Search

Hydrangea macrophylla, renowned for its large inflorescences and a diverse range of colors, highlights a significant limitation in current gene function research, which is the lack of effective molecular genetic tools. This study utilized a tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) system to investigate gene function through posttranscriptional gene silencing in H. macrophylla for the first time. The ortholog of phytoene desaturase (PDS) in H. macrophylla, termed HmPDS, was identified. Infection of tissue-cultured seedlings with TRV-HmPDS led to photobleaching of the leaves. Additionally, infection with TRV containing the HmCHS1 fragment in the flowers resulted in decreased anthocyanin production in sepals and a lightening of sepal coloration in the infected flowers. The phenomena and RT-qPCR results proved that the PDS and CHS genes of hydrangea were successfully silenced via the vacuum infiltration method. Furthermore, the introduction of TRV-HmF3′5′H revealed a decrease in delphinidin-3-glucoside content in sepals and caused a color change in the sepals from blue to pink. This study demonstrated that the TRV-VIGS system was successfully established in H. macrophylla and effectively applied to the function analysis of HmF3′5′H.

Hydrangea macrophylla (H. macrophylla), a species in the genus Hydrangea in the family Hydrangeaceae, is widely valued for its ornamental qualities in both domestic and international markets. Notably, H. macrophylla is known for its ability to accumulate aluminum (Al). Moreover, aluminum ions (Al3+) participate in sepal bluing. However, the underlying mechanisms of Al accumulation in the sepals remain unclear. In this study, we utilized transcriptome data from two cultivars to identify genes associated with Al accumulation. In total, 154 differentially expressed isoforms between the CK and Tr groups in the sepals of both cultivars were screened. Through gene enrichment analysis and similarity identification in the CDS (coding sequence) region, 43 differentially expressed genes were identified, including 30 upregulated and 13 downregulated genes, in the sepals of the Al treatment group. Further analysis revealed that seven of these upregulated genes are related to Al accumulation in sepals. Among the seven, the gene HmALS3.1 was identified as a potential key player in Al transport within the sepals of H. macrophylla. This study lays the groundwork for further exploration into the mechanisms by which HmALS3.1 regulates Al accumulation in H. macrophylla.

Hydrangea macrophylla is popular for its unique physiological characteristics and changeable colors. Previous studies have shown that the pH of the vacuoles of the sepal cells of hydrangea affects the color of the sepals. Located on the vacuolar membrane, NHX1 is an important H+ proton pump that drives the exchange of metal ions. This proton pump affects the physiological environment by controlling the accumulation of H+ in the vacuole. In hydrangea, the HmNHX1 gene has an open reading frame of 1626 bp and encodes a total of 541 amino acids. Bioinformatic analysis showed that HmNHX1, which encodes a Na+/H+ exchanger, is located on the vacuolar membrane. Tissue-specific expression analysis showed that the expression of this gene in the treatment group was higher than that in the control group. The ion flux in the vacuoles of colored hydrangea in the treatment group and the control group were measured, and the results showed that HmNHX1 was indeed a Na+/H+ exchanger. When the results of the HmNHX1 expression analysis and ion flux measurements are combined, it can be seen that HmNHX1 regulates the accumulation of H+ in the vacuole, ultimately affecting the color of the plant.

Under specific cultivation conditions, the sepal color of Hydrangea macrophylla (H. macrophylla) changes from red to blue due to the complexation of aluminum ions (Al3+), delphinidin 3-glucoside, and copigments. However, this phenomenon cannot occur in all cultivars despite the presence of sufficient Al3+ and copigments. To explore the mechanism of sepal bluing in H. macrophylla, there is an urgent need to study the molecular regulation of the anthocyanin biosynthesis pathway. However, the key structural genes, other than CHS, regulating anthocyanin biosynthesis in the sepals of H. macrophylla have not been identified. In this study, based on full-length transcriptome data from H.macrophylla ‘Bailmer’, the key structural genes regulating anthocyanin biosynthesis in the sepals of H. macrophylla were isolated and investigated. Ultimately, seven key structural genes, HmCHS1, HmCHI, HmF3H1, HmF3′H1, HmF3′5′H, HmDFR2, and HmANS3, were demonstrated to show high expression levels in colored sepals. The expression levels of these seven genes increased gradually with the development of sepals and were highest in the full-bloom stage. The trend of gene expression was consistent with the trend of anthocyanin contents. It was concluded that the seven selected genes were involved in anthocyanin biosynthesis in the sepals of H. macrophylla. The full-length sequence data have been deposited into the NCBI Sequence Read Archive (SRA) with accession number PRJNA849710. This study lays a good foundation for the further elucidation of the molecular mechanism of sepal coloration in H. macrophylla.

Recent investigations have shown that the addition of manganese (Mn) sand to constructed wetlands (i.e., Mn-amended CWs) can improve the performance of organic micropollutants (MPs) removal. In addition to the direct oxidation and adsorption of Mn oxides, the indirect role of Mn oxides in MP biotransformation is crucial to the removal of MPs but has seldom been referred to. Herein, we constructed lab-scale CWs with or without the addition of natural Mn sand (∼35% Mn oxides) to decipher the influence of Mn oxides on the biotransformation of the six selected MPs which commonly existed in the wastewater. The experimental results showed that the addition of Mn sand to CWs can improve the removal of MPs (8.48% atrazine, 13.16% atenolol, and 6.27% sulfamethoxazole [pairwise Wilcoxon test p

Abstract Glucoraphanin is a major secondary metabolite found in Brassicaceae vegetables, especially broccoli, and its degradation product sulforaphane plays an essential role in anticancer. The fine mapping of sulforaphane metabolism quantitative trait loci (QTLs) in broccoli florets is necessary for future marker-assisted selection strategies. In this study, we utilized a doubled haploid population consisting of 176 lines derived from two inbred lines (86,101 and 90,196) with significant differences in sulforaphane content, coupled with extensive genotypic and phenotypic data from two independent environments. A linkage map consisting of 438 simple sequence repeats markers was constructed, covering a length of 1168.26 cM. A total of 18 QTLs for sulforaphane metabolism in broccoli florets were detected, 10 were detected in 2017, and the other 8 were detected in 2018. The LOD values of all QTLs ranged from 3.06 to 14.47, explaining 1.74–7.03% of the biochemical variation between two years. Finally, 6 QTLs (qSF-C3-1, qSF-C3-2, qSF-C3-3, qSF-C3-5, qSF-C3-6 and qSF-C7) were stably detected in more than one environment, each accounting for 4.54–7.03% of the phenotypic variation explained (PVE) and a total of 30.88–34.86% of PVE. Our study provides new insights into sulforaphane metabolism in broccoli florets and marker-assisted selection breeding in Brassica oleracea crops.

Gene function analysis is challenging for Lilium due to the lack of an efficient method for stable genetic transformation. Virus-induced gene silencing (VIGS) is an attractive tool for determining gene function in plants. This study reported that Tobacco rattle virus (TRV)-based VIGS of a PHYTOENE DESATURASE (PDS) ortholog (LhPDS) can be achieved in Lilium × formolongi seedlings, with a survival rate of 92%, using the inoculation method of rubbing plus injection. Compared with untreated and mock-treated seedlings, the photobleached leaf phenotype of silenced plants significantly correlates with down-regulation of endogenous LhPDS (P ≤ 0.05). In addition, the silencing phenomenon can be observed in the growing points of plants, indicating that systemic viral infection was achieved using the protocol. The results indicate that TRV-based VIGS can be used to characterize gene function in Lilium × formolongi. This work lays the foundation for gene function analysis and molecular breeding in Lilium spp. Keywords: Lilium, photobleaching, seedling, TRV, VIGS, PDS

Hydrangea spp. is renowned for its variety of color changes in its developmental stage and before and after aluminum treatment. We analyzed gene expression in hydrangeas sepals to study the causes of color change. The accuracy of quantitative RT-qPCR analysis depends on the reliability of reference genes. We selected reference genes for hydrangea of varying cultivars, at different developmental stages, and in aluminum treatment groups. We chose ‘Bailmer’ and ‘Duro’ as subject species. We selected eight candidate genes, all of which were ranked by geNorm, NormFinder, BestKeeper, and RefFinder. CCR, NHX1, and LODX were used to verify the exactitude of reference genes. According to the ranking result of RefFinder, the top-ranked reference genes in each group were different; the top four candidate reference genes in each group mostly included EF1-β, RPL34, GADPH, and RPL10. EF1-β and RPL34 ranked top in the ‘all materials’ group, and their expression trends, obtained from the analysis of CCR, NHX1, and LODX, were consistent. From the results, we gather that EF1-β and RPL34 can be used as reference genes to quantify target gene expression. In this study, we screened for reference genes in hydrangeas to provide a technical basis for hydrangea sepal formation and transformation for further experiments.

Virus-induced gene silencing (VIGS) is an attractive tool for determining gene function in plants. The present study constitutes the first application of VIGS in S. pseudocapsicum, which has great ornamental and pharmaceutical value, using tobacco rattle virus (TRV) vectors. Two marker genes, PHYTOENE DESATURASE (PDS) and Mg-chelatase H subunit (ChlH), were used to test the VIGS system in S. pseudocapsicum. The photobleaching and yellow-leaf phenotypes of the silenced plants were shown to significantly correlate with the down-regulation of endogenous SpPDS and SpChlH, respectively (P ≤ 0.05). Moreover, the parameters potentially affecting the efficiency of VIGS in S. pseudocapsicum, including the Agrobacterium strain and the inoculation method (leaf syringe-infiltration, sprout vacuum-infiltration and seed vacuum-infiltration), were compared. The optimized VIGS parameters were the leaf syringe-infiltration method, the Agrobacterium strain GV3101 and the growth of agro-inoculated plants at 25°. With these parameters, the silencing efficiency of SpPDS and SpChlH could reach approximately 50% in S. pseudocapsicum. Additionally, the suitability of various reference genes was screened by RT-qPCR using three candidate genes, and the results demonstrated that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) can serve as a suitable reference for assessing the gene expression levels of VIGS systems in S. pseudocapsicum. The proven application of VIGS in S. pseudocapsicum and the characterization of a suitable reference gene in the present work will expedite the functional characterization of novel genes in S. pseudocapsicum.

Aerial bulbils are an important propagative organ, playing an important role in population expansion. However, the detailed gene regulatory patterns and molecular mechanism underlying bulbil formation remain unclear. Triploid Lilium lancifolium, which develops many aerial bulbils on the leaf axils of middle-upper stem, is a useful species for investigating bulbil formation. To investigate the mechanism of bulbil formation in triploid L. lancifolium, we performed histological and transcriptomic analyses using samples of leaf axils located in the upper and lower stem of triploid L. lancifolium during bulbil formation. Histological results indicated that the bulbils of triploid L. lancifolium are derived from axillary meristems that initiate de novo from cells on the adaxial side of the petiole base. Transcriptomic analysis generated ~650 million high-quality reads and 11,871 differentially expressed genes (DEGs). Functional analysis showed that the DEGs were significantly enriched in starch and sucrose metabolism and plant hormone signal transduction. Starch synthesis and accumulation likely promoted the initiation of upper bulbils in triploid L. lancifolium. Hormone-associated pathways exhibited distinct patterns of change in each sample. Auxin likely promoted the initiation of bulbils and then inhibited further bulbil formation. High biosynthesis and low degradation of cytokinin might have led to bulbil formation in the upper leaf axil. The present study achieved a global transcriptomic analysis focused on gene expression changes and pathways' enrichment during upper bulbil formation in triploid L. lancifolium, laying a solid foundation for future molecular studies on bulbil formation.

Quantitative real-time PCR (qRT-PCR) is a reliable and high-throughput technique for gene expression studies, but its accuracy depends on the expression stability of reference genes. To date, several reliable reference gene identifications have been reported in Lilium spp., but none has been obtained for lily tepals at different developmental stages. In this study, ten candidate reference genes were selected and evaluated for their expression stability in Lilium ‘Tiny Padhye’ during the process of bicolor tepal development. The expression stability of these candidates was evaluated by three software programs (geNorm, NormFinder, and BestKeeper) and the comparative ΔCt method, and comprehensive stability rankings were generated by RefFinder. As a result, TIP41-like family gene (TIP41) and actin (ACT) were the best combination of reference genes for tepals at different developmental stages; TIP41 and F-box family gene (F-box) for tepals under shading treatment; ACT, actin11 (ACT11), and elongation factor 1-α (EF1-α) for different tissues; and ACT, TIP41, and ACT11 for all samples. The selected optimal reference genes were further verified by analyzing the expression levels of flavonoid 3′-hydroxylase (LhF3′H) and anthocyanidin 3-O-glucosyltransfersae (LhUFGT) in tepals at different developmental stages. This study provides useful information for gene expression characterization in lilies under different experimental conditions, and can serve as a basis for similar research in other closely related species.

Hydrangea macrophylla is popular for its unique physiological characteristics and changeable colors. Previous studies have shown that the pH of the vacuoles of the sepal cells of hydrangea affects the color of the sepals. Located on the vacuolar membrane, NHX1 is an important H+ proton pump that drives the exchange of metal ions. This proton pump affects the physiological environment by controlling the accumulation of H+ in the vacuole. In hydrangea, the HmNHX1 gene has an open reading frame of 1626 bp and encodes a total of 541 amino acids. Bioinformatic analysis showed that HmNHX1, which encodes a Na+/H+ exchanger, is located on the vacuolar membrane. Tissue-specific expression analysis showed that the expression of this gene in the treatment group was higher than that in the control group. The ion flux in the vacuoles of colored hydrangea in the treatment group and the control group were measured, and the results showed that HmNHX1 was indeed a Na+/H+ exchanger. When the results of the HmNHX1 expression analysis and ion flux measurements are combined, it can be seen that HmNHX1 regulates the accumulation of H+ in the vacuole, ultimately affecting the color of the plant. [ABSTRACT FROM AUTHOR]

Pollen viability and stigma receptivity are prerequisites for successful pollination and seed set in flowering plants. In this study, the pollen viabilities and stigma receptivities of nine Lilium genotypes (six cultivars and three species native to China) were assayed by in vitro pollen germination and the benzidine-H2O2 method, respectively. Embryo sac development during anthesis was observed to further ensure the timing of controlled pollination. In addition, the relationship between stigma secretion and stigma receptivity was studied to estimate the pollination time based on phenotype. Anthers cracked on the day of flowering in all genotypes, but pollen germination during anthesis was not observed in Asiatic hybrids excepted for ‘Tiny pudhye’, which exhibited low pollen viability for a short period of time (from 0 to 1 day after anthesis). In the other genotypes, pollen germination rates were highest on anthesis (five of seven genotypes), 0–1 day after anthesis (L. sulphureum), or 0–2 days after anthesis (one Longiflorum hybrid), and then gradually decreased with days after anthesis. While, stigma receptivity first increased and then decreased during anthesis. For most genotypes, stigmas began to be receptive 1 day after anthesis, and all genotypes exhibited stigma receptivity at 2 days after anthesis. The durations of stigma receptivity and strongest stigma receptivity, were genotype dependent, and were 5–8 days and 1–4 days, respectively. Moreover, on the first flowering day, 6 of 7 genotypes had mature embryo sacs, and at the time at which stigmas began to be receptive, all tested genotypes had mature embryo sacs. Some Lilium genotypes showed stigma secretion, which can be a sign of stigma receptivity. Stigmas became receptive and reached highest receptivity within 1 day of the first appearance of secretion on the surface of the stigma and at peaking, respectively. The results of this study are valuable for the implementation of successful Lilium breeding programs. [ABSTRACT FROM AUTHOR]

Quantitative real-time PCR (qRT-PCR) is a reliable and high-throughput technique for gene expression studies, but its accuracy depends on the expression stability of reference genes. To date, several reliable reference gene identifications have been reported in Lilium spp., but none has been obtained for lily tepals at different developmental stages. In this study, ten candidate reference genes were selected and evaluated for their expression stability in Lilium 'Tiny Padhye' during the process of bicolor tepal development. The expression stability of these candidates was evaluated by three software programs (geNorm, NormFinder, and BestKeeper) and the comparative 1Ct method, and comprehensive stability rankings were generated by RefFinder. As a result, TIP41-like family gene (TIP41) and actin (ACT) were the best combination of reference genes for tepals at different developmental stages; TIP41 and F-box family gene (F-box) for tepals under shading treatment; ACT, actin11 (ACT11), and elongation factor 1-α (EF1-α) for different tissues; and ACT, TIP41, and ACT11 for all samples. The selected optimal reference genes were further verified by analyzing the expression levels of flavonoid 3'-hydroxylase (LhF30H) and anthocyanidin 3-O-glucosyltransfersae (LhUFGT) in tepals at different developmental stages. This study provides useful information for gene expression characterization in lilies under different experimental conditions, and can serve as a basis for similar research in other closely related species. [ABSTRACT FROM AUTHOR]

The bicolor Asiatic hybrid lily cultivar "Tiny Padhye" is an attractive variety because of its unique color pattern. During its bicolor tepal development, the upper tepals undergo a rapid color change from green to white, while the tepal bases change from green to purple. However, the molecular mechanisms underlying these changes remain largely uncharacterized. To systematically investigate the dynamics of the lily bicolor tepal transcriptome during development, we generated 15 RNA-seq libraries from the upper tepals (S2-U) and basal tepals (S1-D, S2-D, S3-D, and S4-D) of Lilium "Tiny Padhye." Utilizing the Illumina platform, a total of 295,787 unigenes were obtained from 713.12 million high-quality paired-end reads. A total of 16,182 unigenes were identified as differentially expressed genes during tepal development. Using Kyoto Encyclopedia of Genes and Genomes pathway analysis, candidate genes involved in the anthocyanin biosynthetic pathway (61 unigenes), and chlorophyll metabolic pathway (106 unigenes) were identified. Further analyses showed thatmost anthocyanin biosynthesis genes were transcribed coordinately in the tepal bases, but not in the upper tepals, suggesting that the bicolor trait of "Tiny Padhye" tepals is caused by the transcriptional regulation of anthocyanin biosynthetic genes. Meanwhile, the high expression level of chlorophyll degradation genes and low expression level of chlorophyll biosynthetic genes resulted in the absence of chlorophylls from "Tiny Padhye" tepals after flowering. Transcription factors putatively involved in the anthocyanin biosynthetic pathway and chlorophyll metabolismin lilies were identified using a weighted gene co-expression network analysis and their possible roles in lily bicolor tepal development were discussed. In conclusion, these extensive transcriptome data provide a platform for elucidating the molecular mechanisms of bicolor tepals in lilies and provide a basis for similar research in other closely related species. [ABSTRACT FROM AUTHOR]

Lily tepals have a short lifespan. Once the tepals senesce, the ornamental value of the flower is lost. Some cultivars have attractive purple ovaries and fruits which greatly enhance the ornamental value of Asiatic hybrid lilies. However, little is known about the molecular mechanisms of anthocyanin biosynthesis in Asiatic hybrid lily ovaries. To investigate the transcriptional network that governs purple ovary coloration in Asiatic hybrid lilies, we obtained transcriptome data from green ovaries (S1) and purple ovaries (S2) of Asiatic "Tiny Padhye". Comparative transcriptome analysis revealed 4228 differentially expressed genes. Differential expression analysis revealed that ten unigenes including four CHS genes, one CHI gene, one F3H gene, one F30H gene, one DFR gene, one UFGT gene, and one 3RT gene were significantly up-regulated in purple ovaries. One MYB gene, LhMYB12-Lat, was identified as a key transcription factor determining the distribution of anthocyanins in Asiatic hybrid lily ovaries. Further qPCR results showed unigenes related to anthocyanin biosynthesis were highly expressed in purple ovaries of three purple-ovaried Asiatic hybrid lilies at stages 2 and 3, while they showed an extremely low level of expression in ovaries of three green-ovaried Asiatic hybrid lilies during all developmental stages. In addition, shading treatment significantly decreased pigment accumulation by suppressing the expression of several unigenes related to anthocyanin biosynthesis in ovaries of Asiatic "Tiny Padhye". Lastly, a total of 15,048 Simple Sequence Repeats (SSRs) were identified in 13,710 sequences, and primer pairs for SSRs were designed. The results could further our understanding of the molecular mechanisms of anthocyanin biosynthesis in Asiatic hybrid lily ovaries. [ABSTRACT FROM AUTHOR]

Chromosome doubling of microspore-derived plants is an important factor in the practical application of microspore culture technology because breeding programs require a large number of genetically stable, homozygous doubled haploid plants with a high level of fertility. In the present paper, 29 populations of microspore-derived plantlets from cabbage (Brassica oleracea var. capitata) and broccoli (Brassica oleracea var. italica) were used to study the ploidy level and spontaneous chromosome doubling of these populations, the artificial chromosome doubling induced by colchicine, and the influence of tissue culture duration on the chromosomal ploidy of the microspore-derived regenerants. Spontaneous chromosome doubling occurred randomly and was genotype dependent. In the plant populations derived from microspores, there were haploids, diploids, and even a low frequency of polyploids and mixed-ploidy plantlets. The total spontaneous doubling in the 14 cabbage populations ranged from 0 to 76.9%, compared with 52.2 to 100% in the 15 broccoli populations. To improve the rate of chromosome doubling, an efficient and reliable artificial chromosome doubling protocol (i.e., the immersion of haploid plantlet roots in a colchicine solution) was developed for cabbage and broccoli microspore-derived haploids. The optimal chromosome doubling of the haploids was obtained with a solution of 0.2% colchicine for 9-12 h or 0.4% colchicine for 3-9 h for cabbage and 0.05% colchicine for 6-12 h for broccoli. This protocol produced chromosome doubling in over 50% of the haploid genotypes for most of the populations derived from cabbage and broccoli. Notably, after 1 or more years in tissue culture, the chromosomes of the haploids were doubled, and most of the haploids turned into doubled haploid or mixed-ploidy plants. This is the first report indicating that tissue culture duration can change the chromosomal ploidy of microspore-derived regenerants. [ABSTRACT FROM AUTHOR]

Background: Brassica oleracea encompass a family of vegetables and cabbage that are among the most widely cultivated crops. In 2009, the B. oleracea Genome Sequencing Project was launched using next generation sequencing technology. None of the available maps were detailed enough to anchor the sequence scaffolds for the Genome Sequencing Project. This report describes the development of a large number of SSR and SNP markers from the whole genome shotgun sequence data of B. oleracea, and the construction of a high-density genetic linkage map using a double haploid mapping population. Results: The B. oleracea high-density genetic linkage map that was constructed includes 1,227 markers in nine linkage groups spanning a total of 1197.9 cM with an average of 0.98 cM between adjacent loci. There were 602 SSR markers and 625 SNP markers on the map. The chromosome with the highest number of markers (186) was C03, and the chromosome with smallest number of markers (99) was C09. Conclusions: This first high-density map allowed the assembled scaffolds to be anchored to pseudochromosomes. The map also provides useful information for positional cloning, molecular breeding, and integration of information of genes and traits in B. oleracea. All the markers on the map will be transferable and could be used for the construction of other genetic maps. [ABSTRACT FROM AUTHOR]

Lilium davidii var. unicolor Salisb is a cultivar of Lilium (Liliaceae) with important edible and ornamental characteristic. The application and production of Lilium davidii var. unicolor Salisb were still facing large problems because of its several disadvantages such as narrow range of adaptability, small annual growth increment, and low fertility. To achieve broader environmental adaptability and obtain a more nutritious germplasm, we used colchicine and oryzalin to induce chromosome doubling via the soaking method. Tissue culture bulbs were treated with colchicine at 0.03%, 0.05%, or 0.08%for 32, 40, or 48 hours or with oryzalin at 0.002%, 0.005%, 0.008%, or 0.01% for 3, 6, 9, 12, or 24 hours before being transferred to a differentiating medium. The results showed that colchicine treatment resulted in the highest induction rate when applied at 0.05% for 48 hours, whereas oryzalin treatment produced fewer tetraploid plants. The chromosome number of induced plants with small stoma density and longer guard cells is twice than that of the diploid. The plants were identified as tetraploid. In this study, a new germplasm of Lilium davidii var. unicolor Salisb was innovative and showed novel genetic characteristic. [ABSTRACT FROM AUTHOR]