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.

中文翻译：

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由 AtSWEET13 和 AtSWEET14 运输的蔗糖而不是 GA 支持花药发育后期的花粉适应性

介绍

花粉的发育和形成对于开花植物的繁殖至关重要 (Ma,  2005 )。花粉的发育需要来自花药体细胞层的糖供应，因为未成熟的花粉被花药中的小室很好地包围着。体细胞层由最外层的表皮细胞层、内皮细胞、中间细胞层和最内层的绒毡层组成 (van der Linde & Walbot,  2019 )。雄配子体与花药细胞同质分离，这是由于减数分裂细胞和绒毡层之间的解离（Ma，  2005 年；Borghi 和 Fernie，  2017 年）。糖从花药的体细胞层输出并输入花粉需要质膜定位的载体蛋白 (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 年）。在水稻中，OsSUC1在花药壁中高表达，ossuc1突变体显示花粉功能受损但不影响花粉成熟 (Hirose et al ., 2010 )。OsSWEET11a和OsSWEET11b在花药脉中表达，ossweet11a;11b突变体是雄性不育的 (Wu et al ., 2022 )。在黄瓜中，绒毡层和花粉定位的 CsSUT1 的敲低突变体显示雄性不育（Sun等人，2019 年）。AtSUC1 在花药和成熟花粉的结缔组织中表达，而atsuc1突变体产生有缺陷的花粉，导致花粉发芽率低（Stadler等人，1999 年；Sivitz等人，2008 年）。在拟南芥中，转运葡萄糖的 AtSWEET8（RPG1；破裂的花粉粒 1）和转运蔗糖的 AtSWEET13（RPG2）及其在花药绒毡层中检测到的转录本参与生质素沉积、小孢子发育和随后的种子形成（Guan等人，2008 ; Sun et al ., 2013 ; Xue et al ., 2022 ). 突变rpg1 ( atsweet8) exhibits defective microspore development, and the double mutant rpg1rpg2 (atsweet8;13) results in an almost sterile phenotype (Sun et al., 2013).

除了糖分，发育中的花粉还需要赤霉素 (GA) 来维持其活力和发育（Plackett等人，2011 年）。花药和花粉粒的绒毡层是花发育过程中 GA 合成的主要部位 (Itoh et al ., 1999 )。例如，拟南芥GA生物合成缺陷型突变体ga1-3是雄性不育的(Sun et al ., 1992 )。同样，GA 受体 GID1（赤霉素不敏感矮人 1）的三重突变体gid1a;b;c显示矮化表型和雄性不育（Griffiths等人，2006 年）). GA 跨不同细胞或组织的运动已在许多生物过程中得到报道，这些过程通常涉及 GA 转运蛋白（Binenbaum等人，2018）。GA 运输不足也会影响花药发育和生育力。例如，AtGTR1/NPF2.10（硫代葡萄糖苷转运蛋白 1；硝酸盐转运蛋白 1/肽转运蛋白家族 2.10）除硫代葡萄糖苷外还转运茉莉酸异亮氨酸 (JA-Ile) 和 GA。gtr1突变体由于花丝伸长受损和花药开裂而降低了生育力，这些表型可以通过外源 GA 应用来挽救（Saito等人，2015 年）。

出乎意料的是，质膜定位的 AtSWEET13 和 AtSWEET14 在被充分表征为蔗糖转运蛋白后已被证明可以转运 GA（Kanno等人，2016 年）（Chen等人，2012 年）。除了 AtSWEET 之外，OsSWEET3a 还被发现除了 2-脱氧葡萄糖外还可以转运 GA（Morii等人，2020 年）。atsweet13 ;14双突变体已被证明在花药开裂方面存在缺陷，这可以通过外源施用过量的 GA 来挽救（Kanno等人，2016 年）). 这些结果表明 GA 可能是花药中 AtSWEET13 和 AtSWEET14 的主要底物。然而，不确定这种拯救是 GA 运输改变的结果还是 GA 对糖运输的间接影响的结果，因为 GA 和糖在许多发育过程的信号通路中相互关联，包括开花（Matsoukas，  2014）。为了阐明 AtSWEET13 和 AtSWEET14 的哪种底物对支持花粉发育很重要，我们对atsweet13 进行了补充；14使用转运蔗糖但不转运 GA 的 AtSWEET9 进行了分析（Lin等人，2014 年；Kanno等人，2016 年；Wu等人等，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.