Next-generation ABACUS biosensors reveal cellular ABA dynamics driving root growth at low aerial humidity.

The plant hormone abscisic acid (ABA) accumulates under abiotic stress to recast water relations and development. To overcome a lack of high-resolution sensitive reporters, we developed ABACUS2s-next-generation Förster resonance energy transfer (FRET) biosensors for ABA with high affinity, signal-to-noise ratio and orthogonality-that reveal endogenous ABA patterns in Arabidopsis thaliana. We mapped stress-induced ABA dynamics in high resolution to reveal the cellular basis for local and systemic ABA functions. At reduced foliar humidity, root cells accumulated ABA in the elongation zone, the site of phloem-transported ABA unloading. Phloem ABA and root ABA signalling were both essential to maintain root growth at low humidity. ABA coordinates a root response to foliar stresses, enabling plants to maintain foraging of deeper soil for water uptake.

BIG Regulates Dynamic Adjustment of Circadian Period in Arabidopsis thaliana.

Circadian clocks drive rhythms with a period near 24 h, but the molecular basis of the regulation of the period of the circadian clockis poorly understood. We previously demonstrated that metabolites affect the free-running period of the circadian oscillator of Arabidopsis (Arabidopsis thaliana), with endogenous sugars acting as an accelerator and exogenous nicotinamide acting as a brake. Changes in circadian oscillator period are thought to adjust the timing of biological activities through the process of entrainment, in which the circadian oscillator becomes synchronized to rhythmic signals such as light and dark cycles as well as changes in internal metabolism. To identify the molecular components associated with the dynamic adjustment of circadian period, we performed a forward genetic screen. We identified Arabidopsis mutants that were either period insensitive to nicotinamide (sin) or period oversensitive to nicotinamide (son). We mapped son1 to BIG, a gene of unknown molecular function that was shown previously to play a role in light signaling. We found that son1 has an early entrained phase, suggesting that the dynamic alteration of circadian period contributes to the correct timing of biological events. Our data provide insight into how the dynamic period adjustment of circadian oscillators contributes to establishing a correct phase relationship with the environment and show that BIG is involved in this process.

POLYAMINE OXIDASE2 of Arabidopsis contributes to ABA mediated plant developmental processes.

Polyamines (PA) are catabolised by two groups of amine oxidases, the copper-binding amine oxidases (CuAOs) and the FAD-binding polyamine oxidases (PAOs). Previously, we have shown that CuAO1 is involved in ABA associated growth responses and ABA- and PA-mediated rapid nitric oxide (NO) production. Here we report the differential regulation of expression of POLYAMINE OXIDASE2 of Arabidopsis (AtPAO2) in interaction with ABA, nitrate and ammonium. Without ABA treatment germination, cotyledon growth and fresh weight of pao2 knockdown mutants as well as PAO2OX over-expressor plants were comparable to those of the wild type (WT) plants irrespective of the N source. In the presence of ABA, in pao2 mutants cotyledon growth and fresh weights were more sensitive to inhibition by ABA while PAO2OX over-expressor plants showed a rather similar response to WT. When NO3(-) was the only N source primary root lengths and lateral root numbers were lower in pao2 mutants both without and with exogenous ABA. PAO2OX showed enhanced primary and lateral root growth in media with NO3(-) or NH4(+). Vigorous root growth of PAO2OX and the hypersensitivity of pao2 mutants to ABA suggest a positive function of AtPAO2 in root growth. ABA-induced NO production in pao2 mutants was lower indicating a potential contributory function of AtPAO2 in NO-mediated effects on root growth.

Expression patterns of flagellin sensing 2 map to bacterial entry sites in plant shoots and roots.

Pathogens can colonize all plant organs and tissues. To prevent this, each cell must be capable of autonomously triggering defence. Therefore, it is generally assumed that primary sensors of the immune system are constitutively present. One major primary sensor against bacterial infection is the flagellin sensing 2 (FLS2) pattern recognition receptor (PRR). To gain insights into its expression pattern, the FLS2 promoter activity in ß-glucuronidase (GUS) reporter lines was monitored. The data show that pFLS2::GUS activity is highest in cells and tissues vulnerable to bacterial entry and colonization, such as stomata, hydathodes, and lateral roots. GUS activity is also high in the vasculature and, by monitoring Ca(2+) responses in the vasculature, it was found that this tissue contributes to flg22-induced Ca(2+) burst. The FLS2 promoter is also regulated in a tissue- and cell type-specific manner and is responsive to hormones, damage, and biotic stresses. This results in stimulus-dependent expansion of the FLS2 expression domain. In summary, a tissue- and cell type-specific map of FLS2 expression has been created correlating with prominent entry sites and target tissues of plant bacterial pathogens.

Mutants of phospholipase A (pPLA-I) have a red light and auxin phenotype.

pPLA-I is the evolutionarily oldest patatin-related phospholipase A (pPLA) in plants, which have previously been implicated to function in auxin and defence signalling. Molecular and physiological analysis of two allelic null mutants for pPLA-I [ppla-I-1 in Wassilewskija (Ws) and ppla-I-3 in Columbia (Col) ] revealed pPLA-I functions in auxin and light signalling. The enzyme is localized in the cytosol and to membranes. After auxin application expression of early auxin-induced genes is significantly slower compared with wild type and both alleles show a slower gravitropic response of hypocotyls, indicating compromised auxin signalling. Additionally, phytochrome-modulated responses like abrogation of gravitropism, enhancement of phototropism and growth in far red-enriched light are decreased in both alleles. While early flowering, root coils and delayed phototropism are only observed in the Ws mutant devoid of phyD, the light-related phenotypes observed in both alleles point to an involvement of pPLA-I in phytochrome signalling.

Polyamines, polyamine oxidases and nitric oxide in development, abiotic and biotic stresses.

Nitric oxide (NO), polyamines (PAs), diamine oxidases (DAO) and polyamine oxidases (PAO) play important roles in wide spectrum of physiological processes such as germination, root development, flowering and senescence and in defence responses against abiotic and biotic stress conditions. This functional overlapping suggests interaction of NO and PA in signalling cascades. Exogenous application of PAs putrescine, spermidine and spermine to Arabidopsis seedlings induced NO production as observed by fluorimetry and fluorescence microscopy using the NO-binding fluorophores DAF-2 and DAR-4M. The observed NO release induced by 1 mM spermine treatment in the Arabidopsis seedlings was very rapid without apparent lag phase. These observations pave a new insight into PA-mediated signalling and NO as a potential mediator of PA actions. When comparing the functions of NO and PA in plant development and abiotic and biotic stresses common to both signalling components it can be speculated that NO may be a link between PA-mediated stress responses filing a gap between many known physiological effects of PAs and amelioration of stresses. NO production indicated by PAs could be mediated either by H(2)O(2), one reaction product of oxidation of PAs by DAO and PAO, or by unknown mechanisms involving PAs, DAO and PAO.

COPPER AMINE OXIDASE1 (CuAO1) of Arabidopsis thaliana contributes to abscisic acid- and polyamine-induced nitric oxide biosynthesis and abscisic acid signal transduction.

Polyamines (PA), polyamine oxidases, copper amine oxidases, and nitric oxide (NO) play important roles in physiology and stress responses in plants. NO biosynthesis as a result of catabolism of PA by polyamine oxidases and copper amine oxidases may explain in part PA-mediated responses. Involvement of a copper amine oxidase gene, COPPER AMINE OXIDASE1 (CuAO1), of Arabidopsis was tested for its role in stress responses using the knockouts cuao1-1 and cuao1-2. PA-induced and ABA-induced NO production investigated by fluorometry and fluorescence microscopy showed that the cuao1-1 and cuao1-2 are impaired in NO production, suggesting a function of CuAO1 in PA and ABA-mediated NO production. Furthermore, we found a PA-dependent increase in protein S-nitrosylation. The addition of PA and ABA also resulted in H(2)O(2) increases. cuao1-1 and cuao1-2 showed less sensitivity to exogenous ABA supplementation during germination, seedling establishment, and root growth inhibition as compared to wild-type. In response to ABA treatment, expression levels of the stress-responsive genes RD29A and ADH1 were significantly lower in the knockouts. These observations characterize cuao1-1 and cuao1-2 as ABA-insensitive mutants. Taken together, our findings extend the ABA signal transduction network to include CuAO1 as one potential contributor to enhanced NO production by ABA.

The phosphatidylcholine-hydrolysing phospholipase C NPC4 plays a role in response of Arabidopsis roots to salt stress.

Phosphatidylcholine-hydrolysing phospholipase C, also known as non-specific phospholipase C (NPC), is a new member of the plant phospholipase family that reacts to environmental stresses such as phosphate deficiency and aluminium toxicity, and has a role in root development and brassinolide signalling. Expression of NPC4, one of the six NPC genes in Arabidopsis, was highly induced by NaCl. Maximum expression was observed from 3 h to 6 h after the salt treatment and was dependent on salt concentration. Results of histochemical analysis of P(NPC4):GUS plants showed the localization of salt-induced expression in root tips. On the biochemical level, increased NPC enzyme activity, indicated by accumulation of diacylglycerol, was observed as early as after 30 min of salt treatment of Arabidopsis seedlings. Phenotype analysis of NPC4 knockout plants showed increased sensitivity to salinity as compared with wild-type plants. Under salt stress npc4 plants had shorter roots, lower fresh weight, and reduced seed germination. Expression levels of abscisic acid-related genes ABI1, ABI2, RAB18, PP2CA, and SOT12 were substantially reduced in salt-treated npc4 plants. These observations demonstrate a role for NPC4 in the response of Arabidopsis to salt stress.

Plant phosphatidylcholine-hydrolyzing phospholipases C NPC3 and NPC4 with roles in root development and brassinolide signaling in Arabidopsis thaliana.

Phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC) catalyzes the hydrolysis of phosphatidylcholine (PC) to generate phosphocholine and diacylglycerol (DAG). PC-PLC has a long tradition in animal signal transduction to generate DAG as a second messenger besides the classical phosphatidylinositol splitting phospholipase C (PI-PLC). Based on amino acid sequence similarity to bacterial PC-PLC, six putative PC-PLC genes (NPC1 to NPC6) were identified in the Arabidopsis genome. RT-PCR analysis revealed overlapping expression pattern of NPC genes in root, stem, leaf, flower, and silique. In auxin-treated P(NPC3):GUS and P(NPC4):GUS seedlings, strong increase of GUS activity was visible in roots, leaves, and shoots and, to a weaker extent, in brassinolide-treated (BL) seedlings. P(NPC4):GUS seedlings also responded to cytokinin with increased GUS activity in young leaves. Compared to wild-type, T-DNA insertional knockouts npc3 and npc4 showed shorter primary roots and lower lateral root density at low BL concentrations but increased lateral root densities in response to exogenous 0.05-1.0 µM BL. BL-induced expression of TCH4 and LRX2, which are involved in cell expansion, was impaired but not impaired in repression of CPD, a BL biosynthesis gene, in BL-treated npc3 and npc4. These observations suggest NPC3 and NPC4 are important in BL-mediated signaling in root growth. When treated with 0.1 µM BL, DAG accumulation was observed in tobacco BY-2 cell cultures labeled with fluorescent PC as early as 15 min after application. We hypothesize that at least one PC-PLC is a plant signaling enzyme in BL signal transduction and, as shown earlier, in elicitor signal transduction.