Acknowledgements
We thank Armin Hildebrand and Noureddine Djella for plant care
and the German Research Foundation DFG (ERA-CAPS grant
EVOREPRO DR 334/12-1 to T.D. and S.S.) for financial support.
References
1. Meinke DW (1994) Seed development in Arabidopsis. In: Meyerowitz EM, Somerville CR
(eds) Arabidopsis thaliana. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp
253–295
2. Johnson-Brousseau SA, McCormick S (2004)
A compendium of methods useful for characterizing Arabidopsis pollen mutants and
gametophytically-expressed genes. Plant J
39:761–775
3. Park SK, Howden R, Twell D (1998) The
Arabidopsis thaliana gametophytic mutation
gemini pollen1 disrupts microspore polarity,
division asymmetry and pollen cell fate. Development 125:3789–3799
4. Heslop-Harrison Y (1970) Evaluation of pollen viability by enzymatically induced fluorescence; intracellular hydrolysis of fluorescein
diacetate. Stain Technol 45:115–120
5. Alexander MP (1969) Differential staining of
aborted and non-aborted pollen. Stain Technol
44:117–122
6. Boavida LC, McCormick S (2007) Temperature as a determinant factor for increased and
reproducible in vitro pollen germination in
Arabidopsis thaliana. Plant J 52:570–582
7. Mori T, Kuroiwa H, Higashiyama T, Kuroiwa
T (2005) Generative cell specific 1 is essential
for angiosperm fertilization. Nat Cell Biol
8:64–71
8. Sprunck S, Groß-Hardt R (2011) Nuclear
behavior, cell polarity, and cell specification in
the female gametophyte. Sex Plant Reprod
24:123–136
9. Lawit SJ, Chamberlin MA, Agee A, Caswell ES,
Albertsen MC (2013) Transgenic manipulation of plant embryo sacs tracked through
cell-type-specific fluorescent markers: cell
labeling, cell ablation, and adventitious
embryos. Plant Reprod 26:125–137
10. Vo ¨lz R, Groß-Hardt R (2010) Female gametophytic mutants: diagnosis and characterization.
In: Hennig L, Ko ¨hler C (eds) Plant developmental biology: methods and protocols.
Totowa, Humana Press, pp 143–153
11. Raghavan V (2003) Some reflections on double fertilization, from its discovery to the present. New Phytol 159:565–583
12. Denninger P, Bleckmann A, Lausser A,
Vogler F, Ott T, Ehrhardt DW, Frommer WB,
Sprunck S, Dresselhaus T, Grossmann G
(2014) Male–female communication triggers
calcium signatures during fertilization in Arabidopsis. Nat Commun 5:4645
13. Pereira AM, Masiero S, Nobre MS, Costa ML,
Solı ´s M-T, Testillano PS, Sprunck S, Coimbra S
(2014) Differential expression patterns of arabinogalactan proteins in Arabidopsis thaliana
reproductive tissues. J Exp Bot 65:5459–5471
14. Sprunck S, Hackenberg T, Englhart M, Vogler
F (2014) Same same but different: spermactivating EC1 and ECA1 gametogenesisrelated family proteins. Biochem Soc Trans
42:401–407
15. Vogler F, Schmalzl C, Englhart M,
Bircheneder M, Sprunck S (2014) Brassinosteroids promote Arabidopsis pollen germination and growth. Plant Reprod 27:153–167
16. Mu ¨ller A (1963) Embryonentest zum Nachweis rezessiver Letalfaktoren bei Arabidopsis
thaliana. Biol Zentralbl 82:133–163
17. Meinke DW (1985) Embryo-lethal mutants of
Arabidopsis thaliana: analysis of mutants with a
wide range of lethal phases. Theor Appl Genet
69:543–552
18. Meinke D, Muralla R, Sweeney C, Dickerman
A (2008) Identifying essential genes in Arabidopsis thaliana. Trends Plant Sci 13:483–491
19. Li X (2011) Pollen fertility/viability assay
using FDA staining. Bio-Prot 1:e75
20. Li H, Lin Y, Heath RM, Zhu MX, Yang Z
(1999) Control of pollen tube tip growth by a
Rop GTPase-dependent pathway that leads to
tip-localized calcium influx. Plant Cell
11:1731–1742
21. Coimbra S, Costa M, Mendes MA, Pereira AM,
Pinto J, Pereira LG (2010) Early germination
of Arabidopsis pollen in a double null mutant
for the arabinogalactan protein genes AGP6
and AGP11. Sex Plant Reprod 23:199–205
Identifying Reproductive Mutations in Arabidopsis
389
We thank Armin Hildebrand and Noureddine Djella for plant care
and the German Research Foundation DFG (ERA-CAPS grant
EVOREPRO DR 334/12-1 to T.D. and S.S.) for financial support.
References
1. Meinke DW (1994) Seed development in Arabidopsis. In: Meyerowitz EM, Somerville CR
(eds) Arabidopsis thaliana. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp
253–295
2. Johnson-Brousseau SA, McCormick S (2004)
A compendium of methods useful for characterizing Arabidopsis pollen mutants and
gametophytically-expressed genes. Plant J
39:761–775
3. Park SK, Howden R, Twell D (1998) The
Arabidopsis thaliana gametophytic mutation
gemini pollen1 disrupts microspore polarity,
division asymmetry and pollen cell fate. Development 125:3789–3799
4. Heslop-Harrison Y (1970) Evaluation of pollen viability by enzymatically induced fluorescence; intracellular hydrolysis of fluorescein
diacetate. Stain Technol 45:115–120
5. Alexander MP (1969) Differential staining of
aborted and non-aborted pollen. Stain Technol
44:117–122
6. Boavida LC, McCormick S (2007) Temperature as a determinant factor for increased and
reproducible in vitro pollen germination in
Arabidopsis thaliana. Plant J 52:570–582
7. Mori T, Kuroiwa H, Higashiyama T, Kuroiwa
T (2005) Generative cell specific 1 is essential
for angiosperm fertilization. Nat Cell Biol
8:64–71
8. Sprunck S, Groß-Hardt R (2011) Nuclear
behavior, cell polarity, and cell specification in
the female gametophyte. Sex Plant Reprod
24:123–136
9. Lawit SJ, Chamberlin MA, Agee A, Caswell ES,
Albertsen MC (2013) Transgenic manipulation of plant embryo sacs tracked through
cell-type-specific fluorescent markers: cell
labeling, cell ablation, and adventitious
embryos. Plant Reprod 26:125–137
10. Vo ¨lz R, Groß-Hardt R (2010) Female gametophytic mutants: diagnosis and characterization.
In: Hennig L, Ko ¨hler C (eds) Plant developmental biology: methods and protocols.
Totowa, Humana Press, pp 143–153
11. Raghavan V (2003) Some reflections on double fertilization, from its discovery to the present. New Phytol 159:565–583
12. Denninger P, Bleckmann A, Lausser A,
Vogler F, Ott T, Ehrhardt DW, Frommer WB,
Sprunck S, Dresselhaus T, Grossmann G
(2014) Male–female communication triggers
calcium signatures during fertilization in Arabidopsis. Nat Commun 5:4645
13. Pereira AM, Masiero S, Nobre MS, Costa ML,
Solı ´s M-T, Testillano PS, Sprunck S, Coimbra S
(2014) Differential expression patterns of arabinogalactan proteins in Arabidopsis thaliana
reproductive tissues. J Exp Bot 65:5459–5471
14. Sprunck S, Hackenberg T, Englhart M, Vogler
F (2014) Same same but different: spermactivating EC1 and ECA1 gametogenesisrelated family proteins. Biochem Soc Trans
42:401–407
15. Vogler F, Schmalzl C, Englhart M,
Bircheneder M, Sprunck S (2014) Brassinosteroids promote Arabidopsis pollen germination and growth. Plant Reprod 27:153–167
16. Mu ¨ller A (1963) Embryonentest zum Nachweis rezessiver Letalfaktoren bei Arabidopsis
thaliana. Biol Zentralbl 82:133–163
17. Meinke DW (1985) Embryo-lethal mutants of
Arabidopsis thaliana: analysis of mutants with a
wide range of lethal phases. Theor Appl Genet
69:543–552
18. Meinke D, Muralla R, Sweeney C, Dickerman
A (2008) Identifying essential genes in Arabidopsis thaliana. Trends Plant Sci 13:483–491
19. Li X (2011) Pollen fertility/viability assay
using FDA staining. Bio-Prot 1:e75
20. Li H, Lin Y, Heath RM, Zhu MX, Yang Z
(1999) Control of pollen tube tip growth by a
Rop GTPase-dependent pathway that leads to
tip-localized calcium influx. Plant Cell
11:1731–1742
21. Coimbra S, Costa M, Mendes MA, Pereira AM,
Pinto J, Pereira LG (2010) Early germination
of Arabidopsis pollen in a double null mutant
for the arabinogalactan protein genes AGP6
and AGP11. Sex Plant Reprod 23:199–205
Identifying Reproductive Mutations in Arabidopsis
389