Performance enhancement, bacterial communities optimization and emerging pollutants elimination by microalgal-bacterial consortium for treating aquaculture pond sediments.

Aquaculture pond sediments have a notable influence on the ecosystem balance and farmed animal health. In this study, microalgal-bacterial immobilization (MBI) was designed to improve aquaculture pond sediments via synergistic interactions. The physicochemical characteristics, bacterial communities, and the removal efficiencies of emerging pollutants were systematically investigated. The consortium containing diatom Navicula seminulum and Alcaligenes faecalis was cultivated and established in the free and immobilized forms for evaluating the treatment performance. The results indicated that the immobilized group exhibited superior performance in controlling nutrient pollutants, shaping and optimizing the bacterial community compositions with the enrichment of functional bacteria. Additionally, it showed a stronger positive correlation between the bacterial community shifts and nutrient pollutants removal compared to free cells. Furthermore, the immobilized system maintained the higher removal performance of emerging pollutants (heavy metals, antibiotics, and pathogenic Vibrios) than free group. These findings confirmed that the employment of immobilized N. seminulum and A. faecalis produced more synergistic benefits and exerted more improvements than free cells in ameliorating aquaculture pond sediments, suggesting the potential for engineering application of functional microalgal-bacterial consortium in aquaculture.

Sustainable treatment of aquaculture water employing fungi-microalgae consortium: Nutrients removal enhancement, bacterial communities optimization, emerging contaminants elimination, and mechanism analysis.

Fungi-microalgae consortium (FMC) has emerged as a promising system for advanced wastewater treatment due to its high biomass yield and environmental sustainability. This study aimed to investigate the nutrients removal, bacterial community shift, emerging contaminants elimination, and treatment mechanism of a FMC composed of Cordyceps militaris and Navicula seminulum for aquaculture pond water treatment. The fungi and microalgae were cultured and employed either alone or in combination to evaluate the treatment performance. The results demonstrated that the FMC could improve water quality more significantly by reducing nutrient pollutants and optimizing the bacterial community structures. Furthermore, it exhibited stronger positive correlation between the enrichment of functional bacteria for water quality improvement and pollutants removal performance than the single-species treatments. Moreover, the FMC outperformed other groups in eliminating emerging contaminants such as heavy metals, antibiotics, and pathogenic Vibrios. Superiorly, the FMC also showed excellent symbiotic interactions and cooperative mechanisms for pollutants removal. The results collectively corroborated the feasibility and sustainability of using C. militaris and N. seminulum for treating aquaculture water, and the FMC would produce more mutualistic benefits and synergistic effects than single-species treatments.

Succession and assembly mechanisms of seawater prokaryotic communities along an extremely wide salinity gradient.

Salinity is an important environmental factor in microbial ecology for affecting the microbial communities in diverse environments. Understanding the salinity adaptation mechanisms of a microbial community is a significant issue, while most previous studies only covered a narrow salinity range. Here, variations in seawater prokaryotic communities during the whole salt drying progression (salinity from 3% to 25%) were investigated. According to high-throughput sequencing results, the diversity, composition, and function of seawater prokaryotic communities varied significantly along the salinity gradient, expressing as decreased diversity, enrichment of some halophilic archaea, and powerful nitrate reduction in samples with high salt concentrations. More importantly, a sudden and dramatic alteration of prokaryotic communities was observed when salinity reached 16%, which was recognized as the change point. Combined with the results of network analysis, we found the increasing of complexity but decreasing of stability in prokaryotic communities when salinity exceeded the change point. Moreover, prokaryotic communities became more deterministic when salinity exceeded the change point due to the niche adaptation of halophilic species. Our study showed that substantial variations in seawater prokaryotic communities along an extremely wide salinity gradient, and also explored the underlying mechanisms regulating these changes.

Temporal stability and assembly mechanisms of gut microbiota in sea cucumbers response to nanoplastics treatment.

Aquaculture provides essential food for humans, and the health of farmed species is particularly important for the aquaculture industry. Aquaculture environment could be a sink of plastic debris (PDs) due to the enclosed character and heavy use of plastics. Gut microbiota of aquaculture species could respond to the exogenous pollutants and regulate the health of hosts. Here, variations in gut microbiota of Apostichopus japonicus induced by the ingested nanoplastics (NPs) were investigated by a lab experiment. We selected a NPs concentration gradient of 100 mg/kg and 500 mg/kg to simulate microplastic pollution to A. japonicus, and the significant differences in gut microbiota composition after 21 days of NP exposure were evaluated. According to the high-throughput sequencing from time series samples, a decrease of diversity in gut microbiota of A. japonicus with dietary NPs was observed. In addition, the gut microbiota compositions of sea cucumbers with and without NPs exposure were also distinct, expressing as enrichment of Bacteroidota while reducement of Proteobacteria under NPs stresses. Combined the results of network analysis, the less complexity and stability of gut microbiota in sea cucumbers with dietary NPs were proved. Based on the neutral community model, the ingested NPs elevated the contribution of stochastic processes for the gut microbiota assembly in sea cucumbers. Our study showed that substantial variations in gut microbiota of A. japonicus under NPs stresses, and also explored the underlying mechanisms regulating these changes. This research would offer new meaningful insights into the toxicity of NPs on sea cucumbers, contributing a solid fundament to improve the health of sea cucumbers under NPs stresses.

Adverse effects of polystyrene nanoplastics on sea cucumber Apostichopus japonicus and their association with gut microbiota dysbiosis.

The mariculture environment is a sink of microplastics (MPs) due to its enclosed nature and mass use of plastics. Nanoplastics (NPs) are MPs with a diameter <1 µm that have a more toxic effect on aquatic organisms than other MPs. However, little is known about the underlying mechanisms of NP toxicity on mariculture species. Here, we performed a multi-omics investigation to explore gut microbiota dysbiosis and associated health problems induced by NPs in juvenile sea cucumber Apostichopus japonicus, a commercially and ecologically important marine invertebrate. We observed significant differences in gut microbiota composition after 21 days of NP exposure. Ingestion of NPs significantly increased core gut microbes, especially Rhodobacteraceae and Flavobacteriaceae families. Additionally, gut gene expression profiles were altered by NPs, especially those related to neurological diseases and movement disorders. Correlation and network analyses indicated close relationships between transcriptome changes and gut microbiota variation. Furthermore, NPs induced oxidative stress in sea cucumber intestines, which may be associated with intraspecies variation in Rhodobacteraceae in the gut microbiota. The results suggested that NPs were harmful to the health of sea cucumbers, and they highlighted the importance of the gut microbiota in the responses to NP toxicity in marine invertebrates.

Cloning and characterization of a phosphomevalonate kinase gene that is involved in saponin biosynthesis in the sea cucumber Apostichopus japonicus.

The sea cucumber Apostichopus japonicus is one of the most dominant and economically important aquaculture species in China. Saponin, which possesses notable biological and pharmacological properties, is a key determinant of the nutritional and health value of A. japonicus. In the present study, we amplified the full-length cDNA of a phosphomevalonate kinase (PMK) gene (named AjPMK) using rapid amplification of cDNA ends (RACE). Subsequently, we engineered a recombinant AjPMK (rAjPMK) protein and assessed its enzymatic activity by enzyme-linked immunosorbent assay (ELISA). Proteins that interact with rAjPMK were screened and identified via pull-down assay combined with liquid chromatography with tandem mass spectrometry (LC-MS/MS). We found that the full-length cDNA of AjPMK contained 1354 bp and an open reading frame (ORF) of 612 bp. The AjPMK protein was predicted not to contain a signal peptide but to contain a phosphonolate kinase domain seen in higher eukaryotes and a P-loop with a relatively conserved nucleoside triphosphate hydrolase domain. The molecular weight of the AjPMK protein was estimated to be 23.81 kDa, and its isoelectric point was predicted to be 8.72. Phylogenetic analysis showed that AjPMK had a closer evolutionary relationship with genes from starfish than with those of other selected species. Besides, we found that rAjPMK synthesized mevalonate-5-diphosphate, interacted either directly or indirectly with crucial pattern recognition receptors (PRRs) and was regulated by immune-related processes, including antioxidative reactions, stress resistance responses and enzyme hydrolysis. Moreover, AjPMK also interacted with farnesyl pyrophosphate synthase, an enzyme reported to be involved in saponin biosynthesis. Together, our findings implied that AjPMK may be directly involved in saponin biosynthesis and the regulation of various innate immune processes.

Sea Cucumber Body Vesicular Syndrome Is Driven by the Pond Water Microbiome via an Altered Gut Microbiota.

Apostichopus japonicus (sea cucumber) is one of the most valuable aquaculture species in China; however, different diseases can limit its economic development. Recently, a novel disease, body vesicular syndrome (BVS), was observed in A. japonicus aquaculture. Diseased animals displayed no obvious phenotypic characteristics; however, after boiling at the postharvest stage, blisters, lysis, and body ruptures appeared. In this study, a multiomics strategy incorporating analysis of the gut microbiota, the pond microbiome, and A. japonicus genotype was established to investigate BVS. Detailed analyses of differentially expressed proteins (DEPs) and metabolites suggested that changes in cell adhesion structures, caused by disordered fatty acid ß-oxidation mediated by vitamin B5 deficiency, could be a putative BVS mechanism. Furthermore, intestinal dysbacteriosis due to microbiome variations in pond water was considered a potential reason for vitamin B5 deficiency. Our BVS index, based on biomarkers identified from the A. japonicus gut microbiota, was a useful tool for BVS diagnosis. Finally, vitamin B5 supplementation was successfully used to treat BVS, suggesting an association with BVS etiology. IMPORTANCE Body vesicular syndrome (BVS) is a novel disease in sea cucumber aquaculture. As no phenotypic features are visible, BVS is difficult to confirm during aquaculture and postharvest activities, until animals are boiled. Therefore, BVS could lead to severe economic losses compared with other diseases in sea cucumber aquaculture. In this study, for the first time, we systematically investigated BVS pathogenesis and proposed an effective treatment for the condition. Moreover, based on the gut microbiota, we established a noninvasive diagnostic method for BVS in sea cucumber.

Exploiting the gut microbiota to predict the origins and quality traits of cultured sea cucumbers.

Nowadays, the true economic and nutritional value of food is underpinned by both origin and quality traits, more often expressed as increased quality benefits derived from the origin source. Gut microbiota contribute to food metabolism and host health, therefore, it may be suitable as a qualifying indicator of origin and quality of economic species. Here, we investigated relationships between the gut microbiota of the sea cucumber (Apostichopus japonicus), a valuable aquaculture species in Asia, with their origins and quality metrics. Based on data from 287 intestinal samples, we generated the first biogeographical patterns for A. japonicus gut microbiota from origins across China. Importantly, A. japonicus origins were predicted using the random forest model that was constructed using 20 key gut bacterial genera, with 97.6% accuracy. Furthermore, quality traits such as saponin, fat and taurine were also successfully predicted by random forest models based on gut microbiota, with approximately 80% consistency between predicted and true values. We showed that substantial variations existed in the gut microbiota and quality variables in A. japonicus across different origins, and we also demonstrated the great potential of gut microbiota to track A. japonicus origins and predict their quality traits.

Proteomics reveals gender differences in physiological characteristics of the gonads and tube feet of the sea cucumber, Apostichopus japonicus.

The sea cucumber Apostichopus japonicus is an important aquaculture species in China because of its high nutritional and medicinal values. Gender, as a factor affecting the physiology of organisms, is always considered when improving the breeding efficiency of economically important animals. In the present study, protein expression profiles of the gonads and tube feet of male and female A. japonicus were investigated using a comparative proteomics approach. A set of 7499 proteins were identified, which covered a broad range of functions based on function annotations. A significant difference in protein expression profiles was observed between the gonads and tube feet of A. japonicus; gonads showed more apparent gender differences than tube feet. Moreover, the findings revealed that male A. japonicus had more specific functions and most of these functions were associated with energy consumption. Further analyses suggested that the regulation of ERK activity and the capacity of tyrosine production and virus immunity might be more powerful in male and female A. japonicus, respectively. Some candidate proteins were also recognized as potential targets for gender identification of A. japonicus. Overall, our study provides new insights into the understanding of molecular mechanisms underlying gender-based physiological differences in A. japonicus. SIGNIFICANCE: The current study aimed to reveal gender differences in the physiological characteristics of gonads and tube feet of the sea cucumber A. japonicus. To the best of our knowledge, this is the first proteomics study to analyze the differences in the protein expression profiles of external organs between male and female A. japonicus. The analysis revealed gender differences in the protein expression profiles of both gonads and tube feet of A. japonicus, and the gender differences in gonads were quite apparent. Moreover, according to the recognition of differentially expressed proteins and the enrichment analyses based on Kyoto Encyclopedia of Genes and Genomes, a draft view of how the physiological functions of A. japonicus were affected by gender was obtained. Male A. japonicus could have more specific functions related to energy consumption than females. The regulation of ERK activity and virus immunity might be more robust in male and female A. japonicus, respectively. Some candidate proteins were also recognized as potential targets for gender identification of A. japonicus. The findings presented here will improve the understanding of researchers about the molecular mechanisms underlying gender-based differences in A. japonicus and contribute to the meticulous breeding of A. japonicus.

Divergent metabolic responses to sex and reproduction in the sea cucumber Apostichopus japonicus.

The sea cucumber Apostichopus japonicus is an economically important marine organism, and its aquaculture has rapidly developed in China. The very large market demand puts forward higher requirements for the economically efficient breeding of sea cucumbers. Sex and the associated reproductive processes have been reported to affect the physiological characteristics of sea cucumbers. However, little is known about the metabolism differences that related to sex and the associated reproductive processes and their potential effects on the efficiency of A. japonicus aquaculture. In this study, ultra-performance liquid chromatography was applied to investigate the variations in metabolic profiles in cell-free coelomic fluids (CCFs) of sea cucumbers of different sexes and reproductive states. A total of 4435 metabolites were detected, and the metabolic profiles of A. japonicus were significantly affected by both sexes and reproductive process. The differentially abundant metabolites in CCFs of A. japonicus of different sexes and reproductive states were also screened and analyzed. The findings revealed that unsaturated fatty acid synthesis and phenylalanine metabolism were the most significantly changed pathways. Moreover, the weakest ability to synthesize capsaicin using phenylalanine was found in A. japonicus after spawning. Our study provides new insights into the metabolic response of A. japonicus during the reproductive process, and also provides valuable references for the economically efficient breeding of A. japonicus.

Oleanane-type saponins and prosapogenins from Albizia julibrissin and their cytotoxic activities.

Two undescribed oleanane-type saponins, julibrosides K-L, along with three undescribed oleanane-type prosapogenins, julibrosides M-O, were isolated from the stem bark of Albizia julibrissin Durazz. and the mild alkaline hydrolysate of the total saponin, respectively. Their structures were established by extensive analysis of 1D and 2D NMR experiments (COSY, TOCSY, HSQC, HMBC, and HSQC-TOCSY) and mass spectrometry. Furthermore, the cytotoxic activities of the isolated compounds against BGC-823, A549, HCT-116, and HepG2 cell lines were evaluated, and julibroside L showed significant cytotoxic activities against the four cancer cell lines with IC50 values of 5.77, 4.80, 4.26, and 4.93 µM, respectively.

Proteomics reveals the gender differences in humoral immunity and physiological characteristics associated with reproduction in the sea cucumber Apostichopus japonicus.

Due to the importance of characteristics associated with the immunity and physiology of organisms, gender is always taken into account when improving the breeding efficiency of economically important animals. Protein variations in the cell-free ceolomic fluid (CCF) of male and female Apostichopus japonicus before and after spawning were investigated using a comparative proteomic approach, for the purpose of understanding the influence of gender and gender associated reproductive processes on humoral immunity and physiological characteristics of the sea cucumber, A. japonicus. A total of 6839 peptides were obtained and a set of 1466 proteins were identified in the CCF of male and female A. japonicus before and after spawning. Partial least squares discrimination analysis indicated that protein expression profiles in the CCF of A. japonicus were significantly affected by both gender and reproductive processes. In addition, the differentially expressed proteins (DEPs) in the CCF of different genders and reproductive stages of A. japonicus were determined and immune-related proteins in these DEPs were screened. Findings revealed that the alternative pathway of complement activation in both male and female A. japonicus before spawning might take precedence over that after spawning. Further KEGG enrichment analyses of DEPs suggested that the capacity of muscle contraction in A. japonicus females, and food digestion, nutrition absorption, epithelial differentiation as well as growth in A. japonicus males might be enhanced after spawning. Further, compared with A. japonicus females, males may have an advantage in growth in the stages following spawning. Besides, according to KEGG annotation related to metabolism, arginine biosynthesis and purine metabolism were dominant in the CCF of male and female A. japonicus, respectively, suggesting notable metabolic differences in female and male A. japonicus. Overall, our study provides new insight into the understanding of molecular mechanisms underlying gender-determined immune and physiological differences in A. japonicus. SIGNIFICANCE: The current study aimed to reveal the gender differences in humoral immunity and physiological characteristics associated with reproduction in the sea cucumber Apostichopus japonicus. To the best of our knowledge, this is the first proteomic study analyzing the differences in protein profile between male and female A. japonicus. By analyzing the expression differences of the proteome via label-free proteomic technology, we revealed that both gender and reproduction could alter the protein composition and abundance in the cell-free colemic fluid (CCF) of A. japonicus. Based on the recognition of differentially expressed proteins (DEPs) related to immune function among the CCFs, shifts in humoral immunity of male and female A. japonicus that underwent reproduction were obtained. Moreover, according to the enrichment analyses of DEPs based on Kyoto Encyclopedia of Genes and Genomes, a draft view of how the male and female A. japonicus affected by reproduction physiologically was drawn. The results suggested that the alternative pathway of complement activation in both male and female A. japonicus were weakened after spawning. In addition, after reproduction, the capacity of muscle contraction might be enhanced in female A. japonicus, and the nutrients consumption, epithelial differentiation and growth might be strengthened in males. Further, ipath analysis indicated that A. japonicus with different genders also had notable differences in metabolism that related to arginine biosynthesis and purine metabolism. The findings presented here will improve the cognition of researchers in the molecular mechanism underlying the gender-determined immune and phyisological differences of A. japonicus and contribute for the meticulous breeding of A. japonicus.

Three new triterpenoid saponins from Albizia julibrissin.

Three new triterpenoid saponins, julibrosides A5-A7 (1-3), together with five known saponins (4-8), were isolated from the stem bark of Albizia julibrissin. Their structures were elucidated on the basis of extensive spectroscopic data analysis of MS, 1D and 2D NMR, and chemical methods. Compounds 7 and 8 were isolated from the genus Albizia for the first time. The new compounds showed no cytotoxicity and anti-inflammatory activity.

Influence of Nonfused Cores on the Photovoltaic Performance of Linear Triphenylamine-Based Hole-Transporting Materials for Perovskite Solar Cells.

The core plays a crucial role in achieving high performance of linear hole transport materials (HTMs) toward the perovskite solar cells (PSCs). Most studies focused on the development of fused heterocycles as cores for HTMs. Nevertheless, nonfused heterocycles deserve to be studied since they can be easily synthesized. In this work, we reported a series of low-cost triphenylamine HTMs (M101-M106) with different nonfused cores. Results concluded that the introduced core has a significant influence on conductivity, hole mobility, energy level, and solubility of linear HTMs. M103 and M104 with nonfused oligothiophene cores are superior to other HTMs in terms of conductivity, hole mobility, and surface morphology. PSCs based on M104 exhibited the highest power conversion efficiency of 16.50% under AM 1.5 sun, which is comparable to that of spiro-OMeTAD (16.67%) under the same conditions. Importantly, the employment of M104 is highly economical in terms of the cost of synthesis as compared to that of spiro-OMeTAD. This work demonstrated that nonfused heterocycles, such as oligothiophene, are promising cores for high performance of linear HTMs toward PSCs.

Total saponins of Albiziae Cortex show anti-hepatoma carcinoma effects by inducing S phase arrest and mitochondrial apoptosis pathway activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Albiziae Cortex (AC) is a widely used traditional medicine in China. It is possess various properties to treat insomnia, traumatic injuries, diuresis, sthenia, and confusion. Total saponins of Albiziae Cortex (TSAC) are the most abundant bioactive components of AC, which were reported to show significant anti-tumor effects in vivo and in vitro. But the underlying mechanism of TSAC remained to be revealed. AIM OF STUDY: In this study, we investigated the anti-hepatoma carcinoma effects and the potential mechanism of TSAC in vivo and in vitro. MATERIALS AND METHODS: We first purified TSAC from crude extracts and characterized the major bioactive compounds by high performance liquid chromatography (HPLC). Effects of TSAC on viability of various hepatoma carcinoma cell lines were measured by MTT. Inhibition on cell proliferation was analysed using colony formation assay. Cell cycle distribution was revealed by flow cytometry. The apoptotic cells were observed by Hoechst 33258 staining and acridine orange (AO)/ethidium bromide (EB) double staining. Microstructures of apoptotic cells were examined by Transmission electron microscopy (TEM). The mitochondrial membrane potential were determined by JC-1 staining. Western blot was used to investigate the effects of TSAC on apoptosis-related proteins, B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax), and S-phase related protein cyclin A, cyclin E and cyclin-dependent kinases 2 (CDK2). Effects on tumor growth was assessed by H22-bearing ICR mice. RESULTS: TSAC significantly decreased the hepatoma carcinoma cell viability and inhibited HepG2 cell colony formation in a concentration-dependent manner. We also found that TSAC inhibited HepG2 cell growth via induction of S phase arrest. Further study showed that TSAC significantly down-regulated the expressions of cyclin A, cyclin E and CDK2 in HepG2 cells. Meanwhile, TSAC could effectively induce mitochondria-dependent caspase apoptosis pathway activation. Furthermore, TSAC increased the expression of pro-apoptotic protein Bax and decreased the expression of anti-apoptotic protein Bcl-2. In vivo assay showed that the anti-tumor effects of TSAC were significantly augmented without increasing toxicity in H22-bearing ICR mice. CONCLUSION: TSAC could inhibit cell proliferation through inducing S phase arrest and activate cell apoptosis via mitochondria-dependent apoptosis pathway. Therefore, TSAC could be a promising agent in clinical trials for anti-hepatoma carcinoma treatment.

Cytotoxic oleanane triterpenoid saponins from Albizia julibrissin.

Bioassay-guided fractionation of the ethanolic extract of the stem bark of Albizia julibrissin led to the isolation of ten new oleanane-type triterpenoid saponins, julibrosides J37-J46 (1-10), along with six known analogues (11-16). In addition, 11 prosapogenins (17-27) were prepared by mild or strong alkaline hydrolysis of the total saponin. The structures of 1-27 were determined by spectroscopic and chemical means, and their cytotoxicities against four human cancer cell lines, BGC-823, A549, HCT-116, and HepG2 were evaluated. Compounds 5-16 exhibited significant inhibitory activity with IC50 values ranging from 2.59 to 9.30µM, and 8 turned out to be the most active compound with all IC50 values <5µM. A preliminary structure-activity relationship of these saponins clearly indicated that the outer monoterpenoid moiety (MT') is a crucial substituent for cytotoxicity, and the linkage sites of the MT' unit greatly influenced the activity. It could also be inferred that the existence of 16-OH of the aglycone almost has no effect on cytotoxicity and the N-acetyl-glucosamine moiety at C-3 seems to enhance activity.

[Isolation of the promoter region of HAK gene from Aeluropus littoralis and functional analysis in rice].

The AlHAK1 gene encoding a high-affinity K+ transporter was isolated from Aeluropus littoralis (Gouan) Parl, a graminaceous halophyte, and plays a crucial role in nutrition and ion homeostasis in plant cell. To investigate the regulation role of AlHAK1 on the transcriptional level, an about 1.3 kb 5'-flanking region of the AlHAK1 gene containing a putative promoter was cloned by genome walking method. Cis-regulatory elements analysis showed AlHAK1-promoter region contained typical TATA and CAAT boxes, and some growth and development relative motifs, as well as environmental re-sponsive elements. To reveal the function and regulating role, the AlHAK1 promoter was fused to the ß-glucuronidase (GUS) reporter gene in the pCAMBIA1301 vector and introduced into rice via Agrobacterium-mediated transformation. Histo-chemical staining indicated that the GUS expression directed by AlHAK1 promoter was observed in leaves, stems, roots, anther, lemma, and palea. GUS quantitative fluorometric analysis indicated that GUS activity directed by AlHAK1 promoter was lower than CaMV35S and Ubiquitin constitutive promoters; however, in the roots and stems the GUS activity was rela-tively high and displayed a tissue-specific expression pattern. Under ABA, high temperature or drought stress, the GUS activity directed by AlHAK1 promoter was inducible in the roots and stems, suggesting the elements of HSE (-682 bp) and MybBS (-1 268 bp) might play a role in the inducible regulation.