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.     

The Chromatin-Remodeling Factor PICKLE Integrates Brassinosteroid and Gibberellin Signaling during Skotomorphogenic Growth in Arabidopsis

Plant cell elongation is controlled by endogenous hormones, including brassinosteroid (BR) and gibberellin (GA), and by environmental factors, such as light/darkness. The molecular mechanisms underlying the convergence of these signals that govern cell growth remain largely unknown. We previously showed that the chromatin-remodeling factor PICKLE/ENHANCED PHOTOMORPHOGENIC1 (PKL/EPP1) represses photomorphogenesis in Arabidopsis thaliana. Here, we demonstrated that PKL physically interacted with PHYTOCHROME-INTERACTING FACTOR3 (PIF3) and BRASSINAZOLE-RESISTANT1 (BZR1), key components of the light and BR signaling pathways, respectively. Also, this interaction promoted the association of PKL with cell elongation–related genes. We found that PKL, PIF3, and BZR1 coregulate skotomorphogenesis by repressing the trimethylation of histone H3 Lys-27 (H3K27me3) on target promoters. Moreover, DELLA proteins interacted with PKL and attenuated its binding ability. Strikingly, brassinolide and GA₃ inhibited H3K27me3 modification of histones associated with cell elongation–related loci in a BZR1- and DELLA-mediated manner, respectively. Our findings reveal that the PKL chromatin-remodeling factor acts as a critical node that integrates light/darkness, BR, and GA signals to epigenetically regulate plant growth and development. This work also provides a molecular framework by which hormone signals regulate histone modification in concert with light/dark environmental cues.     

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.     

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.     

Phospholipid:Diacylglycerol Acyltransferase Is a Multifunctional Enzyme Involved in Membrane Lipid Turnover and Degradation While Synthesizing Triacylglycerol in the Unicellular Green Microalga Chlamydomonas reinhardtii

Many unicellular microalgae produce large amounts (∼20 to 50% of cell dry weight) of triacylglycerols (TAGs) under stress (e.g., nutrient starvation and high light), but the synthesis and physiological role of TAG are poorly understood. We present detailed genetic, biochemical, functional, and physiological analyses of phospholipid:diacylglycerol acyltransferase (PDAT) in the green microalga Chlamydomonas reinhardtii, which catalyzes TAG synthesis via two pathways: transacylation of diacylglycerol (DAG) with acyl groups from phospholipids and galactolipids and DAG:DAG transacylation. We demonstrate that PDAT also possesses acyl hydrolase activities using TAG, phospholipids, galactolipids, and cholesteryl esters as substrates. Artificial microRNA silencing of PDAT in C. reinhardtii alters the membrane lipid composition, reducing the maximum specific growth rate. The data suggest that PDAT-mediated membrane lipid turnover and TAG synthesis is essential for vigorous growth under favorable culture conditions and for membrane lipid degradation with concomitant production of TAG for survival under stress. The strong lipase activity of PDAT with broad substrate specificity suggests that this enzyme could be a potential biocatalyst for industrial lipid hydrolysis and conversion, particularly for biofuel production.