Involvement of FLOWERING LOCUS T in microgravity response of Arabidopsis thaliana plants under long- and short-day conditions

Microgravity have an impact on growth and development of higher plants in space at both vegetative stage and reproductive stage. A great deal of information has been available on the vegetative stage in space, but relatively little is known about the influence of microgravity on plants at the reproductive stage. In this study, we constructed a transgenic Arabidopsis thaliana plants expressing flowering control gene, FLOWERING LOCUS T ( FT ), together with green fluorescent protein gene( GFP ) under control of a heat shock-inducible promoter ( HSP17.4 ), by which we induced FT expression inflight through remote controlling heating shock treatment. Inflight photography data showed that induction of FT expression in plants in space could counteract the impact of microgravity and promote flowering. Whole-genome microarray analysis of gene expression changes in leaves of wild-type and these transgenic plants grown under different photoperiod conditions in space indicated that the function of the photoperiod-related microgravity response genes are mainly involved in protein synthesis and post-translation protein modulation, notably protein phosphorylation. In addition, changes of circadian component gene expression in response to microgravity under different photoperiod indicated that role of circadian oscillator could act as integrators of microgravity response and photoperiodic signals in Arabidopsis plant grown in space.


Introduction
1 Microgravity by spaceflight could cause an impact on growth and development of 2 higher plants at both the vegetative stage and the reproductive stage. A great deal of 3 information is available on the vegetative stage in space. For example, alteration of 4 auxin polar transport in etiolated pea seedlings and maize coleoptiles in space (Ueda 5 et al., 2000), inhibition of cell division and mitosis as well as significant 6 karyological disturbances in root-tip cells of oat, mung bean and sunflower 7 seedlings grown in space, modification of cell wall metabolism (Krikorian and 8 O'Conner, 1984;Rasmussen et al., 1994, Sago et al., 2002. Reduction in fresh 9 weight of shoot and photosynthetic function of wheat plants grown onboard space 10 shuttle (Tripathy et al., 1996). The plants grown in space was often smaller than 11 comparably aged ground controls (Kiss et al., 2000;Paul et al., 2012;Wang et al., 12 2018), while others grew faster in space (Matía et al., 2010;Hoson et al., 2014). 13 However, relatively little is known about the influence of microgravity on plants at 14 the reproductive stage. Some early experiments reported failure in seed formation 15 under spaceflight conditions (Nechitatio and Maskinsky, 1993;Kuang et al., 1996;16 Strickland et al., 1997;Soga et al., 1999;Levinskikh et al, 2000;Campbell et al., 17 completion of single reproductive phase, lowering of reproductive success and 23 alteration of seed reserves are still major bottlenecks to maximize the efficiency of 24 plant growth and reproduction in space and to be used to support life in long-term 25 manned missions (Hoson, 2014;reviewed by Zheng 2018). 26 The reproductive success of plants is often dependent on their flowering time being 27 adapted to the growth environment. A number of studies suggest that both biotic and 28 abiotic stress factors play key roles in controlling to alter flowering time in plants. 29 For example, plants often acceleration the flowering process under drought stress 30 (Sherrard and Maherali, 2006;Galbiati et al., 2016) and delays flowering time by 31 salt stress (Achard et al., 2006;Ma et al., 2015). Heat and cold stress can also have a 32 dramatic effect on flowering. In addition, the other stresses, such as, nutrient, sugar 33 budget, geomagnetic field and simulated microgravity, have significant effects on 34 plant development including flowering process time (Lee et al., 2008;Posé et al., 35 2013;Agliassa et al., 2018;Xie et al., 2020). Increasing evidences document that 36 microgravity is a novel stress for plants grown in space (Paul et al., 2001;De Micco 37 et al., 2014;Zhang et al., 2015;Karahara et al., 2020), which cause changes at the 38 physiological, morphological and molecular levels, including altered transcription 39 patterns of many genes. In the space-grown Mizunna, a total of 20 in 32 ROS 40 oxidative maker genes were up-regulated, including common genes response to 41 abiotic and biotic stress (Sugimoto et al., 2014). In Arabidopsis culture cells grown 42 in space, genes associated with heat shock, salt, drought, metals, wounding, 43 phosphate, ethylene, senescence, terpenoids, seed development, cell walls, 44 photosythesis, and auxin were up-regulated by five fold in comparison with their 45 ground controls (Paul et al., 2012;Kwon et al., 2015). The endogenous systems that 46 measure day length was found to interact with stress responses and override 47 interpretation of the signals in plants on ground (Becker et al., 2005). It is however 48 unclear how the photoperiod influence the signals in plants in space. 49 The developmental rate of Arabidopsis plants on ground is directly related to 50 daylength, because Arabidopsis is a long-day (LD) plant, an increase in photoperiod 51 results in an increase in development rate. How photoperiod affect plant 52 development in space has yet known. No space experiments had been carried out to 53 compare the effects of different photoperiod on plant growth and development so far. 54 To exmamine effects of photoperiod signals on the microgravity response of plants 55 in space, we conducted the space experiment by growing Arabidopsis plants under 56 the LD and the short-day (SD), respectively, on board the Chinese recoverable For space experiment on the satellite SJ-10, seeds of WT and FG were germinated 84 and grown in the root modules on ground under the LD condition for 20 days (Fig. 85 1E ; corresponding to stage 1. 06, Boyes et al., 2001). At this age, the plants had 86 formed about 5-6 rossette leaves ( Fig.1E and F), when they were loaded into the 87 plant growth unit (PGU) less than 24h prior to take off. Under the LD condition, 88 floral shoots of WT plants on ground appeared at day 4 after satellite launched, 89 while plants in space initiated floral shoots on day 6 ( Fig. 2A and B). For FG plants, 90 floral shoots appeared at day 2 under the LD on ground were earier than those in 91 space at day 4 ( Fig (Table 1). The genes responding to microgravity under LD and SD 160 conditions with similar behaviors were named 'μg-common ' genes, whereas those 161 in response to μg specific to LD or SD were named 'μg-daylength-related ' genes.  were apparently more than those of 'μg-common ', indicating that daylength is an     Table S11). In addition, several circadian clock genes were abserved among FT 252 interactome ( Fig. 9 B). REV2, which is involved in regulating both photoperiod  Table S10).  conceived of the study, participated in its design and coordination, and drafted the manuscript. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors declare that they have no conflict of interest.

Figure 9
The core photoperiod response genes altered expression levels by expouse to microgravity.
(A) Log2 FC of the 20 core photoperiod genes in the FG under LD or SD on ground (1g) and microgravity in space (µg), respectively, in comparison with these genes in wild-type (WT) under the same condition (FG/WT).
(B) Diagram of the protein interaction networks of the photoperiod response genes.
The genes, which altered expression level in response to microgravity in space in comparison with their controls on ground, were labelled by colour in yellow and log2 FC of these highlighted genes are indicated in C.
(C) Log2 FC of selected core photoperiod genes in WT and FG in response to microgravity under the LD and the SD condition, respectively.
(A and E) 14-day-old wild-type (A) and FG transgenic seedlings (E) were treated by 37C for 1h or under 20C control conditions, respectively.
(B-D) Fluorescence images of leaves from 37C heat treated seedlings and 20C control plants, respectively.
(F-H) image of leaves under differential interence contrast optics microscope.

Supplementary Table S5
Expression data of identified genes of wild-type (WT) plants grown under the short-day (SD) in microgravity (µg) in space were compared with those under the SD on ground(WT-µg -SD versus WT-1g-SD).

Supplementary Table S6
Expression data of identified genes of pHSP::FT, pHSP:GFP (FG) transgenic plants grown under the short-day (SD) in microgravity (µg) in space were compared with those under the SD on ground(FG-µg -SD versus FG-1g-SD).

Supplementary Table S7
Pair-wise comparison of altered expression of genes in response to microgravity in WT under the SD with those under the LD conditions. to C3 indicating in Figure 7E.

Supplementary
Supplementary Table S10 Protein phosphorylation proteins encoded by day-length related microgravity-responsive genes.

Supplementary Table S11
Microgravity response of core photoperiod response genes in WT and/or FG plants grown in space. Genes identified with a significant (FC>2 and p<0.05) change in expersion level.