Plant Signaling and Behavior - Article Addendum Linkage between circadian clock and tricarboxylic acid cycle in Arabidopsis Norihito Nakamichi1,†, Atsushi Fukushima1,†, Miyako Kusano1,†, Hitoshi Sakakibara1, Takeshi Mizuno2, and Kazuki Saito1,3,* 1RIKEN 2School

Plant Science Center; Yokohama; Kanagawa; Japan

of Agriculture; Nagoya University; Nagoya; Aichi; Japan

3Graduate

School of Pharmaceutical Sciences; Chiba University; Chiba;

Japan †These

authors contributed equally to this work.

Acknowledgement We thank Dr. Elaine Tobin at University of California (Los Angeles, CA) for the CCA1-ox seeds. This work is supported in part by grants-in-aid from the Ministry of Education, Science, Culture, Sports, and Technology, Japan to H.S, T.M, and K.S. Key words: Arabidopsis, circadian clock, cold stress, metabolome, TCA, PRR, CCA1

Abstract The transcriptional/translational feedback loop is thought to play a central role in the circadian clock in Arabidopsis. The loop includes close paralogs of MYB transcription factors CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and

LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB

EXPRESSION 1 (TOC1, PSEUDO RESPONSE REGULATOR 1[PRR1]), PRR9, PRR7, and PRR5. The prr9 prr7 prr5 triple mutants (d975) and over-expression line of CCA1 (CCA1-ox) are arrhythmic under continuous light conditions followed by light and dark cycles. We recently demonstrated a tight link between the circadian clock and the tricarboxylic acid (TCA) cycle, from the metabolome analysis of d975. The levels of metabolites belonging to TCA cycle, such as 2-oxoglutarate, succinate, fumarate, citrate, and malate increased in d975, whereas those of arginine and ornithine decreased. In this addendum, we further demonstrate that metabolism belonging to TCA cycle in CCA1-ox was partly similar to that of d975. This profiling also supported the linkage between circadian clock and metabolism associated to TCA cycle. Running title: Metabolism in mutants of clock-associated genes

Introduction Circadian rhythms are endogenous rhythms that allow plants to adapt and adjust to the 24h day-night cycle. They are closely associated to a wide-range of biological processes such as movements of leaves and petals, stomatal opening, hypocotyl elongation, and diurnal changes in photosynthetic activities1. Circadian rhythms are needed for the plants to adjust not only to daily changes, but also to seasonal changes in day length. Many plants monitor seasonal changes in day length to regulate flowering time for successful reproduction2. The mechanisms generating and maintaining circadian rhythms are

called

circadian

clock(s)

or

biological

clock(s).

Multiple

transcriptional/translational feedback loops are thought to play a central role in the circadian clock in Arabidopsis1, 3, 4. The closest paralogs of the MYB transcription factors CCA1 and LHY directly repress TOC1 expression5, 6.

In turn, TOC1 is involved in the activation of CCA1 and LHY expression.

PRR9, PRR7, and PRR5 are also involved in this loop7-9. PRR9, PRR7, and PRR5 repress CCA1 and LHY expression, and CCA1 and LHY activate PRR9 and PRR73, 4, 7, 8. Using genetic approaches, we discovered that Arabidopsis d975 mutants show arrhythmia under all conditions tested7. The mutants also showed pleiotropic phenotypes such as late flowering time, long hypocotyls and petioles, and dark green leaves10, 11. Transcriptome analysis reveals novel phenotype of prr9 prr7 prr5 Expression profiling of d975 using microarrays revealed that the upregulated genes in d975 included a significant number of cold-stress responsive genes

12.

Moreover, d975 is more tolerant to cold stress than the

wild-type12. Expression of three DEHYDRATION RESPONSE ELEMENT

BINDING1 (DREB1)/ C-REPRAT BINDING FAXTOR (CBF) genes are upregulated in d975. Furthermore, immediate target genes of DREB1A are upregulated in d975. Because DREB1 provides cold-stress tolerance to plants,13, 14 upregulation of DREB1 may be one of the causes of cold stress tolerance of d975. The daily peak of expression of the DREB1-downstream

genes is early afternoon15, which might provide Arabidopsis with cold stress tolerance during the nighttime when frost is more likely to occur. The circadian clock is likely to regulate or affect the cold stress responses by mediating the expression of the ‘hub gene’ DREB1. Evidence of a link between clock-associated genes and TCA cycle Metabolite

profiling

using

gas

chromatography-time

of

flight/mass

spectrometry16 (GC-TOF/MS) also provided evidence that PRR genes repress the levels of TCA cycle metabolites, such as 2-oxoglutarate, succinate, fumarate, citrate, and malate17. The levels of osmolytes (e.g., proline, galactinole, and raffinose) were increased in d975, whereas those of arginine and

ornithine

were

decreased.

It

suggests

that

a

shift

in

the

glutamate-metabolic flux to proline production rather than ornithine. Comparison of metabolite profiles of three mutants related to primary metabolism (mto118, sng119, and serat2;1/serat2;220), two mutants related to flavonoid metabolism (tt421 and tt521), CCA1-ox22, and d975 revealed that metabolism related to TCA cycle in CCA1-ox plant was partly similar to that in d975. The levels of fumarate, citrate, and malate were increased both in

d975 and CCA1-ox, but not in the other mutants (asterisk in Figure 1). The levels of ornithine and arginine were decreased both in d975 and CCA1-ox (double asterisk in Figure 1). It should be noted that the degree of changes in metabolite levels between CCA1-ox and the wild-type was less than that between d975 and the wild-type. Based on the result, however, we propose that the circadian clock system including CCA1 is linked to metabolism related to the TCA cycle. The biological meaning of the linkage between circadian clock and TCA cycle remains unclear. TCA cycle provides energy transfer molecule ATP and reducer NADPH. In addition, it also provides precursors for many compounds including amino acids of the glutamate family (i.e., proline and GABA). Metabolite profiling by GC-TOF/MS could not detect diurnal rhythmic patterns for proline; however, Gibon et al., reported diurnal rhythmic pattern for proline with a peak during late daytime23. Thus,

Arabidopsis plants may produce proline late in the day to adapt to cold night

conditions. Since the circadian clock is associated to photoperiodic reactions, it is worth testing whether intermediates in the TCA cycle are responding to changes in photoperiod. Note that the figure (or morphology) and flowering time of d975 in long-day conditions are very similar to those of the wild-type under short-days11,

24.

Arabidopsis may sense shortened day length, and

adjust metabolism to adapt to the winter season. Conclusion and perspective Transcript profiling revealed a role of the circadian clock system in the cold stress response12. Metabolite profiling revealed a tight linkage between circadian clock system and TCA metabolism17. Thus, high throughput (omics) analyses are useful to reveal linkage or interactions between cellular pathways. The exact nature of the link between the circadian clock and the TCA cycle remains to be elucidated. However, we speculate that the downregulation of genes encoding fumarase and putative 2-oxoglutarate delydrogenase in d975 are one of the reasons explaining the metabolic profile of d97517. To verify this hypothesis, the following experiments will need to be performed: (1) Quantitative analysis of fumarase and 2-oxoglutarate dehydrogenase activities in plant extracts and, (2) Tracer experiments using labeled precursors of fumarate or 2-oxoglutarate. In addition, metabolite profiling of mutants or transgenic plants in which expression of clock-associated genes is modified, will help to elucidate how the biological clock system regulates the TCA cycle. Because some metabolites and second messengers modulate circadian clock25,

26,

it is needed to elucidate the

interaction mechanisms between the biological clock system and metabolism such as in the TCA cycle.

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Figure legends Figure 1. Metabolite profiles of mto1, sng1, serat2;1/serat2;2, tt4, tt5,

CCA1-ox, d975, and the wild-type plants. Red and blue colors indicate increased and decreased metabolites levels respectively. Single and double asterisks show the set of increased and decreased metabolites, respectively, both in CCA1-ox and d975. The degree of changes in metabolite levels between CCA1-ox and the wild-type was less than that between d975 and the wild-type. The raw data used for this figure is the same as for Fig. 1-D in our previous paper17.