Calcium Spiking Patterns and the Role of the Calcium/Calmodulin-Dependent Kinase CCaMK in Lateral Root Base Nodulation of Sesbania rostrata

Nodulation factor (NF) signal transduction in the legume-rhizobium symbiosis involves calcium oscillations that are instrumental in eliciting nodulation. To date, Ca²⁺ spiking has been studied exclusively in the intracellular bacterial invasion of growing root hairs in zone I. This mechanism is not the only one by which rhizobia gain entry into their hosts; the tropical legume Sesbania rostrata can be invaded intercellularly by rhizobia at cracks caused by lateral root emergence, and this process is associated with cell death for formation of infection pockets. We show that epidermal cells at lateral root bases respond to NFs with Ca²⁺ oscillations that are faster and more symmetrical than those observed during root hair invasion. Enhanced jasmonic acid or reduced ethylene levels slowed down the Ca²⁺ spiking frequency and stimulated intracellular root hair invasion by rhizobia, but prevented nodule formation. Hence, intracellular invasion in root hairs is linked with a very specific Ca²⁺ signature. In parallel experiments, we found that knockdown of the calcium/calmodulin-dependent protein kinase gene of S. rostrata abolished nodule development but not the formation of infection pockets by intercellular invasion at lateral root bases, suggesting that the colonization of the outer cortex is independent of Ca²⁺ spiking decoding.     

A Two-Dimensional Vibrating Probe Study of Currents around Lateral Roots of Raphanus sativus Developing in Culture

A computer-assisted, two-dimensional vibrating probe was used to study the ionic currents around developing lateral roots of Raphanus sativus in vitro. This system allowed us to superimpose current vectors on the video image of the roots. In a young lateral root, current entered the cap, meristematic, and elongation zones and exited the primary root surface close to the base of the lateral root. As the lateral root grew, current began to exit from its basal (cell maturation zone) end. The densities of currents entering the apical portion of the faster-growing lateral roots in a medium lacking indole 3-acetic acid were about twice as large as those entering the apical region of the slower-growing lateral roots in indole 3-acetic acid-supplemented medium.     

The involvement of the root apex and cytokinins in the control of lateral root emergence in lettuce seedlings

Removal of the apical 3 mm of the primary root of hydroponically-grown lettuce seedlings 3 or 5 days after sowing, prevented further elongation of the root and increased both the number and total length of lateral roots. The length of the lateral zone, i.e. the distance from the base of the parent root to the lateral nearest the tip, except on one occasion, remained the same as the control in both 3 and 5 day treatments, until the length of the decapitated root (which had ceased elongating) became limiting.Zeatin applied via the roots, at a concentration range from 3 × 10^–10 M to 10^–8 M reduced tap root extension growth at all concentrations. Lateral root emergence was enhanced by low zeatin concentrations and retarded by higher ones. In general, the lateral zone length was the same in cytokinin-treated plants as in untreated controls.     

Root growth responses to lead in young maize seedlings

This work was undertaken to follow the appearance and development of symptoms of lead toxicity in growing roots of seedlings. The effects of lead nitrate (10^-2–10⁵ M) were studied on the roots of maize (Zea mays) seedlings, cvs. Diamant and Sterling. The roots were grown on filter paper either on glass in trays or in large Petri dishes. The following characteristics of root growth were studied: seed germination, length of primary and seminal roots, number of seminal and lateral roots, length of branching zone, length of meristem and fully-elongated cells and the number of fully-elongated cells along the daily length increment. 10^-2 M lead nitrate exerted a clear toxic effect on root elongation just after radicle emergence; its influence on shoot growth was weak. However 10^-2 M Pb solution did not affect either radicle emergence itself or seminal root emergence, which can be explained by the impermeability of seed testa to lead salt. The inhibitory effect of 10^-3 M lead nitrate appeared a day later and was not as toxic: the growth of primary and seminal roots proceeded at lower rate due to a partial inhibition of cell division and cell elongation in them. 10^-3 M lead nitrate modified the root system morphology: it exerted no effect on the emergence of lateral roots and their number, but induced a more compact distribution of lateral roots along a shorter branching zone due to a reduced length of mature cells in the primary root. As a result of the more prominent inhibition of primary root growth, a shorter branching zone with more compactly located lateral roots occupied a position much closer to the root tip than in roots grown without the influence of lead.     

Band structure of surface electric potential in growing roots

For growing roots of azuki bean (Phaseolus chrysanthos), an electric potential is measured minutely along the surface of the root, together with the surface pH. It was found that the root begins to display a band-type pattern of potential with a spatial period of about 2 cm in a mature region as soon as it grows to about 10 cm in root length, while the surface potential shows only one convex peak around a position 5-20 mm behind a root tip and a succeeding concave peak around 20-35 mm, providing the length of root is shorter than about 10 cm. Since the surface potential takes a relatively positive value on average at the side of the root base compared with that in an elongation zone near the tip, electric current is supposed to flow into the elongation zone, accompanied by some local current loops in the mature region. The present band-type pattern observed first in multi-cell systems seems to be a kind of dissipative structure appearing far from equilibrium, and hence its relationship to growth is discussed.     

QTLs for nitrate induced elongation and initiation of lateral roots in rice (Oryza sativa L.)

To investigate the genetic background of nitrate-induced elongation and initiation of lateral roots in rice (Oryza sativa L.), a doubled haploid (DH) population, derived from a cross between IR64 and Azucena, which showed different responses to local supplied NO₃ ^– in lateral root elongation and initiation, was used in an agar culture experiment with three separated layers. The second agar layer was supplied with 3 mM NO₃ ^– or without NO₃ ^– as two treatments. Average lateral root length, lateral root number and surface area of lateral roots in the second agar layers with and without nitrate, respectively, were measured. The ratio of the parameters from the two treatments were calculated as derived parameters. Seven putative Quantitative trait loci (QTLs) for the 6 lateral root traits in nitrate-deficient and nitrate-supplied layers were detected. These QTLs individually explained about 9% to 15% of the total phenotypic variations in the traits. Identical QTLs for root traits from other reports with QTLs detected in this case were found, which suggests that the genetic factors responsive to local supplied NO₃ ^– is involved in root growth and development     

Hormonal Regulation of Lateral Bud (Tiller) Release in Oats (Avena sativa L.)

Stem segments containing a single node and quiescent lateral bud (tiller) were excised from the bases of oat shoots (cv. `Victory') and used to study the effects of plant hormones on release of lateral buds and development of adventitious root primordia. Kinetin (10⁻⁵ and 10⁻⁶ molar) stimulates development of tillers and inhibits development of root primordia, whereas indoleacetic acid (IAA) (10⁻⁵ and 10⁻⁶ molar) causes the reverse effects. Abscisic acid strongly inhibits kinetin-induced tiller bud release and elon-gation and IAA-induced adventitious root development. IAA, in combination with kinetin, also inhibits kinetin-induced bud prophyll (outermost leaf of the axillary bud) elongation. The IAA oxidase cofactor p-coumaric acid stimulates lateral bud release; the auxin transport inhibitor 2,3,5-triiodo-benzoic acid and the antiauxin α (p-chlorophenoxy)-isobutyric acid inhibit IAA-induced adventitious root formation. Gibberellic acid is synergistic with kinetin in the elongation of the bud prophyll. In intact oat plants, tiller release is induced by shoot decapitation, geostimulation, or the emergence of the inflorescence. Results shown support the apical dominance theory, namely, that the cytokinin to auxin ratio plays a decisive role in determining whether tillers are released or adventitious roots develop. They also indicate that abscisic acid and possibly gibberellin may act as modulator hormones in this system.     

Influence of Environmental Factors,Cultural Practices,and Herbicide Application on Seed Germination and Emergence Ecology of Ischaemum rugosum Salisb

Ischaemum rugosum Salisb. (Saramolla grass) is a noxious weed of rice that is difficult to control by chemical or mechanical means once established. A study was conducted to determine the effect of light, temperature, salt, drought, flooding, rice residue mulch, burial depth, and pre-emergence herbicides on seed germination and emergence of I. rugosum. Germination was stimulated by light and inhibited under complete darkness. Optimum temperature for germination was 30/20°C (97.5% germination). Germination reduced from 31 to 3.5% when the osmotic potential of the growing medium decreased from -0.1 to -0.6 MPa and no germination occurred at -0.8 MPa. Germination was 18 and 0.5% at 50 and 100 mM NaCl concentrations, respectively, but was completely inhibited at 150 mM or higher. Residue application at 1–6 t ha^-1 reduced weed emergence by 35–88% and shoot biomass by 55–95%. The efficacy of pre-emergence herbicides increased with increasing application rates and decreased with increasing rice residue mulching. The efficacy of herbicides was in the order of oxadiazon> pendimethalin> pretilachlor. At 6 t ha^-1, all herbicides, regardless of rates, did not differ from the control treatment. I. rugosum seeds buried at 2 cm or deeper did not emerge; however, they emerged by 4.5 and 0.5% at 0.5 and 1 cm depths, respectively, compared to the 39% germination for soil surface seeding. Flooding at 4 DAS or earlier reduced seedling emergence and shoot biomass while flooding at 8 DAS reduced only seedling emergence. The depth and timing of flooding independently reduced root biomass. Flooding at 4 and 6 cm depths reduced the root biomass. Relative to flooding on the day of sowing, flooding at 8 DAS increased root biomass by 89%. Similarly, flooding on the day of sowing and at 2 DAS reduced the root–shoot biomass ratio. Under the no-flood treatment, increasing rates of pretilachlor from 0.075 to 0.3 kg ai ha^-1 reduced weed emergence by 61–79%. At the flooding depth of 2–4 cm, pretilachlor reduced weed emergence and shoot and root biomass, but the differences across rates were non-significant. Information generated in this study will be helpful in developing integrated weed management strategies for managing this weed.     

Electrostatic Changes in Lycopersicon esculentum Root Plasma Membrane Resulting from Salt Stress

Salinity-induced alterations in tomato (Lypersicon esculentum Mill. cv Heinz 1350) root plasma membrane properties were studied and characterized using a membrane vesicle system. Equivalent rates of MgATP-dependent H⁺-transport activity were measured by quinacrine fluorescence (ΔpH) in plasma membrane vesicles isolated from control or salt-stressed (75 millimolar salt) tomato roots. However, when bis-[3-phenyl-5-oxoisoxazol-4-yl] pentamethine was used to measure MgATP-dependent membrane potential (ΔΨ) formation, salt-stressed vesicles displayed a 50% greater initial quench rate and a 30% greater steady state quench than control vesicles. This differential probe response suggested a difference in surface properties between control and salt-stressed membranes. Fluorescence titration of vesicles with the surface potential probe, 8-anilino-1-napthalenesulphonic acid (ANS) provided dissociation constants (K_d) of 120 and 76 micromolar for dye binding to control and salt-stressed vesicles, respectively. Membrane surface potentials (Ψ_o) of−26.0 and −13.7 millivolts were calculated for control and salt-stressed membrane vesicles from the measured K_d values and the calculated intrinsic affinity constant, K_i. The concentration of cations and anions at the surface of control and salt-stressed membranes was estimated using Ψ_o values and the Boltzmann equation. The observed difference in membrane surface electrostatic properties was consistent with the measured differences in K⁺-stimulated kinetics of ATPase activity between control and salt-stressed vesicles and by the differential ability of Cl⁻ ions to stimulate H⁺-transport activity. Salinity-induced changes in plasma membrane electrostatic properties may influence ion transport across the plasma membrane.     

Lateral Drug Diffusion in Human Nails

The main objective of the current work is to demonstrate the process of passive lateral diffusion in the human nail plate and its effect on the passive transungual permeation of antifungal drug ciclopirox olamine (CPO). A water soluble dye, methyl red sodium salt (MR) was used to visualize the process of lateral diffusion using a novel suspended nail experiment. The decline in concentration of CPO correlates with that of concentration of MR from the proximal to the distal end of the nail in suspended nail study. Three toenails each were trimmed to 5 mm × 5 mm (25 mm²), 7 mm × 7 mm (49 mm²), and 9 mm × 9 mm (81 mm²) to study the extent and effect of lateral diffusion of the CPO on its in vitro transungual permeation. The permeation flux of CPO decreased as the surface area of the toenail increased. There was a positive correlation between the concentrations of CPO and MR in the area of application and in the peripheral area of the toenails of the three surface areas, confirming the findings in the suspended nail experiment. Profound lateral diffusion of CPO was demonstrated and shown to reduce the in vitro passive transungual drug permeation and prolong the lag-time in human toenails. The study data implies that during passive in vitro transungual permeation experiments, the peripheral nail around the area of drug application has to be kept to a minimum, in order to get reliable data which mimics the in vivo situation.KEY WORDS: ciclopirox olamine, lateral diffusion, passive, topical, transungual     

Alfalfa Controls Nodulation during the Onset of Rhizobium-induced Cortical Cell Division

The formation of first nodules inhibits subsequent nodulation in younger regions of alfalfa (Medicago sativa L.) roots by a feedback regulatory mechanism that controls nodule number systemically (G Caetano-Anollés, WD Bauer [1988] Planta 175: 546-557). Following inoculation with wild-type Rhizobium meliloti, almost all infections associated with cortical cell division developed into mature nodules. While the distribution of Rhizobium- induced cell divisions closely paralleled the distribution of first emergent nodules, only 9 to 15% of total cell division foci failed to become functional nodules. Nodule formation was restricted to the primary root when plants were inoculated before lateral root emergence. Excision of these primary root nodules allowed nodules to reappear in lateral roots clustered around the location of the root tip at the time of nodule removal. Apparently, this region regained susceptibility to infection within the first hours after excision of primary nodules and suppression of nodulation was restored a day later probably due to the development of new infection foci. Our results suggest that alfalfa controls nodulation during the onset of cell division in the root cortex and not during infection development as in soybean.     

Hormonal factors controlling the initiation and development of lateral roots

The decapitated primary root of 3-day-old Alaska pea seedlings has been used as a test system to determine the activities on lateral root formation of six auxins, six cytokinins and several other naturally-occurring compounds. Their effects were assessed on (1) the initiation of lateral root primordia, (2) the emergence of visible lateral roots, and (3) the elongation of these laterals. All the auxins, at the optimum concentration of 10^-4M, promoted the initiation of lateral root primordia, and all except 3-indolylpropionic acid inhibited the elongation of the resulting lateral roots. Their effects on the emergence of laterals were small and varied. All the cytokinins, at 10^-6M and above, inhibited both the initiation and the emergence of lateral roots, zeatin being the most powerful inhibitor. The emergence process was about twice as sensitive as the initiation of primordia to the presence of cytokinins. The cytokinin ribosides were generally less active than the free bases. Abscisic acid and xanthoxin inhibited both emergence and elongation, the concentration for 50% decrease of emergence being about 10^-4M. Gibberellic acid had little clear effect on any of the three criteria. Nicotinic acid and thiamine at 10^-3M promoted both the initiation of primordia and their emergence: pyridoxal phosphate stimulated both emergence and elongation but did not influence the initiation of primordia. Adenine and guanine had little effect but decreased root elongation some 25%. The strong inhibiting effect of the cytokinins may well be the basis for the marked inhibition exerted by the root-tip on lateral root formation, while the promoting effects of auxins may explain the previously observed promotion of lateral root formation by the young shoot and cotyledons.     

High-throughput root phenotyping screens identify genetic loci associated with root architectural traits in Brassica napus under contrasting phosphate availabilities

Background and Aims

Phosphate (Pi) deficiency in soils is a major limiting factor for crop growth worldwide. Plant growth under low Pi conditions correlates with root architectural traits and it may therefore be possible to select these traits for crop improvement. The aim of this study was to characterize root architectural traits, and to test quantitative trait loci (QTL) associated with these traits, under low Pi (LP) and high Pi (HP) availability in Brassica napus.

Methods

Root architectural traits were characterized in seedlings of a double haploid (DH) mapping population (n = 190) of B. napus [‘Tapidor’ × ‘Ningyou 7’ (TNDH)] using high-throughput phenotyping methods. Primary root length (PRL), lateral root length (LRL), lateral root number (LRN), lateral root density (LRD) and biomass traits were measured 12 d post-germination in agar at LP and HP.

Key Results

In general, root and biomass traits were highly correlated under LP and HP conditions. ‘Ningyou 7’ had greater LRL, LRN and LRD than ‘Tapidor’, at both LP and HP availability, but smaller PRL. A cluster of highly significant QTL for LRN, LRD and biomass traits at LP availability were identified on chromosome A03; QTL for PRL were identified on chromosomes A07 and C06.

Conclusions

High-throughput phenotyping of Brassica can be used to identify root architectural traits which correlate with shoot biomass. It is feasible that these traits could be used in crop improvement strategies. The identification of QTL linked to root traits under LP and HP conditions provides further insights on the genetic basis of plant tolerance to P deficiency, and these QTL warrant further dissection.     

Vacuoles from Sugarcane Suspension Cultures : III. PROTONMOTIVE POTENTIAL DIFFERENCE

The electrochemical proton gradient across the tonoplast of isolated (Saccharum sp.) vacuoles and vacuoles in situ was measured. The isolated vacuoles show no significant protonmotive potential difference, the pH gradient of 0.8 (inside acid) was balanced by a membrane potential of about −80 mv (inside negative). From pH and uncoupler insensitivity and K⁺ sensitivity, it was concluded that the experimentally caused K⁺ gradient created the electric potential.     

Characterization of CYCLOPHILLIN38 shows that a photosynthesis-derived systemic signal controls lateral root emergence

Photosynthesis in leaves generates fixed-carbon resources and essential metabolites that support sink tissues, such as roots. Two of these metabolites, sucrose and auxin, promote growth in root systems, but the explicit connection between photosynthetic activity and control of root architecture has not been explored. Through a mutant screen to identify pathways regulating root system architecture, we identified a mutation in the Arabidopsis thaliana CYCLOPHILIN 38 (CYP38) gene, which causes accumulation of pre-emergent stage lateral roots. CYP38 was previously reported to stabilize photosystem II (PSII) in chloroplasts. CYP38 expression is enriched in shoots, and grafting experiments show that the gene acts non-cell-autonomously to promote lateral root emergence. Growth of wild-type plants under low-light conditions phenocopies the cyp38 lateral root emergence defect, as does the inhibition of PSII-dependent electron transport or Nicotinamide adenine dinucleotide phosphate (NADPH) production. Importantly, these perturbations to photosynthetic activity rapidly suppress lateral root emergence, which is separate from their effects on shoot size. Supplementary exogenous sucrose largely rescued primary root (PR) growth in cyp38, but not lateral root growth. Auxin (indole-3-acetic acid (IAA)) biosynthesis from tryptophan is dependent on reductant generated during photosynthesis. Consistently, we found that wild-type seedlings grown under low light and cyp38 mutants have highly diminished levels of IAA in root tissues. IAA treatment rescued the cyp38 lateral root defect, revealing that photosynthesis promotes lateral root emergence partly through IAA biosynthesis. These data directly confirm the importance of CYP38-dependent photosynthetic activity in supporting root growth, and define the specific contributions of two metabolites in refining root architecture under light-limited conditions.

Lateral root emergence is regulated via systemic signaling that incorporates photosynthesis-dependent redox control and auxin biosynthesis.     

Effects of fluridone and abscisic acid on lateral root initiation and root elongation of excised tomato roots cultured in vitro

Roots of tomato (Lycopersicon esculentum Mill. cv. Bonny Best) were excised and cultured in the presence of the abscisic acid synthesis inhibitor fluridone, and with concentrations of exogenous abscisic acid ranging from 10⁻¹⁰to 10⁻⁴M to determine the effects of abscisic acid and its synthesis inhibition on the development of lateral roots in in vitro cultured tomato roots. Exogenous abscisic acid inhibited lateral root initiation and emergence at concentrations of 10⁻⁶M and greater. Fluridone (10⁻⁶M) enhanced the formation of lateral roots even in the presence of abscisic acid, at all concentrations tested except 10⁻⁴M. Abscisic acid increased apical distance, and fluridone reduced it up to 10⁻⁵M abscisic acid. Both fluridone and abscisic acid reduced lateral and primary root lengths. It was concluded the endogenous abscisic acid is probably involved in the regulation of lateral root initiation and root apical dominance, and that abscisic acid may affect lateral root initiation differently than lateral root emergence. This revised version was published online in June 2006 with corrections to the Cover Date.     

Early development and gravitropic response of lateral roots in Arabidopsis thaliana

Root system architecture plays an important role in determining nutrient and water acquisition and is modulated by endogenous and environmental factors, resulting in considerable developmental plasticity. The orientation of primary root growth in response to gravity (gravitropism) has been studied extensively, but little is known about the behaviour of lateral roots in response to this signal. Here, we analysed the response of lateral roots to gravity and, consistently with previous observations, we showed that gravitropism was acquired slowly after emergence. Using a lateral root induction system, we studied the kinetics for the appearance of statoliths, phloem connections and auxin transporter gene expression patterns. We found that statoliths could not be detected until 1 day after emergence, whereas the gravitropic curvature of the lateral root started earlier. Auxin transporters modulate auxin distribution in primary root gravitropism. We found differences regarding PIN3 and AUX1 expression patterns between the lateral root and the primary root apices. Especially PIN3, which is involved in primary root gravitropism, was not expressed in the lateral root columella. Our work revealed new developmental transitions occurring in lateral roots after emergence, and auxin transporter expression patterns that might explain the specific response of lateral roots to gravity.     

Accumulation of 14C from exogenous labelled auxin in lateral root primordia of intact pea seedlings (Pisum sativum L.)

When [¹⁴C]indol-3yl-acetic acid was applied to the apical bud of 5-day old dwarf pea seedlings which possessed unbranched primary roots, a small amount of ¹⁴C was transported into the root system at a velocity of 11–14 mm h^-1. Most of the ¹⁴C which entered the primary root accumulated in the young lateral root primordia, including the smallest detectable (20–30 mm from the primary root tip). In older (8-d old) seedlings in which the primary root bore well-developed lateral roots, ¹⁴C also accumulated in the tertiary root primordia. In contrast, little ¹⁴C was detected in the apical region of the primary root or, in older plants, in the apices of the lateral roots.Abbreviations IAA indol-3yl-acetic acid     

Hormonally Induced Changes in the Stem and Petiole Anatomy and Cellulase Enzyme Patterns in Phaseolus vulgaris L

Time course changes were observed in petiole and stem anatomy and cellulase enzyme patterns in bean (Phaseolus vulgaris L.) explants when 10⁻⁵ or 10⁻²m indoleacetic acid in lanolin paste was applied to acropetal cut surfaces in the presence or absence of ethylene. Auxin (10⁻²m) in the presence of ethylene stimulated rapid ordered cell division and dedifferentiation, with ensuing lateral root formation. Auxin (10⁻⁵m) caused moderate cortical swelling, pit formation in pith parenchyma, and chloroplast development in certain cortical cells. Exogenous ethylene reduced cell division activity and caused cortical cell swelling and separation. Removal of endogenously generated ethylene by mercuric perchlorate resulted in less ordered cell division patterns and no lateral root formation. Auxin treatments enhanced formation of an active acidic pI cellulase, exogenous ethylene-stimulated formation of an active basic pI cellulase. The absence of basic pI cellulase activity by the removal of endogenously generated ethylene suggests a close dependence of basic pI cellulase activity on ethylene.     

Do root hydraulic properties change during the early vegetative stage of plant development in barley (Hordeum vulgare)?

Background and Aims

As annual crops develop, transpirational water loss increases substantially. This increase has to be matched by an increase in water uptake through the root system. The aim of this study was to assess the contributions of changes in intrinsic root hydraulic conductivity (Lp, water uptake per unit root surface area, driving force and time), driving force and root surface area to developmental increases in root water uptake.

Methods

Hydroponically grown barley plants were analysed during four windows of their vegetative stage of development, when they were 9–13, 14–18, 19–23 and 24–28 d old. Hydraulic conductivity was determined for individual roots (Lp) and for entire root systems (Lp_r). Osmotic Lp of individual seminal and adventitious roots and osmotic Lp_r of the root system were determined in exudation experiments. Hydrostatic Lp of individual roots was determined by root pressure probe analyses, and hydrostatic Lp_r of the root system was derived from analyses of transpiring plants.

Key Results

Although osmotic and hydrostatic Lp and Lp_r values increased initially during development and were correlated positively with plant transpiration rate, their overall developmental increases (about 2-fold) were small compared with increases in transpirational water loss and root surface area (about 10- to 40-fold). The water potential gradient driving water uptake in transpiring plants more than doubled during development, and potentially contributed to the increases in plant water flow. Osmotic Lp_r of entire root systems and hydrostatic Lp_r of transpiring plants were similar, suggesting that the main radial transport path in roots was the cell-to-cell path at all developmental stages.

Conclusions

Increase in the surface area of root system, and not changes in intrinsic root hydraulic properties, is the main means through which barley plants grown hydroponically sustain an increase in transpirational water loss during their vegetative development.