PyMYB10 and PyMYB10.1 Interact with bHLH to Enhance Anthocyanin Accumulation in Pears

<div><p>Color is an important agronomic trait of pears, and the anthocyanin content of fruit is immensely significant for pear coloring. In this study, an anthocyanin-activating R2R3-MYB transcription factor gene, <i>PyMYB10</i>.<i>1</i>, was isolated from fruits of red sand pear (<i>Pyrus pyrifolia</i> cv. Aoguan). Alignments of the nucleotide and amino acid sequences suggested that PyMYB10.1 was involved in anthocyanin regulation. Similar to <i>PyMYB10</i>, <i>PyMYB10</i>.<i>1</i> was predominantly expressed in red tissues, including the skin, leaf and flower, but it was minimally expressed in non-red fruit flesh. The expression of this gene could be induced by light. Dual-luciferase assays indicated that both PyMYB10 and PyMYB10.1 activated the <i>AtDFR</i> promoter. The activation of <i>AtDFR</i> increased to a greater extent when combined with a bHLH co-factor, such as PybHLH, MrbHLH1, MrbHLH2, or AtbHLH2. However, the response of this activation depended on the protein complex formed. PyMYB10-AtbHLH2 activated the <i>AtDFR</i> promoter to a greater extent than other combinations of proteins. PyMYB10-AtbHLH2 also induced the highest anthocyanin accumulation in tobacco transient-expression assays. Moreover, PybHLH interacted with PyMYB10 and PyMYB10.1. These results suggest that both PyMYB10 and PyMYB10.1 are positive anthocyanin biosynthesis regulators in pears that act via the formation of a ternary complex with PybHLH. The functional characterization of <i>PyMYB10</i> and <i>PyMYB10</i>.<i>1</i> will aid further understanding of the anthocyanin regulation in pears.</p></div

Activation of the <i>AtDFR</i> promoter by PyMYB10, PyMYB10.1 and anthocyanin-related bHLH transcription factors in a tobacco transient-expression assay.

<p>LUC and REN activities were analyzed three days after transformation. Error bars represent means ± SE (n = 6).Asterisks, * and **, indicate significant differences (P<0.05 and P<0.01).</p

Phylogenetic relationship of PyMYB10.1 to other R2R3-MYBs.

<p>A phylogenetic tree was constructed using the neighbor-joining method by the MEGA3 software. The reliability of the trees was tested using a bootstrapping method with 1000 replicates. PyMYB10.1 is indicated with an asterisk. Putative regulatory functions of the selected R2R3-MYB proteins are indicated. The GenBank accession numbers of some R2R3-MYB protein sequences are as follows: <i>Antirrhinum majus</i> AmROSEA2 (ABB83827); <i>Prunus persica</i> PpMYB10.1 (Ppa026640m), PpMYB10.2 (Ppa016711m), PpMYB10.4 (Ppa018744m), PpMYB10.5(Ppa024617m); <i>Arabidopsis thaliana</i> AtMYB11 (EFH52939), AtMYB12 (AEC10843), AtMYB111 (EFH41988), AtPAP2 (AAG42002), and AtTT2 (AED93980); <i>Fragaria ananassa</i> FaMYB1 (AAK84064); <i>Malus domestica</i>MdMYB3 (AEX08668.1), MdMYB9 (ABB84757), MdMYB11 (AAZ20431), MdMYB17 (ADL36757), and MdMYB111 (ADL36754); <i>Pyrus pyrifolia</i> PyMYB10 (ADN52330); <i>Morella rubra</i> MrMYB1 (ADG21957); <i>Zea mays</i> ZmC1 (AAA33482),ZmPl (AAA19819);and <i>Vitis vinifera</i> VvMYBA2 (BAD18978), VvMYBF1 (ACV81697), and VvMYBPA2 (ACK56131).</p

Patches of anthocyanin production in tobacco leaves infused with <i>Agrobacterium tumefaciens</i>.

<p>Photos of infiltration areas were taken 8 days after transformation with PyMYB10 or PyMYB10.1 together with AtbHLH2, PybHLH, MrbHLH1, or MrbHLH2.</p

Multiple alignments of PyMYB10.1 and anthocyanin R2R3-MYB regulators.

<p>Identical amino acids are shaded in black and similar amino acids in pink or turquoise. The R2- and R3-MYB DNA-binding domains of selected MYB proteins are underlined. The bHLH binding motif is indicated with brackets. The GenBank accession numbers of R2R3-MYB proteins are as follows: <i>Pyrus pyrifolia</i> PyMYB10 (ADN52330); <i>Malus x domestica</i> MdMYB10(ACQ45201) andMdMYB110a (AB743999); <i>Fragaria x ananassa</i> FaMYB10(ABX79947); <i>Petunia x hybrida</i> PhAN2(AAF66727); <i>Arabidopsis thaliana</i> AtPAP1(AAG42001); <i>Vitis vinifera</i> VvMYBA1(BAD18977); and <i>Antirrhinum majus</i> AmROSEA1(ABB83826).</p

Interactions between PyMYB10 or PyMYB10.1 and PybHLH were detected through the yeast two-hybrid assay.

<p>AH109 yeast cells containing plasmids AD+BD, AD-PyMYB10+BD, AD-PyMYB10.1+BD, AD+BD-PybHLH, AD-PyMYB10+BD-PybHLH, or AD-PyMYB10.1+BD-PybHLH were grown on double- and quadruple-selection media. The X-gal assay was performed to confirm positive interactions.</p

Anthocyanin accumulation and expression analysis of <i>PyMYB10</i> and <i>PyMYB10</i>.<i>1</i> in ‘Aoguan’ pears in response to light.

<p>(a) Expression analysis of <i>PyMYB10</i> in ‘Aoguan’ pears during bagging and debagging treatments. (b) Expression analysis of <i>PyMYB10</i>.<i>1</i> in ‘Aoguan’ pears during bagging and debagging treatments. (c) Anthocyanin contents in ‘Aoguan’ pears during bagging and debagging treatments. Fruits were collected 6 days after debagging. The fruits retained in the bags were sampled as controls. Error bars represent means ± SE (n = 3).</p

S, N Codoped Graphene Quantum Dots Embedded in (BiO)₂CO₃: Incorporating Enzymatic-like Catalysis in Photocatalysis

In this study, S, N codoped graphene quantum dots/(BiO)₂CO₃ hollow microspheres have been fabricated by a facile electrostatic self-assembly method. The nanosized S, N:GQDs, which can be obtained by a bottom-up approach, are superior surface modification materials for photocatalytic applications due to their better electron transfer and peroxidase mimetic properties. The excellent oxidation property of the synthesized nanocomposite is confirmed by degradation of different model pollutants, such as rhodamine B, tetracycline, and bisphenol A under light irradiation or dark situation. Based on several experiments, the essential roles of S, N:GQDs can be described as (i) a photocarrier transport center strengthening photoinduced charge carriers (h⁺–e^–) separation and (ii) an enzymatic-like catalysis center to accelerate H₂O₂ decomposition to produce ·OH because the surface accumulation of H₂O₂ is harmful for photocatalytic processes. The present work may pave the way for integrating enzymatic-like cocatalysis into a photocatalytic process to generate more reactive oxygen species, thus advancing the field of environmental remediation and synthetic chemistry

Analysis of the Xyloglucan Endotransglucosylase/Hydrolase Gene Family during Apple Fruit Ripening and Softening

Ethylene and xyloglucan endotransglucosylase/hydrolase (<i>XTH</i>) genes were important for fruit ripening and softening in ‘Taishanzaoxia’ apple. In this study, we found it was <i>ACS1-1/-1</i> homozygotes in ‘Taishanzaoxia’ apple, which determined the higher transcription activity of <i>ACS1</i>. <i>XTH1</i>, <i>XTH3</i>, <i>XTH4</i>, <i>XTH5</i>, and <i>XTH9</i> were mainly involved in the early fruit softening independent of ethylene, while <i>XTH2</i>, <i>XTH6</i>, <i>XTH7</i>, <i>XTH8</i>, <i>XTH10</i>, and <i>XTH11</i> were predominantly involved in the late fruit softening dependent on ethylene. Overexpression of <i>XTH2</i> and <i>XTH10</i> in tomato resulted in the elevated expression of genes involved in ethylene biosynthesis (<i>ACS2</i>, <i>ACO1</i>), signal transduction (<i>ERF2</i>), and fruit softening (<i>XTHs</i>, <i>PG2A</i>, <i>Cel2</i>, and <i>TBG4</i>). In summary, the burst of ethylene in ‘Taishanzaoxia’ apple was predominantly determined by <i>ACS1-1/-1</i> genotype, and the differential expression of <i>XTH</i> genes dependent on and independent of ethylene played critical roles in the fruit ripening and softening. <i>XTH2</i> and <i>XTH10</i> may act as a signal switch in the feedback regulation of ethylene signaling and fruit softening

The comparison of ethylene production and fruit firmness in ‘Taishanzaoxia’ and ‘Liaofu’ cultivar.

<p>(A) Changes of fruit firmness and ethylene production in different apple cultivars during fruit development. (B) Effect of different levers 1-MCP on ethylene production in ‘Taishanzaoxia’. (C) Effect of different levers 1-MCP on fruit firmness in ‘Taishanzaoxia’. TS represent ‘Taishanzaoxia’ cultivar. LF represents ‘Liaofu’ cultivar.</p