Arabidopsis hypocotyl growth in darkness requires the phosphorylation of a microtubule-associated protein.

Rapid hypocotyl elongation allows buried seedlings to emerge, where light triggers de-etiolation and inhibits hypocotyl growth mainly by photoreceptors. Phosphorylation/dephosphorylation events regulate many aspects of plant development. Only recently we have begun to uncover the earliest phospho-signaling responders to light. Here, we reported a large-scale phosphoproteomic analysis and identified 20 proteins that changed their phosphorylation pattern following a 20 min light pulse compared to darkness. Microtubule-associated proteins were highly overrepresented in this group. Among them, we studied CIP7 (COP1-INTERACTING-PROTEIN 7), which presented microtubule (MT) localization in contrast to the previous description. An isoform of CIP7 phosphorylated at Serine915 was detected in etiolated seedlings but was undetectable after a light pulse in the presence of photoreceptors, while CIP7 transcript expression decays with long light exposure. The short hypocotyl phenotype and rearrangement of MTs in etiolated cip7 mutants are complemented by CIP7-YFP and the phospho-mimetic CIP7S915D-YFP, but not the phospho-null CIP7S915A-YFP suggesting that the phosphorylated S915CIP7 isoform promotes hypocotyl elongation through MT reorganization in darkness. Our evidence on Serine915 of CIP7 unveils phospho-regulation of MT-based processes during skotomorphogenic hypocotyl growth.

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.

Synaptic Contributions to Cochlear Outer Hair Cell Ca2+ Dynamics.

For normal cochlear function, outer hair cells (OHCs) require a precise control of intracellular Ca2+ levels. In the absence of regulatory elements such as proteinaceous buffers or extrusion pumps, OHCs degenerate, leading to profound hearing impairment. Influx of Ca2+ occurs both at the stereocilia tips and the basolateral membrane. In this latter compartment, two different origins for Ca2+ influx have been poorly explored: voltage-gated L-type Ca2+ channels (VGCCs) at synapses with Type II afferent neurons, and α9α10 cholinergic nicotinic receptors at synapses with medio-olivochlear complex (MOC) neurons. Using functional imaging in mouse OHCs, we dissected Ca2+ influx individually through each of these sources, either by applying step depolarizations to activate VGCC, or stimulating MOC axons. Ca2+ ions originated in MOC synapses, but not by VGCC activation, was confined by Ca2+-ATPases most likely present in nearby synaptic cisterns. Although Ca2+ currents in OHCs are small, VGCC Ca2+ signals were comparable in size to those elicited by α9α10 receptors, and were potentiated by ryanodine receptors (RyRs). In contrast, no evidence of potentiation by RyRs was found for MOC Ca2+ signals over a wide range of presynaptic stimulation strengths. Our study shows that despite the fact that these two Ca2+ entry sites are closely positioned, they differ in their regulation by intracellular cisterns and/or organelles, suggesting the existence of well-tuned mechanisms to separate the two different OHC synaptic functions.SIGNIFICANCE STATEMENT Outer hair cells (OHCs) are sensory cells in the inner ear operating under very special constraints. Acoustic stimulation leads to fast changes both in membrane potential and in the intracellular concentration of metabolites such as Ca2+ Tight mechanisms for Ca2+ control in OHCs have been reported. Interestingly, Ca2+ is crucial for two important synaptic processes: inhibition by efferent cholinergic neurons, and glutamate release onto Type II afferent fibers. In the current study we functionally imaged Ca2+ at these two different synapses, showing close positioning within the basolateral compartment of OHCs. In addition, we show differential regulation of these two Ca2+ sources by synaptic cisterns and/or organelles, which could result crucial for functional segregation during normal hearing.

Cell-type-specific transcriptome and histone modification dynamics during cellular reprogramming in the Arabidopsis stomatal lineage.

Plant cells maintain remarkable developmental plasticity, allowing them to clonally reproduce and to repair tissues following wounding; yet plant cells normally stably maintain consistent identities. Although this capacity was recognized long ago, our mechanistic understanding of the establishment, maintenance, and erasure of cellular identities in plants remains limited. Here, we develop a cell-type-specific reprogramming system that can be probed at the genome-wide scale for alterations in gene expression and histone modifications. We show that relationships among H3K27me3, H3K4me3, and gene expression in single cell types mirror trends from complex tissue, and that H3K27me3 dynamics regulate guard cell identity. Further, upon initiation of reprogramming, guard cells induce H3K27me3-mediated repression of a regulator of wound-induced callus formation, suggesting that cells in intact tissues may have mechanisms to sense and resist inappropriate dedifferentiation. The matched ChIP-sequencing (seq) and RNA-seq datasets created for this analysis also serve as a resource enabling inquiries into the dynamic and global-scale distribution of histone modifications in single cell types in plants.

A novel strategy to uncover specific GO terms/phosphorylation pathways in phosphoproteomic data in Arabidopsis thaliana.

BACKGROUND: Proteins are the workforce of the cell and their phosphorylation status tailors specific responses efficiently. One of the main challenges of phosphoproteomic approaches is to deconvolute biological processes that specifically respond to an experimental query from a list of phosphoproteins. Comparison of the frequency distribution of GO (Gene Ontology) terms in a given phosphoproteome set with that observed in the genome reference set (GenRS) is the most widely used tool to infer biological significance. Yet, this comparison assumes that GO term distribution between the phosphoproteome and the genome are identical. However, this hypothesis has not been tested due to the lack of a comprehensive phosphoproteome database. RESULTS: In this study, we test this hypothesis by constructing three phosphoproteome databases in Arabidopsis thaliana: one based in experimental data (ExpRS), another based in in silico phosphorylation protein prediction (PredRS) and a third that is the union of both (UnRS). Our results show that the three phosphoproteome reference sets show default enrichment of several GO terms compared to GenRS, indicating that GO term distribution in the phosphoproteomes does not match that of the genome. Moreover, these differences overshadow the identification of GO terms that are specifically enriched in a particular condition. To overcome this limitation, we present an additional comparison of the sample of interest with UnRS to uncover GO terms specifically enriched in a particular phosphoproteome experiment. Using this strategy, we found that mRNA splicing and cytoplasmic microtubule compounds are important processes specifically enriched in the phosphoproteome of dark-grown Arabidopsis seedlings. CONCLUSIONS: This study provides a novel strategy to uncover GO specific terms in phosphoproteome data of Arabidopsis that could be applied to any other organism. We also highlight the importance of specific phosphorylation pathways that take place during dark-grown Arabidopsis development.

Dissection of MAPK signaling specificity through protein engineering in a developmental context.

BACKGROUND: Mitogen-activated protein kinases (MAPK) signaling affects many processes, some of which have different outcomes in the same cell. In Arabidopsis, activation of a MAPK cascade consisting of YODA, MKK4/5 and MPK3/6 inhibits early stages of stomatal developmental, but the ability to halt stomatal progression is lost at the later stage when guard mother cells (GMCs) transition to guard cells (GCs). Rather than downregulating cascade components, stomatal precursors must have a mechanism to prevent late stage inhibition because the same MKKs and MPKs mediate other physiological responses. RESULTS: We artificially activated the MAPK cascade using MKK7, another MKK that can modulate stomatal development, and found that inhibition of stomatal development is still possible in GMCs. This suggests that MKK4/5, but not MKK7, are specifically prevented from inhibiting stomatal development. To identify regions of MKKs responsible for cell-type specific regulation, we used a domain swap approach with MKK7 and a battery of in vitro and in vivo kinase assays. We found that N-terminal regions of MKK5 and MKK7 establish specific signal-to-output connections like they do in other organisms, but they do so in combination with previously undescribed modules in the C-terminus. One of these modules encoding the GMC-specific regulation of MKK5, when swapped with sequences from the equivalent region of MKK7, allows MKK5 to mediate robust inhibition of late stomatal development. CONCLUSIONS: Because MKK structure is conserved across species, the identification of new MKK specificity modules and signaling rules furthers our understanding of how eukaryotes create specificity in complex biological systems.

Manipulation of mitogen-activated protein kinase kinase signaling in the Arabidopsis stomatal lineage reveals motifs that contribute to protein localization and signaling specificity.

When multiple mitogen-activated protein kinase (MAPK) components are recruited recurrently to transduce signals of different origins, and often opposing outcomes, mechanisms to enforce signaling specificity are of utmost importance. These mechanisms are largely uncharacterized in plant MAPK signaling networks. The Arabidopsis thaliana stomatal lineage was previously used to show that when rendered constitutively active, four MAPK kinases (MKKs), MKK4/5/7/9, are capable of perturbing stomatal development and that these kinases comprise two pairs, MKK4/5 and MKK7/9, with both overlapping and divergent functions. We characterized the contributions of specific structural domains of these four "stomatal" MKKs to MAPK signaling output and specificity both in vitro and in vivo within the three discrete cell types of the stomatal lineage. These results verify the influence of functional docking (D) domains of MKKs on MAPK signal output and identify novel regulatory functions for previously uncharacterized structures within the N termini of MKK4/5. Beyond this, we present a novel function of the D-domains of MKK7/9 in regulating the subcellular localization of these kinases. These results provide tools to broadly assess the extent to which these and additional motifs within MKKs function to regulate MAPK signal output throughout the plant.

Modular, programmable RNA sensing using ADAR editing in living cells.

With the increasing availability of single-cell transcriptomes, RNA signatures offer a promising basis for targeting living cells. Molecular RNA sensors would enable the study of and therapeutic interventions for specific cell types/states in diverse contexts, particularly in human patients and non-model organisms. Here we describe a modular, programmable system for live RNA sensing using adenosine deaminases acting on RNA (RADAR). We validate, and then expand, our basic design, characterize its performance, and analyze its compatibility with human and mouse transcriptomes. We identify strategies to boost output levels and improve the dynamic range. Additionally, we show that RADAR enables compact AND logic. In addition to responding to transcript levels, RADAR can distinguish disease-relevant sequence alterations of transcript identities, such as point mutations and fusions. Finally, we demonstrate that RADAR is a self-contained system with the potential to function in diverse organisms.

Mutations in two putative phosphorylation motifs in the tomato pollen receptor kinase LePRK2 show antagonistic effects on pollen tube length.

The tip-growing pollen tube is a useful model for studying polarized cell growth in plants. We previously characterized LePRK2, a pollen-specific receptor-like kinase from tomato (1). Here, we showed that LePRK2 is present as multiple phosphorylated isoforms in mature pollen membranes. Using comparative sequence analysis and phosphorylation site prediction programs, we identified two putative phosphorylation motifs in the cytoplasmic juxtamembrane (JM) domain. Site-directed mutagenesis in these motifs, followed by transient overexpression in tobacco pollen, showed that both motifs have opposite effects in regulating pollen tube length. Relative to LePRK2-eGFP pollen tubes, alanine substitutions in residues of motif I, Ser(277)/Ser(279)/Ser(282), resulted in longer pollen tubes, but alanine substitutions in motif II, Ser(304)/Ser(307)/Thr(308), resulted in shorter tubes. In contrast, phosphomimicking aspartic substitutions at these residues gave reciprocal results, that is, shorter tubes with mutations in motif I and longer tubes with mutations in motif II. We conclude that the length of pollen tubes can be negatively and positively regulated by phosphorylation of residues in motif I and II respectively. We also showed that LePRK2-eGFP significantly decreased pollen tube length and increased pollen tube tip width, relative to eGFP tubes. The kinase activity of LePRK2 was relevant for this phenotype because tubes that expressed a mutation in a lysine essential for kinase activity showed the same length and width as the eGFP control. Taken together, these results suggest that LePRK2 may have a central role in pollen tube growth through regulation of its own phosphorylation status.

Transcriptional Reactivation of Lignin Biosynthesis for the Heterologous Production of Etoposide Aglycone in Nicotiana benthamiana.

Nicotiana benthamiana is a valuable plant chassis for heterologous production of medicinal plant natural products. This host is well suited for the processing of organelle-localized plant enzymes, and the conservation of the primary metabolism across the plant kingdom often provides required plant-specific precursor molecules that feed a given pathway. Despite this commonality in metabolism, limited precursor supply and/or competing host pathways can interfere with yields of heterologous products. Here, we use transient transcriptional reprogramming of endogenous N. benthamiana metabolism to drastically improve flux through the etoposide pathway derived from the medicinal plant Podophyllum spp. Specifically, coexpression of a single lignin-associated transcription factor, MYB85, with pathway genes results in unprecedented levels of heterologous product accumulation in N. benthamiana leaves: 1 mg/g dry weight (DW) of the etoposide aglycone, 35 mg/g DW (-)-deoxypodophyllotoxin, and 3.5 mg/g DW (-)-epipodophyllotoxin─up to two orders of magnitude above previously reported biosynthetic yields for the etoposide aglycone and eight times higher than what is observed for (-)-deoxypodophyllotoxin in the native medicinal plant. Unexpectedly, transient activation of lignin metabolism by transcription factor overexpression also reduces the production of undesired side products that likely result from competing N. benthamiana metabolism. Our work demonstrates that synthetic activation of lignin biosynthesis in leaf tissue is an effective strategy for optimizing the production of medicinal compounds derived from phenylpropanoid precursors in the plant chassis N. benthamiana. Furthermore, our results highlight the engineering value of MYB85, an early switch in lignin biosynthesis, for on-demand modulation of monolignol flux and support the role of MYB46 as a master regulator of lignin polymer deposition.

STIL, a peculiar molecule from styles, specifically dephosphorylates the pollen receptor kinase LePRK2 and stimulates pollen tube growth in vitro.

BACKGROUND: LePRK1 and LePRK2 are two pollen receptor kinases localized to the plasma membrane, where they are present in a high molecular weight complex (LePRK complex). LePRK2 is phosphorylated in mature and germinated pollen, but is dephosphorylated when pollen membranes are incubated with tomato or tobacco style extracts. RESULTS: Here we show that LePRK2 dephosphorylation is mediated by a heat-, acid-, base-, DTT- and protease-resistant component from tobacco styles. Using LePRK2 phosphorylation as a tracking assay for purification, style exudates were subjected to chloroform extraction, anionic exchange, and C18 reverse-phase chromatography columns. We finally obtained a single ~3,550 Da compound (as determined by UV-MALDI-TOF MS) that we named STIL (for Style Interactor for LePRKs). STIL increased pollen tube lengths of in vitro germinated pollen in a dose-dependent manner. CONCLUSION: We propose that the LePRK complex perceives STIL, resulting in LePRK2 dephosphorylation and an increase in pollen tube growth.

Concerted regulation of nuclear and cytoplasmic activities of SR proteins by AKT.

Serine/arginine-rich (SR) proteins are important regulators of mRNA splicing. Several postsplicing activities have been described for a subset of shuttling SR proteins, including regulation of mRNA export and translation. Using the fibronectin gene to study the links between signal-transduction pathways and SR protein activity, we show that growth factors not only modify the alternative splicing pattern of the fibronectin gene but also alter translation of reporter messenger RNAs in an SR protein-dependent fashion, providing two coregulated levels of isoform-specific amplification. These effects are inhibited by specific small interfering RNAs against SR proteins and are mediated by the AKT kinase, which elicits opposite effects to those evoked by overexpressing SR protein kinases Clk and SRPK. These results show how SR protein activity is modified in response to extracellular stimulation, leading to a concerted regulation of splicing and translation.

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.

How many receptor-like kinases are required to operate a pollen tube.

Successful fertilization depends on active molecular dialogues that the male gametophyte can establish with the pistil and the female gametophyte. Pollen grains and stigmas must recognize each other; pollen tubes need to identify the pistil tissues they will penetrate, follow positional cues to exit the transmitting tract and finally, locate the ovules. These molecular dialogues directly affect pollen tube growth rate and orientation. Receptor-like kinases (RLKs) are natural candidates for the perception and decoding of extracellular signals and their transduction to downstream cytoplasmic interactors. Here, we update knowledge regarding how RLKs are involved in pollen tube growth, cell wall integrity and guidance. In addition, we use public data to build a pollen tube RLK interactome that might help direct experiments to elucidate the function of pollen RLKs and their associated proteins.

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.

Oligomerization studies show that the kinase domain of the tomato pollen receptor kinase LePRK2 is necessary for interaction with LePRK1.

LePRK1 and LePRK2 are two pollen-specific receptor-like kinases from Solanum lycopersicum that are involved in signaling during pollen-pistil communication. Previously, we showed that both proteins interact in pollen and when expressed in yeast. We also showed that pollen tube length was regulated by phosphorylation of specific residues in the juxtamembrane domain of LePRK2. To determine the domains responsible for the interaction between LePRK1 and LePRK2, we constructed a series of deletions, expressed them in yeast and determined their association by co-immunoprecipitation assays. We show that deletions containing extracellular domains of LePRK1 and LePRK2 were glycosylated in yeast and were sufficient for interaction with the corresponding full-length receptor. The juxtamembrane domain of LePRK1 was sufficient for its interaction with LePRK2, whereas LePRK2 required its kinase domain for interaction with LePRK1. These findings suggest a role for the juxtamembrane domain of LePRK2 in mediating intracellular dimerization and thus receptor kinase phosphorylation.

The pollen receptor kinase LePRK2 mediates growth-promoting signals and positively regulates pollen germination and tube growth.

In flowering plants, the process of pollen germination and tube growth is required for successful fertilization. A pollen receptor kinase from tomato (Solanum lycopersicum), LePRK2, has been implicated in signaling during pollen germination and tube growth as well as in mediating pollen (tube)-pistil communication. Here we show that reduced expression of LePRK2 affects four aspects of pollen germination and tube growth. First, the percentage of pollen that germinates is reduced, and the time window for competence to germinate is also shorter. Second, the pollen tube growth rate is reduced both in vitro and in the pistil. Third, tip-localized superoxide production by pollen tubes cannot be increased by exogenous calcium ions. Fourth, pollen tubes have defects in responses to style extract component (STIL), an extracellular growth-promoting signal from the pistil. Pollen tubes transiently overexpressing LePRK2-fluorescent protein fusions had slightly wider tips, whereas pollen tubes coexpressing LePRK2 and its cytoplasmic partner protein KPP (a Rop-GEF) had much wider tips. Together these results show that LePRK2 positively regulates pollen germination and tube growth and is involved in transducing responses to extracellular growth-promoting signals.

Kinase partner protein interacts with the LePRK1 and LePRK2 receptor kinases and plays a role in polarized pollen tube growth.

The pollen-specific receptor kinases LePRK1 and LePRK2 have localization and expression profiles that strongly suggest they play roles in pollen germination and tube growth. To identify downstream components of LePRK signaling, we used their cytoplasmic domains (CDs) as baits in yeast two-hybrid screens of a tomato pollen cDNA library. A pollen-specific protein we named kinase partner protein (KPP) interacted with the CDs of both LePRK1 and LePRK2 in yeast and in an in vitro pull-down assay, and with LePRK2 in a co-immunoprecipitation assay. KPP is a peripheral membrane protein and is phosphorylated in pollen. Pollen tubes over-expressing KPP developed balloon-like tips with abnormal cytoplasmic streaming and F-actin arrangements and plants over-expressing KPP exhibited impaired transmission of the transgene through the male. KPP-like genes are found only in plants; the 14 family members in Arabidopsis thaliana exhibit diverse expression patterns and potentially play roles in signaling pathways in other tissues.

The receptor kinases LePRK1 and LePRK2 associate in pollen and when expressed in yeast, but dissociate in the presence of style extract.

After pollen grains germinate on the stigma, pollen tubes traverse the extracellular matrix of the style on their way to the ovules. We previously characterized two pollen-specific, receptor-like kinases, LePRK1 and LePRK2, from tomato (Lycopersicon esculentum). Their structure and immunolocalization pattern and the specific dephosphorylation of LePRK2 suggested that these kinases might interact with signaling molecules in the style extracellular matrix. Here, we show that LePRK1 and LePRK2 can be coimmunoprecipitated from pollen or when expressed together in yeast. In yeast, their association requires LePRK2 kinase activity. In pollen, LePRK1 and LePRK2 are found in an approximately 400-kDa protein complex that persists on pollen germination, but this complex is disrupted when pollen is germinated in vitro in the presence of style extract. In yeast, the addition of style extract also disrupts the interaction between LePRK1 and LePRK2. Fractionation of the style extract reveals that the disruption activity is enriched in the 3- to 10-kDa fraction. A component(s) in this fraction also is responsible for the specific dephosphorylation of LePRK2. The style component(s) that dephosphorylates LePRK2 is likely to be a heat-stable peptide that is present in exudate from the style. The generally accepted model of receptor kinase signaling involves binding of a ligand to extracellular domains of receptor kinases and subsequent activation of the signaling pathway by receptor autophosphorylation. In contrast to this typical scenario, we propose that a putative style ligand transduces the signal in pollen tubes by triggering the specific dephosphorylation of LePRK2, followed by dissociation of the LePRK complex.