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.     

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 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.     

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.     

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.     

Xyloglucan Metabolism Differentially Impacts the Cell Wall Characteristics of the Endosperm and Embryo during Arabidopsis Seed Germination

Cell wall remodeling is an essential mechanism for the regulation of plant growth and architecture, and xyloglucans (XyGs), the major hemicellulose, are often considered as spacers of cellulose microfibrils during growth. In the seed, the activity of cell wall enzymes plays a critical role in germination by enabling embryo cell expansion leading to radicle protrusion, as well as endosperm weakening prior to its rupture. A screen for Arabidopsis (Arabidopsis thaliana) mutants affected in the hormonal control of germination identified a mutant, xyl1, able to germinate on paclobutrazol, an inhibitor of gibberellin biosynthesis. This mutant also exhibited reduced dormancy and increased resistance to high temperature. The XYL1 locus encodes an α-xylosidase required for XyG maturation through the trimming of Xyl. The xyl1 mutant phenotypes were associated with modifications to endosperm cell wall composition that likely impact on its resistance, as further demonstrated by the restoration of normal germination characteristics by endosperm-specific XYL1 expression. The absence of phenotypes in mutants defective for other glycosidases, which trim Gal or Fuc, suggests that XYL1 plays the major role in this process. Finally, the decreased XyG abundance in hypocotyl longitudinal cell walls of germinating embryos indicates a potential role in cell wall loosening and anisotropic growth together with pectin de-methylesterification.Seed germination is a complex process that begins with the absorption of water and ends when the radicle breaks through the seed coat (or testa). In Arabidopsis (Arabidopsis thaliana), as in most angiosperms, the embryo is surrounded by the triploid endosperm and the seed coat of maternal origin (Nonogaki et al., 2010; North et al., 2010). The completion of germination requires the growth potential of the embryo to overcome the resistance of the endosperm and testa layers, which is controlled by the hormonal balance between abscisic acid (ABA) and gibberellins (GAs). During seed development, ABA induces embryo growth arrest at the transition from embryogenesis to the phase of reserve accumulation and then induces primary dormancy, thus preventing vivipary and allowing seed dispersal in a dormant state. Dormancy delays germination until environmental conditions become favorable for seedling survival and growth (Finkelstein et al., 2008; Nambara et al., 2010; Graeber et al., 2012). Dormancy depth varies among plant species and between Arabidopsis accessions; however, seed dormancy of the most commonly used accession Columbia-0 (Col-0) is relatively low and can be released by a few weeks of after-ripening (dry storage) or stratification (cold imbibition). Shortly after hydration, ABA is rapidly degraded in both dormant and nondormant seeds, but ABA catabolism is more active in nondormant seeds, leading to lower ABA levels and thus allowing GA activation of germination processes (Millar et al., 2006). GA increases the elasticity of the wall, thereby reducing the resistance of the endosperm while triggering the elongation of the hypocotyl (Nonogaki et al., 2010). Radicle protrusion through the micropylar endosperm is also stimulated by ethylene, which has an antagonist action with ABA on endosperm cap weakening (Linkies and Leubner-Metzger, 2012). Microarray analyses highlighted the importance of cell wall remodeling processes during germination in various species (Penfield et al., 2006; Morris et al., 2011; Endo et al., 2012; Martínez-Andújar et al., 2012; Dekkers et al., 2013). These studies provided compelling evidence that the tissue-specific expression of genes encoding cell wall biosynthesis or modification enzymes, and their differential response to hormonal signals in the endosperm and embryo, influences the rate of germination.Cell walls are constituted of crystalline cellulose microfibrils that are embedded in an amorphous matrix of complex polysaccharides: pectin and hemicelluloses. Xyloglucan (XyG) is the major hemicellulose polymer in the primary cell walls of gymnosperms and most angiosperms, and its binding to cellulose microfibrils by hydrogen bonding contributes to loosening or stiffening of the wall during cell elongation (Cosgrove, 2005). XyG chains can be cleaved and reconnected by endo-transglycosylases/hydrolases (XTH). Other families of proteins also act on XyG chains, such as expansins, which are thought to nonenzymatically modulate XyG interactions with cellulose microfibrils, thereby controlling the distance between the microfibrils. XyG has a backbone of (1→4)-linked β-d-glucopyranosyl residues, which can be substituted with α-d-xylopyranosyl residues at O-6 (Supplemental Fig. S1). The pattern of XyG substitutions is described using a single-letter nomenclature (Fry et al., 1993). The letter G is used for an unsubstituted Glc and X when it is substituted with a Xyl. In Arabidopsis, like in many other dicots, the xylosylation pattern is in general regular, consisting mainly of XXXG-type units. The xylosyl residue can be further substituted at O-2 with a β-galactosyl (L side chain), which in turn can be substituted at O-2 with α-l-fucosyl (F side chain).Many of the biosynthetic enzymes involved in XyG biosynthesis have been identified, including a glucan synthase, xylosyl, galactosyl, and fucosyltransferases (Scheller and Ulvskov, 2010). Among these, two xylosyltransferases, named XXT1 and XXT2, have been shown to be involved in the synthesis of XyG in Arabidopsis, and the double mutant xxt1 xxt2 lacks detectable XyG (Cavalier et al., 2008). Both belong to the GT34 subfamily of glycosyltransferases, and a third enzyme, XXT5 from a separate clade of GT34, may also be involved in XyG synthesis (Zabotina et al., 2008). These glycosyltransferases are Golgi-localized enzymes, which produce substituted XyG precursors that are secreted into the cell wall. Subsequent trimming of XyG chains is performed by apoplastic glycosidases and determines hemicellulose structure and properties in the wall (Scheller and Ulvskov, 2010). A number of genes involved in the XyG metabolism have been identified, including XYL1, BGAL10, and AXY8 encoding, respectively, α-xylosidase, β-galactosidase, and α-fucosidase (Sampedro et al., 2010; Günl et al., 2011; Günl and Pauly, 2011; Sampedro et al., 2012). Loss of function of these glycosidases results in significant alterations in XyG composition. Although XyG has been proposed to be a major player in cell wall extension and plant growth, mutants with altered XyG composition display only minor growth-related phenotypes. The XyG-deficient double mutant xxt1 xxt2 shows no major growth defect except for deformed root hairs (Cavalier et al., 2008). Nevertheless, it was recently reported that the production of Gal-depleted XyG causes dwarfism in the galactosyltransferase mutant mur3 (Kong et al., 2015) in contrast to xyl1 and bgal10, where increased galactosylation results in shorter but wider siliques (Sampedro et al., 2010; Günl and Pauly, 2011; Sampedro et al., 2012). Phenotypes have not been observed from either reduced or increased fucosylation in the fucosyltransferase mutant mur2 and fucosidase mutant axy8 (Vanzin et al., 2002; Günl et al., 2011). AXY8 overexpression does, however, restore hypocotyl elongation in dwarf AUXIN BINDING PROTEIN1 knockdown seedlings. This demonstrates that in muro removal of Fuc residues can modulate cell elongation (Paque et al., 2014).In contrast to the numerous studies on the impact of XyG composition on plant growth, little information is available on the role of XyG in seed development or germination. A recent study highlighted the slower germination rate of xxt1 xxt2 mutant seeds compared to wild type, whereas germination rates of the arabinan-deficient arad1 arad2 and putative pectin methyltransferase qua2 mutants were not affected (Lee et al., 2012). As mentioned above, XyG chain hydrolysis and linkage is catalyzed by XTH activities, one of which, AtXTH31/XTR8, is encoded by an endosperm-specific gene. Loss of function leads to faster germination, suggesting that AtXTH31/XTR8 is involved in the reinforcement of the cell wall of the endosperm during germination (Endo et al., 2012). Here, we report the identification of an additional xyl1 allele from a screen designed to isolate mutants impaired in the hormonal control of germination, based on their ability to germinate on the GA biosynthesis inhibitor paclobutrazol. To investigate the role of XyG metabolism in seed dormancy and germination characteristics, xyl1 seed phenotypes were correlated with spatio-temporal XyG accumulation during seed development and germination. Comparative studies using mutants impaired in two other apoplastic glycosidases, BGAL10 and AXY8, indicate a major role for XYL1 in XyG remodelling processes that affect germination.     

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.