Omar Pantoja

Salt Stress in Thellungiella halophila Activates Na+ Transport Mechanisms Required for Salinity Tolerance

Salinity is considered one of the major limiting factors for plant growth and agricultural productivity. We are using salt cress (Thellungiella halophila) to identify biochemical mechanisms that enable plants to grow in saline conditions. Under salt stress, the major site of Na+ accumulation occurred in old leaves, followed by young leaves and taproots, with the least accumulation occurring in lateral roots. Salt treatment increased both the H+ transport and hydrolytic activity of salt cress tonoplast (TP) and plasma membrane (PM) H+-ATPases from leaves and roots. TP Na+/H+ exchange was greatly stimulated by growth of the plants in NaCl, both in leaves and roots. Expression of the PM H+-ATPase isoform AHA3, the Na+ transporter HKT1, and the Na+/H+ exchanger SOS1 were examined in PMs isolated from control and salt-treated salt cress roots and leaves. An increased expression of SOS1, but no changes in levels of AHA3 and HKT1, was observed. NHX1 was only detected in PM fractions of roots, and a salt-induced increase in protein expression was observed. Analysis of the levels of expression of vacuolar H+-translocating ATPase subunits showed no major changes in protein expression of subunits VHA-A or VHA-B with salt treatment; however, VHA-E showed an increased expression in leaf tissue, but not in roots, when the plants were treated with NaCl. Salt cress plants were able to distribute and store Na+ by a very strict control of ion movement across both the TP and PM.

Salt stress in Mesembryanthemum crystallinum L. cell suspensions activates adaptive mechanisms similar to those observed in the whole plant

Abstract. A salt-tolerant stable cell-suspension culture from the halophyte Mesembryanthemum crystallinum L. has been established from calli generated from leaves of 6-week-old well-watered plants. Optimal cell growth was observed in the presence of 200 mM NaCl, and within 7 d cells were able to concentrate Na+ to levels exceeding those in the growth medium. Accumulation of Na+ was paralled by increases in the compatible solute pinitol and myo-inositol methyl transferase (IMT), a key enzyme in pinitol biosynthesis. Increasing concentrations of NaCl stimulated the activities of tonoplast and plasma-membrane H+-ATPases. Immunodetection of the ATPases showed that the increased activity was not due to changes in protein amount that could be attributed to treatment conditions. A specific role for these mechanisms in salt-adaptation is supported by the inability of mannitol-induced water stress to elicit the same responses, and the absence of enzyme activity and protein expression associated with Crassulacean acid metabolism in the cells. Results demonstrate that these  M. crystallinum cell suspensions show a halophytic growth response, comparable to that of the whole plant, and thus provide a valuable tool for studying signaling and biochemical pathways involved in salt recognition and response.

Electrophysiological and ultrastructural study of the atrioventricular canal during the development of the chick embryo

The development of the atrioventricular canal (A-V canal) of embryonic chick hearts (35 to 120 h) was studied by morphological and electrophysiological techniques. The earliest identification of the A-V canal action potentials was at 45 to 49 h of incubation coinciding with the atrium formation and the appearance of its corresponding distinctive action potential. At that time the first atrioventricular delay was recorded and its action potentials showed a low rate of rise, particularly at the initiation of the upstroke and a long duration. The conduction velocity was the lowest in the A-V canal. As the development proceeded from 45 to 120 h, the cardiac cells of the A-V canal showed scanty membrane to membrane contacts and large intercellular spaces filled with abundant extracellular matrix components, in striking contrast to the paucity of these components in the atrium and ventricle. The morphological and electrophysiological characteristics described for the A-V canal cells, could help to explain their slow conduction properties and the atrioventricular delay.

Voltage-Dependent Calcium Channels in Plant Vacuoles

Free calcium (Ca2+) in the cytoplasm of plant cells is important for the regulation of many cellular processes and the transduction of stimuli. Control of cytoplasmic Ca2+ involves the activity of pumps, carriers, and possibly ion channels. The patch-clamp technique was used to study Ca2+ channels in the vacuole of sugar beet cells. Vacuolar currents showed inward rectification at negative potentials, with a single-channel conductance of 40 picosiemens and an open probability dependent on potential. Channels were inhibited by verapamil and lanthanum. These channels could participate in the regulation of cytoplasmic Ca2+ by sequestering Ca2+ inside the vacuole.

Identification of a crucial histidine involved in metal transport activity in the Arabidopsis cation/H+ exchanger CAX1

In plants, yeast, and bacteria, cation/H+ exchangers (CAXs) have been shown to translocate Ca2+ and other metal ions utilizing the H+ gradient. The best characterized of these related transporters is the plant vacuolar localized CAX1. We have used site-directed mutagenesis to assess the impact of altering the seven histidine residues to alanine within Arabidopsis CAX1. The mutants were expressed in a Saccharomyces cerevisiae strain that is sensitive to Ca2+ and other metals. By utilizing a yeast growth assay, the H338A mutant was the only mutation that appeared to alter Ca2+ transport activity. The CAX1 His338 residue is conserved among various CAX transporters and may be located within a filter for cation selection. We proceeded to mutate His338 to every other amino acid residue and utilized yeast growth assays to estimate the transport properties of the 19 CAX mutants. Expression of 16 of these His338 mutants could not rescue any of the metal sensitivities. However, expression of H338N, H338Q, and H338K allowed for some growth on media containing Ca2+. Most interestingly, H338N exhibited increased tolerance to Cd2+ and Zn2+. Endomembrane fractions from yeast cells were used to measure directly the transport of H338N. Although the H338N mutant demonstrated 25% of the wild type Ca2+/H+ transport, it showed an increase in transport for both Cd2+ and Zn2+ reflected in a decrease in the Km for these substrates. This study provides insights into the CAX cation filter and novel mechanisms by which metals may be partitioned across membranes.

Progress and challenges for abiotic stress proteomics of crop plants

Plants are continually challenged to recognize and respond to adverse changes in their environment to avoid detrimental effects on growth and development. Understanding the mechanisms that crop plants employ to resist and tolerate abiotic stress is of considerable interest for designing agriculture breeding strategies to ensure sustainable productivity. The application of proteomics technologies to advance our knowledge in crop plant abiotic stress tolerance has increased dramatically in the past few years as evidenced by the large amount of publications in this area. This is attributed to advances in various technology platforms associated with MS‐based techniques as well as the accessibility of proteomics units to a wider plant research community. This review summarizes the work which has been reported for major crop plants and evaluates the findings in context of the approaches that are widely employed with the aim to encourage broadening the strategies used to increase coverage of the proteome

Malate transport and vacuolar ion channels in CAM plants

Malate is a ubiquitous vacuolar anion in terrestrial plants that plays an important role in carbon metabolism and ionic homeostasis. In plants showing crassulacean acid metabolism (CAM), malate is accumulated as a central intermediary in the process of photosynthetic carbon assimilation, and it is also one of the major charge-balancing anions present in the vacuole. During the CAM cycle, malic acid produced as a result of dark CO(2) fixation accumulates in the vacuole at night (2 H(+) per malate), and is remobilized from the vacuole in the following light period. CAM plants thus provide a good model for studying both the mechanism and control of malate transport across the tonoplast. Thermodynamic considerations suggest that malate(2-) (the anionic species transported out of the cytosol) is passively distributed across the tonoplast. Malic acid accumulation could thus be explained by malate(2-) transport into the vacuole occurring electrophoretically in response to the transmembrane electrical potential difference established by the tonoplast H(+)-ATPase and/or H(+)-PPase. Recent studies using the patch-clamp technique have provided evidence for the existence of a vacuolar malate-selective anion channel (VMAL) in both CAM species and C(3) species. The VMAL current has a number of distinctive properties that include strong rectification (opening only at cytosolicside negative membrane potentials that would favour malate uptake into the vacuole), lack of Ca(2+) dependence, and slow activation kinetics. The kinetics of VMAL activation can be resolved into three components, consisting of an instantaneous current and two slower components with voltage-independent time constants of 0.76 s and 5.3 s in Kalanchoë daigremontiana. These characteristics suggest that the VMAL channel represents the major pathway for malate transport into the vacuole, although the strong rectification of the channel means there may be a separate, still-to-be-identified, transport mechanism for malate efflux.

Ion channels in vacuoles from halophytes and glycophytes

The electrical properties of the vacuolar membrane (tonoplast) of a halophyte, sugar beet, and a glycophyte, tomato, have been investigated using the patch‐clamp technique [(1981) Pflügers Arch. 391, 85–100]. Voltage‐dependent ion channels were analyzed using isolated membrane patches. Both species displayed channel activities which were nonselective between sodium and potassium. Beet tonoplast channels displayed inward rectification (65 pS and 10 pS for negative and positive potentials, respectively), while tomato tonoplast channels showed a constant conductance (25 pS) in the range −80 to +80 mV potentials. The observed low channel conductance at positive potentials in halophytes would prevent a significant loss of the Na+ accumulated in the vacuole through the operation of the Na+/H+ antiport [(1987) Physiol. Plant. 69, 731–734], while channel rectification in glycophytes would have no physiological significance.

Na + /H + exchange in the halophyte Mesembryanthemum crystallinum is associated with cellular sites of Na + storage

The tonoplast Na+/H+ exchanger is involved in sequestering Na+ in plant vacuoles, providing solutes for osmotic adjustment while avoiding cytoplasmic Na+ toxicity. As such it is assumed to be one of the key mechanisms involved in salt-tolerance in plants. In this study, we measured tonoplast Na+/H+ exchange in roots and different leaf tissues of adult Mesembryanthemum crystallinum L. plants to determine if activity of the exchanger follows the gradient from roots to leaves previously observed for Na+ and pinitol accumulation. Na+/H+ exchange was absent from roots of control and NaCl-treated plants. In contrast, leaves showed constitutive Na+/H+ exchange that was enhanced by growth of the plants in NaCl. Highest activity was measured in the epidermal bladder cells in agreement with the highest concentrations of Na+ found in this tissue. Tonoplast H+-translocating ATPase activity was also greatest in this tissue, as were protein levels for myo-inositol-O-methyltransferase, a key enzyme in the pinitol biosynthesis pathway. The strong correlation between Na+/H+ exchange and Na+ accumulation confirms the role of this transporter in vacuolar sequestration of Na+ and plant salt tolerance.

PvAMT1;1, a Highly Selective Ammonium Transporter That Functions as H+/NH4+ Symporter

One of the main forms of nitrogen assimilated by microorganisms and plants is ammonium, despite its toxicity at low millimolar concentrations. Ammonium absorption has been demonstrated to be carried out by highly selective plasma membrane-located transporters of the AMT/MEP/Rh family and characterized by the presence of a well conserved hydrophobic pore through which ammonia is proposed to move. However, uncertainties exist regarding the exact chemical species transported by these membrane proteins, which can be in the form of either hydrophobic ammonia or charged ammonium. Here, we present the characterization of PvAMT1;1 from the common bean and demonstrate that it mediates the high affinity (micromolar), rapidly saturating (1 mm) electrogenic transport of ammonium. Activity of the transporter is enhanced by low extracellular pH, and associated with this acidic pH stimulation are changes in the reversal potential and cytoplasm acidification, indicating that PvAMT1;1 functions as an H+/NH4+ symporter. Mutation analysis of a unique histidine present in PvAMT1;1 (H125R) leads to the stimulation of ammonium transport by decreasing the Km value by half and by increasing the Vmax 3-fold, without affecting the pH dependence of the symporter. In contrast, mutation of the first conserved histidine within the channel modifies the properties of PvAMT1;1, increasing its Km and Vmax values and transforming it into a pH-independent mechanism.