Integrative Metabolomic and Transcriptomic Analyses Reveal the Mechanism of Petal Blotch Formation in Rosa persica.

Petal blotch is a specific flower color pattern commonly found in angiosperm families. In particular, Rosa persica is characterized by dark red blotches at the base of yellow petals. Modern rose cultivars with blotches inherited the blotch trait from R. persica. Therefore, understanding the mechanism for blotch formation is crucial for breeding rose cultivars with various color patterns. In this study, the metabolites and genes responsible for the blotch formation in R. persica were identified for the first time through metabolomic and transcriptomic analyses using LC-MS/MS and RNA-seq. A total of 157 flavonoids were identified, with 7 anthocyanins as the major flavonoids, namely, cyanidin 3-O-(6″-O-malonyl) glucoside 5-O-glucoside, cyanidin-3-O-glucoside, cyanidin 3-O-galactoside, cyanidin O-rutinoside-O-malonylglucoside, pelargonidin 3-O-glucoside, pelargonidin 3,5-O-diglucoside, and peonidin O-rutinoside-O-malonylglucoside, contributing to pigmentation and color darkening in the blotch parts of R. persica, whereas carotenoids predominantly influenced the color formation of non-blotch parts. Zeaxanthin and antheraxanthin mainly contributed to the yellow color formation of petals at the semi-open and full bloom stages. The expression levels of two 4-coumarate: CoA ligase genes (Rbe014123 and Rbe028518), the dihydroflavonol 4-reductase gene (Rbe013916), the anthocyanidin synthase gene (Rbe016466), and UDP-flavonoid glucosyltransferase gene (Rbe026328) indicated that they might be the key structural genes affecting the formation and color of petal blotch. Correlation analysis combined with weighted gene co-expression network analysis (WGCNA) further characterized 10 transcription factors (TFs). These TFs might participate in the regulation of anthocyanin accumulation in the blotch parts of petals by modulating one or more structural genes. Our results elucidate the compounds and molecular mechanisms underlying petal blotch formation in R. persica and provide valuable candidate genes for the future genetic improvement of rose cultivars with novel flower color patterns.

Effect of Developmental Stages on Genes Involved in Middle and Downstream Pathway of Volatile Terpene Biosynthesis in Rose Petals.

Terpenoids are economically and ecologically important compounds, and they are vital constituents in rose flower fragrance and rose essential oil. The terpene synthase genes (TPSs), trans-prenyltransferases genes (TPTs), NUDX1 are involved in middle and downstream pathway of volatile terpene biosynthesis in rose flowers. We identified 7 complete RcTPTs, 49 complete RcTPSs, and 9 RcNUDX1 genes in the genome of Rosachinensis. During the flower opening process of butterfly rose (Rosachinensis 'Mutabilis', MU), nine RcTPSs expressed in the petals of opening MU flowers exhibited two main expression trends, namely high and low, in old and fresh petals. Five short-chain petal-expressed RcTPTs showed expression patterns corresponding to RcTPSs. Analysis of differential volatile terpenes and differential expressed genes indicated that higher emission of geraniol from old MU petals might be related to the RcGPPS expression. Comprehensive analysis of volatile emission, sequence structure, micro-synteny and gene expression suggested that RcTPS18 may encode (E,E)-α-farnesene synthase. These findings may be useful for elucidating the molecular mechanism of terpenoid metabolism in rose and are vital for future studies on terpene regulation.

Metabolome-Based Discrimination Analysis of Five Lilium Bulbs Associated with Differences in Secondary Metabolites.

The bulbs of several Lilium species are considered to be both functional foods and traditional medicine in northern and eastern Asia. Considering the limited information regarding the specific bioactive compounds contributing to the functional properties of these bulbs, we compared the secondary metabolites of ten Lilium bulb samples belonging to five different species, using an ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS)-based secondary metabolomics approach. In total, 245 secondary metabolites were detected; further, more metabolites were detected from purple Lilium bulbs (217 compounds) than from white bulbs (123-171 compounds). Similar metabolite profiles were detected in samples within the same species irrespective of where they were collected. By combining herbal analysis and screening differential metabolites, steroid saponins were considered the key bioactive compounds in medicinal lilies. Of the 14 saponins detected, none were accumulated in the bulbs of L. davidii var. willmottiae, also called sweet lily. The purple bulbs of L. regale accumulated more secondary metabolites, and, notably, more phenolic acid compounds and flavonoids. Overall, this study elucidates the differential metabolites in lily bulbs with varying functions and colors and provides a reference for further research on functional foods and the medicinal efficacy of Lilium species.

AtTMEM18 plays important roles in pollen tube and vegetative growth in Arabidopsis.

In flowering plants, pollen tube growth is essential for delivery of male gametes into the female gametophyte or embryo sac for double fertilization. Although many genes have been identified as being involved in the process, the molecular mechanisms of pollen tube growth remains poorly understood. In this study, we identified that the Arabidopsis Transmembrane Protein 18 (AtTMEM18) gene played important roles in pollen tube growth. The AtTMEM18 shares a high similarity with the Transmembrane 18 proteins (TMEM18s) that are conserved in most eukaryotes and may play important roles in obesity in humans. Mutation in the AtTMEM18 by a Ds insertion caused abnormal callose deposition in the pollen grains and had a significant impact on pollen germination and pollen tube growth. AtTMEM18 is expressed in pollen grains, pollen tubes, root tips and other vegetative tissues. The pollen-rescued assays showed that the mutation in AtTMEM18 also caused defects in roots, stems, leaves and transmitting tracts. AtTMEM18-GFP was located around the nuclei. Genetic assays demonstrated that the localization of AtTMEM18 around the nuclei in the generative cells of pollen grains was essential for the male fertility. Furthermore, expression of the rice TMEM18-homologous protein (OsTMEM18) driven by LAT52 promoter could recover the fertility of the Arabidopsis attmem18 mutant. These results suggested that the TMEM18 is important for plant growth in Arabidopsis.

Pollen-expressed transcription factor 2 encodes a novel plant-specific TFIIB-related protein that is required for pollen germination and embryogenesis in Arabidopsis.

Pollen germination and embryogenesis are important to sexual plant reproduction. The processes require a large number of genes to be expressed. Transcription of eukaryotic nuclear genes is accomplished by three conserved RNA polymerases acting in association with a set of auxiliary general transcription factors (GTFs), including B-type GTFs. The roles of B-type GTFs in plant reproduction remain poorly understood. Here we report functional characterization of a novel plant-specific TFIIB-related gene PTF2 in Arabidopsis. Mutation in PTF2 caused failure of pollen germination. Pollen-rescue revealed that the mutation also disrupted embryogenesis and resulted in seed abortion. PTF2 is expressed prolifically in developing pollen and the other tissues with active cell division and differentiation, including embryo and shoot apical meristem. The PTF2 protein shares a lower amino acid sequence similarity with other known TFIIB and TFIIB-related proteins in Arabidopsis. It can interact with TATA-box binding protein 2 (TBP2) and bind to the double-stranded DNA (dsDNA) as the other known TFIIB and TFIIB-related proteins do. In addition, PTF2 can form a homodimer and interact with the subunits of RNA polymerases (RNAPs), implying that it may be involved in the RNAPs transcription. These results suggest that PTF2 plays crucial roles in pollen germination and embryogenesis in Arabidopsis, possibly by regulating gene expression through interaction with TBP2 and the subunits of RNAPs.

WBC27, an adenosine tri-phosphate-binding cassette protein, controls pollen wall formation and patterning in Arabidopsis.

In flowering plants, the exine components are derived from tapetum. Despite its importance to sexual plant reproduction, little is known about the translocation of exine materials from tapetum to developing microspores. Here we report functional characterization of the arabidopsis WBC27 gene. WBC27 encodes an adenosine tri-phosphate binding cassette (ABC) transporter and is expressed preferentially in tapetum. Mutation of WBC27 disrupted the exine formation. The wbc27 mutant microspores began to degenerate once released from tetrads and most of the microspores collapsed at the uninucleate stage. Only a small number of wbc27-1 microspores could develop into tricellular pollen grains. These survival pollen grains lacked exine and germinated in the anther before anthesis. All of these results suggest that the ABC transporter, WBC27 plays important roles in the formation of arabidopsis exine, possibly by translocation of lipidic precursors of sporopollenin from tapetum to developing microspores.

A mutation in Thermosensitive Male Sterile 1, encoding a heat shock protein with DnaJ and PDI domains, leads to thermosensitive gametophytic male sterility in Arabidopsis.

In most flowering plant species, pollination and fertilization occur during the hot summer, so plants must have evolved a mechanism that ensures normal growth of their pollen tubes at high temperatures. Despite its importance to plant reproduction, little is known about the molecular basis of thermotolerance in pollen tubes. Here we report the identification and characterization of a novel Arabidopsis gene, Thermosensitive Male Sterile 1 (TMS1), which plays an important role in thermotolerance of pollen tubes. TMS1 encodes a Hsp40-homologous protein with a DnaJ domain and an a_ERdj5_C domain found in protein disulfide isomerases (PDI). Purified TMS1 expressed in Escherichia coli (BL21 DE3) had the reductive activity of PDI. TMS1 was expressed in pollen grains, pollen tubes and other vegetative tissues, including leaves, stems and roots. Heat shock treatment at 37 degrees C increased its expression levels in growing pollen tubes as well as in vegetative tissues. A knockout mutation in TMS1 grown at 30 degrees C had greatly retarded pollen tube growth in the transmitting tract, resulting in a significant reduction in male fertility. Our study suggests that TMS1 is required for thermotolerance of pollen tubes in Arabidopsis, possibly by functioning as a co-molecular chaperone.