LBD18 uses a dual mode of a positive feedback loop to regulate ARF expression and transcriptional activity in Arabidopsis.

A hierarchy of transcriptional regulators controlling lateral root formation in Arabidopsis thaliana has been identified, including the AUXIN RESPONSE FACTOR 7 (ARF7)/ARF19-LATERAL ORGAN BOUNDARIES DOMAIN 16 (LBD16)/LBD18 transcriptional network; however, their feedback regulation mechanisms are not known. Here we show that LBD18 controls ARF activity using the dual mode of a positive feedback loop. We showed that ARF7 and ARF19 directly bind AuxRE in the LBD18 promoter. A variety of molecular and biochemical experiments demonstrated that LBD18 binds a specific DNA motif in the ARF19 promoter to regulate its expression in vivo as well as in vitro. LBD18 interacts with ARFs including ARF7 and ARF19 via the Phox and Bem1 domain of ARF to enhance the transcriptional activity of ARF7 on AuxRE, and competes with auxin/indole-3-acetic acid (IAA) repressors for ARF binding, overriding the negative feedback loop exerted by Aux/IAA repressors. Taken together, these results show that LBD18 and ARFs form a double positive feedback loop, and that LBD18 uses the dual mode of a positive feedback loop by binding directly to the ARF19 promoter and through the protein-protein interactions with ARF7 and ARF19. This novel mechanism of feedback loops may constitute a robust feedback mechanism that ensures continued lateral root growth in response to auxin in Arabidopsis.

LBD16 and LBD18 acting downstream of ARF7 and ARF19 are involved in adventitious root formation in Arabidopsis.

BACKGROUND: Adventitious root (AR) formation is a complex genetic trait, which is controlled by various endogenous and environmental cues. Auxin is known to play a central role in AR formation; however, the mechanisms underlying this role are not well understood. RESULTS: In this study, we showed that a previously identified auxin signaling module, AUXIN RESPONSE FACTOR(ARF)7/ARF19-LATERAL ORGAN BOUNDARIES DOMAIN(LBD)16/LBD18 via AUXIN1(AUX1)/LIKE-AUXIN3 (LAX3) auxin influx carriers, which plays important roles in lateral root formation, is involved in AR formation in Arabidopsis. In aux1, lax3, arf7, arf19, lbd16 and lbd18 single mutants, we observed reduced numbers of ARs than in the wild type. Double and triple mutants exhibited an additional decrease in AR numbers compared with the corresponding single or double mutants, respectively, and the aux1 lax3 lbd16 lbd18 quadruple mutant was devoid of ARs. Expression of LBD16 or LBD18 under their own promoters in lbd16 or lbd18 mutants rescued the reduced number of ARs to wild-type levels. LBD16 or LBD18 fused to a dominant SRDX repressor suppressed promoter activity of the cell cycle gene, Cyclin-Dependent Kinase(CDK)A1;1, to some extent. Expression of LBD16 or LBD18 was significantly reduced in arf7 and arf19 mutants during AR formation in a light-dependent manner, but not in arf6 and arf8. GUS expression analysis of promoter-GUS reporter transgenic lines revealed overlapping expression patterns for LBD16, LBD18, ARF7, ARF19 and LAX3 in AR primordia. CONCLUSION: These results suggest that the ARF7/ARF19-LBD16/LBD18 transcriptional module via the AUX1/LAX3 auxin influx carriers plays an important role in AR formation in Arabidopsis.

Dimerization in LBD16 and LBD18 Transcription Factors Is Critical for Lateral Root Formation.

LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKEs (hereafter referred to as LBD) are plant-specific transcription factors that play important roles in a plethora of plant growth and development. The leucine (Leu) zipper-like coiled-coil motif in the lateral organ boundaries domain of the class I LBD proteins has been proposed to mediate protein dimerization, but it has not been experimentally assessed yet. LBD16 and LBD18 have been well characterized to play important roles in lateral root development in Arabidopsis (Arabidopsis thaliana). Here, we investigated the role of the coiled-coil motif in the dimerization of LBD16 and LBD18 and in transcriptional regulation and biological function. We built the molecular models of the coiled coil of LBD16 and LBD18, providing the probable Leu zipper models of the helix dimer. Using a variety of molecular techniques, such as bimolecular fluorescence complementation, luciferase complementation imaging, GST pull down, and coimmunoprecipitation assays, we showed that the conserved Leu or valine residues in the coiled-coil motif are critical for the dimerization of LBD16 or LBD18. Using transgenic Arabidopsis plants that overexpress HA:LBD16 or HA:LBD16Q in lbd16 or HA:LBD18 or HA:LBD18Q in lbd18, we demonstrated that the homodimerization of LBD18 mediated by the coiled-coil motif is crucial for transcriptional regulation via promoter binding and for lateral root formation. In addition, we found that the carboxyl-terminal region beyond the coiled-coil motif in LBD18 acts as an additional dimerization domain. These results provide a molecular basis for homodimerization and heterodimerization among the 42 Arabidopsis LBD family members for displaying their biological functions.

Lateral Organ Boundaries Domain16 and 18 Act Downstream of the AUXIN1 and LIKE-AUXIN3 Auxin Influx Carriers to Control Lateral Root Development in Arabidopsis.

Several members of the Lateral Organ Boundaries Domain (LBD)/Asymmetric Leaves2-Like (ASL) gene family have been identified to play important roles in Arabidopsis (Arabidopsis thaliana) lateral root (LR) development during auxin response, but their functional relationship with auxin transporters has not been established yet. Here, we show that the AUXIN1 (AUX1) and LIKE-AUXIN3 (LAX3) auxin influx carriers are required for auxin signaling that activates LBD16/ASL18 and LBD18/ASL20 to control LR development. The lax3 mutant phenotype was not significantly enhanced when combined with lbd16 or lbd18. However, LBD18 overexpression could rescue the defects in LR emergence in lax3 with concomitant expression of the LBD18 target genes. Genetic and gene expression analyses indicated that LBD16 and LBD18 act with AUX1 to regulate LR initiation and LR primordium development, and that AUX1 and LAX3 are needed for auxin-responsive expression of LBD16 and LBD18. LBD18:SUPERMAN REPRESSIVE DOMAIN X in the lbd18 mutant inhibited LR initiation and LR primordium development in response to a gravitropic stimulus and suppressed promoter activities of the cell cycle genes Cyclin-Dependent Kinase A1;1 and CYCLINB1;1. Taken together, these results suggest that LBD16 and LBD18 are important regulators of LR initiation and development downstream of AUX1 and LAX3.

LBD18 acts as a transcriptional activator that directly binds to the EXPANSIN14 promoter in promoting lateral root emergence of Arabidopsis.

Lateral root formation, a developmental process under the control of the plant hormone auxin, is a major determinant of root architecture, and defines the ability of a plant to acquire nutrients and water. The LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) proteins play an important role in the lateral organ development of plants, including lateral root formation. However, their downstream components and signalling mechanisms are largely unknown. Here, we show that auxin-responsive LBD18/ASL20 acts as a specific DNA-binding transcriptional activator that directly regulates EXPANSIN14 (EXP14), a gene encoding a cell wall-loosening factor that promotes lateral root emergence in Arabidopsis thaliana. We showed that LBD18 possesses transcription-activating function in both yeast and Arabidopsis protoplasts. We isolated putative LBD18 target genes by microarray analysis, and identified EXP14 as a direct target of LBD18. Dexamethasone-induced expression of LBD18 under the CaMV 35S promoter in transgenic Arabidopsis resulted in enhanced expression of GUS fused to the EXP14 promoter in primordium and overlaying tissues. In contrast, GUS expression under the EXP14 promoter in the lbd18 mutant background was significantly reduced in the same tissues. Experiments using a variety of molecular techniques demonstrated that LBD18 activates EXP14 by directly binding to a specific promoter element in vitro and in vivo. Overexpression of EXP14 in Arabidopsis resulted in the stimulation of emerged lateral roots, but not primordia, whereas EXP14 loss-of-function plants had reduced auxin-stimulated lateral root formation. This study revealed the molecular function of LBD18 as a specific DNA-binding transcription factor that activates EXP14 expression by directly binding to its promoter.

Developing centrifugal force real-time digital PCR for detecting extremely low DNA concentration.

Digital PCR (dPCR) is a technique for absolute quantification of nucleic acid molecules. To develop a dPCR technique that enables more accurate nucleic acid detection and quantification, we established a novel dPCR apparatus known as centrifugal force real-time dPCR (crdPCR). This system is efficient than other systems with only 2.14% liquid loss by dispensing samples using centrifugal force. Moreover, we applied a technique for analyzing the real-time graph of the each micro-wells and distinguishing true/false positives using artificial intelligence to mitigate the rain, a persistent issue with dPCR. The limits of detection and quantification were 1.38 and 4.19 copies/µL, respectively, showing a two-fold higher sensitivity than that of other comparable devices. With the integration of this new technology, crdPCR will significantly contribute to research on next-generation PCR targeting absolute micro-analysis.

EXPANSINA17 up-regulated by LBD18/ASL20 promotes lateral root formation during the auxin response.

Expansins are non-hydrolytic cell wall-loosening proteins involved in a variety of plant developmental processes during which cell wall modification occurs. Cell wall remodeling proteins including expansins have been suggested to be involved in cell separation to facilitate the emergence of lateral roots (LRs) through the overlaying tissues of the primary root. LBD18/ASL20 activates EXPANSINA14 (EXPA14) expression by directly binding to the EXPA14 promoter to enhance LR emergence in Arabidopsis thaliana. Here we show that EXPA17 is another target gene regulated by LBD18 to promote LR formation in Arabidopsis. We showed that nuclear translocation of the LBD18:GR fusion protein expressed under the Cauliflower mosaic virus (CaMV) 35S promoter or under the LBD18 promoter by dexamethasone treatment results in an increase in EXPA17 transcript levels. ß-Glucuronidase (GUS) expression under the EXPA17 promoter, which is detected only in the roots of the wild type, was reduced in the LR primordium and overlaying tissues in an lbd18 mutant background. The number of emerged LRs of the EXPA17 RNAi (RNA interference) Arabidopsis lines was significantly lower than that of the wild type. Overexpression of EXPA17 in Arabidopsis increased the density of emerged LRs in the presence of auxin compared with the wild type. LR induction experiments with a gravitropic stimulus showed that LR emergence is delayed in the EXPA17 RNAi plants compared with the wild type. In addition, EXPA4 expression was also detected in overlaying tissues of the LR primordium and was inducible by LBD18. Taken together, these results support the notion that LBD18 up-regulates a subset of EXP genes to enhance cell separation to promote LR emergence in Arabidopsis.

The AP2/EREBP gene PUCHI Co-Acts with LBD16/ASL18 and LBD18/ASL20 downstream of ARF7 and ARF19 to regulate lateral root development in Arabidopsis.

The developmental process of lateral root formation consists of priming, initiation, primordium development and the emergence of lateral roots from the primary root. Molecular genetic studies with Arabidopsis have revealed several key transcriptional regulators involved in lateral root development. However, their functional interaction has not been fully characterized yet. Here we utilized a genetic approach to understand some of these interactions, revealing that PUCHI functioning in morphogenesis of early lateral root primordium is regulated downstream of ARF7/ARF19 and acts with LBD16(ASL18)/LBD18(ASL20) to regulate lateral root development. We showed that auxin-responsive expression of PUCHI was significantly reduced in arf7 or arf19 single mutants and completely abolished in arf7 arf19 double mutants. Consistent with this, ß-glucuronidase (GUS) expression under the PUCHI promoter in arf7 arf19 was greatly reduced in the lateral root primordium compared with that in the wild type and did not respond to exogenous auxin. Results of GUS expression analyses under the PUCHI, LBD16 or LBD18 promoter in lbd16, lbd18 single and double mutants or puchi demonstrated that PUCHI and LBD16 or LBD18 do not regulate each other's expression. Lateral root phenotypes of double and triple mutants of lbd16, lbd18 and puchi showed that the puchi mutation in lbd16 and lbd18 mutants synergistically decreased the number of emerged lateral roots. These analyses also showed that puchi affected lateral root primordium development of lbd16 or lbd18 additively but differentially. Taken together, these results suggest that PUCHI co-acts with LBD16 and LBD18 to control lateral root primordium development and lateral root emergence.

Development, evaluation of the PNA RT-LAMP assay for rapid molecular detection of SARS-CoV-2.

Dual-labeled PNA probe used RT-LAMP molecular rapid assay targeting SARS-CoV-2 ORF1ab and N genes was developed, and the analytical, clinical performances for detection of SARS-CoV-2 RNA extracted from clinical nasopharyngeal swab specimens were evaluated in this study. Data showed that this assay is highly specific for SARS-CoV-2, and the absolute detection limit is 1 genomic copy per microliter of viral RNA which can be considered to be comparable to gold-standard molecular diagnostic method real-time reverse transcriptase PCR. Both clinical sensitivity and specificity against a commercial real-time RT-PCR assay were determined as identical. In conclusion, the PNA RT-LAMP assay showed high analytical and clinical accuracy which are identical to real-time RT-PCR which has been routinely used for the detection of SARS-CoV-2.

LBD18/ASL20 regulates lateral root formation in combination with LBD16/ASL18 downstream of ARF7 and ARF19 in Arabidopsis.

The LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) genes encode proteins harboring a conserved amino acid domain, referred to as the LOB (for lateral organ boundaries) domain. While recent studies have revealed developmental functions of some LBD genes in Arabidopsis (Arabidopsis thaliana) and in crop plants, the biological functions of many other LBD genes remain to be determined. In this study, we have demonstrated that the lbd18 mutant evidenced a reduced number of lateral roots and that lbd16 lbd18 double mutants exhibited a dramatic reduction in the number of lateral roots compared with lbd16 or lbd18. Consistent with this observation, significant beta-glucuronidase (GUS) expression in Pro(LBD18):GUS seedlings was detected in lateral root primordia as well as in the emerged lateral roots. Whereas the numbers of primordia of lbd16, lbd18, and lbd16 lbd18 mutants were similar to those observed in the wild type, the numbers of emerged lateral roots of lbd16 and lbd18 single mutants were reduced significantly. lbd16 lbd18 double mutants exhibited additively reduced numbers of emerged lateral roots compared with single mutants. This finding indicates that LBD16 and LBD18 may function in the initiation and emergence of lateral root formation via a different pathway. LBD18 was shown to be localized into the nucleus. We determined whether LBD18 functions in the nucleus using a steroid regulator-inducible system in which the nuclear translocation of LBD18 can be regulated by dexamethasone in the wild-type, lbd18, and lbd16 lbd18 backgrounds. Whereas LBD18 overexpression in the wild-type background induced lateral root formation to some degree, other lines manifested the growth-inhibition phenotype. However, LBD18 overexpression rescued lateral root formation in lbd18 and lbd16 lbd18 mutants without inducing any other phenotypes. Furthermore, we demonstrated that LBD18 overexpression can stimulate lateral root formation in auxin response factor7/19 (arf7 arf19) mutants with blocked lateral root formation. Taken together, our results suggest that LBD18 functions in the initiation and emergence of lateral roots, in conjunction with LBD16, downstream of ARF7 and ARF19.

Genome-wide analysis of the auxin-responsive transcriptome downstream of iaa1 and its expression analysis reveal the diversity and complexity of auxin-regulated gene expression.

The AUXIN RESPONSE FACTORs (ARFs) and the Aux/IAA proteins regulate various auxin responses through auxin perception mediated by the F-box proteins TIR1/AFBs. ARFs are transcription factors that modulate expression of auxin response genes and are negatively regulated by the Aux/IAA proteins. To gain insight into the regulatory mechanisms of Aux/IAA-ARF action at the genome level, the transcriptome regulated downstream of iaa1, a stabilized IAA1 mutant protein, was identified using dexamethasone (DEX)-controlled nuclear translocation of iaa1 during the auxin response. The expression of the iaa1-regulated auxin-responsive genes selected from microarray data was analysed with RNA-gel blot analysis and it was shown that auxin-regulated expression of these genes was significantly inhibited by DEX treatment. While cycloheximide-inducible expression of a majority of these genes was also DEX-suppressible, expression of some genes could not be suppressed by treatment with DEX. Expression analysis in a variety of arf mutant backgrounds suggested that all iaa1-regulated auxin-response genes examined are controlled by ARFs to different extents and that the same ARF protein can regulate the expression of these genes in response to auxin in a positive or a negative manner. However, arf mutations did not affect auxin-mediated down-regulation, indicating that ARFs might not play a critical role in down-regulation. The decrease in auxin-responsive gene expression in arf7 arf19 mutants was more severe than that of tir1/afb quadruple mutants. These results show the diversity and complexity of mechanisms of Aux/IAA-ARF- and auxin-regulated gene expression. These data also provide the opportunity for functional analysis of genes mediating the auxin-response downstream of Aux/IAA-ARFs.

GIP1 may act as a coactivator that enhances transcriptional activity of LBD18 in Arabidopsis.

The LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) gene family encodes a class of transcription factors harboring a conserved plant-specific lateral organ boundaries domain and plays a key role in lateral organ development of plants. Recent studies have revealed developmental functions of some LBD genes in Arabidopsis, rice, and maize. We have shown previously that LBD18/ASL20 promotes the emergence of lateral roots in Arabidopsis. LBD18 induces EXPANSIN14 (EXP14) expression by binding to a specific region of the EXP14 promoter. To further understand the molecular mechanism of LBD18 acting as a transcription factor, we isolated a protein interacting with LBD18 by screening an Arabidopsis cDNA library using the yeast two-hybrid system with LBD18 as bait. We found that GBF INTERACTING PROTEIN1 (GIP1) interacts with LBD18 in yeast and Arabidopsis protoplasts. Reverse-transcription-polymerase chain reaction analysis showed overlapping expression of GIP1 and LBD18 in various tissues of Arabidopsis such as roots, aerial parts, and rosette leaves. Transient gene expression assay results with Arabidopsis protoplasts indicated that GIP1 enhances transcriptional activity of LBD18 in the EXP14 promoter fused to the GUS reporter gene. These results show that GIP1 may act as a transcriptional coactivator of LBD18.

Direct activation of EXPANSIN14 by LBD18 in the gene regulatory network of lateral root formation in Arabidopsis.

Root system architecture is important for plants to adapt to a changing environment. The major determinant of the root system is lateral roots originating from the primary root. The developmental process of lateral root formation can be divided into priming, initiation, primordium development and the emergence of lateral roots, and is well characterized in Arabidopsis. The hormone auxin plays a critical role in lateral root development, and several auxin response modules involving AUXIN RESPONSE FACTORS (ARFs), transcriptional regulators of auxin-regulated genes and Aux/IAA, negative regulators of ARFs, regulate lateral root formation. The LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) gene family encodes a unique class of transcription factors harbouring a conserved plant-specific lateral organ boundary domain and plays a role in lateral organ development of plants including lateral root formation. In our previous study, we showed that LBD18 stimulates lateral root formation in combination with LBD16 downstream of ARF7 and ARF19 during the auxin response. We have recently demonstrated that LBD18 activates expression of EXP14, a gene encoding the cell-wall loosening factor, by directly binding to the EXP14 promoter to promote lateral root emergence. Here we present the molecular function of LBD18 and its gene regulatory network during lateral root formation.