番茄细胞分裂素与生长素响应信号可视化荧光分析

doi:10.16420/j.issn.0513-353x.2023-0715

摘要/Abstract

摘要：

解析细胞分裂素和生长素的响应信号在番茄体内的动态分布，对从细胞水平阐明这两类激素的调控功能及作用机理具有重要意义。利用细胞分裂素响应启动子TCSn1驱动绿色荧光报告基因TCSn1::GFP（GFP）和生长素响应启动子DR5v2驱动红色荧光报告基因DR5v2::tdTomato（tdTomato），实时原位分析了番茄根、茎、叶、花和果实中细胞分裂素和生长素的响应信号，发现两种荧光在发芽3 ~ 7 d幼苗根冠的侧根冠、小柱起始细胞和小柱细胞及根伸长区的原形成层、基本分生组织和原表皮层，发芽5 ~ 10 d的茎尖分生组织，定植23和37 d植株茎段的维管束组织，定植32 d植株的幼嫩叶片，开花当天的花瓣和果梗，开花2 ~ 6 d果实的胚珠、胎座、小柱及表皮等组织/器官中均有分布。两种荧光的强度均在茎尖分生组织和果实胚珠中最高，并且具有类似的变化趋势：在发芽3 ~ 7 d的根冠和伸长区中先下降后有所恢复，茎段中37 d弱于23 d，发芽5 ~ 10 d茎尖分生组织和开花0 ~ 6 d果实中持续上升。本研究为番茄细胞分裂素和生长素响应分析提供了一个灵敏而稳定的可视化工具，并解析了两种激素在番茄不同器官中的分布与强度。

关键词: 番茄, 细胞分裂素响应, 生长素响应, 可视化荧光, 显微成像

Abstract:

Analysis of dynamic distributions of cytokinin and auxin response signals in tomato plants is of great significance for clarifying the regulatory roles and functional mechanisms of these two hormones at the cytological level. A green fluorescent reporter gene TCSn1::GFP（GFP）with a cytokinin-responsive promoter TCSn1，and a red fluorescent reporter gene DR5v2::tdTomato（tdTomato）with an auxin-responsive promoter DR5v2，were used to perform real-time in-situ analyses of the signals of cytokinin and auxin responses in tomato roots，stems，leaves，flowers，and fruits. It is found that the two types of fluorescence distributed in the tissues/organs including：lateral root cap and columella in root cap，as well as the procambium，ground meristerm and protoderm in root elongation zone at three to seven days after germination，the shoot apical meristem at five to ten days after germination，vascular tissues in stems of plants at 23 and 37 days after planting，young leaves of plants at 32 days after planting，petals and fruit peduncles at anthesis，ovules，the placenta，columella and pericarp of fruits at two to six days after anthesis. Intensities of the two types of fluorescence were both highest in the shoot apical meristem and ovules in fruits，and had a similar variation trend：first decreased and then recovered in the root cap and elongation zone during three to seven days of germination；were weaker in stem segments at 37 days than 23 days after planting；continuously rose in the shoot apical meristem at five to ten days after germination and in the fruits at zero to six days after anthesis. This study provides a sensitive and robust visualization tool for analyzing cytokinin and auxin responses in tomato，and has analyzed their distribution and intensity in different organs of tomato.

Key words: Solanum lycopersicum, cytokinin response, auxin response, visible fluorescence, microimaging

赵晓冰, 孙可一, 樊俊苗, 丁静. 番茄细胞分裂素与生长素响应信号可视化荧光分析[J]. 园艺学报, 2024, 51(2): 372-384.

ZHAO Xiaobing, SUN Keyi, FAN Junmiao, DING Jing. Visible Fluorescence Analysis of Cytokinin and Auxin Responses in Tomato[J]. Acta Horticulturae Sinica, 2024, 51(2): 372-384.

图/表 8

图1 ‘Micro-Tom’番茄野生型（WT）和WT × pTCSn1::GFP; pDR5v2::tdTomato（F1）的表型 A：发芽7 d；B：定植29 d；C：定植65 d。

Fig. 1 Phenotype of wild-type tomato‘Micro-Tom’（WT）and WT × pTCSn1::GFP; pDR5v2::tdTomato（F1） A：Seven days after germination；B：Twenty-nine days after planting；C：Sixty-five days after planting.

图2 番茄WT × pTCSn1::GFP; pDR5v2::tdTomato幼苗根冠响应细胞分裂素和生长素的荧光信号 Merge：明场和荧光叠加状态，蓝框为平均荧光强度统计区域；GFP：响应细胞分裂素的绿色荧光信号；tdTomato：响应生长素的红色荧光信号。误差线上的不同字母表示在不同取样天数间差异显著（P < 0.05）。下同。

Fig. 2 Cytokinin- and auxin-responsive fluorescence signals in root caps of WT × pTCSn1::GFP; pDR5v2::tdTomato tomato seedlings Merge：the superposition of bright field and fluorescence，the blue box indicates the area for statistics of the mean fluorescence intensity；GFP：Cytokinin-responsive green fluorescence；tdTomato：Auxin-responsive red fluorescence. Different letters above the error bars indicate significant differences among the different sampling days（P < 0.05）. The same below.

图3 番茄WT × pTCSn1::GFP; pDR5v2::tdTomato幼苗根伸长区响应细胞分裂素和生长素的荧光信号 Pc：原形成层；GM：基本分生组织；Pt：原表皮层。

Fig. 3 Cytokinin- and auxin-responsive fluorescence signals in root elongation zones of WT × pTCSn1::GFP; pDR5v2::tdTomato tomato seedlings Pc：Procambium；GM：Ground meristem；Pt：Protoderm.

图4 番茄WT × pTCSn1::GFP; pDR5v2::tdTomato幼苗茎尖响应细胞分裂素和生长素的荧光信号 CZ：中心区；PZ：周缘区；OC：组织中心；RZ：肋状分生组织区；P：叶原基或原基起始点。

Fig. 4 Cytokinin- and auxin-responsive fluorescence signals in shoot apexes of WT × pTCSn1::GFP; pDR5v2:: tdTomato tomato seedlings CZ：Central zone；PZ：Peripheral zone；OC：Organization center；RZ：Rib zone；P：Leaf primordium or primordium initiation site.

图5 番茄WT × pTCSn1::GFP; pDR5v2::tdTomato植株茎段中响应细胞分裂素和生长素的荧光信号 VB：维管束；Pi：髓。白框所示为荧光分析取样区域。下同。

Fig. 5 Cytokinin- and auxin-responsive fluorescence signals in stems of WT × pTCSn1::GFP; pDR5v2::tdTomato tomato plants VB：Vascular bundle；Pi：Pith. The white box indicates the area for the sampling of fluorescence analysis. The same below.

图6 番茄WT × pTCSn1::GFP; pDR5v2::tdTomato定植32 d叶片和开花当天花瓣中响应细胞分裂素和生长素的荧光信号

Fig. 6 Cytokinin- and auxin-responsive fluorescence signals in WT × pTCSn1::GFP; pDR5v2:: tdTomato tomato leaves at 32 days after planting and petals at anthesis

图7 番茄WT × pTCSn1::GFP; pDR5v2::tdTomato开花的子房/果实中响应细胞分裂素和生长素的荧光信号

Fig. 7 Cytokinin- and auxin-responsive fluorescence signals in WT × pTCSn1::GFP; pDR5v2::tdTomato tomato overies/fruits

图8 番茄WT × pTCSn1::GFP; pDR5v2::tdTomato胚珠/胎座中响应细胞分裂素和生长素的荧光强度

Fig. 8 Intensity of cytokinin- and auxin-responsive fluorescence in WT × pTCSn1::GFP; pDR5v2::tdTomato tomato ovules/placentas

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