Restricted responses of AcMYB68 and AcERF74/75 enhanced waterlogging tolerance in kiwifruit.

Most of kiwifruit cultivars (e.g. Actinidia chinensis cv. Donghong, "DH") were sensitive to waterlogging, thus, waterlogging resistant rootstocks (e.g. Actinidia valvata Dunn, "Dunn") were widely used for kiwifruit industry. Those different species provided ideal materials to understand the waterlogging responses in kiwifruit. Compared to the weaken growth and root activities in "DH", "Dunn" maintained the relative high root activities under the prolonged waterlogging. Based on comparative analysis, transcript levels of pyruvate decarboxylase (PDCs) and alcohol dehydrogenase (ADHs) showed significantly difference between these two species. Both PDCs and ADHs had been significantly increased by waterlogging in "DH", while they were only limitedly triggered by 2 days stress and subsided during the prolonged waterlogging in "Dunn". Thus, 19 differentially expressed transcript factors (DETFs) had been isolated using weighted gene co-expression network analysis combined with transcriptomics and transcript levels of PDCs and ADHs in waterlogged "DH". Among these DETFs, dual luciferase and electrophoretic mobility shift assays indicated AcMYB68 could bind to and trigger the activity of AcPDC2 promoter. The stable over-expression of AcMYB68 significantly up-regulated the transcript levels of PDCs but inhibited the plant growth, especially the roots. Moreover, the enzyme activities of PDC in 35S::AcMYB68 were significantly enhanced during the waterlogging response than that in wild type plants. Most interestingly, comparative analysis indicated that the expression patterns of AcMYB68 and the previously characterized AcERF74/75 (the direct regulator on ADHs) either showed no responses (AcMYB68 and AcERF74) or very limited response (AcERF75) in "Dunn". Taken together, the restricted responses of AcMYB68 and AcERF74/75 in "Dunn" endow its waterlogging tolerance.

MYB transcription factors encoded by diversified tandem gene clusters cause varied Morella rubra fruit color.

Chinese bayberry (Morella rubra) is a fruit tree with a remarkable variation in fruit color, ranging from white to dark red as determined by anthocyanin content. In dark red "Biqi" (BQ), red "Dongkui" (DK), pink "Fenhong" (FH), and white "Shuijing" (SJ), we identified an anthocyanin-related MYB transcription factor-encoding gene cluster of four members, i.e. MrMYB1.1, MrMYB1.2, MrMYB1.3, and MrMYB2. Collinear analysis revealed that the MYB tandem cluster may have occurred in a highly conserved region of many eudicot genomes. Two alleles of MrMYB1.1 were observed; MrMYB1.1-1 (MrMYB1.1n) was a full-length allele and homozygous in "BQ", MrMYB1.1-2 (MrMYB1.1d) was a nonfunctional allele with a single base deletion and homozygous in "SJ", and MrMYB1.1n/MrMYB1.1d were heterozygous in "DK" and "FH". In these four cultivars, expression of MrMYB1.1, MrMYB1.2, and MrMYB2 was enhanced during ripening. Both alleles were equally expressed in MrMYB1.1n/MrMYB1.1d heterozygous cultivars as revealed by a cleaved amplified polymorphic sequence marker. Expression of MrMYB1.3 was restricted to some dark red cultivars only. Functional characterization revealed that MrMYB1.1n and MrMYB1.3 can induce anthocyanin accumulation while MrMYB1.1d, MrMYB1.2, and MrMYB2 cannot. DNA-protein interaction assays indicated that MrMYB1.1n and MrMYB1.3 can directly bind to and activate the promoters of anthocyanin-related genes via interaction with a MYC-like basic helix-loop-helix protein MrbHLH1. We concluded that the specific genotype of MrMYB1.1 alleles, as well as the exclusive expression of MrMYB1.3 in some dark red cultivars, contributes to fruit color variation. The study provides insights into the mechanisms for regulation of plant anthocyanin accumulation by MYB tandem clusters.

AcHZP45 is a repressor of chlorophyll biosynthesis and activator of chlorophyll degradation in kiwifruit.

The degradation of chlorophyll during fruit development is essential to reveal a more 'ripe' color that signals readiness to wild dispersers of seeds and the human consumer. Here, comparative biochemical analysis of developing fruit of Actinidia deliciosa cv. Xuxiang ('XX', green-fleshed) and Actinidia chinensis cv. Jinshi No.1 ('JS', yellow-fleshed) indicated that variation in chlorophyll content is the major contributor to differences in flesh color. Four differentially expressed candidate genes were identified: the down-regulated genes AcCRD1 and AcPOR1 involved in chlorophyll biosynthesis, and the up-regulated genes AcSGR1 and AcSGR2 driving chlorophyll degradation. Prochlorophyllide and chlorophyllide, the metabolites produced by AcCRD1 and AcPOR1, progressively reduced in 'JS', but not in 'XX', indicating that chlorophyll biosynthesis was less active in yellow-fleshed fruit. AcSGR1 and AcSGR2 were verified to be involved in chlorophyll degradation, using both transient expression in tobacco and stable overexpression in kiwifruit. Furthermore, a homeobox-leucine zipper (HD-Zip II), AcHZP45, showed significantly increased expression during 'JS' fruit ripening, which led to both repressed expression of AcCRD1 and AcPOR1 and activated expression of AcSGR1 and AcSGR2. Collectively, the present study indicated that different dynamics of chlorophyll biosynthesis and degradation coordinate the changes in chlorophyll content in kiwifruit flesh, which are orchestrated by the key transcription factor AcHZP45.

Identification of miRNA858 long-loop precursors in seed plants.

MicroRNAs (miRNAs) are a class of nonprotein-coding short transcripts that provide a layer of post-transcriptional regulation essential to many plant biological processes. MiR858, which targets the transcripts of MYB transcription factors, can affect a range of secondary metabolic processes. Although miR858 and its 187-nt precursor have been well studied in Arabidopsis (Arabidopsis thaliana), a systematic investigation of miR858 precursors and their functions across plant species is lacking due to a problem in identifying the transcripts that generate this subclass. By re-evaluating the transcript of miR858 and relaxing the length cut-off for identifying hairpins, we found in kiwifruit (Actinidia chinensis) that miR858 has long-loop hairpins (1,100 to 2,100 nt), whose intervening sequences between miRNA generating complementary sites were longer than all previously reported miRNA hairpins. Importantly, these precursors of miR858 containing long-loop hairpins (termed MIR858L) are widespread in seed plants including Arabidopsis, varying between 350 and 5,500 nt. Moreover, we showed that MIR858L has a greater impact on proanthocyanidin and flavonol levels in both Arabidopsis and kiwifruit. We suggest that an active MIR858L-MYB regulatory module appeared in the transition of early land plants to large upright flowering plants, making a key contribution to plant secondary metabolism.

Abscisic acid biosynthesis, metabolism and signaling in ripening fruit.

Fruits are highly recommended nowadays in human diets because they are rich in vitamins, minerals, fibers and other necessary nutrients. The final stage of fruit production, known as ripening, plays a crucial role in determining the fruit's quality and commercial value. This is a complex physiological process, which involves many phytohormones and regulatory factors. Among the phytohormones involved in fruit ripening, abscisic acid (ABA) holds significant importance. ABA levels generally increase during the ripening process in most fruits, and applying ABA externally can enhance fruit flavor, hasten softening, and promote color development through complex signal regulation. Therefore, gaining a deeper understanding of ABA's mechanisms in fruit ripening is valuable for regulating various fruit characteristics, making them more suitable for consumption or storage. This, in turn, can generate greater economic benefits and reduce postharvest losses. This article provides an overview of the relationship between ABA and fruit ripening. It summarizes the effects of ABA on ripening related traits, covering the biochemical aspects and the underlying molecular mechanisms. Additionally, the article discusses the interactions of ABA with other phytohormones during fruit ripening, especially ethylene, and provides perspectives for future exploration in this field.

Exogenous gibberellin delays maturation in persimmon fruit through transcriptional activators and repressors.

As the harvest season of most fruit is concentrated, fruit maturation manipulation is essential for the fresh fruit industry to prolong sales time. Gibberellin (GA), an important phytohormone necessary for plant growth and development, has also shown a substantial regulatory effect on fruit maturation; however, its regulatory mechanisms remain inconclusive. In this research, preharvest GA3 treatment effectively delayed fruit maturation in several persimmon (Diospyros kaki) cultivars. Among the proteins encoded by differentially expressed genes, 2 transcriptional activators (NAC TRANSCRIPTION FACTOR DkNAC24 and ETHYLENE RESPONSIVE FACTOR DkERF38) and a repressor (MYB-LIKE TRANSCRIPTION FACTOR DkMYB22) were direct regulators of GERANYLGERANYL DIPHOSPHATE SYNTHASE DkGGPS1, LYSINE HISTIDINE TRANSPORTER DkLHT1, and FRUCTOSE-BISPHOSPHATE ALDOLASE DkFBA1, respectively, resulting in the inhibition of carotenoid synthesis, outward transport of an ethylene precursor, and consumption of fructose and glucose. Thus, the present study not only provides a practical method to prolong the persimmon fruit maturation period in various cultivars but also provides insights into the regulatory mechanisms of GA on multiple aspects of fruit quality formation at the transcriptional regulation level.

A dramatic decline in fruit citrate induced by mutagenesis of a NAC transcription factor, AcNAC1.

Citrate is a common primary metabolite which often characterizes fruit flavour. The key regulators of citrate accumulation in fruit and vegetables are poorly understood. We systematically analysed the dynamic profiles of organic acid components during the development of kiwifruit (Actinidia spp.). Citrate continuously accumulated so that it became the predominate contributor to total acidity at harvest. Based on a co-expression network analysis using different kiwifruit cultivars, an Al-ACTIVATED MALATE TRANSPORTER gene (AcALMT1) was identified as a candidate responsible for citrate accumulation. Electrophysiological assays using expression of this gene in Xenopus oocytes revealed that AcALMT1 functions as a citrate transporter. Additionally, transient overexpression of AcALMT1 in kiwifruit significantly increased citrate content, while tissues showing higher AcALMT1 expression accumulated more citrate. The expression of AcALMT1 was highly correlated with 17 transcription factor candidates. However, dual-luciferase and EMSA assays indicated that only the NAC transcription factor, AcNAC1, activated AcALMT1 expression via direct binding to its promoter. Targeted CRISPR-Cas9-induced mutagenesis of AcNAC1 in kiwifruit resulted in dramatic declines in citrate levels while malate and quinate levels were not substantially affected. Our findings show that transcriptional regulation of a major citrate transporter, by a NAC transcription factor, is responsible for citrate accumulation in kiwifruit, which has broad implications for other fruits and vegetables.

Consensus co-expression network analysis identifies AdZAT5 regulating pectin degradation in ripening kiwifruit.

INTRODUCTION: Cell wall degradation and remodeling is the key factor causing fruit softening during ripening. OBJECTIVES: To explore the mechanism underlying postharvest cell wall metabolism, a transcriptome analysis method for more precious prediction on functional genes was needed. METHODS: Kiwifruits treated by ethylene (a conventional and effective phytohormone to accelerate climacteric fruit ripening and softening as kiwifruits) or air were taken as materials. Here, Consensus Coexpression Network Analysis (CCNA), a procedure evolved from Weighted Gene Co-expression Network Analysis (WGCNA) package in R, was applied and generated 85 consensus clusters from twelve transcriptome libraries. Advanced and comprehensive modifications were achieved by combination of CCNA and WGCNA with introduction of physiological traits, including firmness, cell wall materials, cellulose, hemicellulose, water soluble pectin, covalent binding pectin and ionic soluble pectin. RESULTS: As a result, six cell wall metabolisms related structural genes AdGAL1, AdMAN1, AdPL1, AdPL5, Adß-Gal5, AdPME1 and four transcription factors AdZAT5, AdDOF3, AdNAC083, AdMYBR4 were identified as hub candidate genes for pectin degradation. Dual-luciferase system and electrophoretic mobility shift assays validated that promoters of AdPL5 and Adß-Gal5 were recognized and trans-activated by transcription factor AdZAT5. The relatively higher enzyme activities of PL and ß-Gal were observed in ethylene treated kiwifruit, further emphasized the critical roles of these two pectin related genes for fruit softening. Moreover, stable transient overexpression AdZAT5 in kiwifruit significantly enhanced AdPL5 and Adß-Gal5 expression, which confirmed the in vivo regulations between transcription factor and pectin related genes. CONCLUSION: Thus, modification and application of CCNA would be powerful for the precious phishing the unknown regulators. It revealed that AdZAT5 is a key factor for pectin degradation by binding and regulating effector genes AdPL5 and Adß-Gal5.

Transcriptional and post-transcriptional regulation of ethylene biosynthesis by exogenous acetylsalicylic acid in kiwifruit.

Levels of ethylene, implicated in the induction of fruit ripening in a diverse array of plants, are influenced by genetic and environmental factors, such as other plant hormones. Among these, salicylic acid (SA) and its derivative, acetylsalicylic acid (ASA), have been demonstrated to inhibit ethylene biosynthesis in fruit, yet the underlying regulatory mechanisms remain elusive. Here, we showed that treatment with exogenous ASA dramatically reduced ethylene production, as well as activities of ACC synthase (ACS) and ACC oxidase (ACO), in kiwifruit tissues. Comparative transcriptome analysis indicated the differential expression of ethylene biosynthetic genes (AdACS1/2 and AdACO5). A screen of transcription factors indicated that AdERF105L and AdWRKY29 were ASA-responsive regulators of AdACS1/2 and AdACO5, respectively. In addition to these genes, AdACS3 and AdACO3 were abundantly expressed in both ASA-treated and control tissues. AdACS3 protein was phosphorylated and stabilized by AdMPK16, a mitogen-activated protein kinase, while AdACO3 activity was enhanced by AdAP, an aspartic peptidase. Exogenous ASA downregulated AdMPK16 and AdAP, thereby influencing ethylene biosynthesis at a post-transcriptional level. These findings led us to propose a multidimensional system for inhibition of ethylene biosynthesis by ASA, inducing differential expression of some ethylene biosynthesis genes, as well as differential effects on protein activity on other targets.

The Interaction Between CitMYB52 and CitbHLH2 Negatively Regulates Citrate Accumulation by Activating CitALMT in Citrus Fruit.

Citric acid plays significant roles in numerous physiological processes in plants, including carbon metabolism, signal transduction, and tolerance to environmental stress. For fruits, it has a major effect on fruit organoleptic quality by directly influencing consumer taste. Citric acid in citrus is mainly regulated by the balance between synthesis, degradation, and vacuolar storage. The genetic and molecular regulations of citric acid synthesis and degradation have been comprehensively elucidated. However, the transporters for citric acid in fruits are less well understood. Here, an aluminum-activated malate transporter, CitALMT, was characterized. Transient overexpression and stable transformation of CitALMT significantly reduced citrate concentration in citrus fruits and transgenic callus. Correspondingly, transient RNA interference-induced silencing of CitALMT and increased citrate significantly, indicating that CitALMT plays an important role in regulating citrate concentration in citrus fruits. In addition, dual-luciferase assays indicated that CitMYB52 and CitbHLH2 could trans-activate the promoter of CitALMT. EMSA analysis showed that CitbHLH2 could physically interact with the E-box motif in the CitALMT promoter. Bimolecular fluorescence complementation assays, yeast two-hybrid, coimmunoprecipitation and transient overexpression, and RNAi assay indicated that the interaction between CitMYB52 and CitbHLH2 could synergistically trans-activate CitALMT to negatively regulate citrate accumulation.

The red flesh of kiwifruit is differentially controlled by specific activation-repression systems.

Anthocyanins are visual cues for pollination and seed dispersal. Fruit containing anthocyanins also appeals to consumers due to its appearance and health benefits. In kiwifruit (Actinidia spp.) studies have identified at least two MYB activators of anthocyanin, but their functions in fruit and the mechanisms by which they act are not fully understood. Here, transcriptome and small RNA high-throughput sequencing were used to comprehensively identify contributors to anthocyanin accumulation in kiwifruit. Stable overexpression in vines showed that both 35S::MYB10 and MYB110 can upregulate anthocyanin biosynthesis in Actinidia chinensis fruit, and that MYB10 overexpression resulted in anthocyanin accumulation which was limited to the inner pericarp, suggesting that repressive mechanisms underlie anthocyanin biosynthesis in this species. Furthermore, motifs in the C-terminal region of MYB10/110 were shown to be responsible for the strength of activation of the anthocyanic response. Transient assays showed that both MYB10 and MYB110 were not directly cleaved by miRNAs, but that miR828 and its phased small RNA AcTAS4-D4(-) efficiently targeted MYB110. Other miRNAs were identified, which were differentially expressed between the inner and outer pericarp, and cleavage of SPL13, ARF16, SCL6 and F-box1, all of which are repressors of MYB10, was observed. We conclude that it is the differential expression and subsequent repression of MYB activators that is responsible for variation in anthocyanin accumulation in kiwifruit species.

Molecular basis of the formation and removal of fruit astringency.

Astringency is a dry puckering mouthfeel mainly generated by the binding of tannins with proteins in the mouth. Tannins confer benefits such as resistance to biotic stresses and have antioxidant activity, and moderate concentrations of tannins can improve the flavor of fruits or their products. However, fruits with high contents of tannins have excessive astringency, which is undesirable. Thus, the balance of astringency formation and removal is extremely important for human consumption of fruit and fruit-based products. In recent years, the understanding of fruit astringency has moved beyond the biochemical aspects to focus on the genetic characterization of key structural genes and their transcriptional regulators that cause astringency. This article provides an overview of astringency formation and evaluation. We summarize the methods of astringency regulation and strategies and mechanisms for astringency removal, and discuss perspectives for future exploration and modulation of astringency for fruit quality improvement.

Citrus heat shock transcription factor CitHsfA7-mediated citric acid degradation in response to heat stress.

Heat stress is a major abiotic stress for plants, which can generate a range of biochemical and genetic responses. In 'Ponkan' mandarin fruit, hot air treatment (HAT) accelerates the degradation of citric acid. However, the transcriptional regulatory mechanisms of citrate degradation in response to HAT remain to be elucidated. Here, 17 heat shock transcription factor sequences were isolated, and dual-luciferase assays were employed to investigate whether the encoded proteins that could trans-activate the promoters of key genes in the GABA shunt, involved in citrate metabolism. We identified four heat shock transcription factors (CitHsfA7, CitHsfA3, CitHsfA4b and CitHsfA8) that showed trans-activation effects on CitAco3, CitIDH3 and CitGAD4, respectively. Transient expression of the CitHsfs in citrus fruits indicated that CitHsfA7 was the only factor that resulted in a significant lowering of the citric acid content, and these results were confirmed by a virus-induced gene silencing system (VIGS). Sub-cellar localization showed that CitHsfA7 is located in the nucleus and is capable of binding directly to a putative HSE in the CitAco3 promoter and enhance its expression. We proposed that the induction of CitHsfA7 transcript level contributes to citric acid degradation in citrus fruit, via modulation of CitAco3 in response to HAT.

An EjbHLH14-EjHB1-EjPRX12 module is involved in methyl jasmonate alleviation of chilling-induced lignin deposition in loquat fruit.

Loquat fruit are susceptible to chilling injuries induced by postharvest storage at low temperature. The major symptoms are increased lignin content and flesh firmness, which cause a leathery texture. Pretreatment with methyl jasmonate (MeJA) can alleviate this low-temperature-induced lignification, but the mechanism is not understood. In this study, we characterized a novel class III peroxidase, EjPRX12, and studied its relationship to lignification. Transcript levels of EjPRX12 were attenuated following MeJA pretreatment, consistent with the reduced lignin content in fruit. In vitro enzyme activity assay indicated that EjPRX12 polymerized sinapyl alcohol, and overexpression of EjPRX12 in Arabidopsis promoted lignin accumulation, indicating that it plays a functional role in lignin polymerization. We also identified an HD-ZIP transcription factor, EjHB1, repressed by MeJA pretreatment, which directly bound to and significantly activated the EjPRX12 promoter. Overexpression of EjHB1 in Arabidopsis promoted lignin accumulation with induced expression of lignin-related genes, especially AtPRX64. Furthermore, a JAZ-interacting repressor, EjbHLH14, was characterized, and it is proposed that MeJA pretreatment caused EjbHLH14 to be released to repress the expression of EjHB1. These results identified a novel regulatory pathway involving EjbHLH14-EjHB1-EjPRX12 and revealed the molecular mechanism whereby MeJA alleviated lignification of loquat fruit at low temperature.

Transcription factors AcERF74/75 respond to waterlogging stress and trigger alcoholic fermentation-related genes in kiwifruit.

Kiwifruit plants have a fleshy, shallow root system which is sensitive to waterlogging stress, which results in a decrease in crop yield or even plants death. Although the waterlogging stress responses in kiwifruit have attracted much attention, the underlying molecular mechanism remains unclear. In this study, waterlogging led to drastic inhibition of root growth of 'Donghong' kiwifruit (Actinidia chinensis) plants grown in vitro, which was accompanied by significant elevation of endogenous acetaldehyde and ethanol contents. RNA-seq of roots of plants waterlogged for 0, 1 and 2 days revealed that a total of 149 genes were up- or down-regulated, including seven biosynthetic genes related to the glycolysis/gluconeogenesis pathway and 10 transcription factors. Analyses with real-time PCR, dual-luciferase assays and EMSA demonstrated that AcERF74 and AcERF75, two members of the ERF-VII subfamily, directly upregulated AcADH1 (alcohol dehydrogenase). Moreover, the overexpression of AcERF74/75 in transgenic calli resulted in dramatic increase of endogenous ethanol contents through the triggering of AcADH1 and AcADH2 expression. Although the AcPDC2 (pyruvate decarboxylase) expression was also enhanced in transgenic lines, the endogenous acetaldehyde contents showed no significant changes. These results illustrated that AcERF74/75 are two transcriptional activators on alcoholic fermentation related genes and are responsive to waterlogging stress in kiwifruit.

A tomato LATERAL ORGAN BOUNDARIES transcription factor, SlLOB1, predominantly regulates cell wall and softening components of ripening.

Fruit softening is a key component of the irreversible ripening program, contributing to the palatability necessary for frugivore-mediated seed dispersal. The underlying textural changes are complex and result from cell wall remodeling and changes in both cell adhesion and turgor. While a number of transcription factors (TFs) that regulate ripening have been identified, these affect most canonical ripening-related physiological processes. Here, we show that a tomato fruit ripening-specific LATERAL ORGAN BOUNDRIES (LOB) TF, SlLOB1, up-regulates a suite of cell wall-associated genes during late maturation and ripening of locule and pericarp tissues. SlLOB1 repression in transgenic fruit impedes softening, while overexpression throughout the plant under the direction of the 35s promoter confers precocious induction of cell wall gene expression and premature softening. Transcript and protein levels of the wall-loosening protein EXPANSIN1 (EXP1) are strongly suppressed in SlLOB1 RNA interference lines, while EXP1 is induced in SlLOB1-overexpressing transgenic leaves and fruit. In contrast to the role of ethylene and previously characterized ripening TFs, which are comprehensive facilitators of ripening phenomena including softening, SlLOB1 participates in a regulatory subcircuit predominant to cell wall dynamics and softening.

An ethylene-hypersensitive methionine sulfoxide reductase regulated by NAC transcription factors increases methionine pool size and ethylene production during kiwifruit ripening.

Ethylene plays an important role in regulating fruit ripening by triggering dynamic changes in expression of ripening-associated genes, but the functions of many of these genes are still unknown. Here, a methionine sulfoxide reductase gene (AdMsrB1) was identified by transcriptomics-based analysis as the gene most responsive to ethylene treatment in ripening kiwifruit. The AdMsrB1 protein exhibits a stereospecific activity toward the oxidative stress-induced R enantiomer of methionine sulfoxide (MetSO), reducing it to methionine (Met). Stable overexpression of AdMsrB1 in kiwifruit significantly increased the content of free Met and 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, and increased ethylene production. Dual-luciferase assays indicated that the AdMsrB1 promoter was not directly upregulated by ethylene treatment but was modulated by two ethylene-inducible NAM/ATAF/CUC transcription factors (AdNAC2 and AdNAC72) that bind directly to the AdMsrB1 promoter. Overexpression of AdNAC72 in kiwifruit not only enhanced AdMsrB1 expression, but also increased free Met and ACC content and ethylene production rates. This finding establishes an unexpected regulatory loop that enhances ethylene production and the concentration of its biosynthetic intermediates.

Transcription factors DkBZR1/2 regulate cell wall degradation genes and ethylene biosynthesis genes during persimmon fruit ripening.

BRASSINAZOLE RESISTANT (BZR) transcription factors are critical components of the brassinosteroid signalling pathway, but their possible roles in fruit ripening have rarely been reported. In this study, four BZR sequences were isolated from persimmon fruit. Among the four BZR genes, DkBZR1/2 were expressed in persimmon fruit; DkBZR1 protein amount decreased and dephosphorylated DkBZR2 gradually accumulated during the storage period. DkBZR1/2 proteins were localized in both the nucleus and cytoplasm and accumulated in the nucleus after 24-epibrassinolide treatment. DkBZR1 suppressed the transcription of Diospyros kaki endo-1,4-betaglucanase 1 (DkEGase1) and 1-aminocyclopropane-1-carboxylate synthase 1 (DkACS1) by binding to the BR response element (BRRE) in their promoters, and DkBZR2 activated the transcription of pectate lyase 1 (DkPL1) and 1-aminocyclopropane-1-carboxylate oxidase 2 (DkACO2) by binding to the E-box motif in their promoters. Transient overexpression of DkBZR2 promoted the conversion of acid-soluble pectin to water-soluble pectin and increased ethylene production in persimmon fruit. Our findings indicate that DkBZR1 and DkBZR2 serve as repressors and activators of persimmon fruit ripening, respectively.

Genome-wide analysis of the bZIP gene family and the role of AchnABF1 from postharvest kiwifruit (Actinidia chinensis cv. Hongyang) in osmotic and freezing stress adaptations.

Chilling injury (CI) is a barrier to the refrigeration of kiwifruit, resulting in decreased fruit quality and increased nutrient loss during storage. Understanding the molecular basis underlying the cold response and its regulation in refrigerated kiwifruit is therefore highly important. Basic (region) leucine zipper (bZIP) transcription factors (TFs) have been widely studied for their roles in abiotic stress resistance in various species. In this study, we identified 81 bZIP family proteins in kiwifruit and classified them into 11 groups. Further transcriptome analysis revealed that the expression of members of the AREB/ABF family was strongly induced by low temperature and abscisic acid (ABA). Ectopic expression of AchnABF1 enhanced plant cold tolerance by upregulating the expression of several key genes associated with ABA-dependent and ABA-independent pathways in Arabidopsis thaliana. Reactive oxygen species (ROS) metabolism was suggested to be involved in the AchnABF1-mediated osmotic stress response. For instance, enhanced ROS-scavenging ability was observed in transgenic plants with enhanced activity of catalase (CAT) and peroxidase (POD), which resulted in decreased in situ O2.- and H2O2 accumulation, ion leakage, and malondialdehyde (MDA) content under various abiotic stresses. In addition, AchnABF1 also participated in the osmotic stress response during both the germination and postgermination stages. We concluded that AchnABF1 may play an important role in kiwifruit during refrigeration.

The MADS-Box Transcription Factor EjAGL65 Controls Loquat Flesh Lignification via Direct Transcriptional Inhibition of EjMYB8.

Loquat fruit accumulates lignin in its flesh when undergoing chilling injury during postharvest storage, making it a suitable model for the study of flesh lignification. Transcriptional regulation of lignin biosynthesis is principally controlled by the NAC-MYB transcriptional cascade in model plants. Previous research has demonstrated that EjMYB8 activates lignin biosynthesis through direct interaction with the promoter of Ej4CL1. However, the classic NAC-MYB gene regulation network has not been established. Here, the MADS-box gene EjAGL65 was discovered by screening a cDNA library using the EjMYB8 promoter as bait in yeast. A phylogenetic analysis and structural comparisons revealed that EjAGL65 belongs to the Mδ subgroup of the MADS-box family, whose members have not been reported as being involved in the regulation of lignin deposition. EjAGL65 transcription was downregulated at 0°C compared to 5°C, indicating a negative correlation with the change of lignin content. A dual-luciferase assay indicated that EjAGL65 is capable of inhibiting the promoter activity of EjMYB8 in vivo. These results showed that the Mδ MADS-box gene EjAGL65 transcriptionally regulates EjMYB8 during postharvest chilling induced flesh lignification, which differs from the classical regulation model of lignin biosynthesis that has been illustrated for developmental lignin accumulation.