Bioenergy Traits• Yield -• Flowering time• height• leaf size • leaf number • stem diameter• tiller number• seed number• branching• upright growth
• Increased saccharification -• Juvenile leaves• Less lignin• Different linkages• Different sugars
Bioenergy Traits• Yield -• Flowering time indeterminate (Joe Colasanti)• height thick tassel dwarf, knox mutants, GA def• leaf size liguleless, narrow sheath• leaf number terminal ear, td1• stem diameter td1, fea2• tiller number teosinte branched1• seed number ramosa, barren inflorescence, knox• branching ramosa, barren inflorescence, knox• upright growth liguleless
• Increased saccharification -• Juvenile leaves Corngrass• Less lignin brown midrib• Different linkages• Different sugars
Using genetics to tackle the cell wall
• A) screen for mutants that have cell walls with altered saccharificationproperties - identify gene, determine function
• B) transcription factors that regulate growth and differentiation (and thus the making of cell walls), identify the targets that specifically affect cell walls
Streptogyna (2)Oryzoideae (105) - rice
Bambusoideae (970)
Danthonieae (270)Aristidoideae (300)
Chloridoideae (1350) - finger milletArundinoideae (20)
Eriachne (42)
Gynerium (1)Centothecoideae (30)
Micraira (13)
Puelioideae (11)Pharoideae (11)Anomochlooideae (3)
60-70 mya
20-30 mya
Stipeae (400)Brachypodium (16)
Aveneae (1050) - oats
Triti ceae (480) - bar ley, wheat, ryeDiarrhena (4)
Meliceae (130)Nardus + Lygeum (2)
Brachyelytrum (1)
Poeae (1210) - ryegrass, fescue
Paniceae (2240) - common millet, foxtail millet, pearl milletAndropogoneae (1000) - maize, sorghum, sugar cane
}
}Pooideae
Panicoideae
Many biofuel crops are in the grass family, discoveries in one species can easily be transferred
to another
Mutagenize the genome and sort through mutants to find new cell
wall components
Screens for cell wall mutants• A1) brown midrib mutants• Four known loci, bm3 encodes caffeic acid 0-
methyl transferase, bm1 linked to CAD, but could encode a regulator of CAD. 40% increase in of glucose per gram stover after hydrolysis of unpretreated stover (Vermerriset al Crop Science).
• Other bm mutations that are not bm1-4. Map these mutations and see what genes are involved.
• Start with bm and do second screen for enhancer or suppressor
Paul Scott, ARS-USDA, Iowa; Ron Hatfield, ARS-USDA, Wisconsin
Screens for cell wall mutants • A2) Saccarification
mutants• EMS mutants in defined inbred
background • 10 individuals of EMS mutagenized
M2 families are grown. • Family is assayed as pool (with
duplicate samples) and as individuals. Dried leaf tissue is sent to Markus Pauly (Michigan State) for pretreatment, enzyme digestion and analysis
Example of analysis from Pauly, one family in particular stood out (7300-69)
Individuals from family 7300-69 assayed
Family portrait of 7300-69 vs A619
Sibling with different texture of leaves - is this the one with altered cell walls?)
Using genetics to tackle the cell wall
• B) transcription factors that regulate growth and differentiation (and thus the making of cell walls), identify the targets that specifically affect cell walls
• 1) Corngrass• 2) knox genes
Corngrass mutants make many more leaves that have reduced
lignin
Cg WtCg
Cg
Wt
Wt
FT-IR difference spectra of wild-type and Cg1-R freeze dried field samples from blades of leaf 10. The two spectra were subtracted to display differences. Samples were analyzed as KBr pellets on a Perkin Elmer System 2000 FT-IR. Data analysis courtesy of Kay Gregorski(USDA/WRRC).
Corngrassencodes a microRNA that regulates 13 SPL transcription factors. Each of these are decreased in Cg mutants.
Compared gene expression
differences between Cg shoots and normal siblings
In process of obtaining insertions in SPL targets expressed in shoots
Dominant Knotted1 mutantBlade cells have attributes of sheath cells
Dominant mutant has phenotype in leaves due to misexpression in those cells, but gene is normally just expressed in the meristem
Jackson et al., 1994
Recessive knox mutants have increased lignin.
Plants overexpressingknox have decreased lignin
Carried out whole genome microarray analysis using bp1 and control at 2 weeks when no phenotypic difference was visible
Gene # Name Enzyme Function EC # E-value Fold Change
At1g01120 KCS1 Fatty Acid Elongase 3-Ketoacyl-CoA Synthase 1
Wax Biosynthesis 2.3
At1g12780 UDGE Uridine Diphosphate Glucose Epimerase
Cell Wall Biosynthesis 5.1.3.2 Biochemically
tested -1.4
At1g16410 CYP710A2 Cytochrome P450 Lignin Biosynthesis 1.14.13.41 1E-115 1.6
At1g51680 At4CL1 4-Coumarate-CoA Ligase 1
Lignin Biosynthesis 6.2.1.12 Biochemically
tested 1.5
At2g30490 AtC4H Cinnamate 4-monooxygenase
Lignin Biosynthesis 1.14.13.11 Biochemically
tested 1.5
At2g32990 CEL Cellulase Cell Wall Biosynthesis 3.2.1.4 Biochemically
tested 2.1
At2g37040 PAL1 Phenylalanine Ammonia-lyase1
Lignin Biosynthesis 4.3.1.5 Biochemically
tested 1.9
At3g14310 PME3 Pectin Methyl esterase Cell Wall Biosynthesis 3.1.1.11 Biochemically
tested -1.6
At3g21770 AtPRXR9GE Peroxidase Lignin Biosynthesis 1.11.1.7 1E-118 -2.0
At4g23600 TRYT Tyrosine Transaminase Lignin Biosynthesis 2.6.1.5 9E-57 -1.8
At4g23990 AtCSLG1 Cellulose Synthetase Cellulose Biosynthesis 1.6
At4g30270 SEN4 Endo-xyloglucan Transferase
Cell Wall Biosynthesis -1.7
At4g39330 CAD1 Cinnamyl-alcohol Dehydrogenase 1
Lignin Biosynthesis
1.1.1.195
Biochemically tested 1.9
Although there were no phenotypic differences, there were differences in RNA expression
Gene expressed in cambium in stem
• Overexpression of poplar knox gene.
• Thin stems, delayed lignification
• 42% of genes differentially expressed are involved with extracellular matrix functions
Microarray data from maize
What are the genes that are upregulated in Kn1leaves and down-regulated in kn1-e1 tassels?
What are the genes that are down-regulated in Kn1 leaves and up-regulated in kn1-e1 tassels?
Nathalie Bolduc, Wei Huang, Volker Brendel, in progress
Kn1-N : young leaves vs WT siblings (5 biological replicates; 15-20 plants/pool)
kn1-E1 : immature tassels (about 5 mm +/- 2 mm) vs het siblings (5 biological replicates; 5-7 plants/pool)
mRNA amplified, labeled with CY3-CY5 (dye swap among replicates), hybridized, scanned, quantified, analyzed
Data mining and analysis of genomic sequences for the presence of KN1 binding sites in modulated genes
Microarray analysis
transcription factors(9)cell growth andmaintenance (13)cell wall (3)
? (11)
Genes inversely modulated in Kn1-N leaves and kn1-e1 tassels
0.71.2phenylalanine ammonia-lyaselignin synthesis
0.71.4putative receptor-like protein kinasesignal transduction
0.71.9putative MADS-domain transcription factor (m23)transcription
2.30.8Putative EREBP-like protein transcription
1.40.8putative myb protein transcription
1.50.7putative transcription activator RF2a transcription
1.50.7DNA-binding protein RAV2-like (AP2 domain) transcription
1.90.6homeodomain protein-like (HD-Zip)
Transcription
transcription
Kn1-N/wt leaves
kn1-e1/wt tasselannotationcategory
Modulated in KN1-N leaves: about 500 genes
Modulated in kn1-e1 tassels: about 100
Inversely modulated in both experiments: 35
Knotted1 0.4 1.6
myo-inositol1-P synthaseEC 5.5.1.4
EC 5.4.2.2
PhosphoglucoseisomeraseEC 5.3.1.9
Down, p<0.005
Down, p<0.01
Up, p<0.005
Also significantly modulated:
Auxin responsive genes
Cell wall modifications
Expansin
xyloglucan endo-1,4-beta-D-glucanase
beta-D-glucanexohydrolase
Lignin synthesis
phenylalanine ammonia-lyase (PAL)
Cinnamate 4-hydroxylase (C4H)
cinnamyl-alcohol dehydrogenase (CAD)
Modulation of genes involved in biosynthesis of cell wall polysaccharides precursors in Kn1-N leaves
RT-PCR validation
KN1
GA2ox1
GL15
Lg3
Actin
wat
er
wat
er
WT KN1-N WT kn1-E1
leaves tassels
0.89
0.018
3.70
0.006
0.92
0.231
2.34
0.001
0.39
0.168
1.61
0.008
0.41
0.001
1.58
0.056
leaves tassels
pValu
e
pValu
e
Fold
chan
ge
Fold
chan
ge
KN1
GA2ox1
GL15
Lg3
PAL 0.003 1.24 0.001 0.69
Next question, is whether these are direct targets
• Carry out chromatin immunoprecipitation• Use tissue that is highly enriched in KN1
expressed cells• Use KN1 specific antibody
Chu
ck e
t al,
2002
bdT
uE
1/+
E1/
E1
kDa175-62-
47-
33-
25-Ears Shoots
KN1
