Differential structure and function of phosphorus-mineralizing microbial communities in organic and upper mineral soil horizons across a temperate rainforest chronosequence.

Microbial community structure and function were assessed in the organic and upper mineral soil across a ~4000-year dune-based chronosequence at Big Bay, New Zealand, where total P declined and the proportional contribution of organic soil in the profile increased with time. We hypothesized that the organic and mineral soils would show divergent community evolution over time with a greater dependency on the functionality of phosphatase genes in the organic soil layer as it developed. The structure of bacterial, fungal, and phosphatase-harbouring communities was examined in both horizons across 3 dunes using amplicon sequencing, network analysis, and qPCR. The soils showed a decline in pH and total phosphorus (P) over time with an increase in phosphatase activity. The organic horizon had a wider diversity of Class A (phoN/phoC) and phoD-harbouring communities and a more complex microbiome, with hub taxa that correlated with P. Bacterial diversity declined in both horizons over time, with enrichment of Planctomycetes and Acidobacteria. More complex fungal communities were evident in the youngest dune, transitioning to a dominance of Ascomycota in both soil horizons. Higher phosphatase activity in older dunes was driven by less diverse P-mineralizing communities, especially in the organic horizon.

Manipulating exudate composition from root apices shapes the microbiome throughout the root system.

Certain soil microorganisms can improve plant growth, and practices that encourage their proliferation around the roots can boost production and reduce reliance on agrochemicals. The beneficial effects of the microbial inoculants currently used in agriculture are inconsistent or short-lived because their persistence in soil and on roots is often poor. A complementary approach could use root exudates to recruit beneficial microbes directly from the soil and encourage inoculant proliferation. However, it is unclear whether the release of common organic metabolites can alter the root microbiome in a consistent manner and if so, how those changes vary throughout the whole root system. In this study, we altered the expression of transporters from the ALUMINUM-ACTIVATED MALATE TRANSPORTER and the MULTIDRUG AND TOXIC COMPOUND EXTRUSION families in rice (Oryza sativa L.) and wheat (Triticum aestivum L.) and tested how the subsequent release of their substrates (simple organic anions, including malate, citrate, and γ-amino butyric acid) from root apices affected the root microbiomes. We demonstrate that these exudate compounds, separately and in combination, significantly altered microbiome composition throughout the root system. However, the root type (seminal or nodal), position along the roots (apex or base), and soil type had a greater influence on microbiome structure than the exudates. These results reveal that the root microbiomes of important cereal species can be manipulated by altering the composition of root exudates, and support ongoing attempts to improve plant production by manipulating the root microbiome.

Phosphate-solubilising microorganisms for improved crop productivity: a critical assessment.

Phosphate-solubilising microorganisms (PSM) are often reported to have positive effects on crop productivity through enhanced phosphorus (P) nutrition. Our aim was to evaluate the validity of this concept. Most studies that report 'positive effects' of PSM on plant growth have been conducted under controlled conditions, whereas field experiments more frequently fail to demonstrate a positive response. Many studies have indicated that the mechanisms seen in vitro do not translate into improved crop P nutrition in complex soil-plant systems. Furthermore, associated mechanisms are often not rigorously assessed. We suggest that PSM do not mobilise sufficient P to change the crops' nutritional environment under field conditions. The current concept, in which PSM solubilise P 'for the plant' should thus be revised. Although PSM have the capacity to solubilise P to meet their own needs, it is the turnover of the microbial biomass that subsequently provides P to plants over a longer time. Therefore, the existing concept of PSM function is unlikely to deliver a reliable strategy for increasing crop P nutrition. A further mechanistic understanding is needed to determine how P mobilisation by PSM as a component of the whole soil community can be manipulated to become more effective for plant P nutrition.

Critical phosphorus requirements of Trifolium species: The importance of root morphology and root acclimation in response to phosphorus stress.

Differences in root morphology and acclimation to low-phosphorus (P) soil were examined among eight legume species from the Trifolium Section Tricocephalum to understand how these root attributes determine P acquisition. Ornithopus sativus was included as a highly P-efficient benchmark species. Plants were grown as microswards in pots with five rates of P supplied in a topsoil layer to mimic uneven P distribution within a field soil profile. Topsoil and subsoil roots were harvested separately to enable measurement of the nutrient-foraging responses. Critical P requirement (lowest P supply for maximum yield) varied over a threefold range, reflecting differences in root morphology and acclimation of nutrient-foraging roots to P stress. Among the species, there was a 3.2-fold range in root length density, a 1.7-fold range in specific root length, and a 2.1-fold range in root hair length. O. sativus had the lowest critical P requirement, displayed a high root length density, the highest specific root length, and the longest root hairs. Acquisition of P from P-deficient soil was facilitated by development of a large root hair cylinder (i.e. a large root-soil interface). This, in turn, was determined by the intrinsic root morphology attributes of each genotype, and the plasticity of its root morphology response to internal P stress. Root acclimation in low-P soil by all species was mostly associated with preferential allocation of mass to nutrient-foraging roots. Only O. sativus and four of the Trifolium species adjusted specific root length beneficially, and only O. sativus increased its root hair length in low-P soil.

Phosphorus acquisition by citrate- and phytase-exuding Nicotiana tabacum plant mixtures depends on soil phosphorus availability and root intermingling.

Citrate and phytase root exudates contribute to improved phosphorus (P) acquisition efficiency in Nicotiana tabacum (tobacco) when both exudates are produced in a P deficient soil. To test the importance of root intermingling in the interaction of citrate and phytase exudates, Nicotiana tabacum plant-lines with constitutive expression of heterologous citrate (Cit) or fungal phytase (Phy) exudation traits were grown under two root treatments (roots separated or intermingled) and in two soils with contrasting soil P availability. Complementarity of plant mixtures varying in citrate efflux rate and mobility of the expressed phytase in soil was determined based on plant biomass and P accumulation. Soil P composition was evaluated using solution 31 P NMR spectroscopy. In the soil with limited available P, positive complementarity occurred in Cit+Phy mixtures with roots intermingled. Root separation eliminated positive interactions in mixtures expressing the less mobile phytase (Aspergillus niger PhyA) whereas positive complementarity persisted in mixtures that expressed the more mobile phytase (Peniophora lycii PhyA). Soils from Cit+Phy mixtures contained less inorganic P and more organic P compared to monocultures. Exudate-specific strategies for the acquisition of soil P were most effective in P-limited soil and depended on citrate efflux rate and the relative mobility of the expressed phytase in soil. Plant growth and soil P utilization in plant systems with complementary exudation strategies are expected to be greatest where exudates persist in soil and are expressed synchronously in space and time.

Determinants of bacterial communities in Canadian agroforestry systems.

Land-use change is one of the most important factors influencing soil microbial communities, which play a pivotal role in most biogeochemical and ecological processes. Using agroforestry systems as a model, this study examined the effects of land uses and edaphic properties on bacterial communities in three agroforestry types covering a 270 km soil-climate gradient in Alberta, Canada. Our results demonstrate that land-use patterns exert stronger effects on soil bacterial communities than soil zones in these agroforestry systems. Plots with trees in agroforestry systems promoted greater bacterial abundance and to some extent species richness, which was associated with more nutrient-rich soil resources. While Acidobacteria, Actinobacteria and Alphaproteobacteria were the dominant bacterial phyla and subphyla across land uses, Arthrobacter, Acidobacteria_Gp16, Burkholderia, Rhodanobacter and Rhizobium were the keystone taxa in these agroforestry systems. Soil pH and carbon contents emerged as the major determinants of bacterial community characteristics. We found non-random co-occurrence and modular patterns of soil bacterial communities, and these patterns were controlled by edaphic factors and not their taxonomy. Overall, this study highlights the drivers and co-occurrence patterns of soil microbial communities in agroforestry systems.

Rhizosheaths on wheat grown in acid soils: phosphorus acquisition efficiency and genetic control.

Rhizosheaths comprise soil bound to roots, and in wheat (Triticum aestivum L.) rhizosheath size correlates with root hair length. The aims of this study were to determine the effect that a large rhizosheath has on the phosphorus (P) acquisition by wheat and to investigate the genetic control of rhizosheath size in wheat grown on acid soil.Near-isogenic wheat lines differing in rhizosheath size were evaluated on two acid soils. The soils were fertilized with mineral nutrients and included treatments with either low or high P. The same soils were treated with CaCO3 to raise the pH and detoxify Al(3+) Genotypic differences in rhizosheath size were apparent only when soil pH was low and Al(3+) was present. On acid soils, a large rhizosheath increased shoot biomass compared with a small rhizosheath regardless of P supply. At low P supply, increased shoot biomass could be attributed to a greater uptake of soil P, but at high P supply the increased biomass was due to some other factor. Generation means analysis indicated that rhizosheath size on acid soil was controlled by multiple, additive loci. Subsequently, a quantitative trait loci (QTL) analysis of an F6 population of recombinant inbred lines identified five major loci contributing to the phenotype together accounting for over 60% of the total genetic variance. One locus on chromosome 1D accounted for 34% of the genotypic variation. Genetic control of rhizosheath size appears to be relatively simple and markers based on the QTL provide valuable tools for marker assisted breeding.

Archaeal ammonia oxidizers respond to soil factors at smaller spatial scales than the overall archaeal community does in a high Arctic polar oasis.

Archaea are ubiquitous and highly abundant in Arctic soils. Because of their oligotrophic nature, archaea play an important role in biogeochemical processes in nutrient-limited Arctic soils. With the existing knowledge of high archaeal abundance and functional potential in Arctic soils, this study employed terminal restriction fragment length polymorphism (t-RFLP) profiling and geostatistical analysis to explore spatial dependency and edaphic determinants of the overall archaeal (ARC) and ammonia-oxidizing archaeal (AOA) communities in a high Arctic polar oasis soil. ARC communities were spatially dependent at the 2-5 m scale (P < 0.05), whereas AOA communities were dependent at the ∼1 m scale (P < 0.0001). Soil moisture, pH, and total carbon content were key edaphic factors driving both the ARC and AOA community structure. However, AOA evenness had simultaneous correlations with dissolved organic nitrogen and mineral nitrogen, indicating a possible niche differentiation for AOA in which dry mineral and wet organic soil microsites support different AOA genotypes. Richness, evenness, and diversity indices of both ARC and AOA communities showed high spatial dependency along the landscape and resembled scaling of edaphic factors. The spatial link between archaeal community structure and soil resources found in this study has implications for predictive understanding of archaea-driven processes in polar oases.

Evolution of bacterial communities in the wheat crop rhizosphere.

The gap between current average global wheat yields and that achievable through best agronomic management and crop genetics is large. This is notable in intensive wheat rotations which are widely used. Expectations are that this gap can be reduced by manipulating soil processes, especially those that involve microbial ecology. Cross-year analysis of the soil microbiome in an intensive wheat cropping system revealed that rhizosphere bacteria changed much more than the bulk soil community. Dominant factors influencing populations included binding to roots, plant age, site and planting sequence. We demonstrated evolution of bacterial communities within the field rhizosphere. Early in the season, communities tightly bound to the root were simplest. These increased in diversity with plant age and senescence. Loosely bound communities also increased in diversity from vegetative to reproductive plant stages but were more stable than those tightly bound to roots. Planting sequence and, to a lesser extent, wheat genotype also significantly affected rhizosphere bacteria. Plasticity in the rhizosphere generated from crop root system management and genetics offers promise for manipulating the soil ecology of intense cereal systems. Analyses of soil microbiomes for the purpose of developing agronomic benefit should include roots as well as soil loosely adhered to the roots, and the bulk soil.

Network analysis reveals that bacteria and fungi form modules that correlate independently with soil parameters.

Network and multivariate statistical analyses were performed to determine interactions between bacterial and fungal community terminal restriction length polymorphisms as well as soil properties in paired woodland and pasture sites. Canonical correspondence analysis (CCA) revealed that shifts in woodland community composition correlated with soil dissolved organic carbon, while changes in pasture community composition correlated with moisture, nitrogen and phosphorus. Weighted correlation network analysis detected two distinct microbial modules per land use. Bacterial and fungal ribotypes did not group separately, rather all modules comprised of both bacterial and fungal ribotypes. Woodland modules had a similar fungal : bacterial ribotype ratio, while in the pasture, one module was fungal dominated. There was no correspondence between pasture and woodland modules in their ribotype composition. The modules had different relationships to soil variables, and these contrasts were not detected without the use of network analysis. This study demonstrated that fungi and bacteria, components of the soil microbial communities usually treated as separate functional groups as in a CCA approach, were co-correlated and formed distinct associations in these adjacent habitats. Understanding these distinct modular associations may shed more light on their niche space in the soil environment, and allow a more realistic description of soil microbial ecology and function.

C/N ratio drives soil actinobacterial cellobiohydrolase gene diversity.

Cellulose accounts for approximately half of photosynthesis-fixed carbon; however, the ecology of its degradation in soil is still relatively poorly understood. The role of actinobacteria in cellulose degradation has not been extensively investigated despite their abundance in soil and known cellulose degradation capability. Here, the diversity and abundance of the actinobacterial glycoside hydrolase family 48 (cellobiohydrolase) gene in soils from three paired pasture-woodland sites were determined by using terminal restriction fragment length polymorphism (T-RFLP) analysis and clone libraries with gene-specific primers. For comparison, the diversity and abundance of general bacteria and fungi were also assessed. Phylogenetic analysis of the nucleotide sequences of 80 clones revealed significant new diversity of actinobacterial GH48 genes, and analysis of translated protein sequences showed that these enzymes are likely to represent functional cellobiohydrolases. The soil C/N ratio was the primary environmental driver of GH48 community compositions across sites and land uses, demonstrating the importance of substrate quality in their ecology. Furthermore, mid-infrared (MIR) spectrometry-predicted humic organic carbon was distinctly more important to GH48 diversity than to total bacterial and fungal diversity. This suggests a link between the actinobacterial GH48 community and soil organic carbon dynamics and highlights the potential importance of actinobacteria in the terrestrial carbon cycle.

Complex Forms of Soil Organic Phosphorus-A Major Component of Soil Phosphorus.

Phosphorus (P) is an essential element for life, an innate constituent of soil organic matter, and a major anthropogenic input to terrestrial ecosystems. The supply of P to living organisms is strongly dependent on the dynamics of soil organic P. However, fluxes of P through soil organic matter remain unclear because only a minority (typically <30%) of soil organic P has been identified as recognizable biomolecules of low molecular weight (e.g., inositol hexakisphosphates). Here, we use (31)P nuclear magnetic resonance spectroscopy to determine the speciation of organic P in soil extracts fractionated into two molecular weight ranges. Speciation of organic P in the high molecular weight fraction (>10 kDa) was markedly different to that of the low molecular weight fraction (<10 kDa). The former was dominated by a broad peak, which is consistent with P bound by phosphomonoester linkages of supra-/macro-molecular structures, whereas the latter contained all of the sharp peaks that were present in unfractionated extracts, along with some broad signal. Overall, phosphomonoesters in supra-/macro-molecular structures were found to account for the majority (61% to 73%) of soil organic P across the five diverse soils. These soil phosphomonoesters will need to be integrated within current models of the inorganic-organic P cycle of soil-plant terrestrial ecosystems.

The role of gluconate production by Pseudomonas spp. in the mineralization and bioavailability of calcium-phytate to Nicotiana tabacum.

Organic phosphorus (P) is abundant in most soils but is largely unavailable to plants. Pseudomonas spp. can improve the availability of P to plants through the production of phytases and organic anions. Gluconate is a major component of Pseudomonas organic anion production and may therefore play an important role in the mineralization of insoluble organic P forms such as calcium-phytate (CaIHP). Organic anion and phytase production was characterized in 2 Pseudomonas spp. soil isolates (CCAR59, Ha200) and an isogenic mutant of strain Ha200, which lacked a functional glucose dehydrogenase (Gcd) gene (strain Ha200 gcd::Tn5B8). Wild-type and mutant strains of Pseudomonas spp. were evaluated for their ability to solubilize and hydrolyze CaIHP and to promote the growth and assimilation of P by tobacco plants. Gluconate, 2-keto-gluconate, pyruvate, ascorbate, acetate, and formate were detected in Pseudomonas spp. supernatants. Wild-type pseudomonads containing a functional gcd could produce gluconate and mineralize CaIHP, whereas the isogenic mutant could not. Inoculation with Pseudomonas improved the bioavailability of CaIHP to tobacco plants, but there was no difference in plant growth response due to Gcd function. Gcd function is required for the mineralization of CaIHP in vitro; however, further studies will be needed to quantify the relative contribution of specific organic anions such as gluconate to plant growth promotion by soil pseudomonads.

Transposon-mediated alteration of TaMATE1B expression in wheat confers constitutive citrate efflux from root apices.

The TaMATE1B gene (for multidrug and toxic compound extrusion) from wheat (Triticum aestivum) was isolated and shown to encode a citrate transporter that is located on the plasma membrane. TaMATE1B expression in roots was induced by iron deficiency but not by phosphorus deficiency or aluminum treatment. The coding region of TaMATE1B was identical in a genotype showing citrate efflux from root apices (cv Carazinho) to one that lacked citrate efflux (cv Egret). However, sequence upstream of the coding region differed between these two genotypes in two ways. The first difference was a single-nucleotide polymorphism located approximately 2 kb upstream from the start codon in cv Egret. The second difference was an 11.1-kb transposon-like element located 25 bp upstream of the start codon in cv Carazinho that was absent from cv Egret. The influence of these polymorphisms on TaMATE1B expression was investigated using fusions to green fluorescent protein expressed in transgenic lines of rice (Oryza sativa). Fluorescence measurements in roots of rice indicated that 1.5- and 2.3-kb regions upstream of TaMATE1B in cv Carazinho (which incorporated 3' regions of the transposon-like element) generated 20-fold greater expression in the apical 1 mm of root compared with the native promoter in cv Egret. By contrast, fluorescence in more mature tissues was similar in both cultivars. The presence of the single-nucleotide polymorphism alone consistently generated 2-fold greater fluorescence than the cv Egret promoter. We conclude that the transposon-like element in cv Carazinho extends TaMATE1B expression to the root apex, where it confers citrate efflux and enhanced aluminum tolerance.

Can citrate efflux from roots improve phosphorus uptake by plants? Testing the hypothesis with near-isogenic lines of wheat.

Phosphorus (P) deficiency in some plant species triggers the release of organic anions such as citrate and malate from roots. These anions are widely suggested to enhance the availability of phosphate for plant uptake by mobilizing sparingly-soluble forms in the soil. Carazinho is an old wheat (Triticum aestivum) cultivar from Brazil, which secretes citrate constitutively from its root apices, and here we show that it also produces relatively more biomass on soils with low P availability than two recent Australian cultivars that lack citrate efflux. To test whether citrate efflux explains this phenotype, we generated two sets of near-isogenic lines that differ in citrate efflux and compared their biomass production in different soil types and with different P treatments in glasshouse experiments and field trials. Citrate efflux improved relative biomass production in two of six glasshouse trials but only at the lowest P treatments where growth was most severely limited by P availability. Furthermore, citrate efflux provided no consistent advantage for biomass production or yield in multiple field trials. Theoretical modeling indicates that the effectiveness of citrate efflux in mobilizing soil P is greater as the volume of soil into which it diffuses increases. As efflux from these wheat plants is restricted to the root apices, the potential for citrate to mobilize sufficient P to increase shoot biomass may be limited. We conclude that Carazinho has other attributes that contribute to its comparatively good performance in low-P soils.

Towards circular phosphorus: The need of inter- and transdisciplinary research to close the broken cycle.

Phosphorus (P) is an essential element to all living beings but also a finite resource. P-related problems center around broken P cycles from local to global scales. This paper presents outcomes from the 9th International Phosphorus Workshop (IPW9) held 2019 on how to move towards a sustainable P management. It is based on two sequential discussion rounds with all participants. Important progress was reported regarding the awareness of P as finite mineable resource, technologies to recycle P, and legislation towards a circular P economy. Yet, critical deficits were identified such as how to handle legacy P, how climate change may affect ecosystem P cycling, or working business models to up-scale existing recycling models. Workshop participants argued for more transdisciplinary networks to narrow a perceived science-practice/policy gap. While this gap may be smaller in reality as illustrated with a Swiss example, we formulate recommendations how to bridge this gap more effectively.

Effect of soil acidity, soil strength and macropores on root growth and morphology of perennial grass species differing in acid-soil resistance.

It is unclear whether roots of acid-soil resistant plants have significant advantages, compared with acid-soil sensitive genotypes, when growing in high-strength, acid soils or in acid soils where macropores may allow the effects of soil acidity and strength to be avoided. The responses of root growth and morphology to soil acidity, soil strength and macropores by seedlings of five perennial grass genotypes differing in acid-soil resistance were determined, and the interaction of soil acidity and strength for growth and morphology of roots was investigated. Soil acidity and strength altered root length and architecture, root hair development, and deformed the root tip, especially in acid-soil sensitive genotypes. Root length was restricted to some extent by soil acidity in all genotypes, but the adverse impact of soil acidity on root growth by acid-soil resistant genotypes was greater at high levels of soil strength. Roots reacted to soil acidity when growing in macropores, but elongation through high-strength soil was improved. Soil strength can confound the effect of acidity on root growth, with the sensitivity of acid-resistant genotypes being greater in high-strength soils. This highlights the need to select for genotypes that resist both acidity and high soil strength.

Soil microbial communities influencing organic phosphorus mineralization in a coastal dune chronosequence in New Zealand.

The Haast chronosequence in New Zealand is an ∼6500-year dune formation series, characterized by rapid podzol development, phosphorus (P) depletion and a decline in aboveground biomass. We examined bacterial and fungal community composition within mineral soil fractions using amplicon-based high-throughput sequencing (Illumina MiSeq). We targeted bacterial non-specific acid (class A, phoN/phoC) and alkaline (phoD) phosphomonoesterase genes and quantified specific genes and transcripts using real-time PCR. Soil bacterial diversity was greatest after 4000 years of ecosystem development and associated with an increased richness of phylotypes and a significant decline in previously dominant taxa (Firmicutes and Proteobacteria). Soil fungal communities transitioned from predominantly Basidiomycota to Ascomycota along the chronosequence and were most diverse in 290- to 392-year-old soils, coinciding with maximum tree basal area and organic P accumulation. The Bacteria:Fungi ratio decreased amid a competitive and interconnected soil community as determined by network analysis. Overall, soil microbial communities were associated with soil changes and declining P throughout pedogenesis and ecosystem succession. We identified an increased dependence on organic P mineralization, as found by the profiled acid phosphatase genes, soil acid phosphatase activity and function inference from predicted metagenomes (PICRUSt2).

The microbiomes on the roots of wheat (Triticum aestivum L.) and rice (Oryza sativa L.) exhibit significant differences in structure between root types and along root axes.

There is increasing interest in understanding how the microbial communities on roots can be manipulated to improve plant productivity. Root systems are not homogeneous organs but are comprised of different root types of various ages and anatomies that perform different functions. Relatively little is known about how this variation influences the distribution and abundance of microorganisms on roots and in the rhizosphere. Such information is important for understanding how root-microbe interactions might affect root function and prevent diseases. This study tested specific hypotheses related to the spatial variation of bacterial and fungal communities on wheat (Triticum aestivum L.) and rice (Oryza sativa L.) roots grown in contrasting soils. We demonstrate that microbial communities differed significantly between soil type, between host species, between root types, and with position along the root axes. The magnitude of variation between different root types and along individual roots was comparable with the variation detected between different plant species. We discuss the general patterns that emerged in this variation and identify bacterial and fungal taxa that were consistently more abundant on specific regions of the root system. We argue that these patterns should be measured more routinely so that localised root-microbe interactions can be better linked with root system design, plant health and performance.

HvALMT1 from barley is involved in the transport of organic anions.

Members of the ALMT gene family contribute to the Al(3+) resistance of several plant species by facilitating malate efflux from root cells. The first member of this family to be cloned and characterized, TaALMT1, is responsible for most of the natural variation of Al(3+) resistance in wheat. The current study describes the isolation and characterization of HvALMT1, the barley gene with the greatest sequence similarity to TaALMT1. HvALMT1 is located on chromosome 2H which has not been associated with Al(3+) resistance in barley. The relatively low levels of HvALMT1 expression detected in root and shoot tissues were independent of external aluminium or phosphorus supply. Transgenic barley plants transformed with the HvALMT1 promoter fused to the green fluorescent protein (GFP) indicated that expression of HvALMT1 was relatively high in stomatal guard cells and in root tissues containing expanding cells. GFP fused to the C-terminus of the full HvALMT1 protein localized to the plasma membrane and motile vesicles within the cytoplasm. HvALMT1 conferred both inward and outward currents when expressed in Xenopus laevis oocytes that were bathed in a range of anions including malate. Both malate uptake and efflux were confirmed in oocyte assays using [(14)C]malate as a radiotracer. It is suggested that HvALMT1 functions as an anion channel to facilitate organic anion transport in stomatal function and expanding cells.