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Chapter 4. Turfgrass Molecular Genetic Improvement for Abiotic/Edaphic Stress Resistance

Chapter 4. Turfgrass Molecular Genetic Improvement for Abiotic/Edaphic Stress Resistance

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R. R. DUNCAN AND R. N. CARROW



234



7

Pesb (above ground)



* discasea



* tmpmture (low, high)



use (wear, compaction)



* mowing



light (radiation;~ow,high)

* moisture (deficient, excess)

* wind

atmospheric pollutants



cultural practices



* Bases (02,COJ



* low moisture

* excess moisture

macrdmicro pores



o2and co*



* other p w



soil strength

low tempcratun



high temperature

soil stability



Chemical P r o d @

* PH



Al. Mn toxicities



nutrient defiaenaca

nutrient imbalances

dts

low CEC

heavy metals

toxic substances

calureous soils



microorganism types

microorganism

population levels

microorganism

population balances

insscts

nematodes

*'Weeds



animals



Figure 1 The turfgrass system: plant-soil- -atmosphere-man.



sites with marginal soil quality, and increasing usage of recreational sites. These

factors, when coupled with steady environmental pressure toward reduced water,

nutrient, and pesticide inputs, mandate the development of more stress-resistant

grasses.

Research efforts are being channeled into genetic improvement of overall stress

tolerance in grasses, but enhanced efficiency in utilizing gene technology is needed. Conventional breeding techniques can address individual and multiple stresstolerance traits, but advancementsin gene biotechnology can provide new and in-



TURFGRASS MOLECULAR GENETIC IMPROVEMENT



23 5



novative methods for enhancement of individual and more complex, multiple

stress tolerances (Brilman, 1997, Lee et al., 1996). The primary objective of this

review is to address some of the biotechnology methods that could lead turf enhancement programs into the twenty-first century. However, biotechnology is not

an end in itself. As with any scientific methodology, it must fit within a system or

framework with other components if the end result is to significantly improve turfgrasses. Thus, a secondary objective is to present a strategic framework for turfgrass improvement that incorporates gene technology.



II. MOLECULAR GENETIC IMPROVEMENT

A. STRESSADAPTATION

Unpredictable and irregular cyclic environmentalextremes create variable turfgrass performance responses. These responses may be in the form of (i) susceptibility to the stress and diminished turf quality, ultimately leading to death of the

plant, with the duration of exposure and degree of stress dictating the time frame;

(ii) acclimation, which is governed by an increase in turfgrass resistance (or

preadaptation) to harsh environments after exposure to periods of sublethal stress

(i.e., a phenotypic adjustment to gradual seasonal climatic changes) (Hallgren and

Oquist, 1990; Hoffman and Parsons, 1991); or (iii) adaptation, which implies

short- and long-term responses involving adjustments of physiological (Amzallag

and Lerner, 1995) and genetic (Allard, 1988; Orr and Coyne, 1992; Perez de la

Vega, 1996)parameters to environmental stress conditions. Plasticity in response

to stress is a buffering mechanism whereby turf genotypes respond to environmental changes involving genetically based natural selection (Via, 1994). Phenotypic plasticity in unpredictable environments is the variability in phenotypic

expression of individual turf traits within a species gene pool in response to

changeable or varying environments (Bradshaw, 1965; Counts, 1993; Via, 1994).

Quantitative traits that genetically control many of the stress responses exhibit

high plasticity (Perez de la Vega, 1996).

Adaptation mechanisms are not well understood since environmental stress factors are usually complex, several genetic and biochemical/physiological systems

control the turfgrass response, developmental stages are not equally sensitive or

responsive to variable environmental stress factors, and the same genes and associated response mechanisms can function in multiple stress environments (Perez

de la Vega, 1996). Genotypic differences are maximized under extremely stressful environmental conditions (Parsons, 1987); consequently, stress adaptation is

more easily accomplished in less favorable environments in which selection pressure reduces genetic variability for the stress response traits by eliminating less



236



R. R. DUNCAN AND R. N. CARROW



adapted genes (Perez de la Vega, 1996). Thus, poor-performing genotypes can be

excluded from the pool. Multiple severe abiotic and edaphic field stress environments can be utilized in turf breeding programs to enhance turf survivability and

persistence (Duncan and Carrow, 1997) in less favorable environments. However,

these same stress-adapted turf genotypes are capable of performing quite well in

more favorable environments,with better buffering (plasticity) against rapid shifts

in environmental extremes compared to their counterparts.

Genetic variation is a key determinant in successful adaptation to environmental stresses (Stanca et al., 1992). Selection in stress environments may not

necessarily target specific individual genes governing a single component of the

stress response mechanism but may act on multiple loci (Allard, 1988; Allard et

al., 1993). If major (qualitative) genes are functioning in adaptation to rapid environmental changes (Macnair, 1991), the turf population must adapt rapidly or

become locally extinct (Perez de la Vega, 1996). If stress adaptation results from

multiple alleles, each having small effects on various components of the response

mechanism (polygenes or quantitative genes), the breeding program can utilize

sequential exposure to increasing levels of stress and multiple cycles of pollination to enhance adaptation (Duncan and Carrow, 1997) without radically eliminating variability or positive turf traits. Selection indices of 1-5% can be used in

this population breeding program for successful advancement. The key components are a diverse gene pool, one or more discriminatingly severe field stress environments, high plant numbers in the breeding program, and the requirement for

surviving turf plants to go through entire life cycles including the reproductive

stage. Biotechnology can be used to supplement and enhance this strategy. Gene

technology addressing specific abiotic/edaphic stresses is available (Owens,

1995) and its possible application to turfgrass stress-resistance enhancement will

be presented.

Integration of biotechnology in improvementof turf environmental stress adaptation is dependent on

1. Enhancement of genetic-based root plasticity (sufficient root volume to

maintain the plant under cyclic stresses, i.e., rooting depth, secondary and tertiary

roots, and root viability and functionality)

2. Discernment of multiple stress-tolerance mechanisms in highly tolerant genetic resources

3. Mapping and cloning of stress response genes

4. Utilization of marker-assisted selection (MAS) techniques and transformationhegeneration methods to manipulate specific mechanisms across/within

species

5 . Field evaluation in severe multiple stress environments

6. Refinement of management strategies to ensure maximum expression of

stress resistances



TURFGRASS MOLECULAR GENETIC IMPROVEMENT



237



B. MOISTUREDEFICIENCY

1. Drought Resistance



Turfgrass drought resistance encompasses both drought avoidance and tolerance mechanisms. Drought resistance is exhibited through such strategies as developing deep root systems and shoot morphological/biochemical/physiological traits that minimize evapotranspiration (ET) losses (Beard, 1989); enhanced

root viability, functionality, and plasticity (Huang et al., 1997); and greater recoverability from transient drought stress following soil rewetting (BassiriRad

and Caldwell, 1992; Brady ef al., 1995; Nobel and Huang, 1992; Wraith ef al.,

1995).

a. Drought Avoidance

The ideal drought-avoidingturfgrass needs to have low ET during both high and

low soil moisture availability; a viable, functional, and plastic root system through

cyclic stresses; and seasonality (Perez de la Vega, 1996), which is the avoidance

of stress periods by completing the life cycle during favorable conditions (annual

species, e.g., many grassy weeds in turf) or the capacity to go dormant during severe stress periods (perennials, e.g., centipede grass).

Extensive research has been conducted on turfgrass ET rates (Table I). However, this trait is only one component (Nguyen ef al., 1997) of the overall droughtavoidance mechanism and is sensitive to climatic variables (humid vs arid, wellwatered vs limited soil moisture, and greenhouse vs field). These factors challenge

data interpretation and development of useful management strategies (Canow,

1995b). Under dry environmental conditions, turfgrass stomatal aspects (density,

location, and interaction with leaf water potential that influences rate of closure),

delayed or postponed dehydration, and rooting characteristics strongly influence

ET (Carrow, 1995a,b, 1996a,b).

Drought avoidance in tall fescue is associated with the development of high root

length density in the deeper root zone (20-60 cm), maintenance and functionalityhiability of deep roots over the summer months (combination of genetic tolerance to edaphic stresses and indirect high temperature stress), and development of

cultivars with inherently low ET and the ability to maintain low ET during soil

drydown (Carrow, 1996a).

b. Drought Tolerance

A turf plant can possess greater drought tolerance if it has these components:

osmotic adjustment, maintenance of positive turgor pressure, and delayed leaf

rolling (White ef al., 1992, 1993). However, both drought-avoidance and -resistance components contribute to wilt and leaf-firing expression in turf plants under

field conditions (Carrow, 1996a;Nguyen etal., 1997). Some turfcultivars may ex-



'hble I

EvapotranspirptionRanges for Variaus Mgrasses

Species



N

w



m



Tall fescue

Rebel (6.27)

Kenhy (7.09)

Shortstop (8.6)

Alta (10.0)

Rebel II

KY-3 1

KY-3 1

Murieta

Apache

St. Augustine



TX common

Raleigh

Centipede grass

GA common

Common

Buffalo grass

TX common



Location



Range (&day)



or mean



Reference



Arid, field, nonlimiting soil moisture

Field, arid, nonlimiting soil moisture



7.2-12.6

6.3-7.1



Beard (1985)

Kopec et al. (1988)



Greenhouse,arid, nonlimiting soil moisture



Field

Field

Controlled environment

Field

Field

Field

Field

Field



7.1- 10.0

2.7-3.4

3.57

3.69

5.1-7.1

6.6

7.7

6.3-9.6

4.5-5.1

6.7-8.1

4.8-6.3

3.28

4.0-8.7

4.7-5.5

3.8



Bowman and Macaulay (1991)

C m w (1996a)

carrow (199%)

carrow (199%)

Kim and Beard (1988)

Fernandez and Love (1993)

Femandez and Love (1993)

Beard (1985)

Atkins eral. (1991)

Atkins eral. (1991)

Kim and Beard (1988)

C m w (1995b)

Beard (1985)

Kim and Beard (1988)

C m w (1995b)



Field



4.4-5.3



Kim and Beard (1988)



Field, humid, limited soil moisture

Field, humid, limited soil moisture

Field, humid, limited soil moisture

Field, humid, nonlimited soil moisture



Greenhouse

Greenhouse



Zoysia grass



Meyer

Emerald

Meyer

Bermuda grass

Common

Tifway

AZ common

Tifgreen

Tifway

Kentucky bluegrass



Bristol

N

W



\o



Wabash

Perennial ryegrass

Saturn



Accolade

Seashore paspalum

Adalayd



Field

Field

Controlled

Field

Field

Field

Field

Field

Field

Field

Field

Field

Field



4.8-7.6

3.8-4.7

7.6-10.0

4.1-5.8

4.9-6.5

3.54

4.0-8.7

3.03

4.2-5.2

3.11

4.2-5.8

4.6-5.4

4.1-5.9



Beard (1985)

Green et al. (1991)

Greenetal. (1991)

Kim and Beard (1988)

Kim and Beard (1988)

Carrow (1995b)

Beard (1985)

Carrow (1995b)

Beard et al. (1992)

C m w (1995b)

Kim and Beard (1988)

Kim and Beard (1988)

Kim and Beard (1988)



Greenhouse

Greenhouse



5.5

6.0



Fernandez and Love (1993)

Fernandez and Love (1993)



Greenhouse

Greenhouse



6.2

6.3



Fernandez and Love (1993)

Fernandez and Love (1993)



Field



4.7-6.1



Kim and Beard (1988)



240



R. R. DUNCAN AND R. N. CARROW



hibit rapid leaf firing due to a restricted root system and not because they lack

drought tolerance.

Components of drought tolerance in turfgrasses include hardiness, or greater

membrane stability under dehydration; tolerance of protoplasmic constituents to

dehydration;binding of cell water to protoplasmic proteins and carbohydrates;loss

of excess water that contributes to tissue succulence; and accumulation of certain

metabolites (i.e., proline, betaine, and abscisic acid). Genetic engineering of

drought (and salt)-tolerance components is possible (Table 11). The quantitative

trait loci (QTLs) controlling leaf rolling (Champoux et aL, 1995) and osmotic adjustment/dehydrationtolerance (Lilley etal., 1996;Lilley and Ludlow, 1996)have

been located.

lsble II



QTLs or Other Genes Linked to Drought-Tolerance' h i t s

Category



Qn.

QTL



Trait



Reference



Visual leaf rolling during three growth stages

Osmotic adjustment and dehydration tolerance



Champoux er al. (1995)

Lilley er al. (1996)

Lilley and Ludlow (1996)



Mannitol

Glycine betaine

Proline

torn PRO1 (encodes y-glutamyl kinase and

y-glutamyl phosphate reductase)

torn PRO2 (encodes Dl-pyrroline-5-carboxylate

synthetase with GGK and GGPR activity)

Trehalose



Tarczynski et al. (1992, 1993)

Rathinasabapathier al. (1994)

Kishor er al. (1995)

Maggio et al. ( 1996)



Osmolytes

Gene@)

Gene(s)

Gene(s)



Gene(s)

proteins

Gene(s)

Gene(s)

Genes

Genes

GsPM 112

Ose 730

Other

Gene

cDNA

cDNA

cDNA

Gene

Gene



LEA families: D-19, D-113, D-11, D-7

HVAl (LEA or dehydrin)

Rehydrins (Tr 155)

Imm (dehydrin)

LEA D- 113 family

LEA D-1 1 family

cdr 29 (cDNA clone coding for acyl-coenzyme

A oxidase)

Mn-SOD (provides oxidative stress and

water-deficit tolerance)

pCrnPMI/9 (LEA dessication tolerance-soybean)

LEA (dessication tolerance-soybean)

Asr I (tomato)

Rd17 (Arabidopsis-LEA dehydration

stress tolerance)



Maggio er al. (1996)

Holmstrom et al. (1996)

Dure (1993)



Xu et ul. (1996)

Oliver (1996)

Close (1997)

Chiang er al. (1996)

Chen and Chen (1996)

Stanca et al. (1996)

McKersie er al. (1996)

Hsing et al. (1995)

Burns et al. (1997)

Giad er al. (1997)

Iwasaki er al. (1997)



TURFGRASSMOLECULAR GENETIC IMPROVEMENT



241



Genetic manipulation of low-molecular-weight osmolytes [polyols, proline, betaines, and 3-dimethylsulfoniopropionate(DMSP)] can potentially confer osmotolerance to water-deficit (or freezing and salt stress) conditions (Yancey, 1994).

Transgenes controlling production and accumulation of mannitol (Tarczynski et

al., 1993), glycine betaine (Rathinasabapathi et al., 1994), proline (Kishor et al.,

1995),and trehalose (Carninci et al., 1998; Holmstrom et al., 1996) have been engineered into various plant species (Table 11). These osmolytes are functional in

stabilizing dehydrated enzymes and lipid membrane structure/integrity.

Water-deficit (cold and salt) stress-responsive cDNAs encoding different protein classes-late embryogenesis abundant (LEA) dehydrins (Close, 1997), responsive to abscisic acid, and ion channel proteins-have been isolated and characterized, but their role in stress physiology and their physiological functions were

initially not well understood (Bray, 1993).

Dessication (caused by drought, salinity, or extracellular freezing) tolerance incorporatestwo mechanisms: cellular protection and cellular repair/recovery (Oliver, 1996).Cellular protection processes/componentsinclude decreased rate of water loss, stabilization of membranes, water replacement, lipid modifications,

compartmental stabilization,structural modifications, antioxidants, decreased water loss-induced damage, reduced UV light-induced damage, osmotic adjustment,

mRNA conservation, chromatin condensation, greater cell wall elasticity, cell

wall-membrane interactions, and stabilization of sugar, protein, dehydrin, and

polyamine synthesis. Cellular repair processes include compartmental integrity,

membrane reassembly, cytoskeletal reassembly, pH and ion balance maintenance,

consistent electron transport, adequate energy supply, reestablishment of chromatin, DNA repair, lipid synthesis, protein synthesis, RNA synthesis, nutrient uptake, structural integrity, and metabolic reestablishment.

Modified dessication-tolerantplants rely on drying-induced and abscisic acidcontrolled cellular protection strategies for survival. Dessication-tolerant species

utilize a combination of a constitutive protection strategy and a rehydrationinducible recovery mechanism (Oliver, 1996). Transcription and mRNA stability

are essential for activating rehydrins or proteins specific to the rehydration phase

and essential for manifestation of dessication tolerance.

c. Biotechnology

Stress-responsive genes are not necessarily adaptive (may not contribute to

overall adaptation/persistence when transformed) and stress-adaptive genes are

not necessarily responsive (may be nonfunctional when transformed because of

signaling or other problems) (Nguyen et al., 1997). Several QTLs governing visual leaf rolling and osmotic adjustment/dehydration tolerance have been isolated and characterized (Table 11). Genes controlling the production of osmolytes,

such as mannitol, glycine betaine, proline, and trehalose, in response to stress have

been identified and sequenced. Rutgers University (Belanger er al., 1997) is using



242



R. R.DUNCAN AND R. N. CARROW



the gene encoding the enzyme mannitol- 1-phosphate dehydrogenase in creeping

bentgrass (AgrostispafustrisHuds.) transformation studies to accumulate the sugar alcohol mannitol and enhance cell protection from drought stress (Tarczynski

et af., 1992, 1993). In vitro selection with mannitol and subsequent regeneration

of enhanced osmoregulation has been accomplished in Brassica plants (Gangopadhyay et al., 1997).

Recently, a LEA protein gene from barley (HVA1) conferred water deficit and

salt stress tolerance in a transgenic rice (Xuet af., 1996) line that was previously

highly sensitive to both stresses. Additionally, a Mn-superoxide dismutase cDNA

from Nicotiana pfumbaginifoliawas successfully introduced into alfalfa (Medicago sativa L.) to provide oxidative stress tolerance and reduce injury from water-deficit stress. Consequently, alien gene expression for some components of

drought tolerance has been demonstrated.

Another strategy could involve low-molecular-weight antioxidants such as

ascorbate, a-tocopherol, carotenoids, or glutathione and antioxidantenzymes such

as glutathione reductase, ascorbate peroxidase, and superoxide dismutases (Ye et

af., 1997), which have higher activity levels in drought-tolerant plants. There are

also increased amounts of putrescine-generatingenzymes (arginine decarboxylase

and ornathine decarboxylase) in drought-resistantplants, and these enzymes have

an antioxidant defense function in plants. Transformation with myoinositol 0methyltransferase (IMT1) can result in D-ononitol concentrations that might provide better drought tolerance than the use of osmolyte adjustment (Sheveleva et

af., 1997).



C. TEMPERATUREEXTREMES

1. High-Temperature Stress



' h o stress response mechanisms function in turfgrasses, particularly in cool

season species. Direct high-temperature stress occurs when temperatures are sufficiently high to cause immediate death of the plant. Both protein degradation and

membrane deactivation result in catastrophic effects.

Indirect high-temperature stress results in impaired turf growth (with injury levels ranging from slight to extensive), depletion of carbohydrate reserves in the

crown region, and progressive reduction in turf quality and performance. Both protein and membrane deactivation are initiated. Photosynthesis is inhibited (Giardi

et af., 1997) due to degradation of D, protein synthesis, which is initiated by an

increase in oxygen radicals, followed by modification and de novo synthesis of

proteinases, an ATP/ADP ratio change, modification of phosphorylation levels,

and a redox potential change. Carbohydrate accumulation positively affects D,

protein turnover and subsequently photosystem II function and stability (Kilb et



TURFGRASS MOLECULAR GENETIC IMPROVEMENT



243



al., 1996).Consequently, maintaining adequate carbohydrate loads in the turf plant

is essential to the heat stress-tolerance response. The expression of the rbcS genes

(encoding a small subunit of Rubisco) and the expression of the cab genes (encoding the polypeptides of the light-harvesting system) are also controlled by carbohydrate metabolism (Sheen, 1990).

Turf management strategies can have significant impacts on heat (and also

drought and cold) acclimation or hardiness. Poor water drainage, wet or compacted soils, excess N applications, K deficiency, shade, and close mowing can enhance succulence in turf plants and escalate heat stress problems. High N applications, close mowing, shade, mechanical injury, or deficiencies in Fe, Mg, Mn,

S , and N can also decrease carbohydrates and, coupled with prolonged heat stress,

decrease turf quality and persistence.

For high-temperature stress protection, heat-tolerant cultivars need more heatstable photosynthetic systems; greater thermostability of heat-sensitive enzymes,

proteins, and membranes; morphological features (pubescent leaves, waxy leaves,

lighter green color, and vertically oriented leaves) that reduce heat absorption;heat

shock proteins for protection; and high total nonstructural carbohydrates coupled

with an efficient partitioning system for shoot and root growthhiability. A disaccharide such as trehalose provides thermostabilization and thermoactivation of enzymes involved in heat stress protection and cell homeostasis (Caminciet al., 1998).

a. Biotechnology

Genes controlling photosynthetic processes under stress have been identified

(Table 111). Numerous heat shock protein families have been characterized (Neumann et al., 1984), including several turfgrass species (Park et al., 1996). One genetic engineering strategy to increase thermotolerance could involve the biogenic

nonmethane hydrocarbon isoprene, which stabilizes and enhances hydrophobic interactions (lipid-lipid, lipid-protein, and protein-protein) in thykaloid (chloroplast) membranes (Singsaas et al., 1997). Another strategy could utilize high catalytic activity controlled by the catalase gene (CAT-I)to decrease photorespiratory

CO, losses at high (38°C)temperatures while increasing net photosynthesis (Brisson et al., 1998; Schultes et al., 1994). A third strategy could involve heat (cold,

salinity, anoxia, or hypoosmotic) stress-induced changes in cytosolic Ca2+ signal

transduction (Gong et al., 1998).A fourth strategy could include low-molecularweight heat shock proteins that provide photosynthetic and photosystem U (H,Ooxidizing, quinone-reducing complex) thermotolerance and are a major adaptive

mechanism to acute heat stress tolerance in plants (Downs et al., 1998; Heckathorn

et al., 1998). Breeding tactics such as utilizing high bulk density soils, minimal

water, and severe scalping during high heat stress periods can be used in the breeding program to improve root plasticity, carbohydrate load in the crown and subsequent partitioning to roots and shoots, and heat stress recoverability in coolseason grasses under field conditions (Duncan and Carrow, 1997).



R. R.DUNCAN AND R. N. CARROW



244



Table IlI

Genes Linked to Temperature Stress Tolerance

Stress



Category



High temperature Photosynthesis



Calmodulin

Soybean

Potato

Arabidopsis



Gene

psb A (core D , protein

for photosystem II)

rbc S (encode Rubisco)

cab (encode polypeptides

for light harvesting)

HaCaM (sunflower)

Polyubiquitin

ubi3-2 (ubiquitin)



AfJlO (molecular

chaperone and specific

regulator of HSPs)

Yeast

CAJl (molecular

chaperone, binds

calmodulin)

Heat shock

HSP 110 (CL,)

protein families HSP 90 (80-95 kDa)

HSP 70 (63-79 kDa)

HSP 60 (53-62 kDa)

HSP 20 (10-30 kDa)

HSP 8.5 (ubiquitin)

Low temperature Winter hardiness QTL: field survival,

LT,,, growth habit,

crown fructan content

Antifreeze

proteins (AFPs)

Arctic fish

Antifreeze glycoprotein

(ice-binding gene)

Six AFPs (16-35 kDa)

(accumulate. during

acclimation)

Cold-induced

proteins

Barley

COR 14

Arabidopsis

COR 15

Bermuda grass COR 27 (chitinase

protein)

Arubidopsis

COR 47/COR 6.6

Alfalfa

CAS 18 (cold acclimationspecific gene encodes

LEAlABA-responsive/

dehydrin proteins)

Spinach

70-kDa heat shock protein

(metabolic adjustment

during acclimation)



Reference

Giardi et al. (1997)

Sheen (1990)

Sheen (1990)

Courbou e f al. (1997)

Huq ef al. (1997)

Roy0 and SanchezSerrano (1997)

Lin and Lin (1997)



Mukai ef al. (1994)



Neumann et al. (1984)



Hays et al. (1993)



Chen er al. (1997a.b)



Griffith ef al. (1997)



Stanca ef al. (1996)

Lin and Thomashow (1992)

Gatschet et al. (1994, 1996)

Gilmour et al. (1992)

Wolfraim er al. (1993)



Neven ef al. (1992)



continues



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