The feeding preference and bite response between Microtus fortis and Broussonetia papyrifera.

Introduction: Broussonetia papyrifera is a dioecious plant that is rich in various metabolites and widely distribute in Asia. Microtus fortis is a rodent that often causes damage to crops, especially in the Dongting Lake region of China. There is a wide overlap in the distribution areas for the above species and the M. fortis feeds on the leaves of the B. papyrifera. Preliminary experiments have shown that the reproduction of M. fortis is inhibited after feeding on the leaves of the B. papyrifera. Methods: In order to explore the potential of using B. papyrifera to develop botanical pesticides, we investigated the palatability and reactive substances. The feeding frequency of M. fortis on B. papyrifera leaves to that of on daily fodder and Carex brevicuspis that is the primary food for the wild population were compared. We also attempted to identify the responsive substances in B. papyrifera leaves that were bitten by M. fortis using metabolome analysis. Results: In general, B. papyrifera leaves exhibited a stronger attraction to M. fortis. M. fortis foraged B. papyrifera leaves more frequently, and the intake was higher than that of the other two. Differential metabolites were screened by comparing normal leaves and leaves bitten by M. fortis, meanwhile with the intervention of clipped leaves. A total of 269 substances were screened, and many of these were involved in the biosynthesis of secondary metabolites, including terpenoids and alkaloids. These substances may be related to the defense mechanism of B. papyrifera against herbivores. Discussion: These findings support further research examining animal-plant interactions and simultaneously provide insights into the utilisation of B. papyrifera resources and the management of rodents. The good palatability and the defense of B. papyrifera leaves suggest that they have the potential to contribute in development of plant rodenticide.

Effect of Lactiplantibacillus and sea buckthorn pomace on the fermentation quality and microbial community of paper mulberry silage.

Background: Paper mulberry is a promising alternative fodder source due to its high protein and the abundance of active components. However, paper mulberry often faces susceptibility to contamination during silage fermentation, and there is a need to improve the quality of silage fermentation of paper mulberry through exotic additives. Sea buckthorn pomace (BP) is a feed additive containing antimicrobial and antioxidant substances that help to enhance silage fermentation. Therefore, the objective of this study was to evaluate the effects of BP and Lactiplantibacillus as additives on silage fermentation and bacterial community of paper mulberry. Results: The results showed that BP and Lactiplantibacillus significantly reduced the pH and ammonium nitrogen content of paper mulberry silage (P < 0.05) and significantly increased the content of lactic acid and acetic acid (P < 0.05), resulting in more residual water-soluble carbohydrate and crude protein contents and less fiber content relative to the control. The key microorganisms in paper mulberry silage fermentation are Lactiplantibacillus pentosus and Weissella cibaria. Among these, Lactiplantibacillus favored a rapid increase in Lactiplantibacillus pentosus abundance during the pre-silage fermentation period, whereas BP favored the promotion of Lactiplantibacillus pentosus growth, resulting in higher contents of lactic and acetic acid than those of the control. Conclusions: Simultaneously adding Lactiplantibacillus and BP can effectively improve the quality of paper mulberry silage and increase the abundance of beneficial microorganisms in paper mulberry silage.

BpAFP, a Broussonetia papyrifera latex chitinase, exhibits a dual role in resisting to both Verticillium wilt disease and lepidopterous pests, Plutella xylostella and Prodenia litura.

Paper mulberry (Broussonetia papyrifera) is a fast-growing tree known for its tolerance to diverse biotic and abiotic stresses. To explore genes combating Verticillium wilt, a devasting and formidable disease damage to cotton and many economically significant crops, we purified an antifungal protein, named BpAFP, from the latex of paper mulberry. Based on peptide fingerprint, we cloned the full cDNA sequence of BpAFP and revealed that BpAFP belongs to Class I chitinases, sharing 74 % identity with B. papyrifera leaf chitinase, PMAPII. We further introduced BpAFP into Arabidopsis, tobacco, and cotton. Transgenic plants exhibited significant resistance to Verticillium wilt. Importantly, BpAFP also demonstrated insecticidal activity against herbivorous pests, Plutella xylostella, and Prodenia litura, when feeding the larvae with transgenic leaves. Our finding unveils a dual role of BpAFP in conferring resistance to both plant diseases and lepidopterous pests.

Characterization of metallothionein genes from Broussonetia papyrifera: metal binding and heavy metal tolerance mechanisms.

BACKGROUND: Broussonetia papyrifera is an economically significant tree with high utilization value, yet its cultivation is often constrained by soil contamination with heavy metals (HMs). Effective scientific cultivation management, which enhances the yield and quality of B. papyrifera, necessitates an understanding of its regulatory mechanisms in response to HM stress. RESULTS: Twelve Metallothionein (MT) genes were identified in B. papyrifera. Their open reading frames ranged from 186 to 372 bp, encoding proteins of 61 to 123 amino acids with molecular weights between 15,473.77 and 29,546.96 Da, and theoretical isoelectric points from 5.24 to 5.32. Phylogenetic analysis classified these BpMTs into three subclasses: MT1, MT2, and MT3, with MT2 containing seven members and MT3 only one. The expression of most BpMT genes was inducible by Cd, Mn, Cu, Zn, and abscisic acid (ABA) treatments, particularly BpMT2e, BpMT2d, BpMT2c, and BpMT1c, which showed significant responses and warrant further study. Yeast cells expressing these BpMT genes exhibited enhanced tolerance to Cd, Mn, Cu, and Zn stresses compared to control cells. Yeasts harboring BpMT1c, BpMT2e, and BpMT2d demonstrated higher accumulation of Cd, Cu, Mn, and Zn, suggesting a chelation and binding capacity of BpMTs towards HMs. Site-directed mutagenesis of cysteine (Cys) residues indicated that mutations in the C domain of type 1 BpMT led to increased sensitivity to HMs and reduced HM accumulation in yeast cells; While in type 2 BpMTs, the contribution of N and C domain to HMs' chelation possibly corelated to the quantity of Cys residues. CONCLUSION: The BpMT genes are crucial in responding to diverse HM stresses and are involved in ABA signaling. The Cys-rich domains of BpMTs are pivotal for HM tolerance and chelation. This study offers new insights into the structure-function relationships and metal-binding capabilities of type-1 and - 2 plant MTs, enhancing our understanding of their roles in plant adaptation to HM stresses.

Dietary Paper Mulberry Silage Supplementation Improves the Growth Performance, Carcass Characteristics, and Meat Quality of Yangzhou Goose.

There have been few investigations into the health benefits and meat quality of supplementing Yangzhou geese with paper mulberry silage. One hundred and twenty 28-day-old Yangzhou geese were selected for the experiment and randomly divided into two groups: a control group (CON) and a paper mulberry silage group (PM), with six replicates in each group. The experiment lasted for a total of 6 weeks. The experiment found that compared with CON, PM had a promoting effect on the average daily weight gain of Yangzhou geese (p = 0.056). Sensory and nutritional analysis of breast muscles revealed a decrease in a* value (p < 0.05) and an increase in protein content (p < 0.05) following PM treatment. Through untargeted metabolomics analysis of breast muscle samples, it was found that 11 different metabolites, including guanidinoacetic acid and other substances, had a positive effect on amino acid metabolism and lipid antioxidant pathways of PM treatment. Overall, the strategy of feeding Yangzhou geese with paper mulberry silage is feasible, which can improve the sensory quality and nutritional value of goose meat. The experiment provides basic data for the application form of goose breeding, so exploring the impact of substances within paper mulberry on goose meat should be focused on in the future.

Alternative Splicing under Cold Stress in Paper Mulberry.

The paper mulberry is a commonly found tree species with a long history of cultivation. It also serves as a crucial case study for understanding how woody plants adapt to low temperatures. Under cold treatment, we observed a substantial number of alternative splicing (AS) genes, showcasing the intricate landscape of AS events. We have detected all seven types of AS events, with the alternative 3' splice site (A3) having the most. We observed that many genes that underwent differential AS were significantly enriched in starch and sucrose metabolism and circadian rhythm pathways. Moreover, a considerable proportion of differentially spliced genes (DSGs) also showed differential expression, with 20.38% and 25.65% under 12 h and 24 h cold treatments, respectively. This suggests a coordinated regulation between gene AS and expression, playing a pivotal role in the paper mulberry's adaptation to cold stress. We further investigated the regulatory mechanisms of AS, identifying 41 serine/arginine-rich (SR) splicing factors, among which 11 showed differential expression under cold treatment, while 29 underwent alternative splicing. Additionally, genes undergoing AS displayed significantly higher DNA methylation levels under cold stress, while normal splicing (non-AS) genes exhibited relatively lower methylation levels. These findings suggest that methylation may play an important role in governing gene AS. Finally, our research will provide useful information on the role of AS in the cold acclimation tolerance of the paper mulberry.

Competitive Advantage of Broussonetia papyrifera Growing in a Native Area as Suggested by Structural Diversity.

Paper mulberry (Broussonetia papyrifera) is currently an invasive species on several continents. However, little is known about whether paper mulberry has a competitive advantage over its surrounding trees in its native distribution range, subtropical regions of China. Here, we determined the relative intraspecific and interspecific competitive capacity of paper mulberry in three subtropical deciduous broad-leaved forests using the indices of structural diversity including the mixing index, the tree-tree interval index, and the diameter/height differentiation index. It was found that more than 80% of mingling index values were not greater than 0.25, suggesting a stronger competitiveness of paper mulberry relative to other tree species. The tree-tree interval index values ranged between 1 m and 2 m, suggesting a strong competition between paper mulberry and its neighbors. Moreover, more than 60% of the height differentiation index and diameter differentiation index values were positive, suggesting that the reference paper mulberry had a slight competitive advantage over neighboring trees in both the horizontal and vertical planes. These collectively suggest a competitive advantage over other tree species in the native distribution range, which may play a significant role in the ecological invasion of paper mulberry. Our findings not only help to reveal the invasion mechanism of paper mulberry, but also provide an important reference for the management and utilization of paper mulberry in invaded areas.

Exploring the rumen microbiota of Hu lambs in response to diet with paper mulberry.

Paper mulberry (Broussonetia papyrifera), as a new woody forage with high-protein characteristic, is being widely used in ruminant feeding. However, little is known about the comprehensive microbiota picture of whole ruminal niches (liquid, solid, and epithelium) under paper mulberry diet. To gain a better understanding of feeding paper mulberry on the rumen microbiota, the effects of fresh paper mulberry, paper mulberry silage, or a conventional high-protein alfalfa silage on rumen fermentation products and microbiota in rumen niches of Hu lambs were studied. Forty-five Hu lambs were randomly divided into 3 treatments with 15 replicates in each treatment. No significant difference was observed among treatments in the average daily gain (ADG). The fresh paper mulberry treatment had lower (P < 0.05) pH and higher (P < 0.05) total volatile fatty acids (TVFA) compared with silage treatments, but the fermentation parameters did not show significant differences between paper mulberry silage and alfalfa silage treatments. The Shannon index did not show a significant difference (P < 0.05) among treatments except between fresh paper mulberry and alfalfa silage treatment in rumen epithelial niches. Butyrivibrio and Treponema were the predominant genera in the rumen epithelial fraction, while Prevotella and Rikenellaceae_RC9 dominated in both rumen liquid and solid fractions. These results indicated the paper mulberry supplement did not have distinct impact on the microbial diversity and growth performance compared with alfalfa silage, especially for paper mulberry silage, which might help us develop an alternative animal feeding strategy of replacing alfalfa with paper mulberry. KEY POINTS: • Feeding paper mulberry silage did not show significant impact on the growth performance compared with alfalfa silage treatment. • Feeding fresh paper mulberry reduced rumen pH value and increased total volatile fatty acid. • The microbial diversity did not show significant difference among treatments.

Effects of Silage Diet on Meat Quality through Shaping Gut Microbiota in Finishing Pigs.

With increasing demand for high-quality pork, development of green and healthy feed for finishing pigs is urgently needed. In this study, the effects and mechanisms of mulberry and paper mulberry silages on growth performance, meat quality, and intestinal health of finishing pigs were explored. Intestinal microbiota were profiled, and microbially produced short-chain fatty acids (SCFAs) were measured. The average daily gain (ADG) and feed conversion rate (FCR) with mulberry and paper mulberry silages were not significantly different from those of the control. Meat quality as measured by pork marbling and fatty acids in the longissimus dorsi was better with mulberry silage. The highest concentration of SCFAs was also with mulberry silage. According to 16S rRNA sequencing, Clostridium_sensu_stricto_1, Terrisporobacter, and Lachnospiraceae, which are important in SCFA production, were biomarkers of mulberry silage. PICRUSt functional analysis of intestinal microbes indicated that galactose metabolism, starch and sucrose metabolism, and carbohydrate digestion and absorption decreased significantly in silage treatments but increased in the control. Correlations between intestinal microbes and SCFAs and fatty acids indicated Clostridium_sensu_stricto_1, Terrisporobacter, and Lachnospiraceae were closely associated with SCFA and fatty acid contents. The results indicated that mulberry silage could increase SCFA content through shaping intestinal microbes to affect the deposition of fatty acids, which laid a solid theoretical foundation for improving pork quality. IMPORTANCE To avoid competition between people and animals for food, it is essential to develop nontraditional feeds. In this study, the effects of the silages of the unconventional feed resources mulberry and paper mulberry on meat quality of finishing pigs were examined. With mulberry silage in the diet, meat quality improved as indicated by meat color, marbling score, and beneficial fatty acids in the longissimus dorsi muscle. Pigs fed mulberry silage had the highest concentrations of short-chain fatty acids (SCFAs), and 16S rRNA sequencing identified Clostridium_sensu_stricto_1, Terrisporobacter, and Lachnospiraceae as biomarkers, which are important in SCFA production. Functions of intestinal microbes in the two silage groups primarily involved amino acid metabolism and SCFA production. Correlations between intestinal microbes and SCFAs and fatty acids indicated that Clostridium_sensu_stricto-1, Terrisporobacter, and Lachnospiraceae were closely associated with SCFA contents in the intestine and fatty acids in the longissimus dorsi.

Comparative analysis of mitochondrial genomes of Broussonetia spp. (Moraceae) reveals heterogeneity in structure, synteny, intercellular gene transfer, and RNA editing.

The genus Broussonetia (Moraceae) is comprised of three non-hybrid recognized species that all produce high quality fiber essential in the development of papermaking and barkcloth-making technology. In addition, these species also have medicinal value in several countries. Despite their important economical, medicinal, and ecological values, the complete mitogenome of Broussonetia has not been reported and investigated, which would greatly facilitate molecular phylogenetics, species identification and understanding evolutionary processes. Here, we assembled the first-reported three complete Broussonetia (B. papyrifera, B. kaempferi, and B. monoica) mitochondrial genomes (mitogenome) based on a hybrid strategy using Illumina and Oxford Nanopore Technology sequencing data, and performed comprehensive comparisons in terms of their structure, gene content, synteny, intercellular gene transfer, phylogeny, and RNA editing. Our results showed their huge heterogeneities among the three species. Interestingly, the mitogenomes of B. monoica and B. papyrifera consisted of a single circular structure, whereas the B. kaempferi mitogenome was unique and consisted of a double circular structure. Gene content was consistent except for a few transfer RNA (tRNA) genes. The Broussonetia spp. mitogenomes had high sequence conservation but B. monoica with B. kaempferi contained more synteny blocks and were more related, a finding that was well-supported in organellar phylogeny. Fragments that had been transferred between mitogenomes were detected at plastome hotspots that had integrated under potential mediation of tRNA genes. In addition, RNA editing sites showed great differences in abundance, type, location and efficiency among species and tissues. The availability of these complete gap-free mitogenomes of Broussonetia spp. will provide a valuable genetic resource for evolutionary research and understanding the communications between the two organelle genomes.

Effects of Lactiplantibacillus plantarum and Lactiplantibacillus brevis on fermentation, aerobic stability, and the bacterial community of paper mulberry silage.

The present study investigated the dynamic profiles of fermentation quality, aerobic stability, and the bacterial community of paper mulberry silage inoculants without (Control) or with Lactiplantibacillus plantarum (LP), Lactiplantibacillus brevis (LB), or their combination (LPLB), which was screened from naturally fermented paper mulberry. The results showed that the inoculated treatments had significantly reduced neutral detergent fiber, water-soluble carbohydrates, and ammoniacal nitrogen contents compared with the control after 60 days of ensiling (the decreased proportion of LP, LB, and LPLB treatments ranged from 7.33%-11.23%, 9.60%-21.44%, and 21.53%-29.23%, respectively, p < 0.05). The pH value of the LP and LB treatments was significantly lower than that of the control after 60 days of ensiling (4.42 and 4.56 vs. 4.71, p < 0.05). The LP treatment promoted lactic acid accumulation and LAB number compared with the control (66.59% vs. 54.12% and 8.71 log10 CFU/g vs. 8.52 log10 CFU/g, respectively, p < 0.05), and the LB and LPLB treatments inhibited the growth of yeast and mold after 14 days of fermentation. After 5 days of aerobic exposure, both the LB and LPLB treatments increased the aerobic stability time and acetic acid content (from 29 to 75 h and 16.14%-48.72%, respectively, p < 0.05), inhibited the growth of yeast and mold, and did not detect butyric acid. Additionally, the bacteria community of each treatment was dominated by Aerococcus on day 3 of ensilage (accounting for 54.36%-69.31%), while the inoculated treatments reduced the abundance of Aerococcus on day 60 (from 59.73% to 85.16%, p < 0.05), and Lactobacillus became the dominant genus (accounting for 54.57%-70.89%). Inoculation of L. plantarum effectively maintained the acidic environment at the end of the fermentation system by maintaining the abundance of Lactobacillus, maximizing the preservation of dry matter and protein, and reducing protein corruption. Inoculation of L. brevis alone or in combination with L. plantarum significantly inhibited the growth of mold and improved the aerobic stability of paper mulberry silage.

Effect of different organic acid additives on the fermentation quality and bacterial community of paper mulberry (Broussonetia papyrifera) silage.

To investigate the effects of different organic acid additives and their concentrations on the fermentation quality and bacterial community of paper mulberry silage, paper mulberry was left untreated (control) or was treated with ethylenediaminetetraacetic acid (EDTA), propionic acid (PA) or citric acid (CA), the amount of each additive was 2 g.kg-1 FM, 5 g.kg-1 FM and g.kg-1 FM. All groups were ensiled for 3, 7, 15, 30 and 60 days. Compared to the control, adding EDTA reduced protein breakdown, preserved more water-soluble carbohydrates of the silages (WSCs, 24.74 g.kg-1 DM), and high concentrations of EDTA inhibited the activity of undesirable microorganisms. Adding PA increased the abundance of Lactiplantibacillus and decreased the abundance of Enterococcus, and it caused a rapid decrease in the pH of the silage at an early stage (from 6.50 to 5.31) while altering the microbiota, and low concentrations of PA resulted in high LA (66.22 g.kg-1 DM) concentration and low PA (9.92 g.kg-1 DM) concentration at 60 days of ensiling. Different concentrations of additives altered the microbial community of paper mulberry to different degrees. High concentrations of PA and CA can increase the abundance of Lactiplantibacillus. High concentrations of CA resulted in a rapid decrease in silage pH at an early stage and higher WSC concentration. These results suggest that EDTA, PA and CA can be used as additives to improve the quality of paper mulberry silage.

Effects of epiphytic and exogenous lactic acid bacteria on fermentation quality and microbial community compositions of paper mulberry silage.

This study aimed to isolate, characterize, and identify lactic acid bacteria (LAB) strains from various sources and evaluate their effects on the nutritional quality, fermentation characteristics, and microbial compositions of paper mulberry (PM) after 60 days of ensiling. Forty-nine LAB strains were isolated from Phalaris arundinacea silage, pickle, and fresh PM leaves; three of these strains (Lactiplantibacillus plantarum, YC1; Levilactobacillus brevis, PC3; and Lactiplantibacillus plantarum, BP17) and one commercial inoculant Gaofuji (GFJ) were subsequently used. Compared with other treatments, PC3 and BP17 increased (P < 0.05) the LAB count and crude protein content and decreased (P < 0.05) the molds and coliform bacteria counts, pH, and ammonia-N content of PM silages. BP17 and PC3 increased the relative Lactiplantibacillus abundance and decreased that of Lelliottia and Cladosporium, improving PM silage quality. Therefore, PC3 and BP17 can improve the fermentation quality of PM silage and could be used as silage starter cultures.

Effects of different additives on the bacterial community and fermentation mode of whole-plant paper mulberry silage.

The purpose of this study was to investigate the effects of inoculation with two lactic acid bacteria (LAB) strains (Lacticaseibacillus rhamnosus and Lentilactobacillus buchneri) and the addition of four corn flour proportions (0, 3, 6, and 9%) in different treatments, on the composition and function of the bacterial community in whole-plant paper mulberry silage. The different treatments promoted Lactiplantibacillus, Lentilactobacillus, and Lacticaseibacillus growth, but the microbial species responsible for fermentation differed among the treatments. High species diversity and various Gram-negative bacteria, such as Flavitalea sp., Pantoea agglomerans, Acinetobacter pittii, Turicibacter sanguinis, and Ralstonia pickettii, were found in the uninoculated LAB treatments. A beneficial bacterium, Lactobacillus johnsonii, was discovered for the first time in whole-plant paper mulberry silage. LAB inoculation simplifies the microbial community structure, and beneficial Lactobacillus as a key species aggregates in the inoculated treatment group. However, L. rhamnosus inoculation alone may have limited bacteriostatic activity against in whole-plant paper mulberry silage. Compared with silage lacking corn flour, amino sugar and nucleotide sugar metabolism, galactose metabolism, the phosphotransferase system and the pentose phosphate pathway metabolic activity were increased in corn flour-containing silage. Whole-plant paper mulberry can be used as a high-quality silage to provide high-quality feed resources for sustainable ruminant livestock production. Moreover, additive use is necessary for preparing paper mulberry silage.

Effect of Amomum villosum essential oil as an additive on the chemical composition, fermentation quality, and bacterial community of paper mulberry silage.

Paper mulberry (Broussonetia papyrifera L., PM) is being used as a new type of animal protein feed to address the feed crisis. To investigate the effect of additives on the chemical composition, fermentation quality, and bacterial community of PM silage (at room temperature, 25°), paper mulberry was fermented with formic acid (FA), Amomum villosum essential oil (AVEO) and lactic acid bacteria (LAB) inoculant treatments. The results showed that fresh PM had a low water-soluble carbohydrate (WSC) content and large amounts of unclassified bacteria. Compared with the CK and LAB treatments, the FA and AVEO treatments significantly (P < 0.05) decreased the pH and increased the lactic acid content of PM silage after 60 days of ensiling. In the AVEO-treated silages the abundance of Lactococcus in the early stage of ensiling increased by 14.09%, the abundances of Levilactobacillus and Lentilactobacillus in the late stage of ensiling increased by 58.34 and 91.12%, respectively, and the abundance of Stenotrophomonas decreased by 94.71%, resulting in improved PM silage quality. These results confirmed that AVEO could potentially be developed as a new additive for improving the fermentation quality of silage.

Co-production of amino acid-rich xylooligosaccharide and single-cell protein from paper mulberry by autohydrolysis and fermentation technologies.

BACKGROUND: Autohydrolysis is an extensively investigated pretreatment method due to its environmental friendliness. During autohydrolysis, most xylan from hemicellulose can be converted into xylooligosaccharides (XOS), and cellulose in the autohydrolyzed residues can be transformed into glucose after enzymatic hydrolysis. Both of these are value-added biochemicals in the biorefining process. In this work, paper mulberry (PM), which contains abundant protein, was utilized as a raw material to coproduce XOS and single-cell protein (SCP) through autohydrolysis and fermentation technologies. RESULTS: The results showed that 8.3 g of XOS and 1.8 g of amino acids could be recovered in the autohydrolysate (based on 100 g raw material) after autohydrolysis (170 °C, 1 h). Moreover, 5.7 g of low-DP XOS along with 1.8 g of amino acids could be further obtained from the autohydrolysate after hydrolysis with endo-ß-1-4-xylanase. In addition, 20.1 g of fermentable monosaccharides was recovered after hydrolyzing the autohydrolyzed PM with cellulase, which can be used to produce 4.8 g of SCP after fermentation with Candida utilis. CONCLUSION: As a valuable application of PM, a novel process is proposed to coproduce amino acid-rich XOS and SCP through autohydrolysis. The carbohydrate of PM is effectively converted to high value-added products.

Comprehensive Profiling of Paper Mulberry (Broussonetia papyrifera) Crotonylome Reveals the Significance of Lysine Crotonylation in Young Leaves.

Lysine crotonylation is a newly discovered and reversible posttranslational modification involved in various biological processes, especially metabolism regulation. A total of 5159 lysine crotonylation sites in 2272 protein groups were identified. Twenty-seven motifs were found to be the preferred amino acid sequences for crotonylation sites. Functional annotation analyses revealed that most crotonylated proteins play important roles in metabolic processes and photosynthesis. Bioinformatics analysis suggested that lysine crotonylation preferentially targets a variety of important biological processes, including ribosome, glyoxylate and dicarboxylate metabolism, carbon fixation in photosynthetic organisms, proteasome and the TCA cycle, indicating lysine crotonylation is involved in the common mechanism of metabolic regulation. A protein interaction network analysis revealed that diverse interactions are modulated by protein crotonylation. These results suggest that lysine crotonylation is involved in a variety of biological processes. HSP70 is a crucial protein involved in protecting plant cells and tissues from thermal or abiotic stress responses, and HSP70 protein was found to be crotonylated in paper mulberry. This systematic analysis provides the first comprehensive analysis of lysine crotonylation in paper mulberry and provides important resources for further study on the regulatory mechanism and function of the lysine crotonylated proteome.

Genome-Wide Analysis of BpYABs and Function Identification Involving in the Leaf and Silique Development in Transgenic Arabidopsis.

YABs play an important role in the leaf development of the paper mulberry (Broussonetia papyrifera) and of the heterophylly. Thus, we investigated the function of BpYABs. Gene cloning, phylogenetic analysis, motif identification, subcellular localization, transactivation activity assay, qRT-PCR, in situ hybridization, and ectopic expression were used in our study. Six BpYABs were isolated, and four of them had transcriptional activity. BpYAB1, BpYAB3, BpYAB4, and BpYAB5 were localized to the nucleus. BpYAB1 was only expressed in the flower, while BpYAB6 was not expressed in any detected tissues; the four remaining BpYABs were expressed in the bud, leaf and flower, and their expression level decreased with leaf development. Further in situ hybridization showed that BpYAB3 and BpYAB5 were expressed in the vascular tissues and lamina, but neither showed the adaxial-abaxial polarity distribution pattern in the mature leaf lamina. Ectopic expression of BpYAB2, BpYAB3, BpYAB4 and BpYAB5 induced increased expression of AtWOX1 and caused the leaf of Arabidopsis to become smaller and curl downwards. Ectopic expression also led to shorter siliques and smaller seeds, but not for BpYAB5. These results suggest that BpYABs have functional divergency and redundancy in regulating leaf and silique development.

Fructobacillus papyriferae sp. nov., Fructobacillus papyrifericola sp. nov., Fructobacillus broussonetiae sp. nov. and Fructobacillus parabroussonetiae sp. nov., isolated from paper mulberry in Taiwan.

Five Gram-stain-positive strains (M1-10T, M1-13, M1-21T, M2-14T and S1-1T) were isolated from paper mulberry (Broussonetia papyrifera) in Taiwan. Cells were rod-shaped, non-motile, non-haemolytic, asporogenous, facultatively anaerobic, heterofermentative, and did not exhibit catalase and oxidase activities. Phylogenetic analysis of the 16S rRNA gene sequences revealed that these novel strains belonged to the genus Fructobacillus. On the basis of 16S rRNA gene sequence similarities, the type strains of Fructobacillus fructosus and Fructobacillus durionis were the closest neighbours to strains M1-10T, M1-13, M1-21T, M2-14T and S1-1T. Sequence analyses of concatenated two partial housekeeping genes, the RNA polymerase beta subunit (rpoC) and recombinase A (recA) also indicated that the novel strains belonged to the genus Fructobacillus. The 16S rRNA and concatenated rpoC and recA gene sequence similarities between strains M1-10T and M1-13 were 100 %, respectively. The average nucleotide identity values of M1-10T, M1-21T, M2-14T and S1-1T with F. fructosus and F. durionis were 75.1-78.9% and 76.5-77.5 %, respectively. The digital DNA-DNA hybridization values were 19.7-21.5% and 19.6-20.4 %, respectively. Phenotypic and genotypic test results demonstrated that these strains represent four novel species of the genus Fructobacillus, for which the names Fructobacillus papyriferae sp. nov., Fructobacillus papyrifericola sp. nov., Fructobacillus broussonetiae sp. nov. and Fructobacillus parabroussonetiae sp. nov. are proposed with the type strains M1-10T (=BCRC 81237T=NBRC 114433T), M1-21T (=BCRC 81239T=NBRC 114435T), M2-14T (=BCRC 81240T=NBRC 114436T) and S1-1T (=BCRC 81241T=NBRC 114437T), respectively.

Identification of leucine-rich repeat receptor-like protein kinase (LRR-RLK) genes in paper mulberry and their potential roles in response to cold stress.

Leucine-rich repeat receptor-like protein kinases (LRR-RLKs) represent the largest group of receptor-like kinases in plants, which have been previously reported to play vital roles in plant growth, development, stress adaptation and signal transduction. However, there is lack of comprehensive analysis of this family in paper mulberry (Broussonetia papyrifera). In the present investigation, a genome-wide scan revealed the presence of 236 LRR-RLK genes in paper mulberry, which were classified into 21 subgroups based on the maximum-likelihood phylogenetic tree. Gene structure and conserved motif analyses suggested genes in the same subgroup had highly consistent motif composition and intron/exon arrangement, but were divergent among subgroups. Total of 223 BpLRR-RLK genes were unevenly distributed across all 13 chromosomes, while the remaining 13 genes were localized to the unassembled scaffolds. Tandem and segmental duplications were confirmed to contribute to the expansion of BpLRR-RLK family. Further Ka/Ks showed that the duplicated BpLRR-RLKs had experienced strong purifying selection. The global promoter composition, transcriptome and phosphorylation analysis indicated that many of BpLRR-RLKs were associated with plant development, biotic and abiotic stress response, especially for cold stress. Furthermore, protein-protein interaction network was constructed for the 127 and 14 BpLRR-RLKs that responded to cold stress at the transcriptomics and phosphorylation level, respectively. All these findings will facilitate the studies on the evolutionary history of the LRR-RLK gene family in paper mulberry, also establish a solid foundation to further explore the potential functions of LRR-RLK genes in higher plants, particularly with regards to cold resistance.