Summary Harnessing plant microbiota can assist in sustainably increasing primary productivity to meet growing global demands for food and biofuel. However, development of rational microbiome‐based approaches for improving crop yield and productivity is currently hindered by a lack of understanding of the major biotic and abiotic factors shaping the crop microbiome under relevant field conditions. We examined bacterial and fungal communities associated with both aerial (leaves, stalks) and belowground (roots, soil) compartments of four commercial sugarcane varieties (Saccharum spp.) grown in several growing regions in Australia. We identified drivers of the sugarcane microbiome under field conditions and evaluated whether the plants shared a core microbiome. Sugarcane‐associated microbial assemblages were primarily determined by plant compartment, followed by growing region, crop age, variety and Yellow Canopy Syndrome (YCS). We detected a core set of microbiota and identified members of the core microbiome that were influenced by YCS incidence. Our study revealed key hub microorganisms in the core microbiome networks of sugarcane leaves, stalks, roots and rhizosphere soil despite location and time‐associated shifts in the community assemblages. Elucidating their functional roles and identification of the keystone core microbiota that sustain plant health could provide a technological breakthrough for a sustainable increase in crop productivity.

BackgroundDespite the economic importance of sugarcane in sugar and bioenergy production, there is not yet a reference genome available. Most of the sugarcane transcriptomic studies have been based on Saccharum officinarum gene indices (SoGI), expressed sequence tags (ESTs) and de novo assembled transcript contigs from short-reads; hence knowledge of the sugarcane transcriptome is limited in relation to transcript length and number of transcript isoforms.ResultsThe sugarcane transcriptome was sequenced using PacBio isoform sequencing (Iso-Seq) of a pooled RNA sample derived from leaf, internode and root tissues, of different developmental stages, from 22 varieties, to explore the potential for capturing full-length transcript isoforms. A total of 107,598 unique transcript isoforms were obtained, representing about 71% of the total number of predicted sugarcane genes. The majority of this dataset (92%) matched the plant protein database, while just over 2% was novel transcripts, and over 2% was putative long non-coding RNAs. About 56% and 23% of total sequences were annotated against the gene ontology and KEGG pathway databases, respectively. Comparison with de novo contigs from Illumina RNA-Sequencing (RNA-Seq) of the internode samples from the same experiment and public databases showed that the Iso-Seq method recovered more full-length transcript isoforms, had a higher N50 and average length of largest 1,000 proteins; whereas a greater representation of the gene content and RNA diversity was captured in RNA-Seq. Only 62% of PacBio transcript isoforms matched 67% of de novo contigs, while the non-matched proportions were attributed to the inclusion of leaf/root tissues and the normalization in PacBio, and the representation of more gene content and RNA classes in the de novo assembly, respectively. About 69% of PacBio transcript isoforms and 41% of de novo contigs aligned with the sorghum genome, indicating the high conservation of orthologs in the genic regions of the two genomes.ConclusionsThe transcriptome dataset should contribute to improved sugarcane gene models and sugarcane protein predictions; and will serve as a reference database for analysis of transcript expression in sugarcane.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3757-8) contains supplementary material, which is available to authorized users.

SummaryCarbon captured through photosynthesis is transported, and sometimes stored in plants, as sugar. All organic compounds in plants trace to carbon from sugars, so sugar metabolism is highly regulated and integrated with development. Sugars stored by plants are important to humans as foods and as renewable feedstocks for industrial conversion to biofuels and biomaterials. For some purposes, sugars have advantages over polymers including starches, cellulose or storage lipids. This review considers progress and prospects in plant metabolic engineering for increased yield of endogenous sugars and for direct production of higher-value sugars and simple sugar derivatives. Opportunities are examined for enhancing export of sugars from leaves. Focus then turns to manipulation of sugar metabolism in sugar-storing sink organs such as fruits, sugarcane culms and sugarbeet tubers. Results from manipulation of suspected 'limiting' enzymes indicate a need for clearer understanding of flux control mechanisms, to achieve enhanced levels of endogenous sugars in crops that are highly selected for this trait. Outcomes from in planta conversion to novel sugars and derivatives range from severe interference with plant development to field demonstration of crops accumulating higher-value sugars at high yields. The differences depend on underlying biological factors including the effects of the novel products on endogenous metabolism, and on biotechnological fine-tuning including developmental expression and compartmentation patterns. Ultimately, osmotic activity may limit the accumulation of sugars to yields below those achievable using polymers; but results indicate the potential for increases above current commercial sugar yields, through metabolic engineering underpinned by improved understanding of plant sugar metabolism.

l h e temporal relationship between sucrose (Suc) accumulation and carbon partitioning was investigated in developing sugarcane internodes. Radiolabeling studies on tissue slices, which contained Suc concentrations ranging from 14 to 42% of the dry mass, indicated that maturation coincided with a redirection of carbon from water-insoluble matter, respiration, amino acids, organic acids, and phosphorylated intermediates into SUC. It is evident that a cycle of Suc synthesis and degradation exists in all of the internodes. l h e decreased allocation of carbon to respiration coincides with a decreased flux from the hexose pool. 60th the glucose and fructose (Fru) concentrations significantly decrease during maturation. The phosphoenolpyruvate, Fru-6-phosphate (Fru-6-P), and Fru-2,6-bisphosphate (Fru-2, 6-P,) concentrations decrease between the young and older internodal tissue, whereas the inorganic phosphate concentration increases. lhe calculated mass-action ratios indicate that the ATP-dependent phosphofructokinase, pyruvate kinase, and Fru-l,6-bisphosphatase reactions are tightly regulated in all of the internodes, and no evidence was found that major changes in the regulation of any of these enzymes occur. l h e pyrophosphatedependent phosphofructokinase reaction is in apparent equilibrium in ali the internodes. Substrate availability might be one of the prime factors contributing to the observed decrease in respiration.

Sucrose accumulation rates, sucrose-phosphate synthase (SPS, EC 2.4.1.14) and soluble sucrose synthase (SuSy, EC 2.4.1.13) activities were measured in internodal tissue from a sugarcane (Saccharum species hybrids) variety N19. The sucrose accumulation rate sharply increases between internodes 3 to 11. In the older internodes SPS activity was at least three times higher than the SuSy activity. A highly significant positive correlation was found between SPS activity and sucrose content. In contrast, no significant correlation was observed between SuSy and sucrose content. In agreement, when radiolabelled glucose was fed to internodes with a high sucrose accumulation rate, label was equally distributed in the hexose moieties of sucrose. This clearly indicates that SPS is the major sucrose synthesis activity in the culm of sugarcane. Different kinetic forms of SPS apparently exist in the internodal tissue at different stages of development.

The assimilation of NH4 causes a rapid increase in respiration to provided carbon skeletons for amino acid synthesis. In this study we propose a model for the regulation of carbon partitioning from starch to respiration and N assimilation in the green alga Selenastrum minutum. We provide evidence for both a cytosolic and plastidic fructose-1,6-bisphosphatase. The cytosolic form is inhibited by AMP and fructose-1,6-bisphosphate and the plastidic form is inhibited by phosphate. There is only one ATP dependent phosphofructokinase which, based on immunological cross reactivity, has been identified as being localized in the plastid. It is inhibited by phosphoenolpyruvate and activated by phosphate. No pyrophosphate dependent phosphofructokinase was found. The initiation of dark ammonium assimilation resulted in a transient increase in ADP which releases pyruvate kinase from adenylate control. This activation of pyruvate kinase causes a rapid 80% drop in phosphoenolpyruvate and a 2.7-fold increase in pyruvate. The pyruvate kinase mediated decrease in phosphoonolpyruvate correlates with the activation of the ATP dependent phosphofructokinase increasing carbon flow through the upper half of glycolysis. This increased the concentration of triosephosphate and provided substrate for pyruvate kinase. It is suggested that this increase in triosephosphate coupled with the glutamine synthetase mediated decline in glutamate, serves to maintain pyruvate kinase activation once ADP levels recover. The initiation of NH4 assimilation causes a transient 60% increase in fructose-2,6-bisphosphate. Given the sensitivity of the cytosolic fructose-1,6-bisphosphatase to this regulator, its increase would serve to inhibit cytosolic gluconeogenesis and direct the triosephosphate exported from the plastid down glycolysis to amino acid biosynthesis.Over the last decade there has been a significant increase in our knowledge of the control of carbon partitioning from starch to sucrose in photosynthetic tissue (34). In the proposed models, most of the carbon exported from the chloroplast is TP (see Table I for complete list of abbreviations). In the cytosol, Fru-2,6-P2 plays an important role in regulating the conversion of TP to sucrose through its effects on cytosolic It is well established that nitrogen exerts a major influence on respiratory carbon metabolism (1,9,15,16,31,(35)(36)(37)41). The assimilation of NH4' enhances starch breakdown and the flow of carbon into TCA cycle intermediates which are then used in amino acid biosynthesis. At present there is no model which provides an integrative picture of the mechanism by which NH4' assimilation regulates this carbon partitioning. In the present study we report changes in cellular metabolites during the dark assimilation of NH4' by the Nlimited green alga Selenastrum minutum. We also report preliminary data on the localization and regulation of PFK, PFP, and FBPase in this organism. Together, these results allow us to propose an integrative model for the control of carbon partitionin...

Sucrose accumulation in developing sugar cane (Saccharum officinarum) is accompanied by a continuous synthesis and cleavage of sucrose in the storage tissues. Despite numerous studies, the factors affecting sucrose accumulation are still poorly understood, and no consistent pattern has emerged which pinpoints certain enzyme activities as important controlling steps. Here, we develop an approach based on pathway analysis and kinetic modelling to assess the biochemical control of sucrose accumulation and futile cycling in sugar cane. By using the concept of elementary flux modes, all possible routes of futile cycling of sucrose were enumerated in the metabolic system. The available kinetic data for the pathway enzymes were then collected and assembled in a kinetic model of sucrose accumulation in sugar cane culm tissue. Although no data were fitted, the model agreed well with independent experimental results: in no case was the difference between calculated and measured fluxes and concentrations greater than 2-fold. The model thus validated was then used to assess different enhancement strategies for increasing sucrose accumulation. First, the control coefficient of each enzyme in the system on futile cycling of sucrose was calculated. Secondly, the activities of those enzymes with the numerically largest control coefficients were varied over a 5-fold range to determine the effect on the degree of futile cycling, the conversion efficiency from hexoses into sucrose, and the net sucrose accumulation rate. In view of the modelling results, overexpression of the fructose or glucose transporter or the vacuolar sucrose import protein, as well as reduction of cytosolic neutral invertase levels, appear to be the most promising targets for genetic manipulation. This offers a more directed improvement strategy than cumbersome gene-by-gene manipulation. The kinetic model can be viewed and interrogated on the World Wide Web at http://jjj.biochem.sun.ac.za.

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