A chromosome-scale genome of Rhus chinensis Mill. provides new insights into plant-insect interaction and gallotannins biosynthesis.

Rhus chinensis Mill., an economically valuable Anacardiaceae species, is parasitized by the galling aphid Schlechtendalia chinensis, resulting in the formation of the Chinese gallnut (CG). Here, we report a chromosomal-level genome assembly of R. chinensis, with a total size of 389.40 Mb and scaffold N50 of 23.02 Mb. Comparative genomic and transcriptome analysis revealed that the enhanced structure of CG and nutritional metabolism contribute to improving the adaptability of R. chinensis to S. chinensis by supporting CG and galling aphid growth. CG was observed to be abundant in hydrolysable tannins (HT), particularly gallotannin and its isomers. Tandem repeat clusters of dehydroquinate dehydratase/shikimate dehydrogenase (DQD/SDH) and serine carboxypeptidase-like (SCPL) and their homologs involved in HT production were determined as specific to HT-rich species. The functional differentiation of DQD/SDH tandem duplicate genes and the significant contraction in the phenylalanine ammonia-lyase (PAL) gene family contributed to the accumulation of gallic acid and HT while minimizing the production of shikimic acid, flavonoids, and condensed tannins in CG. Furthermore, we identified one UDP glucosyltransferase (UGT84A), three carboxylesterase (CXE), and six SCPL genes from conserved tandem repeat clusters that are involved in gallotannin biosynthesis and hydrolysis in CG. We then constructed a regulatory network of these genes based on co-expression and transcription factor motif analysis. Our findings provide a genomic resource for the exploration of the underlying mechanisms of plant-galling insect interaction and highlight the importance of the functional divergence of tandem duplicate genes in the accumulation of secondary metabolites.

Growth and Physiological Responses of Magnoliaceae to NaCl Stress.

The growth and physiological characteristics of four Magnoliaceae plants (Yulania biondii, Yulania denudata, and two varieties of Magnolia wufengensis (Jiaohong 1 and Jiaohong 2)) were investigated. Four Magnoliaceae plants were subjected to various concentrations of NaCl for 60 days: 0 mM, 60 mM, 120 mM, 180 mM, and 240 mM. The leaf water content (LWC), relative growth rate of plant height and stem diameter, photosynthetic pigments, and photosynthetic rate (Pn) decreased during the NaCl treatments, indicating slowed growth and photosynthesis. Malondialdehyde (MDA), Na+, superoxide dismutase (SOD) activity, peroxidase (POD) activity, ascorbic acid (AsA) content, and soluble sugar content all increased while K+ decreased. Ascorbate peroxidase (APX) activity, glutathione (GSH), soluble protein, and proline first increased after decreasing with increasing NaCl concentration. Principal component 1 (PC1) had larger loading values for growth and photosynthesis indices, while principal component 2 (PC2) exhibited larger loading values for antioxidant substances and osmotic adjustment substances; the correlation analysis showed that PC1 and PC2 had negative correlations. The four Magnoliaceae plants exhibited largely variable growth and physiological activities in response to NaCl. Yulania denudata exhibited greater reductions in growth and photosynthesis and greater decreases in antioxidant enzyme activities and osmotic adjustment substances, which indicated poor tolerance to salt stress. Among the four Magnoliaceae plants, Jiaohong 1 exhibited the greatest salt tolerance, followed by Jiaohong 2, Yulania biondii, and Yulania denudata.

Fine-Scale analysis of both wild and cultivated horned galls provides insight into their quality differentiation.

BACKGROUND: Galla chinensis is a traditional Chinese medicine (TCM) produced due to the interaction between the Fordinae aphids and the Rhus plant species. Horned galls with high tannin content are the most widely cultivated gall type, and Wufeng county of Hubei province in China is the center of cultivation. However, long-term artificial cultivation and domestication of horned galls to meet the increasing production demand have led to quality degradation. Understanding the reasons underlying quality degradation is urgent for horned gall production and application. The present study used a combination of metabolic, genetic, and ecological analyses to investigate the quality and genetic differentiation of the horned galls under long-term domestication as well as the potential relationships between them. RESULTS: Analysis of gallic acid content and other three phenotypic traits (fresh weight, gall size, and wall thickness) revealed quality differentiation of horned galls collected from five locations in Wufeng, in which the cultivated samples from Wang Jiaping (WJP) showed the highest degradation. Genetic differentiation between the cultivated and wild Rhus chinensis trees in WJP, and between WJP and the other populations was detected based on SSR molecular markers, however, no significant difference in genetic structure was seen for the aphid populations. Among the various ecological factors examined, temperature was identified as the primary one affecting the quality of horned galls. CONCLUSIONS: Both genetic and ecological factors caused quality differentiation of horned galls. The collection of diverse germplasm of host trees and aphids will help reduce the quality degradation of horned galls in Wufeng.

First Report of Leaf Spot Caused by Colletotrichum fructicola on Magnolia wufengensis in Hubei, China.

Magnolia wufengensis belongs to the Magnoliaceae family. Its variation-rich flowers (tepal number from 9 to 46, tepal color from pink to bright red) and excellent wood characteristics (strong, straight, texture) have important ornamental and economic value (Duan et al. 2019; Luyi et al. 2006). M. wufengensis is popularly cultivated in parks, courtyards, mountains, and along roadsides. In May 2020, leaf spot symptoms were observed on over 85% of M. wufengensis in Yuyangguan Township, Wufeng County, Hubei Province (110.60°E, 30.21°N). The damaged area was over 18.7 hectares. Early symptoms began as small brown spots with a light-yellow halo. Gradual lesions expanded, and the center was withered, gray, and necrotic with a dark brown border. Eventually, several spots combined with larger irregular lesions, turning the leaves yellow and causing them to fall off. The border of lesions and healthy tissues were cut into small pieces (5×5 mm), and surface sterilized with 1% sodium hypochlorite solution for three minutes, rinsed three times with sterile water, and plated on potato dextrose agar (PDA) medium at 25±2 °C with a 12h photoperiod under fluorescent lighting. Pure isolates (MCS1228.1, MCS1228.4, MCS1228.9) were gray to pale grayish, and their average growth rate was 10.5±1.23 mm/day. Conidiophores were hyaline, aseptate, branched. Conidia were hyaline, aseptate, cylindrical, and 14.00 to 25.17 × 4.74 to 6.56 µm in size (average 17.48 × 5.58 µm) (n=50). Appressoria were brown and showed multivariate shape. The morphological characteristics of the isolates corresponded to the description given for Colletotrichum fructicola (Liu et al. 2015). Molecular identification was accomplished through amplification of the internal transcribed spacer (IST), actin (ACT), calmodulin (CAL), chitin synthase (CHS-1) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin (TUB2) genes (Fu et al. 2018). The ITS (OL800580.1, OL800581.1, OL800582.1), ACT (GenBank accession No. OL873155- OL873157), CAL (GenBank accession No. OL873158- OL873160), CHS-1 (GenBank accession No. OL873161- OL873163), GAPDH (GenBank accession No. OL873164- OL873166) and TUB2 (GenBank accession No. OL873167- OL873169) sequences were deposited in GenBank. A Bayesian inference phylogenetic tree based on multilocus sequences was constructed, and the sequences of the 3 isolations showed the same homology with C. fructicola (Fu et al. 2018). To fulfill Koch's postulates, 30 potted seedlings were inoculated with 1×10^6 conidia/ml suspension of each isolate by spraying the leaves, and 30 potted seedlings were sprayed with sterile distilled water as control. Inoculated and control plants were kept in a greenhouse with 25/15°C (day/night) temperature and 80% relative humidity. In addition, 30 healthy detached leaves free of pests and diseases were washed three times with sterile distilled water, air-dried, and artificially inoculated using a 6 mm (diameter) PDA medium (5 days incubation) with mycelium. 30 leaves were inoculated with sterile PDA medium as control. All leaves were sprayed with sterile water every 24 hours, covered with plastic wrap, and incubated at 25±2 °C, 100% humidity. The experiment was repeated three times. Similar symptoms to those found initially were both observed on all the inoculated potted seedlings and detached leaves after 14 days and 5 days post inoculation (dpi), respectively. Whereas the controls remained symptomless. The reisolated pathogens from symptomatic tissues were identical to the original isolates. In this study, isolated fungi associated with M. wufengensis leaf spot were identified as C. fructicola based on morphological and multiloci phylogenetic analyses, and Koch's postulates. Colletotrichum species are important plant pathogens and cause diseases in a wide variety of woody and herbaceous plants (Cannon et al. 2012). C. fructicola has been identified as a responsible pathogen for apple (Casanova et al. 2016), Fatsia japonica (Shi et al. 2017), and Rubus corchorifolius (Wu et al. 2021) leaf spot. To our knowledge, this is the first report of C. fructicola causing leaf spot in M. wufengensis in China. This research may contribute to the development of management strategies for this disease.

Transcriptomic Analysis Reveals the Positive Role of Abscisic Acid in Endodormancy Maintenance of Leaf Buds of Magnolia wufengensis.

Magnolia wufengensis (Magnoliaceae) is a deciduous landscape species, known for its ornamental value with uniquely shaped and coloured tepals. The species has been introduced to many cities in south China, but low temperatures limit the expansion of this species in cold regions. Bud dormancy is critical for plants to survive in cold environments during the winter. In this study, we performed transcriptomic analysis of leaf buds using RNA sequencing and compared their gene expression during endodormancy, endodormancy release, and ecodormancy. A total of 187,406 unigenes were generated with an average length of 621.82 bp (N50 = 895 bp). In the transcriptomic analysis, differentially expressed genes involved in metabolism and signal transduction of hormones especially abscisic acid (ABA) were substantially annotated during dormancy transition. Our results showed that ABA at a concentration of 100 µM promoted dormancy maintenance in buds of M. wufengensis. Furthermore, the expression of genes related to ABA biosynthesis, catabolism, and signalling pathway was analysed by qPCR. We found that the expression of MwCYP707A-1-2 was consistent with ABA content and the dormancy transition phase, indicating that MwCYP707A-1-2 played a role in endodormancy release. In addition, the upregulation of MwCBF1 during dormancy release highlighted the enhancement of cold resistance. This study provides new insights into the cold tolerance of M. wufengensis in the winter from bud dormancy based on RNA-sequencing and offers fundamental data for further research on breeding improvement of M. wufengensis.

Physiological and transcriptome analysis of Magnolia denudata leaf buds during long-term cold acclimation.

BACKGROUND: Magonlia denudata is an important perennial tree species of the Magnoliaceae family, known for its ornamental value, resistance to smoke pollution and wind, role in air purification, and robust cold tolerance. In this study, a high-throughput transcriptome analysis of leaf buds was performed, and gene expression following artificial acclimation 22 °C, 4 °C and 0 °C, was compared by RNA sequencing. RESULTS: Over 426 million clean reads were produced from three libraries (22 °C, 4 °C and 0 °C). A total of 74,503 non-redundant unigenes were generated, with an average length of 1173.7 bp (N50 = 1548). Based on transcriptional results, 357 and 235 unigenes were identified as being upregulated and downregulated under cold stress conditions, respectively. Differentially expressed genes were annotated using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway analyses. The transcriptomic analysis focused on carbon metabolism and plant hormone signal transduction associated with cold acclimation. Transcription factors such as those in the basic helix-loop-helix and AP2/ERF families were found to play an important role in M. denudata cold acclimation. CONCLUSION: M. denudata exhibits responses to non-freezing cold temperature (4 °C) to increase its cold tolerance. Cold resistance was further strengthened with cold acclimation under freezing conditions (0 °C). Cold tolerance genes, and cold signaling transcriptional pathways, and potential functional key components for the regulation of the cold response were identified in M. denudata. These results provide a basis for further studies, and the verification of key genes involved in cold acclimation responses in M. denudata lays a foundation for developing breeding programs for Magnoliaceae species.

Curcumin Antagonizes Glucose Fluctuation-Induced Renal Injury by Inhibiting Aerobic Glycolysis via the miR-489/LDHA Pathway.

It has been considered that glucose fluctuation (GF) plays a role in renal injury and is related to diabetic nephropathy (DN) development. But the mechanism is still unclear. Aerobic glycolysis has become a topical issue in DN in recent years. There is an internal connection between GF, aerobic glycolysis, and DN. Curcumin (Cur) is a principal curcuminoid of turmeric and possesses specific protective properties in kidney functions. Cur also participates in the regulation of aerobic glycolysis switch. In this study, we first measured the levels of aerobic glycolysis and evaluated Cur's inhibitory ability in a cell model of HEK-293 under the condition of oscillating high glucose. The results indicated that GF exacerbated inflammation injury, oxidative stress, and apoptosis in HEK-293 cell, while Cur alleviated this cytotoxicity induced by GF. We found that GF increased aerobic glycolysis in HEK-293 cells and Cur presented a dose-dependent weakening effect to this exacerbation. Next, we built a panel of 17 miRNAs and 8 lncRNAs that were previously reported to mediate the Warburg effect. Our RT-qPCR results indicated that GF reduced the miR-489 content in the HEK-293 cell model and Cur could prevent this downregulation. Then, we planned to explore the character of miR-489 in Cur-triggered attenuation of the Warburg effect under GF condition. Our findings presented that Cur prevented GF-triggered aerobic glycolysis by upregulating miR-489 in HEK-293 cells. Next, we choose the miR-489/LDHA axis for further investigation. We confirmed that Cur prevented GF-triggered aerobic glycolysis via the miR-489/LDHA axis in HEK-293 cells. In conclusion, this study presented that Cur prevented GF-triggered renal injury by restraining aerobic glycolysis via the miR-489/LDHA axis in the HEK-293 cell model.

Conservation and divergence of ancestral AGAMOUS/SEEDSTICK subfamily genes from the basal angiosperm Magnolia wufengensis.

AGAMOUS/SEEDSTICK (AG/STK) subfamily genes play crucial roles in the reproductive development of plants. However, most of our current knowledge of AG/STK subfamily genes is restricted to core eudicots and grasses, and the knowledge of ancestral exon-intron structures, expression patterns, protein-protein interaction patterns and functions of AG/STK subfamily genes remains unclear. To determine these, we isolated AG/STK subfamily genes (MawuAG1, MawuAG2 and MawuSTK) from a woody basal angiosperm Magnolia wufengensis (Magnoliaceae). MawuSTK arose from the gene duplication event occurring before the diversification of extant angiosperms, and MawuAG1 and MawuAG2 may result from a gene duplication event occurring before the divergence of Magnoliaceae and Lauraceae. Gene duplication led to apparent diversification in their expression and interaction patterns. It revealed that expression in both stamens and carpels likely represents the ancestral expression profiles of AG lineage genes, and expression of STK-like genes in stamens may have been lost soon after the appearance of the STK lineage. Moreover, AG/STK subfamily proteins may have immediately established interactions with the SEPALLATA (SEP) subfamily proteins following the emergence of the SEP subfamily; however, their interactions with the APETALA1/FRUITFULL subfamily proteins or themselves differ from those found in monocots and basal and core eudicots. MawuAG1 plays highly conserved roles in the determinacy of stamen, carpel and ovule identity, while gene duplication contributed to the functional diversification of MawuAG2 and MawuSTK. In addition, we investigated the evolutionary history of exon-intron structural changes of the AG/STK subfamily, and a novel splice-acceptor mode (GUU-AU) and the convergent evolution of N-terminal extension in the euAG and PLE subclades were revealed for the first time. These results further advance our understanding of ancestral AG/STK subfamily genes in terms of phylogeny, exon-intron structures, expression and interaction patterns, and functions, and provide strong evidence for the significance of gene duplication in the expansion and evolution of the AG/STK subfamily.

De novo transcriptome sequencing and gene expression profiling of Magnolia wufengensis in response to cold stress.

BACKGROUND: Magnolia wufengensis is a new species of Magnolia L. and has considerable ornamental and economic value due to its unique characteristics. However, because of its characteristic of poor low temperature resistance, M. wufengensis is hardly popularization and application in the north of China. Furthermore, the mechanisms of gene regulation and signaling pathways involved in the cold-stress response remained unclear in this species. In order to solve the above-mentioned problems, we performed de novo transcriptome assembly and compared the gene expression under the natural (25 °C) and cold (4 °C) conditions for M. wufengensis seedlings. RESULTS: More than 46 million high-quality clean reads were produced from six samples (RNA was extracted from the leaves) and were used for performing de novo transcriptome assembly. A total of 59,764 non-redundant unigenes with an average length of 899 bp (N50 = 1,110) were generated. Among these unigenes, 31,038 unigenes exhibited significant sequence similarity to known genes, as determined by BLASTx searches (E-value ≤1.0E-05) against the Nr, SwissProt, String, GO, KEGG, and Cluster of COG databases. Based on a comparative transcriptome analysis, 3,910 unigenes were significantly differentially expressed (false discovery rate [FDR] < 0.05 and |log2FC (CT/CK)| ≥ 1) in the cold-treated samples, and 2,616 and 1,294 unigenes were up- and down-regulated by cold stress, respectively. Analysis of the expression patterns of 16 differentially expressed genes (DEGs) by quantitative real-time RT-PCR (qRT-PCR) confirmed the accuracy of the RNA-Seq results. Gene Ontology and KEGG pathway functional enrichment analyses allowed us to better understand these differentially expressed unigenes. The most significant transcriptomic changes observed under cold stress were related to plant hormone and signal transduction pathways, primary and secondary metabolism, and photosynthesis. In addition, 113 transcription factors, including members of the AP2-EREBP, bHLH, WRKY, MYB, NAC, HSF, and bZIP families, were identified as cold responsive. CONCLUSION: We generated a genome-wide transcript profile of M. wufengensis and a de novo-assembled transcriptome that can be used to analyze genes involved in biological processes. In this study, we provide the first report of transcriptome sequencing of cold-stressed M. wufengensis. Our findings provide important clues not only for understanding the molecular mechanisms of cold stress in plants but also for introducing cold hardiness into M. wufengensis.

Gene duplication led to divergence of expression patterns, protein-protein interaction patterns and floral development functions of AGL6-like genes in the basal angiosperm Magnolia wufengensis (Magnoliaceae).

The MADS-box family genes play critical roles in the regulation of growth and development of flowering plants. AGAMOUS-LIKE 6 (AGL6)-like genes are one of the most enigmatic subfamilies of the MADS-box family because of highly variable expression patterns and ambiguous functions, which have long puzzled researchers. A lot of AGL6 homologs have been identified from gymnosperms and angiosperms. However, only a few have been characterized, especially for basal angiosperm taxa. Magnolia wufengensis is a woody basal angiosperm from the family Magnoliaceae. In the current study, the phylogenesis, expression and protein-protein interaction (PPI) patterns, and functions of two AGL6 homologs from M. wufengensis, MawuAGL6-1 and MawuAGL6-2, were analyzed. Phylogenetic analysis indicated that the two AGL6 duplicates may have arisen by gene duplication before the divergence of Magnoliaceae and Lauraceae, with the diversification of their expression and PPI patterns after gene duplication. Functional analysis revealed that, in addition to common functions in accelerating flowering, MawuAGL6-1 might be responsible for flower meristem determinacy, while MawuAGL6-2 is preferentially recruited to regulate tepal morphogenesis. These findings further advance our understanding of the evolution of phylogenesis, expression, interaction and functions of AGL6 lineage genes from basal angiosperms, as well as the entire AGL6 lineage genes, and the significance of AGL6 lineage genes in the evolution and biological diversity.