Functional Analyses Of APETALA1/FRUITFULL Subfamily And INDETERMINATE1 Genes In Brachypodium Distachyon L.

Brachypodium distachyon L. is proposed as a model for temperate cereals. B. distachyon belongs to the Brachypodieae tribe, a sister tribe to the Triticeae in the Pooideae subfamily. It possess many advantages as a model species such as a small genome size, a rapid lifecycle, a simple grow condition and self-pollination. We devote to the study of mechanisms of heading time of B. distachyon to facilitate the genetic improvement of temperate cereals. Functional analysis of APETALA1/FUITFULL subclade in B. distachyonThe MADS-box transcript factors play crucial roles in the entire plant development especially in floral organ identity. APETALA1/FUITFULL is a subclade of MADS-box family. APETALA1/FRUITFULL(AP1/FUL) MADS-box transcription factors are angiosperm-specific and have undergone several duplications. The most significant of these duplications, the duplication that produced the euAP1 and euFUL clades, is correlated with the origin of the core eudicots. In core eudicots, euAP1 and euFUL genes together are implicated in proper floral meristem identity and axillary meristem repression. Independently, euAP1 genes play a unique role in the specification of sepal identity whereas euFUL genes function in the reproductive phase transition, proper cauline leaf development, branching, and fruit development as well as compound leaf development. Studies have shown that two major duplication events exist in monocots: the first duplication apparently occurred around the base of the monocots, giving rise to the FUL3/OsMADS18 clade; the other occurred at the base of grasses(Poaceae), giving rise to the FUL1/OsMADS14 and FUL2/OsMADS15 clades. VRN1 functions in vernaliztion response and VRN1 and FUL2 both function in the specification of inflorescence, spikelet and floret meristem identity. So far, the study of grass and especially temperate grass AP1/FUL genes is limited. In this study, we analyzed the expression patterns and functions of four AP1/FUL paralogs in B. distachyon. The main results and conclusions presented in this thesis are as follows:(1) BdVRN1 play a crucial role in vernalization response. Our results showed that, the expression of BdVRN1 was induced by prolonged cold independent of day length while BdFUL2 and BdFUL3 did not respond to it, which suggest that, only BdVRN1 could memory vernalization and promote flowering just like in wheat and barley.(2) Flowering competence is an ancestral function of AP1/FUL genes. Never the less, expression of BdFUL2 and BdFUL3 were also up-regulated in leaves during phase transition and inflorescence development. In addition, vernalized leaves in LD condition accumulated more transcripts of BdFUL2 and BdFUL3 compared with non-vernalized control, although they did not respond vernalization treatment in SD. We speculate that it may be a result of FT enhancing their expression, similar to VRN1. Thus, the positive feedback loop existing between FT and VRN1 may also applies to FUL2 and FUL3 in leaves.(3) The redundant roles of BdVRN1 and BdFUL2 in inflorescence development initiation and floral meristem identity. Consistent with other Pooideae members, in B. distachyon, BdVRN1 and BdFUL2 were expressed in inflorescence meristems, spikelet meristems and floret meristems. It is suggested that VRN1 and FUL2 function redundantly in flowering transition. This suggest is supported by our experiment that ectopic expression of BdVRN1 and BdFUL2 in B. distachyon led to flowering without leaves or with few leaves.(4) BdVRN1 and BdFUL2 function redundantly as A-class genes. In B. distachyon, BdVRN1 was expressed in all floral whorls with a higher level in glumes, while BdFUL2 transcripts accumulated only in outer whorls. In Ubi1::BdVRN1 and Ubi1::BdFUL2 transgenic lines inner three floral whorls were replaced by paleae and some transgenic plants lost the determinacy. In B. distachyon, both BdVRN1 and BdFUL2 could interact with BdSEP and BdLHS1. These results indicate that VRN1/OsMADS14 and FUL2/OsMADS15 may function redundantly to interact with class E proteins to specify the outer whorls identity.(5) The degraded function of FUL3 and FUL4. The expression pattern of BdFUL3 was similar to that of BdVRN1 and BdFUL2, but overexpression of BdFUL3 in B. distachyon did not exhibit a strong early flowering phenotype, and any floral organ alterations, indicating its degraded function in flowering transition and floral organ identity despite its conserved expression pattern. Our results show that, expression of BdFUL4 was not changed during flowering transition and inflorescence development, and no transcripts were detected in young spikeletes and floral organs. Functional analysis of INDETERMINATE1 gene in B. distachyonINDETERMINATE1(ID1) is a heading time regulator first identified in maize. The maize id1 mutant showed an extremely late flowering phenotype. ID1 encodes a transciptor with four zinc fingers, which combines an 11 bp element. ID1 is highly expressed in immature leaves and it regulate heading time upstream ZCN8(a FT homolog). RID1/Ehd2/OsID1, the ID1 homolog in rice also contributes the regulation of heading time. The rice rid1 mutant displayed a neverflowering phenotype. It is reported that, ID1 is specific in grass, and no ID1 homolog is identified in Arabidopsis. Maize and rice are both tropical grasses and their flowering is induced by SD condictions and do not need vernalization. The temperate Pooideae family flower under LD conditions and need vernalization. Here, we analyzed the function of ID1 homolog in B. distachyon(BdID1) to reveal the functions and mechinsms of Pooideae ID1. The main results and conclusions presented in this thesis are as follows:(1) 35S::BdID1 recovered the late flowering of rid1. We expressed BdID1 in rid1 under the 35 S promoter and the transgenic lines recovered heading, which suggest BdID1 could replace the function of RID1.(2) BdID1 is expressed in young tissues. The qRT-PCR experiment showed that, BdID1 transcripts were accumulated in young leaves, young spikelets, which is consistent with maize and rice ID1 genes.(3) BdID1 regulates the flowering time in B. distachyon. We overexpressed BdID1 in B. distachyon and the overexpression plants showed an extremely early flowering phenotype. Then we knocked out BdID1 using CRISPR/Cas9 and the knock-out lines showed an extremely late flowering phenotype. All these results suggest that the function of BdID1 is conserved.(4) The function of BdID1 dependent on the LD conditions. The BdID1 overexpression plants under SD conditions did not show the early flowering phenotype, which suggest that BdID1 could promote flowering only under the LD conditions.(5) Vernalization rescued the late flowering of BdID1 knock-out lines. Our result showed that BdID1 knock-out lines treated with 8 weeks vernalization flowered at 90 days while nontreated controls did not flower within 150 days. Thus the BdID1 Knock-out plant could still respond to vernalization.