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Embryology (発生学)






Mount Usu / Sarobetsu post-mined peatland
From left: Crater basin in 1986 and 2006. Cottongrass / Daylily

Developmental biology

the study of the process by which animals and plants grow and develop, including regeneration, asexual reproduction, metamorphosis and the growth and differentiation of stem cells

animal
plant

Growth and development

Growth (成長)
irreversible change in mass
索引
Development
irreversible change in state

embryogenesis (胚形成)
juvenile
adult vegetative
adult reproductive

Animal developmental biology (動物発生学)


1677 Leeuwenhoek: discovered sperm by a microscope

Ham, Johan: brought in gonorrhea ejaculate to Leeuwenhoek

Preformation and epiformation (前成説と後成説) ☛ view of life

Homeobox (ホメオボックス)

DNA sequence, ≈ 180 base pairs regulating development and differentiation in the early stages of embryonic development
Gehring, Walter Jakob (1939-2014), Drosophila genetics and development

cell determination in the embryo

1983 Gehring et al.: discovered homeobox

Gametogenesis (配偶子形成)


Testis and sperm (精巣・精子)

Sperm or spermatozoon (pl. –a) (精子)
The morphology is different at species level. Sperms, mostly consisting of nucleus, contain least nutrients but have high mobility.
Fig. 32 Fig. 32. Diagram of primitive metazoan spermatozoon (Franzen 1956). A: acrosome, H: head piece, M: mitochondrial body, PP: principal piece, MP: middle piece, T: tail section, EP: end piece, C: centriole, N: nucleus. [Sperms of Gymnosperms: Ginkgo biloba and Cycas revoluta]

Egg ()

Organization of egg cytoplams (卵細胞質)

egg Fig. 63. Redistribution of cytoplasmic inclusions in the egg of Cynthia partita following centrifugation. a, Egg immediately after centrifugation; b, centrifuged egg 22 hours later (the yellow granules, shown black, have taken up a subequational position on one side of the egg). 1st m, Spindle of first meiotic division. (Conklin 1931)

egg
Fig. 1. The four stages of egg maturation at which fertilization occurs in the animal kingdom. (Dalcq 1952)
Table. Chemical composition of the jelly coat (卵ゼリー) of echinoderm and amphibian eggs (% dry weight). +, qualitative estimation only; ?, to be controlled; G, glucose; Gl, galactose; M, mannose; F, fucose; X, xylose; Fr, fructose; Ga, glucosamine; Gla, galactosamine; TN., total nitrogen.

Species                             
Arbacia lixula
Echinus esculentus
Paracentrotus lividus
Strongylocentrotus
    droebachiensis
S. purpuratus
Sphaerechinus grarrularis
Heliocidaris crassispina
Echinarachnius parma
Hemicentrotus pulcherrimus
Pseudocentrotus depressus
Echinocardium cordatum
Brissopsis lyrifera               
Rana temporaria
R. esculenta
R. japonica
R. clamitans
Discoglossus pictus
Bufo bufo
B. vulgaris formosus
Axolotl
Triturus cristatus                  

 G  GL   M     F    X   Fr  Ga   Gla   TN  SO4
  +   +     +     +
       +
4.6        3.8 24.3 0.8                       4.7 20.9
       +            +                                      25.0

 25                                                  5.7   23
  +          +     +
                     +
                                  +
                     +
                     +
                   32.7                             4.1 20.5
                                                              8.1  
  ? 12.7 3.5  7.1   ?         8.9   9.5   8.1
       +            +                  +     ?     9.3
     28.0         ?                       14.0  8.8
  ?   ?      ?    +                         +
  +   +      +   1.7                     16.5 10.0
       +      +  10.4                   20-40 7.6
     30.0         ?                       20.0   8.4
               +    +                         +      8.3
     12.2 1.3   6.2                    20.3 10.0        

Lampbrush chromosome (ランプブラシ染色体)

egg Fig. 3.2. Part of a chromosome from a growing egg cell (oocyte) in the ovary of a salamander showing some of the variety of different kinds of “lampbrush loops” that are formed along the chromosome.

Fertilization (受精)


Theories on fertilization

1. Fertilizin theory (Lillie FR) - negative
2. Lysin corrective factor theory (Loeb)
The issues on Loeb theory: conjunction of sperm and egg (Epel)
Exp. in jelly of sea urchin
sperm → Ca++ permeability (capacitation) → [Ca++ influx] → intracellular Ca++ level, acrosome reaction
→ acrosome

acrosin → dissolution of envelope
lindin → attachment → membrane fusion

→ [Na influx* (phisiological change) ↔ H efflux**]

└* fertilization potential (fast block against polyspermy)
└* intracellular Ca++ level → (a) (b)
└** alkalization of cytoplasm

└ actin polymerization → displacement of cytoplasmic
____________________ constitutions
└ activation of various enzymes
__(ca pH 7.2 = optimum pH for various enzymes)
mRNA is unmasked by this mechanisms (unmasking)

Ex. DNA polymerase → chromosome formation

(a) → cortical vesicle (exocytosis)

└ veteline delaminase → fertilization membran
_____________________(block against polyspermy)
└ sperm receptor hydrolase
_______(attacks binder and slows block against polyspermy)

(b) → oxidation of unsaaturated fatty acid (Ca-activated enzyme)

→ O2 uptake

Table. O2-consumption of unfertilized and fertilized eggs
Species (Stage of mat. division at the time of fertilization) =

(O2 uptake of fertilized egg)/(O2 uptake of unfertilized egg)


Phallusia mamillata = 2.0
Rana platyrrhina = 1.0
Bufo bufo (metaphase of
    2nd mat.) = 1.0
Fundulus heteroclitus (division) = 1.0
Oryzias latipes = 1.0
Fucus vesiculosus = 1.9
Strongylocentrotua purpuratue = 3.7
Psammechinus miliaris = 3.6
Paracentrotus lividus = 4.7
Arbacia punculata = 4.5
Dendraster excentricus = 3.0

Nereis succinea = 1.3
N. limbata Germinal vesicle = 1.35
Mactra laterialis = 1.8
Urechis caupo = 1.2
Cumingia tellinoides = 0.45
Chaetopterus variopedatun = 0.53
Marthasteriaa glacialis = 1.0
Saxostrea commercialis
    (metaphase of 1st mat.) = 1.0
Sabellaria alveolata (division) = 1.1
Asterias glacialis = 1.0
Ciona intestinalis = 1.5

Table. Fertilizability of Asterias eggs at various pH values
              HCl                            NaOH
Concentration  N/   N/   N/   N/  N/ Normal   N/   N/    N/   N/  N/
in sea water  500 1000 2000 4000 104 sea     104  4000  2000 1000 500
                                     water
Cleaved egg %  0   2.5  8   22.5 45    53     84  92.5  88.5  89   0

Cleavage (卵割)


cleavage Fig. 60. Displacement of organ rudiments in an embryo of Cynthia partita as a result of centrifugation of the egg. n, neural system; p, eyp pigment; ch, autochord; ect, ectoderm; ms, muscle cells. (Conklin 1931)







Table. Cleavage of frog eggs by the injection of tissue (testis) extract and of various fractions of the same obtained by centrifugation.

Preparation________Eggs reaching blastula stage (%)
Whole tissue extract__________94
Large granules______________35.2
Medium and small granules____19.5
Supernatant_________________0
Phosphate buffer_____________0.3
Pricked "dry"________________ 0

Table. Nucleic acid content in nuclei during gametogenesis and cleavage in Chaetopterus


Stage             
1st polar body
Sperinatozoid
Cleavage
    interphase
    prophase
    telophase  

Amount
of DNA  
127 ± 3
  61 ± 1

210 ± 9
263 ± 10
124 ± 3  

Condition of
nucleus       
    2n
    1n

    2n-4n
    4n
    2n            

Asymmetric property (非対称性)

1928 Spearman

Spearman
Fig. 13. "Retarded nucleation" in Triton. Zygote nucleus pushed to one side by the constriction of the egg. (a) first cleavage. In the nucleated half; (b) further cleavages In this part, the other half Is still imcleaved; (c) passage of one nucleus from the nucleated into the non-nucleated half, beginning of development In the latter; (d) the two embryo. that developed from the constricted (収縮された, 窄まった) egg; the one produced by the half In whIch nucleation was retarded is considerably younger, but normally built. (Spemann and Tannhauser 1938)

1953 Briggs & King: Rana pipiens

Organogenesis (器官形成)


Development of gonad (生殖腺発達)

1. Origin of primordial germ cell (gamete) (始原生殖細胞の起源)
organogenesis
Fig. Diagrams showing the displacement of the germinal cytoplasm during the early development of Bufo regularis.
2. sex differentiation (性分化)

Pre-embryo (初期胚)

= early embryo or nascent embryo
blastula
Fig. Diagrammatic comparison of blastulae (胞胚) of an echinoderm (a), a frog (b), a bony fish (c) and a bird (d).

Gastrulation process (嚢胚形成過程)


gastrula
Fig. A, B, development of an embryo with animal pole material graft placed in the grey crescent. A, during gastrulation; B, after neurulation. nt, neural tissue, ntt, non-neural tissue, C, D, E, development of an embryo with grey crescent graft at ventral margin. In C embryo shows two neural plates and dorsal lips, one induced, the other normal, D, E show two extreme types of embryo which are produced by these grafts, after neurulation.
sea urchin
Fig. Segmentation and gastrulation in sea urchin. a) fertilized egg, b) 2 blastomere stage, c) 4 blastomere stage, d) 8 blastomere stage, e) 32 blastomere stage, f) blastula (appearance from above), g) blastula (longitudinal section), h) arising of first mesenchyme cells, i) gastrula occurrence. Bld-blastoderm: 1.a.g. first abdominal gap, 1.m.c. first mesenchyme cells.
sea urchin
Table. Chemical composition of sea urchin embryo (% wet weight). (subs = substances. carbs = carbohydrates)

substance           water  dry    protein     total   glycogen   fat    ash
                                      subs  nitrogen  carbs                                  
Unfertilized egg    77.3   22.7    10.7      5.43       5.13     4.82  0.34
Blastula (12 hrs)   77.3   22.7    10.2      5.46        4.8      4.43  2.07
Pluteus                 78.8   21.2      9.7       3.4       traces   3.69  3.56

Table. Chemical composition of the egg and embryo of a frog (mg)

Substance                                                       Egg   Hatched tadpole
Total nitrogen                                                   162            159
Extractable nitrogen (N of active protoplasm)   40            42.8
Nonprotein nitrogen (in nucleic acid?)                4              5.0
Total carbohydrates                                          104             58
Glycogen                                                          73.2            31

Table. Chemical composition of the eggs and later stages of development in three species of dog fish (g)
Species              Scyllium canicula  Mustellus vulgaris  Mustellus laevis
                           (oviparous)           (ovoviviparous)       (viviparous)        
                           Egg    Young         Egg    Young           Egg    Young      
Total weight       1.3      2.7               3.9     60.6              5.5     189.0
Organic
    substances    0.61   0.48             1.9       8.9              2.8       32.0
Water                 0.68   2.15             1.9     49.8              2.6     152.0        
mammal (哺乳類)

Neurula (neurula stage, 神経胚)


ectoderm: epidermis, neural tissues, neural crest cell
mesoderm: 体節somite → muscle, nephorotome → excretory system, lateral plate → somatic layer, parietal l.
extraembryonic structures (胚体外膜)

yolk sac (卵黄嚢), amnion (amniotic sac, 羊膜), chorion (漿膜), allantois (尿膜)

archenteron (primitive gut, 原腸)
enteric (gut tract, 腸管)
neurula
neurula
Fig. 78. Gastrulation of Amphioxus. A series of consecutive stages (median sections). (Conklin 1932)

Neural induction (神経組織誘導)

gastrula
Fig. Diagrammatic illustration of the morphogenetic movements. (a) of normal gastrulation, and (b) of exogastrulation. c, main differentiations in an exogastrulated embryo. (Holtfreter 1933)
gastrula
gastrula
gastrula

Developmental fate (発生運命)


1920-30 Spemann: completed the fate map a few years before

discovered organizer, by using hair loop, glass needle, pipette, etc.

Organizer (形成体)
= dorsal lip
1) neural induction (神経誘導) – mesoderm, endoderm
2) organization center of embryo

neural induction
embryonic induction
primary induction
⇒ not only dorsal lip but also the others can be organizer

Analysis for nature of induction (誘導特性解析)

Technical
grafting in vivo (A, B) → tissue (organ) culture system in vitro (C, D)
fate
Figure 165. Four methods of exposing gastrula ectoderm to induction (diagrammatic). A, Transplantation of part of dorsal lip (stippled) into the ventral marginal zone, where it invaginates and forms a secondary archenteron. B, Transplantation of part of dorsal lip (stippled) into the blastocoele through a slit at the animal pole. Graft is eventually pressed against the ventral ectoderm. C, "Sandwich experiment": inductor (such as a centrifuged tissue homogenate) placed between two pieces of presumptive gastrula ventral ectoderm. D, Cultivation of a piece of presumptive ventral ectoderm of a gastrula in a watch glass filled with fluid containing inducing substance in solution. The reacting ectoderm is held between two layers of supporting material (silk) to prevent it from curling.
fate
Fig. Cell-transfer experiment

formation of limb (or wing) on vertebrate (脊椎動物における肢(羽)形成)


Renal system (腎臓系)

developed from nephrotome, showing the relationship between ontogeny and phylogeny
renal system
pvc: posterior vena cave, bc: blood corpuscles, md: mesonephric (Wolfflan) duct, ga: gonad anlage, pp: presumed path of primordial germ cells to gonad anlage

gonad (♂/♀) (mesodermal) ┅┅┅ different origin
_________↑ incursion_______
_________primodial germ cells (始原生殖細胞) → sperm or egg
┣ ovary ━━━━━┓
┗ testis ━━━ sperm or egg determined by the surrounding tissues
__⇑ hormones
[testis, mesonephros – urinary duct, Wolffian duct]
[ovary, pronephros – Müllarian duct, oviduct]

Metaphros (kidney, 腎臓)
mesodermal epithelium, mesodermal mesenchyme

renal system vreteric bud - mesencyme - branching - collecting duct
(尿管芽)______┗━━━━━┛
_____________condensation (forming duct)
______________________→ uriniferous tubule
renal system
a) epithelium: singular separation (分離単独) → no differentiation / mesenchyme: singular separation → no differentiation
b) ureter: dependent on metanephic mesenchyme
c) uriniferous tuble [replacement is possible to salivary gland epithelium, spinal cord]: not dedepnent on ureter (ureteric bud)
⇒ the formation of urinary duct is "permissive"
mesenchyme: cell adhesion is weak prior to the formation of ureteric bud

Pattern formation (パターン形成)

1) limb field and limb area
1910 Harrison: Salamander tail bud

a) destroyed 1/2 limb bud → normal limb
b) slit limb bud vertically → duplicated limb
c) combined 2 lim buds in harmonous orientation → normal limb
d) rotated limb field at 180o and transplanted →

AP-axis: neural tube closure stage / DB-axis: tail but stage

Proximal-distal: forming limb (need re-consideration)

2) limb bud – polarity

A-P / D-V, somatic layer mesoderm, epidermis epithelium

3) mesodermal-ectodermal interaction

Synthesis (合成)


synthesis
synthesis
Fig. 86. Changes in the synthesis of various classes of nucleic acids in oogenesis, at fertilization, and during early development in the frog embryo. (Gurdon 1968)

+/O (normal)    ×         +/O (normal)
      ┏━━━━╋━━━━┓
     +/+             +/O           O/O
  normal        normal   poor regeneration
                                   (males = sterile / females = maternal effect)
        ♀ O/O         ×         ♂ (+/O)
            ┏━━━┻━━━┓
          +/O                        O/O
arrested gastrula    arrested gastrula

Fig. Maternal inheritancee and manifestation of the o gene in the axolotl (Briggs & Justus 1968)

Immunological response and immune system (免疫反応と免疫システム)


Immunity (免疫): the state or quality of being resistant to a particular infectious disease or pathogen
Table 17. Properties of non-IgM immunoglobulins of lower vertebrates.
Species (Class)                Intact     Light   Heavy CHO(%) Designation  Formula  
Lung fish (dipnoi)             120000  22000  38000    ND           IgN             L2υ2
Bullfrog (amphibian)       (150000) 22000  53000    2.0           IgG?           L2γ(?)2
Marine toad (amphibian) 160000   22500 53000    4.2           IgG?           L2γ(?)2
Xenopus (amphibian)          ND     22000  53000    ND           IgG?           L2γ(?)2
                                          26700  64500
Turtle (reptile)                  180000  22500  67500                    IgG(Y)        L2γ(Y)2
                                        120000  22500  38000     0.9           IgN             L2υ2
Sleeply lizard (reptile)     151000  22400  51000     ND           IgG?           L2γ(?)2
Duck (avian)                    178000  22400  68000     5.0          IgG(Y)         L2γ(Y)2
                                        118000  23000  35000     0.6           IgN             L2υ2
Chicken (avian)               174000  22500  67500  2.2(HEX)    IgG(Y)        L2γ(Y)2
                                             ND       ND      ND       ND           IgA             (L2α2)
Echidna (monotreme)      150000  22500 49000  2.0(HEX)    IgG             L2γ2      

Cell-mediated immunity (cellular immunity)

Ab: overlays on cell surface – Ag-receptor

Humoral immune response: plasma cell → Ig circulation
Cellular immune resposne: lymphocytes → Ab

Genetic basis of cellular immune response


                                              Accept   Reject

    1) isograft (= autograft), isogenic          O
    2) allograft                                         O
    3) AA × BB (P)         Transplant P → F1   O
       → AB (F1)           F1 → P                      O*
    4) → AA, AB, BB        F2 → F1             O

*: reject the substances that have not present in itself

Plant developmental biology (植物発生学)


Morphogenesis (形態発生)

____seed germination
________
________formation and growth of stem and root ←
________↓ senescence and abscission_________
________↓ bud dormancy →→→→→→→→→→→
________formation of flower
________↓ senescence and abscission
________fruit set →→→→→→→→ seed formation
_________________________________
________growth and riping of fruit______
____←←←←←←←←←←←←←← seed dormancy
development

Polarity (極性)

young: shoot apical meristem (root as well as stem)
young: root apical meristem

meristem (meristematic tissue) - absent in animals

Plant movement (植物の運動)

Embryogenesis (胚形成)

= embryo formation

[ hormones ]

Plant hormones (植物ホルモン)


1959 Jacobs WP: PESIGS rules (hypothesis - not always accepted)

P (parallel variation) - correlated to physiological changes
E (excision) - terminated physiological processes after the removal
S (substitution) - physiological processes are resumed by exogenous addition of substances
I (isolation) - the reaction is activated by exogenous substances
G (generality) - the substance and reaction widely observed
S (specificity) - not be replaced by other natural substances
→ When these conditions are satisfied, the hormone (or related substance) works physiological

Hormonal interactions
E1 and E2: effect of horomones 1 and 2 (H1 and H2), respectively

H1 + H2E1 + E2 (additive) vs E1 × E2 (synergistic)

One-way action (interactions among hormones should be clarified)

Ex. high concentration auxin → ethylene production↑

→ the specific enzyme(s)↑ → inactive bound auxin↑
→ maintaining low-leveled auxin in vivo

1. Auxin (オーキシン)

IAA extraction
1934 Kögl, Erxleben & Haagen-Smit (Utrecht): from human urine
1934 Kögl & Kostermans: from yeast
1935 Thimann: from Rhizopus
1946 Haagen-Smit et al.: 27-mg IAA from 100-kg immature tomato

IAA crystallized - confirmed that IAA is a hormone

Natural auxins = endogenous, indole acetic acid (IAA, インドール酢酸)
Synthetic auxins: Ex. 2, 4-dichlorophenoxyacetic acid (2, 4-D), 2-methoxy-3, 6-dichlorobenzoic acid (dicamba)
Synthesis
young developing leaves
terminal buds, growing axillary buds
coleoptile tips
Function
bind receptor protein in plasma membrane
transport into cell
activate ATPase in plasma membrane
H+ ion extrusion
acidify cell wall
break hemicellulose-pectin bonds
cellulose microfibrils slide apart
cell enlarges
Antiauxin (抗オーキシン): a plant substance that opposes or suppresses the natural effect of an auxin
Changes in RNA metabolism induced by auxin
Table. Requirement of nucleoplasimic factor for enhanced RNA synthesis by IAA (Unit = 3H-UMP incorporated (c.p.m))
Incubation system (Unit)                                                                     
complete (336)
complete - DNA (56)                 complete + IAA (328)
nucleoplasm only (72)               nucleoplasm + IAA (68)
complete + nucleoplasm (412)  complete + nucleoplasm + IAA (727)
Table. Simulation of RNA synthesis by IAA and acceptor protein in vivo
Incubation system_β, γ32P-ATP incorporated_3H-UMP incorporated
________________(pmol/mg of enzyme)____(nmol/mg of enzyme)
Complete_______________________60______________6.3
Complete + acceptor protein________35______________4.2
Complete + acceptor protein + IAA__108_____________14.8
Table. Comparison of RNA polymerase activity by incubation of plasma membrane in 0.1 μM 2,4-dichlorophenoxyacetic acid, indoleacetic acid, or 3,5-dichlorophenoxyacetic acid.

[3H]-UMP incorporated (pmol/30 min/mg protein)           
RNA polymearase = 88    + PM-IAA = 146
                                         + PM-2,4-D supernatant = 138
                                         + PM-3,5-D supernatant = 96  

PM, plasma membrane fraction

Table. Enhancement of RNA polymerase activity by incubation of plasma membranes in 2,4-dichlorophenoxyacetic acid. Numeral = [3H]-UMP incorporated (pmol/30 min/mg protein)

RNA polymerase____________________________________ 72
+ PM suspended in TGMED__________________________ 162
+ Repelleted PM resuspended in TGMED________________116
+ TGMED supernatant after PM repelleted________________74
+ PM suspended in TGMED + 0.1 μM 2, 4-D_____________ 236
+ Repelleted PM resuspended in TGMED + 0.1 μM 2, 4-D___162
+ TGMED suspended after PM repelleted with 0.1 μM 2, 4-D_106
+ TGMED + 0.1 μM 2, 4-D_____________________________84


PM, plasma membrane fraction

Table. Effect of α-amanitin on enhancement of RNA polymerase by auxin-related factor.

+ α-amanitin*                                         133
+ PM-2,4-D supernatant                        227
+ PM-2,4-D supernatant + α-amanitin* 140
+ α-amanitin*                                         146
+ PM-IAA supernatant                           267
+ PM-IAA supernatant + α-amanitin*    140

RNA polymeraase 160


RNA polymerase   186


* α-amanitin acts as the inhibitor of mRNA synthesis. Each assay contained 2 μg of α-amanitin

       PLASMA      CYTOPLASM  NUCLEAR    NUCLEUS
       MEMBRANE                        MEMBRANE
Auxin ━>||━━━━> Factor ━━━>||━━> Factor + RNA polymerase
                                                                       ↓ Modified RNA polymerase
                                                                 Altered genome transcription
Fig. A hypothesis in relation to the synthesis of nucleic acids operated by auxin

2. Gibberellin, GA (ジベレリン)

immature seed embryo, young leaves, and roots (tissue localization) → phloem (transport)
α-amylase formation induced by gibberellin
GA
                                            Exposure to GA3 (h)
Fig. (left) α-amylase as % of total cell free protein synthesis. (right) Rate of α-amylase synthesis in vivo (U per 120 min)
Biosynthesis (生合成)
GA
Fig. 1. Early and intermediate steps of GA biosynthesis in higher plants (green arrows) and the fungus Fusarium fujikuroi (red arrows). In plants, ent-kaurene is synthesised in plastids, predominately via the methylerythritol phosphate pathway, while in fungi, it is biosynthesised from mevalonic acid. Conversion of ent-kaurene to GA12 and GA53 (plants) and GA14 (fungi) is catalysed by membrane-associated cytochrome P450 monooxygenases. Arrows running through structures indicate multiple steps catalysed by single enzymes (Hedden & Sponsel 2015)

3. Cytokinin (CK, サイトカイニン)

Root apex (synthesis) → upward in xylem (transport)

4. Abscisic acid (アブシジン酸)

opposes action of gibberellin and auxin
Synthesis
chloroplants - breakdown product of carotenoids
Function
dormancy maintenance - high levels in dormant seeds and buds

5. Ethylene (エチレン)

1901-1930 discovered a gas that is physiologically active = ethylene
1901 Neljubow, Dimitry: active component in illuminating gas = ethylene
1910 Knight, LI: supported Neljubow
1910 Cousins: orange ripnes earlier when stored with banana
1915 Harvey: some effects of ethylene on the metabolism of plants
1930 Chase: unripe, green pears softens fast by mature pear products
1932 Elmer: abnormal germinaton of potato

indcued by a volatile element in matured apple

1933 Kidd & West: a gas promotes ripning of fruits
1934 Gane, Richard: biosynthesis of ethylene by plants
1935 Crocker, William; ethylene = plant hormone
1947 Miller: published "The story of ethylene"
Burg, Stanley P

1959 research on ethylene biosynthesis
1967 auxin is the trigger of ethylene production

1968 discovered ethephon that is ethylene-generating agent

trade name = ethrel → applying research on gardening

1984 Knee: climacteric peak - respiration↑ during maturation

Etylene production (nl/g/hr) on climacteric plants
Ex.                      apple  apricot  kiwi fruit  pear  tomato
pre-climacteric      0.1     0.03      < 0.1    0.5-0.6    0.2
climacteric           100       0.4         50      50-100   120
temperature (°C)   12        20         20         20         20

slightly different among the varieties and/or cultivars

Etylene production (nl/g/hr) on non-climacteric plants
Ex. blueberry = 0.04-0.05. cucumber = 0.02-0.16. orange = 0.02-0.06

Conditions
Temperature: affects C2H4 synthesis largely
Ex. apple: Q10 = 2.7 (0-10°C). 2.8 (10-25°C), 1.74 (10-30°C) ⇒

break point on Arrhenius plot → phase transition (sometimes 11.4°C)

Air: O2↑ → promoting maturation. CO2↑ → supression

O2 is required for ethylene production

Contact stimulus (thigmomorphogenesis): trigger of ethylene production

Ex. wheat treading (麦踏), classically known

1971 Matsukawa (松川) et al. Lilium longiflorum

grew short when the shoots were swept by hand or brush
induced by ethylene productionk - inhibiting shoot elongation

1973 Jaffe MJ: contact stimuli applied to various plants

most plants supressed growth ⇒ thigmomorphogenesis

Disease contraction
1950 Williamson CE: diseased plants produced much etylene
1953 Dimond AE: Fusarium oxysporum f. sp. lycopersici (トマト萎凋病)

→ epinasty (上偏成長), induced by ethylene

Table 1. Plant responses to ethylene (Saltveit 1999)                                

Inhibition responses
    ethylene synthesis in vegetative
        tissue and non-climacteric fruit
    flower development in most plants
    auxin transport
    shoot and root elongation
    normal orientation of cell wall
        microfibrils

Stimulation responses
    ethylene synthesis (positive
        feedback)
    fruit ripening
    pigment synthesis
    cholorophyll destruction
    seed germination
    adventitious root formation
    respiration
    abscission
    senescence
Biosynthesis
1954 Fergus: Penicillium digitatum

mannose, mannitol, citric acid → ethylene

1957 Phan-Chon-Ton M: P. digitatum, glycerol, alanine → ethylene↑
1964 Lieberman M, Mapson LW: genesis and biogenesis of ethylene

                                                            |← hemicellulose
methionine + ATP → SAM1 → ACC2etylene → PG3
______________________________|→ galactose

1: S-adenosyl methionine
2: amino cyclo propane
3: polygalacturonase

1965 Lieberman et al.: radioisotope tracer techniques

C1 ⇒ carbon dioxide (CO2)
C2 ⇒ formic acid
C3 and C4 ⇒ ethylene

  methionine       NH3
                            |
CH3-S-CH2-CH2-CH-COO-
 5          4      3      2     1

1979 Lürssen et al.: Glycine max leaves + ACC → ethylene
1979 Adams & Yang: apple fruit + ACC → ethylene

these two experiments cofirmed: ACC = precursor of ethylene

Ethylene produced not only by plants but also animals, bacteria and fungi
Metabolism
1957 Buhler et al.: mature avocado + green pear + radioactive ethylene

ethylene (14C2H4) abosorved by plants → confirmed the uptake
emitted CO2 = non-radioactive
⇒ CO2 as a resultant of assimilation is not derived from ethylene

1961 Hall et al.: radioactive etylene - uptake by cellulose and lignin
1964 Jansen et al.: avocado + radioactive etylene →

uptake by benzene and toluene

1969 Shimokawa (下川敬之) et al.: juvenile of purple morning glory

radioactive etylene → not moved for long distance

Beyer et al.: ethylene metabolism examined by pea juveniles

                           /O\
H2C=CH2 → H2C=CH2 → HO-CH2-CH2-OH

Ethylene in soil (土壌中エチレン)
1974 Lynch: Mucor hieralis, soil bacterium - producing ethylene
1976 Smith A: ethylene oxygen cycle (酸素-エチレンサイクル)

                        [plant exudes  sugars] ⬅ [nutrients rebind to clay
[micro-organism ⬇                                            and organic matter]
   activity increases]               [micro-organism         ⬆
   ⬇                                         activity resumes]  [iron re-oxidises]
[microsite becomes                                               ⬆
 anaerobic]                [micro-organism     [O2 diffuses back
   ⬇                             activity ceases] into site]
[iron reduces to                                             ⬆
 soluble ferrous] [ethylene gas released]  [plant takes up nutrients]
   ⬇                                                                   ⬆
[releases nutrients bound to clay and organic matter into soil H2O]

Function of ethylene in soil:

adjust energy turnover and nutrient use efficiency
inhibit pathogen growth in soil
response of aboveground organs to immersion ≈ ethylene

6. Florigen (フロリゲン)

Hormonal control at hormone level

Auxin-induced ethylene production - regulation of ethylene biosynthesis -

RA + RB = RAB ← additive response (R: response, AB: hormones)
RA + RB < RAB ← synergistic response
IAA, ABA, Cytokinin havce complementary functions

Cell cycle (細胞周期)


Somatic division or somatic cell division (体細胞分裂)
1953 Pelc & Howard

G1 (gap1) → S (synthesis) → G2 (gap2) → M (mitosis)
common for most eukaryotes

M (mitosis), having four phases

prophase → metaphase → anaphase → telophase

Meiotic division

Chronobiology (時間生物学)


1. Phytochrome (ファイトクローム)
1935 Flint & McAlister: seeds from a cultivar of lettuce

germination rate

a few percent under dark
nearly 100% under light after imbibition duirng 24-48 hrs

exposed to radiation of which wavelengths ≥ 700 nm (far-red, FR)

→ inhibited seed germination

(Institute in Beltsville, Maryland)

1959 Butler WL et al.: isolated and identified from seedlings

Pr ↔ Pfr
Pr → P689 → Pfr → P650 → Pr = circulated
= trigger reaction

1952 Bothwick et al.: seeds of lettuce (cv. Grand Rapids

examined the effects of R/FR on seed germination

(this cultivar does not germinate the seeds under the darkness)

R: exposed to red at 1 W/m2 for 1 min
FR: exposed to far-red at 1 W/m2 for 4 min

Table. Seed germination of Rumex obtsusifolius after R and FR treatments (Bothwick et al. 1952).

light treatment       R   +FR   +R  +FR  +R   +FR  +R  
germination (%)   81.0  4.5  54.5   3.5  65.0  3.5 72.5

R: induced seed germination ⇔ FR: inhibited seed germination

1960 proposed the term phytochrome
Ref. Mycochrome
Table. The effects of blue (B) and near-ultraviolet (NUV) on the spore formation of Helminthosporium oryzae (ごま葉枯病菌, Syn. Cochliobolus miyabeanus) (Honda et al 1968)

Dark(control) NUV : B +NUV +B +NUV +B +NUV
       100           95    11    93    38     86   34    91

⇒ presence of mycochrome

Differentiation (分化)


1968 Harris H

Ⓧ + ◎ + UV irradiation →

differentiation

                                Amoclae   F hoclies
Dry weight                    5-8        2.5-4
Protein                        2-5         1.5-2 ↓
RNA (mg)                      1           0.4
Total carbohydrate       0.5         0.4 =
Cellulose                       0           0.1
Mucopolysaccharide     0          0.08
DNA                             36           30        
de novo Ex. temporal changes in the synthesis patterns of actin (structural protein)

differentiation
Fig. Decrease in dry weight and oxygen consumption

the most of energy during the emergence process is from protei. carbonhyborates are used least
→ expression of genes that are for storage-specific proteins and the RNAs

1972 Firtel RA

3H-DNA → thermal denaturation for single strand → sharing force, ca 400 nucleotides → remnant, return to double strand (= repeated sequence) → hydroxyappite column
3H-labeled single strand = DNA (unique sequence)

→ probe DNA-RNA hybrid

                                          gr    aggr pseudo culm     all  gr + culm
% of genome present
  as RNA transcripts (%)  30.2  27.4    34.2   35.6    56.0  49.0      
Estimated number of
  genes expressed           8600 7800 10000 11000 11000              

gr = growth. aggr = aggregation. culm = culmination. pseudo = pseudo-plasmodium. all = all stage

Growth + (Growth + Culmination) = 65.8: common genes present
2.86 × 107 nucleotide pair → one gene = 103 bp (M.W. = 4300) ⇒ unique gene = 286 × 104

Key enzyme (キーエンザイム)
UDP-galactose (polysaccharide) transferase: spore-specific substances

enzymes related to carbohydrate synthesis (cf. PSV: prespore vacuole) - absent in the stalk

Ex. Blastocladiella emersonii (水生菌), Acetabularia mediterranea

nuclear-cytoplasm interaction after the nuclear transfer

Higher plants (高等植物)
seed: analogy of animal embryo
Ex. α-amylase / β-amylase

de novo: gene = active → m-RNA → protein (α-amylase)

Seed formation (種子形成)


Double fertilization and division pattern

double fertilization
Fig. Life cycle of Angiospermae (Engler’s Syllabus 1964) embryo
Fig. Embryo development in Daucus carota. Longitudinal sections. The lower end of embryo in each drawing is the end directed toward micropyle. A-C, stages in development of linear four-celled embryo. D, E, two common variations in eight-celled embryos: difference in division of cell a of the four-celled embryo. F-I, older embryos varying in cell arrangement. J, embryo differentiated into main body and suspensor. Initial organization of tissue regions is present in J. Relation of parts of certain embryos to cells of the four-celled embryo (C) is indicated by the letters a-d. (Borthwick 1931).
double fertilization
Fig. Embryo development in Lactuca sativa (lettuce). Longitudinal sections. Lower end of embryo in each drawing is the end directed toward micropyle. A, zygote in division. B-G, embryos in successive stages of development, showing establishment of several horizontal tiers of cells. In G, cell h later gives rise to all of the suspensor cells, and the tiers above h develop into the main body of embryo. H-M, further development stages.

Germination and growth (発芽と成長)


embryo: gibberellin synthesis after immersion → active α-amylase synthesis → auxin → differentiation
α-amylase (α-アミラーゼ)
EC 3.2.1.1
hydrolyses alpha bonds of large, α-linked polysaccharides, e.g., starch and glycogen, yielding short chains, dextrins and maltose

[ seed dromancy ]

Dormancy (休眠)


1978 Kreibich G, Ulrich BL & Sabatini DD

Ribophorin in pea epicotyl


             Free              Membrane
             ---------------   ---------------
             M  Sp  Lp  Total  M  Sp  Lp  Total  Total

    Control               480               90     570
    BA                    441              103     544
    GA                    454              126     580
    IAA                   258              323     581
    NAA                   294              272     566

Total amount is constant - (possibility) free-bound induced by hormone
Inhibition factor: Rabit reticulocycle – cell-free system

Heme = globrin, Hemine
in-vitro system does not proceed when hormone is not supplied from the outside

Elongation factor (伸長因子)

1972 Tome et al.: Pea ribosomes: poly(U)-14C-phenyl alanine

                   2 days     4       6  
No addition   816     382    168
2 days         8080      ↔   6580
4                      ↓                 ↓
6                 1953       ↔   1800
S-100: sup of 105 g → translation
No differences of ribosomes + aged S-100 is not effective → presene of inhibitor

Cell differentiation and growth (細胞の分化と成長)


Shoot apex (茎頂)

= the apical part of the stem

characterized by the presence of an apical meristem at the topmost part of the root and the shoot

Relationships between stem and root (茎と根の関係)
1950 Skoog: Tobacco pith

IAA 0.002 ppm → root
Adenine 40 ppm → stem (leaf)
IAA 0.005 ppm + Adenine 40 ppm → stem, leaf, root

1957 Skoog & Miller: tobacco pith → callus

_____________Callus___Roots___Leaves
IAA (mg/l)______3______3________0.03
Kinetin (mg/l)___0.2_____0.02_____ 1

1958 Steward (USA): liquid medium – free cell

1) embryoid (胚様体)
2) origin = unicell (plant): (need to obtain the evidence)

1965 Vasil & Hildebrandt

tobacco pith cells → culture single cell → can form both bud and root

1965 Chen & Gaglston: Pelargonium pith collecting from pith → callus

medium: auxin, kinetin + agar → to liquid: differentiate root, stem and leaf
in general, the differentiation occurs under auxin-free condition ← substances for growth regulation

1970 Backs-Hüsemann & Reinert: unicell → embryoid
1971 Nagata & Takebe (長田・建部): Tobacco mesophyll protoplast

→ mature plant/embryoid → callus mass

Defoliation and shedding (落葉・落花・落果)


Defoliation (落葉)

C2H4: promoting cellulase in the abscission layer (or zone)

Lamina senescence____Abscission layer(zone)
___________________
IAA production →→→→ Stimulate senescence
____________________
_________C2H4(exo) ⇔ C2H4 production
____________________
____________________Increase cellulase
____________________
____________________Cell separation

Defoliation process (落葉過程)

cell differentiation plant hormones
_____________ _______________specific gene activation
functional specification specific enzyme
____________________
specific structure__specific protein ← specific mRNAs _______________ or polypeptide___
______________________________specific transcription
Fig. Conceptual framework for predicted cell differentiation processes

abscission zone
              (a)                        (b)                        (c)
Diagrams of the abscission zone of a leaf. (a) A leaf with the abscission zone indicated at the base of petiole. (b) The abscission zone layers shortly before abscission and (c) the layers after abscission.

Flower shedding (落花)

Fruit shedding (落果)

Flower organ formation (花器官形成)


ABC model

ABC
Fig. ABC model of flower development guided by three groups of homeotic genes.
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