Zajączkowska et al. BMC Plant Biology(2021) 21:136
https://doi.org/10.1186/s12870-021-02917-7

RESEARCH ARTICLE

Open Access

Spikelet movements, anther extrusion andpollen production in wheat cultivars with
contrasting tendencies to cleistogamy
Urszula Zajączkowska1* , Bożena Denisow2 , Barbara Łotocka3 , Alicja Dołkin-Lewko1
Monika Rakoczy-Trojanowska4

andAbstract
Background: Cleistogamic flowers are a main barrier in pollen dispersal for cross-pollination necessary in wheat
hybrid breeding. The aim of our study was to gain new knowledge on the biology of wheat flowering, in particularon the differences between the cleisto- and chasmogamic forms which has certainly cognitive significance, but it
can also be used in practice when seeking a female and male ideotypes for cross hybridization.
Results: We characterized the most significant features defining the flowering specificity in two wheat cultivars
with contrasting tendency to cleistogamy: Piko (chasmogamous) and Dacanto (cleistogamous). In the fieldobservations we assessed diurnal pattern of anther extrusion and anther extrusion capacity. For the first time weadapted the time lapse method for measuring kinetics of the spikelet movement and 3-D image correlation
technique for the non-invasive measurements of potential deformations of the spikelet lemmas. We found that thetwo cultivars differ in the potential of pollen dispersion for-cross-pollination and in the spikelet kinetics. We also
described some anatomical traits that can have potential functional role in floret opening. None of the cultivars
showed any symptoms of lemma surface deformation.
Conclusions: The cleistogamic and chasmogamic wheat cultivars differ significantly in the potential for pollen
dispersion for cross-pollination, which is mainly related to anther extrusion capacity. Although none of thesefeatures differentiated the cultivars clearly, we assume, based on spikelet kinetics and the lack of lemmas surface
deformation, that the water transport and turgor of cells is essential for the floret opening and anther extrusion inwheat. The search for parental ideotype should be supported by marker assisted selection, e.g. based of polymorphisms
in genes related to aquaporin biosynthesis.
Keywords: Cleistogamy, Dacanto, Flower opening, Lemma kinetics, Spikelet anatomy, Piko, Pollen dispersion, Triticum
aestivum, Wheat breeding* Correspondence: urszula_zajaczkowska@sggw.edu.pl
1
Department of Forest Botany, Institute of Forest Sciences, Warsaw Universityof Life Sciences – SGGW, 159 Nowoursynowska Street, 02-776 Warszawa,
PolandFull list of author information is available at the end of the article© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
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The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

Zajączkowska et al. BMC Plant Biology(2021) 21:136

Background
Cleistogamy is a sexual reproduction system, which isdefined as the formation of closed self-pollinatedflowers, has been found in many angiosperm taxa. Several plant species have mixed reproduction strategies
and proportion of cleistogamous and chasmogamous
flowers has been reported to be modified by environmental conditions like soil moisture, light intensity,
fertilization of soil and plant density [1]. Hexaploid common wheat (Triticum aestivum), which is one of themost important world food plant, is a self-pollinatedspecies with mostly cleistogamous florets adapted for
self-pollination and fertilization in closed florets [2–4].
Cleistogamy is the main disadvantage for hybrid breeding [5–8]. The male components of cross hybridization,
regardless of the mechanism on which it is based – natural (e.g. cms-Rf) or artificial (chemical emasculation) should have chasmogamous flowers that are open atanthesis which facilitate anther extrusion and, consequently, pollen shedding for cross-pollination [9–12].
Wheat florets are arranged in a spike inflorescence, typical for Poaceae (Gramineae). The spike main axis bear
spikelets. There are two glumes at the base of each
spikelet. Each spikelet consists of two-five florets. A
floret is perfect and consists of a lemma and palea, threestamens, one pistil, and two lodicules located between
the lemma and the ovary base [2, 13, 14]. The floret
opening at anthesis in cereals is thought to be due to thelodicules swelling [2, 15–18]. In wheat, the pair of lodicules located between the lemma and the ovary base expand rapidly at the time of anthesis, push away the rigid
lemma allowing anthers and stigma to emerge [16, 19].
The process induced by the turgid lodicules called as‘first opening’ is rather short and usually lasts for lessthan 30 min [2, 20, 21]. The ‘second opening’ of wheat
floret was recently described and the authors suggested
that unfertilized ovary increase in radial dimensions andgenerates lateral push of the rigid lemma and palea [22].
However, it is still not clear whether during the ‘first
opening’ the lemma undergoes deformations, which
could increase the efficiency of floret opening and antherextrusion. Also, the kinetics of the floret opening has so
far been poorly studied. It is known that plants have
evolved various mechanisms of movement of certain morphological structures that are important in reproduction
processes, such as floret opening, pollen dispersion or seed
distribution [23, 24]. In many cases of ferns and flowering
plants, the energy that activates the onset of movement isdue to the uneven cell wall thickness or the anisotropy ofa specific structure that is deformed in the evaporation
process [25, 26]. To date, however, there has been no experimental data on the possibility of lemma deformation
during rapid expansion of swelling lodicules and ovarybase that occur at the time of the floret opening at

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anthesis in cereals. There is also no data on the kinetics ofthe flower opening process. Pollen production is also
among the traits reported to be important in hybrid wheat
breeding [4, 10, 27, 28]. However, most cultivated formsof common wheat produce low amounts of pollen, whichis indicated as a significant limitation in wheat hybrid
breeding programs [7, 11, 29].
In this study, we surveyed the kinetics and the micromorphological and anatomical structures of spikelets in
two wheat cultivars with contrasting tendency to cleistogamy. Two winter wheat cultivars - Piko (chasmogamous) and Dacanto (cleistogamous) were used. For the
first time we applied the time lapse technique for measuring the kinetics of spikelets movements and the noninvasive measurements of potential deformations of the
spikelet lemmas during anthesis. Moreover, we analyzed
if the structural features and changes in the pre-anthesis
and post-anthesis spikelets can determine the anther extrusion ability. In addition, we tried to assess to what degree the floral biology (diurnal pattern of antherextrusion, the efficiency for anther extrusion, the pollen
production and pollen dispersion capability) differ between two contrasting cultivars.

Results
Anther extrusion

The diurnal pattern of anther extrusion was similar inboth genotypes grown in the field conditions (Fig. 1,
Additional file 1: Table S1). The peak of anther extrusion
was observed in early morning hours (between 5.00 h and8.00 h). A total lack of anther extrusion was characteristic
for mid-day hours. Towards the end of the day, during
evening hours, a small proportion of anthers have been
extruded (approximately 3–5% of the daily amount in
Dacanto and 10% in Piko). The number of anthers per
spike was statistically non-significant between cultivars(Mann-Whitney U test: Z = − 0.65, P = 0.516; Table 1,
Additional file 1: Tables S3-S4). Considerable year-to-year
differences were documented for the number of anthers
developed in spikes (for cv. Dacanto Z = 3.74, P = 0.000;
for cv. Piko Z = 3.71 P = 0.000) and lower values were
established in 2019 compared to 2018. The proportion of
extruded anthers differed considerably between the cultivars (Z = − 5.39, P = 0.000; Fig. 2, Additional file 1: Table
S2. Approximately two-fold higher proportion of extruded
anthers was noted for the cv. Piko compared to the cv.
Dacanto (42.2% ± 1.9 vs. 18.6% ± 3.0, respectively). The
year effect was found within cultivars for the ability to anther extrusion (for cv. Dacanto Z = 1.96, P = 0.049; for cv.
Piko Z = − 2.88, P = 0.004).
Pollen production

The number of pollen grains produced per anther differedsignificantly between the cultivars (t = 2.00, df = 406,

Zajączkowska et al. BMC Plant Biology(2021) 21:136

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Fig. 1 Diurnal pattern of anther extrusion in two wheat cultivars. The extrusion is expressed as the number of anthers extruded at one hourintervals (GMT + 2 h) in relation of the total number of anthers extruded per day (average from 2018 to 2019). Whiskers indicate ± SD;
hours GMT + 2 h.

P = 0.000; Table 1) and was higher in the cv. Dacanto.
A year-to-year disparity was found in the amount ofpollen in the anthers (Dacanto t = −11.79, df = 202,
P = 0.000; Piko t = −13.43, df = 202, P = 0.000). In
both cultivars, a statistically higher amount of pollen
was produced in 2019 (ca. 2-fold higher than in2018). The total pollen production per spike wassimilar in both cultivars (t = 1.73, df = 78, P = 0.087;
Fig. 3A, Additional file 1: Table S4). However, a year
effect on the number of pollen grains produced in

spikes was found (Dacanto t = − 7.58, df = 38, P =0.000; Piko t = − 5.00, df = 38, P = 0.004). The cultivars differed significantly in the number of pollen
grains released for cross-pollination (t = − 21.15, df =78, P = 0.000), and the cv. Piko exhibited a higher
amount of dispersed pollen (Fig. 3B, Additional file
1: Table S4). The number of pollen grains available
for cross-pollination differed between the seasons(Dacanto t = − 4.24, df = 38, P = 0.000; Piko t = − 7.58,
df = 38, P = 0.000).

Table 1 Components of pollen production in two wheat cultivars across years of study
Cultivar

Dacanto

Piko

Years

Number of flowers per
spikeletNumber of spikelets
per spike

Number of flowers
per spike

Number of anthers
per spike

Number of pollen grains
per anther

mean

±SD

mean

±SD

mean

±SD

mean

±SD

mean

±SD

2018

4.5b

1.7

18.8a

1.7

84.4b

7.8

253.1b23.0

923.0a

418.4

2019

2.7a

1.2

19.3a

1.4

53.0a

3.8

159.5a11.7

1850.5b

583.5

Mean

3.6A

1.8

19.1A

1.6

68.7A

16.8

206.3A

50.8

1386.8B

640.3

2018

4.0b

0.9

21.5a

1,0

86.0b

4.0

258.0b

12.0

885.3a

379.5

2019

2.8a

1.2

20.3a

1.3

56.7a

3.7

169.0a11.1

1600.6b

282.1

Mean

3.4A

1.2

20.9A

1.3

71.35A

15.5

213.5A

46.6

1243.0A

482.3

Means followed by the same small letter are not significantly different between years and that followed by the same capital letters between cultivars at α =0.05

Zajączkowska et al. BMC Plant Biology(2021) 21:136

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Fig. 2 Proportion of extruded anthers in two wheat cultivars observed in 2018 and 2019. Untransformed data are presented. Whiskers indicate ±SD. Means followed by the same small letter are significantly different between the years and those followed by the same capital letter between the cultivars at P < 0.05, according to the Mann-Whitney’s U test

Micromorphology and anatomy of spikelets

The spikelets of both cultivars consisted of two glumes
and 4–6 florets, including 3 fertile ones in cv. Dacanto(Fig. 4a, b), or 4–5 florets, including 2–3 fertile ones in
cv. Piko (Fig. 4c, d) set distichously and alternately on
the rachilla, with antherns positioned higher within theflorets in Piko. Characteristically, in every sectioned
post-anthesis spikelet of the cv. Dacanto, all florets contained some non-extruded anther(s) (Fig. 4b). In the
spikelets of the cv. Piko examined post anthesis, all anthers were absent at least in the basal two florets (Fig.
4d). In the same spikelets, the upper 1 or 2 florets
retained stamens within the lemma and palea, and theirfilaments were slightly or non-elongated. The glumes
were boat-shaped, with a massive base in both cultivars(Fig. 5a-c). In this area, the glume abaxial epidermis inboth cultivars was smooth and composed of elongated
non-turgid pavement cells. In this domain, a minute andshallow transversal groove was discernible before anthesis (Fig. 5a, c). In spikelets fixed post-anthesis, wideningof the grooves within the bases of glumes was visible(Fig. 5b). In both cultivars, the two basal florets formed
the largest pistils with a well-formed gametophyte. The
third flower was smaller but properly built as well, andthe distal 2–3 florets were minute, apparently aborted at
the early developmental stage (Fig. 4a, b). Each floret

Fig. 3 Total pollen production per spike (A) and pollen dispersion capacity per spike (B). Results of the experiments with two wheat cultivars
performed in 2018 and 2019. Whiskers indicate ± SD. Means followed by the same small letter are not significantly different between the years
and those followed by the same capital letter - between the cultivars at P < 0.05, according to t-testZajączkowska et al. BMC Plant Biology(2021) 21:136

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Fig. 4 General anatomy of the spikelet in cv. Dacanto (a, b) and cv. Piko (c, d). Spikelets before anthesis (a, c) and after anther extrusion (b, d).
Arrows, g- glume, le- lemma, p- palea, lo-lodicule, an-anthers, nea- non-extruded anthers, gr - grooves. Scale bars = 1500 μm

was supported by a lemma and palea. The lemmas wereboat-shaped, like the glumes, but wider and longer (Fig.
5d-f). Like in the glumes, a groove was discernible atpre-anthesis also in the lemma bases within a domain of
non-turgid pavement cells of abaxial epidermis. The two
lodicules were both facing lemma. Due to such location,in the dorsoventral longitudinal sections (Fig. 4a-d) onlya single lodicule was visible in every fertile floret. At preanthesis, lodicules were turgid, with basal half convex
and the apical one rather flat and covered with long,thick-walled and spiked trichomes (Fig. 6a, b). The

epidermis of lodicules was composed exclusively of rather short pavement cells, with straight anticlinal walls
and characteristic surface microsculpture (Fig. 6c). Postanthesis, the lodicules were turgor-less and crushed(Fig. 4b, d).
Lemma kinetics

The analysis of time-lapse movies shows that lemma
opening period lasted on average about 5 min, and forindividuals it ranged from 2 to 15 min (Fig. 7, Additional
file 1: Table 5). The maximum lemma opening was onZajączkowska et al. BMC Plant Biology(2021) 21:136

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Fig. 5 Micromorphology of glumes (a-c) and lemmas (d-f). Spikelets of cv. Dacanto (a, b, d, e) and cv. Piko (c, f) before anthesis (a, c, d, f) andafter anther extrusion (b, e). Small grooves (gr) are visible in the basal parts of the glumes and lemmas already before anthesis, ra – rachilla. Scalebars in (a), (b), (d), (e) = 2000 μm, bar in (c) = 500 μm and bar in (f) = 1000 μm

average about 1.5 min from the beginning of the process
in the 2018 experiment and about 1.5–2 min in 2019.
During this first period of flowering, the highest speed oflemmas displacement was also noted. Comparison oflemmas displacement kinetics shows that wider lemmas
opening occurs in the Piko cultivar. In 2018 this was observed for almost the entire lemma opening period andin 2019 it was revealed in the period after the maximum
opening time, when the degree of lemma opening was
gradually decreasing. Additional movie files illustrate theoriginal time-lapse movies recorded during the lemmas
opening in two cultivars (see Additional files 2 and 3).
Potential deformations of lemmas during the flowering
process was measured using a 3-D image correlation
method along two rectangular X-Y axes (Figs. 8, 9, 10, 11).
The longer Y axis was parallel to the lemma venation pattern and the X axis was oriented perpendicularly. The
color maps presented in Figs. 8 c,d, 9, 10 and 11c,d show
the Z values of the points on the whole lemma surface atthe beginning of flowering process before the lemmaZajączkowska et al. BMC Plant Biology(2021) 21:136

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Fig. 6 Micromorphology of cv. Dacanto spikelet before anthesis: palea (cut apically) with a floret attached (a). Lodicules of cv. Piko before
anthesis (b); a criss-cross pattern of the lodicule cell surface in cv. Dacanto (c); pa – palea, lo – lodicules, an – anthers. Scale bar in (a) = 2000 μm,
bar in (b) = 1000 μm, and bars in (c) =100 μm

Fig. 7 Spatial displacement (a, b) and speed of movement (c, d) of the opening lemmas. Results of two experiments during flowering of Piko
and Dacanto wheat cultivars. Carried out in 2018 (a, c) and 2019 (b, d). Data obtained from the time-lapse imaging transformed in the
Tracker programZajączkowska et al. BMC Plant Biology(2021) 21:136

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Fig. 8 Results of the lemma surface deformation test using the 3D image correlation method. Diagrams (a, b) and maps (c, d) of the lemma
surface displacement. The graphs presented in (a) and (b) show the Z values of the points on the lemma surface along X and Y axes,
respectively, at the beginning of the flowering process before the lemma opening (c) and after the first phase of opening (d). Piko cultivar,
lemma No. 1

opening and at the initial phase after opening. The end of
the measurement period was limited by the depth of fieldof the lens of the stereoscopic microscope and occurred
when during the lemma displacement the surface of thelemma became out of focus of the lens. A detailed analysisof the potential lemma surface deformation can be doneon the basis of the data shown in the graphs in Figs. 8a,b,
9, 10 and 11a,b where the Z values of the points along X
and Y axes are presented. The comparison of the patternof the graphs for the same lemma at the beginning andthe end of measurement indicate that during initiation of
the lemma opening there is a spatial position shift ofwhole lemma surface without any noticeable displacement
between the reference points distributed within the lemma
surface which suggests that there are no symptoms of thelemma surface shape deformation.
An additional movie file illustrates, as an example, thechange in the position of the lemma during flowering,recorded from two cameras connected to a stereoscopic

microscope, which were the basis for determining thepotential lemma deformation using 3-D image correlation method (see Additional file 4).

Discussion
In this study we presented a detailed comparative characteristics of flowering biology of two contrasting winterwheat cultivars – cleisto- and chasmogamic, which comprised morpho-anatomical study of spikelet structure including their kinetics, anther extrusion and pollen
production. Such a complex study employing different
analytical approaches was performed in wheat for the
first time. Our anatomical observations revealed that inevery sectioned post-anthesis spikelet of cv. Dacanto, allflorets contained some non-extruded anther(s), while in
cv. Piko spikelets nearly all anthers were absent at leastin the basal two florets. These findings correspond to
field observations that exhibited two-fold higher proportion of anthers extruded from the Piko spikes.

Zajączkowska et al. BMC Plant Biology(2021) 21:136

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Fig. 9 Results of the lemma surface deformation test using the 3D image correlation method. Diagrams (a, b) and maps (c, d) of the lemma
surface displacement. The graphs presented in (a) and (b) show the Z values of the points on the lemma surface along X and Y axes,
respectively, at the beginning of the flowering process before the lemma opening (c) and after the first phase of opening (d). Piko cultivar,
lemma No. 2

Microscopic analysis showed that spikelets anatomy of
the two cultivars, in general is similar to that reported inclassical papers concerning floret anatomy of the breadwheat genotypes [13, 30]. However, certain details of the
spikelet micromorphology were not reported earlier.
This novel trait that we consider as having potentiallyfunctional role in floret opening concerns formation ofgrooves within the glume and lemma bases and deepening of the grooves during anthesis. Although the grooves
seem more pronounced (wider) in cv. Piko, which is abetter extruder of anthers, from the structural point ofview both cultivars can potentially extrude anthers withsimilar efficiency. As it is seemingly not the case, thereasons should be sought in the functioning of the spikelet elements, especially in the water transport and turgor
control [19]. This suggestion is confirmed by our resultsof measuring the kinetics of lemmas openings in which
for the first time the time lapse technique was applied.
The measurements showed that the average length of

this period is about 6–7 min, and for individual spikelets
it is in the range of 2–15 min. The data are consistent
with the reports of earlier authors indicating that the actual opening of the floret in most grasses is caused by
the lodicules becoming suddenly swollen at the base, so
the lemma is levered outwards [22]. The difference between the two cultivars is, however, small, but the kinetics parameters seem to be more favorable for antherextrusion and to the cross- pollination processes for the
cv. Piko.
The diurnal pattern of anther extrusion can also support the importance of hydration of spikelet elements
for the anther extrusion process. The peak of anther release was observed in early morning hours, when theplant cells are usually well hydrated [24]. A fundamentalrole of the water balance, the turgor and its regulation inflower opening have been confirmed in many studies[31]. The key role of water transport and turgor of cells
in lodicules is also confirmed by the results of ourZajączkowska et al. BMC Plant Biology(2021) 21:136

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Fig. 10 Results of the lemma surface deformation test using the 3D image correlation method. Diagrams (a, b) and maps (c, d) of the lemma
surface displacement. The graphs presented in (a) and (b) show the Z values of the points on the lemma surface along X and Y axes,
respectively, at the beginning of the flowering process before the lemma opening (c) and after the first phase of opening (d). Dacanto cultivar,
lemma No.1

studies on the possible deformations of lemmas in theprocess of florets opening using the non-invasive 3-D
image correlation technique, which was for the first time
applied to plant biological material. Due to the limited
range of depth of field of camera lenses, these studies
were limited to the initial lemmas opening period only,
however this period seems to be crucial for the process
and the results obtained can be considered to be reliable
for the whole process. The clearly demonstrated for the
first time the absence of any deformation of the lemma
surface indicates that in the process of wheat florets
opening, the potential mechanical effects associated withlemmas strains do not play a significant role, and themechanisms related to cell turgor and water transport tolodicules should be the crucial factor. These results may
suggest that in future studies on mechanisms associatedwith wheat flowering biology, more attention should befocused on aquaporins. Genes encoding aquaporins inwheat have been already recognized, e.g. Pandey et al.

[32] indicated 13 wheat aquaporin genes, including
genes coding for tonoplast aquaporins. Despite transcripts of two genes (PIP1–2 and TIP2–1, encoding theplasma membrane intrinsic proteins and tonoplast intrinsic proteins, respectively) were detected at a relatively
high level in florets and inflorescences, the authors, however did not indicate the clear role of any of these genes
in the processes related to wheat floret opening.
Our survey has shown that the wheat cultivars with
contrasting tendency to cleistogamy differ in the potential for pollen dispersion. The cultivar Dacanto withhigher pollen production per anther and similar pollen
production per spike compared to Piko, has displayed
lower amount of dispersed pollen (i.e. the number ofpollen grains available for cross-pollination), therefore
we conclude that the anther extrusion capability is particularly important for the provision of air-blown pollen
for cross-pollination in wheat. Several studies have reported that the ability for anther presentation outsideZajączkowska et al. BMC Plant Biology(2021) 21:136

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Fig. 11 Results of the lemma surface deformation test using the 3D image correlation method. Diagrams (a, b) and maps (c, d) of the lemma
surface displacement. The graphs presented in (a) and (b) show the Z values of the points on the lemma surface along X and Y axes,
respectively, at the beginning of the flowering process before the lemma opening (c) and after the first phase of opening (d). Dacanto cultivar,
lemma No.2

the floret is essential for pollen dispersal in wheat [4, 7,
10, 29]. However, the number of pollen grains production per anther can not be ignored, as indicated by deVries [27], who pointed out that pollen donor capacity is
positively related to the total number of produced pollen
grains. In our study, the number of pollen grains established per anther (averaged 1243–1386) is within that reported in literature for wheat cultivars, e.g. 975 to 2773
[33]. However, our values recognized for cv. Piko werelower than that reported for this cultivar by de Vries[27], who documented 2672–2933 of pollen grains peranther. These disparity can be understood as pollen production even in this same species/cultivars is sensitive toexternal factors and can vary greatly spatially and temporally [34, 35]. The differences could be the results of
the human activities, i.e. fertilization, irrigation or their
combination. It was reported in a number of studies that
decrease in nutrient supply may result in the reductionof pollen grains formed per anther [36, 37]. Likewise themethod used for the assessment of pollen grains number
can impact on the records.
Similarly, the temporal (=between years) differences
established in pollen production (per anthers, per spikes)
in both cultivars can be associated, at least partially, withspecific environmental factors occurring during pollen
development. Every stages of pollen development, i.e.
the differentiation of sporogenous cells and meiosis (microsporogenesis) as well as post-meiotic development of
microspores are recognized as the most stress-sensitive
period in plant reproduction [36]. The role of drought
stress, moderate water–deficit stress, suboptimal
temperature and/or oxidative stresses on pollen production has been demonstrated across wheat cultivars [3,38]. According to Monneveux et al. [39], pollen development in wheat is critically affected by imbalance between
photosynthesis and translocation use of photoassimilates(mainly starch). Year-to-year disparity in the pollen production calculated for spikes can be associated with bothZajączkowska et al. BMC Plant Biology(2021) 21:136

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the differences in the pollen production per anther andthe variable number of anthers developed in inflorescences across growing seasons. Such relationships havebeen previously documented in many plants [34, 35].

Conclusions
The aim of our study was to gain new knowledge on the
biology of wheat flowering, in particular on the differences between the cleisto- and chasmogamic forms
which has certainly cognitive significance, but it can also
be used in practice when seeking a female and maleideotypes for cross hybridization. In conclusion, thecleistogamic and chasmogamic wheat cultivars differ significantly in the potential for pollen dispersion for crosspollination, which is mainly related to anther extrusion
capacity. We described several novel anatomical traits
that can have potential functional role in floret opening.
Although none of these features differentiated the cultivars clearly, we assume, based on spikelet kinetics andthe lack of lemmas surface deformation, that the watertransport and turgor of cells is essential for the floret
opening and anther extrusion in wheat. The search forparental ideotype should be supported by marker
assisted selection, e.g. based of polymorphisms in genes
related to aquaporin biosynthesis.

collected for anatomical studies. No fertilization was applied. Standard procedures for winter wheat plant protection were applied. Weeds were removed by hand, if
necessary.

Field study

The field observations were conducted to assess (i) diurnal pattern of anther extrusion and (ii) anther extrusion
capacity. The diurnal pattern of anther extrusion was exhibited based on methods described by Langer et al. [4]and was recorded between 4.00 h (GMT + 2 h) and
20.00 h, in one-hour intervals, using n = 10 spikes percultivar. Diurnal pattern of anther extrusion was
expressed as the percentage of newly extruded anthers
in relation to the total number of anthers extruded during a day. These observations were conducted in full
flowering phase of each cultivar. The anther extrusion
capacity was determined on the basis of the proportionof anthers released out of florets in relation to all anthers
developed per spike. To establish this, we covered spikes(n = 20 per cultivar per year) with isolators (mesh size <1 mm) before flowering and counted the number of released anthers as well as the number of all developed anthers. Isolators were kept until the end of the spike
flowering period.

Materials and methods
Plant material

Two winter wheat cultivars - Piko (chasmogamous, bredby Nordsaat Saatzucht GmbH, Germany), and Dacanto(cleistogamous, bred by KWS Polska Sp. z o.o., Poland)
were used.
For the field study on the floral biology the experimental plants were grown under field conditions at the University of Life Sciences (ULS) in Lublin, Poland (51° 14′
N, 22° 34′ E, altitude 238 m a.s.l) in 2018–2019. The soil
is podzolic, developed from loess. The soil reaction pH
in H2O ranged 5.8–6.8. The average concetrations of
nutriens in soil at depth of 0–20 cm is presented inTable 2. The plants used for anatomical study weregrown in the collection of agricultural crop plants (Department of Agronomy) at the Warsaw University of Life
Sciences (WULS) campus in Ursynów, Warsaw (52° 16′
N, 21° 05′ E) in 2018. Plants were cultivated in podzolic
loam soil at pH 6–7. Caryopses of each cultivar weresown in September (Warsaw) or October (Lublin), inplots (n = 5) 4.0 m long. From each plot 10 plants wereTable 2 Content of nutrient elements in the arable layer (0–20cm) of the soil in the experimental plot
Nutrient (mg/1 dm3 of soil)
N03−

P

K

Ca

Mg

Cl−

9.3

44.0

27.5

473.7

111.5

13.1

Pollen dispersed for cross-pollination

Pollen production was evaluated as (i) total pollen production, i.e. the number of pollen grains produced per
spike, and (ii) capability for pollen dispersion, i.e. thenumber of pollen grains dispersed outside the spike. The
capability for pollen dispersion was calculated based on
total pollen production and the anther extrusion capacity (for definition see Field study subsection).
The number of pollen grains produced per spike wascalculated based on the number of anthers developed
per spike (n = 20 spikes per cultivar per year, collectedfrom different plants) and the number of pollen grains
produced per anther. The number of pollen grains peranther was established using electronic particle counter(Multisizer 4e, Beckman Coulter Counter, Inc., USA)calibrated according to the producer recommendations.
The pollen grains were counted in mature anthers collected just before opening. Anthers were dissected fromflorets randomly selected form different spikes. In total,
408 anthers were sampled (n = 204 for Dacanto and forPiko). Anthers were placed in Eppendorf Tubes 2.0 ml.
Subsequently, pollen was removed from anthers using
ether (2 times × 1 ml) and 70% ethanol (5–7 times × 1–2ml). Finally, ethanol was evaporated, the electrolyte wasadded and counting of pollen grains was made automatically using an electronic sensor.

Zajączkowska et al. BMC Plant Biology(2021) 21:136

Micromorphology and anatomical study

Entire inflorescences (spikes) were taken for microscopic
examination, in two terms (i) before (= pre-anthesis) and(ii) after opening (= post-anthesis) the lemmas. The inflorescences were fixed in pure methanol overnight [40],and next rinsed a few times (and stored) in absoluteethanol. During rinsing, sterile basal spikelets and thebasal fertile spikelet were removed, and the next 3–4 fertile spikelets were later excised for light microscopy(LM), and the next ones for scanning electron microscopy (SEM).
Spikelets intended for LM were stained whole prior toembedding with the safranin-Astra blue (S-AB; Maácz
and Vágás [41]). The most uniform staining wasachieved in rehydrated spikelets, with the staining mixture slowly instilled into the spikelet using a syringe.
Stained spikelets were rinsed a few times with 70% ethanol and embedded for vibratome sectioning. Since theblade pulled the spikelets from the 7% agarose blocks,
the specimens were embedded in Glycid Ether 100
Epoxy Resin (SERVA) grade very soft (without the hardener, prepared according to the manufacturer’s formula),
as follows (i) dehydration in graded ethanol series, witheach stage prolonged to 30 min and overnight final dehydration in absolute ethanol, (ii) propylene oxide, (iii)saturation in resin-propylene oxide mixtures with eachstage prolonged until sinking of most specimens, (iv)overnight saturation with pure resin and placement ofsamples in flat embedding molds for sectioning at theventral-dorsal longitudinal plane, (v) preliminary
polymerization at room temperature overnight to facilitate evaporation of propylene oxide residues (this stage
was tested to be necessary), and final polymerization at
60 °C for 24 h. Blocks were attached to the microtome
holder with commercial two-component epoxy glue andserial-sectioned at 70 μm using VT1000 S vibratome(Leica). Sections were unfolded at 70 °C, mounted in a
glycerol and examined using Provis AX70 (Olympus
Corporation) light microscope equipped with a digital
camera UC90.
Spikelets for SEM were trimmed to expose the basal
part of a glume or lemma/palea, or the adaxial side oflemma or palea. Next the specimens were critical-point
dried using CPD 7501 dryer (Polaron), attached to theholders and gold sputtered in sputter coater JFC-1300(JEOL). They were examined in the Analytical Centre of
WULS using Quanta 200 (FEI Company) environmental
scanning electron microscope operating at 20 kV underhigh vacuum. The images were saved as tiff files at
2048 × 1886 pixels resolution.
Lemma kinetics

The tests were performed in two replicate experiments
done on May 15–31, 2018 and May 20–June 10, 2019.

Page 13 of 15

Just before the start of flowering, the plants were cut off
at the base of the stem, submerged with the basal endsof the stems in a container of water and placed in aplant grow box, under continuous light source (HPS
Phytolite 600 W lamp, photon flux1045 lmol m-2 s-1, luminous flux 100 klm) at 21 °C. Measurements of thelemmas opening kinetics were based on films recorded
using the time-lapse movie method (Ricoh GR cameras,
Japan) and analyzed in the Tracker program (https://www.cabrillo.edu/dbrown/tracker/) based on the Open
Source Physics (OSP) Java framework. Fifteen spikelets
of each of the two cultivars were used in the observations of lemmas opening kinetic in each experiment.
Lemmas potential deformations during the flowering
process were examined using a 3D image correlation
method based on two cameras in a stereoscopic setup
connected to a stereoscopic microscope. Such a technique gives the opportunity to perform measurements of
3D deformation and strain of specimens. Each camera
provides a 2D view and software, through a correlation
algorithm, forms a 3D image combining both camera
views from different angles [42]. Present measurementsof the wheat lemma deformation were made using theDantec Q-400 system based on the Istra 4D softwaremodule with 5 MPix optics connected to the Leica
M125 stereoscopic microscope. Activated carbon particles were sprayed on the lemma to create a stochastic
pattern to the surface. The changes of this pattern due
to deformation or movement of the object are recordedby the cameras. These images were automatically analyzed with special high accuracy correlation algorithms.
As the result a set of data was generated containing thestart contour of the object at beginning of the measurement and the three dimensional displacement vector ofeach object point due to the object deformation. In
order to prevent possible other movements, not related
to the lemma opening process each spikelet was inserted
in a floristic sponge. The deformation measurements
were made in 2019 on two replicate lemmas of each of
the two cultivars.

Statistical analyses

The data are presented as means with standard deviations (SD). Statistical analyses was based on a t-test forindependent groups used to compare the means of analyzed pollen data between cultivars and between years
within cultivars. The pair-wise U Mann-Whitney’s nonparametric test was applied for the number of anthers
per spike and the proportion of extruded anthers as thedata did not fit t-test assumptions. The level of statisticalsignificance for all the analyses was P = 0.05. The analyses were performed using Statistica ver. 13.3 software(Statsoft Polska, Kraków, Poland).

Zajączkowska et al. BMC Plant Biology(2021) 21:136

Abbreviations
LM: Light microscopy; SEM: Scanning electron microscopy; ULS: University ofLife Sciences in Lublin; WULS: Warsaw University of Life Sciences - SGGW

Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12870-021-02917-7.
Additional file 1: Table S1. Diurnal pattern of anther extrusion in two
wheat cultivars. Original data from two experiments in 2018 and 2019 aresummarized in Fig. 1. Table S2. Proportion of extruded anthers in two
wheat cultivars. Original data from two experiments in 2018 and 2019 aresummarized in Fig. 2. Table S3. Number of pollen grains per anther in
two wheat cultivars. Original data from two experiments in 2018 and2019 are summarized in Table 1. Table S3. Number of pollen grains peranther in two wheat cultivars. Original data from two experiments in2018 and 2019 are summarized in Table 1. Table S4. Components ofpollen production (columns C, D,) and the number of pollen grains (E-H)
in two wheat cultivars. Original data from two experiments in 2018 and2019., Data from columns C, D and columns E-H are summarized in Table
1 and Fig. 3, respectively. Table S5. Spatial displacement (mm) of the
opening lemmas. Results of two experiments during flowering ofDacanto and Piko wheat cultivars carried in 2018 and 2019. Data of 15
replicate lemmas obtained from the time-lapse imaging. The data aresummarized in Fig. 7. Table S6. Speed of spatial displacement (mm/min)of the opening lemmas. Results of two experiments during flowering ofDacanto and Piko wheat cultivars carried in 2018 and 2019. Data of 15
replicate lemmas obtained from the time-lapse imaging. The data aresummarized in Fig. 7.
Additional file 2: Movie 1. Example of the original time-lapse movie
recorded during the lemmas opening in cv. Dacanto.
Additional file 3: Movie 2. Example of the original time-lapse movie
recorded during the lemmas opening in cv. Piko.
Additional file 4: Movie 3. Movie illustrating, the change in theposition of the lemma during flowering. Original records from two
cameras connected to a stereoscopic microscope, which were the basis
for determining the potential lemma deformation using 3-D image
correlation method.

Acknowledgements
We are grateful to Grażyna Szymczak PhD for providing the area for theexperimental plots in the UMCS Botanical Garden in Lublin. We thankKrzysztof Pągowski (Department of Agronomy, WULS - SGGW) for expert
technical assistance in growing plants, Agnieszka Ostrowska (AnalyticalCentre, WULS- SGGW) - SEM, Monika Strzałkowska-Abramek, Ernest Stawiarz,
Jacek Jachuła, and Magdalena Kamińska (ULS Lublin) for assistance inflowering observations and pollen production assessment. Our thanks also
go to Agnieszka Kubik-Komar (ULS Lublin) for statistical assistance. Special
thanks are due to an unknown reviewers for substantial improvements ofthe manuscript.
Authors’ contributions
BD performed the anther extrusion and pollen production experiment, BŁ
and AD-L made the microscopic studies, UZ and AD-L conducted the observations of lemma opening kinetics and the lemma deformation experiment,
BD, BŁ, UZ, and MR-T participated in writing the manuscript, UZ prepared
the final edition of the manuscript. MR-T supervised the research. All authors
have read and approved the manuscript.
Funding
The research was financed by BIOSTRATEG3/343665/6/NCBR/2017 grant.
Availability of data and materials
The materials used during the current study are included in the
Supplementary Information: Additional file 1. Tables S1-S6.

Page 14 of 15

Declarations
Ethics approval and consent to participate
Not applicable.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Forest Botany, Institute of Forest Sciences, Warsaw Universityof Life Sciences – SGGW, 159 Nowoursynowska Street, 02-776 Warszawa,
Poland. 2Department of Botany and Plant Physiology, University of Life
Sciences in Lublin, 15 Akademicka Street, 20-950 Lublin, Poland. 3Departmentof Botany, Institute of Biology, Warsaw University of Life Sciences – SGGW,
159 Nowoursynowska Street, 02-776 Warszawa, Poland. 4Department of Plant
Genetics, Breeding and Biotechnology, Institute of Biology, Warsaw Universityof Life Sciences –SGGW, 159 Nowoursynowska Street, 02-776 Warszawa,
Poland.
Received: 3 December 2020 Accepted: 22 February 2021

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