Biology subjects

Sapeta, H.

Publications and source records attributed to Sapeta, H..

4 recordsLinked to original sources

bioRxiv2025

High throughput isolation of male gametophyte cells of Solanum lycopersicum var. Micro-Tom by fluorescence-activated cell sorting.

Efficient isolation of male gametes has enabled unprecedented advances in omics research, crucial for elucidating the molecular mechanisms governing male gametogenesis and fertilization. In this study, we developed a method for isolating generative and sperm cells from the economically important crop Solanum lycopersicum. A double fluorescent marker line was generated in the tomato variety Micro-Tom, employing mTurquoise and mScarlet-I fluorescent proteins under the control of promoters exhibiting preferential activity in generative and sperm cells, respectively. Then we developed a protocol that combines male gamete release from pollen of this line and SYTOX Red live/dead cell stain to obtain viable cells by Fluorescence-activated cell sorting. This allows the isolation of generative cells from mature pollen grains, and of sperm cells from pollen tubes after semi-in vivo growth, both in high quantity and purity. Additionally, an unexpected mScarlet-I signal in the vegetative nucleus, that persists until the sperm cells are formed, allows the sorting of vegetative nuclei. We anticipate that our novel double-marker line will accelerate research into tomato male gametogenesis, thereby enhancing efforts to improve the resilience of fertilization processes to climate change.

plant biology↗

bioRxiv2024

OsbHLH089 and OsbHLH094 Modulate OsSLR1 Levels to Maintain Male Reproductive Fitness in Rice

DELLA proteins are a unique class of transcriptional regulators in plants, playing critical roles in diverse biological processes. Far from being only negative regulators of the Gibberellin (GA) signalling pathway, DELLAs act as central signalling hubs due to their versatile binding capacity and responsiveness to GA fluctuations. This adaptability allows DELLAs to interact with a wide array of proteins but also makes their functional study challenging: disruptions in DELLA function lead to pleiotropic effects across multiple pathways. To address this complexity, understanding DELLA interactors provides valuable insights into DELLAs nuanced functions and regulation. Building on this approach, we investigated novel OsSLR1 interactors, OsbHLH089 and OsbHLH094, members of the basic-helix-loop-helix transcription factor family. Our findings reveal that OsbHLH089 and OsbHLH094 are expressed in mature pollen grains, with the Osbhlh089/94 double mutant displaying smaller, indehiscent anthers and non-viable pollen, indicating their redundant role in pollen development during its late stages. Through ChIP-Seq and RNA-Seq analyses, we identified five target genes repressed by OsbHLH089 and OsbHLH094 (OsTDL1A, OsSPS1, OsDGD2{beta}, OspPGM, and OsDPE2) that are essential for pollen and anther development. Interestingly, we also observed the binding of these TFs to the OsSLR1 promoter. Notably, while other target genes were repressed, OsSLR1 was induced, with a significant protein accumulation in the double mutant compared to the Kitaake background in the late stages of pollen development. This suggests that OsSLR1 accumulation may compromise pollen viability, further highlighting the critical regulatory role of OsbHLH089 and OsbHLH094 in repressing OsSLR1 levels for proper pollen and anther development.

plant biology↗

bioRxiv2023

Drought impact on phellem development: identification of novel gene regulators and evidence of photosynthetic activity

Quercus suber (cork oak) is a sustainably exploited forest resource, producing a unique renewable raw material known as cork. With drought events imposing a negative impact on tree vitality, we need more knowledge on the genetic and environmental regulation of cork development to protect the cork sector. We focused on characterizing long-term drought-induced molecular adaptations occurring in stems, and identifying key genetic pathways regulating phellem development. One-year-old cork oak plants were grown for 6 months under well-watered, or water-deficit (WD) conditions and main stems were targeted for histological characterization and transcriptomic analysis. WD treatment impaired secondary growth, by reducing meristem activity at both vascular cambium and phellogen. We analyzed the transcriptional changes imposed by WD in phellem, inner bark, and xylem, and found a global downregulation of genes related to cell division, differentiation, and cell wall biogenesis. Phellem and inner bark showed upregulation of photosynthesis-related genes, highlighting a determinant role of stem photosynthesis in the adaptation to long-term drought. We show that developing phellem cells contain chloroplasts and their abundance increases under WD. Finally, we propose new candidate regulatory genes involved in the regulation of phellogen activity and demonstrate the involvement of phellem in drought-induced bark photosynthesis in young plants. HighlightPhellem development in cork oak is impaired in drought adaptation, by negative regulation of cell division and differentiation programs, while photosynthesis is induced to contributing to CO2 recycling in the stem.

plant biology↗

bioRxiv2021

Phellem translational landscape throughout secondary development in Arabidopsis roots

O_LIThe phellem is a specialized boundary tissue providing the first line of defense against abiotic and biotic stresses in organs undergoing secondary growth. Phellem cells undergo several differentiation steps, which include cell wall suberization, cell expansion and programmed cell death. Yet, the molecular players acting particularly in phellem cell differentiation remain poorly described, particularly in the widely used model plant Arabidopsis thaliana. C_LIO_LIUsing specific marker lines we followed the onset and progression of phellem differentiation in A. thaliana roots, and further targeted the translatome of new developed phellem cells using Translating Ribosome Affinity Purification followed by mRNA sequencing (TRAP-SEQ). C_LIO_LIWe showed that phellem suberization is initiated early after phellogen (cork cambium) division. The specific translational landscape was organized in three main domains related to energy production, synthesis and transport of cell wall components, and response to stimulus. Novel players in phellem differentiation, related to suberin monomer transport and assembly, as well as novel transcription regulators were identified. C_LIO_LIThis strategy provided an unprecedented resolution of the transcriptome of developing phellem cells, giving a detailed and specific view on the molecular mechanisms controlling cell differentiation in periderm tissues of the model plant Arabidopsis. C_LI Significance statementTo improve the understanding of phellem differentiation into a suberized protective layer, we followed the establishment of periderm in Arabidopsis roots and sequenced the phellem-specific translatome. We found that phellem suberization occurs shortly after pericycle cell divisions with the induction of pivotal suberin biosynthesis genes. In parallel, we detected the activation of three central genetic modules acting throughout the phellem differentiation. This study provides a unique and targeted genetic resource for further functional studies of phellem tissues.

plant biology↗