The BTB-BACK-TAZ domain protein MdBT2 reduces drought resistance by weakening the positive regulatory effect of MdHDZ27 on apple drought tolerance via ubiquitination.

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.

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.

The BTB/TAZ domain-containing protein CmBT1-mediated CmANR1 ubiquitination negatively regulates root development in chrysanthemum.

Roots are fundamental for plants to adapt to variable environmental conditions. The development of a robust root system is orchestrated by numerous genetic determinants and, among them, the MADS-box gene ANR1 has garnered substantial attention. Prior research has demonstrated that, in chrysanthemum, CmANR1 positively regulates root system development. Nevertheless, the upstream regulators involved in the CmANR1-mediated regulation of root development remain unidentified. In this study, we successfully identified bric-a-brac, tramtrack and broad (BTB) and transcription adapter putative zinc finger (TAZ) domain protein CmBT1 as the interacting partner of CmANR1 through a yeast-two-hybrid (Y2H) screening library. Furthermore, we validated this physical interaction through bimolecular fluorescence complementation and pull-down assays. Functional assays revealed that CmBT1 exerted a negative influence on root development in chrysanthemum. In both in vitro and in vivo assays, it was evident that CmBT1 mediated the ubiquitination of CmANR1 through the ubiquitin/26S proteasome pathway. This ubiquitination subsequently led to the degradation of the CmANR1 protein and a reduction in the transcription of CmANR1-targeted gene CmPIN2, which was crucial for root development in chrysanthemum. Genetic analysis suggested that CmBT1 modulated root development, at least in part, by regulating the level of CmANR1 protein. Collectively, these findings shed new light on the regulatory role of CmBT1 in degrading CmANR1 through ubiquitination, thereby repressing the expression of its targeted gene and inhibiting root development in chrysanthemum.

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.

Effect of Clostridium butyricum on intestinal microbiota and resistance to Vibrio alginolyticus of Penaeus vannamei.

In order to evaluate the effect of Clostridium butyricum (C. butyricum) feeding on intestinal microorganisms and protection against infection by Vibrio alginolyticus (V. alginolyticus) in Penaeus vannamei (P. vannamei). We set up two groups, CG30 (fed normal feed) and CB30 (fed feed supplemented with C. butyricum), for the 30d C. butyricum feeding test, and four groups, CG (CG30 group injected with PBS), CB (CB30 group injected with PBS), VACG (CG30 group injected with V. alginolyticus), and VACB (CB30 group injected with V. alginolyticus), for the 24 h infection test. The protective effect of C. butyricum against acute V. alginolyticus infection in P. vannamei was explained in terms of survival, histopathology, changes in enzyme activity, transcriptome analysis, and immune-related genes. We found that feeding C. butyricum significantly altered intestinal microbial populations' abundance and significantly reduced Vibrio spp. In the V. alginolyticus stress test, C. butyricum improved the survival rate and alleviated pathological changes in hepatopancreatic tissues, alleviated the reduction of superoxide dismutase (SOD) and phenoloxidase (PO) activity caused by infection, and increased the lysozyme content in P. vannamei. VACB group compared with the VACG group, 1730 up-regulated differentially expressed genes (DEGs) and 2029 down-regulated DEGs were screened. Quantitative real-time PCR (qRT-PCR) showed that dietary supplementation with C. butyricum suppressed the upregulation of alkaline phosphatase (AKP) transcription factors and the downregulation of prophenoloxidase (proPO), alpha-2-macroglobulin (A2M), and anti-lipopolysaccharide factor (ALF) induced by V. alginolyticus infection. In conclusion, feed supplementation with C. butyricum changed P. vannamei's population ratio of intestinal microorganisms. Moreover, C. butyricum has the potential to act as an inhibitor of V. alginolyticus infection and enhance the resistance of P. vannamei to V. alginolyticus infection.

Glucose sensor MdHXK1 activates an immune response to the fungal pathogen Botryosphaeria dothidea in apple.

Sugars are essential regulatory molecules involved in plant growth and development and defense response. Although the relationship between sugars and disease resistance has been widely discussed, the underlying molecular mechanisms remain unexplored. Ring rot caused by Botryosphaeria dothidea (B. dothidea), which severely affects fruit quality and yield, is a destructive disease of apples (Malus domestica Borkh.). The present study found that the degree of disease resistance in apple fruit was closely related to glucose content. Therefore, the gene encoding a hexokinase, MdHXK1, was isolated from the apple cultivar 'Gala', and characterized during the defense response. Overexpression of MdHXK1 enhanced disease resistance in apple calli, leaves and fruits by increasing the expression levels of genes related to salicylate (SA) synthesis (PHYTOALEXIN DEFICIENT 4, PAD4; PHENYLALANINE AMMONIA-LYASE, PAL; and ENHANCED DISEASE SUSCEPTIBILITY 1, EDS1) and signaling (PR1; PR5; and NONEXPRESSER OF PR GENES 1, NPR1) as well as increasing the superoxide (O2- ) production rate and the hydrogen peroxide (H2 O2 ) content. Overall, the study provides new insights into the MdHXK1-mediated molecular mechanisms by which glucose signaling regulates apple ring rot resistance.

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.

New insights into the role of MADS-box transcription factor gene CmANR1 on root and shoot development in chrysanthemum (Chrysanthemum morifolium).

BACKGROUND: MADS-box transcription factors (TFs) are the key regulators of multiple developmental processes in plants; among them, a chrysanthemum MADS-box TF CmANR1 has been isolated and described as functioning in root development in response to high nitrate concentration signals. However, how CmANR1 affects root and shoot development remains unclear. RESULTS: We report that CmANR1 plays a positive role in root system development in chrysanthemum throughout the developmental stages of in vitro tissue cultures. Metabolomics combined with transcriptomics assays show that CmANR1 promotes robust root system development by facilitating nitrate assimilation, and influencing the metabolic pathways of amino acid, glycolysis, and the tricarboxylic acid cycle (TCA) cycle. Also, we found that the expression levels of TFs associated with the nitrate signaling pathways, such as AGL8, AGL21, and LBD29, are significantly up-regulated in CmANR1-transgenic plants relative to the wild-type (WT) control; by contrast, the expression levels of RHD3-LIKE, LBD37, and GATA23 were significantly down-regulated. These results suggest that these nitrate signaling associated TFs are involved in CmANR1-modulated control of root development. In addition, CmANR1 also acts as a positive regulator to control shoot growth and development. CONCLUSIONS: These findings provide potential mechanisms of MADS-box TF CmANR1 modulation of root and shoot development, which occurs by regulating a series of nitrate signaling associated TFs, and influencing the metabolic pathways of amino acid and glycolysis, as well as TCA cycle and nitrate assimilation.

The apple bHLH transcription factor MdbHLH3 functions in determining the fruit carbohydrates and malate.

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.

BTB-TAZ Domain Protein MdBT2 Modulates Malate Accumulation and Vacuolar Acidification in Response to Nitrate.

Excessive application of nitrate, an essential macronutrient and a signal regulating diverse physiological processes, decreases malate accumulation in apple (Malus domestica) fruit, but the underlying mechanism remains poorly understood. Here, we show that an apple BTB/TAZ protein, MdBT2, is involved in regulating malate accumulation and vacuolar pH in response to nitrate. In vitro and in vivo assays indicate that MdBT2 interacts directly with and ubiquitinates a bHLH transcription factor, MdCIbHLH1, via the ubiquitin/26S proteasome pathway in response to nitrate. This ubiquitination results in the degradation of MdCIbHLH1 protein and reduces the transcription of MdCIbHLH1-targeted genes involved in malate accumulation and vacuolar acidification, including MdVHA-A, which encodes a vacuolar H+-ATPase, and MdVHP1, which encodes a vacuolar H+-pyrophosphatase, as well as MdALMT9, which encodes an aluminum-activated malate transporter. A series of transgenic analyses in apple materials including fruits, plantlets, and calli demonstrate that MdBT2 controls nitrate-mediated malate accumulation and vacuolar pH at least partially, if not completely, via regulating the MdCIbHLH1 protein level. Taken together, these findings reveal that MdBT2 regulates the stability of MdCIbHLH1 via ubiquitination in response to nitrate, which in succession transcriptionally reduces the expression of malate-associated genes, thereby controlling malate accumulation and vacuolar acidification in apples under high nitrate supply.

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.

A basic/helix-loop-helix transcription factor controls leaf shape by regulating auxin signaling in apple.

Climate-driven phenological change across local spatial gradients leads to leaf shape variation. At higher elevations, leaves of broadleaf species tend to become narrower, but the underlying molecular mechanism is largely unknown. In this study, a series of morphometric analyses and biochemical assays, combined with functional identification in apple, were performed. We show that the decrease in apple leaf width with increasing altitude is controlled by a basic/helix-loop-helix transcription factor (bHLH TF), MdbHLH3. The MdbHLH3-overexpressing lines have a lower transcript abundance of MdPIN1 encoding an auxin efflux carrier but a higher transcript abundance of MdGH3-2 encoding a putative auxin amido conjugate synthase, resulting in a lower free auxin concentration; feeding the transgenic leaves with exogenous auxin partially restores leaf width. MdbHLH3 transcriptionally suppresses and activates MdPIN1 and MdGH3-2, respectively, by specifically binding to their promoters. This alters auxin homeostasis and transport, consequently leading to changes in leaf shape. These findings suggest that the bHLH TF MdbHLH3 directly modulates auxin signaling in controlling leaf shape in response to local spatial gradients in apple.

Phosphorylation of a malate transporter promotes malate excretion and reduces cadmium uptake in apple.

Heavy metal contamination is a major environmental and human health hazard in many areas of the world. Organic acids sequester heavy metals and protect plant roots from the effects of toxicity; however, it is largely unknown how these acids are regulated in response to heavy metal stress. Here, protein kinase SOS2L1 from apple was functionally characterized. MdSOS2L1 was found to be involved in the regulation of malate excretion, and to inhibit cadmium uptake into roots. Using the DUAL membrane system in a screen of an apple cDNA library with MdSOS2L1 as bait, a malate transporter, MdALMT14, was identified as an interactor. Bimolecular fluorescence complementation, pull-down, and co-immunoprecipitation assays further indicated the interaction of the two proteins. Transgenic analyses showed that MdSOS2L1 is required for cadmium-induced phosphorylation at the Ser358 site of MdALMT14, a modification that enhanced the stability of the MdALMT14 protein. MdSOS2L1 was also shown to enhance cadmium tolerance in an MdALMT14-dependent manner. This study sheds light on the roles of the MdSOS2L1-MdALMT14 complex in physiological responses to cadmium toxicity.

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.

The apple U-box E3 ubiquitin ligase MdPUB29 contributes to activate plant immune response to the fungal pathogen Botryosphaeria dothidea.

MAIN CONCLUSION: MdPUB29 is a positive regulator of the defense response to the fungal pathogen Botryosphaeria dothidea possibly by directly regulating the salicylic acid (SA) content as well as SA synthesis-related and signaling-related gene transcription. In plants, ubiquitin E3 ligases containing a U-box domain (PUBs, Plant U-box E3 ubiquitin ligase) have been identified as key regulators of fundamental cellular processes, such as cellular growth, development, and apoptosis, as well as biotic and abiotic stress responses. However, the function of PUBs in apple ring rot remains elusive. Here, we isolated the U-box E3 ligase MdPUB29 from the apple cultivar 'Royal Gala' and characterized its function in plant pathogen defense against Botryosphaeria dothidea. qRT-PCR showed that the expression of MdPUB29 was significantly induced in apple fruits after B. dothidea infection. Overexpression of the MdPUB29 gene in apple calli increased the resistance to B. dothidea infection. In contrast, silencing MdPUB29 in apple calli resulted in reduced resistance. Ectopic expression of MdPUB29 in Arabidopsis also exhibited enhanced resistance to B. dothidea infection compared to that of the wild-type (Col) control. In addition, it was found that the increase of plant pathogen defense was correlated with the increased salicylic acid (SA) content, as well as SA synthesis-related and signaling-related gene transcription in comparison to the wild type. We elucidated the mechanism by which MdPUB29 elevates plant pathogen defense against B. dothidea possibly by regulating the SA pathway.

The regulatory module MdPUB29-MdbHLH3 connects ethylene biosynthesis with fruit quality in apple.

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.

The Glucose Sensor MdHXK1 Phosphorylates a Tonoplast Na+/H+ Exchanger to Improve Salt Tolerance.

Glc regulates many vital processes, including plant growth, development, metabolism, and responses to biotic and abiotic stress. However, the molecular mechanism by which Glc acts as a signal to regulate salinity tolerance remains unclear. In this study, we found that the apple (Malus domestica Borkh.) Glc sensor hexokinase1 (MdHXK1) contributes to Glc-mediated salinity tolerance. A combination of split ubiquitin system, pull-down, co-immunoprecipitation, and bimolecular fluorescence complementation assays demonstrated that MdHXK1 interacts with and phosphorylates the Na+/H+ exchanger MdNHX1 at its Ser-275 residue. Phosphorylation improved the stability of MdNHX1 and enhanced its Na+/H+ transport activity in MdNHX1 overexpression transgenic apple and yeast complementation cells. Furthermore, Ser-275 of MdNHX1 was found to be crucial for MdHXK1-mediated phosphorylation. Finally, a series of transgenic analyses demonstrated that salt tolerance mediated by MdHXK1 partially depended on MdNHX1. Overall, our findings provide insights into how sugar recruits and regulates MdNHX1 in response to high salinity in plants.

Glucose Sensor MdHXK1 Phosphorylates and Stabilizes MdbHLH3 to Promote Anthocyanin Biosynthesis in Apple.

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.

The R2R3-MYB transcription factor MdMYB73 is involved in malate accumulation and vacuolar acidification in apple.

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 MdMYB1 modulates anthocyanin accumulation and vacuolar acidification by directly activating vacuolar transporters, including MdVHA-B1, MdVHA-E, MdVHP1 and MdtDT. Interestingly, we isolated and identified a MYB transcription factor, MdMYB73, a distant relative of MdMYB1 in this study. It was subsequently found that MdMYB73 protein bound directly to the promoters of MdALMT9 (aluminum-activated malate transporter 9), MdVHA-A (vacuolar ATPase subunit A) and MdVHP1 (vacuolar pyrophosphatase 1), transcriptionally activating their expression and thereby enhancing their activities. Analyses of transgenic apple calli demonstrated that MdMYB73 influenced malate accumulation and vacuolar pH. Furthermore, MdCIbHLH1 interacted with MdMYB73 and enhanced its activity upon downstream target genes. These findings help to elucidate how MdMYB73 directly modulates the vacuolar transport system to affect malate accumulation and vacuolar pH in apple.

Ectopic expression of the apple nucleus-encoded thylakoid protein MdY3IP1 triggers early-flowering and enhanced salt-tolerance in Arabidopsis thaliana.

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.