Molecular genetic analysis of the maize terminal ear1 gene and in silico analysis of related genes : a thesis presented in partial fulfilment of the requirements for the degree of Doctor in Philosophy in Plant Biology at Massey University, Palmerston Nort

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(2) 1. Molecular genetic analysis of the maize terminal ear1 gene and in silica analysis of related genes. A Thesis Presented in Partial Fulfilment of the requirements for the Degree of Doctor of Philosophy in Plant Biology. at. Massey University Palmerston North, New Zealand.. Daniel Charlton Jeffares. 2001.

(3) 2. . This thesis is dedicated to Ben, Charlie and Alex..

(4) 3. T H E S I S ABSTRACT M utants of the maize terminal ear1 (te 1 ) g e n e have shortened i nternodes, abnormal phyll otaxy, leaf pattern defects and partial fem i nisation of tassels. The te 1 gene e n codes a n R NA recog n ition motif (RRM) prote i n , and is expressed i n the vegetative sh oot apex in semicircular rin g s that l ateral ly oppose the positio n s of leaf pri m o rd i a (Veit 1 998). This project a imed to further characterise t h e molecular b i ology and fun cti o n of the te 1 gene.. Molecular genetic studies aimed to further characterise the genes structure and expres s i o n . Genomic clones were sequenced revea l i ng the i ntron exon structu re. 5' RACE was u sed to predi ct a 5' transcriptio n start site. C o mpetitive RT- P C R showed that te 1 transcri pts were h ig hest in vegetative shoot meristems and embryos, l ower i n ears, roots and tassel s , a nd u ndetectab l e i n leaves. Two te 1 mutant alleles were cloned a n d the j u n cti o n s sequ enced, a further five a lleles were characterised i n co m pl etely .. The T E 1 peptide belongs to a s u bclass of RRM proteins which includes the Schizosaccharomyces pombe protein M E I 2 . More than 30 putative p l a nt Mei2-like. genes were identified in Genbank , no examples have been fou n d in metazo a n s. Seven Mei2-l ike genes were predicted from the completed Arabidopsis genome. Exon structure and amino acid sequence supported three groupings o f Mei2-like genes. Structural p redictions of Mei2-l ike protei n s i n d i cate that the third RRM contained some novel structural features not present i n canonical R R M proteins.. Attempts to study the fun ctio n of the T E 1 protein in vitro were l i m ited by the inability of both E. coli and Pichia pastoris expression systems to express the ful l l ength prote i n , pro b a b ly due to codo n bias. Antibodies produced to a C-termi n a l port i o n of the prote i n did not specifically detect the TE 1 protei n i n plant extracts without i n c u rring n on-specific activity .. The te 1 cDNA was ectopically expressed i n Arabidopsis from a copper-inducible promoter both with and without the SV40 n uclear localisation signal ( N LS ) . Although both te 1 a n d NLS:te 1 tra n sgenes were detected i n tran sformants n o phenotypes consistently correlated with transgene expressi o n ..

(5) 4. A CKNOWLEDGEM E NTS A l ot o f people h a ve helped me with this P h D , but I would l ike to thank David Penny and Paula J ameson encouragi n g m e to stay on after H o n o u rs , encouragi n g me to start my P h D, and for support i n g my applications for the doctoral sch o l a rship. T h a nks for a l l the members of my family for your contin u ed encouragement, and for your attitud e to thi n g s academic. I t really makes a difference.. Thanks to Bruce Veit, for teaching the essentia l techn iques of molecular biology l a b work , h o w t o critique experiments, a l l about meristems, a n d many other things besides. I k n ow we haven't always got o n Bruce, but your attitude to science, and why we d o it, is very coo l . Thanks also to Paul Reynolds for your help at the start of this project.. Thanks also to the many other people who have taught me during my time at M assey, i n particular David Penny, David Fountain , P a u l a J ameson , B i l l Jones, M ike McManus, Barry Scott, Stan Moore . Its been fun . Also to the people who helped me negotiate the tria l s of experimental work; Carolyn Young, Lyn Wats o n , Carmel G i l l m a n , some of the above. C heers also to the Riborg Club, Antho n y Poo/e, a n d David P e n ny for t h e weird a n d wonderful world o f Riborgis eigensis. And to a l l those fel low students and friends around the Department of Plant B io l ogy/ l M BS/D E B Lab-Boffin Lounge and associates - Ive had a darn fine time: Abby, Alicia, Ant, Austin , Bennett, Carm el , Carolyn & David, E m i ly , H u a i Bi, Lyn , Matt (bane of s m a l l mammals), M u rray, Nena, S a n g Dong Yoo, Tony (Yay for the DEB Zeppelin sessions!), Trish McLenachan , Trish M urray, Vern .. Katy. Thanks for everythi n g , for even editing what to you must be mean i ngless jarg o n . © Thanks for all your l ove and affecti o n , you helped me to reject the hegemony of i m p uted needs.. I ack n ow l edge the assistance of the Massey Doctoral Scholarshi p ..

(6) 5. TAB L E OF CONTENTS. TH E S I S A BSTRACT. ...... .. .. ........ ACKN OWLEDG E M E NTS. ............. ............ ... ... ...... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . . . . . 3. ............................. .. .... .. ............. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 4. TABLE O F CO NTENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 LIST O F TABLES. .................. LIST OF F I GURES .. LIST O F GENES ... ..... .. .... ... .... .. ....... ... ......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. .. ............................... . ....................................... . ..................................... . 12. ............................................................................................................................ CHAPTER 1 : I NTRODUCTI O N . . . . . . . . . . . . . .. .. ... .... . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . .. 1 . 1 A BSTRACT . . . . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . .. . ... . . .. . . . . ... . ......... . .... . .. . . . .. . . . . . . ... ... .. .. .. .... 14. 17. . . . .. . . . . . 1 7 .. 1.2. Develop m ental biology . ..... ... .. .. . .. ... . .. 18 . . 1.3. T h ree i m portant aspects of pla n t develop m e n t............................................................... 18 .... ................................. .. ............................ .. .. .. .. ..... .. . ... .. 1 .3a. The control of cell division .......... .................................................................................... 1 9 1 .3 b. The role of intercellular trafficking i n plant development................................................ 20 1.3c. Structures that give rise to the plant body ....., ................................................................ 2 1 1.4. Shoot m e ristems. .. .. .... ... . .. . ..... . ..... . .. . .......... . ......... . ..... . . . ... . . . ...... . . ......... . ....... . ..... 22. .. ...... ......... .. ... 1 .4a. Structu re of shoot m e ristem s ........................., ................................................................ 22 1 .4b. Functions of the shoot m e ristem ........................................................., ................, .......... 25 1.5. I n itiation of lateral organs. . ... .. ... . .. .. .. ........ .... .. . ... .. ...... .. . ...... . ... . ... .. .. ... .. .. ... ... ... .... ... .. ... ... . .. . .... ... ..... 27 .. .. .. 1 .5a. Similarity of lateral org a n i n itiation ..... ................................... ., ...................................... 27 1 .5b. Phy l l otaxy: specification of organ position ...................................................................... 28 i.5c. Lea f initiation and acquisition of identity ............. ....... ...... ..........................., .............. 30 .. 1.6. A n i n t roduction to the terminal ear1 gene and homologous plant g e n es 31 1 .6a. The terminal ear1 gene of maize ................ ............. .......................................,.............. 3 1 1 .6b. Plant homologues of the terminal ear1 gene: Mei2-like genes ............... ........ .... '" ..... 32 .... . ............. 1.7 RNA recog n ition motif protei ns. .. .. .. .. ... ......... ..................... ...................................... ..... ....... . .... 33. 1 .7a Orig i ns and structures of R NA recognition m otif p rote i n s ................................., ............. 3 3 1 .7b Fu nctions o f R NA recog n ition motif p rote i n s ..............................................., ................... 36 1 .7c Biochemical function of the S. pombe M EI2 p rotein ....................................................... 36 1.8. A i m s of this project .. . . . . . . . . .. ... ... .... ... .. .. . ... .. ... .. 1.9. Data produced by oth e r resea rch e rs. . . .. . .. ... .... . .. .......... .. ............ . .. ......... . ....... .. ... ... . . . .. . . . .. . ... . ... ... ..... 37. .. .. . .. . . ...... ..... ............... .......... ... .. .. . .. .. . .. ... .. .... . ... 37. CHAPTER 2: MOLECU LA R G E N ETIC ANALYSIS O F T H E TERMINAL EAR1 . . G E N E O F MAiZE .. ... .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. .. . .. . . . . 4 1 .. 2.1 . A B ST RACT . . . . . .. .. M ETHODS (2) . . . . . . .. .. .. .. .. ... ... ........ .. .. ... .. .... . ... ... ... ...... ... ... .... .. ... ... .... .. .... .... .. .. .. ........... ... ....... ... .. .. ... .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . 4 1. ... . . .. . . . . .. . .. . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . .. . . . . . . . . 42. E. coli methods ....................................................................................................... 42 2.2a. Growth of E. coli .., .............................. ..... ........................ .... ... ............. ...................... 42 2.2b. Preparation of heat shock-competent cells ......... ............... . .............. .......................... 42 . ....... .. . ......................................... 42 2.2c. Transformation of E. coli by heat shock. 2.2d. Preparation of electrocompetent cells .................. ..... ....... .......................................... 43 2.2e. Transformation of E. coli by electroporation ................................................................... 43 ................. . . .................... 4 3 2.2f. Alkaline lysis of E. coli. . . ..................... 2.2g. Lysis of E . coli by b o i l i n g using STET buffer ..................... ....... ................................... 4 4. 2.2 Gen e ral. 5.

(7) 6 2.3 G e n e ral D N A m ethods ............................................................................. ................ ........... 44 .. .. 2.3a. Quantification of DNA a n d R NA... .... .............. ...... ........ 2.3b. Electro p horesis of D NA . .. . ....... ... ... ...... ...... ....... ........ 2.3 c. Stan dard PCR Reaction Protocol.. ........ 2.3d. Colony PCR . ... . 2.3e. Gen e ra l scheme for vector cons truction . . .... . ... ...... .. .. ..... 44 .... 45 ... .... 45 .. 46 ..... ..... 46. 2.4 Seque n c i n g of te1 genom i c clo n e ...................................................................................... 47. 2.4a. Genera l scheme of p roject .... 2.4b. Constr u ction of 5' end geno m i c subclone.. 2.4c. Seq uencing reactions ........... . ..... . ... 2.4d. Assembly of sequence files........... ........... . ... ..... 47 ........ . .. 47 ..... .. ..... .... .... 47 ... .. .. 48. 2.5 G e n e ral R N A m ethodology .................................................................................................. 48. . .................. .......... 48 ........... 49. 2.5a. Precautions to avoi d R N ase contam ination ......... .. 2. 5b. D N as e treatment of RNA p reparations... 2.6 5' Rapid a m plification of c D N A e nds (5' RACE) ............................................................... 49. . . .. .. . . . ........ . '" ... 49 2.6a. S u mmary of 5' RACE method.... .. '" ... 50 2.6b. Special p recautions for 5'RACE experime nts " .. 52 . .. ............ .. 2.6c. Extraction of polyA+ RNA d i rect from tissue Iysates.. . . . .......... . ........... .. 53 ............... 2.6d. Suita b i l ity of R NA sam ples for 5' RACE. . ... .. 53 . ... ........ .... . . ........ 2.6e. Reverse transcription reaction for 5' RACE ..... .. 54 2.6f. Synthesis of homopolymer tail. ............... ............ 2.6g. Secon d strand cDNA sy nthesis and primary PCR Reaction ............ .................. .......... 54 ...... ................ 55 2.6h. Secondary semi-nested PCR reaction. . ....... ...... ....... ... ..... 2.7 Q u antification of te1 transcripts by competitive RT-PCR ............................................... 55. 2.7a. S u m mary of competitive RT-PCR p roced u re. .................. .. . . ........ ............. .. ... 55 2.7b. Extraction of total RNA with Qiagen R N easy Plant Mini Kit ... . ....................... .......... 56 2.7 c. Acid guadi n i u m thiocyanate R N A extraction m ethods ................... ............................... 56 2.7d . Suita b i l ity of R NA sam ples for competitive RT-PCR...................................................... 58 2.7e. Synthesis of altered te1 transcript................................................................................... 58 2.7f. Reverse transcription reaction s for RT-PCR ................................................................... 59 2.7g. Com p etitive PCR ............................................................................................................. 60 2.8. N o rthern blott i n g .................................................................................................................. 61. 2.8a. E lectroph oresis of glyoxylated RNA .. ... ................. .................. ...... .. ......................... 6 1 2.8b.Transfer o f R N A t o memb ranes .................................. ............ ....................................... 62 2.8c. Synthesis of radiolabelled p robes. ........ ......... ... . ....... . . ............ ............ ........... 62 2.8d. H y b ri d isation of R NA m e mbranes................................................................................... 63 2.9a. S u m mary of te1 allele a na lysis ... . ... . .... .. . ... .... 63 . .. ... . .... ... . ... .... 2.9b. Extraction of m aize genomic D NA ........................ ....... .......... ...................................... 64 2.9c. peR analysis of te1 m utant a l leles ................................................... ............................. 65 2.9d. Cloning a n d sequencing of PCR products . . ... . . . .. . . . . . . ...... .. .... . . ... 65 2.ge. Southern Blotting with the D I G System .......................................................................... 66 .. .. . . .. . .. . . . .. . . . .. .. . .. .. . .. . . . . . .. .. . . .. .. .. .. . . . .. .. .. . .. . .. .. . .. . . . .. .. . .. . . .. .. .. .. .. . .. . . . .. .. . . .. .. .. . .. .. .. R E S U LTS ( 2) ...... . . . . .... . .. .. .. . . . ...... . . . ......... . . . ... . .... . .. ............. . ... ..... . . ... . . 6 8 2.11. P redictio n o f transcription sta rt s ite with 5 ' RACE ....................................................... 75 .. ..... .. .. .. .. ... .. .. ... .. ... ..... 2. 1 1 a. The 5' RACE p rotocol was i nconsistent ....................................................................... 75 2. 1 1 b. RT -PC R d i d not confirm that the transcription start site was as p redicted by 5' RACE 75 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 2.12. Qua ntificatio n of te1 tran script levels ............................................................................. 78 2.13 A n alys i s o f te1 m utan t alleles ........................................................................................... 81. '" . ......... . ................... 8 1 2. 1 3a. Analysis of m utant alleles by PCR.. . .. . ........ ..... 8 1 ....................... .... .... .. f leles l o a lots ................................... te1 b 2. 1 3b. Southern 2.1 3c. Analysis of combined Southern a n d PCR data .. . . . .... . . . . . . . .... . . . 86 .. . . . .. .. . .. .. .. . .. . .. . .. . . . . . .. .. . .. ·. . . . .. .. . . . .. DISCU S S I O N ( 2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 2 2.14. A n alys i s o f the te1 g e n o m i c sequence ........................................................................... 92 2.15 Expres s i o n studies ............................................................................................................. 92. .............. ............... 92 2. 1 5a. 5' RACE.................................. 2. 1 Sb. Expression of te1 and m e ristem function .............. ........................................ ........... 93 2.16. M utan t allele a n alysis . ....... . . . .... .... ................................................................................... 96 .. .. 6.

(8) 7. CHAPT E R 3. IN SILICa ANALYSI S OF MEI2-LlKE G EN ES 3. 1 A BSTRACT. .. ... ... ... ............ .. ..... . . . . ... ... .... .... . .. . .... . ... .. . ... .. .. ... ...... ... ..... .. .. .. ...... ....... .. .. ..... ..... . .. . .. ... .... .. ...... .. . 97 .. . . . . . . . . .. .. . .. .. .. ... .. .. .. ... 97. M ET HODS (3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 . 98 . . .. . 99. 3.2 tBLASTn s e a rches t o identify putative Mei2- l ike g e n es . 3.3. Arabidopsis g e n e predictio n m ethodo logy . . . . 3.4. Prom oter a n a ly s i s. ... .................................. ............ ............. ... . . .................... .. ............... .... .... ............................................................................................................... R E S U LTS (3) . .. . . . . . .... . .. .. . ...... . ..... . . . ...... ... . ... . .. . . ...... ... . .... .... 3.4. Seve n Arabidopsis Mei2- l i ke g e n es have been identified ... .. .. ... .. ... ... .. .. ... .. .. .... .. 100. .. . ..... .. . . 102 . . . 102 .. ... .. ...... .................. .. .... ....... .... 3.4a. Prediction of Arabidopsis Mei2-like genes.. 3Ab. Arabidopsis Mei2-l ike cONAs and ESTs .... . . .. ...... . ........ 102 .. ..... 103 . 108 . . . . 116 . . . 116 . . . .. . .. . 119 . 121 . ... . . .. 121. 3.5. Three RRMs can be identified in a l l c o m p l ete Mei2-like g e n es 3.6. The i ntron positio n s of plant Mei2-l ike g enes a re conserved . 3.7 Com p a rison of te1, TEU and TEL2 promoters . . . . 3.8. A l a rg e number of Mei2-like g enes can be identified i n p l a nts . 3.9. Mei2- l ik e g e n es from taxa other than p l a nts . . . . . 3.10. Phy l o g e n etic a n a lysis Mei2-l ike genes . . .. . ........................ .......... ......... ...... .... ............ .. . ..... ...... ................ .. ......... ........ . .. .. .... . . ... ...... . .. ...... . ........ ...... ... ... .... .... ...... ..................... ........ . .. ...... .............................. ...... ........ .. .. .... ... ......... 3 .10a. Phylogenetic a n alysis of com plete Mei2-like genes. . .......... ... ................... ............ 121 3.10b. Phylogen etic a n alysiS of i ncomp lete Mei2-like gene sequences a n d E STs.. ....... .... 122. DISC U S S I O N ( 3 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 25 3.11. Function a n d b iochem istry of MEI2 with respect to plant Mei2-like g e n es .. . . . 125 .. 3.11 a. 3 . 11b. 3.11 c. 3 .11d. 3.11 e.. .. . ..... The biological context of Mei2 and plant Mei2-like genes ................... ...................... 125 R N A depende n t localisation ......... . . ............. . . ..... . ........................................................ 126 R N A binding activity of MEI2 and plan t Mei2-like genes .................. ........... .. ........... 128 Phosphorylation of Mei2-like p rote i n s ... .... . . ... ... . . . . .. .......................................... . . . . . ... 129 I nteraction with M I P1 WO repeat p rotein . ... . ....................... . ............ ......... . . . . .......... . . 130. 3.12. Evolution a nd derivatio n of Mei2-like g enes .. . ... ... . ... 3 .13. Fu nctions of plant Mei2-like genes ....... ... 3.14. Con clud i n g rem arks. .. . .... . . ... ... ......... .. . . .. ... .. .. . ........ .. .... . .. . . ..... . .. . .. .. ........... .. ... . . ...... . 130. ............. . ........ .... ............ ............. . . .. .. 131 .. .. ......... ..... ...... ... .. .. .. . . ... . . ... ............. ..... .. . .. ... ..... .. .. . 132. .... ...... CHAPTER 4. EXPRESSI O N OF T H E TE1 P RO TE I N I N H ETEROLOGOUS HOSTS AND PRODUCTIO N O F A NTI BODI ES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . 1 33 4.1 A BSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 33 M ET HODS (4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 34 ... . ...... . ... . .. .. . .... 134 . ... . . .. . . . .... 4.2 Gen e ral protei n methods. . .. ...... ....... ..... ........... .. .... ... . .. . ........ 4.3 P roduction of m a ltose b i n d i n g protei n fus ion proteins. .... .. .. .. .... . .. .. ..... ......... ........ ...... ... 134 .. .......... ....... ......... ....... 134 .. .. ... 135 . ....... ..... . . ...... .............. 136 . ................ 136. 4 . 2a. B radford assay estimation of p rotein concentration . .. 4.2b. SOS polyacrylam ide gel electrophoreSis (SOS-PAGE) 4.2c. Western blotting. l u m i n escent substrate ..... . ....... ... 4.2d. Western blotting. colorimetric substrate . 4 .2e. E nzym e li nked i m m u no-sorbent assay (ELlSA) .... ........ .. .. . 138. ................ ........... ................. .. 4.3a. Construction of m altose bindi ng protein fusion vectors . .............. ............ . ....... . . ........ 138 4. 3b. Small scale i nduction of E. coli cultures harbouring expression vectors ............... .... 138 1 286 4.3c. Optimisation of M B P:TE1 • expression conditions ... .......... ................... ........ ........... 139 4.3d. Large scale i nductions a nd affinity p urification of M B P:TE fusion p roteins ..... .......... 139. l 286 4.4. P roductio n of anti MBP:TEj . . antibody. .. .. .. . . .. .. . .. .. . 140. ........ ... .. .... ... .. ....... ... ....... .............. .... ... 4.4a I noculation of rabbits and collection of sera ... ............. ........ ... ... .............. ..... ......... . . 140 4Ab. Affi n ity p u rification of polyclonal antibody .... . .... ........ . ........ ...... . .... ................ ... ..... . 141. 4.5 H istidi n e-tagged c onstruct with ArgU tran sfer R N A g e n e .. .. .. . .. . .. . 142. .......... ..... ..... ... . .. .. ..... . .. 4.5a. Construction of h istidin e tagging vector.... ....... .......... ........ ............ . . ... ......... ..... . . . .. 142 .. ·. 7.

(9) 8 4.5b. Subc l o ning the ArgU tRNA gene by peR..... .... 142 4.5c. Expression trials with histidine tagging vector.... . 143 4.5d. Purification of histidine-tagged TE1 p rotein.......... ............. . . .... 143 4.6 Transfor m ation of Pichia pastoris with i ntegratin g expres s i o n constructs .............. 144 4.6a. Overview of Pichia expression system '. . . .. 144 4.6b. Production of integrating expression vector in E coli.... ...... 145 4.6c . Preparation of electro-competant Pichia cells .. .. 145 4.6d. Electroporation of Pici?ia cells.. 145 4.6e. Selection of transform ants with increasing numbers of integration s .. . 146 4.6f. Extraction of Pici?ia genomic DNA .. . 147 4.6g. Southern blotting.... ........ . .. ...... 147 4 .6 h . Induction trials with tran sformed Pichia pastoris . ...... ..... . .. ........... 148 .. . .. .. R ES U LTS (4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 50 4.7 Only the a m i no-ter m in al half of the TE1 protei n was produced as MBP fusions .. 153 4.8 Expres s i o n of f u l l length TE1 protein from the ArgU tRNA construct a nd the A rgU tRNA-su pp l e m ented pSBET vector .. . . ... 155 . ... . 4.9 T h e Pichia pastoris expression system did not prod u c e s i g n ifican t q u antities of the TE1 protei n 159 .... ........ ..... ...................... ............... .... ............... ... .. ....... .................................................................................................................................. 4.9a. AnalysiS of p rotein expression by transformed Pici?ia pastoris lines........................... 159 4.9b. AnalysiS of the integrations of transformed Pichia pastoris lines ................... ............ 161 4.10. C h aracterisatio n of the activity of polyclonal sera .. .. . . ... ..... ... ... ......... .. .. . . ... .. ................... ... 163. 1 286 4.10a. I mm une sera from both rabbits had activity against the MB P:TE1 ' p rotein ........ 163 4.10b. Pre-immune sera B had activity against the M B P p rotein ........................ ...... ....... 164 1 186 4.10c. Antibody activity against MBP and TE1 • portions of the fusion p rotein ............... 165 4 .10d. The affinity purified a ntibody has activity against all maize tissues .......................... 167. DIS C U SS ION (4) . .. . . . . . . . . . . . . . . . . . . . . . .. . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 69 4.11. Why w a s the TE1 protei n not produced i n E. coli? . . 169 ............................ ....... ................. 4.12. Why was t h e TE1 protei n not produced i n Pichia pastoris? . 172 4.13. T h e ' n o n specific' activity of the polyc l o n a l a ntibodies ............................................. 172 4.14. Con c l u d i n g rem a rks and f u t u re prospects . 173 . . .................................... . ...... .......................................................... CHAPTER 5: ECTOP I C EXP RESSI O N OF TEL G EN ES I N ARABIDOPS/S 5 . 1 A B STRACT M ETHODS (5) . .. ..... .... ... ..... .. ...... ... . . .. . ... ........ .... .. .. .. ..... ..... ..... .. .... .. .... .. .... ........................... ... ........ . .. .. ......... . . .. .... . .. . .. . . .. . .. . ........ .. ..... .. .. .. .. .. .. .. .. ... 5.2 Construction of vectors f o r transformation of Arabidopsis. ... .... ....... .. .... 1 74. . . . . . . . . . 1 74 .. .. ......... .... . ... ... ..... ... .. . . . . . 1 75 . 175 .. . .... .... ................ ...... ....... ... ............. 175 5.2a. Construction of copper-inducible transformation vectors . .. 5.2b. Construction of glucocorticoid transformation vectors ............ .................................... 177 5.3 T ra nsform atio n of Agroba c te ri um .................................................................................... 178 5.4 Growth of Arabidopsis . . 178 . . . 5.5 T ra n sformation of Arabidopsis by vacuum i nfi ltratio n ................................................. 179 .......... 179 5.5a. Overview of the p rocedure ................ ................. .... ............... . 5.5b. Vacuum infiltration ...................................... . ... ... . .... ..... ... . ........... ....................... 179 . ........ 180 ...,.. ..... 5.5c. Selection of transformants . .. . ...... ......... ....... ............................. ........................ ........................ 5.6 I n d u ct i o n conditions for p l ants with copper-inducible tra ns g e n es . .. . .. . ... .. ....... ... .......... . 181. ........ 181 5.6a. I nduction in synthetic media.. ................... .. ... ......... . 5.6b. I nduction in soil ..... ......... .... .... ... ........ .. .... .. . ... .......... ..... ................ ............... 181. . 181 5.7. Dexamethasone applicatio n to TEL2:glucocorticoid receptor plan ts 5.8. Assays of transg e n e expressi o n ..................................................................................... 182 ................... ..... .. . ... ........ . . . .......... . ... 5.8a. N orthern blotting .... 5.8b. Western blotting ........ .... R E S U LTS ( 5 ) . . . . ... .. ............ ..... ........ ... . ...... .... ........ .. .. . . .. ... .. ..... . .... . . . . . . .. .. .. .. .. .. ... ............ 182 . .. .. .... ... .. .. 182. .......... .. .......... . . . 1 84 .. ... 8.

(10) 9 5.10. Initial a n alysis of copper-ind ucible transform a n ts. ...................................................... 184. 5. 1 0a. Transgeni c p la nts transforme d with copper inducible constructs contained one or two integrations ofT-DNA . .. . . .. . . . . .. . . . 184 5. 1 0b The te1 transgene was expressed in Arabidopsis from the copper inducibl e p ro m ote r . . ...... ......... ... .. . . . . ... ...... .... .. .. .. . 186 .. 5.11. Expressio n of t h e te1 transgene did not correlate with any observable p h e notypes 188 .......................................................... .......................................................... ..................................... 5.1 1 a. Neither vegetative nor floral morphology was affected by fe 1 transgene expression .............. ....... . . ....... 1 88 5 1 1 b The wnnkled l eaf phenotype did not correlate with transgene expression .. .. ... 1 89 5 1 1 c The antl_M B P·TEi1•286 antibody did not d etect the TEi p rotein In transgenic plants ................. ..... ... .. . .............. . ........ 1% 5. 1 1 d. Aerial rosettes formed, but they were not due to expression of the te 1 transgene . 1 96 .. .. 5.12. I n itial observations of TEL2 glucocorticoid fusion pla nts s h owed n o clea r p h e notype .. ............ . . ... . ................... . ......................... . ............................................. ...................... 198. .... . . . . . . . 199 DISCUS S I O N ( 5) .. . . .. . . . . ....... . . .... . .. .... . . . ... . . . .. . .. . 199 5.12 T h e a e rial rosette p h e notype is the result of g rowth conditions 5.13. Expression of the m aize te1 g e n e or the Arabidopsis TEL2 g e n e i n Arabidopsis did n o t produce any consisten t p h e notype .. 200 . . . . ... .. ...... ..... ... .. .. .. .. ... .. .... .. .. . . . . . . . . ... .. .. .. .. .. .. .. ... ........ ................ ....... ...... .. ... ......... .... .............. ... .......................... .... 5 . 1 3a. Tech nical issues with te1 transgene expression.... . .... 200 5i3b. Technical issues with TEL2:GR transgene expression . . . .. .. . .... 2 01 . ........... 201 5.1 3c. Biological aspects of TEL genes . . ........... . .. . . . 2 0 3 5 . 1 4d. A reinterpretation of overexpression phenotypes based on recent d ata .. . 5.15. Con cluding remarks .. . . . .. . . . . . . . 203 .. .. .. .. .. .. . ............... .... ... .. .... ... ............... ............ ............... .. .... ....... .......... .. . .. . .... . . . ... . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . .. .... . . . . . . . . ... . . . 206 6.1. Recapitulation o f t h e aims o f t h e project . . . . .. . . . .. . . 206 . 6.2. Homology, o rthologs a n d g e n o m e d u plications . . . . . .. .. . .. . . 207 . 6.3. Models of TEL g e n e f u n ction . 208 . . . .. . . . .. 6.4. A fin al s u m m a ry a nd some new q u estion s . . . 209 .. .. CHAPTER 6. SYNOPSIS. .. .. .. .. .. ... .. ... .. .. .... .. ... ... .. .... .. .. . .. ..... ..... .. . ......... .... .... ...... ........ ... . ................................... . .... . ...... . .................... .. .. R E F E R E NCES . . . . . .. .. .. . . . . . .. . ..... . . .. .. .. ... ..... ... . . .. . . . . ... ... ... .. . . . ... .. . ....... ....... ......... ..... .. . ......... ......... . . .. .. ..... .. ... . .. . ... ...... ......... .. .. ... .. ....... .. ....... . ...... .. .... ........ ... ... .... .... ..... ..... . .. .. ... .. .. .. .. .. .. ... .. .. .. .. . 21 1. A P P E NDICES . . . . .. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 Appendix 1. Olig o n ucleotide P ri m e rs . . .. . 227 . . . . .... .. . . .. . .. Appendix 2. PCR P ro g ra ms . . . ... .. . . . . . .... .. 229 . .... .. . . ... . . . . Appendix 3. List of S uppliers . .. . . . . . . . . . . . 232 . . . .. App e n dix 4. Assembly of te1 g e n e sequenci n g reactions . . . . . . .. .. .. . . ... 233 Appendix 5. Subclones of t h e 12.4 kb Kpnl te1 g e n o mic clon e . . .. ... . . . .... . . . 234 App e ndix 7. Pedigrees of m aize stocks ... . . 235 . . . . . . Appendix 8. PCR produ cts from te1 m utant alleles 235 . Appen dix 9. Southern Blot D ata . .. . 236 .. . . . . ....... . .... .. .. .. ..... .. .... . ........ ........ ... ... ...... ..... ....... .... ............ .. . ......... . .. .... .. .... ............. . .. .... . .... .. .. . . . . .... ..... ...... . .. .... .. ................. . .. . ... .. . .......... ... .. .. . .. . ... ... ... ........... .. .. .. ... .. .. .......... .. . . ... ... ... .. .. .. .... ..... .. .. .. . . ..... . ..... . . .................. .. ... .............. ........... .......... ........ ....... ............................ . ..... ................. . .. .. . . ........................ . ... . .......... ... . ... .............................. . ..... Appendix 9. 1 . Southern blot one. . .. . . .. . .. . . . .. . ... . . .... . . ... .. .. . .. . .. . .. .. . .... . .. . 2 36 ....... 2 37 App e ndix 9.2. Southern blot two. . A ppendix 9.3. Southern blot three . . .. .... .. . . . .... . .... . ... . . . . . .. ... ...... .... . .. ... . .. .... . , .... ..... 2 38 .. . .. .. .. .. .. . .. App e ndix 10. Supplem enta ry file disk. .. .. . .. .. .. . .. .. .. .. .. .. .. .. .. .. . .. .. .. .. .. .. .. . .. .. .. .. .. .. .. .. . . .. .. .. . .. .. . . .. .. .. . . .. .. .. .. .. .. . . 239. . .. ...... . ... . ....... . . . ......................................... .. ..... ..... . .. .. .. Gene p rediction s . .. .... .. . ... . . . . .. . . . . . . . . .. .. .. . .. .. .. . .. .. ... . ... . . ... . .... . . . . . . . . ... . . . .. . .. ..... .... .. .. .. Appendix 11. T h e c o n c u rrent RNA world project... . .. . .. .. . .. .. .............. . .. .. .. .. .. .. . .. . .. .. .. .. .......... . .... . .... .. . . .. . ... 2 39 . .. . ........ . .. . ...... . ... . 240. 9.

(11) 10.

(12) 11. LIST OF TABLES. Table 2 . 1 . te 1 mutant a l leles. 56. Table 2 . 2 . S pl i ce sites i n te1 gene. 60. Table 3 . 1 . Arabidopsis Mei2� l i ke genes. 95. Table 3.2. EST h its from Arabidopsis B LASTn searches of genomic sequences. 96. Table 3 . 3. Mei2- l i ke genes from p lants. 1 09. Table 4 . 1 . Expression vectors. 1 39. Table 4 . 2 . Resistance of transgenic Pichia lines G4 1 8. 1 51. Table 5 . 1 . Tra nsgenic Arabidopsis constructs. 1 69. Table 5 . 2 . Leaf n u m ber frequencies i n te 1 -expressing Arabidopsis. 1 78. Table 5 . 3 . C o pper-inducible tran sforma nt l ines; transgene expression and the wrinkled phenotype 1 79.

(13) 12. LIST OF F I GU RES Figure 1 . 1 .. Symplastic fields i n the SAM. 23. Figure 1 . 2 .. Overlay o f five R R M domains. 35. Figure 2 . 1 .. Overview of the 5' RACE protocol. 51. Figu re 2 . 2 .. T h e structure o f t h e te 1 gene. 69. F i g u re 2 . 3 .. Genomic sequence o f t h e te 1 gene. 70. Figu re 2 . 4 .. 5' RAC E g e l s. 77. Figure 2 . 5 .. Competitive RT-PCR quantification o f te 1 transcript. 79. Fig u re 2 . 6 .. RNA samples used for competitive RT- P C R. 80. Figure 2 . 7 .. te1. a l lele S outhern blot 1. 83. Figu re 2 . 8 .. te 1. allele S outhern blot 2. 84. F i g u re 2 . 9 .. t e1 a l l e l e S outhern b l o t 3. 85. Figure 2 . 1 0.. Summary of te1-1 a llele P C R a n d Southern a na lysis. 87. F i g u re 2 . 1 1 .. Summary of te1-ems a ll e l e PCR a n d Southern analysis. 88. Fig u re 2.1 2 .. Summary o f te1-mum4 a l le l e PCR a n d S outhern analysis. 89. Figure 2 . 1 3 .. Summary of te1-mum6 a l le l e PCR a n d Southern analysis. 90. Figu re 2 . 1 4.. Summary o f te1-mum7 allele PCR a n d Southern a n a lysis. 91. Fig u re 2 . 1 5 .. Expression l evel s of t e1 a n d i nternode lengths. 95. F i g u re 3 . 1 .. tBLASTn search for Mei2-l i ke gene. 99. Figure 3 . 2 .. Netgene2 graphica l output for pro m oter a n a lysis. 1 03. Figu re 3 . 3.. Alignment of ful l l en gth Mei2-l i ke proteins. 1 09. Fig u re 3 .4 .. Struct ure-based a l i g n ment of RRM3. 1 14. Figure 3 . 5 .. U nique C-term i n a l ahelix o f R R M 3. 1 15. Figure 3 . 6 .. I ntron positions i n Mei2-li ke genes. 1 17. Figure 3 . 7 .. Promoter elements i n te 1 , TEL 1 a n d TEL2. 1 18. Figure 3 . 8 .. Maxim u m parsimony tree of a l l ful l length Mei2-like genes. 1 23. F i g u re 3 . 9.. Maxim u m parsimony tree including partia l Mei2-like. genes and ESTs. 1 24. Figure 4 . 1 .. Vectors constructed for T E 1 protei n expression. 151. Figure 4 . 2 .. SOS-PAG E g e l of affin ity p u rified M B P:TE 1 proteins. 1 54. Figure 4 . 3 .. Western blot of 6 H : T E 1 i n du ctio n from pOJ06 vector. 1 56.

(14) 13. Figu re 4 . 4 .. Coomassie stai ned S OS-PAG E gel of affin ity purified. 6 H : T E 1 protei n. 1 57. Figure 4 . 5 .. T E 1 expression from the pSBET vector. 1 58. Figure 4 . 6 .. S O S-PAGE gel of Pichia i nd uctions. 1 60. Figure 4 . 7 .. Southern blot of transgenic Pichia lines. 1 62. Figure 4 . 8.. E L l SA data from raw sera. 1 64. Figure 4 . 9 .. Western blot, pre-immune sera B activity against M B P. 1 66. Western blot showing Sera A activity against 2 both portio n s of M B P : T E 1 1 - 86 Figure 4 . 1 0 .. Figure 4 . 1 1 .. 1 66. Western blot showin g n on-specific activity of affin ity. p u rified a ntibody F i g u re 4. 1 2.. The d istribution of rare codo n s i n the fe 1 reading frame. Figu re 5 . 1 .. Vectors constructed for expression of fe 1 a n d. TEL 2 i n A rabidopsis. 1 68 1 71. 1 76. F i gu re 5.2 .. S outhe rn blot of tra nsgeneic A rabidopsis l ines. 1 85. F i g u re 5 . 3 .. N orthern blot of transgeneic A rabidopsis l i nes. 1 87. S.4a.. Wrin kled and normal Arabidopsis siblings i n pot. 191. S.4b.. Wrin kled l eaf phenotype i n te 1 transgenic l i ne. 1 92. S.4c.. Wrin kled l eaf phenotype i n NLS:fe 1 transgenic l i ne. 1 93. F i g u re 5 . 5 .. Northern b l ot showing fe 1 expression i n i nd ividual wri n kled. vs. normal s i b l ings F i g u re 5 . 6 .. 1 94. Northern b l ot showing te 1 expression i n pools of wrin kled. vs. normal s i b l i ng s. 1 95. F i g u re 5 . 7 .. 1 98. The aerial rosette phenotype.

(15) 14. A B B REVIAT I O N S 2-ME Amp BCI P C efo OEPC DIG OMPC. 2-mercaptoethanol (�-mercaptoethanol) ampici l l i n (su perscript, concentrati o n i n �lg/ml) x-phosphate/5-bromo-chloro-indolyl-phosphate Cefotaxi me (superscript, concentration in �g/ml ) diethyl pyrocarbonate Oigoxigeni n d i m ethylpyrocarbonate. OMSO dNTPs OTT E OTA EMS. d imethylsulphoxide deoxyri bonu cleotide triphosphates dithiothreitol disodium ethy lenediaminetetra-acetate ethyl m ethane s ulfon ate. GA3 Gent I PTG Kan LB N a OAc N BT PBS PCR PCR pNPP polyA+ rpm RRM RT-PCR SAM SOS S OS-PAG E Spec SSC. G i bere l l i c acid 3 gentamycin (superscript, concentratio n i n �g/ml) I s o p ro pylthio-/3-0-galactaside kanamycin (superscript, concentration i n �lg/ml) Luria Bertan n i med i u m sodi u m acetate 4 N itroblue tetrazol i u m chlo ride phosphate buffered saline Polym erase chain reactio n polymerase chain reactio n p-Nitrophenyl phosp h ate d isod i u m Polyadenylated RNA rev o l utions per m in ute RNA recogn ition motif Reverse tran scri ption-po lymerase chain reaction shoot a pical m eristem s sod i u m dodecyl sulfate SOS polyacrylaminde gel e lectro p h o resis spectinomycin (superscript, conce ntration i n �g/ml ) sodi u m chloride, sod i u m citrate 20 x SSC is 3 M N a C I , 0 . 3 M s od i u m citrate pH 7 . 0 Tri s (hydroxym ethyl ) a m i nomethane m u ltiples of g ravitati o n a l force. Tris xg.

(16) 15. TABLE OF GEN ES A consistent nomenclature is used throughout this thesis to describe g e n es and p roteins.. G e n es n ames a re a lways ital i cised, protei ns a lways l isted i n block capitals with suffixes d escrib i n g the amino acids incl uded for partial peptides. e . g . the portio n of the te 1 gene 2 was expressed to produce the peptide TE1 1 - 86 G e n e symbol A CE 1 A ML 1, A ML2, A ML3, A ML4, A ML5 ANT CDC2a CLV 1, CLV2, CLV3 C UC 1, C UC2 FIL gn 1 GR kn 1 MBP Mei2 MG0 1, MG02 nptll OSH 1 S TM T7 1 0 te1. TEL 1 , TEL2 UFO WUS YAB 3. Gene meta l l othionein reg u l atory p rote i n Arabidopsis Mei2-li ke. Species Saccharomyces cere visiae Arabidopsis thaliana. AINTEGUMENTA. Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Arabidopsis tha/iana Arabidopsis tha/iana Zea mays mammalian Zea mays E. coli Schizosaccharomyces pombe Arabidopsis thaliana. cel l d ivisio n cycle 2a CLAVA TA CUPSHAPED CO TYLEDONS 1, 2 FILAMENTOUS FLO WER Gnarly g lucocorticoid receptor knotted 1 maltose b i nding protei n Meiosis deficient 2 MGOUN neomycin phoshotransferase Oryza sativa homeobox1 SHOO TMERIS TEMLESS phage T 7 p rotein 1 0 teminal ear 1 terminal ear1-like UNUSUAL FLORAL ORGANS WUSCHEL YABBY3. synthetic Oryza sativa Arabidopsis thaliana phage T7 Zea mays Arabidopsis tha/iana Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana.

(17) Ch a pter 1 . Introduction. C HA PT E R 1: I N T ROD U CTION. 1 . 1 A B S T RACT The terminal ear1 mutant phenotype is characterised by abnormal pos iti o n i n g of l eaf pri mordia in the vegetative shoot meristem of maize (Veit 1 998), i n dicating that the te 1 genes activity is a reg u l ator of the positioning of organ primordia i n the shoot. apex. Analysis of the fu nction of this gene may therefore bring a g reater u n derstanding of the mechanisms that determ i n e phyllotaxy . A genera l i ntroduction to d ev el o p mental b i ology i s presented with s pecific reference to plants, fol lowed by a d iscUs s i o n of some i mportant aspects plant development that relate to this thesis. S hoot meri stems are described with a specific focus o n the e lements that allow the meristem s to b a l a n ce self p ro pagatio n with l ateral differentiatio n . Research i nto the phenomena associated with the positioning and i nitiatio n of l ateral pri m o rd i a is revi ewed to set the scene for the specific context of the terminal ear1 genes activity. The c urrent u nderstanding of the terminal ear1 genes fun ction in the position i ng of leaf prim o rd i a is described . The te 1 gene belongs to a new family of RNA recognition m otif (RRM) prote i n s that a re present i n a l l eukaryotes and i ncludes the S. pombe gene Mei2. The conserved structures of R R M domains described with a brief i ntro d u ction to the wide vari ety of cell ular fun ctio n s they regu l ate.. 17.

(18) Ch apter 1 . Introduction. 18. 1 . 2 . Deve lopme n ta l b io logy At present, the mechanisms that determ i n e phy l l otaxy are not understoo d . Two p ri nciples of developmental biology that relate d i rectly to this probl e m , a n d warrant d iscussion before proceedi n g ; the positi o n a l i nformati o n model (Wo l pert 1 99 8 ) , a n d t h e competence of a tissue t o respond (McDaniel 1 984).. An i m po rtant aspect of develo pmental biology is that s pati a l patterns of gene expression determi n e form. Wol pe rt ( 1 969) formu l ated a model that described cel l s a s g a i n i n g 'knowled ge' of their positio n v i a gradients o f morphogens. C e l l s i nterpret these gradients as positi o n a l inform atio n , and develop accordingly. Positi o n a l i nformatio n i s particularl y i mportant t o plants, because plants cell s a ppear t o alter identity with respect to position to a much greater degree than do a n imal cel l s . Many aspects of shoot meristems rely o n precise position i nformatio n . P h y l l otaxy i s a clear example, i n that certai n populations o f cell s a lter devel opmental fate depen d i n g entirely o n their positi o n .. It i s k n ow n that cell s w i l l n ot a lways respond t o positional i nformation o r m a y respond to the same informati o n i n a d ifferent manner. P l a nt phytohormones, for example, have a l a rge variety of effects on p l a nt growth and development. The contin ued polar transport of auxin i n d u ces g radual d iffere ntiatio n of cortical cell s i nto d i fferent vascul a r tissues (Sachs 1 984) . It appears that a constant signal can g ive rise to further refinement of cell identity. The difference is not in the sign a l , but in the i nterpretatio n of the sig n a l . O n the molecular leve l , this is l i kely to be due to the expression of different receptor molecules, o r d ifferent branch points i n signal transduction cascades. Competence to respond to position a l info rmation is particularly i mportant to the i nitiatio n of l ateral organs i n the shoot meri stems. Whi l e i n t h e central zon e cells m a y receive positi o n a l i nformation b u t they a re not competent to respon d . It appears that as cell s become displaced from the central zone, they l oose some of their i ndetermi nacy , and become able to d iffe rentiate.. 1 . 3 . T h ree i m p o rta n t as pects of p l a n t d eve lopment.

(19) C h a pter 1 . Introduction. 19. 1 . 3a. The control of cell division P l a nt cel l s d o not migrate, and cel l death i s a rare occurrence that u s u a l ly does not affect primary formative development, so the regu lati o n of cel l d ivision would seem to be crucial for the develop ment of plant form . There are three parameters of cel l division that c o u l d a ffe ct de ve lo p me n t significantly;. the frequency. o f division, the. p l a ne of d ivis i o n , a n d the plane and extent of cel l enlargement (elo ngati o n, widening etc. ) .. Alterations i n the overal l frequency o f cel l d ivision do not appear to a lter the tissues o r o rgan isation of tissues, as evidenced by the overexpression of a dominant negative A rabidopsis C DC2a gene i n tobacco ( Hemerly 1 99 8 ) . The resu lting p l a nts had fewer cel l s than norma l , but were otherwise wel l formed . H owever, l o ss of control over the frequency of cel l d ivision in one particular tissue has d ra stic consequences for development, particularly the cel l d ivision of the s h o ot meriste m . M utati o n s i n e ither CLAVA TA 1 or CLA VA TA3 genes result i n a meristem that proliferates beyond n ormal l imits, while shootmeristemless (stm) m utants fai l to proliferate, with significant affects u po n meristem fun ction (Long 1 99 6 , C lark 1 995) . A IN TE GUMENTA appears to promote division s pecifically i n the cel l s of primord i a ,. a n d ectopic overexpression of the gene res ults i n enlarged mature o rg a n s derived fro m the shoot and the root that are normal i n morphology ( Mizukami 2000) , i n dicating that the rel ative control over cel l division rate is i m portant i n mature tis sues also.. The plane of cel l d ivision seem s to be much less important. M utations in the m aize tang/ed1 gene have irregu l a r p lanes of division i n the longitudinal o rientatio n , but. n o rm a l d ivisions i n the tran sverse p l a ne (Smith 1 996). M utant leaves g row s l owly, b ut a re a n o rmal s h a pe. The m utant phenotype of tangled1 s uggests that p l a nes of d ivision are regu lated d ifferently i n transverse vs. longitu d i n a l d i men s i o n . The a nalysis of m utants of ANGUS TlFOLlA and RO TUND/FOLIA in Arabidopsis i n d i cates that cel l expansion may also be regu lated. i ndependently in d ifferent axes (Tsuge. 1 996) . Mosaic a nalysis of the d o m in a nt Gnare/y- 1 R class I homeobox gene also s uggests that there a re d i stinct pathways for communi catio n between the l ateral and transverse d imension (Foster 1 99 9 ) .. Cell enlargement appears t o b e m o re i m portant to esta b l is h i ng the size o f plant organs. ANGUS TIFOLlA m utant leaves are n a rrower than norm a l , while RO TUND/FOLIA m utants are shorter than n o rm al leaves (Tsuge 1 996). Whi le.

(20) Chapte r 1 . Introduction. 20. n a rrowe r/shorter cell s are the cause, the cell s do not seem to compensate, by d ividi n g more frequ ently to produce g reater n um bers of cell s for exa m pl e .. Whi l e the relative rates o f cell d ivision between d ifferent tissues i s very i mportant for d evel opment, neither the plane of cel l division, the overa l l frequency of d iVisi o n , n o r the extent of c e l l expansion needs to be controlled for the development of pattern . As we s h a l l see in the next sectio n , p la nts can be considered a su prace l l u l a r network of cytop l a s m , so it may not matter exactly where the boundaries between cells are forme d .. 1 . 3b. T h e rol e o f i ntercel l u la r traffic k i n g i n plant development I t is k nown fro m studies of pericli n al chimeras that some genes act n on-cell auto n omously, that they affect clonal l ayers other than those i n which they are expressed (e.g. D ud ley 1 993, Hake 1 99 7 , Foster 1 999 ) . Until recently h owever, the mech a nisms underlying this phenomenon were not well u nderstood. One of the most excit i n g findings i n recen t times has been that macromolecul es such a s m R NAs a n d protei n s are transported . T h i s transport is n ot only l i mited t o specific macro molecules, but also to s pecific domains with i n the plant. Few exam p l es have been studied well to date, but two will serve to i l l u strate some of the pri n ciples that a re begi n n i n g to emerge. The K NOTTED 1 ( KN 1 ) homeobox protei n was fou n d to be present i n both the tunica and corpus of maize shoot a pical meristems (SAMs ) , w h i l e knotted1 m R NA w a s l imited to the corpus (Jackson 1 994) , i m p l y i n g t h a t the protei n. w a s a b l e t o move between cells. I t was found that t h e KN 1 prote i n i s a b l e t o traffic k n 1 transcri pts between cell s (Lucas 1 995) . RNA transport also occurs over l o n g. d i stances through the phloem . Experiments w ith pumpkin-cucumber g rafts i n dicated that CmNACP mRNA was transported across the g raft through the phloem, and then thro u g h plasmodesmata i nto the sho ot apex ( R uiz-Medrano 1 99 9 ) . Tra nscri pts with a variety of putative fun ct i o n s (defence, cell cycl e reg u l atio n , flora l develo pment ete.) were found to be present i n cucumber phloem, and some of these tra nscripts were restricted from entry into the a pex.. Tra nsport of macromolecules i n the symplasm a ppears to be contro l l e d . U ltrastructura l stud ies of plasmo desmata indicate that they conta i n some regu l a r complexes, and t h a t b oth cytoplasm and endoplasmic recticu l u m traverse j u n ctions (Ehl ers 1 999) . The molecules that a re able to travel through plasmodesmata a re usually restricted to a ' size exclu s i o n l im it' of a bout 1 kO, but overexpression of vira l move m e nt proteins increases t h e size exclusion limit t o. -. 20 kD (Wolf 1 989). For.

(21) Chapter. 1 . introduction. 21. K N 1 to traverse p lasmodesmata , it must i n duce their expansion, a n d the degree to w h i ch this occurs could well be regu lated by specific domains of the protei n . Experiments with i njecting tracer dyes either directly into the shoot a pex o f b i rch ( R i n n e 1 998), or loading dye i nto the Arabidopsis phloem ( G i sel 1 999) have shown that cells are not u nifo rmly connected by fun ctio n a l plasmodesmata , specific domains are formed. These domains have been m ost extensively studied in the shoot a pex, and they provide a n ew u nderstanding of the structure of the SAM (discussed i n S ectio n 1 .4a) . Two aspects o f plasmodesmata fu nctio n m a y control t h e formation of sym p lastic d o m a i n s . Primary and secondary plasmodesmata may have d ifferences i n tra n s port capabilities, a n d primary plasmodesmata are o n ly formed from clonally derived cells (van der Schoot 1 99 9 ) . Secondary plasmodesmata arise post cytokinesis, a n d the frequency of a ppeara nce a ppears to be deve l o pmentally regu l ated accord i ng to tissue fusion and cell elongation events (Lucas 1 993) . Also , plasmodes m ata may b e selectively closed by the activity of g lucan synthase complexes that form callose plugs in the plasmodesmata l u men ( E h le rs 1 99 6 , Luca s 1 993) .. The p i cture that e merges from these i nvestigatio n s is that a s uprace l l u l a r network a l l ows movement of a variety of small molecules, and certain macro m olecules. The d iscussion of the structu re of the shoot apex will show that the connections of plasmodesmata a re both spatial ly and tempora l l y contro lled.. 1 . 3c. Structu res that g ive rise to the plant body Conceptually, plant development can be d ivided i nto three main stages; e m b ryogenesis, the d evelopment of primary plant tissues from shoot and root m eriste m s , a n d the development of secondary tissues from the vascular camb i u m a n d t h e cork camb i u m . Shoot a n d root meristems are usually morphologically d isti n g u ishable i n the heart-shaped embry o (Steeves 1 98 9 ) , but pro b a b ly begin to be defined at the molecular l evel with the onset of WUS CHEL expression at the 1 6 cell stage. This process is relatively complex, and i s the s u bject of considerable study (see Lenhard 1 99 9 a n d Bowman 2000 for reviews). This i ntrodu ction focuses on vegetative development, and the formatio n of the SAM will not be d escribed i n detail . Two i mportant points w i l l suffice 1 ) the lineage o f the m eristem i s esta b l ished by g ra d u a l stages that begi n very early, and 2) by the completion of embryogenesis a fu lly fun cti o n a l SAM is present. The rem a i nder of the d iscussion of plant development wi 1 1 conce ntrate o n a descripti o n of the structure and fun ctio n of vegetative shoot.

(22) C h a pter 1 . Introduction. 22. m e ristems (Sectio n 1 .4), with a particular focus on the i nitiatio n of l ateral o rgans (Sectio n 1 . 5).. 1.4. S h oot m eristems 1 .4a. Structure of shoot meristems Although shoot m eristems d iffer in externa l d imensions and in vari o u s a spects of cel l u l a r morphology between taxa they share a common structura l org a n i satio n (Steeves 1 989). I n fact shoot meristems a re more similar between d ifferent taxa than a re e mb ryos , and a re a better example of o ntogeny recapitulating phylogeny (Sachs 1 98 1 ) .. Two features o f vegetative m eristem s are visible at the l evel o f cel l h i stology: a n exterio r-interior o rg anisatio n (tu nica-corpus) o rganisatio n , a n d a radi a l zonation (central , peripheral a nd rib meristem zones) . The fu nctio n a l nature of both levels of o rg a n i s ation have support from experimental d ata (see below). The tunica is composed of one or two laye rs of cells (depend i n g o n the taxa) that a p pear i n reg u l a r files. O bserv ations o f pericl i n a l chimeras i n d i cates that each l ayer o f t h e tunica (designated L 1 , a n d L2) is clonally derived , a s i s the corpus (L3) (Steeves 1 989). Cell d ivisions i n the tunica l ayers a re therefore restricted. to the anti c l i n a l p l a n e i n. the m eriste m , until the rel axatio n o f d ivision plane control in i ncipient primord i a (Lyn d o n 1 983).. Another cytological l y visible o rgan isation exists i n the rad i a l d imensi o n between the cells i n the center of the meri ste m , and those at the periphery. Cells of the central zone a re l a rger, more vacuolate, and divide less frequently than the peri pheral zone. The model of activity that is d erived from the o bservations of h isto logy a n d cell d ivision rates is that the cell s of the central zone act a s the progenitors of the perip h e ra l zone cells. Parti c u l a rly l a rge cells can sometimes be observed the a pex of the central zone that a re presumed to be a pical i niti a l s , the u ltimate source of the central zone. The expression patterns of genes such a s CLA VA TA 3 , CLA VA TA 1 , WUS CHEL, UNUS UAL FLORA L ORGANS , a n d SHOO TMERIS TEMLESS have. d e l i m ited central zone cell s of Arabidopsis m ore clearly, and indicated that the cytol ogical zones represent functional d ifferences ( reviewed i n Bowman 2000) ..

(23) Chapter 1 , Introduction. 23. I nvesti gatio n s of the flow of low molecular weight dyes through plasmodes m ata h av e a d d e d considerably to our understanding o f t h e cell u lar structure o f the S A M ( R i n n e 1 998, van d e r Schoot 1 999, G i s e l 1 999), Experi m ents with the i nject i o n o f flo rescent dyes i nto the SAM of birch seed l i n g s i n d icated that there were fou r symplastic zones i n the s h o ot a pex (Fig u re 1 , 1 ) ,. P P CZ-T. CZ-C. F i g u re 1 . 1 . S y m p l astic fie l ds i n the S A M . T h i s d i ag ra m shows an i d e a li s e d meriste m , with two primord i a ( P ) , D y e trace r experiments indicate t h a t there are fou r fie l d s of symp l a st c o n n ection withi n the SAM, The d o m a i n s a p p e a r to c o r re s p o n d to central z o n e t u n i ca (CZ-T) , central zo n e c o r p u s ( C Z-C) , p e r i p h e r a l z o n e t u n ica ( PZ-T) a n d p e ri p h e ra l z o n e c o r p u s ( PZ- C ) . T h e p e r i p h e ra l z o n e d o m a i n s a re c o n nected a ro u n d t h e c i rc u mference o f t h e c e n tral z o n e , I n b i rch vegetative seed l i n g s there i s a tra n s i en t c on n ectio n b etwee n the central zone a n d the peri pheral zone (arrow) , Figure adapted from van der Schoot ( 1 999),.

(24) Cha pter 1 . i ntroduction. 24. Wh i l e these zones were not d i rectly related to the central and peri ph eral zones of b i rch, they appear very s i m i lar. The transient connection between the central and peripheral zones seen i n these studies, which may coincide with the start of a plastochro n . i ndicating that the l i mits of symplastic domains a re dynamically regu l ated . The dyna m i c regu lation of sym p l astic domains was also seen in i nvestig ations of Arabidopsis where the dye was loaded via the leaf phloem (Gisel 1 999) . In this study it was shown that the symplastic pathway from t h e phloem was l i m ited to the t u n ica, a n d that the dye uptake decreased pri o r to the o n set of flowerin g .. B ecau se these experi m ents used low molecul a r weight dyes a s tracers they can p res umably flow though any functi o n a l plasmodesmata. These d o m a i n s therefore d e l i m it the trafficking of low molecular wei g h t compounds (such as p hytoh ormones ) i n the cytoplasm, endoplasmic recticulum (ER) l u m e n , or E R membra n e . I ntrace l l u l a r m acrom olecules m a y h a v e further restricted domains depending o n the facto rs requ i red to i n crease the size exclu s i o n l imit.. The overa l l structure of the s hoot m eristem i s dynamic and changes d u ri n g deve l op m ent. Most notably, the entire meristem becomes l a rger a n d l ooses some of its radi a l patte rn i n g with the o nset of floweri n g , concomitant with a change i n phyllotaxy (Steeves 1 989) . Despite the contin u i n g displacement o f central zone cell s i nto t h e peri pheral zone, the central zone i s m a i n tained throughout vegetative deve l opment. The dynamic m a intenance of central a n d peripheral zones is a central theme of the next sectio n ..

(25) C hapter 1 . I n troduction. 2S. 1 .4b. F u n ctions of the shoot m eriste m S hoot m eristems conti n u e org a nogenesis thro ughout the life of the p la nt, g iv i n g plant org a n s develo pmental plasticity i n response to their environment. Altho u g h there are considerab l e complexities to meristem phenome n a , thei r behaviour can b e s u m m a rised w ith the fol l owing l i st o f features: 1 ) indeterminate; the cel ls can continue to divide ' i nd efinitely' 2) Undifferentiated; meri stem cells are org a nised i nto other structures without prior. d e-differentiation 3) Self propagating; population s of central and periphera l zone cell s a re m a inta i n ed a n d dynamically regu lated 4) Generative; cell s beyond the central zone are i n duced to d ifferentiate. Each of these features w i l l be d iscussed in turn, a lthough they a re i nterdependent. 1 ) M eristems are i ndetermi nate C l o n a l a n a lysis h a s i n d i cated that there may be s m a l l g roups of more or less stab l e c e l l s that a re maintained at t h e s u m m it of t h e meristem that g i v e rise t o t h e e ntire a pical port i o n of the plant (Steeves 1 989). Whi l e the i n d ividual cells that a re i n this position may not b e the same throughout the l ife of the plant the p resence of some cell s i n this state i s m a i ntain ed u ntil the center of the apical dome diffe rentiates at floweri n g . S u ch apical i n itials g ive rise to a l arge n u m ber of cell s , and d o not appear to have a l imitatio n o n the n um ber of cel l d ivisi o n s . D ifferentiated cell s , such as those. of l atera l o rg a n s only u n derg o a l i mited n u m be r of d ivisions. The i ndetermi n ate state is a particul a r feature of the centra l zone, a n d a ppears to be p ro m oted by kn 1-li k e h o m eo b ox g e n e s . Misplaced expression of kn 1 i n m aize leaves resu lts i n 'knots' of n ewly dividing cell s that to some extent are dedifferentiated (Jackson 1 994). The overexpression of an Arabidopsis kn 1-like gene KNA T1 in Arabidopsis produced l obed l eaves and ectopic meristems (Chuck 1 996) .. 2) M eristem cells a re n ot d i fferentiated There are two aspects to thi s feature of meriste m s : 1 ) central zone cells a re a b l e to d iffe re ntiate i nto a variety of o rg a n s directly without dedifferentiati o n , 2) but u ntil that time they are restricted from being recruited to l ateral organ primord i a . Although most plant tissues mainta i n ' p luripotentcy' , the a b i lity to regenerate entire plants, they usually m u st lose structure a n d developmentally regress i nto callus before doing s o ..

(26) C h apter 1 . I ntroduction. 26. The cells of the central zone, and their i mmediate derivatives are able to d iffere ntiate dire ctly. T h e Arabidopsis WUSCHEL ( WUS) gene is requ i red to p revent central zone cell s fro m b e i n g recruited i nto organ primordia , the meristems of w u s m utants are consumed i n leaf primordia (Laux 1 996). I nterestingly, WUS is not req u i red for contin ui n g meri stematic g rowth, since primordia i nitiation re-occurs in mutant apices. S in ce A rabidopsis S TM and the maize orth% g KN 1 a re not expresse d in the site of i ncipient organ primordia their meristem-promoting fu n ct io n s appear to be incompatible with d ifferentiati o n . The Arabidopsis CUP-SHAPED CO TYLEDON 1 and CUP-SHA PED COTYLEDON 2 ( CUC 1 and CUC2) and the P etun i a ortholog No Apical Mer/stem (NAM) are possib ly i nvolved in the separatio n of organ primordia. from the m e ristem (Aida 1 999 , Souer 1 996). These genes a re expressed between the s hoot meri stem and lateral o rg a n primordia. eue. or nam m utatio n s resu lt in fused. coty l ed o n s a n d floral organs.. 3 ) P o p u l ations of centra l a n d peri phera l zon e cells dynam ically self reg ul ate the i r n um be rs S u rgical stUdies have indicated even very small portions of dissected SAMs will rege n e rate entire meristems (reviewed i n Steeves 1 989) . An i nteresting factor of these studi es i s that the meristem reo rganises itself into central and peri pheral zones before new org a n s are formed. These s u rg ical studies a re a d ra m atic example of a natural p ro cess, s ince cells o f the central zone are continually bein g ' removed' o n ce they a re recruited i nto org a n s . The CLAVATA1 /CLAVATA3 complex appears to be requ i red for the correct regu l ation of central zone size since mutatio n s in e ither gene res u lt in fasciated meristems (Clark 1 997) . Both genetic a n d biochemical d ata indi cate that these genes act as a complex with C LAVATA3 as a extracellular sign a l , a n d CLAVATA 1 as a t h e receptor (Clark 1 995, Trotochaud 1 999). S i n ce CLA VA TA 3 m R NA i s p resent i n the L 1 central zone, a n d CLA VA TA 1 i n the L 2 a n d L3, this complex i s a n example of co-ord ination between symplastic boundaries. M utations in MGO UN 1 or MGOUN2 (MG 0 1 , MG02) genes also develop a b n o rmally larg e. meristem s , a n d fasciation o f t h e i nflorescence stem (Laufs 1 998) . A comparison o f t h e cells accumulated i n t h e i ndividual m utants showed that mgo2 apices accu m u l ated cells i n the peripheral zone, whilst clv3- 1 apices accumulated cells i n t h e centra l zone (Laufs 1 998) . A s n oted b y Lenhard ( 1 999) , this suggests that cells m u st progress through two 'checkpoints' that alter fate b efore they are able to be recruited i nto l ateral o rg a n s : the progression from i n itial cell to peri pheral cell , which requ i res the CLA VA TA complex, and then a p rogression peripheral cell i nto prim o rd i a , which requ i res the MGOUN g enes..

(27) C h a pter 1 . Introduction. 27. Leaf p ri m o rd i a also affect the maintenance of the meri ste m . When temperature sensitive m utants of the Antirhinum MYB gene phantastica are g rown in the restrictiv e temperature they develop abaxia l ised leaves (ventralised) , and cease to m ai ntain a meri stem (Waites 1 998).. I n Arabidopsis phabuJosa-1d. m utants l eaves are. adaxialised and the meristems are enlarged . These two observations i mply that the proximity to adaxial (do rsal) primordia cel l s p ro motes meri stems, while proximity to abaxial primordia cell s l imits meristems.. 4) C e l ls i m medi ately beyond the centra l zone d ifferentiate The recent derivatives of central cells proliferate once they exit the central zone, and a re then recruited into organ primord i a . The factors that i nfluence the position of o rg a n primordia (phyl l otaxy) are the subject of the next section. I t appears some aspect of the progression out of the central zone thro u g h the peripheral zone i nd uces o rg a n formation in the absence of any othe r sti m u l i . The nature of the l ateral o rgan formed depends on the identity of the meriste m . Apices that have been cultured in vitro. i l l u strate that the sub-apical portion of the plant i s not required for organ. formatio n (Steeves 1 989). Of p a rticular i nterest here a re the recent experiments of Reinhard t et al. (2000) with vegetative tomato meristems that were cultured i n the presence of the auxin transport i n hi b itor N-1 -naphthylphalamic acid ( N PA) . The res u lting a pices g rew as long ' pi ns' with an appa rently normal apical meristem at their s u m m it, and n o l ateral o rgans. Apices could be cultured to the extent that they contai n ed no previous pattern of leaf primord i a o r s u b-apical tissue. Once these a pices were transfe rred to a medi u m lacking N PA, leaf primordia a rose i n a ran do m phy l l otaxy, i n the a bsence of any predetermined pattern , o r sub-apical tissues. The ran d o m phyllotaxy a/ways sta b i lised to the n o rm a l tomato s p i ral pattern, and this observation serves to i l lustrate an i mportant facet of phyllotaxy: the i nfluence o f existing l eaf primordia u p o n t h e positioning o f new primord i a .. 1 .5 . I n itiatio n of latera l o rg a n s 1 .5a. S i m i l a rity o f lateral o rg a n i n itiatio n T h e types o f l ateral organs produced b y shoot meri stems has been d iversi fied d urin g t h e course of evolution. Molecu l a r studies i n dicate that A, B and C group flo ra l identity genes a re necessary for floral organ identity: i n abc triple mutants aI/ l ateral o rg a n s develop i nto l eaf l i ke structures (Bowman 1 99 1 ) . Ferns do not produce flowers , d o not appear to contai n the specific MADS-box genes that a re i nvolved with.

(28) C h a pter 1 . Introduction. 28. flowerin g ( M O n ster 1 997) . It appears that the commitment of a lateral organ to its identity a s a flo ra l o rg a n , rather than a leaf, occurs after the events of i n itiation, s ince flo ra l o rg a n primord i a develop leaf-like organs when shifted back to vegetative i n ductive conditio n s ( Batty 1 984) . Floral org a n s appear to have evolved by a m od i ficati o n of exi sting l eaf i n itiation mechanisms, a n d would therefore b e expected to s h a re some fun d a me ntal mechanisms.. The n ext two sections will be concerned with the i nitiation of l ateral org a n s , firstly a d i scussion of the factors that determine the positions of lateral organ i n itiatio n (Sect i o n 1 . S b ) , a n d then a discussion of t h e phenomena associated with organ i n itiation p e r s e , and some early events i n the process of leaf differentiation (Section 1 . 5c). Although much of the research i n this area has exami ned the i n itiati o n of vegetative leaves, homologous mechanisms are probably i nvolved i n the i n itiation of othe r l ateral o rgans.. 1 .5b. P hy l l otaxy: specification of organ position The precise arrangements of phyllotaxy have i n s p i re d mathematicians to produce models to accou n t for the vari o u s spatial and volume relationships w ithi n the meristem (e. g . Richards 1 95 1 , Jean 1 98 9 ) . An important fin d i n g of these stu d i es is that volume relationships produce m o re consistent models than surface areas (Richard s 1 95 1 ) . The correct positio n i n g of lateral organs i s i ntimately connected with meristem fun ction, particularly the dimensions of the meristem. The maize abphyl1 mutant, for example h a s wider m eristem, and p rodu ces leaves in opposite p a i rs (decussate phyllotaxy) rather than the usual d i sticho u s phyllotaxy of m a ize (Jackson 1 99 9 ) . S i m i larly sho 1, sh02 and sh03 m utants of rice, produce wider and flatter meristems that are highly variable i n shape, a n d develop m alfo rmed l eaves i n a random phyllotaxy (Itoh 2000). Arabidopsis clavata m utan ts develop fasciated meristems and produce many m o re leaves and flowers (Clark 1 995).. S u rgical studies have indi cated that the positio n of a n ew organ i nitial i s i nfl u e n ced by its proximity to older primord i a (Snow 1 93 1 ) , specifi cally, that existing primordia i n h ib i t the i n i tiation of a n ew prim o rd i a in thei r prox i mity . The l ateral i n h ibition of leaf p ri m o rd i a has give n rise to the field theory of pri mord i a positio n i ng (Snow 1 93 1 , Wardlaw 1 949). Presumably, morphogenetic g radi ents mediate the rad i a l positioning of leaf primord i a . The nature of the repressing morphogen i s u n known, although auxin i s a candidate (see below). Mutants that show a ltered phyllotaxy a s the result of e n l a rged meristems probably do so because the rati o of the d iffusion or.

(29) C h apter 1 . I ntroduction. d e g radation of the lateral i n hi b itor is not in accordance with the extra volume of the m e ristem . The transient connection between the central and periphera l symp/astic d o m a i n s observed i n b i rch SAMs that occurs at the start of a plastochron is a clue to some rad i a l communication i n the SAM at this stage (Rinne 1 998) .. M utants of the m aize te 1 also mi5-regulate the positions of leaf primordia, both i n the longitudinal with regard to the d ivergence angle, althou g h the d i mensions of the meri stem appear to be unchanged (Veit 1 998) . The te 1 expression pattern and m utant phenotype will be further d iscussed i n Sectio n 1 .6 .. A t least two p hytohormones have been implicated i n the control o f phyllotaxy . Applications of exogenous auxin i s able to induce o rg a n formatio n a n d result i n changes i n phyllotaxy that rem a i n for several phytomers (Schwa b e 1 97 1 , Meicemheimer 1 98 1 ) . The culture of tomato meristems i n the presence of the a ux i n transport i n h i bitor N PA has shed more l i g ht o n t h e rol e of auxin (Reinha rdt 2000) . Meriste m s that were cultured i n the presence of the i n h i b itor grew as extended l ea fless 'pin-like' structures. The a pp l i cation of exogenous auxin to the summit o f s u c h p i n s n o with n o s u btend i n g l eaves resulted i n leaf i nitiation at a site below the apex in the radial d imensio n , but constant distance from the apex i rrespective o f the q u an tity of auxin applied. S i n ce there were n o s u btend i n g leaves the meristem itself m u st medi ate the longitudi n a l positio n i n g . I t appears that a uxin influences the rad i a l positi o n i n g of primord i a .. G ibbere l l i n s (GA3 ) h ave a l s o b e e n i m p l i cated i n some aspects of meristem o rg a nisatio n . The applicatio n of exogen o u s GA3 to axillary meristems of the ivy Hedera helix. resu lts in a stable revers i o n of phyllotaxy from the adult spiral pattern to. the j uvenile d istichous a rrangement ( Marc 1 99 1 ) . An exami n ation of the apical m eristem by M a rc et al. ( 1 99 1 ) indicated that the GA3 treatment i ncreased both the radi a l and the l o ng itudi n a l dimensions of the apica l dome. The d i stichous arrangement of leaves formed o n such meristems appears to result from an i ncreased l o ng itudinal d isplacement of meristems from the summit of the apex. The i nvolvement of G i b berell i n s i s also i m p licated by the fin d i n g that over-expressi o n of the rice OSH1 gene in tobacco results in red uced levels of g ibberelin (Kusaba 1 99 8 ) .. 29.

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