SUMMARY
Anthocyanin is one of the most important pigments and nutrients in fruits. Genes encoding R2R3‐MYB transcription factors (TFs) are key to anthocyanin regulation. R2R3‐MYB activators have been ...widely studied in the anthocyanin synthesis pathway. However, the mechanism by which R2R3‐MYB repressors negatively regulate anthocyanin synthesis remains poorly understood. In the present study, we characterized a subfamily 1 R2R3‐MYB anthocyanin repressor gene, MYB306‐like, from apple (Malus × domestica) and demonstrated its significance in anthocyanin regulation. The MdMYB306‐like protein activates the expression of an anthocyanin repressor gene, MdMYB17, and inhibits the expression of MdDFR (an anthocyanin structural gene) by directly binding to the corresponding promoters. The MdMYB306‐like protein interacts with MdMYB17 and MdbHLH33 through its N‐terminus. In addition, MdMYB306‐like interacts with MdbHLH33 and MdMYB17 to enhance its regulatory activities on MdMYB17 and MdDFR, respectively. Our results revealed that the MdMYB306‐like protein, in conjunction with MdMYB17 and MdbHLH33, forms a key regulatory module to fine‐tune anthocyanin synthesis in apple.
Significance Statement
Subfamily 1 R2R3‐MYB TFs might be a new type of anthocyanin repressor, but there is still some controversy over this possibility. Here, we characterized an anthocyanin repressor gene, MdMYB306, from apple that belongs to the R2R3‐MYB subfamily 1, and revealed the repressive mechanisms for MdMYB306 limiting anthocyanin synthesis at the transcriptional level.
Fruit peel colour is an important agronomic trait for fruit quality. Cytosine methylation plays an important role in gene regulation. Although the DNA methylation level of a single gene is important ...to affect the phenotype of mutation, there are large unknown of difference of the DNA methylation in plant and its mutants.
Using bisulfite sequencing (BS-Seq) and RNA-sequencing (RNA-Seq), we analysed three deep-red-skinned apple (Malus × domestica) mutants (Yanfu 3, YF3; Yanfu 8, YF8; Shannonghong, SNH) and their lighter-skinned parents (Nagafu 2, NF2; Yanfu 3, YF3; Ralls, RL) to explore the different changes in methylation patterns associated with anthocyanin concentrations. We identified 13,405, 13,384, and 10,925 differentially methylated regions (DMRs) and 1987, 956, and 1180 differentially expressed genes (DEGs) in the NF2/YF3, YF3/YF8, and RL/SNH comparisons, respectively. And we found two DMR-associated DEGs involved in the anthocyanin pathway: ANS (MD06G1071600) and F3H (MD05G1074200). These genes exhibited upregulated expression in apple mutants, and differences were observed in the methylation patterns of their promoters. These results suggested that both the regulatory and structural genes may be modified by DNA methylation in the anthocyanin pathway. However, the methylation of structural genes was not the primary reason for expression-level changes. The expression of structural genes may be synergistically regulated by transcription factors and methylation changes. Additionally, the expression of the transcription factor gene MYB114 (MD17G1261100) was upregulated in the deep-red-skinned apple.
Through the analysis of global methylation and transcription, we did not find the correlation between gene expression and the DNA methylation. However, we observed that the upregulated expression of ANS (MD06G1071600) and F3H (MD05G1074200) in apple mutants results in increased anthocyanin contents. Moreover, MYB114 (MD17G1261100) is likely another regulatory gene involved in apple coloration. Our data provided a new understanding about the differences in formation of apple colour mutants.
Auxin signaling, which is crucial for normal plant growth and development, mainly depends on ARF-Aux/IAA interactions. However, little is known regarding the regulatory effects of auxin signaling on ...anthocyanin metabolism in apple (
). We investigated the functions of MdARF13, which contains a repression domain and is localized to the nucleus. This protein was observed to interact with the Aux/IAA repressor, MdIAA121, through its C-terminal dimerization domain. Protein degradation experiments proved that MdIAA121 is an unstable protein that is degraded by the 26S proteasome. Additionally, MdIAA121 stability is affected by the application of exogenous auxin. Furthermore, the overexpression of
and
in transgenic red-fleshed apple calli weakened the inhibitory effect of MdARF13 on anthocyanin biosynthesis. These results indicate that the degradation of MdIAA121 induced by auxin treatment can release MdARF13, which acts as a negative regulator of the anthocyanin metabolic pathway. Additionally, yeast two-hybrid, bimolecular fluorescence complementation, and pull-down assays confirmed that MdMYB10 interacts with MdARF13. A subsequent electrophoretic mobility shift assay and yeast one-hybrid assay demonstrated that MdARF13 directly binds to the promoter of
, which is an anthocyanin pathway structural gene. Interestingly, chromatin immunoprecipitation-quantitative real-time PCR results indicated that the overexpression of
clearly inhibits the recruitment of MdARF13 to the
promoter. Our findings further characterized the mechanism underlying the regulation of anthocyanin biosynthesis via Aux/IAA-ARF signaling.
Anthocyanin biosynthesis in callus culture in vitro is strongly influenced by exogenous auxin concentrations. However, the mechanisms by which auxin regulates anthocyanin biosynthesis are largely ...unknown. To understand the molecular basis of this phenomenon, global gene expression was analyzed in red-fleshed apple calli treated with 1-naphthaleneacetic acid (NAA; 0.3 and 10 mg/L) and 2,4-dichlorophenoxyacetic acid (2,4-D; 0.03 and 0.6 mg/L) using RNA-seq. A total of 3070 and 2533 genes were differently expressed (log
2
ratio ≥ 2 at
P
< 0.0001) in the 2,4-D and NAA treatments, respectively. Thereof, 937 genes were up-regulated and 902 genes were both down-regulated. Genes involved in anthocyanin and flavonoid synthesis and transport into the vacuole were generally down-regulated. Higher concentrations of 2,4-D and NAA facilitated the transport of auxin and induced the expressions of genes involved in the homeostatic feedback regulatory loop. In the auxin signaling pathway, nine Aux/IAA family genes and seven ARF family genes were up-regulated. Moreover, 298 transcription factors were differentially expressed in the NAA and 2,4-D treatments. Among them, some members of MYB, bHLH, and WD40 families that directly regulate anthocyanin and flavonoid synthesis, such as
MYB75
(
MdMYB10
),
MYB12
,
MYB111
,
MYB113
,
TT2
, and
TT8
(
MdbHLH3
), were down-regulated by NAA and 2,4-D. Auxin also affected gene expression in other plant hormone signaling pathways, such as the cytokinin, ethylene, and gibberellic acid pathways, which also influenced anthocyanin biosynthesis. This study provides a valuable overview of transcriptome changes and gives insight into the molecular mechanism by which auxin inhibits anthocyanin biosynthesis in red-fleshed apple calli.
Coloring in apple fruit due to anthocyanin accumulation is inhibited by high temperature; however, the underlying mechanism remains unclear. In the present study, total anthocyanin and cyanidin ...3-galactoside contents were determined and compared between cv. 'Redchief Delicious' apple fruits at 25 °C and 35 °C treatments. The high temperature (35 °C) treatment substantially decreased total anthocyanin and cyanidin 3-galactoside contents. The transcriptomes of 25 °C- and 35 °C-treated apples were analyzed by high-throughput RNA sequencing. A total of 8354 differentially expressed genes (DEGs) were detected at four time points corresponding to the two temperature treatments. The up-regulated DEGs were annotated using GO as well as KEGG databases. A network module of 528 genes (including 21 transcription factors) most associated with the total anthocyanin and cyanidin 3-galactoside contents was constructed by weighted correlation network analysis (WGCNA). In the WGCNA module, we unearthed a LOB domain-containing gene designated as
. The expression of
was sharply up-regulated by high temperature and negatively correlated with the total anthocyanin and cyanidin 3-galactoside contents. Overexpression of
in apple fruit and calli decreased the expression of anthocyanin biosynthetic genes, such as
,
,
,
,
, and
, along with anthocyanin accumulation. Our results suggested that
significantly influenced the high-temperature inhibition of anthocyanin accumulation in apples. The findings shed more light on the mechanism of anthocyanin inhibition during high-temperature stress in apples.
We have induced callus tissues from one R6/R6 homozygous genotype red-fleshed apple individual which was the hybrid offspring of Malus sieversii f.niedzwetzkyana and ‘Fuji’, and investigated the ...effect of auxin alone and auxin combined with cytokinin or nitrogen deficiency on anthocyanin synthesis. In callus culture, auxin alone significantly inhibited anthocyanin biosynthesis with the increase of auxin concentration. The inhibitory effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on anthocyanin accumulation was about tenfold stronger than naphthalene acetic acid. Anthocyanin regulatory genes (MdMYB10 and MdbHLH3) and structural genes were dramatically suppressed by 0.6 mg/L 2,4-D. The inhibitory effect of auxin on anthocyanin biosynthesis was influenced by cytokinins 6-benzylaminopurine (BAP) and thidiazuron (TDZ) as well as nitrogen deficiency. Auxin and cytokinin displayed the interaction in controlling anthocyanin biosynthesis. Co-treatment of auxin and cytokinin (BAP or TDZ) significantly enhanced the cytokinin-induced increase in anthocyanin levels but too high auxin concentration strongly inhibited anthocyanin synthesis even in the presence of cytokinin. Nitrogen deficiency could reverse the inhibition of anthocyanin accumulation by auxin.
Aims: To investigate the association between triglyceride-glucose(TyG) index and the risk of hypertension. Method: A cross-sectional study was conducted in Bengbu, China. The participants received ...relevant questionnaire survey, anthropometric tests, and laboratory examination. Multivariate logistic regression analysis was performed to estimate the possible association between TyG index and hypertension risk. The additive interaction evaluated by the relative excess risk due to interaction (RERI), attributable proportion due to interaction (AP), and synergy index(SI) was calculated. Results: A total of 1777 participants (748 men and 1029 women) were investigated. There was a significant increase in the risk of hypertension and isolated systolic hypertension (ISH) when comparing the highest TyG index (the fourth quartile) to the lowest TyG index (the first quartile) and corresponding ORs were 2.446 (95% CI: 1.746-3.426) and 2.621(95%CI: 1.627-4.224), respectively. However, no significant relationship was observed between TyG index and isolated diastolic hypertension (IDH). In males, significant interactions between TyG index and WHtR (RERI:1.978, 95%CI: 0.162-3.792; AP: 0.359, 0.113-0.605; SI:1.782, 1.017-3.122), smoking (AP: 0.437, 95%CI: 0.048-0.825), family history of hypertension (AP:0.433, 95%CI: 0.203-0.662; SI:2.248, 95%CI: 1.333-3.791) were observed. As for females, there were also significant interactions between TyG index and WHtR (RERI:1.415, 95%CI: 0.693-2.136; AP: 0.198, 95%CI: 0.104-0.291; SI:1.298, 95%CI:1.101-1.530), family history of hypertension (RERI:1.744, 95%CI: 0.221-3.267; AP:0.405, 95%CI: 0.113-0.697) on risk of hypertension. Conclusions: Increased TyG index was significantly associated with higher risk of hypertension and ISH, but not for IDH in middle-aged and elderly adults. Our results also demonstrated interactions of TyG index and abdominal obesity and family history of hypertension on hypertension risk.
The eco-environmental problems caused by land desertification are becoming the main restricting factors in regional development. In this study, the engineering eco-environment patterns (EEPs) are ...proposed to evaluate the eco-geological environment fragility to desertification in arid and semiarid areas. A revised zoning method in the variable-weight theory (VWT) was used for EEPs and applied in the Dousitu River Basin (DBR). First, the EEP mechanism was elucidated, of which four types were divided. Second, ten key controlling indicators from climatology, geography, vegetation and social conditions were collected, of which the thematic maps were imported. Third, the weight and its interval of the indicator were determined, establishing the partial stimulation-punishment variable-weight zoning model. Finally, the evaluation result was obtained and mapped in four models or zones. Furthermore, the evaluation via the CWT method was also completed for comparison. Both results from VWT and CWT were validated with the ecological groundwater level (EGL) theory, which proved the accuracy of the VWT study. The method helps with the rational plan and design for the regional economic development and protection of eco-geological environment.
Trimethylamine-N-Oxide (TMAO) is a proatherogenic and prothrombotic metabolite. Our study examined the association of plasma TMAO level with cardiovascular and all-cause mortality in hemodialysis ...(HD) patients.
Patients who were at least 18 years-old and received HD for at least 6 months were enrolled within 6 months. Patients with coronary heart disease, congestive heart failure, arrhythmia, or stroke within 3 months before study onset were excluded. The primary endpoints were cardiovascular and all-cause death, and the secondary endpoint was cerebrovascular death.
We recruited 252 patients and divided them into a high-TMAO group (>4.73 μg/mL) and a low-TMAO group (≤4.73 μg/mL). The median follow-up time was 73.4 months (interquartile range: 42.9, 108). A total of 123 patients died, 39 from cardiovascular disease, 19 from cerebrovascular disease, and 65 from other causes. Kaplan-Meier analysis indicated that the high-TMAO group had a greater incidence of cardiovascular death (Log-Rank: p = 0.006) and all-cause death (Log-Rank: p < 0.001). Cox regression analysis showed that high TMAO level was significantly associated with cardiovascular and all-cause mortality. After adjustment for confounding, this association remained significant for cardiovascular mortality (TMAO as a continuous variable: HR: 1.18, 95%CI: 1.07, 1.294, p < 0.001; TMAO as a dichotomous variable: HR: 3.44, 95%CI: 1.68, 7.08, p < 0.001) and all-cause mortality (TMAO as a continuous variable: HR: 1.14, 95%CI: 1.08, 1.21, p < 0.001; TMAO as a dichotomous variable: HR: 2.54, 95%CI: 1.71, 3.76, p < 0.001).
High plasma TMAO level is significantly and independently associated with cardiovascular and all-cause mortality in HD patients.
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•A re-centering energy-dissipation brace with a pendulum (REBP) is proposed.•The REBP can dissipate energy by friction and re-center the structure once unloaded.•Five REBPs with ...different cable tensions are subjected to cyclic loading tests.•The cable pretension has significant effects on the seismic behaviors of the REBP.•A hysteretic model and design formulas for the REBP are proposed and validated.
In this study, a re-centering energy-dissipation brace with a pendulum (REBP) is proposed for high-rise structures. The REBP can consume energy by friction under the action of a horizontal external load, and then re-center the structure by means of cable pretension after removing the load. Through the lever action of the pendulum, the deformation capability of the REBP is significantly amplified with the small deformation of the cables, and can satisfy the inter-story drift requirement for a severe earthquake. The REBP is optimized from a prestressed re-centering energy-dissipation brace (PTSEB) previously proposed by the authors. In particular, the T-shaped pendulum in the PTSEB is replaced by a truss pendulum. The pendulum-wing, vertical bar, and diagonal bars form a truss with high strength. This increases the initial stiffness and energy consumption capability of the REBP, and decreases the weight of the REBP. Two semicircle rollers are set on the top and bottom of the blocker to facilitate its rotation. An hysteretic model and design formulas are proposed for the REBP. Through cyclic testing and a finite element (FE) analysis of REBPs with five different cable pretensions, the cyclic behaviors of the REBP and the influences of cable pretension on the energy consumption, re-centering, stiffness, friction, and cable tension are determined. The results indicate that the REBP has good energy consumption and a good re-centering capability. An increase in cable pretension leads to an increases in the friction between the blocker and pendulum-wing, energy consumption capability, and stiffness.
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