Genome-wide identification and evolutionary analysis of MLO gene family in Rosaceae plants

Mutants lacking wild-type MLO(Mildew resistance Locus O)proteins show broad-spectrum resistance to the powdery mildew fungus,and dysregulated cell death control,with spontaneous cell death in response to developmental or abiotic stimuli.In order to understand the evolution and divergence patterns of the MLO gene family in Rosaceae plants,we analysed systematically genome-wide data from Fragaria vesca,Prunus persica,Prunus mume,Malus domestica,Pyrus bretschneideri and Rubus occidentalis based on bioinformatics methods.Using three phylogenetic methods(the neighbour-joining,maximum likelihood,and Bayesian methods),we identified 117 MLO genes from 6 Rosaceae species.The results of all three phylogenetic analysis methods supported that these genes were divided into six clades.Conserved motif analysis found that only motif 2 was present in all MLO proteins and had 3 nearly invariant amino acid residues.The findings indicated that motif 2 might be shared by the MLO gene family.The structural features of these genes showed large variations in sequence length among different species,although the lengths and the numbers of exons exhibited high degrees of similarity.Selective pressure analysis showed extremely significant differences in all 6 clades,with 2,1,and 1 site(s)under significant positive selection detected in clades III,IV,and VI,respectively.These positive selection sites were important driving forces for the promotion of the functional differentiation of the MLO genes.Functional divergence analysis showed that the significantly divergent sites were located within the domains of the MLO genes.Functional distance analysis showed that the clade V had more conservative functions and might have retained more original functions during the evolutionary process.However,clade I may have undergone extensive altered functional constraints as a specialised functional role.Moreover,the most original function of the MLO genes in Rosaceae could be related to the evolution of their resistance to powdery mildew,which then gradually evolved into functions such as the regulation of flower development,the control of root morphology,and seed evolution due to the different evolutionary rates after gene duplication.These results provide a theoretical basis for further studies of the molecular evolutionary patterns of the plant MLO gene family.

Comparative transcriptomic and metabonomic analysis revealed the relationships between biosynthesis of volatiles and flavonoid metabolites in Rosa rugosa

Rosa rugosa is not only cultivated as a landscaping plant,but also used in cosmetics,the medical and food industries.However,little information is currently available on the gene regulatory networks involved in its scent and color biosynthesis and metabolism.In this study,R.rugosa Thunb.f.rosea Rehd with red petals(RR)and its white petal variant(WR),were used to study the molecular mechanisms in flower color and scent.Sixty-five differential flavonoid metabolites and 15 volatiles were found to have significant differences between RR and WR.Correspondingly,the key regulators(MYB-bHLH-WD40)of anthocyanin synthesis pathway and their structural genes involved in flavonoid biosynthesis,benzenoid/phenylpropanoid biosynthesis,terpenoid biosynthesis pathways were also found to be differentially expressed by comparative transcriptome.Further,qPCR permitted the identification of some transcripts encoding proteins that were putatively associated with scent and color biosynthesis in roses.Particularly,the results showed that the ACT gene(encoding CoA geraniol/citronellol acetyltransferase,GeneID:112190420),which expressed lower in WR,was involved in three pathways:flavonoid biosynthesis,phenylpropanoid biosynthesis and terpenoid biosynthesis,however,GT5(anthocyanin glycosylation gene,GeneID:112186660),expressed higher in WR,was involved in both flavonoid and phenylpropanoid biosynthesis pathways.These results suggested that ACT and GT5 might play important roles in regulating the relationship of color pigmentation and volatile emission.

Graft-accelerated virus-induced gene silencing facilitates functional genomics in rose flowers

Rose has emerged as a model ornamental plant for studies of flower development, senescence, and morphology, as well as the metabolism of floral fragrances and colors.Virus-induced gene silencing(VIGS) has long been used in functional genomics studies of rose by vacuum infiltration of cuttings or seedlings with an Agrobacterium suspension carrying TRV-derived vectors. However, VIGS in rose flowers remains a challenge because of its low efficiency and long time to establish silencing. Here we present a novel and rapid VIGS method that can be used to analyze gene function in rose,called ‘graft-accelerated VIGS', where axil ary sprouts are cut from the rose plant and vacuum infiltrated with Agrobacterium. The inoculated scions are then grafted back onto the plants to flower and silencing phenotypes can be observed within 5 weeks, post-infiltration. Using this new method, we successfully silenced expression of the RhDFR_1, RhA G, and RhNUDX_1 in rose flowers, and affected their color, petal number, as well as fragrance, respectively. This grafting method will facilitate high-throughput functional analysis of genes in rose flowers. Importantly, it may also be applied to other woody species that are not currently amenable to VIGS by conventional leaf or plantlet/seedling infiltration methods.