Integrated small RNA, transcriptome and physiological approaches provide insight into Taxodium hybrid 'Zhongshanshan' roots in acclimation to prolonged flooding.

Although Taxodium hybrid 'Zhongshanshan' 406 (Taxodium mucronatum Tenore × Taxodium distichum; Taxodium 406) is an extremely flooding-tolerant woody plant, the physiological and molecular mechanisms underlying acclimation of its roots to long-term flooding remain largely unknown. Thus, we exposed saplings of Taxodium 406 to either non-flooding (control) or flooding for 2 months. Flooding resulted in reduced root biomass, which is in line with lower concentrations of citrate, α-ketoglutaric acid, fumaric acid, malic acid and adenosine triphosphate (ATP) in Taxodium 406 roots. Flooding led to elevated activities of pyruvate decarboxylase, alcohol dehydrogenase and lactate dehydrogenase, which is consistent with higher lactate concentration in the roots of Taxodium 406. Flooding brought about stimulated activities of superoxide dismutase and catalase and elevated reduced glutathione (GSH) concentration and GSH/oxidized glutathione, which is in agreement with reduced concentrations of O2- and H2O2 in Taxodium 406 roots. The levels of starch, soluble protein, indole-3-acetic acid, gibberellin A4 and jasmonate were decreased, whereas the concentrations of glucose, total non-structural carbohydrates, most amino acids and 1-aminocyclopropane-1-carboxylate (ACC) were improved in the roots of flooding-treated Taxodium 406. Underlying these changes in growth and physiological characteristics, 12,420 mRNAs and 42 miRNAs were significantly differentially expressed, and 886 miRNA-mRNA pairs were identified in the roots of flooding-exposed Taxodium 406. For instance, 1-aminocyclopropane-1-carboxylate synthase 8 (ACS8) was a target of Th-miR162-3p and 1-aminocyclopropane-1-carboxylate oxidase 4 (ACO4) was a target of Th-miR166i, and the downregulation of Th-miR162-3p and Th-miR166i results in the upregulation of ACS8 and ACO4, probably bringing about higher ACC content in flooding-treated roots. Overall, these results indicate that differentially expressed mRNA and miRNAs are involved in regulating tricarboxylic acid cycle, ATP production, fermentation, and metabolism of carbohydrates, amino acids and phytohormones, as well as reactive oxygen species detoxification of Taxodium 406 roots. These processes play pivotal roles in acclimation to flooding stress. These results will improve our understanding of the molecular and physiological bases underlying woody plant flooding acclimation and provide valuable insights into breeding-flooding tolerant trees.

Physiological and transcriptional regulation in Taxodium hybrid 'Zhongshanshan' leaves in acclimation to prolonged partial submergence.

MAIN CONCLUSION: Taxodium 703 leaves activate fermentation, amino acids metabolism and ROS detoxification, and reduce TCA cycle and ABA biosynthesis in acclimation to prolonged partial submergence stress. Taxodium hybrid 'Zhongshanshan 703' (T. mucronatum × T. distichum; Taxodium 703) is a highly flooding-tolerant woody plant. To investigate the physiological and transcriptional regulatory mechanisms underlying its leaves in acclimation to long-term flooding, we exposed cuttings of Taxodium 703 to either non-flooding (control) or partial submergence for 2 months. The leaf tissues above (AL) and below (BL) flooding-water were separately harvested. Partial submergence decreased concentrations of chlorophyll (a + b) and dehydroascorbate (DHA) and lactate dehydrogenase (LDH) activity in AL, and reduced biomass, concentrations of succinic acid, fumaric acid and malic acid, and transcript levels of genes involved in tricarboxylic acid (TCA) cycle in BL. Under partial submergence, concentrations of starch, malondialdehyde and abscisic acid (ABA) decreased, and also mRNA levels of nine-cis-epoxycarotenoid dioxygenases that are involved in ABA biosynthesis in AL and BL of Taxodium 703. Partial submergence increased O2- content in AL, and improved concentrations of pyruvate and soluble sugars and activities of LDH and peroxidase in BL. In addition, partial submergence increased concentrations of ethanol, lactate, alanine, γ-aminobutyric acid, total amino acids and ascorbic acid (ASA), and ASA/DHA, activities of alcohol dehydrogenases (ADH) and ascorbate peroxidase, as well as transcript levels of ADH1A, ADH1B and genes involved in alanine biosynthesis and starch degradation in AL and BL of Taxodium 703. Overall, these results suggest that Taxodium 703 leaves activate fermentation, amino acids metabolism and reactive oxygen species detoxification, and maintain a steady supply of sugars, and reduce TCA cycle and ABA biosynthesis in acclimation to prolonged partial submergence stress.

Different antioxidant regulation mechanisms in response to aluminum-induced oxidative stress in Eucalyptus species.

Forest ecosystems play an important role in environmental protection and maintaining ecological balance. Understanding the physiological mechanisms of tree species response to aluminum (Al) toxic is crucial to reveal the main causes of plantation decline in acid rain area. As an important afforestation tree species in tropical and subtropical areas, Eucalyptus has high economic value and plays crucial ecological roles. However, continuous fertilization and acid precipitation can exacerbate soil acidification and increase soil active Al, which has a significant negative impact on Eucalyptus growth. Hence, species and genotypes with high Al resistance are required to solve the problem of Al toxicity of acidic soils for sustainable forest production. In this study, E. urophylla was better adapted to Al stress than E. grandis or E. tereticornis; its high Al resistance was attributed to greater antioxidant enzyme activity and non-enzymatic antioxidant content, and a lower degree of membrane lipid peroxidation than E. grandis or E. tereticornis. The differences in adaptability among the three pure species were attributed to their distinct habitats. Eucalyptus urophylla × E. grandis inherited the outstanding adaptability to Al stress from its maternal species (E. urophylla), indicating that Al tolerance is highly heritable and can be selected in Eucalyptus breeding. Our results indicated that the response of Eucalyptus to Al stress may fluctuate according to the time under stress, and might be related to dynamic changes in ROS elimination and accumulation.

Rejuvenation increases leaf biomass and flavonoid accumulation in Ginkgo biloba.

Rejuvenation refers to the transition from an adult state to a juvenile state. Trunk truncation at the base of the tree can result in tree rejuvenation. However, little is known about the association of rejuvenation with leaf biomass and flavonoid accumulation. The results of this study showed that, compared with control leaves, leaves of renewed Ginkgo biloba shoots were larger, thicker, and more lobed and had higher fresh/dry weights and chlorophyll contents. The leaf biomass per hectare of rejuvenated trees was twofold higher than that of the untruncated controls. Moreover, we observed a marked increase in the accumulation of flavonol glycosides via metabolomic analysis and detected upregulated expression of genes involved in flavonoid biosynthesis, including CHS, FLS, F3'H, DFR, and LAR. Overexpression of GbCHS in ginkgo calli confirmed that GbCHS plays an important role in flavonoid biosynthesis. Interestingly, the contents of gibberellins significantly increased in the rejuvenated leaves. Moreover, exogenous gibberellin treatment significantly increased GbCHS expression and flavonoid contents. Our findings show that truncation can stimulate tree rejuvenation by altering hormone levels, representing an effective and feasible approach for enhancing the biomass and flavonoid content of G. biloba leaves.

Hair-growth promoting effect and anti-inflammatory mechanism of Ginkgo biloba polysaccharides.

The aim of this study was to optimize the separation and purification technology of water-soluble Ginkgo biloba leaves polysaccharides (WGBP), analyze its composition characteristics, observe its hair-growth promoting effect in alopecia areata mice, clarify the polysaccharide fraction with bioactive activities, and explore its anti-inflammation mechanism. We isolated acidic polysaccharides (WGBP-A2) and purified a RG-I type polysaccharide (WGBP-A2b) with a molecular weight of 44 kDa. Results showed that WGBP-A2 could significantly increase the contents of VEGF and HGF in the skin tissue of alopecia areata mice, decrease the contents of Inflammatory factors in the serum. On a cellular level, the expressions of p-p65 and p-IκBα, TNF-α and IL-1ß in HUVECs treated with WGBP-A2b were down-regulated. The bioinformatic analysis showed that the inflammation signaling pathway was significantly changed. Its specific mechanism may be related to its regulating the expression of p-p65 p-IκBα, TNF-α and IL-1ß proteins in the inflammation signaling pathway.

Development and Implementation of Couple-Based Collaborative Management Model of Type 2 Diabetes Mellitus for Community-Dwelling Chinese Older Adults: A Pilot Randomized Trial.

Background: To mobilize family's positive involvement in improving and sustaining self-management activities of older adults with diabetes, we developed a couple-based collaborative management model (CCMM) for community-dwelling older Chinese. Methods: The model was developed stepwise through applying theoretical models, interviewing older couples and community healthcare workers, as well as incorporating expert reviews. A 3-month pilot study was conducted to test the model's feasibility and its treatment effects by linear regression on 18 pairs of older couples aged 60 years+, who were equally divided into a couple-based intervention arm and a patient-only control arm. Results: The developed CCMM covered four theory-driven intervention modules: dyadic assessment, dyadic education, dyadic behavior-change training, and dyadic monitoring. Each module was delivered by community healthcare workers and targeted at older couples as the management units. Based on interviews with older couples and healthcare workers, 4 weekly education and training group sessions and 2-month weekly behavior change booster calls were designed to address older adults' main management barriers. These modules and session contents were evaluated as essential and relevant by the expert panel. Furthermore, the CCMM showed good feasibility and acceptability in the pilot, with non-significant yet more positive changes in physiological outcomes of diabetic participants and couples' well-being and exercise levels of these in the intervention arm than their controlled counterparts. Conclusion: We systematically developed a couple-based collaborative management model of diabetes, which was well-received by healthcare practitioners and highly feasible among older Chinese couples living in the community. The model's treatment effects need to be verified in fully powered randomized controlled trials. Clinical Trial Registration: http://www.chictr.org.cn/showproj.aspx?proj=42964, identifier: ChiCTR1900027137.

Identification and characterization of circular RNAs during wood formation of poplars in acclimation to low nitrogen availability.

MAIN CONCLUSION: Circular RNA (circRNA) identification and expression profiles, and construction of circRNAs-miRNAs-mRNAs networks indicates that circRNAs are involved in wood formation of poplars in acclimation to low nitrogen availability. Circular RNAs (circRNAs) are covalently closed non-coding RNAs that play pivotal roles in various biological processes. However, circRNAs' roles in wood formation of poplars in acclimation to low nitrogen (N) availability are currently unknown. Here, we undertook a systematic identification and characterization of circRNAs in the wood of Populus × canescens exposed to either 50 (low N) or 500 (normal N) µM NH4NO3 using rRNA-depleted RNA-sequencing. A total of 2,509 unique circRNAs were identified, and 163 (ca. 6.5%) circRNAs were significantly differentially expressed (DE) under low N condition. We observed a positive correlation between the expression patterns of DE circRNAs and their hosting protein-coding genes. Moreover, circRNAs-miRNAs-mRNAs' networks were identified in the wood of poplars under low N availability. For instance, upregulated several circRNAs, such as circRNA1226, circRNA 1732, and circRNA392 induced increases in nuclear factor Y, subunit A1-A (NFYA1-A), NFYA1-B, and NFYA10 transcript levels via the mediation of miR169b members, which is in line with reduced xylem width and cell layers of the xylem in the wood of low N-supplied poplars. Upregulation of circRNA1006, circRNA1344, circRNA1941, circRNA901, and circRNA146 caused increased transcript level of MYB61 via the mediation of a miR5021 member, corresponding well to the higher lignin concentration in the wood of low N-treated poplars. Overall, these results indicated that DE circRNAs play an essential role in regulating gene expression via circRNAs-miRNAs-mRNAs' networks to modulate wood anatomical and chemical properties of poplars in acclimation to low N availability.

The genetic diversity and population structure of Sophora alopecuroides (Faboideae) as determined by microsatellite markers developed from transcriptome.

Sophora alopecuroides (Faboideae) is an endemic species, mainly distributed in northwest China. However, the limited molecular markers range for this species hinders breeding and genetic studies. A total of 20,324 simple sequence repeat (SSR) markers were identified from 118,197 assembled transcripts and 18 highly polymorphic SSR markers were used to explore the genetic diversity and population structure of S. alopecuroides from 23 different geographical populations. A relatively low genetic diversity was found in S. alopecuroides based on mean values of the number of effective alleles (Ne = 1.81), expected heterozygosity (He = 0.39) and observed heterozygosity (Ho = 0.55). The results of AMOVA indicated higher levels of variation within populations than between populations. Bayesian-based cluster analysis, principal coordinates analysis and Neighbor-Joining phylogeny analysis roughly divided all genotypes into four major groups with some admixtures. Meanwhile, geographic barriers would have restricted gene flow between the northern and southern regions (separated by Tianshan Mountains), wherein the two relatively ancestral and independent clusters of S. alopecuroides occur. History trade and migration along the Silk Road would together have promoted the spread of S. alopecuroides from the western to the eastern regions of the northwest plateau in China, resulting in the current genetic diversity and population structure. The transcriptomic SSR markers provide a valuable resource for understanding the genetic diversity and population structure of S. alopecuroides, and will assist effective conservation management.

Identification and expression analysis under abiotic stress of the R2R3-MYB genes in Ginkgo biloba L.

The R2R3-MYB gene family is the largest MYB subfamily in plants and is involved in the regulation of plant secondary metabolism and specific morphogenesis, as well as the response to biotic and abiotic stress. However, a systematic identification and characterization of this gene family has not been carried out in Ginkgo biloba. In this study, we performed a transcriptome-wide survey from four tissues of G. biloba to determine the genetic variation and expression pattern of the R2R3-MYB genes. We analyzed 45 GbMYBs and identified 42 with a complete coding sequence via conserved motif searches. The MYB domain and other motifs in GbMYBs are highly conserved with Arabidopsis thaliana AtMYBs. Phylogenetic analysis of the GbMYBs and AtMYBs categorized the R2R3-MYBs into 26 subgroups, of which 11 subgroups included proteins from both G. biloba and Arabidopsis, and 1 subgroup was specific to G. biloba. Moreover, the GbMYBs expression patterns were analyzed in different tissues and abiotic stress conditions. The results revealed that GbMYBs were differentially expressed in various tissues and following abiotic stresses and phytohormone treatments, indicating their possible roles in biological processes and abiotic stress tolerance and adaptation. Our study demonstrated the functional diversity of the GbMYBs and will provide a foundation for future research into their biological and molecular functions.

Comparative Proteomic and Physiological Analysis Reveals the Variation Mechanisms of Leaf Coloration and Carbon Fixation in a Xantha Mutant of Ginkgo biloba L.

Yellow-green leaf mutants are common in higher plants, and these non-lethal chlorophyll-deficient mutants are ideal materials for research on photosynthesis and plant development. A novel xantha mutant of Ginkgo biloba displaying yellow-colour leaves (YL) and green-colour leaves (GL) was identified in this study. The chlorophyll content of YL was remarkably lower than that in GL. The chloroplast ultrastructure revealed that YL had less dense thylakoid lamellae, a looser structure and fewer starch grains than GL. Analysis of the photosynthetic characteristics revealed that YL had decreased photosynthetic activity with significantly high nonphotochemical quenching. To explain these phenomena, we analysed the proteomic differences in leaves and chloroplasts between YL and GL of ginkgo using two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF/TOF MS. In total, 89 differential proteins were successfully identified, 82 of which were assigned functions in nine metabolic pathways and cellular processes. Among them, proteins involved in photosynthesis, carbon fixation in photosynthetic organisms, carbohydrate/energy metabolism, amino acid metabolism, and protein metabolism were greatly enriched, indicating a good correlation between differentially accumulated proteins and physiological changes in leaves. The identifications of these differentially accumulated proteins indicates the presence of a specific different metabolic network in YL and suggests that YL possess slower chloroplast development, weaker photosynthesis, and a less abundant energy supply than GL. These studies provide insights into the mechanism of molecular regulation of leaf colour variation in YL mutants.

Expression of selected Ginkgo biloba heat shock protein genes after cold treatment could be induced by other abiotic stress.

Heat shock proteins (HSPs) play various stress-protective roles in plants. In this study, three HSP genes were isolated from a suppression subtractive hybridization (SSH) cDNA library of Ginkgo biloba leaves treated with cold stress. Based on the molecular weight, the three genes were designated GbHSP16.8, GbHSP17 and GbHSP70. The full length of the three genes were predicted to encode three polypeptide chains containing 149 amino acids (Aa), 152 Aa, and 657 Aa, and their corresponding molecular weights were predicted as follows: 16.67 kDa, 17.39 kDa, and 71.81 kDa respectively. The three genes exhibited distinctive expression patterns in different organs or development stages. GbHSP16.8 and GbHSP70 showed high expression levels in leaves and a low level in gynoecia, GbHSP17 showed a higher transcription in stamens and lower level in fruit. This result indicates that GbHSP16.8 and GbHSP70 may play important roles in Ginkgo leaf development and photosynthesis, and GbHSP17 may play a positive role in pollen maturation. All three GbHSPs were up-regulated under cold stress, whereas extreme heat stress only caused up-regulation of GbHSP70, UV-B treatment resulted in up-regulation of GbHSP16.8 and GbHSP17, wounding treatment resulted in up-regulation of GbHSP16.8 and GbHSP70, and abscisic acid (ABA) treatment caused up-regulation of GbHSP70 primarily.