Over 70 years ago, Rosalind Franklin, James Watson, and Francis Crick identified the structure of DNA, the essential blueprint for life. Nowadays, researchers are continually discovering new methods to interpret it.
In 2010, Jianxin Ma, a professor of agronomy, and his colleagues created the first reference genome for soybeans, specifically for the well-studied Williams 82 variety. Since then, numerous scientists and plant breeders have utilized this genome in their research into the genetic factors influencing various traits, such as seed protein and oil levels, plant structure and yield, as well as resistance to diseases and environmental stressors in soybeans.
Throughout the past decade, Ma, who holds the Indiana Soybean Alliance Inc. Endowed Chair in Soybean Improvement, has received global recognition for his advancements in soybean genomics and ongoing innovative research. His latest findings, published in The Plant Cell, leveraged new genomic techniques to resolve missing information from the original soybean reference genome.
“When we first released the reference genome, it was like providing a dictionary,” Ma explained. “Each gene represented a single word. However, we identified a crucial piece of information that was absent: the transcription initiation sites for each gene.”
Transcription initiation sites are spots on the DNA where a specific transcription-factor protein can bind to begin forming an mRNA version of the gene. This mRNA is then interpreted at the cell’s ribosome to produce proteins, essential for the chemical and physical processes of all living organisms.
Understanding where mRNA production starts on the DNA strand is key to knowing how genes are expressed. These initiation sites include regulatory elements which inform the cell about when, where, and how often to transcribe each gene into protein.
Traditionally, it has been understood that each gene possesses a single transcription initiation site, found downstream of a core promoter often near a TATA box—a DNA sequence abundant in thymine and adenine. However, Ma and his team have challenged this notion.
“Our predictions indicate that there are expected transcription start sites for over 50,000 soybean genes, but based on our latest research, fewer than 3% of these predicted transcription initiation sites are actually accurate,” stated Ma.
In 2020, the introduction of the Survey of TRanscription Initiation at Promoter Elements Sequencing (STRIPE-seq) technique enabled Ma’s lab to swiftly and cost-effectively pinpoint transcription initiation sites throughout the soybean genome. This method also revealed the relative abundance of each mRNA copy, providing insights into how often a gene is expressed in different tissues and at various times.
With funding from the USDA’s National Institute of Food and Agriculture (USDA-NIFA) and the National Science Foundation, Ma and his team conducted STRIPE-seq analyses on eight distinct soybean tissues: leaves, stems, stem tips, roots, nodules, flowers, pods, and developing seeds. Despite the DNA being uniform across these tissues, gene expression varied.
In their latest research, Ma’s lab identified transcription initiation sites for approximately 40,000 soybean genes. They uncovered many alternative transcription initiation sites beyond the TATA box region and other presumed promoter sequences. Some newly discovered sites are actually found within the gene’s coding sequence that leads to mRNA production. Thus, transcription-factor proteins can attach to various parts of the gene and initiate mRNA production, resulting in multiple protein variants from a single gene.
Among the specialized transcription initiation sites identified were those located in root nodules, structures on legume roots that facilitate interactions with Rhizobia bacteria. These beneficial soil bacteria fix nitrogen for legumes in exchange for sugars and protection, enhancing the plant’s ability to thrive in nitrogen-poor soils without fertilizers.
“We identified these specific transcription initiation sites in nodules but not in roots or other tissues, suggesting they are involved in tissue-specific transcription related to nodule function,” Ma noted.
To fit inside a cell’s nucleus, DNA coils around histone proteins forming structures known as “chromatin.” The configuration of chromatin—whether tightly or loosely wrapped, depending on chemical markers—determines whether transcription factors can access it. Ma believes that these “epigenetic” alterations work in concert with alternative transcription initiation sites to regulate gene expression. As chromatin structure changes, different transcription initiation sites can become accessible, leading to the production of distinct proteins.
“We’ve found almost 7,000 genes that exhibit alternative transcription initiation within their coding regions. These sites are generally tissue-specific and correspond to histone modifications,” Ma explained.
From an evolutionary standpoint, these alternative sites may have conferred advantages to soybeans and other plants, allowing for greater complexity and flexibility with a limited genome. Soybeans underwent two whole-genome duplication events in their evolutionary history, both occurring millions of years ago. Although some duplicated genes have been lost, Ma suggests that these duplications may have led to the emergence of new or alternative transcription sites.
“Post-duplication, most genes still exist in pairs, but they display different expression patterns and many have functionally diversified to manage various traits,” Ma elaborated. “They often transcribe from different sites, potentially leading to their functional differences.”
Currently, Ma is collaborating with USDA Agricultural Research Service scientists Rex Nelson and Jacqueline Campbell to make this research data available to others, similar to how he did with the original reference genome. They are contributing this information to SoyBase, a shared online resource for soybean research.
Nelson, the curator of SoyBase, emphasized, “Having even a potential transcription start site can greatly enhance the analysis of soybean gene promoter regions, possibly illuminating the proteins that interact with promoters and trigger transcription.”
Campbell, the co-curator of the database, added that “identifying transcription factors that attach to promoter regions will enable researchers to map out gene regulatory networks critical for managing complex gene regulations in agronomically significant traits.”
Ma feels privileged to support the research community once more. “The database serves as a vital tool for both fundamental and applied research,” he stated. “By making our data available there, we encourage further exploration into gene functions, regulations, gene networks, and genetic variations linked to specific traits. As we enhance our understanding of how these alternative transcription sites influence traits, we anticipate advancements in soybean varieties.”
