The HKT1 Na+ transporter protects plant fertility by decreasing Na+ content in stamen filaments

Uchiyama et al., Sci. Adv. 9, eadg5495 (2023) 2 June 2023

盐胁迫会显著降低种子产量，因为植物在其生殖阶段对盐特别敏感。在这里，我们展示拟南芥中的钠离子转运蛋白AtHKT1;1在雄蕊的韧皮部和木质部周围特异表达，以防止盐胁迫导致的种子产量显著减少。在盐胁迫下，AtHKT1;1突变体的雄蕊过度积累Na+，限制了它们的延伸，并导致雄性不育。将AtHKT1;1表达限制在韧皮部，可使种子产量在钠离子胁迫下增加1.5倍。将AtHKT1;1的韧皮部表达扩展到整个植物是增加植物在盐胁迫下生产力的方法。

Fig. Localization of AtHKT1;1 in floral organs. (A) Quantification of AtHKT1;1 transcripts in various organs. White bars, control; orange bars, 100 mM NaCl. n = 3; Student’s t test, *P < 0.05. nd, not determined. (B to E) GUS staining of the flowers without sepals and petals (B), stamen (C), cross section of filament (D). (E) Magnification of part of the filaments in (D). an, anther; fi, filament; se, sieve element; cc, companion cell; ph, phloem; xy, xylem; v, vascular bundle. Scale bars, 1 mm (B), 100 μm (C), 20 μm (D), and 10 μm (E). (F to H) Cross sections of A. thaliana filament were immunostained using an anti-AtHKT1;1 antibody (F). Gold particle–labeled AtHKT1;1 [arrowheads in (G) and (H)] was detected in xylem parenchyma cells (xp) (G) and companion cells (cc) (H). Scale bars, 1 μm.

 图.AtHKT1;1在花细胞器中的定位。(A) 各种器官中AtHKT1;1转录本的定量。白色柱状图，对照组；橙色柱状图，100 mM NaCl。n = 3；学生t检验，*P < 0.05。nd，未确定。(B至E) 不含萼片和花瓣的花的GUS染色(B)、雄蕊(C)、花丝的横截面(D)。(E) (D)中花丝部分的放大图。an，花药；fi，花丝；se，筛管元素；cc，伴随细胞；ph，韧皮部；xy，木质部；v，维管束。比例尺，1毫米(B)、100微米(C)、20微米(D)和10微米(E)。(F至H) 使用抗AtHKT1;1抗体免疫染色A. thaliana花丝的横截面(F)。检测到标记有金颗粒的AtHKT1;1[图(G)和(H)中的箭头头]存在于木质部薄壁细胞(xp) (G)和伴随细胞(cc) (H)中。比例尺，1微米。

Immunoelectron microscopy

Two-week-old WT plants at the postbolting stage were transferred to hydroponic medium with 100 mM NaCl for 4 days. Stamens were collected from stage 15 flowers of each plant according to the protocol by Smyth et al. (24) and used for immunoelectron microscopy. The immunostaining was performed as described previously with some modifications (19, 48). Briefly, the stamens were immersed in 50 mM phosphate buffer (pH 7.2) containing 0.5% glutaraldehyde and 4% paraformaldehyde at 4°C for 2 hours. After washing three times with phosphate buffer, the specimens were dehydrated using an ethanol series (50 to 99.5%, v/v), embedded in LR White resin (Electron Microscopy Sciences, PA, USA) and polymerized at −20°C under ultraviolet light for 2 days. Specimens were sectioned using a ULTRACUT-S ultramicrotome (ReichertNissei, Tokyo, Japan). Ultrathin sections (80 nm) were mounted on nickel grids and blocked with 4% BLOCK-ACE (KAC Co. Ltd., Kyoto, Japan) for 30 min at room temperature. Immunostaining with rabbit anti-AtHKT1;1 antiserum [rabbit immunoglobulin G (IgG); diluted 1:100] was performed on sections overnight at 4°C (19). After washing with blocking buffer (0.4% BLOCK-ACE), Alexa Fluor 546–FluoroNanogold Fab′ goat anti-rabbit IgG (#7404, Nanoprobes Inc., Yaphank, NY, USA) diluted in blocking buffer to 1:100 was added to the sections and incubated for 1 hour at room temperature. After washing with blocking buffer and distilled water, the sections were fixed with 1% glutaraldehyde in 50 mM trisHCl (pH 7.5) containing 137 mM NaCl and 2.7 mM KCl, and the colloidal gold particles were treated with silver enhancement reagents (Nanoprobes Inc., NY, USA) (49). Last, the sections were stained with 4% uranyl acetate for 12 min in the dark, followed by incubation in Lead Stain Solution (18-0875; Sigma Chemical Co., St. Louis, USA) for 5 min, and then washed with distilled water. The stained sections were examined using a JEM-1400 transmission electron microscope (JEOL Ltd., Tokyo, Japan) at 100 kV.

免疫电子显微镜方法。

在后生长期处于后生长阶段的两周龄WT植物被转移到含100 mM NaCl的水培培养基中培养4天。根据Smyth等人的方案（24），从每棵植物的15期花朵中收集了雄蕊，并用于免疫电子显微镜检测。免疫染色按照先前描述的方法进行，稍作修改（19, 48）。简而言之，将雄蕊浸入含有0.5%戊二醛和4%多聚甲醛的50 mM磷酸盐缓冲液（pH 7.2）中，在4°C下固定2小时。用磷酸盐缓冲液洗涤三次后，用乙醇系列（50到99.5%，体积比）脱水，然后在-20°C下用紫外光聚合2天。使用ULTRACUT-S超薄切片机（Reichert-Nissei，东京，日本）进行切片。将超薄切片（80 nm）装在镍网上，并在室温下用4% BLOCK-ACE（KAC Co. Ltd.，京都，日本）阻滞30分钟。在室温下用稀释至1:100的兔抗AtHKT1;1抗血清（兔免疫球蛋白G（IgG））在切片上过夜进行免疫染色（19）。用阻断缓冲液（0.4% BLOCK-ACE）洗涤后，将稀释至1:100的Alexa Fluor 546-FluoroNanogold Fab'山羊抗兔IgG（#7404，Nanoprobes Inc.，Yaphank，NY，USA）添加到切片中，并在室温下孵育1小时。用阻断缓冲液和蒸馏水洗涤后，用含有1%戊二醛的50 mM三（羟甲基）氨基甲烷盐（pH 7.5）固定切片，其中含有137 mM NaCl和2.7 mM KCl，并用银增强试剂处理胶体金颗粒（Nanoprobes Inc.，NY，USA）（49）。最后，将切片在黑暗中用4%醋酸铀染色12分钟，然后在铅染色液中孵育5分钟（18-0875; Sigma Chemical Co.，圣路易斯，美国），然后用蒸馏水洗涤。用JEM-1400透射电子显微镜（JEOL Ltd.，东京，日本）在100 kV下观察。