Local maternal control of seed size by KLUH/CYP78A5-dependent growth signaling

Seed development in plants involves the coordinated growth of the embryo, endosperm, and maternal tissue. Several genes have been identified that influence seed size by acting maternally, such as AUXIN RESPONSE FACTOR2, APETALA2, and DA1. However, given the lack of gain-of-function effects of these genes on seed size, it is unclear whether their activity levels are limiting in WT plants and whether they could thus be used to regulate seed size in development or evolution. Also, whether the altered seed sizes reflect local gene activity or global physiological changes is unknown. Here, we demonstrate that the cytochrome P450 KLUH (KLU) regulates seed size. KLU acts locally in developing flowers to promote seed growth, and its activity level is limiting for seed growth in WT. KLU is expressed in the inner integument of developing ovules, where it non-cell autonomously stimulates cell proliferation, thus determining the growth potential of the seed coat and seed. A KLU-induced increase in seed size leads to larger seedlings and higher relative oil content of the seeds. Genetic analyses indicate that KLU acts independently of other tested maternal factors that influence integument cell proliferation. Thus, the level of KLU-dependent growth factor signaling determines size in ovules and seeds, suggesting this pathway as a target for crop improvement.     

A PHD-polycomb repressive complex 2 triggers the epigenetic silencing of FLC during vernalization

Vernalization, the acceleration of flowering by winter, involves cold-induced epigenetic silencing of Arabidopsis FLC. This process has been shown to require conserved Polycomb Repressive Complex 2 (PRC2) components including the Su(z)12 homologue, VRN2, and two plant homeodomain (PHD) finger proteins, VRN5 and VIN3. However, the sequence of events leading to FLC repression was unclear. Here we show that, contrary to expectations, VRN2 associates throughout the FLC locus independently of cold. The vernalization-induced silencing is triggered by the cold-dependent association of the PHD finger protein VRN5 to a specific domain in FLC intron 1, and this association is dependent on the cold-induced PHD protein VIN3. In plants returned to warm conditions, VRN5 distribution changes, and it associates more broadly over FLC, coincident with significant increases in H3K27me3. Biochemical purification of a VRN5 complex showed that during prolonged cold a PHD-PRC2 complex forms composed of core PRC2 components (VRN2, SWINGER [an E(Z) HMTase homologue], FIE [an ESC homologue], MSI1 [p55 homologue]), and three related PHD finger proteins, VRN5, VIN3, and VEL1. The PHD-PRC2 activity increases H3K27me3 throughout the locus to levels sufficient for stable silencing. Arabidopsis PHD-PRC2 thus seems to act similarly to Pcl-PRC2 of Drosophila and PHF1-PRC2 of mammals. These data show FLC silencing involves changed composition and dynamic redistribution of Polycomb complexes at different stages of the vernalization process, a mechanism with greater parallels to Polycomb silencing of certain mammalian loci than the classic Drosophila Polycomb targets.     

Birth of a new gene on the Y chromosome of Drosophila melanogaster

Contrary to the pattern seen in mammalian sex chromosomes, where most Y-linked genes have X-linked homologs, the Drosophila X and Y chromosomes appear to be unrelated. Most of the Y-linked genes have autosomal paralogs, so autosome-to-Y transposition must be the main source of Drosophila Y-linked genes. Here we show how these genes were acquired. We found a previously unidentified gene (flagrante delicto Y, FDY) that originated from a recent duplication of the autosomal gene vig2 to the Y chromosome of Drosophila melanogaster. Four contiguous genes were duplicated along with vig2, but they became pseudogenes through the accumulation of deletions and transposable element insertions, whereas FDY remained functional, acquired testis-specific expression, and now accounts for ∼20% of the vig2-like mRNA in testis. FDY is absent in the closest relatives of D. melanogaster, and DNA sequence divergence indicates that the duplication to the Y chromosome occurred ∼2 million years ago. Thus, FDY provides a snapshot of the early stages of the establishment of a Y-linked gene and demonstrates how the Drosophila Y has been accumulating autosomal genes.The mammalian Y chromosome has the lowest gene density of any chromosome, and most of its genes have a homolog on the X. This pattern is consistent with the mammalian sex chromosomes having originated from an ordinary pair of chromosomes, followed by massive gene loss from the Y (1–4). In contrast, the closest homologs of all Drosophila melanogaster Y-linked protein-encoding genes are autosomal, strongly suggesting that its Y chromosome has been acquiring genes from the autosomes (5–7). Indeed, gene gains, and not gene losses, have played the major role in shaping the gene content of the Drosophila Y, at least in the last ∼63 million years (My) (8, 9). Hence, the Drosophila Y chromosome seems to be evolving noncanonically (10) and is an ideal model to investigate the dynamics of gene gain on a nonrecombining Y chromosome.The Drosophila Y chromosome has long been known to contain genes essential for male fertility (11, 12). Due to its heterochromatic state, progress in the molecular identification of the Y-linked single-copy genes has been slow. male fertility factor kl5 (kl-5), the first single-copy gene identified, was found serendipitously; it encodes a motor protein (dynein heavy chain) required for flagellar beating (13). More recently, a combination of computational and experimental methods identified 11 single-copy Y-linked genes among the unmapped sequence scaffolds produced by the Drosophila Genome Project (5–7). These genes have two striking features: (i) their closest paralogs are autosomal and not X linked, and (ii) they have male-specific functions, such as the beating of sperm flagella reported for the kl-5 gene (14). The most likely explanation for this pattern is that Y-linked genes were acquired from the autosomes and have been retained because they confer a specific fitness advantage to their carriers. An autosomal origin has previously been reported for a few Y-linked genes in humans and a repetitive gene on the Drosophila Y (4, 15). However, unequivocal evidence of the autosomal origin of Drosophila Y-linked genes, and of the specific mechanism that originated them, is lacking due to their antiquity. The 11 known single-copy genes (kl-2, kl-3, kl-5, ARY, WDY, PRY, Pp1-Y1, Pp1-Y2, Ppr-Y, ORY, and CCY) represent ancient duplications, with amino acid identities to the putative ancestors ranging from 30% to 74%, and poor (if any) alignment at the nucleotide level. Most of them have introns in conserved positions compared with their autosomal paralogs, ruling out retrotransposition and suggesting DNA-based duplication as the mechanism. The original size of these putative duplications is unknown, because the similarity between autosomal and Y-linked regions is restricted to one gene in each case. Flanking sequences and contiguous genes either were not duplicated or were subsequently mutated and deleted beyond recognition.Here we describe flagrante delicto Y (FDY), a single copy Y-linked gene present only in D. melanogaster, and which is 98% identical at the nucleotide level to the autosomal gene vig2. Because its origin is very recent (it occurred after the split between D. melanogaster and Drosophila simulans, ∼4 Mya), it was possible to demonstrate that FDY arose from a DNA-based duplication of chromosome 3R to the Y: the duplicated segment spans 11 kb of autosomal sequence and includes five contiguous genes (vig2, Mocs2, CG42503, Clbn, and Bili); the last four genes became pseudogenes by rapid accumulation of deletions, point mutations, and transposable element insertions or by lack of expression. Thus, FDY unequivocally demonstrates that the Drosophila Y has acquired genes from autosomes. Several Y-linked genes such as kl-2, kl-3, and PRY are shared by distant Drosophila species that diverged ∼60 Mya, implying ancient acquisitions. FDY dates the more recent acquisition to ∼2 My, and hence strongly suggests that Drosophila Y has been continuously acquiring autosomal genes.     

Genetic dissection of the biosynthetic route to gentamicin A2 by heterologous expression of its minimal gene set

Since the first use of streptomycin as an effective antibiotic drug in the treatment of tuberculosis, aminoglycoside antibiotics have been widely used against a variety of bacterial infections for over six decades. However, the pathways for aminoglycoside biosynthesis still remain unclear, mainly because of difficulty in genetic manipulation of actinomycetes producing this class of antibiotics. Gentamicin belongs to the group of 4,6-disubstituted aminoglycosides containing a characteristic core aminocyclitol moiety, 2-deoxystreptamine (2-DOS), and the recent discovery of its biosynthetic gene cluster in Micromonospora echinospora has enabled us to decipher its biosynthetic pathway. To determine the minimal set of genes and their functions for the generation of gentamicin A(2), the first pseudotrisaccharide intermediate in the biosynthetic pathway for the gentamicin complex, various sets of candidate genes from M. echinospora and other related aminoglycoside-producing strains were introduced into a nonaminoglycoside producing strain of Streptomyces venezuelae. Heterologous expression of different combinations of putative 2-DOS biosynthetic genes revealed that a subset, gtmB-gtmA-gacH, is responsible for the biosynthesis of this core aminocyclitol moiety of gentamicin. Expression of gtmG together with gtmB-gtmA-gacH led to production of 2'-N-acetylparomamine, demonstrating that GtmG acts as a glycosyltransferase that adds N-acetyl-d-glucosamine (GLcNA) to 2-DOS. Expression of gtmM in a 2'-N-acetylparomamine-producing recombinant S. venezuelae strain generated paromamine. Expression of gtmE in an engineered paromamine-producing strain of S. venezuelae successfully generated gentamicin A(2), indicating that GtmE is another glycosyltransferase that attaches d-xylose to paromamine. These results represent in vivo evidence elucidating the complete biosynthetic pathway of the pseudotrisaccharide aminoglycoside.     

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.     

On the regulation of photosynthesis by excitonic interactions between carotenoids and chlorophylls

Selective 2-photon excitation (TPE) of carotenoid dark states, Car S₁, shows that in the major light-harvesting complex of photosystem II (LHCII), the extent of electronic interactions between carotenoid dark states (Car S₁) and chlorophyll (Chl) states, _Coupling^{Car S₁−Chl}, correlates linearly with chlorophyll fluorescence quenching under different experimental conditions. Simultaneously, a linear correlation between both Chl fluorescence quenching and _Coupling^{Car S₁−Chl} with the intensity of red-shifted bands in the Chl Q_y and carotenoid absorption was also observed. These results suggest quenching excitonic Car S₁−Chl states as origin for the observed effects. Furthermore, real time measurements of the light-dependent down- and up-regulation of the photosynthetic activity and _Coupling^{Car S₁−Chl} in wild-type and mutant (npq1, npq2, npq4, lut2 and WT+PsbS) Arabidopsis thaliana plants reveal that also in vivo the quenching parameter NPQ correlates always linearly with the extent of electronic Car S₁–Chl interactions in any adaptation status. Our in vivo measurements with Arabidopsis variants show that during high light illumination, _Coupling^{Car S₁−Chl} depends on the presence of PsbS and zeaxanthin (Zea) in an almost identical way as NPQ. In summary, these results provide clear evidence for a very close link between electronic Car S₁–Chl interactions and the regulation of photosynthesis. These findings support a photophysical mechanism in which short-living, low excitonic carotenoid–chlorophyll states serve as traps and dissipation valves for excess excitation energy.     

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.     

Translationally controlled tumor protein is a conserved mitotic growth integrator in animals and plants

The growth of an organism and its size determination require the tight regulation of cell proliferation and cell growth. However, the mechanisms and regulatory networks that control and integrate these processes remain poorly understood. Here, we address the biological role of Arabidopsis translationally controlled tumor protein (AtTCTP) and test its shared functions in animals and plants. The data support a role of plant AtTCTP as a positive regulator of mitotic growth by specifically controlling the duration of the cell cycle. We show that, in contrast to animal TCTP, plant AtTCTP is not implicated in regulating postmitotic growth. Consistent with this finding, plant AtTCTP can fully rescue cell proliferation defects in Drosophila loss of function for dTCTP. Furthermore, Drosophila dTCTP is able to fully rescue cell proliferation defects in Arabidopsis tctp knockouts. Our data provide evidence that TCTP function in regulating cell division is part of a conserved growth regulatory pathway shared between plants and animals. The study also suggests that, although the cell division machinery is shared in all multicellular organisms to control growth, cell expansion can be uncoupled from cell division in plants but not in animals.     

Pseudomonas syringae manipulates systemic plant defenses against pathogens and herbivores

Many pathogens are virulent because they specifically interfere with host defense responses and therefore can proliferate. Here, we report that virulent strains of the bacterial phytopathogen Pseudomonas syringae induce systemic susceptibility to secondary P. syringae infection in the host plant Arabidopsis thaliana. This systemic induced susceptibility (SIS) is in direct contrast to the well studied avirulence/R gene-dependent resistance response known as the hypersensitive response that elicits systemic acquired resistance. We show that P. syringae-elicited SIS is caused by the production of coronatine (COR), a pathogen-derived functional and structural mimic of the phytohormone jasmonic acid (JA). These data suggest that SIS may be a consequence of the previously described mutually antagonistic interaction between the salicylic acid and JA signaling pathways. Virulent P. syringae also has the potential to induce net systemic susceptibility to herbivory by an insect (Trichoplusia ni, cabbage looper), but this susceptibility is not caused by COR. Rather, consistent with its role as a JA mimic, COR induces systemic resistance to T. ni. These data highlight the complexity of defense signaling interactions among plants, pathogens, and herbivores.     

Structure of a mitochondrial supercomplex formed by respiratory-chain complexes I and III

Mitochondria are central to the efficient provision of energy for eukaryotic cells. The oxidative-phosphorylation system of mitochondria consists of a series of five major membrane complexes: NADH-ubiquinone oxidoreductase (commonly known as complex I), succinate-ubiquinone oxidoreductase (complex II), ubiquinol-cytochrome c oxidoreductase (cytochrome bc1 complex or complex III), cytochrome c-O2 oxidoreductase (complex IV), and F1F0-ATP synthase (complex V). Several lines of evidence have recently suggested that complexes I and III-V might interact to form supercomplexes. However, because of their fragility, the structures of these supercomplexes are still unknown. A stable supercomplex consisting of complex I and dimeric complex III was purified from plant mitochondria. Structural characterization by single-particle EM indicates a specific type of interaction between monomeric complex I and dimeric complex III in a 1:1 ratio. We present a model for how complexes I and III are spatially organized within the I+III2 supercomplex.