SINAT E3 Ubiquitin Ligases Mediate FREE1 and VPS23A Degradation to Modulate Abscisic Acid Signaling.

In plants, the ubiquitin-proteasome system, endosomal sorting, and autophagy are essential for protein degradation; however, their interplay remains poorly understood. Here, we show that four Arabidopsis (Arabidopsis thaliana) E3 ubiquitin ligases, SEVEN IN ABSENTIA OF ARABIDOPSIS THALIANA1 (SINAT1), SINAT2, SINAT3, and SINAT4, regulate the stabilities of FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING1 (FREE1) and VACUOLAR PROTEIN SORTING23A (VPS23A), key components of the endosomal sorting complex required for transport-I, to modulate abscisic acid (ABA) signaling. GFP-SINAT1, GFP-SINAT2, and GFP-SINAT4 primarily localized to the endosomal and autophagic vesicles. SINATs controlled FREE1 and VPS23A ubiquitination and proteasomal degradation. SINAT overexpressors showed increased ABA sensitivity, ABA-responsive gene expression, and PYRABACTIN RESISTANCE1-LIKE4 protein levels. Furthermore, the SINAT-FREE1/VPS23A proteins were codegraded by the vacuolar pathway. In particular, during recovery post-ABA exposure, SINATs formed homo- and hetero-oligomers in vivo, which were disrupted by the autophagy machinery. Taken together, our findings reveal a novel mechanism by which the proteasomal and vacuolar turnover systems regulate ABA signaling in plants.

Integrated Transcriptomic and Metabolomic Analysis Reveals the Mechanism of Gibberellic acid Regulates the Growth and Flavonoid Synthesis in Phellodendron chinense Schneid Seedlings.

The phytohormone gibberellic acids (GAs) play a crucial role in the processes of growth, organ development, and secondary metabolism. However, the mechanism of exogenous GA3 regulating the growth and flavonoid synthesis in Phellodendron chinense Schneid (P. chinense Schneid) seedlings remains unclear. In this study, the physicochemical properties, gene expression level, and secondary metabolite of P. chinense Schneid seedlings under GA3 treatment were investigated. The results showed that GA3 significantly improved the plant height, ground diameter, fresh weight, chlorophyll content, soluble substance content, superoxide dismutase, and peroxidase activities. This was accompanied by elevated relative expression levels of Pc(S)-GA2ox, Pc(S)-DELLA, Pc(S)-SAUR50, Pc(S)-PsaD, Pc(S)-Psb 27, Pc(S)-PGK, Pc(S)-CER3, and Pc(S)-FBA unigenes. Conversely, a notable reduction was observed in the carotenoid content, catalase activity and the relative expression abundances of Pc(S)-KAO, Pc(S)-GID1/2, and Pc(S)-GH 3.6 unigenes in leaves of P. chinense Schneid seedlings (p < 0.05). Furthermore, GA3 evidently decreased the contents of pinocembrin, pinobanksin, isosakuranetin, naringin, naringenin, (-)-epicatechin, tricetin, luteolin, and vitexin belonged to flavonoid in stem bark of P. chinense Schneid seedlings (p < 0.05). These results indicated that exogenous GA3 promoted growth through improving chlorophyll content and gene expression in photosynthesis and phytohormone signal pathway and inhibited flavonoid synthesis in P. chinense Schneid seedlings.

Adaptability of Klebsiella pneumoniae 2e, a Newly Isolated 1,3-Propanediol-Producing Strain, to Crude Glycerol as Revealed by Genomic Profiling.

Crude glycerol is largely generated as the main by-product of the biodiesel industry and is unprofitable for industrial application without costly purification. The direct bioconversion of crude glycerol into 1,3-propanediol (1,3-PDO) by microorganisms is a promising alternative for effective and economic utilization. In this study, Klebsiella pneumoniae 2e was newly isolated for the conversion of crude glycerol into 1,3-PDO. Batch fermentation analysis confirmed that crude glycerol and its main impurities had slight impacts on the growth, key enzyme activity, and 1,3-PDO production of K. pneumoniae 2e. The 1,3-PDO yield from crude glycerol by K. pneumoniae 2e reached 0.64 mol 1,3-PDO/mol glycerol, which was higher than that by most reported 1,3-PDO-producing Klebsiella strains. Genomic profiling revealed that K. pneumoniae 2e possesses 30 genes involved in glycerol anaerobic metabolism and 1,3-PDO biosynthesis. Quantitative real-time PCR analysis of these genes showed that the majority of the genes encoding the key enzymes for glycerol metabolism and 1,3-PDO biosynthesis were significantly upregulated during culture in crude glycerol relative to that in pure glycerol. Further comparative genomic analysis revealed a novel glycerol uptake facilitator protein in K. pneumoniae 2e and a higher number of stress response proteins than in other Klebsiella strains. This work confirms the adaptability of a newly isolated 1,3-PDO-producing strain, K. pneumoniae 2e, to crude glycerol and provides insights into the molecular mechanisms involved in its crude glycerol tolerance, which is valuable for industrial 1,3-PDO production from crude glycerol.IMPORTANCE The rapid development of the biodiesel industry has led to tremendous crude glycerol generation. Due to the presence of complex impurities, crude glycerol has low value for industry without costly purification. Obtaining novel microorganisms capable of direct and efficient bioconversion of crude glycerol to value-added products has great economic potential for industrial application. In this work, we characterized a newly isolated strain, Klebsiella pneumoniae 2e, with the capacity to efficiently produce 1,3-propanediol (1,3-PDO) from crude glycerol and demonstrated its adaptation to crude glycerol. Our work provides insights into the molecular mechanisms of K. pneumoniae 2e adaptation to crude glycerol and the expression patterns of its genes involved in 1,3-PDO biosynthesis, which will contribute to the development of industrial 1,3-PDO production from crude glycerol.

Expression of cellobiose dehydrogenase gene in Aspergillus niger C112 and its effect on lignocellulose degrading enzymes.

Cellobiose dehydrogenase (CDH) is one of the cellulase auxiliary proteins, which is widely used in the field of biomass degradation. However, how to efficiently and cheaply apply it in industrial production still needs further research. Aspergillus niger C112 is a significant producer of cellulase and has a relatively complete lignocellulose degradation system, but its CDH activity was only 3.92 U. To obtain a recombinant strain of A. niger C112 with high cellulases activity, the CDH from the readily available white-rot fungus Grifola frondose had been heterologously expressed in A. niger C112, under the control of the gpdA promoter. After cultivation in the medium with alkali-pretreated poplar fiber as substrate, the enzyme activity of recombinant CDH reached 36.63 U/L. Compared with the original A. niger C112, the recombinant A. niger transformed with Grifola frondosa CDH showed stronger lignocellulase activity, the activities of cellulases, ß-1, 4-glucosidase and manganese peroxidase increased by 28.57, 35.07 and 121.69%, respectively. The result showed that the expression of the gcdh gene in A. niger C112 could improve the activity of some lignocellulose degrading enzymes. This work provides a theoretical basis for the further application of gcdh gene in improving biomass conversion efficiency.

Optimization of alkali-treated poplar fiber saccharification using metal ions and surfactants.

In this study, contrary to untreated poplar fiber, processing of alkali-treated poplar fiber was optimized for the enzymatic saccharification. Considering reducing sugar content as the evaluation index, pH, temperature, time, amount of enzyme, and rotational speed of shaker were standardized to optimize the sugar production by enzymatic hydrolysis. Using response surface methodology, the optimum technological condition of enzymatic hydrolysis was found to be utilizing 43 mg cellulase at 46 °C for 50 h. At this, the sugar conversion amount of NaOH or H2O2-NaOH pretreated poplar was 164.62 mg/g and 218.82 mg/g respectively. It was a corresponding increase of 446.73% or 626.75% than that of poplar fiber without a pretreatment. At a low concentration, metal ions and surfactants promoted the conversion of reducing sugar. Especially, 0.01 g/L Mn2+ and 0.50 g/L hexadecyl trimethyl ammonium bromide (CTAB) produced the best effect and increased the conversion rate of reducing sugar by 23.62% and 21.44% respectively. Also, the effect of the combination of metal ions and surfactants was better than that of a single accelerator. By improving the enzymatic process, these findings could enhance the utilization of poplar fiber for the production of reducing sugar.

Facile combination of beta-cyclodextrin host-guest recognition with exonuclease-assistant signal amplification for sensitive electrochemical assay of ochratoxin A.

Smartly coupling exonuclease-induced target recycling signal amplifications with ß-cyclodextrin host-guest recognition, a novel "signal-on" aptamer sensor for sensitive determination of ochratoxin A (OTA) was proposed for the first time. Firstly, the formation of double-strand DNA (dsDNA) was occurred by hybridizing OTA aptamer with its complementary DNA (cDNA) and as the probe DNA the cDNA at its 3' terminal was labeled with methylene blue (MB). Next, when OTA was present, the aptamer tended to form aptamer-OTA complex with conformation of G-quadruplex instead of aptamer-cDNA duplex, leading to thus the probe DNA separating from dsDNA complex. Then the RecJf exonuclease was added, demolishing partially G-quadruplex structure and releasing a certain number of OTA. Sequentially, those released OTA would continue to react with the rest of aptamer in dsDNA, drawn into development of a new round of G-quadruplex complex, where the target cycling was realized. Meanwhile, as a signal molecule, MB modified on cDNA was liberated along with the cDNA being digested into monoucleotides by RecJf exonuclease, capable of diffusing onto the electrode surface due to host-guest recognition with ß-cyclodextrin, whereupon the signal was enriched and yielded. In this way, cycles of target with continuous output of signal indicators were undergone, in which the detection of target was in return fulfilled with signal amplification owing to the joint endeavor of exonuclease and ß-cyclodextrin. Under the optimal conditions, the raising signal maintained a linear relation with the logarithm of the target concentrations ranging from 10 pg/mL to 10.0 ng/mL and the detection limit reached as low as 3 pg/mL. This brand-new strategy was simple and low-cost but satisfactory in terms of detection limit, range and sensitivity, in all possibility to be applied extensively for diverse targets detection by easily alternating the corresponding aptamers.