Two Nucleoporin 98 homologous genes jointly participate in the regulation of starch degradation to enhance growth in Arabidopsis

Background: Starch is synthesized during the day for temporary storage in leaves and then degraded during the subsequent night to support plant growth and development. Impairment of starch degradation leads to stunted growth, even senescence and death. The nuclear pore complex is involved in many cellular processes, but its relationship with starch degradation is unclear until now. We previously identified two Nucleoporin98 ( Nup98 ) genes ( Nup98a and Nup98b ) redundantly regulated flowering through CONSTANS ( CO ) independent pathway. in Arabidopsis thaliana . The nup98a nup98b double mutant also showed severe senescence phenotypes. Results: We found that Nucleoporin 98 ( Nup98 ) participated in the regulation of sugar metabolism in leaves and in turn is involved in senescence regulation in Arabidopsis . We show that Nup98a and Nup98b redundantly function in the different steps of starch degradation, the nup98a nup98b double mutant accumulates more starch than wild type and has a severe early senescence phenotype compared to wild type. The expression of marker genes related to starch degradation was impaired in the nup98a nup98b double mutant, and indicator genes of carbon starvation and senescence expressed earlier in the nup98a nup98b double mutant than that in wild type plants, suggesting abnormality of energy metabolism was the cause of senescence of the nup98a nup98 b double mutant. Addition of sucrose to the growth medium can rescue early senescence phenotype of the nup98a nup98b mutant. Conclusions: Our results provided a line of evidence on a novel role of the nuclear pore complex in energy metabolism related to growth and development, whereas Nup98 functioned in starch degradation conferring growth regulation in Arabidopsis .

Nup98b redundantly function in the different steps of starch degradation, the nup98a nup98b double mutant accumulates more starch than wild type and has a severe early senescence phenotype compared to wild type. The expression of marker genes related to starch degradation was impaired in the nup98a nup98b double mutant, and indicator genes of carbon starvation and senescence expressed earlier in the nup98a nup98b double mutant than that in wild type plants, suggesting abnormality of energy metabolism was the cause of senescence of the nup98a nup98b double mutant. Addition of sucrose to the growth medium can rescue early senescence phenotype of the nup98a nup98b mutant.
Conclusions: Our results provided a line of evidence on a novel role of the nuclear pore complex in energy metabolism related to growth and development, whereas Nup98 functioned in starch degradation conferring growth regulation in Arabidopsis .

Background
The nuclear pore complex (NPC) is the key bridge for communication of macromolecules between the nucleus and cytoplasm and regulates gene expression [1]. NPC is built by at least 30 unique proteins (nucleoporin, Nup) which are highly conserved in eukaryotic cells [2,3]. Nup98 is a mobile and peripheral FG (for Phe-Gly domain) nucleoporin and spans from the nucleus to cytoplasmic of the central channel of the NPC [4,5]. Arabidopsis thaliana Nup98a (also known as DRA2) is also found in different subcellular locations [6]. Nup98 is involved in regulation of cargo export and import, gene expression, transcriptional memory, and multiple developmental processes in animals and yeast [4][5][6][7][8][9]. In Arabidopsis, DRA2 regulates the shade avoidance syndrome [6]. In rice, the Nup98 homolog APIP12 is involved in basal resistance against the pathogen Magnaporthe oryzae and targeted by the Magnaporthe effector AvrPiz-t [10]. Recently in Arabidopsis, the interaction between Nup98 and Nup88/MOS7 is required for plant immunity against the necrotrophic fungal pathogen Botrytis cinerea and mitogen-activated protein kinase signalling [11]. We found Nup98 contributes to flowering regulation in Arabidopsis [12].
Senescence is an important cellular progress sensing developmental and environmental cues [13].
Before death, plants remobilize resources in senescing tissues and translocate them to sink tissues to support growth and development [14][15][16]. To date, at least 200 genes have been identified that regulate or participate in senescence in plants [17]. Starch is synthesized during the day in leaves and then degraded during the subsequent night to support plant growth and development. Impairment of genes functioned in different steps of starch degradation hinders plant growth at different extents [18,19]. Sugar directly or indirectly regulates senescence: sugar accumulation not only triggers and accelerates, but also delayed senescence [20][21][22]. In fact, the response to sugar and senescence of plants is in an age-or condition-dependent manner [21,23,24]. Within the sugar pathway, three control nodes have been identified; glucose sensor gene HEXOKINASE 1 (HXK1), energy sensor genes PROTEIN KINASE (KIN10 and KIN11), and TOR (The target of rapamycin) kinase [22,[25][26][27][28].
Currently, the mechanism of NPC in regulation of starch degradation and senescence in plants is still unknown. However, in animals there are several studies reporting the NPC controls cell senescence through modifying chromosome structure, DNA repair and replication or cell division [29][30][31][32][33]. Impaired NPC results in dysfunction of nucleo-cytoplasm transportation [31]. Both Nup107 [34] and Tpr [35,36] have been linked to cancer cell proliferation and cellular senescence in aging cell, and another Nup98-interacting protein Nup93 was shown to be oxidatively damaged [29].
In this study, we focus on Nup98 in Arabidopsis thaliana. In the Arabidopsis genome, there are two Nup98 homologs, Nup98a and Nup98b, respectively. Single mutants of Arabidopsis, nup98a or nup98b, have no obvious phenotypes. However, the double mutant, nup98a nup98b, showed a significant early-senescence phenotype. Gene expression analysis demonstrated that Nup98a and Nup98b participates in starch degradation conferring to growth regulation. Further analysis indicated the early-senescence may result from a defect in the initial steps of starch degradation resulting in dysfunction in energy supply. Interestingly, the early senescence phenotype in the double mutant could be rescued by sugar in the growth media. Our data suggests that Nup98a and Nup98b function redundantly in regulation of starch degradation, which contribute to normal growth and development in Arabidopsis.

Results
Nup98 mutation results in early senescence in plants Nup98 is a highly conserved nuclear pore protein in eukaryotes. In Arabidopsis thaliana, there are two homologs of the mammalian Nucleoporin 98, Nup98a (At1g10390) and Nup98b (At1g59660), and both share high amino acid sequence similarity in the Phe-Gly (FG)-repeats and autoproteolytic domain (APD, Supplementary Fig. 1) [3,6]. Nup98a was previously reported as DRACULA2 (DRA2), a regulator of the shade avoidance syndrome (SAS) in Arabidopsis [6] and immune responses to a rice fungal pathogen [11]. To investigate Nup98 functions in plant development, we screened mutants of nup98a (SALK_080083, SALK_090744, SALK_023493, SALK_103803, SALK_015016) and nup98b (CS803848 and GABI_288A08) ordered from ABRC and GABI T-DNA mutant center, respectively.
Homozygous lines were isolated for the following insertional mutants of SALK_103803, SALK_015016, GABI_288A08, and among them SALK_103803 and GABI_288A08 were the mutants reported by Parry [9]. The T-DNAs in these homozygous mutants are inserted in the coding regions (Fig. 1A) and RT-PCR results demonstrated that these mutants were null alleles (Fig. 1B) consistent with the Parry's results [9]. We did not observe any obvious phenotypes in either the nup98a or nup98b single mutants when compared to wild type (WT) under long-day photoperiod conditions (Fig. 1C), as previous studies showed [9]. As Nup98a and Nup98b share high amino acid sequence identity ( Supplementary Fig. 1), we tested the hypothesis that Nup98a and Nup98b acted redundantly with the nup98a nup98b double mutants we made by crossing nup98a to nup98b. Strikingly, both double mutants, nup98a1 nup98b1 and nup98a2 nup98b1, displayed similar early senescence phenotypes when compared to WT ( Fig. 1C and 1D). Also, the double mutant plants had additional phenotypes, such as smaller inflorescences, flowers, siliques and short stature and severe sterility when compared to WT (Supplementary Fig. 2 and 3). We recently reported that the nup98a nup98b double mutants had early flowering phenotype .
As expected, the mutant phenotypes observed in the double mutant plants were rescued by expressing only Nup98b (Fig. 1E). Our results demonstrate that Nup98a and Nup98b act redundantly.
To investigate if senescence phenotype was specific to the nup98a nup98b mutant, we selected another three nucleoporin mutants, nup96-1, nup160-1, and nup107-1, which showed flowering phenotypes in our previous report [37], to analyze the effect of other nuclear pore components on senescence. To our surprise, there is no early senescence phenotype observed in these mutants ( Supplementary Fig. 4), suggesting that some of nucleoporins may not be involved in the regulation of senescence and Nup98 had more or less specific functions on this developmental event.
Nup98 gene may be involved at multiple pathways of senescence initiation While the early senescence observed in the nup98a nup98b double mutant could be a secondary effect of altered development, we further explored the role of Nup98a and Nup98b in plant senescence. To date, at least 200 genes have been identified as regulating or participating in senescence in plants [17]. We investigated and summarized literatures in Supplementary Fig. 5 focusing the main genes, which showed that various endogenous and environmental cues, such as different hormones, sugar signalling, light and photoperiod conditions, and multiple stresses, trigger plant senescence and that many genes regulate senescence in multiple cross-talking pathways. To and NAC2 displayed higher transcript abundances at both ZT0 and ZT16 ( Fig.2A), suggesting that stress (WRKY53, WRKY6, and NAC1) and SA (WRKY53, WRKY6, and NAC2) pathways were related to the nup98a1 nup98b1 double mutant phenotype. In the second category, SAG12, NAP, SAG2 and CAT1, also stress and SA pathway genes, were only increased at either ZT0 or ZT16 (Fig. 2B). In contrast, in the third category, SAUR36, WRKY70, ARP4, SEN1, and COI1 were decreased in abundance at either ZT0 and/or ZT16 (Fig. 2C), indicating that auxin (SAUR36) and jasmonate (COI1) may be negatively related to the nup98 phenotypes. The abundance of AGL15, EBP1, RPS6a, and NPR1 had opposite changes at ZT0 and ZT16 (Fig. 2D), suggesting the function of these genes on Nup98 was dependent on circadian rhythm.
This expression profile suggested that there were at least three characteristics of senescence in the nup98 mutant. Firstly, several pathways, mainly stress and SA pathways, were involved in regulation of senescence in the nup98a nup98b double mutant. Secondly, different genes functioned in their own special modes, positively or negatively at different phases (morning or afternoon phases).
The results indicated that the mechanism of senescence regulation in the nup98 double mutant was much complicated. Many genes showed circadian expression pattern, consistent with our previous report that Nup98 participated in regulation circadian clock [12].
Starch metabolism is impaired in the nup98a nup98b double mutant During photosynthesis, glucose is synthesized and stored as starch, which is degraded at night.
Starch synthesis, degradation and metabolism pathways involve a number of genes in plants [18,19].
Firstly, we checked starch homeostasis in the nup98a1 nup98b1 double mutant plants that were grown under 12 h light/12 h dark conditions (at ZT0, dawn, and ZT12, dusk) (Fig. 3). All plants accumulated starch at dusk, however, double mutant plants accumulated much more starch when compared to wild type plants as determined by iodine staining (Fig. 3A) and starch quantitative assay (Fig. 3B). The more intense signals in older, 21 and 28-day, double mutant leaves may be a consequence of starch accumulating over time. Therefore, the double mutant displayed much more starch not only at dawn but also at dusk when compared to wild type plants and this was much clearer in 14 and 21-day old seedlings (Fig. 3A and 3B). Together, these results suggested that starch metabolism was impaired in the nup98a nup98b double mutant.
We measured the transcript abundance of genes involved in starch metabolism [18] by quantitative RT-PCR (Fig. 4) and found that many genes had significantly lower abundant transcripts at least one time point in the double mutant when compared to wild type. These genes encoded enzymes for the degradation of starch not only in the early steps of starch degradation in chloroplasts [18], such as GWD1 (SEX1), β-BAM1, BAM3, BAM5, BAM6, BAM7, BAM8, SEX4, and LSF1, but also in the later steps of starch degradation, such as limit-dextrinase (LDA), α-amylase (AMY1 and AMY2), disproportioning enzyme (DPE1 and DPE2), α-glucan phosphorylase (PHS1 and PHS2). Time-dependent low-expression of these genes suggested they were in the control of circadian clock, since Nup98 is involved in circadian regulation in Arabidopsis [12]. These genes function at different steps [18]. GWD1 is αglucan water, dikinase (also called SEX1) and phosphorylates glucosyl residues of amylopectin at the double mutant (Fig. 3) as these enzyme mutants [18]. We also found that there were some genes, which did not show a significant change in mRNA abundance. These genes included GWD2, GWD3, ISA3 and AMY3, suggesting the effect of Nup98a/b on starch metabolism was more or less specific.
We also measured the abundance of genes related to photosynthesis and sugar metabolism by RT-qPCR in the nup98a1 nup98b1 double mutant and WT plants ( Supplementary Fig. 6). In terms of photosynthesis related genes, the decrease of mRNA abundance of LHCA and LHCB was observed in the nup98a1 nup98b1 double mutant at different time points, e.g., LHCA1/2 and LHCB1.1 at ZT0 and LHCA1 and LHCB1.4 at ZT16. We also observed the decrease of mRNA abundances of KIN10 and KIN11, two sugar signaling genes in the double mutant when compared to WT. Both genes delay plant senescence [22,27,28] and therefore the reduced abundance may be associated with earlier senescence (Supplementary Fig. 5). We also observed slightly-increased mRNA abundance of HXK1 at dusk (ZT16) and this may contribute to earlier senescence via the cytokinin signaling pathway [25].
Unexpectedly, mRNA abundance of TPS1, a senescence activator [21], was reduced in the nup98a nup98b double mutant compared to wild type, suggesting that T6P (trehalose-6-phosphate) was not related to senescence of the nup98a1 nup98b1 double mutant. The results indicated that starch synthesis and sugar signalling were impaired in the double mutant.
Exogenous sugar rescues the early senescence in the nup98a nup98b double mutant Based on our results above, we interpreted that the carbon or energy supply was impaired in nup98a nup98b double mutant plants. We tested the idea by supplying exogenous carbon in the form of sucrose in growing medium to see if the early senescence phenotype in the nup98a nup98b double mutant plants could be rescued. Our results showed that sucrose and MS nutrients can support the double mutant plants growing well even though they were weak compared to WT (Supplementary Fig.   7). Both plants can complete their life cycles on medium containing agarose supplement with sucrose and nutrients. Then, we allowed double mutant and control plants grown in MS medium until inflorescence emergence and then transferred them to soil. As expected, the double mutant grew well as WT did on MS medium (Fig. 5). However, after transferring to soil, senescence symptoms on mutant plants' leaves quickly appear at day 6, and the mutant plants wilted at day 30 (Fig. 5). If seeds were sown on medium containing only MS nutrients or sucrose, both WT and nup98 cannot survive as plants grow agarose medium without any supplements (Supplementary Fig. 7).
To rule out the potential effect of soil on senescence phenotypes observed above, we carried out another experiment to test if exogenous macro and micronutrients would complement the phenotypes observed in the mutant by continuously growing plants on medium at different strengths of sucrose and macro-and micro-nutrients ( Supplementary Fig. 8). To our surprise, not only did sucrose suppress the early senescence phenotype in the double mutant but also macro-and micronutrients in the presence of sucrose. The lower strength nutrients (½ MS) enhanced the lower sucrose effect on suppressing senescence, suggesting that both energy supply and nutrients metabolism were impaired in the nup98a nup98b double mutant. We also tested if sucrose could recue the nup98a nup98b double mutant phenotype in soil. However, such an experiment failed and both the nup98a nup98b double mutant and WT seedlings died, because sucrose enhanced pathogen growing ( Supplementary Fig. 9).
Autophagy is an important event occurring during sugar starvation and senescence [38,43,44], and AUTOPHAGY8a (ATG8a, At4g21980) and ATG8e (At2g45170) are two typical molecular indicators for autophagy in plants [45]. Therefore, we investigated expression changes of these genes in the nup98a nup98b double mutant compared to that in WT, and the results showed that they had different changes in a time-and developmental-dependent mode (Fig. 6). In the double mutant, DRM1 had significantly higher expression at ZT0, but lower at ZT12 from very early stage (day 5 after germination) (Fig. 6A). DIN is a light-repressed and dark-induced gene [46], and its high level of expression at ZT0 in the nup98a nup98b double mutant became obvious at day 15, but at ZT12 higher abundancy appeared earlier from day 10 (Fig. 6A). Compared to WT, the senescence marker WARKY53 in the nup98a nup98b double mutant expressed higher at the early stage when DRM1 expression was in disorder (day 5) (Fig. 6B). SAG12 is a developmental controlled indicator for the later stage of senescence [47,48]. We found that there was no much difference of SAG12 expression in the early stage (day 5) between the nup98a nup98b double mutant and wild type. However, SAG12 had a higher expression level in the nup98a nup98b double mutant at both ZT0 and ZT12 from day 10 ( Fig. 6B). In the meanwhile, the two markers of autophagy, ATG8a and ATG8e, also had higher abundancy of mRNA in most of samples of the double mutant from day 10. Token together, our results showed that the nup98a nup98b double mutant appeared the sign of energy starvation, at least at molecular level, in early developmental stage when plants did not display visible senescence phenotypes. And these expression changes may have circadian and developmental characters. A previous report shows that different sugars (such as sucrose, glucose, and fructose) have different effects on the regulation of senescence [39]. It may be a cue to study the function of Nup98 on senescence regulation.

Nup98 proteins mainly localize to the nuclear membrane and nucleoplasm
Nup98 is one of the mobile and peripheral FG (Phe-Gly domain) nucleoporins and is located at both the nuclear and cytoplasmic sides of the NPC central channel [4,5]. Arabidopsis Nup98a (also known as DRA2) is also found distributing in different subcellular compartments [6]. We constructed transgenic Arabidopsis plants expressing 35S::GFP:Nup98a and 35S::GFP:Nup98b and analyzed the subcellular localization of both translation fusion proteins. Not surprisingly, both proteins were distributed in the cytoplasm, the nucleoplasm and at the nuclear periphery (Fig. 7). We also observed no significant difference in the subcellular distribution of Nup98a and Nup98b and this is consistent with our observations of genetic redundancy. In conclusion, our combined results demonstrated that Nup98a and Nup98b proteins were localized at both the nucleus and cytoplasm similar to their homologs in other organisms.

Discussion
Senescence is a physiological process during the plant life cycle, eventually leading to cell and tissue disintegration and death in plants. In such a physiological process, various nutrients are redistributed from senescing organs, such as leaves, to reproductive organs, for example seeds [13]. However, irregular or premature senescence could lead to organ failure or even whole plant death [49]. Fine tuning senescence could benefit plants by avoiding the deleterious effects of abiotic stresses and thereby lead to an optimal reproductive outcome. A significant number of factors including hormones, developmental age, abiotic stress and light participate in regulation of plant senescence [22,23,[50][51][52][53][54][55][56][57][58][59]. While these factors play important and clear roles in plant senescence, the role of sugar is unclear as different groups have published opposing results [20][21][22]60]. The NPC, is an important gatekeeper for both macromolecular transportation between the nucleus and cytoplasm and gene transcription, and therefore plays an important role in different plant developmental processes [29][30][31][32][33]. Our study provides some additional insight into the plant senescence field as we found that the NPC participates in senescence regulation. Our investigation confirmed that Nup98 genes participated in starch degradation, then conferring to senescence initiation in Arabidopsis.
We identified two homologs of Nup98, Nup98a and Nup98b, in the Arabidopsis genome and both showed high protein sequence similarity and highly similar cellular protein-localization patterns. We observed no obvious phenotypes of senescence in the nup98 single mutants under our growth conditions even though previously nup98a mutant plants, also known as dra2, showed a shade avoid phenotype of longer hypocotyls [6]. However, in the nup98a nup98b double mutant, we observed senescence phenotypes in very early developmental stage and severely reduced seed production.
Molecular evidence showed that key marker genes of plant senescence, such as SAG12, NAP1, WRAY53, WRKY6, WRKY70, NAC1, NAC2 and HXK1, were detected having significant changes in expression, these genes were related to different senescence pathways, for example, ethylene, salicylic acid, ABA, cytokinin, and stress pathways. These genes may play a role in a temporal (circadian) manner as their significant changes only at a specific time point, for example only at dawn or dusk, in the double mutant when compared to wild type plants. Our results suggested that senescence of the nup98a nup98b double mutant was the consequence of impairment of multiple senescence pathways.
We also observed that genes in sugar signaling pathways (TOR, KIN10, KIN11, TPS1, SnRK1) and carbon starvation genes (DRM1 and DIN6) were all mis-regulated in nup98a nup98b double mutant plants. What's more, sucrose could rescue senescence phenotype of the nup98a nup98b double mutant, indicating that sugar availability was hindered in the double mutant.
In plants, sugar is derived from photosynthesis and stored as starch. Starch accumulates in the chloroplast during the day and is degraded at night [18,19]. The nup98a nup98b double mutant accumulated much higher levels of starch that was unlikely due to higher photosynthesis efficiency but impaired starch degradation. This was evident by 1) lower expression levels of genes related to photosynthesis, LHCA1, LHCA2, LHCB1.1 and LHCB1.4, which would lead to reduced starch synthesis in the double mutant; 2) lower expression of starch degradation genes embracing many steps of starch degradation [18], that would lead to starch accumulation in the nup98a nup98b double mutant.
A gradual accumulation of starch over time was also contributed by a partial reduction in degradation pathway gene expression. Impairment of starch degradation directly lead to sugar starvation, and subsequently stunted growth, senescence, finally death.
Beyond this, Nup98a and Nup98b may also have functions in other nutrient metabolism as the concentration of nutrients in the growth medium had a significantly impact on the growth and senescence in nup98a nup98b double mutants. In this case, components (sugar or MS) in growth medium should be taken into consideration, especially for senescence study in future.
Previous studies show that the circadian clock regulate starch metabolism in plants [61,62]. EARLY FLOWERING3 (ELF3) positively regulates starch accumulation and degradation of starch was significantly slower in elf3 mutant plants than in the corresponding wild types [63]. ELF3 and other clock evening genes (ELF4 and LUX ARRHYTHMO) also affect leaf senescence [64,65]. Many genes studied in this study showed time point-dependent changes. We recently found that the expression level of ELF3 and other clock genes significantly reduced in the nup98a nup98b double mutant [12].
Token together, clock genes may participate in signalling pathway of Nup98a/Nup98b regulating starch degradation and senescence in Arabidopsis.
We just reported that the nup98a1 nup98b1 ft-10 triple mutant displayed the late flowering character as the ft-10 mutant but maintained early senescence phenotypes of the nup98a1 nup8b1 double mutant [12], suggesting that Nup98a and Nup98b were involved in regulation of both flowering and senescence, two developmental processes individually regulated by Nup98a and Nup98b.
It is obvious that the function of Nup98 in senescence regulation are indirect. Mutation of Nup98 genes leads to starch degradation hindered firstly, then sugar starvation, and finally senescence. In the future, it will be interesting to determine how Nup98 controls the function of genes related to starch degradation.

Conclusion
Our findings identified a novel function of nuclear pore complex on starch metabolism and verified that Nup98a and Nup98b overlappingly controlled starch degradation and indirectly regulate senescence in Arabidopsis. Gene and promoter cloning, plasmid construction Standard GATEWAY (Invitrogen) methods were employed for cloning and plasmid construction. Most vectors are made by our lab [66]. The full-length of Nup98a and Nup98b open reading frames were PCR amplified with specific primers (Table S1), and then cloned into Fu30 [66]  Semi-quantitative PCR, quantitative real time RT-PCR, and subcellular localization The whole seedlings were harvested at ZT0 (the time point of light on) and ZT16 (the time point of light off) at day 14 after germination. RNA preparation, cDNA synthesis and both quantitative realtime and semi-quantitative RT-PCRs were carried out following Xiao et al. [68], except for the use of At4g34270 as a reference gene in triplicate [69,70]. All gene accession numbers and relevant primer sequences are listed in Table S1. GFP fluorescent signals were visualized by confocal microscopy, and propidium iodide (PI) is for cell wall staining [66]. Declarations Ethics approval and consent to participate Not applicable.

Consent to publish
All Authors read and approved the manuscript.

Plant specimens
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Availability of data and materials
All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).

Competing Interests
The authors declare no competing interests.     double mutant. The seeds of mutant and WT were sown on MS medium. After low temperature treatment for 3 days, plants were grown under long day conditions. Samples were harvested at ZT16, 20 and 24 during the dark phase. All RT-PCR measurements were repeated at least three times, in triplicate. All RT-PCR gene expression measurements were normalized to the control TIP41 (At4g34270) and expressed as a relative expression value.
Student's t test was used to statistically analyze the data. An * indicates measurements that were significantly (*P < 0.05) different from the control. Error bars indicate ± SD of the mean.   mutant. A, Sugar starvation genes. B, Senescence genes. C, Autophagy genes. The seeds of the mutant and WT were sown on MS medium. After low temperature treatment for 3 days, the seedlings were grown for 7 days under long day conditions, then seedlings were transplanted in soil. From that, samples were harvested in day 5, 10, and 15 at both ZT0 and ZT12. All qPCR measurements were repeated at least three times, normalized to the reference gene TIP41 (At4g34270) and expressed as a relative expression value. Student's t test was used to statistically analyze the data. Error bars indicate ± SD of the mean.

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