BTB-BACK-TAZ domain protein MdBT2-mediated MdMYB73 ubiquitination negatively regulates malate accumulation and vacuolar acidification in apple

As an important primary metabolite,malate plays a key role in regulating osmotic pressure,pH homeostasis,stress tolerance,and fruit quality of apple.The R2R3-MYB transcription factor(TF)MdMYB73 was identified as a protein that plays a critical role in determining malate accumulation and vacuolar acidification by directly regulating the transcription of aluminum-activated malate transporter 9(MdALMT9),vacuolar ATPase subunit A(MdVHA-A),and vacuolar pyrophosphatase 1(MdVHP1)in apple.In addition,the bHLH TF MdCIbHLH1 interacts with MdMYB73 and enhances the transcriptional activity of MdMYB73.Our previous studies demonstrated that the BTB-BACK-TAZ domain protein MdBT2 can degrade MdCIbHLH1 to influence malate accumulation and vacuolar acidification.However,the potential upstream regulators of MdMYB73 are currently unknown.In this study,we found that MdBT2 directly interacts with and degrades MdMYB73 through the ubiquitin/26S proteasome pathway to regulate malate accumulation and vacuolar acidification.A series of functional assays with apple calli and fruit showed that MdBT2 controls malate accumulation and vacuolar acidification in an MdMYB73-dependent manner.Overall,our findings shed light on the mechanism by which the BTB-BACK-TAZ domain protein MdBT2 regulates malate accumulation and vacuolar acidification by targeting MdMYB73 and MdCIbHLH1 for ubiquitination in apple.This information may help guide traditional breeding programs and fruit tree molecular breeding,and lead to improvements in fruit quality and stress tolerance.

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 basic helix-loop-helix transcription factor MdbHLH3 modulates leaf senescence in apple via the regulation of dehydratase-enolase-phosphatase complex 1

Basic helix−loop−helix(bHLH)domain-containing transcription factors are known for their roles in regulating various plant growth and developmental processes.Previously,we showed that MdbHLH3 from apple(Malus domestica)has multiple functions,modulating both anthocyanin biosynthesis and cell acidification.Here,we show that MdbHLH3 also regulates ethylene biosynthesis and leaf senescence by promoting the expression of dehydratase-enolasephosphatase complex 1(MdDEP1).Therefore,we propose a model whereby MdbHLH3 acts as a crucial factor that modulates anthocyanin biosynthesis and cell acidification in addition to fruit ripening and leaf senescence by regulating distinct target genes.

The MADS transcription factor CmANR1 positively modulates root system development by directly regulating CmPIN2 in chrysanthemum

Plant root systems are essential for many physiological processes,including water and nutrient absorption.MADS-box transcription factor(TF)genes have been characterized as the important regulators of root development in plants;however,the underlying mechanism is largely unknown,including chrysanthemum.Here,it was found that the overexpression of CmANR1,a chrysanthemum MADS-box TF gene,promoted both adventitious root(AR)and lateral root(LR)development in chrysanthemum.Whole transcriptome sequencing analysis revealed a series of differentially expressed unigenes(DEGs)in the roots of CmANR1-transgenic chrysanthemum plants compared to wild-type plants.Functional annotation of these DEGs by alignment with Gene Ontology(GO)terms and biochemical pathway Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis indicated that CmANR1 TF exhibited“DNA binding”and“catalytic”activity,as well as participated in“phytohormone signal transduction”.Both chromatin immunoprecipitation–polymerase chain reaction(ChIP-PCR)and gel electrophoresis mobility shift assays(EMSA)indicated the direct binding of CmPIN2 to the recognition site CArG-box motif by CmANR1.Finally,a firefly luciferase imaging assay demonstrated the transcriptional activation of CmPIN2 by CmANR1 in vivo.Overall,our results provide novel insights into the mechanisms of MADS-box TF CmANR1 modulation of both AR and LR development,which occurs by directly regulating auxin transport gene CmPIN2 in chrysanthemum.

Deciphering the impact of glucose signaling on fruit quality

Glucose is a preferred source of carbon and energy for plants.In addition to metabolic functions,glucose is a well-known signaling molecule that regulates plant growth and development through multiple pathways.In this review,the mechanisms by which glucose signaling regulates the accumulation of sugars and organic acids,as well as the ripening of fleshy fruit,are examined.An analysis of these complex molecular networks demonstrates the impact of glucose signal perception on fruit quality.

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,CmANR1positively 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 yeasttwo-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 CmBT1mediated 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.

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.

Unraveling a genetic roadmap for improved taste in the domesticated apple

Although taste is an important aspect of fruit quality, an understanding of its genetic control remains elusive in apple and other fruit crops. In this study, we conducted genomic sequence analysis of 497 Malus accessions and revealed erosion of genetic diversity caused by apple breeding and possible independent domestication events of dessert and cider apples. Signatures of selection for fruit acidity and size, but not for fruit sugar content, were detected during the processes of both domestication and improvement. Furthermore, we found that single mutations in major genes affecting fruit taste, including Ma1, MdTDT, and MdSOT2, dramatically decrease malate, citrate, and sorbitol accumulation, respectively, and correspond to important domestication events. Interestingly, Ma1 was identified to have pleiotropic effects on both organic acid content and sugar:acid ratio, suggesting that it plays a vital role in determining fruit taste. Fruit taste is unlikely to have been negatively affected by linkage drag associated with selection for larger fruit that resulted from the pyramiding of multiple genes with minor effects on fruit size. Collectively, our study provides new insights into the genetic basis of fruit quality and its evolutionary roadmap during apple domestication, pinpointing several candidate genes for genetic manipulation of fruit taste in apple.

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.

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 Md PFPβ,a pyrophosphatedependent 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(b HLH)transcription factor,Mdb HLH3,binds to the Md PFPβpromoter and activates its expression,thus promoting soluble sugar accumulation in apple fruit.In addition,Md PFPβoverexpression in tomato influenced photosynthesis and carbon metabolism in the plant.Furthermore,we determined that Mdb HLH3 increases photosynthetic rates and soluble sugar accumulation in apple by activating Md PFPβexpression.Our results thus shed light on the mechanism of soluble sugar accumulation in apple leaves and fruit:Mdb HLH3 regulates soluble sugar accumulation by activating Md PFPβgene expression and coordinating carbohydrate allocation.