CRISPRi-induced transcriptional regulation of IAH1 gene and its influence on volatile compounds profile in Kluyveromyces marxianus DU3.

Mezcal is a traditional Mexican distilled beverage, known for its marked organoleptic profile, which is influenced by several factors, such as the fermentation process, where a wide variety of microorganisms are present. Kluyveromyces marxianus is one of the main yeasts isolated from mezcal fermentations and has been associated with ester synthesis, contributing to the flavors and aromas of the beverage. In this study, we employed CRISPR interference (CRISPRi) technology, using dCas9 fused to the Mxi1 repressor factor domain, to down-regulate the expression of the IAH1 gene, encoding for an isoamyl acetate-hydrolyzing esterase, in K. marxianus strain DU3. The constructed CRISPRi plasmid successfully targeted the IAH1 gene, allowing for specific gene expression modulation. Through gene expression analysis, we assessed the impact of IAH1 down-regulation on the metabolic profile of volatile compounds. We also measured the expression of other genes involved in volatile compound biosynthesis, including ATF1, EAT1, ADH1, and ZWF1 by RT-qPCR. Results demonstrated successful down-regulation of IAH1 expression in K. marxianus strain DU3 using the CRISPRi system. The modulation of IAH1 gene expression resulted in alterations in the production of volatile compounds, specifically ethyl acetate, which are important contributors to the beverage's aroma. Changes in the expression levels of other genes involved in ester biosynthesis, suggesting that the knockdown of IAH1 may generate intracellular alterations in the balance of these metabolites, triggering a regulatory response. The application of CRISPRi technology in K. marxianus opens the possibility of targeted modulation of gene expression, metabolic engineering strategies, and synthetic biology in this yeast strain.

LIPRNAseq: a method to discover lipid interacting RNAs by sequencing.

BACKGROUND: Current biological research extensively describes the interactions of molecules such as RNA with other nucleic acids or proteins. However, the relatively recent discovery of nuclear phospholipids playing biologically relevant processes outside membranes, as well as, RNA-lipid interactions shows the need for new methods to explore the identity of these RNAs. METHODS AND RESULTS: In this study, we describe the method for LIPID-RNA isolation followed by sequencing and analysis of the RNA that has the ability to interact with the selected lipids. Here we utilized specific phospholipid coated beads for selective RNA binding. We tested RNA from organisms belonging to different realms (human, plant, and yeast), and tested their ability to bind a specific lipid. CONCLUSIONS: The results show several RNAs differentially enriched in the pull-down of phosphatidyl Inositol 4,5 bisphosphate coated beads. This method is helpful to screen lipid-binding RNA, which may have relevant biological functions. The method can be used with different lipids and comparison of pull-downs and can narrow the selection of RNAs that interact with a particular lipid for further studies.

Current research on viral proteins that interact with fibrillarin.

The nucleolus is a multifunctional nuclear domain primarily dedicated to ribosome biogenesis. Certain viruses developed strategies to manipulate host nucleolar proteins to facilitate their replication by modulating ribosomal RNA (rRNA) processing. This association interferes with nucleolar functions resulting in overactivation or arrest of ribosome biogenesis, induction or inhibition of apoptosis, and affecting stress response. The nucleolar protein fibrillarin (FBL) is an important target of some plant and animal viruses. FBL is an essential and highly conserved S-adenosyl methionine (SAM) dependent methyltransferase, capable of rRNA degradation by its intrinsically disordered region (IDR), the glycine/arginine-rich (GAR) domain. It forms a ribonucleoprotein complex that directs 2'-O-methylations in more than 100 sites of pre-rRNAs. It is involved in multiple cellular processes, including initiation of transcription, oncogenesis, and apoptosis, among others. The interaction with animal viruses, including human viruses, triggered its redistribution to the nucleoplasm and cytoplasm, interfering with its role in pre-rRNA processing. Viral-encoded proteins with IDRs as nucleocapsids, matrix, Tat protein, and even a viral snoRNA, can associate with FBL, forcing the nucleolar protein to undergo atypical functions. Here we review the molecular mechanisms employed by animal and human viruses to usurp FBL functions and the effect on cellular processes, particularly in ribosome biogenesis.

Evaluation of Natural Pigments Production in Response to Various Stress Signals in Cell Lines of Stenocereus queretaroensis.

Stenocereus queretaroensis (F.A.C. Weber ex Mathes.) Buxb is a cactus that has long been used as a source food in central and northern México. Its fruits, commonly called pitayas, biosynthesize high amounts of betalains. These molecules are water-soluble nitrogenous compounds; that compared to other pigments, such as anthocyanins or carotenoids, stand out for their physicochemical stability in industrial processes. Due to genetic and environmental factors involved in the biosynthesis and accumulation of secondary metabolites in plants, we tested different stress-inducing agents (elicitor, osmotic, salt, and temperature) to induce betalains accumulation in cell culture from fruits of Stenocereus queretaroensis. This work aimed to understand stress conditions that induce the metabolic pathways required for the accumulation of betalains. The results show how betacyanin concentration increases under high sugar conditions, thus affecting the expression of L-DOPA 4, 5 dioxygenase resulting in a strong dark red coloration. This suggests this enzyme is part of a rate-limiting step in betalain production. In addition, we found that betalains accumulation occurs under particular stress conditions. Cells that have a high level of betacyanins show better resistance to stress in the cell culture, as well as an overall different behavior including cell aggregation and alterations in nuclear size. Together the results shown here may provide new strategies to manipulate and mass produce the pigments from Stenocereus queretaroensis in cell culture.

Phylogenomic and Microsynteny Analysis Provides Evidence of Genome Arrangements of High-Affinity Nitrate Transporter Gene Families of Plants.

Nitrate transporter 2 (NRT2) and NRT3 or nitrate-assimilation-related 2 (NAR2) proteins families form a two-component, high-affinity nitrate transport system, which is essential for the acquisition of nitrate from soils with low N availability. An extensive phylogenomic analysis across land plants for these families has not been performed. In this study, we performed a microsynteny and orthology analysis on the NRT2 and NRT3 genes families across 132 plants (Sensu lato) to decipher their evolutionary history. We identified significant differences in the number of sequences per taxonomic group and different genomic contexts within the NRT2 family that might have contributed to N acquisition by the plants. We hypothesized that the greater losses of NRT2 sequences correlate with specialized ecological adaptations, such as aquatic, epiphytic, and carnivory lifestyles. We also detected expansion on the NRT2 family in specific lineages that could be a source of key innovations for colonizing contrasting niches in N availability. Microsyntenic analysis on NRT3 family showed a deep conservation on land plants, suggesting a high evolutionary constraint to preserve their function. Our study provides novel information that could be used as guide for functional characterization of these gene families across plant lineages.

Phase Separation of Intrinsically Disordered Nucleolar Proteins Relate to Localization and Function.

The process of phase separation allows for the establishment and formation of subcompartmentalized structures, thus enabling cells to perform simultaneous processes with precise organization and low energy requirements. Chemical modifications of proteins, RNA, and lipids alter the molecular environment facilitating enzymatic reactions at higher concentrations in particular regions of the cell. In this review, we discuss the nucleolus as an example of the establishment, dynamics, and maintenance of a membraneless organelle with a high level of organization.

Plant viral proteins and fibrillarin: the link to complete the infective cycle.

The interaction between viruses with the nucleolus is already a well-defined field of study in plant virology. This interaction is not restricted to those viruses that replicate in the nucleus, in fact, RNA viruses that replicate exclusively in the cytoplasm express proteins that localize in the nucleolus. Some positive single stranded RNA viruses from animals and plants have been reported to interact with the main nucleolar protein, Fibrillarin. Among nucleolar proteins, Fibrillarin is an essential protein that has been conserved in sequence and function throughout evolution. Fibrillarin is a methyltransferase protein with more than 100 methylation sites in the pre-ribosomal RNA, involved in multiple cellular processes, including initiation of transcription, oncogenesis, and apoptosis, among others. Recently, it was found that AtFib2 shows a ribonuclease activity. In plant viruses, Fibrillarin is involved in long-distance movement and cell-to-cell movement, being two highly different processes. The mechanism that Fibrillarin performs is still unknown. However, and despite belonging to very different viral families, the majority comply with the following. (1) They are positive single stranded RNA viruses; (2) encode different types of viral proteins that partially localize in the nucleolus; (3) interacts with Fibrillarin exporting it to the cytoplasm; (4) the viral protein-Fibrillarin interaction forms an RNP complex with the viral RNA and; (5) Fibrillarin depletion affects the infective cycle of the virus. Here we review the relationship of those plant viruses with Fibrillarin interaction, with special focus on the molecular processes of the virus to sequester Fibrillarin to complete its infective cycle.

Towards an understanding of the role of intrinsic protein disorder on plant adaptation to environmental challenges.

Intrinsic protein disorder is an interesting structural feature where fully functional proteins lack a three-dimensional structure in solution. In this work, we estimated the relative content of intrinsic protein disorder in 96 plant proteomes including monocots and eudicots. In this analysis, we found variation in the relative abundance of intrinsic protein disorder among these major clades; the relative level of disorder is higher in monocots than eudicots. In turn, there is an inverse relationship between the degree of intrinsic protein disorder and protein length, with smaller proteins being more disordered. The relative abundance of amino acids depends on intrinsic disorder and also varies among clades. Within the nucleus, intrinsically disordered proteins are more abundant than ordered proteins. Intrinsically disordered proteins are specialized in regulatory functions, nucleic acid binding, RNA processing, and in response to environmental stimuli. The implications of this on plants' responses to their environment are discussed.

Fibrillarin evolution through the Tree of Life: Comparative genomics and microsynteny network analyses provide new insights into the evolutionary history of Fibrillarin.

Fibrillarin (FIB), a methyltransferase essential for life in the vast majority of eukaryotes, is involved in methylation of rRNA required for proper ribosome assembly, as well as methylation of histone H2A of promoter regions of rRNA genes. RNA viral progression that affects both plants and animals requires FIB proteins. Despite the importance and high conservation of fibrillarins, there little is known about the evolutionary dynamics of this small gene family. We applied a phylogenomic microsynteny-network approach to elucidate the evolutionary history of FIB proteins across the Tree of Life. We identified 1063 non-redundant FIB sequences across 1049 completely sequenced genomes from Viruses, Bacteria, Archaea, and Eukarya. FIB is a highly conserved single-copy gene through Archaea and Eukarya lineages, except for plants, which have a gene family expansion due to paleopolyploidy and tandem duplications. We found a high conservation of the FIB genomic context during plant evolution. Surprisingly, FIB in mammals duplicated after the Eutheria split (e.g., ruminants, felines, primates) from therian mammals (e.g., marsupials) to form two main groups of sequences, the FIB and FIB-like groups. The FIB-like group transposed to another genomic context and remained syntenic in all the eutherian mammals. This transposition correlates with differences in the expression patterns of FIB-like proteins and with elevated Ks values potentially due to reduced evolutionary constraints of the duplicated copy. Our results point to a unique evolutionary event in mammals, between FIB and FIB-like genes, that led to non-redundant roles of the vital processes in which this protein is involved.

The SoNAP gene from sugarcane (Saccharum officinarum) encodes a senescence-associated NAC transcription factor involved in response to osmotic and salt stress.

Climate change has caused serious problems related to the productivity of agricultural crops directly affecting human well-being. Plants have evolved to produce molecular mechanisms in response to environmental stresses, such as transcription factors (TFs), to cope with abiotic stress. The NAC proteins constitute a plant-specific family of TFs involved in plant development processes and tolerance to biotic and abiotic stress. Sugarcane is a perennial grass that accumulates a large amount of sucrose and is a crucial agro-industry crop in tropical regions. Our previous transcriptome analyses on sugarcane that were exposed to drought conditions revealed significant increases in the expression of several NAC TFs through all of the time-point stress conditions. In this work, we characterize all previously detected sugarcane NAC genes, utilizing phylogenetics and expression analyses. Furthermore, we characterized a sugarcane NAC gene orthologous to the senescence-associated genes AtNAP and OsNAP via transient expression in tobacco calluses, from Arabidopsis and rice respectively, thus we named it the SoNAP gene. Transient localization assays on onion epidermal cells confirmed the nuclear localization of the SoNAP. Expression analysis showed that the SoNAP gene was induced by high salinity, drought, and abscisic acid treatments. The overexpression of the SoNAP gene in tobacco calluses caused a senescence associated phenotype. Overall, our results indicated that the SoNAP gene from sugarcane is transcriptionally induced under abiotic stress conditions and conserved the predicted senescence-associated functions when it was overexpressed in a heterologous plant model. This work provides key insights about the senescence mechanisms related to abiotic stress and it provides a benchmark for future work on the improvement of this economically important crop.

Fibrillarin Ribonuclease Activity is Dependent on the GAR Domain and Modulated by Phospholipids.

Fibrillarin is a highly conserved nucleolar methyltransferase responsible for ribosomal RNA methylation across evolution from Archaea to humans. It has been reported that fibrillarin is involved in the methylation of histone H2A in nucleoli and other processes, including viral progression, cellular stress, nuclear shape, and cell cycle progression. We show that fibrillarin has an additional activity as a ribonuclease. The activity is affected by phosphoinositides and phosphatidic acid and insensitive to ribonuclease inhibitors. Furthermore, the presence of phosphatidic acid releases the fibrillarin-U3 snoRNA complex. We show that the ribonuclease activity localizes to the GAR (glycine/arginine-rich) domain conserved in a small group of RNA interacting proteins. The introduction of the GAR domain occurred in evolution in the transition from archaea to eukaryotic cells. The interaction of this domain with phospholipids may allow a phase separation of this protein in nucleoli.

The effect of plant stress on phosphoinositides.

Phosphoinositides are very versatile molecules with a plethora of functions such as cytokinesis, chemotaxis, cell survival, and cell death. Their functions depend on the proteins with which they interact. Thus, when interacting with phospholipases, phosphatases, or kinases, they can be precursors of second messengers in different signalling pathways. They could be second messengers themselves and interact directly with other proteins to modulate their functions trough changing its localization and activity or enhancing its synthesis rate. Because they are more abundant in animal cells and their importance in diseases such as cancer has taken priority, the study of the phosphoinositides in plants has not evolved to the same extent. Nevertheless, several studies have shown the significance of these lipids in plant cells viability and environmental response. This review focuses on phosphoinositides response to abiotic and biotic stress, showing their implication in plant survival during different stages of development. SIGNIFICANCE OF THE STUDY: This review is focused on plant PIPs functions in stress, highlighting in the main differences between plant and mammal PIPs and the novel interactions that could be extrapolated to animal models to contribute in a better understanding of these pivotal molecules.

Global Dynamics in Protein Disorder during Maize Seed Development.

Intrinsic protein disorder is a physicochemical attribute of some proteins lacking tridimensional structure and is collectively known as intrinsically disordered proteins (IDPs). Interestingly, several IDPs have been associated with protective functions in plants and with their response to external stimuli. To correlate the modulation of the IDPs content with the developmental progression in seed, we describe the expression of transcripts according to the disorder content of the proteins that they codify during seed development, from the early embryogenesis to the beginning of the desiccation tolerance acquisition stage. We found that the total expression profile of transcripts encoding for structured proteins is highly increased during middle phase. However, the relative content of protein disorder is increased as seed development progresses. We identified several intrinsically disordered transcription factors that seem to play important roles throughout seed development. On the other hand, we detected a gene cluster encoding for IDPs at the end of the late phase, which coincides with the beginning of the acquisition of desiccation tolerance. In conclusion, the expression pattern of IDPs is highly dependent on the developmental stage, and there is a general reduction in the expression of transcripts encoding for structured proteins as seed development progresses. We proposed maize seeds as a model to study the regulation of protein disorder in plant development and its involvement in the acquisition of desiccation tolerance in plants.

Transcriptomics and co-expression networks reveal tissue-specific responses and regulatory hubs under mild and severe drought in papaya (Carica papaya L.).

Plants respond to drought stress through the ABA dependent and independent pathways, which in turn modulate transcriptional regulatory hubs. Here, we employed Illumina RNA-Seq to analyze a total of 18 cDNA libraries from leaves, sap, and roots of papaya plants under drought stress. Reference and de novo transcriptomic analyses identified 8,549 and 6,089 drought-responsive genes and unigenes, respectively. Core sets of 6 and 34 genes were simultaneously up- or down-regulated, respectively, in all stressed samples. Moreover, GO enrichment analysis revealed that under moderate drought stress, processes related to cell cycle and DNA repair were up-regulated in leaves and sap; while responses to abiotic stress, hormone signaling, sucrose metabolism, and suberin biosynthesis were up-regulated in roots. Under severe drought stress, biological processes related to abiotic stress, hormone signaling, and oxidation-reduction were up-regulated in all tissues. Moreover, similar biological processes were commonly down-regulated in all stressed samples. Furthermore, co-expression network analysis revealed three and eight transcriptionally regulated modules in leaves and roots, respectively. Seventeen stress-related TFs were identified, potentially serving as main regulatory hubs in leaves and roots. Our findings provide insight into the molecular responses of papaya plant to drought, which could contribute to the improvement of this important tropical crop.

New insights into the phylogeny of the TMBIM superfamily across the tree of life: Comparative genomics and synteny networks reveal independent evolution of the BI and LFG families in plants.

The Transmembrane BAX Inhibitor Motif containing (TMBIM) superfamily, divided into BAX Inhibitor (BI) and Lifeguard (LFG) families, comprises a group of cytoprotective cell death regulators conserved in prokaryotes and eukaryotes. However, no research has focused on the evolution of this superfamily in plants. We identified 685 TMBIM proteins in 171 organisms from Archaea, Bacteria, and Eukarya, and provided a phylogenetic overview of the whole TMBIM superfamily. Then, we used orthology and synteny network analyses to further investigate the evolution and expansion of the BI and LFG families in 48 plants from diverse taxa. Plant BI family forms a single monophyletic group; however, monocot BI sequences transposed to another genomic context during evolution. Plant LFG family, which expanded trough whole genome and tandem duplications, is subdivided in LFG I, LFG IIA, and LFG IIB major phylogenetic groups, and retains synteny in angiosperms. Moreover, two orthologous groups (OGs) are shared between bryophytes and seed plants. Other several lineage-specific OGs are present in plants. This work clarifies the phylogenetic classification of the TMBIM superfamily across the three domains of life. Furthermore, it sheds new light on the evolution of the BI and LFG families in plants providing a benchmark for future research.

Transcriptional profiling of sugarcane leaves and roots under progressive osmotic stress reveals a regulated coordination of gene expression in a spatiotemporal manner.

Sugarcane is one of the most important crops worldwide and is a key plant for the global production of sucrose. Sugarcane cultivation is severely affected by drought stress and it is considered as the major limiting factor for their productivity. In recent years, this plant has been subjected to intensive research focused on improving its resilience against water scarcity; particularly the molecular mechanisms in response to drought stress have become an underlying issue for its improvement. To better understand water stress and the molecular mechanisms we performed a de novo transcriptomic assembly of sugarcane (var. Mex 69-290). A total of 16 libraries were sequenced in a 2x100 bp configuration on a HiSeq-Illumina platform. A total of 536 and 750 genes were differentially up-regulated along with the stress treatments for leave and root tissues respectively, while 1093 and 531 genes were differentially down-regulated in leaves and roots respectively. Gene Ontology functional analysis showed that genes related to response of water deprivation, heat, abscisic acid, and flavonoid biosynthesis were enriched during stress treatment in our study. The reliability of the observed expression patterns was confirmed by RT-qPCR. Additionally, several physiological parameters of sugarcane were significantly affected due to stress imposition. The results of this study may help identify useful target genes and provide tissue-specific data set of genes that are differentially expressed in response to osmotic stress, as well as a complete analysis of the main groups is significantly enriched under this condition. This study provides a useful benchmark for improving drought tolerance in sugarcane and other economically important grass species.

Novel Ribonuclease Activity Differs between Fibrillarins from Arabidopsis thaliana.

Fibrillarin is one of the most important nucleolar proteins that have been shown as essential for life. Fibrillarin localizes primarily at the periphery between fibrillar center and dense fibrillar component as well as in Cajal bodies. In most plants there are at least two different genes for fibrillarin. In Arabidopsis thaliana both genes show high level of expression in transcriptionally active cells. Here, we focus on two important differences between A. thaliana fibrillarins. First and most relevant is the enzymatic activity by AtFib2. The AtFib2 shows a novel ribonuclease activity that is not seen with AtFib1. Second is a difference in the ability to interact with phosphoinositides and phosphatidic acid between both proteins. We also show that the novel ribonuclease activity as well as the phospholipid binding region of fibrillarin is confine to the GAR domain. The ribonuclease activity of fibrillarin reveals in this study represents a new role for this protein in rRNA processing.

RAP2.4a Is Transported through the Phloem to Regulate Cold and Heat Tolerance in Papaya Tree (Carica papaya cv. Maradol): Implications for Protection Against Abiotic Stress.

Plants respond to stress through metabolic and morphological changes that increase their ability to survive and grow. To this end, several transcription factor families are responsible for transmitting the signals that are required for these changes. Here, we studied the transcription factor superfamily AP2/ERF, particularly, RAP2.4 from Carica papaya cv. Maradol. We isolated four genes (CpRap2.4a, CpRAap2.4b, CpRap2.1 and CpRap2.10), and an in silico analysis showed that the four genes encode proteins that contain a conserved APETALA2 (AP2) domain located within group I and II transcription factors of the AP2/ERF superfamily. Semiquantitative PCR experiments indicated that each CpRap2 gene is differentially expressed under stress conditions, such as extreme temperatures. Moreover, genetic transformants of tobacco plants overexpressing CpRap2.4a and CpRap2.4b genes show a high level of tolerance to cold and heat stress compared to non-transformed plants. Confocal microscopy analysis of tobacco transgenic plants showed that CpRAP2.4a and CpRAP2.4b proteins were mainly localized to the nuclei of cells from the leaves and roots and also in the sieve elements. Moreover, the movement of CpRap2.4a RNA in tobacco grafting was analyzed. Our results indicate that CpRap2.4a and CpRap2.4b RNA in the papaya tree have a functional role in the response to stress conditions such as exposure to extreme temperatures via direct translation outside the parental RNA cell.

Fibrillarin methylates H2A in RNA polymerase I trans-active promoters in Brassica oleracea.

Fibrillarin is a well conserved methyltransferase involved in several if not all of the more than 100 methylations sites in rRNA which are essential for proper ribosome function. It is mainly localized in the nucleoli and Cajal bodies inside the cell nucleus where it exerts most of its functions. In plants, fibrillarin binds directly the guide RNA together with Nop56, Nop58, and 15.5ka proteins to form a snoRNP complex that selects the sites to be methylated in pre-processing of ribosomal RNA. Recently, the yeast counterpart NOP1 was found to methylate histone H2A in the nucleolar regions. Here we show that plant fibrillarin can also methylate histone H2A. In Brassica floral meristem cells the methylated histone H2A is mainly localized in the nucleolus but unlike yeast or human cells it also localize in the periphery of the nucleus. In specialized transport cells the pattern is altered and it exhibits a more diffuse staining in the nucleus for methylated histone H2A as well as for fibrillarin. Here we also show that plant fibrillarin is capable of interacting with H2A and carry out its methylation in the rDNA promoter.

Comparative Genomics of NAC Transcriptional Factors in Angiosperms: Implications for the Adaptation and Diversification of Flowering Plants.

NAC proteins constitute one of the largest groups of plant-specific transcription factors and are known to play essential roles in various developmental processes. They are also important in plant responses to stresses such as drought, soil salinity, cold, and heat, which adversely affect growth. The current knowledge regarding the distribution of NAC proteins in plant lineages comes from relatively small samplings from the available data. In the present study, we broadened the number of plant species containing the NAC family origin and evolution to shed new light on the evolutionary history of this family in angiosperms. A comparative genome analysis was performed on 24 land plant species, and NAC ortholog groups were identified by means of bidirectional BLAST hits. Large NAC gene families are found in those species that have experienced more whole-genome duplication events, pointing to an expansion of the NAC family with divergent functions in flowering plants. A total of 3,187 NAC transcription factors that clustered into six major groups were used in the phylogenetic analysis. Many orthologous groups were found in the monocot and eudicot lineages, but only five orthologous groups were found between P. patens and each representative taxa of flowering plants. These groups were called basal orthologous groups and likely expanded into more recent taxa to cope with their environmental needs. This analysis on the angiosperm NAC family represents an effort to grasp the evolutionary and functional diversity within this gene family while providing a basis for further functional research on vascular plant gene families.