The tapetal tissue is essential for the maintenance of redox homeostasis during microgametogenesis in tomato

Abstract

The tapetum is a specialized layer of cells within the anther, adjacent to the sporogenous tissue. During its short life, it provides nutrients, molecules and materials to the pollen mother cells and microsporocytes, being essential during callose degradation and pollen wall formation. The interaction between the tapetum and sporogenous cells in Solanum lycopersicum (tomato) plants, despite its importance for breeding purposes, is poorly understood. To investigate this process, gene editing was used to generate loss-of-function mutants that showed the complete and specific absence of tapetal cells. These plants were obtained targeting the previously uncharacterized Solyc03g097530 (SlTPD1) gene, essential for tapetum specification in tomato plants. In the absence of tapetum, sporogenous cells developed and callose deposition was observed. However, sporocytes failed to undergo the process of meiosis and finally degenerated, leading to male sterility. Transcriptomic analysis conducted in mutant anthers lacking tapetum revealed the downregulation of a set of genes related to redox homeostasis. Indeed, mutant anthers showed a reduction in the accumulation of reactive oxygen species (ROS) at early stages and altered activity of ROS-scavenging enzymes. The results obtained highlight the importance of the tapetal tissue in maintaining redox homeostasis during male gametogenesis in tomato plants.

Keywords: Solanum lycopersicum; TPD1; ROS; anther; male sterility; pollen; tapetum.

Figure 1

Solyc03g097530 encodes the ortholog of TPD1, TDL1A and MAC1 in tomato. (a) Amino acid sequence alignment between the Arabidopsis, rice, maize and tomato gene homologs. The putative signal peptides are underlined, the six conserved cysteine residues are in bold and the potential dibasic cleavage site is highlighted with a red rectangle. (b) Relative expression of Solyc03g097530 in different plant tissues analyzed by qRT‐PCR. Data were normalized to the expression of the SlACT10 gene and correspond to the mean (±SD) of three biological replicates. (c–h) Localization of Solyc03g097530 transcript by in situ hybridization on reproductive meristems and developing flowers. Abbreviations: Mp, microspores; MT, microspore tetrads; P, mature pollen; SC, sporogenous cells; St6, floral stage 6; St8, floral stage 8; St10, floral stage 10; St12, floral stage 12; St14, floral stage 14; T, tapetum.

Figure 2

Comparison of wild‐type and Sltpd1 mutant anther and pollen development. Histological sections of anthers from the wild type (a–c and g–i) and Sltpd1 (Sltpd1 ^Del5 ) mutant (d–f and j–l) at different developmental stages. Cross section of anthers from floral stage 6 (a) and (d), floral stage 8 (b) and (e), floral stage 10 (c) and (f), floral stage 12 (g) and (j), floral stage 14 (h) and (k) and floral stage 16 (i) and (l). Floral stages have been named according to Brukhin et al., . Scale bar: 50 μm. Abbreviations: Ep, epidermis; En, endothecium; ML, middle layers; Mp, microspores; P, mature pollen; SC, sporogenous cells; T, tapetum; Td, tetrads. The asterisks (*) in panel (a) mark periclinal cell divisions.

Figure 3

Sltpd1 mutant anthers specifically lack tapetal cells. (a) In situ hybridization of the tapetum marker TomA5B in wild‐type and Sltpd1 (Sltpd1 ^Del5 ) anthers. (b) In situ hybridization of the meiosis marker SlSDS in wild‐type and Sltpd1 (Sltpd1 ^Del5 ) anthers at floral stage 8. (c) Callose deposition in anthers as observed by aniline blue staining of the wild type and Sltpd1 (Sltpd1 ^Del5 ) at floral stages 10 and 12. Abbreviations: St6, floral stage 6; St7, floral stage 7; St8, floral stage 8; St10, floral stage 10; St12, floral stage 12. Scale bars: 50 μm in (a) and (b); 100 μm in (c).

Figure 4

Global gene expression changes in the anthers of Sltpd1 (Sltpd1 ^Del5 ) mutants at floral stage 8 in comparison with the wild type. (a) Total number of differentially expressed genes (DEGs) between wild‐type and mutant anthers. (b) GO biological process enrichment analysis. (c) Expression heat map of DEGs involved in pollen and anther development. (d) Expression heat map of differentially expressed ROS‐related genes. Q < 0.05; P < 0.05.

Figure 5

Expression pattern of genes involved in redox homeostasis during anther development in wild‐type and Sltpd1 (Sltpd1 ^Del5 ) mutant plants. Quantitative RT‐PCR of (a) SlRbohA/Solyc01g099620 gene, (b) SlRbohE/Solyc06g075570 gene, (c) RBOH1SlRbohG/Solyc08g081690 gene, (d) SlGRX9/Solyc08g036570 gene, (e) SlTGA9/Solyc06g074320 gene and (f) SlTGA10/Solyc10g078670 gene. Data correspond to three biological replicates ± SDs. Statistical differences were inferred using a Mann–Whitney test: *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6

SlGRX9 physically interacts with SlTGA9, but not with SlTGA10. BiFC analysis in Nicotiana benthamiana leaves of SlGRX9, SlTGA9 and SlTGA10 fusions to N‐ and C‐terminal fragments of YFP observed by confocal microscopy. SlGRXC9 physically interacted with SlTGA9 in the nuclei. Representative images are shown. Del2GAI_cYFP, which does not interact with SlTGA9, SlTGA10 or SlGRXC9, was used as a negative control. Scale bars: 50 μm.

Figure 7

Redox homeostasis is altered in Sltpd1 mutant anthers. Quantification of superoxide anion (O₂·⁻) levels (a) and H₂O₂ levels (b) in wild‐type and Sltpd1 anthers at different developmental stages (St8–St20). (c) DAB staining of wild‐type and Sltpd1 anthers at the same floral stages analyzed in (a) and (b). Quantification of superoxide dismutase (d) and peroxidase (e) activity in wild‐type and Sltpd1 flowers at different developmental stages (St6–St20). Data correspond to three biological replicates ± SDs. Statistical differences were inferred using a Mann–Whitney test: *P < 0.05; **P < 0.01.

Figure 8

Working model summarizing the genetic elements of the redox network affected by the absence of SlTPD1 and the concomitant tapetum loss, at early stages of tomato anther development. Enzymatic ROS accumulation (orange) is attenuated by an ROS‐scavenging mechanism (pink). Changes in ROS levels activate signaling pathways (blue) that result in the induction of genes involved in anther/pollen development. SlGRX9 physically interacts with SlTGA9 but not with SlTGA10 in planta. Abbreviations: APX, ascorbate peroxidase; GRXs, glutaredoxins; PRXs, peroxidases; RBOH, respiratory burst oxidase homolog; SOD, superoxide dismutase; TGAs, TGA transcription factors.