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Research: Project

Molecular network controlling ovule development in Arabidopsis thaliana

Marta Mendes, University of Milan

In angiosperms, like Arabidopsis thaliana, gametophyte development takes place within the sporophyte. The mature male gametophyte, pollen grain, is released from the anthers, while the mature female gametophyte, the embryo sac, remains embedded within the maternal ovule tissues. The three MADS-box genes SEEDSTICK (STK), SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2) redundantly control ovule development in Arabidopsis thaliana. Protein interaction studies have shown that a multimeric complex, composed of the ovule identity proteins together with the SEPALLATA (SEP) MADS-domain proteins, is necessary to determine ovule identity. Despite the extensive knowledge that has become available about these MADS-domain transcription factors, little is known regarding the genes that they regulate (Fig 1).

Fig 1. SEM images of ovule development in (a,b) wild type Arabidopsis and (c,d) the stk shp1 shp2 triple mutant. (a) Early-stage 12 of flower development. The outer integument exhibits asymmetrical growth. (b) Mature ovules, the integuments envelop the nucellus. (c) In the stk shp1 shp2 triple mutant, ovule integument growth is impaired from the start of mid-stage 12. (d) Some of the stk shp1 shp2 mature ovules are converted into carpel-like structures.

Figure 1

In our lab it has been recently shown that VERDANDI (VDD), a putative transcription factor that belongs to the plant specific transcription factor B3 superfamily, namely the REM family, is directly and redundantly regulated by STK, SHP1 and SHP2. VDD transcripts are present in the same tissues as the ovule identity genes STK, SHP1, SHP2 and SEP. Additionally, VDD expression studies by in situ hybridization using different mutant backgrounds highlighted the redundancy of the ovule identity factors STK, SHP1 and SHP2 in the control of VDD expression within the ovule, since the expression of VDD is strictly dependent on the activity of the three MADS-domain ovule identity factors. Only the absence of all three ovule identity proteins leads to complete absence of VDD expression during ovule development. In VDD regulatory region three CArG boxes were found and data suggests that the ovule identity proteins STK, SHP1, SHP2 and SEP form a protein complex that binds to the first and third CArG. Morphological and genetic analyses of heterozygous plants VDD/vdd showed that the mutation was transmitted through the female gametophyte at a significantly reduced efficiency. This reduction in transmission is due to a defect in fertilization process after the pollen tube guidance and attraction to the female gametophyte. Studies showed also that the embryo sac cells, more particularly the synergids and the antipodals, lost their identity (Fig 2). We intent to further study this transcription factor at cellular/molecular level, to understand its involvement in the fertilization process and to find VDD possible targets.

Fig 2. (A) Pollen tube (pt) staining with aniline blue showed that in the vdd-1 heterozygous background all the embryo-sac are reached by pollen tubes. (B) Detailed image of aniline blue stained pollen tube reaching the micropyle in the vdd-1 heterozygous carpel. In vdd-1 heterozygous carpels we detected the 86% of wild-type pollen tubes that successfully reached the micropyle (n=545). This result is not statistically different compared to wild-type plants where we identified 88% pollen tubes reaching the micropyle (n=229). (C Antipodal-cell marker expression in the vdd-1/VDD plants 72 hours post-emasculation. At this time point vdd-1/VDD heterozygous siliques showed GUS expression in the synergids cells (arrow) (49% n=244).

Figure 2

References:

[1] Colombo L, Battaglia R, Kater MM (2008). Arabidopsis ovule development and its evolutionary conservation. Trends in plant science 13(8): 444-450.

[2] Matias-Hernandez L, Battaglia R, Galbiati F, Rubes M, Eichenberger C, Grossniklaus U, Kater MM, Colombo L (2010). VERDANDI Is a Direct Target of the MADS Domain Ovule Identity Complex and Affects Embryo Sac Differentiation in Arabidopsis. The Plant cell 22: 1702–1715.