Down-regulation of the Genome Uncoupled 4 Retards Starch Biosynthesis in Rice

Background Starch is the major storage carbohydrate in rice, with essential physical functions for plant growth. The starch biosynthesis in rice employs the cooperation of nucleus and plastid, which requires regulation of the signals from nucleus to plastid. However, the plastid-to-nucleus retrograde signals for starch biosynthesis is partly mediated by tetrapyrrole intermediates, i.e., heme, but the underlying mechanism is largely unknown. In previous studies, we revealed that the Genome Uncoupled 4 (OsGUN4) mutation in rice have been revealed to greatly affect tetrapyrrole intermediates but retain a high photosynthetic capacity. Results Here, we further found that down-regulation of OsGUN4 promoted to accumulate sucrose but reduce the total starch, attributing to abnormal performance of metabolisms and enzyme activities of starch biosynthesis in leaves of gun4 epi . Besides, the exogenous sucrose led to induced starch synthesis but reduced sucrose contents in wild-type, while norurazon(NF) treatments could eliminate or weaken these inductions. Nevertheless, no changes were detectedbetween check and sucrose treatments in the gun4 epi ,whereas NF treatment enhanced the trends of increased sucrose but reduced starch,suggesting the roles of OsGUN4 on balance of photosynthesis and starch biosynthesis. Dynamic activity changes of starch biosynthetic enzymes were in accordance with the contents of carbon metabolites. Moreover, RNA sequencing revealed that a great deal DEGs were associated with starch metabolic pathways, with 62 genes being up-regulated and 25 down-regulated in gun4 epi . Many genes involved in starch biosynthesis performed down-regulated expression, including the transcription factor of bZIP58 and its target genes of OsBEIIb and OsSSI, which are vital for the formation of amylopectin and starch granules, while displayed up-regulatedexpression of OsSSIIIa and OsGBSSI that promotes the formation of amylose. OsGUN4 affected expression of many participating in starch and protein biosynthesis in


Abstract Background
Starch is the major storage carbohydrate in rice, with essential physical functions for plant growth. The starch biosynthesis in rice employs the cooperation of nucleus and plastid, which requires regulation of the signals from nucleus to plastid. However, the plastid-to-nucleus retrograde signals for starch biosynthesis is partly mediated by tetrapyrrole intermediates, i.e., heme, but the underlying mechanism is largely unknown. In previous studies, we revealed that the Genome Uncoupled 4 (OsGUN4) mutation in rice have been revealed to greatly affect tetrapyrrole intermediates but retain a high photosynthetic capacity.

Results
Here, we further found that down-regulation of OsGUN4 promoted to accumulate sucrose but reduce the total starch, attributing to abnormal performance of metabolisms and enzyme activities of starch biosynthesis in leaves of gun4 epi . Besides, the exogenous sucrose led to induced starch synthesis but reduced sucrose contents in wild-type, while nor urazon(NF) treatments could eliminate or weaken these inductions. Nevertheless, no changes were detectedbetween check and sucrose treatments in the gun4 epi ,whereas NF treatment enhanced the trends of increased sucrose but reduced starch,suggesting the roles of OsGUN4 on balance of photosynthesis and starch biosynthesis. Dynamic activity changes of starch biosynthetic enzymes were in accordance with the contents of carbon metabolites. Moreover, RNA sequencing revealed that a great deal DEGs were associated with starch metabolic pathways, with 62 genes being up-regulated and 25 down-regulated in gun4 epi . Many genes involved in starch biosynthesis performed down-regulated expression, including the transcription factor of bZIP58 and its target genes of OsBEIIb and OsSSI, which are vital for the formation of amylopectin and starch granules, while displayed up-regulatedexpression of OsSSIIIa and OsGBSSI that promotes the formation of amylose.

Conclusion
In conclusion, these ndings con rm that OsGUN4 play regulatory roles on biosynthetic genes and enzyme activity in starch biosynthesis.

Background
Plants assimilate atmospheric CO 2 during photosynthesis using light energy to produce sugars and chemical energy (ATP) for plant growth [1]. In leaves, sugars are partly retained in chloroplasts during the day to synthesize transitory starch for short term storage, and then exported to non-photosynthetic organs during the subsequent night for long-term storage [2]. Starch is the major storage carbohydrate in higher plants, with essential physical functions and economical importance. As a major factor for plant growth, starch biosynthesis buffers metabolism and growth against the daily light/darkness alternation to avoid a shortfall of carbon at the end of the dark period [3][4].
Transient starch is photosynthetic synthesized during the day but degraded at night to provide carbon and energy under inactive photosynthesis [5]. Leaf starch mainly accumulates in the photosynthetic palisade and mesophyll (M) cells [6], and major mesophyll cells in mature leaves are source for sucrose transport into sink tissues [7][8].
Transient starch in leaves is usually found in the plastid of photosynthetic organs [5]. While enzymatic functionality of the respective plastids depends largely on its own specialized proteome, and corresponding shifts of these proteome determine the transitions of different plastid types along with changes from environmental conditions and tissues [9][10]. The vast majority of plastid proteome is encoded by nucleus, but the expression of plastid genes is essential for metabolic processes such as photosynthesis and lipid biosynthesis [11][12]. Thus, the establishment of plastid multi-subunit protein complexes need a tight cooperation between nucleus and plastid genes [13]. Besides, high morphological and functional diversity of plastids in different tissues of multicellular plants are tightly connected to the function of the corresponding tissue [10], which can explain the manifestations of the same cell organelle in an individual plant.
Development from undifferentiated proplastid to functional plastid is coordinately achieved between plastid and nucleus, requiring cooperation between nucleus-to-plastid antegrade signaling and plastid-to-nucleus retograde signaling [14]. The GUN (genome uncoupled) proteins were identi ed for plastid-tonucleus signaling studies [15]. Thereinto, GUN4 have been found to be involved in the retrograde signaling pathway in Arabidopsis [16] and rice [17]. Besides, the mutation of OsGUN4 in rice have also been revealed to deregulate transcription of PhANGs depending on disruption of 1 O 2 -induced signaling pathway [17]. This model suggested that accumulation of heme in active chloroplast can activate a mechanism to induce the expression of PhANGs [18]. Interestingly, the plastid-to-nucleus retrograde signals is also revealed to regulate expression of nuclear starch biosynthetic genes, which is partly mediated by tetrapyrrole intermediates, i.e., heme [9]. Besides, the mutation of OsGUN4 in rice have also been revealed to greatly affect tetrapyrrole intermediates, including heme, Mg-Proto and Proto [17]. Above on, retrograde signaling may play important roles in starch biosynthesis of leaves, but the underlying mechanism remains largely unknown.
In previous studies, we revealed that the OsGUN4 mutation in rice have been revealed to greatly affect tetrapyrrole intermediates and function in 1 O 2 -induced signaling pathway [17,19]. Here, we further employed the rice epi-genetic mutant gun4 epi to examine carbon metabolites, starch biosynthetic enzymes, genes involved in starch biosynthesis in order to investigate the role of OsGUN4 on starch biosynthesis during vegetative stages. In conclusion, these ndings con rm that OsGUN4 play regulatory roles in starch biosynthesis.

Mutation of OsGUN4 produced aberrant starch metabolism
Previous studies revealed the positive effects of OsGUN4 mutation on photosynthetic capacity during vegetative stages [20][21], but no details were focused on the relationship between photosynthetic products-sucrose and starch biosynthesis. To determine the effects of OsGUN4 mutation on starch biosynthesis during vegetative stage, the carbon metabolites and relative starch biosynthetic enzymes were investigated in 35 days after germination (DAG) seedlings ( Fig. 1-2 and Additional le 5-6: Table S1-S2).
Compared to the wild-type, both of the sucrose and amylose contents were increased in gun4 epi (Fig. 1a, e), while the fructose, glucose, total starch and protein contents were decreased (Fig. 1b, c, d, f), suggesting that the OsGUN4 mutation promoted to accumulate the sucrose but decrease starch synthesis.
To reveal why the OsGUN4 mutation led to abnormal starch metabolism existed, the 35 DAG seedlings were treated with exogenous sucrose (exSuc). In the wild-type, contents of the sucrose and amylose were reduced, whereas the fructose, glucose, total starch and protein contents were increased after treatment ( Fig. 1). However, in gun4 epi , no difference was detected before and after treatment, but both of sucrose and amylose concentration were higher than that in the wild-type, whereas the fructose, glucose, total starch and protein contents were lower, indicating the retardative transformation of sucrose to starch in gun4 epi leaves ( Fig. 1a-f).
We next treated the 35 DAG seedlings were with exSuc added nor urazon (NF; the agent for blocking photosynthesis, causing the gun phenotype). After exposed to exSuc added with NF, contents of carbon metabolites were little induced compared with the control, but greatly inhibited in relative to the single sucrose treatment in wild-type, indicating that NF blocked the sucrose-induced signaling (Fig. 1). Nevertheless, no difference was still detected between the control and the sucrose treatment in gun4 epi , but there were signi cant differences for the treatment of sucrose added with NF compared to the other two treatment ( Fig. 1a-f). All these results suggested that OsGUN4 mutation in uenced the starch biosynthesis in leaves.

Mutation of OsGUN4 deregulated activities of enzymes responsible for starch biosynthesis
Dynamic activity changes of enzymes involved in starch biosynthesis were in accordance with the contents of carbon metabolites ( Fig. 2 and Additional le 6: Table S2). In consistent with the results as shown in Fig. 1, activities of ADP-Glc pyrophosphorylase (AGPase), soluble starch synthase (SSS) and starch branching enzyme (SBE) showed signi cant increases, but activities of sucrose synthase (SS), sucrose phosphate synthase (SPS) and granule-bound starch synthase (GBSS) were increased in gun4 epi ( Fig. 2a-f).
After exposed to exSuc, signi cant increased activities of AGPase, SSS and SBE, but decreased activities of SS, SPS and GBSS were showed in wild-type, whereas no difference was detected in gun4 epi , suggesting the retardative accumulation of starch from sucrose in gun4 epi ( Fig. 2a-f).
After exposed to exSuc added with NF, activities of the related enzymes ( Fig. 2a-f), was little induced compared with the control, but greatly inhibited in relative to the single sucrose treatment in wild-type, indicating that NF blocked the sucrose-induced signaling. Still, no difference was detected between the control and the exSuc treatment in gun4 epi , but, the sucrose added with NF treatment greatly affected the enzyme activities in relative to other treatments ( Fig. 2a-f). All these results suggested that OsGUN4 mutation in uenced activities of the starch biosynthetic enzymes in leaves.

Differently expressed genes related to starch biosynthesis revealed by RNA-seq
To analyze the detailed regulation of OsGUN4 on starch biosynthesis in vegetative leaves, RNA-seq was performed in the wild-type and gun4 epi . According to the mapping results using the metabolism overview pathways in MapMan, a total of 468 differentially expressed genes (DEGs) were identi ed between HYB and LTB by RNA-seq, with 203 genes being up-regulated and 265 down-regulated in gun4 epi (Fig. 3a).
Furthermore, the exogenous sucrose induced the gene expression for AGPase, GBSS, SSS, SBE, whereas reduced the expression of genes for SS and SPS in the wild-type (Fig. 4-6). After exposed to sucrose added with nor urazon, the gene expression for SS, SPS and GBSS still remained higher expression than that of sucrose treatment (Fig. 4-5), whereas gene expression for APGase, SSS and SBE were little increased in the wild-type (Fig. 5). However, sucrose treatment induced no signi cant difference with the control in gun4 epi (Fig. 4-5). But the sucrose supplement with nor urazon treatment intensi ed the trend of gene expression changes, and showed more enhanced dynamics in gun4 epi than that in the wild-type (Fig. 4-5). All these results were consistent with the results as shown in Fig. 1 and Fig. 2, suggesting the regulatory role of OsGUN4 on expression of starch biosynthetic genes.
Abnormal effecting of OsGUN4 mutation on the transcriptional expression of bZIP58 OsGUN4 is localized in plastid in our previous studies, so it is impossible for OsGUN4 to regulate gene expression as transcriptional factors (TFs) in nucleus. Thus, to clarify the signaling of OsGUN4 from plastid to nucleus, the reported TF of bZIP58 was used for further investigation (Fig. 6 and Additional le 7: Table S3).
In 35 DAG seedlings, expression of OsbZIP58 was signi cantly decreased in gun4 epi (Fig. 6). Also, after sucrose treatment, there was no obvious expression difference of OsbZIP58 in gun4 epi (Fig. 6). But in wild-type, expression of OsbZIP58 was signi cantly induced by sucrose, while showed no obvious changes with CK (Fig. 6). These results suggested that OsGUN4 mutation down-regulated the transcriptional expression of bZIP58.

OsGUN4 is involved in regulation of starch biosynthesis
In plant cells, plastids display a high morphological and functional variations, and include four major forms of etioplast, chloroplast, chromoplast and amyloplast [22]. Despite displaying diverse and tissue-dependent functions, each differentiated form of plastid share a set of genome [23]. Chloroplasts are the location for photosynthesis and biosynthesis of transient starch [5]. Also, the aberrant chloroplasts usually would cause abnormal photosynthesis and starch metabolism [5,22]. The OsGUN4 mutation performed aberrant chloroplast morphology as reported previously [20], and also indeed reduced the accumulation of starch at here (Fig. 1d). Nonetheless, the mutation of OsGUN4 did not cause the decrease of sucrose derived from photosynthesis in gun4 epi , and inversely, the OsGUN4 mutation led to the accumulation of sucrose (Fig. 1a), which is partly related to the positive effects of OsGUN4 mutation on photosynthetic capacity during vegetative stages [20][21]. On the other hand, this is due to the deregulated enzyme activities involved in starch biosynthesis (Fig. 2). For example, in gun4 epi , the enhanced activities of SS and SPS was responsible for the accumulation of sucrose (Fig. 2a-b), whereas the decreased AGPase, SSS and SBE activities made neglect effects on starch synthesis (Fig. 2c-f). All these results suggested that OsGUN4 mutation blocked the accumulation of starch from sucrose in leaves.
For example, the mutation of TaSSIVb-D in wheat induced the reduction of starch granule number and photosynthetic e ciency, this may be attributed to high contents of the substrate ADPglucose [24][25]. This is consistent with the results as shown in Fig. 4 and Fig. 6, which could also explain the enhanced AGP activity and increased expression of OsSSIV in gun4 epi . Besides, the addition of exogenous sucrose also indicated that GUN4 played a role in the normal synthesis of starch. Exogenous sucrose could greatly promote the transient starch and protein biosynthesis in wild-type, but could not induce the accumulation of sucrose and amylose in gun4 epi (Fig. 1). This was due to the dynamic activity changes of enzymes involved in starch biosynthesis, which were in accordance with the contents of metabolites (Fig. 2).
Moreover, NF treatment is usually used for explore the uncoupled phenomenon of photosynthesis-associated nuclear genes (PhANGs) transcriptional levels from chlorophyll accumulation in Arabidopsis [15] and C. reinhardtii [26]. Our previous studies revealed that the mutation of OsGUN4 deregulate transcription of PhANGs depending on disruption of 1 O 2 -induced signaling pathway in rice [17]. Here, after NF treatment, the induction by exogenous sucrose were nearly eliminated or weakened in the wild-type, whereas the OsGUN4 mutation aggravated no response to sucrose signals in gun4 epi ( Fig. 1-2, and Additional le 4: Fig. S4). All these results suggested OsGUN4 functions in response to sugar signals during starch biosynthesis.

Roles of OsGUN4 in regulation of starch biosynthesis
The normal functioning of plastids require cooperation of plastid genes and nuclear genes, which could reach the balance of photosynthesis and starch biosynthesis [14]. Although GUN4 have been revealed in the plastid-to-nucleus signaling pathway [17], it has not yet been reported its similar functions on starch biosynthesis. As is shown in preceding part of the text, OsGUN4 indeed function in the starch biosynthesis, and the OsGUN4 mutation also greatly deregulated many genes for key enzymes in starch biosynthesis (Fig. 4-5, and Additional le 4: Fig. S4). Thus, we can conclude that OsGUN4 may regulate the genes encoding key enzymes in starch biosynthesis. There are mainly two ways to employ the regulation, by tetrapyrrole intermediates and by sucrose signals.
Inhibitors of plastid gene expression could repress amyloplast differentiation and starch biosynthesis in tobacco (Nicotiana tabacum) Bright Yellow-2 (BY2) cultured cells [9]. This indicated a plastid-to-nucleus retrograde signals from plastid gene expression to the regulation for expression of nuclear starch biosynthesis genes, partly mediated by tetrapyrrole intermediates, i.e., heme [9]. In our previous studies, the OsGUN4 mutation greatly affect tetrapyrrole intermediates, including heme, Mg-Proto and Proto in rice [17]. The blocking of photosynthesis and starch biosynthesis in gun4 epi also illustrated this from Suc added with NF treatment (Fig. 1-3 and Additional le 3-4: Fig. S3, Fig. S4), suggesting the retardative signals from plastid to nucleus to promote starch biosynthesis.
Regulation of sucrose signals on starch biosynthesis can realized via transcription factors in cereal crops, e.g. NAC36 [27], MYB14 [28]. OsGUN4 is localized in plastid in our previous studies, so it is impossible for OsGUN4 to regulate gene expression as transcriptional factors (TFs) in nucleus. Instead, OsGUN4 might function in regulation of genes participating in starch biosynthesis via transcription factors, e.g. bZIP58 (Fig. 6). RNA-seq and RT-qPCR assays revealed that many genes for key enzymes in starch biosynthesis were signi cantly down-regulated in gun4 epi , including including the transcription factor of bZIP58 and target genes of OsBEIIb and OsSSI, which are vital for the formation of amylopectin and starch granules, while displayed up-regulated expression of OsSSIIIa and OsGBSSI that promotes the formation of amylose (Fig. 3 and Additional le 3: Fig. S3). Thus, the retardative transformation from sucrose to starch mostly depends on the deregulated expression of genes for starch biosynthetic enzymes.

Conclusion
In conclusion, we here found that down-regulation of OsGUN4 promoted to accumulate sucrose but reduce the total starch (Fig. 1), attributing to abnormal performance of metabolisms and enzyme activities of starch biosynthesis (Fig. 2) in leaves of gun4 epi . Moreover, the exogenous sucrose induced starch synthesis but inhibited sucrose contents in wild-type, while nor urazon eliminated the changes ( Fig. 1-2). However, no changes were detected between check and sucrose treatments in the gun4 epi , but NF treatments enhanced the trends of increased sucrose but reduced starch ( Fig. 1-2). Dynamic activity changes of enzymes involved in starch biosynthesis were in accordance with the contents of carbon metabolites, suggesting the roles of OsGUN4 on balance of photosynthesis and starch biosynthesis ( Fig. 1-2). Moreover, RNA sequencing revealed that many of the DEGs were associated with starch metabolic pathways, with 62 genes being up-regulated and 25 down-regulated in gun4 epi (Fig. 3). A great deal of genes involved in starch biosynthesis performed downregulated expression (Fig. 4-6), including the transcription factor of bZIP58 and its target genes of OsBEIIb and OsSSI, which are vital for the formation of amylopectin and starch granules, while displayed up-regulated expression of OsSSIIIa and OsGBSSI that promotes the formation of amylose. All these ndings suggest that OsGUN4 plays vital roles in the biosynthesis of transient starch in leaves.

Plant materials
The following materials were used in the present study: wild-type (Longtepu B, LTB) and its gamma ray-induced xantha mutant line Huangyu B (HYB) [20]. The epigenetic mutation of OsGUN4, gun4 epi , underlies the xantha phenotype of HYB [19].
After germination, seedlings were raised by growing on soil at 30℃ for 35 days under 16 h/8 h light/dark and at low light intensity (LL) of 100 µmol photons m − 2 s − 1 . Above seedlings grown under the LL condition for 28 days were then transferred to 1 × MS liquid medium [29] containing 200 µM sucrose (Suc) or Suc plus with 10 µM nor urazon (Suc + NF) for another 24 hours. The samples at midday were collected for assays for carbon metabolites, enzyme assays and quantitative real-time PCR (RT-qPCR).

Analysis of metabolites
Determination of amylose, starch, and protein were performed as described previously [30]. Sucrose, fructose, and glucose were analyzed using the methods described previously [31]. RNA sequencing analysis RNA extracted from seedlings of 35 DAG were used for the construction of cDNA libraries, which were subsequently sequenced on an Illumina Hiseq 2000 platform (Beijing Novogene Bioinformatics Technology Co., Ltd. Beijing, China). For mapping, the raw reads were cleaned by removing adapter sequences and then aligned to the references genome sequences (www.gramene.org) by using the Tophat v2.0.9 program with E-value 10 − 5 as cut-off point [33]. For detection of differentially expressed genes (DEGs), the DESeq package (ver 2.1.0) was used with a false discovery rate (FDR) ≤ 0.005 and the absolute value of the log2 (fold change) with RPKM ≥ 1 as the threshold to determine signi cant differences of gene expression. For DEG analysis, gene ontology (GO) enrichment were conducted by the GOseq R package with P 0.05 and functionally classi ed by WEGO software, while KEGG pathways were analyzed with a FDR ≤ 0.05 as signi cant levels of differential expression.

Accession numbers
Genes investigated on transcription is were identi ed through homolog search of the following databases: The Rice Annotation Project (RAP) Database (https://rapdb.dna.affrc.go.jp/) and GenBank/EMBL database (https://www.ncbi.nlm.nih.gov/). These genes were subjected to RT-PCR analysis by using gene-speci c primers (Additional le 8: Table S4).

Statistical analysis
All experiments were performed with six independent biological repeats. Values were expressed as means ± standard deviations and analyzed using two-way ANOVA test followed by the Tukey's Multiple Comparison Test with P < 0.05.

Supplementary Files
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