Establishment and validation of a nomogram model for predicting ovulation in the PCOS women.

BACKGROUND: The mechanisms underlying ovulatory dysfunction in PCOS remain debatable. This study aimed to identify the factors affecting ovulation among PCOS patients based on a large sample-sized randomized control trial. METHODS: Data were obtained from a multi-centered randomized clinical trial, the PCOSAct, which was conducted between 2011 and 2015. Univariate and multivariate analysis using binary logistic regression were used to construct a prediction model and nomogram. The accuracy of the model was assessed using receiver operating characteristic (ROC) curves and calibration curves. RESULTS: The predictive variables included in the training dataset model were luteinizing hormone (LH), free testosterone, body mass index (BMI), period times per year, and clomiphene treatment. The ROC curve for the model in the training dataset was 0.81 (95% CI [0.77, 0.85]), while in the validation dataset, it was 0.7801 (95% CI [0.72, 0.84]). The model showed good discrimination in both the training and validation datasets. Decision curve analysis demonstrated that the nomogram designed for ovulation had clinical utility and superior discriminative ability for predicting ovulation. CONCLUSIONS: The nomogram composed of LH, free testosterone, BMI, period times per year and the application of clomiphene may predict the ovulation among PCOS patients.

Is elevated baseline SHBG associated with increased ovulation?

Sexual hormone binding globulin (SHBG) is associated with the endocrine and reproductive systems. We aimed to investigate the role of SHBG in the reproductive process. Therefore, we conducted a secondary analysis of the PCOSAct (Polycystic Ovary Syndrome and Acupuncture Clinical Trial) study, which involved 21 sites in China and a total of 1000 women with PCOS. Out of these, 954 women with SHBG were included in the analysis. Through multivariate analysis of ovulation predictors, we found that age, BMI, estradiol, testosterone, and SHBG all showed a positive predictive value for ovulation (p = 0.0211, 0.0011, 0.0211, 0.0029, 0.0434, respectively). However, the LH to FSH ratio had a negative predictive value (p = 0.0539). Higher quartiles of SHBG were associated with a higher rate of ovulation, and per quartile increased was statistically significant (HR = 1.138, 95%CI [1.054,1.229]). The association remained significant even after adjusting for testosterone (HR = 1.263, 95%CI [1.059, 1.507]). On the other hand, quartiles of testosterone and estradiol did not exhibit any significant tendency toward ovulation. SHBG demonstrated predictive ability for ovulation, conception, pregnancy, and live birth (p < 0.05), and this correlation remained significant after adjusting intervention. Kaplan-Meier curves illustrated that increased levels of SHBG were a factor in high rates of ovulation, conception, and pregnancy. In comparison to other sexual hormones, a higher baseline level of SHBG was related to increased ovulation.

Acupuncture improves the emotion domain and lipid profiles in women with polycystic ovarian syndrome: a secondary analysis of a randomized clinical trial.

Objective: This study aims to evaluate the effect of acupuncture on the emotion domain and metabolic parameters of Chinese women with polycystic ovarian syndrome (PCOS) by secondary analysis of a randomized clinical trial, conducted from 6 July 2012 to 7 October 2015. Method: In this study, we investigated the effects of acupuncture (458 patients) and sham acupuncture (468 patients) on metabolic parameters, serum ions, and all quality-of-life scale scores related to PCOS. The quality of life of patients was evaluated using five relevant scales, operated by the research assistant, namely, PCOSQ, SF-36, and ChiQOL, as well as Zung-SAS and Zung-SDS. Metabolic parameters and serum ions were measured. Results: A reduction in acne score, AN, Hcy, and LDL-C, and an increase in the level of lipoprotein α, Apo A1, and Apo A1/Apo B were observed in the acupuncture group after 4 months' intervention after adjusting clomiphene and reproductive outcome (p< 0.05). An increase in SF-36 total scores, RP and RE scores, ChiQOL total scores, and emotion domain scores was observed in the acupuncture group after 4 months' intervention, while PF and HT scores were decreased (adjusted p< 0.05). Those same changes were observed in sham acupuncture. Meanwhile, the serum levels of Ca, K, and Cl were elevated in the acupuncture group after the interventions (adjusted p< 0.005). There were no significant differences in HOMA-IR, MetS, FPG, FINS, HDL-C, TG, Apo B, and level of serum P, Mg, and Na. Also, no changes in BP, GH, VT, SF, physical form domain, and spirit domain were observed after treatment. Conclusion: Acupuncture can improve not only the emotional changes in SF-36 scores and ChiQOL scores, but also lipid metabolism, implying that it may have a correlation between emotional change and lipid metabolism. Furthermore, acupuncture can also regulate the changes of serum Ca, K, and Cl. Clinical trial registration: ClinicalTrials.gov, identifier NCT01573858.

Low temperature inhibits pectin degradation by PpCBFs to prolong peach storage time.

Low-temperature storage is a widely used method for peach fruit storage. However, the impact of PpCBFs on pectin degradation during low-temperature storage is unclear. As such, in this study, we stored the melting-flesh peach cultivar "Fuli" at low temperature (LT, 6°C) and room temperature (RT, 25°C) to determine the effect of different temperatures on its physiological and biochemical changes. Low-temperature storage can inhibit the softening of "Fuli" peaches by maintaining the stability of the cell wall. It was found that the contents of water-soluble pectin and ionic-soluble pectin in peach fruit stored at RT were higher than those stored at LT. The enzyme activities of polygalacturonase (PG), pectate lyase (PL), and pectin methylesterase (PME) were all inhibited by LT. The expressions of PpPME3, PpPL2, and PpPG were closely related to fruit firmness, but PpCBF2 and PpCBF3 showed higher expression levels at LT than RT. The promoters of PpPL2 and PpPG contain the DER motif, which suggested that PpCBF2 and PpCBF3 might negatively regulate their expression by directly binding to their promoters. These results indicated that LT may maintain firmness by activating PpCBFs to repress pectin-degradation-related enzyme genes during storage.

The MADS-box gene EjAGL15 positively regulates lignin deposition in the flesh of loquat fruit during its storage.

Introduction: Lignification of fruit flesh is a common physiological disorder that occurs during post-harvest storage, resulting in the deterioration of fruit quality. Lignin deposition in loquat fruit flesh occurs due to chilling injury or senescence, at temperatures around 0°C or 20°C, respectively. Despite extensive research on the molecular mechanisms underlying chilling-induced lignification, the key genes responsible for the lignification process during senescence in loquat fruit remain unknown. MADS-box genes, an evolutionarily conserved transcription factor family, have been suggested to play a role in regulating senescence. However, it is still unclear whether MADS-box genes can regulate the lignin deposition that arises from fruit senescence. Methods: Both senescence- and chilling-induced flesh lignification were simulated by applying temperature treatments on loquat fruits. The flesh lignin content during the storage was measured. Transcriptomic, quantitative reverse transcription PCR and correlation analysis were employed to identify key MADS-box genes that may be involved in flesh lignification. The Dual-luciferase assay was utilized to identify the potential interactions between MADS-box members and genes in phenylpropanoid pathway. Results and Discussion: The lignin content of the flesh samples treated at 20°C or 0°C increased during storage, but at different rates. Results from transcriptome analysis, quantitative reverse transcription PCR, and correlation analysis led us to identify a senescence-specific MADS-box gene, EjAGL15, which correlated positively with the variation in lignin content of loquat fruit. Luciferase assay results confirmed that EjAGL15 activated multiple lignin biosynthesis-related genes. Our findings suggest that EjAGL15 functions as a positive regulator of senescence-induced flesh lignification in loquat fruit.

PbO2 materials for electrochemical environmental engineering: A review on synthesis and applications.

Lead dioxide (PbO2) materials have been widely employed in various fields such as batteries, electrochemical engineering, and more recently environmental engineering as anode materials, due to their unique physicochemical properties. Key performances of PbO2 electrodes, such as energy efficiency and space-time yield, are influenced by morphological as well as compositional factors. Micro-nano structure regulation and decoration of metal/non-metal on PbO2 is an outstanding technique to revamp its electrocatalytic activities and enhance environmental engineering efficiency. The aim of this review is to comprehensively summarize the recent research progress in the morphology control, the structure constructions, and the element doping of PbO2 materials, further with many environmental application cases evaluated. Concerning electrochemical environmental engineering, the lead dioxide employed in chemical oxygen demand detection, ozone generators, and wastewater treatment has been comprehensively reviewed. In addition, the future research perspectives, challenges and the opportunities on PbO2 materials for environmental applications are proposed.

Sorbitol induces flower bud formation via the MADS-box transcription factor EjCAL in loquat.

Sorbitol is an important signaling molecule in fruit trees. Here, we observed that sorbitol increased during flower bud differentiation (FBD) in loquat (Eriobotrya japonica Lindl.). Transcriptomic analysis suggested that bud formation was associated with the expression of the MADS-box transcription factor (TF) family gene, EjCAL. RNA fluorescence in situ hybridization showed that EjCAL was enriched in flower primordia but hardly detected in the shoot apical meristem. Heterologous expression of EjCAL in Nicotiana benthamiana plants resulted in early FBD. Yeast-one-hybrid analysis identified the ERF12 TF as a binding partner of the EjCAL promoter. Chromatin immunoprecipitation-PCR confirmed that EjERF12 binds to the EjCAL promoter, and ß-glucuronidase activity assays indicated that EjERF12 regulates EjCAL expression. Spraying loquat trees with sorbitol promoted flower bud formation and was associated with increased expression of EjERF12 and EjCAL. Furthermore, we identified EjUF3GaT1 as a target gene of EjCAL and its expression was activated by EjCAL. Function characterization via overexpression and RNAi reveals that EjUF3GaT1 is a biosynthetic gene of flavonoid hyperoside. The concentration of the flavonoid hyperoside mirrored that of sorbitol during FBD and exogenous hyperoside treatment also promoted loquat bud formation. We identified a mechanism whereby EjCAL might regulate hyperoside biosynthesis and confirmed the involvement of EjCAL in flower bud formation in planta. Together, these results provide insight into bud formation in loquat and may be used in efforts to increase yield.

Research of Maritime Object Detection Method in Foggy Environment Based on Improved Model SRC-YOLO.

An improved maritime object detection algorithm, SRC-YOLO, based on the YOLOv4-tiny, is proposed in the foggy environment to address the issues of false detection, missed detection, and low detection accuracy in complicated situations. To confirm the model's validity, an ocean dataset containing various concentrations of haze, target angles, and sizes was produced for the research. Firstly, the Single Scale Retinex (SSR) algorithm was applied to preprocess the dataset to reduce the interference of the complex scenes on the ocean. Secondly, in order to increase the model's receptive field, we employed a modified Receptive Field Block (RFB) module in place of the standard convolution in the Neck part of the model. Finally, the Convolutional Block Attention Module (CBAM), which integrates channel and spatial information, was introduced to raise detection performance by expanding the network model's attention to the context information in the feature map and the object location points. The experimental results demonstrate that the improved SRC-YOLO model effectively detects marine targets in foggy scenes by increasing the mean Average Precision (mAP) of detection results from 79.56% to 86.15%.

The Effect of Age on IVF-ET Outcome in Infertile Patients with PCOS Based on Tiangui Theory in Chinese Medicine.

Objective: Our aim was to investigate the effect of age on the outcome of IVF-ET and ICSI in infertile PCOS patients under the guidance of Tiangui theory in traditional Chinese medicine. Method: This was a retrospective analysis of 532 infertile women with PCOS and 1,392 women with infertility due to tubal factors as the controls. All of the participants were divided into different age groups-aged 20-28 years, 29-35 years, and ≥36 years-according to the stages of female reproductive development in Tiangui theory as described in the Canon of Internal Medicine-Treatise of Ancient Natural Truth. We explored the effect of age on controlled ovarian hyperstimulation (including the initial dosage and duration of Gn and the estradiol level on the day of human chorionic gonadotropin administration); the numbers of retrieved oocytes, 2PN zygotes, and embryos; and the rates of fertilization, clinical pregnancy, abortion, live birth, and OHSS incidence. Results: Compared to controls, the maximum follicular diameter and the numbers of follicles with d ≥ 20 mm, retrieved oocytes, and 2PN zygotes were greater in the PCOS group with age >28 years (p < 0.05). The abortion rate of PCOS patients with age ≤28 years was higher than that of the controls. All PCOS groups and the control group showed reduced numbers of retrieved oocytes and live births with age. The difference in age was not significant in the PCOS groups but was significant in the control group (p < 0.05), and the trend in the PCOS groups was more gradual. Conclusion: The fertility of all subjects decreased with age, but PCOS patients decreased more slowly than in controls at the same age, which verified the applicability of the guiding principles of Tiangui theory in the clinic.

Physicochemical, digestive and rheological properties of protein from tuna by subcritical dimethyl ether: Focus on process-related indexes.

The process-related physicochemical, digestive and rheological properties of protein prepared by subcritical dimethyl ether extraction (SDEE) were comprehensively investigated and compared with those obtained by pH-shift, to study the industrial potential of SDEE. Two different materials from tuna (meat and liver) were studied in parallel, and SDEE had similar effects on the proteins in them. The protein component was almost unchanged before and after SDEE, while the content of water-soluble protein and alkali-soluble protein was substantially reduced and increased after pH-shift, respectively. We also found that SDEE had superior ability to pH-shift to conserve light metals, remove lipids and heavy metals, and maintain protein structure. Furthermore, SDEE-produced protein powders were easier for humans to digest, and their gelation and emulsification were also superior to those prepared by pH-shift. The aforementioned results suggest that SDEE can remove more impurities, and the obtained protein has outstanding potential in industrial applications.

Keystone taxa-mediated bacteriome response shapes the resilience of the paddy ecosystem to fungicide triadimefon contamination.

The increasing input of fungicides has emerged as a global concern for agroecosystem stability and sustainability. Agroecosystem resilience has been linked to microbiome response, however, is not well understood. Focusing on a widespread triazole-class fungicide triadimefon in the paddy ecosystem, we characterized that the soils and sediments were dominant triadimefon reservoirs with the peak level at 195 µg kg-1 and 31.3 µg kg-1, respectively, but essential for the resilience of paddy ecosystem to triadimefon. In paddy simulation models, the half-life of triadimefon in soil-sediment was 8.4-28.9 days, while it was prolonged to 86.6-115.5 days after elimination of resident microbial community. Phospholipid fatty acid profiling and high-throughput sequencing showed that the distinctive bacterial community responses contributed to variable degradation of triadimefon in paddy soils and sediments. Sphingomonas and Xanthomonas were identified as positive responders of the keystone taxa in the responsive bacteriome, whereas Enterobacter were negative responders that declined over time. Synthetic assemblages combined with quantitative polymerase chain reaction further validated that Sphingomonas and Xanthomonas were involved in sustaining soil-sediment resilience to triadimefon contamination. Collectively, our results revealed that the shaping of soil and sediment bacteriomes was responsible for the resilience of the paddy agroecosystem to fungicide contamination.

The MADS-Box Transcription Factor EjAGL65 Controls Loquat Flesh Lignification via Direct Transcriptional Inhibition of EjMYB8.

Loquat fruit accumulates lignin in its flesh when undergoing chilling injury during postharvest storage, making it a suitable model for the study of flesh lignification. Transcriptional regulation of lignin biosynthesis is principally controlled by the NAC-MYB transcriptional cascade in model plants. Previous research has demonstrated that EjMYB8 activates lignin biosynthesis through direct interaction with the promoter of Ej4CL1. However, the classic NAC-MYB gene regulation network has not been established. Here, the MADS-box gene EjAGL65 was discovered by screening a cDNA library using the EjMYB8 promoter as bait in yeast. A phylogenetic analysis and structural comparisons revealed that EjAGL65 belongs to the Mδ subgroup of the MADS-box family, whose members have not been reported as being involved in the regulation of lignin deposition. EjAGL65 transcription was downregulated at 0°C compared to 5°C, indicating a negative correlation with the change of lignin content. A dual-luciferase assay indicated that EjAGL65 is capable of inhibiting the promoter activity of EjMYB8 in vivo. These results showed that the Mδ MADS-box gene EjAGL65 transcriptionally regulates EjMYB8 during postharvest chilling induced flesh lignification, which differs from the classical regulation model of lignin biosynthesis that has been illustrated for developmental lignin accumulation.

Effect Evaluation of Strychnos nux-vomica L. with Integrative Methods for Bortezomib-Induced Peripheral Neuropathy in Multiple Myeloma Patients: A Self-Controlled Clinical Trial.

OBJECTIVE: To explore the clinical effect and adverse reactions of Strychnos nux-vomica in bortezomib-induced peripheral neuropathy (BIPN) of patients with multiple myeloma (MM). METHODS: A total of 19 MM patients with BIPN were enrolled and Nux Vomica Capsule (NVC, 0.4 g, thrice daily) were orally administrated for 30 days. Comparative analysis on parameters between pre- and post-therapy, including peripheral neuropathy (PN) grade, neurotoxicity score, Chinese medicine (CM) syndrome score, total neuropathy score (TNS), coagulation function, and serum nerve growth factor (NGF) levels were conducted. The adverse events were monitored. RESULTS: In BIPN of MM patients who received NVC, PN grade was lowered, neurotoxicity score was obviously decreased (P⩽0.01), and both CM syndrome score and TNS were remarkably decreased (P<0.01). After the therapy, activated partial thromboplastin time was prolonged (P<0.01) and fibrinogen was declined (P<0.05), showing improvement in the hypercoagulable state of patients. No significant difference of NGF recovery degrees was detected between pre- and post-therapy (P>0.05). No evident adverse reactions were observed during the course of treatment. CONCLUSION: Strychnos nux-vomica L. has significantly effect with a good safety in treatment of BIPN in MM patients.

Effects of hyperhomocysteinaemia and metabolic syndrome on reproduction in women with polycystic ovary syndrome: a secondary analysis.

RESEARCH QUESTION: What is the association between hyperhomocysteinaemia (HHCY), metabolic syndrome, and reproductive outcomes among women with polycystic ovary syndrome (PCOS). DESIGN: A secondary analysis of PCOSAct with 21 sites in China. A total of 1000 women with PCOS were enrolled; 936 women with baseline homocysteine (HCY) were analysed. RESULTS: Higher HCY was associated with higher body mass index, free testosterone and lower FSH, fasting glucose (P < 0.001; P < 0.001; P = 0.005; P < 0.001) and ovulation rate among all participants (OR 0.59, 95% CI 0.41 to 0.86; OR 0.57, 95% CI 0.39 to 0.83 tertiles 2 and 3 versus tertile 1, respectively). The HHCY group had lower oestradiol and higher free testosterone (P = 0.04; P < 0.001) than the controls. In the metabolic syndrome group, LH, LH-FSH ratio and sex hormone-binding globulin were lowest in the metabolic syndrome group (all P < 0.001). In the HHCY group, ovulation rate decreased and the second or third trimester pregnancy loss rate increased compared with controls (OR 1.678, 95% CI 1.04 to 2.70; OR 0.03, 95% CI 0.00 to 0.42) with treatment adjustment. Compared with the controls, ovulation, conception, pregnancy, second or third trimester pregnancy loss and live birth rates were statistically lower in the metabolic syndrome group after adjusting treatment (OR 1.76, 95% CI 1.15 to 2.70; OR 1.75, 95% CI 1.15 to 2.65; OR 2.09, 95% CI 1.27 to 3.44; OR 0.02, 95% CI 0.00 to 0.33; OR 2.42 95% CI 1.42 to 4.10), and pregnancy, pregnancy loss and live birth rates remained significantly different after adjusting for treatment and sex-hormone factors (OR 1.77, 95% CI 1.05 to 2.99; OR 0.14, 95% CI 0.02 to 0.82; OR 2.02, 95% CI 1.16 to 3.50). CONCLUSIONS: In women with PCOS, HHCY contributes to increased pregnancy loss and reduced ovulation, and metabolic syndrome was related to defects in ovulation, conception, pregnancy, pregnancy loss and live birth, indicating that the two conditions lead to defects at various reproductive stages.

EjNAC3 transcriptionally regulates chilling-induced lignification of loquat fruit via physical interaction with an atypical CAD-like gene.

Lignin is an important component of many plant secondary cell walls. In the fruit of loquat (Eriobotrya japonica), lignification of cell walls in the fleshy tissue occurs when fruit are subjected to low-temperature storage, which is commonly used to avoid the rapid senescence that occurs at room temperature. In this study, two NAC domain genes, EjNAC3 and EjNAC4, were isolated and shown to be significantly induced at 0 °C, which was concomitant with an increase in the fruit lignification index. Lignification and expression of both EjNAC3 and EjNAC4 were inhibited by low-temperature conditioning and by heat treatment. In addition, EjNAC3 trans-activated the lignin biosynthesis-related EjCAD-like promoter, which was measured using a dual-luciferase assay. Further analysis with yeast one-hybrid and electrophoretic mobility shift assays indicated that EjNAC3 could physically bind to the promoter of the EjCAD-like gene. Thus, EjNAC3 is a direct regulator of loquat chilling-induced lignification, via regulations of EjCAD-like.

EjODO1, a MYB Transcription Factor, Regulating Lignin Biosynthesis in Developing Loquat (Eriobotrya japonica) Fruit.

Lignin is important for plant secondary cell wall formation and participates in resistance to various biotic and abiotic stresses. Loquat undergoes lignification not only in vegetative tissues but also in flesh of postharvest fruit, which adversely affects consumer acceptance. Thus, researches on lignin biosynthesis and regulation are important to understand loquat fruit lignification. In loquat, a gene encoding an enzyme in the lignin biosynthesis pathway, Ej4CL1, was reported to be regulated by transcription factors, including EjMYB1, EjMYB2, EjMYB8, and EjAP2-1, knowledge of this process is still limited. With the aim of identifying novel transcriptional factors controlling lignin biosynthesis in loquat, the promoter of Ej4CL1 was utilized to screen a cDNA library by yeast one hybrid assay. A novel R2R3 MYB, named EjODO1, was identified. Real-time PCR analyses indicated that EjODO1 is highly expressed in lignified stems and roots. During fruit development, expression of EjODO1 decreased along with the reduction of lignin content and became undetectable in mature ripe fruit. Thus, EjODO1 is likely to be involved in lignification of vegetative organs and early fruit development but not in mature fruit or postharvest lignification. Dual-luciferase assay indicated that EjODO1 could trans-activate promoters of lignin biosynthesis genes, such as EjPAL1, Ej4CL1, and Ej4CL5 and transient overexpression of EjODO1 triggered lignin biosynthesis. These results indicate a role for EjODO1 in regulating lignin biosynthesis in loquat which is different from the previously characterized transcription factors.

EjMYB8 Transcriptionally Regulates Flesh Lignification in Loquat Fruit.

Transcriptional regulatory mechanisms underlying lignin metabolism have been widely studied in model plants and woody trees, but seldom in fruits such as loquat, which undergo lignification. Here, twelve EjMYB genes, designed as EjMYB3-14, were isolated based on RNA-seq. Gene expression indicated that EjMYB8 and EjMYB9 were significantly induced in fruit with higher lignin content resulting from storage at low temperature (0°C), while two treatments (low temperature conditioning, LTC; heat treatment, HT) both alleviated fruit lignification and inhibited EjMYB8 and EjMYB9 expression. Dual-luciferase assays indicated that EjMYB8, but not EjMYB9, could trans-activate promoters of lignin-related genes EjPAL1, Ej4CL1 and Ej4CL5. Yeast one-hybrid assay indicated that EjMYB8 physically bind to Ej4CL1 promoter. Furthermore, the putative functions of EjMYB8 were verified using transient over-expression in both N. tabacum and loquat leaves, which increased lignin content. Moreover, combination of EjMYB8 and previously isolated EjMYB1 generated strong trans-activation effects on the Ej4CL1 promoter, indicating that EjMYB8 is a novel regulator of loquat fruit lignification.

Regulation of loquat fruit low temperature response and lignification involves interaction of heat shock factors and genes associated with lignin biosynthesis.

Transcriptional regulatory mechanisms underlying lignin metabolism have been widely studied in model plants and woody trees, as well as fruit, such as loquat (Eriobotrya japonica). Unlike the well-known NAC, MYB and AP2/ERF transcription factors, the roles of heat shock factors (HSFs) in lignin regulation have been rarely reported. Two treatments (heat treatment, HT; low temperature conditioning, LTC) were applied to alleviate low temperature-induced lignification in loquat fruit. Gene expression analysis indicated that EjHSF1 transcript abundance, in parallel with heat shock protein genes (EjHsp), was induced by HT, while expression of EjHSF3 was repressed by LTC. Using dual-luciferase assays, EjHSF1 and EjHSF3 trans-activated the promoters of EjHsp genes and lignin biosynthesis-related genes, respectively. Thus, two distinct regulatory mechanisms of EjHSF transcription factors in chilling injury-induced fruit lignification are proposed: EjHSF1 transcriptionally regulated EjHsp genes are involved in chilling tolerance, while EjHSF3 transcriptionally regulated lignin biosynthesis. Furthermore, the relations between EjHSF3 and previously characterized fruit lignification regulators, including EjAP2-1, EjMYB1 and EjMYB2, were also investigated. Yeast-two hybrid (Y2H) and biomolecular fluorescence complementation (BiFC) assays demonstrated protein-protein interaction between EjHSF3 and EjAP2-1. Thus, the involvement of EjHSF3 in fruit lignification is via both lignin biosynthetic genes and the regulator, EjAP2-1.

The Citrus transcription factor, CitERF13, regulates citric acid accumulation via a protein-protein interaction with the vacuolar proton pump, CitVHA-c4.

Organic acids are essential to fruit flavor. The vacuolar H(+) transporting adenosine triphosphatase (V-ATPase) plays an important role in organic acid transport and accumulation. However, less is known of V-ATPase interacting proteins and their relationship with organic acid accumulation. The relationship between V-ATPase and citric acid was investigated, using the citrus tangerine varieties 'Ordinary Ponkan (OPK)' and an early maturing mutant 'Zaoshu Ponkan (ZPK)'. Five V-ATPase genes (CitVHA) were predicted as important to citric acid accumulation. Among the genes, CitVHA-c4 was observed, using a yeast two-hybrid screen, to interact at the protein level with an ethylene response factor, CitERF13. This was verified using bimolecular fluorescence complementation assays. A similar interaction was also observed between Arabidopsis AtERF017 (a CitERF13 homolog) and AtVHA-c4 (a CitVHA-c4 homolog). A synergistic effect on citric acid levels was observed between V-ATPase proteins and interacting ERFs when analyzed using transient over-expression in tobacco and Arabidopsis mutants. Furthermore, the transcript abundance of CitERF13 was concomitant with CitVHA-c4. CitERF13 or AtERF017 over-expression leads to significant citric acid accumulation. This accumulation was abolished in an AtVHA-c4 mutant background. ERF-VHA interactions appear to be involved in citric acid accumulation, which was observed in both citrus and Arabidopsis.

Activator- and repressor-type MYB transcription factors are involved in chilling injury induced flesh lignification in loquat via their interactions with the phenylpropanoid pathway.

Lignin biosynthesis and its transcriptional regulatory networks have been studied in model plants and woody trees. However, lignification also occurs in some fleshy fruit and has rarely been considered in this way. Loquat ( Eriobotrya japonica ) is one such convenient tissue for exploring the transcription factors involved in regulating fruit flesh lignification. Firmness and lignin content of 'Luoyangqing' loquat were fund to increase during low-temperature storage as a typical symptom of chilling injury, while heat treatment (HT) and low-temperature conditioning (LTC) effectively alleviated them. Two novel EjMYB genes, EjMYB1 and EjMYB2, were isolated and were found to be localized in the nucleus. These genes responded differently to low temperature, with EjMYB1 induced and EjMYB2 inhibited at 0 °C. They also showed different temperature responses under HT and LTC conditions, and may be responsible for different regulation of flesh lignification at the transcriptional level. Transactivation assays indicated that EjMYB1 and EjMYB2 are a transcriptional activator and repressor, respectively. EjMYB1 activated promoters of both Arabidopsis and loquat lignin biosynthesis genes, while EjMYB2 countered the inductive effects of EjMYB1. This finding was also supported by transient overexpression in tobacco. Regulation of lignification by EjMYB1 and EjMYB2 is likely to be achieved via their competitive interaction with AC elements in the promoter region of lignin biosynthesis genes such as Ej4CL1.