Salicylic Acid Reduces MdPUB24-mediated Ubiquitination of MdWRKY40 to Suppress Ethylene Biosynthesis in Apple Fruit

Apple cultivars exhibit considerable variation in fruit maturation patterns that could influence at-harvest fruit firmness and postharvest storability. Based on the results from our previous transcriptome profiling of apple fruit maturation and well-documented auxin–ethylene crosstalk in other plant processes, the current experiment attempts to get insight into the potential crosstalk between auxin metabolism and ethylene biosynthesis during apple fruit maturation and ripening. Weekly apple fruit samples were collected to include the early maturation stages until 2 weeks after physiological maturity for both ‘Minneiska’ and ‘Scifresh’ cultivars. The expression patterns for genes with annotated functions of auxin transport, conjugation, biosynthesis, and responses were profiled by qRT-PCR along the apple fruit ripening processes and in different apple fruit tissues (seed, core, cortex, and peel). The expression profiles of both auxin metabolism and ethylene biosynthesis genes correlated with the apple fruit maturation process, but with different expression patterns and strength in each cultivar. Temporal and spatial gene expression patterns from seed to the outer fruit tissues corresponded with the center-outward ripening characteristics of apple fruit. It seems that timely reduction of biologically active auxin, in apple fruit cells of specific tissue, is critical for the activation of ethylene biosynthesis, even though auxin is needed for early fruit development. Our results suggest that the regulation of auxin transport and homeostasis may contribute to the time of activation of the ethylene biosynthesis pathway in maturing apple fruit and consequently influence the time of ripening for a specific cultivar.

The plant hormones regulate most physiological processes including apple fruit ripening by integrating diverse developmental cues and environmental signals. In addition to the well-characterized role of ethylene, jasmonic acid (JA) and its derivatives have also been suggested to play an important role during apple fruit maturation and ripening. Till now, the role JA on apple fruit ripening was only observed through exogenous application of JA or its derivatives. The de novo JA biosynthesis and signal transduction in apple fruit tissues have not been studied. In this study, the members of major gene families implicated in JA biosynthesis were identified from apple genome sequences; and their expression profiles were characterized in ‘Golden Delicious’ using quantitative reverse transcription polymerase chain reactions. Our data indicated that the expression patterns of MdLOX23, MdAOS2 and MdJMT2 in JA biosynthesis pathway are coordinated with those of MdACS3, MdERF1 and MdERF2 genes during 12 consecutive weeks of apple fruit maturation toward commercial harvest. Both MdLOX23 and MdAOS2 showed comparable expression patterns in both core and cortex suggesting localized synthesis of JA in different fruit tissues. Most of JA biosynthesis genes were transiently up-regulated in response to exogenous application of methyl jasmonate to on-tree maturing apple fruit, though in a maturity dependent manner. Our results suggested regulating roles of JA biosynthesis and its signaling on apple fruit maturation and ripening.

Fruit ripening is a complex process involving many physiological changes and the dynamic interplay between different phytohormones. In addition to ethylene, jasmonates (JAs) have also been demonstrated to play an important role in the regulation of fruit ripening. However, the mechanisms underlying the interaction between these two pathways during fruit ripening are unknown. In recent years, research has been conducted to illustrate the effects of JAs on the ethylene biosynthesis and signaling pathway, but little is known regarding the effects of ethylene on JA biosynthesis and the signaling pathway during fruit ripening. Herein, we aimed to evaluate the effects of ethylene on JA biosynthesis in ripening apple ( Malus × domestica ) fruit and on the expression of key genes involved in the JA biosynthesis and the signaling pathway. For this purpose, we treated apple fruit with ethephon and 1-methylcyclopropene (1-MCP) at commercial maturity. Our data indicated that endogenous JA content and allene oxide synthase (AOS) activity were reduced by ethephon treatment at the early ripening stage, whereas they were enhanced by 1-MCP treatment at the late ripening stage. Quantitative real-time polymerase chain reaction (PCR) analysis revealed that the expression profiles of three AOS genes ( MdAOS2 , MdAOS3 , and MdAOS5 ) and two lipoxygenase ( LOX ) genes ( MdLOX22 and MdLOX28 ) showed similar trends with the change of AOS activity in all groups during fruit ripening. The expression of MdLOX21 and MdLOX23 was in accordance with the change of ethylene production on ripening, and it was positively regulated by ethylene, whereas the opposite effect was observed for MdLOX39 expression. The transcription of MdLOX310 and MdLOX61 appeared unaffected by ethylene during fruit ripening. Three jasmonate ZIM-domain ( JAZ ) genes ( MdJAZ9 , MdJAZ10 , and MdJAZ18 ) were differentially upregulated by ethephon treatment whereas being downregulated by 1-MCP treatment during fruit ripening. Expression of MdJAZ13 and MdJAZ14 was downregulated at the early ripening stage by both treatments. Our results suggested regulating roles of ethylene on the JA biosynthesis and signaling pathway during fruit ripening and senescence.

Effect of ethylene and 1-MCP on expression of genes involved in ethylene biosynthesis and perception during ripening of apple fruit

The plant hormone ethylene plays a central role in the ripening of climacteric fruits, such as apple (Malus domestica). Ethylene biosynthesis in apple fruit can be suppressed by calcium ions (Ca2+); however, the underlying mechanism is largely unknown. In this study, we identified an apple APETALA2/ETHYLENE-RESPONSIVE FACTOR (AP2/ERF) transcription factor, MdCYTOKININ RESPONSE FACTOR4 (MdCRF4), which functions as a transcriptional activator of ethylene biosynthesis- and signaling-related genes, including Md1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE1 (MdACS1) and MdETHYLENE-RESPONSIVE FACTOR3 (MdERF3), as a partner of the calcium sensor, calmodulin. Ca2+ promoted the Ca2+/CaM2-mediated phosphorylation of MdCRF4, resulting in MdCRF4 recognition by the E3 ubiquitin ligase MdXB3 ORTHOLOG 1 IN ARABIDOPSIS THALIANA (MdXBAT31), and consequently its ubiquitination and degradation via the 26S proteasome pathway. This in turn resulted in lower expression of MdACS1 and MdERF3 and reduced ethylene biosynthesis. Transiently overexpressing various MdCRF4 proteins with specific mutated phosphorylation sites revealed that the phosphorylation state of MdCRF4 affects the ripening of apple fruit. The results reveal that a Ca2+/CaM-MdCRF4-MdXBAT31 module is involved in Ca2+-suppressed ethylene biosynthesis, which delays apple fruit ripening. This provides insights into fruit ripening that may result in strategies for extending fruit shelf life.

Comparative transcriptome analysis of the climacteric of apple fruit uncovers the involvement of transcription factors affecting ethylene biosynthesis

Ethylene, a plant hormone, is essential for apple (Malus domestica) ripening. The precise molecular mechanism by which melatonin (MT) influences ethylene biosynthesis during apple fruit ripening remains unclear. This study found that exogenous MT treatment inhibited ethylene production and postponed apple fruit ripening. The endogenous MT content of apple fruits exhibited an inverse correlation with ethylene production during fruit ripening, suggesting that MT functions as a ripening suppressor in apple fruits. MT treatment suppressed the expression of key ethylene biosynthesis genes, MdACS1 and MdACO1, during apple fruit ripening. MT treatment decreased the expression levels of transcription factors MdREM10 and MdZF32. MdREM10 binds to the MdERF3 promoter, enhancing its expression and subsequently promoting MdACS1 transcription. Furthermore, MdREM10 directly bound to the MdZF32 promoter, promoting its transcription. MdZF32 directly bound to the MdACO1 promoter, inducing its expression. The findings suggested that MT suppresses ethylene biosynthesis and fruit ripening by inhibiting MdREM10, which indirectly promotes MdACS1 transcription via MdERF3 upregulation, and MdACO1 transcription via MdZF32 upregulation.

Effects of methyl jasmonate on expression of genes involved in ethylene biosynthesis and signaling pathway during postharvest ripening of apple fruit

Ripening in climacteric fruit requires the gaseous phytohormone ethylene. Although ethylene signaling has been well studied, knowledge of the transcriptional regulation of ethylene biosynthesis is still limited. Here we show that an apple (Malus domestica) ethylene response factor, MdERF2, negatively affects ethylene biosynthesis and fruit ripening by suppressing the transcription of MdACS1, a gene that is critical for biosynthesis of ripening-related ethylene. Expression of MdERF2 was suppressed by ethylene during ripening of apple fruit, and we observed that MdERF2 bound to the promoter of MdACS1 and directly suppressed its transcription. Moreover, MdERF2 suppressed the activity of the promoter of MdERF3, a transcription factor that we found to bind to the MdACS1 promoter, thereby increasing MdACS1 transcription. We determined that the MdERF2 and MdERF3 proteins directly interact, and this interaction suppresses the binding of MdERF3 to the MdACS1 promoter. Moreover, apple fruit with transiently downregulated MdERF2 expression showed higher ethylene production and faster ripening. Our results indicate that MdERF2 negatively affects ethylene biosynthesis and fruit ripening in apple by suppressing the transcription of MdACS1 via multiple mechanisms, thereby acting as an antagonist of positive ripening regulators. Our findings offer a deep understanding of the transcriptional regulation of ethylene biosynthesis during climacteric fruit ripening.

Genome-wide identification and characterization of long noncoding RNAs involved in apple fruit development and ripening

Apple is one of the most economically important horticultural fruit crops worldwide. It is critical to gain insights into fruit ripening and softening to improve apple fruit quality and extend shelf life. In this study, forward and reverse suppression subtractive hybridization libraries were generated from ‘Taishanzaoxia’ apple fruits sampled around the ethylene climacteric to isolate ripening- and softening-related genes. A set of 648 unigenes were derived from sequence alignment and cluster assembly of 918 expressed sequence tags. According to gene ontology functional classification, 390 out of 443 unigenes (88%) were assigned to the biological process category, 356 unigenes (80%) were classified in the molecular function category, and 381 unigenes (86%) were allocated to the cellular component category. A total of 26 unigenes differentially expressed during fruit development period were analyzed by quantitative RT-PCR. These genes were involved in cell wall modification, anthocyanin biosynthesis, aroma production, stress response, metabolism, transcription, or were non-annotated. Some genes associated with cell wall modification, anthocyanin biosynthesis and aroma production were up-regulated and significantly correlated with ethylene production, suggesting that fruit texture, coloration and aroma may be regulated by ethylene in ‘Taishanzaoxia’. Some of the identified unigenes associated with fruit ripening and softening have not been characterized in public databases. The results contribute to an improved characterization of changes in gene expression during apple fruit ripening and softening.

Fruit ripening is governed by a complex regulatory network, and ethylene plays an important role in this process. MdKING1 is a γ subunit of SNF1-related protein kinases (SnRKs), but the function was unclear. Here, we characterized the role of MdKING1 during fruit ripening, which can promote fruit ripening through the ethylene pathway. Our findings reveal that MdKING1 has higher expression in early-ripening cultivars than late-ripening during the early stage of apple fruit development, and its transcription level significantly increased during apple fruit ripening. Overexpression of MdKING1 (MdKING1 OE) in tomatoes could promote early ripening of fruits, with the increase in ethylene content and the loss of fruit firmness. Ethylene inhibitor treatment could delay the fruit ripening of both MdKING1 OE and WT fruits. However, MdKING1 OE fruits turned fruit ripe faster, with an increase in carotenoid content compared with WT. In addition, the expression of genes involved in ethylene biosynthesis (SlACO1, SlACS2, and SlACS4), carotenoid biosynthesis (SlPSY1 and SlGgpps2a), and fruit firmness regulation (SlPG2a, SlPL, and SlCEL2) was also increased in the fruits of MdKING1 OE plants. In conclusion, our results suggest that MdKING1 plays a key role in promoting tomato fruit ripening, thus providing a theoretical basis for apple fruit quality improvement by genetic engineering in the future.

Zinc finger-homeodomain (ZF-HD) transcription factors play an important role in the regulation of plant growth and development, as well as the regulation of stress responses. Studies on the ZF-HD family genes have been conducted in many plants, however, the characteristics of this family in apple (Malus domestica) fruit remains to be poorly understood. In this study, we identified nineteen ZF-HD family genes in apple at the whole-genome scale, which were unevenly located on ten chromosomes. These MdZF-HD genes were phylogenetically divided into two subfamilies: zinc finger-homeodomain (ZHD) and MINI ZINC FINGER (MIF), and the ZHD subfamily was further classified into five groups (ZHDI–ZHDV). Analysis of the gene structures showed that most MdZF-HD genes lack introns. Gene expression analysis indicated that nine selected MdZF-HD genes were differentially responsive to 1-MCP (1-methylcyclopropene) treatment during the postharvest storage of “Qinguan” apple fruit. Moreover, the transcripts of six genes were further validated in “Golden Delicious” apple fruit, and five genes (MdZHD1/2/6/10/11) were significantly repressed and one gene (MdZHD7) was slightly induced by ethylene treatment. These results indicated that these six MdZF-HD genes may involve in the regulation of ethylene induced ripening process of postharvest apple fruit. These findings provide new clues for further functional investigation of ZF-HD genes, such as their roles in the regulation of fruit ripening.

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.

Histone deacetylase enzymes participate in the regulation of many aspects of plant development. However, the genome-level targets of histone deacetylation during apple (Malus domestica) fruit development have not been resolved in detail, and the mechanisms of regulation of such a process are unknown. We previously showed that the complex of ethylene response factor 4 (MdERF4) and the TOPLESS co-repressor (MdTPL4; MdERF4–MdTPL4) is constitutively active during apple fruit development (Hu et al., 2020), but whether this transcriptional repression complex is coupled to chromatin modification is unknown. Here, we show that a histone deacetylase (MdHDA19) is recruited to the MdERF4–MdTPL4 complex, thereby impacting fruit ethylene biosynthesis. Transient suppression of MdHDA19 expression promoted fruit ripening and ethylene production. To identify potential downstream target genes regulated by MdHDA19, we conducted chromatin immunoprecipitation (ChIP) sequencing of H3K9 and ChIP-quantitative polymerase chain reaction assays. We found that MdHDA19 affects ethylene production by facilitating H3K9 deacetylation and forms a complex with MdERF4–MdTPL4 to directly repress MdACS3a expression by decreasing the degree of acetylation. We demonstrate that an early-maturing-specific acetylation H3K9ac peak in MdACS3a and expression of MdACS3a were specifically up-regulated in fruit of an early-maturing, but not a late-maturing, cultivar. We provide evidence that a C-to-G mutation in the ethylene-responsive element binding factor-associated amphiphilic repression motif of MdERF4 reduces the repression of MdACS3a by the MdERF4–MdTPL4–MdHDA19 complex. Taken together, our results reveal that the MdERF4–MdTPL–MdHDA19 repressor complex participates in the epigenetic regulation of apple fruit ripening.