CELLULOSE SYNTHASE-LIKE A2, a Glucomannan Synthase,Is Involved in Maintaining Adherent Mucilage Structure in Arabidopsis Seed

Mannans are hemicellulosic polysaccharides that are considered to have both structural and storage functions in the plant cell wall. However, it is not yet known how mannans function in Arabidopsis (Arabidopsis thaliana) seed mucilage. In this study, CELLULOSE SYNTHASE-LIKE A2 (CSLA2; At5g22740) expression was observed in several seed tissues, including the epidermal cells of developing seed coats. Disruption of CSLA2 resulted in thinner adherent mucilage halos, although the total amount of the adherent mucilage did not change compared with the wild type. This suggested that the adherent mucilage in the mutant was more compact compared with that of the wild type. In accordance with the role of CSLA2 in glucomannan synthesis, csla2-1 mucilage contained 30% less mannosyl and glucosyl content than did the wild type. No appreciable changes in the composition, structure, or macromolecular properties were observed for nonmannan polysaccharides in mutant mucilage. Biochemical analysis revealed that cellulose crystallinity was substantially reduced in csla2-1 mucilage; this was supported by the removal of most mucilage cellulose through treatment of csla2-1 seeds with endo-β-glucanase. Mutation in CSLA2 also resulted in altered spatial distribution of cellulose and an absence of birefringent cellulose microfibrils within the adherent mucilage. As with the observed changes in crystalline cellulose, the spatial distribution of pectin was also modified in csla2-1 mucilage. Taken together, our results demonstrate that glucomannans synthesized by CSLA2 are involved in modulating the structure of adherent mucilage, potentially through altering cellulose organization and crystallization.Mannan polysaccharides are a complex set of hemicellulosic cell wall polymers that are considered to have both structural and storage functions. Based on the particular chemical composition of the backbone and the side chains, mannan polysaccharides are classified into four types: pure mannan, glucomannan, galactomannan, and galactoglucomannan (Moreira and Filho, 2008; Wang et al., 2012; Pauly et al., 2013). Each of these polysaccharides is composed of a β-1,4-linked backbone containing Man or a combination of Glc and Man residues. In addition, the mannan backbone can be substituted with side chains of α-1,6-linked Gal residues. Mannan polysaccharides have been proposed to cross link with cellulose and other hemicelluloses via hydrogen bonds (Fry, 1986; Iiyama et al., 1994; Obel et al., 2007; Scheller and Ulvskov, 2010). Furthermore, it has been reported that heteromannans with different levels of substitution can interact with cellulose in diverse ways (Whitney et al., 1998). Together, these observations indicate the complexity of mannan polysaccharides in the context of cell wall architecture.CELLULOSE SYNTHASE-LIKE A (CSLA) enzymes have been shown to have mannan synthase activity in vitro. These enzymes polymerize the β-1,4-linked backbone of mannans or glucomannans, depending on the substrates (GDP-Man and/or GDP-Glc) provided (Richmond and Somerville, 2000; Liepman et al., 2005, 2007; Pauly et al., 2013). In Arabidopsis (Arabidopsis thaliana), nine CSLA genes have been identified; different CSLAs are responsible for the synthesis of different mannan types (Liepman et al., 2005, 2007). CSLA7 has mannan synthase activity in vitro (Liepman et al., 2005) and has been shown to synthesize stem glucomannan in vivo (Goubet et al., 2009). Disrupting the CSLA7 gene results in defective pollen growth and embryo lethality phenotypes in Arabidopsis, indicating structural or signaling functions of mannan polysaccharides during plant embryo development (Goubet et al., 2003). A mutation in CSLA9 results in the inhibition of Agrobacterium tumefaciens-mediated root transformation in the rat4 mutant (Zhu et al., 2003). CSLA2, CSLA3, and CSLA9 are proposed to play nonredundant roles in the biosynthesis of stem glucomannans, although mutations in CSLA2, CSLA3, or CSLA9 have no effect on stem development or strength (Goubet et al., 2009). All of the Arabidopsis CSLA proteins have been shown to be involved in the biosynthesis of mannan polysaccharides in the plant cell wall (Liepman et al., 2005, 2007), although the precise physiological functions of only CSLA7 and CSLA9 have been conclusively demonstrated.In Arabidopsis, when mature dry seeds are hydrated, gel-like mucilage is extruded to envelop the entire seed. Ruthenium red staining of Arabidopsis seeds reveals two different mucilage layers, termed the nonadherent and the adherent mucilage layers (Western et al., 2000; Macquet et al., 2007a). The outer, nonadherent mucilage is loosely attached and can be easily extracted by shaking seeds in water. Compositional and linkage analyses suggest that this layer is almost exclusively composed of unbranched rhamnogalacturonan I (RG-I) (>80% to 90%), with small amounts of branched RG-I, arabinoxylan, and high methylesterified homogalacturonan (HG). By contrast, the inner, adherent mucilage layer is tightly attached to the seed and can only be removed by strong acid or base treatment, or by enzymatic digestion (Macquet et al., 2007a; Huang et al., 2011; Walker et al., 2011). As with the nonadherent layer, adherent mucilage is also mainly composed of unbranched RG-I, but with small numbers of arabinan and galactan ramifications (Penfield et al., 2001; Willats et al., 2001; Dean et al., 2007; Macquet et al., 2007a, 2007b; Arsovski et al., 2009; Haughn and Western, 2012). There are also minor amounts of pectic HG in the adherent mucilage, with high methylesterified HG in the external domain compared with the internal domain of the adherent layer (Willats et al., 2001; Macquet et al., 2007a; Rautengarten et al., 2008; Sullivan et al., 2011; Saez-Aguayo et al., 2013). In addition, the adherent mucilage contains cellulose (Blake et al., 2006; Macquet et al., 2007a), which is entangled with RG-I and is thought to anchor the pectin-rich mucilage onto seeds (Macquet et al., 2007a; Harpaz-Saad et al., 2011, 2012; Mendu et al., 2011; Sullivan et al., 2011). As such, Arabidopsis seed mucilage is considered to be a useful model for investigating the biosynthesis of cell wall polysaccharides and how this process is regulated in vivo (Haughn and Western, 2012).Screening for altered seed coat mucilage has led to the identification of several genes encoding enzymes that are involved in the biosynthesis or modification of mucilage components. RHAMNOSE SYNTHASE2/MUCILAGE-MODIFIED4 (MUM4) is responsible for the synthesis of UDP-l-Rha (Usadel et al., 2004; Western et al., 2004; Oka et al., 2007). The putative GALACTURONSYLTRANSFERASE11 can potentially synthesize mucilage RG-I or HG pectin from UDP-d-GalUA (Caffall et al., 2009). GALACTURONSYLTRANSFERASE-LIKE5 appears to function in the regulation of the final size of the mucilage RG-I (Kong et al., 2011, 2013). Mutant seeds defective in these genes display reduced thickness of the extruded mucilage layer compared with wild-type Arabidopsis seeds.RG-I deposited in the apoplast of seed coat epidermal cells appears to be synthesized in a branched form that is subsequently modified by enzymes in the apoplast. MUM2 encodes a β-galactosidase that removes Gal residues from RG-I side chains (Dean et al., 2007; Macquet et al., 2007b). β-XYLOSIDASE1 encodes an α-l-arabinfuranosidase that removes Ara residues from RG-I side chains (Arsovski et al., 2009). Disruptions of these genes lead to defective hydration properties and affect the extrusion of mucilage. Furthermore, correct methylesterification of mucilage HG is also required for mucilage extrusion. HG is secreted into the wall in a high methylesterified form that can then be enzymatically demethylesterified by pectin methylesterases (PMEs; Bosch and Hepler, 2005). PECTIN METHYLESTERASE INHIBITOR6 (PMEI6) inhibits PME activities (Saez-Aguayo et al., 2013). The subtilisin-like Ser protease (SBT1.7) can activate other PME inhibitors, but not PMEI6 (Rautengarten et al., 2008; Saez-Aguayo et al., 2013). Disruption of either PMEI6 or SBT1.7 results in the delay of mucilage release.Although cellulose is present at low levels in adherent mucilage, it plays an important adhesive role for the attachment of mucilage pectin to the seed coat epidermal cells. The orientation and amount of pectin associated with the cellulose network is largely determined by cellulose conformation properties (Macquet et al., 2007a; Haughn and Western, 2012). Previous studies have demonstrated that CELLULOSE SYNTHASE A5 (CESA5) is required for the production of seed mucilage cellulose and the adherent mucilage in the cesa5 mutant can be easily extracted with water (Harpaz-Saad et al., 2011, 2012; Mendu et al., 2011; Sullivan et al., 2011).Despite all of these discoveries, large gaps remain in the current knowledge of the biosynthesis and functions of mucilage polysaccharides in seed coats. In this study, we show that CSLA2 is involved in the biosynthesis of mucilage glucomannan. Furthermore, we show that CSLA2 functions in the maintenance of the normal structure of the adherent mucilage layer through modifying the mucilage cellulose ultrastructure.     

Unidirectional Movement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in Mucilage Extrusion,Adherence, and Ray Formation

CELLULOSE SYNTHASE5 (CESA5) synthesizes cellulose necessary for seed mucilage adherence to seed coat epidermal cells of Arabidopsis (Arabidopsis thaliana). The involvement of additional CESA proteins in this process and details concerning the manner in which cellulose is deposited in the mucilage pocket are unknown. Here, we show that both CESA3 and CESA10 are highly expressed in this cell type at the time of mucilage synthesis and localize to the plasma membrane adjacent to the mucilage pocket. The isoxaben resistant1-1 and isoxaben resistant1-2 mutants affecting CESA3 show defects consistent with altered mucilage cellulose biosynthesis. CESA3 can interact with CESA5 in vitro, and green fluorescent protein-tagged CESA5, CESA3, and CESA10 proteins move in a linear, unidirectional fashion around the cytoplasmic column of the cell, parallel with the surface of the seed, in a pattern similar to that of cortical microtubules. Consistent with this movement, cytological evidence suggests that the mucilage is coiled around the columella and unwinds during mucilage extrusion to form a linear ray. Mutations in CESA5 and CESA3 affect the speed of mucilage extrusion and mucilage adherence. These findings imply that cellulose fibrils are synthesized in an ordered helical array around the columella, providing a distinct structure to the mucilage that is important for both mucilage extrusion and adherence.The epidermal cells of Arabidopsis (Arabidopsis thaliana) seed coats produce two distinct secondary cell walls: pectin-rich mucilage and cellulose-rich columellae (Western et al., 2000). When seeds are hydrated, mucilage expands rapidly, rupturing the outer tangential cell wall and forming a mucilage capsule that surrounds the seed. Seed coat mucilage is composed primarily of rhamnogalacturonan I (RG I) and also contains homogalacturonan (HG), hemicelluloses (such as xylans and glucomannans), and cellulose (for review, see Haughn and Western, 2012). Extruded mucilage consists of an outer, nonadherent fraction and an inner, adherent fraction (Western et al., 2000, 2001; Macquet et al., 2007a). The adherent and nonadherent mucilage layers differ in the amount of methylesterified HG (Rautengarten et al., 2008; Saez-Aguayo et al., 2013; Voiniciuc et al., 2013), galactans (Dean et al., 2007; Macquet et al., 2007b), arabinans (Arsovski et al., 2009), mannans (Yu et al., 2014), and cellulose (Harpaz-Saad et al., 2011; Mendu et al., 2011; Sullivan et al., 2011), all of which influence the physical properties of the layers.Adherent mucilage has a distinct structure, which can be examined using cell wall dyes and antibodies. When treated with cellulose-specific dyes, densely stained rays extend from the top of each columella to the outer edge of the adherent layer, many cell lengths above the seed surface (Mendu et al., 2011; Sullivan et al., 2011). Cytological evidence indicates that cellulose, pectins, and mannans are components of the ray (Haughn and Western, 2012; Griffiths et al., 2014; North et al., 2014; Yu et al., 2014), although the exact manner in which they are assembled is unknown.Cellulose is abundant in mucilage rays and mediates adherence. Loss-of-function mutations in CELLULOSE SYNTHASE5 (CESA5) result in reduced cellulose levels and increased detachment of mucilage from the seed (Harpaz-Saad et al., 2011; Mendu et al., 2011; Sullivan et al., 2011; Griffiths et al., 2014). How a reduction in cellulose results in a loss of adherence is still unknown, but it likely involves interaction with other mucilage components such as pectin and arabinogalactan proteins (Griffiths et al., 2014). Since cesa5 mutants still have some cellulose in the rays of the adherent mucilage halo (Mendu et al., 2011; Sullivan et al., 2011), additional cellulose synthases must be involved in mucilage cellulose biosynthesis.The Arabidopsis genome encodes 10 different CESAs (Delmer, 1999; Richmond and Somerville, 2000). Multiple lines of evidence suggest that three different CESAs are required to form one active cellulose synthase complex (CSC; for review, see Somerville, 2006). CSCs are membrane-bound protein complexes that synthesize cellulose microfibrils in the apoplast (for review, see Somerville, 2006; Endler and Persson, 2011; Lei et al., 2012). CESA1, CESA3, and CESA6 are considered the core components of the primary wall CSC (Desprez et al., 2007; Persson et al., 2007). CESA2, CESA5, and CESA9 are partially redundant to CESA6 in primary wall biosynthesis, and genetic evidence suggests that each of these CESA polypeptides can form a functional CSC with CESA3 and CESA1 (Desprez et al., 2007; Persson et al., 2007). CESA10 is expressed in young plants, stems, floral tissue, and the base of rosette leaves (Beeckman et al., 2002; Doblin et al., 2002), but its function in cellulose biosynthesis is unclear. Other cesa mutant lines have been examined for altered mucilage phenotypes (cesa1, radially swollen1 [Burn et al., 2002; Sullivan et al., 2011], cesa2, cesa6, and cesa9 [Mendu et al., 2011]; CESA3, je5 [Sullivan et al., 2011] and cesa10-1 [Sullivan et al., 2011]); to date, only CESA5 has been shown to be required for cellulose biosynthesis during mucilage deposition.Two mutant alleles of CESA3, isoxaben resistant1-1 (ixr1-1) and ixr1-2, were isolated in a screen for resistance to the herbicide isoxaben (Scheible et al., 2001). Isoxaben inhibits the incorporation of Glc into the emerging cellulose polymer and is considered a potent and specific inhibitor of cellulose biosynthesis (Heim et al., 1990). Homozygous ixr1-1 and ixr1-2 lines show increased resistance to the herbicide, and the mutations causing this resistance were mapped to the genomic locus of CESA3 (Heim et al., 1990; Scheible et al., 2001). The ixr1-1 and ixr1-2 mutations cause amino acid substitutions near the C terminus of the CESA3 protein. ixr1-1 causes a Gly-to-Asn substitution (G998A) located in a transmembrane domain, while ixr1-2 contains a Thr-to-Ile substitution (T942I) in an apoplastic region of the protein between two transmembrane domains (Scheible et al., 2001). Recently, the ixr1-2 allele was shown to affect the velocity of CSCs in the plasma membrane, which consequently modifies cellulose crystallinity in the cell wall (Harris et al., 2012). It is not exactly clear how the ixr1-1 mutation affects cellulose biosynthesis. The effects of either of these mutations on seed coat mucilage have not been investigated.Since mucilage is composed primarily of pectins with smaller amounts of cellulose, seed coat epidermal cells represent an excellent system to study cellulose biosynthesis and interactions between cellulose and other wall components in muro. In this study, we investigated how cellulose is synthesized and deposited in seed coat epidermal cells. We show that at least three different CESA proteins are highly expressed in the seed coat epidermis during mucilage biosynthesis. These CESAs are oriented and move in a linear fashion around the cytoplasmic column of each cell in an identical pattern to cortical microtubules. In addition, we provide evidence that the adherent mucilage has a helical structure that expands and unwinds during extrusion to form the mucilage ray. We propose that during seed coat epidermal cell development, the biosynthesis of cellulose predetermines the structure of rays in the adherent mucilage layer.     

MUCILAGE-RELATED10 Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage

Plants invest a lot of their resources into the production of an extracellular matrix built of polysaccharides. While the composition of the cell wall is relatively well characterized, the functions of the individual polymers and the enzymes that catalyze their biosynthesis remain poorly understood. We exploited the Arabidopsis (Arabidopsis thaliana) seed coat epidermis (SCE) to study cell wall synthesis. SCE cells produce mucilage, a specialized secondary wall that is rich in pectin, at a precise stage of development. A coexpression search for MUCILAGE-RELATED (MUCI) genes identified MUCI10 as a key determinant of mucilage properties. MUCI10 is closely related to a fenugreek (Trigonella foenumgraecum) enzyme that has in vitro galactomannan α-1,6-galactosyltransferase activity. Our detailed analysis of the muci10 mutants demonstrates that mucilage contains highly branched galactoglucomannan (GGM) rather than unbranched glucomannan. MUCI10 likely decorates glucomannan, synthesized by CELLULOSE SYNTHASE-LIKE A2, with galactose residues in vivo. The degree of galactosylation is essential for the synthesis of the GGM backbone, the structure of cellulose, mucilage density, as well as the adherence of pectin. We propose that GGM scaffolds control mucilage architecture along with cellulosic rays and show that Arabidopsis SCE cells represent an excellent model in which to study the synthesis and function of GGM. Arabidopsis natural varieties with defects similar to muci10 mutants may reveal additional genes involved in GGM synthesis. Since GGM is the most abundant hemicellulose in the secondary walls of gymnosperms, understanding its biosynthesis may facilitate improvements in the production of valuable commodities from softwoods.The plant cell wall is the key determinant of plant growth (Cosgrove, 2005) and represents the most abundant source of biopolymers on the planet (Pauly and Keegstra, 2010). Consequently, plants invest a lot of their resources into the production of this extracellular structure. Thus, it is not surprising that approximately 15% of Arabidopsis (Arabidopsis thaliana) genes are likely dedicated to the biosynthesis and modification of cell wall polymers (Carpita et al., 2001). Plant walls consist mainly of polysaccharides (cellulose, hemicellulose, and pectin) but also contain lignin and glycoproteins. While the biochemical structure of each wall component has been relatively well characterized, the molecular players involved in their biogenesis remain poorly understood (Keegstra, 2010). The functions of the individual polymers, and how they are assembled into a three-dimensional matrix, are also largely unknown (Burton et al., 2010; Burton and Fincher, 2012).Significant breakthroughs in cell wall research have been achieved through the examination of specialized plant tissues that contain elevated levels of a single polysaccharide (Pauly and Keegstra, 2010). Some species, particularly legumes, accumulate large amounts of the hemicellulose galactomannan during secondary wall thickening of the seed (Srivastava and Kapoor, 2005). Analysis of the developing fenugreek (Trigonella foenumgraecum) endosperm led to the purification of a GALACTOMANNAN GALACTOSYLTRANSFERASE (TfGMGT), the first glycosyltransferase (GT) whose activity in plant cell wall synthesis was demonstrated in vitro (Scheller and Ulvskov, 2010). TfGMGT catalyzes the decoration of mannan chains with single α-1,6-galactosyl residues (Edwards et al., 1999). A similar approach in guar (Cyamopsis tetragonoloba) seeds revealed that the β-1,4-linked mannan backbone is synthesized by a member of the CELLULOSE SYNTHASE-LIKE A (CSLA) protein family (Dhugga et al., 2004).Galactomannan functions as a storage polymer in the endosperm of the aforementioned seeds, analogous to starch in cereal grains (Dhugga et al., 2004), but it also has important rheological properties in the cell wall that have been exploited to produce valuable stabilizers and gelling agents for human consumption (Srivastava and Kapoor, 2005). The Man-to-Gal ratio is essential for the application of galactomannan gums in the food industry (Edwards et al., 1992). This is because unsubstituted mannan chains can interact via hydrogen bonds to produce crystalline microfibrils similar to cellulose (Millane and Hendrixson, 1994). Indeed, some algae that lack cellulose employ mannan fibrils as a structural material (Preston, 1968). The addition of Gal branches to the smooth, ribbon-like mannan chains creates hairy regions that limit self-association and promote gelation (Dea et al., 1977). All mannans are likely synthesized as highly substituted polymers that are trimmed in the cell wall (Scheller and Ulvskov, 2010).Generally, polysaccharides containing backbones of β-1,4-linked Man units can be classified as heteromannan (HM). Galactoglucomannan (GGM) is the main hemicellulose in gymnosperm secondary walls and, in contrast to galactomannan, has a backbone that contains both Glc and Man units (Pauly et al., 2013). HM is detected in most Arabidopsis cell types (Handford et al., 2003) and facilitates embryogenesis (Goubet et al., 2009), germination (Rodríguez-Gacio et al., 2012), tip growth (Bernal et al., 2008), and vascular development (Benová-Kákosová et al., 2006; Yin et al., 2011). In the last 10 years, in vitro mannan synthase activity has been demonstrated for recombinant CSLA proteins from many land plants (Liepman et al., 2005, 2007; Suzuki et al., 2006; Gille et al., 2011; Wang et al., 2012). HM synthesis may also involve CELLULOSE SYNTHASE-LIKE D (CSLD) enzymes and MANNAN SYNTHESIS-RELATED (MSR) accessory proteins (Yin et al., 2011; Wang et al., 2013), but their precise roles in relation to the CSLAs have not been established. Arabidopsis CSLA2, like most other isoforms, can use both GDP-Man and GDP-Glc as substrates in vitro (Liepman et al., 2005, 2007) and is responsible for stem glucomannan synthesis in vivo along with CSLA3 and CSLA7 (Goubet et al., 2009). CSLA2 also participates in the synthesis of glucomannan present in mucilage produced by seed coat epidermal (SCE) cells (Yu et al., 2014).Arabidopsis SCE cells represent an excellent genetic model in which to study the synthesis, polar secretion, and modification of polysaccharides, since these processes dominate a precise stage of seed coat development but are not essential for seed viability in laboratory conditions (Haughn and Western, 2012; North et al., 2014; Voiniciuc et al., 2015). Hydration of mature seeds in water releases a large gelatinous capsule, rich in the pectic polymer rhamnogalacturonan I, which can be easily stained or extracted (Macquet et al., 2007). Biochemical and cytological experiments indicate that Arabidopsis seed mucilage is more than just pectin and, in addition to cellulose, is likely to contain glycoproteins and at least two hemicellulosic polymers (Voiniciuc et al., 2015). There is mounting evidence that, despite their low abundance, these components play critical functions in seed mucilage architecture. The structure of homogalacturonan (HG), the major pectin in primary cell walls but a minor mucilage component, appears to be a key determinant of gelling properties and mucilage extrusion (Rautengarten et al., 2008; Saez-Aguayo et al., 2013; Voiniciuc et al., 2013). Mucilage attachment to seeds is maintained by the SALT OVERLY SENSITIVE5 glycoprotein and cellulose synthesized by multiple CELLULOSE SYNTHASE (CESA) isoforms (Harpaz-Saad et al., 2011; Mendu et al., 2011; Sullivan et al., 2011; Griffiths et al., 2014, 2015). From more than 35 genes that are reported to affect Arabidopsis seed mucilage properties (Voiniciuc et al., 2015), only CSLA2, CESA3, CESA5, GALACTURONOSYLTRANSFERASE11 (GAUT11; Caffall et al., 2009), and GAUT-LIKE5 (GATL5; Kong et al., 2013) are predicted to encode GTs. This highlights that, despite many detailed studies about mucilage production in SCE cells, the synthesis of its components remains poorly understood.To address this issue, we conducted a reverse genetic search for MUCILAGE-RELATED (MUCI) genes that may be required for polysaccharide biosynthesis. One of these, MUCI10, encodes a member of the Carbohydrate Active Enzymes family, GT34 (Lombard et al., 2014), which includes at least two enzymatic activities and seven Arabidopsis proteins (Keegstra and Cavalier, 2010). Five of them function as XYLOGLUCAN XYLOSYLTRANSFERASES (XXT1–XXT5) in vivo and/or in vitro (Faik et al., 2002; Cavalier et al., 2008; Vuttipongchaikij et al., 2012). MUCI10/GT7 (At2g22900) and its paralog GT6 (At4g37690) do not function as XXTs (Vuttipongchaikij et al., 2012) and are more closely related to the TfGMGT enzyme (Faik et al., 2002; Keegstra and Cavalier, 2010). MUCI10, also called GALACTOSYLTRANSFERASE-LIKE6 (GTL6), served as a Golgi marker in multiple proteomic studies of Arabidopsis callus cultures (Dunkley et al., 2004, 2006; Nikolovski et al., 2012, 2014). Nevertheless, the role of TfGMGT orthologs in Arabidopsis remained unknown. We show that MUCI10 is responsible for the extensive galactosylation of glucomannan in mucilage and influences glucomannan backbone synthesis, cellulose structure, and the distribution of pectin.     

SALT-OVERLY SENSITIVE5 Mediates Arabidopsis Seed Coat Mucilage Adherence and Organization through Pectins

Interactions between cell wall polymers are critical for establishing cell wall integrity and cell-cell adhesion. Here, we exploit the Arabidopsis (Arabidopsis thaliana) seed coat mucilage system to examine cell wall polymer interactions. On hydration, seeds release an adherent mucilage layer strongly attached to the seed in addition to a nonadherent layer that can be removed by gentle agitation. Rhamnogalacturonan I (RG I) is the primary component of adherent mucilage, with homogalacturonan, cellulose, and xyloglucan constituting minor components. Adherent mucilage contains rays composed of cellulose and pectin that extend above the center of each epidermal cell. CELLULOSE SYNTHASE5 (CESA5) and the arabinogalactan protein SALT-OVERLY SENSITIVE5 (SOS5) are required for mucilage adherence through unknown mechanisms. SOS5 has been suggested to mediate adherence by influencing cellulose biosynthesis. We, therefore, investigated the relationship between SOS5 and CESA5. cesa5-1 seeds show reduced cellulose, RG I, and ray size in adherent mucilage. In contrast, sos5-2 seeds have wild-type levels of cellulose but completely lack adherent RG I and rays. Thus, relative to each other, cesa5-1 has a greater effect on cellulose, whereas sos5-2 mainly affects pectin. The double mutant cesa5-1 sos5-2 has a much more severe loss of mucilage adherence, suggesting that SOS5 and CESA5 function independently. Double-mutant analyses with mutations in MUCILAGE MODIFIED2 and FLYING SAUCER1 that reduce mucilage release through pectin modification suggest that only SOS5 influences pectin-mediated adherence. Together, these findings suggest that SOS5 mediates adherence through pectins and does so independently of but in concert with cellulose synthesized by CESA5.Cellulosic cell walls are a defining feature of land plants. Primary cell walls are composed of three major classes of polysaccharides: cellulose, hemicelluloses, and pectins. In addition, approximately 10% of the primary cell wall is composed of protein (Burton et al., 2010). Cell walls provide mechanical support for the cell, and cell wall carbohydrates in the middle lamellae mediate cell-cell adhesion (Caffall and Mohnen, 2009). Current models of cell wall structure depict a cellulose-hemicellulose network embedded in an independent pectin gel (for review, see Albersheim et al., 2011). These components are believed to interact through both covalent and noncovalent bonds to provide structure and strength to the cell wall, although the relative importance of pectin and its interactions with the hemicellulose-cellulose network remain unclear (for review, see Cosgrove, 2005).Another gap in our understanding of cell wall structure and assembly is the role of arabinogalactan proteins (AGPs). AGPs are a family of evolutionarily conserved secreted proteins highly glycosylated with type II arabinogalactans, and they can be localized to the plasma membrane by a C-terminal glycophosphatidylinositol (GPI) lipid anchor (for review, see Schultz et al., 2000; Showalter, 2001; Johnson et al., 2003; Seifert and Roberts, 2007; Ellis et al., 2010). AGPs can be extensively modified in the cell wall; many glycosyl hydrolases can affect AGP function by cleaving their glycosyl side chains (Sekimata et al., 1989; Cheung et al., 1995; Wu et al., 1995; Kotake et al., 2005). The GPI anchor can also be cleaved, releasing the AGPs from the membrane into the cell wall (Schultz et al., 2000). Although their exact roles are still unclear, AGPs have been proposed to interact with cell wall polysaccharides, initiate intracellular signaling cascades, and influence a wide variety of biological processes (for review, see Seifert and Roberts, 2007; Ellis et al., 2010; Tan et al., 2013).Many fasciclin-like AGPs (FLAs), which contain at least one fasciclin domain (FAS) associated with protein-protein interactions, have been suggested to influence cellulose biosynthesis or organization (Seifert and Roberts, 2007; Li et al., 2010; MacMillan et al., 2010). FLA3 RNA interference lines have reduced intine cell wall biosynthesis and loss of Calcofluor white (a fluorescent dye specific for glycan molecules) staining in aborted pollen grains (Li et al., 2010). A fla11 fla12 double mutant was shown to have reduced cellulose deposition, altered cellulose microfibril angle, and reduced cell wall integrity (MacMillan et al., 2010). The fla11 fla12 double mutant also had reductions in arabinans, galactans, and rhamnose (MacMillan et al., 2010). FLA4/SALT-OVERLY SENSITIVE5 (SOS5) was identified in a screen for salt sensitivity in roots. The SOS5 gene encodes an FLA protein with a GPI anchor, two AGP-like domains, and two FAS domains (Shi et al., 2003). Plants homozygous for the loss-of-function conditional allele sos5-1 have thinner root cell walls that appear less organized (Shi et al., 2003). The presence of the two FAS domains has led to the suggestion that SOS5 may interact with other proteins, forming a network that strengthens the cell wall (Shi et al., 2003). SOS5 is involved in regulation of cell wall rheology through a pathway involving two Leu-rich repeat receptor-like kinases, FEI1 and FEI2 (Xu et al., 2008). SOS5 and FEI2 are also required for normal seed coat mucilage adherence and hypothesized to do so by influencing cellulose biosynthesis (Harpaz-Saad et al., 2011, 2012).Arabidopsis (Arabidopsis thaliana) seed coat mucilage is a powerful model for studying cell wall biosynthesis and polysaccharide interactions (Arsovski et al., 2010; Haughn and Western, 2012). Seed coat epidermal cells sequentially produce two distinct types of secondary cell walls with unique morphologies and properties (Western et al., 2000; Windsor et al., 2000). Between approximately 5 and 9 d approximate time of fertilization (DPA), seed coat epidermal cells synthesize mucilage and deposit it in the apoplast, creating a donut-shaped mucilage pocket that surrounds a central cytoplasmic column (Western et al., 2000, 2004; Haughn and Chaudhury, 2005). From 9 to 13 DPA, the cytoplasmic column is gradually replaced by a cellulose-rich, volcano-shaped secondary cell wall called the columella (Beeckman et al., 2000; Western et al., 2000; Windsor et al., 2000; Stork et al., 2010; Mendu et al., 2011).Seed mucilage is composed primarily of relatively unbranched rhamnogalacturonan I (RG I) with minor amounts of homogalacturonan (HG), cellulose, and hemicelluloses (for review, see Haughn and Western, 2012). When mucilage is hydrated, it expands rapidly from the apoplastic pocket, forming a halo that surrounds the seed. Mucilage separates into two fractions: a loose nonadherent fraction and an inner adherent fraction that can only be released by vigorous shaking, strong bases, or glycosidases (for review, see North et al., 2014). Galactans and arabinans are also present in mucilage, and their regulation by glycosidases is required for correct mucilage hydration (Dean et al., 2007; Macquet et al., 2007b; Arsovski et al., 2009). For example, β-XYLOSIDASE1 encodes a bifunctional β-d-xylosidase/α-l-arabinofuranosidase required for arabinan modification in mucilage, and β-xylosidase1 mutant seeds have a delayed mucilage release phenotype (Arsovski et al., 2009). MUCILAGE MODIFIED2 (MUM2) encodes a β-d-galactosidase, and mum2 seeds fail to release mucilage when hydrated in water (Dean et al., 2007; Macquet et al., 2007b). MUM2 is believed to modify RG I galactan side chains but may also affect the galactan component of other mucilage components (Dean et al., 2007; Macquet et al., 2007b). Galactans are capable of binding to cellulose in vitro and could affect mucilage hydration through pectin-cellulose interactions (Zykwinska et al., 2005, 2007a, 2007b; Dick-Pérez et al., 2011; Wang et al., 2012), although carbohydrate linkage analysis suggests that the galactan side chains are very short.Several studies indicate that seed mucilage extrusion and expansion are also influenced by methylesterification of HG. For example, both SUBTILISIN-LIKE SER PROTEASE1.7 and PECTIN METHYLESTERASE INHIBITOR6 are required for proper methyl esterification of mucilage (Rautengarten et al., 2008; Saez-Aguayo et al., 2013). Mutations in another gene, FLYING SAUCER1 (FLY1; a transmembrane E3 ubiquitin ligase), reduce the degree of pectin methylesterification in mucilage and cause increased mucilage adherence and defective mucilage extrusion (Voiniciuc et al., 2013). fly1 seeds have disc-like structures at the edge of the mucilage halo, which are outer primary cell wall fragments that detach from the columella during extrusion and are difficult to separate from the adherent mucilage (Voiniciuc et al., 2013).Recently, CELLULOSE SYNTHASE5 (CESA5) and SOS5 were proposed to facilitate cellulose-mediated mucilage adherence (Harpaz-Saad et al., 2011; Mendu et al., 2011; Sullivan et al., 2011). A simple hypothesis for the role of CESA5 in mucilage adherence is that it synthesizes cellulose, which interacts with the mucilage pectin to mediate adherence. Loss of CESA5 function results in a reduction of mucilage cellulose biosynthesis and a less adherent mucilage cell wall matrix (Mendu et al., 2011; Sullivan et al., 2011). The role of SOS5 in mucilage adherence is more difficult to explain. SOS5 null mutations cause a loss-of-adherence phenotype similar to cesa5-1 seeds, suggesting that SOS5 may regulate mucilage adherence by influencing CESA5 function (Harpaz-Saad et al., 2011). However, the mechanism through which SOS5 could influence CESA5 and/or cellulose biosynthesis is not clear.To better understand the role of SOS5 in mucilage adherence and its relationship to CESA5, we thoroughly investigated the seed coat epidermal cell phenotypes of the cesa5-1 and sos5-2 single mutants as well as those of the cesa5-1 sos5-2 double mutant. We also investigated how cellulose, SOS5, and pectin interact to mediate mucilage adherence by constructing double mutants with either cesa5-1 or sos5-2 together with either mum2-1 or fly1. Our results suggest that SOS5 mediates mucilage adherence independently of CESA5. Furthermore, compared with CESA5, SOS5 has a greater influence on mucilage pectin structure, suggesting that SOS5 mediates mucilage adherence through pectins, not cellulose.     

Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.Drought and salinity cause osmotic stress in plants and severely affect crop productivity throughout the world. Plants respond to osmotic stress by changing a number of cellular processes (Xiong et al., 1999; Xiong and Zhu, 2002; Bartels and Sunkar, 2005; Boudsocq and Lauriére, 2005). Some of these changes include activation of stress-responsive genes, regulation of membrane transport at both plasma membrane (PM) and vacuolar membrane (tonoplast) to maintain water and ionic homeostasis, and metabolic changes to produce compatible osmolytes such as Pro (Stewart and Lee, 1974; Krasensky and Jonak, 2012). It has been well established that a specific calcium (Ca²⁺) signature is generated in response to a particular environmental stimulus (Trewavas and Malhó, 1998; Scrase-Field and Knight, 2003; Luan, 2009; Kudla et al., 2010). The Ca²⁺ changes are primarily perceived by several Ca²⁺ sensors such as calmodulin (Reddy, 2001; Luan et al., 2002), Ca²⁺-dependent protein kinases (Harper and Harmon, 2005), calcineurin B-like proteins (CBLs; Luan et al., 2002; Batistič and Kudla, 2004; Pandey, 2008; Luan, 2009; Sanyal et al., 2015), and other Ca²⁺-binding proteins (Reddy, 2001; Shao et al., 2008) to initiate various cellular responses.Plant CBL-type Ca²⁺ sensors interact with and activate CBL-interacting protein kinases (CIPKs) that phosphorylate downstream components to transduce Ca²⁺ signals (Liu et al., 2000; Luan et al., 2002; Batistič and Kudla, 2004; Luan, 2009). In several plant species, multiple members have been identified in the CBL and CIPK family (Luan et al., 2002; Kolukisaoglu et al., 2004; Pandey, 2008; Batistič and Kudla, 2009; Weinl and Kudla, 2009; Pandey et al., 2014). Involvement of specific CBL-CIPK pair to decode a particular type of signal entails the alternative and selective complex formation leading to stimulus-response coupling (D’Angelo et al., 2006; Batistič et al., 2010).Several CBL and CIPK family members have been implicated in plant responses to drought, salinity, and osmotic stress based on genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants (Zhu, 2002; Cheong et al., 2003, 2007; Kim et al., 2003; Pandey et al., 2004, 2008; D’Angelo et al., 2006; Qin et al., 2008; Tripathi et al., 2009; Held et al., 2011; Tang et al., 2012; Drerup et al., 2013; Eckert et al., 2014). A few CIPKs have also been functionally characterized by gain-of-function approach in crop plants such as rice (Oryza sativa), pea (Pisum sativum), and maize (Zea mays) and were found to be involved in osmotic stress responses (Mahajan et al., 2006; Xiang et al., 2007; Yang et al., 2008; Tripathi et al., 2009; Zhao et al., 2009; Cuéllar et al., 2010).In this report, we examined the role of the Arabidopsis CIPK21 gene in osmotic stress response by reverse genetic analysis. The loss-of-function mutant plants became hypersensitive to salt and mannitol stress conditions, suggesting that CIPK21 is involved in the regulation of osmotic stress response in Arabidopsis. These findings are further supported by an enhanced tonoplast targeting of the cytoplasmic CIPK21 through interaction with the vacuolar Ca²⁺ sensors CBL2 and CBL3 under salt stress condition.     

Enhanced Abscisic Acid-Mediated Responses in nia1nia2noa1-2 Triple Mutant Impaired in NIA/NR- and AtNOA1-Dependent Nitric Oxide Biosynthesis in Arabidopsis

Nitric oxide (NO) regulates a wide range of plant processes from development to environmental adaptation. Despite its reported regulatory functions, it remains unclear how NO is synthesized in plants. We have generated a triple nia1nia2noa1-2 mutant that is impaired in nitrate reductase (NIA/NR)- and Nitric Oxide-Associated1 (AtNOA1)-mediated NO biosynthetic pathways. NO content in roots of nia1nia2 and noa1-2 plants was lower than in wild-type plants and below the detection limit in nia1nia2noa1-2 plants. NIA/NR- and AtNOA1-mediated biosynthesis of NO were thus active and responsible for most of the NO production in Arabidopsis (Arabidopsis thaliana). The nia1nia2noa1-2 plants displayed reduced size, fertility, and seed germination potential but increased dormancy and resistance to water deficit. The increasing deficiency in NO of nia1nia2, noa1-2, and nia1nia2noa1-2 plants correlated with increased seed dormancy, hypersensitivity to abscisic acid (ABA) in seed germination and establishment, as well as dehydration resistance. In nia1nia2noa1-2 plants, enhanced drought tolerance was due to a very efficient stomata closure and inhibition of opening by ABA, thus uncoupling NO from ABA-triggered responses in NO-deficient guard cells. The NO-deficient mutants in NIA/NR- and AtNOA1-mediated pathways in combination with the triple mutant will be useful tools to functionally characterize the role of NO and the contribution of both biosynthetic pathways in regulating plant development and defense.Nitric oxide (NO) is a small ubiquitous molecule derived from nitrogen-containing precursors that is one of the earliest and most widespread signaling molecules in living organisms from metazoans to mammals (Torreilles, 2001). The regulatory functions of NO have been extensively studied in mammals, where it is synthesized from Arg through the activity of NO synthases (Knowles and Moncada, 1994). By contrast, the biosynthesis and function of this molecule in plants are largely unknown. During the last 10 years, NO biosynthesis in plants has been one of the most controversial topics in plant biology (Durner and Klessig, 1999; Wendehenne et al., 2001; del Río et al., 2004; Zeier et al., 2004; Lamotte et al., 2005; Meyer et al., 2005; Modolo et al., 2005; Crawford, 2006; Crawford et al., 2006; Zemojtel et al., 2006a). Despite the controversy about its biosynthesis, it is now clear that NO regulates many physiological processes of plants, including seed germination, cell death, defense responses against pathogens, stomata function, senescence, and flowering (Beligni and Lamattina, 2000; Pedroso et al., 2000; Neill et al., 2002; Lamattina et al., 2003; He et al., 2004; Romero-Puertas et al., 2004; Wendehenne et al., 2004; Delledonne, 2005; Guo and Crawford, 2005; Simpson, 2005; Grün et al., 2006; Melotto et al., 2006; Planchet et al., 2006; Ali et al., 2007; Mishina et al., 2007).The molecular mechanisms underlying the control of seed dormancy and germination are still poorly characterized. Genetic data support a central role of abscisic acid (ABA) in regulating seed dormancy, whereas gibberellins promote germination (Finkelstein et al., 2008; Holdsworth et al., 2008). In addition, NO has been lately characterized as a new component in the signaling pathway leading to dormancy breakage. NO-releasing compounds reduce dormancy in a NO-dependent manner in Arabidopsis (Arabidopsis thaliana), some warm-season grasses, and certain barley (Hordeum vulgare) cultivars (Bethke et al., 2004; Sarath et al., 2006). More recently, the aleurone layer cells have been characterized as responsive to NO, gibberellins, and ABA, thus becoming a primary determinant of seed dormancy in Arabidopsis (Bethke et al., 2007).Two main enzyme-based pathways have been proposed to be functional for NO biosynthesis in plants. One is based on the activity of nitrate reductases (Meyer et al., 2005; Modolo et al., 2005), and another one, yet undefined, is based on the direct or indirect function of the Nitric Oxide-Associated1/Resistant to Inhibition by Fosfidomycin1 (AtNOA1/RIF1) protein. It has been also reported that NO synthesis from nitrite occurs in mitochondria associated with mitochondrial electron transport (Planchet et al., 2005) and also that this pathway is mainly functioning in roots under anoxia (Gupta et al., 2005). Moreover, the balance between mitochondrial nitrite reduction and superoxide-dependent NO degradation seems to be derived from factors controlling NO levels in Arabidopsis (Wulff et al., 2009). It has been recently reported that the synthesis of NO in floral organs requires nitrate reductase activity (Seligman et al., 2008) and also that homologues of AtNOA1 participate in NO biosynthesis in diatoms (Vardi et al., 2008), mammals (Zemojtel et al., 2006b; Parihar et al., 2008a, 2008b), and Nicotiana benthamiana (Kato et al., 2008). Recently, the identification of the rif1 mutant, carrying a null mutation in the AtNOA1 locus (At3g47450), allowed uncovering of a function for AtNOA1/RIF1 in the expression of plastome-encoded proteins (Flores-Pérez et al., 2008). Moreover, another recent report claims that AtNOA1 is not a NO synthase but a cGTPase (Moreau et al., 2008), likely playing a role in ribosome assembly and subsequent mRNA translation to proteins in the chloroplasts.To date, it is not clear if both pathways coexist in plants and, if so, the corresponding contributions of each pathway to NO biosynthesis. In this work, we have addressed the functions of both pathways in Arabidopsis by generating a triple mutant in both nitrate reductases and AtNOA1 that is severely impaired in NO production. Further characterization of NO-deficient plants allowed us to identify a functional cross talk between NO and ABA in controlling seed germination and dormancy as well as plant resistance to water deficit.     

Surveying Rubisco Diversity and Temperature Response to Improve Crop Photosynthetic Efficiency

The threat to global food security of stagnating yields and population growth makes increasing crop productivity a critical goal over the coming decades. One key target for improving crop productivity and yields is increasing the efficiency of photosynthesis. Central to photosynthesis is Rubisco, which is a critical but often rate-limiting component. Here, we present full Rubisco catalytic properties measured at three temperatures for 75 plants species representing both crops and undomesticated plants from diverse climates. Some newly characterized Rubiscos were naturally “better” compared to crop enzymes and have the potential to improve crop photosynthetic efficiency. The temperature response of the various catalytic parameters was largely consistent across the diverse range of species, though absolute values showed significant variation in Rubisco catalysis, even between closely related species. An analysis of residue differences among the species characterized identified a number of candidate amino acid substitutions that will aid in advancing engineering of improved Rubisco in crop systems. This study provides new insights on the range of Rubisco catalysis and temperature response present in nature, and provides new information to include in models from leaf to canopy and ecosystem scale.In a changing climate and under pressure from a population set to hit nine billion by 2050, global food security will require massive changes to the way food is produced, distributed, and consumed (Ort et al., 2015). To match rising demand, agricultural production must increase by 50 to 70% in the next 35 years, and yet the gains in crop yields initiated by the green revolution are slowing, and in some cases, stagnating (Long and Ort, 2010; Ray et al., 2012). Among a number of areas being pursued to increase crop productivity and food production, improving photosynthetic efficiency is a clear target, offering great promise (Parry et al., 2007; von Caemmerer et al., 2012; Price et al., 2013; Ort et al., 2015). As the gatekeeper of carbon entry into the biosphere and often acting as the rate-limiting step of photosynthesis, Rubisco, the most abundant enzyme on the planet (Ellis, 1979), is an obvious and important target for improving crop photosynthetic efficiency.Rubisco is considered to exhibit comparatively poor catalysis, in terms of catalytic rate, specificity, and CO₂ affinity (Tcherkez et al., 2006; Andersson, 2008), leading to the suggestion that even small increases in catalytic efficiency may result in substantial improvements to carbon assimilation across a growing season (Zhu et al., 2004; Parry et al., 2013; Galmés et al., 2014a; Carmo-Silva et al., 2015). If combined with complimentary changes such as optimizing other components of the Calvin Benson or photorespiratory cycles (Raines, 2011; Peterhansel et al., 2013; Simkin et al., 2015), optimized canopy architecture (Drewry et al., 2014), or introducing elements of a carbon concentrating mechanism (Furbank et al., 2009; Lin et al., 2014a; Hanson et al., 2016; Long et al., 2016), Rubisco improvement presents an opportunity to dramatically increase the photosynthetic efficiency of crop plants (McGrath and Long, 2014; Long et al., 2015; Betti et al., 2016). A combination of the available strategies is essential for devising tailored solutions to meet the varied requirements of different crops and the diverse conditions under which they are typically grown around the world.Efforts to engineer an improved Rubisco have not yet produced a “super Rubisco” (Parry et al., 2007; Ort et al., 2015). However, advances in engineering precise changes in model systems continue to provide important developments that are increasing our understanding of Rubisco catalysis (Spreitzer et al., 2005; Whitney et al., 2011a, 2011b; Morita et al., 2014; Wilson et al., 2016), regulation (Andralojc et al., 2012; Carmo-Silva and Salvucci, 2013; Bracher et al., 2015), and biogenesis (Saschenbrecker et al., 2007; Whitney and Sharwood, 2008; Lin et al., 2014b; Hauser et al., 2015; Whitney et al., 2015).A complementary approach is to understand and exploit Rubisco natural diversity. Previous characterization of Rubisco from a limited number of species has not only demonstrated significant differences in the underlying catalytic parameters, but also suggests that further undiscovered diversity exists in nature and that the properties of some of these enzymes could be beneficial if present in crop plants (Carmo-Silva et al., 2015). Recent studies clearly illustrate the variation possible among even closely related species (Galmés et al., 2005, 2014b, 2014c; Kubien et al., 2008; Andralojc et al., 2014; Prins et al., 2016).Until recently, there have been relatively few attempts to characterize the consistency, or lack thereof, of temperature effects on in vitro Rubisco catalysis (Sharwood and Whitney, 2014), and often studies only consider a subset of Rubisco catalytic properties. This type of characterization is particularly important for future engineering efforts, enabling specific temperature effects to be factored into any attempts to modify crops for a future climate. In addition, the ability to coanalyze catalytic properties and DNA or amino acid sequence provides the opportunity to correlate sequence and biochemistry to inform engineering studies (Christin et al., 2008; Kapralov et al., 2011; Rosnow et al., 2015). While the amount of gene sequence information available grows rapidly with improving technology, knowledge of the corresponding biochemical variation resulting has yet to be determined (Cousins et al., 2010; Carmo-Silva et al., 2015; Sharwood and Whitney, 2014; Nunes-Nesi et al., 2016).This study aimed to characterize the catalytic properties of Rubisco from diverse species, comprising a broad range of monocots and dicots from diverse environments. The temperature dependence of Rubisco catalysis was evaluated to tailor Rubisco engineering for crop improvement in specific environments. Catalytic diversity was analyzed alongside the sequence of the Rubisco large subunit gene, rbcL, to identify potential catalytic switches for improving photosynthesis and productivity. In vitro results were compared to the average temperature of the warmest quarter in the regions where each species grows to investigate the role of temperature in modulating Rubisco catalysis.     

Focus on Ethylene: Ethylene and the Regulation of Physiological and Morphological Responses to Nutrient Deficiencies

To cope with nutrient deficiencies, plants develop both morphological and physiological responses. The regulation of these responses is not totally understood, but some hormones and signaling substances have been implicated. It was suggested several years ago that ethylene participates in the regulation of responses to iron and phosphorous deficiency. More recently, its role has been extended to other deficiencies, such as potassium, sulfur, and others. The role of ethylene in so many deficiencies suggests that, to confer specificity to the different responses, it should act through different transduction pathways and/or in conjunction with other signals. In this update, the data supporting a role for ethylene in the regulation of responses to different nutrient deficiencies will be reviewed. In addition, the results suggesting the action of ethylene through different transduction pathways and its interaction with other hormones and signaling substances will be discussed.When plants suffer from a mineral nutrient deficiency, they develop morphological and physiological responses (mainly in their roots) aimed to facilitate the uptake and mobilization of the limiting nutrient. After the nutrient has been acquired in enough quantity, these responses need to be switched off to avoid toxicity and conserve energy. In recent years, different plant hormones (e.g. ethylene, auxin, cytokinins, jasmonic acid, abscisic acid, brassinosteroids, GAs, and strigolactones) have been implicated in the regulation of these responses (Romera et al., 2007, 2011, 2015; Liu et al., 2009; Rubio et al., 2009; Kapulnik et al., 2011; Kiba et al., 2011; Iqbal et al., 2013; Zhang et al., 2014).Before the 1990s, there were several publications relating ethylene and nutrient deficiencies (cited in Lynch and Brown [1997] and Romera et al. [1999]) without establishing a direct implication of ethylene in the regulation of nutrient deficiency responses. In 1994, Romera and Alcántara (1994) published an article in Plant Physiology suggesting a role for ethylene in the regulation of Fe deficiency responses. In 1999, Borch et al. (1999) showed the participation of ethylene in the regulation of P deficiency responses. Since then, evidence has been accumulating in support of a role for ethylene in the regulation of both Fe (Romera et al., 1999, 2015; Waters and Blevins, 2000; Lucena et al., 2006; Waters et al., 2007; García et al., 2010, 2011, 2013, 2014; Yang et al., 2014) and P deficiency responses (Kim et al., 2008; Lei et al., 2011; Li et al., 2011; Nagarajan and Smith, 2012; Wang et al., 2012, 2014c). Both Fe and P may be poorly available in most soils, and plants develop similar responses under their deficiencies (Romera and Alcántara, 2004; Zhang et al., 2014). More recently, a role for ethylene has been extended to other deficiencies, such as K (Shin and Schachtman, 2004; Jung et al., 2009; Kim et al., 2012), S (Maruyama-Nakashita et al., 2006; Wawrzyńska et al., 2010; Moniuszko et al., 2013), and B (Martín-Rejano et al., 2011). Ethylene has also been implicated in both N deficiency and excess (Tian et al., 2009; Mohd-Radzman et al., 2013; Zheng et al., 2013), and its participation in Mg deficiency has been suggested (Hermans et al., 2010).In this update, we will review the information supporting a role for ethylene in the regulation of different nutrient deficiency responses. For information relating ethylene to other aspects of plant mineral nutrition, such as N₂ fixation and responses to excess of nitrate or essential heavy metals, the reader is referred to other reviews (for review, see Maksymiec, 2007; Mohd-Radzman et al., 2013; Steffens, 2014).