Date of Award
Doctor of Philosophy
Plant Pathology and Microbiology
Genetics and Genomics
Steven A. Whitham
Plants, like many other living organisms, evolved circadian clock to increase fitness by synchronizing physiological processes with environmental oscillations. In one hand, the circadian clock helps plants prepare for almost every growth routine, such as germination, flowering, and photosynthesis. On the other hand, the circadian clock regulates plant defense against biotic and abiotic stresses, and these stresses feedback to modulate the clock function. However, most of these findings so far made were from studies of Arabidopsis. Basically, very little is known about soybean (Glycine max) circadian clock and its interactions with stresses.
In Chapter 2, to unveil the impacts of various abiotic stresses on soybean circadian clock, we provided a two-module pipeline consisting of the Molecular Timetable module and the RASL-seq (RNA-Mediated Oligonucleotide Annealing, Selection, and Ligation with Next-Generation Sequencing) module. Using the Molecular Timetable method, we identified 3,695 time-indicating genes in soybean seedlings. Then, using these time-indicating genes, we re-analyzed the publicly available soybean transcriptomes related to abiotic stresses. We found that mild drought caused little changes to the global circadian rhythm while severe drought induced a significant phase shift. And, a 30-minute heat shock was sufficient to cause a phase advancement. However, prolonged heat had a rather mild effect on the global circadian rhythm. These findings suggest that the global circadian rhythm responses to drought and heat are duration-dependent.
In addition to drought and heat, prolonged exposure to iron deficiency caused phase delay of the leaf circadian clock in two near-isogenic lines, Clark and IsoClark. However, in roots, phase of the global circadian clock was advanced in Clark while it was delayed in IsoClark. Alkaline stress also caused organ-specific changes to the global circadian rhythm in the wild soybean, Glycine soja. These findings suggest organ-specific circadian rhythm responses to both iron deficiency and alkaline stresses.
Then, 49 soybean circadian clock genes were surveyed using RASL-seq. It enabled us to identify the clock components targeted by each stress. Lastly, to test whether the stress-induced transcriptional changes of soybean clock genes could lead to changes in clock-controlled physiological outputs, we assessed the effect of abiotic stresses on circadian leaf movement. We found that perturbation in individual or subset of clock components might not necessarily jeopardize clock outputs.
In Chapter 3, I used the two-module pipeline to study the impact of soybean cyst nematode (SCN) on soybean root circadian clock. First, I used the time-indicating genes to re-analyze the publicly available soybean transcriptome data conducted on both SCN feeding and surrounding sites. I found that SCN caused significant changes to the global circadian rhythm in the feeding site. However, circadian rhythm disturbance was not found in the surrounding site. Then, using RASL-seq, I identified that the expression rhythms of 12 circadian genes were modified by SCN. 5 showed significant phase shift and 8 showed significant period changes (1 showed both phase and period changes). Next, I used a live bioluminescence imaging system to confirm the results in soybean hairy roots. The vector carrying a luciferase gene driven by the promoter of the identified circadian gene (promoter-luciferase) was constructed and transformed into soybean hairy roots. I found that only one gene Early Phytochrome Responsive 1 (EPR1) promoter-luciferase activity showed robust circadian rhythm under prolonged constant condition. Then, I performed SCN infection assays and found an obvious phase shift to the EPR1 promoter-luciferase activity after SCN infection. In addition, I also found SCN caused a significant change to the average activity of the CCA1 promoter-luciferase. Overall, I found that the promoter-luciferase activity rhythms of 2 circadian genes were modified by SCN in soybean hairy roots.
On the other hand, I also studied how the root circadian clock regulates soybean defense against SCN. I found that soybean seedlings were more resistant to SCN at dawn than at dusk. Then, I generated the soybean clock mutant hairy root lines by overexpressing one CCA1 gene (OE) and found a disrupted SCN infection rhythm in OE lines. In addition, OE lines are more resistant to SCN than the control lines.
Next, using a time-course RNA sequencing experiment, I identified key circadian rhythmically expressed and non-circadian-rhythm genes in the interaction between soybean root circadian clock and SCN. Roles of these genes still need further investigations.
These results enable us to uncover novel inputs and outputs to the soybean circadian clock. And, these unique responses in soybean demonstrate the necessities to directly study crop circadian clocks, and our discovery may serve as a broadly applicable procedure to facilitate these researches.
Cao, Lijun, "The molecular interactions of soybean circadian clock with abiotic stresses and soybean cyst nematode (Heterodera glycines)" (2019). Graduate Theses and Dissertations. 17415.