Arabidopsis pollen prolyl-hydroxylases P4H4/6 are relevant for correct hydroxylation and secretion of LRX11 in pollen tubes.

Major constituents of the plant cell walls are structural proteins that belong to the hydroxyproline-rich glycoprotein (HRGP) family. Leucine-rich repeat extensin (LRX) proteins contain a leucine-rich domain and a C-terminal domain with repetitive Ser-Pro3-5 motifs that are potentially to be O-glycosylated. It has been demonstrated that pollen-specific LRX8-LRX11 from Arabidopsis thaliana are necessary to maintain the integrity of the pollen tube cell wall during polarized growth. In HRGPs, including classical extensins (EXTs), and probably in LRXs, proline residues are converted to hydroxyproline by prolyl-4-hydroxylases (P4Hs), thus defining novel O-glycosylation sites. In this context, we aimed to determine whether hydroxylation and subsequent O-glycosylation of Arabidopsis pollen LRXs are necessary for their proper function and cell wall localization in pollen tubes. We hypothesized that pollen-expressed P4H4 and P4H6 catalyze the hydroxylation of the proline units present in Ser-Pro3-5 motifs of LRX8-LRX11. Here, we show that the p4h4-1 p4h6-1 double mutant exhibits a reduction in pollen germination rates and a slight reduction in pollen tube length. Pollen germination is also inhibited by P4H inhibitors, suggesting that prolyl hydroxylation is required for pollen tube development. Plants expressing pLRX11::LRX11-GFP in the p4h4-1 p4h6-1 background show partial re-localization of LRX11-green fluorescent protein (GFP) from the pollen tube tip apoplast to the cytoplasm. Finally, immunoprecipitation-tandem mass spectrometry analysis revealed a decrease in oxidized prolines (hydroxyprolines) in LRX11-GFP in the p4h4-1 p4h6-1 background compared with lrx11 plants expressing pLRX11::LRX11-GFP. Taken together, these results suggest that P4H4 and P4H6 are required for pollen germination and for proper hydroxylation of LRX11 necessary for its localization in the cell wall of pollen tubes.

dsRNA-induced immunity targets plasmodesmata and is suppressed by viral movement proteins.

Emerging evidence indicates that in addition to its well-recognized functions in antiviral RNA silencing, dsRNA elicits pattern-triggered immunity (PTI), likely contributing to plant resistance against virus infections. However, compared to bacterial and fungal elicitor-mediated PTI, the mode-of-action and signaling pathway of dsRNA-induced defense remain poorly characterized. Here, using multicolor in vivo imaging, analysis of GFP mobility, callose staining, and plasmodesmal marker lines in Arabidopsis thaliana and Nicotiana benthamiana, we show that dsRNA-induced PTI restricts the progression of virus infection by triggering callose deposition at plasmodesmata, thereby likely limiting the macromolecular transport through these cell-to-cell communication channels. The plasma membrane-resident SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1, the BOTRYTIS INDUCED KINASE1/AVRPPHB SUSCEPTIBLE1-LIKE KINASE1 kinase module, PLASMODESMATA-LOCATED PROTEINs 1/2/3, as well as CALMODULIN-LIKE 41 and Ca2+ signals are involved in the dsRNA-induced signaling leading to callose deposition at plasmodesmata and antiviral defense. Unlike the classical bacterial elicitor flagellin, dsRNA does not trigger a detectable reactive oxygen species (ROS) burst, substantiating the idea that different microbial patterns trigger partially shared immune signaling frameworks with distinct features. Likely as a counter strategy, viral movement proteins from different viruses suppress the dsRNA-induced host response leading to callose deposition to achieve infection. Thus, our data support a model in which plant immune signaling constrains virus movement by inducing callose deposition at plasmodesmata and reveals how viruses counteract this layer of immunity.

Proline-rich extensin-like receptor kinases PERK5 and PERK12 are involved in pollen tube growth.

Proline-rich extensin-like receptor kinases (PERKs) belong to the hydroxyproline-rich glycoprotein (HRGP) superfamily known to be involved in many plant developmental processes. Here, we characterized two pollen-expressed PERKs from Arabidopsis thaliana, PERK5 and PERK12. Pollen tube growth was impaired in single and double perk5-1 perk12-1 loss of function mutants, with an impact on seed production. When the segregation was analysed, a male gametophytic defect was found, indicating that perk5-1 and perk12-1 mutants carry deficient pollen transmission. Furthermore, perk5-1 perk12-1 displayed an excessive accumulation of pectins and cellulose at the cell wall of the pollen tubes. Our results indicate that PERK5 and PERK12 are necessary for proper pollen tube growth, highlighting their role in cell wall assembly and reactive oxygen species homeostasis.

Keeping up with the RALFs: how these small peptides control pollen-pistil interactions in Arabidopsis.

The pollen and pistil RALF peptides, along with multiple receptor-like kinases and leucine-rich repeat extensins, regulate pollen tube growth and the final burst within the ovule, where sperm cells are released for fertilisation to occur. This review introduces some new questions that arose about the regulation of this complex process.

Imaging and Analysis of the Content of Callose, Pectin, and Cellulose in the Cell Wall of Arabidopsis Pollen Tubes Grown In Vitro.

To achieve fertilization, pollen tubes have to protect and properly deliver sperm cells through the pistil to the ovules. Pollen tube growth is a representative example of polarized growth where new components of the cell wall and plasma membrane are continuously deposited at the tip of the growing cell. The integrity of the cell wall is of fundamental importance to maintain apical growth. For this reason, pollen tube growth has become an excellent model to study the role of polysaccharides and structural cell wall proteins involved in polar cell expansion. However, quantification of structural polysaccharides at the pollen tube cell wall has been challenging due to technical complexity and the difficulty of finding specific dyes. Here, we propose simple methods for imaging and quantification of callose, pectin , and cellulose using specific dyes such as Aniline Blue, Propidium Iodide, and Pontamine Fast Scarlet 4B.

The MED30 subunit of mediator complex is essential for early plant development and promotes flowering in Arabidopsis thaliana.

Mediator is a large multiprotein complex that is required for the transcription of most, if not all, genes transcribed by RNA Polymerase II. A core set of subunits is essential to assemble a functional Mediator in vitro and, therefore, the corresponding loss-of-function mutants are expected to be lethal. The MED30 subunit is essential in animal systems, but is absent in yeast. Here, we report that MED30 is also essential for both male gametophyte and embryo development in the model plant Arabidopsis thaliana Mutant med30 pollen grains were viable and some were able to germinate and target the ovules, although the embryos aborted shortly after fertilization, suggesting that MED30 is important for the paternal control of early embryo development. When gametophyte defects were bypassed by specific pollen complementation, loss of MED30 led to early embryo development arrest. Later in plant development, MED30 promotes flowering through multiple signaling pathways; its downregulation led to a phase change delay, downregulation of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 (SPL3), FLOWERING LOCUS T (FTI) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), and upregulation of FLOWERING LOCUS C (FLC).

Filling the Gaps to Solve the Extensin Puzzle.

Extensins (EXTs) are highly repetitive plant O-glycoproteins that require several post-translational modifications (PTMs) to become functional in plant cell walls. First, they are hydroxylated on contiguous proline residues; then they are O-glycosylated on hydroxyproline and serine. After secretion into the apoplast, O-glycosylated EXTs form a tridimensional network organized by inter- and intra-Tyr linkages. Recent studies have made significant progress in the identification of the enzymatic machinery required to process EXTs, which includes prolyl 4-hydroxylases, glycosyltransferases, papain-type cysteine endopeptidases, and peroxidases. EXTs are abundant in plant tissues and are particularly important in rapidly expanding root hairs and pollen tubes, which grow in a polar manner. Small changes in EXT PTMs affect fast-growing cells, although the molecular mechanisms underlying this regulation are unknown. In this review, we highlight recent advances in our understanding of EXT modifications throughout the secretory pathway, EXT assembly in cell walls, and possible sensing mechanisms involving the Catharanthus roseus cell surface sensor receptor-like kinases located at the interface between the apoplast and the cytoplasmic side of the plasma membrane.

Arabidopsis pollen extensins LRX are required for cell wall integrity during pollen tube growth.

Proper cell wall assembly is crucial during pollen tube growth. Leucine-rich repeat extensins (LRXs) are extracellular glycoproteins which belong to the hydroxyproline-rich glycoprotein (HRGP) family. They contain a conserved N-terminal leucine-rich repeat (LRR) domain and a highly variable C-terminal extensin domain. Here, we characterized four LRX proteins (LRX8 through LRX11) from pollen of Arabidopsis thaliana. To investigate the role of LRX8-LRX11 in pollen germination and pollen tube growth, multiple T-DNA lrx mutants were obtained. The lrx mutants display abnormal pollen tubes with an irregular deposition of callose and pectin. They also show serious alterations in pollen germination and segregation ratio. Our results suggest that LRXs are involved in ensuring proper cell wall assembly during pollen tube growth.

An update on cell surface proteins containing extensin-motifs.

In recent years it has become clear that there are several molecular links that interconnect the plant cell surface continuum, which is highly important in many biological processes such as plant growth, development, and interaction with the environment. The plant cell surface continuum can be defined as the space that contains and interlinks the cell wall, plasma membrane and cytoskeleton compartments. In this review, we provide an updated view of cell surface proteins that include modular domains with an extensin (EXT)-motif followed by a cytoplasmic kinase-like domain, known as PERKs (for proline-rich extensin-like receptor kinases); with an EXT-motif and an actin binding domain, known as formins; and with extracellular hybrid-EXTs. We focus our attention on the EXT-motifs with the short sequence Ser-Pro(3-5), which is found in several different protein contexts within the same extracellular space, highlighting a putative conserved structural and functional role. A closer understanding of the dynamic regulation of plant cell surface continuum and its relationship with the downstream signalling cascade is a crucial forthcoming challenge.