Candidate reference genes for quantitative gene expression analysis in Lagerstroemia indica.

Quantitative gene expression analysis by qPCR requires reference genes for normalization. Lagerstroemia indica (crape myrtle) is a popular ornamental plant in the world, but suitable endogenous reference genes are lacking. To find suitable reference genes, we evaluated the stabilities of nine candidate genes in six experimental data sets: six different tissues, three leaf colors, nine flower colors, and under three abiotic stresses (salt, drought, cold) using four statistical algorithms. A target gene LiMYB56 (homolog of Arabidopsis MYB56) was used to verify the authenticity and accuracy of the candidate reference genes. The results showed that the combination of two stably expressed reference genes, rather than a single reference gene, improved the accuracy of the qPCR. LiEF1α-2 + LiEF1α-3 was best for the tissue, salt treatment, and drought treatment sets; LiEF1α-2 + LiEF1α-1 was optimal for leaf color; LiEF1α-2 + LiACT7 was optimal for cold treatment; and LiUBC + LiEF1α-1 was best for the flower color set. Notably, LiEF1α-2 had high expression stability in all six experimental sets, implying it may be a good reference gene for expression studies in L. indica. Our results will facilitate future gene expression studies in L. indica.

Phenolic Compounds and Antioxidant Capacity Comparison of Wild-Type and Yellow-Leaf gl1 Mutant of Lagerstroemia indica.

BACKGROUND: The yellow-leaf gl1 mutant of Lagerstroemia indica exhibits an altered phenylpropanoid metabolism pathway compared to wild-type (WT). However, details on the metabolites associated with leaf color variation, including color-specific metabolites with bioactive constituents, are not fully understood. METHODS: Chemical and metabolomics approaches were used to compare metabolite composition and antioxidant capacity between the gl1 mutant and WT leaves. RESULTS: The mutant exhibited an irregular xylem structure with a significantly lower phenolic polymer lignin content and higher soluble phenolic compounds. Untargeted metabolomics analysis identified phenolic compounds, particularly lignans, as key differential metabolites between gl1 and WT, with a significant increase in the mutant. The neolignan derivative balanophonin-4-O-D-glu was identified as a characteristic metabolite in the gl1 mutant. The soluble phenolic compounds of the gl1 mutant exhibited higher FRAP, ABTS, DPPH, and hydroxyl radical scavenging activity than in WT. Correlation analysis showed a positive relationship between antioxidant capacity and phenolic compounds in L. indica. CONCLUSIONS: Metabolites associated with leaf color variation in the L. indica yellow-leaf gl1 mutant demonstrated high antioxidant capacity, particularly in scavenging hydroxyl radicals.

The oxidative damage of the Lagerstroemia indica chlorosis mutant gl1 involves in ferroptosis.

Photooxidation is the major physiological performance of the Lagerstroemia indica chlorosis mutant gl1 under field conditions. The mechanisms of the progressive symptoms of oxidative damage from the lower older leaves to the upper mature leaves are complicated and still unclear. The aim of this work was to investigate the physiological mechanisms of oxidative stress from the perspective of the photosynthetic metabolites. The phytosynthetic metabolites of gl1 mutant changed significantly compared to wild type (WT) L. indica, such as by increasing phenolics, decreasing soluble sugar, protein and ascorbate, and redistributing antioxidant enzyme activities. The co-accumulation of phenolics and guaiacol-POD in gl1 mutant promote the removal of H2O2, as well the increase of phenoxyl radicals levels. Furthermore, the ion balance was significantly disturbed and Fe accumulated the most among these fluctuating nutrients in the leaves of gl1 mutant. The accumulated Fe was found neither in the chloroplasts nor in the cell wall of the leaves and became unshielded Fe, which favors the Fenton/Haber-Weiss reaction and stabilizes the phenoxyl radicals in metal complexation. The results suggested that the increase of phenolics and Fe accumulation were obviously involved in oxidative damage of gl1 mutant.

Efficient Transformation of Catalpa bungei Shows Crystal Genes Conferring Resistance to the Shoot Borer Omphisa plagialis.

Although Catalpa bungei is a forest plant with considerable economic and ornamental value in China, its wood and decorative qualities are constrained by insect pests such as the shoot borer Omphisa plagialis (Lepidoptera). Overexpressing insect resistance genes such as crystal genes to develop an insect-resistant variety of C. bungei is an environmental and ecological approach. However, genotype limitations and low regeneration rates of embryogenic calli (EC) inhibit the development of transformation and the insect-resistant gene expression system in C. bungei. Here, we first established embryogenic callus induction and regeneration systems of five genotypes using mature seed and stem segment explants; the highest induction and regeneration rates of EC were 39.89 and 100%, respectively. Next, an efficient and stable Agrobacterium-mediated genetic transformation system was developed from EC and its positive frequency was up to 92.31%. Finally, using the transformation system, 15 and 22 transgenic C. bungei lines that expressed Cry2A and Cry9Aa-like were generated, respectively. These transgenic lines that exhibited significantly higher resistance to O. plagialis in the laboratory and field have great promise for meeting the challenge of future pest management under changing climatic conditions. Additionally, this efficient, fast, and stable transformation system could be a potential tool for gene function analysis and forest tree genetic improvement.

[Research of steroidal saponins from the fresh rhizomes of Dioscorea zingiberensis].

OBJECTIVE: To study the steroidal saponins from the fresh rhizomes of Dioscorea zingiberensis and search for new bioactive compounds. METHODS: The steroidal saponins were isolated by normal phase silica gel and RP-C18 column chromatography, their chemical structures were elucidated by MS and NMR methods. RESULTS: One steroidal saponin was isolated from EtOH extract of the fresh rhizomes of D. zingiberensis and identified as 26-O-beta-D-glucopyranosyl-3beta, 22alpha, 26-trihydroxy-25(R)-furosta-5-en-3-O-[alpha-L-rhamnopyranosyl-(1 --> 4) ]-beta-D-glucopyranoside. CONCLUSION: This compound is isolated from Dioscorea L. the first time.

Label-free comparative proteomic and physiological analysis provides insight into leaf color variation of the golden-yellow leaf mutant of Lagerstroemia indica.

GL1 is a golden-yellow leaf mutant that cultivated from natural bud-mutation of Lagerstroemia indica and has a very low level of photosynthetic pigment under sunlight. GL1 can gradually increase its pigment content and turn into pale-green leaf when shading under sunshade net (referred as Re-GL1). The mechanisms that cause leaf color variation are complicated and are not still unclear. Here, we have used a label-free comparative proteomics to investigate differences in proteins abundance and analyze the specific biological process associated with mechanisms of leaf color variation in GL1. A total of 245 and 160 proteins with different abundance were identified in GL1 vs WT and GL1 vs Re-GL1, respectively. Functional classification analysis revealed that the proteins with different abundance mainly related to photosynthesis, heat shock proteins, ribosome proteins, and oxidation-reduction. The proteins that the most significantly contributed to leaf color variation were photosynthetic proteins of PSII and PSI, which directly related to photooxidation and determined the photosynthetic performance of photosystem. Further analysis demonstrated that low jasmonic acid content was needed to golden-yellow leaf GL1. These findings lay a solid foundation for future studies into the molecular mechanisms that underlie leaf color formation of GL1. BIOLOGICAL SIGNIFICANCE: The natural bud mutant GL1 of L. indica is an example through changing leaf color to cope with complex environment. However, the molecular mechanism of leaf color variation are largely elusive. The proteins with different abundance identified from a label-free comparative proteomics revealed a range of biological processes associated with leaf color variation, including photosynthesis, oxidation-reduction and jasmonic acid signaling. The photooxidation and low level of jasmonic acid played a primary role in GL1 adaptation in golden-yellow leaf. These findings provide possible pathway or signal for the molecular mechanism associated with leaf color formation and as a valuable resource for signal transaction of chloroplast.

Electrochemical Voltammogram Recording for Identifying Varieties of Ornamental Plants.

An electrochemical voltammogram recording method for plant variety identification is proposed. Electrochemical voltammograms of Vistula, Andromeda, Danuta, Armandii 'Apple Blossom,' Proteus, Hagley Hybrid, Violet Elizabeth, Kiri Te Kanawa, Regina, and Veronica's Choice were recorded using leaf extracts with two solvents under buffer solutions. The voltametric data recorded under different conditions were derived as scatter plots, 2D density patterns, and hot maps for variety identification. In addition, the voltametric data were further used for genetic relationship studies. The dendrogram deduced from the voltammograms was used as evidence for relationship study. The dendrogram deduced from voltametric data suggested the Andromeda, Danuta, Proteus, Regina, and Hagley Hybrid were closely related, while Violet Elizabeth and Veronica's Choice were closely related. In addition, Vistula and Armandii 'Apple Blossom' could be considered outliers among the varieties.

Preparation of RGO/TiO2/Ag Aerogel and Its Photodegradation Performance in Gas Phase Formaldehyde.

To increase the utilization ratio and catalytic efficiency of the nano TiO2, The RGO/TiO2/(Ag) powders and RGO/TiO2/Ag aerogel photocatalyst were designed and prepared. The composition and microstructure of RGO/TiO2/(Ag) powders and RGO/TiO2/Ag aerogel were studied, in addition, the photocatalytic activity of RGO/TiO2/(Ag) powders and RGO/TiO2/Ag aerogel was researched by the photocatalytic degradation behavior of formaldehyde solution and formaldehyde gas respectively. The result indicate that TiO2 is uniformly loaded on the surface of RGO with a particle size of 10 nm to 20 nm. When the amount of graphene oxide added is 1 wt%, RGO/TiO2 powder has the highest degradation effect on formaldehyde solution, in addition, the introduction of Ag can greatly improve the photocatalytic effect of the sample. The results also show that the pore size of RGO/TiO2/Ag aerogel is between 7.6 nm and 12.1 nm, and the degradation rate of formaldehyde gas is 77.08% within 2 hours.

Identification and Analysis of a Candidate WRKY Transcription Factor Gene Affecting Adventitious Root Formation Using Association Mapping in Catalpa Scop.

The capacity to develop adventitious roots (AR) from cuttings is a key factor for the mass deployment of superior genotypes in the woody plant, including Catalpa Scop. Catalpa Scop. was characterized as having excellent woody qualities in China. However, the knowledge of the molecular mechanisms of AR formation is limited in Catalpa Scop. In this study, for the first time, association mapping for AR formation was performed on a selected sample of 108 Catalpa accessions. Genetic diversity and population structure was estimated on the basis of 54 gene-derived simple sequence repeat markers. Genetic diversity analysis revealed that four accessions belonging to Catalpa duclouxii and eight belonging to Catalpa fargesii formed one clade, providing molecular evidence for C. duclouxii belonging to C. fargesii. Marker-trait association analysis revealed four genes associated with three rooting traits, namely AR rating, adventitious root numbers (ARN), and maximal AR length, with phenotypic variation explained for these traits of 10.77-18.49% in experiments in 2 years. Among the four genes, a WRKY transcription factor gene CbNN1 was the only gene that showed association with the ARN in both years, and expression of this gene (determined by analysis by real-time quantitative polymerase chain reaction) increased with increasing rooting ability. These results indicated that the gene CbNN1 might play a positive role in AR formation. The findings from this study will not only be beneficial to the research of AR formation, but also contribute to the phylogeny of interspecies in Catalpa Scop.

Transcriptome Profiling of Clematis apiifolia: Insights into Heat-Stress Responses.

Clematis apiifolia, belonging to the Clematis L., is a woody vine native to China. It is characterized as heat resistant and fast growing. To better understand potential mechanisms involved in heat-stress responses in Clematis, we characterized the digital gene expression signatures of C. apiifolia under heat-stress conditions. Using RNA sequencing technology, we sequenced six libraries, three biological replicates of control samples and three of heat-stressed samples. In total, 61,708 unigenes were obtained, 36,447 (59.06%) of which were annotated. There were 1941 differentially expressed genes (DEGs) under heat stress, including 867 upregulated and 1074 downregulated genes. Gene ontology enrichment of DEGs revealed that "metabolic process," "cellular process," and "single organism" were the top three functional terms under heat stress. A Kyoto Encyclopedia of Genes and Genomes analysis led to the identification of "protein processing in metabolic pathways," "phenylpropanoid biosynthesis," and "biosynthesis of secondary metabolites" as significantly enriched pathways. Among the upregulated genes, heat-shock factors and heat-shock proteins, especially small heat-shock proteins, were particularly abundant under heat stress. The data will aid in elucidating the molecular events underlying heat-stress responses in Clematis L.

Characterization and Complementation of a Chlorophyll-Less Dominant Mutant GL1 in Lagerstroemia indica.

Crape myrtle (Lagerstroemia indica) is a woody ornamental plant popularly grown because of its long-lasting, midsummer blooms and beautiful colors. The GL1 dominant mutant is the first chlorophyll-less mutant identified in crape myrtle. It was obtained from a natural yellow leaf bud mutation. We previously revealed that leaf color of the GL1 mutant is affected by light intensity. However, the mechanism of the GL1 mutant on light response remained unclear. The acclimation response of mutant and wild-type (WT) plants was assessed in a time series after transferring from low light (LL) to high light (HL) by analyzing chlorophyll synthesis precursor content, photosynthetic performance, and gene expression. In LL conditions, coproporphyrinogen III (Coprogen III) content had the greatest amount of accumulation in the mutant compared with WT, increasing by 100%. This suggested that the yellow leaf phenotype of the GL1 dominant mutant might be caused by disruption of coproporphyrinogen III oxidase (CPO) biosynthesis. Furthermore, the candidate gene, oxygen-independent CPO (HEMN), might only affect expression of upstream genes involved in chlorophyll metabolism in the mutant. Moreover, two genes, photosystem II (PSII) 10 kDa protein (psbR) and chlorophyll a/b binding protein gene (CAB1), had decreased mRNA levels in the GL1 mutant within the first 96 h following LL/HL transfer compared with the WT. Hierarchical clustering revealed that these two genes shared a similar expression trend as the oxygen-dependent CPO (HEMF). These findings provide evidence that GL1 is highly coordinated with PSII stability and chloroplast biogenesis.

Development and characterization of EST-SSR markers for Catalpa bungei (Bignoniaceae).

PREMISE OF THE STUDY: Catalpa bungei (Bignoniaceae) is a deciduous tree native to China. We developed microsatellite markers for C. bungei to investigate its population genetics. METHODS AND RESULTS: One hundred seventy-seven expressed sequence tag (EST)-simple sequence repeat (SSR) primer pairs were isolated and characterized using next-generation sequencing. Thirty of these primer pairs were polymorphic loci in 52 individuals of C. bungei. The number of alleles ranged from two to 18 with observed and expected heterozygosity values of 0.05-1.00 and 0.18-0.95, respectively. The fixation index ranged from -1.00 to 1.00 with an average of 0.32. No linkage disequilibrium was detected in any pair of loci. All markers showed good amplification results in four species (C. bungei, C. fargesii, C. duclouxii, and C. ovata) except three loci. CONCLUSIONS: These polymorphic markers are expected to be helpful in further studies on the systematics and phylogeography of C. bungei and related species.

Global transcriptome analysis and identification of the flowering regulatory genes expressed in leaves of Lagerstroemia indica.

Flowering time is an important trait for ornamental plants, and flowering regulation has thus been both a focus of and challenge to researchers. Lagerstroemia indica is an important summer flowering tree in China and has been introduced abroad as a key parent of new cultivars; no previous reports have addressed the regulation of flowering time in this species. In this study, 28,567,778×2 reads were obtained from leaves of L. indica. A total of 37,325 unigenes were assembled with an average length of 849.56 bp, and 17,506 (46.90%) unigenes were significantly matched to known genes in the nr database of GenBank. The annotated sequences were clustered into putative functional categories using the Gene Ontology framework. Potential genes and their functions were predicted by the Cluster of Orthologous Groups analysis and Kyoto Encyclopedia of Genes and Genomes pathway mapping. A total of 115 unigenes related to flowering time control were discovered. Ten homologous genes of the CONSTANS-like (COL) gene family were identified based on transcript data. Phylogenetic analysis of the CONSTANS and COL genes from L. indica and other species grouped them into three clades. The transcriptome dataset and outcome of the analysis provide a valuable new resource for research on the functional genomics and molecular mechanisms of flowering control in L. indica.