Ethylene Biosynthesis Enzymes Research Articles

Alleviating dormancy in Brassica oleracea seeds using NO and KAR1 with ethylene biosynthetic pathway, ROS and antioxidant enzymes modifications

BackgroundSeed dormancy is a prevailing condition in which seeds are unable to germinate, even under favorable environmental conditions. Harvested Brassica oleracea (Chinese cabbage) seeds are dormant and normally germinate (poorly) at 21 °C. This study investigated the connections between ethylene, nitric oxide (NO), and karrikin 1 (KAR1) in the dormancy release of secondary dormant Brassica oleracea seeds.ResultsNO and KAR1 were found to induce seed germination, and stimulated the production of ethylene and 1-aminocyclopropane-1-carboxylic acid (ACC), and both ethylene biosynthesis enzyme ACC oxidase (ACO) [1] and ACC synthase (ACS) [2]. In the presence of NO and KAR1, ACS and ACO activity reached maximum levels after 36 and 48 h, respectively. The inhibitor of ethylene 2,5-norbornadiene (NBD) had an adverse effect on Brassica oleracea seed germination (inhibiting nearly 50% of germination) in the presence of NO and KAR1. The benefits from NO and KAR1 in the germination of secondary dormant Brassica oleracea seeds were also associated with a marked increase in reactive oxygen species (ROS) (H2O2 and O2˙ˉ) and antioxidant enzyme activity at early germination stages. Catalase (CAT) and glutathione reductase (GR) activity increased 2 d and 4 d, respectively, after treatment, while no significant changes were observed in superoxide dismutase (SOD) activity under NO and KAR1 applications. An increase in H2O2 and O2˙ˉ levels were observed during the entire incubation period, which increasing ethylene production in the presence of NO and KAR1. Abscisic acid (ABA) contents decreased and glutathione reductase (GA) contents increased in the presence of NO and KAR1. Gene expression studies were carried out with seven ethylene biosynthesis ACC synthases (ACS) genes, two ethylene receptors (ETR) genes and one ACO gene. Our results provide more evidence for the involvement of ethylene in inducing seed germination in the presence of NO and KAR1. Three out of seven ethylene biosynthesis genes (BOACS7, BOACS9 and BOACS11), two ethylene receptors (BOETR1 and BOETR2) and one ACO gene (BOACO1) were up-regulated in the presence of NO and KAR1.ConclusionConsequently, ACS activity, ACO activity and the expression of different ethylene related genes increased, modified the ROS level, antioxidant enzyme activity, and ethylene biosynthesis pathway and successfully removed (nearly 98%) of the seed dormancy of secondary dormant Brassica olereace seeds after 7 days of NO and KAR1 application.

CRISPR/Cas9-mediated editing of 1-aminocyclopropane-1-carboxylate oxidase1 enhances Petunia flower longevity.

SummaryThe genes that encode the ethylene biosynthesis enzyme 1‐aminocyclopropane‐1‐carboxylate oxidase (ACO) are thought to be involved in flower senescence. Hence, we investigated whether the transcript levels of PhACO genes (PhACO1, PhACO3 and PhACO4) in Petunia cv. Mirage Rose are associated with ethylene production at different flowering stages. High transcript levels were detected in the late flowering stage and linked to high ethylene levels. PhACO1 was subsequently edited using the CRISPR/Cas9 system, and its role in ethylene production was investigated. PhACO1‐edited T0 mutant lines, regardless of mutant type (homozygous or monoallelic), exhibited significantly reduced ethylene production and enhanced flower longevity compared with wild‐type. Flower longevity and the reduction in ethylene production were observed to be stronger in homozygous plants than in their monoallelic counterparts. Additionally, the transmission of the edited gene to the T1 (lines 6 and 36) generation was also confirmed, with the results for flower longevity and ethylene production proving to be identical to those of the T0 mutant lines. Overall, this study increases the understanding of the role of PhACO1 in petunia flower longevity and also points to the CRISPR/Cas9 system being a powerful tool in the improvement of floricultural quality.

Light-induced stabilization of ACS contributes to hypocotyl elongation during the dark-to-light transition inArabidopsis seedlings.

Hypocotyl growth during seedling emergence is a crucial developmental transition influenced by light and phytohormones such as ethylene. Ethylene and light antagonistically control hypocotyl growth in either continuous light or darkness. However, how ethylene and light regulate hypocotyl growth, including seedling emergence, during the dark-to-light transition remains elusive. Here, we show that ethylene and light cooperatively stimulate a transient increase in hypocotyl growth during the dark-to-light transition via the light-mediated stabilization of 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACSs), the rate-limiting enzymes in ethylene biosynthesis. We found that, in contrast to the known inhibitory role of light in hypocotyl growth, light treatment transiently increases hypocotyl growth in wild-type etiolated seedlings. Moreover, ACC, the direct precursor of ethylene, accentuates the effects of light on hypocotyl elongation during the dark-to-light transition. We determined that light leads to the transient elongation of hypocotyls by stabilizing the ACS5 protein during the dark-to-light transition. Furthermore, biochemical analysis of an ACS5 mutant protein bearing an alteration in the C-terminus indicated that light stabilizes ACS5 by inhibiting the degradation mechanism that acts through the C-terminus of ACS5. Our study reveals that plants regulate hypocotyl elongation during seedling establishment by coordinating light-induced ethylene biosynthesis at the post-translational level. Moreover, the stimulatory role of light on hypocotyl growth during the dark-to-light transition provides additional insights into the known inhibitory role of light in hypocotyl development.

Genetic Regulation of Ethylene Dosage for Cucumber Fruit Elongation.

Plant organ growth and development are determined by a subtle balance between growth stimulation and inhibition. Fruit size and shape are important quality traits influencing yield and market value; however, the underlying mechanism regulating the balance of fruit growth to achieve final size and shape is not well understood. Here, we report a mechanistic model that governs cucumber (Cucumis sativus) fruit elongation through fine-tuning of ethylene homeostasis. We identified a cucumber mutant that bears short fruits owing to repressed cell division. SF1 (Short Fruit 1) encodes a cucurbit-specific RING-type E3 ligase, and the mutation resulted in its enhanced self-ubiquitination and degradation, but accumulation of ACS2 (1-aminocyclopropane-1-carboxylate synthase 2), a rate-limiting enzyme for ethylene biosynthesis. The overproduction of ethylene contributes to the short-fruit phenotype of sf1 Dysfunction of ACS2 resulted in reduced ethylene production, but still repressed cell division and shorter fruit, suggesting that ethylene is still required for basal fruit elongation. SF1 ubiquitinates and degrades both itself and ACS2 to control ethylene synthesis for dose-dependent effect on cell division and fruit elongation. Our findings reveal the mechanism by which ethylene dosage is regulated for the control of cell division in developing fruit.

Re-localization of hormone effectors is associated with dormancy alleviation by temperature and after-ripening in sunflower seeds

Temperature is the primary factor that affects seed dormancy and germination. However, the molecular mechanism that underlies its effect on dormancy alleviation remained largely unknown. In this study, we investigate hormone involvement in temperature induced germination as compared to that caused by after-ripening. Dormant (D) sunflower seeds cannot germinate at 10 °C but fully germinate at 20 °C. After-ripened seeds become non-dormant (ND), i.e. able to germinate at 10 °C. Pharmacological experiments showed the importance of abscisic acid (ABA), gibberellins (GAs) and ethylene in temperature- and after-ripening-induced germination of sunflower seeds. Hormone quantification showed that after-ripening is mediated by a decline in both ABA content and sensitivity while ABA content is increased in D seeds treated at 10 or 20 °C, suggesting that ABA decrease is not a prerequisite for temperature induced dormancy alleviation. GAs and ethylene contents were in accordance with germination potential of the three conditions (GA1 was higher in D 20 °C and ND 10 °C than in D 10 °C). Transcripts analysis showed that the major change concerns ABA and GAs metabolism genes, while ABA signalling gene expression was significantly unchanged. Moreover, another level of hormonal regulation at the subcellular localization has been revealed by immunocytolocalization study. Indeed, ABA, protein Abscisic acid-Insensitive 5 (ABI5), involved in ABA-regulated gene expression and DELLA protein RGL2, a repressor of the gibberellins signalling pathway, localized mainly in the nucleus in non-germinating seeds while they localized in the cytosol in germinating seeds. Furthermore, ACC-oxidase (ACO) protein, the key ethylene biosynthesis enzyme, was detected in the meristem only in germinating seeds. Our results reveal the importance of hormone actors trafficking in the cell and their regulation in specialized tissue such as the meristem in dormancy alleviation and germination.

Preharvest Fruit Drop in Huanglongbing-affected ‘Valencia’ Sweet Orange

For field-grown ‘Valencia’ sweet orange (Citrus sinensis) affected by Huanglongbing [HLB (Candidatus Liberibacter asiaticus (CLas)], trees that displayed more severe HLB symptoms (severe trees) had 74% fruit drop before harvest; however, the drop rate for less symptomatic trees (mild trees) was 45%. For mature fruit (3 weeks before harvest) still attached to the branches, 60% of them from severe trees were “loose fruit” [fruit detachment force (FT) < 6 kgf]. In contrast, only 13% of the attached fruit from the mild trees were loose. Overall, fresh weight and size of loose fruit were lower than “tight fruit” (FT > 6 kgf). Irrespective of the symptom levels of trees, the concentrations of glucose, fructose, and inositol in juice of loose fruit were the same or larger than those of tight fruit, suggesting that the shortage of carbohydrates is not the dominant cause of HLB-associated preharvest fruit drop. Expression levels of the cell wall modification genes encoding cellulase (endo-1,4-β-glucanase), polygalacturonase, and pectate lyase were greater in the calyx abscission zones of loose fruit compared to tight fruit, indicating that cell separation was occurring in the former at the time of collection. No differences in the expression levels of genes encoding the ethylene biosynthesis enzymes, including 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACC oxidase (ACO), and an ethylene-responsive transcription factor 1 (ERF1) were observed in tissues of loose and tight fruit. Interestingly, ACS, ACO, and EFR1 expressions were lower in calyx abscission zones and in leaves of the severe trees compared with those of mild trees, suggesting an ostensible, HLB-dependent reduction in ethylene biosynthesis and/or signaling close to harvest time. However, the role of ethylene in HLB-associated preharvest fruit drop remains to be determined. The results leave open the possibility of early ethylene production and action before the initiation of fruit abscission.

Editing of the OsACS locus alters phosphate deficiency-induced adaptive responses in rice seedlings.

Phosphate (Pi) deficiency severely influences the growth and reproduction of plants. To cope with Pi deficiency, plants initiate morphological and biochemical adaptive responses upon sensing low Pi in the soil, and the plant hormone ethylene plays a crucial role during this process. However, how regulation of ethylene biosynthesis influences the Pi-induced adaptive responses remains unclear. Here, we determine the roles of rice 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS), the rate-limiting enzymes in ethylene biosynthesis, in response to Pi deficiency. Through analysis of tissue-specific expression of OsACS in response to Pi deficiency and OsACS mutants generated by CRISPR/Cas9 [clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9] genome editing, we found that two members of the OsACS family, i.e. OsACS1 and OsACS2, are involved but differed in their importance in controlling the remodeling of root system architecture, transcriptional regulation of Pi starvation-induced genes, and cellular phosphorus homeostasis. Interestingly, in contrast to the known inhibitory role of ethylene on root elongation, both OsACS mutants, especially OsACS1, almost fail to promote lateral root growth in response to Pi deficiency, demonstrating a stimulatory role for ethylene in lateral root development under Pi-deficient conditions. Together, this study provides new insights into the roles of ethylene in Pi deficiency response in rice seedlings and the isoform-specific function of OsACS genes in this process.

The Phytophthora sojae RXLR effector Avh238 destabilizes soybean Type2 GmACSs to suppress ethylene biosynthesis and promote infection.

Phytophthora pathogens secrete many effector proteins to manipulate host innate immunity. PsAvh238 is a Phytophthora sojae N-terminal Arg-X-Leu-Arg (RXLR) effector, which evolved to escape host recognition by mutating one nucleotide while retaining plant immunity-suppressing activity to enhance infection. However, the molecular basis of the PsAvh238 virulence function remains largely enigmatic. By using coimmunoprecipitation and liquid chromatography-tandem mass spectrometry analysis, we identified the 1-aminocyclopropane-1-carboxylate synthase (ACS) isoforms, the key enzymes in ethylene (ET) biosynthesis, as a host target of PsAvh238. We show that PsAvh238 interacts with soybean ACSs (GmACSs) invivo and invitro. By destabilizing Type2 GmACSs, PsAvh238 suppresses Type2 ACS-catalyzed ET biosynthesis and facilitates Phytophthora infection. Silencing of Type2 GmACSs, and inhibition of ET biosynthesis or signaling, increase soybean susceptibility to P.sojae infection, supporting a role for Type2 GmACSs and ET in plant immunity against P.sojae. Moreover, wild-type P.sojae but not the PsAvh238-disrupted mutants, inhibits ET induction and promotes P.sojae infection in soybean. Our results highlight the ET biosynthesis pathway as an essential part in plant immunity against P.sojae and a direct effector target.

MaMADS2 repression in banana fruits modifies hormone synthesis and signalling pathways prior to climacteric stage

BackgroundWhile the role of ethylene in fruit ripening has been widely studied, the contributions of additional plant hormones are less clear. Here we examined the interactions between the transcription factor MaMADS2-box which plays a major role in banana fruit ripening and hormonal regulation. Specifically, we used MaMADS2 repressed lines in transcriptome and hormonal analyses throughout ripening and assessed hormone and gene expression perturbations as compared to wild-type (WT) control fruit.ResultsOur analyses revealed major differences in hormones levels and in expression of hormone synthesis and signaling genes mediated by MaMADS2 especially in preclimacteric pulp. Genes encoding ethylene biosynthesis enzymes had lower expression in the pulp of the repressed lines, consistent with reduced ethylene production. Generally, the expression of other hormone (auxin, gibberellins, abscisic acid, jasmonic acid and salicylic acid) response pathway genes were down regulated in the WT pulp prior to ripening, but remained high in MaMADS2 repressed lines. Hormone levels of abscisic acid were also higher, however, active gibberellin levels were lower and auxin levels were similar with MaMADS2 repression as compared to WT. Although abscisic level was higher in MaMADS2 repression, exogenous abscisic acid shortened the time to ethylene production and increased MaMADS2 mRNA accumulation in WT. Exogenous ethylene did not influence abscisic acid level. CRE - a cytokinin receptor, increased its expression during maturation in WT and was lower especially at prebreaker in the repressed line and zeatin level was lower at mature green of the repressed line in comparison to WT.ConclusionsIn addition to previously reported effects of MaMADS2 on ethylene, this transcription factor also influences other plant hormones, particularly at the pre-climacteric stage. The cytokinin pathway may play a previously unanticipated role via MaMADS2 in banana ripening. Finally, abscisic acid enhances MaMADS2 expression to promote ripening, but the transcription factor in turn auto inhibits ABA synthesis and signaling. Together, these results demonstrate a complex interaction of plant hormones and banana fruit ripening mediated by MaMADS2.

Involvement of ethylene biosynthesis and perception during germination of dormant Avena fatua L. caryopses induced by KAR1 or GA3

Main conclusionGermination of primary dormant wild oat caused by KAR1 or GA3 is associated with ACC accumulation and increased ethylene production shortly before radicle protrusion as a result of the non-transcriptional and transcriptional activation of ACS and ACO enzymes, respectively. Response to both compounds involves the modulation of ethylene sensitivity through ethylene receptor genes.Harvested Avena fatua caryopses are primary dormant and, therefore, germinated poorly at 20 °C. Karrikin 1 (KAR1), which action probably requires endogenous gibberellins (GAs), and gibberellin A3 (GA3) was found to induce dormant caryopses to germinate. The stimulatory effects were accompanied by the activation of the ethylene biosynthesis pathway and depended on undisturbed ethylene perception. KAR1 and GA3 promoted 1-aminocyclopropane-1-carboxylic acid (ACC) accumulation during coleorhizae emergence and ethylene production shortly prior to the radicle protrusion, which resulted from the enhanced activity of two ethylene biosynthesis enzymes, ACC synthase (ACS) and ACC oxidase (ACO). The inhibitor of ACS adversely affected beneficial impacts of both KAR1 and GA3 on A. fatua caryopses germination, while the inhibitor of ACO more efficiently impeded the GA3 effect. The inhibitors of ethylene action markedly lowered germination in response to KAR1 and GA3. Gene expression studies preceded by the identification of several genes related to ethylene biosynthesis (AfACS6, AfACO1, and AfACO5) and perception (AfERS1b, AfERS1c, AfERS2, AfETR2, AfETR3, and AfETR4) provided further evidence for the engagement of ethylene in KAR1 and GA3 induced germination of A. fatua caryopses. Both AfACO1 and AfACO5 were upregulated, whereas AfACS6 remained unaffected by the treatment. This suggests the existence of different regulatory mechanisms of enzymatic activity, transcriptional for ACO and non-transcriptional for ACS. During imbibition in water, AfERS1b was stronger expressed than other receptor genes. In the presence of KAR1 or GA3, the expression of AfETR3 was substantially induced. Differential expression of ethylene receptor genes implies the modulation of caryopses sensitivity adjusted to ethylene availability and suggests the functional diversification of individual receptors.

Regulation of the protein and gene expressions of ethylene biosynthesis enzymes under different temperature during peach fruit ripening

Ethylene has profound effect on fruit development and ripening, and the role of ethylene biosynthesis enzymes involving 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS), ACC oxidase (ACO), and S-Adenosyl-l-methionine synthetase (SAMS) in peach fruit (cv. Xiahui-8) was characterized under 25 and 4 °C, respectively. All these enzymes in ethylene synthesis pathway were identified using 2-DE and real-time PCR. Both protein and gene expressions of ACO and SAMS were much higher at 25 °C than at 4 °C. Among five members of ACS family, PpaACS4 may belong to system II ethylene biosynthesis, while PpaACS3 involved in system I during development stage, and low temperature can induce PpaACS1 expression. The ethylene release and low expressions of proteins and genes of most enzymes indicated that low temperature can effectively postpone ripening stage by reducing ethylene evolution. High gene expression of PpaSAMS did not cause excessive expression of SAMS protein under low temperature, and over-expression of PpaACS1 at low temperature still did not induce increase of ethylene production. The mechanism underlying the phenomenon about how temperature affects ethylene release was also discussed.

Molecular aspects of flower senescence and strategies to improve flower longevity.

Flower longevity is one of the most important traits for ornamental plants. Ethylene plays a crucial role in flower senescence in some plant species. In several species that show ethylene-dependent flower senescence, genetic modification targeting genes for ethylene biosynthesis or signaling has improved flower longevity. Although little is known about regulatory mechanisms of petal senescence in flowers that show ethylene-independent senescence, a recent study of Japanese morning glory revealed that a NAC transcription factor, EPHEMERAL1 (EPH1), is a key regulator in ethylene-independent petal senescence. EPH1 is induced in an age-dependent manner irrespective of ethylene signal, and suppression of EPH1 expression dramatically delays petal senescence. In ethylene-dependent petal senescence, comprehensive transcriptome analyses revealed the involvement of transcription factors, a basic helix-loop-helix protein and a homeodomain-leucine zipper protein, in the transcriptional regulation of the ethylene biosynthesis enzymes. This review summarizes molecular aspects of flower senescence and discusses strategies to improve flower longevity by molecular breeding.

A comparison of the molecular mechanisms underpinning high-intensity, pulsed polychromatic light and low-intensity UV-C hormesis in tomato fruit

Postharvest treatment of tomato fruit with high-intensity, pulsed polychromatic light (HIPPL) has previously been shown to induce delayed ripening and disease resistance comparable to that of low-intensity UV-C (LIUV). Little, however, is known of the mechanisms underpinning postharvest HIPPL hormesis in tomato fruit. Expression of genes involved in plant hormone biosynthesis, defence, secondary metabolism and ripening were monitored 24h post treatment (24 HPT), 10 d post treatment (10 DPT) and 12h post inoculation with Botrytis cinerea (12 HPI). All genes monitored were constitutively expressed and changes in expression profiles following treatment were highly similar for both HIPPL and LIUV treatments. Expression of pathogenesis-related proteins P4, β-1,3,-Glucanase and Chitinase 9 and a jasmonate biosynthesis enzyme (OPR3), were significantly upregulated at 10 DPT and 12 HPI. Both treatments significantly downregulated the expression of polygalacturonase and flavonol synthase at 10 DPT and 12 HPI. Ethylene biosynthesis enzyme ACO1 and β-carotene hydroxylase were significantly upregulated at 24 HPT, and phenylalanine ammonia-lyase (PAL) was significantly upregulated at 12 HPI. Both HIPPL and LIUV treatments stimulate defence responses that are mediated by salicylic acid, jasmonic acid and ethylene. This may lead to broad range resistance against both necrotrophic and biotrophic pathogens as well as abiotic stresses and herbivorous pests. Following inoculation with B. cinerea only PAL showed indication of a gene priming response for HIPPL- and LIUV-treated fruit.

Ethylene Mediates Alkaline-Induced Rice Growth Inhibition by Negatively Regulating Plasma Membrane H+-ATPase Activity in Roots.

pH is an important factor regulating plant growth. Here, we found that rice was better adapted to low pH than alkaline conditions, as its growth was severely inhibited at high pH, with shorter root length and an extreme biomass reduction. Under alkaline stress, the expression of genes for ethylene biosynthesis enzymes in rice roots was strongly induced by high pH and exogenous ethylene precursor ACC and ethylene overproduction in etol1-1 mutant aggravated the alkaline stress-mediated inhibition of rice growth, especially for the root elongation with decreased cell length in root apical regions. Conversely, the ethylene perception antagonist silver (Ag+) and ein2-1 mutants could partly alleviate the alkaline-induced root elongation inhibition. The H+-ATPase activity was extremely inhibited by alkaline stress and exogenous ACC. However, the H+-ATPase-mediated rhizosphere acidification was enhanced by exogenous Ag+, while H+ efflux on the root surface was extremely inhibited by exogenous ACC, suggesting that ethylene negatively regulated H+-ATPase activity under high-pH stress. Our results demonstrate that H+-ATPase is involved in ethylene-mediated inhibition of rice growth under alkaline stress.

Persistence of plant hormone levels in rice shoots grown under microgravity conditions in space: its relationship to maintenance of shoot growth.

We investigated the effects of microgravity environment on growth and plant hormone levels in dark-grown rice shoots cultivated in artificial 1 g and microgravity conditions on the International Space Station (ISS). Growth of microgravity-grown shoots was comparable to that of 1 g-grown shoots. Endogenous levels of indole-3-acetic acid (IAA) in shoots remained constant, while those of abscisic acid (ABA), jasmonic acid (JA), cytokinins (CKs) and gibberellins (GAs) decreased during the cultivation period under both conditions. The levels of auxin, ABA, JA, CKs and GAs in rice shoots grown under microgravity conditions were comparable to those under 1 g conditions. These results suggest microgravity environment in space had minimal impact on levels of these plant hormones in rice shoots, which may be the cause of the persistence of normal growth of shoots under microgravity conditions. Concerning ethylene, the expression level of a gene for 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, the key enzyme in ethylene biosynthesis, was reduced under microgravity conditions, suggesting that microgravity may affect the ethylene production. Therefore, ethylene production may be responsive to alterations of the gravitational force.

1-Aminocyclopropane-1-Carboxylate Oxidase Induction in Tomato Flower Pedicel Phloem and Abscission Related Processes Are Differentially Sensitive to Ethylene.

Ethylene has impact on several physiological plant processes, including abscission, during which plants shed both their vegetative and reproductive organs. Cell separation and programmed cell death are involved in abscission, and these have also been correlated with ethylene action. However, the detailed spatiotemporal pattern of the molecular events during abscission remains unknown. We examined the expression of two tomato ACO genes, LeACO1, and LeACO4 that encode the last enzyme in ethylene biosynthesis, 1-aminocyclopropane-1-carboxylate oxidase (ACO), together with the expression of other abscission-associated genes involved in cell separation and programmed cell death, during a period of 0–12 h after abscission induction in the tomato flower pedicel abscission zone and nearby tissues. In addition, we determined their localization in specific cell layers of the flower pedicel abscission zone and nearby tissues obtained by laser microdissection before and 8 h after abscission induction. The expression of both ACO genes was localized to the vascular tissues in the pedicel. While LeACO4 was more uniformly expressed in all examined cell layers, the main expression site of LeACO1 was in cell layers just outside the abscission zone in its proximal and distal part. We showed that after abscission induction, ACO1 protein was synthesized in phloem companion cells, in which it was localized mainly in the cytoplasm. Samples were additionally treated with 1-methylcyclopropene (1-MCP), a competitive inhibitor of ethylene actions, and analyzed 8 h after abscission induction. Cell-layer-specific changes in gene expression were observed together with the specific localization and ethylene sensitivity of the hallmarks of cell separation and programmed cell death. While treatment with 1-MCP prevented separation of cells through inhibition of the expression of polygalacturonases, which are the key enzymes involved in degradation of the middle lamella, this had less impact on the occurrence of different kinds of membrane vesicles and abscission-related programmed cell death. In the flower pedicel abscission zone, the physical progressions of cell separation and programmed cell death are perpendicular to each other and start in the vascular tissues.

Construction of an RNAi vector for knockdown of GM-ACS genes in the cotyledonary nodes of soybean

800x600 Background : Ethylene plays an important role in the regulation of floral organ development in soybean, and ACC synthase is a rate-limiting enzyme for ethylene biosynthesis. However, whether ACC synthase also regulates floral organ differentiation in soybean remains unknown. To address this, we constructed an RNAi vector to inhibit ACC synthase expression in cotyledonary nodes. Linear DNA cassettes of RNAi-ACS obtained by PCR were used to transform soybean cotyledonary nodes. Results : One hundred thirty-one of 139 transiently transformed plants acquired herbicide resistance and displayed GUS activities in the new buds. In comparison to untransformed seedling controls, a greater number of flower buds were differentiated at the cotyledonary node; GM-ACS1 mRNA expression levels and ethylene emission in the transformed buds were reduced. Conclusion : These results indicate that the cotyledonary node transient transformation system may be suitable for stable transformation, and that inhibition of ACC synthase expression may be an effective strategy for promoting floral organ differentiation in soybean.

Expression and functional analysis of PhEOL1 and PhEOL2 during flower senescence in petunia.

The ethylene biosynthesis pathway controls flower senescence. Previous studies have shown that Arabidopsis ETHYLENE-OVERPRODUCER1 (ETO1) interacts specifically with and negatively regulates type 2 1-aminocyclopropane-1-carboxylate synthases (ACSs), the rate-limiting enzymes of ethylene biosynthesis. The ethylene biosynthesis pathway controls flower senescence in petunias (Petunia hybrida Juss.). However, the role of ETO1-like genes (EOLs) during flower senescence has not been investigated. Here, two full-length petunia EOL cDNAs, PhEOL1 and PhEOL2, were isolated. RT-PCR assays indicated that the expression of PhEOL1 and PhEOL2 increased after exogenous ethylene treatment. The VIGS-mediated silencing of PhEOL1 accelerated flower senescence and produced more ethylene than the control condition, whereas the silencing of PhEOL2 did not. Notably, the effects caused by PhEOL1 suppression were not enhanced by PhEOL2 suppression in corollas. In addition, the expression of two petunia type 2 PhACS genes increased during flower senescence and after ethylene treatment. A yeast two-hybrid assay showed that PhEOL1 interacts with both PhACS2 and PhACS3. It is possible that PhEOL1 is involved in flower senescence by interacting with type 2 PhACSs in petunias.

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.

Isolation and molecular characterization of 1-aminocyclopropane-1-carboxylic acid synthase genes in Hevea brasiliensis.

Ethylene is an important factor that stimulates Hevea brasiliensis to produce natural rubber. 1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is a rate-limiting enzyme in ethylene biosynthesis. However, knowledge of the ACS gene family of H. brasiliensis is limited. In this study, nine ACS-like genes were identified in H. brasiliensis. Sequence and phylogenetic analysis results confirmed that seven isozymes (HbACS1–7) of these nine ACS-like genes were similar to ACS isozymes with ACS activity in other plants. Expression analysis results showed that seven ACS genes were differentially expressed in roots, barks, flowers, and leaves of H. brasiliensis. However, no or low ACS gene expression was detected in the latex of H. brasiliensis. Moreover, seven genes were differentially up-regulated by ethylene treatment.These results provided relevant information to help determine the functions of the ACS gene in H. brasiliensis, particularly the functions in regulating ethylene stimulation of latex production.