[Plant Signaling & Behavior 3:4, 246-247; April 2008]; ©2008 Landes Bioscience
Article Addendum
Ovule integument identity determination in Arabidopsis Vittoria Brambilla, Martin Kater and Lucia Colombo* Dipartimento di Biologia; Università degli Studi di Milano; Milano, Italy
Key words: gametophytes, integument development, MADS-box genes, ovules, transcription factors
Ovules are the organs in which the female gametophyte develops. They develop in Arabidopsis and many other plant species from carpel tissue as new meristematic formations. In ovules we can distinguish three major parts which are the funiculus that attaches the ovule to the placenta, the integuments and the nucellus which contains the female gametophyte. Little is known about the molecular genetic regulatory cues that control the development of these ovule tissues. In the August issue of The Plant Cell, we have shown that there are genetic and molecular interactions between BELL1 and the MADS‑box genes AGAMOUS, SEEDSTICK, SHATTERPROOF1 and SHATTERPROOF2 to control integu‑ ment identity.1 We have shown that BEL1 directly interacts with a MADS‑box dimer composed of AG and SEPALLATA3 and we proposed that this interaction is essential to prevent that integu‑ ments turn into carpels. Furthermore, we have shown that during ovule development BEL1 is important for the regulation of the stem cell maintenance gene WUSCHEL. In this Addendum we discuss addition literature data that sustain our model for integument development in Arabidopsis. Arabidopsis ovules originate from the carpel placental tissue as finger‑like protrusions. Within the ovule primordia three regions are distinguishable: the distal part, the nucellus that will originate the embryo‑sac, the medial part, named chalaza, from which the two integuments develop and the proximal region that will originate the funiculus that will connect the ovule to the placenta. Studies in Petunia and Arabidopsis have shown that genes belonging to the AGAMOUS subfamily of MADS‑box genes are controlling ovule identity. In Petunia cosuppression of both FLORAL BINDING PROTEIN7 and FBP11 resulted in the conversion of ovules into carpelloid structures and more recently the group of Martin Yanofsky showed that in the stk shp1 shp2 triple mutant ovules are converted into leaf‑like or carpel‑like structures.2,3 We analysed this Arabidopsis *Correspondence to: Lucia Colombo; Dipartimento di Biologia; Università di Milano; via Celoria 26; 20133 Milano, Italy; Tel.: +39.02.50314772; Fax: +39.02.50314763; Email:
[email protected] Submitted: 10/09/07; Accepted: 10/16/07 Previously published online as a Plant Signaling & Behavior E-publication: www.landesbioscience.com/journals/psb/article/5175 Addendum to: Brambilla V, Battaglia R, Colombo M, Masiero S, Bencivenga S, Kater MM, Colombo L. Genetic and molecular interactions between BELL1 and MADS box factors support ovule development in Arabidopsis. Plant Cell 2007; 19:2544–56; PMID: 17693535; doi: 10.1105/tpc.107.051797. 246
triple mutant in more detail and showed that ovule development is normal until stage 11 of flower development. However, from stage 12 integument development clearly switched to a different developmental programme forming wide carpel‑like structures. Experiments that compared ectopic ovule formation on sepals between the ap2 single mutant and the ap2 ag double mutant suggested that AG also plays a role in ovule identity determination. These data were supported by our experiments in which we reduced AG gene activity in the stk shp1 shp2 triple mutant. When growing the ag/AG stk shp1 shp2 mutant at 30°C the phenotype resembled the bel1 mutant: the outer integument did not develop and the inner integument was replaced by cells that proliferate into carpelloid structures. These data show that the four AG subfamily members are all playing a role in ovule identity determination. The bel1 mutant has been well studied and it has been suggested that in ovules BEL1 negatively regulates AG and that therefore in the bel1 mutant the loss of AG regulation results in the conversion of integuments into carpelloid structures.4,5 However, expression analysis has shown that the regulation of AG by BEL1 is probably not at the transcript level.6 Our experiments provide evidence that this regulation of AG carpel identity activity might be at the protein level since we observed a strong interaction between BEL1 and the AG‑SEPALLATA dimer. Based on our data and those of other laboratories we have formulated a hypothetical model for integument identity determination and development. In this model we propose that the carpel identity determination activity of the AG‑SEP dimer is repressed by binding to BEL1. Knocking‑out BEL1 will release AG from the complex and integuments will develop into carpel tissue. The AG‑SEP‑BEL1 complex is not only to control AG carpel activity but has probably also other functions, like the regulation of WUS expression. It is important for correct ovule formation to restrict WUS expression to the nucellus, from where the WUS‑induced signaling pathway induces integument formation in the underlying chalaza.7 We have shown that in the bel1 mutant WUS expression is deregulated since expression was also found in the chalaza and funiculus, which might cause the enlargement of and irregular shape of the integument structure of bel1 ovules. This phenotype was further enhanced in the bel1 stk shp1 shp2 quadruple mutant. These observations, together with the fact that the ag/AG stk shp1 shp2 mutant at 30°C phenocopies the bel1 mutant, suggest that all these factors form a complex to control integument identity and development. What is the role of the STK, SHP1 and SHP2 factors? Integument development requires the control of AG activity. However, the correct
Plant Signaling & Behavior
2008; Vol. 3 Issue 4
Ovule integument identity determination in Arabidopsis
Figure 1. A Model for Integument Identity Determination and Development. In wild‑type ovules, the BEL1 protein interacts with the AG‑SEP dimmer to repress WUS in the chalaza and to regulate outer integument development. This complex is stabilized by the ovule identity complex. When STM is highly expressed during ovule development, the STM protein will bind BEL1 and the AG‑SEP dimer concentration that is not bound to BEL1 increases leading to the transformation of integuments into carpel tissue.
development of integuments also requires the expression of the STK, SHP1 and SHP2 integument identity genes since in the stk shp1 shp2 triple mutant integuments are transformed into carpelloid structures. This indicates that there has to be a clear balance between carpel identity and integument identity activity. Our model proposes that the STK and SHP factors in a complex with SEP stabilise the AG‑SEP‑BEL complex.8 When STK, SHP1 and SHP2 are not present than the AG‑SEP dimer is less bound to BEL1 and the carpel pathway is activated though the phenotype is not completely penetrant. When AG activity is further reduced in this triple mutant there is still enough AG‑SEP‑BEL complex available to establish inner and outer integument development (since this complex is probably also necessary to activate INNER NO OUTER a gene necessary for outer integument development9). However, when the ag/AG stk shp1 shp2 mutant is grown at higher temperature than the integument identity complex is getting more instable due to the absence of STK, SHP1 and SHP2, the complex AG‑SEP‑BEL is not made and only one carpelloid integument develops like in the bel1 single mutant. Scofield et al (2007)10 recently published interesting data that support our hypothesis that the interaction between BEL1 and the AG‑SEP dimer is important for controlling the carpel identity activity of AG (Fig. 1).10 They showed that expression of the class 1 KNOX homeodomain transcription factor gene SHOOT MERISTEMLESS in gynoecia results in the formation of carpelloid ovules that are similar to those of the bel1 mutant. They analysed BEL1 expression and found no change in expression in response to transient overexpression of STM, which means that the carpelloid ovules are not due to silencing of BEL1. However, since BEL1 can heterodimerise with KNOX proteins including STM the most likely explanation is that STM competes for BEL1 binding with the AG‑SEP complex.11 When STM levels are high enough BEL1 will not be bound to the AG‑SEP complex and this will lead to AG turning ovule integuments into carpelloid structures just as in the bel1 mutant occurs. The model that we propose will have to be further tested by molecular genetic experiments and other components will probably have to be added to the proposed complexes. Our model is meant as starting point for a better understanding of the molecular mechanisms underlying ovule development in Arabidopsis.
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References 1. Brambilla V, Battaglia R, Colombo M, Masiero S, Bencivenga S, Kater MM, Colombo L. Genetic and molecular interactions between BELL1 and MADS box factors support ovule development in Arabidopsis. Plant Cell 2007; 19:2544‑56. 2. Colombo L, Franken J, Koetje E, van Went J, Dons HJ, Angenent GC, van Tunen AJ. The petunia MADS box gene FBP11 determines ovule identity. Plant Cell 1995; 7:1859‑68. 3. Pinyopich A, Ditta GS, Savidge B, Liljegren SJ, Baumann E, Wisman E, Yanofsky MF. Assessing the redundancy of MADS‑box genes during carpel and ovule development. Nature 2003; 424:85‑8. 4. Modrusan Z, Reiser L, Feldmann KA, Fischer RL, Haughn GW. Homeotic transformation of ovules into carpel‑like structures in Arabidopsis. Plant Cell 1994; 6:333‑49. 5. Ray A, Robinson‑Beers K, Ray S, Baker SC, Lang JD, Preuss D, Milligan SB, Gasser CS. Arabidopsis floral homeotic gene BELL (BEL1) controls ovule development through negative regulation of AGAMOUS gene (AG). Proc Natl Acad Sci USA 1994; 91:5761‑5. 6. Reiser L, Modrusan Z, Margossian L, Samach A, Ohad N, Haughn GW, Fischer RL. The BELL1 gene encodes a homeodomain protein involved in pattern formation in the Arabidopsis ovule primordium. Cell 1995; 83:735‑42. 7. Gross‑Hardt R, Lenhard M, Laux T. WUSCHEL signaling functions in interregional communication during Arabidopsis ovule development. Genes Dev 2002; 16:1129‑38. 8. Favaro R, Pinyopich A, Battaglia R, Kooiker M, Borghi L, Ditta G, Yanofsky MF, Kater MM, Colombo L. MADS‑box protein complexes control carpel and ovule development in Arabidopsis. Plant Cell 2003; 15:2603‑11. 9. Villanueva JM, Broadhvest J, Hauser BA, Meister RJ, Schneitz K, Gasser CS. INNER NO OUTER regulates abaxial‑adaxial patterning in Arabidopsis ovules. Genes Dev 1999; 13:3160‑9. 10. Scofield S, Dewitte W, Murray JA. The KNOX gene SHOOT MERISTEMLESS is required for the development of reproductive meristematic tissues in Arabidopsis. Plant J 2007; 50:767‑81. 11. Bellaoui M, Pidkowich MS, Samach A, Kushalappa K, Kohalmi SE, Modrusan Z, Crosby WL, Haughn GW. The Arabidopsis BELL1 and KNOX TALE homeodomain proteins interact through a domain conserved between plants and animals. Plant Cell 2001; 13:2455‑70.
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