Shoot Apex Research Articles

Chromatin Immunoprecipitation from Shoot Apices and Young Panicles of Rice.

Chromatin immunoprecipitation (ChIP) assay is a standard method to examine protein-DNA interactions in vivo. Here, I describe a basic procedure for ChIP assay in rice shoot apices using an antibody against the homeodomain transcription factor Oryza sativa homeobox1 (OSH1). I also introduce the tips to overcome several obstacles when performing this assay for the first time and potential pitfalls that could impact the result.

Preparation and Sectioning of Paraffin-Embedded Tissue for Histology and Histochemistry.

Cutting thin sections of plant tissues enables observation of internal structures and inner cell layers, and it facilitates various histochemical staining methods. Paraffin embedding gives the tissues a uniform stiffness that is sufficient for sectioning with a microtome. Here, we describe the procedures for preparation of paraffin-embedded tissues and the techniques for sectioning with a microtome. This method is applicable to various types of tissues, including rice shoot apex and immature inflorescence.

Purification of Anti-OSH1 IgG and Immunohistochemical Staining of Rice Shoot Apical Meristem.

Immunohistochemical staining (IHC) enables the visualization of protein localization within tissues and organs. Here, we describe IHC to detect the ORYZA SATIVA HOMEOBOX1 (OSH1) protein in sections of rice shoot apical meristems (SAMs). First, anti-OSH1 IgG is purified from OSH1 polyclonal rabbit antisera. For subsequent IHC, rice shoot apices are fixed and embedded in paraffin and sections are prepared for IHC. In this IHC procedure, two different labeling methods, fluorescein isothiocyanate and alkaline phosphatase, are utilized for secondary antibody detection.

Hormonal profiles in shoot apices in (sub)tropical populations of the aquatic carnivorous plant Aldrovanda vesiculosa in summer and winter

Hormonal profiles in shoot apices in (sub)tropical populations of the aquatic carnivorous plant Aldrovanda vesiculosa in summer and winter

Efficacy of Plant Tissue Culture Techniques for Eliminating Black Mulberry Idaeovirus (BMIV) from Infected Black Mulberry (Morus nigra).

Obtaining virus-free plants is a crucial step in disease management that enables reliable and profitable fruit farming. The present study applied various in vitro virus elimination protocols, including apical shoot culture, chemotherapy, thermotherapy, cryotherapy, and their combination, to eliminate black mulberry Idaeovirus (BMIV) from sour black mulberry. First, a shoot tip (0.5-2 mm) culture protocol was optimized, and four ribavirin concentrations (0, 10, 20, and 30 mg/L) were investigated over five weeks as a form of chemotherapy (ch). For the first thermotherapy treatment (Ch + Th1st), chemotherapy treatment was followed by a gradual increase in the temperature (24-33 °C). In another experiment (Th2nd + Ch), in vitro shoots were incubated in the dark for two weeks at two different temperatures (35 ± 1 °C and 37 ± 1 °C, for one week each). Subsequently, the shoot tips were incubated with various ribavirin doses. Finally, cryotherapy (Cr) was used with or without immersing the shoot tips in liquid nitrogen. A two-step RT-PCR was performed to assess the presence of the virus in 7-8-week-old in vitro plants. Th2nd + Ch significantly increased the shoot tip burst and plant survival/morphogenesis compared to the other treatments. Except for the application of cryotherapy, the protocols eliminated BMIV in different proportions, and the highest virus elimination rate (50%) was obtained by applying 30 mg/L ribavirin during the Ch + Th1st treatment. These findings are essential in preventing the dissemination of the virus and enabling the safe movement of germplasm around the world.

Development of an efficient in vitro shoot regeneration method for carnation cultivars using cotyledons with apical shoot meristems

Development of an efficient in vitro shoot regeneration method for carnation cultivars using cotyledons with apical shoot meristems

Design and evaluation of an active vineyard heating system to simulate temperature increase in the context of climate change

The current study introduces an innovative direct and active heating system designed for precise temperature control in vineyards. This system serves as a valuable tool for investigating the influence of climate change on grapevine physiology and, consequently, the characteristics of the resulting wine. The research took place in an experimental vineyard located in Mendoza, Argentina, with V. vinifera cvs. trained to a vertical shoot positioning trellis system over two consecutive growing seasons. The system design utilized electric hot water tanks and polypropylene pipes attached to the foliage catch wires. Over two growing seasons, the system consistently elevated the ambient air temperatures within the canopy by 2.5 ± 0.12 °C compared to the control group. This temperature increase emulated the temperature projections for Mendoza as forecasted by the IPCC by the end of this century. The system displayed heating uniformity, as evidenced by the absence of both vertical and horizontal temperature gradients. Additionally, the significant variation in mean daytime and night-time temperatures between the control and heated treatments highlighted the effectiveness of the system in modifying temperature conditions on a diurnal basis. The heated treatment applied with this system proved to have an effective biological impact on the physiology of grapevines. In both seasons, plants under the heated treatment advanced their bud break and harvest dates. The study showed a significant growth enhancement in the heated treatment, with apical shoots extending significantly longer than those in the control treatment. Additionally, the total soluble solids content increased in the heating treatment, while yield decreased, for both experimental seasons. These results illustrate the robust performance of the system throughout the entire growth period, regardless of fluctuations in atmospheric conditions. This study establishes a new foundation for future research on grapevine responses to climate change. It also opens the door to the implementation of effective adaptation strategies in vineyards, promising a more resilient and adaptable future for grape cultivation.

Bioenergy sorghum nodal root bud development: morphometric, transcriptomic and gene regulatory network analysis.

Bioenergy sorghum's large and deep nodal root system and associated microbiome enables uptake of water and nutrients from and deposition of soil organic carbon into soil profiles, key contributors to the crop's resilience and sustainability. The goal of this study was to increase our understanding of bioenergy sorghum nodal root bud development. Sorghum nodal root bud initiation was first observed on the stem node of the 7th phytomer below the shoot apex. Buds were initiated near the upper end of the stem node pulvinus on the side of the stem opposite the tiller bud, then additional buds were added over the next 6-8 days forming a ring of 10-15 nascent nodal root buds around the stem. Later in plant development, a second ring of nodal root buds began forming on the 17th stem node immediately above the first ring of buds. Overall, nodal root bud development can take ~40 days from initiation to onset of nodal root outgrowth. Nodal root buds were initiated in close association with vascular bundles in the rind of the pulvinus. Stem tissue forming nascent nodal root buds expressed sorghum homologs of genes associated with root initiation (WOX4), auxin transport (LAX2, PIN4), meristem activation (NGAL2), and genes involved in cell proliferation. Expression of WOX11 and WOX5, genes involved in root stem niche formation, increased early in nodal root bud development followed by genes encoding PLTs, LBDs (LBD29), LRP1, SMB, RGF1 and root cap LEAs later in development. A nodal root bud gene regulatory network module expressed during nodal root bud initiation predicted connections linking PFA5, SPL9 and WOX4 to genes involved in hormone signaling, meristem activation, and cell proliferation. A network module expressed later in development predicted connections among SOMBRERO, a gene involved in root cap formation, and GATA19, BBM, LBD29 and RITF1/RGF1 signaling. Overall, this study provides a detailed description of bioenergy sorghum nodal root bud development and transcriptome information useful for understanding the regulation of sorghum nodal root bud formation and development.

Overexpression of lily MicroRNA156-resistant SPL13A stimulates stem elongation and flowering in Lilium formosanum under non-inductive (non-chilling) conditions.

Flowering plants undergo juvenile vegetative, adult vegetative, and reproductive phases. Lily plants (Lilium spp.) develop scaly leaves during their juvenile vegetative phase. Stem elongation occurs in the adult vegetative phase and is followed by floral transition. As the duration of the juvenile vegetative phase is long in lilies, the microRNA156 (miR156) and SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) modules are expected to play a major role in vegetative phase change and flower induction. In the present study, we aimed to explore the functions of lily SLP13A. We evaluated phenotypic changes and gene expression in L. formosanum plants overexpressing miR156-resistant SPL13A (rSPL13A) and examined the accumulation levels of gene transcripts and mature miRNAs in non-transformed L. longiflorum plants. Lily plants overexpressing rSPL13A exhibited stem elongation under non-inductive conditions, and FLOWERING LOCUS T (FT) genes were poorly involved in this stem elongation. Flowering was induced in the transformed plants with elongated stems, and the accumulation of MADS5 (APETALA1) transcripts and mature miR172 was elevated in these plants. In non-transformed lilies, SPL13A transcripts were highly accumulated in the shoot apices of both juvenile and adult plants. As mature miR156 was poorly accumulated in the shoot apices of the adult plants, SPL13A was active enough to stimulate stem elongation and flower induction. In contrast, mature miR156 was reliably detected in shoot apices of the juvenile plants. Because our transient assay using tobacco plants expressing a SPL13A-GFP fusion protein indicated that miR156 repressed SPL13A expression mainly at the translational level, SPL13A activity should be insufficient to stimulate stem elongation in the juvenile plants. In addition, the accumulation of MADS5 transcripts and mature miR172 in the shoot apices increased with plant growth and peaked before the transition to the reproductive phase. Therefore, we conclude that SPL13A regulates stem elongation in the adult vegetative phase, which differs from the mechanisms evaluated in Arabidopsis and rice, wherein stem elongation proceeds in a reproductive phase and FT genes are heavily involved in it, and that SPL13A induces flowering by the activation of genes related to the age pathway underlying floral transition, as APETALA1 and primary-MIR172 are mainly involved in this pathway.

RETINOBLASTOMA-RELATED Has Both Canonical and Noncanonical Regulatory Functions During Thermo-Morphogenic Responses in Arabidopsis Seedlings.

Warm temperatures accelerate plant growth, but the underlying molecular mechanism is not fully understood. Here, we show that increasing the temperature from 22°C to 28°C rapidly activates proliferation in the apical shoot and root meristems of wild-type Arabidopsis seedlings. We found that one of the central regulators of cell proliferation, the cell cycle inhibitor RETINOBLASTOMA-RELATED (RBR), is suppressed by warm temperatures. RBR became hyper-phosphorylated at a conserved CYCLIN-DEPENDENT KINASE (CDK) site in young seedlings growing at 28°C, in parallel with the stimulation of the expressions of the regulatory CYCLIN D/A subunits of CDK(s). Interestingly, while under warm temperatures ectopic RBR slowed down the acceleration of cell proliferation, it triggered elongation growth of post-mitotic cells in the hypocotyl. In agreement, the central regulatory genes of thermomorphogenic response, including PIF4 and PIF7, as well as their downstream auxin biosynthetic YUCCA genes (YUC1-2 and YUC8-9) were all up-regulated in the ectopic RBR expressing line but down-regulated in a mutant line with reduced RBR level. We suggest that RBR has both canonical and non-canonical functions under warm temperatures to control proliferative and elongation growth, respectively.

Chronological events unfolding the vegetative and floral phenology of apical bud in Crocus sativus.

Saffron (Crocus sativus L.) is an infertile perennial geophyte considered the most expensive spice in the world. Seasonal fluctuations and climate change have significant impact on the growth, development, and yield of saffron stigma, which is the economically valued part of plant. The stigma being part of the flower, the knowledge of phenotypic transition from dormant apical bud up to flowering is vital, yet, not explored properly. The complexity of flowering in C. sativus further accentuates by the lack of clear demarcation between flowering and non-flowering corms in terms of weight and sizes, as small corms are known to be vegetative only, while large ones produce flower. Therefore, chronological phenotyping on a weekly and quarterly basis of apical shoot and flowering primordia between June and October was carried out to understand the organogenesis sequentially. In large corms, the stamen was the first floral organ to initiate followed by the formation of tepal from the base of the stamen. The plants exhibited both synanthous and hysteranthous flowering. Untargeted metabolome analysis of dormant apical bud just before dormancy break from flowering buds from large corms as well as non-flowering buds from small corms identified the presence of many differentially accumulated metabolites including sphingosine and meglutol. Key metabolites such as phytosphingosine, 3-hydroxy-3-methyl glutaric acid, 3-acetamidopropanal, 6-hydroxykynurenic acid, D-serine, and 1-D-myo-inositol 3-phosphate were also detected having associated with isoprenoid biosynthesis, lignin pathway regulation, and carbohydrate metabolism that participates in flowering. The integration of morphological, histological, and metabolomic data offers a comprehensive view of the flowering process that can be utilised in future biotechnological interventions in C. sativus.

A regulatory module mediating temperature control of cell-cell communication facilitates tree bud dormancy release.

The control of cell-cell communication via plasmodesmata (PD) plays a key role in plant development. In tree buds, low-temperature conditions (LT) induce a switch in plasmodesmata from a closed to an open state, which restores cell-to-cell communication in the shoot apex and releases dormancy. Using genetic and cell-biological approaches, we have identified a previously uncharacterized transcription factor, Low-temperature-Induced MADS-box 1 (LIM1), as an LT-induced, direct upstream activator of the gibberellic acid (GA) pathway. The LIM1-GA module mediates low temperature-induced plasmodesmata opening, by negatively regulating callose accumulation to promote dormancy release. LIM1 also activates expression of FT1 (FLOWERING LOCUS T), another LT-induced factor, with LIM1-FT1 forming a coherent feedforward loop converging on low-temperature regulation of gibberellin signaling in dormancy release. Mathematical modeling and experimental validation suggest that negative feedback regulation of LIM1 by gibberellin could play a crucial role in maintaining the robust temporal regulation of bud responses to low temperature. These results reveal genetic factors linking temperature control of cell-cell communication with regulation of seasonally-aligned growth crucial for adaptation of trees.

Comparative Photosynthetic Capacity, Respiration Rates, and Nutrient Content of Micropropagated and Wild-Sourced Sphagnum

The rapid, effective restoration of degraded peatlands is urgently needed to reduce their current high levels of carbon loss. The re-introduction of Sphagnum moss, along with re-wetting, is key to returning carbon sequestration and retention capabilities to northern degraded bogs. Micropropagated Sphagnum has already been applied in large quantities, and more is planned, for restoration projects in Britain and parts of Europe. A comparison with wild-sourced Sphagnum material is therefore pertinent to demonstrate its safety and suitability for wide-scale application. Six Sphagnum species of both micropropagated and wild-sourced origin were assessed for photosynthetic capacity, nutrient content, form parity, chlorocyst size, and chloroplast numbers. Micropropagated Sphagnum had significantly higher light-saturated photosynthesis (Pmax) rates, little color expression, an open growth habit, greater chloroplast numbers, and more numerous, smaller shoot apices than wild-sourced Sphagnum. Higher Pmax rates were associated with a lower bulk density and higher tissue nutrient concentrations. Potentially, greater chloroplast numbers in micropropagated Sphagnum facilitate higher photosynthesis rates, driving rapid growth in early-stage plants, particularly in optimum moisture conditions. Micropropagated Sphagnum can be used confidently, propagated in large quantities, and will likely establish well on application to sites where re-wetting has already occurred, therefore making it highly beneficial for the restoration of degraded bogs.

BnaC09.tfl1 controls determinate inflorescence trait in Brassica napus

Determinate inflorescence is indeed a pivotal agricultural characteristic in crops, notably impacting the architecture modification of Brassica napus (AACC, 2n = 38). Previous study identified a crucial gene Bnsdt2 that encodes the transcription factor BnaC09.TFL1 (Terminal Flower 1). Here by two alleles were cloned and sequenced from indeterminate 2982 and determinate 4769, respectively, we found that BnaC09.TFL1 harbors two T/C and G/C non-synonymous mutations in exon 1, and contains sixty-six differences in a 1.9 Kb promoter sequence. Subsequently, BnaC09.TFL1 was introduced into B. napus 571 line by genetic complementation and overexpression, transgenic plants 571CTO lines and 571TClines were all restored to the indeterminate inflorescence. Interestingly, after BnaC09.TFL1 was knocked out in ‘Westar’, transgenic plants WestarTcr lines were mutated to determinate inflorescences. Additionally, a NIL-4769 line was constructed to evaluate the effect of BnaC09.TFL1 on agronomic traits of Brassica napus, the results demonstrated that BnaC09.tfl1 reduced the plant height and increased the branch number and branch thousand grain weight of Brassica napus. Finally, we performed RT-qPCR, GUS staining and subcellular localization experiments to analyze the expression pattern of BnaC09.TFL1, the results showed that the expression of BnaC09.TFL1 at shoot apex of NIL-4769 was higher than that of 4769, GUS activity was detected at apical of Arabidopsis thaliana and BnC09.TFL1-GFP was detected in cell membrane, nucleus and cytoplasm. Our findings provide a firm molecular foundation for the study of rapeseed’s molecular mechanism of determinate inflorescence formation, as well as theoretical guidance for the application of determinate inflorescence in rapeseed breeding.

Impact of salt strength on in vitro propagation and rebaudioside A content in Stevia rebaudiana under semi-solid and liquid MS media.

Stevia rebaudiana (Bertoni), commonly known as stevia, is a sought-after natural sweetener, but its conventional propagation methods are slow and inefficient. This study aims to enhance the in vitro culture for stevia by investigating the impact of different Murashige and Skoog (MS) medium salt strengths and plant growth hormones on growth and rebaudioside A content. Apical bud-containing shoot segments were used as explants and cultured on various semi-solid and liquid MS media formulations, incorporating cytokinins (BAP and Kin), auxins (NAA and IAA), and different MS major salt concentrations (MS full, ½ MS, and ¼ MS). Assessments of shoot growth parameters, root formation, and HPLC analysis for rebaudioside A content were conducted. The optimal conditions for in vitro growth was found to be in the ¼ MS + Kin 3mg/L + NAA 0.1mg/L (semi-solid) medium, resulting in significantly improved shoot growth and enhanced 30.04% rebaudioside A content. Genetic fidelity of regenerated plants was confirmed using RAPD and ISSR markers. These findings offer valuable insights for optimizing in vitro propagation of stevia and potentially enhancing rebaudioside A content.

Positive contribution of shoot apex to the growth and flooding tolerance of Hemarthria altissima upon complete submergence

ABSTRACT Flooding events tend to destroy the original flood-intolerant vegetation in riparian zones, but the flood-tolerant species can confront the stress, and contribute to the riparian ecosystem. Grass species, Hemarthria altissima, are usually dominant in the riparian zones. This species is considered as good forage which is usually grazed by livestock or mowed by local people. Therefore, the apical tissues of the plants are often removed, and the plants have to grow without stem apexes, during their life cycle. In this study, we aimed to examine the differences in growth performance of intact versus apex-cut individuals of H. altissima upon complete submergence. Two groups of H. altissima plants (with and without shoot apexes) were treated with dark non-submergence and dark complete submergence conditions for 200 days. During the experiment, we measured plant growth, biomass changes in plant organs, and the consumption of non-structural carbohydrates (NSC) by different tissues. During submergence, shoot elongation stopped, and around six lateral buds were developed averagely by each plant without apexes. This growth performance finally caused 60% decline of NSC in underground parts. The relatively intensive consumption of carbohydrates in submerged apex-removed plants induced the 21% stem length decreased under water, which indicated the decreasing submergence tolerance of plants with shoot apex removed. Therefore, we suggest that when using H. altissima for restoring degraded riparian ecosystems, the shoot apexes should be protected from grazing by livestock or harvesting by local people in order to maintain the submergence tolerance of H. altissima.

Inflorescence development in female cannabis plants is mediated by photoperiod and gibberellin.

In cannabis seedlings, the initiation of solitary flowers is photoperiod-independent. However, when cannabis reaches the adult stage, short-day photoperiod (SD) triggers branching of the shoot apex and a reduction in internode length, leading to development of a condensed inflorescence. We demonstrate that SD affects cannabis plants in two distinct phases: the first includes rapid elongation of the internodes and main stem, and occurring from Day 5 to Day 10 of plant cultivation under SD; in the second phase, elongation of newly developed internodes ceases, and a condensed inflorescence is formed. Exposure of plants to alternating photoperiods revealed that inflorescence onset requires at least three consecutive days of SD, and SD is consistently required throughout inflorescence maturation to support its typical condensed architecture. This photoperiod-dependent morphogenesis was associated with a decrease in gibberellin (GA4) and auxin levels in the shoot apex. Reverting the plants to a long-day photoperiod (LD) increased GA4 and auxin levels, leading to inflorescence disassembly, internode elongation, and subsequent resumption of LD growth patterns. Similar developmental patterns were observed under SD following the application of exogenous GA (and not auxin), which also impeded inflorescence development. Nevertheless, additional studies will help to further evaluate auxin's role in these developmental changes. We propose a crucial role for GA in sexual reproduction and inflorescence development in female cannabis by mediating photoperiod signaling in the inflorescence tissues.

Development of a Robust In vitro Regeneration Protocol in Cassava (Manihot esculenta C.) Using Axillary Bud Explants for Cryopreservation

Cassava is an important crop as it is the source of energy in the diet for the most of the tropical countries. Field gene bank is the only viable option for the maintenance of cassava germplasm. Cryopreservation offers the long-term conservation of vegetatively propagates crops without losing its viability through an effective in vitro regeneration system. In vitro regeneration response in YTP-1 and Sree Athulya genotypes of cassava was standardized through optimization of various factors influencing organogenesis. Among the different media tested, highest shoot induction frequencies of 81.4% and 77.8% were recorded in medium supplemented with 0.5 mg/l BAP + 0.2 mg/l NAA (CMSI 2) for YTP 1 and Sree Athulya respectively in the present study. 3 to 5 multiple shoots from a single shoot apex obtained from 70-80% of explants within 50-60 days. Highest regeneration frequencies of 56.6%and 46.6% were recorded in medium supplemented with 1 mg/l IAA (CRM 2) for YTP1 and Sree Athulya. As a result, an easy in vitro regeneration method that is genotype independent was created and may be effectively used to preserve cassava germplasm in the cryogen bank.

ScRNA-seq reveals the spatiotemporal distribution of camptothecin pathway and transposon activity in Camptotheca acuminata shoot apexes and leaves.

Camptotheca acuminata Decne., a significant natural source of the anticancer drug camptothecin (CPT), synthesizes CPT through the monoterpene indole alkaloid (MIA) pathway. In this study, we used single-cell RNA sequencing (scRNA-seq) to generate datasets encompassing over 60,000 cells from C. acuminata shoot apexes and leaves. After cell clustering and annotation, we identified five major cell types in shoot apexes and four in leaves. Analysis of MIA pathway gene expression revealed that most of them exhibited heightened expression in proliferating cells (PCs) and vascular cells (VCs). In contrast to MIA biosynthesis in Catharanthus roseus, CPT biosynthesis in C. acuminata did not exhibit multicellular compartmentalization. Some putative genes encoding enzymes and transcription factors (TFs) related to the biosynthesis of CPT and its derivatives were identified through co-expression analysis. These include 19 cytochrome P450 genes, 8 O-methyltransferase (OMT) genes, and 62 TFs. Additionally, these pathway genes exhibited dynamic expression patterns during VC and EC development. Furthermore, by integrating gene and transposable element (TE) expression data, we constructed novel single-cell transcriptome atlases for C. acuminata. This approach significantly facilitated the identification of rare cell types, including peripheral zone cells (PZs). Some TE families displayed cell type specific, tissue specific, or developmental stage-specific expression patterns, suggesting crucial roles for these TEs in cell differentiation and development. Overall, this study not only provides novel insights into CPT biosynthesis and spatial-temporal TE expression characteristics in C. acuminata, but also serves as a valuable resource for further comprehensive investigations into the development and physiology of this species.

Anatomic adaptation of stems of aquatic Utricularia species dragging turions to the bottom of their habitats

Turions are vegetative, dormant, overwintering storage organs formed in aquatic plants. We have observed that shoots with maturating turions of three robust aquatic Utricularia species possess an anatomical adaptation of the central cylinder in apical shoot segments. The senescent mother shoots drag turions in these species to the bottom of their habitat. This adaptation leads to impregnation of the central cylinder by substances preventing the stems from early decomposing. Using anatomical staining methods, we have aimed to elucidate the substances impregnating the central cylinder - partly lignin and mainly polyphenols.