Reactive oxygen species mediate tapetal programmed cell death in tobacco and tomato
© The Author(s). 2017
Received: 8 December 2016
Accepted: 6 April 2017
Published: 20 April 2017
Hybrid vigor is highly valued in the agricultural industry. Male sterility is an important trait for crop breeding. Pollen development is under strict control of both gametophytic and sporophytic factors, and defects in this process can result in male sterility. Both in the dicot Arabidopsis and in the moncot rice, proper timing of programmed cell death (PCD) in the tapetum ensures pollen development. Dynamic ROS levels have been reported to control tapetal PCD, and thus pollen development, in Arabidopsis and rice. However, it was unclear whether it is evolutionarily conserved, as only those two distantly related species were studied.
Here, we performed histological analyses of anther development of two economically important dicot species, tobacco and tomato. We identified the same ROS amplitude during anther development in these two species and found that dynamic ROS levels correlate with the initiation and progression of tapetal PCD. We further showed that manipulating ROS levels during anther development severely impaired pollen development, resulting in partial male sterility. Finally, real-time quantitative PCR showed that several tobacco and tomato RBOHs, encoding NADPH oxidases, are preferentially expressed in anthers.
This study demonstrated evolutionarily conserved ROS amplitude during anther development by examining two commercially important crop species in the Solanaceae. Manipulating ROS amplitude through genetic interference of RBOHs therefore may provide a practical way to generate male sterile plants.
Hybrid vigor is highly valued in agricultural industry, and thus male sterile lines are favored in generating hybrids commercially. In addition, male sterility is a trait critical for biomass production, as plant senescence proceeds much faster in fertile strains than in sterile strains [1, 2]. Therefore, breeding new varieties of male sterile lines is not only important for the seed industry to generate hybrids at low cost but also critical for delaying senescence and increasing plant biomass.
Defective pollen development is a major factor causing male sterility. Pollen development can be affected by tapetal irregularities [3–6], cytoskeletal alterations [7, 8], aberrations in auxin metabolism [9, 10], and altered sugar utilization. Among all the factors controlling pollen development, the tapetum, a layer of sporophytic cells surrounding the pollen sac, plays an essential role . The tapetum undergoes a programmed cell death (PCD) process during pollen development, to provide enzymes to release microspores from tetrads, to mediate microspore development, and to deposit components for the pollen coat [3, 12, 13]. The timing and progression of tapetal PCD is tightly controlled by evolutionarily conserved transcriptional networks [3, 12–17].
In addition to the transcriptional networks regulating tapetal PCD, recently intracellular factors have been identified during this process. Reactive oxygen species (ROS), including hydrogen peroxide and superoxide, affect a large number of proteins through post-translational modifications and ROS also act as critical signaling molecules [18–20]. ROS plays a key role in tapetal PCD both in Arabidopsis and in rice [21–23]. Failure to remove ROS in the tapetum resulted in male sterility, due to precocious tapetal cell death [21–23] whereas reducing ROS levels during anther development by mutation of RBOHs, genes encoding NADPH oxidases, delayed tapetal PCD . In both situations, pollen development was severely impaired [21–23].
Although the essential role of dynamic ROS levels for tapetal PCD and pollen development has been proven in Arabidopsis and rice, it was unknown whether such a mechanism is conserved in the Solanaceae. Here, we examined anther development of two economically important dicots, tobacco (Nicotiana benthamiana) and tomato (Lycopersicon esculentum, cv. ‘moneymaker’), in detail. We identified the same ROS amplitude during anther development in these two species and found that dynamic ROS level correlates with the initiation and progression of tapetal PCD. We further showed that manipulating ROS levels during anther development severely impaired pollen development, resulting in partial male sterility. Finally, real-time quantitative PCRs (qPCRs) showed that several tobacco and tomato RBOHs, encoding NADPH oxidases, are preferentially expressed in anthers. Manipulating the ROS amplitude through genetic interference of RBOHs therefore may provide a practical way to generate male sterile plants in the Solanaceae.
Anther development of tobacco and tomato accompanies with dynamic ROS levels
To determine whether anthers of tobacco and tomato exhibited dynamic ROS amplitude during development, similar to that shown in Arabidopsis , we first classified anthers into six groups (Additional file 1: Table S1), based on the correlation of developmental stages from transverse-sections of anthers with the sizes of floral buds and anthers (Additional file 1: Figures S1 and S2). These six groups represent pre-meiosis, meiosis, tetrad, microspore, mitosis, and dehiscence (Additional file 1: Figures S1, S2 and Table S1).
Pollen development in tobacco anthers
To examine the timing and progression of tapetal PCD during pollen development, it was necessary to finely dissect pollen developmental stages. Unlike Arabidopsis and rice, for which extensive studies have been conducted regarding pollen developmental processes within anthers [27, 28], pollen development of both tobacco and tomato has not been thoroughly described, although there are a few histological studies [29, 30].
Tapetal PCD in tobacco
Pollen development in tomato
Tapetal PCD in tomato
Pharmacological interference of ROS amplitude impaired pollen development
Anther-preferential expression of RBOHs in tobacco and tomato
ROS can be generated by mitochondria, peroxisomes, the cytoplasm, and the plasma membrane (PM) . Extensive studies have demonstrated the critical roles of the PM-localized NADPH oxidases in ROS signaling [20, 33], including tapetal PCD . We were thus interested in determining whether expression changes of the NADPH oxidase-encoding genes, RBOHs, contribute to the dynamic ROS amplitude during anther development in tobacco and tomato.
Another developmental stages of tobacco and tomato were divided into 20 stages beginning from stamen primordial formation to mature pollen [29, 30]. Based on the clearly defined anther developmental stages in Arabidopsis  and histological analyses (Figs. 2 and 5), we classified tobacco and tomato anther development into six major stages, i.e. pre-meiosis, meiosis, tetrad, microspore, mitosis, and dehiscence. These stages are characterized by major events occurring during pollen maturation. Anther development of tomato and tobacco differs slightly from that in Arabidopsis or rice, especially regarding tapetal cells. In the crescent-shaped anther locules of tobacco and tomato anthers, the inner, i.e. proximal tapetal cells, were more vacuolated than the outer, i.e. distal tapetal cells, which are more electron-dense (Figs. 2 and 5). By comparison, in the round anther locules of Arabidopsis and rice, tapetal cells are not dimorphic [27, 28].
By using NBT and H2DCF-DA staining of ROS in anthers of tobacco and tomato at various developmental stages, we demonstrated that dynamic ROS amplitude during anther development. Although the gradual increase of ROS during anther development occurs slightly earlier in tomato (early microspore stage) than in tobacco (Fig. 1, Additional file 1: Figure S3), the highest level of ROS was detected at the microspore stage in both species (Fig. 1), suggesting that conserved ROS amplitude during anther development. Although dynamic ROS levels were reported in developing anthers of now four different plant species, slight differences exist regarding timing and amplitude. In both Arabidopsis and rice, the ROS level rapidly increased at stage 8 and reached the highest level at stage 9 i [21, 22]. After that, it decreased rapidly, achieving a basal level at stage 11 [21, 22]. By comparison, in tomato and tobacco the ROS level slowly and gradually increased starting from stage 5–6 (Fig. 1, Additional file 1: Figure S3), reaching the highest at stage 8–9, followed by a decrease starting from stage 10 (Fig. 1).
By using transverse sections as well as TUNEL assays, we analyzed the initiation and progression of tapetal PCD in tobacco and tomato anthers. As reported for Arabidopsis , the tapetal layer of tomato anthers began condensation as early as the tetrad stage (Fig. 5). Soon after the release of microspores (Fig. 5), positive TUNEL signals were detected in the tapetal layers of tomato anthers (Fig. 6), suggesting massive tapetal PCD. At the mitotic stage, the PM of tapetal cells lost its integrity (Fig. 5), indicating the end of tapetal degeneration. By contrast, tapetal cells of tobacco anthers contained enlarged nuclei and cell volume at the tetrad stage (Fig. 2). Cell condensation was observed only at late microspore stage (Fig. 2), which led to the appearance of TUNEL signals (Fig. 4). Massive tapetal cell death was detected at the mitotic stage in tobacco anthers (Fig. 4), consistent with a rapid reduction of the tapetal layer at the same stage based on histological analysis (Fig. 2). In comparison, tapetal PCD started at the dyad stage (stage 8a) and finished at the middle microspore stage (stage 9) in rice anthers , indicating that distinct timing or progression of tapetal PCD among different plant species is possible.
The slight difference in the timing and progression of tapetal PCD between tobacco and tomato correlates well with the dynamic ROS amplitude. The increase of ROS in tomato anthers started earlier but progressed slowly, consistent with the early initiation and long duration of tapetal PCD (Fig. 6). In comparison, ROS in tobacco anthers increased abruptly at the mitotic stage, followed by a sudden drop (Fig. 1, Additional file 1: Figure S3), consistent with the late initiation and short duration of tapetal PCD (Fig. 4). Pharmacologically scavenging ROS by applying DPI during anther development severely compromised the ROS amplitude (Fig. 7). Consequently, tapetal degeneration was delayed and pollen development impaired (Fig. 7). These results suggest that the dynamic ROS amplitude during anther development is intimately linked with the initiation and progression of tapetal PCD.
Although ROS can be produced from different intracellular sources , we consider the PM-localized NADPH oxidases essential for the dynamic ROS amplitude during anther development. First, Arabidopsis RBOHs have been demonstrated to mediate the dynamic ROS amplitude, and mutations in these genes interfered with tapetal PCD . Second, as PM-associated ROS source [37, 38], NADPH oxidases are localized at the interface between developing microspores and the tapetal cells, and therefore would be good candidates to facilitate intercellular communication. Third, among several RBOHs in tobacco and tomato, we identified a few whose preferential expression in anthers suggests their potential functionality during tapetal PCD (Fig. 8). Indeed, the expression of these RBOHs not only showed tissue specificity but also displayed a temporal expression pattern during anther development (Fig. 8), correlating with the temporal pattern of ROS (Fig. 8). Reverse genetics by the newly developed genome-editing technologies, such as CRISPR-Cas9 , on these anther-preferential RBOHs may provide a feasible way to generate male-sterile tobacco and tomato plants.
Plant materials and growth conditions
Tobacco (Nicotiana benthamiana) and tomato (Lycopersicon esculentum, cv. ‘moneymaker’) were grown in an incubator at 25/20 °C (day/night) under long days.
RNA-extraction and real-time quantitative PCR (qPCRs)
Total RNAs were extracted from various tissues or anthers at different developmental stages of tobacco and tomato using the Ultrapure RNA kit according to the manufacturer’s instructions (CWBIO). Reverse transcription was performed using Reverse Transcriptase M-MLV (Takara). The qRT-PCR analyses were performed with the BioRad CFX96 real-time system using SYBR Green real-time PCR master mix (Toyobo) as described . Sizing of anthers from tobacco or tomato was used to determine developmental stages, as shown in Additional file 1: Table S1. All primers used are listed in Additional file 1: Table S2.
TUNEL assays (in situ nick-end labeling of nuclear DNA fragmentation) were performed using the In Situ Cell Death Detection Kit TUNEL system (Roche) according to the supplier’s instructions. Samples were analyzed with Axio Observer D1 microscope equipped with a CCD camera (Zeiss). Emission/excitation for the TUNEL signals and PI signals are 488 nm/505–550 nm and 561 nm/575–650 nm, respectively.
Histology of anthers and histochemical assays for ROS
Semi-thin transverse sections as well as TEMs of floral buds at different development stages were performed as described . NBT staining  and H2DCFDA staining of anthers  were performed as described. Fluorescence imaging of H2DCFDA stained anthers was performed with an Axio Observer D1 microscope equipped with a CCD camera (Zeiss) at the same parameters for all samples to facilitate comparative quantification. Anthers were classified into five groups based on their sizes as described in Additional file 1: Table S1. Three independent experiments were conducted, and each experiment involved 20 to 25 anthers in one group. Fluorescence intensity of whole anthers was quantified using ImageJ software (http://rsbweb.nih.gov/ij/).
DPI was dissolved in DMSO to make a 10 mM stock solution. The stock solution was diluted with 0.02% (w/v) Triton X-100 as a 10 μM working solution. Same dilution was made for DMSO as the control. One sepal of floral buds corresponding to anther developmental stage 5–7, as day 0 (D0), was removed. DPI or DMSO was sprayed on the floral buds once per day for 5 days until anther stage 10–11. Anthers were harvested at D0, D2, D4, D6, and D8, which corresponds to the stage of meiosis, tetrad, microspore, mitosis, and dehiscence, respectively, for NBT staining and transverse sections. Pollen grains from anthers harvested at D8 were stained with PI to determine pollen viability.
Phylogenetic analysis of RBOHs in Arabidopsis, tobacco, and tomato
Protein sequences of Arabidopsis RBOHs were obtained from the TAIR website (http://www.arabidopsis.org/). Protein sequences of Arabidopsis RBOHs were used in BLAST search at NCBI to identify tobacco RBOHs. Protein sequences of tomato RBOHs were obtained from the tomato genomic website (http://www.solgenomics.net/). The software Vector NTI (Invitrogen) was used to perform phylogenetic analysis.
In this study, we demonstrated that pollen development of two economically important dicot species, tobacco and tomato, requires dynamic ROS amplitude during anther development. Such ROS amplitude correlates with the onset and progression of tapetal PCD. Furthermore, we provide evidence supporting a key role of anther-preferentially expressed RBOH genes in the generation of tapetal ROS amplitude. Results presented here provide theoretical bases to generate commercially valuable male sterile plants.
We thank Prof. Sheila McCormick for English editing of this article.
This work was supported by Shandong Provincial Natural Science Foundation (ZR2014CM027 to S.L.) and by National Natural Science Foundation of China (31,471,304 and 31,625,003 to Y.Z.). Y.Z.’s laboratory is partially supported by Tai-Shan Scholar Program by Shandong Provincial Government.
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information files.
SY, QF, and HX performed the experiments and acquired the data; SY and SL analyzed the data; YZ designed the experiments; YZ and SY wrote the article. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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