A NAC transcription factor ZaNAC93 confers floral initiation, fruit development, and prickle formation in Zanthoxylum armatum.

Zanthoxylum armatum is a dioecious prickly plant which developed apomictic reproduction. The increases in male flowers and prickle density in female plants lead to low yield and picking efficiency. However, little is known concerning the mechanisms of floral development and prickle formation. NAC is a well-known transcription factor that participates in multiple aspects of plant growth and development. Herein, we characterize the functions and regulatory mechanisms of candidate NACs controlling both traits in Z. armatum. A total of 159 ZaNACs were identified, and 16 of these were male-biased, represented by the NAP subfamily members ZaNAC93 and ZaNAC34, orthologs of AtNAC025 and AtNARS1/NAC2 respectively. Overexpression of ZaNAC93 in tomato led to modifications in flower and fruit development, including earlier flowering, increased numbers of lateral shoots and flowers, accelerated plant senescence, and reduced size and weight of fruits and seeds. In addition, the trichome density in leaves and inflorescences was dramatically reduced in ZaNAC93-OX lines. Overexpression of ZaNAC93 resulted in the up-/downregulation of genes associated with GA, ABA and JA signaling pathways, such as GAI, PYL and JAZ, as well as several TFs, including bZIP2, AGL11, FBP24 and MYB52. Yeast two-hybrid analysis revealed that ZaNAC93 protein could interact with AP1, GAI, bZIP2 and AGL11 in Z. armatum, which might contribute to floral induction, fruit growth, and trichome initiation. This work provides new insights into the molecular mechanisms of ZaNAC93 in reproductive development and prickle formation in Z. armatum.

Comparative transcriptome analysis reveals hormone, transcriptional and epigenetic regulation involved in prickle formation in Zanthoxylum armatum.

Zanthoxylum armatum is an evergreen plant with high economical and medicinal values. The presence of prickles on stems and leaves is undesirable for them make picking difficult. To date, little is known of prickle formation in Z. armatum. Herein, the morphological and molecular features of prickle initiation in prickless (WC) and three types of prickly Z. armatum were characterized. Compared to WC, the levels of cytokinin and auxin were increased, while GA and JA declined in prickly Z. armatum. Transcriptome analysis identified 6258 differentially expressed genes (DEGs) between prickless and prickly Z. armatum. Among them, several DEGs related to hormone metabolism and signaling, including LOG7, CKX3, AHK1, three DELLAs, six JAZs and TIR1, were candidate genes involved in prickle formation. Transcription factors associated with prickle formation were screened, including MYB6-1/MYB6-2, WER, GL3-2, SPL4/5, SOC1, and SCL32. Of them, MYB6-1 and WER might negatively regulate prickles initiation via interacting with GL3-2. Additionally, the histone acetylation and DNA methylation levels, the transcripts of histone acetyltransferase/deacetylase and DNA methyltransferases showed significant differences between prickless and prickly plants, indicating their involvements in prickle initiation. These findings illustrate the regulation of prickle formation might be mediated by phytohormones (especially cytokinin), transcription factors and epigenetic modifications in Z. armatum.

Genome-wide identification, interaction of the MADS-box proteins in Zanthoxylum armatum and functional characterization of ZaMADS80 in floral development.

As a typical dioecious species, Zanthoxylum armatum establishes apomictic reproduction, hence only female trees are cultivated. However, male and hermaphrodite flowers have recently appeared in female plants, resulting in a dramatic yield reduction. To date, the genetic basis underlying sex determination and apomixis in Z. armatum has been largely unknown. Here, we observed abortion of the stamen or carpel prior to primordium initiation, thus corroborating the potential regulation of MADS-box in sex determination. In Z. armatum, a total of 105 MADS-box genes were identified, harboring 86 MIKC-type MADSs with lack of FLC orthologues. Transcriptome analysis revealed candidate MADSs involved in floral organ identity, including ten male-biased MADSs, represented by ZaMADS92/81/75(AP3/PI-like), and twenty-six female-specified, represented by ZaMADS80/49 (STK/AGL11-like) and ZaMADS42 (AG-like). Overexpressing ZaMADS92 resulted in earlier flowering, while ZaMADS80 overexpression triggered precocious fruit set and parthenocarpy as well as dramatic modifications in floral organs. To characterize their regulatory mechanisms, a comprehensive protein-protein interaction network of the represented MADSs was constructed based on yeast two-hybrid and bimolecular fluorescence complementation assays. Compared with model plants, the protein interaction patterns in Z. armatum exhibited both conservation and divergence. ZaMADS70 (SEP3-like) interacted with ZaMADS42 and ZaMADS48 (AP3-like) but not ZaMADS40 (AP1-like), facilitating the loss of petals in Z. armatum. The ZaMADS92/ZaMADS40 heterodimer could be responsible for accelerating flowering in ZaMADS92-OX lines. Moreover, the interactions between ZaMADS80 and ZaMADS67(AGL32-like) might contribute to apomixis. This work provides new insight into the molecular mechanisms of MADS-boxes in sex organ identity in Z. armatum.

Transcriptome and miRNA sequencing analyses reveal the regulatory mechanism of α-linolenic acid biosynthesis in Paeonia rockii.

Paeonia rockii is a promising woody oil crop because its seeds are rich in polyunsaturated fatty acids especially α-linolenic acid (ALA). ALA is an essential fatty acid that the human body cannot synthesize and is the direct synthetic precursor of eicosapentaenoic and docosahexaenoic acids, which play crucial roles in the development of the blood vessels, brain and nervous system of humans. However, the mechanisms underlying the dynamic changes in ALA during seed development are unknown. In this study, we found that the fatty acid content gradually increased with P. rockii seed development, with ALA being the main unsaturated acid component (37-44%). The content of ALA reached the peak value of 306.26 mg/g DW 20 days before the seeds had fully maturated. Seeds from three different developmental stages were selected for transcriptome and miRNA sequencing analyses to explore the molecular mechanism of ALA accumulation in P. rockii seeds. A total of 39 differentially expressed genes were screened for their involvement in ALA biosynthesis, among which FAD2/8, GPAT, PDAT, LACS, LPAAT, and KAS II might be the key structural genes of ALA accumulation. The differential expression of these genes was dependent on the regulation of five miRNAs (mdm_miR156b, novel miR_91, novel miR_133, novel miR_291, and novel miR_405) and four transcription factors (AP2, SNL2, TGA-like, and SPL). This study reveals the mechanism behind the dynamic changes of ALA contents in P. rockii during seed development, and also provides an important theoretical basis for the breeding of excellent varieties of P. rockii.

Transcriptome and metabolite profiling to identify genes associated with rhizome lignification and the function of ZoCSE in ginger (Zingiber officinale).

Ginger (Zingiber officinale Roscoe) is an important spice crop in China, and fresh ginger rhizomes are consumed as vegetable in Sichuan and Chongqing. However, tissue lignification accelerates with rhizome maturation, resulting in the loss of edible quality. To understand the molecular mechanisms of texture modification during rhizome development, we investigated lignin accumulation patterns and identified the key genes associated with lignin biosynthesis using gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and RNA-sequencing (RNA-Seq). Results showed that the contents of total lignin and its precursors exhibited notable declines with tissue maturation. However, the lignin composition was remarkably modified and syringyl lignin was deposited in mature rhizomes, leading to ginger lignification. Transcriptome analysis displayed 32 lignin biosynthetic genes were dramatically downregulated with rhizome development, including caffeoylshikimate esterase (CSE ), 4-coumarate-CoA ligase , laccase , cinnamoyl-CoA reductase , cinnamyl-alcohol dehydrogenase , peroxidase and caffeic acid 3-O-methyltransferase , indicating that lignin reduction might be attributed to deficiency in intermediates or the downregulation of key biosynthetic enzymes. Furthermore, overexpressing ZoCSE in Nicotiana benthamiana L. enhanced the total lignin content, suggesting its fundamental role in lignin biosynthesis. RNA-Seq also identified candidate lignin production regulators, including hormone-related genes and NAC/MYB transcription factors (ZoNAC1 , ZoNAC4 , ZoMYB14 and ZoMYB17 ). This result provides a molecular basis for lignin accumulation in ginger.

Transcriptome-wide characterization of the WRKY family genes in Lonicera macranthoides and the role of LmWRKY16 in plant senescence.

BACKGROUND: Lonicera macranthoides is an important woody plant with high medicinal values widely cultivated in southern China. WRKY, one of the largest transcription factor families, participates in plant development, senescence, and stress responses. However, a comprehensive study of the WRKY family in L. macranthoides hasn't been reported previously. OBJECTIVE: To establish an extensive overview of the WRKY family in L. macranthoides and identify senescence-responsive members of LmWRKYs. METHODS: RNA-Seq and phylogenetic analysis were employed to identify the LmWRKYs and their evolutionary relationships. Quantitative real-time (qRT-PCR) and transgenic technology was utilized to investigate the roles of LmWRKYs in response to developmental-, cold-, and ethylene-induced senescence. RESULTS: A total of 61 LmWRKY genes with a highly conserved motif WRKYGQK were identified. Phylogenetic analysis of LmWRKYs together with their orthologs from Arabidopsis classified them into three groups, with the number of 15, 39, and 7, respectively. 17 LmWRKYs were identified to be differentially expressed between young and aging leaves by RNA-Seq. Further qRT-PCR analysis showed 15 and 5 LmWRKY genes were significantly induced responding to tissue senescence in leaves and stems, respectively. What's more, five LmWRKYs, including LmWRKY4, LmWRKY5, LmWRKY6, LmWRKY11, and LmWRKY16 were dramatically upregulated under cold and ethylene treatment in both leaves and stems, indicating their involvements commonly in developmental- and stress-induced senescence. In addition, function analysis revealed LmWRKY16, a homolog of AtWRKY75, can accelerate plant senescence, as evidenced by leaf yellowing during reproductive growth in LmWRKY16-overexpressing tobaccos. CONCLUSION: The results lay the foundation for molecular characterization of LmWRKYs in plant senescence.

SMRT and Illumina RNA sequencing reveal the complexity of terpenoid biosynthesis in Zanthoxylum armatum.

Sichuan pepper (Zanthoxylum armatum DC) is a popular spice and is often prescribed in traditional Chinese medicine to treat vomiting, diarrhea, ascariasis and eczema, among other conditions. Volatile oils from Z. armatum leaves contain active ingredients, with terpenoids being one of the main components. In the present study, the combination of sequencing data of Z. armatum from PacBio single molecule real time (SMRT) and Illumina RNA sequencing (RNA-Seq) platforms facilitated an understanding of the gene regulatory network of terpenoid biosynthesis in pepper leaves. The leaves of three developmental stages from two Z. armatum cultivars, 'Rongchangwuci' (WC) and 'Zhuye' (ZY), were selected as test materials to construct sequencing libraries. A total of 143,122 predictions of unique coding sequences, 105,465 simple sequence repeats, 20,145 transcription factors and 4719 long non-coding RNAs (lncRNAs) were identified, and 142,829 transcripts were successfully annotated. The occurrence of alternative splicing events was verified by reverse transcription PCR, and quantitative real-time PCR was used to confirm the expression pattern of six randomly selected lncRNAs. A total of 96,931 differentially expressed genes were filtered from different samples. According to functional annotation, a total of 560 candidate genes were involved in terpenoid synthesis, of which 526 were differentially expressed genes (DEGs). To identify the key genes involved in terpenoid biosynthesis, the module genes in different samples, including structural and transcription factors genes, were analyzed using the weighted gene co-expression network method, and the co-expression network of genes was constructed. Thirty-one terpenoids were identified by gas chromatography-mass spectrometry. The correlation between 18 compounds with significantly different contents and genes with high connectivity in the module was jointly analyzed in both cultivars, yielding 12 candidate DEGs presumably involved in the regulation of terpenoid biosynthesis. Our findings showed that full-length transcriptome SMRT and Illumina RNA-Seq can play an important role in studying organisms without reference genomes and elucidating the gene regulation of a biosynthetic pathway.

A R2R3-MYB transcriptional activator LmMYB15 regulates chlorogenic acid biosynthesis and phenylpropanoid metabolism in Lonicera macranthoides.

Lonicera macranthoides Hand-Mazz is an important medicinal plant widely distributed in southern China that has long been used in Chinese traditional medicines. Chlorogenic acid (CGA, 3-caffeoylquinic acid) is the major biologically active ingredient in L. macranthoides. Although key CGA biosynthetic genes have been well documented, their transcriptional regulation remains largely unknown. In this study, we observed that a R2R3 MYB transcription factor LmMYB15 showed a significant correlation with CGA content, indicating its potential role in CGA biosynthesis. A yeast two-hybrid assay suggested that LmMYB15 functions as a transcriptional activator. Overexpression of LmMYB15 in tobacco led to increased accumulation of CGA compared to those in wild-type leaves. To elucidate its functional mechanism, genome-wide DAP-seq was employed and identified the conserved binding motifs of LmMYB15, that is [(C/T) (C/T) (C/T) ACCTA(C/A) (C/T) (A/T)], as well as its direct downstream target genes, including 4CL, MYB3, MYB4, KNAT6/7, IAA26, and ETR2. Subsequently, yeast one-hybrid and dual-luciferase reporter assays verified that LmMYB15 could bind and activate the promoters of 4CL, MYB3 and MYB4, thereby facilitating CGA biosynthesis and phenylpropanoid metabolism. Our findings provide a new track for breeding strategies aiming to enhance CGA content in L. macranthoides that can significantly contribute to better mechanical properties.