Auxins reverse plant male sterility caused by high temperatures
叶黄素能逆转高温导致的植物雄性不育

To define the relationship between auxin and HT injury, we measured endogenous auxin levels and auxin signal transduction activity. In barley, we observed that the endogenous auxin levels of developing anthers are reduced in response to HT (Fig. 1). In untreated controls (5-mm-long panicles), auxin accumulated abundantly throughout the developing anther cells, i.e., parietal, epidermal, and sporogenous cells, and in rachis cells around vascular bundles (Fig. 1 B and D). This stage of development is just before development of tapetum, middle layer, and endothecial cells from the parietal cells (4, 5). In plants exposed to HT for <3 days, no morphological abnormalities were observed, and after a temperature downshift, male fertility was recovered (3, 4). On the other hand, when 5-mm-long panicles were examined after HT treatment for 3 days, anther parietal and epidermal cells significantly decreased auxin levels, showing 52.9% intensity of fluorescent signals per area in each anther locule compared with levels of control plant (Fig. 1 H and J). However, HT increased the signal intensity in the rachis cells around vascular bundles by >148.7% (Fig. 1 H and J). When 10-mm-long panicles were examined after exposure to HT for 5 days, auxin levels had decreased even further (39.4% intensity) in the pollen mother and tapetum cells at center of the anthers (Fig. 1 F and L).
为了确定辅助素与 HT 损伤之间的关系，我们测量了内源辅助素水平和辅助素信号转导活性。在大麦中，我们观察到正在发育的花药中的内源辅助素水平会因 HT 而降低（图 1）。在未处理的对照组（5 毫米长的圆锥花序）中，整个发育中的花药细胞，即顶叶细胞、表皮细胞和孢子细胞，以及维管束周围的轴细胞中都大量积累了辅酶（图 1 B 和 D）。这一发育阶段恰好是顶叶细胞的绦层、中层和内皮细胞发育之前（4, 5）。在暴露于高温下 <3 天的植株中，未观察到形态异常，温度降低后雄性繁殖力恢复 ( 3, 4)。另一方面，当 HT 处理 3 天后，检查 5 毫米长的圆锥花序时，花药顶叶和表皮细胞的辅助素水平显著下降，与对照植株的水平相比，每个花药子房每面积的荧光信号强度下降了 52.9%（图 1 H 和 J）。然而，HT 使维管束周围轴细胞的信号强度增加了 148.7% 以上（图 1 H 和 J）。当将 10 毫米长的圆锥花序暴露于 HT 5 天后进行检测时，花药中心的花粉母细胞和花粉块细胞中的辅助素水平进一步降低（强度降低 39.4%）（图 1 F 和 L）。

To study whether the phenomenon of auxin reduction could be observed in the anther cells of other plant species, we tested a recombinant Arabidopsis line that expressed β-glucuronidase (GUS), with the gene under control of a synthetic auxin response element (DR5-GUS) and the natural auxin response gene (ARF19-GUS) (20, 21). The disappearance of DR5-GUS signals has been reported in anther cells of auxin perception defective tir1 and afb multiple mutants (19). In addition, the significant reduction of DR5-GUS signals was observed in young leaves of triple mutants of YUCCA genes yuc1, -2, and -6 or yuc1, -4, and -6 (17). HT injury was observed in recombinant Arabidopsis grown at 33 °C for >7 days, with plants forming short stamens and rarely producing any pollen because of premature abortion of microsporogenesis (Fig. 2 A, B, G, and I). In the case of recombinants exposed to 31 °C for 7 days, microspores could be observed in anthers at stage 10; however, pollen maturation and filament elongation were ultimately repressed (Fig. 2 C and M). The strongest GUS activity in DR5-GUS line appeared in pollen mother and tapetum cells at stage 10 of floral development (ref. 19; Fig. 2 D and G and Fig. S1). In recombinants exposed to HT (31 °C or 33 °C) for >1 day, the DR5-GUS signals in anther cells decreased significantly at stage 10 (Fig. 2). In developing anther cells, GUS activity in ARF19-GUS line were lower than that in DR5-GUS line, and these weak signals disappeared completely after 1 day of 33 °C HT treatment (Fig. 2 N and O).

In contrast to the GUS staining in anther cells, the signals in the top of gynoecium and around vascular cells of petals were amplified with increased temperatures not only at stage 10 (Fig. 2) but also at earlier stages (Fig. 3 A–C). In mature flowers at later stage, the HT-caused induction around vascular cells of carpel was still observed (Fig. 3 D–F). However, the signals in anther cells at earlier and later stages were silent under HT condition (Fig. 3). In seedlings, HT treatment increased DR5-GUS signals in petiol of cotyledon, hypocotyl, and central cylinder of root (Fig. 3 G–I). These results indicate clearly that HT treatment represses the auxin signaling in an anther cell-specific manner, leading to similar abortion of pollen development and filament elongation in Arabidopsis as observed in barley.
与花药细胞中的 GUS 染色不同，雌蕊群顶部和花瓣维管细胞周围的信号不仅在第 10 期（图 2）随温度升高而放大，而且在早期也是如此（图 3 A-C）。在后期的成熟花朵中，仍能观察到 HT 在心皮维管细胞周围的诱导作用（图 3 D-F）。然而，在 HT 条件下，早期和晚期花药细胞中的信号均消失（图 3）。在幼苗中，HT 处理增加了子叶叶柄、下胚轴和根中心圆柱体中的 DR5-GUS 信号（图 3 G-I）。这些结果清楚地表明，HT 处理以花药细胞特异性的方式抑制了辅助素信号转导，导致拟南芥花粉发育和花丝伸长与在大麦中观察到的类似流产。

To study the effect of HT on expression of certain auxin-related genes, we used stereomicroscopy to dissect Arabidopsis anthers at stage 9. Real-time RT-PCR of the YUCCA genes YUC2 and -6 showed significant repression 1 day after temperature upshift to 33 °C (Fig. 4A). After 3 days of 33 °C, YUC6 expression was even more severely reduced. TAA1/TIR2 expression was reduced after 3 days (Fig. 4A). In control plants, expression of the auxin-induced gene IAA1 (22) is weak at stage 9 (19). However, IAA1 expression decreased by ≈50% under HT conditions (Fig. 4A). Although treated with HT for 3 days, we did not detect any change in expression of the auxin receptor gene TIR1 (23), or ARF6 and 8, which are involved in jasmonic acid production and flower maturation (24), in anthers at stage 9 (Fig. 4A). Under normal conditions, the barley YUCCA genes unigene Nos. 31993 and 5729 in HarvEST: Barley Version 1.68 Assembly No. 35, show increased expression during the early development of panicles, but the up-regulation of No. 31993 expression could not be detected and the No. 5729 expression was severely repressed to ≈60% normal levels by increased temperatures (Fig. 4 B and C).
为了研究高温对某些辅助素相关基因表达的影响，我们使用立体显微镜对第 9 期拟南芥花药进行了解剖。对YUCCA基因YUC2和-6的实时RT-PCR检测显示，温度升高到33 °C后1天，YUC2和-6的表达明显受到抑制（图4A）。33 ℃ 3 天后，YUC6 的表达量减少得更为严重。TAA1/TIR2 的表达在 3 天后也有所降低（图 4A）。在对照植株中，辅助素诱导基因 IAA1 ( 22) 的表达在第 9 阶段很弱 ( 19)。然而，在 HT 条件下，IAA1 的表达量减少了≈50%（图 4A）。虽然用 HT 处理了 3 天，但我们没有检测到第 9 期花药中的辅助素受体基因 TIR1 ( 23) 或参与茉莉酸产生和花成熟的 ARF6 和 ARF8 ( 24) 的表达有任何变化（图 4A）。在正常条件下，HarvEST：Barley Version 1.68 Assembly No.35中的大麦YUCCA基因unigene 31993号和5729号在圆锥花序早期发育过程中的表达量增加，但在温度升高的情况下，无法检测到31993号基因表达量的上调，而5729号基因的表达量则被严重抑制至正常水平的60%以下（图4 B和C）。

Recent Arabidopsis studies have reported that auxin affects pollen maturation, filament growth, and anther dehiscence (17, 19). Auxin is most likely biosynthesized in developing anther cells at floral stages 8–11, with the auxin response beginning to appear after stage 9 (19). The auxin biosynthesis genes YUC2 and -6 are strongly expressed in anther cells; their inactivation leads to short stamens and rarely produced any pollen (17). Thus, in the developing anther cells of Arabidopsis and barley plants, expression of auxin biosynthesis genes is susceptible to increased temperatures. Reduced expression of these genes probably causes anther-specific auxin depletion, and reduction of the auxin response.
最近的拟南芥研究报告称，叶绿素会影响花粉成熟、花丝生长和花药开裂 ( 17, 19)。在花期 8-11 期，最有可能在发育中的花药细胞中生物合成辅酶，辅酶反应在花期 9 期后开始出现 ( 19)。辅助素生物合成基因 YUC2 和 -6 在花药细胞中强烈表达；它们的失活导致雄蕊短小，很少产生花粉（17）。因此，在拟南芥和大麦植物发育中的花药细胞中，辅助素生物合成基因的表达易受温度升高的影响。这些基因表达的减少可能会导致花药特异性的辅酶耗竭，并降低辅酶反应。

Our previous result also shows that HT induces expression of auxin response genes in barley seedlings but not in panicles (5). In addition, Figs. 1–3 show opposite effects of HT on both endogenous auxin level and auxin signaling between developing anther cells and other tissues, especially around vascular cells; namely, they decrease in the former and increase in the latter. Thus, HT effects on auxin level appear to differ among plant tissues. Furthermore, auxin transport and perception mutants show a reduction in filament length but only exert a moderate effect on pollen maturation and anther dehiscence in Arabidopsis (19). Likewise, application of an auxin transport inhibitor does not affect the DR5-GUS staining in anthers (19). These results suggest that biosynthesis of endogenous auxin in developing anthers is a major factor responsible for the HT-caused abortion of pollen and auxin reduction. Accordingly, HT-tolerant plants might be obtained by controlling anther-specific auxin biosynthesis genes.
我们之前的研究结果也表明，HT 能诱导大麦幼苗中的辅助素响应基因表达，但不能诱导圆锥花序中的辅助素响应基因表达 ( 5)。此外，图 1- 3 显示 HT 对发育中的花药细胞和其他组织（尤其是维管细胞周围）的内源辅素水平和辅素信号转导的影响相反，即前者降低，后者升高。因此，HT 对植物组织间的辅素水平的影响似乎是不同的。此外，在拟南芥中，辅助素转运和感知突变体显示出花丝长度的减少，但对花粉成熟和花药开裂的影响不大（19）。同样，使用辅助素运输抑制剂也不会影响花药中的 DR5-GUS 染色（19）。这些结果表明，正在发育的花药中内源辅素的生物合成是导致 HT 引起的花粉流产和辅素减少的主要因素。因此，可以通过控制花药特异性的辅助素生物合成基因来获得耐 HT 植物。

To assess how an exogenous application of auxin would affect HT injury to anther early development, we applied 10⁻⁶, 10⁻⁵, or 10⁻⁴ M indole-3-acetic acid (IAA; natural auxin), or the synthetic auxins 1-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Control or auxin-containing solutions contained 0.1% DMSO and 0.1% (vol/vol) Tween 20. Solutions were applied to barley plants in the mornings of days 18, 19, 21, and 23 (Fig. 5A). At the heading stage, control anthers grew to 2.99 ± 0.05 mm in length, whereas anthers exposed to high temperatures were only 1.48 ± 0.04 mm in length and contained no pollen grains (Fig. 5 B and C). Irrespective of auxinic compound used, auxin application restored anther length (1.8–2.5 mm), mature pollen grains (Fig. 5), and seed setting rate (Fig. 6) in a dose-dependent manner. Although anthers developed normally with application of 10⁻⁴ M 2,4-D, negative effects of auxin were detected, including premature blighting of leaves and loss of mature seeds (Fig. 6). Two applications of auxin (at days 19 and 21) were sufficient to restore anther development under HT conditions (Fig. S2). We have observed transcriptional repression of certain replication related genes including DNA replication licensing factor MCM5, and cell-proliferation arrest in anther wall cells and sporogenous cells, under HT conditions (5). The application of exogenous auxin could completely restore the MCM5 gene expression (Fig. S3), and it probably conducts normal proliferation and development of anther cells.
为了评估外源施用的辅助素如何影响 HT 对花药早期发育的伤害，我们施用了 10 ⁻⁶ 、10 ⁻⁵ 或 10 ⁻⁴ M 吲哚-3-乙酸（IAA；天然辅助素）或合成辅助素 1-萘乙酸（NAA）或 2,4-二氯苯氧乙酸（2,4-D）。对照组或含辅助剂的溶液含有 0.1% DMSO 和 0.1% （体积分数）吐温 20。溶液在第 18、19、21 和 23 天上午施用到大麦植株上（图 5A）。在头状花序阶段，对照花药的长度为 2.99 ± 0.05 毫米，而暴露在高温下的花药长度仅为 1.48 ± 0.04 毫米，且不含花粉粒（图 5 B 和 C）。无论使用哪种助长化合物，施用助长素都能以剂量依赖的方式恢复花药长度（1.8-2.5 毫米）、成熟花粉粒（图 5）和结实率（图 6）。虽然施用 10 ⁻⁴ M 2,4-D 后花药发育正常，但仍发现了辅助剂的负面影响，包括叶片过早枯萎和成熟种子的损失（图 6）。在 HT 条件下，两次施用叶绿素（第 19 天和第 21 天）足以恢复花药的发育（图 S2）。在 HT 条件下，我们观察到某些复制相关基因（包括 DNA 复制许可因子 MCM5）的转录抑制以及花药壁细胞和孢子细胞的细胞增殖停滞 ( 5)。施用外源辅助素可以完全恢复 MCM5 基因的表达（图 S3），它很可能能促进花药细胞的正常增殖和发育。

The apex of each Arabidopsis inflorescence was sprayed once with 10⁻⁷ or 10⁻⁶ M IAA or NAA solution containing 0.1% DMSO just before increasing the temperatures to 31 °C. Seven days after the temperature upshift, HT injury resulted in significantly reduced stamen length, whereas in auxin-treated plants, this reduction could be suppressed in a dose-dependent manner (Fig. 7 A and B). In the stamens >1.0 mm length, mature pollen stained normally with an iodine solution. These results clearly suggest that a reduction in male tissue-specific auxin is the primary cause of HT injury. Furthermore, the resulting abortion of pollen development and male sterility can be reversed by the application of exogenous auxin. These phenomena are highly conserved in not only monocots but also dicots. To date, auxins have been used widely as potent and selective herbicides. Our results show that auxin is useful for the promotion of plant fertility and maintenance of crop yields under the global warming conditions.
在将温度升高到 31 °C之前，用 10 ⁻⁷ 或 10 ⁻⁶ M IAA 或含有 0.1% DMSO 的 NAA 溶液喷洒每个拟南芥花序的先端一次。温度升高七天后，高温热损伤导致雄蕊长度显著减少，而在辅助素处理的植株中，这种减少可以剂量依赖的方式被抑制（图 7 A 和 B）。在长度大于 1.0 毫米的雄蕊中，成熟花粉在碘溶液中染色正常。这些结果清楚地表明，雄性组织特异性辅助素的减少是 HT 损伤的主要原因。此外，由此导致的花粉发育中止和雄性不育可以通过施用外源辅助素来逆转。这些现象不仅在单子叶植物中高度保守，在双子叶植物中也是如此。迄今为止，辅酶已被广泛用作强效的选择性除草剂。我们的研究结果表明，在全球变暖的条件下，辅助素对促进植物生育和保持作物产量非常有用。

Under normal conditions, double-rowed barley plants (H. vulgare L. cv “Haruna-nijyo”) were maintained for the entire growth period in a growth cabinet (LH350S, NK system) at 20 °C during the day and 15 °C at night, with a 16-h photoperiod. As described, HT treatment was started at the five-leaf stage (2-mm-long panicle), when the tip of the fifth leaf had emerged. Plants were grown at 30 °C during the day and 25 °C at night for 5 days (3–5). During this period, pollen mother and tapetum cells developed from anther sporogenous and parietal cells, respectively (4, 5). Arabidopsis thaliana Columbia (wild-type) and its derivatives expressing the GUS reporter, were grown at 23 °C (day and night) in a growth chamber with a 16-h photoperiod. Light conditions were 60–80 μE/m² per s. To study the effects of elevated temperature on reproductive development, we monitored the primary inflorescence of plants transferred to growth chambers at 31 °C or 33 °C under a 16-h photoperiod.
在正常条件下，双行大麦植株（H. vulgare L. cv "Haruna-nijyo"）的整个生长期都被置于生长柜（LH350S，NK 系统）中，白天温度为 20 °C，夜间温度为 15 °C，光周期为 16 小时。如上所述，在五叶期（2 毫米长的圆锥花序），即第五片叶的顶端出现时开始进行 HT 处理。植物在白天 30 °C、夜间 25 °C的条件下生长 5 天（3- 5）。在此期间，花粉母细胞和花粉块细胞分别从花药的孢子细胞和顶叶细胞发育而来 ( 4, 5)。拟南芥哥伦比亚（野生型）及其表达 GUS 报告基因的衍生物在 23 °C（昼夜）的生长室中生长，光周期为 16 小时。为了研究温度升高对生殖发育的影响，我们对转移到 31 °C 或 33 °C 生长室、光周期为 16 小时的植株的初级花序进行了监测。

We assessed the effects of exogenously applied auxin on HT injury of anthers during early development. The entire barley shoot was sprayed with 6 mL of control or 10⁻⁷, 10⁻⁶, 10⁻⁵, or 10⁻⁴ M auxin-containing solution [0.1% DMSO and 0.1% (vol/vol) Tween 20]. The natural auxin IAA, or the synthetic auxins NAA or 2,4-D, were applied in the morning of days 18, 19, 21, and 23 (Fig. 4). Tween 20 was eliminated from auxin solutions for Arabidopsis treatments. A single application of ≈0.1 mL of auxin solution was sprayed on the inflorescence of each Arabidopsis plant in the morning of the day that the temperature upshift occurred. Stamen length and pollen morphology were observed in the mature flowers 7–11 days after the temperature upshift. Ten to 12 plants were grown into a single pot, and the three pots were used in a series of experiments for each treatment. Each series of experiments was carried out independently in triplicate.
我们评估了外源施用的辅助素对花药早期发育过程中 HT 损伤的影响。用 6 mL 含有辅助素的溶液 [0.1% DMSO 和 0.1% (vol/vol) Tween 20] 喷洒整个大麦嫩枝，这些溶液分别是对照溶液或 10 ⁻⁷ 、10 ⁻⁶ 、10 ⁻⁵ 或 10 ⁻⁴ M 溶液。在第 18、19、21 和 23 天上午施用天然辅助剂 IAA 或合成辅助剂 NAA 或 2,4-D（图 4）。拟南芥处理的助剂溶液中剔除了吐温 20。在发生温度上移的当天上午，在每株拟南芥的花序上喷洒一次≈0.1毫升的助长素溶液。温度上移 7-11 天后，观察成熟花朵的雄蕊长度和花粉形态。在一个花盆中种植 10 到 12 株植物，每个处理使用三个花盆进行一系列实验。每组实验均独立进行，一式三份。

Barley anthers and Arabidopsis stamens were measured at the heading stage and anthesis, respectively, after dissection with a stereo microscope and CCD camera (SZX12 and DP20; Olympus). Pollen grains in the anthers were stained with an iodine solution (Lugol solution; MERCK). Dark brown pollen grains were scored as mature pollen. To observe mature Arabidopsis flowers with the scanning electron microscope (SEM: JEOL JSM-5800LV at 5 kV), excised flowers were fixed in FAA [3.7% formaldehyde, 50% ethanol, and 5% glacial acetic acid (vol/vol)] for 3 h at room temperature, dehydrated with ethanol, replaced with isoamyl acetate, and dried with a critical point drier (JEOL JCPD-5).
使用体视显微镜和 CCD 摄像机（SZX12 和 DP20；奥林巴斯）对大麦花药和拟南芥雄蕊进行解剖后，分别在头状花序期和开花期进行测量。花药中的花粉粒用碘溶液（Lugol solution; MERCK）染色。深棕色的花粉粒即为成熟花粉。用扫描电子显微镜（SEM：JEOL JSM-5800LV，5 kV）观察拟南芥成熟的花朵，将切除的花朵在 FAA [3.7% 甲醛、50% 乙醇和 5% 冰醋酸（体积分数）] 中室温固定 3 小时，用乙醇脱水，换上乙酸异戊酯，再用临界点干燥器（JEOL JCPD-5）干燥。

We examined the IAA distribution in early developing barley panicles (5-mm-long, i.e., just before development of tapetum, middle layer, and endothecial cells from the parietal cells), after treatment with or without HT stress for 3 days. As described, IAA was detected by using an anti-IAA monoclonal antibody (25), with the modifications indicated below. To cross-link IAA, excised panicles were immediately prefixed for 2 h in 3% (wt/vol) EDAC (Sigma-Aldrich) at room temperature and then transferred to FAA for 24 h at 4 °C. Fixed panicles were dehydrated in a series of ethyl alcohol and tertiary butyl alcohol washes, and then embedded in paraffin (PARAPLAST Plus, Oxford Labware). Samples were serially sectioned (10 μm thick) with a microtome and then affixed onto MAS-coated slides (Matsunami Glass Industry). After overnight drying at 42 °C, sections were deparaffinized with xylene and hydrated by using an ethanol-water series. Specimens were incubated in 10 mM PBS (PBS; 2.68 mM KCl, 0.15 M Na₂HPO₄, and 0.086 M KH₂PO₄) containing 0.1% (vol/vol) Tween 20, 1.5% glycine, and 5% BSA for 45 min at 22 °C. Samples were then rinsed in a regular salt rinse solution [RSRS; 10 mM PBS, 0.88% NaCl, 0.1% (vol/vol) Tween 20 and 0.8% BSA] and washed briefly with 10 mM PBS containing 0.8% BSA solution (PBS+BSA) to remove the Tween 20. After the application of anti-IAA monoclonal antibody (No. A0855, Sigma-Aldrich; 400 μL of a 1:1,000 dilution from 2.1 mg/mL stock) to each slide, samples were incubated overnight in a humidity chamber at room temperature. After hybridization, samples were subjected to a series of vigorous washes, twice with a high-salt rinse solution [HSRS; 10 mM PBS, 2.9% NaCl, 0.1% (vol/vol) Tween 20 and 0.1% BSA] for 10 min, once with RSRS for 10 min, and briefly with PBS+BSA. The Alexa 488-conjugated goat anti-mouse IgG antibody (Invitrogen; 400 μL of a 1:300 dilution of 2 mg/mL stock) was then placed on each slide, and these were incubated for 4–6 h in a humidity chamber at room temperature. After washing with RSRS twice for 15 min, specimens were mounted with an anti-fade reagent (ProLong Gold; Molecular Probes), covered with a coverslip, and observed under a fluorescent microscope (BX51; Olympus). The intensity of fluorescent signal was measured by Image J software.
我们研究了在施加或不施加 HT 胁迫 3 天后，早期发育的大麦圆锥花序（5 毫米长，即顶叶细胞、中层细胞和内皮细胞发育之前）中 IAA 的分布情况。如前所述，使用抗 IAA 单克隆抗体（25）检测 IAA，并做如下修改。为了交联 IAA，切除的圆锥花序立即在室温下用 3%（重量/体积）EDAC（Sigma-Aldrich）预固定 2 小时，然后转移到 FAA 中在 4 °C 下固定 24 小时。固定的圆锥花序在一系列乙醇和叔丁醇洗涤中脱水，然后包埋在石蜡中（PARAPLAST Plus，Oxford Labware）。用显微切片机对样本进行连续切片（10 微米厚），然后贴在涂有 MAS 的载玻片上（Matsunami Glass Industry）。在 42 °C 下干燥过夜后，用二甲苯对切片进行脱石蜡处理，并用乙醇-水系列进行水合处理。将样本在含有 0.1% （体积分数）吐温 20、1.5% 甘氨酸和 5% BSA 的 10 mM PBS（PBS；2.68 mM KCl、0.15 M Na ₂ HPO ₄ 和 0.086 M KH ₂ PO ₄ ）中于 22 ℃ 温育 45 分钟。然后用常规盐冲洗液[RSRS；10 mM PBS、0.88% NaCl、0.1%（体积分数）吐温 20 和 0.8% BSA]冲洗样品，并用含 0.8% BSA 溶液的 10 mM PBS（PBS+BSA）短暂洗涤以去除吐温 20。在每张载玻片上涂抹抗-IAA 单克隆抗体（编号 A0855，Sigma-Aldrich；400 μL 1:1,000 稀释液，来自 2.1 mg/mL 储存液）后，将样品置于室温湿度箱中孵育过夜。杂交后，对样本进行一系列剧烈冲洗，两次用高盐冲洗液[HSRS；10 mM PBS、2.9% NaCl、0.1%（体积分数）Tween 20 和 0.1% BSA]冲洗 10 分钟，一次用 RSRS 冲洗 10 分钟，再用 PBS+BSA 短时间冲洗。然后将 Alexa 488 结合的山羊抗小鼠 IgG 抗体（Invitrogen；400 μL 1:300 稀释的 2 mg/mL 储存液）置于每张载玻片上，并在室温湿度箱中孵育 4-6 小时。用 RSRS 冲洗两次，每次 15 分钟，然后用抗褪色试剂（ProLong Gold；Molecular Probes）装片，盖上盖玻片，在荧光显微镜（BX51；Olympus）下观察。荧光信号的强度由 Image J 软件测量。

GUS staining was performed as follows: Arabidopsis inflorescences were fixed in acetone (90%) for 2 h at −20 °C, and then infiltrated for 15 min with 2 mM ferricyanide and 2 mM ferrocyanide in Na phosphate buffer (staining buffer). Samples were then incubated in 2 mM X-Gluc (in staining buffer) at 37 °C for 15 h (for inflorescences) or 0.5 h (for seedlings), and the whole tissues were cleared by using Hoyer's medium. Observations were performed by using DIC optics (BX51; Olympus). To examine the GUS expression pattern in tissues, stained samples were fixed in FAA for 30 min at room temperature. Then samples were dehydrated, embedded in paraffin, and serially sectioned at a thickness of 10 μm.
GUS 染色方法如下：拟南芥花序在-20 °C下用丙酮（90%）固定 2 小时，然后用 2 mM 铁氰化钾和 2 mM 铁氰化钾在 Na 磷酸盐缓冲液（染色缓冲液）中浸润 15 分钟。然后将样品放入 2 mM X-Gluc（染色缓冲液）中于 37 °C下孵育 15 小时（花序）或 0.5 小时（幼苗），并用霍耶氏培养基清除整个组织。使用 DIC 光学仪器（BX51；奥林巴斯）进行观察。为了检测组织中的 GUS 表达模式，染色后的样品在室温下用 FAA 固定 30 分钟。然后将样本脱水，包埋在石蜡中，并以 10 μm 厚度连续切片。

Isolation of total RNA from barley panicles and Arabidopsis stamens was carried out by using TRIzol Reagent (Invitrogen). Real-time quantitative RT-PCR was performed by using the SYBER ExScript RT-PCR Kit (TaKaRa), and the primer sets are listed in Table S1. Triplicate PCRs in each sample were carried out in a series of experiments, and each series was performed in biological triplicate.
使用 TRIzol Reagent（Invitrogen 公司）从大麦圆锥花序和拟南芥雄蕊中分离总 RNA。使用 SYBER ExScript RT-PCR Kit（TaKaRa）进行实时定量 RT-PCR，引物组见表 S1。在一系列实验中对每个样本进行了一式三份的 PCR，每一系列实验均以生物三联法进行。

Statistics were calculated with MS Excel. Statistical significance was assessed by an unpaired Student two-tailed t test. Values were considered statistically significant at P < 0.05.
统计数据用 MS Excel 计算。统计显著性采用非配对的学生双尾 t 检验。当 P < 0.05 时，结果具有统计学意义。