Arabidopsis thaliana CYCLIC NUCLEOTIDE-GATED CHANNEL2 mediates extracellular ATP signal transduction in root epidermis.

Damage can be signalled by extracellular ATP (eATP) using plasma membrane (PM) receptors to effect cytosolic free calcium ion ([Ca2+ ]cyt ) increase as a second messenger. The downstream PM Ca2+ channels remain enigmatic. Here, the Arabidopsis thaliana Ca2+ channel subunit CYCLIC NUCLEOTIDE-GATED CHANNEL2 (CNGC2) was identified as a critical component linking eATP receptors to downstream [Ca2+ ]cyt signalling in roots. Extracellular ATP-induced changes in single epidermal cell PM voltage and conductance were measured electrophysiologically, changes in root [Ca2+ ]cyt were measured with aequorin, and root transcriptional changes were determined by quantitative real-time PCR. Two cngc2 loss-of-function mutants were used: cngc2-3 and defence not death1 (which expresses cytosolic aequorin). Extracellular ATP-induced transient depolarization of Arabidopsis root elongation zone epidermal PM voltage was Ca2+ dependent, requiring CNGC2 but not CNGC4 (its channel co-subunit in immunity signalling). Activation of PM Ca2+ influx currents also required CNGC2. The eATP-induced [Ca2+ ]cyt increase and transcriptional response in cngc2 roots were significantly impaired. CYCLIC NUCLEOTIDE-GATED CHANNEL2 is required for eATP-induced epidermal Ca2+ influx, causing depolarization leading to [Ca2+ ]cyt increase and damage-related transcriptional response.

Phosphate-deprivation and damage signalling by extracellular ATP.

Phosphate deprivation compromises plant productivity and modulates immunity. DAMP signalling by extracellular ATP (eATP) could be compromised under phosphate deprivation by the lowered production of cytosolic ATP and the need to salvage eATP as a nutritional phosphate source. Phosphate-starved roots of Arabidopsis can still sense eATP, indicating robustness in receptor function. However, the resultant cytosolic free Ca2+ signature is impaired, indicating modulation of downstream components. This perspective on DAMP signalling by extracellular ATP (eATP) addresses the salvage of eATP under phosphate deprivation and its promotion of immunity, how Ca2+ signals are generated and how the Ca2+ signalling pathway could be overcome to allow beneficial fungal root colonization to fulfill phosphate demands. Safe passage for an endophytic fungus allowing root colonization could be achieved by its down-regulation of the Ca2+ channels that act downstream of the eATP receptors and by also preventing ROS accumulation, thus further impairing DAMP signalling.

Annexin 1 Is a Component of eATP-Induced Cytosolic Calcium Elevation in Arabidopsis thaliana Roots.

Extracellular ATP (eATP) has long been established in animals as an important signalling molecule but this is less understood in plants. The identification of Arabidopsis thaliana DORN1 (Does Not Respond to Nucleotides) as the first plant eATP receptor has shown that it is fundamental to the elevation of cytosolic free Ca2+ ([Ca2+]cyt) as a possible second messenger. eATP causes other downstream responses such as increase in reactive oxygen species (ROS) and nitric oxide, plus changes in gene expression. The plasma membrane Ca2+ influx channels involved in eATP-induced [Ca2+]cyt increase remain unknown at the genetic level. Arabidopsis thaliana Annexin 1 has been found to mediate ROS-activated Ca2+ influx in root epidermis, consistent with its operating as a transport pathway. In this study, the loss of function Annexin 1 mutant was found to have impaired [Ca2+]cyt elevation in roots in response to eATP or eADP. Additionally, this annexin was implicated in modulating eATP-induced intracellular ROS accumulation in roots as well as expression of eATP-responsive genes.

Phosphate Deprivation Can Impair Mechano-Stimulated Cytosolic Free Calcium Elevation in Arabidopsis Roots.

The root tip responds to mechanical stimulation with a transient increase in cytosolic free calcium as a possible second messenger. Although the root tip will grow through a heterogeneous soil nutrient supply, little is known of the consequence of nutrient deprivation for such signalling. Here, the effect of inorganic phosphate deprivation on the root's mechano-stimulated cytosolic free calcium increase is investigated. Arabidopsisthaliana (cytosolically expressing aequorin as a bioluminescent free calcium reporter) is grown in zero or full phosphate conditions, then roots or root tips are mechanically stimulated. Plants also are grown vertically on a solid medium so their root skewing angle (deviation from vertical) can be determined as an output of mechanical stimulation. Phosphate starvation results in significantly impaired cytosolic free calcium elevation in both root tips and whole excised roots. Phosphate-starved roots sustain a significantly lower root skewing angle than phosphate-replete roots. These results suggest that phosphate starvation causes a dampening of the root mechano-signalling system that could have consequences for growth in hardened, compacted soils.

DORN1/P2K1 and purino-calcium signalling in plants: making waves with extracellular ATP.

BACKGROUND AND AIMS: Extracellular ATP governs a range of plant functions, including cell viability, adaptation and cross-kingdom interactions. Key functions of extracellular ATP in leaves and roots may involve an increase in cytosolic free calcium as a second messenger ('calcium signature'). The main aim here was to determine to what extent leaf and root calcium responses require the DORN1/P2K1 extracellular ATP receptor in Arabidopsis thaliana. The second aim was to test whether extracellular ATP can generate a calcium wave in the root. METHODS: Leaf and root responses to extracellular ATP were reviewed for their possible links to calcium signalling and DORN1/P2K1. Leaves and roots of wild type and dorn1 plants were tested for cytosolic calcium increase in response to ATP, using aequorin. The spatial abundance of DORN1/P2K1 in the root was estimated using green fluorescent protein. Wild type roots expressing GCaMP3 were used to determine the spatial variation of cytosolic calcium increase in response to extracellular ATP. KEY RESULTS: Leaf and root ATP-induced calcium signatures differed markedly. The leaf signature was only partially dependent on DORN1/P2K1, while the root signature was fully dependent. The distribution of DORN1/P2K1 in the root supports a key role in the generation of the apical calcium signature. Root apical and sub-apical calcium signatures may operate independently of each other but an apical calcium increase can drive a sub-apical increase, consistent with a calcium wave. CONCLUSION: DORN1 could underpin several calcium-related responses but it may not be the only receptor for extracellular ATP in Arabidopsis. The root has the capacity for a calcium wave, triggered by extracellular ATP at the apex.

Iron availability modulates the Arabidopsis thaliana root calcium signature evoked by exogenous ATP.

Plants use changes in cytosolic free Ca2+ ("signatures") to encode information from the specific signals generated in development, immunity and stress perception. Phosphate availability has a significant impact on the Arabidopsis thaliana root calcium signatures generated in response to abiotic stress stimuli and exogenous purine nucleotides. In the case of the response to exogenous ATP, the effect of low phosphate availability is linked to abnormal iron and reactive oxygen species accumulation with iron deprivation's restoring normal signature dynamics. Here, the effect of iron deprivation with normal phosphate availability has been examined. Iron deprivation significantly alters the root calcium signature evoked by exogenous ATP and may link to levels of reactive oxygen species and callose deposition.

Phosphate Starvation Alters Abiotic-Stress-Induced Cytosolic Free Calcium Increases in Roots.

Phosphate (Pi) deficiency strongly limits plant growth, and plant roots foraging the soil for nutrients need to adapt to optimize Pi uptake. Ca2+ is known to signal in root development and adaptation but has to be tightly controlled, as it is highly toxic to Pi metabolism. Under Pi starvation and the resulting decreased cellular Pi pool, the use of cytosolic free Ca2+ ([Ca2+]cyt) as a signal transducer may therefore have to be altered. Employing aequorin-expressing Arabidopsis (Arabidopsis thaliana), we show that Pi starvation, but not nitrogen starvation, strongly dampens the [Ca2+]cyt increases evoked by mechanical, salt, osmotic, and oxidative stress as well as by extracellular nucleotides. The altered root [Ca2+]cyt response to extracellular ATP manifests during seedling development under chronic Pi deprivation but can be reversed by Pi resupply. Employing ratiometric imaging, we delineate that Pi-starved roots have a normal response to extracellular ATP at the apex but show a strongly dampened [Ca2+]cyt response in distal parts of the root tip, correlating with high reactive oxygen species levels induced by Pi starvation. Excluding iron, as well as Pi, rescues this altered [Ca2+]cyt response and restores reactive oxygen species levels to those seen under nutrient-replete conditions. These results indicate that, while Pi availability does not seem to be signaled through [Ca2+]cyt, Pi starvation strongly affects stress-induced [Ca2+]cyt signatures. These data reveal how plants can integrate nutritional and environmental cues, adding another layer of complexity to the use of Ca2+ as a signal transducer.

Impairment in karrikin but not strigolactone sensing enhances root skewing in Arabidopsis thaliana.

Roots form highly complex systems varying in growth direction and branching pattern to forage for nutrients efficiently. Here mutations in the KAI2 (KARRIKIN INSENSITIVE) α/ß-fold hydrolase and the MAX2 (MORE AXILLARY GROWTH 2) F-box leucine-rich protein, which together perceive karrikins (smoke-derived butenolides), caused alteration in root skewing in Arabidopsis thaliana. This phenotype was independent of endogenous strigolactones perception by the D14 α/ß-fold hydrolase and MAX2. Thus, KAI2/MAX2 effect on root growth may be through the perception of endogenous KAI2-ligands (KLs), which have yet to be identified. Upon perception of a ligand, a KAI2/MAX2 complex is formed together with additional target proteins before ubiquitination and degradation through the 26S proteasome. Using a genetic approach, we show that SMAX1 (SUPPRESSOR OF MAX2-1)/SMXL2 and SMXL6,7,8 (SUPPRESSOR OF MAX2-1-LIKE) are also likely degradation targets for the KAI2/MAX2 complex in the context of root skewing. In A. thaliana therefore, KAI2 and MAX2 act to limit root skewing, while kai2's gravitropic and mechano-sensing responses remained largely unaffected. Many proteins are involved in root skewing, and we investigated the link between MAX2 and two members of the SKS/SKU family. Though KLs are yet to be identified in plants, our data support the hypothesis that they are present and can affect root skewing.

Responses of contrasting rice genotypes to excess manganese and their implications for lignin synthesis.

Manganese (Mn) toxicity is frequently encountered in crops grown on soils with low pH or low redox potential, and harmful to plant development and growth. This study aimed at exploring adaptive mechanisms to Mn toxicity in rice, and investigated the effects of Mn toxicity on shoot lignification. Sixteen rice genotypes were grown in hydroponic solutions and exposed to normal (0.5 mg dm-3) or toxic (5 mg dm-3) Mn concentrations for three weeks. Morphological responses to Mn toxicity included a significant reduction in shoot length and the formation of visible symptoms scored as leaf damage index (LDI). Based on shoot Mn concentrations in the Mn toxic treatment, genotypes were classified as Mn includers and excluders. Across different genotypes, shoot Mn concentrations were significantly negatively correlated with relative shoot length and positively correlated with LDI. Consequently, the most tolerant genotypes in terms of morphology were all excluders, while the most sensitive genotypes were includers. The sensitive genotypes were also more responsive to manganese in terms of lipid peroxidation than tolerant genotypes. Shoots of rice plants grown in the high Mn treatment showed a higher level of lignification measured as thioglycolic acid lignin (TGAL), especially among Mn includers. TGAL was positively correlated with shoot Mn concentration and the levels of phenolics. In contrast, peroxidase activity was not responsive to the Mn treatment and was not significantly correlated with shoot lignification. In conclusion, exclusion is a dominant tolerance mechanism to Mn toxicity in rice. Further, Mn stimulated lignin biosynthesis in rice, especially in genotypes that were unable to exclude Mn.

Calcium-Mediated Abiotic Stress Signaling in Roots.

Roots are subjected to a range of abiotic stresses as they forage for water and nutrients. Cytosolic free calcium is a common second messenger in the signaling of abiotic stress. In addition, roots take up calcium both as a nutrient and to stimulate exocytosis in growth. For calcium to fulfill its multiple roles must require strict spatio-temporal regulation of its uptake and efflux across the plasma membrane, its buffering in the cytosol and its sequestration or release from internal stores. This prompts the question of how specificity of signaling output can be achieved against the background of calcium's other uses. Threats to agriculture such as salinity, water availability and hypoxia are signaled through calcium. Nutrient deficiency is also emerging as a stress that is signaled through cytosolic free calcium, with progress in potassium, nitrate and boron deficiency signaling now being made. Heavy metals have the capacity to trigger or modulate root calcium signaling depending on their dose and their capacity to catalyze production of hydroxyl radicals. Mechanical stress and cold stress can both trigger an increase in root cytosolic free calcium, with the possibility of membrane deformation playing a part in initiating the calcium signal. This review addresses progress in identifying the calcium transporting proteins (particularly channels such as annexins and cyclic nucleotide-gated channels) that effect stress-induced calcium increases in roots and explores links to reactive oxygen species, lipid signaling, and the unfolded protein response.

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.

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.

Genetic and physiological analysis of tolerance to acute iron toxicity in rice.

BACKGROUND: Fe toxicity occurs in lowland rice production due to excess ferrous iron (Fe(2+)) formation in reduced soils. To contribute to the breeding for tolerance to Fe toxicity in rice, we determined quantitative trait loci (QTL) by screening two different bi-parental mapping populations under iron pulse stresses (1,000 mg L(-1) = 17.9 mM Fe(2+) for 5 days) in hydroponic solution, followed by experiments with selected lines to determine whether QTLs were associated with iron exclusion (i.e. root based mechanisms), or iron inclusion (i.e. shoot-based mechanisms). RESULTS: In an IR29/Pokkali F8 recombinant inbred population, 7 QTLs were detected for leaf bronzing score on chromosome 1, 2, 4, 7 and 12, respectively, individually explaining 9.2-18.7% of the phenotypic variation. Two tolerant recombinant inbred lines carrying putative QTLs were selected for further experiments. Based on Fe uptake into the shoot, the dominant tolerance mechanism of the tolerant line FL510 was determined to be exclusion with its root architecture being conducive to air transport and thus the ability to oxidize Fe(2+) in rhizosphere. In line FL483, the iron tolerance was related mainly to shoot-based mechanisms (tolerant inclusion mechanism). In a Nipponbare/Kasalath/Nipponbare backcross inbred population, 3 QTLs were mapped on chromosomes 1, 3 and 8, respectively. These QTLs explained 11.6-18.6% of the total phenotypic variation. The effect of QTLs on chromosome 1 and 3 were confirmed by using chromosome segment substitution lines (SL), carrying Kasalath introgressions in the genetic background on Nipponbare. The Fe uptake in shoots of substitution lines suggests that the effect of the QTL on chromosome 1 was associated with shoot tolerance while the QTL on chromosome 3 was associated with iron exclusion. CONCLUSION: Tolerance of certain genotypes were classified into shoot- and root- based mechanisms. Comparing our findings with previously reported QTLs for iron toxicity tolerance, we identified co-localization for some QTLs in both pluse and chronic stresses, especially on chromosome 1.