Articulating the effect of food systems innovation on the Sustainable Development Goals.

Food system innovations will be instrumental to achieving multiple Sustainable Development Goals (SDGs). However, major innovation breakthroughs can trigger profound and disruptive changes, leading to simultaneous and interlinked reconfigurations of multiple parts of the global food system. The emergence of new technologies or social solutions, therefore, have very different impact profiles, with favourable consequences for some SDGs and unintended adverse side-effects for others. Stand-alone innovations seldom achieve positive outcomes over multiple sustainability dimensions. Instead, they should be embedded as part of systemic changes that facilitate the implementation of the SDGs. Emerging trade-offs need to be intentionally addressed to achieve true sustainability, particularly those involving social aspects like inequality in its many forms, social justice, and strong institutions, which remain challenging. Trade-offs with undesirable consequences are manageable through the development of well planned transition pathways, careful monitoring of key indicators, and through the implementation of transparent science targets at the local level.

Overexpression of TaCML20, a calmodulin-like gene, enhances water soluble carbohydrate accumulation and yield in wheat.

Calcium (Ca2+ ) is a universal messenger that mediates intracellular responses to extracellular stimuli in living organisms. Calmodulin (CaM) and calmodulin-like (CML) proteins are the important Ca2+ sensors in plants that decode Ca2+ -signatures to execute downstream intracellular level responses. Several studies indicate the interlinking of Ca2+ and sugar signaling in plants; however, no genes have been functionally characterized to provide molecular evidence. Our study found that expression of TaCML20 was significantly correlated with water soluble carbohydrate (WSC) concentrations in recombinant inbred lines in wheat. TaCML20 has four EF-hand motifs that may facilitate the binding of Ca2+ . To explore the role of CML20, we generated TaCML20 overexpressing transgenic lines in wheat. These lines accumulated higher WSC concentrations in the shoots, and we also found a significantly increased transcript level of sucrose:sucrose-1-fructosyltransferase (1-SST) in the internodes compared with the control plants. In addition, TaCML20 overexpressing plants showed significantly increased tillers per plant and also increased about 19% of grain weight per plant compared with control plants. The results also suggested a role for TaCML20 in drought stress, as its transcripts significantly increased in the shoots of wild-type plants under water deficit. These results uncovered the role of CML20 in determining multiple traits in wheat.

An analysis of the role of the ShSUT1 sucrose transporter in sugarcane using RNAi suppression.

The role of ShSUT1 in sucrose mobilisation and storage in sugarcane was investigated by employing RNAi technology to reduce the expression of this gene. Transcript profiling in non-transformed plants showed an alignment between expression and sucrose concentration, with strongest expression in source leaves and increasing expression through the daylight period of a diurnal cycle. Five transgenic plant lines were produced with reduced ShSUT1 expression ranging from 52 to 92% lower than control plants. Differential suppression of ShSUT1 sequence variants in the highly polyploid sugarcane genome were also investigated. Amplicon sequencing of the ShSUT1 variants within the transgenic lines and controls showed no preferential suppression with only minor differences in the proportional expression of the variants. A range of altered sugar, fibre and moisture contents were measured in mature leaf and internode samples, but no phenotype was consistently exhibited by all five transgenic lines. Phenotypes observed indicate that ShSUT1 does not play a direct role in phloem loading. ShSUT1 is likely involved with retrieving sucrose from intercellular spaces for transport and storage.

Tissue-specific transcriptome analysis within the maturing sugarcane stalk reveals spatial regulation in the expression of cellulose synthase and sucrose transporter gene families.

Sugarcane (Saccharum spp. hybrids) accumulates high concentrations of sucrose in its mature stalk and a considerable portion of carbohydrate metabolism is also devoted to cell wall synthesis and fibre production. We examined tissue-specific expression patterns to explore the spatial deployment of pathways responsible for sucrose accumulation and fibre synthesis within the stalk. We performed expression profiling of storage parenchyma, vascular bundles and rind dissected from a maturing stalk internode of sugarcane, identifying ten cellulose synthase subunit genes and examining significant differences in the expression of their corresponding transcripts and those of several sugar transporters. These were correlated with differential expression patterns for transcripts of genes encoding COBRA-like proteins and other cell wall metabolism-related proteins. The sugar transporters genes ShPST2a, ShPST2b and ShSUT4 were significantly up-regulated in storage parenchyma while ShSUT1 was up-regulated in vascular bundles. Two co-ordinately expressed groups of cell wall related transcripts were also identified. One group, associated with primary cell wall synthesis (ShCesA1, ShCesA7, ShCesA9 and Shbk2l3), was up-regulated in parenchyma. The other group, associated with secondary cell wall synthesis (ShCesA10, ShCesA11, ShCesA12 and Shbk-2), was up-regulated in rind. In transformed sugarcane plants, the ShCesA7 promoter conferred stable expression of green fluorescent protein preferentially in the storage parenchyma of the maturing stalk internode. Our results indicate that there is spatial separation for elevated expression of these important targets in both sucrose accumulation and cell wall synthesis, allowing for increased clarity in our understanding of sucrose transport and fibre synthesis in sugarcane.

Poor Fertility, Short Longevity, and Low Abundance in the Soil Seed Bank Limit Volunteer Sugarcane from Seed.

The recent development of genetically modified sugarcane, with the aim of commercial production, requires an understanding of the potential risks of increased weediness of sugarcane as a result of spread and persistence of volunteer sugarcane. As sugarcane is propagated vegetatively from pieces of stalk and the seed plays no part in the production cycle, the fate of seed in the environment is yet to be studied. In this study, sugarcane seed samples, collected in fields over a 2-year period, were used to determine the overall level of sugarcane fertility, seed dormancy, and longevity of seed under field conditions. A survey of the soil seed bank in and around sugarcane fields was used to quantify the presence of sugarcane seeds and to identify and quantify the weeds that would compete with sugarcane seedlings. We demonstrated that under field conditions, sugarcane has low fertility and produces non-dormant seed. The viability of the seeds decayed rapidly (half-life between 1.5 and 2.1 months). This means that, in Australia, sugarcane seeds die before they encounter climatic conditions that could allow them to germinate and establish. Finally, the soil seed bank analysis revealed that there were very few sugarcane seeds relative to the large number of weed seeds that exert a large competitive effect. In conclusion, low fertility, short persistence, and poor ability to compete limit the capacity of sugarcane seed spread and persistence in the environment.

Identification of drought-response genes and a study of their expression during sucrose accumulation and water deficit in sugarcane culms.

BACKGROUND: The ability of sugarcane to accumulate high concentrations of sucrose in its culm requires adaptation to maintain cellular function under the high solute load. We have investigated the expression of 51 genes implicated in abiotic stress to determine their expression in the context of sucrose accumulation by studying mature and immature culm internodes of a high sucrose accumulating sugarcane cultivar. Using a sub-set of eight genes, expression was examined in mature internode tissues of sugarcane cultivars as well as ancestral and more widely related species with a range of sucrose contents. Expression of these genes was also analysed in internode tissue from a high sucrose cultivar undergoing water deficit stress to compare effects of sucrose accumulation and water deficit. RESULTS: A sub-set of stress-related genes that are potentially associated with sucrose accumulation in sugarcane culms was identified through correlation analysis, and these included genes encoding enzymes involved in amino acid metabolism, a sugar transporter and a transcription factor. Subsequent analysis of the expression of these stress-response genes in sugarcane plants that were under water deficit stress revealed a different transcriptional profile to that which correlated with sucrose accumulation. For example, genes with homology to late embryogenesis abundant-related proteins and dehydrin were strongly induced under water deficit but this did not correlate with sucrose content. The expression of genes encoding proline biosynthesis was associated with both sucrose accumulation and water deficit, but amino acid analysis indicated that proline was negatively correlated with sucrose concentration, and whilst total amino acid concentrations increased about seven-fold under water deficit, the relatively low concentration of proline suggested that it had no osmoprotectant role in sugarcane culms. CONCLUSIONS: The results show that while there was a change in stress-related gene expression associated with sucrose accumulation, different mechanisms are responding to the stress induced by water deficit, because different genes had altered expression under water deficit.

Co-ordinated synthesis of gentiobiitol and sorbitol, evidence of sorbitol glycosylation in transgenic sugarcane.

Sugarcane (a Saccharum spp. interspecific hybrid) was previously engineered to synthesize sorbitol (designated as sorbitolcane). Motivated by the atypical development of the leaves in some sorbitolcane, the polar metabolite profiles in the leaves of those plants were compared against a group of control sugarcane plants. Eighty-six polar metabolites were detected in leaf extracts by GC-MS. Principal component analysis of the metabolites indicated that three compounds were strongly associated with sorbitolcane. Two were identified as sorbitol and gentiobiose and the third was unknown. Gentiobiose and the unknown compound were positively correlated with sorbitol accumulation. The unknown compound was only abundant in sorbitolcane. This compound was structurally characterized and found to be a sorbitol-glucose conjugate. (13)C NMR analysis indicated that the glucopyranose and glucitol moieties were 1,6-linked. Ligand exchange chromatography confirmed that the compound was a beta-anomer, thus identifying the compound as 6-O-beta-d-glucopyranosyl-D-glucitol, or gentiobiitol.

Efficient silencing of reporter transgenes coupled to known functional promoters in sugarcane, a highly polyploid crop species.

Sugarcane is a crop of great interest for engineering of sustainable biomaterials and biofuel production. Isolated sugarcane promoters have generally not maintained the expected patterns of reporter transgene expression. This could arise from defective promoters on redundant alleles in the highly polyploid genome, or from efficient transgene silencing. To resolve this question we undertook detailed analysis of a sugarcane gene that combines a simple pattern in genomic Southern hybridization analysis with potentially useful, sink-specific, expression. Sequence analysis indicates that this gene encodes a member of the SHAQYF subfamily of MYB transcription factors. At least eight alleles were revealed by PCR analysis in sugarcane cultivar Q117 and a similar level of heterozygosity was seen in BAC clones from cultivar Q200. Eight distinct promoter sequences were isolated from Q117, of which at least three are associated with expressed alleles. All of the isolated promoter variants were tested for ability to drive reporter gene expression in sugarcane. Most were functional soon after transfer, but none drove reporter activity in mature stems of regenerated plants. These results show that the ineffectiveness of previously tested sugarcane promoters is not simply due to the isolation of non-functional promoter copies from the polyploid genome. If the unpredictable onset of silencing observed in most other plant species is associated with developmental polyploidy, approaches that avoid efficient transgene silencing in polyploid sugarcane are likely to have much wider utility in molecular improvement.

Changes in the sugarcane metabolome with stem development. Are they related to sucrose accumulation?

Sucrose content increases with internode development down the stem of sugarcane. In an attempt to determine which other changes in metabolites may be linked to sucrose accumulation gas chromatography-mass spectrometry was used to obtain metabolic profiles from methanol/water extracts of four samples of different age down the stem of cultivar Q117. Extracts were derivatized with either N-methyl-N-(trimethylsilyl) trifluoracetamide (TMS) or N-methyl N-(tert-butyldimethylsilyl) trifluoroacetamide (TBS) separately in order to increase the number of metabolites that could be detected. This resulted in the measurement of 121 and 71 metabolites from the TMS and TBS derivatization, respectively. Fifty-five metabolites were identified using commercial and publicly available libraries. Statistical analysis of the metabolite profiles resulted in clustering of tissue types. Particular metabolites were correlated with the level of sucrose accumulation, which as expected increased down the stem. Metabolites, such as tricarboxylic acid cycle intermediates and amino acids, were more abundant in the M2 sample (meristem to internode 2) that was actively growing and decreased in an apparently coordinated developmentally programmed manner in more mature internodes down the stem. However, other metabolites such as trehalose and raffinose showed positive correlations with sucrose concentration. Here we discuss the technique used to measure metabolites in sugarcane and the changes in metabolite abundance down the sugarcane stem.

Growth and metabolism in sugarcane are altered by the creation of a new hexose-phosphate sink.

An efficient in planta sugarcane-based production system may be realized by coupling the synthesis of alternative products to the metabolic intermediates of sucrose metabolism, thus taking advantage of the sucrose-producing capability of the plant. This was evaluated by synthesizing sorbitol in sugarcane (Saccharum hybrids) using the Malus domestica sorbitol-6-phosphate dehydrogenase gene (mds6pdh). Mature transgenic sugarcane plants were compared with untransformed sugarcane variety Q117 by evaluation of the growth, metabolite levels and extractable activity of relevant enzymes. The average amounts of sorbitol detected in the most productive line were 120 mg/g dry weight (equivalent to 61% of the soluble sugars) in the leaf lamina and 10 mg/g dry weight in the stalk pith. The levels of enzymes involved in sucrose synthesis and cleavage were elevated in the leaves of plants accumulating sorbitol, but this did not affect sucrose accumulation in the culm. The activity of oxidative reactions in the pentose phosphate pathway and the non-reversible glyceraldehyde-3-phosphate dehydrogenase reaction were elevated to replenish the reducing power consumed by sorbitol synthesis. Sorbitol-producing sugarcane generated 30%-40% less aerial biomass and was 10%-30% shorter than control lines. Leaves developed necrosis in a pattern characteristic of early senescence, and the severity was related to the relative quantity of sorbitol accumulated. When the Zymomonas mobilis glucokinase (zmglk) gene was co-expressed with mds6pdh to increase the production of glucose-6-phosphate, the plants were again smaller, indicating that glucose-6-phosphate deficiency was not responsible for the reduced growth. In summary, sorbitol hyperaccumulation affected sugarcane growth and metabolism, but the outcome was not lethal for the plant. This work also demonstrated that impressive yields of alternative products can be generated from the intermediates of sucrose metabolism in Saccharum spp.

Identification of transcripts associated with cell wall metabolism and development in the stem of sugarcane by Affymetrix GeneChip Sugarcane Genome Array expression profiling.

Sugarcane is an important crop in tropical regions of the world, producing a very large biomass and accumulating large amounts of sucrose in the stem. In this study, we present the first report of transcript profiling using the GeneChip Sugarcane Genome Array. We have identified transcripts that are differentially expressed in the sugarcane stem during development by expression profiling using the array and total RNA derived from three disparate stem tissues (meristem, internodes 1-3, 8, and 20) from four replicates of the sugarcane variety Q117 grown in the field. We have identified 119 transcripts that were highly differentially expressed with development and have characterised members of the cellulose synthase (CesA) and cellulose synthase-like (Csl) gene families, which displayed coordinated expression during stem development. In addition, we determined that many other transcripts involved in cell wall metabolism and lignification were also co-expressed with members of the CesA and Csl gene families, offering additional insights into the dynamics of primary and secondary cell wall synthesis in the developing sugarcane stem.

A bioinformatic approach to the identification of a conserved domain in a sugarcane legumain that directs GFP to the lytic vacuole.

Sugarcane is an ideal candidate as a biofactory for the production of alternate higher value products. One way of achieving this is to direct useful proteins into the vacuoles within the sugarcane storage parenchyma tissue. By bioinformatic analysis of gene sequences from putative sugarcane vacuolar proteins a motif has been identified that displays high conservation across plant legumain homologues that are known to function within vacuolar compartments. This five amino acid motif, represented by the sequence IRLPS in sugarcane is shown to direct an otherwise secreted GFP fusion protein into a large acidic and proteolytic vacuole in sugarcane callus cells as well as in diverse plant species. In mature sugarcane transgenic plants, the stability of GFP appeared to be dependent on cell type, suggesting that the vacuolar environment can be hostile to introduced proteins. This targeting motif will be a valuable tool for engineering plants such as sugarcane for production of novel products.

Identification of differentially expressed transcripts from maturing stem of sugarcane by in silico analysis of stem expressed sequence tags and gene expression profiling.

Sugarcane accumulates high concentrations of sucrose in the mature stem and a number of physiological processes on-going in maturing stem tissue both directly and indirectly allow this process. To identify transcripts that are associated with stem maturation, we compared patterns of gene expression in maturing and immature stem tissue by expression profiling and bioinformatic analysis of sets of stem ESTs. This study complements a previous study of gene expression associated directly with sugar metabolism in sugarcane. A survey of sequences derived from stem tissue identified an abundance of several classes of sequence that are associated with fibre biosynthesis in the maturing stem. A combination of EST analyses and microarray hybridization revealed that genes encoding homologues of the dirigent protein, a protein that assists in the stereospecificity of lignin assembly, were the most abundant and most strongly differentially expressed transcripts in maturing stem tissue. There was also evidence of coordinated expression of other categories of fibre biosynthesis and putative defence- and stress-related transcripts in the maturing stem. This study has demonstrated the utility of genomic approaches using large-scale EST acquisition and microarray hybridization techniques to highlight the very significant transcriptional investment the maturing stem of sugarcane has placed in fibre biosynthesis and stress tolerance, in addition to its already well-documented role in sugar accumulation.

Fructan exohydrolase from grasses.

In grasses, fructan reserves are mobilized from vegetative plant parts during seasonal growth, after defoliation during grazing and from stems during seed filling. Well-illuminated leaves show a diurnal pattern of fructan accumulation during the light and mobilization during the dark. In expanding leaves, fructans are accumulated in cells of the elongation zone and when mobilized are considered to contribute assimilate for synthetic processes. Even in leaves which do not contain high fructan concentrations, high rates of fructan turnover occur. The process of fructan mobilization appears to be regulated in relation to ontogenic events, demand for assimilate during growth and in response to environmental stress. Hydrolysis of fructans in bacteria is catalyzed by both endo- and exohydrolases. However, in higher plants only fructan exohydrolases (FEH) (EC 3.2.1.80) have been reported. FEH has been extracted from only a limited number of grass species. The pH optimum of FEH activities varies between pH 45-5-5, the temperature optimum ranges from 25-40 °C and FEH is considered to be entirely localized in vacuoles. Estimates of the Km for FEH assayed using high molecular weight fructan substrates vary widely and should be considered carefully because most substrates are ill-defined. Many studies indicate that crude and partially-purified FEH activity is highest when assayed using a fructan substrate extracted from the species that was the source of the enzyme activity. Inulin extracted from members of the Asteraceae is generally less readily hydrolyzed and levans from bacteria are relatively poor substrates for FEH from grasses. Glycosidic-linkage-specific hydrolysis has been demonstrated for an FEH activity extracted from barley. This FEH activity hydrolyzed ß-2,1-glycosidic linkages more rapidly than ß-2,6-linkages. Most other studies are less conclusive because ill-defined fructan substrates were used. Two isoforms of FEH are reported in leaves of Lolium spp., but the roles of isoforms and their kinetic characteristics are not known. FEH activity in different tissues may be regulated by metabolic concentrations, sucrose (5-10 mw) being a strong inhibitor in vitro of FEH from some species. Results of experiments with Dactylis glomerata indicate control of expression of FEH activity at the gene level. In stem bases, FEH activity increased after defoliation. The increase was abolished by applications of inhibitors of protein synthesis and was apparently repressed by application of various sugars. Although the rates of fructan hydrolysis measured in vitro are sufficient to explain the in vivo rates of fructan hydrolysis, it is yet to be shown whether fructan hydrolysis in vivo is due to the activity of FEH exclusively, or FEH and invertase-like activities. The overriding conclusion is that the various studies of FEH from grasses present a confusing and incomplete picture of the function, activity and kinetics of this enzyme. This is due in part to the lack of defined, commercially-available substrates. The chromatographic techniques available to most laboratories do not permit purification of sufficient quantities of high molecular weight fructans of specific degree of polymerization, or fructan oligosaccharides with glycosidic linkages which differ from that of the inulin series for enzyme characterization. It is recommended that a few well-defined oligosaccharides be adopted as substrate standards for future research.