De-Yu Xie

Features of anthocyanin biosynthesis in pap1-D and wild-type Arabidopsis thaliana plants grown in different light intensity and culture media conditions

The number of different anthocyanin molecules potentially produced by Arabidopsis thaliana and which anthocyanin molecule is the first product of anthocyanidin modification remain unknown. To accelerate the understanding of these questions, we investigated anthocyanin biosynthesis in rosette leaves of both pap1-D and wild-type (WT) A. thaliana plants grown in nine growth conditions, which were composed of three light intensities (low light, middle light, and high light) and three media derived from MS medium (medium-1, 2, and 3). These nine growth conditions differentially affected the levels of anthocyanins and pigmentation patterns of rosette leaves, which were closely related to the diversification levels of cyanin structures. The combined growth conditions of high light and either medium-2 or medium-1 induced the most molecular diversity of anthocyanin structures in rosette leaves of pap1-D plants. Twenty cyanin molecules, including five that were previously unknown, were characterized by HPLC-ESI-MS and HPLC-TOF-MS analyses. We detected that the A. thaliana anthocyanin molecule A11 was most likely the first cyanin derived from the multiple modification steps of cyanidin. In addition, in the same growth condition, rosette leaves of pap1-D plants produced much higher levels and more diverse molecular profiling of cyanins than those of WT plants. The transcript levels of PAP1, PAL1, CHS, DFR, and ANS cDNAs were much higher in pap1-D rosette leaves than in WT ones. Furthermore, on the same agar-solidified medium, an enhancement of light intensity increased levels and molecular diversity of cyanins in both pap1-D and WT rosette leaves. In the same light intensity condition, the responses of anthocyanin levels and profiling to medium alternation were different between pap1-D and WT plants.

Engineering of red cells of Arabidopsis thaliana and comparative genome-wide gene expression analysis of red cells versus wild-type cells

Plants express genes that encode enzymes that catalyse reactions to form plant secondary metabolites in specific cell types. However, the mechanisms of how plants decide their cellular metabolic fate and how cells diversify and specialise their specific secondary metabolites remains largely unknown. Additionally, whether and how an established metabolic program impacts genome-wide reprogramming of plant gene expression is unclear. We recently isolated PAP1-programmed anthocyanin-producing (red) and -free (white) cells from Arabidopsis thaliana; our previous studies have indicated that the PAP1 expression level is similar between these two different cell types. Transcriptional analysis showed that the red cells contain the TTG1-GL3/TT8-PAP1 regulatory complex, which controls anthocyanin biosynthesis; in contrast, the white cells and the wild-type cells lack this entire complex. These data indicate that different regulatory programming underlies the different metabolic states of these cells. In addition, our previous transcriptomic comparison indicated that there is a clear difference in the gene expression profiles of the red and wild-type cells, which is probably a consequence of cell-specific reprogramming. Based on these observations, in this report we discuss the potential mechanisms that underlie the programming and reprogramming of gene expression involved in anthocyanin biosynthesis.We report metabolic engineering of Arabidopsis red cells and genome-wide gene expression analysis associated with anthocyanin biosynthesis and other metabolic pathways between red cells and wild-type (WT) cells. Red cells of A. thaliana were engineered for the first time from the leaves of production of anthocyanin pigment 1-Dominant (pap1-D). These red cells produced seven anthocyanin molecules including a new one that was characterized by LC–MS analysis. Wild-type cells established as a control did not produce anthocyanins. A genome-wide microarray analysis revealed that nearly 66 and 65% of genes in the genome were expressed in the red cells and wild-type cells, respectively. In comparison with the WT cells, 3.2% of expressed genes in the red cells were differentially expressed. The expression levels of 14 genes involved in the biosynthetic pathway of anthocyanin were significantly higher in the red cells than in the WT cells. Microarray and RT-PCR analyses demonstrated that the TTG1–GL3/TT8–PAP1 complex regulated the biosynthesis of anthocyanins. Furthermore, most of the genes with significant differential expression levels in the red cells versus the WT cells were characterized with diverse biochemical functions, many of which were mapped to different metabolic pathways (e.g., ribosomal protein biosynthesis, photosynthesis, glycolysis, glyoxylate metabolism, and plant secondary metabolisms) or organelles (e.g., chloroplast). We suggest that the difference in gene expression profiles between the two cell lines likely results from cell types, the overexpression of PAP1, and the high metabolic flux toward anthocyanins.

Evolution of bract development and B‐class MADS box gene expression in petaloid bracts of Cornus s. l. (Cornaceae)

Despite increasing interest in the molecular mechanisms of floral diversity, few studies have investigated the developmental and genetic bases of petaloid bracts. This study examined morphological patterns of bract initiation and expression patterns of B-class MADS-box genes in bracts of several Cornus species. We suggest that petaloid bracts in this genus may not share a single evolutionary origin. Developmental pathways of bracts and spatiotemporal expression of B-class genes in bracts and flowers were examined for four closely related dogwood species. Divergent morphological progressions and gene expression patterns were found in the two sister lineages with petaloid bracts, represented by Cornus florida and Cornus canadensis. Phylogeny-based analysis identified developmental and gene expression changes that are correlated with the evolution of petaloid bracts in C. florida and C. canadensis. Our data support the existence of independent evolutionary origins of petaloid bracts in C. canadensis and C. florida. Additionally, we suggest that functional transference within B-class gene families may have contributed to the origin of bract petaloidy in C. florida. However, the underlying mechanisms of petaloid bract development likely differ between C. florida and C. canadensis. In the future this hypothesis can be tested by functional analyses of Cornus B-class genes.

Salt stress induces differential regulation of the phenylpropanoid pathway in Olea europaea cultivars Frantoio (salt-tolerant) and Leccino (salt-sensitive)

Olive tree (Olea europaea L.) is an important crop in the Mediterranean Basin where drought and salinity are two of the main factors affecting plant productivity. Despite several studies have reported different responses of various olive tree cultivars to salt stress, the mechanisms that convey tolerance and sensitivity remain largely unknown. To investigate this issue, potted olive plants of Leccino (salt-sensitive) and Frantoio (salt-tolerant) cultivars were grown in a phytotron chamber and treated with 0, 60 and 120mM NaCl. After forty days of treatment, growth analysis was performed and the concentration of sodium in root, stem and leaves was measured by atomic absorption spectroscopy. Phenolic compounds were extracted using methanol, hydrolyzed with butanol-HCl, and quercetin and kaempferol quantified via high performance liquid-chromatography-electrospray-mass spectrometry (HPLC-ESI-MS) and HPLC-q-Time of Flight-MS analyses. In addition, the transcripts levels of five key genes of the phenylpropanoid pathway were measured by quantitative Real-Time PCR. The results of this study corroborate the previous observations, which showed that Frantoio and Leccino differ in allocating sodium in root and leaves. This study also revealed that phenolic compounds remain stable or are strongly depleted under long-time treatment with sodium in Leccino, despite a strong up-regulation of key genes of the phenylpropanoid pathway was observed. Frantoio instead, showed a less intense up-regulation of the phenylpropanoid genes but overall higher content of phenolic compounds. These data suggest that Frantoio copes with the toxicity imposed by elevated sodium not only with mechanisms of Na+ exclusion, but also promptly allocating effective and adequate antioxidant compounds to more sensitive organs.

An integrated approach to demonstrating the ANR pathway of proanthocyanidin biosynthesis in plants

Proanthocyanidins (PAs) are oligomers or polymers of plant flavan-3-ols and are important to plant adaptation in extreme environmental conditions. The characterization of anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) has demonstrated the different biogenesis of four stereo-configurations of flavan-3-ols. It is important to understand whether ANR and the ANR pathway widely occur in the plant kingdom. Here, we report an integrated approach to demonstrate the ANR pathway in plants. This includes different methods to extract native ANR from different tissues of eight angiosperm plants (Lotus corniculatus, Desmodium uncinatum, Medicago sativa, Hordeum vulgare, Vitis vinifera, Vitis bellula, Parthenocissus heterophylla, and Cerasus serrulata) and one fern plant (Dryopteris pycnopteroides), a general enzymatic analysis approach to demonstrate the ANR activity, high-performance liquid chromatography-based fingerprinting to demonstrate (−)-epicatechin and other flavan-3-ol molecules, and phytochemical analysis of PAs. Results demonstrate that in addition to leaves of M. sativa, tissues of other eight plants contain an active ANR pathway. Particularly, the leaves, flowers and pods of D. uncinatum, which is a model plant to study LAR and the LAR pathways, are demonstrated to express an active ANR pathway. This finding suggests that the ANR pathway involves PA biosynthesis in D. uncinatum. In addition, a sequence BLAST analysis reveals that ANR homologs have been sequenced in plants from both gymnosperms and angiosperms. These data show that the ANR pathway to PA biosynthesis occurs in both seed and seedless vascular plants.

Analysis of two TFL1 homologs of dogwood species (Cornus L.) indicates functional conservation in control of transition to flowering.

AbstractMain conclusionTwoTFL1-like genes,CorfloTFL1andCorcanTFL1cloned fromCornus floridaandC. canadensis,function in regulating the transition to reproductive development inArabidopsis. TERMINAL FLOWER 1 (TFL1) is known to regulate inflorescence development in Arabidopsis thaliana and to inhibit the transition from a vegetative to reproductive phase within the shoot apical meristem. Despite the importance, TFL1 homologs have been functionally characterized in only a handful eudicots. Here we report the role of TFL1 homologs of Cornus L. in asterid clade of eudicots. Two TFL1-like genes, CorfloTFL1 and CorcanTFL1, were cloned from Cornus florida (a tree) and C. canadensis (a subshrub), respectively. Both are deduced to encode proteins of 175 amino acids. The amino acid sequences of these two Cornus TFL1 homologs share a high similarity to Arabidopsis TFL1 and phylogenetically more close to TFL1 paralogous copy ATC (Arabidopsis thaliana CENTRORADIALIS homologue). Two genes are overexpressed in wild-type and tfl1 mutant plants of A. thaliana. The over-expression of each gene in wild-type Arabidopsis plants results in delaying flowering time, increase of plant height and cauline and rosette leaf numbers, excessive shoot buds, and secondary inflorescence branches. The over-expression of each gene in the tfl1 mutant rescued developmental defects, such as the early determinate inflorescence development, early flowering time, and other vegetative growth defects, to normal phenotypes of wild-type plants. These transgenic phenotypes are inherited in progenies. All data indicate that CorfloTFL1 and CorcanTFL1 have conserved the ancestral function of TFL1 and CEN regulating flowering time and inflorescence determinacy.

Characterization of Flavan-3-ols and Expression of MYB and Late Pathway Genes Involved in Proanthocyanidin Biosynthesis in Foliage of Vitis bellula.

Proanthocyanidins (PAs) are fundamental nutritional metabolites in different types of grape products consumed by human beings. Although the biosynthesis of PAs in berry of Vitis vinifera has gained intensive investigations, the understanding of PAs in other Vitis species is limited. In this study, we report PA formation and characterization of gene expression involved in PA biosynthesis in leaves of V. bellula, a wild edible grape species native to south and south-west China. Leaves are collected at five developmental stages defined by sizes ranging from 0.5 to 5 cm in length. Analyses of thin layer chromatography (TLC) and high performance liquid chromatography-photodiode array detector (HPLC-PAD) show the formation of (+)-catechin, (−)-epicatechin, (+)-gallocatechin and (−)-epigallocatechin during the entire development of leaves. Analyses of butanol-HCl boiling cleavage coupled with spectrometry measurement at 550 nm show a temporal trend of extractable PA levels, which is characterized by an increase from 0.5 cm to 1.5 cm long leaves followed by a decrease in late stages. TLC and HPLC-PAD analyses identify cyanidin, delphinidin and pelargonidin produced from the cleavage of PAs in the butanol-HCl boiling, showing that the foliage PAs of V. bellula include three different types of extension units. Four cDNAs, which encode VbANR, VbDFR, VbLAR1 and VbLAR2, respectively, are cloned from young leaves. The expression patterns of VbANR and VbLAR2 but not VbLAR1 and VbDFR follow a similar trend as the accumulation patterns of PAs. Two cDNAs encoding VbMYBPA1 and VbMYB5a, the homologs of which have been demonstrated to regulate the expression of both ANR and LAR in V. vinifera, are also cloned and their expression profiles are similar to those of VbANR and VbLAR2. In contrast, the expression profiles of MYBA1 and 2 homologs involved in anthocyanin biosynthesis are different from those of VbANR and VbLAR2. Our data show that both ANR and LAR branches are involved in PA biosynthesis in leaves of V. bellula.

Overexpression of Populus × canescens isoprene synthase gene in Camelina sativa leads to alterations in its growth and metabolism

Isoprene (2-methyl-1,3-butadiene) is a hemiterpene molecule. It has been estimated that the plant kingdom emits 500-750 million tons of isoprene in the environment, half of which results from tropical broadleaf trees and the remainder from shrubs. Camelina (Camelina sativa (L.) Crantz) is an emerging bioenergy plant for biodiesel. In this study, we characterized isoprene formation following a diurnal/nocturnal cycle in wild-type Camelina plants. To understand the potential effects of isoprene emission on this herbaceous plant, a gray poplar Populus×canescens isoprene synthase gene (PcISPS) was overexpressed in Camelina. Transgenic plants showed increased isoprene production, and the emissions were characterized by a diurnal/nocturnal cycle. Measurements of the expression of six genes of the plastidial 2-C-methyl-d-erythriol-4-phosphate (MEP) pathway revealed that the expression patterns of three key genes were associated with isoprene formation dynamics in the three genotypic plants. Conversely, dissimilar gene expression levels existed in different genotypes, indicating that dynamics and variations occurred among plants. Moreover, transgenic plants grew shorter and developed smaller leaves than the wild-type and empty vector control transgenic plants. Photosynthetic analysis showed that the CO2 assimilation rate, intracellular CO2 concentration, mesophyll conductance and contents of chlorophylls a and b were similar among PcISPS transgenic, empty-vector control transgenic, and wild-type plants, indicating that the transgene did not negatively affect photosynthesis. Based on these results, we suggest that the reduced biomass was likely a trade-off consequence of the increased isoprene emission.

Cloning and characterization of a monoterpene synthase gene from flowers of Camelina sativa

Main conclusionCsTPS1encodes for a monoterpene synthase that contributes to the emission of a blend of volatile compounds emitted from flowers ofCamelina sativa.The work describes the in vitro characterization of a monoterpene synthase and its regulatory region that we cloned from Camelina sativa (Camelina). Here, we named this gene as C. sativa terpene synthase 1 (CsTPS1). In vitro experiments performed with the CsTPS1 protein after expression and purification from Escherichia coli (E. coli) showed production of a blend of monoterpene volatile organic compounds, of which the emission was also detected in the floral bouquet of wild-type Camelina plants. Quantitative-PCR measurements revealed a high abundance of CsTPS1 transcripts in flowers and experiments performed with the GUS reporter showed high CsTPS1 expression in the pistil, in the cells of the wall of the ovary and in the stigma. Subcellular localization of the CsTPS1 protein was investigated with a GFP reporter construct that showed expression in plastids. The CsTPS1 gene identified in this study belongs to a mid-size family of 60 genes putatively codifying for TPS enzymes. This enlarged family of TPS genes suggests that Camelina has the structural framework for the production of terpenes and other secondary metabolites of relevance for the consumers.

Correction to: Cloning and characterization of a monoterpene synthase gene from flowers of Camelina sativa

In the original publication, the order of figures and citations was incorrect. The corrections are listed below: