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.     

Functional Analysis of the Hydrophilic Loop in Intracellular Trafficking of Arabidopsis PIN-FORMED Proteins

Different PIN-FORMED proteins (PINs) contribute to intercellular and intracellular auxin transport, depending on their distinctive subcellular localizations. Arabidopsis thaliana PINs with a long hydrophilic loop (HL) (PIN1 to PIN4 and PIN7; long PINs) localize predominantly to the plasma membrane (PM), whereas short PINs (PIN5 and PIN8) localize predominantly to internal compartments. However, the subcellular localization of the short PINs has been observed mostly for PINs ectopically expressed in different cell types, and the role of the HL in PIN trafficking remains unclear. Here, we tested whether a long PIN-HL can provide its original molecular cues to a short PIN by transplanting the HL. The transplanted long PIN2-HL was sufficient for phosphorylation and PM trafficking of the chimeric PIN5:PIN2-HL but failed to provide the characteristic polarity of PIN2. Unlike previous observations, PIN5 showed clear PM localization in diverse cell types where PIN5 is natively or ectopically expressed and even polar PM localization in one cell type. Furthermore, in the root epidermis, the subcellular localization of PIN5 switched from PM to internal compartments according to the developmental stage. Our results suggest that the long PIN-HL is partially modular for the trafficking behavior of PINs and that the intracellular trafficking of PIN is plastic depending on cell type and developmental stage.     

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.     

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.     

Phosphatidylinositol 4,5-Bisphosphate Influences PIN Polarization by Controlling Clathrin-Mediated Membrane Trafficking in Arabidopsis

The functions of the minor phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂] during vegetative plant growth remain obscure. Here, we targeted two related phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) PIP5K1 and PIP5K2, which are expressed ubiquitously in Arabidopsis thaliana. A pip5k1 pip5k2 double mutant with reduced PtdIns(4,5)P₂ levels showed dwarf stature and phenotypes suggesting defects in auxin distribution. The roots of the pip5k1 pip5k2 double mutant had normal auxin levels but reduced auxin transport and altered distribution. Fluorescence-tagged auxin efflux carriers PIN-FORMED (PIN1)–green fluorescent protein (GFP) and PIN2-GFP displayed abnormal, partially apolar distribution. Furthermore, fewer brefeldin A–induced endosomal bodies decorated by PIN1-GFP or PIN2-GFP formed in pip5k1 pip5k2 mutants. Inducible overexpressor lines for PIP5K1 or PIP5K2 also exhibited phenotypes indicating misregulation of auxin-dependent processes, and immunolocalization showed reduced membrane association of PIN1 and PIN2. PIN cycling and polarization require clathrin-mediated endocytosis and labeled clathrin light chain also displayed altered localization patterns in the pip5k1 pip5k2 double mutant, consistent with a role for PtdIns(4,5)P₂ in the regulation of clathrin-mediated endocytosis. Further biochemical tests on subcellular fractions enriched for clathrin-coated vesicles (CCVs) indicated that pip5k1 and pip5k2 mutants have reduced CCV-associated PI4P 5-kinase activity. Together, the data indicate an important role for PtdIns(4,5)P₂ in the control of clathrin dynamics and in auxin distribution in Arabidopsis.     

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.     

Aspen Tension Wood Fibers Contain β-(1→4)-Galactans and Acidic Arabinogalactans Retained by Cellulose Microfibrils in Gelatinous Walls

Contractile cell walls are found in various plant organs and tissues such as tendrils, contractile roots, and tension wood. The tension-generating mechanism is not known but is thought to involve special cell wall architecture. We previously postulated that tension could result from the entrapment of certain matrix polymers within cellulose microfibrils. As reported here, this hypothesis was corroborated by sequential extraction and analysis of cell wall polymers that are retained by cellulose microfibrils in tension wood and normal wood of hybrid aspen (Populus tremula × Populus tremuloides). β-(1→4)-Galactan and type II arabinogalactan were the main large matrix polymers retained by cellulose microfibrils that were specifically found in tension wood. Xyloglucan was detected mostly in oligomeric form in the alkali-labile fraction and was enriched in tension wood. β-(1→4)-Galactan and rhamnogalacturonan I backbone epitopes were localized in the gelatinous cell wall layer. Type II arabinogalactans retained by cellulose microfibrils had a higher content of (methyl)glucuronic acid and galactose in tension wood than in normal wood. Thus, β-(1→4)-galactan and a specialized form of type II arabinogalactan are trapped by cellulose microfibrils specifically in tension wood and, thus, are the main candidate polymers for the generation of tensional stresses by the entrapment mechanism. We also found high β-galactosidase activity accompanying tension wood differentiation and propose a testable hypothesis that such activity might regulate galactan entrapment and, thus, mechanical properties of cell walls in tension wood.Contractile cell walls found in plant organs and tissues such as tendrils, contractile roots, and tension wood (TW) have remarkable functions and properties. Their traits have been most intensely studied in TW of hardwoods, where they provide negative gravitropic response capacities to stems with secondary growth, as recently reviewed by Mellerowicz and Gorshkova (2012). These properties are conferred by TW fibers, which in many species contain a so-called gelatinous cell wall layer (G-layer; Norberg and Meier, 1966; Clair et al., 2008). G-layers are formed following the deposition of xylan-type secondary cell wall layer(s) and, thus, can be considered tertiary layers (Wardrop and Dadswell, 1948). They are almost or completely devoid of xylan and lignin and have very high cellulose contents (up to 85%). However, several other polymers appear to be present in TW G-layers, according to recent chemical analyses of isolated G-layers (Nishikubo et al., 2007; Kaku et al., 2009) and immunohistochemical labeling of TW sections (Arend, 2008; Bowling and Vaughn, 2008). Notably, xyloglucan (XG) has been found in G-layers of poplar (Populus spp.) TW (Nishikubo et al., 2007) and at the boundary between secondary cell wall layers (S-layers) and G-layers (Baba et al., 2009; Sandquist et al., 2010). It is also important for tension creation (Baba et al., 2009). However, it is not detectable in mature G-layers by monoclonal antibodies or XG-binding modules (Nishikubo et al., 2007; Baba et al., 2009; Sandquist et al., 2010).Structurally similar G-layers have been also identified in phloem fibers in many fibrous crops, such as flax (Linum usitatissimum), hemp (Cannabis sativa), and ramie (Boehmeria nivea; Gorshkova et al., 2012). These fibers occur in bundles that can be isolated for biochemical analysis. G-layers in fibers from diverse sources have a very similar structure, being largely composed of cellulose (with axial microfibril orientation, high degrees of crystallinity, and large crystallite sizes) lacking xylan and lignin (Mellerowicz et al., 2001; Pilate et al., 2004; Gorshkova et al., 2010, 2012) and having high water contents (Schreiber et al., 2010). In phloem fibers, the G-layers become very prominent, reaching thicknesses up to 15 µm and occupying over 90% of the cell wall’s total cross-sectional areas (Crônier et al., 2005). Pectic β-(1→4)-galactan with complex structures has been shown to be the major matrix polysaccharide of isolated phloem fibers in flax (Gorshkova et al., 2004; Gorshkova and Morvan, 2006; Gurjanov et al., 2007). Some of it is so strongly retained within cellulose that it cannot be extracted by concentrated alkali and can only be obtained after cellulose dissolution (Gurjanov et al., 2008). Such galactan, therefore, is a prime candidate for a polymer entrapped by cellulose microfibrils during crystallization that could substantially contribute to the contractile properties of cellulose in G-layers, according to recently formulated models (Mellerowicz et al., 2008; Mellerowicz and Gorshkova 2012). Furthermore, Roach et al. (2011) have shown that trimming of β-(1→4)-galactan by β-galactosidase is important for final cellulose crystallization, the formation of G-layer structure, and, hence, the stem’s mechanical properties.There is also immunocytochemical evidence for the presence of β-(1→4)-galactan and type II arabinogalactan (AG-II) in G-layers of TW fibers (Arend, 2008; Bowling and Vaughn, 2008). In addition, high-M_r branched galactans have been isolated from TW of Fagus sylvestris (Meier, 1962) and Fagus grandifolia (Kuo and Timell, 1969), with estimated degrees of polymerization (DP) of approximately 300 and complex structure, probably including both β-(1→4) and β-(1→6) linkages, although their exact nature remains unknown. Furthermore, Gal has been identified as one of the major sugars after Glc and Xyl in hydrolysates of isolated Populus spp. G-layers (Furuya et al., 1970; Nishikubo et al., 2007), and the Gal content of cell walls is a proposed indicator of the extent of TW development in beech (Fagus spp.; Ruel and Barnoud, 1978). However, subsequent linkage analyses identified only 2- and 3,6-linked Gal in poplar TW G-layers (Nishikubo et al., 2007), while in flax fibers, 4-linked Gal is the main component (Gorshkova et al., 1996, 2004; Gurjanov et al., 2007, 2008). Thus, the type(s) of galactans present in poplar TW remains unclear, and the galactans have not been shown previously either to have a rhamnogalacturonan-I (RG-I) backbone or to be strongly retained by cellulose microfibrils, as demonstrated for flax gelatinous fibers.To improve our understanding of cell wall properties in TW and their contraction mechanism, in the study presented here, we tested aspects of the recently proposed entrapment model (Mellerowicz et al., 2008; Mellerowicz and Gorshkova, 2012). According to this model, contraction is driven by the formation of larger cellulose structures, sometimes called macrofibrils, via interactions of cellulose microfibrils in the G-layer with each other and forming inclusions containing matrix polymers. This would induce tension within cellulose through the stretching of microfibrils required to surround the inclusions. The model is compatible with available data on the structure and action of gelatinous walls, but the main assumption, that polymers are trapped inside crystalline cellulose, such as that found in flax, has not been tested previously. Therefore, we compared matrix polymers retained by cellulose microfibrils in normal wood (NW) and TW of the model hardwood species hybrid aspen (Populus tremula × Populus tremuloides) that forms TW with gelatinous fibers. For this purpose, we used a combination of sequential cell wall extractions, similar to those used previously to characterize flax gelatinous fibers (Gurjanov et al., 2008), followed by fractionation of polymers by size-exclusion chromatography, immunological analyses, and oligosaccharide profiling by polysaccharide analysis using carbohydrate gel electrophoresis (PACE). The results reveal the main polymers of cellulose-retained fractions and key differences between NW and TW. Comparison of our results and previous findings also indicates that there are both similarities and differences in the constitution of gelatinous fibers in aspen and flax. An updated model of the contractile G-layer of TW fibers based on the data is presented.     

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.     

Clathrin Light Chains Regulate Clathrin-Mediated Trafficking,Auxin Signaling,and Development in Arabidopsis

Plant clathrin-mediated membrane trafficking is involved in many developmental processes as well as in responses to environmental cues. Previous studies have shown that clathrin-mediated endocytosis of the plasma membrane (PM) auxin transporter PIN-FORMED1 is regulated by the extracellular auxin receptor AUXIN BINDING PROTEIN1 (ABP1). However, the mechanisms by which ABP1 and other factors regulate clathrin-mediated trafficking are poorly understood. Here, we applied a genetic strategy and time-resolved imaging to dissect the role of clathrin light chains (CLCs) and ABP1 in auxin regulation of clathrin-mediated trafficking in Arabidopsis thaliana. Auxin was found to differentially regulate the PM and trans-Golgi network/early endosome (TGN/EE) association of CLCs and heavy chains (CHCs) in an ABP1-dependent but TRANSPORT INHIBITOR RESPONSE1/AUXIN-BINDING F-BOX PROTEIN (TIR1/AFB)-independent manner. Loss of CLC2 and CLC3 affected CHC membrane association, decreased both internalization and intracellular trafficking of PM proteins, and impaired auxin-regulated endocytosis. Consistent with these results, basipetal auxin transport, auxin sensitivity and distribution, and root gravitropism were also found to be dramatically altered in clc2 clc3 double mutants, resulting in pleiotropic defects in plant development. These results suggest that CLCs are key regulators in clathrin-mediated trafficking downstream of ABP1-mediated signaling and thus play a critical role in membrane trafficking from the TGN/EE and PM during plant development.     

Studies on the Tempo of Bubble Formation in Recently Cavitated Vessels: A Model to Predict the Pressure of Air Bubbles

A cavitation event in a vessel replaces water with a mixture of water vapor and air. A quantitative theory is presented to argue that the tempo of filling of vessels with air has two phases: a fast process that extracts air from stem tissue adjacent to the cavitated vessels (less than 10 s) and a slow phase that extracts air from the atmosphere outside the stem (more than 10 h). A model was designed to estimate how water tension (T) near recently cavitated vessels causes bubbles in embolized vessels to expand or contract as T increases or decreases, respectively. The model also predicts that the hydraulic conductivity of a stem will increase as bubbles collapse. The pressure of air bubbles trapped in vessels of a stem can be predicted from the model based on fitting curves of hydraulic conductivity versus T. The model was validated using data from six stem segments each of Acer mono and the clonal hybrid Populus 84K (Populus alba × Populus glandulosa). The model was fitted to results with root mean square error less than 3%. The model provided new insight into the study of embolism formation in stem tissue and helped quantify the bubble pressure immediately after the fast process referred to above.Vulnerability curves (VCs) have been viewed as a good measure of the drought resistance of woody stems (Cochard et al., 2013). Increasing drought increases the xylem tension (T) and eventually induces cavitation of the water in conduits when the T exceeds a certain threshold (Sperry and Tyree, 1988; Sperry et al., 1996). A cavitated vessel first fills with water vapor and eventually fills with air at atmospheric pressure because of Henry’s law, which describes gas equilibrium at the water/air interface. The time required for the progress mainly depends on the penetration rate of air into the recently cavitated vessel lumen via diffusion through the liquid phase.Previous studies were made about how fast bubbles disappear in embolized stems because of the solubility of air in water when water pressure exceeds atmospheric pressure, and the process takes 10 to 100 h depending on conditions (Tyree and Yang, 1992; Yang and Tyree, 1992). The tempo of bubble disappearance was measured by following the rise in stem hydraulic conductivity (k_h) versus time. The theory of Yang and Tyree (1992) relied on the same principles used in this article (Henry’s law, Fick’s law, and the ideal gas law), but modeling and experiments were done at pressures between 1 and 3 times atmospheric pressure rather than subatmospheric pressure (negative pressure). However, much less is known about the tempo of bubble formation in recently cavitated vessels (Brodersen et al., 2013). If the progress of embolus formation takes several minutes, then no changes in conductivity could be observed with available techniques, but if it takes hours, then the tempo of bubbles can be studied by rapidly inducing cavitation with increasing T and after cavitation induction measuring the influence of T on stem k_h as T is reduced gradually to zero. If air bubbles are at a pressure (bubble pressure [P_b*]) lower than a threshold near atmospheric pressure, bubbles ought to collapse when T decreases according to the ideal gas law and Henry’s law (see theory below). The consequence of bubble collapse will be partial filling of vessels with water and the rest with air bubbles. The partial filling of water in a recently cavitated vessel ought to increase the lumen conductivity from zero and connect the embolized vessel to adjacent conductive vessels and, hence, ought to increase the conductivity of the stem by an additional flow pathway (Wheeler et al., 2005; Hacke et al., 2006). The vascular system of stems is a complicated network with vessels of different lengths, diameters, and orientation (Evert, 2006), and the complex vessel network makes the additional pathway possible. Therefore, bubble collapse could be detected through the impact of T on the k_h of the stems in a way that is very similar to the methods used by Yang and Tyree (1992) but requires a more sophisticated centrifuge technique to induce embolism.Many studies have assumed that the bubble pressure in newly cavitated vessels ought to be near atmospheric pressure, and no corrections for bubble pressure have been taken in measuring percentage loss of conductivity (PLC) when T is lower than a critical threshold (Li et al., 2008; Wang et al., 2014a). As a result of bubble collapse, the measured k_h under a mild T should be higher than that under high T (greater than 0.5 MPa). And the lower the initial bubble pressure, the more bubbles collapse with decreasing T.The aim of this study is to construct a model that estimates average bubble pressure in partly embolized stems from the functional dependence of k_h on T, and with this model, we can further our understanding of the tempo of bubble formation in stems. Here, we will argue that the tempo of bubble formation is in two phases: an initial rapid phase (seconds to minutes to complete) followed by a much slower phase (many hours to complete). Since there is no method for measuring the rapid phase, the rapid phase will be described theoretically below. Next, a theory will be developed that allows the estimation of the pressure of air in recently formed bubbles in vessels during the slow phase. An experimental validation of the model will follow that will yield values of bubble pressure within the first 1 to 2 h following the fast phase of embolism formation in vessels.