Abstract
BackgroundStarch serves as a temporal storage of carbohydrates in plant leaves during day/night cycles. To study transcriptional regulatory modules of this dynamic metabolic process, we conducted gene regulation network analysis based on small-sample inference of graphical Gaussian model (GGM).ResultsTime-series significant analysis was applied for Arabidopsis leaf transcriptome data to obtain a set of genes that are highly regulated under a diurnal cycle. A total of 1,480 diurnally regulated genes included 21 starch metabolic enzymes, 6 clock-associated genes, and 106 transcription factors (TF). A starch-clock-TF gene regulation network comprising 117 nodes and 266 edges was constructed by GGM from these 133 significant genes that are potentially related to the diurnal control of starch metabolism. From this network, we found that β-amylase 3 (b-amy3: At4g17090), which participates in starch degradation in chloroplast, is the most frequently connected gene (a hub gene). The robustness of gene-to-gene regulatory network was further analyzed by TF binding site prediction and by evaluating global co-expression of TFs and target starch metabolic enzymes. As a result, two TFs, indeterminate domain 5 (AtIDD5: At2g02070) and constans-like (COL: At2g21320), were identified as positive regulators of starch synthase 4 (SS4: At4g18240). The inference model of AtIDD5-dependent positive regulation of SS4 gene expression was experimentally supported by decreased SS4 mRNA accumulation in Atidd5 mutant plants during the light period of both short and long day conditions. COL was also shown to positively control SS4 mRNA accumulation. Furthermore, the knockout of AtIDD5 and COL led to deformation of chloroplast and its contained starch granules. This deformity also affected the number of starch granules per chloroplast, which increased significantly in both knockout mutant lines.ConclusionsIn this study, we utilized a systematic approach of microarray analysis to discover the transcriptional regulatory network of starch metabolism in Arabidopsis leaves. With this inference method, the starch regulatory network of Arabidopsis was found to be strongly associated with clock genes and TFs, of which AtIDD5 and COL were evidenced to control SS4 gene expression and starch granule formation in chloroplasts.
Highlights
Starch serves as a temporal storage of carbohydrates in plant leaves during day/night cycles
From the graphical Gaussian model (GGM) network construction, we found that the sub-networks of two starch metabolic genes encoding granule-bound starch synthase (GBSS) (At1g32900) and disproportionating enzyme (DPE1: At5g64860) and their transcription factor (TF) neighbours are entirely separated from the rest of the network (Figure 2)
The results suggest that reduction of synthase 4 (SS4) expression in the Atidd5 and col mutant lines leads to a significant increase in starch granule numbers, while their distributions of granule number per chloroplast are differently affected among the mutants (Figure 8)
Summary
Starch serves as a temporal storage of carbohydrates in plant leaves during day/night cycles. Starch granules in leaves are decomposed to sugars to be transported to seeds or storage organs and stored as reserved carbohydrates or used as precursors in other metabolic pathways [1,2,3]. Additional finding in Arabidopsis indicates that expression of the GBSS-I gene is controlled by 2 main clock TFs, circadian clock associated 1 (CCA1: At2g46830) and late elongated hypocotyl (LHY: At1g01060) [24]. The roles of these TFs suggest the significance of transcriptional mechanisms, gene regulatory networks of starch metabolism remain largely uncharacterized
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