Skip to content

Advertisement

  • Short communication
  • Open Access

Rice UCL8, a plantacyanin gene targeted by miR408, regulates fertility by controlling pollen tube germination and growth

Contributed equally
Rice201811:60

https://doi.org/10.1186/s12284-018-0253-y

  • Received: 28 August 2018
  • Accepted: 7 November 2018
  • Published:

Abstract

Background

Pollen tube formation and growth are crucial steps that lead to seed production. Despite the importance of pollen tube growth, the molecular mechanisms implicated in its spatial and temporal control are not fully known. In this study, we found an uclacyanin gene, OsUCL8, that regulates pollen intine deposition and pollen tube growth.

Findings

The overexpression of OsUCL8 led to a striking irregularity in pollen tube growth and pollination and thus affected the seed setting rate in rice; many pollen tubes appeared to lose the ability to grow directly into the style. Conversely, plants with OsUCL8 knocked out and plants overexpressing miR408, a negative regulator of OsUCL8, had vigorous pollens with a higher germination rate. We further demonstrated that OsUCL8 mainly affects pollen intine formation. The addition of Vitamin B1 (VB1) significantly contributed to the germination of OXUCL8 pollen grains, suggesting that OsUCL8 could be associated with VB1 production. Using a yeast two-hybrid system, we revealed that OsUCL8 interacts with the protein OsPKIWI, a homolog of the Arabidopsis FNRL protein. We thus hypothesized that OsUCL8 might regulate the production of VB components by interacting with OsPKIWI. This study revealed a novel molecular mechanism of pollen tube growth regulation.

Conclusions

The rice plantacyanin family member OsUCL8 plays an important role in pollen tube formation and growth and, in turn, regulates fertility and the seed setting rate.

Findings

Double fertilization is a crucial step in flowering plant reproduction. Highly orchestrated pollen-pistil interactions and signaling events enable plant species to avoid inbreeding and outcrossing with other species. This process starts with the adhesion of pollen to the stigma, followed by grain hydration, tube generation, stigma penetration, and tube elongation inside the style (Berger et al., 2008). Pollen grains induce the formation of pollen tubes after germination on the stigma and begin the long journey to deliver their sperm cells. Despite the importance of this crucial step in seed production, the molecular mechanisms implicated in the spatial and temporal control of pollen tube growth are not fully known (Johnson and Lord, 2006; Palanivelu and Tsukamoto, 2012). Plantacyanins belong to a subfamily of blue copper proteins (Ryden and Hunt, 1993) and have been proposed to be involved in the oxidative burst that occurs during pathogen infection and in the cross-linking and solubilization of cell wall materials (Nersissian et al., 1998). Arabidopsis plantacyanins can regulate reproduction by affecting anther development and pollination(Dong et al., 2005). Recently, we also showed that a rice plantacyanin gene, OsUCL8(Oryza sativa Uclacyanin like protein 8), could regulate grain yield and photosynthesis(Zhang et al., 2017). Further studies have revealed that the cleavage of OsUCL8 by miR408 affects copper homeostasis in the plant cell, which in turn affects the abundance of plastocyanin proteins and photosynthesis. These studies suggest that the plantacyanin family can have multiple functions during plant development. We also found that plants overexpressing OsUCL8 showed semi-dwarf phenotypes, and the effective grains per panicle were dramatically lower than in WT plants. In crops, the seed setting rate is controlled by pistil and stamen development, including pollination. Thus, we asked whether OsUCL8 also participates in pollination or sexual-reproduction-associated processes.

To demonstrate how OsUCL8 affects the seed setting rate, we used three OsUCL8 mutants, including transgenic plants that ectopically expressed OsUCL8 (OXUCL8), transgenic plants with OsUCL8 knocked out via CRISPR-Cas9 (ucl8), and transgenic plants that ectopically expressed a microRNA, miR408, which has been shown to negatively regulate OsUCL8. Figure 1a showed the genomic structure of rice OsUCL8. We observed the floret structure and pollen fertility of the plants mentioned above. As shown in Fig. 1b and Additional file 1: Figure S1, the floret structures of three OsUCL8 mutants are similar to those of the WT; only the stigmas of OsUCL8-overexpressing plants appear slightly smaller than those of wild-type, and the OXUCL8 plants have twisted anthers. We also observed sporogenesis and the mature pollen; no obvious differences were found between the three mutants and the WT from the meiosis stage to mature microspore development (Additional file 1: Figure S2), suggesting that OsUCL8 did not affect male gametogenesis. To clarify the cause of the observed semisterility, mature embryo sacs were examined using whole-mount confocal laser scanning microscopy (WE-CLSM) of eosin B staining (Zeng et al., 2007). The results showed that almost all of the mature mutant embryo sacs were of the typical Polygonum type and had complete inner components with apparent polarity (Additional file 1: Figure S1c), suggesting that the mature mutant embryo sacs are functional and that the low grain setting rate of OXUCL8 may not result from a defect in female gametogenesis. In crops, the seed setting rate is controlled by pistil and stamen development, as well as pollination. Because neither male nor female gametogenesis showed defects, we speculated that the cause of the sterility in OXUCL8 plants might be associated with pollination or other sexual-reproduction-associated processes.
Fig. 1
Fig. 1

OsUCL8 affected pollination and pollen germination. (a) Genomic structure of rice OsUCL8 and constructions of the transgenic plants. (b) Mature spikelet of WT and transgenic plants. Scale bars, 1 mm. (c) Aniline blue staining of pollen tube growth in WT and mutants at 10 min, 20 min, 30 min, 1 h, 2 h after pollination (AP). Scale bars, 100 μm. (d) Reciprocal crosses between OXUCL8 and WT 2hs after artificial pollination, Scale bars, 500 μm. (e) Reciprocal crosses between OXUCL8 and WT 3 d after pollination, Scale bars, 3 mm

To validate this hypothesis, we performed in vivo pollen grain germination experiments using aniline blue staining(Kho et al., 1968) to detect pollen tube formation. The results showed that in WT and ucl8, the pollen grains attached to the stigmas and generated pollen tubes 10 min after pollination (AP); then, the pollen tubes entered the stigmas and grew rapidly 20 min AP (Fig. 1c). However, in OXUCL8, fewer pollen grains were observed on the stigmas at 20 min AP, and few pollen tubes were generated until 30 min AP. One hour AP, pollen tubes in the WT and ucl8 plants easily entered the style tract and grew rapidly, whereas pollen tubes in the OXUCL8 mutants appeared to experience difficulty entering the style tract and grew extremely slowly. In the WT, several pollen tubes reached the ovary at 2 h AP; however, pollen tubes in the OXUCL8 mutant were abnormally delayed in the style tract. Consequently, no pollen tubes were found entering the embryo sac in the mutant, even at 2 h AP (Additional file 1: Figure S3).

To further confirm that the decreased seed setting rate of OXUCL8 plants was caused by abnormal germination of pollen grains on stigma in OXUCL8 plants, we then crossed the OXUCL8 and WT plants (the OXUCL8 plants served as male parent or female parent respectively). The female parent were first treated by hot water (42 °C, 5 min) to emasculation, then the pollens from the male parent were placed on the stigma of emasculated flower to bring about fertilization. We found that lots of pollen grains fixed to the stigma of both WT and OXUCL8 plants 2 h after artificial pollination when using WT as male parent. In contrast, when we placed OXUCL8 pollens on the WT stigma, there were only few pollen grains fixed to the stigma 2 h after artificial pollination (Fig. 1d). The cross success rate when using OXULC8 as female parent is 2.5 folds higher than that using OXUCL8 as male parent (Fig. 1e). These results indicate that the stigmas of OXUCL8 plants are ready for pollination, but the pollen grains of OXUCL8 failed to germinate on the stigma, thus the cause of the sterility in OXUCL8 plants is associated with abnormal pollen grain germination..

We then monitored the pollen tube germination process in vitro in two types of media. In the optimal GM1 (Mizuta et al., 2010), 60.3% of wild-type pollen grains germinated after 12 h of culture (Fig. 2a). By contrast, the percentage of germinated OXUCL8 pollen grains was drastically lower; only 19.0% of the pollen grains had germinated after 12 h of culture. In GM2 (Dai et al., 2007), 62.9% of wild-type pollen grains had germinated after 12 h of culture. By contrast, only 32.9% of OXUCL8 pollen grains had germinated after 12 h. Interestingly, the germination rate of OXUCL8 pollen grains was higher in liquid medium containing only extra PEG and VB1 than on solid medium. To determine whether PEG or VB1 affect the germination rate of OXUCL8 pollen, the same concentration of PEG or VB1 was added to the solid GM. In the +PEG group, 78.7% of the WT pollen germinated, and OXUCL8 germination improved to 27.6%. However, in the +VB1 group, the WT pollen germination dropped to 52%. This is possibly the reason that VB1 could induced the higher levels of reactive oxygen species as reported(Ahn et al., 2006; Maksimov et al., 2018). By contrast, the pollen germination of OXUCL8 still improved to 26.7% at a low concentration of VB1 (Fig. 2a), and very notably, the OXUCL8 pollen germination increased from 26.7% to 77.2% when the VB1 in the solid GM was increased from 0.3 mg/L to 30 mg/L (Fig. 2b and c), indicating that the higher VB1 concentration significantly contributed to the germination of OXUCL8 pollen grains. For comparison, we also investigated ucl8. The results clearly showed ucl8 phenotypes are negatively correlated with those of OXUCL8, further supporting the observation that OsUCL8 plays a regulatory role in pollen germination. The in vitro experiment also suggested that OsUCL8 might affect the VB1 composition.
Fig. 2
Fig. 2

OsUCL8 regulates pollen intine and interacts with OsPKIWI protein. (a) Pollen germination rate of WT and mutants in different germination mediums,values are the means ± s.d. (n = 480 pollens from 3 plants). (b) Pollen germination of WT, ucl8 and OXUCL8 pollens in GM1 with 0 or 30 mg/L VB1. Scale bars, 100 μm. (c) Pollen germination rate of WT, ucl8 and OXUCL8 pollens in GM1 with 0, 3 and 30 mg/L VB1, values are the means ± s.d. (n = 480 pollens from 3 plants). (d) Observation of the ultrastructure of pollen wall for WT, OXUCL8, ucl8 and OXmiR408 by TEM. The parts surrounded by red lines are intine, Scale bars, 2 μm. (e) The statistical results of intine thinkness, values are the means ± s.d. (n 77 pollens). (f) Detection of OsUCL8-OsPKIWI interaction with a yeast two-hybrid assay. The combinations of AD/BD-OsUCL8 and AD-OsPKIWI/BD were used as negative controls. (g) Verification of the interaction between OsUCL8 and OsPKIWI by BiFC assay in rice protoplasts. Empty YC and YN were used as negative controls. Scale bars, 10 μm. Asterisks (***) indicate P value < 0.0001 (t tests), Asterisks (**) indicate P value < 0.01 (t tests)

Pollen tubes can be identified based on the intine, which is the inside structure of the pollen wall(Edlund et al., 2004). Thus, we observed pollen wall structure using transmission electron microscopy (TEM) (Liu et al., 2013). The ultrastructure of the pollen walls of the WT, OXUCL8, ucl8 and OXmiR408 plants was investigated. All plants developed an intact exine, but the intine was thinner in OXUCL8 pollen and thicker in the OXmiR408 pollen than in WT pollen (Fig. 2d and e), indicating that OsUCL8 regulates pollen intine formation and affects pollen tube elongation in the style, consequently affecting fertility. To dissect the mechanism by which OsUCL8 regulates pollen germination and pollen tube growth in rice, yeast two-hybrid (Y2H) screening was performed to identify OsUCL8-interacting proteins from a rice panicle cDNA Y2H library. After several screens, several proteins were identified (Additional file 1: Table S1). A fruit protein, PKIWI502 (Os02g0328300), was further validated using the full-length coding sequence of the PKIWI502 protein. The results indicated that OsPKIWI indeed interacted with OsUCL8 (Fig. 2f and g).

OsPKIWI is a homolog of the Arabidopsis FNRL protein (AT1G15140). FNR is a flavoenzyme that catalyzes the last step in linear photosynthetic electron transfer and produces the coenzyme NADPH (Koskela et al.,2018). The coenzyme NADPH has been reported to be involved in cell wall integrity and growth at the pollen tube tip (Boisson-Dernier et al., 2013; Potocky et al., 2007). Very interestingly, VB1 is also an essential coenzyme for carbohydrate metabolism and is involved in energy generation(Bazurto et al., 2015). We have found that the high VB1 concentrations significantly contributed to the germination of OXUCL8 pollen grains (Fig. 2a), suggesting that the germination defect in OsUCL8-overexpressing pollen grains might be related to the components of the VB1 sink in the intine wall. We hypothesized that OsUCL8 might regulate the production of VB1 components by interacting with OsPKIWI. This hypothesis deserved to be further validated.

In conclusion, we discovered an uclacyanin gene OsUCL8 which regulates pollen germination and pollen tube growth, and in turn regulates fertility and seed setting rate. We further demonstrated that OsUCL8 mainly affects pollen intine formation. Addition of VB1 could significantly contribute to the germination of OXUCL8 pollen grains, suggesting that VB1 could be the important component of pollen intine. We revealed that OsUCL8 interacts with OsPKIWI, the homolog of Arabdopsis FNRL protein. We thus hypothesized that OsUCL8 might regulate the production of VB1 components through interaction with OsPKIWI. The study revealed a novel molecular mechanism in regulating pollen tube growth.

Notes

Abbreviations

AP: 

After pollination

DAPI: 

4′,6-diamidino-2-phenylindole

GM: 

Germination mediums

TEM: 

Transmission electron microscopy

VB1: 

Vitamin B1

WE-CLSM: 

Whole-mount eosin B staining confocal laser scanning microscopy

WT: 

Wild type

Y2H: 

Yeast two-hybrid

Declarations

Acknowledgements

Not applicable.

Funding

This research was supported by the National Natural Science Foundation of China (No. 91640202, 31770883 and 31801082), the grants from Guangdong Province (No. 2014 T70833 and 2016A030308015) and Guangzhou (201707020018 and 201710010029).

Availability of data and materials

All data generated or analysed during this study are included in this published article [and its supplementary information files].

Authors’ contributions

FZ and YCZ carried out the functional analysis and drafted the manuscript. JPZ, YY,YFZ and FYZ carried out mutant screening and validation experiments. YWY, MQL JPL performed BiFC and yeast two-hybrid assays. YQC conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors’ Affiliations

(1)
Guangdong Provincial Key Laboratory of Plant Resources, State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, People’s Republic of China

References

  1. Ahn IP, Kim S, Lee YH, Suh SC (2006) Vitamin B1-induced priming is dependent on hydrogen peroxide and the NPR1 gene in Arabidopsis. Plant Physiol 143:838–848.View ArticleGoogle Scholar
  2. Bazurto JV, Heitman NJ, Downs DM (2015) Aminoimidazole carboxamide ribotide exerts opposing effects on thiamine synthesis in Salmonella enterica. J Bacteriol 197:2821–2830.View ArticleGoogle Scholar
  3. Berger F, Hamamura Y, Ingouff M, Higashiyama T (2008) Double fertilization – caught in the act. Trends Plant Sci 13:437–443.View ArticleGoogle Scholar
  4. Boisson-Dernier A, Lituiev DS, Nestorova A, Franck CM, Thirugnanarajah S (2013) ANXUR receptor-like kinases coordinate cell wall integrity with growth at the pollen tube tip via NADPH oxidases. PLoS Biol. https://doi.org/10.1371/journal.pbio.1001719.View ArticleGoogle Scholar
  5. Dai SJ, Chen TT, Chong K (2007) Proteomics identification of differentially expressed proteins associated with pollen germination and tube growth reveals characteristics of germinated Oryza sativa pollen. Mol Cell Proteomics 6:207–230.View ArticleGoogle Scholar
  6. Dong J, Sun TK, Elizabeth ML (2005) Plantacyanin plays a role in reproduction in Arabidopsis. Plant Physiol 138:778–789.View ArticleGoogle Scholar
  7. Edlund AF, Robert S, Daphne P (2004) Pollen and stigma structure and function: the role of diversity in pollination. Plant Cell 16:S84–S97.View ArticleGoogle Scholar
  8. Johnson MA, Lord E (2006) Extracellular guidance cues and intracellular signaling pathways that direct pollen tube growth. Plant Cell Monographs 3:223–242.View ArticleGoogle Scholar
  9. Kho YO, Baer J (1968) Observing pollen tubes by means of fluorescence. Euphytica 17:298–302.Google Scholar
  10. Koskela MM, Dahlström KM, Goñi G, Lehtimäki N, Nurmi M, Velazquez-Campoy A, Hanke G, Bölter B, Salminen TA, Medina M, Mulo P (2018) Arabidopsis FNRL protein is an NADPH-dependent chloroplast oxidoreductase resembling bacterial ferredoxin-NADP+ reductases. Physiol Plantarum 162:177–190.View ArticleGoogle Scholar
  11. Liu Y, Cui SJ, Wu F, Yan S, Lin XL, Du SQ, Chong K, Schilling S, Theißen G, Zheng M (2013) Functional conservation of MIKC*-type MADS box genes in arabidopsis and rice pollen maturation. Plant Cell 25:1288–1303.View ArticleGoogle Scholar
  12. Ma XL, Zhang QY, Zhu QL, Liu W, Chen Y, Qin R, Wang B, Yang ZF, Li HY, Lin YR, Xie YY, Shen RX, Chen SF, Wang Z, Chen YL, Guo JX, Chen LT, Zhao XC, Liu YG (2015) A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant 8:1274–1284.View ArticleGoogle Scholar
  13. Maksimov N, Evmenyeva A, Breygina M, Yermakov I (2018) The role of reactive oxygen species in pollen germination in Picea pungens (blue spruce). Plant Reproduction. https://doi.org/10.1007/s00497-018-0335-4.View ArticleGoogle Scholar
  14. Mizuta Y, Harushima Y, Kurata N (2010) Rice pollen hybrid incompatibility caused by reciprocal loss of duplicated genes. PNAS 107:20417–20422.View ArticleGoogle Scholar
  15. Nersissian AM, Valentine JS, Immoos C, Hill MG, Hart PJ, Williams G, Herrmann RG (1998) Uclacyanins, stellacyanins, and plantacyanins are distinct subfamilies of phytocyanins: plant-specific mononuclear blue copper proteins. Protein Sci 7:1915–1929.View ArticleGoogle Scholar
  16. Palanivelu R, Tsukamoto T (2012) Pathfinding in angiosperm reproduction: pollen tube guidance by pistils ensures successful double fertilization. WIREs Dev Biol 1:96–113.View ArticleGoogle Scholar
  17. Potocký M, Jones MA, Bezvoda R, Smirnoff N, Žárský V (2007) Reactive oxygen species produced by NADPH oxidase are involved in pollen tube growth. New Phytol 174:742–751.View ArticleGoogle Scholar
  18. Ross KJ, Fransz P, Jones GH (1996) A light microscopic atlas of meiosis in Arabidopsis thaliana. Chromosom Res 4:507–516.View ArticleGoogle Scholar
  19. Rydén LG, Hunt LT (1993) Evolution of protein complexity: the blue copper-containing oxidases and related proteins. J Mol Evol 36:41.View ArticleGoogle Scholar
  20. Zeng YX, Hu CY, Lu YG, Li JQ, Liu XD (2007) Diversity of abnormal embryo sacs in indica/japonica hybrids in rice demonstrated by confocal microscopy of whole ovary. Plant Breed 126:574–580.View ArticleGoogle Scholar
  21. Zhang JP, Yu Y, Feng YZ, Zhou YF, Zhang F, Yang YY, Lei MQ, Zhang YC, Chen YQ (2017) MiR408 regulates grain yield and photosynthesis via a phytocyanin protein. Plant Physiol 175:1175.View ArticleGoogle Scholar

Copyright

© The Author(s). 2018

Advertisement