A comparison between water uptake and root length density in winter wheat: effects of root density and rhizosphere properties.

BACKGROUND AND AIMS: We aim to quantify the variation in root distribution in a set of 35 experimental wheat lines. We also compared the effect of variation in hydraulic properties of the rhizosphere on water uptake by roots. METHODS: We measured the root length density and soil drying in 35 wheat lines in a field experiment. A 3D numerical model was used to predict soil drying profiles with the different root length distributions and compared with measured soil drying. The model was used to test different scenarios of the hydraulic properties of the rhizosphere. RESULTS: We showed that wheat lines with no detectable differences in root length density can induce soil drying profiles with statistically significant differences. Our data confirmed that a root length density of at least 1 cm/cm3 is needed to drain all the available water in soil. In surface layers where the root length density was far greater than 1 cm/cm3 water uptake was independent of rooting density due to competition for water. However, in deeper layers where root length density was less than 1 cm/cm3, water uptake by roots was proportional to root density. CONCLUSION: In a set of wheat lines with no detectable differences in the root length density we found significant differences in water uptake. This may be because small differences in root density at depth can result in larger differences in water uptake or that the hydraulic properties of the rhizosphere can greatly affect water uptake.

Molybdate toxicity in Chinese cabbage is not the direct consequence of changes in sulphur metabolism.

In polluted areas, plants may be exposed to supra-optimal levels of the micronutrient molybdenum. The physiological basis of molybdenum phytotoxicity is poorly understood. Plants take up molybdenum as molybdate, which is a structural analogue of sulphate. Therefore, it is presumed that elevated molybdate concentrations may hamper the uptake and subsequent metabolism of sulphate, which may induce sulphur deficiency. In the current research, Chinese cabbage (Brassica pekinensis) seedlings were exposed to 50, 100, 150 and 200 µm Na2 MoO4 for 9 days. Leaf chlorosis and a decreased plant growth occurred at concentrations ≥100 µm. Root growth was more affected than shoot growth. At ≥100 µm Na2 MoO4 , the sulphate uptake rate and capacity were increased, although only when expressed on a root fresh weight basis. When expressed on a whole plant fresh weight basis, which corrects for the impact of molybdate on the shoot-to-root ratio, the sulphate uptake rate and capacity remained unaffected. Molybdate concentrations ≥100 µm altered the mineral nutrient composition of plant tissues, although the levels of sulphur metabolites (sulphate, water-soluble non-protein thiols and total sulphur) were not altered. Moreover, the levels of nitrogen metabolites (nitrate, amino acids, proteins and total nitrogen), which are generally strongly affected by sulphate deprivation, were not affected. The root water-soluble non-protein thiol content was increased, and the tissue nitrate levels decreased, only at 200 µm Na2 MoO4 . Evidently, molybdenum toxicity in Chinese cabbage was not due to the direct interference of molybdate with the uptake and subsequent metabolism of sulphate.

Multiple abiotic stress, nitrate availability and the growth of wheat.

In the field, wheat experiences a combination of physical and nutrient stresses. There has been a tendency to study root impedance and water stress in separation and less is known about how they might interact. In this study, we investigated the effect of root impedance on the growth of three wheat varieties (Cadenza, Xi19 and Battalion) at different levels of nitrate availability, from 0-20 mM nitrate, in sand culture. This model system allows soil strength to be increased while maintaining adequate water availability. In a separate pot experiment, we grew the same wheat varieties in a loamy sand where soil was allowed to dry sufficiently to both reduce water potential and increase root impedance. This pot experiment also had a range of nitrate availabilities 0-20 mM nitrate. Once the seedlings were established we limited water supply to apply a matric potential of approximately -200 kPa to the roots. Soil drying increased the penetrometer resistance from approximately 300 kPa to more than 1 MPa. There were differences between the two experimental systems; growth was smaller in the soil-based experiment compared to the sand culture. However, the effects of the experimental treatment, root impedance or water withholding, relative to the control were comparable. Our data confirmed that leaf elongation in Cadenza (carrying the tall Rht allele) was the most sensitive to root impedance. Leaf stunting occurred irrespective of nitrate availability. Leaf elongation in the Xi19 and Battalion (carrying the semi-dwarf Rht allele) was less sensitive to root impedance and drought than Candenza. We suggest that the critical stress in a pot experiment where the soil was allowed to dry to approximately -200 kPa was root impedance and not water availability.

Identification and expression profiling of Pht1 phosphate transporters in wheat in controlled environments and in the field.

Phosphorus (P) is an important macronutrient with critical functions in plants. Phosphate (Pi) transporters, which mediate Pi acquisition and Pi translocation within the plant, are key factors in Pi deficiency responses. However, their relevance for adaptation to long-term Pi limitation under agronomic conditions, particularly in wheat, remains unknown. Here, we describe the identification of the complete Pi transporter gene family (Pht1) in wheat (Triticum aestivum). Gene expression profiles were compared for hydroponic and field-grown plant tissues of wheat at multiple development stages. Cis-element analysis of selected Pht1 promoter regions was performed. A broad range of expression patterns of individual TaPht1 genes was observed in relation to tissue specificity and the nutrient supply in the soil or in liquid culture, as well as an influence of the experimental system. The expression patterns indicate the involvement of specific transporters in Pi uptake, and in Pi transport and remobilisation within the plant, at different growth developmental stages. Specifically, the influence of Pi nutrition indicates a complex regulatory pattern of TaPht1 gene transcript abundances as a response to low Pi availability in different culture systems, correlating with the existence of different cis-acting promoter elements.

Methods to estimate changes in soil water for phenotyping root activity in the field.

BACKGROUND AND AIMS: There is an urgent need to develop new high throughput approaches to phenotype roots in the field. Excavating roots to make direct measurements is labour intensive. An alternative to excavation is to measure soil drying profiles and to infer root activity. METHODS: We grew 23 lines of wheat in 2013, 2014 and 2015. In each year we estimated soil water profiles with electrical resistance tomography (ERT), electromagnetic inductance (EMI), penetrometer measurements and measurements of soil water content. We determined the relationships between the measured variable and soil water content and matric potential. RESULTS: We found that ERT and penetrometer measurements were closely related to soil matric potential and produced the best discrimination between wheat lines. We found genotypic differences in depth of water uptake in soil water profiles and in the extent of surface drying. CONCLUSIONS: Penetrometer measurements can provide a reliable approach to comparing soil drying profiles by different wheat lines, and genotypic rankings are repeatable across years. EMI, which is more sensitive to soil water content than matric potential, and is less effective in drier soils than the penetrometer or ERT, nevertheless can be used to rapidly screen large populations for differences in root activity.

Temperature determines size and direction of effects of elevated CO2 and nitrogen form on yield quantity and quality of Chinese cabbage.

Rising atmospheric CO2 concentrations (e[CO2 ]) are presumed to have a significant impact on plant growth and yield and also on mineral nutrient composition, and therefore, on nutritional quality of crops and vegetables. To assess the relevance of these effects in future agroecosystems it is important to understand how e[CO2 ] interacts with other environmental factors. In the present study, we examined the interactive effects of e[CO2 ] with temperature and the form in which nitrogen is supplied (nitrate or ammonium nitrate) on growth, amino acid content and mineral nutrient composition of Chinese cabbage (Brassica pekinensis Rupr.), a crop characterised by its high nutritional value and increasing relevance for human nutrition in many developing countries. Higher temperature, ammonium nitrate and e[CO2 ] had a positive impact on net photosynthesis and growth. A stimulating effect of e[CO2 ] on growth was only observed if the temperature was high (21/18 °C, day/night), and an interaction of e[CO2 ] with N form was only observed if the temperature was ambient (15/12 °C, day/night). Mineral nutrient composition was affected in a complex manner by all three factors and their interaction. These results demonstrate how much the effect of e[CO2 ] on mineral quality of crops depends on other environmental factors. Changes in temperature, adapting N fertilisation and the oxidation state of N have the potential to counteract the mineral depletion caused by e[CO2 ].

Overexpression of a NAC transcription factor delays leaf senescence and increases grain nitrogen concentration in wheat.

Increasing the duration of leaf photosynthesis during grain filling using slow-senescing functional stay-green phenotypes is a possible route for increasing grain yields in wheat (Triticum aestivum L.). However, delayed senescence may negatively affect nutrient remobilisation and hence reduce grain protein concentrations and grain quality. A novel NAC1-type transcription factor (hereafter TaNAC-S) was identified in wheat, with gene expression located primarily in leaf/sheath tissues, which decreased during post-anthesis leaf senescence. Expression of TaNAC-S in the second leaf correlated with delayed senescence in two doubled-haploid lines of an Avalon × Cadenza population (lines 112 and 181), which were distinct for leaf senescence. Transgenic wheat plants overexpressing TaNAC-S resulted in delayed leaf senescence (stay-green phenotype). Grain yield, aboveground biomass, harvest index and total grain N content were unaffected, but NAC over-expressing lines had higher grain N concentrations at similar grain yields compared to non-transgenic controls. These results indicate that TaNAC-S is a negative regulator of leaf senescence, and that delayed leaf senescence may lead not only to increased grain yields but also to increased grain protein concentrations.

Effects of genotype, season, and nitrogen nutrition on gene expression and protein accumulation in wheat grain.

Six commercial U.K. cultivars of winter wheat selected to represent different abilities to partition nitrogen into grain protein were grown in replicated field trials at five different sites over three seasons. The proportion of LMW glutenin subunits decreased and the proportion of gliadins increased during grain development and in response to N application. Differences were observed between the proportions of LMW glutenin subunits and gliadins in low- and high-protein grain, these two fractions being decreased and increased, respectively. There was little effect of grain protein content on the proportions of either the HMW glutenin subunits or large glutenin polymers, which are enriched in these subunits, with the latter increasing during development in all cultivars. The proportion of total protein present in polymers in the mature grain decreased with increasing N level. Correlations were also observed between the abundances of gliadin protein transcripts and the corresponding proteins.

Copper toxicity in Chinese cabbage is not influenced by plant sulphur status, but affects sulphur metabolism-related gene expression and the suggested regulatory metabolites.

The toxicity of high copper (Cu) concentrations in the root environment of Chinese cabbage (Brassica pekinensis) was little influenced by the sulphur nutritional status of the plant. However, Cu toxicity removed the correlation between sulphur metabolism-related gene expression and the suggested regulatory metabolites. At high tissue Cu levels, there was no relation between sulphur metabolite levels viz. total sulphur, sulphate and water-soluble non-protein thiols, and the expression and activity of sulphate transporters and expression of APS reductase under sulphate-sufficient or-deprived conditions, in the presence or absence of H2 S. This indicated that the regulatory signal transduction pathway of sulphate transporters was overruled or by-passed upon exposure to elevated Cu concentrations.

Remobilization of leaf S compounds and senescence in response to restricted sulphate supply during the vegetative stage of oilseed rape are affected by mineral N availability.

The impact of sulphur limitation on the remobilization of endogenous S compounds during the rosette stage of oilseed rape, and the interactions with N availability on these processes, were examined using a long-term (34)SO(4)(2-) labelling method combined with a study of leaf senescence progression (using SAG12/Cab as a molecular indicator) and gene expression of the transporters, BnSultr4;1 and BnSultr4;2, involved in vacuolar sulphate efflux. After 51 d on hydroponic culture at 0.3 mM (34)SO(4)(2-) (1 atom% excess), the labelling was stopped and plants were subject for 28 d to High S-High N (HS-HN, control), Low S-High N (LS-HN) or Low S-Low N (LS-LN) conditions. Compared with the control, LS-HN plants showed delayed leaf senescence and, whilst the shoot growth and the foliar soluble protein amounts were not affected, S, (34)S, and SO(4)(2-) amounts in the old leaves declined rapidly and were associated with the up-regulation of BnSultr4;1. In LS-LN plants, shoot growth was reduced, leaf senescence was accelerated, and the rapid S mobilization in old leaves was accompanied by decreased (34)S and SO(4)(2-), higher protein mobilization, and up-regulation of BnSultr4;2, but without any change of expression of BnSultr4;1. The data suggest that to sustain the S demand for growth under S restriction (i) vacuolar SO(4)(2-) is specifically remobilized in LS-HN conditions without any acceleration of leaf senescence, (ii) SO(4)(2-) mobilization is related to an up-regulation of BnSultr4;1 and/or BnSultr4;2 expression, and (iii) the relationship between sulphate mobilization and up-regulation of expression of BnSultr4 genes is specifically dependent on the N availability.

Leaf developmental stage affects sulfate depletion and specific sulfate transporter expression during sulfur deprivation in Brassica napus L.

BRASSICA NAPUS was grown under hydroponic conditions and responses to the removal of the external supply of sulfur (S) were analysed in roots and in leaves of different developmental age. The concentrations of sulfate and nitrate were greatest in the older leaves and least in younger leaves, whilst phosphate was greatest in roots and youngest leaves and least in old leaves. S-deprivation resulted in decreases in tissue sulfate concentrations at variable rates in the order: roots and young leaves > middle-aged leaves > oldest leaves. Phosphate concentrations were unaffected and nitrate concentrations were only depleted in the oldest leaves. Expression of representative members of the sulfate transporter gene family was assessed by Northern blotting in the respective tissues. Group 1 transporters (high affinity type) were induced in response to S-deprivation in all tissues except old leaves, where no expression was detected, and to the greatest extent in roots. Groups 2 and 5 (a BRASSICA Group 5 sulfate transporter is reported here, accession number: AJ311389) transporters showed either no or only a small induction by S-deprivation. Group 4 transporters (localised in the tonoplast membrane and thought to be involved in vacuolar sulfate efflux) were induced by S-deprivation with a complex pattern: 4;1 was expressed in root and mature leaves, was strongly induced by sulfur-deprivation in roots, and was also induced in the middle-aged leaves alone; 4;2 was only expressed under S-deprivation in parallel with the observed pattern of tissue sulfate concentrations. Expression patterns indicated that both differences in intracellular sulfate pools and localised aspects of the signal transduction pathway link tissue sulfate-status and sulfur-nutrition regulated gene expression.

The characteristic high sulfate content in Brassica oleracea is controlled by the expression and activity of sulfate transporters.

The uptake and distribution of sulfate in BRASSICA OLERACEA, a species characterised by its high sulfate content in root and shoot, are coordinated and adjusted to the sulfur requirement for growth, even at external sulfate concentrations close to the K (m) value of the high-affinity sulfate transporters. Plants were able to grow normally and maintain a high sulfur content when grown at 5 or 10 microM sulfate in the root environment. Abundance of mRNAs for the high affinity sulfate transporters, BolSultr1;1 and BolSultr1;2, were enhanced at

Coordinated expression of sulfate uptake and components of the sulfate assimilatory pathway in maize.

A high-affinity-type sulfate transporter (Group 1: ZmST1;1, Accession No. AF355602) has been cloned from maize seedlings by RT-PCR. Tissue and cell specific localisation of this sulfate transporter has been determined along the developmental gradient of the root and in leaves of different ages. In S-sufficient conditions there was uniform low expression of ZmST1;1 in the root and very low expression in the leaves. Increased mRNA abundance and sulfate influx capacity indicated that S-starvation increased ZmST1;1 expression in roots, especially at the top of the root (just behind the seed, the area possessing most laterals and root hairs) compared to the root tip. Similarly a group 2, probable low affinity-type sulfate transporter, ZmST2;1, and also ATP-sulfurylase and APS-reductase but not OAS(thiol)lyase were induced by S-starvation and showed highest expression in the upper section of the root. S-starvation increased root/shoot ratio by 20 % and increased root lateral length and abundance in the region closest to the root tip. As the increase in root proliferation was not as great as the increase in mRNA pools, it was clear that there was a higher cellular abundance of the mRNAs for sulfate transporters, ATP-sulfurylase, and APS-reductase in response to sulfur starvation. In the leaves, the sulfate transporters, ATP-sulfurylase and APS-reductase were induced by S-starvation with the most mature leaf showing increased mRNA abundance first. In situ hybridization indicated that ZmST1;1 was expressed in epidermal and endodermal cell layers throughout the root whilst OAS(thiol)lyase was highly expressed in the root cortex.

Aerenchyma formation in roots of maize during sulphate starvation.

Young maize ( Zea mays L., Poaceae) plants were grown in a complete, well-oxygenated nutrient solution and then deprived of their external source of sulphate. This treatment induced the formation of aerenchyma in roots. In addition to the effect of sulphate starvation on root anatomy, the presence and location of superoxide anions and hydrogen peroxide, and changes in calcium and pH were examined. By day 6 of sulphate deprivation, aerenchyma started to form in the roots of plants and the first aerenchymatous spaces were apparent in the middle of the cortex. S-starvation also induced thickening of the cell walls of the endodermis. Active oxygen species appeared in groups of intact mid-cortex cells. Formation of superoxide anion and hydrogen peroxide was found in degenerating cells of the mid-cortex. Very few nuclei in the cortex of S-starved roots fluoresced, being shrunken and near to the cell wall. By day 12 of S-deprivation, a fully developed aerenchyma was apparent and there were only a few 'chains' of cells bridging hypodermis to endodermis and stele of roots. Cell walls of endodermis of S-starved roots increased 68% in thickness. Intensive fluorescence in the cell walls of the endodermal, hypodermal and to a lesser extent of epidermal cells was observed due to the formation of active oxygen species, while there was no fluorescence in the cortical cells. There was a higher Ca concentration in the cells walls of the endodermis and epidermis, compared to the rest of the S-starved root tissues. A higher pH was observed, mainly in the cell walls of the hypodermis and to a lesser extent in the cell walls of the endodermis. Superoxide anion and hydrogen peroxide was found in degenerating cells of the root cortex. There was no fluorescence of nuclei in the cortex of S-starved roots.

The roles of three functional sulphate transporters involved in uptake and translocation of sulphate in Arabidopsis thaliana.

To investigate the uptake and long-distance translocation of sulphate in plants, we have characterized three cell-type-specific sulphate transporters, Sultr1;1, Sultr2;1 and Sultr2;2 in Arabidopsis thaliana. Heterologous expression in the yeast sulphate transporter mutant indicated that Sultr1;1 encodes a high-affinity sulphate transporter (Km for sulphate 3.6 +/- 0.6 microM), whereas Sultr2;1 and Sultr2;2 encode low-affinity sulphate transporters (Km for sulphate 0.41 +/- 0.07 mM and >/= 1.2 mM, respectively). In Arabidopsis plants expressing the fusion gene construct of the Sultr1;1 promoter and green fluorescent protein (GFP), GFP was localized in the lateral root cap, root hairs, epidermis and cortex of roots. beta-glucuronidase (GUS) expressed with the Sultr2;1 promoter was specifically accumulated in the xylem parenchyma cells of roots and leaves, and in the root pericycles and leaf phloem. Expression of the Sultr2;2 promoter-GFP fusion gene showed specific localization of GFP in the root phloem and leaf vascular bundle sheath cells. Plants continuously grown with low sulphate concentrations accumulated high levels of Sultr1;1 and Sultr2;1 mRNA in roots and Sultr2;2 mRNA in leaves. The abundance of Sultr1;1 and Sultr2;1 mRNA was increased remarkably in roots by short-term stress caused by withdrawal of sulphate. Addition of selenate in the sulphate-sufficient medium increased the sulphate uptake capacity, tissue sulphate content and the abundance of Sultr1;1 and Sultr2;1 mRNA in roots. Concomitant decrease of the tissue thiol content after selenate treatment was consistent with the suggested role of glutathione (GSH) as a repressive effector for the expression of sulphate transporter genes.

Cysteine synthase (O-acetylserine (thiol) lyase) substrate specificities classify the mitochondrial isoform as a cyanoalanine synthase.

A cyanoalanine synthase and two isoforms (A, cytosolic and B, chloroplastic) of cysteine synthase (O:-acetylserine (thiol) lyase) were isolated from spinach. N-terminal amino acid sequence analysis of the cyanoalanine synthase gave 100% homology for the determined 12 residues with a published sequence for the mitochondrial cysteine synthase isoform. All three enzymes catalysed both the cysteine synthesis and cyanoalanine synthesis reactions, although with different efficiencies. Michaelis-Menten kinetics were observed for all three enzymes when substrate saturation experiments were performed varying O:-acetylserine, chloroalanine and cysteine. Negative co-operative kinetics were observed for cysteine synthases A and B when substrate saturation experiments were performed varying sulphide and cyanide, compared with the Michaelis-Menten kinetics observed for cyanoalanine synthase. The exception was negative co-operativity observed towards sulphide for cyanoalanine synthase with O:-acetylserine as co-substrate. The optimum sulphide concentration was dependent on the alanyl co-substrate used. The amino acid sequence similarity places these three enzymes in the same gene family, and whilst the close kinetic similarities support this, they also indicate distinct roles for the isoforms.

Plant responses to sulphur deficiency and the genetic manipulation of sulphate transporters to improve S-utilization efficiency.

Decreased inputs of S have increased the incidence of S-deficiency in crops, resulting in decreased yields and quality. Remediation by fertilizer application is not always successful because this often results in an uneven supply of S. The ability to respond to S-deficiency stress varies between crops and this is a target for the genetic improvement of S-utilization efficiency. Improved capture of resources, the accumulation of greater reserves of S and improved mechanisms for the remobilization of these reserves are required. It is an inability to over-accumulate S and subsequently, effectively remobilize S-reserves, which restricts optimum S-use efficiency. Genetic manipulation of the transporters and their expression will contribute to overcoming these limitations. Control of gene expression limits excess uptake and activity of the assimilatory pathway: the endogenous expression of sulphate transporters is regulated by S-supply, with negative regulation from reduced S-containing compounds and positive regulation by O-acetylserine, the C/N skeleton precursor of cysteine. Constitutive expression of the transporter will remove this control and may enable the accumulation of sulphate reserves. Sulphate in the vacuole and other pools of reduced sulphur, such as glutathione or protein may be remobilized under S-limiting conditions. Low efficiencies of these remobilization processes, particularly the remobilization of vacuolar sulphate, suggest that the transporters involved in the remobilization are a target for modification. Transporters are involved in facilitating the multiple trans-membrane transport steps between uptake of sulphate from the soil solution, and delivery to the site of reduction in the chloroplast or plastid. A gene family has been identified and phylogenetic relationships based on primary sequence information indicate multiple sub-groups. Groups which are expressed in roots, in shoots and in both tissue types are postulated, however, the functional roles for these groups and the identification of transporters involved in recycling remain to be confirmed.

Genomic and functional characterization of the oas gene family encoding O-acetylserine (thiol) lyases, enzymes catalyzing the final step in cysteine biosynthesis in Arabidopsis thaliana.

The final step of cysteine biosynthesis in plants is catalyzed by O-acetylserine (thiol) lyase (OAS-TL), which occurs as several isoforms found in the cytosol, the plastids and the mitochondria. Genomic DNA blot hybridization and isolation of genomic clones indicate single copy genes (oasA1, oasA2, oasB and oasC) that encode the activities of OAS-TL A, B and C found in separate subcellular compartments in the model plant Arabidopsis thaliana. Sequence analysis reveals that the newly discovered oasA2 gene represents a pseudogene that is still transcribed, but is not functionally translated. The comparison of gene structures suggests that oasA1/oasA2 and oasB/oasC are closely related and may be derived from a common ancestor by subsequent duplications. OAS-TL A, B and C were overexpressed in an Escherichia coli mutant lacking cysteine synthesis and exhibited bifunctional OAS-TL and beta-cyanoalanine synthase (CAS) activities. However, all three proteins represent true OAS-TLs according to kinetic analysis and are unlikely to function in cyanide detoxification or secondary metabolism. In addition, it was demonstrated that the mitochondrial OAS-TL C exhibits in vivo protein-protein interaction capabilities with respect to cysteine synthase complex formation similar to cytosolic OAS-TL A and plastid OAS-TL B. Multiple database accessions for each of the A. thaliana OAS-TL isoforms can thus be attributed to a specified number of oas genes to which functionally defined gene products are assigned, and which are responsible for compartment-specific cysteine synthesis.

Molecular mechanisms of phosphate and sulphate transport in plants.

The application of molecular techniques in recent years has advanced our understanding of phosphate and sulphate transport processes in plants. Genes encoding phosphate and sulphate transporters have been isolated from a number of plant species. The transporters encoded by these genes are related to the major facilitator superfamily of proteins. They are predicted to contain 12 membrane-spanning domains and function as H(+)/H(2)PO(-4) or H(+)/SO(2/-4) cotransporters. Both high-affinity and low-affinity types have been identified. Most research has concentrated on genes that encode transporters expressed in roots. The expression of many of these genes is transcriptionally regulated by signals that respond to the nutrient status of the plant. Nutrient demand and the availability of precursors needed in the assimilatory pathways also regulate transcription of some of these genes. Information on the cell types in which phosphate and sulphate transporters are expressed is becoming available. These data, together with functional characterisation of the transporters, are enabling the roles of various transporters in the overall phosphate and sulphate nutrition of plants to be defined.