Showing papers on "Arabidopsis published in 2004"

Novel and Stress-Regulated MicroRNAs and Other Small RNAs from Arabidopsis

Abstract: MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are small noncoding RNAs that have recently emerged as important regulators of mRNA degradation, translational repression, and chromatin modification. In Arabidopsis thaliana, 43 miRNAs comprising 15 families have been reported thus far. In an attempt to identify novel and abiotic stress regulated miRNAs and siRNAs, we constructed a library of small RNAs from Arabidopsis seedlings exposed to dehydration, salinity, or cold stress or to the plant stress hormone abscisic acid. Sequencing of the library and subsequent analysis revealed 26 new miRNAs from 34 loci, forming 15 new families. Two of the new miRNAs from three loci are members of previously reported miR171 and miR319 families. Some of the miRNAs are preferentially expressed in specific tissues, and several are either upregulated or downregulated by abiotic stresses. Ten of the miRNAs are highly conserved in other plant species. Fifty-one potential targets with diverse function were predicted for the newly identified miRNAs based on sequence complementarity. In addition to miRNAs, we identified 102 other novel endogenous small RNAs in Arabidopsis. These findings suggest that a large number of miRNAs and other small regulatory RNAs are encoded by the Arabidopsis genome and that some of them may play important roles in plant responses to environmental stresses as well as in development and genome maintenance.

A MicroRNA as a Translational Repressor of APETALA2 in Arabidopsis Flower Development

Abstract: Plant microRNAs (miRNAs) show a high degree of sequence complementarity to, and are believed to guide the cleavage of, their target messenger RNAs. Here, I show that miRNA172, which can base-pair with the messenger RNA of a floral homeotic gene, APETALA2, regulates APETALA2 expression primarily through translational inhibition. Elevated miRNA172 accumulation results in floral organ identity defects similar to those in loss-of-function apetala2 mutants. Elevated levels of mutant APETALA2 RNA with disrupted miRNA172 base pairing, but not wild-type APETALA2 RNA, result in elevated levels of APETALA2 protein and severe floral patterning defects. Therefore, miRNA172 likely acts in cell-fate specification as a translational repressor of APETALA2 in Arabidopsis flower development.

The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana

Abstract: Background Most genes in Arabidopsis thaliana are members of gene families. How do the members of gene families arise, and how are gene family copy numbers maintained? Some gene families may evolve primarily through tandem duplication and high rates of birth and death in clusters, and others through infrequent polyploidy or large-scale segmental duplications and subsequent losses.

Isolation and Functional Analysis of Arabidopsis Stress-Inducible NAC Transcription Factors That Bind to a Drought-Responsive cis-Element in the early responsive to dehydration stress 1 Promoter

Abstract: The MYC-like sequence CATGTG plays an important role in the dehydration-inducible expression of the Arabidopsis thaliana EARLY RESPONSIVE TO DEHYDRATION STRESS 1 (ERD1) gene, which encodes a ClpA (ATP binding subunit of the caseinolytic ATP-dependent protease) homologous protein. Using the yeast one-hybrid system, we isolated three cDNA clones encoding proteins that bind to the 63-bp promoter region of erd1, which contains the CATGTG motif. These three cDNA clones encode proteins named ANAC019, ANAC055, and ANAC072, which belong to the NAC transcription factor family. The NAC proteins bound specifically to the CATGTG motif both in vitro and in vivo and activated the transcription of a beta-glucuronidase (GUS) reporter gene driven by the 63-bp region containing the CATGTG motif in Arabidopsis T87 protoplasts. The expression of ANAC019, ANAC055, and ANAC072 was induced by drought, high salinity, and abscisic acid. A histochemical assay using P(NAC)-GUS fusion constructs showed that expression of the GUS reporter gene was localized mainly to the leaves of transgenic Arabidopsis plants. Using the yeast one-hybrid system, we determined the complete NAC recognition sequence, containing CATGT and harboring CACG as the core DNA binding site. Microarray analysis of transgenic plants overexpressing either ANAC019, ANAC055, or ANAC072 revealed that several stress-inducible genes were upregulated in the transgenic plants, and the plants showed significantly increased drought tolerance. However, erd1 was not upregulated in the transgenic plants. Other interacting factors may be necessary for the induction of erd1 in Arabidopsis under stress conditions.

The ubiquitin 26S proteasome proteolytic pathway

Abstract: Much of plant physiology, growth, and development is controlled by the selective removal of short-lived regulatory proteins. One important proteolytic pathway involves the small protein ubiquitin (Ub) and the 26S proteasome, a 2-MDa protease complex. In this pathway, Ub is attached to proteins destined for degradation; the resulting Ub-protein conjugates are then recognized and catabolized by the 26S proteasome. This review describes our current understanding of the pathway in plants at the biochemical, genomic, and genetic levels, using Arabidopsis thaliana as the model. Collectively, these analyses show that the Ub/26S proteasome pathway is one of the most elaborate regulatory mechanisms in plants. The genome of Arabidopsis encodes more than 1400 (or >5% of the proteome) pathway components that can be connected to almost all aspects of its biology. Most pathway components participate in the Ub-ligation reactions that choose with exquisite specificity which proteins should be ubiquitinated. What remains to be determined is the identity of the targets, which may number in the thousands in plants.

Genome-Wide Analysis of Arabidopsis Pentatricopeptide Repeat Proteins Reveals Their Essential Role in Organelle Biogenesis

Abstract: The complete sequence of the Arabidopsis thaliana genome revealed thousands of previously unsuspected genes, many of which cannot be ascribed even putative functions. One of the largest and most enigmatic gene families discovered in this way is characterized by tandem arrays of pentatricopeptide repeats (PPRs). We describe a detailed bioinformatic analysis of 441 members of the Arabidopsis PPR family plus genomic and genetic data on the expression (microarray data), localization (green fluorescent protein and red fluorescent protein fusions), and general function (insertion mutants and RNA binding assays) of many family members. The basic picture that arises from these studies is that PPR proteins play constitutive, often essential roles in mitochondria and chloroplasts, probably via binding to organellar transcripts. These results confirm, but massively extend, the very sparse observations previously obtained from detailed characterization of individual mutants in other organisms.

Photoreceptor regulation of CONSTANS protein in photoperiodic flowering

Abstract: Many plants flower in response to seasonal fluctuations in day length. The CONSTANS (CO) gene of Arabidopsis promotes flowering in long days. Flowering is induced when CO messenger RNA expression coincides with the exposure of plants to light. However, how this promotes CO activity is unknown. We show that light stabilizes nuclear CO protein in the evening, whereas in the morning or in darkness the protein is degraded by the proteasome. Photoreceptors regulate CO stability and act antagonistically to generate daily rhythms in CO abundance. This layer of regulation refines the circadian rhythm in CO messenger RNA and is central to the mechanism by which day length controls flowering.

Bacterial Volatiles Induce Systemic Resistance in Arabidopsis

Abstract: Plant growth-promoting rhizobacteria, in association with plant roots, can trigger induced systemic resistance (ISR). Considering that low-molecular weight volatile hormone analogues such as methyl jasmonate and methyl salicylate can trigger defense responses in plants, we examined whether volatile organic compounds (VOCs) associated with rhizobacteria can initiate ISR. In Arabidopsis seedlings exposed to bacterial volatile blends from Bacillus subtilis GB03 and Bacillus amyloliquefaciens IN937a, disease severity by the bacterial pathogen Erwinia carotovora subsp. carotovora was significantly reduced compared with seedlings not exposed to bacterial volatiles before pathogen inoculation. Exposure to VOCs from rhizobacteria for as little as 4 d was sufficient to activate ISR in Arabidopsis seedlings. Chemical analysis of the bacterial volatile emissions revealed the release of a series of low-molecular weight hydrocarbons including the growth promoting VOC (2R,3R)-(-)-butanediol. Exogenous application of racemic mixture of (RR) and (SS) isomers of 2,3-butanediol was found to trigger ISR and transgenic lines of B. subtilis that emitted reduced levels of 2,3-butanediol and acetoin conferred reduced Arabidopsis protection to pathogen infection compared with seedlings exposed to VOCs from wild-type bacterial lines. Using transgenic and mutant lines of Arabidopsis, we provide evidence that the signaling pathway activated by volatiles from GB03 is dependent on ethylene, albeit independent of the salicylic acid or jasmonic acid signaling pathways. This study provides new insight into the role of bacteria VOCs as initiators of defense responses in plants.

The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine in Arabidopsis.

Abstract: Despite its importance in a variety of plant defense responses, our understanding of how jasmonic acid (JA) functions at the biochemical level is limited. Several amino acid conjugates of JA were tested for their ability to complement the JA-insensitive Arabidopsis thaliana mutant jar1-1. Unlike free JA, JA-Ile inhibited root growth in jar1-1 to the same extent as in the wild type, whereas JA-Val, JA-Leu, and JA-Phe were ineffective inhibitors in both genotypes. Thin-layer chromatography and gas chromatography–mass spectrometry (GC-MS) analysis of products produced in vitro by recombinant JAR1 demonstrated that this enzyme forms JA-amido conjugates with several amino acids, including JA-Ile. JA-Val, -Leu, -Ile, and -Phe were each quantified in Arabidopsis seedlings by GC-MS. JA-Ile was found at 29.6 pmole g−1 fresh weight (FW) in the wild type but was more than sevenfold lower in two jar1 alleles. JA-Leu, -Val, and -Phe were present at only low levels in both genotypes. Expression of wild-type JAR1 in transgenic jar1-1 plants restored sensitivity to JA and elevated JA-Ile to the same level as in the wild type. The ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) conjugated to JA was also found in plant tissue at 18.4 pmole g−1 FW. JA-ACC was determined not be an effective jasmonate root inhibitor, and surprisingly, was twofold higher in the mutants than in the wild type. This suggests that another JA-conjugating enzyme(s) is present in Arabidopsis. Synthesis of JA-ACC might provide a mechanism to coregulate the availability of JA and ACC for conversion to the active hormones JA-Ile and ethylene, respectively. We conclude that JAR1 is a JA-amino synthetase that is required to activate JA for optimal signaling in Arabidopsis. Plant hormone activation by conjugation to amino acids and the enzymes involved in their formation were previously unknown.

Comparative Analysis of the Receptor-Like Kinase Family in Arabidopsis and Rice

Abstract: Receptor-like kinases (RLKs) belong to the large RLK/Pelle gene family, and it is known that the Arabidopsis thaliana genome contains >600 such members, which play important roles in plant growth, development, and defense responses. Surprisingly, we found that rice (Oryza sativa) has nearly twice as many RLK/Pelle members as Arabidopsis does, and it is not simply a consequence of a larger predicted gene number in rice. From the inferred phylogeny of all Arabidopsis and rice RLK/Pelle members, we estimated that the common ancestor of Arabidopsis and rice had >440 RLK/Pelles and that large-scale expansions of certain RLK/Pelle members and fusions of novel domains have occurred in both the Arabidopsis and rice lineages since their divergence. In addition, the extracellular domains have higher nonsynonymous substitution rates than the intracellular domains, consistent with the role of extracellular domains in sensing diverse signals. The lineage-specific expansions in Arabidopsis can be attributed to both tandem and large-scale duplications, whereas tandem duplication seems to be the major mechanism for recent expansions in rice. Interestingly, although the RLKs that are involved in development seem to have rarely been duplicated after the Arabidopsis–rice split, those that are involved in defense/disease resistance apparently have undergone many duplication events. These findings led us to hypothesize that most of the recent expansions of the RLK/Pelle family have involved defense/resistance-related genes.

The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development.

Abstract: MicroRNAs (miRNAs) are endogenous 21–24-nt RNAs that can down-regulate gene expression by pairing to the messages of protein-coding genes to specify mRNA cleavage or repression of productive translation. They act within the RNA-induced silencing complex (RISC), which in animals contains a member of the Argonaute family of proteins. In the present study, we show that Arabidopsis ago1 mutants have increased accumulation of mRNAs known to be targeted for cleavage by miRNAs. In hypomorphic ago1 alleles, this compromised miRNA function occurs without a substantial change in miRNA accumulation, whereas in null alleles it is accompanied by a drop in some of the miRNAs. Therefore, AGO1 acts within the Arabidopsis miRNA pathway, probably within the miRNA-programmed RISC, such that the absence of AGO1 destabilizes some of the miRNAs. We also show that targeting of AGO1 mRNA by miR168 is needed for proper plant development, illustrating the importance of feedback control by this miRNA. Transgenic plants expressing a mutant AGO1 mRNA with decreased complementarity to miR168 overaccumulate AGO1 mRNA and exhibit developmental defects partially overlapping with those of dcl1, hen1, and hyl1 mutants showing a decrease in miRNA accumulation. miRNA targets overaccumulate in miR168-resistant plants, suggesting that a large excess of AGO1 protein interferes with the function of RISC or sequesters miRNAs or other RISC components. Developmental defects induced by a miR168-resistant AGO1 mRNA can be rescued by a compensatory miRNA that is complementary to the mutant AGO1 mRNA, proving the regulatory relationship between miR168 and its target and opening the way for engineering artificial miRNAs in plants.

Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions

Abstract: Micro-RNAs (miRNAs) are small, noncoding RNAs of 18-25 nt in length that negatively regulate their complementary mRNAs at the posttranscriptional level. Previous work has shown that some RNase III-like enzymes such as Drosha and Dicer are known to be involved in miRNA biogenesis in animals. However, the mechanism of plant miRNA biogenesis still remains poorly understood. In this article, the process of Arabidopsis miR163 biogenesis was examined. The results revealed that two types of miR163 primary transcripts (pri-miR163s) are transcribed from a single gene by RNA polymerase II and that miR163 biogenesis requires at least three cleavage steps by RNase III-like enzymes at 21-nt-long intervals. The first step is from pri-miR163 to long miR163 precursor (premiR163), the second step is from long pre-miR163 to short premiR163, and the last step is from short pre-miR163 to mature miR163 and the remnant. It is interesting that, during the process, four small RNAs including miR163 are released. By using dcl1 mutants, it was demonstrated that Arabidopsis Dicer homologue Dicer-like 1 (DCL1) catalyzes at least the first and second cleavage steps and that double-stranded RNA-binding domains of DCL1 are involved in positioning of the cleavage sites. Our result is direct evidence that DCL1 is involved in processing of pri- and pre-miRNA.

The Arabidopsis cytochrome P450 CYP707A encodes ABA 8′‐hydroxylases: key enzymes in ABA catabolism

Abstract: The hormonal action of abscisic acid (ABA) in plants is controlled by the precise balance between its biosynthesis and catabolism. In plants, ABA 8′-hydroxylation is thought to play a predominant role in ABA catabolism. ABA 8′-hydroxylase was shown to be a cytochrome P450 (P450); however, its corresponding gene had not been identified. Through phylogenetic and DNA microarray analyses during seed imbibition, the candidate genes for this enzyme were narrowed down from 272 Arabidopsis P450 genes. These candidate genes were functionally expressed in yeast to reveal that members of the CYP707A family, CYP707A1–CYP707A4, encode ABA 8′-hydroxylases. Expression analyses revealed that CYP707A2 is responsible for the rapid decrease in ABA level during seed imbibition. During drought stress conditions, all CYP707A genes were upregulated, and upon rehydration a significant increase in mRNA level was observed. Consistent with the expression analyses, cyp707a2 mutants exhibited hyperdormancy in seeds and accumulated six-fold greater ABA content than wild type. These results demonstrate that CYP707A family genes play a major regulatory role in controlling the level of ABA in plants.

The N Terminus of Bacterial Elongation Factor Tu Elicits Innate Immunity in Arabidopsis Plants

Abstract: Innate immunity is based on the recognition of pathogen-associated molecular patterns (PAMPs). Here, we show that elongation factor Tu (EF-Tu), the most abundant bacterial protein, acts as a PAMP in Arabidopsis thaliana and other Brassicaceae. EF-Tu is highly conserved in all bacteria and is known to be N-acetylated in Escherichia coli. Arabidopsis plants specifically recognize the N terminus of the protein, and an N-acetylated peptide comprising the first 18 amino acids, termed elf18, is fully active as inducer of defense responses. The shorter peptide, elf12, comprising the acetyl group and the first 12 N-terminal amino acids, is inactive as elicitor but acts as a specific antagonist for EF-Tu-related elicitors. In leaves of Arabidopsis plants, elf18 induces an oxidative burst and biosynthesis of ethylene, and it triggers resistance to subsequent infection with pathogenic bacteria.

A dehydration‐induced NAC protein, RD26, is involved in a novel ABA‐dependent stress‐signaling pathway

Abstract: *Summary Arabidopsis thaliana RD26 cDNA, isolated from dehydrated plants, encodes a NAC protein. Expression of the RD26 gene was induced not only by drought but also by abscisic acid (ABA) and high salinity. The RD26 protein is localized in the nucleus and its C terminal has transcriptional activity. Transgenic plants overexpressing RD26 were highly sensitive to ABA, while RD26-repressed plants were insensitive. The results of microarray analysis showed that ABA- and stress-inducible genes are upregulated in the RD26-overexpressed plants and repressed in the RD26-repressed plants. Furthermore, RD26 activated a promoter of its target gene in Arabidopsis protoplasts. These results indicate that RD26 functions as a transcriptional activator in ABAinducible gene expression under abiotic stress in plants.

Phosphorylation of 1-Aminocyclopropane-1-Carboxylic Acid Synthase by MPK6, a Stress-Responsive Mitogen-Activated Protein Kinase, Induces Ethylene Biosynthesis in Arabidopsis

Abstract: Mitogen-activated protein kinases (MAPKs) are implicated in regulating plant growth, development, and response to the environment. However, the underlying mechanisms are unknown because of the lack of information about their substrates. Using a conditional gain-of-function transgenic system, we demonstrated that the activation of SIPK, a tobacco (Nicotiana tabacum) stress-responsive MAPK, induces the biosynthesis of ethylene. Here, we report that MPK6, the Arabidopsis thaliana ortholog of tobacco SIPK, is required for ethylene induction in this transgenic system. Furthermore, we found that selected isoforms of 1-aminocyclopropane-1-carboxylic acid synthase (ACS), the rate-limiting enzyme of ethylene biosynthesis, are substrates of MPK6. Phosphorylation of ACS2 and ACS6 by MPK6 leads to the accumulation of ACS protein and, thus, elevated levels of cellular ACS activity and ethylene production. Expression of ACS6DDD, a gain-of-function ACS6 mutant that mimics the phosphorylated form of ACS6, confers constitutive ethylene production and ethylene-induced phenotypes. Increasing numbers of stress stimuli have been shown to activate Arabidopsis MPK6 or its orthologs in other plant species. The identification of the first plant MAPK substrate in this report reveals one mechanism by which MPK6/SIPK regulates plant stress responses. Equally important, this study uncovers a signaling pathway that modulates the biosynthesis of ethylene, an important plant hormone, in plants under stress.

Modulation of floral development by a gibberellin-regulated microRNA

Abstract: Floral initiation and floral organ development are both regulated by the phytohormone gibberellin (GA). For example, in short-day photoperiods, the Arabidopsis floral transition is strongly promoted by GA-mediated activation of the floral meristem-identity gene LEAFY. In addition, anther development and pollen microsporogenesis depend on GA-mediated opposition of the function of specific members of the DELLA family of GA-response repressors. We describe the role of a microRNA (miR159) in the regulation of short-day photoperiod flowering time and of anther development. MiR159 directs the cleavage of mRNA encoding GAMYB-related proteins. These proteins are transcription factors that are thought to be involved in the GA-promoted activation of LEAFY, and in the regulation of anther development. We show that miR159 levels are regulated by GA via opposition of DELLA function, and that both the sequence of miR159 and the regulation of miR159 levels by DELLA are evolutionarily conserved. Finally, we describe the phenotypic consequences of transgenic over-expression of miR159. Increased levels of miR159 cause a reduction in LEAFY transcript levels, delay flowering in short-day photoperiods, and perturb anther development. We propose that miR159 is a phytohormonally regulated homeostatic modulator of GAMYB activity, and hence of GAMYB-dependent developmental processes.

Integration of transcriptomics and metabolomics for understanding of global responses to nutritional stresses in Arabidopsis thaliana

Abstract: Plant metabolism is a complex set of processes that produce a wide diversity of foods, woods, and medicines. With the genome sequences of Arabidopsis and rice in hands, postgenomics studies integrating all "omics" sciences can depict precise pictures of a whole-cellular process. Here, we present, to our knowledge, the first report of investigation for gene-to-metabolite networks regulating sulfur and nitrogen nutrition and secondary metabolism in Arabidopsis, with integration of metabolomics and transcriptomics. Transcriptome and metabolome analyses were carried out, respectively, with DNA macroarray and several chemical analytical methods, including ultra high-resolution Fourier transform-ion cyclotron MS. Mathematical analyses, including principal component analysis and batch-learning self-organizing map analysis of transcriptome and metabolome data suggested the presence of general responses to sulfur and nitrogen deficiencies. In addition, specific responses to either sulfur or nitrogen deficiency were observed in several metabolic pathways: in particular, the genes and metabolites involved in glucosinolate metabolism were shown to be coordinately modulated. Understanding such gene-to-metabolite networks in primary and secondary metabolism through integration of transcriptomics and metabolomics can lead to identification of gene function and subsequent improvement of production of useful compounds in plants.

Ehd1, a B-type response regulator in rice, confers short-day promotion of flowering and controls FT-like gene expression independently of Hd1

Abstract: Two evolutionarily distant plant species, rice (Oryza sativa L.), a short-day (SD) plant, and Arabidopsis thaliana, a long-day plant, share a conserved genetic network controlling photoperiodic flowering. The orthologous floral regulators—rice Heading date 1 (Hd1) and Arabidopsis CONSTANS (CO)—integrate circadian clock and external light signals into mRNA expression of the FLOWERING LOCUS T (FT) group floral inducer. Here, we report that the rice Early heading date 1 (Ehd1) gene, which confers SD promotion of flowering in the absence of a functional allele of Hd1, encodes a B-type response regulator that might not have an ortholog in the Arabidopsis genome. Ehd1 mRNA was induced by 1-wk SD treatment, and Ehd1 may promote flowering by inducing FT-like gene expression only under SD conditions. Microarray analysis further revealed a few MADS box genes downstream of Ehd1. Our results indicate that a novel two-component signaling cascade is integrated into the conserved pathway in the photoperiodic control of flowering in rice.

Roles of the CBF2 and ZAT12 transcription factors in configuring the low temperature transcriptome of Arabidopsis.

Abstract: Summary The CBF cold response pathway has a prominent role in cold acclimation. The pathway includes action of three transcription factors, CBF1, 2 and 3 (also known as DREB1b, c and a, respectively), that are rapidly induced in response to low temperature followed by expression of the CBF-targeted genes (the CBF regulon) that act in concert to increase plant-freezing tolerance. The results of transcriptome profiling and mutagenesis experiments, however, indicate that additional cold response pathways exist and may have important roles in life at low temperature. To further understand the roles that the CBF proteins play in configuring the low temperature transcriptome and to identify additional transcription factors with roles in cold acclimation, we used the Affymetrix GeneChip containing probe sets for approximately 24,000 Arabidopsis genes to define a core set of cold-responsive genes and to determine which genes were targets of CBF2 and 6 other transcription factors that appeared to be coordinately regulated with CBF2. A total of 514 genes were placed in the core set of cold-responsive genes, 302 of which were upregulated and 212 downregulated. Hierarchical clustering and bioinformatic analysis indicated that the 514 cold-responsive transcripts could be assigned to one of seven distinct expression classes and identified multiple potential novel cis-acting cold-regulatory elements. Eighty-five cold-induced genes and eight cold-repressed genes were assigned to the CBF2 regulon. An additional nine cold-induced genes and 15 cold-repressed genes were assigned to a regulon controlled by ZAT12. Of the 25 core cold-induced genes that were most highly upregulated (induced over 15-fold), 19 genes (84%) were induced by CBF2 and another two genes (8%) were regulated by both CBF2 and ZAT12. Thus, the large majority (92%) of the most highly induced genes belong to the CBF and ZAT12 regulons. Constitutive expression of ZAT12 in Arabidopsis caused a small, but reproducible, increase in freezing tolerance, indicating a role for the ZAT12 regulon in cold acclimation. In addition, ZAT12 downregulated the expression of the CBF genes indicating a role for ZAT12 in a negative regulatory circuit that dampens expression of the CBF cold response pathway.

Naturally occurring genetic variation in Arabidopsis thaliana.

Abstract: Currently, genetic variation is probably the most important basic resource for plant biology. In addition to the variation artificially generated by mutants in model plants, naturally occurring genetic variation is extensively found for most species, including Arabidopsis. In many cases, natural variation present among accessions is multigenic, which has historically hampered its analysis. However, the exploitation of this resource down to the molecular level has now become feasible, especially in model species like Arabidopsis, where several genes accounting for natural variation have already been identified. Dissecting this variation requires first a quantitative trait locus (QTL) analysis, which in Arabidopsis has proven very effective by using recombinant inbred lines (RILs). Second, identifying the particular gene and the nucleotide polymorphism underlying QTL is the major challenge, and is now feasible by combining high-throughput genetics and functional genomic strategies. The analysis of Arabidopsis natural genetic variation is providing unique knowledge from functional, ecological, and evolutionary perspectives. This is illustrated by reviewing current research in two different biological fields: flowering time and plant growth. The analysis of Arabidopsis natural variation for flowering time revealed the identity of several genes, some of which correspond to genes with previously unknown function. In addition, for many other traits such as those related to primary metabolism and plant growth, Arabidopsis QTL analyses are detecting loci with small effects that are not easily amenable by mutant approaches, and which might provide new insights into the networks of gene regulation.

The Essential Basic Helix-Loop-Helix Protein FIT1 Is Required for the Iron Deficiency Response

Abstract: Regulation of iron uptake is critical for plant survival. Although the activities responsible for reduction and transport of iron at the plant root surface have been described, the genes controlling these activities are largely unknown. We report the identification of the essential gene Fe-deficiency Induced Transcription Factor 1 (FIT1), which encodes a putative transcription factor that regulates iron uptake responses in Arabidopsis thaliana. Like the Fe(III) chelate reductase FRO2 and high affinity Fe(II) transporter IRT1, FIT1 mRNA is detected in the outer cell layers of the root and accumulates in response to iron deficiency. fit1 mutant plants are chlorotic and die as seedlings but can be rescued by the addition of supplemental iron, pointing to a defect in iron uptake. fit1 mutant plants accumulate less iron than wild-type plants in root and shoot tissues. Microarray analysis shows that expression of many (72 of 179) iron-regulated genes is dependent on FIT1. We demonstrate that FIT1 regulates FRO2 at the level of mRNA accumulation and IRT1 at the level of protein accumulation. We propose a new model for iron uptake in Arabidopsis where FRO2 and IRT1 are differentially regulated by FIT1.

Transcriptome analysis of haploid male gametophyte development in Arabidopsis

Abstract: Background: The haploid male gametophyte generation of flowering plants consists of two- or three-celled pollen grains. This functional specialization is thought to be a key factor in the evolutionary success of flowering plants. Moreover, pollen ontogeny is also an attractive model in which to dissect cellular networks that control cell growth, asymmetric cell division and cellular differentiation. Our objective, and an essential step towards the detailed understanding of these processes, was to comprehensively define the male haploid transcriptome throughout development. Results: We have developed staged spore isolation procedures for Arabidopsis and used Affymetrix ATH1 genome arrays to identify a total of 13,977 male gametophyte-expressed mRNAs, 9.7% of which were male-gametophyte-specific. The transition from bicellular to tricellular pollen was accompanied by a decline in the number of diverse mRNA species and an increase in the proportion of male gametophyte-specific transcripts. Expression profiles of regulatory proteins and distinct clusters of coexpressed genes were identified that could correspond to components of gametophytic regulatory networks. Moreover, integration of transcriptome and experimental data revealed the early synthesis of translation factors and their requirement to support pollen tube growth. Conclusions: The progression from proliferating microspores to terminally differentiated pollen is characterized by large-scale repression of early program genes and the activation of a unique late gene-expression program in maturing pollen. These data provide a quantum increase in knowledge concerning gametophytic transcription and lay the foundations for new genomic-led studies of the regulatory networks and cellular functions that operate to specify male gametophyte development.

Phosphate transport in Arabidopsis: Pht1;1 and Pht1;4 play a major role in phosphate acquisition from both low- and high-phosphate environments.

Abstract: Of the mineral nutrients essential for plant growth, phosphorus plays the widest diversity of roles and a lack of phosphorus has profound effects on cellular metabolism. At least eight members of the Arabidopsis Pht1 phosphate (Pi) transporter family are expressed in roots and Pht1;1 and Pht1;4 show the highest transcript levels. The spatial and temporal expression patterns of these two genes show extensive overlap. To elucidate the in planta roles of Pht1;1 and Pht1;4, we identified loss-of-function mutants and also created a double mutant, lacking both Pht1;1 and Pht1;4. Consistent with their spatial expression patterns, membrane location and designation as high-affinity Pi transporters, Pht1;1 and Pht1;4 contribute to Pi transport in roots during growth under low-Pi conditions. In addition, during growth under high-Pi conditions, the double mutant shows a 75% reduction in Pi uptake capacity relative to wildtype. Thus, Pht1;1 and Pht1;4 play significant roles in Pi acquisition from both low- and high-Pi environments.

A prominent role for the CBF cold response pathway in configuring the low-temperature metabolome of Arabidopsis

Abstract: The Arabidopsis CBF cold response pathway has a central role in cold acclimation, the process whereby plants increase in freezing tolerance in response to low nonfreezing temperatures. Here we examined the changes that occur in the Arabidopsis metabolome in response to low temperature and assessed the role of the CBF cold response pathway in bringing about these modifications. Of 434 metabolites monitored by GC-time-of-flight MS, 325 (75%) were found to increase in Arabidopsis Wassilewskija-2 (Ws-2) plants in response to low temperature. Of these 325 metabolites, 256 (79%) also increased in nonacclimated Ws-2 plants in response to overexpression of C-repeat/dehydration responsive element-binding factor (CBF)3. Extensive cold-induced changes also occurred in the metabolome of Arabidopsis Cape Verde Islands-1 (Cvi-1) plants, which were found to be less freezing tolerant than Ws-2 plants. However, low-temperature-induced expression of CBF1, CBF2, CBF3, and CBF-targeted genes was much lower in Cvi-1 than in Ws-2 plants, and the low-temperature metabolome of Cvi-1 plants was depleted in metabolites affected by CBF3 overexpression. Taken together, the results indicate that the metabolome of Arabidopsis is extensively reconfigured in response to low temperature, and that the CBF cold response pathway has a prominent role in this process.

Type-A Arabidopsis response regulators are partially redundant negative regulators of cytokinin signaling.

Abstract: Type-A Arabidopsis (Arabidopsis thaliana) response regulators (ARRs) are a family of 10 genes that are rapidly induced by cytokinin and are highly similar to bacterial two-component response regulators. We have isolated T-DNA insertions in six of the type-A ARRs and constructed multiple insertional mutants, including the arr3,4,5,6,8,9 hextuple mutant. Single arr mutants were indistinguishable from the wild type in various cytokinin assays; double and higher order arr mutants showed progressively increasing sensitivity to cytokinin, indicating functional overlap among type-A ARRs and that these genes act as negative regulators of cytokinin responses. The induction of cytokinin primary response genes was amplified in arr mutants, indicating that the primary response to cytokinin is affected. Spatial patterns of ARR gene expression were consistent with partially redundant function of these genes in cytokinin signaling. The arr mutants show altered red light sensitivity, suggesting a general involvement of type-A ARRs in light signal transduction. Further, morphological phenotypes of some arr mutants suggest complex regulatory interactions and gene-specific functions among family members.