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The WRKY transcription factor (TF) genes form a large family in higher plants, with 72 members in Arabidopsis (Arabidopsis thaliana). The gaseous phytohormone ethylene (ET) regulates multiple physiological processes in plants. It is known that 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACSs, EC 4.4.1.14) limit the enzymatic reaction rate of ethylene synthesis. However, whether WRKY TFs regulate the expression of ACSs and/or ACC oxidases (ACOs, EC 1.14.17.4) remains largely elusive. Here, we demonstrated that Arabidopsis WRKY22 positively regulated the expression of a few ACS and ACO genes, thus promoting ethylene production. Inducible overexpression of WRKY22 caused shorter hypocotyls without ACC treatment. A qRT-PCR screening demonstrated that overexpression of WRKY22 activates the expression of several ACS and ACO genes. The promoter regions of ACS5, ACS11, and ACO5 were also activated by WRKY22, which was revealed by a dual luciferase reporter assay. A follow-up chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) and electrophoretic mobility shift assay (EMSA) showed that the promoter regions of ACS5 and ACO5 could be bound by WRKY22 directly. Moreover, wrky22 mutants had longer primary roots and more lateral roots than wild type, while WRKY22-overexpressing lines showed the opposite phenotype. In conclusion, this study revealed that WRKY22 acts as a novel TF activating, at least, the expression of ACS5 and ACO5 to increase ethylene synthesis and modulate root development., (© 2024 Scandinavian Plant Physiology Society.)

Summary: Ethylene (ETH) controls climacteric fruit ripening and can be triggered by osmotic stress. However, the mechanism regulating ETH biosynthesis during fruit ripening and under osmotic stress is largely unknown in apple (Malus domestica).Here, we explored the roles of SnRK2 protein kinases in ETH biosynthesis related to fruit ripening and osmoregulation. We identified the substrates of MdSnRK2‐I using phosphorylation analysis techniques. Finally, we identified the MdSnRK2‐I‐mediated signaling pathway for ETH biosynthesis related to fruit ripening and osmoregulation.The activity of two MdSnRK2‐I members, MdSnRK2.4 and MdSnRK2.9, was significantly upregulated during ripening or following mannitol treatment. Overexpression of MdSnRK2‐I increased ETH biosynthesis under normal and osmotic conditions in apple fruit. MdSnRK2‐I phosphorylated the transcription factors MdHB1 and MdHB2 to enhance their protein stability and transcriptional activity on MdACO1. MdSnRK2‐I also interacted with MdACS1 and increased its protein stability through two phosphorylation sites. The increased MdACO1 expression and MdACS1 protein stability resulted in higher ETH production in apple fruit. In addition, heterologous expression of MdSnRK2‐I or manipulation of SlSnRK2‐I expression in tomato (Solanum lycopersicum) fruit altered fruit ripening and ETH biosynthesis.We established that MdSnRK2‐I functions in fruit ripening and osmoregulation, and identified the MdSnRK2‐I‐mediated signaling pathway controlling ETH biosynthesis. [ABSTRACT FROM AUTHOR]

Background: The phytohormone ethylene controls many processes in plant development and acts as a key signaling molecule in response to biotic and abiotic stresses: it is rapidly induced by flooding, wounding, drought, and pathogen attack as well as during abscission and fruit ripening. In kiwifruit (Actinidia spp.), fruit ripening is characterized by two distinct phases: an early phase of system-1 ethylene biosynthesis characterized by absence of autocatalytic ethylene, followed by a late burst of autocatalytic (system-2) ethylene accompanied by aroma production and further ripening. Progress has been made in understanding the transcriptional regulation of kiwifruit fruit ripening but the regulation of system-1 ethylene biosynthesis remains largely unknown. The aim of this work is to better understand the transcriptional regulation of both systems of ethylene biosynthesis in contrasting kiwifruit organs: fruit and leaves. Results: A detailed molecular study in kiwifruit (A. chinensis) revealed that ethylene biosynthesis was regulated differently between leaf and fruit after mechanical wounding. In fruit, wound ethylene biosynthesis was accompanied by transcriptional increases in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS), ACC oxidase (ACO) and members of the NAC class of transcription factors (TFs). However, in kiwifruit leaves, wound-specific transcriptional increases were largely absent, despite a more rapid induction of ethylene production compared to fruit, suggesting that post-transcriptional control mechanisms in kiwifruit leaves are more important. One ACS member, AcACS1, appears to fulfil a dominant double role; controlling both fruit wound (system-1) and autocatalytic ripening (system-2) ethylene biosynthesis. In kiwifruit, transcriptional regulation of both system-1 and -2 ethylene in fruit appears to be controlled by temporal up-regulation of four NAC (NAM, ATAF1/2, CUC2) TFs (AcNAC1–4) that induce AcACS1 expression by directly binding to the AcACS1 promoter as shown using gel-shift (EMSA) and by activation of the AcACS1 promoter in planta as shown by gene activation assays combined with promoter deletion analysis. Conclusions: Our results indicate that in kiwifruit the NAC TFs AcNAC2–4 regulate both system-1 and -2 ethylene biosynthesis in fruit during wounding and ripening through control of AcACS1 expression levels but not in leaves where post-transcriptional/translational regulatory mechanisms may prevail. [ABSTRACT FROM AUTHOR]

Summary: The gaseous plant hormone ethylene is produced by a fairly simple two‐step biosynthesis route. Despite this pathway's simplicity, recent molecular and genetic studies have revealed that the regulation of ethylene biosynthesis is far more complex and occurs at different layers. Ethylene production is intimately linked with the homeostasis of its general precursor S‐adenosyl‐l‐methionine (SAM), which experiences transcriptional and posttranslational control of its synthesising enzymes (SAM synthetase), as well as the metabolic flux through the adjacent Yang cycle. Ethylene biosynthesis continues from SAM by two dedicated enzymes: 1‐aminocyclopropane‐1‐carboxylic (ACC) synthase (ACS) and ACC oxidase (ACO). Although the transcriptional dynamics of ACS and ACO have been well documented, the first transcription factors that control ACS and ACO expression have only recently been discovered. Both ACS and ACO display a type‐specific posttranslational regulation that controls protein stability and activity. The nonproteinogenic amino acid ACC also shows a tight level of control through conjugation and translocation. Different players in ACC conjugation and transport have been identified over the years, however their molecular regulation and biological significance is unclear, yet relevant, as ACC can also signal independently of ethylene. In this review, we bring together historical reports and the latest findings on the complex regulation of the ethylene biosynthesis pathway in plants. [ABSTRACT FROM AUTHOR]

Summary: Fruit ripening is a developmental process that is spatio‐temporally tuned at multiple levels. Molecular dissections of the mechanisms underlying the ripening process have revealed a network encompassed by hormones, transcriptional regulators, epigenomic modifications and other regulatory elements that directly determine fruit quality and the postharvest commodity of fresh produce. Many studies have addressed the important roles of ethylene, abscisic acid (ABA) and other hormones in regulating fruit ripening. Recent studies have shown that some spontaneous mutants for tomato transcription factors (TFs) have resulted from loss‐of‐function or dominant‐negative mutations. Unlike in DNA methylation variation, the histone mark H3K27me3 may be conserved and prevents the transcriptional feedback circuit from generating autocatalytic ethylene. These observations of a network of partially redundant component indicate the need to improve our current understanding. Here, we focussed on the recent advances and future challenges in investigations of the molecular mechanisms of fruit ripening. We also identified several issues that still need to be addressed in future studies. [ABSTRACT FROM AUTHOR]

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

Main conclusion: The overexpression of SlbHLH22 functioned in controlling flowering time, accelerated fruit ripening, and produced more ethylene-producing phenotypes in tomato. Flowering and fruit ripening are two complex transition processes regulated by various internal and external factors that ultimately lead to fruit maturation and final seed dispersal. The basic helix–loop–helix (bHLH) transcription factor is the largest TF gene family in plants that controls various biological and developmental aspects, but the actual roles of these genes have not been fully studied. Here, we performed a functional characterization of the bHLH gene SlbHLH22 in tomato. SlbHLH22 was fully expressed in tomato flowers, while a moderate expression level was also observed in fruits at different developmental stages. Overexpression of the SlbHLH22 gene revealed that it is highly involved in controlling flowering time, through the activation of the SlSFT or SlLFY genes, and promoting fruit ripening and improved carotenoid accumulation. The expression patterns of carotenoid-related genes (SlPYS1) were also upregulated in transgenic tomato fruits. In transgenic tomato fruit, we observed clear changes in colour from green to orange with enhanced expression of the SlbHLH22 gene. SlbHLH22 was upregulated under exogenous ACC, IAA, ABA, and ethephon. Overexpression of SlbHLH22 also promotes ethylene production. Moreover, ethylene biosynthesis and perception genes (SlACO3, SlACS1, SlACS2, SlACS4, SlACS1a, SlEIN1, SlEIN2, SlEIN3, SlEIN4, SlETR2, SlETR3, SlSAM3, and SlSAMS) were upregulated. Ripening-related genes (SlAP2a, SlCNR, SlNOR, SlMYB, and SlTAG) were consistent in their expression pattern in transgenic plants. Finally, our study provides evidence that tomato bHLH genes play an important role in flowering, fruit ripening, and development. [ABSTRACT FROM AUTHOR]

Fruits result from complex biological processes that begin soon after fertilization. Among these processes are cell division and expansion, accumulation of secondary metabolites, and an increase in carbohydrate biosynthesis. Later fruit ripening is accomplished by chlorophyll degradation and cell wall lysis. Fruit maturation is an essential step to optimize seed dispersal, and is controlled by a complex network of transcription factors and genetic regulators that are strongly influenced by phytohormones. Abscisic acid (ABA) and ethylene are the major regulators of ripening and senescence in both dry and fleshy fruits, as demonstrated by numerous ripening-defective mutants, effects of exogenous hormone application, and transcriptome analyses. While ethylene is the best characterized player in the final step of a fruit's life, ABA also has a key regulatory role, promoting ethylene production and acting as a stress-related hormone in response to drought and pathogen attack. In this review, we focus on the role of ABA and ethylene in relation to the interconnected biotic and abiotic phenomena that affect ripening and senescence. We integrate and discuss the most recent data available regarding these biological processes, which are crucial for post-harvest fruit conservation and for food safety. [ABSTRACT FROM AUTHOR]

Banana fruit (Musa acuminate L.) ripening is a complex genetical process affected by multiple phytohormones and expression of various genes. However, whether plant hormone brassinosteroid (BR) is involved in this process remains obscure. In this work, three genes that encode BR core signaling components brassinazole resistant (BZR) proteins, namely MaBZR1 to MaBZR3, were characterized from banana fruit. MaBZR1‐MaBZR3 exhibited both nuclear and cytoplasmic localization and behaved as transcription inhibitors. Expression analysis showed that MaBZR1/2/3 were continuously decreased as fruit ripening proceeded, indicating their negative roles in banana ripening. Moreover, gel shift and transient expression assays demonstrated that MaBZR1/2 could suppress the transcription of ethylene biosynthetic genes, including MaACS1, MaACO13 and MaACO14, which increased gradually during the banana ripening, via specifically binding to CGTGT/CG sequence in their promoters. Importantly, exogenous application of BRs promotes banana ripening, which is presumably due to the accelerated expression of MaACS1 and MaACO13/14, and consequently the ethylene production. Our study indicates that MaBZR1/2 act as transcriptional repressors of ethylene biosynthetic genes during banana fruit ripening. [ABSTRACT FROM AUTHOR]

Background Fruit maturation and ripening are genetically regulated processes that involve a complex interplay of plant hormones, growth regulators and multiple biological and environmental factors. Tomato (Solanum lycopersicum) has been used as a model of biological and genetic studies on the regulation of specific ripening pathways, including ethylene, carotenoid and cell wall metabolism. This model has also been used to investigate the functions of upstream signalling and transcriptional regulators. Thus far, many ripening-associated transcription factors that influence fruit development and ripening have been reported. NAC transcription factors are plant specific and play important roles in many stages of plant growth and development, such as lateral root formation, secondary cell wall synthesis, and embryo, floral organ, vegetative organ and fruit development. Results Tissue-specific analysis by quantitative real-time PCR showed that SlNAC1 was highly accumulated in immature green fruits; the expression of SlNAC1 increased with fruit ripening till to the highest level at 7 d after the breaker stage. The overexpression of SlNAC1 resulted in reduced carotenoids by altering carotenoid pathway flux and decreasing ethylene synthesis mediated mainly by the reduced expression of ethylene biosynthetic genes of system-2, thus led to yellow or orange mature fruits. The results of yeast one-hybrid experiment demonstrated that SlNAC1 can interact with the regulatory regions of genes related lycopene and ethylene synthesis. These results also indicated that SlNAC1 inhibited fruit ripening by affecting ethylene synthesis and carotenoid accumulation in SlNAC1 overexpression lines. In addition, the overexpression of SlNAC1 reduced the firmness of the fruits and the thickness of the pericarp and produced more abscisic acid, resulting in the early softening of fruits. Hence, in SlNAC1 overexpression lines, both ethylene-dependent and abscisic acid-dependent pathways are regulated by SlNAC1 in fruit ripening regulatory network. Conclusions SlNAC1 had a broad influence on tomato fruit ripening and regulated SlNAC1 overexpression tomato fruit ripening through both ethylene-dependent and abscisic acid-dependent pathways. Thus, this study provided new insights into the current model of tomato fruit ripening regulatory network. [ABSTRACT FROM AUTHOR]

Background: During ripening, climacteric fruits increase their ethylene level and subsequently undergo various physiological changes, such as softening, pigmentation and development of aroma and flavor. These changes occur simultaneously and are caused by the highly synchronized expression of numerous genes at the onset of ripening. In tomatoes, the MADS-box transcription factor RIN has been regarded as a key regulator responsible for the onset of ripening by acting upstream of both ethylene- and non-ethylene-mediated controls. However, except for LeACS2, direct targets of RIN have not been clarified, and little is known about the transcriptional cascade for ripening. Results: Using immunoprecipitated (IPed) DNA fragments recovered by chromatin immunoprecipitation (ChIP) with anti-RIN antibody from ripening tomato fruit, we analyzed potential binding sites for RIN (CArG-box sites) in the promoters of representative ripening-induced genes by quantitative PCR. Results revealed nearly a 5- to 20-fold enrichment of CArG boxes in the promoters of LeACS2, LeACS4, PG, TBG4, LeEXP1, and LeMAN4 and of RIN itself, indicating direct interaction of RIN with their promoters in vivo. Moreover, sequence analysis and genome mapping of 51 cloned IPed DNAs revealed potential RIN binding sites. Quantitative PCR revealed that four of the potential binding sites were enriched 4- to 17-fold in the IPed DNA pools compared with the controls, indicating direct interaction of RIN with these sites in vivo. Near one of the four CArG boxes we found a gene encoding a protein similar to thioredoxin y1. An increase in the transcript level of this gene was observed with ripening in normal fruit but not in the rin mutant, suggesting that RIN possibly induces its expression. Conclusions: The presented results suggest that RIN controls fruit softening and ethylene production by the direct transcriptional regulation of cell-wall-modifying genes and ethylene biosynthesis genes during ripening. Moreover, the binding of RIN to its own promoter suggests the presence of autoregulation for RIN expression. ChIP-based analyses identified a novel RIN-binding CArG-box site that harbors a gene associated with RIN expression in its flanking region. These findings clarify the crucial role of RIN in the transcriptional regulation of ripening initiation and progression. [ABSTRACT FROM AUTHOR]