Journal of Plant Physiology 248 (2020) 153156

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Journal of Plant Physiology

journal homepage: www.elsevier.com/locate/jplph

The higher availability of nutrients increases the production but decreases T

the quality of pollen grains in Juniperus communis L.
Emilia Pers-Kamczycb,*, Żanna Tyrała-Wieruckaa, Mariola Rabskab, Dorota Wrońska-Pilarekc,
Jacek Kamczycd
a
University of Zielona Góra, Faculty of Biological Sciences, Licealna 9, 65-417 Zielona Góra, Poland
b
Institute of Dendrology, Polish Academy of Sciences, Parkowa 5, 62-035 Kórnik, Poland
c
Poznan University of Life Sciences, Faculty of Forestry, Department of Forest Botany, Wojska Polskiego 71E, 60-625 Poznań, Poland
d
Poznan University of Life Sciences, Faculty of Forestry, Department of Game Management and Forest Protection, Wojska Polskiego 71C, 60-625 Poznań, Poland

A R T I C LE I N FO A B S T R A C T

Keywords: Next to global warming, excessive deposition of nitrogen has an alarming environmental impact on forest
C:N ratio ecology, especially within dioecious species. Resource availability affects seed quality and can affect the dis-
Coniferous plants tribution of plant species. Lower seed productivity can also be a result of limited pollen availability or lower
Fertilizer deposition pollen quality. A few studies have assessed the effect of nutrient availability on the quantity and quality of pollen
Pollen development
grains produced. Therefore, rooted shoots of dioecious, male Juniperus communis L. grown in different nutritional
Pollen morphology
conditions were used to assess the impact on productivity and quality attributes of produced pollen grains
Pollen quality
(pollen volume, morphology, germination, and chemical composition). The results indicated that nutrient
availability impacts pollen grain development. Male plants growing in nutrient-rich environments appear to
compensate for the lower quality of produced pollen grains by producing a higher number of male cones and
thus a greater quantity of pollen. In contrast, the opposite was observed in plants growing in nutrient-poor
environments.
The availability of nitrogen and other nutrients will probably continue to impact soils in the foreseeable
future due to anthropogenic activity and can be one of the drivers that can impact the reproduction and dis-
tribution of plants.

1. Introduction DeBoer, 2008). Although the dramatic increase in fertilizer production

The availability of nitrogen in soils in the majority of the continental
and northern forests of Europe is low and is considered to be a primary
growth-limiting nutrient (Nybakken et al., 2018). In addition to ni-
trogen levels, however, the availability of phosphorus (Lun et al.,
2018), potassium (Sardans and Peñuelas, 2015), and other nutrients
may also, jointly or separately, represent a limiting factor or factors for
plant growth (Sinclair, 1992). In modern times, chemical fertilizers
have been used to overcome this potential problem in agriculture.
Commercial chemical fertilizers can provide nutrients in optimum
forms as they can be species-specific and quantities as they are used
properly, however, it is not always like that. Based on evaluations and
projections of fertilizer demand, it appears that the fertilizer use is in-
creasing and will continue to do so soon (Lun et al., 2018; Sardans and
Peñuelas, 2015; Galloway et al., 2004; Tenkorang and Lowenberg-
is justified by the need to increase food production to reduce world
hunger, the excessive and unwarranted use of fertilizers can also cause
environmental and ecological problems. Specifically, air pollution, soil
degradation, and acidification; as well as water eutrophication and the
excessive accumulation of N, phosphorus, potassium, and magnesium
in soils are possible outcomes of excessive fertilizer usage. Many Eur-
opean arctic, boreal, and temperate ecosystems have already been af-
fected by excessive levels of nitrogen (Bobbink et al., 2010).
Similar to agriculture, there has been increased interest in the use of
fertilization in forests, mainly for increasing timber production, which
is an important part of the economy of northern countries (Bergh et al.,
2014). Although the amount of applied fertilizer during forest seedling
production is lower than in agriculture, almost half of the applied ni-
trogen annually leaches into the environment (Juntunen and Rikala,
2001; Velthof et al., 2011). In natural ecosystems, the accumulation of

⁎
Corresponding author.
E-mail addresses: epk@man.poznan.pl (E. Pers-Kamczyc), zannat@wp.pl (Ż. Tyrała-Wierucka), mrabska@man.poznan.pl (M. Rabska),
pilarekd@up.poznan.pl (D. Wrońska-Pilarek), jacek.kamczyc@up.poznan.pl (J. Kamczyc).

https://doi.org/10.1016/j.jplph.2020.153156
Received 5 November 2019; Received in revised form 11 March 2020; Accepted 11 March 2020
Available online 19 March 2020
0176-1617/ © 2020 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
E. Pers-Kamczyc, et al.
nutrients, especially nitrogen, has several effects on soil quality and
causes undesirable changes in plant diversity (Bobbink et al., 2010;
Velthof et al., 2011). Recent studies have demonstrated that the addi-
tion of nitrogen in forest stands increases aboveground biomass of
forest trees (Meunier et al., 2016), including rapid foliar growth
(Takemoto et al., 2001), but also harms the natural chemical defense
compounds typically found in the needles of coniferous trees
(Nybakken et al., 2018). Importantly, excessive nutrients also affect
both the diversity and functioning of mycorrhizal fungi, by altering the
abundance and diversity of mutualistic species associated with the
health of many terrestrial plants (Fenn et al., 2003). The impact of
nutrient availability on the physiology of a plant can vary depending on
the species, with some species being better adapted to poor nutrient
conditions while others prefer richer nutrient habitats.
According to the growth-differentiation balance hypothesis, growth
and defense processes compete for available nutrients and a trade-off
occurs between allocating nutrients for growth vs. defense (Meunier
et al., 2016). Species adapted to resource-rich environments grow faster
and invest less in defense than species adapted to lower resource ha-
bitats (Endara and Coley, 2011). In addition to growth and main-
tenance, plants also allocate available resources into reproduction, in-
cluding pollen and seed production (Matyssek et al., 2012).
Recent studies have shown that mineral soil composition can affect
seed quality and thus change the distribution of plant species
(Bogdziewicz et al., 2017). Moreover, the interactions between nutri-
tional status and pollen quality had been discussed for nitrogen (Lau
and Stephenson, 1993; Atasay et al., 2013), phosphorus (Lau and
Stephenson, 1994) and some micronutrients (Pandey, 2010). Viable
pollen is crucial for effective sexual plant reproduction. Both micro-
sporogenesis and microgametogenesis in male reproductive organs are
extremely sensitive to adverse environmental conditions. Stress-in-
duced male sterility is a significant problem for seed production and the
production of grains (De Storme and Geelen, 2014). Pollen quality can
be evaluated by either in vivo or in vitro germination assays and by the
analysis of pollen tube growth, pollen tube enzyme activity and mem-
brane integrity (Shivanna and Rangaswamy, 1992). The assessment of
pollen quality is an essential aspect of plant breeding and horticultural
production (Dafni and Firmage, 2000). Reductions in pollen viability
are often attributable to air pollutions, mainly NO2 (Gottardini et al.,
2008); but also sulphur, trace metals, and environmental factors such as
heat stress, UV-B radiation, and drought/dehydration (Gottardini et al.,
2008; Nguyen et al., 2009). The above factors can impact the bio-
chemistry, ultrastructure, morphology, and/or physiology of pollen
grains (Pukacki and Chałupka, 2003; Bist et al., 2004; Majd et al.,
2004). While many studies have investigated the influence of abiotic
factors on the growth and physiology of herbaceous plants (Dafni and
Firmage, 2000; Gottardini et al., 2008; Lau et al., 1995; Delph et al.,
1997), little is known about the effect of environmental resources on
the physiology and reproductive fitness of coniferous shrubs and trees.
This includes pollen and seed viability, and rates of germination and
seed abortion (Owens et al., 1998; Gruwez et al., 2013, 2014, 2017); as
well as other reproductive traits and reproductive capacity (García
et al., 2000; Callahan et al., 2008).
Our previous studies performed on Taxus baccata L. male plants
indicated that a higher level of nutrient availability can negatively
impact on the pollen grain germination (Pers-Kamczyc et al., 2019). As
T. baccata is evolutionally adapt to rich nutrient availability and we
know that higher nutrient availability increases the number of pro-
duced male cones leading to increases in pollen grain production but
negatively impact on the pollen germination rates, we wanted to test if
that higher nutrient availability impacts also the reproductive fitness of
species evolutionally adapt to low nutrient availability. We used
common juniper (Juniperus communis L.) as a model plant. This is a
dioecious, non-flowering seed plant and is considered to be a woody
pioneer and colonizing plant (García et al., 2000; Faliński, 1980;
Oostermeijer and Knegt, 2004). It is adapted to low nutrient availability
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Journal of Plant Physiology 248 (2020) 153156
in soil and it has one of the widest distribution ranges among plant
species (Gruwez et al., 2013, 2017; Oostermeijer and Knegt, 2004;
Verheyen et al., 2009; Broome et al., 2017). Populations in some parts
of the Alps, Scandinavia, and Poland exhibit good regeneration
(Faliński, 1980; Rosén and Bakker, 2005), but some populations are
targeted of conservation efforts mainly within the northwest European
lowlands and Mediterranean mountain regions (Verheyen et al., 2009).
The decline of populations of tree species has been attributed to habitat
degradation and destruction, limited recruitment of new individuals,
climate warming, and nutrient status (Vanden-Broeck et al., 2011).
Studies conducted on J. communis have indicated that climate warming
and the enhanced airborne deposition of nitrogen and sulphur have
both affected seed viability (Gruwez et al., 2013, 2017; Verheyen et al.,
2009). Since common juniper is a dioecious plant (García et al., 2000;
Faliński, 1980), stresses that decrease reproductive fitness can also be a
source of juniper decline (Gruwez et al., 2013, 2014). Further, the
potential impact of environmental changes on pollen production was
mentioned but not discussed in detail (Verheyen et al., 2009).
Therefore, rooted shoots of plants collected from a natural popula-
tion and maintained under relatively homogenous environmental con-
ditions (water and light conditions, soil type, plants density) were used
to determine the impact of nutrient availability on male reproductive
features. The following questions were addressed in the study: Do
higher levels of nutrient availability supplied by fertilizer increase the
production of male cones? Do pollen grains produced by fertilized
plants have higher germination potential than pollen grains produced
in nutrient-poor habitats? Do the morphology and chemical composi-
tion of pollen grains differ between fertilized and non-fertilized (con-
trol) groups of plants?
2. Material and methods
2.1. Experimental design
The study was conducted at the Institute of Dendrology, Polish
Academy of Sciences, Kórnik, Poland. Rooted shoots of Juniperus com-
munis L. were used in the study. Fifty shoots were collected and rooted
in 2012 from each of 10 male trees of J. communis growing in the Rokita
forest district in West Pomerania, Poland. Cuttings of a similar size were
taken from the middle part of each crown, growing under similar,
partially-shaded light conditions. In March 2013, rooted cuttings were
placed in five-litre pots and then transferred to ten-litre pots in May
2016. This transferring was done to minimalize the negative effect of
pot size on plant growth (Poorter et al., 2012). We used barren and gray
forest soil collected at the our forest station. Soil was sieved on sieve
(1.0 × 1.0 cm), mixed and used for planting and mycorrhizal in-
oculation of the pots was accomplished by adding soil (10 % of the soil
volume) collected from a forest stand dominated by J. communis. The
main soil in pots contained the percentage (g/kg) of elements:
36.0 ± 0.9 total carbon, 2.0 ± 0.4 total nitrogen, 0.3 ± 0.06 total
phosphorus, 0.46 ± 0.05 total calcium, 0.84 ± 0.13 magnesium, and
250.0 ± 25.0 mg/kg sodium. Soil samples were analysed by JARS S.A.
(Mysłowice, Poland) according to Polish Committee for Standardization
(PKN, 2012, 2009, 2002). During this time, the cuttings were grown
under a two-meter-high scaffolding with a shading net resulting in a 50
% reduction of full sunlight, to create light conditions similar to con-
ditions under the conopy layer in the forest. The degree of light re-
duction was confirmed by measurements of relative photosynthetic
photon flux density using a line quantum sensor (Apogee Instruments
Inc., Logan, UT, USA), following the methods described by Messier and
Puttonen Messier and Puttonen (1995). Each pot was irrigated sepa-
rately with an automatic irrigation system. As fertilization increases the
growth of the plant and it is related to water availability, plants from
both groups were irrigated with different amounts of water. Therefore
fertilized plants got twice the volume of water as nonfertilized plants
during irrigation and plants were watered each day to keep the medium
E. Pers-Kamczyc, et al.
soil moisture during the whole vegetation season.
The study was conducted over four years, from March, 2013 to May,
2017. All plants were placed in pots with the same soil and were ran-
domly divided into two groups in March, 2013 and assigned one of two
levels of nutrients. Pots were placed at using the same distance between
rows and within a row to avoid density effects. Fertilizer was applied to
half of the plants each year in spring (March/April). Osmocote Exact
5−6 M (ICL, Tel Aviv, Israel) fertilizer was used at the highest to the
coniferous species doses recommended by fertilizer producer (six grams
per litre). The fertilized plants received 0.75 g N (N-NO3 – 0.33 g, N-
NH4 – 0.42 g), 0.45 g P2O5, 0.6 g K2O, 0.125 g MgO, and microelements
(22.5 mg Fe, 3.0 mg Mn, 1.0 mg B, 2.5 mg Cu, 1.0 mg Mo, 0.75 mg Zn)
per litre of soil. The control group of non-fertilized plants was grown
without the use of any fertilizer or other supplements.
During the study, measurements of air temperature and relative air
humidity were recorded each hour using four EL-USB-2+ data loggers
(EasyLog, Inc.). Monthly mean values of air temperature and air re-
lative humidity during the period of male flower development (from
August to April/May) were similar to long-term averages in this region
of Poland.
In September 2017, soil samples were collected from randomly
chosen pots of the non-fertilized and fertilized plants and their chemical
composition was analysed. Soil of fertilized (F) and non-fertilized (NF)
plants contained the following percentage (g/kg) of the elements, re-
spectively: total carbon (F: 40.4 ± 1.2 and NF: 42.3 ± 0.7), total ni-
trogen (F: 1.8 ± 0.12 and NF: 1.7 ± 0.06), C:N ratio (F: 22.4 and NF:
25.1), total phosphorus (F: 0.39 ± 0.05 and NF: 0.15 ± 0.02), total
calcium (F: 0.69 ± 0.02 and NF: 0.53 ± 0.06), magnesium (F:
0.38 ± 0.04 and NF: 0.33 ± 0.02), and sodium (F: 303.67 mg/
kg ± 3.84 and NF: 257.7 mg/kg ± 10.6).
2.2. Pollen productivity - the number of male cones
Sixty plants were randomly selected and tagged from each of the
two groups in March, 2013 and subsequently monitored for the pre-
sence of male cones until the end of the study in 2017. Equal numbers
of plants from fertilized and non-fertilized groups, all of which were
derived from the same parental line, were used in the analysis. Plants
were evaluated in May, 2014 to characterize male cones. The number of
male cones was counted on three shoots located in the middle part of
the crown in both treatment groups (F and NF). The number of male
cones per shoot length and the total height of the plants from the
ground level to the apical meristem were assessed during flowering
(May) in three successive years (2015, 2016, and 2017). The number of
cones produced per one cm of shoot length was determined and ex-
pressed in relation to the total height of each plant. Two F plants and
two comparable, NF plants of the same parental line were removed in
2016 to maintain an equal number of trees in both groups thus two
fertilized plants were broken.
2.3. Pollen quality – pollen development in vitro
Pollen grains were collected in 2017 from 15 flowering plants (three
randomly selected individuals per five randomly selected parental
plants) in each of the treatment groups (F and NF). Freshly released
pollen grains were collected from each plant when pollen sacs began to
open, placed in an Eppendorf tube, and stored at 10 °C. Pollen used for
in vitro analysis was stored at 10 °C for no longer than 24 h.
Germination was assessed in a Brewbaker-Kwack liquid medium
(Brewbaker and Kwack, 1963) supplemented with 5 % sucrose. The
optimum energy source was determined in pre-experiment in which
Brewbaker-Kwack liquid medium without and with supplementation of
a different source of energy (5 and 10 % of fructose, glucose or sucrose)
were tested. Incubation was conducted without antibiotics or fungi-
cides. Pollen concentration was adjusted to 10 mg/ml and the pollen
suspensions (8–10 ml) were placed in sterile pots (3 cm in diameter)
3
Journal of Plant Physiology 248 (2020) 153156
and maintained in the dark at 21 °C. Three replication of three samples
were analyzed and pollen morphology was described after 48 h of in-
cubation, however pollen development was inspected every 24 h up to
10 days of incubation or evidence of fungal growth.
Finally, two experiments were conducted over a two-day period. In
each experiment, pollen grains were collected and used from single
individuals derived from five parental plants from both treatment
groups. In total, three replications of ten samples were analyzed. Pollen
grain development was examined after 48 and 96 h of in vitro incuba-
tion. The analysis was performed using an AxioVision microscope
(Zeiss, Germany).
A minimum of 300 random pollen grains was examined at each time
point from the three technical replications of each parental line. Pollen
grains were considered as viable if they met two criteria: a) they were
able to rupture their exine, and b) the main body of the pollen grain had
a regular, spherical shape. The size of the main body of each pollen
grain was assessed after 96 h of in vitro incubation. The length of the
vertical and horizontal axes of a minimum of 300 randomly-selected,
viable pollen grains without an exine, were measured using AxioVision
software (Zeiss). Pollen grains were considered as elongated when the
ratio of the measured axes was at least 1.41, while germinated pollen
had a ratio of at least 1.81. Pollen grain volume was calculated as de-
scribed by Pacini (1990). The volume of a spherical pollen grain was
calculated using the formula 4/3π r3, where r was the radius of pollen
grains, For ellipsoidal pollen grains the formula 4/3π a2 b was used,
where a is the equatorial axis/2 and b is the longitudinal axis/2.
Pollen grains (Fig. 1) were placed into one of two main categories
after in vitro incubation; those remaining within their exine and those
that were able to rupture their exine. Subclasses were distinguished
within the second category based on the absence or presence of
shrinkage and/or fragmentation of the main body of the pollen grain.
Based on measurements made of the main body of the pollen grain,
those with regular granulation of the main body were further divided
into spherical, elongated or germinated subclasses.
2.4. Pollen morphology
Pollen grains collected from each plant were analyzed separately.
Measurements from 30 mature, randomly-selected, correctly-formed
pollen grains were made using light microscopy (LM). A total of 580
pollen grains were measured and pollen grains were acetolysed as de-
scribed by Erdtman (1960). Briefly, pollen grains were mixed with the
acetolysis solution, which consisted of nine parts acetic anhydrite and
one-part concentrated sulphuric acid. The mixture was heated to
boiling in a water bath and kept there for 2–3 min. Samples were then
centrifuged in the acetolysis mixture, washed with acetic acid, and
centrifuged again. The pollen grain samples were then mixed with 96 %
alcohol and centrifuged. This step was repeated 4 times. The processed
grains were subsequently divided into two groups. One half of the
processed sample was immersed in an alcohol-based solution of gly-
cerin for light microscopy, and the other was placed in 96 % ethyl al-
cohol in preparation for scanning electron microscopy (SEM). The SEM
observations were made using a Zeiss Evo 40 (Zeiss, Germany) and the
LM using a Biolar 2308 (Nikon HFX-DX). LM measurements of acet-
olysed pollen grains were made at a 640x magnification. The mor-
phological features of pollen grains that were assessed included the
length of the polar axis (P), equatorial diameter (E), exine thickness
measured along the polar axis (Exp), and the ratios of P/E and Exp/P. A
qualitative assessment (pollen outline, shape and exine ornamentation)
was also made as described by (Punt et al. (2007) and Hesse et al.
(2009).
2.5. The content of C, N and C:N ratio in the pollen grains
Dry pollen grains collected from ten plants grown in the F and NF
groups were used to determine the carbon to nitrogen ratio (n = 20).
E. Pers-Kamczyc, et al. Journal of Plant Physiology 248 (2020) 153156

Fig. 1. Morphological classes of Juniperus

communis L. pollen grains observed after 48 h
(A, B, C, D) and 8 days (E, F) of in vitro in-
cubation. A - re-hydrated pollen within (inEx)
and without (outEx) the exine; B - pollen grains
without proper re-hydration within the exine
(inEx); C - pollen grain escaping from the
exine, without evidence of shrinkage of the
main body of the pollen grain; D - elongated
pollen grain without an exine; and germinating
pollen grains with (E) and without (F) evidence
of shrinkage.

Table 1 fertilization treatment as fixed effects and an individual plant as a

The effect of nutrient availability (F – fertilized plants, NF – non fertilized random effect. Data for P/E, Exp/P, and Exe/E were square-root
plants) on various morphological features of Juniperus communis L. plants. Data transformed before statistical analysis.
shown represent the mean ± SE. Means within a column differ significantly at Data obtained from the analysis of C:N ratios in pollen grains were
P < 0.05. transformed by arcsine and then subsequently analyzed with a
Treatment 2015 2016 2017 Student’s t-test.
The presented data represent the mean ± SE (1 SE). Means were
Plant height (cm)
considered significantly different at P < 0.05.
F 78.00 ± 2.97 a 103.19 ± 3.24 a 242.74 ± 7.38 a
NF 51.22 ± 3.75 b 89.36 ± 3.95 b 216.57 ± 9.01 b
Number of flowers per one cm of shoot lenght 3. Results
F 1.51 ± 0.29 a 4.46 ± 0.55 a 7.94 ± 0.68 a
NF 0.57 ± 0.14 b 1.44 ± 0.28 b 3.65 ± 0.41 b
Shoot length (cm) 3.1. Production of male cones
F 13.19 ± 0.66 a 16.16 ± 0.77 a 20.98 ± 0.97 a
NF 5.87 ± 0.48 b 11.19 ± 0.81 b 14.57 ± 0.82 b All measurements were done on plants that were kept under nu-
tritionally differentiated conditions (fertilized and non-fertilized)
starting in 2013 and the production of male cones within the crown
Four mg of dry pollen grains was analyzed separately from individual beginning in 2014. NF plants were smaller than F plants, producing
samples. Carbon and nitrogen content (% of dry mass) was determined shoots of shorter length and a lower number of male cones per one cm
using an Elemental Combustion System CHNS-O 4010 analyzer of shoot length in all of the analyzed years (Table 1). In F plants, male
(Costech Instruments, Italy/USA; http://www.costechanalytical.com). cones were numerous and located within the entire crown. On the other
hand, male cones in NF plants were less numerous and mainly located
2.6. Statistical analysis in the upper three-quarters of the crown. Regression analysis indicated
that the number of male cones produced per one cm of the shoot was
Data describing plant morphology were subjected to an analysis of correlated with plant growth and taller plants produced more male
variance (ANOVA) for height and diameter, as well as for the number of cones. This relationship, however, was dependent on the year
male cones, with the fertilization treatment and genotype as fixed ef- (P(F=56.39, DF=1) < 0.001) and treatment (P(F=56.99, DF=1) < 0.001)
fects and individual plants as a random effect. Correlations between the (Fig. 2).
number of male cones produced per one cm of shoot and plant height
were also analyzed concerning fertilization treatment and year. 3.2. Pollen quality – in vitro development of pollen grains
Data describing the features of pollen grains in the two treatment
groups after 48 and 96 h of in vitro incubation were analyzed using the In total, 5005 pollen grains were used during optimization of in vitro
chi-square test for independent data. Data describing in vitro parameters conditions and pollen grains re-hydrated differentially in a different
of viable pollen development after 48 h were analyzed using an ANOVA type of medium. After 48 h, numerous pollen grains were extruded from
for the longitudal axis, equatorial diameter, the ratio of the longitudal exine in medium without any supplementation (95.3 %, 624/655) and
axis to equatorial diameter and pollen volume (with genotype and within medium supplemented with both doses of sucrose (5 %: 95.7 %,
fertilization treatment as fixed effects and individual plant as a random 620/648 and 10 %: 91.7%, 684/746). Moreover, pollen grains started
effect). to germinate only in medium supplemented with 5% (3.22 %, 20/620)
Data describing the parameters of pollen grain morphology from LM and in medium without any of the supplementation (1.44 %; 9/624),
and SEM were statistically analyzed with an ANOVA for length of polar however incidental evidence of germination (one germinating pollen
axis (P), equatorial diameter (E), exine thickness (Exp and Exe), P/E grains) was also observed in other media (Additional data – Table 1).
ratio, Exp/P and Exe/E of dry pollen grains; with genotype and On the base of this observation medium supplemented with 5 % of

4
E. Pers-Kamczyc, et al. Journal of Plant Physiology 248 (2020) 153156

Fig. 2. Juniperus communis L. male cone production per one cm of shoot in relation to plant height (cm) in fertilized (A, B, C) and non-fertilized (D, E, F) plants over
three consecutive years (2015-2017).

sucrose was used for further analysis. pollen grains from F plants (63.5 %, 1671/2631). Also, a significantly
In total, 9725 pollen grains of Juniperus communis were analyzed. greater number of NF (72.5 %, 1093/1507, P(χ2=5.7537, DF =1) < 0.05)
After 48 h, more than 50 % (3185/5464) of all pollen grains emerged pollen grains exhibited a lack of shrinkage of the main body of the
out of their exine. Significantly more NF pollen grains ruptured their pollen grain (F: 68.6 %, 1147/1671). Notably, ten times more NF pollen
exines (78.3 %, 2103/2684) compared to pollen collected from F plants grains classified as good were elongated and showed signs of germi-
(38.9 %, 1082/2780; P(χ2=232.47, DF=1) < 0.05). However, significantly nation (37.14 %, 406/1093) than pollen from F plants (3.1 %, 36/1147)
more F than NF pollen grains did not exhibit evidence of shrinkage of (Table 2, Figure 3). Again, this difference was highly significant
the main body of the pollen grain indicating a lack of degradation (NF: (P(χ2=408.6509, DF=1) < 0.001). Based on the ratio of the length of the
87.5 %, 1841/2103 vs F: 93.99 % (1017/1082); P(χ2=1.87, polar and equatorial axes, germinating viable pollen grains were ob-
DF=1) < 0.05) (Table 2). Importantly, almost six times more pollen served within all genotypes of NF plants, while no pollen grain ger-
grains that were classified as good began to germinate from NF (9.97 %, mination was observed in any of the F plant genotypes (Table 2).
176/1765) plants compared to F plants (1.77 %, 18/1017; P(χ2=62.8331, After 96 h of in vitro incubation, NF pollen grains had a longer
DF=1) < 0.001). After 96 h of incubation, approximately 75 % (3178/ average, minimum and maximum polar axis length (48.00 ± 0.48 μm,
4261) of the pollen grains were released from their exines. As with the 23.56, 87.25 μm – mean, min. and max., respectively) and average,
48 h samples, significantly more NF pollen grains had ruptured their min., and max. equatorial diameter (43.00 ± 0.53 μm, 19.75, 78.56 μm
exine (92.45 %, 1507/1630, P(χ2=62.82, DF=1) < 0.05) as compared to – average, min. and max, respectively) than F pollen grain polar axis
length (25.03 ± 0.12 μm, 18.35, 44.39 μm – average, min. and max,
respectively) and equatorial diameter (21.52 ± 0.07 μm, 16.17, 30.58
Table 2
μm – average, min., and max, respectively) (Fig. 4A, B, Table 3). The
The effect of nutrient availability (F – fertilized plants, NF – non fertilized
plants) on the number of pollen grains classified in different morphological
differences in polar length (P(F=272.656,DF=1) < 0.001) and equatorial
classes after 48th and 96th hours of in vitro incubation. diameter (P(F=10.8622, DF=1) < 0.001) were both highly significant.
Nutrient availability also affected the volume of developing pollen
Treatment without exine with exine All* (100%)
grains after 96 h of in vitro incubation, with the volume of the main
degraded good body of NF pollen grains 60,386 ± 1819 μm3) being significantly
greater (P(F=959.1799, DF=1) < 0.001) than the main body of F pollen
rounded germinating grains (8,202 ± 104 μm3) (Fig. 4C, Table 3. The volume of developing
NF pollen grains was highly variable, ranging from 6631 μm3 to
48th hours of pollen incubation in vitro
F 65 (2.34 %) 999 (35.91 18 (0.65 %) 1698 2780 278,842 μm3, while the volume of F pollen grains ranged from 3108
%) (61.1 %) μm3 to 24,600 μm3.
NF 262 (9.76 1665 176 (6.56 %) 581 2684
%) (62.03 %) (21.65 %)
Total 327 2664 194 2279 5464
3.3. Pollen morphology (SEM)
96th hours of pollen incubation in vitro
F 524 (19.92 1111 36 (1.36 %) 960 2631 The measured morphological parameters of J. communis pollen
%) (42.23 %) (36.49 %) grains were affected by genotype and nutrient availability (Table 4,
NF 414 (25.40 687 (42.15 406 (24.9 %) 123 (7.55 1630
Fig. 5). The length of the polar axis (P) of all the analyzed pollen grains
%) %) %)
Total 938 1798 442 1083 4261 ranged from 22 μm to 34 μm, while the equatorial diameter (E) ranged
from 18 μm to 30 μm. Low nutrient availability (NF) resulted in pollen
* Percentage of all pollen grains. grains with a significantly longer polar axis (27.12 ± 0.132 μm vs

5
E. Pers-Kamczyc, et al.
Fig. 3. Features of fertilized (A, C, E) and non-fertilized (B, D, F) Juniperus communis L plants including male cone production (A, B) and the morphology of pollen
grains after 48 h (C, D) and 96 h (E, F) of in vitro incubation.
25.48 ± 0.117 μm, ; P(F=95.1172, DF=1) < 0.001, NF vs F) and sig-
nificantly wider equatorial diameter (23.933 ± 0.106 μm vs
22.475 ± 0.106 NF vs F) P(F=100.1623, DF=1) < 0.001) but did not affect
the P/E ratio (1.136 ± 0.004 vs 1.135 ± 0.005; P(F=0.2161,
DF=1) > 0.05). The exine thickness on the polar axis was affected by
genotype but not by nutrient availability. A significant interaction was
observed, however, between genotype and nutrient availability
(Table 4, Fig. 5). Based on the P/E ratio, more than 90 % of the J.
communis pollen grains were classified as elongated pollen grains
(prolate-spheroidal, subprolate and prolate) and rarely as spheroidal
(Table 5).
The proximal and distal outline of the pollen grains was irregular,
mostly circular, but also pentagonal or triangular with the presence of
obtuse apices. Nutrient availability did not appear to affect this ap-
pearance (Fig. 6). J. communis has pollen grains that open due to
swelling of the middle exine layer, which splits the exine almost com-
pletely into two hemispherical parts. The two halves of the burst grain
were often found more or less rolled into spindle shapes. Scanning
electron microscopy (SEM) revealed that the ornamentation of the
exine was granulate with adhering Ubisch-bodies having diameters
from 0.3 μm to 0.8 μm. Ubisch-bodies were distinctive, orbicular
6
Journal of Plant Physiology 248 (2020) 153156
granules of sporopollenins produced by the tapetum. Granules, are el-
lipsoidal or globular with diameters up to 0.5 μm. Granules and Ubisch-
bodies were both arranged irregularly on the pollen grain surface, being
dense in some places and loosely-arranged in others, and completely
absent on some pollen grains.
All of the examined pollen grains were ulcerated. Each grain had a
single ectoaperture – ulcus (rounded – circular or elliptical), situated at
the distal pole of the pollen grain. The ulcus area diameter was 6.30
(2.00–8.00) μm and their membranes were usually psilate. The ulcus
diameter ranged from 1.00 to 2.00 μm.
3.4. Carbon, nitrogen, and the carbon to nitrogen ratio in pollen grains
Application of fertilizer did not affect carbon content (F:
41.61 ± 0.3 vs NF: 38.16 ± 2.1 % dry mass, P(F=2.7888, DF=1) > 0.05);
however, fertilization did significantly increase nitrogen content (F:
1.79 ± 0.06 vs NF: 1.38 ± 0.08 % dry mass, P(F=17.0781, DF=1)
=0.0033) of pollen grains. Consequently, a significantly lower carbon
to nitrogen ratio was observed in pollen grains collected from F
(23.37 ± 0.91 dry mass) plants than in pollen grains from NF
(27.79 ± 0.8 % dry mass, P(F=13.0641, DF=1) =0.0068; Fig. 7).
E. Pers-Kamczyc, et al. Journal of Plant Physiology 248 (2020) 153156

Fig. 4. Features of viable pollen grains after 96 h of in vitro incubation relative to genotype and fertilization treatment (fertilized – dark green; non-fertilized – red). A
- length of the polar axis (μm); B - equatorial diameter (μm); C - pollen volume (103 μm3). Data are presented by quartiles as hollow rectangles with the median, and
outliers are indicated by points. Significant differences within a genotype are indicated by a star (P < 0.001).

Table 3 Table 4
A number of Juniperus communis L. pollen grains classified as viable based on ANOVA results for the length of the polar axis, equatorial diameter, and volume
the ratio of the length of the longitudal axis to the equatorial diameter in pollen of freshly collected Juniperus communis L. pollen grains after 96 h of in vitro
after 96 h of in vitro incubation. incubation with genotype and fertilization treatment as fixed effects and pollen
grains as a random effect.
Treatment Genotype Total ≤1.400 1.401 ≥1.800
number of do Parameter Source DF F-value P-value
pollen 1.800
longitudal axis treatment 1 2723.656 < .0001
non-fertilized (NF) 4 60 56 3 1 genotype 4 27.3823 < .0001
10 100 90 6 4 genotype*treatment 4 20.7112 < .0001
14 100 99 1 0 equatorial diameter treatment 1 10.8622 < .0001
20 73 45 14 14 genotype 4 8.3431 < .0001
24 100 92 5 3 genotype*treatment 4 10.8622 < .0001
All (%*) 433 382 (88.2) 29 22 (5.1) pollen volume treatment 1 959.1799 < .0001
(6.7) genotype 4 15.4219 < .0001
fertilized (F) 4 150 150 0 0 genotype*treatment 4 15.5256 < .0001
10 150 143 7 0
14 150 150 0 0
20 150 137 12 1
pioneer species, Juniperus communis L. These is in agreement with our
24 150 144 4 2
All (%*) 750 724 (96.5) 23 3 (0.4) previous studies done on the Taxus baccata L. plants (Pers-Kamczyc
(3.1) et al., 2019). Although those species are evolutionally adapt to different
Total (%*) 1183 1106 (93.5) 52 25 (2.1) nutritional environments as the natural population of J. communis occur
(4.4)
on poor whereas T. baccata on rich soil, we observed a similar pattern of
* Percentage of a total number of pollen grains represented within the
nutritional availability effect on the reproductive features of male
treatment group. plants. Male plants grown in a nutrient-rich environment, grow taller
and produce more male cones than male plants grown in a nutrient-
4. Discussion limited environment, however, they produced lower quality pollen
grains described by a lower in vitro germination potential and smaller
The current study demonstrated that long-term nutrient availability volume of the developing pollen grain.
affects several features of plant growth and reproduction in the woody In woody dioecious species, male plants are taller than female in-
dividuals, which is in contrast to the situation in herbaceous plants;

7
E. Pers-Kamczyc, et al. Journal of Plant Physiology 248 (2020) 153156

Fig. 5. Features of pollen grains relative to fertilization treatment (fertilized – dark green; non-fertilized – red) and genotype. A - length of the polar axis (P, μm); B -
the ratio of P to equatorial diameter (P/E); C - the ratio of exine thickness measured along the polar axis to the length of the polar axis (Exp/P). Data are presented by
quartiles as a hollow rectangle with the median and outliers indicated by points. Significant differences (P < 0.05) within a genotype are indicated by a star.

Table 5 production was observed in Alnus rubra under lower plant density in-
Pollen shape classes of the Juniperus communis L. pollen grains with respect to dicating that competition for resources (light, water, nutrients) among
fertilization treatment. The listed number represents the percentage whereas plants may have an impact on plant reproduction (Harrington and
the number in parentheses represents the actual number of pollen grains. Debell, 1995). It has to be pointed that we tried to minimize the impact
Treatment Percentage of pollen within shape class (n) of other environmental factors between F and NF plants by creating
stable and similar soil moisture, enabling plant growth by replanting to
spheroidal prolate- subprolate prolate All* larger pots when needed, light availability and meteorological condi-
(1.0) spheroidal (1.15–1.33) (1.34–2.0)
tions, therefore the fertilizer availability can be treated as the main
(1.01–1.14)
factor influencing plant respond. Thus we observed a significant rela-
fertilized 8.15 (22) 40.74 (110) 51.11 (138) − (-) 270 tion between F plant growth and the number of male cones produced
non-fertilized 6.3 (17) 40.0 (108) 52.6 (142) 1.11 (3) 270 per 1 cm of the shoot but not always in NF plants, it can be suggested
Total 7.22 (39) 40.37 (218) 51.86 (280) 0.55 (3) 540
that resource allocation in woody plants can be differentially dis-
tributed between growth, reproduction, and maintenance and is related
where male plants are typically smaller than female plants (Obeso, with nutrient availability. It has to be remember that wooden plants
and one-year plants differ in resource allocation and the main differ-
2002). Moreover, there is a relationship between limited mineral nu-
trient availability and plant growth (Sinclair, 1992), as well as with the ence is that trees still grow even after entering the breeding phase. Our
observations have revealed an important aspect of the relationship
observation that the height of plants has a direct impact on male fitness
in wind-pollinated species. Therefore taller plants can disperse pollen between environmental resources and the development of reproductive
structures and growth not only in female plants (Gruwez et al., 2013,
grains more effectively what is in line with our observations that male
cones were located within the upper part of the crown in NF in J. 2014; Messier and Puttonen, 1995) but also in male individuals. These
data are in agreement with previous studies on T. baccata where the use
communis plants. Although F plants were characterized by vigorous
growth and the increase in flowers production per branch length may of a fertilizer enhanced plant growth and photosynthetic rates, de-
creased oxidative stress and increased pollen production; however, the
easily compensate for the reduced pollen viability, however, production
of huge amount of produced pollen grains with decreased quality and pollen had a significantly lower rate of germination (Pers-Kamczyc
et al., 2019; Robakowski et al., 2018). The amount of available re-
observed retraction of pollination drop due to induction not only by
viable pollen grains but also by non-biological particles of appropriate sources and/or energy allocated to reproduction (number of male cones
production, quality of pollen grains), however, may be similar in both
size or by dead juniper pollen (Mugnaini et al., 2007), can cause an
increase of the empty seeds production. Notably, increased flower environments and needs to be further analyzed (Wenk and Falster,

8
E. Pers-Kamczyc, et al.
2015).
Soil composition can affect plant growth and reproduction and both,
macronutrients and micronutrient deficiency are known to affect the
reproductive development of plants (Pandey, 2010; Marschner, 1995).
The macronutrients such as nitrogen, sulfur, and phosphorus serve as
constituents of protein and nucleic acids, therefore, their functions are
quite evident, whereas the micronutrients may function as constituents
of organic structures, predominantly of enzyme molecules, and thus be
involved in the catalytic functions of the enzymes. Deficiency of mi-
cronutrient can lead to ultrastructural changes (Cu, Zn), decrease pollen
productivity and pollen grain size (Mn, Fe, Zn, Mo), impact on viability
and pollen tube growth (Mn, Cu, Zn, B) as well as lower starch content
in pollen grains (Mo, Cu). Although we did not analyzed the micro-
nutrient in soil used for planting, the obtained results allowed us to
predict that the minimal concentration was provided for all of them
thus NF plants produced an even better quality of pollen grains when
compared to F plants. On the other hand, the negative effect of higher
doses of both macro and micronutrients on the quality of pollen grains
produced by F plants cannot be neglected as high soil concentrations of
even the most essentials nutrients can be toxic to plants.
Plant exhibit different nutrient requirement, thus some of them
grown on rich and some on the poor soil stands. Moreover, soil nu-
trients play a crucial role in sustaining soil quality, crop or timber
production and the environment in general and it is closely related to
9
Journal of Plant Physiology 248 (2020) 153156
Fig. 6. SEM micrographs of Juniperus communis
L. pollen grain. A - Polar proximal view of a
pollen grain. Granules and Ubisch-bodies are
irregularly arranged on the surface of the
pollen grain; B - Granulate exine ornamenta-
tion with numerous granules and adhering
Ubisch-bodies; C - Exine surface with darker
and finer granules and a few brighter, large
Ubisch-bodies; D - Darker and finer granules
with very numerous, densely distributed,
bright Ubisch-bodies.
management practices used to agricultural land. Maximizing economic
profit drives farmers to increase the chemical fertilizer consumptions
and lead to an increase the nutrient contents in soil under orchard and
vegetable production. Intensive agriculture is known to emit more
nutrients, particularly N and P to the environment, which may result in
serious water quality impairment and higher nutrient depositions
within the globe (Chen et al., 2011). Element ratios (e.g. N:P, N:K,
N:Mg) are often taken as a measure to identify nutrient imbalances and
are used to assess the nutrition of plants. As indicated by long-term
monitoring, the forest ecosystems suffered from nitrogen saturation and
impaired nutrient balances. Although nutrient ratios should be in the
normal range from 10 to 17 for N:P ratios, 2.1–3.8 for N:K ratios and
10–21.5 for N:Mg ratios, an increase of N:P and N:K ratios over time
could be observed in many forest areas (Flückiger and Braun, 2003;
Franzaring et al., 2010). Further, C/N ratio can be used as an indicator
of soil fertility and degradability of organic material in the upper hor-
izons, and C/N ratios from 8 to 10 are indicative for good quality
“stabilized” humus (Schulte and Ruhiyat, 1998). In a forest, the highest
C/N ratios were observed in stands dominated by pine (25.3) and the
lowest (15.8) in mineral soil under the canopy of a broadleaved forest,
whereas in agriculture C/N ratio is mostly lower than in forest and
ranged from 13 to 22 (Wellbrock and Bolte, 2019). As it was expected,
plants grown in pots affected nutrient ratios in the soil in both treat-
ment group. Fertilizer application led to decrease of N:P ratio (4.6)
Fig. 7. Chemical composition of pollen grains.
A - percentage nitrogen relative to dry mass; B
- percentage nitrogen relative to dry mass; C -
carbon to nitrogen ratio (C:N ratio) in dry
pollen grains of Juniperus communis L. collected
from fertilized (dark green) and non-fertilized
(red) plants. Data are presented by quartiles as
hollow rectangles with a median and outliers
are indicated by points. Significant differences
(P < 0.05) between treatments are indicated
by a star.
E. Pers-Kamczyc, et al.
together with increase of N:Mg (4.7) and C:N (22.2) ratios, whereas
increase of all of those ratios (N:P - 11.3; N:Mg - 5.15; and C:N - 24.9)
was observed in soil of NF plants when compared to soil used for the
experiment (N:P - 6.7; N:Mg - 2.3 and C:N - 18). Lower germination
potential of pollen grains obtained from F plants grown in soil char-
acterized by a higher level of nitrogen but deficiency of P (low N:P
ratio) demonstrates that soil phosphorus availability could affect the
male function of a plant. Our results are in agreement with Lau and
Stephenson (1994) who showed that phosphorus levels were positively
correlated with pollen grain number and size in zucchini, but not with
Erel et al. (2016) who observed considerably and consistently reduction
pollen viability associated with P levels increase in olive trees (Olea
europaea L.). These suggest that both N and P nutrient availability and
their relations should be taken into consideration when the re-
productive potential of trees would be studied.
Under non-fertilized conditions (high N:P ratio but also high C:N
ratio), male plants of the pioneer woody species, J. communis, produced
pollen grains with better viability. This resembles the production of a
higher quality of seeds produced by female plants grown under stress
conditions (Herman and Sultan, 2011; Abeli et al., 2017). The greater
developmental potential of pollen grains produced by non-fertilized
plants may be connected to the level and/or type of accumulated en-
ergy present in the pollen grains. The energy in mature pollen grains is
mainly represented by the accumulation of sugars (glucose, fructose,
sucrose), which serve as energy for microsporogenesis pollen matura-
tion but also pollen tube growth. As an osmolyte, sugars protect pollen
from desiccation and other abiotic stresses (De Storme and Geelen,
2014). A decrease in carbohydrate reserves is often associated with
defective pollen development that may or may not lead to plant male
sterility (Nguyen et al., 2009; Sheoran and Saini, 1996; Pressman et al.,
2002; Jain et al., 2007). Although the use of fertilizer did not change
the carbon content in pollen grains within our study, an increase in
nitrogen content and a concomitant reduction in the C:N ratio was
observed in pollen grains of fertilized plants. Modification of the C:N
ratio was recently reported to be associated with a change in the
phosphorylation status of invertase and sucrose synthase and their ac-
tivity, subcellular localization, stability, and signal transduction activity
(Huarancca Reyes et al., 2016). Thus, plants that grown under high C
and limited N conditions can accumulate carbohydrates by the co-
ordinated regulation of these enzymes. This is in line with reports of
improved pollen production (Ladeau and Clark, 2006), quality (Koti
et al., 2005) and viability (Prasad et al., 2011; Schaeffer et al., 2013) by
elevated CO2. Collectively, the data suggest that the nutrient status of a
plant can affect not only whole plant growth and seed production
(Hanley et al., 2007) but can also alter the probability of the partici-
pation of pollen grains in a successful fertilization event. The earlier
initiation of pollen germination in non-fertilized plants can be viewed
as an adaptation to environmental conditions that increase the prob-
ability that pollen grains will successfully participate in fertilization,
with only fast germinating pollen grains winning this competition
(Gruwez et al., 2013).
Environmental conditions are one of the major driving forces of
evolution, resulting in the selection of individuals within a species that
are better adapted to the prevailing conditions, as well as the selection
of the best-adapted species within a community of species. An increase
in the availability of nitrogen and other nutrients due to anthropogenic
activity has significantly changed the soil environment in cropland
(Bouwman et al., 2017), mountainous areas (Bytnerowicz et al., 2019)
and forests, and will probably continue to impact soils in the foresee-
able future. Male reproductive success is tightly associated with plant
reproduction and survival plants, requiring proper pollen grain devel-
opment and proper pollen tube growth (Selinski and Scheibe, 2014;
Nguyen et al., 2010). Therefore, in addition to the impact of air pol-
lutants, and other abiotic stresses (such as heat and drought) on pollen
grain development, the effect of the nutrient status of an environment
should also be considered in future studies. We hope that this study
10
Journal of Plant Physiology 248 (2020) 153156
promotes further discussion on the impact of fertilizer deposition on
woody plant reproduction and species conservation.
Author contributions statement
Study design: EPK; acquisition of data: EPK, MR, DWP, ŻTW; ana-
lysis and interpretation of data: EPK, JK; manuscript preparation: EPK;
final review: EPK, JK, DWP, ŻTW, MR.
Funding information
This work was supported by the statutory research of the Institute of
Dendrology, Polish Academy of Sciences and by the Polish National
Science Centre [grant number 2015/19/N/NZ8/03850].
Declaration of Competing Interest
The authors state that there is no conflict of interest regarding the
publication of this article.
Acknowledgments
The authors would like to thank Kinga Nowak, Stanisław Zymon
and Grzegorz Iszkuło for their assistance in rooting shoots.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jplph.2020.153156.
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