Brassinosteroid Regulates Cell Elongation by Modulating Gibberellin Metabolism in Rice

Brassinosteroid (BR) and gibberellin (GA) are two predominant hormones regulating plant cell elongation. A defect in either of these leads to reduced plant growth and dwarfism. However, their relationship remains unknown in rice (Oryza sativa). Here, we demonstrated that BR regulates cell elongation by modulating GA metabolism in rice. Under physiological conditions, BR promotes GA accumulation by regulating the expression of GA metabolic genes to stimulate cell elongation. BR greatly induces the expression of D18/GA3ox-2, one of the GA biosynthetic genes, leading to increased GA₁ levels, the bioactive GA in rice seedlings. Consequently, both d18 and loss-of-function GA-signaling mutants have decreased BR sensitivity. When excessive active BR is applied, the hormone mostly induces GA inactivation through upregulation of the GA inactivation gene GA2ox-3 and also represses BR biosynthesis, resulting in decreased hormone levels and growth inhibition. As a feedback mechanism, GA extensively inhibits BR biosynthesis and the BR response. GA treatment decreases the enlarged leaf angles in plants with enhanced BR biosynthesis or signaling. Our results revealed a previously unknown mechanism underlying BR and GA crosstalk depending on tissues and hormone levels, which greatly advances our understanding of hormone actions in crop plants and appears much different from that in Arabidopsis thaliana.     

Fruit Growth in Arabidopsis Occurs via DELLA-Dependent and DELLA-Independent Gibberellin Responses

Fruit growth and development depend on highly coordinated hormonal activities. The phytohormone gibberellin (GA) promotes growth by inducing degradation of the growth-repressing DELLA proteins; however, the extent to which DELLA proteins contribute to GA-mediated gynoecium and fruit development remains to be clarified. Here, we provide an in-depth characterization of the role of DELLA proteins in Arabidopsis thaliana fruit growth. We show that DELLA proteins are key regulators of reproductive organ size and important for ensuring optimal fertilization. We demonstrate that the seedless fruit growth (parthenocarpy) observed in della mutants can be directly attributed to the constitutive activation of GA signaling. It has been known for >75 years that another hormone, auxin, can induce formation of seedless fruits. Using mutants with complete lack of DELLA activity, we show here that auxin-induced parthenocarpy occurs entirely through GA signaling in Arabidopsis. Finally, we uncover the existence of a DELLA-independent GA response that promotes fruit growth. This response requires GIBBERELLIN-INSENSITIVE DWARF1–mediated GA perception and a functional 26S proteasome and involves the basic helix-loop-helix protein SPATULA as a key component. Taken together, our results describe additional complexities in GA signaling during fruit development, which may be particularly important to optimize the conditions for successful reproduction.     

The Calcium-Dependent Protein Kinase CPK28 Regulates Development by Inducing Growth Phase-Specific,Spatially Restricted Alterations in Jasmonic Acid Levels Independent of Defense Responses in Arabidopsis

Phytohormones play an important role in development and stress adaptations in plants, and several interacting hormonal pathways have been suggested to accomplish fine-tuning of stress responses at the expense of growth. This work describes the role played by the CALCIUM-DEPENDENT PROTEIN KINASE CPK28 in balancing phytohormone-mediated development in Arabidopsis thaliana, specifically during generative growth. cpk28 mutants exhibit growth reduction solely as adult plants, coinciding with altered balance of the phytohormones jasmonic acid (JA) and gibberellic acid (GA). JA-dependent gene expression and the levels of several JA metabolites were elevated in a growth phase-dependent manner in cpk28, and accumulation of JA metabolites was confined locally to the central rosette tissue. No elevated resistance toward herbivores or necrotrophic pathogens was detected for cpk28 plants, either on the whole-plant level or specifically within the tissue displaying elevated JA levels. Abolishment of JA biosynthesis or JA signaling led to a full reversion of the cpk28 growth phenotype, while modification of GA signaling did not. Our data identify CPK28 as a growth phase-dependent key negative regulator of distinct processes: While in seedlings, CPK28 regulates reactive oxygen species-mediated defense signaling; in adult plants, CPK28 confers developmental processes by the tissue-specific balance of JA and GA without affecting JA-mediated defense responses.     

The Arabidopsis DELLA RGA-LIKE3 Is a Direct Target of MYC2 and Modulates Jasmonate Signaling Responses

Gibberellins (GAs) are plant hormones involved in the regulation of plant growth in response to endogenous and environmental signals. GA promotes growth by stimulating the degradation of nuclear growth–repressing DELLA proteins. In Arabidopsis thaliana, DELLAs consist of a small family of five proteins that display distinct but also overlapping functions in repressing GA responses. This study reveals that DELLA RGA-LIKE3 (RGL3) protein is essential to fully enhance the jasmonate (JA)-mediated responses. We show that JA rapidly induces RGL3 expression in a CORONATINE INSENSITIVE1 (COI1)– and JASMONATE INSENSITIVE1 (JIN1/MYC2)–dependent manner. In addition, we demonstrate that MYC2 binds directly to RGL3 promoter. Furthermore, we show that RGL3 (like the other DELLAs) interacts with JA ZIM-domain (JAZ) proteins, key repressors of JA signaling. These findings suggest that JA/MYC2-dependent accumulation of RGL3 represses JAZ activity, which in turn enhances the expression of JA-responsive genes. Accordingly, we show that induction of primary JA-responsive genes is reduced in the rgl3-5 mutant and enhanced in transgenic lines overexpressing RGL3. Hence, RGL3 positively regulates JA-mediated resistance to the necrotroph Botrytis cinerea and susceptibility to the hemibiotroph Pseudomonas syringae. We propose that JA-mediated induction of RGL3 expression is of adaptive significance and might represent a recent functional diversification of the DELLAs.     

Focus on Water: Leaf Shrinkage with Dehydration: Coordination with Hydraulic Vulnerability and Drought Tolerance

Leaf shrinkage with dehydration has attracted attention for over 100 years, especially as it becomes visibly extreme during drought. However, little has been known of its correlation with physiology. Computer simulations of the leaf hydraulic system showed that a reduction of hydraulic conductance of the mesophyll pathways outside the xylem would cause a strong decline of leaf hydraulic conductance (K_leaf). For 14 diverse species, we tested the hypothesis that shrinkage during dehydration (i.e. in whole leaf, cell and airspace thickness, and leaf area) is associated with reduction in K_leaf at declining leaf water potential (Ψ_leaf). We tested hypotheses for the linkage of leaf shrinkage with structural and physiological water relations parameters, including modulus of elasticity, osmotic pressure at full turgor, turgor loss point (TLP), and cuticular conductance. Species originating from moist habitats showed substantial shrinkage during dehydration before reaching TLP, in contrast with species originating from dry habitats. Across species, the decline of K_leaf with mild dehydration (i.e. the initial slope of the K_leaf versus Ψ_leaf curve) correlated with the decline of leaf thickness (the slope of the leaf thickness versus Ψ_leaf curve), as expected based on predictions from computer simulations. Leaf thickness shrinkage before TLP correlated across species with lower modulus of elasticity and with less negative osmotic pressure at full turgor, as did leaf area shrinkage between full turgor and oven desiccation. These findings point to a role for leaf shrinkage in hydraulic decline during mild dehydration, with potential impacts on drought adaptation for cells and leaves, influencing plant ecological distributions.As leaves open their stomata to capture CO₂ for photosynthesis, water is lost to transpiration, which needs to be replaced by flow through the hydraulic system. The leaf hydraulic system has two components, which act essentially in series: the pathways for water movement through the xylem from the petiole to leaf minor veins, and those through the living bundle sheath and mesophyll cells to the sites of evaporation (Tyree and Zimmermann, 2002; Sack et al., 2004; Sack and Holbrook, 2006). The decline in leaf hydraulic conductance (K_leaf) with dehydration may thus depend on both components. The importance of the xylem component is well established. Vein xylem embolism and cell collapse have been observed in dehydrating leaves (Salleo et al., 2001; Cochard et al., 2004a; Johnson et al., 2009), and computer modeling and experimental work showed that species with high major vein length per leaf area (VLA; i.e. for the first three vein-branching orders) were more resistant to hydraulic decline, providing more pathways around embolisms (Scoffoni et al., 2011). However, the physical impacts of dehydration on the extraxylem pathways have not been studied, even though in turgid leaves these pathways account for 26% to 88% of leaf hydraulic resistance (i.e. of 1/K_leaf), depending on species (Sack et al., 2003a; Cochard et al., 2004b). The aim of this study was to determine whether leaf shrinkage during dehydration relates to the decline of K_leaf as well as the structural determinants of leaf shrinkage.The shrinkage of leaves with dehydration has drawn attention for over 100 years. Leaves shrink in their area (Bogue, 1892; Gardner and Ehlig, 1965; Jones, 1973; Tang and Boyer, 2007; Blonder et al., 2012) and, considered in relative terms, even more strongly in their thickness (Fig. 1; Meidner, 1952; Gardner and Ehlig, 1965; Downey and Miller, 1971; Syvertsen and Levy, 1982; Saini and Rathore, 1983; Burquez, 1987; McBurney, 1992; Sancho-Knapik et al., 2010, 2011). Leaves fluctuate in thickness daily and seasonally according to transpiration (Kadoya et al., 1975; Tyree and Cameron, 1977; Fensom and Donald, 1982; Rozema et al., 1987; Ogaya and Peñuelas, 2006; Seelig et al., 2012). Indeed, the relation of leaf thickness to water status is so tight that using leaf thickness to guide irrigation has led to water savings of up to 45% (Seelig et al., 2012).Open in a separate windowFigure 1.Sketches of a fully turgid leaf (A) versus a strongly dehydrated leaf (B; drawings based on leaf cross sections of sunflower in Fellows and Boyer, 1978). Note the strong reduction in leaf thickness, cell thickness, and intercellular airspaces in the dehydrated leaf. Epidermal cells are shrunk in the dehydrated leaf, inducing whole-leaf area shrinkage. Note that this sketch represents shrinkage for a typical drought-sensitive species. Many species such as oaks (Quercus spp.) will experience less thickness shrinkage and an increase in intercellular airspace (see “Discussion”). [See online article for color version of this figure.]Previous studies of leaf shrinkage with progressive dehydration have tended to focus on single or few species. These studies showed that thickness declines with water status in two phases. Before the bulk leaf turgor loss point (TLP; leaf water potential [Ψ_leaf] at TLP) is reached, the slope of leaf thickness versus Ψ_leaf or relative water content (RWC) is shallower than past TLP for most species (Meidner, 1955, Kennedy and Booth, 1958, Burquez, 1987, McBurney, 1992, Sancho-Knapik et al., 2010, 2011). This is because before TLP, declining Ψ_leaf is strongly driven by declines in turgor pressure, which have a relatively low impact on cell and airspace volume, whereas past the TLP, declining Ψ_leaf depends only on solute concentration, which increases in inverse proportion as cell water volume declines while airspaces may shrink or expand (Tyree and Hammel, 1972, Sancho-Knapik et al., 2011). However, the steepness of the slope of leaf thickness versus Ψ_leaf before TLP seems to vary strongly across species (Meidner, 1955; Kennedy and Booth, 1958; Fellows and Boyer, 1978; Burquez, 1987; Colpitts and Coleman, 1997; Sancho-Knapik et al., 2010).A high leaf cell volume and turgor is crucial to physiological processes (Boyer, 1968; Lawlor and Cornic, 2002). Shrinkage may affect cell connectivity and water transport (Sancho-Knapik et al., 2011). However, no studies have tested for a possible relationship of leaf shrinkage with the decline of K_leaf during dehydration. Such an association would arise if, across species, shrinkage occurred simultaneously with vein xylem embolism or if tissue shrinkage led to declines in the extraxylem hydraulic conductance.To refine our hypotheses, we modified a computer model of the leaf hydraulic system (Cochard et al., 2004b; McKown et al., 2010; Scoffoni et al., 2011) to predict the impact of losses of xylem and extraxylem conductance on the response of K_leaf to dehydration. We characterized the degree of leaf shrinkage in thickness, in the thickness of cells and airspaces within the leaf, and in leaf area for 14 species diverse in phylogeny, leaf traits, and drought tolerance. We hypothesized that loss of extraxylem hydraulic conductance should have a greater impact on K_leaf at less negative water potentials when xylem tensions are too weak to trigger embolism and induce dramatic declines in K_leaf. We hypothesized that species with greater degrees of shrinkage before TLP would experience greater loss of K_leaf. Furthermore, we hypothesized that species from moist habitats would have greater degrees of shrinkage.For insight into the mechanisms and consequences of leaf shrinkage, we also investigated the relationships of 18 indices of leaf shrinkage with a wide range of aspects of leaf structure and composition, including gross morphology, leaf venation architecture, parameters of pressure-volume curves, and leaf water storage. We hypothesized that, across species, shrinkage in whole leaf, cell, and intercellular airspace thickness would be lower for species with greater allocation to structural rigidity and osmotic concentration, and thus shrinkage would be positively correlated with a lower modulus of elasticity (ε), less negative osmotic pressure at full turgor (π_o), lower leaf mass per area (LMA), and lower leaf density. Additionally, we tested the longstanding hypothesis that species with higher major VLA and/or minor VLA (i.e. the fourth and higher vein-branching orders) would shrink less in area and/or thickness with dehydration (Gardner and Ehlig, 1965). Finally, we tested the ability of dehydrated leaves to recover in size with rehydration. We hypothesized that recovery would be greater for mildly than for strongly dehydrated leaves and that species with greater leaf shrinkage would be better able to recover from shrinkage.     

Evidence That Chlorinated Auxin Is Restricted to the Fabaceae But Not to the Fabeae

Auxin is a pivotal plant hormone, usually occurring in the form of indole-3-acetic acid (IAA). However, in maturing pea (Pisum sativum) seeds, the level of the chlorinated auxin, 4-chloroindole-3-acetic acid (4-Cl-IAA), greatly exceeds that of IAA. A key issue is how plants produce halogenated compounds such as 4-Cl-IAA. To better understand this topic, we investigated the distribution of the chlorinated auxin. We show for the first time, to our knowledge, that 4-Cl-IAA is found in the seeds of Medicago truncatula, Melilotus indicus, and three species of Trifolium. Furthermore, we found no evidence that Pinus spp. synthesize 4-Cl-IAA in seeds, contrary to a previous report. The evidence indicates a single evolutionary origin of 4-Cl-IAA synthesis in the Fabaceae, which may provide an ideal model system to further investigate the action and activity of halogenating enzymes in plants.The chlorinated form of auxin, 4-chloroindole-3-acetic acid (4-Cl-IAA), is a highly active hormone that is thought to play a key role in early pericarp growth (Reinecke et al., 1995, 1999; Ozga et al., 2009). Exogenous 4-Cl-IAA, for example, has been shown to promote the pericarp elongation of deseeded pea (Pisum sativum) pods (Reinecke et al., 1999). Johnstone et al. (2005) reported that 4-Cl-IAA and bioactive GA (GA₃ or GA₁) act synergistically on pericarp growth when applied simultaneously, and a growth regulatory role has been proposed for 4-Cl-IAA through induction of GA biosynthesis and inhibition of ethylene action. In other species, e.g. tomato (Solanum lycopersicum), the nonchlorinated form of auxin, indole-3-acetic acid (IAA), also stimulates fruit growth via GAs (Serrani et al., 2008; Tang et al., 2015). The chlorinated auxin is mainly found in reproductive structures (Katayama et al., 1988), in which its levels often exceed those of the more widespread IAA (Tivendale et al., 2012). The chlorinated form is thought to be restricted to members of the leguminous tribe Fabeae (Reinecke 1999), which includes the genera Vicia, Pisum, Lathyrus, Lens, and Vavilovia (Schaefer et al., 2012). However, there is a curious exception: 4-Cl-IAA has been reported also from Scots pine (Pinus sylvestris; Ernstsen and Sandberg, 1986).We previously published evidence that most 4-Cl-IAA in maturing pea seeds is synthesized from 4-Cl-tryptophan (4-Cl-Trp) via 4-Cl-indole-3-pyruvic acid (Tivendale et al., 2012, 2014). 4-Cl-Trp has been identified in extracts from pea and broad bean (Vicia faba) seeds (Kettner et al., 1992; Manabe et al., 1999), but whether the precursors of Trp can be chlorinated is unknown.Virtually nothing is known about the enzymes that catalyze halogenation reactions in plants. In bacteria, fungi, and marine algae, there are six types of enzymes responsible for the addition of halogen atoms to organic molecules. These include heme haloperoxidases, vanadium-dependent haloperoxidases, mononuclear nonheme iron halogenases, flavin-dependent halogenases, S-adenosyl-l-Met-dependent chlorinases and fluorinases, and methyl halide transferases (Butler and Sandy, 2009; Wagner et al., 2009). However, in the genomes of angiosperms, the only type of halogenating enzyme that has been annotated are haloperoxidases, but very little is known about these enzymes. To further understand the activity and action of halogenating enzymes in plants, a comparative system is required.In this study, we investigated the distribution of 4-Cl-IAA and 4-Cl-Trp in the Fabaceae by monitoring these compounds in the seeds of representative species spanning the phylogeny of this family. Most of these species have not been previously tested for the presence of the chlorinated compounds. In addition, we reexamined the reported occurrence of 4-Cl-IAA outside the Fabaceae, namely in Scots pine; several other Pinus species were investigated here as well. We also examined the endogenous levels of 4-Cl-IAA in both vegetative tissues and seeds of broad bean to address the question of whether 4-Cl-IAA is largely restricted to seeds (Pless et al., 1984; Katayama et al., 1988).     

Nitrogen Stress Affects the Turnover and Size of Nitrogen Pools Supplying Leaf Growth in a Grass

The effect of nitrogen (N) stress on the pool system supplying currently assimilated and (re)mobilized N for leaf growth of a grass was explored by dynamic ¹⁵N labeling, assessment of total and labeled N import into leaf growth zones, and compartmental analysis of the label import data. Perennial ryegrass (Lolium perenne) plants, grown with low or high levels of N fertilization, were labeled with ¹⁵NO₃⁻/¹⁴NO₃⁻ from 2 h to more than 20 d. In both treatments, the tracer time course in N imported into the growth zones fitted a two-pool model (r² > 0.99). This consisted of a “substrate pool,” which received N from current uptake and supplied the growth zone, and a recycling/mobilizing “store,” which exchanged with the substrate pool. N deficiency halved the leaf elongation rate, decreased N import into the growth zone, lengthened the delay between tracer uptake and its arrival in the growth zone (2.2 h versus 0.9 h), slowed the turnover of the substrate pool (half-life of 3.2 h versus 0.6 h), and increased its size (12.4 μg versus 5.9 μg). The store contained the equivalent of approximately 10 times (low N) and approximately five times (high N) the total daily N import into the growth zone. Its turnover agreed with that of protein turnover. Remarkably, the relative contribution of mobilization to leaf growth was large and similar (approximately 45%) in both treatments. We conclude that turnover and size of the substrate pool are related to the sink strength of the growth zone, whereas the contribution of the store is influenced by partitioning between sinks.This article examines the nitrogen (N) supply system of growing grass leaves, and it investigates how functional and kinetic properties of this system are affected by N stress. The N supply of growing leaves is a dominant target of whole-plant N metabolism. This is primarily related to the high N demand of the photosynthetic apparatus and the related metabolic machinery of new leaves (Evans, 1989; Makino and Osmond, 1991; Grindlay, 1997; Lemaire, 1997; Wright et al., 2004; Johnson et al., 2010; Maire et al., 2012). The N supply system, as defined here, is an integral part of the whole plant: it includes all N compounds that supply leaf growth. Hence, it integrates all events between the uptake of N from the environment (source), intermediate uses in other processes of plant N metabolism, and the eventual delivery to the leaf growth zone (sink; Fig. 1). N that does not ultimately serve leaf growth is not included in this system; all N that serves leaf growth is included, irrespective of its localization in the plant. Conceptually, two distinct sources supply N for leaf growth: N from current uptake and assimilation that is directly transferred to the growing leaf (“directly transferred N”) and N from turnover/redistribution of organic compounds (“mobilized N”).Open in a separate windowFigure 1.Schematic representation of N fluxes in the leaf growth zone and in the N supply system of leaf growth in a grass plant. A, Scheme of a growing leaf, with its growth zone (including zones of cell division, expansion, and maturation) and recently produced tissue (RPT). N import (I; μg h⁻¹) into the growth zone is mostly in the form of amino acids. Inside the growth zone, the nitrogenous substrate is used in new tissue construction. Then, N export (E; μg h⁻¹) is in the form of newly formed, fully expanded nitrogenous tissue (tissue-bound export with RPT) and is calculated as leaf elongation rate (L_ER; mm h⁻¹) times the lineal density of N in RPT (ρ; μg mm⁻¹): E = L_ER × ρ (Lattanzi et al., 2004). In a physiological steady state, import equals export (I = E) and the N content of the growth zone (G; μg [not shown]) is constant. Labeled N import into the growth zone (I_lab) commences shortly after labeling of the nutrient solution with ¹⁵N. The labeled N content of the growth zone (G_lab; μg) increases over time (dG_lab/dt) until it eventually reaches isotopic saturation (Fig. 2B). Similarly, the lineal density of labeled N in RPT (ρ_lab) increases until it approaches ρ. At any time, the export of labeled N in RPT (E_lab) equals the concurrent ρ_lab × L_ER. The import of labeled N is obtained as I_lab = E_lab + dG_lab/dt (Lattanzi et al., 2005) and considers the increasing label content in the growth zone during labeling. The fraction of labeled N in the import flux (f_{lab I}) is calculated as f_{lab I} = I_lab/I. The time course of f_{lab I} (Fig. 3) reflects the kinetic properties of the N supply system of leaf growth (C). B, Scheme of a vegetative grass plant (reduced to a rooted tiller with three leaves) with leaf growth zone. N import into the growth zone (I) originates from (1) N taken up from the nutrient solution that is transferred directly to the growth zone following assimilation (directly transferred N) and (2) N derived from turnover/redistribution of stores (mobilized N). The store potentially includes proteins in all mature and senescing tissue in the shoot and root of the entire plant. As xylem, phloem, and associated transfer cells/tissue provide for a vascular network that connects all parts of the plant, the mobilized N may principally originate from any plant tissue that exhibits N turnover/mobilization. The fraction of total N uptake that is allocated to the N supply system of the growth zone equals U (see model in C). The fraction of total mobilized N allocated to the growth zone equals M (see model in C). C, Compartmental model of the source-sink system supplying N to the leaf growth zone, as shown by Lattanzi et al. (2005) and used here. Newly absorbed N (U; μg h⁻¹) enters a substrate pool (Q₁); from there, the N is either imported directly into the growth zone (I) or exchanged with a store (Q₂). Q₁ integrates the steps of transport and assimilation that precede the translocation to the growth zone. Q₂ includes all proteins that supply N for leaf growth during their turnover and mobilization. The parameters of the model, including the (relative) size and turnover of pools Q₁ and Q₂, the deposition into the store (D; μg h⁻¹), and the mobilization from the store (M; μg h⁻¹), and the contribution of direct transfer relative to mobilization to the N supply of the growth zone are obtained by fitting the compartmental model to the f_{lab I} data (A) obtained in dynamic ¹⁵N labeling experiments (for details, see “Materials and Methods”). During physiological steady state, the sizes of Q₁ and Q₂ are constant, I = U, and M = D. [See online article for color version of this figure.]Amino acids are the predominant form in which N is supplied for leaf growth in grasses, and incorporation in new leaf tissue occurs mainly in the leaf growth zone (Gastal and Nelson, 1994; Amiard et al., 2004). This is a heterotrophic piece of tissue that includes the zones of cell division and elongation, is located at the base of the leaf, and is encircled by the sheath of the next older leaf (Volenec and Nelson, 1981; MacAdam et al., 1989; Schnyder et al., 1990; Kavanová et al., 2008). As most N is taken up in the form of nitrate but supplied to the growth zone in the form of amino acids, the path of directly transferred N includes a series of metabolic and transport steps. These include transfer to and loading into the xylem, xylem transport and unloading, reduction and ammonium assimilation, cycling through photorespiratory N pools, amino acid synthesis, loading into the phloem, and transport to the growth zone (Hirel and Lea, 2001; Novitskaya et al., 2002; Stitt et al., 2002; Lalonde et al., 2003; Dechorgnat et al., 2011). The time taken to pass through this sequence is unknown at present, as is the effect of N deficiency on that time. Also, it is not known how much N is contained in, and moving through, the different compartments that supply leaf growth with currently assimilated N.At the level of mature organs, mainly leaves, there is considerable knowledge about N turnover and redistribution. Much less is known about the fate of the mobilized N and its actual use in sink tissues like the leaf growth zone. The processes in mature organs are associated with the maintenance metabolism of proteins, organ senescence, and adjustments in leaf protein levels to decreasing irradiance inside growing canopies when leaves become shaded by overtopping newer ones (Evans, 1993; Vierstra, 1993; Hikosaka et al., 1994; Anten et al., 1995; Hirel et al., 2007; Jansson and Thomas, 2008; Moreau et al., 2012). N mobilization in shaded leaves supports the optimization of photosynthetic N use efficiency at plant and canopy scale (Field, 1983; Evans, 1993; Anten et al., 1995), it reduces the respiratory burden of protein maintenance costs (Dewar et al., 1998; Amthor, 2000; Cannell and Thornley, 2000), and it provides a mechanism for the conservation of the most frequently growth-limiting nutrient (Aerts, 1996). Mobilization of N involves protein turnover and net degradation (Huffaker and Peterson, 1974), redistribution in the form of amino acids (Simpson and Dalling, 1981; Simpson et al., 1983; Hörtensteiner and Feller, 2002), and (at least) some of the mobilized N is supplied to new leaf growth (Lattanzi et al., 2005).N fertilizer supply has multiple direct and indirect effects on plant N metabolism (Stitt et al., 2002; Schlüter et al., 2012). In particular, it modifies the N content of newly produced leaves, leaf longevity/senescence, and the dynamics of light distribution inside expanding canopies (Evans, 1983, 1989; Lötscher et al., 2003; Moreau et al., 2012). Thus, N fertilization influences the availability of recyclable N. At the same time, it augments the availability of directly transferable N to leaf growth. The net effect of these factors on the importance of mobilized versus directly transferred N substrate for leaf growth is not known. Also, it is unknown how N fertilization influences the functional characteristics of the N supply system, such as the size and turnover of its component pools.The assessment of the importance of directly transferred versus mobilized N for leaf growth requires studies at the sink end of the system (i.e. investigations of the N import flux into the leaf growth zone). Directly transferred N and mobilized N can be distinguished on the basis of their residence time in the plant, the time between uptake from the environment and import into the leaf growth zone: direct transfer involves a short residence time (fast transfer), whereas mobilized N resides much longer in the plant before it is delivered to the growth zone (slow transfer; De Visser et al., 1997; Lattanzi et al., 2005). Such studies require dynamic labeling of the N taken up by the plant (Schnyder and de Visser, 1999) and monitoring of the rate and isotopic composition/label content of N import into the leaf growth zone (Lattanzi et al., 2005). For grass plants in a physiological steady state, N import and the isotopic composition of the imported N are calculated from the leaf elongation rate and the lineal density of N in newly formed tissue (Fig. 1A; Lattanzi et al., 2004) and the change of tracer content in the leaf growth zone and recently produced leaf tissue over time (Lattanzi et al., 2005). Such data reveal the temporal change of the fraction of labeled N in the N import flux (f_{lab I}), which then can be used to characterize the N supply system of leaf growth via compartmental modeling. So far, there is only one study that has partially characterized this system (Lattanzi et al., 2005): this work was conducted with a C₃ grass, perennial ryegrass (Lolium perenne), and a C₄ grass, Paspalum dilatatum, growing in mixed stands and indicated that two interconnected N pools supplied the leaf growth zone in both species: a “substrate pool” (Q₁), which provided a direct route for newly absorbed and assimilated N import into the leaf growth zone (directly transferred N), and a mobilizing “store” (Q₂), which supplied N to the leaf growth zone via the substrate pool (Fig. 1C). The relative contribution of mobilization from the store was least important in the fast-growing, dominant individuals and most important in subordinate, shaded individuals. That work did not address the role of N deficiency, and the limited short-term resolution of the study (labeling intervals of 24 h or greater) precluded an analysis of the fast-moving parts of the system.Accordingly, this work addresses the following questions. How does N deficiency influence the substrate supply system of the leaf growth sink in terms of the number, size, and turnover (half-life) of its kinetically distinct pools? How does N deficiency affect the relationship between directly transferred and mobilized N for leaf growth? And what additional insight on the compartmental structure of the supply system is obtained when the short-term resolution of the analysis is increased by 1 order of magnitude? The work was performed with vegetative plants of perennial ryegrass grown in constant conditions with either a low (1.0 mm; termed low N) or high (7.5 mm; high N) nitrate concentration in the nutrient solution. In both treatments, a large number of plants were dynamically labeled with ¹⁵N over a wide range of time intervals (2 h to more than 20 d). The import of total N and ¹⁵N tracer into growth zones was estimated at the end of each labeling interval. Tracer data were analyzed with compartmental models following principles detailed by Lattanzi et al. (2005, 2012) and Lehmeier et al. (2008) to address the specific questions. Previous articles reported on root and shoot respiration (Lehmeier et al., 2010) and cell division and expansion in leaf growth zones (Kavanová et al., 2008) in the same experiment.     

Arabidopsis ERG28 Tethers the Sterol C4-Demethylation Complex to Prevent Accumulation of a Biosynthetic Intermediate That Interferes with Polar Auxin Transport

Sterols are vital for cellular functions and eukaryotic development because of their essential role as membrane constituents. Sterol biosynthetic intermediates (SBIs) represent a potential reservoir of signaling molecules in mammals and fungi, but little is known about their functions in plants. SBIs are derived from the sterol C4-demethylation enzyme complex that is tethered to the membrane by Ergosterol biosynthetic protein28 (ERG28). Here, using nonlethal loss-of-function strategies focused on Arabidopsis thaliana ERG28, we found that the previously undetected SBI 4-carboxy-4-methyl-24-methylenecycloartanol (CMMC) inhibits polar auxin transport (PAT), a key mechanism by which the phytohormone auxin regulates several aspects of plant growth, including development and responses to environmental factors. The induced accumulation of CMMC in Arabidopsis erg28 plants was associated with diagnostic hallmarks of altered PAT, including the differentiation of pin-like inflorescence, loss of apical dominance, leaf fusion, and reduced root growth. PAT inhibition by CMMC occurs in a brassinosteroid-independent manner. The data presented show that ERG28 is required for PAT in plants. Furthermore, it is accumulation of an atypical SBI that may act to negatively regulate PAT in plants. Hence, the sterol pathway offers further prospects for mining new target molecules that could regulate plant development.     

Antiphase Light and Temperature Cycles Affect PHYTOCHROME B-Controlled Ethylene Sensitivity and Biosynthesis,Limiting Leaf Movement and Growth of Arabidopsis

In the natural environment, days are generally warmer than the night, resulting in a positive day/night temperature difference (+DIF). Plants have adapted to these conditions, and when exposed to antiphase light and temperature cycles (cold photoperiod/warm night [−DIF]), most species exhibit reduced elongation growth. To study the physiological mechanism of how light and temperature cycles affect plant growth, we used infrared imaging to dissect growth dynamics under +DIF and −DIF in the model plant Arabidopsis (Arabidopsis thaliana). We found that −DIF altered leaf growth patterns, decreasing the amplitude and delaying the phase of leaf movement. Ethylene application restored leaf growth in −DIF conditions, and constitutive ethylene signaling mutants maintain robust leaf movement amplitudes under −DIF, indicating that ethylene signaling becomes limiting under these conditions. In response to −DIF, the phase of ethylene emission advanced 2 h, but total ethylene emission was not reduced. However, expression analysis on members of the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase ethylene biosynthesis gene family showed that ACS2 activity is specifically suppressed in the petiole region under −DIF conditions. Indeed, petioles of plants under −DIF had reduced ACC content, and application of ACC to the petiole restored leaf growth patterns. Moreover, acs2 mutants displayed reduced leaf movement under +DIF, similar to wild-type plants under −DIF. In addition, we demonstrate that the photoreceptor PHYTOCHROME B restricts ethylene biosynthesis and constrains the −DIF-induced phase shift in rhythmic growth. Our findings provide a mechanistic insight into how fluctuating temperature cycles regulate plant growth.In nature, during the day (light), temperatures are usually higher than during the night (dark). Correspondingly, most plants show optimal growth under such synchronous light and temperature cycles. Increasing the difference between day and night temperature (+DIF) results in increased elongation growth in various species, a phenomenon referred to as thermoperiodism (Went, 1944). The opposite regime, when the temperature of the day (DT) is lower than the temperature of the night (NT), is called −DIF (negative DT/NT difference). Under −DIF conditions, the elongation growth of stems and leaves of various plant species is reduced (Maas and van Hattum, 1998; Carvalho et al., 2002; Thingnaes et al., 2003). Arabidopsis (Arabidopsis thaliana) plants grown under −DIF (DT/NT 12°C/22°C) displayed a reduction in leaf elongation of approximately 20% compared with the control (DT/NT 22°C/12°C; Thingnaes et al., 2003). −DIF is frequently applied in horticulture to produce crops with a desirable compact architecture without the need for growth-retarding chemicals (Myster and Moe, 1995). Despite the economic importance of the application of such temperature regimes in horticulture, the mechanistic basis of the growth reduction under −DIF is still poorly understood.Previously, it was demonstrated that −DIF affects phytohormone signaling in plants. In pea (Pisum sativum), for instance, the −DIF growth reduction correlated with increased catabolism of the phytohormone GA (Stavang et al., 2005). In contrast to pea, active GA levels did not decrease in response to −DIF in Arabidopsis (Thingnaes et al., 2003). On the other hand, the −DIF growth response in Arabidopsis was associated with reduced auxin levels (Thingnaes et al., 2003). The photoreceptor PHYTOCHROME B (PHYB) has been shown to be important for the response to −DIF, as phyB mutants of Arabidopsis (Thingnaes et al., 2008) and cucumber (Cucumis sativus; Patil et al., 2003) are insensitive to −DIF.In this work, the growth-related movement of mature Arabidopsis rosette leaves was analyzed under control (+DIF) and −DIF conditions. Under −DIF, the amplitude of leaf movement was decreased and the phase of movement was later, compared with control plants. The altered leaf growth patterns observed in −DIF could be restored by the application of ethylene. −DIF reduced the expression of 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE2 (ACS2) in the petiole, which correlated with reduced 1-aminocyclopropane-1-carboxylic acid (ACC) levels and decreased amplitude and delayed phase of leaf movement. Our results indicate that local ACS activity plays an important biological role, despite the fact that ethylene is a gaseous and fast-diffusing hormone. In addition, we demonstrate that in the phyB9 mutant, the phase of leaf movement is almost fully temperature entrained. Finally, ethylene levels and sensitivity are increased in phyB9, suggesting a role for PHYB in constraining temperature-induced shifts in the phase of leaf movement and dampening of leaf movement amplitude by controlling ethylene production and sensitivity.     

Trafficking of Vacuolar Proteins: The Crucial Role of Arabidopsis Vacuolar Protein Sorting 29 in Recycling Vacuolar Sorting Receptor

The retromer is involved in recycling lysosomal sorting receptors in mammals. A component of the retromer complex in Arabidopsis thaliana, vacuolar protein sorting 29 (VPS29), plays a crucial role in trafficking storage proteins to protein storage vacuoles. However, it is not known whether or how vacuolar sorting receptors (VSRs) are recycled from the prevacuolar compartment (PVC) to the trans-Golgi network (TGN) during trafficking to the lytic vacuole (LV). Here, we report that VPS29 plays an essential role in the trafficking of soluble proteins to the LV from the TGN to the PVC. maigo1-1 (mag1-1) mutants, which harbor a knockdown mutation in VPS29, were defective in trafficking of two soluble proteins, Arabidopsis aleurain-like protein (AALP):green fluorescent protein (GFP) and sporamin:GFP, to the LV but not in trafficking membrane proteins to the LV or plasma membrane or via the secretory pathway. AALP:GFP and sporamin:GFP in mag1-1 protoplasts accumulated in the TGN but were also secreted into the medium. In mag1-1 mutants, VSR1 failed to recycle from the PVC to the TGN; rather, a significant proportion was transported to the LV; VSR1 overexpression rescued this defect. Moreover, endogenous VSRs were expressed at higher levels in mag1-1 plants. Based on these results, we propose that VPS29 plays a crucial role in recycling VSRs from the PVC to the TGN during the trafficking of soluble proteins to the LV.     

Dynamic Changes in the Distribution of Minerals in Relation to Phytic Acid Accumulation during Rice Seed Development

Phytic acid (inositol hexakisphosphate [InsP₆]) is the storage compound of phosphorus in seeds. As phytic acid binds strongly to metallic cations, it also acts as a storage compound of metals. To understand the mechanisms underlying metal accumulation and localization in relation to phytic acid storage, we applied synchrotron-based x-ray microfluorescence imaging analysis to characterize the simultaneous subcellular distribution of some mineral elements (phosphorus, calcium, potassium, iron, zinc, and copper) in immature and mature rice (Oryza sativa) seeds. This fine-imaging method can reveal whether these elements colocalize. We also determined their accumulation patterns and the changes in phosphate and InsP₆ contents during seed development. While the InsP₆ content in the outer parts of seeds rapidly increased during seed development, the phosphate contents of both the outer and inner parts of seeds remained low. Phosphorus, calcium, potassium, and iron were most abundant in the aleurone layer, and they colocalized throughout seed development. Zinc was broadly distributed from the aleurone layer to the inner endosperm. Copper localized outside the aleurone layer and did not colocalize with phosphorus. From these results, we suggest that phosphorus translocated from source organs was immediately converted to InsP₆ and accumulated in aleurone layer cells and that calcium, potassium, and iron accumulated as phytic acid salt (phytate) in the aleurone layer, whereas zinc bound loosely to InsP₆ and accumulated not only in phytate but also in another storage form. Copper accumulated in the endosperm and may exhibit a storage form other than phytate.The transport of nutrients into developing seeds has received considerable attention. During the grain-filling stage, plants remobilize and transport nutrients distributed throughout the vegetative source organs into seeds. Plant seeds contain large amounts of phosphorus (P) in organic form, which supports growth during the early stages of seedling development. Most of the P in seeds is stored in the form of phytic acid (inositol hexakisphosphate [InsP₆]). Seeds also accumulate mineral nutrients such as potassium (K), magnesium (Mg), calcium (Ca), iron (Fe), zinc (Zn), copper (Cu), and manganese (Mn), which are used in seedling growth. Phytic acid acts as a strong chelator of metal cations and binds them to form phytate, a salt of InsP₆ (Lott et al., 2002; Raboy, 2009). During germination, phytate is decationized and hydrolyzed by phytases, and then inorganic phosphates, inositol, and various minerals are released from the phytate (Loewus and Murthy, 2000). Phytate accumulates within protein bodies, generally of vacuolar origin, in seed storage cells and is usually concentrated in spherical inclusions called globoids. Many studies of the elemental composition of phytate in seeds have been published. Energy-dispersive x-ray microanalyses of many plant species have revealed that, other than P, globoids contain mainly K and Mg as well as low levels of Ca, Mn, Fe, and Zn (Lott, 1984; Lott et al., 1995; Wada and Lott, 1997). This indicates that phytate is a mixed salt of these cations.Whether all storage metal elements can bind equally to InsP₆ is not known, although most elements are thought to exist in seeds in the form of phytate (Raboy, 2009). To form phytate, P and the other elements must be present in the same place. Therefore, determination of the precise locations of P and other elements in seed tissues makes it possible to judge whether an element exists in the form of phytate. Differences in metal distribution with P might suggest a storage form other than phytate. For determining distributions, synchrotron-based x-ray microfluorescence (µ-XRF) imaging utilizing an x-ray microbeam is a powerful tool. The microbeam excites the elements, thereby revealing the details of their spatial distribution. The development of focusing optics for high-energy x-rays using a Kirkpatrick-Baez mirror raises the imaging resolution of elements in µ-XRF analysis. A focal spot size smaller than 1 µm with x-ray energy as high as 100 keV enables detection of the subcellular distribution of elements in plant tissues (Fukuda et al., 2008; Takahashi et al., 2009).Whether there is an order in the affinity of elements for phytic acid in plant cells remains unknown. The stability of InsP₆-metal complexes has been estimated by in vitro titration (Maddaiah et al., 1964; Vohra et al., 1965; Persson et al., 1998). The binding strength of InsP₆ with metal is stronger for Zn and Cu than for Fe, Mn, and Ca. We also do not know if the mineral composition of phytate in seeds is determined by the relative abundance of these elements in the seed or by their biochemical characteristics. As a first step to address these issues, we examined the simultaneous changes in the distribution of P and metal elements during seed development using µ-XRF imaging analysis.Our objective in this study was to observe the dynamic changes in the distribution of some nutritionally important minerals (P, Ca, K, Fe, Zn, and Cu) in relation to the accumulation of phytic acid during rice (Oryza sativa) seed development.