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Arabidopsis glt1- T mutant defines a role for NADH-GOGAT in the non-photorespiratory ammonium assimilatory pathway

Arabidopsis glt1- T mutant defines a role for NADH-GOGAT in the non-photorespiratory ammonium assimilatory pathway. Muriel Lancien, et al. 2002. Presented by: Arin, Artin, Judy, and Ryan. Introduction. Ammonium Sources:. Nitrate assimilation. N 2 fixation in root nodules of legumes.

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Arabidopsis glt1- T mutant defines a role for NADH-GOGAT in the non-photorespiratory ammonium assimilatory pathway

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  1. Arabidopsis glt1-T mutant defines a role for NADH-GOGAT in the non-photorespiratory ammonium assimilatory pathway Muriel Lancien, et al. 2002. Presented by: Arin, Artin, Judy, and Ryan

  2. Introduction Ammonium Sources: • Nitrate assimilation • N2 fixation in root nodules of legumes • phenylpropanoid pathway • (leads to colored anthocyanins) • Nitrogen release during transport • Breakdown of nitrogenous compounds • Photorespiration

  3. Introduction NH4+ released during photorespiration 2-phosphoglycolate To 3-phosphoglycerate

  4. Introduction Air: favors photorespiration 1% CO2: represses photorespiration • Release of ammonium during photorespiration: • toxic in excess • required component of macromolecules Plants do not excrete nitrogen Plants possess mechanisms for re-assimilating photorespiratory ammonium

  5. Introduction • Glutamate Synthase • a.k.a GOGAT • (Glutamine • 2-Oxoglutarate Aminotransferase) GS and GOGAT work in pairs to assimilate NH4+ into useful amino acids Glutamine Synthetase (GS) oxoglutarate = -ketoglutarate

  6. Introduction • Glutamate Synthase • a.k.a GOGAT • (Glutamine • 2-Oxoglutarate Aminotransferase) 2 forms found in chloroplast: • Ferredoxin-dependent (Fd-GOGAT) • NADH-dependent (NADH-GOGAT) Isoforms that differ in source of reductant

  7. Introduction Lots is known about Fd-GOGAT. Phenotype described previously. • 95% of GOGAT activity in leaves is Fd-GOGAT • Conditional lethality in Fd-GOGAT knock-outs: • chlorotic in air • rescued in 1% CO2 • Mutants lack ability to re-assimilate NH4+ under photorespiratory conditions • Mutants still competent in primary N assimilation Fd-GOGAT function: re-assimilation of photorespiratory NH4+

  8. Introduction Why are N-assimilation mutants chlorotic? When N is lacking, plants don’t want to tie it up in chlorophyll No chlorophyll = yellow plant

  9. Introduction Much less is known about NADH-GOGAT Questions: What does NADH-dependent GOGAT do??? Does NADH-GOGAT have a distinct function??? Does the function of NADH-GOGAT overlap with Fd-GOGAT??? Note: This is NOT a paper about Fd-GOGAT. A Fd-GOGAT mutant is included for comparison, to determine if NADH-GOGAT has same or different phenotype.

  10. Introduction What was known about NADH-GOGAT before this paper? • Encoded by GLT1 gene • Expressed mainly in non-photosynthetic tissues • seeds • roots • Less photorespiration in these tissues • Different tissue expression suggest different roles for • Fd-GOGAT and NADH-GOGAT • Activity of NADH-GOGAT can’t replace Fd-GOGAT in knock-outs

  11. Introduction Model proposed by the authors: non-redundant functions Figure 5

  12. Introduction We’ll present evidence that supports non-redundant purposes for Fd-GOGAT and NADH-GOGAT • Verification of tissue expression patterns • Characterization of NADH-GOGAT phenotype using knock-out mutant • Comparison to Fd-GOGAT knock-out to verify different phenotypes

  13. Expression of NADH-GOGAT mRNA in Arabidopsis First step to understanding the physiological role of NADH-GOGAT: Identify expression pattern of GLT1 gene

  14. Expression of NADH-GOGAT mRNA in Arabidopsis Gene sequence of GLT1 is known. • Used to design oligonucleotide primers for Northern blot Total RNA isolated from: • roots • leaves 6-week-old wild type Arabidopsis plants

  15. Expression of NADH-GOGAT mRNA in Arabidopsis Figure 1 • Total RNA probed for different enzymes involved in N-assimilation • Allows comparison of expression patterns Ethidium bromide control: RNA is present on gel.

  16. Expression of NADH-GOGAT mRNA in Arabidopsis Figure 1 Glutamine Synthetase Isoforms GLN2: chloroplastic Glutamine Synthetase 2 (GS2) GLN1: cytosolic Glutamine Synthetase 1 (GS1)

  17. Expression of NADH-GOGAT mRNA in Arabidopsis Figure 1 Glutamine Synthetase Isoforms Fd-GOGAT Isoforms GLU1: Fd-GOGAT (expressed in leaves) GLU2: Fd-GOGAT (expressed in roots)

  18. Expression of NADH-GOGAT mRNA in Arabidopsis Figure 1 Glutamine Synthetase Isoforms Fd-GOGAT Isoforms NADH-GOGAT GLT1: NADH-GOGAT

  19. Expression of NADH-GOGAT mRNA in Arabidopsis Figure 1 Glutamine Synthetase Isoforms Fd-GOGAT Isoforms NADH-GOGAT Genes expressed mainly in leaves.

  20. Expression of NADH-GOGAT mRNA in Arabidopsis Figure 1 Glutamine Synthetase Isoforms Fd-GOGAT Isoforms NADH-GOGAT Genes expressed mainly in leaves. Genes expressed mainly in roots.

  21. Expression of NADH-GOGAT mRNA in Arabidopsis Figure 1 Glutamine Synthetase Isoforms Fd-GOGAT Isoforms NADH-GOGAT Notice: GLN1 and GLT1 have similar expression in roots Genes expressed mainly in leaves. Genes expressed mainly in roots.

  22. Expression of NADH-GOGAT mRNA in Arabidopsis Results??? Suggests that NADH-GOGAT (GLT1) may play a role in N-assimilation in roots in concert with Glutamine Synthetase 1 (GLN1)

  23. Identification of a T-DNA insertion mutant (glt1-T) in the NADH-GOGAT GLT1 gene Before we start: • Complete sequence of Arabidopsis thaliana genome available • NADH-GOGAT is a single-copy gene • Alignment with cDNA reveals: • 17 exons • 16 introns • ORF is ~ 7.7 kb. Protein is ~ 240 kDa

  24. Identification of a T-DNA insertion mutant (glt1-T) in the NADH-GOGAT GLT1 gene • glt1-T mutant identified from Arabidopsis T-DNA line • (T-DNA line had been previously generated) • PCR strategy using nested T-DNA-specific primers • PCR products sequenced and compared to GLT1 gene • Result: Line CS21349 had T-DNA inserted into GLT1 gene • Crosses of this line generated 4 progeny homozygous for glt1-T mutation • Still need to verify that T-DNA insert causes loss-of-function of NADH-GOGAT

  25. Identification of a T-DNA insertion mutant (glt1-T) in the NADH-GOGAT GLT1 gene • Location of T-DNA insertion • inside exon 9 • between amino acids D315 and G316 Figure 2a

  26. Identification of a T-DNA insertion mutant (glt1-T) in the NADH-GOGAT GLT1 gene Domains important to function of NADH-GOGAT: Figure 2a

  27. Identification of a T-DNA insertion mutant (glt1-T) in the NADH-GOGAT GLT1 gene Identification of a T-DNA insertion mutant (glt1-T) in the NADH-GOGAT GLT1 gene • Domains important to function of NADH-GOGAT: • 1, 2, 3, 4: PurF-type amidotransferase regions Figure 2a

  28. Identification of a T-DNA insertion mutant (glt1-T) in the NADH-GOGAT GLT1 gene Identification of a T-DNA insertion mutant (glt1-T) in the NADH-GOGAT GLT1 gene • Domains important to function of NADH-GOGAT: • 1, 2, 3, 4: PurF-type amidotransferase regions • 5 & 6: flavin mononucleotide (FMN) binding regions Figure 2a

  29. Identification of a T-DNA insertion mutant (glt1-T) in the NADH-GOGAT GLT1 gene Identification of a T-DNA insertion mutant (glt1-T) in the NADH-GOGAT GLT1 gene • Domains important to function of NADH-GOGAT: • 1, 2, 3, 4: PurF-type amidotransferase regions • 5 & 6: flavin mononucleotide (FMN) binding regions • 7: 3Fe-4S cluster binding domain Figure 2a

  30. Identification of a T-DNA insertion mutant (glt1-T) in the NADH-GOGAT GLT1 gene Identification of a T-DNA insertion mutant (glt1-T) in the NADH-GOGAT GLT1 gene • Domains important to function of NADH-GOGAT: • 1, 2, 3, 4: PurF-type amidotransferase regions • 5 & 6: flavin mononucleotide (FMN) binding regions • 7: 3Fe-4S cluster binding domain • 8: NADH-binding domain Figure 2a

  31. Identification of a T-DNA insertion mutant (glt1-T) in the NADH-GOGAT GLT1 gene Identification of a T-DNA insertion mutant (glt1-T) in the NADH-GOGAT GLT1 gene • Domains important to function of NADH-GOGAT: • 1, 2, 3, 4: PurF-type amidotransferase regions • 5 & 6: flavin mononucleotide (FMN) binding regions • 7: 3Fe-4S cluster binding domain • 8: NADH-binding domain If truncated by T-DNA insertion: loss-of-function Figure 2a

  32. Identification of a T-DNA insertion mutant (glt1-T) in the NADH-GOGAT GLT1 gene To test whether mRNA is truncated by T-DNA insertion: • 2 oligonucleotide probes designed for Northern blot • Probed total leaf RNA from WT and glt1-T mutants Figure 2a

  33. Identification of a T-DNA insertion mutant (glt1-T) in the NADH-GOGAT GLT1 gene To test whether mRNA is truncated by T-DNA insertion: • 2 oligonucleotide probes designed for Northern blot • Probed total leaf RNA from WT and glt1-T mutants • p1 is upstream of T-DNA insertion Figure 2a

  34. Identification of a T-DNA insertion mutant (glt1-T) in the NADH-GOGAT GLT1 gene To test whether mRNA is truncated by T-DNA insertion: • 2 oligonucleotide probes designed for Northern blot • Probed total leaf RNA from WT and glt1-T mutants • p1 is upstream of T-DNA insertion • p2 is downstream of T-DNA insertion Figure 2a

  35. Identification of a T-DNA insertion mutant (glt1-T) in the NADH-GOGAT GLT1 gene Both p1 and p2detect 7.6 kb GLT1 mRNA in WT GLN2 is a control Figure 2

  36. Identification of a T-DNA insertion mutant (glt1-T) in the NADH-GOGAT GLT1 gene Both p1 and p2 detect 7.6 kb GLT1 mRNA in WT No 7.6 kb GLT1 mRNA in glt1-T mutant GLN2 is a control Figure 2

  37. Identification of a T-DNA insertion mutant (glt1-T) in the NADH-GOGAT GLT1 gene Both p1 and p2 detect 7.6 kb GLT1 mRNA in WT No 7.6 kb GLT1 mRNA in glt1-T mutant p1 and p2 detect 2 and 5 kb GLT1 mRNA in glt1-T mutant GLT1 mRNA is truncated in mutant GLN2 is a control Figure 2

  38. Identification of a T-DNA insertion mutant (glt1-T) in the NADH-GOGAT GLT1 gene Results show NADH-GOGAT GLT1 gene was in fact truncated in the glt1-T mutants Suggests that mutant can NOT produce function NADH-GOGAT protein One last step to verify that glt1-T is a null mutant: • Assays of NADH-GOGAT activity in WT and glt1-T mutant • Performed on WT and mutant plants grown in air and 1% CO2

  39. Identification of a T-DNA insertion mutant (glt1-T) in the NADH-GOGAT GLT1 gene No detectable NADH-GOGAT activity in glt1-T mutants Verifies that truncation has generated a functional null mutant

  40. Analysis of NADH-GOGAT T-DNA mutant (glt1-T) phenotype grown under photorespiratory conditions What exactly is the function of NADH-GOGAT? Specifically, is it different than that of Fd-GOGAT?

  41. Analysis of NADH-GOGAT T-DNA mutant (glt1-T) phenotype grown under photorespiratory conditions What exactly is the function of NADH-GOGAT? Specifically, is it different than that of Fd-GOGAT? How did Lancien et al. determine this?

  42. Analysis of NADH-GOGAT T-DNA mutant (glt1-T) phenotype grown under photorespiratory conditions What exactly is the function of NADH-GOGAT? Specifically, is it different than that of Fd-GOGAT? How did Lancien et al. determine this? • Compared NADH-GOGAT mutant (glt1-T) with Fd-GOGAT mutant (gls113) and wild type plants • Looked at each plant’s phenotype under two conditions: in airand in high CO2 • First, let’s look at the results when grown in air, when photorespiration occurs.

  43. Analysis of NADH-GOGAT T-DNA mutant (glt1-T) phenotype grown under photorespiratory conditions • As expected, the Fd-GOGAT mutant, gls113, shows severe growth defects when grown in air. Figure 3a

  44. Analysis of NADH-GOGAT T-DNA mutant (glt1-T) phenotype grown under photorespiratory conditions • As expected, the Fd-GOGAT mutant, gls113, shows severe growth defects when grown in air. • Chlorosis occurs because the Fd-GOGAT mutant is unable to reassimilate the ammonium released by photorespiration. Figure 3a **Not only is the excess ammonium toxic, but the nitrogen level falls. In order to conserve nitrogen, the plant stops producing chlorophyll.**

  45. Analysis of NADH-GOGAT T-DNA mutant (glt1-T) phenotype grown under photorespiratory conditions • In contrast, the NADH-GOGAT mutant shows normal growth in air based on appearance alone. Figure 3a

  46. Analysis of NADH-GOGAT T-DNA mutant (glt1-T) phenotype grown under photorespiratory conditions • In contrast, the NADH-GOGAT mutant shows normal growth in air based on appearance alone. • Their distinct phenotypes when grown in air supports the model that NADH-GOGAT and Fd-GOGAT have similarly distinct functions. Figure 3a However, this is not a quantitative measure or comparison of growth

  47. Analysis of NADH-GOGAT T-DNA mutant (glt1-T) phenotype grown under photorespiratory conditions • In contrast, the NADH-GOGAT mutant shows normal growth in air based on appearance alone. • Their distinct phenotypes when grown in air supports the model that NADH-GOGAT and Fd-GOGAT have similarly distinct functions. Figure 3a However, this is not a quantitative measure or comparison of growth

  48. Analysis of NADH-GOGAT T-DNA mutant (glt1-T) phenotype grown under photorespiratory conditions Analysis of NADH-GOGAT T-DNA mutant (glt1-T) phenotype grown under photorespiratory conditions To quantify growth: Fresh weight of plant material is measured and reported relative to wild type Figure 3b

  49. Analysis of NADH-GOGAT T-DNA mutant (glt1-T) phenotype grown under photorespiratory conditions Analysis of NADH-GOGAT T-DNA mutant (glt1-T) phenotype grown under photorespiratory conditions To quantify growth: Fresh weight of plant material is measured and reported relative to wild type Fd-GOGAT mutant has significantly reduced fresh weight Figure 3b

  50. Analysis of NADH-GOGAT T-DNA mutant (glt1-T) phenotype grown under photorespiratory conditions Analysis of NADH-GOGAT T-DNA mutant (glt1-T) phenotype grown under photorespiratory conditions To quantify growth: Fresh weight of plant material is measured and reported relative to wild type Wild type and NADH-GOGAT mutant have nearly identical fresh weights Figure 3b

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