Ethylene Biosynthesis Enzymes Research Articles

Identification of Key Ubiquitination Sites Involved in the Proteasomal Degradation of AtACS7 in Arabidopsis.

The gaseous hormone ethylene plays pivotal roles in plant growth and development. The rate-limiting enzyme of ethylene biosynthesis in seed plants is 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS). ACS proteins are encoded by a multigene family and the expression of ACS genes is highly regulated, especially at a post-translational level. AtACS7, the only type III ACS in Arabidopsis, is degraded in a 26S proteasome-dependent pathway. Here, by using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis, two lysine residues of AtACS7, lys285 (K285) and lys366 (K366), were revealed to be ubiquitin-modified in young, light-grown Arabidopsis seedlings but not in etiolated seedlings. Deubiquitylation-mimicking mutations of these residues significantly increased the stability of the AtACS7K285RK366R mutant protein in cell-free degradation assays. All results suggest that K285 and K366 are the major ubiquitination sites on AtACS7, providing deeper insights into the post-translational regulation of AtACS7 in Arabidopsis.

Genome-Wide Identification and Expression Analysis of 1-Aminocyclopropane-1-Carboxylate Synthase (ACS) Gene Family in Chenopodium quinoa

Ethylene plays an important role in plant development and stress resistance. The rate-limiting enzyme in ethylene biosynthesis is 1-aminocyclopropane-1-carboxylic acid synthase (ACS). C. quinoa (Chenopodium quinoa) is an important food crop known for its strong tolerance to abiotic stresses. However, knowledge regarding the ACS gene family in C. quinoa remains restricted. In this study, we successfully identified 12 ACS genes (CqACSs) from the C. quinoa genome. Through thorough analysis of their sequences and phylogenetic relationships, it was verified that 8 out of these 12 CqACS isozymes exhibited substantial resemblance to ACS isozymes possessing ACS activity. Furthermore, these eight isozymes could be categorized into three distinct groups. The four remaining CqACS genes grouped under category IV displayed notable similarities with AtACS10 and AtACS12, known as amido transferases lacking ACS activity. The CqACS proteins bore resemblance to the AtACS proteins and had the characteristic structural features typically observed in plant ACS enzymes. Twelve CqACS genes were distributed across 8 out of the 18 chromosomes of C. quinoa. The CqACS genes were expanded from segment duplication. Many cis-regulatory elements related with various abiotic stresses, phytohormones, and light were found. The expression patterns of ACS genes varied across different tissues of C. quinoa. Furthermore, the analysis of gene expression patterns under abiotic stress showed that CqACS genes can be responsive to various stresses, implying their potential functions in adapting to various abiotic stresses. The findings from this research serve as a foundation for delving deeper into the functional roles of CqACS genes.

Postharvest application of carrageenan oligosaccharides on quality attributes and activities of defense-related enzymes in strawberry (Fragaria×ananassa Duch)

The carrageenan oligosaccharides (COS) possessed versatile biological activities such as antibacterial, antioxidant, anti-inflammatory activities. Herein, we aimed to explore the effects of carrageenan oligosaccharides (COS) on the preservation of strawberries (Fragaria×ananassa Duch) and factors including decay percentage, hardness, soluble solids, titratable acids, vitamin C (VC) content, the activity of peroxidase (POD), polyphenol oxidase (PPO) were judged as criteria. Also, the expression of genes related to fruit-softening enzymes were detected by real-time qPCR. Soaking treatment of oligosaccharides could significantly (P < 0.05) improve the physical and chemical factors compared with the control group. As for the preservation mechanism of strawberries, the Real-time qPCR results showed that COS coating treatment significantly prolonged the shelf life of strawberries by suppressing the gene expression of cell wall degraded enzymes and ethylene biosynthesis enzymes, with the most obvious effects observed in the 10 mg/mL concentration group. This experiment explores the possibility of COS as a strawberry preservative, which lays a theoretical foundation for future practical application.

Characterization of MicroRNAs and Gene Expression in ACC Oxidase RNA Interference-Based Transgenic Bananas.

Banana (Musa acuminata, AAA group) is a typically respiratory climacteric fruit. Previously, genes encoding ACC oxidase, one of the key enzymes in ethylene biosynthesis, Mh-ACO1 and Mh-ACO2 in bananas were silenced individually using RNAi interference technology, and fruit ripening of transgenic bananas was postponed. Here, the differential expression of miRNAs and their targeted mRNAs were analyzed in the transcriptomes of fruits at the third ripening stage, peel color more green than yellow, from the untransformed and RNAi transgenic bananas. Five significantly differentially expressed miRNAs (mac-miR169a, mac-miR319c-3p, mac-miR171a, mac-miR156e-5p, and mac-miR164a-5p) were identified. The predicted miRNA target genes were mainly enriched in six KEGG pathways, including 'sulfur relay system', 'protein digestion and absorption', 'histidine metabolism', 'pathogenic E. coli infection', 'sulfur metabolism', and 'starch and sucrose metabolism'. After ethylene treatment, the expression of ACC oxidase silencing-associated miRNAs was down-regulated, and that of their target genes was up-regulated along with fruit ripening. The evolutionary clustering relationships of miRNA precursors among 12 gene families related to fruit ripening were analyzed. The corresponding expression patterns of mature bodies were mainly concentrated in flowers, fruits, and leaves. Our results indicated that ethylene biosynthesis is associated with miRNAs regulating the expression of sulfur metabolism-related genes in bananas.

GhXB38D represses cotton fibre elongation through ubiquitination of ethylene biosynthesis enzymes GhACS4 and GhACO1.

Ethylene plays an essential role in the development of cotton fibres. Ethylene biosynthesis in plants is elaborately regulated by the activities of key enzymes, 1-aminocyclopropane-1-carboxylate oxidase (ACO) and 1-aminocyclopropane-1-carboxylate synthase (ACS); however, the potential mechanism of post-translational modification of ACO and ACS to control ethylene synthesis in cotton fibres remains unclear. Here, we identify an E3 ubiquitin ligase, GhXB38D, that regulates ethylene biosynthesis during fibre elongation in cotton. GhXB38D gene is highly expressed in cotton fibres during the rapid elongation stage. Suppressing GhXB38D expression in cotton significantly enhanced fibre elongation and length, accompanied by the up-regulation of genes associated with ethylene signalling and fibre elongation. We demonstrated that GhXB38D interacts with the ethylene biosynthesis enzymes GhACS4 and GhACO1 in elongating fibres and specifically mediates their ubiquitination and degradation. The inhibition of GhXB38D gene expression increased the stability of GhACS4 and GhACO1 proteins in cotton fibres and ovules, resulting in an elevated concentration of ethylene. Our findings highlight the role of GhXB38D as a regulator of ethylene synthesis by ubiquitinating ACS4 and ACO1 proteins and modulating their stability. GhXB38D acts as a negative regulator of fibre elongation and serves as a potential target for enhancing cotton fibre yield and quality through gene editing strategy.

Genome-Wide Analysis and Identification of 1-Aminocyclopropane-1-Carboxylate Synthase (ACS) Gene Family in Wheat (Triticum aestivum L.).

Ethylene has an important role in regulating plant growth and development as well as responding to adversity stresses. The 1-aminocyclopropane-1-carboxylate synthase (ACS) is the rate-limiting enzyme for ethylene biosynthesis. However, the role of the ACS gene family in wheat has not been examined. In this study, we identified 12 ACS members in wheat. According to their position on the chromosome, we named them TaACS1-TaACS12, which were divided into four subfamilies, and members of the same subfamilies had similar gene structures and protein-conserved motifs. Evolutionary analysis showed that fragment replication was the main reason for the expansion of the TaACS gene family. The spatiotemporal expression specificity showed that most of the members had the highest expression in roots, and all ACS genes contained W box elements that were related to root development, which suggested that the ACS gene family might play an important role in root development. The results of the gene expression profile analysis under stress showed that ACS members could respond to a variety of stresses. Protein interaction prediction showed that there were four types of proteins that could interact with TaACS. We also obtained the targeting relationship between TaACS family members and miRNA. These results provided valuable information for determining the function of the wheat ACS gene, especially under stress.

Targeted system approach to ethylene biosynthesis and signaling of a heat tolerant tomato cultivar; the impact of growing season on fruit ripening.

Growing tomato in hot weather conditions is challenging for fruit production and yield. Tomato cv. Savior is a heat-tolerant cultivar which can be grown during both the Vietnamese winter (mild condition) and summer (hot condition) season. Understanding the mechanisms of ethylene biosynthesis and signaling are important for agriculture, as manipulation of these pathways can lead to improvements in crop yield, stress tolerance, and fruit ripening. The objective of this study was to investigate an overview of ethylene biosynthesis and signaling from target genes to proteins and metabolites and the impact of growing season on a heat tolerant tomato cultivar throughout fruit ripening and postharvest storage. This work also showed the feasibility of absolute protein quantification of ethylene biosynthesis enzymes. Summer fruit showed the delayed peak of ethylene production until the red ripe stage. The difference in postharvest ethylene production between winter and summer fruit appears to be regulated by the difference in accumulation of 1-aminocyclopropane-1-carboxylic acid (ACC) which depends on the putative up-regulation of SAM levels. The lack of differences in protein concentrations between winter and summer fruit indicate that heat stress did not alter the ethylene biosynthesis-related protein abundance in heat tolerant cultivar. The analysis results of enzymatic activity and proteomics showed that in both winter and summer fruit, the majority of ACO activity could be mainly contributed to the abundance of ACO5 and ACO6 isoforms, rather than ACO1. Likewise, ethylene signal transduction was largely controlled by the abundance of ethylene receptors ETR1, ETR3, ETR6, and ETR7 together with the constitute triple response regulator CTR1 for both winter and summer grown tomatoes. Altogether our results indicate that in the heat tolerant tomato cv. Savior, growing season mainly affects the ethylene biosynthesis pathway and leaves the signaling pathway relatively unaffected.

Transcriptional regulation of MbACO2-mediated ethylene synthesis during postharvest banana ripening

Ethylene plays a central role in climacteric fruit ripening. 1-Aminocyclopropane-1-carboxylic acid oxidase (ACO) is a crucial enzyme for ethylene biosynthesis. Edible cultivated banana varieties are mainly composed of the A- or A/B-genome. Nevertheless, the function and regulatory mechanism of ACO genes in the B-genome of banana fruit are unknown. The present study revealed the function and regulatory mechanism of the B-genome-specific gene MbACO2 in ethylene biosynthesis during postharvest banana ripening. MbACO2 expression significantly increased at 6 d postharvest. MbACO2 overexpression in tomato resulted in an increase in ethylene biosynthesis and the acceleration of fruit ripening. MaNAC29 and MbERF71/MbERF113 showed induction during the postharvest periods and directly activated the transcription of MbACO2 through the CGTC-element and GCC-box in its promoter, respectively. In addition, MaMAPK1 interacted with MaNAC29 to further enhance the transcription of MbACO2. Collectively, our findings demonstrate a novel regulatory cascade involving MaMAPK1, MaNAC29, and MbERF71/MbERF113 that promotes MbACO2 transcription during postharvest banana ripening, which helps design breeding strategies to extend the shelf life of fruit.

Phytohormone ethylene mediates oligogalacturonic acid-induced growth inhibition in tomato etiolated seedlings

Plant growth and immunity are tightly interconnected. Oligogalacturonic acids (OGs) are pectic fragments and have been well investigated in plant immunity as a damage-associated molecular pattern. However, little is known regarding how OGs affect plant growth. Here, we reveal that OGs inhibit the growth of intact etiolated seedling by using the horticultural crop tomato as a model. This inhibitory effect is partially suppressed by the action of ethylene biosynthesis inhibitors, or the gene silencing of SlACS2, an essential rate-limiting enzyme for ethylene biosynthesis, suggesting that SlACS2-mediated ethylene production promotes OG-induced growth inhibition. Furthermore, OGs treatment elevates the SlACS2 protein phosphorylation, and its decrease by the kinase inhibitor K252a partially rescue OG-induced growth inhibition, indicating that SlACS2 phosphorylation involves in OG-induced growth inhibition. Moreover, the mitogen-activated protein kinase SlMPK3 could be activated by OGs treatment and can directly phosphorylate SlACS2 in vitro, and the bimolecular fluorescence complementation combining with the yeast two-hybrid assay shows that SlMPK3 interacts with SlACS2, indicating that SlMPK3 may participate in modulating the OG-induced SlACS2 phosphorylation and growth inhibition. Our results reveal a regulatory mechanism at both the transcriptional and post-transcriptional levels by which OGs inhibit the growth of intact plant seedlings.

Exogenous Ca2+ promotes transcription factor phosphorylation to suppress ethylene biosynthesis in apple.

Ethylene biosynthesis in apple (Malus domestica) fruit can be suppressed by calcium ions (Ca2+) during storage; however, the underlying mechanisms are unclear. In this study, we identified the apple transcription factor MCM1-AGAMOUS-DEFICIENS-SRF5 (MdMADS5), which functions as a transcriptional activator of the ethylene biosynthesis-related gene 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE1 (MdACS1), a partner of the calcium sensor CALCIUM-DEPENDENT PROTEIN KINASES7 (MdCDPK7). Ca2+ promoted the MdCDPK7-mediated phosphorylation of MdMADS5, which resulted in the degradation of MdMADS5 via the 26S proteasome pathway. MdCDPK7 also phosphorylated 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID OXIDASE1 (MdACO1), the key enzyme in ethylene biosynthesis, leading to MdACO1 degradation and inhibition of ethylene biosynthesis. Our results reveal that Ca2+/MdCDPK7-MdMADS5 and Ca2+/MdCDPK7-MdACO1 are involved in Ca2+-suppressed ethylene biosynthesis, which delays apple fruit ripening. These findings provide insights into fruit ripening, which may lead to the development of strategies for extending the shelf life of fruit.

Advances in Physiological, Transcriptomic, Proteomic, Metabolomic, and Molecular Genetic Approaches for Enhancing Mango Fruit Quality.

Mango (Mangifera indica L.) is a nutritionally important fruit of high nutritive value, delicious in taste with an attractive aroma. Due to their antioxidant and therapeutic potential, mango fruits are receiving special attention in biochemical and pharmacognosy-based studies. Fruit quality determines consumer's acceptance, and hence, understanding the physiological, biochemical, and molecular basis of fruit development, maturity, ripening, and storage is essential. Transcriptomic, metabolomic, proteomic, and molecular genetic approaches have led to the identification of key genes, metabolites, protein candidates, and quantitative trait loci that are associated with enhanced mango fruit quality. The major pathways that determine the fruit quality include amino acid metabolism, plant hormone signaling, carbohydrate metabolism and transport, cell wall biosynthesis and degradation, flavonoid and anthocyanin biosynthesis, and carotenoid metabolism. Expression of the polygalacturonase, cutin synthase, pectin methyl esterase, pectate lyase, β-galactosidase, and ethylene biosynthesis enzymes are related to mango fruit ripening, flavor, firmness, softening, and other quality processes, while genes involved in the MAPK signaling pathway, heat shock proteins, hormone signaling, and phenylpropanoid biosynthesis are associated with diseases. Metabolomics identified volatiles, organic acids, amino acids, and various other compounds that determine the characteristic flavor and aroma of the mango fruit. Molecular markers differentiate the mango cultivars based on their geographical origins. Genetic linkage maps and quantitative trait loci studies identified regions in the genome that are associated with economically important traits. The review summarizes the applications of omics techniques and their potential applications toward understanding mango fruit physiology and their usefulness in future mango breeding.

Epigenetic regulation for delaying tomato fruit ripening through histone modification by specific sound wave treatment

Controlling fruit ripening is crucial for maintaining its quality during delivery. A specific sound wave treatment to delay fruit ripening without genetic modifications has been identified as an effective method in tomatoes. However, the molecular mechanisms of the sound wave-triggered suppression of fruit ripening remain largely unknown. In this study, we demonstrated that sound waves influence histone modifications, particularly at ethylene biosynthesis-related genetic loci, inhibiting transcription. Sound waves reduced histone modifications involved in transcriptional activation but increased repressive histone modifications at the RIN, NOR, and TAGL1 genes, which are positive regulators of fruit ripening. Similar epigenetic events have also been observed in genes encoding ethylene biosynthesis enzymes. Sound wave treatment upregulated the expression of histone modification enzyme genes involved in H3 methylation and deacetylation but downregulated those involved in H3 demethylation and acetylation. In addition, histone-modifying enzymes may directly associate with promoters of RIN, NOR, and TAGL1 genes and indirectly associate with promoters of ethylene biosynthesis enzyme genes through physical interactions with transcription factors. Our findings provide the basis for the practical use of sound waves to delay fruit ripening.

Integration of Transcriptome and Metabolome Analyses Reveals the Mechanistic Basis for Cadmium Accumulation in Maize.

Cadmium (Cd) pollution in soil has become a major environmental issue worldwide. However, the underlying molecular mechanism of low grain-Cd accumulation (GCA) in maize is still largely unknown. Herein, we report the mechanistic basis for low GCA in maize by a multiomics approach. The low GCA genotype L63 showed normal vacuolar formation and a lower capacity of xylem loading of Cd than the high-accumulator L42 under Cd stress. Transcriptomic sequencing identified 84 low-GCA-associated genes which are mainly involved in the S-adenosylmethionine (SAM) cycle, metal transport, and vacuolar sequestration. A metabolome analysis revealed that L63 plants had a more active SAM cycle and a greater capacity for terpenoid synthesis and phenylalanine metabolism than L42. Combining the analysis of transcriptome and metabolome characterized several genes as key genes involved in the determination of Cd accumulation. Our study identifies a mechanistic basis for low Cd accumulation in maize grains and provides candidate genes for genetic improvement of crops.

WRKY29 transcription factor regulates ethylene biosynthesis and response in arabidopsis

The gaseous phytohormone ethylene participates in a lot of physiological processes in plants. 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS, EC 4.4.1.14) and the ACC oxidase (ACO, EC 1.14.17.4) are key enzymes in ethylene biosynthesis. However, how ACSs and ACOs are regulated at the transcriptional level is largely unknown. In the present study, we showed that an Arabidopsis (Arabidopsis thaliana) WRKY-type transcription factor (TF), WRKY29 positively regulated the expression of ACS5, ACS6, ACS8, ACS11 and ACO5 genes and thus promoted basal ethylene production. WRKY29 protein was localized in nuclei and was a transcriptional activator. Overexpression of WRKY29 caused pleiotropic effect on plant growth, development and showed obvious response even without ACC treatment. Inducible overexpression of WRKY29 also reduced primary root elongation and lateral root growth. A triple response assay of overexpression and mutant seedlings of WRKY29 showed that overexpression seedlings had shorter hypocotyls than the transgenic GFP (Green Fluorescence Protein) control, while mutants had no difference from wild-type. A qRT-PCR assay demonstrated that expression of multiple ACSs and ACO5 was up-regulated in WRKY29 overexpression plants. A transactivation assay through dual luciferase reporter system confirmed the regulation of promoters of ACS5, ACS6, ACS8, ACS11 and ACO5 by WRKY29. Both in vivo chromatin immunoprecipitation (ChIP)- quantitative PCR and in vitro electrophoretic mobility shift assay (EMSA) revealed that WRKY29 directly bound to the promoter regions of its target genes. Taken together, these results suggest that WRKY29 is a novel TF positively regulating ethylene production by modulating the expression of ACS and ACO genes.

The HY5 transcription factor negatively regulates ethylene production by inhibiting ACS1 expression under blue light conditions in pear

Ethylene is the main factor controlling fruit ripening of pear (Pyrus ussuriensis). Ethylene production rate is negatively correlated with fruit shelf life; therefore, it is important to decrease the ethylene levels for optimal fruit storage. Here, we observed that blue light treatment could inhibit ethylene production and promote the expression of ELONGATED HYPOCOTYL 5 (PuHY5), a basic leucine zipper domain (bZIP) transcription factor. The following studies showed that PuHY5 could bind to the promoter of ACC synthase 1 (PuACS1), a rate-limiting enzyme in ethylene biosynthesis, and inhibit its expression. For pears in which PuHY5 was silenced, the ethylene production and PuACS1 expression were much higher than those in the control fruit. These results demonstrated that blue light inhibited ethylene production through the induction of PuHY5 in pear. Our finding provides a new method for prolonging fruit shelf life.

Nonclimacteric ripening characteristics of ‘Jen-Ju Bar’ guava conferred by a defect in the expression of the system-2 ACC synthase gene PgACS1

Nonclimacteric varieties of guava (Psidium guajava L.), such as ‘Jen-Ju Bar’ (‘JJB’), have been cultivated in tropical areas due to their crispy texture and long storability. However, the molecular mechanism resulting in different ripening behaviors among guava varieties is still obscure. To elucidate the underlying mechanism, four cDNA clones of two ACC synthases (ACS) and two ACC oxidases (ACO), the key enzymes of ethylene biosynthesis, were isolated based on the conserved nucleotide sequences. Their enzymatic activities were confirmed by heterologous expression in Escherichia coli. The expression levels of PgACS1, PgACO1, and PgACO2 increased dramatically with the advance of ripening in the climacteric cultivar ‘Li-Tzy Bar’ (‘LTB’), whereas the transcripts of these genes, especially PgACS1 and PgACO2, were barely detected in ‘JJB’ throughout the phase. In addition, PgACS1 and PgACO2 transcription were induced by ethylene and repressed by 1-methylcyclopropene, an ethylene action inhibitor, which exhibited the features of system-2 ethylene production genes. These results suggested that PgACS1 and PgACO2 are two important coordinators for controlling autocatalytic ethylene biosynthesis in guava fruit. The major defect of system-2 ethylene production in ‘JJB’ is caused by the silencing of PgACS1 during ripening. Consequently, ‘JJB’ behaves nonclimacterically and has a long storability.

Melatonin stimulates postharvest ripening of apples by up-regulating gene expression of ethylene synthesis enzymes

Melatonin has been implicated in the regulation of ripening in several types of climacteric fruits, but its role in apple is poorly understood. In this study, the effectiveness of melatonin in influencing postharvest ripening in apples and interacting with ethylene biosynthesis process was investigated. Apples of four local cultivars (Golden, Reineta, Blanquina and Rabiosa) were harvest at an immature green stage and immersed for 120 min in a solution with water (control), melatonin 50 µM, ACC 1 mM, and melatonin 50 µM + ACC 1 mM. The evolution of apple quality parameters and ethylene production was assessed in all cultivars for 35 days after treatment. In addition, Golden apple was used to examine the transcript abundance of ethylene biosynthesis enzymes genes in the skin and flesh. Exogenous melatonin improved apple quality during postharvest ripening and stimulated ethylene production in all cultivars studied, advancing the timing of the climacteric burst. Melatonin-induced changes in ethylene production correlated well with changes in fruit quality and were in part similar to those induced by the ethylene precursor ACC, so ethylene appears to be a target of melatonin. In Golden apple, melatonin stimulated the expression of MdACS1 and MdACO1 genes, which showed a climacteric profile similar to that of ethylene. The transcriptional effects of exogenous melatonin on ethylene-related genes was also more notable in the skin than in the flesh, suggesting an initiating role of the former in the biosynthesis of the phytohormone. Taken together, the results point to the fact that exogenous melatonin may be useful in improving apple quality during postharvest ripening by promoting colour and flavour change and extending fruit firmness. The effect of melatonin appears to be mediated in part by up-regulation of the transcriptional activity of the MdACS1 and MdACO1 genes during the climacteric peak, thus contributing to an increase in ethylene availability.

Genome-Wide Identification and Characterization of Members of the ACS Gene Family in Cucurbita maxima and Their Transcriptional Responses to the Specific Treatments.

Ethylene biosynthesis and signal transduction play critical roles in plant sex differentiation. ACS (1-aminocyclopropane-1-carboxylic acid synthase) is a rate-limiting enzyme in ethylene biosynthesis. However, the understanding of the ACS gene family in Cucurbita maxima is limited. Here, we identified and characterized 13 ACS genes in the C. maxima genome. All ACS genes could be divided into three groups according to a conserved serine residue at the C-terminus. Thirteen CmaACS genes were found to be randomly distributed on 10 of the 20 chromosomes of C. maxima. The ACS gene exhibits different tissue-specific expression patterns in pumpkin, and four ACS genes (CmaACS1, CmaACS4, CmaACS7, and CmaACS9) were expressed specifically in both the female and male flowers of C. maxima. In addition, the expression levels of CmaACS4 and CmaACS7 were upregulated after ethephon and IAA treatments, which ultimately increased the number of female flowers, decreased the position of the first female flower and decreased the number of bisexual flowers per plant. These results provide relevant information for determining the function of the ACS genes in C. maxima, especially for regulating the function of ethylene in sex determination.

Wheat Elongator Subunit 4 Negatively Regulates Freezing Tolerance by Regulating Ethylene Accumulation.

Freezing stress is a major factor limiting production and geographical distribution of temperate crops. Elongator is a six subunit complex with histone acetyl-transferase activity and is involved in plant development and defense responses in Arabidopsis thaliana. However, it is unknown whether and how an elongator responds to freezing stress in plants. In this study, we found that wheat elongator subunit 4 (TaELP4) negatively regulates freezing tolerance through ethylene signaling. TaELP4 promoter contained cold response elements and was up-regulated in freezing stress. Subcellular localization showed that TaELP4 and AtELP4 localized in the cytoplasm and nucleus. Silencing of TaELP4 in wheat with BSMV-mediated VIGS approach significantly elevated tiller survival rate compared to control under freezing stress, but ectopic expression of TaELP4 in Arabidopsis increased leaf damage and survival rate compared with Col-0. Further results showed that TaELP4 positively regulated ACS2 and ACS6 transcripts, two main limiting enzymes in ethylene biosynthesis. The determination of ethylene content showed that TaELP4 overexpression resulted in more ethylene accumulated than Col-0 under freezing stress. Epigenetic research showed that histone H3K9/14ac levels significantly increased in coding/promoter regions of AtACS2 and AtACS6 in Arabidopsis. RT-qPCR assays showed that the EIN2/EIN3/EIL1-CBFs-COR pathway was regulated by TaELP4 under freezing stress. Taken together, our results suggest that TaELP4 negatively regulated plant responses to freezing stress via heightening histone acetylation levels of ACS2 and ACS6 and increasing their transcription and ethylene accumulation.

Reprint of: 1-Aminocyclopropanecarboxylate Synthase, a Key Enzyme in Ethylene Biosynthesis

1-Aminocyclopropanecarboxylate (ACC) synthase, which catalyzes the conversion of S-adenosylmethionine (SAM) to ACC and methylthioadenosine, was demonstrated in tomato extract. Methylthioadenosine was then rapidly hydrolyzed to methylthioribose by a nucleosidase present in the extract. ACC synthase had an optimum pH of 8.5, and a Km of 20 μM with respect to SAM. S-Adenosylethionine also served as a substrate for ACC synthase, but at a lower efficiency than that of SAM. Since S-adenosylethionine had a higher affinity for the enzyme than SAM, it inhibited the reaction of SAM when both were present. S-Adenosylhomocysteine was, however, an inactive substrate. The enzyme was activated by pyridoxal phosphate at a concentration of 0.1 μM or higher, and competitively inhibited by aminoethoxyvinylglycine and aminooxyacetic acid, which are known to inhibit pyridoxal phosphate-mediated enzymic reactions. These results support the view that ACC synthase is a pyridoxal enzyme. The biochemical role of pyridoxal phosphate is catalyzing the formation of ACC by α,γ-elimination of SAM is discussed.