Several studies have focused on cold tolerance in multiple regulated levels. However, a genome-scale molecular analysis of the regulated network under the control of transcription factors (TFs) is still lacking, especially for trees. To comprehensively identify the TFs that regulate cold stress response in the paper mulberry and understand their regulatory interactions, transcriptomic data was used to assess changes in gene expression induced by exposure to cold. Results indicated that 794 TFs, belonging to 47 families and comprising more than 59% of the total TFs of this plant, were involved in the cold stress response. They were clustered into three groups, namely early, intermediate and late responsive groups which contained 95, 550 and 149 TFs, respectively. Among of these differentially expressed TFs, one bHLH, two ERFs and three CAMTAs were considered to be the key TFs functioning in the primary signal transduction. After that, at the intermediate stage of cold stress, there were mainly two biological processes that were regulated by TFs, namely cold stress resistance (including 5 bHLH, 14 ERFs, one HSF, 4 MYBs, 3 NACs, 11 WRKYs and so on) and growth and development of lateral organ or apical meristem (including ARR-B, B3, 5 bHLHs, 2 C2H2, 4 CO-like, 2 ERF, 3 HD-ZIP, 3 YABBYs, G2-like, GATA, GRAS and TCP). In late responsive group, 3 ARR-B, C3H, 6 CO-like, 2 G2-like, 2 HSFs, 2 NACs and TCP. Most of them presented the up-regulated expression at 12 or 24 hours after cold stress implied their important roles for the new growth homeostasis under cold stress. Our study identified the key TFs that function in the regulatory cascades mediating the activation of downstream genes during cold tress tolerance in the paper mulberry. Based on the analysis, we found that the AP2/ERF, bHLH, MYB, NAC and WRKY families might play the central and significant roles during cold stress response in the paper mulberry just as in other species. Meanwhile, many other TF families previously reported as involving in regulation of growth and development, including ARF, DBB, G2-like, GRF, GRAS, LBD, WOX and YAABY exhibited their important potential function in growth regulation under cold stress.

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Transcriptional regulation of the paper mulberry under cold stress as revealed by a comprehensive analysis of transcription factors.

Most studies on the paper mulberry are mainly focused on the medicated and pharmacology, fiber quality, leaves feed development, little is known about its mechanism of adaptability to abiotic stress. Physiological measurement, transcriptomics and proteomic analysis were employed to understand its response to cold stress in this study. The second to fourth fully expanded leaves from up to down were harvested at different stress time points forthe transmission electron microscope (TEM) observation. Physiological characteristics measurement included the relative electrolyte leakage (REL), SOD activity assay, soluble sugar content, and Chlorophyll fluorescence parameter measurement. For screening of differentially expressed genes, the expression level of every transcript in each sample was calculated by quantifying the number of Illumina reads. To identify the differentially expressed protein, leaves of plants under 0, 6, 12, 24, 48 and 72 h cold stress wereharvested for proteomic analysis. Finally, real time PCR was used to verify the DEG results of the RNA-seq and the proteomics data. Results showed that at the beginning of cold stress, respiratory metabolism was decreased and the transportation and hydrolysis of photosynthetic products was inhibited, leading to an accumulation of starch in the chloroplasts. Total of 5800 unigenes and 38 proteins were affected, including the repressed expression of photosynthesis and the enhanced expression in signal transduction, stress defense pathway as well as secondary metabolism. Although the transcriptional level of a large number of genes has been restored after 12 h, sustained cold stress brought more serious injury to the leaf cells, including the sharp rise of the relative electrolyte leakage, the declined Fv/Fm value, swelled chloroplast and the disintegrated membrane system. The starch accumulation and the photoinhibition might be the main adaptive mechanism of the paper mulberry responded to cold stress. Most of important, enhancing the transport and hydrolysis of photosynthetic products could be the potential targets for improving the cold tolerance of the paper mulberry.

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The cold responsive mechanism of the paper mulberry: decreased photosynthesis capacity and increased starch accumulation

Artemisia argyi Lev. et Vant. (A. argyi) is widely utilized for moxibustion in Chinese medicine, and the mechanism underlying terpenoid biosynthesis in its leaves is suggested to play an important role in its medicinal use. However, the A. argyi transcriptome has not been sequenced. Herein, we performed RNA sequencing for A. argyi leaf, root and stem tissues to identify as many as possible of the transcribed genes. In total, 99,807 unigenes were assembled by analysing the expression profiles generated from the three tissue types, and 67,446 of those unigenes were annotated in public databases. We further performed differential gene expression analysis to compare leaf tissue with the other two tissue types and identified numerous genes that were specifically expressed or up-regulated in leaf tissue. Specifically, we identified multiple genes encoding significant enzymes or transcription factors related to terpenoid synthesis. This study serves as a valuable resource for transcriptome information, as many transcribed genes related to terpenoid biosynthesis were identified in the A. argyi transcriptome, providing a functional genomic basis for additional studies on molecular mechanisms underlying the medicinal use of A. argyi.

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De novo assembly and analysis of the Artemisia argyi transcriptome and identification of genes involved in terpenoid biosynthesis.

Vicia faba (L.) is an important cool-season grain legume species used widely in agriculture but also in plant physiology research, particularly as an experimental model to study transfer cell (TC) development. TCs are specialized nutrient transport cells in plants, characterized by invaginated wall ingrowths with amplified plasma membrane surface area enriched with transporter proteins that facilitate nutrient transfer. Many TCs are formed by trans-differentiation from differentiated cells at apoplasmic/symplasmic boundaries in nutrient transport. Adaxial epidermal cells of isolated cotyledons can be induced to form functional TCs, thus providing a valuable experimental system to investigate genetic regulation of TC trans-differentiation. The genome of V. faba is exceedingly large (ca. 13 Gb), however, and limited genomic information is available for this species. To provide a resource for future transcript profiling of epidermal TC differentiation, we have undertaken de novo assembly of a genome-wide transcriptome map for V. faba. Illumina paired-end sequencing of total RNA pooled from different tissues and different stages, including isolated cotyledons induced to form epidermal TCs, generated 69.5 M reads, of which 65.8 M were used for assembly following trimming and quality control. Assembly using a De-Bruijn graph-based approach generated 21,297 contigs, of which 80.6% were successfully annotated against GO terms. The assembly was validated against known V. faba cDNAs held in GenBank, including transcripts previously identified as being specifically expressed in epidermal cells across TC trans-differentiation. This genome-wide transcriptome map therefore provides a valuable tool for future transcript profiling of epidermal TC trans-differentiation, and also enriches the genetic resources available for this important legume crop species.

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De novo assembly of a genome-wide transcriptome map of Vicia faba (L.) for transfer cell research

Paper mulberry is a valuable woody species with a good chilling tolerance. In this study, phosphoproteomic analysis, physiological measurement, and mRNA quantification were employed to explore the molecular mechanism of chilling (4 °C) tolerance in paper mulberry. After chilling for 6 h, 427 significantly changed phosphoproteins were detected in paper mulberry seedlings without obvious physiological injury. When obvious physiological injury occurred after chilling for 48 h, a total of 611 phosphoproteins were found to be significantly changed at the phosphorylation level. Several protein kinases, especially CKII, were possibly responsible for these changes according to conserved sequence analysis. The results of Gene Ontology analysis showed that phosphoproteins were mainly responsible for signal transduction, protein modification, and translation during chilling. Additionally, transport and cellular component organization were enriched after chilling for 6 and 48 h, respectively. On the basis of the prot...

Phosphoproteomic Analysis of Paper Mulberry Reveals Phosphorylation Functions in Chilling Tolerance.

The paper mulberry is one of the multifunctional tree species in agroforestry systems and is also commonly utilized in traditional medicine in China and other Asian countries. However, little is known about its molecular genetics, which hinders research on and exploitation of this valuable resource. To discern the correlation between gene expression and the essential properties of the paper mulberry, we performed a transcriptomics analysis, assembling a total of 37,725 unigenes from 54,638,676 reads generated by RNA-seq. Among these, 22,692 unigenes showed greater than 60% similarity with genes from other species. The lengths of 13,566 annotated unigenes were longer than 1,000 bp. Functional clustering analysis with COG (Cluster of Orthologous Groups) revealed that 17,184 unigenes are primarily involved in transcription, translation, signal transduction, carbohydrate metabolism, secondary metabolism, and energy metabolism. GO (Gene Ontology) annotation suggests enrichment of genes encoding antioxidant activity, transporter activity, biosynthesis, metabolism and stress response, with a total of 30,659 unigenes falling in these categories. KEGG (Kyoto Encyclopedia of Genes and Genomes) metabolic pathway analysis showed that 7,199 unigenes are associated with 119 metabolic pathways. In addition to the basic metabolism, these genes are enriched for plant pathogen interaction, flavonoid metabolism and other secondary metabolic processes. Furthermore, differences in the transcriptomes of leaf, stem and root tissues were analyzed and 7,233 specifically expressed unigenes were identified. This global expression analysis provided novel insights about the molecular mechanisms of the biosynthesis of flavonoid, lignin and cellulose, as well as on the response to biotic and abiotic stresses including the remediation of contaminated soil by the paper mulberry.

De Novo assembly of expressed transcripts and global transcriptomic analysis from seedlings of the paper mulberry (Broussonetia kazinoki x Broussonetia papyifera).

Green macroalgae Chaetomorpha aerea and C. linum are taxonomically confused. In this paper, we tried morphological and molecular analyses to separate these two species. C. aerea and C. linum can be distinguished from morphological characteritics, such as frond dimension, cells size and shape, their mean length/width ratios (LWR), and cell walls constriction. Thalli of C. aerea attenuate basipetally, with diameter 270-500 mu m at upper portion, 160-360 mu m at middle portion, 100-160 mu m at basal portion. For the upper part, the length of cells is less than their diameter. Cell walls usually constrict at the dissepiments, which are pellucid or colorless and give the filament beaded appearance. In contrast, thalli of C. linum often have a constant diameter of 90-300 mu m within the same individual, cell walls usually do not constrict and cells are cylindrical or barrel shaped. The LWR is larger than that of C. aerea. Results show that the pairwise distance between two species is 3.6%-3.7% for 18S rRNA gene and 53.5%-54.3% for ITS region. In phylogeny, they distribute at distant clades, which confirms a genetic divergence at molecular level. In addition, morphological data indicates that filament diameter of C. linum samples is highly variable, ranging from 90 mu m to 300 mu m. Then these two species can be considered as separate species.

Teng Linhong

Papers

De Novo assembly of expressed transcripts and global transcriptomic analysis from seedlings of the paper mulberry (Broussonetia kazinoki x Broussonetia papyifera).

Morphological and molecular discrimination of green macroalgae Chaetomorpha aerea and C. linum