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JXB Advance Access originally published online on March 10, 2006
Journal of Experimental Botany 2006 57(6):1315-1321; doi:10.1093/jxb/erj106
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© The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

RESEARCH PAPER

Developmental regulation of K accumulation in pollen, anthers, and papillae: are anther dehiscence, papillae hydration, and pollen swelling leading to pollination and fertilization in barley (Hordeum vulgare L.) regulated by changes in K concentration?

S. Rehman and S. J. Yun*

Division of Biological Resources Science, College of Agriculture and Life Science, Chonbuk National University, Jeonju 561-756, Republic of Korea

* To whom correspondence should be addressed. E-mail: sjyun{at}chonbuk.ac.kr

Received 31 May 2005; Accepted 22 December 2005


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The presence of potassium (K) in pollen, anthers and papillae from barley (Hordeum vulgare L.) flowers with different levels of developmental stages starting from boot stage to fully mature flower, was studied by using the K-sensitive fluorescent dye PBFI (potassium-binding benzofuran isophthalate) and confocal laser scanning microscopy. The presence of heavy K fluorescence was detected only at the aperture area of the mature pollen. Similarly, the presence of K increased with the progression from immature to mature anther and papillae. In addition, a higher concentration of K was observed only at the stomium area (the place of anther dehiscence) of mature anthers. Keeping in view the role of K as an active osmoticum and the consistent and synchronized appearance of K in mature pollen, anthers, and papillae, it was concluded that K may regulate anther dehiscence, pollen imbibition, and papillae hydration leading to pollination and fertilization.

Key words: Anther, confocal microscopy, papillae, PBFI, pollen, potassium


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Next to nitrogen, K is the mineral nutrient required in the largest amount by plants. It has an outstanding role in plant water relations as well as its role in enzyme activation, protein synthesis, photosynthesis, and other function (Marschner, 1995Go). It is widely known for its rapid action as an osmotic regulator (Fischer, 1971Go; Heslop-Harrison and Heslop-Harrison, 1996Go). For example, it has been well reported that the movement of water into and out of guard cells is an osmotic response governed by K levels in the guard cells. If K concentration in guard cells increases, more negative water potential is created there and water moves osmotically into the guard cells, making them turgid. If the K levels drop, the water potential becomes more positive and water osmotically exits the guard cells, making them flaccid (Moore et al., 1995Go).

It has been reported that mature barley (Hordeum vulgare L.) pollen swells in a fraction of a second upon hydration and the presence of potassium (K) at the aperture area of pollen was considered responsible for the rapid hydration of pollen (Rehman et al., 2004Go). Furthermore, K is an essential constituent of the pollen-germinating medium; therefore, it may also be directly involved in the processes of pollen germination and tube growth. Fan et al. (2001)Go highlighted the physiological importance of K in Arabidopsis pollen germination and tube growth.

Anther dehiscence is an important step in pollination and the failure of anther dehiscence causes loss of yield. Previously, anther dehiscence was considered solely a desiccatory process (Schmid, 1976Go), which was later rejected by reports that anther dehiscence was not merely an act of desiccation, but it was a multi-step process (Keijzer, 1987Go). Furthermore, Matsui et al. (1999Go, 2000aGo) reported that the rapid swelling of pollen inside the anther soon after floret opening was one of the causes of anther dehiscence in rice and barley. They suggested that the swelling of pollen exerted pressure to bulge theca and rupture septa. Matsui et al. (1999)Go also reported that anther dehiscence in rice was a combination of a moisture requiring and a desiccatory process. They concluded that the rapid swelling of pollen was caused by the presence of potassium (K) in pollen, which is known for its function as a turgor regulator.

The stigma is the receptive surface of the style that collects the pollen and enables its hydration and germination. It is believed that the initial interchanges between pollen and stigma are extremely rapid and they lead to early germination. Normally, in the grasses, the stigma remained turgid to capture and hydrate pollen, and to allow penetration of pollen tube into the intercellular spaces of the pollen tube transmitting tract. Heslop-Harrison and Reger (1986)Go reported that K is responsible for maintaining the stigma turgidity due to its role as an osmoticum.

The success of pollination and fertilization in plants depends on a series of co-ordinated events starting from pollen production, its transfer to the stigma, and details of pollen–pistil interaction. The failure or breakdown in any of these sequential events affects the seed and fruit set. It was hypothesized, bearing in mind these synchronized events involved in pollination and fertilization, that there should be certain factor(s), which regulate these progressive steps. Moreover, considering the critical role of pollen, anther, and papillae hydration in pollen release and germination, it was assumed that K might be involved in the process of hydration due to its role as an active osmotic regulator. Therefore, in the present work, experiments were designed to trace the changes in K distribution in pollen, anthers, and papillae of barley flowers at different developmental stages.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Six-rowed barley (Hordeum vulgare L. cv. Saegang-bori) plants were grown in a greenhouse at Chonbuk National University. Anthers, pollen grains and papillae were freshly collected from the central spikelets of plants when (i) the head (spike) was completely covered in the flag leaf sheath (booting stage); (ii) half of the head had emerged from the flag leaf sheath; (iii) the head was fully emerged, but without visible anthers, and (iv) the head with visible anthers after complete extrusion from the flag leaf sheath. For the convenience of description and understanding, the developmental stages are denoted as stage 1, stage 2, stage 3, and stage 4, respectively (Fig. 1). PBFI a K+-sensing fluorescent probe (P1267) was purchased from Molecular Probes (Eugene, OR, USA). The presence of K at the barley pollen aperture was examined by loading PBFI as described by Halperin and Lynch (2003)Go and Rehman et al. (2005)Go. Barley pollen was placed in a 200 µl micro-centrifuge tube and 20 µl of 20 µM PBFI dissolved in dimethyl sulphoxide (DMSO) was added. The micro-centrifuge tube was incubated in the dark at 4 °C for 1 h, followed by incubation at 20 °C for 1 h in a dye-free solution. The samples were observed under a confocal laser scanning microscope (Carl Zeiss LSM 510, Jena, Germany) with an optical filter BP 385-470 (excitation 364 nm). SBFI and Fluo-4 dyes, a Na+ and Ca2+-sensing fluorescent probe, respectively, were purchased from Molecular Probes (Eugene, OR, USA). Both of the dyes were used for the control treatment and the procedure was followed as for PBFI except Fluo-4 was illuminated with 488 nm light. The intensity of K-specific fluorescence was measured in pixels, in pollen, anthers, and papillae by using the GAIA Blue image analyser (http://www.gaia-zone.com/). Each data point represents the accumulated value of 11 randomly chosen points and their corresponding standard deviation values.


Figure 1
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  		Fig. 1. The developmental stages of barley flowers. Stage 1, head (spike) was completely covered in the flag leaf sheath (boot stage) (A); stage 2, half of the head had emerged from flag leaf sheath (B); stage 3, fully emerged head, but without visible anthers (C), and stage 4, head with visible anthers after complete extrusion from the flag leaf sheath (D).

 

   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Control treatment
Barley pollen in a dry and hydrated condition, without loading with PBFI, was illuminated with 364 nm (Fig. 2) to confirm the PBFI-specific fluorescence. A very weak auto-fluorescence or no fluorescence was observed in the absence of PBFI treatment, which indicated that the fluorescence that appeared in a later treatment with PBFI (Fig. 3) was K-specific. Furthermore, no significant fluorescence was observed when pollen was loaded with Fluo-4 (a calcium-specific dye) and SBFI (a sodium-specific dye) illuminated with 488 and 364 nm light, respectively (Fig. 2).


Figure 2
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  		Fig. 2. Control treatment—confocal potassium imaging: fluorescent images of barley pollen in dry and hydrated conditions without loading with PBFI excited with 364 nm and hydrated pollen loaded with Fluo-4 and SBFI and excited with 488 and 364 nm light, respectively. (A–F) Bright field images; (G–L) corresponding fluorescent images of pollen from mature barley flowers. Faint auto-fluorescence is visible all around the pollen (G, H). However, no PBFI-specific fluorescence was observed at the aperture area of pollen as seen in Fig. 3 after loading with PBFI. Furthermore, no significant fluorescence was observed in the case of Fluo-4 (I, J) and SBFI (K, L) dyes which show that the fluorescence appearing in Fig. 3 is specific to K. The pollen was observed with a x40 water immersion lens (C-Apochromat, NA=1.2, Carl Zeiss) and an image was captured by a confocal microscope equipped with a x10 ocular lens. Arrowheads indicate the aperture of pollen. Bar: 10 µm.

 

Figure 3
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  		Fig. 3. Confocal potassium imaging: fluorescent image of barley pollen loaded with PBFI and excited with 364 nm light. Pollen from barley flowers at different stages of development as mentioned in Fig. 1. (A–D) bright field images while (E–H) are their corresponding fluorescent images. Heavy K-specific fluorescence is visible around the wall during the early stages of growth (E, F) and at the aperture area during the later mature stages of pollen (G, H). Higher fluorescence indicates the higher presence of K which is measured in pixels (I), in the wall (circle) and the aperture area of pollen by using an image analyser. The pollen was observed with a x40 water immersion lens (C-Apochromat, NA=1.2, Carl Zeiss) and an image was captured by a confocal microscope equipped with a x10 ocular lens. Arrowheads indicate the aperture of pollen. Bar: 10 µm.

 
Changing K in pollen
Fluorescent images of barley pollen resulting from the dye distribution of PBFI, illuminated with 364 nm light, are shown in Fig. 3. Barley pollen has only one aperture. It was found that the intensity of K-specific fluorescence changed with the progression of pollen development. Heavy fluorescence was detected only at the aperture area of the mature pollen (Fig. 3H), indicating the high concentration of K in this area compared with faint fluorescence at stage 3 (Fig. 3G) and no traces of fluorescence at the aperture area of pollen at developmental stages 1 and 2 (Fig. 3E, F). By contrast, in the early stages of pollen development, heavy fluorescence was detected in the surrounding wall area of pollen (Fig. 3E, F), that became fainter or disappeared altogether from the wall of mature pollen (Fig. 3G, H). The intensity of K-specific fluorescence was measured in pixels, in the wall (circle) and the aperture area of pollen by using an image analyser (Fig. 3I). A significant drop and a significant increase in K around the wall and at the aperture area, respectively, was observed with the progression of pollen maturity. The fluorescence at the aperture area for pollen at stage 2 could not be quantified due to the sitting position of pollen which made it difficult to differentiate between the wall and aperture fluorescence.

Changing K in anthers
The changing intensity of K fluorescence with the maturity of barley anthers is shown in Fig. 4. No fluorescence was observed anywhere in the early stage (stage 1) of anther development. However, it progressed with the maturity of the anther and a high intensity of K fluorescence was found in mature anthers (stage 4). Furthermore, K fluorescence was observed only at the stomium area of the anther. Figure 4I elaborates these results further which shows that K concentration significantly increased in the anther at the mature stage.


Figure 4
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  		Fig. 4. Anther from barley flowers at different stages of development as mentioned in Fig. 1. (A–D) Bright field images while (E–H) are their corresponding fluorescent images. The intensity of K-specific fluorescence increases as the maturity of the anther progresses from stage 1 to stage 4, i.e. no fluorescent signal was visible at booting stage (E) and a higher signal was evident at the stage of full maturity (H). Higher fluorescence indicates the higher presence of K which corresponds with the results shown in Fig. I. The anther was observed with a x40 water immersion lens (C-Apochromat, NA=1.2, Carl Zeiss) and an image was captured by a confocal microscope equipped with a x10 ocular lens. Bar: 10 µm.

 
Changing K in papillae (stigma)
Pollination initiates when pollen is captured by the stigma. Barley stigma has finger-like papillae and pollen germinate after adhering to the stigma. Fluorescent images of barley papillae (stigma) resulting from the K-specific dye distribution of PBFI, are shown in Fig. 5. The intensity of fluorescence increased from none at the immature papillae (stage 1) (Fig. 5E) to very high at the mature papillae (stage 4) (Fig. 5H), indicating the greater presence of K in mature papillae compared with immature ones. Furthermore, the quantification of the K-specific fluorescence in papillae showed a significant increase in K as the papillae matured (Fig. 5I).


Figure 5
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  		Fig. 5. Papillae from barley flowers at different stages of development as mentioned in Fig. 1. (A–D) Bright field images while (E–H) are their corresponding fluorescent images. The pattern of K-specific fluorescence distribution was the same as seen in the case of anthers (Fig. 2). In the early stage (stage 1), no fluorescence was observed but it progressed slowly and heavier fluorescent signals were observed later at the fully mature stage (stage 4) (H). Higher fluorescence indicates the higher presence of K (I). The papillae were observed with a x40 water immersion lens (C-Apochromat, NA=1.2, Carl Zeiss) and an image was captured by a confocal microscope equipped with a x10 ocular lens. Bar: 10 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Confocal laser scanning microscopy has wider applications in morphological studies and ion imaging in plants. The potassium binding fluorescent dye PBFI has mainly been used to measure intracellular K in animal cells (Kasner and Ganz, 1992Go) and rarely utilized with plant cells (Halperin and Lynch, 2003Go). However, Rehman et al. (2005)Go used PBFI for the first time to determine K distribution in barley pollen. The application of PBFI in current work could become a valuable tool to analyse K in plant reproductive organs. Moreover, to the best of our knowledge, the use of PBFI has not yet been tested for its suitability to determine K in the anther and stigma.

It is not possible to get K-free plant material that could be used as a control to test PBFI specificity. However, the absence of fluorescence in dry and hydrated pollen without treatment with PBFI (Fig. 2) was considered as the control and it was found that the PBFI was a K-specific dye and fluorescence could only be observed after treatment with PBFI as shown in Fig. 3. The absence of high intensity fluorescence at the aperture area of mature barley pollen after treatment with Fluo-4 (Ca2+-specific) and SBFI (Na+-specific) dyes further support the specificity of PBFI for K in this study.

One of the important functions of pollen aperture is believed to be regulating the water balance of the pollen when it is subjected to changes in humidity (Shukla et al., 1998Go). Barley has only one aperture and, therefore, it should be the only way to regulate the water uptake and protrude pollen tube. In current work, in the early stages of pollen development, heavy traces of K were found in the surrounding wall area which disappeared from the wall area with maturity and appeared heavily only at the aperture area of mature pollen (Fig. 3). These results suggest that in the early stage of development, K was still in the wall area of pollen, being absorbed from inter-pollen spaces, which was later translocated to the aperture area upon the maturity of pollen. Similar results were reported by Matsui et al. (2000b)Go. They found K on the surface of pollen grains and in the tapetum of indehisced anther and, by contrast, detected K only inside the pollen of a dehisced anther. They concluded that pollen swells even whilst still in the anther and, furthermore, they attribute swelling of pollen to the presence of K in pollen. Previously, Rehman et al. (2002Go, 2004Go) reported a heavy accumulation of K only at the aperture area, regardless of the number of apertures, for example, one in barley pollen and 12 apertures in sesame pollen. Furthermore, the rapid swelling of pollen was attributed to the heavy presence of K at the aperture area of pollen.

The stronger presence of K at the dehiscing area of the mature anther suggests a greater role of K as an osmoticum in the dehiscence of anthers (Fig. 3). When rice anthers were exposed to 20, 60, and 100% relative humidity (RH) at 24 °C, the dehiscence of anthers increased with the increase of RH (Matsui et al., 1999Go). They suggested that desiccation of anthers was unnecessary and even disturbed the dehiscence. Furthermore, Matsui et al. (1999Go, 2000aGo, bGo) reported rapid swelling of barley and rice pollen inside the anther after opening of the florets. They suggested that the swelling of pollen exerted pressure to bulge theca, rupture septa, and unfold the locule. They concluded that pollen swelling inside the anther, caused by K in pollen, was indispensable for anther dehiscence.

However, if it is believed that the pollen inside the anther sufficiently hydrates to swell due to water driven by K, resulting in the dehiscence of the anther and the release of pollen, then the question arises—where did the pollen get the moisture from? The possible answer could be from/through the anther. This study's results (Fig. 3) suggest that the heavy presence of K at the stomium area of the mature anther could attract moisture from the surrounding environment. The moisture absorbed by the anther could be directly involved in anther dehiscence, as well as indirectly, which is subsequently absorbed by pollen resulting in swelling and forcing locules to unfold.

The next step in pollination after releasing pollen from the anther and landing on the stigma is the hydration and germination of pollen. The stigma is the receptive surface of the style, which is the most short-lived structure of a plant. Pollen further hydrates rapidly once landing on stigma. The rapid imbibition may be a prerequisite for rapid pollen tube emergence because, in most cases, the emergence of pollen tube takes a few seconds to a few hours after being placed in conditions favourable for germination (Anthony and Harlan, 1920Go; Stone et al., 2004Go). Heslop-Harrison and Reger (1986)Go reported that, in the grasses, the stigma remained turgid to capture and hydrate pollen and that K is responsible for maintaining the stigma turgidity due to its role as an osmoticum. In the present work, K was not traced in papillae during the early stages (immature) of development but heavy accumulation was found later in fully mature papillae (Fig. 4). These results suggest that hydration of pollen probably comes from the hydration of papillae, which is caused by the osmotic gradient created by the accumulation of a higher level of K in mature papillae. Moreover, K was accumulated in papillae at the time when it was accumulated in the aperture of pollen and stomium of anther.

It is well known that the success of fertilization in plants depends on the success of pollination and, furthermore, successful pollination requires simultaneous maturation of the stamens and carpels. It is important that the stigma must be receptive before the pollen lands on it and, similarly, pollen must be available before the stigma is receptive. These events suggest the existence of synchronised action leading to successful pollination and fertilization. The present results provide consistent and solid evidence of the simultaneous and heavy presence of K only when pollen, anther, and stigma reach maturity. Therefore, it is fairly safe to say that K may regulate all the events of anther dehiscence, pollen and stigma hydration leading to successful pollination and germination of pollen. In addition, K may also be playing an important role in pollen germination and pollen tube growth because it is an essential ingredient of the pollen germinating medium.


   Acknowledgements
 
This work was supported by a grant (CG2213) from the Crop Functional Genomics Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology (MOST) and Rural Development Administration (RDA), Republic of Korea.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Anthony S, Harlan HV. 1920. Germination of barley pollen. Journal of Agricultural Research 18, 525–539.

Fan L-M, Wang Y-F, Hong W, Wu W-H. 2001. In vitro Arabidopsis pollen germination and characterization of the inward potassium currents in Arabidopsis pollen grain protoplasts. Journal of Experimental Botany 52, 1603–1614.[Abstract/Free Full Text]

Fischer RA. 1971. Role of potassium in stomatal opening in the leaf of Vicia faba. Plant Physiology 47, 555–558.[Abstract/Free Full Text]

Halperin SJ, Lynch JP. 2003. Effects of salinity on cytosolic Na+ and K+ in root hairs of Arabidopsis thaliana in vitro measurements using the fluorescent dyes SBFI and PBFI. Journal of Experimental Botany 54, 2035–2043.[Abstract/Free Full Text]

Heslop-Harrison JS, Reger BJ. 1986. Chloride and potassium ions and turgidity in the grass stigma. Journal of Plant Physiology 124, 55–60.

Heslop-Harrison Y, Heslop-Harrison JS. 1996. Lodicule function and filament extension in the grasses: potassium ion movement and tissue specialization. Annals of Botany 77, 573–582.

Kasner SE, Ganz MB. 1992. Regulation of intracellular potassium in mesangial cells: a fluorescence analysis using the dye, PBFI. American Journal of Physiology 262, 462–467.

Keijzer CJ. 1987. The process of anther dehiscence and pollen dispersal. I. The opening mechanism of longitudinally dehiscing anthers. New Phytologist 105, 487–498.[CrossRef][ISI]

Marschner H. 1995. Mineral nutrition of higher plants. London: Academic Press: Harcourt Brace & Company, Publishers.

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Matsui T, Omasa A, Horie T. 2000b. Mechanism of septum opening in anthers of two-rowed barley (Hordeum vulgare L.). Annals of Botany 86, 47–51.[Abstract/Free Full Text]

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Rehman S, Lee KJ, Rha ES, Yun SJ, Kim JK. 2002. Mechanisms involved in rapid swelling of sesame (Sesamus indicum L.) pollen. New Zealand Journal of Crop and Horticultural Science 30, 209–213.

Rehman S, Rha ES, Ashraf M, Lee KJ, Yun SJ, Kwak YG, Yoo NH, Kim J-K. 2004. Does barley (Hordeum vulgare L.) pollen swell in fractions of a second? Plant Science 167, 137–142.[CrossRef]

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