Aide Wang

The Methylation of the PcMYB10 Promoter Is Associated with Green-Skinned Sport in Max Red Bartlett Pear

In MRB-G fruits, the expression of PcMYB10 was reduced by the methylation of regions −604 to −911 bp and −1,218 to −1,649 bp in its promoter. As a result, the expression of PcUFGT, a key gene involved in anthocyanin biosynthesis and regulated by PcMYB10, was also reduced. This might cause the inhibition of anthocyanin biosynthesis and the formation of green-skinned sport. Varieties of the European pear (Pyrus communis) can produce trees with both red- and green-skinned fruits, such as the Max Red Bartlett (MRB) variety, although little is known about the mechanism behind this differential pigmentation. In this study, we investigated the pigmentation of MRB and its green-skinned sport (MRB-G). The results suggest that a reduction in anthocyanin concentration causes the MRB-G sport. Transcript levels of PcUFGT (for UDP-glucose:flavonoid 3-O-glucosyltransferase), the key structural gene in anthocyanin biosynthesis, paralleled the change of anthocyanin concentration in both MRB and MRB-G fruit. We cloned the PcMYB10 gene, a transcription factor associated with the promoter of PcUFGT. An investigation of the 2-kb region upstream of the ATG translation start site of PcMYB10 showed the regions −604 to −911 bp and −1,218 to −1,649 bp to be highly methylated. A comparison of the PcMYB10 promoter methylation level between the MRB and MRB-G forms indicated a correlation between hypermethylation and the green-skin phenotype. An Agrobacterium tumefaciens infiltration assay was conducted on young MRB fruits by using a plasmid constructed to silence endogenous PcMYB10 via DNA methylation. The infiltrated fruits showed blocked anthocyanin biosynthesis, higher methylation of the PcMYB10 promoter, and lower expression of PcMYB10 and PcUFGT. We suggest that the methylation level of PcMYB10 is associated with the formation of the green-skinned sport in the MRB pear. The potential mechanism behind the regulation of anthocyanin biosynthesis is discussed.

Apple MdABCF assists in the transportation of S-RNase into pollen tubes

Self-incompatibility (SI) is a reproductive isolation mechanism in flowering plants. Plants in the Solanaceae, Rosaceae and Plantaginaceae belong to the gametophytic self-incompatibility type. S-RNase, which is encoded by a female-specific gene located at the S locus, degrades RNA in the pollen tube and causes SI. Recent studies have provided evidence that S-RNase is transported non-selectively into the pollen tube, but have not specified how this transportation is accomplished. We show here that the apple (Malus domestica) MdABCF protein, which belongs to group F of the ABC transporter family, assists in transportation of S-RNase into the pollen tube. MdABCF is located in the pollen tube membrane and interacts with S-RNase. S-RNase was unable to enter the pollen tube when MdABCF was silenced by antisense oligonucleotide transfection. Our results show that MdABCF assists in transportation of either self or non-self S-RNase into the pollen tube. Moreover, MdABCF coordinates with the cytoskeleton to transport S-RNase. Blockage of S-RNase transport disrupts self-incompatibility in this system.

Apple (Malus domestica) MdERF2 negatively affects ethylene biosynthesis during fruit ripening by suppressing MdACS1 transcription

Ripening in climacteric fruit requires the gaseous phytohormone ethylene. Although ethylene signaling has been well studied, knowledge of the transcriptional regulation of ethylene biosynthesis is still limited. Here we show that an apple (Malus domestica) ethylene response factor, MdERF2, negatively affects ethylene biosynthesis and fruit ripening by suppressing the transcription of MdACS1, a gene that is critical for biosynthesis of ripening-related ethylene. Expression of MdERF2 was suppressed by ethylene during ripening of apple fruit, and we observed that MdERF2 bound to the promoter of MdACS1 and directly suppressed its transcription. Moreover, MdERF2 suppressed the activity of the promoter of MdERF3, a transcription factor that we found to bind to the MdACS1 promoter, thereby increasing MdACS1 transcription. We determined that the MdERF2 and MdERF3 proteins directly interact, and this interaction suppresses the binding of MdERF3 to the MdACS1 promoter. Moreover, apple fruit with transiently downregulated MdERF2 expression showed higher ethylene production and faster ripening. Our results indicate that MdERF2 negatively affects ethylene biosynthesis and fruit ripening in apple by suppressing the transcription of MdACS1 via multiple mechanisms, thereby acting as an antagonist of positive ripening regulators. Our findings offer a deep understanding of the transcriptional regulation of ethylene biosynthesis during climacteric fruit ripening.

The Microtubule Cytoskeleton and Pollen Tube Golgi Vesicle System are Required for in Vitro S-RNase Internalization and Gametic Self-Incompatibility in Apple

S-RNase is the female determinant of gametophytic self-incompatibility in apple and is usually considered to be the reason for rejection of pollen. In this study, we investigated the role of microtubules (MTs) in internalization of S-RNases by pollen tubes cultured in vitro. The results showed that S-RNase was imported into the pollen tube where it inhibits pollen tube growth, and that S-RNase is co-localized with the Golgi vesicle during the internalization process. Moreover, MT depolymerization is observed following accumulation of S-RNases in the pollen cytosol. On the other hand, S-RNase was prevented from entering the pollen tube when the pollen was treated with the actin filament (AF) inhibitor latrunculin A (LatA), the MT inhibitor oryzalin, or the MT stabilizer taxol at subtoxic concentrations. These hindered the construction of the MT, with pollen tubes capable of growth under these conditions. Pollen tubes showed improved growth in self-pollinated styles that were pre-treated with taxol. This suggests that cytoskeleton antagonists can prevent S-RNase-mediated inhibition of pollen tubes in vivo by blocking S-RNase internalization. These results suggest that an intact and dynamic cytoskeleton is required for the in vitro internalization of S-RNase, as shown by the effects of various cytoskeleton inhibitors. S-RNase internalization takes place via a membrane/cytoskeleton-based Golgi vesicle system, which can also affect self-incompatibility in apple.

A novel gene, MdSSK1, as a component of the SCF complex rather than MdSBP1 can mediate the ubiquitination of S-RNase in apple

Summary The related components of the SCF complex in apple were cloned and it was proved that an SCF complex containing MdSSK1 rather than MdSBP1 can mediate the ubiquitination of S-RNase.

The Jasmonate-Activated Transcription Factor MdMYC2 Regulates ETHYLENE RESPONSE FACTOR and Ethylene Biosynthetic Genes to Promote Ethylene Biosynthesis during Apple Fruit Ripening

Jasmonate (JA) regulates an important transcription factor in the JA signaling pathway, thereby activating genes involved in ethylene biosynthesis, shedding light on fruit ripening in apple. The plant hormone ethylene is critical for ripening in climacteric fruits, including apple (Malus domestica). Jasmonate (JA) promotes ethylene biosynthesis in apple fruit, but the underlying molecular mechanism is unclear. Here, we found that JA-induced ethylene production in apple fruit is dependent on the expression of MdACS1, an ACC synthase gene involved in ethylene biosynthesis. The expression of MdMYC2, encoding a transcription factor involved in the JA signaling pathway, was enhanced by MeJA treatment in apple fruits, and MdMYC2 directly bound to the promoters of both MdACS1 and the ACC oxidase gene MdACO1 and enhanced their transcription. Furthermore, MdMYC2 bound to the promoter of MdERF3, encoding a transcription factor involved in the ethylene-signaling pathway, thereby activating MdACS1 transcription. We also found that MdMYC2 interacted with MdERF2, a suppressor of MdERF3 and MdACS1. This protein interaction prevented MdERF2 from interacting with MdERF3 and from binding to the MdACS1 promoter, leading to increased transcription of MdACS1. Collectively, these results indicate that JA promotes ethylene biosynthesis through the regulation of MdERFs and ethylene biosynthetic genes by MdMYC2.

Apple MdACS6 Regulates Ethylene Biosynthesis During Fruit Development Involving Ethylene-Responsive Factor.

Ethylene biosynthesis in plants involves different 1-aminocyclopropane-1-carboxylic acid synthase (ACS) genes. The regulation of each ACS gene during fruit development is unclear. Here, we characterized another apple (Malus×domestica) ACS gene, MdACS6. The transcript of MdACS6 was observed not only in fruits but also in other tissues. During fruit development, MdACS6 was initiated at a much earlier stage, whereas MdACS3a and MdACS1 began to be expressed at 35 d before harvest and immediateley after harvest, respectively. Moreover, the enzyme activity of MdACS6 was significantly lower than that of MdACS3a and MdACS1, accounting for the low ethylene biosynthesis in young fruits. Overexpression of MdACS6 (MdACS6-OE) by transient assay in apple showed enhanced ethylene production, and MdACS3a was induced in MdACS6-OE fruits but not in control fruits. In MdACS6 apple fruits silenced by the virus-induced gene silencing (VIGS) system (MdACS6-AN), neither ethylene production nor MdACS3a transcript was detectable. In order to explore the mechanism through which MdACS3a was induced in MdACS6-OE fruits, we investigated the expression of apple ethylene-responsive factor (ERF) genes. The results showed that the expression of MdERF2 was induced in MdACS6-OE fruits and inhibited in MdACS6-AN fruits. Yeast one-hybrid assay showed that MdERF2 protein could bind to the promoter of MdACS3a. Moreover, down-regulation of MdERF2 in apple flesh callus led to a decrease of MdACS3a expression, demonstrating the regulation of MdERF2 on MdACS3a. The mechanism through which MdACS6 regulates the action of MdACS3a was discussed.

Screening and characterization of apple Rho-like GTPase (MdROPs) genes related to S-RNase mediated self-incompatibility

Abstract ROP/Rac GTPase is a conserved class of proteins which play diverse signalling roles in plants. They regulate many fundamental cellular processes such as F-actin dynamics, cell polarity and polar growth. Using apple genomic database analyses, nine ROP family members were cloned for the first time in a fruit tree (apple). Phylogenetic analyses indicated that the nine MdROPs were distributed into two groups, as previously described in the literature for model plants. Expression analyses show all MdROPs were highly expressed in pollen, in particular MdROP1, 3, 4 and 8. Yeast two hybrid and bimolecular fluorescence complementation analyses indicated MdROP8 interacts with S-RNase, a pistil determinate factor in gametophyte self-incompatibility. The pollen tube microtubule is shown to depolymerize in response to S-RNase treatment, during which the expression of MdROP8 rapidly decreased. These results indicate MdROP8 is related to S-RNase mediated self-incompatibility, and gives some useful evidence in modeling the relationship between cytoskeleton depolymerization and pollen tube growth inhibition during the apple SI reaction.

A CBL gene, MdCBL5, controls the calcium signal and influences pollen tube growth in apple

As a signaling molecule in plants and animals, calcium plays important roles in cell growth and development. A concentration gradient of calcium in the apex of tip-growing cells regulates polarized growth. In this study, we cloned the gene encoding a calcineurin B subunit protein, MdCBL5, in “Ralls Janet” apple. We found that MdCBL5 was strongly expressed in pollen and that the encoded protein contained the conserved calcineurin EF hand domain. Antisense-mediated silencing of MdCBL5 disrupted the calcium ion concentration gradient in the pollen tube apex and inhibited pollen tube growth. During the self-incompatibility response in apple, S-RNase uptake disrupts the calcium gradient in the pollen tube apex and inhibits pollen tube growth. Quantitative PCR analysis indicated that MdCBL5 was downregulated in pollen tubes treated with S-RNase. We confirmed the interaction between MdCBL5 and S-RNase using yeast two-hybrid, pull-down, and bimolecular fluorescence complementation (BiFC) experiments. These results show that MdCBL5 is a calcium signaling factor that interacts with S-RNases and regulates pollen tube growth and is itself likely regulated by S-RNase during the self-incompatibility response.

Characterization of the Pear (Pyrus ussuriensis) Actin-depolymerizingFactor PuADF During Fruit Ripening

The polymerization or depolymerization of the cytoskeleton is very important in various aspects of plant development. ADF (actin-depolymerizing factor) is a typical actin binding protein that can mediate the polymerization or depolymerization of the actin cytoskeleton, but its role in fruit ripening remains unclear. Here, we characterized an ADF gene, PuADF. The expression of PuADF was evaluated in young leaves, stems, flowers, and roots as well as in fruits. The expression of PuADF was induced by ethephon treatment but inhibited by 1-MCP treatment. To explore the network of PuADF function in ‘Nanguo’ pear fruits ripening, we screened a cDNA library from ‘Nanguo’ pear fruits using PuADF as bait. Two potential interactors of PuADF were identified, PuAS (Asparagine Synthetase) and PuDAD1 (Defender Against Death 1), both of which have been associated with PCD (Programmed Cell Death). This direct interaction was further confirmed by yeast two-hybrid analyses. The expression levels of PuAS and PuDAD1 were affected by ethylene. Our results indicated that PuADF is involved in ethylene-mediated fruit ripening and might be related to PCD during fruit ripening. The possible mechanisms are discussed.