♦ Profiling the Transcriptomes of Every Seed Region, Subregion, and Tissue Throughout Soybean and Arabidopsis Seed Development – An Atlas of Seed Gene Activity
We used laser capture microdissection (LCM) to isolate specific soybean and Arabidopsis seed regions (e.g., seed coat, endosperm, and embryo) subregions (e.g., embryo proper and suspensor), and tissues (e.g., cotyledon adaxial and abaxial parenchyma) at different stages of development. RNA-Seq (soybean) and microarray (Arabidopsis) transcriptome profiling experiments were carried out to determine (1) the spectrum of genes that are active in different parts of the seed during development, (2) what transcription factors are localized in specific seed regions and subregions, and (3) the biological processes that are partitioned within a seed which may play important roles in seed differentiation and/or function.
We profiled the mRNA sets present in 40 soybean and 42 Arabidopsis seed compartments captured using LCM - from shortly after fertilization through the early maturation stage of development. All transcriptome data have been deposited on the interactive Database. This database contains analysis tools that allows users to browse the database by gene identification, gene ontology, and gene function, as well as compare gene activity in different seed regions during development. Click the buttons below to access the interactive database.
To study the functions of transcription factor genes active during soybean seed development, we collaborated with Dr. David Somers (Monsanto) to generate a collection of soybean seed RNAi knock-out lines for 63 transcription factor genes that are expressed in specific seed regions at the globular, heart, cotyledon, and early-maturation stages of development.
The soybean and Arabidopsis LCM transcriptome datasets (1) provide one of the most comprehensive descriptions of gene activity within seeds, (2) contain unique insights into the functions of understudied seed regions, such as the suspensor, and (3) are a unique resource for uncovering the gene networks for controlling seed form and function. Functional knockout studies were carried with 53 soybean region- and subregion-specific transcription factors (TFs) in order to determine what role they might play during seed development.
We compared the globular stage embryo region transcriptomes of Soybean with those of the Arabidopsis, Scarlet Runner Bean, and Common Bean, and identified a set of embryo proper- and suspensor-specific transcription factors (TFs) that are shared by these species and might play a role in the specification processes controlling embryo proper and suspensor development shortly after fertilization. In addition, these experiments showed that giant suspensors in Scarlet Runner Bean and Common Bean carry out highly specialized metabolic functions, particularly those involved in hormone function, as compared with the less specialized soybean and Arabidopsis suspensors.
In this project, we have generated 246 RNA-Seq and 190 GeneChip Datasets which have been deposited to GEO. Click here to download the datasets.
Publications:
Danzer, J., Mellott, E., Bui, A.Q., Le, B.H., Martin, P., Hashimoto, M., Perez-Lesher, J., Chen, M., Pelletier, J.M., Somers, D.A., Goldberg, R.B., Harada, J.J. (2015) Down-regulating the expression of 53 soybean transcription factor genes uncovers a role for SPEECHLESS in initiating stomatal cell lineages during embryo development, Plant Physiology, 168:3, 1025-35.[DOWNLOAD PDF]
Belmonte, M., Kirkbride, R.C., Stone, S.L., Pelletier, J.M., Bui, A.Q., Yeung, E.C., Hashimoto, M., Fei, J., Harada, C.M., Munoz, M.D., Le, B.H., Drews, G.N., Brady, S.M., Goldberg, R.B., and Harada, J.J. (2013) Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed. Proc. Natl. Acad. Sci. USA, 110:5, E434-44. [DOWNLOAD PDF]
Le, B.H., Cheng, C., Bui, A.Q., Wagmeister, J.A., Henry, K.F., Pelletier, J., Kwong, L., Belmonte, M., Kirkbride, R., Horvath, S., Drews, G.N., Fischer, R.L., Okamuro, J.K., Harada, J.J., and Goldberg, R.B. (2010) Global analysis of gene activity during Arabidopsis seed development and identification of seed-specific transcription factors. Proc. Natl. Acad. Sci USA, 170:18, 8063-70.[DOWNLOAD PDF]
♦ Transcriptional Profiling of lec1 Mutant Seed Development
To obtain insight into the developmental processes that are controlled by LEC1, we profiled mRNA populations in seeds homozygous for the lec1-1 null mutation at five different stages of seed development using Affymetrix ATH1 GeneChips –24 h after pollination (24H), globular (GLOB), linear cotyledon (LCOT), mature green (MG), and postmature green (PMG). Comparing the transcriptional profiles of lec1 mutant seeds to those of LEC1 wild-type seeds showed that LEC1 directly or indirectly activates a number of distinct cellular processes during seed development, including genes involved in seed maturation and photosynthesis. By contrast, many genes up-regulated in mutant lec1 seeds are normally expressed during post-embryonic seedling germination and are repressed during wild-type seed development. These results indicate that in addition to being an activator, LEC1 plays a major role in repressing seed germination genes during the terminal stages of seed development – explaining how lec1 mutant seeds precociously germinate and fail to undergo desiccation. The molecular processes by which LEC1 acts as a repressor late in seed development are not yet understood.
In this project, we have generated 48 GeneChip Datasets which have been deposited to GEO. Click here to download the datasets.
Publications:
Pelletier, J.M., Kwong, R. Park, S., Le, B.H., Baden, R., Cagliari, A., Hashimoto, M., Munoz, M., Fischer, R.L., Goldberg, R.B., Harada J.J. (2017) LEC1 sequentially regulates the transcription of genes involved in diverse developmental processes during seed development. Proc. Natl. Acad. Sci. USA, 114:32, E6710–E6719. [DOWNLOAD PDF]
Le, B.H., Cheng, C., Bui, A.Q., Wagmeister, J.A., Henry, K.F., Pelletier, J., Kwong, L., Belmonte, M., Kirkbride, R., Horvath, S., Drews, G.N., Fischer, R.L., Okamuro, J.K., Harada, J.J., and Goldberg, R.B. (2010) Global analysis of gene activity during Arabidopsis seed development and identification of seed-specific transcription factors. Proc. Natl. Acad. Sci USA, 170 :18, 8063-70. [DOWNLOAD PDF]