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Transgenic lines of Begonia maculata generated by ectopic expression of PttKN1

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KNOX (KNOTTED1-like homeobox) genes encode homeodomain-containing transcription factors which play crucial roles in meristem maintenance and proper patterning of organ initiation. PttKN1 gene, isolated from the vascular cambium of hybrid aspen (Populus tremula × P. tremuloides), is a member of class I KNOX gene family. In order to understand the roles of PttKN1 gene in meristem activity and morphogenesis as well as to explore the possibility to generate novel ornamental lines via its ectopic expression, it was introduced into the genome of Begonia maculata Raddi by Agrobacterium tumefasciens-mediated gene transformation here. Four types of transgenic plants were observed, namely coral-like (CL) type, ectopic foliole (EF) type, phyllotaxy-irregular (IP) type and cup-shaped (CS) type, which were remarkably different from corresponding wild type and were not also observed in the regenerated plantlets of wild type plant. Among these four types of transgenic plants, the phenotype of coral-like was observed for the first time in the transformants ectopically expressed KNOX genes. The observation of scanning electron microscope (SEM) showed ectopic meristems on the adaxial leaf surface of the transformants. Interestingly, the plantlets with ectopic foliole could generate new ectopic folioles from the original ectopic folioles again, and the plants regenerated from the EF-type transformants could also maintain the original morphology. The same specific RT-PCR band of the four types of transgenic plantlets showed that PttKN1 was ectopically expressed. All these data demonstrated that the ectopic expression of PttKN1 caused a series of alterations in morphology which provided possibilities producing novel ornamental lines and that PttKN1 played important roles in meristem initiation, maintenance and organogenesis events as other class I KNOX genes. Key words Begonia maculata –ectopic expression–morphogenesis– KNOX – PttKN1
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Biologia 66/2: 251—257, 2011
Section Botany
DOI: 10.2478/s11756-011-0008-3
Transgenic lines of Begonia maculata generated by ectopic
expression of PttKN1
Quan-le Xu1,2, Jiang-ling Dong1,NanGao1,Mei-yuRuan1, Hai-yan Jia1,LiangZhang1,
& Chong-ying Wang1*
1Institute of Cel l Biology, School of Life Sciences, Lanzhou University, Lanzhou 730000,P.R.China;e-mail:
wangcy@lzu.edu.cn
2College of Life sciences, Northwest A&F University, Yangling 712100,P.R.China
Abstract: KNOX (KNOTTED1-like homeobox) genes encode homeodomain-containing transcription factors which play
crucial roles in meristem maintenance and proper patterning of organ initiation. PttKN1gene, isolated from the vascular
cambium of hybrid aspen (Populus tremula ×P. tremuloides), is a member of class I KNOX gene family. In order to under-
stand the roles of PttKN1gene in meristem activity and morphogenesis as well as to explore the possibility to generate novel
ornamental lines via its ectopic expression, it was introduced into the genome of Begonia maculata Raddi by Agrobacterium
tumefasciens–mediated gene transformation here. Four types of transgenic plants were observed, namely coral-like (CL)
type, ectopic foliole (EF) type, phyllotaxy-irregular (IP) type and cup-shaped (CS) type, which were remarkably different
from corresponding wild type and were not also observed in the regenerated plantlets of wild type plant. Among these
four types of transgenic plants, the phenotype of coral-like was observed for the first time in the transformants ectopically
expressed KNOX genes. The observation of scanning electron microscope (SEM) showed ectopic meristems on the adaxial
leaf surface of the transformants. Interestingly, the plantlets with ectopic foliole could generate new ectopic folioles from the
original ectopic folioles again, and the plants regenerated from the EF-type transformants could also maintain the original
morphology. The same specific RT–PCR band of the four types of transgenic plantlets showed that PttKN1was ectopically
expressed. All these data demonstrated that the ectopic expression of PttKN1caused a series of alterations in morphology
which provided possibilities producing novel ornamental lines and thatPttKN1played important roles in meristem initiation,
maintenance and organogenesis events as other class I KNOX genes.
Key words: Begonia maculata; ectopic expression; morphogenesis; KNOX;PttKN1
Introduction
Plant developmental and morphological traits were
greatly related to the roles and regulations of tran-
scription factors (Peng et al. 1999; Wang et al.1999;
Zhang 2003). Among all transcription factors, KNOT-
TED1(KN1)-like homeobox genes (KNOX) were well
studied since they had been widely identified in mono-
cot and dicot species (Scofield & Murray 2006, Hay
et al. 2009), especially the class I KNOX genes,
such as KNOTTEDI (KN1), SHOOTMERISTEMLESS
(STM), knotted1-like homeobox gene from Arabidopsis
thaliana1(KNAT1), Oryza sativa homeobox 1 (OSH1),
Oryza sativa homeobox 15(OSH15) and Potato home-
obox 1 (POTH1). Their functions in morphogenesis
have been extensively studied by analyzing transgenic
plants. Sinha et al. (1993) found that the leaves of
KN1-transgenic tobacco produced numerous shoots.
Chuck et al. (1996) found that the ectopic expression of
KNAT1was able to transform simple leaves into lobed
leaves in Arabidopsis. In transgenic potato, the overex-
pression of POTH1produced dwarf plants with abnor-
mal leaves (Rosin et al. 2003). These reports confirm
that KNOX gene is a key player in plant morphogen-
esis which derived from the balance of the stem-cells
proliferation and maintenance in SAM.
Most of the KNOX genes that have been exten-
sively studied so far are isolated from annual herbaceous
plants such as maize, Arabidopsis,Oryza sativa and
potato, however, KNOX genes from woody plants (tree)
have been scarcely investigated. A class I KNOX gene
from the palm species Elaeis guineensis was thought
to be associated with meristem function and a distinct
mode of leaf dissection (Jouannic et al. 2007). Later,
Barth et al. (2009) found that overexpressionof KNAP1
(KN1-like class I homologues from apple tree) in Koh-
leria drastically altered the leaf shape. Although there
have been several researches, it is still necessary to ex-
plore whether class I KNOXgenes from woody plants
play the same role as those from herbaceous plants in
morphological morphogenesis.
PttKN1is a class I KNOX gene isolated from
the vascular cambial region of a hybrid aspen (Pop-
ulus tremula×tremuloides). To understand whether the
PttKN1gene from the aspen functions as the class I
KNOX genes from herbaceous plants in meristematic
c
2011 Institute of Botany, Slovak Academy of Sciences
Author's copy
252 Q. Xu et al.
initiation and maintenance, it was transformed into
petunia (Hu et al. 2005) and cockscomb (Meng et al.
2009). The PttKN1transgenic petunia showed altered
phenotypes such as dwarf and bushy, a loss of apical
dominance, lobed leaves and changes of flower colour
(Hu et al. 2005). Similar phenotypes were observed
in PttKN1transgenic cockscomb (Meng et al. 2009).
Those results demonstrated that PttKN1played a sim-
ilar role as the class I KNOX genes from herbaceous
plants in morphogenesis.
In order to develop new ornamental lines and fur-
ther investigate the role of PttKN1in meristem activ-
ity and morphogenesis, PttKN1was introduced into
Begonia maculata, which is one of the best evergreen
perennial cultivars with cane-like stem, bold foliage and
large clusters of red pendulous flowers, and belongs to
the fibrous rooted type of Begonia. Begonia is one of
the most popular ornamental plants grown in gardens,
pots, hanging baskets and greenhouses. Meanwhile, ge-
netic engineering has provided a valuable means of ex-
panding the horticulture gene pool and promoting the
generation of new ornamental varieties. In this study,
we reported for the first time that the ectopic expres-
sion of PttKN1in B. maculata caused the alteration of
leaf shape and plant architecture, including four novel
PttKN1-transgenic lines.
Material and methods
Plant species, bacterial strain and vector
Begonia maculata Raddi, purchased from a garden com-
pany as small plants, were grown in pots filled with mixed
soil containing vermiculite, perlite and peat moss (1:1:1) in
growth chamber with photoperiod of 16 h light of 24 µmol
m2s1. The used bacterial strain and vector were Agrobac-
terium tumefasciens GV3101 and pPCV702, respectively,
supplied friendly by Dr. Olof Olsson (G¨oteberg University,
Sweden). The pPCV702, containing a copy of CaMV 35S
and neomycin phosphotransferase gene (NPT II ), was intro-
duced into A. tumefasciens GV3101 by electroporation. Pt-
tKN1gene was integrated under the downstream of CaMV
35S promoter.
Callus induction and plant regeneration from B. maculata
20-day-old young leaves were surface-sterilized with 70%
(v/v) ethanol for 1 min and then with 0.1 % (w/v) mer-
curic chloride for 8 min. After being rinsed thoroughly
with sterile distilled water, they were cut into 0.5 ×0.5
cm2pieces and cultured on Murashige & Skoog (MS)
medium supplemented with 6-benzylaminopurine (BA) 4.0
mg L1,α-naphthaleneacetic acid (NAA) 0.2 mg L1and
2,4-dichlorophenoxy acetic acid (2,4-D) 0.1 mg L1under
the cool white fluorescent light of 24 µmol m2s1with a
photoperiod of 16 h light and 8 h dark in growth chamber.
About one month later, the regenerated adventitious
buds were inserted into 1/2 MS with NAA 0.3 mg L1for
rooting. Another month later, the regenerated shoots with
well-developed roots were transplanted into a 1:1:1 mixture
of vermiculite, perlite, and peat moss to be cultured under
dim light for one week and then under natural light and
temperature.
Transformation of PttKN1 gene to B. maculata
20-day-old young leaves were surface sterilized by sequential
soaking in 70% (v/v) ethanol (1 min) and 0.1% (w/v) mer-
curic chloride (8 min). After being rinsed thoroughly with
sterile distilled water, they were cut into 0.5 ×0.5 cm2pieces
and then soaked in the cultures of A. tumefasciens GV3101
harbouring pPCV702 with different optical density (0.205,
0.220 and 0.340 OD600 ) for 1 min. Then they were trans-
ferred onto MS solid medium containing 4.0 mg L1BA,
0.2mgL
1NAA and 0.1 mg L12,4-D and co-cultured in
the dark for 2 d, followed by culturing on MS solid medium
with4.0mgL
1BA, 0.2 mg L1NAA, 0.1 mg L12,4-
D and 500 mg L1cefotaxime. Seven days later they were
transferred to MS solid medium with 4.0 mg L1BA, 0.2
mg L1NAA, 0.1 mg L12,4-D, 500 mg L1cefotaxime
and 100 mg L1kanamycin for transformant selection, cal-
lus induction and plant regeneration. The newly generated
buds and shoots were treated following the same method as
the above.
RT-PCR analysis
Total RNA was isolated from leaves of putative trans-
genic and wild type plants using Trizol Isolation Reagent
of Invitrogen. RT-PCR was performed with one step
RNA PCR kit (TaKaRa Biotechnology, Dalian, China).
A 300bp fragment of PttKN1gene was amplified using
primers: forward 5’-gctgctcgtcaagagtttgg-3’ and reverse 5’-
aatctcaggtagttcagtctccc-3’ (Hu et al. 2005) under the fol-
lowing conditions: one cycle of 50
C for 30 min; one cycle
of 94
C for 2 min; 30 cycles of 94
C for 30 s, 55
C for 30
sand72
C for 1 min; and finally elongated at 72
Cfor5
min. RT-PCR products were electrophoresis-separated on
1% agarose gel and photographed with Alpha ImagerTM
2000 Documentation Analysis System. All above kits were
used according to the manufacturer’s protocol.
Scanning electron microscopy
Leaves of transgenic and wild type plants were fixed with
4% glutaraldehyde (v/v) (Sigma) dissolved in phosphate
buffer, dehydrated in ethanol series, desiccated in critical
point dryer, coated with gold, and finally observed under
JEOL1600 scanning electron microscope (Japan).
Induction and plant regeneration from the transgenic plants
To understand whether the traits of the PttKN1transgenic
plant above could be transmitted through micropropaga-
tion, the leaf segments of the transgenic plants grown in
pots in a greenhouse were re-cultured on MS agar medium
with BA 4.0 mg L1, NAA 0.2 mg L1and 2,4-D 0.1 mg L1
under the same illumination conditions as above. About one
month later, the morphology of the regenerated adventitious
buds and shoots were observed and also photographed.
Results
Callus induction and plant regeneration of B. maculata
The leaf explants of B. maculata which produced white
and loosen callus on their surface after about 15 days
being cultured on MS medium supplemented with 4.0
mg L1BA, 0.2 mg L1NAA and 0.1 mg L12,4-
D (Fig. 1A). Another 30 d later, these calli differenti-
ated into adventitious buds (Fig. 1B). 20-day-old ad-
ventitious buds, about 2 cm high, were transferred to
rooting medium (1/2 MS with NAA 0.3 mg L1), sub-
sequently well-developed roots were formed (Fig. 1C).
Author's copy
Transformation of Begonia maculata 253
Fig. 1. Plantlet regeneration of Begonia maculata from leaf explants. A – callus formed on the surface (cut ends) of leaf explants;
B – adventitious buds formed on the callus; C – regenerated plantlet with roots; D – regenerated plantlet growing in mixed soil.
Table 1. Influence of A. tumefasciens density (OD600) on transformation efficiency of leaf explants in Begonia maculate.
Bacterial
density
Number of
infected
explants(I)
Number of
explants
developing
callus(C)
Number of
explants
formed
adventitious
buds (B)
Rate of
callus
induction
(C/I, %)
Rate of bud
differentia-
tion
(B/C, %)
Transfor-
mation
efficiency
(B/I, %)
Number of
buds per
callus
Number of
regenerated
plantlets
0.205 579 13 10 2.25 76.92 1.73 5.53 48
0.220 579 33 25 5.70 75.76 4.32 3.11 63
0.340 413 39 24 9.44 61.54 5.81 2.65 57
The shoots with roots could survive and grow well after
being transplanted into a mixture of vermiculite, perlite
and peat moss (1:1:1) (Fig. 1D).
Of a total of 540 inoculated leaf explants, 438 could
form callus. All of callus produced adventitious buds;
13.50 buds per explant on average, and the adventitious
buds all could root in rooting medium. After being ex-
planted to soil, about 93% of the shoots survived and
exhibited normal development.
Introduction of PttKN1 gene to B. maculata
The leaf explants were infected with cultures of A.
tumefasciens at different densities (0.205, 0.220 and
0.340 OD600) to get high transformation efficiency. The
final result showed that the explants infected with 0.340
OD600 of A. tumefasciens cultures shared the highest
survival rate of 9.44% on the selection medium (Ta-
ble 1). In addition, it could be seen that the number
of survived explants of A. tumefasciens infected on the
selection medium increased with increased OD600 value
(Table 1). From 1571 leaf explants infected by A. tume-
fasciens, we obtained a total of 85 explants/callus re-
sistant to kanamycin.
Morphological features of PttKN1-transgenic plants
Normal B. maculata plants have strong main stem and
alternate phyllotaxy. Their leaves are simple, ovate and
entire with oblique base. Leaf blade is not eudipleu-
ral and its adaxial surface is dark green with regularly
arrayed white speckles, but the dorsal one is dark red
without speckles (Figs 2A, 2H).
Of a total of 168 putative PttKN1-transgenic plants
obtained here, 72 showed no visible differences from
wild type plants, and 96 showed abnormality to differ-
ent extent. Based on morphological features, the plants
with visible abnormality were roughly divided into four
groups, i.e. coral-like (CL) type, ectopic foliole (EF)
type, phyllotaxy-irregular (IP) type and cup-shaped
(CS) type. These different types counted for 17.26%,
21.43%, 1.79% and 0.89% of total, respectively.
The CL-typed transformants showed dwarfism and
had no clear stem and apical dominance, both stem and
leaves were not easily distinguishable. The leaves (or
stems) appeared needle-like, had multilevel branches
(2–4) and lost original dorsoventrality (Figs 2B, 2C).
The EF-typed transformants had distinctive stems
and roughly normal leaves, but 1–8 small ectopic folioles
(EFs) were formed on the leaf surface. The folioles were
round and mostly located at the centre or close to the
main vein of leaves. Interestingly, these EFs could pro-
duce smaller folioles on their surface again compared to
original EFs (Fig. 4D). All EFs were mostly curled, and
Author's copy
254 Q. Xu et al.
Fig. 2. Phenotype of wild type Begonia maculata and its transformants. A – wild-type plant; B – CL-type transformant; C – local
magnifications of B showed multilevel needle-like branches; D – EF-type transformant; E – local magnifications of D showed the EFs
produced smaller folioles that were located at the centre or close to the main vein of round leaves; F – IP-typed transformant, with
two leaves at single node (arrows); G – IP-type transformant, showing fused stem (arrows); H – young leaf of wild-type plant; I –
CS-type leaf; J – regenerative ectopic foliole producing leaves from ectopic foliole. CL – coral like; EF – ectopic foliole; IP – irregular
phyllotaxy; CS – cup shaped.
had dark green colour, unclear dorsoventrality and ir-
regular white speckles compared to the wild type. Some
EFs also displayed a petiole-like structure (Figs 2D,
2E).
The phyllotaxy of the IP-typed transformants dif-
fered from the normal one, and there were two leaves on
each side. The two leaves exhibited identical size and
shape, and did not show notable difference from nor-
mal leaves. In addition, the stem of the IP-type trans-
formants seemed to consist of two stems fused together
(Figs 2F, 2G), thus it looked flatter and thicker than
the normal one.
The transformants of the fourth type, CS type, dis-
played nearly completely the same phenotype as the
wild type, but a few leaves were changed into cup-
shaped (Fig. 2I).
The phenotypes identical with that of the above
transformants were not observed among the regener-
ated plants from leaf explants of the same plant, which
were not infected by A. tumefasciens.
In order to confirm that PttKN1was expressed in
Fig. 3. RT-PCR analysis of the putative PttKN1-transgenic
plants. M, marker; lane 1, non-transformed control; lane 2, coral-
like transformant; lane 3, ectopic foliole transformant; lane 4,
phyllotaxy-irregular; lane 5, cup shaped typed transformant. The
300 bp indicates PttKN1-specific bands.
the transgenic plants, at least two plants from each of
the four types of transformants were subjected to RT-
PCR analysis. As shown in Fig. 3, each of them dis-
played one completely identical specific band of about
300bp, indicating that PttKN1gene had been expressed
in transformed plants.
Author's copy
Transformation of Begonia maculata 255
Fig. 4. SEM micrographs of the adaxial leaf surface from wild and PttKN1-transgenic plants (ectopic foliole-type). A – wild type,
showing very flat and smooth upper leaf surface; B-D – transgenic plants. B – numerous protuberances with different size and shape
on leaf surface; C – single small protuberance on flat leaf surface; D – several pieces of ectopic folioles on flat adaxial leaf surface,
which had a very big outgrowth (white arrows) and smaller folioles generating from ectopic folioles (black arrows).
Histological and anatomical characteristics of the
PttKN1-transgenic plants
To understand the origin of the ectopic foliole in the
EF-typed transformants, a SEM examination was per-
formed. The upper surface of the leaves of the wild
B. maculata appeared very flat and smooth. Polyg-
onal cells with uniform size were regularly arranged
(Fig. 4A). For the EF-typed transgenic plants, how-
ever, some adaxial leaf surfaces were extremely acci-
dented, showing numerous protuberances with differ-
ent size and shape. The cell shape and arrangement
were extremely irregular. And some adaxial leaf sur-
faces looked like that of the wild type, but single small
spherical protuberance could be observed (Fig. 4B-D);
The feature of the in vitro regenerated plants from the
PttKN1 transgenic plants
To understand whether the phenotypes of transgenic
plants could be transmitted by micropropagation, the
leaf explants from the EF-type were cultured on MS
medium with BA 4.0 mg L1, NAA 0.2 mg L1and
2,4-D 0.1 mg L1. Similarly, white and loosen callus
formed on the leaf explants when cultured around 15
d, and they differentiated into adventitious buds 30 d
later.
The plants regenerated from the EF-type transfor-
mants still maintained the original morphology: there
were many circular folioles across the adaxial surface of
large leaves, and on these circular folioles, smaller fo-
lioles in comparison to original EFs could be observed
(Fig. 2J). The initial folioles were produced at the top of
the petiole and the subsequent ones at the sites nearby
midveins and secondary veins, which was the same as
in the EF-type transgenic plants.
Discussion
In the present study, four types of PttKN1-transgenic
B. maculata, i.e. coral-like (CL), ectopic foliole (EF),
phyllotaxy-irregular (IP) and cup-shaped (CS), were
obtained via gene introduction in B. maculata medi-
ated by A. tumefasciens. These transformants were re-
markably different in morphology from the wild typeB.
maculata. RT-PCR assay confirmed that PttKN1gene
was expressed at transcription level in each of the four-
typed transgenic plantlets.
Among the four types of PttKN1-transgenic plant-
lets, the EF type is notably interesting. The transfor-
mants have normal stems and leaves, but there are nu-
merous small round folioles across the adaxial surface
of the leaves. The folioles can produce smaller round
folioles on their surface again. Although it had been
well reported that ectopic meristems formed on the sur-
face of the transgenic leaves (Sinha et al. 1993; Lin-
coln et al. 1994; Chuck et al. 1996; Hu et al. 2005), EF
typed transgenic plants in this experiment could gen-
erate ectopic folioles from the original ectopic folioles
again. This has not been reported neither among the
class I KNOX transgenic plants nor among transgenic
plants of other genes, to our knowledge. This fact in-
dicated that PttKN1probably had quite a strong role
in meristematic activity and ability of morphogenesis.
Furthermore, EF typed transgenic plants were beauti-
ful in shape and could be transmitted via micropropa-
gation, which suggested a potential ornamental value.
The phenotype with changed phyllotaxy appeared
in fasciata1(fas1), fasciata 2(fas2), tonsoku (tsk)and
pinoid (pid) mutants of Arabidopsis (Leyser & Furner
1992; Bennett et al. 1995; Suzuki et al. 2004), aber-
Author's copy
256 Q. Xu et al.
rant phyllotaxy (abphyll) of maize (Jackson & Hake
1999; Giulini et al. 2004), low auxin transport (lat)
mutant of tobacco (Naderi et al. 1997) and in some
transgenic plants expressing KNOX genes (Lincon et al.
1994; Chuck et al. 1996; Tamaoki et al. 1997; Nishimura
et al. 2000). In the present IP-type transformants, their
phyllotaxy still remained different from the normal one,
but the number of leaves per node has doubled. The
pair of leaves looked similar in size and shape and had
a separated petiole. In addition, their stems were fas-
ciated and looked like a fusion of two stems (Fig. 2F,
2G). Phyllotaxy alteration was also observed in PttKN1
transgenic petunia (Hu et al. 2005). This phenomenon
suggested a stronger role of PttKN1in the remodelling
of phyllotaxy and plant morphogenesis.
Meristems are closely connected with the postem-
bryonic plant growth and initiation of organs. It has
been studied extensively that KNOX genes play vi-
tal roles in the shoot apical meristem (SAM) function
(Hake et al. 2004; Norberg et al. 2005; Wang et al.
2006) and lateral meristem activity (Ko & Han 2004;
Schrader et al. 2004; Groover et al. 2006) via maintain-
ing the stem cell population. Class I KNOX genes were
found to be necessary during all stages of the plant life
(Jasinski et al. 2006; Sakamoto et al. 2006). Primordia
of lateral organs, such as leaves, emerge from peripheral
regions of the SAM. Leaf shapes are highly correlated
with expression patterns of class I KNOX genes in leaf
promordia (Uchida et al. 2010). Ectopic expression of
class I KNOX genes resulted in morphologically altered
leaves and flowers in transgenic Arabidopsis (Chuck et
al. 1996; Liu et al. 2008), tobacco (Nishimura et al.
2000), tomato (Kim et al. 2003; Kimura et al. 2008).
From our research, it could be concluded that as a
member of class IKNOX gene family, PttKN1played
a similar role in meristem as the other class I KNOX
genes. The morphological, histological and anatomical
analysis of EF-type transgenic plants indicated that Pt-
tKN1played a role in meristem initiation and mainte-
nance; ectopic expression of PttKN1could greatly en-
hance the meristematic activity not only in SAM but
also in other tissues, which further resulted in the mor-
phologic changes. Ectopic expression of PttKN1gene in
petunia (Hu et al. 2005), and cockscomb (Meng et al.
2009) also proved the function of the gene in meristems.
Taken together, ectopic expression of PttKN1gene
in B. maculata caused alterations of leaf shape and
plant architecture. Most importantly, it was the ability
of the ectopic foliole on the surface of the transgenic B.
maculata to generate new ectopic foliole again, as well
as the phyllotaxy alteration with two identical leaves at
each side of one internode. These data showed that the
PttKN1gene from vascular cambium of the tree played
an important role in meristem activity and morphogen-
esis, and its ectopic expression might have a possibility
to develop novel ornamental lines.
Acknowledgements
This work was supported by the National Natural Science
Foundation, P. R. China (30370087), by City gardening de-
partment of Xi’an, P. R. China (XA081023) and the Special
Foundation for Young Scholars of Northwest A & F Univer-
sity, P. R. China (Z111020912). The authors thank Dr. Olof
Olsson (G¨oteberg University, G¨oteberg, Sweden) for kindly
providing the plasmid containing 35S::Pttkn1.
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Received September 7, 2009
Accepted June 22, 2010
Author's copy
... The PttKN1 gene (Populus tremula × P. tremuloides was isolated from the vascular cambium of a hybrid aspen (Hu et al., 2005;Meng et al., 2009a). To investigate the gene function, it was introduced into Begonia maculata (Xu et al., 2011), carnation (Meng et al., 2009a), cockscomb (Meng et al., 2009b), coleus (Xu et al., 2013), Petunia hybrida (Hu et al., 2005) and tobacco (Ding et al., 2008;Xu et al., 2015) plants via Agrobacterium tumefaciens transformation. Generally, the PttKN1 gene transformed plants exhibit a series of morphological alterations like dwarfish and fascicular plants, lobed leaves, altered leaf surfaces, flattened stems, ectopic meristems, etc. (Hu et al., 2005;Ding et al., 2008;Meng et al., 2009aMeng et al., , 2009bXu et al., 2011;Xu et al., 2013;Xu et al., 2015). ...
... To investigate the gene function, it was introduced into Begonia maculata (Xu et al., 2011), carnation (Meng et al., 2009a), cockscomb (Meng et al., 2009b), coleus (Xu et al., 2013), Petunia hybrida (Hu et al., 2005) and tobacco (Ding et al., 2008;Xu et al., 2015) plants via Agrobacterium tumefaciens transformation. Generally, the PttKN1 gene transformed plants exhibit a series of morphological alterations like dwarfish and fascicular plants, lobed leaves, altered leaf surfaces, flattened stems, ectopic meristems, etc. (Hu et al., 2005;Ding et al., 2008;Meng et al., 2009aMeng et al., , 2009bXu et al., 2011;Xu et al., 2013;Xu et al., 2015). These morphological alterations were very similar to those described for the class I KNOX (KNOTTED1-like homeobox genes) gene transformed plants (Lincoln et al., 1994;Chuck et al., 1996). ...
... These morphological alterations were very similar to those described for the class I KNOX (KNOTTED1-like homeobox genes) gene transformed plants (Lincoln et al., 1994;Chuck et al., 1996). Hence, it was speculated that the gene belongs to the class I KNOX gene family (Hu et al., 2005), and plays roles in plant development (typically in meristem initiation), maintenance and organogenesis (Xu et al., 2011). However, the sequence alignment of the PttKN1 gene was never conducted to make sure it's a close homolog. ...
Article
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PttKN1 gene (Populus tremula × P. tremuloides KNOTTED1) was isolated from the vascular cambium of a hybrid aspen. Previous studies on transformed plants with the PttKN1 gene suggested that it plays roles in plant development (typically in meristem initiation), maintenance and organogenesis in simple-leaved species. To investigate the gene functions further, sequence analysis of the deduced amino acid was conducted. The results suggested that the gene belongs to the class I KNOX gene (KNOTTED1-like homeobox genes) family and might play important roles in plant development by coding a transcription factor. The gene was introduced into Cardamine hirsuta using the floral dip method mediated via Agrobacterium tumefaciens. The primary transformed plants were obtained via kanamycin selection. Compared to the wild type, the kanamycin resistant plants demonstrated several morphological alterations, such as abnormal cotyledons, abnormal shoot meristem, flattened stem, and lobed and cup-shaped leaves. RT-PCR results showed that the above five types of kanamycin resistant plants expressed the same specific PttKN1 gene band. This suggested that the morphological alterations were caused by the insertion and expression of the gene. However, these phenotypes were similar to other PttKN1 transformed plants, despite the fact that C. hirsuta is a species with compound leaves and the other species have simple leaves. Therefore, the functions of the PttKN1 gene on compound-leaf species have yet to be investigated via the comparison between related species such as Arabidopsis thaliana and C. hirsuta.
... PttKN1 (Populus tremula × tremuloides KNOTTED1), which is a hybrid aspen homeobox gene (Hu et al., 2005;Meng et al., 2009a) plays an important role in plant development, especially in meristem initiation, maintenance and organogenesis (Xu et al.. 2011). It is well reported that the PttKN1 transformants of Begonia maculata (Xu et al., 2011), carnation (Meng et al., 2009a), cockscomb (Meng et al., 2009b), Petunia hybrida (Hu et al., 2005) and tobacco (Xu et PCR assay DNA was extracted from the leaves of wild type and putative transgenic plants by improved CTAB method (Doyle, 1990). ...
... PttKN1 (Populus tremula × tremuloides KNOTTED1), which is a hybrid aspen homeobox gene (Hu et al., 2005;Meng et al., 2009a) plays an important role in plant development, especially in meristem initiation, maintenance and organogenesis (Xu et al.. 2011). It is well reported that the PttKN1 transformants of Begonia maculata (Xu et al., 2011), carnation (Meng et al., 2009a), cockscomb (Meng et al., 2009b), Petunia hybrida (Hu et al., 2005) and tobacco (Xu et PCR assay DNA was extracted from the leaves of wild type and putative transgenic plants by improved CTAB method (Doyle, 1990). Transgenic plants were verified via PCR of the PttKN1 gene, NPTII gene and 35S-Promoter. ...
... The pigment concentration was calculated using extinction value E (1 cm/1%) al., 2014) produced various morphological alterations compared with their wild type plant. Those phenotypes were mainly focused on altered leaf arrangement (Hu et al., 2005;Meng et al., 2009b;Xu et al., 2011) and leaf morphology like lobed leaf (Hu et al., 2005;Xu et al., 2011;Xu et al., 2014), cup-shaped leaf (Xu et al., 2011) and distorted leaf (Hu et al., 2005;Meng et al., 2009a;Xu et al., 2011;Xu et al., 2014) etc. However, transgenic PttKN1 P. hybrida showed color modification of flower (Hu et al., 2005) and B. maculata showed color modification of leaf (Xu et al., 2011). ...
Article
Full-text available
PttKN1 (Populus tremula × tremuloides KNOTTED1) gene belongs to the KNOXI gene family. It plays an important role in plant development, typically in meristem initiation, maintenance and organogenesis, and potentially in plant coloration. To investigate the gene functions further, it was introduced into red leaf beet by the floral dip method mediated via Agrobacterium tumefaciens. The transformants demonstrated typical phenotypes as with other PttKN1 transformants. These alterations were very different from the morphology of the wild type. Among them, morphological modification of changed color throughout the entire plant from claret of wild type to yellowish green was the highlight in those transgenic PttKN1-beet plants. The result of spraying selection showed that the PttKN1-beet plants had kanamycin resistance. PCR assay of the 35S-Promoter, NPTII and PttKN1 gene, PCR-Southern analysis of the NPTII and PttKN1 gene showed that the foreign PttKN1 gene had successfully integrated into the genome of beet plant. Furthermore, the results of RT-PCR analysis showed that the gene was ectopic expressed in transgenic plants. These data suggested that there is a correlation between the ectopic expression of PttKN1 gene and morphological alterations of beet plants. Pigment content assay showed that betaxanthins concentrations shared little difference between wild type and transgenic lines, while betacyanins content in transgenic plants was sharply decreased, indicating that the altered plant coloration of the transgenic beet plants may be caused by the changed betacyanins content. The tyrosinase study suggested that the sharply decreased of betacyanins content in transgenic plants was caused via the decreased tyrosinase level. Therefore, the reason for the altered plant coloration may be due to partial inhibition of betacyanin biosynthesis that was induced via the pleiotropic roles of PttKN1 gene.
... PttKN1 (Populus tremula × tremuloides KNOTTED1) is a class I KNOX gene isolated from the cambial region of a hybrid aspen (Hu et al. 2005;Meng et al. 2009a). The PttKN1 transformants displayed typical phenotypes of class I KNOX gene transformants, such as ectopic meristems formation and altered leaf morphologies (Hu et al. 2005;Ding et al. 2008;Meng et al. 2009a;Xu et al. 2011;Xu et al. 2013). This circumstance indicated the roles of PttKN1 gene in plant development, especially in meristem initiation and organogenesis (Xu et al. 2011). ...
... The PttKN1 transformants displayed typical phenotypes of class I KNOX gene transformants, such as ectopic meristems formation and altered leaf morphologies (Hu et al. 2005;Ding et al. 2008;Meng et al. 2009a;Xu et al. 2011;Xu et al. 2013). This circumstance indicated the roles of PttKN1 gene in plant development, especially in meristem initiation and organogenesis (Xu et al. 2011). To get more insight into the functions of the gene in regulating plant morphology, it was introduced into the genome of tobacco through the leaf disc method mediated via Agrobacterium tumefaciens (Ding et al. 2008). ...
... e-h HiTAIL-PCR from WT and transgenic tobacco and identification before the amplified fragment sequencing. Lane 1, 3, 5, results of the pre-amplification, primary and secondary hiTAIL-PCR of WT; Lane 2, 4, 6, results of the pre-amplification, primary and secondary hiTAIL-PCR of transgenic tobacco; Lane 7-10, further validity identification before sequencing; Lane 7-8, PCR products of WT and transgenic plant with BR2 and the specific primer; Lane 9-11, PCR products of E. coli for sequencing with BR2 and AC1, LAD and AC1, BR2 and the specific primer, respectively; M marker, PC positive control, WT wild type, TT transgenic tobacco The PttKN1 gene had been confirmed to have a strong role in plant development via the analysis of its transgenic plants, such as the Begonia maculata (Xu et al. 2011), coleus (Xu et al. 2013), Petunia hybrid (Hu et al. 2005) and tobacco (Ding et al. 2008). Among them, most showed morphological alterations sporadically in transgenic plants. ...
Article
Full-text available
PttKN1 (Populus tremula × tremuloides KNOTTED1) gene plays an important role in plant morphological formation. To investigate the gene functions, it was introduced into tobacco using the leaf disc method. The transgenic tobacco lines displayed various morphologies different from those of wild type. The results of RT-PCR suggested that these morphological alterations were caused by the ectopic expression of the PttKN1 gene. Notably, almost all the transgenic lines displayed phenotypes sporadically except for one, which appeared with wrinkled leaves throughout the plant growth. The plant grow two round stems compared to one of the wild type, and have mild, intermediate or severe wrinkled leaves distributed on both of the stems. The results of RT-PCR, hiTAIL-PCR and Southern blot analysis indicated that the foreign PttKN1 gene was integrated into the genome of the plant by the way of direct repeats with two copies. Further study on endogenous hormonal levels from HPLC showed that the CK (cytokinin) level was increased and GA (gibberellins) level was decreased in the transgenic line. Correspondingly, the expression of GA 20-oxidase gene that related to GA biosynthesis was down-regulated. These data demonstrated that the ectopic expression of the PttKN1 gene induced aberrant leaf morphology and endogenous hormonal levels in transgenic tobacco, which would enhance our understanding on the functions of PttKN1 gene in leaf development.
... It belongs to KNOXI gene family and play roles in plant development by means of coding transcription factor [1]. The PttKN1 over-expression plants generally exhibited series of morphological alterations like dwarf and fascicular plant, lobed leaves, accidented leaf surfaces, flatted stem and ectopic meristems, etc. [1][2][3][4][5]. These typical phenotypes suggested the pleiotropic roles of PttKN1 gene on plant development as a member of KNOXI gene family. ...
... These typical phenotypes suggested the pleiotropic roles of PttKN1 gene on plant development as a member of KNOXI gene family. However, PttKN1 gene over-expression in Petunia hybrida showed flower morphology and color modification [1], and Begonia maculata Raddi showed leaf color and phyllotaxy modification [5]. Those alterations of ornamental characters were absolutely different from the above typical phenotypes of KNOXI gene over-expression plants, which provide the possibility of breeding new ornamental lines. ...
... As the ectopic expression of PttKN1 gene causes pleiotropic morphological alteration in transgenic plants including coleus (Table 1), we deduced that it may function as a transcription factor to involve in plant morphogenesis and development [1,5]. Among those phenotypes, morphological alterations in local of the transgenic plant hold the most. ...
Article
Full-text available
The foreign PttKN1 gene was introduced into the genome of coleus (Solenostemon scutellarioides) using the floral dip method mediated by Agrobacterium tumefaciens. The seeds that generated from the dipped mother plant were screened on MS medium with a kanamycin concentration of 100 mg l-1. Those kanamycin-resistant plantlets displayed various morphological alterations different from the wild type plant. Among those morphological alterations, the most were appeared in the local of the transgenic plant, including (1) the leaf morphological changes: knots formation, accidented leaf surface, lobed leaf, twisted and asymmetric leaf, etc. (2) the floral morphological changes like receptacle and flower color. Moreover, alterations throughout the plant were also observed such as clustered plant, plant with whorled phyllotaxy or faded leaf coloration, etc. Among them, the altered morphologies of whorled phyllotaxy and faded leaf coloration were scarcely appeared in other PttKN1 gene transformants. The results of PCR and PCR-Southern showed that the foreign PttKN1 gene had integrated into the genome of coleus, and was responsible for the above morphological alterations. Further comparison of the anthocyanin content in transgenic and wild type coleus showed that the biosynthesis of anthocyanin was inhibited in the faded-anthocyanin coleus.
... PttKN1 (Populus tremula × tremuloides KNOTTED1) is a class I KNOX gene isolated from the cambial region of a hybrid aspen (Hu et al. 2005; Meng et al. 2009a). The PttKN1 transformants displayed typical phenotypes of class I KNOX gene transformants, such as ectopic meristems formation and altered leaf morphologies (Hu et al. 2005; Ding et al. 2008; Meng et al. 2009a; Xu et al. 2011; Xu et al. 2013). This circumstance indicated the roles of PttKN1 gene in plant development, especially in meristem initiation and organogenesis (Xu et al. 2011). ...
... The PttKN1 transformants displayed typical phenotypes of class I KNOX gene transformants, such as ectopic meristems formation and altered leaf morphologies (Hu et al. 2005; Ding et al. 2008; Meng et al. 2009a; Xu et al. 2011; Xu et al. 2013). This circumstance indicated the roles of PttKN1 gene in plant development, especially in meristem initiation and organogenesis (Xu et al. 2011). To get more insight into the functions of the gene in regulating plant morphology, it was introduced into the genome of tobacco through the leaf disc method mediated via Agrobacterium tumefaciens (Ding et al. 2008). ...
... The PttKN1 gene had been confirmed to have a strong role in plant development via the analysis of its transgenic plants, such as the Begonia maculata (Xu et al. 2011), coleus (Xu et al. 2013), Petunia hybrid (Hu et al. 2005) and tobacco (Ding et al. 2008). Among them, most showed morphological alterations sporadically in transgenic plants. ...
... Similar phenotypic abnormalities were observed in overexpressing O. sativa OSH1, A. thaliana KNAT1, and Populus tremula× tremuloides PttKN1. [41][42][43] The proliferation and differentiation of cells directly affect the leaf, and this is a key step driving the development of leaves. 44 The class I KNOX is the key transcription factor supporting the SAM maintenance including the indeterminate cell fates. ...
Article
Full-text available
Leaves are the main vegetative organs of the aboveground part of plants and play an important role in plant morphogenesis. KNOTTED-LIKE HOMEOBOX (KNOX) plays a crucial role in regulating leaf cell fate and maintaining leaf development. In this study, we analyzed LtKNOX1 from Lilium tsingtauense and illustrated its function in transgenic plants. Tissue-specific expression analysis indicated that LtKNOX1 was highly expressed in stems, young flower buds, and shoot apical meristems (SAMs). Ectopic overexpression of LtKNOX1 in Nicotiana benthamiana suggested that transformants with mild phenotypes were characterized by foliar wrinkles and mildly curled leaves; transformants with intermediate phenotypes showed severely crimped blades and narrow leaf angles, and the most severe phenotypes lacked normal SAMs and leaves. Moreover, the expression levels of genes involved in the regulation of KNOX in transgenic plants were detected, including ASYMMETRIC LEAVES1, PIN-FORMED 1, GA20-oxidase, CUP-SHAPED COTYLEDON 2, CLAVATA 1 and WUSCHEL(WUS), and the expression of other genes were down-regulated except WUS. This study contributes to our understanding of the LtKNOX1 function.
... Begonia is also a major horticultural crop, being important in both bedding plants and indoor plants markets (Hvoslef-Eide and Munster 2007) and consequently large, accessible collections of species and hybrids are available (Tebbit 2005). Additionally, methods of in-vitro propagation and genetic transformation that have been developed for commercial exploitation of Begonia can also be applied to research (Kishimoto et al. 2002;Xu et al. 2011). ...
Article
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Begonia is one of the ten largest angiosperm genera with over 1,500 species found throughout the tropics. To use this group as a model for the evolution of diversity in tropical herbaceous plants, we have produced three species transcriptomes, physical genome size measures, and two backcross genetic maps. We chose to focus on two Central American species, B. conchifolia and B. plebeja, and one SE Asian species, B. venusta, allowing us to pose questions at widely different evolutionary scales within the genus. We used next generation sequencing of cDNA libraries to produce annotated transcriptome databases for each of the three species. Though Begonia is functionally diploid, transcriptome analysis suggested a genome duplication occurred at or near the base of the Begonia clade. The genetic maps were built from first generation backcrosses in both directions between B. plebeja and B. conchifolia using 105 SNP markers in genes known to regulate development that were identified from the transcriptomes and the map bulked out with 226 AFLP loci. The genetic maps had 14 distinct linkage groups each and mean marker densities of between 3.6 and 5.8 cM providing between 96 and 99 % genomic coverage within 10 cM. We measured genome size 1C value of 0.60 and 0.63 pg for B. conchifolia and B. plebeja corresponding to recombination rates of between 441 and 451 Kb per cM in the genetic maps. Altogether, these new data represent a powerful new set of molecular genetic tools for evolutionary study in the genus Begonia.
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SummaryA series of mutants of Arabidopsis thaliana was selected in which the inflorescence stem elongates but loses the ability to produce flower primordia on its flanks. Mutants fell into two classes, further occurrences of pin-formed mutants and mutations at a new locus named pinoid. As well as causing inflorescence defects, pinoid mutations result in pleiotropic defects in the development of floral organs, cotyledons and leaves. Most changes involve the number of organs produced rather than their differentiation suggesting that PINOID controls an early general step in meristem development. pinoid mutant defects are similar to those seen in pin-formed mutants for inflorescences and flowers, but different for cotyledons and leaves indicating that the two genes have separate but overlapping functions. A defect in polar auxin transport is implicated in the pin-formed mutant phenotype, but in young inflorescence stems of even the strongest pinoid mutants it occurs at close to wild-type levels. It is markedly reduced only after stems have ceased elongating. Thus, it is likely that polar auxin transport is secondarily affected in pinoid mutants rather than being directly controlled by the PINOID gene product. Even so, double mutant studies indicate that the process controlled by PINOID overlaps with that specified by the AUXIN RESISTANT1 gene, suggesting that PINOID plays some role in an auxin-related process.
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The KNAT1 gene is a member of the Class I KNOX homeobox gene family and is thought to play an important role in meristem development and leaf morphogenesis. Recent studies have demonstrated that KNAT1/BP regulates the architecture of the inflorescence by affecting pedicle development in Arabidopsis thaliana. Herein, we report the characterization of an Arabidopsis T-DNA insertion mutant that shares considerable phenotypic similarity to the previously identified mutant brevipedicle (bp). Molecular and genetic analyses showed that the mutant is allelic to bp and that the T-DNA is located within the first helix of the KNAT1 homeodomain (HD). Although the mutation causes a typical abnormality of short pedicles, propendent siliques, and semidwarfism, no obvious defects are observed in the vegetative stage. A study on cell morphology showed that asymmetrical division and inhibition of cell elongation contribute to the downward-pointing and shorter pedicle phenotype. Loss of KNAT/BP function results in the abnormal development of abscission zones. Microarray analysis of gene expression profiling suggests that KNAT1/BP may regulate abscission zone development through hormone signaling and hormone metabolism in Arabidopsis. (Managing editor: Ping He)
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We have established a shoot regeneration system and genetic transformation of cockscomb (Celosia cristata and Celosia plumosus). The best results in terms of frequency of shoot regeneration and number of shoot buds per explant are observed on media supplemented with 0.5mgl−1 6-BA (for explants of apical meristems of C. cristata) or 2.0mgl−16-BA, 0.5 mgl−1 NAA and 0.5 mgl−1 IAA (for hypocotyls explants of C. plumosus). We use apical meristems of C. cristata and hypocotyls of C. plumosus as the starting material for transformation. A novel KNOTTED1-like homeobox1 (KNOX), PttKN1 (Populus tremula×P. tremuoides knotted1) isolated from the vascular cambial region of hybrid aspen, is introduced into cockscomb by Agrobacterium. A series of novel phenotypes are obtained from the transgenic cockscomb plants, including lobed or rumpled leaves, partite leaves and two or three leaves developed on the same petiole, on the basis of their leaf phenotypes. Transformants are selected by different concentrations of kanamycin. Transformants are confirmed by PCR of the NptII gene and PCR or RT-PCR of PttKN1 gene. Furthermore, RT-PCR shows that 35S:: PttKN1 RNA levels do not correlate with phenotypic severity. It is discussed that our results bring elements on possible function of PttKN1 gene. To our knowledge, genetic transformation of cockscomb is first reported.
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Plant development depends on the activity of apical meristems, which are groups of indeterminate cells whose derivatives elaborate the organs of the mature plant. Studies of knotted1 (kn1) and related gene family members have determined potential roles for homeobox genes in the function of shoot meristems. The Arabidopsis kn1-like gene, KNAT1, is expressed in the shoot apical meristem and not in determinate organs. Here, we show that ectopic expression of KNAT1 in Arabidopsis transforms simple leaves into lobed leaves. The lobes initiate in the position of serrations yet have features of leaves, such as stipules, which form in the sinus, the region at the base of two lobes. Ectopic meristems also arise in the sinus region close to veins. Identity of the meristem, that is, vegetative or floral, depends on whether the meristem develops on a rosette or cauline leaf, respectively. Using in situ hybridization, we analyzed the expression of KNAT1 and another kn1-like homeobox gene, SHOOT MERISTEMLESS, in cauliflower mosaic virus 35S::KNAT1 transformants. KNAT1 expression is strong in vasculature, possibly explaining the proximity of the ectopic meristems to veins. After leaf cells have formed a layered meristem, SHOOT MERISTEMLESS expression begins in only a subset of these cells, demonstrating that KNAT1 is sufficient to induce meristems in the leaf. The shootlike features of the lobed leaves are consistent with the normal domain of KNAT1's expression and further suggest that kn1-related genes may have played a role in the evolution of leaf diversity.
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The domestication of all major crop plants occurred during a brief period in human history about 10,000 years ago. During this time, ancient agriculturalists selected seed of preferred forms and culled out seed of undesirable types to produce each subsequent generation. Consequently, favoured alleles at genes controlling traits of interest increased in frequency, ultimately reaching fixation. When selection is strong, domestication has the potential to drastically reduce genetic diversity in a crop. To understand the impact of selection during maize domestication, we examined nucleotide polymorphism in teosinte branched1, a gene involved in maize evolution. Here we show that the effects of selection were limited to the gene's regulatory region and cannot be detected in the protein-coding region. Although selection was apparently strong, high rates of recombination and a prolonged domestication period probably limited its effects. Our results help to explain why maize is such a variable crop. They also suggest that maize domestication required hundreds of years, and confirm previous evidence that maize was domesticated from Balsas teosinte of southwestern Mexico.
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Recent work on species with simple leaves suggests that the juxtaposition of abaxial (lower) and adaxial (upper) cell fates (dorsiventrality) in leaf primordia is necessary for lamina outgrowth. However, how leaf dorsiventral symmetry affects leaflet formation in species with compound leaves is largely unknown. In four non-allelic dorsiventrality-defective mutants in tomato, wiry, wiry3, wiry4 and wiry6, partial or complete loss of ab-adaxiality was observed in leaves as well as in lateral organs in the flower, and the number of leaflets in leaves was reduced significantly. Morphological analyses and expression patterns of molecular markers for ab-adaxiality [LePHANTASTICA (LePHAN) and LeYABBY B (LeYAB B)] indicated that ab-adaxial cell fates were altered in mutant leaves. Reduction in expression of both LeT6 (a tomato KNOX gene) and LePHAN during post-primordial leaf development was correlated with a reduction in leaflet formation in the wiry mutants. LePHAN expression in LeT6 overexpression mutants suggests that LeT6 is a negative regulator of LePHAN. KNOX expression is known to be correlated with leaflet formation and we show that LeT6 requires LePHAN activity to form leaflets. These phenotypes and gene expression patterns suggest that the abaxial and adaxial domains of leaf primordia are important for leaflet primordia formation, and thus also important for compound leaf development. Furthermore, the regulatory relationship between LePHAN and KNOX genes is different from that proposed for simple-leafed species. We propose that this change in the regulatory relationship between KNOX genes and LePHAN plays a role in compound leaf development and is an important feature that distinguishes simple leaves from compound leaves.
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A novel knottedl-like homeobox (knox) gene, Pttknl (Populus tremula tremuloides knottedl), isolated from the cambial region of hybrid aspen, was introduced into Petunia hybrida Vilm. using the leaf-disc method mediated by Agrobacterium. A series of novel phenotypes was observed in transgenic petunia plants, including the formation of ectopic spikes on the adaxial surface of corollas and small pet als on the abaxial surface of corollas, fusion of floral organs, shortening of corolla midribs, the formation of tumor-like knots along the midrib on the abaxial surface and serrated lobs of corolla margins, and alterations in pet al color; except for changes in the leaves and plant architecture, RT-PCR showed that the Pttknl gene was expressed in the leaves of different petunia transgenic plants, whereas no signal was detected in wild-type plants. The possible function of Pttknl in leaf and flower development is discussed. (Managing editor: Li-Hui ZHAO)
Article
A mutation in tobacco (Nicotiana tabacum L. cv `Xanthi') called lat (low auxin transport) that changes many morphogenic features throughout the life of the plant has been isolated. Abnormalities were observed in seed development, embryogenesis, cotyledon formation, leaf initiation and development, leaf veination pattern, and flower development. Selfed R2 lat mutant plants set between 60% and 90% fewer seeds than wild-type tobacco, and about 10% of these seeds did not germinate. Non-germinating seeds contained either abnormal embryos or abnormal endosperm tissues. There was no uniformity in the stage at which embryonic development ceased in the aberrant seeds. Seedlings often revealed abnormal and highly varied phenotypes after germination. In some of these cases, cotyledons were heart-shaped, fused, cup-shaped, or cylindrical. Leaf morphology ranged from normal to cup-shaped, and some leaves occasionally produced shoots from the leaf midvein. Flowers ranged from normal to compound with occasional fused floral parts or split petals. Stamens were sometimes petal-like. This unusual assortment of phenotypic changes suggested that the mutation might affect a basic component of plant metabolism. We found that polar transport of indole-3-acetic acid (IAA) was reduced to about 9–19% of the wild-type level in the inflorescence axis of selfed R2 lat mutants. In addition, supplementation of 1-naphthaleneacetic acid (NAA) to sterile media suppressed some of the abnormalities of the lat mutation so long as the plants grew there. Similarities in the phenotype of embryos, cotyledon and leaf shapes, translocation of labeled IAA, and response to applied NAA indicate that the lat locus of tobacco may be analogous to the pin locus of Arabidopsis, or produce a protein that functions in the same auxin-transport pathway.