ARTICLES
LaboratoryExperimentto Assess Plant Responses
to EnvironmentalStresses
Allen V. Barker*
ABSTRACT
and ethylene evolution by tomato plants that have been
subjected to stresses induced by ammonium
toxicity and that
Plant responsesto environmentalstresses are expressed
have
had
these
stresses
alleviated
by
K
fertilization.
Fromthe
throughchangesin plant morphology
and chemicalcomposianalyses
of
K,
a
critical
concentration
to
inhibit
lesion
formation. Theobjective of this experiment
is to teach studentsto
tion
and
evolution
of
ethylene
was
estimated
(Ulrich,
1952,
accurately identify and measuresuch changes, in this case
1976).
Students
also
can
correlate
plant
responses
to
the
modeled
by ammonium
toxicity. Studentsutilized potassium(K)
amount
of
K
that
is
supplied
in
the
fertilization
regimen.
fertilization to alleviate stresses imparted
by ammonium
toxicPlant responses to ammonium
toxicity are predictable with
ity. In this experiment,
studentsmeasured
growth,leafepinasty,
respect to time and intensity of symptomdevelopment. Belesion formation,K
accumulation,and
ethyleneevolutionthrough
cause of the reliability of this experiment,the instructor does
visual indexingandquantitativeanalyses.Classprocedures
and
not haveto take chanceson conditions that fail to materialize
results of an experiment
conducted
by a class in plantnutrition
are reported. Youngtomatoplants (Lycopersiconesculentum
within the constraints of an academiccalendar. Withvarying
Mill.), startingat the five-leaf stage, weregrown
in a soil-based
amountsof K nutrition, a progressivearray of stress intensimedium
with ammonium
nutrition and variable amountsof K.
ties can be developedand controlled.
After 3 wkof treatment,the responseswererecorded.Growth
Stemlesions develop on tomatoplants that are grownwith
wasmeasured
as fresh weightsof shoots; epinastywasmeasured high ammonium
nutrition in K-deficient soils (Barker et al.,
as percentof total leaves showingdownward
bendingandby the
1967). The formation of lesions has been attributed to accuangleof bending;lesion formationon stemswasassessedby a
mulation of toxic amountsofputrescine, a polyaminethat is
standardindex; ethyleneevolutionfromleaves wasmeasured
by
synthesized in K-deficient plants and that is increased by
gas chromatography;
andKin stemsor leaves wasanalyzedby
ammonium
nutrition (Hohlt et al., 1970; Smith and Richards,
flame photometry.Fromthe analyses of Kin tissues andthe
1962). Polyamineanalysis was not included in this experigrowthresponses,students determined
the critical concentramentsince the analysis involves skills and instrumentation
tion neededto alleviate toxic responsesof plants to ammonium that are not commonlyavailable in laboratories in plant
nutrition.Thisexperiment
gives studentsan experientiallearnnutrition (Flores and Galston, 1982; Slocumet al., 1989).
ing opportunityandbuilds on knowledge
gainedthroughpreviEthylene biosynthesis and polyaminebiosynthesis are correous studiesin plantscience.
lated processes in environmentallystressed plants (Coreyand
Barker, 1989; Flores et al., 1990; Miyazakiand Yang,1987).
Ethylene evolution increases in stressed plants and can be
HROUGH
measurements of morphological changes and
measuredeasily by gas chromatography(Corey et al., 1987;
analyses of constituents in tomato (Lycopersicon
Tingey, 1980). Ethylene is a plant hormonethat causes
esculentum Mill.), this experiment teaches students to obmultiplicity of plant responses, of which epinasty is one
serve, measure,record, and interpret plant responsesto envi(Burg, 1968; Yang, 1985). Tomatoplants stressed by ammoronmental stresses. This experiment integrates a numberof
niumtoxicity are epinastic and evolve ethylene at enhanced
qualitative and quantitative assessments of plant growth,
rates relative to unstressedplants (Coreyet ai., 1987).Potasdevelopment,and compositionin experiential learning through
sium fertilization restricts lesion developmentand restricts
laboratory training. Utilization of all the measurements
and
evolution of ethylene by ammonium-stressedplants (Barker
techniquesreported in this presentation are not necessaryfor
et al., 1967;Coreyand Barker, 1989; Coreyet al., 1987).
this experimentto be a successfulteachingtool. OtherinstrucThe experimentthat is presented here has been conducted
tors can adapt the procedures according to the available
at
least
14 times in Plant Nutrition, a coursetaken by graduate
facilities.
students
and upper-level undergraduate students. Although
Visual indexing and scalar measurementspermit recordthe
basic
treatments have remained the same, the numberof
ing of morphologicalobservations that include assessments
parameters
measuredhas increased with time. The original
of growth, lesion formation on stems, and leaf epinasty.
experiment
involved only correlations amongplant growth,
Chemicaldeterminations involve analyses of K accumulation
lesion development, and K fertilization.
Measurementsof
epinasty, K composition, and ethylene evolution were added
Dep.of Plant andSoil Sciences,Bowditch
Hall,Univ.of Massachusetts, in responseto student interest and the availabiltiy of equipAmherst,MA
01003.Contributionof Massachusetts
Agric. Exp.Sn.,
ment. Details of procedures and results of an experiment
JournalSeries no. 3158.Received
4 June1993.*Corresponding
author
conductedby the class in the fall of 1990are presented. This
([email protected]).
experimentwas chosenbecause the class measuredthe largest
numberof responses of plants to treatments in recent years.
Published
in J. Nat.Resour.Life Sci. Educ.24:145-149
(1995).
T
J. Nat. Resour.
Life ScLEduc.,VoL24, no. 2, 1995¯ 145
In this course, students write five reports, choosingamong
the results of eight experiments.This experimentis the sixth
one in the sequenceof eight, and students comment
that it, as
well as other experimentsin the course, allows themto use
knowledgegained from the previous five experiments. Students whoreport on this experiment complimentits simple
design, extensive analyses, and unambiguousresults. All
reports are presentedas journal articles. This journal article is
written to provide a model that students may follow in
preparation of their reports.
MATERIALS AND METHODS
Establishment of Treatments
Essentially any tomato cultivar maybe used, because all
cultivars are expected to be sensitive to ammonium
toxicity
(Maynardet al., 1966; Barker and Lachman,1986). ’Heinz
1350’wasused in this experiment.Plants at the five-true-leaf
stage of growth were set individually in 15-cmdiameter by
15-cmdeep pots, holding about 1.8 kg of potting medium(7
parts sandy loam/3parts peat moss/2parts builder’s sand, by
volume)and placed in a greenhouse. Stem lesions appear in
14 to 21 d, dependingon the soil, the greenhouseenvironment, and the treatments applied. Stresses develop rapidly
with the followingconditions: infertile soils, warmtemperatures, treatments of high ammonium
concentrations, or combinations of these factors. Withthe treatments used in this
experiment, an array of symptomsappeared on the plants in
21 d, fitting well into a semester time-frame.
Application of treatments of ammonium
and K salts was
delayed for 1 wk after transplanting to permit plants to
overcometransplanting shock. In the present study, 0.02 M
(NH4)2SO
4 was applied daily in 100-mLportions. The ammoniumsalt can be chloride or sulfate to supply 0.01 to 0.08 M
NH~.The higher the ammonium
concentration, the quicker
the stress develops in the plants. Potassiumconcentrations
ranged from nil to 0.08 MKCi(Table 1), and these solutions
were applied daily in 100-mLportions.
All treatments were duplicated. This level of replication
has beensatisfactory, providedthat a uniformsupply of plants
was available and environmental conditions in the greenhouse were uniform where the experiment was conducted. A
randomizedcomplete block design with replication is recommended.Replication is limited often to two blocks because
space is at a premiumin greenhouses, and roommust be made
available for several other simultaneousexperiments. Three
or four replications add more precision to the results, but
students seemto concentrate their observations on one replicate and routinely record observations from the other blocks.
Also, increasing the numberof replicates prolongs the time
needed to makemeasurements, makingit unlikely that all
measurements can be made in a standard 2-h laboratory
period.
The eight concentrations of K salt used in this experiment
are the minimum
numberrequired to give an array of treatments for determination of critical K concentrations. The
increment between concentrations should be small at low
concentrations to give gooddefinition of plant responses in
the transition zone betweennutrient deficiency and sufficiency (Ulrich, 1976). If additional concentrations of K are
included, they should be below 0.02 MK÷ to define plant
146
¯ J. Nat. Resour. Life ScL Educ., VoL 24, no. 2, 1995
Table 1. Variation of several
plants to K fertilization.
responses of ammonium-stressed
Measurements
K cone.
Leaf epinasty
Fresh Lesion
Ethylene
Treatment weights rating Stems Leaves Showing Angle evolutionS"
-~
g-~ kg
M KC1 g/plant visuals
%§
degrees
nl,{gh)
dry wt
0
94
3
12.5 21.0
100
34
47
0.005
160
3
25.0 42.5
76
39
14
0.010
178
2
27.0 50.5
65
28
4
0.015
198
2
42.0 62.0
52
35
6
0.020
213
1
45.5 65.5
52
24
7
0.030
208
0
49.0 65.5
65
30
10
0.040
205
0
55.5 71.0
41
33
7
0.080
220
0
66.5 84.5
33
33
8
Regression
**
**
**
**
*
NS
**
a
Q
Q
Q
Q
Q
**,*,NSTrendssignificant atp < 0.05, p -< 0.01, or nonsignificant by polynomialregression, respectively. L, linear; Q, quadratic.
Ethylene evolution by first, fully expandedleaf from the tip of plants.
Visual rating: 3 = severe, 2 = moderate; 1 = slight; 0 = no lesion development on stems.
Total numberof leaves per plant: Treatment1, 8 leaves; all other treatments, ll or 12 leaves.
responsesin the zones of deficiency and transition. At 0.08 M
K÷, toxic responses, possibly due to salinity or CI- toxicity,
often are noted as suppressions of growth.
Measurements
All of the measurementsof plant responses are conducted
by students in the class. Dividing the class into teams that
rotate to perform measurementson replicates permits each
studentto participate in collecting data for all of the measured
responses.
Growth. In this experiment, growth was measured as
shoot fresh weights, and as such was madeavailable immediately to the class. If dry weightsare used to assess growth,
stems and leaves should be separated so that the stems can be
saved for K analyses.
Lesions. Lesions appear as dark browndepressions on the
stems. Theywere rated on a index of 0 to 3, with 0 being no
lesions, 1 being a few scattered lesions per stem, 2 being
widespread lesion developmentwith individual lesions remainingdistinguishable, and 3 being widespreadlesions with
coalescence of lesions into large necrotic areas (Barker,
1976).
Epinasty. The numberofepinastic leaves amongthe total
numberof fully expandedleaves was counted on each plant.
The angle ofepinasty was expressed in degrees of depression
of the petiole from the perpendicular of the junction of the
petiole and the stem. Measurement
of the angle in degrees is
preferred to measurementin radians. Determiningthe angles
in incrementsof 15° without the use of a protractor is easy,
comparedto expression of the angles in fractions ofradians.
In this experiment,the angle ofepinasty of all epinastic leaves
was estimated and averagedfor each plant. Epinasty develops
from the bottom of the plant upward.
Ethylene. If ethylene evolution is to be measured,preparations for this determinationshould be started at the beginning of the plant-harvesting period. Timeis neededfor sampling and for incubation of samples from whichethylene is
collected and measured. In this experiment, the top fully
expandedleaf was excised with a razor blade and inserted into
a wide-mouth,1-L canningjar, the lid of whichhad beenfitted
with a rubber serumcap. The petiole was inserted in 25 to 50
mLof deionized water to keep the lamina turgid. Ethylene
maybe producedin response to excision of the leaf. A delay
of 15 to 30 minbefore sealing the jars permitsthis responseto
dissipate. Theprecedingstep canbe omittedif timeis limiting,
for the amountof ethylene producedin response to excision
is small comparedwith that induced by ammonium
stress. The
sealed jars with leaves werekept in light for 1.5 h. Photosynthetic photon flux was 50 ~mol/s per m2 from cool-white,
fluorescent lamps in a controlled-environmentroomat 22°C.
Natural light with ambienttemperaturesin the greenhousecan
be used if controlled chambersare not available. After incubation of the leaves, a portion of the gases in the jars was
removed through the septum with a hypodermic syringe.
Students determinedethylene in this sample by gas chromatography (Coreyet al., 1987).
Potassium.The leaves that were used for determination of
ethylene evolution and the stems from the measurementof
growth were dried at 60°C and ground. Potassium was extracted from the ground tissues with 1 MHCI,using a 250:1
ratio (w/w)of extractant to sample(Sahrawat,1980). Potassium in the extract was measuredby flame emission spectrophotometry. Since K analyses are performed on dried material, these tests are conductedin the next laboratory session,
meeting1 wkafter the samplesare taken.
~ t80-
/-
t5o-
/r
I
120-
90
’,
t0
~o ~0 ~0
,,
~8 60
POTA$SIUHCONCENTRATION
Fig. 1. Fresh weights of tomato shoots as a function of potassium
concentrations in stems or leaves. The arrows indicate the critical
concentrations of potassiumnecessary to give 90%of the maximum
growth.
mm
¯
Analysis of Data
In this experiment,the design wasa replicated randomized
complete block. Data for the effects of treatments on the
aboveparameterswere processed by analysis of variance, and
trends wereassessedby polynomialregression if the effects of
treatments were significant (P < 0.05) (Steele and Torrie,
1980). Regressions of growth, lesion formation, or ethylene
evolution against K concentration in stems or leaves were
plotted. Theseregressions were interpreted by the conceptof
critical concentrationof nutrient (Ulrich, 1976).The critical
K concentration in the stems or leaves was defined as the
concentration occurring at 90%of the maximum
growth in the
zoneof transition.
RESULTS
Theconceptof critical concentrationof nutrients is difficult to present and to understand. Obtaining and pondering
these results allows students to make two assessments of
critical concentration,one that is basedon the Kthat accumulates in the plant in responseto fertilization, and one that is
basedon the concentrationof Kfertilizer applied. Theinstructor can emphasizethat for universal application, the critical
concentration should be based on the K accumulationin the
tissue.
Growth.Fresh weights increased as K fertilization and
accumulationof K in stems or leaves increased (Table 1).
Thesetrends are ideal for determinationof critical concentrations with regression of growth against K concentration in
tissues. For shoot growth,the critical K concentrationin stems
was41 g/kg (4.1%)and in leaves was61 g/kg (6. 1%)(Fig.
Theconcentrationof Kin the fertilizer solution to give these
concentrations in plant tissues was about 0.015 M.
o
Io
~o
]0
~o
~o
~o
POTAS~;IUH
CONCENTHAIION
IN STEt4~(~/k~)
Fig. 2. Lesion formation as a function of potassium concentration in
tomato stems. The arrow indicates the critical
concentration of
potassium necessary to give 90% inhibition
of lesion formation.
Lesions. Students will observethat lesion developmentis
suppressed by increased K fertilization and accumulationin
the stems(Table 1). The relationship betweenlesion development and K accumulation is curvilinear and and allows for
determination of critical concentrations (Ulrich 1952, 1976)
(Fig. 2). The critical K concentration in this experimentwas
estimated to be 44 g/kg (4.4%) of the stem dry weight. The
external supply of K necessaryto give this concentration was
between 0.015 and 0.02 M(Table1).
Ethylene. Students will observe that ethylene evolution
falls sharply with increased K fertilization and K accumulation in leaves (Table1). In this experiment,ethyleneevolution
declined curvilinearly with increased K accumulationin the
leaves, giving goodresults for determinationof critical concentration (Fig. 3). Thecritical K concentrationwasestimated
to be 46 g/kg (4.6%) of the leaf dry weight. The external
supply of K necessary to give this concentration was between
0.005 and 0.010 M(Table1).
Epinasty. The percentage of epinastic leaves declined
with increased K fertilization and accumulationin leaves and
with decreasedrates of ethylene evolution (Table 1). Regression analysis showeda highly significant linear relation
J. Nat.
Resour.
Life
ScL Educ.,
VoL 24, no. 2, 1995 ¯ 147
SO
~
30-
~
~O-
POTASSIUMCONCENTRATION
IN LEAVES(g/kg}
Fig. 3. Ethylene evolution gs ~ [un~lion o[ potassium ~on~entr~Iion in
potassium ne~essgry To ~ive ~0% inhibition
o[eIhylene e~olufion.
be~eenpercen[~eo~ epin~s[ic leaves and ethyleneevolution. The~n~le o~ epinas~w~sno[ ~ffec~edby trea~men[
(T~bleI).
DISCUSSION
Experiential ]e~in~ is used[o refine studen[s’ undersmndin~o~ abs[rac~concep[s.This experimen[~llows studenisin pl~n~nu~ifionor in other pl~n~response
courses
exploreideasthai are presen[ed
~s [heo~in lectures. Experiential le~in~ from Ibis experimen[~oHows
th~ Lewinian
modelo~c~ionresearch~ndI~bor~[o~tr~inin~ (Ko]b, 1984).
FoIlowin~this modeI~
abstrac~concep[ssuch~s c~i[ical concentre[iono~ nutrien[s ~ndplan[ responses
~o environmen[al
stress are [es[ed ~or wlidi~ in ~ l~bor~[o~wherestudenLs
sherethe work~ndtheir experience.Thefeedbackespect
the Lewini~nmodelimplied ~romthis experimenL
is thai the
experimen[
servesas ~ ~uide~or ~esfin~otherhypothesiswi[h
similiarly stickled Iearnin~ experiences.~or example,studenismaydesi~nexpe~imen[s
[o assesseffec[s o~incremen[~l
levels o~ s~Iini~ o~diseasein~ec[iononpl~n[ responses
th~
can be measured
by ~echniqueslearned in this labora[o~.
Smdems
~Iso m~ysee ~pplicafions o~ this experienc~
problemsolvin~ in unreImedinvestigations. Another
ma[ion~Ifeedbackin this experimen[occurs from [he studen~s’use o~ knowledge
~ainedin previousexperimen[sin
Ibis course.
Studen[soften have~ difficuh ~imerepoffin~andin[e~re[in~ I~bora[o~resuffs. ~e ~oHowin~
discussion is directed
toward(i) heIpin~studen[sev~lua[eda[~such~s [hosecollec[ed in Ibis expe~imen~
~nd(ii) offerin~ suggestions
how~n instructor might modi~the experimenL
Shoot~row[hincreasedin ~esponse
[o incre~sin~~ accumulafionin the leaves~nds[ems,~lIowin~~or de~e~in~ion
og cri[ical concen~aUons
o~ K ~or op[imum
~owtho~ plants
receivin~ hi~h ~mmonium
nutri[Jon (~i~. l). ~e differences
in critical concen~mions
~or leaves~nds~emsemph~size
the
impo~ance
o~choosin~
[he properpl~n[ Ussues
~o~die,noses.
~e suppressions
o~lesion~orma[ion,e[hyleneevolu~ion,
epinas~in concu~ence
with enhanced~rowth demonstrate
the ameliorativeeffects of K.
Re~essJon
o~ lesion ~orm~[iona~ains[ K accumulation
(Fi~. 2) showsconsiderablescoffer o~d~at low K concen148
¯ J. Nat. Resour. Life
ScL Educ.,
VoL 24, no. 2, 1995
trations in the stems.This scatter is attributable to the subjective rating of lesion formation by a visual index that is
saturated at a maxium
value of 3. Including moregrades in the
index, perhaps up to 5, mayimprove the regression, but
assigning too manygrades imposesdifficulty in distinguishing amonggrades.
The suppression of ethylene evolution by increased K
fertilization and accumulationin the leaves is well demonstrated in this experiment. Theregression of ethylene evolution against K concentration in the leaves gives a plot that
lends itself well to assessmentof critical concentrations(Fig.
3).
Laboratoriesthat do not havefacilities for determinationof
ethylene evolution can use measurements of epinasty to
assess ethylenebiosynthesis in stressed plants. Theregression
of numberor percent of epinastic leaves against ethylene
evolution was linear. This response showsthat intensity of
epinasty is correlated with ethylene evolution. Percentageof
epinastic leaves has been a consistently goodparameter for
assessing stress. Petiolar angle has beenused as a parameter
to assess stress (McNamara
and Mitchell, 1991). In 1990, the
class did not note a trend for wider angles of epinasty with
higher levels of stress (lower concentrations of K), but this
trend has been noted in previous and subsequentclasses.
Intensities of other environmentalstresses on plants can be
measuredby techniques learned in this experiment. Flooding
or anoxia (Bradford and Dilley, 1978), drought and salinity
(Fengand Barker, 1992), pathogenicinfections (Glazer et al.,
1983; Bashan et al., 1980), heavy metals (Tingey, 1980),
herbicides (Merseyet al., 1990), and nutrient deficiencies
(Barker and Corey, 1988) are amongfactors that induce
biosynthesis of stress ethylene or ammonium
accumulation.
Ethylene evolution and leaf epinasty will assess plant response to a progressive array of stress from these factors.
However,ameliorative factors, such as those demonstratedby
K fertilization in this experiment,are not available in each
case, and a progressive array of stresses maybe difficult to
developin somecases. Experienceof the instructor will be an
important factor in choosing an environmental stress for
demonstrationin this experiment.
Other determinations can be madeto enhancethe value or
expand this experiment. Ammonium
accumulation often occurs in plant tissues that are stressed by unfavorableenvironmental factors. Ammonium
can be determined volumetrically in extracts of plant tissues (Barker and Volk, 1964).
Potassium fertilization ameliorates the toxic responses of
plants to ammonium
and restricts ammonium
accumulation in
plant tissues (Barker, 1967;Ajayi et al., 1970).
Statistical analysesfor this experiment,other than determination of critical concentrations, should involve regression
analyses to assess plant responsesto increasedK fertilization
or accumulation.Analysis of variance in this experimentand
linear regression can be performedeasily with hand calculators. Higher order polynomialregression is performedbest
with statistical software and computers(Dixon, 19831). Many
undergraduate students and beginning graduate students are
not knowledgeableof regression or trend analyses. The response phenomena
tested in this experimentare sufficiently
dramatic to showthat data processing is not needed in this
learning process. Tests for significance of trends gives feedbackfor students whoare able to run these tests.
J. Nat. Resour. Life Sci. Educ., Vol. 24, no. 2, 1995
149