Here we propose a protocol for embryogenic cultures induction, proliferation and maturation for the Brazilian conifer Podocarpus lambertii, and investigated the effect of abscisic acid (ABA) and glutathione (GSH) supplementation on the maturation phase. ABA, zeatin (Z) and salicylic acid (SA) endogenous levels were quantified. Number of somatic embryos obtained in ABA-supplemented treatment was significant higher than in ABA-free treatment, showing the relevance of ABA supplementation during somatic embryos maturation. Histological analysis showed the stereotyped sequence of developmental stages in conifer somatic embryos, reaching the late torpedo-staged embryo. GSH supplementation in maturation culture medium improved the somatic embryos number and morphological features. GSH 0mM and GSH 0.1mM treatments correlated with a decreased ABA endogenous level during maturation, while GSH 0.5mM treatment showed constant levels. All treatments resulted in decreased Z endogenous levels, supporting the concept that cytokinins are important during the initial cell division but not for the later stages of embryo development. The lowest SA levels found in GSH 0.5mM treatment were coincident with early embryonic development, and this treatment resulted in the highest development of somatic embryos. Thus, a correlation between lower SA levels and improved somatic embryo formation can be hypothesized.

Winter dormancy is an adaptative mechanism that temperate and boreal trees have developed to protect their meristems against low temperatures. In apple trees (Malus domestica), cold temperatures induce bud dormancy at the end of summer/beginning of the fall. Apple buds stay dormant during winter until they are exposed to a period of cold, after which they can resume growth (budbreak) and initiate flowering in response to warmer temperatures in spring. It is well-known that small RNAs modulate temperature responses in many plant species, but however, how small RNAs are involved in genetic networks of temperature-mediated dormancy control in fruit tree species remains unclear. Here, we have made use of a recently developed ARGONAUTE (AGO)-purification technique to isolate small RNAs from apple buds. A small RNA-seq experiment resulted in the identification of 17 micro RNAs (miRNAs) that change their pattern of expression in apple buds during dormancy. Furthermore, the functional analysis of their predicted target genes suggests a main role of the 17 miRNAs in phenylpropanoid biosynthesis, gene regulation, plant development and growth, and response to stimulus. Finally, we studied the conservation of the Arabidopsis thaliana regulatory miR159-MYB module in apple in the context of the plant hormone abscisic acid homeostasis.

SUMMARY: Winter bud dormancy is a critical adaptive process in temperate fruit trees, safeguarding meristems from freezing temperatures and aligning growth with seasonal cues. Dormancy encompasses two primary phases: endodormancy, where internal signals, particularly elevated abscisic acid (ABA), block growth and necessitate chilling for release; and ecodormancy, where buds regain growth competence but remain quiescent until external conditions are favourable. In apple ( Malus domestica ), we demonstrated that BRANCHED1 ( MdBRC1 ) serves as a central inhibitor of bud growth specifically during ecodormancy. In many plant species, BRC1-like transcription factors integrate environmental and hormonal signals, activating gene networks linked to growth repression, notably those involved in ABA biosynthesis and signalling. Gain-of-function studies in poplar confirmed that MdBRC1 robustly suppresses shoot growth in trees. Our comparative transcriptomic analyses revealed that MdBRC1 directly regulates a suite of dormancy-associated genes, reinforcing its role as a molecular brake on bud break. Importantly, we show that the apple gene FLOWERING LOCUS T2 ( MdFT2 ) is transcriptionally upregulated after dormancy and might act as a key inducer of bud break. Our data reveal that MdFT2 physically interacts with MdBRC1, reducing MdBRC1 activity during ecodormancy. This antagonistic interaction acts as a molecular switch, facilitating the transition from ecodormancy to active bud growth as spring approaches. Together, these findings uncover a regulatory module that finely tunes bud break timing in apple trees and provide a foundation for breeding strategies to enhance fruit tree resilience and adaptability in the context of climate change.