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Agricultural residues (e.g. corn stover (CS)) representing a huge lignocellulosic biomass waste are regarded as a promising renewable resource that can be converted to fuels and chemicals via biochemical route. Nevertheless, the unfavorable properties, such as low bulk density, contamination by microorganisms, fluffy and thereby difficult to handle, cause huge problems for biomass logistics and biomass conversion. Furthermore, traditional biomass pretreatment methods often use severe conditions, consume much energy, difficult to scale up and generate a feedstock with many toxic degradation products that inhibit fermentation. In this study, we developed a novel, low-cost and easy-to-implement pretreatment method: “Densifying Lignocellulosic biomass with acidic Chemicals (DLC)” on CS. The DLC-CS owning a uniform shape showed a bulk density 4 times higher compared to loose CS and was highly resistant to microbial contamination, which greatly facilitates biomass handling, transportation and storage. DLC-CS after regular steam autoclave treatment at 121 ○C exhibited high enzymatic digestibility and much higher fermentability compared to traditional dilute acid pretreatment. An ethanol titer as high as 68.1 g/L was achieved without washing or detoxification of the pretreated biomass. The superior performances of DLC for biomass logistics and biomass conversion render it very promising for industrial use.

Anthocyanins are common secondary metabolites in plants that impart red coloration to fruits and flowers. The important WRKY transcription factor family plays multifaceted roles in plant growth and development. In this study, we found a WRKY family gene, Pyrus bretschneideri WRKY75, that may be involved in anthocyanin synthesis in pear. Unlike Arabidopsis thaliana WRKY75, PbWRKY75 may be a positive regulator of anthocyanin synthesis. A transient expression assay indicated that PbWRKY75 promoted pear anthocyanin synthesis. The structural genes (PbANS, PbDFR, and PbUFGT) and positive regulators (PbMYB10 and PbMYB10b) of anthocyanin synthesis were significantly upregulated in the fruitlet skins of PbWRKY75-overexpressing "Zaosu" pears. Subsequently, yeast one-hybrid and dual-luciferase assays indicated that PbWRKY75 promoted PbDFR, PbUFGT, and PbMYB10b expression by activating their promoters. These results revealed that PbWRKY75 may promote the expression of both PbMYB10b and anthocyanin late biosynthetic genes (PbDFR and PbUFGT) by activating their promoters, thereby inducing anthocyanin synthesis in pear. This study enhanced our understanding of the mechanism of pear anthocyanin synthesis, which will be beneficial in the improvement of pear peel color.

Background Parthenocarpy results in traits attractive to both consumers and breeders, and it overcomes the obstacle of self-incompatibility in the fruit set of horticultural crops, including pear (Pyrus bretshneider). However, there is limited knowledge regarding the genetic and molecular mechanisms that regulate parthenogenesis. Results Here, in a transcriptional comparison between pollination-dependent fruit and GA4-induced parthenocarpy, PbCYP78A6 was identified and proposed as a candidate gene involved in parthenocarpy. PbCYP78A6 is similar to Arabidopsis thaliana CYP78A6 and highly expressed in pear hypanthia. The increased PbCYP78A6 expression, as assessed by RT-qPCR, was induced by pollination and GA4 exposure. The ectopic overexpression of PbCYP78A6 contributed to parthenocarpic fruit production in tomato. The PbCYP78A6 expression coincided with fertilized and parthenocarpic fruitlets development and the expression of fruit development-related genes as assessed by cytological observations and RT-qPCR, respectively. PbCYP78A6 RNA interference and overexpression in pear calli revealed that the gene is an upstream regulator of specific fruit development-related genes in pear. Conclusions Our findings indicate that PbCYP78A6 plays a critical role in fruit formation and provide insights into controlling parthenocarpy.

Cellulosic ethanol has attracted much attention as it is of great benefit to social and environmental sustainability. However, the adverse properties of lignocellulosic biomass (i.e. low bulk density, fluffy/difficult to handle, and being easily contaminated by microbes), and issues related to biomass pretreatment (i.e. high energy consumption, difficulty in scale-up and low fermentability of pretreated biomass), largely impede the commercialization of cellulosic ethanol. To address these issues, we invented a novel pretreatment method, Densifying Lignocellulosic biomass with alkaline Chemicals (DLC), and studied DLC on corn stover (CS). DLC-CS has high density and high durability, with contamination prevented and sugar fully preserved, which greatly facilitates biomass logistics. DLC-CS showed high enzymatic digestibility and high fermentability after storage. DLC-CS containing calcium hydroxide yielded 21.4 g of ethanol per 100 g of CS, which was further enhanced to 25.3 g of ethanol per 100 g of CS using a regular steam autoclave. An ethanol titer as high as 70.6 g L−1 was achieved, for the first time, without washing or detoxification of the pretreated biomass. These promising results demonstrated the great potential of DLC pretreatment for lignocellulosic biofuel production.

Background Decrease in anthocyanin content results in the loss of red color in leaves, petals and receptacles during development. The content of anthocyanin was affected by the biosynthesis and degradation of anthocyanin. Compared with the known detailed mechanism of anthocyanin biosynthesis, the degradation mechanism is not fully investigated. It is vital to study the degradation mechanism of anthocyanin in pear for promoting the accumulation of anthocyanin and inhibiting the red fading in pear. Results Here, we reported that laccase encoded by PbLAC4-like was associated with anthocyanin degradation in pear. The expression pattern of PbLAC4-like was negatively correlated with the content of anthocyanin during the color fading process of pear leaves, petals and receptacles. Phylogenetic analysis and sequence alignment revealed that PbLAC4-like played a vital role in anthocyanin degradation. Thus, the degradation of anthocyanin induced by PbLAC4-like was further verified by transient assays and prokaryotic expression. More than 80% of anthocyanin compounds were degraded by transiently over-expressed PbLAC4-like in pear fruitlet peel. The activity of crude enzyme to degrade anthocyanin in leaves at different stages was basically consistent with the expression of PbLAC4-like. The anthocyanin degradation ability of prokaryotic induced PbLAC4-like protein was also verified by enzyme activity assay. Besides, we also identified PbMYB26 as a positive regulator of PbLAC4-like. Yeast one-hybrid and dual luciferase assay results showed that PbMYB26 activated PbLAC4-like expression by directly binding to the PbLAC4-like promoter. Conclusions Taken together, the PbLAC4-like activated by PbMYB26, was involved in the degradation of anthocyanin, resulting in the redness fading in different pear tissues.

Seedless fruit is a feature appreciated by consumers. The ovule abortion process is highly orchestrated and controlled by numerous environmental and endogenous signals. However, the mechanisms underlying ovule abortion in pear remain obscure. Here, we found that gibberellins (GAs) have diverse functions during ovules development between seedless pear ‘1913’ and seeded pear, and that GA4+7 activates a potential programmed cell death process in ‘1913’ ovules. After hormone analyses, strong correlations were determined among jasmonic acid (JA), ethylene and salicylic acid (SA) in seedless and seeded cultivars, and GA4+7 treatments altered the hormone accumulation levels in ovules, resulting in significant correlations between GA and both JA and ethylene. Additionally, SA contributed to ovule abortion in ‘1913’. Exogenously supplying JA, SA or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid promoted ‘Bartlett’ seed death. The regulatory mechanism in which ethylene controls ovule death has been demonstrated, therefore, JA’s role in regulating ‘1913’ ovule abortion was investigated. A further study identified that the JA signaling receptor MYC2 bound the SENESCENCE-ASSOCIATED 39 promoter and triggered its expression to regulate ovule abortion. Thus, we established ovule abortion-related relationships between GA and the hormones JA, ethylene and SA, and we determined their synergistic functions in regulating ovule death.

Flavonoids play important roles in regulating plant growth and development. In this study, three kaempferol 3-O-glycosides were identified and mainly accumulated in flowers but not in leaves or fruits ofMalus. InMalus, flower petal color is normally white, but some genotypes have red flowers containing anthocyanin. Anthocyanin biosynthesis appears to be in competition with kaempferol 3-O-glycosides production and controlled by the biosynthetic genes. The white flowerMalusgenotypes had better-developed seeds than the red flower genotypes. In flowers, the overexpression ofMYB10inMalus domesticaenhanced the accumulation of anthocyanin, but decreased that of kaempferol 3-O-glycosides. After pollination the transgenic plants showed slower pollen tube growth and fewer developed seeds. Exogenous application of different flavonoid compounds suggested that kaempferol 3-O-glycosides, especially kaempferol 3-O-rhamnoside, regulated pollen tube growth and seed set rather than cyanidin or quercetin 3-O-glycosides. It was found that kaempferol 3-O-rhamnoside might regulate pollen tube growth through effects on auxin, the Rho of plants (ROP) GTPases, calcium and the phosphoinositides signaling pathway. With the inhibition of auxin transport, the transcription levels of Heat Shock Proteins (HSPs) and ROP GTPases were downregulated while the levels were not changed or even enhanced when blocking calcium signaling, suggesting that HSPs and ROP GTPases were downstream of auxin signaling, but upstream of calcium signaling. In summary, kaempferol glycoside concentrations in pistils correlated with auxin transport, the transcription of HSPs and ROP GTPases, and calcium signaling in pollen tubes, culminating in changes to pollen tube growth and seed set.

Understanding ripening and senescence processes in postharvest stored fruit is key to the identification and implementation of effective treatment methods. Here, we explored the effects of exogenous applications of melatonin (MT) and nitric oxide (NO) on ripening and softening processes in three cultivars of European pear ( Pyrus communis L.). The results showed that MT and NO played important roles in the two processes: they decreased the rate of upregulation of PcCel and PcPG, inhibited expression of ethylene synthetase genes ( PcACS and PcACO), and reduced rates of respiration and ethylene production. MT increased activity of NO synthase through upregulation of expression of PcNOS that subsequently led to an increase in NO content. However, when NO synthesis was inhibited, the delaying effect of MT on fruit senescence was almost eliminated. These findings indicate that MT acted on the upstream process of NO synthesis that then delayed senescence in pear fruit.

Lignin is the most abundant aromatic substrate on Earth and its valorization technologies are still under developed. Depolymerization and fragmentation are the predominant preparatory strategies for valorization of lignin to chemicals and fuels. However, due to the structural heterogeneity of lignin, depolymerization and fragmentation typically result in diverse product species, which require extensive separation and purification procedures to obtain target products. For lignin valorization, bacterial-based systems have attracted increasing attention because of their diverse metabolisms, which can be used to funnel multiple lignin-based compounds into specific target products. Here, recent advances in lignin valorization using bacteria are critically reviewed, including lignin-degrading bacteria that are able to degrade lignin and use lignin-associated aromatics, various associated metabolic pathways, and application of bacterial cultures for lignin valorization. This review will provide insight into the recent breakthroughs and future trends of lignin valorization based on bacterial systems.

Parthenocarpy, the productions of seedless fruit without pollination or fertilization, is a potentially desirable trait in many commercially grown fruits, especially in pear, which is self-incompatible. Phytohormones play important roles in fruit set, a process crucial for parthenocarpy. In this study, 2,4-dichlorophenoxyacetic acid (2,4-D), an artificially synthesized plant growth regulator with functions similar to auxin, was found to induce parthenocarpy in pear. Histological observations revealed that 2,4-D promoted cell division and expansion, which increased cortex thickness, but the effect was weakened by paclobutrazol (PAC), a gibberellin (GA) biosynthesis inhibitor. Phenotypic differences in pear may therefore be due to different GA contents. Hormone testing indicated that 2,4-D mainly induced the production of bioactive GA4 , rather than GA3. Three key oxidase genes function in the GA biosynthetic pathway: GA20ox, GA3ox and GA2ox. In a pear group treated with only 2,4-D, PbGA20ox2-like and PbGA3ox-1 were significantly upregulated. When treated with 2,4-D supplemented with PAC, however, expression levels of these genes were significantly downregulated. Additionally, PbGA2ox1-like and PbGA2ox2-like expression levels were significantly downregulated in pear treated with either 2,4-D only or 2,4-D supplemented with PAC. We thus hypothesize that 2,4-D can induce parthenocarpy by enhancing GA4 biosynthesis.

Alkali and acid pretreatment based processes for lignocellulosic biofuel production typically consume substantial amount of alkali and acid. In addition, alkali pretreatment generates compounds with high toxicity rendering washing/detoxification inevitable. In this work, alkali (AL) and dilute acid (DA) pretreatment based processes were compared side by side for ethanol production from corn stover (CS) using commercial enzymes and Zymomonas mobilis 8b strain. Next, AL and DA pretreated CS slurries (without removing liquid stream) were mixed to neutralize for enzymatic hydrolysis and ethanol fermentation. The digestibility and fermentability of the mixed pretreated biomass were systematically studied and compared to unmixed pretreated biomass. Results showed that mixed biomass process saved 40–50% alkali/acid use and enhanced fermentability compared to AL-CS. This work suggests that it could be beneficial to have both alkali and acid pretreatments operating in a biorefinery, which could accommodate various feedstocks.

The N-terminal of Myc-like basic helix-loop-helix transcription factors (bHLH TFs) contains an interaction domain, namely the MYB-interacting region (MIR), which interacts with the R2R3-MYB proteins to regulate genes involved in the anthocyanin biosynthetic pathway. However, the functions of MIR-domain bHLHs in this pathway are not fully understood. In this study, PbbHLH2 containing the MIR-domain was identified and its function investigated. The overexpression of PbbHLH2 in ”Zaosu” pear peel increased the anthocyanin content and the expression levels of late biosynthetic genes. Bimolecular fluorescence complementation showed that PbbHLH2 interacted with R2R3-MYB TFs PbMYB9, 10, and 10b in onion epidermal cells and confirmed that MIR-domain plays important roles in the interaction between the MIR-domain bHLH and R2R3-MYB TFs. Moreover, PbbHLH2 bound and activated the dihydroflavonol reductase promoter in yeast one-hybrid (Y1H) and dual-luciferase assays. Taken together these results suggested that the MIR domain of PbbHLH2 regulated anthocyanin biosynthesis in pear fruit peel.

The N-terminal of Myc-like basic helix-loop-helix transcription factors (bHLH TFs) contains an interaction domain, namely the MYB-interacting region (MIR), which interacts with the R2R3-MYB proteins to regulate genes involved in the anthocyanin biosynthetic pathway. However, the functions of MIR-domain bHLHs in this pathway are not fully understood. In this study, PbbHLH2 containing the MIR-domain was identified and its function investigated. The overexpression of PbbHLH2 in ”Zaosu” pear peel increased the anthocyanin content and the expression levels of late biosynthetic genes. Bimolecular fluorescence complementation showed that PbbHLH2 interacted with R2R3-MYB TFs PbMYB9, 10, and 10b in onion epidermal cells and confirmed that MIR-domain plays important roles in the interaction between the MIR-domain bHLH and R2R3-MYB TFs. Moreover, PbbHLH2 bound and activated the dihydroflavonol reductase promoter in yeast one-hybrid (Y1H) and dual-luciferase assays. Taken together these results suggested that the MIR domain of PbbHLH2 regulated anthocyanin biosynthesis in pear fruit peel.

Microbial lipids derived from lignocellulosic biomass are attractive feedstocks for biodiesel production. However, toxic degradation products generated by pretreatment greatly inhibited lipid fermentation. Herein, two pretreatment methods, i.e. densifying lignocellulosic biomass with sulfuric acid (DLC(sa)) and densifying lignocellulosic biomass with sulfuric acid followed by autoclave (DLCA(sa)), were developed on rice straw (RS) for lipid production. The conditions of DLCA(sa) pretreatment, including sulfuric acid dosage, dry biomass loading and autoclave time, were optimized by response surface methodology. High fermentable sugar yields with low inhibitor concentrations were obtained on DLC(sa) pretreated RS (DLC(sa)-RS) and DLCA(sa) pretreated RS (DLCA(sa)-RS) via enzymatic hydrolysis at a high solid loading of 30 wt%. Lipid titers of as high as 28.9 g/L and 31.0 g/L, with lipid yields of 0.22 g/g and 0.24 g/g consumed sugar, were achieved when RS was pretreated by DLC(sa) and DLCA(sa), respectively. This study demonstrated efficient microbial lipid production from lignocellulosic biomass without washing or detoxification steps.

Anthocyanin contributes to the coloration of pear fruit and enhances plant defenses. Members of the ethylene response factor (ERF) family play vital roles in hormone and stress signaling and are involved in anthocyanin biosynthesis. Here, PbERF22 was identified from the lanolin-induced red fruit of &lsquo, Zaosu&rsquo, pear (Pyrus bretschneideri Rehd.) using a comparative transcriptome analysis. Its expression level was up- and down-regulated by methyl jasmonate and 1-methylcyclopropene plus lanolin treatments, respectively, which indicated that PbERF22 responded to the jasmonate- and ethylene-signaling pathways. In addition, transiently overexpressed PbERF22 induced anthocyanin biosynthesis in &lsquo, fruit, and a quantitative PCR analysis further confirmed that PbERF22 facilitated the expression of anthocyanin biosynthetic structural and regulatory genes. Moreover, a dual luciferase assay showed that PbERF22 enhanced the activation effects of PbMYB10 and PbMYB10b on the PbUFGT promoter. Therefore, PbERF22 responses to jasmonate and ethylene signals and regulates anthocyanin biosynthesis. This provides a new perspective on the correlation between jasmonate&ndash, ethylene crosstalk and anthocyanin biosynthesis.

Fruit set and development occur following successful fertilization. Parthenocarpy, a valuable trait in some self-incompatible species, produces seedless fruit without fertilization. Gibberellin (GA) is a crucial hormone in fruit-set regulation and development. While investigating the development of parthenocarpy in pear (Pyrus bretschneideri Rehd.), we determined that GA 20-oxidases (GA20ox) may play key roles in seedless pear fruit development. Sequence analysis revealed three PbGA20ox genes: PbGA20ox1, PbGA20ox2, and PbGA20ox3. We analyzed the expression patterns of candidate genes and found that PbGA20ox2 levels significantly changed in pollinated fruits. Tissue-specific expression assays revealed that PbGA20ox2 is highly expressed in young fruit and leaves. Subcellular localization assays showed it was located in the cytoplasm, nucleus, and plasma membrane. Overexpressed PbGA20ox2 tomato plants were taller and had longer hypocotyls and internodes, and the emasculated flowers produced parthenocarpic fruit. In pear, the transient overexpression of PbGA20ox2 promoted fruit development and delayed the drop of nonpollinated fruit. Furthermore, the fruit of PbGA20ox2-overexpressing tomato and transient PbGA20ox2-overexpressing pear had increased GA4 (but not GA3 and GA1) contents. This result provided evidence that PbGA20ox2 was necessary for GA4-dependent pear fruit development. Our study revealed that PbGA20ox2 altered the GA biosynthetic pathway and enhanced GA4 synthesis, thereby promoting fruit set and parthenocarpic fruit development.

Anthocyanin contributes to the coloration of pear fruit and enhances plant defenses. Members of the ethylene response factor (ERF) family play vital roles in hormone and stress signaling and are involved in anthocyanin biosynthesis. Here, PbERF22 was identified from the lanolin-induced red fruit of ‘Zaosu’ pear (Pyrus bretschneideri Rehd.) using a comparative transcriptome analysis. Its expression level was up- and down-regulated by methyl jasmonate and 1-methylcyclopropene plus lanolin treatments, respectively, which indicated that PbERF22 responded to the jasmonate- and ethylene-signaling pathways. In addition, transiently overexpressed PbERF22 induced anthocyanin biosynthesis in ‘Zaosu’ fruit, and a quantitative PCR analysis further confirmed that PbERF22 facilitated the expression of anthocyanin biosynthetic structural and regulatory genes. Moreover, a dual luciferase assay showed that PbERF22 enhanced the activation effects of PbMYB10 and PbMYB10b on the PbUFGT promoter. Therefore, PbERF22 responses to jasmonate and ethylene signals and regulates anthocyanin biosynthesis. This provides a new perspective on the correlation between jasmonate–ethylene crosstalk and anthocyanin biosynthesis.

Anthocyanin biosynthesis exhibits a rhythmic oscillation pattern in some plants. To investigate the correlation between the oscillatory regulatory network and anthocyanin biosynthesis in pear, the anthocyanin accumulation and the expression patterns of anthocyanin late biosynthetic genes (ALBGs) were investigated in fruit skin of ‘Red Zaosu’ (Pyrus bretschneideri Rehd.). The anthocyanin accumulated mainly during the night over three continuous days in the fruit skin, and the ALBGs’ expression patterns in ‘Red Zaosu’ fruit skin were oscillatory. However, the expression levels of typical anthocyanin-related transcription factors did not follow this pattern. Here, we found that the expression patterns of four PbREVEILLEs (PbRVEs), members of a class of atypical anthocyanin-regulated MYBs, were consistent with those of ALBGs in ‘Red Zaosu’ fruit skin over three continuous days. Additionally, transient expression assays indicated that the four PbRVEs promoted anthocyanin biosynthesis by regulating the expression of the anthocyanin biosynthetic genes encoding dihydroflavonol-4-reductase (DFR) and anthocyanidin synthase (ANS) in red pear fruit skin, which was verified using a dual-luciferase reporter assay. Moreover, a yeast one-hybrid assay indicated that PbRVE1a, 1b and 7 directly bound to PbDFR and PbANS promoters. Thus, PbRVEs promote anthocyanin accumulation at night by up-regulating the expression levels of PbDFR and PbANS in ‘Red Zaosu’ fruit skin.

Fruit with stripes, which are generally longitudinal, can occur naturally, but the bioprocesses underlying this phenomenon are unclear. Previously, we observed an atypical anthocyanin distribution that caused red-striped fruit on the spontaneous pear bud sport “Red Zaosu” (Pyrus bretschneideri Rehd.). In this study, comparative transcriptome analysis of the sport and wild-type “Zaosu” revealed that this atypical anthocyanin accumulation was tightly correlated with abnormal overexpression of the gene-encoding gibberellin (GA) 2-beta-dioxygenase 8, PbGA2ox8. Consistently, decreased methylation was also observed in the promoter region of PbGA2ox8 from “Red Zaosu” compared with “Zaosu”. Moreover, the GA levels in “Red Zaosu” seedlings were lower than those in “Zaosu” seedlings, and the application of exogenous GA4 reduced abnormal anthocyanin accumulation in “Red Zaosu”. Transient overexpression of PbGA2ox8 reduced the GA4 level and caused anthocyanin accumulation in pear fruit skin. Moreover, the presence of red stripes indicated anthocyanin accumulation in the hypanthial epidermal layer near vascular branches (VBs) in “Red Zaosu”. Transient overexpression of PbGA2ox8 resulting from vacuum infiltration induced anthocyanin accumulation preferentially in calcium-enriched areas near the vascular bundles in pear leaves. We propose a fruit-striping mechanism, in which the abnormal overexpression of PbGA2ox8 in “Red Zaosu” induces the formation of a longitudinal array of anthocyanin stripes near vascular bundles in fruit.

Aroma affects the sensory quality of fruit and, consequently, consumer satisfaction. Melatonin (MT) is a plant growth regulator used to delay senescence in postharvest fruit during storage, however, its effect on aroma of pear fruit remains unclear. In this study, we assessed the effects of 0.1 mmol L&minus, 1 MT on volatiles and associated gene expression in the fruit of pear cultivars &lsquo, Korla&rsquo, (Pyrus brestschneideri Rehd) and &lsquo, Abb&eacute, Fetel&rsquo, (Pyrus communis L.). MT mainly affected the production of C6 aromatic substances in the two varieties. In &lsquo, MT inhibited expression of PbHPL, and reduced hydroperoxide lyase (HPL) activity and content of hexanal and (E)-hex-2-enal. In contrast, MT inhibited activity of lipoxygenase (LOX), reduced expression of PbLOX1 and PbLOX2, promoted PbAAT gene expression, increased alcohol acyltransferase (AAT) activity, and increased propyl acetate, and hexyl acetate content in &lsquo, that similarly led to the reduction in content of hexanal and (E)-hex-2-enal. Content of esters in &lsquo, pear increased with increasing postharvest storage period. Although mechanisms differed between the two varieties, effects on aroma volatiles mediated by MT were driven by expression of genes encoding LOX, HPL, and AAT enzymes.

The presence of toxic degradation products in lignocellulosic hydrolysate typically reduced fermentation rates and xylose consumption rate, resulting in a decreased ethanol productivity. In the present study, Zymomonas mobilis 8b was investigated for high cell density fermentation with cell recycling to improve the ethanol productivity in lignocellulosic hydrolysate. The fermentation performances of Z. mobilis 8b at various conditions were first studied in yeast extract-tryptone medium. It was found that nutrient level was essential for glucose and xylose co-fermentation by Z. mobilis 8b and high cell density fermentation with cell recycling worked well in yeast extract-tryptone medium for 6 rounds fermentation. Z. mobilis 8b was then studied in enzymatic hydrolysates derived from dilute acid (DA) pretreated corn stover (CS) and ammonia pretreated CS for high cell density fermentation with cell recycling. Ethanol productivity obtained was around three times higher compared to traditional fermentation. Ethanol titer and metabolic yield were also enhanced with high cell density fermentation. Z. mobilis 8b cells showed high recyclability in ammonia pretreated CS hydrolysate.

Some pear (Pyrus communis L.) fruit show a certain resistance to ripening. Thus, postharvest cold storage is commonly used to accelerate their ripening process by promoting ethylene production. However, the enhanced ethylene biosynthesis also accelerates pear fruit senescence. Here, the effects of melatonin on the senescence process in pear fruit was investigated. In a rapidly softening pear ‘Starkrimson’, melatonin delayed the ethylene burst. In ‘Abbe Fetel’ and ‘Red Anjou’, normally softening pears, melatonin inhibited ethylene production during the entire senescence process. The limited ethylene production resulted in a lower loss of firmness in melatonin-treated fruit than in water treated fruit. PcPG, a major cell wall degradation-related gene, was inhibited by melatonin in all three cultivars. The expressions of ethylene biosynthesis genes PcACS1 and PcACO1 are correlated with the senescence process. The former was inhibited by melatonin in ‘Starkrimson’, both were inhibited in ‘Red Anjou’, but neither was inhibited in ‘Abbe Fetel’. Moreover, the antioxidant systems in ‘Starkrimson’ and ‘Abbe Fetel’ were enhanced by melatonin and their fruit did not undergo water soaking or core browning. Thus, melatonin has the potential to retain the commercial value of postharvest pear fruit and delay senescence by limiting ethylene production and the reactive oxygen burst.

Microbial lipid production from lignocellulosic biomass is largely impeded by unsatisfactory fermentation performance due to the presence of toxic degradation products generated by pretreatment. DLC (Densifying Lignocellulosic biomass with Chemicals) pretreatment completed at room temperature, generating fewer degradation products, showed great potentials for lignocellulosic biorefineries. This work developed DLC pretreatment with sodium hydroxide and calcium hydroxide on rice straw (RS) for microbial lipid production. Conditions for steam autoclave on DLC-RS (i.e. DLCA treatment) were optimized using response surface methodology. High solid loading enzymatic hydrolysis and lipid fermentations by Trichosporon dermatis 32,903 together with mass balances were performed. Lipid fermentations were successfully conducted, for the first time, on pretreated biomass at a solid loading as high as 35 wt.% without washing or detoxification. Lipid titers as high as 28.3 g/L and 29.5 g/L were achieved on DLC-RS and DLCA-RS, respectively, with lipid yields of 0.24 and 0.23 g/g consumed sugar.

The presence of toxic degradation products in lignocellulosic hydrolysate typically reduced fermentation rates and xylose consumption rate, resulting in a decreased ethanol productivity. In the present study, Zymomonas mobilis 8b was investigated for high cell density fermentation with cell recycling to improve the ethanol productivity in lignocellulosic hydrolysate. The fermentation performances of Z. mobilis 8b at various conditions were first studied in yeast extract-tryptone medium. It was found that nutrient level was essential for glucose and xylose co-fermentation by Z. mobilis 8b and high cell density fermentation with cell recycling worked well in yeast extract-tryptone medium for 6 rounds fermentation. Z. mobilis 8b was then studied in enzymatic hydrolysates derived from dilute acid (DA) pretreated corn stover (CS) and ammonia pretreated CS for high cell density fermentation with cell recycling. Ethanol productivity obtained was around three times higher compared to traditional fermentation. Ethanol titer and metabolic yield were also enhanced with high cell density fermentation. Z. mobilis 8b cells showed high recyclability in ammonia pretreated CS hydrolysate.

Background As a class of natural antioxidants in plants, fruit flavonol metabolites are beneficial to human health. However, the regulatory networks for flavonol biosynthesis in most fruits are largely unknown. Previously, we reported a spontaneous pear bud sport ‘Red Zaosu’ (Pyrus bretschneideri Rehd.) with a high flavonoid content in its fruit. The identification of the flavonol biosynthetic regulatory network in this mutant pear fruit is crucial for elucidating the flavonol biosynthetic mechanism in fruit. Results Here, we demonstrated the PbMYB12b positively regulated flavonols biosynthesis in ‘Red Zaosu’ fruit. Initially, we investigated the accumulation patterns of four major quercetin glycosides and two major isorhamnetin glycosides in the fruit of ‘Red Zaosu’ and its wild-type ‘Zaosu’. A PRODUCTION OF FLAVONOL GLYCOSIDES (PFG)-type MYB transcription factor PbMYB12b was also screened for because of its correlation with flavonol accumulation in pear fruit. The biofunction of PbMYB12b was verified by transient overexpression and RNAi assays in pear fruit and young leaves. Overexpression of PbMYB12b enhanced the biosynthesis of quercetin glycosides and isorhamnetin glycosides by positively regulating a general flavonoids biosynthesis gene PbCHSb and a flavonol biosynthesis gene PbFLS. This finding was also supported by dual-luciferase transient expression assay and transient β-glucuronidase (GUS) reporter assay. Conclusions Our study indicated that PbMYB12b positively regulated flavonol biosynthesis, including four major quercetin glycosides and two major isorhamnetin glycosides, by promoting the expression of PbCHSb and PbFLS in pear fruit. Electronic supplementary material The online version of this article (10.1186/s12870-019-1687-0) contains supplementary material, which is available to authorized users.

Microbes can produce not only commodity fatty acids, such as palmitic acid (16:0) and stearic acid (18:0), but also high-value fatty acids (essential fatty acids). Most high value fatty acids belong to long chain polyunsaturated fatty acids (PUFA), such as omega-3 fatty acids (e.g., eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) and omega-6 fatty acids (e.g., arachidonic acid (ARA) and γ-linolenic acid (GLA)). EPA (20:5n-3) is a 20-carbon fatty acid with five double bonds, and the first double bond is in the n-3 position. DHA (22:6n-3) is a 22-carbon fatty acid with 6 double bonds and the first double bond is also in the n-3 position. Both EPA and DHA play an essential role in cardiovascular health including prevention of atherosclerotic disease development (Zehr and Walker, Prostaglandins Other Lipid Mediat 134:131-140, 2018). ARA (20:4n-6) is a 20-carbon fatty acid with four double bonds, and the first double bond is in the n-6 position. GLA (18:3n-6) is an 18-carbon fatty acid with three double bonds, and the first double bond is in the n-6 position. ARA and GLA have multiple biological effects, such as lowering blood cholesterol, and lowering cardiovascular mortality (Poli and Visioli, Eur J Lipid Sci Technol 117(11):1847-1852, 2015). This chapter provides details on microbial production of EAP, DHA, ARA, and GLA.

The biosynthetic gene cluster of the fungal meroterpenoid chrodrimanin B (4) was discovered in Penicillium verruculosum TPU1311, and the complete biosynthetic pathway of 4 has been elucidated by heterologous reconstitution of its biosynthesis in Aspergillus oryzae, as well as by in vitro characterizations of selected enzymes. The present study has identified the polyketide synthase that produces 6-hydroxymellein (3) and also provided a biosynthetic platform of chrodrimanins for further bioengineering.

Postharvest softening is an important physiological process that influences pear fruit quality. ‘Starkrimson’ pears soften rapidly, resulting in fruits with a low tolerance for storage and transportation. To better characterize the mechanism regulating the rapid softening of pears, cell wall-related changes were compared among three soft-fleshed and rapid-softening cultivars (‘Starkrimson’, ‘Bartlett’, and ‘Abbe Fetel’) and a crisp-fleshed and non-softening cultivar (‘Dangshansuli’). Cell wall polysaccharide content measurements revealed that rapid fruit softening was accompanied by extensive pectin degradation in the three soft-fleshed cultivars. Fifteen genes encoding two polygalacturonases (PG; EC 3.2.1.15), four pectin methylesterases (EC 3.1.1.11), four β-galactosidases (EC 3.2.1.23), one α-L-arabinofuranosidase (EC 3.2.1.55) and four cellulases (EC 3.2.1.4) were isolated from pear fruit flesh. Gene expression analyses indicated that almost all of the genes were involved in ripening. Furthermore, PG2 exhibited the most significant expression-level differences between the two types of pears, suggesting it was responsible for the rapid softening of the three soft-fleshed pears. PG1 was highly expressed only in ‘Starkrimson’ pears, which may explain the considerably faster softening rate of ‘Starkrimson’ fruits compared with those of other cultivars. Additionally, 1-methylcyclopropene (1-MCP) was used to control the softening rate of ‘Starkrimson’ pears, PG1 and PG2 expression was effectively inhibited by 1-MCP. The results of this study suggest that PG genes have crucial roles in the rapid softening of ‘Starkrimson’ pears.

Flavonoid compounds play important roles in the modern diet, and pear fruits are an excellent dietary source of these metabolites. However, information on the regulatory network of flavonoid biosynthesis in pear fruits is rare. In this work, 18 putative flavonoid-related MYB transcription factors (TFs) were screened by phylogenetic analysis and four of them were correlated with flavonoid biosynthesis patterns in pear fruits. Among these MYB-like genes, the specific functions of two novel MYB TFs, designated as PbMYB10b and PbMYB9, were further verified by both overexpression and RNAi transient assays. PbMYB10b, a PAP-type MYB TF with atypical motifs in its conserved region, regulated the anthocyanin and proanthocyanidin pathways by inducing the expression of PbDFR, but its function could be complemented by other MYB TFs. PbMYB9, a TT2-type MYB, not only acted as the specific activator of the proanthocyanidin pathway by activating the PbANR promoter, but also induced the synthesis of anthocyanins and flavonols by binding the PbUFGT1 promoter in pear fruits. The MYBCORE-like element has been identified in both the PbUFGT1 promoter and ANR promoters in most species, but it was not found in UFGT promoters isolated from other species. This finding was also supported by a yeast one-hybrid assay and thus enhanced the likelihood of the interaction between PbMYB9 and the PbUFGT1 promoter.

Anthocyanin biosynthesis exhibits a rhythmic oscillation pattern in some plants. To investigate the correlation between the oscillatory regulatory network and anthocyanin biosynthesis in pear, the anthocyanin accumulation and the expression patterns of anthocyanin late biosynthetic genes (ALBGs) were investigated in fruit skin of &lsquo, Red Zaosu&rsquo, (Pyrus bretschneideri Rehd.). The anthocyanin accumulated mainly during the night over three continuous days in the fruit skin, and the ALBGs&rsquo, expression patterns in &lsquo, fruit skin were oscillatory. However, the expression levels of typical anthocyanin-related transcription factors did not follow this pattern. Here, we found that the expression patterns of four PbREVEILLEs (PbRVEs), members of a class of atypical anthocyanin-regulated MYBs, were consistent with those of ALBGs in &lsquo, fruit skin over three continuous days. Additionally, transient expression assays indicated that the four PbRVEs promoted anthocyanin biosynthesis by regulating the expression of the anthocyanin biosynthetic genes encoding dihydroflavonol-4-reductase (DFR) and anthocyanidin synthase (ANS) in red pear fruit skin, which was verified using a dual-luciferase reporter assay. Moreover, a yeast one-hybrid assay indicated that PbRVE1a, 1b and 7 directly bound to PbDFR and PbANS promoters. Thus, PbRVEs promote anthocyanin accumulation at night by up-regulating the expression levels of PbDFR and PbANS in &lsquo, fruit skin.

Subgroup 4 R2R3 MYBs play vital roles in the regulation of anthocyanin biosynthesis. However, there is limited knowledge regarding the functions of MYB repressors in pear (Pyrus &times, bretschneideri). Here, PbMYB120 was identified as a potential regulator of anthocyanin biosynthesis. A phylogenetic analysis revealed that PbMYB120 was clustered into the FaMYB1-like clade of the subgroup 4 R2R3 MYBs. PbMYB120 was expressed higher in red peels than in green peels in five pear cultivars. PbMYB120 expression was positively correlated with anthocyanin accumulation. However, the transient overexpression of PbMYB120 led to the inhibition of anthocyanin accumulation and PbUFGT1 expression. Promoter binding and activation assays indicated that PbMYB120 binds to the promoter of PbUFGT1 and represses the promoter&rsquo, s activity. Thus, the inhibition of anthocyanin accumulation by PbMYB120 may be correlated with the repression of PbUFGT1. Furthermore, during anthocyanin induction, the expression levels of anthocyanin activators and PbMYB120 were upregulated. This study demonstrated that PbMYB120 was highly expressed in pear tissues having higher anthocyanin accumulations but acted as a repressor in the regulation of anthocyanin accumulation. PbMYB120 may work coordinately with anthocyanin activators and serve as a balancer of anthocyanin accumulation.

Parthenocarpy, the production of seedless fruit without fertilization, has a variety of valuable qualities, especially for self-incompatible species, such as pear. To explore whether melatonin (MT) induces parthenocarpy, we used ‘Starkrimson’ pear as a material for morphological observations. According to our results, exogenous MT promoted the expansion and division of the mesocarp cells in a manner similar to hand pollination. However, the seeds of exogenous MT-treated fruit were undeveloped and aborted later in the fruit-setting stage. To further investigate how MT induced parthenocarpy, we studied changes of related hormones in the ovaries and found that MT significantly increased the contents of the gibberellins (GAs) GA3 and GA4. Thus, paclobutrazol (PAC), a GA-biosynthesis inhibitor, was used to study the relationship between GAs and MT. In addition, spraying MT after treatment with PAC did not increase GA content nor lead to parthenocarpy. Through a transcriptome analysis, we discovered that MT can cause significant upregulation of PbGA20ox and downregulation of PbGA2ox. However, no significant difference was observed in PbGA2ox compared with the control after PAC and MT applications. Thus, MT induces parthenocarpy by promoting GA biosynthesis along with cell division and mesocarp expansion in pear.

Phytohormones play crucial roles in fruit set regulation and development. Here, gibberellins (GA4+7), but not GA3, induced pear parthenocarpy. To systematically investigate the changes upon GA4+7 induced pear parthenocarpy, dynamic changes in histology, hormone and transcript levels were observed and identified in unpollinated, pollinated and GA4+7-treated ovaries. Mesocarp cells continued developing in both GA4+7-treated and pollinated ovaries. In unpollinated ovaries, mesocarp cells stopped developing 14 days after anthesis. During fruit set process, GA4+7, but not GA1+3, increased after pollination. Abscisic acid (ABA) accumulation was significantly repressed by GA4+7 or pollination, but under unpollinated conditions, ABA was produced in large quantities. Moreover, indole-3-acetic acid biosynthesis was not induced by GA4+7 or pollination treatments. Details of this GA–auxin–ABA cross-linked gene network were determined by a comparative transcriptome analysis. The indole-3-acetic acid transport-related genes, mainly auxin efflux carrier component genes, were induced in both GA4+7-treated and pollinated ovaries. ABA biosynthetic genes of the 9-cis-epoxycarotenoid dioxygenase family were repressed by GA4+7 and pollination. Moreover, directly related genes in the downstream parthenocarpy network involved in cell division and expansion (upregulated), and MADS-box family genes (downregulated), were also identified. Thus, a model of GA-induced hormonal balance and its effects on parthenocarpy were established., Asian pear: Plant hormones induce seedless fruit Various hormonal, anatomical and genetic changes explain how a plant hormone can induce pears to make seedless fruit without fertilization. Lingfei Xu and colleagues from Northwest A&F University in Yangling, China, studied the effect of plant growth hormones known as gibberellins on fruit development in the Dangshansuli pear, Pyrus bretschneideri Rehd. They found that spraying a mixture of two gibberellins, known as GA4+7, on unpollinated flowers produced seedless mature fruits of a normal size, whereas treatment with another gibberellin, GA3, yielded fruit that died before harvest. Analyses of gene expression, hormone levels and cell growth in the developing pear fruits showed that applying GA4+7 to unpollinated flowers generated many of the same effects as pollination. The findings could aid in future commercial production of this popular Asian fruit.

Parthenocarpy, the production of seedless fruit without fertilization, has a variety of valuable qualities, especially for self-incompatible species, such as pear. To explore whether melatonin (MT) induces parthenocarpy, we used ‘Starkrimson’ pear as a material for morphological observations. According to our results, exogenous MT promoted the expansion and division of the mesocarp cells in a manner similar to hand pollination. However, the seeds of exogenous MT-treated fruit were undeveloped and aborted later in the fruit-setting stage. To further investigate how MT induced parthenocarpy, we studied changes of related hormones in the ovaries and found that MT significantly increased the contents of the gibberellins (GAs) GA3 and GA4. Thus, paclobutrazol (PAC), a GA-biosynthesis inhibitor, was used to study the relationship between GAs and MT. In addition, spraying MT after treatment with PAC did not increase GA content nor lead to parthenocarpy. Through a transcriptome analysis, we discovered that MT can cause significant upregulation of PbGA20ox and downregulation of PbGA2ox. However, no significant difference was observed in PbGA2ox compared with the control after PAC and MT applications. Thus, MT induces parthenocarpy by promoting GA biosynthesis along with cell division and mesocarp expansion in pear.

The synthesis of anthocyanin in pear (Pyrus bretschneideri) fruit is regulated by light. However, little is known about the molecular mechanisms of pear fruit coloring mediated by upstream light-signaling regulators. Here, the photoresponse factors CONSTITUTIVE PHOTOMORPHOGENIC (COP) 1.1 and 1.2 were cloned from &lsquo, Red Zaosu&rsquo, peel to study their functions in pear fruit coloring. The overexpression vectors pBI121-PbCOP1.1 and pBI121-PbCOP1.2 were constructed to analyze their effects on anthocyanin synthesis in pear fruit. A protein sequence alignment and phylogenetic tree analysis revealed that PbCOP1 proteins are highly homologous with those of other species. An analysis of tissue differential expression showed that the greatest expression levels of PbCOP1s occurred in the leaves. Their expression levels increased in the leaves during development, when the leaves changed from red to green. The overexpression of PbCOP1s in the peel resulted in reduced anthocyanin synthesis at the injection sites. A quantitative PCR analysis of the injection sites showed that PbCOP1.1 significantly inhibited the expression of the anthocyanin synthesis-related genes CHI, DFR, UFGT2, bHLH3, HY5 and GST. Based on the above results, we hypothesize that PbCOP1.1 is an anthocyanin synthetic inhibitory factor of pear coloration.

In order to investigate the nutritive and ensiling characteristics of banana stems and leaves, the chemical compositions of banana stems and leaves were analyzed and the effects of additives on fermentation quality were measured. The research results showed: Banana stems and leaves had high fiber content (stem>58% DM, leaf >72% DM), nitrogen free extract content (>50% DM), and water soluble carbohydrate contents (WSC>5% DM), with certain nutritive value. Tannins of banana stem and leaf were 0.11% DM and 0.24% DM. There were lactic acid bacteria (>105cfu/g FM) in the banana stems and leaves; they might be well preserved with some treatments.

Four lactic acid bacteria (LAB) strains named HN78, HN87, HN91 and HN113 were separated fromStylosanthes guianensis SW. of Hainan province, which were identified bacterially using the classical classification and the sequences of 16S rDNA. The results showed that four strains wereEnterococcus hirae,Enterococcus faecalis,Lactococcus lactisandLactobacillus plantarum. The capability of lactic acid production of HN113 and HN91 were better than HN78 and HN87.

Cassava residues were taken as main raw material, Aspergillus oryzae and Aspergillus usamii were chosen as glucoamylase preparation, and response surface was used to optimize and analyze saccharification temperature, pH, rate of enzyme and total amount of Aspergillus (g/100g cassava residues) so order to find out the best operation conditon for cassava residues saccharification. It was found that the best operation condition for Aspergillus to saccharify cassava residues were as follows: saccharification temperature 70°C, pH 4, ratio of Aspergillus oryzae and Aspergillus usamii was 3:9, total amount of Aspergillus 10%, and it turned out to be a total reducing sugar content of 2.70g/L.

Flavonoid biosynthesis profile was clarified by fruit bagging and re-exposure treatments in the green Chinese pear ‘Zaosu’ (Pyrus bretschneideri Rehd.) and its red mutant ‘Red Zaosu’. Two distinct biosynthesis patterns of flavonoid 3-glycosides were found in ‘Zaosu’ pear. By comparison with ‘Red Zaosu’, the biosynthesis of flavonoid 3-galactosides and flavonoid 3-arabinosides were inhibited by bagging and these compounds only re-accumulated to a small degree in the fruit peel of ‘Zaosu’ after the bags were removed. In contrast, the biosynthesis of flavonoid 3-gluctosides and flavonoid 3-rutinosides was reduced by bagging and then increased when the fruits were re-exposed to sunlight. A combination of correlation, multicollinearity test and partial-correlation analyses among major flavonoid metabolites indicated that biosynthesis of each phenolic compound was independent in ‘Zaosu’ pear, except for the positive correlation between flavonoid 3-rutincosides and flavanols. In contrast with the green pear cultivar, almost all phenolic compounds in the red mutant had similar biosynthesis patterns except for arbutin. However, only the biosynthesis of flavonoid 3-galactosides was relatively independent and strongly affected the synthesis of the other phenolic compounds. Therefore, we propose a hypothesis that the strong accumulation of flavonoid 3-galactosides stimulated the biosynthesis of other flavonoid compounds in the red mutant and, therefore, caused systemic variation of flavonoid biosynthesis profiles between ‘Zaosu’ and its red mutant. This hypothesis had been further demonstrated by the enzyme activity of UFGT, and transcript levels of flavonoid biosynthetic genes and been well tested by a stepwise linear regression forecasting model. The gene that encodes flavonoid 3-galacosyltransferase was also identified and isolated from the pear genome.