Quiescent Center Research Articles

Characterisation of the ERF102 to ERF105 genes of Arabidopsis thaliana and their role in the response to cold stress

Key messageThe four phylogenetically closely related ERF102 to ERF105 transcription factors of Arabidopsis thaliana are regulated by different stresses and are involved in the response to cold stress.The ETHYLENE RESPONSE FACTOR (ERF) genes of Arabidopsis thaliana form a large family encoding plant-specific transcription factors. Here, we characterise the four phylogenetically closely related ERF102/ERF5, ERF103/ERF6, ERF104 and ERF105 genes. Expression analyses revealed that these four genes are similarly regulated by different hormones and abiotic stresses. Analyses of tissue-specific expression using promoter:GUS reporter lines revealed their predominant expression in root tissues including the root meristem (ERF103), the quiescent center (ERF104) and the root vasculature (all). All GFP-ERF fusion proteins were nuclear-localised. The analysis of insertional mutants, amiRNA lines and 35S:ERF overexpressing transgenic lines indicated that ERF102 to ERF105 have only a limited impact on regulating shoot and root growth. Previous work had shown a role for ERF105 in the cold stress response. Here, measurement of electrolyte leakage to determine leaf freezing tolerance and expression analyses of cold-responsive genes revealed that the combined activity of ERF102 and ERF103 is also required for a full cold acclimation response likely involving the CBF regulon. These results suggest a common function of these ERF genes in the response to cold stress.

Intersected functional zone of transcriptional regulators patterns stemness within stem cell niche of root apical meristem.

Root stem cell niche (SCN) consists of a quiescent center (QC) and surrounding stem cells. Disrupted symplastic communication leads to loss of stemness in the whole SCN. Several SCN regulators were reported to move between cells for SCN maintenance. However, single mutant of these regulators is insufficient to abolish QC stemness despite the high differentiation rate in surrounding stem cells. To dissect the mechanism behind such distinct stemness in SCN, we combined the mis-expression strategy with pWOX5:icals3m system in which QC is symplastically isolated. We found the starch accumulation in QC could be synergistically repressed by WUSCHEL-RELATED HOMEOBOX 5 (WOX5), SHORT-ROOT (SHR), SCARCROW (SCR), and PLETHORA (PLT). Like PLTs, other core regulators also exhibited dimorphic functions by inhibiting differentiation at a higher dose while promoting cell division at a low protein level. Being located in the center of the intersected expression zones, QC cells receive the highest level of core regulators, forming the most robust stemness within SCN. WUSCHEL-RELATED HOMEOBOX 5 was sufficient to activate PLT1/2 expression, contributing to the QC-enriched PLTs. Our results provide experimental evidence supporting the long-standing hypothesis that the combination of spatial expression, synergistic function and dosage effect of core regulators result in spatially distinct stemness in SCN.

A system-level mechanistic explanation for asymmetric stem cell fates: Arabidopsis thaliana root niche as a study system

Asymmetric divisions maintain long-term stem cell populations while producing new cells that proliferate and then differentiate. Recent reports in animal systems show that divisions of stem cells can be uncoupled from their progeny differentiation, and the outcome of a division could be influenced by microenvironmental signals. But the underlying system-level mechanisms, and whether this dynamics also occur in plant stem cell niches (SCN), remain elusive. This article presents a cell fate regulatory network model that contributes to understanding such mechanism and identify critical cues for cell fate transitions in the root SCN. Novel computational and experimental results show that the transcriptional regulator SHR is critical for the most frequent asymmetric division previously described for quiescent centre stem cells. A multi-scale model of the root tip that simulated each cell’s intracellular regulatory network, and the dynamics of SHR intercellular transport as a cell-cell coupling mechanism, was developed. It revealed that quiescent centre cell divisions produce two identical cells, that may acquire different fates depending on the feedback between SHR’s availability and the state of the regulatory network. Novel experimental data presented here validates our model, which in turn, constitutes the first proposed systemic mechanism for uncoupled SCN cell division and differentiation.

Defective Quiescent Center/AtTRS85 Encoding a TRAPPIII-specific Subunit Required for the Trans-golgi Network/Early Endosome Integrity is Essential for the Proper Root Development in Arabidopsis

Tethering factors mediate the initial contact between donor and acceptor membranes. Transport protein particle (TRAPP)III, a multisubunit tethering complex, controls both secretory pathway and autophagy in yeast. However, the roles of TRAPPIII is poorly understood in plants. We have isolated a recessive mutant displaying retarded root growth, defective root hair elongation, and compromised organization of quiescent center by introducing UAS tag in Arabidopsis thaliana and named defective quiescent center (dqc)-1. The tag localized in the 25th exon of At5g16280 encoding AtTRS85 specifying the TRAPPIII complex. Three additional T-DNA insertion mutants of dqc/attrs85 exhibited the defective gravitropic responses in addition to the weak dqc-1-like phenotypes. In dqc-2, the auxin-responsive DR5 expression in the columella was extended and an auxin efflux carrier PIN-FORMED1-GFP abnormally aggregated in the vacuole-like subcellular compartments. In the root epidermis of dqc-2, an early endosome (EE) marker was dispersed in the cytoplasm, a trans-Golgi network (TGN) marker localized in tonoplast, and a vacuole marker localized in the fragmented tonoplast. Furthermore, the endocytosis of FM4-64 in dqc-2 was compromised as evidenced by the decrease in the EE punctum formation, the rapid accumlation into the tonoplast, and the infrequent brefeldin A body formation. Together, DQC/AtTRS85 is essential for the integrity of TGN/EE and the vacuole fusion.

Root apical meristem diversity and the origin of roots: insights from extant lycophytes.

The independent origin of roots in lycophytes and euphyllophytes has been proposed, mainly based on paleobotanical records. However, the question of how roots evolved within these lineages remains unresolved. Root apical meristem (RAM) organization in lycophytes would provide a clue toward understanding the early evolution of roots. Recently, we examined RAM organization in lycophytes (Lycopodiaceae, Isoetaceae, and Selaginellaceae) in terms of cell division activity and anatomy, comparing RAM among vascular plants. Lycophyte RAM exhibited four organization types (I, II, III, and apical); thus, RAM organization in extant lycophytes was more diverse than expected. Type I RAM contained a region with very low cell division frequency, reminiscent of the quiescent center (QC) in seed plant RAM. Although some euphyllophyte RAMs were structurally similar to types II and III and apical cell-type RAM, lycophyte RAM of types II and III had no QC-like area. These results support the paleobotanical predictions that roots evolved several times in lycophytes, as well as in euphyllophytes. In this review, we also introduce recent findings on RAM organization in extant lycophytes and discuss the origin of roots in vascular plants.

CLE40 Signaling Regulates Root Stem Cell Fate.

The quiescent center (QC) of the Arabidopsis (Arabidopsis thaliana) root meristem acts as an organizer that promotes stem cell fate in adjacent cells and patterns the surrounding stem cell niche. The stem cells distal from the QC, the columella stem cells (CSCs), are maintained in an undifferentiated state by the QC-expressed transcription factor WUSCHEL RELATED HOMEOBOX5 (WOX5) and give rise to the columella cells. Differentiated columella cells provide a feedback signal via secretion of the peptide CLAVATA3/ESR-RELATED40 (CLE40), which acts through the receptor kinases ARABIDOPSIS CRINKLY4 (ACR4) and CLAVATA1 (CLV1) to control WOX5 expression. Previously, it was proposed that WOX5 protein movement from the QC into CSCs is required for CSC maintenance, and that the CLE40/CLV1/ACR4 signaling module restricts WOX5 mobility or function. Here, these assumptions were tested by exploring the function of CLE40/CLV1/ACR4 in CSC maintenance. However, no role for CLE40/CLV1/ACR4 in constricting the mobility of WOX5 or other fluorescent test proteins was identified. Furthermore, in contrast to previous observations, WOX5 mobility was not required to inhibit CSC differentiation. We propose that WOX5 acts mainly in the QC, where other short-range signals are generated that not only inhibit differentiation but also promote stem cell division in adjacent cells. Therefore, the main function of columella-derived CLE40 signal is to position the QC at a defined distance from the root tip by repressing QC-specific gene expression via the ACR4/CLV1 receptors in the distal domain and promoting WOX5 expression via the CLV2 receptor in the proximal meristem.

Analysis of Cell Division Frequency in the Root Apical Meristem of Lycophytes, Non-seed Vascular Plants, Using EdU Labeling.

The organization of the root apical meristem (RAM) provides insights into the evolution of roots in vascular plants. The RAM of seed plants has a quiescent center (QC), in which the cells divide infrequently and function to maintain neighboring stem cells. However, the existence of a QC and the mechanisms of RAM maintenance in non-seed plants are poorly understood. We analyzed the RAM organization of lycophytes focusing on cell division activity using the EdU labeling method and showed that the RAM of Lycopodium species has a region with a very low cell division frequency, which was named the QC-like region. Here, we describe an in situ EdU labeling method for the RAM of growing roots in nature.

ULTRAPETALA1 maintains Arabidopsis root stem cell niche independently of ARABIDOPSIS TRITHORAX1.

During plant development, morphogenetic processes rely on the activity of meristems. Meristem homeostasis depends on a complex regulatory network constituted by different factors and hormone signaling that regulate gene expression to coordinate the correct balance between cell proliferation and differentiation. ULTRAPETALA1, a transcriptional regulatory protein described as an Arabidopsis Trithorax group factor, has been characterized as a regulator of the shoot and floral meristems activity. Here, we highlight the role of ULTRAPETALA1 in root stem cell niche maintenance. We found that ULTRAPETALA1 is required to regulate both the quiescent center cell division rate and auxin signaling at the root tip. Furthermore, ULTRAPETALA1 regulates columella stem cell differentiation. These roles are independent of the ARABIDOPSIS TRITHORAX1, suggesting a different mechanism by which ULTRAPETALA1 can act in the root apical meristem of Arabidopsis. This work introduces a new component of the regulatory network needed for the root stem cell niche maintenance.

Stem cell fate in hypoxic root apical meristems is influenced by phytoglobin expression.

Root survival to flooding-induced hypoxic stress is dependent upon maintaining the functionality of the root apical meristem quiescent center (QC), a process that is governed by the basipetal flow of auxin leading to the formation of an auxin maximum, which is needed for the establishment of a highly oxidized environment specifying the QC niche. Perturbations in auxin flow and distribution along the root profile occurring during hypoxia can shift the redox state of the QC towards a more reduced environment, leading to the activation of the QC, degradation of the meristem, and root abortion. The maize phytoglobin gene ZmPgb1.1 is involved in minimizing these damaging effects during hypoxia in processes that result in sustaining the PIN-mediated auxin maximum and an oxidized environment in the QC. The oxidized environment is accomplished by maintaining the activity of redox enzymes oxidizing ascorbate and glutathione. These events, compromised in QCs suppressing ZmPgb1.1, ensure the functionality of the QC and root meristems under conditions of low oxygen, resulting in stable root performance.

The SMO1 Family of Sterol 4α-Methyl Oxidases Is Essential for Auxin- and Cytokinin-Regulated Embryogenesis.

In the plant sterol biosynthetic pathway, sterol 4α-methyl oxidase1 (SMO1) and SMO2 enzymes are involved in the removal of the first and second methyl groups at the C-4 position, respectively. SMO2s have been found to be essential for embryonic and postembryonic development, but the roles of SMO1s remain unclear. Here, we found that the three Arabidopsis (Arabidopsis thaliana) SMO1 genes displayed different expression patterns. Single smo1 mutants and smo1-1 smo1-3 double mutants showed no obvious phenotype, but the smo1-1 smo1-2 double mutant was embryo lethal. The smo1-1 smo1-2 embryos exhibited severe defects, including no cotyledon or shoot apical meristem formation, abnormal division of suspensor cells, and twin embryos. These defects were associated with enhanced and ectopic expression of auxin biosynthesis and response reporters. Consistently, the expression pattern and polar localization of PIN FORMED1, PIN FORMED7, and AUXIN RESISTANT1 auxin transporters were dramatically altered in smo1-1 smo1-2 embryos. Moreover, cytokinin biosynthesis and response were reduced in smo1-1 smo1-2 embryos. Tissue culture experiments further demonstrated that homeostasis between auxin and cytokinin was altered in smo1-1 smo1-2 heterozygous mutants. This disturbed balance of auxin and cytokinin in smo1-1 smo1-2 embryos was accompanied by unrestricted expression of the quiescent center marker WUSCHEL-RELATED HOMEOBOX5 Accordingly, exogenous application of either auxin biosynthesis inhibitor or cytokinin partially rescued the embryo lethality of smo1-1 smo1-2 Sterol analyses revealed that 4,4-dimethylsterols dramatically accumulated in smo1-1 smo1-2 heterozygous mutants. Together, these data demonstrate that SMO1s function through maintaining correct sterol composition to balance auxin and cytokinin activities during embryogenesis.

Comparison of Promeristem Structure and Ontogeny of Procambium in Primary Roots of Zea mays ssp. Mexicana and Z. mays 'Honey Bantam' with Emphasis on Metaxylem Vessel Histogenesis.

Classical histology describes the histological organization in Zea mays as having a “closed organization” that differs from Arabidopsis with the development of xylem conforming to predictable rules. We speculated that root apical meristem organization in a wild subspecies of Z. mays (a teosinte) would differ from a domestic sweetcorn cultivar (‘Honey Bantam’). Careful comparison could contribute to understanding how evolutionary processes and the domestication of maize have affected root development. Root tips of seedlings were prepared and sectioned for light microscopy. Most sections were treated with RNase before staining to increase contrast between the walls and cytoplasm. Longitudinal and serial transverse sections were analyzed using computer imaging to determine the position and timing of key xylem developmental events. Metaxylem development in mexicana teosinte differed from sweetcorn only in that the numbers of late-maturing metaxylem vessels in the latter are typically two-fold greater and the number of cells in the transverse section of procambium were greater in the latter, but parenchymatous cell sizes were not statistically different. Promeristems of both were nearly identical in size and organization, but did not operate quite as previously described. Mitotic activity was rare in the quiescent centers, but occasionally a synchronized pulse of mitoses was observed there. Our reinterpretation of histogen theory and procambium development should be useful for future detailed studies of regulation of development, and perhaps its evolution, in this species.

Abscisic Acid Regulates Auxin Distribution to Mediate Maize Lateral Root Development Under Salt Stress.

Roots are important plant organs. Lateral root (LR) initiation (LRI) and development play a central role in environmental adaptation. The mechanism of LR development has been well investigated in Arabidopsis. When we evaluated the distribution of auxin and abscisic acid (ABA) in maize, we found that the mechanism differed from that in Arabidopsis. The distribution of ABA and auxin within the primary roots (PRs) and LRs was independent of each other. Auxin localization was observed below the quiescent center of the root tips, while ABA localized at the top of the quiescent center. Furthermore, NaCl inhibited LRI by increasing ABA accumulation, which mainly regulates auxin distribution, while auxin biosynthesis was inhibited by ABA in Arabidopsis. The polar localization of ZmPIN1 in maize was disrupted by NaCl and exogenous ABA. An inhibitor of ABA biosynthesis, fluridone (FLU), and the ABA biosynthesis mutant vp14 rescued the phenotype under NaCl treatment. Together, all the evidence suggested that NaCl promoted ABA accumulation in LRs and that ABA altered the polar localization of ZmPIN1, disrupted the distribution of auxin and inhibited LRI and development.

Cystathionine beta-lyase is crucial for embryo patterning and the maintenance of root stem cell niche in Arabidopsis.

The growth and development of roots in plants depends on the specification and maintenance of the root apical meristem. Here, we report the identification of CBL, a gene required for embryo and root development in Arabidopsis, and encodes cystathionine beta-lyase (CBL), which catalyzes the penultimate step in methionine (Met) biosynthesis, and which also led to the discovery of a previous unknown, but crucial, metabolic contribution by the Met biosynthesis pathway. CBL is expressed in embryos and shows quiescent center (QC)-enriched expression pattern in the root. cbl mutant has impaired embryo patterning, defective root stem cell niche, stunted root growth, and reduces accumulation of the root master regulators PLETHORA1 (PLT1) and PLT2. Furthermore, mutation in CBL severely decreases abundance of several PIN-FORMED (PIN) proteins and impairs auxin-responsive gene expression in the root tip. cbl seedlings also exhibit global reduction in histone H3 Lys-4 trimethylation (H3K4me3) and DNA methylation. Importantly, mutation in CBL reduces the abundance of H3K4me3 modification in PLT1/2 genes and downregulates their expression. Overexpression of PLT2 partially rescues cbl root meristem defect, suggesting that CBL acts in part through PLT1/2. Moreover, exogenous supplementation of Metalso restores the impaired QC activity and the root growth defects of cbl. Taken together, our results highlight the unique role of CBL to maintain the root stem cell niche by cooperative actions between Met biosynthesis and epigenetic modification of key developmental regulators.

Week-long imaging of cell divisions in the Arabidopsis root meristem

BackgroundCharacterizing the behaviors of dynamic systems requires capturing them with high temporal and spatial resolution. Owing to its transparency and genetic tractability, the Arabidopsis thaliana root lends itself well to live imaging when combined with cell and tissue-specific fluorescent reporters. We developed a novel 4D imaging method that utilizes simple confocal microscopy and readily available components to track cell divisions in the root stem cell niche and surrounding region for up to 1 week.ResultsUsing this method, we performed a direct measurement of cell division intervals within and around the root stem cell niche. The results reveal a short, steep gradient of cell division rates in proximal stem cells, with progressively more rapid cell division rates from quiescent center (QC), to cells in direct contact with the QC (initials), to their immediate daughters, after which division rates appear to become more homogeneous.ConclusionsThese results provide a baseline to study how perturbations in signaling could affect cell division patterns in the root meristem. This new setup further allows us to finely analyze meristematic cell division rates that lead to patterning.

MicroRNA Sequencing Revealed Citrus Adaptation to Long-Term Boron Toxicity through Modulation of Root Development by miR319 and miR171.

Boron (B) toxicity in Citrus is a common physiological disorder leading to reductions in both productivity and quality. Studies on how Citrus roots evade B toxicity may provide new insight into plant tolerance to B toxicity. Here, using Illumina sequencing, differentially expressed microRNAs (miRNAs) were identified in B toxicity-treated Citrus sinensis (tolerant) and C. grandis (intolerant) roots. The results showed that 37 miRNAs in C. grandis and 11 miRNAs in C. sinensis were differentially expressed when exposed to B toxicity. Among them, miR319, miR171, and miR396g-5p were confirmed via 5′-RACE and qRT-PCR to target a myeloblastosis (MYB) transcription factor gene, a SCARECROW-like protein gene, and a cation transporting ATPase gene, respectively. Maintenance of SCARECROW expression in B treated Citrus roots might fulfill stem cell maintenance, quiescent center, and endodermis specification, thus allowing regular root elongation under B-toxic stress. Down-regulation of MYB due to up-regulation of miR319 in B toxicity-treated C. grandis roots might decrease the number of root tips, thereby dramatically changing root system architecture. Our findings suggested that miR319 and miR171 play a pivotal role in Citrus adaptation to long-term B toxicity by targeting MYB and SCARECROW, respectively, both of which are responsible for root growth and development.

Identification of Periplasmic Root-Cap Mucilage in Developing Columella Cells of Arabidopsis thaliana.

Plant roots secrete various substances with diverse functions against both plants and microbes in the rhizosphere. A major secretory substance is root-cap mucilage, whose functions have been well characterized, albeit mainly in crops. However, little is currently known about the developmental mechanisms of root-cap mucilage. Here, we show the accumulation and extrusion of root-cap mucilage in Arabidopsis. We found propidium iodide (PI) stainable structures between the plasma membrane and cell wall in the sixth layer of columella cells (c6) from the quiescent center. Ruthenium red staining and PI staining with calcium ions suggested that the structure comprises in part pectin polysaccharides. Electron microscopy revealed that the structure had a meshwork of electron-dense filaments that resembled periplasmic mucilage in other plants. In the c6 cells, we also observed many large vesicles with denser meshwork filaments to periplasmic mucilage, which likely mediate the transport of mucilage components. Extruded mucilage was observed outside a partially degraded cell wall in the c7 cells. Moreover, we found that the Class IIB NAC transcription factors BEARSKIN1 (BRN1) and BRN2, which are known to regulate the terminal differentiation of columella cells, were required for the efficient accumulation of root-cap mucilage in Arabidopsis. Taken together, our findings reveal the accumulation of and dynamic changes in periplasmic mucilage during columella cell development in Arabidopsis.

Temporal changes in cell division rate and genotoxic stress tolerance in quiescent center cells of Arabidopsis primary root apical meristem

Plant roots provide structural support and absorb nutrients and water; therefore, their proper development and function are critical for plant survival. Extensive studies on the early stage of ontogenesis of the primary root have revealed that the root apical meristem (RAM) undergoes dynamic structural and organizational changes during early germination. Quiescent center (QC) cells, a group of slowly dividing cells at the center of the stem-cell niche, are vital for proper function and maintenance of the RAM. However, temporal aspects of molecular and cellular changes in QC cells and their regulatory mechanisms have not been well studied. In the present study, we investigated temporal changes in QC cell size, expression of QC cell-specific markers (WOX5 and QC25), and genotoxic tolerance and division rate of QC cells in the Arabidopsis primary root. Our data revealed the decreased size of QC cells and the decreased expression of the QC cell-specific markers with root age. We also found that QC cell division frequency increased with root age. Furthermore, our study provides evidence supporting the link between the transition of QC cells from a mitotically quiescent state to the frequently dividing state and the decrease in tolerance to genotoxic stress.

Spatiotemporal Developmental Trajectories in the Arabidopsis Root Revealed Using High-Throughput Single-Cell RNA Sequencing

High-throughput single-cell RNA sequencing (scRNA-seq) is becoming a cornerstone of developmental research, providing unprecedented power in understanding dynamic processes. Here, we present a high-resolution scRNA-seq expression atlas of the Arabidopsis root composed of thousands of independently profiled cells. This atlas provides detailed spatiotemporal information, identifying defining expression features for all major cell types, including the scarce cells of the quiescent center. These reveal key developmental regulators and downstream genes that translate cell fate into distinctive cell shapes and functions. Developmental trajectories derived from pseudotime analysis depict a finely resolved cascade of cell progressions from the niche through differentiation that are supported by mirroring expression waves of highly interconnected transcription factors. This study demonstrates the power of applying scRNA-seq to plants and provides an unparalleled spatiotemporal perspective of root cell differentiation.

ARF5/MONOPTEROS directly regulates miR390 expression in the Arabidopsis thaliana primary root meristem.

The root meristem is organized around a quiescent center (QC) surrounded by stem cells that generate all cell types of the root. In the transit‐amplifying compartment, progeny of stem cells further divides prior to differentiation. Auxin controls the size of this transit‐amplifying compartment via auxin response factors (ARFs) that interact with auxin response elements (AuxREs) in the promoter of their targets. The microRNA miR390 regulates abundance of ARF2, ARF3, and ARF4 by triggering the production of trans‐acting (ta)‐siRNA from the TAS3 precursor. This miR390/TAS3/ARF regulatory module confers sensitivity and robustness to auxin responses in diverse developmental contexts and organisms. Here, we show that miR390 is expressed in the transit‐amplifying compartment of the root meristem where it modulates response to exogenous auxin. We show that a single AuxRE located in miR390 promoter is necessary for miR390 expression in this compartment and identify that ARF5/MONOPTEROS (MP) binds miR390 promoter via the AuxRE. We show that interfering with ARF5/MP‐dependent auxin signaling attenuates miR390 expression in the transit‐amplifying compartment of the root meristem. Our results show that ARF5/MP regulates directly the expression of miR390 in the root meristem. We propose that ARF5, miR390, and the ta‐siRNAs‐regulated ARFs modulate the response of the transit‐amplifying region of the meristem to exogenous auxin.

Single-cell expression noise and gene-body methylation in Arabidopsis thaliana

Gene-body methylation (gbM) refers to an increased level of methylated cytosines specifically in a CG sequence context within genes. gbM is found in plant genes with intermediate expression level, which evolve slowly, and is often broadly conserved across millions of years of evolution. Intriguingly however, some plants lack gbM, and thus it remains unclear whether gbM has a function. In animals, there is support for a role of gbM in reducing erroneous transcription and transcription noise, but so far most studies in plants have tested for an effect of gbM on expression level, not noise. Here, we therefore tested whether gbM was associated with reduced expression noise in Arabidopsis thaliana, using single-cell transcriptome sequencing data from root quiescent centre cells. We find that gbM genes have lower expression noise levels than unmethylated genes. However, an analysis of covariance revealed that, if other genomic features are taken into account, this association disappears. Nonetheless, gbM genes were more consistently expressed across single-cell samples, supporting previous inference that gbM genes are constitutively expressed. Finally, we observed that fewer RNAseq reads map to introns of gbM genes than to introns of unmethylated genes, which indicates that gbM might be involved in reducing erroneous transcription by reducing intron retention.