Showing papers by "Klaus Palme published in 2021"

Flavonol-mediated stabilization of PIN efflux complexes regulates polar auxin transport.

Abstract: The transport of auxin controls the rate, direction and localization of plant growth and development. The course of auxin transport is defined by the polar subcellular localization of the PIN proteins, a family of auxin efflux transporters. However, little is known about the composition and regulation of the PIN protein complex. Here, using blue-native PAGE and quantitative mass spectrometry, we identify native PIN core transport units as homo- and heteromers assembled from PIN1, PIN2, PIN3, PIN4 and PIN7 subunits only. Furthermore, we show that endogenous flavonols stabilize PIN dimers to regulate auxin efflux in the same way as does the auxin transport inhibitor 1-naphthylphthalamic acid (NPA). This inhibitory mechanism is counteracted both by the natural auxin indole-3-acetic acid and by phosphomimetic amino acids introduced into the PIN1 cytoplasmic domain. Our results lend mechanistic insights into an endogenous control mechanism which regulates PIN function and opens the way for a deeper understanding of the protein environment and regulation of the polar auxin transport complex.

Auxin biosynthesis and cellular efflux act together to regulate leaf vein patterning.

Abstract: Our current understanding of vein development in leaves is based on canalization of the plant hormone auxin into self-reinforcing streams which determine the sites of vascular cell differentiation. By comparison, how auxin biosynthesis affects leaf vein patterning is less well understood. Here, after observing that inhibiting polar auxin transport rescues the sparse leaf vein phenotype in auxin biosynthesis mutants, we propose that the processes of auxin biosynthesis and cellular auxin efflux work in concert during vein development. By using computational modeling, we show that localized auxin maxima are able to interact with mechanical forces generated by the morphological constraints which are imposed during early primordium development. This interaction is able to explain four fundamental characteristics of midvein morphology in a growing leaf: (i) distal cell division; (ii) coordinated cell elongation; (iii) a midvein positioned in the center of the primordium; and (iv) a midvein which is distally branched. Domains of auxin biosynthetic enzyme expression are not positioned by auxin canalization, as they are observed before auxin efflux proteins polarize. This suggests that the site-specific accumulation of auxin, as regulated by the balanced action of cellular auxin efflux and local auxin biosynthesis, is crucial for leaf vein formation.

AtNSF regulates leaf serration by modulating intracellular trafficking of PIN1 in Arabidopsis thaliana.

Abstract: In eukaryotes, N-ethylmaleimide-sensitive factor (NSF) is a conserved AAA+ ATPase and a key component of the membrane trafficking machinery that promotes the fusion of secretory vesicles with target membranes. Here, we demonstrate that the Arabidopsis thaliana genome contains a single copy of NSF, AtNSF, which plays an essential role in the regulation of leaf serration. The AtNSF knock-down mutant, atnsf-1, exhibited more serrations in the leaf margin. Moreover, polar localization of the PIN-FORMED1 (PIN1) auxin efflux transporter was diffuse around the margins of atnsf-1 leaves and root growth was inhibited in the atnsf-1 mutant. More PIN1-GFP accumulated in the intracellular compartments of atnsf-1 plants, suggesting that AtNSF is required for intracellular trafficking of PIN between the endosome and plasma membrane. Furthermore, the serration phenotype was suppressed in the atnsf-1 pin1-8 double mutant, suggesting that AtNSF is required for PIN1-mediated polar auxin transport to regulate leaf serration. The CUP-SHAPED COTYLEDON2 (CUC2) transcription factor gene is up-regulated in atnsf-1 plants and the cuc2-3 single mutant exhibits smooth leaf margins, demonstrating that AtNSF also functions in the CUC2 pathway. Our results reveal that AtNSF regulates the PIN1-generated auxin maxima with a CUC2-mediated feedback loop to control leaf serration. This article is protected by copyright. All rights reserved.

Estimation of cell cycle kinetics in higher plant root meristem with cellular fate and positional resolution.

Abstract: Root development is a complex spatial-temporal process that originates in the root apical meristem (RAM). To keep the organ’s shape in harmony, the different cell files’ growth must be coordinated. Thereby, diverging kinetics of cell growth in these files may be obtained not only by differential cell growth but also by local differences in cell proliferation frequency. Understanding potential local differences in cell cycle duration in the RAM requires a quantitative estimation of the underlying mitotic cell cycle phases’ timing at every cell file and every position. However, so far, precise methods for such analysis are missing. This study presents a robust and straightforward method to determine the duration of the cell cycle’s key stages in all cell layers in a plant’s root simultaneously. The technique combines marker-free experimental techniques based on detection of incorporation of 5-ethynyl-2’-deoxyuridine (EdU) and mitosis with a high-resolution plant phenotyping platform to analyze all key cell cycle events’ kinetics. In the Arabidopsis thaliana L. RAM S-phase duration was found to be as short as 18-20 minutes in all cell files. But subsequent G2-phase duration depends on the cell type/position and varies from 3,5 hours in the pericycle to more than 4,5 hours in the epidermis. Overall, S+G2+M duration in Arabidopsis under our condition is 4 hours in the pericycle and up to 5,5 hours in the epidermis. Endocycle duration was determined as the time required to achieve 100% EdU index in the transition zone and estimated as 3-4 hours. Besides Arabidopsis, we show that the presented technique is applicable also to root tips of other dicot and monocot plants (tobacco (Nicotiana tabacum L.), tomato (Lycopersicon esculentum L.) and wheat (Triticum aestivum L.)).

The role of AUX1 during lateral root development in the domestication of the model C4 grass Setaria italica

Abstract: C4 photosynthesis increases the efficiency of carbon fixation by spatially separating high concentrations of molecular oxygen from rubisco. The specialized leaf anatomy required for this separation evolved independently many times. C4 root systems are highly branched, an adaptation thought to support high rates of photosynthesis; however, little is known about the molecular mechanisms that have driven the evolution of C4 root system architecture (RSA). Using a mutant screen in the C4 model plant Setaria italica, we identify siaux1-1 and siaux1-2 as RSA mutants, and use CRISPR/cas9-mediated genome editing and overexpression to confirm the importance of the locus. As AUX1 is not necessary for lateral root emergence in S. viridis, the species from which S. italica was domesticated, we conducted an analysis of auxin responsive elements in the promoters of auxin-responsive gene families in S. italica, and explore the molecular basis of SiAUX1’s role in seedling development using an RNAseq analysis of wild type and siaux1-1 plants. Finally, we use a root coordinate system to compare cell-by-cell meristem structures in siaux1-1 and wild type Setaria plants, observing changes in the distribution of cell volumes in all cell layers and a dependence in the frequency of protophloem and protoxylem strands on siAUX1.

Estimation of cell cycle kinetics in higher plant root meristem links organ position with cellular fate and chromatin structure

Abstract: Plant root development is a complex spatial-temporal process that originates in the root apical meristem (RAM). To shape the organ’s structure signaling between the different cells and cell files must be highly coordinated. Thereby, diverging kinetics of chromatin remodeling and cell growth in these files need to be integrated and balanced by differential cell growth and local differences in cell proliferation frequency. Understanding the local differences in cell cycle duration in the RAM and its correlation with chromatin organization is crucial to build a holistic view on the different regulatory processes and requires a quantitative estimation of the chromatin geometry and underlying mitotic cell cycle phases’ timing at every cell file and every position. Unfortunately, so far precise methods for such analysis are missing. This study presents a robust and straightforward pipeline to determine in parallel the duration of cell cycle’s key stages in all cell layers of a plant’s root and their nuclei organization. The methods combine marker-free techniques based on the detection of the nucleus, deep analysis of the chromatin phase transition, incorporation of 5-ethynyl-2′-deoxyuridine (EdU), and mitosis with a deep-resolution plant phenotyping platform to analyze all key cell cycle events’ kinetics. In the Arabidopsis thaliana L. RAM S-phase duration was found to be as short as 20-30 minutes in all cell files. The subsequent G2-phase duration however depends on the cell type/position and varies from 3.5 hours in the pericycle to more than 4.5 hours in the epidermis. Overall, S+G2+M duration in Arabidopsis under our condition is 4 hours in the pericycle and up to 5.5 hours in the epidermis. Endocycle duration was determined as the time required to achieve 100% EdU index in the transition zone and estimated to be in the range of 3-4 hours. Besides Arabidopsis, we show that the presented technique is applicable also to root tips of other dicot and monocot plants (tobacco (Nicotiana tabacum L.), tomato (Lycopersicon esculentum L.) and wheat (Triticum aestivum L.).

Estimation of differential cell cycle kinetics in higher plant root meristem with cellular fate and positional resolution

Abstract: Plant root development is a complex spatial-temporal process that originates in the root apical meristem (RAM). To shape the organ9s structure signaling within the different cells and the different cell files must be coordinated. Thereby, diverging kinetics of cell growth in these files needs to be integrated with differential cell growth and local differences in cell proliferation frequency. Understanding the local differences in cell cycle duration in the RAM is crucial to build a holistic view on the different regulatory processes and requires a quantitative estimation of the underlying mitotic cell cycle phases9 timing at every cell file and every position. Unfortunately, so far precise methods for such analysis are missing. This study presents a robust and straightforward pipeline to determine simultaneously the duration of the cell cycle9s key stages in all cell layers of a plant9s root. The technique combines marker-free experimental techniques based on detection of incorporation of 5-ethynyl-2′-deoxyuridine (EdU) and mitosis with a deep-resolution plant phenotyping platform to analyze all key cell cycle events9 kinetics. In the Arabidopsis thaliana L. RAM S-phase duration was found to be as short as 18-20 minutes in all cell files. The subsequent G2-phase duration however depends on the cell type/position and varies from 3.5 hours in the pericycle to more than 4.5 hours in the epidermis. Overall, S+G2+M duration in Arabidopsis is 4 hours in the pericycle and up to 5.5 hours in the epidermis. Endocycle duration was determined as the time required to achieve 100% EdU index in the transition zone and estimated to be in the range of 3-4 hours. Besides Arabidopsis, we show that the presented technique is applicable also to root tips of other dicot and monocot plants (tobacco (Nicotiana tabacum L.), tomato (Lycopersicon esculentum L.) and wheat (Triticum aestivum L.)).