Uptake and translocation of phosphate by pho2 mutant and wild-type seedlings of Arabidopsis thaliana

The pho2 mutant of Arabidopsis thaliana (L.) Heynh. accumulates excessive Pi (inorganic phosphate) concentrations in shoots compared to wild-type plants (E. Delhaize and P. Randall, 1995, Plant Physiol. 107: 207–213). In this study, a series of experiments was conducted to compare the uptake and translocation of Pi by pho2 with that of wild-type plants. The pho2 mutants had about a twofold greater Pi uptake rate than wild-type plants under P-sufficient conditions and a greater proportion of the Pi taken up accumulated in shoots of pho2. When shoots were removed, the uptake rate by roots was found to be similar for both genotypes, suggesting that the greater Pi uptake by the intact pho2 mutant is due to a greater shoot sink for Pi. Although pho2 mutants could recycle ³²Pi from shoots to roots through phloem the proportion of ³²Pi translocated to roots was less than half of that found in wild-type plants. When transferred from P-sufficient to P-deficient solutions, Pi concentrations in pho2 roots had a similar depletion rate to wild-type roots despite pho2 shoots having a fourfold greater Pi concentration than wild-type shoots throughout the experiment. We suggest that the pho2 phenotype could result from a partial defect in Pi transport in the phloem between shoots and roots or from an inability of shoot cells to regulate internal Pi concentrations. Received: 20 August 1997 / Accepted: 4 October 1997     

Characterization of a Phosphate-Accumulator Mutant of Arabidopsis thaliana

We have characterized a novel mutation of Arabidopsis thaliana at a locus designated pho2. pho2 mutants accumulated up to 3-fold more total P in leaves, mostly as inorganic phosphate (Pi), than wild-type seedlings. In addition, we isolated a mutant (locus designated pho1-2, an allelle of pho1-1 described by Y. Poirier, S. Thoma, C. Somerville, J. Schiefelbein [1991] Plant Physiol 97: 1087-1093) with low Pi concentrations in leaves. When grown under high transpiration conditions, leaves of pho2 seedlings became severely P intoxicated, whereas shoots of pho1-2 mutants were P deficient and wild-type seedlings were normal. A pho1/pho2 double mutant resulting from a cross between the single mutants was identified in the F2 generation and shown to have a pho1 phenotype. Prior to the development of P toxicity symptoms, P was the only mineral nutrient whose concentration was greater in pho2 mutants than wild-type seedlings. Compared to wild-type, pho2 mutants had greater Pi concentrations in stems, siliques, and seeds, but roots of pho2 mutants had similar or lower Pi concentrations than either pho1 mutants or wild-type seedlings. We suggest that the pho2 mutation affects a function normally involved in regulating the concentration of Pi in shoots of Arabidopsis.     

Regulatory network of microRNA399 and PHO2 by systemic signaling

Recently, we showed that microRNA399s (miR399s) control inorganic phosphate (Pi) homeostasis by regulating the expression of PHO2 encoding a ubiquitin-conjugating E2 enzyme 24. Arabidopsis (Arabidopsis thaliana) plants overexpressing miR399 or the pho2 mutant overaccumulate Pi in shoots. The association of Pi translocation and coexpression of miR399s and PHO2 in vascular tissues suggests their involvement in long-distance signaling. In this study, we used reciprocal grafting between wild-type and miR399-overexpressing transgenic plants to dissect the systemic roles of miR399 and PHO2. Arabidopsis rootstocks overexpressing miR399 showed high accumulation of Pi in the wild-type scions because of reduced PHO2 expression in the rootstocks. Although miR399 precursors or expression was not detected, we found a small but substantial amount of mature miR399 in the wild-type rootstocks grafted with transgenic scions, which indicates the movement of miR399 from shoots to roots. Suppression of PHO2 with miR399b or c was less efficient than that with miR399f. Of note, findings in grafted Arabidopsis were also discovered in grafted tobacco (Nicotiana benthamiana) plants. The analysis of the pho1 mutant provides additional support for systemic suppression of PHO2 by the movement of miR399 from Pi-depleted shoots to Pi-sufficient roots. We propose that the long-distance movement of miR399s from shoots to roots is crucial to enhance Pi uptake and translocation during the onset of Pi deficiency. Moreover, PHO2 small interfering RNAs mediated by the cleavage of miR399s may function to refine the suppression of PHO2. The regulation of miR399 and PHO2 via long-distance communication in response to Pi deficiency is discussed.     

Nitrate uptake by bean (Phaseolus vulgaris L.) roots under phosphate deficiency

Bean (Phaseolus vulgaris L.) plants were cultured for 19 d on complete or on phosphate deficient culture media. Low inorganic phosphate concentration in the roots decreased ATP level and nitrate uptake rate. The mechanisms which may control nitrate uptake rate during phosphate deficiency were examined. Plasma membrane enriched fractions from phosphate sufficient and phosphate deficient plants were isolated and compared. The decrease in total phospholipid content was observed in plasma membranes from phosphate deficient roots, but phospholipid composition was similar. No changes in ATPase and proton pumping activities measured in isolated plasma membrane of phosphate sufficient and phosphate deficient bean roots were noted. The electron microscope observations carried out on cortical meristematic cells of the roots showed that active ATPases were found in plasma membrane of both phosphate sufficient and phosphate deficient plants. The decrease in inorganic phosphate concentration in roots led to increased nitrate accumulation in roots, accompanied by a corresponding alterations in NO₃ distribution between shoots and roots. Nitrate reductase activity in roots of phosphate deficient plants estimated in vivo and in vitro was reduced to 50–60% of the control. The increased NO₃ concentration in root tissue may be explained by decreased NR activity and lower transport of nitrate from roots to shoots. Therefore, the reduction of nitrate uptake during phosphate starvation is mainly a consequence of nitrate accumulation in the roots.     

Arsenic uptake, translocation and speciation in pho1 and pho2 mutants of Arabidopsis thaliana

Arsenate [As (V)] is taken up by phosphate [P (V)] transporters in the plasma membrane of roots cells, but the translocation of As from roots to shoots is not well understood. Two mutants of Arabidopsis thaliana (L.) [( pho1 , P deficient) and ( pho2 , P accumulator)], with defects in the regulation and translocation of P (V) from roots to shoots, were therefore used in this study to investigate uptake, translocation and speciation of As in roots and shoots of plants grown in soil or nutrient solution. The shoots of the pho2 mutant contained higher P concentrations, but similar or slightly higher As concentrations, in comparison with the wild type. In the pho1 mutant, the P concentration in the shoots was lower, and the As concentration was higher, in comparison with the wild type. Both pho2 and the wild type contained mainly As (III) in roots and shoot (67–90% of total As). Arsenic was likely to be translocated by a different pathway to P (V) in the pho2 and pho1 mutants . Therefore, it is suggested that As (III) is the main As species translocated from roots to shoots in Arabidopsis thaliana.     

Uncoupling phosphate deficiency from its major effects on growth and transcriptome via PHO1 expression in Arabidopsis

Inorganic phosphate (Pi) is one of the most limiting nutrients for plant growth in both natural and agricultural contexts. Pi‐deficiency leads to a strong decrease in shoot growth, and triggers extensive changes at the developmental, biochemical and gene expression levels that are presumably aimed at improving the acquisition of this nutrient and sustaining growth. The Arabidopsis thaliana PHO1 gene has previously been shown to participate in the transport of Pi from roots to shoots, and the null pho1 mutant has all the hallmarks associated with shoot Pi deficiency. We show here that A. thaliana plants with a reduced expression of PHO1 in roots have shoot growth similar to Pi‐sufficient plants, despite leaves being strongly Pi deficient. Furthermore, the gene expression profile normally triggered by Pi deficiency is suppressed in plants with low PHO1 expression. At comparable levels of shoot Pi supply, the wild type reduces shoot growth but maintains adequate shoot vacuolar Pi content, whereas the PHO1 underexpressor maintains maximal growth with strongly depleted Pi reserves. Expression of the Oryza sativa (rice) PHO1 ortholog in the pho1 null mutant also leads to plants that maintain normal growth and suppression of the Pi‐deficiency response, despite the low shoot Pi. These data show that it is possible to unlink low shoot Pi content with the responses normally associated with Pi deficiency through the modulation of PHO1 expression or activity. These data also show that reduced shoot growth is not a direct consequence of Pi deficiency, but is more likely to be a result of extensive gene expression reprogramming triggered by Pi deficiency.     

The Down-Regulation of Mt4-Like Genes by Phosphate Fertilization Occurs Systemically and Involves Phosphate Translocation to the Shoots

Mt4 is a cDNA representing a phosphate-starvation-inducible gene from Medicago truncatula that is down-regulated in roots in response to inorganic phosphate (Pi) fertilization and colonization by arbuscular mycorrhizal fungi. Split-root experiments revealed that the expression of the Mt4 gene in M. truncatula roots is down-regulated systemically by both Pi fertilization and colonization by arbuscular mycorrhizal fungi. A comparison of Pi levels in these tissues suggested that this systemic down-regulation is not caused by Pi accumulation. Using a 30-bp region of the Mt4 gene as a probe, Pi-starvation-inducible Mt4-like genes were detected in Arabidopsis and soybean (Glycine max L.), but not in corn (Zea mays L.). Analysis of the expression of the Mt4-like Arabidopsis gene, At4, in wild-type Arabidopsis and pho1, a mutant unable to load Pi into the xylem, suggests that Pi must first be translocated to the shoot for down-regulation to occur. The data from the pho1 and split-root studies are consistent with the presence of a translocatable shoot factor responsible for mediating the systemic down-regulation of Mt4-like genes in roots.     

Fine characterization of OsPHO2 knockout mutants reveals its key role in Pi utilization in rice

Previous research using forward genetics approaches demonstrated that OsPHO2 regulates multiple phosphate-starvation responses in rice. In this work, we finely characterized two independent OsPHO2 knockout rice mutants under inorganic phosphate (Pi)-sufficient conditions. The ospho2 mutants exhibited defects in growth and reproductive development in the whole growing period. The cells in the elongation zone of ospho2 seedling roots were much shorter than those of the wild type. The phosphorus concentration in the blades of ospho2 mutants was 5.8-fold higher than those of wild-type plants, whereas it was only slightly higher in the sheaths, culms, spikelets, and seeds. Furthermore, Pi levels in the ospho2 mutants were highest in the oldest leaf and lowest in the youngest leaf, whereas there was no significant difference in the corresponding leaves of wild-type plants. These results suggest that ospho2 mutant phenotype results from a partial defect in Pi translocation and remobilization in the shoot of rice. This study thus provides evidence that OsPHO2, which functions at the downstream of OsPHF1, modulates Pi utilization by regulating the expression of Pht1 transporters in rice.     

OsZIP5 is a plasma membrane zinc transporter in rice

Zinc is essential for normal plant growth and development. To understand its transport in rice, we characterized OsZIP5, which is inducible under Zn deficiency. OsZIP5 complemented the growth defect of a yeast Zn-uptake mutant, indicating that OsZIP5 is a Zn transporter. The OsZIP5-GFP fusion protein was localized to the plasma membrane. Transgenic plants overexpressing the gene grew less well. Overexpression of the gene decreased the Zn concentration in shoots, but caused it to rise in the roots. Knockout plants showed no visible phenotypic changes under either normal or deficient conditions. However, they were tolerant to excess Zn and contained less Zn. In contrast, overexpressing transgenics were sensitive to excess Zn. These results indicate that OsZIP5 plays a role in Zn distribution within rice.     

The effect of phosphate nutrition of young rape plants on nitrate reductase activity and xylem exudation,and their relation to H ion efflux from the roots

Levels of nitrate reductase activity (N.R.A.) were measured in shoots and roots of P sufficient and P deficient rape plants and changes in N.R.A. examined in relation to the onset of H ion efflux from the roots. Rates of xylem exudation were measured and the sap analysed for nitrate, amino-N and phosphate content. The optimum concentration of phosphate in the leaves for N.R.A. was about 0.7%. Both high and low concentrations of phosphate within the leaves inhibited N.R.A in those leaves. This inhibition of N.R.A led to the accumulation of nitrate in the older parts of the shoots of P sufficient plants. Less accumulation of nitrate occurred in the P deficient plants since nitrate uptake by the plants decreased before any fall in N.R.A. Xylem exudation rates halved within 18 hours of depriving the plants of phosphate, and, since the composition of the sap remained constant, this indicated a reduced flux of nitrate into the xylem. The rate of xylem exudation continued to fall and by the end of the experiment was approximately one tenth of the rate in the P sufficient plants. The onset of H ion efflux from the terminal portions of the root preceded any effect on N.R.A by 2 days.     

Nitrate Assimilation Contributes to Ralstonia solanacearum Root Attachment,Stem Colonization,and Virulence

Ralstonia solanacearum, an economically important plant pathogen, must attach, grow, and produce virulence factors to colonize plant xylem vessels and cause disease. Little is known about the bacterial metabolism that drives these processes. Nitrate is present in both tomato xylem fluid and agricultural soils, and the bacterium''s gene expression profile suggests that it assimilates nitrate during pathogenesis. A nasA mutant, which lacks the gene encoding the catalytic subunit of R. solanacearum''s sole assimilatory nitrate reductase, did not grow on nitrate as a sole nitrogen source. This nasA mutant exhibited reduced virulence and delayed stem colonization after soil soak inoculation of tomato plants. The nasA virulence defect was more severe following a period of soil survival between hosts. Unexpectedly, once bacteria reached xylem tissue, nitrate assimilation was dispensable for growth, virulence, and competitive fitness. However, nasA-dependent nitrate assimilation was required for normal production of extracellular polysaccharide (EPS), a major virulence factor. Quantitative analyses revealed that EPS production was significantly influenced by nitrate assimilation when nitrate was not required for growth. The plant colonization delay of the nasA mutant was externally complemented by coinoculation with wild-type bacteria but not by coinoculation with an EPS-deficient epsB mutant. The nasA mutant and epsB mutant did not attach to tomato roots as well as wild-type strain UW551. However, adding either wild-type cells or cell-free EPS improved the root attachment of these mutants. These data collectively suggest that nitrate assimilation promotes R. solanacearum virulence by enhancing root attachment, the initial stage of infection, possibly by modulating EPS production.     

PHO1 expression in guard cells mediates the stomatal response to abscisic acid in Arabidopsis

Stomatal opening and closing are driven by ion fluxes that cause changes in guard cell turgor and volume. This process is, in turn, regulated by environmental and hormonal signals, including light and the phytohormone abscisic acid (ABA). Here, we present genetic evidence that expression of PHO1 in guard cells of Arabidopsis thaliana is required for full stomatal responses to ABA. PHO1 is involved in the export of phosphate into the root xylem vessels and, as a result, the pho1 mutant is characterized by low shoot phosphate levels. In leaves, PHO1 was found expressed in guard cells and up‐regulated following treatment with ABA. The pho1 mutant was unaffected in production of reactive oxygen species following ABA treatment, and in stomatal movements in response to light cues, high extracellular calcium, auxin, and fusicoccin. However, stomatal movements in response to ABA treatment were severely impaired, both in terms of induction of closure and inhibition of opening. Micro‐grafting a pho1 shoot scion onto wild‐type rootstock resulted in plants with normal shoot growth and phosphate content, but failed to restore normal stomatal response to ABA treatment. PHO1 knockdown using RNA interference specifically in guard cells of wild‐type plants caused a reduced stomatal response to ABA. In agreement, specific expression of PHO1 in guard cells of pho1 plants complemented the mutant guard cell phenotype and re‐established ABA sensitivity, although full functional complementation was dependent on shoot phosphate sufficiency. Together, these data reveal an important role for phosphate and the action of PHO1 in the stomatal response to ABA.     

pho3: a phosphorus-deficient mutant of Arabidopsis thaliana (L.) Heynh

A novel P-deficient mutant of Arabidopsis thaliana, pho3, was isolated by screening for root acid phosphatase (APase) activity in plants grown under low-P conditions. pho3 had 30% less APase activity in roots than the wild type and, in contrast to wild-type plants, root APase activity did not increase in response to growth in low P. However, shoot APase activity was higher in pho3 than in the wild-type plants. In addition, the pho3 mutant had a P-deficient phenotype, even when grown in P-sufficient conditions. The total P content of 11-d-old pho3 plants, grown in agar media with a plentiful supply of P, was about 25% lower than the wild-type level in the shoot, and about 65% lower in the roots. In the rosette leaves of mature soil-grown pho3 plants the total P content was again reduced, to about 50% of wild-type levels. pho3 exhibited a number of characteristics normally associated with low-P stress, including severely reduced growth, increased anthocyanin content (at least 100-fold greater than the wild type in soil-grown plants) and starch accumulation. The results suggest that the mutant is unable to respond to low internal P levels, and may lack a transporter or a signalling component involved in regulating P nutrition. Received: 21 March 2000 / Accepted: 15 August 2000     

Effects of wheat plant inoculation with cytokinin-producing microorganisms on plant growth at increasing level of mineral nutrition

Triticum durum Desf. plants were grown for 11 days in sand culture on nutrient solutions with optimum or lowered content of mineral nutrients. Thereafter, the level of mineral nutrition was increased in some portion of deficient plants. Two days before, plants were inoculated with cytokinin-producing microorganisms of the Bacillus genus. Nutrition deficiency resulted in a decrease in the rate of plant biomass accumulation, which was correlated with the level of active cytokinins in both roots and shoots. After improving the mineral nutrition of noninoculated plants, the rate of their biomass accumulation increased and, by the end of experiment, their shoot fresh weight was 1.5-fold higher than in deficient plants; however, it was still by 20% lower than in plants continuously growing at optimum mineral nutrition. Inoculation resulted in the considerable increase in the cytokinin content in shoots as compared with all other treatments. In this case, after the improvement of plant mineral nutrition, the rates of growth and relative biomass accumulation increased sharply; as a result, these plants had the highest dry and fresh weights. Thus, inoculation with cytokinin-producing bacteria was beneficial for plant growth after their transfer from deficient to sufficient mineral nutrition.     

PHO2-dependent degradation of PHO1 modulates phosphate homeostasis in Arabidopsis

The Arabidopsis thaliana pho2 mutant, which is defective in a ubiquitin-conjugating E2 enzyme, displays inorganic phosphate (Pi) toxicity as a result of enhanced uptake and root-to-shoot translocation of Pi. To elucidate downstream components of the PHO2-dependent regulatory pathway, we identified two pho2 suppressors as carrying missense mutations in PHO1, which has been implicated in Pi loading to the xylem. In support of the genetic interaction between PHO1 and PHO2, we found that the protein level of PHO1 is increased in pho2, whereas such accumulation is ameliorated in both pho2 suppressors. Results from cycloheximide and endosomal Cys protease inhibitor E-64d treatments further suggest that PHO1 degradation is PHO2 dependent and involves multivesicular body-mediated vacuolar proteolysis. Using the transient expression system of tobacco (Nicotiana tabacum) leaves, we demonstrated that PHO1 and PHO2 are partially colocalized and physically interact in the endomembranes, where the ubiquitin conjugase activity of PHO2 is required for PHO1 degradation. In addition, reduced PHO1 expression caused by PHO1 mutations impede Pi uptake, indicating a functional association between xylem loading and acquisition of Pi. Together, our findings uncover a pivotal molecular mechanism by which PHO2 modulates the degradation of PHO1 in the endomembranes to maintain Pi homeostasis in plants.     

Role of phytochrome B in organ formation processes in Cucumis sativus L.

The role of phytochrome B in the organogenesis process in the apical and axillary shoot meristems during early ontogenesis stages in cucumber Cucumis sativus L. at photoperiods (day/night) 10/14, 16/8 h, and continuous light in comparison with wild type plants and phytochrome B-deficient mutant (lh-mutant) was investigated. In mutant phytochrome B, deficiency caused inhibition of initiation of leaves both in the main shoot and lateral shoots and increased the number of flower buds (IV stage of organogenesis). With continuous light, the number of lateral shoots and flowers during stage IV of organogenesis in wild-type plants increased twofold in comparison with the mutant. Short-term temperature drops did not induce floral ontogenesis in mutants but increased the number of off-shoots in both experimental variants during a long photoperiod and continuous lighting. We propose that phytochrome B, by increasing the compactness of chromatin, may facilitate coordination of ontogenesis processes with changing environmental conditions.     

The role of photorespiration in redox and energy balance of photosynthetic plant cells: A study with a barley mutant deficient in glycine decarboxylase

Protoplasts and mitochondria were isolated from leaves of homozygous barley ( Hordeum vulgare L.) mutant deficient in glycine decarboxylase complex (GDC, EC 2.1.2.10) and wild-type plants. The photosynthetic rates of isolated protoplasts from the mutant and wild-type plants under saturating CO₂ were similar, but the respiratory rate of the mutant was two-fold higher. Respiration in the mutant plants was much more strongly inhibited by antimycin A than in wild-type plants and a low level of the alternative oxidase protein was found in mitochondria. The activities of NADP- and NAD-dependent malate dehydrogenases were also increased in mutant plants, suggesting an activation of the malate-oxaloacetate exchange for redox transfer between organelles. Mutant plants had elevated activities of NADH- and NADPH-dependent glyoxylate/hydroxypyruvate reductases, which may be involved in oxidizing excess NAD(P)H and the scavenging of glyoxylate. We estimated distribution of pools of adenylates, NAD(H) and NADP(H) between chloroplasts, cytosol and mitochondria. Under photorespiratory conditions, ATP/ADP and NADPH/NADP ratios in the mutant were higher in chloroplasts as compared to wild-type plants. The cytosolic NADH/NAD ratio was increased, whereas the ratio in mitochondria decreased. It is concluded that photorespiration serves as an effective redox transfer mechanism from the chloroplast. Plants with a lowered GDC content are deficient in this mechanism, which leads to over-reduction and over-energization of the chloroplasts.