Cloning and Developmental Expression of a Nodule-Enhanced Sucrose Synthase cDNA from Alfalfa

Robinson, David L.; Trepp, Gian B.; Gregerson, Robert G.; Twary, Scott N.; Roeven, Robert; Gantt, J. Stephen; Vance, Carroll P.

doi:10.1007/978-1-4615-4795-2_5

Cited by 5 publications

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“…A sequence correction has been submitted to GenBank for the PEPC-7 gene. In an earlier study, the cDNA from a nodule-enhanced sucrose synthase gene ( MsSUS1 ; AF049487) was isolated and cloned into pBluescript [ 41 ]. For this study, the gusA sequence in the PEPC7-P4::GUS construct was replaced with an Xma I- Sac I fragment containing the MsSUS1 cDNA to produce the PEPC7-P4:: MsSUS1 expression vector.…”

Section: Methodsmentioning

confidence: 99%

Transgene silencing of sucrose synthase in alfalfa (Medicago sativa L.) stem vascular tissue suggests a role for invertase in cell wall cellulose synthesis

et al. 2015

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BackgroundAlfalfa (Medicago sativa L.) is a widely adapted perennial forage crop that has high biomass production potential. Enhanced cellulose content in alfalfa stems would increase the value of the crop as a bioenergy feedstock. We examined if increased expression of sucrose synthase (SUS; EC 2.4.1.13) would increase cellulose in stem cell walls.ResultsAlfalfa plants were transformed with a truncated alfalfa phosphoenolpyruvate carboxylase gene promoter (PEPC7-P4) fused to an alfalfa nodule-enhanced SUS cDNA (MsSUS1) or the β-glucuronidase (GUS) gene. Strong GUS expression was detected in xylem and phloem indicating that the PEPC7-P4 promoter was active in stem vascular tissue. In contrast to expectations, MsSUS1 transcript accumulation was reduced 75–90 % in alfalfa plants containing the PEPC7-P4::MsSUS1 transgene compared to controls. Enzyme assays indicated that SUS activity in stems of selected down-regulated transformants was reduced by greater than 95 % compared to the controls. Although SUS activity was detected in xylem and phloem of control plants by in situ enzyme assays, plants with the PEPC7-P4::MsSUS1 transgene lacked detectable SUS activity in post-elongation stem (PES) internodes and had very low SUS activity in elongating stem (ES) internodes. Loss of SUS protein in PES internodes of down-regulated lines was confirmed by immunoblots. Down-regulation of SUS expression and activity in stem tissue resulted in no obvious phenotype or significant change in cell wall sugar composition. However, alkaline/neutral (A/N) invertase activity increased in SUS down-regulated lines and high levels of acid invertase activity were observed. In situ enzyme assays of stem tissue showed localization of neutral invertase in vascular tissues of ES and PES internodes.ConclusionsThese results suggest that invertases play a primary role in providing glucose for cellulose biosynthesis or compensate for the loss of SUS1 activity in stem vascular tissue.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0649-4) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Transgene silencing of sucrose synthase in alfalfa (Medicago sativa L.) stem vascular tissue suggests a role for invertase in cell wall cellulose synthesis

et al. 2015

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“…5). In comparison, GS, AAT, AS, Vance et al (1994), Shi et al (1997), Miller et al (1998 and Robinson et al (1999). See text for abbreviations.…”

Section: Nadh-glutamate Synthasementioning

confidence: 99%

“…Our postulate that NADH-GOGAT occupies perhaps the key step in assimilation of symbiotically fixed N in alfalfa is based upon several findings: (i) NADH-GOGAT in alfalfa Table 5. Activity and relative abundance of alfalfa nodule enzymes involved in nitrogen and carbon assimilation Data derived from Egli et al (1989), Gantt et al (1992), Pathirana et al (1992), Gregerson et al (1993), Shi et al (1997), Miller et al (1998, Robinson et al (1999) occurs as a single gene encoding a single enzyme while other enzymes in nodule N and C assimilation are encoded by multiple genes encoding multiple isozymes; (ii) the protein comprises only a small, 0.1-0.4%, of nodule soluble protein while the other enzymes are more abundant; (iii) the enzyme is the junction for channeling amino acid synthesis to Asp in amyloplasts and Glu in the cytosol (Fig. 8); (iv) selection for reduced NADH-GOGAT enzyme activity and expression of antisense NADH-GOGAT transcripts have a negative effect on plant growth but can be rescued by nitrate fertilisation; and (v) all other genes involved in primary N and C metabolism are expressed at fairly high levels in ineffective nodules, while NADH-GOGAT expression has a requirement for effective nodules.…”

Section: Nadh-glutamate Synthasementioning

confidence: 99%

Application of biochemical studies to improving nitrogen fixation

Vance¹,

Lamb²

2001

Aust. J. Exp. Agric.

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Improvement of symbiotic nitrogen fixation requires a multidisciplinary approach with a comprehensive program ranging from microbial ecology to plant breeding and genomics. Achievement of symbiotic nitrogen fixation requires at least 100 genes from each partner interacting in a favorable environment. The more information that we obtain from applied and fundamental studies of Rhizobium–legume and Frankia–non-legume symbioses, the greater are our chances to extend nitrogen fixation to non-fixing species. Studies with alfalfa (Medicago sativa L.) aimed at improving symbiotic nitrogen fixation have resulted in significant advances in germplasm development, plant biochemistry, microbial ecology and the understanding of plant genes involved in nodule nitrogen and carbon metabolism. However, translation to field improvement of symbiotic nitrogen fixation has proven elusive.

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“…Συγκεκριμένα, στα καθορισμένα φυμάτια φαίνεται να λαμβάνει χώρα μεγαλύτερο ποσοστό του μεταβολισμού του άνθρακα στα μη μολυσμένα κύτταρα της αζωτοδεσμεύουσας ζώνης και του εσωτερικού φλοιού του φυματίου σε σχέση με το αντίστοιχο ποσοστό στα μη καθορισμένα φυμάτια. Αντίθετα τα μη καθορισμένα φυμάτια φαίνεται να παρουσιάζουν μια πιο ομοιόμορφη κατανομή μεταξύ μολυσμένων και μη μολυσμένων κυττάρων (Day and Copeland, 1991;Robinson et al, 1999;Hohnjec et al, 2003).…”

Section: γενικάunclassified

Αναπρογραμματισμός του μεταβολισμού του Lotus japonicus σε μεταγραφικό, βιοχημικό και μεταβολομικό επίπεδο κατά τη συμβιωτική αζωτοδέσμευση

Καλλονιάτη¹

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Symbiotic nitrogen fixation in legumes takes place in specialized organs called nodules,which become the main source of assimilated nitrogen for the whole plant. Symbiotic nitro‐gen fixation requires exquisite integration of plant and bacterial metabolism and involvesglobal changes in gene expression and metabolite accumulation in both rhizobia and thehost plant. In order to study the metabolic changes mediated by symbiotic nitrogen fixationon a whole‐plant level, metabolite levels were profiled by gas chromatography–mass spec‐trometry in nodules and non‐symbiotic organs of Lotus japonicus plants uninoculated or in‐oculated with M. loti wt, ΔnifA or ΔnifH fix‐ strains. Furthermore, transcriptomic andbiochemical approaches were combined to study sulfur metabolism in nodules, its link tosymbiotic nitrogen fixation, and the effect of nodules on whole‐plant sulfur partitioning andmetabolism. It is well established that nitrogen and sulfur (S) metabolism are tightly en‐twined and sulfur is required for symbiotic nitrogen fixation, however, little is known aboutthe molecular and biochemical mechanisms governing sulfur uptake and assimilation duringsymbiotic nitrogen fixation. Transcript profiling in Lotus japonicus was combined with quan‐tification of S‐metabolite contents and APR activity in nodules and in non‐symbiotic organsof plants uninoculated or inoculated with M. loti wt, ΔnifA or ΔnifH fix‐ strains. Moreover,sulfate uptake and its distribution into different plant organs were analyzed and 35S‐flux intodifferent S‐pools was monitored. Metabolite profiling revealed that symbiotic nitrogen fixa‐tion results in dramatic changes of many aspects of primary and secondary metabolism innodules which leads to global reprogramming of metabolism of the model legume on awhole‐plant level. Moreover, our data revealed that nitrogen fixing nodules represent athiol‐rich organ. Their high APR activity and 35S‐flux into cysteine and its metabolites in com‐bination with the transcriptional up‐regulation of several genes involved in sulfur assimila‐tion highlight the function of nodules as a new site of sulfur assimilation. The higher thiolcontent observed in non‐symbiotic organs of nitrogen fixing plants in comparison touninoculated plants cannot be attributed to local biosynthesis, indicating that nodules couldserve as a novel source of reduced sulfur for the plant, which triggers whole‐plant repro‐gramming of sulfur metabolism. Interestingly, the changes in metabolite profiling and theenhanced thiol biosynthesis in nodules and their impact on the whole‐plant sulfur, carbonand nitrogen economy are dampened in fix‐ plants, which in most respects metabolically re‐sembled uninoculated plants, indicating a strong interaction between nitrogen fixation andsulfur and carbon metabolism.

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Cloning and Developmental Expression of a Nodule-Enhanced Sucrose Synthase cDNA from Alfalfa

Cited by 5 publications

References 42 publications

Transgene silencing of sucrose synthase in alfalfa (Medicago sativa L.) stem vascular tissue suggests a role for invertase in cell wall cellulose synthesis

Transgene silencing of sucrose synthase in alfalfa (Medicago sativa L.) stem vascular tissue suggests a role for invertase in cell wall cellulose synthesis

Application of biochemical studies to improving nitrogen fixation

Αναπρογραμματισμός του μεταβολισμού του Lotus japonicus σε μεταγραφικό, βιοχημικό και μεταβολομικό επίπεδο κατά τη συμβιωτική αζωτοδέσμευση

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