Cui-Hui Sun

MdMYB1 Regulates Anthocyanin and Malate Accumulation by Directly Facilitating Their Transport into Vacuoles in Apples.

An MYB transcription factor influences organ coloration and acidity by activating the expression of the genes encoding vacuolar proton pump subunits as well as anthocyanin transporters and malate transporters. Tonoplast transporters, including proton pumps and secondary transporters, are essential for plant cell function and for quality formation of fleshy fruits and ornamentals. Vacuolar transport of anthocyanins, malate, and other metabolites is directly or indirectly dependent on the H+-pumping activities of vacuolar H+-ATPase (VHA) and/or vacuolar H+-pyrophosphatase, but how these proton pumps are regulated in modulating vacuolar transport is largely unknown. Here, we report a transcription factor, MdMYB1, in apples that binds to the promoters of two genes encoding the B subunits of VHA, MdVHA-B1 and MdVHA-B2, to transcriptionally activate its expression, thereby enhancing VHA activity. A series of transgenic analyses in apples demonstrates that MdMYB1/10 controls cell pH and anthocyanin accumulation partially by regulating MdVHA-B1 and MdVHA-B2. Furthermore, several other direct target genes of MdMYB10 are identified, including MdVHA-E2, MdVHP1, MdMATE-LIKE1, and MdtDT, which are involved in H+-pumping or in the transport of anthocyanins and malates into vacuoles. Finally, we show that the mechanism by which MYB controls malate and anthocyanin accumulation in apples also operates in Arabidopsis (Arabidopsis thaliana). These findings provide novel insights into how MYB transcription factors directly modulate the vacuolar transport system in addition to anthocyanin biosynthesis, consequently controlling organ coloration and cell pH in plants.

Overexpression of MdSOS2L1, a CIPK protein kinase, increases the antioxidant metabolites to enhance salt tolerance in apple and tomato

Soil salinity hinders the growth of most higher plants and becomes a gradually increasing threat to the agricultural production of such crops as the woody plant apple. In this study, a calcineurin B-like protein (CBL)-interacting protein kinase, MdCIPK24-LIKE1 (named as MdSOS2L1), was identified. Quantitative real-time polymerase chain reaction (qRT-PCR) assay revealed that the expression of MdSOS2L1 was upregulated by CaCl2 . Yeast two-hybrid (Y2H) assay and transiently transgenic analysis demonstrated that the MdSOS2L1 protein kinase physically interacted with MdCBL1, MdCBL4 and MdCBL10 proteins to increase salt tolerance in apple. Furthermore, iTRAQ proteome combined with liquid chromatography-tandem mass spectrometry (LC/MS) analysis found that several proteins, which are involved in reactive oxygen species (ROS) scavenging, procyanidin biosynthesis and malate metabolism, were induced in MdSOS2L1-overexpressing apple plants. Subsequent studies have shown that MdSOS2L1 increased antioxidant metabolites such as procyanidin and malate to improve salt tolerance in apple and tomato. In summary, our studies provide a mechanism in which SOS2L1 enhances the salt stress tolerance in apple and tomato.

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.

MdSOS2L1 phosphorylates MdVHA-B1 to modulate malate accumulation in response to salinity in apple

Key messageSalt-induced phosphorylation of MdVHA-B1 protein was mediated by MdSOS2L1 protein kinase, and thereby increasing malate content in apple.AbstractSalinity is an important environmental factor that influences malate accumulation in apple. However, the molecular mechanism by which salinity regulates this process is poorly understood. In this work, we found that MdSOS2L1, a novel AtSOS2-LIKE protein kinase, interacts with V-ATPase subunit MdVHA-B1. Furthermore, MdSOS2L1 directly phosphorylates MdVHA-B1 at Ser396 site to modulate malate accumulation in response to salt stress. Meanwhile, a series of transgenic analyses in apple calli showed that the MdSOS2L1–MdVHAB1 pathway was involved in the regulation of malate accumulation. Finally, a viral vector-based transformation approach demonstrated that the MdSOS2L1–MdVHAB1 pathway also modulated malate accumulation in apple fruits with or without salt stress. Collectively, our findings provide a new insight into the mechanism by which MdSOS2L1 phosphorylates MdVHA-B1 to modulate malate accumulation in response to salinity in apple.

Chrysanthemum MADS-box transcription factor CmANR1 modulates lateral root development via homo-/heterodimerization to influence auxin accumulation in Arabidopsis

Root system architecture is an important agronomic trait by which plants both acquire water and nutrients from the soil and adapt to survive in a complex environment. The adaptation of plant root systems to environmental constraints largely depends on the growth and development of lateral roots (LRs). MADS-box transcription factors (TFs) are important known regulators of plant growth, development, and response to environmental stimuli. However, the potential mechanisms by which they regulate LRs development remain poorly understood. Here, we identified a MADS-box chrysanthemum gene CmANR1, homologous to the Arabidopsis gene AtANR1, which plays a key role in the regulation of LR development. qRT-PCR assays indicated that CmANR1 was primarily expressed in chrysanthemum roots and was rapidly induced by exposure to high nitrate concentrations. Ectopic expression of CmANR1 in Arabidopsis significantly increased the number and length of emerged LRs compared to the wild-type (col) control, but had no obvious affect on primary root (PR) development. We also found that CmANR1 positively influenced auxin accumulation in LRs at least partly by improving auxin biosynthesis and transport, thereby promoting LR development. Furthermore, we found that ANR1 formed homo- and heterodimers through interactions with itself and AGL21 at its C-terminal domain. Overall, our findings provide considerable new information about the mechanisms by which the chrysanthemum MADS-box TF CmANR1 mediates LR development by directly altering auxin accumulation.

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 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.Crop genetics: The genes that make chrysanthemum rootsResearchers in China have elucidated the genetic components controlling root development and architecture in chrysanthemum. A team at Shandong Agricultural University led by Da-gang Hu showed that the ANR1 gene promotes root branching as well as the development of additional roots from stem tissue. To identify other genes in the pathway, the team sequenced the RNA of plants with increased ANR1 expression to identify. They found 7,600 genes regulated by ANR1, including genes related to the plant hormone auxin, which is known to regulate root development. Further experiments revealed that ANR1 directly activates the PIN2 gene, which plays a role in determining the auxin distribution in the root. These findings not only advance our understanding of root development but will be valuable in efforts to breed varieties with improved root systems.HighlightMADS-box TF CmANR1 modulates both AR and LR development by directly regulating auxin transport gene CmPIN2 in chrysanthemum.

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

BackgroundThe 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.ResultsWe 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.ConclusionsThese 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.

MdSOS2L1 forms a complex with MdMYB1 to control vacuolar pH by transcriptionally regulating MdVHA-B1 in apples

ABSTRACT Vacuolar pH is important and involves in many different physiological processes in plants. A recent paper published in Plant Physiology reveals that MdMYB1 regulates vacuolar pH by directly transcriptionally regulating proton pump genes and malate transporters genes, such as V-ATPase subunit gene MdVHA-B1. Here, we found that MdSOS2L1 in vitro did not directly interact with MdMYB1, however, in vivo formed a complex with MdMYB1 in the nucleus to regulate MdVHA-B1-mediated vacuolar acidification. This finding shed light on the role of MdSOS2L1 in transcriptionally regulating MdVHA-B1 in addition to its post-modified function in apples.