The Laforin-Like Dual-Specificity Phosphatase SEX4 from Arabidopsis Hydrolyzes Both C6- and C3-Phosphate Esters Introduced by Starch-Related Dikinases and Thereby Affects Phase Transition of α-Glucans

The biochemical function of the Laforin-like dual-specific phosphatase AtSEX4 (EC 3.1.3.48) has been studied. Crystalline maltodextrins representing the A- or the B-type allomorph were prephosphorylated using recombinant glucan, water dikinase (StGWD) or the successive action of both plastidial dikinases (StGWD and AtPWD). AtSEX4 hydrolyzed carbon 6-phosphate esters from both the prephosphorylated A- and B-type allomorphs and the kinetic constants are similar. The phosphatase also acted on prelabeled carbon-3 esters from both crystalline maltodextrins. Similarly, native starch granules prelabeled in either the carbon-6 or carbon-3 position were also dephosphorylated by AtSEX4. The phosphatase did also hydrolyze phosphate esters of both prephosphorylated maltodextrins when the (phospho)glucans had been solubilized by heat treatment. Submillimolar concentrations of nonphosphorylated maltodextrins inhibited AtSEX4 provided they possessed a minimum of length and had been solubilized. As opposed to the soluble phosphomaltodextrins, the AtSEX4-mediated dephosphorylation of the insoluble substrates was incomplete and at least 50% of the phosphate esters were retained in the pelletable (phospho)glucans. The partial dephosphorylation of the insoluble glucans also strongly reduced the release of nonphosphorylated chains into solution. Presumably, this effect reflects fast structural changes that following dephosphorylation occur near the surface of the maltodextrin particles. A model is proposed defining distinct stages within the phosphorylation/dephosphorylation-dependent transition of α-glucans from the insoluble to the soluble state.The metabolism of starch, the most prominent storage carbohydrate in plants, is assumed to require approximately 30 to 40 distinct (iso)enzymes (Deschamps et al., 2008), but, presumably, the list of the starch-related proteins is not yet complete. Several novel proteins (and protein functions) essential for the normal starch metabolism have recently been identified among which are two α-glucan phosphorylating dikinases. One dikinase (glucan, water dikinase [GWD], EC 2.7.9.4) utilizes ATP as dual phosphate donor and esterifies the C6 position of amylopectin-related glucosyl residues, whereas the other dikinase (phosphoglucan, water dikinase [PWD], EC 2.7.9.5) selectively transfers the β-phosphate group from ATP to the C3 position of glucosyl residues (Ritte et al., 2006).Two other previously unknown starch-related enzymes were designated as SEX4 protein (EC 3.1.3.48; At3g52180; previous designations PTPKIS1 and DSP4) and as Like Sex Four1 (LSF1) protein (At3g01510; previously named PTPKIS2; Comparot-Moss et al., 2010). Both proteins are predicted to contain a noncatalytic carbohydrate-binding module (CBM; Boraston et al., 2004; Shoseyov et al., 2006) and a catalytic dual-specificity phosphatase (DSP) domain. The latter is shared by the large family of DSPs that dephosphorylate distinct target phosphoproteins both at phosphotyrosine and phosphoserine/phosphothreonine residues. Some DSPs also act on various nonproteinaceous substrates, such as phospholipids or phosphorylated polyglycans (Pulido and Hooft van Huijsduijnen, 2008).Arabidopsis (Arabidopsis thaliana) mutants lacking a functional SEX4 protein contain both elevated starch levels and significant amounts of soluble phosphooligoglucans that are below the limit of detection in wild-type plants and probably originate from starch. However, the precise biochemical function of SEX4 is far from being clear (Kötting et al., 2009). The phenotype of the SEX4-deficient mutant is complex: Transitory starch possesses an elevated amylose-to-amylopectin ratio but the phosphate content of amylopectin is not increased. It has been hypothesized that SEX4 and LSF1 selectively hydrolyze C6- and C3-phosphate esters, respectively, but experimental evidence is lacking (Kötting et al., 2009). Likewise, it is unknown whether SEX4 preferentially acts on particulate starch or on soluble phosphoglucans.Crystalline maltodextrins (MD_cryst) have recently been introduced as a model mimicking some structural features of the native starch granules (Hejazi et al., 2008). They can be crystallized as either the A- or the B-type allomorph (Gallant et al., 1997; Gérard et al., 2001). Recombinant StGWD phosphorylates both maltodextrin allomorphs with a far higher rate than native starch granules and thereby initiates solubilization of both phosphorylated and nonphosphorylated maltodextrins. In vitro both allomorphs act also as substrate for PWD provided a prephosphorylation by GWD (Hejazi et al., 2009).In this study, we used the prephosphorylated A- and B-type allomorphs of MD_cryst to study biochemical functions of AtSEX4. As highly ordered α-glucans are the preferred sites of the dikinase-mediated phosphorylation, we designed experiments to answer the following questions: Does SEX4 preferentially act on phosphorylated insoluble or soluble glucans? If insoluble α-glucans are the preferred substrate, does the phosphatase distinguish between the A- and the B-type allomorph? Does SEX4 preferentially or selectively hydrolyze C6-phosphate esters? Does SEX4 also interact with nonphosphorylated oligoglucans? Finally, assuming that SEX4 acts on insoluble phosphoglucans, does the removal of phosphate esters affect the phase transition and/or the physical order of the glucans?     

A Suite of Lotus japonicus Starch Mutants Reveals Both Conserved and Novel Features of Starch Metabolism

The metabolism of starch is of central importance for many aspects of plant growth and development. Information on leaf starch metabolism other than in Arabidopsis (Arabidopsis thaliana) is scarce. Furthermore, its importance in several agronomically important traits exemplified by legumes remains to be investigated. To address this issue, we have provided detailed information on the genes involved in starch metabolism in Lotus japonicus and have characterized a comprehensive collection of forward and TILLING (for Targeting Induced Local Lesions IN Genomes) reverse genetics mutants affecting five enzymes of starch synthesis and two enzymes of starch degradation. The mutants provide new insights into the structure-function relationships of ADP-glucose pyrophosphorylase and glucan, water dikinase1 in particular. Analyses of the mutant phenotypes indicate that the pathways of leaf starch metabolism in L. japonicus and Arabidopsis are largely conserved. However, the importance of these pathways for plant growth and development differs substantially between the two species. Whereas essentially starchless Arabidopsis plants lacking plastidial phosphoglucomutase grow slowly relative to wild-type plants, the equivalent mutant of L. japonicus grows normally even in a 12-h photoperiod. In contrast, the loss of GLUCAN, WATER DIKINASE1, required for starch degradation, has a far greater effect on plant growth and fertility in L. japonicus than in Arabidopsis. Moreover, we have also identified several mutants likely to be affected in new components or regulators of the pathways of starch metabolism. This suite of mutants provides a substantial new resource for further investigations of the partitioning of carbon and its importance for symbiotic nitrogen fixation, legume seed development, and perenniality and vegetative regrowth.Recent studies in Arabidopsis (Arabidopsis thaliana) have greatly enhanced our knowledge about pathways of transitory starch metabolism (Zeeman et al., 2007; Keeling and Myers, 2010; Kötting et al., 2010; Zeeman et al., 2010). The pathway of synthesis is well established for several species, but the degradative pathway is understood only in Arabidopsis. During synthesis, the plastidial isoforms of phosphoglucoisomerase (PGI1) and phosphoglucomutase (PGM1), together with ADP-Glc pyrophosphorylase (AGPase), catalyze the conversion of the Calvin cycle intermediate Fru 6-P to ADPGlc, the substrate for starch synthases (Supplemental Fig. S1). Leaves of mutants lacking any of these three enzymes either have strongly reduced starch contents or lack starch almost completely (Caspar et al., 1985; Hanson and McHale, 1988; Lin et al., 1988a, 1988b; Kruckeberg et al., 1989; Harrison et al., 1998; Yu et al., 2000; Streb et al., 2009). In contrast, the phenotypes of mutants lacking individual enzymes that convert ADPGlc into starch vary between species and are often much less pronounced (starch synthases [Delvallé et al., 2005; Zhang et al., 2005] and starch-branching enzymes [Tomlinson et al., 1997; Blauth et al., 2001; Dumez et al., 2006]).The degradation of the starch granule in Arabidopsis leaves is catalyzed primarily by β-amylases and isoamylase 3 (Wattebled et al., 2005; Delatte et al., 2006; Fulton et al., 2008). Normal rates of degradation require phosphorylation of the starch polymers by two glucan, water dikinases, GWD1 (Ritte et al., 2002) and GWD3 (or PWD, for phosphoglucan water, dikinase; Baunsgaard et al., 2005; Kötting et al., 2005), followed by dephosphorylation by a phosphoglucan phosphatase, STARCH EXCESS4 (SEX4; Kötting et al., 2009). Maltose produced by starch degradation is exported from the chloroplast by a maltose transporter and further metabolized to hexose phosphates in the cytosol (Zeeman et al., 2007; Supplemental Fig. S1). Mutations in numerous components of this pathway result in a starch-excess phenotype, in which the starch content of leaves at the end of the night is higher than that of wild-type plants.These studies have also revealed the importance of starch turnover for the productivity of the plant. Mutants of Arabidopsis that are essentially unable to synthesize transitory starch, or with reduced rates of starch degradation at night, have a reduced rate of growth and delayed flowering time relative to wild-type plants under most conditions (Caspar et al., 1985, 1991; Eimert et al., 1995; Corbesier et al., 1998; Smith and Stitt, 2007). However, it is not known whether information about the nature and importance of starch turnover in Arabidopsis is widely applicable. Plant species differ considerably in the extent to which starch is stored in leaves at night as well as in diurnal patterns of growth and metabolic demand. The function and regulation of starch metabolism in heterotrophic organs and its importance in major physiological and developmental processes such as perenniality, vegetative regrowth, symbiotic nitrogen fixation, and the accumulation of seed storage reserves cannot be studied easily in Arabidopsis and remain largely unknown. These processes represent traits of agronomic value in legumes (Fabaceae), a family that includes some of the most agriculturally important forage (e.g. alfalfa [Medicago sativa] and clover [Trifolium spp.]), grain (e.g. pea [Pisum sativum] and common bean [Phaseolus vulgaris]), and oilseed (e.g. soybean [Glycine max]) crops.Some information is already available about starch metabolism in pea and other legume crops (Martin and Smith, 1995; Wang et al., 1998b, and refs. therein). However, characteristics including large genome sizes and recalcitrant transformation and regeneration have limited progress on these species. There is insufficient information to allow either an overview of the nature and importance of starch metabolism in legumes or a meaningful comparison with the detailed picture emerging for Arabidopsis. The development of both Lotus japonicus and Medicago truncatula as legume model systems, and the wide range of genetic and genomic resources generated for them, offer the opportunity for a systematic analysis.To elucidate the pathway of starch synthesis and degradation in legumes and provide resources for future experimentation, we screened an ethyl methanesulfonate (EMS)-mutagenized population of L. japonicus (Perry et al., 2003) for mutants altered in transitory starch metabolism and carried out genetic mapping to identify the mutation responsible for their phenotype. We also used TILLING (for Targeting Induced Local Lesions IN Genomes; McCallum et al., 2000) to confirm that the mutations identified were indeed responsible for the mutant phenotype and to obtain additional mutations in genes known to affect leaf starch content in other species. We present the results of molecular and phenotypic analyses on the mutants that provide novel insights into the structure-function relationship of the AGPase and GWD1 enzymes. In addition, our analyses reveal new information on the nature and importance of starch metabolism for plant growth and development in L. japonicus. The importance of starch accumulation and degradation and a comparison with pathways in other plant species are also discussed.     

Decreasing the Mitochondrial Synthesis of Malate in Potato Tubers Does Not Affect Plastidial Starch Synthesis, Suggesting That the Physiological Regulation of ADPglucose Pyrophosphorylase Is Context Dependent

Modulation of the malate content of tomato (Solanum lycopersicum) fruit by altering the expression of mitochondrially localized enzymes of the tricarboxylic acid cycle resulted in enhanced transitory starch accumulation and subsequent effects on postharvest fruit physiology. In this study, we assessed whether such a manipulation would similarly affect starch biosynthesis in an organ that displays a linear, as opposed to a transient, kinetic of starch accumulation. For this purpose, we used RNA interference to down-regulate the expression of fumarase in potato (Solanum tuberosum) under the control of the tuber-specific B33 promoter. Despite displaying similar reductions in both fumarase activity and malate content as observed in tomato fruit expressing the same construct, the resultant transformants were neither characterized by an increased flux to, or accumulation of, starch, nor by alteration in yield parameters. Since the effect in tomato was mechanistically linked to derepression of the reaction catalyzed by ADP-glucose pyrophosphorylase, we evaluated whether the lack of effect on starch biosynthesis was due to differences in enzymatic properties of the enzyme from potato and tomato or rather due to differential subcellular compartmentation of reductant in the different organs. The results are discussed in the context both of current models of metabolic compartmentation and engineering.Starch is the most important carbohydrate used for food and feed purposes and represents the major resource for our diet (Smith, 2008). The total yield of starch in rice (Oryza sativa), corn (Zea mays), wheat (Triticum aestivum), and potato (Solanum tuberosum) exceeds 10⁹ tons per year (Kossmann and Lloyd, 2000; Slattery et al., 2000). In addition to its use in a nonprocessed form, extracted starch is processed in many different ways, for instance as a high-Fru syrup, as a food additive, or for various technical purposes. As a result of this considerable importance, increasing the starch content of plant tissues has been a major goal for many years, with both classical breeding and biotechnological approaches being taken extensively over the last few decades (Martin and Smith, 1995; Regierer et al., 2002).The pathway by which carbon is converted from Suc to starch in the potato tuber is well established (Kruger, 1997; Fernie et al., 2002; Geigenberger et al., 2004; Geigenberger, 2011). Imported Suc is cleaved in the cytosol by Suc synthase, resulting in the formation of UDP-Glc and Fru; the UDP-Glc is subsequently converted to Glc-1-P by UDP-Glc pyrophosphorylase. The second product of the Suc synthase reaction, Fru, is efficiently phosphorylated to Fru-6-P by fructokinase (Renz et al., 1993; Davies et al., 2005). Fru-6-P is freely converted to Glc-6-P, in which form it normally enters the amyloplast (Kammerer et al., 1998; Tauberger et al., 2000; Zhang et al., 2008), and once in the plastid, it is converted to starch via the concerted action of plastidial phosphoglucomutase, ADP-Glc pyrophosphorylase (AGPase), and the various isoforms of starch synthase (Martin and Smith, 1995; Geigenberger, 2011). Of these reactions, although some of the control of starch synthesis resides in the plastidial phosphoglucomutase reaction (Fernie et al., 2001b), the AGPase reaction harbors the highest proportion of control within the linear pathway (Sweetlove et al., 1999; Geigenberger et al., 1999, 2004). In addition, considerable control resides in both the Glc-6-P phosphate antiporter (Zhang et al., 2008) and the amyloplastidial adenylate transporter (Tjaden et al., 1998; Zhang et al., 2008) as well as in reactions external to the pathways, such as the amyloplastidial adenylate kinase (Regierer et al., 2002), cytosolic UMP synthase (Geigenberger et al., 2005), and mitochondrial NAD-malic enzyme (Jenner et al., 2001).As part of our ongoing study of the constituent enzymes of the tricarboxylic acid (TCA) cycle, we made an initially surprising observation that increasing or decreasing the content of malate via a fruit-specific expression of antisense constructs targeted against the mitochondrial malate dehydrogenase or fumarase, respectively, resulted in opposing changes in the levels of starch (Centeno et al., 2011). We were able to demonstrate that these plants were characterized by an altered cellular redox balance and that this led to changes in the activation state of the AGPase reaction. Given that starch only accumulates transiently in tomato (Solanum lycopersicum; Beckles et al., 2001) as a consequence of this activation, the fruits were characterized by altered sugar content at ripening, a fact that dramatically altered their postharvest characteristics (Centeno et al., 2011). Here, we chose to express the antisense fumarase construct in potato in order to ascertain the effect of the manipulation in an organ that linearly accumulates starch across its development. The results obtained are compared and contrasted with those of the tomato fruit and within the context of current models of subcellular redox regulation.     

Characterization of the Possible Roles for B Class MADS Box Genes in Regulation of Perianth Formation in Orchid

To investigate sepal/petal/lip formation in Oncidium Gower Ramsey, three paleoAPETALA3 genes, O. Gower Ramsey MADS box gene5 (OMADS5; clade 1), OMADS3 (clade 2), and OMADS9 (clade 3), and one PISTILLATA gene, OMADS8, were characterized. The OMADS8 and OMADS3 mRNAs were expressed in all four floral organs as well as in vegetative leaves. The OMADS9 mRNA was only strongly detected in petals and lips. The mRNA for OMADS5 was only strongly detected in sepals and petals and was significantly down-regulated in lip-like petals and lip-like sepals of peloric mutant flowers. This result revealed a possible negative role for OMADS5 in regulating lip formation. Yeast two-hybrid analysis indicated that OMADS5 formed homodimers and heterodimers with OMADS3 and OMADS9. OMADS8 only formed heterodimers with OMADS3, whereas OMADS3 and OMADS9 formed homodimers and heterodimers with each other. We proposed that sepal/petal/lip formation needs the presence of OMADS3/8 and/or OMADS9. The determination of the final organ identity for the sepal/petal/lip likely depended on the presence or absence of OMADS5. The presence of OMADS5 caused short sepal/petal formation. When OMADS5 was absent, cells could proliferate, resulting in the possible formation of large lips and the conversion of the sepal/petal into lips in peloric mutants. Further analysis indicated that only ectopic expression of OMADS8 but not OMADS5/9 caused the conversion of the sepal into an expanded petal-like structure in transgenic Arabidopsis (Arabidopsis thaliana) plants.The ABCDE model predicts the formation of any flower organ by the interaction of five classes of homeotic genes in plants (Yanofsky et al., 1990; Jack et al., 1992; Mandel et al., 1992; Goto and Meyerowitz, 1994; Jofuku et al., 1994; Pelaz et al., 2000, 2001; Theißen and Saedler, 2001; Pinyopich et al., 2003; Ditta et al., 2004; Jack, 2004). The A class genes control sepal formation. The A, B, and E class genes work together to regulate petal formation. The B, C, and E class genes control stamen formation. The C and E class genes work to regulate carpel formation, whereas the D class gene is involved in ovule development. MADS box genes seem to have a central role in flower development, because most ABCDE genes encode MADS box proteins (Coen and Meyerowitz, 1991; Weigel and Meyerowitz, 1994; Purugganan et al., 1995; Rounsley et al., 1995; Theißen and Saedler, 1995; Theißen et al., 2000; Theißen, 2001).The function of B group genes, such as APETALA3 (AP3) and PISTILLATA (PI), has been thought to have a major role in specifying petal and stamen development (Jack et al., 1992; Goto and Meyerowitz, 1994; Krizek and Meyerowitz, 1996; Kramer et al., 1998; Hernandez-Hernandez et al., 2007; Kanno et al., 2007; Whipple et al., 2007; Irish, 2009). In Arabidopsis (Arabidopsis thaliana), mutation in AP3 or PI caused identical phenotypes of second whorl petal conversion into a sepal structure and third flower whorl stamen into a carpel structure (Bowman et al., 1989; Jack et al., 1992; Goto and Meyerowitz, 1994). Similar homeotic conversions for petal and stamen were observed in the mutants of the AP3 and PI orthologs from a number of core eudicots such as Antirrhinum majus, Petunia hybrida, Gerbera hybrida, Solanum lycopersicum, and Nicotiana benthamiana (Sommer et al., 1990; Tröbner et al., 1992; Angenent et al., 1993; van der Krol et al., 1993; Yu et al., 1999; Liu et al., 2004; Vandenbussche et al., 2004; de Martino et al., 2006), from basal eudicot species such as Papaver somniferum and Aquilegia vulgaris (Drea et al., 2007; Kramer et al., 2007), as well as from monocot species such as Zea mays and Oryza sativa (Ambrose et al., 2000; Nagasawa et al., 2003; Prasad and Vijayraghavan, 2003; Yadav et al., 2007; Yao et al., 2008). This indicated that the function of the B class genes AP3 and PI is highly conserved during evolution.It has been thought that B group genes may have arisen from an ancestral gene through multiple gene duplication events (Doyle, 1994; Theißen et al., 1996, 2000; Purugganan, 1997; Kramer et al., 1998; Kramer and Irish, 1999; Lamb and Irish, 2003; Kim et al., 2004; Stellari et al., 2004; Zahn et al., 2005; Hernandez-Hernandez et al., 2007). In the gymnosperms, there was a single putative B class lineage that duplicated to generate the paleoAP3 and PI lineages in angiosperms (Kramer et al., 1998; Theißen et al., 2000; Irish, 2009). The paleoAP3 lineage is composed of AP3 orthologs identified in lower eudicots, magnolid dicots, and monocots (Kramer et al., 1998). Genes in this lineage contain the conserved paleoAP3- and PI-derived motifs in the C-terminal end of the proteins, which have been thought to be characteristics of the B class ancestral gene (Kramer et al., 1998; Tzeng and Yang, 2001; Hsu and Yang, 2002). The PI lineage is composed of PI orthologs that contain a highly conserved PI motif identified in most plant species (Kramer et al., 1998). Subsequently, there was a second duplication at the base of the core eudicots that produced the euAP3 and TM6 lineages, which have been subject to substantial sequence changes in eudicots during evolution (Kramer et al., 1998; Kramer and Irish, 1999). The paleoAP3 motif in the C-terminal end of the proteins was retained in the TM6 lineage and replaced by a conserved euAP3 motif in the euAP3 lineage of most eudicot species (Kramer et al., 1998). In addition, many lineage-specific duplications for paleoAP3 lineage have occurred in plants such as orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009), Ranunculaceae, and Ranunculales (Kramer et al., 2003; Di Stilio et al., 2005; Shan et al., 2006; Kramer, 2009).Unlike the A or C class MADS box proteins, which form homodimers that regulate flower development, the ability of B class proteins to form homodimers has only been reported in gymnosperms and in the paleoAP3 and PI lineages of some monocots. For example, LMADS1 of the lily Lilium longiflorum (Tzeng and Yang, 2001), OMADS3 of the orchid Oncidium Gower Ramsey (Hsu and Yang, 2002), and PeMADS4 of the orchid Phalaenopsis equestris (Tsai et al., 2004) in the paleoAP3 lineage, LRGLOA and LRGLOB of the lily Lilium regale (Winter et al., 2002), TGGLO of the tulip Tulipa gesneriana (Kanno et al., 2003), and PeMADS6 of the orchid P. equestris (Tsai et al., 2005) in the PI lineage, and GGM2 of the gymnosperm Gnetum gnemon (Winter et al., 1999) were able to form homodimers that regulate flower development. Proteins in the euAP3 lineage and in most paleoAP3 lineages were not able to form homodimers and had to interact with PI to form heterodimers in order to regulate petal and stamen development in various plant species (Schwarz-Sommer et al., 1992; Tröbner et al., 1992; Riechmann et al., 1996; Moon et al., 1999; Winter et al., 2002; Kanno et al., 2003; Vandenbussche et al., 2004; Yao et al., 2008). In addition to forming dimers, AP3 and PI were able to interact with other MADS box proteins, such as SEPALLATA1 (SEP1), SEP2, and SEP3, to regulate petal and stamen development (Pelaz et al., 2000; Honma and Goto, 2001; Theißen and Saedler, 2001; Castillejo et al., 2005).Orchids are among the most important plants in the flower market around the world, and research on MADS box genes has been reported for several species of orchids during the past few years (Lu et al., 1993, 2007; Yu and Goh, 2000; Hsu and Yang, 2002; Yu et al., 2002; Hsu et al., 2003; Tsai et al., 2004, 2008; Xu et al., 2006; Guo et al., 2007; Kim et al., 2007; Chang et al., 2009). Unlike the flowers in eudicots, the nearly identical shape of the sepals and petals as well as the production of a unique lip in orchid flowers make them a very special plant species for the study of flower development. Four clades (1–4) of genes in the paleoAP3 lineage have been identified in several orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009). Several works have described the possible interactions among these four clades of paleoAP3 genes and one PI gene that are involved in regulating the differentiation and formation of the sepal/petal/lip of orchids (Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009). However, the exact mechanism that involves the orchid B class genes remains unclear and needs to be clarified by more experimental investigations.O. Gower Ramsey is a popular orchid with important economic value in cut flower markets. Only a few studies have been reported on the role of MADS box genes in regulating flower formation in this plant species (Hsu and Yang, 2002; Hsu et al., 2003; Chang et al., 2009). An AP3-like MADS gene that regulates both floral formation and initiation in transgenic Arabidopsis has been reported (Hsu and Yang, 2002). In addition, four AP1/AGAMOUS-LIKE9 (AGL9)-like MADS box genes have been characterized that show novel expression patterns and cause different effects on floral transition and formation in Arabidopsis (Hsu et al., 2003; Chang et al., 2009). Compared with other orchids, the production of a large and well-expanded lip and five small identical sepals/petals makes O. Gower Ramsey a special case for the study of the diverse functions of B class MADS box genes during evolution. Therefore, the isolation of more B class MADS box genes and further study of their roles in the regulation of perianth (sepal/petal/lip) formation during O. Gower Ramsey flower development are necessary. In addition to the clade 2 paleoAP3 gene OMADS3, which was previously characterized in our laboratory (Hsu and Yang, 2002), three more B class MADS box genes, OMADS5, OMADS8, and OMADS9, were characterized from O. Gower Ramsey in this study. Based on the different expression patterns and the protein interactions among these four orchid B class genes, we propose that the presence of OMADS3/8 and/or OMADS9 is required for sepal/petal/lip formation. Further sepal and petal formation at least requires the additional presence of OMADS5, whereas large lip formation was seen when OMADS5 expression was absent. Our results provide a new finding and information pertaining to the roles for orchid B class MADS box genes in the regulation of sepal/petal/lip formation.     

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.     

Analysis of the Rice ADP-Glucose Transporter (OsBT1) Indicates the Presence of Regulatory Processes in the Amyloplast Stroma That Control ADP-Glucose Flux into Starch

Previous studies showed that efforts to further elevate starch synthesis in rice (Oryza sativa) seeds overproducing ADP-glucose (ADPglc) were prevented by processes downstream of ADPglc synthesis. Here, we identified the major ADPglc transporter by studying the shrunken3 locus of the EM1093 rice line, which harbors a mutation in the BRITTLE1 (BT1) adenylate transporter (OsBt1) gene. Despite containing elevated ADPglc levels (approximately 10-fold) compared with the wild-type, EM1093 grains are small and shriveled due to the reduction in the amounts and size of starch granules. Increases in ADPglc levels in EM1093 were due to their poor uptake of ADP-[¹⁴C]glc by amyloplasts. To assess the potential role of BT1 as a rate-determining step in starch biosynthesis, the maize ZmBt1 gene was overexpressed in the wild-type and the GlgC (CS8) transgenic line expressing a bacterial glgC-TM gene. ADPglc transport assays indicated that transgenic lines expressing ZmBT1 alone or combined with GlgC exhibited higher rates of transport (approximately 2-fold), with the GlgC (CS8) and GlgC/ZmBT1 (CS8/AT5) lines showing elevated ADPglc levels in amyloplasts. These increases, however, did not lead to further enhancement in seed weights even when these plant lines were grown under elevated CO₂. Overall, our results indicate that rice lines with enhanced ADPglc synthesis and import into amyloplasts reveal additional barriers within the stroma that restrict maximum carbon flow into starch.Cereal grains contribute a significant portion of worldwide starch production. Unlike other plant tissue, starch biosynthesis in the endosperm storage organ of cereal grains is unique in its dependence on two ADP-Glc pyrophosphorylase (AGPase) isoforms (Denyer et al., 1996; Thorbjørnsen et al., 1996; Sikka et al., 2001), a major cytosolic enzyme and a minor plastidial one, to generate ADP-glucose (ADPglc), the sugar nucleotide utilized by starch synthases in the amyloplast (Cakir et al., 2015). The majority of ADPglc in cereal endosperm is generated in the cytosol from AGPase (Tuncel and Okita, 2013) as well as by Suc synthase (Tuncel and Okita, 2013; Bahaji et al., 2014) and subsequently transported into amyloplasts by the BRITTLE-1 (BT1) protein located at the plastid envelope (Cao et al., 1995; Shannon et al., 1998).The Bt1 gene, first identified in maize (Zea mays; Mangelsdorf, 1926) and isolated by Sullivan et al. (1991), encodes a major amyloplast membrane protein ranging from 39 to 44 kD (Cao et al., 1995). The BT1 protein and its homologs belong to the mitochondrial carrier family (Sullivan et al., 1991; Haferkamp, 2007), which has a diverse range of substrates (Patron et al., 2004; Leroch et al., 2005; Kirchberger et al., 2008). The assignment of BT1 protein as the ADPglc transporter in cereal endosperms was first proposed by Sullivan et al. (1991), and then it was characterized based on the increased ADPglc levels and reduced ADPglc import rate in endosperms of BT1-deficient maize and barley (Hordeum vulgare) mutants (Tobias et al., 1992; Shannon et al., 1996, 1998; Patron et al., 2004). Biochemical transport studies of the maize BT1 showed that it imported ADPglc by counter exchanging with ADP (Kirchberger et al., 2007). The wheat (Triticum aestivum) BT1 homolog also transports ADPglc but has similar affinities for ADP and AMP as the counter-exchange substrate (Bowsher et al., 2007).Evidence from previous studies by our laboratory (Sakulsingharoj et al., 2004; Nagai et al., 2009) suggested the potential role of BT1 as well as other downstream processes as a rate-limiting step in starch biosynthesis in the transgenic rice (Oryza sativa) GlgC (CS8) lines overexpressing an up-regulated AGPase (Escherichia coli glgC-TM). In GlgC (CS8) rice lines, grain weights (starch) are elevated up to 15% compared with wild-type plants, indicating that the AGPase-catalyzed reaction is a rate-limiting step in starch biosynthesis under normal conditions. When transgenic GlgC (CS8) plants were grown under elevated CO₂ levels, no further increases in grain weight were evident compared with those grown at ambient CO₂. As Suc levels are elevated in leaf blades, leaf sheaths, culms (Rowland-Bamford et al., 1990), and peduncle exudates (Chen et al., 1994) in rice plants grown under elevated CO₂, developing GlgC (CS8) grains were unable to convert the increased levels of sugars into starch. This lack of increase indicated that the AGPase-catalyzed reaction (ADPglc synthesis) was no longer rate limiting and that one or more downstream processes regulated carbon flux from source tissues in developing GlgC (CS8) endosperm (Sakulsingharoj et al., 2004). This view is also supported by a subsequent metabolite study in which several GlgC (CS8) lines were found to contain up to 46% higher ADPglc levels than wild-type plants (Nagai et al., 2009). As this increase in ADPglc levels was nearly 3-fold higher than the increase in grain weight, starch biosynthesis is saturated with respect to ADPglc levels and carbon flow into starch is restricted by one or more downstream steps. Potential events that may limit the utilization of ADPglc in starch in GlgC (CS8) lines are the import of this sugar nucleotide via the BT1 transporter into amyloplasts and/or the utilization of ADPglc by starch synthases. Mutant analysis of the two major starch synthases indicated no significant impact on grain weight when one of these starch synthases was nonfunctional, suggesting that this enzyme activity, contributed by multiple enzyme isoforms, is present at excessive levels (Fujita et al., 2006, 2007). Therefore, we suspected that BT1 is the likely candidate limiting carbon flow into starch in GlgC (CS8) endosperms.The aim of this study was to investigate the role of BT1 in mediating the transport of ADPglc into amyloplast and to determine whether this transport activity is rate limiting in rice endosperm. In order to address these questions, we show that BT1 is the major transporter of ADPglc by analysis of the EM1093 rice line, which contains a mutation at the shrunken3 (shr3) locus and, specifically, in the OsBt1-1 gene. Second, we assessed the impact of the expression of the maize ZmBt1 gene in wild-type and GlgC (CS8) seeds to determine the potential limiting role of BT1 transport activity on starch biosynthesis. Our results indicate that BT1 is essential for starch synthesis but is not rate limiting and that one or more stroma-localized processes limit maximum carbon flow into starch.     

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).