Climate change is forecasted to have an impact in agricultural productivity. To ensure security of crop yields, we must develop a better understanding of flowering mechanisms in plants. Research on flowering mechanism has been performed mostly under simplified lab growing conditions. However, in the natural environment, plants experience dynamic changes in abiotic factors like temperature throughout the day. To better understand how flowering is regulated under natural conditions, we focused on the expression profiles of FLOWERING LOCUS T (FT), which correlates with the flowering time in plants. FT expression is known to peak in the evening under long day lab conditions (16 hours of light, 8 hours of darkness, 22^oC constant). However, under natural long day conditions (Summer Solstice in Seattle, 16 hours of light, 8 hours of darkness, average highest temperature 21^oC) we have observed an additional morning peak. We have been able to recreate the observed expression of FT in the lab by modulating temperature oscillation and light quality of long day lab conditions. In this project, we first asked if the observed FT pattern is widely conserved in different wild type accessions in Arabidopsis thaliana. We grew different accessions under re-created lab growth conditions and analyzed FT expression using quantitative PCR. So far we have seen similar FT expression patterns among some accessions, suggesting that morning peak of FT is conserved among these lines. Second, we studied which timing of the day temperature acts as a cue to affect morning FT expression. We grew plants under various temperature conditions and compared results to the observed FT expression in nature. The results from this project will contribute to the development of new modified lab growth conditions that represent natural conditions and give a better understanding of the mechanism that controls FT regulation in nature.

Plants respond to abiotic factors in natural settings in order to regulate physical responses at the most appropriate timing. The timing of flowering affects seed and/or fruit production in many agriculturally relevant plants, so understanding its mechanism is essential for addressing agricultural needs as climate changes. To address this aim, we have used Arabidopsis thaliana as a model plant to understand the molecular mechanism of seasonal flowering. It has been shown that the expression of FLOWERING LOCUS T (FT), which encodes florigen, peaks in the evening under long-day lab conditions (16 hours light, 8 hours dark, 22°C) and promotes flowering in a day-length specific manner. However, our lab found that FT also peaks in the morning under outside long-day conditions over the summer solstice in Seattle (16 hours light, 8 hours dark, highest average temperature 21°C). This double-peak of FT can be re-created by changing the light quality and temperature settings of long-day lab conditions. In addition, we found that a mutant of PHYTOCHROME A (PHYA), which encodes a red/far-red light photoreceptor in plants, lacks FT expression in a morning-specific manner, suggesting that PHYA is involved in regulating morning expression of FT. In order to understand the molecular mechanism of PHYA-mediated FT regulation, we are in the process of identifying transcription factors and other molecules that work with PHYA to induce FT expression. We have successfully cloned various transcription factors that are reported to act in the flowering regulatory pathway and are in the process of testing protein-protein interaction against PHYA by yeast-two-hybrid methods. Identifying the components that act in the PHYA-FT pathway will allow us to further understand how light affects flowering regulation.

FLAVIN-BINDING KELCH REPEAT F-BOX 1 (FKF1) is a blue light photoreceptor which induces photoperiodic flowering in Arabidopsis. Depending on blue light, FKF1, along with GIGANTEA, forms a complex to induce flowering by degrading a transcriptional repressor, CYCLING DOF FACTOR 1 (CDF1). FKF1 stays in light status for a long time, allowing plants to continue induce flowering. ZEITLUPE (ZTL), a close homolog of FKF1, is a circadian clock regulator with a faster photocycle LOV domain. A photocycle is the sequence of structural changes that takes place in molecules when exposed to light. ZTL has high affinity with GI in presence of light, but also degrades its targets quickly in the dark. The faster ZTL photocycle allows it to act as a sensor to the night periods in circadian oscillation phases. Therefore, we hypothesize that slower photocycles of FKF1 is necessary for FKF1 to keep light information and induce photoperiodic flowering. To test the effect of photocycle on the FKF1 LOV domain in photoperiodic flowering, we generated mutations on the FKF1 LOV domain based upon sequence information of the ZTL LOV domain, to speed up the photocycle kinetics. Quantitative real time PCR (qPCR) was used to select stable, transgenic lines expressing comparable levels of mutated FKF1. Then we analyzed samples for expressions of downstream genes CONSTANS, FLOWERING LOCUS T, along with flowering times to measure FKF1 functionality. As a control, wild type plants were used to provide regular flowering time data. We gathered samples to establish lines to test the effect of each mutation on FKF1 function. We expect to find that the mutations’ faster conversion to dark status decreases its affinity to GIGANTEA and CDF1 degradation, delaying the flowering time. This suggests the slow conversion of FKF1 LOV domain to dark status is necessary to induce photoperiodic flowering in Arabidopsis.

Properly timed seasonal flowering in plants is necessary for reproductive viability in natural and agricultural settings. We use Arabidopsis thaliana as a model organism to study the molecular mechanisms that underlie the developmental transition to flowering, a process that is mediated by both internally propagated circadian cycles as well as information about day length (photoperiod) detected via photoreception. Transcriptional regulation of the FT (FLOWERING LOCUS T) gene is the primary mechanism of controlling the flowering response, and flowering occurs when requisite levels of FT proteins are transported from the leaves to apical stem cell tissue. FT expression is initiated by CO (CONSTANS), a transcription factor whose own expression is tightly regulated by molecular outputs of the endogenous circadian clock. One such output, CDF1 (CYCLING DOF FACTOR 1) is responsible for repression of CO and FT expression. We established that CDF1 forms a complex with the TPL (TOPLESS) protein, an interaction that is dependent on a conserved peptide sequence at the N-terminal of CDF1. We generated phloem companion tissue-specific mutants of TPL in a variety of flowering time genetic backgrounds and found that mutants of TPL consistently flowered earlier than nonmutants, similar to the phenotype witnessed in cdf mutants. tpl mutation had a similar effect on CO and FT expression across 24-hour timecourse to cdf-1,2,3,5 mutants in both long and short day conditions. We have shown that TPL protein associates with CO and FT promoters during the mornings of long days, and that this recruitment of TPL to the promoters is enhanced by the addition of excess CDF1 protein. We demonstrate that TPL protein is required for the repressive activity of CDF1 and are working to better understand the complex role it plays in regulating flowering time in Arabidopsis.