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Transcriptome Analysis for identification of
genes involved in cassava tuberization
Yeetoh Chaweewan
www.iita.org/cms/articlefiles/267-cassava.jpg
The molecular mechanism underlying initiation
and development of the storage root
 To improve the long-term cassava productivity
 Leading to the efficient genetic modification
 Understanding of the regulating factors in
tuberization mechanism in cassava
Specific aims
1.
Determination the initiation time of tuber formation in cassava
2.
Transcriptome profiling of the tuberization process using microarrays
3. Identify candidate genes regulating the cassava root tuberization
process
Determination the storage root initiation time using
anatomical studies
Fibrous root
stem
node
Nodal-derived root
Storage root
-Storage Roots develop from node
(nodal-derived root)
-Storage root connects to stem through junction
structure (peduncle)
-Fibrous root system can be developed from tuber
Morphology and histology of stem and cassava storage roots
Development of secondary
xylem parenchyma
functioning as starch
storage cell
2.5 mm
2.5 mm
1.5 mm
1.5 mm
1.5 mm
1.5 mm
1 mm
Junction structure
Initial point of starch storage cell formation is at the center of the
determined roots (junction structure).
Conclusions
1. The initiation of starch storage cells formation in tuber is
developed at 1 months after transfer into the soil (before the
swelling of storage root was visually detected).
2. The secondary growth of xylem in junction structure is
formed as the starch storage cell initiated from the center to
disperse the xylem vessel around.
3. Node-derived tuberizing roots are structured ab initio for
starch accumulation in their secondary growth of xylem.
Transcriptome profiling of the tuberization process using microarrays
To identify the major biochemical pathways and/or subset of gene involved
in the tuberization process
Dr. James Anderson (2007):
-Isolate cDNA library from the model cultivar 60444 (USDA, Fargo)
4000 cassava cDNAs + 23,000 leafy spurge cDNAs
 Developmental-stage-specific gene expression profiling
- Comparison between 4 developmental stages of tuberization
1.
2.
3.
4.
Before visual tuber formation (2 weeks)
Initiation of tuber formation (1 months)
Filling stage of tuber (2 months)
Late stage of tuber or Maturation (3 months)
Identification of developmental stage-specific gene expression
profiling of cassava tuberization
Developmental stages
Before tuberization
Rooting system
Beginning
Starch filling
TMS 60444
Mature
Putative biological pathway
involved in tuber formation in
different developmental stages
Time
2 weeks
1 months
2 months
3 months
Microarray
analysis
Balance rolling cycle dye swap
4 developmental stages
4 biological replication
(total 16 hybridization events)
Developmental stages of
tuber formation
2 weeks:
1 month:
2 months:
3 months:
2 w
1 m
2 m
Before tuber formation
Initiation of tuber formation
Starch filling of tuber
Maturation of tuber
3 m
*Time count after planting into the soil
Putative regulatory pathway involved in tuberization
Initiation
X
X
X
X
X
Starch filling
Maturation
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Pathway
Jasmonic acid biosynthesis
Systemin signaling
Sucrose degradation
Glycolysis II (plant plastids)
Superpathway of sucrose degradation to pyruvate
Superpathway of starch degradation to pyruvate
Sucrose degradation to ethanol and lactate (anaerobic)
Glycolysis I (plant cytosol)
Suberin biosynthesis
Flavonoid biosynthesis
Superpathway of isoleucine and valine biosynthesis
Superpathway of leucine, valine, and isoleucine biosynthesis
Oxidative ethanol degradation
Fatty acid omega-oxidation
Glyoxylate cycle
Superpathway of pantothenate and coenzyme A biosynthesis
Photosynthesis, light reaction
Leucine biosynthesis
Phospholipases
Photosynthesis
Auxin Signaling
Jasmonic acid biosynthesis pathway is shown to highly express in the initiation stage
of tuber formation together with the sucrose degradation and glycolysis.
Systemin Signaling
Glycolysis II (plant plastids)
Superpathway of starch
degradation to pyruvate
Sucrose degradation
Superpathway of sucrose
degradation to pyruvate
Jasmonic acid biosynthesis
Sucrose degradation to
ethanol and lactate
(anaerobic)
35%
Up-regulated pathway in the initiation stage of tuber formation
30%
number of genes (%)
25%
20%
15%
10%
5%
0%
Number of the gene involved in jasmonate response and sucrose
degradation is highly found in the early stage of tuber formation
Gene expression profiles of the gene involved in
cassava tuberization
Before
tuberization
Tuber
initiation
Starch
filling
Tuber
maturation
allergenic-related protein Pt2L4
[Manihot esculenta]
major latex allergen Hev b 4
[Hevea brasiliensis]
(high homology to patatin-like
phospholipase gene)
Level of expression
max
min
Starch filling and tuber maturation : DV445495 – Allergenic-related protein Pt2L4
Early stage of tuberization : DV127294 – Major latex allergen Hev b 4
Validation of putative upregulated genes (DV445495) in late tuberization stage
EU249994.1 Manihot esculenta allergenicrelated protein mRNA (Pt2L4), complete cds
-Related to secondary growth and storage
root formation in cassava
-A Homologue to Hev b 5 from Rubber tree
514 Cassava root cDNA library
allergenic-related protein Pt2L4 (c1)
r19 Cassava root cDNA library
DV445495
allergenic-related protein mRNA
glutamic acid-rich protein (c54)
cassava4.1_018170m
cassava4.1_017439m
Souza et al.(2002) : Identified the minor cassava root protein genes that have a
parenchyma-specific expression pattern in the storage roots
- A glutamic acid-rich protein (Pt2L4)
Souza et al.(2006) : Report the RNA expression patterns of Mec1, encoding a glutamic
acid-rich protein (Pt2L4)
- Pt2L4 correlated with maturation of the secondary xylem parenchyma in storage roots
Beltran et al. (2010): Characterized Mec1 promoter in cassava
DNA Cis-elements (Motifs) for transcription factors binding sites:
#
DNA sequence
TF
Function
Reference
1
ACGT
-------
Stress response
Yamamoto et al 2007
2
TTGAC
WRKY
SA-Induced
Yamamoto et al 2007
3
CNGTTR
MYB
Binding to Myb
Yamamoto et al 2007
4
TAACAAR
-------
GA-Responsive
Yamamoto et al 2007
5
GATAAG
I-Box
Zinc-Finger (root specific)
Baum et al., 1997
6
ATATT
POX1
Root specific
Karpichev et al., 1996
7
AATAGAAA
STK
Sucrose induced
Sun et al., 2003
8
TATCCA
G-box
Binding to Myb factors
Lu et al., 2002
9
TATAA
TATA
Transcription initiation
-------------
10
Intron
Expressing pattern of this promoter showed in a wide range of tissue types
Expression analysis of DV445495 in cassava
Northern blot
Young tuber
(green house)
L SR FR ST PR
mature tuber
(field)
L SR ST PR
Regulation of Mec1 gene coding for the Pt2L4
glutamic acid rich protein
- No up-regulation in leaf and fibrous root
- Highly up-regulated in storage root at
starch filling stage
- Up-regulation in stem depends on the
developmental stage of tuberization
L- Leaf
SR-Storage root
FR-Fibrous root
ST-Stem
PR- Storage root peel
* DV445495 gene encoding Pt2L4 protein
expresses in stems and tuberized roots
Tissue printing technique to analyze gene expression of Pt2L4
diferent section of storage root comparing with stem
Stem
Sectioning of storage roots
2mm
2mm
2mm
RNA of Pt2L4 protein up-regulated in starchy tissue of storage
root and in some part of stem xylem
Validation of candidate gene regulated initiation of tuberization in cassava
• DV127294 (Leafy spurge cDNA)
• Blast hit : major latex allergen Hev b 4 [Hevea brasiliensis]
Putative Arabidopsis_Orthologue : At1g54030
• 7 paralogues from Cassava genome (http://Phytozome.net)
- contain GDSL-like lipase acylhydrolase
*Previous observation of Patatin-related tuberization
• Beezhold (1998) : a latex allergen (Hev b 7) have a homology to patatin, a plant PLA2
• Souza (2008) : a latex allergen (Hev b 5) have a homology to Pt2L4
(cassava)
(cassava)
(cassava)
(cassava)
(cassava)
Hev b 8
(cassava)
(cassava)
Some genes in cassava contain the same conserve motif as Hev b 4 may
be a gene involve in initiation stage of tuberization
Identify the homolog of patatin-related phopholipase in cassava
Location: scaffold12753
Homology of gene involved tuberization
(latex allergen genes, Pt2L4 and patatin-related tuberization)
100.0
74.1
72.9
45.6
92.1
27.4
NA
25.9
68.4
164.3
160
140
120
100
80
60
Nucleotide Substitutions (x100)
Bootstrap Trials = 1000, seed = 111
40
DV445495
Pt2L4
99.2 Hev b 5
Hev b 7
latex patatin
potato patatin
76.3 Hev b 8.2
97.6 Hev b 8.10
82.4
Hev b 8.8
SC12753
SC07520
97.7
SC03686
69.2
SC05934
67.6
Hev-b-4
SC10504
DV127294
98.2
SC05875
SC06700
SC04175
20
0
• SC12753 is one homologue to DV127294
• SC12753 also is in the group of Pt2L4 and patatin genes that is known to express in tuber
Patatin-like phospholipase may be one regulator in tuberization mechanism in cassava
Conclusions
1. Response to jasmonic acid is found to play an important role in the
initiation of tuber formation
2. Candidate genes involved in tuberization in cassava
1.1. Manihot esculenta allergenic-related protein mRNA (Pt2L4)
1.2. Patatin-like genes (scaffold12753: 32743 – 41921)
- Patatin-like phospholipase : Lipid matabolism
- Armadillo/beta-catenin-like repeat : Protein binding
- Leucine rich repeat
3. Candidate genes are being characterized to determine their role in cassava
tuberization
Acknowledgement
Committee:
Dr. Claude Fauquet (ILTAB, DDPSC)
Dr. Nigel Taylor (ILTAB, DDPSC)
Dr. Bethany Zolman (Biology dept., UMSL)
Dr. Xuemin Wang (DDPSC and Biology dept, UMSL)
Dr. Wendy Olivas (Biology dept, UMSL)
USDA-ARS, Plant Science Research Unit, Fargo
Dr. James V. Anderson
Royal Thai government
Dr. David Horvath
Donald Danforth Plant Science Center
DDPSC
Dr. Howard Berg
Dr. Eliana Gaitan-Solis
Dr. Valentina Carballo Portela
ILTAB members
Cynthia Trembley
Donald Danforth
Plant Science Center