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Physiological and interactomic analysis reveals versatile functions of Arabidopsis 14-3-3 quadruple mutants in response to Fe deficiency.

ABSTRACT: To date, few phenotypes have been described for Arabidopsis 14-3-3 mutants or the phenotypes showing the role of 14-3-3 in plant responding to abiotic stress. Although one member of the 14-3-3 protein family (14-3-3 omicron) was shown to be involved in the proper operation of Fe acquisition mechanisms at physiological and gene expression levels in Arabidopsis thaliana, it remains to be explored whether other members play a role in regulating iron acquisition. To more directly and effectively observe whether members of 14-3-3 non-epsilon group have a function in Fe-deficiency adaptation, three higher order quadruple KOs, kappa/lambda/phi/chi (klpc), kappa/lambda/upsilon/nu(klun), and upsilon/nu/phi/chi (unpc) were generated and studied for physiological analysis in this study. The analysis of iron-utilization efficiency, root phenotyping, and transcriptional level of Fe-responsive genes suggested that the mutant with kl background showed different phenotypes from Wt when plants suffered Fe starved, while these phenotypes were absent in the unpc mutant. Moreover, the absence of the four 14-3-3 isoforms in the klun mutant has a clear impact on the 14-3-3 interactome upon Fe deficiency. Dynamics of 14-3-3-client interactions analysis showed that 27 and 17 proteins differentially interacted with 14-3-3 in Wt and klun roots caused by Fe deficiency, respectively. Many of these Fe responsive proteins have a role in glycolysis, oxidative phosphorylation and TCA cycle, the FoF1-synthase and in the cysteine/methionine synthesis. A clear explanation for the observed phenotypes awaits a more detailed analysis of the functional aspects of 14-3-3 binding to the target proteins identified in this study.

SUBMITTER: Gao J

PROVIDER: S-EPMC8324900 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Due to its ease to donate or accept electrons, iron (Fe) plays a crucial role in respiration and metabolism, including tetrapyrrole synthesis, in virtually all organisms. In plants, Fe is a component of the photosystems and thus essential for photosynthesis. Fe deficiency compromises chlorophyll (Chl) synthesis, leading to interveinal chlorosis in developing leaves and decreased photosynthetic activity. To gain insights into the responses of photosynthetically active cells to Fe deficiency, we conducted transcriptional profiling experiments on leaves from Fe-sufficient and Fe-deficient plants using the RNA-seq technology. As anticipated, genes associated with photosynthesis and tetrapyrrole metabolism were dramatically down-regulated by Fe deficiency. A sophisticated response comprising the down-regulation of HEMA1 and NYC1, which catalyze the first committed step in tetrapyrrole biosynthesis and the conversion of Chl b to Chl a at the commencement of Chl breakdown, respectively, and the up-regulation of CGLD27, which is conserved in plastid-containing organisms and putatively involved in xanthophyll biosynthesis, indicates a carefully orchestrated balance of potentially toxic tetrapyrrole intermediates and functional end products to avoid photo-oxidative damage. Comparing the responses to Fe deficiency in leaves to that in roots confirmed subgroup 1b bHLH transcription factors and POPEYE/BRUTUS as important regulators of Fe homeostasis in both leaf and root cells, and indicated six novel players with putative roles in Fe homeostasis that were highly expressed in leaves and roots and greatly induced by Fe deficiency. The data further revealed down-regulation of organ-specific subsets of genes encoding ribosomal proteins, which may be indicative of a change in ribosomal composition that could bias translation. It is concluded that Fe deficiency causes a massive reorganization of plastid activity, which is adjusting leaf function to the availability of Fe.

Project description:Essential metals such as iron are required for healthy plant growth. Fe is an important cofactor and catalytic element in many biological processes. Fe and other metals can also be toxic when present in excess. Therefore plants have mechanisms of metal homeostasis which involve coordination of metal ion transporters for uptake, translocation and compartmentalisation. The NAS genes are supposed to play an important role in Fe homeostasis. They are coding for enzymes called nicotianaminesynthase (NAS), which synthesize nicotianamine (NA) by a one-step condensation reaction of three molecules S-adenosyl-methionine. NA acts as a chelator for Fe, Cu, Ni and Zn and might be involved in the transport and allocation of Fe throughout the plant. We generated quadruple T-DNA insertion mutant nas plants to investigate NA function as described in Klatte et al., 2009, Plant Physiol. The nas4x-1 plants show an interveinal leaf chlorosis when turning from vegetative to reproductive stage, which intensifies when growing under Fe deficiency conditions. nas4x-1 plants have strongly reduced NA contents and show an elevated Fe deficiency response in roots. By performing microarray experiments we want to reveal global changes on transcriptional level in roots and leaves of nas4x-1 mutant compared to wild type plants grown under Fe supply and Fe deficiency conditions, respectively. The loss of NAS genes has a strong impact on the regulation of other metal homeostasis genes and allows to draw conclusions about nicotianamine function in metal homeostasis of A.thaliana. For this study, four-week old nas4x-1 mutant and wild type plants were exposed for 7 days to plant medium with and without Fe supply. These conditions have been established previously and have resulted in a reproducibly strong interveinal leaf chlorosis of nas4x-1 plants compared to wild type, especially upon Fe deficiency conditions. The experiment was repeated three times in consecutive weeks to obtain three independent biological repetitions. Rosette leaves and roots of five week-old plants were harvested, RNA was isolated and microarray hybridization was performed. 24 Total samples were analyzed. We generated the following pairwise comparisons: WT + Fe vs. – Fe, nas4x-1 + Fe vs. – Fe, + Fe WT vs. + Fe nas4x-1, - Fe WT vs. - Fe nas4x-1, roots and leaves

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Physiological and interactomic analysis reveals versatile functions of Arabidopsis 14-3-3 quadruple mutants in response to Fe deficiency.

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