Apple <scp>MdABCF</scp> assists in the transportation of <i>S</i>‐<scp>RN</scp>ase into pollen tubes

Meng, Dong; Gu, Zhennan; Li, Wei; Wang, Aide; Yuan, Hui; Yang, Qing; Li, Tianzhong

doi:10.1111/tpj.12524

Cited by 75 publications

(77 citation statements)

References 50 publications

(71 reference statements)

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“…The transient expression vector pEZS-NL harboring an EGFP-tag [31] was used to study the transformation efficiency in response to PEG concentrations and transfection time (Fig. 3).…”

Section: Resultsmentioning

confidence: 99%

“…In order to select the optimal concentration of PEG4000, concentrations of 10, 15, 20, 25 and 30% PEG4000, w/v were tested. Next, 20 µg of the pEZS-NL plasmid, which contained a 35S promoter and the coding sequence fused to EGFP and OCS 3′ terminator which can used for transient expression [31] (Isolated by PureLink ® Quick Plasmid Miniprep Kit, Invitrogen, cat. no.…”

Section: Methodsmentioning

confidence: 99%

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Efficient isolation of Magnolia protoplasts and the application to subcellular localization of MdeHSF1

et al. 2017

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Background Magnolia is a woody ornamental plant, which is widely used in urban landscaping. However, its lengthy juvenile period and recalcitrance to regeneration impedes functional characterization of its genes.ResultsWe developed an efficient protoplast isolation and transient expression system for Magnolia denudata × Magnolia acuminata ‘Yellow River’. The highest yield of protoplasts was obtained from young leaves digested in 3% Cellulase R10, 0.8% Macerozyme R10, 0.04% pectinase and 0.4 M mannitol enzymolysis solution for 6 h. For transfection of protoplasts, 20% PEG4000 for 5 min was optimal. To verify the protoplast system and begin to understand heat tolerance in Magnolia, a heat shock transcription factor MdeHSF1 was cloned from ‘Yellow River’, which belongs to the HSF subfamily A and has significant homology with AtHSFA1A. Subcellular localization analysis indicated that MdeHSF1 was expressed in the cell nucleus. Furthermore, qPCR analysis of the MdeHSF1 transcript level in response to high temperature stress suggested that MdeHSF1 might be involved in regulating heat stress tolerance in ‘Yellow River’.ConclusionThe described protocol provides a simple and straightforward method for isolating protoplast and exploring gene subcellular localization of MdeHSF1 in Magnolia. This expands the new research of protoplast isolation and transfection in Magnolia.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-017-0193-3) contains supplementary material, which is available to authorized users.

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“…The transient expression vector pEZS-NL harboring an EGFP-tag [31] was used to study the transformation efficiency in response to PEG concentrations and transfection time (Fig. 3).…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Efficient isolation of Magnolia protoplasts and the application to subcellular localization of MdeHSF1

et al. 2017

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“…The endogenous cytosolic PPase activity decreased and the PPi levels increased in SPI‐treated pollen tubes compared with those that were CPI‐treated, suggesting that MdPPa is involved in the SI response. Interestingly, we found that non‐self S ‐RNase is largely absent from the cytosol of growing pollen tubes after 120 min of culture, implying that non‐self S ‐RNase is degraded by the 26S proteasome, as we have shown previously that both self and non‐self S ‐RNases are taken up by growing apple pollen tubes (Meng et al ., ,b). Moreover, there may be some low‐level interactions between MdPPa and non‐self S ‐RNases in CPI treatment, resulting in a weak reduction in pollen growth and MdPPa activity.…”

Section: Discussionmentioning

confidence: 99%

“…A recent model has proposed that, when taken up nonspecifically through the action of an ABCF protein, S ‐RNase is recognized by collaborative non‐self SLF/SFBB proteins and degraded via the ubiquitin‐26S‐proteasome pathway in a non‐self‐pollen tube. By contrast, when recognition does not occur, the S ‐RNase has a cytotoxic effect inside the self‐pollen tube, resulting in an inhibition of pollen tube growth (Luu et al ., ; Qiao et al ., ; Hua & Kao, ; Kubo et al ., ; Minamikawa et al ., ; De Franceschi et al ., ; Meng et al ., ; Yuan et al ., ). Another compartmentalization model has also explained why S‐RNase has a specific cytotoxic effect on self‐pollen tubes, but not on non‐self‐pollen tubes.…”

Section: Introductionmentioning

confidence: 99%

Apple S‐RNase triggers inhibition of tRNA aminoacylation by interacting with a soluble inorganic pyrophosphatase in growing self‐pollen tubes in vitro

Meng

et al. 2018

New Phytologist

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Apple exhibits S-RNase-based self-incompatibility (SI), in which S-RNase plays a central role in rejecting self-pollen. It has been proposed that the arrest of pollen growth in SI of Solanaceae plants is a consequence of the degradation of pollen rRNA by S-RNase; however, the underlying mechanism in Rosaceae is still unclear. Here, we used S -RNase as a bait to screen an apple pollen cDNA library and characterized an apple soluble inorganic pyrophosphatase (MdPPa) that physically interacted with S-RNases. When treated with self S-RNases, apple pollen tubes showed a marked growth inhibition, as well as a decrease in endogenous soluble pyrophosphatase activity and elevated levels of inorganic pyrophosphate (PPi). In addition, S-RNase was found to bind to two variable regions of MdPPa, resulting in a noncompetitive inhibition of its activity. Silencing of MdPPa expression led to a reduction in pollen tube growth. Interestingly, tRNA aminoacylation was inhibited in self S-RNase-treated or MdPPa-silenced pollen tubes, resulting in the accumulation of uncharged tRNA. Furthermore, we provide evidence showing that this disturbance of tRNA aminoacylation is independent of RNase activity. We propose an alternative mechanism differing from RNA degradation to explain the cytotoxicity of the S-RNase apple SI process.

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“…S-RNase was observed to be non-selectively taken up by in vitro cultured apple pollen tubes. The uptake appeared to be mediated by the cytoskeleton and an ABC transporter family protein (MdABCF) localized in the pollen tube membrane (Meng et al, 2014a). This ABCF-mediated S-RNaseuptake pathway was also required for SI in Petunia species.…”

Section: Self-incompatibility Events That Arrest Self-pollen Tube Gromentioning

confidence: 99%

Distinct Self-recognition in the <i>Prunus</i> S-RNase-based Gametophytic Self-incompatibility System

Matsumoto

Tao

2016

The Hortic J

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Prunus fruit tree species exhibit S-ribonuclease (S-RNase)-based gametophytic self-incompatibility (GSI). This system is also present in the families Plantaginaceae and Solanaceae and the tribe Maleae of the family Rosaceae. In S-RNase-based GSI, self/nonself-recognition between the pistil and pollen is controlled by the pistil S determinant S-ribonuclease gene (S-RNase) and the pollen S determinant F-box gene(s). Accumulated evidence indicates the Prunus pollen S locus contains a single F-box gene, while that of other plants consists of multiple F-box genes. The pollen S F-box genes are called S haplotype-specific F-box (SFB), S-locus F-box brothers (SFBB), and S-locus F-box (SLF) in Prunus, Maleae, and Solanaceae species, respectively. The consequences of pollen S gene mutations and heterodiallelic pollen production differ between Prunus species and other plants, suggesting there are different pollen S functions during self/nonself-recognition. The GSI systems of Prunus and other plants are believed to include the ubiquitin proteasome system for protein degradation. However, Prunus SFB is assumed to facilitate the S-RNase cytotoxic effects during selfrecognition, while SLFs and SFBBs are thought to function collaboratively during nonself-recognition to avoid S-RNase cytotoxicity. This review summarizes the distinct features of the S-RNase-based GSI mechanism in Prunus species, with special references to the recent advances in our understanding of S-RNase-based GSI.

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

Cited by 75 publications

References 50 publications

Efficient isolation of Magnolia protoplasts and the application to subcellular localization of MdeHSF1

Efficient isolation of Magnolia protoplasts and the application to subcellular localization of MdeHSF1

Apple S‐RNase triggers inhibition of tRNA aminoacylation by interacting with a soluble inorganic pyrophosphatase in growing self‐pollen tubes in vitro

Distinct Self-recognition in the <i>Prunus</i> S-RNase-based Gametophytic Self-incompatibility System

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