Conserved structure and varied expression reveal key roles of phosphoglucan phosphatase gene starch excess 4 in barley

As one of the phosphoglucan phosphatases, starch excess 4 (SEX4) encoded by SEX4 gene has recently been intensively studied because of its vital role in the degradation of leaf starch. In this study, we isolated and chromosomally mapped barley SEX4, characterized its gene and protein structure, predicted the cis-elements of its promoter, and analysed its expression based on real-time quantitative PCR and publically available microarray data. The full length of barely SEX4 (HvSEX4) was 4,598 bp and it was mapped on the long arm of chromosome 4H (4HL). This gene contained 14 exons and 13 introns in all but two of the species analysed, Arabidopsis (13 exons and 12 introns) and Oryza brachyantha (12 exons and 11 introns). An exon–intron junction composed of intron 4 to intron 7 and exon 5 to exon 8 was highly conserved among the analysed species. SEX4 is characterized with conserved functional domains (dual specificity phosphatase domain and carbohydrate-binding module 48) and varied chloroplast transit peptide and C-terminal. Expression analyses indicated that: (1) SEX4 was mainly expressed in anthers of barley, young leaf and anthers of rice, and leaf of Arabidopsis; (2) it exhibited a diurnal pattern in barley, rice and Arabidopsis; (3) significant difference in the expression of SEX4 was not detected for either barley or rice under any of the investigated stresses; and (4) it was significantly down-regulated at middle stage and up-regulated at late stage under cold treatment, down-regulated at early stage under heat treatment, and up-regulated at late stage under salt treatment in Arabidopsis. The strong relationships detected in the current study between SEX4 and glucan, water dikinases (GWD) or phosphoglucan, water dikinases (PWD) were discussed. Collectively, our results provide insights into genetic manipulation of SEX4, especially in monocotyledon and uncovering the possible roles of SEX4 in plant development.     

Double Knockout Mutants of Arabidopsis Grown under Normal Conditions Reveal that the Plastidial Phosphorylase Isozyme Participates in Transitory Starch Metabolism

In leaves of two starch-related single-knockout lines lacking either the cytosolic transglucosidase (also designated as disproportionating enzyme 2, DPE2) or the maltose transporter (MEX1), the activity of the plastidial phosphorylase isozyme (PHS1) is increased. In both mutants, metabolism of starch-derived maltose is impaired but inhibition is effective at different subcellular sites. Two constitutive double knockout mutants were generated (designated as dpe2-1 × phs1a and mex1 × phs1b) both lacking functional PHS1. They reveal that in normally grown plants, the plastidial phosphorylase isozyme participates in transitory starch degradation and that the central carbon metabolism is closely integrated into the entire cell biology. All plants were grown either under continuous illumination or in a light-dark regime. Both double mutants were compromised in growth and, compared with the single knockout plants, possess less average leaf starch when grown in a light-dark regime. Starch and chlorophyll contents decline with leaf age. As revealed by transmission electron microscopy, mesophyll cells degrade chloroplasts, but degradation is not observed in plants grown under continuous illumination. The two double mutants possess similar but not identical phenotypes. When grown in a light-dark regime, mesophyll chloroplasts of dpe2-1 × phs1a contain a single starch granule but under continuous illumination more granules per chloroplast are formed. The other double mutant synthesizes more granules under either growth condition. In continuous light, growth of both double mutants is similar to that of the parental single knockout lines. Metabolite profiles and oligoglucan patterns differ largely in the two double mutants.During the last two decades, biochemical analyses of starch metabolism in higher plants have been favored by the availability of large sets of insertion mutants deficient in a single starch-related gene product. Based on phenotypical characterization of these mutants followed by the identification of the respective locus in the genome, novel starch-related proteins were discovered that reside inside the plastid, in the cytosol, in the nucleus, and in the plastidial envelope membranes. Taken together, these results have largely altered the current view on starch metabolism (Zeeman et al., 2010; Fettke et al., 2012a; Smith, 2012).Despite this progress, phenotypical analyses of starch-related mutants are complex and, under certain circumstances, yield misleading conclusions. Loss of function of metabolic steps may cause the entire starch synthesizing or degrading process to become nonfunctional. In this case, mutants are expected to have starch levels that are significantly altered. If, however, single knockout mutants are capable of partially or fully compensating the loss of function by other routes, the resulting phenotypes are less obvious and more difficult to predict. Carbon fluxes through existing paths may be enhanced, or novel metabolic routes may be established that compensate the lost function. As an example, leaves of Arabidopsis (Arabidopsis thaliana) mutants constitutively lacking the plastidial hexose-phosphate isomerase strongly express a distinct plastidial Glc-6-P/orthophosphate antiporter isoform that in wild-type plants is found only in heterotrophic tissues (Kunz et al., 2010). In mesophyll cells of the mutant, the reductive pentose phosphate cycle cannot drive assimilatory starch biosynthesis, as chloroplasts are unable to convert Fru-6-P to Glc-6-P. However, their capacity of transporting Glc-6-P between the cytosolic and the chloroplastic compartment is strongly increased. Furthermore, nonfunctionality of some starch-related proteins can lead to enlarged or diminished metabolite pools that via sensing processes, lead to cellular alterations distant from central carbon metabolism. This complexity is evidenced by several starch-related Arabidopsis mutants that possess a largely altered plastidial ultrastructure and exhibit premature degradation of the entire chloroplast (Stettler et al., 2009; Cho et al., 2011).Furthermore, several starch-related enzymes are capable of forming homomeric or heteromeric complexes that are functionally relevant but, to some extent, variable (Delatte et al., 2005; Utsumi and Nakamura, 2006; Kubo et al., 2010; Emes and Tetlow, 2012; Nakamura et al., 2012; Streb et al., 2012).In starch or glycogen storing prokaryotic and eukaryotic cells, α-glucan phosphorylase (EC 2.4.1.1) is common. Initially, this enzyme was considered to be the main starch synthesizing activity (Hanes, 1940). Later, both starch and glycogen synthases have been discovered that utilize either ADPglucose or UDPglucose (or both; Deschamps et al., 2006) as hexosyl donor. Ample evidence has been presented that these enzymes are essential biosynthetic enzymes (Ballicora et al., 2003; Zeeman et al., 2010; Roach et al., 2012; Palm et al., 2013). Furthermore, it is widely accepted that in glycogen-storing cells, phosphorylase is indispensible for the degradation of the storage polysaccharide (Hwang et al., 1989; Alonso-Casajús et al., 2006; Wilson et al., 2010; Roach et al., 2012; Gazzerro et al., 2013).In plant cells, the metabolic function of phosphorylase is more complex and far from being clear. In lower and higher plants, two distinct phosphorylase types exist as plastid- and cytosol-specific isozymes and are designated as Pho1 (or, in Arabidopsis, PHS1) and Pho2 (PHS2), respectively. Based on the large differences in the affinities for glycogen, the plastidial and the cytosolic phosphorylases are also named as low-affinity (L-type) and high-affinity (H-type) isozymes, respectively. As starch is restricted to the plastids, only the Pho1 (PHS1) type appears to possess direct access to native starch and/or plastidial starch-derived α-glucans.Conflicting phenotypical features have been reported for several mutants possessing altered levels of the plastidial phosphorylase isozyme(s). In the starch-related mutant4 of the unicellular green alga Chlamydomonas reinhardtii, the lack of one plastidial Pho1 isozyme (designated as PhoB) was associated with a lower cellular starch content, abnormally shaped granules, a modified amylopectin structure, and an elevated amylose-to-amylopectin ratio when the cells were kept under nitrogen limitation (Dauvillée et al., 2006). These phenotypical features suggest an involvement of the plastidial phosphorylase PhoB in the biosynthesis of a storage polysaccharide resembling the reserve starch of higher plants. Similarly, a rapid incorporation of ¹⁴C into starch was observed when tuber discs from various transgenic potato lines were incubated with [U-¹⁴C]Glc-1-P. The rate of starch labeling was found to reflect the activity of the plastidial phosphorylase isozyme Pho1 (Fettke et al., 2010, 2012b). By contrast, transgenic potato (Solanum tuberosum) lines have been generated that due to expression of an antisense construct, possess a largely diminished total Pho1 activity in leaves. Leaf starch content is essentially unchanged compared with that of the wild-type plants, suggesting that under normal growth conditions, the plastidial phosphorylase is not necessarily involved in starch metabolism or, alternatively, can easily be replaced by other enzymes (Sonnewald et al., 1995). Likewise, the phenotype (including leaf starch content) of an Arabidopsis mutant lacking functional PHS1 has been reported not to differ from the wild type when the plants were grown under normal conditions. However, under water stress conditions, significantly more local leaf lesions have been reported to occur (Zeeman et al., 2004).When leaf discs from bean (Phaseolus vulgaris) or Arabidopsis plants were exposed to conditions favoring photorespiration (i.e. an atmosphere consisting of 30% [v/v] O₂ and 70% [v/v] N₂ but lacking CO₂), transitory starch was degraded in the light at a high rate and the plastidial Glc-6-P pool increased. In Arabidopsis mutants deficient in PHS1, the Glc monophosphate pool did not respond to photorespiratory conditions (Weise et al., 2006). These data lead to the conclusion that in illuminated leaves with very high rates of photorespiration, PHS1 is involved in the conversion of starch to Glc monophosphates but does not to participate in the nocturnal starch degradation.When studying several starch-related Arabidopsis mutants, we noticed that two single knockout mutations that both affect the maltose metabolism but differ in the subcellular location of the target protein possess a significantly increased PHS1 activity (Malinova et al., 2011a, 2011b). One mutant constitutively lacks the functional cytosolic transglucosidase (also designated as disproportionating enzyme2; DPE2) and, therefore, the cytosolic route of starch-derived maltose metabolism is impaired (Chia et al., 2004; Lu and Sharkey, 2004). The other mutant does not express the plastidial maltose transporter MEX1, resulting in a massively enlarged maltose pool (Niittylä et al., 2004). Thus, in the two mutants, the metabolism of starch-derived maltose is blocked at different subcellular sites, i.e. the cytosol and the chloroplast. The enhanced PHS1 activity as observed for the two mutants is difficult to explain unless a more general function of the phosphorylase isozyme in starch metabolism is assumed.For a detailed functional analysis of PHS1-related processes, we generated two types of constitutive PHS1-deficient double knockout mutants (DPE2 plus PHS1 or MEX1 plus PHS1) and studied their phenotypes in more detail under various experimental conditions. Shoot growth and leaf chlorophyll content are reduced when the plants are grown under a light-dark regime, but under continuous illumination, both effects are far less pronounced. Based on these data, we propose that the plastidial phosphorylase participates in both the turnover of transitory starch and in the maintenance of intact chloroplasts.     

Rice endosperm-specific plastidial α-glucan phosphorylase is important for synthesis of short-chain malto-oligosaccharides

Previous genetic studies have indicated that the type L α-glucan phosphorylase (Pho1) has an essential role during the initiation process of starch biosynthesis during rice seed development. To gain insight into its role in starch metabolism, we characterized the enzymatic properties of the Pho1 recombinant form. Pho1 has significantly higher catalytic efficiency toward both linear and branched α-glucans in the synthesis direction than in the degradation direction with equilibrium constants for the various substrates ranging from 13 to 45. Pho1 activity is strongly inhibited by its own reaction product (Pi) in the synthesis reaction (K_i = 0.69 mM) when amylopectin is the primer substrate, but this inhibition is less pronounced (K_i = 14.2 mM) when short α-glucan chains are used as primers. Interestingly, even in the presence of Pi alone, Pho1 not only degrades maltohexaose but also extends them to synthesize longer MOSs. Production of a broad spectrum of MOSs (G4-G19) was stimulated by both Pi and Glc1P in an additive fashion. Thus, even under physiological conditions of high Pi/Glc1P, Pho1 extends the chain length of short MOSs which can then be used as subsequent primer by starch synthase activities. As ADP-glucose strongly inhibits Pho1activity, Pho1 likely operates only during the initial stage and not during maturation phase of starch synthesis.     

Starch-related cytosolic heteroglycans in roots from Arabidopsis thaliana

Both photoautotrophic and heterotrophic plant cells are capable of accumulating starch inside the plastid. However, depending on the metabolic state of the respective cell the starch-related carbon fluxes are different. The vast majority of the transitory starch biosynthesis relies on the hexose phosphate pools derived from the reductive pentose phosphate cycle and, therefore, is restricted to ongoing photosynthesis. Transitory starch is usually degraded in the subsequent dark period and mainly results in the formation of neutral sugars, such as glucose and maltose, that both are exported into the cytosol. The cytosolic metabolism of the two carbohydrates includes reversible glucosyl transfer reactions to a heteroglycan that are mediated by two glucosyl transferases, DPE2 and PHS2 (or, in all other species, Pho2).In heterotrophic cells, accumulation of starch mostly depends on the long distance transport of reduced carbon compounds from source to sink organs and, therefore, includes as an essential step the import of carbohydrates from the cytosol into the starch forming plastids.In this communication, we focus on starch metabolism in heterotrophic tissues from Arabidopsis thaliana wild type plants (and in various starch-related mutants as well). By using hydroponically grown A. thaliana plants, we were able to analyse starch-related biochemical processes in leaves and roots from the same plants. Within the roots we determined starch levels and the morphology of native starch granules. Cytosolic and apoplastic heteroglycans were analysed in roots and compared with those from leaves of the same plants. A. thaliana mutants lacking functional enzymes either inside the plastid (such as phosphoglucomutase) or in the cytosol (disproportionating isoenzyme 2 or the phosphorylase isozyme, PHS2) were included in this study. In roots and leaves from the three mutants (and from the respective wild type organ as well), starch and heteroglycans as well as enzyme patterns were analysed.     

Characterization of plastidial starch phosphorylase in Triticum aestivum L. endosperm

Starch phosphorylase (Pho) catalyses the reversible transfer of glucosyl units from glucose1-phosphate to the non-reducing end of an α-1,4-linked glucan chain. Two major isoforms of Pho exist in the plastid (Pho1) and cytosol (Pho2). In this paper it is proposed that Pho1 may play an important role in recycling glucosyl units from malto-oligosaccharides back into starch synthesis in the developing wheat endosperm. Pho activity was observed in highly purified amyloplast extracts prepared from developing wheat endosperms, representing the first direct evidence of plastidial Pho activity in this tissue. A full-length cDNA clone encoding a plastidial Pho isoform, designated TaPho1, was also isolated from a wheat endosperm cDNA library. The TaPho1 protein and Pho1 enzyme activity levels were shown to increase throughout the period of starch synthesis. These observations add to the growing body of evidence which indicates that this enzyme class has a role in starch synthesis in wheat endosperm and indeed all starch storing tissues.     

Cloning and Characterization of the Wx Gene Encoding a Granule-Bound Starch Synthase in Lotus (Nelumbo nucifera Gaertn)

The granule-bound starch synthase (GBSS) proteins were widely considered as one of the most important enzymes in plant amylose synthesis. However, understanding of the molecular basis of the GBSS protein in lotus remains fragmented. In this work, a lotus Wx gene, encoding a GBSS (GenBank accession no. EU938541), was isolated and characterized. This gene comprises 13 exons and 12 introns and covers 4152?bp (GenBank accession no. FJ602702). The exons of Wx gene have similar lengths, while the introns vary greatly. Phylogenetic tree indicated that the lotus GBSS protein belongs to a GBSS I subgroup. The expression of the Wx gene varies in different organs of the lotus during its development process and is also expressed differently in different cultivars. The Wx gene is expressed at a higher level in the rhizomes of cultivar Meirenhong than in those of cultivar Elian 4. This study elucidates more molecular information about the Wx gene in lotus and provides a theoretical foundation for the genes regulation and the modification of starch quality.     

Genomic and functional characterization of StCDPK1

StCDPK1 is a calcium dependent protein kinase expressed in tuberizing potato stolons and in sprouting tubers. StCDPK1 genomic sequence contains eight exons and seven introns, the gene structure is similar to Arabidopsis, rice and wheat CDPKs belonging to subgroup IIa. There is one copy of the gene per genome and it is located in the distal portion of chromosome 12. Western blot and immunolocalization assays (using confocal and transmission electron microscopy) performed with a specific antibody against StCDPK1 indicate that this kinase is mainly located in the plasma membrane of swelling stolons and sprouting tubers. Sucrose (4–8%) increased StCDPK1 protein content in non-induced stolons, however the amount detected in swelling stolons was higher. Transgenic lines with reduced expression of StCDPK1 (β7) did not differ from controls when cultured under multiplication conditions, but when grown under tuber inducing conditions some significant differences were observed: the β7 line tuberized earlier than controls without the addition of CCC (GA inhibitor), developed more tubers than wild type plants in the presence of hormones that promote tuberization in potato (ABA and BAP) and was more insensitive to GA action (stolons were significantly shorter than those of control plants). StCDPK1 expression was induced by GA, ABA and BAP. Our results suggest that StCDPK1 plays a role in GA-signalling and that this kinase could be a converging point for the inhibitory and promoting signals that influence the onset of potato tuberization.     

Structural organisation,expression, and promoter analysis of a 16R isoform of 14-3-3 protein gene from potato

Six full-length cDNAs encoding 14-3-3 proteins from potato (Solanum tuberosum L. cv. Desiree) plants have been recently isolated and sequenced. Screening of a potato genomic library with the 16R cDNA encoding 14-3-3 protein isoform resulted in the identification and isolation of the respective genomic clone. The gene contains four exons and three introns. Inspection of the promoter sequence of the 16R gene revealed several boxes important for the regulation of the gene expression. The induction of the promoter activity by sucrose, IAA, ABA and salicylic acid has been shown. Dof protein-binding sequences, E-boxes and sequences responsible for developmental regulation are most frequently represented. Northern blot and fluorometric analyses, as well as the microscopic examination of transgenic potato plants transformed with GUS reporter under 14-3-3 protein promoter, provide evidence for tissue-specific expression and age-dependent promoter activity. Significant GUS expression was observed in young organs or organ portions, as well as in minor vascular bundles of mature organs.     

Genomic organization and expression analysis of a farnesyl diphosphate synthase gene (FPPS2) in apples (Malus domestica Borkh.)

A farnesyl diphosphate synthase gene (FPPS2), which contains 11 introns and 12 exons, was isolated from the apple cultivar “White Winter Pearmain”. When it was compared to our previously reported FPPS1, its each intron size was different, its each exon size was the same as that of FPPS1 gene, 30 nucleotide differences were found in its coding sequence. Based on these nucleotide differences, specific primers were designed to perform expression analysis; the results showed that it expressed in both fruit and leaf, its expression level was obviously lower than that of FPPS1 gene in fruit which was stored at 4 °C for 5 weeks. This is the first report concerning two FPPS genes and their expression comparison in apples.     

Characterization of rat 17β-hydroxysteroid dehydrogenase type 1 gene and mRNA transcripts

In the present study, the gene encoding rat 17β-hydroxysteroid dehydrogenase type 1 (rHSD17B1 gene) was cloned and characterized. Like the analogous human gene (hHSD17B1), rHSD17B1 contains six exons and five introns spanning approximately 2.2 kb. The identity between the exons and introns of the two genes ranges from 58% to 82% and 42% to 57%, respectively. In contrast to hHSD17B1, rHSD17B1 is not duplicated. The cap site for rHSD17B1 was localized to position −41 upstream of the ATG translation initiation codon. Sequence comparison of the first 200 bp upstream of the cap site showed 72% identity between the human and rat HSD17B1 genes, including a conserved GC-rich area. Further upstream, no significant identity between the two genes was observed and several, cis-acting elements known to modulate the expression of hHSD17B1 are not conserved in the rat gene. Rat HSD17B1 unlike hHSD17B1 with two cap sites, possesses two polyadenylation signals, thus resulting in two mRNAs.     

Structure and expression of the <Emphasis Type="Italic">TaGW7</Emphasis> in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

OsGW7 (also known as OsGL7) is homologous to the Arabidopsis thaliana gene that encodes LONGIFOLIA protein, which regulates cell elongation, and is involved in regulating grain length in rice. However, our knowledge on its ortholog in wheat, TaGW7, is limited. In this study, we identified and mapped TaGW7 in wheat, characterized its nucleotide and protein structures, predicted the cis-elements of its promoter, and analysed its expression patterns. The GW7 orthologs in barley (HvGW7), rice (OsGW7), and Brachypodium distachyon (BdGW7) were also identified for comparative analyses. TaGW7 mapped onto the short arms of group 2 chromosomes (2AS, 2BS, and 2DS). Multiple alignments indicated GW7 possesses five exons and four introns in all but two of the species analysed. An exon–intron junction composed of introns 3–4 and exons 4–5 was highly conserved. GW7 has a conserved domain (DUF 4378) and two neighbouring low complexity regions. GW7 was mainly expressed in wheat spikes and stems, in barley seedling crowns, and in rice anthers and embryo-sacs during early development. Drought and heat significantly increased and decreased GW7 expression in wheat, respectively. In barley, GW7 was significantly down-regulated in paleae and awns but up-regulated in seeds under drought treatment and down-regulated under Fusarium and stem rust inoculation. In rice, OsGW7 expression differed significantly under drought treatments. Collectively, these results provide insights into GW7 structure and expression in wheat, barley and rice. The GW7 sequence structure and expression data are the foundation for manipulating GW7 and uncovering its roles in plants.