The rapid rise in mean global temperature as a result of global warming threatens plant productivity (Li et al., 2015). Chloroplasts and chloroplast proteins are associated with environmental stresses (Alexia et al., 2019; Hong et al., 2020). Many heat‐shock proteins (HSPs) associate with chloroplast development and improve plant tolerance to heat stress at a high temperature (Shen et al., 2015; Zhong et al., 2013), whereas no gene is reported to promote chloroplast development and enhance tolerance to high temperature synchronously. The impact of high temperature on chloroplast is of particular significance since photosynthesis is often inhibited before other cell functions are impaired (Zhang et al., 2010). Thus, promoting chloroplast biogenesis and photosynthesis is a potential method to enhance heat tolerance of plants. We previously found that overexpression of SOC1 or SOC1‐like genes in heat‐stressed plants induces chloroplast biogenesis in petals (Wang et al., 2019). However, it is unknown whether the photosynthesis apparatus is impaired and whether the plant thermotolerance is enhanced in transgenic plants. In our present study, the transplastomic (harbouring GFP reporter gene driven by psbA promoter of chloroplast), multigene transgenic tobacco (Fbp21 gene was introduced to the genome of the pure line of GFP transplastomic tobacco‐labelling nFbp21*pGFP) and transgenic petunia‐overexpressing FBP21 gene were produced by chloroplast and nuclear transformation. Additionally, the transgenic plants (Fbp21‐labelling F21 and Fbp21*22‐labelling F21_22 in this paper) harbouring SOC1‐like genes and RNA‐Seq data of petals, previously reported (Wang et al., 2019), were also integrated. Finally, a series of experiments related to RNA sequencing in leaves, biological and physiological, anatomical and phenotypic determination were undertaken.

When plants were grown at high temperature (40°C days/28°C nights), it showed that only nonphotosynthetic plastids containing plastoglobules were seen in pink petals of control tobacco plants. We observed morphologically normal chloroplasts in green petals of the SOC1‐like gene transgenic tobacco plants (Figure 1a). Chloroplasts in green petals of nFbp21*pGFP transplastomic tobacco were observed to emit red and green fluorescence simultaneously at high temperature (Figure 1b). It indicates that chloroplast genes were expressing in these heat stress‐induced plastids in petals. Maximum photochemical efficiency values (F_v/F_m) determination also showed that photosynthesis took place in chloroplast‐containing petals (Figure 1c and d). Most of the photosynthesis genes were dramatically up‐regulated in chloroplast‐containing green petals (Figure 1e). Immunoblot analysis also showed many photosynthesis associated proteins were synthesized in green petals (Figure 1f).

Heat‐resistant assay showed that the SOC1‐like gene transgenic tobacco (F21and F21_22) was substantially different from the control tobacco in their tolerance to prolonged extreme heat stress. For 2‐week‐old tobacco plants, more light yellow seedlings were seen in the wild‐type tobacco than in transgenic lines after heat stress (Figure 1g). F_v/F_m values in transgenic lines were also notably higher than that of wild type (Figure 1g and h). The lower electrolyte leakage (EL) (Figure 1i) and higher survival rate (Figure 1j) suggest that these 2‐week‐old transgenic tobacco had enhanced heat tolerance. When plants were grown at normal temperature, more chloroplasts in cells of leaves of the transgenic tobacco (including F21 or F21_22 tobacco) also observed in the nFbp21*pGFP transplastomic tobacco according to red and green fluorescence compared to GFP transplastomic tobacco (Figure 1k). These results suggest that overexpression of Soc1‐like genes promotes chloroplast biogenesis in transgenic leaves. When grown at high temperature, the leaf chloroplasts of the transgenic tobacco (also seen in nFbp21*pGFP) maintained normal appearance and orderly distribution and emitted more green fluorescence (Figure 1k). These observations indicate that the chloroplast genes can normally express at high temperature, whereas the chloroplasts in leaves of control tobacco became swollen, globular and irregular. It was consistent with what reported by Kwon and colleagues in GFP transplastomic tobacco (Kwon et al., 2013). The structural changes of chloroplasts and their scattered distribution in control tobacco (Figure 1k) suggest higher instability of varied cell membranes and cell damages by heat at high temperature.

The response to high temperature of 6‐week‐old tobacco plants was also markedly different between control and transgenic tobacco (Figure 1l). Many photosynthesis genes were dramatically up‐regulated in leaves of transgenic plants growing at high temperature (Figure 1m). Immunoblot analysis showed that photosynthesis‐associating proteins were accumulated in transgenic plants (Figure 1n). Under continuous heat stress (45°C for 9 h), F_v/F_m values were also significantly higher in leaves of transgenic tobacco plants (Figure 1o). A time series of net photosynthetic rate (Pn) determination indicated that the leaf Pn rate of transgenic tobacco plants was higher than that of wild type (Figure 1p). Magnesium is part of the chlorophyll and essential for photosynthesis (Leonard, 1954), and higher magnesium content was also detected in leaves of F21 transgenic tobacco plants (Figure 1q). Taking together, these results suggested that SOC1‐like gene transgenic tobacco plants possess enhanced photosynthetic capacity under heat stress conditions.

Leaf EL value of 6‐week‐old transgenic tobacco was lower than that of wild type after heat stress (Figure 1r). RNA‐seq analysis showed that genes encoding heat‐shock proteins were also greatly up‐regulated in leaves of transgenic tobacco plants under heat stress (Figure 1s). GC‐MS analysis showed the proline content was significantly higher in SOC1‐like gene transgenic tobacco plants (Figure 1t). It is worth noticing that during budding phase, the transgenic tobacco plants were not impaired and flowered normally under heat stress (12‐h light cycle for 15 days). In contrast, wild‐type tobacco plants did not flower or flowered poorly under the same heat stress condition (Figure 1u).

Additionally, the transgenic petunia‐overexpressing Fbp21 gene was also more heat tolerant at varied growth phases than wild‐type petunia plants (Figure 1v and w). The highest survival rate (96.60%) was recorded for 2‐week‐old transgenic petunia after heat stress (Figure 1v). For 6‐week‐old petunia seedlings, none of the wild‐type petunia seedlings survived after 3 days under high temperature (Figure 1w). Similar to tobacco‐overexpressing SOC1‐like genes, the transgenic petunia‐overexpressing Fbp21 gene also produced light green petals under heat stress (Figure 1x).

In an earlier study, we reported for the first time that SOC1‐like genes promote chloroplast biogenesis in heat‐stressed petals (Wang et al., 2019). In the present study, we show that cell containing increased number of chloroplasts is observed more frequently in leaves of Soc1‐like gene transgenic plants than in wild‐type plants and that SOC1‐like genes up‐regulate photosynthesis and heat‐shock‐associated genes, improve plant photosynthesis and alleviate heat stress damage to the chloroplast. Our results demonstrated that the super plants having chloroplast‐containing petals, higher chlorophyll contents, increasing photosynthesis and enhancing heat tolerance could be synchronously achieved by genetic engineering. We showed that plant flowers can perform photosynthesis to further improve carbon utilization efficiency under heat stress and that overexpression of SOC1‐like genes reduce the deleterious effects of heat stress on chloroplast and enhance photosynthesis in plants. Our observation provides a novel insight into the crosstalk mechanism between high temperature, plant functional chloroplast biogenesis, plant photosynthesis and plant heat tolerance. Producing heat‐tolerant plants will be of great ecological and economic significance under the increasing threat of global warming.

全球变暖导致全球平均温度的迅速上升威胁着植物的生产力（Li等，2015）。叶绿体和叶绿体蛋白与环境压力有关（Alexia等， 2019； Hong等，2020）。许多热休克蛋白（HSP）与叶绿体的发育有关，并提高了植物在高温下对热胁迫的耐受性（Shen等，2015； Zhong等，2013）。），而尚无相关基因可促进叶绿体发育并同步增强对高温的耐受性。高温对叶绿体的影响尤为重要，因为光合作用通常在其他细胞功能受损之前就被抑制了（Zhang等，2010）。因此，促进叶绿体的生物发生和光合作用是增强植物耐热性的潜在方法。我们先前发现热胁迫植物中SOC1或SOC1样基因的过表达诱导花瓣中的叶绿体生物发生（Wang等人，2019）。然而，尚不清楚在转基因植物中光合作用装置是否受损以及植物耐热性是否增强。在我们目前的研究中，转基因组（携带叶绿体的psbA启动子驱动的GFP报告基因），多基因转基因烟草（Fbp21基因被引入GFP转质子烟草标记nFbp21 * pGFP纯系的基因组）和转基因矮牵牛过表达FBP21基因是通过叶绿体和核转化产生的。此外，转基因植物（本文报道的Fbp21标记为F21和Fbp21 * 22标记为F21_22）具有类似SOC1的基因和花瓣的RNA-Seq数据，以前已有报道（Wang等人（2019）。最后，进行了一系列与叶片RNA测序，生物学和生理学，解剖学和表型测定有关的实验。

当植物在高温（40°C天/ 28°C晚上）下生长时，表明在对照烟草植物的粉红色花瓣中仅观察到含有质体球的非光合质体。我们在SOC1类基因转基因烟草植物的绿色花瓣中观察到形态正常的叶绿体（图1a）。在高温下，观察到nFbp21 * pGFP转质体烟草的绿色花瓣中的叶绿体同时发出红色和绿色荧光（图1b）。这表明叶绿体基因在这些热应激诱导的花瓣质体中表达。最大光化学效率值（F _v / F _m）测定还表明，光合作用发生在含叶绿体的花瓣中（图1c和d）。大多数光合作用基因在含有叶绿体的绿色花瓣中显着上调（图1e）。免疫印迹分析还显示，许多与光合作用相关的蛋白质均在绿色花瓣中合成（图1f）。

耐热性分析表明，类似于SOC1的基因转基因烟草（F21和F21_22）与对照烟草在延长的极端高温胁迫下的耐受性显着不同。对于2周龄的烟草植物，在野生型烟草中发现的浅黄色幼苗比受热胁迫的转基因品系中的种子多（图1g）。˚F _v / F_米转基因品系中的值也显着高于野生型（图1g和h）。较低的电解质渗漏（EL）（图1i）和较高的存活率（图1j）表明，这些2周龄的转基因烟草具有增强的耐热性。当植物在常温下生长时，与GFP转质体烟草相比，根据红色和绿色荧光，在nFbp21 * pGFP质体烟草中转基因烟草（包括F21或F21_22烟草）的叶片细胞中也观察到更多的叶绿体（图1k）。这些结果表明，Soc1类基因的过表达促进转基因叶片中叶绿体的生物发生。当在高温下生长时，转基因烟草的叶片叶绿体（也见于nFbp21 * pGFP）保持正常外观和有序分布，并发出更多的绿色荧光（图1k）。这些观察结果表明叶绿体基因可以在高温下正常表达，而对照烟草叶片中的叶绿体变得肿胀，球形和不规则。这与Kwon及其同事在GFP转质子烟草中的报道一致（Kwon等，2013）。叶绿体的结构变化及其在对照烟草中的分散分布（图1k）表明，高温下各种细胞膜的不稳定性更高，细胞受热破坏也更高。

对照和转基因烟草对6周龄烟草植株的高温反应也有显着差异（图1l）。在高温下生长的转基因植物的叶子中，许多光合作用基因被显着上调（图1m）。免疫印迹分析表明，与光合作用相关的蛋白质在转基因植物中积累（图1n）。在连续热应力下（45°C持续9 h），F _v / F _m转基因烟草植株的叶片中的值也显着较高（图1o）。净光合速率（Pn）的时间序列确定表明，转基因烟草植物的叶片Pn速率高于野生型（图1p）。镁是叶绿素的一部分，是光合作用所必需的（Leonard，1954年），在F21转基因烟草植物的叶片中也检测到较高的镁含量（图1q）。综上所述，这些结果表明，在热胁迫条件下，类似于SOC1的基因转基因烟草植物具有增强的光合作用能力。

热应激后6周龄转基因烟草的叶片EL值低于野生型（图1r）。RNA序列分析表明，在热胁迫下，转基因烟草植物叶片中编码热休克蛋白的基因也大大上调（图1s）。GC‐MS分析表明，SOC1类基因转基因烟草植株中脯氨酸含量显着较高（图1t）。值得注意的是，在发芽阶段，转基因烟草植株在热胁迫下（12 h光照周期为15天）没有受到损害并正常开花。相反，在相同的热胁迫条件下，野生型烟草植物没有开花或开花不良（图1u）。

此外，转基因矮牵牛过表达的Fbp21基因在不同的生长阶段也比野生型矮牵牛植物具有更高的耐热性（图1v和w）。热应激后2周龄的转基因矮牵牛的最高成活率（96.60％）（图1v）。对于6周龄的矮牵牛苗，在高温下3天后，没有野生型矮牵牛苗存活（图1w）。与烟草过表达SOC1样基因相似，转基因矮牵牛过表达的Fbp21基因在热胁迫下也产生浅绿色的花瓣（图1x）。

在较早的研究中，我们首次报道了SOC1样基因促进热应激花瓣中的叶绿体生物发生（Wang等，2019）。在本研究中，我们表明，与野生型植物和SOC1类似的植物相比，在Soc1类基因转基因植物的叶片中观察到的叶绿体数量增加的细胞更为常见。这些基因上调了光合作用和热休克相关基因，改善了植物的光合作用并减轻了热胁迫对叶绿体的损害。我们的结果表明，通过基因工程可以同时实现具有叶绿体花瓣，较高叶绿素含量，增加的光合作用和增强耐热性的超级植物。我们证明了植物花可以在热胁迫下进行光合作用以进一步提高碳的利用效率，并且SOC1样的过表达基因减少了热胁迫对叶绿体的有害影响并增强了植物的光合作用。我们的观察结果为高温，植物功能性叶绿体生物发生，植物光合作用和植物耐热性之间的串扰机制提供了新颖的见解。在全球变暖威胁日益严重的情况下，生产耐热植物将具有重要的生态和经济意义。