Evolution of the WRKY Family in Angiosperms and Functional Diversity under Environmental Stress.

The transcription factor is an essential factor for regulating the responses of plants to external stimuli. The WRKY protein is a superfamily of plant transcription factors involved in response to various stresses (e.g., cold, heat, salt, drought, ions, pathogens, and insects). During angiosperm evolution, the number and function of WRKY transcription factors constantly change. After suffering from long-term environmental battering, plants of different evolutionary statuses ultimately retained different numbers of WRKY family members. The WRKY family of proteins is generally divided into three large categories of angiosperms, owing to their conserved domain and three-dimensional structures. The WRKY transcription factors mediate plant adaptation to various environments via participating in various biological pathways, such as ROS (reactive oxygen species) and hormone signaling pathways, further regulating plant enzyme systems, stomatal closure, and leaf shrinkage physiological responses. This article analyzed the evolution of the WRKY family in angiosperms and its functions in responding to various external environments, especially the function and evolution in Magnoliaceae plants. It helps to gain a deeper understanding of the evolution and functional diversity of the WRKY family and provides theoretical and experimental references for studying the molecular mechanisms of environmental stress.

Genome-wide characterization of the SPL gene family involved in the age development of Jatropha curcas.

BACKGROUND: SPL (SQUAMOSA-promoter binding protein-like) proteins form a large family of plant-specific transcription factors that play essential roles in various aspects of plant growth and development. They are potentially important candidates for genetic improvement of agronomic traits. However, there were limited information about the SPL genes in Jatropha curcas, an important biofuel plant. RESULTS: In Jatropha, 15 JcSPL genes were identified. Phylogenetic analysis revealed that most of the JcSPLs were closely related to SPLs from woody plant rather than herbaceous plant and distantly related to monocotyledon SPLs. Gene structure, conserved motif and repetitive sequence analysis indicated diverse and specific functions of some JcSPL genes. By combination of target prediction and degradome sequencing analysis, 10 of the 15 JcSPLs were shown to be targets of JcmiR156. Quantitative PCR analysis showed diversified spatial-temporal expression patterns of JcSPLs. It is interesting that the expression levels of JcSPL3 were the highest in all tissues examined in 7- or 10-year-old plants and exhibited increasing trend with plant age, suggesting its important role in the regulation of age development in Jatropha. Overexpression of JcSPL3 in Arabidopsis resulted in earlier flowering time, shorter silique length and reduced biomass of roots. CONCLUSIONS: Through comprehensive and systematic analysis of phylogenetic relationships, conserved motifs, gene structures, chromosomal locations, repetitive sequence and expression patterns, 15 JcSPL genes were identified in Jatropha and characterized in great detail. These results provide deep insight into the evolutionary origin and biological significance of plant SPLs and lay the foundation for further functional characterization of JcSPLs with the purpose of genetic improvement in Jatropha.

Regulation of flowering time by SPL10/MED25 module in Arabidopsis.

Several SQUAMASA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors are involved in plant developmental transition from vegetative to reproductive growth. However, the function of SPL10 in regulating floral transition is largely unknown. It is also not known which Mediator subunit mediates SPL10 transcriptional activity. Here, we used overexpression lines and knockout mutants to examine the role of SPL10 in flowering-time regulation and we investigated possible interactions of SPL10 with several mediator subunits in vitro and in vivo. Plants overexpressing SPL10 showed precocious flowering, whereas the triple loss-of-function mutants of SPL10 and its two homologous genes, SPL2 and SPL11, flowered late compared with wild-type plants. We found that SPL10 interacts with MED25, a subunit of the Mediator complex, which bridges transcription factors and RNA polymerase II to facilitate transcription initiation. Genetic analysis showed that MED25 acts downstream of SPL10 to execute SPL10-regulated floral transition. Furthermore, SPL10 was required for MED25 association with the promoters of two target genes, FUL and LFY. We provide evidence that SPL10 recruits MED25 to the promoters of target genes to regulate flowering time. Our results on the SPL10/MED25 module are relevant to the molecular mechanism of other SPL family members.

Identification and characterization of a novel esterase from Thauera sp.

A novel esterase gene TLip was identified from the strain Thauera sp. and expressed at high levels in Escherichia coli. The TLip protein shared the highest identity (48%) to esterase TesA from Pseudomonas aeruginosa when compared to enzymes with reported properties. Phylogenetic analysis showed that TLip belongs to the GDSL family of bacterial lipolytic enzymes. TLip was an alkaline esterase with a broad optimal temperature range 37-50 °C and an optimal pH of 8.0. Substrate specificity assays showed that TLip preferred medium chain p-nitrophenyl esters (C6 -C12 ). Besides, the activity of TLip was strongly inhibited by Cu2+ but greatly enhanced by Triton X-100 and Tween 80. Thermostability assay revealed that TLip was stable without loss of activity at 37 °C and still retained 69% activity at 50 °C after 2 H of incubation. Together, these provided a good candidate for further exploration of TLip as a promising biocatalyst in industry.

The role of miR156/SPLs modules in Arabidopsis lateral root development.

miR156 is an evolutionarily highly conserved miRNA in plants that defines an age-dependent flowering pathway. The investigations thus far have largely, if not exclusively, confined to plant aerial organs. Root branching architecture is a major determinant of water and nutrients uptake for plants. We show here that MIR156 genes are differentially expressed in specific cells/tissues of lateral roots. Plants overexpressing miR156 produce more lateral roots whereas reducing miR156 levels leads to fewer lateral roots. We demonstrate that at least one representative from the three groups of miR156 targets SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes: SPL3, SPL9 and SPL10 are involved in the repression of lateral root growth, with SPL10 playing a dominant role. In addition, both MIR156 and SPLs are responsive to auxin signaling suggesting that miR156/SPL modules might be involved in the proper timing of the lateral root developmental progression. Collectively, these results unravel a role for miR156/SPLs modules in lateral root development in Arabidopsis.

Analysis of non-coding transcriptome in rice and maize uncovers roles of conserved lncRNAs associated with agriculture traits.

Long non-coding RNAs (lncRNAs) have recently been found to widely exist in eukaryotes and play important roles in key biological processes. To extend our knowledge of lncRNAs in crop plants we performed both non-directional and strand-specific RNA-sequencing experiments to profile non-coding transcriptomes of various rice and maize organs at different developmental stages. Analysis of more than 3 billion reads identified 22 334 long intergenic non-coding RNAs (lincRNAs) and 6673 pairs of sense and natural antisense transcript (NAT). Many lincRNA genes were associated with epigenetic marks. Expression of rice lincRNA genes was significantly correlated with that of nearby protein-coding genes. A set of NAT genes also showed expression correlation with their sense genes. More than 200 rice lincRNA genes had homologous non-coding sequences in the maize genome. Much more lincRNA and NAT genes were derived from conserved genomic regions between the two cereals presenting positional conservation. Protein-coding genes flanking or having a sense-antisense relationship to these conserved lncRNA genes were mainly involved in development and stress responses, suggesting that the associated lncRNAs might have similar functions. Integrating previous genome-wide association studies (GWAS), we found that hundreds of lincRNAs contain trait-associated SNPs (single nucleotide polymorphisms [SNPs]) suggesting their putative contributions to developmental and agriculture traits.

Age-dependent modulation of oleoresin production in the stem of Sindora glabra.

Plants produce specialized metabolites in various organs which serve important functions in defense and development. However, the molecular regulatory mechanisms of oleoresin production in stems from broadleaved tree species are not fully understood. To determine whether endogenous developmental cues play a role in the regulation of oleoresin biosynthesis in tree stems, anatomy, multi-omics and molecular experiments were utilized to investigate the change of secretory structures, chemical profiles and gene expression in different ontogenetic stages of Sindora glabra tree, which accumulates copious amount of sesquiterpene-rich oleoresin in stems. The size of secretory canals and the concentration of five sesquiterpenes in Sindora stems exhibited obvious increase with plant age, from 0.5- to 20-year-old plants. Moreover, α-copaene and ß-copaene were found to be stem-specific sesquiterpenes. Metabolomic analysis revealed that salicylic acid highly accumulated in mature stems, but the content of triterpenes was greatly decreased. The expression of three repressors AUX/IAA, DELLA and JAZ involved in hormone signaling transduction pathways was significantly downregulated in stems of 10- and 20-year-old plants. Two key genes SgTPS3 and SgTPS5 were identified, whose expression was highly correlated with the accumulation patterns of specific sesquiterpenes and their enzymatic products were consistent with the chemical profiles in the stem. The promoters of three SgTPSs exhibiting high activity were isolated. Furthermore, we demonstrated that SgSPL15 directly interacts with SgTPS3 and SgTPS5 promoters and activates SgTPS5 expression but SgSPL15 inhibits SgTPS3 expression. In addition, SgSPL15 enhanced sesquiterpene levels by upregulating AtTPSs expression in Arabidopsis. These results suggested that sesquiterpene biosynthesis in S. glabra stem was dependent on the regulation of endogenous hormones as well as plant age, and SgSPL15 might act as a buffering factor to regulate sesquiterpene biosynthesis by targeting SgTPS genes.

The Diesel Tree Sindora glabra Genome Provides Insights Into the Evolution of Oleoresin Biosynthesis.

Sindora glabra is an economically important tree that produces abundant oleoresin in the trunk. Here, we present a high-quality chromosome-scale assembly of S. glabra genome by combining Illumina HiSeq, Pacific Biosciences sequencing, and Hi-C technologies. The size of S. glabra genome was 1.11 Gb, with a contig N50 of 1.27 Mb and 31,944 predicted genes. This is the first sequenced genome of the subfamily Caesalpinioideae. As a sister taxon to Papilionoideae, S. glabra underwent an ancient genome triplication shared by core eudicots and further whole-genome duplication shared by early-legume in the last 73.3 million years. S. glabra harbors specific genes and expanded genes largely involved in stress responses and biosynthesis of secondary metabolites. Moreover, 59 terpene backbone biosynthesis genes and 64 terpene synthase genes were identified, which together with co-expressed transcription factors could contribute to the diversity and specificity of terpene compounds and high terpene content in S. glabra stem. In addition, 63 disease resistance NBS-LRR genes were found to be unique in S. glabra genome and their expression levels were correlated with the accumulation of terpene profiles, suggesting potential defense function of terpenes in S. glabra. These together provide new resources for understanding genome evolution and oleoresin production.

Integrated transcriptomic and metabolomic analyses reveal regulation of terpene biosynthesis in the stems of Sindora glabra.

Sesquiterpenes are important defensive secondary metabolites that are synthesized in various plant organs. Methyl jasmonate (MeJA) plays a key role in plant defense responses and secondary metabolism. Sindora glabra Merr. ex de Wit produces abundant sesquiterpenes in its trunks, and was subjected to investigation after MeJA treatment in order to characterize the molecular mechanisms underlying the regulation of sesquiterpene biosynthesis in plant stems and further our understanding of oleoresin production in trees. A total of 14 types of sesquiterpenes in the stems of mature S. glabra trees were identified. The levels of two sesquiterpenes, α-copaene and ß-caryophyllene, significantly increased after MeJA treatment. Differentially expressed genes involved in terpenoid backbone biosynthesis were significantly enriched over time, while the expression of JAZ genes involved in the jasmonic acid signaling pathway and TGA genes involved in the salicylic acid signaling pathway was significantly enriched at later time points after treatment. Two new terpene synthase genes, SgSTPS4 and SgSTPS5, were also identified. Following MeJA treatment, the expression levels of SgSTPS1, SgSTPS2 and SgSTPS4 decreased, while SgSTPS5 expression increased. The major enzymatic products of SgSTPS4 were identified as ß-elemene and cyperene, while SgSTPS5 was identified as a bifunctional mono/sesquiterpene synthase that could catalyze farnesyl pyrophosphate to produce nine types of sesquiterpenes, including α-copaene and ß-caryophyllene, while SgSTPS5 could also use geranyl pyrophosphate to produce geraniol. Dramatic changes in the amounts of α-copaene and ß-caryophyllene in response to MeJA were correlated with transcriptional expression changes of SgSTPS5 in the wood tissues. In addition, the transcription factors MYB, NAC, ARF, WRKY, MYC, ERF and GRAS were co-expressed with terpene biosynthesis genes and might potentially regulate terpene biosynthesis. Metabolite changes were further investigated with UPLC-TOF/MS following MeJA treatment. These results contribute to the elucidation of the molecular mechanisms of terpene biosynthesis and regulation as well as to the identification of candidate genes involved in these processes.

Transcriptome Analysis of Oleoresin-Producing Tree Sindora Glabra and Characterization of Sesquiterpene Synthases.

Terpenes serve important physiological and ecological functions in plants. Sindora glabra trees accumulate copious amounts of sesquiterpene-rich oleoresin in the stem. A transcriptome approach was used to determine the unique terpene biosynthesis pathway and to explore the different regulatory mechanisms responsible for the variation of terpene content among individuals. Analysis of de novo-assembled contigs revealed a complete set of genes for terpene biosynthesis. A total of 23,261 differentially expressed unigenes (DEGs) were discovered between high and low oil-yielding plants. DEG enrichment analysis suggested that the terpene biosynthesis process and the plant hormone signal transduction pathway may exert a major role in determining terpene variation in S. glabra. The expression patterns of candidate genes were further verified by quantitative RT-PCR experiments. Key genes involved in the terpene biosynthesis pathway were predominantly expressed in phloem and root tissues. Phylogenetic analysis and subcellular localization implied that S. glabra terpene synthases may evolve from a common ancestor. Furthermore, two sesquiterpene synthase genes, SgSTPS1 and SgSTPS2, were functionally characterized. SgSTPS1 mainly generated ß-caryophyllene from farnesyl pyrophosphate. SgSTPS2 is a versatile enzyme that catalyzes the formation of 12 sequiterpenes from farnesyl pyrophosphate and synthesis of three monoterpenes using geranyl pyrophosphate. Together, these results provide large reservoir for elucidating the molecular mechanism of terpene biosynthesis and for exploring the ecological function of sesquiterpenes in S. glabra.

Electron beam melting in the fabrication of three-dimensional mesh titanium mandibular prosthesis scaffold.

The study was designed to fulfill effective work-flow to fabricate three-dimensional mesh titanium scaffold for mandibular reconstruction. The 3D titanium mesh scaffold was designed based on a volunteer with whole mandible defect. (1) acquisition of the CT data; (2) design with computer aided design (CAD) and finite element analysis (FEA). The pore size and intervals with the best mechanic strength was also calculated using FEA. (3) fabrication of the scaffold using electron beam melting (EBM); (4) implantation surgery. The case recovered well, without loosening and rejection. Additionally, 12 mandibular defect model beagles were used to verify the results. The model was established via tooth extraction and mandibular resection surgeries, and the scaffold was designed individually based on CT data obtained at 2 weeks after extraction operation. Then scaffolds were fabricated using 3D EBM, and the implantation surgery was performed at 2 months after extraction operation. All the animals healed well after implantation, and the grafted mandibular recovered well with time. The relevant parameters of the grafted mandibular were nearly to the native mandibular at postoperative 12 months. It is feasible to fabricate mesh titanium scaffold for repairing mandibular defects individually using reverse engineering, CAD and EBM techniques.