Biology subjects

Loubery, S.

Publications and source records attributed to Loubery, S..

7 recordsLinked to original sources

bioRxiv2026

Plant Kelch phosphatases are Ser/Thr phosphatases involved in cell cycle regulation

Brassinosteroids (BRs) are plant steroid hormones sensed by the membrane receptor kinase BRI1. Activation of BRI1 leads to the dephosphorylation of BZR1/BES1 transcription factors. Overexpression of the Kelch phosphatase BRI1 SUPPRESSOR 1 (BSU1) rescued the growth defects of bri1 mutants. Subsequent studies identified BSU1 as a protein tyrosine phosphatase, which promotes BR signaling by dephosphorylating a phosphotyrosine in the glycogen synthase kinase 3 BIN2. Crystal structures of the BSU1 phosphatase domain now reveal a high degree of structural similarity to protein phosphatase 1 (PP1), a eukaryotic serine/threonine phosphatase. Consistently, BSU1 efficiently dephosphorylated phosphothreonine- and phosphoserine-containing substrate peptides, but showed no detectable activity toward BIN2 and other phosphotyrosine substrates. A catalytically inactive BSU1 phosphatase domain suppresses the growth phenotypes of the Arabidopsis bri1-5 mutant and binds the BSU1 homologs BSL1-3. bsu1 and bsu1 bsl1 bsl2/3 loss-of-function mutants display wild-type-like BR responses, but exhibit stomatal patterning and fertility defects. Importantly, the PP1-like C-terminal tail of BSU1 is phosphorylated at Thr785 by a cyclin-dependent kinase complex. The phosphorylated tail binds to the BSU1 substrate-binding grooves, blocking access to the active site. Mutation of Thr785 to alanine activates BSU1, suggesting that Kelch phosphatases and PP1 share a common regulatory mechanism. Deletion of the Marchantia polymorpha Kelch phosphatase MpBSLM results in an undifferentiated cell mass phenotype, associated with the over-activation of a cell cycle reporter. Taken together, our experiments suggest that plant Kelch phosphatases act as PP1-like cell cycle regulators, rather than as tyrosine phosphatases in brassinosteroid signaling. (243 words) Significance StatementBrassinosteroid hormones initiate a well-characterized signaling pathways in plants. While the early signaling events at the plasma membrane have been thoroughly examined, the precise sequence of molecular events that comprise the cytoplasmic signaling cascade is less well-understood. Here, the molecular function of the Kelch protein phosphatase BSU1 is re-evaluated. Previous reports have indicated that BSU1 dephosphorylates the GSK3 kinase BIN2 on a central tyrosine residue. However, structural, quantitative enzyme kinetic, and genetic analyses now suggest that BSU1 is a PP1-like serine/threonine phosphatase involved in cell cycle control rather than a tyrosine phosphatase involved in BIN2 de-phosphorylation. The regulation of BSU1 by a cyclin-dependent kinase complex and the role of Kelch phosphatases in cell cycle progression in Marchantia polymorpha are experimentally characterized. (121 words)

plant biology↗

bioRxiv2024

Directional Cell-to-cell Transport in Plant Roots

Cell-to-cell communication is critical for multicellular organisms. In plants, plasmodesmata--cytoplasmic channels--enable molecular transport between adjacent cells. In roots, this transport is predicted to be essential in nutrient acquisition and delivery to the vasculature. We demonstrate that plasmodesmatal transport persists in differentiated roots, despite apoplastic barriers such as Casparian strips and suberin lamellae in the endodermis, suggesting plasmodesmata as the sole pathway for water and nutrient flow at this stage. We also reveal a developmental switch in plasmodesmata function resulting in an unidirectional transport in differentiated roots. A genetic screen identified mutations that disrupt this directionality, leading to bidirectional transport. These mutations correlate with larger plasmodesmatal apertures, linked to defects in pectin composition and cell wall organization. This discovery underscores the role of plasmodesmatal aperture regulation and pectin in controlling directional transport. Our findings provide insights into plasmodesmata function and their regulation in roots.

plant biology↗

bioRxiv2024

Temperature-dependent polar lignification of a seed coat suberin layer promoting dormancy in Arabidopsis thaliana

The seed is a landmark plant adaptation where the embryo is sheltered by a protective seed coat to facilitate dispersion. In Arabidopsis, the seed coat, derived from ovular integuments, plays a critical role in maintaining dormancy, ensuring germination occurs during a favorable season. Dormancy is enhanced by cold temperatures during seed development by affecting seed coat permeability through changes in apoplastic barriers. However, the localization and composition of these apoplastic barriers are poorly understood. This study identifies and investigates a polar barrier in the seed coats outer integument (oi1) cells. We present histological, biochemical, and genetic evidence showing that cold promotes polar seed coat lignification of the outer integument 1 (oi1) cells and suberization throughout the entire oi1 cell boundary. The polar oi1 barrier is regulated by the transcription factors MYB107 and MYB9. MYB107, in particular, is crucial for the lignified polar oi1 barrier formation under cold temperatures. The absence of the oi1 barrier in mutant seeds correlates with increased permeability and reduced dormancy. Our findings elucidate how temperature-induced modifications in seed coat composition regulate dormancy, highlighting the roles of suberin and lignin in this process. Significance statementOur study uncovers how cold temperatures during seed development in the mother plant influence seed dormancy through apoplastic modifications in the Arabidopsis thaliana seed coat. We identified a polar lignin barrier in the outer integument 1 (oi1) cells, which are also suberized. Lignification and suberization are regulated by transcription factors MYB107 and MYB9. Cold promotes lignification and suberization of oi1 cells through MYB107, thus creating a "memory" that reduces seed permeability and strengthens dormancy. Mutants defective in the oi1 barrier exhibit lower dormancy, highlighting the adaptive importance of this barrier. These findings advance our understanding of temperature-induced seed coat adaptations and their agricultural implications, particularly in the context of climate change, offering valuable insights for improving crop resilience and yield.

plant biology↗

bioRxiv2024

Inositol pyrophosphate catabolism by three families of phosphatases controls plant growth and development

Inositol pyrophosphates (PP-InsPs) are nutrient messengers whose cellular concentration must be tightly regulated. Diphosphoinositol pentakisphosphate kinases (PPIP5Ks) generate the active signaling molecule 1,5-InsP8. PPIP5Ks contain additional phosphatase domains involved in PP-InsP catabolism. Plant and Fungi Atypical Dual Specificity Phosphatases (PFA-DSPs) and NUDIX phosphatases (NUDTs) also hydrolyze PP-InsPs. Here we dissect the relative contributions of the three different phosphatase families to plant PP-InsP catabolism and nutrient signaling. We report the biochemical characterization of inositol pyrophosphate phosphatases from Arabidopsis and Marchantia polymorpha. Overexpression of different PFA-DSP and NUDT enzymes affects PP-InsP levels and leads to stunted growth phenotypes in Arabidopsis. nudt17/18/21 knock-out mutants have altered PP-InsP pools and gene expression patterns, but no apparent growth defects. In contrast, Marchantia polymorpha Mppfa-dsp1ge, Mpnudt1ge and Mpvip1ge mutants display severe growth and developmental phenotypes associated with changes in cellular PP-InsP levels. Analysis of Mppfa-dsp1geand Mpvip1ge supports a role for PP-InsPs in Marchantia phosphate signaling, and additional functions in nitrate homeostasis and cell wall biogenesis. Simultaneous removal of two phosphatase activities enhances the observed growth phenotypes. Taken together, PPIP5K, PFA-DSP and NUDT inositol pyrophosphate phosphatases play important roles in growth and development by collectively shaping plant PP-InsP pools. Author summaryOrganisms must maintain adequate levels of nutrients in their cells and tissues. One such nutrient is phosphorus, an essential building block of cell membranes, nucleic acids and energy metabolites. Plants take up phosphorus in the form of inorganic phosphate and require sufficient cellular phosphate levels to support their growth and development. It has been shown that plants and other eukaryotic organisms "measure" cellular phosphate levels using inositol pyrophosphate signaling molecules. The concentration of inositol pyrophosphates serves as a proxy for the cellular concentration of inorganic phosphate, and therefore inositol pyrophosphate synthesis and degradation must be tightly regulated. Here, we report that three different families of enzymes contribute to the degradation of inositol pyrophosphates in plants. The different phosphatases together shape cellular inositol pyrophosphate pools and thereby affect inorganic phosphate levels. Loss-of-function mutants of the different enzymes display additional defects in nitrate levels and cell wall architecture, suggesting that inositol pyrophosphates regulate cellular processes beyond inorganic phosphate homeostasis.

plant biology↗

bioRxiv2022

Structural and functional insight into the plant unique multimodular triphosphosphate tunnel metalloenzymes of Arabidopsis thaliana

Triphosphate tunnel metalloenzymes (TTMs) are found in all biological kingdoms and have been characterized in microorganisms and animals. Members of the TTM family already characterized have divergent biological functions and act on a range of triphosphorylated substrates (RNA, thiamine triphosphate, inorganic polyphosphate). TTM proteins in plants have received considerably less attention and are unique in that some homologs harbor additional domains including a P-loop kinase and transmembrane domain. Here we report on structural and functional aspects of the multimodular TTM1 and TTM2 of Arabidopsis thaliana. Tissue and cellular microscopy studies show that both AtTTM1 and AtTTM2 are expressed in actively dividing (meristem) tissue and are tail-anchored proteins at the outer mitochondrial membrane - mediated by the single transmembrane domain at the C-terminus, supporting earlier studies. Crystal structures of AtTTM1 in the presence and absence of a non-hydrolyzable ATP analog reveal a catalytically incompetent TTM tunnel domain tightly interacting with the P-loop kinase domain that is locked in an inactive conformation. Structural comparison reveals that a helical hairpin may facilitate movement of the TTM domain thereby activating the kinase. Genetic studies show that AtTTM2 is important for the developmental transition from the vegetative to the reproductive phase in Arabidopsis, whereas its closest paralog AtTTM1 is not. Rational design of mutations based on the 3D structure demonstrates that both the P-loop kinase and TTM tunnel modules of AtTTM2 are required for the developmental switch.

plant biology↗

bioRxiv2020

Tannic cell walls form a continuous apoplastic barrier sustaining Arabidopsis seed coat biophysical properties

Seeds are a late land plant evolution innovation that promoted the striking spread and diversity of angiosperms. The seed coat is a specialized dead tissue protecting the plant embryo from mechanical damage. In many species, including Arabidopsis thaliana, the seed coat also achieves a remarkable balancing act: it limits oxygen uptake, avoiding premature embryo oxidative damage, but not entirely so as to enable seed dormancy release. The seed coat biophysical features implementing the striking physiological properties of the seed remain poorly understood. Tannins, a type of flavonoids, are antioxidants known to accumulate in the Arabidopsis seed coat and transparent testa (tt) mutant seeds, deficient in flavonoid synthesis, exhibit low dormancy and viability. However, their precise contribution to seed coat architecture and biophysics remains evasive. A seed coat cuticle, covering the endosperm outer surface was, intriguingly, previously shown to be more permeable in tt mutants deficient not in cuticular component synthesis, but rather in flavonoid synthesis. Investigating the role of flavonoids in cuticle permeability led us to identify cell walls, originating from the seed coat inner integument 1 cells, impregnated with tannins. We found that tannic cell walls are tightly associated with the cuticle, forming two fused layers that regulate endosperm permeability. In addition, we show that tannic cell walls are prominent building blocks of the seed coat, constituting a continuous barrier around the seed living tissues. Altogether our findings reveal the existence of tannic cell walls as a previously unrecognized biological barrier sustaining the seeds key physiological properties. One sentence summaryThe seed coat is largely composed of plant cell walls impregnated with tannins, forming a thick and continuous protective barrier surrounding the embryo promoting seed viability and dormancy.

plant biology↗

bioRxiv2019

A genetically validated approach to detect inorganic polyphosphates in plants.

Inorganic polyphosphates (polyPs) are linear polymers of orthophosphate units linked by phosphoanhydride bonds. PolyPs represent important stores of phosphate and energy, and are abundantly found in many pro- and eukaryotic organisms. In plants, the existence of polyPs has been established using microscopy and biochemical extraction methods that are now known to produce artifacts. Here we use a polyP-specific dye and a polyP binding domain to detect polyPs in plant and algal cells. To develop the staining protocol, we induced polyP granules in Nicotiana benthamiana and Arabiopsis cells by heterologous expression of E. coli polyphosphate kinase 1 (PPK1). Over-expression of PPK1 but not of a catalytically impaired version of the enzyme lead to severe growth phenotypes, suggesting that ATP-dependent synthesis and accumulation of polyPs in the plant cytosol is toxic. We next crossed stable PPK1 expressing Arabidopsis lines with plants expressing the polyP-binding domain of E. coli exopolyphosphatase (PPX1c), which co-localized with PPK1-generated polyP granules. These granules were stained by the polyP-specific dye JC-D7 and appeared as electron dense structures in transmission electron microscopy (TEM) sections. Using the polyP staining protocol derived from these experiments, we screened for polyP stores in different organs and tissues of both mono- and dicotyledonous plants. While we could not detect polyP granules in higher plants, we could visualize the polyP-rich acidocalicisomes in the green algae Chlamydomonas reinhardtii. Together, our experiments suggest that higher plants may not contain large polyPs stores.\n\nSignificance StatementA chemical dye and an inorganic polyphosphate binding domain are shown to specifically label inorganic polyphosphate granules in transgenic Arabidopsis lines and Chlamydomonas acidocalcisomes. Using these tools, we show that in contrast to many prokaryotic and eukaryotic organisms, higher plants do not seem to contain large inorganic polyphosphate stores.

plant biology↗