Transcriptomic analysis reveals potential pathways associated with salt resistance in pecan (Carya illinoensis K. Koch).

Soil salinity is a serious abiotic stress worldwide. Pecan plants (Carya illinoensis K. Koch) have been suggested for cultivation in soils with high levels of salinity owing to their huge demand. To understand the potential molecular mechanisms in pecan in response to salt stress, RNA-sequencing technology was used to compare the transcriptomes of pecan plants treated with 0, 0.3 %, or 0.6 % NaCl solutions. The results indicated that 170,086 unigenes were obtained from pecan leaf cDNA samples. Based on the assembled de novo transcriptome, 53, 535, and 7358 differentially expressed genes (DEGs) were detected between untreated and salt-treated leaves at 8, 24, and 48 h, respectively. Because of the large number of DEGs across different contrasts, a Gene Set Enrichment Analysis was selected to identify gene pathways associated with salt treatment. A total of 1858 DEGs were enriched in 66 gene sets, including 22 up-regulated and 47 down-regulated gene sets in the salt treatment groups, compared with those in the control groups. The up-regulated gene sets were mainly involved in the response to salicylic acid; the regulation of the jasmonic acid-mediated signalling pathway during the short-term treatment (8 h); and the cellular response to hypoxia, cellular respiration, and RNA modification during the long-term treatment (24-48 h). The down-regulated gene sets were predominately associated with photosynthesis, water transport, and the metabolic biosynthetic process under salt stress. Genes related to the Really Interesting New Gene superfamily protein and F-box domain protein in the ubiquitin-dependent degradation pathway were significantly up-regulated or down-regulated in different periods of the regulating process. Overall, these results not only enrich genomic resources but also provide insights into the molecular mechanism in pecan under salt stress.

Improving enzymatic digestibility of wheat straw pretreated by a cellulase-free xylanase-secreting Pseudomonas boreopolis G22 with simultaneous production of bioflocculants.

BACKGROUND: Xylan removal by bacterial pretreatments has been confirmed to increase the digestibility of biomass. Here, an effective xylan removal technique has been developed to enhance the digestibility of wheat straw and simultaneously produce bioflocculants by a cellulase-free xylanase-secreting strain, Pseudomonas boreopolis G22. RESULTS: The results indicated that P. boreopolis G22 is an alkaliphilic strain which can secrete abundant amounts of xylanase. This xylanase had activity levels of 2.67-1.75 U mL-1 after an incubation period of 5-25 days. The xylanase showed peak activity levels at pH 8.6, and retained more than 85% relative activity in the pH range of 7.2-9.8. After 15 days of cultivation, the hemicellulose contents of the wheat straw were significantly decreased by 32.5%, while its cellulose contents were increased by 27.3%, compared to that of the control. The maximum reducing sugars released from the 15-day-pretreated wheat straw were 1.8-fold higher than that of the untreated wheat straw, under optimal enzymatic hydrolysis conditions. In addition, a maximum bioflocculant yield of 2.08 g L-1 was extracted from the fermentation broth after 15 days of incubation. The aforementioned bioflocculants could be used to efficiently decolorize a dye solution. CONCLUSIONS: The results indicate that the cellulase-free xylanase-secreting P. boreopolis G22 may be a potential strain for wheat straw pretreatments. The strain G22 does not only enhance the enzymatic digestibility of wheat straw, but also simultaneously produces a number of bioflocculants that can be used for various industrial applications.

Bioflocculants' production from a cellulase-free xylanase-producing Pseudomonas boreopolis G22 by degrading biomass and its application in cost-effective harvest of microalgae.

The major problem for industrial application of bioflocculants is its high production cost. Here, a novel bacterium Pseudomonas boreopolis G22, which can secret a cellulase-free xylanase and simultaneously produce bioflocculants (MBF-G22) through directly converting untreated biomass, was isolated. The bioflocculants' production of G22 was closely related to its xylanase activity, hydrolysis ability of biomass and the hemicellulose loss caused by G22. The optimal fermentation conditions with the highest bioflocculants' yield (3.75 mg g-1 dry biomass) were obtained at the fermentation time of 96 h, incubation temperature of 30 °C, inoculum concentration of 1.0% and biomass concentration of 1.0% in an initial pH value of 7.0. MBF-G22 mainly consisted of polysaccharides (63.3%) with a molecular weight of 3.982 × 106 Da and showed the highest flocculating efficiency of 97.1% at a dosage of 3.5 mg L-1. In addition, MBF-G22 showed high flocculating efficiency of microalgae (95.7%) at a dosage of 80 mg L-1.

Bioflocculants' production in a biomass-degrading bacterium using untreated corn stover as carbon source and use of bioflocculants for microalgae harvest.

BACKGROUND: Bioflocculation has been developed as a cost-effective and environment-friendly method to harvest multiple microalgae. However, the high production cost of bioflocculants makes it difficult to scale up. In the current study, low-cost bioflocculants were produced from untreated corn stover by a biomass-degrading bacterium Pseudomonas sp. GO2. RESULTS: Pseudomonas sp. GO2 showed excellent production ability of bioflocculants through directly hydrolyzing various biomasses. The untreated corn stover was selected as carbon source for bioflocculants' production due to its highest flocculating efficiency compared to that when using other biomasses as carbon source. The effects of fermentation parameters on bioflocculants' production were optimized via response surface methodology. According to the optimal model, an ideal flocculating efficiency of 99.8% was obtained with the fermentation time of 130.46 h, initial pH of 7.46, and biomass content of 0.64%. The relative importance of carboxymethyl cellulase and xylanase accounted for 51.8% in the process of bioflocculants' production by boosted regression tree analysis, further indicating that the bioflocculants were mainly from the hydrolysates of biomass. Biochemical analysis showed that it contained 59.0% polysaccharides with uronic acid (34.2%), 32.1% protein, and 6.1% nucleic acid in the bioflocculants, which had an average molecular weight as 1.33 × 106 Da. In addition, the bioflocculants showed the highest flocculating efficiency at a concentration of 12.5 mg L-1 and were stable over broad ranges of pH and temperature. The highest flocculating efficiencies obtained for Chlorella zofingiensis and Neochloris oleoabundans were 77.9 and 88.9%, respectively. CONCLUSIONS: The results indicated that Pseudomonas sp. GO2 can directly utilize various untreated lignocellulolytic biomasses to produce low-cost bioflocculants, which showed the high efficiency to harvest two green microalgae in a low GO2 fermentation broth/algal culture ratio.

Enhancing digestibility of Miscanthus using lignocellulolytic enzyme produced by Bacillus.

In this study an effective bacterial pretreatment method was developed to improve digestibility of Miscanthus. Seven new bacterial isolates, which showed excellent xylanase production ability using Miscanthus as carbon source, were used to perform the pretreatment experiments. After pretreatment, the hemicellulose content and crystallinity index of Miscanthus were decreased, while the reducing sugars released from Miscanthus were significantly increased by 30.8-87.8% after enzymatic hydrolysis. Bacillus sp. G0 was selected to optimize the pretreatment parameters via response surface methodology due to its high reducing sugars released from Miscanthus. According to the optimal model, the pretreatment parameters were set as citrate buffer/G0 fermentation broth ratio at 0.34, pretreatment time at 100h and Tween-20 concentration at 1.73%. The reducing sugars released from Miscanthus pretreated by optimal parameters were 305mgg-1 dry biomass. The results suggested our bacterial pretreatment approaches have great potential to increase digestibility of bioenergy crops.

Exogenous malic acid alleviates cadmium toxicity in Miscanthus sacchariflorus through enhancing photosynthetic capacity and restraining ROS accumulation.

Malic acid (MA) plays an important role in the regulation of plant growth, stomatal aperture, nutrition elements homeostasis and toxic metals tolerance. However, little is known about the effects of exogenous MA on physiological and biochemical responses to toxic metals in plants. To measure the alleviation roles of exogenous MA against cadmium (Cd), we determined the effects of MA on plant growth, net photosynthetic rate (Pn), reactive oxygen species (ROS) accumulation and the activities of anti-oxidant enzymes in the leaves of Miscanthus sacchariflorus (M. sacchariflorus) under Cd stress. The Cd exposure alone significantly inhibited plant growth and Pn, but increased the accumulation of ROS even though the anti-oxidant enzymes were markedly activated in the leaves of M. sacchariflorus. Treatment with MA significantly enhanced plant growth and decreased Cd accumulation accompanied by increasing Pn under Cd stress as compared to Cd stress alone, especially when treatment with high concentration of MA (200µM) was used. In addition, Cd and MA indicated synergistic effects by further increasing the activities and genes expression of partial anti-oxidant enzymes, thus resulting in higher glutathione accumulation and reduction of ROS production. The results showed that application of MA alleviated Cd-induced phytotoxicity and oxidant damage through the regulation of both enzymatic and non-enzymatic anti-oxidants under Cd stress in M. sacchariflorus.

Malate secretion from the root system is an important reason for higher resistance of Miscanthus sacchariflorus to cadmium.

Miscanthus is a vigorous perennial Gramineae genus grown throughout the world as a promising bioenergy crop and generally regarded as heavy metal tolerant due to its ability to absorb heavy metals. However, little is known about the mechanism for heavy metal tolerance in Miscanthus. In this study, two Miscanthus species (Miscanthus sacchariflorus and Miscanthus floridulus) exhibiting different cadmium (Cd) sensitivity were used to address the mechanisms of Cd tolerance. Under the same Cd stress, M. sacchariflorus showed higher Cd tolerance with better growth and lower Cd accumulation in both shoots and roots than M. floridulus. The malate (MA) content significantly increased in root exudates of M. sacchariflorus following Cd treatment while it was almost unchanged in M. floridulus. Cellular Cd analysis and flux data showed that exogenous MA application markedly restricted Cd influx and accumulation while an anion-channel inhibitor (phenylglyoxal) effectively blocked Cd-induced MA secretion and increased Cd influx in M. sacchariflorus, indicating that MA secretion could alleviate Cd toxicity by reducing Cd uptake. The genes of malate dehydrogenases (MsMDHs) and Al-activated malate transporter 1 (MsALMT1) in M. sacchariflorus were highly upregulated under Cd stress, compared with that in M. floridulus. The results indicate that Cd-induced MA synthesis and secretion efficiently alleviate Cd toxicity by reducing Cd influx in M. sacchariflorus.

Real-time kinetics of cadmium transport and transcriptomic analysis in low cadmium accumulator Miscanthus sacchariflorus.

MAIN CONCLUSION: The molecular mechanism of low Cd influxes and accumulation in Miscanthus sacchariflorus is revealed by RNA sequencing technique. Soil cadmium (Cd) pollution has posed a serious threat to our soil quality and food security as well as to human health. Some wild plants exhibit high tolerance to heavy metals stress. However, mechanisms of Cd tolerance of wild plants remain to be fully clarified. In this study, we found that two Miscanthus species, Miscanthus (M.) sacchariflorus and M. floridulus, showed different Cd-tolerant mechanisms. M. sacchariflorus accumulated less Cd in both root and leaf by limiting Cd uptake from root and showed superior Cd tolerance, while M. floridulus not only absorbs more Cd from root but also transports more Cd to shoot. To investigate the molecular mechanism of different Cd uptake patterns in the two Miscanthus species, we analyzed the transcriptome of M. sacchariflorus and identified transcriptional changes in response to Cd in roots by high-throughput RNA-sequencing technology. A total of 92,985 unigenes were obtained from M. sacchariflorus root cDNA samples. Based on the assembled de novo transcriptome, 681 DEGs which included 345 upregulated and 336 downregulated genes were detected between two libraries of untreated and Cd-treated roots. Gene ontology (GO) and pathway enrichment analysis revealed that upregulated DEGs under Cd stress are predominately involved in metabolic pathway, starch and sucrose and biosynthesis of secondary metabolites and metal ion transporters. Quantitative RT-PCR was employed to compare the expression levels of some metal transport genes in roots of two Miscanthus species, and the genes involved in Cd uptake from root and transfer from root to shoot were extremely different. The results not only enrich genomic resource but also help to better understand the molecular mechanisms of Cd accumulation and tolerance in wild plants.

Different Growth and Physiological Responses to Cadmium of the Three Miscanthus Species.

Miscanthus has been proposed as a promising crop for phytoremediation due to its high biomass yield and remarkable adaptability to different environments. However, little is known about the resistance of Miscanthus spp. to cadmium (Cd). To determine any differences in resistance of Miscanthus to Cd, we examined plant growth, net photosynthetic rate (Pn), activities of anti-oxidant and C4 photosynthetic enzymes, concentrations of Cd in leaves and roots, and observed the chloroplast structure in three Miscanthus species treated with 0, 10, 50, 100 or 200 µM Cd in solutions. Miscanthus sinensis showed more sensitivity to Cd, including sharp decreases in growth, Pn, PEPC activity and damage to chloroplast structure, and the highest H2O2 and Cd concentrations in leaves and roots after Cd treatments. Miscanthus sacchariflorus showed higher resistance to Cd and better growth, had the highest Pn and phosphoenolpyruvate carboxylase (PEPC) activities and integrative chloroplast structure and the lowest hydrogen peroxide (H2O2) and leaf and root Cd concentrations. The results could play an important role in understanding the mechanisms of Cd tolerance in plants and in application of phytoremediation.