Additive and Specific Effects of Elicitor Treatments on the Metabolic Profile of Arabidopsis thaliana.

Several elicitors of plant defense have been identified and numerous efforts to use them in the field have been made. Exogenous elicitor treatments mimic the in planta activation of pattern-triggered immunity (PTI), which relies on the perception of pathogen-associated molecular patterns (PAMPs) such as bacterial flg22 or fungal chitins. Early transcriptional responses to distinct PAMPs are mostly overlapping, regardless of the elicitor being used. However, it remains poorly known if the same patterns are observed for metabolites and proteins produced later during PTI. In addition, little is known about the impact of a combination of elicitors on PTI and the level of induced resistance to pathogens. Here, we monitored Arabidopsis thaliana resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) following application of flg22 and chitosan elicitors, used individually or in combination. A slight, but not statistically significant increase in induced resistance was observed when the elicitors were applied together when compared with individual treatments. We investigated the effect of these treatments on the metabolome by using an untargeted analysis. We found that the combination of flg22 and chitosan impacted a higher number of metabolites and deregulated specific metabolic pathways compared with the elicitors individually. These results contribute to a better understanding of plant responses to elicitors, which might help better rationalize their use in the field. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Pollen viability, longevity, and function in angiosperms: key drivers and prospects for improvement.

Pollen grains are central to sexual plant reproduction and their viability and longevity/storage are critical for plant physiology, ecology, plant breeding, and many plant product industries. Our goal is to present progress in assessing pollen viability/longevity along with recent advances in our understanding of the intrinsic and environmental factors that determine pollen performance: the capacity of the pollen grain to be stored, germinate, produce a pollen tube, and fertilize the ovule. We review current methods to measure pollen viability, with an eye toward advancing basic research and biotechnological applications. Importantly, we review recent advances in our understanding of how basic aspects of pollen/stigma development, pollen molecular composition, and intra- and intercellular signaling systems interact with the environment to determine pollen performance. Our goal is to point to key questions for future research, especially given that climate change will directly impact pollen viability/longevity. We find that the viability and longevity of pollen are highly sensitive to environmental conditions that affect complex interactions between maternal and paternal tissues and internal pollen physiological events. As pollen viability and longevity are critical factors for food security and adaptation to climate change, we highlight the need to develop further basic research for better understanding the complex molecular mechanisms that modulate pollen viability and applied research on developing new methods to maintain or improve pollen viability and longevity.

The Light-Controlled Release of 2-fluoro-l-fucose, an Inhibitor of the Root Cell Elongation, from a nitrobenzyl-caged Derivative.

Glycan metabolic engineering is a powerful tool for studying the glycosylation in living plant cells. The use of modified monosaccharides such as deoxy or fluorine-containing glycosides has been reported as a powerful pharmacological approach for studying the carbohydrate metabolism. 1,3,4-tri-O-acetyl-2-fluoro-l-fucose (2F-Fuc) is a potent inhibitor of the plant cell elongation. After feeding plant seedlings with 2F-Fuc, this monosaccharide derivative is deacetylated and converted by the endogenous metabolic machinery into the corresponding nucleotide-sugar, which then efficiently inhibits Golgi-localized fucosyltransferases. Among plant cell wall polymers, defects in the fucosylation of the pectic rhamnogalacturonan-II cause a decrease in RG-II dimerization, which in turn induce the arrest of the cell elongation. In order to perform the inhibition of the cell elongation process in a spatio-temporal manner, we synthesized a caged 3,4-di-O-acetyl-1-hydroxy-2-fluoro-l-fucose (1-OH-2F-Fuc) derivative carrying a photolabile ortho-nitrobenzyl alcohol function at the anomeric position: 3,4-di-O-acetyl-1-ortho-nitrobenzyl-2-fluoro-l-fucose (2F-Fuc-NB). The photorelease of the trapped 1-OH-2F-Fuc was performed under a 365 nm LED illumination. We demonstrated that the in planta elimination by photoexcitation of the photolabile group releases free 2F-Fuc in plant cells, which in turn inhibits in a dose-dependent manner and, reversibly, the root cell elongation.

Rapid evolution of pollen and pistil traits as a response to sexual selection in the post-pollination phase of mating.

Sexual selection is the basis of some of the most striking phenotypic variation in nature.1,2 In animals, sexual selection in males can act on traits that improve access to mates prior to copulation,3-8 but also on sperm traits filtered by sperm competition,9-14 or female choice expressed simply by the morphology and physiology of genital tracts.14-16 Although long overlooked as a mode of selection on plant traits, sexual selection should act on land plants too because they are anisogamous: males produce more, and smaller, gametes than females.17-19 Numerical asymmetry in gamete production is thought to play a central role in selection on traits that affect pollen transfer to mates,20,21 but very little is known about how pollen competition or cryptic female choice might affect the evolution of traits expressed after pollination.22,23 Here, we report the divergence of pollen and pistil traits of the dioecious wind-pollinated annual herb Mercurialis annua during evolution over three generations between populations at low versus high plant density, corresponding to low versus higher levels of polyandry;24 we expected selection under higher polyandry to strengthen competition among pollen donors for fertilizing ovules. We found that populations at high density evolved faster-growing pollen tubes (an equivalent of greater sperm velocity), greater expression of pollen proteins involved in pollen growth, and larger stigmas (a trait likely enhancing the number of pollen donors and thus competition for ovules). Our results identify the post-pollination phase of plant mating as an important arena for the action of sexual selection.

Dynamic imaging of cell wall polysaccharides by metabolic click-mediated labeling of pectins in living elongating cells.

Protein tracking in living plant cells has become routine with the emergence of reporter genes encoding fluorescent tags. Unfortunately, this imaging strategy is not applicable to glycans because they are not directly encoded by the genome. Indeed, complex glycans result from sequential additions and/or removals of monosaccharides by the glycosyltransferases and glycosidases of the cell's biosynthetic machinery. Currently, the imaging of cell wall polymers mainly relies on the use of antibodies or dyes that exhibit variable specificities. However, as immunolocalization typically requires sample fixation, it does not provide access to the dynamics of living cells. The development of click chemistry in plant cell wall biology offers an alternative for live-cell labeling. It consists of the incorporation of a carbohydrate containing a bio-orthogonal chemical reporter into the target polysaccharide using the endogenous biosynthetic machinery of the cell. Once synthesized and deposited in the cell wall, the polysaccharide containing the analog monosaccharide is covalently coupled to an exogenous fluorescent probe. Here, we developed a metabolic click labeling approach which allows the imaging of cell wall polysaccharides in living and elongating cells without affecting cell viability. The protocol was established using the pollen tube, a useful model to follow cell wall dynamics due to its fast and tip-polarized growth, but was also successfully tested on Arabidopsis root cells and root hairs. This method offers the possibility of imaging metabolically incorporated sugars of viable and elongating cells, allowing the study of the long-term dynamics of labeled extracellular polysaccharides.

Two Carbohydrate-Based Natural Extracts Stimulate in vitro Pollen Germination and Pollen Tube Growth of Tomato Under Cold Temperatures.

To date, it is widely accepted by the scientific community that many agricultural regions will experience more extreme temperature fluctuations. These stresses will undoubtedly impact crop production, particularly fruit and seed yields. In fact, pollination is considered as one of the most temperature-sensitive phases of plant development and until now, except for the time-consuming and costly processes of genetic breeding, there is no immediate alternative to address this issue. In this work, we used a multidisciplinary approach using physiological, biochemical, and molecular techniques for studying the effects of two carbohydrate-based natural activators on in vitro tomato pollen germination and pollen tube growth cultured in vitro under cold conditions. Under mild and strong cold temperatures, these two carbohydrate-based compounds significantly enhanced pollen germination and pollen tube growth. The two biostimulants did not induce significant changes in the classical molecular markers implicated in pollen tube growth. Neither the number of callose plugs nor the CALLOSE SYNTHASE genes expression were significantly different between the control and the biostimulated pollen tubes when pollens were cultivated under cold conditions. PECTIN METHYLESTERASE (PME) activities were also similar but a basic PME isoform was not produced or inactive in pollen grown at 8°C. Nevertheless, NADPH oxidase (RBOH) gene expression was correlated with a higher number of viable pollen tubes in biostimulated pollen tubes compared to the control. Our results showed that the two carbohydrate-based products were able to reduce in vitro the effect of cold temperatures on tomato pollen tube growth and at least for one of them to modulate reactive oxygen species production.

Ethylene signaling modulates tomato pollen tube growth through modifications of cell wall remodeling and calcium gradient.

Ethylene modulates plant developmental processes including flower development. Previous studies have suggested ethylene participates in pollen tube (PT) elongation, and both ethylene production and perception seem critical at the time of fertilization. The full gene set regulated by ethylene during PT growth is unknown. To study this, we used various EThylene Receptor (ETR) tomato (Solanum lycopersicum) mutants: etr3-ko, a loss-of-function (LOF) mutant; and NR (NEVER RIPE), a gain-of-function (GOF) mutant. The etr3-ko PTs grew faster than wild-type (WT) PTs. Oppositely, NR PT elongation was slower than in WT, and PTs displayed larger diameters. ETR mutations result in feedback control of ethylene production. Furthermore, ethylene treatment of germinating pollen grains increased PT length in etr-ko mutants and WT, but not in NR. Treatment with the ethylene perception inhibitor 1-methylcyclopropene decreased PT length in etr-ko mutants and WT, but had no effect on NR. This confirmed that ethylene regulates PT growth. The comparison of PT transcriptomes in LOF and GOF mutants, etr3-ko and NR, both harboring mutations of the ETR3 gene, revealed that ethylene perception has major impacts on cell wall- and calcium-related genes as confirmed by microscopic observations showing a modified distribution of the methylesterified homogalacturonan pectic motif and of calcium load. Our results establish links between PT growth, ethylene, calcium, and cell wall metabolism, and also constitute a transcriptomic resource.

The Scope for Postmating Sexual Selection in Plants.

Sexual selection is known to shape plant traits that affect access to mates during the pollination phase, but it is less well understood to what extent it affects traits relevant to interactions between pollen and pistils after pollination. This is surprising, because both of the two key modes of sexual selection, male-male competition and female choice, could plausibly operate during pollen-pistil interactions where physical male-female contact occurs. Here, we consider how the key processes of sexual selection might affect traits involved in pollen-pistil interactions, including 'Fisherian runaway' and 'good-genes' models. We review aspects of the molecular and cellular biology of pollen-pistil interactions on which sexual selection could act and point to research that is needed to investigate them.

Effect of a Bacillus subtilis strain on flax protection against Fusarium oxysporum and its impact on the root and stem cell walls.

In Normandy, flax is a plant of important economic interest because of its fibres. Fusarium oxysporum, a telluric fungus, is responsible for the major losses in crop yield and fibre quality. Several methods are currently used to limit the use of phytochemicals on crops. One of them is the use of plant growth promoting rhizobacteria (PGPR) occurring naturally in the rhizosphere. PGPR are known to act as local antagonists to soil-borne pathogens and to enhance plant resistance by eliciting the induced systemic resistance (ISR). In this study, we first investigated the cell wall modifications occurring in roots and stems after inoculation with the fungus in two flax varieties. First, we showed that both varieties displayed different cell wall organization and that rapid modifications occurred in roots and stems after inoculation. Then, we demonstrated the efficiency of a Bacillus subtilis strain to limit Fusarium wilt on both varieties with a better efficiency for one of them. Finally, thermo-gravimetry was used to highlight that B. subtilis induced modifications of the stem properties, supporting a reinforcement of the cell walls. Our findings suggest that the efficiency and the mode of action of the PGPR B. subtilis is likely to be flax variety dependent.

Identification of two compounds able to improve flax resistance towards Fusarium oxysporum infection.

Plants are surrounded by a diverse range of microorganisms that causes serious crop losses and requires the use of pesticides. Flax is a major crop in Normandy used for its fibres and is regularly challenged by the pathogenic fungus Fusarium oxysporum (Fo) f. sp. lini. To protect themselves, plants use "innate immunity" as a first line of defense level against pathogens. Activation of plant defense with elicitors could be an alternative for crop plant protection. A previous work was conducted by screening a chemical library and led to the identification of compounds able to activate defense responses in Arabidopsis thaliana. Four compounds were tested for their abilities to improve resistance of two flax varieties against Fo. Two of them, one natural (holaphyllamine or HPA) and one synthetic (M4), neither affected flax nor Fo growth. HPA and M4 induced oxidative burst and callose deposition. Furthermore, HPA and M4 caused changes in the expression patterns of defense-related genes coding a glucanase and a chitinase-like. Finally, plants pre-treated with HPA or M4 exhibited a significant decrease in the disease symptoms. Together, these findings demonstrate that HPA and M4 are able to activate defense responses in flax and improve its resistance against Fo infection.

The exogenous application of AtPGLR, an endo-polygalacturonase, triggers pollen tube burst and repair.

Plant cell wall remodeling plays a key role in the control of cell elongation and differentiation. In particular, fine-tuning of the degree of methylesterification of pectins was previously reported to control developmental processes as diverse as pollen germination, pollen tube elongation, emergence of primordia or elongation of dark-grown hypocotyls. However, how pectin degradation can modulate plant development has remained elusive. Here we report the characterization of a polygalacturonase (PG), AtPGLR, the gene for which is highly expressed at the onset of lateral root emergence in Arabidopsis. Due to gene compensation mechanisms, mutant approaches failed to determine the involvement of AtPGLR in plant growth. To overcome this issue, AtPGLR has been expressed heterologously in the yeast Pichia pastoris and biochemically characterized. We showed that AtPGLR is an endo-PG that preferentially releases non-methylesterified oligogalacturonides with a short degree of polymerization (< 8) at acidic pH. The application of the purified recombinant protein on Amaryllis pollen tubes, an excellent model for studying cell wall remodeling at acidic pH, induced abnormal pollen tubes or cytoplasmic leakage in the subapical dome of the pollen tube tip, where non-methylesterified pectin epitopes are detected. Those leaks could either be repaired by new ß-glucan deposits (mostly callose) in the cell wall or promoted dramatic burst of the pollen tube. Our work presents the full biochemical characterization of an Arabidopsis PG and highlights the importance of pectin integrity in pollen tube elongation.

Evolution of Cell Wall Polymers in Tip-Growing Land Plant Gametophytes: Composition, Distribution, Functional Aspects and Their Remodeling.

During evolution of land plants, the first colonizing species presented leafy-dominant gametophytes, found in non-vascular plants (bryophytes). Today, bryophytes include liverworts, mosses, and hornworts. In the first seedless vascular plants (lycophytes), the sporophytic stage of life started to be predominant. In the seed producing plants, gymnosperms and angiosperms , the gametophytic stage is restricted to reproduction. In mosses and ferns, the haploid spores germinate and form a protonema, which develops into a leafy gametophyte producing rhizoids for anchorage, water and nutrient uptakes. The basal gymnosperms (cycads and Ginkgo) reproduce by zooidogamy. Their pollen grains develop a multi-branched pollen tube that penetrates the nucellus and releases flagellated sperm cells that swim to the egg cell. The pollen grain of other gymnosperms (conifers and gnetophytes) as well as angiosperms germinates and produces a pollen tube that directly delivers the sperm cells to the ovule (siphonogamy). These different gametophytes, which are short or long-lived structures, share a common tip-growing mode of cell expansion. Tip-growth requires a massive cell wall deposition to promote cell elongation, but also a tight spatial and temporal control of the cell wall remodeling in order to modulate the mechanical properties of the cell wall. The growth rate of these cells is very variable depending on the structure and the species, ranging from very slow (protonemata, rhizoids, and some gymnosperm pollen tubes), to a slow to fast-growth in other gymnosperms and angiosperms. In addition, the structural diversity of the female counterparts in angiosperms (dry, semi-dry vs wet stigmas, short vs long, solid vs hollow styles) will impact the speed and efficiency of sperm delivery. As the evolution and diversity of the cell wall polysaccharides accompanied the diversification of cell wall structural proteins and remodeling enzymes, this review focuses on our current knowledge on the biochemistry, the distribution and remodeling of the main cell wall polymers (including cellulose, hemicelluloses, pectins, callose, arabinogalactan-proteins and extensins), during the tip-expansion of gametophytes from bryophytes, pteridophytes (lycophytes and monilophytes), gymnosperms and the monocot and eudicot angiosperms.

A chemical screen identifies two novel small compounds that alter Arabidopsis thaliana pollen tube growth.

BACKGROUND: During sexual reproduction, pollen grains land on the stigma, rehydrate and produce pollen tubes that grow through the female transmitting-tract tissue allowing the delivery of the two sperm cells to the ovule and the production of healthy seeds. Because pollen tubes are single cells that expand by tip-polarized growth, they represent a good model to study the growth dynamics, cell wall deposition and intracellular machineries. Aiming to understand this complex machinery, we used a low throughput chemical screen approach in order to isolate new tip-growth disruptors. The effect of a chemical inhibitor of monogalactosyldiacylglycerol synthases, galvestine-1, was also investigated. The present work further characterizes their effects on the tip-growth and intracellular dynamics of pollen tubes. RESULTS: Two small compounds among 258 were isolated based on their abilities to perturb pollen tube growth. They were found to disrupt in vitro pollen tube growth of tobacco, tomato and Arabidopsis thaliana. We show that these 3 compounds induced abnormal phenotypes (bulging and/or enlarged pollen tubes) and reduced pollen tube length in a dose dependent manner. Pollen germination was significantly reduced after treatment with the two compounds isolated from the screen. They also affected cell wall material deposition in pollen tubes. The compounds decreased anion superoxide accumulation, disorganized actin filaments and RIC4 dynamics suggesting that they may affect vesicular trafficking at the pollen tube tip. CONCLUSION: These molecules may alter directly or indirectly ROP1 activity, a key regulator of pollen tube growth and vesicular trafficking and therefore represent good tools to further study cellular dynamics during polarized-cell growth.

Desiccation tolerance in plants: Structural characterization of the cell wall hemicellulosic polysaccharides in three Selaginella species.

Drought-induced dehydration of vegetative tissues in lycopods affects growth and survival. Different species of Selaginella have evolved a series of specialized mechanisms to tolerate desiccation in vegetative tissues in response to water stress. In the present study, we report on the structural characterization of the leaf cell wall of the desiccation-tolerant species S. involvens and two desiccation-sensitive species, namely S. kraussiana and S. moellendorffii. Isolated cell walls from hydrated and desiccated leaves of each species were fractionated and the resulting oligosaccharide fragments were analyzed to determine their structural features. Our results demonstrate that desiccation induces substantial modifications in the cell wall composition and structure. Altogether, these data highlight the fact that structural remodeling of cell wall hemicellulosic polysaccharides including XXXG-rich xyloglucan, arabinoxylan and acetylated galactomannan is an important process in order to mitigate desiccation stress in Selaginella.

TBL10 is required for O-acetylation of pectic rhamnogalacturonan-I in Arabidopsis thaliana.

O-Acetylated pectins are abundant in the primary cell wall of plants and growing evidence suggests they have important roles in plant cell growth and interaction with the environment. Despite their importance, genes required for O-acetylation of pectins are still largely unknown. In this study, we showed that TRICHOME BIREFRINGENCE LIKE 10 (AT3G06080) is involved in O-acetylation of pectins in Arabidopsis (Arabidopsis thaliana). The activity of the TBL10 promoter was strong in tissues where pectins are highly abundant (e.g. leaves). Two homozygous knock-out mutants of Arabidopsis, tbl10-1 and tbl10-2, were isolated and shown to exhibit reduced levels of wall-bound acetyl esters, equivalent of ~50% of the wild-type level in pectin-enriched fractions derived from leaves. Further fractionation revealed that the degree of acetylation of the pectin rhamnogalacturonan-I (RG-I) was reduced in the tbl10 mutant compared to the wild type, whereas the pectin homogalacturonan (HG) was unaffected. The degrees of acetylation in hemicelluloses (i.e. xyloglucan, xylan and mannan) were indistinguishable between the tbl10 mutants and the wild type. The mutant plants contained normal trichomes in leaves and exhibited a similar level of susceptibility to the phytopathogenic microorganisms Pseudomonas syringae pv. tomato DC3000 and Botrytis cinerea; while they displayed enhanced tolerance to drought. These results indicate that TBL10 is required for O-acetylation of RG-I, possibly as an acetyltransferase, and suggest that O-acetylated RG-I plays a role in abiotic stress responses in Arabidopsis.

Holaphyllamine, a steroid, is able to induce defense responses in Arabidopsis thaliana and increases resistance against bacterial infection.

MAIN CONCLUSION: A chemical screen of plant-derived compounds identified holaphyllamine, a steroid, able to trigger defense responses in Arabidopsis thaliana and improve resistance against the pathogenic bacterium Pseudomonas syringae pv tomato DC3000. A chemical screen of 1600 plant-derived compounds was conducted and allowed the identification of a steroid able to activate defense responses in A. thaliana at a concentration of 1 µM without altering growth. The identified compound is holaphyllamine (HPA) whose chemical structure is similar to steroid pregnanes of mammals. Our data show that HPA, which is not constitutively present in A. thaliana, is able to trigger the formation of reactive oxygen species, deposition of callose and expression of several pathogenesis-related genes of the salicylic and jasmonic acid pathways. In addition, the results show that pre-treatment of A. thaliana seedlings with HPA before infection with the pathogenic bacterium Pseudomonas syringae pv tomato DC3000 results in a significant reduction of symptoms (i.e., reduction of bacterial colonies). Using A. thaliana mutants, we have found that the activation of defense responses by HPA does not depend on BRI1/BAK1 receptor kinases. Finally, a structure/function study reveals that the minimal structure required for activity is a 5-pregnen-20-one steroid with an equatorial nucleophilic group in C-3. Together, these findings demonstrate that HPA can activate defense responses that lead to improved resistance against bacterial infection in A. thaliana.

AtPME3, a ubiquitous cell wall pectin methylesterase of Arabidopsis thaliana, alters the metabolism of cruciferin seed storage proteins during post-germinative growth of seedlings.

AtPME3 (At3g14310) is a ubiquitous cell wall pectin methylesterase. Atpme3-1 loss-of-function mutants exhibited distinct phenotypes from the wild type (WT), and were characterized by earlier germination and reduction of root hair production. These phenotypical traits were correlated with the accumulation of a 21.5-kDa protein in the different organs of 4-day-old Atpme3-1 seedlings grown in the dark, as well as in 6-week-old mutant plants. Microarray analysis showed significant down-regulation of the genes encoding several pectin-degrading enzymes and enzymes involved in lipid and protein metabolism in the hypocotyl of 4-day-old dark grown mutant seedlings. Accordingly, there was a decrease in proteolytic activity of the mutant as compared with the WT. Among the genes specifying seed storage proteins, two encoding CRUCIFERINS were up-regulated. Additional analysis by RT-qPCR showed an overexpression of four CRUCIFERIN genes in the mutant Atpme3-1, in which precursors of the α- and ß-subunits of CRUCIFERIN accumulated. Together, these results provide evidence for a link between AtPME3, present in the cell wall, and CRUCIFERIN metabolism that occurs in vacuoles.

UUAT1 Is a Golgi-Localized UDP-Uronic Acid Transporter That Modulates the Polysaccharide Composition of Arabidopsis Seed Mucilage.

UDP-glucuronic acid (UDP-GlcA) is the precursor of many plant cell wall polysaccharides and is required for production of seed mucilage. Following synthesis in the cytosol, it is transported into the lumen of the Golgi apparatus, where it is converted to UDP-galacturonic acid (UDP-GalA), UDP-arabinose, and UDP-xylose. To identify the Golgi-localized UDP-GlcA transporter, we screened Arabidopsis thaliana mutants in genes coding for putative nucleotide sugar transporters for altered seed mucilage, a structure rich in the GalA-containing polysaccharide rhamnogalacturonan I. As a result, we identified UUAT1, which encodes a Golgi-localized protein that transports UDP-GlcA and UDP-GalA in vitro. The seed coat of uuat1 mutants had less GalA, rhamnose, and xylose in the soluble mucilage, and the distal cell walls had decreased arabinan content. Cell walls of other organs and cells had lower arabinose levels in roots and pollen tubes, but no differences were observed in GalA or xylose contents. Furthermore, the GlcA content of glucuronoxylan in the stem was not affected in the mutant. Interestingly, the degree of homogalacturonan methylation increased in uuat1 These results suggest that this UDP-GlcA transporter plays a key role defining the seed mucilage sugar composition and that its absence produces pleiotropic effects in this component of the plant extracellular matrix.

Combined Experimental and Computational Approaches Reveal Distinct pH Dependence of Pectin Methylesterase Inhibitors.

The fine-tuning of the degree of methylesterification of cell wall pectin is a key to regulating cell elongation and ultimately the shape of the plant body. Pectin methylesterification is spatiotemporally controlled by pectin methylesterases (PMEs; 66 members in Arabidopsis [Arabidopsis thaliana]). The comparably large number of proteinaceous pectin methylesterase inhibitors (PMEIs; 76 members in Arabidopsis) questions the specificity of the PME-PMEI interaction and the functional role of such abundance. To understand the difference, or redundancy, between PMEIs, we used molecular dynamics (MD) simulations to predict the behavior of two PMEIs that are coexpressed and have distinct effects on plant development: AtPMEI4 and AtPMEI9. Simulations revealed the structural determinants of the pH dependence for the interaction of these inhibitors with AtPME3, a major PME expressed in roots. Key residues that are likely to play a role in the pH dependence were identified. The predictions obtained from MD simulations were confirmed in vitro, showing that AtPMEI9 is a stronger, less pH-independent inhibitor compared with AtPMEI4. Using pollen tubes as a developmental model, we showed that these biochemical differences have a biological significance. Application of purified proteins at pH ranges in which PMEI inhibition differed between AtPMEI4 and AtPMEI9 had distinct consequences on pollen tube elongation. Therefore, MD simulations have proven to be a powerful tool to predict functional diversity between PMEIs, allowing the discovery of a strategy that may be used by PMEIs to inhibit PMEs in different microenvironmental conditions and paving the way to identify the specific role of PMEI diversity in muro.

Salicylic Acid Regulates Pollen Tip Growth through an NPR3/NPR4-Independent Pathway.

Tip growth is a common strategy for the rapid elongation of cells to forage the environment and/or to target to long-distance destinations. In the model tip growth system of Arabidopsis pollen tubes, several small-molecule hormones regulate their elongation, but how these rapidly diffusing molecules control extremely localized growth remains mysterious. Here we show that the interconvertible salicylic acid (SA) and methylated SA (MeSA), well characterized for their roles in plant defense, oppositely regulate Arabidopsis pollen tip growth with SA being inhibitory and MeSA stimulatory. The effect of SA and MeSA was independent of known NPR3/NPR4 SA receptor-mediated signaling pathways. SA inhibited clathrin-mediated endocytosis in pollen tubes associated with an increased accumulation of less stretchable demethylated pectin in the apical wall, whereas MeSA did the opposite. Furthermore, SA and MeSA alter the apical activation of ROP1 GTPase, a key regulator of tip growth in pollen tubes, in an opposite manner. Interestingly, both MeSA methylesterase and SA methyltransferase, which catalyze the interconversion between SA and MeSA, are localized at the apical region of pollen tubes, indicating of the tip-localized production of SA and MeSA and consistent with their effects on the apical cellular activities. These findings suggest that local generation of a highly diffusible signal can regulate polarized cell growth, providing a novel mechanism of cell polarity control apart from the one involving protein and mRNA polarization.