The use of newly isolated fungal cultures for the selective delignification of bamboo culms.

The screening of ligninolytic enzyme-producing fungal species in samples led to the identification of Paracremonium sp. LCB1, Clonostachys compactiuscula LCD1 and C. compactiuscula LCN1. Both these strains produced high levels of hemicellulase and ligninolytic enzyme production over a relatively short fermentation period of 3-5 days while exhibiting very low levels of cellulase activity. The results of the tests indicated that co-culturing LCB1 and LCN1 enhanced the ability to degrade lignin, and the ideal degrading circumstances and internal degrading mechanism of combined fungi were examined. The results showed that under conditions of temperature (30°C), pH (5), culture time (40 d), solid-liquid ratio (1:2.5), the pretreatment of bamboo culms with a co-culture of LCB1 and LCN1 resulted in a pronounced 76.37% drop in lignin weight and a high lignin/cellulose loss ratio (>10). Fourier transform infrared spectroscopy, X-ray diffractometry, and scanning electron microscopy were used to characterize the physicochemical properties of these bio-pretreated bamboo culms, further confirming that LCB1 and LCN1 co-culture represents an effective approach to bamboo delignification.

Dynamic Changes in Flavor and Microbiota in Traditionally Fermented Bamboo Shoots (Chimonobambusa szechuanensis (Rendle) Keng f.).

Dissecting flavor formation and microbial succession during traditional fermentation help to promote standardized and large-scale production in the sour shoot industry. The principal objective of the present research is to elucidate the interplay between the physicochemical attributes, flavor, and microbial compositions of sour bamboo shoots in the process of fermentation. The findings obtained from the principal component analysis (PCA) indicated notable fluctuations in both the physicochemical parameters and flavor components throughout the 28 day fermentation process. At least 13 volatile compounds (OAV > 1) have been detected as characteristic aroma compounds in sour bamboo shoots. Among these, 2,4-dimethyl Benzaldehyde exhibits the highest OAV (129.73~668.84) and is likely the primary contributor to the sour odor of the bamboo shoots. The analysis of the microbial community in sour bamboo shoots revealed that the most abundant phyla were Firmicutes and Proteobacteria, while the most prevalent genera were Enterococcus, Lactococcus, and Serratia. The results of the correlation analysis revealed that Firmicutes exhibited a positive correlation with various chemical compounds, including 3,6-nonylidene-1-ol, 2,4-dimethyl benzaldehyde, silanediol, dimethyl-, nonanal, and 2,2,4-trimethyl-1,3-pentylenediol diisobutyrate. Similarly, Lactococcus was found to be positively correlated with several chemical compounds, such as dimethyl-silanediol, 1-heptanol, 3,6-nonylidene-1-ol, nonanal, 2,2,4-trimethyl-1,3-pentanediol diisobutyrate, dibutyl phthalate, and TA. This study provides a theoretical basis for the standardization of traditional natural fermented sour bamboo production technology, which will help to further improve the flavor and quality of sour bamboo.

Genome-Wide Identification and Expression Analysis of Dendrocalamus farinosus CCoAOMT Gene Family and the Role of DfCCoAOMT14 Involved in Lignin Synthesis.

As the main component of plant cell walls, lignin can not only provide mechanical strength and physical defense for plants, but can also be an important indicator affecting the properties and quality of wood and bamboo. Dendrocalamus farinosus is an important economic bamboo species for both shoots and timber in southwest China, with the advantages of fast growth, high yield and slender fiber. Caffeoyl-coenzyme A-O-methyltransferase (CCoAOMT) is a key rate-limiting enzyme in the lignin biosynthesis pathway, but little is known about it in D. farinosus. Here, a total of 17 DfCCoAOMT genes were identified based on the D. farinosus whole genome. DfCCoAOMT1/14/15/16 were homologs of AtCCoAOMT1. DfCCoAOMT6/9/14/15/16 were highly expressed in stems of D. farinosus; this is consistent with the trend of lignin accumulation during bamboo shoot elongation, especially DfCCoAOMT14. The analysis of promoter cis-acting elements suggested that DfCCoAOMTs might be important for photosynthesis, ABA/MeJA responses, drought stress and lignin synthesis. We then confirmed that the expression levels of DfCCoAOMT2/5/6/8/9/14/15 were regulated by ABA/MeJA signaling. In addition, overexpression of DfCCoAOMT14 in transgenic plants significantly increased the lignin content, xylem thickness and drought resistance of plants. Our findings revealed that DfCCoAOMT14 can be a candidate gene that is involved in the drought response and lignin synthesis pathway in plants, which could contribute to the genetic improvement of many important traits in D. farinosus and other species.

Bio-organic fertilizers improve Dendrocalamus farinosus growth by remolding the soil microbiome and metabolome.

Organic and microbial fertilizers have potential advantages over inorganic fertilizers in improving soil fertility and crop yield without harmful side-effects. However, the effects of these bio-organic fertilizers on the soil microbiome and metabolome remain largely unknown, especially in the context of bamboo cultivation. In this study, we cultivated Dendrocalamus farinosus (D. farinosus) plants under five different fertilization conditions: organic fertilizer (OF), Bacillus amyloliquefaciens bio-fertilizer (Ba), Bacillus mucilaginosus Krassilnikov bio-fertilizer (BmK), organic fertilizer plus Bacillus amyloliquefaciens bio-fertilizer (OFBa), and organic fertilizer plus Bacillus mucilaginosus Krassilnikov bio-fertilizer (OFBmK). We conducted 16S rRNA sequencing and liquid chromatography/mass spectrometry (LC-MS) to evaluate the soil bacterial composition and soil metabolic activity in the different treatment groups. The results demonstrate that all the fertilization conditions altered the soil bacterial community composition. Moreover, the combination of organic and microbial fertilizers (i.e., in the OFBa and OFBmK groups) significantly affected the relative abundance of soil bacterial species; the largest number of dominant microbial communities were found in the OFBa group, which were strongly correlated with each other. Additionally, non-targeted metabolomics revealed that the levels of soil lipids and lipid-like molecules, and organic acids and their derivatives, were greatly altered under all treatment conditions. The levels of galactitol, guanine, and deoxycytidine were also markedly decreased in the OFBa and OFBmK groups. Moreover, we constructed a regulatory network to delineated the relationships between bamboo phenotype, soil enzymatic activity, soil differential metabolites, and dominant microbial. The network revealed that bio-organic fertilizers promoted bamboo growth by modifying the soil microbiome and metabolome. Accordingly, we concluded that the use of organic fertilizers, microbial fertilizers, or their combination regulated bacterial composition and soil metabolic processes. These findings provide new insights into how D. farinosus-bacterial interactions are affected by different fertilization regiments, which are directly applicable to the agricultural cultivation of bamboo.

Epigenetic and somaclonal divergence in Dendrocalamus farinosus for physiological augmentation and lignin degradation.

Epigenetic and molecular variation is a key approach for the improvement of plants. From in vitro tissue culture of Dendrocalamus farinosus; 30 plants were regenerated and after 2 years, the physiological, biochemical, and genomic studies were conducted. The results highlighted that for all phenotypic characteristics, the 30 regenerated plants were superior in comparison with the control (CK). From genetical analysis, a total of 97 bands were witnessed ranging between 212 bp and 2.2 kb. The results for OPU14 showed one additional specific band (723 bp) and one band (700 bp) were missing. The 10 plants were having genetic variability and can be termed as somaclones while the other plants have epigenetic variations. The cellulose and lignin analysis highlighted that somaclone No. 30 has the least cellulose content of 35%; while the somaclones No. 102 and No. 213 have the least 3.21% and 3.48% of lignin contents. Therefore, somaclones No. 30, No. 102, and No. 213 were selected for the histochemical localization. The lignin investigation revealed that somaclone No. 30 is greater while somaclones No. 102 and No. 213 were reduced in vascular bundles in comparison with the CK along with the high expression level of 4CL, C3H, C4H, COMT, and CCoAOMT1 genes.

Genome-wide analysis and characterization of Dendrocalamus farinosus SUT gene family reveal DfSUT4 involvement in sucrose transportation in plants.

Sucrose is the main transported form of photosynthetic products. Sucrose transporter (SUT) participates in the translocation of sucrose from source to sink, which is important for the growth and development of plants. Dendrocalamus farinosus is an important economic crop in southwestern China because of its high growth rate, high fiber content, and dual usage for food and timber, but the mechanism of sucrose transportation in D. farinosus is unclear. In this study, a total of 12 SUT transporter genes were determined in D. farinosus by whole-genome identification. DfSUT2, DfSUT7, and DfSUT11 were homologs of rice OsSUT2, while DfSUT4 was a homolog of OsSUT4, and these four DfSUT genes were expressed in the leaf, internode, node, and bamboo shoots of D. farinosus. In addition, DfSUT family genes were involved in photosynthetic product distribution, ABA/MeJA responses, and drought resistance, especially DfSUT4. The function of DfSUT4 was then verified in Nicotiana tabacum. DfSUT4 was localized mainly in the leaf mesophyll and stem phloem of pDfSUT4::GUS transgenic plant. The overexpression of DfSUT4 gene in transgenic plant showed increases of photosynthetic rate, above-ground biomass, thousand grain weight, and cellulose content. Our findings altogether indicate that DfSUT4 can be a candidate gene that can be involved in phloem sucrose transportation from the source leaves to the sink organs, phytohormone responses, abiotic stress, and fiber formation in plants, which is very important in the genetic improvement of D. farinosus and other crops.

Efficient enzymatic saccharification of alkaline and ionic liquid-pretreated bamboo by highly active extremozymes produced by the co-culture of two halophilic fungi.

Herein, we studied two strains of halophilic fungi (Aspergillus flavus and Aspergillus penicillioides) as potential potent sources of hydrolases under solid-state fermentation conditions. We found that the co-culture of these two fungal species was associated with maximal CMCase, FPase, xylanase, and ß-xylosidase activity under optimized fermentation conditions. These enzymes functioned optimally at pH values from 9.0 to 10.0, at temperatures from 50 °C to 60 °C, and in the presence of 15-20% NaCl. These enzymes were also stable in metal salt solutions and the presence of ionic liquids. Reducing sugar yields following the cellulase-hemicellulase co-treatment of untreated, alkaline-pretreated, and ionic liquid-pretreated bamboo were higher than those associated with separate cellulase and hemicellulase treatments, thus confirming the synergistic activity of cellulase-hemicellulase co-treatment in the context of bamboo saccharification. These results indicate that these two fungi are promising hydrolase producers that can facilitate the bioconversion of bamboo biomass.

Differential responses of soil hydrolytic and oxidative enzyme activities to the natural forest conversion.

Effects of natural forest conversion (NFC) on soil nutrient turnover are substantially mediated by soil microbial extracellular hydrolytic enzymatic activities (Hy-EEAs) and oxidative enzymatic activities (Ox-EEAs). Yet it remains largely unknown the indicative links between soil Hy- and Ox-EEAs and soil carbon (C), nitrogen (N) and phosphorus (P) supplies based on economic theories of microbial metabolism under NFC. Here we used a meta-analysis approach to synthesize the responses of the soil C-, N-, P-degrading Hy-EEAs and Ox-EEAs, soil microbial biomass, soil organic C, total N, P and available P parameters to natural forest conversion from 51 peer-reviewed studies. Our results showed that NFC notably decreased soil Hy-EEAs but statistically insignificant reduction of soil Ox-EEAs. The changes of soil Hy- and Ox-EEAs were significantly and positively associated with soil organic C, available P as well as microbial biomass C and N but significantly and negatively correlated with soil pH, whereas the changes of soil C/N impacted on soil Ox-EEAs remarkably but not for soil Hy-EEAs. The depletion of soil organic carbon stimulated soil microbial secretion of Hy- and Ox-EEAs. The soil total N scarcity only provoked soil microbial Hy-EEAs rather than Ox-EEAs. The soil total P dearth quickened the soil Ox-EEAs, however, the plenitude of soil available P suppressed soil Hy- and Ox-EEAs. Moreover, the eco-enzymatic stoichiometry of soil Hy-EEAs indicated that soil N and P nutrient limitation after NFC restricted soil microbial N- and P-acquiring enzymes secretion, which ultimately reduced resources availability for C acquisition. Altogether, the distinct responses of soil Hy- and Ox-EEAs depended on substrate availability peculiarly for soil N and P gains of microorganisms for further enzymatic ability on soil C decomposition and highlighted the abundant or absent supply of soil N and P for positive or negative enzymatic activities on metabolic requirement of soil edaphons.

Phytoremediation through Bidens pilosa L., a nonhazardous approach for uranium remediation of contaminated water.

A few plant species are recognized for uranium bioaccumulation, particularly as upper accumulator. Uranium has a dynamic impact on the physiological, biochemical, and photochemical reactions. Therefore, the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), soluble sugar, protein, photochemical reactions, and accumulation of uranium characteristics were studied in Bidens pilosa L. while applying altered concentrations of uranium in the form of C4H6O6U. It was done to measure the capacity of B. pilosa L. to remediate uranium from wastewater. In this study, the results showed that B. pilosa L. not only has the ability of uranium accumulation but it can accumulate in the upper parts i.e. leaves and stem. Overall it can accumulate as much as 1538 mg/kg on a dry weight basis. Uranium accumulation is a complex process which changes both physiological and biochemical index in plant species under different treatments. SOD decreased in leaves and stem in response to all treatments whereas POD and CAT increased at treatment 3 and 72 h up to 1335 µ/g-FW and 47 µ/g-FW at 72 h, respectively. This increase was followed by a downward trend. The correlation coefficient between fluorescence ratio Fv/Fm and the concentrations of uranium treatment were significantly negative i.e. -0.928. The Fourier transform infrared spectroscopy (FTIR) analysis also highlighted that uranium does not change the basic chemical composition of B. pilosa L., but has an effect on the contents of chemical constituents. From the study, it is concluded that B. Pilosa L. has shown a capacity for uranium enrichment, especially as an upper accumulator.

Effects of exotic plantation forests on soil edaphon and organic matter fractions.

There is uncertainty and limited knowledge regarding soil microbial properties and organic matter fractions of natural secondary forest accompanying chemical environmental changes of replacement by pure alien plantation forests in a hilly area of southwest of Sichuan province China. The aim of this study was to evaluate the impact of natural secondary forest (NSF) to pure Cryptomeria fortunei forest (CFF) and Cunninghamia lanceolata forest (CLF) on soil organic fractions and microbial communities. The results showed that the soil total phospholipid fatty acids (PLFAs), total bacteria and fungi, microbial carbon pool, organic recalcitrant carbon (C) and (N) fractions, soil microbial quotient and labile and recalcitrant C use efficiencies in each pure plantation were significantly decreased, but their microbial N pool, labile C and N pools, soil carbon dioxide efflux, soil respiratory quotient and recalcitrant N use efficiency were increased. An RDA analysis revealed that soil total PLFAs, total bacteria and fungi and total Gram-positive and Gram-negative bacteria were significantly associated with exchangeable Al3+, exchangeable acid, Al3+, available P and Mg2+ and pH, which resulted into microbial functional changes of soil labile and recalcitrant substrate use efficiencies. Modified microbial C- and N-use efficiency due to forest conversion ultimately meets those of rapidly growing trees in plantation forests. Enlarged soil labile fractions and soil respiratory quotients in plantation forests would be a potential positive effect for C source in the future forest management. Altogether, pure plantation practices could provoke regulatory networks and functions of soil microbes and enzyme activities, consequently leading to differentiated utilization of soil organic matter fractions accompanying the change in environmental factors.

[Transcriptome analysis and gene function annotation of Bambusa emeiensis shoots based on high-throughput sequencing technology].

Bambusa emeiensis is one of the preponderant species of sympodial bamboos in Sichuan province of China, and has excellent fiber length and quality as raw materials for papermaking, textile and other industries. In this study, with the application of Illumina HiSeq™ 2000 platform, we analyzed transcriptome in B. emeiensis with different heights of 10, 50, 100 and 150 cm. A total of 69.28 M reads were obtained, and a sum up of 111 137 bands of Unigenes were acquired following de novo stitching, assembly and clustering, among which there were 63 094 bands that had been integrated in the COG, GO, KEGG, Swiss-Prot and Nr databases using annotated methods. These Unigenes not only had general functions, such as transcription and signal transduction, but were also involved in sucrose transport and metabolism, secondary metabolites and cell wall biosynthesis. There was significant difference regarding the expression of cellulose synthase gene in B. emeiensis at different heights, relevant genes were found that might be responsible for the regulation of the growth and development of B. emeiensis as well as the biosynthesis of cellulose and lignin. Our findings could provide some elementary theories for breed improvement of B. emeiensis.

Tomato terpene synthases TPS5 and TPS39 account for a monoterpene linalool production in tomato fruits.

Recombinant tomato terpene synthases, TPS5/37/39, catalyze the formation of linalool or nerolidol in vitro. However, little is known about their actual biological activities in tomato plants, especially in their fruits. Here, when all three TPSs were induced in tomato fruits by a chemical elicitor, geraniol, a significant linalool peak was detected in fruit tissues but not in control fruits. Considering the compartments of these TPS proteins and available substrates, the linalool peak induced by geraniol might be attributed to TPS5 and TPS37, both of them putatively localized in the plastids where high levels of monoterpene substrate geranyl diphosphate exist. In addition, application of geraniol also triggered jasmonic acid (JA)-related defense genes suggesting that the inducible TPSs might be correlated with JA-signaled defense responses.

Molecular cloning, expression pattern, and putative cis-acting elements of a 4-coumarate: CoA ligase gene in bamboo (Neosinocalamus affinis)

Background: 4-coumarate:CoA ligase (4CL) plays an important role at the divergence point from general phenylpropanoid metabolism to several branch pathways. Although 4CL sin higher plants have been extensively studied, little has known about the 4CL gene of bamboo. Results: In current study, a Na4CL gene putative encoding 4-coumarate:CoA ligase (4CL) and its 5’-flanking region were isolated from bamboo (Neosinocalamus affinis) by RACE-PCR and genomic DNA walker, respectively. Na4CL encodes a predicted protein of 557 amino acids, with conserved motifs of adenylate-forming enzymes. Phylogenetic analysis showed that Na4CL shared 62~85 percent identity with other known plant 4CLs, and cluster closely with some known 4CLs in monocots. Sequence analysis revealed conserved cis-acting elements (Box A and AC-II element) present in the Na4CL promoter. Additionally, a Na4CL RNAi construct was transformed into tobacco. Transgenic tobaccos displayed significant down-expression of endogenesis 4CL and reduced lignin contents. Conclusion: These results contribute to the knowledge of the presence of Na4CL gene and its possible role in phenylpropanoid metabolism.

Single Walled Carbon Nanotubes Exhibit Dual-Phase Regulation to Exposed Arabidopsis Mesophyll Cells.

Herein we are the first to report that single-walled carbon nanotubes (SWCNTs) exhibit dual-phase regulation to Arabidopsis mesophyll cells exposed to different concentration of SWCNTs. The mesophyll protoplasts were prepared by enzyme digestion, and incubated with 15, 25, 50, 100 µg/ml SWCNTs for 48 h, and then were observed by optical microscopy and transmission electron microscopy, the reactive oxygen species (ROS) generation was measured. Partial protoplasts were stained with propidium iodide and 4'-6- diamidino-2-phenylindole, partial protoplasts were incubated with fluorescein isothiocyanate-labeled SWCNTs, and observed by fluorescence microscopy. Results showed that SWCNTs could traverse both the plant cell wall and cell membrane, with less than or equal to 50 µg/ml in the culture medium, SWCNTs stimulated plant cells to grow out trichome clusters on their surface, with more than 50 µg/ml SWCNTs in the culture medium, SWCNTs exhibited obvious toxic effects to the protoplasts such as increasing generation of ROS, inducing changes of protoplast morphology, changing green leaves into yellow, and inducing protoplast cells' necrosis and apoptosis. In conclusion, single walled carbon nanotubes can get through Arabidopsis mesophyll cell wall and membrane, and exhibit dose-dependent dual-phase regulation to Arabidopsis mesophyll protoplasts such as low dose stimulating cell growth, and high dose inducing cells' ROS generation, necrosis or apoptosis.

Characterization of the HMW glutenin subunits from Aegilops searsii and identification of a novel variant HMW glutenin subunit.

High molecular weight (HMW) glutenin subunits are conserved seed storage proteins in wheat and related species. Here we describe a more detailed characterization of the HMW glutenin subunits from Aegilops searsii, which is diploid and contains the S(s) genome related to the S genome of Aegilops speltoides and the A, B and D genomes of hexaploid wheat. SDS-PAGE experiments revealed two subunits (one x and one y) for each of the nine Ae. searsii accessions analyzed, indicating that the HMW glutenin subunit gene locus of Ae. searsii is similar to the Glu-1 locus found in wheat in containing both x and y genes. The primary structure of the four molecularly cloned subunits (from two Ae. searsii accessions) was highly similar to that of the previously reported x and y subunits. However, in one accession (IG49077), the last 159 residues of the x subunit (1S(s)x49077), which contained the sequence element GHCPTSPQQ, were identical to those of the y subunit (1S(s)y49077) from the same accession. Consequently, 1S(s)x49077 contains an extra cysteine residue located at the C-terminal part of its repetitive domain, which is novel compared to the x-type subunits reported so far. Based on this and previous studies, the structure and expression of the Glu-1 locus in Ae. searsii is discussed. A hypothesis on the genetic mechanism generating the coding sequence for the novel 1S(s)x49077 subunit is presented.