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SUMMARY: Drought stress is one of the dominating challenges to the growth and productivity in crop plants. Elucidating the molecular mechanisms of plants responses to drought stress is fundamental to improve fruit quality. However, such molecular mechanisms are poorly understood in apple (Malus domestica Borkh.). In this study, we explored that the BTB‐BACK‐TAZ protein, MdBT2, negatively modulates the drought tolerance of apple plantlets. Moreover, we identified a novel Homeodomain‐leucine zipper (HD‐Zip) transcription factor, MdHDZ27, using a yeast two‐hybrid (Y2H) screen with MdBT2 as the bait. Overexpression of MdHDZ27 in apple plantlets, calli, and tomato plantlets enhanced their drought tolerance by promoting the expression of drought tolerance‐related genes [responsive to dehydration 29A (MdRD29A) and MdRD29B]. Biochemical analyses demonstrated that MdHDZ27 directly binds to and activates the promoters of MdRD29A and MdRD29B. Furthermore, in vitro and in vivo assays indicate that MdBT2 interacts with and ubiquitinates MdHDZ27, via the ubiquitin/26S proteasome pathway. This ubiquitination results in the degradation of MdHDZ27 and weakens the transcriptional activation of MdHDZ27 on MdRD29A and MdRD29B. Finally, a series of transgenic analyses in apple plantlets further clarified the role of the relationship between MdBT2 and MdHDZ27, as well as the effect of their interaction on drought resistance in apple plantlets. Collectively, our findings reveal a novel mechanism by which the MdBT2‐MdHDZ27 regulatory module controls drought tolerance, which is of great significance for enhancing the drought resistance of apple and other plants. Significance Statement: Collectively, our findings clearly decipher a novel regulatory network for drought tolerance in the apple plant, which centers on the regulatory module MdBT2‐MdHDZ27 and its roles in transcriptional regulation and post‐transcriptional modification, which will be useful for improving the drought tolerances of apple and other economically important plant species. [ABSTRACT FROM AUTHOR]

Summary: 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. See also the Commentary on this article by Martinoia & Neuhaus, 239: 821–823. [ABSTRACT FROM AUTHOR]

Malate, the predominant organic acid in many fruits, is a crucial component of the organoleptic quality of fruit, including taste and flavor. The genetic and environmental mechanisms affecting malate metabolism in fruit cells have been studied extensively. However, the transcriptional regulation of malate-metabolizing enzymes and vacuolar transporters remains poorly understood. Our previous studies demonstrated that Md MYB1 modulates anthocyanin accumulation and vacuolar acidification by directly activating vacuolar transporters, including Md VHA-B1, Md VHA-E, Md VHP1 and Mdt DT. Interestingly, we isolated and identified a MYB transcription factor, Md MYB73, a distant relative of Md MYB1 in this study. It was subsequently found that Md MYB73 protein bound directly to the promoters of Md ALMT9 (aluminum-activated malate transporter 9), Md VHA-A (vacuolar ATPase subunit A) and Md VHP1 (vacuolar pyrophosphatase 1), transcriptionally activating their expression and thereby enhancing their activities. Analyses of transgenic apple calli demonstrated that Md MYB73 influenced malate accumulation and vacuolar pH. Furthermore, Md CIb HLH1 interacted with Md MYB73 and enhanced its activity upon downstream target genes. These findings help to elucidate how Md MYB73 directly modulates the vacuolar transport system to affect malate accumulation and vacuolar pH in apple. [ABSTRACT FROM AUTHOR]

Glucose induces anthocyanin accumulation in many plant species; however, the molecular mechanism involved in this process remains largely unknown. Here, we found that apple hexokinase MdHXK1, a glucose sensor, was involved in sensing exogenous glucose and regulating anthocyanin biosynthesis. In vitro and in vivo assays suggested that MdHXK1 interacted directly with and phosphorylated an anthocyanin-associated bHLH transcription factor (TF) MdbHLH3 at its Ser361 site in response to glucose. Furthermore, both the hexokinase_2 domain and signal peptide are crucial for the MdHXK1-mediated phosphorylation of MdbHLH3. Moreover, phosphorylation modification stabilized MdbHLH3 protein and enhanced its transcription of the anthocyanin biosynthesis genes, thereby increasing anthocyanin biosynthesis. Finally, a series of transgenic analyses in apple calli and fruits demonstrated that MdHXK1 controlled glucose-induced anthocyanin accumulation at least partially, if not completely, via regulating MdbHLH3. Overall, our findings provide new insights into the mechanism of the glucose sensor HXK1 modulation of anthocyanin accumulation, which occur by directly regulating the anthocyanin-related bHLH TFs in response to a glucose signal in plants. [ABSTRACT FROM AUTHOR]

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 MdPFPβ, 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 MdPFPβ promoter and activates its expression, thus promoting soluble sugar accumulation in apple fruit. In addition, MdPFPβ 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 MdPFPβ 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 MdPFPβ gene expression and coordinating carbohydrate allocation. [ABSTRACT FROM AUTHOR]

Summary: Changes in carbohydrates and organic acids largely determine the palatability of edible tissues of horticulture crops. Elucidating the potential molecular mechanisms involved in the change in carbohydrates and organic acids, and their temporal and spatial crosstalk are key steps in understanding fruit developmental processes. Here, we used apple (Malus domestica Borkh.) as research materials and found that MdbHLH3, a basic helix–loop–helix transcription factor (bHLH TF), modulates the accumulation of malate and carbohydrates. Biochemical analyses demonstrated that MdbHLH3 directly binds to the promoter of MdcyMDH that encodes an apple cytosolic NAD‐dependent malate dehydrogenase, activating its transcriptional expression, thereby promoting malate accumulation in apple fruits. Additionally, MdbHLH3 overexpression increased the photosynthetic capacity and carbohydrate levels in apple leaves and also enhanced the carbohydrate accumulation in fruits by adjusting carbohydrate allocation from sources to sinks. Overall, our findings provide new insights into the mechanism of how the bHLH TF MdbHLH3 modulates the fruit quality. It directly regulates the expression of cytosolic malate dehydrogenase MdcyMDH to coordinate carbohydrate allocation and malate accumulation in apple. [ABSTRACT FROM AUTHOR]

Highlights • MdDEP1 is a Yang's cycle gene and involved in ethylene biosynthesis. • The apple Yang cycle's gene MdDEP1 is involved in the stress responses to salinity and drought. • MdDEP1 triggers early flowering in Arabidopsis. Abstract DEHYDRATASE-ENOLASE-PHOSPHATASE−COMPLEX1 (DEP1), a trifunctional enzyme with dehydratase, enolase, and phosphatase activities, initially characterizes with its enzymatic function in the recycling of the ethylene precursor methionine (Met). Here, we identified an apple MdDEP1 gene, which encodes a protein highly homologous to Arabidopsis Yang's cycle gene AtDEP1. GUS staining of 35S::pMdDEP1-GUS transgenic Arabidopsis showed that MdDEP1 is widely expressed in Arabidopsis tissues especially in vascular systems. Additionally, ectopic expression of MdDEP1 alters salt and drought tolerance, and triggers early flowering in Arabidopsis on the medium. The increase of salt tolerance in MdDEP1 -expressing plants was realized through upregulation of the typical salt overly sensitive (SOS) pathway-related genes, including AtSOS1 , AtSOS2 , and AtSOS3 ; and that MdDEP1 controlled flowering time mainly by influencing the expression of flowering-associated genes. Collectively, these findings provide new functions of the DEP1 protein and its underlying mechanisms, in which MdDEP1 confers salt and drought-tolerance, and early flowering phenotypes. [ABSTRACT FROM AUTHOR]

Summary: The plant hormone ethylene is critical for climacteric fruit ripening, while glucose and anthocyanins determine the fruit quality of climacteric fruits such as apple. Understanding the exact molecular mechanism for this process is important for elucidating the interconnection of ethylene and fruit quality.Overexpression of apple MdbHLH3 gene, an anthocyanin‐related basic helix–loop–helix transcription factor (bHLH TF) gene, promotes ethylene production, and transgenic apple plantlets and trees exhibit ethylene‐related root developmental abnormalities, premature leaf senescence, and fruit ripening. Biochemical analyses demonstrate that MdbHLH3 binds to the promoters of three genes that are involved in ethylene biosynthesis, including MdACO1, MdACS1, and MdACS5A, activating their transcriptional expression, thereby promoting ethylene biosynthesis.High glucose‐inhibited U‐box‐type E3 ubiquitin ligase MdPUB29, the ortholog of Arabidopsis AtPUB29 in apple, influences the expression of ethylene biosynthetic genes and ethylene production by direct ubiquitination of the MdbHLH3 protein.Our findings provide new insights into the ubiquitination of MdbHLH3 by glucose‐inhibited ubiquitin E3 ligase MdPUB29 in the regulation of ethylene biosynthesis as well as indicate that the regulatory module MdPUB29‐MdbHLH3 connects ethylene biosynthesis with fruit quality in apple. [ABSTRACT FROM AUTHOR]

Background: The roles in photosystem I (PSI) assembly of the nucleus-encoded thylakoid protein Y3IP1 who interacts with the plastid-encoded Ycf3 protein that has been well-characterized in plants. However, its function and potential mechanisms in other aspects remain poorly understood. Results: We identified the apple MdY3IP1 gene, which encodes a protein highly homologous to the Arabidopsis Y3IP1 (AtY3IP1). Ectopic expression of MdY3IP1 triggered early-flowering and enhanced salt tolerance in Arabidopsis plants. MdY3IP1 controlled floral transition by accelerating sugar metabolism process in plant cells, thereby influencing the expression of flowering-associated genes. The increase in salt stress tolerance in MdY3IP1-expressing plants correlated with reduced reactive oxygen species (ROS) accumulation, and an increase in lateral root development by regulating both auxin biosynthesis and transport, as followed by enhancement of salt tolerance in Arabidopsis. Overall, these findings provide new evidences for additional functions of Y3IP1-like proteins and their underlying mechanisms of which Y3IP1 confers early-flowering and salt tolerance phenotypes in plants. Conclusions: These observations suggest that plant growth and stress resistance can be affected by the regulation of the MdY3IP1 gene. Further molecular and genetic approaches will accelerate our knowledge of MdY3IP1 functions in PSI complex formation and plants stress resistance, and inform strategies for creating transgenic crop varieties with early maturity and high-resistant to adverse environmental conditions. [ABSTRACT FROM AUTHOR]

Vacuolar H + -ATPase (V-H + -ATPase) is highly conserved, multisubunit proton pumps that play key roles in adaptation to abiotic stress in plants. Function of apple V-ATPase subunit B1 ( MdVHA-B1 ) has been identified in drought tolerance, however, it remains unclear whether MdVHA-B1 is involved in salt tolerance. In this work, we characterized the function of MdVHA-B1 gene in salt stress response. GUS and Western blotting assays showed that MdVHA-B1 was highly induced by salt stress. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays demonstrated that MdVHA-B1 interacts with MdSOS2L1, a Arabidopsis SOS2-like protein kinase, in apple. Compared with wild-type (WT) apple calli, V-ATPase activities in the transgenic calli were significantly enhanced under salt stress condition. Furthermore, Na + and K + were accumulated much higher in transgenic tomato plants than the WT control. These results suggest that MdVHA-B1 may confer salt tolerance through salt overly sensitive (SOS) signal pathway in apple to regulate the ion balance. [ABSTRACT FROM AUTHOR]

• Ethylene signaling pathway is involved in the plant defense response to pathogens. • ERFs are located downstream of the ethylene signaling pathway, and involved in the ethylene signaling pathway resistance to pathogens. • The apple ethylene response factor MdERF11 plays a significant role in activating phytoimmunity response to 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. [ABSTRACT FROM AUTHOR]