Nice to meet you: genetic, epigenetic and metabolic controls of plant perception of beneficial associative and endophytic diazotrophic bacteria in non-leguminous plants.

A wide range of rhizosphere diazotrophic bacteria are able to establish beneficial associations with plants, being able to associate to root surfaces or even endophytically colonize plant tissues. In common, both associative and endophytic types of colonization can result in beneficial outcomes to the plant leading to plant growth promotion, as well as increase in tolerance against biotic and abiotic stresses. An intriguing question in such associations is how plant cell surface perceives signals from other living organisms, thus sorting pathogens from beneficial ones, to transduce this information and activate proper responses that will finally culminate in plant adaptations to optimize their growth rates. This review focuses on the recent advances in the understanding of genetic and epigenetic controls of plant-bacteria signaling and recognition during beneficial associations with associative and endophytic diazotrophic bacteria. Finally, we propose that "soil-rhizosphere-rhizoplane-endophytes-plant" could be considered as a single coordinated unit with dynamic components that integrate the plant with the environment to generate adaptive responses in plants to improve growth. The homeostasis of the whole system should recruit different levels of regulation, and recognition between the parties in a given environment might be one of the crucial factors coordinating these adaptive plant responses.

Nitrogen signalling in plant interactions with associative and endophytic diazotrophic bacteria.

Some beneficial plant-interacting bacteria can biologically fix N2 to plant-available ammonium. Biological nitrogen fixation (BNF) is an important source of nitrogen (N) input in agriculture and represents a promising substitute for chemical N fertilizers. Diazotrophic bacteria have the ability to develop different types of root associations with different plant species. Among the highest rates of BNF are those measured in legumes nodulated by endosymbionts, an already very well documented model of plant-diazotrophic bacterial association. However, it has also been shown that economically important crops, especially monocots, can obtain a substantial part of their N needs from BNF by interacting with associative and endophytic diazotrophic bacteria, that either live near the root surface or endophytically colonize intercellular spaces and vascular tissues of host plants. One of the best reported outcomes of this association is the promotion of plant growth by direct and indirect mechanisms. Besides fixing N, these bacteria can also produce plant growth hormones, and some species are reported to improve nutrient uptake and increase plant tolerance against biotic and abiotic stresses. Thus, this particular type of plant-bacteria association consists of a natural beneficial system to be explored; however, the regulatory mechanisms involved are still not clear. Plant N status might act as a key signal, regulating and integrating various metabolic processes that occur during association with diazotrophic bacteria. This review will focus on the recent progress in understanding plant association with associative and endophytic diazotrophic bacteria, particularly on the knowledge of the N networks involved in BNF and in the promotion of plant growth.

Members of the ethylene signalling pathway are regulated in sugarcane during the association with nitrogen-fixing endophytic bacteria.

Nitrogen-fixing bacteria have been isolated from sugarcane in an endophytic and beneficial interaction that promotes plant growth. In this work, for the first time, the involvement of ethylene signalling in this interaction was investigated by molecular characterizing members of this pathway in sugarcane. The expression pattern of a putative ethylene receptor (SCER1) and two putative ERF transcription factors (SCERF1 and SCERF2) show exclusive modulation in plants inoculated with the diazotrophic endophytes. The gene expression profile of SCER1, SCERF1, and SCERF2 is differentially regulated in sugarcane genotypes that can establish efficient or inefficient associations with diazotrophic micro-organisms, exhibiting high or low biological nitrogen fixation (BNF) rates, respectively. In addition, SCER1, SCERF1, and SCERF2 expression is different in response to interactions with pathogenic and beneficial micro-organisms. Taken together, that data suggest that SCER1, SCERF1, and SCERF2 might participate in specific ethylene signalling cascade(s) that can identify a beneficial endophytic association, modulating sugarcane responses toward the diazotrophic endophytes.

SHR5: a novel plant receptor kinase involved in plant-N2-fixing endophytic bacteria association.

Endophytic nitrogen-fixing bacteria have been isolated from graminaceous plants such as maize, rice, and sugarcane. They are thought to promote plant growth, not only by fixing nitrogen, but also by the production of plant hormones. The molecular mechanisms involved in this interaction are not yet clear. In this work, the identification of a receptor-like kinase (RLK), named SHR5, which may participate in signal transduction involved in the establishment of plant-endophytic bacteria interaction is described for the first time. SHR5 seems to be part of a novel subclass of RLKs present in a wide range of plant species. The expression of this gene is down-regulated in sugarcane plants associated exclusively with beneficial endophytic bacteria and is not a general response caused by micro-organisms or abiotic stress. In addition, more successful sugarcane-endophytic bacteria associations have a more pronounced decrease in SHR5 expression, suggesting that SHR5 mRNA levels in plant cells are inversely related to the efficiency of the association.

Genome based identification and analysis of the pre-replicative complex of Arabidopsis thaliana.

Eukaryotic DNA replication requires an ordered and regulated machinery to control G1/S transition. The formation of the pre-replicative complex (pre-RC) is a key step involved in licensing DNA for replication. Here, we identify all putative components of the full pre-RC in the genome of the model plant Arabidopsis thaliana. Different from the other eukaryotes, Arabidopsis houses in its genome two putative homologs of ORC1, CDC6 and CDT1. Two mRNA variants of AtORC4 subunit, with different temporal expression patterns, were also identified. Two-hybrid binary interaction assays suggest a primary architectural organization of the Arabidopsis ORC, in which AtORC3 plays a central role in maintaining the complex associations. Expression profiles differ among pre-RC components suggesting the existence of various forms of the complex, possibly playing different roles during development. In addition, the expression of the putative pre-RC genes in non-proliferating plant tissues suggests that they might have roles in processes other than DNA replication licensing.

DNA replication in plants: characterization of a cdc6 homologue from Arabidopsis thaliana.

Cdc6 is a key regulator of DNA replication in eukaryotes. In this work, the expression pattern of an Arabidopsis cdc6 homologue is characterized by RT-PCR and in situ hybridization. The data suggest that cdc6At expression is cell cycle regulated. During development, high cdc6At mRNA levels are found in regular cycling cells. In addition, cdc6At expression is also observed in cells that are probably undergoing endoreduplication, suggesting a possible role of Cdc6At in this process in plants.

Cell division events are essential for embryo patterning and morphogenesis: studies on dominant-negative cdc2aAt mutants of arabidopsis.

During plant development, cell division events are coordinately regulated, leading to specific growth patterns. Experimental evidence indicates that the morphogenetic controls that act at the vegetative plant growth stage are flexible and tolerate distortions in patterns and frequencies of cell division. To address questions concerning the relationship between cell division and embryo formation, a novel experimental approach was used. The frequencies of cell division were reduced exclusively during embryo development of Arabidopsis by the expression of a dominant cdc2a mutant. The five independent transgenic lines with the highest levels of the mutant cdc2a affected embryo formation. In the C13 line, seeds failed to germinate. The C1, C5 and C12 lines displayed a range of distortions on the apical-basal embryo pattern. In the C3 line, the shoot apical meristem of the seedlings produced leaves defective in growth and with an incorrect phyllotactic pattern. The results demonstrate that rates of cell division do not dictate cellular differentiation of embryos. Nevertheless, whereas cell divisions are uncoupled from vegetative development, they are instrumental in elaborating embryo structures and modulating embryo and seedling morphogenesis.

Adaptation of the differential display RT-PCR technique to isolate sugarcane genes induced by plant association with endophytic nitrogen-fixing bacteria.

Differential Display RT-PCR is a powerful technique that has been used to isolate differentially expressed genes. This technique was first described by Liang & Pardee, in 1992. Afterwards, several modifications were introduced in the original version, including a simplification described by Sokolov & Prockop, in 1994. In this work, we describe an adaptation of the Sokolov & Prockop technique, in order to isolate sugarcane genes induced by plant association with endophytic nitrogen-fixing bacteria. Several modifications were introduced: the use of oligo-dT primer in the first strand cDNA synthesis, replicates of the PCR reactions, analysis of the amplified fragments on silver stained polyacrilamide gel and confirmation of cloning the differentially amplified selected band prior to its molecular characterisation. The methodology established was successfully used to identify a large number of differentially amplified sugarcane cDNAs. So far, one of these cDNAs was already isolated and characterized.

Induction of cdc2a and cyc1At expression in Arabidopsis thaliana during early phases of nematode-induced feeding cell formation.

Root-knot and cyst nematodes are plant parasites that induce large multinucleated feeding cells in the roots of their hosts. Cytological observations have shown that root-knot nematodes induce giant cells by cycles of mitosis without cytokinesis whereas cyst nematodes provoke cell wall degradation leading to the formation of a large syncytium. This study was intended to characterize and compare the ability of both types of nematodes to induce progression through the cell cycle. For this purpose, the expression, upon nematode infection, of two cell cycle markers was followed: a marker for division competence, the cyclin-dependent kinase cdc2a and a marker for the G2 phase, the mitotic cyclin cyc1At. For both types of nematodes, transcriptional activation of these markers was correlated with early phases of feeding cell development. Using molecular markers, it was thus possible to confirm and extend the observations of repeated mitosis in root-knot nematode-induced giant cells. Surprisingly, promoter activation of both cdc2a and cyc1At markers was also found upon cyst nematode infection, in feeding cells in which mitosis has not been clearly reported. Incorporation of tritiated thymidine in these syncytia confirms that they progress through the S phase of the cell cycle. One possibility is that cyst nematodes induce cycles of DNA endoreduplication shunting the M phase. Despite obvious differences in ontogeny, common molecular mechanisms, involving cycles of DNA endoreduplication and cdc2a and cyc1At expression, might thus be involved in the formation of a giant cell or a syncytium.

Estudos cromossômicos em indivíduos portadores de retardo mental e atraso psicomotor / Chromosomal investigation in mentally handicapped and psychomotor retardation patients

O retardo mental é uma característica comum aos pacientes que buscam o Serviço de Genética Médica, apresentando heterogeneidade clínica e etiológica. Neste trabalho, estudos cromossômicos foram realizados em 30 indivíduos portadores de retardo mental e atraso psicomotor. Verificamos que 40 por cento dos indivíduos investigados apresentaram anomalias cromossômicas. Nos demais casos (60 por cento) o cariótipo foi normal. Este estudo reforça a importância da investigaçåo citogenética em indivíduos portadores de retardo mental e atraso psicomotor rastreando aberraçöes cromosssômicas numéricas ou estruturais e auxiliando no diagnóstico, prognóstico e aconselhamento gewnético das famílias

Dominant negative mutants of the Cdc2 kinase uncouple cell division from iterative plant development.

Because plant cells do not move and are surrounded by a rigid cell wall, cell division rates and patterns are believed to be directly responsible for generating new structures throughout development. To study the relationship between cell division and morphogenesis, transgenic tobacco and Arabidopsis plants were constructed expressing dominant mutations in a key regulator of the Arabidopsis cell cycle, the Cdc2a kinase. Plants constitutively overproducing the wild-type Cdc2a or the mutant form predicted to accelerate the cell cycle did not exhibit a significantly altered development. In contrast, a mutation expected to arrest the cell cycle abolished cell division when expressed in Arabidopsis, whereas some tobacco plants constitutively producing this mutant protein were recovered. These plants had a reduced histone H1 kinase activity and contained considerably fewer cells. These cells were, however, much larger and underwent normal differentiation. Morphogenesis, histogenesis and developmental timing were unaffected. The results indicate that, in plants, the developmental controls defining shape can act independently from cell division rates.

Developmental expression of the arabidopsis cyclin gene cyc1At.

In eukaryotes, the control of cell cycle progression is exercised by heteromeric protein kinase complexes composed of a cell cycle-dependent, kinase-related subunit (Cdc2) and a cyclin subunit. To explore the possibility that cyclin transcription plays a role in the developmental regulation of cell division, we examined the spatial and temporal expression of a cyclin gene (cyc1At) in Arabidopsis. In root and shoot apical meristems and during embryogenesis, cyc1At expression is almost exclusively confined to dividing cells. A cell-specific pattern of cyc1At expression was noticed in root meristems. We examined the effects of induction of cell division of differentiated cells on cyc1At expression. During lateral root formation, induction of cyc1At expression is a very early event and was detected before anatomical modifications were visible. Treatment of roots with oryzalin, which blocks cell division in metaphase, did not inhibit the auxin induction of cyc1At, suggesting that induction of cyc1At expression precedes the completion of the first division cycle after induction of lateral roots. In tobacco protoplasts, an increase in cyc1At expression was observed only when cell division was induced. Together, the results suggest that Cyc1At accumulation in Arabidopsis is transcriptionally regulated and might be one of the limiting factors for the activation of cell division.

Control of cell proliferation during plant development.

Knowledge of the control of cell division in eukaryotes has increased tremendously in recent years. The isolation and characterization of the major players from a number of systems and the study of their interactions have led to a comprehensive understanding of how the different components of the cell cycle apparatus are brought together and assembled in a fine-tuned machinery. Many parts of this machine are highly conserved in organisms as evolutionary distant as yeast and animals. Some key regulators of cell division have also been identified in higher plants and have been shown to be functional homologues of the yeast or animal proteins. Although still in its early days, investigations into the regulation of these molecules have provided some clues on how cell division is coupled to plant development.

Three discrete classes of Arabidopsis cyclins are expressed during different intervals of the cell cycle.

We have isolated cDNAs encoding four additional mitotic-like cyclins from Arabidopsis: cyc2aAt, cyc2bAt, cyc3aAt, and cyc3bAt. Examination of amino acid sequences deduced from plant cyclin cDNAs isolated so far showed that they can be grouped into three distinct classes. The members of each plant cyclin family are more related to each other than to any animal or yeast cyclin. Reverse transcription-PCR analysis demonstrated that cyc2aAt was expressed in all plant organs, whereas cyc2bAt mRNAs were found only in roots; cyc3aAt was not expressed in leaves and was barely expressed in flowers. On the other hand, cyc3bAt transcripts were observed in all organs. Whole-mount in situ hybridizations on roots showed that the cyclin mRNAs were confined to parts of the roots with mitotic activity. Furthermore, results of whole-mount in situ hybridizations on roots treated with either oryzalin or hydroxyurea suggest that the different cyclin classes have distinct functions in the cell cycle.

cdc2a expression in Arabidopsis is linked with competence for cell division.

A key regulator of the cell cycle is a highly conserved protein kinase whose catalytic subunit, p34(cdc2), is encoded by the cdc2 gene. We studied the control of the expression of the Arabidopsis cdc2a gene in cell suspensions and during plant development. In cell cultures, arrest of the cell cycle did not significantly affect cdc2a mRNA levels, but nutrient conditions were important for cdc2a expression. During plant development, the pattern of cdc2a expression was strongly correlated with the cell proliferation potential. The effects of external signals on cdc2a expression were analyzed. Wounding induced expression in leaves. Lack of light altered temporal regulation of cdc2a in the apical but not root meristem of seedlings. Differential cdc2a responses were obtained after different hormone treatments. Signals present only in intact plants were necessary to mediate these responses. Although other control levels have yet to be analyzed, these results suggest that the regulation of cdc2a expression may contribute greatly to spatial and temporal regulation of cell division in plants. Our results also show that cdc2a expression is not always coupled with cell proliferation but always precedes it. We propose that cdc2a expression may reflect a state of competence to divide, and that the release of other controls is necessary for cell division to occur.

A protein phosphatase 1 from Arabidopsis thaliana restores temperature sensitivity of a Schizosaccharomyces pombe cdc25ts/wee1- double mutant.

There is increasing evidence that the mechanisms controlling the eukaryotic cell cycle are regulated by protein phosphorylation/dephosphorylation cascades. The catalytic subunit of the protein phosphatase 1 is implicated genetically and biochemically in the complex network that regulates mitosis. To investigate further the cell division in plants, we have isolated and characterized two full-length cDNAs from Arabidopsis thaliana, PP1A-At1 and PP1A-At2, encoding polypeptides highly homologous to known protein phosphatase 1 (PP1). DNA gel blot analysis suggests that the protein phosphatases 1 might form a small gene family in Arabidopsis. Northern analysis shows that transcripts are present in all plant organs. In cell cultures, the PP1 mRNA levels are differentially affected by treatment with drugs that block cell division. The expression of PP1A-At1 in a Schizosaccharomyces pombe cdc25ts/wee1- double-mutant strain restores temperature sensitivity, showing that the Arabidopsis phosphatase gene is capable of interacting with genes that regulate the fission yeast mitotic apparatus. However, the dis2-11 S. pombe strain, which has a cold-sensitive allele of the phosphatase 1 gene, is not rescued by expression of the PP1A-At1 gene, suggesting that the plant cDNA is not a functional homolog of the fission yeast gene.

A cdc2 gene of Petunia hybrida is differentially expressed in leaves, protoplasts and during various cell cycle phases.

Analysis of p34cdc2 kinase in higher eukaryotes has demonstrated that p34cdc2 function is conserved in all eukaryotic cells. The p34cdc2 kinase (the product of the cdc2 gene) is required during the G1 cell cycle phase at the initiation of DNA replication and also in G2-M phases for entry into mitosis. In this paper we report the isolation and characterization of a cdc2 Petunia hybrida PCR fragment (cdc2Pet). Using a DNA probe based on this fragment and a p34cdc2-specific antibody, cdc2Pet transcript and p34 protein levels were found to be constant both in 2C nuclei of highly proliferating mesophyll 2C cells derived from protoplasts and in 2C nuclei isolated directly from expanded petunia leaves. Both the cdc2Pet transcript and p34cdc2 protein levels were found to be higher in nuclei at 4C than in those at 2C, even when these 4C nuclei were from non-proliferating tissue. Thus cdc2Pet mRNA and protein levels measured in different tissues should not be interpreted to reflect exclusively the proliferative state of the tissue but also the frequency of G2 cells including those in the differentiated state.

Genes regulating the plant cell cycle: isolation of a mitotic-like cyclin from Arabidopsis thaliana.

A key element of cell cycle control in eukaryotes is the M-phase kinase, composed of p34cdc2 and cyclin. To dissect the plant cell cycle, we have previously isolated a cdc2 gene homolog from Arabidopsis thaliana. We have now cloned an Arabidopsis cDNA corresponding to cyclins. This gene (cyc1At) encodes a protein with a predicted molecular mass of 48.4 kDa and a domain homologous to the cyclin box of mitotic cyclins. However, by sequence comparison the cyc1At gene could not be assigned to the A- or B-type group. The mRNA accumulates preferentially in actively dividing cells and when these cells are blocked during the cell cycle, the amount of transcripts decreases dramatically. cyc1At mRNA is found mainly in G2-phase nuclei, suggesting that its expression is periodic in the cell cycle. Microinjection of synthetic cyc1At mRNA induced meiotic maturation in Xenopus oocytes. Cyc1At is encoded by a single gene, but the amplification by the polymerase chain reaction of other fragments homologous to cyclins indicates the presence of a family of cyclins in Arabidopsis.

The Arabidopsis functional homolog of the p34cdc2 protein kinase.

The p34cdc2 protein kinase is a key component of the eukaryotic cell cycle, which is required for G1 to S-phase transition and for entry into mitosis. Using a 380-base pair DNA fragment obtained by polymerase chain reaction amplification from an Arabidopsis thaliana flower cDNA library as a probe, we isolated and sequenced a cdc2-homologous cDNA from Arabidopsis. The encoded polypeptide has extensive homology with cdc2-like kinases. Furthermore, when expressed in a CDC28ts Saccharomyces strain, it partially restores the capacity to grow at 36 degrees C, indicating that the plant cDNA is a functional homolog of the p34cdc2 kinase. Genomic hybridization demonstrated that there is one copy of the cdc2 gene per Arabidopsis haploid genome. Using RNA gel blot analysis, we found that cdc2 mRNA is present in all plant organs.