The loss of carbon from roots (rhizodeposition) and the consequent proliferation of microorganisms in the surrounding soil, coupled with the physical presence of a root and processes associated with nutrient uptake, gives rise to a unique zone of soil called the rhizosphere. In this review, we bring together evidence to show that roots can directly regulate most aspects of rhizosphere C flow either by regulating the exudation process itself or by directly regulating the recapture of exudates from soil. Root exudates have been hypothesized to be involved in the enhanced mobilization and acquisition of many nutrients from soil or the external detoxification of metals. With few exceptions, there is little mechanistic evidence from soil-based systems to support these propositions. We conclude that much more integrated work in realistic systems is required to quantify the functional significance of these processes in the field. We need to further unravel the complexities of the rhizosphere in order to fully engage with key scientific ideas such as the development of sustainable agricultural systems and the response of ecosystems to climate change. Contents I. Introduction 460 II. What is rhizodeposition? 460 III. Regulation of rhizodeposition 460 IV. How large is the root exudation C flux? 463 V. How responsive is the root exudation C flux? 463 VI. How responsive is the microbial community to root exudation? 464 VII. The role of root exudates in nutrient acquisition 464 VIII. Mycorrhizal fungi and rhizodeposition 471 IX. Future thoughts 474 Acknowledgements 474 References 474.

https://nph.onlinelibrary.wiley.com/doi/epdf/10.1111/j.1469-8137.2004.01130.x

Plant and mycorrhizal regulation of rhizodeposition

Root systems of most land plants form arbuscular mycorrhizal (AM) symbioses in the field, and these contribute to nutrient uptake. AM roots have two pathways for nutrient absorption, directly through the root epidermis and root hairs and via AM fungal hyphae into root cortical cells, where arbuscules or hyphal coils provide symbiotic interfaces. New physiological and molecular evidence shows that for phosphorus the mycorrhizal pathway (MP) is operational regardless of plant growth responses (positive or negative). Amounts delivered cannot be determined from plant nutrient contents because when responses are negative the contribution of the direct pathway (DP) is reduced. Nitrogen (N) is also delivered to roots via an MP, but the contribution to total N requirement and the costs to the plant are not clear. The functional interplay between activities of the DP and MP has important implications for consideration of AM symbioses in ecological, agronomic, and evolutionary contexts.

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Roles of Arbuscular Mycorrhizas in Plant Nutrition and Growth: New Paradigms from Cellular to Ecosystem Scales

Most transport proteins in plant cells are energized by electrochemical gradients of protons across the plasma membrane. The formation of these gradients is due to the action of plasma membrane H+ pumps fuelled by ATP. The plasma membrane H+-ATPases share a membrane topography and general mechanism of action with other P-type ATPases, but differ in regulatory properties. Recent advances in the field include the identification of the complete H+-ATPase gene family in Arabidopsis, analysis of H+-ATPase function by the methods of reverse genetics, an improved understanding of the posttranslational regulation of pump activity by 14-3-3 proteins, novel insights into the H+ transport mechanism, and progress in structural biology. Furthermore, the elucidation of the three-dimensional structure of a related Ca2+ pump has implications for understanding of structure-function relationships for the plant plasma membrane H+-ATPase.

PLANT PLASMA MEMBRANE H+-ATPases: Powerhouses for Nutrient Uptake.

Many plants have the capacity to obtain phosphate via a symbiotic association with arbuscular mycorrhizal (AM) fungi. In AM associations, the fungi release phosphate from differentiated hyphae called arbuscules, that develop within the cortical cells, and the plant transports the phosphate across a symbiotic membrane, called the periarbuscular membrane, into the cortical cell. In Medicago truncatula, a model legume used widely for studies of root symbioses, it is apparent that the phosphate transporters known to operate at the root–soil interface do not participate in symbiotic phosphate transport. EST database searches with short sequence motifs shared by known phosphate transporters enabled the identification of a novel phosphate transporter from M. truncatula, MtPT4. MtPT4 is significantly different from the plant root phosphate transporters cloned to date. Complementation of yeast phosphate transport mutants indicated that MtPT4 functions as a phosphate transporter, and estimates of the Km suggest a relatively low affinity for phosphate. MtPT4 is expressed only in mycorrhizal roots, and the MtPT4 promoter directs expression exclusively in cells containing arbuscules. MtPT4 is located in the membrane fraction of mycorrhizal roots, and immunolocalization revealed that MtPT4 colocalizes with the arbuscules, consistent with a location on the periarbuscular membrane. The transport properties and spatial expression patterns of MtPT4 are consistent with a role in the acquisition of phosphate released by the fungus in the AM symbiosis.

A Phosphate Transporter from Medicago truncatula Involved in the Acquisition of Phosphate Released by Arbuscular Mycorrhizal Fungi

The plasma membrane

The plasma membrane proton pump ATPase (H(+)-ATPase) plays a central role in transport across the plasma membrane. As a primary transporter, it mediates ATP-dependent H(+) extrusion to the extracellular space, thus creating pH and potential differences across the plasma membrane that activate a large set of secondary transporters. In several species, the H(+)-ATPase is encoded by a family of approximately 10 genes, classified into 5 gene subfamilies and we might ask what can this tell us about the concept, and the evolution, of gene families in plants. All the highly expressed H(+)-ATPase genes are classified into only two gene subfamilies, which diverged before the emergence of present plant species, raising the questions of the significance of the existence of these two well-conserved subfamilies and whether this is related to different kinetic or regulatory properties. Finally, what can we learn from experimental approaches that silence specific genes? In this review, we would like to discuss these questions in the light of recent data.

The plasma membrane proton pump ATPase: the significance of gene subfamilies

In arbuscular mycorrhizas, H+-ATPase is active in the plant membrane around arbuscules but absent from plant mutants defective in arbuscule development (Gianinazzi-Pearson et al. 1995, Can J Bot 73: S526–S532). The proton-pumping H+-ATPase is encoded by a family of genes in plants. Immunocytochemical studies and promoter-gusA fusion assays were performed in transgenic tobacco (Nicotiana tabacum L.) to determine whether the periarbuscular enzyme activity results from de-novo activation of plant genes by an arbuscular mycorrhizal fungus. The H+-ATPase protein was localized in the plant membrane around arbuscule hyphae. The enzyme was absent from non-colonized cortical cells. Regulation of seven H+-ATPase genes (pma) was compared in non-mycorrhizal and mycorrhizal roots by histochemical detection of β-glucuronidase (GUS) activity. Two genes (pma2, pma4) were induced in arbuscule-containing cells of mycorrhizal roots but not in non-mycorrhizal cortical tissues or senescent mycorrhiza. It is concluded that de-novo H+-ATPase activity in the periarbuscular membrane results from selective induction of two H+-ATPase genes, which can have diverse roles in plant-fungal interactions at the symbiotic interface.

https://www.jstor.org/stable/pdfplus/23386040.pdf

Differential activation of H+-ATPase genes by an arbuscular mycorrhizal fungus in root cells of transgenic tobacco.

To analyze in detail the multigene family encoding the plasma-membrane H(+)-ATPase (pma) in Nicotiana plumbaginifolia Viv., five new pma genes (pma 5-9) were isolated. Three of these (pma 6, 8, 9) were fully characterized and classified into new and independent subfamilies. Their cell-type expression was followed by the beta-glucuronidase (gusA) reporter-gene method. While the pma8-gusA transgene was not expressed in transgenic tobacco, expression of the two other transgenes (pma6- and pma9-gusA) was found to be restricted to particular cell types. In the vegetative tissues, pma6-gusA expression was limited to the head cells of the leaf short trichomes, involved in secretion, and to the cortical parenchyma of the young nodes where the developing leaves and axillary flowering stalks join the stem. In the latter tissues, gene expression was enhanced by mechanical stress, suggesting that H(+)-ATPase might be involved in the strength of the tissues and their resistance to mechanical trauma. The pma9-gusA transgene was mainly expressed in the apical meristem of adventitious roots and axillary buds as well as in the phloem tissues of the stem, in which expression depended on the developmental stage. In flowers, pma9-gusA expression was limited to the mature pollen grains and the young fertilized ovules, while that of pma6-gusA was identified in most of the organs. Reverse transcription-polymerase chain reaction of leaf and stem RNA confirmed the expression of pma 6 and 9, while pma8 was found to be expressed in both organs at a lower level. In conclusion, although pma 6 and 9 had a more restricted expression pattern than the previously characterized pma genes, they were nevertheless expressed in cell types in which H(+)-ATPase had not been previously detected.

Identification and expression of three new Nicotiana plumbaginifolia genes which encode isoforms of a plasma-membrane H(+)-ATPase, and one of which is induced by mechanical stress.

The plasma membrane H+-ATPase couples ATP hydrolysis to proton transport, thereby establishing the driving force for solute transport across the plasma membrane. In Nicotiana plumbaginifolia, this enzyme is encoded by at least nine pma (plasma membrane H+-ATPase) genes. Four of these are classified into two gene subfamilies, pma1-2-3 and pma4, which are the most highly expressed in plant species. We have isolated genomic clones for pma2 and pma4. Mapping of their transcript 5' end revealed the presence of a long leader that contained small open reading frames, regulatory features typical of other pma genes. The gusA reporter gene was then used to determine the expression of pma2, pma3 and pma4 in N. tabacum. These data, together with those obtained previously for pma1, led to the following conclusions. (i) The four pma-gusA genes were all expressed in root, stem, leaf and flower organs, but each in a cell-type specific manner. Expression in these organs was confirmed at the protein level, using subfamily-specific antibodies. (ii) pma4-gusA was expressed in many cell types and notably in root hair and epidermis, in companion cells, and in guard cells, indicating that in N. plumbaginifolia the same H+-ATPase isoform might be involved in mineral nutrition, phloem loading and control of stomata aperture. (iii) The second gene subfamily is composed, in N. plumbaginifolia, of a single gene (pma4) with a wide expression pattern and, in Arabidopsis thaliana, of three genes (aha1, aha2, aha3), at least two of them having a more restrictive expression pattern. (iv) Some cell types expressed pma2 and pma4 at the same time, which encode H+-ATPases with different enzymatic properties.

Expression analysis of two gene subfamilies encoding the plasma membrane H+‐ATPase in Nicotiana plumbaginifolia reveals the major transport functions of this enzyme

A deletion and mutagenesis study was performed on the mitochondrial presequence of the beta-subunit of the F(1)-ATP synthase from Nicotiana plumbaginifolia linked to the green fluorescent protein (GFP). The various constructs were tested in vivo by transient expression in tobacco protoplasts. GFP distribution in transformed cells was analysed in situ by confocal microscopy, and in vitro in subcellular fractions by Western blotting. Despite its being highly conserved in different species, deletion of the C-terminal region (residues 48-54) of the presequence did not affect mitochondrial import. Deletion of the conserved residues 40-47 and the less conserved intermediate region (residues 18-39) resulted in 60% reduction in GFP import, whereas mutation of conserved residues within these regions had little effect. Further shortening of the presequence progressively reduced import, with the construct retaining the predicted N-terminal amphiphilic alpha-helix (residues 1-12) being unable to mediate mitochondrial import. However, point mutation showed that this last region plays an important role through its basic residues and amphiphilicity, but also through its hydrophobic residues. Replacing Arg4 and Arg5 by alanine residues and shifting the Arg5 and Leu6 (in order to disturb amphiphilicity) resulted in reduction of the presequence import efficiency. The most dramatic effects were seen with single or double mutations of the four Leu residues (positions 5, 6, 10 and 11), which resulted in marked reduction or abolition of GFP import, respectively. We conclude that the N-terminal helical structure of the presequence is necessary but not sufficient for efficient mitochondrial import, and that its hydrophobic residues play an essential role in in vivo mitochondrial targeting.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-313X.2001.01098.x

Hydrophobic residues within the predicted N-terminal amphiphilic alpha-helix of a plant mitochondrial targeting presequence play a major role in in vivo import.

Mohammed Oufattole

Papers

The plasma membrane proton pump ATPase: the significance of gene subfamilies

Differential activation of H+-ATPase genes by an arbuscular mycorrhizal fungus in root cells of transgenic tobacco.

Identification and expression of three new Nicotiana plumbaginifolia genes which encode isoforms of a plasma-membrane H(+)-ATPase, and one of which is induced by mechanical stress.

Expression analysis of two gene subfamilies encoding the plasma membrane H+‐ATPase in Nicotiana plumbaginifolia reveals the major transport functions of this enzyme

Hydrophobic residues within the predicted N-terminal amphiphilic alpha-helix of a plant mitochondrial targeting presequence play a major role in in vivo import.