Identification of Novel Candidate Genes Involved in Apple Cuticle Integrity and Russeting-Associated Triterpene Synthesis Using Metabolomic, Proteomic, and Transcriptomic Data

Abstract

Apple russeting develops on the fruit surface when skin integrity has been lost. It induces a modification of fruit wax composition, including its triterpene profile. In the present work, we studied two closely related apple varieties, ‘Reinette grise du Canada’ and ‘Reinette blanche du Canada’, which display russeted and non-russeted skin phenotypes, respectively, during fruit development. To better understand the molecular events associated with russeting and the differential triterpene composition, metabolomics data were generated using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) and combined with proteomic and transcriptomic data. Our results indicated lower expression of genes linked to cuticle biosynthesis (cutin and wax) in russet apple throughout fruit development, along with an alteration of the specialized metabolism pathways, including triterpene and phenylpropanoid. We identified a lipid transfer protein (LTP3) as a novel player in cuticle formation, possibly involved in the transport of both cutin and wax components in apple skin. Metabolomic data highlighted for the first time a large diversity of triterpene-hydroxycinnamates in russeted tissues, accumulation of which was highly correlated with suberin-related genes, including some enzymes belonging to the BAHD (HXXXD-motif) acyltransferase family. Overall, this study increases our understanding about the crosstalk between triterpene and suberin pathways.

MdMYB52 regulates lignin biosynthesis upon the suberization process in apple

“…Alternatively, using NCBI blastX against the A. thaliana database, the highest identity level (83.64%) was obtained against AtMYB52 with 31% coverage, the remaining part corresponding, again, to the intrinsically disordered region. Interestingly, AtMYB52 and other members of the same clade (MYB54, MYB69) were previously described as regulators of secondary cell wall biogenesis (Zhong et al, 2008), thus we first suspected that MdMYB52 might contribute to the cell wall m od ifi c a t io n / l o os e n in g ob s e r v e d i n o u r p r e v i o u s transcriptomics studies (Legay et al, 2015;Andréet al, 2022).…”

“…Nevertheless, multiple orthologous genes of the above-cited Arabidopsis suberin regulatory MYB transcription factors displayed an increased expression in russeted apple fruit skin, suggesting that these might be involved in the suberization process in apple as well (Legay et al, 2015;Andréet al, 2022). Using our transcriptomic datasets, we also identified number of other characterized and uncharacterized MYB transcription factors.…”

“…Shortly, from each tree, 10 apples were sampled from the south side of the tree at a height ranging from 1.20 to 2.20 m. An additional sampling has been performed for 'Cox Orange Pippin' variety (COX-L) from a private orchard in Luxembourg in 2013. Finally, samples collected during the fruit growth kinetic of the semi-russeted 'Reinette du Canada Blanc' and heavily russeted 'Reinette du Canada Grise' varieties described in Andréet al (2022) were also used. Finally, Cox Orange Pippin, the russeted and waxy patches (areas) were carefully excised with scalpels taking care to remove the flesh as much as possible.…”

“…In Arabidopsis, this includes MYB41, MYB53, MYB92, MYB93, MYB9, MYB102, SUBERMAN, whereas in apple only MdMYB93 has been described so far ( Kosma et al., 2014 ; Lashbrooke et al., 2016 ; Legay et al., 2016 ; Cohen et al., 2020 ; Shukla et al., 2021 ). Nevertheless, multiple orthologous genes of the above-cited Arabidopsis suberin regulatory MYB transcription factors displayed an increased expression in russeted apple fruit skin, suggesting that these might be involved in the suberization process in apple as well ( Legay et al., 2015 ; André et al., 2022 ). Using our transcriptomic datasets, we also identified number of other characterized and uncharacterized MYB transcription factors.…”

“…Using our transcriptomic datasets, we also identified number of other characterized and uncharacterized MYB transcription factors. Among these, we selected MD05G1011100 ( Daccord et al., 2017 ) or MDP0000852158 ( Velasco et al., 2010 ), which is similar to the Arabidopsis MYB52 transcription factor and displayed a significant increased expression in russeted (suberized) apple fruit skin ( Legay et al., 2015 ; Legay et al., 2017 ; André et al., 2022 ). In a previous work, we showed that MD05G1011100 is able to positively regulate the expression of MdOSC5, an oxidosqualene cyclase, which is involved in the synthesis of the lupan series of pentacyclic triterpenes, the latter being specifically accumulated in suberized fruit skin ( Falginella et al., 2021 ).…”