2023 Solved Old Paper (BOT

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Osmotic potential:-

> It is the potential of water molecules to move from a hypotonic solution to a hypertonic solution across a semi-permeable membrane.

> The value of the osmotic potential can be determined using the Van't Hoff equation.

> The water potential of pure water is zero by convention.

> It is zero because there are no dissolved salts.

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Transpiration is Necessary Evil:- Transpiration and photosynthesis occur simultaneously due to the opening of stomata. - However, transpiration also causes loss of water unnecessarily. Due to the process of transpiration, there is pressure on the plant for the absorption of water. Therefore, the process of transpiration is called a necessary evil.

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Donnan Equilibrium Theory:- The membrane is selectively permeable in nature. Some are permeable to ions and some are impermeable.

The ions for which the membrane is permeable enter the cell by diffusion. This disturbs the equilibrium. To re-establish the equilibrium, there is an exchange of cations and anions again.

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Salt Respiration:- The term 'salt respiration' will be used here simply to mean an observed increase of the rate of oxygen uptake or carbon dioxide emission when salts are added to tissue respiring in water.

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Four non-leguminous Nitrogen Fixing plants:-

Some non-leguminous plants have been reported to fix atmospheric nitrogen.

i. Association between Sugarcane and endophytic bacteria Gluconacetobacter diazotrophicus.

ii. Association of tropical grasses such as Digitaria sp. and Paspalum sp. with Azospirillum sp.

iii. association of Coffee and Maize with Burkholderia sp.

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Hup genes:-

The hup genes play an important role in plant productivity by recycling the considerable quantities of reducing equivalents (in the form of H2) wasted during symbiotic nitrogen fixation of leguminous crops such as soybean.

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Apical dominance:- It is the dominance of shoot apex over its lateral branches. Here shoot apex inhibit the growth of lateral axillary buds. It is caused by the apical bud producing IAA (auxin) in abundance. The IAA causes the lateral buds to remain dormant.

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Discovery of Gibberellins:- It was discovered by Kurosawa. In 1926, Japanese scientist Eiichi Kurosawa identified that foolish seedling disease was caused by the fungus Gibberella fujikuroi. Later work at the University of Tokyo showed that a substance produced by this fungus triggered the symptoms of foolish seedling disease and they named this substance "gibberellin".

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Day Neutral Plants:- A plant that flowers regardless of the length of the period of light it is exposed to, is called day neutral plant.

Example:- Maize, Cucumber, Tomato, Cotton, Dandelion, Sunflower

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Senescence:- Plant senescence is the process of aging in plants. Plants have both stress-induced and age-related developmental aging. Chlorophyll degradation during leaf senescence reveals the carotenoids, such as anthocyanin and xanthophylls, which are the cause of autumn leaf color in deciduous trees.

> Ethylene enhances senescence of leaves and flowers in plants. In senescence, concentration of endogenous ethylene increases with decrease in conc. of cytokinins and it is now generally held that a balance between these two phytohormones controls senescence.

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Soil Plant Atmosphere Continuum (SPAC):-

> It is proposed by Huber in 1924.

> All components of field environment i.e. soil, plant and atmosphere, form a physically unified and dynamics networking system in which various water flow processes occur independently. This unified system is called the soil-Plant-Atmosphere-Continuum or SPAC.

> The fundamental principle of SPAC is water moves from higher total water potential to regions of lower total water potential at a rate depending on the hydraulic resistance of the medium.

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Internal Water Deficit:-

1. Internal Water deficit:- It is defined as being when plant water status is reduced sufficiently to affect normal plant functioning.

2. Causes of Water deficit:-

i. A combination of limited water absorption and high evaporative demand.

ii. Generally, plant water deficits may be considered as being induced by either insufficient available soil water or a high atmospheric evaporative demand.

3. Physiological implications of water deficit:-

i. Water deficit has an adverse effect on plant growth. Therefore, drought stress is the most severe environmental stress for plant growth and crop production.

ii. Water deficit reduces photosynthesis by closing stomata, decreasing the efficiency of the carbon fixation process, suppressing leaf formation and expansion, and inducing the shedding of leaves.

iii. Response of leaves to water stress is by inward curling or exhibiting a wilted appearance as a result of an absence of turgor in leaves.

iv. A notable response to the deficit of water is stomata closing themselves to scale back water loss by transpiration.

v. However, it increases the leaves’ internal temperature leading to heat stress.

vi. Closure of stomata decreases greenhouse emission diffusion, finally reducing the expansion of the plant.

Ans.

Cytochrome Pump Theory:-

Ø Lundegardh and Burstrom (1933) believed that there was a definite correlation between respiration and anion absorption. Thus when a plant is transferred from water to a salt solution the rate of respiration increases. This increase in rate of respiration over the normal respiration has been called as anion respiration or salt respiration.

Ø The inhibition of salt respiration and the accompanying absorption of anions by CO and cyanides (which are known inhibitors of cytochrome oxidase of electron transport chain in mitochondria), later on led Lundegardh (1950, 54) to propose cytochrome pump theory. This is based on the following assumptions:-

i. The mechanism of anion and cation absorption is different.

ii. Anions are absorbed through cytochrome chain by an active process.

iii. Cations are absorbed passively.

Ø According to this theory:-

i. ehydrogenase reactions on inner side of the membrane give rise to protons (H⁺) and electrons (e^–).

ii. The electron travels over the cytochrome chain towards outside the membrane, so that the Fe of the cytochrome becomes reduced (Fe⁺⁺) on the outer surface and oxidized (Fe⁺⁺⁺) on the inner surface.

iii. On the outer surface, the reduced cytochrome is oxidized by oxygen releasing the electron (e^–) and taking an anion (A^–).

iv. The electron thus released unites with H⁺ and oxygen to form water.

v. The anion (A^–) travels over the cytochrome chain towards inside.

vi. On the inner surface the oxidized cytochrome becomes reduced by taking an electron produced through the dehydrogenase reactions, and the anion (A^–) is released.

vii. As a result of anion absorption, a cation (M⁺) moves passively from outside to inside to balance the anion.

Ø Main defects of the above theory are:-

i. It envisages active absorption of only anions.

ii. It does not explain selective uptake of ions.

iii. It has been found that cations also stimulate respiration.

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Mass flow hypothesis:-The Mass flow hypothesis is also known as the pressure flow hypothesis. It describes the movement of sap through the phloem, proposed by the German physiologist Ernst Munch in 1930.

> The following steps take place:-

i. Active transport of sucrose from source to companion cells

ii. Phloem loading

iii. Movement of water from xylem to sieve tubes

iv. Transport in sieve tube

v. Phloem unloading

i. Source to companion cells:- Some amount of sucrose from the leaf (source) enters the companion cells through the plasmodesmata across the concentration gradient.

ii. Phloem loading:- The transport of sucrose from the companion cells to the sieve tube is called phloem loading. This happens through active transport as the sucrose moves against the concentration gradient.

iii. Movement of water from xylem:- As the water potential of the sieve tube has decreased compared to the water potential in the xylem, water from the xylem enters the sieve tube through osmosis

iv. Transport in the sieve tube- pressure flow:- The turgor pressure in the sieve tube of phloem increases due to the presence of water. As a result pressure flow begins, and the sap moves through the phloem sieve plates towards the sink.

v. Phloem unloading:- The sucrose from the phloem sieve tube, through the process of active transport enters the sink ( storage organs like roots or actively metabolising organs like fruits). This is called phloem unloading.

As the sucrose has been unloaded the water potential of the sieve tube has now increased compared to the nearby xylem vessel. The water from the sieve tube now enters the xylem vessel.

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Physical Nitrogen Fixation:- The process of converting nitrogen gas into a useful form for plants and other organisms in a biological process is called physical nitrogen fixation. Ammonia is produced industrially by direct mixing of nitrogen with hydrogen at high temperatures and pressures. Later, it is converted into various types of fertilizers like urea, etc.

i. Natural Nitrogen Fixation:- Under the influence of lightning (i.e., electric discharge in the clouds) and thunder, N2 and O2 of the air react to form nitric oxide (NO). The nitric oxides are again oxidized with oxygen to form nitrogen peroxide (NO2).

> The reactions are as follows:

N2 + O2 Lightning → Thunder 2N0 (Nitric Oxide); 2NO + O2 → 2NO2 Oxidation (Nitrogen peroxide)

> During the rains, NO2 combines with rain water to form nitrous acid (HNO2) and nitric acid (HNO3). The acids fall on the soil along with rain water and react with the alkaline radicals to form water soluble nitrates (NO3-) and nitrites (N02-).

2NO2 + H2O → HNO2 + HNO3; HNO3 + Ca or K salts → Ca or K nitrates

> The nitrates are soluble in water and are directly absorbed by the roots of the plants.

ii. Industrial Nitrogen Fixation:- Ammonia is produced industrially by direct combination of nitrogen with hydrogen (obtained from water) at high temperature and pressure. Later, it is converted into various kinds of fertilizers, such as urea etc.

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Factors Affecting Nodule Formation:- Nodulation is affected by both external and internal factors. External factors include heat, acidity, nitrate content of the soil, etc.

i. If soil is rich in nitrogen content, it interferes with the nodule formation and symbiotic association as plants already have enough nitrogen and they do not need more.

ii. Nitrogen fixation is an oxygen-sensitive process. The root nodules contain a heme pigment called leghaemoglobin, which facilitates the diffusion of oxygen.

iii. Nodule formation is auto regulated by leaf tissues. Plants have evolved defense mechanisms to check the infection.

iv. Ethylene also regulates nodule formation internally. Exogenous application of ethylene has shown to inhibit nodule formation.

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Bioassay of Auxins:-

It is testing of a biological activity like growth response of a substance by employing a living material like plant or plant part. Auxin bioassay is quantitative test as it measures concentration of auxin to produce the effect and the amount of effect.

a. Avena Curvature Test:-

> The test is based upon experiments of Went (1928). 10° curvature is produced by auxin concentration of 150 µg/litre at 25° C and 90% relative humidity. The test can measure auxin upto 300 pg/litre.

> Auxin from a shoot tip or any other plant organ is allowed to diffuse in a standard size agar block (generally 2 x 2 x 1 mm). Auxin can also be dissolved directly in agar. 15-30 mm long oat coleoptile grown in dark is held vertically over water. 1 mm tip of coleoptile is removed without injuring the primary leaf.

> After 3 hours a second decapitation is carried out for a distance of 4 mm. Primary leaf is now pulled loose and agar block supported against it at the tip of decapitated coleoptile. After 90-110 minutes, the coleoptile is found to have bent. The curvature is measured. It can also be photographed and the curvature known from shadow graph.

b. Root Growth Inhibition:-

Sterilized seeds of Cress are allowed to germinate on moist filter paper. As the roots reach a length of 1 cm or so, root lengths are measured. 50% of the seedlings are placed in a test solution while the remaining are allowed to grow over moist paper.

Lengths of the roots are measured after 48 hours. It is seen that the seedlings placed in test solution show very little root growth while root growth is normal in control seedlings.

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Physiological effects of Ethylene:-

i. Fruit Ripening:- Its main function is to ripen fruits. The most commonly used chemical is called ethephon (2-chloro ethylphosphonic acid). It penetrates into the fruit and decomposes ethylene.

ii. Triple Response:- Ethylene causes plants to have –

i. Short shoots:- Inhibition of stem elongation.

ii. Fat shoots:- Stimulation of radial swelling of stems.

iii. Diageotropism:- Increased lateral root growth and horizontal growth of stems with respect to gravity.

iii. Formation of Adventitious Roots and Root Hairs:- Ethylene induces formation of adventitious roots in plants from different plant parts such as leaf, stem, peduncle and even other roots. In many plants especially Arabidopsis, ethylene treatment promotes initiation of root hairs.

iv. Inhibition of Root Growth:- Ethylene is known to inhibit linear growth of roots of dicotyledonous plants.

v. Leaf Epinasty:- When upper side (adaxial side) of the petiole of the leaf grows faster than the lower side (abaxial side), the leaf curves downward. This is called as epinasty. Ethylene causes leaf epinasty in tomato and other dicot plants such as potato, pea and sunflower. Young leaves are more sensitive than the older leaves. However, monocots do not exhibit this response.

vi. Flowering:- Ethylene is known to inhibit flowering in plants.

vii. Sex Expression:- In monoecious species especially some cucurbits like cucumber, pumpkin, squash and melon; ethylene strongly promotes formation of female flowers thereby suppressing the number of male flowers considerably.

viii. Senescence:- Ethylene enhances senescence of leaves and flowers in plants. In senescence, concentration of endogenous ethylene increases with decrease in conc. of cytokinins and it is now generally held that a balance between these two phytohormones controls senescence.

ix. Abscission of Leaves:- Ethylene promotes abscission of leaves in plants. Older leaves are more sensitive than the younger ones.

x. Breaking Dormancy of Seeds and Buds:- Ethylene is known to break dormancy and initiate germination of seeds in barley and other cereals. Seed dormancy is also overcome in strawberry, apple and other plants by treatment with ethylene. Non-dormant varieties of seeds produce more ethylene than those of dormant varieties.

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Circadian Rhythm:-

> A circadian rhythm, or circadian cycle, is a natural oscillation that repeats roughly every 24 hours.

> Circadian rhythms can refer to any process that originates within an organism (i.e., endogenous) and responds to the environment (is entrained by the environment).

> Circadian rhythms have been widely observed in animals, plants, fungi and cyanobacteria and there is evidence that they evolved independently in each of these kingdoms of life.

> Its primary function is to rhythmically co-ordinate biological processes so they occur at the correct time to maximise the fitness of an individual.

> The term circadian comes from the Latin circa, meaning "approximately", and dies, meaning "day".

Circadian Rhythm in Plants:-

- Plant circadian rhythms tell the plant what season it is and when to flower for the best chance of attracting pollinators.

- Behaviors showing rhythms include leaf movement (Nyctinasty), growth, germination, stomatal/gas exchange, enzyme activity, photosynthetic activity, and fragrance emission, among others.

- Circadian rhythms occur as a plant entrains to synchronize with the light cycle of its surrounding environment.

- These rhythms are endogenously generated, self-sustaining and are relatively constant over a range of ambient temperatures. I

-mportant features include two interacting transcription-translation feedback loops: proteins containing

i. PAS domains, which facilitate protein-protein interactions.

ii. Several photoreceptors, that fine-tune the clock to different light conditions.

- A better understanding of plant circadian rhythms has applications in agriculture, such as helping farmers stagger crop harvests to extend crop availability and securing against massive losses due to weather.

- Light is the signal by which plants synchronize their internal clocks to their environment and is sensed by a wide variety of photoreceptors.

- Red and blue light are absorbed through several phytochromes and cryptochromes.

- Phytochrome A, phyA, is light labile and allows germination and de-etiolation when light is scarce.

- Phytochromes B–E are more stable with phyB, the main phytochrome in seedlings grown in the light.

- The cryptochrome (cry) gene is also a light-sensitive component of the circadian clock and is thought to be involved both as a photoreceptor and as part of the clock's endogenous pacemaker mechanism.

- Cryptochromes 1–2 (involved in blue–UVA) help to maintain the period length in the clock through a whole range of light conditions.

Ans.

Autonomic Movements in Plants:-

1. Growth movements

2. Variation movements

1. Autonomic growth movements:- These movements are due to unequal growth in different parts of an organ and are irreversible. They are independent of the surroundings of plants or external stimuli. They are further divided into types:

a. Nutation:- These movements occur in the growing stem of twiners (peas or beans). The stem exhibits a kind of nodding movements in two directions. As, the stem apex shows more growth on one side at one time and a little later there is a greater growth on the opposite side. It is called nutation. This circular movement of growing stem takes place always in the same direction for the same plant species. Since, the more rapid rate of growth travels around the tip which, as it grows upwards must therefore rotate.

b. Circumnutation:- In spirally growing stems and tendrils, the region of greater growth passes gradually around the growing point resulting in the spiral coiling of stem and tendrils. Such a movement is called circumnutation.

c. Epinasty and Hyponasty:- They are non-directional movements in which the response is determined by the structure of the responsive organ and not the direction of the stimulus. This kind of movement is exhibited mostly by young leaves, organs having dorsiventral symmetry.

> Epinasty is caused due to rapid growth on the upper surface (abaxial side), such as occurs at the time of the opening of leaves.

> Hyponasty is greater growth on the lower side (adaxial side) e.g., circinate coiling and closed sepals and petals in a floral bud.

2. Movements of variation:-

> They are autonomous curvature movements and are independent of external stimuli.

> They are not growth movements hence are reversible.

> These movements are also known as turgor movements because these are brought about by changes in the turgor pressure of certain sensitive cells of the plant organs. Such movements are exhibited by lateral leaflets in Desmodium gyrans.

Ans.

Mechanism of Stomatal Movement:-

> Stomata function as turgor-operated valves because their opening and closing movement is governed by turgor changes of the guard cells.

> Whenever, the guard cells swell up due to increased turgor, a pore is created between them. With the loss of turgor the stomatal pores are closed.

> Stomata generally open during the day and close during the night with a few exceptions.

>The important factors which govern the stomatal opening are light, high pH or reduced CO2 and availability of water. The opposite factors govern stomatal closure, viz., darkness, low pH or high CO2 and dehydration.

> There are three main theories about the mechanism of stomatal movements:

1. Classical Starch Hydrolysis Theory:-

- The main features of the theory were spelled out by Sayre (1923).

- It was modified by Steward (1964).

- The guard cells contain starch.

- At low carbon dioxide concentration (in the morning achieved through photosynthesis by mesophyll and guard cells), pH of guard cells rises. It stimulates enzyme phosphorylase. Phosphorylase converts starch into glucose 1- phosphate. The latter is changed to glucose 6-phosphate which undergoes hydrolysis to produce glucose and phosphoric acid. Glucose increases osmotic concentration of guard cells. On account of it, the guard cells absorb water from neighbouring cells, swell up and create a pore in between them.

- Evening closure of stomata is brought about by increased carbon dioxide content (due to stoppage of photosynthesis) of leaf. It decreases pH of guard cells and brings about phosphorylation of glucose. In the presence of phosphorylase, glucose 1-phosphate is changed into starch. As a result, osmotic concentration of guard cells falls. They lose water to adjacent epidermal cells. With the loss of turgidity, the guard cells shrink and close the pore in between them.

Objections:-

(i) Glucose is not found in guard cells at the time of stomatal opening.

(ii) Starch ↔ Sugar changes are chemically slow while opening and closing of stomata are quite

rapid.

(iii) Wide changes in pH of guard cells cannot be explained on the basis of carbon dioxide

concentration.

(iv) Onion and some of its relatives do not possess starch or related polysaccharide that can be

hydrolysed to the level of glucose.

(v) Blue light has been found to be more effective than other wavelengths for opening of

stomata. The same cannot be explained by starch hydrolysis theory.

(vi) Hydrolysis of starch theory cannot account for high rise in osmotic pressure found in guard

cells.

2. Malate or K+ ion Pump Hypothesis (Modern Theory):- The main features of the theory were put forward by Levitt (1974).

i. During Stomatal Opening:-

- According to this theory, pH of the guard cell can rise due to active H+ uptake by guard cell chloroplasts or mitochondria, CO2 assimilation by mesophyll and guard cells.

- A rise in pH causes hydrolysis of starch to form organic acids, especially phosphoenol pyruvate. Starch → Hexose Phosphate → Phosphoenol Pyruvate.

- Phosphoenol pyruvate can also be formed by pyruvic acid of respiratory pathway. With

the help of PEP carboxylase (PEP case), it combines with available CO2 to produce oxalic acid

which gets changed into malic acid.

- Malic acid dissociates into H+ and malate. H+ ions pass out of the guard cells actively. In exchange, K+ ions pass inwardly. Same CI– ions may also enter guard cells along with K+ ions.

- Guard cells maintain their electroneutrality by balancing K+ with malate and Cl–.

- In the combined state they pass into the small vacuoles and increase the osmotic concentration of the guard cells. As a result guard cells absorb water from the nearby epidermal cells through endosmosis, swell up and create a pore in between them.

ii. During Stomatal Closing:-

- The H+ ions diffuse out of the guard cell chloroplasts. It decreases pH of the guard cell cytoplasm.

- Any malate present in the cytoplasm combines with H+ to form malic acid.

- Excess of malic acid inhibits its own biosynthesis. High CO2 concentration also has a similar effect.

- Un-dissociated malic acid promotes leakage of ions. As a result K+ ions dissociate from malate and pass out of the guard cells.

- Formation of abcisic acid (as during drought or midday) also promotes reversal of H+ = ↔ K+ pump and increases availability of H+ inside the guard cell cytoplasm.

- Loss of K+ ions decreases osmotic concentration of guard cells as compared to adjacent epidermal cells. This causes exosmosis and hence turgidity of the guard cells decreases. It closes the pore

between the guard cells. Simultaneously the organic acids are metabolised to produce starch.

Ans.

Active Absorption (Active Uptake):-

Ø It has often been observed that the cell sap in plants accumulates large quantities of mineral salts ions

against the concentration gradient. It is an active process which involves the expenditure of metabolic energy through respiration.

Ø Following evidences favor this view:-

i. The factors like low temp., deficiency of O₂, metabolic inhibitors etc. which inhibit metabolic activities like respiration in plants also inhibit accumulation of ions.

ii. Rate of respiration is increased when a plant is transferred from water to salt solution. It is called salt respiration.

a. The Carrier Concept:- According to this theory the plasma membrane is impermeable to free ions. But some compounds present in it acts as carrier and combines with ions to form carrier-ion-complex which can move across the membrane. On the inner surface of the membrane this complex breaks releasing ions into the cell while the carrier goes back to the outer surface to pick up fresh ions.

b. Cytochrome Pump Theory:-

i. The mechanism of anion and cation absorption is different.

ii. Anions are absorbed through cytochrome chain by an active process.

iii. Cations are absorbed passively.

Ø According to this theory:-

i. ehydrogenase reactions on inner side of the membrane give rise to protons (H⁺) and electrons (e^–).

iii. On the outer surface, the reduced cytochrome is oxidized by oxygen releasing the electron (e^–) and taking an anion (A^–).

iv. The electron thus released unites with H⁺ and oxygen to form water.

v. The anion (A^–) travels over the cytochrome chain towards inside.

vi. On the inner surface the oxidized cytochrome becomes reduced by taking an electron produced through the dehydrogenase reactions, and the anion (A^–) is released.

vii. As a result of anion absorption, a cation (M⁺) moves passively from outside to inside to balance the anion.

Ø Main defects of the above theory are:-

i. It envisages active absorption of only anions.

ii. It does not explain selective uptake of ions.

iii. It has been found that cations also stimulate respiration.

c. Protein-Lecithin Theory:-

Ø In 1956, Bennet-Clark suggested that because the cell membranes chiefly consist of phospholipids and proteins and certain enzymes seem to be located on them, the carrier could be a protein associated with the phosphatide called as lecithin.

Ø He also assumed the presence of different phosphatides to correspond with the number of known competitive groups of cations and anions (which will be taken inside the cell).

Ø According to this theory:-

i. The phosphate group in the phosphatide is regarded as the active centre binding the cations, and the basic choline group as the anion binding center.

ii. The ions are liberated on the inner surface of the membrane by decomposition of the lecithin by the enzyme lecithinase.

iii. The regeneration of the carrier lecithin from phosphatidic acid and choline takes place in the presence of the enzymes choline acetylase and choline esterase and ATP. The ATP acts as a source of energy.

Ans.

Biological Nitrogen Fixation:-

The conversion of atmospheric nitrogen into the nitrogenous compounds by living organisms is called biological nitrogen fixation. Only prokaryotes can fix nitrogen. Nitrogen fixation require anaerobic conditions because oxygen inactivates nitrogenase enzyme.

Hence for obligate anaerobes nitrogen fixation is easy, but in case of facultative anaerobes the nitrogen fixation occurs only in anaerobic conditions. In case of obligate aerobes the oxygen level inside the cell must be kept low for nitrogen fixation.

1. Nitrogen Fixers (Diazotrophs):- Among the earth’s organisms, only some prokaryotes like bacteria and cyanobacteria can fix atmosphere nitrogen. They are called nitrogen fixers or diazotrophs. They fix about 95% of the total global nitrogen fixed annually by natural process.

a. Asymbionts (Free living)

b. Symbionts

a. Asymbionts (Free living):-

i. Bacteria:- They add up to 10-25 kg, of nitrogen/ha/annum.

> Azotobacter (Aerobic, Saprophytic)

> Beijerinckia (Aerobic, Saprophytic)

> Clostridium (Anaerobic, Saprophytic)

> Desulphovibrio (Chemotrophic)

> Rhodopseudomonas (Photoautotrophic)

> Rhodospirillum (Photoautotrophic)

> Chromatium (Photoautotrophic)

ii. Blue Green Algae (Cyanobacteria):- Heterocysts are the special cells that fix nitrogen. They add 20-30 kg Nitrogen/ha/annum.

> Nostoc

> Anabaena

> Aulosira:- A. fertilissima is the most active nitrogen fixer in Rice fields.

> Cylindrospermum:- It is active in sugarcane and maize fields.

> Trichodesmium

b. Symbionts:- Live in close symbiotic association with other plants.

i. Bacteria:-

> Rhizobium:- It is aerobic, gram negative nitrogen fixing bacterial symbionts of legume roots. Sesbania rostrata has Rhizobium in root nodules and Aerorhizobium in stem nodules.

> Frankia:- It is symbiont in root nodules of many non-leguminous plants like Casuarina and Alnus.

> Xanthomonas and Mycobacterium:- They occur as symbiont in the leaves of some members of the families Rubiaceae and Myrsinaceae (e.g., Ardisia).

ii. Blue Green Algae (Cyanobacteria):-

> Nostoc and Anabaena:- They are common symbionts in lichens, Anthoceros, Azolla and Cycas roots.

> Anabaena azollae:- It is found in fronds of Azolla pinnata (a water fern). It is often inoculated to Rice fields for nitrogen fixation.

2. Rhizobium Nitrogen Fixation:-

> Rhizobium bacteria:-

i. Free living

ii. Gram negative

iii. Aerobic

iv. Soil bacteria

> Rhizobium becomes anaerobic upon entry into roots.

> Leghaemoglobin (legHb or symbiotic Hb):-

- It is a pink coloured pigment.

- It occurs in the root nodules of leguminous plants.

- It acts as an oxygen scavenger. It provides anaerobic conditions for the nitrogenase enzyme and protects the enzyme from inactivation.

> Two main steps:-

a. Nodule formation

b. Nitrogen fixation

a. Nodule formation:- Root nodule formation is initiated, when the soil contains a low level of nitrogen. Steps of nodulation are:

i. Aggregation:- Roots of legumes secrete flavonoids, which attracts rhizobia towards the root. Rhizobia aggregate around root hairs.

ii. Developmental changes:- Rhizobia secrete nod factors, which causes stimulate many developmental changes:

- Membrane depolarization

- Curling of root hairs

- Cell division in the root cortex

- Intracellular calcium movement

iii. Infection thread:- The nod factor attaches to receptors present on the plasma membrane of the root hairs, which leads to the formation of the infection thread.

iv. Entry:- Infection thread provides the passage to bacteria to enter epidermal cells. Rhizobia then enter cortex cells, each bacterium gets surrounded by a plant-derived membrane known as symbiosome.

v. Nodulation:- Nodule formation is initiated by chemicals produced by rhizobia. It is a result of calcium dependent signal transduction pathway, which triggers biochemical changes leading to cell division and nodule formation. Cytokinin also plays an important role in nodules formation.

vi. Bacteroids:- Within nodules, bacteria get differentiated into bacteroids, which fix nitrogen. The Rhizobia stop dividing, loose cell wall and become nitrogen fixing cells as bacteroids . Vascular tissues are developed for nodules for exchange of nutrients.

b. Nitrogen fixation:-

- The nodule serves as site for N2 fixation.

- Nodule contains nitrogenase and leghaemoglobin.

- The nitrogenase has 2 components:

i. Molybdoferredoxin (Mo-Fe protein)

ii. Azoferredoxin (Fe-protein)

- The free di-nitrogen first bound to MoFe protein and is not released until completely reduced to ammonia.

- In this process ferredoxin serves as an electron donor to Fe-protein (nitrogenase reductase) which in turn hydrolyzes ATP and reduce Mo-Fe protein, the Mo-Fe protein in Turn reduce the substrate N2. The electrons and ATP are provided by photosynthesis and respiration of the host cells.

- Many intermediates are formed to form ammonia (NH3).

Dinitrogen → Hydrazine → Diamine → Ammonia

- Ammonia (NH3) is immediately protonated at physiological pH to form ammonium ion (NH4+). As NH4+ is toxic to plants, it is rapidly used near the site of generation to synthesize amino acids.

Ans.

Abscisic Acid (ABA):-

> It is found in older leaves, apical buds and seeds.

> Both GA and ABA hormones work opposite to each other.

> ABA inhibits the biosynthesis of the hormone GA.

1. Discovery of Abscisic Acid:-

- Torsten Hemberg (1940) reported that dormant potato tubers and buds of ash (Fraxinus excelsior) contained inhibitor (rather than stimulators) that blocked the effects of IAA. When the buds germinated, the amount of these inhibitors decreased.

- Eagles and Wareing (1963) isolated an inhibitor from the birch (Betula pubescens) leaves held under short day conditions. When this substance was reapplied to the leaves of birch seedlings, apical growth was completely arrested. As this substance induced dormancy, they named it as ‘dormin’.

- Cornus and Addicot (1963) isolated this compound from the shedding bolls of cotton plant.

- Ohkum (1965) isolated an inhibitor from cotton fruits and named it ‘abscisin II’.

- Cornforth (1965) demonstrated that both Dormin and Abscisin- II were chemically identical and given a common name Abscisic acid (ABA) because of its known effects on stimulating abscission.

2. Chemical nature of Abscisic Acid:- This phytohormone is a 15-carbon sesquiterpene and its synonyms are dormin and abscisin-II.

3. Biosynthesis of Abscisic Acid:- Two pathways for the biosynthesis of ABA have been identified:

a. By direct synthesis from mevalonic acid:-

> Direct synthesis of abscisic acid from mevalonic acid through farnesyl pyrophosphate has been demonstrated in many cases, especially in the water stressed tissues.

> The water stress increases Abscisic acid formation.

> The early reactions in the synthesis of abscisic acid are identical to those of gibberellins, sterols, carotenoids and other isoprenoid compounds.

Mevalonic acid→ Isopentenyl pyrophosphate→ Farnesyl pyrophosphate→ Abscisic acid

b. By indirect synthesis from the oxidation of carotenoids:-

> Indirect synthesis of abscisic acid takes place by the oxidation of carotenoids or oxidized carotenoids (xanthophyll such as violaxanthin or neoxanthin).

> The xanthophyll, violaxanthin has been known to be the precursor of ABA.

> Violaxanthin in synthesized from zeaxantin (also a 40-C xanthophyll) in a reaction that is catalyzed by the enzyme zeaxanthin epoxidase (ZEP).

> Violaxanthin is converted into 9’ –cis-neoxanthin which is then cleaved into a 15-C compound Xanthoxal (previously called Xanthoxin) and a 25-C epoxy aldehyde in the presence of the enzyme 9′-cis-epoxycarotenoid dioxygenase (NCED). (This enzyme can also catalyse cleavage of violaxanthin into xanthoxal and a 25-C allenic apoaldehyde).

> Xanthoxal (xanthoxin) is finally converted into ABA in cytosol via two oxidation steps catalysed by the enzymes aldehyde oxidases involving abscisyl aldehyde (and/or possibly xanthoxic acid) as intermediates.

> The enzymes aldehyde oxidases require Mo as cofactor.

> The initial steps of ABA biosynthesis take place in chloroplasts or other plastids while final steps occur in cytosol.

4. Bioassay of Abscisic Acid:-

> ABA now ranks in importance with auxins, gibberellins and cytokinins as a controlling factor in physiological processes.

> ABA can decrease, overcome, reverse, counteract, and inhibit the responses of plant materials to each of the growth-promoting hormones.

> The choice of bioassay is dictated by the type of process believed to be influenced by the substance.

> The bioassay methods which have been extensively used in connection with ABA are:

a. Acceleration of Abscission in Excised Abscission Zones (Explants):-

- Addicott et al. who first detected ABA by its effect in promoting leaf abscission, used this action in a biological test.

- Test object was young cotton seedlings from which roots, stem tips and blades of cotyledons (seed leaves) were removed leaving an explant consisting of a section of stem to which the stumps of the petioles, i.e., leaf stalks were still attached.

- ABA as lanolin paste may be applied either to the proximal or to the distal end of the abscission zone.

- Time taken by the petiolar stump to abscise in response to the application of a definite concentration of ABA is taken as the standard.

b. Inhibition of Coleoptile Section Growth:-

- Inhibitors have usually been detected by their ability to reduce the extension growth of oat or wheat coleoptiles.

- Since such growth is stimulated by auxins, a common practice has been to add a standard amount of IAA to the test solution and to observe the reduction by inhibitor of growth stimulated by auxin (Rothwell and Wain, 1964).

- Wheat seeds are thoroughly washed, soaked for 3-4 hours and then germinated in dark humid chamber for 72 hours.

- When the coleoptiles are about 3 cm in length, they are cut at the bases, placed vertically with the tips upward in specimen tubes containing distilled water.

- The tubes are then placed in a light-tight box, first incubated at 37°C for one hour and then kept in cold at 4°C for 24 hours. This pretreatment has been found to minimise the residual endogenous auxin content.

- After this, the sub-apical 6 mm lengths of coleoptiles 2 mm below the apices are cut with razor blades.

- Such coleoptile sections are placed in Petri dishes with ABA to be assayed, standard IAA, 3 per cent sucrose in phosphate buffer (0.006 M, pH 5.2).

- These are incubated at 25°C for 20 hours and the lengths measured accurately.

c. Retardation of Growth of Cultures of Small Aquatic Plants:-

- This assay has been described by Van Overbeek et al.

- Cultures of Lemna minor (duckweed) are the sensitive materials to respond to ABA. Therefore, sterile cultures of L. minor grown under constant fluorescent light and constant temperature are used as bioassay materials.

- Growth is vegetative by budding and is determined as increase of fresh weight.

- ABA concentration as low as 1 part per billion (ppb) causes detectable inhibition.

d. Barley, Rice and Wheat Endosperm Bioassay:-

- Paleg (1960) studied GA-induced production of a-amylase in barley and the subsequent release of reducing sugars into the medium.

- ABA inhibits the production of a-amylase which is triggered by GA in isolated aleurone layers or de-embryonated seeds.

- Seeds are cut, embryo-containing portion discarded, placed in vials with test solutions (GA + ABA) and antibiotic Streptomycin sulphate to prevent bacterial growth.

- After incubation for a definite period, reducing sugars are assayed.

- Instead of measuring reducing sugar in the medium, α-amylase may be directly assayed.

- Since enzyme synthesis is a stage closer to the primary site of action of GA and ABA in this system than is sugar release, the authors regard this an advantage in itself.

e. Detection of Antitranspirant Activity:-

- A greatly improved method has been described for the bioassay of ABA and other compounds that possess antitranspirant activity.

- The stomatal responses are observed on pieces of isolated epidermis of Commelina sp. immersed in small volumes of solution containing the compounds to be assayed.

- It is possible to obtain linear responses to ABA concentrations over the range 10-7 to 10-10M in citrate buffer at pH 5.5.

- The extent of stomatal closure in response to a definite concentration of ABA is the basis for this assay and it is possible to detect as little as 26 picogram (pg) of ABA present in the medium.

- Lack of interference from other regulators is a feature unique to this assay.

5. Physiological effects of Abscisic Acid:-

i. Seed and bud dormancy:- Its main function is to maintain seed dormancy. Abscisic acid induces dormancy of buds towards the approach of winter. Abscisic acid accumulates in many seeds during maturation and apparently contributes to seed dormancy.

ii. Senescence:- ABA acts as a general inducer of senescence (Thimann). The onset of senescence is correlated with stomatal closure. The ABA content of aging leaves increases markedly as senescence is initiated.

iii. Abscission:- It is the process of shedding old or unwanted organs such as leaves, flower, floral organs, and fruits. ABA promotes abscission through ethylene.

iv. Flowering:- In long-day plants, the effect of gibberellins on flowering is counteracted by ABA, which accumulated in the leaves during the short winter days. This ABA acts as inhibitor of flowering in long-day plants. On the other hand ABA induces flowering in short-day plants.

v. Starch hydrolysis:- The GA-induced synthesis of a-amylase and other hydrolytic enzymes in barley aleurone cells is inhibited by abscisic acid. This inhibition can be reversed by increasing the amount of GA supplied.

vi. Geotropism:- ABA controls geotropic responses of roots. It stimulates positive geotropism in roots.

vii. Stress response:- It also close the stomata of the leaves in dry conditions. Hence it is also called stress hormone.

6. Applications of Abscisic Acid:-

i. Antitranspirant:- Application of minute quantity of abscisic acid to leaves shall reduce transpiration to a great extent through partial closure of stomata. It conserves water and reduces the requirement of irrigation. Photosynthesis is reduced to a lesser extent (Transpiration 56%: Photosynthesis 14%).

ii. Flowering:- It is useful in introducing flowering in some short day plants kept under un-favourable photoperiods.

iii. Rooting:- Use of abscisic acid promotes rooting in many stem cuttings.

iv. Dormancy:- Abscisic acid can be used in prolonging dormancy of buds, storage organs and seeds.

7. Mode of Action of Abscisic Acid:-

a. Regulation of Gene Expression and Enzyme Synthesis:-

> ABA has inhibitory effects on protein synthesis. The effect of ABA on protein synthesis appears to be selective since it has been shown to affect directly the synthesis of those proteins whose synthesis is under hormonal control.

> The best example of such translational control is the inhibition by ABA of the GA-promoted synthesis of α-amylase and other hydrolases like protease and ribonuclease in barley aleurone layers.

> Ho and Varner suggested that the inhibition of a-amylase synthesis by ABA is due to an effect on translation, since they found that ABA still inhibited the formation of α-amylase at 12 h, a time when RNA synthesis inhibitor cordycepin had no longer any effect.

> They have postulated that ABA might de-repress a regulator gene or interact with a regulator RNA protein species to inhibit the translation of α-amylase mRNA.

> ABA has regulatory effects at the levels of both transcription and translation. The expression of numerous genes is stimulated by ABA under various stress conditions. Such conditions include heat shock, low temperature and salinity stress as well as the period of seed maturation.

> It is well known that gene activation is mediated by DNA-binding proteins, which acts as transcription factors. ABA has been shown to stimulate the transcription of the genes, which encode these binding proteins.

> Distinct DNA segments have been identified that are involved in stimulation of transcription by ABA, thought to be ABA-response elements (ABAREs). Contrary to the stimulation of gene expression, ABA is also involved in repression of gene transcription.

> The common example is ABA-induced repression of barley a-amylase gene which is expressed by GA. In this case, a few DNA elements which mediate ABA-induced gene repression are quite similar to the gibberellin response elements (GAREs).

b. Stomatal Complexes and ABA:-

> Turgor changes within the guard cells regulate stomatal aperture. Such turgor changes are caused by movement of K+, H+, Cl– ions and the synthesis, metabolism and movement of organic anions, particularly malate. The role of ABA in these movements and synthesis is still a matter of conjecture.

> One important factor in regulating guard-cell turgor is thought to be the operation of an active H+/K+ exchange process. ABA inhibits K+ uptake into the guard cells and also proton release. The fungal toxin fusicoccin (FC) overcomes the effect of ABA on the stomata by stimulating H+/K+ exchange. ABA may also affect the distribution of malate.

> During stomatal closure in dark, malate leaks from the epidermal strips of Commelina communis, Vicia faba floating on water. Addition of ABA increases both the rate of closure and the rate of malate leakage.

> Malate serves as a source of protons for H+/K+ exchange process during opening and malate leakage from guard cells during closure is required to reduce turgor. ABA would inhibit H+/K+ exchange and promote specific leakage of malate, thus inhibiting opening and promoting closure.

c. Membrane Depolarization by Increasing Ca2+ and pH of Cytosol:-

> Although stomatal closure is mediated by a reduction in guard cell turgor pressure caused by massive outward movement of K+ and anions (CI– and malate) from the cell, ABA also induces a net influx of positive charge.

> ABA has been shown to induce an increase in cytosolic Ca2+ concentration by activation of calcium channels leading to membrane depolarization. Cytosolic calcium concentration is further increased by ABA by causing release of calcium from an internal store like vacuole, and this increase is sufficient to cause stomatal closing.

> In addition to increasing cytosolic Ca2+, ABA causes alkalization of the cytosol. The increase in cytosolic pH triggers the voltage-gated K+ efflux channels to open resulting in K+ loss and stomatal closure.

> The inhibition of plasma membrane H+-ATPase has also been held responsible for membrane depolarization. ABA inhibits the plasma membrane proton pump and favours depolarization in an indirect manner. It is presumed that the increase in Ca2+ concentration and pH of the cytosol in the presence of ABA results in the inhibition of H+ -ATPase.

> ABA not only causes stomatal closure by activating outward ion channels but also prevents stomatal opening. Under normal conditions, the inward K+ channels are open when the membrane is polarized by the proton pump and K+ ions get inside through H+/K+ exchange causing stomatal opening. In this case, ABA inhibits inward K+ channels through an increase in Ca2+ and pH, thus preventing stomatal opening.

Ans.

Vernalization:- It is the artificial exposure of plants or seeds to low temperatures in order to stimulate flowering or to enhance seed production. Gibberellin is a hormone that replaces vernalization. The metabolically active apical meristems are the sites of perception of temperature to initiate flowering. The younger leaves are more susceptible to the process of vernalization. The shoot apex of mature stems or embryo of seeds receives low temperature stimulus.

Mechanism of Vernalization:- Through vernalization, there is an advancement in the process of blooming as a result of the delayed period of low temperatures, for instance, that which is attained in winter. To describe the mechanism of vernalization, there are two main hypotheses –

a. Phasic development theory

b. Hormonal theories

a. Phasic Development Theory:- As per this hypothesis, there is organization of stages in the plant’s improvement. Each stage is under the impact of environmental elements such as light, temperature etc. Here, in turn, there are two main stages –

i.Thermostage:- It depends on temperature, wherein vernalization accelerates thermostat. Thermostage is the vegetative phase requiring low heat, aeration and enough dampness

ii. Photostage:- It necessitates high temperature. Here, vernalin assists in producing florigen.

b. Hormonal theories:- As per this hypothesis, the freezing treatment propels the development of a floral hormone referred to as vernalin. Such a hormone is imparted to various parts of the plant. The vernalin hormone diffuses from the vernalized plants to the unvernalized plants, prompting blooming.

M.SC. BOTANY SEM - II MGSU, BIKANER

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