Network Inference of Transcriptional Regulation in Germinating Low Phytic Acid Soybean Seeds

The low phytic acid trait in soybeans can be conferred by loss-of-function mutations in genes encoding myo-inositol phosphate synthase and two epistatically interacting genes encoding multidrug-resistance protein ABC transporters. However, perturbations in phytic acid biosynthesis are associated with poor seed vigor. Since the benefits of the low phytic acid trait, in terms of end-use quality and sustainability, far outweigh the negatives associated with poor seed performance, a fuller understanding of the molecular basis behind the negatives will assist crop breeders and engineers in producing variates with low phytic acid and better germination rate. The gene regulatory network for developing low and normal phytic acid soybean seeds was previously constructed, with genes modulating a variety of processes pertinent to phytic acid metabolism and seed viability being identified. In this study, a comparative time series analysis of low and normal phytic acid soybeans was carried out to investigate the transcriptional regulatory elements governing the transitional dynamics from dry seed to germinated seed. Gene regulatory networks were reverse engineered from time series transcriptomic data of three distinct genotypic subsets composed of low phytic acid soybean lines and their normal phytic acid sibling lines. Using a robust unsupervised network inference scheme, putative regulatory interactions were inferred for each subset of genotypes. These interactions were further validated by published regulatory interactions found in Arabidopsis thaliana and motif sequence analysis. Results indicate that low phytic acid seeds have increased sensitivity to stress, which could be due to changes in phytic acid levels, disrupted inositol phosphate signaling, disrupted phosphate ion homeostasis, and altered myo-inositol metabolism. Putative regulatory interactions were identified for the latter two processes. Changes in abscisic acid signaling candidate transcription factors putatively regulating genes in this process were identified as well. Analysis of the gene regulatory networks reveal altered regulation in processes that may be affecting the germination of low phytic acid soybean seeds. Therefore, this work contributes to the ongoing effort to elucidate molecular mechanisms underlying altered seed viability, germination and field emergence of low phytic acid crops, understanding of which is necessary in order to mitigate these problems. Overall design: The four genotypic lines used in this study are designated as “MRP,” contains four near isogenic lines, the lpa line “2mlpa” (mrp-l and mrp-n mutations) and three normal phytic acid lines, “2MWT” (no mutation), “2MWT-L” (mrp-n mutation only), and “2MWT-N” (mrp-l mutation only). These lines were developed from a cross between the lpa lines “CX-1834” (mrp-1 and mrp-n mutations) and the normal phytic acid line V99-5089. The mutations conferring the lpa trait in 2mlpa and CX-1834 are the result of point mutations in the epistatically interacting loci, MRP-L and MRP-N, on chromosomes 19 and 3, respectively (Wilcox et al., 2000; Walker et al., 2006; Maroof et al., 2009). In the associated manuscript, eight experimental lines were used – 1mlpa, 1MWT, 2mlpa, 2MWT, 2MWT-L, 2MWT-N, 3mlpa, and 3MWT. The eight experimental lines represent three distinct subsets of genotypes and data from four lines are included in this dataset. Other four lines have been released from our previous publication (data in GSE101692). Line "mips1/mrp-l/mrp-n" is also called "3mlpa" which contains mips1, mrp-1, and mrp-n mutations and has low phytic acid content. The normal phytic acid line "MIPS1/MRP-L/MRP-N" (no mutation) is also called 3MWT. These lines were developed from a cross between CX-1834 and V99-5089. These files also include the lpa line "1mlpa" (mips1 mutation) and the normal phytic acid line "1MWT" (no mutation). These lines are isogenic and were developed from a cross between the normal phytic acid line "Essex"" (no MIPS1 mutation) and the lpa line "V99-5089" (mips1 mutation).

大豆低植酸（low phytic acid, LPA）性状可由编码肌醇磷酸合酶（myo-inositol phosphate synthase）的基因功能丧失突变，以及两个上位性互作的编码多药耐药蛋白ABC转运体（multidrug-resistance protein ABC transporters）的基因突变赋予。然而，植酸生物合成的扰动会导致种子活力下降。尽管低植酸性状在终端使用品质与可持续性方面的优势远大于其伴随的种子表现不佳的劣势，但更全面地解析这一劣势背后的分子机制，将有助于作物育种者与工程师培育出兼具低植酸特性与更佳发芽率的品种。

此前已有研究构建了发育中的低植酸与正常植酸大豆种子的基因调控网络（gene regulatory network, GRN），并鉴定出调控植酸代谢与种子活力相关多种过程的基因。本研究对低植酸与正常植酸大豆开展了比较时间序列分析，以探究调控从干种子到萌发种子转变动态过程的转录调控元件（transcriptional regulatory elements）。

研究从三组不同基因型亚群的时间序列转录组（transcriptomic）数据中逆向工程构建基因调控网络，这三组亚群分别由低植酸大豆品系与其正常植酸姊妹系构成。采用稳健的无监督网络推断（unsupervised network inference）方案，本研究为每个基因型亚群推断出潜在调控互作。这些互作进一步通过拟南芥（Arabidopsis thaliana）已发表的调控互作与基序序列分析（motif sequence analysis）得到验证。

结果显示，低植酸种子对胁迫的敏感性升高，这可能源于植酸水平改变、肌醇磷酸信号通路紊乱、磷酸盐离子稳态失衡以及肌醇代谢改变。本研究还鉴定出参与后两个过程的潜在调控互作，以及调控该过程中相关基因的脱落酸（abscisic acid, ABA）信号通路候选转录因子（transcription factor）。

对基因调控网络的分析揭示，可能影响低植酸大豆种子萌发的过程出现了调控异常。因此，本研究为阐明低植酸作物种子活力、萌发与田间出苗率改变背后的分子机制提供了新的见解，而解析这些机制正是缓解上述问题的必要前提。

实验设计概述：本研究使用的4个基因型品系被命名为“MRP”组，包含4个近等基因系（near isogenic lines）：低植酸品系“2mlpa”（携带mrp-l与mrp-n突变），以及3个正常植酸品系：“2MWT”（无突变）、“2MWT-L”（仅携带mrp-n突变）与“2MWT-N”（仅携带mrp-l突变）。这些品系由低植酸品系“CX-1834”（携带mrp-1与mrp-n突变）与正常植酸品系V99-5089杂交选育获得。2mlpa与CX-1834中赋予低植酸性状的突变分别为19号与3号染色体上的上位性互作位点（epistatically interacting loci）MRP-L与MRP-N的点突变（Wilcox等，2000；Walker等，2006；Maroof等，2009）。

在相关发表论文中，共使用了8个实验品系：1mlpa、1MWT、2mlpa、2MWT、2MWT-L、2MWT-N、3mlpa与3MWT。这8个实验品系对应3个不同的基因型亚群，本数据集仅包含其中4个品系的数据，其余4个品系的数据已在我们此前的研究中发表（数据编号GSE101692）。品系“mips1/mrp-l/mrp-n”亦被称为“3mlpa”，携带mips1、mrp-1与mrp-n突变，表现为低植酸含量。正常植酸品系“MIPS1/MRP-L/MRP-N”（无突变）亦被称为3MWT。这些品系由CX-1834与V99-5089杂交选育获得。本数据集还包含低植酸品系“1mlpa”（仅携带mips1突变）与正常植酸品系“1MWT”（无突变），这些品系为近等基因系，由正常植酸品系“Essex”（无MIPS1突变）与低植酸品系“V99-5089”（携带mips1突变）杂交选育获得。