Abstract
Our previous studies suggested that FLOWERING LOCUS (FT) can integrate microgravity signals into the photoperiodic flowering pathway in Arabidopsis. However, the exact mechanisms remain unclear. In this study, we carried out transcriptomic analysis of wild-type (WT), gigantea (gi) mutant, CONSTANS overexpressing (35S:CO), and transgenic plants expressing FT with the green fluorescent protein gene controlled by a heat shock-inducible promoter (pHSP:GFP;pHSP:FT, FG) grown on the Chinese space station in comparison with their ground controls. The findings revealed that transcriptional microgravity response signature was not only conserved across the GI-CO-FT module disruption genotypes and the WT, but also displayed GI-CO-FT module disruption specific associated responses. Transcription factors belonging to the ERF, bZIP, bHLH, and BES1 families were significantly overrepresented among the GCC- and CACGTG-elements across GI-CO-FT specific microgravity responsive genes, suggesting that disruption of GI-CO-FT module could influence plant response to microgravity through integration pathways.
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Introduction
For extended human space exploration, a tightly integrated biological life support system (BLSS) is imperative, with plants playing pivotal roles in air regeneration, food production, and water recycling. Given the constrained volume and energy supply aboard space missions designated for plant cultivation, there is a pressing need not only to successfully grow edible plants in space but also to optimize the harvest index while minimizing production time and energy consumption. However, spaceflight presents a unique environment with complex stressors, such as microgravity, that affect the absorption, transport, and distribution of water and nutrients in plants, as well as transpiration rates1,2,3. The growth of plants at both the vegetative and reproductive stages is affected by complex responses to microgravity. For example, many plant seedlings grown in space have been observed to be smaller than their ground controls4,5, whereas some exhibited accelerated growth6,7. In addition, reduced shoot fresh weight, compromised photosynthetic functions8, delays in the reproductive phase9 and alterations in seed reserves10,11 have been observed7,12,13. The effects of microgravity on the vegetative growth phase have been relatively well documented at the molecular level14,15. Successful seed-to-seed experiments under microgravity have been performed with both dicotyledonous plants such as Brassica rapa L10,16, Arabidopsis thaliana L9,15,17,18, Pisum sativum L19. and monocotyledonous plants, such as wheat20 and rice21. These experiments led to the fair conclusion that completion of the seed-to-seed cycle in space is possible if suitable support conditions are provided. However, we are going beyond plant survival in space11, but the development of higher resource-use efficiency BLSS requires higher-yielding plants.
Reproductive growth directly affects plant grain yield both on Earth and in space. A growing body of evidence underscores the influence of microgravity on the reproductive growth of plants in space13,22. For example, plants grown in space mature more slowly, with reproductive phases often being compromised22,23. Although advancements in cultivation chambers have facilitated successful seed production in both dicotyledonous and monocotyledonous plants during space missions15,17,24,25, space plants still face abiotic stressors induced directly or indirectly by microgravity. For instance, plants grown in space produced fewer flowers, altered inflorescence structures, and smaller seeds with changed storage reserves10,20,26. During the plant life cycle, the floral transition, which is one of the most decisive developmental processes between vegetative and reproductive growth phases, determines the environmental adaptation, survival, and grain yield of plants and can be interrupted by microgravity in space more easily than other growth phases. Thus, understanding how microgravity interacts with regulatory networks involved in floral transition and reproductive development in space is necessary.
Plants have evolved complex regulatory networks to control flowering processes and reproductive development in response to environmental signals, including the photoperiod, vernalization, aging, autonomous and gibberellin pathways. In A. thaliana, the photoperiod pathway regulates floral transition mainly via the GIGANTEA-CONSTANS-FLOWERING LOCUS T(GI-CO-FT) module in which CO receives a signal from GI and subsequently affects the expression of FT27,28,29. Mutations in any of the GI-CO-FT module genes can cause a delay in flowering under LDs, whereas their overexpression accelerates flowering independent of day length30. Both GI and CO serve as nexus points connecting various stress pathways with the activation of FT transcription. The gi knockout mutants were observed to flower late and exhibited increased tolerance to abiotic stresses31,32. In contrast, overexpressing CO and FT led to early flowering and diminished stress tolerance33,34, because FT overexpression could influence various biological processes in response to environmental stimuli. The mechanism underlying FT-controlled flowering has been extensively studied. The application of FT should be of particular use in increasing the input/output ratio of resources obtainable by plants, as it could greatly accelerate and enable new kinds of breeding and research for BLSS.
In earlier space experiments, we studied the interplay between the induced expression of FT and photoperiodic signaling in space-grown Arabidopsis plants9,13. Floral initiation in space-grown Arabidopsis under long-day (LD) conditions was delayed by ~20 days compared with that in ground controls. Using transgenic plants with heat shock-inducible FT expression, we showed that plant transcriptional responses to microgravity are related to day-length conditions, and that the induced overexpression of FT during the reproductive phase could mitigate spaceflight stresses, thereby accelerating reproductive development in space13. However, the molecular mechanisms through which microgravity influences plant development and flowering remain unknown. To assess the contribution of the GI-CO-FT module to the functions of the plant microgravity response and flowering control in space, we present findings from our most recent experiments aboard the Chinese Space Station (CSS), where Arabidopsis wild-type (WT), gi-2 mutant (gi), CO overexpression plants (35S:CO), and transgenic plants expressing the FT with the green fluorescent protein (GFP) gene controlled by a heat shock-inducible (HSP17.4) promoter (pHSP:GFP; pHSP:FT, named “FG”) as described by Wang et al. 13. were cultivated and their transcriptomic adjustments in space were analyzed by comparison with those of their controls on ground. Notably, for our transcription analysis, plant samples were collected in orbit and frozen at –80 °C before examination. Thus, the differentially expressed genes (DEGs) identified in plants grown in space in comparison with their control on ground in this study could represent spaceflight microgravity-responsive genes, that are not affected by altered gravity during landing.
Results
Spaceflight microgravity affects Arabidopsis growth and flowering
The General Biological Modular Culture System (GBMCS) on the CSS is equipped with subsystems for atmospheric, temperature, and hydration regulation and a lighting system as described21. This facility allowed the Arabidopsis plants to germinate and mature on the CSS in space (Fig. 1a–c). Additionally, the GBMCS is fitted with four digital cameras to facilitate image capture and to visually monitor the ongoing experiment. Ground control experiments were carried out in parallel with culture chambers (CCs) in an engineering reproduction of the spaceflight GBMCS. Dry seeds of Arabidopsis WT, gi, 35S:CO and FG were grown in CCs under GBMCS on the CSS (Fig. 1d, e). In addition to the WT, a FT promoter construct (pFT:GFP) as described by Xie et al.9 was also used as a control both in space and on ground (Fig. 2a–f, a1–f1).
a Diagram of the outside view of the life ecology experimental cabinet. b, c The external view and the internal composition of the GBMCS, showing water recycling, air recycling, the CO2 and ethylene regulators (Et. reg.), a culture medium delivery system, an illumination system, an image acquisition subsystem that includes two cameras (Cam 1 and Cam 4) for acquiring visible images and two cameras (Cam 2 and Cam 3) for acquiring fluorescence images. d Images of culture chambers (CC1 and CC2) for Arabidopsis culture. e Example images showing the distribution of samples on the installation panels of CCs. WT wild-type, gi gi-2, 35S:CO CONSTANS overexpression, FG transgenic plants pHSP:FT, pHSP:GFP.
Representative images of plants grown on ground (a–x) and in space (a1–x1) from germination to the inflorescence emergence stage. Note that the first flower opened approximately 30 days (d30) after sowing for WT, d25 and d26 for FG and 35S:CO, respectively, on ground, and on d53, d35 and d40 for WT, FG and 35S:CO in space respectively. WT, wild-type; ft, ft-10 mutant (Col-0 background); pFT:GFP, transgenic plants expressing the green fluorescent protein (GFP) coding region under control of the FT promoter in the Col-0 background; FG, transgenic plants expressing FT with the GFP gene controlled by a heat shock-protein (HSP) promoter (pHSP:GFP; pHSP:FT); 35S:CO, transgenic plants overexpressing CONSTANS(CO). Bars=10 mm.
Downlinked images revealed that the germination of the seeds of both the WT and the GI-CO-FT module disruption genotypes initiated ~3 days after sowing (DAS) at the same rate in space and on ground (Fig. 2 and Supplementary Fig. S1), but the timing of floral initiation significantly differed between the ground control and spaceflight materials (Figs. 2 and 3a, b). Compared with the ground controls, WT and pFT:GFP plants grown in space exhibited significantly later bolting times (Figs. 2a–f and a1–f1, and 3a, b) as we previously reported9. After germination, all the plants developed rosette leaves under either in space or on ground conditions (Fig. 2), but the space plants grew slowly with smaller cotyledons than their ground controls did. Definitions of the growth stage of space-growing plants in the culture chamber presented a challenge. To characterize the growth stage, the change in rosette leaf area per culture hole was used to determine the plant growth rate. The average rosette leaf area of WT plants at ~25 DAS both on ground and in space was more than 50% of the final size (corresponding to an Arabidopsis growth stage of 3.5 at floral induction stage)35 (Supplementary Fig. S2). Mutants gi and ft-10 grown in space and on ground didn’t flower (corresponding to Arabidopsis growth stage number 1.06 at the leaf production stage), whereas 35S:CO and FG flowered (corresponding to Arabidopsis growth stage number 6.1 at the flowering stage) at 25 DAS (Fig. 2). Thus, the samples used in this study represent plant response to microgravity at three different developmental stages: floral induction stage (WT), seedling growth stage (gi mutant) and flowering stage (35S:CO and FG), respectively. The 35S:CO transgenic plants consistently flowered early under both space and ground conditions (Figs. 2m–r and m1–r1, and 3a, b), whereas compared with the control plants, the FG plants flowered slightly earlier under spaceflight conditions (Figs. 2g–l and g1–l1, and 3a, b), indicating that the transcriptional regulation of CO and FT may differ under spaceflight conditions. Because of the limitation of the volume of the freezer in the SZ-14 spacecraft for returning samples, only WT, gi, 35S:CO and FG samples at 25 DAS were collected for further mRNA analysis, ft-10 and pFT:GFP plants were kept in orbit and did not return.
a Quantification of the spaceflight response of each genotype detailed in comparison with their ground controls. Values represent average bolting time of plants grown in four different cultivate chambers (n = 8~12 per genotypes) and error bars represent standard deviation (SD). Student’s t-test values are as follows: **P ≤ 0.03; ***P ≤ 0.001; ns, not significant. b Relative bolting time variation. The numbers indicate the percentage variations in bolting time in plants grown in the sp condition relative to those in the gr condition. Different letters indicate significant difference between samples (Student’s t-test, P < 0.05). c RNA-seq data for comparisons of changes in the transcript abundance of GI, CO and FT in WT, gi, 35S:CO and FG transgenic plants grown in space and on ground, respectively, are shown as three independent biological replicates. Absolute expression levels, transcripts per million reads. Student’s t-test values are shown as follows: **P ≤ 0.03; ***P ≤ 0.001; ns, not significant.
The transcript abundances of GI, CO and FT in the WT, gi, 35S:CO and FG samples in space and on ground were also examined by quantitative real time polymerase chain reaction (qRT-PCR) and RNA-sequence. Compared with those in the ground controls, the transcript abundances of GI in the space samples of the WT, 35S:CO and FG did not significantly change (Fig. 3c). The gi (also called gi-2 as described27) knock-out mutant exhibited later flowering (Supplementary Fig. S3a–c). There was no change in GI abundance in the gi mutant both in space and on ground, but the phenotype of the gi mutant grown in space exhibited exclusively elongated hypocotyls in comparison with that of the controls on ground (Fig. 2s1–x1 and Supplementary Fig. S1e). The reason for this difference remains unknown. The expression levels of CO in WT and the gi in space was not different from those on ground, but its expression slightly decreased in the 35S:CO and increased in the FG in space, respectively, compared with its control on ground (Fig. 3c). In contrast, the abundance of FT in the FG in space was significantly greater than that on ground, but there was no difference in the expression levels of FT in the WT, gi and 35S:CO in space from those on ground (Fig. 3c). FT is constitutively overexpressed in 35S:CO plants, whereas FT expression in FG plants apparently induced by microgravity could be caused by the inducible promoter HSP17.4, which could be activated by spaceflight microgravity as we previously reported13 (Fig. 3c). In summary, the differential expression of GI, CO and FT in WT, gi, 35S:CO and FG plants in response to spaceflight represented three different types: (1) GI mutation (in the gi mutant); (2) overexpression of CO (in 35S:CO) and (3) overexpression of FT (in FG). The complex genetic regulation and redundancy functions of genes in the GI-CO-FT pathway make identifying key gene functions in response to spaceflight challenging13. These different spaceflight response types of GI-CO-FT disruption plants present an alternative strategy for studying GI-CO-FT function in plants grown in space in response to spaceflight microgravity.
Global transcriptome analysis reveals the role of the GI-CO-FT module in response to spaceflight microgravity condition
To elucidate the global transcriptional effects of spaceflight on Arabidopsis, we performed RNA sequencing (RNA-seq) of leaves from plants grown in space and compared them with those from the ground controls. RNA was extracted from the leaves of plants at 25 DAS in CCs-space (sp) and CCs-ground (gr), respectively (Figs. 1 and 2). The mean expression levels of genes in the space-grown samples significantly differed from those in the ground controls when we applied a false discovery rate (FDR) threshold of 0.01. These genes were selected as potential DEGs (Supplementary Table S1). Among these genes, we further refined our list by considering those whose expression levels varied more than fivefold (fold change [FC] ≥ 5) when we compared space samples with their ground controls. These analyses revealed that 4901 genes of the WT in space in comparison with the controls on ground (WT, sp vs. gr, named ‘WT-comparison’), 4952 genes of the gi mutant in sp vs. gr (gi-comparison), 3871 genes of 35S:CO in sp vs. gr (35S:CO-comparison), and 3832 genes of FG in sp vs. gr (FG-comparison) were differentially expressed (Fig. 4a and Supplementary Table S2). To validate these RNA-seq data, we designed sequence-specific primers and performed qRT-PCR. The results of qRT-PCR analysis of the differential expression levels of selected genes, NUDT 21 (AT1G73540), DIC2 (AT4G24570) and glycine-rich protein (AT1G07135), matched those of the RNA-seq results (Supplementary Fig. S4 and Supplementary Table S3).
a Numbers of differentially expressed genes (DEGs) identified in WT, gi, 35S:CO and FG (p < 0.01) in response to spaceflight. FC, fold change. b Venn diagram of transcriptomic data (FC ≥ 5), showing the spaceflight responses of DEGs between gi, 35S:CO and FG in comparison with WT, respectively. Red arrows indicate upregulation and downregulation. Density scatter plots show comparisons of the spaceflight microgravity response (sp vs. gr) between gi, 35S:CO, FG and WT. The log2 fold changes (FCs) are plotted for both the x and y variables. The grey dotted lines represent y = x, and the black dotted lines represent the regression of the data for the gi (y = 0.72x + 0.69, r2 = 0.54), the 35S:CO (y = 0.51x + 0.80, r2 = 0.40), the FG (y = 0.53x + 1.13, r2 = 0.46). The differences in fold changes (sp vs gr) between the gi, 35S:CO or FG and WT, respectively, are indicated by red dots (>5 fold) and grey dots (≤5 fold). sp, spaceflight, gr, ground control. c Principal component analysis (PCA) was done with R package (https://bio-cloud.aptbiotech.com/) and demonstrated a strong difference between transcriptomes of the sp- and the gr-samples. The horizontal axis represents the contribution of principal component 1 (PC1) to distinguishing samples in the two-dimensional graph, whereas the vertical axis represents the contribution of principal component 2 (PC2). d PCA of four different genotypes, WT, gi, 35S:CO and FG, with distance based on fold changes of DEGs in space samples (top-500 genes) in comparison with their controls on ground. sp spaceflight condition, gr ground condition.
To determine whether altered expression profiles of GI, CO and FT could affect the response of plants to spaceflight microgravity, we compared the DEGs in the gi-, 35S:CO- and FG-comparisons, respectively, with those in the WT-comparison. The comparisons indicated that 2869 DEGs (45%), 3678 DEGs (59%) and 3461 DEGs (57%) in the gi-, 35S:CO- and FG-comparison, respectively, differed from the DEGs in the WT-comparison (Fig. 4b), indicating the apparent influence of altered expression profiles of GI, CO and FT on the response of plants to microgravity. Overlap of DEGs between the gi, 35S:CO and FG with that of WT, respectively, was also observed (Fig. 4b). These findings suggest that the responses of plants to spaceflight microgravity include both GI-CO-FT -independent response and the GI-CO-FT -specific responses. Furthermore, principal component analysis (PCA) highlighted marked differences between the transcriptomes of space-grown samples and their ground controls (Fig. 4c). In addition, a separate PCA of the genotype transcriptome in response to spaceflight revealed that the WT- and the gi-comparisons were similar, but the 35S:CO- and the FG-comparisons were significantly different (Fig. 4d). These results are consistent with the fact that the overlap of DEGs between the 35S:CO- or the FG-comparison and the WT-comparison was relatively lower than that between the gi- and the WT-comparisons, whereas the number of total DEGs between the 35S:CO or FG-comparison and the WT-comparison was apparently greater than that between the gi and WT-comparisons (Fig. 4b), suggesting that overexpression of CO and FT could induce different responses of Arabidopsis plants to spaceflight microgravity in comparison with the gi mutant.
To understand the biological roles of these DEGs in response to microgravity, we undertook GO analysis for the WT-, gi-, 35S:CO-, and FG-comparisons. We emphasized the top ten GO enrichment terms for both up- and down-regulated genes. The upregulated genes were predominantly associated with phosphate transport and response, secondary cell wall, phenylpropanoid metabolism and oxidant detoxification in the WT- comparison. Except for the oxidant detoxification term, the gi-, 35S:CO- and FG-comparisons were all marked by flavanol biosynthesis and metabolism terms, indicating that overexpression of either CO or FT could stimulate the flavonoid biosynthesis pathway, whereas downregulated genes were linked to the photosynthetic process, and these trends were consistent across all four comparisons (Fig. 5 and Supplementary Table S4). Several genes related to the flavonoid biosynthetic process (Supplementary Fig. S5) responded primarily or solely to spaceflight in the gi, 35S:CO, and FG, relative to those in the WT. These results indicated that CO and FT overexpression could stimulate the biosynthesis of flavonoid, which can act as antioxidants to increase plant stress tolerance.
Overrepresentation was determined for genes that exhibited differential expression in response to spaceflight in comparison with their control on ground (P < 0.01 and FC ≥ 5). a Number of genes per gene ontology in WT (sp vs. gr). b Number of genes per gene ontology in gi (sp vs. gr). c Number of genes per gene ontology in 35S:CO (sp vs. gr). d Number of genes per gene ontology in FG (sp vs. gr). The black and white bars indicate the GO terms of the up- and down-regulated genes, respectively. Gene Ontology (GO) enrichment analyses (term type: biological process, FDR P-value < 0.001) were performed using the Goatools (https://github.com/tanghaibao/GOatools). The GO terms with low P values are listed first (from left to right). In cases of identical P values, the term with high enrichment (Ratio in study/Ratio in pop) was ranked first. Red triangles indicate flavone metabolic processes.
The responses of flowering pathway genes to microgravity
Our previous studies demonstrated that the spaceflight environment notably altered the expression of FT in Arabidopsis9,13 and suggested that spaceflight microgravity signals could converge on FT expression. To gain a comprehensive understanding of the altered expression profiles of flowering time regulator genes in the whole genome transcriptional response to microgravity, DEGs that were involved in different flowering control pathways (http://www.flor-id.org) were identified36. The numbers of DEGs involved in the flowering time pathway identified in the WT-, gi-, 35S:CO-, and FG-comparisons (sp vs. gr) were 127, 115, 109, and 115, respectively (Fig. 6a and Supplementary Table S5). We categorized these transcripts based on their established or predicted functions in the flowering regulation pathway36. The DEGs in the four comparisons were all functionally categorized to be related to different flowering regulation pathways, including photoperiodic response (accounting for 29–31% of all DEGs in the flowering pathway). Subsequent classifications highlighted genes functioning in general (15–21%), hormone regulation (11–17%), circadian clock (7–11%), vernalization (5–7%), aging (7–9%) and flower development (7–9%), sugar metabolism (3–5%), and ambient temperature response (3–4%) (Fig. 6a). These findings indicate that spaceflight microgravity response signaling pathways concurrently integrate into various flowering regulation pathways. Among them, the photoperiod pathway was most strongly affected by spaceflight microgravity, followed by the general, hormone, and circadian clock pathways (Fig. 6b). To further contextualize the functions of these key flowering regulators in response to microgravity, we generated STRING-DB associated microgravity regulatory networks (minimum edge score≥0.5) among different flowering pathways (Fig. 7a–d).
a The pie charts represent the percentages of DEGs (including up- and down-regulated DEGs) involved in flowering time pathways (FLOR-ID database, http://www.flor-id.org). b Clustering of the flowering signaling pathway-specific behavior for microgravity responses, in which up- and down-regulated expression levels under spaceflight conditions were compared to those of their controls on ground. Values represent the log2 fold change (FC) ratios of altered expression levels of DEGs in WT, gi, 35S:CO and FG in space compared with those on ground, respectively. Changes in transcript levels are colored as indicated in the legend. Red, upregulation (log2FC ≥ 1); blue, downregulation (log2FC ≤ –1). No significant change (–1 < log2FC < 1) is absent (in white). All log2 FC values are shown in Supplementary Table S5. “+” indicating positive effect on flowering time; “-” indicating negative effect on flowering time (FLOR-ID database, http://www.flor-id.org).
a–d Protein interaction networks were generated using STRINR-DB (https://cn.string-db.org/, version12.0, minimum edge score≥0.5). Changes in the transcript abundances of core flowering control genes of WT, gi, 35S:CO and FG plants grown in space (sp) in comparison with their controls on ground (gr) as shown in Fig. 6 are indicated by color. Red, upregulation; Blue, downregulation. White, no significant change. e Schematic diagram illustrating the regulatory networks of different flowering controlling pathways involved in the spaceflight response of the WT plants. Red characters, upregulation. Blue characters, downregulation. Note, differential responses of gi, 35S:CO and FG plants in comparison with that of WT are indicated with symbols of triangles in green (gi), blue (35S:CO) and red (FG) color, respectively.
With respect to the photoperiod pathway, 44 key genes exhibited changes in expression levels (Fig. 6b). Among them, the expression levels of four genes (CIB2, APL, FBH3 and FKF1), whose expression levels have positive effects on flowering time36, and nine genes (ATC, MYR2, CDF2/3/4/5, PHYD, AGL15 and SPA3), whose expression levels have negative effects on flowering time, were upregulated by more than fivefold in response to spaceflight microgravity in WT-, gi-, 35S:CO- and/or FG-comparison. The photoperiod regulator genes that downregulated by more than fivefold include five positive effect genes (AG17, NF-YB3, NF-YC4, CRY1 and COL5) and one negative effect gene (AT1G19330) (Fig. 6b and Supplementary Fig. S6). Notably, among the positive effect genes, the expression levels of cryptochrome-interacting basic-helix-loop-helix 1 (CIB1) in the 35S:CO- and the FG-comparisons apparently increased (~278- and 22-fold, respectively), whereas the expression levels in the gi- and WT-comparisons significantly decreased (~100- and 16.6-fold, respectively) (Fig. 6b). In contrast, the expression levels of CIB2 were upregulated by spaceflight microgravity in the four comparisons, but the increases in expression levels in the 35S:CO- and FG-comparisons were significantly lower than those in the WT- and gi-comparisons. CIB1 and CIB2 were reported to act redundantly to activate the transcription of FT37. The overexpression of FT in 35S:CO and FG plants caused significant increases in CIB1 and CIB2 expression levels in response to spaceflight microgravity (Fig. 6b), indicating that feedback regulation could occur between FT and CIB1/2. In addition, among the DEGs, the expression of ATC, which directly interact with FT (Fig. 7), showed most significantly upregulated in the four comparisons (Fig. 6b). NF-Y transcription factor genes (NF-YA4, -YC3 and YC4) exhibited altered expression in response to spaceflight in all four comparisons. In particularly, the expression level of NF-YC4 decreased in all four comparisons, whereas most significantly increased (about 1812-fold) in the gi-comparison in comparison with in the WT-, 35S:CO- and FG-comparison (about 33-, 12.5- and 8.3-fold, respectively) (Fig. 6b and Supplementary Fig. S6).
Gibberellin (GA) has been shown to be a major phytohormone involved in floral transition in Arabidopsis. Microgravity caused consistent alterations in the transcription levels of GA “activating enzymes”, GA20ox2/3 and GA3ox2, and “deactivating enzymes” GA2ox3/4, GID-1B and DELLA proteins (RGA1, RGL 2/3) in the four comparisons (Figs. 6b and 7e; Supplementary Fig. S6). In the FG-comparison, the transcription levels of both GA20ox1/2/3 and GA3ox1/2 were notably upregulated (Fig. 6b and Supplementary Fig. S6). The expression levels of ATH1, a GA20ox1 negative regulator, were downregulated by microgravity in the WT- and gi- comparisons relative to those in the 35S:CO- and FG-comparisons. In contrast, the expression of FPF1, a GA response regulator, increased in all four genotypes and was greatest in the gi in response to microgravity (Figs. 6b and 7e).
Our previous space experiments suggested that the circadian oscillator might integrate microgravity with photoperiodic signals in Arabidopsis grown in space13. In this study, the expression levels of the clock transcriptional repressors CCA1, LHY and REV2 were consistently upregulated, whereas the expression levels of the transcriptional activators PRR3 and PRR7 were downregulated in all four comparisons (Figs. 6b and 7e and Supplementary Fig. S6). The expression of TOC1 was significantly downregulated (above twofold) exclusively in the WT-comparison. PRR5 was significantly upregulated in the gi and the FG-comparisons but did not change in the 35S:CO-comparison compared with the WT-comparison. In addition, LWD1/2 expression was consistently downregulated in the 35S:CO- and the FG-comparisons, but differed between the gi- and WT-comparisons (Figs. 6b and 7).
With respect to the vernalization pathway, the expression levels of genes such as WRKY34, VIN3 and AGL19 were significantly upregulated in all four comparisons, whereas degrees of upregulation WRKY34 and VIN3 in the gi-comparison were clearly greater than those in the other three comparisons (Figs. 6b and 7). The ability of CCA1 and LHY to induce VIN3 transcription has been reported38, which is consistent with our findings that both CCA1/LHY and VIN3 are upregulated in response to spaceflight (Figs. 6b and 7). Mutation of the GI gene caused photoperiod-insensitive flowering and reduced CCA1 and LHY transcription levels28. Our results revealed that the expression level of VIN3 in the gi mutant increased the most, whereas the expression of AGL19, an upstream activator of FT and a target gene of VIN3 in the FLC-independent39, increased the least among the four genotypes in response to microgravity (Supplementary Fig. S6 and Supplementary Table S5), indicating the influence of the gi mutant on the response to microgravity in the vernalization pathway.
In the aging pathway, spaceflight caused notable changes in the expression levels of nine genes across the four comparisons. Among these genes, the expression levels of miR156 a/e, TOE3 and SMZ were upregulated, whereas the expression levels of SPL3 and SPL15 were downregulated in the four comparisons (Fig. 6b). The altered expression levels of SPL4 and SPL5 in response to microgravity in the WT-comparison differed from those in the gi-, the 35S:CO- or the FG-comparison (Fig. 7). For example, among the four genotypes, the greatest decrease of SNZ expression occurred in the gi-comparison, whereas the greatest decrease of SPL15 expression occurred in the 35S:CO. In addition, the expression of SPL4 was downregulated less in the gi-comparison and not changed in the 35S:CO- and the FG-comparisons compared with that in WT-comparison (Fig. 6b and Supplementary Fig. S6). These results indicated important roles for miR156/SNZ/SMZ/SPLs in the microgravity response networks.
In summary, the results indicate that, like other environmental stresses, microgravity, as a stress environmental factor in space, affects flowering time9,13 and has a very complex regulatory network in both GI-CO-FT -independent and GI-CO-FT dependent ways (Fig. 7). The mechanism that configures the transcriptome of plants under microgravity should be further elucidated.
Organization of cis-acting regulatory elements in microgravity responsive promoters
To elucidate the regulatory mechanism of microgravity on plant flowering pathway control in space, the major potential cis-regulatory elements (CREs) for the putative promoter regions of the microgravity responsive DEGs in the four genotypes (Fig. 4) were predicted using Plant Regulomics (bioinfo.cemps.ac.cn/plant-regulomics/, P adjust < 0.001)40, which provides a direction for coregulation among sets of genes. Analysis of the overrepresented motifs in the 1-kb sequence (–1000 to +100) upstream of the transcription start sites of DEGs in the WT-, gi-, 35S:CO- and FG-comparisons, respectively, revealed a total of 214 potential CREs corresponding to 22 transcription factor (TF) families (named “microgravity responsive TFs”, Fig. 8a and Supplementary Table S6 a–d), among which we defined a TF by its inclusion in the Plant Transcription Factor Database (PlnTFDB)41.
a Transcript factor family were detected by plant regulomics (bioinfo.cemps.ac.cn/plant-n, FDR P-value < 0.001). b Venn diagrams summarizing the number of differentially expressed transcription factor (TF) genes (enrichment score>150 and FDR P-value < 0.01) in response to spaceflight among WT, gi, 35S:CO and FG. c Log2 fold changes (FC) of potential overrepresented TF gene expression in WT, gi, 35S:CO and FG in response to spaceflight microgravity. TFs are grouped as described in Table 1. G1, group 1; G2,1, group 2.1; G2.2, group 2.2; G2.3, group 2.3. Changes in transcript levels of the TFs are colored as indicated in the legend.
Among the microgravity responsive DEGs identified above (Fig. 4), clusters of coregulated genes were closely related to known motif elements linked to stress-responsive and developmental TFs, such as ethylene responsive factor (ERF), basic (region) leucine zipper (bZIP), bHLH, MYB, squamosa promoter binding protein (SBP), and WRKY (Fig. 8a and Supplementary Table S6). To distinguish between motifs found by chance and those that were meaningful, we selected only highly enriched motifs. A total of 57 TFs were identified with potential significance (enrichment score ≥150 and Padjust < 0.001) for the putative CREs among the DEGs in the four genotypes (Fig. 8b, c and Supplementary Table S6). Based on the enrichment score of the CREs in the putative promotes of DEGs among the four genotypes, the potential TFs can be divided into two groups (Table 1 and Fig. 8b, c).
Group 1 (G1) encompasses 26 identified TFs, all of which are members of only the ERF family, exhibiting high enrichment scores for the associated GCC-box like elements (Table 1 and Fig. 9a), a conserved motif presents in the promoter regions of ethylene-inducible genes42,43. This group represents a universal microgravity responsive regulatory network across the four genotypes independent of the GI-CO-FT module. The GCC-box was reported to be well conserved in ethylene-inducible genes44. In this study, the GCC-box was exclusively overrepresented in the target genes of TFs in G1, suggesting that the GCC-box might be particularly important in mediating ethylene signaling induced microgravity-responsive transcriptional regulation during the Arabidopsis flowering, similar to the findings from responses during the seedling and embryo developmental stages26,45,46.
a–c Standard stacked bar charts show log2 fold changes in the relative transcript abundances of transcription factors (TFs) in WT, gi, 35S:CO and FG plants in space (sp) in comparison with the ground controls (gr). The 57 TF genes associated with potentially highly enriched (enrichment score>150 and FDR < 0.001) cis-elements (GCC and CACGTG) in WT, gi, 35S:CO and FG, respectively, are listed in Table 1. G1, Group 1; G2,1, group 2.1; G2.2, group2.2; G2.3, group 2.3. d GO (Gene Ontology) terms overrepresented in the target genes of the GCC - and the CACGTG-motif-bound TFs in (a–c). The significant GO terms (PANTHER statistical overrepresentation test, GO-Slim biological process, FDR P-value < 0.01) are shown as colored bars.
Group 2 (G2) contained TFs from the BES1, bHLH, bZIP, ERF, and SBP families and presented high enrichment scores (enrichment score ≥150) in the gi, 35S:CO and/or FG but was not overrepresent (enrichment score <150) in the WT (Table 1). Both the GCC- and the CACGTG-box motifs were detected at high levels in the promoter regions of microgravity responsive genes in G2 (Table 1 and Fig. 9b, c) indicating the important roles of these motifs in the GI-CO-FT pathway-specific microgravity response. The G2 TFs can be further divided into three subgroups. Subgroup 2.1 (G2.1) includes highly enriched TFs that overlap in the gi, 35S:CO and FG. The expression levels of four CACGTG-elements associated with bZIP genes, ABF1, ABI5, AREB3 and bZIP28 significantly changed in response to microgravity in the gi, 35S:CO and FG (Fig. 9c). In addition, the GCC-elements associated with seven ERF TFs (e.g., CEJ1, CRF7, DREB26/2F, ERF015/027 and RAP2.1), which have been reported to function in the regulation of ethylene and jasmonate signaling during plant development and defense response47,48, were also highly overrepresented in G2.1 (Table 1 and Fig. 9b), suggesting potential roles for ethylene, ABA and jasmonate signaling in the GI-CO-FT -dependent microgravity response at the flowering stage.
In subgroup 2.2 (G2.2), the GCC-elements associated with two ERF TFs (e.g., RAP2.10 and ERF4), which have been reported to act as transcriptional repressors capable of modulating ethylene and ABA responses49, were highly enriched exclusively in the gi (Table 1), suggesting the potential roles of GI in regulating ethylene and ABA signaling under spaceflight condition.
Analysis of the highly enriched TFs exclusively in 35S:CO and FG in subgroup 2.3 (G2.3) revealed seven TFs associated with the CACGTG-elements and eleven TFs associated with the GCC-elements (Table 1 and Fig. 9b, c). TFs belonging to bZIP (e.g., ABF2 and bZIP68), BES1(e.g., BZR1 and BZR2) and bHLH (e.g., PIF1, PIF7 and bHLH31), respectively, which were reported to link ABA, brassinosteroid (BR), GA and light signaling in the regulation of flowering time and stress acclimation50,51,52,53, were significantly related to the CACGTG-elements exclusively in 35S:CO and FG (Table 1 and Supplementary Table S6). In addition, the GCC-elements associated with the ERF (e.g., ERF109) and SBP family TFs (e.g., SPL11) were also highly enriched in G2.3 (Table 1, Fig. 9b and Supplementary Table S7).
To contextualize the functions of these highly enriched TFs (Table 1), their target genes among microgravity responsive DEGs with the GCC- and CACGTG-boxes in the promoter regions were identified (Supplementary Table S6 and Supplementary Fig. S7). Biological process enrichment of these target genes was performed by GO analysis. The GO terms with high significance levels converged into the phase transition from vegetative to reproductive pathways (Fig. 9d). In addition, “water transport”, “response to light stimulus” and “photosynthesis” the terms “water transport”, “response to light stimulus” and “photosynthesis” were overrepresented only among the DEGs with GCC-boxes (Fig. 9d). Interestingly, 62 of the 3853 DEGs identified in response to microgravity (Supplementary Table S6e) for biological process enrichment were involved in the phase transition from vegetative to reproductive pathways (Fig. 9d). For example, we found that the GCC-box with core motifs (5′-CGCCGCC-3′ or 5′-CGTACGG-3′) recognized by TFs ERF109 or SPL11 in G2.3 were uniquely associated with circadian and photoperiodic flowering genes (LWD1, CDF1, CIB2, ATX1 and CLF). In contrast, nine genes (LWD2, PHYD, CRY1, GID1B, J3, PIL6, WDR5a, AGL16 and PUB12) showed at least one CACGTG-box core motif (5′-CACGTG-3′), which is potentially recognized by eleven CACGTG-box transcription factors in G2.1 (ABF1, ABI5, AREB3 and bZIP28) and G2.3 (ABF2, PIF1, PIF7, bZIP68, BZR1, BZR2 and bHLH31) (Fig. 9b, c and Supplementary Table S6). The location of the GCC-box and the CACGTG-box core motif in the promoter of target genes is given in Supplementary Table S7. For example, CRY1, which showed less downregulation in the FG-comparison than other three comparisons (Fig. 6b), has the CACGTG-box on the promoter (–190 to –195, –11 to –16) (Supplementary Table S7). The differential expression profiles of eight CACGTG-box TFs (ABI5, BZR1/2, ABF2, bHLH31, AREB3, bZIP68 and PIF1) exhibited similar expression behaviors, whereas the expression levels of PIF7, bZIP28 and ABF1 tend to differ between early-flowering genotypes (35S:CO and FG) and later-flowering genotypes (WT and gi) in space (Fig. 8c).
Furthermore, Protein-Protein interaction (PPI) network, from which could infer the group of genes that are differentially expressed and co-expressed at the same time, was constructed for finding the hub genes in response to microgravity. The STRING PPI network (minimum edge score≥0.5) based on 62 DEGs, which are overrepresented in phase transition GO term (Fig. 9d and Supplementary Table S6e), and 58 highly enriched TFs in G1 and/or G2 (Table 1) are shown (Fig. 10). According to the enrichment of the CREs in the putative promoters of DEGs as described above (Table 1), the PPI partners include key interacting nodes like AP2/ERF TFs (e.g., ERF2, ERF104, ESE and RAP2.1) in G1 only, while COL5/CRF2 and LFY/PUCHL (in G1 and G2), GI/SPL11/SOC1 (in G2.3 only), WDR5A/bZIP28/ABF1/ABI5/SOC1/PIL6 (in G2.1 only), and GID1B/ BZR1/BZR2/PIF1/PIF7/PIL6 (in G2.3 only). For example, WDR5a, which showed increased expression exclusively in the FG (Fig. 6b), has the complete the CACGTG-box on the promoter (–97 to –75) (Supplementary Table S7), connected with a TF bZIP28 (Fig. 10). In addition, PIL6 and GID1B, which function as negative regulator of PHYB and GA receptor, respectively, connected other flowering proteins (e.g., SOC1 and GI) as well as TFs associated with the CACGTG-box in G2.3 only (Fig. 10). Together, comprehensive analysis suggests that AP2 could act as hub genes in common microgravity response pathway (G1), while a component of histone WDR5a, photoreceptors PHYD, CRY1 and PIL6, GA receptor GID1B and flowering integrator SOC1 could be hub genes in GI-CO-FT specific microgravity response pathways (G2.1 and G2.3). In addition, COL5 and LFY could be act as hub genes in connection both common (G1) and GI-CO-FT disruption related microgravity response (G2).
Potential links of overrepresented transcriptional modules with flowering phase transition signals are shown for flowering time regulatory pathway. G1: Group 1, G2.1: group 2.1; G2.3: group 2.3. Flowering genes directly regulated by the transcription factors (TFs), which associated with GCC- and/or CACGTG-box are shown in protein interaction networks, which were generated using STRINR-DB (https://cn.string-db.org/, Version12.0, minimum edge score≥0.5).
Discussion
The significant interruption of flowering time by microgravity is the major barrier to maximizing the efficiency of plant growth and reproduction in the construction of a BLSS for long time stay of human in space1. These issues pose two important challenges. The first is the need to obtain additional information on the link between flowering time and microgravity response and adaptation to establish the single or multiple pathways involved. The second is to search for countermeasures to the disturbance of flowering time and the low efficiency of seed production in space.
With respect to the first challenge, our previous space experiments revealed that FT could serve as a hub, integrating spaceflight microgravity stress signals into the photoperiodic flowering pathway in space-grown Arabidopsis9,13. However, the precise mechanism by which FT regulates the response to spaceflight microgravity remains elusive. In our current investigation, we conducted transcriptional analyses to compare spaceflight microgravity responses among various Arabidopsis flowering time genotypes with different FT expression levels, namely gi mutant (late flowering), 35S:CO (constitutionally early flowering), and FG (stress inducible early flowering), against the WT (Figs. 2 and 3, and Supplementary Figs. S1, and S3). Our data revealed that the transcriptional microgravity response signature was not only conserved across these flowering time disruption genotypes and the WT (Fig. 4b), but also displayed GI-CO-FT module-specific responses (Figs. 4c, d, 6 and 7). The GCC-elements were highly enriched in both common and GI-CO-FT specific microgravity responsive genes, whereas the CACGTG-elements were highly conserved across GI-CO-FT specific microgravity responsive genes (Fig. 9 and Table 1). TFs belonging to the ERF, bZIP, bHLH, and BES1 families were significantly overrepresented among the GCC- and CACGTG-elements, respectively, across GI-CO-FT specific microgravity responsive genes, suggesting important roles for ABA, BR, GA and light signaling in coordinating microgravity with flowering time.
Our previous studies elucidated the regulatory networks involved in the response to microgravity and contributed to the identification of the molecular basis for the integrating microgravity signals into the photoperiod flowering pathway in Arabidopsis under spaceflight condition9,13. In this study, GO group analyses highlighted responses common across WT, gi, 35S:CO, and FG, involving metabolic processes (e.g., phenylpropanoids, flavonoids, glutathione) tied to detoxification (e.g., H2O2) and phosphate starvation in upregulated groups, with photosynthesis being dominant in downregulated groups.
Notably, the most overrepresented GO terms for the CO/FT overexpression lines (35S:CO and FG) were apparently upregulated expression levels of genes involved in flavonoids biosynthesis and metabolic processes, revealing a pronounced enrichment in genes related to the upregulation of cell wall and vacuole function (Fig. 5 and Supplementary Table S4). Our transcription analysis also revealed clearly increased expression levels of key genes involved in flavonoid metabolism pathways in CO/FT overexpressing plants under spaceflight conditions (Supplementary Fig. S6). In particular, compared with the late flowering genotypes (gi and WT), the overexpression of FT in the earlier-flowering genotypes (35S:CO and FG) resulted in the upregulation of several flavonoid biosynthesis regulators, such as MYB114, TT10 and TT12. Blue light is suggested to stimulate the biosynthesis of compounds from the phenylpropanoid pathway such as flavonoids and anthocyanins54. Compared with those in the WT and gi plants, the expression levels of the photoperiod flowering regulator CIB1 in the 35S:CO and FG increased in response to microgravity (Fig. 6b). CIB1 was reported to physically interact with the blue light receptors CRY1 and CRY255. An increase in CIB1 expression indicates that FT overexpressing plants became more sensitive to blue light in space and resulted in the stimulation of flavonoid biosynthesis. Blue light-emitting diodes (LEDs) are commonly used as the main components of light sources in space plant culture experiments15,17,56 as well as in this study. Thus, the synergistic effects of microgravity and blue light on the flowering and development of plants growing in space should be considered.
In this study, altered expression levels of GI, CO and FT in 35S:CO and FG lines caused apparent changes in the expression of flowering regulatory genes and plant development under microgravity (Figs. 2 and 6), suggesting that GI, CO and FT play important roles in the responses of plants to microgravity. However, no difference in transcript levels was detected for GI, CO and FT in WT plants under microgravity in comparison with those on ground in this study (Figs. 3c and 6). This is different from our previous space experiments, which found upregulation of FT and downregulation of SOC1 transcript levels in Arabidopsis WT plants at flowering transition stage under spaceflight conditions9,13. SOC1 was also downregulated in the WT-comparison in this study (Fig. 6). The reason for the difference in FT expression in this study compared with that in our previous studies could be that samples from previous studies were collected 2 h post landing and experienced hypergravity during landing, whereas the samples used in this study were collected in orbit and frozen at –80 °C before analysis, which excluded the influence of altered gravity during landing on gene expression and represented the real microgravity response. In addition, decrease of SOC1 levels in FT overexpression lines 35S:CO (fivefold) and FG- (3.7-fold)-comparison in this study was much less than that in WT-comparison (13.7-fold), indicating possibly role of FT and SOC1 coordinately in stabilizing plant response to microgravity during flowering to adapt to long-term spaceflight condition. Furthermore, expression pattern of pFT:GFP in the leaves of Arabidopsis in our pervious study showed that peak levels of FT for induction flowering in space plants was apparently higher than their control on the ground9, indicating that higher peak level of FT expression may be needed for remodeling microgravity-induced changes in flowering transition. Thus, we assume that similar expression levels of FT could cause different responses in WT plants in space from their controls on ground.
Transcriptome analysis using microarray or RNA seq technologies is a powerful tool that has been used in the discovery of many microgravity-inducible genes, which are available in public databases5,57,58,59. In these studies, transcriptional responses to abiotic stress, oxidative stress, cell wall reorganization and secondary metabolism remodeling are commonly reported in microgravity. However, the transcriptional regulation of the responses of plants flowering to microgravity is still unknown. Our previous studies revealed that potential common cis-regulatory elements (CREs) are present among the daylength-spaceflight responsive genes of Arabidopsis plants grown in space9,13. This study revealed 22 TF families, that interacted with CREs in the promoter regions of microgravity responsive genes in the WT, gi, 35S:CO and FG grown in space (Fig. 8 and Supplementary Table S6). Interestingly, the GCC-box motif was detected mainly in common spaceflight microgravity responsive genes (Group 1) across the four genotypes, whereas the CACGTG-box motif was overrepresented mainly in the GI-CO-FT specific spaceflight microgravity responsive genes (Group 2).
The GCC element is well conserved in ethylene-inducible genes44. This result is consistent with previous investigations that showed the concentration of ethylene in growth chambers increased markedly due to disrupted convective air movement, which hindered plant reproduction in space25. Technological advancements in growth chamber environments have addressed the ethylene issue, but the enclosed gaseous environment surrounding plant tissues in spaceflight microgravity could still stress the plants10,26. In our research setting, ethylene was eliminated from the growth chamber. The pronounced enrichment of GCC-box ERF family motifs, which are common across microgravity-responsive genes of the four genotypes examined, might also be associated with the enclosed gaseous microenvironment surrounding plant tissues under spaceflight. Ethylene response factors could play important roles in regulating the expression profiles of common microgravity-responsive genes, possibly via interactions with the GCC-box.
The CACGTG-box motif is conserved bound by TFs in bHLH, bZIP and BES1 families in plants60. bZIPs and bHLHs can act antagonistically, competing for binding to the same sites, such as the competition for binding to targets shared between PIF3 and HY561. In this study, bZIP (e.g., AREB3, ABF1, ABI5, bZIP28 and bZIP68), bHLH (e.g., PIF1 and PIF7) and BES1(e.g., BRZ2) transcription factors, which were especially higher enriched in 35S:CO and/or FG were detected to bind to promoters of nine key flowering regulator genes that contain one or several CACGTG-boxes (Table 1 and Fig. 10). Among these target genes, the expression levels of LWD2 and PIL6 (circadian-controlled protein), CRY1 and PHYD (photoreceptor), GID1B (GA receptor), WDR5a and J3 containing common CACGTG-motifs in the promoter regions were coregulated by key bZIP, bHLH and BES1 transcriptional regulators, suggesting that the GI-CO-FT module could integrate ABA, BR, light and microgravity signals into the flowering pathways. Notably, WDR5a, which is a subcomplex component for histone H3 lysine H4 (H3H4) methylation during vegetative and reproductive development62, was apparently upregulated by microgravity exclusively in FG (Fig. 6b), but whether overexpression of FT could affect H3H4 methylation to activate transcribed genes in response to microgravity in space remains to be studied.
Regarding the second challenge, an acceleration of flowering time in seed crops, such as rice and wheat, while preventing of bolting in vegetable plants, such as sugar beet, many Brassica species, potato, spinach, and lettuce, would significantly increase yield. Thus, control of flowering time by manipulation will be clearly an important biotechnological method to increase the production of BLSS. FT, as a major component of florigen, is highly conserved across flowering plants63. Transgenic plants with increased amounts of FT flowered earlier and plants with less FT flowered later. Thus, the overexpression of FT could facilitate the design of early-maturing crops for accelerating breeding to increase resource-use efficiency in BLSS.
However, FT gene overexpression also has adverse consequences for the suppression of vegetative growth, including reduced leaf number and size and stem thinning, ultimately affecting seed yield64. Our results indicate that the negative effects of FT overexpression on vegetative growth became even more serious in space. For example, compared with the ground controls, all the examined plants grown in space exhibited significantly smaller rosette leaf size, shorter inflorescence stem and less flower at the flowering stage. Among them, the FT overexpression lines (35S:CO and FG) in space were even smaller than the WT and gi mutant under the same conditions (Fig. 2). In addition, FT overexpression in plants grown in space could elevate changes in expression of some flowering key genes, which are potentially regulated by transcription factors associated the GCC- and/or CACGTG-box motifs (Fig. 10). Our knowledge of the subject of flowering in space begun to increase in recent years because of the increase in long-term space experiments from seed-to-seed9,13,15 and the basic outline of FT function on ground is becoming clear. However, there is still no way to utilize FT to accelerate breeding and avoid the weaknesses in vegetative growth in space.
Genetic manipulation of gene expression at key developmental stages using a gene switch may be a practical and feasible approach to design efficient production and resilient crop cultivation in BLSS in the future. The heat shock protein (HSP) gene promoters are well-suited for this purpose. The HSP promoter can be activated in space plants by the direct application of a localized thermal stimulus by remote control13. HSP promoters, such as HSP17.4, exhibit extremely low basal activity and high heat inducibility in the vegetative tissues of ground plants13,65. However, previous studies reported that HSP genes were highly induced in response to prolonged microgravity in space66,67. Our results showed that FT overexpression using the CO and FT genes driven by 35S and a heat-inducible (HSP17.4) promoter, respectively, exhibited substantial variations in response to microgravity. Compared with that on the ground, the HSP17.4 promoter in the FG plants was specifically activated by microgravity and caused early expression of FT (Fig. 3c). Thus, attempts to control gene activity in plants grown in space by heat shock could suffer from leakiness from effects caused by the induction of microgravity. Optimal HS-inducible promoters need to be designed to eliminate the influence of microgravity to generate transgenic plants that accumulate only transgenic products at the expected time in future space experiments.
GI-CO-FT specific microgravity responses regulated by specific TFs associated with GCC- and CACGTG-elements were observed. CREs in stress-responsive promoters, which function as terminal points of stress signals in cascades, have been considered as important molecular switches for the design of efficient promoters that enable the precise control of transgene activity under spaceflight conditions. In the future, optimal HS-inducible promoters for space plants could be developed, based on an understanding of how plants use special molecular switches to initiate reproduction and avoid their negative effects on vegetative growth in space. This is also of fundamental importance for the use of functional genes, such as the hub genes found in this study, as tools to design more tolerant and highly productive varieties for BLSS.
Methods
Plant materials and spaceflight experiments
A. thaliana wild-type (WT) ecotype Columbia (Col-0) and the mutant ft-10 have been previously described68. The seeds of the mutant gi (gi-2, CS3124, Col-0 background) obtained from the Arabidopsis Resource Center at Ohio State University (https://abrc.osu.edu) as described27. The 35S:CO plants (pro35S:CO-6FALG in a Col-0 background), which were kindly provided by Dr. Xiangyang Hu (Shanghai University, China) as described69. The expression level of CO in 35S:CO plants was confirmed (Supplementary Fig. S3). The transgenic plants pFT:GFP and pHSP:FT; pHSP:GFP (FG, Col-0 background) were developed by Xie et al. and Wang et al., respectively9,13. The seeds were prepared and planted in two culture chambers (CCs) as described by Wu et al.70 and Xie et al.9. In short, the seeds underwent a sterilization process, which began with 1-min of exposure to 75% (v/v) ethanol, followed by 20 min of soaking in a 2% (v/v) NaClO bleach solution containing 0.01% (v/v) Triton X-100 detergent. After five sterile water rinses, the seeds were attached to cheesecloth pieces (5 × 10 mm, ten seeds per piece) with sterile 0.1% (v/v) methyl cellulose solution. These seeds were then dried on a flow bench and stored for the spaceflight experiments.
The GBMCS was used for the spaceflight experiments aboard the CSS (Fig. 1a, b); it is equipped with subsystems for atmospheric, temperature, and hydration regulation and a lighting system (Fig. 1c). Additionally, it is fitted with four video cameras to facilitate image capture and to visually monitor the ongoing experiment. Arabidopsis plants were cultured in CC1 and CC2. Each CC measured 60 × 300 × 260 mm externally. The internal space measured 60 × 200 × 210 mm, and the root module was 60 × 200 × 50 mm (Fig. 1d). The cheesecloth seed carriers were set in holes on a panel of root modules filled with commercially available vermiculite (Fig. 1e). Prior to the launch of the Wentian experiment module of the CSS on 24 July 2022, the seeds in the CCs were stored in a dry state for ~45 days. The space experiment began on 29 July 2022, with the hydration of the seeds, controlled remotely from Earth. This date was designated as Day 0. At the start of the experiment, 280 ml of sterile MS medium (PhytoTech, Beijing XMJ Scientific Co. Ltd) was supplied via a pump system (Fig. 1c). The germinated plants were grown at 22 ± 1 °C, under light from white/red (2:1) LEDs with an intensity of 120 µmol m–2s–1 on the surface plane of the root module for an LD photoperiod (16 h light/8 h dark) within the GBMCS. Inside the CCs, the relative humidity was maintained between 88 and 98%. Readings for temperature, humidity, and light levels were taken every minute throughout the flight. The GBMCS environment was maintained by a slow air exchange system (20 mL min–1) that directed filtered air from an external system into the CCs. The system’s atmospheric pressure was stabilized at one atmosphere. The ethylene in growth chamber was removed following the methods in Zheng et al.56.
For the ground control experiments, a GBMCS facility mirroring the one used in space was established. The ground experiments began on 6 September 2022 in Shanghai. Ground-control plants were organized as described above for the space flight samples. The ground-control CCs were maintained in the mirror GBMCS facility for the duration of the shuttle mission. The ground mirror GBMCS provided an environmental profile that corresponded with the profile of the orbiter with regard to temperature, humidity, light and air, except for microgravity and possible other physical factors experienced during spaceflight, such as possible space radiation, vibration and noise from spacecraft. In addition, simulation experiments on Arabidopsis plants grown in the CCs in the mirror GBMCS on ground have been performed to simulate culture conditions (temperature, humidity, light and air) under spaceflight before took off21 and the influences of simulation microgravity on expression of flowering genes were examined by the 3-D clinostat rotation71.
For RNA-Seq analysis, plants grown in space were harvested at 25 DAS (Fig. 2 and Supplementary Fig. S1) by an astronaut. Each sample of the indicated phenotypes included all rosette leaves of six seedlings grown in two different culture chambers (i.e., three seedlings of the indicated phenotype from each chamber) that were randomly collected and placed into a designated sample vial. Three such samples for each phenotype were collected and stored in three different sample vials. After being harvested, the sample vials were immediately frozen in the Experiment Refrigerator (–80 °C) onboard the CSS. Prior to their return to Earth, these samples were transferred to a low-temperature freezer (–20 °C) aboard the Shenzhou-14 spacecraft on 4 December 2022. Once the frozen seedlings reached Earth, they were immediately transported on dry ice and promptly preserved using RNAater-ICE solution (Invitrogen TM RNAlater TM -ICE, Ambion, Life Technologies, CA, USA) for subsequent RNA-seq analyses. The ground control experiment procedures were set to match the data collected from the space experiment.
RNA extraction and sequencing
Total RNA was extracted using TRIzol® reagent according to the manufacturer’s instructions as described in our previous papers9,13. Briefly, total RNA was extracted individually from the leaves of WT, gi, 35S:CO and FG plants frozen in RNlater solution. The samples for each phenotype were stored in three different vials as we described above. Thus, samples in one vial comprised a biological replicate. Three biological replicates were used for RNA-seq. After DNA removal, the quantity and quality of RNA were determined using the Nanodrop 2000 (Thermo Scientific). RNA integrity was detected by agarose gel electrophoresis and the RNA integrity number was determined using Agilent 2100 instrument. Total RNA for single database construction≥1 µg; RNA concentration≥35 ng/µl; OD260/280 ≥ 1.8; OD260/230 ≥ 1.0. mRNA isolation was achieved through the use of Oligo(dT) beads. Once the fragmented mRNA (300 bp) was prepared, an RNA-seq library was constructed. Double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA) with random hexamer primers (Illumina). The synthesized cDNA was subjected to end-repair, phosphorylation and ‘A’ base addition according to Illumina’s library construction protocol. Sequencing was executed using the Illumina Nova seq 6000 system. The RNA-seq generated 165.75 Gb of clean data with an average quality score (Q30) exceeding 94% and an average yield of 6.03 Gb of clean data per sample (Supplementary Table S1). The quality of the raw data was checked to ensure the reliability of the high-throughput sequence data. Approximately 98% of the cleaned reads were mapped to the Arabidopsis TAIR10 genomes using HISAT2 software (available at http://ccb.jhu.edu/software/hisat2/index.shtml). Approximately 90% of these reads were uniquely mapped, counted, and subsequently used for gene-level differential expression analysis with the R software for statistical computations. The raw data were uploaded successfully to the NCBI Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra) with accession number SRP488015. For further analysis, only those features with read counts exceeding one per million in at least three samples were retained. This resulted in the expression of a total of 28,080 genes across all the conditions (Supplementary Table S1). The significantly differentially expressed transcripts between space- and ground-grown samples were identified by controlling the FDR at 1% and filtering for fold change (FC) values either below or exceeding 2. Gene Ontology (GO) enrichment analyses were conducted using the Goatools website72 (https://github.com/tanghaibao/GOatools/). Gene expression profile clustering was performed by the application of fuzzy c-means clustering73.
Real-time qRT-PCR
Total RNA was extracted from the leaves of both the space and ground samples as previously described. A total of 1.0 μg of DNase-treated total RNA was reverse transcribed using HiScript QRT SuperMix (Vazyme). The cDNA was diluted 1:1 prior to use. Each qPCR analysis was completed using 1.0-μL of cDNA, ChamQ SYBR Color qPCR Master Mix(2X) (Vazyme) and an Applied Biosystems 7300 (Thermo Fisher Scientific, America) real-time PCR instrument according to the manufacturer’s instructions. The genes, along with their respective qRT-PCR primers, are listed in Supplementary Table S3. The Arabidopsis APT1 and ACTIN7genes served as the loading controls for all real-time qRT-PCR experiments. Each biological replicate was represented by at least four technical replicates in the real-time qRT-PCR analyses.
Analysis of cis-regulatory elements
The base sequences (DNA) 1000 bp upstream of the transcription start sites (–1000 to +100) corresponding to microgravity responsive genes in the WT, gi, 35S:CO and FG Arabidopsis genomes were queried from Plant Requlomics (www/bioinfo.cemps.ac.cn/plant-regulomics/), respectively, and a search for overrepresented motifs was performed according to Ran et al.40. Overrepresented motifs (P value < 0.05) were selected and analyzed. Briefly, cis-regulatory element enrichment was used to determine whether the occurrence of a given motif in input genes was significantly greater than that in random genomic regions. The total target motif enrichment score in this study is the percentage occurrence of a given motif in the genomic region in upstream of the transcription start site (−1000 to +100) of DEGs compared with that in random genomic regions.
Data availability
The data presented in the study are deposited in the (https://www.ncbi.nlm.nih.gov/sra) repository, accession code (SRP488015).
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Acknowledgements
The authors are indebted to Prof. Tao Zhang’s group at the Shanghai Institute of Technique Physics for culture chamber construction, and the Technology and Engineering Center for Space Utilization for supporting the space experiment.This work was supported by the Space Application System of China Manned Space program (YYWT-0801-EXP-02, SM1-0057), the National Natural Science Foundation of China (32271277 and 32471283), the Strategic Pioneer of CAS (XDB37020104), the Young Scientist Fund of the National Natural Science Foundation of China(32301038), and the Science and Technology Commission of Shanghai Municipality (22JC1401300).
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H.Z. conceived and designed the project and wrote the manuscript. J.X. and J.Z. performed the data curation, formal analysis, and investigation. L.W., Y.J., Y.D., Z.Z., B.L., and M.H. carried out the space plant culture experiment and data curation and investigated gene expression. All the authors have read and agreed to the published version of the manuscript.
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Xie, J., Zhu, J., Wang, L. et al. Potential regulatory modules to integrate microgravity signals into flowering pathways in Arabidopsis thaliana grown in space. npj Microgravity 11, 87 (2025). https://doi.org/10.1038/s41526-025-00539-z
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DOI: https://doi.org/10.1038/s41526-025-00539-z
