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INTRODUCTION: Sugarcane is a C4 grass that is able to accumulate great amounts of sucrose in its shoot. The circadian clock modulates major pathways associated to sucrose synthesis, such as photosynthesis, sucrose synthesis and sugar transport. The circadian clock can be divided into three parts: the central oscillator, that generates circadian rhythms; the input pathways, that brings information from the environment to the central oscillator; and the output pathways that take the temporal information generated by the central oscillator to the rest of the plant. The most known input pathways are associated with light perception, even though temperature is also involved. There are at least three families of photoreceptors that regulate the circadian clock: the red/far-red light-receptor PHYTOCHROMES (PHY) and the blue light-receptors CRYPTOCHROME (CRY) and ZEITLUPE (ZTL). The central oscillator is mainly a series of interlocked negative feedback loops based on the regulation of gene expression. One loop consists of CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), CCA1 HIKING EXPEDITION (CHE) and TIMING OF CAB EXPRESSION 1(TOC1). CCA1 and LHY are transcription factors of the same family and have redundant function. Both proteins repress TOC1 and CHE expression. In turn, TOC1 activates CCA1 and LHY expression and also inactivates CHE, which acts as a repressor of CCA1 and LHY expression. Another loop, called “morning loop” is composed of PSEUDO RESPONSE REGULATOR 5 (PRR5), PRR7 and PRR9 that interact with CCA1 and LHY. A final loop, called “evening loop” is composed of GIGANTEA (GI), PRR3 and ZTL, which interact with each other in order to regulate TOC1 degradation. AIM: The aim of this study is to identify transcripts associated with the generation of circadian rhythms in sugarcane and test if their pattern of expression under constant light is consistent with a role in the circadian clock. METHODS: Putative circadian genes were identified in sugarcane from the SUCEST, a sugarcane EST database. Each SAS (sugarcane assembled sequence) was selected using tBLASTx with an Arabidopsis thaliana circadian gene as an input. SAS with highest identity were selected (usually e-value < e-30) and used as input in a tBLASTx query against an Arabidopsis database. If the original Arabidopsis gene was the top hit, the SAS was confirmed as a putative circadian gene. The expression levels of putative genes associated with the circadian clock was estimated using custom oligoarrays. A commercial sugarcane variety (RB855453), which still has a functional flowering apparatus, was grown in soil for three months in a growth room in a 12 h light/ 12 h dark photoperiod and constant temperature. The plants were kept in constant light and temperature for 24 h then leaves from nine individuals were harvested every 4 h for 48 h. RNA from the samples was extracted and samples from each time points were hybridized against a RNA pool containing equimolar amounts of each sample. The oligoarray contains 21902 elements of which 7380 are specific for the antisense sequences sugarcane ESTs. RESULTS AND CONCLUSION: We have identified a total of 2354 circadian transcripts (~16%). A. thaliana has five versions of PHY (AtPHYA, AtPHYB,At PHYC, AtPHYD and AtPHYE) while grasses seem to have only three (PHYA, PHYB and PHYC). Five SAS were found to have high identity to AtPHYs but they could be grouped in three isoforms, like found in grasses: ScPHYA-1, ScPHYA-2, ScPHYB, ScPHYC-1 and ScPHYC-2. Of these SAS, only ScPHYB is not circadian regulated. ScPHYA-1 and ScPHYA-2 peaked at ZT16 (16 h after subjective dawn) and ScPHYC-1 and ScPHYC-2 peaked at ZT12. In Arabidopsis, only AtPHYA - peaking at ZT0 and AtPHYE - peaking at ZT20 – were circadian regulated. Sugarcane has five SAS with high identity to AtCRY1 (ZT8): ScCRY1-1 to ScCRY1-5. Of these, only two are circadian regulated: ScCRY1-2 (ZT4) and ScCRY1-3 (ZT12). In contrast, only one SAS had high identity to AtCRY2 (ZT12) but it was not circadian regulated. Three sugarcane ZTL were found, one with a peak at ZT4 (ScZTL-1) and one with a peak at ZT20 (ScZTL-2) while AtZTL is not circadian regulated. In the central oscillator, only one SAS (ScLHY, ZT0) had high identity to AtCCA1 (ZT0) and AtLHY (ZT0). In contrast, two SAS were found with high identity to AtTOC1, ScTOC1-1 and ScTOC1-2, both with a peak at ZT12. ScPRR3, ScPRR59 and ScPRR7 had all peaks at ZT8. Finally, there were four SAS with high identity to AtGI. Of those, ScGI-1 was not circadian-regulated, ScGI-2 had a peak at ZT12 and ScGI-3 had a peak at ZT8. ScGI-4 was not represented in the oligoarrays but real-time PCR showed that it was also circadian-regulated with a peak at ZT8. AtGI is found to have a peak that varies between ZT8 and ZT12 depending on the growth conditions. The circadian clock is thought to be conserved among the plants. Sugarcane has robust circadian rhythms, despite its aneuploidy and polyploidy. Sugarcane photoreceptors associated to the input pathways to the circadian clock are also circadian regulated but their regulation differs to their Arabidopsis counterpart, as seen by their time of peak. At the same time, genes associated to the central oscillator of the circadian clock all peak at the similar times in sugarcane and Arabidopsis, probably because they are essential to the maintenance of circadian function. Finally, the number of components of the central oscillator seems to vary in sugarcane in comparison to Arabidopsis: sugarcane has only one CCA1/LHY, two TOC1, one PRR5/PRR9 and four GI. In conclusion, the sugarcane circadian clock has small differences in regulation and composition when compared to the Arabidopsis circadian clock but it is uncertain how those differences impact the function of sugarcane circadian clock and whether they impact sugarcane capacity to accumulate sucrose.ഀ Supported by FAPESPഀ ഀ