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Organo 1
Organo 1
Secara bersama-sama, baik konsentrasi dan rasio hormon eksogen adalah penting
untuk
menentukan sel nasib selama de novo organogenesis
The link between distribution of hormones and de novo organogenesis
Polar auxin transport is a unique feature of auxin action and mediates vectorial
gradients that
provide positional cues for organogenesis and are crucial to pattern formation
(Reinhardt et al.
2000, 2003; Benkova et al. 2003; Heisler et al. 2005; Sauer et al. 2006; Scarpella et
al. 2006).
Although the data on the direct links are very poor, the asymmetric distribution of
hormones is
essential for the formation of stem cell because the disruption of this distribution
pattern inhibits
the formation of new organs (Okada et al. 1991; Liu et al. 1993; Hadfi et al. 1998;
Mattsson et al.
1999; Sabatini et al. 1999; Reinhardt et al. 2000; Tanaka et al. 2006; Larsson et al.
2008;
Dhonukshe et al. 2008). Arabidopsis PIN proteins are asymmetrically localized at
the plasma
membrane, which attributes to the establishment of the local auxin gradients
(Tanaka et al. 2006). PIN1, one of auxin efflux carrier proteins, initiates and
maintains the auxin concentration
gradients (Friml et al. 2003; Heisler et al. 2005). The artificial promoter DR5
(Ulmasov et al. 1997)
is highly responsive to active auxin, and furthermore, its expression can be induced
by
exogenously applied auxin (Sabatini et al. 1999; Friml et al. 2002, 2003; Benkov et
al. 2003;
Ottenschlger et al. 2003). Therefore, the reporter driven by DR5 can be used as
reasonable
approximations of auxin accumulation in different cells of tissues, allowing
visualization of the
spatiotemporal pattern of auxin distribution (Sabatini et al. 1999; Casimiro et al.
2001;
Avsian-Kretchmer et al. 2002; Benkov et al. 2003; Friml et al. 2003; Ottenschlger
et al. 2003; de
Reuille et al. 2006). In Arabidopsis, all organs originate from apical meristems
including shoot
meristem and root meristem, whereas the in vitro organs come from a group of
undifferentiated
somatic cells, namely calli that are produced throughout the dedifferentiation of
explant cells.
Interesting questions arise that how the exogenous auxin is transported from
medium into explant
cells, and what is the distribution pattern of auxin during in vitro organogenesis.
Hubungan antara distribusi hormon dan de novo organogenesis
transportasi auksin kutub adalah fitur unik dari aksi auksin dan memediasi gradien
vectorial yang
memberikan isyarat posisi untuk organogenesis dan sangat penting untuk
pembentukan pola (Reinhardt et al.
2000, 2003; Benkova et al. 2003; Heisler et al. 2005; Sauer et al. 2006; Scarpella et
al. 2006).
Meskipun data pada link langsung sangat miskin, distribusi asimetris hormon adalah
penting untuk pembentukan sel induk karena gangguan pola distribusi ini
menghambat
pembentukan organ baru (Okada et al 1991;. Liu et al 1993;. Hadfi et al 1998;.
Mattsson et al.
1999; Sabatini et al. 1999; Reinhardt et al. 2000; Tanaka et al. 2006; Larsson et al.
2008;
Dhonukshe et al. 2008). protein Arabidopsis PIN asimetris lokal di plasma
membran, yang atribut untuk pembentukan gradien auksin lokal (Tanaka et al.
2006). PIN1, salah satu protein pembawa auksin penghabisan, memulai dan
mempertahankan konsentrasi auksin
gradien (. Friml et al 2003; Heisler et al 2005.). Buatan promotor DR5 (Ulmasov et
al. 1997)
sangat responsif terhadap auksin aktif, dan lebih jauh lagi, ekspresinya dapat
diinduksi oleh
eksogen diterapkan auksin (Sabatini et al 1999;. Friml et al, 2002, 2003;. Benkov
et al 2003.;
Ottenschlger et al. 2003). Oleh karena itu, reporter didorong oleh DR5 dapat
digunakan sebagai wajar
perkiraan akumulasi auksin di sel yang berbeda dari jaringan, memungkinkan
visualisasi
Pola spatiotemporal distribusi auksin (Sabatini et al 1999;. Casimiro et al, 2001.;
Avsian-Kretchmer et al. 2002; Benkov et al. 2003; Friml et al. 2003; Ottenschlger
et al. 2003; de
Reuille et al. 2006). Dalam Arabidopsis, semua organ berasal dari meristem apikal
termasuk menembak
meristem dan akar meristem, sedangkan in vitro organ berasal dari sekelompok
undifferentiated
sel somatik, yaitu kalus yang dihasilkan seluruh dedifferentiation sel eksplan.
pertanyaan menarik muncul bahwa bagaimana auksin eksogen diangkut dari media
ke dalam eksplan
sel, dan apa pola distribusi auksin selama organogenesis in vitro.
kalus dan de novo perbungaan induksi, DR5 :: GUS digunakan sebagai penanda
untuk mendeteksi
distribusi auksin. Hasil penelitian menunjukkan bahwa distribusi GUS terkonsentrasi
di beberapa daerah di
kalus, yang mungkin membentuk promeristem perbungaan, pada 2 hari setelah
induksi (Cheng et al.
unpubl. data).
In addition to inflorescence regeneration, we induce the somatic embryogenesis in
vitro by
auxin (Su et al. unpubl. data). DR5::GUS expression pattern was analyzed during
somatic
embryogenesis, and the results indicate that the SE (somatic embryo) induction is
correlated with
the auxin asymmetric distribution. Further analysis of pPIN1::PIN1-GFP expression
pattern shows that efflux-dependent auxin gradients are established in the cells
those will outgrow to form
somatic proembryo (Figure 1D, E). In contrast, somatic embryos can be inhibited by
the
application of naphthylphthalamic acid (NPA), a type of auxin transport inhibitor
(data not shown).
Thus, auxin polar transport occurs during inflorescence and somatic embryo
induction, and
asymmetric distribution of auxin is necessary to de novo initiation of organ
primordia under
culture conditions.
Besides auxin, cytokinin is believed to be another important factor for
organogenesis. Therefore,
it is an interested question if cytokinin response is partitioned during organogenesis
in a fashion
like auxin. During de novo shoot regeneration, the expression of cytokinin
responsive
ARABIDOPSIS RESPONSE REGULATOR 5 (ARR5) reporter shows a dynamic change
pattern.
Accumulation of ARR5 mRNA is increased not only in callus initiated from root
explants but also
during the early stages of incubation on shoot-induction medium (SIM). Of course,
its expression
peaks at 6 days after incubation, and then begin to diminish at approximately the
time of cells fate
switch to shoot primordia, which is correlated to the presumptive site of shoot
formation (Che et al.
2002; Gordon et al. 2007). Thus, the distribution of cytokinin during in vitro shoot
organogenesis
is dynamic and asymmetric. More important, hormonal distribution pattern is
essential for cell fate
switch, although the response patterns of auxin and cytokinin during organ
regeneration may be
divergent
Selain regenerasi perbungaan, kami menginduksi embriogenesis somatik in vitro
oleh
auksin (Su et al. unpubl. Data). pola ekspresi DR5 :: GUS dianalisis selama somatik
embriogenesis, dan hasilnya menunjukkan bahwa SE (somatik embrio) induksi
berkorelasi dengan
auksin distribusi asimetris. Analisis lebih lanjut dari pola ekspresi pPIN1 :: PIN1-GFP
menunjukkan bahwa penghabisan tergantung gradien auksin yang didirikan pada
sel-sel yang akan mengatasi untuk membentuk
somatik proembrio (Gambar 1D, E). Sebaliknya, embrio somatik dapat dihambat
oleh
aplikasi asam naphthylphthalamic (NPA), jenis inhibitor transportasi auksin (data
tidak ditampilkan).
Dengan demikian, transportasi polar auksin terjadi selama perbungaan dan somatik
embrio induksi, dan
distribusi asimetris auksin diperlukan untuk de inisiasi novo dari primordia organ di
bawah
kondisi budaya.
Selain auksin, sitokinin diyakini menjadi faktor penting untuk organogenesis. Karena
itu,
itu adalah pertanyaan tertarik jika respon sitokinin dipartisi selama organogenesis
dalam mode
seperti auksin. Selama de novo tunas regenerasi, ekspresi sitokinin responsif
ARABIDOPSIS RESPONSE REGULATOR 5 (ARR5) reporter menunjukkan pola
perubahan dinamis.
Akumulasi ARR5 mRNA meningkat tidak hanya di kalus dimulai dari eksplan akar
tetapi juga
selama tahap awal inkubasi pada medium shoot-induksi (SIM). Tentu saja, ekspresi
puncak pada 6 hari setelah inkubasi, dan kemudian mulai berkurang kira-kira pada
saat sel nasib
beralih untuk menembak primordia, yang berkorelasi ke situs dugaan pembentukan
tunas (Che et al.
2002; Gordon et al. 2007). Dengan demikian, distribusi sitokinin selama in vitro
menembak organogenesis
adalah dinamis dan asimetris. , Pola distribusi hormonal yang lebih penting adalah
penting untuk nasib sel
beralih, meskipun pola respon auksin dan sitokinin selama regenerasi organ
mungkin
berbeda
Hormone signal transduction and its relation to cell fate determination
In most cases, the cells involved organ regeneration are not directly contacted with
the hormones
in the induction medium, thereby the hormone signal transduction through cells
might play a role
in the response of explant cells to hormones. The in vitro shoot induction from root
explants was
carried out by Valvekens et al. (1988). Explants acquire competence to respond to
shoot formation
signals on callus-induction medium (CIM) containing low levels of cytokinin to auxin.
After
transferred to SIM containing higher cytokinin levels for 6 days, explants become
committed to
shoot formation; and shoots emerge two days later (Valvekens et al. 1988). Gene
expression
profiles during in vitro shoot morphogenesis were investigated by a genomic
approach (Cary et al.
2002; Che et al. 2002, 2006). During preincubation on CIM, a number of auxinresponsive genes,
e.g., IAA1, IAA5, IAA9, and IAA10, are upregulated, followed by downregulation
upon transfer
from CIM to SIM (Che et al. 2002), suggesting that these genes involve in auxin
transduction during shoot induction.
Transcript levels of CKI1 and CRE1/AHK4/WOL are increased by transferred onto SIM
(Che et
al. 2002), implying both genes activities may be related to the acquisition of
competence. The
expression of two cytokinin primary response genes, ARR4 (ARABIDOPSIS
RESPONSE
REGULATOR 4) and ARR5, are also highly increased during shoot development (Che
et al. 2002).
During in vitro inflorescence induction, genes involved in cytokinin signaling, such
as AHK2,
AHK3, and several A-type ARRs, show inductive expression patterns, which may
participate in
inflorescence primordial commitment (Cheng et al. unpubl. data). Thus, it is likely
that a number
of genes, involved in auxin transporting and cytokinin signalling, are regulated by
hormones,
which might be important for in vitro organogenesis.
Stem cells are a group of cells that not only are able to form many differentiated
cell types, but
also to self-renew to maintain the relative stable cell number. In planta, stem cells
in meristems are
the origin of all organs including leaves, stems, and flowers during postembryonic
development
(Weigel and Jrgens 2002). The molecular mechanism of stem cell formation and
their
proliferation are reviewed in several papers (Weigel and Jrgens 2002; Scheres
2007). The WUS
gene encodes a homeodomain protein, and its protein activity results in signaling to
the overlaying
stem cells, which induces CLV3 expression. In turn, CLV3 protein interacts with the
CLV1/CLV2
receptor complex to limit the area of WUS expression in the underlying organizing
center. Thus,
the organization and functioning of the embryonic shoot apical meristem (SAM) in
Arabidopsis is
regulated by CLV3/WUS negative-feedback loop, which ensures homeostasis of the
SAM by
controlling the number of stem cells present in the central zones (Fletcher et al.
1999; Brand et al.
2000; Schoof et al. 2000; Weigel and Jrgens 2002). Different with that of
embryonic meristem,
both WUS and STM are needed for CLV3 expression during post-embryo
organogenesis (Brand et
al. 2002).The WUS and CLV3 genes are usually considered as markers for
organizing center cell
and stem cell identities in the SAM, respectively.
Apakah ada sel induk selama in vitro tanaman organogenesis?
Sel punca adalah sekelompok sel yang tidak hanya mampu membentuk berbagai
jenis sel dibedakan, tetapi
juga untuk memperbaharui diri untuk mempertahankan jumlah sel stabil relatif.
Dalam planta, sel punca di meristem adalah
asal dari semua organ termasuk daun, batang, dan bunga selama pengembangan
postembryonic
(Weigel dan Jurgens 2002). Mekanisme molekuler pembentukan sel induk dan
mereka
proliferasi ditelaah dalam beberapa makalah (Weigel dan Jurgens 2002; Scheres
2007). WUS yang
gen mengkode protein homeodomain, dan hasil aktivitas protein dalam sinyal untuk
overlay tersebut
sel induk, yang menginduksi ekspresi CLV3. Pada gilirannya, protein CLV3
berinteraksi dengan CLV1 / CLV2
kompleks reseptor untuk membatasi area ekspresi WUS di pusat pengorganisasian
yang mendasari. Demikian,
organisasi dan fungsi embrio menembak meristem apikal (SAM) di Arabidopsis
adalah
diatur oleh CLV3 / WUS negatif-umpan balik, yang menjamin homeostasis dari SAM
oleh
mengendalikan jumlah sel induk hadir dalam zona sentral (Fletcher et al 1999;.
Merek et al.
2000; Schoof et al. 2000; Weigel dan Jurgens 2002). Berbeda dengan yang dari
meristem embrio,
baik WUS dan STM diperlukan untuk ekspresi CLV3 selama pasca-embrio
organogenesis (Merek et
Al. 2002) .suatu WUS dan CLV3 gen biasanya dianggap sebagai penanda untuk
mengorganisir sel pusat
dan batang identitas sel di SAM, masing-masing.
Previous studies demonstrated that the shoot organs during postembryonic
development
ultimately derive from as little as 6-9 founder cells, namely the stem cells (Stewart
and Dermen
1970; Laux 2003). During de novo organogenesis, the competent cells in cultured
explants are
canalized and determined for specific organ formation under cultured conditions.
The comparison between organ formation in vivo and in vitro raises the question of
whether there are the stem cells
during in vitro organogenesis. If it is case, how the stem cell does form in callus?
During in vitro
shoot organogenesis, WUS expression is upregulated in the callus at 3 days after
induction on SIM
(Cary et al. 2002; Gordon et al. 2007). Further observation shows that WUS is
initially expressed
in cells peripheral to shoot meristem progenitor cells, but is later upregulated within
the center of
the phyllotactic shoot meristem (Cary et al. 2002; Gordon et al. 2007). The
expression pattern of
CLV3 was also investigated during de novo organogenesis. No signal of the CLV3
reporter was
observed in shoot progenitor cells. However, when the WUS reporter was
upregulated within the
center of the new meristem, CLV3 reporter was also detected. These studies
suggest that shoot
mekanisme pembentukan sel-sel induk tidak jelas di kalus, jelas bahwa sel-sel induk
merupakan kontribusi
untuk menembak organogenesis. Dalam embriogenesis somatik, gen CLV3
dinyatakan dalam somatik
bakal embrio pada 2 hari setelah induksi, dan kemudian, sinyal yang dibatasi untuk
promeristem yang
antara kotiledon promodia. Akhirnya sel-sel ini diposisikan di SAM embrio somatik.
Menjelang gen CLV3, gen WUS diaktifkan dalam jaringan embrio pada 24 ~ 36 jam
setelah
induksi. Dan kemudian transkrip yang terakumulasi dalam sel promeristem
menembak dan sel-sel meristem dari
embrio somatik (Su et al. unpubl data.). Domain ekspresi WUS adalah tepat di
bawah CLV3 sebuah
Ekspresi satu (Gambar. 1F).
During the in vitro inflorescence regeneration, Both WUS and CLV3 reporter signals
were
observed in callus at 4~5 days after induction. Further co-localization analysis of
both genes
shows that the WUS expression domain is just below the CLV3 expression one,
suggesting that
both WUS and CLV3 expression may imply promeristem formation within the callus
(Cheng et al.
unpubl. data). These results indicate that both genes are responsible for new stem
cell formation.
In addition, the cell competences to WUS activity in different microenvironment are
various. And
the combination of WUS signal with different environmental factors could activate
different
downstream genes (Xu and Chong 2005), leading to produce various types of cells.
Selama in vitro perbungaan regenerasi, Kedua WUS dan reporter CLV3 sinyal yang
diamati pada kalus pada 4 ~ 5 hari setelah induksi. analisis co-lokalisasi lebih lanjut
dari kedua gen
menunjukkan bahwa domain ekspresi WUS adalah tepat di bawah CLV3 ekspresi
satu, menunjukkan bahwa
baik WUS dan ekspresi CLV3 mungkin menyiratkan pembentukan promeristem
dalam kalus (Cheng et al.
unpubl. data). Hasil ini menunjukkan bahwa kedua gen bertanggung jawab untuk
pembentukan sel induk baru.
Selain itu, kompetensi sel untuk aktivitas WUS di lingkungan mikro yang berbeda
berbagai. Dan
kombinasi sinyal WUS dengan faktor lingkungan yang berbeda bisa mengaktifkan
berbeda
gen hilir (Xu dan Chong 2005), yang mengarah untuk memproduksi berbagai jenis
sel.
Genes involved in organ identity
Organ identity genes play a key role during organogenesis in planta. Molecular
analysis of de novo
organogenesis shows that inductive expression of organ identity genes is essential
for organ
regeneration under cultured conditions (Li et al. 2002; An et al. 2004; Xu et al.
2004; Guan et al. 2006; Gordon et al. 2007). During shoot regeneration, a number
of genes are required for SAM
formation, such as SHOOT MERISTEMLESS (STM), CUP-SHAPED COTYLEDON 1 and 2
(CUC1 and CUC2) (Cary et al. 2002; Gordon et al. 2007). Cytokinin can stimulate the
expression
of STM which is involved in shoot meristem formation in tissue culture (Rupp et al.
1999; Teo et
al. 2001). CUC1 and CUC2 are upregulated during callus formation on CIM (Cary et
al. 2002;
Gordon et al. 2007). CUC2 is also upregulated after the callus is transferred onto
SIM, and its
expression reaches peak at 6 days. At this time, clusters of small dividing CUC2positive cells,
which will develop into new shoot meristems with high frequency, were observed in
some regions
of callus while absent from others (Cary et al. 2002; Gordon et al. 2007)
Gen yang terlibat dalam identitas organ
gen identitas organ memainkan peran kunci selama organogenesis di planta.
analisis molekuler de novo
organogenesis menunjukkan bahwa ekspresi induktif gen identitas organ penting
bagi organ
regenerasi dalam kondisi berbudaya (Li et al, 2002;.. Sebuah et al 2004; Xu et al
2004;. Guan et al 2006;. Gordon et al 2007.). Selama regenerasi tunas, sejumlah
gen yang diperlukan untuk SAM
pembentukan, seperti SHOOT MERISTEMLESS (STM), CUP-BENTUK kotiledon 1 dan 2
(CUC1 dan CUC2) (Cary et al, 2002;. Gordon et al 2007.). Sitokinin dapat
merangsang ekspresi
STM yang terlibat dalam pembentukan meristem tunas dalam kultur jaringan (Rupp
et al 1999;. Teo et
Al. 2001). CUC1 dan CUC2 yang diregulasi selama pembentukan kalus pada CIM
(Cary et al 2002.;
Gordon et al. 2007). CUC2 juga diregulasi setelah kalus ditransfer ke SIM, dan yang
Ekspresi mencapai puncaknya pada 6 hari. Pada saat ini, kelompok membagi kecil
sel CUC2-positif,
yang akan berkembang menjadi meristem tunas baru dengan frekuensi tinggi, yang
diamati di beberapa daerah
kalus sementara absen dari orang lain (Cary et al, 2002;.. Gordon et al 2007)
In Hyacinthus, induced expression of HAG1 by high level phytohormones promotes
the
regeneration of floral buds, whereas, low level phytohormones could activate
HoMADS1 that is
responsible to formation of in vitro ovule (Li et al. 2002; Xu et al. 2004). The
induction of TFL1 is
critical to Arabidopsis inflorescence regeneration (Guan et al. 2006). Promoter
analysis shows that
there are three auxin response elements, suggesting that auxin may activate TFL1
expression
(Woodward et al. 2005; Guan et al. 2006). Although the direct link between
phytohormones and
organ identity genes is not elucidated, the organ identity genes might be activated
by exogenous
phytohormones, and then, their expression regulates the cell fate of progenitor
cells. The study on
a versatile floral induction system provides further evidence for this conclusion. For
example,
ectopic overexpression of LEAFY in callus directly induces formation of flowers from
root
explants bypassing normal vegetative development, indicating that the genes
involved in organ
identity play an important role during the in vitro organogenesis. But the pathway
throughout
which the organ identity genes are acitivated by phytohormones remains unclear.
Dalam Hyacinthus, ekspresi diinduksi dari HAG1 oleh phytohormones tingkat tinggi
mempromosikan
regenerasi tunas bunga, sedangkan, phytohormones tingkat rendah bisa
mengaktifkan HoMADS1 yang
bertanggung jawab untuk pembentukan in vitro ovula (Li et al, 2002;. Xu et al
2004.). Induksi TFL1 adalah
penting untuk Arabidopsis perbungaan regenerasi (Guan et al. 2006). Analisis
promotor menunjukkan bahwa
ada tiga unsur respon auksin, menunjukkan auksin yang mungkin mengaktifkan
ekspresi TFL1
(Woodward et al 2005;. Guan et al 2006.). Meskipun hubungan langsung antara
phytohormones dan
gen identitas organ tidak dijelaskan, gen identitas organ mungkin diaktifkan oleh
eksogen
phytohormones, dan kemudian, ekspresi mereka mengatur nasib sel sel progenitor.
Studi pada
sistem induksi bunga serbaguna memberikan bukti lebih lanjut untuk kesimpulan
ini. Sebagai contoh,
berlebih ektopik dari LEAFY di kalus langsung menginduksi pembentukan bunga dari
akar
eksplan melewati pengembangan vegetatif normal, menunjukkan bahwa gen yang
terlibat dalam organ
Identitas memainkan peran penting selama in vitro organogenesis. Namun jalur
sepanjang
yang organ gen identitas yang acitivated oleh phytohormones masih belum jelas.