Banana MabHLH28 positively regulates the expression of softening-related genes to mediate fruit ripening independently or via cooperating with MaWRKY49/111.

Texture softening is a physiological indicator of fruit ripening, which eventually contributes to fruit quality and the consumer's acceptance. Despite great progress having been made in identification of the genes related to fruit softening, the upstream transcriptional regulatory pathways of these softening-related genes are not fully elucidated. Here, a novel bHLH gene, designated as MabHLH28, was identified because of its significant upregulation in banana fruit ripening. DAP-Seq analysis revealed that MabHLH28 bound to the core sequence of 'CAYGTG' presented in promoter regions of fruit softening-associated genes, such as the genes related to cell wall modification (MaPG3, MaPE1, MaPL5, MaPL8, MaEXP1, MaEXP2, MaEXPA2, and MaEXPA15) and starch degradation (MaGWD1 and MaLSF2), and these bindings were validated by EMSA and DLR assays. Transient overexpression and knockdown of MabHLH28 in banana fruit resulted in up- and down-regulation of softening-related genes, thereby hastening and postponing fruit ripening. Furthermore, overexpression of MabHLH28 in tomato accelerated the ripening process by elevating the accumulation of softening-associated genes. In addition, MabHLH28 showed interaction withMaWRKY49/111 and itself to form protein complexes, which could combinatorically strengthen the transcription of softening-associated genes. Taken together, our findings suggest that MabHLH28 mediates fruit softening by upregulating the expression of softening-related genes either alone or in combination with MaWRKY49/111.

High temperature elevates carotenoid accumulation of banana fruit via upregulation of MaEIL9 module.

Banana is a good source of carotenoids, which are bioactive metabolites with health beneficial properties for human. However, the molecular mechanism of carotenoid accumulation in banana fruit is largely unclear. In this study, we found that high temperature elevated carotenoid production in banana pulp, which is presumably due to upregulation of a subset of carotenogenic genes as well as a carotenoid biosynthesis regulator MaSPL16. Moreover, an ethylene signaling component MaEIL9 was identified, whose transcript and protein contents were also induced by high temperature. In addition, MaEIL9 positively regulates transcription of MaDXR1, MaPDS1, MaZDS1 and MaSPL16 through directly targeting their promoters. Overexpression of MaEIL9 in tomato fruit substantially increased the expression of carotenoid formation genes and elevated carotenoid content. Importantly, transiently silencing MaEIL9 in banana fruit weakened carotenoid production caused by high temperature. Taken together, these results indicate that high temperature induces carotenoid production in banana fruit, at least in part, through MaEIL9-mediated activation of MaDXR1, MaPDS1, MaZDS1 and MaSPL16 expression.

Methionine oxidation and reduction of the ethylene signaling component MaEIL9 are involved in banana fruit ripening.

The ethylene insensitive 3/ethylene insensitive 3-like (EIN3/EIL) plays an indispensable role in fruit ripening. However, the regulatory mechanism that links post-translational modification of EIN3/EIL to fruit ripening is largely unknown. Here, we studied the expression of 13 MaEIL genes during banana fruit ripening, among which MaEIL9 displayed higher enhancement particularly in the ripening stage. Consistent with its transcript pattern, abundance of MaEIL9 protein gradually increased during the ripening process, with maximal enhancement in the ripening. DNA affinity purification (DAP)-seq analysis revealed that MaEIL9 directly targets a subset of genes related to fruit ripening, such as the starch hydrolytic genes MaAMY3D and MaBAM1. Stably overexpressing MaEIL9 in tomato fruit hastened fruit ripening, whereas transiently silencing this gene in banana fruit retarded the ripening process, supporting a positive role of MaEIL9 in fruit ripening. Moreover, oxidation of methionines (Met-129, Met-130, and Met-282) in MaEIL9 resulted in the loss of its DNA-binding capacity and transcriptional activation activity. Importantly, we identified MaEIL9 as a potential substrate protein of methionine sulfoxide reductase A MaMsrA4, and oxidation of Met-129, Met-130, and Met-282 in MaEIL9 could be restored by MaMsrA4. Collectively, our findings reveal a novel regulatory network controlling banana fruit ripening, which involves MaMsrA4-mediated redox regulation of the ethylene signaling component MaEIL9.

Hydrolytic enzyme of cellulose for complex formulation applied research.

To improve the enzymatic hydrolytic efficiency and reduce the supplementation of enzymes, the mixture designed experimental approach was used to optimize the composition of enzyme mixture and promote the hydrolysis of ball-milled corn stover. From the experimental results, a synergistic effect was found when combinations of the three enzymes, two kinds of cellulases and a kind of xylanase, were used. The optimal hydrolysis of pretreated corn stover accorded with enzymes activity ration of FPU/CMCase/ß-glucosidase/xylanase = 4.4:1:75:829, and the hydrolysis efficiency of corn stover increased significantly compared with using individual enzyme. The results indicated that the mixture design experiment could be an effective tool for optimized enzyme mixture for lignocellulose hydrolysis.

Ball milling pretreatment of corn stover for enhancing the efficiency of enzymatic hydrolysis.

Ethanol can be produced from lignocellulosic biomass with the usage of ball milling pretreatment followed by enzymatic hydrolysis and fermentation. The sugar yields from lignocellulosic feed stocks are critical parameters for ethanol production process. The research results from this paper indicated that the yields of glucose and xylose were improved by adding any of the following dilute chemical reagents: H(2)SO(4), HCl, HNO(3), CH(3)COOH, HCOOH, H(3)PO(4), and NaOH, KOH, Ca(OH)(2), NH(3)·H(2)O in the ball milling pretreatment of corn stover. The optimal enzymatic hydrolysis efficiencies were obtained under the conditions of ball milling in the alkali medium that was due to delignification. The data also demonstrated that ball milling pretreatment was a robust process. From the microscope image of ball milling-pretreated corn stover, it could be observed that the particle size of material was decreased and the fiber structure was more loosely organized. Meanwhile, the results indicate that the treatment effect of wet milling is better than that of dry milling. The optimum parameters for the milling process were ball speed of 350 r/min, solid/liquid ratio of 1:10, raw material particle size with 0.5 mm, and number of balls of 20 (steel ball, Φ = 10 mm), grinding for 30 min. In comparison with water milling process, alkaline milling treatment could increase the enzymatic hydrolysis efficiency of corn stover by 110%; and through the digestion process with the combination of xylanase and cellulase mixture, the hydrolysis efficiency could increase by 160%.

Dilute sulfuric acid cycle spray flow-through pretreatment of corn stover for enhancement of sugar recovery.

A cycle spray flow-through reactor was designed and used to pretreat corn stover in dilute sulfuric acid medium. The dilute sulfuric acid cycle spray flow-through (DCF) process enhanced xylose sugar yields and cellulose digestibility while increasing the removal of lignin. Within the DCF system, the xylose sugar yields of 90-93% could be achieved for corn stover pretreated with 2% (w/v) dilute sulfuric acid at 95 degrees C during the optimal reaction time (90 min). The remaining solid residue exhibited enzymatic digestibility of 90-95% with cellulase loading of 60 FPU/g glucan that was due to the effective lignin removal (70-75%) in this process. Compared with flow-through and compress-hot water pretreatment process, the DCF method produces a higher sugar concentration and higher xylose monomer yield. The novel DCF process provides a feasible approach for lignocellulosic material pretreatment.