SYNAPTOTAGMIN 4 is expressed mainly in the phloem and participates in abiotic stress tolerance in Arabidopsis.

Plant synaptotagmins structurally resemble animal synaptotagmins and extended-synaptotagmins. Animal synaptotagmins are well-characterized calcium sensors in membrane trafficking, and extended-synaptotagmins mediate lipid transfer at the endoplasmic reticulum-plasma membrane contact sites. Here, we characterize SYNAPTOTAGMIN 4 (SYT4), which belongs to the six-member family in Arabidopsis. Fluorometric GUS assay showed that the SYT4 promoter was strongest in roots and the least active in rosettes and cauline leaves, which was confirmed by qPCR. In seedlings, promoter activity was influenced by several factors, such as plant growth regulators, mannitol, sucrose, polyethylene glycol and cold. GUS histochemistry revealed SYT4 promoter activity in the phloem of all organs and even almost exclusively in sieve element precursors and differentiating sieve elements. Accordingly, the SYT-GFP fusion protein also accumulated in these cells with maximal abundance in sieve element precursors. The protein formed a network in the cytoplasm, but during sieve tube differentiation, it deposited at the cell periphery and disappeared from mature tubes. Using photoconvertible fluorescence technology, we showed that a high abundance of SYT4 protein in meristematic protophloem cells was due to its extensive synthesis. SYT4 protein synthesis was interrupted in differentiating sieve elements, but protein degradation was also reduced. In addition to phloem, the fusion protein was detected in shoot and root stem cell niche as early as the late heart stage of the embryo. We isolated and molecularly and biologically characterized five syt4 T-DNA insertion alleles and subjected them to phenotype analysis. The allele with the C2B domain interrupted by an T-DNA insertion exhibits increased sensitivity to factors such as auxins, osmotics, salicylic acid, sodium chloride, and the absence of sucrose in the root growth test.

Nitric oxide in plants: an insight on redox activity and responses toward abiotic stress signaling.

Plants, as sessile organisms, are subjected to diverse abiotic stresses, including salinity, desiccation, metal toxicity, thermal fluctuations, and hypoxia at different phases of plant growth. Plants can activate messenger molecules to initiate a signaling cascade of response toward environmental stresses that results in either cell death or plant acclimation. Nitric oxide (NO) is a small gaseous redox-active molecule that exhibits a plethora of physiological functions in growth, development, flowering, senescence, stomata closure and responses to environmental stresses. It can also facilitate alteration in protein function and reprogram the gene profiling by direct or indirect interaction with different target molecules. The bioactivity of NO can be manifested through different redox-based protein modifications including S-nitrosylation, protein nitration, and metal nitrosylation in plants. Although there has been considerable progress in the role of NO in regulating stress signaling, still the physiological mechanisms regarding the abiotic stress tolerance in plants remain unclear. This review summarizes recent advances in understanding the emerging knowledge regarding NO function in plant tolerance against abiotic stresses. The manuscript also highlighted the importance of NO as an abiotic stress modulator and developed a rational design for crop cultivation under a stress environment.

PIN2/3/4 auxin carriers mediate root growth inhibition under conditions of boron deprivation in Arabidopsis.

The mechanistic basis by which boron (B) deprivation inhibits root growth via the mediation of root apical auxin transport and distribution remains elusive. This study showed that B deprivation repressed root growth of wild-type Arabidopsis seedlings, which was related to higher auxin accumulation (observed with DII-VENUS and DR5-GFP lines) in B-deprived roots. Boron deprivation elevated the auxin content in the root apex, coinciding with upregulation of the expression levels of auxin biosynthesis-related genes (TAA1, YUC3, YUC9, and NIT1) in shoots, but not in root apices. Phenotyping experiments using auxin transport-related mutants revealed that the PIN2/3/4 carriers are involved in root growth inhibition caused by B deprivation. B deprivation not only upregulated the transcriptional levels of PIN2/3/4, but also restrained the endocytosis of PIN2/3/4 carriers (observed with PIN-Dendra2 lines), resulting in elevated protein levels of PIN2/3/4 in the plasma membrane. Overall, these results suggest that B deprivation not only enhances auxin biosynthesis in shoots by elevating the expression levels of auxin biosynthesis-related genes but also promotes the polar auxin transport from shoots to roots by upregulating the gene expression levels of PIN2/3/4, as well as restraining the endocytosis of PIN2/3/4 carriers, ultimately resulting in auxin accumulation in root apices and root growth inhibition.

Membrane-anchored calpains - hidden regulators of growth and development beyond plants?

Calpains are modulatory proteases that modify diverse cellular substrates and play essential roles in eukaryots. The best studied are animal cytosolic calpains. Here, we focus on enigmatic membrane-anchored calpains, their structural and functional features as well as phylogenetic distribution. Based on domain composition, we identified four types of membrane-anchored calpains. Type 1 and 2 show broad phylogenetic distribution among unicellular protists and streptophytes suggesting their ancient evolutionary origin. Type 3 and 4 diversified early and are present in brown algae and oomycetes. The plant DEK1 protein is the only representative of membrane-anchored calpains that has been functionally studied. Here, we present up to date knowledge about its structural features, putative regulation, posttranslational modifications, and biological role. Finally, we discuss potential model organisms and available tools for functional studies of membrane-anchored calpains with yet unknown biological role. Mechanistic understanding of membrane-anchored calpains may provide important insights into fundamental principles of cell polarization, cell fate control, and morphogenesis beyond plants.

The Absence of the AtSYT1 Function Elevates the Adverse Effect of Salt Stress on Photosynthesis in Arabidopsis.

Arabidopsis thaliana SYNAPTOTAGMIN 1 (AtSYT1) was shown to be involved in responses to different environmental and biotic stresses. We investigated gas exchange and chlorophyll a fluorescence in Arabidopsis wild-type (WT, ecotype Col-0) and atsyt1 mutant plants irrigated for 48 h with 150 mM NaCl. We found that salt stress significantly decreases net photosynthetic assimilation, effective photochemical quantum yield of photosystem II (ΦPSII), stomatal conductance and transpiration rate in both genotypes. Salt stress has a more severe impact on atsyt1 plants with increasing effect at higher illumination. Dark respiration, photochemical quenching (qP), non-photochemical quenching and ΦPSII measured at 750 µmol m-2 s-1 photosynthetic photon flux density were significantly affected by salt in both genotypes. However, differences between mutant and WT plants were recorded only for qP and ΦPSII. Decreased photosynthetic efficiency in atsyt1 under salt stress was accompanied by reduced chlorophyll and carotenoid and increased flavonol content in atsyt1 leaves. No differences in the abundance of key proteins participating in photosynthesis (except PsaC and PsbQ) and chlorophyll biosynthesis were found regardless of genotype or salt treatment. Microscopic analysis showed that irrigating plants with salt caused a partial closure of the stomata, and this effect was more pronounced in the mutant than in WT plants. The localization pattern of AtSYT1 was also altered by salt stress.

Specific expression of AtIRT1 in phloem companion cells suggests its role in iron translocation in aboveground plant organs.

IRON-REGULATED TRANSPORTER 1 (IRT1) is a central iron transporter responsible for the uptake of iron from the rhizosphere to root epidermal cells. This study uses immunohistochemistry, histochemistry, and fluorometry to show that this gene's promoter is also active in the aboveground parts, specifically in phloem companied cells. Promoter activity here was regulated by iron as it was in the roots. The promoter of the close IRT2 homolog was root-specific and only weakly active in the stem pits. RT-PCR showed the presence of a long splicing form exclusively in iron-deficient roots. The short splicing form was present in all organs regardless of the presence of iron. Immunohistology exhibited labeling on the periphery of the epidermal cells in matured root zone and intracellular patches in the meristematic cells. In the aboveground organs, the protein was seen in the whole volume of companion cells and in neighboring sieve elements as bodies. The fluorescent protein technique revealed the short IRT1 form to be present in the patches accumulated mainly around the nucleus and the long form as a continuous layer along the cells periphery. These results suggest that IRT1 has a role also in the aboveground organs.

Endosidin 2 accelerates PIN2 endocytosis and disturbs intracellular trafficking of PIN2, PIN3, and PIN4 but not of SYT1.

We established that Endosidin2 (ES2) affected the trafficking routes of both newly synthesized and endocytic pools of PIN-FORMED2 (PIN2) in Arabidopsis root epidermal cells. PIN2 populations accumulated in separated patches, which gradually merged into large and compact ES2 aggregates (ES2As). FM4-64 endocytic tracer labeled ES2As as well. Both PIN2 pools also appeared in vacuoles. Accelerated endocytosis of PIN2, its aggregation in the cytoplasm, and redirection of PIN2 flows to vacuoles led to a substantial reduction of the abundance of this protein in the plasma membrane. Whereas PIN-FORMED3 and PIN-FORMED4 also aggregated in the cytoplasm, SYT1 was not sensitive to ES2 treatment and did not appear either in the cytoplasmic aggregates or vacuoles. Ultrastructural analysis revealed that ES2 affects the Golgi apparatus so that stacks acquired cup-shape and even circular shape surrounded by several vesicles. Abnormally shaped Golgi stacks, stack remnants, multi-lamellar structures, separated Golgi cisterna rings, tubular structures, and vesicles formed discrete clusters.

The Photoconvertible Fluorescent Protein Dendra2 Tag as a Tool to Investigate Intracellular Protein Dynamics.

Fluorescence proteins changing spectral properties after exposure to light with a specific wavelength have recently become outstanding aids in the study of intracellular protein dynamics. Herein we show using Arabidopsis SYNAPTOTAGMIN 1 as a model protein that the Dendra2 green to red photoconvertible protein tag in combination with confocal scanning laser microscopy is a useful tool to study membrane protein intracellular dynamics.

Effects of Auxins on PIN-FORMED2 (PIN2) Dynamics Are Not Mediated by Inhibiting PIN2 Endocytosis.

By using the photoconvertible fluorescence protein Dendra2 as a tag we demonstrated that neither the naturally occurring auxins indole-3-acetic acid and indole-3-butyric acid, nor the synthetic auxin analogs 1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid nor compounds inhibiting polar auxin transport such as 2,3,5-triiodobenzoic acid and 1-N-naphthylphthalamic acid, were able to inhibit endocytosis of the putative auxin transporter PIN-FORMED2 (PIN2) in Arabidopsis (Arabidopsis thaliana) root epidermis cells. All compounds, except Indole-3-butyric acid, repressed the recovery of the PIN2-Dendra2 plasma membrane pool after photoconversion when they were used in high concentrations. The synthetic auxin analogs 1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid showed the strongest inhibition. Auxins and auxin transport inhibitors suppressed also the accumulation of both newly synthesized and endocytotic PIN2 pools in Brefeldin A compartments (BFACs). Furthermore, we demonstrated that all compounds are also interfering with BFAC formation. The synthetic auxin analogs caused the highest reduction in the number and size of BFACs. We concluded that auxins and inhibitors of auxin transport do affect PIN2 turnover in the cells, but it is through the synthetic rather than the endocytotic pathway. The study also confirmed inappropriateness of the BFA-based approach to study PIN2 endocytosis because the majority of PIN2 accumulating in BFACs is newly synthesized and not derived from the plasma membrane.

Actin3 promoter reveals undulating F-actin bundles at shanks and dynamic F-actin meshworks at tips of tip-growing pollen tubes.

The dynamic actin cytoskeleton of pollen tubes is both the driver of the tip growth and the organizer of cell polarity. In order to understand this fast re-arranging cytoskeletal system, we need reliable constructs expressed under relevant promoters. Here we are reporting that the Lifeact reporter, expressed under the pollen-specific Actin3 promoter, visualizes very dynamic F-actin elements both in germinating pollen grains and tip-growing pollen tubes. Importantly, we have documented very active actin polymerization at the cell periphery, especially in the bulging area during pollen germination and in the apical clear zone. Expression of the Lifeact reporter under control of the pollen-specific Actin3 promoter revealed 2 new aspects: (i) long F-actin bundles in pollen tube shanks are dynamic, showing undulating movements, (ii) subapical 'actin collars' or 'fringes' are absent.

Arabidopsis Synaptotagmin 2 Participates in Pollen Germination and Tube Growth and Is Delivered to Plasma Membrane via Conventional Secretion.

Arabidopsis synaptotagmin 2 (SYT2) has been reported to participate in an unconventional secretory pathway in somatic cells. Our results showed that SYT2 was expressed mainly in the pollen of Arabidopsis thaliana. The pollen of syt2 T-DNA and RNA interference mutant lines exhibited reduced total germination and impeded pollen tube growth. Analysis of the expression of SYT2-GFP fusion protein in the pollen tube indicates that SYT2 was localized to distinct, patchy compartments but could co-localize with the Golgi markers, BODIPY TR C5 ceramide and GmMan1-mCherry. However, SYT2-DsRed-E5 was localized to the plasma membrane in Arabidopsis suspension cells, in addition to the Golgi apparatus. The localization of SYT2 at the plasma membrane was further supported by immunofluorescence staining in pollen tubes. Moreover, brefeldin A treatment inhibited the transport of SYT2 to the plasma membrane and caused SYT2 to aggregate and form enlarged compartments. Truncation of the SYT2-C2AB domains also resulted in retention of SYT2 in the Golgi apparatus. An in vitro phospholipid-binding assay showed that SYT2-C2AB domains bind to the phospholipid membrane in a calcium-dependent manner. Take together, our results indicated that SYT2 was required for pollen germination and pollen tube growth, and was involved in conventional exocytosis.

PIN2 turnover in Arabidopsis root epidermal cells explored by the photoconvertible protein Dendra2.

The steady state level of integral membrane proteins is dependent on a strictly controlled delivery and removal. Here we show that Dendra2, a green-to-red photoconvertible fluorescent protein, is a suitable tool to study protein turnover in plants. We characterized the fluorescence properties of Dendra2 expressed either as a free protein or as a tag in Arabidopsis thaliana roots and optimized photoconversion settings to study protein turnover. Dendra2 was fused to the PIN2 protein, an auxin transporter in the root tip, and by time-lapse imaging and assessment of red and green signal intensities in the membrane after photoconversion we quantified directly and simultaneously the rate of PIN2 delivery of the newly synthesized protein into the plasma membrane as well as the disappearance of the protein from the plasma membrane due to degradation. Additionally we have verified several factors which are expected to affect PIN2 protein turnover and therefore potentially regulate root growth.

NBP, a zebrafish homolog of human Kank3, is a novel Numb interactor essential for epidermal integrity and neurulation.

Numb is an adaptor protein implicated in diverse basic cellular processes. Using the yeast-two hybrid system we isolated a novel Numb interactor in zebrafish called NBP which is an ortholog of human renal tumor suppressor Kank. NBP interacts with the PTB domain of Numb through a region well conserved among vertebrate Kanks containing the NGGY sequence. Similar NBP and Numb morphant phenotype such as impaired convergence and extension movements during gastrulation, neurulation and epidermis defects and enhanced phenotypic aberrations in double morphants suggest that the genes interact genetically. We demonstrate that the expression of NBP undergoes quantitative and qualitative changes during embryogenesis and that the protein accumulates at the cell periphery to sites of cell-cell contact during gastrulation and later in development it concentrates at the basal poles of differentiated cells. These findings imply a possible role of NBP in establishing and maintaining cell adhesion and tissue integrity.

The signal transducer NPH3 integrates the phototropin1 photosensor with PIN2-based polar auxin transport in Arabidopsis root phototropism.

Under blue light (BL) illumination, Arabidopsis thaliana roots grow away from the light source, showing a negative phototropic response. However, the mechanism of root phototropism is still unclear. Using a noninvasive microelectrode system, we showed that the BL sensor phototropin1 (phot1), the signal transducer NONPHOTOTROPIC HYPOCOTYL3 (NPH3), and the auxin efflux transporter PIN2 were essential for BL-induced auxin flux in the root apex transition zone. We also found that PIN2-green fluorescent protein (GFP) localized to vacuole-like compartments (VLCs) in dark-grown root epidermal and cortical cells, and phot1/NPH3 mediated a BL-initiated pathway that caused PIN2 redistribution to the plasma membrane. When dark-grown roots were exposed to brefeldin A (BFA), PIN2-GFP remained in VLCs in darkness, and BL caused PIN2-GFP disappearance from VLCs and induced PIN2-GFP-FM4-64 colocalization within enlarged compartments. In the nph3 mutant, both dark and BL BFA treatments caused the disappearance of PIN2-GFP from VLCs. However, in the phot1 mutant, PIN2-GFP remained within VLCs under both dark and BL BFA treatments, suggesting that phot1 and NPH3 play different roles in PIN2 localization. In conclusion, BL-induced root phototropism is based on the phot1/NPH3 signaling pathway, which stimulates the shootward auxin flux by modifying the subcellular targeting of PIN2 in the root apex transition zone.

Subtissue-specific evaluation of promoter efficiency by quantitative fluorometric assay in laser microdissected tissues of rapeseed.

ß-glucuronidase (GUS) is a useful reporter for the evaluation of promoter characteristics in transgenic plants. Here, we introduce an original technique to quantify the strength of promoters at subtissue resolution of cell clusters. The method combines cryotomy, laser microdissection, and improved fluorometric analysis of GUS activity using 6-chloro-4-methylumbelliferyl-ß-D-glucuronide as an efficient fluorogenic substrate for kinetic studies in plants. The laser microdissection/6-chloro-4-methylumbelliferyl-ß-D-glucuronide method is robust and reliable in a wide range of GUS expression levels and requires extremely low (few cells) tissue amounts. Suitability of the assay was demonstrated on rapeseed (Brassica napus) plants transformed with a P35S2::GUS construct. GUS expression patterns were visualized and quantified in approximately 30 tissues of vegetative and generative organs. Considerable differences in promoter activity within the tissues are discussed in relation to the cell type and developmental state.

Arabidopsis synaptotagmin 1 is required for the maintenance of plasma membrane integrity and cell viability.

Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca(2+)-activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca(2+)-regulated exocytosis. Here, we show that loss of function of the homologous Arabidopsis thaliana Synaptotagmin 1 protein (SYT1) reduces the viability of cells as a consequence of a decrease in the integrity of the plasma membrane. This reduced integrity is enhanced in the syt1-2 null mutant in conditions of osmotic stress likely caused by a defective plasma membrane repair. Consistent with a role in plasma membrane repair, SYT1 is ubiquitously expressed, is located at the plasma membrane, and shares all domains characteristic of animal synaptotagmins (i.e., an N terminus-transmembrane domain and a cytoplasmic region containing two C2 domains with phospholipid binding activities). Our analyses support that membrane trafficking mediated by SYT1 is important for plasma membrane integrity and plant fitness.

Duplicated P5CS genes of Arabidopsis play distinct roles in stress regulation and developmental control of proline biosynthesis.

Delta-1-pyrroline-5-carboxylate synthetase enzymes, which catalyse the rate-limiting step of proline biosynthesis, are encoded by two closely related P5CS genes in Arabidopsis. Transcription of the P5CS genes is differentially regulated by drought, salinity and abscisic acid, suggesting that these genes play specific roles in the control of proline biosynthesis. Here we describe the genetic characterization of p5cs insertion mutants, which indicates that P5CS1 is required for proline accumulation under osmotic stress. Knockout mutations of P5CS1 result in the reduction of stress-induced proline synthesis, hypersensitivity to salt stress, and accumulation of reactive oxygen species. By contrast, p5cs2 mutations cause embryo abortion during late stages of seed development. The desiccation sensitivity of p5cs2 embryos does not reflect differential control of transcription, as both P5CS mRNAs are detectable throughout embryonic development. Cellular localization studies with P5CS-GFP gene fusions indicate that P5CS1 is sequestered into subcellular bodies in embryonic cells, where P5CS2 is dominantly cytoplasmic. Although proline feeding rescues the viability of mutant embryos, p5cs2 seedlings undergo aberrant development and fail to produce fertile plants even when grown on proline. In seedlings, specific expression of P5CS2-GFP is seen in leaf primordia where P5CS1-GFP levels are very low, and P5CS2-GFP also shows a distinct cell-type-specific and subcellular localization pattern compared to P5CS1-GFP in root tips, leaves and flower organs. These data demonstrate that the Arabidopsis P5CS enzymes perform non-redundant functions, and that P5CS1 is insufficient for compensation of developmental defects caused by inactivation of P5CS2.

SWI3 subunits of putative SWI/SNF chromatin-remodeling complexes play distinct roles during Arabidopsis development.

SWITCH/SUCROSE NONFERMENTING (SWI/SNF) chromatin-remodeling complexes mediate ATP-dependent alterations of DNA-histone contacts. The minimal functional core of conserved SWI/SNF complexes consists of a SWI2/SNF2 ATPase, SNF5, SWP73, and a pair of SWI3 subunits. Because of early duplication of the SWI3 gene family in plants, Arabidopsis thaliana encodes four SWI3-like proteins that show remarkable functional diversification. Whereas ATSWI3A and ATSWI3B form homodimers and heterodimers and interact with BSH/SNF5, ATSWI3C, and the flowering regulator FCA, ATSWI3D can only bind ATSWI3B in yeast two-hybrid assays. Mutations of ATSWI3A and ATSWI3B arrest embryo development at the globular stage. By a possible imprinting effect, the atswi3b mutations result in death for approximately half of both macrospores and microspores. Mutations in ATSWI3C cause semidwarf stature, inhibition of root elongation, leaf curling, aberrant stamen development, and reduced fertility. Plants carrying atswi3d mutations display severe dwarfism, alterations in the number and development of flower organs, and complete male and female sterility. These data indicate that, by possible contribution to the combinatorial assembly of different SWI/SNF complexes, the ATSWI3 proteins perform nonredundant regulatory functions that affect embryogenesis and both the vegetative and reproductive phases of plant development.