LBD18 and IAA14 antagonistically interact with ARF7 via the invariant Lys and acidic residues of the OPCA motif in the PB1 domain.

MAIN CONCLUSION: LBD18 and IAA14 antagonistically interact with ARF7 through the electrostatic faces in the ARF7PB1 domain, modulating ARF7 transcriptional activity. Auxin Response Factor 7 (ARF7)/ARF19 control lateral root development by directly activating Lateral Organ Boundaries Domain 16 (LBD16)/LBD18 genes in Arabidopsis. LBD18 upregulates ARF19 expression by binding to the ARF19 promoter. It also interacts with ARF7 through the Phox and Bem1 (PB1) domain to enhance the ARF7 transcriptional activity, forming a dual mode of positive feedback loop. LBD18 competes with the repressor indole-3-acetic acid 14 (IAA14) for ARF7 binding through the PB1 domain. In this study, we examined the molecular determinant of the ARF7 PB1 domain for interacting with LBD18 and showed that the electronic faces in the ARF7 PB1 domain are critical for interacting with LBD18 and IAA14/17. We used a luminescence complementation imaging assay to determine protein-protein interactions. The results showed that mutation of the invariant lysine residue and the OPCA motif in the PB1 domain in ARF7 significantly reduces the protein interaction between ARF7 and LBD18. Transient gene expression assays with Arabidopsis protoplasts showed that IAA14 suppressed transcription-enhancing activity of LBD18 on the LUC reporter gene fused to the ARF19 promoter harboring an auxin response element, but mutation of the invariant lysine residue and OPCA motif in the PB1 domain of IAA14 reduced the repression capability of IAA14 for transcription-enhancing activity of LBD18. We further showed that the same mutation in the PB1 domain of IAA14 reduces its repression capability, thereby increasing the LUC activity induced by both ARF7 and LBD18 compared with IAA14. These results suggest that LBD18 competes with IAA14 for ARF7 binding via the electrostatic faces of the ARF7 PB1 domain to modulate ARF7 transcriptional activity.

Understanding the Modus Operandi of Class II KNOX Transcription Factors in Secondary Cell Wall Biosynthesis.

Lignocellulosic biomass from the secondary cell walls of plants has a veritable potential to provide some of the most appropriate raw materials for producing second-generation biofuels. Therefore, we must first understand how plants synthesize these complex secondary cell walls that consist of cellulose, hemicellulose, and lignin in order to deconstruct them later on into simple sugars to produce bioethanol via fermentation. Knotted-like homeobox (KNOX) genes encode homeodomain-containing transcription factors (TFs) that modulate various important developmental processes in plants. While Class I KNOX TF genes are mainly expressed in the shoot apical meristems of both monocot and eudicot plants and are involved in meristem maintenance and/or formation, Class II KNOXTF genes exhibit diverse expression patterns and their precise functions have mostly remained unknown, until recently. The expression patterns of Class II KNOX TF genes in Arabidopsis, namely KNAT3, KNAT4, KNAT5, and KNAT7, suggest that TFs encoded by at least some of these genes, such as KNAT7 and KNAT3, may play a significant role in secondary cell wall formation. Specifically, the expression of the KNAT7 gene is regulated by upstream TFs, such as SND1 and MYB46, while KNAT7 interacts with other cell wall proteins, such as KNAT3, MYB75, OFPs, and BLHs, to regulate secondary cell wall formation. Moreover, KNAT7 directly regulates the expression of some xylan synthesis genes. In this review, we summarize the current mechanistic understanding of the roles of Class II KNOX TFs in secondary cell wall formation. Recent success with the genetic manipulation of Class II KNOX TFs suggests that this may be one of the biotechnological strategies to improve plant feedstocks for bioethanol production.

Recent advances in peptide signaling during Arabidopsis root development.

Roots provide the plant with water and nutrients and anchor it in a substrate. Root development is controlled by plant hormones and various sets of transcription factors. Recently, various small peptides and their cognate receptors have been identified as controlling root development. Small peptides bind to membrane-localized receptor-like kinases, inducing their dimerization with co-receptor proteins for signaling activation and giving rise to cellular signaling outputs. Small peptides function as local and long-distance signaling molecules involved in cell-to-cell communication networks, coordinating root development. In this review, we survey recent advances in the peptide ligand-mediated signaling pathways involved in the control of root development in Arabidopsis. We describe the interconnection between peptide signaling and conventional phytohormone signaling. Additionally, we discuss the diversity of identified peptide-receptor interactions during plant root development.

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.

LBD13 positively regulates lateral root formation in Arabidopsis.

MAIN CONCLUSION: Lateral Organ Boundaries Domain 13 (LBD13), which is expressed in emerged lateral roots and encodes a transcriptional activator, plays an important role in lateral root formation in Arabidopsis. Lateral roots (LRs) are major determinants of root system architecture, contributing to the survival strategies of plants. Members of the LBD gene family encode plant-specific transcription factors that play key roles in plant organ development. Several LBD genes, such as LBD14, 16, 18, 29, and 33, have been shown to play important roles in regulating LR development in Arabidopsis. In the present study, we show that LBD13 is expressed in emerged LRs and LR meristems of elongated LRs and regulates LR formation in Arabidopsis. Transient gene expression assays with Arabidopsis protoplasts showed that LBD13 is localized to the nucleus and harbors transcription-activating potential. Knock-down of LBD13 expression by RNA interference resulted in reduced LR formation, whereas overexpression of LBD13 enhanced LR formation in transgenic Arabidopsis. Analysis of ß-glucuronidase (GUS) expression under the control of the LBD13 promoter showed that GUS staining was detected in LRs emerged from the primary root, but not in LR primordia. Moreover, both the distribution of LR primordium number and developmental kinetics of LR primordia were not affected either by knock-down or by overexpression of LBD13. Taken together, these results suggest that LBD13 is a nuclear-localized transcriptional activator and controls LR formation during or after LR emergence.

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.

Coiled-coil motif in LBD16 and LBD18 transcription factors are critical for dimerization and biological function in arabidopsis.

The LATERAL ORGAN BOUNDARIES (LOB) DOMAIN (LBD) gene family members encode a class of plant-specific transcription factors that play important roles in many different aspects of plant growth and development. The LBD proteins contain a conserved LOB domain harboring a Leu zipper-like coiled-coil motif, which has been predicted to mediate protein-protein interactions among the LBD family members. Dimerization of transcription factors is crucial for the modulation of their DNA-binding affinity, specificity, and diversity, contributing to the transcriptional regulation of distinct cellular and biological responses. Our various molecular and biochemical experiments with genetic approaches on LBD16 and LBD18, which are known to control lateral root development in Arabidopsis, demonstrated that the conserved Leu or Val residues in the coiled-coil motifs of these transcription factors are critical for their dimerization as well as the transcriptional regulation to display their biological functions during lateral root formation. We further showed that beside the coiled-coil motif, the carboxyl-terminal region in LBD18 acts as an additional dimerization domain. These findings provide a molecular framework for the homo- and hetero-dimerization of the LBD family proteins for displaying their distinct and diverse biological functions in plants.

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.

Virus-induced gene silencing (VIGS)-mediated functional characterization of two genes involved in lignocellulosic secondary cell wall formation.

KEY MESSAGE: Functional characterization of two tobacco genes, one involved in xylan synthesis and the other, a positive regulator of secondary cell wall formation, is reported. Lignocellulosic secondary cell walls (SCW) provide essential plant materials for the production of second-generation bioethanol. Therefore, thorough understanding of the process of SCW formation in plants is beneficial for efficient bioethanol production. Recently, we provided the first proof-of-concept for using virus-induced gene silencing (VIGS) approach for rapid functional characterization of nine genes involved in cellulose, hemicellulose and lignin synthesis during SCW formation. Here, we report VIGS-mediated functional characterization of two tobacco genes involved in SCW formation. Stems of VIGS plants silenced for both selected genes showed increased amount of xylem formation but thinner cell walls than controls. These results were further confirmed by production of stable transgenic tobacco plants manipulated in expression of these genes. Stems of stable transgenic tobacco plants silenced for these two genes showed increased xylem proliferation with thinner walls, whereas transgenic tobacco plants overexpressing these two genes showed increased fiber cell wall thickness but no change in xylem proliferation. These two selected genes were later identified as possible members of DUF579 family involved in xylan synthesis and KNAT7 transcription factor family involved in positive regulation of SCW formation, respectively. Glycome analyses of cell walls showed increased polysaccharide extractability in 1 M KOH extracts of both VIGS-NbDUF579 and VIGS-NbKNAT7 lines suggestive of cell wall loosening. Also, VIGS-NbDUF579 and VIGS-NbKNAT7 lines showed increased saccharification rates (74.5 and 40 % higher than controls, respectively). All these properties are highly desirable for producing higher quantities of bioethanol from lignocellulosic materials of bioenergy plants.

Enhanced accumulation of fatty acids and triacylglycerols in transgenic tobacco stems for enhanced bioenergy production.

KEY MESSAGE: We report a novel approach for enhanced accumulation of fatty acids and triacylglycerols for utilization as biodiesel in transgenic tobacco stems through xylem-specific expression of Arabidopsis DGAT1 and LEC2 genes. The use of plant biomass for production of bioethanol and biodiesel has an enormous potential to revolutionize the global bioenergy outlook. Several studies have recently been initiated to genetically engineer oil production in seeds of crop plants to improve biodiesel production. However, the "food versus fuel" issues have also sparked some studies for enhanced accumulation of oils in vegetative tissues like leaves. But in the case of bioenergy crops, use of woody stems is more practical than leaves. Here, we report the enhanced accumulation of fatty acids (FAs) and triacylglycerols (TAGs) in stems of transgenic tobacco plants expressing Arabidopsis diacylglycerol acyltransferase 1 (DGAT1) and leafy cotyledon2 (LEC2) genes under a developing xylem-specific cellulose synthase promoter from aspen trees. The transgenic tobacco plants accumulated significantly higher amounts of FAs in their stems. On an average, DGAT1 and LEC2 overexpression showed a 63 and 80% increase in total FA production in mature stems of transgenic plants over that of controls, respectively. In addition, selected DGAT1 and LEC2 overexpression lines showed enhanced levels of TAGs in stems with higher accumulation of 16:0, 18:2 and 18:3 TAGs. In LEC2 lines, the relative mRNA levels of the downstream genes encoding plastidic proteins involved in FA synthesis and accumulation were also elevated. Thus, here, we provide a proof of concept for our approach of enhancing total energy yield per plant through accumulation of higher levels of FAs in transgenic stems for biodiesel production.