Over-Expression of Rose RrLAZY1 Negatively Regulates the Branch Angle of Transgenic Arabidopsis Inflorescence.

Branch angle is a key shoot architecture trait that strongly influences the ornamental and economic value of garden plants. However, the mechanism underlying the control of branch angle, an important aspect of tree architecture, is far from clear in roses. In the present study, we isolated the RrLAZY1 gene from the stems of Rosa rugosa 'Zilong wochi'. Sequence analysis showed that the encoded RrLAZY1 protein contained a conserved GΦL (A/T) IGT domain, which belongs to the IGT family. Quantitative real-time PCR (qRT-PCR) analyses revealed that RrLAZY1 was expressed in all tissues and that expression was highest in the stem. The RrLAZY1 protein was localized in the plasma membrane. Based on a yeast two-hybrid assay and bimolecular fluorescence complementation experiments, the RrLAZY1 protein was found to interact with auxin-related proteins RrIAA16. The over-expression of the RrLAZY1 gene displayed a smaller branch angle in transgenic Arabidopsis inflorescence and resulted in changes in the expression level of genes related to auxin polar transport and signal transduction pathways. This study represents the first systematic analysis of the LAZY1 gene family in R. rugosa. The results of this study will provide a theoretical basis for the improvement of rose plant types and molecular breeding and provide valuable information for studying the regulation mechanism of branch angle in other woody plants.

Identification of Two Novel R2R3-MYB Transcription factors, PsMYB114L and PsMYB12L, Related to Anthocyanin Biosynthesis in Paeonia suffruticosa.

Flower color is a charming phenotype with very important ornamental and commercial values. Anthocyanins play a critical role in determining flower color pattern formation, and their biosynthesis is typically regulated by R2R3-MYB transcription factors (TFs). Paeonia suffruticosa is a famous ornamental plant with colorful flowers. However, little is known about the R2R3-MYB TFs that regulate anthocyanin accumulation in P. suffruticosa. In the present study, two R2R3-MYB TFs, namely, PsMYB114L and PsMYB12L, were isolated from the petals of P. suffruticosa 'Shima Nishiki' and functionally characterized. Sequence analysis suggested that PsMYB114L contained a bHLH-interaction motif, whereas PsMYB12L contained two flavonol-specific motifs (SG7 and SG7-2). Subsequently, the in vivo function of PsMYB114L and PsMYB12L was investigated by their heterologous expression in Arabidopsis thaliana and apple calli. In transgenic Arabidopsis plants, overexpression of PsMYB114L and of PsMYB12L caused a significantly higher accumulation of anthocyanins, resulting in purple-red leaves. Transgenic apple calli overexpressing PsMYB114L and PsMYB12L also significantly enhanced the anthocyanins content and resulted in a change in the callus color to red. Meanwhile, gene expression analysis in A. thaliana and apple calli suggested that the expression levels of the flavonol synthase (MdFLS) and anthocyanidin reductase (MdANR) genes were significantly downregulated and the dihydroflavonol 4-reductase (AtDFR) and anthocyanin synthase (AtANS) genes were significantly upregulated in transgenic lines of PsMYB114L. Moreover, the expression level of the FLS gene (MdFLS) was significantly downregulated and the DFR (AtDFR/MdDFR) and ANS (AtANS/MdANS) genes were all significantly upregulated in transgenic lines plants of PsMYB12L. These results indicate that PsMYB114L and PsMYB12L both enhance anthocyanin accumulation by specifically regulating the expression of some anthocyanin biosynthesis-related genes in different plant species. Together, these results provide a valuable resource with which to further study the regulatory mechanism of anthocyanin biosynthesis in P. suffruticosa and for the breeding of tree peony cultivars with novel and charming flower colors.

RrGT2, A Key Gene Associated with Anthocyanin Biosynthesis in Rosa rugosa, Was Identified Via Virus-Induced Gene Silencing and Overexpression.

In this study, a gene with a full-length cDNA of 1422 bp encoding 473 amino acids, designated RrGT2, was isolated from R. rugosa 'Zizhi' and then functionally characterized. RrGT2 transcripts were detected in various tissues and were proved that their expression patterns corresponded with anthocyanins accumulation. Functional verification of RrGT2 in R. rugosa was performed via VIGS. When RrGT2 was silenced, the Rosa plants displayed a pale petal color phenotype. The detection results showed that the expression of RrGT2 was significantly downregulated, which was consistent with the decrease of all anthocyanins; while the expression of six key upstream structural genes was normal. Additionally, the in vivo function of RrGT2 was investigated via its overexpression in tobacco. In transgenic tobacco plants expressing RrGT2, anthocyanin accumulation was induced in the flowers, indicating that RrGT2 could encode a functional GT protein for anthocyanin biosynthesis and could function in other species. The application of VIGS in transgenic tobacco resulted in the treated tobacco plants presenting flowers whose phenotypes were lighter in color than those of normal plants. These results also validated and affirmed previous conclusions. Therefore, we speculated that glycosylation of RrGT2 plays a crucial role in anthocyanin biosynthesis in R. rugosa.

Comparative analysis of headspace volatiles of Chinese Rosa rugosa.

The floral headspace compounds of Chinese Rosa rugosa germplasms that were isolated by an automated headspace sampler with built-in trap, and followed by gas chromatography-mass spectrometry for identification and quantification. Up to 33 volatile compounds were identified from the 23 rose germplasms, including nine alcohols, five esters, three alkanes, 10 terpenes, three aldehydes, two ketones, and one ether. The main floral components identified were 2-phenylethanol, ß-citronellol, ethanol, and n-hexane. 'xizi', 'miaofengshan', 'xiangciguo', and 'tangbai' contained the highest amounts of 2-phenylethanol at 84.66 µg·g⁻¹, ß-citronellol at 70.98 µg·g⁻¹, ethanol at 83.87 µg·g⁻¹, and n-hexane at 18.23 µg·g⁻¹, respectively. 'Rongchengyesheng', 'tanghong', 'xizi', 'miaofengshan', and 'baizizhi' could be considered good materials for extracting rose oil and breeding new cultivars.

RrGT1, a key gene associated with anthocyanin biosynthesis, was isolated from Rosa rugosa and identified via overexpression and VIGS.

At present, research on the flower color of Rosa rugosa requires very innovative and practical studies. Glycosylation plays an important role in increasing the stability and solubility of anthocyanins in plants. In this study, a gene with a full-length cDNA of 1161 bp encoding 386 amino acids, designated RrGT1 (MK034140), was isolated from the flowers of R. rugosa 'Zizhi' and then functionally characterized. Sequence alignment revealed that the coding regions of RrGT1 were highly specific among different species but still contained typical conserved amino acid residues that are crucial for RrGT1 enzyme activity. RrGT1 transcripts were detected in various tissues of R. rugosa 'Zizhi' and Rosa davurica, and their expression patterns corresponded with the accumulation of anthocyanins. Additionally, the in vivo function of RrGT1 was investigated via its overexpression in Arabidopsis thaliana. Transgenic Arabidopsis plants expressing RrGT1 regained red color pigmentation of their leaves and flower stems, indicating that RrGT1 could encode a functional glycosyltransferase (GT) protein for anthocyanin biosynthesis and could function in other species. The functional verification of RrGT1 for anthocyanin biosynthesis in R. rugosa was performed via virus-induced gene silencing (VIGS). This was the first time that a VIGS system was developed for use with perennial Rosa plants grown naturally in the field as experimental materials to study a key color-controlling gene in Rosa. When the RrGT1 gene was silenced, the Rosa plants displayed a pale petal color phenotype. The detection results showed that the expression of the endogenous RrGT1 gene was significantly downregulated while the six key structural genes in its upstream were normally expressed, and the contents of all anthocyanins also decreased significantly. Therefore, we speculated that glycosylation of RrGT1 plays a crucial role in anthocyanin biosynthesis in R. rugosa.

Transcriptome sequencing of Paeonia suffruticosa 'Shima Nishiki' to identify differentially expressed genes mediating double-color formation.

Paeonia suffruticosa 'Shima Nishiki' is one of extremely rare double-color cultivars in the world. It usually shows the two beautiful colors of red and white in the same flower, and this trait undoubtedly makes the flowers more charming for the ornamental market. However, few studies have been done to unravel the molecular mechanisms of double-color formation in P. suffruticosa 'Shima Nishiki'. In this study, we measured the anthocyanin composition and concentration, and sequenced the transcriptomes of the red and white petals. We found that the total content of Pg-based glycosides was at a significantly higher level in the red petals. Furthermore, we assembled and annotated 92,671 unigenes. Comparative analyses of the two transcriptomes showed 227 differentially expressed genes (DEGs), among which 57 were up-regulated, and 170 were down-regulated in the red petals. Subsequently, we identified 3 DEGs and the other 6 structural genes in the anthocyanin biosynthetic pathway including PsCHS, PsCHI, PsF3H, PsF3'H, PsDFR, PsANS, PsAOMT, PsMYB, and PsWD40. Among them, PsDFR and PsMYB expressed at a significantly higher level and showed positive correlations between their expression and anthocyanin concentration in the red petals. However, PsWD40 expressed at a significantly lower level and exhibited an inverse relationship in the red petals. Furthermore, we further confirmed the relative expression of the 9 candidate genes using quantitative real-time PCR. Based on the above results, we concluded that the significant differential expression of PsDFR, PsMYB and PsWD40 may play a key role in anthocyanin concentration in the red and white petals, thereby mediating double-color formation. These data will provide a valuable resource to better understand the molecular mechanisms of double-color formation of P. suffruticosa 'Shima Nishiki'.

Anthocyanins and their biosynthetic genes in three novel-colored Rosa rugosa cultivars and their parents.

The petals of Rosa rugosa are generally pink and purple, never yellow. Although new varieties of R. rugosa have been bred, no yellow variety has ever been obtained. Therefore, the use of roses in garden settings has been restricted. Three R. rugosa hybrid cultivars (R. rugosa 'Miaoyu', 'Rudiepianpian' and 'Jiaomeisanbian') were bred in our laboratory using wild R. rugosa 'Hunchun' as the female parent and Rosa xanthina as the male parent. The petals of these cultivars appear yellow, at least in part; thus, these cultivars represent the first R. rugosa with yellow flowers. To investigate the causes of this yellow petal color, the petals of these materials were studied at both the physiological and molecular levels. Anthocyanins are the most important chromogenic substances in plants. In this study, six types of anthocyanins, cyanidin-3-O-glucoside (Cy3G), cyanidin-3,5-di-O-glucoside (Cy3G5G), pelargonidin-3-O-glucoside (Pg3G), pelargonidin-3,5-di-O-glucoside (Pg3G5G), peonidin-3-O-glucoside (Pn3G) and peonidin-3,5-di-O-glucoside (Pn3G5G), were analyzed in the petals of the new R. rugosa cultivars and their parents. All of the above anthocyanins were found in the petals of 'Hunchun', and a small amount of Cy3G5G was present in 'Miaoyu' and 'Jiaomeisanbian', but no anthocyanins were found in R. xanthina or 'Rudiepianpian'. Moreover, the expression levels of seven structural genes (RrCHS, RrCHI, RrF3H, RrFLS, RrF3'H, RrDFR and RrANS) in the flavonoid biosynthetic pathway were quantitatively analyzed via qRT-PCR. We concluded that RrFLS, RrDFR and RrF3'H are the key genes controlling petal color in these different rose varieties.

Anatomical and biochemical analyses reveal the mechanism of double-color formation in Paeonia suffruticosa 'Shima Nishiki'.

Paeonia suffruticosa 'Shima Nishiki' is a very precious double-color cultivar because of its distinctive and colorful flowers. However, our understanding of the underlying mechanisms of its double-color formation is limited. The present study investigated the soluble sugar content, cell sap pH value and anatomical structure, anthocyanin composition and content and expression patterns of genes related to anthocyanin biosynthesis in the red and pink petals of the 'Shima Nishiki' cultivar. Here, we found that soluble sugar content, cell sap pH and the shape of outer epidermal cells were not the key factors that determine double-color formation. Five different anthocyanins were detected in both the red and pink petals, and the pelargonidin-3,5-di-O-glucoside (Pg3G5G) and pelargonidin-3-O-glucoside (Pg3G) contents in the red petals were significantly higher than those in the pink petals at every developmental stage. In addition, these gene expression patterns suggested that the significant differential expression of the dihydroflavonol 4-reductase gene (PsDFR) gene might play a key role in double-color formation. These results will provide a valuable resource for further studies unraveling the underlying genetic mechanisms of double-color formation in P. suffruticosa 'Shima Nishiki'.

[Impacts of salt stress on the growth and physiological characteristics of Rosa rugosa].

Taking 1-year-old cuttings of a wild type and three cultivars of Rosa rugosa as test materials, this paper studied their biomass, photosynthesis, osmotic adjustment substance contents, root activity, and ion contents under the stress of different concentration NaCl. Salt stress inhibited the growth of the cuttings, and root was more sensitive than shoot. Under salt stress, wild rose had significantly higher contents of free proline and soluble sugar than the cultivars, and the contents of free proline and soluble sugar in cultivar 'Ziyan' were higher than those in cultivars 'Zhongke 2' and 'Purple Branch'. Compared with rose cultivars, the wild rose under salt stress had smaller changes in its photosynthetic characteristics and root activity. It was suggested that wild rose had a higher resistance to salt stress than the cultivars, and cultivar 'Ziyan' had a higher resistance than 'Purple Branch' and 'Zhongke 2'. All the test indices could be used as the indicators of R. rugosa salt-tolerance.