KNOTTED1-like Homeobox Research Articles

The Arabidopsis JAGGED LATERAL ORGANS (JLO) gene sensitizes plants to auxin.

Plant growth and development of new organs depend on the continuous activity of the meristems. In the shoot, patterns of organ initiation are determined by PINFORMED (PIN)-dependent auxin distribution, while the undifferentiated state of meristem cells requires activity of KNOTTED LIKE HOMEOBOX (KNOX) transcription factors. Cell proliferation and differentiation of the root meristem are regulated by the largely antagonistic functions of auxin and cytokinins. It has previously been shown that the transcription factor JAGGED LATERAL ORGANS (JLO), a member of the LATERAL ORGAN BOUNDARY DOMAIN (LBD) family, coordinates KNOX and PIN expression in the shoot and promotes root meristem growth. Here we show that JLO is required for the establishment of the root stem cell niche, where it interacts with the auxin/PLETHORA pathway. Auxin signaling involves the AUX/IAA co-repressor proteins, ARF transcription factors and F-box receptors of the TIR1/AFB1-5 family. Because jlo mutants fail to degrade the AUX/IAA protein BODENLOS, root meristem development is inhibited. We also demonstrate that the expression levels of two auxin receptors, TIR1 and AFB1, are controlled by JLO dosage, and that the shoot and root defects of jlo mutants are alleviated in jlo plants expressing TIR1 and AFB1 from a transgene. The finding that the auxin sensitivity of a plant can be differentially regulated through control of auxin receptor expression can explain how different developmental processes can be integrated by the activity of a key transcription factor.

KNOTTED1 Cofactors, BLH12 and BLH14, Regulate Internode Patterning and Vein Anastomosis in Maize.

Monocot stems lack the vascular cambium and instead have characteristic structures in which intercalary meristems generate internodes and veins remain separate and scattered. However, developmental processes of these unique structures have been poorly described. BELL1-like homeobox (BLH) transcription factors (TFs) are known to heterodimerize with KNOTTED1-like homeobox TFs to play crucial roles in shoot meristem maintenance, but their functions are elusive in monocots. We found that maize (Zea mays) BLH12 and BLH14 have redundant but important roles in stem development. BLH12/14 interact with KNOTTED1 (KN1) in vivo and accumulate in overlapping domains in shoot meristems, young stems, and provascular bundles. Similar to kn1 loss-of-function mutants, blh12 blh14 (blh12/14) double mutants fail to maintain axillary meristems. Unique to blh12/14 is an abnormal tassel branching and precocious internode differentiation that results in dwarfism and reduced veins in stems. Micro-computed tomography observation of vascular networks revealed that blh12/14 double mutants had reduced vein number due to fewer intermediate veins in leaves and precocious anastomosis in young stems. Based on these results, we propose two functions of BLH12/14 during stem development: (1) maintaining intercalary meristems that accumulate KN1 and prevent precocious internode differentiation and (2) preventing precocious anastomosis of provascular bundles in young stems to ensure the production of sufficient independent veins.

Comparative Transcriptome Analysis between Gynoecious and Monoecious Plants Identifies Regulatory Networks Controlling Sex Determination in Jatropha curcas.

Most germplasms of the biofuel plant Jatropha curcas are monoecious. A gynoecious genotype of J. curcas was found, whose male flowers are aborted at early stage of inflorescence development. To investigate the regulatory mechanism of transition from monoecious to gynoecious plants, a comparative transcriptome analysis between gynoecious and monoecious inflorescences were performed. A total of 3,749 genes differentially expressed in two developmental stages of inflorescences were identified. Among them, 32 genes were involved in floral development, and 70 in phytohormone biosynthesis and signaling pathways. Six genes homologous to KNOTTED1-LIKE HOMEOBOX GENE 6 (KNAT6), MYC2, SHI-RELATED SEQUENCE 5 (SRS5), SHORT VEGETATIVE PHASE (SVP), TERMINAL FLOWER 1 (TFL1), and TASSELSEED2 (TS2), which control floral development, were considered as candidate regulators that may be involved in sex differentiation in J. curcas. Abscisic acid, auxin, gibberellin, and jasmonate biosynthesis were lower, whereas cytokinin biosynthesis was higher in gynoecious than that in monoecious inflorescences. Moreover, the exogenous application of gibberellic acid (GA3) promoted perianth development in male flowers and partly prevented pistil development in female flowers to generate neutral flowers in gynoecious inflorescences. The arrest of stamen primordium at early development stage probably causes the abortion of male flowers to generate gynoecious individuals. These results suggest that some floral development genes and phytohormone signaling pathways orchestrate the process of sex determination in J. curcas. Our study provides a basic framework for the regulation networks of sex determination in J. curcas and will be helpful for elucidating the evolution of the plant reproductive system.

Screening of Candidate Leaf Morphology Genes by Integration of QTL Mapping and RNA Sequencing Technologies in Oilseed Rape (Brassica napus L.).

Leaf size and shape play important roles in agronomic traits, such as yield, quality and stress responses. Wide variations in leaf morphological traits exist in cultivated varieties of many plant species. By now, the genetics of leaf shape and size have not been characterized in Brassica napus. In this study, a population of 172 recombinant inbred lines (RILs) was used for quantitative trait locus (QTL) analysis of leaf morphology traits. Furthermore, fresh young leaves of extreme lines with more leaf lobes (referred to as ‘A’) and extreme lines with fewer lobes (referred to as ‘B’) selected from the RIL population and leaves of dissected lines (referred to as ‘P’) were used for transcriptional analysis. A total of 31 QTLs for the leaf morphological traits tested in this study were identified on 12 chromosomes, explaining 5.32–39.34% of the phenotypic variation. There were 8, 6, 2, 5, 8, and 2 QTLs for PL (petiole length), PN (lobe number), LW (lamina width), LL (Lamina length), LL/LTL (the lamina size ratio) and LTL (leaf total length), respectively. In addition, 74, 1,166 and 1,272 differentially expressed genes (DEGs) were identified in ‘A vs B’, ‘A vs P’ and ‘B vs P’ comparisons, respectively. The Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to predict the functions of these DEGs. Gene regulators of leaf shape and size, such as ASYMMETRIC LEAVES 2, gibberellin 20-oxidase 3, genes encoding gibberellin-regulated family protein, genes encoding growth-regulating factor and KNOTTED1-like homeobox were also detected in DEGs. After integrating the QTL mapping and RNA sequencing data, 33 genes, including a gene encoding auxin-responsive GH3 family protein and a gene encoding sphere organelles protein-related gene, were selected as candidates that may control leaf shape. Our findings should be valuable for studies of the genetic control of leaf morphological trait regulation in B. napus.

Nonreciprocal complementation of KNOX gene function in land plants.

Summary Class I KNOTTED‐LIKE HOMEOBOX (KNOX) proteins regulate development of the multicellular diploid sporophyte in both mosses and flowering plants; however, the morphological context in which they function differs.In order to determine how Class I KNOX function was modified as land plants evolved, phylogenetic analyses and cross‐species complementation assays were performed.Our data reveal that a duplication within the charophyte sister group to land plants led to distinct Class I and Class II KNOX gene families. Subsequently, Class I sequences diverged substantially in the nonvascular bryophyte groups (liverworts, mosses and hornworts), with moss sequences being most similar to those in vascular plants. Despite this similarity, moss mutants were not complemented by vascular plant KNOX genes. Conversely, the Arabidopsis brevipedicellus (bp‐9) mutant was complemented by the PpMKN2 gene from the moss Physcomitrella patens. Lycophyte KNOX genes also complemented bp‐9 whereas fern genes only partially complemented the mutant. This lycophyte/fern distinction is mirrored in the phylogeny of KNOX‐interacting BELL proteins, in that a gene duplication occurred after divergence of the two groups.Together, our results imply that the moss MKN2 protein can function in a broader developmental context than vascular plant KNOX proteins, the narrower scope having evolved progressively as lycophytes, ferns and flowering plants diverged.

Ectopic KNOX Expression Affects Plant Development by Altering Tissue Cell Polarity and Identity.

Plant development involves two polarity types: tissue cell (asymmetries within cells are coordinated across tissues) and regional (identities vary spatially across tissues) polarity. Both appear altered in the barley (Hordeum vulgare) Hooded mutant, in which ectopic expression of the KNOTTED1-like Homeobox (KNOX) gene, BKn3, causes inverted polarity of differentiated hairs and ectopic flowers, in addition to wing-shaped outgrowths. These lemma-specific effects allow the spatiotemporal analysis of events following ectopic BKn3 expression, determining the relationship between KNOXs, polarity, and shape. We show that tissue cell polarity, based on localization of the auxin transporter SISTER OF PINFORMED1 (SoPIN1), dynamically reorients as ectopic BKn3 expression increases. Concurrently, ectopic expression of the auxin importer LIKE AUX1 and boundary gene NO APICAL MERISTEM is activated. The polarity of hairs reflects SoPIN1 patterns, suggesting that tissue cell polarity underpins oriented cell differentiation. Wing cell files reveal an anisotropic growth pattern, and computational modeling shows how polarity guiding growth can account for this pattern and wing emergence. The inverted ectopic flower orientation does not correlate with SoPIN1, suggesting that this form of regional polarity is not controlled by tissue cell polarity. Overall, the results suggest that KNOXs trigger different morphogenetic effects through interplay between tissue cell polarity, identity, and growth.

Transcription factors AS1 and AS2 interact with LHP1 to repress KNOX genes in Arabidopsis.

Polycomb group proteins are important repressors of numerous genes in higher eukaryotes. However, the mechanism by which Polycomb group proteins are recruited to specific genes is poorly understood. In Arabidopsis, LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), also known as TERMINAL FLOWER 2, was originally proposed as a subunit of polycomb repressive complex 1 (PRC1) that could bind the tri-methylated lysine 27 of histone H3 (H3K27me3) established by the PRC2. In this work, we show that LHP1 mainly functions with PRC2 to establish H3K27me3, but not with PRC1 to catalyze monoubiquitination at lysine 119 of histone H2A. Our results show that complexes of the transcription factors ASYMMETRIC LEAVES 1 (AS1) and AS2 could help to establish the H3K27me3 modification at the chromatin regions of Class-I KNOTTED1-like homeobox (KNOX) genes BREVIPEDICELLUS and KNAT2 via direct interactions with LHP1. Additionally, our transcriptome analysis indicated that there are probably more common target genes of AS1 and LHP1 besides Class-I KNOX genes during leaf development in Arabidopsis.

Protocorms and Protocorm-Like Bodies Are Molecularly Distinct from Zygotic Embryonic Tissues in Phalaenopsis aphrodite.

The distinct reproductive program of orchids provides a unique evolutionary model with pollination-triggered ovule development and megasporogenesis, a modified embryogenesis program resulting in seeds with immature embryos, and mycorrhiza-induced seed germination. However, the molecular mechanisms that have evolved to establish these unparalleled developmental programs are largely unclear. Here, we conducted comparative studies of genome-wide gene expression of various reproductive tissues and captured the molecular events associated with distinct reproductive programs in Phalaenopsis aphrodite Importantly, our data provide evidence to demonstrate that protocorm-like body (PLB) regeneration (the clonal regeneration practice used in the orchid industry) does not follow the embryogenesis program. Instead, we propose that SHOOT MERISTEMLESS, a class I KNOTTED-LIKE HOMEOBOX gene, is likely to play a role in PLB regeneration. Our studies challenge the current understanding of the embryonic identity of PLBs. Taken together, the data obtained establish a fundamental framework for orchid reproductive development and provide a valuable new resource to enable the prediction of gene regulatory networks that is required for specialized developmental programs of orchid species.

A class II KNOX gene, KNOX4, controls seed physical dormancy

Physical dormancy of seed is an adaptive trait that widely exists in higher plants. This kind of dormancy is caused by a water-impermeable layer that blocks water and oxygen from the surrounding environment and keeps embryos in a viable status for a long time. Most of the work on hardseededness has focused on morphological structure and phenolic content of seed coat. The molecular mechanism underlying physical dormancy remains largely elusive. By screening a large number of Tnt1 retrotransposon-tagged Medicago truncatula lines, we identified nondormant seed mutants from this model legume species. Unlike wild-type hard seeds exhibiting physical dormancy, the mature mutant seeds imbibed water quickly and germinated easily, without the need for scarification. Microscopic observations of cross sections showed that the mutant phenotype was caused by a dysfunctional palisade cuticle layer in the seed coat. Chemical analysis found differences in lipid monomer composition between the wild-type and mutant seed coats. Genetic and molecular analyses revealed that a class II KNOTTED-like homeobox (KNOXII) gene, KNOX4, was responsible for the loss of physical dormancy in the seeds of the mutants. Microarray and chromatin immunoprecipitation analyses identified CYP86A, a gene associated with cutin biosynthesis, as one of the downstream target genes of KNOX4 This study elucidated a novel molecular mechanism of physical dormancy and revealed a new role of class II KNOX genes. Furthermore, KNOX4-like genes exist widely in seed plants but are lacking in nonseed species, indicating that KNOX4 may have diverged from the other KNOXII genes during the evolution of seed plants.

Regulation, overexpression, and target gene identification ofPotato Homeobox 15(POTH15) – a class-IKNOXgene in potato

Potato Homeobox 15 (POTH15) is a KNOX-I (Knotted1-like homeobox) family gene in potato that is orthologous to Shoot Meristemless (STM) in Arabidopsis. Despite numerous reports on KNOX genes from different species, studies in potato are limited. Here, we describe photoperiodic regulation of POTH15, its overexpression phenotype, and identification of its potential targets in potato (Solanum tuberosum ssp. andigena). qRT-PCR analysis showed a higher abundance of POTH15 mRNA in shoot tips and stolons under tuber-inducing short-day conditions. POTH15 promoter activity was detected in apical and axillary meristems, stolon tips, tuber eyes, and meristems of tuber sprouts, indicating its role in meristem maintenance and leaf development. POTH15 overexpression altered multiple morphological traits including leaf and stem development, leaflet number, and number of nodes and branches. In particular, the rachis of the leaf was completely reduced and leaves appeared as a bouquet of leaflets. Comparative transcriptomic analysis of 35S::GUS and two POTH15 overexpression lines identified more than 6000 differentially expressed genes, including 2014 common genes between the two overexpression lines. Functional analysis of these genes revealed their involvement in responses to hormones, biotic/abiotic stresses, transcription regulation, and signal transduction. qRT-PCR of selected candidate target genes validated their differential expression in both overexpression lines. Out of 200 randomly chosen POTH15 targets, 173 were found to have at least one tandem TGAC core motif, characteristic of KNOX interaction, within 3.0kb in the upstream sequence of the transcription start site. Overall, this study provides insights to the role of POTH15 in controlling diverse developmental processes in potato.

Evolution, diversification, and expression of KNOX proteins in plants.

The KNOX (KNOTTED1-like homeobox) transcription factors play a pivotal role in leaf and meristem development. The majority of these proteins are characterized by the KNOX1, KNOX2, ELK, and homeobox domains whereas the proteins of the KNATM family contain only the KNOX domains. We carried out an extensive inventory of these proteins and here report on a total of 394 KNOX proteins from 48 species. The land plant proteins fall into two classes (I and II) as previously shown where the class I family seems to be most closely related to the green algae homologs. The KNATM proteins are restricted to Eudicots and some species have multiple paralogs of this protein. Certain plants are characterized by a significant increase in the number of KNOX paralogs; one example is Glycine max. Through the analysis of public gene expression data we show that the class II proteins of this plant have a relatively broad expression specificity as compared to class I proteins, consistent with previous studies of other plants. In G. max, class I protein are mainly distributed in axis tissues and KNATM paralogs are overall poorly expressed; highest expression is in the early plumular axis. Overall, analysis of gene expression in G. max demonstrates clearly that the expansion in gene number is associated with functional diversification.

Light-induced indeterminacy alters shade avoiding tomato leaf morphology

Plants sense the foliar shade of competitors and alter their developmental programs through the shade-avoidance response. Internode and petiole elongation, and changes in overall leaf area and leaf mass per area, are the stereotypical architectural responses to foliar shade in the shoot. However, changes in leaf shape and complexity in response to shade remain incompletely, and qualitatively, described. Using a meta-analysis of more than 18,000 previously published leaflet outlines, we demonstrate that shade avoidance alters leaf shape in domesticated tomato (Solanum lycopersicum) and wild relatives. The effects of shade avoidance on leaf shape are subtle with respect to individual traits but are combinatorially strong. We then seek to describe the developmental origins of shade-induced changes in leaf shape by swapping plants between light treatments. Leaf size is light responsive late into development, but patterning events, such as stomatal index, are irrevocably specified earlier. Observing that shade induces increases in shoot apical meristem size, we then describe gene expression changes in early leaf primordia and the meristem using laser microdissection. We find that in leaf primordia, shade avoidance is not mediated through canonical pathways described in mature organs but rather through the expression of KNOTTED1-LIKE HOMEOBOX and other indeterminacy genes, altering known developmental pathways responsible for patterning leaf shape. We also demonstrate that shade-induced changes in leaf primordium gene expression largely do not overlap with those found in successively initiated leaf primordia, providing evidence against classic hypotheses that shaded leaf morphology results from the prolonged production of juvenile leaf types.

Extended expression ofMaKN1contributes to the leaf morphology in aquatic forms ofMyriophyllum aquaticum

Myriophyllum aquaticum (Vell.) Verdc. is heterophyllous in nature with highly dissected simple leaves consisting of several lobes. KNOX (KNOTTED1-LIKE HOMEOBOX) genes are believed to have played an important role in the evolution of leaf diversity. Up-regulation of KNOX during leaf primordium initiation can lead to leaf dissection in plants with simple leaves and, if overexpressed, can produce ectopic meristems on leaves. A previous study on KNOX gene expression in the aerial form of this species showed that this gene is expressed in the shoot apical meristem (SAM), as well as in leaf primordia P0 to P8. Based on these results, it was hypothesized that the prolonged expression of the MaKN1 (Myriophyllum aquaticum Knotted1-like homeobox) gene beyond P8, might play an important role in the generation of more lobes, longer lobes, and hydathode formation in the aquatic leaves of M. aquaticum. The technique of in situ hybridization was carried out using a previously sequenced 300 bp fragment of MaKN1 to determine the expression patterns of this gene in the shoot of aquatic forms of the plant. Expression patterns of MaKN1 revealed that the SAM and leaf primordia of aquatic forms of M. aquaticum at levels P0 (youngest) to P4 were distributed throughout these structures. The level of expression of this MaKN1 gene progressively became more localized to lobes in older leaf primordia (levels P5 to P12). Previous studies of aerial forms of this plant showed MaKN1 expression until P8. Our results with aquatic forms show that the highly dissected leaf morphology in aquatic forms was the result of the prolonged expression of MaKN1 beyond P8. This resulted in the formation of elongated and slightly more numerous lobes, and hydathodes in aquatic forms. These findings support the view that KNOX genes are important developmental regulators of leaf morphogenesis and have played an important role in the evolution of leaf forms in the plant kingdom.

OsARID3, an AT-rich Interaction Domain-containing protein, is required for shoot meristem development in rice.

The shoot apical meristem (SAM) produces all of the plant's aerial organs. The SAM is established either during embryogenesis or experimentally in invitro tissue culture. Although several factors including the Class I KNOTTED1-LIKE HOMEOBOX (KNOXI) proteins, auxin, and cytokinin are known to play essential roles in SAM development, the underlying mechanisms of SAM formation and maintenance are still largely not understood. Herein we demonstrate that OsARID3, a member of the rice (Oryza sativa) AT-rich Interaction Domain (ARID) family, is required for SAM development. Disruption of OsARID3 leads to a defective SAM, early seedling lethality, and impaired capacity of invitro shoot regeneration. We show that the expression levels of several KNOXI genes and the biosynthetic genes for auxin and cytokinin are significantly altered in the Osarid3 mutant calli. Moreover, we determine that auxin concentrations are increased, whereas cytokinin levels are decreased, in Osarid3 calli. Furthermore, chromatin immunoprecipitation results demonstrate that OsARID3 binds directly to the KNOXI gene OSH71, the auxin biosynthetic genes OsYUC1 and OsYUC6, and the cytokinin biosynthetic genes OsIPT2 and OsIPT7. We also show through electrophoretic mobility shift assays that OsARID3 specifically binds to the AT-rich DNA sequences of the identified target genes. We conclude that OsARID3 is an AT-rich specific DNA-binding protein and that it plays a major role in SAM development in rice.

RNA Interference Knockdown of BRASSINOSTEROID INSENSITIVE1 in Maize Reveals Novel Functions for Brassinosteroid Signaling in Controlling Plant Architecture.

Brassinosteroids (BRs) are plant hormones involved in various growth and developmental processes. The BR signaling system is well established in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) but poorly understood in maize (Zea mays). BRASSINOSTEROID INSENSITIVE1 (BRI1) is a BR receptor, and database searches and additional genomic sequencing identified five maize homologs including duplicate copies of BRI1 itself. RNA interference (RNAi) using the extracellular coding region of a maize zmbri1 complementary DNA knocked down the expression of all five homologs. Decreased response to exogenously applied brassinolide and altered BR marker gene expression demonstrate that zmbri1-RNAi transgenic lines have compromised BR signaling. zmbri1-RNAi plants showed dwarf stature due to shortened internodes, with upper internodes most strongly affected. Leaves of zmbri1-RNAi plants are dark green, upright, and twisted, with decreased auricle formation. Kinematic analysis showed that decreased cell division and cell elongation both contributed to the shortened leaves. A BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1-yellow fluorescent protein (BES1-YFP) transgenic line was developed that showed BR-inducible BES1-YFP accumulation in the nucleus, which was decreased in zmbri1-RNAi. Expression of the BES1-YFP reporter was strong in the auricle region of developing leaves, suggesting that localized BR signaling is involved in promoting auricle development, consistent with the zmbri1-RNAi phenotype. The blade-sheath boundary disruption, shorter ligule, and disrupted auricle morphology of RNAi lines resemble KNOTTED1-LIKE HOMEOBOX (KNOX) mutants, consistent with a mechanistic connection between KNOX genes and BR signaling.

Beyond the meristems: similarities in the CLAVATA3 and INFLORESCENCE DEFICIENT IN ABSCISSION peptide mediated signalling pathways.

Plants form new organs throughout their lives; this requires a balance between cell proliferation and differentiation, and between the generation and loss of organs. To do this, plants must maintain a population of stem cells within the meristems, and at the same time, closely control the identity and position of cells at the meristem boundaries as they differentiate to new leaf or flower primordia. Once developed, organs may need to be shed, either as a controlled developmental decision-such as floral abscission after pollination, or as a response to disease, environmental stress, and predators. Cell wall degradation at specialized abscission zone (AZ) cells needs to occur for this to take place, but since there is little cell rearrangement in plants, cell separation events are also important for plant architecture. In this Opinion paper we discuss the role of two peptide ligand signalling systems that control stem cell homeostasis and cell separation, respectively. We draw parallels between the signalling pathways and explore on the commonalities of the downstream components activated and controlled by the signalling peptides. We provide evidence for AZ cells having a meristem identity and discuss the role of identical KNOTTED-LIKE HOMEOBOX (KNOX) transcription factors in meristem maintenance and abscission. Lastly we explore the evolutionary relationship between the pathways.

Arabidopsis BREVIPEDICELLUS interacts with the SWI2/SNF2 chromatin remodeling ATPase BRAHMA to regulate KNAT2 and KNAT6 expression in control of inflorescence architecture.

BREVIPEDICELLUS (BP or KNAT1), a class-I KNOTTED1-like homeobox (KNOX) transcription factor in Arabidopsis thaliana, contributes to shaping the normal inflorescence architecture through negatively regulating other two class-I KNOX genes, KNAT2 and KNAT6. However, the molecular mechanism of BP-mediated transcription regulation remains unclear. In this study, we showed that BP directly interacts with the SWI2/SNF2 chromatin remodeling ATPase BRAHMA (BRM) both in vitro and in vivo. Loss-of-function BRM mutants displayed inflorescence architecture defects, with clustered inflorescences, horizontally orientated pedicels, and short pedicels and internodes, a phenotype similar to the bp mutants. Furthermore, the transcript levels of KNAT2 and KNAT6 were elevated in brm-3, bp-9 and brm-3 bp-9 double mutants. Increased histone H3 lysine 4 tri-methylation (H3K4me3) levels were detected in brm-3, bp-9 and brm-3 bp-9 double mutants. Moreover, BRM and BP co-target to KNAT2 and KNAT6 genes, and BP is required for the binding of BRM to KNAT2 and KNAT6. Taken together, our results indicate that BP interacts with the chromatin remodeling factor BRM to regulate the expression of KNAT2 and KNAT6 in control of inflorescence architecture.

Antagonistic roles for KNOX1 and KNOX2 genes in patterning the land plant body plan following an ancient gene duplication.

Neofunctionalization following gene duplication is thought to be one of the key drivers in generating evolutionary novelty. A gene duplication in a common ancestor of land plants produced two classes of KNOTTED-like TALE homeobox genes, class I (KNOX1) and class II (KNOX2). KNOX1 genes are linked to tissue proliferation and maintenance of meristematic potentials of flowering plant and moss sporophytes, and modulation of KNOX1 activity is implicated in contributing to leaf shape diversity of flowering plants. While KNOX2 function has been shown to repress the gametophytic (haploid) developmental program during moss sporophyte (diploid) development, little is known about KNOX2 function in flowering plants, hindering syntheses regarding the relationship between two classes of KNOX genes in the context of land plant evolution. Arabidopsis plants harboring loss-of-function KNOX2 alleles exhibit impaired differentiation of all aerial organs and have highly complex leaves, phenocopying gain-of-function KNOX1 alleles. Conversely, gain-of-function KNOX2 alleles in conjunction with a presumptive heterodimeric BELL TALE homeobox partner suppressed SAM activity in Arabidopsis and reduced leaf complexity in the Arabidopsis relative Cardamine hirsuta, reminiscent of loss-of-function KNOX1 alleles. Little evidence was found indicative of epistasis or mutual repression between KNOX1 and KNOX2 genes. KNOX proteins heterodimerize with BELL TALE homeobox proteins to form functional complexes, and contrary to earlier reports based on in vitro and heterologous expression, we find high selectivity between KNOX and BELL partners in vivo. Thus, KNOX2 genes confer opposing activities rather than redundant roles with KNOX1 genes, and together they act to direct the development of all above-ground organs of the Arabidopsis sporophyte. We infer that following the KNOX1/KNOX2 gene duplication in an ancestor of land plants, neofunctionalization led to evolution of antagonistic biochemical activity thereby facilitating the evolution of more complex sporophyte transcriptional networks, providing plasticity for the morphological evolution of land plant body plans.

A KNOTTED1-LIKE HOMEOBOX protein regulates abscission in tomato by modulating the auxin pathway.

A gene encoding a KNOTTED1-LIKE HOMEOBOX PROTEIN1 (KD1) is highly expressed in both leaf and flower abscission zones. Reducing the abundance of transcripts of this gene in tomato (Solanum lycopersicum) by both virus-induced gene silencing and stable transformation with a silencing construct driven by an abscission-specific promoter resulted in a striking retardation of pedicel and petiole abscission. In contrast, Petroselinum, a semidominant KD1 mutant, showed accelerated pedicel and petiole abscission. Complementary DNA microarray and quantitative reverse transcription-polymerase chain reaction analysis indicated that regulation of abscission by KD1 was associated with changed abundance of genes related to auxin transporters and signaling components. Measurement of auxin content and activity of a DR5::β-glucuronidase auxin reporter assay showed that changes in KD1 expression modulated the auxin concentration and response gradient in the abscission zone.

Ectopic expression of the PttKN1 gene in Cardamine hirsuta mediated via the floral dip method

PttKN1 gene (Populus tremula × P. tremuloides KNOTTED1) was isolated from the vascular cambium of a hybrid aspen. Previous studies on transformed plants with the PttKN1 gene suggested that it plays roles in plant development (typically in meristem initiation), maintenance and organogenesis in simple-leaved species. To investigate the gene functions further, sequence analysis of the deduced amino acid was conducted. The results suggested that the gene belongs to the class I KNOX gene (KNOTTED1-like homeobox genes) family and might play important roles in plant development by coding a transcription factor. The gene was introduced into Cardamine hirsuta using the floral dip method mediated via Agrobacterium tumefaciens. The primary transformed plants were obtained via kanamycin selection. Compared to the wild type, the kanamycin resistant plants demonstrated several morphological alterations, such as abnormal cotyledons, abnormal shoot meristem, flattened stem, and lobed and cup-shaped leaves. RT-PCR results showed that the above five types of kanamycin resistant plants expressed the same specific PttKN1 gene band. This suggested that the morphological alterations were caused by the insertion and expression of the gene. However, these phenotypes were similar to other PttKN1 transformed plants, despite the fact that C. hirsuta is a species with compound leaves and the other species have simple leaves. Therefore, the functions of the PttKN1 gene on compound-leaf species have yet to be investigated via the comparison between related species such as Arabidopsis thaliana and C. hirsuta.