Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Arabidopsis Aquaporins AtTIPI Slide 1 AtTIPK AtTIPD AtTIPC AtTIP2C AtTIPH AtTIP2G AtTIPG AtMIPH AtMIPK AtTMP2c AtMIPL AtPIP2a PIP TIP Clustal-X/TreeView AtTIPA AtTIPB GLPG F E. coli AtMIPG AtPIP3 AtMIPI AtPIP1a AtPIP1b AtMIPF AtTMPB AtNLM6 AtMIPM AtNLM14 BoMIP AtNLM3 AtNLM1 AtNLM2 AtNLM12 AtNLM10 AtNLM9 AtNLM4 AtNLM13 NLM-B Quigley F (Arizona) AtNLM5 0.1 AtNLM11 NLM-A Slide 2 Quigley F (Arizona) Location of AQP Genes on Chromosomes 10 5 Mb TMPB TIPB 20 NLM14 30 TIPL TIPA NLM4 (15) Ch-I MIPI Ch-II NLM8 TIPK NLM7 NLM3 MipH PIP1b (4) rDNA TIPg MIPL TMP2c NLM13 NLM6 TIPD TIP2G TIPI Ch-III MIPF Ch-IV (14) TIPH NLM5 TIPC NLM1 NLM2 MIPM PIP3 MIPK PIP1a (3) NLM12 Ch-V PIP2a NLM11 NLM9 NLM10 TIP2c MIPG (12) * - NLM7 and TipL encode for 3 transmembrane domains. Loci identified by the same color scheme encode closely related genes. - duplicated chromosome portions with AQPs. - position of centromers (Mb from end). Slide 3 Exon-Intron Structure of Arabidopsis AQP Genes NLM-B ++ # NLM-A H1 TIP PIP H2 LB H3 H4 H5 H6 NPA NPA * LE + ** + Arrows show the position of introns within aquaporin sub-classes H1-6 are transmembrane segments. LB, LE: Loops B and E with conserved N-P-A motif. *Intron absent from TIPc, TIP2c, TIPg, TIP2g and TIPh. +Intron ++Intron **Intron absent from TIPh. sliding in MIPg (appears where intron 2 in TIPs is found). absent from NLM3 & NLM5. #Intron absent from NLM14. Quigley F (Arizona) Slide 4 Structure of the putative Pseudogene TIPL* NLM-B ++ # NLM-A H1 TIP PIP H2 H3 H5 H6 NPA NPA * ** TIPL TIP sub-family H4 H4 ATG H5 H6 STOP H3 missing, truncated AQP with 2 TM, no EST reported. *We had considered NLM14 a Pseudogene before, but closer inspection indicates that is an authentic gene. Quigley F (Arizona) Slide 5 Structure of the putative Pseudogene NLM7* NLM-B ++ # NLM-A H1 TIP PIP NLM7 H2 H3 H4 H5 H6 NPA NPA * ** ATG Attila Tn-like sequence fragment H3 H4 H5 H6 frame shift, homology continues NLM-A sub-family Protein start after H3; frame-shift (sequence error?) Would lead to short peptide, but sequence homology Continues (5’ and 3’ including the C-terminus; no ESTs reported *We had considered NLM14 a Pseudogene before, but closer inspection indicates that is an authentic gene. Quigley F (Arizona) Slide 6 Structure of the putative Pseudogene NLM8* NLM-B ++ # NLM-A H1 TIP PIP H2 H3 H4 H5 H6 NPA NPA * ** STOP NLM8 H1 H2 H3 H5 H6 Different reading frame ATG NLM-A sub-family Predicted longer N-terminal sequence; TM H3,4, and 5 are missing; Homology exists but a different reading frame is used Protein with longer C-terminal end. No ESTs reported. *We had considered NLM14 a Pseudogene before, but closer inspection indicates that is an authentic gene. (Computer prediction missed Exon 1 - but close inspection indicates its presence - no ESTs reported. Quigley F (Arizona) Slide 7 AtTIP2g is close to a Mutator-like Element ORF mudrA ORF at3g26530 AtTIP2g ORF 1Kb 9 bp target site duplication TTAAAAAAA Chromosome 3 BAC, MFE16; MULE-23 (Mutator-like element, group 23*, 12,268 bp) The region 5’ of the ATG to the insertion site of MULE-23 is 178 bp in length. * Zhihui Yu, Stephen I. Wright and Thomas E.Bureau, Genetics 156: 2019-2031 (2000) Quigley F (Arizona) Slide 8 Mesembryanthemum AQPs are found in different Locations membrane localization TO PM MIP-A * 41kD * MIP-B 32 kD # MIP-C MIP-F 26 kD 33 kD To - tonoplast; PM - plasma membrane. Antibodies raised against peptides. MIP-A, B, C align with PIPs. MIP-F aligns with TIPs. *dimer Even in the unstressed state some PIPs are found in more than one membrane. Analysis by continuous sucrose gradient fractionation confirmed this fact (using antibodies to markers of PM, To, mitochondria, chloroplasts, nuclei and the endoplasmic reticulum Under stress conditions (drought, salinity, ABA) the redistribution is additionally affected. Barkla et al., 1999 Kirch et al., 2000 Vera-Estrella et al., 2001 and unpublished data. forms (~41 kD) product #degradation Barkla, B, Vera-Estrella, R. & Pantoja, O. (UNAM, Mexico) Slide 9 Expression of GFP under control of the Ice plant promoter MipB in Arabidopsis. For components of the vector see Grebenok et al., 1997. The promoter is not active in the primary root meristem But in the elongation zone. MipB is active, as in the ice plant, in lateral root meristems in Arabidopsis. Shuhua Yuan, MS thesis, (Arizona) 2001 Slide 10 We have been using the stopped flow photometer to determine the osmotic permeability of the tonoplast from the halophyte Mesembryanthemum crystallinum, as well as changes induced by salt and drought stress. The figure below shows original recordings of the changes in light scattering resulting from the imposition of a 100 mOsmol osmotic gradient (hyperosmotic) or under iso-osmotic conditions (iso-osmotic) in tonoplast vesicles from M. crystallinum leaves. The rate constant (kos) was obtained by fitting a single exponential to the curve, from where we have determined a Pos 0.6 hyperosmotic (100 mOsmol) Light Intensity (au) 0.5 0.4 0.3 0.2 0.1 Analysis of tonoplast vesicles using stopped-flow photometric determination of light-scattering. iso-osmotic 0.0 -0.1 0.0 0.2 0.4 0.6 0.8 1.0 Time (s) of 1205 µm s -1, a value twice that of tobacco cell cultures (Maurel et al., 1997) and 14-times higher than that for wheat roots (Niemietz and Tyerman, 1997). From similar experiments carried out at different temperatures, we have calculated a value for the activation energy for water movement across the tonoplast vesicles of M. crystallinum of 8.97 kJ/mol -1 K -1, a smaller value than that reported for the tonoplast of wheat Roots (23.32 kJ mol -1 K -1) and tobacco cell cultures (10.47 kJ mol-1 K -1). Vera-Estrella, R. & Pantoja, O. (UNAM, Mexico) Slide 11 Conductance of a Na+/K+-Transporter in Xenopus Oocytes 1 Na+ 1 Na+; 0.3 K+ 0.3 K+ 50 nA water injected oocyte 2.5 min 1 Na+ 1 Na+; 0.3 K+ 0.3 K+ 50 nA cRNA injected oocyte 5 min Carlos Muñoz-Garay and Omar Pantoja (IBT, UNAM, Cuernavaca, Mexico) Slide 12 Internal Concentration as a Fraction of External Influx and Efflux of Deuterium Tracer to Follow Water Movement in Zea Maize Normal Tissue Fit of Normal Tissue Stressed Tissue Fit of Stressed Tissue The flux of deuterated water into and out of corn roots, +/- 150 mM NaCl, was measured by NMR. _____ 1.2 1 0.8 0.6 0.4 0.2 0 0 500 1000 1500 Time (sec) Exodermal Layer 2000 2500 3000 The analysis, using models for N-layers (N-= 1, 2, etc.), indicated a major influence of the endodermis and trans-cellular flux of water through all cells of the cortex as the most likely route for water movement. ______ In stress tissues water flux is reduced most likely by reduced amounts of AQPs See: Rosenberg et al. (2000) ASPP Annual Meeting, abstract #454 Endodermal Layer Rosenberg, J & Shachar-Hill, Y (New Mexico State) Slide 13 Rice AQPs - Salt Shock Water Channel ESTs - Time Course in Rice OC104E01 - WCP-I Decline & recovery of transcripts during NaCl stress Water channels (1 PIP; 3 TIP) OC03G03 - WCP-II OC104A02 - WCP-IV 3.0 OC03F03 - WCP-III 2.0 1.0 Complete recovery after 7d (+) +1.6-fold 0.0 PIP-AQP -1.6-fold -1.0 -2.0 6 hours 24 hours 1 week -3.0 10.5 Decline after 15 min ( 11.5 12.5 13.5 14.5 Log(2) Signal Intensity ) 15 minutes 1 hour after 3 hours Kawasaki et al (U. Arizona, 2001) 15.5 NaCl shock Arrays can report strength & relative strength of expression/abundance