Progress 08/01/24 to 07/31/25

Outputs
Target Audience:The audience for the research component of the project during this reporting year was the scientific community focused on agricultural research, especially researchers who areinterested in studying abiotic stress responses in crop plants, including cereals. Also, the audience for our research activities was researchers interested in dissecting molecular responses to stresses in crop plants. The audience for the educational component of the project during this reporting period was graduate and undergraduate students at the host institution located in South Texas. Additionally, our outreach activities were targeted towards local high-school and middle-school students in South Texas. Changes/Problems:In our outreach activities, we originally planned to offer 8 weeks of summer internship to two local high-school students per year. During this reporting period, we had the opportunity to host a very meritorious high-school student. Hence, instead of offering very short internships to two students, we decided to provide an internship to the same student in the Fall of 2024 and Spring of 2025, to ensure considerable direct training in the field of agricultural genomics and biotechnology. We made another change in our outreach activities. We proposed to conduct four 'ag-biotech day' events per year and to cater to 24 school students per event. Due to logistical reasons, we conducted two events with multiple sessions on the same day in the same school, which allowed us to reach more students while utilizing our resources judiciously. Altogether, we conducted multiple sessions on two such events in two local schools, and 140 students (mostly middle-school) participated in those events. Considering the huge success of the events, we would like to follow the same format in the next reporting cycle. What opportunities for training and professional development has the project provided?To address Objective-B, during this reporting period, one graduate student (Mr.Javier Ramos) and two undergraduate students (Mr.Nicolas Torres and Mr.Dayvin Rodriguez) were recruited and trained through this project. The PI designed a specific curriculum to train graduate students (including students not supported by this award) in the field of agricultural biotechnology. This involves the selection of specific courses to allow graduate students to acquire robust knowledge in the field of molecular genetics, biotechnology, plant biology, data analysis, and scientific communication. Mr.Javier Ramos is pursuing an MS degree in Biology at the host institution. Mr.Nicolas Torres was a BS student with a Biology major, and Mr.Dayvin Rodriguez is a BS student with a Biology Major at the host institution. The graduate student, Mr. Javier Ramos, received in-depth training on carrying out abiotic stress treatments, total RNA isolation, and quality assessment, and hands-on training on setting up bioinformatic pipelines to analyze genomic datasets. Mr. Javier Ramos also received training on scientific experiment design and on improving soft skills necessary for future success in a scientific career, such as strengthening both written and oral scientific communication skills. Mr. Javier Ramos also received opportunities to mentor undergraduate students, thus acquiring key mentoring skills, and to gain teaching skills by instructing a laboratory course at the host institution. Two undergraduate students received direct training on agricultural biotechnology, especially utilizing bioinformatics tools to analyze agricultural genomic datasets. Additionally, Mr.Javier Roams (Graduate student) and Mr.Dayvin Rodriguez (Undergraduate student) received one week of training on different aspects of plant protection research, including the usage of agricultural biotechnology at USDA-APHIS-PPQ-S&T located in Edinburg, Texas. During this week-long training, students also received valuable first-hand experience on research conducted by USDA scientists. Mr.Javier Roams (Graduate student) and Mr.Dayvin Rodriguez (Undergraduate student) also received one-week hands-on training in the Co-PI's lab at the University of Texas-Austin. During this one-week training, students received direct training on using an HPC (High-Performance Computing) system and implementing different bioinformatic pipelines using a HPC system.To expand the knowledge base of the students at the host institution in broad areas of agriculture, food, and biotechnology, we organized a webinar series, where we invited expert speakers to give lectures on relevant topics. During this reporting period, we organized two webinars. In the first webinar, Mr.Stephen Sturdivant, Environmental Engineer at the US Environmental Protection Agency (EPA), gave a lecture on ways to reduce environmental impacts through food choices. In the second webinar, Dr.Rafael Loureiro, Associate Professor, Winston-Salem State University, North Carolina, gave a lecture on the importance of Lunar and Martian regolith as a substrate for space agriculture. These events were extremely well received by the students and were attended by 52 and 17 attendees, primarily consisting of undergraduate students at the host institution. We will keep organizing this webinar series in the next reporting period, and we will invite speakers with expertise in diverse areas of agriculture, especially in the field of agricultural biotechnology. To address Objective-C, we undertook multiple outreach activities. As part of our outreach effort, we provided an internship for a whole academic year to one high-school student, Ms.Sreeja Mullapudi from the STISD Science Academy, Mercedes, Texas. The high-school student received extensive training on agricultural biotechnology and genomics, including handling of next-generation sequencing datasets. Additionally, to promote awareness of agricultural biotechnology among local middle-school students, we conducted 'ag-biotech' day events, where school students get hands-on training on agricultural biotechnology. On February 28th, 2025, 40 students from the Mozart Secondary School participated in the 'ag-biotech day' event. On May 8th, 2025, 100 students from the 'Central Middle School' attended the 'ag-biotech day' event. Altogether, during this reporting cycle, we provided exposure to agricultural biotechnology to 140 local school students in South Texas. In the future, to promote the interest of local youth in the emerging field of agricultural biotechnology, we will conduct similar outreach events. How have the results been disseminated to communities of interest?The results from this project were disseminated to the scientific communities at the host institution and in the South Texas region through two oral and one poster presentations. The graduate student, Mr.Javier Ramos, gave two oral presentations on his research findings, one at the Annual Research Conference of the College of Sciences at the University of Texas Rio Grande Valley (UTRGV). Mr. Javier Ramos also gave an oral presentation at the 79thAnnual Conference of the Subtropical Agriculture & Environments Society (SAES) on February 27-28 in Mission, Texas, and received 2nd prize in the graduate student category. The SAES is the oldest professional society focusing on agricultural and environment research in the South Texas. Undergraduate student Dayvin Rodriguez also gave a poster presentation at the same conference. What do you plan to do during the next reporting period to accomplish the goals?During the second year of the project, we will finish bioinformatic analyses of the small RNA sequencing dataset of the combined stress-treated and corresponding control samples. We will also compare the effects of combined stress and individual stresses at the small RNA level. We will also conduct bioinformatic analyses of the direct RNA sequencing dataset to decipher combined stress-induced changes in RNA modifications and their interconnection. We will continue training one graduate student (Mr.Javier Ramos) and a new undergraduate student in the field of agricultural biotechnology. We are currently in the process of recruiting the new undergraduate student for the second year of the project. We will modernize and offer the undergraduate 'Biotechnology' course in the Spring of 2026. We will also continue to provide internship opportunities to local high-school students in the fields of agricultural biotechnology and genomics. We will also continue to conduct our highly successful Ag-Biotech Day events for the local middle-school students.

Impacts
What was accomplished under these goals? To address Objective-A, we have standardized and conducted combined abiotic stress treatment using sorghum seedlings. For the stress treatment, seeds were surface sterilized with a 20% bleach treatment for 30 min and then rinsed thoroughly with water. Seeds were allowed to germinate for 48 hours on a wet germination paper inside a growth chamber with 50% humidity, 26°C temperature, and 16-8 hours of day-night cycle. Uniformly germinated seedlings were transferred to 0.5x Hoagland solution in culture tubes and were grown in a growth chamber. Combined drought and heat stress was exerted by replacing 0.5x Hoagland solution with 0.5x Hoagland solution supplemented with 30% PEG (to simulate drought-induced osmotic stress) and by incubating culture tubes in a growth chamber at 45°C under constant light for 6 hours. For control samples, 0.5x Hoagland solution was replenished with new 0.5x Hoagland solution, and samples were incubated in a growth chamber at 26°C under constant light for 6 hours. The experiment was done in triplicate, and each replicate consisted of seven uniform seedlings. Sample collection, total RNA isolation, RNA quality assessment, and next-generation sequencing: After the stress treatment, tissue samples were immediately flash frozen in liquid nitrogen and stored at -80°C until further use. Total RNA was isolated using a commercial kit, and RNA quality and quantity were assessed using Qubit. Total RNA samples were used to prepare so-called RNAseq libraries, and libraries were sequenced on the Illumina platform (150nt, paired-end). From the raw reads, low-quality reads, and Illumina adapter sequences were removed, and roughly 36 to 51 million clean reads were obtained for each stress-treated and control sample. Transcriptome analysis was conducted using Salmon and DEseq2. Our transcriptome analysis identified genes displaying up- or down-regulation in response to combined stress. We also compared differentially expressed genes (DEGs) obtained in response to combined stress with DEGs obtained in response to individual stresses and identified shared and unique DEGs in response to combined stress. We have also generated libraries for small RNA sequencing for the combined stress-treated and corresponding control samples and sequenced libraries on the Illumina platform. Altogether, we obtained 9.9 to 14.8 million clean reads per library. We have conducted preliminary bioinformatic analysis of the small RNA seq dataset. Detailed bioinformatic analysis of the small RNAseq data will be conducted in the second year of the project.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: 79th Annual Conference of the Subtropical Agriculture & Environments Society (SAES) in Mission, Texas. miRNAome of Sorghum Seedlings Subjected to Individual and Combined Drought and Heat Stress.
• Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: 79th Annual Conference of the Subtropical Agriculture & Environments Society (SAES) in Mission, Texas. Investigating the role of small RNAs in combined drought and heat stress in sorghum.