Corinne Renner Professor Assmann Biology 240M 27 February 2013

Florigen: The Fascinatingly Misunderstood Hormone in Rice The physiological functioning and life cycles of many plant species are synchronized to the changing seasons throughout the year. Plants have the ability to perceive changes in day length, which is just one of the major ways in which they can detect these seasonal changes (Allard and Garner, 1920). This characteristic, formally known as photoperiodism, is essential to proper plant functioning, specifically flowering. Recently, researchers have produced tangible evidence of the existence of a plant hormone, florigen, which complements this process, as well as many other functioning systems related to flowering. This hormone is not specific to a single plant, but rather plays an appreciable role in the life cycles of a variety of organisms. One such organism, rice, possesses several genes pertaining to florigen (Shimamoto et al., 2011). The implications of this mobile stimulus are imperative to breeding successful rice crops and, thus, play a large role in society due to the utilization of rice as a staple crop throughout the world. While substantial research concerning florigen has already been conducted, there is still a great deal of information about this chemical entity that remains uncertain and subject to exploration. The discovery of photoperiodism fueled researchers interest in a flowering stimulus. Julius von Sachs, a German chemist, first considered the concept of a flowering hormone in 1865. Sachs demonstrated that sap transferred from a flowering

2 plant induced a non-flowering plant to flower (Sachs, 1865). This discovery did not spur immediate recognition or further research for some time. In the 1930s, physiological experiments demonstrated that leaves were capable of sensing inductive photoperiods (Knott 1934). Russian plant physiologist, Mikhail Chailakhyan, revealed that floral induction could be transmitted through a graft from an induced plant to a non-induced plant (Chalakhyan 1985). This research posed several fundamental questions to the entire botanical community: what component of plants motivated these responses, and what was the explanation behind the biochemistry for this phenomenon? For several decades these questions remained unanswered. Researchers battled inconsistent results, failed experiments, and skepticism during this time period, and almost deemed the flowering hormone nonexistent (Zeevaart, 1976). However, in 2005 three separate groups of researchers solved the mystery by identifying FD, a bZIP transcription factor that was preferentially expressed in the shoot apex, within the model organism Arabidopsis. These scientists proved that FD interacted with FT, a component expressed primarily in the leaves of plants in response to photoperiodic signals, to promote flowering. Their experiments demonstrated that mRNA of the FT gene moved from the leaf to the shoot apex in plants to induce the response (Abe et al. 2005; Bohlenius et al. 2005; Busche et al. 2005). The identification of the molecular nature of florigen was considered one of the 10 scientific breakthroughs in 2005 by the journal Science. Since its discovery, researchers have sought to better understand florigen and its role in a variety of different organisms. A significant amount research concerning florigen and the various flowering mechanisms of rice exists. While Arabidopsis thaliana is the popular model organism for

3 a variety of other experimental studies, it does not accurately depict florigens role in all plants. Flowering time is controlled by many genes, like florigen, which are either expressed or suppressed in close interaction with environmental factors such as day length and temperature. Arabidopsis thaliana is a good representative model of long-day plants. However, the mechanisms of flowering in short-day plants are very different from those of long-day plants, so rice serves as the ideal model organism for study (Greenup et al. 2009). Flowering time in rice is controlled by day length, which helps to ensure reproductive success. For rice plants, flowering accelerates when day length becomes shorter. The florigen gene expression in rice has a critical day length threshold of approximately 13.5 hours (Itoh et al., 2010). Several plant photoreceptors play roles in measuring this environmental cue. Induction of the flowering hormone is based on whether the given external light signal overlaps with the photosensitive phase that is established by an internal circadian rhythm (Ikeda-Kawakatsu et al., 2011). The various photoreceptors and the rice plants corresponding reactions are of great interest in many experiments. Japanese researchers studying the affects of photoreceptors on florigen subjected several rice plants to varying blue-light conditions. The first of two experiments analyzed the induction of Ehd1, one of the genes that induces the flowering hormone, Hd3a, in rice, in se5 and se5 osgi-1 rice plants under blue-light conditions. The second experiment subjected se5 plants to blue light pulses at various circadian phases after entrainment under long-day or short-day conditions. The scientists extracted the RNA from each plant in the separate experiments, and analyzed the relative mRNA levels of Ehd1 as 3

4 determined by quantitative polymerase chain reaction (Itoh et al., 2010).

Figure 1: (a) Diurnal expression patterns of Ehd1 in se5 and se5 osgi-1 rice plants grown under short-day conditions (10 h light, 14 h dark) in blue light. The blue bar represents the blue-light period and the black bar represents the period of darkness. The gray bar represents dark treatment instead of blue light. (b) Gated expression of Ehd1 expression responsive to blue light. se5 and se5 osgi-1 plants were entrained under long-day (left) or short-day conditions (right) for 14 d. Plants were transferred to continuous dark at dusk (0). Replicate samples were then each exposed once to 2 h of blue light at times differing by 2 h. Black and gray bars in panels to left of graphs represent subjective night and day. The blue bands in the panels represent the 2 h blue-light exposure periods. Arrowheads represent the timing of harvest for RNA extraction. Average values and s.d. from three RT-PCR data are shown. All data are representative of two independent experiments. LD, long-day conditions; SD, short-day conditions (Itoh et al. 2010). In the first experiment, the se5 plants subjected to blue-light and short-day conditions demonstrated strong Ehd1 expression 2 hours after dawn. However, Ehd1 induction was not detected in continuous dark conditions. Additionally, morning expression of Ehd1 was not observed in se5 osgi-1 mutants. In the second experiment, the mRNA levels of Edh1 were up-regulated when exposed to blue-light. However, these levels varied depending on when the plants were exposed to blue light. In se5 osgi-1 mutants, an absence of Ehd1 induction by blue light pulses was consistently observed. This observation indicated that the OsGI protein does not directly contribute to Ehd1 induction as it neither inhibited nor attributed to the response. The study also demonstrated that Ehd1 experienced peak expression between 10 to 12 hours after dusk,

5 regardless of the entrained day-length conditions. This research reaffirmed the critical day length threshold for rice, as well as indicated the induction of the flowering stimulus as a result of blue-light photoreceptors in the plant, even under varying day-length conditions (Itoh et al. 2010). This experiment is just one of many that illustrates the complex relationship between the flowering stimulus, its components, and photoreceptors. While blue-light and day-length definitely contribute to the induction of florigen in rice, much more is occurring at the molecular level. The quantitative trait locus and protein, Heading date 3a (Hd3a), was first detected as a promoter for flowering in rice under short-day conditions. The Hd3a protein is a member of a large gene family consisting of at least 13 genes in the rice genome (Chardon and Damerval, 2005). In rice, as well as in many other short-day plants, Hd3a accumulates in the vascular tissue of the leaf blades. It is then transported through the phloem tissue to the shoot apical meristem, where it is unloaded from the vascular tissue into the cells just below the meristem. A second component of flowering, the rice flowering locus T1 gene (RFT1), complements the activity of Hd3a. The RFT1 gene lies adjacent to Hd3a on chromosome 6 in rice, and is only separated from the corresponding gene by 11.5 kb (Izawa et al., 2003). The sequences of the two genes are very similar, suggesting that they were likely the products of a recent tandem duplication event (Chardon and Damerval, 2005). RFT1 is expressed in a similar manner to that of Hd3a, and complements the overall process of flowering in short-day plants. Under short-day conditions, the expression of Hd3a occurs gradually and then peaks approximately thirty days before flowering, about the time that floral transition occurs (Araki et al. 2002). This peak of Hd3a promoter activity and mRNA accumulation 5

6 is a diurnal process, suggesting that the major point of Hd3a regulation is transcriptional. The primary factors involved in this process are the genes Heading date 1 (Hd1) and Early heading date 1 (Ehd1). Hd1 is composed of two exons that encode a 395-amino acid protein with a zinc finger domain. Hd1 up-regulates the amount of Hd3a mRNA under short-day conditions. Detailed phylogenetic analyses indicate that Hd3a is the sole ortholog of Arabidopsis CONSTANS (CO), an important factor that regulates flowering time in the same manner in long-day plants such as the model organism, Arabidopsis thaliana (Izawa et al 2003). Similar to CO expression in Arabidopsis thaliana, Hd1 is regulated by OsGI, a large protein present in both the cytosol and nucleus of plants, which is another ortholog of an Arabidopsis flowering component. OsGI expression demonstrates regulation by the circadian clock. Suppression of this protein results in reduced Hd3a accumulation in leaf blades and late flowering. The second Hd3a activator, Ehd1 is similarly regulated by OsGI and maintains an identical relationship with the Hd3a gene in that it up-regulates its mRNA levels the during the day just as Hd1 does (Hayama et al. 2002). These interconnecting processes at the molecular level of the rice plant all contribute to flowering time during the day. When these molecular components are collectively at compatible concentrations, and the plant has been exposed to an uninterrupted and appropriately timed dark period, Hd3a and RFT1 will interact with a bZIP transcription factor in the shoot apical meristem to induce phase transition from the vegetative state to the reproductive state. The apical meristem is converted into a floral meristem because the transcription factor induces the expression of the protein that is required for the formation of the first floral bud (Komiya et al. 2008).

7 The similarities between the flowering mechanisms and involved components for Arabidopsis and rice are noteworthy. Although there are different genes, proteins, and processes involved, florigen proves to play a significant role in the flowering of both rice and Arabidopsis. The culmination of research comprised for Arabidopsis over time is much more substantial, yet it is this research that paved the road for understanding similar mechanisms in rice. An examination of both flowering systems, those of shortday and long-day plants, indicates the importance of researching the two processes separately.

Figure 1: This figure presents a simplified model of the florigen cycling in rice and compares it to that of Arabidopsis. The clock genes refer to those comparable to OsG1, as explained earlier. Both the proposed florigen genes, FT in Arabidopsis and Hd3a in rice, oscillate in a circadian fashion, but undergo different phases at the molecular level with different components due to varying photoperiods (Greenup et al. 2009). Research regarding florigen and the flowering mechanisms in rice is of immense interest to agriculturists worldwide. Rice serves as a dietary staple for more than half of the global population. Unfortunately, climate change, diseases, and inability to sustain its 7

8 demand currently jeopardize the worlds rice supply. If researchers develop a deeper understanding of florigen and its function in rice, they may be able to isolate the gene and utilize it to adapt rice plants that are subjected to different environmental conditions. Rice plants that are modified to flower earlier will have increased reproductive fitness and produce higher crop yields (Weixun et al. 2012). However, the research necessary to infiltrate this practice into everyday agriculture is still in its preliminary stages. The topic of florigen and its molecular identity and role in various species of plants is a relatively recent matter that must still undergo a great deal of exploration. Researchers have developed a very basic understanding of flowering, but have yet to comprehend the variation in the process that confers the diversity in flowering behaviors observed in nature. It is unclear whether or not the Hd3a protein is sufficient to induce this process on its own or whether it requires other molecules to accompany it during its transportation through the phloem. Similarly, little is actually known of the manner in which Hd3a is transported through the phloem and loaded into the shoot apical meristem. All of these matters, and countless others require the careful analysis of future research. And even after the flowering mechanisms in Arabidopsis and rice are completely clarified, the broader context in which photoperiodic response functions in other angiosperms will require further examination (Coupland et al. 2008). The recent discovery of the mobile flowering stimulus, florigen, marks a significant milestone in scientific history. In the past few decades, researchers have explored the role of this hormone at the molecular level in hopes of understanding the process of flowering in a variety of plants. Rice, a model organism of short-day plants, has produced an abundance of useful experimental results, which if researched further 8

9 could potentially lead to increasing crop yields and a solution to sustaining this important dietary staple. While the past few decades of research have provided a wealth of insightful data regarding florigen, there is still a substantial amount of unclear information. With the use of rice as a model organism, researchers are one step closer to uncovering the unsolved mysteries of this influential plant flowering hormone.

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Works Cited Abe, M., Daimon, Y., Ikeda, Y., Kobayashi, Y., Yamaguchi, A., Yamamoto, S. et al. (2005). FD, a bZIP protein mediating signals from the floral pathway integrator FT at the shoot apex. Science 309: 10521056. Allard, H.A., Garner, W.W. (1920) Effect of the relative length of day and night and other factors of the environment on growth and reproduction in plants. Journal of Agriculture Research, 18: 553-606. Araki, T., Kobayashi, Y., Kojima, S., Monna, L., Sasaki, T., Takahashi, Y., Yano, M. (2002) Hd3a, a rice ortholog of the Arabidopsis FT gene, promotes transition to flowering downstream of Hd1 under short- day conditions. Plant Cell Physiology, 43:1096105. Bohlenius, H., Eriksson, S., Huang, T., Nilsson, O., Parcy, F. (2005). The mRNA of the Arabidopsis gene FT moves from leaf to shoot apex and induces flowering. Science 309: 16941696. Busch, W., Jaeger, K.E., Kim, M.C., Lohmann, J.U., Schmid, M., Wigge, P.A., et al. (2005). Integration of spatial and temporal information during floral induction in Arabidopsis. Science 309: 10561059. Chalakhyan, M.K. (1985). "Hormonal regulation of reproductive development in higher plants". Biologia Plantarium, 27 (45): 292302 Chardon, F., Damerval, C. (2005) Phylogenomic analysis of the PEBP gene family in cereals. J Mol Evol. 61:57990. Coupland, G., Fornara, Fabio., Turck, Franziska. (2008) Regulation and identity of florigen: FLOWERING LOCUS T moves center stage. Annual Review of Plant Biology. 59: 573-594. Greenup, A., et al. (2009) The molecular biology of seasonal flowering-responses in Arabidopsis and the cereals. Annals of Botany, 103: 1165-1172. Hayama, R., Izawa, T., Shimamoto, K. (2002) Isolation of rice genes possibly involved in the photoperiodic control of flowering by a fluorescent differential display method. Plant Cell Physiology, 43:494504. Ikeda-Kawakatsu, K., Itoh, H., Izawa, T., Osugi, A., Takano, M. (2011) Molecular dissection of the roles of phytochrome in photoperiodic flowering in rice. Plant Physiology, 157: 1128-1137 Itoh, H., Izawa, T., Nonoue, Y., Yano, M. (2010) A pair of floral regulators sets critical day length for Hd3a florigen expression in rice. Nat. Genet., 42: 635638

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11 Izawa, T. (2007) Daylength measurements by rice plants in photoperiodic short-day flowering. Int Rev Cytol., 256:191222. Izawa, T., Takahashi, Y., Yano, M. (2003) Comparative biology comes into bloom: genomic and genetic comparison of flowering pathways in rice and Arabidopsis. Current Opinion Plant Biology, 6:11320. Komiya, R., Tamaki, S., Tsuji, S. (2008) Florigen and the photoperiodic control of flowering in rice. Rice, 1: 25-35. Knott, J. E. (1934). Effect of a localized photoperiod on spinach. Proc. Soc. Hort. Sci, 31: 152-154. Sachs J (1865) Wirkung des Lichtes auf die Bltenbildung unter Vermittlung der Laubbltter. Bot Ztg, 23: 117121; 125131; 133139. Shimamoto, K., Taoko, K., Tsuji, H. (2011) Regulation of flowering in rice: two florigen genes, a complex gene network, and natural variation. Current Opinion in Plant Biology, 14: 45-52 Wu, Weixun. (2012) Association of functional nucleotide polymorphisms at DTH2 with the northward expansion of rice cultivation in Asia. Proceedings of the National Academy of Sciences of the United States of America, 110: 2775-2780. Zeevaart JAD (1976). Physiology of flower formation. Annu, Rev. Plant Physiology, 27: 321348.

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