Nitrogen (N) and phosphorus (P) are the most limiting factors for plant growth. Some microorganisms improve the uptake and availability of N and P, minimizing chemical fertilizer dependence. It has been published that the RD64 strain, a
Sinorhizobium meliloti 1021 strain engineered to overproduce indole-3-acetic acid (IAA), showed improved nitrogen fixation ability compared to the wild-type 1021 strain. Here, we present data showing that RD64 is also highly effective in mobilizing P from insoluble sources, such as phosphate rock (PR). Under P-limiting conditions, the higher level of P-mobilizing activity of RD64 than of the 1021 wild-type strain is connected with the upregulation of genes coding for the high-affinity P transport system, the induction of acid phosphatase activity, and the increased secretion into the growth medium of malic, succinic, and fumaric acids.
Medicago truncatula plants nodulated by RD64 (
Mt-RD64), when grown under P-deficient conditions, released larger amounts of another P-solubilizing organic acid, 2-hydroxyglutaric acid, than plants nodulated by the wild-type strain (
Mt-1021). It has already been shown that
Mt-RD64 plants exhibited higher levels of dry-weight production than
Mt-1021 plants. Here, we also report that P-starved
Mt-RD64 plants show significant increases in both shoot and root fresh weights when compared to P-starved
Mt-1021 plants. We discuss how, in a
Rhizobium-legume model system, a balanced interplay of different factors linked to bacterial IAA overproduction rather than IAA production
per se stimulates plant growth under stressful environmental conditions and, in particular, under P starvation.Compared with the other major nutrients, such as nitrogen, phosphorus (P) is by far the least mobile and available to plants under most soil conditions. Although P is abundant in soils in both organic and inorganic forms, it is frequently a major or even the prime limiting factor for plant growth. Many soils throughout the world are P deficient, because the free concentration (the form available to the plant), even in fertile soils, is generally low due to high reactivity of soluble P with calcium, iron, or aluminum that leads to P precipitation (
36,
41). In addition, in developing countries, chemical fertilizers, which provide the three major plant nutrients (N, P, and potassium), are not widely used, due to cost limitations. In these areas, the direct application of ground phosphate rock (PR) is increasingly used, even if the level of P released from PR is often too low for crop growth (
9,
38). It is known that many microorganisms, in particular those of the genera
Pseudomonas,
Bacillus, and
Rhizobium, have the ability to change their metabolism in response to the phosphorus available for cellular growth. The switch in metabolism is mediated through the repression and induction of various genes whose products are involved in processes ranging from uptake and acquisition of P sources to
de novo synthesis of new cellular components (
18,
36). Furthermore,
in vitro studies showed that for some of these bacteria, the P-solubilizing activity and the production of the auxin indole-3-acetic acid (IAA) were coexpressed (
17,
39), although a direct correlation linking IAA production to P solubilization was not demonstrated.P uptake in various microorganisms has been investigated. Many bacterial species, including
Sinorhizobium meliloti, have at least two P transport systems, consistent with the high- and low-affinity transport systems. The high-affinity system is encoded by the
phoCDET operon, and the low-affinity system is encoded by
pit (in the
orfA-pit operon). In
S. meliloti, the expression of genes encoding both P transport systems is controlled by the PhoB activator. Under P excess conditions, PhoB is inactive, and the
phoCDET genes are not expressed. Under P-limiting conditions, the low-affinity Pit permease system is repressed by activated PhoB, while the high-affinity PhoCDET system is induced and becomes the primary mechanism of P transport (
10). Many bacterial strains contain PstSCAB homologs that function as high-affinity phosphate transporters. For
S. meliloti 1021, a 1-bp deletion in the
pstC open reading frame (ORF) is probably responsible (via PhoB) for the moderate constitutive activation of 12 phosphate starvation-inducible genes, observed in the absence of phosphate stress (
24,
43).In both plants and microorganisms, the primary mechanisms of PR solubilization are H
+ excretion, organic acid production, and acid phosphatase biosynthesis (
2,
3). Organic acids, including acetate, lactate, malate, oxalate, succinate, citrate, gluconate, ketogluconate, etc., can form complexes with the iron or aluminum in ferric and aluminum phosphates, thus releasing plant-available phosphate into the soil (
18,
22). Organic acids may also increase P availability by blocking P absorption sites on soil particles or by forming complexes with cations on the soil mineral surface (
36).Mineralization of most organic phosphorus compounds is carried out by means of phosphatase enzymes. The major source of these enzymes in soil is considered to be of microbial origin. In particular, phosphatase activity is substantially increased in the rhizosphere. The pHs of most soils range from acid to neutral values. Thus, acid phosphatases should play the major role in this process (
36).In the present study, the P-solubilizing ability of an
S. meliloti 1021 strain, RD64, and its effect on the growth of a
Medicago host plant were analyzed. We used the
S. meliloti-Medicago truncatula system since the microarrays were available for the bacterium and
Medicago is a well-recognized model system for indeterminate nodule development. The RD64 strain has previously been engineered to overproduce IAA (
11,
35), showing that this strain is able to release into liquid growth medium up to 78-fold more IAA than wild-type 1021 (
12,
21). It was also previously reported that, as found for IAA-treated
Escherichia coli cells (
7), RD64 is more resistant to salinity and other abiotic stresses than 1021 (
5). Medicago plants nodulated by this strain have a higher degree of protection against oxidative damage induced by salt stress than 1021-nodulated plants (
5).It was previously shown that IAA triggers induction of tricarboxylic acid (TCA) cycle enzymes in quite-distant systems, such as transformed human cells (
15),
E. coli (
8) and
S. meliloti (
21), with a mechanism not yet understood. To evaluate the global effects triggered by IAA overproduction in
S. meliloti RD64, the gene expression pattern of wild-type 1021 was compared with those of RD64 and 1021 treated with IAA and four other chemically or functionally related molecules by microarray analysis.Among the genes differentially expressed in RD64 and IAA-treated 1021 cells, we found two genes of the
pho operon:
phoT, coding for the phosphate uptake ABC transporter permease protein, and
phoC, coding for the phosphate uptake ABC transporter ATP binding protein. This unexpected finding led us to examine the mechanisms for mineral P solubilization in RD64 and the potential ability of this strain to improve
Medicago growth under P-starved conditions. Increases in acid phosphatase activity and organic acid excretion were observed for the RD64 strain under free-living conditions. Furthermore, the amount of organic acids exuded from the roots of
Medicago plants nodulated by this strain was larger than that measured for plants nodulated by the 1021 wild-type strain. This effect was connected to the enhanced P solubilization and plant dry weight production observed for these plants.
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