MdPRX34L, a class III peroxidase gene, activates the immune response in apple to the fungal pathogen Botryosphaeria dothidea.

MAIN CONCLUSION: MdPRX34L enhanced resistance to Botryosphaeria dothidea by increasing salicylic acid (SA) and abscisic acid (ABA) content as well as the expression of related defense genes. The class III peroxidase (PRX) multigene family is involved in complex biological processes. However, the molecular mechanism of PRXs in the pathogen defense of plants against Botryosphaeria dothidea (B. dothidea) remains unclear. Here, we cloned the PRX gene MdPRX34L, which was identified as a positive regulator of the defense response to B. dothidea, from the apple cultivar 'Royal Gala.' Overexpression of MdPRX34L in apple calli decreased sensitivity to salicylic acid (SA) and abscisic acid（ABA). Subsequently, overexpression of MdPRX34L in apple calli increased resistance to B. dothidea infection. In addition, SA contents and the expression levels of genes related to SA synthesis and signaling in apple calli overexpressing MdPRX34L were higher than those in the control after inoculation, suggesting that MdPRX34L enhances resistance to B. dothidea via the SA pathway. Interestingly, infections in apple calli by B. dothidea caused an increase in endogenous levels of ABA followed by induction of ABA-related genes expression. These findings suggest a potential mechanism by which MdPRX34L enhances plant-pathogen defense against B. dothidea by regulating the SA and ABA pathways.

Optimization of apple fruit flavor by MdVHP1-2 via modulation of soluble sugar and organic acid accumulation.

For fleshy fruits, the content and ratio of organic acids and soluble sugars are key factors for their flavor. Therefore, a better understanding of soluble sugar and organic acid accumulation in vacuoles is essential to the improvement of fruit quality. Vacuolar-type inorganic pyrophosphatase (V-PPase) has been found in various plants with crucial functions based on the hydrolysis of PPi. However, the effects of V-PPase on the soluble sugar and organic acid accumulation in apple fruit remain unclear. In this study, MdVHP1-2, a V-PPase protein in the vacuolar membrane, was identified. The results showed a positive correlation between the expression of MdVHP1-2 and the sugar/acid ratio during ripening of apple fruits. A series of transgenic analyses showed that overexpression of MdVHP1-2 significantly elevated the contents of soluble sugars and organic acids as well as the sugar/acid ratio in apple fruits and calli. Additionally, transient interference induced by MdVHP1-2 expression inhibited the accumulation of soluble sugars and organic acids in apple fruits. In summary, this study provides insight into the mechanisms by which MdVHP1-2 modulates fruit flavor through mediation of soluble sugar and organic acid accumulation, thereby facilitating improvement of the overall quality of apple and other fruits.

Regulation of a vacuolar proton-pumping P-ATPase MdPH5 by MdMYB73 and its role in malate accumulation and vacuolar acidification.

As the main organic acid in fruits, malate is produced in the cytoplasm and is then transported into the vacuole. It accumulates by vacuolar proton pumps, transporters, and channels, affecting the taste and flavor of fruits. Among the three types of proton pumps (V-ATPases, V-PPases, and P-ATPases), the P-ATPases play an important role in the transport of malate into vacuoles. In this study, the transcriptome data, collected at different stages after blooming and during storage, were analyzed and the results demonstrated that the expression of MdPH5, a vacuolar proton-pumping P-ATPase, was associated with both pre- and post-harvest malate contents. Moreover, MdPH5 is localized at the tonoplast and regulates malate accumulation and vacuolar pH. In addition, MdMYB73, an upstream MYB transcription factor of MdPH5, directly binds to its promoter, thereby transcriptionally activating its expression and enhancing its activity. In this way, MdMYB73 can also affect malate accumulation and vacuolar pH. Overall, this study clarifies how MdMYB73 and MdPH5 act to regulate vacuolar malate transport systems, thereby affecting malate accumulation and vacuolar pH. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-023-00115-7.

Ethylene inhibits malate accumulation in apple by transcriptional repression of aluminum-activated malate transporter 9 via the WRKY31-ERF72 network.

Malic acid accumulation in the vacuole largely determines acidity and perception of sweetness of apple. It has long been observed that reduction in malate level is associated with increase in ethylene production during the ripening process of climacteric fruits, but the molecular mechanism linking ethylene to malate reduction is unclear. Here, we show that ethylene-modulated WRKY transcription factor 31 (WRKY31)-Ethylene Response Factor 72 (ERF72)-ALUMINUM ACTIVATED MALATE TRANSPORTER 9 (Ma1) network regulates malate accumulation in apple fruit. ERF72 binds to the promoter of ALMT9, a key tonoplast transporter for malate accumulation of apple, transcriptionally repressing ALMT9 expression in response to ethylene. WRKY31 interacts with ERF72, suppressing its transcriptional inhibition activity on ALMT9. In addition, WRKY31 directly binds to the promoters of ERF72 and ALMT9, transcriptionally repressing and activating ERF72 and ALMT9, respectively. The expression of WRKY31 decreases in response to ethylene, lowering the transcription of ALMT9 directly and via its interactions with ERF72. These findings reveal that the regulatory complex WRKY31 forms with ERF72 responds to ethylene, linking the ethylene signal to ALMT9 expression in reducing malate transport into the vacuole during fruit ripening.

MdbHLH3 modulates apple soluble sugar content by activating phosphofructokinase gene expression.

Sugars are involved in plant growth, fruit quality, and signaling perception. Therefore, understanding the mechanisms involved in soluble sugar accumulation is essential to understand fruit development. Here, we report that MdPFPß, a pyrophosphate-dependent phosphofructokinase gene, regulates soluble sugar accumulation by enhancing the photosynthetic performance and sugar-metabolizing enzyme activities in apple (Malus domestica Borkh.). Biochemical analysis revealed that a basic helix-loop-helix (bHLH) transcription factor, MdbHLH3, binds to the MdPFPß promoter and activates its expression, thus promoting soluble sugar accumulation in apple fruit. In addition, MdPFPß overexpression in tomato influenced photosynthesis and carbon metabolism in the plant. Furthermore, we determined that MdbHLH3 increases photosynthetic rates and soluble sugar accumulation in apple by activating MdPFPß expression. Our results thus shed light on the mechanism of soluble sugar accumulation in apple leaves and fruit: MdbHLH3 regulates soluble sugar accumulation by activating MdPFPß gene expression and coordinating carbohydrate allocation.

Yang cycle enzyme DEP1: its moonlighting functions in PSI and ROS production during leaf senescence.

Ethylene-mediated leaf senescence and the compromise of photosynthesis are closely associated but the underlying molecular mechanism is a mystery. Here we reported that apple DEHYDRATASE-ENOLASE-PHOSPHATASE-COMPLEX1 (MdDEP1), initially characterized to its enzymatic function in the recycling of the ethylene precursor SAM, plays a role in the regulation of photosystem I (PSI) activity, activating reactive oxygen species (ROS) homeostasis, and negatively regulating the leaf senescence. A series of Y2H, Pull-down, CO-IP and Cell-free degradation biochemical assays showed that MdDEP1 directly interacts with and dephosphorylates the nucleus-encoded thylakoid protein MdY3IP1, leading to the destabilization of MdY3IP1, reduction of the PSI activity, and the overproduction of ROS in plant cells. These findings elucidate a novel mechanism that the two pathways intersect at MdDEP1 due to its moonlighting role in destabilizing MdY3IP1, and synchronize ethylene-mediated leaf senescence and the compromise of photosynthesis.

Genome-Wide Analysis of the Glutathione S-Transferase (GST) Genes and Functional Identification of MdGSTU12 Reveals the Involvement in the Regulation of Anthocyanin Accumulation in Apple.

Anthocyanins have essential biological functions, affecting the development of horticultural production. They are synthesized in the cytoplasm through flavonoid metabolic pathways and finally transported into vacuoles for storage. Plant glutathione S-transferases (GSTs) are multifunctional enzymes involved in anthocyanin transportation. In this study, we identified 38 GSTs from the apple (Malus domestica) genome (HFTH1 Whole Genome v1.0) based on the sequence similarity with the GST family proteins of Arabidopsis. These MdGST genes could be grouped into nine chief subclasses: U, F, L, Z, T, GHR, EF1Bγ, TCHQD, and DHAR. The structures, motifs, three-dimensional models, and chromosomal distribution of MdGST genes were further analyzed. Elements which are responsive for some hormones and stress, and others that involve genes related to flavonoid biosynthesis were forecast in the promoter of MdGST. In addition, we identified 32 orthologous gene pairs between apple and Arabidopsis. These genes indicated that numerous apple and Arabidopsis counterparts appeared to be derived from a common ancestor. Amongst the 38 MdGST genes, MdGSTU12 was considerably correlated with anthocyanin variation in terms of extracting expression profiles from reported. Finally, further functional identification in apple transgenic calli and subcellular localization confirmed that MdGSTU12 was of great significance in anthocyanin accumulation in apple.

Anthocyanin stability and degradation in plants.

Anthocyanins, a flavonoid group of polyphenolic compounds, have evolved in plants since the land was colonized by plants. These bioactive compounds play critical roles in diverse physiological processes. They are synthesized in the cytosol and transported into the vacuole for storage or to other destinations, where they function as bioactive molecules. The mechanisms of anthocyanin synthesis and transport have been well studied. However, the precise regulation of the mechanisms of anthocyanin degradation remains to be elucidated. In this review, we highlight recent progress in the understanding of the characteristics and functions of anthocyanins and class III peroxidases, as well as of the existing evidence of the effects of class III peroxidases on the degradation of anthocyanins and the possible regulatory mechanisms involved.

Mechanisms and regulation of organic acid accumulation in plant vacuoles.

In fleshy fruits, organic acids are the main source of fruit acidity and play an important role in regulating osmotic pressure, pH homeostasis, stress resistance, and fruit quality. The transport of organic acids from the cytosol to the vacuole and their storage are complex processes. A large number of transporters carry organic acids from the cytosol to the vacuole with the assistance of various proton pumps and enzymes. However, much remains to be explored regarding the vacuolar transport mechanism of organic acids as well as the substances involved and their association. In this review, recent advances in the vacuolar transport mechanism of organic acids in plants are summarized from the perspectives of transporters, channels, proton pumps, and upstream regulators to better understand the complex regulatory networks involved in fruit acid formation.

The C2H2-type zinc finger transcription factor MdZAT10 negatively regulates drought tolerance in apple.

Various abiotic stressors, particularly drought stress, affect plant growth and yield. Zinc finger proteins play an important role in plant abiotic stress tolerance. Here, we isolated the apple MdZAT10 gene, a C2H2-type zinc finger protein, which is a homolog of Arabidopsis STZ/ZAT10. MdZAT10 was localized to the nucleus and highly expressed in leaves and fruit. Promoter analysis showed that MdZAT10 contained several response elements and the transcription level of MdZAT10 was induced by abiotic stress and hormone treatments. MdZAT10 was responsive to drought treatment both at the transcriptional and post-translational levels. MdZAT10-overexpressing apple calli decreased the expression level of MdAPX2 and increased sensitivity to PEG 6000 treatment. Moreover, ectopically expressed MdZAT10 in Arabidopsis reduced the tolerance to drought stress, and exhibited higher water loss, higher malondialdehyde (MDA) content and higher reactive oxygen species (ROS) accumulation under drought stress. In addition, MdZAT10 reduced the sensitivity to abscisic acid in apple. Ectopically expressed MdZAT10 in Arabidopsis promoted seed germination and seedling growth. These results indicate that MdZAT10 plays a negative regulator in the drought resistance, which can provide theoretical basis for further molecular mechanism research.

R2R3-MYB Transcription Factor MdMYB73 Confers Increased Resistance to the Fungal Pathogen Botryosphaeria dothidea in Apples via the Salicylic Acid Pathway.

MYB transcription factors (TFs) participate in many biological processes. However, the molecular mechanisms by which MYB TFs affect plant resistance to apple ring rot remain poorly understood. Here, the R2R3-MYB gene MdMYB73 was cloned from "Royal Gala" apples and functionally characterized as a positive regulator of the defense response to Botryosphaeria dothidea. qRT-PCR and GUS staining demonstrated that MdMYB73 was strongly induced in apple fruits and transgenic calli after inoculation with B. dothidea. MdMYB73 overexpression improved resistance to B. dothidea in apple calli and fruits, while MdMYB73 suppression weakened. Increased resistance to B. dothidea was also observed in MdMYB73-expressing Arabidopsis thaliana. Interestingly, salicylic acid (SA) contents and the expression levels of genes related with SA synthesis and signaling were greater in MdMYB73-overexpressing plant materials compared to wild-type controls after inoculation, suggesting that MdMYB73 might enhance resistance to B. dothidea via the SA pathway. Finally, we discovered that MdMYB73 interacts with MdWRKY31, a positive regulator of B. dothidea. Together, MdWRKY31 and MdMYB73 enhanced B. dothidea resistance in apples. Our results clarify the mechanisms by which MdMYB73 improves resistance to B. dothidea and suggest that resistance may be affected by regulating the SA pathway.

The BTB-TAZ protein MdBT2 negatively regulates the drought stress response by interacting with the transcription factor MdNAC143 in apple.

Drought stress is a severe source of abiotic stress that can affect apple yield and quality, yet the underlying molecular mechanism of the drought stress response and the role of MdBT2 in the process remain unclear. Here, we find that MdBT2 negatively regulates the drought stress response. Both in vivo and in vitro assays indicated that MdBT2 interacted physically with and ubiquitinated MdNAC143, a member of the NAC TF family that is a positive regulator under drought stress. In addition, MdBT2 promotes the degradation of MdNAC143 via the 26S proteasome system. A series of transgenic assays in apple calli and Arabidopsis verify that MdBT2 confers susceptibility to drought stress at least in part by the regulation of MdNAC143. Overall, our findings provide new insight into the mechanism of MdBT2, which functions antagonistically to MdNAC143 in regulating drought stress by regulating the potential downstream target protein MdNAC143 for proteasomal degradation in apple.

Auxin regulates anthocyanin biosynthesis through the auxin repressor protein MdIAA26.

Auxin plays an important role in plant growth and development; for example, it regulates the elongation and division of plant cells, the formation of plantlet's geotropism and phototropism, and the growth of main lateral roots and hypocotyl. IAA gene is associated with auxin and can response to biotic and abiotic stress in plants. However, the regulatory effect of auxin on anthocyanin accumulation has been rarely reported. In this study, we show that auxin inhibites the accumulation of anthocyanin and decreases the expression of genes related to anthocyanin synthesis in calli, leaves, and seedlings of apple. The expression levels of MdIAA family genes were determined, and we found that MdIAA26 significantly responded to auxin, which also induced MdIAA26 degradation. Functional analysis of MdIAA26 showed that overexpressing MdIAA26 in apple calli and Arabidopsis could promote the accumulation of anthocyanin and up-regulate the genes related to anthocyanin synthesis. Furthermore, the MdIAA26-overexpressing Arabidopsis could counteract auxin-induced inhibition on anthocyanin accumulation, which indicates that auxin inhibits the accumulation of anthocyanin in apple by degrading MdIAA26 protein.

Apple ethylene response factor MdERF11 confers resistance to fungal pathogen Botryosphaeria dothidea.

Ethylene response factor (ERF) is a plant-specific transcription factor involved in many biological processes including root formation, hypocotyl elongation, fruit ripening, organ senescence and stress responses, as well as fruit quality formation. However, its underlying mechanism in plant pathogen defense against Botryosphaeria dothidea (B. dothidea) remains poorly understood. Here, we isolate MdERF11, an apple nucleus-localized ERF transcription factor, from apple cultivar 'Royal Gala'. qRT-PCR assays show that the expression of MdERF11 is significantly induced in apple fruits after B. dothidea infection. Overexpression of MdERF11 gene in apple calli significantly increases the resistance to B.dothidea infection, while silencing MdERF11 in apple calli results in reduced resistance. Ectopic expression of MdERF11 in Arabidopsis also exhibits enhanced resistance to B. dothidea infection compared to that of wild type. Infections in apple calli and Arabidopsis leaves by B. dothidea respectively cause an increase in endogenous levels of salicylic acid (SA) followed by induction of SA synthesis-related and signaling-related gene expression. Taken together, these findings illustrate a potential mechanism by which MdERF11 elevates plant pathogen defense against B. dothidea by regulating SA synthesis pathway.

BTB-BACK Domain E3 Ligase MdPOB1 Suppresses Plant Pathogen Defense against Botryosphaeria dothidea by Ubiquitinating and Degrading MdPUB29 Protein in Apple.

Apple ring rot is a severe disease that affects the yield and quality of apple fruits worldwide. However, the underlying molecular mechanism that involved in this process still remains largely unexplored. Here, we report that apple POZ/BTB CONTAINING-PROTEIN 1 (MdPOB1), a BTB-BACK domain E3 ligase protein, functions to suppress apple pathogen defense against Botryosphaeria dothidea (B. dothidea). Both in vitro and in vivo assays indicated that MdPOB1 interacted directly with and degraded apple U-box E3 ligase MdPUB29, a well-established positive regulator of plant innate immunity, through the ubiquitin/26S proteasome pathway. A series of transgenic analyses in apple fruits demonstrated that MdPOB1 affected apple pathogen defense against B. dothidea at least partially, if not completely, via regulating MdPUB29. Additionally, it was found that the apple pathogen defense against B. dothidea was correlated with the H2O2 contents and the relative expression of salicylic acid (SA) synthesis- and SA signaling-related genes, which might be regulated via degradation of MdPUB29 by MdPOB1. Overall, our findings provide new insights into the mechanism of the MdPOB1 modulation of apple ring rot resistance, which occur by directly regulating potential downstream target protein MdPUB29 for proteasomal degradation in apple.