Auxin-dependent cell cycle reactivation through transcriptional regulation of Arabidopsis E2Fa by lateral organ boundary proteins

Multicellular organisms depend on cell production, cell fate specification, and correct patterning to shape their adult body. In plants, auxin plays a prominent role in the timely coordination of these different cellular processes. A well-studied example is lateral root initiation, in which auxin triggers founder cell specification and cell cycle activation of xylem pole-positioned pericycle cells. Here, we report that the E2Fa transcription factor of Arabidopsis thaliana is an essential component that regulates the asymmetric cell division marking lateral root initiation. Moreover, we demonstrate that E2Fa expression is regulated by the LATERAL ORGAN BOUNDARY DOMAIN18/LATERAL ORGAN BOUNDARY DOMAIN33 (LBD18/LBD33) dimer that is, in turn, regulated by the auxin signaling pathway. LBD18/LBD33 mediates lateral root organogenesis through E2Fa transcriptional activation, whereas E2Fa expression under control of the LBD18 promoter eliminates the need for LBD18. Besides lateral root initiation, vascular patterning is disrupted in E2Fa knockout plants, similarly as it is affected in auxin signaling and lbd mutants, indicating that the transcriptional induction of E2Fa through LBDs represents a general mechanism for auxin-dependent cell cycle activation. Our data illustrate how a conserved mechanism driving cell cycle entry has been adapted evolutionarily to connect auxin signaling with control of processes determining plant architecture

Activation of AtMPK9 through autophosphorylation that makes it independent of the canonical MAPK cascades.

Mitogen activated protein kinases (MAPKs) are part of conserved signal transduction modules in eukaryotes that are typically organised into three-tiered kinase cascades. The activation of MAPKs in these pathways is fully dependent on the bisphosphorylation of the TXY motif in the T-loop by the pertinent dual-specificity MAPK kinases (MAPKKs). The plant AtMPK9 is a member of an atypical class of MAPKs. Representatives of this MAPK family have TDY phosphoacceptor site, a long C-terminal extension, and lack the common MAPKK binding docking motif. Here, we present multiple in vitro and in vivo data that AtMPK9 is activated independently of any upstream MAPKKs but it is activated through autophosphorylation. We mapped the autophosphorylation sites by mass spectrometry to the TDY motif and to the C-terminal regulatory extension. We mutated the phosphoacceptor sites on the TDY, which confirmed the requirement for bisphorylation at this site for full kinase activity. Next, we demonstrated that the kinase inactive mutant form of AtMPK9 is not transphosphorylated on the TDY site when mixed with an active AtMPK9, implying that the mechanism of the autocatalytic phosphorylation is intramolecular. Furthermore, we show that in vivo AtMPK9 is activated by salt and is regulated by okadaic acid-sensitive phosphatases. We conclude that the plant AtMPK9 shows similarities to the mammalian atypical MAPKs, ERK7/8 in terms of MAPKK-independent activation mechanism

Cellular and transcriptomic analyses reveal two-staged chloroplast biogenesis underpinning photosynthesis build-up in the wheat leaf

Background The developmental gradient in monocot leaves has been exploited to uncover leaf developmental gene expression programs and chloroplast biogenesis processes. However, the relationship between the two is barely understood, which limits the value of transcriptome data to understand the process of chloroplast development. Results Taking advantage of the developmental gradient in the bread wheat leaf, we provide a simultaneous quantitative analysis for the development of mesophyll cells and of chloroplasts as a cellular compartment. This allows us to generate the first biologically-informed gene expression map of this leaf, with the entire developmental gradient from meristematic to fully differentiated cells captured. We show that the first phase of plastid development begins with organelle proliferation, which extends well beyond cell proliferation, and continues with the establishment and then the build-up of the plastid genetic machinery. The second phase is marked by the development of photosynthetic chloroplasts which occupy the available cellular space. Using a network reconstruction algorithm, we predict that known chloroplast gene expression regulators are differentially involved across those developmental stages. Conclusions Our analysis generates both the first wheat leaf transcriptional map and one of the most comprehensive descriptions to date of the developmental history of chloroplasts in higher plants. It reveals functionally distinct plastid and chloroplast development stages, identifies processes occurring in each of them, and highlights our very limited knowledge of the earliest drivers of plastid biogenesis, while providing a basis for their future identification

Genomic organization and evolutionary conservation of plant D-type cyclins

Plants contain more genes encoding core cell cycle regulators than other organisms but it is unclear whether these represent distinct functions. D-type cyclins (CYCD) play key roles in the G1-to-S-phase transition, and Arabidopsis (Arabidopsis thaliana) contains 10 CYCD genes in seven defined subgroups, six of which are conserved in rice (Oryza sativa). Here, we identify 22 CYCD genes in the poplar (Populus trichocarpa) genome and confirm that these six CYCD subgroups are conserved across higher plants, suggesting subgroup-specific functions. Different subgroups show gene number increases, with CYCD3 having three members in Arabidopsis, six in poplar, and a single representative in rice. All three species contain a single CYCD7 gene. Despite low overall sequence homology, we find remarkable conservation of intron/exon boundaries, because in most CYCD genes of plants and mammals, the first exon ends in the conserved cyclin signature. Only CYCD3 genes contain the complete cyclin box in a single exon, and this structure is conserved across angiosperms, again suggesting an early origin for the subgroup. The single CYCD gene of moss has a gene structure closely related to those of higher plants, sharing an identical exon/intron structure with several higher plant subgroups. However, green algae have CYCD genes structurally unrelated to higher plants. Conservation is also observed in the location of potential cyclin-dependent kinase phosphorylation sites within CYCD proteins. Subgroup structure is supported by conserved regulatory elements, particularly in the eudicot species, including conserved E2F regulatory sites within CYCD3 promoters. Global expression correlation analysis further supports distinct expression patterns for CYCD subgroups

Arabidopsis RETINOBLASTOMA RELATED directly regulates DNA damage responses through functions beyond cell cycle control

The rapidly proliferating cells in plant meristems must be protected from genome damage. Here, we show that the regulatory role of the Arabidopsis RETINOBLASTOMA RELATED (RBR) in cell proliferation can be separated from a novel function in safeguarding genome integrity. Upon DNA damage, RBR and its binding partner E2FA are recruited to heterochromatic γH2AX-labelled DNA damage foci in an ATM- and ATR-dependent manner. These γH2AX-labelled DNA lesions are more dispersedly occupied by the conserved repair protein, AtBRCA1, which can also co-localise with RBR foci. RBR and AtBRCA1 physically interact in vitro and in planta. Genetic interaction between the RBR-silenced amiRBR and Atbrca1 mutants suggests that RBR and AtBRCA1 may function together in maintaining genome integrity. Together with E2FA, RBR is directly involved in the transcriptional DNA damage response as well as in the cell death pathway that is independent of SOG1, the plant functional analogue of p53. Thus, plant homologs and analogues of major mammalian tumour suppressor proteins form a regulatory network that coordinates cell proliferation with cell and genome integrity

Expression of a nondegradable cyclin B1 affects plant development and leads to endomitosis by inhibiting the formation of a phragmoplast

In plants after the disassembly of mitotic spindle, a specific cytokinetic structure called the phragmoplast is built, and after cytokinesis, microtubules populate the cell cortex in an organized orientation that determines cell elongation and shape. Here, we show that impaired cyclin B1 degradation, resulting from a mutation within its destruction box, leads to an isodiametric shape of epidermal cells in leaves, stems, and roots and retarded growth of seedlings. Microtubules in these misshaped cells are grossly disorganized, focused around the nucleus, whereas they were entirely missing or abnormally organized along the cell cortex. A high percentage of cells expressing nondestructible cyclin B1 had doubled DNA content as a result of undergoing endomitosis. During anaphase the cytokinesis-specific syntaxin KNOLLE could still localize to the midplane of cell division, whereas NPK1-activating kinesin-like protein 1, a cytokinetic kinesin-related protein, was unable to do so, and instead of the formation of a phragmoplast, the midzone microtubules persisted between the separated nuclei, which eventually fused. In summary, our results show that the timely degradation of mitotic cyclins in plants is required for the reorganization of mitotic microtubules to the phragmoplast and for proper cytokinesis. Subsequently, the presence of nondegradable cyclin B1 leads to a failure in organizing properly the cortical microtubules that determine cell elongation and shape

Increase in fruit size of a spontaneous mutant of ‘Gala’ apple (Malus×domestica Borkh.) is facilitated by altered cell production and enhanced cell size

Fruit size regulation was studied in the apple cultivar ‘Gala’ and a large fruit size spontaneous mutant of ‘Gala’, ‘Grand Gala’ (GG). GG fruits were 15% larger in diameter and 38% heavier than ‘Gala’ fruits, largely due to an increase in size of the fruit cortex. The mutation in GG altered growth prior to fruit set and during fruit development. Prior to fruit set, the carpel/floral-tube size was enhanced in GG and was associated with higher cell number, larger cell size, and increased ploidy through endoreduplication, an altered form of the cell cycle normally absent in apple. The data suggest that the mutation in GG promotes either cell production or endoreduplication in the carpel/floral-tube cells depending on their competence for division. Ploidy was not altered in GG leaves. During fruit growth, GG fruit cells exited cell production earlier, and with a DNA content of 4C suggesting G2 arrest. Cell size was higher in GG fruits during exit from cell production and at later stages of fruit growth. Final cell diameter in GG fruit cortex cells was 15% higher than that in ‘Gala’ indicating that enhanced fruit size in GG was facilitated by increased cell size. The normal progression of cell expansion in cells arrested in G2 may account for the increase in cell size. Quantitative RT-PCR analysis indicated higher MdCDKA1 expression and reduced MdCYCA2 expression during early fruit development in GG fruits. Together, the data indicate an important role for cell expansion in regulating apple fruit size

As estratégias de sobrevivência e de busca de emprego adotadas pelos desempregados

Este trabalho tem por objetivo conhecer como trabalhadores desempregados lidam com a situação de desemprego, de modo a caracterizar suas estratégias de sobrevivência e recolocação no mercado de trabalho. Parte-se do pressuposto de que estas estratégias estão orientadas pelas representações sociais destes trabalhadores a respeito da questão do desemprego. Para tanto realizou-se um estudo qualitativo em que foram entrevistados trabalhadores desempregados em duas instituições: o Sistema Nacional de Empregos de São Paulo (Sinesp) e o Sindicato dos Metalúrgicos de Osasco. A análise constituiu-se no levantamento e exploração de alguns temas básicos presentes nas entrevistas: estratégias de sobrevivência adotadas pelos trabalhadores desempregados, representação do mercado de trabalho, representação de emprego e concepção do próprio desemprego, manutenção do vínculo de trabalho, repercussão do desemprego e perspectivas de futuro. Estes temas constituíram-se objeto de análise de cada entrevista, de modo que se pudessem conhecer as formas particulares através das quais os trabalhadores lidam com estas questões. As estratégias de sobrevivência e recolocação são influenciadas pelas redes de relações que o trabalhador faz parte. Embora as estratégias atuais sejam basicamente as mesmas do passado, estas se mostram menos eficazes devido às maiores dificuldades que o mercado de trabalho tem imposto nas últimas décadas

Regulation of Translation by TOR, eIF4E and eIF2 alpha in Plants:Current Knowledge, Challenges and Future Perspectives

An important step in eukaryotic gene expression is the synthesis of proteins from mRNA, a process classically divided into three stages, initiation, elongation, and termination. Translation is a precisely regulated and conserved process in eukaryotes. The presence of plant-specific translation initiation factors and the lack of well-known translational regulatory pathways in this kingdom nonetheless indicate how a globally conserved process can diversify among organisms. The control of protein translation is a central aspect of plant development and adaptation to environmental stress, but the mechanisms are still poorly understood. Here we discuss current knowledge of the principal mechanisms that regulate translation initiation in plants, with special attention to the singularities of this eukaryotic kingdom. In addition, we highlight the major recent breakthroughs in the field and the main challenges to address in the coming years