Genetic manipulation of trehalose-6-phosphate synthase results in changes in the soluble sugar profile in transgenic sugarcane stems.

Trehalose is a non-reducing disaccharide widely distributed in nature. The trehalose biosynthetic intermediate, trehalose 6-phosphate (Tre6P) is an essential regulatory and signaling molecule involved in both regulation of carbon metabolism and photosynthesis. To investigate the effect of altered trehalose synthesis on sucrose accumulation in sugarcane (Saccharum spp. hybrid), we independently overexpressed the Escherichia coli otsA (trehalose-6-phosphate synthase; TPS) and otsB (trehalose-6-phosphate phosphatase; TPP) genes and additionally partially silenced native TPS expression. In mature cane, sucrose levels in the otsA transgenic plants were lowered, whereas sucrose levels in the otsB transgenic plants were increased. Partial silencing of TPS expression in sugarcane transformed with a TPS-targeted microRNA recombinant construct was confirmed in leaf and mature internode tissue of transgenic plants. Most of the silencing transgenic lines accumulated trehalose at lower levels than the wild-type (WT) plants. The immature stalk tissue of these transgenic lines had lower levels of glucose and fructose, whereas the mature internode tissue had lower sucrose and glucose levels, when compared with the WT. Furthermore, various minor metabolites and sugars were detected in the sugarcane plants, which mostly decreased as the stalk tissue of the cane matured. The results demonstrate that manipulation of Tre6P/trehalose metabolism has the potential to modify the profile of soluble sugars accumulated in sugarcane stems.

Mutations in Glucan, Water Dikinase Affect Starch Degradation and Gametophore Development in the Moss Physcomitrella patens.

The role of starch degradation in non-vascular plants is poorly understood. To expand our knowledge of this area, we have studied this process in Physcomitrella patens. This has been achieved through examination of the step known to initiate starch degradation in angiosperms, glucan phosphorylation, catalysed by glucan, water dikinase (GWD) enzymes. Phylogenetic analysis indicates that GWD isoforms can be divided into two clades, one of which contains GWD1/GWD2 and the other GWD3 isoforms. These clades split at a very early stage within plant evolution, as distinct sequences that cluster within each were identified in all major plant lineages. Of the five genes we identified within the Physcomitrella genome that encode GWD-like enzymes, two group within the GWD1/GWD2 clade and the others within the GWD3 clade. Proteins encoded by both loci in the GWD1/GWD2 clade, named PpGWDa and PpGWDb, are localised in plastids. Mutations of either PpGWDa or PpGWDb reduce starch phosphate abundance, however, a mutation at the PpGWDa locus had a much greater influence than one at PpGWDb. Only mutations affecting PpGWDa inhibited starch degradation. Mutants lacking this enzyme also failed to develop gametophores, a phenotype that could be chemically complemented using glucose supplementation within the growth medium.

Modification of Cassava Root Starch Phosphorylation Enhances Starch Functional Properties.

Cassava (Manihot esculenta Crantz) is a root crop used as a foodstuff and as a starch source in industry. Starch functional properties are influenced by many structural features including the relative amounts of the two glucan polymers amylopectin and amylose, the branched structure of amylopectin, starch granule size and the presence of covalent modifications. Starch phosphorylation, where phosphates are linked either to the C3 or C6 carbon atoms of amylopectin glucosyl residues, is a naturally occurring modification known to be important for starch remobilization. The degree of phosphorylation has been altered in several crops using biotechnological approaches to change expression of the starch-phosphorylating enzyme GLUCAN WATER DIKINASE (GWD). Interestingly, this frequently alters other structural features of starch beside its phosphate content. Here, we aimed to alter starch phosphorylation in cassava storage roots either by manipulating the expression of the starch phosphorylating or dephosphorylating enzymes. Therefore, we generated transgenic plants in which either the wild-type potato GWD (StGWD) or a redox-insensitive version of it were overexpressed. Further plants were created in which we used RNAi to silence each of the endogenous phosphoglucan phosphatase genes STARCH EXCESS 4 (MeSEX4) and LIKE SEX4 2 (MeLSF), previously discovered by analyzing leaf starch metabolism in the model species Arabidopsis thaliana. Overexpressing the potato GWD gene (StGWD), which specifically phosphorylates the C6 position, increased the total starch-bound phosphate content at both the C6 and the C3 positions. Silencing endogenous LSF2 gene (MeLSF2), which specifically dephosphorylates the C3 position, increased the ratio of C3:C6 phosphorylation, showing that its function is conserved in storage tissues. In both cases, other structural features of starch (amylopectin structure, amylose content and starch granule size) were unaltered. This allowed us to directly relate the physicochemical properties of the starch to its phosphate content or phosphorylation pattern. Starch swelling power and paste clarity were specifically influenced by total phosphate content. However, phosphate position did not significantly influence starch functional properties. In conclusion, biotechnological manipulation of starch phosphorylation can specifically alter certain cassava storage root starch properties, potentially increasing its value in food and non-food industries.

Repression of Sex4 and Like Sex Four2 Orthologs in Potato Increases Tuber Starch Bound Phosphate With Concomitant Alterations in Starch Physical Properties.

To examine the roles of starch phosphatases in potatoes, transgenic lines were produced where orthologs of SEX4 and LIKE SEX FOUR2 (LSF2) were repressed using RNAi constructs. Although repression of either SEX4 or LSF2 inhibited leaf starch degradation, it had no effect on cold-induced sweetening in tubers. Starch amounts were unchanged in the tubers, but the amount of phosphate bound to the starch was significantly increased in all the lines, with phosphate bound at the C6 position of the glucosyl units increased in lines repressed in StSEX4 and in the C3 position in lines repressed in StLSF2 expression. This was accompanied by a reduction in starch granule size and an alteration in the constituent glucan chain lengths within the starch molecule, although no obvious alteration in granule morphology was observed. Starch from the transgenic lines contained fewer chains with a degree of polymerization (DP) of less than 17 and more with a DP between 17 and 38. There were also changes in the physical properties of the starches. Rapid viscoanalysis demonstrated that both the holding strength and the final viscosity of the high phosphate starches were increased indicating that the starches have increased swelling power due to an enhanced capacity for hydration.

Physcomitrella patens MAX2 characterization suggests an ancient role for this F-box protein in photomorphogenesis rather than strigolactone signalling.

Strigolactones (SLs) are key hormonal regulators of flowering plant development and are widely distributed amongst streptophytes. In Arabidopsis, SLs signal via the F-box protein MORE AXILLARY GROWTH2 (MAX2), affecting multiple aspects of development including shoot branching, root architecture and drought tolerance. Previous characterization of a Physcomitrella patens moss mutant with defective SL synthesis supports an ancient role for SLs in land plants, but the origin and evolution of signalling pathway components are unknown. Here we investigate the function of a moss homologue of MAX2, PpMAX2, and characterize its role in SL signalling pathway evolution by genetic analysis. We report that the moss Ppmax2 mutant shows very distinct phenotypes from the moss SL-deficient mutant. In addition, the Ppmax2 mutant remains sensitive to SLs, showing a clear transcriptional SL response in dark conditions, and the response to red light is also altered. These data suggest divergent evolutionary trajectories for SL signalling pathway evolution in mosses and vascular plants. In P. patens, the primary roles for MAX2 are in photomorphogenesis and moss early development rather than in SL response, which may require other, as yet unidentified, factors.

Cell division and turgor mediate enhanced plant growth in Arabidopsis plants treated with the bacterial signalling molecule lumichrome.

MAIN CONCLUSION: Transcriptomic analysis indicates that the bacterial signalling molecule lumichrome enhances plant growth through a combination of enhanced cell division and cell enlargement, and possibly enhances photosynthesis. Lumichrome (7,8 dimethylalloxazine), a novel multitrophic signal molecule produced by Sinorhizobium meliloti bacteria, has previously been shown to elicit growth promotion in different plant species (Phillips et al. in Proc Natl Acad Sci USA 96:12275-12280, https://doi.org/10.1073/pnas.96.22.12275 , 1999). However, the molecular mechanisms that underlie this plant growth promotion remain obscure. Global transcript profiling using RNA-seq suggests that lumichrome enhances growth by inducing genes impacting on turgor driven growth and mitotic cell cycle that ensures the integration of cell division and expansion of developing leaves. The abundance of XTH9 and XPA4 transcripts was attributed to improved mediation of cell-wall loosening to allow turgor-driven cell enlargement. Mitotic CYCD3.3, CYCA1.1, SP1L3, RSW7 and PDF1 transcripts were increased in lumichrome-treated Arabidopsis thaliana plants, suggesting enhanced growth was underpinned by increased cell differentiation and expansion with a consequential increase in biomass. Synergistic ethylene-auxin cross-talk was also observed through reciprocal over-expression of ACO1 and SAUR54, in which ethylene activates the auxin signalling pathway and regulates Arabidopsis growth by both stimulating auxin biosynthesis and modulating the auxin transport machinery to the leaves. Decreased transcription of jasmonate biosynthesis and responsive-related transcripts (LOX2; LOX3; LOX6; JAL34; JR1) might contribute towards suppression of the negative effects of methyl jasmonate (MeJa) such as chlorophyll loss and decreases in RuBisCO and photosynthesis. This work contributes towards a deeper understanding of how lumichrome enhances plant growth and development.

Starch Trek: The Search for Yield.

Starch is a plant storage polyglucan that accumulates in plastids. It is composed of two polymers, amylose and amylopectin, with different structures and plays several roles in helping to determine plant yield. In leaves, it acts as a buffer for night time carbon starvation. Genetically altered plants that cannot synthesize or degrade starch efficiently often grow poorly. There have been a number of successful approaches to manipulate leaf starch metabolism that has resulted in increased growth and yield. Its degradation is also a source of sugars that can help alleviate abiotic stress. In edible parts of plants, starch often makes up the majority of the dry weight constituting much of the calorific value of food and feed. Increasing starch in these organs can increase this as well as increasing yield. Enzymes involved in starch metabolism are well known, and there has been much research analyzing their functions in starch synthesis and degradation, as well as genetic and posttranslational regulatory mechanisms affecting them. In this mini review, we examine work on this topic and discuss future directions that could be used to manipulate this metabolite for improved yield.

A Comparative Study of Selected Physical and Biochemical Traits of Wild-Type and Transgenic Sorghum to Reveal Differences Relevant to Grain Quality.

Transgenic sorghum featuring RNAi suppression of certain kafirins was developed recently, to address the problem of poor protein digestibility in the grain. However, it was not firmly established if other important quality parameters were adversely affected by this genetic intervention. In the present study several quality parameters were investigated by surveying several important physical and biochemical grain traits. Important differences in grain weight, density and endosperm texture were found that serve to differentiate the transgenic grains from their wild-type counterpart. In addition, ultrastructural analysis of the protein bodies revealed a changed morphology that is indicative of the effect of suppressed kafirins. Importantly, lysine was found to be significantly increased in one of the transgenic lines in comparison to wild-type; while no significant changes in anti-nutritional factors could be detected. The results have been insightful for demonstrating some of the corollary changes in transgenic sorghum grain, that emerge from imposed kafirin suppression.

Analysis of genes involved in glycogen degradation in Escherichia coli.

Escherichia coli accumulate or degrade glycogen depending on environmental carbon supply. Glycogen phosphorylase (GlgP) and glycogen debranching enzyme (GlgX) are known to act on the glycogen polymer, while maltodextrin phosphorylase (MalP) is thought to remove maltodextrins released by GlgX. To examine the roles of these enzymes in more detail, single, double and triple mutants lacking all their activities were produced. GlgX and GlgP were shown to act directly on the glycogen polymer, while MalP most likely catabolised soluble malto-oligosaccharides. Interestingly, analysis of a triple mutant lacking all three enzymes indicates the presence of another enzyme that can release maltodextrins from glycogen.

Diverse Exopolysaccharide Producing Bacteria Isolated from Milled Sugarcane: Implications for Cane Spoilage and Sucrose Yield.

Bacterial deterioration of sugarcane during harvesting and processing is correlated with significant loss of sucrose yield and the accumulation of bacterial polysaccharides. Dextran, a homoglucan produced by Leuconostoc mesenteroides, has been cited as the primary polysaccharide associated with sugarcane deterioration. A culture-based approach was used to isolate extracellular polysaccharide (EPS) producing bacterial strains from milled sugarcane stalks. Ribosomal RNA sequencing analysis grouped 25 isolates into 4 genera. This study identified 2 bacterial genera not previously associated with EPS production or sucrose degradation. All isolates produced polysaccharide when grown in the presence of sucrose. Monosaccharide analysis of purified polymers by Gas Chromatography revealed 17 EPSs consisting solely of glucose (homoglucans), while the remainder contained traces of mannose or fructose. Dextranase treatment of polysaccharides yielded full digestion profiles for only 11 extracts. Incomplete hydrolysis profiles of the remaining polysaccharides suggest the release of longer oligosaccharides which may interfere with sucrose crystal formation.

Transitory and storage starch metabolism: two sides of the same coin?

The industrially important polymer, starch, is manufactured through a complex process involving multiple isoforms of several different enzymes. These contribute to alter the structure of starch which, in turn, affects its downstream industrial use. This review compares recent advances in our knowledge of starch metabolism in leaves and storage organs. Starch granule initiation and formation, enzyme complexes involved in starch metabolism and control of flux in starch synthesis and degradation are examined.

The reallocation of carbon in P deficient lupins affects biological nitrogen fixation.

It is not known how phosphate (P) deficiency affects the allocation of carbon (C) to biological nitrogen fixation (BNF) in legumes. The alteration of the respiratory and photosynthetic C costs of BNF was investigated under P deficiency. Although BNF can impose considerable sink stimulation on host respiratory and photosynthetic C, it is not known how the change in the C and energy allocation during P deficiency may affect BNF. Nodulated Lupinus luteus plants were grown in sand culture, using a modified Long Ashton nutrient solution containing no nitrogen (N) for ca. four weeks, after which one set was exposed to a P-deficient nutrient medium, while the other set continued growing on a P-sufficient nutrient medium. Phosphorus stress was measured at 20 days after onset of P-starvation. During P stress the decline in nodular P levels was associated with lower BNF and nodule growth. There was also a shift in the balance of photosynthetic and respiratory C toward a loss of C during P stress. Below-ground respiration declined under limiting P conditions. However, during this decline there was also a shift in the proportion of respiratory energy from maintenance toward growth respiration. Under P stress, there was an increased allocation of C toward root growth, thereby decreasing the amount of C available for maintenance respiration. It is therefore possible that the decline in BNF under P deficiency may be due to this change in resource allocation away from respiration associated with direct nutrient uptake, but rather toward a long term nutrient acquisition strategy of increased root growth.

In vitro selection of transgenic sugarcane callus utilizing a plant gene encoding a mutant form of acetolactate synthase.

Selection genes are routinely used in plant genetic transformation protocols to ensure the survival of transformed cells by limiting the regeneration of non-transgenic cells. In order to find alternatives to the use of antibiotics as selection agents, we followed a targeted approach utilizing a plant gene, encoding a mutant form of the enzyme acetolactate synthase, to convey resistance to herbicides. The sensitivity of sugarcane callus (Saccharum spp. hybrids, cv. NCo310) to a number of herbicides from the sulfonylurea and imidazolinone classes was tested. Callus growth was most affected by sulfonylurea herbicides, particularly 3.6 µg/l chlorsulfuron. Herbicide-resistant transgenic sugarcane plants containing mutant forms of a tobacco acetolactate synthase (als) gene were obtained following biolistic transformation. Post-bombardment, putative transgenic callus was selectively proliferated on MS medium containing 3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 20 g/l sucrose, 0.5 g/l casein, and 3.6 µg/l chlorsulfuron. Plant regeneration and rooting was done on MS medium lacking 2,4-D under similar selection conditions. Thirty vigorously growing putative transgenic plants were successfully ex vitro-acclimatized and established under glasshouse conditions. Glasshouse spraying of putative transgenic plants with 100 mg/l chlorsulfuron dramatically decreased the amount of non-transgenic plants that had escaped the in vitro selection regime. PCR analysis showed that six surviving plants were als-positive and that five of these expressed the mutant als gene. This report is the first to describe a selection system for sugarcane transformation that uses a selectable marker gene of plant origin targeted by a sulfonylurea herbicide.

Starches--from current models to genetic engineering.

As the world's second most abundant biopolymer, starch serves as food, feed and renewable resource for bioenergy production and other industrial applications. Unlike storage lipids, starch is stored in the form of semi-crystalline granules, which are tissue- and species-specific in number, shape and size. Over the last decades, most biosynthetic and degradative enzymes of starch metabolism have been identified in the model species Arabidopsis thaliana. Based on this, biotechnological applications have arisen that led to a number of transgenic crop plants with elevated starch content or improved starch quality. Irrespective of this great success, there are still numerous open questions including the regulation of starch metabolism, the initiation of granule formation, the regulation of granule shape and size and many more, which will be tackled over the next decades. Here, we briefly summarize current knowledge concerning starch metabolism and its regulation and biotechnological use.

Reuteran and levan as carbohydrate sinks in transgenic sugarcane.

The present study reports the effect of high molecular weight bacterial fructan (levan) and glucan (reuteran) on growth and carbohydrate partitioning in transgenic sugarcane plants. These biopolymers are products of bacterial glycosyltransferases, enzymes that catalyze the polymerization of glucose or fructose residues from sucrose. Constructs, targeted to different subcellular compartments (cell wall and cytosol) and driven by the Cauliflower mosaic virus-35S: maize-ubiquitin promoter, were introduced into sugarcane by biolistic transformation. Polysaccharide accumulation severely affected growth of callus suspension cultures. Regeneration of embryonic callus tissue into plants proved problematic for cell wall-targeted lines. When targeted to the cytosol, only plants with relative low levels of biopolymer accumulation survived. In internodal stalk tissue that accumulate reuteran (max 0.03 mg/g FW), sucrose content (ca 60 mg/g FW) was not affected, while starch content (<0.4 mg/g FW) was increased up to four times. Total carbohydrate content was not significantly altered. On the other hand, starch and sucrose levels were significantly reduced in plants accumulating levan (max 0.01 mg/g FW). Heterologous expression resulted in a reduction in total carbohydrate assimilation rather than a simple diversion by competition for substrate.

The plant growth promoting substance, lumichrome, mimics starch, and ethylene-associated symbiotic responses in lotus and tomato roots.

Symbiosis involves responses that maintain the plant host and symbiotic partner's genetic program; yet these cues are far from elucidated. Here we describe the effects of lumichrome, a flavin identified from Rhizobium spp., applied to lotus (Lotus japonicus) and tomato (Solanum lycopersicum). Combined transcriptional and metabolite analyses suggest that both species shared common pathways that were altered in response to this application under replete, sterile conditions. These included genes involved in symbiosis, as well as transcriptional and metabolic responses related to enhanced starch accumulation and altered ethylene metabolism. Lumichrome priming also resulted in altered colonization with either Mesorhizobium loti (for lotus) or Glomus intraradices/G. mossea (for tomato). It enhanced nodule number but not nodule formation in lotus; while leading to enhanced hyphae initiation and delayed arbuscule maturation in tomato.

Virus-induced multiple gene silencing to study redundant metabolic pathways in plants: silencing the starch degradation pathway in Nicotiana benthamiana.

Virus-induced gene silencing (VIGS) is a rapid technique that allows for specific and reproducible post-transcriptional degradation of targeted mRNA. The method has been proven efficient for suppression of expression of many single enzymes. The metabolic networks of plants, however, often contain isoenzymes and gene families that are able to compensate for a mutation and mask the development of a silencing phenotype. Here, we show the application of multiple gene VIGS repression for the study of these redundant biological pathways. Several genes in the starch degradation pathway [disproportionating enzyme 1; (DPE1), disproportionating enzyme 2 (DPE2), and GWD] were silenced. The functionally distinct DPE enzymes are present in alternate routes for sugar export to the cytoplasm and result in an increase in starch production when silenced individually. Simultaneous silencing of DPE1 and DPE2 in Nicotiana benthamiana resulted in a near complete suppression in starch and accumulation of malto-oligosaccharides.