Early Bud Development Research Articles

Molecular mechanism of synovial joint site specification and induction in developing vertebrate limbs.

The vertebrate appendage comprises three primary segments, the stylopod, zeugopod and autopod, each separated by joints. The molecular mechanisms governing the specification of joint sites, which define segment lengths and thereby limb architecture, remain largely unknown. Existing literature suggests that reciprocal gradients of retinoic acid (RA) and fibroblast growth factor (FGF) signaling define the expression domains of the putative segment markers Meis1, Hoxa11 and Hoxa13. Barx1 is expressed in the presumptive joint sites. Our data demonstrate that RA-FGF signaling gradients define the expression domain of Barx1 in the first presumptive joint site. When misexpressed, Barx1 induces ectopic interzone-like structures, and its loss of function partially blocks interzone development. Simultaneous perturbations of RA-FGF signaling gradients result in predictable shifts of Barx1 expression domains along the proximo-distal axis and, consequently, in the formation of repositioned joints. Our data suggest that during early limb bud development in chick, Meis1 and Hoxa11 expression domains are overlapping, whereas the Barx1 expression domain resides within the Hoxa11 expression domain. However, once the interzone is formed, the expression domains are refined and the Barx1 expression domain becomes congruent with the border of these two putative segment markers.

Morphological and physiological mechanism of cytoplasmic inheritance stigma exsertion trait expression in tobacco (Nicotiana tabacum)

Stigma exsertion is an essential outcrossing trait that can improve hybrid seed production efficiencies. In this study, the morphological and physiological mechanisms of cytoplasmic inheritance stigma exsertion trait expression in a tobacco line (MSK326SE) which generated from a stigma exsertion tobacco mutant through continuous backcross were investigated. Compared with its homonuclear-heteroplasmic lines (MSK326 and K326 with inserted stigmas), the exserted stigma phenotype of MSK326SE was mainly caused by corolla shortening, while was stable under different environmental temperature. The different responses of mainly endogenous hormones and expression of cell division- and expansion-related genes caused the differences in cell division and expansion in different flower organs, which further determined the lengths of the corolla. Furthermore, the significant decrease of MSK326SE corolla epidermal cell size caused corolla shortening and finally resulting in stigma exsertion. Exogenous JA could shorten the corolla and more effective increased stigma exsertion degree of MSK326SE, suggesting a potential relationship between stigma exsertion and high JA levels during early bud development. The hybrid seed production efficiency could be improved in tobacco. Our results provide a basis for elucidating the cytoplasmic inheritance stigma exsertion trait expression in tobacco while helping to improve hybrid seed production efficiency.

En1 and Lmx1b do not recapitulate embryonic dorsal-ventral limb patterning functions during mouse digit tip regeneration.

The mouse digit tip regenerates following amputation. How the regenerate is patterned is unknown, but a long-standing hypothesis proposes developmental patterning mechanisms are re-used during regeneration. The digit tip bone exhibits dorsal-ventral (DV) polarity, so we focus on En1 and Lmx1b, two factors necessary for DV patterning during limb development. We investigate whether they are re-expressed during regeneration in a developmental-like pattern and whether they direct DV morphology of the regenerate. We find that both En1 and Lmx1b are expressed in the regenerating digit tip epithelium and mesenchyme, respectively, but without DV polarity. Conditional genetics and quantitative analysis of digit tip bone morphology determine that genetic deletion of En1 or Lmx1b in adult digit tip regeneration modestly reduces bone regeneration but does not affect DV patterning. Collectively, our data suggest that, while En1 and Lmx1b are re-expressed during mouse digit tip regeneration, they do not define the DV axis during regeneration.

Early flower bud development and plant growth regulators to improve return bloom of pears

Early flower bud development and plant growth regulators to improve return bloom of pears

Mechanical Regulation of Limb Bud Formation.

Early limb bud development has been of considerable interest for the study of embryological development and especially morphogenesis. The focus has long been on biochemical signalling and less on cell biomechanics and mechanobiology. However, their importance cannot be understated since tissue shape changes are ultimately controlled by active forces and bulk tissue rheological properties that in turn depend on cell–cell interactions as well as extracellular matrix composition. Moreover, the feedback between gene regulation and the biomechanical environment is still poorly understood. In recent years, novel experimental techniques and computational models have reinvigorated research on this biomechanical and mechanobiological side of embryological development. In this review, we consider three stages of early limb development, namely: outgrowth, elongation, and condensation. For each of these stages, we summarize basic biological regulation and examine the role of cellular and tissue mechanics in the morphogenetic process.

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 identify the SMAD4 target genes in mouse limb buds using an epitope-tagged Smad4 allele for ChIP-seq analysis in combination with transcription profiling. This analysis shows that SMAD4 predominantly mediates BMP signal transduction during early limb bud development. Unexpectedly, the expression of cholesterol biosynthesis enzymes is precociously downregulated and intracellular cholesterol levels are reduced in Smad4-deficient limb bud mesenchymal progenitors. Most importantly, our analysis reveals a predominant function of SMAD4 in upregulating target genes in the anterior limb bud mesenchyme. Analysis of differentially expressed genes shared between Smad4- and Shh-deficient limb buds corroborates this function of SMAD4 and also reveals the repressive effect of SMAD4 on posterior genes that are upregulated in response to SHH signaling. This analysis uncovers opposing trans-regulatory inputs from SHH- and SMAD4-mediated BMP signal transduction on anterior and posterior gene expression during the digit patterning and outgrowth in early limb buds.

New genomic resources and comparative analyses reveal differences in floral gene expression in selfing and outcrossing Collinsia sister species.

The evolutionary transition from outcross- to self-fertilization is one of the most common in angiosperms and is often associated with a parallel shift in floral morphological and developmental traits, such as reduced flower size and pollen to ovule ratios, known as the “selfing syndrome.” How these convergent phenotypes arise, the extent to which they are shaped by selection, and the nature of their underlying genetic basis are unsettled questions in evolutionary biology. The genus Collinsia (Plantaginaceae) includes seven independent transitions from outcrossing or mixed mating to high selfing rates accompanied by selfing syndrome traits. Accordingly, Collinsia represents an ideal system for investigating this parallelism, but requires genomic resource development. We present a high quality de novo genome assembly for the highly selfing species Collinsia rattanii. To begin addressing the basis of selfing syndrome developmental shifts, we evaluate and contrast patterns of gene expression from floral transcriptomes across three stages of bud development for C. rattanii and its outcrossing sister species Collinsia linearis. Relative to C. linearis, total gene expression is less variable among individuals and bud stages in C. rattanii. In addition, there is a common pattern among differentially expressed genes: lower expression levels that are more constant across bud development in C. rattanii relative to C. linearis. Transcriptional regulation of enzymes involved in pollen formation specifically in early bud development may influence floral traits that distinguish selfing and outcrossing Collinsia species through pleiotropic functions. Future work will include additional Collinsia outcrossing-selfing species pairs to identify genomic signatures of parallel evolution.

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.

RNA-Seq Time Series of Vitis vinifera Bud Development Reveals Correlation of Expression Patterns with the Local Temperature Profile.

Plants display sophisticated mechanisms to tolerate challenging environmental conditions and need to manage their ontogenesis in parallel. Here, we set out to generate an RNA-Seq time series dataset throughout grapevine (Vitis vinifera) early bud development. The expression of the developmental regulator VviAP1 served as an indicator of the progression of development. We investigated the impact of changing temperatures on gene expression levels during the time series and detected a correlation between increased temperatures and a high expression level of genes encoding heat-shock proteins. The dataset also allowed the exemplary investigation of expression patterns of genes from three transcription factor (TF) gene families, namely MADS-box, WRKY, and R2R3-MYB genes. Inspection of the expression profiles from all three TF gene families indicated that a switch in the developmental program takes place in July which coincides with increased expression of the bud dormancy marker gene VviDRM1.

Defoliation, not gibberellin, induces tree peony autumn reflowering regulated by carbon allocation and metabolism in buds and leaves

Short and concentrated natural fluorescence hinders tree peony (Paeonia suffruticosa) annual production, and defoliation and gibberellin (GA) application is used to induce its reflowering in autumn. Here, the individual roles of defoliation and GA treatment were determined by monitoring morphological and soluble sugar changes in buds and leaves, and by investigating carbon allocation- and metabolism-related gene expression. Both defoliation and GA treatment induced early bud development, but induction was faster using the GA treatment. Only defoliation, not GA treatment, induced the final reflowering, although their combination accelerated it. Furthermore, defoliation decreased the sucrose content in buds much faster than the GA treatment. This sucrose reduction may play a key role in tree peony reflowering, and the higher carbon metabolism activity in young leaves after defoliation may further help the reflowering process. Defoliation enhanced the expression of sucrose transporters PsSUT4 and PsSWEET12 in buds, and their expression in young leaves was greater than after GA treatment. This indicated that PsSUT4 and PsSWEET12 may help transport carbon into buds after defoliation. In addition, the invertases, PsCIN2 and PsCWIN1 in young leaves were more highly expressed after defoliation, indicating that they may contribute to reflowering after defoliation by accelerating sucrose hydrolysis in young leaves. In addition, the expression levels of PsVIN1 and PsVIN2 in leaves, and PsVIN2 in buds were more highly induced by GA treatment than by defoliation, indicating that PsVINs may mainly respond to GA treatment. These results may help improve the tree peony forcing culture technology and related industrial production.

MiR156b from Soybean CMS Line Modulates Floral Organ Development

The miR156 and plant specific transcription factor SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) are known for their function regulating plant growth and development. In this study, we identified 20 GmSPLs which are targeted by gma-miR156b via plant small RNA target and degradome analysis. And we found high transcript levels of gma-miR156b and its targeted GmSPLs in the flower of soybean cytoplasmic male sterility (CMS) line and its maintainer. The gma-miR156b direct cleavage of GmSPL2b and GmSPL9b, and have opposite expression levels during early flower buds development. We observed a high expression level of GUS protein in the anthers of the line with pgma-MIR156b::GUS reporter. Over-expression of the gma-miR156b precursor in Arabidopsis inhibited floral organ development, including reduced anther size and the amount of pollen grains per anther etc. Like miR156-targeted SPL genes, non-targeted GmSPL8s were also down-regulated in early flower bud development of soybean CMS line compared with its maintainer line, which might act in concert with miR156-targeted SPL genes to participate in the floral organ development. Quantitative real time PCR (qRT-PCR) suggested that miR156/SPL modulates floral organ development by regulating the expression of LATERAL ORGAN BOUNDARIES DOMAIN22 (LBD22), LBD36, AGAMOUS-LIKE30 (AGL30) and AGL104. Our findings will facilitate understanding of the biological functions of miR156/SPL in floral organ development of soybean CMS.

Termination of Insecticide Applications for Tarnished Plant Bug (Hemiptera: Miridae) Management in Cotton

Tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), is the most important insect pest of cotton, Gossypium hirsutum L., in the states of Arkansas, Louisiana, Mississippi, Tennessee, and Missouri. Foliar insecticide sprays are an important tactic within an integrated pest management (IPM) program for this pest which occurs from early flower bud development to cut out, a stage where the plant has reached the capacity for supporting fruiting positions and equivalent to when there are five nodes above the uppermost first position white flower (NAWF 5). Currently, NAWF and heat unit accumulation monitoring is limited, resulting in a need for a more simplified insecticide termination method that has the potential for wider adoption. Experiments were conducted in 2015 and 2016 in the previously mentioned states to determine when to terminate insecticide sprays for tarnished plant bug based on week of flowering. Treatments included terminating sprays after the second through sixth weeks of flowering, a season-long control, and an untreated control. In general, insecticides reduced densities of tarnished plant bugs in the sprayed treatments. When analyzed by location, treatments that resulted in cotton yields similar to the season-long control ranged from weeks two through five of flowering. These data suggest that treatments can be terminated after the fifth week of flowering. Data from this study indicate applications terminated after the fifth week of flowering would be similar to the current recommended termination timing of NAWF 5 plus 350 heat units. Results from this experiment will be used to better define a simplified IPM strategy for tarnished plant bug in cotton.

Spike Activator 1, Encoding a bHLH, Mediates Axillary Bud Development and Spike Initiation in Phalaenopsis aphrodite.

Double-spikes Phalaenopsis orchids have greater market value than those with single-spike. In this study, a gene designated as Spike Activator 1 (SPK1), which encodes a basic helix-loop-helix (bHLH) transcription factor, was isolated and characterized from Phalaenopsis aphrodite (moth orchid). SPK1 was highly expressed in the meristematic tissues. In the axillary bud, SPK1 was highly upregulated by a moderately low temperature of 20 °C but downregulated by a spike inhibition temperature of 30 °C. SPK1 protein is localized in the nucleus. Another bHLH, bHLH35, which is also highly expressed in young tissues in the same way as SPK1 was also identified. In contrast to SPK1, bHLH35 transcripts are downregulated at 20 °C but upregulated at 30 °C. Bimolecular florescence complementation assay and yeast two-hybrid assays indicated that SPK1 interacts with bHLH35 and forms a heterodimer. Virus-induced gene silencing (VIGS) showed that 7 out of 15 vector control plants produced double spikes but that only 1 out of 15 VIGS-spk1 plants produced double spikes. RT-qPCR results indicated that VIGS-spk1 downregulated gene expression levels of SPK1, FT, CYCB, and EXPA8. Overall, we propose that SPK1 plays an essential role in early axillary bud development and spike initiation of P. aphrodite.

Rabbit Whole Embryo Culture.

The rabbit is a mainstay of regulatory developmental toxicity testing; however, due to the historic absence of experimental tools for this species, there is a dearth of information about its fundamental embryology and the mechanisms underlying developmental toxicity. Relatively recently, there have been advances in the methods of rabbit whole embryo culture (WEC), and this has prompted an increase in understanding of rabbit embryogenesis. Described herein are the methods used to remove early somite-stage embryos (gestation day 9) and sustain their growth for 48h. Although there are similarities to the well-described rodent WEC, there are also important differences. Akin to rodent WEC, the major phases of organogenesis can be investigated, including neural tube development, cardiac looping, segmentation, and the development of the anlagen of the optic and otic regions, craniofacial development, somites, and early limb bud development. Unlike the rodent, rabbit WEC requires the use of an apparatus that allows for the continuous gassing of embryos, and one may observe the expansion and closure of the visceral yolk sac around the embryo. After completion of the culture period, embryos are examined across several growth and developmental parameters including a quantitative morphological scoring system. Embryonic growth and development in the absence of maternal influences allows for the study of the direct action of agents or their metabolites on the embryo. The use of both rodent and rabbit WEC together is a powerful strategy with which to investigate species-specific vulnerabilities to specific agents.

Integrative Analysis of Three RNA Sequencing Methods Identifies Mutually Exclusive Exons of MADS-Box Isoforms During Early Bud Development in Picea abies.

Recent efforts to sequence the genomes and transcriptomes of several gymnosperm species have revealed an increased complexity in certain gene families in gymnosperms as compared to angiosperms. One example of this is the gymnosperm sister clade to angiosperm TM3-like MADS-box genes, which at least in the conifer lineage has expanded in number of genes. We have previously identified a member of this sub-clade, the conifer gene DEFICIENS AGAMOUS LIKE 19 (DAL19), as being specifically upregulated in cone-setting shoots. Here, we show through Sanger sequencing of mRNA-derived cDNA and mapping to assembled conifer genomic sequences that DAL19 produces six mature mRNA splice variants in Picea abies. These splice variants use alternate first and last exons, while their four central exons constitute a core region present in all six transcripts. Thus, they are likely to be transcript isoforms. Quantitative Real-Time PCR revealed that two mutually exclusive first DAL19 exons are differentially expressed across meristems that will form either male or female cones, or vegetative shoots. Furthermore, mRNA in situ hybridization revealed that two mutually exclusive last DAL19 exons were expressed in a cell-specific pattern within bud meristems. Based on these findings in DAL19, we developed a sensitive approach to transcript isoform assembly from short-read sequencing of mRNA. We applied this method to 42 putative MADS-box core regions in P. abies, from which we assembled 1084 putative transcripts. We manually curated these transcripts to arrive at 933 assembled transcript isoforms of 38 putative MADS-box genes. 152 of these isoforms, which we assign to 28 putative MADS-box genes, were differentially expressed across eight female, male, and vegetative buds. We further provide evidence of the expression of 16 out of the 38 putative MADS-box genes by mapping PacBio Iso-Seq circular consensus reads derived from pooled sample sequencing to assembled transcripts. In summary, our analyses reveal the use of mutually exclusive exons of MADS-box gene isoforms during early bud development in P. abies, and we find that the large number of identified MADS-box transcripts in P. abies results not only from expansion of the gene family through gene duplication events but also from the generation of numerous splice variants.

Photosynthetic Efficiency and Antioxidant Activity of Cotton under Drought Stress during Early Floral Bud Development

Photosynthetic Efficiency and Antioxidant Activity of Cotton under Drought Stress during Early Floral Bud Development

Strigolactone Promotes Degradation of DWARF14, an α/β Hydrolase Essential for Strigolactone Signaling inArabidopsis

Strigolactones (SLs) are phytohormones that play a central role in regulating shoot branching. SL perception and signaling involves the F-box protein MAX2 and the hydrolase DWARF14 (D14), proposed to act as an SL receptor. We used strong loss-of-function alleles of the Arabidopsis thaliana D14 gene to characterize D14 function from early axillary bud development through to lateral shoot outgrowth and demonstrated a role of this gene in the control of flowering time. Our data show that D14 distribution in vivo overlaps with that reported for MAX2 at both the tissue and subcellular levels, allowing physical interactions between these proteins. Our grafting studies indicate that neither D14 mRNA nor the protein move over a long range upwards in the plant. Like MAX2, D14 is required locally in the aerial part of the plant to suppress shoot branching. We also identified a mechanism of SL-induced, MAX2-dependent proteasome-mediated degradation of D14. This negative feedback loop would cause a substantial drop in SL perception, which would effectively limit SL signaling duration and intensity.

Characterization and expression analysis of somatic embryogenesis receptor-like kinase genes from Phalaenopsis.

Somatic embryogenesis receptor-like kinase (SERK) genes have been found to be involved in the somatic embryogenesis of several plant species. We identified and characterized 5 PhSERK genes in the Phalaenopsis orchid. The amino acid sequences of PhSERKs and other SERK proteins are highly conserved, with the highest homology observed in the leucine-rich repeat-receptor-like kinase domain. All 5 PhSERKs were expressed in all Phalaenopsis organs examined (root, leaf, shoot apical meristem, and flower), with the strongest expression, particularly for PhSERK1 and 3, in the shoot apical meristem of mature plants. Expression of all PhSERKs was downregulated during early floral bud development and was upregulated gradually until the semi-open flower stage was reached. All 5 PhSERKs were expressed during both seed germination and protocorm-like-body (PLB) development. In germinated seeds, quantitative real-time PCR revealed upregulation of all PhSERKs except PhSERK4 at 1 week and downregulation after 4 weeks. The 5 PhSERKs were differentially expressed in the early stage of PLB development and maintained substantial levels during PLB formation, with PhSERK1 and 5 upregulated 1 week after culture and PhSERK2, 3, and 4 downregulated over this period. Because physical wounding of PLB stimulates secondary PLB formation, the PhSERK5 expression peak at week 3 coincided with visible and fully developed secondary PLBs. PhSERK5 may be important in PLB induction and subsequent development. Our PhSERK expression analysis revealed that these genes have a broad role during orchid plant development.

Spreading Dogbane (Apocynum androsaemifolium) Development in Wild Blueberry Fields

Spreading dogbane is a common perennial weed in wild blueberry fields. It is highly competitive and spreads rapidly once established. Herbicides can provide effective control of spreading dogbane, but application timing is important. The emergence pattern, ramet height, and flowering time of spreading dogbane were observed in 2008 and 2009, and thermal-based emergence, growth, and development models were developed and used to estimate optimum herbicide application timing. Spreading dogbane emergence and height were described with a three-parameter, sigmoid, nonlinear regression model, whereas flowering was described with a four-parameter, Weibull, nonlinear regression model. Spreading dogbane ramets initiated emergence soon after the biofix date of April 1. Peak emergence tended to occur at 420 growing degree days (GDD). Spreading dogbane reached its peak height by about 558 GDD. The maximum number of flowers per plant was reached at approximately 750 GDD. This study suggested that POST herbicides should be applied between 486 and 535 GDD to maximize efficacy. This time frame occurs after peak emergence and during early floral bud development.

Genes expressed in cotton (Gossypium hirsutum) buds isolated with a subtractive library

A subtractive cDNA library from cotton buds was constructed to prospect for differentially expressed genes related to early bud development. A library was constructed and 768 cDNA sequences were obtained, comprising 168 clusters, with 126 contigs and 42 singlets. Both the Gossypium as well as Arabidopsis databases were utilized for the in silico analysis, since some genes identified in cotton have not yet been studied for functionality, although they have homology with genes from other species. The transcriptome revealed a large number of transcripts, some of them with unknown function, and others related to pollen development, pollen tubes, ovules, and fibers at different stages. The most populated contig was identified as fiber from 0-10 days after anthesis, with 12 reads. The success and novelty rates generated from the library were 67 and 51%, respectively. The information obtained here will provide a framework for research on functional cotton genomics.