air environment and are blocked by active aeration, air-insulating materials and a decrease in temperature. The stronger the damaging effect on seeds is caused by the stress factor, the more intense is the accumulation of stress ethylene. An increase in the duration of exposure of intact by stressed seeds is accompanied by a more pronounced physiological modifications in seedlings of intact seeds with a sequential change in time in their stimulation of the initial growth processes and a sharp deterioration in sowing qualities.

Homeobox transcription factor OsZHD2 promotes root meristem activity in rice by inducing ethylene biosynthesis.

Root meristem activity is the most critical process influencing root development. Although several factors that regulate meristem activity have been identified in rice, studies on the enhancement of meristem activity in roots are limited. We identified a T-DNA activation tagging line of a zinc-finger homeobox gene, OsZHD2, which has longer seminal and lateral roots due to increased meristem activity. The phenotypes were confirmed in transgenic plants overexpressing OsZHD2. In addition, the overexpressing plants showed enhanced grain yield under low nutrient and paddy field conditions. OsZHD2 was preferentially expressed in the shoot apical meristem and root tips. Transcriptome analyses and quantitative real-time PCR experiments on roots from the activation tagging line and the wild type showed that genes for ethylene biosynthesis were up-regulated in the activation line. Ethylene levels were higher in the activation lines compared with the wild type. ChIP assay results suggested that OsZHD2 induces ethylene biosynthesis by controlling ACS5 directly. Treatment with ACC (1-aminocyclopropane-1-carboxylic acid), an ethylene precursor, induced the expression of the DR5 reporter at the root tip and stele, whereas treatment with an ethylene biosynthesis inhibitor, AVG (aminoethoxyvinylglycine), decreased that expression in both the wild type and the OsZHD2 overexpression line. These observations suggest that OsZHD2 enhances root meristem activity by influencing ethylene biosynthesis and, in turn, auxin.

Auxin-activated MdARF5 induces the expression of ethylene biosynthetic genes to initiate apple fruit ripening.

Summary The gaseous plant hormone ethylene induces the ripening of climacteric fruit, including apple (Malus domestica). Another phytohormone, auxin, is known to promote ethylene production in many horticultural crops, but the regulatory mechanism remains unclear.Here, we found that auxin application induces ethylene production in apple fruit before the stage of commercial harvest, when they are not otherwise capable of ripening naturally.The expression of MdARF5, a member of the auxin response factor transcription factor (TF) family involved in the auxin signaling pathway, was enhanced by treatment with the synthetic auxin naphthaleneacetic acid (NAA). Further studies revealed that MdARF5 binds to the promoter of MdERF2, encoding a TF in the ethylene signaling pathway, as well as the promoters of two 1‐aminocyclopropane‐1‐carboxylic acid synthase (ACS) genes (MdACS3a and MdACS1) and an ACC oxidase (ACO) gene, MdACO1, all of which encode key steps in ethylene biosynthesis, thereby inducing their expression. We also observed that auxin‐induced ethylene production was dependent on the methylation of the MdACS3a promoter.Our findings reveal that auxin induces ethylene biosynthesis in apple fruit through activation of MdARF5 expression.

Evidence for pre-climacteric activation of AOX transcription during cold-induced conditioning to ripen in European pear (Pyrus communis L.).

European pears (Pyrus communis L.) require a range of cold-temperature exposure to induce ethylene biosynthesis and fruit ripening. Physiological and hormonal responses to cold temperature storage in pear have been well characterized, but the molecular underpinnings of these phenomena remain unclear. An established low-temperature conditioning model was used to induce ripening of 'D'Anjou' and 'Bartlett' pear cultivars and quantify the expression of key genes representing ripening-related metabolic pathways in comparison to non-conditioned fruit. Physiological indicators of pear ripening were recorded, and fruit peel tissue sampled in parallel, during the cold-conditioning and ripening time-course experiment to correlate gene expression to ontogeny. Two complementary approaches, Nonparametric Multi-Dimensional Scaling and efficiency-corrected 2-(ΔΔCt), were used to identify genes exhibiting the most variability in expression. Interestingly, the enhanced alternative oxidase (AOX) transcript abundance at the pre-climacteric stage in 'Bartlett' and 'D'Anjou' at the peak of the conditioning treatments suggests that AOX may play a key and a novel role in the achievement of ripening competency. There were indications that cold-sensing and signaling elements from ABA and auxin pathways modulate the S1-S2 ethylene transition in European pears, and that the S1-S2 ethylene biosynthesis transition is more pronounced in 'Bartlett' as compared to 'D'Anjou' pear. This information has implications in preventing post-harvest losses of this important crop.

Effect of the host-specific toxin SnTOX3 from Stagonospora nodorum on ethylene signaling pathway regulation and redox-state in common wheat

The fungus Stagonospora nodorum Berk. is the causative agent of Septoria nodorum blotch (SNB) of wheat. The most important factors of Stagonospora nodorum virulence include numerous fungal necrotrophic effectors (NEs) encoded by SnTox genes. They interact with the matching products of host susceptibility genes (Snn). SnTox-Snn interactions are mirror images of classical gene-for-gene interactions and lead to the development of disease. We have studied the SnTox3-Snn3 interaction, resulting in the development of infection on leaves and formation of extensive lesions. The mechanism of SnTox3 action is likely to be linked to the regulation of redox metabolism and the influence on ethylene synthesis in the wheat plants, although the molecular mechanisms are not fully unveiled. To characterize the SnTox3-Snn3 interaction, we used S. nodorum isolates differing in the expression of the NEs genes SnTox3 (SnB (Tox3+), Sn4VD (Tox3–)) and two soft spring wheat (Triticum aestivum L.) cultivars, contrasting in resistance to the SNB agent and differing in the allelic composition of the susceptibility locus Snn3-B1: Kazakhstanskaya 10 (susceptible) and Omskaya 35 (resistant). We carried out a comparative assessment of the transcriptional activity patterns of genes responsible for ethylene biosynthesis (TaACS1, TaACО) and signaling pathway (TaEIL1, TaERF1) by real-time PCR and estimated the redox state of wheat plants infected with different isolates of S. nodorum by spectrometry. The induction of ethylene biosynthesis and signaling has been shown to result from gene-for-gene interaction between Snn3-B1 and SnTox3. The results of plant redox status estimation showed that ethylene inhibited accumulation of hydrogen peroxide in SnTox3-sensitive genotypes by regulating the operation of various pro-/antioxidant enzymes at the transcriptional and posttranslational levels. Our results suggest that NE SnTox3 influences ethylene biosynthesis and signaling, thereby regulating redox metabolism in infected wheat plants as necessary for successful host colonization at the initial phases of infection, which ultimately leads to extensive lesions due to fast pathogen reproduction.

Ethylene mediates the branching of the jasmonate-induced flavonoid biosynthesis pathway by suppressing anthocyanin biosynthesis in red Chinese pear fruits.

SummaryFlavonoid accumulation in most fruits is enhanced by ethylene and jasmonate. However, little is known about the hormone functions related to red pear fruit coloration or their combined effects and potential underlying mechanisms. Various treatments were used to investigate the flavonoid metabolite profile and pear transcriptome to verify the effects of ethylene and jasmonate on flavonoid biosynthesis in red pear fruits as well as the mechanism behind this. Ethylene inhibits anthocyanin biosynthesis in red Chinese pear fruits, whereas jasmonate increases anthocyanin and flavone/isoflavone biosyntheses. The branching of the jasmonate‐induced flavonoid biosynthesis pathway is determined by ethylene. Co‐expression network and Mfuzz analyses revealed 4,368 candidate transcripts. Additionally, ethylene suppresses PpMYB10 and PpMYB114 expression via TF repressors, ultimately decreasing anthocyanin biosynthesis. Jasmonate induces anthocyanin accumulation through transcriptional or post‐translational regulation of TFs‐like MYB and bHLH in the absence of ethylene. However, jasmonate induces ethylene biosynthesis and the associated signalling pathway in pear, thereby decreasing anthocyanin production, increasing the availability of the precursors for flavone/isoflavone biosynthesis and enhancing deep yellow fruit coloration. We herein present new phenotypes and fruit coloration regulatory patterns controlled by jasmonate and ethylene, and confirm that the regulation of fruit coloration is complex.

Deciphering Auxin-Ethylene Crosstalk at a Systems Level.

The auxin and ethylene pathways cooperatively regulate a variety of developmental processes in plants. Growth responses to ethylene are largely dependent on auxin, the key regulator of plant morphogenesis. Auxin, in turn, is capable of inducing ethylene biosynthesis and signaling, making the interaction of these hormones reciprocal. Recent studies discovered a number of molecular events underlying auxin-ethylene crosstalk. In this review, we summarize the results of fine-scale and large-scale experiments on the interactions between the auxin and ethylene pathways in Arabidopsis. We integrate knowledge on molecular crosstalk events, their tissue specificity, and associated phenotypic responses to decipher the crosstalk mechanisms at a systems level. We also discuss the prospects of applying systems biology approaches to study the mechanisms of crosstalk between plant hormones.

Arabidopsis Transcription Factor MYB102 Increases Plant Susceptibility to Aphids by Substantial Activation of Ethylene Biosynthesis.

Induction of ethylene biosynthesis by aphids increases the susceptibility of several plant species to aphids. Recent studies have indicated that some MYB transcription factors regulate the phloem-based defense against aphid infestation by modulating ethylene (ET) signaling. Arabidopsis MYB102 has previously been shown to be induced by wound signaling and regulate defense response against chewing insects. However, it remains unclear whether Arabidopsis MYB102 takes part in the defense response of plants to aphids. Here, we investigated the function of MYB102 in the response of Arabidopsis to aphid infestation. Arabidopsis MYB102 was primarily expressed in vascular tissues, and its transcription was remarkably induced by green peach aphids (GPA; Myzus persicae). The results of RNA-Sequencing revealed that overexpression of MYB102 in Arabidopsis promoted ET biosynthesis by upregulation of some 1-aminocyclopropane-1-carboxylate synthase (ACS) genes, which are rate-limiting enzymes of the ET-synthetic pathway. Enhanced ET levels led to reduced Arabidopsis resistance to GPA. Furthermore, dominant suppression of MYB102 inhibited aphid-induced increase of ET levels in Arabidopsis. In agreement with a negative regulatory role for ET in aphid defense responses, the MYB102-overexpressing lines were more susceptible to GPA than wild-type (WT) plants. Overexpression of MYB102 in Arabidopsis obviously repressed aphid-induced callose deposition. Conversely, overexpression of MYB102 failed to increase aphid susceptibility in both the ET-insensitive mutants and plants treated with inhibitors of ET signaling pathways, demonstrating that the ET was critical for promoting aphid performance conferred by overexpression of MYB102. Collectively, our findings indicate that the Arabidopsis MYB102 increases host susceptibility to GPA through the ET-dependent signaling pathways.

Brassinosteroids regulate root growth by controlling reactive oxygen species homeostasis and dual effect on ethylene synthesis in Arabidopsis.

The brassinosteroids (BRs) represent a class of phytohormones, which regulate numerous aspects of growth and development. Here, a det2-9 mutant defective in BR synthesis was identified from an EMS mutant screening for defects in root length, and was used to investigate the role of BR in root development in Arabidopsis. The det2-9 mutant displays a short-root phenotype, which is result from the reduced cell number in root meristem and decreased cell size in root maturation zone. Ethylene synthesis is highly increased in the det2-9 mutant compared with the wild type, resulting in the hyper-accumulation of ethylene and the consequent inhibition of root growth. The short-root phenotype of det2-9 was partially recovered in the det2-9/acs9 double mutant and det2-9/ein3/eil1-1 triple mutant which have defects either in ethylene synthesis or ethylene signaling, respectively. Exogenous application of BR showed that BRs either positively or negatively regulate ethylene biosynthesis in a concentration-dependent manner. Different from the BR induced ethylene biosynthesis through stabilizing ACSs stability, we found that the BR signaling transcription factors BES1 and BZR1 directly interacted with the promoters of ACS7, ACS9 and ACS11 to repress their expression, indicating a native regulation mechanism under physiological levels of BR. In addition, the det2-9 mutant displayed over accumulated superoxide anions (O2-) compared with the wild-type control, and the increased O2- level was shown to contribute to the inhibition of root growth. The BR-modulated control over the accumulation of O2- acted via the peroxidase pathway rather than via the NADPH oxidase pathway. This study reveals an important mechanism by which the hormone cross-regulation between BRs and ethylene or/and ROS is involved in controlling root growth and development in Arabidopsis.

Molecular regulation of aluminum resistance and sulfur nutrition during root growth

Aluminum toxicity and sulfate deprivation both regulate microRNA395 expression, repressing its low-affinity sulfate transporter ( SULTR2;1 ) target. Sulfate deprivation also induces the high-affinity sulfate transporter gene ( SULTR12 ), allowing enhanced sulfate uptake. Few studies about the relationships between sulfate, a plant nutrient, and aluminum, a toxic ion, are available; hence, the molecular and physiological processes underpinning this interaction are poorly understood. The Al-sulfate interaction occurs in acidic soils, whereby relatively high concentrations of trivalent toxic aluminum (Al3+) may hamper root growth, limiting uptake of nutrients, including sulfur (S). On the other side, Al3+ may be detoxified by complexation with sulfate in the acid soil solution as well as in the root-cell vacuoles. In this review, we focus on recent insights into the mechanisms governing plant responses to Al toxicity and its relationship with sulfur nutrition, emphasizing the role of phytohormones, microRNAs, and ion transporters in higher plants. It is known that Al3+ disturbs gene expression and enzymes involved in biosynthesis of S-containing cysteine in root cells. On the other hand, Al3+ may induce ethylene biosynthesis, enhance reactive oxygen species production, alter phytohormone transport, trigger root growth inhibition and promote sulfate uptake under S deficiency. MicroRNA395, regulated by both Al toxicity and sulfate deprivation, represses its low-affinity Sulfate Transporter 2;1 (SULTR2;1) target. In addition, sulfate deprivation induces High Affinity Sulfate Transporters (HAST; SULTR1;2), improving sulfate uptake from low-sulfate soil solutions. Identification of new microRNAs and cloning of their target genes are necessary for a better understanding of the role of molecular regulation of plant resistance to Al stress and sulfate deprivation.

Assay Methods for ACS Activity and ACS Phosphorylation by MAP Kinases In Vitro and In Vivo.

Ethylene, a gaseous phytohormone, has profound effects on plant growth, development, and adaptation to the environment. Ethylene-regulated processes begin with the induction of ethylene biosynthesis. There are two key steps in ethylene biosynthesis. The first is the biosynthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) from S-Adenosyl-Methionine (SAM), a common precursor in many metabolic pathways, which is catalyzed by ACC synthase (ACS). The second is the oxidative cleavage of ACC to form ethylene under the action of ACC oxidase (ACO). ACC biosynthesis is the committing and generally the rate-limiting step in ethylene biosynthesis. As a result, characterizing the cellular ACS activity and understanding its regulation are important. In this chapter, we detail the methods used to measure, (1) the enzymatic activity of both recombinant and native ACS proteins, and (2) the phosphorylation of ACS protein by mitogen-activated protein kinases (MAPKs) in vivo and in vitro.