GENETIC ANALYSES OF ROOT SYSTEM DEVELOPMENT IN THE TOMATO CROP MODEL Kelsey Hoth 1 Dr. Maria Ivanchenko 2 Bioresourse Research 1, Department of Botany and Plant Physiology 2, Oregon State University, Corvallis, OR, 97331
UNDERSTANDING ROOT ARCHITECTURE Increase crop yields Increase plant quality Reduce irrigation and fertilization needs Aid in industry and research Micro propagation Transformation
TOMATO AS A CROP MODEL May have less redundancy than Arabidopsis in some genes New genomic resources and tools available Results applicable to other commercially important crops (e.g. pepper, eggplant, potato, and coffee)
ROOT ARCHITECTURE MUTANTS Four tomato mutants exhibiting altered root architecture- ara 1 ara 2 ara 3 diageotropica (dgt) Figure 1- Tomato mutants seedlings grown on agar media
ROOT GROWTH EFFECTORS Auxin Vital plant hormone Involved in many plant processes Available Nutrition Photosynthesis produces sugars Sugar must be transported to roots Auxin Sucrose
HYPOTHESES Alterations in auxin response Lack sufficient nutrient supply from shoot Dgt mutation needs further characterization in cell type-specific effects need to know precise expression pattern of the DGT protein
EXPERIMENTS Test auxin response in adventitious root formation Assess auxin response at tissue level Analyze starch accumulation and export from leaves Analyze nutritional responses Identify cell-type specific expression of dgt gene
ARA 1
JUVENILE ARA 1 Figure 2- Juvenile WT compared to juvenile ara 1 grown on agar media for 13 days
MATURE ARA 1 WT F1 ara 1 Figure 3- Comparison of adventitious root formation on the stem of mature AC (left), F1 heterozygote (center), and ara 1 (right).
HORMONAL RESPONSES IN ARA 1 Seedlings grown on agar media Stems cut in 4 pieces and placed on media containing different concentrations of auxin Cut stems left on media for two weeks Adventitious roots counted
WT ara 1 0 0.01 0.05 0.1 0.5 1.0 Auxin concentration (µm) Figure 4- Adventitious root formation of ara 1 and WT on stems after 2 weeks exposure to auxin
# of roots on stem # of roots on leaf 6 Wild Type ara 1 7 Wild Type ara 1 5 4 3 2 1 6 5 4 3 2 1 0 0 0.01 0.05 0.1 0.5 1 0 0 0.01 0.05 0.1 0.5 1 Auxin Concentration (µm) Figure 5- Nearly normal response in adventitious root formation on the leaves and stems of ara 1 after 2 weeks exposure to auxin
NUTRITIONAL RESPONSES Will removing sucrose from media differentially affect ara 1? Plants grown on optimally nutritional media and media lacking sucrose Roots measured at 7 days old
Wild Type ara 1 No Sucrose 1% Sucrose Figure 6- Root growth on media containing standard sucrose and no sucrose
Lateral Root Length (mm) Root Length (mm) WT, WT(-) vs WT(+): p 0.05 120 ara 1, ara 1(-) vs ara 1(+): p 0.01 100 Figure 7- Root growth (top) and lateral root growth (bottom) on media containing standard sucrose and no sucrose 80 60 40 20 0 25 20 15 10 5 No Sucrose 1% Sucrose WT, WT(-) vs WT(+): p 0.131 ara 1, ara 1(-) vs ara 1(+): p 0.054 0 No Sucrose 1% Sucrose
STARCH ACCUMULATION AND EXPORT FROM LEAVES Main product of photosynthesis in leaves is sucrose Sucrose is converted to starch Starch is converted back to sucrose at night and transported to other organs
17h Dark 2h Light 4h Light Uncovered Control Wild type ara 1 Trail 1 ara 1 Trial 2 Figure 8- Starch accumulation and export throughout the day in ara 1
ARA 1 DISCUSSION Small to no difference in auxin response, not a probable explanation for the mutation Root growth insensitive to nutritional depletion Leaves may not be transporting sucrose Possible mutation in sucrose biosynthesis
ARA 2
JUVENILE ARA 2 Figure 9- Juvenile WT compared to juvenile ara 2 grown on agar media for 13 days
ARA 2 PHENOTYPE A ara 2 WT B ara 2 WT Figure 10- Mature ara 2 grown in soil (A) and comparison of wild type and ara 2 seed (B)
HORMONAL RESPONSES IN ARA 2 Seedlings grown on agar media Stems cut in 4 pieces and placed on media containing different concentrations of auxin Cut stems left on media for two weeks Adventitious roots counted
WT WT ara 2 WT ara 2 0 0.01 0.05 0.1 0.5 1.0 Auxin concentration (µm) Figure 11- Adventitious root formation of ara 2 and WT on leaves and stems after 2 weeks exposure to auxin
# of roots on stem # of roots on leaf 7 6 5 4 3 2 1 0 Wild Type ara 2 0 0.01 0.05 0.1 0.5 1 3 Wild Type ara 2 2.5 2 1.5 1 0.5 0 0 0.01 0.05 0.1 0.5 1 Auxin Concentration (µm) Figure 12- Decreased response in adventitious root formation on leaves and stems of ara 2 after 2 weeks exposure to auxin
AUXIN RESPONSE AT THE TISSUE LEVEL Can ara 2 respond to auxin? Selectively breed ara 2 to incorporate DR5:GUS into it s genome DR5:GUS allows visualization of auxin response at the tissue level
DR5:GUS Plants with the DR5:GUS construct produce the GUS protein in cells where auxin-regulated genes are expressed When incubated with a substrate, DR5:GUS creates a blue stain that remains within the cell Auxin expression can therefore be seen at the tissue level as it will appear blue Auxin Promoters (DR5) GUS Protein Sequence
AUXIN RESPONSE AT THE TISSUE LEVEL Cross homozygous WT plant with the DR5:GUS construct to ara 2 Selectively breed plants to create homozygous WT and ara 2 plants that are also homozygous for DR5:GUS Compare WT and ara2 staining intensity and pattern
AUXIN RESPONSE AT THE TISSUE LEVEL WT and ara 2 seedlings selected Seedling root tips incubated with substrate Plants with strongly stained blue root tips grown to maturity in greenhouse Seed from mature plants used to verify homozygosity WT ara 2
A tip lrp lrp B tip 30µM 100µM Figure 13- Comparable DR5 response between the WT and ara 2 in root tips and a lack of DR5 response in lateral root formation in ara 2
ARA 2 DISCUSSION Exogenous auxin application creates no response Auxin response within the tissue appears functional Further testing needed to determine whether auxin signaling or transport is disrupted
ARA 3
JUVENILE ARA 3 Figure 9- Juvenile WT compared to juvenile ara 3 grown on agar media for 13 days
ARA 3 PHENOTYPE A WT B WT ara 3 ara 3 WT ara 3 Figure 14- Decreased apical dominance (A) and comparison of wild type and ara 3 fruit (B)
ISOLATION OF ROOT MUTATION IN ARA 3 Ara 3 phenotypic characteristics segregating in progeny Ara 3 contains more than one mutation ara 3 WT Fruit segregation in F3 progeny red striped red sl. striped pink red Fruit segregation in F3 progeny red red striped pink Figure 15- Segregation between WT and ara 3 of fruit color and striping
ISOLATION OF ROOT MUTATION IN ARA 3 Results with ara 3 may not be related to the root mutation, experimentation haulted Selective breeding of ara 3 to isolate root mutation new focus Once ara 3 contains only the root mutation, experimentation could continue
ARA 3 DISCUSSION Isolation of ara 3 root mutation not yet successful, further selective breeding needed Pink fruit due to colorless fruit epidermis (Y) gene on chromosome 1 Striping due to green stripe (GS) gene on chromosome 7 Apical dominance largely controlled by auxin, multiple genes could be responsible
DGT
DGT PHENOTYPE Juvenile dgt has no lateral roots Auxin resistant Dgt gene responsible for mutation Viewing dgt gene expression pattern may help understand it s role in root development Figure 16- AC (WT) and dgt grown on agar media at 13 days old
TOMATO TRANSFORMATION (DGT) Transform dgt mutant with tagged DGT protein DGT protein expression identified as red fluorescence Similar to how DR5:GUS works, but no selective breeding Figure 17- Arabidopsis transformed with DGT:mCherry-DGT construct
TOMATO TRANSFORMATION Young stems cut and exposed to Agrobacterium previously transformed with DGT:mCherry-DGT Stems placed on containing hormones Organogenesis forms new stem that may have the DGT:mCherry-DGT construct Figure 18- Cut dgt stems growing on hormone media
TOMATO TRANSFORMATION Newly formed stems cut and placed on different hormone media Stem produces roots and becomes self sustaining Full plants grown in greenhouse Seed from this plant grown and dgt expression pattern viewed Figure 19- Cut dgt stems formed by organogenesis growing on different hormone media
TOMATO TRANSFORMATION Limited success in transformation; only wild type transformed Construct is effective, dgt gene is highly expressed within the root tip Dgt may be difficult to transform; auxin resistant Several crosses performed to selectively breed construct into dgt Figure 19- DGT:mCherry-DGT expression in transformed seedling
SUMMARY Ara 1 may have a mutation effecting sucrose biosynthesis and does not appear to have abnormal auxin sensitivity Ara 2 has an altered auxin response, but auxin expression within the root tip is normal Ara 3 contains multiple mutations and further selective breeding is needed to isolate the root mutation The dgt gene is highly expressed in the root tip and the DGT:mCherry-DGT construct is effective
CONCLUSIONS Ara 1 homozygousity needs to be confirmed, starch accumulation needs more repeat experiments. If starch accumulation results confirmed, sucrose biosynthesis patway and sucrose effects on root development could be better understood
CONCLUSIONS Ara 2 could elucidate genes involved in auxin signaling or transport, further experimentation in auxin response is needed Results could broaden the understanding of how auxin regulates root development
CONCLUSIONS Ara 3 could help illuminate regulation of lateral root initiation Once root gene isolation is successful experimentation can continue Dgt currently being crossed with transformed WT, results could reveal importance of dgt gene Better understanding the dgt gene could also increase understanding of the regulation lateral root initiation
ACKNOWLEDGMENTS Dr. Maria Ivanchenko, Botany and Plant Pathology Dr. Molly Megraw, Botany and Plant Pathology Wanda Crannell, College of Agriculture Administration Dr. John Fowler, Botany and Plant Pathology Oregon State University, Corvallis, Oregon
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