Lateral Organ Boundaries Domain Transcription Factor Research Articles

Genome-wide identification and gene expression networks of LBD transcription factors in Populus trichocarpa

The Lateral Organ Boundaries Domain (LBD) proteins, an exclusive family of transcription factors (TFs) found solely in plants, play pivotal roles in lateral organogenesis, stress adaptation, secondary growth, and hormonal signaling responses. In this study, a total of 55 PtLBD TFs from Populus trichocarpa were identified and systematically classified into two subfamilies, designated as subfamily-I and subfamily-II with seven distinct groups based on phylogenetic analysis. Gene structure detection indicated that the difference of phase numbers linking adjacent exons contribute to the variations in splicing patterns among different PtLBD groups. Numerous transcription factor binding sites and cis-elements pertinent to hormone signaling pathways and stress response mechanisms were identified within the upstream promoter regions of the PtLBD genes. Thirty-five PtLBDs were found to be engaged in either tandem or segmental duplications, and genomic collinearity analysis revealed a stronger alignment between PtLBD genes and eudicots plants compared to their relationship with monocots. GO enrichment and temporal-spatio expression patterns showed that PtLBD7 from subfamily-I and PtLBD20 from subfamily-II, along with other 13 PtLBDs, were involved in plant growth and development biological processes. The multilayered hierarchical gene networks (ML-hGRN) mediated by PtLBD7 and PtLBD20 indicated that PtLBDs were mainly function in poplar growth and stress tolerance through a multifaceted and intricate regulatory machinery. This study lays a solid groundwork for delving deeper into the roles and underlying mechanisms of LBD transcription factors in poplar, specifically those related to plant hormones and stress tolerance.

VvLBD39, a grape LBD transcription factor, regulates plant response to salt and drought stress

Grape (Vitis vinifera L.), as an important deciduous perennial fruit tree, constantly confronts various abiotic stresses such as salinity and drought. The lateral organ boundaries domain (LBD) proteins are a class of plant-specific transcription factors that play pivotal roles in regulating plant growth and responding to abiotic stress. However, the biological function of the LBD transcription factor in grape remains poorly understood. Here, we cloned and characterized the VvLBD39 gene from grape, which contained a highly conserved LBD domain and localized to the cell nucleus. qRT-PCR analyses showed that the expression of VvLBD39 was downregulated upon exposure to NaCl, polyethylene glycol 6000 (PEG6000) and abscisic acid (ABA) treatments, respectively. Overexpression of VvLBD39 in grape calli and Arabidopsis resulted in hypersensitivity to PEG6000 and NaCl stress. Moreover, VvLBD39-overexpressing transgenic tobacco exhibited decreased tolerance to drought and salt stress, as well as insensitivity to exogenous ABA. After drought and salt stress treatments, the chlorophyll content, root length and antioxidant enzyme activity of the transgenic tobacco were lower than those of the wild-type (WT). Conversely, malonic dialdehyde (MDA) content, electronic conductivity, hydrogen peroxide (H2O2) content and superoxide anion (O2-) productivity were markedly elevated in the transgenic tobacco compared to the WT. Further investigations found that VvLBD39 had a negative impact on stomatal closure, ABA biosynthesis and ABA signaling under drought and salt treatments. In addition, the expression of genes related to reactive oxygen species (ROS) scavenging and stress response were significantly downregulated in VvLBD39 transgenic tobacco. Taken together, these results indicated that VvLBD39 functions as a negative regulator of salt and drought tolerance, making it a promising target for drought and salt resistance breeding.

Regulatory mechanisms of the LBD40 transcription factor in Arabidopsis thaliana somatic embryogenesis

AbstractSomatic embryogenesis (SE) is a process by which an embryo is derived from somatic tissue. Transcription factors (TFs) have been identified that control this process. One such TF that promotes SE is AGAMOUS‐like 15 (AGL15). Prior work has shown that AGL15 can both induce and repress gene expression. One way this type of dual function TF works is via protein interactions, so a yeast 2‐hybrid (Y2H) screen was undertaken. One intriguing protein with which AGL15 interacted in Y2H was LBD40. LBD40 encodes a LATERAL ORGAN BOUNDARIES (LOB)‐domain TF that is unique to plants and is primarily expressed during seed development. Here, we confirm the AGL15‐LBD40 interaction by quantitative assays and in planta co‐immunoprecipation. We also document a role for LBD40, and the closely related protein LBD41, in supporting SE. To determine downstream genes potentially controlled by LBD40, chromatin immunoprecipitation followed by high throughput sequencing (ChIP‐seq) was used. More than 400 binding regions for LBD40 were consistently found genome‐wide. To determine genes responsive to LBD40/41 accumulation, RNA‐seq analysis of transcriptomes of wild‐type control and loss‐of‐function lbd40/lbd41 was performed. Combining these datasets provides insight into genes directly and indirectly controlled by these LOB domain TFs. The gene ontology (GO) enrichment analysis of these regulated genes showed an overrepresentation of biological processes that are associated with SE, further indicating the importance of LBD40 in SE. This work provides insight into SE, a poorly understood, but essential process to generate transgenic plants to meet agricultural demands or test gene function. This manuscript reports on experiments to understand the role that LDB40, a TF, plays in support of SE by investigating genes directly and indirectly controlled by LBD40 and examining physical and genetic interactions with other TFs active in SE. We uncover targets of LBD40 and an interacting TF of the MADS family and investigate targets involvement in SE.

Genome-wide characterization of LBD transcription factors in switchgrass (Panicum virgatum L.) and the involvement of PvLBD12 in salt tolerance.

PvLBD12 enhanced the salt tolerance by increasing proline accumulation, improving K+ accumulation, and decreasing reactive oxygen species level in switchgrass. Abiotic stresses are the serious factors which limit plant development and productivity and restrict the agricultural economy. It is important, therefore, to understand the mechanism of abiotic tolerance in plants. Lateral organ boundaries domain (LBD) proteins as plant-specific transcription factors play important function in plant lateral organ development, plant regeneration, and abiotic stress. In our study, we identify 69 LBD members from switchgrass genome-wide sequences and classify them based on their homology with LBD proteins in Arabidopsis. RT-qPCR showed that PvLBD genes had different expression patterns under abiotic stress conditions, indicating that they play important roles in various stress. PvLBD12 was selected as a candidate gene for further functional analysis because it had the highest expression level under salt stress. Overexpression of PvLBD12 enhanced salt tolerance by altering a wide range of physiological responses (like increased proline accumulation, reduced malondialdehyde production, improved K+ accumulation, and reduced Na+ absorption) in switchgrass. Some stress response genes such as proline biosynthesis gene PvP5CS1, vacuolar Na+(K+)/H+ antiporter gene PvNHX1, two key ROS-scavenging enzyme genes PvCAT and PvSOD were all upregulated in PvLBD12 overexpression lines. Taken together, PvLBD12 plays a pivotal role in response to salt stress by increasing proline accumulation, improving K+ accumulation, reducing Na+ absorption, and decreasing reactive oxygen species level. It will be better to understand the potential biological functions of LBD genes in other plants.

PheLBD29, an LBD transcription factor from Moso bamboo, causes leaf curvature and enhances tolerance to drought stress in transgenic Arabidopsis

The lateral organ boundaries domain (LBD), a unique family of transcription factors in higher plants, plays a key role in plant growth and development, and stress response. Here, we report on the novel lateral organ boundaries domain (LBD) gene PheLBD29, a nuclear localization protein that can bind the conserved GCCCCG sequence, as determined by electrophoretic mobility shift assay (EMSA). PheLBD29 was highly expressed in blade leaf and significantly induced by polyethylene glycol (PEG). Overexpression of PheLBD29 leads to small and abaxially rolled leaves in Arabidopsis, and anatomically, 35S:PheLBD29 Arabidopsis leaves showed transformation of adaxial cells into abaxial cells. Moreover, overexpression of PheLBD29 in Arabidopsis increased plant tolerance to drought stress, by accumulation of more soluble sugars, less malondialdehyde (MDA), and had lower REL levels under drought stress. Transient expression assay revealed PheLBD29 directly bound to the promoter region of RAB18. In addition, 35S:PheLBD29 Arabidopsis showed higher sensitivity to abscisic acid (ABA) than the wild type. Therefore, we conclude that PheLBD29 may participate in the ABA-dependent signaling pathway to improve drought tolerance. Our study provides new evidence for a Moso bamboo LBD protein regulatory module in leaf curvature and drought resistance.

Genome-Wide Identification of the Ginkgo (Ginkgo biloba L.) LBD Transcription Factor Gene and Characterization of Its Expression.

Lateral organ boundaries domain (LBD) proteins are plant-specific transcription factors involved in various transcriptional regulation processes. We identified a total of 37 GbLBD genes in ginkgo, and based on gene structure and phylogenetic analysis, the GbLBD gene family was classified into class I (33, with the largest number of Id genes (16)) and class II (4). The ginkgo LBD gene was also analyzed regarding its chromosomal distributions, gene duplications, promoters, and introns/exons. In addition, gene expression profiling and real-time quantitative PCR analysis showed that the expression of 14 GbLBD genes differed in six different tissues and three developmental stages. The GbLBD gene of class II were highly expressed relative to the class I gene in all tissues and developmental stages, while class Id gene were generally at low levels or were not expressed, especially in seed developmental stages. The expression pattern analysis of cold/drought treatment and IAA/ABA hormone treatment showed that abiotic stress treatment could significantly induce the expression of GbLBD gene, of which class II genes played a key role in stress treatment. Our study provides a solid foundation for further evolutionary and functional analysis of the ginkgo LBD gene family.

Transcription factor PagLBD21 functions as a repressor of secondary xylem development in <i>Populus</i>

During secondary growth in trees, xylem cells differentiated from cambium cells begin to synthesize secondary cell walls that are primarily composed of cellulose, hemicellulose and lignin, and are deposited between primary cell walls and plasma membranes, leading to in wood formation. Identification of regulatory genes functioning during this developmental process is valuable for increasing wood production. In this study, we functionally characterized an LBD (LATERAL ORGAN BOUNDARIES DOMAIN) transcription factor PagLBD21 as a repressor of secondary xylem development in <italic>Populus</italic>. Compared to wild type plants, transgenic plants overexpressing <italic>PagLBD21</italic> (<italic>PagLBD21OE</italic>) exhibited decreased xylem widths in cross-sections. Consistent with the functional analysis, RNA sequencing (RNA-seq) analysis revealed that genes functioning in xylem development and secondary cell wall biosynthesis pathways were significantly down-regulated in <italic>PagLBD21OE</italic> plants. We also performed DNA affinity purification followed by sequencing (DAP-seq) to identify genome-wide target genes of PagLBD21. Furthermore, we compared the RNA-seq and DAP-seq datasets of PagLBD21 and PagLBD3, and the results showed that there was a significant overlap between their target genes, suggesting these two LBD transcription factors are functionally redundant during secondary growth.

Genome-Wide Identification, Evolution, and Expression Analysis of LBD Transcription Factor Family in Bread Wheat (Triticum aestivum L.).

The lateral organ boundaries domain (LBD) genes, as the plant-specific transcription factor family, play a crucial role in controlling plant architecture and stress tolerance. Although it has been thoroughly characterized in many species, the LBD family was not well studied in wheat. Here, the wheat LBD family was systematically investigated through an in silico genome-wide search method. A total of 90 wheat LBD genes (TaLBDs) were identified, which were classified into class I containing seven subfamilies, and class II containing two subfamilies. Exon–intron structure, conserved protein motif, and cis-regulatory elements analysis showed that the members in the same subfamily shared similar gene structure organizations, supporting the classification. Furthermore, the expression patterns of these TaLBDs in different types of tissues and under diverse stresses were identified through public RNA-seq data analysis, and the regulation networks of TaLBDs involved were predicted. Finally, the expression levels of 12 TaLBDs were validated by quantitative PCR (qPCR) analysis and the homoeologous genes showed differential expression. Additionally, the genetic diversity of TaLBDs in the landrace population showed slightly higher than that of the genetically improved germplasm population while obvious asymmetry at the subgenome level. This study not only provided the potential targets for further functional analysis but also contributed to better understand the roles of LBD genes in regulating development and stress tolerance in wheat and beyond.

Genome-wide identification and expression analysis of LBD transcription factor genes in Moso bamboo (Phyllostachys edulis)

BackgroundMoso bamboo, the fastest growing plant on earth, is an important source for income in large areas of Asia, mainly cultivated in China. Lateral organ boundaries domain (LBD) proteins, a family of transcription factors unique to plants, are involved in multiple transcriptional regulatory pathways and play important roles in lateral organ development, pathogen response, secondary growth, and hormone response. The LBD gene family has not previously been characterized in moso bamboo (Phyllostachys edulis).ResultsIn this study, we identified 55 members of the LBD gene family from moso bamboo and found that they were distributed non-uniformly across its 18 chromosomes. Phylogenetic analysis showed that the moso bamboo LBD genes could be divided into two classes. LBDs from the same class share relatively conserved gene structures and sequences encoding similar amino acids. A large number of hormone response–associated cis-regulatory elements were identified in the LBD upstream promoter sequences. Synteny analysis indicated that LBDs in the moso bamboo genome showed greater collinearity with those of O. sativa (rice) and Zea mays (maize) than with those of Arabidopsis and Capsicum annuum (pepper). Numerous segmental duplicates were found in the moso bamboo LBD gene family. Gene expression profiles in four tissues showed that the LBD genes had different spatial expression patterns. qRT–PCR assays with the Short Time-series Expression Miner (STEM) temporal expression analysis demonstrated that six genes (PeLBD20, PeLBD29, PeLBD46, PeLBD10, PeLBD38, and PeLBD06) were consistently up-regulated during the rapid growth and development of bamboo shoots. In addition, 248 candidate target genes that function in a variety of pathways were identified based on consensus LBD binding motifs.ConclusionsIn the current study, we identified 55 members of the moso bamboo transcription factor LBD and characterized for the first time. Based on the short-time sequence expression software and RNA-seq data, the PeLBD gene expression was analyzed. We also investigated the functional annotation of all PeLBDs, including PPI network, GO, and KEGG enrichment based on String database. These results provide a theoretical basis and candidate genes for studying the molecular breeding mechanism of rapid growth of moso bamboo.

Molecular and in silico characterization of tomato LBD transcription factors reveals their role in fruit development and stress responses

LATERAL ORGAN BOUNDARIES DOMAIN (LBD) proteins represent a family of plant specific transcription factors associated with roles in plant organ development, pollen development, regeneration, disease susceptibility, photomorphogenesis, secondary growth and metabolism. LBD gene family has been studied in Arabidopsis thaliana, Glycine max, Medicago truncatula, Oryza sativa and Zea mays; however, there is no detail information about expression pattern and roles of LBD genes in tomato. In present study, a total of 47 SlLBD genes from tomato were in silico characterized by conserved motif analysis, cis-regulatory element analysis and interaction network analysis. Motif analysis of SlLBD proteins revealed close evolutionary relationship or similar motif compositions. Cis-regulatory elements analysis of promoters of SlLBD genes suggested their diverse functions associated with hormone and stress response, tissue specific expression, metabolite biosynthesis and light response. The interaction network analysis of SlLBD proteins suggests its interaction with ARF, GRAS, WRKY, MADS, MYB CONSTANS TFs as well as with other SlLBD members viz. SlLBD41-SlLBD3-SlLBD36 and SlLBD7-SlLBD19-SlLBD14. The combinatorial interaction between SlLBDs and other TFs is responsible to play role in regulating diverse functions in tomato. Further quantitative RT-PCR expression analysis of a total of 33 SlLBD genes in different vegetative and fruit tissues revealed spatio-temporal expression in different plant parts and fruit developing stages. Two of the SlLBD genes SlLBD20 and SlLBD47 were found exclusively expressing in fruit, suggesting their role in fruit development and ripening. Expression of SlLBD genes under different hormone treatments and stress (abiotic & biotic) conditions were analyzed using TomExpress database, suggested their role in defense and hormonal regulation.

Genome-wide analysis of LBD(lateral organ boundaries domain) gene family in Cannabis sativa of traditional Chinese medicine hemp seed

LBD(lateral organ boundaries)transcription factors play an important role in the regulation of plant growth, development and secondary metabolism. In order to explore the function of LBD genes in cannabis, the Cannabis sativa genome and transcriptome were used to identify the C. sativa LBD gene family, and analyzed their expression patterns. Our results showed that the cannabis LBD contains 32 members, which were divided into two major categories, seven sub-families. Class Ⅰ was divided into 5 sub-families, named Class Ⅰ_a to Class Ⅰ_e, while Class Ⅱ was divided into 2 sub-families, including Class Ⅱ_a and Class Ⅱ_b. Analysis showed that the number of amino acids encoded LBDs was between 172 and 356, and the isoelectric point was between 4.92 and 9.43. The mole-cular weight of LBD was between 18 862.92 Da and 40 081.33 Da, and most members are located in the nucleus. Chromosome positioning of LBD showed that 32 members were unevenly distributed on 10 chromosomes of C. sativa LBD transcription factor domain, gene structure and motifs are relatively conservative, and the characteristics of different class members are similar. The upstream promoter region of the gene contains a variety of cis-acting elements related to plant hormones and environmental factors, C. sativa LBD genes have different expression patterns in the stems, leaves, and flowers of ZYS varieties(low tetrahydrocannabinol, high cannabidiol). The members of the LBD gene family are mainly expressed in the flowers and stems of ZYS varieties, while members expressed in the leaves very few; Class Ⅱ members CsLBD21 and CsLBD23 are expressed in flowers and stems, and CsLBD8 and CsLBD18 are expressed in flowers, stems and leaves. These genes may participate in the growth and development of cannabis and affect the biosynthesis of cannabinoids. This study laid the foundation for the subsequently functional research of the cannabis LBD gene family.

Genome-wide characterisation and expression profiling of the LBD family in Salvia miltiorrhiza reveals the function of LBD50 in jasmonate signaling and phenolic biosynthesis

Jasmonates (JAs) regulate growth, development, and secondary metabolism in many economically significant crops. The lateral organ boundaries domain (LBD) protein family is a class of plant-specific transcription factors involved in the regulation of lateral organ development and jasmonate signaling. However, the functions of LBD genes in Salvia miltiorrhiza Bunge, an emerging model medicinal plant, are unclear. In this study, a total of 51 LBD transcription factor genes were identified in the genome of S. miltiorrhiza, and were classified by phylogenetic analysis into seven subfamilies. The expression profile revealed that 10 of the LBD genes are tissue-specific and JA-inducible. Of the 51 LBD transcription factor genes, 11 % (six out of 51) were potentially involved in the biosynthesis of tanshinones or phenolic acids. Correlation analysis showed that the expression of SmLBD9, SmLBD13, SmLBD21, and SmLBD50 were observably regulated in JA zim domain (JAZ) proteins transgenic S. miltiorrhiza, indicating their critical roles in the JA signaling pathway. Further functional analysis indicated that the overexpression of SmLBD50 inhibited the synthesis of total phenolics, total flavonoids, and anthocyanins in S. miltiorrhiza. Consistent with these phytochemical changes, the expression of phenolic biosynthetic genes was also stimulated. Yeast two-hybrid (Y2H) assays indicated that the SmLBD50 protein interacts with SmJAZ1, SmMYB36/97, SmbHLH37 and SmMYC2a/2b, four important transcription factors involved in JA signaling. In conclusion, our study off ;ers the first genome-wide overview and annotation of the LBD transcription factor family in S. miltiorrhiza and provides a foundation for understanding the molecular basis and regulatory mechanisms of SmLBD50 in relation to the JA response and phenolic metabolism in S. miltiorrhiza.

Massive up-regulation of LBD transcription factors and EXPANSINs highlights the regulatory programs of rhizomania disease.

Rhizomania of sugar beet, caused by Beet necrotic yellow vein virus (BNYVV), is characterized by excessive lateral root (LR) formation leading to dramatic reduction of taproot weight and massive yield losses. LR formation represents a developmental process tightly controlled by auxin signaling through AUX/IAA-ARF responsive module and LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcriptional network. Several LBD transcription factors play central roles in auxin-regulated LR development and act upstream of EXPANSINS (EXPs), cell wall (CW)-loosening proteins involved in plant development via disruption of the extracellular matrix for CW relaxation and expansion. Here, we present evidence that BNYVV hijacks these auxin-regulated pathways resulting in formation LR and root hairs (RH). We identified an AUX/IAA protein (BvAUX28) as interacting with P25, a viral virulence factor. Mutational analysis indicated that P25 interacts with domains I and II of BvAUX28. Subcellular localization of co-expressed P25 and BvAUX28 showed that P25 inhibits BvAUX28 nuclear localization. Moreover, root-specific LBDs and EXPs were greatly upregulated during rhizomania development. Based on these data, we present a model in which BNYVV P25 protein mimics action of auxin by removing BvAUX28 transcriptional repressor, leading to activation of LBDs and EXPs. Thus, the evidence highlights two pathways operating in parallel and leading to uncontrolled formation of LRs and RHs, the main manifestation of the rhizomania syndrome.

The LATERAL ORGAN BOUNDARIES Domain gene family in grapevine: genome-wide characterization and expression analyses during developmental processes and stress responses

LATERAL ORGAN BOUNDARIES (LOB) DOMAIN (LBD) constitute a family of plant-specific transcription factors with key roles in the regulation of plant organ development, pollen development, plant regeneration, pathogen response, and anthocyanin and nitrogen metabolisms. However, the role of LBDs in fruit ripening and in grapevine (Vitis vinifera L.) development and stress responses is poorly documented. By performing a model curation of LBDs in the latest genome annotation 50 genes were identified. Phylogenetic analysis showed that LBD genes can be grouped into two classes mapping on 16 out of the 19 V. vinifera chromosomes. New gene subclasses were identified that have not been characterized in other species. Segmental and tandem duplications contributed significantly to the expansion and evolution of the LBD gene family in grapevine as noticed for other species. The analysis of cis-regulatory elements and transcription factor binding sites in the VviLBD promoter regions suggests the involvement of several hormones in the regulation of LBDs expression. Expression profiling suggest the involvement of LBD transcription factors in grapevine development, berry ripening and stress responses. Altogether this study provides valuable information and robust candidate genes for future functional analysis aiming to clarify mechanisms responsible for the onset of fruit ripening and fruit defense strategies.

Primitive Auxin Response without TIR1 and Aux/IAA in the Charophyte Alga Klebsormidium nitens.

The phytohormone auxin regulates many aspects of growth and development in land plants, but the origin and evolution of auxin signaling and response mechanisms remain largely unknown. Indeed, it remains to be investigated whether auxin-related pathways diverged before the emergence of land plants. To address this knowledge deficit, we analyzed auxin responses in the charophyte alga Klebsormidium nitens NIES-2285, whose ancestor diverged from a green algal ancestor during the evolution of land plants. This strain is the same as Klebsormidium flaccidum NIES-2285, for which the draft genome was sequenced in 2014, and was taxonomically reclassified as K. nitens This genome sequence revealed genes involved in auxin responses. Furthermore, the auxin indole-3-acetic acid (IAA) was detected in cultures of K. nitens, but K. nitens lacks the central regulators of the canonical auxin-signaling pathway found in land plants. Exogenous IAA inhibited cell division and cell elongation in K. nitens Inhibitors of auxin biosynthesis and of polar auxin transport also inhibited cell division and elongation. Moreover, exogenous IAA rapidly induced expression of a LATERAL ORGAN BOUNDARIES-DOMAIN transcription factor. These results suggest that K. nitens has acquired the part of the auxin system that regulates transcription and cell growth without the requirement for the central players that govern auxin signaling in land plants.

Characterization of the LBD gene family in Brachypodium: a phylogenetic and transcriptional study.

An unambiguous nomenclature is proposed for the twenty-eight-member LOB domain transcription factor family in Brachypodium . Expression analysis provides unique transcript patterns that are characteristic of a wide range of organs and plant parts. LOB (lateral organ boundaries)-domain proteins define a family of plant-specific transcription factors involved in developmental processes from embryogenesis to seed production. They play a crucial role in shaping the plant architecture through coordinating cell fate at meristem to organ boundaries. Despite their high potential importance, our knowledge of them is limited, especially in the case of monocots. In this study, we characterized LOB domain protein coding genes (LBDs) of Brachypodium distachyon, a model plant for grasses, and present their phylogenetic relationships and an overall spatial expression study. In the Brachypodium genome database, 28 LBDs were found and then classified based on the presence of highly conserved LOB domain motif. Their transcript amounts were measured via quantitative real-time RT-PCR in 37 different plant parts from root tip to generative organs. Comprehensive phylogenetic analysis suggests that there are neither Brachypodium- nor monocot-specific lineages among LBDs, but there are differences in terms of complexity of subclasses between monocots and dicots. Although LBDs in Brachypodium have wide variation of tissue-specific expression and relative transcript levels, overall expression patterns show similarity to their counterparts in other species. The varying transcript profiles we observed support the hypothesis that Brachypodium LBDs have diverse but conserved functions in plant organogenesis.

Expression and Protein Interaction Analyses Reveal Combinatorial Interactions of LBD Transcription Factors During Arabidopsis Pollen Development.

LATERAL ORGAN BOUNDARIES DOMAIN (LBD) transcription factor gene family members play key roles in diverse aspects of plant development. LBD10 and LBD27 have been shown to be essential for pollen development in Arabidopsis thaliana. From the previous RNA sequencing (RNA-Seq) data set of Arabidopsis pollen, we identified the mRNAs of LBD22, LBD25 and LBD36 in addition to LBD10 and LBD27 in Arabidopsis pollen. Here we conducted expression and cellular analysis using GFP:GUS (green fluorescent protein:β-glucuronidase) reporter gene and subcellular localization assays using LBD:GFP fusion proteins expressed under the control of their own promoters in Arabidopsis. We found that these LBD proteins display spatially and temporally distinct and overlapping expression patterns during pollen development. Bimolecular fluorescence complementation and GST (glutathione S-transferase) pull-down assays demonstrated that protein-protein interactions occur among the LBDs exhibiting overlapping expression during pollen development. We further showed that LBD10, LBD22, LBD25, LBD27 and LBD36 interact with each other to form heterodimers, which are localized to the nucleus in Arabidopsis protoplasts. Taken together, these results suggest that combinatorial interactions among LBD proteins may be important for their function in pollen development in Arabidopsis.

Identification and expression of Aux/IAA, ARF, and LBD family transcription factors in Boehmeria nivea

Auxin controls numerous processes in plant development and auxin/indoleacetic acid (Aux/IAA), an auxin response factor (ARF), and a lateral organ boundaries domain (LBD) were considered as early auxin response transcription factors (TFs). Till now, no Aux/IAA, ARF, and LBD TFs were identified in ramie (Boehmeria nivea L. Gaud). In this study, we used Arabidopsis and mulberry sequences as query to search against the ramie transcriptome database and the searched sequences were analyzed for a full-length coding sequence. In total, we obtained 16 BnAux/IAA, 14 BnARF, and 16 BnLBD TFs on which evolutionary analysis and expression profiling were conducted. Analysis of sequence conservation revealed close evolution relationships between ramie and mulberry. Expression analysis shows these genes were actively expressed in major ramie tissues, and several were auxin responsive. The expressions of these genes were also investigated under drought and a high temperature, main abiotic stresses during ramie life cycle. We found that most genes of the three families were stress-responsive and showed distinct expression patterns under the drought and high temperature stresses.

Chrysanthemum transcription factor CmLBD1 direct lateral root formation in Arabidopsis thaliana.

The plant-specific LATERAL ORGAN BOUNDARIES DOMAIN (LBD) genes are important regulators of growth and development. Here, a chrysanthemum class I LBD transcription factor gene, designated CmLBD1, was isolated and its function verified. CmLBD1 was transcribed in both the root and stem, but not in the leaf. The gene responded to auxin and was shown to participate in the process of adventitious root primordium formation. Its heterologous expression in Arabidopsis thaliana increased the number of lateral roots formed. When provided with exogenous auxin, lateral root emergence was promoted. CmLBD1 expression also favored callus formation from A. thaliana root explants in the absence of exogenously supplied phytohormones. In planta, CmLBD1 probably acts as a positive regulator of the response to auxin fluctuations and connects auxin signaling with lateral root formation.

LATERAL ORGAN BOUNDARIES DOMAIN transcription factors direct callus formation in Arabidopsis regeneration

The remarkable regeneration capability of plant tissues or organs under culture conditions has underlain an extensive practice for decades. The initial step in plant in vitro regeneration often involves the induction of a pluripotent cell mass termed callus, which is driven by the phytohormone auxin and occurs via a root development pathway. However, the key molecules governing callus formation remain unknown. Here we demonstrate that Arabidopsis LATERAL ORGAN BOUNDARIES DOMAIN (LBD)/ASYMMETRIC LEAVES2-LIKE (ASL) transcription factors are involved in the control of callus formation program. The four LBD genes downstream of AUXIN RESPONSE FACTORs (ARFs), LBD16, LBD17, LBD18 and LBD29, are rapidly and dramatically induced by callus-inducing medium (CIM) in multiple organs. Ectopic expression of each of the four LBD genes in Arabidopsis is sufficient to trigger spontaneous callus formation without exogenous phytohormones, whereas suppression of LBD function inhibits the callus formation induced by CIM. Moreover, the callus triggered by LBD resembles that induced by CIM by characteristics of ectopically activated root meristem genes and efficient regeneration capacity. These findings define LBD transcription factors as key regulators in the callus induction process, thereby establishing a molecular link between auxin signaling and the plant regeneration program.