An Arabidopsis Prolyl 4 Hydroxylase Is Involved in the Low Oxygen Response

Konkina, Anna; Klepadlo, Mariola; Lakehal, Abdellah; Zein, Zein El; Krokida, Afroditi; Mina, Botros; Iakovidis, Michail; Chernobavskiy, Pavel; Zerroumda, Mohamed Elfatih; Tsanakas, George; Petrakis, Nikos; Dourou, Athanasia-Maria; Kalaitzis, Panagiotis

doi:10.3389/fpls.2021.637352

Cited by 10 publications

(11 citation statements)

References 68 publications

(97 reference statements)

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“…This is an indication that the deleted P4H contributes considerably to the prolyl-hydroxylation of the respective HRGP. An effect of a P4H KO on cell wall protein expression was also observed in Arabidopsis where AGP12 was downregulated in a P4H3 mutant, indicating that the presence of P4Hs is linked with transcriptional regulation of AGPs (Konkina et al, 2021).…”

Section: Discussionmentioning

confidence: 91%

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Differential prolyl hydroxylation by six Physcomitrella prolyl-4 hydroxylases

Rempfer,

Hoernstein,

van Gessel

et al. 2024

Preprint

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The hydroxylation of proline residues to 4-trans-hydroxyproline (Hyp) is a common post-translational protein modification in plants, mediated by prolyl 4-hydroxylases (P4Hs). Hyps predominantly occur in a group of cell wall proteins, the Hydroxyproline-rich glycoproteins (HRGPs), where they are frequently O-glycosylated. While prolyl-hydroxylation and O-glycosylation are important, e.g. for cell wall stability, they are undesired on plant-made pharmaceuticals. Sequence motifs recognized for prolyl-hydroxylation derived from vascular plants were proposed but did not include data from mosses, such as Physcomitrella. Here, a phylogenetic reconstruction of plant P4Hs identified six P4Hs in four subfamilies in mosses. We analysed the amino acid sequences and structural environments around Hyps in Physcomitrella utilizing 73 Hyp sites in 24 secretory proteins from multiple MS/MS datasets, and found that prolines in close proximity to other prolines, alanine, serine, threonine and valine were preferentially hydroxylated. About 95% of the Hyp sites were predictable with a combination of previously defined motifs and methods. In our data, AOV (Ala-Hyp-Val) was the most frequent prolyl-hydroxylation pattern. Additionally, short arabinose chains were attached to Hyps in two cell-wall pectinesterases. A combination of 443 AlphaFold structure models and our MS data of peptides with nearly 3000 proline sites found Hyps predominantly on protein surfaces in disordered regions. Moss-produced human erythropoietin (EPO) exhibited plant-specific O-glycosylation with arabinose chains on two Hyps. This modification was significantly reduced in a P4H1 single knock-out (KO) Physcomitrella mutant. Quantitative proteomics after isotope labelling with different P4H-KO moss mutants revealed specific changes in the amount of proteins, including HRGPs, and modified prolyl-hydroxylation pattern from the mutants, suggesting a differential function of the six Physcomitrella P4Hs. Quantitative RT-PCR proved a differential effect of single P4H KOs on the expression of the other five p4h genes, suggesting a partial compensation of the mutation. AlphaFold-Multimer models for Physcomitrella P4H1 and its target EPO peptide superposed with the crystal structure of Chlamydomonas P4H1 and a peptide substrate suggested significant amino acids in the active centre of the enzyme that form H-bonds with the peptide substrate, and revealed differences between P4H1 and the other five Physcomitrella P4Hs.

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Section: Discussionmentioning

confidence: 91%

“…Arabidopsis P4H5 clusters with Physcomitrella P4H5 and P4H6 and hydroxylates all except the last proline in Ser-(Pro) 4 extensin motifs (Velasquez et al, 2015). Another P4H from this cluster is Arabidopsis P4H3 that plays a role in the response to low oxygen (Konkina et al, 2021).…”

Section: Discussionmentioning

confidence: 99%

Differential prolyl hydroxylation by six Physcomitrella prolyl-4 hydroxylases

Rempfer,

Hoernstein,

van Gessel

et al. 2024

Preprint

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“…Thus, even a triple A. thaliana mutant p4h2 , 5 , 13 was not more affected in the root hairs growth than a single p4h5 mutant showing redundancy of prolyl-4-hydroxylases. Furthermore, until now, no mutants in prolyl-4-hydroxylases were shown to be lethal [ 64 , 65 ]. Since 3,4-DHP is a selective inhibitor of prolyl-4-hydroxylases, inactivation of all of them may result in significant changes in the cell wall structure, which may lead to cell death.…”

Section: Discussionmentioning

confidence: 99%

3,4-Dehydro-L-proline Induces Programmed Cell Death in the Roots of Brachypodium distachyon

Piński

Betekhtin

Kwaśniewska

et al. 2021

IJMS

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As cell wall proteins, the hydroxyproline-rich glycoproteins (HRGPs) take part in plant growth and various developmental processes. To fulfil their functions, HRGPs, extensins (EXTs) in particular, undergo the hydroxylation of proline by the prolyl-4-hydroxylases. The activity of these enzymes can be inhibited with 3,4-dehydro-L-proline (3,4-DHP), which enables its application to reveal the functions of the HRGPs. Thus, to study the involvement of HRGPs in the development of root hairs and roots, we treated seedlings of Brachypodium distachyon with 250 µM, 500 µM, and 750 µM of 3,4-DHP. The histological observations showed that the root epidermis cells and the cortex cells beneath them ruptured. The immunostaining experiments using the JIM20 antibody, which recognizes the EXT epitopes, demonstrated the higher abundance of this epitope in the control compared to the treated samples. The transmission electron microscopy analyses revealed morphological and ultrastructural features that are typical for the vacuolar-type of cell death. Using the TUNEL test (terminal deoxynucleotidyl transferase dUTP nick end labelling), we showed an increase in the number of nuclei with damaged DNA in the roots that had been treated with 3,4-DHP compared to the control. Finally, an analysis of two metacaspases’ gene activity revealed an increase in their expression in the treated roots. Altogether, our results show that inhibiting the prolyl-4-hydroxylases with 3,4-DHP results in a vacuolar-type of cell death in roots, thereby highlighting the important role of HRGPs in root hair development and root growth.

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“…Among the required enzymes during the AGP protein domain modification, an important function is performed by prolyl 4 hydroxylases (P4Hs). P4Hs catalyse the hydroxylation of proline residues in the polypeptide to form hydroxyproline ( Vlad et al., 2007 ; Perrakis et al., 2019 ; Konkina et al., 2021 ). After proper hydroxylation of proline catalysed by P4Hs, AGPs undergo glycosylation, and this molecular mechanism is well known, but the effect of changes in this mechanism on AGPs as well as the mechanism of disruption of the subsequent glycosylation process by changes in the protein domain need to be elucidated.…”

Section: Introductionmentioning

confidence: 99%

The modified activity of prolyl 4 hydroxylases reveals the effect of arabinogalactan proteins on changes in the cell wall during the tomato ripening process

Kutyrieva-Nowak,

Leszczuk,

Ezzat

et al. 2024

Front. Plant Sci.

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Arabinogalactan proteins (AGPs) are proteoglycans with an unusual molecular structure characterised by the presence of a protein part and carbohydrate chains. Their specific properties at different stages of the fruit ripening programme make AGPs unique markers of this process. An important function of AGPs is to co-form an amorphous extracellular matrix in the cell wall-plasma membrane continuum; thus, changes in the structure of these molecules can determine the presence and distribution of other components. The aim of the current work was to characterise the molecular structure and localisation of AGPs during the fruit ripening process in transgenic lines with silencing and overexpression of SlP4H3 genes (prolyl 4 hydroxylase 3). The objective was accomplished through comprehensive and comparative in situ and ex situ analyses of AGPs from the fruit of transgenic lines and wild-type plants at specific stages of ripening. The experiment showed that changes in prolyl 4 hydroxylases (P4H3) activity affected the content of AGPs and the progress in their modifications in the ongoing ripening process. The analysis of the transgenic lines confirmed the presence of AGPs with high molecular weights (120–60 kDa) at all the examined stages, but a changed pattern of the molecular features of AGPs was found in the last ripening stages, compared to WT. In addition to the AGP molecular changes, morphological modifications of fruit tissue and alterations in the spatio-temporal pattern of AGP distribution at the subcellular level were detected in the transgenic lines with the progression of the ripening process. The work highlights the impact of AGPs and their alterations on the fruit cell wall and changes in AGPs associated with the progression of the ripening process.

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An Arabidopsis Prolyl 4 Hydroxylase Is Involved in the Low Oxygen Response

Cited by 10 publications

References 68 publications

Differential prolyl hydroxylation by six Physcomitrella prolyl-4 hydroxylases

Differential prolyl hydroxylation by six Physcomitrella prolyl-4 hydroxylases

3,4-Dehydro-L-proline Induces Programmed Cell Death in the Roots of Brachypodium distachyon

The modified activity of prolyl 4 hydroxylases reveals the effect of arabinogalactan proteins on changes in the cell wall during the tomato ripening process

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