RESEARCH New Phytol.

(2000), 146, 47–57

Physiological impacts of Mg deficiency in

Pinus radiata : growth and photosynthesis

W I L L I A M L A I N G"*, D E N N I S G R E ER # , O S B E R T S UN $ ,
P E T E R B E E TS % , A L I S O N L O W E%    T I M P A Y N%
"The Horticultural and Food Research Institute of New Zealand, Private Bag 92169,
Auckland, New Zealand
#The Horticultural and Food Research Institute of New Zealand, Private Bag 11030,
Palmerston North, New Zealand
$The New Zealand Forest Research Institute, PO Box 29237, Christchurch, New Zealand
%The New Zealand Forest Research Institute, Private Bag 3020, Rotorua, New Zealand
Received 23 July 1999 ; accepted 15 December 1999


This paper reports the effects of nutrient magnesium (Mg) concentrations on the growth and photosynthetic
physiology of clonal Pinus radiata from four female parents (families) known to differ in their tolerance to Mg
deficiency and in their needle Mg concentrations. Plants were grown in flowing nutrient solutions with 2 mg l−"
(control) and 0.8 mg l−" (low) Mg. Plant growth, needle Mg concentration, photosynthesis, chlorophyll
fluorescence and carotenoid pigment content were measured. At low Mg, needle Mg concentration was about half
that of control plants, height growth was reduced 15–25%, and the needles showed strong visual characteristics
of Mg deficiency. Photosynthesis was also halved, and was associated with closure of the stomata under low Mg
and with reductions in the residual conductance. In needles from plants grown at low Mg, photochemical yield
was reduced both in the light and in the dark, and was strongly dependent on needle Mg concentrations below a
threshold concentration of 0.02–0.025% (d. wt basis). The electron transport rate (ETR) at saturating photon flux
density in low-Mg-grown needles was reduced to about half that of their Mg controls, but the photon efficiency
of ETR was unaffected by the Mg concentration the plants were grown in. Photosynthetic quenching was
markedly reduced and non-photosynthetic quenching was increased following growth in low Mg. Growth under
low Mg also increased levels of zeaxanthin. Although family differences in growth and photosynthetic physiology
were present, few familyiMg interactions were significant. We conclude that Mg deficiency probably affects
growth through severe reductions in photosynthesis.

Key words : Pinus radiata, magnesium, growth, photosynthesis, photochemical quenching, non-photochemical
quenching.

thought to be caused by magnesium deficiency


Pinus radiata, an economically important forest tree
species in New Zealand, is subject to a physio-
logical disorder called upper mid-crown yellowing
(UMCY). UMCY, a form of crown dieback, is
*Author for correspondence (tel j64 9 8154200 ; fax j64 9
8154202 ; e-mail wlaing!hort.cri.nz).
Abbreviations : ETR, electron transport rate ; Ft, fluorescence
during illumination ; F , initial fluorescence after dark ; F , initial
! !
fluorescence after far-red illumination ; Fm, maximum fluorescence
after dark ; Fm, maximum fluorescence during illumination ; Fv, Fm
– F ; Fv, Fm – Ft ; Fv\Fm, intrinsic efficiency of photosystem II in
!
dark ; Fv\Fm, efficiency of photosystem II during illumination ;
NPQ, non-photochemical quenching ; PFD, photon flux density ;
PSII, photosystem II ; qp, photochemical quenching.
(Forest Research Institute, 1991 ; Beets et al., 1993).
The symptoms of UMCY include the yellowing of
needles in the central portion of the upper crown
while needles in the lower portion of the tree remain
green (Forest Research Institute, 1991). The yellow-
ing is first seen in older needles, with loss of needles
leading to a thinning of the upper mid-crown. The
needles can initially develop small yellow or orange
spots, which extend and merge, beginning from the
needle tip. Orange deposits form under the en-
dodermis. New needles can either be healthy, show
pale and dark green bands, or in severe cases are
yellow and stunted. One-yr-old foliage from affected
trees shows reduced Mg and calcium concentrations
48 RESEARCH W. Laing et al.
compared with healthy needles, and potassium
concentrations can be high. No other nutrients
appear to be markedly different, and no Ca deficiency
symptoms have been noted (Beets & Jokela, 1994).
The most favoured hypothesis is that UMCY is a
result of nutritional imbalance, caused by Mg
deficiency and K excess (Beets & Jokela, 1994).
Antagonism between Mg and cations such as K has
been observed in plants (Mengel & Kirby, 1978 ;
Diem & Godbold, 1993), although the reasons for
this are unclear (Sun & Payn, 1999). There is also a
significant genetic component to the disorder. In the
field, the severity of the symptoms of UMCY varied
significantly with clone, with a broad sense estimate
of heritability of 59% (Beets & Jokela, 1994). It
appears that the supply of Mg is the overriding
factor in development of symptoms of UMCY, with
K excess assuming greater importance when plants
are under Mg stress. The response of UMCY in P.
radiata to Mg fertilizer is variable and depends on
environmental conditions (Payn et al., 1995) which
might reflect long lag periods between fertilizer
application and large tree responses. Magnesium
deficiency in pines also results in a reduction in
allocation of carbohydrate to the roots (Payn et al.,
1995), resulting in increased shoot : root ratios (Sun
& Payn, 1999) exacerbating the problem of Mg
supply and therefore preventing or slowing a
fertilizer response.
Magnesium is an essential cellular constituent
(Marschner, 1983, 1995). Many metabolic processes
are directly affected by Mg, including enzymatic
reactions that depend on the presence of Mg (e.g. the
enzymes of photosynthesis, ATPases, kinases, cellu-
lar pH control ; Mengel & Kirby, 1978 ; Marschner,
1983, 1995 ; Wyn Jones & Pollard, 1983). In addition,
the chlorophyll molecule forms a complex with Mg
(Wyn Jones & Pollard, 1983). Magnesium deficiency
in plants often results in ultrastructural changes
(Fink, 1993 ; Puech & Mehne-Jakobs, 1997), es-
pecially in the chloroplast, well before visible foliage
symptoms are obvious. This is accompanied by
impairment of photosynthesis (Fischer & Bremer,
1993 ; Sun & Payn, 1999). These symptoms include
build-up of starch in the chloroplast, perhaps
because of collapse of the phloem cells preventing
translocation (Puech & Mehne-Jakobs, 1997). Pot-
assium has been implicated in cellular osmotic
regulation, and also has a function in maintaining
protein conformation and enzyme activity (Flowers
& Lauchli, 1983 ; Wyn Jones & Pollard, 1983).
Chlorophyll fluorescence has been frequently used
as an indicator of stress in plants (e.g. Demmig-
Adams & Adams, 1992 ; Greer & Laing, 1992 ; Greer
et al., 1993 ; Schreiber et al., 1995 ; Greer, 1998).
The photochemical yield is a direct measure of the
efficiency of photosystem II (PSII) and is correlated
with the photon efficiency of oxygen evolution
in photosynthesis (Bjo$ rkman & Demmig, 1987 ;
Schreiber et al., 1995). Using fluorescence, it is
possible to measure photochemical yield under
ambient light as well as under totally relaxed
dark conditions. Fluorescence quenching can be
partitioned into photochemical (qp) and non-
photochemical (NPQ) quenching (Demmig-Adams
& Adams, 1992), providing information about re-
action centre activity and the protective mechanisms
of photosynthesis.
The aim of this work was to induce Mg deficiency
in clones of P. radiata derived from parents
expressing different susceptibilities to UMCY, and
to measure growth, photosynthesis, fluorescence and
pigment contents of the plants. The objectives were
to develop a physiological understanding of the
photosynthetic processes that occur during devel-
opment of Mg deficiency, and to examine genetic
variation. We were especially interested in how low-
Mg-grown plants handled excess light energy.
  
Plant material and growth conditions
Seed of Pinus radiata L. was collected from four
open-pollinated female parents grown at the Puruki
trial site near Rotorua, New Zealand (Beets &
Brownlie, 1987). These parents were selected from a
larger population of trees as examples of the four
extremes of different combinations of either high or
low foliar Mg concentration with either high or low
needle chlorosis (UMCY rating : see Forest Research
Institute, 1991 ; Table 1). Seedlings were raised from
the four female parents. Four seedlings were ran-
domly selected from each parent for propagation of
fascicle cuttings. These four seedlings from the same
female parent are called a family in this paper, each
family thus having four half-siblings. Multiple
fascicle cuttings were taken from the 16 genotypes.
At mid-winter 1997, when the plants were 15–18 cm
high, they were lifted, the roots rinsed with distilled
water, then transplanted individually into 4 l pots
filled with perlite and placed in a glasshouse. Eight
fascicle cuttings of similar height from each genotype
Table 1. Degree of yellowing (UMCY) and foliar
magnesium concentration of the mature Pinus radiata
female parent trees from the Puruki trial site
Family UMCY Foliar Mg
(female parent) value* (% d. wt)
2 1 0n11
7 7 0n05
16 3 0n06
23 8 0n09
*Increasing UMCY values (1–8) relate to increasing
severity of UMCY yellowing symptoms observed in the
upper crown (Beets & Jokela, 1994).
 RESEARCH Magnesium deficiency in Pinus radiata
were selected for the Mg nutrition experiment.
Throughout the entire growth period a fan operated
to aid cooling when the temperature was over 21mC,
and throughout the three summer months evap-
orative cooling was also deployed.
Trial design
The glasshouse trial was laid out as a completely
randomized design with a split-plot treatment struc-
ture. The main plot consisted of two troughs with
flowing nutrient solution, each trough containing a
single individual of the 16 genotypes belonging to
the four half-sibling families (four clones per family).
Each of two Mg treatments, control (2.0 mg l−") and
low (0.8 mg l−") were randomly allocated to two main
plot replicates. The four half-sibling families made
up the subplot treatment. Therefore each subplot
treatment can be considered as replicated twice (one
in each trough) within the main plot. All plants
belonging to a given family were raised from cuttings
of the same half-sibling seedlings, hence the eight
subplots were genetically identical. This plot struc-
ture meant that family and Mg main effect means
were based on 16 (from four half-siblings) and 32
pine seedlings, respectively.
Flowing nutrient solution treatments
Plants were grown in a flowing hydroponic system of
complete essential nutrients (based on that of
Ingestad & A/ gren, 1988, modified by adjusting the K
concentration to 20 mg l−" and the N concentration
to 100 mg l−" ; Sun & Payn, 1999). The nutrient
solutions were contained in 200 l tanks and pumped
around the troughs through a spray nozzle to deliver
solution to each pot for 1 h each day.
Nutrient solutions were sampled weekly and
analysed using standard procedures (Nicholson,
1984) to determine the concentration of Mg, pH and
conductivity. Concentrated ammonia solution, nu-
trient stock solutions and distilled water were added
fortnightly in order to maintain pH between 4 and 6
and to bring nutrient concentrations back to the set
concentration and volume.
Physiological measurements
Nutrition, height growth and chlorosis. Foliage sam-
ples were taken from plants after 5 months in the
glasshouse under the two Mg treatments when the
fully expanded stem needles had developed since the
treatments were applied. Foliage samples were dried
at 70mC and analysed for K, Ca and Mg by atomic
spectrophotometry, after digestion in nitric\
perchloric acids (Nicholson, 1984). These samples
were a subset of 56 plants selected representatively
from the four families and two Mg concentrations,
49
but randomly from troughs. These needles were also
evaluated for photosynthetic fluorescence photon
flux density (PFD) response curves.
The height of each plant was measured every 2 wk.
Each plant was visually assessed for the degree of
chlorosis of the entire plant after 5 months, on a scale
of 1–5, where 1 l 0 to 20% of needles chlorotic ;
5 l
80 to 100% needles chlorotic.
Photosynthesis and leaf conductance. Rates of photo-
synthesis (A), stomatal conductance to diffusion of
water vapour (gsw) and intercellular CO concen-
#
tration (Ci) were measured with a portable photo-
synthesis system (LI-6400, LI-COR Inc., Lincoln,
NE, USA) on every plant after 5 months in the
glasshouse. The measurements were made on six
attached needles per plant developed after the
treatment began. During measurements, tempera-
ture and reference CO concentration in the leaf
#
cuvette were maintained at 25mC and 350 ppm,
respectively. Irradiance (PFD, 400–700 nm) was
maintained at 1500 µmol m−# s−" using a red LED
light source (LI-6400–02). Needle surface area for
measurements of A was determined following the
method described by Beets (1977). Leaf conductance
was partitioned into stomatal (gsc) and non-stomatal
or residual conductance (grc) to diffusion of CO
#
according to Farquhar & Sharkey (1982).
Fluorescence. Chlorophyll fluorescence was measured
at 5 months in the glasshouse on attached needles
that had developed since the imposition of the
Mg treatments using either a PAM-2000 or a
MINIPAM (Waltz, Effeltrich, Germany) fluori-
meter (measuring light 0.6 kHz at 0.5 µmol m−# s−",
saturating light 8000 µmol m−# s−" for 0.8 s).
Fluorescence was measured on a bundle of needles
that created an ‘ artificial leaf ’ completely occupying
the measuring area of the fluorescence instrument.
Light response curves of fluorescence were measured
on a subset of 39 plants pre-adapted to the dark for
30 min before measurements using an external
actinic light (Greer, 1998). Fluorescence yield of
every plant was surveyed at midday under natural
solar irradiance, and correction was made for the
effect of incident PFD on yield using a general linear
model statistical analysis of the relationship between
fluorescence yield and PFD. A similar survey was
also carried out in the dark at 22 : 00 hours. In the
PFD response curves, electron transport rate (ETR),
qp and NPQ were calculated according to Bilger &
Bjo$ rkman (1990).
Carotenoid pigment analysis. Three youngest mature
fascicles, each with three to four needles after 5
months in the glasshouse, were randomly selected
from 48 plants from both Mg concentrations and all
four families, placed in a cryotube and immediately
frozen, and stored in liquid nitrogen for later
analysis. These samples were weighed, ground to a
50 RESEARCH W. Laing et al.
fine powder in liquid nitrogen using a mortar and
pestle, and then half their weight of dry polyvinyl
poly pyrrolidone was added and the powders mixed
by further grinding. Ten volumes of extraction
buffer (100 mM Hepes, 2 mM EDTA, 2 mM
EGTA, 10 mM DTT, 0.1% (w\v) PEG 6000, 1%
(v\v) Tween 20 pH 7.5) were added per g f. wt
needle, and the mixture ground to ensure complete
extraction. Sufficient acetone was added to a 200 µl
aliquot of this uncentrifuged mix to bring it to 85%
(v\v) acetone (Thayer & Bjorkman, 1990) and the
sample was mixed and centrifuged. A 25 µl aliquot of
the supernatant was separated on an HPLC column
to measure carotenoid pigments (Gilmore & Yama-
moto, 1991). Absorbance on the column eluate was
monitored at 440 nm as described by Laing et al.
(1995).
Statistical analysis. Plant heights were analysed by
fitting linear regression slopes to each individual
plant after the fifth measurement, by which time
growth was increasing linearly with time. These
slopes, as well as other measures, were analysed by
the GLM procedure (SAS, 1989) for a completely
randomized design with the split-plot treatment
structure as described earlier. Strictly, there were
only two replicates for each Mgifamily mean,
although each subplot was in two ‘ quasi-exper-
imental units ’, one in each trough. The two troughs
were connected together for practical convenience,
and in controlled environments it was unlikely that
these troughs were more similar than those between
main-plot replicates. The variation would have come
mainly from variable plant response. However, in
the analysis we assumed two replicates, with main
plot and subplot errors of 2 and 18 df, respectively.
Where only a subsample of plants was measured,
plants were randomly selected with the restriction
that similar numbers of each family and each Mg
level were represented. Where overall F tests
indicated significance in the family effects or
Mgifamily interactions, means separation tests
were carried out using the LSD (5%).

Height growth, mineral nutrition and needle chlorosis
The growth curve of plant heights showed an initial
lag, followed by a linear increase with time (Fig. 1).
The lag was still apparent when the data were plotted
on a log scale (data not shown). Linear regressions of
the increase in plant height with time were calculated
for each individual plant over the linear growth
period, and analysis of slopes showed a significant
effect of Mg and family but no significant interaction
between these two factors (Table 2). Families 7 and
16 grew the slowest at both Mg concentrations, with
low Mg slowing height growth by 15–25%.
Plants grown in low-Mg conditions had about half
the concentration of Mg in their needles compared
with the plants grown in normal Mg (Table 3). The
levels of Ca were reduced by c. 30% through growth
in low Mg, but K was unaffected. There was also a
significant family effect on Mg and Ca concen-
trations. Family 7 was lowest in Mg at each Mg
growth concentration.
Visual assessment (chlorosis score, Table 3)
showed significant differences between families and
between control and low Mg. Chlorosis was an
excellent predictor of Mg concentration, with a
calculated r# value of 0.72 (n l 56) from regression
on individual plants (using integer scores, data not
shown). When the same relationship was plotted
between the mean score for families and needle Mg
concentrations, an essentially straight line was
observed (r# l 0.96, n l 8, data not shown).
Magnesium and Ca concentrations were also corre-
lated (r# l 0.52, n l 55).
Photosynthetic gas exchange and conductance
All three variables relating to gas exchange were
affected by the Mg treatment, and differed signif-
icantly between the four families (Table 4). Family 7
showed the lowest photosynthesis rate. The reduc-
tions in photosynthetic rate were associated with the
reduction of both gsc and grc in Mg-deficient plants.
Fluorescence
In the light, photochemical yield was significantly
reduced (average 11%) by low Mg nutrition (Table
5). Differences between families were also significant
(both P 0.0001), as was the interaction between
family and Mg nutrition. However this interaction
had little effect on the ability to discriminate tolerant
and intolerant genotypes. This interaction was
caused because, while family 7 always had the lowest
yield, this was especially so in low Mg. These
changes in yield were generated from marked
changes in Ft with both Mg and family. However, Ft
was little affected by Mg, and was similar over the
four families (data not shown).
Fluorescence during the day was also significantly
affected by the incident irradiance (data not shown).
When fluorescence yield during the day (corrected
for the incident PFD using a linear model) was
compared with needle Mg concentration, there was
an initial rapid increase in yield with small changes
in Mg concentration at around 0.01–0.02% Mg (Fig.
2). However,
0.025% Mg photochemical yield was
essentially independent of needle Mg content.
The intrinsic photochemical yield in the dark was
also significantly affected by Mg treatment, family
and by the interaction between these two effects (P
0.0003, Table 5). F was unaffected by any of these
!
 RESEARCH Magnesium deficiency in Pinus radiata 51

60 60
Family 2 Family 7
50 50

40 40

30 30

20 20

10 10
Plant height (cm)

0 0
0 50 100 150 0 50 100 150
60 60
Family 16 Family 23
50 50

40 40

30 30

20 20

10 10

0 0
0 50 100 150 0 50 100 150
Time (d) Time (d)
Fig. 1. Mean height growth of Pinus radiata seedlings for each family after application of two different Mg
concentrations. Squares with continuous line, control (2 mg l−") Mg ; triangles with broken line, low Mg (0.8
mg l−"). Height growth rates were calculated from regressions over the part of the curve where a linear increase
in height with time occurred (continuous line).

Table 2. Effect of magnesium concentration and family Table 3. Effects of magnesium supply and family on
on the mean height growth rate of seedlings over the mineral concentration after 5 months’ growth
linear period of growth
Growth Chlorosis Mg Ca K
Rate of increase in height (mg l−") score (mg g−") (mg g−") (mg g−")
Main effect (cm d−")
Mg
Mg 0n8 4n09 0n193 0n880 8n33
0n8 0n252 2n0 1n69 0n393 1n21 8n46
2n0 0n327 P 0n001 0n05 0n013 ns
P 0n005 Family
Family 2 2n44a 0n313a 1n19a 8n40a
2 0n320a 7 3n50b 0n220b 0n98b 8n54a
7 0n275b 16 3n16c 0n313a 0n92b 8n38a
16 0n242b 23 2n47a 0n325a 1n09ab 8n27a
23 0n320a
Mineral concentrations are expressed on a dry weight
Mean values were calculated from slopes of the linear part basis. Chlorosis was visually assessed as described in the
of the relationship between height and time for individual Materials and Methods section. Data represent the least
plants (see Fig. 1). Data represent the least squares mean squares mean (based on a subsample of 56 plants for Mg
using all 128 plants. For Mg means, the P value represents measurements and all 128 plants for chlorosis assess-
the statistical significance of the difference of the means. ments). The Mgifamily interaction was not significant
For the family means, values within a column with the in either case. For Mg means, the P value represents the
same letter are not significantly different (P 0n005). statistical significance of the difference of the means. For
family means, values within a column are not significantly
different (P 0n05) if they share a common letter.
factors, and yield changes were driven by changes in
Fm, a similar result to that observed in the light. A actual photochemical yield in the light (Fig. 2),
plot of intrinsic yield versus Mg content (data not except with higher values of yield for any Mg
shown) showed a very similar relationship to the content.
52 RESEARCH W. Laing et al.

Table 4. Effects of Mg supply and family on photosynthetic rates (A), stomatal conductance (gsc), and residual
conductance (grc) to diffusion of CO in four famillies of Pinus radiata known to differ in susceptibility to UMCY
#
Main A gsc grc
effect (µmol m−# s−") (mmol m−# s−") (mmol m−# s−")

Mg
0n8 mg l−" 2n12 33 8n2
2n0 mg l−" 4n86 78 18
P 0n059 0n005 0n09
Family
2 3n94ab 60n3 17n5a
7 2n46c 44n2b 9n9b
16 4n18a 65n5a 17n8
23 3n40b 48n8b 13n9a

Data represent the least squares means measuring all 128 plants. For Mg means, the P value represents the statistical
significance of the difference of the means. For family means, values within columns with the same letter are not
significantly different from each other (P 0n05).

Fluorescence was also used to analyse the utiliz-

Table 5. Fluorescence yield in the light and in the dark
ation of light energy by the needles at the two Mg
Yield concentrations. The response of fluorescence yield,
Mg qp, NPQ and ETR to incident PFD are shown for
Family (mg l−") Light values Dark Values one family in Fig. 3. At both Mg concentrations, the
photochemical yield decreased curvilinearly with
2 0n8 0n680a 0n799a
7 0n8 0n568b 0n746b
increasing PFD, but for the low-Mg-grown plants
16 0n8 0n653c 0n803a yield was always lower than for the control Mg-
23 0n8 0n651c 0n798a grown plants. By contrast, ETR increased in a
2 2n0 0n740d 0n836c curvilinear fashion with increasing PFD, with simi-
7 2n0 0n705e 0n821c lar initial slopes at low PFD between plants from the
16 2n0 0n734d 0n835c
23 2n0 0n732d 0n835c
two Mg concentrations. However, the low-Mg-
grown plants had lower maximal ETRs than the
Photochemical yield (l Fv\Fm l (FmkF \Fm) was deter- control Mg-grown plants and were evidently satu-
! rated at PFDs
c. 1000 µmol m−# s−" where, for the
minated in the dark at approx. 22 : 00 hours and during the
day at approx. 12 : 00 hours. Values are the least squares control Mg-grown plants, ETR was apparently not
means (based on one measurement from all 128 plants for saturated at 2200 µmol m−# s−". Partitioning of
night measurements, two measurements per plant for all
plants for 1 d). In the analysis of yield values measured
fluorescence quenching between qp and NPQ showed
during the day, light levels were included as a covariate. that the PSII reaction centres of the control Mg-
Values within columns with the same letter are not grown needles were relatively resistant to closure
significantly different from each other (P 0n05). with increasing PFD (80% open at 2200 µmol m−#
s−") while needles of the low-Mg-grown plants were
0.85
significantly more susceptible (50% open at 2200
µmol m−# s−"). In keeping with the higher ETR and
0.80 qp in the control Mg-grown needles, NPQ increased
proportionally less in control Mg-grown needles
Fluorescence yield

0.75
than in the low-Mg-grown needles. Thus, in needles
0.70 of plants from both Mg concentrations the PFD-
dependent decline in photochemical yield was at-
0.65 Family 2 tributable both to some reaction centre closure and
Family 7 to increased non-radiative heat dissipation, but the
0.60 Family 16
Family 23
effects were markedly greater in the low-Mg-grown
0.55 needles.
0.50
When these parameters are analysed for all the
0.0 0.1 0.2 0.3 0.4 0.5 0.6 families and Mg concentrations, similar trends are
Needle Mg (mg g–1 d. wt) observed (Tables 6 and 7). In the case of ETR, the
Fig. 2. Relationship between photochemical yield and response is curvilinear, saturating at high PFD, and
needle Mg content in Pinus radiata. Needle Mg was a simple hyperbolic two-parameter curve was fitted
measured independently of fluorescence, and fluorescence to the data as
was corrected for the incident photon flux density (PFD)
as described in the text. ETR l ETRmaxiTanh(PFDiα\ETRmax)
 RESEARCH Magnesium deficiency in Pinus radiata 53

0.8

(a) (b)
0.7 400

0.6

300
0.5

ETR (µmol m–2 s–1)

0.4
Yield

200
0.3

0.2
100

0.1

0.0 0

(c) (d)
3
0.8

0.6 2

NPQ
qp

0.4

0.2

0
0.0

0 500 1000 1500 2000 0 500 1000 1500 2000

PFD (µmol m–2 s–1)
Fig. 3. Response of photochemical yield (a), electron transport rate (ETR) (b), photochemical quenching (qp)
(c) and non-photochemical quenching (NPQ) (d) to photon flux density (PFD) for Pinus radiata needles from
plants of family 16 grown at 2 and 0.8 mg l−" Mg. Fluorescence was measured and the parameters calculated
as described in the text. Open symbols, 0.8 mg l−" ; closed symbols, 2 mg l−" Mg. The continuous lines in the
ETR panel are the fitted curve described in the text. The other continuous lines are arbitrary best-fit curves
to the data.

One parameter, α, or the initial slope of the
response curve, corresponds to the photon efficiency
of electron transport. The second parameter,
ETRmax, is the maximum rate of electron transport
at saturating light. Both these parameters were
calculated for all plants using non-linear regression,
and the fitted parameters compared statistically.
Least-square means of the data are shown in Table
6. There was a strong and very significant effect of
growth Mg on ETRmax, while there were significant
differences between families for ETRmax, with family
7 having a lower ETRmax than the others. However,
54 RESEARCH W. Laing et al.

Table 6. Effect of magnesium and family on the calculated least square mean values of parameters for the response
of the electron transport rate to photon flux density

Maximum ETR Maximum ETR

Mg α (µmol m−# s−") Family α (µmol m−# s−")

0n8 0n225 180 2 0n239ab 340a

2 0n239 417 7 0n204b 231b
P ns 0n006 16 0n234ab 349a
23 0n251a 275ab

Light curves of fluorescence versus PFD were measured as described in the text for a subset of 39 plants over the four
clones. α (the slope of the relationship when light is limiting photosynthesis) and maximum ETR (ETRmax) were
calculated by non-linear regression to a model of the form
ETR l ETRmaxitanh(PFDiα\ETRmax)
For Mg means, the P value represents the statistical significance of the difference of the means. For family means, values
within columns which have different letters are significantly different at P 0n05.

Table 7. Effect of magnesium on the quenching coefficients NPQ and qP

Mg qP NPQ qP NPQ
(mg l−") Low PFD Low PFD High PFD High PFD

0n8 0n880 0n961 0n609 2n70

2n0 0n872 0n500 0n722 1n86
P ns 0n001 0n001 0n001

Least square mean values were calculated from pine plants exposed at a light
similar to that in which they grew (low PFD : 100–500 µmol m−# s−") or at near-
saturating PFD (high PFD :
1500 µmol m−# s−"), as described in the Materials
and Methods section, on a subsample of 39 plants. There were no significant
family or Mgifamily interactions. For Mg means, the P value represents the
statistical significance of the difference of the means.

(
1500 µmol m−# s−") (Table 7). When averaged

Table 8. The effect of growth at control and low
over all families (Table 7), there was a near doubling
magnesium on the xanthophyll pigment levels of pine
of NPQ at low Mg compared with control Mg at the
needles
low PFD. On the other hand, qp was unaffected by
Mg Mg at low PFD. At high PFD, these relative
differences in NPQ were reduced while qp was
Pigment 2n0 mg l−" 0n8 mg l−" P c. 20% lower than control at low Mg.
Violaxanthin (V) 82 41 0n001
Antheraxanthin (A) 4n8 6n5 ns Carotenoid pigments
Zeaxanthin (Z) 2n9 6n8 0n0004
(ZjA)\(ZjAjV) 0n091 0n27 0n001 While most carotenoid concentrations and chloro-
phyll were reduced significantly in plants grown at
Carotenoids were separated by HPLC as described under low Mg compared with the control (data not shown),
the Materials and Methods section. Individual carotenoids
were identified by their retention time on the HPLC antheraxanthin was unaffected and zeaxanthin was
column (Laing et al., 1995). They are expressed as relative increased by growth in low Mg (Table 8). This
units normalized on a g f. wt basis (on a subsample of 48 resulted in the xanthophyll ratio (zeaxanthinj
plants). The P value represents the statistical significance antheraxanthin)\(zeaxanthinjantheraxanthinj
of the difference of the means. ns, not significant. violaxanthin) rising from 0.09 in the control plants to
0.27 in the plants grown in low Mg (Table 8).
α was not significantly affected by Mg, and only
family 7 showed a reduced α compared with the

other families. There were no significant interactions
between Mg and family in either case. This paper reports the effects of Mg nutrient solution
Quenching coefficients were also calculated at a concentrations on the growth, mineral content and
PFD close to the natural light of growth (100–500 photosynthesis of P. radiata from four female
µmol m−# s−") and also at a near-saturating PFD parents known to differ in their response to low Mg.
 RESEARCH Magnesium deficiency in Pinus radiata
The parents represented the four extremes of
phenotypes (Table 1) : low UMCY with high and
low needle Mg (families 2 and 16), and high UMCY
with high and low needle Mg (families 7 and 23).
However, because the male parent of each seed was
unknown (from open pollination), the trial was
established by clonally propagating randomly selec-
ted seedlings from each of the female parents. This
ensured that a wide range of genetic material was
represented in each family.
One aim was to test the hypothesis that there was
genetic variation in response to solution Mg con-
centration and that this was related to the origin of
the family. In general, the results show families 7
and 16 were the poorest performers in terms of
height growth, and this was reflected in their high
chlorosis scores. However, performance related best
to the Mg concentration in the needles of the parent
trees (Table 1) rather than to their recorded
susceptibility to UMCY. In general, interaction
between Mg treatment and family was not signif-
icant, showing that poor performers at low Mg were
also poor at control concentrations of Mg nutrition.
Although growth in plant height was reduced by
only 15–25% at low Mg concentration, the con-
centration of needle Mg was reduced by c. 50%
compared with control Mg-grown plants and the
needles showed strong visual characteristics of Mg
deficiency. However, biomass might be more
strongly reduced by Mg deficiency than plant height
(T. W. Payn et al., unpublished). There appeared to
be a threshold for needle Mg concentration of
0.02–0.025% d. wt below which growth was severely
reduced (data not shown). Similar growth reductions
were observed by Sun & Payn (1999) in their studies
with 10 different clones of P. radiata, especially at
Mg concentrations below 1 mg l−". They also
observed that marked chlorosis occurred at low Mg
concentrations before there were significant reduc-
tions in growth.
Along with the reductions in height growth,
photosynthetic rates were reduced by about half by
Mg deficiency. These reductions were accompanied
by both closure of the stomata and reductions in the
residual resistance. These are comparable results to
those observed in P. radiata by Sun & Payn (1999),
who found a saturating relationship between shoot
Mg and photosynthetic rate. At Mg concentrations
below c. 0.06% (d. wt), photosynthesis began to
become impaired. Mehne-Jakobs (1995, 1997) also
showed strong reductions in photosynthesis in low-
Mg-grown spruce seedlings, especially when needle
Mg was less than c. 0.04% d. wt. In addition she
reported a reduction in carboxylation efficiency at
low Mg, but no change in the apparent photon yield
of photosynthesis (as measured by the light response
of photosynthesis).
Chlorophyll fluorescence has been frequently used
as an indicator of stress in plants (e.g. Demmig-
55
Adams & Adams, 1992 ; Greer & Laing, 1992 ; Greer
et al., 1993 ; Schreiber et al., 1995 ; Greer, 1998).
The photochemical yield is a direct measure of the
efficiency of PSII and is correlated with the photon
efficiency of oxygen evolution in photosynthesis
(Bjo$ rkman & Demmig, 1987 ; Schreiber et al., 1995).
In the current work, photochemical yield was a very
sensitive indicator of Mg deficiency associated with
reduced height growth. On average, growth of the
plants under Mg deficiency reduced photochemical
yield of needles by c. 8% of that of control Mg-
grown needles. However, when the photochemical
yield at the ambient PFD conditions was compared
with needle Mg concentration (Fig. 2), there were no
changes in yield above Mg concentrations of 0.02–
0.025%. Below this concentration of Mg, photo-
chemical yield decreased precipitously. This shows
similarity to the growth response to Mg (data not
shown). When photochemical yield was measured at
night, after complete re-oxidation of the PSII
reaction centre pool would have occurred, there were
still significant differences (5%) in intrinsic yield
between Mg-deficient needles and the needles with
sufficient Mg. This suggests that some of the
reduction in photochemical yield attributable to Mg
deficiency was probably due to a permanent closure
of at least some PSII reaction centres and indicative
of chronic photo-inhibition (Greer & Laing, 1989).
The dark yield values observed in Mg-sufficient
needles were typical of healthy leaves (Bjo$ rkman &
Demmig, 1987).
The differences between photochemical yield
response to PFD of plants grown at the two Mg
concentrations resulted in marked differences in
ETR light response. However, closer analysis
showed that the initial slope of the light response of
ETR (photon yield of electron transport) was
essentially unaffected by growth Mg (Table 6),
similar to that reported by Mehne-Jakobs (1997). On
the other hand, the maximum rate of ETR in plants
grown in low Mg was reduced to c. 50% of that in
plants grown at control Mg, in agreement with the
gas-exchange data (Table 4). These responses to Mg
are in agreement with those recorded by Mehne-
Jakobs (1997) and O. J. Sun et al. (unpublished) who
both observed reductions in photosynthesis in plants
grown in low Mg concentrations. Interestingly,
family 7 with the lowest photon yield and lowest
ETR also had the lowest photosynthetic rate, while
family 16 with the highest photosynthetic rate had
the highest ETR.
The differences between plants grown at control
and low Mg in fluorescence quenching (Fig. 3)
depended on the Mg concentration in which the
plants were grown, and on the PFD. In ambient
growth conditions (low PFD, Table 7) there were
only small differences in qp between the plants at the
two Mg concentrations, and qp was relatively high
(approx. 0.88), indicating that the effects of differ-
56 RESEARCH W. Laing et al.
ences in Mg concentration on reaction centre closure
are small under these conditions. By contrast, there
were marked differences in NPQ, with the needles of
the low-Mg-grown plants having a significantly
higher capacity for non-radiative heat loss compared
with the needles on control Mg-grown plants. Thus
the differences in photochemical yield between the
plants induced by differences in Mg concentration
were predominantly caused by differences in thermal
dissipation.
At high PFD, in needles of control Mg plants, qp
continued to remain relatively high (approx. 0.72),
indicating that the pool of functional PSII reaction
centres in these needles was relatively resistant to
closure. Both high rates of electron transport and
high carboxylation demand in the control Mg-grown
plants would have ensured a low probability of these
reaction centres experiencing photoinhibition at the
high PFD. By contrast, for the needles of low-Mg-
grown plants, qp was reduced markedly more with
increasing PFD (approx. 0.6), suggesting that a high
proportion of reaction centres on these needles was
closed, that is, photochemically inactivated through
photoinhibition.
Consistent with the higher capacity to maintain a
pool of functional reaction centres across a range of
PFDs, the needles grown at control Mg also had a
concomitantly lower requirement for non-radiative
heat dissipation, as indicated by lower NPQ, than
occurred in the low-Mg-grown plants. Conforming
to this conclusion, xanthophyll cycle activity was
significantly higher in the low-Mg-grown plants
compared with those grown in control Mg (Table 8).
Thus with increased PFD, changes in photochemical
yield and electron transport in pine needles were
dependent on both closure of PSII reaction centres
and xanthophyll cycle-mediated thermal dissipation,
and low Mg concentration markedly exacerbated
these effects.
Magnesium deficiency has been observed to
reduce the root : shoot ratio in plants (e.g. Cakmak
et al., 1994 ; Ericsson & Kahr, 1995 ; Sun & Payn,
1999), contrary to what might be expected of a plant
low in Mg supply from the roots. One interpretation
might be that signals from needles to increase needle
biomass because of reduced photosynthesis under
Mg deficiency might outweigh signals to increase
root biomass.
In summary, this paper demonstrates that Mg
deficiency reduces plant growth and impairs mature
needle photosynthesis. We show that Mg deficiency
in combination with high PFDs predisposes P.
radiata needles to photoinhibition of photosynthesis,
that is, reaction centre inactivation, despite the
photoprotective processes involving xanthophyll
cycle-mediated heat dissipation. These photobleach-
ing effects are possibly extensive enough to give rise
to the visible symptoms of UMCY. Although family
differences in susceptibility to Mg deficiency were
evident in many of the parameters measured,
interaction between Mg nutrition and family was
rare in this study.

Beets PN. 1977. Determination of the fascicle surface area for
Pinus radiata. New Zealand Journal of Forestry Science 7 :
397–407.
Beets PN, Brownlie RK. 1987. Puruki experimental catchment :
site, climate, forest management, and research. New Zealand
Journal of Forestry Science 17 : 137–160.
Beets PN, Jokela EJ. 1994. Upper mid-crown yellowing in Pinus
radiata : some genetic and nutritional aspects associated with its
occurrence. New Zealand Journal of Forestry Science 24 : 35–50.
Beets PN, Payn TW, Jokela EJ. 1993. Upper mid-crown
yellowing (UMCY) in Pinus radiata forests. New Zealand
Journal of Forestry 38 : 24–28.
Bilger W, Bjo$ rkman O. 1990. Role of the xanthophyll cycle in
photoprotection elucidated by measurements of light-induced
absorbance changes, fluorescence and photosynthesis in leaves
of Hedera canariensis. Photosynthesis Research 25 : 173–185.
Bjo$ rkman O, Demmig B. 1987. Photon yield of O evolution
#
and chlorophyll fluorescence characteristics at 77 K among
vascular plants of diverse origins. Planta 170 : 489–504.
Cakmak I, Hengeler C, Marschner H. 1994. Partioning of
shoot and root dry matter and carbohydrates in bean plants
suffering from phosphorus, potassium and magnesium def-
iciency. Journal of Experimental Botany 45 : 1245–1250.
Demmig-Adams B, Adams WW III. 1992. Photoprotection
and other responses of plants to high light stress. Annual Review
of Plant Physiology and Plant Molecular Biology 43 : 599–626.
Diem B, Godbold DL. 1993. Potassium, calcium and magnesium
antagonism in clones of Populus trichocarpa. In : Barrow NJ, ed.
Plant nutrition – from genetic engineering to field practice.
Dordrecht, The Netherlands : Kluwer Academic, 613–616.
Ericsson T, Kahr M. 1995. Growth and nutrition of birch
seedlings at varied relative addition rates of magnesium. Tree
Physioliology 15 : 85–93.
Farquhar GD, Sharkey TD. 1982. Stomatal conductance and
photosynthesis. Annual Review of Plant Physiology 33 : 317–345.
Fink S. 1993. Microscopic criteria for the diagnosis of abiotic
injuries to conifer needles. In : Huettl RF, Mueller-Dombois
D, eds. Forest decline in the Atlantic and Pacific region. Berlin,
Germany : Springer Verlag, 175–189.
Fischer ES, Bremer E. 1993. Magnesium deficiency in ex-
panding leaves of Phaseolus vulgaris – gas exchange and nutrient
concentrations. In : Barrow NJ, ed. Plant nutrition – from genetic
engineering to field practice. Dordrecht, The Netherlands :
Kluwer Academic, 621–624.
Flowers TJ, Lauchli A. 1983. Sodium versus potassium :
substitution and compartmentation. In : Lauchli A, Bieleski
RL, eds. Encyclopedia of plant physiology, NS 15B. Berlin,
Germany : Springer Verlag, 651–681.
Forest Research Institute. 1991. Mid-crown yellowing – on the
increase. What’s New in Forest Research, Series No. 206.
Rotorua, New Zealand : New Zealand Ministry of Forestry.
Gilmore AM, Yamamoto HY. 1991. Resolution of lutein and
zeaxanthin using a non-endcapped lightly carbon loaded C18
high performance liquid chromatography column. Journal of
Chromatography 543 : 137–145.
Greer DH. 1998. Photoinhibition of photosynthesis in dwarf
bean (Paseolus vulgaris L.) leaves : effects of sink-limitations
induced by changes in daily photon receipt. Planta 205 :
189–196.
Greer DH, Laing WA. 1989. Photoinhibition of photosynthesis
in intact kiwifruit (Actinidia deliciosa) leaves : effect of growth
temperature on photoinhibition and recovery. Planta 180 :
32–39.
Greer DH, Laing WA. 1992. Photoinhibition of photosynthesis
in intact kiwifruit (Actinidia deliciosa) leaves : changes in
susceptibility to photoinhibition and recovery during the
growth season. Planta 186 : 418–425.
Greer DH, Laing WA, Woolley DJ. 1993. The effect of
chloramphenicol on photoinhibition of photosynthesis and its
 RESEARCH Magnesium deficiency in Pinus radiata
recovery in intact kiwifruit (Actinidia deliciosa) leaves. Aus-
tralian Journal of Plant Physiology 20 : 33–43.
Ingestad T, A/ gren GI. 1988. Nutrient uptake and allocation at
steady-state nutrition. Physiologia Plantarum 72 : 450–459.
Laing WA, Greer DH, Schnell TA. 1995. Photoinhibition of
photosynthesis causes a reduction in vegetative growth rates
of dwarf bean (Phaseolus vulgaris) plants. Australian Journal of
Plant Physiology 22 : 511–520.
Marschner H. 1983. General introduction to the mineral
nutrition of plants. In : Lauchli A, Bieleski RL, eds. Encyclo-
pedia of plant physiology, NS 15A. Berlin, Germany : Springer
Verlag, 5–49.
Marschner H. 1995. Mineral nutrition of higher plants, 2nd edn.
London, UK : Academic Press.
Mehne-Jakobs B. 1995. Seasonal development of the photo-
synthetic performance of Norway spruce (Picea abies [L] Karst.)
under magnesium deficiency. Plant and Soil 168–169 : 255–261.
Mehne-Jakobs B. 1997. Physiological consequences of mag-
nesium deficiency. In : Rennenberg H, Eschrich W, Ziegler H,
eds. Trees : contributions to modern tree physiology. Leiden, The
Netherlands : Backhuys, 313–325.
Mengel K, Kirby EA. 1978. Principles of plant nutrition. Berne,
Switzerland : International Potash Institute.
Nicholson G, ed. 1984. Methods of soil, plant and water analysis.
57
NZ FRI Bulletin No. 70. Rotorua, New Zealand : New Zealand
Forest Research Institute.
Payn TW, Mead DJ, Will GM, Hunter IR. 1995. Magnesium
nutrition and dry matter allocation patterns in Pinus radiata.
New Zealand Journal of Forestry Science 25 : 39–48.
Puech L, Mehne-Jakobs B. 1997. Histology of magnesium-
deficient Norway spruce needles influenced by nitrogen source.
Tree Physiology 17 : 301–310.
SAS. 1989. SAS\STAT Guide for personal computers, Version 6,
12th edn. Cary, NC, USA : SAS Institute.
Schreiber U, Hormann H, Neubauer C, Klughammer C.
1995. Assessment of photosystem II photochemical quantum
yield by chlorophyll fluorescence quenching analysis. Aus-
tralian Journal of Plant Physiology 22 : 209–220.
Sun OJ, Payn TW. 1999. Magnesium nutrition and leaf
photosynthesis in Pinus radiata : clonal variation and influence
of potassium. Tree Physiology 19 : 535–540.
Thayer SS, Bjorkman O. 1990. Leaf xanthophyll content and
composition in sun and shade determined by HPLC. Photo-
synthesis Research 23 : 331–343.
Wyn Jones RG, Pollard A. 1983. Proteins, enzymes and
inorganic ions. In : Lauchli A, Bieleski RL, eds. Encyclopedia of
plant physiology, NS 15B. Berlin, Germany : Springer Verlag,
528–562.