Publikationen - Molekulare Signalverarbeitung

BackgroundPlant adaptation to limited phosphate availability comprises a wide range of responses to conserve and remobilize internal phosphate sources and to enhance phosphate acquisition. Vigorous restructuring of root system architecture provides a developmental strategy for topsoil exploration and phosphate scavenging. Changes in external phosphate availability are locally sensed at root tips and adjust root growth by modulating cell expansion and cell division. The functionally interacting Arabidopsis genes, LOW PHOSPHATE RESPONSE 1 and 2 (LPR1/LPR2) and PHOSPHATE DEFICIENCY RESPONSE 2 (PDR2), are key components of root phosphate sensing. We recently demonstrated that the LOW PHOSPHATE RESPONSE 1 - PHOSPHATE DEFICIENCY RESPONSE 2 (LPR1-PDR2) module mediates apoplastic deposition of ferric iron (Fe3+) in the growing root tip during phosphate limitation. Iron deposition coincides with sites of reactive oxygen species generation and triggers cell wall thickening and callose accumulation, which interfere with cell-to-cell communication and inhibit root growth.ResultsWe took advantage of the opposite phosphate-conditional root phenotype of the phosphate deficiency response 2 mutant (hypersensitive) and low phosphate response 1 and 2 double mutant (insensitive) to investigate the phosphate dependent regulation of gene and protein expression in roots using genome-wide transcriptome and proteome analysis. We observed an overrepresentation of genes and proteins that are involved in the regulation of iron homeostasis, cell wall remodeling and reactive oxygen species formation, and we highlight a number of candidate genes with a potential function in root adaptation to limited phosphate availability. Our experiments reveal that FERRIC REDUCTASE DEFECTIVE 3 mediated, apoplastic iron redistribution, but not intracellular iron uptake and iron storage, triggers phosphate-dependent root growth modulation. We further highlight expressional changes of several cell wall-modifying enzymes and provide evidence for adjustment of the pectin network at sites of iron accumulation in the root.ConclusionOur study reveals new aspects of the elaborate interplay between phosphate starvation responses and changes in iron homeostasis. The results emphasize the importance of apoplastic iron redistribution to mediate phosphate-dependent root growth adjustment and suggest an important role for citrate in phosphate-dependent apoplastic iron transport. We further demonstrate that root growth modulation correlates with an altered expression of cell wall modifying enzymes and changes in the pectin network of the phosphate-deprived root tip, supporting the hypothesis that pectins are involved in iron binding and/or phosphate mobilization.

Auxin coordinates plant development largely via hierarchical control of gene expression. During the past decades, the study of early auxin genes paired with the power of Arabidopsis genetics have unraveled key nuclear components and molecular interactions that perceive the hormone and activate primary response genes. Recent research in the realm of structural biology allowed unprecedented insight into: (i) the recognition of auxin-responsive DNA elements by auxin transcription factors; (ii) the inactivation of those auxin response factors by early auxin-inducible repressors; and (iii) the activation of target genes by auxin-triggered repressor degradation. The biophysical studies reviewed here provide an impetus for elucidating the molecular determinants of the intricate interactions between core components of the nuclear auxin response module.

Plants have evolved two major strategies to cope with phosphate (Pi) limitation. The systemic response, mainly comprising increased Pi uptake and metabolic adjustments for more efficient Pi use, and the local response, enabling plants to explore Pi-rich soil patches by reorganization of the root system architecture. Unlike previous reports, this study focused on root exudation controlled by the local response to Pi deficiency. To approach this, a hydroponic system separating the local and systemic responses was developed. Arabidopsis thaliana genotypes exhibiting distinct sensitivities to Pi deficiency could be clearly distinguished by their root exudate composition as determined by non-targeted reversed-phase ultraperformance liquid chromatography electrospray ionization quadrupole-time-of-flight mass spectrometry metabolite profiling. Compared with wild-type plants or insensitive low phosphate root 1 and 2 (lpr1 lpr2) double mutant plants, the hypersensitive phosphate deficiency response 2 (pdr2) mutant exhibited a reduced number of differential features in root exudates after Pi starvation, suggesting the involvement of PDR2-encoded P5-type ATPase in root exudation. Identification and analysis of coumarins revealed common and antagonistic regulatory pathways between Pi and Fe deficiency-induced coumarin secretion. The accumulation of oligolignols in root exudates after Pi deficiency was inversely correlated with Pi starvation-induced lignification at the root tips. The strongest oligolignol accumulation in root exudates was observed for the insensitive lpr1 lpr2 double mutant, which was accompanied by the absence of Pi deficiency-induced lignin deposition, suggesting a role of LPR ferroxidases in lignin polymerization during Pi starvation.

BarJey leaf segments treated with jasmonate respond with the synthesis of specific proseins, referred to as jasmonate‐induced proteins (JIPs). Application of abscisic acid (ABAl also induced JIP synthesis (Weidhase et al. 1987). In this study the effects of inhibitors on sorbitol‐induced increases of endogenous jasmonates and ABA were investigated. The promotion of jasmonates by sorbitol was inhibited by the growth retardant tetcyclacis at concentrations as low as 1 ftM. In parallel with the decrease of jasmonates, JIP gene expression was reduced as reflected by a decline in the level of a 23‐kDa protein UIP‐23) and mRNAs of JIP‐6 and JIP‐23. 12‐Oxo‐phytodienoic acid, an inlermediale in the lipoxygenase (LOX) pathway leading to jasmonic acid was able to overcome the inhibition by tetcyclacis and increases both the endogenous jasmonate content and transcript accumulation. This suggests that tetcyclacis acts upstream of 12‐oxo‐phytodienoic acid and in keeping with this proposal, an increase in relative LOX activity was detected after tetcyclacis treatment. Although tetcyclacis was shown to inhibit the degradation of ABA to phaseic acid, its effect on jasmonate synthesis is much more pronounced.

The accumulation of abundant proteins and their respective transcripts, induced by 10−4 M cisabscisic acid or 10−5 M jasmonic acid methyl ester, was studied in barley (Hordeum vulgare L.) leaf segments and compared to that resulting from osmotic stress caused by floating the segments on solutions of sorbitol, glucose, polyethyleneglycol (PEG)-6000 or NaCl. Osmotic stress or treatment with abscisic acid led to the synthesis of novel proteins which were identical to jasmonateinduced proteins (JIPs) with respect to immunological properties and molecular masses. The most prominent polypeptides were characterized by molecular masses of 66, 37 and 23 kDa and were newly synthesized. Whereas sorbitol, mannitol, sucrose, glucose and PEG provoked the synthesis of JIPs, 2deoxyglucose and NaCl did not. We provide evidence that the synthesis of JIPs induced by osmotic stress is directly correlated with a preceding rise in endogenous jasmonates. These jasmonates, quantified by an enzyme immunoassay specific for (−)jasmonic acid and its aminoacid conjugates, increased remarkably in leaf segments treated with sorbitol, glucose or other sugars. In contrast, no increase in jasmonates could be observed in tissues exposed to salts (NaCl). The results strengthen the hypothesis that the accumulation of jasmonates, probably by de-novo synthesis, is an intermediate and essential step in a signalling pathway between (osmotic) stress and activation of genes coding for polypeptides of high abundance.

The plant hormone auxin transcriptionally activates early genes. We have isolated a 14-member family of DNA sequences complementary to indoleacetic acid (IAA)-inducible transcripts inArabidopsis thaliana. The corresponding genes, IAA1 and IAA14, are homologs of PS-1AA4/5 and PS-IAA6 from pea, AUX22 and AUX28 from soybean, ARG3 and ARG4from mungbean, and AtAux2-11 and AtAux2-27 from Arabidopsis. The members of the family are differentially expressed in mature Arabidopsis plants. Characterization of IAA gene expression in etiolated seedlings demonstrates specificity for auxin inducibility. The response of most family members to IAA is rapid (within 4 to 30 minutes) and insensitive to cyclohexamide. Cyclohexamide alone induces all the early genes. Auxin-induction of two late genes, IAA7 and IAA8, is inhibited by cyclohexamide, indicating requirement of protein synthesis for their activation. All IAA genes display a biphasic dose response that is optimal at 10 μM IAA. However, individual genes respond differentially between 10 nM and 5μM IAA. Expression of all genes is defective in the Arabidopsis auxin-resistant mutant lines axr1, axr2, and aux1.The encoded polypeptides share four conserved domains, and seven invariant residues in the intervening regions. The spaces vary considerably in length, rendering the calculated molecular mass of IAA proteins to range from 19 kDa to 36 kDa. Overall sequence identity between members of the family is highly variable (36 to 87%). Their most significant structural features are functional nuclear transport signals, and a putative βαα-fold whose modeled three dimensional structure appears to be compatible with the prokaryotic β-ribbon DNA recognition motif. The data suggest that auxin induces in a differential and hierarchical fashion a large family of early genes that encode a structurally diverse class of nuclear proteins. These proteins are proposed to mediate tissue-specific and cell-type restricted responses to the hormone during plant growth and development.

The plant hormone, indoleacetic acid (IAA), transcriptionally activates two early genes in pea, PS‐IAA4/5 and PS‐IAA6 , that encode short‐lived nuclear proteins. The identification of the nuclear localization signals (NLS) in PS‐IAA4 and PS‐IAA6 using progressive deletion analysis and site‐directed mutagenesis is reported. A C‐terminal SV40‐type NLS is sufficient to direct the β‐glucuronidase reporter to the nucleus of transiently transformed tobacco protoplasts, but is dispensible for nuclear localization of both proteins. The dominant and essential NLS in PS‐IAA4 and PS‐IAA6 overlap with a bipartite basic motif which is polymorphic and conserved in related proteins from other plant species, having the consensus sequence (KKNEK)KR‐X(24–71)‐(RSXRK)/(RK/RK). Both basic elements of this motif in PS‐IAA4, (KR‐X41‐RSYRK), function interdependently as a bipartite NLS. However, in PS‐IAA6 (KKNEKKR‐X36‐RKK) the upstream element of the corresponding motif contains additional basic residues which allow its autonomous function as an SV40‐type monopartite NLS. The spacer‐length polymorphism, X(24–70), in respective bipartite NLS peptides of several PS‐IAA4‐like proteins from Arabidopsis thaliana does not affect nuclear targeting function. The structural and functional variation of the bipartite basic motif in PS‐IAA4‐like proteins supports the proposed integrated consensus of NLS.