Download Mutation of Rice BC12/GDD1, Which Encodes a Kinesin

The Plant Cell, Vol. 23: 628–640, February 2011
Mutation of Rice BC12/GDD1, Which Encodes a Kinesin-Like
Protein That Binds to a GA Biosynthesis Gene Promoter,
Leads to Dwarfism with Impaired Cell Elongation
Summary: Gibberellin-deficient dwarf, a kinesin-like protein with transcriptional activ
ity of GA biosynthesis gene thereby regulates GA production and mediates stem elo
ngation in Oryza sativa
By
Muralidharan Mani
Virus Lab,
Department of Biological Sciences,
University of Ulsan
A and B the gross morphology of gdd1 Mutant was shorter than wild type plant at the third
-leaf and heading stage
C. spike and grains of gdd1 Mutant were also shorter to that of wild type plants
D. Internode of mutant gdd 1 was also shorter to
that of wild type plants
E. & F. Parenchyma of m
utant gdd 1was also shor
ter to that of wild type pl
ants
G. Gross morphological features were different from wild type over all it is shorter. This p
henomenon was may be due to either GA insensitivity or GA deficiency. So to assess this
phenomenon they test the second-leaf sheath length upon GA induction. Eventually they
find GA induction rescue the mutant phenotype into wild-type
Figure 1. Phenotypic Characterization of the gdd1 Mutant.
A) Seedling phenotype of the gdd1 mutant and wild type (WT). Arrows indicate the second leaf sheath. Bar = 1 cm.
(B) The gdd1 mutant and wild type at 20 d after heading stage. Bar = 10 cm.
(C) Spike and seeds of the gdd1 mutant and wild type. Bottom bar = 3 cm (spike). Top bar = 5 mm (seeds).
(D) Internode lengths of the gdd1 mutant and wild type. The average values were calculated from measurement of at least 20 plants.
(E) Parenchyma cells in the third internode in the gdd1 mutant and wild type. Bars = 50 mm.
(F) Quantitative measurement of the cell length of gdd1 and the wild type (n = 20). Data are mean 6 SD. Asterisks indicate significant difference at
P # 0.01 compared with the wild type by Student’s t test.
(G) Elongation of the second leaf sheath in gdd1 and the wild type in response to GA3. Data are mean 6 SD (n = 25). Asterisks indicate significant
difference at P # 0.01 compared with the wild type by Student’s t test.
A. First step of map-based cloning is to identify a marker tigh
tly linked to gdd 1 gene in a large population. So, they map
ped GDD1 locus between the molecular markers M5909 & M
5744 from 3300 dwarf plants from F2 generation. Because, gd
d1 mutant resulted in dwarf phenotype. Second step is findin
g a BAC clone to which the marker probe hybridizes. They fo
und, it is localized to BAC3. they compared ORF of wt to that
of mutant and found out one putative gene 27 bp deletion,
Os09g02650 (gdd 1 mutant)
This deletion may disrupt the splicing of the premature RNA, an
additional 31 bp deletion in GDD1 transcript was also demonstr
ated by sequencing and matching comparison of the cDNA prod
ucts of gdd 1 and the wild type (2c & 2D), causing a premature
protein of 717 amino acid residues.
A fragment containing the GDD1 promoter region (~2 kb) and the en
tire ORF from the wild type was transformed into the gdd1 mutants b
y Agrobacterium-mediated transformation. 10 in-dependent transgeni
c lines rescued the mutant phenotypes, whereas the control lines with
the empty vector failed to complement the gdd1 mutant. Therefore,
Os09g02650 is the GDD1 gene.
Figure 2. Map-Based Cloning of GDD1 and Complementation Test.
(A) Physical mapping of GDD1. GDD1 was localized on BAC3. Accession numbers of the five BAC clones (B) to (E) Mutation site of the gdd1
mutant.
(B) The 27-bp deletion in the gdd1 DNA sequence (underlined) corresponded to 26 bp in the 19th intron (bold italics) and 1 bp in the 20th
exon (not italic).
(C) The 31-bp deletion in the cDNA (underlined) and the stop codon (asterisk).
(D) and (E) The different sizes of amplified fragments for the wild type (WT) and gdd1 are shown using genomic DNA (D) and cDNA (E).
They found 116 down-regulated and 125 up-regulated genes in the gdd1mutant compared wit
h the
wild type (more than fourfold expression change). Of note, the genes involved in cell wall expa
nsion were significantly altered in expression in the gdd1 mutant. Those encoding xylanase inh
ibitor protein and cellulose synthase (CESA6) were greatly up-regulated.
Using quantitative RT-PCR, they further compared the expression of representative
GA biosynthesis-related genes, such as ent-copalyl diphosphate synthase (CPS), ent
-kaurene synthase (KS), KO2, KAO, GA2ox1, and GA2ox3, as well as GA20ox2/SD1an
d GA3ox2/D18 in gdd1
Interestingly, the expression of KO2 was down-reg
ulated in gdd1.
They have found no significant diffe
rences between the mutant and wil
d type in expression patterns of CP
S, KS, GA2ox1, and GA2ox3.
By contrast, the mutant and the
wild type did not differ in expres
sion of GA3ox2/D18, although b
oth showed negative induced pa
tterns with GA3 treatment.
Figure 3. Mutation of GDD1 Altered GA Metabolic Gene Expression.
(A) Classification of the genes up- or downregulated fourfold or more in gdd1 plants by whole-genome DNA microarray analysis.
(B) Real-time RT-PCR analysis of transcript levels of the eight genes in the GA synthesis pathway. Expression was normalized to that of Actin. Tr
anscript levels from the wild type (WT) were set to 1. Data are means 6 SD (n = 3). Asterisk indicates significant difference at P # 0.05 compare
d with the wild type by Student’s t test.
(C) and (D) Effect of GA3 on the relative expression of GA20ox2 (C) and GA3ox2 (D) in the gdd1 mutant and wild type. Transcript levels from th
Motif analysis revealed a Leu zipper motif, characterized by conserved Leu residu
es in the bZIP transcription factors, located within the amino acid residues 413 to
434 in the GDD1 protein
Alignment analysis revealed that the region at 2121 nucleotides of the KO02 promoter shared a ho
mologous sequence.
To determine whether GDD1 could bind to the KO2 promoter, EMSA was performed using the His f
usion protein (His-GDD1DC701-1035) containing
the Leu zipper domain expressed in E. coli and affinity purified. The nucleotide sequences of the GD
D1 binding element of KO2 (K2) and its mutated form (M1) were used as probes. His-GDD1DC701-1
035 bound to the sequence ACCAACTTGAA (K2) that corresponded to the sequence in the promoter
of KO2 but not bind to the mutated version M1, with ACCAACTTGAA changed to ACTTACTCCAA. Be
sides, the formation of a complex of K2 with His-GDD1DC701-1035 was not inhibited in the presenc
e of an excess amount of M1 sequence or unrelated DNA sequences.
Real-time PCR showed that the fragment “a,” including the binding site in the KO2 pro
moter.
By contrast, the remaining regions from b to g, as well as the negative control, were le
ss amplified in the ChIP assay. Thus, GDD1 binds directly to the promoter of KO2 in viv
o.
Figure 4. GDD1 Has DNA Binding and Transactivation Activity.
(A) Alignment of amino acid sequences of the bZIP domain of tobacco RSG and rice GDD1. Highlighted residues indicate the position of
Leu residues
conserved in the bZIP proteins.
(B) EMSA showing His-GDD1DC701-1035 fusion protein binding to the KO2 promoter. Oligonucleotides containing K2, representing the K
O2 promoter
binding site, or M1, a mutation of K2, were used as the biotin-labeled probes. The K2 sequence is boxed and the mutated bases are highl
ighted in M1.
CK indicates the sonicated extract of E. coli expressing His-GDD1-N without the bZIP domain as a negative control (lane 5). The (+) prese
nce or (-)
D) Schematic representation of the reporter and the effector constructs in t
he transcription activity assay. The reporter consisted of five copies of bindi
ng sites for GAL4 in tandem fused upstream of a firefly luciferase gene (LU
control
C). The effector constructs contained the cauliflower mosaic virus 35S prom
oter, the coding region of the GAL4DB-GDD1 fusion, and the nos polyaden
ylation signal (Nos ter). A translational enhancer sequence, V, from tobacco
mosaic virus was located upstream of the sites of translation initiation.
The activity of luciferase using the pLUC/pGAL4DBGDD1
construct was much higher than that in the negative
control (pLUC/pGAL4DB) and mock treatment.
deletion of amino acids 718 to 1035 on the C terminus, like t
he truncated GDD1 in gdd1, had no transactivation activity
Neither GDD1nN1-717 nor GDD1nN1-849 conferred transac
tivation activity
By contrast, GDD1nN1-370 still had the same activity asGDD
1.
Therefore, the C terminus (371 to 1035) could be indispensable for the transactivation activity, and mutation of the GDD1 pr
otein in the gdd1 plant led a deficiency of its transactivation activity.
Figure 4. GDD1 Has DNA Binding and Transactivation Activity.
(E) Relative luciferase activities in Arabidopsis mesophyll protoplasts after transfection with reporter plasmids and effectors of various constructs.
MOCK1 was a negative control without the reporter and effector. MOCK2 was a negative control with only the pLUC reporter. MOCK3 was a negative
control with only pGAL4DB. pARF5M was a positive control. Data are the mean of three independent experiments. RLU, relative light units.
Length of the seminal root in gdd1 mutant was rescued upon
GA3 induction
Microtubule disorientation was also recu
ed by upon GA3 induction on gdd1 mut
ant cells
Figure 6. Visualization of Cortical MicroTubule Orientation in Cells of the gdd1 Mutant and Wild Type.
(A) Seminal root phenotype of gdd1 mutant can be rescued by GA3. Seeds were germinated in hydroponic culture media for 14 d and then transfered t
o hydroponic culture media containing 1 mM GA3 for 10 d. Bar = 10 mm. WT, wild type.
(B) Elongation of the seminal root in gdd1 and the wild type in response to GA3. Seeds were germinated in hydroponic culture media for 14 d and the
n transfered to hydroponic culture media containing different GA3 concentrations. The lengths of seminal roots were measured 10 d after treatment. Da
ta are means 6 SD (n = 20).
(C) MT orientation patterns in the elongating root cells of the wild type and gdd1. The wild type shows transverse CMTs along the long axis. The gdd1
mutant shows obliquely oriented CMTs and shows transversely oriented CMTs after 1 mM GA3 treatment. Bars = 5 mm.
(D) Frequency of MT orientation patterns in the elongating root cells of the wild type and gdd1 (n ≥30).
Conclusion
They demonstrated that GDD1/BC12, a kinesin-like protein, plays a novel role in directly regulating the expr
ession of the Ient-kaurene oxidase gene in the Gibberellic acid biosynthesis pathway for regulation of Micro
tubule arrangement and cell elongation to modulate development in rice.
BE3 stable clone use Gli1 gene targeting shRNA.
Gli shRNA is knock down Gli1.
Fig.4
mRNA level of Gli target gene(GLI1, PTCH1)
measure via RT-PCR.
“Gli1” transcription level decreased to Gli1-shRNA
compared to parental and control-shRNA.
Showed the same results in “PTCH1”
Cell seeding and cell count after 48h.
Cell number increase 2 times in patental, control
shRNA transfection cell.
But, cell proliferation decrease in Gli1-shRNA
compare to patental, control shRNA cell.
Measuring live cell to use MTT assay.
Observe relative cell proliferation between control and
shRNA transfection cell(Gli1 knock down)
Fig.5
Stable cells, which are transformated Gli1 and S84E,
has higher Gli protein level than BE3.
S84E is ser84 mutated into glutamine.
S84E much higher Gli1 transcriptional activity
among all the stable clones.
mRNA level of Gli target gene also showed the same
result. Measurement though RT-PCR.
Stable cells, which are overexpressing Gli1, has higher
mRNA level of Gli1.