Ca2+-dependent phosphorylation of NRAMP1 by CPK21 and CPK23 facilitates manganese uptake and homeostasis in Arabidopsis.

Homeostasis of the essential micronutrient manganese (Mn) is crucially determined through availability and uptake efficiency in all organisms. Mn deficiency of plants especially occurs in alkaline and calcareous soils, seriously restricting crop yield. However, the mechanisms underlying the sensing and signaling of Mn availability and conferring regulation of Mn uptake await elucidation. Here, we uncover that Mn depletion triggers spatiotemporally defined long-lasting Ca2+ oscillations in Arabidopsis roots. These Ca2+ signals initiate in individual cells, expand, and intensify intercellularly to transform into higher-order multicellular oscillations. Furthermore, through an interaction screen we identified the Ca2+-dependent protein kinases CPK21 and CPK23 as Ca2+ signal-decoding components that bring about translation of these signals into regulation of uptake activity of the high-affinity Mn transporter natural resistance associated macrophage proteins 1 (NRAMP1). Accordingly, a cpk21/23 double mutant displays impaired growth and root development under Mn-limiting conditions, while kinase overexpression confers enhanced tolerance to low Mn supply to plants. In addition, we define Thr498 phosphorylation within NRAMP1 as a pivot mechanistically determining NRAMP1 activity, as revealed by biochemical assays and complementation of yeast Mn uptake and Arabidopsis nramp1 mutants. Collectively, these findings delineate the Ca2+-CPK21/23-NRAMP1 axis as key for mounting plant Mn homeostasis.

Manganese in Plants: From Acquisition to Subcellular Allocation.

Manganese (Mn) is an important micronutrient for plant growth and development and sustains metabolic roles within different plant cell compartments. The metal is an essential cofactor for the oxygen-evolving complex (OEC) of the photosynthetic machinery, catalyzing the water-splitting reaction in photosystem II (PSII). Despite the importance of Mn for photosynthesis and other processes, the physiological relevance of Mn uptake and compartmentation in plants has been underrated. The subcellular Mn homeostasis to maintain compartmented Mn-dependent metabolic processes like glycosylation, ROS scavenging, and photosynthesis is mediated by a multitude of transport proteins from diverse gene families. However, Mn homeostasis may be disturbed under suboptimal or excessive Mn availability. Mn deficiency is a serious, widespread plant nutritional disorder in dry, well-aerated and calcareous soils, as well as in soils containing high amounts of organic matter, where bio-availability of Mn can decrease far below the level that is required for normal plant growth. By contrast, Mn toxicity occurs on poorly drained and acidic soils in which high amounts of Mn are rendered available. Consequently, plants have evolved mechanisms to tightly regulate Mn uptake, trafficking, and storage. This review provides a comprehensive overview, with a focus on recent advances, on the multiple functions of transporters involved in Mn homeostasis, as well as their regulatory mechanisms in the plant's response to different conditions of Mn availability.

Loci, genes, and mechanisms associated with tolerance to ferrous iron toxicity in rice (Oryza sativa L.).

KEY MESSAGE: A genome-wide association study in rice yielded loci and candidate genes associated with tolerance to iron toxicity, and revealed biochemical mechanisms associated with tolerance in contrasting haplotypes. Iron toxicity is a major nutrient disorder affecting rice. Therefore, understanding the genetic and physiological mechanisms associated with iron toxicity tolerance is crucial in adaptive breeding and biofortification. We conducted a genome-wide association study (GWAS) by exposing a population of 329 accessions representing all subgroups of rice to ferrous iron stress (1000 ppm, 5 days). Expression patterns and sequence polymorphisms of candidate genes were investigated, and physiological hypotheses related to candidate loci were tested using a subset of contrasting haplotypes. Both iron including and excluding tolerant genotypes were observed, and shoot iron concentrations explained around 15.5 % of the variation in foliar symptom formation. GWAS for seven traits yielded 20 SNP markers exceeding a significance threshold of -log10 P > 4.0, which represented 18 distinct loci. One locus mapped for foliar symptom formation on chromosome 1 contained two putative glutathione-S-transferases, which were strongly expressed under iron stress and showed sequence polymorphisms in complete linkage disequilibrium with the most significant SNP. Contrasting haplotypes for this locus showed significant differences in dehydroascorbate reductase activity, which affected the plants' redox status under iron stress. We conclude that maintaining foliar redox homeostasis under iron stress represented an important tolerance mechanism associated with a locus identified through GWAS.

Ascorbate biosynthesis and its involvement in stress tolerance and plant development in rice (Oryza sativa L.).

Ascorbic acid (AsA) biosynthesis and its implications for stress tolerance and plant development were investigated in a set of rice knock-out (KO) mutants for AsA biosynthetic genes and their wild-types. KO of two isoforms of GDP-D-mannose epimerase (OsGME) reduced the foliar AsA level by 20-30%, and KO of GDP-L-galactose phosphorylase (OsGGP) by 80%, while KO of myo-inositol oxygenase (OsMIOX) did not affect foliar AsA levels. AsA concentration was negatively correlated with lipid peroxidation in foliar tissue under ozone stress and zinc deficiency, but did not affect the sensitivity to iron toxicity. Lack of AsA reduced the photosynthetic efficiency as represented by the maximum carboxylation rate of Rubisco (Vmax), the maximum electron transport rate (Jmax) and the chlorophyll fluorescence parameter ΦPSII. Mutants showed lower biomass production than their wild-types, especially when OsGGP was lacking (around 80% reductions). All plants except for KO mutants of OsGGP showed distinct peaks in foliar AsA concentrations during the growth, which were consistent with up-regulation of OsGGP, suggesting that OsGGP plays a pivotal role in regulating foliar AsA levels during different growth stages. In conclusion, our data demonstrate multiple roles of AsA in stress tolerance and development of rice.

Genetic dissection of ozone tolerance in rice (Oryza sativa L.) by a genome-wide association study.

Tropospheric ozone causes various negative effects on plants and affects the yield and quality of agricultural crops. Here, we report a genome-wide association study (GWAS) in rice (Oryza sativa L.) to determine candidate loci associated with ozone tolerance. A diversity panel consisting of 328 accessions representing all subgroups of O. sativa was exposed to ozone stress at 60 nl l(-1) for 7h every day throughout the growth season, or to control conditions. Averaged over all genotypes, ozone significantly affected biomass-related traits (plant height -1.0%, shoot dry weight -15.9%, tiller number -8.3%, grain weight -9.3%, total panicle weight -19.7%, single panicle weight -5.5%) and biochemical/physiological traits (symptom formation, SPAD value -4.4%, foliar lignin content +3.4%). A wide range of genotypic variance in response to ozone stress were observed in all phenotypes. Association mapping based on more than 30 000 single-nucleotide polymorphism (SNP) markers yielded 16 significant markers throughout the genome by applying a significance threshold of P<0.0001. Furthermore, by determining linkage disequilibrium blocks associated with significant SNPs, we gained a total of 195 candidate genes for these traits. The following sequence analysis revealed a number of novel polymorphisms in two candidate genes for the formation of visible leaf symptoms, a RING and an EREBP gene, both of which are involved in cell death and stress defence reactions. This study demonstrated substantial natural variation of responses to ozone in rice and the possibility of using GWAS in elucidating the genetic factors underlying ozone tolerance.

Zinc deficiency differentially affects redox homeostasis of rice genotypes contrasting in ascorbate level.

Zinc (Zn) deficiency is an important mineral disorder affecting rice production, and is associated with the formation of oxidative stress in plant tissue. In this study we investigated processes of oxidative stress formation as affected by ascorbate (AsA) in two pairs of contrasting rice genotypes: (i) two indica lines differing in field tolerance to Zn deficiency and AsA metabolism, i.e. RIL46 (tolerant) and IR74 (sensitive); (ii) the japonica wild-type Nipponbare (tolerant) and the AsA deficient TOS17 mutant line ND6172 (sensitive) having a 20-30% lower AsA level due to the knockout of an AsA biosynthetic gene (OsGME1). Plants were grown hydroponically under +Zn and -Zn conditions for 21 days and samples were investigated after 7, 14, and 21 days of treatment. Tissue Zn concentrations below 20mg kg(-1) in the -Zn treatment induced the formation of visible symptoms of Zn deficiency from day 14 in all genotypes, but especially in the sensitive IR74. Significant increases in lipid peroxidation were observed in the leaves of the sensitive genotypes IR74 and ND6172, and in the roots of IR74, but not in the tolerant genotypes. At day 21, the tolerant genotypes RIL46 and Nipponbare had significantly higher AsA levels in both shoots and roots compared to the sensitive lines. Consistently, higher levels of hydrogen peroxide formation in leaves and roots of the sensitive genotypes were detected using staining methods. Differences in foliar hydrogen peroxide formation between IR74 and RIL46 became apparent on day 7 and between ND6172 and Nipponbare on day 14. Similarly, genotypic differences in hydrogen peroxide formation in the roots were seen on day 21. In conclusion, our data demonstrate that Zn deficiency leads to a redox imbalance in roots and shoots prior to the occurrence of visible symptoms, and that the antioxidant AsA plays an important role in maintaining the redox homeostasis under Zn deficiency.

Ascorbate metabolism in rice genotypes differing in zinc efficiency.

Effects of zinc (Zn) deficiency on shoot metabolites were investigated in contrasting rice (Oryza sativa L.) genotypes with special focus on ascorbic acid (AsA) biosynthesis, recycling, and catabolism. The genotypes IR74 (sensitive) and RIL46 (tolerant) were subjected to -Zn and control treatments for 3 weeks, and samples were taken at three different stages representing the pre-stress phase, emergence of visible stress, and severe visible stress. The emergence of visible symptoms was paralleled by an increase in lipid peroxidation and a decrease in AsA concentration in the sensitive, but not in the tolerant genotype. The tolerant RIL46 showed enhanced transcript levels of several genes involved in the mannose/L-galactose pathway to AsA biosynthesis, and significant up-regulation of a gene involved in the putative alternative myo-inositol pathway under low Zn stress. The level of most AsA precursors was negatively affected by Zn deficiency, but RIL46 had a constitutively higher level of non-phosphorylated precursors. Products of AsA catabolism such as oxalate and threonate did not accumulate in either genotype, suggesting that AsA degradation did not contribute to the stress-induced decline of the AsA pool in IR74. Further factors possibly contributing to tolerance in RIL46 included an almost fivefold higher proline level under -Zn stress and significantly higher trehalose content. The implications of these compounds in AsA metabolism and Zn efficiency thus deserve further attention.