A polycomb repressive complex 2 gene regulates apogamy and gives evolutionary insights into early land plant evolution

Land plants have distinct developmental programs in haploid (gametophyte) and diploid (sporophyte) generations. Although usually the two programs strictly alternate at fertilization and meiosis, one program can be induced during the other program. In a process called apogamy, cells of the gametophyte other than the egg cell initiate sporophyte development. Here, we report for the moss Physcomitrella patens that apogamy resulted from deletion of the gene orthologous to the Arabidopsis thaliana CURLY LEAF (PpCLF), which encodes a component of polycomb repressive complex 2 (PRC2). In the deletion lines, a gametophytic vegetative cell frequently gave rise to a sporophyte-like body. This body grew indeterminately from an apical cell with the character of a sporophytic pluripotent stem cell but did not form a sporangium. Furthermore, with continued culture, the sporophyte-like body branched. Sporophyte branching is almost unknown among extant bryophytes. When PpCLF was expressed in the deletion lines once the sporophyte-like bodies had formed, pluripotent stem cell activity was arrested and a sporangium-like organ formed. Supported by the observed pattern of PpCLF expression, these results demonstrate that, in the gametophyte, PpCLF represses initiation of a sporophytic pluripotent stem cell and, in the sporophyte, represses that stem cell activity and induces reproductive organ development. In land plants, branching, along with indeterminate apical growth and delayed initiation of spore-bearing reproductive organs, were conspicuous innovations for the evolution of a dominant sporophyte plant body. Our study provides insights into the role of PRC2 gene regulation for sustaining evolutionary innovation in land plants.     

Control of seed mass and seed yield by the floral homeotic gene APETALA2

APETALA2 (AP2) is best known for its role in the regulation of flower meristem and flower organ identity and development in Arabidopsis. We show here that AP2 also plays an important role in determining seed size, seed weight, and the accumulation of seed oil and protein. We demonstrate genetically that AP2 acts through the maternal sporophyte and endosperm genomes to control seed weight and seed yield. Thus, AP2 functions outside the boundaries of flower meristem and flower organ development to affect agronomically relevant traits in Arabidopsis.     

Distinct deregulation of the hypoxia inducible factor by PHD2 mutants identified in germline DNA of patients with polycythemia

Background

Congenital secondary erythrocytoses are due to deregulation of hypoxia inducible factor resulting in overproduction of erythropoietin. The most common germline mutation identified in the hypoxia signaling pathway is the Arginine 200-Tryptophan mutant of the von Hippel-Lindau tumor suppressor gene, resulting in Chuvash polycythemia. This mutant displays a weak deficiency in hypoxia inducible factor α regulation and does not promote tumorigenesis. Other von Hippel-Lindau mutants with more deleterious effects are responsible for von Hippel-Lindau disease, which is characterized by the development of multiple tumors. Recently, a few mutations in gene for the prolyl hydroxylase domain 2 protein (PHD2) have been reported in cases of congenital erythrocytosis not associated with tumor formation with the exception of one patient with a recurrent extra-adrenal paraganglioma.

Design and Methods

Five PHD2 variants, four of which were novel, were identified in patients with erythrocytosis. These PHD2 variants were functionally analyzed and compared with the PHD2 mutant previously identified in a patient with polycythemia and paraganglioma. The capacity of PHD2 to regulate the activity, stability and hydroxylation of hypoxia inducible factor α was assessed using hypoxia-inducible reporter gene, one-hybrid and in vitro hydroxylation assays, respectively.

Results

This functional comparative study showed that two categories of PHD2 mutants could be distinguished: one category with a weak deficiency in hypoxia inducible factor α regulation and a second one with a deleterious effect; the mutant implicated in tumor occurrence belongs to the second category.

Conclusions

As observed with germline von Hippel-Lindau mutations, there are functional differences between the PHD2 mutants with regards to hypoxia inducible factor regulation. PHD2 mutation carriers do, therefore, need careful medical follow-up, since some mutations must be considered as potential candidates for tumor predisposition.     

Helicobacter pylori-induced inflammation and epigenetic changes during gastric carcinogenesis

The sequence of events associated with the development of gastric cancer has been described as “the gastric precancerous cascade”. This cascade is a dynamic process that includes lesions, such as atrophic gastritis, intestinal metaplasia and dysplasia. According to this model, Helicobacter pylori (H. pylori) infection targets the normal gastric mucosa causing non-atrophic gastritis, an initiating lesion that can be cured by clearing H. pylori with antibiotics or that may then linger in the case of chronic infection and progress to atrophic gastritis. The presence of virulence factors in the infecting H. pylori drives the carcinogenesis process. Independent epidemiological and animal studies have confirmed the sequential progression of these precancerous lesions. Particularly long-term follow-up studies estimated a risk of 0.1% for atrophic gastritis/intestinal metaplasia and 6% in case of dysplasia for the long-term development of gastric cancer. With this in mind, a better understanding of the genetic and epigenetic changes associated with progression of the cascade is critical in determining the risk of gastric cancer associated with H. pylori infection. In this review, we will summarize some of the most relevant mechanisms and focus predominantly but not exclusively on the discussion of gene promoter methylation and miRNAs in this context.     

Endosidin1 defines a compartment involved in endocytosis of the brassinosteroid receptor BRI1 and the auxin transporters PIN2 and AUX1

Although it is known that proteins are delivered to and recycled from the plasma membrane (PM) via endosomes, the nature of the compartments and pathways responsible for cargo and vesicle sorting and cellular signaling is poorly understood. To define and dissect specific recycling pathways, chemical effectors of proteins involved in vesicle trafficking, especially through endosomes, would be invaluable. Thus, we identified chemicals affecting essential steps in PM/endosome trafficking, using the intensely localized PM transport at the tips of germinating pollen tubes. The basic mechanisms of this localized growth are likely similar to those of non-tip growing cells in seedlings. The compound endosidin 1 (ES1) interfered selectively with endocytosis in seedlings, providing a unique tool to dissect recycling pathways. ES1 treatment induced the rapid agglomeration of the auxin translocators PIN2 and AUX1 and the brassinosteroid receptor BRI1 into distinct endomembrane compartments termed "endosidin bodies"; however, the markers PIN1, PIN7, and other PM proteins were unaffected. Endosidin bodies were defined by the syntaxin SYP61 and the V-ATPase subunit VHA-a1, two trans-Golgi network (TGN)/endosomal proteins. Interestingly, brassinosteroid (BR)-induced gene expression was inhibited by ES1 and treated seedlings displayed a brassinolide (BL)-insensitive phenotype similar to a bri1 loss-of-function mutant. No effect was detected in auxin signaling. Thus, PIN2, AUX1, and BRI1 use interactive pathways involving an early SYP61/VHA-a1 endosomal compartment.     

Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action

The plant hormone abscisic acid (ABA) is produced in response to abiotic stresses and mediates stomatal closure in response to drought via recently identified ABA receptors (pyrabactin resistance/regulatory component of ABA receptor; PYR/RCAR). SLAC1 encodes a central guard cell S-type anion channel that mediates ABA-induced stomatal closure. Coexpression of the calcium-dependent protein kinase 21 (CPK21), CPK23, or the Open Stomata 1 kinase (OST1) activates SLAC1 anion currents. However, reconstitution of ABA activation of any plant ion channel has not yet been attained. Whether the known core ABA signaling components are sufficient for ABA activation of SLAC1 anion channels or whether additional components are required remains unknown. The Ca(2+)-dependent protein kinase CPK6 is known to function in vivo in ABA-induced stomatal closure. Here we show that CPK6 robustly activates SLAC1-mediated currents and phosphorylates the SLAC1 N terminus. A phosphorylation site (S59) in SLAC1, crucial for CPK6 activation, was identified. The group A PP2Cs ABI1, ABI2, and PP2CA down-regulated CPK6-mediated SLAC1 activity in oocytes. Unexpectedly, ABI1 directly dephosphorylated the N terminus of SLAC1, indicating an alternate branched early ABA signaling core in which ABI1 targets SLAC1 directly (down-regulation). Furthermore, here we have successfully reconstituted ABA-induced activation of SLAC1 channels in oocytes using the ABA receptor pyrabactin resistant 1 (PYR1) and PP2C phosphatases with two alternate signaling cores including either CPK6 or OST1. Point mutations in ABI1 disrupting PYR1-ABI1 interaction abolished ABA signal transduction. Moreover, by addition of CPK6, a functional ABA signal transduction core from ABA receptors to ion channel activation was reconstituted without a SnRK2 kinase.     

ROTUNDA3 function in plant development by phosphatase 2A-mediated regulation of auxin transporter recycling

The shaping of organs in plants depends on the intercellular flow of the phytohormone auxin, of which the directional signaling is determined by the polar subcellular localization of PIN-FORMED (PIN) auxin transport proteins. Phosphorylation dynamics of PIN proteins are affected by the protein phosphatase 2A (PP2A) and the PINOID kinase, which act antagonistically to mediate their apical–basal polar delivery. Here, we identified the ROTUNDA3 (RON3) protein as a regulator of the PP2A phosphatase activity in Arabidopsis thaliana. The RON3 gene was map-based cloned starting from the ron3-1 leaf mutant and found to be a unique, plant-specific gene coding for a protein with high and dispersed proline content. The ron3-1 and ron3-2 mutant phenotypes [i.e., reduced apical dominance, primary root length, lateral root emergence, and growth; increased ectopic stages II, IV, and V lateral root primordia; decreased auxin maxima in indole-3-acetic acid (IAA)-treated root apical meristems; hypergravitropic root growth and response; increased IAA levels in shoot apices; and reduced auxin accumulation in root meristems] support a role for RON3 in auxin biology. The affinity-purified PP2A complex with RON3 as bait suggested that RON3 might act in PIN transporter trafficking. Indeed, pharmacological interference with vesicle trafficking processes revealed that single ron3-2 and double ron3-2 rcn1 mutants have altered PIN polarity and endocytosis in specific cells. Our data indicate that RON3 contributes to auxin-mediated development by playing a role in PIN recycling and polarity establishment through regulation of the PP2A complex activity.Organ growth is determined by cell numbers produced by meristems and by cell expansion to reach final volume. Plant hormones steer the extent and timing of growth and mediate signals of various types that are transmitted within the cell, between cells, or at a long distance within the plant. The phytohormone auxin is a major regulator of cell division and expansion during plant growth and development. The molecular mechanisms by which auxin controls these essential cellular responses are roughly understood thanks to the recent progress in the identification of auxin receptors and components of auxin signaling, transport, and metabolism (1). Auxin gradients between the cells are generated and maintained by intercellular auxin transport mediated by efflux carriers from the PIN-FORMED (PIN) family (2). PIN proteins contain transmembrane domains and continuously cycle between the basal (rootward) and apical (shootward) plasma membranes and endosomes, allowing rapid and dynamic changes in the PIN localization (3). The sorting of PIN proteins into the apical or basal trafficking pathway depends on the PIN phosphorylation status, which is controlled by the PINOID (PID) protein kinase and phosphatase 2A (PP2A) (4, 5), a heterotrimeric complex consisting of a C-catalytic subunit together with A- and B-regulatory subunits. One of the A-subunit isoforms, ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1 (RCN1), acts as a key positive regulator of the PP2A activity in seedlings. The rcn1 mutant that lost part of the PP2A activity displays abnormalities related to defective auxin transport, such as altered gravity response and lateral root growth (6, 7).In an ethyl methanesulfonate-induced collection of Arabidopsis thaliana leaf mutants (8), we identified ROTUNDA3 (RON3) as a proline-rich, plant-specific single-copy gene with a function in auxin-related processes in all organs throughout the plant’s lifecycle. Affinity purification of the PP2A complex with RON3 as bait, and genetic and cell biology analyses support the hypothesis that RON3 affects the cellular dynamics of PIN proteins through interference with the PP2A activity.     

Mutations of cellulose synthase (CESA1) phosphorylation sites modulate anisotropic cell expansion and bidirectional mobility of cellulose synthase

The CESA1 component of cellulose synthase is phosphorylated at sites clustered in two hypervariable regions of the protein. Mutations of the phosphorylated residues to Ala (A) or Glu (E) alter anisotropic cell expansion and cellulose synthesis in rapidly expanding roots and hypocotyls. Expression of T166E, S686E, or S688E mutants of CESA1 fully rescued the temperature sensitive cesA1-1 allele (rsw1) at a restrictive temperature whereas mutations to A at these positions caused defects in anisotropic cell expansion. However, mutations to E at residues surrounding T166 (i.e., S162, T165, and S167) caused opposite effects. Live-cell imaging of fluorescently labeled CESA showed close correlations between tissue or cell morphology and patterns of bidirectional motility of CESA complexes in the plasma membrane. In the WT, CESA complexes moved at similar velocities in both directions along microtubule tracks. By contrast, the rate of movement of CESA particles was directionally asymmetric in mutant lines that exhibited abnormal tissue or cell expansion, and the asymmetry was removed upon depolymerizing microtubules with oryzalin. This suggests that phosphorylation of CESA differentially affects a polar interaction with microtubules that may regulate the length or quantity of a subset of cellulose microfibrils and that this, in turn, alters microfibril structure in the primary cell wall resulting in or contributing to the observed defect in anisotropic cell expansion.