Evidence of differential pH regulation of the Arabidopsis vacuolar Ca2+/H+ antiporters CAX1 and CAX2.

The Arabidopsis Ca(2+)/H(+) antiporters cation exchanger (CAX) 1 and 2 utilise an electrochemical gradient to transport Ca(2+) into the vacuole to help mediate Ca(2+) homeostasis. Previous whole plant studies indicate that activity of Ca(2+)/H(+) antiporters is regulated by pH. However, the pH regulation of individual Ca(2+)/H(+) antiporters has not been examined. To determine whether CAX1 and CAX2 activity is affected by pH, Ca(2+)/H(+) antiport activity was measured in vacuolar membrane vesicles isolated from yeast heterologously expressing either transporter. Ca(2+) transport by CAX1 and CAX2 was regulated by cytosolic pH and each transporter had a distinct cytosolic pH profile. Screening of CAX1/CAX2 chimeras identified an amino acid domain within CAX2 that altered the pH-dependent Ca(2+) transport profile so that it was almost identical to the pH profile of CAX1. Results from mutagenesis of a specific His residue within this domain suggests a role for this residue in pH regulation.     

Expression of Arabidopsis CAX1 in tobacco: altered calcium homeostasis and increased stress sensitivity

Calcium (Ca(2)+) efflux from the cytosol modulates Ca(2+) concentrations in the cytosol, loads Ca(2+) into intracellular compartments, and supplies Ca(2+) to organelles to support biochemical functions. The Ca(2+)/H(+) antiporter CAX1 (for CALCIUM EXCHANGER 1) of Arabidopsis is thought to be a key mediator of these processes. To clarify the regulation of CAX1, we examined CAX1 RNA expression in response to various stimuli. CAX1 was highly expressed in response to exogenous Ca(2+). Transgenic tobacco plants expressing CAX1 displayed symptoms of Ca(2+) deficiencies, including hypersensitivity to ion imbalances, such as increased magnesium and potassium concentrations, and to cold shock, but increasing the Ca(2+) in the media abrogated these sensitivities. Tobacco plants expressing CAX1 also demonstrated increased Ca(2+) accumulation and altered activity of the tonoplast-enriched Ca(2+)/H(+) antiporter. These results emphasize that regulated expression of Ca(2+)/H(+) antiport activity is critical for normal growth and adaptation to certain stresses.     

The role of CAX1 and CAX3 in elemental distribution and abundance in Arabidopsis seed

The ability to alter nutrient partitioning within plants cells is poorly understood. In Arabidopsis (Arabidopsis thaliana), a family of endomembrane cation exchangers (CAXs) transports Ca(2+) and other cations. However, experiments have not focused on how the distribution and partitioning of calcium (Ca) and other elements within seeds are altered by perturbed CAX activity. Here, we investigate Ca distribution and abundance in Arabidopsis seed from cax1 and cax3 loss-of-function lines and lines expressing deregulated CAX1 using synchrotron x-ray fluorescence microscopy. We conducted 7- to 10-μm resolution in vivo x-ray microtomography on dry mature seed and 0.2-μm resolution x-ray microscopy on embryos from lines overexpressing deregulated CAX1 (35S-sCAX1) and cax1cax3 double mutants only. Tomograms showed an increased concentration of Ca in both the seed coat and the embryo in cax1, cax3, and cax1cax3 lines compared with the wild type. High-resolution elemental images of the mutants showed that perturbed CAX activity altered Ca partitioning within cells, reducing Ca partitioning into organelles and/or increasing Ca in the cytosol and abolishing tissue-level Ca gradients. In comparison with traditional volume-averaged metal analysis, which confirmed subtle changes in seed elemental composition, the collection of spatially resolved data at varying resolutions provides insight into the impact of altered CAX activity on seed metal distribution and indicates a cell type-specific function of CAX1 and CAX3 in partitioning Ca into organelles. This work highlights a powerful technology for inferring transport function and quantifying nutrient changes.     

High boron-induced ubiquitination regulates vacuolar sorting of the BOR1 borate transporter in Arabidopsis thaliana

Boron homeostasis is important for plants, as boron is essential but is toxic in excess. Under high boron conditions, the Arabidopsis thaliana borate transporter BOR1 is trafficked from the plasma membrane (PM) to the vacuole via the endocytic pathway for degradation to avoid excess boron transport. Here, we show that boron-induced ubiquitination is required for vacuolar sorting of BOR1. We found that a substitution of lysine 590 with alanine (K590A) in BOR1 blocked degradation. BOR1 was mono- or diubiquitinated within several minutes after applying a high concentration of boron, whereas the K590A mutant was not. The K590A mutation abolished vacuolar transport of BOR1 but did not apparently affect polar localization to the inner PM domains. Furthermore, brefeldin A and wortmannin treatment suggested that Lys-590 is required for BOR1 translocation from an early endosomal compartment to multivesicular bodies. Our results show that boron-induced ubiquitination of BOR1 is not required for endocytosis from the PM but is crucial for the sorting of internalized BOR1 to multivesicular bodies for subsequent degradation in vacuoles.     

OsAGAP, an ARF-GAP from rice, regulates root development mediated by auxin in Arabidopsis

Arf (ADP-ribosylation factor) proteins, which mediate vesicular transport, have little or no intrinsic GTPase activity. They rely on the action of GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) for their function. In the present study the OsAGAP gene in rice, which encoded a protein with predicted structure similar to ArfGAP, was identified. The purified OsAGAP-GST fusion protein was able to stimulate the GTPase activity of rice Arf. Furthermore, OsAGAP can rescue the defect of vesicular transport in the yeast gcs1 delta glo3 delta double-mutant cells. Transgenic Arabidopsis with OsAGAP constitutively expression showed reduced apical dominance, shorter primary roots, increasing number of longer adventitious roots. Many of the phenotypes can be phenocopied by treatment of exogenous indoleacetic acid level (IAA) in wild-type plants. Determination of whole-plant IAA level showed that there is a sharp increase of free IAA in OsAGAP transgenic Arabidopsis seedlings. In addition, removal of the 4-day-old shoot apex could inhibit the adventitious root formation in the transgenic seedlings. These results suggest OsAGAP, an ARF-GAP of rice, maybe involved in the mediation of plant root development by regulating auxin level.     

CBL1, a calcium sensor that differentially regulates salt,drought, and cold responses in Arabidopsis

Although calcium is a critical component in the signal transduction pathways that lead to stress gene expression in higher plants, little is known about the molecular mechanism underlying calcium function. It is believed that cellular calcium changes are perceived by sensor molecules, including calcium binding proteins. The calcineurin B-like (CBL) protein family represents a unique group of calcium sensors in plants. A member of the family, CBL1, is highly inducible by multiple stress signals, implicating CBL1 in stress response pathways. When the CBL1 protein level was increased in transgenic Arabidopsis plants, it altered the stress response pathways in these plants. Although drought-induced gene expression was enhanced, gene induction by cold was inhibited. In addition, CBL1-overexpressing plants showed enhanced tolerance to salt and drought but reduced tolerance to freezing. By contrast, cbl1 null mutant plants showed enhanced cold induction and reduced drought induction of stress genes. The mutant plants displayed less tolerance to salt and drought but enhanced tolerance to freezing. These studies suggest that CBL1 functions as a positive regulator of salt and drought responses and a negative regulator of cold response in plants.     

Polarized cell growth in Arabidopsis requires endosomal recycling mediated by GBF1-related ARF exchange factors

Polarized tip growth is a fundamental cellular process in many eukaryotic organisms, mediating growth of neuronal axons and dendrites or fungal hyphae. In plants, pollen and root hairs are cellular model systems for analysing tip growth. Cell growth depends on membrane traffic. The regulation of this membrane traffic is largely unknown for tip-growing cells, in contrast to cells exhibiting intercalary growth. Here we show that in Arabidopsis, GBF1-related exchange factors for the ARF GTPases (ARF GEFs) GNOM and GNL2 play essential roles in polar tip growth of root hairs and pollen, respectively. When expressed from the same promoter, GNL2 (in contrast to the early-secretory ARF GEF GNL1) is able to replace GNOM in polar recycling of the auxin efflux regulator PIN1 from endosomes to the basal plasma membrane in non-tip growing cells. Thus, polar recycling facilitates polar tip growth, and GNL2 seems to have evolved to meet the specific requirement of fast-growing pollen in higher plants.     

Analysis of the Ca2+ domain in the Arabidopsis H+/Ca2+ antiporters CAX1 and CAX3

Ca²⁺ levels in plants are controlled in part by H⁺/Ca²⁺ exchangers. Structure/function analysis of the Arabidopsis H⁺/cation exchanger, CAX1, revealed that a nine amino acid region (87–95) is involved in CAX1-mediated Ca²⁺ specificity. CAX3 is 77% identical (93% similar) to CAX1, and when expressed in yeast, localizes to the vacuole but does not suppress yeast mutants defective in vacuolar Ca²⁺ transport. Transgenic tobacco plants expressing CAX3 containing the 9 amino acid Ca²⁺ domain (Cad) from CAX1 (CAX3-9) displayed altered stress sensitivities similar to CAX1-expressing plants, whereas CAX3-9-expressing plants did not have any altered stress sensitivities. A single leucine-to-isoleucine change at position 87 (CAX3-I) within the Cad of CAX3 allows this protein to weakly transport Ca²⁺ in yeast (less than 10% of CAX1). Site-directed mutagenesis of the leucine in the CAX3 Cad demonstrated that no amino acid change tested could confer more activity than CAX3-I. Transport studies in yeast demonstrated that the first three amino acids of the CAX1 Cad could confer twice the Ca²⁺ transport capability compared to CAX3-I. The entire Cad of CAX3 (87–95) inserted into CAX1 abolishes CAX1-mediated Ca²⁺ transport. However, single, double, or triple amino acid replacements within the native CAX1 Cad did not block CAX1 mediated Ca²⁺ transport. Together these findings suggest that other domains within CAX1 and CAX3 influence Ca²⁺ transport. This study has implications for the ability to engineer CAX-mediated transport in plants by manipulating Cad residues.     

Precipitation regulates plant gas exchange and its long-term response to climate change in a temperate grassland

Aims Climate change largely impacts ecosystem carbon and water cycles by changing plant gas exchange, which may further cause positive or negative feedback to global climate change. However, long-term global change manipulative experiments are seldom conducted to reveal plant ecophysiological responses to climatic warming and altered precipitation regimes.Methods An 8-year field experiment with both warming and increased precipitation was conducted in a temperate grassland in northern China. We measured leaf gas exchange rates (including plant photosynthesis, transpiration and instantaneous water use efficiency [WUE]) of two dominant plant species (Stipa sareptana var. krylovii and Agropyron cristatum) from 2005 to 2012 (except 2006 and 2010) and those of other six species from 2011 to 2012.Important findings Increased precipitation significantly stimulated plant photosynthetic rates (A) by 29.5% and 19.9% and transpiration rates (E) by 42.2% and 51.2% for both dominant species S. sareptana var. krylovii and A. cristatum, respectively, across the 8 years. Similarly, A and E of the six plant functional types were all stimulated by increased precipitation in 2011 and 2012. As the balance of A and E, the instantaneous WUEs of different plant species had species-specific responses to increased precipitation. In contrast, neither warming nor its interaction with increased precipitation significantly affected plant leaf gas exchange rates. Furthermore, A and E of the two dominant species and their response magnitudes to water treatments positively correlated with rainfall amount in July across years. We did not find any significant difference between the short-term versus long-term responses of plant photosynthesis, suggesting the flexibility of leaf gas exchange under climate change. The results suggest that changing precipitation rather than global warming plays a prominent role in determining production of this grassland in the context of climate change.     

Retarded Embryo Development 1(RED1) regulates embryo development,seed maturation and plant growth in Arabidopsis

Plant seeds accumulate large amounts of protein and carbohydrate as storage reserves during maturation.Thus,understanding the genetic control of embryo and seed development may provide bioengineering tools for yield improvement.In this study,we report the identification of Retarded Embryo Development 1(RED1) gene in Arabidopsis,whose two independent T-DNA insertion mutant lines,SALK_085642(red1-1) and SALK_022583(red1-2),show a retarded embryo development phenotype.The embryogenesis process ceases at the late heart stage in red 1-1 and at the bent-cotyledon stage in red 1-2,respectively,resulting in seed abortion in both lines.The retarded embryo development and seed abortion phenotypes reverted to normal when RED1 complementation constructs were introduced into mutant plants.Small red 1-2 homozygous plants can be successfully rescued by culturing immature seeds,indicating that seed abortion likely results from compromised tolerance to the desiccation process associated with seed maturation.Consistent with this observation,red 1-2 seeds accumulate less protein,and the expression of two late embryo development reporter transgenes,LEA::GUS and β-conglycinin::GUS,was significantly weak and started relatively late in the red 1-2 mutant lines compared to the wild type.The RED1 gene encodes a plant specific novel protein that is localized in the nucleus.These results indicate that RED1 plays important roles in embryo development,seed maturation and plant growth.     

The TIP GROWTH DEFECTIVE1 S-acyl transferase regulates plant cell growth in Arabidopsis

TIP GROWTH DEFECTIVE1 (TIP1) of Arabidopsis thaliana affects cell growth throughout the plant and has a particularly strong effect on root hair growth. We have identified TIP1 by map-based cloning and complementation of the mutant phenotype. TIP1 encodes an ankyrin repeat protein with a DHHC Cys-rich domain that is expressed in roots, leaves, inflorescence stems, and floral tissue. Two homologues of TIP1 in yeast (Saccharomyces cerevisiae) and human (Homo sapiens) have been shown to have S-acyl transferase (also known as palmitoyl transferase) activity. S-acylation is a reversible hydrophobic protein modification that offers swift, flexible control of protein hydrophobicity and affects protein association with membranes, signal transduction, and vesicle trafficking within cells. We show that TIP1 binds the acyl group palmitate, that it can rescue the morphological, temperature sensitivity, and yeast casein kinase2 localization defects of the yeast S-acyl transferase mutant akr1Delta, and that inhibition of acylation in wild-type Arabidopsis roots reproduces the Tip1- mutant phenotype. Our results demonstrate that S-acylation is essential for normal plant cell growth and identify a plant S-acyl transferase, an essential research tool if we are to understand how this important, reversible lipid modification operates in plant cells.     

Influences of PAR, temperature and water vapor concentration on gas exchange by ferns

The effects of photosynthetically active radiation (PAR), leaf temperature and the leaf-to-air water vapor concentration drop on net CO₂ uptake and water vapor conductance were surveyed for 14 species of ferns. Most previous studies indicated that ferns have extremely low maximal rates of net CO₂ uptake, below 2 umol m^?2 s^?1, whereas the average maximal rate observed here at 25⁰ C was 7 umol m^?2 s^?1. Net CO₂ uptake reached 90% of saturation at an average PAR (400 to 700 nm) of only 240 umol m^?2 s^?1, consistent with the typically shaded habitats of most ferns. Maximal CO₂ uptake rates were positively correlated with the PAR for 90% saturation (r²=0.59), the chlorophyII per unit leaf area (r²=0.30), the water vapor conductance (r²=0.65), and the CO₂ residual conductance (r²=0.69). A higher water vapor conductance (gwv) was correlated with a greater fractional change in gwv as the leaf-to-air water vapor concentration drop (Δc_wv) was raised from 5to20 g m^?3 (r²=0.90). Specifically, for species with low g_wv of about I mm s^?1 the ratio of g_wv at Δc_wv= 5 g m^?3 to that at Δc_wv= 20 g m^?3 was near 1, but it was near 2 for species with g_wv of about 4 mm s^?1. Such a relationship, which can prevent excessive transpiration, has apparently not previously been pointed out in surveys of other plant groups.     

Populus alba cationic cell-wall-bound peroxidase (CWPO-C) regulates the plant growth and affects auxin concentration in Arabidopsis thaliana

The poplar cationic cell-wall-bound peroxidase (CWPO-C) mediates the oxidative polymerization of lignin precursors, especially sinapyl alcohols, and high molecular weight compounds that cannot be oxidized by other plant peroxidases, including horseradish peroxidase C. Therefore, CWPO-C is believed to be a lignification-specific peroxidase, but direct evidence of its function is lacking. Thus, the CWPO-C expression pattern in Arabidopsis thaliana (Arabidopsis) was determined using the β-glucuronidase gene as a reporter. Our data indicated that CWPO-C  was expressed in young organs, including the meristem, leaf, root, flower, and young xylem in the upper part of the stem. Compared with the wild-type control, transgenic Arabidopsis plants overexpressing CWPO-C had shorter stems. Approximately 60% of the plants in the transgenic line with the highest CWPO-C content had curled stems. These results indicate that CWPO-C plays a role in cell elongation. When plants were placed horizontally, induced CWPO-C expression was detected in the curved part of the stem during the gravitropic response. The stem curvature associated with gravitropism is controlled by auxin localization. The time needed for Arabidopsis plants overexpressing CWPO-C placed horizontally to bend by 90° was almost double the time required for the similarly treated wild-type controls. Moreover, the auxin content was significantly lower in the CWPO-C-overexpressing plants than in the wild-type plants. These results strongly suggest that CWPO-C has pleiotropic effects on plant growth and indole-3-acetic acid (IAA) accumulation. These effects may be mediated by altered IAA concentration due to oxidation.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-022-01241-0.     

Regulation of vacuolar Na+/H+ exchange in Arabidopsis thaliana by the salt-overly-sensitive (SOS) pathway

For plants growing in highly saline environments, accumulation of sodium in the cell cytoplasm leads to disruption of metabolic processes and reduced growth. Maintaining low levels of cytoplasmic sodium requires the coordinate regulation of transport proteins on numerous cellular membranes. Our previous studies have linked components of the Salt-Overly-Sensitive pathway (SOS1-3) to salt tolerance in Arabidopsis thaliana and demonstrated that the activity of the plasma membrane Na+/H+ exchanger (SOS1) is regulated by SOS2 (a protein kinase) and SOS3 (a calcium-binding protein). Current studies were undertaken to determine if the Na+/H+ exchanger in the vacuolar membrane (tonoplast) of Arabidopsis is also a target for the SOS regulatory pathway. Characterization of tonoplast Na+/H+ exchange demonstrated that it represents activity originating from the AtNHX proteins since it could be inhibited by 5-(N-methyl-N-isobutyl)amiloride and by anti-NHX1 antibodies. Transport activity was selective for sodium (apparent Km=31 mm) and electroneutral (one sodium ion for each proton). When compared with tonoplast Na+/H+-exchange activity in wild type, activity was significantly higher, greatly reduced, and unchanged in sos1, sos2, and sos3, respectively. Activated SOS2 protein added in vitro increased tonoplast Na+/H+-exchange activity in vesicles isolated from sos2 but did not have any effect on activity in vesicles isolated from wild type, sos1, or sos3. These results demonstrate that (i) the tonoplast Na+/H+ exchanger in Arabidopsis is a target of the SOS regulatory pathway, (ii) there are branches to the SOS pathway, and (iii) there may be coordinate regulation of the exchangers in the tonoplast and plasma membrane.     

TNO1 is involved in salt tolerance and vacuolar trafficking in Arabidopsis

The Arabidopsis (Arabidopsis thaliana) soluble N-ethylmaleimide-sensitive factor attachment protein receptor SYP41 is involved in vesicle fusion at the trans-Golgi network (TGN) and interacts with AtVPS45, SYP61, and VTI12. These proteins are involved in diverse cellular processes, including vacuole biogenesis and stress tolerance. A previously uncharacterized protein, named TNO1 (for TGN-localized SYP41-interacting protein), was identified by coimmunoprecipitation as a SYP41-interacting protein. TNO1 was found to localize to the TGN by immunofluorescence microscopy. A tno1 mutant showed increased sensitivity to high concentrations of NaCl, KCl, and LiCl and also to mannitol-induced osmotic stress. Localization of SYP61, which is involved in the salt stress response, was disrupted in the tno1 mutant. Vacuolar proteins were partially secreted to the apoplast in the tno1 mutant, suggesting that TNO1 is required for efficient protein trafficking to the vacuole. The tno1 mutant had delayed formation of the brefeldin A (BFA) compartment in cotyledons upon application of BFA, suggesting less efficient membrane fusion processes in the mutant. Unlike most TGN proteins, TNO1 does not relocate to the BFA compartment upon BFA treatment. These data demonstrate that TNO1 is involved in vacuolar trafficking and salt tolerance, potentially via roles in vesicle fusion and in maintaining TGN structure or identity.