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Pollen germination (PG) and pollen tube growth (PTG) play crucial roles in sexual reproduction of flowering plants by sending sperm cells to the ovule. These two processes are regarded as ideal model system for the study of cell signaling and cell polarized growth. It has been considered for a long time that ion transports across the pollen tube membranes are essential for pollen tube navigation and growth. Previous transcriptome analyses for Arabidopsis have shown that the transcripts related to cellular transport are correspondingly overrepresented during the process of pollen tube growth. Here, we showed that 459 transporter genes expressed during PG and PTG in Arabidopsis. In addition, the gene expression profiles of ion (including Ca2+, H+, K+, Cl−) channels and transporters were further analyzed. This analysis provides novel information for the potential candidate genes involving in ion fluxes across the pollen tube membranes and in regulation of pollen tube tip growth.
The membrane transporters have been considered as crucial proteins that participate in regulating intracellular ion gradients, maintaining turgor pressure and supplying sufficient materials for the rapid growing pollen tubes.1,2 In Arabidopsis genome, 1,269 genes encoding transporters have been classified according to their potential functions.3 The transcriptome analyses using Affymetrix ATH1 Genome Arrays have shown that there were 459 transporter genes expressed during the processes of pollen germination and pollen tube growth.4 The number of expressed transporter genes was gradually increased from desiccated mature pollen grains (MP, 332 genes) to hydrated pollen grains (HP, 371 genes) and to pollen tubes (PT, 396 genes). It was also notable that the expression levels of these genes were significantly elevated along with pollen tube growth. The number of upregulated genes (more than 1.6-fold) during PTG was 91, which was much more than that of PG (12 genes). The increments of both expressed numbers and expression levels of transporter genes may be related to their potential physiological roles during pollen germination and tube growth.
The tip-focused [Ca2+]cyt5–7 and pH gradients2,8,9 at the apex region of growing pollen tubes are essential in controlling the polarized growth of pollen tubes.10,11 Furthermore, these ion gradients displayed remarkable periodic oscillations, which are correlated with the growth oscillations of pollen tubes.8,10,12,13 It has been suggested that these intracellular ion gradients mainly derive from the influx of extracellular Ca2+ and H+, which is mediated by the Ca2+ channels and/or non-selective cation channels (NSCC) located at the plasma membrane of pollen tube tips.1,2,7,8 But the molecular identities of these channels are still unclear.
In Arabidopsis genome, genes from two gene families CNGC (Cyclic Nucleotide Gated ion Channel) and GLR (Glutamate Receptor) are suggested to encode putative weakly selective cation channels with a range of Ca2+ permeability.14 Our microarray data showed that CNGC18 was highly expressed during PG and PTG.4 Recent study has also shown that CNGC18 was permeable to Ca2+ and it localized to the plasma membrane at the growing tip of pollen tube.15 More importantly, loss function of CNGC18 caused the male sterility by disrupting the normal tip growth of pollen tubes, suggesting its role in mediating the Ca2+ influx at pollen tube tips.15 Here, we observed five other CNGC genes (CNGC7, 8, 9, 10 and 16) and 1 GLR gene (GLR2.1) expressed in MP and also during PG and PTG (Suppl. Data), which suggests that these genes may also involve in Ca2+ and H+ fluxes across the pollen tube membrane.
The Ca2+-pumps and P-type H+-pumps are also regarded to control the ion gradients and pollen tube growth.8,16 A Ca2+-pump ACA9 has been found localized at plasma membrane of pollen tubes.16 Mutation of ACA9 resulted in reduction of pollen tube growth and high frequency of aborted fertilization, which indicated the roles of ACA9 in regulation of pollen development and fertilization in plants via controlling the cytoplasmic Ca2+ dynamics.16 In our microarray experiments, the transcripts of 5 Ca2+-pump genes (AtACAs) and 7 P-type H+-pump genes (AtAHAs) were detected during PG and PTG (Suppl. Data). In order to further characterize the functions of these pumps may increase our understanding on complex mechanisms of ion fluxex across the pollen membranes.
In addition, some antiporters from CaCA (Ca2+ Cation Antiporter) family and CHX (Cation-Hydrogen Exchanger) family may be important components involving in ion flux regulation in pollen. Sze et al.17 has demonstrated that there were 18 AtCHX genes specifically or preferentially expressed in the male gametophyte. We have also observed that 18 AtCHX genes were expressed during PG and PTG (Suppl. Data), and the transcriptional levels of 9 AtCHX genes were specifically or preferentially upregulated during PTG.4 These AtCHXs may involve in regulating the local pH gradient immediately adjacent to the PM of pollen tubes, in adjusting ion homeostasis as well as in maintaining turgor and membrane potential of pollen tubes.1
Potassium (K+) and chloride (Cl−) ions were also accumulated abundantly in growing pollen tubes. K+ and Cl− may play important roles in controlling the turgor pressure and membrane potential for normal pollen tube growth.1 Previous studies have shown that K+ fluxes across the pollen or pollen tube plasma membrane are critical for PTG and/or PG.18–20 A pollen specifi- cally expressed K+ channel SPIK has been identified as an essential channel mediating the K+ influx across pollen plasma membrane.21 The mutation of SPIK (in the spik mutant) resulted in the decrease of pollen tube growth, suggesting that SPIK functions as a K+ uptake channel during pollen tube growth and thereby influences the tube growth.21 Our microarray data showed that SPIK was highly transcribed during PG and PTG.4 Several other putative K+ channels and transporters were also transcriptionally expressed during PG and PTG, such as AKT5, KEA5, KUP5 and so on (Suppl. Data).
Efflux of Cl− at pollen tube tips was essential for turgor regulation and water uptake in pollen tube.22,23 However, the identity and characteristics of the channels responsible for this efflux remain unknown. The microarray data showed that three Cl− channels were expressed during PG and PTG (Suppl. Data), which may be the candidates responsible for the Cl− efflux and participate in the regulation of pollen tip growth.
The phenomena of ion fluxes and ion gradient oscillations in growing pollen tubes have been observed for more than a decade.1 However, the molecular identifications for these channels and transporters mediating the ion fluxes are still limited. The transcriptome analysis for the processes of PG and PTG provide us the information for identifying candidate channel and transporter genes possibly involving in the regulation of PG and PTG. Obviously, detailed functional characterization of these transporters and investigation of their interaction or coordination are required for elucidating complex regulatory mechanisms of ion fluxes across the pollen and pollen tube membranes as well as the related regulatory mechanism of pollen tube tip growth.
Previously published online: www.landesbioscience.com/journals/psb/article/10063