MicroRNAs in the axon locally mediate the effects of chondroitin sulfate proteoglycans and cGMP on axonal growth

Axonal miRNAs locally regulate axonal growth by modulating local protein composition. Whether localized miRNAs in the axon mediate the inhibitory effect of Chondroitin sulfate proteoglycans (CSPGs) on the axon remains unknown. We showed that in cultured cortical neurons, axonal application of CSPGs inhibited axonal growth and altered axonal miRNA profiles, whereas elevation of axonal cyclic guanosine monophosphate (cGMP) levels by axonal application of sildenafil reversed the effect of CSPGs on inhibition of axonal growth and on miRNA profiles. Specifically, CSPGs elevated and reduced axonal levels of miR‐29c and integrin β1 (ITGB1) proteins, respectively, while elevation of cGMP levels overcame these CSPG effects. Gain‐of‐ and loss‐of‐function experiments demonstrated that miR‐29c in the distal axon mediates axonal growth downstream of CSPGs and cGMP by regulating axonal protein levels of ITGB1, FAK, and RhoA. Together, our data demonstrate that axonal miRNAs play an important role in mediating the inhibitory action of CSPGs on axonal growth and that miR‐29c at least partially mediates this process. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1402–1419, 2015     

Calcium/calmodulin-dependent protein kinase II regulates mammalian axon growth by affecting F-actin length in growth cone

While axon regeneration is a key determinant of functional recovery of the nervous system after injury, it is often poor in the mature nervous system. Influx of extracellular calcium (Ca²⁺) is one of the first phenomena that occur following axonal injury, and calcium/calmodulin-dependent protein kinase II (CaMKII), a target substrate for calcium ions, regulates the status of cytoskeletal proteins such as F-actin. Herein, we found that peripheral axotomy activates CaMKII in dorsal root ganglion (DRG) sensory neurons, and inhibition of CaMKII impairs axon outgrowth in both the peripheral and central nervous systems (PNS and CNS, respectively). Most importantly, we also found that the activation of CaMKII promotes PNS and CNS axon growth, and regulatory effects of CaMKII on axon growth occur via affecting the length of the F-actin. Thus, we believe our findings provide clear evidence that CaMKII is a critical modulator of mammalian axon regeneration.     

Exosomes derived from smooth muscle cells ameliorate diabetes‐induced erectile dysfunction by inhibiting fibrosis and modulating the NO/cGMP pathway

Erectile dysfunction (ED) is a major health issue among men with diabetes, and ED induced by diabetes mellitus (DMED) is particularly difficult to treat. Therefore, novel therapeutic approaches for the treatment of DMED are urgently needed. Exosomes, nanosized particles involved in many physiological and pathological processes, may become a promising tool for DMED treatment. In this study, we investigated the therapeutic effect of exosomes derived from corpus cavernosum smooth muscle cells (CCSMC‐EXOs) on erectile function in a rat model of diabetes and compared their effect with that of exosomes derived from mesenchymal stem cells (MSC‐EXOs). We incubated labelled CCSMC‐EXOs and MSC‐EXOs with CCSMCs and then observed uptake of the exosomes at different time points using laser confocal microscopy. CCSMC‐EXOs were more easily taken up by CCSMCs. The peak concentration and retention time of labelled CCSMC‐EXOs and MSC‐EXOs in the corpus cavernosum of DMED rats after intracavernous injection were compared by in vivo imaging techniques. Intracavernous injection of CCSMC‐EXOs was associated with a relatively high peak concentration and long retention time. Our data showed that CCSMC‐EXOs could improve erectile function in DMED rats. Meanwhile, CCSMC‐EXOs could exert antifibrotic effects by increasing the smooth muscle content and reducing collagen deposition. CCSMC‐EXOs also increased the expression of eNOS and nNOS, followed by increased levels of NO and cGMP. These findings initially identify the possible role of CCSMC‐EXOs in ameliorating DMED through inhibiting corporal fibrosis and modulating the NO/cGMP signalling pathway, providing a theoretical basis for a breakthrough in the treatment of DMED.     

Neuronal NADPH oxidase 2 regulates growth cone guidance downstream of slit2/robo2

NADPH oxidases (Nox) are membrane‐bound multi‐subunit protein complexes producing reactive oxygen species (ROS) that regulate many cellular processes. Emerging evidence suggests that Nox‐derived ROS also control neuronal development and axonal outgrowth. However, whether Nox act downstream of receptors for axonal growth and guidance cues is presently unknown. To answer this question, we cultured retinal ganglion cells (RGCs) derived from zebrafish embryos and exposed these neurons to netrin‐1, slit2, and brain‐derived neurotrophic factor (BDNF). To test the role of Nox in cue‐mediated growth and guidance, we either pharmacologically inhibited Nox or investigated neurons from mutant fish that are deficient in Nox2. We found that slit2‐mediated growth cone collapse, and axonal retraction were eliminated by Nox inhibition. Though we did not see an effect of either BDNF or netrin‐1 on growth rates, growth in the presence of netrin‐1 was reduced by Nox inhibition. Furthermore, attractive and repulsive growth cone turning in response to gradients of BDNF, netrin‐1, and slit2, respectively, were eliminated when Nox was inhibited in vitro. ROS biosensor imaging showed that slit2 treatment increased growth cone hydrogen peroxide levels via mechanisms involving Nox2 activation. We also investigated the possible relationship between Nox2 and slit2/Robo2 signaling in vivo. astray/nox2 double heterozygote larvae exhibited decreased area of tectal innervation as compared to individual heterozygotes, suggesting both Nox2 and Robo2 are required for establishment of retinotectal connections. Our results provide evidence that Nox2 acts downstream of slit2/Robo2 by mediating growth and guidance of developing zebrafish RGC neurons.     

Nt-RhoGDI2 regulates Rac/Rop signaling and polar cell growth in tobacco pollen tubes

Rac/Rop-type Rho-family small GTPases accumulate at the plasma membrane in the tip of pollen tubes and control the polar growth of these cells. Nt-RhoGDI2, a homolog of guanine nucleotide dissociation inhibitors (GDIs) regulating Rho signaling in animals and yeast, is co-expressed with the Rac/Rop GTPase Nt-Rac5 specifically in tobacco (Nicotiana tabacum) pollen tubes. The two proteins interact with each other in yeast two-hybrid assays, preferentially when Nt-Rac5 is prenylated. Transient over-expression of Nt-Rac5 and Nt-RhoGDI2 depolarized or inhibited tobacco pollen tube growth, respectively. Interestingly, pollen tubes over-expressing both proteins grew normally, demonstrating that the two proteins functionally interact in vivo. Nt-RhoGDI2 was localized to the pollen tube cytoplasm and effectively transferred co-over-expressed YFP-Nt-Rac5 fusion proteins from the plasma membrane to this compartment. A single amino acid exchange (R69A), which abolished binding to Nt-RhoGDI2, caused Nt-Rac5 to be mis-localized to the flanks of pollen tubes and strongly compromised its ability to depolarize pollen tube growth upon over-expression. Based on these observations, we propose that Nt-RhoGDI2-mediated recycling of Nt-Rac5 from the flanks of the tip to the apex has an essential function in the maintenance of polarized Rac/Rop signaling and cell expansion in pollen tubes. Similar mechanisms may generally play a role in the polarized accumulation of Rho GTPases in specific membrane domains, an important process whose regulation has not been well characterized in any cell type to date.     

Regulation of synaptic vesicle accumulation and axon terminal remodeling during synapse formation by distinct Ca2+ signaling

The synaptic vesicle accumulation and subsequent morphological remodeling of axon terminals are characteristic features of presynaptic differentiation of zebrafish olfactory sensory neurons. The synaptic vesicle accumulation and axon terminal remodeling are regulated by protein kinase A and calcineurin signaling, respectively. To investigate upstream signals of presynaptic differentiation, we focused on Ca²⁺ signaling as Ca²⁺/calmodulin is required for the activation of both calcineurin and some adenylyl cyclases. We here showed that application of Ca²⁺/calmodulin inhibitor or olfactory sensory neuron-specific expression of calmodulin inhibitory peptide suppressed both synaptic vesicle accumulation and axon terminal remodeling. Thus, the trigger of presynaptic differentiation could be Ca²⁺ release from intracellular stores or Ca²⁺ influx. Application of a phospholipase C inhibitor or olfactory sensory neuron-specific expression of inositol 1,4,5-trisphosphate (IP₃) 5-phosphatase suppressed synaptic vesicle accumulation, but not morphological remodeling. In contrast, application of a voltage-gated Ca²⁺ channel blocker or expression of Kir2.1 inward rectifying potassium channel prevented the morphological remodeling. We also provided evidence that IP₃ signaling acted upstream of protein kinase A signaling. Our results suggest that IP₃-mediated Ca²⁺/calmodulin signaling stimulates synaptic vesicle accumulation and subsequent neuronal activity-dependent Ca²⁺/calmodulin signaling induces the morphological remodeling of axon terminals.     

Modeling squid axon Na+ channel by a nucleation and growth kinetic mechanism

A kinetic model accounting for all salient features of the Na⁺ channel of the squid giant axon is provided. The model furnishes explanations for the Cole-Moore-like effect, the rising phase of the ON gating current and the slow ‘intermediate component’ of its decaying phase, as well as the gating charge immobilization. Experimental ON ionic currents are semi-quantitatively simulated by the use of only three free parameters, upon assuming that the Na⁺ channel opening proceeds along with the stepwise aggregation of its four domains, while they are moving their gating charge outward under depolarizing conditions. The inactivation phase of the ON ionic current is interpreted by a progressive electrostatic attraction between the positively charged ‘hinged lid’ containing the hydrophobic IFM triad and its receptor inside the channel pore, as the stepwise outward movement of the S4 segments of the Na⁺ channel progressively increases the negative charge attracting the triad to its receptor. The Na⁺ channel closing is assumed to proceed by repolarization-induced disaggregation of its domains, accompanied by inward movement of their gating charge. The phenomenon of ‘gating charge immobilization’ can be explained by assuming that gradual structural changes of the receptor over the time course of depolarization strengthen the interaction between the IFM triad and its receptor, causing a slow release of the gating charge during the subsequent repolarization.     

Dual-frequency ultrasound enhances functional neuron differentiation from neural stem cells by modulating Ca2+ dynamics and the ERK1/2 signaling pathway

Our previous study demonstrated that ultrasound is able to promote differentiation on neural stem cells (NSCs), and dual-frequency ultrasound promotes this effect due to enhanced acoustic cavitation compared with single-frequency ultrasound. However, the underlying biological reasons have not been well disclosed. The purpose of this study was to investigate the underlying bioeffects, mechanisms and signaling pathways of dual-frequency ultrasound on NSC differentiation. The morphology, neurite outgrowth, and differentiation percentages were investigated under various dual-frequency simulation parameters with exposure periods varying from 5 to 15 min. Morphological observations identified that dual-frequency ultrasound stimulation promoted ultrasound dose-dependent neurite outgrowth. In particular, cells exposed for 10 min/2 days showed optimal neurite outgrowth and neuron differentiation percentages. In addition, live cell calcium images showed that dual-frequency ultrasound enhanced the internal calcium content of the cells, and calcium ions entering cells from the extracellular environment could be observed. Dual frequency ultrasound exposure enhanced extracellular calcium influx and upregulated extracellular signal-regulated kinases 1/2 (ERK1/2) expression. Observations from immunostaining and protein expression examinations also identified that dual-frequency ultrasound promoted brain-derived neurotrophic factor (BDNF) secretion from astrocytes derived from NSCs. In summary, evidence supports that dual-frequency ultrasound effectively enhances functional neuron differentiation via calcium channel regulation via the downstream ERK1/2 pathway and promotes BDNF secretion to serve as feedback to cascade neuron differentiation. The results may provide an alternative for cell-based therapy in brain injury.     

Profiling of anti-fibrotic signaling by hepatocyte growth factor in renal fibroblasts

Hepatocyte growth factor (HGF) is a multifunctional growth factor affecting cell proliferation and differentiation. Due to its mitogenic potential, HGF plays an important role in tubular repair and regeneration after acute renal injury. However, recent reports have shown that HGF also acts as an anti-inflammatory and anti-fibrotic factor, affecting various cell types such as renal fibroblasts and triggering tubulointerstitial fibrosis of the kidney.The present study provides evidence that HGF stimulation of renal fibroblasts results in the activation of both the Erk1/2 and the Akt pathways. As previously shown, Erk1/2 phosphorylation results in Smad-linker phosphorylation, thereby antagonizing cellular signals induced by TGFβ. By siRNA mediated silencing of the Erk1/2-Smad linkage, however, we now demonstrate that Akt signaling acts as an auxiliary pathway responsible for the anti-fibrotic effects of HGF. In order to define the anti-fibrotic function of HGF we performed comprehensive expression profiling of HGF-stimulated renal fibroblasts by microarray hybridization. Functional cluster analyses and quantitative PCR assays indicate that the HGF-stimulated pathways transfer the anti-fibrotic effects in renal interstitial fibroblasts by reducing expression of extracellular matrix proteins, various chemokines, and members of the CCN family.     

DAF‐2/insulin IGF‐1 receptor regulates motility during aging by integrating opposite signaling from muscle and neuronal tissues

During aging, preservation of locomotion is generally considered an indicator of sustained good health, in elderlies and in animal models. In Caenorhabditis elegans, mutants of the insulin‐IGF‐1 receptor DAF2/IIRc represent a paradigm of healthy aging, as their increased lifespan is accompanied by a delay in age‐related loss of motility. Here, we investigated the DAF‐2/IIRc‐dependent relationship between longevity and motility using an auxin‐inducible degron to trigger tissue‐specific degradation of endogenous DAF‐2/IIRc. As previously reported, inactivation of DAF‐2/IIRc in neurons or intestine was sufficient to extend the lifespan of worms, whereas depletion in epidermis, germline, or muscle was not. However, neither intestinal nor neuronal depletion of DAF‐2/IIRc prevented the age‐related loss of motility. In 1‐day‐old adults, DAF‐2/IIRc depletion in neurons reduced motility in a DAF‐16/FOXO dependent manner, while muscle depletion had no effect. By contrast, DAF‐2 depletion in the muscle of middle‐age animals improved their motility independently of DAF‐16/FOXO but required UNC‐120/SRF. Yet, neuronal or muscle DAF‐2/IIRc depletion both preserved the mitochondria network in aging muscle. Overall, these results show that the motility pattern of daf‐2 mutants is determined by the sequential and opposing impact of neurons and muscle tissues and can be dissociated from the regulation of the lifespan. This work also provides the characterization of a versatile tool to analyze the tissue‐specific contribution of insulin‐like signaling in integrated phenotypes at the whole organism level.     

Paracrine regulation of growth factor signaling by shed leucine-rich repeats and immunoglobulin-like domains 1

Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is a recently discovered negative regulator of growth factor signaling. The LRIG1 integral membrane protein has been demonstrated to regulate various oncogenic receptor tyrosine kinases, including epidermal growth factor (EGF) receptor (EGFR), by cell-autonomous mechanisms. Here, we investigated whether LRIG1 ectodomains were shed, and if LRIG1 could regulate cell proliferation and EGF signaling in a paracrine manner. Cells constitutively shed LRIG1 ectodomains in vitro, and shedding was modulated by known regulators of metalloproteases, including the ADAM17 specific inhibitor TAPI-2. Furthermore, shedding was enhanced by ectopic expression of Adam17. LRIG1 ectodomains appeared to be shed in vivo, as well, as demonstrated by immunoblotting of mouse and human tissue lysates. Ectopic expression of LRIG1 in lymphocytes suppressed EGF signaling in co-cultured fibroblastoid cells, demonstrating that shed LRIG1 ectodomains can function in a paracrine fashion. Purified LRIG1 ectodomains suppressed EGF signaling without any apparent downregulation of EGFR levels. Taken together, the results show that the LRIG1 ectodomain can be proteolytically shed and can function as a non-cell-autonomous regulator of growth factor signaling. Thus, LRIG1 or its ectodomain could have therapeutic potential in the treatment of growth factor receptor-dependent cancers.     

Functional receptor for platelet-derived growth factor in rat embryonic heart-derived myocytes: role of sequestered Ca2+ stores in receptor signaling and antagonism by arginine vasopressin

Platelet-derived growth factor (PDGF) is established to function importantly in the growth, development, and function of most cardiovascular tissues. However, evidence that the factor participates directly in the growth and development of the mammalian myocardium is lacking. H9c2 rat embryonic ventricular myocytes were found to respond to PDGF-BB with a rapid mobilization of cell-associated Ca2+ and increased rates of protein synthesis, followed by markedly increased rates of DNA synthesis. PDGF acted as a full mitogen for these myocytes. Evidence is provided that documents the expression of classical PDGF-beta, but not PDGF-alpha, receptors in H9c2 cells. Scatchard analysis revealed the presence of 44,000 beta-receptors per myocyte. Cell shortening and clustering of plasmalemmal beta-receptors occurred within 30 min of exposure to PDGF-BB. Treatment was also associated with a transient increase in the rate of synthesis of GRP78/BiP, consistent with a transitory release of Ca2+ from the sarcoplasmic/endoplasmic reticulum [S(E)R]. Increased rates of protein synthesis at early times of PDGF treatment were additive with those occurring in response to arginine vasopressin, indicating different mechanisms of translational upregulation by these agents. The mitogenic effects of PDGF were delayed by vasopressin, which causes H9c2 myocytes to undergo hypertrophy while promoting the persistent depletion of S(E)R Ca2+ stores. In the presence of PDGF, vasopressin did not induce hypertrophy. As compared to untreated myocytes, DNA synthesis in PDGF-treated myocytes was optimized at lower extracellular Ca2+ concentrations and was significantly less sensitive to inhibition by ionomycin. H9c2 cells appear to provide a useful embryonic cardiomyocyte model in which to examine both PDGF-activated proliferative and vasopressin-activated hypertrophic events and the importance of transient vs. sustained Ca2+ release in these events.     

Epidermal growth factor- and stress-induced loss of gap junctional communication is mediated by ERK-1/ERK-2 but not ERK-5 in rat liver epithelial cells

Extracellular signal-regulated kinases (ERK) 1 and 2 as well as ERK-5 were previously suggested to phosphorylate connexin-43 and to contribute to the modulation of gap junctional intercellular communication (GJC). Exposure of rat liver epithelial cells to epidermal growth factor (EGF) or the redox cycling and alkylating agent menadione resulted in phosphorylation of connexin-43 and loss in GJC, both of which were abrogated by pharmacological inhibitors of ERK-1/2 activation, if used in concentrations that selectively abrogate phosphorylation of ERK-1/2 but not of ERK-5. Thus, EGF- or menadione-induced loss of GJC is mediated by ERK-1/2 but not ERK-5 in rat liver epithelial cells.     

Suppression of proliferation and migration in highly-metastatic lung cancer cells as well as tumor growth by a new synthesized compound TBrC and its molecular mechanisms of action

To develop new anticancer agents has been considered as a useful and necessary strategy to suppress highly-metastatic lung cancer, the leading cause of cancer-related deaths in the world. In this study, we synthesized a new compound ethyl 6-bromocoumarin-3-carboxylyl L-theanine (TBrC) and studied the anticancer activity of TBrC and its molecular mechanisms of action. Our results show that TBrC remarkably inhibits the proliferation and migration in highly-metastatic lung cancer cells by inducing apoptosis and cell cycle arrest as well as regulating related protein expressions. Further study indicated that TBrC not only enhances the protein levels of Bax, cytosolic cytochrome c, caspase-3 and PARP-1 but also reduces the protein expressions of Bcl-2, cyclin D1, VEGFR1 and NF-κB as well as inhibits the phosphorylation and expressions of VEGFR2 and Akt in the cancer cells. More importantly, TBrC displays strong suppression of highly-metastatic tumor growth and reduces the tumor weight by 61.6 % in tumor-bearing mice without toxicity to the mice. Our results suggest that TBrC suppresses the proliferation and migration of lung cancer cells via VEGFR-Akt-NF-κB signaling pathways; TBrC may have a wide therapeutic and/or adjuvant therapeutic application in the treatment of lung cancer.     

Epac1‐induced cellular proliferation in prostate cancer cells is mediated by B‐Raf/ERK and mTOR signaling cascades

cAMP‐dependent, PKA‐independent effects on cell proliferation are mediated by cAMP binding to EPAC and activation of Rap signaling. In this report, we employed the analogue 8‐CPT‐2‐O‐Me‐cAMP to study binding to EPAC and subsequent activation of B‐Raf/ERK and mTOR signaling in human cancer cells. This compound significantly stimulated DNA synthesis, protein synthesis, and cellular proliferation of human 1‐LN prostate cancer cells. By study of phosphorylation‐dependent activation, we demonstrate that EPAC‐mediated cellular effects require activation of the B‐Raf/ERK and mTOR signaling cascades. RNAi directed against EPAC gene expression as well as inhibitors of ERK, PI 3‐kinase, and mTOR were employed to further demonstrate the role of these pathways in regulating prostate cancer cell proliferation. These studies were then extended to several other human prostate cancer cell lines and melanoma cells with comparable results. We conclude that B‐Raf/ERK and mTOR signaling play an essential role in cAMP‐dependent, but PKA‐independent, proliferation of cancer cells. J. Cell. Biochem. 108: 998–1011, 2009. © 2009 Wiley‐Liss, Inc.     

Exposure to novel environment is characterized by an interaction of D1/NMDA receptors underlined by phosphorylation of the NMDA and AMPA receptor subunits and activation of ERK1/2 signaling, leading to epigenetic changes and gene expression in rat hippocampus

Interactions between dopamine and glutamate receptors are essential for prefrontal cortical (PFC) and hippocampal cognitive functions. The hippocampus has been identified as a detector of a novel stimulus, where an association between incoming information and stored memories takes place. Further to our previous results which showed a strong synergistic interaction of dopamine D1 and glutamate NMDA receptors, the present study is going to investigate the functional status of that interaction in rats, following their exposure to a novel environment. Our results showed that the “spatial” novelty induced in rat hippocampus and PFC (a) a significant increase in phosphorylation of NMDA and AMPA receptor subunits, as well as a robust phosphorylation/activation of ERK1/2 signaling, which are both dependent on the concomitant stimulation of D1/NMDA receptors and are both abolished by habituation procedure, (b) chromatin remodeling events (phosphorylation-acetylation of histone H3) and (c) an increase in the immediate early genes (IEGs) c-Fos and zif-268 expression in the CA1 region of hippocampus, which is dependent on the co-activation of D1/NMDA and acetylcholine muscarinic receptors. In conclusion, our results clearly show that a strong synergistic interaction of D1/NMDA receptor is required for the novelty-induced phosphorylation of NMDA and AMPA receptor subunits and for the robust activation of ERK1/2 signaling, leading to chromatin remodeling events and the expression of the IEGs c-Fos and zif-268, which are involved in the regulation of synaptic plasticity and memory consolidation.     

Carbon dioxide diffusion across stomata and mesophyll and photo-biochemical processes as affected by growth CO2 and phosphorus nutrition in cotton

Nutrients such as phosphorus may exert a major control over plant response to rising atmospheric carbon dioxide concentration (CO₂), which is projected to double by the end of the 21st century. Elevated CO₂ may overcome the diffusional limitations to photosynthesis posed by stomata and mesophyll and alter the photo-biochemical limitations resulting from phosphorus deficiency. To evaluate these ideas, cotton (Gossypium hirsutum) was grown in controlled environment growth chambers with three levels of phosphate (Pi) supply (0.2, 0.05 and 0.01 mM) and two levels of CO₂ concentration (ambient 400 and elevated 800 μmol mol⁻¹) under optimum temperature and irrigation. Phosphate deficiency drastically inhibited photosynthetic characteristics and decreased cotton growth for both CO₂ treatments. Under Pi stress, an apparent limitation to the photosynthetic potential was evident by CO₂ diffusion through stomata and mesophyll, impairment of photosystem functioning and inhibition of biochemical process including the carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase/oxyganase and the rate of ribulose-1,5-bisphosphate regeneration. The diffusional limitation posed by mesophyll was up to 58% greater than the limitation due to stomatal conductance (g_s) under Pi stress. As expected, elevated CO₂ reduced these diffusional limitations to photosynthesis across Pi levels; however, it failed to reduce the photo-biochemical limitations to photosynthesis in phosphorus deficient plants. Acclimation/down regulation of photosynthetic capacity was evident under elevated CO₂ across Pi treatments. Despite a decrease in phosphorus, nitrogen and chlorophyll concentrations in leaf tissue and reduced stomatal conductance at elevated CO₂, the rate of photosynthesis per unit leaf area when measured at the growth CO₂ concentration tended to be higher for all except the lowest Pi treatment. Nevertheless, plant biomass increased at elevated CO₂ across Pi nutrition with taller plants, increased leaf number and larger leaf area.