Spatiotemporally distinct responses to mechanical forces shape the developing seed of Arabidopsis.

Organ morphogenesis depends on mechanical interactions between cells and tissues. These interactions generate forces that can be sensed by cells and affect key cellular processes. However, how mechanical forces, together with biochemical signals, contribute to the shaping of complex organs is still largely unclear. We address this question using the seed of Arabidopsis as a model system. We show that seeds first experience a phase of rapid anisotropic growth that is dependent on the response of cortical microtubule (CMT) to forces, which guide cellulose deposition according to shape-driven stresses in the outermost layer of the seed coat. However, at later stages of development, we show that seed growth is isotropic and depends on the properties of an inner layer of the seed coat that stiffens its walls in response to tension but has isotropic material properties. Finally, we show that the transition from anisotropic to isotropic growth is due to the dampening of cortical microtubule responses to shape-driven stresses. Altogether, our work supports a model in which spatiotemporally distinct mechanical responses control the shape of developing seeds in Arabidopsis.

Minimizing IP issues associated with gene constructs encoding the Bt toxin - a case study.

BACKGROUND: As part of a publicly funded initiative to develop genetically engineered Brassicas (cabbage, cauliflower, and canola) expressing Bacillus thuringiensis Crystal (Cry)-encoded insecticidal (Bt) toxin for Indian and Australian farmers, we designed several constructs that drive high-level expression of modified Cry1B and Cry1C genes (referred to as Cry1BM and Cry1CM; with M indicating modified). The two main motivations for modifying the DNA sequences of these genes were to minimise any licensing cost associated with the commercial cultivation of transgenic crop plants expressing CryM genes, and to remove or alter sequences that might adversely affect their activity in plants. RESULTS: To assess the insecticidal efficacy of the Cry1BM/Cry1CM genes, constructs were introduced into the model Brassica Arabidopsis thaliana in which Cry1BM/Cry1CM expression was directed from either single (S4/S7) or double (S4S4/S7S7) subterranean clover stunt virus (SCSV) promoters. The resulting transgenic plants displayed a high-level of Cry1BM/Cry1CM expression. Protein accumulation for Cry1CM ranged from 5.18 to 176.88 µg Cry1CM/g dry weight of leaves. Contrary to previous work on stunt promoters, we found no correlation between the use of either single or double stunt promoters and the expression levels of Cry1BM/Cry1CM genes, with a similar range of Cry1CM transcript abundance and protein content observed from both constructs. First instar Diamondback moth (Plutella xylostella) larvae fed on transgenic Arabidopsis leaves expressing the Cry1BM/Cry1CM genes showed 100% mortality, with a mean leaf damage score on a scale of zero to five of 0.125 for transgenic leaves and 4.2 for wild-type leaves. CONCLUSIONS: Our work indicates that the modified Cry1 genes are suitable for the development of insect resistant GM crops. Except for the PAT gene in the USA, our assessment of the intellectual property landscape of components presents within the constructs described here suggest that they can be used without the need for further licensing. This has the capacity to significantly reduce the cost of developing and using these Cry1M genes in GM crop plants in the future.

Trees need closure too: Wound-induced secondary vascular tissue regeneration.

Trees play a pivotal role in terrestrial ecosystems as well as being an important natural resource. These attributes are primarily associated with the capacity of trees to continuously produce woody tissue from the vascular cambium, a ring of stem cells located just beneath the bark. Long-lived trees are exposed to a myriad of biological and environmental stresses that may result in wounding, leading to a loss of bark and the underlying vascular cambium. This affects both wood formation and the quality of timber arising from the tree. In addition, the exposed wound site is a potential entry point for pathogens that cause disease. In response to wounding, trees have the capacity to regenerate lost or damaged tissues at this site. Investigating gene expression changes associated with different stages of wound healing reveals complex and dynamic changes in the activity of transcription factors, signalling pathways and hormone responses. In this review we summarise these data and discuss how they relate to our current understanding of vascular cambium formation and xylem differentiation during secondary growth. Based on this analysis, a model for wound healing that provides the conceptual foundations for future studies aimed at understanding this intriguing process is proposed.

Precision genome editing in plants using gene targeting and prime editing: existing and emerging strategies.

Precise modification of plant genomes, such as seamless insertion, deletion, or replacement of DNA sequences at a predefined site, is a challenging task. Gene targeting (GT) and prime editing are currently the best approaches for this purpose. However, these techniques are inefficient in plants, which limits their applications for crop breeding programs. Recently, substantial developments have been made to improve the efficiency of these techniques in plants. Several strategies, such as RNA donor templating, chemically modified donor DNA template, and tandem-repeat homology-directed repair, are aimed at improving GT. Additionally, improved prime editing gRNA design, use of engineered reverse transcriptase enzymes, and splitting prime editing components have improved the efficacy of prime editing in plants. These emerging strategies and existing technologies are reviewed along with various perspectives on their future improvement and the development of robust precision genome editing technologies for plants.

Overexpressing Arabidopsis thaliana ACBP6 in transgenic rapid-cycling Brassica napus confers cold tolerance.

BACKGROUND: Rapid-cycling Brassica napus (B. napus-RC) has potential as a rapid trait testing system for canola (B. napus) because its life cycle is completed within 2 months while canola usually takes 4 months, and it is susceptible to the same range of diseases and abiotic stress as canola. However, a rapid trait testing system for canola requires the development of an efficient transformation and tissue culture system for B. napus-RC. Furthermore, effectiveness of this system needs to be demonstrated by showing that a particular trait can be rapidly introduced into B. napus-RC plants. RESULTS: An in-vitro regeneration protocol was developed for B. napus-RC using 4-day-old cotyledons as the explant. High regeneration percentages, exceeding 70%, were achieved when 1-naphthaleneacetic acid (0.10 mg/L), 6-benzylaminopurine (1.0 mg/L), gibberellic acid (0.01 mg/L) and the ethylene antagonist silver nitrate (5 mg/L) were included in the regeneration medium. An average transformation efficiency of 16.4% was obtained using Agrobacterium-mediated transformation of B. napus-RC cotyledons using Agrobacterium strain GV3101 harbouring a plasmid with an NPTII (kanamycin-selectable) marker gene and the Arabidopsis thaliana cDNA encoding ACYL-COA-BINDING PROTEIN6 (AtACBP6). Transgenic B. napus-RC overexpressing AtACBP6 displayed better tolerance to freezing/frost than the wild type, with enhanced recovery from cellular membrane damage at both vegetative and flowering stages. AtACBP6-overexpressing B. napus-RC plants also exhibited lower electrolyte leakage and improved recovery following frost treatment, resulting in higher yields than the wild type. Ovules from transgenic AtACBP6 lines were better protected from frost than those of the wild type, while the developing embryos of frost-treated AtACBP6-overexpressing plants showed less freezing injury than the wild type. CONCLUSIONS: This study demonstrates that B. napus-RC can be successfully regenerated and transformed from cotyledon explants and has the potential to be an effective trait testing platform for canola. Additionally, AtACBP6 shows potential for enhancing cold tolerance in canola however, larger scale studies will be required to further confirm this outcome.

Combining GAL4 GFP enhancer trap with split luciferase to measure spatiotemporal promoter activity in Arabidopsis.

In multicellular organisms different types of tissues have distinct gene expression profiles associated with specific function or structure of the cell. Quantification of gene expression in whole organs or whole organisms can give misleading information about levels or dynamics of expression in specific cell types. Tissue- or cell-specific analysis of gene expression has potential to enhance our understanding of gene regulation and interactions of cell signalling networks. The Arabidopsis circadian oscillator is a gene network which orchestrates rhythmic expression across the day/night cycle. There is heterogeneity between cell and tissue types of the composition and behaviour of the oscillator. In order to better understand the spatial and temporal patterns of gene expression, flexible tools are required. By combining a Gateway®-compatible split luciferase construct with a GAL4 GFP enhancer trap system, we describe a tissue-specific split luciferase assay for non-invasive detection of spatiotemporal gene expression in Arabidopsis. We demonstrate the utility of this enhancer trap-compatible split luciferase assay (ETSLA) system to investigate tissue-specific dynamics of circadian gene expression. We confirm spatial heterogeneity of circadian gene expression in Arabidopsis leaves and describe the resources available to investigate any gene of interest.

Extracellular endonucleases in the midgut of Myzus persicae may limit the efficacy of orally delivered RNAi.

Myzus persicae is a major pest of many crops including canola and Brassica vegetables, partly because it vectors plant viruses. Previously it has been reported that double-stranded RNA delivered to aphids by injection, artificial diet or transgenic plants has knocked down target genes and caused phenotypic effects. While these studies suggest that RNA interference (RNAi) might be used to suppress aphid populations, none have shown effects sufficient for field control. The current study analyses the efficacy of dsRNA directed against previously reported gene-targets on Green peach aphid (Myzus persicae) strains. No silencing effect was observed when dsRNA was delivered in artificial diet with or without transfection reagents. dsRNA produced in planta also failed to induce significant RNAi in M. persicae. Transcriptome analyses of the midgut suggested other potential targets including the Ferritin heavy chain transcripts, but they also could not be knocked down with dsRNA. Here we show that dsRNA is rapidly degraded by midgut secretions of Myzus persicae. Analysis of the transcriptome of the M. persicae midgut revealed that an ortholog of RNases from other insects was abundant.

Control of leaf blade outgrowth and floral organ development by LEUNIG, ANGUSTIFOLIA3 and WOX transcriptional regulators.

Plant lateral organ development is a complex process involving both transcriptional activation and repression mechanisms. The WOX transcriptional repressor WOX1/STF, the LEUNIG (LUG) transcriptional corepressor and the ANGUSTIFOLIA3 (AN3) transcriptional coactivator play important roles in leaf blade outgrowth and flower development, but how these factors coordinate their activities remains unclear. Here we report physical and genetic interactions among these key regulators of leaf and flower development. We developed a novel in planta transcriptional activation/repression assay and suggest that LUG could function as a transcriptional coactivator during leaf blade development. MtLUG physically interacts with MtAN3, and this interaction appears to be required for leaf and flower development. A single amino acid substitution at position 61 in the SNH domain of MtAN3 protein abolishes its interaction with MtLUG, and its transactivation activity and biological function. Mutations in lug and an3 enhanced each other's mutant phenotypes. Both the lug and the an3 mutations enhanced the wox1 prs leaf and flower phenotypes in Arabidopsis. Our findings together suggest that transcriptional repression and activation mediated by the WOX, LUG and AN3 regulators function in concert to promote leaf and flower development, providing novel mechanistic insights into the complex regulation of plant lateral organ development.

Layers of regulation - Insights into the role of transcription factors controlling mucilage production in the Arabidopsis seed coat.

A polysaccharide-rich mucilage is released from the seed coat epidermis of numerous plant species and has been intensively studied in the model plant Arabidopsis. This has led to the identification of a large number of genes involved in the synthesis, secretion and modification of cell wall polysaccharides such as pectin, hemicellulose and cellulose being identified. These genes include a small network of transcription factors (TFs) and transcriptional co-regulators, that not only regulate mucilage production, but epidermal cell differentiation and in some cases flavonoid biosynthesis in the internal endothelial layer of the seed coat. Here we focus on the function of these regulators and propose a simplified model where they are assigned to a hierarchical gene network with three regulatory levels (tiers) as a means of assisting in the interpretation of the complexity. We discuss limitations of current methodologies and highlight some of the problems associated with defining the function of TFs, particularly those that perform different functions in adjacent layers of the seed coat. We suggest approaches that should provide a more accurate picture of the function of transcription factors involved with mucilage production and release.

Class C ARFs evolved before the origin of land plants and antagonize differentiation and developmental transitions in Marchantia polymorpha.

A plethora of developmental and physiological processes in land plants is influenced by auxin, to a large extent via alterations in gene expression by AUXIN RESPONSE FACTORs (ARFs). The canonical auxin transcriptional response system is a land plant innovation, however, charophycean algae possess orthologues of at least some classes of ARF and AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) genes, suggesting that elements of the canonical land plant system existed in an ancestral alga. We reconstructed the phylogenetic relationships between streptophyte ARF and AUX/IAA genes and functionally characterized the solitary class C ARF, MpARF3, in Marchantia polymorpha. Phylogenetic analyses indicate that multiple ARF classes, including class C ARFs, existed in an ancestral alga. Loss- and gain-of-function MpARF3 alleles result in pleiotropic effects in the gametophyte, with MpARF3 inhibiting differentiation and developmental transitions in multiple stages of the life cycle. Although loss-of-function Mparf3 and Mpmir160 alleles respond to exogenous auxin treatments, strong miR-resistant MpARF3 alleles are auxin-insensitive, suggesting that class C ARFs act in a context-dependent fashion. We conclude that two modules independently evolved to regulate a pre-existing ARF transcriptional network. Whereas the auxin-TIR1-AUX/IAA pathway evolved to repress class A/B ARF activity, miR160 evolved to repress class C ARFs in a dynamic fashion.

LEUNIG_HOMOLOG transcriptional co-repressor mediates aluminium sensitivity through PECTIN METHYLESTERASE46-modulated root cell wall pectin methylesterification in Arabidopsis.

A major factor determining aluminium (Al) sensitivity in higher plants is the binding of Al to root cell walls. The Al binding capacity of cell walls is closely linked to the extent of pectin methylesterification, as the presence of methyl groups attached to the pectin backbone reduces the net negative charge of this polymer and hence limits Al binding. Despite recent progress in understanding the molecular basis of Al resistance in a wide range of plants, it is not well understood how the methylation status of pectin is mediated in response to Al stress. Here we show in Arabidopsis that mutants lacking the gene LEUNIG_HOMOLOG (LUH), a member of the Groucho-like family of transcriptional co-repressor, are less sensitive to Al-mediated repression of root growth. This phenotype is correlated with increased levels of methylated pectin in the cell walls of luh roots as well as altered expression of cell wall-related genes. Among the LUH-repressed genes, PECTIN METHYLESTERASE46 (PME46) was identified as reducing Al binding to cell walls and hence alleviating Al-induced root growth inhibition by decreasing PME enzyme activity. seuss-like2 (slk2) mutants responded to Al in a similar way as luh mutants suggesting that a LUH-SLK2 complex represses the expression of PME46. The data are integrated into a model in which it is proposed that PME46 is a major inhibitor of pectin methylesterase activity within root cell walls.

Arabidopsis DEFECTIVE KERNEL1 regulates cell wall composition and axial growth in the inflorescence stem.

Axial growth in plant stems requires a fine balance between elongation and stem mechanical reinforcement to ensure mechanical stability. Strength is provided by the plant cell wall, the deposition of which must be coordinated with cell expansion and elongation to ensure that integrity is maintained during growth. Coordination of these processes is critical and yet poorly understood. The plant-specific calpain, DEFECTIVE KERNEL1 (DEK1), plays a key role in growth coordination in leaves, yet its role in regulating stem growth has not been addressed. Using plants overexpressing the active CALPAIN domain of DEK1 (CALPAIN OE) and a DEK1 knockdown line (amiRNA-DEK1), we undertook morphological, biochemical, biophysical, and microscopic analyses of mature inflorescence stems. We identify a novel role for DEK1 in the maintenance of cell wall integrity and coordination of growth during inflorescence stem development. CALPAIN OE plants are significantly reduced in stature and have short, thickened stems, while amiRNA-DEK1 lines have weakened stems that are unable to stand upright. Microscopic analyses of the stems identify changes in cell size, shape and number, and differences in both primary and secondary cell wall thickness and composition. Taken together, our results suggest that DEK1 influences primary wall growth by indirectly regulating cellulose and pectin deposition. In addition, we observe changes in secondary cell walls that may compensate for altered primary cell wall composition. We propose that DEK1 activity is required for the coordination of stem strengthening with elongation during axial growth.

Ascorbate-Deficient vtc2 Mutants in Arabidopsis Do Not Exhibit Decreased Growth.

In higher plants the L-galactose pathway represents the major route for ascorbate biosynthesis. The first committed step of this pathway is catalyzed by the enzyme GDP-L-galactose phosphorylase and is encoded by two paralogs in Arabidopsis - VITAMIN C2 (VTC2) and VTC5. The first mutant of this enzyme, vtc2-1, isolated via an EMS mutagenesis screen, has approximately 20-30% of wildtype ascorbate levels and has been reported to have decreased growth under standard laboratory conditions. Here, we show that a T-DNA insertion into the VTC2 causes a similar reduction in ascorbate levels, but does not greatly affect plant growth. Subsequent segregation analysis revealed the growth defects of vtc2-1 mutants segregate independently of the vtc2-1 mutation. These observations suggest that it is the presence of an independent cryptic mutation that affects growth of vtc2-1 mutants, and not the 70-80% decrease in ascorbate levels that has been assumed in past studies.

A Glycosyltransferase from Nicotiana alata Pollen Mediates Synthesis of a Linear (1,5)-α-L-Arabinan When Expressed in Arabidopsis.

The walls of Nicotiana alata pollen tubes contain a linear arabinan composed of (1,5)-α-linked arabinofuranose residues. Although generally found as a side chain on the backbone of the pectic polysaccharide rhamnogalacturonan I, the arabinan in N. alata pollen tubes is considered free, as there is no detectable rhamnogalacturonan I in these walls. Carbohydrate-specific antibodies detected arabinan epitopes at the tip and along the shank of N. alata pollen tubes that are predominantly part of the primary layer of the bilayered wall. A sequence related to ARABINAN DEFICIENT1 (AtARAD1), a presumed arabinan arabinosyltransferase from Arabidopsis (Arabidopsis thaliana), was identified by searching an N alata pollen transcriptome. Transcripts for this ARAD1-like sequence, which we have named N. alata ARABINAN DEFICIENT-LIKE1 (NaARADL1), accumulate in various tissues, most abundantly in the pollen grain and tube, and encode a protein that is a type II membrane protein with its catalytic carboxyl terminus located in the Golgi lumen. The NaARADL1 protein can form homodimers when transiently expressed in Nicotiana benthamiana leaves and heterodimers when coexpressed with AtARAD1 The expression of NaARADL1 in Arabidopsis led to plants with more arabinan in their walls and that also exuded a guttation fluid rich in arabinan. Chemical and enzymatic characterization of the guttation fluid showed that a soluble, linear α-(1,5)-arabinan was the most abundant polymer present. These results are consistent with NaARADL1 having an arabinan (1,5)-α-arabinosyltransferase activity.

FILAMENTOUS FLOWER Is a Direct Target of JAZ3 and Modulates Responses to Jasmonate.

The plant hormone jasmonate (JA) plays an important role in regulating growth, development, and immunity. Activation of the JA-signaling pathway is based on the hormone-triggered ubiquitination and removal of transcriptional repressors (JASMONATE-ZIM DOMAIN [JAZ] proteins) by an SCF receptor complex (SCF(COI1)/JAZ). This removal allows the rapid activation of transcription factors (TFs) triggering a multitude of downstream responses. Identification of TFs bound by the JAZ proteins is essential to better understand how the JA-signaling pathway modulates and integrates different responses. In this study, we found that the JAZ3 repressor physically interacts with the YABBY (YAB) family transcription factor FILAMENTOUS FLOWER (FIL)/YAB1. In Arabidopsis thaliana, FIL regulates developmental processes such as axial patterning and growth of lateral organs, shoot apical meristem activity, and inflorescence phyllotaxy. Phenotypic analysis of JA-regulated responses in loss- and gain-of-function FIL lines suggested that YABs function as transcriptional activators of JA-triggered responses. Moreover, we show that MYB75, a component of the WD-repeat/bHLH/MYB complex regulating anthocyanin production, is a direct transcriptional target of FIL. We propose that JAZ3 interacts with YABs to attenuate their transcriptional function. Upon perception of JA signal, degradation of JAZ3 by the SCF(COI1) complex releases YABs to activate a subset of JA-regulated genes in leaves leading to anthocyanin accumulation, chlorophyll loss, and reduced bacterial defense.

SEUSS and SEUSS-LIKE 2 coordinate auxin distribution and KNOXI activity during embryogenesis.

In Arabidopsis, SEUSS (SEU) and SEUSS-LIKE 2 (SLK2) are components of the LEUNIG (LUG) repressor complex that coordinates various aspects of post-embryonic development. The complex also plays a critical role during embryogenesis, as seu slk2 double mutants have small, narrow cotyledons and lack a shoot apical meristem (SAM). Here we show that seu slk2 double mutant embryos exhibit delayed cotyledon outgrowth and that this is associated with altered PIN-FORMED1 (PIN1) expression and localisation during the early stages of embryogenesis. These observations suggest that SEU and SLK2 promote the transition to bilateral symmetry by modulating auxin distribution in the embryonic shoot. This study also shows that loss of SAM formation in seu slk2 mutants is associated with reduced expression of the class I KNOX (KNOXI) genes SHOOTMERISTEMLESS (STM), BREVIPEDICELLUS and KNAT2. Furthermore, elevating STM expression in seu slk2 mutant embryos was sufficient to restore SAM formation but not post-embryonic activity, while both SAM formation and activity were rescued when SLK2 expression was restored in either the cotyledons or boundary regions. These results demonstrate that SEU and SLK2 function redundantly to promote embryonic shoot development and likely act through a non-cell autonomous pathway to promote KNOXI activity.

FILAMENTOUS FLOWER controls lateral organ development by acting as both an activator and a repressor.

BACKGROUND: The YABBY (YAB) family of transcription factors participate in a diverse range of processes that include leaf and floral patterning, organ growth, and the control of shoot apical meristem organisation and activity. How these disparate functions are regulated is not clear, but based on interactions with the LEUNIG-class of co-repressors, it has been proposed that YABs act as transcriptional repressors. In the light of recent work showing that DNA-binding proteins associated with the yeast co-repressor TUP1 can also function as activators, we have examined the transcriptional activity of the YABs. RESULTS: Of the four Arabidopsis YABs tested in yeast, only FILAMENTOUS FLOWER (FIL) activated reporter gene expression. Similar analysis with Antirrhinum YABs identified the FIL ortholog GRAMINIFOLIA as an activator. Plant-based transactivation assays not only confirmed the potential of FIL to activate transcription, but also extended this property to the FIL paralog YABBY3 (YAB3). Subsequent transcriptomic analysis of lines expressing a steroid-inducible FIL protein revealed groups of genes that responded either positively or negatively to YAB induction. Included in the positively regulated group of genes were the polarity regulators KANADI1 (KAN1), AUXIN RESPONSE FACTOR 4 (ARF4) and ASYMMETRIC LEAVES1 (AS1). We also show that modifying FIL to function as an obligate repressor causes strong yab loss-of-function phenotypes. CONCLUSIONS: Collectively these data show that FIL functions as a transcriptional activator in plants and that this activity is involved in leaf patterning. Interestingly, our study also supports the idea that FIL can act as a repressor, as transcriptomic analysis identified negatively regulated FIL-response genes. To reconcile these observations, we propose that YABs are bifunctional transcription factors that participate in both positive and negative regulation. These findings fit a model of leaf development in which adaxial/abaxial patterning is maintained by a regulatory network consisting of positive feedback loops.

Diverse roles of Groucho/Tup1 co-repressors in plant growth and development.

Transcriptional regulation involves coordinated and often complex interactions between activators and repressors that together dictate the temporal and spatial activity of target genes. While the study of developmental regulation has often focused on positively acting transcription factors, it is becoming increasingly clear that transcriptional repression is a key regulatory mechanism underpinning many developmental processes in both plants and animals. In this review, we focus on the plant Groucho (Gro)/Tup1-like co-repressors and discuss their roles in establishing the apical-basal axis of the developing embryo, maintaining the stem cell population in the shoot apex and determining floral organ identity.  As well as being developmental regulators, recent studies have shown that these co-repressors play a central role in regulating auxin and jasmonate signalling pathways and are also linked to the regulation of pectin structure in the seed coat. These latest findings point to the Gro/Tup1-like co-repressors playing a much broad role in plant growth and development than previously thought; an observation that underlines the central importance of transcriptional repression in plant gene regulation.

The transcriptional regulator LEUNIG_HOMOLOG regulates mucilage release from the Arabidopsis testa.

Exposure of the mature Arabidopsis (Arabidopsis thaliana) seed to water results in the rapid release of pectinaceous mucilage from the outer cells of the testa. Once released, mucilage completely envelops the seed in a gel-like capsule. The physical force required to rupture the outer cell wall of the testa comes from the swelling of the mucilage as it expands rapidly following hydration. In this study, we show that mutations in the transcriptional regulator LEUNIG_HOMOLOG (LUH) cause a mucilage extrusion defect due to altered mucilage swelling. Based on sugar linkage and immunomicroscopic analyses, we show that the structure of luh mucilage is altered, having both an increase in substituted rhamnogalacturonan I and in methyl-esterified homogalacturonan. Also correlated with the structural modification of luh mucilage is a significant decrease in MUCILAGE MODIFIED2 (MUM2; a ß-galactosidase) expression in the luh seed coat, raising the possibility that reduced activity of this glycosidase is directly responsible for the luh mucilage defects. Consistent with this is the structural similarity between mum2 and luh mucilage as well as the observation that elevating MUM2 expression in luh mutants completely suppresses the mucilage extrusion defect. Suppression of the luh mutant phenotype was also observed when LEUNIG, a transcriptional corepressor closely related to LUH, was introduced in luh mutants under the control of the LUH promoter. Based on these data, we propose a new model for the regulation of pectin biosynthesis during plant growth and development.

YABBYs and the transcriptional corepressors LEUNIG and LEUNIG_HOMOLOG maintain leaf polarity and meristem activity in Arabidopsis.

In Arabidopsis thaliana, FILAMENTOUS FLOWER (FIL) and YABBY3 (YAB3) encode YABBY domain proteins that regulate abaxial patterning, growth of lateral organs, and inflorescence phyllotaxy. In this study, we show that YABs physically interact with components of a transcriptional repressor complex that include LEUNIG (LUG), LEUNIG_HOMOLOG (LUH), the LUG-associated coregulator SEUSS, and related SEUSS-LIKE proteins. Consistent with the formation of a LUG-YAB complex, we find that lug mutants enhance the polarity and growth defects of fil yab3 mutant leaves and that this enhancement is due to a loss of LUG activity from the abaxial domain. We performed a more extensive genetic analysis, which included the characterization of yab triple and quadruple mutants, lug luh/+ (heterozygous only for luh) mutants, and plants expressing artificial microRNAs targeting LUG or LUH. These analyses showed that the LUG-YAB complex also promotes adaxial cell identity in leaves as well as embryonic shoot apical meristem (SAM) initiation and postembryonic SAM maintenance. Based on the likely formation of the LUG-YAB complex in the abaxial domain of cotyledons and leaves, we propose that this complex has numerous non-cell-autonomous functions during plant development.