A stress recovery signaling network for enhanced flooding tolerance in Arabidopsis thaliana.

Abiotic stresses in plants are often transient, and the recovery phase following stress removal is critical. Flooding, a major abiotic stress that negatively impacts plant biodiversity and agriculture, is a sequential stress where tolerance is strongly dependent on viability underwater and during the postflooding period. Here we show that in Arabidopsis thaliana accessions (Bay-0 and Lp2-6), different rates of submergence recovery correlate with submergence tolerance and fecundity. A genome-wide assessment of ribosome-associated transcripts in Bay-0 and Lp2-6 revealed a signaling network regulating recovery processes. Differential recovery between the accessions was related to the activity of three genes: RESPIRATORY BURST OXIDASE HOMOLOG D, SENESCENCE-ASSOCIATED GENE113, and ORESARA1, which function in a regulatory network involving a reactive oxygen species (ROS) burst upon desubmergence and the hormones abscisic acid and ethylene. This regulatory module controls ROS homeostasis, stomatal aperture, and chlorophyll degradation during submergence recovery. This work uncovers a signaling network that regulates recovery processes following flooding to hasten the return to prestress homeostasis.

The effect of auxin (indole-3-acetic acid) on the growth rate and tropism of the sporangiophore of Phycomyces blakesleeanus and identification of auxin-related genes.

The roles of fungal auxins in the regulation of elongation growth, photo-, and gravitropism are completely unknown. We analyzed the effects of exogenous IAA (indole-3-acetic acid), various synthetic auxins including 1-NAA (1-naphthaleneacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid), and the auxin transport inhibitor NPA (N-1-naphtylphtalamic acid) on the growth rate and bending of the unicellular sporangiophore of the zygomycete fungus, Phycomyces blakesleeanus. Sporangiophores that were submerged in an aqueous buffer responded to IAA with a sustained enhancement of the growth rate, while 1-NAA, 2,4-D, and NPA elicited an inhibition. In contrast, sporangiophores kept in air responded to IAA with a 20 to 40% decrease of the growth rate, while 1-NAA and NPA elicited an enhancement. The unilateral and local application of IAA in the growing zone of the sporangiophore elicited in 30 min a moderate negative tropic bending in wild type C2 and mutant C148madC, which was, however, partially masked by a concomitant avoidance response caused by the aqueous buffer. Auxin transport-related genes ubiquitous in plants were found in a BLAST search of the Phycomyces genome. They included members of the AUX1 (auxin influx carrier protein 1), PILS (PIN-LIKES, auxin transport facilitator protein), and ABCB (plant ATP-binding cassette transporter B) families while members of the PIN family were absent. Our observations imply that IAA represents an intrinsic element of the sensory transduction of Phycomyces and that its mode of action must very likely differ in several respects from that operating in plants.

Protoplast Swelling and Hypocotyl Growth Depend on Different Auxin Signaling Pathways.

Members of the TRANSPORT INHIBITOR RESPONSE1/AUXIN SIGNALING F-BOX PROTEIN (TIR1/AFB) family are known auxin receptors. To analyze the possible receptor function of AUXIN BINDING PROTEIN1 (ABP1), an auxin receptor currently under debate, we performed different approaches. We performed a pharmacological approach using α-(2,4-dimethylphenylethyl-2-oxo)-indole-3-acetic acid (auxinole), α-(phenylethyl-2-oxo)-indole-3-acetic acid (PEO-IAA), and 5-fluoroindole-3-acetic acid (5-F-IAA) to discriminate between ABP1- and TIR1/AFB-mediated processes in Arabidopsis (Arabidopsis thaliana). We used a peptide of the carboxyl-terminal region of AtABP1 as a tool. We performed mutant analysis with the null alleles of ABP1, abp1-c1 and abp1-TD1, and the TILLING mutant abp1-5 We employed Coimbra, an accession that exhibits an amino acid exchange in the auxin-binding domain of ABP1. We measured either volume changes of single hypocotyl protoplasts or hypocotyl growth, both at high temporal resolution. 5-F-IAA selectively activated the TIR1/AFB pathway but did not induce protoplast swelling; instead, it showed auxin activity in the hypocotyl growth test. In contrast, PEO-IAA induced an auxin-like swelling response but no hypocotyl growth. The carboxyl-terminal peptide of AtABP1 induced an auxin-like swelling response. In the ABP1-related mutants and Coimbra, no auxin-induced protoplast swelling occurred. ABP1 seems to be involved in mediating rapid auxin-induced protoplast swelling, but it is not involved in the control of rapid auxin-induced growth.

The Physiology of Adventitious Roots.

Adventitious roots are plant roots that form from any nonroot tissue and are produced both during normal development (crown roots on cereals and nodal roots on strawberry [Fragaria spp.]) and in response to stress conditions, such as flooding, nutrient deprivation, and wounding. They are important economically (for cuttings and food production), ecologically (environmental stress response), and for human existence (food production). To improve sustainable food production under environmentally extreme conditions, it is important to understand the adventitious root development of crops both in normal and stressed conditions. Therefore, understanding the regulation and physiology of adventitious root formation is critical for breeding programs. Recent work shows that different adventitious root types are regulated differently, and here, we propose clear definitions of these classes. We use three case studies to summarize the physiology of adventitious root development in response to flooding (case study 1), nutrient deficiency (case study 2), and wounding (case study 3).

The role of ethylene and ROS in salinity, heavy metal, and flooding responses in rice.

Plant growth and developmental processes as well as abiotic and biotic stress adaptations are regulated by small endogenous signaling molecules. Among these, phytohormones such as the gaseous alkene ethylene and reactive oxygen species (ROS) play an important role in mediating numerous specific growth or cell death responses. While apoplastic ROS are generated by plasma membrane-located respiratory burst oxidase homolog proteins, intracellular ROS are produced mainly in electron transfer chains of mitochondria and chloroplasts. Ethylene accumulates in plants due to physical entrapment or by enhanced ethylene biosynthesis. A major crop that must endure high salt and heavy metal concentrations upon flooding in regions of Asia is rice. Ethylene and ROS have been identified as the major signals that mediate salinity, chromium, and flooding stress in rice. This mini review focuses on (i) what is known about ethylene and ROS level control during these abiotic stresses in rice, (ii) how the two signals mediate growth or death processes, and (iii) feedback mechanisms that in turn regulate ethylene and ROS signaling.

Reactive oxygen species mediate growth and death in submerged plants.

Aquatic and semi-aquatic plants are well adapted to survive partial or complete submergence which is commonly accompanied by oxygen deprivation. The gaseous hormone ethylene controls a number of adaptive responses to submergence including adventitious root growth and aerenchyma formation. Reactive oxygen species (ROS) act as signaling intermediates in ethylene-controlled submergence adaptation and possibly also independent of ethylene. ROS levels are controlled by synthesis, enzymatic metabolism, and non-enzymatic scavenging. While the actors are by and large known, we still have to learn about altered ROS at the subcellular level and how they are brought about, and the signaling cascades that trigger a specific response. This review briefly summarizes our knowledge on the contribution of ROS to submergence adaptation and describes spectrophotometrical, histochemical, and live cell imaging detection methods that have been used to study changes in ROS abundance. Electron paramagnetic resonance (EPR) spectroscopy is introduced as a method that allows identification and quantification of specific ROS in cell compartments. The use of advanced technologies such as EPR spectroscopy will be necessary to untangle the intricate and partially interwoven signaling networks of ethylene and ROS.

The Clubroot Pathogen (Plasmodiophora brassicae) Influences Auxin Signaling to Regulate Auxin Homeostasis in Arabidopsis.

The clubroot disease, caused by the obligate biotrophic protist Plasmodiophora brassicae, affects cruciferous crops worldwide. It is characterized by root swellings as symptoms, which are dependent on the alteration of auxin and cytokinin metabolism. Here, we describe that two different classes of auxin receptors, the TIR family and the auxin binding protein 1 (ABP1) in Arabidopsis thaliana are transcriptionally upregulated upon gall formation. Mutations in the TIR family resulted in more susceptible reactions to the root pathogen. As target genes for the different pathways we have investigated the transcriptional regulation of selected transcriptional repressors (Aux/IAA) and transcription factors (ARF). As the TIR pathway controls auxin homeostasis via the upregulation of some auxin conjugate synthetases (GH3), the expression of selected GH3 genes was also investigated, showing in most cases upregulation. A double gh3 mutant showed also slightly higher susceptibility to P. brassicae infection, while all tested single mutants did not show any alteration in the clubroot phenotype. As targets for the ABP1-induced cell elongation the effect of potassium channel blockers on clubroot formation was investigated. Treatment with tetraethylammonium (TEA) resulted in less severe clubroot symptoms. This research provides evidence for the involvement of two auxin signaling pathways in Arabidopsis needed for the establishment of the root galls by P. brassicae.

Emerging roots alter epidermal cell fate through mechanical and reactive oxygen species signaling.

A central question in biology is how spatial information is conveyed to locally establish a developmental program. Rice (Oryza sativa) can survive flash floods by the emergence of adventitious roots from the stem. Epidermal cells that overlie adventitious root primordia undergo cell death to facilitate root emergence. Root growth and epidermal cell death are both controlled by ethylene. This study aimed to identify the signal responsible for the spatial control of cell death. Epidermal cell death correlated with the proximity to root primordia in wild-type and ADVENTITIOUS ROOTLESS1 plants, indicating that the root emits a spatial signal. Ethylene-induced root growth generated a mechanical force of ~18 millinewtons within 1 h. Force application to epidermal cells above root primordia caused cell death in a dose-dependent manner and was inhibited by 1-methylcyclopropene or diphenylene iodonium, an inhibitor of NADPH oxidase. Exposure of epidermal cells not overlying a root to either force and ethylene or force and the catalase inhibitor aminotriazole induced ectopic cell death. Genetic downregulation of the reactive oxygen species (ROS) scavenger METALLOTHIONEIN2b likewise promoted force-induced ectopic cell death. Hence, reprogramming of epidermal cell fate by the volatile plant hormone ethylene requires two signals: mechanosensing for spatial resolution and ROS for cell death signaling.

Phytosulfokine-α controls hypocotyl length and cell expansion in Arabidopsis thaliana through phytosulfokine receptor 1.

The disulfated peptide growth factor phytosulfokine-α (PSK-α) is perceived by LRR receptor kinases. In this study, a role for PSK signaling through PSK receptor PSKR1 in Arabidopsis thaliana hypocotyl cell elongation is established. Hypocotyls of etiolated pskr1-2 and pskr1-3 seedlings, but not of pskr2-1 seedlings were shorter than wt due to reduced cell elongation. Treatment with PSK-α did not promote hypocotyl growth indicating that PSK levels were saturating. Tyrosylprotein sulfotransferase (TPST) is responsible for sulfation and hence activation of the PSK precursor. The tpst-1 mutant displayed shorter hypocotyls with shorter cells than wt. Treatment of tpst-1 seedlings with PSK-α partially restored elongation growth in a dose-dependent manner. Hypocotyl elongation was significantly enhanced in tpst-1 seedlings at nanomolar PSK-α concentrations. Cell expansion was studied in hypocotyl protoplasts. WT and pskr2-1 protoplasts expanded in the presence of PSK-α in a dose-dependent manner. By contrast, pskr1-2 and pskr1-3 protoplasts were unresponsive to PSK-α. Protoplast swelling in response to PSK-α was unaffected by ortho-vanadate, which inhibits the plasma membrane H(+)-ATPase. In maize (Zea mays L.), coleoptile protoplast expansion was similarly induced by PSK-α in a dose-dependent manner and was dependent on the presence of K(+) in the media. In conclusion, PSK-α signaling of hypocotyl elongation and protoplast expansion occurs through PSKR1 and likely involves K(+) uptake, but does not require extracellular acidification by the plasma membrane H(+)-ATPase.

Aerenchyma formation in the rice stem and its promotion by H2O2.

• Gas spaces (aerenchyma) form as an adaptation to submergence to facilitate gas exchange. In rice (Oryza sativa), aerenchyma develop by cell death and lysis, which are poorly understood at the cellular level. • Aerenchyma formation was studied in rice stems by light microscopy. It was analyzed in response to submergence, ethylene and hydrogen peroxide (H(2)O(2)) treatment, and in the MT2b::Tos17 mutant. O(2)·(-) was detected with nitroblue tetrazolium and an epinephrine assay. H(2)O(2) was detected with 3,3'-diaminobenzidine. • Aerenchyma develop constitutively in all internodes of the deep-water rice variety Pin Gaew 56, but are absent from the nodes. Constitutive aerenchyma formation was also observed in two lowland rice varieties, albeit to a lesser degree. A larger number of aerenchyma are present in older internodes, and at the top of each internode, revealing developmental gradients. Submergence or treatment with the ethylene-releasing compound ethephon promoted aerenchyma formation in all genotypes analyzed. Pre-aerenchymal cells contain less starch, no chloroplasts, thinner cell walls and produce elevated levels of O(2)·(-) and H(2)O(2) compared with other parenchymal cells. Ethephon promotes O(2)·(-) formation and H(2)O(2) promotes aerenchyma formation in a dose-dependent manner. Further-more, genetic downregulation of the H(2)O(2) scavenger MT2b enhances aerenchyma formation. • Aerenchyma formation is mediated by reactive oxygen species.

G proteins as regulators in ethylene-mediated hypoxia signaling.

Waterlogging or flooding are frequently or constitutively encountered by many plant species. The resulting reduction in endogenous O2 concentration poses a severe threat. Numerous adaptations at the anatomical, morphological and metabolic level help plants to either escape low oxygen conditions or to endure them. Formation of aerenchyma or rapid shoot elongation are escape responses, as is the formation of adventitious roots. The metabolic shift from aerobic respiration to anaerobic fermentation contributes to a basal energy supply at low oxygen conditions. Ethylene plays a central role in hypoxic stress signaling, and G proteins have been recognized as crucial signal transducers in various hypoxic signaling pathways. The programmed death of parenchyma cells that results in hypoxia-induced aerenchyma formation is an ethylene response. In maize, aerenchyma are induced in the absence of ethylene when G proteins are constitutively activated. Similarly, ethylene induced death of epidermal cells that cover adventitious roots at the stem node of rice is strictly dependent on heterotrimeric G protein activity. Knock down of the unique Gα gene RGA1 in rice prevents epidermal cell death. Finally, in Arabidopsis, induction of alcohol dehydrogenase with resulting increased plant survival relies on the balanced activities of a small Rop G protein and its deactivating protein RopGAP4. Identifying the general mechanisms of G protein signaling in hypoxia adaptation of plants is one of the tasks ahead.

ABP1: an auxin receptor for fast responses at the plasma membrane.

Auxin-binding protein 1 (ABP1) is an auxin receptor for responses not primarily regulated by gene regulation. One fast response is protoplast swelling. By using immunological ABP1 tools we showed that the highly conserved box a is not alone important for auxin binding. Box c is another part of the auxin binding domain.1 Here we present a novel method to analyze auxin-induced, ABP1-mediated effects at the plasma membrane on single cell level in vivo. The fluorescence of FM4-64 in the plasma membrane is reduced by auxin and this response is mediated by ABP1. This method indicates a functional role of ABP1 at the plasma membrane.

A role for PSK signaling in wounding and microbial interactions in Arabidopsis.

PSK-alpha is a disulfated peptide that acts as a growth factor in plants. PSK-alpha is derived from preproproteins which are encoded by five PSK precursor genes in Arabidopsis thaliana (L.) Heynh and is perceived by leucine-rich repeat receptor kinases. Arabidopsis has two PSK receptor genes, PSKR1 and PSKR2. Although ligand and receptor are well characterized, the biological functions of PSK signaling are not well understood. Using reporter lines and receptor knockout mutants of Arabidopsis, a role for PSK signaling in biotic interactions and in wounding was analyzed. Treatment of Arabidopsis leaves with the fungal elicitor E-Fol, or the fungal pathogens Alternaria brassicicola and Sclerotinia sclerotiorum resulted in induction of PSK2 and PSKR1 as shown by promoter:GUS analysis. Wounding of hypocotyls or leaves induced PSK3:GUS, PSK5:GUS and PSKR1:GUS expression indicating that PSK precursor genes are differentially regulated in response to specific stresses. The receptor knockout lines pskr1-3 and pskr2-1 showed significantly reduced photosynthesis in response to the fungal elicitor E-Fol which indicates that fungal defence is impaired. pskr1-3 plants further showed reduced growth of crown galls after infection with Agrobacterium tumefaciens. A role for PSK signaling in Agrobacterium tumefaciens tumor growth was supported by the finding that PSK precursor genes and PSKR1 are expressed in crown galls. Overall, the results indicate that PSK signaling may play a previously undescribed role in pathogen or herbivore interactions and is crucial for Agrobacterium-induced cell proliferation in crown gall formation.

The auxin-binding pocket of auxin-binding protein 1 comprises the highly conserved boxes a and c.

The auxin-binding protein 1 (ABP1) has already been proved to be an extracellular receptor of auxin in single cell systems. Protoplasts of maize coleoptiles respond to auxin with an increase in volume. The 2-naphthaleneacetic acid (2-NAA), an inactive auxin analog, acts as an anti-auxin in protoplast swelling, as it suppresses the effect of indole-3-acetic acid (IAA). Antibodies raised against box a of ABP1 induce protoplast swelling in the absence of auxin. This response is inhibited by pre-incubation with 2-NAA. The effect of 2-NAA on swelling induced by agonistic antibodies appears to depend on the binding characteristics of the antibody. ScFv12, an antibody directed against box a, box c and the C-terminal domain of ABP1 also exhibits auxin-agonist activity which is, however, not abolished by 2-NAA. Neither does 2-NAA affect the activity of the C-terminal peptide of ABP1, which is predicted to interact with putative binding proteins of ABP1. These results support the view that box a and box c of ABP1 are auxin-binding domains.

Heterotrimeric G protein signaling is required for epidermal cell death in rice.

In rice (Oryza sativa) adventitious root primordia are formed at the nodes as part of normal development. Upon submergence of rice plants, adventitious roots emerge from the nodes preceded by death of epidermal cells above the root primordia. Cell death is induced by ethylene and mediated by hydrogen peroxide (H(2)O(2)). Pharmacological experiments indicated that epidermal cell death was dependent on signaling through G proteins. Treatment with GTP-gamma-S induced epidermal cell death, whereas GDP-beta-S partially inhibited ethylene-induced cell death. The dwarf1 (d1) mutant of rice has repressed expression of the Galpha subunit RGA1 of heterotrimeric G protein. In d1 plants, cell death in response to ethylene and H(2)O(2) was nearly completely abolished, indicating that signaling through Galpha is essential. Ethylene and H(2)O(2) were previously shown to alter gene expression in epidermal cells that undergo cell death. Transcriptional regulation was not generally affected in the d1 mutant, indicating that altered gene expression is not sufficient to trigger cell death in the absence of Galpha. Analysis of genes encoding proteins related to G protein signaling revealed that four small GTPase genes, two GTPase-activating protein genes, and one GDP dissociation inhibitor gene but not RGA1 were differentially expressed in epidermal cells above adventitious roots, indicating that Galpha activity is regulated posttranscriptionally.

Epidermal cell death in rice is confined to cells with a distinct molecular identity and is mediated by ethylene and H2O2 through an autoamplified signal pathway.

Rice (Oryza sativa) forms adventitious root primordia at stem nodes during normal development. Root emergence is preceded by ethylene-induced, H(2)O(2)-mediated local death of epidermal cells. Exogenous H(2)O(2) or enhancement of endogenous H(2)O(2) promoted epidermal cell death in a dose-dependent manner. Inhibition of NADPH oxidase lowered ethylene-induced cell death rates. Inhibition of ethylene perception by 1-methylcyclopropene did not abolish H(2)O(2)-induced cell death, indicating that H(2)O(2) acts downstream of ethylene. Microarray studies of epidermal cells that undergo cell death identified 61 genes coregulated by the ethylene-releasing compound ethephon and by H(2)O(2), supporting a joint signaling pathway. Regulation of the ethylene biosynthetic genes 1-Aminocyclopropane-1-Carboxylate Oxidase1 and Ethylene Overproducer-Like1 and downregulation of Metallothionein2b (MT2b), which encodes a reactive oxygen scavenger, indicated mutual enhancement of ethylene and H(2)O(2) signaling. Analysis of MT2b knockdown mutants showed that cell death rates were inversely related to MT2b transcript abundance. Epidermal cells above adventitious roots have a morphological and molecular identity distinct from other epidermal cells. Pro-death signals regulated several transcription factor genes with a proposed function in cell type specification. It is hypothesized that induction of cell death is dependent on epidermal cell identity.

Interactions between ethylene, gibberellin and abscisic acid regulate emergence and growth rate of adventitious roots in deepwater rice.

Growth of adventitious roots is induced in deepwater rice (Oryza sativa L.) when plants become submerged. Ethylene which accumulates in flooded plant parts is responsible for root growth induction. Gibberellin (GA) is ineffective on its own but acts in a synergistic manner together with ethylene to promote the number of penetrating roots and the growth rate of emerged roots. Studies with the GA biosynthesis inhibitor paclobutrazol revealed that root emergence was dependent on GA activity. Abscisic acid (ABA) acted as a competitive inhibitor of GA activity. Root growth rate on the other hand was dependent on GA concentration and ABA acted as a potent inhibitor possibly of GA but also of ethylene signaling. The results indicated that root emergence and elongation are distinct phases of adventitious root growth that are regulated through different networking between ethylene, GA and ABA signaling pathways. Adventitious root emergence must be coordinated with programmed death of epidermal cells which cover root primordia. Epidermal cell death is also controlled by ethylene, GA and ABA albeit with cell-type specific cross-talk. Different interactions between the same hormones may be a means to ensure proper timing of cell death and root emergence and to adjust the growth rate of emerged adventitious roots.

Epidermal cell death in rice is regulated by ethylene, gibberellin, and abscisic acid.

Programmed cell death (PCD) of epidermal cells that cover adventitious root primordia in deepwater rice (Oryza sativa) is induced by submergence. Early suicide of epidermal cells may prevent injury to the growing root that emerges under flooding conditions. Induction of PCD is dependent on ethylene signaling and is further promoted by gibberellin (GA). Ethylene and GA act in a synergistic manner, indicating converging signaling pathways. Treatment of plants with GA alone did not promote PCD. Treatment with the GA biosynthesis inhibitor paclobutrazol resulted in increased PCD in response to ethylene and GA presumably due to an increased sensitivity of epidermal cells to GA. Abscisic acid (ABA) was shown to efficiently delay ethylene-induced as well as GA-promoted cell death. The results point to ethylene signaling as a target of ABA inhibition of PCD. Accumulation of ethylene and GA and a decreased ABA level in the rice internode thus favor induction of epidermal cell death and ensure that PCD is initiated as an early response that precedes adventitious root growth.

The diageotropica mutation of tomato disrupts a signalling chain using extracellular auxin binding protein 1 as a receptor.

The diageotropica ( dgt) mutant of tomato ( Lycopersicon esculentum Mill.) is known to lack a number of typical auxin responses. Here we show that rapid auxin-induced growth of seedling hypocotyls is completely abolished by the mutation over the full range of auxin concentrations tested, and also in early phases of the time course. Protoplasts isolated from wild-type hypocotyls respond to auxin by a rapid increase in cell volume, which we measured by image analysis at a high temporal resolution. A similar swelling could be triggered by antibodies directed against a part of the putative auxin-binding domain (box-a) of the auxin-binding protein 1 (ABP1). Induction of swelling both by auxin and by the antibody was not observed in the protoplasts isolated from the dgt mutant. However, dgt protoplasts are able to respond to the stimulator of the H(+)-ATPase, fusicoccin, with normal swelling. We propose that dgt is a signal-transduction mutation interfering with an auxin-signalling pathway that uses ABP1 as a receptor.