| Other Abstract |
Floral symmetry is a characteristic feature of floral structure and diversity among
angiosperm. There are three main types of floral symmetry including actinomorphy,
zygomorphy and asymmetry. Among these, zygomorphy is considered the more
specialized form. Advances in molecular evolution of zygomorphy have shown the
key regulatory role of CYC-like gene, TCP gene family members, in determining the
development of floral zygomorphy. Studies found that CYC genes from Lotus
japonicus and Pisum sativum are essential for the establishment of dorsoventral (DV)
development in legume plants.
In this study, we isolated and identified MtCYC1 and MtCYC2 genes mutants with
Tnt1 insertion in M. truncatula. The MtCYC2 mutant plants exhibited the deficiency in
dorsal petals development (Lob Standard 1, ls1). The genetic analysis showed the
defect phenotype of ls1 was controlled by one recessive allele. In contrast, the mutant
of MtCYC1 gene did not give rise to a detectable phenotype in flower, similar to wild
plants (Normal Petals, npe). Intriguingly, the double mutant npe/ls1 results in strong
phenotypic changes such as more strong developmental deficiency in dorsal petals
and loss of internal asymmetry that was similar to internal asymmetrical lateral petals
in floral shape and size, ultimately resulting in the loss of whole floral symmetry.
Gene expression analysis showed that MtCYC1 and MtCYC2 have a similar
expression pattern, highly transcripted in dorsal petals. These results suggested that
MtCYC1 and MtCYC2 play a critical role in the control of dorsal petals development
in M. truncatula, particularly MtCYC2, and have partially functional redundancy.
Besides, we also isolated an additional CYC gene, MtCYC3, which might play a role
in lateral petals development. The MtCYC3 gene showed the increased expression
level in the dorsal petals of double mutant npe/ls1 compared with the wild type. This
implied that MtCYC3 may be repressed by MtCYC2 and MtCYC1 in the dorsal petals.
Subsequently, we further dissected the regulatory mechanism underlying the
molecular basis of MtCYC1 and MtCYC2 in dorsal petals in M. truncatula. We
initially investigated the known TCP gene family binding sites in the promoter region
of MtCYC2 and found the presence of GGGCCCT sequence for TCP protein binding,
implying that the MtCYC2 gene may be regulated by itself or MtCYC1 protein. Yeast
one-hybrid were carried out and the result showed that either MtCYC1 or MtCYC2
can recognize the GGGCCCT sequence from MtCYC2 promoter region. In order to
confirm this fact in vivo, we constructed a effector-reporter transient expression assay
in tobacco. The promoter sequence of MtCYC2 gene fused with luciferase gene were
considered as reporter, and coding sequences of MtCYC1 or MtCYC2 with CaMV35S
promoter were designed as effector. Co-injection of the reporter and effector in
tobacco mature leaves found that MtCYC1 and MtCYC2 protein may be negative
regulators. In addition, as previously reported in other plants, our finding showed that
MtCYC1 and MtCYC2 can form homodimer or heterodimer by protein interaction
that was confirmed by yeast two-hybrid and bimolecular fluorescence
complementation (BiFC) experiment. Besides, the MtCYC1 and MtCYC2 protein
were found to locate in cell nucleus. These results suggested MtCYC1 and MtCYC2
protein exert their function in cell nucleus by homo- or hetero-dimer protein complex.
Additionally, we identified a novel mutant in M. truncatula mutant library, in which
all five petals of the mutant were changed into dorsal petals, called dsp1 (Dorsalized
Petal 1). Compared with wild type, this mutant displayed some distinct features such
as stigma and stamens erection, scattered anthers unable to enclose the stigma. The
genetic analysis showed that the phenotype of dsp1 was controlled by one dominant
allele. Combined with the phenotype characterization of npe and ls1, we proposed a
hypothesis that DSP1 might be a key negative regulator for dorsal petals development
via repressing the MtCYC1 and MtCYC2. To test it, we crossed the dsp1 and ls1, and
obtained the double mutant dsp1/ls1. The dsp1/ls1 mutant had a quite similar floral
phenotype to dsp1, strongly implying that DSP1 may be a upstream regulator of
MtCYC2. The expression levels of MtCYC genes were performed in different mutants
and wild type by quantitative RT-PCR. The results showed that the expression levels
of both MtCYC1 and MtCYC2 in flower bud were significantly up-regulated in dsp1
mutant, compared with that in wild plant. Consistent with in situ hybird results,
MtCYC1 and MtCYC2 just expressed in dorsal petal of wild type, whereas they
ABSTRACT
VI
ectopic expressed in all five petals in dsp1. The expression of MtCYC3 gene in dsp1
mutant reduced to the half of that in wild plant. In dsp1/ls1, MtCYC1 was
up-regulated compared with wild plant, while MtCYC3 was similar to its expression
in dsp1. These observations suggested that DSP1 also play essential role in dorsal
petals determination by repressing the MtCYC1 and MtCYC2 in early flower
development except for dorsal petals.
The surface wax covers the various tissues and organs of terrestrial plant primary
aerial, which serves as an important defense barrier and plays important roles in a
variety of abiotic and biotic stresses. Plant cuticular waxes are complex mixture and
primarily comprised of long-chain fatty acids that were derived from 16- or 18-
carbon fatty acids in endoplasmic reticulum. The key genes involved into biosynthesis
pathway of long-chain fatty acids and wax formation were well-characterized in
Arabidopsis, but the key factors and molecular mechanism in M. truncatula remain
unknown. In our work, we screened the M. truncatula Tnt1-insertion mutant library
and identified a novel mutant with one recessive allele, called wfl1 (winkled flower
and leaf 1). The mutant had a strongly phenotypic defect, such as extremely fused and
wrinkled leaves, wrinkled petals that are unable to open, exposed pistil and infertility.
Combined linkage analysis and reverse genetic screen, we finally identified that the
target gene can encode a 3-ketoacyl-CoA synthase (KCS), which catalyzed initiation
step in long-chain fatty acids extension.
In this study, we primarily characterized the phenotype of wfl1 mutant, identified
the candidate gene and analyzed its function. The morphology of leaf cuticular waxes
of wfl1 mutant and wild plants was characterized by using scanning electron
microscope (SEM). The result showed that wfl1 leaves exhibited a more short and thin
crystal wax structure. Toluidine blue test performed on plants showed that wfl1 was
more easily to be dyed, indicating the reduction of cuticular waxes in wfl1 leaves that
would resulted in the increase of permeability of leaf epidermis. Genetic analysis for
selfing offspring of wfl1 heterozygote showed the numbers of the mutant and wild
type plants was followed 3:1 segregation ratio, suggesting that the wfl1 was controlled
by one recessive allele. Based on the protein sequence analysis, WFL1 gene encodes a
3-ketoacyl-CoA synthase, belonging to the KCS gene family.Based on the extensive
screen, we found 28 KCS genes in Medicago truncatula genome. Phylogenetic
analysis showed that these genes can be distinctly divided into four subfamilies:
FAE1-like, FDH-like, CER6 and KCS1-like. The WFL1 gene was homologous to the
FDH gene (AT2G26250) in Arabidopsis. The wfl1 mutant exhibited a similarly
phenotypic deficiency to fdh mutant. The result of quantitative RT-PCR showed the
WFL1 gene displayed a constitutive expression. High expression levels were found in
vegetative and reproductive shoots, stigma and pistil, followed by stem, leaf and
sepals. The temporal and spatial expression patterns were further confirmed by RNA
in situ hybridization. Transcripts of wfl1 gene were detected in outer cells of early leaf
primordium. At early developmental stage of floral primordium, the WFL1 can be
detected in the floral organs including sepals, petals, stigma and stamens. WFL1 was
highly transcripted in the stigma and ovule through the whole developmental stages,
but sharply reduced in the mature stamen. Analysis of methyl esters prepared from
total fatty acids of leaves showed that the content of fatty acid (FA) was significantly
decreased in the wfl1 plants. Compared to WT, the FA profiles were altered by the
decrease of linolenic acid (18C:3) proportions and dramatic increase of
very-long-chain FAs (>18C) proportions. The unsaturated FAs to saturated FAs ratio
(US/S) showed a significant reduction in wfl1.
Keywords: Flower development, Dorsoventral, TCP gene, KCS gene, Medicago
truncatula |
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