OBJECTIVE: SPATULA (SPT) encodes a basic Helix-Loop-Helix transcription factor in Arabidopsis thaliana that functions in the development of the style, stigma and replum tissues, all of which arise from the carpel margin meristem (CMM) of the gynoecium. Here, we use a comparative approach to investigate the evolutionary history of SPT and identify changes that potentially contributed to its role in gynoecium development.
METHODS: We investigate SPT\'s molecular and functional evolution using phylogenetic reconstruction, yeast-2-hybrid analyses of protein-protein interactions, microarray-based analyses of protein-DNA interactions, plant transformation assays, RNA in-situ hybridization, and in-silico analyses of promoter sequences.
RESULTS: We demonstrate the SPT lineage to have arisen at the base of euphyllophytes from a clade of potentially light-regulated transcription factors through gene duplication followed by the loss of an Active Phytochrome Binding (APB) domain. We also clarify the more recent evolutionary history of SPT and its paralog ALCATRAZ (ALC), which appear to have arisen through a large-scale duplication within Brassicales. We find that SPT orthologs from diverse groups of seed plants share strikingly similar capacities for protein-protein and protein-DNA interactions, and that SPT coding regions from a wide taxonomic range of plants are able to complement loss-of-function spt mutations in transgenic Arabidopsis. However, the expression pattern of SPT appears to have evolved significantly within angiosperms, and we identify structural changes in SPT\'s promoter region that correlate with the acquisition of high expression levels in tissues arising from the CMM in Brassicaeae.
CONCLUSIONS: We conclude that changes to SPT\'s expression pattern made a major contribution to the evolution of its developmental role in the gynoecium of Brassicaeae. By contrast, the main biochemical capacities of SPT, as well as many of its immediate transcriptional targets, appear to have been conserved at least since the base of living angiosperms.

BACKGROUND: Gynostemma pentaphyllum, an ancient Chinese herbal medicine, serves as a natural source of gypenosides with significant medicinal properties. Basic helix-loop-helix (bHLH) transcription factors play pivotal roles in numerous biological processes, especially in the regulation of secondary metabolism in plants. However, the characteristics and functions of the bHLH genes in G. pentaphyllum remain unexplored, and their regulatory role in gypenoside biosynthesis remains poorly elucidated.
RESULTS: This study identified a total of 111 bHLH members in G. pentaphyllum (GpbHLHs), categorizing them into 26 subgroups based on shared conserved motif compositions and gene structures. Collinearity analysis illustrated that segmental duplications predominately lead to the evolution of GpbHLHs, with most duplicated GpbHLH gene pairs undergoing purifying selection. Among the nine gypenoside-related GpbHLH genes, two GpbHLHs (GpbHLH15 and GpbHLH58) were selected for further investigation based on co-expression analysis and functional prediction. The expression of these two selected GpbHLHs was dramatically induced by methyl jasmonate, and their nuclear localization was confirmed. Furthermore, yeast one-hybrid and dual-luciferase assays demonstrated that GpbHLH15 and GpbHLH58 could bind to the promoters of the gypenoside biosynthesis pathway genes, such as GpFPS1, GpSS1, and GpOSC1, and activate their promoter activity to varying degrees.
CONCLUSIONS: In conclusion, our findings provide a detailed analysis of the bHLH family and valuable insights into the potential use of GpbHLHs to enhance the accumulation of gypenosides in G. pentaphyllum.

Basic helix-loop-helices (bHLHs) are present in all eukaryotes and form one of the largest families of transcription factors (TFs) found in plants. bHLHs function as transcriptional activators and/or repressors of genes involved in key processes involved in plant growth and development in interaction with the environment (e.g., stomata and root hair development, iron homeostasis, and response to heat and shade). Recent studies have improved our understanding of the functioning of bHLH TFs in complex regulatory networks where a series of post-translational modifications (PTMs) have critical roles in regulating their subcellular localization, DNA-binding capacity, transcriptional activity, and/or stability (e.g., protein-protein interactions, phosphorylation, ubiquitination, and sumoylation). Further elucidating the function and regulation of bHLHs will help further understanding of the biology of plants in general and for the development of new tools for crop improvement.

The basic helix-loop-helix (bHLH) family is one of the most well-known transcription factor families in plants, and it regulates growth, development, and abiotic stress responses. However, systematic analyses of the bHLH gene family in Prunus sibirica have not been reported to date. In this study, 104 PsbHLHs were identified and classified into 23 subfamilies that were unevenly distributed on eight chromosomes. Nineteen pairs of segmental replication genes and ten pairs of tandem replication genes were identified, and all duplicated gene pairs were under purifying selection. PsbHLHs of the same subfamily usually share similar motif compositions and exon-intron structures. PsbHLHs contain multiple stress-responsive elements. PsbHLHs exhibit functional diversity by interacting and coordinating with other members. Twenty PsbHLHs showed varying degrees of expression. Eleven genes up-regulated and nine genes down-regulated in -4°C. The majority of PsbHLHs were highly expressed in the roots and pistils. Transient transfection experiments demonstrated that transgenic plants with overexpressed PsbHLH42 have better cold tolerance. In conclusion, the results of this study have significant implications for future research on the involvement of bHLH genes in the development and stress responses of Prunus sibirica.

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Continual advances in our understanding of the human genome have led to exponential increases in known single nucleotide variants. The characterization of each of the variants lags behind. For researchers needing to study a single gene, or multiple genes in a pathway, there must be ways to narrow down pathogenic variants from those that are silent or pose less pathogenicity. In this study, we use the NHLH2 gene which encodes the nescient helix-loop-helix 2 (Nhlh2) transcription factor in a systematic analysis of all missense mutations to date in the gene. The NHLH2 gene was first described in 1992. Knockout mice created in 1997 indicated a role for this protein in body weight control, puberty, and fertility, as well as the motivation for sex and exercise. Only recently have human carriers of NHLH2 missense variants been characterized. Over 300 missense variants for the NHLH2 gene are listed in the NCBI single nucleotide polymorphism database (dbSNP). Using in silico tools, predicted pathogenicity of the variants narrowed the missense variants to 37 which were predicted to affect NHLH2 function. These 37 variants cluster around the basic-helix-loop-helix and DNA binding domains of the transcription factor, and further analysis using in silico tools provided 21 SNV resulting in 22 amino acid changes for future wet lab analysis. The tools used, findings, and predictions for the variants are discussed considering the known function of the NHLH2 transcription factor. Overall use of these in silico tools and analysis of these data contribute to our knowledge of a protein which is both involved in the human genetic syndrome, Prader-Willi syndrome, and in controlling genes involved in body weight control, fertility, puberty, and behavior in the general population, and may provide a systematic methodology for others to characterize variants for their gene of interest.

[This corrects the article DOI: 10.3389/fpls.2022.903793.].

The development of functional neural circuits in the central nervous system (CNS) requires the production of sufficient numbers of various types of neurons and glial cells, such as astrocytes and oligodendrocytes, at the appropriate periods and regions. Hence, severe neuronal loss of the circuits can cause neurodegenerative diseases such as Huntington\'s disease (HD), Parkinson\'s disease (PD), Alzheimer\'s disease (AD), and Amyotrophic Lateral Sclerosis (ALS). Treatment of such neurodegenerative diseases caused by neuronal loss includes some strategies of cell therapy employing stem cells (such as neural progenitor cells (NPCs)) and gene therapy through cell fate conversion. In this report, we review how bHLH acts as a regulator in neuronal differentiation, reprogramming, and cell fate determination. Moreover, several different researchers are conducting studies to determine the importance of bHLH factors to direct neuronal and glial cell fate specification and differentiation. Therefore, we also investigated the limitations and future directions of conversion or transdifferentiation using bHLH factors.

Triterpene saponins (TS) are a structurally diverse group of metabolites that are widely distributed in plants. They primarily serve as defense compounds and their production is often triggered by biotic stresses through signaling cascades that are modulated by phytohormones such as the jasmonates (JA). Two JA-modulated basic helix-loop-helix (bHLH) transcription factors (TFs), triterpene saponin biosynthesis activating regulator 1 (TSAR1) and TSAR2, have previously been identified as direct activators of TS biosynthesis in the model legume Medicago truncatula. Here, we report on the involvement of the core endoplasmic reticulum (ER) stress-related basic leucine zipper (bZIP) TFs bZIP17 and bZIP60 in the regulation of TS biosynthesis. Expression and processing of M. truncatula bZIP17 and bZIP60 proteins were altered in roots with perturbed TS biosynthesis or treated with JA. Accordingly, such roots displayed an altered ER network structure. M. truncatula bZIP17 and bZIP60 proteins were shown to localize in the nucleus and appeared to be capable of interfering with the TSAR-mediated transactivation of TS biosynthesis genes. Furthermore, interference between ER stress-related bZIP and JA-modulated bHLH TFs in the regulation of JA-dependent terpene biosynthetic pathways may be widespread in the plant kingdom, as we demonstrate that it also occurs in the regulation of monoterpene indole alkaloid biosynthesis in the medicinal plant Catharanthus roseus.

Clade II basic helix-loop-helix transcription factors (bHLH TFs) are essential for pollen production and tapetal nursing functions in angiosperm anthers. As pollen has been suggested to be related to bryophyte spores by descent, we characterized two Physcomitrium (Physcomitrella) patens clade II bHLH TFs (PpbHLH092 and PpbHLH098), to test if regulation of sporogenous cells and the nursing cells surrounding them is conserved between angiosperm anthers and bryophyte sporangia. We made CRISPR-Cas9 reporter and loss-of-function lines to address the function of PpbHLH092/098. We sectioned and analyzed WT and mutant sporophytes for a comprehensive stage-by-stage comparison of sporangium development. Spore precursors in the P. patens sporangium are surrounded by nursing cells showing striking similarities to tapetal cells in angiosperms. Moss clade II bHLH TFs are essential for the differentiation of these tapetal-like cells and for the production of functional spores. Clade II bHLH TFs provide a conserved role in controlling the sporophytic somatic cells surrounding and nursing the sporogenous cells in both moss sporangia and angiosperm anthers. This supports the hypothesis that such nursing functions in mosses and angiosperms, lineages separated by c. 450 million years, are related by descent.