1. Plant Secondary metabolites
Dr. Abdul Latif Khan

2. Primary and Secondary metabolism
Secondary metabolites
\figures\ch13\pp13040.jpg

6. The Growth Hormone
- efficient communication among cells, tissues, and organs of multicellular organisms
- In higher plants, regulation and coordination of metabolism, growth, and morphogenesis depend on chemical signals from one part of the plant to another.
- Chemical messengers “hormones” are responsible for the formation and growth of different plant organs.
- External factors such as gravity could affect the distribution of these chemical messengers within a plant

7. - Hormones are chemical messengers that are produced in one cell or tissue and modulate cellular processes in another cell by interacting with specific protein receptors
- Most plant hormones are synthesized in one tissue and act on specific target sites in another tissue at vanishingly low concentrations.
- Endocrine hormones: hormones that are transported to sites of action in tissues distant from their site of synthesis.
- Paracrine hormones: hormones that act on cells “sites” adjacent to the source “site” of synthesis.

8. - Plant development is regulated by six major types of hormones: auxins, gibberellins, cytokinins, ethylene, abscisic acid, and brassinosteroids.
- Other signaling molecules that play roles in resistance to pathogens and defense against herbivores have also been identified in plants (e.g. jasmonic acid, salicylic acid, polypeptide systemin)

9. Function of Hormones Light - phototropism Touch - thigmotropism
Gravity – gravitropism
Turgor movements
Biological clock - circadian rhythms
When to open and close stomata
Control of flowering - photoperiodism

10. Response of Hormones

11. Auxins

12. - Auxin was the first growth hormone to be studied in plants; the early physiological work on the mechanism of plant cell expansion was carried out in relation to auxin action.
- Auxin and cytokinin differ from the other plant hormones in one important aspect: they are required for viability.
(so far, no mutants lacking either auxin or cytokinin have been found, suggesting that mutations that eliminate them are lethal)

13. - points to be covered:
- history of auxins discovery
- description of the chemical structures of auxins
- detection methods of auxins in plant tissues
- pathways of auxins biosynthesis
- developmental processes controlled by auxin:
---- stem elongation
---- apical dominance
---- root initiation
---- fruit development
---- meristem development
---- oriented or tropic growth

14. Auxin research has been continually advanced by new technologies
The black triangle shows dark-grown Arabidopsis and pea seedlings, two of the plants that have contributed most to our understanding of auxin action.
Abel, S., and Theologis, A. (2010). Odyssey of auxin. Cold Spring Harb Perspect Biol. doi: /cshperspect.a004572

15. Darwin (1890s) studied phototropism – movement towards light
Darwin and others studied coleoptiles –tissues that protect monocot leaves during germination
Darwin, C., and Darwin, F. (1881) The power of movement in plants. Appleton and Co., New York.; Photos courtesy of Dr. R.L. Nielsen

16. Cutting off or covering the coleoptile tip interferes with the response
These experiments showed that the light signal is perceived at the tip, although the bending occurs at the base
Untreated coleoptile bends
Coleoptiles with tips shielded from light or removed do not bend

17. Darwins’ (Charles and son) experiment
Under normal conditions, shoot tips bend towards the light
Without light on the tip, no bending
When not at tip, collar doesn’t prevent bending
Conclusion: Light is sensed at the tip, but response not at tip
New hypothesis: A substance or chemical is transported
Auxin later isolated from shoot tips and established to be involved in cell elongation
Drawings depicting seedlings of Zea (Gramineae family)

18. Darwin concluded that a signal moves from tip to base
“We must therefore conclude that when seedlings are freely exposed to a lateral light some influence is transmitted from the upper to the lower part, causing the latter to bend.”

19. 1- The Emergence of the Auxin Concept
- Charles Darwin and his son Francis and their studies on plant tropisms
- Phototropism, the phenomenon of bending of plants toward light due to differential growth.
- Darwin’s experimental observations on Coleoptiles:
--- If coleoptiles illuminated on one side with short pulse of dim blue light, they will bend (grow) toward the source of the light pulse within an hour.

20. --- If the tips of the coleoptiles covered with foil, the coleoptiles would not bend.
(i.e. the tip of the coleoptile perceived the light)
--- The region of the coleoptile that is responsible for the bending toward the light (growth zone) is several millimeters below the tip.

21. - Darwins conclusion:
- some sort of signal is produced in the tip, travels to the growth zone, and causes the shaded side to grow faster than the illuminated side
- Followed research experimentation on the nature of the growth stimulus in coleoptiles culminated in the presence of a growth-promoting chemical in the tip of coleoptiles
- If the tip of a cleoptile was removed, coleoptile growth ceased.

22. Boysen-Jensen (1913) showed that the transmitted influence can move through a gelatin block
Left to right – solid, butter, gelatin, control. The base of the coleoptile was shaded.
Before
The signal cannot move through a solid block or butter, demonstrating that it is a water- soluble chemical.
After
Left to right – solid, butter, gelatin, control (no cutting). By shading the base, he ensured that the signal had to move from the site of perception above the cut surface through the butter or block and stimulate bending in the base.

23. Repositioning the tip can induce bending in uniform light
Before
After
Paal (1919) showed that removing the tip and replacing it on one side of the base is sufficient to cause bending.
Control
Tip removed
Tip removed and replaced
Tip removed and replaced to one side
Asymmetric tip placement causes bending
Leftmost coleoptile is the uncut control, second is cut coleoptile, third is coleoptile with tip replaced symmetrically, and fourth is coleoptile with tip replaced to the side, which initiates bending. Note that these experiments are done in uniform, not unidirectional, light. These experiments suggest that the signal from the tip promotes growth and that the light changes its distribution.

24. In the 1930s, auxin was purified and shown to promote growth
Frits Went collected auxin from shoot tips into agar blocks...
...and showed that the material collected in the agar blocks was the growth-promoting substance.
This bending assay for the growth-promoting effect of auxin was used as a basis for its purification.
Angle of curvature is proportional to amount of auxin in block
Indole-3-acetic acid, IAA
Redrawn from Went, F.W. (1935) Auxin, the plant growth-hormone. Bot. Rev. 1:

25. - Major breakthrough by Went (1926):
--- allowing the material to diffuse out of excised cleoptile tips into gelatin blocks
--- placing these gelatin blocks asymmetrically on top of a decapitated coleoptile, then
--- testing for the ability of these gelatin blocks to cause bending of the coleoptile in the absence of a unilateral light source.
- (see Figures 19.1 and 19.2)

27. Auxin’s root-promoting properties were also known by the 1930s
Adventitious roots are initiated from grape stems treated with auxin
A more recent experiment: auxin-treated radish roots initiate lateral roots at a frequency proportional to auxin concentration
µM IAA
Thimann, K.V. (1938). Hormones and the analysis of growth. Plant Physiol. 13: Kerk, N.M., Jiang, K., and Feldman, L.J. (2000). Auxin metabolism in the root apical meristem. Plant Physiol. 122:

28. Auxin’s role in apical dominance was also known in the 1930s
Decapitate
Replace apex with agar block: without or with auxin.
Auxin suppresses bud outgrowth
No auxin
Auxin
Bud Length
Thimann, K.V., and Skoog, F. (1934). On the inhibition of bud development and other functions of growth substance in Vicia faba. Proceedings of the Royal Society of London B. 114: with permission; Went, F.W. and Thimann, K.V. (1937) Phytohormones. The Macmillan Company, New York.

29. Evidence for the role of auxin in apical dominance
High auxin concentration
Low auxin concentration
Drawings depicting Coleus (Lamiaceae family)

30. Different tissues were recognized to have different sensitivities to auxin
Auxin concentrations that promote elongation in stems can be inhibitory in roots.
Thimann, K.V. (1938). Hormones and the analysis of growth. Plant Physiol. 13:

31. Polar transport of auxin was recognized in the 1930s
A segment cut from a coleoptile can move auxin from tip to base.
This experiment, carried out by H.G. Van der Weij revealed that auxin in the shoot is translocated from tip to base.
The upper agar block was loaded with auxin, which the segment translocated to the lower block.
When the segment is inverted, it is unable to transport auxin from base to tip.
Adapted from Went, F.W. (1935) Auxin, the plant growth-hormone. Bot. Rev. 1:

32. What classical studies told us
What we didn’t know was how auxin promoted growth, moved in a polar direction, limited bud outgrowth and stimulated root formation.
This lecture describes how the tools of molecular biology, genetics, and cell biology have continued the auxin story, and revealed more about how auxin regulates growth.
The chemical structure of auxin
That auxin promotes root formation and inhibits bud outgrowth
That auxin moves through the shoot from tip to base
That different tissues have different sensitivities
That auxin affects growth in a concentration and tissue-specific way

33. \figures\ch19\pp19030.jpg

34. \figures\ch19\pp19040.jpg

35. Auxin: a 21st century perspective
Auxin homeostasis
Tools in auxin research
Polar auxin transport
Perception and signaling
Auxin action in whole-plant processes
Interactions with other signals

36. Auxin signaling pathway
Synthesis
IAA
Transport
Perception (receptor)
TF activation/ inactivation
Target genes
Biological Functions
Catabolism
Conjugation
Auxin’s effects depend upon its synthesis, transport, perception, signaling, and target gene responses. Most of these functions are controlled by many genes with differing cell specificities.
TF indicates transcription factor
Adapted from Kieffer, M., Neve, J., and Kepinski, S. (2010). Defining auxin response contexts in plant development. Curr. Opin. Plant Biol.13:

37. Auxin response pathway – feedback regulation
Synthesis
IAA
Transport
Perception (receptor)
TF activation/ inactivation
Target genes
Biological Functions
Catabolism
Conjugation
The pathway is extensively self-regulated through positive and negative feedback.
Adapted from Kieffer, M., Neve, J., and Kepinski, S. (2010). Defining auxin response contexts in plant development. Curr. Opin. Plant Biol.13:

38. Regulation by environmental factors and other hormones
Synthesis
IAA
Transport
Perception (receptor)
TF activation/ inactivation
Target genes
Biological Functions
Catabolism
Conjugation
Gravity, Nutrient status, Ionic environment, Pathogens,
Light: directionality, intensity, wavelength
Gravity, nutrient status, ionic environment, pathogens,
Ethylene, brassinosteroids, cytokinins, gibberellins, jasmonates, strigolactones....
Adapted from Kieffer, M., Neve, J., and Kepinski, S. (2010). Defining auxin response contexts in plant development. Curr. Opin. Plant Biol.13:

39. 2- Identification, Biosynthesis, and Metabolism of Auxin
The principal auxin in higher plants is indole-3-acetic acid (IAA):
- (see figures 19.3 and 19.4 for structures of natural and synthetic auxins, respectively)
- Early definition of auxins: all natural and synthetic chemical substances that stimulate elongation in coleoptiles and stem sections.
- Auxins can be defined as compounds with developmental biological activities similar to those of (or associated with) IAA.

40. Biosynthesis and homeostasis
Indole
Tryptophan
IAA
Indole-3-pyruvic acid
(IPA)
Indole-3-acetaldehyde
Indole-3-acetamide
(IAM)
Indole-3-acetaldoximine
(IAOx)
Tryptamine
IAA is produced from tryptophan (Trp) via several pathways and one Trp-independent pathway (black arrow).
The IAOx pathway may be restricted to Arabidopsis and its close relatives.
Adapted from Quittenden, L.J., Davies, N.W., Smith, J.A., Molesworth, P.P., Tivendale, N.D., and Ross, J.J. (2009). Auxin biosynthesis in pea: Characterization of the tryptamine pathway. Plant Physiol. 151:

44. IAA is synthesized in meristems and young dividing tissues:
Biosynthesis of IAA is associated with rapidly dividing and rapidly growing tissues.
Sites of auxin synthesis:
--- Shoot apical meristems
--- young leaves
--- Root apical meristems
--- Young fruits and seeds ?
Basipetal shift in auxin production in very young leaf primordia correlates closely with the basipetal maturation sequence of leaf development

45. Auxin synthesis is developmentally and environmentally controlled
Indole
Tryptophan
IAA
Indole-3-pyruvic acid
Indole-3-acetaldehyde
Indole-3-acetamide
Indole-3-acetaldoximine
Tryptamine
Ethylene
Methyl Jasmonate
Red / Far-red light ratio
Temperature
Auxin biosynthesis is influenced by other hormones and environmental conditions
Adapted from Quittenden, L.J., Davies, N.W., Smith, J.A., Molesworth, P.P., Tivendale, N.D., and Ross, J.J. (2009). Auxin biosynthesis in pea: Characterization of the tryptamine pathway. Plant Physiol. 151:

46. Auxin synthesis promotes shade-avoidance hypocotyl-elongation response
An environment enriched with far-red light (which simulates shading by other plants) promotes hypocotyl elongation, but not in a mutant blocked in an auxin synthesis pathway.
Wild-type
taa1
In wild-type plants but not loss-of-function taa1 mutants, auxin synthesis is increased in shade conditions.
Auxin synthesis
White light
Shade
Tryptophan
Indole-3-pyruvic acid
TAA1
Reprinted from Tao, Y., et al. (2008) Rapid synthesis of auxin via a new tryptophan-dependent pathway
is required for shade avoidance in plants. Cell 133: 164–176, with permission from Elsevier.

47. Auxin homeostasis is also regulated by conjugation and degradation
IAA
(GH3 genes)
Overexpression of an auxin conjugating enzyme encoded by a GH3 gene reduces auxin levels in the plant and causes a dwarfed phenotype.
GH3.13 overexpression
Wild-type
GH3 genes are auxin-induced
Arabidopsis
Rice
Zhang, S.-W., et al., (2009) Altered architecture and enhanced drought tolerance in rice via the cown-regulation of indole-3-acetic acid by TLD1/OsGH3.13 activation. Plant Physiol. 151: Staswick, P.E., et al., (2005) Characterization of an Arabidopsis enzyme family that conjugates amino acids to indole-3-acetic acid. Plant Cell 17:

48. 3- Auxin Transport
The main axes of shoots and roots show apex-base structural polarity.
The apex-base structural polarity of shoots and roots is dependent on the polarity of auxin transport.
In excised oat coleoptile sections, IAA moves mainly from the apical to the basal end (basipetally), i.e. unidirectional transport (polar transport).
Polar auxin transport was reported in 375 million year old fossil wood.
- If polar auxin transport is disrupted in some regions (by the presence of buds or branches), the tracheary elements that differentiate in these regions form circular patterns.

49. - Since the shoot apex is the primary source of auxin in the plant, polar transport is the principal cause of an auxin gradient extending from the shoot tip to the root tip.
- The longitudinal gradient of auxin from the shoot to the root affects various developmental processes (e.g. embryonic development, stem elongation, apical dominance, wound healing, and leaf senescence).
- In roots, acropetal transport of auxin occurs in phloem, and phloem-based movement, driven by “source-sink” translocation of sugars.

50. Mechanisms responsible for the distribution of auxin in the plant
(Cellular mechanisms underlying and regulating auxin transport and their roles in plant adaptation to various environmental signals)
Polar transport requires energy and is gravity independent
- Donor- receiver agar block method (Fig ):
-- a donor block contains radioisotope- labeled auxin
-- a tissue segment
-- a receiver block
-- measuring the radioactivity in the receiver block

51. General properties of polar IAA transport:
1- Tissues differ in the degree of polarity of IAA transport.
-- Basipetal transport of IAA predominates in cleoptiles, vegetative stems, and leaf petioles.
-- Acropetal transport of IAA occurs in the stelar tissues of the root.
-- Polar transport is not affected by the orientation of the tissue, so it is independent of gravity.
-- See Fig for the lack of gravity effects on basipetal auxin transport in the inverted or upright orientation of stem cuttings.

52. -- Roots form at the base end because root differentiation is stimulated by auxin accumulation due to basipetal transport.
-- Shoots form at the apical end where the auxin concentration is lowest.
2- Polar transport proceeds in a cell-to-cell fashion
-- Auxin exits the cell through the plasma membrane, diffuses across the compound middle lamella, and enter the next cell through its plasma membrane.
-- Auxin efflux is the loss of auxin from cells.
-- Auxin influx or uptake is the entry of auxin into cells.
-- The overall process requires metabolic energy.

53. 3- The velocity of polar auxin transport ranges from 2 to 20 cm/h.
-- Higher rates of polar transport are observed in tissue immediately adjacent to the shoot and root apical meristems.
-- Polar transport is specific for active auxin; auxin is recognized by protein carriers on the plasma membrane.
4- The major sites of polar auxin transport in stems, leaves, and roots is the vascular parenchyma tissue (the xylem).
-- In vascular parenchyma, the overall direction of auxin transport is downward; basipetally in the shoot and acropetally in the root.

54. -- In the coleoptile of grasses, basipetal polar transport of auxin occurs mainly in the nonvascular parenchyma tissues.
-- Auxin translocated in the phloem sieve tubes contributes to transport from shoot tissues to the growing root tips.
-- Basipetal auxin transport from the apex occurs in root as well.
-- Basipetal auxin transport in the root occurs in the epidermal and cortical tissues, and is important in gravitropism.

55. Evidence for the role of auxin in adventitious root formation
With synthetic auxin
Without synthetic auxin
Adventitious roots growing from stem tissue
Saintpaulia (Gesneriaceae family)
Another example of misleading common name
The African violet is not in the violet family

56. Band of achenes removed
Evidence for the role of auxin in formation of fruit and structures of similar function (e.g. receptacle in strawberry)
Normal conditions
All achenes removed
Band of achenes removed
Without seed formation, fruits do not develop. Developing seeds are a source of auxin.
What do you expect?
Not shown: Auxin replacement restores normal fruit formation and can be used commercially to produce seedless fruits
However, too much auxin can kill the plant and thus synthetic auxins used commercially as herbicides
Fragaria (Rosaceae family)

57. Perception (receptor) TF activation/ inactivation
Summary
Synthesis
IAA
Transport
Perception (receptor)
TF activation/ inactivation
Target genes
Biological Functions
Catabolism
Conjugation
In the past 30 years we have identified many of the molecular characters in the auxin story, and have a pretty good idea of its major themes, but the story is far from complete.
Adapted from Kieffer, M., Neve, J., and Kepinski, S. (2010). Defining auxin response contexts in plant development. Current Opinion in Plant Biology 13:

58. Ongoing investigations
Synthesis
IAA
Transport
Perception (receptor)
TF activation/ inactivation
Target genes
Biological Functions
Catabolism
Conjugation
What regulates auxin synthesis?
What do all those transport proteins do? What controls their activity and distribution??
Where does auxin elimination fit in?
What are the target genes, and what do they do?
What are the other TIR1 like proteins doing, and what does ABP1 do?
How do all these pieces fit together to make a functioning plant????
Why so many ARFs and Aux/IAAs?
Adapted from Kieffer, M., Neve, J., and Kepinski, S. (2010). Defining auxin response contexts in plant development. Current Opinion in Plant Biology 13: