Dormancy in Daylilies

Abscisic Acid (Abscicic Acid)

by Tom Hart

Introduction:

There has been much discussion among daylily aficionados about what causes dormancy of both the plant and the seed. It is my hope that this paper will present some information and then allow us to make predictions based on this information. Those interested in the subject may then test these predictions thus furthering our knowledge about daylily dormancy. This is not meant to be the answer to all our questions, but hopefully it will be a start.

In many plants Abscisic Acid (ABA) is essential for seed maturation and enforces a period of both seed and plant dormancy. Could this be the compound that causes dormancy in daylilies? We may assume that it is, at least, one of the compounds responsible for plant and seed dormancy. Abscisic acid was first discovered back in 1963 by Ohkuma et al. who were studying factors influencing the leaf abscission. Shortly afterwards, Comfort et al. 1965, isolated the same dormancy compound from Sycamore.¹

When infrared spectra were compared they were found to be identical. Since then this dormancy influencing compound has been found in many plants, both monocots and dicots.

It is important that seeds not germinate prior to their normal growth period. ABA in the seed enforces this dormancy. Not until the seed has been exposed to a prolonged cold spell and/or sufficient water to support germination is dormancy lifted. (Water will tend to leach or remove the ABA from the seed or dormant bud, allowing for growth.) ABA mediates the conversion of an actively growing apical meristem into a dormant bud. ABA in the bud or growing point acts to enforce dormancy so if an unseasonably warm spell occurs before winter is over, the buds will not sprout prematurely.

Functions of Plant Hormones:

Plant hormones (phytohormones) are compounds produced by the plant and are moved to a location where they act on other compounds or cells. Auxins, cytokinins, and gibberellins stimulate growth in the tissues. Abscisic acid, acting as a growth inhibitor, suspends growth producing dormancy the opposite of the three growth stimulators. Other hormones may have other functions.

Auxins - Auxins promote stem elongation, cell enlargement and inhibit growth of lateral buds (maintains apical dominance). They are produced in the stem, buds, and root tips. Example: Indole Acetic Acid (IAA) and a number of synthetic forms. Auxin is a natural plant hormone produced in the stem tip that promotes cell enlargement. Auxin moves to the darker side of the plant, causing the cells there to grow larger than corresponding cells on the lighter side of the plant. This produces a curving of the plant stem tip toward the light, a plant movement known as phototropism. The auxin produced by the apical bud (or growing tip) diffuses downwards and inhibits the development of lateral bud growth, which would otherwise compete with the apical tip for light and nutrients. Removing the apical tip and its suppressive hormone allows the lower dormant lateral buds to develop, and the buds between the leaf stalk and stem produce new shoots which compete to become the lead growing stem. The plant hormone strigolactone has also been found to inhibit shoot branching. Auxins also influence the production of gibberellic acid.

Cytokinins - Cytokinins promote cell division. They are produced in growing areas, such as meristems at tip of the shoot.

Gibberellic Acid – Gibberellins cause an increase in cell elongation and the stimulation of growth. • Stimulates stem elongation by stimulating cell division and elongation. • Stimulates bolting/flowering in response to long days. • Breaks seed dormancy in some plants which require stratification (cold periods) or light to induce germination. Works in opposition to Abscisic Acid. • Stimulates enzyme production (a-amylase) in germinating cereal grains for mobilization of seed reserves.

Abscisic Acid Functions:

The actual means of action in most of the phytohormones is in question. In humans, abscisic acid causes an increase in cyclic ADP-ribose and an increase of intracellular calcium which then act on the cells. The action of abscisic acid in plants is still in doubt, but it has been reported that abscisic acid acts to stimulate calcium ions to cause physiological actions in plants².

Growth will begin either when there is an increase of activity of gibberellins or when abscisic acid is removed or inactivated. Cold temperatures will cause an increase in gibberellins activity. The ratio between abscisic acid and gibberellins regulate dormancy of the terminal buds. Abscisic acid prevents gibberellic acid from causing an increase in cell elongation. Heavy rain or high amounts of water can cause the abscisic acid to be washed out. Light and other stimulates can trigger the degradation of abscisic acid.

Cold temperatures will stimulate the production of ABA causing dormancy of the plants. In many cases the temperatures must be below 10oC for a specific period of time to produce enough ABA to cause dormancy.³ ABA can substitute for the low temperature stimulus, if there is an adequate supply of sugars. This Abscisic Acid may be produced when the plants are exposed to short day (SD) conditions; it is actually the long night which causes ABA production – not the short days. The combination of a SD photoperiod and cold temperatures will greatly increase the amount of ABA produced. In Alfalfa different varieties will have different concentrations of phytochrome and associated ABA. More dormant varieties will have higher concentrations of phytochrome and Abscisic Acid.⁴

Some physiological responses known to be associated with abscisic acid ⁵,⁶, ⁷

• Stimulates the closure of stomata (water stress brings about an increase in ABA synthesis).

• Inhibits shoot growth but will not have as much affect on roots or may even promote growth of roots.

• Induces seeds to synthesize storage proteins.

• Inhibits seed germination – causes seed dormancy • Inhibits the affect of gibberellins on stimulating synthesis of a-amylase (stored starch).

• Effects induction and maintenance of dormancy.

• Induces gene transcription (DNA - RNA - protein) especially for proteinase inhibitors (enzymes which inhibit the enzymes which break down proteins) in response to wounding.

• Acts by inhibiting indole acetic acid (auxin), gibberellin and cytokinin (growth promoters)

• Abscisic acid increases through time during temperatures below 10oC

• Abscisic acid increases under short day conditions in many trees and grasses

• Evidence suggests ABA can substitute for the low temperature stimulus, provided there is also an adequate supply of sugars

• Inhibits fruit ripening • Other growth inhibitors also present

• Probably a partially dominant gene (actually controlled by two genes)

Synthesis of Abscisic Acid:

Genes are composed of DNA which is used by the plants to produce RNA. The RNA, in turn, is used to produce proteins, which, for our purposes, act as enzymes. The enzymes allow specific compounds to be changed to particular other compounds. Carotenoids such as zeaxanthin and violaxanthin in daylilies give the yellow color to the flowers, but more importantly supply light energy to chlorophyll in the leaves.

The breakdown of these carotenoids to xanthoxal (xanthoxin) in the leaves will cause a decrease in the yellow pigment in the leaves. This decrease in yellow color may be enough to give a blue green cast to the leaf in daylilies. However, in tomatoes the concentration of carotenoids is 20 to 100 times the concentration of ABA, which might make any decrease in carotenoid concentration due to an increase in ABA difficult to detect ⁸.

Previous studies have indicated that the conversion of violaxanthin to xanthoxal occurs in the chloroplast and the conversion of xanthoxal to abscisic acid occurs in the cytoplasm. Recently it has been noted that spinach has the ability to produce ABA within the chloroplasts themselves.⁹ To my knowledge, no one has yet determined if washed chloroplast can produce ABA by themselves in daylilies. We can, for now, assume that xanthoxal is produced by the chloroplast (possible gene is cis-isomerase) and is determined by the pod parent. Abscisic acid would be produced from Xanthoxal within the daylily cytoplasm and would be controlled by both the pod and pollen parent. Xanthoxal (xanthoxin) has a slight ability to cause dormancy in plants. Abscisic Acid has a much stronger potential to inhibit plant growth and therefore cause dormancy.¹⁰ Note that there is one chloroplast gene to change violoxanthin to Xanthoxal (xanthoxin) and it takes two genes to change xanthoxal to Abscisic Acid. The yellowish cast to the leaves which is produced by the carotenoids zeaxanthin and violaxanthin would be decreased if Xanthoxal is produced causing a decrease in the carotenoid concentration.

A diploid monohybrid cross would involve one gene and two alleles. A heterozygous individual for ABA production would have the both an "A" gametes or "a" gametes. Homozygous individuals would either have two dominant alleles (AA) or two recessive alleles (aa). The heterozygous individual then would produce gametes either containing a dominant "A" or a recessive "a".

The following example crosses are assuming no activity of the pod parent produced xanthoxal:
Semi-evergreen x Semi-evergreen (Aa x Aa) – produce gametes either A (dominant) or a (recessive) in about equal numbers

The results from this monohybrid partially dominant cross of a semi-evergreen plant with another semi-evergreen plant would be around half semi-evergreen while the other half would be roughly split evenly between dormant and evergreens.

Xanthoxal must be present before Abscisic Acid can be produced.  Two enzymes are necessary to convert Xanthoxal to Abscisic Acid.  Therefore two separate genes must have the dominant allele present.  We can assume that "B" stands for the dominant allele and "b" stands for the recessive allele.  If a large "B" gene is present, Abscisic Aldehyde will be produced. With "A" standing for the dominant allele which produces Abscisic Acid from Abscisic Aldehyde. ABA would be produced only when both "B" and "A" genes are present. Assuming partial dominance of both genes, The more dominant genes (B) present the more Abscisic Aldehyde would be produced and the more dominant genes (A) present the more Abscisic Acid would be produced from the Abscisic Aldehyde.

Possible cross between two semi-evergreen daylilies - AaBb x AaBb
Diploid Dihybrid Cross (possible gametes are AB, Ab, aB, ab)

Possible cross between two semi-evergreen daylilies - (AABb x AaBB)
Diploid Dihybrid Cross - possible gametes are Female (AB, Ab) and male (AB, aB)

Possible cross between two dormant daylilies - AABb x AABb
Diploid Dihybrid Cross - possible gametes are Female (AB, Ab) and male (AB, Ab)

Possible cross between two evergreen daylilies - AAbb x aaBB
Diploid Dihybrid Cross - possible gametes are Female (Ab,) and male (aB)

When examining two gene characteristics in diploid daylilies we have the potential of getting up to nine different genotypes (allele combinations) and five different appearances (phenotypes) if both genes are partially dominant.  In tetraploids there would be over twice as many allele combinations.  Of these combinations nine would produce evergreen plants.  The rest would be variations which would be hard to distinguish.

Conclusions:
If we, as daylily growers/hybridizers can assume that ABA is involved with dormancy we can then make a number of further assumptions.
1. Spraying Abscisic Acid on the plants may temporarily increase dormancy. As no synthetics are known, ABA is quite expensive.
2. Spraying Gibberellic Acid on plants may temporarily counteract dormancy.
3. Blue-green foliage may indicate increased Abscisic Acid (dormancy) because the concentration of carotenoids would be lower than normal in the leaves.
4. The decrease of yellow leaf color is actually due to Xanthoxal production, so it will be possible to have evergreen/semi-evergreen daylilies which have blue-green leaves.
5. Zeaxanthin and violaxanthin and, in a few species, Abscisic Acid concentrations are produced in the chloroplast and therefore controlled by the pod parent.
6. Xanthoxal, which may cause some dormancy, is produced in the chloroplasts and is therefore controlled by the pod parent.
7. Abscisic Acid enzymes are typically thought to be found in the cytoplasm and therefore would be controlled by both the pod and pollen parent.
8. Soils deficient in molybdenum may not allow Abscisic Acid to be produced from Abscisic Aldehyde because of a deficient amount of the molybdenum cofactor.
9. As the Abscisic Acid concentration is increased by lack of water or water stress, the degree of plant dormancy may vary somewhat from location to location and from year to year.
10. As ABA is soluble in water, and may be leached from the plant/seed, plant dormancy may vary from location to location and from year to year depending on water present.
11. Cold temperatures increase Gibberellic Acid production therefore stimulating seed/plant growth, seed/plant dormancy may vary from location to location and from year to year depending on the temperatures.
12. Abscisic Acid increases under short day conditions and may be controlled by photoperiod.
13. As daylilies originated in different latitudes, it would be logical that the different species would have evolved different photoperiod requirements (different required lengths of darkness).  Today some varieties might require nights of 18 hours, 12 hours, 8 hours, etc to go dormant.
14. The terms dormant, semi-evergreen and evergreen becomes less valuable in tetraploids because there are more than three possibilities.  With two genes influencing the production of Abscisic Acid we might expect many more variations which would be difficult to tell apart.

A few questions:
A. Are evergreen daylilies really more tender or do the plants tend to run out of stored food because they "attempt" to grow all winter long?  Can we really say daylily dormants are hardy while evergreen daylilies are tender?
B. Do dormant plants always have seeds which require a dormant period?  It would be possible for an evergreen plant to produce dormant seeds requiring a resting period or a dormant plant produce seeds which will grow without a rest period.  Different parts of the plant may, at times, be affected by different genes or have specific genes turned on/off and different times and therefore “behave” differently.
C. Water stress increases ABA concentration and abundant water causes lower levels.  Do dayliles tend to go into normal dormancy when entering into the dormant period under conditions of high water?
D. How important is xanthoxal (xanthoxin) in the dormancy of daylilies?  How much dormancy does this pod parent produced compound cause by itself? 
E. Are Gene A and Gene B completely dominant or partially dominant?
F. Can we produce hard dormant plants by crossing two fully evergreen diploid plants?  If hard dormant plants are commonly produced we might include multiple genes or other compounds as part of the explanation for dormancy.
G. Which particular effects of Abscisic Acid are not useful in our understanding of dormancy in daylilies?  As other compounds may also be involved in daylily dormancy, it is important for us to know which characteristics of daylily dormancy cannot be explained by the actions of the Abscisic Acid compounds alone.

Sources:

1. Milborrow, B.V. 2001. The pathway of biosynthesis of abscisic acid in vascular plants: a review of the present state of knowledge of ABA biosynthesis. Journal of Experimental Botany, Vol. 52, No. 359, pp. 1145-1164, June 1, 2001, Oxford University Press.  http://jxb.oxfordjournals.org/cgi/content/full/52/359/1145

2. Wei-Yi Song, Zheng-Bin Zhang, Hong-Bo Shao, Xiu-Lin Guo, Hong-Xing Cao, Hong-Bin, ZhaoZheng-Yan Fu, Xiao-Jun Hu.  2008.  Relationship between calcium decoding elements and plant abiotic-stress Resistance.  International Journal of Biological Sciences,4(2): 116-125. http://www.biolsci.org/v04p0116.htm  

3. L. V. Gusta, R. Trischuk, and C. J. Weiser.    2005.  Plant Cold Acclimation: The Role of
Abscisic Acid.  J Plant Growth Regul ,24:308–318 http://www.plantstress.com/Articles/up_cold_files/Cold%20accl-Gusta.pdf

4. CHENGZHANG WANG; MA B. L. ; JINFENG HAN ; YANHUA WANG ; YONGGE GAO ; XIFENG HU ; CHUNMEI .  2008 .   Photoperiod Effect on Phytochrome and Abscisic Acid in Alfalfa Varieties Differing in Fall Dormancy.   Journal of plant, vol. 31,  pp. 1257-1269  http://www.informaworld.com/smpp/content~content=a794141369

5. Kimball's Biology Pages. http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/ABA.html

6. Mendipweb. Plant Hormones. http://www.plant-hormones.info/abscisicacid.htm

7. L. V. Gusta, R. Trischuk, and C. J. Weiser.  2005.    Plant Cold Acclimation: The Role of Abscisic Acid.  J Plant Growth Regul  24:308–318 http://www.plantstress.com/Articles/up_cold_files/Cold%20accl-Gusta.pdf

8. Milborrow, B.V.   2001. The pathway of biosynthesis of abscisic acid in vascular plants: a review of the present state of knowledge of ABA biosynthesis. Journal of Experimental Botany, Vol. 52, No. 359, pp. 1145-1164, June 1, 2001, Oxford University Press http://jxb.oxfordjournals.org/cgi/content/full/52/359/1145

9. Milborrow BV, Lee H-S.  1998.  Endogenous biosynthetic precursors of (+)-abscisic acid. VI. Carotenoids and ABA are formed by the ‘non-mevalonate’ triose-pyruvate pathway in chloroplasts. Australian Journal of Plant Physiology 25, 507–512.

10. Milborrow, B.V.  2001.  The pathway of biosynthesis of abscisic acid in vascular plants: a review of the present state of knowledge of ABA biosynthesis. Journal of Experimental Botany, Vol. 52, No. 359, pp. 1145-1164, June 1, 2001, Oxford University Press http://jxb.oxfordjournals.org/cgi/content/full/52/359/1145

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