RCB-mediated chlorophagy caused by oversupply of nitrogen suppresses phosphate-starvation stress in plants

Inorganic phosphate (Pi) and nitrogen (N) are essential nutrients for plant growth. We found that a five-fold oversupply of nitrate rescues Arabidopsis (Arabidopsis thaliana) plants from Pi-starvation stress. Analyses of transgenic plants that overexpressed GFP-AUTOPHAGY8 showed that an oversupply of nitrate induced autophagy flux under Pi-depleted conditions. Expression of DIN6 and DIN10, the carbon (C) starvation-responsive genes, was upregulated when nitrate was oversupplied under Pi starvation, which suggested that the plants recognized the oversupply of nitrate as C starvation stress because of the reduction in the C/N ratio. Indeed, formation of Rubisco-containing bodies (RCBs), which contain chloroplast stroma and are induced by C starvation, was enhanced when nitrate was oversupplied under Pi starvation. Moreover, autophagy-deficient mutants did not release Pi (unlike wild-type plants), exhibited no RCB accumulation inside vacuoles, and were hypersensitive to Pi starvation, indicating that RCB-mediated chlorophagy is involved in Pi starvation tolerance. Thus, our results showed that the Arabidopsis response to Pi starvation is closely linked with N and C availability and that autophagy is a key factor that controls plant growth under Pi starvation.

Disturbance of the carbon/nitrogen ratio induces partial chloroplast degradation via autophagy under phosphate starvation and rescues phosphate starvation stress.     

Phosphate Starvation Inducible Metabolism in Lycopersicon esculentum: I. Excretion of Acid Phosphatase by Tomato Plants and Suspension-Cultured Cells

Both tomato (Lycopersicon esculentum cv VF 36) plants and suspension cultured cells show phosphate starvation inducible (psi) excretion of acid phosphatase (Apase). Apase excretion in vitro was proportional to the level of exogenous orthophosphate (Pi). Intracellular Apase activity remained the same in both Pi-starved and sufficient cells, while Apase excreted by the starved cells increased by as much as six times over unstressed control cells on a dry weight basis. At peak induction, 50% of total Apase was excreted. Ten day old tomato seedlings grown without Pi showed slight growth reduction versus unstressed control plants. The Pi-depleted roots showed psi enhancement of Apase activity. Severely starved seedlings (17 days) reached only one-third of the biomass of unstressed control plants but, because of a combination of psi Apase excretion by roots and a shift in biomass to this organ, they excreted 5.5 times the Apase activity of the unstressed control. Observed psi Apase excretion may be part of a phosphate starvation rescue system in plants. The utility of the visible indicator dye 5-bromo-4-chloro-3-indolyl-phosphate-p-toluidine as a phenotypic marker for plant Apase excretion is demonstrated.     

Non-growing Escherichia coli cells starved for glucose or phosphate use different mechanisms to survive oxidative stress

Recent data suggest that superoxide dismutases are important in preventing lethal oxidative damage of proteins in Escherichia coli cells incubated under aerobic, carbon starvation conditions. Here, we show that the alkylhydroperoxide reductase AhpCF (AHP) is specifically required to protect cells incubated under aerobic, phosphate (Pi) starvation conditions. Additional loss of the HP-I (KatG) hydroperoxidase activity dramatically accelerated the death rate of AHP-deficient cells. Investigation of the composition of spent culture media indicates that DeltaahpCF katG cells leak nutrients, which suggests that membrane lipids are the principal target of peroxides produced in Pi-starved cells. In fact, the introduction of various mutations inactivating repair activities revealed no obvious role for protein or DNA lesions in the viability of ahp cells. Because the death of ahp cells was directly related to ongoing aerobic glucose metabolism, we wondered how glycolysis, which requires free Pi, could proceed. 31P nuclear magnetic resonance spectra showed that Pi-starved cells consumed Pi but were apparently able to liberate Pi from phosphorylated products, notably through the synthesis of UDP-glucose. Whereas expression of the ahpCF and katG genes is enhanced in an OxyR-dependent manner in response to H2O2 challenge, we found that the inactivation of oxyR and both oxyR and rpoS genes had little effect on the viability of Pi-starved cells. In stark contrast, the inactivation of both oxyR and rpoS genes dramatically decreased the viability of glucose-starved cells.     

Protein as Chemical Cue: Non-Nutritional Growth Enhancement by Exogenous Protein in Pseudomonas putida KT2440

Research pertaining to microbe-microbe and microbe-plant interactions has been largely limited to small molecules like quorum sensing chemicals. However, a few recent reports have indicated the role of complex molecules like proteins and polysaccharides in microbial communication. Here we demonstrate that exogenous proteins present in culture media can considerably accelerate the growth of Pseudomonas putida KT2440, even when such proteins are not internalized by the cells. The growth enhancement is observed when the exogenous protein is not used as a source of carbon or nitrogen. The data show non-specific nature of the protein inducing growth; growth enhancement was observed irrespective of the protein type. It is shown that growth enhancement is mediated via increased siderophore secretion in response to the exogenous protein, leading to better iron uptake. We highlight the ecological significance of the observation and hypothesize that exogenous proteins serve as chemical cues in the case of P.putida and are perceived as indicator of the presence of competitors in the environment. It is argued that enhanced siderophore secretion in response to exogenous protein helps P.putida establish numerical superiority over competitors by way of enhanced iron assimilation and quicker utilization of aromatic substrates.     

OsARF16, a transcription factor,is required for auxin and phosphate starvation response in rice (Oryza sativa L.)

Plant responses to auxin and phosphate (Pi) starvation are closely linked. However, the underlying mechanisms connecting auxin to phosphate starvation (?Pi) responses are largely unclear. Here, we show that OsARF16, an auxin response factor, functions in both auxin and ?Pi responses in rice (Oryza sativa L.). The knockout of OsARF16 led to primary roots (PR), lateral roots (LR) and root hair losing sensitivity to auxin and ?Pi response. OsARF16 expression and OsARF16::GUS staining in PR and LR of rice Nipponbare (NIP) were induced by indole acetic acid and ?Pi treatments. In ?Pi conditions, the shoot biomass of osarf16 was slightly reduced, and neither root growth nor iron content was induced, indicating that the knockout of OsARF16 led to loss of response to Pi deficiency in rice. Six phosphate starvation‐induced genes (PSIs) were less induced by ?Pi in osarf16 and these trends were similar to a knockdown mutant of OsPHR2 or AtPHR1, which was a key regulator under ?Pi. These data first reveal the biological function of OsARF16, provide novel evidence of a linkage between auxin and ?Pi responses and facilitate the development of new strategies for the efficient utilization of Pi in rice.     

Strigolactone Regulates Anthocyanin Accumulation,Acid Phosphatases Production and Plant Growth under Low Phosphate Condition in Arabidopsis

Phosphate is an essential macronutrient in plant growth and development; however, the concentration of inorganic phosphate (Pi) in soil is often suboptimal for crop performance. Accordingly, plants have developed physiological strategies to adapt to low Pi availability. Here, we report that typical Pi starvation responses in Arabidopsis are partially dependent on the strigolactone (SL) signaling pathway. SL treatment induced root hair elongation, anthocyanin accumulation, activation of acid phosphatase, and reduced plant weight, which are characteristic responses to phosphate starvation. Furthermore, the expression profile of SL-response genes correlated with the expression of genes induced by Pi starvation. These results suggest a potential overlap between SL signaling and Pi starvation signaling pathways in plants.     

PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 is a plant-specific SEC12-related protein that enables the endoplasmic reticulum exit of a high-affinity phosphate transporter in Arabidopsis

PHOSPHATE TRANSPORTER1 (PHT1) genes encode phosphate (Pi) transporters that play a fundamental role in Pi acquisition and remobilization in plants. Mutation of the Arabidopsis thaliana PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 (PHF1) impairs Pi transport, resulting in the constitutive expression of many Pi starvation-induced genes, increased arsenate resistance, and reduced Pi accumulation. PHF1 expression was detected in all tissues, particularly in roots, flowers, and senescing leaves, and was induced by Pi starvation, thus mimicking the expression patterns of the whole PHT1 gene family. PHF1 was localized in endoplasmic reticulum (ER), and mutation of PHF1 resulted in ER retention and reduced accumulation of the plasma membrane PHT1;1 transporter. By contrast, the PIP2A plasma membrane protein was not mislocalized, and the secretion of Pi starvation-induced RNases was not affected in the mutant. PHF1 encodes a plant-specific protein structurally related to the SEC12 proteins of the early secretory pathway. However, PHF1 lacks most of the conserved residues in SEC12 proteins essential as guanine nucleotide exchange factors. Although it functions in early secretory trafficking, PHF1 likely evolved a novel mechanism accompanying functional specialization on Pi transporters. The identification of PHF1 reveals that plants are also endowed with accessory proteins specific for selected plasma membrane proteins, allowing their exit from the ER, and that these ER exit cofactors may have a phylum-specific origin.     

Proteomic analysis of Arabidopsis thaliana ecotypes with contrasted root architecture in response to phosphate deficiency

Owing to a weak availability in soil, plants have developed numerous morphological, physiological and biochemical adaptations to acquire phosphate (Pi). Identification and characterisation of key genes involved in the initial steps of Pi-signalling might provide clues about the regulation of the complex Pi deficiency adaptation mechanism. A two-dimensional gel electrophoresis approach was performed to investigate proteome responses to Pi starvation in Arabidopsis. Two ecotypes were selected according to contrasting responses of their root system architecture to low availability of Pi. Thirty protein spots were shown to be affected by Pi deficiency. Fourteen proteins appeared to be up-regulated and ten down-regulated with ecotype Be-0, wheras only thirteen proteins were observed as down-regulated for ecotype Ll-0. Furthermore, systematic and opposite responses to Pi deficiency were observed between the two ecotypes. The sequences of these 30 differentially expressed protein spots were identified using mass spectrometry, and most of the proteins were involved in oxidative stress, carbohydrate and proteins metabolism. The results suggested that the modulation of alcohol dehydrogenase, malic enzyme and aconitate hydratase may contribute to the contrasted adaptation strategy to Pi deficiency of Be-0 and Ll-0 ecotypes. A focus on aconitate hydratase highlighted a complex reverse response of the pattern of corresponding spots between the two ecotypes. This protein, also potentially involved in iron homeostasis, was speculated to contribute, at least indirectly, to the root architecture response of these ecotypes.     

Inhibition of phosphate uptake in corn roots by aluminum-fluoride complexes

F forms stable complexes with Al at conditions found in the soil. Fluoroaluminate complexes (AlF(x)) have been widely described as effective analogs of inorganic phosphate (Pi) in Pi-binding sites of several proteins. In this work, we explored the possibility that the phytotoxicity of AlF(x) reflects their activity as Pi analogs. For this purpose, (32)P-labeled phosphate uptake by excised roots and plasma membrane H(+)-ATPase activity were investigated in an Al-tolerant variety of maize (Zea mays L. var. dwarf hybrid), either treated or not with AlF(x). In vitro, AlF(x) competitively inhibited the rate of root phosphate uptake as well as the H(+)-ATPase activity. Conversely, pretreatment of seedlings with AlF(x) in vivo promoted no effect on the H(+)-ATPase activity, whereas a biphasic effect on Pi uptake by roots was observed. Although the initial rate of phosphate uptake by roots was inhibited by AlF(x) pretreatment, this situation changed over the following minutes as the rate of uptake increased and a pronounced stimulation in subsequent (32)Pi uptake was observed. This kinetic behavior suggests a reversible and competitive inhibition of the phosphate transporter by fluoroaluminates. The stimulation of root (32)Pi uptake induced by AlF(x) pretreatment was tentatively interpreted as a phosphate starvation response. This report places AlF(3) and AlF(4)(-) among Al-phytotoxic species and suggests a mechanism of action where the accumulation of Pi-mimicking fluoroaluminates in the soil may affect the phosphate absorption by plants. The biochemical, physiological, and environmental significance of these findings is discussed.     

Optimization of medium composition for improving biomass production of Lactobacillus plantarum Pi06 using the Taguchi array design and the Box-Behnken method

An immune-enhancing strain, Lactobacillus plantarum Pi06, isolated from a healthy infant was used for biomass production following optimization of the medium in shake-flask culture. Preliminary studies showed that commercial MRS medium and cultivation under static conditions generated higher biomass production than four other tested media with or without a shaking condition. The selected medium composition, consisting of glucose, yeast extract, soy peptone, ammonium citrate, and corn steep liquor, was further optimized using a systematic method that integrated the Taguchi array design and the Box-Behnken method. The response effects of these factors were first investigated using Taguchi design under an L ₁₆ (4⁵) array. The suggested medium composition, derived from Statistica 7.1 using the Taguchi design, was applied to cultivate cells and a biomass of 7.16 g dry cell weight (DCW)/L was obtained. Response surface methodology based on the Box-Behnken method for the three response variables of glucose, yeast extract, and corn steep liquor was then used to further increase the biomass level to 8.94 g DCW/L. The resulting optimum medium consisted of 35 g/L glucose, 35 g/L yeast extract, and 40 mL/L corn steep liquor. Compared with the initial medium, the biomass yield was improved from 4.31 to 8.94 g DCW/L, an enhancement of approximately 107%.