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Plant Physiol. 2006 Apr; 140(4): 1151–1168.
PMCID: PMC1435806

Integrating Membrane Transport with Male Gametophyte Development and Function through Transcriptomics1,[W]


Male fertility depends on the proper development of the male gametophyte, successful pollen germination, tube growth, and delivery of the sperm cells to the ovule. Previous studies have shown that nutrients like boron, and ion gradients or currents of Ca2+, H+, and K+ are critical for pollen tube growth. However, the molecular identities of transporters mediating these fluxes are mostly unknown. As a first step to integrate transport with pollen development and function, a genome-wide analysis of transporter genes expressed in the male gametophyte at four developmental stages was conducted. Approximately 1,269 genes encoding classified transporters were collected from the Arabidopsis (Arabidopsis thaliana) genome. Of 757 transporter genes expressed in pollen, 16% or 124 genes, including AHA6, CNGC18, TIP1.3, and CHX08, are specifically or preferentially expressed relative to sporophytic tissues. Some genes are highly expressed in microspores and bicellular pollen (COPT3, STP2, OPT9), while others are activated only in tricellular or mature pollen (STP11, LHT7). Analyses of entire gene families showed that a subset of genes, including those expressed in sporophytic tissues, was developmentally regulated during pollen maturation. Early and late expression patterns revealed by transcriptome analysis are supported by promoter∷β-glucuronidase analyses of CHX genes and by other methods. Recent genetic studies based on a few transporters, including plasma membrane H+ pump AHA3, Ca2+ pump ACA9, and K+ channel SPIK, further support the expression patterns and the inferred functions revealed by our analyses. Thus, revealing the distinct expression patterns of specific transporters and unknown polytopic proteins during microgametogenesis provides new insights for strategic mutant analyses necessary to integrate the roles of transporters and potential receptors with male gametophyte development.

Male fertility in plants depends on several critical events, beginning with the proper development of the male gametophyte, the transfer of mature pollen to a compatible stigma, and subsequent germination and pollen tube growth through the style. When the pollen tube reaches the ovule, it ruptures and delivers two sperm cells that result in double fertilization of egg and central cells (Lord and Russell, 2002). Although the major events of male reproductive development are well known, the molecular and cellular bases of these processes are still poorly understood (Twell, 2002; McCormick, 2004). Microgametogenesis starts with a single haploid microspore that divides and develops into a mature pollen grain consisting of a large vegetative cell, plus one generative cell or two sperm cells. Pollen germination and tube growth are manifestations of a single vegetative cell as it carries and delivers the sperm cells to the ovule. Thus, the development and the physiology of the male gametophyte is an attractive model to study the molecular and cellular bases of development, stress tolerance, signal transduction, growth, and morphogenesis (Twell, 2002; McCormick, 2004).

It is well recognized that the transport of ions and metabolites is required not only for nutrient uptake and sorting, metabolism, and energy production during plant growth, but also is integrated with signaling, movement, development, and stress tolerance (e.g. Franklin-Tong, 1999; Schwacke et al., 1999). This idea is especially well illustrated in the establishment and maintenance of polarity in the growing pollen tube (Weisenseel et al., 1975; Hepler et al., 2001). The current picture emerging is that growth depends on a tip-focused Ca2+ gradient and other ion fluxes (for review, see Holdaway-Clarke and Hepler, 2003). Cytosolic [Ca2+] at the tip reaches as high as 5 μm and drops to approximately 0.1 μm away from the apex. However, the Ca2+ gradient and the tip-focused acidification are not static and undergo regular oscillations that appear to be in phase with tube growth. Moreover, Ca2+ and H+ influx are maintained as the tube grows. These and other studies suggest that signaling networks modulate a variety of pumps, porters, and channels in time and in space to orchestrate the ion gradients, oscillations, and fluxes (Feijo et al., 2001). Despite this knowledge of pollen tube physiology, we know almost nothing about the molecular nature of the transporters involved and those required for the proper development of the male gametophyte.

To begin integrating transport activities with microgametogenesis and pollen tube growth, we wanted to identify membrane transporters that are used by the male gametophyte. The complete Arabidopsis (Arabidopsis thaliana) genome revealed the presence of more than 800 transporters (Arabidopsis Genome Initiative, 2000); however, the biological roles of most genes and their integration with the plant life cycle remain unknown. The availability of the pollen transcriptome using the whole-genome Arabidopsis ATH1 chip reveals for the first time all the genes expressed in the mature male gametophyte (Honys and Twell, 2004; Pina et al., 2005). Moreover, a unique dataset for four stages of male gametophyte development enables developmental analysis of gene expression (Honys and Twell, 2004). These resources present a golden opportunity to study the molecular and cellular bases of cell proliferation, tip growth, gene expression, cell communication, and signaling through analysis of developmental gene-expression profiles.

Although genome-wide transcriptome data are publicly available or published, the wealth of information is obscured by the immense datasets for several reasons. The datasets include genes that are not organized in a biologically relevant manner and often are not well annotated. The data are not normalized using the same method and sometimes show irreproducibility from one laboratory to another. Data-mining tools, though available (e.g. Genevestigator; Zimmermann et al., 2004), are inadequate for bulk analysis of functionally related genes. Therefore, data mining and analyses by experts in the field present a powerful approach to distill significant insights from the large datasets.

Here we have focused on membrane transporters and have identified all those expressed in the male gametophyte, as well as those that are preferentially transcribed in pollen. By following expression over different stages of microgametogenesis, we distinguish between early microspore and pollen-expressed versus late pollen-expressed genes. This analysis provides comprehensive information about which transporter genes within multigene families are developmentally regulated and insight into their potential roles in microspore proliferation, pollen maturation, or pollen tube growth. Recent genetic evidence of transporter function further supports the inferred functions revealed by our analyses. These results therefore generate working ideas for streamlining efforts to uncover the roles of many transporters and putative receptors not only in the male gametophyte but also in sporophytic tissues.


To conduct a meaningful and useful analysis of the pollen transcriptome for all transporters, it was necessary (1) to compile a comprehensive list of all known and putative transporters from Arabidopsis, and (2) to organize the genes according to their gene families and their transporter classification (TC). At the time this project was started, a comprehensive list was not available in the public databases, including the Arabidopsis Membrane Protein Library (AMPL; www.cbs.umn.edu/arabidopsis/), Aramemnon (aramemnon.botanik.uni-koeln.de/), and PlantsT (plantst.genomics.purdue.edu).

Identifying Transporter Genes in Arabidopsis and in Pollen

We used three databases to obtain a comprehensive list of genes encoding polytopic membrane proteins from the completed Arabidopsis genome (Arabidopsis Genome Initiative, 2000). The initial list obtained from AMPL included genes predicting proteins with two or more transmembrane spans (Ward, 2001). Other sequences were added or removed based upon the isospecific homolog clusters from release 3.0 of the Aramemnon membrane protein database (Schwacke et al., 2003) and protein family membership in the TC system at the PlantsT database (Tchieu et al., 2003). Genes encoding proteins with nontransport activities were removed from the list. The final number of transporters is still uncertain as a large number of genes encode proteins annotated as “expressed” or “hypothetical.” Many genes encoding unclassified proteins were retained for analysis as they could encode potential transporters. In a few cases, peripheral subunits of known multimeric transporter complexes were also included. The final master list of 1,751 sequences includes 1,269 transporters and 482 membrane proteins of unknown function (Supplemental Table I). For simplicity, we refer to this list as the “master” list of transporters and unknown polytopic proteins, though the list will be revised as functional studies uncover new transporters. Transporter genes are defined as those encoding proteins that have a TC number or are related to proteins with a TC number. This master sheet is an updated and comprehensive list of all known and potential transporters from Arabidopsis organized using TC system (http://www.tcdb.org/).

Developmental pollen transcriptome data were incorporated into the master list of transporter and unknown protein genes using Microsoft Office Access 2003 SP1, which extracted the normalized data from the pollen transcriptome of Honys and Twell (2004) and linked them to the corresponding genes. Unlike other studies that looked at the genome-wide transcriptome of mature pollen alone (Honys and Twell, 2003; Becker et al., 2003; Zimmermann et al., 2004; Pina et al., 2005), the dataset of Honys and Twell (2004) included expression of all genes at four stages of microgametogenesis, including microspores, bicellular pollen, tricellular pollen, and mature pollen. The expression level of each gene at any stage of pollen development was compared with datasets from 12 sporophytic organs or tissues. “Pollen preferential” was assigned to those genes showing at least a 3-fold increase for the maximum expression signal at any stage of pollen development relative to the highest level in any sporophytic tissue (Supplemental Table I). “Pollen specific” was assigned to genes that showed positive expression at any stage of pollen development and a normalized value of zero for all sporophytic tissues examined.

Of 1,751 total transporter and unknown protein-encoding genes in the Arabidopsis genome, 1,511 were on the ATH1 chip, and 1,046 genes (or 69%) were expressed in developing or mature pollen (Table I). This value seems surprisingly high considering that these genes are expressed by one or two cell types, yet it is consistent with the previous estimation that 62% (or 13,977 genes) of the genome on the ATH1 chip (22,591 genes) is expressed in developing or mature pollen (Honys and Twell, 2004). The total number will increase slightly as some genes are absent from the ATH1 chip or were undetected due to very low expression. Nearly 10% (1,355) of all pollen-expressed genes are specific to the male gametophyte. The list of 1,046 genes expressed in the male gametophyte includes 757 genes that encode channels (11%), porters (61%), or pumps (19%), according to the TC system, plus 289 genes of unknown identity. Approximately 14% of the pollen-expressed transporter and unknown protein genes (1,046) are specifically or preferentially expressed in the male gametophyte using the criteria described above. When genes encoding classified transporters are considered independently, transporters make up 7.1% of the genes expressed in mature pollen relative to 5.3% of the total genes expressed in microspores or bicellular pollen. Furthermore, 13.6% of transporters are specifically or preferentially expressed in microspores; remarkably, the proportion of specific plus preferentially expressed transporter genes increases to approximately 21% (or 109 genes) in tricellular and mature pollen (Table I). Thus, transporter genes as a whole, including pollen-specific ones, are overrepresented as pollen matures.

Table I.
Number of transporter genes expressed in the Arabidopsis male gametophyte during microgametogenesis

Transporters Specifically or Preferentially Expressed in Developing Pollen

Table II shows a working list of 124 transporter genes that are defined as specifically or preferentially expressed in developing pollen. The list includes members of well known transport families, such as P-type H+ pumps and Ca2+ pumps, several ABC transporters, and only two vacuolar H+-ATPase subunits, E2 (At3g08560) and G3 (At4g25950). H+-coupled cotransporters include members of the monosaccharide/H+ symporter (STP), amino acid transporter (AAAP), and putative oligopeptide and nitrate transporter (OPT and POT) families. Channels include two K+ channels of the voltage-gated ion channel (VIC) family, four members of the cyclic nucleotide and calmodulin-regulated ion channel (CNGC) subfamily, and two putative tonoplast water channels of the major integral protein (MIP) family (Table II). Anion transporters include a putative borate transporter, sulfate transporters, and phosphate translocators. Although this working list may include genes that are expressed in sporophytic tissues (e.g. meristem cells) not included in our analysis, the table highlights those that have potential functions in pollen development. The transporters are diverse and in general include a few members of each gene family. However, a striking presence is the CPA2 family of putative cation/proton antiporters, in which 14 of 28 genes are specifically or preferentially expressed in pollen.

Table II.
Genes that are specifically or preferentially expressed in pollen include classified transporters and unknown membrane proteins

Twenty-six of the genes specifically or preferentially expressed in pollen either are not classified as transporters in the TC system or are not annotated (Table II). Examples include the DHHC-type zinc (Zn)-finger domain-containing proteins, rhomboid family proteins, nodulin MtN3-like proteins, and unknown expressed proteins.

Coexpression of Pollen-Specific Genes Late or Early in Development

Strong clues to function can be inferred from the pattern of expression during microgametogenesis. Nearly half (64 out of 150) of the pollen-specific and pollen-preferential transporter genes are coexpressed late in pollen development, first appearing at the tricellular stage or in the mature pollen (Fig. 1, A and B; Supplemental Fig. 1A). These expression patterns belong to clusters 1, 2, and 3, according to the coexpression groupings of Honys and Twell (2004). This group includes SPIK (At2g25600); several CNGCs, including CNGC18 (At5g14870), AtRbohH (At5g60010), AHA08 (At3g42640), ACA9 (At3g21180), OPT1 (At5g55930), STP11 (At5g23270), TIP5.1 (At3g47440), and BOR1 homolog (At5g25430); and several CHXs, including CHX08 (At2g28180). Many unclassified or unknown genes are also expressed late in pollen development. Thirty-one genes belonging to clusters 18 to 22 show a similar late pollen-expression pattern, except that the level of message peaked in tricellular stage and dropped dramatically in mature pollen (Fig. 1C; Supplemental Fig. 1C). This group included genes encoding plasma membrane (PM)-localized H+ pumps (AHA6, At2g07560; AHA9, At1g80660), autoinhibited calcium pump (ACA7, At2g22950), vacuolar H+-ATPase subunits (VHA-E and VHA-G), putative tonoplast water channel (TIP1.3, At4g01470), monosaccharide/H+ symporter (STP9, At1g50310), putative receptor MLO9 (At1g42560), cation/proton exchangers (CHX13, At2g30240; CHX19, At3g17630), and putative K+ transporter (KUP9, At4g19960).

Figure 1.
Coexpression of genes encoding transporters revealed many genes are expressed either early or late during microgametogenesis. Shown is the relative expression of each gene at the four stages of pollen development: microspore (MS), bicellular (BC), tricellular ...

In contrast, a group of only 22 pollen-specific and pollen-preferential genes showed peak expression in the microspore and bicellular pollen, with expression low or undetectable in tricellular or mature pollen (Cluster 29 of Honys and Twell, 2004; Fig. 1D). These genes are referred to as early pollen-expressed genes. This group included putative ABC transporter WBC09 (At4g27420), ammonium transporter (Amt1.4, At4g28700), copper transporter (COPT3, At5g59040), magnesium transporter (MGT5, At4g28580), STP2 (At1g07340), OPT9 (At5g53510), phosphate transporter (AtPHT6/AtPHT1-6, At5g43340), ACA13 (At3g22910), and AHA12 (At4g11730). Approximately 23 other genes showed variations of other expression patterns (Fig. 1, B and D).

Identifying Additional Transporter Genes with Roles in Pollen Biology

Over 80% of pollen-expressed genes are expressed in sporophytic tissues, so we examined the expression pattern of all members of a transporter gene family over the four pollen developmental stages. In most cases, this approach revealed differential expression of additional genes within each family during microgametogenesis, regardless of whether they were expressed highly in sporophyte. In many cases, the pollen-specific or -preferential genes identified in Table II are also the most highly expressed members of their gene families at a certain developmental stage (e.g. CNGC18, BOR1 homolog, CHX08).

Here we highlight a few gene families that show distinct increases or decreases in expression during microgametogenesis. For instance, AHA6, AHA8, and AHA9 of the PM H+-ATPase family are late pollen-specific genes; however, AHA3 (At5g57350), known to function in phloem, is highly expressed in the early stages of pollen development (Fig. 2, F.1) when other AHA genes show little or no expression. These results show that discrete members of the AHA family are developmentally regulated during microspore proliferation and pollen maturation.

Figure 2.Figure 2.
Discrete subsets of genes within selected gene families are expressed in male gametophyte in a developmentally regulated manner. Relative expression of all genes within a gene family was monitored at the microspore (MS), bicellular (BC), tricellular (TC), ...

Among autoinhibited Ca2+-pumping ATPases, ACA2 (At4g37640), ACA7, and ACA9 are late pollen-expressed genes, though only ACA9 expression is especially high in mature pollen (Fig. 2, F.2). In contrast, ACA10 (At4g29900) and ACA13 are early pollen-expressed genes. Multiple ACAs are likely localized at different subcellular membranes, including the endoplasmic reticulum (ER) ACA2, the vacuolar ACA4, and a PM ACA8 (Sze et al., 2000). Curiously, ER-type Ca2+ pumps (ECA1–4) are expressed in pollen but do not display differential patterns of expression (Fig. 2F).

Two K+ channels, SPIK and SKOR (At3g02850), are highly expressed late in pollen development, though SKOR, an outward-rectifying channel, is also expressed in the stele (Fig. 2, A.1). AKT5 (At4g32500) is constitutively expressed. Except for two cyclic nucleotide and calmodulin-regulated ion channels that are expressed early in pollen development, most of those (CNGC7, At1g15990; CNGC8, At1g19780; CNGC16, At3g48010; and CNGC18) activated late in pollen development are also preferentially or specifically expressed in the gametophyte (Fig. 2, A.2). Several putative Cl channels are expressed in pollen at all stages, though only CLCc (At5g49890) showed enhanced expression in the mature pollen grain (Fig. 2A). Interestingly, only six of more than 30 MIPs are highly expressed in the male gametophyte (Fig. 2, A.3). Three of these genes (TIP1.3; TIP5.1, At3g47440; NIP4.1, At5g37810) are also pollen specific, indicating that expression of aquaporins in pollen is under strict regulation by the gametophytic program.

The expression of monosaccharide/H+ symporters of the STP family is particularly striking during microgametogenesis. STP2 is an early pollen-expressed gene, whereas STP11 is expressed late in pollen maturation. STP4 (At3g19930), STP6 (At3g05960), and STP9 are coexpressed late in pollen development, yet their expression profiles are distinct from STP11 (Fig. 2, B.1). All of these, except for STP4, are specifically or preferentially expressed in pollen.

Fourteen members of the cation/proton exchanger (CHX) gene family are expressed late in pollen development (Supplemental Fig. 2C), raising questions about their roles and multiplicity. It is possible they are localized to different intracellular compartments and are modulated by different signaling molecules. A few CHX genes are preferentially expressed in vegetative tissues, such as CHX17 (At4g23700) in the root (Sze et al., 2004). So it was surprising to find that only CHX17 appears to be expressed at the microspore and bicellular pollen stages from transcriptomic data. This low level gametophytic expression was questioned until it was verified by promoter∷β-glucuronidase (GUS) staining (Fig. 3).

Figure 3.
Differential expression of CHX genes in developing pollen is confirmed by promoter∷GUS analyses of the inflorescence. A, CHX17 promoter∷GUS. Flowers at different stages (S9–S13) were taken from one inflorescence that was stained ...

Among cation transporters, Cu2+ transporter COPT3 is expressed early in microspore development, whereas COPT1 (At5g59030) appears late in pollen maturation (Fig. 2, C.1). COPT1 is also highly expressed in root tips, trichomes, and guard cells (Sancenon et al., 2004). A heavy-metal pump with specificity for Zn2+ and Cd2+, HMA4 (At2g19110), is expressed early in pollen development (Fig. 2, F.4). In contrast, the Cu2+ pump RAN1/HMA7 (At5g44790; Fig. 2, F.4) is switched on later in tricellular or mature pollen stages. These and other results (Supplemental Fig. 2) strongly suggest that specific members of each gene family play distinct roles in microgametogenesis and in postpollination events.

Several amino acid transporter genes that are specific or preferentially expressed in pollen (LHT7, At4g35180; LHT8, At1g71680) appear late in development (Fig. 2, E.1). Interestingly, AtProT1 (At2g39890), a Pro/H+ symporter with specificity for betaine and γ-aminobutyrate, is also highly expressed late in pollen development. Pro is the most abundant amino acid in mature tomato (Lycopersicon esculentum) pollen, suggesting that AtProT1, like LeProT1, may accumulate compatible osmolytes to tolerate dessication as pollen matures (Schwacke et al., 1999). Among genes in the oligopeptide transporter family, OPT9 (At5g53510) expression is exclusively high in microspores and bicellular pollen, in contrast to OPT1 expression in tricellular and mature pollen grains. This pattern suggests these two pollen-specific OPT genes are differentially regulated to transport oligopeptides in the proliferating microspore or in mature pollen (Fig. 2, E.1).

It is important to point out that many genes do not show any specific pattern of expression. Examples include the vacuolar H+-pumping ATPase subunits (VHA; Fig. 2, F.3), H+-pumping pyrophosphatases (AVP1–3), and the mitochondrial carrier (MC) family proteins. These proteins probably support essential cellular processes, such as energy production and vesicle trafficking during microgametogenesis. Expression patterns of most other genes are presented in Supplemental Figure 2.

Promoter∷GUS Activity Confirms Late and Early Pollen-Expression Patterns

We verified microarray results by analyzing promoter∷GUS activity of several CHX genes during flower development. The upstream regulatory regions were transcriptionally fused with the GUS gene and introduced into Arabidopsis plants by floral dip. Transgenic plants were analyzed for promoter activity in flowers at stages 9 to 14, which spans the period from microspore development to mature pollen (Bowman, 1994). All the flower stages shown for each gene were derived from a single inflorescence stained for GUS activity under the same conditions.

CHX24 promoter-driven GUS is active in pollen of flowers at stages 12 through 14; however, stage 10 flowers showed no GUS staining (Fig. 3). These results agree with the microarray data that show that CHX24 (At5g37060) is preferentially expressed in tricellular and mature pollen (Supplemental Table I). Therefore, CHX24 is classified as a late pollen-expressed gene. Similar results were observed for CHX06a (At1g08140) and CHX08 (data not shown). By contrast, we detected CHX17 promoter activity in the anther of stage 10 flowers, but not in pollen of stage 12 to 13 flowers. These results parallel those found in the whole-genome microarray, which indicated low levels of expression in the microspore stage. CHX17 transcript was undetectable in total RNA isolated from mature pollen (Sze et al., 2004). These results support the conclusion that CHX17 is an early pollen-expressed gene.

Comparison of Transcriptome Analyses and Published Functional Studies

To determine if the developmental pollen transcriptome can provide useful insights into transporter gene function, we compared microarray expression data for several genes that have been functionally characterized by other groups (summarized in Table III). Both STP2 and AHA3 are early pollen-expressed genes based on the microarray data (Fig. 2, B.1 and F.1). STP2 is pollen specific, though AHA3 is not. Each belongs to a sizable gene family with more than 10 members; however, other members of the family show little or no expression at the microspore stage, suggesting a primary role of STP2 and AHA3 in microgametogenesis. STP2 was shown to be a monosaccharide/H+ symporter on the PM. Although a stp2 knockout mutant has not been reported, the expression pattern of STP2 clearly points to a role in microspore nutrition. In situ hybridization and immunolocalization show RNA and protein are expressed in the microspore at the beginning of callose degradation before tetrad release. STP2 was suggested to function in sugar import after the microspore is symplastically cut off from tapetal cells (Truernit et al., 1999). AHA3 promoter∷GUS staining confirmed the microarray data that AHA3 is expressed in microspores. Strikingly, heterozygous AHA3/aha3 mutants produce pollen with a 1:1 ratio of full round pollen and smaller, misshapen grains (Robertson et al., 2004). Results would support a model in which the PM AHA3 produces a proton gradient that drives the uptake of sugars and other nutrients required to support the developing microspore.

Table III.
Comparing pollen transcriptome analyses with functional studies of transport genes expressed at different stages of pollen development

STP11, SPIK, and ACA9 genes are specifically or preferentially expressed in pollen late in development according to microarray analyses (Supplemental Table I). Schneidereit et al. (2005) showed that STP11 promoter∷green fluorescent protein is active in the pollen tube. Furthermore, specific antibodies showed STP11 protein is exclusively found in the pollen tube. STP11 is a high-affinity, broad-spectrum monosaccharide/H+ symporter localized at the PM and is thought to supply monosaccharides to the growing pollen tube. Mouline et al. (2002) showed that Shaker K+ channel, SPIK/AKT6, is highly expressed in the grain and tubes according to promoter∷GUS and reverse transcription-PCR analyses of pollen RNA. Moreover, protoplast of homozygous spik-1 mutant grain showed reduced equation M1 channel activity after hyperpolarization relative to wild-type pollen. Knockout mutants also showed decreased pollen tube growth, lending support to the electrophysiological results that SPIK channels provide the main pathway for K+ uptake during pollen tube growth. Recently, the Ca2+-ATPase ACA9 protein fused to yellow fluorescent protein was localized to the PM of pollen tubes (Schiott et al., 2004). Significantly, pollen from homozygous aca9 plants showed reduced tube growth in vivo and a high frequency of aborted fertilization. As PM-bound Ca2+ pumps extrude Ca2+ to the walls, perturbation of extracellular [Ca2+] plus possibly disruption of [Ca2+] dynamics in the cytosol could lead to decreased tube elongation and reduced seed set.

Together, these few examples demonstrate that some early pollen-expressed genes (e.g. AHA3) are critical for development of the microspore to the bicellular stage. Interestingly, some late pollen-expressed genes (STP11, ACA9), with high levels of RNA in mature pollen grains, are expressed as proteins later in the growing pollen tube. These observations support the idea that late pollen-expressed genes influence postpollination events, such as tube growth, fertilization, and seed set.


The male gametophyte presents a simple model for systems biology studies, as its short life span from a microspore to a pollen tube depends on nearly all the major cellular processes of a living organism. Key processes from nutrition, cell division, growth, development, and signaling can be studied as the microspore divides and differentiates to form a mature pollen grain that germinates to deliver sperms to the ovule. A first step in systems biology requires the identification and localization of all the functional components at different stages (Minorsky, 2003). We have presented the first genome-wide analyses of membrane transporters expressed in the male gametophyte at four developmental stages. This type of transcriptomic analysis is particularly significant as nearly all transcripts can be considered expressed during the ontogeny of essentially a single cell type. We have assumed that most of the pollen-expressed genes represent messages found in the microspore and the vegetative cell, though it remains possible that a minor fraction of messages expressed late in pollen development is the product of generative or sperm cells (Engel et al., 2003). A systematic identification of transporters is an important first step to discover how transport of ions and metabolites is integrated with the diverse phases of pollen development. We show that the expression of genes encoding known and putative membrane transporters are overrepresented in mature pollen relative to the microspore, underscoring the significance of transporters in the maturation and progamic phases of pollen development (Honys and Twell, 2004; Pina et al., 2005).

Validity of Pollen Transcriptome Analyses

Microarray results of Arabidopsis plants often yield variable results in different studies, so the transporter genes that we identified as specifically or preferentially expressed in pollen (Table II) were compared with those identified recently in mature pollen alone by Pina et al. (2005). Instead of using Gene Ontology terms, which collected 671 transport genes (Pina et al., 2005), we compiled a list of 1,269 classified transporter genes based on gene family classification obtained from three transport databases (AMPL, Aramemnon, and PlantsT), published papers, and investigator Web sites. The criterion used to define pollen-specific genes was similar in both studies, though the methods differed. Exclusive calculation of detection calls in biological replicates were used in this study, whereas Pina et al. (2005) scored genes as expressed when a gene gave a positive detection call in at least one of several replicates. Furthermore, we assigned genes as preferentially expressed in pollen if they showed at least 3-fold higher expression relative to the highest level in any sporophytic tissue, rather than the 1.2-fold minimum used by Pina et al. (2005) for pollen-enriched genes. Thus, approximately 43 of 94 pollen-selective and/or -enriched genes identified by Pina et al. (2005) were absent from our Table II, probably due to the small number of sporophyte datasets used (four arrays and two arrays from siliques). We used a large number of sporophyte datasets consisting of 75 microarrays (Supplemental Table I) from 12 tissues; consequently, our estimate of pollen-specific or -preferential genes is decreased. By analyzing only mature pollen, Pina et al. (2005) missed the early pollen-specific genes. The different methods used to select pollen-specific or pollen-enriched genes probably override variations resulting from the different algorithms (i.e. MAS4 or MAS5 detection calls) used to compute the normalized expression levels of the pollen transcriptome data and from use of two different ecotypes, Columbia (Col-0) and Landsberg erecta (Ler). Regardless of the methods used, 51 genes overlapped in both studies as specific or enriched in mature pollen (see asterisk in Table II).

Other lines of evidence provide strong support for the validity of the normalized pollen transcriptome results used in our analyses. First, a portion of the pollen-preferential genes has been verified by PCR amplification of reverse-transcribed messages isolated from mature pollen and by promoter∷GUS analyses (Honys and Twell, 2003; Sze et al., 2004). Second, we showed that promoter activity of the CHX17 and CHX24 genes correspond to early and late expressed genes, respectively, which is consistent with the coexpression clusters of the pollen transcriptome. Third, differential expression of discrete genes in pollen seen in microarrays confirms previous studies by others. For instance, STP2 mRNA and protein were localized in microspores by in situ hybridization and immunohistochemical staining but not in tricellular or mature pollen (Truernit et al., 1999). In contrast, STP11 protein is exclusively in the pollen tube, but not in pollen grains (Schneidereit et al., 2005). As transcriptomic results indicated STP11 peaks at the mature pollen stage, these results support the idea that many messages are stored until pollen germination. Moreover, AHA3 promoter-driven GUS activity occurred in early and vacuolated microspores, as well as microspores undergoing the first mitosis, but was absent from the mature grains (Robertson et al., 2004), consistent with the early pollen expression of AHA3 in transcriptome data. Together, these results verify the developmentally regulated expression seen in the pollen transcriptome for genes encoding channel, cotransporters, and pumps, and emphasize the value of microarray data derived from highly purified populations of viable spores at defined stages (Honys and Twell, 2004).

Distinct Functions of Early and Late Genes

Although the functions of only a few of the transporter genes have been studied in pollen so far, the results strongly indicate that genes specifically or preferentially expressed in pollen (Table II) serve critical roles for pollen maturation or pollen tube growth. Examples include (1) knockout mutants of an inward-rectifying K+ channel, SPIK/AKT6, which showed decreased pollen tube growth (Mouline et al., 2002); (2) impaired Ca2+ efflux from the pollen tube decreased pollen tube growth, fertility, and seed set in homozygous aca9 mutants (Schiott et al., 2004); (3) a monosaccharide/H+ symporter that was exclusively expressed in pollen tube PM (Schneidereit et al., 2005); and (4) loss of function in a late pollen-expressed Cu2+ pump gene, RAN1, resulted in male gametophyte infertility (Woeste and Kieber, 2000). These few examples (Table III) show that late pollen-expressed genes play critical roles in K+ and monosaccharide nutrient uptake for tube growth, and that maintaining Cu2+ homeostasis, extracellular [Ca2+], and cytosolic Ca2+ dynamics are important for tube growth, fertilization, and seed set.

If so, other genes showing specific or preferential expression in pollen from Table II are promising candidates for detailed functional studies. Ca2+ gradients and oscillations accompany tip growth, suggesting that putative Ca2+ channels, like CNGC proteins, and Ca2+ pumps, like ACA7, are involved. Given the role of pH oscillation in tube growth (Messerli and Robinson, 1998; Feijo et al., 1999), the specific roles of PM H+ pumps (e.g. AHA6, AHA8, and AHA9), H+-coupled cotransporters (e.g. CHX), and anion channels (e.g. CLC) that alter pH and/or membrane potential across the PM or intracellular compartments are particularly exciting. Boron is essential for in vitro pollen germination; thus, At5g25430 (Table II), a gene related to BOR1 (Takano et al., 2002), is a prime candidate for a pollen tube boron exporter. The roles of tonoplast water channels, such as TIP1.3 and 5.1, in pollen grain dessication and tube growth also need to be investigated.

In addition, transporter genes expressed in sporophytic tissues are also crucial, especially when they are selectively expressed in pollen relative to other members of the family at a developmental stage. For instance, SUC1 (At1g71880) is a PM-localized H+/Suc symporter widely expressed in vegetative organs. Yet transcriptome results show it is the only member of the SUC family highly expressed at the tricellular pollen stage (Fig. 2, B.2). Pollen expression was shown previously by in situ hybridization and promoter∷GUS (Stadler et al., 1999). hap3 mutant pollen germinates but the pollen tube fails to exit the style (Johnson et al., 2004), suggesting a critical role of SUC1 in accumulating Suc for pollen tube growth. The PM proton pump AHA3, known to reside in phloem companion cells (DeWitt and Sussman, 1995), is selectively expressed in microspores. Only half the microspores from heterozygous aha3 mutants develop into mature pollen grains. This example suggests that a distinct role of the PM AHA3 is to produce a proton gradient to drive uptake of sugars and other nutrients (Robertson et al., 2004) to nourish developing microspores.

Many other transporter genes expressed early in pollen development are most likely important for microspore proliferation or cell division. For instance, STP2 is preferentially expressed in microspores and bicellular pollen, and this H+-coupled monosaccharide symporter protein appears at the beginning of callose degradation when the microspores are released from the tetrad (Truernit et al., 1999). Other genes corepressed with STP2 identified in our study include WBC09, COPT3, and OPT9, as well as other non-pollen-specific genes.

However, functional analyses using mutants may depend on the absence of similar transporters and the sensitivity of the functional assay. We have begun testing single homozygous chx mutants containing a single T-DNA insertion. So far, there are little or no obvious phenotypic changes in flowering, in vitro pollen germination, or seed set. This may not be surprising given the multiplicity of CHX genes in mature pollen, so double mutants are being tested. A voltage-independent K+ channel, KCO4/TPK4 (At1g02510), was recently localized to the PM of pollen tubes. The channel is modulated by Ca2+ and pH and is thought to have roles in K+ homeostasis and membrane voltage control. However, knockout mutants did not show any apparent growth phenotype, though the ratio of the instantaneous to steady-state current was slightly lower in mutant than in wild type (Becker et al., 2004).

Constitutively Expressed Transporter Genes

Notably, many genes are constitutively expressed during microgametogenesis, suggesting they serve essential needs at all stages and have high turnover rates. For instance, many transporters in the MC family are highly expressed at all stages, most likely to supply a constant need for energy and metabolites. Proton pumps, V-ATPase and pyrophosphatase, acidify endomembrane compartments of the secretory system, including the vacuole, Golgi, and intracellular vesicles (Sze et al., 1999), so are probably required through all stages of gametophyte development. Several ion channels, including putative mechanosensitive channels (MscS) and putative Glu ionotropic channels (GLR), appear to be expressed at all stages. Multiple genes encoding nearly identical (e.g. VHA-c1–c5) or homologous proteins with similar membrane localization may compensate for defects in one gene. However, a mutant of a single gene is predicted to compromise male fertility or cause male gametophyte lethality as seen with vacuolar H+-ATPase peripheral subunit A, vha-A, mutant (Dettmer et al., 2005) and with mia mutants of a P(5) P-type cation-pumping ATPase (Jakobsen et al., 2005). Our analysis showed that several singletons are expressed at all stages of pollen development. One example is TPC1, the vacuolar Ca2+-activated calcium channel known from electrophysiological studies as the slow vacuolar channel (Peiter et al., 2005). These transporters are probably integrated with male gametophyte development through signaling networks that modulate their transport activity in a temporal and spatial manner.

Unknown Proteins and Potential Receptors in Pollen

In the process of classifying proteins on the master list, more than 100 proteins that were previously unclassified or labeled as hypothetical or expressed have been tentatively assigned as members of a protein family based on results of BLASTP at the Transport Classification Database (TCDB) Web site (see “Materials and Methods” and Supplemental Table I). However, there are still 482 predicted proteins (out of 1,751) that are unclassified within the TC system. Approximately 300 of the unknown proteins are expressed in pollen, and 9% (26 genes) are specifically or preferentially expressed in pollen. These represent novel polytopic proteins with potentially unique roles in pollen tube guidance or growth. For instance, At3g20300 predicts a protein with approximately seven transmembrane domains. According to the Universal Protein Resource (UniProt; http://www.pir.uniprot.org/), it is 23% similar and 12% identical to olfactory receptors from Drosophila (DmOR83c, or Locus Tag CG15581; Vosshall et al., 1999). A homolog in rice (Oryza sativa), Os08g41000, has an e value of 2e-43 and 45% similarity with At3g20300. Regardless of whether these act as odorant receptors, the pollen tube depends on diverse chemical cues to guide its path to the ovule, raising the exciting possibility that several unknown proteins, such as At3g20300, the ADIPOR-like At4g30850, and MLO05 (At2g33670) and MLO09 (At1g42560; Devoto et al., 2003), encode novel receptors in the male gametophyte.


Transcriptomic analysis of all transporter genes is an important first step to identify transport proteins that participate in pollen development, postpollination events, or both. Our analysis has revealed many specific transporter genes, including (1) those that are specifically or preferentially expressed in the male gametophyte relative to the sporophyte; (2) gene(s) preferentially expressed in pollen relative to other members of its gene family; and (3) those that are expressed early or late in pollen development. Expression and phenotypic analyses of a few transport genes support the idea that early and late pollen-expressed genes have distinct functions in pollen development, maturation, or postpollination events. Thus, these findings provide the groundwork to streamline functional studies of many specific transporter genes and an opportunity to discover the functions of unknown proteins, including putative membrane receptors. Transport of ions and metabolites is intimately connected with signal transduction, cell wall metabolism, cytoskeleton rearrangement, and vesicle trafficking. Thus, this type of analysis, coupled with studies to determine transport activity, protein spatial distribution, and interacting partners, will provide insights toward understanding the systems biology of the male gametophyte.


Plant Materials and Growth Conditions

Arabidopsis (Arabidopsis thaliana) ecotypes Col-0 and Ler were used in this study. Wild-type and transgenic seeds were sterilized according to published procedures (Boyes et al., 2001). In general, growth conditions in the light incubators were as follows: light/dark cycle 16 h/8 h, light intensity 150 μmol s−1 m−2 photosynthetically active radiation, and temperature 22°C/20°C.

Genome-Wide Microarray Analyses of Pollen

Spore Isolation

Mature pollen was isolated according to Honys and Twell (2003). Isolated spores from three stages of immature male gametophyte were obtained from Arabidopsis ecotype Ler as described before (Honys and Twell, 2004; Sze et al., 2004). Briefly, mixed spores from inflorescences removed of open flowers were separated with two sequential Percoll step gradients. The relative purity and viability of isolated fractions was determined by light and fluorescence microscopy using 4′,6-diamino-phenylindole and fluorescein 3′,6′-diacetate staining.

DNA-Chip Hybridization

Briefly, total RNA extracted from spores at each developmental stage using the RNeasy plant kit (Qiagen) was used to synthesize double-stranded cDNA. The cDNA was then transcribed in vitro to obtain biotin-labeled complementary RNA in the presence of biotinylated UTP and CTP as described (Honys and Twell, 2004). Arabidopsis ATH1 genome arrays were hybridized with biotinylated target complementary RNA for 16 h at 45°C. Microarrays were stained with Streptavidin-Phycoerythrin solution and scanned with an Agilent 2500A GeneArray scanner (Agilent Technologies).

Data Analysis

For gametophytic developmental stages, two biological replicates from independent plant populations were used to ensure reliability and reproducibility. Seventy-five sporophytic datasets (see supplemental “Materials and Methods” section), representing 12 vegetative tissues (seedlings, whole plants, shoots, leaves, guard cell-enriched leaf extracts, petioles, stems, hypocotyls, xylem, cork, root hair zone, and roots), from public baseline GeneChip experiments were downloaded from the NASCArray database (http://www.arabidopsis.info; Craigon et al., 2004) for comparison to the pollen transcriptome (Honys and Twell, 2004). Duplicates or triplicates were used in each experiment. All gametophytic and sporophytic datasets were normalized using the freely available sChip 1.3 software (http://www.dchip.org/) as described previously (Honys and Twell, 2004). The reliability was ensured by the normalization of all 75 arrays to the median probe intensity level and the use of normalized CEL intensities of all arrays for the calculation of model-based gene-expression values based on the Perfect Match-only model. For the treatment of biological replicates, an exclusive approach was used. If a specific gene had a Detection call “P” in all replicates of an experiment, then it was scored as expressed. When expression was absent in any of the replicates, a value of 0 was given to that gene for that particular dataset (Honys and Twell, 2004). A mean value was then calculated for sporophytic tissues for which multiple datasets were available. Suspension-cultured cell was not included as a sporophytic tissue in our analysis.

Analyses of Transporters in the Pollen Transcriptome

Identifying and Classifying Transporters

To obtain a comprehensive list of predicted membrane proteins in Arabidopsis, Ward (2001) assembled the AMPL (http://www.cbs.umn.edu/arabidopsis/) by utilizing a series of protein sequence analysis programs. Of the 27,288 predicted proteins available from The Institute for Genomic Research at the time, 4,752 were found to have at least two transmembrane domains as predicted by HMMTOP (Tusnady and Simon, 1998). The Needleman and Wunsch (1970) programming method was used to create a similarity matrix for all pairs of the 4,752 proteins (Ward, 2001). A Perl program called cluster.mem read the resulting matrix and extracted groups of protein sequences. These groups were collected into distinct protein families by a Perl program called cfoo5; 607 families were numbered using this method (Ward, 2001).

Proteins were then added or subtracted based upon three factors: (1) the presence or absence in isospecic homolog clusters from the Aramemnon membrane protein database, release 3.0 (http://aramemnon.botanik.uni-koeln.de/; Schwacke et al., 2003); (2) protein family membership in the TC system at the PlantsT database (Tchieu et al., 2003); and (3) additional transmembrane domain predictions, specifically ConPred_II and Aramemnon's consensus prediction (the latter combines the results of 16 programs). With exceptions made for single, truncated proteins of larger families, three transmembrane domains were chosen as a suitable threshold for separating putative transporters from membrane-associated proteins. This threshold was based upon predictions for known transporters. Using those approaches, a first master list of 1,604 proteins was obtained. This original list was then compared to a list of 2,286 membrane proteins from Aramemnon, which included 960 proteins classified by the TC system and 1,326 polytopic proteins (three or more transmembrane domains) that were unclassified. Using both the protein descriptions and isospecic homolog clusters from Aramemnon, 147 proteins were extracted from the Aramemnon list. The final master list (Supplemental Table I) of transporter proteins contains 1,751 sequences, including 482 that are labeled as unknown or without a family designation from either AMPL or Aramemnon.

Using AMPL protein family numbers as a base, proteins in the master worksheet were further classified using Aramemnon and Saier's TCDB (http://www.tcdb.org/; Busch and Saier, 2004). The Aramemnon database groups proteins as isospecic homologs when a minimum threshold of 20% similarity (and at least 20% coverage) is reached in alignments performed using the Smith-Waterman algorithm (FASTA 3 tools; Pearson, 1996; Schwacke et al., 2003). Occasionally, two or more family numbers from AMPL were combined (into one family number) if representative members of each family were grouped as isospecic homologs by Aramemnon with at least 25% similarity, the same threshold used by Ward (2001).

TC codes and descriptions were also originally obtained from Aramemnon for all proteins from the master worksheet. Unknown proteins listed as “unclassified” by Aramemnon were searched against protein sequences in the TCDB using BLASTP (default parameters) at the TCDB Web site. E values below e-20 were considered significant, and those proteins were classified as members of the family with the highest-scoring BLAST result. Proteins were nominated as putative members of a family (designated with a superscript “a” next to their TC code) when e values between e-04 and e-20 were achieved. All remaining unclassified proteins were blasted at UniProt (Bairoch et al., 2005), in order to gather information about putative functions from several resources, such as Inter-Pro-EMBL (http://www.ebi.ac.uk/interpro/), PFAM (http://www.sanger.ac.uk/Software/Pfam/), and Protein Information Resource (PIR; http://pir.georgetown.edu/). Revised information on the list of classified transporter genes from Arabidopsis and their expression in pollen will be available under Arabidopsis 2010 at http://www.life.umd.edu/CBMG/faculty/sze/lab/index.html.

Transporter Genes Expressed in Pollen

Expression data from the pollen and sporophyte transcriptomes were incorporated into the master sheet by creating a query in Microsoft Office Access 2003, SP1, which extracted columns of data from Honys and Twell's updated supplementary data file 1 (July 2005), and inserted this data into the corresponding rows for each gene in the master sheet. Some of the putative transporter genes were not included on the ATH1 chip and have no data shown. To identify genes with specific and preferential expression in the male gametophyte, the expression data were analyzed in Microsoft Office Excel 2003, SP1. First, maximum pollen (“MaxPollen”) and maximum sporophytic (“MaxSpor”) expression signals were extracted for each gene on the ATH1 chip. The MaxPollen:MaxSpor ratio was then calculated for each gene to determine the fold difference in expression between pollen and sporophytic tissues. Expression was defined as specific if an expression signal was present in any stage of the male gametophyte (MaxPollen > 0.00) and an expression signal was absent from all 12 sporophytic tissues (MaxSpor = 0.00). Preferential expression was defined as maximum pollen expression being at least 3 times greater than maximum sporophytic expression (MaxPollen:MaxSpor > 3.00).

The 3× increase in expression was arbitrarily chosen as a suitable cutoff to indicate genes with preferential expression in pollen for the following reasons. When the commonly used 10×, 5×, and 3× cutoffs were applied to determine pollen-preferential expression, the number of detected genes was 42, 72, and 93, respectively. However, when a 2× cutoff was used, the number of pollen-preferential transporters was 135, which is disproportionately high. Furthermore, due to cell-type heterogeneity in sporophytic tissues or organs, transcriptomic data of pollen and of complex sporophytic organs are not strictly comparable by statistical means, even when normalized with the best available method. Expression in pollen is mainly from a single cell type, whereas expression in organs includes multiple cell types. Thus, the relative transcript level from one cell type could be considerably diluted in sporophytic tissues. Given this uncertainty, pollen specific and pollen preferential used in this article should be viewed as relative working terms. The normalized data are provided in Supplemental Table I for other users to apply their own criteria for analyzing a subset of genes.

To group male gametophyte-expressed genes with similar expression patterns and therefore discover those most likely to be coregulated, all pollen-expressed genes were clustered using the EPCLUST program with a threshold value of 0.05 (Honys and Twell, 2004). This method yielded 39 unique clusters. Cluster numbers, from Honys and Twell's supplemental table I, were added to the master sheet by creating another query in Microsoft Office Access.

Promoter∷GUS Reporter Analyses

To examine the precise gene-expression patterns of AtCHX members, promoter regions upstream of the ATG start codon were transcriptionally fused with GUS to generate the CHX∷GUS reporters. Promoter fragments of CHX17 and CHX24 were amplified by PCR from Col-0 genomic DNA isolated from 3-week-old seedlings using Expand High Fidelity PCR system (Roche). The primers used to generate the 2,034-bp CHX17 promoter region were 5′-CGCGTCGACTTCGTCAAGTGTCAACGAGCTTCATAG-3′ (appended SalI site is underlined) and 5′-CGCGGATCCGCTTTAAAGATCTGACAAATGATGAAT-3′ (appended BamHI site is underlined). The primers used to generate CHX24 (1,638 bp) are 5′-CGCGTCGACTCTGGAAAGTGTAGTAGTCATGCGTACCG-3′ (appended SalI site is underlined) and 5′-CGCGGATCCGCAAACTAATCTTTCATAATAAGATTTGA-3′ (appended BamHI site is underlined). The SalI-BamHI PCR fragments of CHX17 and CHX24 promoters were cloned into the plasmid pRITA I, and then subcloned into the binary vector pMLBart as described (Sze et al., 2004). Using the floral-dip method of Agrobacterium-mediated plant transformation (Clough and Bent, 1998), these binary vectors were transformed into Arabidopsis Col-0 ecotype plants. Transgenic progenies were selected in soil by spraying 0.05% phosphoinothricine (BASTA) on 1-week-old seedlings. Ten independent T1 lines for each construct were obtained, and at least five independent homozygous T2 lines for each construct were examined for GUS expression.

Histochemical assays for GUS activity in T2 or T3 generation of Arabidopsis transgenic plants were performed according to the protocol described previously (Sze et al., 2004). Inflorescence from 6- to 8-week transgenic plants were rinsed with staining buffer lacking 5-bromo-4-chloro-3-indolyl β-d-glucuronide (50 mm sodium phosphate, pH 7.2, 0.5 mm K4Fe[CN]6, 0.5 mm K3Fe[CN]6), and then incubated for 24 to 48 h at 37°C in staining buffer containing 1 mm 5-bromo-4-chloro-3-indolyl β-d-glucuronide. To clear chlorophyll from the green tissues, the stained tissues were incubated in 70% ethanol overnight. GUS staining patterns were recorded using a Nikon Eclipse E600 microscope (Nikon Instruments) equipped with a differential interference contrast lens. Images were processed using Adobe Photoshop software (version 5.5; Adobe Systems).

Supplementary Material

Supplemental Data:


We thank Lai-su Lee and Raja Mazumder from the PIR for help in identifying several unannotated proteins. We gratefully acknowledge Rainer Schwacke for providing the list of Aramemnon membrane proteins from release 3.0; Yi-Fang Tsay (Academia Sinica, Taipei), Anke Reinders (University of Minnesota), and Norbert Sauer (Erlangen University) for help in classifying H+-coupled transporters; and Sue Rhee (Carnegie Institute of Washington) for providing valuable suggestions.


1This work was supported in part by the National Science Foundation (Arabidopsis 2010 grant nos. 0209792 to J.M.W., 020977 to K.D.H., and IBN0209788 to H.S.) and by the U.S. Department of Energy (grant no. DEFG0295ER20200 to H.S.). D.T. and D.H. gratefully acknowledge support from the Royal Society Joint Project (grant no. 2004/R3–EU). D.H. was supported through a Grant Agency of the Academy of Sciences of the Czech Republic (grant no. KJB6038409). D.T. gratefully acknowledges support from the Biotechnology and Biological Sciences Research Council and the GARNet transcriptome center at the Nottingham Arabidopsis Stock Centre for performing pollen microarray hybridizations.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Heven Sze (ude.dmu@ezsh).

[W]The online version of this article contains Web-only data.



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