ESCRT components ISTL1 andLIP5 are required for tapetal function and pollen viability.

Pollen wall assembly is crucial for pollen development and plant fertility. The durable biopolymer sporopollenin and the constituents of the tryphine coat are delivered to developing pollen grains by the highly coordinated secretory activity of the surrounding tapetal cells. The role of membrane trafficking in this process, however, is largely unknown. In this study, we used Arabidopsis thaliana to characterize the role of two late-acting endosomal sorting complex required for transport (ESCRT) components, ISTL1 and LIP5, in tapetal function. Plants lacking ISTL1 and LIP5 form pollen with aberrant exine patterns, leading to partial pollen lethality. We found that ISTL1 and LIP5 are required for exocytosis of plasma membrane and secreted proteins in the tapetal cells at the free microspore stage, contributing to pollen wall development and tryphine deposition. Whereas the ESCRT machinery is well known for its role in endosomal trafficking, the function of ISTL1 and LIP5 in exocytosis is not a typical ESCRT function. The istl1 lip5 double mutants also show reduced intralumenal vesicle concatenation in multivesicular endosomes in both tapetal cells and developing pollen grains as well as morphological defects in early endosomes/trans-Golgi networks, suggesting that late ESCRT components function in the early endosomal pathway and exocytosis.

Tonoplast-localized Ca2+ pumps regulate Ca2+ signals during pattern-triggered immunity in Arabidopsis thaliana.

One of the major events of early plant immune responses is a rapid influx of Ca2+ into the cytosol following pathogen recognition. Indeed, changes in cytosolic Ca2+ are recognized as ubiquitous elements of cellular signaling networks and are thought to encode stimulus-specific information in their duration, amplitude, and frequency. Despite the wealth of observations showing that the bacterial elicitor peptide flg22 triggers Ca2+ transients, there remain limited data defining the molecular identities of Ca2+ transporters involved in shaping the cellular Ca2+ dynamics during the triggering of the defense response network. However, the autoinhibited Ca2+-ATPase (ACA) pumps that act to expel Ca2+ from the cytosol have been linked to these events, with knockouts in the vacuolar members of this family showing hypersensitive lesion-mimic phenotypes. We have therefore explored how the two tonoplast-localized pumps, ACA4 and ACA11, impact flg22-dependent Ca2+ signaling and related defense responses. The double-knockout aca4/11 exhibited increased basal Ca2+ levels and Ca2+ signals of higher amplitude than wild-type plants. Both the aberrant Ca2+ dynamics and associated defense-related phenotypes could be suppressed by growing the aca4/11 seedlings at elevated temperatures. Relocalization of ACA8 from its normal cellular locale of the plasma membrane to the tonoplast also suppressed the aca4/11 phenotypes but not when a catalytically inactive mutant was used. These observations indicate that regulation of vacuolar Ca2+ sequestration is an integral component of plant immune signaling, but also that the action of tonoplast-localized Ca2+ pumps does not require specific regulatory elements not found in plasma membrane-localized pumps.

Class III Peroxidases PRX01, PRX44, and PRX73 Control Root Hair Growth in Arabidopsis thaliana.

Root hair cells are important sensors of soil conditions. They grow towards and absorb water-soluble nutrients. This fast and oscillatory growth is mediated by continuous remodeling of the cell wall. Root hair cell walls contain polysaccharides and hydroxyproline-rich glycoproteins, including extensins (EXTs). Class-III peroxidases (PRXs) are secreted into the apoplastic space and are thought to trigger either cell wall loosening or polymerization of cell wall components, such as Tyr-mediated assembly of EXT networks (EXT-PRXs). The precise role of these EXT-PRXs is unknown. Using genetic, biochemical, and modeling approaches, we identified and characterized three root-hair-specific putative EXT-PRXs, PRX01, PRX44, and PRX73. prx01,44,73 triple mutation and PRX44 and PRX73 overexpression had opposite effects on root hair growth, peroxidase activity, and ROS production, with a clear impact on cell wall thickness. We use an EXT fluorescent reporter with contrasting levels of cell wall insolubilization in prx01,44,73 and PRX44-overexpressing background plants. In this study, we propose that PRX01, PRX44, and PRX73 control EXT-mediated cell wall properties during polar expansion of root hair cells.

Role of SKD1 Regulators LIP5 and IST1-LIKE1 in Endosomal Sorting and Plant Development.

SKD1 is a core component of the mechanism that degrades plasma membrane proteins via the Endosomal Sorting Complex Required for Transport (ESCRT) pathway. Its ATPase activity and endosomal recruitment are regulated by the ESCRT components LIP5 and IST1. How LIP5 and IST1 affect ESCRT-mediated endosomal trafficking and development in plants is not known. Here we use Arabidopsis mutants to demonstrate that LIP5 controls the constitutive degradation of plasma membrane proteins and the formation of endosomal intraluminal vesicles. Although lip5 mutants were able to polarize the auxin efflux facilitators PIN2 and PIN3, both proteins were mis-sorted to the tonoplast in lip5 root cells. In addition, lip5 root cells over-accumulated PIN2 at the plasma membrane. Consistently with the trafficking defects of PIN proteins, the lip5 roots showed abnormal gravitropism with an enhanced response within the first 4 h after gravistimulation. LIP5 physically interacts with IST1-LIKE1 (ISTL1), a protein predicted to be the Arabidopsis homolog of yeast IST1. However, we found that Arabidopsis contains 12 genes coding for predicted IST1-domain containing proteins (ISTL1-12). Within the ISTL1-6 group, ISTL1 showed the strongest interaction with LIP5, SKD1, and the ESCRT-III-related proteins CHMP1A in yeast two hybrid assays. Through the analysis of single and double mutants, we found that the synthetic interaction of LIP5 with ISTL1, but not with ISTL2, 3, or 6, is essential for normal plant growth, repression of spontaneous cell death, and post-embryonic lethality.

Constitutive and Companion Cell-Specific Overexpression of AVP1, Encoding a Proton-Pumping Pyrophosphatase, Enhances Biomass Accumulation, Phloem Loading, and Long-Distance Transport.

Plant productivity is determined in large part by the partitioning of assimilates between the sites of production and the sites of utilization. Proton-pumping pyrophosphatases (H(+)-PPases) are shown to participate in many energetic plant processes, including general growth and biomass accumulation, CO2 fixation, nutrient acquisition, and stress responses. H(+)-PPases have a well-documented role in hydrolyzing pyrophosphate (PPi) and capturing the released energy to pump H(+) across the tonoplast and endomembranes to create proton motive force (pmf). Recently, an additional role for H(+)-PPases in phloem loading and biomass partitioning was proposed. In companion cells (CCs) of the phloem, H(+)-PPases localize to the plasma membrane rather than endomembranes, and rather than hydrolyzing PPi to create pmf, pmf is utilized to synthesize PPi. Additional PPi in the CCs promotes sucrose oxidation and ATP synthesis, which the plasma membrane P-type ATPase in turn uses to create more pmf for phloem loading of sucrose via sucrose-H(+) symporters. To test this model, transgenic Arabidopsis (Arabidopsis thaliana) plants were generated with constitutive and CC-specific overexpression of AVP1, encoding type 1 ARABIDOPSIS VACUOLAR PYROPHOSPHATASE1. Plants with both constitutive and CC-specific overexpression accumulated more biomass in shoot and root systems. (14)C-labeling experiments showed enhanced photosynthesis, phloem loading, phloem transport, and delivery to sink organs. The results obtained with constitutive and CC-specific promoters were very similar, such that the growth enhancement mediated by AVP1 overexpression can be attributed to its role in phloem CCs. This supports the model for H(+)-PPases functioning as PPi synthases in the phloem by arguing that the increases in biomass observed with AVP1 overexpression stem from improved phloem loading and transport.

Arabidopsis type I proton-pumping pyrophosphatase expresses strongly in phloem, where it is required for pyrophosphate metabolism and photosynthate partitioning.

Phloem loading is a critical process in plant physiology. The potential of regulating the translocation of photoassimilates from source to sink tissues represents an opportunity to increase crop yield. Pyrophosphate homeostasis is crucial for normal phloem function in apoplasmic loaders. The involvement of Arabidopsis (Arabidopsis thaliana) type I proton-pumping pyrophosphatase (AVP1) in phloem loading was analyzed at genetic, histochemical, and physiological levels. A transcriptional AVP1 promoter::GUS fusion revealed phloem activity in source leaves. Ubiquitous AVP1 overexpression (35S::AVP1 cassette) enhanced shoot biomass, photoassimilate production and transport, rhizosphere acidification, and expression of sugar-induced root ion transporter genes (POTASSIUM TRANSPORTER2 [KUP2], NITRATE TRANSPORTER2.1 [NRT2.1], NRT2.4, and PHOSPHATE TRANSPORTER1.4 [PHT1.4]). Phloem-specific AVP1 overexpression (Commelina Yellow Mottle Virus promoter [pCOYMV]::AVP1) elicited similar phenotypes. By contrast, phloem-specific AVP1 knockdown (pCoYMV::RNAiAVP1) resulted in stunted seedlings in sucrose-deprived medium. We also present a promoter mutant avp1-2 (SALK046492) with a 70% reduction of expression that did not show severe growth impairment. Interestingly, AVP1 protein in this mutant is prominent in the phloem. Moreover, expression of an Escherichia coli-soluble pyrophosphatase in the phloem (pCoYMV::pyrophosphatase) of avp1-2 plants resulted in severe dwarf phenotype and abnormal leaf morphology. We conclude that the Proton-Pumping Pyrophosphatase AVP1 localized at the plasma membrane of the sieve element-companion cell complexes functions as a synthase, and that this activity is critical for the maintenance of pyrophosphate homeostasis required for phloem function.

The VASCULATURE COMPLEXITY AND CONNECTIVITY gene encodes a plant-specific protein required for embryo provasculature development.

The molecular mechanisms by which vascular tissues acquire their identities are largely unknown. Here, we report on the identification and characterization of VASCULATURE COMPLEXITY AND CONNECTIVITY (VCC), a member of a 15-member, plant-specific gene family in Arabidopsis (Arabidopsis thaliana) that encodes proteins of unknown function with four predicted transmembrane domains. Homozygous vcc mutants displayed cotyledon vein networks of reduced complexity and disconnected veins. Similar disconnections or gaps were observed in the provasculature of vcc embryos, indicating that defects in vein connectivity appear early in mutant embryo development. Consistently, the overexpression of VCC leads to an unusually high proportion of cotyledons with high-complexity vein networks. Neither auxin distribution nor the polar localization of the auxin efflux carrier were affected in vcc mutant embryos. Expression of VCC was detected in developing embryos and procambial, cambial, and vascular cells of cotyledons, leaves, roots, hypocotyls, and anthers. To evaluate possible genetic interactions with other genes that control vasculature patterning in embryos, we generated a double mutant for VCC and OCTOPUS (OPS). The vcc ops double mutant embryos showed a complete loss of high-complexity vascular networks in cotyledons and a drastic increase in both provascular and vascular disconnections. In addition, VCC and OPS interact physically, suggesting that VCC and OPS are part of a complex that controls cotyledon vascular complexity.

Enhanced proton translocating pyrophosphatase activity improves nitrogen use efficiency in Romaine lettuce.

Plant nitrate (NO3(-)) acquisition depends on the combined activities of root high- and low-affinity NO3(-) transporters and the proton gradient generated by the plasma membrane H(+)-ATPase. These processes are coordinated with photosynthesis and the carbon status of the plant. Here, we present the characterization of romaine lettuce (Lactuca sativa 'Conquistador') plants engineered to overexpress an intragenic gain-of-function allele of the type I proton translocating pyrophosphatase (H(+)-PPase) of Arabidopsis (Arabidopsis thaliana). The proton-pumping and inorganic pyrophosphate hydrolytic activities of these plants are augmented compared with control plants. Immunohistochemical data show a conspicuous increase in H(+)-PPase protein abundance at the vasculature of the transgenic plants. Transgenic plants displayed an enhanced rhizosphere acidification capacity consistent with the augmented plasma membrane H(+)-ATPase proton transport values, and ATP hydrolytic capacities evaluated in vitro. These transgenic lines outperform control plants when challenged with NO3(-) limitations in laboratory, greenhouse, and field scenarios. Furthermore, we report the characterization of a lettuce LsNRT2.1 gene that is constitutive up-regulated in the transgenic plants. Of note, the expression of the LsNRT2.1 gene in control plants is regulated by NO3(-) and sugars. Enhanced accumulation of (15)N-labeled fertilizer by transgenic lettuce compared with control plants was observed in greenhouse experiments. A negative correlation between the level of root soluble sugars and biomass is consistent with the strong root growth that characterizes these transgenic plants.

Microautophagy Mediates Vacuolar Delivery of Storage Proteins in Maize Aleurone Cells.

The molecular machinery orchestrating microautophagy, whereby eukaryotic cells sequester autophagic cargo by direct invagination of the vacuolar/lysosomal membrane, is still largely unknown, especially in plants. Here, we demonstrate microautophagy of storage proteins in the maize aleurone cells of the endosperm and analyzed proteins with potential regulatory roles in this process. Within the cereal endosperm, starchy endosperm cells accumulate storage proteins (mostly prolamins) and starch whereas the peripheral aleurone cells store oils, storage proteins, and specialized metabolites. Although both cell types synthesize prolamins, they employ different pathways for their subcellular trafficking. Starchy endosperm cells accumulate prolamins in protein bodies within the endoplasmic reticulum (ER), whereas aleurone cells deliver prolamins to vacuoles via an autophagic mechanism, which we show is by direct association of ER prolamin bodies with the tonoplast followed by engulfment via microautophagy. To identify candidate proteins regulating this process, we performed RNA-seq transcriptomic comparisons of aleurone and starchy endosperm tissues during seed development and proteomic analysis on tonoplast-enriched fractions of aleurone cells. From these datasets, we identified 10 candidate proteins with potential roles in membrane modification and/or microautophagy, including phospholipase-Dα5 and a possible EUL-like lectin. We found that both proteins increased the frequency of tonoplast invaginations when overexpressed in Arabidopsis leaf protoplasts and are highly enriched at the tonoplast surface surrounding ER protein bodies in maize aleurone cells, thus supporting their potential connections to microautophagy. Collectively, this candidate list now provides useful tools to study microautophagy in plants.

Expression of an Arabidopsis vacuolar H+-pyrophosphatase gene (AVP1) in cotton improves drought- and salt tolerance and increases fibre yield in the field conditions.

The Arabidopsis gene AVP1 encodes a vacuolar pyrophosphatase that functions as a proton pump on the vacuolar membrane. Overexpression of AVP1 in Arabidopsis, tomato and rice enhances plant performance under salt and drought stress conditions, because up-regulation of the type I H+-PPase from Arabidopsis may result in a higher proton electrochemical gradient, which facilitates enhanced sequestering of ions and sugars into the vacuole, reducing water potential and resulting in increased drought- and salt tolerance when compared to wild-type plants. Furthermore, overexpression of AVP1 stimulates auxin transport in the root system and leads to larger root systems, which helps transgenic plants absorb water more efficiently under drought conditions. Using the same approach, AVP1-expressing cotton plants were created and tested for their performance under high-salt and reduced irrigation conditions. The AVP1-expressing cotton plants showed more vigorous growth than wild-type plants in the presence of 200 mM NaCl under hydroponic growth conditions. The soil-grown AVP1-expressing cotton plants also displayed significantly improved tolerance to both drought and salt stresses in greenhouse conditions. Furthermore, the fibre yield of AVP1-expressing cotton plants is at least 20% higher than that of wild-type plants under dry-land conditions in the field. This research indicates that AVP1 has the potential to be used for improving crop's drought- and salt tolerance in areas where water and salinity are limiting factors for agricultural productivity.

Cell-Free Protein Translation System for Expression of Lipid-Binding Proteins Tagged with Small epitopes and Their Use in Protein-Lipid Overlay Assays.

We adapted an efficient cell-free protein synthesis-based protocol for the production of lipid-binding proteins. The experimental procedures are based on the following steps: (1) cell-free synthesis of soluble, lipid-binding proteins fused to small tags; (2) analysis by dot blot of the accessibility of antibodies to the small tags. (3) protein lipid overlay assay with, immunodetection of bound protein by either chemiluminescence or fluorescence. We also provide a fast and inexpensive protocol for homemade lipid nitrocellulose strips spotted with acidic lipids (mostly phosphoinositides) extracted from plant tissues. These homemade lipid strips can be used for preliminary screen and characterization of putative phosphoinositide-binding proteins.

Reticulon proteins modulate autophagy of the endoplasmic reticulum in maize endosperm.

Reticulon (Rtn) proteins shape tubular domains of the endoplasmic reticulum (ER), and in some cases are autophagy receptors for selective ER turnover. We have found that maize Rtn1 and Rtn2 control ER homeostasis and autophagic flux in endosperm aleurone cells, where the ER accumulates lipid droplets and synthesizes storage protein accretions metabolized during germination. Maize Rtn1 and Rtn2 are expressed in the endosperm, localize to the ER, and re-model ER architecture in a dose-dependent manner. Rtn1 and Rtn2 interact with Atg8a using four Atg8-interacting motifs (AIMs) located at the C-terminus, cytoplasmic loop, and within the transmembrane segments. Binding between Rtn2 and Atg8 is elevated upon ER stress. Maize rtn2 mutants display increased autophagy and up-regulation of an ER stress-responsive chaperone. We propose that maize Rtn1 and Rtn2 act as receptors for autophagy-mediated ER turnover, and thus are critical for ER homeostasis and suppression of ER stress.

Purification of Plant ESCRT Proteins for Polyclonal Antibody Production.

Most endosomal sorting complex required for transport (ESCRT)-III proteins are not fully functional when expressed as fusion of fluorescent or epitope tags, frequently making the use of specific antibodies the only available method for their detection. Heterologous expression of ESCRT-III proteins in bacteria often results in the formation of insoluble aggregates or inclusion bodies that interfere with their purification. However, inclusion bodies are usually pure protein aggregates with high antigenicity. In addition, since proteins within inclusion bodies are presented in a range of folding states, immunization with inclusion bodies can potentially result in antibodies with specificity for different folding states of the protein under study. We describe here a protocol to isolate bacterial inclusion bodies of plant ESCRT-III proteins for production of polyclonal antibodies. We also describe a nitrocellulose-based immunoaffinity purification method that allows the immobilization of ESCRT-III proteins and the subsequent isolation of specific antibodies from a crude serum.

Identification of Fructose-1,6-bisphosphate aldolase cytosolic class I as an NMH7 MADS domain associated protein.

We are interested in identifying proteins that interact with the MADS domain protein NMH7 of Medicago sativa. We use an affinity column with a synthetic peptide derived from the MADS domain of NMH7 which has been reported to mediate protein-protein interaction with non-MADS domain interacting proteins. We identified approximately 40 and approximately 80kDa specifically bound proteins as the monomeric and dimeric forms of Fructose-1,6-bisphosphate aldolase cytosolic class I. NiNTA pull down assays revealed that K- and C-terminus regions of NMH7 are not required for the interaction with aldolase. Aldolase enzymatic activity is not required for the interaction with NMH7. NMH7 and aldolase were coimmunoprecipitated from non-inoculated seed and seedlings extracts. Colocalization studies using confocal microscopy showed that aldolase and NMH7 are localized in the cytoplasm and the nucleus of the cortical cells. These data together show that M. sativa aldolase is a novel MADS domain binding protein, and suggest a broader functional repertory for this enzyme, as has been proposed for other glycolytic enzymes.

The Diverse Iron Distribution in Eudicotyledoneae Seeds: From Arabidopsis to Quinoa.

Seeds accumulate iron during embryo maturation stages of embryogenesis. Using Arabidopsis thaliana as model plant, it has been described that mature embryos accumulate iron within a specific cell layer, the endodermis. This distribution pattern was conserved in most of the analyzed members from Brassicales, with the exception of the basal Vasconcellea pubescens that also showed elevated amounts of iron in cortex cells. To determine whether the V. pubescens iron distribution was indicative of a wider pattern in non-Brassicales Eudicotyledoneae, we studied iron distribution pattern in different embryos belonging to plant species from different Orders from Eudicotyledoneae and one basal from Magnoliidae. The results obtained indicate that iron distribution in A. thaliana embryo is an extreme case of apomorphic character found in Brassicales, not-extensive to the rest of Eudicotyledoneae.

ESCRT-mediated vesicle concatenation in plant endosomes.

Ubiquitinated plasma membrane proteins (cargo) are delivered to endosomes and sorted by endosomal sorting complex required for transport (ESCRT) machinery into endosome intralumenal vesicles (ILVs) for degradation. In contrast to the current model that postulates that ILVs form individually from inward budding of the endosomal limiting membrane, plant ILVs form as networks of concatenated vesicle buds by a novel vesiculation mechanism. We ran computational simulations based on experimentally derived diffusion coefficients of an ESCRT cargo protein and electron tomograms of Arabidopsis thaliana endosomes to measure cargo escape from budding ILVs. We found that 50% of the ESCRT cargo would escape from a single budding profile in 5-20 ms and from three concatenated ILVs in 80-200 ms. These short cargo escape times predict the need for strong diffusion barriers in ILVs. Consistent with a potential role as a diffusion barrier, we find that the ESCRT-III protein SNF7 remains associated with ILVs and is delivered to the vacuole for degradation.

Endocytosis and Endosomal Trafficking in Plants.

Endocytosis and endosomal trafficking are essential processes in cells that control the dynamics and turnover of plasma membrane proteins, such as receptors, transporters, and cell wall biosynthetic enzymes. Plasma membrane proteins (cargo) are internalized by endocytosis through clathrin-dependent or clathrin-independent mechanism and delivered to early endosomes. From the endosomes, cargo proteins are recycled back to the plasma membrane via different pathways, which rely on small GTPases and the retromer complex. Proteins that are targeted for degradation through ubiquitination are sorted into endosomal vesicles by the ESCRT (endosomal sorting complex required for transport) machinery for degradation in the vacuole. Endocytic and endosomal trafficking regulates many cellular, developmental, and physiological processes, including cellular polarization, hormone transport, metal ion homeostasis, cytokinesis, pathogen responses, and development. In this review, we discuss the mechanisms that mediate the recognition and sorting of endocytic and endosomal cargos, the vesiculation processes that mediate their trafficking, and their connection to cellular and physiological responses in plants.

Arabidopsis sodium dependent and independent phenotypes triggered by H⁺-PPase up-regulation are SOS1 dependent.

Coordinate regulation of transporters at both the plasma membrane and vacuole contribute to plant cell's ability to adapt to a changing environment and play a key role in the maintenance of the chemiosmotic circuits required for cellular growth. The plasma membrane (PM) Na⁺/H⁺ antiporter (SOS1) is involved in salt tolerance, presumably in sodium extrusion; the vacuolar type I H⁺-PPase AVP1 is involved in vacuolar sodium sequestration, but its overexpression has also been shown to alter the abundance and activity of the PM H⁺-ATPase. Here we investigate the relationship between these transporters utilizing loss-of-function mutants of SOS1 (sos1) and increased expression of AVP1 (AVP1OX). Heightened expression of AVP1 enhances pyrophosphate-dependent proton pump activity, salt tolerance, ion vacuolar sequestration, K⁺ uptake capacity, root hair development, osmotic responses, and PM ATPase hydrolytic and proton pumping activities. In sos1 lines overexpressing AVP1, these phenotypes are negatively affected demonstrating that sos1 is epistatic to AVP1. Enhanced AVP1 protein levels require SOS1 and this regulation appears to be post-translational.