Xyloglucan Endotransglucosylase-Hydrolase17 Interacts with Xyloglucan Endotransglucosylase-Hydrolase31 to Confer Xyloglucan Endotransglucosylase Action and Affect Aluminum Sensitivity in Arabidopsis

Previously, we reported that although the Arabidopsis (Arabidopsis thaliana) Xyloglucan Endotransglucosylase-Hydrolase31 (XTH31) has predominately xyloglucan endohydrolase activity in vitro, loss of XTH31 results in remarkably reduced in vivo xyloglucan endotransglucosylase (XET) action and enhanced Al resistance. Here, we report that XTH17, predicted to have XET activity, binds XTH31 in yeast (Saccharomyces cerevisiae) two-hybrid and coimmunoprecipitations assays and that this interaction may be required for XTH17 XET activity in planta. XTH17 and XTH31 may be colocalized in plant cells because tagged XTH17 fusion proteins, like XTH31 fusion proteins, appear to target to the plasma membrane. XTH17 expression, like that of XTH31, was substantially reduced in the presence of aluminum (Al), even at concentrations as low as 10 µm for 24 h or 25 µm for just 30 min. Agrobacterium tumefaciens-mediated transfer DNA insertion mutant of XTH17, xth17, showed low XET action and had moderately shorter roots than the wild type but was more Al resistant than the wild type. Similar to xth31, xth17 had low hemicellulose content and retained less Al in the cell wall. These data suggest a model whereby XTH17 and XTH31 may exist as a dimer at the plasma membrane to confer in vivo XET action, which modulates cell wall Al-binding capacity and thereby affects Al sensitivity in Arabidopsis.Soil acidity (pH < 5.5) affects about 40% of the world’s arable land (von Uexküll and Mutert, 1995) and more than 50% of land that is potentially arable, particularly in the tropics and subtropics (Kochian et al., 2004; Eticha et al., 2010). Al is the most growth-limiting factor for crop production on acid soils worldwide (Foy, 1988; Kochian, 1995), especially when the pH drops below 5 (Eswaran et al., 1997).To survive in an Al-toxic environment, Al-resistant plants have evolved two mechanisms to cope with Al toxicity. One is to restrict Al uptake from the root, while the other is to cope with internalized Al (Taylor, 1991; Kochian et al., 2004). The latter is usually employed by Al-accumulating species such as Hydrangea macrophylla (Ma et al., 1997a) and buckwheat (Fagopyrum esculentum; Ma et al., 1997b). In most cases, secretion of the organic acid anions is the most important Al exclusion mechanism (Kochian, 1995; Ryan et al., 2001; Ma and Furukawa, 2003), although it does not explain all the Al resistance in some plants such as signalgrass (Brachiaria decumbens Stapf cv Basilisk; Wenzl et al., 2001), maize (Zea mays; Piñeros et al., 2005), buckwheat (Zheng et al., 2005), rice (Oryza sativa; Ma et al., 2005; Yang et al., 2008), or Fagopyrum tataricum (Yang et al., 2011a). Therefore, it is possible that for some plant species (such as buckwheat), Al tolerance is a combination of mechanisms including organic anion efflux.Recently, evidence has accumulated that the cell wall, especially the hemicellulose component, may impact Al resistance. For example, Al induces significant changes in the hemicellulose fraction of wheat (Triticum aestivum; Tabuchi and Matsumoto, 2001), triticale (× Triticosecale Wittmack; Liu et al., 2008), rice (Yang et al., 2008), and Arabidopsis (Arabidopsis thaliana; Zhu et al., 2012), especially the Al-sensitive cultivars. Moreover, we found that Arabidopsis hemicellulose is not only very sensitive to Al stress (the content of hemicellulose increased quickly under Al stress), but is also the principal binding site for Al (Yang et al., 2011b). Furthermore, loss of Xyloglucan Endotransglucosylase-Hydrolase31 (XTH31) function resulted in lower xyloglucan content and cell wall Al-binding capacity in Arabidopsis (Zhu et al., 2012). Thus, xyloglucan may be a major Al-binding site in Arabidopsis, and any effects leading to xyloglucan modifications may regulate Al sensitivity.XTHs are a family of enzymes that play principal roles in the construction and restructuring of the load-bearing cross links among cellulose microfibrils (Osato et al., 2006) through catalyzing the molecular grafting or hydrolyzing of the xyloglucans to form the framework (Fry et al., 1992; Nishitani and Tominaga, 1992; Okazawa et al., 1993; Rose et al., 2002). There are 33 identified XTH genes in the Arabidopsis genome, and one-third occur as clusters resulting from genome duplication (Blanc et al., 2000; Yokoyama and Nishitani, 2001); XTH1-11 are classified in subgroup 1, XTH12-26 are in subgroup 2, and XTH27-33 are in subgroup 3 (Rose et al., 2002). Each member of the XTH gene family is likely regulated by specific cues and committed to cell wall dynamics specific to certain tissues or cell types (Nishitani, 2002; Becnel et al., 2006; Osato et al., 2006). For example, XTH27 is involved in the cell wall modification of tracheary elements at a specific stage of rosette leaf development and is essential for tertiary vein development (Matsui et al., 2005), whereas XTH31 is involved in cell wall modification and cell elongation through modulating xyloglucan endotransglucosylase (XET) action under Al stress (Zhu et al., 2012). However, XTH31 is an XTH for which xyloglucan endohydrolase (XEH) activity has been predicted (Baumann et al., 2007), and in our previous report, we demonstrated that XTH31 produced heterologously in Pichia pastoris has high XEH activity but low XET activity in vitro (Zhu et al., 2012), which is in accordance with Kaewthai et al. (2013), who reported that XTH31 is a predominant hydrolase using the in vitro activity assays and enzyme product analysis, as well as the use of a fluorogenic substrate in vivo. Unexpectedly, however, the xth31 mutant has very low XET action and activity (Zhu et al., 2012). One possible explanation for this result is that XTH31 may interact with and be required for activity of XET-active XTHs.In this study, we demonstrate that XTH17 can bind to XTH31 in vitro and in vivo and that a transfer DNA (T-DNA) insertional mutant of XTH17 has elevated Al resistance and exhibits a phenotype very similar to xth31. Together, these data are consistent with the interpretation that XTH17 and XTH31 may interact with each other and thereby contribute to Al-inhibited XET action in Arabidopsis.     

One Divinyl Reductase Reduces the 8-Vinyl Groups in Various Intermediates of Chlorophyll Biosynthesis in a Given Higher Plant Species,But the Isozyme Differs between Species

Divinyl reductase (DVR) converts 8-vinyl groups on various chlorophyll intermediates to ethyl groups, which is indispensable for chlorophyll biosynthesis. To date, five DVR activities have been detected, but adequate evidence of enzymatic assays using purified or recombinant DVR proteins has not been demonstrated, and it is unclear whether one or multiple enzymes catalyze these activities. In this study, we systematically carried out enzymatic assays using four recombinant DVR proteins and five divinyl substrates and then investigated the in vivo accumulation of various chlorophyll intermediates in rice (Oryza sativa), maize (Zea mays), and cucumber (Cucumis sativus). The results demonstrated that both rice and maize DVR proteins can convert all of the five divinyl substrates to corresponding monovinyl compounds, while both cucumber and Arabidopsis (Arabidopsis thaliana) DVR proteins can convert three of them. Meanwhile, the OsDVR (Os03g22780)-inactivated 824ys mutant of rice exclusively accumulated divinyl chlorophylls in its various organs during different developmental stages. Collectively, we conclude that a single DVR with broad substrate specificity is responsible for reducing the 8-vinyl groups of various chlorophyll intermediates in higher plants, but DVR proteins from different species have diverse and differing substrate preferences, although they are homologous.Chlorophyll (Chl) molecules universally exist in photosynthetic organisms. As the main component of the photosynthetic pigments, Chl molecules perform essential processes of absorbing light and transferring the light energy in the reaction center of the photosystems (Fromme et al., 2003). Based on the number of vinyl side chains, Chls are classified into two groups, 3,8-divinyl (DV)-Chl and 3-monovinyl (MV)-Chl. The DV-Chl molecule contains two vinyl groups at positions 3 and 8 of the tetrapyrrole macrocycle, whereas the MV-Chl molecule contains a vinyl group at position 3 and an ethyl group at position 8 of the macrocycle. Almost all of the oxygenic photosynthetic organisms contain MV-Chls, with the exceptions of some marine picophytoplankton species that contain only DV-Chls as their primary photosynthetic pigments (Chisholm et al., 1992; Goericke and Repeta, 1992; Porra, 1997).The classical single-branched Chl biosynthetic pathway proposed by Granick (1950) and modified by Jones (1963) assumed the rapid reduction of the 8-vinyl group of DV-protochlorophyllide (Pchlide) catalyzed by a putative 8-vinyl reductase. Ellsworth and Aronoff (1969) found evidence for both MV and DV forms of several Chl biosynthetic intermediates between magnesium-protoporphyrin IX monomethyl ester (MPE) and Pchlide in Chlorella spp. mutants. Belanger and Rebeiz (1979, 1980) reported that the Pchlide pool of etiolated higher plants contains both MV- and DV-Pchlide. Afterward, following the further detection of MV- and DV-tetrapyrrole intermediates and their biosynthetic interconversion in tissues and extracts of different plants (Belanger and Rebeiz, 1982; Duggan and Rebeiz, 1982; Tripathy and Rebeiz, 1986, 1988; Parham and Rebeiz, 1992, 1995; Kim and Rebeiz, 1996), a multibranched Chl biosynthetic heterogeneity was proposed (Rebeiz et al., 1983, 1986, 1999; Whyte and Griffiths, 1993; Kolossov and Rebeiz, 2010).Biosynthetic heterogeneity refers to the biosynthesis of a particular metabolite by an organelle, tissue, or organism via multiple biosynthetic routes. Varieties of reports lead to the assumption that Chl biosynthetic heterogeneity originates mainly in parallel DV- and MV-Chl biosynthetic routes. These routes are interconnected by 8-vinyl reductases that convert DV-tetrapyrroles to MV-tetrapyrroles by conversion of the vinyl group at position 8 of ring B to the ethyl group (Parham and Rebeiz, 1995; Rebeiz et al., 2003). DV-MPE could be converted to MV-MPE in crude homogenates from etiolated wheat (Triticum aestivum) seedlings (Ellsworth and Hsing, 1974). Exogenous DV-Pchlide could be partially converted to MV-Pchlide in barley (Hordeum vulgare) plastids (Tripathy and Rebeiz, 1988). 8-Vinyl chlorophyllide (Chlide) a reductases in etioplast membranes isolated from etiolated cucumber (Cucumis sativus) cotyledons and barley and maize (Zea mays) leaves were found to be very active in the conversion of exogenous DV-Chlide a to MV-Chlide a (Parham and Rebeiz, 1992, 1995). Kim and Rebeiz (1996) suggested that Chl biosynthetic heterogeneity in higher plants may originate at the level of DV magnesium-protoporphyrin IX (Mg-Proto) and would be mediated by the activity of a putative 8-vinyl Mg-Proto reductase in barley etiochloroplasts and plastid membranes. However, since these reports did not use purified or recombinant enzyme, it is not clear whether the reductions of the 8-vinyl groups of various Chl intermediates are catalyzed by one enzyme of broad specificity or by multiple enzymes of narrow specificity, which actually has become one of the focus issues in Chl biosynthesis.Nagata et al. (2005) and Nakanishi et al. (2005) independently identified the AT5G18660 gene of Arabidopsis (Arabidopsis thaliana) as an 8-vinyl reductase, namely, divinyl reductase (DVR). Chew and Bryant (2007) identified the DVR BciA (CT1063) gene of the green sulfur bacterium Chlorobium tepidum, which is homologous to AT5G18660. An enzymatic assay using a recombinant Arabidopsis DVR (AtDVR) on five DV substrates revealed that the major substrate of AtDVR is DV-Chlide a, while the other four DV substrates could not be converted to corresponding MV compounds (Nagata et al., 2007). Nevertheless, a recombinant BciA is able to reduce the 8-vinyl group of DV-Pchlide to generate MV-Pchlide (Chew and Bryant, 2007). Recently, we identified the rice (Oryza sativa) DVR encoded by Os03g22780 that has sequence similarity with the Arabidopsis DVR gene AT5G18660. We also confirmed that the recombinant rice DVR (OsDVR) is able to not only convert DV-Chlide a to MV-Chlide a but also to convert DV-Chl a to MV-Chl a (Wang et al., 2010). Thus, it is possible that the reductions of the 8-vinyl groups of various Chl biosynthetic intermediates are catalyzed by one enzyme of broad specificity.In this report, we extended our studies to four DVR proteins and five DV substrates. First, ZmDVR and CsDVR genes were isolated from maize and cucumber genomes, respectively, using a homology-based cloning approach. Second, enzymatic assays were systematically carried out using recombinant OsDVR, ZmDVR, CsDVR, and AtDVR as representative DVR proteins and using DV-Chl a, DV-Chlide a, DV-Pchlide a, DV-MPE, and DV-Mg-Proto as DV substrates. Third, we examined the in vivo accumulations of various Chl intermediates in rice, maize, and cucumber. Finally, we systematically investigated the in vivo accumulations of Chl and its various intermediates in the OsDVR (Os03g22780)-inactivated 824ys mutant of rice (Wang et al., 2010). The results strongly suggested that a single DVR protein with broad substrate specificity is responsible for reducing the 8-vinyl groups of various intermediate molecules of Chl biosynthesis in higher plants, but DVR proteins from different species could have diverse and differing substrate preferences even though they are homologous.     

Focus on Weed Control: A Novel Rice Cytochrome P450 Gene,CYP72A31, Confers Tolerance to Acetolactate Synthase-Inhibiting Herbicides in Rice and Arabidopsis

Target-site and non-target-site herbicide tolerance are caused by the prevention of herbicide binding to the target enzyme and the reduction to a nonlethal dose of herbicide reaching the target enzyme, respectively. There is little information on the molecular mechanisms involved in non-target-site herbicide tolerance, although it poses the greater threat in the evolution of herbicide-resistant weeds and could potentially be useful for the production of herbicide-tolerant crops because it is often involved in tolerance to multiherbicides. Bispyribac sodium (BS) is an herbicide that inhibits the activity of acetolactate synthase. Rice (Oryza sativa) of the indica variety show BS tolerance, while japonica rice varieties are BS sensitive. Map-based cloning and complementation tests revealed that a novel cytochrome P450 monooxygenase, CYP72A31, is involved in BS tolerance. Interestingly, BS tolerance was correlated with CYP72A31 messenger RNA levels in transgenic plants of rice and Arabidopsis (Arabidopsis thaliana). Moreover, Arabidopsis overexpressing CYP72A31 showed tolerance to bensulfuron-methyl (BSM), which belongs to a different class of acetolactate synthase-inhibiting herbicides, suggesting that CYP72A31 can metabolize BS and BSM to a compound with reduced phytotoxicity. On the other hand, we showed that the cytochrome P450 monooxygenase CYP81A6, which has been reported to confer BSM tolerance, is barely involved, if at all, in BS tolerance, suggesting that the CYP72A31 enzyme has different herbicide specificities compared with CYP81A6. Thus, the CYP72A31 gene is a potentially useful genetic resource in the fields of weed control, herbicide development, and molecular breeding in a broad range of crop species.The mechanism of herbicide tolerance can be classified roughly into two groups: target-site and non-target-site herbicide tolerance (Powles and Yu, 2010). Target-site herbicide tolerance is caused by the prevention of herbicide binding to the target enzyme, caused by point mutations occurring in the latter. It is relatively easy to elucidate the molecular mechanisms of target-site herbicide tolerance, because it is regulated mostly by a single gene encoding a target enzyme harboring point mutations. On the other hand, non-target-site herbicide tolerance is caused by reduction to a nonlethal dose of herbicide reaching the target enzyme, caused by mechanisms such as activation of herbicide detoxification, decrease of herbicide penetration, and herbicide compartmentation in plant cells (Yuan et al., 2007). Among these mechanisms, the oxidization of herbicides by endogenous cytochrome P450 monooxygenase is thought to be a major pathway in plants (Werck-Reichhart et al., 2000; Siminszky, 2006; Powles and Yu, 2010). From the point of view of weed control, non-target-site herbicide tolerance is a greater threat to crop production and in the evolution of herbicide-resistant weeds, because it is often involved in resistance to multiherbicides that inhibit different target proteins, including never-used and potential plant growth regulators (Yuan et al., 2007; Powles and Yu, 2010). Conversely, it is expected that multiherbicide-tolerant crops could be produced easily by the application of non-target-site herbicide tolerance. Moreover, information gained from study of the molecular mechanisms of non-target-site herbicide tolerance can be applied to the research and development of novel herbicides and plant growth regulators.Acetolactate synthase (ALS; also known as acetohydroxy acid synthase) plays a key role in the biosynthesis of branched-chain amino acids such as Val, Leu, and Ile in many organisms. ALS is the primary target site for at least four classes of herbicides: sulfonylurea, imidazolinone, pyrimidinyl carboxylates, and triazolopyrimidine herbicides (Shimizu et al., 2002, 2005). These herbicides can inhibit ALS activity, resulting in plant death caused by a deficiency of branched-chain amino acids. ALS-inhibiting herbicides control many weed species in addition to exhibiting high selectivity in major crops and low toxicity to mammals, which lack the branched-chain amino acid biosynthetic pathway. However, various mutations in ALS that confer ALS-inhibiting herbicide tolerance have been found in many weeds (Shimizu et al., 2005; Powles and Yu, 2010). Similar mutations in ALS have also been reported in crops (Shimizu et al., 2005). To date, crops that show tolerance to ALS-inhibiting herbicides have been produced by various approaches, such as conventional mutation breeding, conventional transformation, and pinpoint mutagenesis via gene targeting based on information obtained from analyses of ALS mutants (Shimizu et al., 2005; Endo and Toki, 2013). On the other hand, weeds that show tolerance to ALS-inhibiting herbicides by cytochrome P450-mediated detoxification have also been reported (Powles and Yu, 2010). However, compared with target-site herbicide tolerance, little is known of the molecular mechanism of herbicide metabolism mediated by cytochrome P450. In rice (Oryza sativa), an herbicide-sensitive mutant has been produced by γ-ray irradiation (Zhang et al., 2002). This mutant showed 60-fold higher sensitivity to bensulfuron-methyl (BSM), a sulfonylurea herbicide, compared with wild-type rice (Pan et al., 2006). Genetic mapping and complementation tests revealed that a cytochrome P450, CYP81A6, is involved in BSM tolerance (Pan et al., 2006). As far as we know, this is the only example of the isolation and characterization of a cytochrome P450 gene involved in nontarget herbicide tolerance in rice.Bispyribac sodium (BS), a pyrimidinyl carboxylate herbicide, is effective in controlling many annual and perennial weeds, with excellent selectivity on direct-seeded rice (Shimizu et al., 2002). Recently, it was reported that japonica rice varieties show higher sensitivity to BS compared with indica rice varieties at the early stages of plant growth (Ohno et al., 2008; Taniguchi et al., 2010). A mutated ALS gene confers BS tolerance in plants including rice (Shimizu et al., 2005; Endo and Toki, 2013). However, the deduced amino acid sequences were shown to be highly conserved among japonica and indica rice varieties, and ALS levels of sensitivity to BS were similar in japonica and indica rice varieties (Taniguchi et al., 2010). These results suggest the possibility that indica rice varieties might show higher tolerance to BS due to the acquisition of nontarget herbicide tolerance.In this study, we isolated and characterized a novel cytochrome P450 gene, CYP72A31, involved in BS tolerance in rice. We also demonstrated that overexpression of CYP72A31 confers tolerance to ALS-inhibiting herbicides, including BS and BSM, in Arabidopsis (Arabidopsis thaliana).     

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.     

TAA1-Regulated Local Auxin Biosynthesis in the Root-Apex Transition Zone Mediates the Aluminum-Induced Inhibition of Root Growth in Arabidopsis

The transition zone (TZ) of the root apex is the perception site of Al toxicity. Here, we show that exposure of Arabidopsis thaliana roots to Al induces a localized enhancement of auxin signaling in the root-apex TZ that is dependent on TAA1, which encodes a Trp aminotransferase and regulates auxin biosynthesis. TAA1 is specifically upregulated in the root-apex TZ in response to Al treatment, thus mediating local auxin biosynthesis and inhibition of root growth. The TAA1-regulated local auxin biosynthesis in the root-apex TZ in response to Al stress is dependent on ethylene, as revealed by manipulating ethylene homeostasis via the precursor of ethylene biosynthesis 1-aminocyclopropane-1-carboxylic acid, the inhibitor of ethylene biosynthesis aminoethoxyvinylglycine, or mutant analysis. In response to Al stress, ethylene signaling locally upregulates TAA1 expression and thus auxin responses in the TZ and results in auxin-regulated root growth inhibition through a number of auxin response factors (ARFs). In particular, ARF10 and ARF16 are important in the regulation of cell wall modification–related genes. Our study suggests a mechanism underlying how environmental cues affect root growth plasticity through influencing local auxin biosynthesis and signaling.     

Distinct Cell Wall Architectures in Seed Endosperms in Representatives of the Brassicaceae and Solanaceae

In some species, a crucial role has been demonstrated for the seed endosperm during germination. The endosperm has been shown to integrate environmental cues with hormonal networks that underpin dormancy and seed germination, a process that involves the action of cell wall remodeling enzymes (CWREs). Here, we examine the cell wall architectures of the endosperms of two related Brassicaceae, Arabidopsis (Arabidopsis thaliana) and the close relative Lepidium (Lepidium sativum), and that of the Solanaceous species, tobacco (Nicotiana tabacum). The Brassicaceae species have a similar cell wall architecture that is rich in pectic homogalacturonan, arabinan, and xyloglucan. Distinctive features of the tobacco endosperm that are absent in the Brassicaceae representatives are major tissue asymmetries in cell wall structural components that reflect the future site of radicle emergence and abundant heteromannan. Cell wall architecture of the micropylar endosperm of tobacco seeds has structural components similar to those seen in Arabidopsis and Lepidium endosperms. In situ and biomechanical analyses were used to study changes in endosperms during seed germination and suggest a role for mannan degradation in tobacco. In the case of the Brassicaceae representatives, the structurally homogeneous cell walls of the endosperm can be acted on by spatially regulated CWRE expression. Genetic manipulations of cell wall components present in the Arabidopsis seed endosperm demonstrate the impact of cell wall architectural changes on germination kinetics.Angiosperms are a diverse group of seed plants that reproduce by a double fertilization event; the first produces a zygote and the second a specialized nutritive tissue known as the endosperm. The endosperm and the maternally derived testa (seed coat) evolved to protect the embryo until conditions are favorable for germination and establishment of the next generation (Rajjou and Debeaujon, 2008; Linkies et al., 2010). Endosperm from cereals/grasses, such as maize (Zea mays), barley (Hordeum vulgare), and wheat (Triticum aestivum), is vital for human and animal nutrition and is therefore of global economic importance (Olsen, 2007). In many seeds, such as some representatives of the Brassicaceae, the endosperm is entirely absent at seed maturity, the storage reserves having been absorbed by the cotyledons during embryo development. Arabidopsis (Arabidopsis thaliana) and Lepidium (Lepidium sativum) are notable exceptions in that they have retained a thin layer of endosperm tissue in the mature seed (Müller et al., 2006; Linkies and Leubner-Metzger, 2012).Some seeds exhibit primary dormancy at maturity that has been induced by abscisic acid (ABA; Hilhorst, 1995; Kucera et al., 2005). In its simplest sense, dormancy can be thought of as a block to germination of an intact viable seed under favorable conditions (Hilhorst, 1995; Bewley, 1997). A more sophisticated definition was proposed by Baskin and Baskin (2004), who state that a dormant seed does not have the capacity to germinate in a specified period of time under any combination of normal physical environmental factors that are otherwise favorable for its germination. Seed dormancy can be imposed by the embryo, the seed coat (including the endosperm), or a combination of both depending on the plant species (Bewley, 1997).The endosperm has been shown to be an important regulator of germination potential in several systems, including tomato (Solanum lycopersicum; Groot et al., 1988; Toorop et al., 2000), tobacco (Nicotiana tabacum; Leubner-Metzger et al., 1995; Petruzzelli et al., 2003), Arabidopsis (Bethke et al., 2007), and Lepidium (Müller et al., 2006; Linkies et al., 2009; Voegele et al., 2011). Arabidopsis continues to be an important model for elucidating the hormonal and genetic networks that regulate dormancy and germination (Kucera et al., 2005; Holdsworth et al., 2008), and new bioinformatic methods are providing insights into the evolutionary conservation of such networks in angiosperms (Bassel et al., 2011). Research using the close relative Lepidium, whose larger size makes it amenable to biomechanical techniques, has given insight into the hormonal control of endosperm weakening during germination and established that the mechanism of control is conserved between Arabidopsis, Lepidium, and tobacco (Müller et al., 2006; Linkies et al., 2009; Voegele et al., 2011). It has been reported that ABA is a key regulator of germination in tobacco, Arabidopsis, and Lepidium, controlling the process of endosperm rupture but not testa rupture (Leubner-Metzger et al., 1995; Petruzzelli et al., 2003; Müller et al., 2006). Microarray analyses of ABA-treated Arabidopsis and Lepidium seeds revealed that many cell wall remodeling enzyme (CWRE) genes are down-regulated upon exogenous application of ABA (Penfield et al., 2006; Linkies et al., 2009). Therefore, it follows that ABA impacts cell wall remodeling, which influences germination kinetics. The endosperm is therefore an important control tissue for seed germination and represents a useful model to investigate cell wall architectures and their remodeling.Cell walls are robust, multifunctional structures that not only protect cells from biotic and abiotic stresses, but also regulate growth, physiology and development (Albersheim et al., 2010). Cell walls are fibrous composites in which cellulose microfibrils are coextensive with/cross-linked by noncellulosic polysaccharides. In dicotyledonous plants, xyloglucan (XG) is a major polymer that can cross-link cellulose (Cosgrove, 2000). Load-bearing fibrous networks impart tensile strength to cell walls and are embedded in more soluble, gel-like matrices of pectic polysaccharides, glycoproteins, proteins, ions, and water. The constituent pectic polymers are currently classified as homogalacturonan (HG), rhamnogalacturonan I [RG-I; also comprising arabinans and type 1 (arabino)galactans as side branches] and rhamnogalacturonan II, and xylogalacturonan (XGA) (Willats et al., 2001; Caffall and Mohnen, 2009). Pectins are involved in a diverse range of processes, including the regulation of intercellular adhesion/cell separation at the middle lamella, regulating the ionic status, and the porosity of cell walls that influences the access of CWREs to substrates (Willats et al., 2001). Noncellulosic polysaccharides exhibit numerous structural elaborations and differ in their glycan, methyl, and acetyl substitution (Caffall and Mohnen, 2009; Burton et al., 2010). Such modifications have the potential to impact their functionality, including their ability to interact with other wall components and their susceptibility to degradation and modification by CWREs.Studies using Arabidopsis (Iglesias-Fernández et al., 2011), Lepidium (Morris et al., 2011), and tomato (Groot et al., 1988) have highlighted a role for endo-β-mannanases (EBMs), enzymes that degrade heteromannan polysaccharides, during seed germination. In hard seeds with heteromannan-rich endosperms, such as carob (Ceratonia siliqua), date (Phoenix dactylifera), Chinese senna (Senna obtusifolia), and fenugreek (Trigonella foenum-graecum), however, it has been proposed that thinner walls in the micropylar endosperm (ME) and not EBM activity are responsible for allowing radicle protrusion during germination (Gong et al., 2005). Therefore, enzymatic cell wall remodeling and native cell wall architectural asymmetries both have the potential to impact on germination.Although studies on the molecular networks controlling germination have indicated a role for several classes of CWREs in endosperm remodeling and the promotion of germination (Penfield et al., 2006; Kanai et al., 2010; Morris et al., 2011), there is a paucity of information relating to the characterization of such changes at the cell wall level and, indeed, cell wall structures themselves. This study focuses on the targets of CWRE genes currently thought to be involved in seed germination (i.e. cellulose, XG, heteromannan, and pectic polysaccharides). We show that all three seeds possess a similar core cell wall architecture containing unesterified HG, arabinan, and XG. In tobacco, the core cell wall architecture is restricted to the ME, whereas in Arabidopsis and Lepidium, this architecture is observed throughout the endosperm. A further unique feature of the tobacco endosperm is abundant heteromannan. We also outline, using Arabidopsis, to what extent cell wall components contribute to the regulation of seed germination.     

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.     

Identification of the Primary Lesion of Toxic Aluminum in Plant Roots

Despite the rhizotoxicity of aluminum (Al) being identified over 100 years ago, there is still no consensus regarding the mechanisms whereby root elongation rate is initially reduced in the approximately 40% of arable soils worldwide that are acidic. We used high-resolution kinematic analyses, molecular biology, rheology, and advanced imaging techniques to examine soybean (Glycine max) roots exposed to Al. Using this multidisciplinary approach, we have conclusively shown that the primary lesion of Al is apoplastic. In particular, it was found that 75 µm Al reduced root growth after only 5 min (or 30 min at 30 µm Al), with Al being toxic by binding to the walls of outer cells, which directly inhibited their loosening in the elongation zone. An alteration in the biosynthesis and distribution of ethylene and auxin was a second, slower effect, causing both a transient decrease in the rate of cell elongation after 1.5 h but also a longer term gradual reduction in the length of the elongation zone. These findings show the importance of focusing on traits related to cell wall composition as well as mechanisms involved in wall loosening to overcome the deleterious effects of soluble Al.Acid soils, in which soluble aluminum (Al) is elevated, comprise approximately 4 billion ha of the global ice-free land or approximately 40% of the world’s total arable land (Eswaran et al., 1997). Although it has been known for over a century that Al decreases plant root growth, the underlying reasons for its toxic effects remain elusive. In a highly cited review of literature, Horst et al. (2010) stated that the “mechanism of Al-induced inhibition of root elongation is still not well understood, and it is a matter of debate whether the primary lesions of Al toxicity are apoplastic or symplastic.” For example, in the symplast, Al has been reported to cause interference with DNA synthesis and mitosis, disrupt the function of the Golgi apparatus, damage membrane integrity, and inhibit mitochondrial functions. In the apoplast, Al may rigidify the cell wall (prevent loosening), inhibit cell wall enzymes, such as expansin, and cause cell rupturing (for review, see Rengel, 1997; Horst et al., 2010). The identification of numerous processes influenced by Al (such as those listed above) has occurred for a number of reasons. First, it is almost certain that Al does, indeed, have multiple mechanisms by which it reduces growth in both the short and long term. Second, there has been a lack of studies that have related the changes observed in these processes to the actual underlying changes in root elongation rate (RER). Thus, there typically has been no clear separation of the primary and secondary toxic effects of Al. Although some studies have examined the speed with which Al reduces RER, these studies have generally (1) been at comparatively coarse time steps and (2) not taken the additional step of relating these changes in RER to the underlying mechanism of toxicity (for example, see Llugany et al., 1995; Parker, 1995; Kidd et al., 2001; and Blamey et al., 2004).In this study, we used kinematic analyses as the basis for elucidating the underlying mechanisms by which Al exerts toxic effects on the growth of soybean (Glycine max) roots. First, after exposure to Al, we captured digital images every 0.5 to 1 min so as to calculate changes in overall RER with a resolution of 5 to 10 min. Second, we examined whether these changes in overall RER resulted from either or both (1) changes in the length of the root elongation zone (LEZ) or (2) changes in elemental elongation rate (EER), which is defined as the change in length per unit length of a small portion of tissue (Silk, 1992), as a measure of the rate at which individual cells elongate. Based upon these data, it seemed that Al is toxic by at least three separate but interrelated mechanisms. To provide additional information on these mechanisms, we: (1) used kinematic analyses to investigate the effects of aminoethoxyvinyl-Gly (AVG), an ethylene synthesis inhibitor; (2) examined changes in auxin distribution and movement in the root apex using a highly active synthetic auxin-response element (referred to as DR5) with a minimal promoter-GUS reporter gene (DR5::GUS); (3) investigated rapid changes in the mechanical properties of root cell walls using creep analysis; and (4) used synchrotron-based low-energy x-ray fluorescence spectromicroscopy (LEXRF) and high-resolution secondary ion mass spectroscopy (NanoSIMS) to examine the spatial distribution of Al on cellular and subcellular levels in roots exposed to Al for only 30 min. This integrated approach has allowed us to identify the sequence of processes whereby Al reduces the growth of soybean roots in the short term.     

Focus on Water: Polarity of Water Transport across Epidermal Cell Membranes in Tradescantia virginiana

Using the automated cell pressure probe, small and highly reproducible hydrostatic pressure clamp (PC) and pressure relaxation (PR) tests (typically, applied step change in pressure = 0.02 MPa and overall change in volume = 30 pL, respectively) were applied to individual Tradescantia virginiana epidermal cells to determine both exosmotic and endosmotic hydraulic conductivity (L_pOUT and L_pIN, respectively). Within-cell reproducibility of measured hydraulic parameters depended on the method used, with the PR method giving a lower average coefficient of variation (15.2%, 5.8%, and 19.0% for half-time, cell volume [V_o], and hydraulic conductivity [L_p], respectively) than the PC method (25.4%, 22.0%, and 24.2%, respectively). V_o as determined from PC and PR tests was 1.1 to 2.7 nL and in the range of optically estimated V_o values of 1.5 to 4.9 nL. For the same cell, V_o and L_p estimates were significantly lower (about 15% and 30%, respectively) when determined by PC compared with PR. Both methods, however, showed significantly higher L_pOUT than L_pIN (L_pOUT/L_pIN ≅ 1.20). Because these results were obtained using small and reversible hydrostatically driven flows in the same cell, the 20% outward biased polarity of water transport is most likely not due to artifacts associated with unstirred layers or to direct effects of externally applied osmotica on the membrane, as has been suggested in previous studies. The rapid reversibility of applied flow direction, particularly for the PR method, and the lack of a clear increase in L_pOUT/L_pIN over a wide range of L_p values suggest that the observed polarity is an intrinsic biophysical property of the intact membrane/protein complex.The conductivity of membranes to water (hydraulic conductivity [L_p]) is an important property of the cells of all organisms, and whether plant cell membranes exhibit a polarity in this property has been debated for a number of decades (Dainty and Hope, 1959; Steudle, 1993). Most early evidence for polarity was based on transcellular osmotic experiments using giant algal cells in the Characeae, in which the relative areas of cell membrane exposed to conditions of osmotic inflow (endosmosis) or outflow (exosmosis) could be varied and, hence, L_p for both directions determined (Tazawa and Shimmen, 2001). Interpretation of these experiments is complicated by unstirred layer (USL) effects (Dainty, 1963), but even after accounting for these, it was concluded that inflow L_p (L_pIN) was higher than outflow L_p (L_pOUT) in these cells, with L_pOUT/L_pIN of about 0.65 (Dainty, 1963). When using osmotic driving forces in algal cells, L_pOUT/L_pIN values of between 0.5 and 0.91 have been reported in many studies (Steudle and Zimmermann, 1974; Steudle and Tyerman, 1983; Tazawa et al., 1996), and the same direction of polarity was also reported using osmotic driving forces in whole roots of maize (Zea mays; Steudle et al., 1987). When applying hydrostatic driving forces in algal cells using the pressure probe (Steudle, 1993), which is less influenced by USL effects (Steudle et al., 1980), L_pOUT/L_pIN has been closer to 1 (0.83–1; Steudle and Zimmermann, 1974; Steudle and Tyerman, 1983). However, in higher plant cells, an analysis of the data presented by Steudle et al. (1980, 1982) and Tomos et al. (1981) indicates the opposite polarity, with L_pOUT/L_pIN averaging from 1.2 to 1.4. Moore and Cosgrove (1991) used two contrasting hydrostatic methods to measure L_p in sugarcane (Saccharum spp.) stem cells: (1) the most commonly used pressure relaxation (PR) method, in which cell turgor pressure (P_cell) changes during the measurement, and (2) the more technically demanding pressure clamp (PC) method, in which P_cell is maintained constant. Consistent with other studies in higher plant cells, Moore and Cosgrove (1991) reported average L_pOUT/L_pIN from 1.15 (PC) to 1.65 (PR). Using the PR method in epidermal cells of barley (Hordeum vulgare), Fricke (2000) reported only a modest L_pOUT/L_pIN (based on reported half-time [T_1/2]) of 1.08. In view of the contribution of proteins (e.g. aquaporins) to overall membrane L_p, Tyerman et al. (2002) suggested that polarity may result either from asymmetry in the pores themselves or from an active regulation of the conductive state of the pores in response to the experimental conditions that cause inflow or outflow. Either of these mechanisms may explain the wide range of values reported in the literature for L_pOUT/L_pIN. Cosgrove and Steudle (1981) reported that a substantial (6-fold) and rapid (within 20 s) reduction in L_p could occur in the same cell, and in hindsight, this presumably reflected the influence of aquaporins. Cosgrove and Steudle (1981) did not consider the lower L_p as indicative of the L_p in situ, and Wan et al. (2004) reported that a reduction in L_p was associated with perturbations to P_cell on the order of 0.1 MPa. Hence, if measured membrane L_p itself can exhibit substantial changes over relatively short periods of time in the same cell, then further study of systematic differences between L_pOUT and L_pIN will require a robust hydrostatic methodology (PC or PR) that can reversibly and reproducibly apply small perturbations in pressure (P) to individual cells over short periods of time.For the PR method, a T_1/2 of water exchange is measured by fitting an exponential curve to the observed decay in P_cell over time following a step change in volume, and membrane L_p can be calculated if cell surface area (A), cell volume (V_o), and volumetric elastic modulus (ε) are known (Steudle, 1993). In practice, A and V_o are typically calculated from optical measurements of individual cell dimensions or estimates using average values, and ε is calculated based on V_o and an empirical change in pressure (dP) to change in volume (dV) relation for each cell (Steudle, 1993; Tomos and Leigh, 1999). In the PC method, first developed by Wendler and Zimmermann (1982), V_o (and, given reasonable assumptions about cell geometry, A) is estimated without the need for optical measurements, and L_p can be measured without the need to determine dP/dV or ε. However, this method is technically more demanding because it requires precise P control as well as a continuous record of the volume flow of water across the cell membrane (as measured by changes in the position of the cell solution/oil meniscus within the glass capillary over time) and has rarely been used (Wendler and Zimmermann, 1982, 1985; Cosgrove et al., 1987; Moore and Cosgrove, 1991; Zhang and Tyerman, 1991; Murphy and Smith, 1998). Since volume (V) is continuously changing over time, this approach may also be influenced by the hydraulic conductance of the capillary tip (K_h) used to make the measurements as well as surface tension effects due to the progressive changes in capillary diameter with meniscus position, and these influences have not been quantitatively addressed.Automation of the pressure probe operation, particularly automatic tracking of the meniscus location in the glass microcapillary tip, would address many of the above-mentioned issues, and to date, several attempts have been made to monitor the meniscus location using electrical resistance (Hüsken et al., 1978) or hardware-based image analysis (Cosgrove and Durachko, 1986; Murphy and Smith, 1998). Recently, Wong et al. (2009) redesigned the automated cell pressure probe (ACPP), originally proposed by Cosgrove and Durachko (1986), using a software-based meniscus detection system and a precise pressure control system. In the new ACPP system, both the position of the meniscus and oil pressure (P_oil) are recorded frequently (typically at 10 Hz), and P_oil is controlled with a resolution of ±0.002 MPa. We have combined the ACPP with a new technique to reproducibly fabricate microcapillary tips of known hydraulic properties (Wada et al., 2011) in order to correct for K_h and surface tension effects in both PC and PR estimates of the water relations parameters of Tradescantia virginiana epidermal cells and have determined the relation of L_pOUT to L_pIN in these cells.     

Identification of a Hydrolyzable Tannin,Oenothein B,as an Aluminum-Detoxifying Ligand in a Highly Aluminum-Resistant Tree,Eucalyptus camaldulensis

Eucalyptus camaldulensis is a tree species in the Myrtaceae that exhibits extremely high resistance to aluminum (Al). To explore a novel mechanism of Al resistance in plants, we examined the Al-binding ligands in roots and their role in Al resistance of E. camaldulensis. We identified a novel type of Al-binding ligand, oenothein B, which is a dimeric hydrolyzable tannin with many adjacent phenolic hydroxyl groups. Oenothein B was isolated from root extracts of E. camaldulensis by reverse-phase high-performance liquid chromatography and identified by nuclear magnetic resonance and mass spectrometry analyses. Oenothein B formed water-soluble or -insoluble complexes with Al depending on the ratio of oenothein B to Al and could bind at least four Al ions per molecule. In a bioassay using Arabidopsis (Arabidopsis thaliana), Al-induced inhibition of root elongation was completely alleviated by treatment with exogenous oenothein B, which indicated the capability of oenothein B to detoxify Al. In roots of E. camaldulensis, Al exposure enhanced the accumulation of oenothein B, especially in EDTA-extractable forms, which likely formed complexes with Al. Oenothein B was localized mostly in the root symplast, in which a considerable amount of Al accumulated. In contrast, oenothein B was not detected in three Al-sensitive species, comprising the Myrtaceae tree Melaleuca bracteata, Populus nigra, and Arabidopsis. Oenothein B content in roots of five tree species was correlated with their Al resistance. Taken together, these results suggest that internal detoxification of Al by the formation of complexes with oenothein B in roots likely contributes to the high Al resistance of E. camaldulensis.Aluminum (Al) toxicity is a major factor that limits plant growth in acid soils and affects approximately 30% of the total ice-free land area of the world (von Uexküll and Mutert, 1995). Although Al in soils exist in nonphytotoxic silicate or oxide forms at neutral pH, it is solubilized into a phytotoxic form, mainly as Al³⁺, at a pH of less than 5 (Kinraide, 1991; Kochian, 1995). The accumulation of Al in root tips causes rapid inhibition of root elongation, which is a characteristic symptom of Al toxicity in plants (Delhaize and Ryan, 1995; Ma, 2007). In general, plants exhibit an inhibition of root elongation as early as 30 to 120 min after exposure to excessive Al (Barceló and Poschenrieder, 2002). Inhibition of root elongation leads to decreased water and nutrient uptake and, eventually, to restriction of growth of the whole plant.Plants have evolved different levels of Al resistance mediated by two distinct classes of mechanisms (Kochian et al., 2004; Ma, 2007). One strategy is the exclusion of Al from the root tips (exclusion mechanism), and the other is tolerance to Al that enters the root tips (internal tolerance mechanism). The secretion of organic acid anions from roots in response to exposure to Al is the best-documented mechanism for Al exclusion. Organic acid anions (i.e. malate, citrate, and oxalate) can form a complex with Al in the rhizosphere and thereby prevent Al from entering the root tips. The genes encoding transporters for the Al-induced secretion of malate and citrate have been identified and characterized in several plant species (Ryan et al., 2011; Delhaize et al., 2012). Organic acid anions also play a role in the detoxification of Al that enters the roots by means of internal formation of complexes with Al (Ma et al., 1998). However, findings in recent studies increasingly suggest that the Al resistance of some plant species and cultivars cannot be explained solely by these two functions of organic acid anions (Wenzl et al., 2001, 2002; Piñeros et al., 2005; Zheng et al., 2005; Famoso et al., 2010). In addition to organic acid anions, flavonoid-type phenolics (Kidd et al., 2001), phenolic compounds (Ofei-Manu et al., 2001), cyclic hydroxamates (Poschenrieder et al., 2005), and proanthocyanidins (Osawa et al., 2011) in roots or root exudates are proposed as potential organic ligands for Al. The mechanisms by which these additional ligands confer Al resistance remain poorly understood.Eucalyptus camaldulensis is an evergreen tree belonging to the Myrtaceae family and is cultivated in tropical and subtropical regions of the world on account of its superior growth, broad adaptability, and multipurpose wood properties. E. camaldulensis can grow in acid soils and even in acid sulfate soils, where the pH is often lower than 3.5 and the Al concentration in the soil solution often reaches the millimolar level (van Breemen and Pons, 1978). Indeed, seedlings of this species show no inhibition of root elongation and plant growth when exposed to 1 mm Al for 20 d under hydroponic conditions (Tahara et al., 2005). Such Al resistance is considerably higher than that reported for a variety of herbaceous crops and model plants in studies of Al resistance mechanisms; such plants exhibit an inhibition of root elongation at 1 to 50 μm Al (Wenzl et al., 2001). Although our understanding of Al resistance mechanisms in some crops and model plants has improved recently, that for extremely Al-resistant species such as E. camaldulensis is limited.In E. camaldulensis, citrate secretion from roots and its content in the root tips are increased by exposure to Al, suggesting that citrate may contribute to its Al resistance (Tahara et al., 2008a). However, the amounts of organic acid anions, including citrate, secreted from roots and contained within the root tips are lower than those of more sensitive species (Tahara et al., 2008a). Therefore, the high Al resistance of E. camaldulensis cannot be explained only by the presence of organic acid anions. Roots of E. camaldulensis can accumulate large amounts of Al (11 mg g⁻¹ dry weight) with no symptoms of Al toxicity (Tahara et al., 2005), suggesting the existence of additional mechanisms for internal tolerance. In this study, we investigated the presence of novel Al-binding ligands other than organic acid anions in E. camaldulensis roots and identified a hydrolyzable tannin, oenothein B, as a novel type of Al-binding ligand. We also examined the role of the ligand in the internal Al tolerance of E. camaldulensis.     

Structural Studies of Cinnamoyl-CoA Reductase and Cinnamyl-Alcohol Dehydrogenase,Key Enzymes of Monolignol Biosynthesis

The enzymes cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the two key reduction reactions in the conversion of cinnamic acid derivatives into monolignol building blocks for lignin polymers in plant cell walls. Here, we describe detailed functional and structural analyses of CCRs from Medicago truncatula and Petunia hybrida and of an atypical CAD (CAD2) from M. truncatula. These enzymes are closely related members of the short-chain dehydrogenase/reductase (SDR) superfamily. Our structural studies support a reaction mechanism involving a canonical SDR catalytic triad in both CCR and CAD2 and an important role for an auxiliary cysteine unique to CCR. Site-directed mutants of CAD2 (Phe226Ala and Tyr136Phe) that enlarge the phenolic binding site result in a 4- to 10-fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2. This finding demonstrates the potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants. Thermal denaturation measurements and structural comparisons of various liganded and unliganded forms of CCR and CAD2 highlight substantial conformational flexibility of these SDR enzymes, which plays an important role in the establishment of catalytically productive complexes of the enzymes with their NADPH and phenolic substrates.     

Xyloglucan Deficiency Disrupts Microtubule Stability and Cellulose Biosynthesis in Arabidopsis,Altering Cell Growth and Morphogenesis

Xyloglucan constitutes most of the hemicellulose in eudicot primary cell walls and functions in cell wall structure and mechanics. Although Arabidopsis (Arabidopsis thaliana) xxt1 xxt2 mutants lacking detectable xyloglucan are viable, they display growth defects that are suggestive of alterations in wall integrity. To probe the mechanisms underlying these defects, we analyzed cellulose arrangement, microtubule patterning and dynamics, microtubule- and wall-integrity-related gene expression, and cellulose biosynthesis in xxt1 xxt2 plants. We found that cellulose is highly aligned in xxt1 xxt2 cell walls, that its three-dimensional distribution is altered, and that microtubule patterning and stability are aberrant in etiolated xxt1 xxt2 hypocotyls. We also found that the expression levels of microtubule-associated genes, such as MAP70-5 and CLASP, and receptor genes, such as HERK1 and WAK1, were changed in xxt1 xxt2 plants and that cellulose synthase motility is reduced in xxt1 xxt2 cells, corresponding with a reduction in cellulose content. Our results indicate that loss of xyloglucan affects both the stability of the microtubule cytoskeleton and the production and patterning of cellulose in primary cell walls. These findings establish, to our knowledge, new links between wall integrity, cytoskeletal dynamics, and wall synthesis in the regulation of plant morphogenesis.The primary walls of growing plant cells are largely constructed of cellulose and noncellulosic matrix polysaccharides that include hemicelluloses and pectins (Carpita and Gibeaut, 1993; Somerville et al., 2004; Cosgrove, 2005). Xyloglucan (XyG) is the most abundant hemicellulose in the primary walls of eudicots and is composed of a β-1,4-glucan backbone with side chains containing Xyl, Gal, and Fuc (Park and Cosgrove, 2015). XyG is synthesized in the Golgi apparatus before being secreted to the apoplast, and its biosynthesis requires several glycosyltransferases, including β-1,4-glucosyltransferase, α-1,6-xylosyltransferase, β-1,2-galactosyltransferase, and α-1,2-fucosyltransferase activities (Zabotina, 2012). Arabidopsis (Arabidopsis thaliana) XYLOGLUCAN XYLOSYLTRANSFERASE1 (XXT1) and XXT2 display xylosyltransferase activity in vitro (Faik et al., 2002; Cavalier and Keegstra, 2006), and strikingly, no XyG is detectable in the walls of xxt1 xxt2 double mutants (Cavalier et al., 2008; Park and Cosgrove, 2012a), suggesting that the activity of XXT1 and XXT2 are required for XyG synthesis, delivery, and/or stability.Much attention has been paid to the interactions between cellulose and XyG over the past 40 years. Currently, there are several hypotheses concerning the nature of these interactions (Park and Cosgrove, 2015). One possibility is that XyGs bind directly to cellulose microfibrils (CMFs). Recent data indicating that crystalline cellulose cores are surrounded with hemicelluloses support this hypothesis (Dick-Pérez et al., 2011). It is also possible that XyG acts as a spacer-molecule to prevent CMFs from aggregating in cell walls (Anderson et al., 2010) or as an adapter to link cellulose with other cell wall components, such as pectin (Cosgrove, 2005; Cavalier et al., 2008). XyG can be covalently linked to pectin (Thompson and Fry, 2000; Popper and Fry, 2005, 2008), and NMR data demonstrate that pectins and cellulose might interact to a greater extent than XyG and cellulose in native walls (Dick-Pérez et al., 2011). Alternative models exist for how XyG-cellulose interactions influence primary wall architecture and mechanics. One such model posits that XyG chains act as load-bearing tethers that bind to CMFs in primary cell walls to form a cellulose-XyG network (Carpita and Gibeaut, 1993; Pauly et al., 1999; Somerville et al., 2004; Cosgrove, 2005). However, results have been accumulating against this tethered network model, leading to an alternative model in which CMFs make direct contact, in some cases mediated by a monolayer of xyloglucan, at limited cell wall sites dubbed “biomechanical hotspots,” which are envisioned as the key sites of cell wall loosening during cell growth (Park and Cosgrove, 2012a; Wang et al., 2013; Park and Cosgrove, 2015). Further molecular, biochemical, and microscopy experiments are required to help distinguish which aspects of the load-bearing, spacer/plasticizer, and/or hotspot models most accurately describe the functions of XyG in primary walls.Cortical microtubules (MTs) direct CMF deposition by guiding cellulose synthase complexes in the plasma membrane (Baskin et al., 2004; Paredez et al., 2006; Emons et al., 2007; Sánchez-Rodriguez et al., 2012), and the patterned deposition of cellulose in the wall in turn can help determine plant cell anisotropic growth and morphogenesis (Baskin, 2005). Disruption of cortical MTs by oryzalin, a MT-depolymerizing drug, alters the alignment of CMFs, suggesting that MTs contribute to CMF organization (Baskin et al., 2004). CELLULOSE SYNTHASE (CESA) genes, including CESA1, CESA3, and CESA6, are required for normal CMF synthesis in primary cell walls (Kohorn et al., 2006; Desprez et al., 2007), and accessory proteins such as COBRA function in cellulose production (Lally et al., 2001). Live-cell imaging from double-labeled YFP-CESA6; CFP-ALPHA-1 TUBULIN (TUA1) Arabidopsis seedlings provides direct evidence that cortical MTs determine the trajectories of cellulose synthesis complexes (CSCs) and patterns of cellulose deposition (Paredez et al., 2006). Additionally, MT organization affects the rotation of cellulose synthase trajectories in the epidermal cells of Arabidopsis hypocotyls (Chan et al., 2010). Recently, additional evidence for direct guidance of CSCs by MTs has been provided by the identification of CSI1/POM2, which binds to both MTs and CESAs (Bringmann et al., 2012; Li et al., 2012). MICROTUBULE ORGANIZATION1 (MOR1) is essential for cortical MT organization (Whittington et al., 2001), but disruption of cortical MTs in the mor1 mutant does not greatly affect CMF organization (Sugimoto et al., 2003), and oryzalin treatment does not abolish CSC motility (Paredez et al., 2006).Conversely, the organization of cortical MTs can be affected by cellulose synthesis. Treatment with isoxaben, a cellulose synthesis inhibitor, results in disorganized cortical MTs in tobacco cells, suggesting that inhibition of cellulose synthesis affects MT organization (Fisher and Cyr, 1998), and treatment with 2,6-dichlorobenzonitrile, another cellulose synthesis inhibitor, alters MT organization in mor1 plants (Himmelspach et al., 2003). Cortical MT orientation in Arabidopsis roots is also altered in two cellulose synthesis-deficient mutants, CESA6^52-isx and kor1-3, suggesting that CSC activity can affect MT arrays (Paredez et al., 2008). Together, these results point to a bidirectional relationship between cellulose synthesis/patterning and MT organization.MTs influence plant organ morphology, but the detailed mechanisms by which they do so are incompletely understood. The dynamics and stability of cortical MTs are also affected by MT-associated proteins (MAPs). MAP18 is a MT destabilizing protein that depolymerizes MTs (Wang et al., 2007), MAP65-1 functions as a MT crosslinker, and MAP70-1 functions in MT assembly (Korolev et al., 2005; Lucas et al., 2011). MAP70-5 stabilizes existing MTs to maintain their length, and its overexpression induces right-handed helical growth (Korolev et al., 2007); likewise, MAP20 overexpression results in helical cell twisting (Rajangam et al., 2008). CLASP promotes microtubule stability, and its mutant is hypersensitive to microtubule-destabilizing drug oryzalin (Ambrose et al., 2007). KATANIN1 (KTN1) is a MT-severing protein that can sever MTs into short fragments and promote the formation of thick MT bundles that ultimately depolymerize (Stoppin-Mellet et al., 2006), and loss of KTN1 function results in reduced responses to mechanical stress (Uyttewaal et al., 2012). In general, cortical MT orientation responds to mechanical signals and can be altered by applying force directly to the shoot apical meristem (Hamant et al., 2008). The application of external mechanical pressure to Arabidopsis leaves also triggers MT bundling (Jacques et al., 2013). Kinesins, including KINESIN-13A (KIN-13A) and FRAGILE FIBER1 (FRA1), have been implicated in cell wall synthesis (Cheung and Wu, 2011; Fujikura et al., 2014). The identification of cell wall receptors and sensors is beginning to reveal how plant cell walls sense and respond to external signals (Humphrey et al., 2007; Ringli, 2010); some of them, such as FEI1, FEI2, THESEUS1 (THE1), FERONIA (FER), HERCULES RECEPTOR KINASE1 (HERK1), WALL ASSOCIATED KINASE1 (WAK1), WAK2, and WAK4, have been characterized (Lally et al., 2001; Decreux and Messiaen, 2005; Kohorn et al., 2006; Xu et al., 2008; Guo et al., 2009; Cheung and Wu, 2011). However, the relationships between wall integrity, cytoskeletal dynamics, and wall synthesis have not yet been fully elucidated.In this study, we analyzed CMF patterning, MT patterning and dynamics, and cellulose biosynthesis in the Arabidopsis xxt1 xxt2 double mutant that lacks detectable XyG and displays altered growth (Cavalier et al., 2008; Park and Cosgrove, 2012a). To investigate whether and how XyG deficiency affects the organization of CMFs and cortical MTs, we observed CMF patterning in xxt1 xxt2 mutants and Col (wild-type) controls using atomic force microscopy (AFM), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), and confocal microscopy (Hodick and Kutschera, 1992; Derbyshire et al., 2007; Anderson et al., 2010; Zhang et al., 2014). We also generated transgenic Col and xxt1 xxt2 lines expressing GFP-MAP4 (Marc et al., 1998) and GFP-CESA3 (Desprez et al., 2007), and analyzed MT arrays and cellulose synthesis using live-cell imaging. Our results show that the organization of CMFs is altered, that MTs in xxt1 xxt2 mutants are aberrantly organized and are more sensitive to external mechanical pressure and the MT-depolymerizing drug oryzalin, and that cellulose synthase motility and cellulose content are decreased in xxt1 xxt2 mutants. Furthermore, real-time quantitative RT-PCR measurements indicate that the enhanced sensitivity of cortical MTs to mechanical stress and oryzalin in xxt1 xxt2 plants might be due to altered expression of MT-stabilizing and wall receptor genes. Together, these data provide insights into the connections between the functions of XyG in wall assembly, the mechanical integrity of the cell wall, cytoskeleton-mediated cellular responses to deficiencies in wall biosynthesis, and cell and tissue morphogenesis.