# Patent

## Title
Plant tubby-like proteins

## Abstract

An isolated polypeptide containing an amino acid sequence at least 70% identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, and an isolated nucleic acid encoding the polypeptide are disclosed. Disclosed is an isolated nucleic acid that, under stringent conditions, hybridizes to a probe containing SEQ ID NO:20; or its complementary sequence. Also disclosed are (1) a transformed cell or a transgenic plant containing such a nucleic acid and (2) a transformed cell or a transgenic plant lacking the polypeptide encoded by the nucleic acid. Also within the scope of the invention are methods for making the transformed cells or transgenic plants.

## Background
RELATED APPLICATION
This application claims priority to U.S. Provisional Application Ser. No. 60/441,380, filed on Jan. 21, 2003, the contents of which are incorporated by reference in their entirety.


BACKGROUND
Various environmental factors, e.g., high salinity, pathogens, and chilling, cause stress and adverse effects on growth and productivity of crops. It is therefore desirable to produce transgenic crops that are tolerant to such factors. Genetic engineering can be used to modify proteins that are involved in regulating responses of plants to environmental factors, thereby improving stress-tolerance.
TUBBY proteins, a group of membrane-bound transcription regulators, were first identified from obese mice via positional cloning (Kleyn et al., 1996, Cell 85: 281-290 and Noben-Trauth et al., 1996, Nature 380: 534-538.). Mutations in the TUBBY genes lead to maturity-onset obesity, insulin resistance, retinal degeneration, and neurosensory hearing loss. TUBBY-like proteins (TLPs) were subsequently discovered in other mammals and were found to be activated through G-proteins, which, in higher plants, are involved in the response to environmental factors and hormone regulation (Warpeha et al., 1991, Proc. Natl. Acad. Sci. 88: 8925-8929, and Ueguchi-Tanaka et al., Proc. Natl. Acad. Sci. 97: 11638-11643).
SUMMARY
This invention is based on the discovery of eleven Arabidopsis TUBBY-like proteins, designated as AtTLPs 1-11. These proteins regulate the response of Arabidopsis to environmental factors. The full-length AtTLPs 1-11 polypeptides (designated as SEQ ID NOs: 1-11), and cDNAs encoding the polypeptides (designated as SEQ ID NOs: 12-22), are shown below:








AtTLP1:



polypeptide:











  1
MSFRSIVRDV RDSIGSLSRR SFDFKLSSLN KEGGKSRGSV QDSHEEQLVV
(SEQ ID NO: 1)



 


 51
TIQETPWANL PPELLRDVIK RLEESESVWP ARRHVVACAS VCRSWRDMCK


 


 101
EIVQSPELSG KITFPVSLKQ PGPRDATMQC FIKRDKSNLT YHLYLCLSPA


 


 151
LLVENGKFLL SAKRIRRTTY TEYVISMHAD TISRSSNTYI GKIRSNFLGT


 


 201
KFIIYDTQPA YNSNIARAVQ PVGLSRRFYS KRVSPKVPSG SYKIAQVSYE


 


 251
LNVLGTRGPR RMHCAMNSIP ASSLAEGGTV PGQPDIIVPR SILDESFRSI


 


 301
TSSSSRKITY DYSNDFSSAR FSDILGPLSE DQEVVLEEGK ERNSPPLVLK


 


 351
NKPPRWHEQL QCWCLNFRGR VTVASVKNFQ LIAANQPQPQ PQPQPQPQPL


 


 401
TQPQPSGQTD GPDKIILQFG KVGKDMFTMD FRYPLSAFQA FAICLSSFDT


 


 451
KLACE


 


cDNA:


  1
ATGTCGTTCC GTAGCATAGT TCGTGATGTG AGAGATAGTA TAGGAAGTCT
(SEQ ID NO: 12)


 


 51
ATCGAGGCGT AGTTTCGACT TTAAGTTAAG CAGCTTGAAC AAAGAAGGTG


 


 101
GTAAATCCCG TGGTTCGGTT CAAGATTCTC ATGAGGAACA ACTTGTAGTA


 


 151
ACGATTCAAG AAACACCGTG GGCGAATCTA CCTCCAGAGT TATTACGTGA


 


 201
TGTGATCAAA AGACTTGAAG AGAGTGAAAG TGTGTGGCCT GCTCGTAGAC


 


 251
ATGTTGTTGC TTGTGCTTCT GTTTGCAGGT CATGGAGAGA TATGTGTAAA


 


 301
GAGATTGTTC AAAGTCCGGA GCTCTCAGGC AAAATCACAT TTCCTGTTTC


 


 351
GTTGAAACAG CCTGGACCAA GAGATGCAAC AATGCAATGC TTTATCAAAA


 


 401
GGGATAAATC TAACTTGACT TATCATTTAT ATCTTTGTCT CAGTCCTGCT


 


 451
TTGTTGGTTG AGAATGGAAA GTTTCTTCTT TCTGCAAAAC GCATAAGAAG


 


 501
AACTACATAC ACCGAGTACG TGATCTCTAT GCACGCCGAC ACCATTTCGA


 


 551
GATCAAGCAA TACCTACATT GGCAAAATCA GGTCTAATTT TCTGGGGACG


 


 601
AAGTTTATAA TATACGATAC ACAACCAGCA TACAACAGCA ACATCGCTCG


 


 651
AGCGGTCCAA CCGGTAGGTC TTAGCCGCAG ATTCTACTCA AAGAGAGTCT


 


 701
CTCCCAAAGT ACCTAGTGGG AGCTACAAAA TTGCGCAGGT TTCTTATGAG


 


 751
CTAAACGTTC TTGGTACCCG TGGTCCGAGG AGAATGCATT GTGCGATGAA


 


 801
CTCAATTCCC GCCTCTTCCC TTGCGGAAGG CGGAACTGTG CCTGGACAGC


 


 851
CCGATATCAT TGTCCCGCGC TCTATTCTCG ACGAATCGTT CCGCAGCATT


 


 901
ACCTCTTCGT CATCGAGAAA AATCACTTAC GATTACTCGA ATGATTTTAG


 


 951
CAGTGCACGC TTTTCCGACA TTCTTGGCCC GTTAAGCGAA GACCAAGAAG


 


1001
TGGTATTAGA AGAAGGGAAA GAGCGGAATT CGCCACCACT TGTGCTTAAG


 


1051
AACAAGCCGC CGAGGTGGCA TGAACAGCTT CAGTGTTGGT GTTTAAACTT


 


1101
CAGGGGACGT GTAACAGTCG CATCAGTTAA GAACTTTCAG CTCATTGCAG


 


1151
CAAACCAACC ACAGCCTCAG CCTCAGCCTC AACCGCAACC TCAACCCCTA


 


1201
ACTCAGCCGC AACCGTCTGG TCAGACCGAT GGTCCCGACA AGATCATATT


 


1251
GCAGTTTGGG AAAGTGGGAA AAGACATGTT CACGATGGAT TTCCGGTATC


 


1301
CGCTCTCTGC GTTTCAGGCT TTCGCTATCT GTTTGAGCAG TTTCGACACA


 


1351
AAACTTGCTT GCGAA


 


AtTLP2:


polypeptide:


  1
MSLKSILRDL KEVRDGLGGI SKRSWSKSSH IAPDQTTPPL DNIPQSPWAS
(SEQ ID NO: 2)


 


 51
LPPELLHDII WRVEESETAW PARAAVVSCA SVCKSWRGIT MEIVRIPEQC


 


 101
GKLTFPISLK QPGPRDSPIQ CFIKRNRATA TYILYYGLMP SETENDKLLL


 


 151
AARRIRRATC TDFIISLSAK NFSRSSSTYV GKLRSGFLGT KFTIYDNQTA


 


 201
SSTAQAQPNR RLHPKQAAPK LPTNSSTVGN ITYELNVLRT RGPRRMHCAM


 


 251
DSIPLSSVIA EPSVVQGIEE EVSSSPSPKG ETITTDKEIP DNSPSLRDQP


 


 301
LVLKNKSPRW HEQLQCWCLN FKGRVTVASV KNFQLVAEID ASLDAPPEEH


 


 351
ERVILQFGKI GKDIFTMDYR YPLSAFQAFA ICISSFDTKP ACEG


 


cDNA:


  1
ATGTCTTTGA AAAGCATCCT TCGTGATCTG AAGGAAGTGA GGGATGGACT
(SEQ ID NO: 13)


 


 51
TGGAGGCATC TCCAAGAGAA GCTGGTCAAA GTCGTCTCAC ATTGCTCCTG


 


 101
ATCAAACAAC TCCACCACTG GATAACATAC CACAGAGCCC ATGGGCTTCT


 


 151
TTGCCGCCTG AGTTGCTTCA TGACATTATC TGGAGGGTTG AAGAGAGTGA


 


 201
GACAGCTTGG CCCGCTCGAG CTGCCGTTGT CTCTTGTGCT TCAGTATGTA


 


 251
AATCATGGAG AGGAATCACT ATGGAGATTG TGAGGATCCC TGAGCAGTGT


 


 301
GGGAAGCTCA CTTTTCCAAT CTCATTGAAA CAGCCGGGGC CTCGAGACTC


 


 351
TCCAATTCAA TGTTTTATTA AGAGGAACAG AGCAACAGCT ACATACATTC


 


 401
TCTATTATGG TTTGATGCCT TCGGAGACTG AGAACGACAA ACTGTTGTTA


 


 451
GCAGCAAGAA GGATTAGAAG AGCGACATGC ACAGACTTTA TAATCTCCCT


 


 501
ATCTGCAAAG AACTTCTCAC GGAGCAGCAG TACTTATGTT GGCAAATTAA


 


 551
GGTCTGGTTT TCTGGGAACC AAGTTCACAA TATATGACAA CCAAACAGCA


 


 601
TCATCCACAG CACAAGCCCA ACCTAACCGA AGACTCCACC CGAAACAAGC


 


 651
GGCTCCTAAA CTACCTACGA ATAGCTCTAC CGTAGGAAAC ATAACCTACG


 


 701
AGCTCAATGT TCTTCGCACA AGGGGACCTA GAAGAATGCA CTGCGCTATG


 


 751
GATTCTATAC CCCTCTCTTC TGTTATTGCT GAACCGTCAG TAGTTCAAGG


 


 801
CATAGAAGAG GAAGTCTCTT CCTCTCCTTC ACCAAAAGGA GAAACCATCA


 


 851
CAACAGACAA AGAGATTCCT GATAATTCTC CAAGCTTAAG GGACCAACCG


 


 901
CTAGTTCTCA AAAACAAATC CCCAAGATGG CATGAGCAGT TGCAGTGCTG


 


 951
GTGCCTCAAC TTCAAGGGAA GAGTGACTGT GGCTTCAGTT AAGAATTTCC


 


1001
AGCTTGTTGC AGAGATTGAC GCTTCTTTGG ATGCGCCGCC TGAAGAACAT


 


1051
GAGAGGGTGA TCTTACAGTT TGGCAAAATC GGTAAGGATA TTTTCACCAT


 


1101
GGATTATCGC TACCCTCTAT CTGCTTTTCA AGCCTTTGCT ATATGCATTA


 


1151
GCAGCTTTGA CACCAAACCG GCATGTGAAG GG


 


AtTLP3:


polypeptide:


  1
MSFKSLIQDM RGELGSISRK GFDVRFGYGR SRSQRVVQDT SVPVDAFKQS
(SEQ ID NO: 3)


 


 51
CWASMPPELL RDVLMRIEQS EDTWPSRKNV VSCAGVCRNW REIVKEIVRV


 


 101
PELSSKLTFP ISLKQPGPRG SLVQCYIMRN RSNQTYYLYL GLNQAASNDD


 


 151
GKFLLAAKRF RRPTCTDYII SLNCDDVSRG SNTYIGKLRS NFLGTKFTVY


 


 201
DAQPTNPGTQ VTRTRSSRLL SLKQVSPRIP SGNYPVAHIS YELNVLGSRG


 


 251
PRRMQCVMDA IPASAVEPGG TAPTQTELVH SNLDSFPSFS FFRSKSIRAE


 


 301
SLPSGPSSAA QKEGLLVLKN KAPRWHEQLQ CWCLNFNGRV TVASVKNFQL


 


 351
VAAPENGPAG PEHENVILQF GKVGKDVFTM DYQYPISAFQ AFTICLSSFD


 


 401
TKIACE


 


cDNA:


  1
ATGTCCTTCA AGAGTCTCAT TCAGGACATG AGAGGAGAGC TTGGGAGTAT
(SEQ ID N0:14)


 


 51
ATCCAGAAAG GGATTCGATG TCAGATTCGG GTATGGTAGA TCCAGGTCTC


 


 101
AACGTGTTGT TCAGGATACT TCTGTTCCTG TTGATGCTTT CAAGCAGAGC


 


 151
TGCTGGGCTA GTATGCCTCC GGAGCTCCTG AGAGATGTTC TTATGAGGAT


 


 201
TGAGCAATCC GAAGACACTT GGCCGTCTAG GAAAAATGTT GTTTCTTGCG


 


 251
CTGGTGTCTG CAGGAACTGG CGAGAAATCG TCAAAGAGAT CGTCAGAGTT


 


 301
CCTGAGCTTT CTAGCAAACT CACTTTTCCT ATCTCCCTCA AACAGCCGGG


 


 351
TCCTAGAGGA TCACTTGTTC AATGCTATAT TATGAGAAAC CGCAGCAATC


 


 401
AAACCTACTA TCTATACCTC GGGTTAAACC AAGCAGCTTC AAATGATGAT


 


 451
GGAAAGTTCC TTCTTGCTGC CAAGAGGTTT CGGAGGCCAA CTTGCACTGA


 


 501
CTACATCATC TCCTTAAACT GCGATGATGT CTCTCGAGGA AGCAATACCT


 


 551
ATATCGGAAA GCTTAGATCT AACTTTCTGG GGACCAAGTT CACTGTCTAT


 


 601
GACGCTCAGC CGACGAATCC TGGAACTCAG GTTACCAGAA CCCGTTCAAG


 


 651
CAGACTTCTC AGTTTGAAAC AAGTGAGCCC GAGAATTCCA TCTGGCAACT


 


 701
ATCCTGTAGC ACATATCTCA TATGAGCTTA ACGTCTTGGG TTCCAGAGGA


 


 751
CCGAGGAGGA TGCAGTGTGT CATGGATGCC ATCCCTGCAT CAGCTGTAGA


 


 801
ACCTGGAGGA ACAGCTCCAA CTCAGACGGA ACTTGTCCAT AGCAATCTTG


 


 851
ATAGTTTCCC CTCATTCTCC TTCTTCAGGT CGAAATCAAT TCGTGCAGAG


 


 901
AGTCTCCCTT CTGGTCCATC ATCTGCTGCT CAGAAGGAAG GACTGCTTGT


 


 951
CCTGAAAAAC AAAGCGCCCA GATGGCACGA ACAGCTCCAG TGCTGGTGCC


 


1001
TCAACTTCAA TGGGAGAGTC ACAGTTGCTT CCGTCAAAAA CTTTCAGCTG


 


1051
GTAGCTGCTC CTGAGAATGG ACCTGCAGGA CCTGAGCACG AAAACGTGAT


 


1101
TCTCCAGTTT GGAAAAGTCG GAAAAGATGT GTTCACAATG GATTATCAGT


 


1151
ACCCTATCTC TGCCTTCCAG GCCTTCACCA TTTGCCTCAG CAGTTTCGAC


 


1201
ACCAAGATAG CATGTGAA


 


AtTLP4:


polypeptide:


  1
MPPELLRDVL MRIERSEDTW PSRKNVVSCV GVCKNWRQIF KEIVNVPEVS
(SEQ ID NO:4)


 


 51
SKFTFPISLK QPGPGGSLVQ CYVKRNRSNQ TFYLYLGGEA KIFCQSEPSD


 


 101
IYLVPYSYRE THCVMDAISA SAVKPGGTAT TQTELDNFVS FRSPSGQKEG


 


 151
VLVLKSKVPR LEEQSWCLDF NGSPENEPEN ENDIFQFAKV GNLHKLFSLY


 


 201
EAEWIPLVRT SVFAVIARVC RDKKHTPSYE LKLALYFAKN SAILKKFVLR


 


 251
GYTREEDLLA LPVAN


 


cDNA:


  1
ATGCCTCCTG AGCTTCTGAG AGATGTTCTG ATGAGGATAG AGCGATCCGA
(SEQ ID NO:15)


 


 51
AGACACTTGG CCTTCTAGGA AGAATGTTGT TTCTTGTGTA GGTGTGTGTA


 


 101
AGAACTGGCG ACAAATATTC AAAGAGATCG TTAACGTTCC TGAGGTTTCT


 


 151
AGCAAATTCA CTTTTCCAAT CTCCTTGAAA CAGCCTGGTC CAGGAGGATC


 


 201
ACTTGTTCAA TGCTATGTTA AGAGAAACCG TAGCAATCAA ACTTTCTATC


 


 251
TATACCTTGG AGGTGAAGCA AAAATATTTT GTCAGTCTGA ACCAACTGAT


 


 301
ATTTATCTCG TTCCTTACAG TTACAGAGAG ACGCATTGCG TCATGGATGC


 


 351
CATCTCTGCA TCAGCAGTAA AACCTGCAGG AACAGCTACA ACTCAGACAG


 


 401
AACTCGATAA TTTCGTGTCA TTCAGGTCTC CTTCTGGTCA AAAGGAAGGA


 


 451
GTGCTTGTTC TTAAGAGCAA AGTGCCTAGA TTGGAAGAAC AGAGCTGGTG


 


 501
TCTCGACTTC AATGGCTCTC CTGAGAACGA ACCTGAGAAT GAAAACGACA


 


 551
TTTTCCAGTT TGCGAAAGTC GGAAACTTGC ACAAACTCTT CAGTTTATAT


 


 601
GACCCTGAAT GCATTCCTCT CGTTCGCACC TCAGTGTTTG CTGTCATTGC


 


 651
TCGAGTTTGT AGAGATAAAA AGCATACACC ATCGTATGAA TTGAAACTTG


 


 701
CATTGTACTT TGCAAAAAAC TCTGCAATCC TCAAGAAATT CGTTCTCCGC


 


 751
GGTTACACTC GAGAAGAAGA TTTACTCGCA TTGCCCGTGG CTAAC


 


AtTLP5:


polypeptide:


  1
MSFLSIVRDV RDTVGSFSRR SFDVRVSNGT THQRSKSHGV EAHIEDLIVI
(SEQ ID NO: 5)


 


 51
KNTRWANLPA ALLRDVMKKL DESESTWPAR KQVVACAGVC KTWRLMCKDI


 


 101
VKSPEFSGKL TFPVSLKQPG PRDGIIQCYI KRDKSNMTYH LYLSLSPAIL


 


 151
VESGKFLLSA KRSRRATYTE YVISMDADNI SRSSSTYIGK LKSNFLGTKF


 


 201
IVYDTAPAYN SSQILSPPNR SRSFNSKKVS PKVPSGSYNI AQVTYELNLL


 


 251
GTRGPRRMNC IMHSIPSLAL EPGGTVPSQP EFLQRSLDES FRSIGSSKIV


 


 301
NHSGDFTRPK EEEGKVRPLV LKTKPPRWLQ PLRCWCLNFK GRVTVASVKN


 


 351
FQLMSAATVQ PGSGSDGGAL ATRPSLSPQQ PEQSNHDKII LHFGKVGKDM


 


 401
FTMDYRYPLS AFQAFAISLS TFDTKLACE


 


cDNA:


  1
ATGTCGTTTC TGAGTATTGT TCGTGATGTT AGAGATACTG TAGGAAGCTT
(SEQ ID NO: 16)


 


 51
TTCGAGACGT AGTTTCGACG TGAGAGTATC TAATGGGACG ACTCATCAGA


 


 101
GGAGTAAATC TCACGGTGTT GAGGCACATA TTGAAGATCT TATTGTAATC


 


 151
AAGAACACTC GTTGGGCTAA TTTACCGGCT GCGCTATTAC GAGATGTGAT


 


 201
GAAAAAGTTG GATGAAAGCG AGAGTACTTG GCCTGCACGT AAACAAGTCG


 


 251
TTGCTTGTGC TGGTGTCTGC AAGACATGGA GACTAATGTG CAAAGATATT


 


 301
GTGAAAAGTC CTGAGTTCTC AGGCAAACTC ACATTTCCAG TTTCGTTGAA


 


 351
ACAGCCCGGG CCTAGGGATG GAATCATACA ATGTTATATC AAAAGAGACA


 


 401
AGTCTAACAT GACTTACCAC CTTTACCTTT CTCTTAGTCC TGCCATACTT


 


 451
GTTGAAAGTG GGAAGTTTCT TCTCTCGGCA AAGCGCTCAC GGAGAGCTAC


 


 501
ATACACAGAG TATGTAATAT CAATGGATGC AGACAACATT TCAAGATCAA


 


 551
GCAGCACTTA CATTGGCAAA CTGAAGTCTA ACTTTCTAGG GACAAAATTT


 


 601
ATAGTATATG ATACGGCTCC TGCGTACAAC AGTAGCCAGA TATTGTCCCC


 


 651
ACCAAACCGG AGTCGTAGTT TCAACTCCAA GAAAGTGTCT CCCAAAGTCC


 


 701
CTTCTGGAAG TTACAACATT GCTCAAGTTA CATACGAGCT GAACTTGCTT


 


 751
GGAACCCGTG GACCTCGTAG AATGAACTGC ATTATGCACT CTATCCCCTC


 


 801
CTTAGCTCTA GAACCCGGAG GTACTGTCCC TAGCCAACCT GAGTTTCTAC


 


 851
AACGTTCCCT TGATGAATCT TTCCGCAGCA TCCGTTCCTC AAAGATAGTC


 


 901
AACCACTCGG GAGATTTCAC CCGACCGAAA GAGGAAGAAG GAAAGGTGCG


 


 951
ACCTTTGGTA CTGAAAACTA AACCGCCAAG GTGGCTCCAA CCGTTGCGAT


 


1001
GTTGGTGCCT TAACTTCAAA GGGAGAGTGA CTGTAGCTTC TGTCAAGAAC


 


1051
TTCCAGTTGA TGTCCGCTGC AACGGTTCAG CCCGGTAGTG GTAGTGATGG


 


1101
TGGAGCATTG GCTACGAGGC CATCGTTATC ACCACAGCAG CCAGAGCAAT


 


1151
CAAACCATGA TAAGATAATA CTACACTTTG GGAAAGTGGG TAAGGATATG


 


1201
TTCACTATGG ACTATCGTTA TCCTCTCTCT GCCTTTCAAG CGTTTGCCAT


 


1251
TTCCCTGAGC ACCTTTGATA CTAAATTGGC ATGTGAA


 


AtTLP6:


polypeptide:


  1
MSLKNIVKNK YKAIGRRGRS HIAPEGSSVS SSLSTNEGLN QSIWVDLPPE
(SEQ ID NO:6)


 


 51
LLLDIIQRIE SEQSLWPGRR DVVACASVCK SWREMTKEVV KVPELSGLIT


 


 101
FPISLRQPGP RDAPIQCFIK RERATGIYRL YLGLSPALSG DKSKLLLSAK


 


 151
RVRRATGAEF VVSLSGNDFS RSSSNYIGKL RSNFLGTKFT VYENQPPPFN


 


 201
RKLPPSMQVS PWVSSSSSSY NIASILYELN VLRTRGPRRM QCIMHSIPIS


 


 251
AIQEGGKIQS PTEFTNQGKK KKKPLMDFCS GNLGGESVIK EPLILKNKSP


 


 301
RWHEQLQCWC LNFKGRVTVA SVKNFQLVAA AAEAGKNMNI PEEEQDRVIL


 


 351
QFGKIGKDIF TMDYRYPISA FQAFAICLSS FDTKPVCE


 


cDNA:


  1
ATGTCATTGA AGAACATAGT GAAGAACAAA TACAAAGCTA TTGGTAGAAG
(SEQ ID NO:17)


 


 51
AGGGAGGTCA CACATTGCAC CAGAAGGATC ATCTGTGTCT TCTTCTTTAT


 


 101
CAACTAATGA AGGTTTAAAC CAGAGTATTT GGGTTGATTT GCCTCCAGAG


 


 151
TTACTTCTTG ATATAATCCA AAGGATTGAG TCTGAACAGA GTTTATGGCC


 


 201
GGGGAGGAGA GATGTTGTTG CTTGTGCTTC GGTTTGTAAG TCATGGAGGG


 


 251
AGATGACTAA AGAAGTTGTT AAAGTTCCTG AGCTCTCTGG TTTGATCACG


 


 301
TTTCCGATTT CTTTAAGACA GCCTGGACCT AGAGATGCTC CAATTCAATG


 


 351
CTTTATTAAA CGTGAAAGAG CTACGGGGAT ATACCGTCTC TATCTTGGTT


 


 401
TAAGCCCTGC TCTTTCCGGT GACAAGAGTA AGTTGTTGTT ATCTGCAAAG


 


 451
AGAGTCAGGA GAGCGACGGG TGCGGAGTTT GTTGTATCGT TATCGGGGAA


 


 501
TGACTTCTCG AGAAGTAGTA GTAATTACAT AGGAAAACTG AGATCAAATT


 


 551
TCCTGGGAAC GAAGTTCACA GTCTACGAAA ACCAACCTCC TCCGTTTAAC


 


 601
CGAAAGCTCC CACCATCGAT GCAAGTGTCT CCATGGGTAT CGTCGTCATC


 


 651
TAGTAGTTAC AACATAGCTT CAATCTTGTA TGAGCTGAAT GTTCTGAGAA


 


 701
CCAGAGGTCC AAGAAGAATG CAATGTATAA TGCACAGTAT CCCGATTTCA


 


 751
GCGATTCAAG AAGGCGGCAA AATCCAGTCG CCAACGGAGT TCACAAACCA


 


 801
AGGAAAGAAG AAGAAGAAGC CGCTGATGGA TTTCTGCTCA GGGAACCTGG


 


 851
GAGGAGAATC CGTTATAAAA GAACCATTAA TTCTGAAAAA CAAGTCGCCG


 


 901
AGATGGCACG AACAGCTTCA GTGCTGGTGT CTAAACTTCA AAGGTCGAGT


 


 951
CACAGTCGCC TCGGTGAAAA ACTTCCAGCT AGTGGCAGCT GCTGCAGAAG


 


1001
CAGGGAAGAA CATGAACATA CCAGAAGAGG AACAAGATAG AGTGATATTA


 


1051
CAGTTTGGGA AGATAGGCAA AGACATTTTC ACAATGGATT ATCGTTACCC


 


1101
GATCTCTGCA TTCCAAGCTT TTGCTATTTG TTTAAGCAGC TTCGACACGA


 


1151
AGCCAGTCTG CGAA


 


AtTLP7:


polypeptide:


  1
MPLSRSLLSR RISNSFRFHQ GETTTAPESE SIPPPSNMAG SSSWSAMLPE
(SEQ ID NO:7)


 


 51
LLGEIIRRVE ETEDRWPQRR DVVTCACVSK KWREITHDFA RSSLNSGKIT


 


 101
FPSCLKLPGP RDFSNQCLIK RNKKTSTFYL YLALTPSFTD KGKFLLAARR


 


 151
FRTGAYTEYI ISLDADDFSQ GSNAYVGKLR SDFLGTNFTV YDSQPPHNGA


 


 201
KPSNGKASRR FASKQISPQV PAGNFEVGHV SYKFNLLKSR GPRRMVSTLR


 


 251
CPSPSPSSSS AGLSSDQKPC DVTKIMKKPN KDGSSLTILK NKAPRWHEHL


 


 301
QCWCLNFHGR VTVASVKNFQ LVATVDQSQP SGKGDEETVL LQFGKVGDDT


 


 351
FTMDYRQPLS AFQAFAICLT SFGTKLACE


 


cDNA:


  1
ATGCCTTTGT CACGGTCCCT CCTTTCGCGG AGGATCTCGA ACTCTTTTAG
(SEQ ID NO:18)


 


 51
GTTTCATCAG GGAGAGACAA CGACGGCACC GGAATCCGAA TCGATTCCTC


 


 101
CGCCGTCGAA TATGGCCGGT TCTTCGTCAT GGTCGGCGAT GCTCCCTGAA


 


 151
TTATTAGGCG AGATCATTCG TCGCGTGGAG GAGACTGAGG ACCGTTGGCC


 


 201
TCAACGTCGT GATGTAGTTA CTTGCGCTTG CGTTTCTAAG AAATGGAGAG


 


 251
AAATCACTCA CGATTTCGCT AGATCCTCTC TTAACTCTGG CAAAATTACT


 


 301
TTCCCTTCTT GCCTCAAATT GCCAGGTCCT AGAGACTTTT CTAATCAGTG


 


 351
CTTGATAAAG AGGAACAAGA AGACATCAAC GTTTTACTTG TATCTTGCTC


 


 401
TAACACCATC ATTCACTGAT AAGGGAAAGT TTCTTCTGGC GGCGCGOAGG


 


 451
TTTAGGACCG GTGCTTACAC TGAGTACATC ATATCACTTG ATGCTGATGA


 


 501
TTTCTCTCAA GGAAGTAATG CCTACGTCGG AAAATTAAGA TCAGATTTTC


 


 551
TTGGGACCAA CTTTACAGTA TACGATAGCC AACCACCACA CAACGGAGCA


 


 601
AAACCTTCAA ATGGCAAAGC CAGTCGCAGA TTTGCATCAA AGCAGATAAG


 


 651
CCCTCAAGTT CCAGCAGGCA ACTTTGAAGT CGGTCATGTT TCTTATAAAT


 


 701
TCAACCTTTT GAAATCAAGA GGTCCAAGAA GAATGGTAAG CACACTCCGA


 


 751
TGCCCATCAC CATCACCTTC ATCATCATCC GCTGGACTCT CGTCTGACCA


 


 801
AAAGCCATGT GATGTAACCA AGATAATGAA AAAACCCAAC AAGGATGGTT


 


 851
CCAGCTTGAC AATACTAAAG AACAAAGCTC CTAGATGGCA CGAGCACTTG


 


 901
CAGTGCTGGT GTCTGAACTT CCATGGACGA GTTACTGTTC CTTCGGTCAA


 


 951
GAACTTTCAG CTGGTTGCGA CCGTTGACCA AAGTCAACCG AGCGGTAAAG


 


1001
GCGATGAAGA AACAGTTCTT CTACAGTTTG GTAAAGTGGG AGATGACACT


 


1051
TTCACTATGG ATTATAGACA GCCTCTCTCT GCATTTCAGG CTTTTGCTAT


 


1101
CTGTCTCACA AGTTTCGGCA CTAAACTTGC CTGCGAG


 


AtTLP8:


polypeptide:


  1
MAGSRKVNDL LEENKGNVDT ITGSLSTQKG EDKENVSPEK VSTSVETRKL
(SEQ ID NO:8)


 


 51
DRALKSQSMK GNSGFPTEVT NFKSFSTGGR TALKQSSLQA CMQKNSEVDK


 


 101
SSFGMKTWTS VDSEHSSSLK VWEFSDSEAA PASSWSTLPN RALLCKTLPL


 


 151
DVGRCTCLIV KEQSPEGLSH GSVYSLYTHE GRGRKDRKLA VAYHSRRNGK


 


 201
SIFRVAQNVK GLLCSSDESY VGSMTANLLG SKYYIWDKGV RVGSVGKMVK


 


 251
PLLSVVIFTP TITTWTGSYR RMRTLLPKQQ PMQKNNNKQV QQASKLPLDW


 


 301
LENKEKIQKL CSRIPHYNKI SKQHELDFRD RGRTGLRIQS SVKNFQLTLT


 


 351
ETPRQTILQM GRVDKARYVI DFRYPFSGYQ AFCICLASID SKLCCTV


 


cDNA:


  1
ATGGCTGGTT CGAGAAAAGT GAATGATTTG TTGGAGGAAA ATAAGGGAAA
(SEQ ID NO:19)


 


 51 
TGTGGACACA ATTACAGGGT CTTTATCCAC TCAAAAGGGA GAGGATAAGG


 


 101
AGAATGTGTC GCCGGAGAAA GTCTCTACCT CTGTGGAAAC TCGGAAACTA


 


 151
GATCGAGCTT TGAAGTCTCA ATCGATGAAG GGTAACTCTG GGTTTCCAAC


 


 201
GGAAGTTACA AATTTCAAAT CTTTCTCAAC TGGTGGTCGA ACAGCTCTGA


 


 251
AGCAGTCATC ACTGCAAGCG TGTATGCAGA AGAACAGTGA GGTTGATAAG


 


 301
AGTAGTTTCG GAATGAAAAC TTGGACTAGT GTTGATTCAG AGCATTCAAG


 


 351
TTCGTTGAAA GTGTGGGAGT TTTCGGATTC TGAAGCTGCC CCTGCTTCCT


 


 401
CTTGGTCTAC TTTGCCCAAC AGGGCTTTGT TGTGCAAGAC ACTACCTTTG


 


 451
GATGTGGGAA GATGCACTTG TCTGATTGTG AAAGAACAAT CACCTGAAGG


 


 501
CTTGAGCCAC GGATCTGTAT ATTCACTTTA TACACATGAA GGTCGGGGGC


 


 551
GTAAAGACCG GAAGTTAGCA GTTGCTTACC ATAGCCGACG TAATGGGAAA


 


 601
TCTATATTTA GGGTGGCACA GAATGTTAAG GGATTGCTGT GCAGTTCGGA


 


 651
TGAAAGTTAT GTCGGTTCCA TGACGGCTAA TCTCTTGGGT TCCAAGTACT


 


 701
ACATATGGGA CAAGGGAGTT CGAGTTGGTT CTGTAGGTAA AATGGTGAAG


 


 751
CCGCTTCTTT CGGTTGTAAT ATTCACACCC ACCATAACAA CTTGGACAGG


 


 801
GAGCTACAGA AGAATGAGAA CTTTGCTACC AAAGCAGCAG CCAATGCAGA


 


 851
AAAACAACAA TAAGCAGGTT CAACAAGCTA GTAAACTACC GCTTGATTGG


 


 901
CTTGAGAATA AGGAAAAAAT TCAGAAGCTA TGCTCAAGGA TACCACATTA


 


 951
CAACAAAATC TCCAAGCAGC ATGAGTTAGA CTTCAGAGAC AGAGGAAGAA


 


1001
CAGGACTGAG AATACAGAGC TCGGTGAAGA ACTTTCAGCT AACACTCACG


 


1051
GAGACTCCAA GGCAGACAAT TCTTCAAATG GGGAGAGTTG ACAAAGCAAG


 


1101
ATATGTAATC GACTTCAGGT ATCCATTCTC AGGCTACCAA GCATTCTGCA


 


1151
TTTGCTTGGC TTCTATTGAT TCCAAGCTTT GTTGTACTGT T


 


AtTLP9:


polypeptide:


  1
MTFRSLLQEM RSRPHRVVHA AASTANSSDP FSWSELPEEL LREILIRVET
(SEQ ID NO:9)


 


 51
VDGGDWPSRR NVVACAGVCR SWRILTKEIV AVPEFSSKLT FPISLKQSGP


 


 101
RDSLVQCFIK RNRNTQSYHL YLGLTTSLTD NGKFLLAASK LKRATCTDYI


 


 151
ISLRSDDISK RSNAYLGRMR SNFLGTKFTV FDGSQTGAAK MQKSRSSNFI


 


 201
KVSPRVPQGS YPIAHISYEL NVLGSRGPRR MRCIMDTIPM SIVESRGVVA


 


 251
STSISSFSSR SSPVFRSHSK PLRSNSASCS DSGNNLGDPP LVLSNKAPRW


 


 301
HEQLRCWCLN FHGRVTVASV KNFQLVAVSD CEAGQTESRI ILQFGKVGKD


 


 351
MFTMDYGYPI SAFQAFAICL SSFETRIACE


 


cDNA:


  1
ATGACGTTCC GAAGTTTACT CCAGGAAATG CGGTCTAGGC CACACCGTGT
(SEQ ID NO:20)


 


 51
AGTTCACGCC GCCGCCTCAA CCGCTAATAG TTCAGACCCT TTCAGCTGGT


 


 101
CGGAGCTCCC GGAGGAGCTG CTTAGAGAAA TCCTGATTAG GGTTGAGACT


 


 151
GTTGACGGCG GCGATTGGCC GTCGCGGCGA AACGTGGTGG CTTGTGCCGG


 


 201
CGTTTGTCGT AGCTGGAGGA TTCTCACCAA GGAGATTGTA GCTGTTCCTG


 


 251
AATTCTCCTC TAAATTGACT TTCCCTATCT CCCTCAAGCA GTCTGGTCCA


 


 301
AGAGATTCTC TAGTTCAATG CTTTATAAAA CGTAATCGAA ATACTCAATC


 


 351
GTATCATCTC TATCTCGGAT TAACTACCTC TTTGACGGAT AACGGGAAGT


 


 401
TTCTTCTTGC TGCTTCTAAG CTGAAGCGCG CAACTTGCAC TGATTACATC


 


 451
ATCTCTTTGC GTTCAGACGA TATCTCAAAG AGAAGCAACG CGTATCTTGG


 


 501
GAGAATGAGA TCGAACTTCC TTGGAACAAA ATTCACGGTC TTTGATGGTA


 


 551
GTCAGACCGG AGCAGCGAAG ATGCAGAAGA GCCGCTCTTC TAATTTCATC


 


 601
AAAGTTTCAC CTAGAGTTCC TCAGGGAAGT TACCCCATCG CTCACATTTC


 


 651
ATACGAGTTA AACGTCTTAG GCTCTCGGGG ACCGAGAAGA ATGCGTTGCA


 


 701
TCATGGATAC AATACCTATG AGCATCGTGG AGTCGCGAGG AGTAGTAGCT


 


 751
TCAACATCCA TAAGCTCTTT TTCCAGTCGG TCATCACCAG TCTTTAGGTC


 


 801
TCACTCAAAA CCATTGCGCA GTAATAGTGC ATCATGTAGC GACTCAGGCA


 


 851
ACAACCTGGG AGATCCACCA TTGGTGCTGA GCAACAAAGC TCCACGGTGG


 


 901
CATGAGCAGT TACGTTGCTG GTGCTTAAAT TTCCATGGTC GAGTCACAGT


 


 951
GGCTTCGGTT AAGAACTTTC AGCTTGTGGC AGTTAGTGAC TGTGAAGCAG


 


1001
GGCAGACATC TGAGAGGATC ATACTCCAGT TTGGGAAAGT TGGGAAGGAC


 


1051
ATGTTTACCA TGGATTATGG ATATCCGATT TCTGCGTTTC AAGCGTTTGC


 


1101
TATCTGCCTG AGCAGTTTTG AAACCAGAAT TGCCTGTGAA


 


AtTLP10:


polypeptide:


  1
MSFRGIVQDL RDGFGSLSRR SFDFRLSSLH KGKAQGSSFR EYSSSRDLLS
(SEQ ID NO:10)


 


 51
PVIVQTSRWA NLPPELLFDV IKRLEESESN WPARKHVVAC ASVCRSWRAM


 


 101
CQEIVLGPEI CGKLTFPVSL KQPGPRDAMI QCFIKRDKSK LTFHLFLCLS


 


 151
PALLVENGKF LLSAKRTRRT TRTEYIISMD ADNISRSSNS YLGKLRSNFL


 


 201
GTKFLVYDTQ PPPNTSSSAL ITDRTSRSRF HSRRVSPKVP SGSYNIAQIT


 


 251
YELNVLGTRG PRRMHCIMNS IPISSLEPGG SVPNQPEKLV PAPYSLDDSF


 


 301
RSNISFSKSS FDHRSLDFSS SRFSEMGISC DDNEEEASFR PLILKNKQPR


 


 351
WHEQLQCWCL NFRGRVTVAS VKNFQLVAAR QPQPQGTGAA AAPTSAPAHP


 


 401
EQDKVILQFG KVGKDMFTMD YRYPLSAFQA FAICLSSFDT KLACE


 


cDNA:


  1
ATGTCGTTTC GAGGCATTGT TCAAGATTTG AGAGATGGGT TTGGGAGCTT
(SEQ ID NO:21)


 


 51
GTCAAGGAGG AGTTTCGATT TTAGGCTCTC GAGTCTTCAT AAAGGGAAAG


 


 101
CTCAGGGTTC TTCGTTCCGT GAGTATTCGT CATCCCGTGA TCTCTTGTCG


 


 151
CCTGTGATAG TTCAGACAAG TAGATGGGCT AATCTTCCTC CAGAGTTACT


 


 201
CTTTGATGTG ATCAAAAGAT TAGAGGAAAG TGAGAGTAAT TGGCCTGCAA


 


 251
GAAAACATGT TGTGGCTTGT GCTTCGGTTT GTCGGTCTTG GAGAGCTATG


 


 301
TGCCAAGAGA TTGTTTTGGG GCCTGAAATC TGTGGGAAAC TCACTTTCCC


 


 351
TGTTTCCCTC AAACAGCCAG GGCCTCGTGA TGCAATGATT CAGTGTTTCA


 


 401
TCAAAAGGGA TAAATCAAAG CTAACATTTC ACCTTTTTCT TTGTTTAAGT


 


 451
CCCGCTCTAT TAGTGGAGAA TGGGAAATTT CTTCTTTCAG CTAAAAGAAC


 


 501
TCGTAGAACT ACTCGAACCG AGTACATTAT CTCCATGGAT GCTGATAACA


 


 551
TCTCAAGATC CAGCAACTCT TACCTCGGAA AGCTCAGATC AAACTTCCTT


 


 601
GGGACAAAGT TCTTGGTGTA CGACACGCAA CCACCACCAA ACACATCTTC


 


 651
GAGCGCACTT ATCACTGATC GAACAAGCCG AAGCAGGTTT CACTCCAGAC


 


 701
GAGTTTCTCC TAAAGTACCA TCCGGAAGCT ACAACATTGC TCAAATCACC


 


 751
TATGAGCTCA ACGTGTTGGG CACACGCGGG CCACGACGAA TGCACTGCAT


 


 801
CATGAACTCC ATCCCAATTT CATCGCTCGA ACCAGGCGGT TCAGTCCCTA


 


 851
ACCAACCCGA GAAACTCGTC CCTGCACCAT ACTCTCTCGA CGACTCATTC


 


 901
CGCAGTAACA TCTCCTTCTC CAAATCATCA TTTGACCACC GCTCCCTCGA


 


 951
TTTCAGCAGT TCTAGATTCT CCGAAATGGG AATATCCTGC GACGACAACG


 


1001
AAGAAGAAGC GAGTTTCAGA CCGTTGATTC TAAAGAACAA GCAGCCAAGG


 


1051
TGGCACGAGC AGTTGCAATG CTGGTGTTTG AATTTCCGCG GACGTGTGAC


 


1101
AGTTGCATCG GTTAAGAATT TCCAGCTTGT AGCAGCAAGA CAGCCGCAGC


 


1151
CTCAAGGGAC AGGTGCAGCA GCAGCACCAA CAAGTGCACC TGCTCACCCT


 


1201
GAGCAAGACA AGGTGATTCT CCAGTTTGGT AAAGTAGGGA AAGATATGTT


 


1251
CACAATGGAC TATAGGTATC CATTATCGGC GTTTCAGGCG TTTGCGATAT


 


1301
GCTTAAGCAG CTTTGACACC AAGCTTGCTT GTGAA


 


AtTLP11:


polypeptide:


  1
MRSRPHRVVH DLAAAAAADS TSVSSQDYRW SEIPEELLRE ILIRVEAADG
(SEQ ID NO:11)


 


 51
GGWPSRRSVV ACAGVCRGWR LLMNETVVVP EISSKLTFPI SLKQPGPRDS


 


 101
LVQCFIKRNR ITQSYHLYLG LTNSLTDDGK FLLAACKLKH TTCTDYIISL


 


 151
RSDDMSRRSQ AYVGKVRSNF LGTKFTVFDG NLLPSTGAAK LRKSRSYNPA


 


 201
KVSAKVPLGS YPVAHITYEL NVLGSRGPRK MQCLMDTIPT STMEPQGVAS


 


 251
EPSEFPLLGT RSTLSRSQSK PLRSSSSHLK ETPLVLSNKT PRWHEQLRCW


 


 301
CNLFHGRVTV ASVKNFQLVA AGASCGSGTG MSPERQSERI ILQFGKVGKD


 


 351
MFTMDYGYPI SAFQAFAICL SSFETRIACE


 


cDNA:


  1
ATGCGTTCGA GACCGCATCG TGTGGTCCAC GACCTTGCCG CCGCCGCAGC
(SEQ ID NO:22)


 


 51
TGCCGATTCC ACTTCTGTGT CATCGCAAGA TTATCGCTGG TCAGAGATTC


 


 101
CTGAAGAGCT TCTTAGGGAG ATTCTGATTC GTGTTGAAGC GGCGGACGGT


 


 151
GGCGGATGGC CGTCACGACG CAGCGTGGTG GCTTGTGCCG GCGTTTGTCG


 


 201
TGGCTGGCGG CTACTTATGA ACGAAACCGT CGTTGTCCCT GAGATCTCTT


 


 251
CTAAGTTGAC TTTCCCCATC TCTCTCAAGC AGCCTGGTCC AAGGGATTCA


 


 301
CTGGTTCAAT GCTTTATCAA ACGTAATCGA ATTACGCAAT CATATCATCT


 


 351
CTATCTCGGA TTAACCAACT CTTTAACGGA TGATGGGAAG TTTTTGCTTG


 


 401
CTGCGTGTAA GTTGAAGCAC ACAACTTGTA CGGATTACAT TATCTCTTTA


 


 451
CGTTCTGATG ATATGTCGAG AAGAAGCCAA GCTTATGTTG GCAAAGTGAG


 


 501
ATCGAACTTC CTAGGAACGA AATTCACTGT CTTTGATGGA AATCTGCTGC


 


 551
CTTCAACGGG AGCCGCAAAG TTGAGAAAGA GCCGATCTTA TAATCCCGCA


 


 601
AAAGTTTCAG CAAAAGTTCC TCTTGGAAGT TATCCTGTCG CTCATATCAC


 


 651
ATATGAGCTG AATGTCTTAG GATCCCGGGG ACCAAGAAAG ATGCAATGTC


 


 701
TTATGGACAC AATACCTACA AGCACAATGG AGCCTCAAGG AGTAGCTTCA


 


 751
GAACCATCAG AGTTTCCCTT ACTCGGTACT CGGTCAACCT TATCCAGGTC


 


 801
TCAGTCAAAA CCATTACGCA GTAGCTCAAG CCACCTGAAA GAAACACCAT


 


 851
TAGTGCTGAG CAACAAGACA CCACGGTGGC ACGAGCAGCT ACGCTGCTGG


 


 901
TGCTTGAATT TCCATGGCCG TGTCACAGTA GCGTCAGTGA AGAACTTTCA


 


 951
GCTCGTGGCA GCAGGAGCTA GCTGTGGCAG TGGCACGGGA ATGTCACCGG


 


1001
AGAGGCAGAG CGAGCGGATT ATATTGCAGT TTGGGAAAGT CGGGAAAGAT


 


1051
ATGTTCACGA TGGATTATGG ATACCCGATC TCAGCTTTCC AGGCTTTTGC


 


1101
CATTTGCTTG AGCAGCTTTG AGACTAGAAT CGCTTGTGAA






Accordingly, one aspect of the invention features an isolated polypeptide containing an amino acid sequence at least 70% (i.e., any number between 70% and 100%, inclusive) identical to one of SEQ ID NOs: 1-11. When expressed in a plant cell, e.g., an Arabidopsis cell, the polypeptide regulates the transcription of genes, in response to environmental stimuli. The polypeptide of the invention can be used to identify DNA elements, such as promoters, enhances, or silencers, which it binds to. Such DNA elements mediate the response of plants to various environmental factors. The polypeptide of the invention can also be used for producing anti-AtTLP antibodies (either monoclonal or polyclonal). These antibodies in turn are useful for detecting the presence and distribution of AtTLP proteins in tissues and in cellular compartments. For example, such antibodies can be used to verify the expression of TLP proteins in a transgenic plant.
An isolated polypeptide refers to a polypeptide substantially free from naturally associated molecules, i.e., it is at least 75% (i.e., any number between 75% and 100%, inclusive) pure by dry weight. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
The percent identity of two amino acid sequences is determined using the algorithm of Karlin and Altschul ((1990) Proc. Natl. Acad. Sci. USA 87, 2264-2268), modified as in Karlin and Altschul ((1993) Proc. Natl. Acad. Sci. USA 90, 5873-5877). Such an algorithm is incorporated into the XBLAST programs of Altschul, et al. ((1990) J. Mol. Biol. 215, 403-410). BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3. Where gaps exist between two sequences, Gapped BLAST is utilized as described in Altschul, et al. ((1997) Nucleic Acids Res. 25, 3389-3402). When employing BLAST and Gapped BLAST programs, one can conveniently use the default parameters (e.g., XBLAST). See ncbi.nlm.nih.gov.
The invention further features (1) an isolated nucleic acid encoding a polypeptide of the invention and (2) an isolated nucleic acid that, under a high stringent condition, hybridizes to a probe containing a sequence selected from the group consisting of SEQ ID NOs: 12-22, or a complement of any selected sequence. Such a nucleic acid is at least 15 (e.g., at least 30, 50, 100, 200, 500, or 1000) nucleotides in length. By hybridization under a high stringent condition is meant hybridization at 65° C., 0.5×SSC, followed by washing at 45° C., 0.1×SSC. The nucleic acids of the invention can be used to determine whether an AtTLP mRNA is expressed in a tissue or cell. The nucleic acids can be used as primers in PCR-based detection methods, or as labeled probes in nucleic acid blots (e.g., Northern blots).
An isolated nucleic acid refers to a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein.
The invention also features a vector and a host cell containing a nucleic acid of the invention. The host cell can be an E. coli., a yeast, an insect, a plant (e.g., Arabidopsis), or a mammalian cell. The vector and host cell can be used for producing a polypeptide of the invention. For this purpose, one can culture the host cell in a medium under conditions permitting expression of the polypeptide, and isolate the polypeptide.
The just-described vector and host cell can also be used for generating a transformed plant cell or a transgenic plant containing a recombinant nucleic acid that encodes a heterologous polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. One can generate such a transformed plant cell by introducing into a plant cell a recombinant nucleic acid encoding such a heterologous polypeptide and expressing the polypeptide in the cell. To generate a transgenic plant, one can (1) introduce into a plant cell a recombinant nucleic acid encoding one just-described heterologous polypeptide; (2) expressing the polypeptide in the cell, and (3) cultivating the cell to generate a plant. The transformed plant cell or transgenic plant is more sensitive to environmental factors, such as high salinity, pathogens, and chilling, and therefore can be used as a sensor to detect and monitor small changes in environment, such as soil and air.
Also within the scope of this invention are a homozygous transformed plant cell (e.g., an Arabidopsis cell) and a transgenic plant (e.g. Arabidopsis) that lack a polypeptide containing a sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. The transformed plant cell or transgenic plant, compared with the wild type cell or plant, has a higher (by at least 30%, e.g., 50%, 90%, 100%, 200%) tolerance to salt, chilling, pathogens, oxidative stress, or water-deficit due to absence of or lowered level of the polypeptide. In addition, the invention features method of making the transformed plant cell and the transgenic plant. Both methods include introducing into a plant cell a nucleic acid (e.g., a T-DNA, an anti-sense RNA, and an iRNA) that decreases the expression of a gene encoding a polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. The method for making the plant further includes cultivating the plant cell to generate a plant.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.


DETAILED DESCRIPTION
This invention is based on an unexpected discovery that (1) overexpression of AtTLPs in Arabidopsis increases sensitivity of the plant to various environmental factors, such as salt, chilling, oxidative stress, or water-deficit; and (2) lack of expression of AtTLPs increases tolerance of the plant to several environmental factors.
Accordingly, in one aspect, the invention features a transformed plant cell containing a recombinant nucleic acid that encodes a heterologous AtTLP. The AtTLP proteins useful for this invention include, Arabidopsis AtTLPs 1-11 and TLPs of other species. The plant cell can be a dicot plant cell (e.g., a tomato cell, a brassica cell, or a potato cell) or a monocot plant cell (e.g. a rice cell, a wheat cell, or a barley cell).
A transformed plant cell of the invention can be produced by introducing into a plant cell a recombinant nucleic acid that encodes a heterologous AtTLP protein and expressing the protein in the cell. Techniques for transforming a wide variety of plant cells are well known in the art and can be found in technical and scientific literature. See, for example, Weising et al., 1988, Ann. Rev. Genet. 22:421-477. To express a heterologous AtTLP gene in a plant cell, the gene can be combined with transcriptional and translational initiation regulatory sequences that direct the transcription of the gene and translation of the encoded protein in the plant cell.
For overexpression, a constitutive plant promoter may be employed. A constitutive promoter is active under most environmental conditions and states of cell differentiation. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumafaciens, the ACT11 and Cat3 promoters from Arabidopsis (Huang et al., 1996, Plant Mol. Biol. 33:125-139 and Zhong et al., 1996, Mol. Gen. Genet. 251:196-203), the stearoyl-acyl carrier protein desaturase gene promoter from Brassica napus (Solocombe et al., 1994, Plant Physiol. 104:1167-1176), and the GPc1 and Gpc2 promoters from maize (Martinez et al., 1989, J. Mol. Biol. 208:551-565 and Manjunath et al., 1997, Plant Mol. Biol. 33:97-112).
Alternatively, a tissue-specific promoter or an inducible promoter may be employed to direct expression of the AtTLP gene in a specific cell type or under more precise environmental or developmental control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobicity, elevation of temperature, presence of light, spray with chemicals or hormones, or infection by a pathogen. Examples of a tissue-specific promoter or an inducible promoter include the root-specific ANR1 promoter (Zhang and Forde, 1998, Science 279:407) and the photosynthetic organ-specific RBCS promoter (Khoudi et al., 1997, Gene 197:343).
For proper polypeptide expression, a polyadenylation region at the 3′-end of the coding region should be included. The polyadenylation region can be derived from the same gene, from a variety of other genes, or from T-DNA.
A marker gene can also be included to confer a selectable phenotype on plant cells. For example, the marker gene may encode a protein that confers biocide resistance, antibiotic resistance (e.g., resistance to kanamycin, G418, bleomycin, hygromycin), or herbicide resistance (e.g., resistance to chlorosulfuron or Basta).
A recombinant nucleic acid that encodes a heterologous AtTLP protein may be introduced into the genome of a desired plant host cell by a variety of conventional techniques. For example, the recombinant nucleic acid may be introduced directly into the genomic DNA of a plant cell using techniques such as polyethylene glycol precipitation, electroporation, microinjection, or ballistic methods (e.g., DNA particle bombardment). See, e.g., Paszkowski et al., 1984, EMBO J. 3:2717-2722, Fromm et al., 1985, Proc. Natl. Acad. Sci. USA 82:5824, and Klein et al., 1987, Nature 327:70-73. Alternatively, the recombinant nucleic acid may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host direct the insertion of the AtTLP gene and adjacent marker into the plant cell DNA when the cell is infected by the bacteria. Agrobacterium tumefaciens-mediated transformation techniques, including disarming and use of binary vectors, are well known in the art. See, e.g., Horsch et al., 1984, Science 233:496-498; Fraley et al., 1983, Proc. Natl. Acad. Sci. USA 80:4803; and Gene Transfer to Plants, Potrykus, ed., Springer-Verlag, Berlin, 1995.
The presence and copy number of a heterologous AtTLP gene in a transgenic plant can be determined using standard methods, e.g., Southern blotting. Expression of the heterologous AtTLP gene in a transgenic plant can be confirmed by detecting and quantifying the heterologous AtTLP mRNA or protein in the transgenic plant.
The transformed plant cells thus obtained can then be cultured to regenerate a whole plant. Regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide or herbicide marker that has been introduced together with a heat shock factor gene. Plant regeneration from cultured protoplasts is described in Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, New York, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al., 1987, Ann. Rev. Plant Phys. 38:467-486. Once the heterologous AtTLP gene has been confirmed to be stably incorporated in the genome of a transgenic plant, it can be introduced into other plants by sexual crossing. Depending upon the species to be crossed, one or more standard breeding techniques can be used to generate the whole plant.
In another aspect, the invention feature a homozygous transformed plant cell that lack one or more of AtTLPs 1-11. Absence of the AtTLP(s) enhances tolerance of the cell to various environmental factors, e.g., high salinity. Such a transformed cell can be made by introducing into a plant cell a nucleic acid that lowers the expression of a gene encoding a polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. The nucleic acid, e.g., T-DNA, antisense RNA, or iRNA, can be introduced into the cell using one of the standard transforming techniques described above. Stable transformants can be selected using the marker genes and selection methods also described above. A whole plant can then be regenerated from the transformed plant cells. It can be further crossed using conventional breeding techniques to generate homozygous plant.
The specific example below is to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety.
Identification of the AtTLP Family
A tubby consensus sequence (Pfam PF01167, Kleyn et al., 1996, Cell 85: 281-290 and Noben-Trauth et al., 1996, Nature 380: 534-538) was used to search the Arabidopsis thaliana expressed sequence tag (EST) database and the completed Arabidopsis genome sequence (The Institute of Genome Research, TIGR) with multiple BLAST algorithms to locate all the sequences sharing significant similarities with the tubby domain (P-value <0.0085). The search results revealed eleven TUBBY-like protein genes, termed AtTLP1 to AtTLP11, in the Arabidopsis genome. For each of the 11 genes, the corresponding BAC locus (The Arabidopsis Information Resource), Tentative Consensus (TC) group, AGI gene code, cDNA GenBank accession number, and predicted protein length (No. of amino acid) are summarized in Table 1 below.







TABLE 1







AtTLP family members
















cDNA
Predicted





AGI Gene
GenBank
Protein


Gene Name
BAG Locus
TC group
Code
Accession No.
Length





AtTLP1
F22K20.1
TC95487
At1g76900
AF487267
455


AtTLP2
T30D6.21
TC86308
At2g18280
AY045773
394


AtTLP3
F17A22.29
TC86633
At2g47900
AY045774
406


AtTLP4
F8K4.13
—
At1g61940
—
265


AtTLP5
T10P12.9
TC102456
At1g43640
AY046921
429


AtTLP6
F8G22.1
TC90700
At1g47270
AF487268
388


AtTLP7
F12M16.22
TC88599
At1g53320
AY092403
379


AtTLP8
T24D18.17
—
At1g16070
AF487269
397


AtTLP9
F24P17.15
TC102624
At3g06380
AF487270
380


AtTLP10
F4F7.13.
TC101291
At1g25280
AF487271
445


AtTLP11
T1A4.60
—
At5g18680
AY046922
380









Gene-specific 5′ and 3′ primers were designed based on the sequence of the predicted open reading frame (ORF) and the corresponding EST in the database. The primer pairs used are listed below:








AtTLP1-5′ (5′-ATGTCGTTCCGTAGCATAGTTCGT-3′)



(SEQ ID NO:23)


 


AtTLP1-3′ (5′-TTATTCGCAAGCAAGTTTTGTGTCG-3′)


(SEQ ID NO:24)


 


AtTLP2-5′ (5′-ATGTCTTTGAAAAGCATCCTTCGTGATC-3′)


(SEQ ID NO:25)


 


AtTLP2-3′ (5′-TTACCCTTCACATGCCGGTTTGGTGTCA-3′)


(SEQ ID NO:26)


 


AtTLP3-5′ (5′-ATGTCCTTCAAGAGTCTCATTCAG-3′)


(SEQ ID NO:27)


 


AtTLP3-3′ (5′-TCATTCACATGCTATCTTGGTGTC-3′)


(SEQ ID NO:28)


 


AtTLP5-5′ (5′-ATGTCGTTTCTGAGTATTGTTCG-3′)


(SEQ ID NO:29)


 


AtTLP5-3′ (5′-TTATTCACATGCCAATTTAGTAT-3′)


(SEQ ID NO:30)


 


AtTLP6-5′ (5′-ATGTCATTGAAGAACATAGTGAA-3′)


(SEQ ID NO:31)


 


AtTLP6-3′ (5′-TCATTCGCAGACTGGCTTCGTGT-3′)


(SEQ ID NO:32)


 


AtTLP7-5′ (5′-ATGCCTTTGTCACGGTCCCTC-3′)


(SEQ ID NO:33)


 


AtTLP7-3′ (5′-TCACTCGCAGGCAAGTTTAGTG-3′)


(SEQ ID NO:34)


 


AtTLP8-5′ (5′-ATGGCTGGTTCGAGAAAAGTGAA-3′)


(SEQ ID NO:35)


 


AtTLP8-3′ (5′-TCAAACAGTACAACAAAGCTTGG-3′)


(SEQ ID NO:36)


 


AtTLP9-5′ (5′-ATGACGTTCCGAAGTTTACTCCA-3′)


(SEQ ID NO:37)


 


AtTLP9-3′ (5′-TTATTCACAGGCAATTCTGGTTT-3′)


(SEQ ID NO:38)


 


AtTLP10-5′ (5′-ATGTCGTTTCGAGGCATTGTTCA-3′)


(SEQ ID NO:39)


 


AtTLP10-3′ (5′-CTATTCACAAGCAAGCTTGGTGT-3′)


(SEQ ID NO:40)


 


AtTLP11-5′ (5′-ATGTCGTTTCTGAGTATTGTTCG-3′)


(SEQ ID NO:41)


 


AtTLP11-3′ (5′-TTATTCACATGCCAATTTAGTAT-3′)


(SEQ ID NO:42)






RT-PCR was then performed using total RNA from-2-week-old Arabidopsis seedlings. The total RNA was isolated using the TRIZOL reagent (Invitrogen) according the manufacture's direction. PolyA+-mRNA was isolated using oligo (dT)-coated magnetic beads and the PolyATract system (Promega, Madison, Wis.). First strand cDNA was synthesized from 0.5 μg PolyA+-mRNA using SuperScript II RNase H Reverse Transcriptase (Invitrogen) according to the protocol of the supplier.
The above-described gene-specific primer pairs were used for amplifying cDNA of each AtTLP gene from first-strand cDNA. PCR conditions were as follows: 3 min at 94° C.; 25 cycles of 1 min denaturation at 94° C./1 min annealing at 55° C./1 min 30 s extension at 72° C. The PCR products were purified using the QIAquick PCR purification kit (Qiagen) and subcloned into a T-easy vector (Promega). Each of these clones was verified by sequencing. Ten AtTLP cDNAs, AtTLPs 1-3 and AtTLPs 5-11, were successfully amplified.
It was found that, except for AtTLPs 2 and 11, the amino acid sequences deduced from the cDNA sequences of AtTLPs 1, 3, and 5-10 are identical to the predicted ORFs in the database. The analysis of the AtTLP2 cDNA sequence indicated that its intron3 was located between 708-781 bp whereas the predicted splicing sites for this intron located were 663 and 766 bp. The analysis of AtTLP11 cDNA sequence showed that intron2 and intron4 were located at 669-803 bp and 1334-1575 bp, respectively, whereas the computer predicted intron2 was at 621-803 bp and there was no predicted intron4. All cDNA sequences obtained from this study were submitted to GenBank.
Sequence Analysis of AtTLP Proteins
The search for all known motifs in the deduced AtTLP amino acid sequences was conducted by MOTIF SCANNING (Pagni et al., 2001, Nucleic Acids Res 29: 148-151). Multiple sequence alignment was performed using ClustalW (Thomopson et al., 1994). This analysis reveled that each AtTLP gene, except AtTLPs 4 and 8, had a well-conserved tubby domain at its C-terminus. Unlike animal TLPs, which have highly diverse N-terminal sequences, each AtTLP, except AtTLP8, had a conserved F-box (51-57 residues) containing domain (Pfam PF00646).
Pair-wise comparisons among the AtTLP proteins revealed that their tubby domains shared 30% to 80% similarities. Further analyzing the tubby domain revealed two PROSITE signature patterns: TUB1 (Prosite Accession No. PS01200) and TUB2 motif (Prosite Accession No. PS01201). The TUB1 and TUB2 motifs were located at the C-terminal of each AtTLP protein and contain 14 and 16 amino acid residues, respectively. These two TUB motifs are highly conserved among TLPs from various organisms. Though AtTLP4 and 8 do not have obvious TUB1 and TUB2 motifs, their C-terminal tubby domains are recognizable by MOTIF SCANNING (N-score >15) (Pagni et al., 1993, Nucleic Acids Res 29: 148-151).
An obvious feature of AtTLPs is the tubby domain. In the tubby domain of a mouse TUBBY protein, three positively-charged amino acid residues, R332, R363 and K330, were thought to be crucial for PI (4,5) P2 binding (Santagata et al., 2001, Science 292: 2041-2050). A sequence alignment of AtTLP tubby domains with the mouse TUBBY domain revealed a putative PI (4,5) P2 binding domain in each AtTLP, except AtTLPs 4 and 8. This suggests that AtTLPs 1-3, 4-7, and 8-11 may bind to PI (4,5) P2. It is known that the mouse TUBBY protein is a bipartite transcription regulator. Its tubby domain possesses double-stranded DNA binding activity, and its N-terminal segment seems to modulate transcription (Boggon et al., 1999, Science 286: 2119-2125). In plants, the N-terminal region of TLPs is quite different from that in mammal TLPs as AtTLP9-GAL4 DNA binding domain fusion protein failed to activate transcription from a GAL4 promoter in a heterologous system.
Location and Gene Structure Comparison of the AtTLP Gene Family
Chromosome localizations of each AtTLP genes were determined using Map View available at the website of arabidopsis.org/servlets/mapper (Huala et al., 2001, Nucleic Acids Res 29: 102-105). It was found that the genes were not evenly distributed on chromosomes I, II, III, and V. Seven genes (AtTLPs 1, 4, 5, 6, 7, 8, and 10) were located on chromosome I, and two genes (AtTLPs 2 and 3) were located on chromosomes II. The other two, AtTLPs 9 and 11, were located on chromosomes II, III respectively. Although most of the AtTLP genes were located on chromosome I, no local tandem repeats or gene clusters were identified.
By comparing the sequences of the RT-PCR products and the Arabidopsis genome, the corrected exon-intron organizations of the AtTLP genes (except for AtTLP4) were determined. It was found that exon 1 contained the sequences encoding each protein's N-terminal leading sequence, the F-box, and a nine-residue spacer between the F-box and tubby domain. This result indicated that the genes might have arisen from the same ancestral gene. The sequence encoding the C-terminal tubby domain was found to distribute in exons interrupted by 2 or 3 introns. On the basis of the exon and intron composition, the AtTLP genes were classified into three groups. Each gene of the first group (AtTLPs 1, 2, 5, 6, 7, and 10) contains three introns. Each of the second group, AtTLPs 3, 9 and 11, contains an additional intron in the region encoding the C-terminal part of the tubby domain. The third and the most distinct group (AtTLPs 4 and 8) contain 5 and 8 introns, respectively.
Expression of AtTLP Genes
A coupled RT-PCR based assay was conducted to determine the expression pattern of AtTLP genes. Total RNA was isolated from roots, main and lateral stems, rosette leaves, flower clusters, and green siliques of 42-days-old soil-grown Arabidopsis. For each gene, a pair of gene-specific primers was chosen, and PCR amplifications were carried out using 15 ng of first strand cDNA synthesized as described above. Primers of ubiquitin gene, UBQ10, (5′-ATTTCTCAAAATCTTAAAAACTT-3′ (SEO ID NO:43) and 5′-TGATAGTTTTCC CAGTCAAC-3′ (SEQ ID NO:44)) were used to amplify ubiquitin, which served as an internal loading standard (Norris et al., 1993, Plant Mol. Biol. 21: 895-906).
The results showed that AtTLPs 1, 2, 3, 6, 7, 9, 10 and 11 were expressed in all organs tested, with slight variations in mRNA accumulation. In contrast, AtTLPs 5 and 8 were primarily expressed in the root, flower, and silique. The organ-specific expressions of AtTLPs 5 and 8 indicate their specific roles in particular organs.
Although the expression of AtTLP1, 2, 3, 6, 7, 9, 10 and 11 is present in all tissues tested, the possibility that these genes are expressed with cell type specificity could not be excluded. It is possible that differential expression of these AtTLP genes could only be observed when internal developmental programming was altered or specific environmental stimuli were applied to the plants. To test this hypothesis, the public Arabidopsis Functional Genomics Consortium (AFGC) microarray expression database (the Stanford Microarray Database, available at the website of stanford.edu/MicroArray/SMD/) (Wu et al., 2001, Plant Physiol Biochem 39: 917-926) was searched. Twofold expression was used as the difference cutoff. Based on the search, the expression profiles of DNA fragments corresponding to AtTLP2, 7, 9 and 10 were summarized in Table 2 below.







TABLE 3







Microarray analysis of AtTLP genes expression











Channel 1
Channel 2
Ch2/Ch1 Normalized (Mean)b,c













Experiment
Description
Description
AtTLP2
AtTLP7
AtTLP9
AtTLP10
















Hormone Effect








Auxin Response
msg seedlings,
msg seedlings, 10 uM
0.32
2.21



untreated
IAA for 30 min


Auxin Induction
Mock-treated
NAA-treated

0.46

2.22



Columbia roots
Columbia roots


Cytokinin response
Control
15 min cytokinin
2.19




treatment


Abscisic acid
Wild type control
Abscisic acid
0.49
2.6
0.35


Insensitive 1

insensitive 1 mutant


edrl Mutant
Wild type leaves
edrl mutant leaves
2.33


Downstream genes of KN1
Control
Overexpression of
0.43
2.78

4.73




KN1-GR in Columbia-0




background


Stress


Effects of Elevated
Columbia leaves
Columbia leaves
0.33


atmospheric CO2
360 ppm CO2
1000 ppm CO2


Genes involved in chilling
Cold treated Columbia
Cold treated cls8
0.22

0.15


tolerance
wild type tissue
mutant tissue


Genes involved in
[K+] = 120 uM
[K+] = 2 mM

0.34

0.2


potassium nutrition


Cadmium
Control
10 uM cadmium treated


2.72




plant


Light Signaling


Circadian rhythm time = 12.0
Time = 0 hrs
Time = 12 hrs
0.37


Phototropic stimulation
Seedlings grown in the
nph4–2 seedlings grown
2.08



dark and exposed to
in the dark and exposed



1 hr blue light
to 1 hr blue light


Protein import into
Wild type
cia-2 (mutant)



3.46


chloroplasts: CIA-2


Identification of genes in
WT leaves after
cch1 leaves after



2.02


chlorophyll starvation
exposure to 230 uE
exposure to 230 uE



for 2 days
for 2 days


Stress


Effects of Elevated
Columbia leaves
Columbia leaves
0.33


Atmospheric CO2
360 ppm CO2
1000 ppm CO2


Genes involved
Cold treated Columbia
Cold treated cls8
0.22

0.15


In chilling tolerance
wild type tissue
mutant tissue


Genes involved in
[K+] = 120 uM
[K+] = 2 mM

0.34

0.2


potassium nutrition


Cadmium
Control
10 uM cadmium


2.72




treated plant





aThese data are obtained from the website of afgc.stanford.edu/afgc_html/site2.htm


bAll data are corresponding with fluorescence intensities greater than 500 in both channels and ch2/ch1 normalized ratio ≧2.0 or ≦0.5


cWhen searching dbEST with blastn, we find Arabidopsis EST corresponding to fragments of four AtTLPs represented on microarray data generated by AFGC. AtTLP2 is corresponding to the EST clone 289B10T7 and 173K22T7. AtTLP7 AtTLP9 and AtTLP10 are corresponding to the EST clone 173G1T7, 201E19T7 and F3E6T7, respectively.






The resulted show that factors like hormone fluctuation and environmental stimuli modulate the expression of the four AtTLP genes. As shown in Table 2, the four AtTLP genes had different responses to treatments of various hormones. AtTLP2 gene expression instantaneously increased more than twofold with cytokinin treatment but decreased to one-third after being treated with IAA. This suggests that Cytokinin and auxin play antagonistic roles in regulating AtTLP2 gene expression.
Another cytokinin-related experiment was aimed at identifying downstream genes of KN1. KN1-like protein is a homeobox transcription factor. Its overexpression upregulates cytokinin production and leads to delayed senescence (Vollbrecht et al., 1991, Nature 350: 241-243). The expression of AtTLP7 and 10 is upregulated in KN1 overexpression transgenic plant while AtTLP2 is down-regulated.
The different responses of AtTLPs 7, 2, and 10 to ABA treatment is also worth noticing. In abscisic acid insensitive 1 mutant (Pei et al., 1997, Plant Cell 9: 409-423), the expression of AtTLP2 and AtTLP10 decreases by two to threefold, but AtTLP7 expression increases over twofold. Interestingly, AtTLP2 and AtTLP7 behaved oppositely to auxin treatment and in abscisic acid insensitive 1 mutant and KN1 overexpression transgenic plant. These two AtTLPs therefore may function antagonistically in regulating phytohormone-signaling pathways.
The expression level of AtTLP2 rose in the edr1 (enhanced disease resistance 1) mutant leaves. The EDR1 gene encodes a putative MAP kinase similar to CTR1, a negative regulator of ethylene response in Arabidopsis (Frye et al., 2001, Proc. Nat. Acad. Sci. 98: 373-378). The edr1 mutation of Arabidopsis also confers resistance to powdery mildew disease (Frye and Innes, 1998, Plant Cell 10: 947-956). Thus, the regulation of AtTLP2 gene expression may be associated with SA-inducible and ethylene defense mechanism.
Environmental stresses also impose influences on the expression of AtTLP genes. For example, similar to the cold treatment on cls8 mutant, elevated CO2 level inhibited the expression of AtTLP2. K+ deficiency augmented the expression of AtTLPs 7 and 10 by threefold and fivefold, respectively. Heavy metal cadmium treatment stimulated the expression of AtTLP9.
In conclusion, the expression data of these four AtTLP genes indicate their involvement in phytohormone and environmental stress signaling.
AtTLP9 Interacts with ASK1 Protein
Homology searches in the public databases reveal that TLPs were also present in multiple plant species, including Lemna paucicostata, Oryza sativa, Cicer arietinum, maize, and Arabidopsis. Unlike animal TLPs having highly diverse N-terminal sequences, plant TLPs had conserved F-box-containing domain. Sequence alignment of the F-box cores from AtTLP, TIR, UFO, COI1 and the human F-box protein Skp2 revealed conserved islands separated by regions with weak homology. Many of the conserved residues correspond with those known to be important for Skp association (Schulman et al., 2000, Nature 408: 381-386 and Zheng et al., 2002, Nature 416: 703-709).
The F-box domain, first found in cyclin-F, interacts with the protein SKP1, which interacts with the Cdc53 (Cullin) proteins, to form a so-called SCF complex. The F-box is involved in recruiting specific proteins (e.g., transcription activators or repressors) and targeting them for ubiquitin-mediated proteolysis by 26S proteosome. Analysis of the Arabidopsis genome revealed that Arabidopsis had 21 Skp1-like, or ASK, protein, which exhibited different expression patterns. Among them, ASK1 is involved in vegetative growth and reproductive development (Zhao et al., 2003, Plant Physiology 133: 203-217).
To test whether AtTLP could interact with ASK1, AtTLP9 was examined by the yeast two-hybrid analysis. Yeast two-hybrid vectors, pAD-GAL4-2.1 and pBD-GAL4 Cam (Stratagene, La Jolla, Calif.), were used for C-terminal GAL4 AD and BD fusion constructions, respectively. A 1.1-kb SalI-PstI fragment containing the entire coding region of AtTLP9 was cloned into the SalI-PstI site of the pBD-GAL4 Cam vector. A 480-bp EcoRI-PstI fragment containing the entire coding region of ASK1 (Atg175950) was cloned into the EcoRI-PstI site of the pAD-GAL4-2.1 vector. The yeast strain YRG-2 [MATa ura3-52 his3-200 ade2-101 lys2-801 trp1-901 leu2-3,112 gal4-542 gal80-538 LYS2::GAL1UAS-GAL1TATA-HIS3 URA3::(GAL43×17mer)-CYC1TATA-lacZ] was co-transformed with the two vectors. The Y2H analysis was performed according to the manufacturer's recommendations (Stratagene). The result suggested that AtTLP9 physically interacts with ASK1 to form SCF complex and acts as a factor for substrate recognition in the ubiquitin-mediated proteolysis.
Attlp9 Null Mutants and Overexpression Lines
ATLP9 was analyzed to investigate for it in vivo functions. Both loss-of-function and overexpression approaches were taken to address its biological roles.
To identify attlp9 T-DNA insertion mutant, AtTLP9 (At3g06380) was used to search the T-DNA Express database at the website of signal.salk.edu/cgi-bin/tdnaexpress. Two attlp9 T-DNA insertion mutants (ABRC seed stock SALK—016678 and 051138) were identified and designated as attlp9-1 and attlp9-2. T3 seeds of attlp9-1 and attlp9-2 were obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus). The position of the T-DNA within the AtTLP9 gene was re-confirmed by sequencing a PCR-amplified fragment amplified by primer pairs corresponding to the T-DNA left borders and the AtTLP9 gene specific primer. The following primer pairs were used for attlp9-1 and attlp9-2 specific amplification,







(SEQ ID NO:45)









attlp9-1: N1, 5′-ATGACGTTCCGAAGTTTACTC-3′;



 







(SEQ ID NO:46)









LBa1, 5′-TGGTTCACGTAGTGGGCCATC-3′;



 







(SEQ ID NO:47)









attlp9-2: C1, 5′-TTATTCACAGGCAATTCTGGT-3′;



 



and







(SEQ ID NO:46)









LBa1, 5′-TGGTTCACGTAGTGGGCCATC-3′.






It was found that Attlp9-1 had a T-DNA insertion in the coding sequence at codon 705, whereas attlp9-2 had an insertion in the 5′ distal region of this gene. The T-DNA insertion site of attlp9-1 was identical to that originally described in the T-DNA Express database. However, the T-DNA insertion site of attlp9-2 was in the promoter region instead of exon1 as predicted in the database (the latter is supported by a potential full length cDNA corresponding to At3g06380 generated in RIKEN, accession number BT004092).
Southern blot was conducted with the nptII marker gene to determine the T-DNA insertion number in attlp9-1 and 9-2 knockout mutants. It was found that one and three T-DNA insertion events in the T4 attlp9-1 and attlp9-2 T-DNA insertion mutants, respectively.
The T-DNAs in attlp9-1 and attlp9-2 carried a gene leading to resistance to kanamycin. Homozygous analyses of attlp9-1 and attlp9-2 plants were carried out by kanamycin selection and PCR based method. RT-PCR analyses of T4 homozygous of attlp9-1 and attlp9-2 plants indicated that attlp9-1 was a null allele, whereas attlp9-2 was somewhat leaky. For the phenotype investigation, attlp9-1 and attlp9-2 T4 homozygous lines were used for detailed analysis.
Transgenic plants with overexpressing AtTLP9 were generated. An XbaI-SmaI fragment of AtTLP9 was inserted into an XbaI-SmaI site of the pBI121 Ti-vector (Clontech) to generate a 35S::AtTLP9 sense construct. The XbaI-SmaI fragment contained the entire AtTLP9 coding region and was under the control of the 35S promoter of cauliflower mosaic virus. The constructs were introduced into Agrobacterium strain LBA4404 by electroporation and transformed into wild-type plants by the floral dip method (Clough et al., 1998, Plant J 16: 735-743). 38 independent transgenic lines (T0 generation) were obtained. Among them, seven independent homozygous lines from the T3 sense transgenic plants were analyzed for the AtTLP9 expression. Each of these lines contained a single copy of the transgene. Two independent transgenic lines (S13-2 and S16-1) showed dramatic increases in the AtTLP9 transcript levels and were further analyzed. A number of control transgenic lines were generated by transforming with Agrobacterium with PBI121 vector alone.
The wild type Arabidopsis thaliana ecotype Columbia-0 (Col-0) and mutant abi4-1 (obtained from Dr Wan-Hsing Cheng, Institute of Botany, Academia Sinica Taipei) were used. The phenotypes of abi4-1 were confirmed as described (Söderman et al., 2000, Plant Physiol. 124: 1752-1765) prior to use.
All seeds of the above-described lines were surface sterilized with 70% ethanol for 30 s and then with 6% household bleach for 5 min before being washed five times with sterile water. For aseptic growth, they were plated on solid medium containing Murashige and Skoog salts (Invitrogen), vitamins (Duchefa), 0.7% phytoagar (Invitrogen), and 1% sucrose and transferred to a tissue culture room. For soil growth, seedlings were transferred into individual pots 8-10 days after germination and maintained in the growth chamber. Plants were grown at 22° C. under a 16-hr-light/8-hr-dark photoperiod aseptically or on soil.
The general development and growth phenotypes of the attlp9-1 and attlp9-2 knockout plants appear to be similar to those of the wild type plants. However, when seeds were plated on nutrient agar media, the germination time of mutant attlp9-1 and attlp9-2 seeds was advanced several hours compared with that of the wild type plants, whereas the selected sense line seeds (i.e., S13-2 and S16-1) germinated later than vector control seeds. It was found that 50% of the wild type seeds geminated after about 37 hour after plating. In contrast, 50% of the attlp9-1 and attlp9-2 knockout seeds geminated at hours 26-28 after plating, and 50% of the S13-2 and S16-1 seeds geminated around hours 40-42 hours after plating.
Effect of ABA on Seed Germination of Attlp9 Mutants and Overexpression Lines
It is known that seed germination is the outcome of an integration of many signals coordinated by the interactions of stage-specific developmental regulators and the competing effects of hormonal signals (Finkelstein et al., 2002, Curr. Opin. Plant. Biol. 5: 26-32). The most critical hormone promoting embryo maturation and preventing germination is ABA.
To determine whether the transgenic plants display altered ABA responses, the above-described lines were germinated on media containing various concentrations of ABA. Seeds collected at the same or similar times were used. After surface-sterilization, sterile seeds were suspended in 0.15% agarose, and kept in the dark at 4° C. for 3 days to break residual dormancy. The seeds were then plated on agar plates in six replicates containing no ABA or 0.25, 0.5, 0.75, or 1.0 μM ABA in 12-cm plastic petri dishes. Each agar plate was divided into seven sections, and 50 seeds of WT and AtTLP9 transgenic seeds were plated on each section. A seed was regarded as germinated when the radicle protruded through the seed coat.
In the presence of 1 μM ABA, the germination of sense lines seeds was further delayed and the germination rate was reduced to less than 10%. In contrast, the germination rate of attlp9-1 and attlp9-2 mutant seeds nearly reached 50%, and about 30% of wild-type seeds were able to germinate in the presence of 1 μM ABA. These results suggest that the disruption of the AtTLP9 gene affects the sensitivity of seeds to exogenous ABA.
In addition to reducing seed germination rate, ABA also inhibited the growth and the greening process in cotyledons of the sense transgenic lines. In MS agar medium containing 1 μM ABA and 1% sucrose, 90% of the 10-d-old seedlings showed developmental arrest although the radicles of most sense lines seeds emerged. In contrast, under the same conditions, attlp9-1 and attlp9-2 plants continued to grow and about 45% of the seedlings continued to develop true leaves, although at slower rates than abi4-1 mutant does. These results indicate that the alteration of AtTLP9 modulate plant's sensitivity to ABA during seed germination and early seedling development.
AtTLP9 Expression is Transiently Up-regulated During Imbibition of Seeds
Real-time PCR experiments were conducted to quantify AtTLP9 transcript levels at seed maturation, seed germination, and early development stage. UBQ10 was used as the endogenous control (Norris et al., 1993, Plant. Mol. Biol. 21: 895-906). Primers were designed using Primer Express 1.0 software (Applied Biosystems). The primers used were:










AtTLP9 forward primer,
(SEQ ID NO:48)









5′-TAGGCCACACCGTGTAGTTCA-3′;



 










AtTLP9 reverse primer,
(SEQ ID NO:49)









5′-CGTCAACAGTCTCAACCCTAATCA-3′;



 



UBQ10 forward primer,
(SEQ ID NO:50)



5′-AGAAGTTCAATGTTTCGTTTCATGTAA-3′; and



 










UBQ10 reverse primer,
(SEO ID NO:51)









5′-GAACGGAAACATAGTAGAACACTTATTCA-3′.






The real-time PCR was performed in a 50 μL reaction mixture containing 500 ng first strand cDNA, 2.5 μM each primers and 1× SYBR Green PCR Master Mix (Applied Biosystems). PCR cycling was 50° C. for 2 min, 95° C. for 10 min, followed by 40 cycles of 15 sec at 95° C./1 min at 60° C. The UBQ10 mRNA quantity was set at ‘1’ and AtTLP9 expression was determined relative to control samples. Threshold cycles were determined by Sequence Detection System V. 1.7a software (Applied Biosystems). The products were quantified by the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Scoresby, Victoria, Australia).
Seed germination is divided into three phases: imbibition, increased metabolic activity, and initiation of growth (Bewley. 1997, Plant Cell 9: 1055-1066). It was found that during seed maturation and seed imbibition at 4° C. for 72 h, AtTLP9 transcripts remained at a relatively low level. When the seeds were transferred to 22° C. for further incubation, the levels rose after 8 h, peaked at 16 h, and fell rapidly after 24 h when the radicle emerged. The AtTLP9 transcripts were barely detectable afterwards. The transient expression of AtTLP9 indicated that AtTLP9 functions at stage II of seed germination as a checkpoint before radicle protrusion.
OTHER EMBODIMENTS
All of the features disclosed in this specification may be combined in any combination.
Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the scope of the following claims.

## Claims

1. A transformed plant cell comprising a recombinant nucleic acid that encodes the heterologous polypeptide of SEQ ID NO: 9.

2. A transgenic plant comprising a recombinant nucleic acid that encodes the heterologous polypeptide of SEQ ID NO: 9.

3. A method of producing a transformed plant cell, the method comprising:
introducing into a plant cell a recombinant nucleic acid encoding the heterologous polypeptide of SEQ ID NO: 9, and
expressing the polypeptide in the cell.

4. A method of producing a transgenic plant, the method comprising:
introducing into a plant cell a recombinant nucleic acid encoding the heterologous polypeptide of SEQ ID NO:9,
expressing the polypeptide in the cell, and
cultivating the cell to regenerate a plant.

5. A transformed plant cell comprising a heterologous DNA sequence that encodes a polypeptide having at least 95% sequence identity to SEQ ID NO:9, and wherein expression of the DNA sequence in said plant cell confers increased sensitivity to ABA relative to an untransformed plant cell of the same species.

6. A method of producing a transformed plant cell, the method comprising introducing into a plant cell a heterologous DNA sequence that encodes a polypeptide having at least 95% sequence identity to SEQ ID NO:9, and wherein expression of the DNA sequence in said plant cell confers increased sensitivity to ABA relative to an untransformed plant cell of the same species.

7. A method of producing a transgenic plant, the method comprising introducing into a plant cell a heterologous DNA sequence that encodes a polypeptide having at least 95% sequence identity to SEQ ID NO:9, and cultivating the transformed cell to regenerate the transgenic plant, wherein expression of the DNA sequence in said plant confers increased sensitivity to ABA relative to an untransformed plant of the same species.