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.     

Salicylic Acid Regulates Arabidopsis Microbial Pattern Receptor Kinase Levels and Signaling

In Arabidopsis thaliana, responses to pathogen-associated molecular patterns (PAMPs) are mediated by cell surface pattern recognition receptors (PRRs) and include the accumulation of reactive oxygen species, callose deposition in the cell wall, and the generation of the signal molecule salicylic acid (SA). SA acts in a positive feedback loop with ACCELERATED CELL DEATH6 (ACD6), a membrane protein that contributes to immunity. This work shows that PRRs associate with and are part of the ACD6/SA feedback loop. ACD6 positively regulates the abundance of several PRRs and affects the responsiveness of plants to two PAMPs. SA accumulation also causes increased levels of PRRs and potentiates the responsiveness of plants to PAMPs. Finally, SA induces PRR- and ACD6-dependent signaling to induce callose deposition independent of the presence of PAMPs. This PAMP-independent effect of SA causes a transient reduction of PRRs and ACD6-dependent reduced responsiveness to PAMPs. Thus, SA has a dynamic effect on the regulation and function of PRRs. Within a few hours, SA signaling promotes defenses and downregulates PRRs, whereas later (within 24 to 48 h) SA signaling upregulates PRRs, and plants are rendered more responsive to PAMPs. These results implicate multiple modes of signaling for PRRs in response to PAMPs and SA.     

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.     

The SUD1 Gene Encodes a Putative E3 Ubiquitin Ligase and Is a Positive Regulator of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Arabidopsis

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity. We show that mutations in SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) gene recover most developmental defects in dry2 through changes in HMGR activity. SUD1 encodes a putative E3 ubiquitin ligase that shows sequence and structural similarity to yeast Degradation of α factor (Doα10) and human TEB4, components of the endoplasmic reticulum–associated degradation C (ERAD-C) pathway. While in yeast and animals, the alternative ERAD-L/ERAD-M pathway regulates HMGR activity by controlling protein stability, SUD1 regulates HMGR activity without apparent changes in protein content. These results highlight similarities, as well as important mechanistic differences, among the components involved in HMGR regulation in plants, yeast, and animals.     

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.     

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.     

Focus Issue on Calcium Signaling: Recent Advances in Calcium/Calmodulin-Mediated Signaling with an Emphasis on Plant-Microbe Interactions

Calcium/calmodulin-mediated signaling contributes in diverse roles in plant growth, development, and response to environmental stimuli.During calcium (Ca2+) signaling, decoding the stimulus-response coupling involves a set of Ca2+ sensor proteins or Ca2+-binding proteins (DeFalco et al., 2010a; Kudla et al., 2010). These proteins usually possess one or more classical helix-loop-helix elongation factor (EF) hand motifs. Three major types of Ca2+-sensor proteins in plants are calmodulin (CaM)/CaM-like proteins, calcium-dependent protein kinases (CDPKs), and calcineurin B-like proteins. As compared with animals, plant genomes encode more diversified Ca2+ sensors; with the exception of canonic CaM, all other types of Ca2+ sensors (CaM-like proteins, CDPKs, and calcineurin B-like proteins) are plant specific. The large population and unique structural composition of Ca2+-binding proteins and the diversity of the target proteins regulated by the Ca2+ sensors reflect the complexity of Ca2+ signaling, which helps plants adapt to the changing environment. This update will be limited primarily to discussions on CaM and CaM-binding proteins and the recent advances in Ca2+/CaM-mediated signaling.CaM is a conserved Ca2+-binding protein found in all eukaryotes. The discovery of CaM can be traced back to the 1970s. An activator of cyclic nucleotide phosphodiesterase was shown to be involved in the regulation of cAMP concentration, which was stimulated by Ca2+ (Kakiuchi and Yamazaki, 1970; Cheung, 1971). The activator was found to bind Ca2+ and was eventually named “calmodulin,” an abbreviation of Ca2+-modulated protein. Since its discovery over 40 years ago, CaM has been regarded as a model Ca2+-binding protein and has been subjected to intensive studies in biochemistry, cell biology, and molecular biology because of its importance in almost all aspects of cellular regulation (Poovaiah and Reddy, 1987, 1993; Bouche et al., 2005; DeFalco et al., 2010a; Du et al., 2011; Reddy et al., 2011b). Disruption or depletion of the single copy of the CaM gene in yeast (Saccharomyces cerevisiae) results in a recessive lethal mutation (Davis et al., 1986), suggesting that CaM has a critical role in eukaryotic cells.The structure of CaM has been well studied, and the prototype of CaM found in all eukaryotes has 149 amino acids with two globular domains, each containing two EF hands connected by a long flexible helix (Meador et al., 1993; Zhang et al., 1995; Yun et al., 2004; Ishida et al., 2009). As more and more genomes are sequenced, it is becoming clear that CaM belongs to a small gene family in plants. In the model plant Arabidopsis (Arabidopsis thaliana), seven CaM genes encode for four highly conserved isoforms (CaM1/4, CaM2/3/5, CaM6, and CaM7) that differ in only one to five amino acid residues. Loss-of-function mutations of individual CaMs indicate that the different CaMs may have overlapping yet different functions. For example, a loss of function in Arabidopsis AtCaM2 affects pollen germination (Landoni et al., 2010). Phenotypic analysis showed that in normal growth conditions, atcam2-2 plants were indistinguishable from the wild type, while genetic analysis showed a reduced transmission of the atcam2-2 allele through the male gametophyte, and in vitro pollen germination revealed a reduced level of germination in comparison with the wild type. However, the atcam3 knockout mutant showed a clear reduction in thermotolerance after heat treatment at 45°C for 50 min (Zhang et al., 2009). Overexpression of AtCaM3 in either the atcam3 knockout or wild-type background significantly rescued or increased the thermotolerance, respectively. Further analysis of individual CaM mutants under different stress conditions should reveal more on the functional significance of individual CaM genes.     

Microtubules Contribute to Tubule Elongation and Anchoring of Endoplasmic Reticulum,Resulting in High Network Complexity in Arabidopsis

The endoplasmic reticulum (ER) is a network of tubules and sheet-like structures in eukaryotic cells. Some ER tubules dynamically change their morphology, and others form stable structures. In plants, it has been thought that the ER tubule extension is driven by the actin-myosin machinery. Here, we show that microtubules also contribute to the ER tubule extension with an almost 20-fold slower rate than the actin filament-based ER extension. Treatment with the actin-depolymerizing drug Latrunculin B made it possible to visualize the slow extension of the ER tubules in transgenic Arabidopsis (Arabidopsis thaliana) plants expressing ER-targeted green fluorescent protein. The ER tubules elongated along microtubules in both directions of microtubules, which have a distinct polarity. This feature is similar to the kinesin- or dynein-driven ER tubule extension in animal cells. In contrast to the animal case, ER tubules elongating with the growing microtubule ends were not observed in Arabidopsis. We also found the spots where microtubules are stably colocalized with the ER subdomains during long observations of 1,040 s, suggesting that cortical microtubules contribute to provide ER anchoring points. The anchoring points acted as the branching points of the ER tubules, resulting in the formation of multiway junctions. The density of the ER tubule junction positively correlated with the microtubule density in both elongating cells and mature cells of leaf epidermis, showing the requirement of microtubules for formation of the complex ER network. Taken together, our findings show that plants use microtubules for ER anchoring and ER tubule extension, which establish fine network structures of the ER within the cell.The endoplasmic reticulum (ER) is a complex network composed of tubules and sheet structures. The ER network’s morphology changes dynamically by elongation and shrinkage of tubules, sheet expansion, and sliding junctions. For example, an ER tubule elongates straight forward from a cisterna and subsequently, fuses to another cisterna, producing a linkage between two cisternae. If an elongating tubule fails to fuse to another cisterna, the tubule contracts into the original cisterna. However, the ER has stable anchoring points that associate with other cellular structures, such as the plasma membrane or cytoskeleton. When an elongating ER tubule reaches an association point, it forms a stable ER anchor (i.e. establishment of the ER anchoring points forms stable ER tubules). Hence, increasing the number of ER anchoring points produces fine ER meshwork.ER dynamics in eukaryotes depend on the cytoskeleton. In plants, major contributors for ER organization are actin filaments (Quader et al., 1989; Knebel et al., 1990; Lichtscheidl and Hepler, 1996; Sparkes et al., 2009a) and the actin-associated motor proteins (myosins; Prokhnevsky et al., 2008; Peremyslov et al., 2010; Ueda et al., 2010). However, it had generally been thought that microtubules are not involved in ER organization in plants, because microtubule-depolymerizing drugs do not induce obvious changes in the ER network (Quader et al., 1989; Knebel et al., 1990; Lichtscheidl and Hepler, 1996; Sparkes et al., 2009a). Nevertheless, involvement of microtubules in plant ER organization has been suspected from several electron microscopy observations that showed microtubules located close to the ER membrane in Vicia faba guard cells, Nicotiana alata pollen tubes, and Funaria hygrometrica caulonemata (Lancelle et al., 1987; Hepler et al., 1990; McCauley and Hepler, 1992).Foissner et al. (2009) have suggested that microtubules are involved in motility and orientation of cortical ER in Characean algae (Nitella translucens, Nitella flexilis, Nitella hyalina, and Nitella pseudoflabellata) internodal cells. Characean cortical ER is spatially separated from inner cytoplasmic streaming by the middle layer of fixed chloroplasts. The cortical ER forms a tight meshwork of predominantly transverse ER tubules that frequently coalign with microtubules, and microtubule depolymerization reduces the transverse ER tubules and increases mesh size (Foissner et al., 2009). Consistently, Hamada et al. (2012) have shown in Arabidopsis (Arabidopsis thaliana) that microtubule depolymerization increases mesh size in young elongating cells. In addition, stable ER tubule junctions are often colocalized with cortical microtubules (Hamada et al., 2012), suggesting that microtubules stabilize ER tubule junctions to form fine ER meshes. Oryzalin-induced ER nodulation (Langhans et al., 2009) was not observed in our experimental conditions.Here, we showed that ER tubules elongate along microtubules in plant cells. In addition, we revealed that the ER is stably anchored to defined points on cortical microtubules. The stable anchoring points are the basis of various ER shapes, such as three-way, two-way, or dead-end ER tubules. These microtubule-ER interactions, together with the actin-myosin system, contribute to ER network organization.