Biology subjects

Robert, H. S.

Publications and source records attributed to Robert, H. S..

8 recordsLinked to original sources

bioRxiv2026

EPF/EPFL Peptide Signaling as a Regulatory Node Linking Stomatal Development to Drought and Heat Tolerance in Tomato (Solanum lycopersicum L.)

Stomatal development and function are tightly regulated by signaling peptides with distinct, overlapping, or antagonistic roles in gas exchange and water balance. EPIDERMAL PATTERNING FACTOR (EPF) / EPF-LIKE (EPF/EPFL) family is a founding member of the secreted peptide family that regulates stomatal patterning. The roles of EPF/EPFL genes in tomato responses to drought, heat, and combined drought-heat stress remain largely unexplored. To characterize the EPF/EPFL gene family and elucidate its potential regulatory roles in stomatal development, 15 family members were identified in S. lycopersicum L. All 15 candidate SlEPF proteins contained an EPF-type domain. Of these, 13 exhibited specific matches to the EPF domain, whereas SlEPF6 was classified within the EPF superfamily and SlEPF11 within the STOMAGEN-like superfamily. Phylogenetic analysis grouped EPF genes from diverse plant species into two major clades (A and B), each comprising distinct evolutionary subgroups. GO annotation indicated that most SlEPF genes were involved in stomatal complex development and guard cell differentiation, with molecular functions associated with MAP kinase activity, receptor serine/threonine kinase binding, and receptor ligand activity. Promoter analysis of SlEPF genes identified diverse cis-regulatory elements potentially involved in the regulation of environmental stress responses and plant development. Expression analysis of five putative stress-responsive genes (SlEPF5, SlEPF8, SlEPF9, SlEPF10, and SlEPF13) revealed distinct transcriptional patterns in the heat-tolerant drought-sensitive Money Maker (MM) and drought-tolerant Ailsa Craig (AC) tomato genotypes under drought, heat, and combined drought-heat stress. In MM, SlEPF5 and SlEPF13 were consistently downregulated across all stress conditions, whereas SlEPF8 was strongly induced under drought and combined stress. In contrast, AC showed broader transcriptional activation, particularly under heat and combined stress. Overall, these contrasting expression profiles suggest genotype- and stress-dependent regulation of SlEPF genes, highlighting their potential involvement in tomato abiotic stress responses.

plant biology↗

bioRxiv2026

Development of auxin reporters in oilseed rape (Brassica napus)

Auxin is a key phytohormone that regulates all aspects of plant growth, development, and environmental responses, making the precise analysis of its distribution and signaling essential for understanding plant adaptation and physiological processes. However, despite the agricultural importance of oilseed rape (Brassica napus), the lack of robust, species-specific molecular tools limits detailed studies of hormone signaling in this crop. Here, we developed and characterized reporter systems for the sensitive visualization and quantification of auxin distribution and signaling in B. napus. The DR5cc auxin signaling reporter and a novel synthetic auxin-responsive reporter, BIP3, assembled from promoter fragments of three oilseed rape IAA genes, were generated to drive GUS expression. In hairy roots, both reporters showed auxin-responsive expression in the root apical meristem that became broader after auxin treatment. In transgenic seedlings, flowers at anthesis, and 12-day-old embryos, DR5cc exhibited a more defined expression pattern than BIP3. To monitor real-time auxin dynamics under abiotic stress, DR5cc fluorescent reporters were employed in hairy roots. Mannitol and NaCl treatments induced a time-dependent increase in fluorescence, peaking at 6-12 h before returning to basal levels after 24 h. Furthermore, dual-reporter assays enabled simultaneous monitoring of auxin and cytokinin signaling, revealing distinct hormone-specific spatial responses in hairy roots. Finally, we established a quantitative DII (qDII) reporter system using degron domains from B. napus Aux/IAA proteins, providing a high-resolution quantitative readout of auxin depletion. Together, these reporter systems enable spatial, temporal, and quantitative analyses of auxin dynamics during development and stress adaptation in oilseed rape.

plant biology↗

bioRxiv2026

Adaptive response to long-term high temperatures during the reproductive development in Arabidopsis thaliana

Increasing global temperatures and the rising frequency of heat waves pose a significant threat to plant reproduction. The reproductive phase is particularly sensitive to heat stress, yet the underlying mechanisms regulating thermotolerance during this stage remain insufficiently understood, despite significant advcances in its understanding during vegetative growth. Heat stress responses are largely controlled by heat shock factors (HSFs) and their downstream targets, including heat shock proteins (HSPs). Among these, HSP101 is essential for acquired thermotolerance and recovery from stress, while HEAT SHOCK BINDING PROTEIN (HSBP) acts as a negative regulator of HSF activity, modulating the heat shock response. Here, we investigated the impact of elevated temperature regimes on the reproductive development of Arabidopsis thaliana, with a particular focus on pollen development and fertility. Our results show that heat stress negatively affects pollen development in a dose-dependent manner, leading to reduced reproductive success. We confirmed the critical role of HSP101 in reproductive thermotolerance using the hot1-3 mutant, deficient in HSP101. Furthermore, we provide evidence that the hot1-3 mutant is tetraploid. The origin of this event is unknown, but it is tempting to speculate that disruption of heat stress responses and interference with meiotic processes may lead to whole genome duplication. Overall, this study provides new insights into the regulation of plant reproductive development under heat stress and highlights the importance of HSP101 in maintaining fertility. These findings contribute to a better understanding of plant responses to rising temperatures and may inform strategies to enhance crop resilience under climate change. Main ConclusionFlowering Arabidopsis plants adapt to long-term high temperature by shortening the flowering period and reducing their fertility. The study also demonstrated that the commonly used hot1-3 mutant is tetraploid.

plant biology↗

bioRxiv2024

Long-term high temperatures affect seed maturation and seed coat integrity in Brassica napus

Temperatures above the optimum growth threshold affect seed development, accelerating embryo development and producing seeds with ruptured seed coats. However, the underlying mechanisms of this effect remain unclear. To investigate temperature-induced seed coat rupture, we used a multidisciplinary approach combining detailed phenotyping, transcriptomics, histology, immunolabelling, cell wall profiling and nanoindentation-based mechanics of the seed coat in oilseed rape (Brassica napus). Our data reveal that high temperatures accelerate embryo growth, resulting in larger embryos without a compensatory increase in overall seed size. This rapid embryo expansion may exert elevated mechanical stress on the seed coat cells, significantly thinning the seed coat layers. Concurrently, prolonged exposure to high temperatures drives premature biochemical maturation of the seed coat, characterized by accumulation of demethylesterified pectin. Nanoindentation analysis demonstrated that these changes may have compromised the structural integrity of seed coat cell walls. Ultimately, the weakened seed coat may not withstand the internal tension imposed by the fast-growing embryo, leading to seed coat rupture and reduced seed quality. Our work on seed maturation and the mechanism of heat-induced seed coat rupture provides valuable insights for future research into breeding thermotolerant Brassica napus, crucial in the context of a changing climate. HIGHLIGHTSO_LIGrowth of Brassica napus under long-term high temperatures accelerated embryo growth and led to seed coat rupture in approximately half of the seeds. C_LIO_LIPathways linked to cell wall modification were found to be significantly differentially regulated at high temperatures, accompanied by increased pectin demethylesterification in the seed coat. C_LIO_LIA lower reduced modulus and hardness indicate that the surface of HT seeds is mechanically more compliant and softer. Together with the reduced thickness of the seed coat layers, such a seed coat, as a whole structure, is mechanically weaker and less able to resist deformation imposed by embryo growth, resulting in seed coat rupture. C_LI

plant biology↗

bioRxiv2024

MONOPTEROS isoform MP11ir role during somatic embryogenesis in Arabidopsis thaliana

Auxin is crucial for plant morphogenesis, including in vivo and in vitro embryo development. Exogenous auxin application is necessary for inducing embryogenic responses in in vitro cultured explants of Arabidopsis and other plants. Thus, components of auxin transport, signaling, and metabolism are key to somatic embryogenesis. AUXIN RESPONSE FACTOR (ARF) transcription factors bind to auxin response elements to control auxin-responsive gene expression and are vital in somatic embryo regeneration. ARFs are often repressed by AUXIN/INDOLE-3-ACETIC ACIDs (Aux/IAAs). MONOPTEROS (MP)/ARF5 is especially important in the embryogenic transition, being highly expressed during somatic embryogenesis; its mutant cannot develop somatic embryos. The MP11ir transcript, an alternatively spliced variant of MP, produces a truncated protein missing the Phox and Bem1p (PB1) domain, crucial for ARF-Aux/IAA dimerization. This renders the MP11ir isoform insensitive to Aux/IAA repression, suggesting an auxin-independent regulation. High levels of MP11ir transcript are observed during auxin- and trichostatin A-dependent induction of somatic embryogenesis. Both MP and MP11ir are essential for embryo regeneration in the mpS319 mutant. However, overexpression of a truncated MP protein ({Delta}ARF5) lacking the PB1 domain inhibits somatic embryogenesis, resulting in callus formation instead of somatic embryos. Overexpression of {Delta}ARF5, lack of MP protein (mp mutant), and interference with MP action by the auxin-resistant BODENLOS (BDL) protein affect the expression of the genes TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1), TAA1-RELATED 1 (TAR1), YUCCA3 (YUC3), YUC5 and YUC8, which are involved in auxin biosynthesis. The partial complementation of mpS319 by the expression of MP11ir may be due to the restoration of the expression of TAA1, TAR1, YUC3, YUC5, and YUC8 to the levels observed in the wild-type genotype Col-0. The presence of MP protein alone or in combination with MP11ir in mpS319 restored the expression of all analyzed genes compared to Col-0. Our results suggest that the analyzed auxin-related genes could be potential targets of MP11ir and/or MP. Consequently, {Delta}ARF5 overexpression alters auxin homeostasis and endogenous auxin levels, hindering embryogenic transition.

plant biology↗

bioRxiv2024

Genome editing in almond: A CRISPR-based approach through hairy root transformation

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) technology has revolutionized genome manipulation for crop enhancement, providing a powerful toolkit. However, the tissue culture and plant regeneration steps that are critical to the CRISPR/Cas editing framework are often challenging, especially in some woody plant species that exhibit substantial resistance to these procedures. To address this, we have developed an injection-based protocol for inducing hairy roots in almond (Prunus dulcis, syn. Prunus amygdalus), a species known for its recalcitrance to conventional transformation methods. Notably, the hairy root induction method also proved effective in almond x peach hybrids. To evaluate its utility for gene functional analysis, we combined the hairy root transformation system with CRISPR/Cas9 gene editing technology, targeting two transcription factor genes (ERF74 and GAI). Our efforts resulted in transformants with target knock-out, suggesting the potential of this genetic transformation technology as a valuable tool for future routine gene function studies in almond.

plant biology↗

bioRxiv2022

Comparing the efficiency of six clearing methods in developing seeds of Arabidopsis thaliana

Tissue clearing methods eliminate the need for sectioning, thereby helping better understand the 3D organization of tissues and organs. In the past fifteen years, clearing methods have been developed to preserve endogenous fluorescent protein tags. Some of these methods (ClearSee, TDE, PEA-Clarity, etc.) were adapted to clear various plant species, with the focus on roots, leaves, shoot apical meristems, and floral parts. However, these methods have not been used in developing seeds beyond the early globular stage. Tissue clearing is problematic in post-globular seeds due to various apoplastic barriers and secondary metabolites. In this study, we compared six methods for their efficiency in clearing Arabidopsis thaliana seeds at post-globular embryonic stages. Three methods (TDE, ClearSee, and ClearSee alpha) have been already reported in plants whereas the others (fsDISCO, FAST9, and CHAPS clear) are used in this context for the first time. These methods were assessed for seed morphological changes, clearing capacity, removal of tannins, and spectral properties. We tested each method in seeds from globular to mature stages. The pros and cons of each method are listed herein. ClearSee alpha appears to be the method of choice as it preserves seed morphology and prevents tannin oxidation. However, FAST9 with 60% iohexol as a mounting medium is faster, clears better, and appears suitable for embryonic shape imaging. Our results may guide plant researchers to choose a suitable method for imaging fluorescent protein-labeled embryos in intact Arabidopsis seeds. Key messageClearSee alpha and FAST9 were optimized for imaging Arabidopsis seeds up to the torpedo stages. The methods preserve the fluorescence of reporter proteins and seed shape, allowing phenotyping embryos in intact seeds.

plant biology↗

bioRxiv2021

Effects of long-term high-temperature stress on reproductive growth and seed development in development in Brassica napus

Brassica napus is the second most important oilseed crop worldwide. Increasing average temperatures and extreme weather have a severe impact on rapeseed yield. We determined the response of three cultivars to different temperature regimes (21/18 {degrees}C, 28/18 {degrees}C and 34/18 {degrees}C), focusing on the plant appearance, seed yield, seed quality, seed viability, and embryo development. Our microscopic observations identified that embryo development is affected by high temperatures. We noticed an acceleration of its development, in addition to pattern defects. Reduced fertilization rate, increased abortion rate, and preharvest sprouting would be responsible for the low seed yield at the high-temperature regime. Hormone profiling indicates that reduced auxin levels in young seeds may cause the observed embryo pattern defects. Moreover, reduced seed dormancy may result from low ABA and IAA levels in mature seeds. Glucosinolates and oil composition measurements suggest reduced seed quality. These identified cues help understand seed thermomorphogenesis and pave the way to the development of thermoresilient rapeseed plants. HighlightBrassica napus flowering plants growth at high temperatures accelerates embryo development, causing a high seed abortion rate and reduced seed quality. Temperature-reduced ABA levels cause premature seed sprouting.

plant biology↗