Periodic Reporting for period 1 - Clock-SAM (Transcriptional reprogramming of Clock at Shoot Apical Meristem in regulating plant organogenesis and growth.)
Período documentado: 2019-10-01 hasta 2021-09-30
The increase on food consumption due to current global population growth places a higher demand to the agriculture sector to produce high-yielding crops with reduced inputs. Studies performed in Arabidopsis has demonstrated that circadian clock, which is an endogenous 24-hour time keeping machinery in plants, has a prominent effect on the physiology and development of plants. The circadian clock contributes to a vast developmental output such as flowering time, biomass allocation, photosynthesis, water uptake, temperature and stress responses. Plants with properly functioning clock match the day and night cycle to fix more carbon and grow faster. After germination, the shoot apical meristem produces leaves. The first leaves have a juvenile appearance and the leaves produced later gradually develop more adult characteristics as a result of robust meristem activity. Plant shoot apical meristems (SAM) are responsible for the production and regeneration of the different organs; factors that regulate both biomass and seed production. Despite the importance of stem cell function, the mechanisms controlling the timing of meristem activity in synchronization with the environment remain essentially unknown.
The project provides novel information into the interactions between circadian clock and meristem function. It gives us great insights that the pace at which plants transition from juvenile to adult is tightly regulated by the transcriptional reprogramming controlled by the circadian clock at the shoot meristem. Our studies have shown that the shoot apex clocks are highly coupled or interconnected, as compared to clocks from other tissues. Consistently, clock mutant and over-expressing plants display alterations in SAM function manifested, for instance, in small organ sizes with a considerable reduction of cell numbers in the aerial organs. Clock miss-expression affects the pace of primordia appearance and the control of cell size and number during primordia initiation.
Figure:A simplified model showing the importance of clock synchrony in plants through the SAM. Alteration of clock at SAM regulates various developmental processes throughout the life cycle of plants including early seedling, development, phenology and flowering.
The project provides novel information into the interactions between circadian clock and meristem function. It gives us great insights that the pace at which plants transition from juvenile to adult is tightly regulated by the transcriptional reprogramming controlled by the circadian clock at the shoot meristem. Our studies have shown that the shoot apex clocks are highly coupled or interconnected, as compared to clocks from other tissues. Consistently, clock mutant and over-expressing plants display alterations in SAM function manifested, for instance, in small organ sizes with a considerable reduction of cell numbers in the aerial organs. Clock miss-expression affects the pace of primordia appearance and the control of cell size and number during primordia initiation.
Figure:A simplified model showing the importance of clock synchrony in plants through the SAM. Alteration of clock at SAM regulates various developmental processes throughout the life cycle of plants including early seedling, development, phenology and flowering.
We first studied clock function in plants with altered meristematic activities and analyzed whether it had an effect on clock function at the shoot apex. Circadian rhythms at the shoot apexes were monitored by in vivo luminescence assays to analyze the oscillatory patterns of the promoter activities of clock genes. Our results indicate that perturbations in the meristem activity affect the rhythms of TIMING OF CAB EXPRESSION 1 (TOC1) promoter activity at the shoot apex suggesting that the evening phase clock genes might have a higher correlation with the meristem activity at the shoot apex. The results also suggest that the regulatory morning and evening loops might be disengaged, or at least, the ones connecting TOC1 and CIRCADIAN CLOCK ASSOCIATED (CCA1). Study of plants expressing TOC1 at different tissue layers of shoot meristem in toc1-2 mutant background showed that specific expression of TOC1 in central zone (CZ) was able to partially rescue the long hypocotyl phenotype of toc1-2 mutant, while expressing TOC1 in the organizing center (OC) was not able to rescue the hypocotyl phenotype of the toc1-2 mutant. Thus, these results indicate that expression of TOC1 in specific SAM regions renders different results in the control of clock outputs such as hypocotyl growth. Examination of leaf emergence as a proxy of meristem activity further strengthened the studies on hypocotyl elongation and confirm our conclusions on the specificity of TOC1 function at different SAM zones. Time course analyses of clock gene expression studied by RT-qPCR showed that the targeted expression of TOC1 in CZ cells was able to partially restore the expression of the oscillator genes at the shoot apex. In vivo bioluminescence analyses of rhythms at the shoot apex and leaves showed that the short period phenotype of toc1-2 mutant compared to WT was also restored not only at the shoot apex but also in leaves in plants expressing TOC1 at CZ. Expression of TOC1 at the OC led to arrhythmicity both at the shoot apex and in leaves. These transgenic lines also showed significant alteration in flowering time, one key agronomic trait in agriculture. Overall, our results suggest a crucial connection between meristem function and circadian clock which is tightly regulated to control many agronomically important traits such as plant biomass and flowering. On the other hand, some objectives are delayed and partly accomplished due to the COVID-19 outbreak and subsequent restricted access to research facilities at the host centre due to lockdown and mobility restrictions, measures implemented to help control the Covid-19 pandemic situation in Spain. The lockdown period and the following policies at the host center related to the maximal capacity in the laboratory had a direct negative impact on the generation of the engineered plant resources and their subsequent analyses. Therefore, some work related to the project is still underway and we have also identified some new avenues which holds great potential to be further explored.
The results generated during the progression of the project have been presented at internal seminars and at the “Development retreat” at the host institute. The experimental results will be published in a peer-reviewed paper and the primary data will be made available through public repositories. From the researchers’ point of view, the management of the project and its funds, training activities performed in form of several courses/workshops, and students supervision, has brought valuable leadership qualities and knowledge transfer skills which will help him to become an independent group leader.
The results generated during the progression of the project have been presented at internal seminars and at the “Development retreat” at the host institute. The experimental results will be published in a peer-reviewed paper and the primary data will be made available through public repositories. From the researchers’ point of view, the management of the project and its funds, training activities performed in form of several courses/workshops, and students supervision, has brought valuable leadership qualities and knowledge transfer skills which will help him to become an independent group leader.
Virtually all the aerial parts of the plant derive from stem cells at the SAM. The outcome of this research will act as a foundation to addressing key questions about organ formation and how it can be regulated by circadian clock. The results from this proposal define the role of the circadian clock in the regulation of the SAM function. The innovative nature and impact of our proposal is that our studies have established a direct connection of the circadian timing with SAM-dependent physiological outputs. We have uncovered how the circadian function is able to regulate the timing of leaf emergence and that cell-specific changes in clock function at SAM niches are able to modulate these responses. We have also studied the time of flowering and found that SAM cell-type specific changes in clock function trigger flowering. The impact of our results not only rely on the advancement of knowledge but also, and due to the importance of meristem activity in plant organogenesis and productivity, our studies can be applied in crops of agronomical interest. This is particularly relevant to improve crop productivity in the context to meet the food demand of an increasing population. Once we have the cell specific transcriptional landscape, it can become an important target for breeders to improve crops and also allow plant biologists to further explore how day length information can be translated to crop productivity. The project outcome will serve as a new beginning for future studies in developing resilient crops in response to environmental changes.