Biology subjects

Dobrev, P. I.

Publications and source records attributed to Dobrev, P. I..

10 recordsLinked to original sources

bioRxiv2026

Decarboxylation of indoleacetic acid illuminates its evolution into a plant hormone

The evolution of indoleacetic acid (IAA) into the plant hormone auxin remains unclear. Here, we compared IAA response and metabolism in land plants and their closest living relatives, the streptophyte algae, using chemical treatments, phytohormone profiling, and targeted and untargeted metabolomics. Streptophyte algae showed neither plant-like auxin responses nor canonical IAA metabolism. Here, we demonstrate IAA side-chain decarboxylation as a novel pathway of cellular IAA metabolism, operating in streptophyte algae and land plants alike. Decarboxylation also underpinned IAA's innate photolability and yielded cytotoxic intermediates, which also accumulated endogenously under simulated sunlight. Together, these findings establish IAA decarboxylation as a major, conserved metabolic pathway, and suggest that the metabolic constraints arising from IAA's instability and indirect cytotoxicity contributed to its adoption as a plant hormone.

plant biology↗

bioRxiv2025

The CATION CALCIUM EXCHANGER 4 (CCX4) regulates LRX1-related root hair development through Ca2+ homeostasis

Calcium, as a cellular second messenger, is essential for plant growth. A tip-focused Ca2+ gradient in polarized cells is considered to drive cell expansion. The cell wall polysaccharide pectin is a major Ca2+ binding structure and Ca2+ homeostasis is influenced by the cell wall architecture. LRR-extensin (LRX) proteins are extracellular regulators of cell wall development that are anchored in the cell wall by their extensin domain. The extensin-less LRX1{Delta}E14 variant of the root hair-expressed LRX1 of Arabidopsis induces a dominant-negative effect resulting in aberrant root hairs. In an effort to identify the underlying mechanism of the root hair defect caused by LRX1{Delta}E14, we isolated a suppressor of dominant-negative effect mutant, sune42. It codes for the CATION CALCIUM EXCHANGER 4 (CCX4) that localizes to the Golgi apparatus and was shown to have Ca2+ transport activity. A detailed investigation of the Ca2+ dynamics revealed that LRX1{Delta}E14 coincides with a defect in tip-focused cytoplasmic Ca2+ oscillation, and this effect is alleviated by the sune42 mutation. Additionally, reducing Ca2+ availability influences the LRX1{Delta}E14-induced root hair defect. We conclude that sune42 suppresses the root hair defect in LRX1{Delta}E14 through modulating cytoplasmic Ca2+ dynamics, pointing at the importance of the Golgi apparatus for cellular Ca2+ homeostasis.

plant biology↗

bioRxiv2024

Co-cultivating rice plants with Azolla filiculoides modifies root architecture and timing of developmental stages

Strategies for increasing the yield of rice, the staple food for more than half of the global population, are needed to keep pace with the expected worldwide population increase, and sustainably forefront the challenges posed by climate change. In Southern-East Asian countries, rice farming benefits from the use of Azolla spp. for nitrogen supply. In virtue of the symbiosis with the nitrogen-fixing cyanobacterium Trichormus azollae, Azolla spp. are ferns that release nitrogen into the environment upon decomposition of their biomass. However, if and to what extent actively growing Azolla plants impact on the development of co-cultivated rice plantlets remains to be understood. Here, we show that actively growing Azolla filiculoides plants alter the architecture of the roots and accelerates the differentiation and proliferation of leaves and tillers in co-cultivated rice plants. These changes result from an intimate cross-talk between rice and A. filiculoides, in which hormones and other metabolites released by the fern in the growth medium trigger an alteration in the rice root transcriptome and the hormonal profiles of both roots and leaves. Overall, the present data let us argue that co-cultivation with A. filiculoides might prime rice plants to better deal with both abiotic and biotic stress. HighlightAzolla filiculoides alters the root transcriptome and hormonal balance in both roots and leaves of co-cultivated rice plantlets, thereby interfering with the progression of their developmental programs

plant biology↗

bioRxiv2024

Physiological and Molecular Responses of Projected Future Temperatures on Potato Tuberization

Potato (Solanum tuberosum L.) is one of the most important food crops globally and is especially vulnerable to heat stress. Significant knowledge gaps remain however, in our understanding of the developmental mechanisms associated with tuber responses to heat stress. This study uses whole-plant physiology, transcriptomics, and hormone profiling to gain insights into the mechanisms associated with heat stress impacts on potato tuber development. When plants were grown in projected future temperature conditions, levels of abscisic acid (ABA) were significantly decreased in leaf and tuber tissues while rates of leaf carbon assimilation and stomatal conductance were not significantly affected. While plants grown in elevated temperature conditions initiated more tubers on average per plant, there was a significant decrease (66%) in mature tubers at final harvest. We hypothesize that reduced tuber yields at elevated temperatures are not due to reductions in tuber initiation, but due to impaired tuber filling. Transcriptomic analysis found significant changes in transcript expression for genes related to response to ABA, heat and auxin biosynthetic process. The known tuberization repressor genes SELF PRUNING 5G (StSP5G) and CONSTANS-LIKE1 (StCOL1) were found to be differentially expressed in tubers grown in elevated temperatures. IDENTITY OF TUBER 1 (StIT1) and TIMING OF CAB EXPRESSION 1 (StTOC1) are other known tuberization genes that displayed distinct expression patterns in elevated versus ambient temperatures but were not differentially expressed. This work highlights potential gene targets and key developmental stages associated with tuberization to development more heat tolerant potatoes.

plant biology↗

bioRxiv2024

ER-located PIN5 transporter generates potent auxin sinks driven by the IAA decarboxylation pathway

Auxin is an essential and well-investigated regulator of plant development. Still, many aspects determining its (sub-)cellular distribution, as well as its metabolic turnover, are obscure. PIN5 is a transporter that resides on the endoplasmic reticulum and is presumed to influence internal auxin homeostasis by direct sequestration and subsequent degradation. Due to its distinct expression pattern and incomplete metabolomics analyses, the exact role of PIN5 protein and the identity of downstream auxin degradation products show significant gaps. To this end, we utilized morphologically homogeneous tobacco BY-2 cell cultures. We show that the expression of Arabidopsis thaliana AtPIN5 in the BY-2 system phenocopies so-called auxin starvation defects. Moreover, we reveal that the activity of AtPIN5 leads, in extreme cases, to the broad range of processes accompanying programmed cell death (PCD). Notably, based on the recently updated knowledge on auxin metabolism, we also show that a significant part of auxin metabolites downstream of the AtPIN5 activity are part of the re-emerged auxin decarboxylation pathway. Taking together, we report the direct induction of PCD by auxin stimulus and propose the physiological framework of the auxin decarboxylation route.

plant biology↗

bioRxiv2023

Cytokinin Dehydrogenase in Xylem Sap Reveals A Direct Link Between Cytokinin Metabolism and Long-Distance Transport

Metabolic degradation of plant hormones cytokinins (CKs) co-regulates their homeostasis and signalling. In this work, we employed a large-scale bioinformatical analysis to address a diversity of cytokinin oxidase/dehydrogenase (CKX) substrate specificities previously described in several case studies. We present a three-way correlation of the entire CKX amino acid sequences, a variable motif involved in substrate binding, and subcellular localizations predicted by a deep learning model. This correlation is conserved in monocotyledonous plants, suggesting that the CKX diversity in a single species allows a precise tuning of the CK homeostasis. Following these findings, we detected CKX activity in xylem sap for the first time, using the oat (Avena sativa) as a model plant. Further investigation of the substrate specificity and glycosylation of this xylem-located CKX suggested that it originates in roots. We have identified 27 putative CKXs in oats and attributed the xylem-located activity to the extracellular isoforms AsCKX1a,c,d. Finally, we show that the xylem-located CKX activity responds to the nitrate supply, highlighting its physiological relevance. Taken together, we show that CKX directly modulates root-to-shoot CK translocation through metabolic degradation of the transported CKs.

plant biology↗

bioRxiv2023

Study of auxin metabolism using stable isotope labeling and LCMS; evidence for in planta auxin decarboxylation pathway.

The natural plant hormone auxin indole-3-acetic acid (IAA) influences many physiological processes in plants. Here, the metabolism of IAA was studied in detail using tobacco BY-2 cells as a model and compared with the in planta metabolism in several plant species. A combination of labeled/unlabeled substrate feeding, global untargeted mass spectrometric (MS) scanning, and selective MS filtering allowed the detection of 17 auxin metabolites, 15 of which were identified. Subsequent study of intermediate metabolism and dynamics revealed eight major pathways: three amino acid conjugation pathways with aspartate, glutamate, and glutamine, followed by their 2-oxidation with the help of the DAO enzyme; side-chain glucosyl ester formation; direct 2-oxidation; two decarboxylation pathways; and a pathway producing an unidentified metabolite. Interestingly, the first intermediates of the two decarboxylation pathways, indole-3-carbinol and oxoindole-3-carbinol, were formed outside the cells. We found that the majority of the detected auxin metabolites occur naturally in several plant species and that IAA is their precursor, indicating that the auxin metabolic pathways observed in BY-2 cells also occur in planta. Our finding that the IAA decarboxylation pathway occurs in planta, and the previous reports of auxin activity of some metabolites of this pathway, suggest that at least some of the biological effects of IAA may be explained by its conversion to decarboxylative metabolites.

plant biology↗

bioRxiv2023

Auxin coreceptor IAA17/AXR3 controls cell elongation in Arabidopsis thaliana root by modulation of auxin and gibberellin perception

The nuclear TIR1/AFB - Aux/IAA auxin pathway plays a crucial role in regulating plant growth and development. Specifically, the IAA17/AXR3 protein participates in root development, and the accumulation of its mutant variant, AXR3-1, which cannot bind auxin, leads to severe root growth phenotype and agravitropism. However, the mechanism by which AXR3 regulates cell elongation is not fully understood. Here we show that the inducible expression of AXR3-1 in the Arabidopsis thaliana root triggers excessive cell elongation that is followed by growth arrest of the root. We exploited this effect to reveal the underlying molecular mechanism of AXR3 action. We show that AXR3-1 acts exclusively in the nucleus where it interferes with the nuclear auxin transcriptional pathway, while the rapid cytoplasmic auxin root growth response is not affected. The analysis of the transcriptome of the induced AXR3-1 roots revealed changes in phytohormone perception and homeostasis. We show that the accumulation of AXR3-1 disturbs auxin homeostasis which leads to excessive auxin accumulation. At the same time, the reaction of the AXR3-1 roots to gibberellin is altered. These results show that the IAA17/AXR3 maintains an optimal cell elongation rate by controlling the auxin response, auxin homeostasis and the interplay with gibberellin signaling.

plant biology↗

bioRxiv2021

NON-SPECIFIC EFFECTS OF A CINNAMATE-4-HYDROXYLASE INHIBITOR ON AUXIN HOMEOSTASIS

Chemical inhibitors are often implemented for the functional characterization of genes to overcome the limitations associated with genetic approaches. Although being a powerful tool, off-target effects of these inhibitors are easily overlooked in a complex biological setting. Here we illustrate the implications of such secondary effects by focusing on piperonylic acid (PA), an inhibitor of CINNAMATE-4-HYDROXYLASE (C4H) that is often used to investigate the involvement of lignin during plant growth and development. When supplied to plants, we found that PA is recognized as a substrate by GRETCHEN HAGEN 3.6 (GH3.6), an amido synthetase involved in the formation of the auxin catabolite indole-3-acetic acid (IAA)-Asp. By competing for the same enzyme, PA interferes with auxin conjugation, resulting in an increase in cellular auxin concentrations. These increased auxin levels likely further contribute to an increase in adventitious rooting previously observed upon PA-treatment. Despite the focus on GH3.6 in this report, PA is conjugated by an array of enzymes and their subsequent reduced activity on native substrates could potentially affect a whole set of physiological processes in the plant. We conclude that surrogate occupation of the endogenous conjugation machinery in the plant by exogenous compounds is likely a more general phenomenon that is rarely considered in pharmacological studies. Our results hereby provide an important basis for future reference in studies using chemical inhibitors.

plant biology↗

bioRxiv2020

A role of DAO1 in oxidation of IAA amino acid conjugates revealed through metabolite, high throughput transcript and protein level analysis.

Auxin metabolism is, together with auxin transport, a key determinant of auxin signalling output in plant cells, yet details on the underlying mechanisms and factors involved are still largely unknown. Processes involved in the auxin metabolism are subject to regulation based on numerous signals, including auxin concentration itself. Altered auxin availability and the subsequent changes of auxin metabolite profiles can therefore elucidate the function and regulatory role of individual elements in the auxin metabolic machinery. After analysing auxin metabolism in auxin dependent tobacco BY-2 cell line grown in presence or absence of synthetic auxin 2,4-D we found that both conditions were similarly characterized by very low levels of endogenous indole-3-acetic acid (IAA) and its metabolites. However, metabolic profiling after exogenous application of IAA uncovered that the concentration of N-(2-oxindole-3-acetyl)-L-aspartic acid (oxIAA-Asp), the most abundantly formed auxin metabolite in the control culture, dramatically decreased in auxin-starved conditions. To describe the molecular mechanism behind this regulation, we analysed transcriptome and proteome changes caused by auxin starvation. While no changes in the expression of auxin biosynthetic machinery were observed, many genes related to auxin conjugation and degradation showed differential expression. Selected putative auxin glycosylating enzymes as well as members of the Gretchen Hagen 3 gene family involved in auxin amino acid conjugation showed both up- and down-regulation. Contrarily to that, all tobacco homologs of Arabidopsis thaliana DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1), known to be responsible for the formation of oxIAA from IAA, showed significant downregulation at both transcript and protein levels. To validate the role of DAO1 in auxin metabolism, we performed auxin metabolite profiling in BY-2 mutants carrying either siRNA-silenced or CRISPR-Cas9-mutated NtDAO1, as well as in dao1-1 Arabidopsis thaliana plants. Both mutants showed not only expectedly lower levels of oxIAA, but also significantly lower abundance of oxidated amino acid conjugates of IAA (oxIAA-Asp). Our results thus represent the first direct evidence on DAO1 activity on IAA amino acid conjugates. Statement of significanceHere we present an analysis of auxin metabolism on metabolite, transcript and protein levels in tobacco BY-2 cell line, collectively identifying oxidation of IAA amino acid conjugates as a new role of DIOXYGENASE FOR AUXIN OXIDATION 1 within an auxin-level-responsive metabolic system.

plant biology↗