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. 2022 Jul 29;236(2):525–537. doi: 10.1111/nph.18368

Sucrose rather than GA transported by AtSWEET13 and AtSWEET14 supports pollen fitness at late anther development stages

Jiang Wang 1,*, Xueyi Xue 1,*, Houqing Zeng 1,2, Jiankun Li 1, Li‐Qing Chen 1,✉
PMCID: PMC9795879  PMID: 35811428

Summary

  • Both sugar and the hormone gibberellin (GA) are essential for anther‐enclosed pollen development and thus for plant productivity in flowering plants. Arabidopsis (Arabidopsis thaliana) AtSWEET13 and AtSWEET14, which are expressed in anthers and associated with seed yield, transport both sucrose and GA. However, it is still unclear which substrate transported by them directly affects anther development and seed yield.

  • Histochemical staining, cross‐sectioning and microscopy imaging techniques were used to investigate and interpret the phenotypes of the atsweet13;14 double mutant during anther development. Genetic complementation of atsweet13;14 using AtSWEET9, which transports sucrose but not GA, and the GA transporter AtNPF3.1, respectively, was conducted to test the substrate preference relevant to the biological process.

  • The loss of both AtSWEET13 and AtSWEET14 resulted in reduced pollen viability and therefore decreased pollen germination. AtSWEET9 fully rescued the defects in pollen viability and germination of atsweet13;14, whereas AtNPF3.1 failed to do so, indicating that AtSWEET13/14‐mediated sucrose rather than GA is essential for pollen fertility.

  • AtSWEET13 and AtSWEET14 function mainly at the anther wall during late anther development stages, and they probably are responsible for sucrose efflux into locules to support pollen development to maturation, which is vital for subsequent pollen viability and germination.

Keywords: GA transport, pollen germination, pollen viability, sucrose transport, SWEET13, SWEET14

Introduction

Pollen development and formation are vital for the reproduction of flowering plants (Ma, 2005). Developing pollen requires a sugar supply from the somatic cell layers of anthers because immature pollen is well‐surrounded by locules in the anthers. The somatic cell layers consist of the outermost epidermal cell layer, the endothecium, the middle cell layer and the innermost tapetum (van der Linde & Walbot, 2019). The male gametophyte is symplasmically isolated from the anther cells, resulting from dissociation between meiotic cells and the tapetum (Ma, 2005; Borghi & Fernie, 2017). Plasma membrane‐localized carrier proteins are needed for sugar export from the somatic cell layers of the anther and import into pollen (Borghi & Fernie, 2017). Sucrose is symplasmically unloaded into the connective tissues of the anthers from the phloem, largely following a long‐distance sugar transport starting from source tissues. Sucrose is either exported to the locules directly or hydrolyzed into hexoses by invertase before being exported to the locules. However, sugars have to cross different layers in the anther wall to reach the locules. Tapetal cells are structurally disconnected from the adjacent middle cell layer (Clément & Audran, 1995), which indicates that sugars must be apoplasmically transported from the middle cell layer into the tapetum cells, and from there, exported into the locules, finally reaching the pollen.

Sugars Will Eventually Be Exported Transporters (SWEETs), primarily transporting both mono‐ and di‐saccharides, are involved in many biological processes associated with apoplasmic transport routes, such as phloem loading and nectar secretion (Chen et al., 2012; Lin et al., 2014; Xue et al., 2022). Sucrose Uptake Transporters (SUTs/SUCs), mainly transporting sucrose, often are paired with SWEETs in many processes (Braun, 2022). In tomato plants, SlSWEET5b mediates hexose export into the locules; unsurprisingly, the SlSWEET5b silencing mutant exhibits reduced pollen germination and seed production (Ko et al., 2022). In rice, OsSUC1 is highly expressed in the wall of the anther, and the ossuc1 mutant shows impaired pollen function without affecting pollen maturation (Hirose et al., 2010). OsSWEET11a and OsSWEET11b are expressed in anther veins, and ossweet11a;11b mutant is male sterile (Wu et al., 2022). In cucumber, the knockdown mutant of tapetum‐ and pollen‐localized CsSUT1 shows male sterility (Sun et al., 2019). AtSUC1 is expressed in the connective tissue of anther and mature pollen, and the atsuc1 mutant produces defective pollen resulting in a low pollen germination rate (Stadler et al., 1999; Sivitz et al., 2008). In Arabidopsis, AtSWEET8 (RPG1; Ruptured Pollen Grain 1) that transports glucose and AtSWEET13 (RPG2) that transports sucrose with their transcripts detected in the tapetum of anthers are involved in primexine deposition, microspore development and subsequent seed formation (Guan et al., 2008; Sun et al., 2013; Xue et al., 2022). The mutant rpg1 (atsweet8) exhibits defective microspore development, and the double mutant rpg1rpg2 (atsweet8;13) results in an almost sterile phenotype (Sun et al., 2013).

Besides sugars, developing pollen also requires gibberellin (GA) for its viability and development (Plackett et al., 2011). The tapetum of the anthers and pollen grains are positioned as major sites for GA synthesis during flower development (Itoh et al., 1999). For example, the Arabidopsis GA biosynthesis‐deficient mutant ga1‐3 is male sterile (Sun et al., 1992). Likewise, the triple mutant gid1a;b;c of GA receptor GID1 (Gibberellin‐Insensitive Dwarf 1) showed a dwarf phenotype and male sterility (Griffiths et al., 2006). GA movement across different cells or tissues has been reported in many biological processes, which often involve GA transporters (Binenbaum et al., 2018). Deficiency in GA transport also affects anther development and fertility. For instance, AtGTR1/NPF2.10 (Glucosinolate Transporter 1; Nitrate transporter 1/Peptide transporter Family 2.10) transports jasmonoyl‐isoleucine (JA‐Ile) and GA in addition to glucosinolates. The gtr1 mutant has reduced fertility due to impairment in filament elongation and anther dehiscence, and these phenotypes can be rescued by exogenous GA application (Saito et al., 2015).

Unexpectedly, plasma membrane‐localized AtSWEET13 and AtSWEET14 have been shown to transport GA (Kanno et al., 2016) after they were well‐characterized as sucrose transporters (Chen et al., 2012). Besides AtSWEETs, OsSWEET3a also has been found to transport GA in addition to 2‐deoxy‐glucose (Morii et al., 2020). The atsweet13;14 double mutant has been shown to have a defect in anther dehiscence, which can be rescued by exogenous application of an excess amount of GA (Kanno et al., 2016). These results suggested that GA might be the predominant substrate of AtSWEET13 and AtSWEET14 in anther. However, it is uncertain whether this rescue is a consequence of altered GA transport or the result of an indirect effect of GA on sugar transport, because GA and sugar are interconnected in signaling pathways of many developmental processes, including flowering (Matsoukas, 2014). To clarify which substrate of AtSWEET13 and AtSWEET14 is important in anther to support pollen development, we conducted complementation of atsweet13;14 assays using AtSWEET9 that transports sucrose but not GA (Lin et al., 2014; Kanno et al., 2016; Wu et al., 2022) and using a GA transporter AtNPF3.1 individually under the control of the AtSWEET14 promoter (David et al., 2016; Tal et al., 2016). As a result, AtSWEET9 was able to fully rescue the defective pollen germination and viability phenotypes, whereas AtNPF3.1 failed to do so. Our data suggest that sucrose transported by AtSWEET13 and AtSWEET14 is vital to pollen viability and male fertility in Arabidopsis. In addition, our cross‐section results from the GUS reporter line and starch accumulation comparison between Col‐0 and atsweet13;14 support that AtSWEET13/14 mediate sucrose release from endothecium to locule at the late stages of anther development when the tapetum has degenerated.

Materials and Methods

Plant materials and growth conditions

Arabidopsis (Arabidopsis thaliana (L.) Heynh.) Col‐0 plants were grown under a controlled condition with a constant temperature of 22°C, light intensity of 100–150 μmol m−2 s−1, and a 16 h : 8 h, light : dark photoperiod. T‐DNA mutants of atsweet13 (SALK_087791) and atsweet14 (SALK_010224) were obtained from ABRC. Homozygous lines were genotyped using primers of P1‐P4 (Supporting Information Table S1) and used in related experiments. The floral dip transformation method (Clough & Bent, 1998) was used to generate all of the transgenic lines described in this study. At least 16 T1 lines were generated for each construct, and at least two randomly selected lines were propagated to generate T3 homozygous seeds.

Constructs for localization and complementation

The 5′ upstream promoter fragments before ATG of AtSWEET13 (2083 bp) and AtSWEET14 (3798 bp) were amplified using gene‐specific primers (P5–P8) with an XbaI site added to the reverse primers. The promoter fragments each were cloned into the entry vector pDONR221‐f1 via the BP reaction to make pDONR221‐f1/pSWEET13 and pDONR221‐f1/pSWEET14. The CDS of AtSWEET13, AtSWEET14, AtSWEET9 and AtNPF3.1 each were amplified using their own specific primers (P9‐P16) and then seamlessly subcloned to generate the corresponding constructs pDONR221‐f1/pSWEET13:SWEET13, pDONR221‐f1/pSWEET14:SWEET14, pDONR221‐f1/pSWEET14:SWEET9 and pDONR221‐f1/pSWEET14:NPF3.1 by In‐Fusion® (TaKaRa, Shiga, Japan) after the linearization at the XbaI restriction site. The resulting entry vectors were subjected to LR reaction with the destination vectors pBGGUS and pHGY (Kubo et al., 2005) to make pBGGUS/pSWEET13:SWEET13‐GUS, pBGGUS/pSWEET14:SWEET14‐GUS, pHGY/pSWEET13:SWEET13‐YFP, pHGY/pSWEET14:SWEET14‐YFP, pHGY/pSWEET14:SWEET9‐YFP and pHGY/pSWEET14:NPF3.1‐YFP (GUS, β‐glucuronidase; YFP, yellow fluorescent protein). For the GUS localization assay, pBGGUS constructs were transformed into Col‐0. For complementation and fluorescence assay, pHYG constructs were transformed into atsweet13;14 double mutant.

Reciprocal crossing and silique imaging

Pollen from either Col‐0 or atsweet13;14 was taken to cross with the stigma of either Col‐0 or atsweet13;14. Siliques were cleared with a solution containing 1% SDS and 0.2 M NaOH for 2 d, followed by imaging using a dissecting microscope after they were collected 14 d from crossing. At least five siliques were generated from each combination for each of the three independent trials. Two representative siliques for each combination were shown.

In vitro pollen germination

The in vitro pollen germination assay was conducted according to a previously described method (Wang et al., 2022b). Three independent repeats were conducted.

Soluble sugar quantification

Pollen grains from 100 flowers at stage 13 were used for soluble sugar quantification. Each sample was treated with 1 ml ethanol (80% v/v) and then mixed by vortexing for 30 s. The samples were kept at 80°C for 40 min and inverted to mix every 10 min. The supernatant was transferred into a new tube after being centrifuged at 16 000  g for 15 min. The extraction process was repeated twice for each sample. The sugar‐containing supernatant was kept at 42°C for evaporation before being re‐dissolved in 0.15 ml milli‐Q water. After filtering through a nylon membrane (0.22‐μm), the samples were analyzed by high‐performance liquid chromatography (HPLC) (1200 series; Agilent Technologies, Santa Clara, CA, USA) using a separation column containing Rezex™ RCM‐Monosaccharide Ca2+ (8%) (Phenomenex, Torrance, CA, USA). The average of three independent repeats was used for calculation.

Fatty acids measurement

The total fatty acid profiles from 0.5 mg freeze‐dried pollen samples were determined using a direct acid‐catalyzed transmethylation method with minor modifications (Browse et al., 1986). Samples were placed in Teflon‐lined screw‐capped glass tubes containing 1 ml 5% H2SO4 (v/v) in methanol (freshly prepared), 50 μg butylated hydroxytoluene (BHT) and 5 μg C15:0 pentadecanoic acid as internal standard, and then mixed with 300 μl of toluene as cosolvent followed by vortexing for 60 s. Samples were heated at 85°C for 2 h. After cooling down to room temperature, 1 ml 0.9% NaCl (w/v) and 1 ml hexane were used to extract fatty acid methyl esters (FAME). Samples were vortexed for 60 s and centrifuged to accelerate phase separation. The upper organic phase (FAME containing) was transferred into a 2‐ml injection vial. Samples were injected into a gas chromatography (GC) apparatus (Agilent) with a flame ionization detector (FID) using DB‐Wax 30 m column (30 m × 0.25 mm × 0.25 μm; J&W Scientific, Folsom, CA, USA). The GC conditions were set as follows: split mode injection (1 : 2); injector and flame ionization detector temperature, 260°C; oven temperature program 150°C for 3 min, then increasing at 10°C min−1 to 240°C and holding this temperature for 5 min. The average of four independent repeats was calculated.

GUS histochemical analysis

GUS staining was performed as described previously (Chen et al., 2012). Entire inflorescences of 6‐wk‐old plants were collected for histochemical GUS staining.

Paraffin‐embedded cross‐sectioning

Paraffin‐embedded cross‐sectioning was performed as described with minor modifications (Zhang et al., 2021). GUS‐stained samples of entire inflorescences from pSWEET13:SWEET13‐GUS and pSWEET14:SWEET14‐GUS plants were washed three times with PBS (pH 7.2) and fixed with cold 4% paraformaldehyde and 2.5% sucrose buffer overnight. Samples then were embedded in paraffin, followed by sectioning and imaging as described previously (Zhang et al., 2021).

Microscopy imaging

A Zeiss Apotome.2 (Carl Zeiss) was used for fluorescent signal acquisition. As a result of the strong autofluorescence observed under the YFP filter set, the green fluorescent protein (GFP) filter set was used to lower the autofluorescence background. Different FL filter sets (GFP: 470/20 nm excitation, 505–530 nm emission; RFP: 546 nm excitation, 590 nm emission) were used to image samples as needed. Image acquisition parameters were held consistent. For the confocal microscope, all images were taken using a LSM 710 (Carl Zeiss). Argon laser excitation wavelength and emission bandwidths were 514 nm (10% intensity) and 518–565 nm for YFP (gain 750), and 514 nm (10% intensity) and 619–697 nm for chlorophyll autofluorescence (gain 450), respectively. A Field‐Emission Environmental Scanning Electron Microscope (ESEM‐FEI; Thermo Fisher Scientific, Waltham, MA, USA) with Energy‐Dispersive Spectroscopy (EDS) on Wet mode was used for SEM imaging of anthers at different stages.

Pollen viability assay

Pollen viability assay was conducted as described previously (Muhlemann et al., 2018) with minor modifications. For each repeat, 50 flowers at stage 13 for each genotype were collected into a 2‐ml tube, immersed in 1 ml of liquid pollen viability solution (PVS, 290 mM sucrose, 1.27 mM Ca(NO3)2, 0.16 mM boric acid, 1 mM KNO3) and vortexed vigorously for 60 s to release mature pollen grains into the solution. Pollen from each genotype was collected after being centrifuged at 15 000  g for 1 min. The pollen pellet was resuspended in 1 ml PVS containing 0.001% (w/v) fluorescein diacetate (FDA), and 10 μM propidium iodide (PI). Pollen was stained for 15 min at 28°C and then centrifuged. The pollen pellet was washed using PVS followed by imaging using a fluorescence microscope. FDA was imaged using the GFP filter set, and PI was imaged using the RFP filter set. Three independent repeats were conducted.

Starch staining assay

Flowers at each stage were collected 4 h after lights were on. Samples were fixed in Carnoy fixative (ethanol : acetic acid, 3 : 1, v/v) overnight before moving to 70% ethanol and storage at 4°C. After sepals and petals were removed, extra ethanol was absorbed using tissue wipes. Samples were stained using 40 μl of clearing‐staining solution (Image‐iT™ Plant Tissue Clearing Reagent (Invitrogen) with 4% (w/v) potassium iodide and 1.27% (w/v) iodine) before placing the cover slip. Samples were imaged by Apotome.2 (Carl Zeiss) microscopy. More than six flowers at each stage were assayed for one repeat and four independent repeats were conducted. For starch staining on wax embedded cross‐sections, flower buds after stage 12 were fixed in Carnoy fixative overnight before moving to 70% ethanol, samples were dehydrated and embedded in wax as described (previously Zhang et al., 2021). Cross‐sections (after wax was removed by HistoChoice® Clearing Agent) were stained using 100 μl potassium iodide solution (composed of 4% (w/v) potassium iodide and 1.27% (w/v) iodine) kept in the dark for 5 min and washed twice using ddH2O before being imaged with a compound microscope (Nikon, Melville, NY, USA).

Statistical analysis

The differences between the two subjects were determined using the two‐tailed Student's t‐test with equal variance. The differences among multiple subjects were assessed using one‐way analysis of variance (ANOVA) followed by multiple comparison tests (Fisher's least significant difference (LSD) method). All statistical analysis was performed using Origin 2021b statistical software (OriginLab Corp., Northampton, MA, USA).

Accession numbers

Sequence information from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: AtSWEET9 (AT2G39060), AtSWEET13 (AT5G50800), AtSWEET14 (AT4G25010), AtNPF3.1 (AT1G68570).

Results

AtSWEET13 and AtSWEET14 expressed at late anther development stages

AtSWEET13 and AtSWEET14 transcripts were detected in stamens at late‐stage flower development stages using GUS reporter under the control of either AtSWEET13 or AtSWEET14 promoter (Kanno et al., 2016), but the exact anther development stages were yet to be determined. We examined the spatial and temporal protein accumulation of AtSWEET13 and AtSWEET14 using transgenic lines harboring a construct with either AtSWEET13 or AtSWEET14 translational fusion with GUS or with YFP driven by its own native AtSWEET13 or AtSWEET14 promoter (named as pSWEET13:SWEET13‐GUS, pSWEET14:SWEET14‐GUS, pSWEET13:SWEET13‐YFP, and pSWEET14:SWEET14‐YFP). Both SWEET13‐GUS protein and SWEET14‐GUS were detected at late anther development stages in both unopened flower buds (anther development stage 12 and below) and fully opened flowers (anther development stages 13) (Fig. 1a) (Sanders et al., 1999). AtSWEET14 also was detected at the silique and pedicel junctions after fertilization, suggesting that it may be involved in sugar transport from the pedicel to the silique. To determine which anther development stage AtSWEET13 and AtSWEET14 start to function, we conducted the GUS staining of inflorescence tissues from pSWEET13:SWEET13‐GUS and pSWEET14:SWEET14‐GUS and embedded them in wax for cross‐sectioning examination. Both SWEET13‐GUS and SWEET14‐GUS proteins were not detected at stage 11 (Fig. 1b), at which the tapetum is not fully degenerated, and pollen grains can be observed easily (Sanders et al., 1999). At stage 13 when the flower fully opens and pollen dehiscence occurs, both AtSWEET13 and AtSWEET14 were strongly accumulated in connective, epidermis and endothecium cells (Fig. 1b). All stages stated here were confirmed by examining the flowers using pSWEET13:SWEET13‐YFP and pSWEET14:SWEET14‐YFP transgenic lines based on defined anther development stages (Bowman, 1994). Both AtSWEET13 and AtSWEET14 were only strongly detected at flower stages 12 and 13, corresponding to anther stages 11–13 (Sanders et al., 1999), and there were no signals observed in released pollen grains at anther stage 13 for both AtSWEET13 and AtSWEET14 (Fig. 1c). The anthers at stage 12 were further examined under confocal microscopy; AtSWEET13 and AtSWEET14 were strongly detected in the anther wall, including endothecium and epidermis cells (Fig. 1d), consistent with the detected GUS signal. In short, AtSWEET13 and AtSWEET14 were expressed mainly in connective, epidermis and endothecium cells at anther development stages 12 and 13.

Fig. 1.

Fig. 1

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Spatial and temporal localization of Arabidopsis thaliana Sugars Will Eventually Be Exported Transporters (AtSWEET)13 and AtSWEET14. (a) Tissue‐specific accumulation of AtSWEET13 and AtSWEET14 was evaluated. Inflorescences from 6‐wk‐old Arabidopsis plants carrying pSWEET13:SWEET13‐GUS and pSWEET14:SWEET14‐GUS were histochemically stained for 12 h for β‐glucuronidase (GUS) activity analysis. GUS staining patterns were consistent from two independent T2 lines for each construct. (b) Cross‐sections of GUS‐stained anther samples from pSWEET13:SWEET13‐GUS and pSWEET14:SWEET14‐GUS lines. C, connective; E, epidermis; En, endothecium; P, pollen grains; St, stomium; T, tapetum; V, vascular region. Section thickness: 6 μm. (c) AtSWEET13 and AtSWEET14 accumulation in different floral stages of Arabidopsis were examined using a fluorescence microscope. The signals were detected in anthers at floral stages 12 and 13 of transgenic lines carrying pSWEET13:SWEET13‐YFP and pSWEET14:SWEET14‐YFP. All images in this panel were acquired using identical imaging parameters. (d) AtSWEET13 and AtSWEET14 accumulation in the anther wall of transgenic lines carrying pSWEET13:SWEET13‐YFP and pSWEET14:SWEET14‐YFP visualized with confocal microscopy. Stage 12 anthers from two independent lines were examined, and representative pictures from one line were shown.

The reduced fertility phenotype of atsweet13;14 is caused by pollen defectiveness

The double mutant of atsweet13;14 has been reported to show a reduced fertility phenotype (Kanno et al., 2016). First, we confirmed the T‐DNA insertion sites of atsweet13 and atsweet14 each single mutant was located in the second to the last exon, and no full‐length AtSWEET13 and AtSWEET14 transcripts were detected in stage 13 flowers of atsweet13;14 using reverse transcription (RT)‐PCR (Fig. S1). As AtSWEET13 and AtSWEET14 were highly accumulated in anther, we speculated that the paternal effect on this phenotype is dominant. To test this, we reciprocally crossed atsweet13;14 with Col‐0. As shown in Fig. 2(a), reduced fertility was exhibited when atsweet13;14 pollen was crossed with Col‐0 stigma or atsweet13;14 stigma, whereas a normal seed set was observed when atsweet13;14 stigma was crossed with Col‐0 pollen. These data suggest that the reduced fertility phenotype of atsweet13;14 is caused by defected pollen. We further investigated whether the affected pollen is the result of a defect in pollen viability or pollen germination. Pollen viability of atsweet13;14, was severely reduced compared to Col‐0 (Fig. 2b), as demonstrated by FDA and PI staining because viable pollen can be stained green by FDA and nonviable pollen can be stained red by PI. The defective atsweet13;14 pollen viability can be fully complemented by either AtSWEET13 or AtSWEET14 under their own native promoters, as represented from two independent homozygous complementation lines (Fig. 2b,c). Around 65% of Col‐0 pollen was alive, but < 3% of atsweet13;14 pollen was alive (Fig. 2c). Subsequently, in vitro pollen germination of the atsweet13;14 pollen was analyzed. Similar to the pollen viability assay, pollen germination rate of atsweet13;14 was severely reduced compared to Col‐0 (Fig. 2d). Around 60% of Col‐0 pollen germinated in vitro, whereas only 1% of atsweet13;14 germinated (Fig. 2e). The defected atsweet13;14 pollen germination also can be fully complemented by AtSWEET13 or AtSWEET14 under their own native promoters, as represented from two independent homozygous complementation lines (Fig. 2d,e). Thus, the reduced fertility phenotype of atsweet13;14 is the result of low pollen viability and in turn low pollen germination.

Fig. 2.

Fig. 2

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atsweet13;14 pollen showed low viability and barely germinated in vitro (AtSWEET, Arabidopsis thaliana Sugars Will Eventually Be Exported Transporters). (a) Reciprocal crossing using pollen of Col‐0 or atsweet13;14 with the stigma of Col‐0 or atsweet13;14. Fourteen‐day‐old siliques were imaged using a dissecting microscope. (b) Fluorescein diacetate (FDA) and propidium iodide (PI) staining of pollen from Col‐0, atsweet13;14 and complementation lines. Pollen was collected directly from fresh flowers. Live pollen was stained green by FDA, and dead pollen was stained red by PI. (c) Statistical analysis of FDA positive rates of pollen from Col‐0, atsweet13;14, and complementation lines. The means were calculated from multiple repeats (±SE, n = 6), with > 900 pollen grains counted in total. Black circles, individual repeats of the FDA positive rate for each genotype. (d) In vitro pollen germination analysis of Col‐0, atsweet13;14 and complementation lines. The pictures were taken 8 h post‐germination. (e) Statistical analysis of pollen germination rates for various genotypes. The means were calculated from multiple repeats (±SE, n = 6), with > 450 pollen grains/tubes counted in total. Black circles, individual repeats of the pollen germination rate for each genotype. The statistically significant differences among different samples in panels (c, e) were determined using one‐way ANOVA followed by multiple comparison tests and were represented by different letters (P < 0.05).

Disrupted sucrose transport is responsible for the defected atsweet13;14 pollen fertility

Both sucrose and GA have been demonstrated as the substrates of AtSWEET13 or AtSWEET14 (Chen et al., 2012; Kanno et al., 2016). To address which substrate is responsible for the defective atsweet13;14 pollen, we used the AtSWEET14 promoter to drive AtSWEET9 and AtNPF3.1 each to complement atsweet13;14, because AtSWEET9 transports sucrose but not GA, whereas AtNPF3.1 has been well‐characterized to transport GA (David et al., 2016; Tal et al., 2016). We started with evaluating their developmental accumulation. Both SWEET9‐YFP and NPF3.1‐YFP were detected in the anthers at flower stages 12 and 13 (Fig. S2), and SWEET9‐YFP and NPF3.1‐YFP were visualized in the anther wall at anther stage 12 using confocal microscopy (Fig. 3a), similar to the temporospatial accumulation of SWEET14‐YFP under its own promoter (Fig. 1c,d). As a result, AtSWEET9 fully complemented atsweet13;14 pollen viability and germination phenotype as represented in two independent homozygous complementation lines, whereas AtNPF3.1 failed to do so (Fig. 3b–e). These data support that sucrose transport mediated by AtSWEET13 and AtSWEET14 is essential for supporting pollen viability and subsequent pollen germination.

Fig. 3.

Fig. 3

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Defective pollen of atsweet13;14 was rescued by AtSWEET9 but not by AtNPF3.1 (AtNPF3.1, Arabidopsis thaliana Nitrate transporter 1/Peptide transporter Family 3.1; AtSWEET, Sugars Will Eventually Be Exported Transporters). (a) AtSWEET9 and AtNPF3.1 accumulation detected in anther wall of atsweet13;14 complementation lines carrying pSWEET14:SWEET9‐YFP or pSWEET14:NPF3.1‐YFP visualized with confocal microscopy (YFP, yellow fluorescent protein). Stage 12 anthers of atsweet13;14 complementation lines were examined. Two independent complementation lines were examined, and representative pictures from one line were shown. (b) Complementation analysis of pollen viability of atsweet13;14 by pSWEET14:SWEET9‐YFP or pSWEET14:NPF3.1‐YFP. AtSWEET9 rather than AtNPF3.1 rescued low pollen viability of atsweet13;14. Live pollen was stained green by fluorescein diacetate (FDA), and dead pollen was stained red by propidium iodide (PI). (c) Statistical analysis of FDA positive rates for AtSWEET9 and AtNPF3.1 complementation lines. The means were calculated from multiple repeats (±SE, n = 12), with > 1400 pollen grains counted in total. Black circles, individual repeats of the FDA positive rate for each genotype. (d) Complementation analysis of in vitro pollen germination of atsweet13;14 by pSWEET14:SWEET9‐YFP or pSWEET14:NPF3.1‐YFP. AtSWEET9 rather than AtNPF3.1 rescued low pollen germination of atsweet13;14. The pictures were taken a 8 h post‐germination. (e) Statistical analysis of pollen germination rates for AtSWEET9 and AtNPF3.1 complementation lines. The means were calculated from multiple repeats (±SE, n = 6), with > 300 pollen grains/tubes counted in total. Black circles, individual repeats of the pollen germination rate for each genotype. The statistically significant differences among different samples in panels (c, e) were determined using one‐way ANOVA followed by multiple comparison tests and were represented by different letters (P < 0.05).

AtSWEET13 and AtSWEET14 function as gatekeepers to regulate sugar distribution between anther and pollen at late anther stages

Pollen is symplasmically isolated from the somatic cell layers of anther wall where AtSWEET13 and AtSWEET14 proteins are located. Sucrose probably is exported by AtSWEET13 and AtSWEET14 from the endothecium into locules before being taken up by pollen. We speculated that more sugar would accumulate in the anther wall of atsweet13;14 if the endothecium was the site to export sugar. Sugar availability is tightly associated with starch accumulation, and starch dynamics have been observed in the stamen envelope (Hedhly et al., 2016). Thus, instead of measuring soluble sugar content directly from stamen cell walls, which is more challenging, we compared starch staining between Col‐0 and atsweet13;14. As expected, more starch accumulation was observed in anthers of atsweet13;14 than that of Col‐0 at stage 12 (Fig. S3). To confirm the differential starch accumulation at anther stage 12, we embedded flower buds of Col‐0 and atsweet13;14 in wax for cross‐sectioning examination followed by starch staining. Substantial starch granules were clearly observed in connective, epidermis, and endothecium cells of atsweet13;14 compared with Col‐0 at anther stage 12 (Fig. 4a,b). Although there was higher starch with an indication of high soluble sugar in the anther wall of atsweet13;14, no structural differences of anthers were observed when scanned using the SEM at stages 12 and 13 (Fig. 4c,d). Consistent with the reported observation (Kanno et al., 2016), atsweet13;14 delayed anther dehiscence at stage 12 (Fig. 4c). Unsurprisingly, we found that the size of atsweet13;14 pollen was affected as demonstrated by the significantly reduced length and width compared to Col‐0 pollen at both stages 12 and 13 (Fig. 4e,f), probably resulting from the lack of sugar supply from the anther wall, which is consistent with the low pollen viability (Fig. 2b,c). Notably, the width of Col‐0 pollen was significantly decreased from stage 12 to stage 13 (Fig. 4f), in agreement with the report that changes in pollen size can be caused by dehydration occurring before anthesis (Shi & Yang, 2010). To confirm that insufficient sugar supply probably is the cause of pollen defects, we measured soluble sugar concentrations from the mature pollen. All sugars, including sucrose (Fig. 4g), glucose (Fig. 4h), and fructose (Fig. 4i), substantially decreased in atsweet13;14 pollen compared to Col‐0 pollen. Lipid is another major form of carbon storage in mature pollen grains, especially for Arabidopsis (Shi & Yang, 2010; Wang et al., 2022a). Sugar is the primary source for lipid body biogenesis in pollen grains. Defects in lipid body accumulation affect pollen fitness and successful fertilization (Zheng et al., 2018). Thus, we wondered whether the total fatty acids altered, which in turn could contribute to the low pollen germination in atsweet13;14. Surprisingly, the total fatty acids in atsweet13;14 remained similar to that in Col‐0 (Fig. 4j). In addition, no significant differences were observed in each fatty acid species commonly found in Arabidopsis pollen (Wang et al., 2022a), including C16:0, C18:0, C18:1, C18:2 and C18:3 (Fig. S4).

Fig. 4.

Fig. 4

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Phenotypic effects of atsweet13;14 on anthers and pollen (AtSWEET, Arabidopsis thaliana Sugars Will Eventually Be Exported Transporters). (a, b) Comparison of starch accumulation in anther cross‐sections between Col‐0 (a) and atsweet13;14 (b) at stage 12. Starch granules were accumulated in endothecium, epidermis and connective cells of atsweet13;14 anther compared with those in Col‐0. C, connective; E, epidermis; En, endothecium; P, pollen grains; V, vascular region. Section thickness: 6 μm. (c, d) Comparison of anther and pollen morphological phenotypes imaged by scanning electron microscopy between Col‐0 and atsweet13;14 at stage 12 (c) and stage 13 (d). The close‐up pictures at the bottom were taken in the marked rectangle region from the corresponding top image. (e, f) Quantitative comparison of pollen size between Col‐0 and atsweet13;14 at stages 12 and 13. (e) Comparison in length and (f) comparison in width measured by ImageJ from 99 pollen grains with near‐flat orientation. The boxes were drawn from the 25th percentile to the 75th percentile; whiskers extend to 1.5× the interquartile range from the 25th and 75th percentiles; the medians are shown in line, and the means in a black square. The small open circles represent the length of individual pollen grains (e) and the width of individual pollen grains (f), respectively. The statistical analysis was done using one‐way ANOVA followed by multiple comparison tests, and differences were represented by different letters (P < 0.05). (g–i) Comparison of soluble sugar contents measured from Col‐0 and atsweet13;14 mutant pollen grains collected directly from fresh flowers. Sucrose (g), glucose (h) and fructose (i) in pollen were quantified from fresh open flowers collected in three independent repeats (means ± SE, n = 3). Black circles, sucrose content (g), glucose content (h) and fructose content (i) from the individual repeats, respectively. Glucose and fructose content in atsweet13;14 was below the detection limit using our established method. ND, not detected. (j) Comparison of total fatty acid content from Col‐0 and atsweet13;14 mutant pollen. Total fatty acid content of Col‐0 and atsweet13;14 pollen collected directly from fresh flowers. Fatty acid methyl esters extracted from four independent repeats (±SE, n = 4) were quantified using gas chromatography with a flame ionization detector. Black circles, fatty acid content of the individual repeats from Col‐0 or atsweet13;14. The statistically significant differences in panels (g, j) were determined using Student's t‐test and were represented by different letters (P < 0.05).

Discussion

Pollen fertility phenotypes are explained by sucrose rather than GA as a substrate of AtSWEET13 and AtSWEET14

Transport proteins are essential for cells to selectively exchange solutes across the cell membrane in many physiological processes, including nutrient transport. It is not uncommon for a single transporter to transport multiple substrates that are structurally distant. For example, AtGTR1/NPF2.10 transports glucosinolates, JA‐Ile and GA (Saito et al., 2015); AtNPF3.1/NRT1.1 transports nitrate, GA and ABA (David et al., 2016; Tal et al., 2016); AtNPF6.3 transports both nitrate and auxin (Krouk et al., 2010); AtNPF4.1/AIT3 transports both ABA and GA (Kanno et al., 2012); AtPHT4.4 transports phosphate and ascorbate (Guo et al., 2008; Miyaji et al., 2015); AtABCG36 transports indole‐3‐butyric acid and cadmium (Strader & Bartel, 2009); and a classical sucrose/H+ symporter AtSUC5 transports biotin in addition to sucrose (Ludwig et al., 2000) – this was the first report of a sugar transporter transporting a structurally unrelated substrate from sugar.

It has been over a decade since SWEETs were characterized as bidirectional uniporters transporting sugars (Chen et al., 2010; Xue et al., 2022). In 2016, Kanno et al. reported that AtSWEET10, AtSWEET12, AtSWEET13 and AtSWEET14 were able to mediate GA transport using a modified yeast two‐hybrid system containing BD‐GID1a (GA receptor) and AD‐GAI (a DELLA protein). The same system has been used to detect weak GA transport activity for OsSWEET3a, OsSWEET11a and OsSWEET12 (Kanno et al., 2016; Morii et al., 2020; Wu et al., 2022). However, the biological relevance of GA transport mediated by SWEETs was only indicated by partial restoration of anther dehiscence of the atsweet13;14 mutant through exogenous GA3 application (Kanno et al., 2016), and restoration of defects in germination and early shoot development for ossweet3a (Morii et al., 2020).

In addition, only a few members of SWEETs from Arabidopsis and rice transport GA, which offers a chance to study the preferred substrate for a given biological process. Kanno et al. (2016) indicated that AtSWEET13 and AtSWEET14 mediated GA transport in the anther. AtSWEET9 and AtSWEET12 show a similar sucrose transport activity to AtSWEET13 and AtSWEET14 in a biosensor‐based HEK293T detection system (Chen et al., 2012), but AtSWEET9 does not show any GA transport activity (Kanno et al., 2016). In addition, AtSWEET12, under the control of the AtSWEET9 promoter, is sufficient to rescue the defect in the nectar secretion phenotype of the atsweet9 mutant, further supporting the conclusion that they are exchangeable in a physiological context when expressed at the same site (Lin et al., 2014). Following this concept, we generated transgenic plants with AtSWEET9 expression driven by the promoter of AtSWEET14, and found that this combination can fully complement the compromised pollen viability and germination phenotypes of atsweet13;14, whereas AtNPF3.1 is unable to rescue defective pollen phenotypes of atsweet13;14 (Fig. 3). One would argue that AtNPF3.1, AtSWEET13 and AtSWEET14 may have different transport activities to GA. In fact, they have a similar range of K m for GA transport: AtNPF3.1 has a K m of c. 500 μM to GA4 (Tal et al., 2016), and both AtSWEET13 and AtSWEET14 have a K m of several hundred μM to GA3 (Kanno et al., 2016). However, unlike SWEETs that are bidirectional uniporters (Xue et al., 2022), most NPFs characterized to date are proton‐dependent symporters (Niño‐González et al., 2019). Although we could not exclude the possibility that the failure of complementing atsweet13;14 by AtNPF3.1 was a result of the mechanistic differences in transporting substrates between the two transporter families, the results from SWEET9 complementation support that sucrose, not GA, is critical in rescuing the atsweet13;14 defective phenotypes of pollen viability and pollen germination. Consistent with our findings, one parallel study shows that the AtSWEET13 mutant that prefers sucrose selection was able to fully rescue the pollen viability and germination phenotype of atsweet13;14 but AtSWEET13 mutant that prefers GA selection was not (Isoda et al., 2022). Moreover, the soluble sugar content (including sucrose, glucose and fructose) was dramatically reduced in pollen of atsweet13;14, and starch was substantially accumulated in the anther wall anther of atsweet13;14 compared with those in Col‐0 (Fig. 4), whereas the content of all measured GA species was not significantly affected in anthers between Col‐0 and atsweet13;14 (Kanno et al., 2016).

Notably, externally provided GA can partially restore the delayed dehiscence phenotype of atsweet13,14 (Kanno et al., 2016). However, it is uncertain whether exogenous GA can be directly transported by other GA transporters to compensate for the local loss of GA transported by AtSWEET13/14, or whether exogenous GA triggers components in GA signaling pathway to impact sugar metabolism and/or transport during anther dehiscence of the atsweet13,14 mutant. GA plays an important role in regulating filament elongation and anther dehiscence (Marciniak & Przedniczek, 2019). Examples include Arabidopsis gibberellin 20‐oxidase mutants (ga20ox1;ga20ox2) exhibited impaired filament elongation and delayed dehiscence, but the ga20ox1;ga20ox2 pollen is fully viable (Rieu et al., 2008), suggesting that GA‐regulated anther dehiscence may not necessarily be linked to pollen viability. The role of sugar during anther dehiscence is poorly understood in Arabidopsis. Silencing of Petunia hybrida PhNEC1 that is a homolog of AtSWEET9 and is highly expressed in nectary and anther stomium cells results in an early dehiscence phenotype and reduced pollen germination (Ge et al., 2001). Different steps of anther development may require specific GA transporters, whereas GA transported by AtSWEET13/14 may only be restricted to anther dehiscence. However, we do not have conclusive results of complementing atsweet13,14 dehiscence phenotypes by either AtSWEET9 or AtNPF3.1 resulting from the considerable variation of anther dehiscence that is highly sensitive to the environment under our settings (J. Wang, pers. obs.), which precludes a reliable interpretation of the results.

Endothecium is the major site where sucrose is transported by AtSWEET13 and AtSWEET14

Pollen development requires nutrient supply from locules of the anthers. The question of how sugars reach locules is poorly understood. The anther structure changes dynamically in a development‐dependent manner. The anther wall has four layers – the epidermis, endothecium, middle layers and tapetum. The epidermis plays a protective role during the anther development. The endothecium, probably serving as an energy storage tissue, stores starch and is associated with anther dehiscence for pollen grain release (van der Linde & Walbot, 2019). Little is known about the function of the middle layer, which disappears entirely at stage 11 (Xue et al., 2021). The tapetum facilitates the flow of nutrients and water for microspore development and secretes exine components for pollen formation (van der Linde & Walbot, 2019). The tapetum initiates degeneration at stage 10 and completely disappears at stage 12 (Sanders et al., 1999). Our results showed that AtSWEET13 and AtSWEET14 proteins were detected at stages 12 and 13 (Fig. 1b,c) when both the middle layer and tapetum degenerated. AtSWEEET13‐GUS/YFP and AtSWEET14‐GUS/YFP were observed mainly in the wall of the anther (Fig. 1b,d). These data suggest that sucrose efflux mediated by AtSWEET13 and AtSWEET14 from endothecium cells into the cavity of the anther occurs mainly at stages 12 and 13, when the endothecium is the innermost layer of the anther wall. Thus, it was reasonable to predict that impaired sucrose efflux from the endothecium in atsweet13;14 mutant can lead to higher sucrose accumulation and, in turn, that more starch would be synthesized in the anther, which was demonstrated by the excess starch accumulation in the connective, epidermis and endothecium cells of atsweet13;14 at stage 12 (Fig. 4a,b). Consistently, pollen grains were detected to have a lower concentration of sugar, resulting from less sugar being available from the wall of the anther in atsweet13;14. Although pollen viability and pollen germination were reduced dramatically, there still was a low rate of viable pollen in atsweet13;14, which may be due to the expression of clade III other AtSWEETs transport sucrose (Chen et al., 2015; Mergner et al., 2020) or the degeneration of starch synthesized in pollen from earlier developmental stages (Hedhly et al., 2016).

Interestingly, the total fatty acid content of atsweet13;14 remains comparable with that of Col‐0 (Figs 4j, S4), which could be synthesized before the stages when AtSWEET13 and AtSWEET14 start to function during anther development. Tapetum accumulates lipidic components for pollen coat formation (Piffanelli et al., 1998; Ariizumi et al., 2004), whereas AtSWEET13 and AtSWEET14 are highly expressed after tapetum degeneration, which indicates that mature pollen grains have received some lipidic components from tapetum even in atsweet13;14. In addition, six sugar transporters are expressed at earlier stages of anther development (Feng et al., 2012), which may provide pollen with sugar for carbon storage. mRNA encoding a functional acyl‐CoA:diacylglycerol acyltransferase (AtDGAT2) (Zhou et al., 2013) is strongly transcribed in Arabidopsis microspore before gradually decreasing in mature pollen grains (Honys & Twell, 2004). The carbon needed for fatty acid synthesis at early anther stages probably is directed through tapetum‐expressed sugar transporters, such as AtSWEET8 (Guan et al., 2008). Sucrose transporter AtSWEET15 also is accumulated in the anthers of early buds (Chen et al., 2015). This possible explanation also is supported by the observation that lipid bodies were already visible in microspores of tobacco pollen (Rotsch et al., 2017). Likewise, the total fatty acid content of atsuc1 pollen also was comparable to that of Col‐0, even though the pollen germination of atsuc1 was substantially compromised (Sivitz et al., 2008). Further investigation is needed to elucidate the underlying mechanism. It would be most helpful to conduct metabolite analysis of soluble sugar, amino‐acid and fatty acid contents across pollen development stages in Arabidopsis, which produces tricellular pollen, different from the bicellular pollen from tobacco. In tobacco, sucrose content increases substantially right before pollen matures and peaks in mature pollen grains (Rotsch et al., 2017), which could be the consequence of increased sucrose unloading and/or reduced storage conversion. It is worth addressing whether any SWEETs, such as homologs of AtSWEET13 and AtSWEET14, are involved in the increased sucrose concentrations.

To summarize, we proposed a model to illustrate the role of sugar transporters during the late anther developmental stage (Fig. 5). Photoassimilates are symplasmically unloaded from the phloem of the filament to the anther connective tissue (Imlau et al., 1999). AtSUC1 that is accumulated in the connective tissue at late anther stages may retrieve sugar from the apoplasmic space (Stadler et al., 1999). AtSWEET13 and AtSWEET14 proteins are found in the connective tissue, epidermis and endothecium at the late anther stage (Fig. 1b), and participate in sucrose apoplasmic unloading into the locules. The released sucrose exported by AtSWEET13 and AtSWEET14 probably is partially cleaved into glucose and fructose by cell wall invertase 2 (AtcwINV2) or other AtcwINVs, because the AtcwINV2 antisense mutant displays defective pollen and reduced fertility phenotypes similar to atsweet13;14 (Hirsche et al., 2009). The resulting hexoses and sucrose may be taken up by pollen grain‐accumulated AtSWEET5 (Ko et al., 2022; Wang et al., 2022b), AtSWEET8 (Zhang et al., 2022), AtSUC1 (Stadler et al., 1999; Sivitz et al., 2008; Mergner et al., 2020) and AtSWEET11/12 (Mergner et al., 2020).

Fig. 5.

Fig. 5

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Schematic representation of the role of AtSWEET13/14 and other sugar transporters at the late anther development stages. C, connective cells; CC, companion cells; En, endothecium; L, locule; P, pollen grains; PP, phloem parenchyma cells; SE, sieve element.

Overall, the lack of sucrose exported by AtSWEET13/14 from the anther wall to locules leads to the defective pollen phenotypes of atsweet13;14 at late anther development stages, and impaired pollen viability is responsible for reduced fertility of atsweet13;14. Therefore, we conclude that sucrose mediated by AtSWEET13/14 is vital for Arabidopsis pollen viability and thereby fertility.

Author contributions

L‐QC oversaw the whole project; JW, XX and L‐QC designed the work; JW, XX, HZ and JL conducted the experiments; JW, XX and HZ analyzed the data; JW and L‐QC wrote the manuscript; JW and XX contributed equally to this work.

Supporting information

Fig. S1 Genotyping of atsweet13;14 double mutant.

Fig. S2 AtSWEET9 and AtNPF3.1 accumulation in different floral stages of Arabidopsis were examined using fluorescence microscopy.

Fig. S3 Comparison of starch accumulation in anthers between Col‐0 and atsweet13;14 at various stages.

Fig. S4 Comparison of fatty acid species between Col‐0 and atsweet13;14 pollen.

Table S1 Primers used in this study.

Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

Click here for additional data file. (1.1MB, pdf)

Acknowledgements

We thank Yaxin Li for the help on soluble sugar measurement and also Catherine Wallace for help with SEM imaging. This work was supported by startup funds from the University of Illinois at Urbana‐Champaign to Li‐Qing Chen (to JW, XX, JL). HZ was supported by a scholarship from the China Scholarship Council (201708330168).

Data availability

All of the data and materials that support the findings of this study are available upon request from the corresponding author.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1 Genotyping of atsweet13;14 double mutant.

Fig. S2 AtSWEET9 and AtNPF3.1 accumulation in different floral stages of Arabidopsis were examined using fluorescence microscopy.

Fig. S3 Comparison of starch accumulation in anthers between Col‐0 and atsweet13;14 at various stages.

Fig. S4 Comparison of fatty acid species between Col‐0 and atsweet13;14 pollen.

Table S1 Primers used in this study.

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Data Availability Statement

All of the data and materials that support the findings of this study are available upon request from the corresponding author.

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