Disruption of NTPHAN function in antisense transgenics produces

MYB Proteins Are: Transcription Factors Found in Eukaryotic Organisms A Small Gene Family in

Sequence Homology: MYB Domain Nicotiana: MRERQRWRSEEDALLRAYVKQYGPKEWHLVSQRMNTALNRDAKSCLERWKNYLKP MKERQRWRPEEDALLRAYVKEYGPRDWHLVTQRMNKPLNRDAKSCLERWKNYLKP Arabidopsis: MKERQRWSGEEDALLRAYVRQFGPREWHLVSERMNKPLNRDAKSCLERWKNYLKP Antirrhinum: Nicotiana: RIKKGSLTQEEQRSVIHLQAKHGNKWKKIAAEVPGRTAKRLGKWWEVFKEKQHRE GIKKESLTQEEQISVINLQAKHGNKWKKIAAEVPGRTAKRLGKWWEVFKEKKHRE Arabidopsis:

Phantastica. . . • Is a MYB Protein • Is involved in leaf dorsoventrality

p. FF 19 4. 2 kb Bam. H 1 Xba 1 Sal 1 Pst

Antisense NTPHAN leaves are abnormal, but maintain normal dorsoventrality. • Juvenile leaves produce ectopic

In wild-type plants, cauline leaves become progressively smaller and the petiole becomes shorter. In

Does NTPHAN regulate KNOX genes? ELK homeo Helix 1 Helix 2 turn Helix 3

RT-PCR = Reverse Transcription Polymerase Chain Reaction • RNA is extracted from plant tissue

RT-PCR Analysis of NTH Expression PHAN and NTH 23 are controls for comparing “loading”

Conclusions: • PHAN is expressed at all stages of leaf development. • Loss of

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Disruption of NTPHAN function in antisense transgenics produces ectopic leaf blades and hyperelongation of the leaf petiole in Nicotiana sylvestris Neil A. Mc. Hale 1, Ross Koning 2, and Hsu-Liang Hsieh 1. 1 The Connecticut Agricultural Experiment Station, New Haven, CT 06504 2 Eastern Connecticut State University, Willimantic, CT 06226

MYB Proteins Are: Transcription Factors Found in Eukaryotic Organisms A Small Gene Family in Mammals A Large Gene Family in Plants Have 3 Repeat Domains in Mammals: R 1 R 2 R 3 Have 2 Repeat Domains in Plants…including Phantastica: R 2 R 3 C terminus

Sequence Homology: MYB Domain Nicotiana: MRERQRWRSEEDALLRAYVKQYGPKEWHLVSQRMNTALNRDAKSCLERWKNYLKP MKERQRWRPEEDALLRAYVKEYGPRDWHLVTQRMNKPLNRDAKSCLERWKNYLKP Arabidopsis: MKERQRWSGEEDALLRAYVRQFGPREWHLVSERMNKPLNRDAKSCLERWKNYLKP Antirrhinum: Nicotiana: RIKKGSLTQEEQRSVIHLQAKHGNKWKKIAAEVPGRTAKRLGKWWEVFKEKQHRE GIKKESLTQEEQISVINLQAKHGNKWKKIAAEVPGRTAKRLGKWWEVFKEKKHRE Arabidopsis: GIKKGSLTEEEQRSVIRLQEKHGNKWKKIAAEVPGRTAKRLGKWWEVFKEKQHRE Antirrhinum: C terminus Nicotiana: PFVTELVECCKELDEGHRAWAAHKKEAAWRLRRVELQLESEKICKVREKMEEIEA HIVPELLECCKELEEGQRAWAAHRKEAAWRLKRVELQLESEKACRRREKMEEIEA Arabidopsis: VFLSELVECCKELEEGHRAWADHKKEAAWRLRRVELQLESEKTCRQREKMEEIEA Antirrhinum: Nicotiana: KMKALREEQKATLDRIEAEYKEQLAGLRRDAEAKEQKLAEQWASKHLRLSKFLEQ KMKALREEQKASLDRIEAEYREQLAGLRREAEVKEQKLAEQWAAKHLRLTKFLEQ Arabidopsis: KMKALREEQKNAMEKIEAEYREQLVGLRRDAEAKDQKLADQWTSKHIRLTKFLEQ Antirrhinum: The PHAN genes of three species have two conserved domains

Phantastica. . . • Is a MYB Protein • Is involved in leaf dorsoventrality • Regulates shoot apical meristem function • Produces bladeless leaves in Antirrhinum • Produces lobed leaves in Arabidopsis We cloned the orthologous gene (NTPHAN) and generated antisense transgenics in Nicotiana sylvestris

p. FF 19 4. 2 kb Bam. H 1 Xba 1 Sal 1 Pst 1 Sph 1 Nsi 1 35 S poly. A (203 bp) Sst 1 Nru 1 35 S prom (462 bp) Eco. R 1 NTPHAN Bam. H 1 35 S enh (329 bp) Sst 1 The coding sequence of NTPHAN with its 5’ UTR was cloned as a Bam/Sst fragment in antisense orientation to the 35 S Ca. MV promoter in p. FF 19. Hind III Eco. R 1 Sac 1 Kpn 1 Sma 1 Bam. H 1 Xba 1 Sal 1 Pst 1 Hind. III (1092)………………. MYB…………. (-348) in amyc Gent LB Spec p. PZP 221 RB The Hind. III/Eco. R 1 fragment from p. FF 19 was inserted into the multiple cloning site of p. PZP 221, and introduced into Agrobacterium for leaf disc transformation of WT Nicotiana sylvestris.

Antisense NTPHAN leaves are abnormal, but maintain normal dorsoventrality. • Juvenile leaves produce ectopic leaf blades on the dorsal surface on both sides of the midrib. • These ectopic blades become vascularized and have normal leaf anatomy. • The dorsal surface always faces outward toward the leaf margins. • Adult leaves have an elongated petiole/midrib similar to PHAN mutants in Antirrhinum. • The vascular system is cylindrical but a dorsal rib is present in a normal position. • The blades of adult leaves are reduced in area, become peltate, and ultimately bladeless on cauline leaves.

In wild-type plants, cauline leaves become progressively smaller and the petiole becomes shorter. In antisense NTPHAN plants, cauline leaves remain long and the petiole elongates as blade is reduced…sometimes disappearing!

Does NTPHAN regulate KNOX genes? ELK homeo Helix 1 Helix 2 turn Helix 3 Gene expression studies in maize and Arabidopsis suggest that PHAN functions as a negative transcriptional regulator of class I KNOX genes. Maize RSI and Arabidopsis KNATI are expressed ectopically in developing leaves. Does loss of NTPHAN produce ectopic expression of genes in the NTH (Nicotiana tabacum homeobox) genes? We are examining expression of NTH 15 and NTH 20, the KNOX genes most similar to Arabidopsis STM and KNAT 1, respectively.

RT-PCR = Reverse Transcription Polymerase Chain Reaction • RNA is extracted from plant tissue of specific age • Reverse transcriptase, d. NTPs, buffer are added • Incubated to produce DNA from RNA • Custom oligonucleotide primers for specific gene added • Taq DNA polymerase, d. NTPs, buffer are added • Thermal Cycling used to amplify specific gene • Electrophoresis with ethidium bromide staining

RT-PCR Analysis of NTH Expression PHAN and NTH 23 are controls for comparing “loading” factors Juvenile leaves express NTH 15 and 20 Young leaves stop producing NTH 15 Young antisense PHAN leaves continue to produce NTH 20 while young WT leaves stop. Maturing leaves stop producing both NTH 15 and NTH 20 Even in antisense PHAN leaves.

Conclusions: • PHAN is expressed at all stages of leaf development. • Loss of PHAN causes ectopic blade formation. • Loss of PHAN correlates with extended NTH 20 expression…but this extension disappears rapidly. • Because ectopic blade development continues through all of leaf development, it is unlikely to be caused directly by ectopic NTH 20 expression.