Jasmonate (JA) critically regulates plant development and stress response, but its spatio-temporal distribution at the cellular level remains unclear. A JA biosensor consisting of a JA degron motif Jas9 fused with the fluorescent protein VENUS was developed in Arabidopsis (Larrieu et al., 2015), but its 35S promoter has low activity in reproductive tissues and does not express well in monocotyledons, thus limiting its application in crops and reproductive development.

To develop a JA biosensor in rice, we generated a synthetic construct based on Jas9-VENUS (Figure 1a), containing (i) a single optimal maize ubiquitin-1 (Ubi-1) promoter (Cornejo et al., 1993), (ii) a nuclear-localized JA sensor module (Jas-VENUS) with an optimized JA-dependent degradation sequence, VENUS, a N7 nuclear localization signal (NLS) (Cutler et al., 2000) and a 6x Hemagglutinin (HA) tag, (iii) a nuclear normalization element (H2B-mCherry) containing a fusion of the Histone H2B protein and the red fluorescent protein mCherry (Shaner et al., 2004) and (iv) a F2A ribosomal skipping peptide as linker, allowing stoichiometric co-production of Jas-VENUS-HA and H2B-mCherry (Liu et al., 2017). JA responses can thus be inferred ratiometrically by comparing fluorescence signals of VENUS and mCherry.

We selected OsJAZ3 and OsJAZ6 as sensor constructs as they were expressed ubiquitously and were JA sensitive (Figure 1b), and they interacted with OsCOI1b in the presence of coronatin (COR, JA analog) (≥0.5 μm; Figure 1c). We used their Jas degron sequences to make J3V-HM and J6V-HM (Ubi-1:Jas3/6-VENUS-6HA:F2A:H2B-mCherry) (Figure 1b). Jas motif mutants (mJas), having two amino acids substitutions (RK->AA) that block JA-dependent Jas degradation, were used as controls (Figure 1a) (Cai et al., 2014). For each construct, we obtained at least three independent transgenic lines, in which the sensor and the normalization element proteins were properly expressed and translated in tandem (Figure 1d). Robust VENUS fluorescence signals were only observed in the J6V-HM transgenic lines, with line 6 chosen for further analysis.

We next assessed whether the J6V-HM transgenic lines are suitable as JA indicators in rice. We first characterized JA content in the root tip since the fluorescence was clearest in this region. Upon MeJA treatment, VENUS fluorescence was rapidly (20 min) suppressed in J6V-HM seedling roots, but not in mJ6V-HM roots (Figure 1e). Immunoblot analyses confirmed that the decrease in fluorescence correlated with the degradation of the J6V-VENUS protein (Figure 1f). Treatment with MG132, an inhibitor of the 26S proteasome, blocked the fluorescence change in J6V-HM (Figure 1e), demonstrating that the J6V-HM response was due to JA-induced protein degradation through the 26S proteasome. Further analyses revealed that J6V-HM degradation responded to four active jasmonic molecules (Figures 1g,h). Finally, fluorescence quantification showed that the relatively rapid decrease in J6V-HM fluorescence was induced by bioactive JA, and to a lesser extent by GA3 (Figure 1i), confirming cross talks between JA and GA signalling (Hou et al., 2010).

We next explored whether J6V-HM can measure cellular JA responses upon environmental challenges. VENUS fluorescence, and thus JA, was significantly reduced in all root cells 8 min after the addition of 200 mm NaCl, which continued until 60 min after treatment (Figure 1j). These results correlated well with JA levels, that is JA and JA-Ile levels (Figure 1k), analysed via high-performance liquid chromatography–tandem mass spectrometry (HPLC-QQQMS), and the expression of the JA-responsive gene OsOPR7 (Figure 1l). We also tested the efficiency of J6V-HM in response to wounding at the root tip after damaging the roots 1 cm above the root tip with tweezers. Here, the VENUS signal was significantly reduced within 30 min (Figure 1m), and the expression of the wounding marker gene OsAOS2, which encodes a JA biosynthetic enzyme, was induced (Figure 1n).

We next assessed JA content in different root tip cells. Since degradation of J6V-HM is proteasome-dependent and OsCOI1-mediated, and that most of the OsJAZ proteins only interact with OsCOI1b (Cai et al., 2014), we generated OsCOI1b-GFP plants as control. By comparing the fluorescence of OsCOI1b-GFP and mJ6V-HM (Figures 1o,p), we found higher JA levels in root epidermis, root cap and the quiescent centre, and relatively lower JA levels in stele and cortex cells, especially in exodermis (Figure 1q).

Jasmonate is pivotal for reproduction (Acosta and Przybyl, 2019); however, JA distribution in anthers during reproduction is unclear. We observed J6V-HM fluorescence in the anther at stage 8, with the strongest signals in tetrads. At stage 9, the signals subsided in epidermis (E) and endothecium (En), but remained strong in microspore (MSP). Fluorescence decreased in E, En and MSP at stage 11, and no above-background fluorescence was detected in any anther cells at stage 12 (Figure 1r). The VENUS fluorescence was homogenous in all filament cells, increased continuously from stages 8 to 11 and started to decrease at stage 12 before completely disappearing at stage 13 (Figure 1s). These results demonstrate that JA levels peaked at stage 13 in filaments and at stage 12 in anthers during rice anthesis. It should be noted that some cell and tissue types may be better suited to visualize the JA content via J6V-HM, largely due to protein and expression levels. Nevertheless, J6V-HM is a powerful tool to detect JA levels during reproduction and to monitor dynamic JA responses in rice.

茉莉酸 (JA) 严格调节植物发育和胁迫反应，但其在细胞水平上的时空分布仍不清楚。在拟南芥中开发了一种由 JA degron 基序 Jas9 与荧光蛋白 VENUS 融合而成的 JA 生物传感器(Larrieu et al ., 2015 )，但其35S启动子在生殖组织中活性低，在单子叶植物中表达不佳，从而限制了其在作物和生殖发育中的应用。

为了在水稻中开发 JA 生物传感器，我们生成了基于 Jas9-VENUS 的合成构建体（图 1a），包含（i）单个最佳玉米泛素-1（Ubi-1）启动子（Cornejo等，1993），（ ii) 核定位 JA 传感器模块 (Jas-VENUS)，具有优化的 JA 依赖性降解序列、VENUS、N7 核定位信号 (NLS) (Cutler et al ., 2000 ) 和 6x 血凝素 (HA) 标签， (iii) 包含组蛋白 H2B 蛋白和红色荧光蛋白 mCherry 融合的核标准化元件 (H2B-mCherry) (Shaner et al ., 2004 ) 和 (iv) F2A 核糖体跳跃肽作为接头，允许化学计量共生产 Jas-VENUS-HA 和 H2B-mCherry (Liu等人，2017 年）。因此，可以通过比较 VENUS 和 mCherry 的荧光信号来按比例推断 JA 响应。

我们选择 OsJAZ3 和 OsJAZ6 作为传感器构建体，因为它们普遍表达并且对 JA 敏感（图 1b），并且它们在 coronatin（COR，JA 类似物）存在下与 OsCOI1b 相互作用（≥0.5 μ m；图 1c）。我们使用他们的 Jas degron 序列制造 J3V-HM 和 J6V-HM ( Ubi-1:Jas3/6-VENUS-6HA:F2A:H2B-mCherry ) (图 1b)。Jas 基序突变体 (mJas)，具有两个氨基酸取代 (RK->AA)，可阻止 JA 依赖性 Jas 降解，用作对照 (图 1a) (Cai et al ., 2014）。对于每个构建体，我们获得了至少三个独立的转基因系，其中传感器和标准化元件蛋白被正确表达和串联翻译（图 1d）。仅在 J6V-HM 转基因株系中观察到强 VENUS 荧光信号，选择第 6 株进行进一步分析。

我们接下来评估了 J6V-HM 转基因品系是否适合作为水稻中的 JA 指标。我们首先表征了根尖中的 JA 含量，因为该区域的荧光最清晰。在 MeJA 处理后，VENUS 荧光在 J6V-HM 幼苗根中被迅速抑制（20 分钟），但在 mJ6V-HM 根中没有（图 1e）。免疫印迹分析证实荧光的降低与 J6V-VENUS 蛋白的降解相关（图 1f）。用 26S 蛋白酶体抑制剂 MG132 处理阻断 J6V-HM 中的荧光变化（图 1e），证明 J6V-HM 反应是由于 JA 诱导的通过 26S 蛋白酶体的蛋白质降解。进一步的分析表明，J6V-HM 降解对四种活性茉莉分子有反应（图 1g，h）。最后，等人，2010 年）。

我们接下来探讨了 J6V-HM 是否可以测量细胞对环境挑战的 JA 反应。在添加 200 mm NaCl后 8 分钟，所有根细胞中的 VENUS 荧光和因此 JA 显着降低，持续到处理后 60 分钟（图 1j）。这些结果与 JA 水平密切相关，即 JA 和 JA-Ile 水平（图 1k），通过高效液相色谱 - 串联质谱（HPLC-QQQMS）分析，以及 JA 响应基因OsOPR7的表达（图1升）。我们还测试了 J6V-HM 在用镊子损坏根尖上方 1 厘米的根部后对根尖损伤的反应效率。在这里，VENUS 信号在 30 分钟内显着降低（图 1m），并且伤人标记基因的表达编码 JA 生物合成酶的OsAOS2被诱导（图 1n）。

我们接下来评估了不同根尖细胞中的 JA 含量。由于 J6V-HM 的降解是蛋白酶体依赖性和 OsCOI1 介导的，并且大多数 OsJAZ 蛋白仅与 OsCOI1b 相互作用（Cai等人，2014 年），我们生成了 OsCOI1b-GFP 植物作为对照。通过比较 OsCOI1b-GFP 和 mJ6V-HM 的荧光（图 1o，p），我们发现根表皮、根冠和静止中心的 JA 水平较高，而中石细胞和皮层细胞的 JA 水平相对较低，尤其是在外皮层（图 1o，p）。图 1q)。

茉莉酸是繁殖的关键（Acosta 和 Przybyl，2019); 然而，生殖过程中花药中的 JA 分布尚不清楚。我们在第 8 阶段观察到花药中的 J6V-HM 荧光，在四分体中信号最强。在第 9 阶段，信号在表皮 (E) 和内皮 (En) 中减弱，但在小孢子 (MSP) 中仍然很强。在第 11 阶段，E、En 和 MSP 的荧光降低，在第 12 阶段的任何花药细胞中均未检测到高于背景的荧光（图 1r）。VENUS 荧光在所有细丝细胞中是均匀的，从第 8 阶段到第 11 阶段连续增加，并在第 12 阶段开始减少，然后在第 13 阶段完全消失（图 1s）。这些结果表明，在水稻开花期，JA 水平在花丝的第 13 阶段和花药的第 12 阶段达到峰值。应该注意的是，某些细胞和组织类型可能更适合通过 J6V-HM 可视化 JA 内容，主要是由于蛋白质和表达水平。然而，J6V-HM 是检测繁殖过程中 JA 水平和监测水稻动态 JA 反应的强大工具。