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The development of angiosperm flowers is regulated by homeotic MIKC-type MADS-domain transcription factors that activate or repress target genes via the formation of DNA-bound, organ-specific tetrameric complexes. The protein--protein interaction (PPI) capabilities differ considerably between different MIKC-type proteins. In Arabidopsis thaliana the floral homeotic protein SEPALLATA3 (SEP3) acts as a hub that incorporates numerous other MADS-domain proteins into tetrameric complexes that would otherwise not form. However, the molecular mechanisms that underlie these promiscuous interactions remain largely unknown. In this study, we created a collection of amino acid substitution mutants of SEP3 to quantify the contribution of individual residues on protein tetramerization during DNA-binding, employing methods of molecular biophysics. We show that leucine residues at certain key positions form a leucine-zipper structure that is essential for tetramerization of SEP3, whereas the introduction of physicochemically very similar residues at respective sites impedes the formation of DNA-bound tetramers. Comprehensive molecular evolutionary analyses of MADS-domain proteins from a diverse set of flowering plants revealed exceedingly high conservation of the identified leucine residues within SEP3-subfamily proteins throughout angiosperm evolution. In contrast, MADS-domain proteins that are unable to tetramerize among themselves exhibit preferences for other amino acids at homologous sites. Our findings indicate that the subfamily-specific conservation of amino acid residues at just a few key positions accounts for subfamily-specific interaction capabilities of MADS-domain transcription factors and this has shaped the present-day structure of the PPI network controlling flower development. [ABSTRACT FROM AUTHOR]

DWARF1 (DWF1) is a sterol C-24 reductase that catalyses the conversion of 24-methylenecholesterol (24-MCHR) to campesterol (CR) in Arabidopsis. A loss-of-function mutant, dwf1, showed similar phenotypic abnormalities to brassinosteroid (BR)-deficient mutants. These abnormalities were reversed in the wild-type phenotype by exogenous application of castasterone (CS) and brassinolide (BL), but not dolichosterone (DS). Accumulation of DS and decreased CS were found in quantitative analysis of endogenous BRs in dwf1. The enzyme solution prepared from dwf1 was unable to convert 6-deoxoDS to 6-deoxoCS and DS to CS, as seen in either wild-type or 35S:DWF1 transgenic plants. This suggests that DWF1 has enzyme activity not only for a sterol C-24 reductase, but also for a BR C-24 reductase that catalyses C-24 reduction of 6-deoxoDS to 6-deoxoCS and of DS to CS in Arabidopsis. Overexpression of DWF1 in a BR-deficient mutant (det2 35S:DWF1) clearly rescued abnormalities found in det2, indicating that DWF1 functions in biosynthesis of active BRs in Arabidopsis. Expression of DWF1 is down-regulated by application of CS and BL and in a BR-dominant mutant, bes1-D. E-boxes in the putative promoter region of DWF1 directly bind to a BR transcription factor, BES1, implying that DWF1 expression is feedback-regulated by BR signaling via BES1. Overall, biosynthesis of 24-methylene BR is an alternative route for generating CS, which is mediated and regulated by DWF1 in Arabidopsis. [ABSTRACT FROM AUTHOR]

As a consequence of global change processes, plants will increasingly be challenged by extreme climatic events, against a background of elevated atmospheric CO2. We analysed responses of Arabidopsis thaliana to periods of a combination of elevated heat and water deficit at ambient and elevated CO2 in order to gain mechanistic insights regarding changes in primary metabolism. Metabolic changes induced by extremes of climate are dynamic and specific to different classes of molecules. Concentrations of soluble sugars and amino acids increased transiently after short (4-d) exposure to heat and drought, and readjusted to control levels under prolonged (8-d) stress. In contrast, fatty acids showed persistent changes during the stress period. Elevated CO2 reduced the impact of stress on sugar and amino acid metabolism, but not on fatty acids. Integrating metabolite data with transcriptome results revealed that some of the metabolic changes were regulated at the transcriptional level. Multivariate analyses grouped metabolites on the basis of stress exposure time, indicating specificity in metabolic responses to short and prolonged stress. Taken together, the results indicate that dynamic metabolic reprograming plays an important role in plant acclimation to climatic extremes. The extent of such metabolic adjustments is less under high CO2, further pointing towards the role of high CO2 in stress mitigation. [ABSTRACT FROM AUTHOR]

Brassinosteroids (BRs) are plant-specific steroidal hormones; BR homeostasis is crucial for various aspects of plant growth and development. However, to date, the BR inactivation process has not been thoroughly elucidated. In this study, we identified and characterized a novel BAHD family acyltransferase gene, BRASSINOSTEROID INACTIVATOR2 (BIA2), involved in BR inactivation. BIA2-overexpressing (OE-BIA2) plants displayed typical BR-deficient phenotypes, which were rescued by exogenous BR treatment. Real-time qRT-PCR and transcriptome analyses showed that expression levels of virtually all of the BR biosynthetic genes were increased, whereas the expression of many BR inactivation genes was reduced in OE-BIA2 plants. Root inhibition assays showed that the root growth of OE-BIA2 plants was inhibited. We obtained plants with an intermediate phenotype by crossing the OE-BIA2 plants with BRASSINOSTEROID-INSENSITIVE1 (BRI1)-overexpressing plants. The null BIA2 mutants had longer hypocotyls in the dark. BIA2 was predominantly expressed in roots, and its expression was induced by 24-epibrassinolide or dark treatment, but it exhibited a differential expression pattern compared with its homologue, BIA1. Furthermore, genetic transformation with point-mutant and deleted-BIA2 constructs confirmed that the HXXXD motif is essential for the function of BIA2. Taken together, these findings indicate that BIA2 is a typical BAHD acyltransferase that is involved in BR homeostasis and may inactivate bioactive BRs by esterification, particularly in roots and hypocotyls under dark conditions. [ABSTRACT FROM AUTHOR]

As a consequence of global change processes, plants will increasingly be challenged by extreme climatic events, against a background of elevated atmospheric CO2. We analysed responses of Arabidopsis thaliana to periods of a combination of elevated heat and water deficit at ambient and elevated CO2 in order to gain mechanistic insights regarding changes in primary metabolism. Metabolic changes induced by extremes of climate are dynamic and specific to different classes of molecules. Concentrations of soluble sugars and amino acids increased transiently after short (4-d) exposure to heat and drought, and readjusted to control levels under prolonged (8-d) stress. In contrast, fatty acids showed persistent changes during the stress period. Elevated CO2 reduced the impact of stress on sugar and amino acid metabolism, but not on fatty acids. Integrating metabolite data with transcriptome results revealed that some of the metabolic changes were regulated at the transcriptional level. Multivariate analyses grouped metabolites on the basis of stress exposure time, indicating specificity in metabolic responses to short and prolonged stress. Taken together, the results indicate that dynamic metabolic reprograming plays an important role in plant acclimation to climatic extremes. The extent of such metabolic adjustments is less under high CO2, further pointing towards the role of high CO2 in stress mitigation. [ABSTRACT FROM AUTHOR]

The phytohormone auxin regulates virtually every developmental process in land plants. This regulation is mediated via de-repression of DNA-binding auxin response factors (ARFs). ARFs bind TGTC-containing auxin response ciselements (AuxREs), but there is growing evidence that additional cis-elements occur in auxin-responsive regulatory regions. The repertoire of auxin-related cis-elements and their involvement in different modes of auxin response are not yet known. Here we analyze the enrichment of nucleotide hexamers in upstream regions of auxin-responsive genes associated with auxin up- or down-regulation, with early or late response, ARF-binding domains, and with different chromatin states. Intriguingly, hexamers potentially bound by basic helix–loop–helix (bHLH) and basic leucine zipper (bZIP) factors as well as a family of A/T-rich hexamers are more highly enriched in auxin-responsive regions than canonical TGTC-containing AuxREs. We classify and annotate the whole spectrum of enriched hexamers and discuss their patterns of enrichment related to different modes of auxin response. [ABSTRACT FROM AUTHOR]

In natural environments, nitrogen (N) concentration in the soil fluctuates greatly and is often limiting for plant growth and development. The ability of plants to respond to changes in N availability is therefore essential for adaptation. The response of plants to N variations consists in particular of adjusting root N uptake systems and root architecture. To do so, plants integrate local sensing and signaling of external N availability with systemic sensing and signaling of their internal N status, in order to tune the functional and structural properties of the root system in accordance with the N demand for growth of the whole plant. Transcriptional regulation of gene expression is one of the most important processes plants use to adapt the properties of the root system in response to local and long-distance N pathways. This review focuses on the mechanisms that give rise to transcriptional responses in Arabidopsis roots under N fluctuations, with an emphasis on those associated with the regulation of nitrate uptake and transport systems. We discuss the putative long-distance signals triggering the gene expression responses, as well as the molecular players that locally induce transcriptional changes. We also highlight several observations revealing the importance of adopting an integrative approach in the regulation of N signaling. [ABSTRACT FROM AUTHOR]

Calcium (Ca2+) mobilization is a central theme in various plant signal transduction pathways. We demonstrate that Arabidopsis thaliana cyclic nucleotide-gated channel 2 (AtCNGC2) is involved in jasmonic acid (JA)-induced apoplastic Ca2+ influx in Arabidopsis epidermal cells. Ca2+ imaging results showed that JA can induce an elevation in the cytosolic cAMP concentration ([cAMP]cyt), reaching a maximum within 3 min. Dibutyryl cAMP (db-cAMP), a cell membrane-permeable analogue of cAMP, induced an increase in the cytosolic Ca2+ concentration ([Ca2+]cyt), with a peak at 4 min. This [Ca2+]cyt increase was triggered by the JA-induced increase in [cAMP]cyt. W-7[N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], an antagonist of calmodulin, positively modulated the JA-induced increase in [Ca2+]cyt, while W-5[N-(6-aminohexyl)-1-naphthalenesulfonamide], an inactive antagonist of calmodulin, had no apparent effect. db-cAMP and JA positively induced the expression of primary (i.e. JAZ1 and MYC2) and secondary (i.e. VSP1) response genes in the JA signalling pathway in wild-type Arabidopsis thaliana, whereas they had no significant effect in the AtCNGC2 mutant 'defense, no death (dnd1) plants. These data provide evidence that JA first induces the elevation of cAMP, and cAMP, as an activating ligand, activates the AtCNGC2 channel, resulting in apoplastic Ca2+ influx through AtCNGC2. [ABSTRACT FROM AUTHOR]

Copper homeostasis under deficiency is regulated by the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (SPL7) transcription factor. The daily oscillating expression of two SPL7-dependent copper deficiency markers, COPPER TRANSPORTER (COPT2) and IRON SUPEROXIDE DISMUTASE (FSD1), has been followed by quantitative PCR and in promoter:LUCIFERASE transgenic plants. Both genes showed circadian and diurnal regulation. Under copper deficiency, their expression decreased drastically in continuous darkness. Accordingly, total copper content was slightly reduced in etiolated seedlings under copper deficiency. The expression of SPL7 and its targets COPT2 and FSD1 was differently regulated in various light signalling mutants. On the other hand, increased copper levels reduced the amplitude of nuclear circadian clock components, such as GIGANTEA (GI). The alteration of copper homeostasis in the COPT1 overexpression line and spl7 mutants also modified the amplitude of a classical clock output, namely the circadian oscillation of cotyledon movements. In the spl7 mutant, the period of the oscillation remained constant. These results suggest a feedback of copper transport on the circadian clock and the integration of rhythmic copper homeostasis into the central oscillator of plants. [ABSTRACT FROM AUTHOR]

The molecular mechanisms of abscisic acid (ABA) signalling have been studied for many years; however, how mitochondria-localized proteins play roles in ABA signalling remains unclear. Here an Arabidopsis mitochondria-localized protein RRL (RETARDED ROOT GROWTH-LIKE) was shown to function in ABA signalling. A previous study had revealed that the Arabidopsis mitochondria-localized protein RRG (RETARDED ROOT GROWTH) is required for cell division in the root meristem. RRL shares 54% and 57% identity at the nucleotide and amino acid sequences, respectively, with RRG; nevertheless, RRL shows a different function in Arabidopsis. In this study, disruption of RRL decreased ABA sensitivity whereas overexpression of RRL increased ABA sensitivity during seed germination and seedling growth. High expression levels of RRL were found in germinating seeds and developing seedlings, as revealed by ß-glucuronidase (GUS) staining of ProRRL-GUS transgenic lines. The analyses of the structure and function of mitochondria in the knockout rrl mutant showed that the disruption of RRL causes extensively internally vacuolated mitochondria and reduced ABA-stimulated reactive oxygen species (ROS) production. Previous studies have revealed that the expression of alternative oxidase (AOX) in the alternative respiratory pathway is increased by mitochondrial retrograde regulation to regain ROS levels when the mitochondrial electron transport chain is impaired. The APETALA2 (AP2)-type transcription factor ABI4 is a regulator of ALTERNATIVE OXIDASE1a (AOX1a) in mitochondrial retrograde signalling. This study showed that ABA-induced AOX1a and ABI4 expression was inhibited in the rrl mutant, suggesting that RRL is probably involved in ABI4-mediated mitochondrial retrograde signalling. Furthermore, the results revealed that ABI4 is a downstream regulatory factor in RRL-mediated ABA signalling in seed germination and seedling growth. [ABSTRACT FROM AUTHOR]

Over 130 years ago, Charles Darwin recognized that sensory functions in the root tip influence directional root growth. Modern plant biology has unravelled that many of the functions that Darwin attributed to the root tip are actually accomplished by a particular organ--the root cap. The root cap surrounds and protects the meristematic stem cells at the growing root tip. Due to this vanguard position, the root cap is predisposed to receive and transmit environmental information to the root proper. In contrast to other plant organs, the root cap shows a rapid turnover of short-lived cells regulated by an intricate balance of cell generation, differentiation, and degeneration. Thanks to these particular features, the root cap is an excellent developmental model system, in which generation, differentiation, and degeneration of cells can be investigated in a conveniently compact spatial and temporal frame. In this review, we give an overview of the current knowledge and concepts of root cap biology, focusing on the model plant Arabidopsis thaliana. [ABSTRACT FROM AUTHOR]

High salinity has negative impacts on plant growth through altered water uptake and ion-specific toxicities. Plants have therefore evolved an intricate regulatory network in which plant hormones play significant roles in modulating physiological responses to salinity. However, current understanding of the plant peptides involved in this regulatory network remains limited. Here, we identified a salt-regulated peptide in Arabidopsis. The peptide was 11 aa and was derived from the C terminus of a cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins (CAP) superfamily. This peptide was found by searching homologues in Arabidopsis using the precursor of a tomato CAP-derived peptide (CAPE) that was initially identified as an immune signal. In searching for a CAPE involved in salt responses, we screened CAPE precursor genes that showed salt-responsive expression and found that the PROAtCAPE1 (AT4G33730) gene was regulated by salinity. We confirmed the endogenous Arabidopsis CAP-derived peptide 1 (AtCAPE1) by mass spectrometry and found that a key amino acid residue in PROAtCAPE1 is critical for AtCAPE1 production. Moreover, although PROAtCAPE1 was expressed mainly in the roots, AtCAPE1 was discovered to be upregulated systemically upon salt treatment. The salt-induced AtCAPE1 negatively regulated salt tolerance by suppressing several salt-tolerance genes functioning in the production of osmolytes, detoxification, stomatal closure control, and cell membrane protection. This discovery demonstrates that AtCAPE1, a homologue of tomato immune regulator CAPE1, plays an important role in the regulation of salt stress responses. Our discovery thus suggests that the peptide may function in a trade-off between pathogen defence and salt tolerance. [ABSTRACT FROM AUTHOR]

The root meristem of Arabidopsis thaliana harbours a pool of stem cells, which divide to give rise to the differentiated cells of the various root tissues. Regulatory networks of inter-cellular signalling molecules control the homeostasis of stem cell number and position so that both stem and differentiated cells are consistently available. This work focuses on the transcription factor WUSCHEL-RELATED HOMEOBOX 5 (WOX5), the signalling peptide CLAVATA3/EMBRYOSURROUNDING REGION 40 (CLE40) and the feedback loops involving them, which maintain the columella stem cells (CSCs). WOX5 signals from the quiescent centre (QC) to promote stem cell fate, while CLE40 is secreted from the differentiated columella cells (CCs) to promote differentiation. Our analyses of mathematical models of this network show that, when cell fate is controlled primarily by antagonistic factors, homeostasis requires a spatial component and inter-cellular signalling. We have also found that WOX5 contributes to, but is not absolutely necessary for, CSC maintenance. Furthermore, our modelling led us to postulate an additional signalling molecule that promotes CSC maintenance. We propose that this WOX5-independent signal originates in the QC, is targeted by CLE40 signalling and is capable of maintaining CSCs. [ABSTRACT FROM AUTHOR]

A balanced supply of essential nutrients is an important factor influencing root architecture in many plants, yet data related to the interactive effects of two nutrients on root growth are limited. Here, we investigated the interactive effect between phosphorus (P) and magnesium (Mg) on root growth of Arabidopsis grown in pH-buffered agar medium at different P and Mg levels. The results showed that elongation and deviation of primary roots were directly correlated with the amount of P added to the medium but could be modified by the Mg level, which was related to the root meristem activity and stem-cell division. High P enhanced while low P decreased the tip-focused fluorescence signal of auxin biosynthesis, transport, and redistribution during elongation of primary roots; these effects were greater under low Mg than under high Mg. The altered root growth in response to P and Mg supply was correlated with AUX1, PIN2, and PIN3 mRNA abundance and expression and the accumulation of the protein. Application of either auxin influx inhibitor or efflux inhibitor inhibited the elongation and increased the deviation angle of primary roots, and decreased auxin level in root tips. Furthermore, the auxin-transport mutants aux1-22 and eir1-1 displayed reduced root growth and increased the deviation angle. Our data suggest a profound effect of the combined supply of P and Mg on the development of root morphology in Arabidopsis through auxin signals that modulate the elongation and directional growth of primary root and the expression of root differentiation and development genes. [ABSTRACT FROM AUTHOR]

Most mitochondrial proteins contain an N-terminal targeting signal that is removed by specific proteases following import. In plant mitochondria, only mitochondrial processing peptidase (MPP) has been characterized to date. Therefore, we sought to determine the substrates and cleavage sites of the Arabidopsis thaliana homologues to the yeast Icp55 and Oct1 proteins, using the newly developed ChaFRADIC method for N-terminal protein sequencing. We identified 88 and seven putative substrates for Arabidopsis ICP55 and OCT1, respectively. It was determined that the Arabidopsis ICP55 contains an almost identical cleavage site to that of Icp55 from yeast. However, it can also remove a far greater range of amino acids. The OCT1 substrates from Arabidopsis displayed no consensus cleavage motif, and do not contain the classical -10R motif identified in other eukaryotes. Arabidopsis OCT1 can also cleave presequences independently, without the prior cleavage of MPP. It was concluded that while both OCT1 and ICP55 were probably acquired early on in the evolution of mitochondria, their substrate profiles and cleavage sites have either remained very similar or diverged completely. [ABSTRACT FROM AUTHOR]

In this study, evidence is provided for the role of fructose-1,6-bisphosphatases (FBPases) in plant development and carbohydrate synthesis and distribution by analysing two Arabidopsis thaliana T-DNA knockout mutant lines, cyfbp and cfbp1, and one double mutant cyfbp cfbp1 which affect each FBPase isoform, cytosolic and chloroplastic, respectively. cyFBP is involved in sucrose synthesis, whilst cFBP1 is a key enzyme in the Calvin-Benson cycle. In addition to the smaller rosette size and lower rate of photosynthesis, the lack of cFBP1 in the mutants cfbp1 and cyfbp cfbp1 leads to a lower content of soluble sugars, less starch accumulation, and a greater superoxide dismutase (SOD) activity. The mutants also had some developmental alterations, including stomatal opening defects and increased numbers of root vascular layers. Complementation also confirmed that the mutant phenotypes were caused by disruption of the cFBP1 gene. cyfbp mutant plants without cyFBP showed a higher starch content in the chloroplasts, but this did not greatly affect the phenotype. Notably, the sucrose content in cyfbp was close to that found in the wild type. The cyfbp cfbp1 double mutant displayed features of both parental lines but had the cfbp1 phenotype. All the mutants accumulated fructose-1,6-bisphosphate and triose-phosphate during the light period. These results prove that while the lack of cFBP1 induces important changes in a wide range of metabolites such as amino acids, sugars, and organic acids, the lack of cyFBP activity in Arabidopsis essentially provokes a carbon metabolism imbalance which does not compromise the viability of the double mutant cyfbp cfbp1. [ABSTRACT FROM AUTHOR]

Phosphite is a less oxidized form of phosphorus than phosphate. Phosphite is considered to be taken up by the plant through phosphate transporters. It can mimic phosphate to some extent, but it is not metabolized into organophosphates. Phosphite could therefore interfere with phosphorus signalling networks. Typical physiological and transcriptional responses to low phosphate availability were investigated and the short-term kinetics of their reversion by phosphite, compared with phosphate, were determined in both roots and shoots of Arabidopsis thaliana. Phosphite treatment resulted in a strong growth arrest. It mimicked phosphate in causing a reduction in leaf anthocyanins and in the expression of a subset of the phosphate-starvation-responsive genes. However, the kinetics of the response were slower than for phosphate, which may be due to discrimination against phosphite by phosphate transporters PHT1;8 and PHT1;9 causing delayed shoot accumulation of phosphite. Transcripts encoding PHT1;7, lipid-remodelling enzymes such as SQD2, and phosphocholine- producing NMT3 were highly responsive to phosphite, suggesting their regulation by a direct phosphate-sensing network. Genes encoding components associated with the 'PHO regulon' in plants, such as At4, IPS1, and PHO1;H1, generally responded more slowly to phosphite than to phosphate, except for SPX1 in roots and MIR399d in shoots. Two uncharacterized phosphate-responsive E3 ligase genes, PUB35 and C3HC4, were also highly phosphite responsive. These results show that phosphite is a valuable tool to identify network components directly responsive to phosphate. [ABSTRACT FROM AUTHOR]

Recent research has linked the non-coding intronic regions of plant genes to the production of small RNAs (sRNAs). Certain introns, called 'mirtrons' and 'sirtrons', could serve as the single-stranded RNA precursors for the generation of microRNA and small interfering RNA, respectively. However, whether the intronic regions could serve as the template for double-stranded RNA synthesis and then for sRNA biogenesis through an RDR (RNA-dependent RNA polymerase)-dependent pathway remains unclear. In this study, a genome-wide search was made for the RDRdependent sRNA loci within the intronic regions of the Arabidopsis genes. Hundreds of intronic regions encoding three or more RDR-dependent sRNAs were found to be covered by dsRNA-seq (double-stranded RNA sequencing) reads, indicating that the intron-derived sRNAs were indeed generated from long double-stranded RNA precursors. More interestingly, phase-distributed sRNAs were discovered on some of the dsRNA-seq read-covered intronic regions, and those sRNAs were largely 24 nt in length. Based on these results, the opinion is put forward that the intronic regions might serve as the genomic origins for the RDR-dependent sRNAs. This opinion might add a novel layer to the current biogenesis model of the intron-derived sRNAs. [ABSTRACT FROM AUTHOR]

Lipid remodeling is one of the most dramatic metabolic responses to phosphorus (P) starvation. It consists of the degradation of phospholipids to release the phosphate needed by the cell and the accumulation of glycolipids to replace phospholipids in the membranes. It is shown that PHR1, a well-described transcriptional regulator of P starvation of the MYB family, largely controls this response. Glycerolipid composition and the expression of most lipid-remodeling gene transcripts analysed were altered in the phr1 mutant under phosphate starvation in comparison to wild-type plants. In addition to these results, the lipidomic characterization of wild-type plants showed two novel features of the lipid response to P starvation for Arabidopsis. Triacylglycerol (TAG) accumulates dramatically under P starvation (by as much as ~20-fold in shoots and ~13-fold in roots), a response known to occur in green algae but hardly known in plants. Surprisingly, there was an increase in phosphatidylglycerol (PG) in P-starved roots, a response that may be adaptive as it was suppressed in the phr1 mutant. [ABSTRACT FROM AUTHOR]