Biomedical subjects

Jose M Alonso

Publications and source records attributed to Jose M Alonso.

At least 19 recordsLinked to original sources

PubMed2006

The Arabidopsis histidine phosphotransfer proteins are redundant positive regulators of cytokinin signaling.

Arabidopsis thaliana histidine phosphotransfer proteins (AHPs) are similar to bacterial and yeast histidine phosphotransfer proteins (HPts), which act in multistep phosphorelay signaling pathways. A phosphorelay pathway is the current model for cytokinin signaling. To assess the role of AHPs in cytokinin signaling, we isolated T-DNA insertions in the five AHP genes that are predicted to encode functional HPts and constructed multiple insertion mutants, including an ahp1,2,3,4,5 quintuple mutant. Single ahp mutants were indistinguishable from wild-type seedlings in cytokinin response assays. However, various higher-order mutants displayed reduced sensitivity to cytokinin in diverse cytokinin assays, indicating both a positive role for AHPs in cytokinin signaling and functional overlap among the AHPs. In contrast with the other four AHPs, AHP4 may play a negative role in some cytokinin responses. The quintuple ahp mutant showed various abnormalities in growth and development, including reduced fertility, increased seed size, reduced vascular development, and a shortened primary root. These data indicate that most of the AHPs are redundant, positive regulators of cytokinin signaling and affect multiple aspects of plant development.

Arabidopsis↗

PubMed2006

Downregulation of ClpR2 leads to reduced accumulation of the ClpPRS protease complex and defects in chloroplast biogenesis in Arabidopsis.

Plastids contain tetradecameric Clp protease core complexes, with five ClpP Ser-type proteases, four nonproteolytic ClpR, and two associated ClpS proteins. Accumulation of total ClpPRS complex decreased twofold to threefold in an Arabidopsis thaliana T-DNA insertion mutant in CLPR2 designated clpr2-1. Differential stable isotope labeling of the ClpPRS complex with iTRAQ revealed a fivefold reduction in assembled ClpR2 accumulation and twofold to fivefold reductions in the other subunits. A ClpR2:(his)(6) fusion protein that incorporated into the chloroplast ClpPRS complex fully complemented clpr2-1. The reduced accumulation of the ClpPRS protease complex led to a pale-green phenotype with delayed shoot development, smaller chloroplasts, decreased thylakoid accumulation, and increased plastoglobule accumulation. Stromal ClpC1 and 2 were both recruited to the thylakoid surface in clpr2-1. The thylakoid membrane of clpr2-1 showed increased carotenoid content, partial inactivation of photosystem II, and upregulated thylakoid proteases and stromal chaperones, suggesting an imbalance in chloroplast protein homeostasis and a well-coordinated network of proteolysis and chaperone activities. Interestingly, a subpopulation of PsaF and several light-harvesting complex II proteins accumulated in the thylakoid with unprocessed chloroplast transit peptides. We conclude that ClpR2 cannot be functionally replaced by other ClpP/R homologues and that the ClpPRS complex is central to chloroplast biogenesis, thylakoid protein homeostasis, and plant development.

Amino Acid Sequence↗

PubMed2006

Moving forward in reverse: genetic technologies to enable genome-wide phenomic screens in Arabidopsis.

Genome sequencing, in combination with various computational and empirical approaches to sequence annotation, has made possible the identification of more than 30,000 genes in Arabidopsis thaliana. Increasingly sophisticated genetic tools are being developed with the long-term goal of understanding how the coordinated activity of these genes gives rise to a complex organism. The combination of classical forward genetics with recently developed genome-wide, gene-indexed mutant collections is beginning to revolutionize the way in which gene functions are studied in plants. High-throughput screens using these mutant populations should provide a means to analyse plant gene functions--the phenome--on a genomic scale.

Arabidopsis↗

PubMed2006

A practical ruthenium-catalyzed cleavage of the allyl protecting group in amides, lactams, imides, and congeners.

A convenient methodology for the deprotection of N-allylic amide-like moieties was developed. The first examples accounting for the ruthenium-catalyzed deallylation of amides, lactams, imides, pyrazolidones, hydantoins, and oxazolidinones have been achieved by the sequential use of Grubbs carbene (isomerization step) and RuCl(3) (oxidation step). A variety of substrates, including enantiopure multifunctional beta- and gamma-lactams, can be employed.

Allyl Compounds↗

PubMed2006

Molecular mechanisms of ethylene signaling in Arabidopsis.

Ethylene is a gaseous plant hormone involved in several important physiological processes throughout a plant's life cycle. Decades of scientific research devoted to deciphering how plants are able to sense and respond to this key molecule have culminated in the establishment of one of the best characterized signal transduction pathways in plants. The ethylene signaling pathway starts with the perception of this gaseous hormone by a family of membrane-anchored receptors followed by a Raf-like kinase CTR1 that is physically associated with the receptors and actively inhibits downstream components of the pathway. A major gap is represented by the mysterious plant protein EIN2 that genetically works downstream of CTR1 and upstream of the key transcription factor EIN3. Transcriptional regulation by EIN3 and EIN3-family members has emerged as a key aspect of ethylene responses. The major components of this transcriptional cascade have been characterized and the involvement of post-transcriptional control by ubiquitination has been determined. Nevertheless, many aspects of this pathway still remain unknown. Recent genomic studies aiming to provide a more comprehensive view of modulation of gene expression have further emphasized the ample role of ethylene in a myriad of cellular processes and particularly in its crosstalk with other important plant hormones. This review aims to serve as a guide to the main scientific discoveries that have shaped the field of ethylene biology in the recent years.

Arabidopsis↗

PubMed2006

CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-type anion- and Ca(2+)-permeable channels and stomatal closure.

Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca(2+) in guard cell ion channel regulation. However, genetic mutants in Ca(2+) sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca(2+)-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell-expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca(2+) activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca(2+)-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca(2+)-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca(2+) oscillation experiments revealed that Ca(2+)-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca(2+)-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca(2+)-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling.

Abscisic Acid↗

PubMed2006

PCR-based screening for insertional mutants.

In Arabidopsis, where homologous recombination is highly inefficient, insertional mutagenesis has provided a great alternative means for identifying loss-of-function mutants in genes of interest. Many collections of T-DNA and transposon-tagged plants have been generated and several have been made available to the research community. With recent sequencing of the insertion sites and the creation of public databases, identification of knockouts in most of the genes of interest has become a matter of a few minutes. However, because of the random selection of the insertion lines to be sequenced, knockouts of the remaining several thousand genes may not be easily found by the mere continuation of these types of approaches. Therefore, alternative methods such as polymerase chain reaction (PCR)-based screening of pooled genomic DNA from mutant lines using a combination of tag- and gene-specific primers, once again, are becoming valuable for obtaining loss-of-function alleles in the genes "missed" by the sequencing projects. In this chapter, we provide a detailed description of the design and implementation of a PCR-based screen for insertional knockouts using T-DNA-mutagenized lines as an example.

Alleles↗

PubMed2006

[Chlamydia trachomatis and papillomavirus infection in women with cytohistological abnormalities in uterine cervix].

The presence of Chlamydia trachomatis and human papillomavirus (HPV) was evaluated by PCR technique in 189 sexually active women, between 15 and 58 years old, with cytological abnormalities in their uterine cervical epithelium and belonging to a region of Argentina with high incidence of cervical cancer. Risk factors in relation to chlamydial infection were also analyzed. Total prevalence for C. trachomatis infection was 24.9%, but there was a significant difference between prevalence in low socio-economical level (32.9%) and high or medium socio-economical level (17.7%). Total prevalence for DNA of HPV was 52.9%, but women infected with C. trachomatis showed a higher risk for viral infection than non-infected ones (OR = 2.27 / CI 95% = 1.10-4.73), with statistical significant difference (p = 0.016).

Adolescent↗

PubMed2005

Multiple type-B response regulators mediate cytokinin signal transduction in Arabidopsis.

Type-B Arabidopsis thaliana response regulators (ARRs) are transcription factors that function in the final step of two-component signaling systems. To characterize their role in plant growth and development, we isolated T-DNA insertions within six of the genes (ARR1, ARR2, ARR10, ARR11, ARR12, and ARR18) from the largest subfamily of type-B ARRs and also constructed various double and triple combinations of these mutations. Higher order mutants revealed progressively decreased sensitivity to cytokinin, including effects on root elongation, lateral root formation, callus induction and greening, and induction of cytokinin primary response genes. The triple mutant arr1,10,12 showed almost complete insensitivity to cytokinin under many of the assay conditions used. By contrast, no significant change in the sensitivity to ethylene was found among the mutants examined. These results indicate that there is functional overlap among the type-B ARRs and that they act as positive regulators of cytokinin signal transduction.

Arabidopsis↗

PubMed2005

A Link between ethylene and auxin uncovered by the characterization of two root-specific ethylene-insensitive mutants in Arabidopsis.

The plant hormone ethylene participates in the regulation of a variety of developmental processes and serves as a key mediator of plant responses to biotic and abiotic stress factors. The diversity of ethylene functions is achieved, at least in part, by combinatorial interactions with other hormonal signals. Here, we show that ethylene-triggered inhibition of root growth, one of the classical effects of ethylene in Arabidopsis thaliana seedlings, is mediated by the action of the WEAK ETHYLENE INSENSITIVE2/ANTHRANILATE SYNTHASE alpha1 (WEI2/ASA1) and WEI7/ANTHRANILATE SYNTHASE beta1 (ASB1) genes that encode alpha- and beta-subunits of a rate-limiting enzyme of Trp biosynthesis, anthranilate synthase. Upregulation of WEI2/ASA1 and WEI7/ASB1 by ethylene results in the accumulation of auxin in the tip of primary root, whereas loss-of-function mutations in these genes prevent the ethylene-mediated auxin increase. Furthermore, wei2 and wei7 suppress the high-auxin phenotypes of superroot1 (sur1) and sur2, two auxin-overproducing mutants, suggesting that the roles of WEI2 and WEI7 in the regulation of auxin biosynthesis are not restricted to the ethylene response. Together, these findings reveal that ASA1 and ASB1 are key elements in the regulation of auxin production and an unexpected node of interaction between ethylene responses and auxin biosynthesis in Arabidopsis. This study provides a mechanistic explanation for the root-specific ethylene insensitivity of wei2 and wei7, illustrating how interactions between hormones can be used to achieve response specificity.

Anthranilate Synthase↗

PubMed2005

Ethylene signaling pathway.

The structural simplicity of the plant hormone ethylene contrasts with its dramatic effects in various developmental processes, as well as in the cellular processes that ethylene initiates in response to a diversity of environmental signals. A single well-conserved signaling cascade mediates this broad spectrum of responses. Ethylene is perceived by a family of two-component histidine kinase receptors that become inactivated upon ethylene binding. In the absence of the hormone, the receptors activate CTR1, a negative regulator of ethylene responses. Sequence similarity between CTR1 and the Raf protein kinases implies involvement of a mitogen-activated protein kinase cascade in this signaling pathway. The protein EIN2 acts downstream of CTR1 and the possible kinase cascade. Although the biochemical function of EIN2 is not understood, its critical role is manifested by the complete ethylene insensitivity of EIN2 loss-of-function mutants. Downstream of EIN2, a family of plant-specific EIN3-like transcription factors mediate ethylene responses. The regulation of EIN3 stability by ethylene is accomplished by F-box-containing proteins that participate in the formation of a SKP1/cullin/F-box complex that targets proteins for degradation by the proteasome. A large number of ethylene-regulated genes have been identified, including the APETALA2 domain-containing transcription factor genes ERF1 and EDF1 to 4, which suggests the participation of a transcriptional cascade in the ethylene response. The differential regulation of some components of this complex nuclear cascade by other signaling pathways provides a possible mechanism for interaction and signal integration. As new points of intersection with other pathways and additional participants in the pathway are identified, the Connections Map will be updated to include this new information.

Arabidopsis↗

PubMed2005

Arabidopsis ethylene signaling pathway.

In plants, ethylene gas functions as a potent endogenous growth regulator. In the model system Arabidopsis thaliana, the molecular mechanisms that underlie perception and transduction of the ethylene signal to the nucleus, where the transcription of hundreds of genes is altered, are being elucidated. In the current view, ethylene is sensed by a family of five receptors that show similarity to the bacterial two-component histidine kinases, and in plants function as negative regulators of the pathway. Binding of the ethylene gas turns off the receptors, resulting in the inactivation of another negative regulator of ethylene signaling, CTR1, a Raf-like protein kinase that directly interacts with the receptors. EIN2, a protein of unknown biochemical activity that functions as a positive regulator of the pathway, acts downstream of CTR. Derepression of EIN2 by ethylene upon disabling of the receptors and CTR1 leads to the activation of EIN3 and EIN3-like transcription factors. In the absence of ethylene, the levels of EIN3 protein are extremely low because of the function of two F-box-containing proteins, EBF1 and EBF2, that target EIN3 for proteosome-mediated degradation. In the presence of ethylene, the EIN3 protein accumulates in the nucleus and initiates a transcriptional cascade, resulting in the activation and repression of hundreds of genes. To date, the only empirically demonstrated direct target of EIN3 is the APETALA2 (AP2)-domain-containing transcription factor gene ERF1. The coregulation of ERF1 by another plant hormone, jasmonic acid, illustrates how a transcriptional cascade could be utilized in a combinatorial fashion to generate a large diversity of responses using a limited number of input signals. As new components and points of intersection with other pathways are identified, the Connections Map will be updated.

Arabidopsis↗

PubMed2005

Phytochrome-specific type 5 phosphatase controls light signal flux by enhancing phytochrome stability and affinity for a signal transducer.

Environmental light information such as quality, intensity, and duration in red (approximately 660 nm) and far-red (approximately 730 nm) wavelengths is perceived by phytochrome photoreceptors in plants, critically influencing almost all developmental strategies from germination to flowering. Phytochromes interconvert between red light-absorbing Pr and biologically functional far-red light-absorbing Pfr forms. To ensure optimal photoresponses in plants, the flux of light signal from Pfr-phytochromes should be tightly controlled. Phytochromes are phosphorylated at specific serine residues. We found that a type 5 protein phosphatase (PAPP5) specifically dephosphorylates biologically active Pfr-phytochromes and enhances phytochrome-mediated photoresponses. Depending on the specific serine residues dephosphorylated by PAPP5, phytochrome stability and affinity for a downstream signal transducer, NDPK2, were enhanced. Thus, phytochrome photoreceptors have developed an elaborate biochemical tuning mechanism for modulating the flux of light signal, employing variable phosphorylation states controlled by phosphorylation and PAPP5-mediated dephosphorylation as a mean to control phytochrome stability and affinity for downstream transducers.

Arabidopsis↗

PubMed2005

Functional genomic analysis of the AUXIN RESPONSE FACTOR gene family members in Arabidopsis thaliana: unique and overlapping functions of ARF7 and ARF19.

The AUXIN RESPONSE FACTOR (ARF) gene family products, together with the AUXIN/INDOLE-3-ACETIC ACID proteins, regulate auxin-mediated transcriptional activation/repression. The biological function(s) of most ARFs is poorly understood. Here, we report the identification and characterization of T-DNA insertion lines for 18 of the 23 ARF gene family members in Arabidopsis thaliana. Most of the lines fail to show an obvious growth phenotype except of the previously identified arf2/hss, arf3/ett, arf5/mp, and arf7/nph4 mutants, suggesting that there are functional redundancies among the ARF proteins. Subsequently, we generated double mutants. arf7 arf19 has a strong auxin-related phenotype not observed in the arf7 and arf19 single mutants, including severely impaired lateral root formation and abnormal gravitropism in both hypocotyl and root. Global gene expression analysis revealed that auxin-induced gene expression is severely impaired in the arf7 single and arf7 arf19 double mutants. For example, the expression of several genes, such as those encoding members of LATERAL ORGAN BOUNDARIES domain proteins and AUXIN-REGULATED GENE INVOLVED IN ORGAN SIZE, are disrupted in the double mutant. The data suggest that the ARF7 and ARF19 proteins play essential roles in auxin-mediated plant development by regulating both unique and partially overlapping sets of target genes. These observations provide molecular insight into the unique and overlapping functions of ARF gene family members in Arabidopsis.

Arabidopsis↗

PubMed2005

An Arabidopsis NPR1-like gene, NPR4, is required for disease resistance.

The Arabidopsis genome contains six NPR1-related genes. Given the pivotal role played by NPR1 in controlling salicylic acid (SA)-mediated gene expression and disease resistance, functional characterization of other family members appears to be justified. Reverse genetics was used to analyze the role of one NPR1-like gene, which we called NPR4. The NPR4 protein shares 36% identity with NPR1 and interacts with the same spectrum of TGA transcription factors in yeast two-hybrid assays. Plants with T-DNA insertions in NPR4 are more susceptible to the virulent bacterial pathogen Pseudomonas syringe pv. tomato DC3000. This phenotype is complemented by expression of the wild type NPR4 coding region. As determined by the parasite reproduction, the npr4-1 mutant is more susceptible to the fungal pathogen Erysiphe cichoracearum, but does not differ markedly from wild type in its interaction with virulent and avirulent strains of the oomycete Peronospora parasitica. In leaves of wild-type plants, NPR4 mRNA levels increase following pathogen challenge or SA treatment, and decrease rapidly following methyl jasmonic acid (MeJA) treatment. Transcripts of the pathogenesis-related (PR) genes PR-1, PR-2, and PR-5 are only marginally reduced in the npr4-1 mutant following pathogen challenge or SA treatment. This reduction of PR gene expression is more pronounced when leaves are challenged with the bacterial pathogen following SA treatment. Expression of the jasmonic acid-dependent pathway marker gene PDF1.2 is compromised in npr4-1 leaves following application of MeJA or a combination of SA and MeJA. These results indicate that NPR4 is required for basal defense against pathogens, and that it may be implicated in the cross-talk between the SA- and JA-dependent signaling pathways.

Amino Acid Sequence↗

PubMed2005

NPH4/ARF7 and ARF19 promote leaf expansion and auxin-induced lateral root formation.

Auxin response factors (ARFs) bind auxin response promoter elements and mediate transcriptional responses to auxin. Five of the 22 ARF genes in Arabidopsis thaliana encode ARFs with glutamine-rich middle domains. Four of these can activate transcription and have been ascribed developmental functions. We show that ARF19, the fifth Q-rich ARF, also activates transcription. Mutations in ARF19 have little effect on their own, but in combination with mutations in NPH4/ARF7, encoding the most closely related ARF, they cause several phenotypes including a drastic decrease in lateral and adventitious root formation and a decrease in leaf cell expansion. These results indicate that auxin induces lateral roots and leaf expansion by activating NPH4/ARF7 and ARF19. Auxin induces the ARF19 gene, and NPH4/ARF7 and ARF19 together are required for expression of one of the arf19 mutant alleles, suggesting that a positive feedback loop regulates leaf expansion and/or lateral root induction.

Arabidopsis↗

PubMed2004

The ethylene signaling pathway.

Plants use a structurally very simple gas molecule, the hydrocarbon ethylene, to modulate various developmental programs and coordinate responses to a multitude of external stress factors. How this simple molecule generates such a diverse array of effects has been the subject of intense research for the past two decades. A fascinating signaling pathway, with classical as well as novel plant-specific signaling elements, is emerging from these studies. We describe the four main modules that constitute this signaling pathway: a phosphotransfer relay, an EIN2-based unit, a ubiquitin-mediated protein degradation component, and a transcriptional cascade. The canonical and Arabidopsis ethylene signaling pathways in the Signal Transduction Knowledge Environment Connections Maps provide a complete panoramic view of these signaling events in plants.

Arabidopsis↗

PubMed2004

The phytochrome-interacting transcription factor, PIF3, acts early, selectively, and positively in light-induced chloroplast development.

The phytochrome (phy) family of sensory photoreceptors transduce informational light signals to selected nuclear genes, inducing plant growth and developmental responses appropriate to the environment. Existing data suggest that one signaling pathway by which this occurs involves direct, intranuclear interaction of the photoactivated phy molecule with PIF3, a basic helix-loop-helix transcription factor. Here, we provide evidence from recently identified pif3 mutant alleles that PIF3 is necessary for early chloroplast greening and rapid phy-induced expression of nuclear genes encoding chloroplast components upon first exposure of seedlings to light. Therefore, these data indicate that PIF3 functions to transduce phy signals to genes involved in a critical facet of the early seedling deetiolation process, the generation of a functional photosynthetic apparatus. When transgenically expressed GUS:PIF3 fusion protein constructs were used, we found that PIF3 protein levels are rapidly and reversibly modulated by the photoreceptor over diurnal cycles in Arabidopsis seedlings. The PIF3 protein declines rapidly to a basal steady-state level upon initial light exposure, but reaccumulates to preirradiation levels in darkness during the subsequent night period. These data suggest that PIF3 may function in early phy signaling at the dark-to-light transition, not only during initial seedling deetiolation, but daily at dawn under diurnal light-dark cycles.

Arabidopsis↗