Ahmed Ismail’s Work in the Hibberd Lab

C₄ photosynthesis allows increased water use efficiency and hence underpins significant improvements in crop yield. A full understanding of this remarkable phenomenon would facilitate water efficient and productive crops to be engineered in the future. In this current H2020 project (#735105), C₄ Sorghum bicolor was used to better understand molecular events associated with the induction of C₄ photosynthesis. To do so, changes in the transcriptome of sorghum leaves were assessed during de-etiolation at 0, 0.5, 2, 4, 6, and 12 h after light exposure. The data were subjected to the DESeq2 pipeline, and through pairwise comparisons between consecutive time-points, 9091 transcripts were identified as being differentially expressed. Of these, 1032 transcripts showed a response within 30 minutes of exposure to light, and the greatest response was seen between 0.5 and 2 hours when 6323 transcripts were differentially expressed. At each time point, the number of up-regulated and down-regulated transcripts were similar, except after 0.5 hours of exposure to light when transcripts that increased in abundance were approximately 5 times more common than those showed a reduction in transcript abundance. Web-based Clust tool classified 4064 genes into 20 clusters, which could then be manually placed into five groups. To provide a broad overview of the types of genes that were found in each of these clusters, Gene Ontology (GO) analyses were performed. Consistent with previous knowledge, GO terms for biological processes of photosynthesis (GO:0015979) were over-represented in the light-upregulated Clusters 4 and 9. In addition, Cluster 9 was enriched in GO terms for plastid organization (GO:0009657), chloroplast organization (GO:0009658), light reactions (GO:0019684), plastid membrane organization (GO:0009668), thylakoid membrane organization (GO:0010027), photosynthetic electron transport chain (GO:0009767), chlorophyll metabolic process (GO:0015994), chloroplast RNA modification (GO:1900865), plastid translation (GO:0032544), and chloroplast RNA processing (GO:0031425). In agreement with these GO terms relating to biological processes, Clusters 4, 5, 9, and 10 were enriched in cellular components GO terms for plastid (GO:0009536), plastid part (GO:0044435), chloroplast (GO:0009507 and GO:0044434) and other plastid/chloroplast components GO terms. Lastly, GO terms for the photosystem II oxygen evolving complex (GO:0009654), photosystem (GO:0009521), photosystem II (GO:0009523), plastid membrane (GO:0042170), NAD(P)H dehydrogenase complex (plastoquinone) (GO:0010598), and photosystem I reaction center (GO:0009538) were over-represented in Cluster 9. In addition, the comparison of these new data with existing publicly available datasets for specific cell types of the sorghum leaf (Covshoff et al., 2013, Emms et al., 2016). Finally, the complementary datasets were then interrogated for transcription factors that showed behaviours that could explain the induction of photosynthesis gene expression in sorghum. This has generated a list of candidate transcription factors that may well control this fundamental process in this important crop. Analysis of such lists of candidates is non-trivial in model organisms such as A. thaliana, and even less simple in crops such as sorghum. Thus, to provide greater insight into which transcription factors may be important, we implemented ATAC-seq across the same time course to define the DNA regions that were accessible to transcription factor binding, and the actual cis-elements bound by transcription factors in vivo. Here, purification of total nuclei was carried out using sucrose sedimentation, and for each biological sample ~50,000 nuclei used for ATAC-seq that is currently being analysed. These data will be interrogated to test the hypothesis that genes of the C₄ pathway evolved to become induced by light in C₄ leaves.

A review article was published “Salt Stress Signals on Demand: Cellular Events in the Right Context” (Int. J. Mol. Sci. 2020, 21(11), 3918; https://doi.org/10.3390/ijms21113918).