Wounding-Related Signaling Is Integrated within the Auxin-Response Framework to Induce Adventitious Rooting in Chestnut.

Wounding and exogenous auxin are needed to induce adventitious roots in chestnut microshoots. However, the specific inductive role of wounding has not been characterized in this species. In the present work, two main goals were established: First, we prompted to optimize exogenous auxin treatments to improve the overall health status of the shoots at the end of the rooting cycle. Second, we developed a time-series transcriptomic analysis to compare gene expression in response to wounding alone and wounding plus auxin, focusing on the early events within the first days after treatments. Results suggest that the expression of many genes involved in the rooting process is under direct or indirect control of both stimuli. However, specific levels of expression of relevant genes are only attained when both treatments are applied simultaneously, leading to the successful development of roots. In this sense, we have identified four transcription factors upregulated by auxin (CsLBD16, CsERF113, Cs22D and CsIAA6), with some of them also being induced by wounding. The highest expression levels of these genes occurred when wounding and auxin treatments were applied simultaneously, correlating with the rooting response of the shoots. The results of this work clarify the genetic nature of the wounding response in chestnut, its relation to adventitious rooting, and might be helpful in the development of more specific protocols for the vegetative propagation of this species.

Proliferation of Axillary Shoots of Chestnut in Temporary Immersion Systems.

Shoots from European chestnut and hybrids of European and Asian chestnuts can be efficiently proliferated in liquid medium by temporary immersion (TIS) using RITA® and Plantform™ bioreactors. The main challenges of applying TIS to these species were the lack of growth and hyperhydricity; problem solutions included the manipulation of the hormone concentration, the explant type, immersion frequency, and a support for maintaining shoots in a vertical position. After protocol optimization, explants cultured by TIS produced more rootable shoots than explants growing in semisolid medium, enabling increased number of rooted and acclimatable shoots. In this chapter, we will describe the protocols for proliferating chestnut by TIS in RITA® and Plantform™ bioreactors, together with tips for avoiding the main pitfalls of the technique. The strategies applied to chestnut can be useful for culturing other woody plants in bioreactors.

Expression of CsSCL1 and Rooting Response in Chestnut Leaves Are Dependent on the Auxin Polar Transport and the Ontogenetic Origin of the Tissues.

The mechanisms underlying the de novo regeneration of adventitious roots are still poorly understood, particularly in trees. We developed a system for studying adventitious rooting (AR) at physiological and molecular levels using leaves excised from chestnut microshoots of the same genotype but with two distinct ontogenetic origins that differ in rooting competence. Leaves were treated with auxin and N-1-naphthyl-phthalamic acid (NPA), an inhibitor of auxin polar transport (PAT). The physiological effects were investigated by recording rooting rates and the number and quality of the roots. Molecular responses were examined by localizing and monitoring the changes in the expression of CsSCL1, an auxin-inducible gene in juvenile and mature shoots during AR. The rooting response of leaves was ontogenetic-stage dependent and similar to that of the donor microshoots. Initiation of root primordia and root development were inhibited by application of NPA, although its effect depended on the timing of application. CsSCL1 was upregulated by auxin only in rooting-competent leaves during the novo root organogenesis, and the expression was reduced by NPA. The inhibitory effect on gene expression was detected during the reprograming of rooting competent cells towards root initials in response to auxin, indicating that PAT-mediated upregulation of CsSCL1 is required in the initial steps of AR in chestnut leaves. The localized expression of CsSCL1 in the quiescent center (QC) also suggests a role for this gene in the maintenance of meristematic competence and root radial patterning.

Transcriptomics Analysis Reveals a Putative Role for Hormone Signaling and MADS-Box Genes in Mature Chestnut Shoots Rooting Recalcitrance.

Maturation imposes several changes in plants, which are particularly drastic in the case of trees. In recalcitrant woody species, such as chestnut (Castanea sativa Mill.), one of the major maturation-related shifts is the loss of the ability to form adventitious roots in response to auxin treatment as the plant ages. To analyze the molecular mechanisms underlying this phenomenon, an in vitro model system of two different lines of microshoots derived from the same field-grown tree was established. While juvenile-like shoots root readily when treated with exogenous auxin, microshoots established from the crown of the tree rarely form roots. In the present study, a transcriptomic analysis was developed to compare the gene expression patterns in both types of shoots 24 h after hormone and wounding treatment, matching the induction phase of the process. Our results support the hypothesis that the inability of adult chestnut tissues to respond to the inductive treatment relies in a deep change of gene expression imposed by maturation that results in a significant transcriptome modification. Differences in phytohormone signaling seem to be the main cause for the recalcitrant behavior of mature shoots, with abscisic acid and ethylene negatively influencing the rooting ability of the chestnut plants. We have identified a set of related MADS-box genes whose expression is modified but not suppressed by the inductive treatment in mature shoots, suggesting a putative link of their activity with the rooting-recalcitrant behavior of this material. Overall, distinct maturation-derived auxin sensibility and homeostasis, and the related modifications in the balance with other phytohormones, seem to govern the outcome of the process in each type of shoots.

Plant Development and Crop Yield: The Role of Gibberellins.

Gibberellins have been classically related to a few key developmental processes, thus being essential for the accurate unfolding of plant genetic programs. After more than a century of research, over one hundred different gibberellins have been described. There is a continuously increasing interest in gibberellins research because of their relevant role in the so-called "Green Revolution", as well as their current and possible applications in crop improvement. The functions attributed to gibberellins have been traditionally restricted to the regulation of plant stature, seed germination, and flowering. Nonetheless, research in the last years has shown that these functions extend to many other relevant processes. In this review, the current knowledge on gibberellins homeostasis and mode of action is briefly outlined, while specific attention is focused on the many different responses in which gibberellins take part. Thus, those genes and proteins identified as being involved in the regulation of gibberellin responses in model and non-model species are highlighted. The present review aims to provide a comprehensive picture of the state-of-the-art perception of gibberellins molecular biology and its effects on plant development. This picture might be helpful to enhance our current understanding of gibberellins biology and provide the know-how for the development of more accurate research and breeding programs.

A Temporary Immersion System to Improve Cannabis sativa Micropropagation.

The aim of this study was to propagate axillary shoots of Cannabis sativa L. using liquid medium in temporary immersion bioreactors. The effect of immersion frequency (3 or 6 immersions per day), explant type (apical or basal sections), explant number (8, 10, and 16 explants), mineral medium (Murashige and Skoog half-strength nitrates, ß-A and ß-H, all supplemented with 2-µM metatopoline), sucrose supplementation (2, 0.5, and 0% sucrose), culture duration (4 and 6 weeks), and bioreactor type (RITA® and Plantform™) were investigated. As a result, we propose a protocol for the proliferation of cannabis apical segments in RITA® or Plantform™ bioreactors. The explants (8 per RITA® and 24 per Plantform™) are immersed for 1 min, 3 times per day in ß-A medium supplemented with 2-µM metatopoline and 0.5% of sucrose and subcultured every 4 weeks. This is the first study using temporary immersion systems in C. sativa production, and our results provide new opportunities for the mass propagation of this species.

Effect of Sucrose on Growth and Stress Status of Castanea sativa x C. crenata Shoots Cultured in Liquid Medium.

Current breeding programs aim to increase the number of ink-tolerant chestnut trees using vegetative propagation of selected genotypes. However, the commercial vegetative propagation of chestnut species is still a bottleneck for the forest industry, mainly due to problems in the rooting and acclimation of propagules. This study aimed to explore the potential benefits of decreasing sucrose supplementation during chestnut micropropagation. Explants were cultured with high light intensity and CO2-enriched air in temporary or continuous immersion bioreactors and with different sucrose supplementation to evaluate the impact of these treatments on growth, rooting and physiological status (monosaccharide content, soluble phenolics and antioxidant activity). The proliferation and rooting performance of shoots cultured by continuous immersion decreased sharply with sucrose concentrations lower than 1%, whereas shoots cultured by temporary immersion grew and rooted successfully with 0.5% sucrose. These results suggest this system is appropriate to culture chestnut with low sucrose concentration and to explore photoautotrophic propagation of this species.

Exogenous application of stress-related signaling molecules affect growth and cannabinoid accumulation in medical cannabis (Cannabis sativa L.).

Medical cannabis (Cannabis sativa L.) is a source of bioactive phytochemicals with promising pharmacological and therapeutic applications. Enhancing the accumulation of valuable bioactive compounds is potentially a way of increasing the economic importance of this crop. Signaling molecules like salicylic acid (SA), jasmonic acid (JA), and γ-aminobutyric acid (GABA) are involved in the regulation of plant development and responses to biotic and abiotic stresses. Moreover, several phytohormones regulate plant trichome formation and elicit the synthesis of secondary metabolites in many plant species in both in vitro and in vivo systems. Therefore, exogenously delivered plant signaling molecules have the potential to modify the chemical profiles of medical cannabis. In this study, we found that the foliar application of SA, methyl jasmonate (MeJA), and GABA produces changes in the accumulation of the two major cannabinoids, cannabidiolic acid (CBDA) and Δ9- tetrahydrocannabinolic acid (THCA), in leaves and inflorescences of a medical cannabis variety. MeJA at 0.1 mM increased the CBDA content in inflorescences by 15.6%, while SA and MeJA at 0.1 mM increased CBDA and THCA accumulation in leaves by up to 57.3%. Treatments did not change the expression of genes participating in the final steps of the biosynthetic pathway of cannabinoids: olivetolic acid cyclase (CsOAC-1 and CsOAC-2), 2-acylphloroglucinol 4-prenyltransferase (CsPT4), cannabidiolic acid synthase (CsCBDAS), and tetrahydrocannabinolic acid synthase (CsTHCAS). Trichome density was not significantly different from the control plants in any treatment. Besides, we found strong correlations between several plant growth parameters and cannabinoid yields, showing a direct link between plant fitness and the production of cannabinoids.

Effect of Soil Type and In Vitro Proliferation Conditions on Acclimation and Growth of Willow Shoots Micropropagated in Continuous Immersion Bioreactors.

Salix viminalis L. is a species with high capacity for micropropagation and acclimation and could therefore be used to evaluate emergent techniques in the field of plant propagation. The aims of this study were to propagate willow in liquid medium with a continuous immersion system, to explore the application of photoautotrophic conditions and to investigate the adaptation of willow plantlets to different soils that could be used as alternatives to commercial peat. For proliferation, we used 3% sucrose or sugar-free medium, and as substrates, we used commercial peat, a soil from an oak forest with high organic matter content and a crop soil with low organic matter content. The effect of sugar supplementation during proliferation and the soil characteristics during acclimation and growth were evaluated on the basis of aerial and root growth and the hydrolytic and dehydrogenase enzymatic activities of the soils. The results indicate that under photoautotrophic conditions, the supplementation of sucrose during micropropagation did not affect the subsequent growth of the plantlets. All plants acclimated without loss, but the type of soil influenced the height and vigor. Plants produced the highest shoots in peat, whereas the most root development occurred in crop soil. Soil enzyme activities were more influenced by the type of soil than by the presence of plants.

Recent Advances in Adventitious Root Formation in Chestnut.

The genus Castanea includes several tree species that are relevant because of their geographical extension and their multipurpose character, that includes nut and timber production. However, commercial exploitation of the trees is hindered by several factors, particularly by their limited regeneration ability. Regardless of recent advances, there exists a serious limitation for the propagation of elite genotypes of chestnut due to decline of rooting ability as the tree ages. In the present review, we summarize the research developed in this genus during the last three decades concerning the formation of adventitious roots (ARs). Focusing on cuttings and in vitro microshoots, we gather the information available on several species, particularly C. sativa, C. dentata and the hybrid C.sativa × C. crenata, and analyze the influence of several factors on the achievements of the applied protocols, including genotype, auxin treatment, light regime and rooting media. We also pay attention to the acclimation phase, as well as compile the information available about biochemical and molecular related aspects. Furthermore, we considerate promising biotechnological approaches that might enable the improvement of the current protocols.

Use of bioreactor systems in the propagation of forest trees.

Plant biotechnology can be used to conserve the germplasm of natural forests, and to increase the productivity and sustainability of plantations. Both goals imply working with mature trees, which are often recalcitrant to micropropagation. Conventional in vitro culture uses closed containers and gelled medium with sugar supplementation. Bioreactor culture uses liquid medium and usually incorporates aeration. The increased absorption of nutrients via the liquid medium together with the renewal of the air inside the bioreactors may improve the physiological state of the explants. In this review, we will explore the feasibility of using bioreactors to overcome the recalcitrance of many trees to micropropagation and/or to decrease the cost of large-scale propagation. We will focus on the recent use of bioreactors during the multiplication, rooting (plant conversion in the case of somatic embryos), and acclimation stages of the micropropagation of axillary shoots and somatic embryos of forest trees (including some shrubs of commercial interest), in both temporary and continuous immersion systems. We will discuss the advantages and the main obstacles limiting the widespread implementation of bioreactor systems in woody plant culture, considering published scientific reports and contributions from the business sector.

Combinatorial targeting of nuclear export and translation of RNA inhibits aggressive B-cell lymphomas.

Aggressive double- and triple-hit (DH/TH) diffuse large B-cell lymphomas (DLBCLs) feature activation of Hsp90 stress pathways. Herein, we show that Hsp90 controls posttranscriptional dynamics of key messenger RNA (mRNA) species including those encoding BCL6, MYC, and BCL2. Using a proteomics approach, we found that Hsp90 binds to and maintains activity of eIF4E. eIF4E drives nuclear export and translation of BCL6, MYC, and BCL2 mRNA. eIF4E RNA-immunoprecipitation sequencing in DLBCL suggests that nuclear eIF4E controls an extended program that includes B-cell receptor signaling, cellular metabolism, and epigenetic regulation. Accordingly, eIF4E was required for survival of DLBCL including the most aggressive subtypes, DH/TH lymphomas. Indeed, eIF4E inhibition induces tumor regression in cell line and patient-derived tumorgrafts of TH-DLBCL, even in the presence of elevated Hsp90 activity. Targeting Hsp90 is typically limited by counterregulatory elevation of Hsp70B, which induces resistance to Hsp90 inhibitors. Surprisingly, we identify Hsp70 mRNA as an eIF4E target. In this way, eIF4E inhibition can overcome drug resistance to Hsp90 inhibitors. Accordingly, rational combinatorial inhibition of eIF4E and Hsp90 inhibitors resulted in cooperative antilymphoma activity in DH/TH DLBCL in vitro and in vivo.