2023-04-25

Agrobacterium을이용한 binary vector

• Crown gall disease à Auxin과 cyokinin이 계속 발현됨
• 1. chemoattraction &virulence activation
• 2. T-DNA excision à T4SS를 통해 plant cell로 진입 Water movement
• 3. nuclear import and integration 1. Water potential
2. Absorption in root – casparian strip
3. Movement through stem – tension-cohesion model
Vir gene product를 이용해plant
nucleus로transfer될수 있음. 4. Transpiration – guard cell, Cartenoid, blue light

이 외에도PEG나 particle gun을

이용해서transformation 가능

Water movement in roots Transpiration

• Apoplast transport – Cell wall. Membrane 지나가지 않음.
• Symplast transport – plasmodesmata
• Transmembrane transport – cytoplasm and cell mb.
• Casparian strip: back diffusion을 억제함.
• Plasmodesmata: channels for virus
• Solute의 농도 차이로 stomatal movement가 변화함
• à H+pump가 hyperpolarized 시킴: cell 외부가 산성으로변화함
• Voltage-gated potassium channel이 열림 à K+ influx
• Malate2-made by GC, Cl-diffuses to balance K+
• à water enters the cells, increasing turgor pressure
• Blue light가 많이 관여함 – blue light 세기가 강할수록 H+ pump가 더
일을 잘하는 듯함... (pH가 감소함)
• Cartenoid: light photoreceptor로써 활동함.

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 2023-04-25

1. ROS (Reactive Oxygen Species)
: 대사중간에발생. 원치 않은반응을일으킴.
Antioxidant systems are induced by dehydration stress
antioxidant —> photosynthesis동안excited electron들을
2. Water deficit
Salinity, flooding, high temperature, chilling, freezing
The severity of water deficit affects plant responses
soil moisture가critical potential threshold 아래로내려가자
증산작용과광합성이작용이확 낮아짐
Perception → signaling → response

Abiotic stress 만들어냄;

dehydration stress이광합성을방해함—> excess energy
accumulate가 ROS를 형성함
signaling: ABA
water deficit —> turgor sensor —> ABA biosynthetic
enzyme —> ABA —> altered ion channel activity /
광합성을방해했을때 ROS가 형성됨 transcription factor activation —> altered hydraulic
Enzymatic and nonenzymatic antioxidants detoxify conductnace
ROS ⇒ growth (cell division and expansion)
SOD, catalase, APX, GPX, glutathione reductase, Vitamin ABA → SnRK2(kinase) —> AREB/ABF phosphorylation —
C, Vitamin E, Carotenoids > stress tolerance
ABA —> SAPK/OSRK1(kinase) —>
TRAB1/OsABI5/OREB1 phosphorylation —> stress
tolerance

3. Light stress -shade

under the shade condition —> low R/FR ratio, low red light, low blue light Low K+ stress à calcium signaling
—> shade avoidance
Shade avoidance response CIPK23-CBL1 or AKT1
K+ tolerant
ATHB2(= Arabidopsis Thaliana Homeobox) Low K+ stress
CBL9 phosphorylation Iks1
phenotype
*calcium sensor:
—> light quality change에 따라조절됨. CBL1 or CBL9
(TF)

Low R/FR일 때 많이발현됨.

Yeast two-hybrid 실험

Chloroplast-to-nucleus signaling
Plant responses to water deficit affect many processes Retrograde signaling: chloroplast에서nucleus로의signaling. Stress가있을때 전달됨.
광합성감소 In chloroplast, at
shot meristem 감소, leaf growth 감소 least four distinct
GUN1으로 신호 LHCB expression 억
plastid processes GUN1, 핵으로 이동 ABI4 발현
root growth 증가 generate plastid
converge됨 제됨

stomatal aperture 감소 signal.

여기에서ABI4나, CAB 유전자들은retrograde signaling의screening 용으로사용할수 있음.

A유전자관여여부확인위해돌연변이식물, 와일드타입식물에CAB 도입. 돌연변이A식물이

정상작동하면A 유전자는retrograde signaling 관여.

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Epigenetic mechanism – flowering

FLC
Florigen(FT) à leaf에서 apical meristem으로이동하는mobile protein. 꽃 피는부위에서자극함.
FT와FD 모두TF임.

몇몇식물들은꽃을피우기위해vernalization (춘화처리, cold period) 과정이필요함. (FLC때문)

FLC: inhibitor of flowering. Vernalization 과정이후silence됨.
The Rhizobia-legume
왜냐하면FLC gene에 epigenetic modification이 일어나기때문인데, heterochromatin을 함.
H3Acà H3K27me, H3K9me symbiosis
ABA signaling in stomatal guard cell closing
PYR à PP2C 활성화 억제 OST1 activated Anion channel
ABA OCPK activated à
Activated NADPH oxidase activated à Stomatal
binds to PP2C는 원래 phosphatase --> NADPH oxidase
ROS formation efflux of anion, closure
PYR SnRK2의 phosphorylation activation
[Ca2+] increase K+ efflux

Infection processes – early signal exchange

1.soybean의 flavonoid를 rhizobia에 노출시킴.
식물의 flavonoid(luteolin)가rhizobia의NodD gene과결합하여 Nod gene을발현시킴. 2.flavonoid가 rhizobia안에 들어가 Nod D와 결합하여 nod
gene을 발현시킨다.
Nod Factor는lipid moiety, chitin, sugar reside가존재함. 이 다양한 backbone은host specificity를결정함.
3.gel shift assay를 실행하여 nod D의 양을 늘려줄수록 free
DNA의 양보다 binding 된 DNA가 많아지는 것을 알 수 있다.
Nod Factor는식물에게 분비되고, 이는 NFR(node factor receptor) root hair curling을유도하게 됨.
결론: nod D의 양을 늘려주었을 때 nod gene이 더 많이 발현되어
Specificity: root hair curling에들어가서, infection thread를만들 수 있는박테리아는 한정적임. nod D의 양이 증가한다는 것을 알 수 있다.

이후 SYMRK gene이발현되고, Ca2+spiking이 일어남.

CCaMK(Ca2+binding protein)이cytokinin을발현시킴 à cell division이일어남.

internalization of Rhizobium: endocytosis via Rab7

Bacteroid로 분화하고, 이는symbiosome이라는식물세포에둘러싸여있다.
Symbiosome은 peribacterial membrane으로이루어져있음.
Nitrogen fixation: bacteroid 내부에서일어나고, 암모니아를형성함. 암모니아는A.a.로 assimilation됨
(GS, GOGAT).
박테리아의Nitrogenase는oxygen에 의해inactivation되기 때문에, 식물의Leghemoglobin을 사용하여
산소분압을낮추면서도산소를공급받음.