The high affinity K+ transporter AtHAK5 plays a physiological role in planta at very low K+ concentrations and provides a caesium uptake pathway in Arabidopsis

Caesium (Cs⁺) is a potentially toxic mineral element that isreleased into the environment and taken up by plants. AlthoughCs⁺ is chemically similar to potassium (K⁺), and much is knownabout K⁺ transport mechanisms, it is not clear through whichK⁺ transport mechanisms Cs⁺ is taken up by plant roots. In thisstudy, the role of AtHAK5 in high affinity K⁺ and Cs⁺ uptakewas characterized. It is demonstrated that AtHAK5 is localizedto the plasma membrane under conditions of K⁺ deprivation, whenit is expressed. Growth analysis showed that AtHAK5 plays arole during severe K⁺ deprivation. Under K⁺-deficient conditionsin the presence of Cs⁺, Arabidopsis seedlings lacking AtHAK5had increased inhibition of root growth and lower Cs⁺ accumulation,and significantly higher leaf chlorophyll concentrations thanwild type. These data indicate that, in addition to transportingK⁺ in planta, AtHAK5 also transports Cs⁺. Further experimentsshowed that AtHAK5 mediated Cs⁺ uptake into yeast cells andthat, although the K⁺ deficiency-induced expression of AtHAK5was inhibited by low concentrations of NH     

Exchangers man the pumps: Functional interplay between proton pumps and proton-coupled Ca2+ exchangers

Tonoplast-localised proton-coupled Ca²⁺ transporters encoded by cation/H⁺ exchanger (CAX) genes play a critical role in sequestering Ca²⁺ into the vacuole. These transporters may function in coordination with Ca²⁺ release channels, to shape stimulus-induced cytosolic Ca²⁺ elevations. Recent analysis of Arabidopsis CAX knockout mutants, particularly cax1 and cax3, identified a variety of phenotypes including sensitivity to abiotic stresses, which indicated that these transporters might play a role in mediating the plant''s stress response. A common feature of these mutants was the perturbation of H⁺-ATPase activity at both the tonoplast and the plasma membrane, suggesting a tight interplay between the Ca²⁺/H⁺ exchangers and H⁺ pumps. We speculate that indirect regulation of proton flux by the exchangers may be as important as the direct regulation of Ca²⁺ flux. These results suggest cautious interpretation of mutant Ca²⁺/H⁺ exchanger phenotypes that may be due to either perturbed Ca²⁺ or H⁺ transport.Key words: abiotic stress, Ca²⁺ transport, Ca²⁺/H⁺ exchanger, H⁺-ATPase, Na⁺ transport, pH, salt stress, vacuoleCa²⁺ plays a fundamental role in the plant cell, functioning as a highly versatile second messenger controlling a multitude of cellular reactions and adaptive responses.^1,2 Ca²⁺ dynamics are maintained by precise interplay among transporters involved in its release from or uptake into Ca²⁺ stores. The vacuole, as the largest internal Ca²⁺ pool, is assumed to play a major role in Ca²⁺ signalling, and has been shown to be the source of Ca²⁺ release following various abiotic stresses such as cold and osmotic stress.^3,4 Rapid, stimulus-induced release of Ca²⁺ from the vacuole is attributable to selectively permeable Ca²⁺ channels, however, the identity of candidate genes encoding this mechanism remains contested.^5,6 Better understood, are the two major vacuolar uptake mechanisms; P-type Ca²⁺ pumps, including ACA4 and ACA11, which mediate high-affinity Ca²⁺ uptake, and a family of cation/H⁺ exchangers (CAX), responsible for lower-affinity but high-capacity Ca²⁺ uptake.^7,8 While Ca²⁺ pumps rely directly on the hydrolysis of ATP to drive Ca²⁺ uptake, Ca²⁺/H⁺ exchangers are energized indirectly by the pH gradient generated by electrogenic H⁺ pumps located on the tonoplast, including the vacuolar-type H⁺-ATPase (V-ATPase).⁹With the aim of further understanding the role of specific CAX isoforms in Arabidopsis, we and others have recently characterized CAX mutants and overexpression lines and observed a variety of phenotypes, including altered response to abiotic stresses.^10–14 While some phenotypes are identical among different CAX mutants, others are specific to individual lines.¹⁴ Moreover, these analyses have highlighted the interplay of these transporters with H⁺ pumps at both the tonoplast and the plasma membrane. Overexpression of CAX1 in Arabidopsis results in increased activity of the V-ATPase, whereas mutations in CAX1 cause a concomitant decrease in measured V-ATPase activity (Fig. 1).¹¹ Similar reductions in V-ATPase activity are also observed in cax2 and cax3 mutant plants but to a lesser extent,^12,13 and a significant reduction is observed in a cax1 cax3 double knockout line.¹³ At the plasma membrane, P-type H⁺-ATPase (P-ATPase) activity is increased in cax1 but decreased in cax3 (Fig. 1).¹⁴ Indeed cax3 lines appeared more sensitive to changes in the pH of the growth media.¹⁴ This implies that unlike cax1, cax3 is less efficient at cytoplasmic pH adjustment. Another intriguing observation is that activity of the H⁺-pyrophosphatase (H⁺-PPase) at the tonoplast is largely unaltered following CAX gene deletion. While overexpression of the Arabidopsis H⁺-PPase AVP1 leads to increased Ca²⁺/H⁺ exchange activity,^15,16 there is little alteration in H⁺-PPase activity following perturbed expression of CAX1 or CAX2.^11,12 Thus, this feedback interplay appears to exist only between exchangers and H⁺-ATPases.Open in a separate windowFigure 1Tonoplast H⁺-ATPase (V-ATPase) activity and plasma membrane H⁺-ATPase (P-ATPase) activity in wild type Arabidopsis (ecotype Col-0) and Arabidopsis lines with manipulated tonoplast Ca²⁺/H⁺ exchange activity. 35S::CAX1 and 35S::CAX2 denote lines that overexpress a constitutively active N-terminally truncated CAX1 or CAX2 construct driven by the CaMV 35S promoter in the cax1-1 or cax2-1 mutant background, respectively. V-ATPase H⁺-transport activity was measured by the ATP-dependent quenching of quinacrine fluorescence, and rates of bafilomycin-sensitive, vanadate-resistant hydrolytic activity of the V-ATPase were determined in isolated tonoplast membranes, as described in refs. ¹¹ and ¹³. Rates of vanadate-sensitive, bafilomycin- and azide-resistant hydrolytic activity of the P-ATPase were determined in isolated plasma membranes, as described in ref. ¹⁴. Results are shown as % of wild type (Col-0) ATPase activity and are means ± SE of 3–4 independent experiments. Data taken and modified from refs. ^11–14.The V-ATPase is important not only for maintenance of a pH gradient across the tonoplast, but also in maintenance of Golgi structure, endocytosis and secretory trafficking.^17,18 The V-ATPase is localised at the Golgi, endoplasmic reticulum and endosomes, in addition to the tonoplast.⁹ The det3 mutant, with a mutation in subunit C (VHA-C), has a 40–60% reduction in V-ATPase activity, but numerous severe developmental phenotypes.¹⁹ In contrast, the cax1 and cax1 cax3 mutants have a reduction in V-ATPase activity equivalent to det3 (Fig. 1), but the morphological phenotypes are not as pronounced.¹³ It is therefore likely that reduction of tonoplast Ca²⁺/H⁺ exchange primarily affects tonoplast V-ATPase activity, while V-ATPase activity in the secretory pathway is unperturbed. The V-ATPase is a multi-subunit protein and some of these subunit gene products appear to be either tonoplast-specific or tonoplast-enriched. Mutations in tonoplast subunits may cause defective V-ATPase activity only at the tonoplast.⁹ It will be of interest to see whether such tonoplast-specific V-ATPase mutants phenocopy the cax mutants, and possess perturbed Ca²⁺/H⁺ exchange activity and altered abiotic stress responses.CAX-mediated transport may alter both cytoplasmic and lumenal pH, as well as intracellular Ca²⁺ gradients. In the case of the V-ATPase, evidence is emerging for a role not only in the generation of a pH gradient across membranes, but also in the direct sensing of pH within the compartment,^20,21 creating a feedback mechanism which regulates pump activity. Thus, in cax1 lines, abnormal acidification of the lumen is detected by the V-ATPase resulting in a dampening of its activity. This would conserve ATP, which we postulate could be utilized to drive the tonoplast Ca²⁺ pump which itself is upregulated in cax1 as a compensatory mechanism to correct perturbations in the Ca²⁺ gradient.¹¹ In the case of cax1, this in turn may signal the P-ATPase to remove surplus H⁺ from the cytoplasm, triggering its upregulation (Fig. 1). However, not all CAX mutants show this complex H⁺ feedback mechanism.Co-ordinate downregulation of the V-ATPase in the cax1 mutant lines may also be a result of activity of the SOS2 kinase. This Ser/Thr kinase, which specifically interacts with the N-terminus of CAX1 resulting in Ca²⁺/H⁺ exchange activation,²² upregulates V-ATPase activity through interactions with the VHA-B regulatory subunit.²³ Loss of CAX1 may be signalling to the V-ATPase through changes in SOS2 activity resulting in a compensatory downregulation of the pump. It is tempting to speculate that SOS2 may signal the alteration in P-ATPase activity, as it is known to regulate other plasma membrane proteins, notably the Na⁺/H⁺ exchanger SOS1.²⁴ It will be interesting to determine if SOS2 and the P-ATPase interact directly. It is notable, however, that SOS2 does not appear to interact with CAX3,²² while P-ATPase activity is reduced in cax3 plants.¹⁴Our recent results indicate there are at least two modes by which Ca²⁺/H⁺ exchangers can mediate adaptive responses to stress: direct manipulation of cytosolic Ca²⁺ and indirect feedback of H⁺ flux (Fig. 2). For example, salt stress responses are likely controlled via the generation of a specific cytosolic Ca²⁺ signature, which mediates a downstream signalling pathway. CAX3 appears to be the principle isoform providing tonoplast Ca²⁺/H⁺ exchange in response to salt stress.¹⁴ Disruption of CAX3-mediated tonoplast Ca²⁺ transport and the alteration of cytosolic Ca²⁺ dynamics may therefore alter the plant''s normal response to salt stress (Fig. 2). Maintenance of H⁺ gradients at both the vacuole and plasma membrane are also critical for salt tolerance, such that salt treatment upregulates V-ATPase and P-ATPase activity.²⁵ This energizes Na⁺ efflux from the cytosol mediated by Na⁺/H⁺ exchangers at the plasma membrane and the tonoplast.^24,26 Therefore downregulation of H⁺ pumps at both membranes in the cax3 mutant is likely to perturb the ability of the cell to remove Na⁺ (Fig. 2). Further analysis of cax mutants, P-ATPase mutants, and tonoplast-specific V-ATPase mutants will be required to determine whether many of the phenotypes resulting from lack of Ca²⁺/H⁺ exchange activity are due to altered Ca²⁺ transport or H⁺ transport.Open in a separate windowFigure 2Model of tonoplast Ca²⁺/H⁺ exchanger interaction with H⁺ pumps in response to salt stress. (A) In response to NaCl treatment, an elevation in cytosolic Ca²⁺ will occur, possibly due to vacuolar Ca²⁺ release.³ Increased CAX3-mediated Ca²⁺/H⁺ exchange activity¹⁴ will sequester excess Ca²⁺ into the vacuole. CAX3 may be involved in the generation of a specific Ca²⁺ signature that is recognised by the cell to mediate downstream stress responses. In addition, salt stress will lead to upregulation of H⁺ pumps at both the plasma membrane and the tonoplast (P-ATPase and V-ATPase)²⁵ which will in turn energize Na⁺/H⁺ exchange activity encoded by SOS1 and NHX1, promoting Na⁺ efflux from the cell. Increased V-ATPase activity may also upregulate Ca²⁺/H⁺ exchange. Activity of SOS1 requires activation by the kinase SOS2²⁴ which may also regulate tonoplast Na⁺/H⁺ exchange and V-ATPase activity.^23,24 (B) In a cax3 knockout mutant experiencing salt stress, the cytosolic Ca²⁺ elevation may be sustained due to reduced vacuolar Ca²⁺ sequestration and normal salinity-induced Ca²⁺ signalling pathways may be perturbed. Lack of CAX3 downregulates both P-ATPase and V-ATPase activity¹⁴ thereby reducing energization of the plasma membrane and tonoplast Na⁺/H⁺ exchangers and reducing Na⁺ efflux from the cell. Some energization of H⁺-coupled processes at the vacuole may be maintained by residual H⁺-pyrophosphatase (V-PPase) activity.The phenomenon observed between tonoplast Ca²⁺/H⁺ exchangers and H⁺ pumps at both the tonoplast and plasma membranes, suggesting a co-ordinate regulation between several transporters, is not solely restricted to this family of transporters. It is a common observation emerging from recent research on the manipulation of tonoplast transporters. Several labs have reported unpredictable phenotypes associated with ectopic expression of tonoplast proteins.^26–28 Until we understand the significance of these types of unexpected interactions, it is naïve to believe that engineering plants will provide predictable results.     

Tonoplast Na+/H+ Antiport Activity and Its Energization by the Vacuolar H+-ATPase in the Halophytic Plant Mesembryanthemum crystallinum L

Tonoplast vesicles were isolated from leaf mesophyll tissue of the inducible Crassulacean acid metabolism plant Mesembryanthemum crystallinum to investigate the mechanism of vacuolar Na+ accumulation in this halophilic species. In 8-week-old plants exposed to 200 mM NaCl for 2 weeks, tonoplast H+-ATPase activity was approximately doubled compared with control plants of the same age, as determined by rates of both ATP hydrolysis and ATP-dependent H+ transport. Evidence was also obtained for the presence of an electroneutral Na+/H+ antiporter at the tonoplast that is constitutively expressed, since extravesicular Na+ was able to dissipate a pre-existing transmembrane pH gradient. Initial rates of H+ efflux showed saturation kinetics with respect to extravesicular Na+ concentration and were 2.1-fold higher from vesicles of salt-treated plants compared with the controls. Na+-dependent H+ efflux also showed a high selectivity for Na+ over K+, was insensitive to the transmembrane electrical potential difference, and was more than 50% inhibited by 200 [mu]M N-amidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride. The close correlation between increased Na+/H+ antiport and H+-ATPase activities in response to salt treatment suggests that accumulation of the very high concentrations of vacuolar Na+ found in M. crystallinum is energized by the H+ electrochemical gradient across the tonoplast.     

A covariotide model explains apparent phylogenetic structure of oxygenic photosynthetic lineages

The aims of the work were (1) to develop statistical tests to identify whether substitution takes place under a covariotide model in sequences used for phylogenetic inference and (2) to determine the influence of covariotide substitution on phylogenetic trees inferred for photosynthetic and other organisms. (Covariotide and covarion models are ones in which sites that are variable in some parts of the underlying tree are invariable in others and vice versa.) Two tests were developed. The first was a contingency test, and the second was an inequality test comparing the expected number of variable sites in two groups with the observed number. Application of these tests to 16S rDNA and tufA sequences from a range of nonphotosynthetic prokaryotes and oxygenic photosynthetic prokaryotes and eukaryotes suggests the occurrence of a covariotide mechanism. The degree of support for partitioning of taxa in reconstructed trees involving these organisms was determined in the presence or absence of sites showing particular substitution patterns. This analysis showed that the support for splits between (1) photosynthetic eukaryotes and prokaryotes and (2) photosynthetic and nonphotosynthetic organisms could be accounted for by patterns arising from covariotide substitution. We show that the additional problem of compositional bias in sequence data needs to be considered in the context of patterns of covariotide/covarion substitution. We argue that while covariotide or covarion substitution may give rise to phylogenetically informative patterns in sequence data, this may not always be so.      

禾木科植物染色体消除型远缘杂交的研究进展

植物远缘杂交是作物育种中广泛应用的技术。除了核型稳定的种间杂交可以获得杂种以外,还可以利用核型不稳定的种间杂交后父本染色体消除的现象,通过胚培养和染色体加倍处理获得加倍单倍体(     

Serum-free organ culture of suckling rat jejunum: Effect of regulatory hormones

Summary To facilitate the study of regulators of differentiation and proliferation of small intestinal epithelium in the suckling rat we have developed a serum-free organ culture system and used it to examine epithelial responsiveness to various regulatory hormones. These hormones included the insulin-like growth factors (IGFs) whose action can be blocked by binding proteins in serum. Jejunal explants from 5-day-old suckling rats maintained better brush border enzyme activity and better histology when cultured under hyperbaric conditions for 24 h in serum-free Dulbecco’s modified Eagle’s medium/F12 medium than in RPMI 1640 plus 10% fetal bovine serum. Tissue responsiveness to various regulatory hormones was then tested in the serum-free medium. Insulin had no significant effect on morphology, proliferation rate, or enzyme activity in 5-day explants after 24 h in culture. However, insulin did increase lactase activity and induce the early appearance of sucrase in 10- and 12-day explants after 48 h culture. Dexamethasone increased specific activities of alkaline phosphatase (30%,P<0.001) and lactase (15%,P<0.001), and reduced shedding of alkaline phosphatase into the medium (P<0.001), in explants of 5-day-old rats cultured over 24 h. Dexamethasone combined with insulin had no obvious effect on the rate of protein or DNA synthesis but did increase villus height (P=0.04) and crypt depth (P=0.001) and acted synergistically to further increase lactase activity above levels obtained by either alone. IGF-I and IGF-II, des-(1–3)IGF-I, fibroblast growth factor (FGF), and growth hormone (GH) had no effect on morphology or biochemical activity of explants after 24 or 48 h culture. In conclusion, histology, enzyme activity, protein, and DNA synthesis of suckling rat jejunal explants were equivalent or better in serum-free than in serum-containing organ culture systems. Furthermore, biological responsiveness was demonstrated by dexamethasone and insulin altering the explants morphologically or biochemically. None of the IGFs or GH had any biological effects, raising doubts about their direct biological action on the developing intestinal epithelium.     

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     

Influence of nutrients and currents on the genomic composition of microbes across an upwelling mosaic

Metagenomic data sets were generated from samples collected along a coastal to open ocean transect between Southern California Bight and California Current waters during a seasonal upwelling event, providing an opportunity to examine the impact of episodic pulses of cold nutrient-rich water into surface ocean microbial communities. The data set consists of ∼5.8 million predicted proteins across seven sites, from three different size classes: 0.1–0.8, 0.8–3.0 and 3.0–200.0 μm. Taxonomic and metabolic analyses suggest that sequences from the 0.1–0.8 μm size class correlated with their position along the upwelling mosaic. However, taxonomic profiles of bacteria from the larger size classes (0.8–200 μm) were less constrained by habitat and characterized by an increase in Cyanobacteria, Bacteroidetes, Flavobacteria and double-stranded DNA viral sequences. Functional annotation of transmembrane proteins indicate that sites comprised of organisms with small genomes have an enrichment of transporters with substrate specificities for amino acids, iron and cadmium, whereas organisms with larger genomes have a higher percentage of transporters for ammonium and potassium. Eukaryotic-type glutamine synthetase (GS) II proteins were identified and taxonomically classified as viral, most closely related to the GSII in Mimivirus, suggesting that marine Mimivirus-like particles may have played a role in the transfer of GSII gene functions. Additionally, a Planctomycete bloom was sampled from one upwelling site providing a rare opportunity to assess the genomic composition of a marine Planctomycete population. The significant correlations observed between genomic properties, community structure and nutrient availability provide insights into habitat-driven dynamics among oligotrophic versus upwelled marine waters adjoining each other spatially.