The LaCEP1 peptide modulates cluster root morphology in Lupinus albus

White lupin cluster roots are specialized brush‐like root structures that are formed in some species under phosphorus (P)‐deficient conditions. They intensely secrete protons and organic acid anions for solubilization and acquisition of sparingly soluble phosphates. Phytohormones and sucrose modulate cluster root number, but the molecular mechanisms of cluster root formation have been elusive. Here, a novel peptide phytohormone was identified that affects cluster root development. It belongs to the C‐TERMINALLY-ENCODED PEPTIDE (CEP) family. Members of that family arrest root growth and modulate branching in model species. LaCEP1 was highly expressed in the pre‐emergence zone of clusters. Over‐expression of the gene encoding the LaCEP1 propeptide resulted in moderate inhibition of cluster root formation. The primary and lateral root lengths of lupin were little affected by the overexpression, but LaCEP1 reduced cluster rootlet and root hair elongation. Addition of a 15‐mer core peptide derived from LaCEP1 similarly altered root morphology and modified cluster activity, suggesting that a core sequence of the propeptide is functionally sufficient. Stable overexpression in Arabidopsis confirmed the LaCEP1 function in root growth inhibition across species. Taken together, the root inhibitory effects of the LaCEP1 phytohormone suggest a role as of a regulatory module involved in cluster root development in white lupin.     

Phosphorus acquisition from soil by white lupin (Lupinus albus L.) and soybean (Glycine max L.), species with contrasting root development

White lupin and soybean have contrasting root morphologies: white lupin develops proteoid or cluster roots, roots with discreet clusters of short, determinate branch roots (rootlets) while soybean develops a more fibrous root system with evenly distributed, longer branch roots. Growth and P acquisition by white lupin and soybean were compared in a soil high in bound, total P, with or without additional inorganic P applied in solution. Additional P increased biomass by 25% and doubled total P in soybean. In contrast, white lupin did not respond to additional P in biomass or total P. However added P decreased cluster development on proteoid roots indicating that white lupin sensed the added P. The reduction in cluster weight per plant was exactly countered by an increase in dry weight of other roots. Soybean root development responded to P application, proliferating branch roots with active meristems in the upper portion of the soil profile where P was applied, and reducing root weight to plant weight by 13%. White lupin did not proliferate roots in response to P application. When P was not added to soil, soybean and lupin acquired similar P per unit root dry weight. However, white lupin accumulated 4.8 times more P per unit root length, suggesting that P acquisition in these plants involved other mechanisms such as the exudation of P solubilizing compounds. Soybean accessed P by developing more root length thus colonising more soil volume than white lupin and, therefore, was better able to take advantage of the added P. Pericycle and root tip meristem activities were critical to the differences in root development between white lupin and soybean, and therefore their responses to plant and soil P.     

SIZ1 regulation of phosphate starvation-induced root architecture remodeling involves the control of auxin accumulation

Phosphate (Pi) limitation causes plants to modulate the architecture of their root systems to facilitate the acquisition of Pi. Previously, we reported that the Arabidopsis (Arabidopsis thaliana) SUMO E3 ligase SIZ1 regulates root architecture remodeling in response to Pi limitation; namely, the siz1 mutations cause the inhibition of primary root (PR) elongation and the promotion of lateral root (LR) formation. Here, we present evidence that SIZ1 is involved in the negative regulation of auxin patterning to modulate root system architecture in response to Pi starvation. The siz1 mutations caused greater PR growth inhibition and LR development of seedlings in response to Pi limitation. Similar root phenotypes occurred if Pi-deficient wild-type seedlings were supplemented with auxin. N-1-Naphthylphthalamic acid, an inhibitor of auxin efflux activity, reduced the Pi starvation-induced LR root formation of siz1 seedlings to a level equivalent to that seen in the wild type. Monitoring of the auxin-responsive reporter DR5::uidA indicated that auxin accumulates in PR tips at early stages of the Pi starvation response. Subsequently, DR5::uidA expression was observed in the LR primordia, which was associated with LR elongation. The time-sequential patterning of DR5::uidA expression occurred earlier in the roots of siz1 as compared with the wild type. In addition, microarray analysis revealed that several other auxin-responsive genes, including genes involved in cell wall loosening and biosynthesis, were up-regulated in siz1 relative to wild-type seedlings in response to Pi starvation. Together, these results suggest that SIZ1 negatively regulates Pi starvation-induced root architecture remodeling through the control of auxin patterning.     

ATP citrate lyase: cloning, heterologous expression and possible implication in root organic acid metabolism and excretion

In order to cope with phosphate deficiency, white lupin produces bottle‐brushed like roots, so‐called cluster or proteoid roots which are specialized in malate and citrate excretion. Young, developing cluster roots mainly excrete malate whereas mature cluster roots mainly release citrate. Mature proteoid roots excrete four to six times more carboxylates compared with juvenile proteoid roots. Using a cDNA‐amplified restriction fragment length polymorphism (AFLP) approach we identified a gene coding for a putative ATP‐citrate lyase (ACL) up‐regulated in young cluster roots. Cloning of the lupin ACL revealed that plant ACL is constituted by two polypeptides (ACLA and ACLB) encoded by two different genes. This contrasts with the animal ACL, constituted of one polypeptide which covers ACLA and ACLB. The ACL function of the two lupin gene products has been demonstrated by heterologous expression in yeast. Both subunits are required for ACL activity. In lupin cluster roots, our results suggest that ACL activity could be responsible for the switch between malate and citrate excretion in the different developmental stages of cluster roots. In primary roots of lupin and maize, ACL activity was positively correlated with malate exudation. These results show that ACL is implicated in root exudation of organic acids and hence plays a novel role in addition to lipid synthesis. Our results suggest that in addition to lipid biosynthesis, in plants, ACL is implicated in malate excretion.     

Physiological adaptations to phosphorus deficiency during proteoid root development in white lupin

Release of large amounts of citric acid from specialized root clusters (proteoid roots) of phosphorus (P)-deficient white lupin (Lupinus albus L.) is an efficient strategy for chemical mobilization of sparingly available P sources in the rhizosphere. The present study demonstrates that increased accumulation and exudation of citric acid and a concomitant release of protons were predominantly restricted to mature root clusters in the later stages of P deficiency. Inhibition of citrate exudation by exogenous application of anion-channel blockers such as ethacrynic- and anthracene-9-carboxylic acids may indicate involvement of an anion channel. Phosphorus-deficiency-induced accumulation and subsequent exudation of citric acid seem to be a consequence of both increased biosynthesis and reduced metabolization of citric acid in the proteoid root tissue, indicated by increased in-vitro activity and enzyme protein levels of phosphoenolpyruvate carboxylase (EC 4.1.1.31), and reduced activity of aconitase (EC 4.2.1.3) and root respiration. Similar to citric acid, acid phosphatase, which is secreted by roots and involved in the mobilization of the organic soil P fraction, was released predominantly from proteoid roots of P-deficient plants. Also ³³Pi uptake per unit root fresh-weight was increased by approximately 50% in juvenile and mature proteoid root clusters compared to apical segments of non-proteoid roots. Kinetic studies revealed a K _m of 30.7 μM for Pi uptake of non-proteoid root apices in P-sufficient plants, versus K _m values of 8.5–8.6 μM for non-proteoid and juvenile proteoid roots under P-deficient conditions, suggesting the induction of a high-affinity Pi-uptake system. Obviously, P-deficiency-induced adaptations of white lupin, involved in P acquisition and mobilization of sparingly available P sources, are predominantly confined to proteoid roots, and moreover to distinct stages during proteoid root development. Received: 10 September 1998 / Accepted: 22 December 1998     

Transport and metabolism of glycerophosphodiesters produced through phospholipid deacylation

Phospholipid deacylation results in the formation of glycerophosphodiesters and free fatty acids. In Saccharomyces cerevisiae, four gene products with phospholipase B (deacylating) activity have been characterized (PLB1, PLB2, PLB3, NTE1), and those activities account for most, if not all, of the glycerophosphodiester production observed to date. The glycerophosphodiesters themselves are hydrolyzed into glycerol-3-phosphate and the corresponding alcohol by glycerophosphodiester phosphodiesterases. Although only one glycerophosphodiester phosphodiesterase-encoding gene (GDE1) has been characterized in S. cerevisiae, others certainly exist. Both internal and external glycerophosphodiesters (primarily glycerophosphocholine and glycerophosphoinositol) are formed as a result of phospholipid turnover in S. cerevisiae. A permease encoded by the GIT1 gene imports extracellular glycerophosphodiesters across the plasma membrane, where their hydrolytic products can provide crucial nutrients such as inositol, choline, and phosphate to the cell. The importance of this metabolic pathway in various aspects of S. cerevisiae cell physiology is being explored.     

Isoflavonoid exudation from white lupin roots is influenced by phosphate supply, root type and cluster-root stage

The internal concentration of isoflavonoids in white lupin (Lupinus albus) cluster roots and the exudation of isoflavonoids by these roots were investigated with respect to the effects of phosphorus (P) supply, root type and cluster-root developmental stage.To identify and quantify the major isoflavonoids exuded by white lupin roots, we used high-pressure liquid chromatography (HPLC) coupled to electrospray ionization (ESI) in mass spectrometry (MS).The major exuded isoflavonoids were identified as genistein and hydroxygenistein and their corresponding mono- and diglucoside conjugates. Exudation of isoflavonoids during the incubation period used was higher in P-deficient than in P-sufficient plants and higher in cluster roots than in noncluster roots. The peak of exudation occurred in juvenile and immature cluster roots, while exudation decreased in mature cluster roots.Cluster-root exudation activity was characterized by a burst of isoflavonoids at the stage preceding the peak of organic acid exudation. The potential involvement of ATP-citrate lyase in controlling citrate and isoflavonoid exudation is discussed, as well as the possible impact of phenolics in repelling rhizosphere microbial citrate consumers.     

The effect of nitrogen nutrition on cluster root formation and proton extrusion by Lupinus albus

Nitrogen nutrition can influence cluster root formation in many wild species, but the effect of N form on cluster root formation and root exudation by white lupin is not known. In a solution culture study, we examined the effect of N nutrition (ammonium, nitrate, both or N2 fixation) on cluster root formation and H+ extrusion by white lupin plants under deficient and adequate P supply. The number of cluster roots increased greatly when plants were supplied with I microM P compared with 50 microM P, the increase being 7.8-fold for plants treated with (NH4)2SO4, 3-fold for plants treated with KNO3 and NH4NO3, and 2-4-fold for N2-fixing plants. Under P deficiency. NH4+-N supply resulted in production of a greater number and biomass of cluster roots than other N sources. Dry weight of cluster roots was 30 % higher than that of non-cluster roots in P-deficient plants treated with (NH4)2SO4 and NH4NO3. In plants treated with sufficient P (50 microM), the weight of non-cluster roots was approx. 90 % greater than that of cluster roots. Both total (micromol per plant h(-1)) and specific (micromol g(-1) root d. wt h(-1)) H+ extrusions were greatest from roots of plants supplied with (NH4)2SO4, followed by those supplied with NH4NO3 and N2 fixation, whereas plants receiving KNO3 had negative net H+ extrusion between the third and fifth week of growth (indicating uptake of protons or release of OH- ions). The rate of proton extrusion by NH4+-N-fed plants was similar under P-deficient and P-sufficient conditions. In contrast, proton exudation by N2-fixing plants and KNO3-treated plants was ten-fold greater under P deficiency than under P sufficiency. In comparison with P deficiency, plants treated with 50 microM P had a significantly higher concentration of P in roots, shoots and youngest expanded leaves (YEL). Compared with the N2 fixation and KNO3 treatments, total N concentration was highest in roots, shoots and YEL of plants supplied with (NH4)2SO4 and NH4NO3, regardless of P supply. Under P deficiency, K concentrations in roots decreased at all N supplies, especially in plants treated with (NH4)2SO4 and NH4NO3, which coincided with the greatest H+ extrusion at these P and N supplies. In conclusion, NH4-N nutrition stimulated cluster root formation and H+ extrusion by roots of P-deficient white lupin.     

Different effects of phospholipase Dζ2 and non‐specific phospholipase C4 on lipid remodeling and root hair growth in Arabidopsis response to phosphate deficiency

Phosphate (Pi) deficiency in soils is a major limiting factor for plant growth. In response to Pi deprivation, one prominent metabolic adaptation in plants is the decrease in membrane phospholipids that consume approximately one‐third cellular Pi. The level of two phospholipid‐hydrolyzing enzymes, phospholipase Dζ2 (PLDζ2) and non‐specific phospholipase C4 (NPC4), is highly induced in Pi‐deprived Arabidopsis. To determine the role of PLDζ2 and NPC4 in plant growth under Pi limitation, Arabidopsis plants deficient in both PLDζ2 and NPC4 (npc4pldζ2) were generated and characterized. Lipid remodeling in leaves and roots was analyzed at three different durations of Pi deficiency. NPC4 affected lipid changes mainly in roots at an early stage of Pi deprivation, whereas PLDζ2 exhibited a more overt effect on lipid remodeling in leaves at a later stage of Pi deprivation. Pi deficiency‐induced galactolipid increase and phospholipid decrease were impeded in pldζ2 and npc4pldζ2 plants. In addition, seedlings of npc4pldζ2 had the same root hair density as pldζ2 but shorter root hair length than pldζ2 in response to Pi deficiency. The loss of NPC4 decreased root hair length but had no effect on root hair density. These data suggest that PLDζ2 and NPC4 mediate the Pi deprivation‐induced lipid remodeling in a tissue‐ and time‐specific manner. PLDζ2 and NPC4 have distinctively different roles in root hair growth and development in response to Pi deprivation; PLDζ2 negatively modulates root hair density and length, whereas NPC4 promotes root hair elongation.     

Analysis of physiological and miRNA responses to Pi deficiency in alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis> L.)

Key message

The induction of miR399 and miR398 and the inhibition of miR156, miR159, miR160, miR171, miR2111, and miR2643 were observed under Pi deficiency in alfalfa. The miRNA-mediated genes involved in basic metabolic process, root and shoot development, stress response and Pi uptake.

Abstract

Inorganic phosphate (Pi) deficiency is known to be a limiting factor in plant development and growth. However, the underlying miRNAs associated with the Pi deficiency-responsive mechanism in alfalfa are unclear. To elucidate the molecular mechanism at the miRNA level, we constructed four small RNA (sRNA) libraries from the roots and shoots of alfalfa grown under normal or Pi-deficient conditions. In the present study, alfalfa plants showed reductions in biomass, photosynthesis, and Pi content and increases in their root-to-shoot ratio and citric, malic, and succinic acid contents under Pi limitation. Sequencing results identified 47 and 44 differentially expressed miRNAs in the roots and shoots, respectively. Furthermore, 909 potential target genes were predicted, and some targets were validated by RLM-RACE assays. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed prominent enrichment in signal transducer activity, binding and basic metabolic pathways for carbohydrates, fatty acids and amino acids; cellular response to hormone stimulus and response to auxin pathways were also enriched. qPCR results verified that the differentially expressed miRNA profile was consistent with sequencing results, and putative target genes exhibited opposite expression patterns. In this study, the miRNAs associated with the response to Pi limitation in alfalfa were identified. In addition, there was an enrichment of miRNA-targeted genes involved in biological regulatory processes such as basic metabolic pathways, root and shoot development, stress response, Pi transportation and citric acid secretion.

     

Double knockouts of phospholipases Dzeta1 and Dzeta2 in Arabidopsis affect root elongation during phosphate-limited growth but do not affect root hair patterning

Root elongation and root hair formation are important in nutrient absorption. We found that two Arabidopsis (Arabidopsis thaliana) phospholipase Ds (PLDs), PLDzeta1 and PLDzeta2, were involved in root elongation during phosphate limitation. PLDzeta1 and PLDzeta2 are structurally different from the majority of plant PLDs by having phox and pleckstrin homology domains. Both PLDzetas were expressed more in roots than in other tissues. It was reported previously that inducible suppression or inducible overexpression of PLDzeta1 affected root hair patterning. However, gene knockouts of PLDzeta1, PLDzeta2, or the double knockout of PLDzeta1 and PLDzeta2 showed no effect on root hair formation. The expression of PLDzetas increased in response to phosphate limitation. The elongation of primary roots in PLDzeta1 and PLDzeta2 double knockout mutants was slower than that of wild type and single knockout mutants. The loss of PLDzeta2, but not PLDzeta1, led to a decreased accumulation of phosphatidic acid in roots under phosphate-limited conditions. These results indicate that PLDzeta1 and PLDzeta2 play a role in regulating root development in response to nutrient limitation.     

Transgenic proteoid roots of white lupin: a vehicle for characterizing and silencing root genes involved in adaptation to P stress

White lupin (Lupinus albus L.) has become an illuminating model for the study of plant adaptation to phosphorus (P) deficiency. It adapts to -P stress with a highly coordinated modification of root development and biochemistry resulting in short, densely clustered secondary roots called proteoid (or cluster) roots. In order to characterize genes involved in proteoid root formation and function in a homologous system, we have developed an Agrobacterium rhizogenes-based transformation system for white lupin roots that allows rapid analysis of reporter genes as well as RNA interference (RNA(i))-based gene silencing. We used this system to characterize a lupin multidrug and toxin efflux (Lupinus albus MULTIDRUG AND TOXIN EFFLUX, LaMATE) gene previously shown to have enhanced expression under -P stress. Here, we show that LaMATE had high expression in proteoid roots not only under -P, but also under -Fe, -N, -Mn and +Al stress. A portion containing the putative LaMATE promoter was fused to GUS and enhanced green fluorescence protein (EGFP) reporter genes, and a translational LaMATE::EGFP fusion was constructed under control of the LaMATE promoter. The LaMATE promoter directed P-dependent GUS and EGFP expression to proteoid roots. Confocal microscopy in white lupin and Arabidopsis point to the plasma membrane as the likely location of the LaMATE protein. LaMATE displayed homology to FRD3 in Arabidopsis, but did not complement an Arabidopsis ferric reductase defective 3 (FRD3) mutant. RNA(i)-based gene silencing was shown to effectively reduce LaMATE expression in transformed white lupin roots. LaMATE RNAi-silenced plants displayed an about 20% reduction in dry weight.     

Role of phosphorus nutrition in development of cluster roots and release of carboxylates in soil-grown Lupinus albus

The present study examined the effect of phosphorus (P) limitation on cluster root formation and exudation of carboxylates by N₂-fixing white lupin (Lupinus albus L. cv. Kiev) grown in a P-deficient sandy soil. Plants received 10 (limited P) or 200 g P g^–1 soil as FePO₄ (adequate P) and were grown in a phytotron at 20/12 °C (12/12 h) for 76 days in soil columns. Cluster root formation was assessed and root exudates were collected at 9-day intervals. Shoot and root dry weights were higher in plants grown in the adequate-P compared to the limited-P treatment for 67 days. No clear difference in the total root length was observed between two P treatments before day 58. However, the specific root length increased rapidly from 17 m g^–1 DW at day 40 to 28 m g^–1 at day 49 in the P-limited plants, but decreased in the P-adequate plants. The effect of P limitation on enhancement of cluster root formation was observed from day 40 and reached the maximum at day 58. The number of cluster roots was negatively correlated with the P concentration in both roots and shoots. Phosphorus limitation increased exudation of citrate from day 40. The exudation of citrate displayed a cyclic pattern throughout the experiment, and appeared related to internal P concentration in plants, particularly P concentration in shoots. The sorption of exogenously added citrate in the soil was also examined. The amount of extractable citrate remained unchanged for 2 h, but decreased thereafter, suggesting that the soil had a low capacity to sorb citrate, and the rate of its decomposition by microorganisms was slow. Collecting solution leached through a soil column is a simple and reliable method to acquire root exudates from white lupin grown in soil. The results suggest that formation of cluster roots and exudation of citrate in white lupin are regulated by P concentration in shoots.     

White lupin has developed a complex strategy to limit microbial degradation of secreted citrate required for phosphate acquisition

White lupins (Lupinus albus L.) respond to phosphate deficiency by producing special root structures called cluster roots. These cluster roots secrete large amounts of carboxylates into the rhizosphere, mostly citrate and malate, which act as phosphate solubilizers and enable the plant to grow in soils with sparingly available phosphate. The success and efficiency of such a P-acquisition strategy strongly depends on the persistence and stability of the carboxylates in the soil, a parameter that is influenced to a large extent by biodegradation through rhizosphere bacteria and fungi. In this study, we show that white lupin roots use several mechanisms to reduce microbial growth. The abundance of bacteria associated with cluster roots was decreased at the mature state of the cluster roots, where a burst of organic acid excretion and a drastic pH decrease is observed. Excretion of phenolic compounds, mainly isoflavonoids, induced fungal sporulation, indicating that vegetative growth, and thus potential citrate consumption, is reduced. In addition, the activity of two antifungal cell wall-degrading enzymes, chitinase and glucanase, were highest at the stage preceding the citrate excretion. Therefore, our results suggest that white lupin has developed a complex strategy to reduce microbial degradation of the phosphate-solubilizing agents.     

Burkholderia species are major inhabitants of white lupin cluster roots

The formation of cluster roots by plants represents a highly efficient strategy for acquisition of sparingly available phosphate. This particular root type is characterized by a densely branched structure and high exudation of organic acids and protons, which are likely to influence the resident bacterial community. Until now, the identity of the bacterial populations living in cluster roots has not been investigated. We applied cultivation-dependent and cultivation-independent methods to characterize the dominant bacterial genera inhabiting the growing cluster roots of white lupin. We observed a high relative abundance of Burkholderia species (up to 58% of all isolated strains and 44% of all retrieved 16S rRNA sequences) and a significant enrichment with increasing cluster root age. Most of the sequences retrieved clustered together with known plant- or fungus-associated Burkholderia species, while only one of 98 sequences was affiliated with the Burkholderia cepacia complex. In vitro assays revealed that Burkholderia strains were much more tolerant to low pH than non-Burkholderia strains. Moreover, many strains produced large amounts of siderophores and were able to utilize citrate and oxalate as carbon sources. These features seem to represent important traits for the successful colonization and maintenance of Burkholderia species in white lupin cluster roots.