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SUMMARY: Aporphine alkaloids are a large group of natural compounds with extensive pharmaceutical application prospects. The biosynthesis of aporphine alkaloids has been paid attentions in the past decades. Here, we determined the contents of four 1‐benzylisoquinoline alkaloids and five aporphine alkaloids in root, stem, leaf, and flower of Aristolochia contorta Bunge, which belongs to magnoliids. Two CYP80 enzymes were identified and characterized from A. contorta. Both of them catalyze the unusual C‐C phenol coupling reactions and directly form the aporphine alkaloid skeleton. AcCYP80G7 catalyzed the formation of hexacyclic aporphine corytuberine. AcCYP80Q8 catalyzed the formation of pentacyclic proaporphine glaziovine. Kingdom‐wide phylogenetic analysis of the CYP80 family suggested that CYP80 first appeared in Nymphaeales. The functional divergence of hydroxylation and C‐C (or C‐O) phenol coupling preceded the divergence of magnoliids and eudicots. Probable crucial residues of AcCYP80Q8 were selected through sequence alignment and molecular docking. Site‐directed mutagenesis revealed two crucial residues E284 and Y106 for the catalytic reaction. Identification and characterization of two aporphine skeleton‐forming enzymes provide insights into the biosynthesis of aporphine alkaloids. [ABSTRACT FROM AUTHOR]

SUMMARY: Prunella vulgaris is one of the bestselling and widely used medicinal herbs. It is recorded as an ace medicine for cleansing and protecting the liver in Chinese Pharmacopoeia and has been used as the main constitutions of many herbal tea formulas in China for centuries. It is also a traditional folk medicine in Europe and other countries of Asia. Pentacyclic triterpenoids are a major class of bioactive compounds produced in P. vulgaris. However, their biosynthetic mechanism remains to be elucidated. Here, we report a chromosome‐level reference genome of P. vulgaris using an approach combining Illumina, ONT, and Hi‐C technologies. It is 671.95 Mb in size with a scaffold N50 of 49.10 Mb and a complete BUSCO of 98.45%. About 98.31% of the sequence was anchored into 14 pseudochromosomes. Comparative genome analysis revealed a recent WGD in P. vulgaris. Genome‐wide analysis identified 35 932 protein‐coding genes (PCGs), of which 59 encode enzymes involved in 2,3‐oxidosqualene biosynthesis. In addition, 10 PvOSC, 358 PvCYP, and 177 PvUGT genes were identified, of which five PvOSCs, 25 PvCYPs, and 9 PvUGTs were predicted to be involved in the biosynthesis of pentacyclic triterpenoids. Biochemical activity assay of PvOSC2, PvOSC4, and PvOSC6 recombinant proteins showed that they were mixed amyrin synthase (MAS), lupeol synthase (LUS), and β‐amyrin synthase (BAS), respectively. The results provide a solid foundation for further elucidating the biosynthetic mechanism of pentacyclic triterpenoids in P. vulgaris. Significance Statement: The first chromosome‐level reference genome of Prunella vulgaris and the Prunella genus was reported. Fifty‐nine 2,3‐oxidosqualene biosynthesis‐related genes and 39 pentacyclic triterpenoid biosynthesis‐related PvOSCs, PvCYPs, and PvUGTs genes were systematically analyzed. PvOSC2, PvOSC4, and PvOSC6 were experimentally verified to be mixed amyrin synthase, lupeol synthase, and β‐amyrin synthase, respectively. The results provide a solid foundation for further elucidating the biosynthetic mechanism of bioactive compounds in the widely used medicinal plant, P. vulgaris. [ABSTRACT FROM AUTHOR]

Increasing evidence suggests that long non-coding RNAs (lncRNAs) play significant roles in plants. However, little is known about lncRNAs in Panax ginseng C. A. Meyer, an economically significant medicinal plant species. A total of 3,688 mRNA-like non-coding RNAs (mlncRNAs), a class of lncRNAs, were identified in P. ginseng. Approximately 40% of the identified mlncRNAs were processed into small RNAs, implying their regulatory roles via small RNA-mediated mechanisms. Eleven miRNA-generating mlncRNAs also produced siRNAs, suggesting the coordinated production of miRNAs and siRNAs in P. ginseng. The mlncRNA-derived small RNAs might be 21-, 22-, or 24-nt phased and could be generated from both or only one strand of mlncRNAs, or from super long hairpin structures. A full-length mlncRNA, termed MAR (multiple-function-associated mlncRNA), was cloned. It generated the most abundant siRNAs. The MAR siRNAs were predominantly 24-nt and some of them were distributed in a phased pattern. A total of 228 targets were predicted for 71 MAR siRNAs. Degradome sequencing validated 68 predicted targets involved in diverse metabolic pathways, suggesting the significance of MAR in P. ginseng. Consistently, MAR was detected in all tissues analyzed and responded to methyl jasmonate (MeJA) treatment. It sheds light on the function of mlncRNAs in plants. [ABSTRACT FROM AUTHOR]

SQUAMOSA promoter binding protein-likes (SPLs) are plant-specific transcription factors playing vital regulatory roles in plant growth and development. There is no information about SPLs in Salvia miltiorrhiza (Danshen), a significant medicinal plant widely used in Traditional Chinese medicine (TCM) for >1,700 years and an emerging model plant for TCM studies. Through genome-wide identification and subsequent molecular cloning, we identified a total 15 SmSPLs with divergent sequence features, gene structures, and motifs. Comparative analysis showed sequence conservation between SmSPLs and their Arabidopsis counterparts. A phylogenetic tree clusters SmSPLs into six groups. Many of the motifs identified commonly exist in a group/subgroup, implying their functional redundancy. Eight SmSPLs were predicted and experimentally validated to be targets of miR156/157. SmSPLs were differentially expressed in various tissues of S. milltiorrhiza. The expression of miR156/157-targeted SmSPLs was increased with the maturation of S. miltiorrhiza, whereas the expression of miR156/157 was decreased, confirming the regulatory roles of miR156/157 in SmSPLs and suggesting the functions of SmSPLs in S. miltiorrhiza development. The expression of miR156/157 was negatively correlated with miR172 during the maturation of S. miltiorrhiza. The results indicate the significance and complexity of SmSPL-, miR156-, and miR172-mediated regulation of developmental timing in S. miltiorrhiza. [ABSTRACT FROM AUTHOR]

Chinese fir ( Cunninghamia lanceolata), a commercially important tree for the timber and pulp industry, is widely distributed in southern China and northern Vietnam, but its large and complex genome has hindered the development of genomic resources. Few efforts have focused on analysis of the modulation of transcriptional networks in vascular cambium during the transition from active growth to dormancy in conifers. , Here, we used Illumina sequencing to analyze the global transcriptome alterations at the different stages of vascular cambium development in Chinese fir., By analyzing dynamic changes in the transcriptome of vascular cambium based on our RNA sequencing ( RNA- Seq) data at the dormant, reactivating and active stages, many potentially interesting genes were identified that encoded putative regulators of cambial activity, cell division, cell expansion and cell wall biosynthesis and modification. In particular, the genes involved in transcriptional regulation and hormone signaling were highlighted to reveal their biological importance in the cambium development and wood formation., Our results reveal the dynamics of transcriptional networks and identify potential key components in the regulation of vascular cambium development in Chinese fir, which will contribute to the in-depth study of cambial differentiation and wood-forming candidate genes in conifers. [ABSTRACT FROM AUTHOR]

Plants develop important regulatory networks to adapt to the frequently-changing availability of copper (Cu). However, little is known about miRNA-associated Cu-regulatory networks in plant species other than Arabidopsis. Here, we report that Cu-responsive miRNAs in Populus trichocarpa (Torr. & Gray) include not only conserved miR397, miR398 and miR408, but also Populus-specific miR1444, suggesting the conservation and diversity of Cu-responsive miRNAs in plants. Copper-associated suppression of mature miRNAs is in company with the up-regulation of their target genes encoding Cu-containing proteins in Populus. The targets include miR397-targeted PtLAC5, PtLAC6 and PtLAC110a, miR398-targeted PtCSD1, PtCSD2a and PtCSD2b, miR408-targeted PtPCL1, PtPCL2, PtPCL3 and PtLAC4, and miR1444-targeted PtPPO3 and PtPPO6. Consistently, P. trichocarpa miR408 promoter-directed GUS gene expression is down-regulated by Cu in transgenic tobacco plants. Cu-response elements (CuREs) are found in the promoters of Cu-responsive miRNA genes. We identified 34 SQUAMOSA-promoter binding protein-like (SPL) genes, of which 17 are full-length PtSPL proteins or partial sequences with at least 300 amino acids. Phylogenetic analysis indicates that PtSPL3 and PtSPL4 are CuRE-binding proteins controlling Cu-responsive gene expression. Cu appears to be not involved in the regulation of these transcription factors because neither PtSPL3 nor PtSPL4 is Cu-regulated and no CuRE exists in their promoters. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs), a group of small non-coding RNAs, have recently become the subject of intense study. They are a class of post-transcriptional negative regulators playing vital roles in plant development and growth. However, little is known about their regulatory roles in the responses of trees to the stressful environments incurred over their long-term growth. Here, we report the cloning of small RNAs from abiotic stressed tissues of Populus trichocarpa (Ptc) and the identification of 68 putative miRNA sequences that can be classified into 27 families based on sequence homology. Among them, nine families are novel, increasing the number of the known Ptc-miRNA families from 33 to 42. A total of 346 targets was predicted for the cloned Ptc-miRNAs using penalty scores of ≤2.5 for mismatched patterns in the miRNA:mRNA duplexes as the criterion. Six of the selected targets were validated experimentally. The expression of a majority of the novel miRNAs was altered in response to cold, heat, salt, dehydration, and mechanical stresses. Microarray analysis of known Ptc-miRNAs identified 19 additional cold stress-responsive Ptc-miRNAs from 14 miRNA gene families. Interestingly, we found that individual miRNAs of a family responded differentially to stress, which suggests that the members of a family may have different functions. These results reveal possible roles for miRNAs in the regulatory networks associated with the long-term growth of tree species and provide useful information for developing trees with a greater level of stress resistance. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are endogenous small RNAs that can have large-scale regulatory effects on development and on stress responses in plants. The endemic rust fungus Cronartium quercuum f. sp. fusiforme causes fusiform rust disease in pines, resulting in the development of spindle-shaped galls (cankers) on branches or stems. This disease is the most destructive disease of pines in the southern USA. To test whether miRNAs play roles in fusiform rust gall development, we cloned and identified 26 miRNAs from stem xylem of loblolly pine ( Pinus taeda), which belong to four conserved and seven loblolly pine-specific miRNA families. Forty-three targets for nine of these 11 families were experimentally validated in vivo. Sequence analysis suggested that the target cleavage site may be determined not only by the miRNA sequence but also by the target sequence. Members of three loblolly pine-specific miRNA families target a large number of non-protein coding transcripts, and one of these families could also initiate secondary phased production from its target of a putative trans-acting short interfering RNA (ta-siRNA). Expression of 10 of these 11 miRNA families was significantly repressed in the galled stem. PCR-based transcript quantification showed complex expression patterns of these miRNAs and their targets in the galled tissues and in tissues surrounding the gall. We further predict 82 plant disease-related transcripts that may also response to miRNA regulation in pine. These results reveal a new genetic basis for host–pathogen interactions in the development of fusiform rust gall. [ABSTRACT FROM AUTHOR]