Picky eaters: selective autophagy in plant cells.

Autophagy, a fundamental cellular process, plays a vital role in maintaining cellular homeostasis by degrading damaged or unnecessary components. While selective autophagy has been extensively studied in animal cells, its significance in plant cells has only recently gained attention. In this review, we delve into the intriguing realm selective autophagy in plants, with specific focus on its involvement in nutrient recycling, organelle turnover, and stress response. Moreover, recent studies have unveiled the interesting interplay between selective autophagy and epigenetic mechanisms in plants, elucidating the significance of epigenetic regulation in modulating autophagy-related gene expression and finely tuning the selective autophagy process in plants. By synthesizing existing knowledge, this review highlights the emerging field of selective autophagy in plant cells, emphasizing its pivotal role in maintaining nutrient homeostasis, facilitating cellular adaptation, and shedding light on the epigenetic regulation that governs these processes. Our comprehensive study provides the way for a deeper understanding of the dynamic control of cellular responses to nutrient availability and stress conditions, opening new avenues for future research in this field of autophagy in plant physiology.

Chlorophyll deficiency in Agave angustifolia Haw.: unveiling the impact on secondary metabolite production.

MAIN CONCLUSIONS: The albino phenotype of Agave angustifolia Haw. accumulates higher levels of phenylalanine and phenylpropanoids, while the green phenotype has a greater concentration of phenolic compounds. The metabolic consequences of chlorophyll deficiency in plants continue to be a captivating field of research, especially in relation to production of metabolic compounds. This study conducts a thorough analysis of the metabolome in green (G), variegated (V), and albino (A) phenotypes of Agave angustifolia Haw. Specifically, it examines the differences in the accumulation of compounds related to the phenylpropanoid and flavonoid biosynthesis pathways. Methanol extracts of leaf and meristem tissues from the three phenotypes grown in vitro were analyzed using liquid chromatography coupled with quadrupole time-of-flight high-resolution mass spectrometry (UPLC-MS-QTOF) for untargeted metabolomics and triple quadrupole (QqQ) mass spectrometry for targeted metabolomic analyses. By employing these methods, we discovered notable differences in the levels of important metabolites such as L-phenylalanine, 4-hydroxyphenylpyruvic acid, and various flavonoids among the different phenotypes. The results of our study indicate that the A phenotype shows a significant increase in the levels of phenylalanine and phenylpropanoids in both leaf and meristem tissues. This is in contrast to a decrease in flavonoids, suggesting a metabolic reprogramming to compensate for the lack of chlorophyll. Significantly, compounds such as kaempferol-3-O-glucoside and rutin exhibited significant quantitative reduction in the A leaves, suggesting a subtle modification in the production of flavonols and potentially a changed mechanism for antioxidant protection. This study emphasizes the complex metabolic changes in A. angustifolia´s chlorophyll-deficient phenotypes, providing insight into the complex interplay between primary and secondary metabolism in response to chlorophyll deficiency. Our research not only enhances the comprehension of plant metabolism in albino phenotypes but also opens new avenues for exploring the biochemical and genetic basis of such adaptations, with potential biotechnological applications of these distinct plant variants.

Transcriptome analysis reveals molecular mechanisms underlying chloroplast biogenesis in albino Agave angustifolia plantlets.

Albino plants display partial or complete loss of photosynthetic pigments and defective thylakoid membrane development, consequently impairing plastid function and development. These distinctive attributes render albino plants excellent models for investigating chloroplast biogenesis. Despite their potential, limited exploration has been conducted regarding the molecular alterations underlying these phenotypes, extending beyond photosynthetic metabolism. In this study, we present a novel de novo transcriptome assembly of an albino somaclonal variant of Agave angustifolia Haw., which spontaneously emerged during the micropropagation of green plantlets. Additionally, RT-qPCR analysis was employed to validate the expression of genes associated with chloroplast biogenesis, and plastome copy numbers were quantified. This research aims to gain insight into the molecular disruptions affecting chloroplast development and ascertain whether the expression of critical genes involved in plastid development and differentiation is compromised in albino tissues of A. angustifolia. Our transcriptomic findings suggest that albino Agave plastids exhibit high proliferation, activation of the protein import machinery, altered transcription directed by PEP and NEP, dysregulation of plastome expression genes, reduced expression of photosynthesis-associated nuclear genes, disruption in the tetrapyrrole and carotenoid biosynthesis pathway, alterations in the plastid ribosome, and an increased number of plastome copies, among other alterations.

Same modification, different location: the mythical role of N6-adenine methylation in plant genomes.

MAIN CONCLUSION: The present review summarizes recent advances in the understanding of 6mA in DNA as an emergent epigenetic mark with distinctive characteristics, discusses its importance in plant genomes, and highlights its chemical nature and functions. Adenine methylation is an epigenetic modification present in DNA (6mA) and RNA (m6A) that has a regulatory function in many cellular processes. This modification occurs through a reversible reaction that covalently binds a methyl group, usually at the N6 position of the purine ring. This modification carries biophysical properties that affect the stability of nucleic acids as well as their binding affinity with other molecules. DNA 6mA has been related to genome stability, gene expression, DNA replication, and repair mechanisms. Recent advances have shown that 6mA in plant genomes is related to development and stress response. In this review, we present recent advances in the understanding of 6mA in DNA as an emergent epigenetic mark with distinctive characteristics. We discuss the key elements of this modification, focusing mainly on its importance in plant genomes. Furthermore, we highlight its chemical nature and the regulatory effects that it exerts on gene expression and plant development. Finally, we emphasize the functions of 6mA in photosynthesis, stress, and flowering.

Auxins, the hidden player in chloroplast development.

Throughout decades of plant research, the plant hormones known as auxins have been found to be of vital importance in most plant development processes. Indole-3-acetic acid (IAA) represents the most common auxin in plants and can be synthesized from its tryptophan precursor, which is synthesized in the chloroplast. The chloroplast constitutes an organelle of great relevance to plants since the photosynthesis process by which plants get most of their energy is carried out there. The role of auxins in photosynthesis has been studied for at least 50 years, and in this time, it has been shown that auxins have an effect on several of the essential components and structure of the chloroplast. In recent decades, a high number of genes have been reported to be expressed in the chloroplast and some of their mutants have been shown to alter different auxin-mediated pathways. Genes in signaling pathways such as IAA/AUX, ARF, GH.3, SAUR and TIR, biosynthesis-related genes such as YUCCA and transport-related genes such as PIN have been identified among the most regulated genes in mutants related to alterations in the chloroplast. This review aims to provide a complete and updated summary of the relationship between auxins and several processes that involve the chloroplast, including chloroplast development, plant albinism, redox regulation and pigment synthesis.

A peak in global DNA methylation is a key step to initiate the somatic embryogenesis of coconut palm (Cocos nucifera L).

KEY MESSAGE: DNA methylation, morphogenesis and gene expression during the somatic embryogenesis of Coconut are affected by 5-Azacytidine pretreatments, indicating that DNA methylation is an important factor throughout this process. Somatic embryogenesis (SE) is a process that can aid in the production of elite Cocos nucifera palms. It has been well established that epigenetic mechanisms are regulators of cell differentiation programs; however, their role in the coconut somatic embryogenesis has not yet been addressed. To this end, the morphogenetic changes, the global DNA methylation and the expression profiles of the SE-related genes and DNA methyltransferases genes were evaluated during the SE process, with and without the presence of 5-Azacytidine (AzaC). The results show that three days of pretreatments with 15 µM and 20 µM of AzaC significantly increased early somatic embryo formation (four- and tenfold, respectively). A clear peak of the global percentage of DNA methylation (approximately 13%) was determined at the beginning of the culture, followed by a re-establishing stage and a steady increase thereafter; in all cases, the levels of DNA methylation were lower after the pretreatments with AzaC. Additionally, the expression profiles of the SERK, WUS, BBM and LEC genes are modulated during the SE process and the pretreatments with AzaC affect the expression profiles of these genes, even at early stages. Furthermore, increased levels of expression were observed for the genes encoding for DNA methyltransferases (MET, CMT and DRM) at early and late stages of SE, indicating that DNA methylation is an important factor throughout the SE.

5-Azacytidine: A Promoter of Epigenetic Changes in the Quest to Improve Plant Somatic Embryogenesis.

Somatic embryogenesis (SE) is a widely studied process due to its biotechnological potential to generate large quantities of plants in short time frames and from different sources of explants. The success of SE depends on many factors, such as the nature of the explant, the microenvironment generated by in vitro culture conditions, and the regulation of gene expression, among others. Epigenetics has recently been identified as an important factor influencing SE outcome. DNA methylation is one of the most studied epigenetic mechanisms due to its essential role in gene expression, and its participation in SE is crucial. DNA methylation levels can be modified through the use of drugs such as 5-Azacytidine (5-AzaC), an inhibitor of DNA methylation, which has been used during SE protocols. The balance between hypomethylation and hypermethylation seems to be the key to SE success. Here, we discuss the most prominent recent research on the role of 5-AzaC in the regulation of DNA methylation, highlighting its importance during the SE process. Also, the molecular implications that this inhibitor might have for the increase or decrease in the embryogenic potential of various explants are reviewed.

Physiological differences and changes in global DNA methylation levels in Agave angustifolia Haw. albino variant somaclones during the micropropagation process.

KEY MESSAGE: Global DNA methylation changes caused by in vitro conditions are associated with the subculturing and phenotypic variation in Agave angustifolia Haw. While the relationship between the development of albinism and in vitro culture is well documented, the role of epigenetic processes in this development leaves some important questions unanswered. During the micropropagation of Agave angustifolia Haw., we found three different phenotypes, green (G), variegated (V) and albino (A). To understand the physiological and epigenetic differences among the somaclones, we analyzed several morphophysiological parameters and changes in the DNA methylation patterns in the three phenotypes during their in vitro development. We found that under in vitro conditions, the V plantlets maintained their CAM photosynthetic capacity, while the A variant showed no pigments and lost its CAM photosynthetic ability. Epigenetic analysis revealed that global DNA methylation increased in the G phenotype during the first two subcultures. However, after that time, DNA methylation levels declined. This hypomethylation correlated with the appearance of V shoots in the G plantlets. A similar correlation occurred in the V phenotype, where an increase of 2 % in the global DNA methylation levels was correlated with the generation of A shoots in the V plantlets. This suggests that an "epigenetic stress memory" during in vitro conditions causes a chromatin shift that favors the generation of variegated and albino shoots.

Biotic interactions in the rhizosphere: a diverse cooperative enterprise for plant productivity.

Microbes and plants have evolved biochemical mechanisms to communicate with each other. The molecules responsible for such communication are secreted during beneficial or harmful interactions. Hundreds of these molecules secreted into the rhizosphere have been identified, and their functions are being studied in order to understand the mechanisms of interaction and communication among the different members of the rhizosphere community. The importance of root and microbe secretion to the underground habitat in improving crop productivity is increasingly recognized, with the discovery and characterization of new secreting compounds found in the rhizosphere. Different omic approaches, such as genomics, transcriptomics, proteomics, and metabolomics, have expanded our understanding of the first signals between microbes and plants. In this review, we highlight the more recent discoveries related to molecules secreted into the rhizosphere and how they affect plant productivity, either negatively or positively. In addition, we include a survey of novel approaches to studying the rhizosphere and emerging opportunities to direct future studies.

Protocol for Generating Arabidopsis thaliana Cell Suspension Cultures Under Different Light Conditions.

Plant cell suspension cultures (PCSCs) are in vitro-cultured cells that can divide indefinitely in a sterile growth medium. These PCSCs can be derived from various plant tissues, such as the root, stem, leaves, or seeds, and are maintained in a suitable culture medium containing nutrients, vitamins, hormones, and other essential components necessary for their growth. PCSCs have extensive applications in biotechnology, particularly in producing pharmaceutical and chemical compounds. This chapter presents a protocol for generating cell lines from Arabidopsis thaliana root callus under different light conditions, which can be used to investigate the effects of light on plant cell growth and development. The protocol described in this chapter is a valuable tool for researchers interested in utilizing PCSCs in their studies.

Chloroplast Isolation in Agavaceas.

Chloroplast isolation protocols have been extensively developed for various species of plants, particularly model organisms with easily manipulable physical characteristics. However, succulent plants, such as Agave angustifolia Haw., which possess adaptations for arid environments like the Crassulacean acid metabolism (CAM) and a thicker cuticle, have received less attention, resulting in a potential knowledge gap. This chapter presents a specialized protocol focusing on isolating chloroplast from A. angustifolia, a species exhibiting adaptations to arid conditions and holding ecological and economic significance due to its role in producing bacanora and mezcal beverages. By successfully isolating chloroplast from A. angustifolia plant growth in ex vitro and in vitro conditions, this protocol enables comprehensive future analyses to elucidate metabolic processes and explore potential applications in related species. Consequently, this research aims to bridge this knowledge gap in chloroplast isolation for succulent plants, providing new insights for future investigations in the field.

Epigenetic Regulation in Heterosis and Environmental Stress: The Challenge of Producing Hybrid Epigenomes to Face Climate Change.

Epigenetic regulation has the potential to revolutionize plant breeding and improve crop yields by regulating gene expression in plants. DNA methylation and histone modifications are key epigenetic modifications that can impact plant development, stress responses, productivity, and yields. Higher-yielding crops not only generate greater profits for farmers and seed producers, but also require less land, water, fuel, and fertilizer than traditional crops for equivalent yields. The use of heterosis in crops can influence productivity and food quality, but producing hybrids with superior agronomic traits to their parents remains challenging. However, epigenetic markers, such as histone methylation and acetylation, may help select parental and hybrid combinations with better performances than the parental plants. This review assesses the potential applications of epigenetics in crop breeding and improvement, rendering agriculture more efficient, sustainable, and adaptable to changing environmental conditions.

Physiological, epigenetic, and proteomic responses in Pfaffia glomerata growth in vitro under salt stress and 5-azacytidine.

Plants adjust their complex molecular, biochemical, and metabolic processes to overcome salt stress. Here, we investigated the proteomic and epigenetic alterations involved in the morphophysiological responses of Pfaffia glomerata, a medicinal plant, to salt stress and the demethylating agent 5-azacytidine (5-azaC). Moreover, we investigated how these changes affected the biosynthesis of 20-hydroxyecdysone (20-E), a pharmacologically important specialized metabolite. Plants were cultivated in vitro for 40 days in Murashige and Skoog medium supplemented with NaCl (50 mM), 5-azaC (25 µM), and NaCl + 5-azaC. Compared with the control (medium only), the treatments reduced growth, photosynthetic rates, and photosynthetic pigment content, with increase in sucrose, total amino acids, and proline contents, but a reduction in starch and protein. Comparative proteomic analysis revealed 282 common differentially accumulated proteins involved in 87 metabolic pathways, most of them related to amino acid and carbohydrate metabolism, and specialized metabolism. 5-azaC and NaCl + 5-azaC lowered global DNA methylation levels and 20-E content, suggesting that 20-E biosynthesis may be regulated by epigenetic mechanisms. Moreover, downregulation of a key protein in jasmonate biosynthesis indicates the fundamental role of this hormone in the 20-E biosynthesis. Taken together, our results highlight possible regulatory proteins and epigenetic changes related to salt stress tolerance and 20-E biosynthesis in P. glomerata, paving the way for future studies of the mechanisms involved in this regulation.

KNOX1 is expressed and epigenetically regulated during in vitro conditions in Agave spp.

BACKGROUND: The micropropagation is a powerful tool to scale up plants of economical and agronomical importance, enhancing crop productivity. However, a small but growing body of evidence suggests that epigenetic mechanisms, such as DNA methylation and histone modifications, can be affected under the in vitro conditions characteristic of micropropagation. Here, we tested whether the adaptation to different in vitro systems (Magenta boxes and Bioreactors) modified epigenetically different clones of Agave fourcroydes and A. angustifolia. Furthermore, we assessed whether these epigenetic changes affect the regulatory expression of KNOTTED1-like HOMEOBOX (KNOX) transcription factors. RESULTS: To gain a better understanding of epigenetic changes during in vitro and ex vitro conditions in Agave fourcroydes and A. angustifolia, we analyzed global DNA methylation, as well as different histone modification marks, in two different systems: semisolid in Magenta boxes (M) and temporary immersion in modular Bioreactors (B). No significant difference was found in DNA methylation in A. fourcroydes grown in either M or B. However, when A. fourcroydes was compared with A. angustifolia, there was a two-fold difference in DNA methylation between the species, independent of the in vitro system used. Furthermore, we detected an absence or a low amount of the repressive mark H3K9me2 in ex vitro conditions in plants that were cultured earlier either in M or B. Moreover, the expression of AtqKNOX1 and AtqKNOX2, on A. fourcroydes and A. angustifolia clones, is affected during in vitro conditions. Therefore, we used Chromatin ImmunoPrecipitation (ChIP) to know whether these genes were epigenetically regulated. In the case of AtqKNOX1, the H3K4me3 and H3K9me2 were affected during in vitro conditions in comparison with AtqKNOX2. CONCLUSIONS: Agave clones plants with higher DNA methylation during in vitro conditions were better adapted to ex vitro conditions. In addition, A. fourcroydes and A. angustifolia clones displayed differential expression of the KNOX1 gene during in vitro conditions, which is epigenetically regulated by the H3K4me3 and H3K9me2 marks. The finding of an epigenetic regulation in key developmental genes will make it important in future studies to identify factors that help to find climate-resistant micropropagated plants.

Root secretion of defense-related proteins is development-dependent and correlated with flowering time.

Proteins found in the root exudates are thought to play a role in the interactions between plants and soil organisms. To gain a better understanding of protein secretion by roots, we conducted a systematic proteomic analysis of the root exudates of Arabidopsis thaliana at different plant developmental stages. In total, we identified 111 proteins secreted by roots, the majority of which were exuded constitutively during all stages of development. However, defense-related proteins such as chitinases, glucanases, myrosinases, and others showed enhanced secretion during flowering. Defense-impaired mutants npr1-1 and NahG showed lower levels of secretion of defense proteins at flowering compared with the wild type. The flowering-defective mutants fca-1, stm-4, and co-1 showed almost undetectable levels of defense proteins in their root exudates at similar time points. In contrast, root secretions of defense-enhanced cpr5-2 mutants showed higher levels of defense proteins. The proteomics data were positively correlated with enzymatic activity assays for defense proteins and with in silico gene expression analysis of genes specifically expressed in roots of Arabidopsis. In conclusion, our results show a clear correlation between defense-related proteins secreted by roots and flowering time.

Methyl jasmonate induces ATP biosynthesis deficiency and accumulation of proteins related to secondary metabolism in Catharanthus roseus (L.) G. hairy roots.

Jasmonates are specific signal molecules in plants that are involved in a diverse set of physiological and developmental processes. However, methyl jasmonate (MeJA) has been shown to have a negative effect on root growth and, so far, the biochemical mechanism for this is unknown. Using Catharanthus roseus hairy roots, we were able to observe the effect of MeJA on growth inhibition, cell disorganization and cell death of the root cap. Hairy roots treated with MeJA induced the perturbation of mitochondrial membrane integrity and a diminution in ATP biosynthesis. Furthermore, several proteins were identified that were involved in energy and secondary metabolism; the changes in accumulation of these proteins were observed with 100 µM MeJA. In conclusion, our results suggest that a switch of the metabolic fate of hairy roots in response to MeJA could cause an increase in the accumulation of secondary metabolites. This is likely to have important consequences in the production of specific alkaloids important for the pharmaceutical industry.

Effects on the Post-translational Modification of H3K4Me3, H3K9ac, H3K9Me2, H3K27Me3, and H3K36Me2 Levels in Cerebral Cortex, Hypothalamus and Pons of Rats after a Systemic Administration of Cannabidiol: A Preliminary Study.

BACKGROUND: Cannabidiol (CBD), a non-psychotropic constituent of Cannabis sativa, has shown therapeutic promises by modulating several pathological conditions, including pain, epilepsy autism, among others. However, the molecular mechanism of action of CBD remains unknown and recent data suggest the engagement on CBD´s effects of nuclear elements, such as histone activity. AIM: This study assessed the changes in the post-translational modification (PTM) on the histones H3K4Me3, H3K9ac, H3K9Me2, H3K27Me3, and H3K36Me2 in several brain regions of rats after the administration of CBD (20 mg/Kg/i.p.). OBJECTIVE: To evaluate the effects on the PTM of histones H3K4Me3, H3K9ac, H3K9Me2, H3K27Me3, and H3K36Me2 levels in the cerebral cortex, hypothalamus and pons of CBD-treated rats. METHODS: Ten adult rats were randomly assigned into 2 groups: Control or CBD (20 mg/Kg/i.p). Animals were sacrificed after treatments and brains were collected for dissections of the cerebral cortex, hypothalamus and pons. Samples were analyzed for PTM on the histones H3K4Me3, H3K9ac, H3K9Me2, H3K27Me3, and H3K36Me2 levels by Western blot procedure. RESULTS: CBD increased the PTM levels on the histones H3K4Me3, H3K9ac, and H3K27Me3 in the cerebral cortex whereas no significant differences were found in H3K9Me2 and H3K36Me2. In addition, in the hypothalamus, CBD decreased the contents of H3K9ac while no significant effects were observed in H3K4Me3, H3K9Me2, H3K27Me3, and H3K36Me2. Lastly, in the pons, CBD- treated rats showed a significant decline on the PTM levels of H3K4Me3 whereas no statistical differences were found in H3K9ac, H3K9Me2, H3K27Me3, and H3K36Me2. CONCLUSION: The study showed that CBD induced differential effects in levels of PTM on the histones H3K4Me3, H3K9ac, H3K9Me2, H3K27Me3, and H3K36Me2 in several brain regions.

Plant neighbor identity influences plant biochemistry and physiology related to defense.

BACKGROUND: Chemical and biological processes dictate an individual organism's ability to recognize and respond to other organisms. A small but growing body of evidence suggests that plants may be capable of recognizing and responding to neighboring plants in a species specific fashion. Here we tested whether or not individuals of the invasive exotic weed, Centaurea maculosa, would modulate their defensive strategy in response to different plant neighbors. RESULTS: In the greenhouse, C. maculosa individuals were paired with either conspecific (C. maculosa) or heterospecific (Festuca idahoensis) plant neighbors and elicited with the plant defense signaling molecule methyl jasmonate to mimic insect herbivory. We found that elicited C. maculosa plants grown with conspecific neighbors exhibited increased levels of total phenolics, whereas those grown with heterospecific neighbors allocated more resources towards growth. To further investigate these results in the field, we conducted a metabolomics analysis to explore chemical differences between individuals of C. maculosa growing in naturally occurring conspecific and heterospecific field stands. Similar to the greenhouse results, C. maculosa individuals accumulated higher levels of defense-related secondary metabolites and lower levels of primary metabolites when growing in conspecific versus heterospecific field stands. Leaf herbivory was similar in both stand types; however, a separate field study positively correlated specialist herbivore load with higher densities of C. maculosa conspecifics. CONCLUSIONS: Our results suggest that an individual C. maculosa plant can change its defensive strategy based on the identity of its plant neighbors. This is likely to have important consequences for individual and community success.

Auxin perception in Agave is dependent on the species' Auxin Response Factors.

Auxins are one of the most important and studied phytohormones in nature. Auxin signaling and perception take place in the cytosol, where the auxin is sensed. Then, in the nucleus, the auxin response factors (ARF) promote the expression of early-response genes. It is well known that not all plants respond to the same amount and type of auxins and that the response can be very different even among plants of the same species, as we present here. Here we investigate the behavior of ARF in response to various auxins in Agave angustifolia Haw., A. fourcroydes Lem. and A. tequilana Weber var. Azul. By screening the available database of A. tequilana genes, we have identified 32 ARF genes with high sequence identity in the conserved domains, grouped into three main clades. A phylogenetic tree was inferred from alignments of the 32 Agave ARF protein sequences and the evolutionary relationship with other species was analyzed. AteqARF 4, 15, 21, and 29 were selected as a representative diverse sample coming from each of the different subclades that comprise the two main clades of the inferred phylogenetic reconstruction. These ARFs showed differential species-specific expression patterns in the presence of indole-3-acetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4-D). Interestingly, A. angustifolia showed different phenotypes in the presence and absence of auxins. In the absence of auxin, A. angustifolia produces roots, while shoots are developed in the presence of IAA. However, in the presence of 2,4-D, the plant meristem converts into callus. According to our results, it is likely that AteqARF15 participates in this outcome.