Quantitative analysis of lateral root development with time-lapse imaging and deep neural network.

During lateral root (LR) development, morphological alteration of the developing single LR primordium occurs continuously. Precise observation of this continuous alteration is important for understanding the mechanism involved in single LR development. Recently, we reported that very long-chain fatty acids are important signalling molecules that regulate LR development. In the study, we developed an efficient method to quantify the transition of single LR developmental stages using time-lapse imaging followed by a deep neural network (DNN) analysis. In this 'insight' paper, we discuss our DNN method and the importance of time-lapse imaging in studies on plant development. Integrating DNN analysis and imaging is a powerful technique for the quantification of the timing of the transition of organ morphology; it can become an important method to elucidate spatiotemporal molecular mechanisms in plant development.

AtMYB50 regulates root cell elongation by upregulating PECTIN METHYLESTERASE INHIBITOR 8 in Arabidopsis thaliana.

Plant root development involves multiple signal transduction pathways. Notably, phytohormones like auxin and cytokinin are well characterized for their molecular mechanisms of action. Reactive oxygen species (ROS) serve as crucial signaling molecules in controlling root development. The transcription factor, UPBEAT1 (UPB1) is responsible for maintaining ROS homeostasis at the root tip, influencing the transition from cell proliferation to differentiation. While UPB1 directly regulates peroxidase expression to control ROS homeostasis, it targets genes other than peroxidases, suggesting its involvement in root growth through non-ROS signals. Our investigation focused on the transcription factor MYB50, a direct target of UPB1, in Arabidopsis thaliana. By analyzing multiple fluorescent proteins and conducting RNA-seq and ChIP-seq, we unraveled a step in the MYB50 regulatory gene network. This analysis, in conjunction with the UPB1 regulatory network, demonstrated that MYB50 directly regulates the expression of PECTIN METHYLESTERASE INHIBITOR 8 (PMEI8). Overexpressing PMEI8, similar to the MYB50, resulted in reduced mature cell length. These findings establish MYB50 as a regulator of root growth within the UPB1 gene regulatory network. Our study presents a model involving transcriptional regulation by MYB50 in the UPB1 regulated root growth system and sheds light on cell elongation via pectin modification.

Early differentiation of Casparian strip mediated by nitric oxide is required for efficient K transport under low K conditions in Arabidopsis.

The Casparian strip (CS) is a cell wall modification made of lignin that functions as an apoplastic barrier in the root endodermis to restrict nutrient and water transport between the soil and stele. CS formation is affected by nutritional conditions, and its physiological roles have been discussed. This study found that low K condition affects CS permeability, lignin deposition, and MYB36 mRNA accumulation. To understand the mechanism underlying these findings, we focused on nitric oxide (NO). NO is known to act as a signaling molecule and participates in cell wall synthesis, especially for lignin composition. However, the mechanism by which NO affects lignin deposition and corrects CS formation in the plant roots remains unclear. Through combining fluorescent observation with histological stains, we demonstrated that the root endodermal cell lignification response to low-potassium (K) conditions is mediated by NO through the MYB36-associated lignin-polymerizing pathway. Furthermore, we discovered the noteworthy ability of NO to maintain nutrient homeostasis for adaptation to low K conditions by affecting the correct apoplastic barrier formation of CS. Collectively, our results suggest that NO is required for the lignification and apoplastic barrier formation in the root endodermis during adaptation to low K conditions, which revealing the novel physiological roles of CS under low nutrient conditions and making a significant contribution to CS biology.

A very long chain fatty acid responsive transcription factor, MYB93, regulates lateral root development in Arabidopsis.

Lateral roots (LRs) are critical to root system architecture development in plants. Although the molecular mechanisms by which auxin regulates LR development have been extensively studied, several additional regulatory systems are hypothesized to be involved. Recently, the regulatory role of very long chain fatty acids (VLCFAs) has been shown in LR development. Our analysis showed that LTPG1 and LTPG2, transporters of VLCFAs, are specifically expressed in the developing LR primordium (LRP), while the number of LRs is reduced in the ltpg1/ltpg2 double mutant. Moreover, late LRP development was hindered when the VLCFA levels were reduced by the VLCFA synthesis enzyme mutant, kcs1-5. However, the details of the regulatory mechanisms of LR development controlled by VLCFAs remain unknown. In this study, we propose a novel method to analyze the LRP development stages with high temporal resolution using a deep neural network and identify a VLCFA-responsive transcription factor, MYB93, via transcriptome analysis of kcs1-5. MYB93 showed a carbon chain length-specific expression response following treatment of VLCFAs. Furthermore, myb93 transcriptome analysis suggested that MYB93 regulated the expression of cell wall organization genes. In addition, we also found that LTPG1 and LTPG2 are involved in LR development through the formation of root cap cuticle, which is different from transcriptional regulation by VLCFAs. Our results suggest that VLCFA is a regulator of LRP development through transcription factor-mediated regulation of gene expression and the transportation of VLCFAs is also involved in LR development through root cap cuticle formation.

Biochemical Characterization of a Pectate Lyase AnPL9 from Aspergillus nidulans.

Pectinolytic enzymes have diverse industrial applications. Among these, pectate lyases act on the internal α-1,4-linkage of the pectate backbone, playing a critical role in pectin degradation. While most pectate lyases characterized thus far are of bacterial origin, fungi can also be excellent sources of pectinolytic enzymes. In this study, we performed biochemical characterization of the pectate lyase AnPL9 belonging to the polysaccharide lyase family 9 (PL9) from the filamentous fungus Aspergillus nidulans. Recombinant AnPL9 was produced using a Pichia pastoris expression system and purified. AnPL9 exhibited high activity on homogalacturonan (HG), pectin from citrus peel, pectin from apple, and the HG region in rhamnogalacturonan-I. Although digalacturonic acid and trigalacturonic acid were not degraded by AnPL9, tetragalacturonic acid was converted to 4,5-unsaturated digalacturonic acid and digalacturonic acid. These results indicate that AnPL9 degrades HG oligosaccharides with a degree of polymerization > 4. Furthermore, AnPL9 was stable within a neutral-to-alkaline pH range (pH 6.0-11.0). Our findings suggest that AnPL9 is a candidate pectate lyase for biotechnological applications in the food, paper, and textile industries. This is the first report on a fungal pectate lyase belonging to the PL9 family.

Systemic Regulation of Iron Acquisition by Arabidopsis in Environments with Heterogeneous Iron Distributions.

Nutrient distribution within the soil is generally heterogeneous. Plants, therefore, have evolved sophisticated systemic processes enabling them to optimize their nutrient acquisition efficiency. By organ-to-organ communication in Arabidopsis thaliana, for instance, iron (Fe) starvation in one part of a root drives the upregulation of a high-affinity Fe-uptake system in other root regions surrounded by sufficient levels of Fe. This compensatory response through Fe-starvation-triggered organ-to-organ communication includes the upregulation of Iron-regulated transporter 1 (IRT1) gene expression on the Fe-sufficient side of the root; however, the molecular basis underlying this long-distance signaling remains unclear. Here, we analyzed gene expression by RNA-seq analysis of Fe-starved split-root cultures. Genome-wide expression analysis showed that localized Fe depletion in roots upregulated several genes involved in Fe uptake and signaling, such as IRT1, in a distant part of the root exposed to Fe-sufficient conditions. This result indicates that long-distance signaling for Fe demand alters the expression of a subset of genes responsible for Fe uptake and coumarin biosynthesis to maintain a level of Fe acquisition sufficient for the entire plant. Loss of IRON MAN/FE-UPTAKE-INDUCING PEPTIDE (IMA/FEP) leads to the disruption of compensatory upregulation of IRT1 in the root surrounded by sufficient Fe. In addition, our split-root culture-based analysis provides evidence that the IMA3/FEP1-MYB10/72 pathway mediates long-distance signaling in Fe homeostasis through the regulation of coumarin biosynthesis. These data suggest that the signaling of IMA/FEP, a ubiquitous family of metal-binding peptides, is critical for organ-to-organ communication in response to Fe starvation under heterogeneous Fe conditions in the surrounding environment.

Suppression of MYC transcription activators by the immune cofactor NPR1 fine-tunes plant immune responses.

Plants tailor immune responses to defend against pathogens with different lifestyles. In this process, antagonism between the immune hormones salicylic acid (SA) and jasmonic acid (JA) optimizes transcriptional signatures specifically to the attacker encountered. Antagonism is controlled by the transcription cofactor NPR1. The indispensable role of NPR1 in activating SA-responsive genes is well understood, but how it functions as a repressor of JA-responsive genes remains unclear. Here, we demonstrate that SA-induced NPR1 is recruited to JA-responsive promoter regions that are co-occupied by a JA-induced transcription complex consisting of the MYC2 activator and MED25 Mediator subunit. In the presence of SA, NPR1 physically associates with JA-induced MYC2 and inhibits transcriptional activation by disrupting its interaction with MED25. Importantly, NPR1-mediated inhibition of MYC2 is a major immune mechanism for suppressing pathogen virulence. Thus, NPR1 orchestrates the immune transcriptome not only by activating SA-responsive genes but also by acting as a corepressor of JA-responsive MYC2.

Reactive Oxygen Species Link Gene Regulatory Networks During Arabidopsis Root Development.

Plant development under altered nutritional status and environmental conditions and during attack from invaders is highly regulated by plant hormones at the molecular level by various signaling pathways. Previously, reactive oxygen species (ROS) were believed to be harmful as they cause oxidative damage to cells; however, in the last decade, the essential role of ROS as signaling molecules regulating plant growth has been revealed. Plant roots accumulate relatively high levels of ROS, and thus, maintaining ROS homeostasis, which has been shown to regulate the balance between cell proliferation and differentiation at the root tip, is important for proper root growth. However, when the balance is disturbed, plants are unable to respond to the changes in the surrounding conditions and cannot grow and survive. Moreover, ROS control cell expansion and cell differentiation processes such as root hair formation and lateral root development. In these processes, the transcription factor-mediated gene expression network is important downstream of ROS. Although ROS can independently regulate root growth to some extent, a complex crosstalk occurs between ROS and other signaling molecules. Hormone signals are known to regulate root growth, and ROS are thought to merge with these signals. In fact, the crosstalk between ROS and these hormones has been elucidated, and the central transcription factors that act as a hub between these signals have been identified. In addition, ROS are known to act as important signaling factors in plant immune responses; however, how they also regulate plant growth is not clear. Recent studies have strongly indicated that ROS link these two events. In this review, we describe and discuss the role of ROS signaling in root development, with a particular focus on transcriptional regulation. We also summarize the crosstalk with other signals and discuss the importance of ROS as signaling molecules for plant root development.

Root system architecture analysis in Mesembryanthemum crystallinum (ice plant) seedlings reveals characteristic root halotropic response.

One of the major environmental stress factors that affect root growth is salinity. Arabidopsis thaliana, a glycophyte, shows halotropism, whereby it alters the direction of root growth in a non-gravitropic pattern to evade high soil salinity. Asymmetric auxin distribution regulated by the relocation of auxin-efflux carrier proteins is a key cellular event in the halotropic response. However, there are no reports of halotropism in halophytes. Here, we investigated root growth traits in Mesembryanthemum crystallinum (ice plant), under high salinity conditions. We hypothesized that ice plant roots would show halotropic responses different from those of Arabidopsis Notably, similar to halotropism observed in Arabidopsis, ice plant roots showed continuous root bending under salinity stress. However, the root elongation rate did not change in ice plants. Expression analyses of several genes revealed that auxin transport might be partially involved in ice plant halotropism. This study enhances our understanding of halophyte root adaptation to high salinity stress.

Rapid and easy method for in vitro determination of transcription factor binding core motifs.

We introduce a rapid method for easily elucidating transcription factor (TF) cis-elements by adopting a highly efficient in vitro protein synthesis method and identifying protein-DNA interactions using PCR. We determined two cis-elements for plant TFs using this method, and the results confirmed our method as an easy and time-saving alternative for elucidating TF cis-elements using common laboratory procedures.

ANAC032 regulates root growth through the MYB30 gene regulatory network.

Reactive oxygen species (ROS) play important roles as root growth regulators. We previously reported a comprehensive transcriptomic atlas, which we named ROS-map, that revealed ROS-responsible genes in Arabidopsis root tips. By using ROS-map, we have characterised an early ROS response key transcription factor, MYB30, as a regulator of root cell elongation under ROS signals. However, there are other ROS-responsible transcription factors which have the potential to regulate root growth. In the present study, we characterised the function of another early ROS-responsible transcription factor, ANAC032, that was selected from ROS-map. Overexpression of ANAC032 fused with the transcriptional activation domain, VP16, inhibited root growth, especially decreasing cell elongation. By transcriptome analysis, we revealed that ANAC032 regulated many stress-responsible genes in the roots. Intriguingly, ANAC032 upregulated MYB30 and its target genes. The upregulation of MYB30 target genes was completely abolished in the ANAC032-VP16x2 OX and ANAC032 estradiol-inducible line in myb30-2 mutants. Moreover, root growth inhibition was alleviated in ANAC032-OX in myb30-2 mutants. Overall, we characterised an upstream transcription factor, ANAC032, of the MYB30 transcriptional cascade which is a key regulator for root cell elongation under ROS signalling.

In vitro Protein-DNA Binding Assay (AlphaScreen® Technology).

Identification of specific DNA binding sites of transcription factors is important in understanding their functions. Recent techniques allow us to investigate genome-wide in vivo binding positions by chromatin immunoprecipitation combined with high-throughput sequencing. However, to further explore the binding motifs of transcription factors, in-depth biochemical analysis is required. Here, we describe an efficient protocol of protein-DNA interactions based on a combination of our in vitro transcription/translation system and AlphaScreen® technology. The in vitro transcription/translation system supports an efficient and quick way of protein synthesis by alleviating cumbersome cloning steps. In addition, AlphaScreen® system provides a highly sensitive, quick, and easy handling platform to investigate the protein-DNA interactions in vitro. Thus, our method largely contributes to comprehensive analysis of the biochemical properties of transcription factors.

MYB30 regulates root cell elongation under abscisic acid signaling.

Reactive oxygen species (ROS) and plant hormones play important roles in regulating plant growth and stress responses as signaling molecules. Abscisic acid (ABA) is known as the key regulator of both abiotic and biotic stress responses. During stress responses, ABA is known to regulate ROS production, indicating that important crosstalk occurs between ROS and ABA signaling. We recently reported that MYB30, an MYB-type transcription factor, regulates root cell elongation under ROS signaling. In this study, we analyzed the molecular interaction between ROS and ABA signal during for root development, which is mediated through MYB30 transcriptional regulation. We showed that MYB30-regulated root cell elongation was mediated by ROS production under ABA signaling. Our findings will provide one piece of evidence of the complex cross talk between ROS and hormone signaling that regulates root development.

MYB30 links ROS signaling, root cell elongation, and plant immune responses.

Reactive oxygen species (ROS) are known to be important signal molecules that are involved in biotic and abiotic stress responses as well as in growth regulation. However, the molecular mechanisms by which ROS act as a growth regulator, as well as how ROS-dependent growth regulation relates to its roles in stress responses, are not well understood. We performed a time-course microarray analysis of Arabidopsis root tips upon treatment with hydrogen peroxide, which we named "ROS-map." Using the ROS-map, we identified an MYB transcription factor, MYB30, which showed a strong response to ROS treatment and is the key regulator of a gene network that leads to the hydrogen peroxide-dependent inhibition of root cell elongation. Intriguingly, this network contained multiple genes involved in very-long-chain fatty acid (VLCFA) transport. Finally, we showed that MYB30 is necessary for root growth regulation during defense responses, thus providing a molecular link between these two ROS-associated processes.

Ectopic expression of Mesembryanthemum crystallinum sodium transporter McHKT2 provides salt stress tolerance in Arabidopsis thaliana.

Most plants do not tolerate highly saline environments; the development of salt stress tolerance is crucial for improving crop yield. An efficient way of finding genes involved in salt tolerance is to study and use data from halophytes. In this study, we used the Mesembryanthemum crystallinum (ice plant) expression data-set and selected for further study the gene McHKT2, which encodes for the Arabidopsis sodium transporter ortholog AtHKT1. In comparison with the HKT1 amino acid sequences from other plants, McHKT2 has several unique features. It seems to be localized to the plasma membrane, and its overexpression confers strong salt tolerance in Arabidopsis thaliana. Our results indicate that McHKT2 is a suitable candidate protein that can induce salt tolerance in non-halophytes. Like McHKT2, using transcriptome data-sets from halophytes such as ice plant give us an efficiency way to obtain new gene resources that might involve in plant salt tolerance.

Control of root growth and development by reactive oxygen species.

Reactive oxygen species (ROS) are relatively simple molecules that exist within cells growing in aerobic conditions. ROS were originally associated with oxidative stress and seen as highly reactive molecules that are injurious to many cell components. More recently, however, the function of ROS as signal molecules in many plant cellular processes has become more evident. One of the most important functions of ROS is their role as a plant growth regulator. For example, ROS are key molecules in regulating plant root development, and as such, are comparable to plant hormones. In this review, the molecular mechanisms of ROS that are mainly associated with plant root growth are discussed. The molecular links between root growth regulation by ROS and other signals will also be briefly discussed.

RNA-seq analysis of the response of the halophyte, Mesembryanthemum crystallinum (ice plant) to high salinity.

Understanding the molecular mechanisms that convey salt tolerance in plants is a crucial issue for increasing crop yield. The ice plant (Mesembryanthemum crystallinum) is a halophyte that is capable of growing under high salt conditions. For example, the roots of ice plant seedlings continue to grow in 140 mM NaCl, a salt concentration that completely inhibits Arabidopsis thaliana root growth. Identifying the molecular mechanisms responsible for this high level of salt tolerance in a halophyte has the potential of revealing tolerance mechanisms that have been evolutionarily successful. In the present study, deep sequencing (RNAseq) was used to examine gene expression in ice plant roots treated with various concentrations of NaCl. Sequencing resulted in the identification of 53,516 contigs, 10,818 of which were orthologs of Arabidopsis genes. In addition to the expression analysis, a web-based ice plant database was constructed that allows broad public access to the data. The results obtained from an analysis of the RNAseq data were confirmed by RT-qPCR. Novel patterns of gene expression in response to high salinity within 24 hours were identified in the ice plant when the RNAseq data from the ice plant was compared to gene expression data obtained from Arabidopsis plants exposed to high salt. Although ABA responsive genes and a sodium transporter protein (HKT1), are up-regulated and down-regulated respectively in both Arabidopsis and the ice plant; peroxidase genes exhibit opposite responses. The results of this study provide an important first step towards analyzing environmental tolerance mechanisms in a non-model organism and provide a useful dataset for predicting novel gene functions.

Wound-induced expression of DEFECTIVE IN ANTHER DEHISCENCE1 and DAD1-like lipase genes is mediated by both CORONATINE INSENSITIVE1-dependent and independent pathways in Arabidopsis thaliana.

Endogenous JA production is not necessary for wound-induced expression of JA-biosynthetic lipase genes such as DAD1 in Arabidopsis. However, the JA-Ile receptor COI1 is often required for their JA-independent induction. Wounding is a serious event in plants that may result from insect feeding and increase the risk of pathogen infection. Wounded plants produce high amounts of jasmonic acid (JA), which triggers the expression of insect and pathogen resistance genes. We focused on the transcriptional regulation of DEFECTIVE IN ANTHER DEHISCENCE1 and six of its homologs including DONGLE (DGL) in Arabidopsis, which encode lipases involved in JA biosynthesis. Plants constitutively expressing DAD1 accumulated a higher amount of JA than control plants after wounding, indicating that the expression of these lipase genes contributes to determining JA levels. We found that the expression of DAD1, DGL, and other DAD1-LIKE LIPASE (DALL) genes is induced upon wounding. Some DALLs were also expressed in unwounded leaves. Further experiments using JA-biosynthetic and JA-response mutants revealed that the wound induction of these genes is regulated by several distinct pathways. DAD1 and most of its homologs other than DALL4 were fully induced without relying on endogenous JA-Ile production and were only partly affected by JA deficiency, indicating that positive feedback by JA is not necessary for induction of these genes. However, DAD1 and DGL required CORONATINE INSENSITIVE1 (COI1) for their expression, suggesting that a molecule other than JA might act as a regulator of COI1. Wound induction of DALL1, DALL2, and DALL3 did not require COI1. This differential regulation of DAD1 and its homologs might explain their functions at different time points after wounding.

Defective root growth triggered by oxidative stress is controlled through the expression of cell cycle-related genes.

Reactive oxygen species (ROS) have many functions in aerobic organisms. High levels of ROS can have a negative impact on plant cells leading to senescence and cell death. ROS accumulates in cells subjected to environmental stress and induces a cellular response to this external stimulus. To protect cells from the negative impacts of excess ROS, plants also possess a ROS detoxifying system to maintain normal ROS levels. The regulation of ROS levels is particularly important as ROS also functions as an important signal molecule and can regulate plant growth by modulating gene expression. Despite the functional importance of ROS signaling, little is known about the molecular mechanisms involved in the regulation of gene expression through ROS. Therefore, the present study investigated the effect of hydrogen peroxide (H(2)O(2)), a ROS compound, on cell cycle-related gene expression. Gene expression analyses coupled with microdissected sections of the developmental zone of Arabidopsis root tips revealed that H(2)O(2) affects the expression of cell cycle-related genes. Additionally, ROS scavenging enzymes were found to play an important role in the root growth phenotype induced by H(2)O(2). Specifically, root growth inhibition by H(2)O(2) was diminished in transgenic Arabidopis overexpressing peroxidase but increased in a catalase2 (cat2) mutant. The strong root growth inhibition observed in the cat2 mutant upon H(2)O(2) treatment indicated that CAT2 has an essential role in maintaining root meristem activity in the presence of oxidative stress. Overall, these results confirm that ROS function not only as stress-related compounds but that they also function as signaling molecules to regulate the progression of the cell cycle in root tips.