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 file 5–6: Table S1-S2).
Compared to the wild-type, both of the sucrose and amylose contents were increased in gun4epi (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 gun4epi, 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 gun4epi leaves (Fig. 1a-f).
We next treated the 35 DAG seedlings were with exSuc added norflurazon (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 gun4epi, but there were significant 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 influenced 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 file 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 significant increases, but activities of sucrose synthase (SS), sucrose phosphate synthase (SPS) and granule-bound starch synthase (GBSS) were increased in gun4epi (Fig. 2a-f).
After exposed to exSuc, significant 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 gun4epi, suggesting the retardative accumulation of starch from sucrose in gun4epi (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 gun4epi, 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 influenced 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 gun4epi. According to the mapping results using the metabolism overview pathways in MapMan, a total of 468 differentially expressed genes (DEGs) were identified between HYB and LTB by RNA-seq, with 203 genes being up-regulated and 265 down-regulated in gun4epi (Fig. 3a).
To investigate the expression profiles in lodicules of the wild-type and gun4epi, DEGs with adjusted P < 0.001 were selected for further analysis, and many of the DEGs were associated with sucrose and starch metabolic pathways, with 62 genes being up-regulated and 25 down-regulated in gun4epi (Fig. 3b and Additional file 1: Fig. S1), including genes for fructose-1,6-bisphosphate aldolase (FBA, OsFEA), fructose 1,6-bisphosphate (FBP), phosphoglucoseisomerase (PGI), phosphoglucomutase (PGM), sucrose-phosphate-synthase (SPS), sucrose phosphate phosphatase (SPP), sucrose synthase (SuS) and GBSS (OsGBSSI), as well as genes for fructose and glucose synthesis, such as fructokinase (Frk) and hexokinase (HxK), soluble starch synthase (SS) and branching enzyme (BE). Besides, genes for isoamylases (ISA), pollulanase (PUL) and α-amylase (Amy3B, 3C, 3D), and representative genes for storage proteins, including protein disulfate isomerase (PDI), prolamin (CysR10), PPDKB, glutelins (GluA1, GluA2, GluA3), alanine aminotransferase (AlaAT1, AlaAT4), major allergenic protein (RA16, RA17, RA5B, RAG2 and RG21), globulins (globulin1, globulin2, 11 s-globulin, 10kD-, 13kD-, 17kD-, 19kD-globulin) also deregulated expression in gun4epi in relative to WT (Fig. 3b and Additional file 1: Fig. S1). These results suggested that OsGUN4 mutation affected expression of many genes participating in starch and protein biosynthesis in leaves.
Mutation of OsGUN4 deregulated gene expression responsible for starch metabolism in leaves
To ensure the sequencing results, expression for genes of log2 fold changes more than 0.5 folds were further detected by RT-qPCR (Fig. 4–5 and Additional file 7: Table S3). Compared to the wild-type, gene expression for sucrose synthesis, including genes for FBA (OsFEA), FBP (OsCFR, OsFBP2, OsFBP3), PGI (OsPGI), PGM (OspPGM), SPS (OsSPS2, OsSPS11), SPP (OsSPP1, OsSPP2), SuS (OsSUS1, 2, 5, 6, 7) and GBSS (OsGBSSI) were significantly increased (Additional file 2: Fig. S2 and Additional file 3: Fig. S3), while genes for fructose and glucose synthesis, such as Frk (OsFrK1) and HxK (OsHxK1, OsHxK4 and OsHxK7), were greatly reduced in gun4epi (Fig. 4). Moreover, gene expression for AGPase (OsAGPS1), SS (OsSSIVa, OsSSV) and BE (OsBEIIa, OsBEIIb) were also decreased in gun4epi compared to that in wild-type (Fig. 5 and Additional file 2–3: Fig. S2 and Fig. S3). Nonetheless, OsSUS4, OsAGPL1, OsAGPL3, OsAGPL4 and OsSSIIc showed increased expression in gun4epi (Fig. 4–5). All these results were consistent with the results as shown in Fig. 3b, indicating that the mutation of OsGUN4 affected expression of starch biosynthetic genes.
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 norflurazon, 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 significant difference with the control in gun4epi (Fig. 4–5). But the sucrose supplement with norflurazon treatment intensified the trend of gene expression changes, and showed more enhanced dynamics in gun4epi 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 file 7: Table S3).
In 35 DAG seedlings, expression of OsbZIP58 was significantly decreased in gun4epi (Fig. 6). Also, after sucrose treatment, there was no obvious expression difference of OsbZIP58 in gun4epi (Fig. 6). But in wild-type, expression of OsbZIP58 was significantly 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.