Unraveling the Contribution of MulSOS2 in Conferring Salinity Tolerance in Mulberry (Morus atropurpurea Roxb).

Salinity is one of the most serious threats to sustainable agriculture. The Salt Overly Sensitive (SOS) signaling pathway plays an important role in salinity tolerance in plants, and the SOS2 gene plays a critical role in this pathway. Mulberry not only has important economic value but also is an important ecological tree species; however, the roles of the SOS2 gene associated with salt stress have not been reported in mulberry. To gain insight into the response of mulberry to salt stress, SOS2 (designated MulSOS2) was cloned from mulberry (Morus atropurpurea Roxb), and sequence analysis of the amino acids of MulSOS2 showed that it shares some conserved domains with its homologs from other plant species. Our data showed that the MulSOS2 gene was expressed at different levels in different tissues of mulberry, and its expression was induced substantially not only by NaCl but also by ABA. In addition, MulSOS2 was exogenously expressed in Arabidopsis, and the results showed that under salt stress, transgenic MulSOS2 plants accumulated more proline and less malondialdehyde than the wild-type plants and exhibited increased tolerance to salt stress. Moreover, the MulSOS2 gene was transiently overexpressed in mulberry leaves and stably overexpressed in the hairy roots, and similar results were obtained for resistance to salt stress in transgenic mulberry plants. Taken together, the results of this study are helpful to further explore the function of the MulSOS2 gene, which provides a valuable gene for the genetic breeding of salt tolerance in mulberry.

Large-Scale Quantitative Proteomic Analysis during Different Stages of Somatic Embryogenesis in Larix olgensis.

Larix olgensis is an economically important tree species native to northeastern China. The use of somatic embryogenesis (SE) is efficient and enables the rapid production of varieties with desirable qualities. Here, isobaric labeling via tandem mass tags was used to conduct a large-scale quantitative proteomic analysis of proteins in three critically important stages of SE in L. olgensis: the primary embryogenic callus, the single embryo, and the cotyledon embryo. We identified 6269 proteins, including 176 shared differentially expressed proteins across the three groups. Many of these proteins are involved in glycolipid metabolism, hormone response/signal transduction, cell synthesis and differentiation, and water transport; proteins involved in stress resistance and secondary metabolism, as well as transcription factors, play key regulatory roles in SE. The results of this study provide new insights into the key pathways and proteins involved in SE in Larix. Our findings have implications for the expression of totipotency, the preparation of synthetic seeds, and genetic transformation.

Comparison of guidelines for biological ancillary materials used for the manufacture of gene and cellular therapy products in Asia.

BACKGROUND AIMS: Although biologiocal ancillay materials (AMs) have specific risk associated with their derivations, it plays key role to manufature cell and gene therapy (CGT) products. It is important to understand the regulation relevant to AMs for developers. METHODS: The authors investigated the guidelines and pharmacopeia (hereinafter referred to as "guidelines") for biological AMs used for the manufacture of CGT products in Asia (China, India, Japan, Korea and Taiwan). In addition, the authors benchmarked the relevant guidelines in the United States (US) and European Union (EU). RESULTS AND DISCUSSIONS: The guidelines could be classified into two types based on whether specific AMs are scoped: (i) general guidelines for risk assessment of AMs and (ii) guidelines for specific AMs. The authors compared the risk categories for each type of AM provided in the general guidelines between the US and China and the specific requirements for bovine serum and trypsin in the guidelines of China, Japan, Taiwan, US and EU. The authors further compiled in-depth descriptions of the respective regulations in China, India, Japan, Korea and Taiwan. There is limited availability of some guidelines for specific AMs. Moreover, there are no common requirements established across the surveyed countries and regions. Therefore, the authors suggest a risk assessment approach for AMs with consideration of their biological origin and traceability, production steps applied and ability to control or remove AMs from the final medicinal product over the CGT manufacturing process.

Fusion gene 4CL-CCR promotes lignification in tobacco suspension cells.

KEY MESSAGE: The fusion gene 4CL-CCR promotes lignification and activates lignin-related MYB expression in tobacco but inhibits auxin-related gene expression and hinders the auxin absorption of cells. Given the importance of lignin polymers in plant growth and their industrial value, it is necessary to investigate how plants synthesize monolignols and regulate the level of lignin in cell walls. In our previous study, expression of the Populus tomentosa fusion gene 4CL-CCR significantly promoted the production of 4-hydroxycinnamyl alcohols. However, the function of 4CL-CCR in organisms remains poorly understood. In this study, the fusion gene 4CL-CCR was heterologously expressed in tobacco suspension cells. We found that the transgenic suspension cells exhibited lignification earlier. Furthermore, 4CL-CCR significantly reduced the content of phenolic acids and increased the content of aldehydes in the medium, which led to an increase in lignin deposition. Moreover, transcriptome results showed that the genes related to lignin synthesis, such as PAL, 4CL, CCoAOMT and CAD, were significantly upregulated in the 4CL-CCR group. The expression of genes related to auxin, such as ARF3, ARF5 and ARF6, was significantly downregulated. The downregulation of auxin affected the expression of transcription factor MYBs. We hypothesize that the upregulated genes MYB306 and MYB315 are involved in the regulation of cell morphogenesis and lignin biosynthesis and eventually enhance lignification in tobacco suspension cells. Our findings provide insight into the function of 4CL-CCR in lignification and how secondary cell walls are formed in plants.

Hyposterolactone A, a 3α-Hydroxy Steroidal Lactone from the Deep-Sea-Derived Fungus Hypocrea sp. ZEN14.

Chemical investigation of the deep-sea-derived fungus Hypocrea sp. ZEN14 afforded a new 3α-hydroxy steroidal lactone, hyposterolactone A (1) and 25 known secondary metabolites (2-26). The structure of the new compound was established by detailed spectroscopic analysis, electronic circular dichroism (ECD) calculation as well as a J-based configuration analysis. Compound 10 showed potent cytotoxicity against Huh7 and Jurkat cells with IC50 values of 1.4 µM and 6.7 µM, respectively.

Anti-HIV Compounds from the Deep-Sea-Derived Fungus Chaetomium globosum.

Chemical investigation on the deep-sea-derived fungus Chaetomium globosum led to the isolation of nine compounds. By extensive analyses of the 1D and 2D NMR as well as HR-ESI-MS spectra, their structures were elucidated as xylariol A (1), 1,3-dihydro-4,5,6-trihydroxy-7-methylisobenzofuran (2), epicoccone B (3), epicoccolide B (4), chaetoglobosin G (5), chaetoglobosin Fex (6), cochliodone A (7), cochliodone B (8), and chaetoviridin A (9), assorting as four phenolics (1-4), two cytochalosans (5-6), and three azaplilones (7-9). Compounds 1-3 were firstly reported from C. globosum. Under the concentrations of 20 µg/mL, 1, 2, and 3 exhibited potent in vitro anti-HIV activity with the inhibition rates of 70 %, 75 %, and 88 %, respectively.

Discovery of andrastones from the deep-sea-derived Penicillium allii-sativi MCCC 3A00580 by OSMAC strategy.

Andrastones are unusual 6,6,6,5-tetracyclic meroterpenoids that are rarely found in nature. Previously, three andrastones were obtained from the rice static fermentation extract of the deep-sea-derived fungus Penicillium allii-sativi MCCC 3A00580. Inspired by one strain many compounds (OSMAC) approach, the oat static fermentation on P. allii-sativi was conducted. As a result, 14 andrastones were isolated by UV-guided isolation. The chemical structures of the nine new compounds (1-9) was established by comprehensive analysis of the NMR, MS, ECD, and X-ray crystallography and the five known ones (10-14) were assigned by comparing their NMR, MS, and OR data with those reported in literature. Compound 1 bears a novel hemiketal moiety while 2 is the first example to possess a novel tetrahydrofuran moiety via C-7 and C-15. All isolates were tested for anti-allergic bioactivity. Compound 10, 3-deacetylcitreohybridonol, significantly decreased degranulation with the IC50 value of 14.8 µM, compared to that of 92.5 µM for the positive control, loratadine. Mechanism study indicated 10 could decrease the generation of histamine and TNF-α by reducing the accumulation of Ca2+ in RBL-2H3 cells. These findings indicate andrastones could be potential to discover new anti-allergic candidate drugs.

Systematic Analysis and Biochemical Characterization of the Caffeoyl Shikimate Esterase Gene Family in Poplar.

Caffeoyl shikimate esterase (CSE) hydrolyzes caffeoyl shikimate into caffeate and shikimate in the phenylpropanoid pathway. In this study, we performed a systematic analysis of the CSE gene family and investigated the possible roles of CSE and CSE-like genes in Populus. We conducted a genome-wide analysis of the CSE gene family, including functional and phylogenetic analyses of CSE and CSE-like genes, using the poplar (Populus trichocarpa) genome. Eighteen CSE and CSE-like genes were identified in the Populus genome, and five phylogenetic groups were identified from phylogenetic analysis. CSEs in Group Ia, which were proposed as bona fide CSEs, have probably been lost in most monocots except Oryza sativa. Primary functional classification showed that PoptrCSE1 and PoptrCSE2 had putative function in lignin biosynthesis. In addition, PoptrCSE2, along with PoptrCSE12, might also respond to stress with a function in cell wall biosynthesis. Enzymatic assay of PoptoCSE1 (Populus tomentosa), -2 and -12 showed that PoptoCSE1 and -2 maintained CSE activity. PoptoCSE1 and 2 had similar biochemical properties, tissue expression patterns and subcellular localization. Most of the PoptrCSE-like genes are homologs of AtMAGL (monoacylglycerol lipase) genes in Arabidopsis and may function as MAG lipase in poplar. Our study provides a systematic understanding of this novel gene family and suggests the function of CSE in monolignol biosynthesis in Populus.

Functional Characteristics Analysis of Dehydrins in Larix kaempferi under Osmotic Stress.

Dehydrins (DHN) belong to the late embryogenesis abundant II family and have been found to enhance plant tolerance to abiotic stress. In the present study, we reported four DHNs in Larix kaempferi (LkDHN) which were identified from the published transcriptome. Alignment analysis showed that these four LkDHNs shared close relationships and belonged to SK3-type DHNs. The electrophoretic mobility shift assay indicated that these four LkDHNs all possess sequence-independent binding capacity for double-strands DNAs. The subcellular localizations of the four LkDHNs were in both the nucleus and cytoplasm, indicating that these LkDHNs enter the nucleus to exert the ability to bind DNA. The preparation of tobacco protoplasts with different concentrations of mannitol showed that LkDHNs enhanced the tolerance of plant cells under osmotic stress. The overexpression of LkDHNs in yeasts enhanced their tolerance to osmotic stress and helped the yeasts to survive severe stress. In addition, LkDHNs in the nucleus of salt treated tobacco increased. All of these results indicated that the four LkDHNs help plants survive from heavy stress by participating in DNA protection. These four LKDHNs played similar roles in the response to osmotic stress and assisted in the adaptation of L. kaempferi to the arid and cold winter of northern China.

Integrated Phloem Sap mRNA and Protein Expression Analysis Reveals Phytoplasma-infection Responses in Mulberry.

To gain insight into the response of mulberry to phytoplasma-infection, the expression profiles of mRNAs and proteins in mulberry phloem sap were examined. A total of 955 unigenes and 136 proteins were found to be differentially expressed between the healthy and infected phloem sap. These differentially expressed mRNAs and proteins are involved in signaling, hormone metabolism, stress responses, etc. Interestingly, we found that both the mRNA and protein levels of the major latex protein-like 329 (MuMLPL329) gene were increased in the infected phloem saps. Expression of the MuMLPL329 gene was induced by pathogen inoculation and was responsive to jasmonic acid. Ectopic expression of MuMLPL329 in Arabidopsis enhances transgenic plant resistance to Botrytis cinerea, Pseudomonas syringae pv tomato DC3000 (Pst. DC3000) and phytoplasma. Further analysis revealed that MuMLPL329 can enhance the expression of some defense genes and might be involved in altering flavonoid content resulting in increased resistance of plants to pathogen infection. Finally, the roles of the differentially expressed mRNAs and proteins and the potential molecular mechanisms of their changes were discussed. It was likely that the phytoplasma-responsive mRNAs and proteins in the phloem saps were involved in multiple pathways of mulberry responses to phytoplasma-infection, and their changes may be partially responsible for some symptoms in the phytoplasma infected plants.

Gibberellins Play a Role in Regulating Tomato Fruit Ripening.

Although exogenous applications of gibberellins (GAs) delay tomato ripening, the regulatory mechanisms of GAs in the process have never been well recognized. Here, we report that the concentration of endogenous GAs is declined before the increase of ethylene production in mature-green to breaker stage fruits. We further demonstrate that reductions in GA levels via overexpression of a GA catabolism gene SlGA2ox1 specifically in fruit tissues lead to early ripening. Consistently, we have also observed that application of a GA biosynthetic inhibitor, prohexadione-calcium, at the mature-green stage accelerates fruit ripening, while exogenous GA3 application delays the process. Furthermore, we demonstrate that ethylene biosynthetic gene expressions and ethylene production are activated prematurely in GA-deficient fruits but delayed/reduced in exogenous GA3-treated WT fruits. We also show that the GA deficiency-mediated activation of ethylene biosynthesis is due to the activation of the ripening regulator genes RIN, NOR and CNR. In conclusion, our results demonstrate that GAs play a negative role in tomato fruit ripening.

Characterization of NPR1 and NPR4 genes from mulberry (Morus multicaulis) and their roles in development and stress resistance.

The quality and quantity of mulberry leaves are often affected by various environmental factors. The plant NPR1 and its homologous genes are important for plant systemic acquired resistance. Here, the full-length cDNAs encoding the NPR1 and NPR4 genes (designated MuNPR1 and MuNPR4, respectively) were isolated from Morus multicaulis. Sequence analysis of the amino acids and protein modeling of the MuNPR1 and MuNPR4 proteins showed that MuNPR1 shares some conserved characteristics with its homolog MuNPR4. MuNPR1 was shown to have different expression patterns than MuNPR4 in mulberry plants. Interestingly, MuNPR1 or MuNPR4 transgenic Arabidopsis produced an early flowering phenotype, and the expression of the pathogenesis-related 1a gene was promoted in MuNPR1 transgenic Arabidopsis. The MuNPR1 transgenic plants showed more resistance to Pseudomonas syringae pv. tomato DC3000 (Pst. DC3000) than did the wild-type Arabidopsis. Moreover, the ectopic expression of MuNPR1 might lead to enhanced scavenging ability and suppress collase accumulation. In contrast, the MuNPR4 transgenic Arabidopsis were hypersensitive to Pst. DC3000 infection. In addition, transgenic Arabidopsis with the ectopic expression of either MuNPR1 or MuNPR4 showed sensitivity to salt and drought stresses. Our data suggest that both the MuNPR1 and MuNPR4 genes play a role in the coordination between signaling pathways, and the information provided here enables the in-depth functional analysis of the MuNPR1 and MuNPR4 genes and may promote mulberry resistance breeding in the future.

Functional Characteristics of Caffeoyl Shikimate Esterase in Larix Kaempferi and Monolignol Biosynthesis in Gymnosperms.

Caffeoyl shikimate esterase (CSE) has been reported to be involved in lignin biosynthesis; however, studies of CSE in gymnosperms are lacking. In this study, CSE was successfully cloned from Larix kaempferi (LkCSE) based on Larix laricina transcriptome screening. LkCSE was likely to have catalytic activity based on homologous sequence alignment and phylogenetic analyses of CSEs from different species. In vitro assays with the recombinant enzyme validated the catalytic activity of LkCSE, indicating its function in converting caffeoyl shikimate into caffeate and shikimate. Additionally, the optimum reaction pH and temperature of LkCSE were determined to be 6.0 and 30 °C, respectively. The values of Km and Vmax of CSE for caffeoyl shikimate were 98.11 µM and 14.44 nM min-1, respectively. Moreover, LkCSE was observed to have tissue expression specificity and was abundantly expressed in stems and leaves, especially stems, which was 50 times higher than the expression levels of roots. Lastly, translational fusion assays using LkCSE fused with green fluorescent proteins (GFP) in tobacco leaves indicated that LkCSE was localized in the plasma membrane and endoplasmic reticulum (ER). These results revealed that CSE clearly functions in gymnosperms and it is possible for LkCSE to interact with other ER-resident proteins and regulate mass flux in the monolignol biosynthesis pathway.

Characterization of the cinnamoyl-CoA reductase (CCR) gene family in Populus tomentosa reveals the enzymatic active sites and evolution of CCR.

MAIN CONCLUSION: Two distinct cinnamoyl-coenzyme A reductases (CCRs) from Populus tomentosa were cloned and studied and active sites in CCRs were further identified based on sequence divergence, molecular simulation, and site-directed mutants. Cinnamoyl-coenzyme A (CoA) reductase (CCR) is the first committed gene in the lignin-specific pathway and plays a role in the lignin biosynthesis pathway. In this study, we cloned 11 genes encoding CCR or CCR-like proteins in Populus tomentosa. An enzymatic assay of the purified recombinant P. tomentosa (Pto) CCR and PtoCCR-like proteins indicated that only PtoCCR1 and PtoCCR7 had detectable activities toward hydroxycinnamoyl-CoA esters. PtoCCR1 exhibited specificity for feruloyl-CoA, with no detectable activity for any other hydroxycinnamoyl-CoA esters. However, PtoCCR7 catalyzed p-coumaroyl-CoA, caffeoyl-CoA, feruloyl-CoA, and sinapoyl-CoA with a preference for feruloyl-CoA. Site-directed mutations of selected amino acids divergent between PtoCCR1 and 7, combined with modeling and docking, showed that A132 in CCR7 combined with the catalytic triad might comprise the catalytic center. In CCR7, L192, F155, and H208 were identified as the substrate-binding sites, and site-directed mutations of these amino acids showed obvious changes in catalytic efficiency with respect to both feruloyl-CoA and sinapoyl-CoA. Mutant F155Y exhibited greater catalytic efficiency for sinapoyl-CoA compared with that of wild-type PtoCCR7. Finally, recent genome duplication events provided the foundation for CCR divergence. This study further identified the active sites in CCRs and the evolutionary process of CCRs in terrestrial plants.

Two distinct cinnamoyl-CoA reductases in Selaginella moellendorffii offer insight into the divergence of CCRs in plants.

MAIN CONCLUSION: Two distinct cinnamoyl-coenzyme A reductases (CCRs) from Selaginella moellendorffii were evaluated, and of these, SmCCR2-1, which has both distinct sequence motifs and catalytic properties, was clustered into a new CCR subgroup. Cinnamoyl-coenzyme A reductases (CCRs) have been reported in many land plants to have critical functions in monolignol biosynthesis. In this study, we performed a genome-wide screen and obtained two distinct SmCCRs from S. moellendorffii. Phylogenetic analysis indicated that SmCCR2 (both SmCCR2-1 and 2-2) and SmCCR3 together with PpaCCR belong to a distinct subgroup of genuine CCRs with variations in the NAD(P)H-binding motif. Enzymatic assays showed detectable activity by both SmCCR1 and SmCCR2-1 toward four hydroxycinnamoyl-CoA esters. SmCCR1, which clustered with reported CCRs from angiosperms and gymnosperms, exhibited specificity toward feruloyl-CoA, while SmCCR2-1 showed a preference for sinapoyl-CoA. Interestingly, the reaction temperature profiles for SmCCR1 and SmCCR2-1 are complementary. Homology models and molecular simulations suggest that the variations in NADPH-binding motifs, especially R(X)6K instead of R(X)5K, affect the NADP+ conformation. Notably, the signature motif NWYCY was replaced with NGYCL in SmCCR1 and with EWYCL in SmCCR2-1, while the signature residues H202 and R253, reported in a previous study, were conserved in SmCCR1 and SmCCR2-1 but varied in SmCCR-like genes. It is likely that NWYCY is not a reliable signature for CCRs in plants. The detectable activity of site-direct mutant S123T of SmCCR1 suggested that S123 which consists of catalytic triad is changeable. Possible evolution process for the emergence of two subgroups of genuine CCRs was also revealed. Altogether, these findings revise our understanding of CCRs with regard to divergence and active sites.

Three steps in one pot: biosynthesis of 4-hydroxycinnamyl alcohols using immobilized whole cells of two genetically engineered Escherichia coli strains.

BACKGROUND: 4-Hydroxycinnamyl alcohols are a class of natural plant secondary metabolites that include p-coumaryl alcohol, caffeyl alcohol, coniferyl alcohol and sinapyl alcohol, and have physiological, ecological and biomedical significance. While it is necessary to investigate the biological pathways and economic value of these alcohols, research is hindered because of their limited availability and high cost. Traditionally, these alcohols are obtained by chemical synthesis and plant extraction. However, synthesis by biotransformation with immobilized microorganisms is of great interest because it is environmentally friendly and offers high stability and regenerable cofactors. Therefore, we produced 4-hydroxycinnamyl alcohols using immobilized whole cells of engineered Escherichia coli as the biocatalyst. RESULTS: In this study, we used the recombinant E. coli strain, M15-4CL1-CCR, expressing the fusion protein 4-coumaric acid: coenzyme A ligase and the cinnamoyl coenzyme A reductase and a recombinant E. coli strain, M15-CAD, expressing cinnamyl alcohol dehydrogenase from Populus tomentosa (P. tomentosa). High performance liquid chromatography and mass spectrometry showed that the immobilized whole cells of the two recombinant E. coli strains could effectively convert the phenylpropanoic acids to their corresponding 4-hydroxycinnamyl alcohols. Further, the optimum buffer pH and the reaction temperature were pH 7.0 and 30 °C. Under these conditions, the molar yield of the p-coumaryl alcohol, the caffeyl alcohol and the coniferyl alcohol was around 58, 24 and 60%, respectively. Moreover, the highly sensitive and selective HPLC-PDA-ESI-MSn method used in this study could be applied to the identification and quantification of these aromatic polymers. CONCLUSIONS: We have developed a dual-cell immobilization system for the production of 4-hydroxycinnamyl alcohols from inexpensive phenylpropanoic acids. This biotransformation method is both simple and environmental-friendly, which is promising for the practical and cost effective synthesis of natural products. Graphical abstract Biotransformation process of phenylpropanoic acids by immobilized whole-cells.

Overexpression of artificially fused bifunctional enzyme 4CL1-CCR: a method for production of secreted 4-hydroxycinnamaldehydes in Escherichia coli.

BACKGROUND: 4-Hydroxycinnamaldehydes are important intermediates in several secondary metabolism pathways, including those involved in the biosynthesis of phenolic acids, flavonoids, terpenoids and monolignols. They are also involved in the biosynthesis and degradation of lignins, which are important limiting factors during the processes of papermaking and biofuel production. Access to these aromatic polymers is necessary to explore the secondary biometabolic pathways they are involved in. Coniferaldehyde, sinapaldehyde, p-coumaraldehyde and caffealdehyde are members of the 4-hydroxycinnamaldehyde family. Although coniferaldehyde and sinapaldehyde can be purchased from commercial sources, p-coumaraldehyde and caffealdehyde are not commercially available. Therefore, there is increasing interest in producing 4-hydroxycinnamaldehydes. Here, we attempted to produce 4-hydroxycinnamaldehydes using engineered Escherichia coli. RESULTS: 4-Coumaric acid: coenzyme A ligase (4CL1) and cinnamoyl coenzyme A reductase (CCR) were fused by means of genetic engineering to generate an artificial bifunctional enzyme, 4CL1-CCR, which was overexpressed in cultured E. coli supplemented with phenylpropanoic acids. Three 4-hydroxycinnamaldehydes, p-coumaraldehyde, caffealdehyde and coniferaldehyde, were thereby biosynthesized and secreted into the culture medium. The products were extracted and purified from the culture medium, and identically characterized by the HPLC-PDA-ESI-MSn. The productivity of this new metabolic system were 49 mg/L for p-coumaraldehyde, 19 mg/L for caffealdehyde and 35 mg/L for coniferaldehyde. Extracellular hydroxycinnamoyl-coenzyme A thioesters were not detected, indicating that these thioesters could not pass freely through the cellular membrane. The fusion enzyme 4CL1-CCR can catalyze sequential multistep reactions, thereby avoiding the permeability problem of intermediates, which reveals its superiority over a mixture of individual native enzymes. Moreover, we have described a highly sensitive and selective method for separation and identification of phenylpropanoic acids and their corresponding cinnamaldehydes in the present paper. The feasibility of this method has been proven in the application of the method to the analysis of the metabolites of whole-cell catalysts. CONCLUSIONS: We have established a bioconversion pathway for the microbial production of valuable 4-hydroxycinnamaldehydes from phenylpropanoic acids. This biotransformation method is both convenient and environmentally friendly, and provides new insights into the biosynthesis of natural plant secondary products.

Molecular cloning and functional analysis of nine cinnamyl alcohol dehydrogenase family members in Populus tomentosa.

MAIN CONCLUSION: Nine CAD/CAD-like genes in P. tomentosa were classified into four classes based on expression patterns, phylogenetic analysis and biochemical properties with modification for the previous claim of SAD. Cinnamyl alcohol dehydrogenase (CAD) functions in monolignol biosynthesis and plays a critical role in wood development and defense. In this study, we isolated and cloned nine CAD/CAD-like genes in the Populus tomentosa genome. We investigated differential expression using microarray chips and found that PtoCAD1 was highly expressed in bud, root and vascular tissues (xylem and phloem) with the greatest expression in the root. Differential expression in tissues was demonstrated for PtoCAD3, PtoCAD6 and PtoCAD9. Biochemical analysis of purified PtoCADs in vitro indicated PtoCAD1, PtoCAD2 and PtoCAD8 had detectable activity against both coniferaldehyde and sinapaldehyde. PtoCAD1 used both substrates with high efficiency. PtoCAD2 showed no specific requirement for sinapaldehyde in spite of its high identity with so-called PtrSAD (sinapyl alcohol dehydrogenase). In addition, the enzymatic activity of PtoCAD1 and PtoCAD2 was affected by temperature. We classified these nine CAD/CAD-like genes into four classes: class I included PtoCAD1, which was a bone fide CAD with the highest activity; class II included PtoCAD2, -5, -7, -8, which might function in monolignol biosynthesis and defense; class III genes included PtoCAD3, -6, -9, which have a distinct expression pattern; class IV included PtoCAD12, which has a distinct structure. These data suggest divergence of the PtoCADs and its homologs, related to their functions. We propose genes in class II are a subset of CAD genes that evolved before angiosperms appeared. These results suggest CAD/CAD-like genes in classes I and II play a role in monolignol biosynthesis and contribute to our knowledge of lignin biosynthesis in P. tomentosa.

A transcriptomic network underlies microstructural and physiological responses to cadmium in Populus x canescens.

Bark tissue of Populus × canescens can hyperaccumulate cadmium, but microstructural, transcriptomic, and physiological response mechanisms are poorly understood. Histochemical assays, transmission electron microscopic observations, energy-dispersive x-ray microanalysis, and transcriptomic and physiological analyses have been performed to enhance our understanding of cadmium accumulation and detoxification in P. × canescens. Cadmium was allocated to the phloem of the bark, and subcellular cadmium compartmentalization occurred mainly in vacuoles of phloem cells. Transcripts involved in microstructural alteration, changes in nutrition and primary metabolism, and stimulation of stress responses showed significantly differential expression in the bark of P. × canescens exposed to cadmium. About 48% of the differentially regulated transcripts formed a coregulation network in which 43 hub genes played a central role both in cross talk among distinct biological processes and in coordinating the transcriptomic regulation in the bark of P. × canescens in response to cadmium. The cadmium transcriptome in the bark of P. × canescens was mirrored by physiological readouts. Cadmium accumulation led to decreased total nitrogen, phosphorus, and calcium and increased sulfur in the bark. Cadmium inhibited photosynthesis, resulting in decreased carbohydrate levels. Cadmium induced oxidative stress and antioxidants, including free proline, soluble phenolics, ascorbate, and thiol compounds. These results suggest that orchestrated microstructural, transcriptomic, and physiological regulation may sustain cadmium hyperaccumulation in P. × canescens bark and provide new insights into engineering woody plants for phytoremediation.

Metabolomic analysis reveals the potential metabolites and pathogenesis involved in mulberry yellow dwarf disease.

To analyse the molecular mechanisms of phytoplasma pathogenicity, the comprehensive metabolomic changes of mulberry leaf and phloem sap in response to phytoplasma infection were examined using gas chromatography-mass spectrometry. The metabolic profiles obtained revealed that the metabolite compositions of leaf and phloem sap were different, and phytoplasma infection has a greater impact on the metabolome of phloem sap than of leaf. Phytoplasma infection brought about the content changes in various metabolites, such as carbohydrates, amino acids, organic acids, etc. Meanwhile, the results of biochemical analysis showed that the degradation of starch was repressed, and the starch content was increased in the infected leaves. In addition, we found that phytoplasma infection changed the levels of abscisic acid and cytokinin and break phytohormone balance. Interestingly, our data showed that the contents of H2O2 and superoxide were increased in the infected leaves, but not in the phloem saps. Based on the results, the expression levels of the genes involved in the metabolism of some changed metabolites were examined, and the potential molecular mechanisms of these changes were discussed. It can be concluded that both the leaf and phloem saps have a complicated metabolic response to phytoplasma infection, but their response mechanisms were different.