LS3121 Plant Physiology Lec 15, 2001 T. Y. Lin

Plant Hormones and Growth Regulators Part II

Cytokinins

I. Cell Division in Plant Development

1. Differentiated plant cells can resume division

(1). Self-limiting mitotic activity: cortex and phloem, abscission, wounding

(2). Neoplastically mitotic activity: Agrobacterium tumefaciens

2. Plant tissues and organs can be cultured

(1). Differences in the regulation of cell division in root and shoot meristem.

(2). Crown gall tissue is an exception (callus).

II. The Discovery, Identification and Properties of Cytokinin

1. The discovery of Kinetin

(1). Haberlandt (1913-1921) observed:

a. Phloem diffusates could cause cell division in potato parenchyma.

b. Wounding-induced cell division was prevented by rinsing wound surface.

(2). Skoog (1948-1956) indicated:

a. Specific cell division inducing factors exist in vascular tissue.

b. In the presence of auxin, pith explants from tobacco showed extensive cell enlargement but no cell division. However, when the pith explants were placed in contact with vascular tissue, division of pith cells was observed.

c. Potential sources of cell division promoting substances: coconut milk, malt extract, yeast extract, extracts of vascular tissue and autoclaved DNA.

(3). Miller isolated a pure, highly active, cell-division-inducing factor from autoclaved herring sperm DNA, kinetin (6-(furfurylamino)purine.

2. Naturally occurring cytokinins

(1). Cytokinins are N6-substituted derivatives of the nitrogenous purine base adenine, characterized by the ability to stimulate cell division in tissue culture.

(2). In 1963, Letham isolated zeatin (6-(4-hydroxy-3-methylbut-trans-2-enylamino) purine from maize kernels.

(3). Some cytokinins occur in tRNA or rRNA molecules of seed plants, yeasts, bacteria and even primates.

3. Identifications and Measurement of Cytokinins

(1). Bioassays: 4 groups, promotion of cell division, promotion of cell expansion, retardation of senescence, induction of pigment synthesis.

Commonly used bioassays Time Min·det·conc

(days) (mg l^-1)

Tobacco pith callus 21 <1

Soybean callus 21 1

Carrot phloem 21 0.5

Radish leaf disk expansion 1 2

Radish cotyledon expansion 3 10

Barley leaf senescence 2 3

Oat leaf senescence 4 3

Cucumber greening 1 1

Amaranthus betacyanin 2 5

(2). Quantitative measurement of cytokinins: paper chromatography of a crude plant extract is followed by bioassay of the Rf zones of the chromatogram.

(3). Radio-immunoassay

Cytokinins can be linked to a protein to raise specific antibodies. Cytokinin concentration may be measured using the competitive binding between cytokinins in extract and cytokinins added as a radioactive ligand.

(4). Mass spectrometry (GCMS)

A known quantity of internal standard (deuterium or ¹⁵N labeled cytokinin) is added to crude extract. After converted to volatile derivatives, a specific ion from the endogenous cytokinin and the corresponding ion from IS is monitored.

III. The Biological Role of Cytokinins

1. Hormones may regulate plant cell cycle.

(1). Auxin may regulate events leading to DNA replication.

(2). Cytokinin may regulate events leading to mitosis.

2. The auxin/cytokinin ratio regulates morphogenesis in cultured tissues.

(1). High auxin/kinetin ratio® stimulates the formation of roots.

(2). High kinetin/auxin ratio® stimulates the formation of shoots.

(3). The effects of T-DNA mutations on crown gall tumor morphology.

a. Hormones play a central role in maintaining the tumor phenotype.

b. Three loci have been defined (tms, tmr, tml)

3. Delay of senescence and increase of nutrient-sink activities.

4. Cytokinins promote chloroplast development and chlorophyll synthesis.

(1). Addition of cytokinins to etiolated leaves or cotyledons several hours before they are exposed to light,

a. Enhance development of etioplasts into chloroplasts (especially grana).

b. Increase the rate of chlorophyll formation.

(2). Cytokinins activate synthesis of a protein that binds chlorophyll a and b.

5. Cytokinins can stimulate cell enlargement.

(1). Cytokinins enhance cell expansion in dicot cotyledons and in leaves.

(2). Promote elongation in wheat coleoptiles and intact watermelon hypocotyls.

IV. Biosynthesis, Metabolism and Transport of Cytokinins

1. Biosynthesis: cytokinin synthase, the key enzyme in cytokinin biosynthesis, catalyzes the first step in cytokinin biosynthesis.

2. Transport

(1). Cytokinins synthesized in the root are transported to the shoot in the transpiration stream through the xylem (cytokinin nucleotides).

(2). Cytokinins are concentrated in the apical meristem of both root and stem.

3. Habituation: when normal callus tissues have been subcultured over a long period, they become hormone autonomous.

4. Cytokinin synthesis by crown gall tissues

(1). Crown galls (caused by Agrobacterium tumefaciens) can be grown in sterile culture without addition of cytokinin or auxin.

(2). In the T-DNA region of Ti plasmid, there is one gene codes for the enzyme isopentenyl AMP synthase and two genes convert tryptophan to IAA.

V. Cellular and Molecular Modes of Cytokinin Action

1. A possible cytokinin receptor: cytokinin binds specifically to cytokinin-binding factor, CBF-1, possibly a ribosomal protein.

2. Cytokinins regulate protein synthesis: cytokinin stimulates polyribosome formation in cultured soybean tissues.

3. Cytokinins affect a posttranscriptional step in Lemna (SSU and LHCP).

4. The cytokinins in tRNA may regulate protein synthesis.

5. Cytokinins regulate calcium concentration in the cytosol.

Calcium ionophore A23187 can substitute for cytokinin to initiate bud development. But these buds cannot develop normally without cytokinin.

Ethylene

I. Introduction

1. Neljubow (1901) identified ethylene in illuminating gas. Ethylene causes triple response on etiolated pea seedlings: reduce stem elongation, increase lateral growth of stem (swelling) and abnormal horizontal growth.

2. Ethylene trapping systems: the storage of fruits, vegetables and flowers.

II. Ethylene synthesis

1. Biosynthesis and catabolism determine physiological activity of ethylene.

(1). Ethylene biosynthesis and its regulation

a. Methionine SAM ACC ethylene.

b. ACC (1-amino-cyclopropane-1-carboxylic acid) is a close precursor of ethylene. The CH₃S group is recycled back to methionine. The 3,4-carbon moiety is ultimately replenished from the ribose moiety of ATP.

(2). ACC synthase (rate limiting) may be a pyridoxal enzyme. Conversion from AdoMet to ACC is blocked by AVG (aminoethoxyvinylglycine) and AOA (aminooxyacetic acid), inhibitors of pyridoxal phosphate enzymes.

(3). ACC oxidase: the conversion from ACC to ethylene is oxygen-dependent. Fe²⁺ and ascorbate are required for activity. Cobalt is an inhibitor.

2. Environmental stresses and auxins promote ethylene biosynthesis.

(1). Fruit ripening.

(2). Stress-induced ethylene production: drought, flooding, chilling, wounding.

(3). Auxin-induced ethylene production.

III. Measurement of Ethylene

1. Bioassay: the triple response (sensitivity: 0.1 ml L^-1).

2. Gas chromatography: sensitivity: 5 part per billion (ppb).

IV. Developmental and Physiological Effects of Ethylene

1. Fruit ripening

(1). Addition of ethylene to fruits hastens ripening.

(2). Inhibitors of ethylene biosynthesis delay or prevent ripening.

(3). Ripening is characterized by a climacteric rise in respiration and ethylene production.

2. Abscission

(1). The hormonal balance during leaf abscission

a. Leaf maintenance phase: high auxin prevents leaf shedding.

b. Shedding induction phase

(a). An abscission signal is perceived and transduced into a message.

(b). The level of auxin decreases, and the level of ethylene increases.

(c). Ethylene reduces auxin transport and promotes senescence and leaf abscission.

c. Shedding phase: the actual abscission events.

(2). Application of exogenous auxin to petioles from which the leaf blade has been removed delays the abscission process.

3. Epinasty

(1). The downward curvature of leaves occurs when the adaxial side of the petiole grows faster than the abaxial side.

(2). Environmental stimuli are sensed by the roots, and a signal (ACC) from the roots is transported to the shoots.

4. Seedling growth: the triple response.

5. Hook opening

(1). A transient red-light exposure produces a transient decrease in ethylene production and an increase in plumule growth (opening the hook).

(2). Exogenous ethylene will reclose the hook even in light.

6. Ethylene breaks bud dormancy, increases the rate of seed germination and stem elongation, and induces root formation (10 ml L^-1).

7. Ethylene inhibits flowering in many species, it induces flowering in pineapple, it promotes the female flower formation in cucumber, and it hastens the onset of leaf and flower senescence.

8. Ethephon (2-chloroethylphosphonic acid) is a commercial compound.

V. The Mechanism of Ethylene Action

1. Bound radioactive ethylene may be associated with Golgi bodies or ER.

2. The binding is heat-labile and is inhibited by proteolytic enzymes.

3. Ethylene receptor may be a copper-containing protein.

Abscisic Acid (ABA)

I. Introduction

1. In 1963, F. A. Addicott identified two compounds, abscisin I and abscisin II (ABA), responsible for abscision of cotton fruits.

2. Wareing's group (Wareing, Cornforth, Milborrow etc., 1963-1966) found that an inhibitor was present in dormant buds of deciduous trees such sycamore (Acer pseudoplatanus) and birch (Betula pendula) and that the amounts were correlated to the depth of dormancy.

3. The Chemical Structure of ABA: a 15-carbon sesquiterpenoid.

II. Biosynthesis, Metabolism and Transport of ABA

1. Derived from violaxanthin (40-carbon).

Violaxanthin xanthoxin ABA aldehyde ABA

2. ABA can be inactivated by forming ABA-glucose ester or oxidized to phaseic acid and dihydrophaseic acid.

III. ABA Assay

1. Bioassay

(1). Coleoptile growth inhibition, linear response: 10^-7-10^-5 M.

(2). Inhibition of germination.

(3). Inhibition of GA-induced a-amylase synthesis in aleurone layers.

(4). Stomatal closure, high sensitivity: 10^-9 M.

2. Gas chromatography: 10^-13 g ABA (quantitative analysis).

3. Immunoassay.

IV. Developmental and Physiological Effects of ABA

1. ABA levels in seeds peak during embryogenesis

2. ABA promotes the desiccation tolerance of the embryo

(1). Late-embryogenesis-abundant protein sequences are highly conserved.

(2). Lea are related to RAB (responsive to ABA) and DHN (dehydrin) proteins.

(3). Lea is water soluble, basic, rich in gly & lys, low in hydrophobic residues.

3. ABA promotes the accumulation of seed storage protein during embryogenesis

Accumulation of storage protein in At aba/abi3-1 mutant strongly reduced.

4. ABA maintains the mature embryo in a dormant state.

(1). Seed dormancy

a. Types of seed dormancy: coat-imposed dormancy and embryo dormancy

b. Environmental factors control the release from seed dormancy

c. Seed dormancy is controlled by the ratio of ABA to GA.

d. ABA inhibits precocious germination and vivipary.

(2). ABA accumulates in dormant buds.

5. ABA inhibits GA-induced enzymes.

6. ABA closes stomata in response to water stress.

(1). Wilting, water logging, chilling, heat, salinity cause ABA increase.

(2). Applied ABA can reduce the plant's reaction to the stress factor.

a. ABA hardens plants against frost damage.

b. In tobacco, both salt stress and ABA induce formation of osmotin (a low-molecular-weight protein induced by salt stress) synthesis.

(3). Influence of turgor: Pierce and Raschke (1978-1983) found (in cockle burr) the onset of ABA accumulation occurred exactly at zero turgor. Water stress may act by turgor loss to activate genes that control ABA synthesis.

(4). Effects of ABA on stomata (Wright and Hiron)

a. Foliar application of ABA causes stomatal closure in light or darkness.

b. Foliar levels of ABA often rise from ~20 mg kg^-1 fresh weight to 500 mg kg^-1. Water stress causes 20-fold increase (~8 fg per cell).

c. ABA inhibits an ATP-dependent H⁺ pump in plasma membrane of guard cells and causes stomates to close. This H⁺ pump normally pumps out H⁺ and leads to K⁺ influx. However, ABA in free space of the outer surface of guard cell plasma membrane, shuts off K⁺ influx, reduces turgor and leads to stomates closure.

(5). Model: decrease turgor ® signal to plasma membrane ® Ca²⁺ into cell and PI signal transduction ® Activates genes for ABA synthesis.

7. ABA increases the hydraulic conductivity and ion flux of roots.

8. ABA promotes root growth and inhibits shoot growth at low water potentials.

9. ABA promotes leaf senescence independently of ethylene.

(1). ABA probably acts indirectly by causing premature senescence of cells in the organ that is shed.

(2). This senescence provokes a rise in production in ethylene.

V. Cellular and Molecular Modes of ABA Action

1. Extracellular and intracellular ABA receptors.

2. ABA increases cytosolic Ca²⁺ and pH, causes membrane depolarization.

3. ERA1 (the b subunit of farnesyl transferase) and a negative regulator.

Salicylic Acid

I. Introduction

1. Salicylic acid (active ingredient of willow bark): Raffaele Piria in 1838.

2. In 1898, aspirin (acetylsalicylic acid) was introduced by Bayer Company.

3. Conversion of cinnamic acid to SA is likely via benzoic or coumaric acid.

4. The highest level of SA was found in the inflorescence of thermogenic plants and in plants infected with necrotizing pathogens.

II. SA Induces Flowering

1. It was first found in tobacco tissue culture.

2. Flower-inducing substance from Xanthium strumarum (5.6 mM) induced Lemna gibba flowering. However, applied SA did not induce flowering in X. strumarum. Besides, levels of SA were not different in vegetative and flowering plants.

III. SA Promotes Cyanide-resistant Respiration

1. In the male reproductive structures of cycads and in the inflorescences of some angiosperm (Araceae), thermogenicity (heat production) and scent production is associated with a large increase in cyanide-resistant respiratory pathway.

2. Van Herk (1937) suggested that the burst of voodoo lily (Sauromatum guttatum) is triggered by "calorigen" produced in staminate flowers. Applications of SA (0.13 mg g^-1 fw) to sections of immature appendix led to temp increases of 12°C.

3. SA causes the induction of alternative oxidase (~38.9 kD) in S. guttatum.

IV. SA and Disease Resistance in Plants

1. Hypersensitive response (HR): some disease-resistant plants restrict the spread of pathogens to a small necrotic lesion around the point of initial penetration.

2. System acquired resistance (SAR) response: a resistance to subsequent pathogen attack that develops in the uninfected, pathogen-free part of the plant after the initial inoculation.

3. Low molecular weight pathogenesis-related (PR) proteins and HR and SAR.

4. SA and aspirin can be inducing resistance to pathogens and some PR proteins.

5. SA levels

(1). In TMV-resistant (Xanthi-nc) tobacco inoculated leaves increased 50-fold.

(2). 10-fold in uninfected leaves of Xanthi-nc, but none in susceptible tobacco.

6. SA can be exported but the SA-glucoside is immobile.

Jasmonic Acid (JA) and Methyl jasmomate (MeJA)

I. Introduction

1. In plants, JA is synthesized from a-linolenic acid by a lipoxygenase-mediated oxidation, followed by additional modifications.

2. JA is ~ 10 ng to 3 mg g^-1 fw in plants with JA as the dominant form. When applied exogenously, MeJA is more active than JA (volatile and not ionized).

3. Plant responses to JA

(1). Promotion of senescence.

(2). Ethylene synthesis and b-carotene synthesis.

(3). Inhibit seed germination, callus growth, root growth, chlorophyll production.

II. JA Induces Gene Expression

1. JA induces gene expression of VSPs (vegetative storage proteins) of soybeans: wound-induced proteinase inhibitors in tomato and potato, seed storage proteins and oil body membrane protein (oleosins) in developing Brassica napus embryo.

2. Leaf proteinase inhibitors are induced by MeJA at 40 to 80 nM.

3. Sagebrush (Artemesia tridentata) can release MeJA to induce proteinase inhibitors in plants incubated in the same chamber (not applied for JA).

4. Wounding is proposed to activate systemin (an 18 aa peptide) by releasing systemin from an inactive propeptide. Systemin serves as a systemic signal; it binds to a plasma membrane receptor and releases linolenic acid from membrane. Therefore, JA is synthesized and activates genes through another receptor.