Li Guohui,Song Cheng,Manzoor Muhammad Aamir,Li Daoyuan,Cao Yunpeng,Cai Yongping
Background The enzyme phenylalanine ammonia lyase (PAL) controls the transition from primary to secondary metabolism by converting L-phenylalanine (L-Phe) to cinnamic acid. However, the function of PAL in pear plants ( Pyrus bretschneideri ) has not yet been fully elucidated. Results We identified three PAL genes ( PbPAL1 , PbPAL2 and PbPAL3 ) from the pear genome by exploring pear genome databases. The evolutionary tree revealed that three PbPAL s were classified into one group. We expressed PbPAL1 and PbPAL2 recombinant proteins, and the purified PbPAL1 and PbPAL2 proteins showed strict substrate specificity for L-Phe, no activity toward L-Tyr in vitro, and modest changes in kinetics and enzyme characteristics. Furthermore, overexpression of PbAL1 and PbPAL1 -RNAi, respectively, and resulted in significant changes in stone cell and lignin contents in pear fruits. The results of yeast one-hybrid (Y1H) assays that PbWLIM1 could bind to the conserved PAL box in the PbPAL promoter and regulate the transcription level of PbPAL2. Conclusions Our findings not only showed PbPAL’s potential role in lignin biosynthesis but also laid the foundation for future studies on the regulation of lignin synthesis and stone cell development in pear fruit utilizing molecular biology approaches.
Summary Stone cells are often present in pear fruit, and they can seriously affect the fruit quality when present in large numbers. The plant growth regulator NAA, a synthetic auxin, is known to play an active role in fruit development regulation. However, the genetic mechanisms of NAA regulation of stone cell formation are still unclear. Here, we demonstrated that exogenous application of 200 μ M NAA reduced stone cell content and also significantly decreased the expression level of PbrNSC encoding a transcriptional regulator. PbrNSC was shown to bind to an auxin response factor, PbrARF13. Overexpression of PbrARF13 decreased stone cell content in pear fruit and secondary cell wall (SCW) thickness in transgenic Arabidopsis plants. In contrast, knocking down PbrARF13 expression using virus-induced gene silencing had the opposite effect. PbrARF13 was subsequently shown to inhibit PbrNSC expression by directly binding to its promoter, and further to reduce stone cell content. Furthermore, PbrNSC was identified as a positive regulator of PbrMYB132 through analyses of co-expression network of stone cell formation-related genes. PbrMYB132 activated the expression of gene encoding cellulose synthase ( PbrCESA4b/7a/8a) and lignin laccase ( PbrLAC5 ) binding to their promotors. As expected, overexpression or knockdown of PbrMYB132 increased or decreased stone cell content in pear fruit and SCW thickness in Arabidopsis transgenic plants. In conclusion, our study shows that the ‘PbrARF13-PbrNSC-PbrMYB132’ regulatory cascade mediates the biosynthesis of lignin and cellulose in stone cells of pear fruit in response to auxin signals and also provides new insights into plant SCW formation.
Ke Le Yan,Hong Xing Zhang,Ying Li,Bing Zou,An Shan Xiao,Ming Ma
A new kind of kinetic hydrate inhibitor (KHI) named KL-1 as the ramification of poly (N-vinyl caprolactam) (PVCap), was synthesized successfully by use of precipitation polymerization. The hydrate inhibition performance of KL-1 was assessed in a high pressure sapphire cell, and the onset time of hydrate formation and maximum subcooling were determined by the visual observation method and compared with the commercial KHIs, including Inhibex 501 and VC-713. Meanwhile, the synergic effect between ethanol and KL-1 developed was also studied in this work. The experimental results show that the onset time of KL-1 measured increases with the increase of the dosage and decrease of subcooling. Compared with the system without kinetic hydrate inhibitor, the morphological behavior of hydrate crystals in the systems containing KL-1 is different, and the hydrate crystals only grow to the gas phase with the hydrate formation. Additionally, based on the measurement of inhibition time, the inhibition performance of KL-1 is superior to Inhibex 501 and VC-713, and shows higher maximum subcooling at the similar conditions. Finally, we also demonstrated that ethanol can be used as synergist to improve the performance of KL-1 remarkably at suitable dosage.
危险品化学品经营许可证（带存储）