Transcriptome profiling based on Illumina- and SMRT-based RNA-seq reveals circadian regulation of key pathways in flower bud development in walnut

Flower bud development is a defining feature of walnut, which contributes to the kernel yield, yield stability, fruit quality and commodity value. However, little is known about the mechanism of the flower bud development in walnut. Here, the stages of walnut female flower bud development were divided into five period (P01-05) by using histological observation. They were further studied through PacBio Iso-Seq and RNA-seq analysis. Accordingly, we obtained 52,875 full-length transcripts, where 4,579 were new transcripts, 3,065 were novel genes, 1,437 were consensus lncRNAs and 20,813 were alternatively spliced isoforms. These transcripts greatly improved the current genome annotation and enhanced our understanding of the walnut transcriptome. Next, RNA sequencing of female flower buds at five periods revealed that circadian rhythm-plant was commonly enriched along with the flower bud developmental gradient. A total of 14 differentially expressed genes (DEGs) were identified, and six of them were confirmed by real-time quantitative analysis. Additionally, six and two differentially expressed clock genes were detected to be regulated by AS events and lncRNAs, respectively. All these detected plant circadian genes form a complex interconnected network to regulate the flower bud development. Thus, investigation of key genes associated with the circadian clock could clarify the process of flower bud development in walnut.

In vitro germination and vegetative propagation through bud development of sacha inchi (Plukenetia volubilis L.)

This study describes the in vitro seed germination and micropropagation of Plukenetia volubilis (sacha inchi), an oilseed crop rich in omega-3 fatty acids, with health benefits and several industrial applications. Seed germination was evaluated in different culture media (MS and 1/2 MS), seed coat presence/absence and culture temperature (18 °C and 28 °C). Micropropagation was performed using axillary bud development (ABD) on nodal segments from in vitro seedlings. KIN, BAP and 2-ip were evaluated for ABD, and the effect of modified MS in 453 mg L-1 CaCl2 and 351.62 mg L-1 MgSO4 on ABD and shoot survival was assessed to improve the process. Finally, six treatments were evaluated to optimize ABD and shoot leaf formation. Seed germination of 91.6 % was achieved in MS at 28 °C when the seed coat was removed. ABD was obtained in 45 % and 40 % with 0.4 mg L-1 KIN and 0.6 mg L-1 2-ip, respectively, with the least CAL. The modification in 453 mg L-1 CaCl2 then allowed 76 % ABD and 82 % explant survival. ABD response was optimized to 95 % and 2.45 leaves with MS medium + CaCl2 modification + 10 % coconut water + 0.4 mg L-1 KIN. The same results were obtained by replacing the latter with 0.6 mg L-1 2-ip. Rooting was achieved in MS without PGR, and acclimatization was successful. The results indicate that plant production via germination and vegetative propagation is effective for commercial purposes.

Spatial regulation by multiple Gremlin1 enhancers provides digit development with cis-regulatory robustness and evolutionary plasticity

Precise cis-regulatory control of gene expression is essential for normal embryogenesis and tissue development. The BMP antagonist Gremlin1 (Grem1) is a key node in the signalling system that coordinately controls limb bud development. Here, we use mouse reverse genetics to identify the enhancers in the Grem1 genomic landscape and the underlying cis-regulatory logics that orchestrate the spatio-temporal Grem1 expression dynamics during limb bud development. We establish that transcript levels are controlled in an additive manner while spatial regulation requires synergistic interactions among multiple enhancers. Disrupting these interactions shows that altered spatial regulation rather than reduced Grem1 transcript levels prefigures digit fusions and loss. Two of the enhancers are evolutionary ancient and highly conserved from basal fishes to mammals. Analysing these enhancers from different species reveal the substantial spatial plasticity in Grem1 regulation in tetrapods and basal fishes, which provides insights into the fin-to-limb transition and evolutionary diversification of pentadactyl limbs.

SMAD4 target genes are part of a transcriptional network that integrates the response to BMP and SHH signaling during early limb bud patterning

ABSTRACTSMAD4 regulates gene expression in response to BMP and TGFβ signal transduction and is required for diverse morphogenetic processes, but its target genes have remained largely elusive. Here, we use an epitope-tagged Smad4 allele for ChIP-seq analysis together with transcriptome analysis of wild-type and mouse forelimb buds lacking Smad4 in the mesenchyme. This analysis identifies the SMAD4 target genes during establishment of the feedback signaling system and establishes that SMAD4 predominantly mediates BMP signal-transduction during early limb bud development. Unexpectedly, the initial analysis reveals that the expression of cholesterol biosynthesis enzymes is precociously down-regulated and intracellular cholesterol levels reduced in Smad4-deficient limb bud mesenchymal progenitors. The SMAD4 target GRNs includes genes, whose expression in the anterior limb bud is up-regulated by interactions of SMAD4 complexes with enhancers active in the anterior mesenchyme. This reveals a predominant function of SMAD4 in up-regulating target gene expression in the anterior limb bud mesenchyme. Analysis of differentially expressed genes that are shared between Smad4- and Shh-deficient limb buds corroborates the positive role of SMAD4 in transcriptional regulation of anterior genes and reveals a repressive effect on posterior genes that are positively regulated by SHH signaling. This analysis uncovers the overall opposing effects of SMAD4-mediated BMP and SHH signalling on transcriptional regulation during early limb bud development. In summary, this analysis indicates that during early digit patterning and limb bud outgrowth, the anterior/proximal and proximo/distal expression dynamics of co-regulated genes are controlled by distinct and contrasting trans-regulatory inputs from SHH and SMAD4-mediated BMP signal transduction.

Molecular mechanism of lateral bud differentiation of Pinus massoniana based on high-throughput sequencing

Knot-free timber cultivation is an important goal of forest breeding, and lateral shoots affect yield and stem shape of tree. The purpose of this study was to analyze the molecular mechanism of lateral bud development by removing the apical dominance of Pinus massoniana young seedlings through transcriptome sequencing and identify key genes involved in lateral bud development. We analyzed hormone contents and transcriptome data for removal of apical dominant of lateral buds as well as apical and lateral buds of normal development ones. Data were analyzed using an comprehensive approach of pathway- and gene-set enrichment analysis, Mapman visualization tool, and gene expression analysis. Our results showed that the contents of auxin (IAA), Zea and strigolactone (SL) in lateral buds significantly increased after removal of apical dominance, while abscisic acid (ABA) decreased. Gibberellin (GA) metabolism, cytokinin (CK), jasmonic acid, zeatin pathway-related genes positively regulated lateral bud development, ABA metabolism-related genes basically negatively regulated lateral bud differentiation, auxin, ethylene, SLs were positive and negative regulation, while only A small number of genes of SA and BRASSINOSTEROID, such as TGA and TCH4, were involved in lateral bud development. In addition, it was speculated that transcription factors such as WRKY, TCP, MYB, HSP, AuxIAA, and AP2 played important roles in the development of lateral buds. In summary, our results provided a better understanding of lateral bud differentiation and lateral shoot formation of P. massoniana from transcriptome level. It provided a basis for molecular characteristics of side branch formation of other timber forests, and contributed to knot-free breeding of forest trees.

Comparative proteomics analysis reveals differentially accumulated proteins associated with male and female A. chinensis var. chinensis bud development

Background Kiwifruit (Actinidia chinensis var. Chinensis) is abundant with vitamin C and is a rapidly developing crop in China, New Zealand, and other countries. It has been widely used as a raw material for food and kiwifruit wine. Among these, A. chinensis var. chinensis and A. chinensis var. deliciosa are the most valuable kiwifruit in production. Kiwifruit is a typical dioecious plant and its female and male plants have different economic values. Therefore, sex identification, especially at the seedling stage, has important implications for the scientific planning of its production and economic benefits. However, the kiwifruit sex regulation mechanism is very complex and molecular studies are in the initial stages. Currently, there is not a universal and effective sex identification method for A. chinensis. Methods In this study, we used a label-free quantitative proteomics approach to investigate differentially accumulated proteins, including their presence/absence and significantly different levels of abundances during A. chinensis var. chinensis male and female flower bud development. Results A total of 6485 proteins were identified, among which, 203 were identified in male buds, which were mainly associated with phenylalanine metabolism, tyrosine metabolism, and plant hormone signal transduction. In female buds, 241 were identified, which were mainly associated with the ErbB signaling pathway, growth hormone synthesis, secretion and action, and mRNA surveillance pathway. A total of 373 proteins were significantly differentially accumulated proteins (fold change > 2; P < 0.05), of which, 168 were upregulated and 205 were downregulated. Significant differences between proteins involved 13 signaling pathways, most of which were involved in flavonoid biosynthesis, phenylpropanoid biosynthesis, and starch and sucrose metabolism. Protein interaction analysis showed that enriched protein nodes included cell division cycle 5-like protein, 40S ribosomal protein S8, ribosomal protein, and 40S ribosomal protein like, which interact with 35, 25, 22, and 22 proteins, respectively. Conclusions This study provide valuable information for cloning key genes that control sex traits and functionally analyze their roles, which lay a foundation to the development of molecular markers for male and female kiwifruit identification.

Toward Systematic Understanding of Flower Bud Induction in Apple: A Multi-Omics Approach

The induction of flower buds in apple (Malus × domestica Borkh.) is tightly connected to biennial bearing, which is characterized by alternating years with high (ON) and low or no (OFF) crop loads. In order to study this irregular cropping behavior, spur buds from ON- and OFF-trees of the biennial-bearing cultivar ‘Fuji’ and the regular bearing cultivar ‘Gala’ were collected. First, the time of flower bud initiation was precisely determined for both cultivars by histological analysis. Moreover, for a systematic understanding of flower bud induction in apple, the physiological and molecular mechanisms within the bud tissue were evaluated over four weeks prior to flower bud initiation by employing a multi-omics approach, including RNA sequencing, proteomic and metabolic profiling. Gene and protein enrichment analysis detected physiological pathways promoting and inhibiting early flower bud development. Metabolic profiles from the cropping treatments revealed a greater abundance of thiamine, chlorogenic acid, and an adenine derivative in spur buds from OFF-trees, whereas tryptophan was more abundant in the buds collected from ON-trees. Cultivar comparison indicated that chlorogenic acid was more abundant in ‘Gala’ than in ‘Fuji’ spur buds, whereas the opposite effect was found for tryptophan. Genes controlling tryptophan biosynthesis were not affected by ON- and OFF-treatments, but genes assigned to the metabolism of tryptophan into indoleacetate were differentially expressed between cultivars and treatments. The multi-omics approach permitted analyzing complex plant metabolic processes involved in early flower bud development and more specifically presumably in flower bud induction by tracing some pathways from gene to product level.