Presentation on theme: "Disruption of NTPHAN function in antisense transgenics produces ectopic leaf blades and hyperelongation of the leaf petiole in Nicotiana sylvestris Neil."— Presentation transcript:
Disruption of NTPHAN function in antisense transgenics produces ectopic leaf blades and hyperelongation of the leaf petiole in Nicotiana sylvestris Neil A. McHale 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
Transcription Factors Found in Eukaryotic Organisms A Small Gene Family in Mammals A Large Gene Family in Plants Have 3 Repeat Domains in Mammals: Have 2 Repeat Domains in Plants…including Phantastica: R1R2R3 R2R3 C terminus MYB Proteins Are:
Nicotiana: MRERQRWRSEEDALLRAYVKQYGPKEWHLVSQRMNTALNRDAKSCLERWKNYLKP Antirrhinum: MKERQRWRPEEDALLRAYVKEYGPRDWHLVTQRMNKPLNRDAKSCLERWKNYLKP Arabidopsis: MKERQRWSGEEDALLRAYVRQFGPREWHLVSERMNKPLNRDAKSCLERWKNYLKP Nicotiana: RIKKGSLTQEEQRSVIHLQAKHGNKWKKIAAEVPGRTAKRLGKWWEVFKEKQHRE Antirrhinum: GIKKESLTQEEQISVINLQAKHGNKWKKIAAEVPGRTAKRLGKWWEVFKEKKHRE Arabidopsis: GIKKGSLTEEEQRSVIRLQEKHGNKWKKIAAEVPGRTAKRLGKWWEVFKEKQHRE Nicotiana: PFVTELVECCKELDEGHRAWAAHKKEAAWRLRRVELQLESEKICKVREKMEEIEA Antirrhinum: HIVPELLECCKELEEGQRAWAAHRKEAAWRLKRVELQLESEKACRRREKMEEIEA Arabidopsis: VFLSELVECCKELEEGHRAWADHKKEAAWRLRRVELQLESEKTCRQREKMEEIEA Nicotiana: KMKALREEQKATLDRIEAEYKEQLAGLRRDAEAKEQKLAEQWASKHLRLSKFLEQ Antirrhinum: KMKALREEQKASLDRIEAEYREQLAGLRREAEVKEQKLAEQWAAKHLRLTKFLEQ Arabidopsis: KMKALREEQKNAMEKIEAEYREQLVGLRRDAEAKDQKLADQWTSKHIRLTKFLEQ Sequence Homology: MYB Domain C terminus The PHAN genes of three species have two conserved domains
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 Phantastica...
Hind III BamH1 Sst1 BamH1 Sst1 Nru1 Xba1 Sal1 Pst1 Sph1 Nsi1 (1092)……………….MYB………….(-348) NTPHAN EcoR1 pFF19 4.2 kb 35S enh (329 bp) 35S prom (462 bp) 35S polyA (203 bp) The coding sequence of NTPHAN with its 5 UTR was cloned as a Bam/Sst fragment in antisense orientation to the 35S CaMV promoter in pFF19. The HindIII/EcoR1 fragment from pFF19 was inserted into the multiple cloning site of pPZP221, and introduced into Agrobacterium for leaf disc transformation of WT Nicotiana sylvestris. pPZP221 Spec Gentamycin EcoR1 Sac1 Kpn1 Sma1 BamH1 Xba1 Sal1 Pst1 HindIII LB RB
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!
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 KNAT1, respectively. ELK Helix 1 homeo Helix 2 Helix 3 turn Does NTPHAN regulate KNOX genes?
RT-PCR = Reverse Transcription Polymerase Chain Reaction RNA is extracted from plant tissue of specific age Reverse transcriptase, dNTPs, buffer are added Incubated to produce DNA from RNA Custom oligonucleotide primers for specific gene added Taq DNA polymerase, dNTPs, buffer are added Thermal Cycling used to amplify specific gene Electrophoresis with ethidium bromide staining
RT-PCR Analysis of NTH Expression PHAN and NTH23 are controls for comparing loading factors Juvenile leaves express NTH15 and 20 Young leaves stop producing NTH15 Young antisense PHAN leaves continue to produce NTH20 while young WT leaves stop. Maturing leaves stop producing both NTH 15 and NTH 20 Even in antisense PHAN leaves.
PHAN is expressed at all stages of leaf development. Loss of PHAN causes ectopic blade formation. Loss of PHAN correlates with extended NTH20 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 NTH20 expression. Conclusions: