對於動物及植物而言，鐵是不可或缺的營養素之一。然而，高濃度的鐵卻會對植物產生毒害。目前在植物中，於高濃度鐵的環境下，調控植物生長之分子機制尚未明瞭。因此，本論文將把目標定在過量的重金屬鐵對水稻根尖細胞的影響及其相關訊息傳遞路徑。首先，對水稻處理由低至高濃度的鐵，結果顯示隨著鐵濃度愈高，水稻根尖細胞死亡愈明顯，當濃度為5 mM時，可抑制水稻根部生長。而且我們發現過量鐵可於水稻根部細胞誘發MBP(myelin basic protein) kinase之活性，進一步透過免疫沉澱法(IP)證實此MBP kinase為ERK-like MAP kinase。在植物中，鐵透過Fenton reaction，會產生活性氧化物質，若處理抗氧化劑GSH，可降低鐵造成水稻根部細胞的死亡及降低鐵誘發的MAP kinase，顯然活性氧化物質為鐵誘發訊息傳遞分子之一。此外，當我們利用鉀離子通道抑制劑4-AP，可降低鐵及銅造成水稻根部細胞死亡，但卻會加強鐵所誘發的MAPK活性。利用蛋白質酪氨酸去磷酸酶及H+-ATPase之抑制劑，也可發現其可抑制鐵誘發水稻根部細胞之死亡。經由序列比對分析，我們在水稻中發現十一個雙特異性去磷酸酶基因，其表現量會受鐵及釩逆境之影響。综合以上所述，MAP kinase，活性氧化物質，鉀離子通道，蛋白質酪氨酸酶，H+-ATPase及雙特異性去磷酸酶參與鐵在水稻根部誘發之訊息傳遞途徑。

　Iron is an essential micronutrient for normal growth and development of plants. However, at high concentrations iron can become toxic to plants. Very little information is known about the molecular mechanism responsible for the regulation of plant growth by excess iron. The aim of this study was to investigate the signal transduction pathway activated by increasing concentrations (0.5, 1.0, and 2.5 mM) of iron. We showed that iron elicited a remarkable MBP kinase activity. By western blot and immunoprecipitation analysis, we suggested that iron-activated 42-kDa MBP kinase is a mitogen-activated protein kinase (MAPK). Cell death in rice roots due to iron toxicity was investigated using inhibitors of signal molecules known to regulate programmed cell death in plants. Phenylarsine oxide (PAO) and sodium orthovanadate, known inhibitors of tyrosine phosphatase, reduced iron-induced root cell death, but, cantharidin and endothall two-serine/threonine phosphatase inhibitor enhanced iron-induced root cell death. Moreover, our results revealed that H+-ATPase might participate in iron-induced cell death. These results suggested that the MAPK, reactive oxygen species (ROS), potassium channel, protein phosphatase, and H+-ATPase might function in the plant iron-triggered signalling pathway in rice roots. With analysis of the rice genome database, we identified eleven dual specificity phosphotases in rice genome. We found some dual-specificity phosphatases of gene expression pattern could be induced by vanadate. The pyronitrophenyl phosphatase (pNPP) was used as substrate and the phosphatase activity of OsDSP11 were measured in the present study.

Baker CJ and Mock NM. 1994. An improved method for monitoring cell death in
cell suspension and leaf disc assays using Evans blue. Plant Cell Tissue
Organ Cult 39: 7-12
Barizza E, Schiavo FL, Terzi M and Filippini F. 1999. Evidence suggesting
protein tyrosine phosphorylation in plants depends on the developmental
conditions. FEBS Lett 447: 191-194
Berset T, Frohli Hoier E, Battu G, Canevascini S and Hajnal A. 2001. Notch
inhibition of RAS signaling through MAP kinase phosphatase LIP-1 during C.
elegans vulval development. Science 291: 1055-1058
Briat JF and Lobreaux S. 1997. Iron transport and storage in plants. Trends
Plant Sci 2: 187-193
Caro A and Puntarulo S. 1996. Effect of in vivo iron supplementation on oxygen
radical production by soybean roots. Biochim Biophys Acta 1291: 245-251
Comporti M, Signorini C, Buonocore G and Ciccoli L. 2002. Iron release,
oxidative stress and erythrocyte ageing. Free Radic Biol Med 32: 568-576
Dai J, Huang C, Wu J, Yang C, Frenkel K and Huang X. 2004. Iron-induced
interleukin-6 gene expression: possible mediation through the extracellular
signal-regulated kinase and p38 mitogen-activated protein kinase pathways.
Toxicology 203: 199-209
Decroocg-Ferrant V, Decroocq S, Van Went J, Schmidt E and Kreis M. 1995. A
homologue of the MAP/ERK family of protein kinase genes is expressed in
vegetative and in female reproductive organs of Petunia hybrida. Plant Mol
Biol 27: 339-350
Demidchik V, Shabala SN, Coutts KB, Tester MA, and Davies JM. 2003. Free
oxygen radicals regulate plasma membrane Ca2+- and K+-permeable channels in
plant root cells. J Cell Sci 116: 81-88
Desikan R, Clarke A, Hancock JT and Neill SJ. 1999. H2O2 activates a MAP
kinase-like enzyme in Arabidopsis thaliana suspension cultures. J Exp Bot
50: 1863-1866
Elstner EF. 1991. Oxygen radicals-biochemical basis for their efficacy. Klin
Wochenschr 69: 949-956
Evans NH, McAinsh MR and Hetherington AM. 2001. Calcium oscillations in higher
plants. Curr Opin Plant Biol 4: 415-420
Fang W and Kao CH. 2000. Enhanced peroxidase activity in rice leaves in
response to excess iron, copper and zinc. Plant Sci 158: 71-76
Franklin CC, Srikanth S, Kraft AS. 1998. Conditional expression of mitogen-
activated protein kinase phosphatase-1, MKP-1, is cytoprotective against UV-
induced apoptosis. Proc Natl Acad Sci USA 95: 3014-3019
Fu SF, Chow WC, Huang DD and Huang HJ. 2002. Transcriptional regulation of a
rice mitogen-activated protein kinase gene, OsMAPK4, in response to
environmental stresses. Plant Cell Physiol 43: 958-963
Gallego SM, Benavides MP and Tomaro ML. 1996. Effect of heavy metal ion excess
on sunflower leaves: evidence for involvement of oxidative stress. Plant Sci
121: 151-159
Gupta R, Huang Y, Kieber J and Luan S. 1998. Identification of a dual-
specificity protein phosphatase that inactivates a MAP kinase from
Arabidopsis. Plant J 16: 581-589
Halliwell B and Gutteridge JMC. 1988. Free Radicals in Biology and Medicine.
Clarendon Press, Oxford
Hancock JT, Desikan R and Neill SJ. 2001. Role of reactive oxygen species in
cell signalling pathways. Biochem Soc Trans 29: 345-350
Herskowitz I. 1995. MAP kinase pathways in yeast: for mating and more. Cell
80: 187-197
Hopkins WG. 1997. Introduction to Plant Physiology, John Wiley & Sons, London,
pp. 61-76
Huang HJ, Lin YM, Huang DD, Takahashi T and Sugiyama M. 2003. Protein tyrosine
phosphorylation during phytohormone-stimulated cell proliferation in
Arabidopsis hypocotyls. Plant Cell Physiol 44: 770-775
Ichimura K, Shinozaki K and Tena G. 2002. Mitogen-activated protein kinase
cascades in plants: a new nomenclature. Trends Plant Sci 7: 301-308
Iryo Y, Matsuoka M, Wispriyono B, Sugiura T and Igisu H. 2000. Involvement of
the extracellular signal-regulated protein kinase (ERK) pathway in the
induction of apoptosis by cadmium chloride in CCRF-CEM cells. Biochem
Pharmacol 60: 1875-1882
Jonak C, Ligterink W and Hurt H. 1999. MAP kinases in plant signal
transduction. Cell Mol Life Sci 55: 204-213
Kampfenkel K, Van Montagu M and Inze D. 1995. Effects of Iron Excess on
Nicotiana plumbaginifolia Plants (Implications to Oxidative Stress). Plant
Physiol 107: 725-735
Kerk D, Bulgrien J, Smith DW, Barsam B, Veretnik S and Gribskov M. 2002. The
complement of protein phosphatase catalytic subunits encoded in the genome
of Arabidopsis. Plant Physiol 129: 908-925
Klumpp S and Krieglstein J. 2002. Serine/threonine protein phosphatases in
apoptosis. Curr Opin Pharmacol 2: 458-62
Knight H and Knight MR. 2001. Abiotic stress signalling pathways: specificity
and cross-talk. Trends Plant Sci 6: 262-267
Kourie JI. 1998. Interaction of reactive oxygen species with ion transport
mechanisms. Am J Physiol 44: 1-24
Kovtun Y, Chiu WL, Tena G and Sheen J. 2000. Functional analysis of oxidative
stress-activated mitogen-activated protein kinase cascade in plants. Proc
Natl Acad Sci USA 97: 2940-2945
Kuperstein F and Yavin E. 2003. Pro-apoptotic signaling in neuronal cells
following iron and amyloid beta peptide neurotoxicity. J Neurochem 86: 114-
125
Kyriakis JM and Avruch J. 1996. Sounding the alarm: protein kinase cascades
activated by stress and inflammation. J Biol Chem 271: 24313-24316
Lobreaux S, Thoiron S and Briat JF. 1995. Induction of ferritin synthesis in
maize leaves by an iron-mediated oxidative stress. Plant J 8: 443-449
Mackintosh C and Mackintosh RW. 1994. Inhibitors of protein kinases and
phosphatases. Trends Biochem Sci 19: 444-448
MacRobbie EA. 2002. Evidence for a role for protein tyrosine phosphatase in
the control of ion release from the guard cell vacuole in stomatal closure.
Proc Natl Acad Sci USA 99: 11963-11968
Monroe-Augustus M, Zolman BK and Bartel B. 2003. IBR5, a dual-specificity
phosphatase-like protein modulating auxin and abscisic acid responsiveness
in Arabidopsis. Plant Cell 15: 2979-2991
Murphy AS, Eisinger WR, Shaff JE, Kochian LV and Taiz L. 1999. Early copper-
induced leakage of K+ from Arabidopsis seedlings in mediated by ion channels
and coupled to citrate efflux. Plant Physiol 121: 1375-1382
Naoi K and Hashimoto T. 2004. A semidominant mutation in an Arabidopsis
mitogen-activated protein kinase phosphatase-like gene compromises cortical
microtubule organization. Plant Cell 16: 1841-1853
Nishizaki Y. 1994. Vanadate and dicyclohexylcarbodiimide inhibit the blue
light-induced depolarization of the membrane in pulvinar motor cells of
Phaseolus. Plant Cell Physiol 35: 841-844
Olivari C, Albumi C, Pugliarello MC and De Michelis MI. 2000. Phenylarsine
oxide inhibits the fusicoccin-induced activation of plasma membrane H(+)-
ATPase. Plant Physiol 122: 463-470
Osawa H and Matsumoto H. 2002. Aluminium triggers malate-independent potassium
release via ion channels from the root apex in wheat. Planta 215: 405-412
Pekker I, Tel-Or E and Miltter R. 2002. Reactive oxygen intermediates and
glutathione regulate the expression of cytosolic ascorbate peroxidase during
iron-mediated oxidative stress in bean. Plant Mol Biol 49: 429-438
Rakwal R, Kumar Agrawal G and Jwa NS. 2001. Characterization of a rice (Oryza
sativa L.) Bowman-Birk proteinase inhibitor: tightly light regulated
induction in response to cut, jasmonic acid, ethylene and protein
phosphatase 2A inhibitors. Gene 263: 189-198
Roux PP and Blenis J. 2004. ERK and p38 MAPK-activated protein kinases: a
family of protein kinases with diverse biological functions. Microbiology
and Molecular Biology Reviews 68: 320-344
Ryan PR, Delhaize E and Randall PJ. 1995 Characterisation of Al-stimulated
efflux of malate from the apices of Al-tolerant wheat roots. Planta 196: 103-
110
Saijo Y, Hata S, Kyozuka J, Shimamoto K and Izui K. 2000. Over-expression of a
single Ca2+-dependent protein kinase confers both cold and salt/drought
tolerance on rice plants. Plant J 23: 319-327
Sander D, Brownlee C and Harper JF. 1999. Communicating with calcium. Plant
Cell 11: 691-706
Savino G, Briat JF and Lobreaux S. 1997. Inhibition of the iron-induced ZmFer1
maize ferritin gene expression by antioxidants and serine/threonine
phosphatase inhibitors. J Biol Chem. 272: 33319-33326
Schmidt W. 2003. Iron solutions: acquisition strategies and signaling pathways
in plants. Trends Plant Sci 8: 188-193
Shih YW, Chou WC, Lin YM, Huang DD, Liu ZH and Huang HJ. 2004. Changes in
protein tyrosine phosphorylation during mannose and senescence induced cell
death in rice. Plant Growth Regul 42: 271-282
Shiozaki K, Shiozaki M and Russell P. 1998. Heat stress activates fission
yeast Spc1/StyI MAPK by a MEKK-independent mechanism. Mol Biol Cell 9: 1339-
1349
Smith and Walker. 1996. Plant protein phosphatases. Annu Rev Plant Physiol
Plant Mol Biol 47: 101-125
Tahara S, Fukuda K, Kodama H, Kato T, Miyoshi S, and Ogawa S. 2001. Potassium
channel blocker activates extracellular signal-regulated kinases through
Pyk2 and epidermal growth factor receptor in rat cardiomyocytes. J Am Coll
Cardiol 38: 1554-1563
Ulm R, Revenkova E, di Sansebastiano G-P, Bechtold N and Paszkowski K. 2001.
Mitogen-activated protein kinase phosphatase is required for genotoxic
stress relief in Arabidopsis. Genes Dev 15: 699-709
Van Hoof C and Goris J. 2003. Phosphatases in apoptosis: to be or not to be,
PP2A is in the heart of the question. Biochim Biophys Acta 1640: 97-104
Walter PB, Knutson MD, Paler-Martinez A, Lee S, Xu Y, Viteri FE and Ames BN.
2002. Iron deficiency and iron excess damage mitochondria and mitochondrial
DNA in rats. Proc Natl Acad Sci USA 99: 2264-269
Wang X, Martindale JL, Liu Y and Holbrook NJ. 1998. The cellular response to
oxidative stress: influences of mitogen-activated protein kinase signaling
pathways on cell survival. Biochem J 333: 291-300
Welch KD, Davis TZ, Van Eden ME and Aust SD. 2002. Deleterious iron-mediated
oxidation of biomolecules. Free Radic Biol Med 32: 577-583
Widmann C, Gibson S, Jarpe MB and Johnson GL. 1999. Mitogen-activated protein
kinase: conservation of a three-kinase module from yeast to human. Physiol
Rev 79: 143-180
Williams LE, Pittman JK and Hall JL. 2000. Emerging mechanisms for heavy metal
transport in plants. Biochim Biophys Acta 1465: 104-126
Wilson MA, Burt AR, Milligan G and Anderson NG. 1997. Mitogenic signalling by opioid receptors expressed in rat-1 fibroblasts involves activation of the
p70s6k/p85s6k S6 kinase. Biochem J 325: 217-222
Xu Q, Fu HH, Gupta R and Luan S. 1998. Molecular characterization of a
tyrosine-specific protein phosphatase encoded by a stress-response gene in
Arabidopsis. Plant Cell 10: 849-857
Yeh CM, Hung WC and Huang HJ. 2003. Copper treatment activates mitogen-
activated protein kinase signaling in rice. Physiol Plant 119: 392-399
Yeh CM, Hsiao LJ and Huang HJ. 2004. Cadmium activates a mitogen-activated
protein kinase gene and MBP kinases in rice. Plant Cell Physiol 45: 1306-1312
Yoo YE, Hong JH, Hur KC, Oh ES and Chung JM. 2004. Iron enhances NGF-induced
neurite outgrowth in PC12 cells. Mol Cells 17: 340-346
Yuasa T, Ichimura K, Mizoguchi T and Shinozaki K. 2001. Oxidative stress
activates ATMPK6, an Arabidopsis homologue of MAP kinase. Plant Cell Physiol
42: 1012-1016
Zenk MH. 1996. Heavy metal detoxification in higher plants-a review. Gene 179:
21-30
Zhang S and Klessing DF. 1997. Salicylic acid activates a 48-kD MAP kinase in
Tobacco. Plant Cell 9: 809-824