Ethylene Biosynthesis-related Genes Research Articles

Hydrogen sulfide inhibits tomato fruit ripening by restraining fruit softening and ethylene production

Hydrogen sulfide (H2S) plays various roles in plant growth, development and stress response. However, the mechanism of action of H2S in fruit ripening remains unclear. This study investigated the roles and mechanisms of H2S in tomato (Solanum lycopersicum) fruit repining. The results showed that compared with the control (distilled water), H2S treatment significantly delayed the color and hardness transition of tomato fruit; while an H2S scavenger hypotaurine (HT) treatment promoted the color and hardness transition. H2S increased the endogenous chlorophyll content, activity of chlorophyll synthesis-related enzymes, and expression of chlorophyll synthesis-related genes and decreased the expression of chlorophyll degradation-related genes and lycopene content. In contrast, HT decreased the activity of chlorophyll synthesis-related enzymes and expression of related genes; and increased the expression of chlorophyll degradation-related genes. In addition, the activity of the softening-related enzymes, ACS and ACO, ethylene content, and the expression of softening- and ethylene biosynthesis-related genes were decreased by H2S but increased by HT. Exogenous H2S increases endogenous H2S levels. Therefore, H2S may delay tomato fruit ripening by down-regulating the expression of softening-related and ethylene synthesis-related genes and reducing the activity of softening-related and ethylene synthesis-related enzymes.

Unravelling the role of adventitious roots under priming-induced tolerance to waterlogging stress in wheat

Waterlogging is a significant environmental stressor that can have detrimental effects on crop yields. Waterlogging priming has emerged as a promising technique for enhancing plant waterlogging tolerance. However, the underlying mechanisms behind the regulation of root resistance, particularly adventitious roots (ARs), in wheat under waterlogging stress remain unclear. In this study, we subjected plants to waterlogging priming for two days at the two-leaf stage, followed by seven days of waterlogging stress after a ten-day recovery period to investigate the morphological and anatomical structure and physiological functions of ARs in wheat. Our results demonstrated that primed plants under waterlogging stress (PW) had increased numbers of ARs, improved gas exchange, and enhanced aerenchyma formation in ARs, leading to normal plant respiration when compared to non-primed plants under waterlogging stress treatment (CW). PW up-regulated the expression of cell wall remodeling genes (CEL, XET), reactive oxygen species (ROS) production-related gene (RBOH1), and ethylene biosynthesis-related genes (ACS2, ACS4) in ARs, and increased anaerobic respiratory enzyme activity, thus preserving the supply of soluble sugars in ARs and providing more materials and energy for adventitious root regeneration compared to CW. Overall, waterlogging priming improved the waterlogging tolerance of both above-ground and ARs in wheat, reducing the severity of damage caused by waterlogging. Our findings provide new insights into the mechanisms underlying waterlogging priming and suggest that it may be a promising strategy for mitigating the adverse effects of waterlogging on crop productivity.

1-MCP application in ‘Xuxiang’ kiwifruit at appropriate firmness avoids hard-core and prolongs the shelf life

The lack of synchrony in softening among the tissue zones and the increase in the production of autocatalytic ethylene lead to the hard-core and a short eating window in ready-to-eat kiwifruit. The present study analyzed the differences between the outer pericarp and the core during ripening to identify the cause of hard-core in ‘Xuxiang’ kiwifruit and aimed to maintain a soft texture and extend the eating window of ready-to-eat kiwifruit using 1-methylcyclopropene (1-MCP). Initial analysis showed that the outer pericarp had a few small spherical cells and many elongated cells, while the core had large homogeneous parenchyma cells. The core maintained higher firmness and cell wall polysaccharide content than the outer pericarp. During ripening, the pericarp followed a sigmoidal trend in softening, while the core followed a linear trend. We also found that the outer pericarp preceded the core in expressing the cell wall degradation-related and ethylene biosynthesis-related genes (AcXET1, AcEXP1, AcPG, AcPL2, AcACS1, and AcACO2). Moreover, the increase in ethylene production was highly correlated with the decrease in core firmness. These differences between the outer pericarp and the core probably led to the hard-core in kiwifruit. Subsequent texture analysis and sensory evaluation revealed 75–100 N as the acceptable core firmness limit for ‘Xuxiang’ kiwifruit. The fruit treatment experiments based on these observations showed that applying 1-MCP on ready-to-eat ‘Xuxiang’ kiwifruit at ∼12 N firmness resulted in best preservation, with an extra 15 d of shelf life and an acceptable core firmness. In conclusion, these findings suggest that treating ‘Xuxiang’ kiwifruit at ∼12 N firmness after harvest with 1-MCP will help avoid hard-core and prolong the eating window, improving consumer acceptance.

Physiological changes besides the enhancement of pigmentation in Petunia hybrida caused by overexpression of PhAN2, an R2R3-MYB transcription factor.

Ectopic expression of PhAN2 in vegetative tissue can improve regeneration and adventitious rooting but inhibit axillary bud outgrowth of petunia, while overexpression specifically in flowers could shorten longevity. Anthocyanin 2 has been only treated as a critical positive regulation factor of anthocyanin biosynthesis in petunia flowers. To determine if this gene had other functions in plant growth, we overexpressed this gene in an an2 mutant petunia cultivar driven by promoters with different strengths or tissue specificity. Various physiological processes of transformants in different growth stages and environments were analyzed. Besides the expected pigmentation improvement in different tissues, the results also showed that ectopic expression of AN2 could improve the regeneration skill but inhibit the axillary bud germination of in vitro plants. Moreover, the rooting ability of shoot tips of transformants was significantly improved, while some transgenic lines' flower longevity was shortened. Gene expression analysis showed that the transcripts level of AN2, partner genes anthocyanin 1 (AN1), anthocyanin 11 (AN11), and target gene dihydroflavonol 4-reductase (DFR) was altered in the different transgenic lines. In addition, ethylene biosynthesis-related genes 1-aminocyclopropane-1-carboxylic acid synthase (ACS1) and ACC oxidase (ACO1) were upregulated in rooting and flower senescence processes but at different time points. Overall, our data demonstrate that the critical role of this AN2 gene in plant growth physiology may extend beyond that of a single activator of anthocyanin biosynthesis.

Endogenous melatonin delays sepal senescence and extends the storage life of broccoli florets by decreasing ethylene biosynthesis

Although previous work has revealed that exogenous melatonin (MT) improves the storage life and quality of broccoli, whether endogenous MT has similar effects and the mechanism of MT action remains uncertain. We discovered that during storage of broccoli, endogenous MT was impaired. Supplementation with 100 μmol L-1 MT enhanced endogenous MT production, whereas 10 μmol L-1 luzindole (LD, an MT receptor inhibitor) decreased endogenous MT production. Differences in sepal yellowing, storage life, and chlorophyll degradation were observed, such that the senescence of broccoli was delayed by MT but accelerated by LD. Further results revealed that the decreased storage life and accelerated senescence induced by LD were blocked by MT administration, and endogenous MT homeostasis was re-established. These beneficial roles of MT were verified by the alleviation of senescence-induced ethylene production and the increased activities of ethylene biosynthesis enzymes. Compared with those of LD alone, the expressions of chlorophyll degradation-related and ethylene biosynthesis-related genes were also downregulated when MT was added. Overall, the above results imply that endogenous MT homeostasis prolongs the storage life and delays the senescence of broccoli by decreasing ethylene biosynthesis.

Transcriptomics and metabolomics reveal the possible mechanism by which 1-methylcyclopropene regulates the postharvest senescence of Zizania latifolia

Abstract To understand the mechanism governing the postharvest senescence of Zizania latifolia (Z. latifolia), and the regulatory mechanism induced by 1-methylcyclopropene (1-MCP) during storage at 25 °C, physiobiochemical and conjoint analyses of the transcriptome and metabolome were performed. The results indicated that 1-MCP treatment engendered changes in the expression of genes and metabolites during the postharvest storage of Z. latifolia. The 1-MCP treatment maintained a good visual appearance, preserved the cell structure and membrane integrity of Z. latifolia by keeping the expression of membrane-related lipolytic enzymes (and related genes) low and the amount of phosphatidylethanolamine high. Compared to the control group, 1-MCP treatment enhanced the activities of antioxidant enzymes, resulting in a decrease of reactive oxygen species and malondialdehyde contents, and thus inhibition of oxidative damage and loss of membrane integrity. In addition, 1-MCP treatment retarded the senescence of Z. latifolia by downregulating the expression of ethylene biosynthesis-related genes and promoting upregulation of brassinosteroid insensitive 1 kinase inhibitor 1, calmodulin, glutathione reductase, jasmonate amino acid synthase, and mitogen-activated protein kinase-related genes. Moreover, 1-MCP retarded Z. latifolia senescence by inducing the activity of adenosine triphosphate (ATP) biosynthesis-related genes and metabolites. Our findings should facilitate future research on the postharvest storage of Z. latifolia, and could help delay senescence and prolong the storage time for commercial applications.

Integrating physiological and metabolites analysis to identify ethylene involvement in petal senescence in Tulipa gesneriana

Flower senescence is classified into ethylene-dependent and ethylene-independent manners and determines the flower longevity which is valuable for ornamental plants. However, the manner of petal senescence in tulip is still less defined. In this study, we characterized the physiological indexes in the process of petal senescence, as well as metabolic and ethylene responses in tulip cultivar ‘American Dream’, and further identified the role of ethylene biosynthesis genes TgACS by transgenic and transient assays. Primary metabolites profiling revealed that sugars, amino acids and organic acids preferentially accumulated in senescent petals. Additionally, senescence-associated genes were identified and significantly up-regulated, coupled with increased ROS contents, rapid water loss and accelerated cell membrane breakdown. Moreover, ethylene production was stimulated as evidenced by increasing in ACS activity and ethylene biosynthesis-related genes expression. Exogenous treatment of cutting flowers with 1-MCP or ethephon resulted in delayed or enhanced petal senescence, respectively. Transient down-regulation of TgACS by VIGS assay in tulip petals delayed senescence, while over-expressed TgACS1 in tobacco promoted leaf senescence. Taken together, this study provides evidences to certify ethylene roles and TgACS functions during flower senescence in tulip.

Abscisic acid and ethylene biosynthesis-related genes are associated with anthocyanin accumulation in purple ornamental cabbage (Brassica oleracea var. acephala).

Purple ornamental cabbage (Brassica oleracea var. acephala) is a popular decorative plant, cultivated for its colorful leaf rosettes that persist in cool weather. It is characterized by green outer leaves and purple inner leaves, whose purple pigmentation is due to the accumulation of anthocyanin pigments. Phytohormones play important roles in anthocyanin biosynthesis in other species. Here, we identified 14 and 19 candidate genes putatively involved in abscisic acid (ABA) and ethylene (ET) biosynthesis, respectively, in B. oleracea. We determined the expression patterns of these candidate genes by reverse-transcription quantitative PCR (RT-qPCR). Among candidate ABA biosynthesis-related genes, the expressions of BoNCED2.1, BoNCED2.2, BoNCED6, BoNCED9.1, and BoAAO3.2 were significantly higher in purple compared to green leaves. Likewise, most of the ET biosynthetic genes (BoACS6, BoACS9.1, BoACS11, BoACO1.1, BoACO1.2, BoACO3.1, BoACO4, and BoACO5) had significantly higher expression in purple compared to green leaves. Among these genes, BoNCED2.1, BoNCED2.2, BoACS11, and BoACO4 showed particularly strong associations with total anthocyanin content of the purple inner leaves. Our results suggest that ABA and ET might promote the intense purple pigmentation of the inner leaves of purple ornamental cabbage.

Ethylene and expansin biosynthesis related genes polymorphism in local apple (Malus domestica Borkh.) cultivars from VIR Collection of plant genetic resources

At Pushkin and Pavlovsk Laboratories of the N.I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR) a diverse collec tion of local apple cultivars is maintained. Some of the cultivars are widely used in breeding programs for their ecological plasticity, increased adaptation to abiotic stress and disease resistance, still there have been no large-scale studies of these local cultivars for fruit storage ability. Fruit softening during storage is an important problem for apple production. Retention of desirable firmness after prolonged storage is one of the key requirements for new apple cultivars. Expansin and ethy lene biosynthesis related genes are known to be involved in control of fruit softening in apple, and gene specific molecular markers have been reported. In this study the polymorphism and allelic configuration of ethylene and expansin biosynthesis related genes Md-ACS1, Md-ACO1 and Md-Exp7 involved in control of fruit softening in 87 local apple cultivars from VIR Collection of Plant Genetic Resources were analyzed. PCR markers Md-ACS1, Md-ACO1 and SSR-marker Md-Exp7 were used in the study. The allele frequencies in the collection generally coincided with the data from previous studies. Md-ACS1 allele 2 associated with reduced ethylene production was found only in three local cultivars, while all the studied local cultivars were heterozygous for the Md-ACO1 locus, as well as most modern Russian apple cultivars. Half of the studied local cultivars were heterozygous for Md-Exp7 (198 : 202). Thirteen local cultivars with rare Md-Exp7 alleles (206, 210 and 212) were detected. No association was found between the Md-Exp7 genotype and the cultivars’ maturation time. The obtained results can be used for additional evaluation of the cultivars’ potential for breeding.

Ethylene biosynthesis controlled by NON-RIPENING: A regulatory conflict between wounding and ripening

The phytohormone ethylene is involved in multiple aspects of morphological and physiological processes in plants. Tomato rapidly and transiently increases ethylene production during fruit ripening and in plant defense responses. The transcription factor non-ripening (NOR) has significant effects on fruit ripening via regulation of ethylene biosynthesis-related genes. The nor loss-of-function allele produces a basal level of ethylene during ripening, in contrast to the induced ethylene evolution observed upon Agrobacterium tumefaciens infection. The use of ACC deaminase represses ethylene production and significantly improves the efficiency of Agrobacterium-mediated T-DNA transfer in nor plants. Analyses of the transcription levels of the ethylene biosynthesis genes ACC synthase (ACS) and ACC oxidase (ACO) in nor plants revealed that the induced ethylene production was largely due to transcriptional accumulation of ACS2 and ACO1. Accumulation of ACS2 and ACO1 mRNA opposes NOR-mediated regulation in tomato fruit during ripening, and the feedback regulation of NOR is rendered ineffective by defense responses, thereby precluding the control of its own expression. The ethylene synthesis mechanisms respond properly to NOR-mediated transcriptional regulation that is differed through the wound-induced and ripening-induced signaling pathway.

The novel ethylene-responsive factor CsERF025 affects the development of fruit bending in cucumber.

Overexpression of CsERF025 induces fruit bending by promoting the production of ethylene. Cucumber fruit bending critically affects cucumber quality, but the mechanism that causes fruit bending remains unclear. To better understand this mechanism, we performed transcriptome analyses on tissues from the convex (C1) and concave (C2) sides of bending and straight (S) fruit at 2 days post anthesis (DPA). We identified a total of 281 differentially expressed genes (DEGs) from both the convex and concave sides of bent fruit that showed significantly different expression profiles relative to straight fruits. Of these 281 DEGs, 196 were up-regulated (C1/S_C2/S) and 85 were down-regulated (C1/S_C2/S). Among the 196 up-regulated DEGs, the transcriptional levels of genes related to ethylene biosynthesis and signaling pathways were significantly higher in bending fruit compared with straight fruit. CsERF025 showed the largest difference in expression between bending and straight fruit. CsERF025 is an AP2/ERF gene encoding a protein that localizes to the nucleus. Overexpression of this gene increased the bending rate of cucumber fruits and increased the angle of bending. CsERF025 increased both the expression of ethylene biosynthesis-related genes and the production of ethylene. The application of exogenous 1-aminocyclopropane-l-carboxylic acid (ACC) to straight fruits from control plants promoted fruit bending. Thus, CsERF025 enhances the production of ethylene and thereby promotes fruit bending in cucumber.

Sound wave affects the expression of ethylene biosynthesis-related genes through control of transcription factors RIN and HB-1

We previously reported that sound wave treatment (1 kHz) delays fruit ripening in tomato (Solanum lycopersicum), affecting the expression of ethylene biosynthesis-related genes encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACS) and ACC oxidases (ACO). In this study, we investigated the activity of the transcription factors RIN and HB-1, which function in the ethylene biosynthetic pathway, in response to sound treatment. To investigate whether RIN and HB-1 directly activate the transcription of ACS and ACO, we performed transcriptional activation analysis in Arabidopsis thaliana leaf protoplasts, transiently expressing RIN or HB-1 and using reporter constructs with promoters of the tomato ACS and ACO genes. Activation of the endogenous AtACS and AtACO genes was also measured by qPCR. The RIN- and HB-1-induced expression of these genes decreased, but the HB-1-induced expression of some genes increased after sound treatment. To confirm these results, we performed transient assays in Nicotiana tabacum, which produced results similar to those observed in Arabidopsis. The major ethylene biosynthesis-related genes harbor a CArG-box as a RIN-binding motif. These findings indicate that RIN and HB-1 affect the expression of ethylene biosynthesis-related genes in response to sound treatment, and they suggest that RIN may regulate the ethylene biosynthesis-related genes by binding to their CArG-boxes.

Oil palm EgCBF3 conferred stress tolerance in transgenic tomato plants through modulation of the ethylene signaling pathway

CBF/DREB1 is a group of transcription factors that are mainly involved in abiotic stress tolerance in plants. They belong to the AP2/ERF superfamily of plant-specific transcription factors. A gene encoding a new member of this group was isolated from ripening oil palm fruit and designated as EgCBF3. The oil palm fruit demonstrates the characteristics of a climacteric fruit like tomato, in which ethylene has a major impact on the ripening process. A transgenic approach was used for functional characterization of the EgCBF3, using tomato as the model plant. The effects of ectopic expression of EgCBF3 were analyzed based on expression profiling of the ethylene biosynthesis-related genes, anti-freeze proteins (AFPs), abiotic stress tolerance and plant growth and development. The EgCBF3 tomatoes demonstrated altered phenotypes compared to the wild type tomatoes. Delayed leaf senescence and flowering, increased chlorophyll content and abnormal flowering were the consequences of overexpression of EgCBF3 in the transgenic tomatoes. The EgCBF3 tomatoes demonstrated enhanced abiotic stress tolerance under in vitro conditions. Further, transcript levels of ethylene biosynthesis-related genes, including three SlACSs and two SlACOs, were altered in the transgenic plants’ leaves and roots compared to that in the wild type tomato plant. Among the eight AFPs studied in the wounded leaves of the EgCBF3 tomato plants, transcript levels of SlOSM-L, SlNP24, SlPR5L and SlTSRF1 decreased, while expression of the other four, SlCHI3, SlPR1, SlPR-P2 and SlLAP2, were up-regulated. These findings indicate the possible functions of EgCBF3 in plant growth and development as a regulator of ethylene biosynthesis-related and AFP genes, and as a stimulator of abiotic stress tolerance.

A basic helix-loop-helix transcription factor, PhFBH4, regulates flower senescence by modulating ethylene biosynthesis pathway in petunia.

The basic helix-loop-helix (bHLH) transcription factors (TFs) play important roles in regulating multiple biological processes in plants. However, there are few reports about the function of bHLHs in flower senescence. In this study, a bHLH TF, PhFBH4, was found to be dramatically upregulated during flower senescence. Transcription of PhFBH4 is induced by plant hormones and abiotic stress treatments. Silencing of PhFBH4 using virus-induced gene silencing or an antisense approach extended flower longevity, while transgenic petunia flowers with an overexpression construct showed a reduction in flower lifespan. Abundance of transcripts of senescence-related genes (SAG12, SAG29) was significantly changed in petunia PhFBH4 transgenic flowers. Furthermore, silencing or overexpression of PhFBH4 reduced or increased, respectively, transcript abundances of important ethylene biosynthesis-related genes, ACS1 and ACO1, thereby influencing ethylene production. An electrophoretic mobility shift assay showed that the PhFBH4 protein physically interacted with the G-box cis-element in the promoter of ACS1, suggesting that ACS1 was a direct target of the PhFBH4 protein. In addition, ectopic expression of this gene altered plant development including plant height, internode length, and size of leaves and flowers, accompanied by alteration of transcript abundance of the gibberellin biosynthesis-related gene GA2OX3. Our results indicate that PhFBH4 plays an important role in regulating plant growth and development through modulating the ethylene biosynthesis pathway.

Ethylene is involved in leafy mustard systemic resistance to Turnip mosaic virus infection through the mitochondrial alternative oxidase pathway

The role of the mitochondrial alternative oxidase (AOX) pathway and the relationship between AOX and ethylene in resistance to Turnip mosaic virus (TuMV) were investigated in systemic infected mustard leaves. Expressions of AOX1a and ethylene biosynthesis-related genes were enhanced in the upper uninoculated leaves at 6 days of postinoculation (dpi). Increased accumulation of AOX1a transcript and reduced levels of TuMV-CP were also observed after pretreatment with AA (a cytochrome pathway inhibitor) or ACC (an ethylene precursor) at non-lethal concentrations. Meanwhile, application of AVG (an ethylene biosynthesis inhibitor) mostly blocked TuMV-induced AOX activity and substantially increased TuMV susceptibility in the upper leaves. SHAM (AOX pathway inhibitor) pretreatment partially reduced the ACC-induced cyanide-resistant respiration and TuMV resistance. Conversely, AA and SHAM pretreatments had little effect on generation of ethylene. These results suggested that the TuMV-induced increase of AOX was partly involved in the ethylene pathway, and ethylene unregulated AOX in upper uninoculated mustard leaves.

Proteome analysis of Arabidopsis seedlings exposed to bacterial volatiles

Plant root-associated bacteria (rhizobacteria) elicit plant basal immunity referred to as induced systemic resistance (ISR) against multiple pathogens. Among multi-bacterial determinants involving such ISR, the induction of ISR and promotion of growth by bacterial volatile compounds was previously reported. To exploit global de novo expression of plant proteins by bacterial volatiles, proteomic analysis was performed after exposure of Arabidopsis plants to the rhizobacterium Bacillus subtilis GB03. Ethylene biosynthesis enzymes were significantly up-regulated. Analysis by quantitative reverse transcriptase polymerase chain reaction confirmed that ethylene biosynthesis-related genes SAM-2, ACS4, ACS12, and ACO2 as well as ethylene response genes, ERF1, GST2, and CHIB were up-regulated by the exposure to bacterial volatiles. More interestingly, the emission of bacterial volatiles significantly up-regulated both key defense mechanisms mediated by jasmonic acid and salicylic acid signaling pathways. In addition, high accumulation of antioxidant proteins also provided evidence of decreased sensitivity to reactive oxygen species during the elicitation of ISR by bacterial volatiles. The present results suggest that the proteomic analysis of plant defense responses in bacterial volatile-mediated ISR can reveal the mechanisms of plant basal defenses orchestrated by endogenous ethylene production pathways and the generation of reactive oxygen species.