Although abundant in soils, iron (Fe) is poorly bioavailable for plants. Improving Fe uptake in crops, enabling them to grow in Fe-depleted soils, has become a major focal interest. The secretion of ...Fe-mobilizing coumarins by plant roots recently emerged as an important factor allowing nongrass species to cope with low Fe bioavailability. The main molecular actors involved in the biosynthesis and secretion of coumarins have been identified, but the precise regulatory mechanisms that tune their production remain poorly understood. Here, we review the recent progress in coumarin synthesis and transport in plants and future research directions to gain knowledge of these mechanisms, which will offer novel opportunities for improving plant growth and health and for generating Fe-fortified crops.
Iron is an important factor conditioning biomass production, crop yield, and the quality of plant products.Although iron is the fourth most abundant element on Earth, iron is poorly available for plants.The secretion of coumarins is a crucial mechanism for iron uptake in nongrass species when iron availability is low, particularly in alkaline soils.Recent studies have identified the main coumarins involved in iron uptake and the main biochemical steps and regulatory processes controlling their biosynthesis.These findings provide novel targets for improving plant growth and health together with iron content in the edible part of plants and thus human diet.
Since their discovery as a substrate for ribonucleotide reductase (RNR), the role of thioredoxin (Trx) and glutaredoxin (Grx) has been largely extended through their regulatory function. Both ...proteins act by changing the structure and activity of a broad spectrum of target proteins, typically by modifying redox status. Trx and Grx are members of families with multiple and partially redundant genes. The number of genes clearly increased with the appearance of multicellular organisms, in part because of new types of Trx and Grx with orthologs throughout the animal and plant kingdoms. The function of Trx and Grx also broadened as cells achieved increased complexity, especially in the regulation arena. In view of these progressive changes, the ubiquitous distribution of Trx and the wide occurrence of Grx enable these proteins to serve as indicators of the evolutionary history of redox regulation. In so doing, they add a unifying element that links the diverse forms of life to one another in an uninterrupted continuum. It is anticipated that future research will embellish this continuum and further elucidate the properties of these proteins and their impact on biology. The new information will be important not only to our understanding of the role of Trx and Grx in fundamental cell processes but also to future societal benefits as the proteins find new applications in a range of fields.
Iron (Fe) homeostasis is crucial for all living organisms. In mammals, an integrated posttranscriptional mechanism couples the regulation of both Fe deficiency and Fe excess responses. Whether in ...plants an integrated control mechanism involving common players regulates responses both to deficiency and to excess is still to be determined.
In this study, molecular, genetic and biochemical approaches were used to investigate transcriptional responses to both Fe deficiency and excess.
A transcriptional activator of responses to Fe shortage in Arabidopsis, called bHLH105/ILR3, was found to also negatively regulate the expression of ferritin genes, which are markers of the plant’s response to Fe excess. Further investigations revealed that ILR3 repressed the expression of several structural genes that function in the control of Fe homeostasis. ILR3 interacts directly with the promoter of its target genes, and repressive activity was conferred by its dimerisation with bHLH47/PYE. Last, this study highlighted that important facets of plant growth in response to Fe deficiency or excess rely on ILR3 activity.
Altogether, the data presented herein support that ILR3 is at the centre of the transcriptional regulatory network that controls Fe homeostasis in Arabidopsis, in which it acts as both transcriptional activator and repressor.
Iron-sulfur (Fe-S) proteins are crucial for many cellular functions, particularly those involving electron transfer and metabolic reactions. An essential monothiol glutaredoxin GRXS15 plays a key ...role in the maturation of plant mitochondrial Fe-S proteins. However, its specific molecular function is not clear, and may be different from that of the better characterized yeast and human orthologs, based on known properties. Hence, we report here a detailed characterization of the interactions between
GRXS15 and ISCA proteins using both in vivo and in vitro approaches. Yeast two-hybrid and bimolecular fluorescence complementation experiments demonstrated that GRXS15 interacts with each of the three plant mitochondrial ISCA1a/1b/2 proteins. UV-visible absorption/CD and resonance Raman spectroscopy demonstrated that coexpression of ISCA1a and ISCA2 resulted in samples with one 2Fe-2S
cluster per ISCA1a/2 heterodimer, but cluster reconstitution using as-purified 2Fe-2S-ISCA1a/2 resulted in a 4Fe-4S
cluster-bound ISCA1a/2 heterodimer. Cluster transfer reactions monitored by UV-visible absorption and CD spectroscopy demonstrated that 2Fe-2S-GRXS15 mediates 2Fe-2S
cluster assembly on mitochondrial ferredoxin and 4Fe-4S
cluster assembly on the ISCA1a/2 heterodimer in the presence of excess glutathione. This suggests that ISCA1a/2 is an assembler of 4Fe-4S
clusters, via two-electron reductive coupling of two 2Fe-2S
clusters. Overall, the results provide new insights into the roles of GRXS15 and ISCA1a/2 in effecting 2Fe-2S
to 4Fe-4S
cluster conversions for the maturation of client 4Fe-4S cluster-containing proteins in plants.
Glutaredoxins (GRXs) have at least three major identified functions. In apoforms, they exhibit oxidoreductase activity controlling notably protein glutathionylation/deglutathionylation. In holoforms, ...i.e., iron-sulfur (Fe-S) cluster-bridging forms, they act as maturation factors for the biogenesis of Fe-S proteins or as regulators of iron homeostasis contributing directly or indirectly to the sensing of cellular iron status and/or distribution. The latter functions seem intimately connected with the capacity of specific GRXs to form 2Fe-2S cluster-bridging homodimeric or heterodimeric complexes with BOLA proteins. In yeast species, both proteins modulate the localization and/or activity of transcription factors regulating genes coding for proteins involved in iron uptake and intracellular sequestration in response notably to iron deficiency. Whereas vertebrate GRX and BOLA isoforms may display similar functions, the involved partner proteins are different. We perform here a critical evaluation of the results supporting the implication of both protein families in similar signaling pathways in plants and provide ideas and experimental strategies to delineate further their functions.
Proteins incorporating iron–sulfur (Fe-S) co-factors are required for a plethora of metabolic processes. Their maturation depends on three Fe-S cluster assembly machineries in plants, located in the ...cytosol, mitochondria, and chloroplasts. After de novo formation on scaffold proteins, transfer proteins load Fe-S clusters onto client proteins. Among the plastidial representatives of these transfer proteins, NFU2 and NFU3 are required for the maturation of the 4Fe-4S clusters present in photosystem I subunits, acting upstream of the high-chlorophyll fluorescence 101 (HCF101) protein. NFU2 is also required for the maturation of the 2Fe-2S-containing dihydroxyacid dehydratase, important for branched-chain amino acid synthesis. Here, we report that recombinant Arabidopsis thaliana NFU1 assembles one 4Fe-4S cluster per homodimer. Performing co-immunoprecipitation experiments and assessing physical interactions of NFU1 with many 4Fe-4S-containing plastidial proteins in binary yeast two-hybrid assays, we also gained insights into the specificity of NFU1 for the maturation of chloroplastic Fe-S proteins. Using bimolecular fluorescence complementation and in vitro Fe-S cluster transfer experiments, we confirmed interactions with two proteins involved in isoprenoid and thiamine biosynthesis, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase and 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase, respectively. An additional interaction detected with the scaffold protein SUFD enabled us to build a model in which NFU1 receives its Fe-S cluster from the SUFBC2D scaffold complex and serves in the maturation of specific 4Fe-4S client proteins. The identification of the NFU1 partner proteins reported here more clearly defines the role of NFU1 in Fe-S client protein maturation in Arabidopsis chloroplasts among other SUF components.
Nfu-type proteins are essential in the biogenesis of iron–sulfur (Fe-S) clusters in numerous organisms. A number of phenotypes including low levels of Fe-S cluster incorporation are associated with ...the deletion of the gene encoding a chloroplast-specific Nfu-type protein, Nfu2 from Arabidopsis thaliana (AtNfu2). Here, we report that recombinant AtNfu2 is able to assemble both 2Fe-2S and 4Fe-4S clusters. Analytical data and gel filtration studies support cluster/protein stoichiometries of one 2Fe-2S cluster/homotetramer and one 4Fe-4S cluster/homodimer. The combination of UV–visible absorption and circular dichroism and resonance Raman and Mössbauer spectroscopies has been employed to investigate the nature, properties, and transfer of the clusters assembled on Nfu2. The results are consistent with subunit-bridging 2Fe-2S2+ and 4Fe-4S2+ clusters coordinated by the cysteines in the conserved CXXC motif. The results also provided insight into the specificity of Nfu2 for the maturation of chloroplastic Fe-S proteins via intact, rapid, and quantitative cluster transfer. 2Fe-2S cluster-bound Nfu2 is shown to be an effective 2Fe-2S2+ cluster donor for glutaredoxin S16 but not glutaredoxin S14. Moreover, 4Fe-4S cluster-bound Nfu2 is shown to be a very rapid and efficient 4Fe-4S2+ cluster donor for adenosine 5′-phosphosulfate reductase (APR1), and yeast two-hybrid studies indicate that APR1 forms a complex with Nfu2 but not with Nfu1 and Nfu3, the two other chloroplastic Nfu proteins. This cluster transfer is likely to be physiologically relevant and is particularly significant for plant metabolism as APR1 catalyzes the second step in reductive sulfur assimilation, which ultimately results in the biosynthesis of cysteine, methionine, glutathione, and Fe-S clusters.
Plant chloroplasts have versatile thioredoxin systems including two thioredoxin reductases and multiple types of thioredoxins. Plastid-localized NADPH-dependent thioredoxin reductase (NTRC) contains ...both reductase (NTRd) and thioredoxin (TRXd) domains in a single polypeptide and forms homodimers. To study the action of NTRC and NTRC domains in vivo, we have complemented the ntrc knockout line of Arabidopsis with the wild type and full-length NTRC genes, in which 2-Cys motifs either in NTRd, or in TRXd were inactivated. The ntrc line was also transformed either with the truncated NTRd or TRXd alone. Overexpression of wild-type NTRC promoted plant growth by increasing leaf size and biomass yield of the rosettes. Complementation of the ntrc line with the full-length NTRC gene containing an active reductase but an inactive TRXd, or vice versa, recovered wild-type chloroplast phenotype and, partly, rosette biomass production, indicating that the NTRC domains are capable of interacting with other chloroplast thioredoxin systems. Overexpression of truncated NTRd or TRXd in ntrc background did not restore wild-type phenotype. Modeling of the three-dimensional structure of the NTRC dimer indicates extensive interactions between the NTR domains and the TRX domains further stabilize the dimeric structure. The long linker region between the NTRd and TRXd, however, allows flexibility for the position of the TRXd in the dimer. Supplementation of the TRXd in the NTRC homodimer model by free chloroplast thioredoxins indicated that TRXf is the most likely partner to interact with NTRC. We propose that overexpression of NTRC promotes plant biomass yield both directly by stimulation of chloroplast biosynthetic and protective pathways controlled by NTRC and indirectly via free chloroplast thioredoxins. Our data indicate that overexpression of chloroplast thiol redox-regulator has a potential to increase biofuel yield in plant and algal species suitable for sustainable bioenergy production.
In plants RNA silencing is a host defense mechanism against viral infection, in which double-strand RNA is processed into 21-24-nt short interfering RNA (siRNA). Silencing spreads from cell to cell ...and systemically through a sequence-specific signal to limit the propagation of the virus. To counteract this defense mechanism, viruses encode suppressors of silencing. The P1 protein encoded by the rice yellow mottle virus (RYMV) displays suppression activity with variable efficiency, according to the isolates that they originated from. Here, we show that P1 proteins from two RYMV isolates displaying contrasting suppression strength reduced local silencing induced by single-strand and double-strand RNA in Nicotiana benthamiana leaves. This suppression was associated with a slight and a severe reduction in 21- and 24-nt siRNA accumulation, respectively. Unexpectedly, cell-to-cell movement and systemic propagation of silencing were enhanced in P1-expressing Nicotiana plants. When transgenically expressed in rice, P1 proteins induced specific deregulation of DCL4-dependent endogenous siRNA pathways, whereas the other endogenous pathways were not affected. As DCL4-dependent pathways play a key role in rice development, the expression of P1 viral proteins was associated with the same severe developmental defects in spikelets as in dcl4 mutants. Overall, our results demonstrate that a single viral protein displays multiple effects on both endogenous and exogenous silencing, not only in a suppressive but also in an enhancive manner. This suggests that P1 proteins play a key role in maintaining a subtle equilibrium between defense and counter-defense mechanisms, to insure efficient virus multiplication and the preservation of host integrity.
In monocotyledons, the root system is mostly composed of postembryonic shoot‐borne roots called crown roots. In rice (Oryza sativa), auxin promotes crown root initiation via the LOB‐domain ...transcription factor (LBD) transcription factor CROWN ROOTLESS1 (CRL1); however, the gene regulatory network downstream of CRL1 remains largely unknown. We tested CRL1 transcriptional activity in yeast and in planta, identified CRL1‐regulated genes using an inducible gene expression system and a transcriptome analysis, and used in situ hybridization to demonstrate coexpression of a sample of CRL1‐regulated genes with CRL1 in crown root primordia. We show that CRL1 positively regulates 277 genes, including key genes involved in meristem patterning (such as QUIESCENT‐CENTER SPECIFIC HOMEOBOX; QHB), cell proliferation and hormone homeostasis. Many genes are homologous to Arabidopsis genes involved in lateral root formation, but about a quarter are rice‐specific. Our study reveals that several genes acting downstream of LBD transcription factors controlling postembryonic root formation are conserved between monocots and dicots. It also provides evidence that specific genes are involved in the formation of shoot‐derived roots in rice.
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