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Tree Physiology 23, 217–226
© 2003 Heron Publishing—Victoria, Canada
Chilling-dependent photoinhibition, nutrition and growth analysis of
Eucalyptus nitens seedlings during establishment
1–4 1,5
DUGALD C. CLOSE and CHRISTOPHER L. BEADLE
1 Cooperative Research Centre for Sustainable Production Forestry, GPO Box 252-12, Hobart, Tasmania 7001, Australia
2 School of Plant Science, University of Tasmania, GPO Box 252-55, Hobart, Tasmania 7001, Australia
3 School of Agricultural Science, University of Tasmania, GPO Box 252-54, Hobart, Tasmania 7001, Australia Downloaded from https://academic.oup.com/treephys/article/23/4/217/1645013 by guest on 13 January 2023
4 Author to whom correspondence should be addressed (Dugald.Close@ffp.csiro.au)
5 CSIROForestry and Forest Products, GPO Box 252-12,Hobart, Tasmania 7001, Australia
Received April 30, 2002; accepted August 17, 2002; published online February 3, 2003
Summary Effectsofchilling-dependent photoinhibition on Keywords: biomass, chlorophyll fluorescence, frost, gas ex-
gas exchange, chlorophyll fluorescence, growth and nutrition change, N- and P-fractionation, nitrogen, NPQ, phosphorus,
of Eucalyptus nitens (Deane and Maiden) Maiden seedlings shade, shadecloth tree shelters.
were assessed for 70 weeks after transplanting 9-month-old
seedlings in early winter. One month before transplanting, the
seedlingswereassignedtofertilizedornutrient-deprivedtreat- Introduction
ments. Immediately after transplanting, half the seedlings in Exposure of leaves to high irradiance and low temperature
eachnutrienttreatmentwereplacedinshadeclothtreeshelters. leads to sustained decreases in the efficiency of Photosystem
Theexperimentalsitewasatanaltitudeof700m,whichiscon- II (Adamsetal.1994),whichismanifestasreducedphotosyn-
sideredmarginalforthe establishmentofE.nitensplantations thetic capacity (Berry and Björkman 1980). This reduction in
in Tasmania because of low mean annual minimum tempera- photosynthetic capacity is termed photoinhibition. Photo-
tures. inhibition is defined as the light-dependent and slowly revers-
Overnight frosts followed by sunny morning conditions in ible retardation of photosynthesis, independent of develop-
thefirst 20 weeksaftertransplanting(earlyJunetoearlyOcto- mental change (Long and Humphries 1994).
ber) caused severe photoinhibition. Predawn maximal photo- Exposure to chilling-dependent photoinhibition can affect
chemicalefficiency(F /F )andmaximumnetphotosynthesis the establishment and distribution of regenerating tree seed-
v m
(Amax) were depressed in nutrient-deprived seedlings com- lings.Forexample,seedlingsofEucalyptuspaucifloraSiebex
pared with fertilized seedlings, although shading partially al- Spreng(Balletal.1991),PiceaenglemanniiParryexEngelm.
leviated this difference. Neither Fv/Fm nor Amax recovered to and Abies lasiocarpa (Hook.) Nutt. (Germino and Smith
values observed before transplanting until > 20 weeks after 1999)arerestrictedtoparticularmicrositesneartheirlowtem-
transplanting. During this period, non-photochemical quench- -
perature limits of distribution, and this is linked to high inci
ing (NPQ) was high in seedlings in all treatments, although dent irradiance. Eucalyptus pauciflora regenerates under the
NPQwas lower in shaded, fertilized seedlings than in seed- southwestern aspect of established tree canopies where the
lings in the other treatments. -
seedlings are protected from exposure to high light and night
Total foliar nitrogen (N) concentration increased up to timeradiationfrosts (Ball et al. 1991). Growth of transplanted
42weeksaftertransplantinginthenutrient-deprivedseedlings Eucalyptus polyanthemos Schau. seedlings was greater when
in parallel with increasing relative growth rate (RGR). Frac- chilling-dependent photoinhibition was alleviated using
-
tionation of N- and phosphorus (P)-containing compounds in -
shadecloth tree shelters (Holly et al. 1994). Chilling-depend
dicated that differences in protein N accounted for the treat- -
ent photoinhibition detrimentally affected growth of Eucalyp
mentdifferences in total seedling N. Nucleic acid P increased tus globulus Labill. in a nursery (Close et al. 1999), and of
andinorganicPdecreasedduringgrowthperiods,althoughto- E. globulus and Eucalyptus nitens (Deane and Maiden)
tal seedling P remained constant. Among treated seedlings, Maiden established in plantations (Close et al. 2000a, 2001a,
heightgrowthwasgreatestinshadedseedlings:thiswasprob- 2001b); shading alleviated photoinhibition (Close et al. 1999,
ably a result of apical dominance effects because RGR was Close et al. 2002).
higher in unshaded seedlings than in shaded seedlings. Thus, In southeastern Australia, eucalypts are planted over large
the shade treatment alleviated chilling-dependent photoin- areas at high altitudes with low mean annual temperatures
hibition and maximized growth during winter, but limited (<10°C).Eucalyptusnitensisthepreferredspeciesforplant-
growth during warmer periods and therefore overall growth. ing in these cold environments (Tibbits 1986). The seedlings
218 CLOSE AND BEADLE
to be planted are often produced in nurseries at low altitudes, tively. The soil type was lithic eutrudox (or brown ferrosol)
and so, are not acclimated to the conditions of high light and with an average depth of about 30 cm. The area was sur
-
low temperature prevalent at planting sites (Close et al. rounded by an electrified fence to exclude browsing mam
-
2000a). Nutrient deprivation, a nursery practice used to mals. The approximately flat site was prepared with a
harden eucalypt seedlings to cold planting sites prior to trans Savannah (Yandina, Queensland) mound plough. No fertiliz
- -
planting, provides a degree of acclimation to low temperature ers, herbicides or insecticides were applied during the study.
(Close et al. 2000a). The treatment induces the synthesis of
large amounts of anthocyanins in the leaves (Close et al. Experimental treatments and layout
2000a, 2001a) that attenuate light under conditions of chill
- Seedlings were transplanted on June 10, 1998. Between and
ing-dependent photoinhibition (Krol et al. 1995, Pietrini and within row spacings were 3.5 and 0.6 m, respectively. Seed
-
Massacci 1998, Close et al. 2001b). Another nursery practice lings were planted in blocks, four seedlings per block, with
is nutrient loading. This is the application of nutrients at rates eachseedlingreceivingoneofthefourtreatments:unshaded+ Downloaded from https://academic.oup.com/treephys/article/23/4/217/1645013 by guest on 13 January 2023
higher than that required for growth just before the seedlings unfertilized (NF); unshaded+fertilized(F);shaded+unfertil
-
leave the nursery. This has been shown to increase relative ized (Sh-NF); and shaded + fertilized (Sh-F). Shaded seed-
growthratesofconiferseedlingsonbothherbicide-treatedand lings were surrounded by a shelter consisting of three wooden
untreated planting sites compared with non-nutrient-loaded stakes that supported 50% shadecloth to a height of 1.8 m
seedlings (Miller and Timmer 1994, Timmer 1999). above ground. Seedling treatments were randomly allocated
Correlations of total foliar nitrogen (N) and phosphorus (P) within blocks. One hundred and four replicate blocks of seed
-
with photosynthesis have been reported in various eucalypt lings were planted in a completely randomized design of three
species(FieldandMooney1986,Mulligan1989,Kirschbaum rows of 25 seedling groups and a fourth row of 29 seedling
and Tompkins 1990, Sheriff and Nambiar 1991, Medhurst groups. A buffer of one row of seedlings surrounded the ex-
2001). Seedling foliar N is the major variable explaining periment. A completely randomized design was implemented
growth of E. globulus seedlings after planting (Close et al. on the basis of climate, slope, aspect and uniformity of soil
2000a). However, carbon assimilation, and therefore growth, conditions across the experimental site.
dependsonNpartitioningwithintheleaf(Evans1989),which Four blocks were randomly selected from the fourth row
is affected by environmentalfactors(Fieldetal.1983).Analy- (for ease of access) and used for gas exchange and chlorophyll
sis of N- and P-containing chemical fractions (Kedrowski fluorescence measurements. The 50 blocks in the inner two
1983)allowsinvestigationofphysiologicallyactiveNandP. rowswereusedforheightmeasurement.Thus,measurements
The objective of this study was to investigate the effect of of gas exchange, chlorophyll fluorescence and height were
pre-planting nutrition on post-planting chilling-dependent made on the same seedlings throughout the experiment. The
pho-toinhibition of E. nitens seedlings. We hypothesized that other 50 blocks in the outer two rows were used as harvesting
theseverityofchilling-dependentphotoinhibitionisgreaterin material for growth analysis and nutrient analysis.
unshaded and nutrient-deprived seedlings than in shaded and The mean initial seedling height was 26.3 and 27.0 cm for
fertilized seedlings, and that chilling-dependent photoinhi- the unfertilized and fertilized seedlings, respectively.
bition adversely affects growth of unshaded and nutrient-de-
prived seedlings relative to shaded and fertilized seedlings. Micrometeorology
Air temperature was monitored with a thermocouple shielded
by a 12-Plate Gill Radiation Shield (Vaisala, Helsinki, Fin-
Materials and methods land)atareferenceheightof1.3m.Additionalthermocouples
Plant material wereplacedwithinshieldsinsideandoutsideeachtreeshelter
at a height of 0.9 m. These shields comprised two white PVC
Eucalyptus nitens seedlings from Rubicon provenance, seed pipesofdiameters50and90mm(with20mmholesdrilledfor
lot 79 3324 (North Forest Products, Burnie, Tasmania im- ventilation), with the smaller pipe secured within the larger
3 containers in the North
proved seed) were raised in 115-cm pipetopreventdirectradiationofthethermocouples(Closeet
Forest Products’ Somerset nursery, which is situated at sea al. 2000b). The thermocouples were calibrated immediately
level. The potting mix was saturated with 1 g l–1 Aquasol -
before installation. Data were recorded by a CR10X data log
every 10 days, each seedling receiving about 1.25 mg of ging system (Campbell Scientific, Logan, UT). The quantum
N:P:K,20:2.2:6.6. After 8 months, seedlings were moved to a -
sensor on the chlorophyll fluorometer, which had been cali
-
second nursery at 350 m altitude for 1 month. Half the seed brated against an LI-190 quantum sensor (Li-Cor, Lincoln,
lings were fed twice weekly with 1 g l–1 Aquasol (fertilized NE), was used to estimate mean light attenuation through the
treatment), whereas the remaining seedlings were deprived of shadecloth tree shelters.
nutrients (non-fertilized treatment).
Chlorophyll fluorescence
Experimental site Predawn maximal photochemical efficiency (F /F ) and
v m
The0.75hasitewasabout60kmsouthofRidgley,Tasmania steady-state fluoresence (non-photochemical quenching,
(41°03′S,145°49′E).Thealtitudewas700mwithameanan- NPQ) were assessed with a PAM-2000 fluorometer and
- -
nual rainfall and temperature of 2400 mm and 7.9 °C, respec 2030-B leaf-clip holder (Heinz Walz GmbH, Effeltrich, Ger
TREE PHYSIOLOGY VOLUME 23, 2003
COLD-INDUCEDPHOTOINHIBITIONOFE.NITENSSEEDLINGS 219
many). Single measurements were made on each leaf of the 107-06-2E, Lachat Instruments, Madison, WI) and P (Quik
-
mostrecentfully expanded leaf pair and on a single older leaf Chemmethod10-115-01-1d,LachatInstruments)onacontin
-
from the leaf pair one node back from the apex of three seed uous flow injection analyzer (QuikChem 800, Lachat
-
lings. PredawnF /F andinitialchlorophyllfluorescence(F ) Instruments). Standard samples of known N and P concentra
v m 0 -
weremeasuredonJune2,17and30,July21,August6and31, tion and blank samples were included to validate the effi
-
October8,November11,1998andJanuary20,1999(–1,1,3, ciency of digestion and elemental analysis.
6,8,12,16,20and32weeksaftertransplanting,respectively). The TCA fractionation method of Kedrowski (1983) as
Measurement of NPQ was conducted at 0800 h on clear sky modifiedbyPolglaseetal.(1992)wasusedtoextractthevari-
daysandwasrestrictedtoMay21(beforetransplanting),June ous N and P fractions from foliage samples (Hooda and Wes
-
23, August 13, September 21, November 3, 1998 and January ton 1999). Oven-dried samples (0.4 g) were initially extracted
20, 1999(–2,2,8,15,19and32weeksaftertransplanting,re- in 50 ml of 0.3 M TCA at 4 °C for 1 h, with shaking every
10 min. This was followed by centrifugation, supernatant re Downloaded from https://academic.oup.com/treephys/article/23/4/217/1645013 by guest on 13 January 2023
spectively). Ambient air temperatures during measurement -
were 12.8, 2.1, 3.7, 9.0, 13.8 and 14.7 °C, respectively. movalandre-extractionoftheresiduein0.15MTCAat90°C
for 1 h. A subsample (20 ml) of the supernatant from each of
Gas exchange thecold-andhot-extractionprocedureswasevaporatedtodry
-
Maximumnet photosynthesis (Amax) was determined with an ness at 100 °C and digested for total N and P determination as
open-flow gas-analysis system incorporating a Parkinson described above.
PLC-B leaf chamber (area 6.25 cm2) and lamp comprising Fractionation of N and P was performed according to Ked-
rowski’s(1983)classification.Themorelabileinorganicfrac
four 150 W Wotan xenon quartz globes (LCA2, Analytical -
+ 2–
tionswereextractedinTCAat4°C( andHPO )whereas
Development, Hoddesdon, Herts, UK). Measurements were NH4 4
–2 –1 thosethatwerelesslabilewereextractedinTCAat90°C(nu
made at saturating light, determined as 1500 µmol m s -
from photosynthetic light response curves, and ambient CO cleic acid related N and P). The residual N and P following
2
–1 TCA extraction comprises relatively insoluble P complexes
concentration (350 to 370 µmol mol ).
Single measurementsweremadeoneachofthemostrecent andproteinN(Polglaseetal.1992).InorganicNwasaninsig-
fully expanded leaf pairs of the three seedlings measured for nificantcomponentofthetotalNpool(asreportedbyPolglase
predawnF /F .Measurementsweremadeonthesamedaysas et al. 1992) and was omitted from further analysis. Seedlings
v m
for Fv/Fm between 1100 and 1400 h Australian Eastern Stan- were sampled on June 4, October 8, December 2, 1998 and
dard Time. March25,1999(0,18,26and42weeksafterplanting,respec-
tively).
Growth analysis
Growth analysis was conducted up to 70 weeks after trans- Statistical analysis
planting. Data were collected from all seedlings used for Two-wayanalysisofvariance(ANOVA)wasperformedwith
height measurements and separated into four height classes. the SAS statistical software package (1989; SAS Institute,
Ten seedlings representative of each of the classes were se- Cary,NC)andindicatedsignificantinteractionsbetweentime
lected for harvesting on June 4, October 8, December 2, 1998, and treatment on chlorophyll fluorescence, gas exchange, nu-
-
March25andOctober8,1999(0,18,26,42and70weeksaf trient concentrations and growth analyses. One-way
ter transplanting, respectively). Soil was washed from the root ANOVAs were then conducted to test changes (α = 0.05)
systems and total single-sided leaf area measured (Delta-T within treatment with time and between treatments within
Devices,Cambridge,U.K.).Leaf,stem,branchandrootswere time.
oven-driedat70°Candthedrymasswasdetermined.Growth
analyses were done according to Beadle (1993).
Results
Nutrient analysis
For nutrient analyses, foliage from 10 seedlings from each Micrometeorology
-
harvest used for growth analyses were mixed and divided into Frosts were frequent and occurred throughout the experimen
three samples. Thesesampleswereusedasthreereplicatesfor tal period, even in summer (Table 1). There was no difference
total N, P and trichloroacetic acid (TCA) nutrient analyses. inminimumtemperatureexperiencedbyshadedandunshaded
Plant material was dried at 70 °C to constant weight and then seedlings (data not shown). Irradiance on clear days after
–2 –1
ground in a hammer mill. frosts was typically between 900 and 1100 µmol m s .Tree
About 0.16 g of oven-dried leaf material of seedlings of shelters reduced incident photosynthetically active radiation
- by about 54%.
each treatment was ground and digested in 4 ml of concen
tratedH SO and2mlofhydrogenperoxide(H O )(30%w/v)
2 4 2 2 Gas exchange
at 360 °C for 30 min (Lowther 1980). After cooling to 150 °C,
HO wasaddeddropwise until the solution cleared to a pale For seedlings in all treatments, A generally decreased for
2 2 max
yellow.Thesampleswerethendigestedat360°Cforafurther 6 weeks following transplanting and then increased to reach
–2 –1
60 min, resulting in a clear digest. The digest was diluted, maximal rates (Amax > 15 µmol m s ) by 32 weeks after
and colorimetrically analyzed for N (QuikChem method 10- transplanting (Figure 1). Before transplanting, Amax was
TREE PHYSIOLOGY ONLINE athttp://heronpublishing.com
220 CLOSE AND BEADLE
Table1.Numberoffrosts,andabsoluteandmeanminimumandmax- Chlorophyll fluorescence
imum air temperatures (°C) measured at 1.3 m during the period of Predawn Fv/Fm of Sh-F seedlings was maintained at optimal
gas exchange and chlorophyll fluorescence measurements. values (> 0.7) throughout the establishment period (Fig
-
Weeks after Number of Air temperature (°C) ure 2a). Values of Fv/Fm of seedlings in the other treatments
generally declined until 6 weeks after transplanting and in
planting frosts -
creasedtooptimalvaluesby32weeksaftertransplanting.The
Absolute Mean Mean decline in Fv/Fm was most pronounced in NF seedlings. The
minimum minimum maximum minimumF /F measuredwas0.16,0.44and0.56intheNF,
v m
1 1 –3.9 2.2 8.4 F, and Sh-NF seedlings, respectively. Values of F0 remained
2 0 0.5 2.4 6.8 similar in seedlings in all treatments within sampling periods
3 3 –4.6 –0.2 5.1 (Figure 2b) except for 32 weeks after planting when F0 was Downloaded from https://academic.oup.com/treephys/article/23/4/217/1645013 by guest on 13 January 2023
4 1 –2.4 2.0 6.8 higher in shaded seedlings.
5 4 –3.3 1.2 5.9 In seedlings in all treatments, NPQ was high (15–25) from
6 3 –3.4 0.5 7.2 before transplanting until Week 15 after transplanting, after
7 3 –0.9 0.7 7.7 whichNPQremainedlow(Figure3).InSh-Fseedlings,NPQ
8 1 –0.3 2.6 6.7
9 3 –2.3 1.0 8.8 decreased significantly after Week 8, whereas NPQ of F,
10 6 –5.5 –2.1 7.5 Sh-NF and NF seedlings only decreased significantly after
11 4 –4.3 –0.3 8.3 Week15.Beforetransplanting, NPQwassignificantly higher
12 3 –3.2 0.4 10.6 in NF seedlings than in F seedlings. At 15 weeks after trans-
16 7 –2.2 2.7 9.7 planting, NPQ was significantly lower in Sh-F seedlings than
20 6 –1.1 2.9 10.3 in seedlings in the other treatments.
24 9 –4.1 2.1 13.7
28 1 –0.6 5.6 15.3 Nutrient analysis
32 1 –0.7 7.8 16.2
Total N concentration decreased for the first 18 weeks after
transplanting in seedlings in all treatments (Table 2). Concen-
greaterinFseedlingsthaninNFseedlings(seeWeek0inFig-
ure 1). At Weeks 1 and 3 after planting, shaded seedlings had
greater Amax than the corresponding unshaded seedlings. At
Weeks6,8and12aftertransplanting, Amax was similar in NF
andSh-NFseedlings,butgreaterinFseedlingsthaninNFand
Sh-NFseedlings, and Amax in Sh-F seedlings was greater than
in F seedlings. Sixteen weeks after transplanting, Amax varied
amongtreatmentsasSh-F>Sh-NF=F>NF,butwasnotsig-
nificantly different between treatments thereafter.
–2 –1 -
Figure 1. Maximum net photosynthesis (A ; µmol m s ) with Figure 2. (a) Photochemical efficiency (F /F ) and (b) initial chloro
max v m
time after transplanting for unfertilized (NF), fertilized (F), shaded + phyllfluorescence(F )versustimeaftertransplantingforunfertilized
0
- -
unfertilized (Sh-NF) and shaded + fertilized (Sh-F) E. nitens seed (NF),fertilized(F),shaded+unfertilized(Sh-NF)andshaded+fertil
lings. Bars indicate ± standard error. ized (Sh-F) E. nitens seedlings. Bars indicate ± standard error.
TREE PHYSIOLOGY VOLUME 23, 2003
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