Regulation of Jasmonate-Induced Leaf Senescence by Antagonism between bHLH Subgroup IIIe and IIId Factors in Arabidopsis

Plants initiate leaf senescence to relocate nutrients and energy from aging leaves to developing tissues or storage organs for growth, reproduction, and defense. Leaf senescence, the final stage of leaf development, is regulated by various environmental stresses, developmental cues, and endogenous hormone signals. Jasmonate (JA), a lipid-derived phytohormone essential for plant defense and plant development, serves as an important endogenous signal to activate senescence-associated gene expression and induce leaf senescence. This study revealed one of the mechanisms underlying JA-induced leaf senescence: antagonistic interactions of the bHLH subgroup IIIe factors MYC2, MYC3, and MYC4 with the bHLH subgroup IIId factors bHLH03, bHLH13, bHLH14, and bHLH17. We showed that MYC2, MYC3, and MYC4 function redundantly to activate JA-induced leaf senescence. MYC2 binds to and activates the promoter of its target gene SAG29 (SENESCENCE-ASSOCIATED GENE29) to activate JA-induced leaf senescence. Interestingly, plants have evolved an elaborate feedback regulation mechanism to modulate JA-induced leaf senescence: The bHLH subgroup IIId factors (bHLH03, bHLH13, bHLH14, and bHLH17) bind to the promoter of SAG29 and repress its expression to attenuate MYC2/MYC3/MYC4-activated JA-induced leaf senescence. The antagonistic regulation by activators and repressors would mediate JA-induced leaf senescence at proper level suitable for plant survival in fluctuating environmental conditions.     

Arabidopsis DELLA and JAZ Proteins Bind the WD-Repeat/bHLH/MYB Complex to Modulate Gibberellin and Jasmonate Signaling Synergy

Integration of diverse environmental and endogenous signals to coordinately regulate growth, development, and defense is essential for plants to survive in their natural habitat. The hormonal signals gibberellin (GA) and jasmonate (JA) antagonistically and synergistically regulate diverse aspects of plant growth, development, and defense. GA and JA synergistically induce initiation of trichomes, which assist seed dispersal and act as barriers to protect plants against insect attack, pathogen infection, excessive water loss, and UV irradiation. However, the molecular mechanism underlying such synergism between GA and JA signaling remains unclear. In this study, we revealed a mechanism for GA and JA signaling synergy and identified a signaling complex of the GA pathway in regulation of trichome initiation. Molecular, biochemical, and genetic evidence showed that the WD-repeat/bHLH/MYB complex acts as a direct target of DELLAs in the GA pathway and that both DELLAs and JAZs interacted with the WD-repeat/bHLH/MYB complex to mediate synergism between GA and JA signaling in regulating trichome development. GA and JA induce degradation of DELLAs and JASMONATE ZIM-domain proteins to coordinately activate the WD-repeat/bHLH/MYB complex and synergistically and mutually dependently induce trichome initiation. This study provides deep insights into the molecular mechanisms for integration of different hormonal signals to synergistically regulate plant development.     

Auxin Resistant1 and PIN-FORMED2 Protect Lateral Root Formation in Arabidopsis under Iron Stress

A stunted root system is a significant symptom of iron (Fe) toxicity, yet little is known about the effects of excess Fe on lateral root (LR) development. In this work, we show that excess Fe has different effects on LR development in different portions of the Arabidopsis (Arabidopsis thaliana) root system and that inhibitory effects on the LR initiation are only seen in roots newly formed during excess Fe exposure. We show that root tip contact with Fe is both necessary and sufficient for LR inhibition and that the auxin, but not abscisic acid, pathway is engaged centrally in the initial stages of excess Fe exposure. Furthermore, Fe stress significantly reduced PIN-FORMED2 (PIN2)-green fluorescent protein (GFP) expression in root tips, and pin2-1 mutants exhibited significantly fewer LR initiation events under excess Fe than the wild type. Exogenous application of both Fe and glutathione together increased PIN2-GFP expression and the number of LR initiation events compared with Fe treatment alone. The ethylene inhibitor aminoethoxyvinyl-glycine intensified Fe-dependent inhibition of LR formation in the wild type, and this inhibition was significantly reduced in the ethylene overproduction mutant ethylene overproducer1-1. We show that Auxin Resistant1 (AUX1) is a critical component in the mediation of endogenous ethylene effects on LR formation under excess Fe stress. Our findings demonstrate the relationship between excess Fe-dependent PIN2 expression and LR formation and the potential role of AUX1 in ethylene-mediated LR tolerance and suggest that AUX1 and PIN2 protect LR formation in Arabidopsis during the early stages of Fe stress.Iron (Fe) is an essential trace element for plants (Pilon et al., 2009), and species differ greatly in how much Fe they require for optimal growth (Wheeler and Power, 1995; Batty and Younger, 2003). As Fe is frequently limiting, Fe deficiency is more commonly studied than toxicity arising from excess Fe exposure (Lei et al., 2014; Bashir et al., 2015; Briat et al., 2015). Fe is also a major focus for efforts in biofortification by targeting Fe transporters (Zhai et al., 2014; Pinto and Ferreira, 2015). However, the excessive presence of Fe in soils is equally common, in particular in soils characterized by low pH and hypoxic or anoxic conditions (Connolly and Guerinot, 2002). Toxicity arising from excess Fe exposure is recognized as one of the major plant diseases attributable to abiotic factors that impact the development and yield potential in the world’s leading cereal crops, rice (Oryza sativa) and wheat (Triticum aestivum; Becker and Asch, 2005; Khabaz-Saberi et al., 2012). Understanding the mechanisms underlying excess Fe toxicity is therefore essential.Plastic responses in the plant’s root system architecture are known to constitute a major mechanism by which plants cope with fluctuating environments. Lateral roots (LRs), which typically comprise the majority of the root system, contribute pivotally to nutrient acquisition from soil, and modulating LR development is a very important avoidance strategy for plants when confronted with unfavorable edaphic conditions, such as high salinity or heavy metals (Ivanov et al., 2003). In the case of excess exposure to Fe, stunting of the root system is among the chief symptoms of toxicity (Becker and Asch, 2005). However, while some information has been emerging on the primary root axis (Li et al., 2015), the specific role of the plant’s LR apparatus remains poorly studied. Yamauchi and Peng (1995) reported retardation of root growth and a reduction in LR length and number under excess Fe conditions. Recently, Reyt et al. (2015) showed that excess Fe had no significant effect on LR initiation in the LR branching zone and that ferritins play an important role in LR emergence under excess Fe in this portion of the root, although the authors had not investigated LR development in the root portions near the growing tip of the primary root. Because LR initiation is restricted to specific pericycle cell files adjacent to a xylem pole in the basal region of the meristem (De Smet et al., 2007; Fukaki and Tasaka, 2009), and LR formation in this new growing root portion may be more susceptible to stress stimuli, such as observed with exposure to high NH₄⁺ and salt (Duan et al., 2013; Li et al., 2013), it is reasonable to suggest that modulation of LR formation near the growing tip of the primary root is critical to the response to excess Fe stress.In Arabidopsis (Arabidopsis thaliana), the development of LRs proceeds through the following stages: lateral root primordia (LRP) initiation, establishment, emergence, activation into mature LRs, and final maintenance of LR elongation (Fukaki and Tasaka, 2009; Péret et al., 2009). The hormones abscisic acid (ABA) and auxin are important internal negative and positive regulators during LR development, respectively (Fukaki and Tasaka, 2009). ABA has been implicated in LRP emergence and meristem activation independent of auxin (De Smet et al., 2003). Auxin is an important internal positive regulator during LR development (Fukaki and Tasaka, 2009), and auxin transport is critical (Blilou et al., 2005). Mutants in auxin efflux carriers such as PIN-FORMED (PIN) and P-Glycoprotein show significant defects in LR formation (Fukaki and Tasaka, 2009; Péret et al., 2009). For example, LR initiation frequency was significantly reduced in pin2 and pin3 mutants (Dubrovsky et al., 2009), and PIN2 was also shown to be involved in exogenous and endogenous signal-mediated LR development (by brassinosteroid, jasmonate, and fungal challenge; Li et al., 2005; Felten et al., 2009; Sun et al., 2009). Similarly, Auxin Resistant1 (AUX1), an auxin influx carrier, also regulates LRP positioning and initiation (De Smet et al., 2007). While both AUX1 and PIN2 are required specifically for the basipetal transport of auxin through the outer root cell layers (Fukaki and Tasaka, 2009), PIN1 localized at the basal end of vascular cells is responsible for direct acropetal auxin flow in the root stele (Blilou et al., 2005). Recently, the roles of ethylene on LR development have also been highlighted, and the ethylene-mediated LR formation is dependent on the auxin pathway (Ivanchenko et al., 2008; Lewis et al., 2011). Ethylene treatment could mediate fluorescence of AUX1 and PIN2 fluorescent protein fusions at the root tip (Růzicka et al., 2007; Lewis et al., 2011). Although ABA, auxin, and ethylene signals have been implicated as important for LR development, it is not known whether and how the three hormones are involved in the response of LR formation to Fe stress.The previously described phenotypes and physiological processes related to Fe toxicity do not clarify the effect of excess Fe on LR formation. In this study, we employed the Arabidopsis wild type and ABA-, auxin-, and ethylene-related mutants to explore the LR formation response to Fe toxicity and to elucidate the roles of ABA, auxin, and ethylene. Potential mechanisms involved in the early stress response to Fe stress are discussed.     

Putrescine Alleviates Iron Deficiency via NO-Dependent Reutilization of Root Cell-Wall Fe in Arabidopsis

Plants challenged with abiotic stress show enhanced polyamines levels. Here, we show that the polyamine putrescine (Put) plays an important role to alleviate Fe deficiency. The adc2-1 mutant, which is defective in Put biosynthesis, was hypersensitive to Fe deficiency compared with wild type (Col-1 of Arabidopsis [Arabidopsis thaliana]). Exogenous Put decreased the Fe bound to root cell wall, especially to hemicellulose, and increased root and shoot soluble Fe content, thus alleviating the Fe deficiency-induced chlorosis. Intriguingly, exogenous Put induced the accumulation of nitric oxide (NO) under both Fe-sufficient (+Fe) and Fe-deficient (-Fe) conditions, although the ferric-chelate reductase (FCR) activity and the expression of genes related to Fe uptake were induced only under -Fe treatment. The alleviation of Fe deficiency by Put was diminished in the hemicellulose-level decreased mutant-xth31 and in the noa1 and nia1nia2 mutants, in which the endogenous NO levels are reduced, indicating that both NO and hemicellulose are involved in Put-mediated alleviation of Fe deficiency. However, the FCR activity and the expression of genes related to Fe uptake were still up-regulated under -Fe+Put treatment compared with -Fe treatment in xth31, and Put-induced cell wall Fe remobilization was abolished in noa1 and nia1nia2, indicating that Put-regulated cell wall Fe reutilization is dependent on NO. From our results, we conclude that Put is involved in the remobilization of Fe from root cell wall hemicellulose in a process dependent on NO accumulation under Fe-deficient condition in Arabidopsis.Iron is an essential element for plant growth and development, and iron deficiency is the most common micronutrient deficiency in the world. To cope with iron deficiency, plants have evolved two distinct mechanisms for Fe acquisition from the rhizosphere. Strategy I, found in all dicots and monocots with the exception of graminaceous species, is characterized by (1) release of protons to acidify the rhizosphere, which is mediated in Arabidopsis (Arabidopsis thaliana) by the proton-translocating ATPase AHA2 (ARABIDOPSIS PLASMA MEMBRANE H⁺-ATPASE ISOFORM 2; Curie and Briat, 2003; Santi and Schmidt, 2009); (2) inducing ferric chelate reductase activity mediated by FRO2 (FERRIC REDUCTASE OXIDASE2; Robinson et al., 1999); and (3) uptake of Fe²⁺ by the metal transporter IRT1 (IRON REGULATED TRANSPORTER1; Eide et al., 1996; Vert et al., 2002). Strategy II, utilized by graminaceous monocots (Römheld and Marschner, 1986), is characterized by enhanced release of phytosiderophores that form chelates with Fe(III) (Curie and Briat, 2003). However, in addition to Fe acquisition, the mechanisms underlying the mobilization of Fe(III) also are a major challenge for us to understand.Recently, accumulating evidence has shown that phenolic compounds are important for iron mobilization. Rodríguez-Celma et al. (2013) showed that secretion of phenolics is critical for Arabidopsis Fe acquisition from low bioavailability sources, and then Fourcroy et al. (2014) and Schmidt et al. (2014) demonstrated that coumarins are the active compounds in this process. Schmid et al. (2014) confirmed that secretion of coumarins is an essential aspect of Arabidopsis Fe acquisition and provided extensive information on metabolomic changes elicited by Fe deficiency. However, under certain conditions Fe is not readily available, and Fe is difficult to mobilize; thus, Fe stored in the plant needs to be reutilized. For example, phenolics are secreted to remobilize the root apoplastic Fe and improve Fe nutrition in red clover (Trifolium pratense) and rice (Oryza sativa) (Jin et al., 2007; Bashir et al., 2011). Moreover, Lei et al. (2014) reported that the cell wall can be an important Fe source during periods of limited Fe supply. As the first barrier to encounter the soil environment, the cell wall is a pivotal site for most cationic ions in plants (Lozano-Rodríguez et al., 1997; Carrier et al., 2003). Hemicellulose contributes to the overall Al/Cd accumulation in the cell wall of Arabidopsis (Zhu et al., 2012, 2013) and also acts as a Fe pool (Lei et al., 2014). Over 75% of Fe in the root is retained in the cell wall (Bienfait et al., 1985), especially in the hemicellulose fraction (Lei et al., 2014). Thus, the cell wall is not only a site to immobilize an element and restrict its entrance into the cell, but also can serve as a pool to provide the nutrient when the supply from the growth medium is limited. However, the upstream mechanism of Fe reutilization through the cell wall, especially hemicellulose, is still far from clear.The responses to Fe deficiency in plants involve numerous phytohormones and signaling molecules, including auxin (Römheld and Marschner, 1981; Chen et al., 2010), ethylene (García et al., 2010; Wu et al., 2011), and NO (Graziano and Lamattina, 2007; Chen et al., 2010). Polyamines share common substrates with nitric oxide (NO) (Shi and Chan, 2014), and polyamines like spermidine and spermine rapidly induce a burst of NO in various plant species, indicating that NO is a potential intermediate of polyamine-mediated signaling.Polyamines, including putrescine (Put), spermidine, and spermine, are low M_r natural compounds with nitrogen-containing aliphatic structure and influence basic physiological and developmental events, such as cell division and differentiation, rhizogenesis, leaf senescence, zygotic, somatic embryogenesis, and development of flowers and fruits (Feirer et al., 1984; Galston et al., 1995; Bouchereau et al., 1999; Kakkar et al., 2000; Tun et al., 2001; Shi and Chan, 2014). The metabolism of polyamines in plant tissues is subject to strict regulation, and polyamine levels in plant roots change upon exposure to abiotic stress such as salt, drought, low and high temperature, heavy metals (Cu, Cr, Fe, and Ni), and oxidative stresses (Liu et al., 2005; Cheng et al., 2009; Wimalasekera et al., 2011; Tavladoraki et al., 2012).Ample evidence demonstrates the involvement of Put in responses to various types of abiotic stress, such as mineral deficiency in barley (Hordeum vulgare) leaves (Smith, 1973), high osmotic pressure in barley, corn, wheat, and wild oat leaves (Flores and Galston, 1982a), low pH in peeled oat (Avena sativa L. var Victory) leaf (Young and Galston, 1983), potassium deficiency in oat shoot and Arabidopsis thaliana (L.) Heynh (Young and Galston, 1984; Watson and Malmberg, 1996), and cadmium toxicity in oat and bean leaves (Weinstein et al., 1986). In animals, Put is produced either from Orn by Orn decarboxylase or from Arg by Arg decarboxylase (ADC) (Hanfrey et al., 2001). As there is no detectable Orn decarboxylase activity in Arabidopsis, the ADC route is critical for Put biosynthesis. Although there are two genes responsible for ADC activity, Urano et al. (2004) reported that the expression of ADC2 correlates well with the increment of free Put, indicating ADC2 plays an important role in Put biosynthesis in Arabidopsis. However, the role of Put under Fe deficiency in plants remains unknown.In this study, we found that Fe deficiency results in enhanced Put levels. Further, whereas exogenous Put alleviated Fe deficiency, the adc2-1 mutant, in which endogenous Put is decreased, exhibited a Fe deficiency-sensitive phenotype. We demonstrated that Put acts upstream of NO to decrease the Fe binding capacity of the cell wall, especially that of hemicellulose, thus resulting in greater Fe reutilization.