Endoplasmic reticulum membrane receptors of the GET pathway are conserved throughout eukaryotes.

Type II tail-anchored (TA) membrane proteins are involved in diverse cellular processes, including protein translocation, vesicle trafficking, and apoptosis. They are characterized by a single C-terminal transmembrane domain that mediates posttranslational targeting and insertion into the endoplasmic reticulum (ER) via the Guided-Entry of TA proteins (GET) pathway. The GET system was originally described in mammals and yeast but was recently shown to be partially conserved in other eukaryotes, such as higher plants. A newly synthesized TA protein is shielded from the cytosol by a pretargeting complex and an ATPase that delivers the protein to the ER, where membrane receptors (Get1/WRB and Get2/CAML) facilitate insertion. In the model plant Arabidopsis thaliana, most components of the pathway were identified through in silico sequence comparison, however, a functional homolog of the coreceptor Get2/CAML remained elusive. We performed immunoprecipitation-mass spectrometry analysis to detect in vivo interactors of AtGET1 and identified a membrane protein of unknown function with low sequence homology but high structural homology to both yeast Get2 and mammalian CAML. The protein localizes to the ER membrane, coexpresses with AtGET1, and binds to Arabidopsis GET pathway components. While loss-of-function lines phenocopy the stunted root hair phenotype of other Atget lines, its heterologous expression together with the coreceptor AtGET1 rescues growth defects of Δget1get2 yeast. Ectopic expression of the cytosolic, positively charged N terminus is sufficient to block TA protein insertion in vitro. Our results collectively confirm that we have identified a plant-specific GET2 in Arabidopsis, and its sequence allows the analysis of cross-kingdom pathway conservation.

Marchantia TCP transcription factor activity correlates with three-dimensional chromatin structure.

Information in the genome is not only encoded within sequence or epigenetic modifications, but is also found in how it folds in three-dimensional space. The formation of self-interacting genomic regions, named topologically associated domains (TADs), is known as a key feature of genome organization beyond the nucleosomal level. However, our understanding of the formation and function of TADs in plants is extremely limited. Here we show that the genome of Marchantia polymorpha, a member of a basal land plant lineage, exhibits TADs with epigenetic features similar to those of higher plants. By analysing various epigenetic marks across Marchantia TADs, we find that these regions generally represent interstitial heterochromatin and their borders are enriched with Marchantia transcription factor TCP1. We also identify a type of TAD that we name 'TCP1-rich TAD', in which genomic regions are highly accessible and are densely bound by TCP1 proteins. Transcription of TCP1 target genes differs on the basis gene location, and those in TCP1-rich TADs clearly show a lower expression level. In tcp1 mutant lines, neither TCP1-bound TAD borders nor TCP1-rich TADs display drastically altered chromatin organization patterns, suggesting that, in Marchantia, TCP1 is dispensable for TAD formation. However, we find that in tcp1 mutants, genes residing in TCP1-rich TADs have a greater extent of expression fold change as opposed to genes that do not belong to these TADs. Our results suggest that, besides standing as spatial chromatin-packing modules, plant TADs function as nuclear microcompartments associated with transcription factor activities.

Neofunctionalisation of basic helix-loop-helix proteins occurred when embryophytes colonised the land.

ROOT HAIR DEFECTIVE SIX-LIKE (RSL) genes control the development of structures from single cells at the surface of embryophytes (land plants) such as rhizoids and root hairs. RSL proteins constitute a subclass (VIIIc) of the basic helix-loop-helix (bHLH) class VIII transcription factor family. The Charophyceae form the only class of streptophyte algae with tissue-like structures and rhizoids. To determine if the function of RSL genes in the control of cell differentiation in embryophytes was inherited from a streptophyte algal ancestor, we identified the single class VIII bHLH gene from the charophyceaen alga Chara braunii (CbbHLHVIII). CbbHLHVIII is sister to the RSL proteins; they constitute a monophyletic group. Expression of CbbHLHVIII does not compensate for loss of RSL functions in Marchantia polymorpha or Arabidopsis thaliana. In C. braunii CbbHLHVIII is expressed at sites of morphogenesis but not in rhizoids. This finding indicates that C. braunii class VIII protein is functionally different from land plant RSL proteins. This result suggests that the function of RSL proteins in cell differentiation at the plant surface evolved by neofunctionalisation in the land plants lineage after its divergence from its last common ancestor with C. braunii, at or before the colonisation of the land by embryophytes.

Evolutionary and Functional Analysis of a Chara Plasma Membrane H+-ATPase.

H+-ATPases are the main transporters in plant and fungal plasma membranes (PMs), comparable to the Na+/K+ ATPases in animal cells. At the molecular level, most studies on the PM H+-ATPases have been focused on land plants and fungi (yeast). The research of PM H+-ATPases in green algae falls far behind due to the lack of genetic information. Here we studied a potential PM H+-ATPase (CHA1) from Chara australis, a species of green algae belonging to the division Charophyta, members of which are considered to be one of the closest ancestors of land plants. The gene encodes a 107 kDa protein with all 6 P-type ATPase-specific motifs and a long, diverse C-terminal domain. A new amino acid sequence motif R*****Q in transmembrane segment 5 was identified among the known PM H+-ATPases from Charophyta and Chlorophyta algae, which is different from the typical PM H+-ATPases in yeast or land plants. Complementation analysis in yeast showed that CHA1 could successfully reach the PM, and that proton pump activity was obtained when the last 77 up to 87 amino acids of the C-terminal domain were deleted. PM localization was confirmed in Arabidopsis protoplasts; however, deletion of more than 55 amino acids at the N-terminus or more than 98 amino acids at the C-terminus resulted in failure of CHA1 to reach the PM in yeast. These results suggest that an auto-inhibition domain is located in the C-terminal domain, and that CHA1 is likely to have a different regulation mechanism compared to the yeast and land plant PM H+-ATPases.

The Chara Genome: Secondary Complexity and Implications for Plant Terrestrialization.

Land plants evolved from charophytic algae, among which Charophyceae possess the most complex body plans. We present the genome of Chara braunii; comparison of the genome to those of land plants identified evolutionary novelties for plant terrestrialization and land plant heritage genes. C. braunii employs unique xylan synthases for cell wall biosynthesis, a phragmoplast (cell separation) mechanism similar to that of land plants, and many phytohormones. C. braunii plastids are controlled via land-plant-like retrograde signaling, and transcriptional regulation is more elaborate than in other algae. The morphological complexity of this organism may result from expanded gene families, with three cases of particular note: genes effecting tolerance to reactive oxygen species (ROS), LysM receptor-like kinases, and transcription factors (TFs). Transcriptomic analysis of sexual reproductive structures reveals intricate control by TFs, activity of the ROS gene network, and the ancestral use of plant-like storage and stress protection proteins in the zygote.

Functional PTB phosphate transporters are present in streptophyte algae and early diverging land plants.

Two inorganic phosphate (Pi) uptake mechanisms operate in streptophytes and chlorophytes, the two lineages of green plants. PHOSPHATE TRANSPORTER B (PTB) proteins are hypothesized to be the Na+ /Pi symporters catalysing Pi uptake in chlorophytes, whereas PHOSPHATE TRANSPORTER 1 (PHT1) proteins are the H+ /Pi symporters that carry out Pi uptake in angiosperms. PHT1 proteins are present in all streptophyte lineages. However, Pi uptake in streptophyte algae and marine angiosperms requires Na+ influx, suggesting that Na+ /Pi symporters also function in some streptophytes. We tested the hypothesis that Na+ /Pi symporters exist in streptophytes. We identified PTB sequences in streptophyte genomes. Core PTB proteins are present at the plasma membrane of the liverwort Marchantia polymorpha. The expression of M. polymorpha core PTB proteins in the Saccharomyces cerevisiae pho2 mutant defective in high-affinity Pi transport rescues growth in low-Pi environments. Moreover, levels of core PTB mRNAs of M. polymorpha and the streptophyte alga Coleochaete nitellarum are higher in low-Pi than in Pi-replete conditions, consistent with a role in Pi uptake from the environment. We conclude that land plants inherited two Pi uptake mechanisms - mediated by the PTB and PHT1 proteins, respectively - from their streptophyte algal ancestor. Both systems operate in parallel in extant early diverging land plants.

Presence versus absence of CYP734A50 underlies the style-length dimorphism in primroses.

Heterostyly is a wide-spread floral adaptation to promote outbreeding, yet its genetic basis and evolutionary origin remain poorly understood. In Primula (primroses), heterostyly is controlled by the S-locus supergene that determines the reciprocal arrangement of reproductive organs and incompatibility between the two morphs. However, the identities of the component genes remain unknown. Here, we identify the Primula CYP734A50 gene, encoding a putative brassinosteroid-degrading enzyme, as the G locus that determines the style-length dimorphism. CYP734A50 is only present on the short-styled S-morph haplotype, it is specifically expressed in S-morph styles, and its loss or inactivation leads to long styles. The gene arose by a duplication specific to the Primulaceae lineage and shows an accelerated rate of molecular evolution. Thus, our results provide a mechanistic explanation for the Primula style-length dimorphism and begin to shed light on the evolution of the S-locus as a prime model for a complex plant supergene.

Diversification of a Transcription Factor Family Led to the Evolution of Antagonistically Acting Genetic Regulators of Root Hair Growth.

Streptophytes colonized the land some time before 470 million years ago [1-3]. The colonization coincided with an increase in morphological and cellular diversity [4-7]. This increase in diversity is correlated with a proliferation in transcription factors encoded in genomes [8-10]. This suggests that gene duplication and subsequent diversification of function was instrumental in the generation of land plant diversity. Here, we investigate the diversification of the streptophyte-specific Lotus japonicus ROOTHAIRLESS LIKE (LRL) transcription factor (TF) [11, 12] subfamily of basic loop helix (bHLH) proteins by comparing gene function in early divergent and derived land plant species. We report that the single Marchantia polymorpha LRL gene acts as a general growth regulator required for rhizoid development, a function that has been partially conserved throughout multicellular streptophytes. In contrast, the five relatively derived Arabidopsis thaliana LRL genes comprise two antagonistically acting groups of differentially expressed genes. The diversification of LRL genes accompanied the evolution of an antagonistic regulatory element controlling root hair development.

Atkinesin-13A modulates cell-wall synthesis and cell expansion in Arabidopsis thaliana via the THESEUS1 pathway.

Growth of plant organs relies on cell proliferation and expansion. While an increasingly detailed picture about the control of cell proliferation is emerging, our knowledge about the control of cell expansion remains more limited. We demonstrate here that the internal-motor kinesin AtKINESIN-13A (AtKIN13A) limits cell expansion and cell size in Arabidopsis thaliana, with loss-of-function atkin13a mutants forming larger petals with larger cells. The homolog, AtKINESIN-13B, also affects cell expansion and double mutants display growth, gametophytic and early embryonic defects, indicating a redundant role of the two genes. AtKIN13A is known to depolymerize microtubules and influence Golgi motility and distribution. Consistent with this function, AtKIN13A interacts genetically with ANGUSTIFOLIA, encoding a regulator of Golgi dynamics. Reduced AtKIN13A activity alters cell wall structure as assessed by Fourier-transformed infrared-spectroscopy and triggers signalling via the THESEUS1-dependent cell-wall integrity pathway, which in turn promotes the excess cell expansion in the atkin13a mutant. Thus, our results indicate that the intracellular activity of AtKIN13A regulates cell expansion and wall architecture via THESEUS1, providing a compelling case of interplay between cell wall integrity sensing and expansion.

Recruitment and remodeling of an ancient gene regulatory network during land plant evolution.

The evolution of multicellular organisms was made possible by the evolution of underlying gene regulatory networks. In animals, the core of gene regulatory networks consists of kernels, stable subnetworks of transcription factors that are highly conserved in distantly related species. However, in plants it is not clear when and how kernels evolved. We show here that RSL (ROOT HAIR DEFECTIVE SIX-LIKE) transcription factors form an ancient land plant kernel controlling caulonema differentiation in the moss Physcomitrella patens and root hair development in the flowering plant Arabidopsis thaliana. Phylogenetic analyses suggest that RSL proteins evolved in aquatic charophyte algae or in early land plants, and have been conserved throughout land plant radiation. Genetic and transcriptional analyses in loss of function A. thaliana and P. patens mutants suggest that the transcriptional interactions in the RSL kernel were remodeled and became more hierarchical during the evolution of vascular plants. We predict that other gene regulatory networks that control development in derived groups of plants may have originated in the earliest land plants or in their ancestors, the Charophycean algae.

Expression of the central growth regulator BIG BROTHER is regulated by multiple cis-elements.

BACKGROUND: Much of the organismal variation we observe in nature is due to differences in organ size. The observation that even closely related species can show large, stably inherited differences in organ size indicates a strong genetic component to the control of organ size. Despite recent progress in identifying factors controlling organ growth in plants, our overall understanding of this process remains limited, partly because the individual factors have not yet been connected into larger regulatory pathways or networks. To begin addressing this aim, we have studied the upstream regulation of expression of BIG BROTHER (BB), a central growth-control gene in Arabidopsis thaliana that prevents overgrowth of organs. Final organ size and BB expression levels are tightly correlated, implying the need for precise control of its expression. BB expression mirrors proliferative activity, yet the gene functions to limit proliferation, suggesting that it acts in an incoherent feedforward loop downstream of growth activators to prevent over-proliferation. RESULTS: To investigate the upstream regulation of BB we combined a promoter deletion analysis with a phylogenetic footprinting approach. We were able to narrow down important, highly conserved, cis-regulatory elements within the BB promoter. Promoter sequences of other Brassicaceae species were able to partially complement the A. thaliana bb-1 mutant, suggesting that at least within the Brassicaceae family the regulatory pathways are conserved. CONCLUSIONS: This work underlines the complexity involved in precise quantitative control of gene expression and lays the foundation for identifying important upstream regulators that determine BB expression levels and thus final organ size.

Control of tissue and organ growth in plants.

Plant organs grow to characteristic, species-specific sizes and shapes. At the cellular level, organ growth is initially characterized by cell proliferation, which gives way to cell expansion at later stages. Using mainly Arabidopsis thaliana as a model species, a number of factors have been isolated in recent years that promote or restrict organ growth, with the altered organ size being associated with changes in cell number, in cell size, or in both. However, cells in an organ do not appear to follow a strictly autonomous program of proliferation and expansion, and their behavior is coordinated in at least three different respects: normally sized organs can be formed consisting of altered numbers of cells with compensatory changes in the size of the individual cells, suggesting that cellular behavior is subject to organ-wide control; the growth of cells derived from more than one clonal origin is coordinated within a plant lateral organ with its different histological layers; and growth of cells in different regions of an organ is coordinated to generate a reasonably flat leaf or floral organ. Organ growth is strongly modulated by environmental factors, and the molecular basis for this regulation is beginning to be understood. Given the complexity of organ growth as a dynamic four-dimensional process, precise quantification of growth parameters and mathematical modeling are increasingly used to understand this fascinating problem of plant biology.

KLUH/CYP78A5 promotes organ growth without affecting the size of the early primordium.

Mobile signals play a key role in controlling the growth of organisms. In Arabidopsis, the cytochrome P450 CYP78A5/KLUH (KLU) non-cell autonomously stimulates cell proliferation in developing organs. In a recent study, we determined the range of KLU action, using a widely applicable system to generate predictable chimaeric plants. We showed that KLU acts not only within individual floral organs or flowers, but that its overall activity level is integrated across an inflorescence to determine organ size. Here, we address the question at which stage of petal development KLU acts to promote growth. We demonstrate that the size of the very young petal primordium in klu mutants is not altered, supporting the conclusion that KLU acts during later stages of organ outgrowth and a correspondingly longer range of the presumed KLU-dependent growth signal.

KLUH/CYP78A5-dependent growth signaling coordinates floral organ growth in Arabidopsis.

Growth control in animals and plants involves mobile signals. Depending on their range of action, these signals coordinate the growth of cells within an organ or the growth of different organs in a larger, functionally integrated structure. In plants, flowers are such integrated structures, yet it remains poorly understood how growth of the constituent organs is coordinated to ensure their correct relative sizes. The cytochrome P450 KLUH/CYP78A5 and its homolog CYP78A7 promote organ growth via a non-cell-autonomous signal; however, the range of this signal and thus its developmental function are unknown. Here we use a system for the predictable generation of chimeric plants to determine the range of the KLUH-dependent signal. In contrast with the largely autonomous behavior of another tested growth-control gene, we find that KLUH activity extends beyond individual organs and flowers. Its overall activity is integrated across an inflorescence to determine final organ size, which is largely independent of the genotype of the individual organs. Thus, the KLUH-dependent signal appears to move beyond individual organs in a flower, providing a mechanism for coordinating their growth and ensuring floral symmetry as an important determinant of a plant's attractiveness to pollinators.

SLOW MOTION is required for within-plant auxin homeostasis and normal timing of lateral organ initiation at the shoot meristem in Arabidopsis.

The regular arrangement of leaves and flowers around a plant's stem is a fascinating expression of biological pattern formation. Based on current models, the spacing of lateral shoot organs is determined by transient local auxin maxima generated by polar auxin transport, with existing primordia draining auxin from their vicinity to restrict organ formation close by. It is unclear whether this mechanism encodes not only spatial information but also temporal information about the plastochron (i.e., the interval between the formation of successive primordia). Here, we identify the Arabidopsis thaliana F-box protein SLOW MOTION (SLOMO) as being required for a normal plastochron. SLOMO interacts genetically with components of polar auxin transport, and mutant shoot apices contain less free auxin. However, this reduced auxin level at the shoot apex is not due to increased polar auxin transport down the stem, suggesting that it results from reduced synthesis. Independently reducing the free auxin level in plants causes a similar lengthening of the plastochron as seen in slomo mutants, suggesting that the reduced auxin level in slomo mutant shoot apices delays the establishment of the next auxin maximum. SLOMO acts independently of other plastochron regulators, such as ALTERED MERISTEM PROGRAM1 or KLUH/CYP78A5. We propose that SLOMO contributes to auxin homeostasis in the shoot meristem, thus ensuring a normal rate of the formation of auxin maxima and organ initiation.

Differential expression of WOX genes mediates apical-basal axis formation in the Arabidopsis embryo.

Axis formation is one of the earliest patterning events in plant and animal embryogenesis. In Arabidopsis, the main axis of the embryo is evident at the asymmetric division of the zygote into a small, embryonic apical cell and a large extraembryonic basal cell. Here we show that the homeobox genes WOX2 and WOX8, which are initially coexpressed in the zygote, act as complementary cell fate regulators in the apical and basal lineage, respectively. Furthermore, WOX8 expression in the basal cell lineage is required for WOX2 expression and normal development of the proembryo, suggesting an inductive mechanism. The identified WOX cascade is required for normal expression of a reporter gene of the auxin efflux carrier PIN1 and for the formation of auxin response maxima in the proembryo. Thus, our results link the spatial separation of WOX transcription factors to localized auxin response and the formation of the main body axis in the embryo.

Polycomb group proteins function in the female gametophyte to determine seed development in plants.

Polycomb group (PcG) proteins are evolutionary conserved proteins that stably maintain established transcriptional patterns over cell generations. The FERTILIZATION INDEPENDENT SEED (FIS) PcG complex from plants has a similar composition to the Polycomb repressive complex 2 from animals. Mutations in FIS genes cause parent-of-origin-dependent seed abortion. Every seed inheriting a mutant fis allele from the mother is destined to abort, regardless of the presence of a wild-type paternal allele. We tested in Arabidopsis whether the parent-of-origin-dependent seed abortion caused by lack of the FIS subunit MSI1 is caused by parental imprinting of the MSI1 gene. Our data show that MSI1 is not an imprinted gene and that early paternal MSI1 expression is not sufficient to rescue msi1 mutant seeds. By contrast, expression of MSI1 in msi1 female gametophytes is necessary to restore normal seed development, strongly arguing that the female gametophytic effect of fis mutants is caused by a functional requirement for an intact FIS complex in the female gametophyte. Thus, FIS-mediated expression patterns established in the female gametophyte can impact on seed development, establishing fis mutants as true female gametophytic maternal-effect mutants.

Expression dynamics of WOX genes mark cell fate decisions during early embryonic patterning in Arabidopsis thaliana.

During embryonic pattern formation, the main body axes are established and cells of different developmental fates are specified from a single-cell zygote. Despite the fundamental importance of this process, in plants, the underlying mechanisms are largely unknown. We show that expression dynamics of novel WOX (WUSCHEL related homeobox) gene family members reveal early embryonic patterning events in Arabidopsis. WOX2 and WOX8 are co-expressed in the egg cell and zygote and become confined to the apical and basal daughter cells of the zygote, respectively, by its asymmetric division. WOX2 not only marks apical descendants of the zygote, but is also functionally required for their correct development, suggesting that the asymmetric division of the plant zygote separates determinants of apical and basal cell fates. WOX9 expression is initiated in the basal daughter cell of the zygote and subsequently shifts into the descendants of the apical daughter apparently in response to signaling from the embryo proper. Expression of WOX5 shows that identity of the quiescent center is initiated very early in the hypophyseal cell, and highlights molecular and developmental similarities between the stem cell niches of root and shoot meristems. Together, our data suggest that during plant embryogenesis region-specific transcription programs are initiated very early in single precursor cells and that WOX genes play an important role in this process.