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.     

Coexistence but Independent Biosynthesis of Catechyl and Guaiacyl/Syringyl Lignin Polymers in Seed Coats

Lignins are phenylpropanoid polymers, derived from monolignols, commonly found in terrestrial plant secondary cell walls. We recently reported evidence of an unanticipated catechyl lignin homopolymer (C lignin) derived solely from caffeyl alcohol in the seed coats of several monocot and dicot plants. We previously identified plant seeds that possessed either C lignin or traditional guaiacyl/syringyl (G/S) lignins, but not both. Here, we identified several dicot plants (Euphorbiaceae and Cleomaceae) that produce C lignin together with traditional G/S lignins in their seed coats. Solution-state NMR analyses, along with an in vitro lignin polymerization study, determined that there is, however, no copolymerization detectable (i.e., that the synthesis and polymerization of caffeyl alcohol and conventional monolignols in vivo is spatially and/or temporally separated). In particular, the deposition of G and C lignins in Cleome hassleriana seed coats is developmentally regulated during seed maturation; C lignin appears successively after G lignin within the same testa layers, concurrently with apparent loss of the functionality of O-methyltransferases, which are key enzymes for the conversion of C to G lignin precursors. This study exemplifies the flexible biosynthesis of different types of lignin polymers in plants dictated by substantial, but poorly understood, control of monomer supply by the cells.     

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

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

MTV1 and MTV4 Encode Plant-Specific ENTH and ARF GAP Proteins That Mediate Clathrin-Dependent Trafficking of Vacuolar Cargo from the Trans-Golgi Network

Many soluble proteins transit through the trans-Golgi network (TGN) and the prevacuolar compartment (PVC) en route to the vacuole, but our mechanistic understanding of this vectorial trafficking step in plants is limited. In particular, it is unknown whether clathrin-coated vesicles (CCVs) participate in this transport step. Through a screen for modified transport to the vacuole (mtv) mutants that secrete the vacuolar protein VAC2, we identified MTV1, which encodes an EPSIN N-TERMINAL HOMOLOGY protein, and MTV4, which encodes the ADP ribosylation factor GTPase-activating protein NEVERSHED/AGD5. MTV1 and NEV/AGD5 have overlapping expression patterns and interact genetically to transport vacuolar cargo and promote plant growth, but they have no apparent roles in protein secretion or endocytosis. MTV1 and NEV/AGD5 colocalize with clathrin at the TGN and are incorporated into CCVs. Importantly, mtv1 nev/agd5 double mutants show altered subcellular distribution of CCV cargo exported from the TGN. Moreover, MTV1 binds clathrin in vitro, and NEV/AGD5 associates in vivo with clathrin, directly linking these proteins to CCV formation. These results indicate that MTV1 and NEV/AGD5 are key effectors for CCV-mediated trafficking of vacuolar proteins from the TGN to the PVC in plants.     

MicroRNA Superfamilies Descended from miR390 and Their Roles in Secondary Small Interfering RNA Biogenesis in Eudicots

Trans-acting small interfering RNAs (tasiRNAs) are a major class of small RNAs performing essential biological functions in plants. The first reported tasiRNA pathway, that of miR173-TAS1/2, produces tasiRNAs regulating a set of pentatricopeptide repeat (PPR) genes and has been characterized only in Arabidopsis thaliana to date. Here, we demonstrate that the microRNA (miRNA)-trans-acting small interfering RNA gene (TAS)-pentatricopeptide repeat-containing gene (PPR)-small interfering RNA pathway is a highly dynamic and widespread feature of eudicots. Nine eudicot plants, representing six different plant families, have evolved similar tasiRNA pathways to initiate phased small interfering RNA (phasiRNA) production from PPR genes. The PPR phasiRNA production is triggered by different 22-nucleotide miRNAs, including miR7122, miR1509, and fve-PPRtri1/2, and through distinct mechanistic strategies exploiting miRNA direct targeting or indirect targeting through TAS-like genes (TASL), one-hit or two-hit, or even two layers of tasiRNA–TASL interactions. Intriguingly, although those miRNA triggers display high sequence divergence caused by the occurrence of frequent point mutations and splicing shifts, their corresponding MIRNA genes show pronounced identity to the Arabidopsis MIR173, implying a common origin of this group of miRNAs (super-miR7122). Further analyses reveal that super-miR7122 may have evolved from a newly defined miR4376 superfamily, which probably originated from the widely conserved miR390. The elucidation of this evolutionary path expands our understanding of the course of miRNA evolution, especially for relatively conserved miRNA families.     

Engineering Monolignol p-Coumarate Conjugates into Poplar and Arabidopsis Lignins

Lignin acylation, the decoration of hydroxyls on lignin structural units with acyl groups, is common in many plant species. Monocot lignins are decorated with p-coumarates by the polymerization of monolignol p-coumarate conjugates. The acyltransferase involved in the formation of these conjugates has been identified in a number of model monocot species, but the effect of monolignol p-coumarate conjugates on lignification and plant growth and development has not yet been examined in plants that do not inherently possess p-coumarates on their lignins. The rice (Oryza sativa) p-COUMAROYL-Coenzyme A MONOLIGNOL TRANSFERASE gene was introduced into two eudicots, Arabidopsis (Arabidopsis thaliana) and poplar (Populus alba × grandidentata), and a series of analytical methods was used to show the incorporation of the ensuing monolignol p-coumarate conjugates into the lignin of these plants. In poplar, specifically, the addition of these conjugates did not occur at the expense of the naturally incorporated monolignol p-hydroxybenzoates. Plants expressing the p-COUMAROYL-Coenzyme A MONOLIGNOL TRANSFERASE transgene can therefore produce monolignol p-coumarate conjugates essentially without competing with the formation of other acylated monolignols and without drastically impacting normal monolignol production.Lignification of plant cell walls prototypically involves the polymerization of the monolignols (MLs), p-coumaryl alcohol, coniferyl alcohol (CA), and sinapyl alcohol (SA), predominantly by stepwise radical coupling of each monomer to the phenolic end of the growing polymer (Sarkanen and Ludwig, 1971; Boerjan et al., 2003; Ralph et al., 2004). The contribution of various MLs to the lignins depends on plant species, cell type, plant tissue, and tissue age. Although the majority of the lignin polymer is derived from these three MLs, the lignification process has a high degree of metabolic plasticity (Boerjan et al., 2003; Ralph et al., 2004; Ralph, 2007; Vanholme et al., 2012). Of particular interest are ML conjugates in which the ester group can be acetate (Ac; Sarkanen et al., 1967; Ralph, 1996; Ralph and Lu, 1998; Del Río et al., 2007; del Río et al., 2008; Martínez et al., 2008), p-hydroxybenzoate (pBz; Venverloo, 1971; Monties and Lapierre, 1981; Landucci et al., 1992; Tomimura, 1992a, 1992b; Hibino et al., 1994; Sun et al., 1999; Kuroda et al., 2001; Lu et al., 2004, 2015; Morreel et al., 2004; Rencoret et al., 2013), p-coumarate (pCA; Monties and Lapierre, 1981; Ralph et al., 1994; Crestini and Argyropoulos, 1997; del Río et al., 2008, 2012a, 2012b; Withers et al., 2012; Rencoret et al., 2013; Petrik et al., 2014), or ferulate (FA; Grabber et al., 2008; Ralph, 2010; Wilkerson et al., 2014). In all cases, the MLs are acylated before polymerization as proven by the presence in the lignins of unique β-β coupling products that only arise when one or both of the MLs are acylated, preventing the formation of the typical resinols from internal trapping of the quinone methide intermediates by the γ-OH (Lu and Ralph, 2002, 2008; Del Río et al., 2007; Lu et al., 2015).The BAHD acyltransferase, FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT), was recently identified in Angelica sinensis and transformed into poplar (Populus alba × grandidentata), which naturally incorporates other acylated MLs, namely ML-pBz conjugates, into its lignin (Wilkerson et al., 2014). Plants that incorporate ML-FAs into their lignins have the potential to be particularly important economically, because their lignin backbones are permeated with readily cleavable ester bonds, facilitating lignin breakdown and removal under alkaline pretreatment conditions. Determining the extent to which ML-FAs are incorporated into the lignin polymer is, however, extremely difficult because of the diversity of products generated during the polymerization events, which is described in the supplemental information in Wilkerson et al., 2014.There is currently only one technique, derivatization followed by reductive cleavage (DFRC), that can release diagnostic chemical marker compounds from lignins containing ML-FAs (Lu and Ralph, 2014; Wilkerson et al., 2014). The DFRC method selectively cleaves β-ethers while leaving ester linkages intact. This technique was recently used to show that ML-FA conjugates are fully incorporated into the lignin of the FMT poplar (Wilkerson et al., 2014), but the extent of incorporation, the spatial distribution, the exact mechanism of delivery to the developing cell wall, and the efficiency of incorporation remain largely unknown.The biological role of pCA in lignin has been highly speculative. It is hypothesized that the pCA moieties may function as a radical sensitizer (Takahama and Oniki, 1996, 1997; Takahama et al., 1996; Ralph et al., 2004; Hatfield et al., 2008; Ralph, 2010). Peroxidases and/or laccases readily oxidize pCA to a radical but are poor oxidizers for SA. Free radicals of pCA readily undergo radical transfer to SA, which in turn, forms a homodimer or couples to the end of a growing polymer chain. Conjugating pCA to an ML, like SA, to form SA-pCA, the most prevalent ML-pCA conjugate in grasses, creates a compound with a built-in radical sensitizer that can participate in the polymerization event. The prevalence of these conjugates in potential biofuel crops and the impact that these ester-linked conjugates have on the lignin polymer during pretreatment and downstream fermentation processes have driven the search to find the genes and their enzymes responsible for acylating MLs in monocots (Withers et al., 2012; Marita et al., 2014; Petrik et al., 2014; Wilkerson et al., 2014).In rice (Oryza sativa), enzymes have been characterized that function specifically in the addition of pCA onto hemicelluloses (Bartley et al., 2013) or lignin (Withers et al., 2012; Petrik et al., 2014). The p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE (PMT) was identified as one of many grass-specific BAHD acyltransferases produced by rice and found to coexpress with many ML biosynthetic enzymes (Withers et al., 2012). The enzyme preferentially forms a γ-ester through its specificity toward p-coumaroyl-CoA and an ML, and has kinetic efficiency with p-coumaryl alcohol > SA > CA. In most grasses, the PMT enzyme predominantly produces SA-pCA conjugates that are then incorporated into the lignin polymer (Petrik et al., 2014).To test the role of PMT during cell wall lignification, genetic manipulation of PMT genes has been performed in Brachypodium distachyon and maize (Zea mays), two model monocots. The suppression and overexpression of a BdPMT revealed the PMT to be involved only in the acylation of MLs before polymerization and not in the acylation of hemicelluloses (Petrik et al., 2014). RNA interference-mediated suppression of BdPMT resulted in decreased incorporation of ML-pCA conjugates into the cell wall without adversely affecting growth, height, or digestibility of the mature plants. Even deleterious mutations in the BdPMT gene, which resulted in a complete absence of pCA-acylating B. distachyon lignins, did not affect plant growth or development (Petrik et al., 2014). The arabinose-bound FA and pCA levels remained virtually unchanged in the PMT-misregulated plants, illustrating the specificity of the PMT enzyme for the p-coumaroyl-CoA substrate and its ML acylation. The PMT enzyme identified in maize (pCAT = ZmPMT) also displayed the highest catalytic efficiency with p-coumaroyl-CoA and SA as substrates (Marita et al., 2014). RNAi-mediated suppression of ZmPMT also resulted in decreased production of the ML conjugates. The effect on the lignin polymer when introducing PMT into plants that do not normally express a homologous enzyme is, however, unknown.pCAs, because they favor radical transfer over radical coupling, are overwhelmingly seen as free-phenolic pendant entities on the lignin polymer (Ralph et al., 1994; Ralph, 2010). As a result, the pCA itself can be completely quantified by simple saponification. The units to which the pCA is attached are, like their normal ML-derived counterparts, not fully releasable from lignin as identifiable monomers (during degradative reactions), but the pCA’s terminal location makes p-coumaroylated units more readily releasable and detectable than if they participated in lignification (as FAs do). Examining the effect of PMT and its resulting conjugates on lignification in plants that do not naturally produce such conjugates will contribute to our understanding of the role of PMT in lignification in general.In this study, we aimed to assess the ability of the model eudicot plants Arabidopsis (Arabidopsis thaliana) and poplar, neither of which naturally produces ML-pCA conjugates, to express a PMT gene and incorporate these novel conjugates into their cell wall lignins. We also investigated the effect that the introduction of PMT has on the native levels of ML-pBz conjugates in poplar lignin. Various analytical techniques were optimized and used to examine the cell walls of the transgenic plants for pCA conjugates and determine whether they were specifically incorporated into the lignin polymer in the cell wall.     

KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis

Humans are unable to synthesize l-ascorbic acid (AsA), yet it is required as a cofactor in many critical biochemical reactions. The majority of human dietary AsA is obtained from plants. In Arabidopsis thaliana, a GDP-mannose pyrophosphorylase (GMPP), VITAMIN C DEFECTIVE1 (VTC1), catalyzes a rate-limiting step in AsA synthesis: the formation of GDP-Man. In this study, we identified two nucleotide sugar pyrophosphorylase-like proteins, KONJAC1 (KJC1) and KJC2, which stimulate the activity of VTC1. The kjc1kjc2 double mutant exhibited severe dwarfism, indicating that KJC proteins are important for growth and development. The kjc1 mutation reduced GMPP activity to 10% of wild-type levels, leading to a 60% reduction in AsA levels. On the contrary, overexpression of KJC1 significantly increased GMPP activity. The kjc1 and kjc1kjc2 mutants also exhibited significantly reduced levels of glucomannan, which is also synthesized from GDP-Man. Recombinant KJC1 and KJC2 enhanced the GMPP activity of recombinant VTC1 in vitro, while KJCs did not show GMPP activity. Yeast two-hybrid assays suggested that the stimulation of GMPP activity occurs via interaction of KJCs with VTC1. These results suggest that KJCs are key factors for the generation of GDP-Man and affect AsA level and glucomannan accumulation through the stimulation of VTC1 GMPP activity.     

Regulating the Redox Gatekeeper: Vacuolar Sequestration Puts Glutathione Disulfide in Its Place

The case is made for the potential importance of compartmentalization in redox signaling with new data on the transporters that may be involved.The nucleophilic properties of reduced glutathione (GSH) can be harnessed to produce glutathione S- (GS) conjugates or to reduce oxidants such as peroxides or dehydroascorbate (Dixon et al., 2009). When GSH is used as a reductant, glutathione disulfide (GSSG) is produced as a stable product from which the reduced form can be regenerated by NADPH-dependent glutathione reductase. In plants and some fungi, GS conjugates are imported into vacuoles, where they are degraded (Rea, 2007). Structurally, GSSG can be considered to be a glutathione S-autoconjugate, and this compound could also be transported into vacuoles. Indeed, in vitro studies show that isolated barley (Hordeum vulgare) vacuoles can take up GSSG and that certain Arabidopsis (Arabidopsis thaliana) tonoplast-localized proteins are competent in both GS conjugate and GSSG transport when expressed in yeast (Martinoia et al., 1993; Tommasini et al., 1993; Lu et al., 1997, 1998).Despite these observations, it remained unclear whether GSSG accumulation in the vacuole is ever a significant phenomenon in vivo. Evidence that this is the case comes from a study of a catalase-deficient Arabidopsis mutant (cat2; Queval et al., 2011). In this system, the decreased capacity for catalase-dependent hydrogen peroxide (H₂O₂) metabolism increases the oxidative burden on the cellular reducing system, triggering well-defined changes in tissue glutathione status that are qualitatively similar to those that can be driven by certain external stresses. In cat2 leaf extracts, the GSH-GSSG ratio is close to 1 (compared with over 20 in wild-type leaves) and glutathione is typically increased about 3-fold, so that leaf GSSG contents are much higher than in the wild type (Mhamdi et al., 2010). To date, in plants, as in other organisms, the cytosolic redox potential of glutathione, estimated using thiol-dependent redox-sensitive green fluorescent protein (roGFP), implies a very low concentration of GSSG in optimal conditions. Here, we propose that compartmentalization of GSSG can explain some of these apparently conflicting observations. Although the cytosolic glutathione pool is significantly increased in cat2, much more marked changes are observed in the chloroplast and, especially, the vacuole, where concentrations are increased at least 10 times compared with ecotype Columbia (Col-0; Queval et al., 2011).     

From Endoplasmic Reticulum to Mitochondria: Absence of the Arabidopsis ATP Antiporter Endoplasmic Reticulum Adenylate Transporter1 Perturbs Photorespiration

The carrier Endoplasmic Reticulum Adenylate Transporter1 (ER-ANT1) resides in the endoplasmic reticulum (ER) membrane and acts as an ATP/ADP antiporter. Mutant plants lacking ER-ANT1 exhibit a dwarf phenotype and their seeds contain reduced protein and lipid contents. In this study, we describe a further surprising metabolic peculiarity of the er-ant1 mutants. Interestingly, Gly levels in leaves are immensely enhanced (26×) when compared with that of wild-type plants. Gly accumulation is caused by significantly decreased mitochondrial glycine decarboxylase (GDC) activity. Reduced GDC activity in mutant plants was attributed to oxidative posttranslational protein modification induced by elevated levels of reactive oxygen species (ROS). GDC activity is crucial for photorespiration; accordingly, morphological and physiological defects in er-ant1 plants were nearly completely abolished by application of high environmental CO₂ concentrations. The latter observation demonstrates that the absence of ER-ANT1 activity mainly affects photorespiration (maybe solely GDC), whereas basic cellular metabolism remains largely unchanged. Since ER-ANT1 homologs are restricted to higher plants, it is tempting to speculate that this carrier fulfils a plant-specific function directly or indirectly controlling cellular ROS production. The observation that ER-ANT1 activity is associated with cellular ROS levels reveals an unexpected and critical physiological connection between the ER and other organelles in plants.