Physiological Effects of the Synthetic Strigolactone Analog GR24 on Root System Architecture in Arabidopsis: Another Belowground Role for Strigolactones?

In this study, the role of the recently identified class of phytohormones, strigolactones, in shaping root architecture was addressed. Primary root lengths of strigolactone-deficient and -insensitive Arabidopsis (Arabidopsis thaliana) plants were shorter than those of wild-type plants. This was accompanied by a reduction in meristem cell number, which could be rescued by application of the synthetic strigolactone analog GR24 in all genotypes except in the strigolactone-insensitive mutant. Upon GR24 treatment, cells in the transition zone showed a gradual increase in cell length, resulting in a vague transition point and an increase in transition zone size. PIN1/3/7-green fluorescent protein intensities in provascular tissue of the primary root tip were decreased, whereas PIN3-green fluorescent protein intensity in the columella was not affected. During phosphate-sufficient conditions, GR24 application to the roots suppressed lateral root primordial development and lateral root forming potential, leading to a reduction in lateral root density. Moreover, auxin levels in leaf tissue were reduced. When auxin levels were increased by exogenous application of naphthylacetic acid, GR24 application had a stimulatory effect on lateral root development instead. Similarly, under phosphate-limiting conditions, endogenous strigolactones present in wild-type plants stimulated a more rapid outgrowth of lateral root primordia when compared with strigolactone-deficient mutants. These results suggest that strigolactones are able to modulate local auxin levels and that the net result of strigolactone action is dependent on the auxin status of the plant. We postulate that the tightly balanced auxin-strigolactone interaction is the basis for the mechanism of the regulation of the plants' root-to-shoot ratio.     

Strigolactones promote nodulation in pea

Strigolactones are recently defined plant hormones with roles in mycorrhizal symbiosis and shoot and root architecture. Their potential role in controlling nodulation, the related symbiosis between legumes and Rhizobium bacteria, was explored using the strigolactone-deficient rms1 mutant in pea (Pisum sativum L.). This work indicates that endogenous strigolactones are positive regulators of nodulation in pea, required for optimal nodule number but not for nodule formation per se. rms1 mutant root exudates and root tissue are almost completely deficient in strigolactones, and rms1 mutant plants have approximately 40% fewer nodules than wild-type plants. Treatment with the synthetic strigolactone GR24 elevated nodule number in wild-type pea plants and also elevated nodule number in rms1 mutant plants to a level similar to that seen in untreated wild-type plants. Grafting studies revealed that nodule number and strigolactone levels in root tissue of rms1 roots were unaffected by grafting to wild-type scions indicating that strigolactones in the root, but not shoot-derived factors, regulate nodule number and provide the first direct evidence that the shoot does not make a major contribution to root strigolactone levels.     

The potential roles of strigolactones and brassinosteroids in the autoregulation of nodulation pathway

Background and Aims

The number of nodules formed on a legume root system is under the strict genetic control of the autoregulation of nodulation (AON) pathway. Plant hormones are thought to play a role in AON; however, the involvement of two hormones recently described as having a largely positive role in nodulation, strigolactones and brassinosteroids, has not been examined in the AON process.

Methods

A genetic approach was used to examine if strigolactones or brassinosteroids interact with the AON system in pea (Pisum sativum). Double mutants between shoot-acting (Psclv2, Psnark) and root-acting (Psrdn1) mutants of the AON pathway and strigolactone-deficient (Psccd8) or brassinosteroid-deficient (lk) mutants were generated and assessed for various aspects of nodulation. Strigolactone production by AON mutant roots was also investigated.

Key Results

Supernodulation of the roots was observed in both brassinosteroid- and strigolactone-deficient AON double-mutant plants. This is despite the fact that the shoots of these plants displayed classic strigolactone-deficient (increased shoot branching) or brassinosteroid-deficient (extreme dwarf) phenotypes. No consistent effect of disruption of the AON pathway on strigolactone production was found, but root-acting Psrdn1 mutants did produce significantly more strigolactones.

Conclusions

No evidence was found that strigolactones or brassinosteroids act downstream of the AON genes examined. While in pea the AON mutants are epistatic to brassinosteroid and strigolactone synthesis genes, we argue that these hormones are likely to act independently of the AON system, having a role in the promotion of nodule formation.     

Ethylene Controls Adventitious Root Initiation Sites in <Emphasis Type="Italic">Arabidopsis</Emphasis> Hypocotyls Independently of Strigolactones

Adventitious root formation is essential for cutting propagation of diverse species; however, until recently little was known about its regulation. Strigolactones and ethylene have both been shown to inhibit adventitious roots and it has been suggested that ethylene interacts with strigolactones in root hair elongation. We have investigated the interaction between strigolactones and ethylene in regulating adventitious root formation in intact seedlings of Arabidopsis thaliana. We used strigolactone mutants together with 1-aminocyclopropane-1-carboxylic acid (ACC) (ethylene precursor) treatments and ethylene mutants together with GR24 (strigolactone agonist) treatments. Importantly, we conducted a detailed mapping of adventitious root initiation along the hypocotyl and measured ethylene production in strigolactone mutants. ACC treatments resulted in a slight increase in adventitious root formation at low doses and a decrease at higher doses, in both wild-type and strigolactone mutants. Furthermore, the distribution of adventitious roots dramatically changed to the top third of the hypocotyl in a dose-dependent manner with ACC treatments in both wild-type and strigolactone mutants. The ethylene mutants all responded to treatments with GR24. Wild type and max4 (strigolactone-deficient mutant) produced the same amount of ethylene, while emanation from max2 (strigolactone-insensitive mutant) was lower. We conclude that strigolactones and ethylene act largely independently in regulating adventitious root formation with ethylene controlling the distribution of root initiation sites. This role for ethylene may have implications for flood response because both ethylene and adventitious root development are crucial for flood tolerance.     

The role of strigolactones in photomorphogenesis of pea is limited to adventitious rooting

The recently discovered group of plant hormones, the strigolactones, have been implicated in regulating photomorphogenesis. We examined this extensively in our strigolactone synthesis and response mutants and could find no evidence to support a major role for strigolactone signaling in classic seedling photomorphogenesis (e.g. elongation and leaf expansion) in pea (Pisum sativum), consistent with two recent independent reports in Arabidopsis. However, we did find a novel effect of strigolactones on adventitious rooting in darkness. Strigolactone‐deficient mutants, Psccd8 and Psccd7, produced significantly fewer adventitious roots than comparable wild‐type seedlings when grown in the dark, but not when grown in the light. This observation in dark‐grown plants did not appear to be due to indirect effects of other factors (e.g. humidity) as the constitutively de‐etiolated mutant, lip1, also displayed reduced rooting in the dark. This role for strigolactones did not involve the MAX2 F‐Box strigolactone response pathway as Psmax2 f‐box mutants did not show a reduction in adventitious rooting in the dark compared with wild‐type plants. The auxin‐deficient mutant bushy also reduced adventitious rooting in the dark, as did decapitation of wild‐type plants. Rooting was restored by the application of indole‐3‐acetic acid (IAA) to decapitated plants, suggesting a role for auxin in the rooting response. However, auxin measurements showed no accumulation of IAA in the epicotyls of wild‐type plants compared with the strigolactone synthesis mutant Psccd8, suggesting that changes in the gross auxin level in the epicotyl are not mediating this response to strigolactone deficiency.     

Phosphorus deficiency in red clover promotes exudation of orobanchol,the signal for mycorrhizal symbionts and germination stimulant for root parasites

Plant derived sesquiterpene strigolactones, which have previously been characterized as germination stimulants for root parasitic plants, have recently been identified as the branching factors which induce hyphal branching morphogenesis, a critical step in host recognition by arbuscular mycorrhizal (AM) fungi. We show here that, in red clover plants (Trifolium pratense L.), which is known as a host for both AM fungi and the root holoparasitic plant Orobanche minor Sm., reduced supply of phosphorus (P) but not of other elements examined (N, K, Mg, Ca) in the culture medium significantly promotes the release of a strigolactone, orobanchol, by the roots of this plant. In red clover plants, the level of orobanchol exudation appeared to be regulated by P availability and was in good agreement with germination stimulation activity of the root exudates. This implies that under P deficiency, plant roots attract not only symbiotic fungi but also root parasitic plants through the release of strigolactones. This is the first report demonstrating that nutrient availability influences both symbiotic and parasitic interactions in the rhizosphere.     

Indole glucosinolate and auxin biosynthesis in Arabidopsis thaliana (L.) Heynh. glucosinolate mutants and the development of clubroot disease

Mutants and wild type plants of Arabidopsis thaliana were analysed for differences in glucosinolate accumulation patterns, indole-3-acetic acid (IAA) biosynthesis and phenotype. A previously identified series of mutants, termed TU, with altered glucosinolate patterns was used in this study. Only the line TU8 was affected in shoot phenotype (shorter stems, altered branching pattern). Synthesis of IAA and metabolism were not much affected in the TU8 mutant during seedling development, although the content of free IAA peaked earlier in TU8 during plant development than in the wild type. Indole glucosinolates and IAA may, however, be involved in the development of clubroot disease caused by the obligate biotrophic fungus Plasmodiophora brassicae since the TU3 line had a lower infection rate than the wild type, and lines TU3 and TU8 showed decreased symptom development. The decline in clubroot formation was accompanied by a reduced number of fungal structures within the root cortex and slower development of the fungus. Indole glucosinolates were lower in infected roots of TU3 and TU8 than in control roots of these lines, whereas in wild-type plants the differences were not as prominent. Free IAA and indole-3-acetonitrile (IAN) were increased in infected roots of the wild type and mutants with normal clubroot symptoms, whereas they were reduced in infected roots of mutants TU3 and TU8. These results indicate a role for indole glucosinolates and IAN/IAA in relation to symptom development in clubroot disease. Received: 23 July 1998 / Accepted: 12 January 1999     

Studies on sodium bypass flow in lateral rootless mutants lrt1 and lrt2, and crown rootless mutant crl1 of rice (Oryza sativa L.)

An apoplastic pathway, the so‐called bypass flow, is important for Na⁺ uptake in rice (Oryza sativa L.) under saline conditions; however, the precise site of entry is not yet known. We report the results of our test of the hypothesis that bypass flow of Na⁺ in rice occurs at the site where lateral roots emerge from the main roots. We investigated Na⁺ uptake and bypass flow in lateral rootless mutants (lrt1, lrt2), a crown rootless mutant (crl1), their wild types (Oochikara, Nipponbare and Taichung 65, respectively) and in seedlings of rice cv. IR36. The results showed that shoot Na⁺ concentration in lrt1, lrt2 and crl1 was lower (by 20–23%) than that of their wild types. In contrast, the bypass flow quantified using trisodium‐8‐hydroxy‐1,3,6‐pyrenetrisulphonic acid (PTS) was significantly increased in the mutants, from an average of 1.1% in the wild types to 3.2% in the mutants. Similarly, bypass flow in shoots of IR36 where the number of lateral and crown roots had been reduced through physical and hormonal manipulations was dramatically increased (from 5.6 to 12.5%) as compared to the controls. The results suggest that the path of bypass flow in rice is not at the sites of lateral root emergence.     

Systemic Resistance in Tomato Induced by Biocontrol Bacteria Against the Root-Knot Nematode, Meloidogyne javanica is Independent of Salicylic Acid Production

Salicylic acid (SA)‐mediated induction of systemic resistance by Pseudomonas aeruginosa strain 7NSK2 and P. fluorescens strain CHA0 against soil‐borne fungi and viruses have been reported. The role of SA biosynthesis in the enhancement of defence mechanism against plant‐parasitic nematodes by these bacterial strains in tomato is not known. To better understand the importance of SA in rhizobacteria‐mediated suppression of root‐knot nematodes, biocontrol potential of SA‐negative or SA‐overproducing mutants against Meloidogyne javanica was evaluated with their respective wild type counter parts. Culture supernatant of 7NSK2, CHA0 and their respective mutants caused significant mortality of M. javanica juveniles in vitro. SA deletion in 7NSK2 and SA overproduction in CHA0 did not influence bacterial efficacy to cause nematode deaths. Similarly, culture supernatants resulting from King's B liquid medium amended with FeCl₃ did not influence nematicidal activity of the bacterial strains. Strain CHA0 induced juvenile deaths more than 7NSK2 did. In pot experiments, the bacterial strains applied in unsterilized sandy loam soil markedly reduced final nematode population densities in roots and subsequent root‐knot infection in tomato seedlings. SA‐negative or overproducing derivatives prevented tomato roots in kinetics similar to those with their respective wild types. When soil iron concentration was lowered by the addition of ethylenediamine di(o‐hydroxyphenylacetic acid), nematode biocontrol by the bacterial strains (both wild type and mutants) remained unaltered. To understand the mechanism involved in rhizobacteria‐mediated suppression of root‐knot nematode in tomato, bacterial performance was assessed in a split root trial in which one‐half of the root system was treated with bacterium while the other inoculated with nematode. Compared with the controls, application of the bacterial cell suspension to one‐half of the root system lowered the populations of root‐knot nematode in non‐bacterized nematode‐treated sections indicating enhanced defence in the non‐bacterized half. With respect to nematode infection, mutants induced systemic resistance to a similar extent as that caused by the wild types in both wild type tomato and NahG tomato plants. It is concluded that fluorescent pseudomonads induce systemic resistance against root‐knot nematode via a signal transduction pathway, which is independent of SA accumulation in roots.     

Nitrogen deficiency as well as phosphorus deficiency in sorghum promotes the production and exudation of 5-deoxystrigol,the host recognition signal for arbuscular mycorrhizal fungi and root parasites

Strigolactones released from plant roots induce hyphal branching of symbiotic arbuscular mycorrhizal (AM) fungi and germination of root parasitic weeds, Striga and Orobanche spp. We already demonstrated that, in red clover plants (Trifolium pratense L.), a host for both AM fungi and the root holoparasitic plant Orobanche minor Sm., reduced supply of phosphorus (P) but not of other elements examined (N, K, Ca, Mg) in the culture medium significantly promoted the secretion of a strigolactone, orobanchol, by the roots of this plant. Here we show that in the case of sorghum [Sorghum bicolor (L.) Moench], a host of both the root hemiparasitic plant Striga hermonthica and AM fungi, N deficiency as well as P deficiency markedly enhanced the secretion of a strigolactone, 5-deoxystrigol. The 5-deoxystrigol content in sorghum root tissues also increased under both N deficiency and P deficiency, comparable to the increase in the root exudates. These results suggest that strigolactones may be rapidly released after their production in the roots. Unlike the situation in the roots, neither N nor P deficiency affected the low content of 5-deoxystrigol in sorghum shoot tissues.     

Do strigolactones contribute to plant defence?

Strigolactones are multifunctional molecules involved in several processes outside and within the plant. As signalling molecules in the rhizosphere, they favour the establishment of arbuscular mycorrhizal symbiosis, but they also act as host detection cues for root parasitic plants. As phytohormones, they are involved in the regulation of plant architecture, adventitious rooting, secondary growth and reproductive development, and novel roles are emerging continuously. In the present study, the possible involvement of strigolactones in plant defence responses was investigated. For this purpose, the resistance/susceptibility of the strigolactone‐deficient tomato mutant Slccd8 against the foliar fungal pathogens Botrytis cinerea and Alternaria alternata was assessed. Slccd8 was more susceptible to both pathogens, pointing to a new role for strigolactones in plant defence. A reduction in the content of the defence‐related hormones jasmonic acid, salicylic acid and abscisic acid was detected by high‐performance liquid chromatography coupled to tandem mass spectrometry in the Slccd8 mutant, suggesting that hormone homeostasis is altered in the mutant. Moreover, the expression level of the jasmonate‐dependent gene PinII, involved in the resistance of tomato to B. cinerea, was lower than in the corresponding wild‐type. We propose here that strigolactones play a role in the regulation of plant defences through their interaction with other defence‐related hormones, especially with the jasmonic acid signalling pathway.     

Identification of mutants in phosphorus metabolism

Phosphorus availability is often limiting for plant growth. However, little is known of the pathways and mechanisms that regulate phosphorus (P) uptake and distribution in plants. We have developed a screen based on the induction of secreted root acid phosphatase activity by low‐P stress to identify mutants of Arabidopsis thaliana with defects in P metabolism. Acid phosphatase activity was detected visually in the roots of A. thaliana seedlings grown in vitro on low‐P medium, using the chromogenic substrate, 5‐bromo‐4‐chloro‐3‐indolyl‐phosphate (BCIP). In low‐P stress conditions the roots of wild‐type plants stained blue, as the induced root acid phosphatase cleaved BCIP to release the coloured product. Potential mutants were identified as having white, or pale blue, roots under these conditions. Out of approximately 79 000 T‐DNA mutagenised seedlings screened, two mutants with reduced acid phosphatase staining were further characterised. Both exhibited reduced growth and differences in their P contents when compared to wild‐type A. thaliana. The mutant with the most severe phenotype, pho3, accumulated high levels of anthocyanins and starch in a distinctive visual pattern within the leaves. The phenotypes of these mutants are distinct from two previously identified phosphorus mutants (phol and pho2) and from an acid phosphatase deficient mutant (pupl) of A. thaliana. This suggested that the screening method was robust and might lead to the identification of further mutants with the potential for increasing our understanding of P nutrition.     

Genes controlling legume nodule numbers affect phenotypic plasticity responses to nitrogen in the presence and absence of rhizobia

We investigated the role of three autoregulation of nodulation (AON) genes in regulating of root and shoot phenotypes when responding to changing nitrogen availability in the model legume, Medicago truncatula. These genes, RDN1‐1 (ROOT DETERMINED NODULATION1‐1), SUNN (SUPER NUMERIC NODULES), and LSS (LIKE SUNN SUPERNODULAOR), act in a systemic signalling pathway that limits nodule numbers. This pathway is also influenced by nitrogen availability, but it is not well known if AON genes control root and shoot phenotypes other than nodule numbers in response to nitrogen. We conducted a controlled glasshouse experiment to compare root and shoot phenotypes of mutants and wild type plants treated with four nitrate concentrations. All AON mutants showed altered rhizobia‐independent phenotypes, including biomass allocation, lateral root length, lateral root density, and root length ratio. In response to nitrogen, uninoculated AON mutants were less plastic than the wild type in controlling root mass ratio, root length ratio, and lateral root length. This suggests that AON genes control nodulation‐independent root architecture phenotypes in response to nitrogen. The phenotypic differences between wild type and AON mutants were exacerbated by the presence of nodules, pointing to resource competition as an additional mechanism affecting root and shoot responses to nitrogen.     

RCN1/OsABCG5, an ATP‐binding cassette (ABC) transporter,is required for hypodermal suberization of roots in rice (Oryza sativa)

Suberin is a complex polymer composed of aliphatic and phenolic compounds. It is a constituent of apoplastic plant interfaces. In many plant species, including rice (Oryza sativa), the hypodermis in the outer part of roots forms a suberized cell wall (the Casparian strip and/or suberin lamellae), which inhibits the flow of water and ions and protects against pathogens. To date, there is no genetic evidence that suberin forms an apoplastic transport barrier in the hypodermis. We discovered that a rice reduced culm number1 (rcn1) mutant could not develop roots longer than 100 mm in waterlogged soil. The mutated gene encoded an ATP‐binding cassette (ABC) transporter named RCN1/OsABCG5. RCN1/OsABCG5 gene expression in the wild type was increased in most hypodermal and some endodermal roots cells under stagnant deoxygenated conditions. A GFP‐RCN1/OsABCG5 fusion protein localized at the plasma membrane of the wild type. Under stagnant deoxygenated conditions, well suberized hypodermis developed in wild types but not in rcn1 mutants. Under stagnant deoxygenated conditions, apoplastic tracers (periodic acid and berberine) were blocked at the hypodermis in the wild type but not in rcn1, indicating that the apoplastic barrier in the mutant was impaired. The amount of the major aliphatic suberin monomers originating from C₂₈ and C₃₀ fatty acids or ω‐OH fatty acids was much lower in rcn1 than in the wild type. These findings suggest that RCN1/OsABCG5 has a role in the suberization of the hypodermis of rice roots, which contributes to formation of the apoplastic barrier.     

Overexpression of hns in the plant growth‐promoting bacterium Enterobacter cloacae UW5 increases root colonization

Aims: Plant growth‐promoting rhizobacteria (PGPR) introduced into soil often do not compete effectively with indigenous micro‐organisms for plant colonization. The aim of this study was to identify novel genes that are important for root colonization by the PGPR Enterobacter cloacae UW5. Methods and Results: A library of transposon mutants of Ent. cloacae UW5 was screened for mutants with altered ability to colonize canola roots using a thermal asymmetric interlaced (TAIL)‐PCR‐based approach. A PCR fragment from one mutant was reproducibly amplified at greater levels from genomic DNA extracted from mutant pools recovered from seedling roots 6 days after seed inoculation compared to that from the cognate inoculum cultures. Competition assays confirmed that the purified mutant designated Ent. cloacae J28 outcompetes the wild‐type strain on roots but not in liquid cultures. In Ent. cloacae J28, the transposon is inserted upstream of the hns gene. Quantitative RT‐PCR showed that transposon insertion increased expression of hns on roots. Conclusions: These results indicate that increased expression of hns in Ent. cloacae enhances competitive colonization of roots. Significance and Impact of the Study: A better understanding of the genes involved in plant colonization will contribute to the development of PGPR that can compete more effectively in agricultural soils.     

Differential IAA dose response relations of the axr1 and axr2 mutants of Arabidopsis

In comparison to wild type Arabidopsis thaliana, the auxin resistant mutants axr1 and axr2 exhibit reduced inhibition of root elongation in response to auxins. Several auxin-regulated physiological processes are also altered in the mutant plants. When wild-type, axr1 and axr2 seedlings were grown in darkness on media containing indoleacetic acid (IAA), promotion of root growth was observed at low concentrations of IAA (10^?11 to 10^?7M) in 5-day-old axr2 seedlings, but not in axr1 or wild-type seedlings. In axr1 there was little or no measurable root growth response over the same concentration range. In wild type, root growth was inhibited at concentrations greater than 10^?10M and no detectable root growth response was observed at lower concentrations. In addition, production of lateral roots in response to IAA increased in axr2 seedlings and decreased in axr1 seedlings relative to wild type. Promotion of root elongation and initiation of lateral roots in axr2 seedlings in response to auxin indicate that axr2 seedlings are able to perceive and respond to IAA.     

Changes in the acquisition and partitioning of carbon and nitrogen in the gibberellin-deficient mutants A70 and W335 of tomato (Solanum lycopersicum L.)

Even though the growth‐promoting effects of gibberellins (GAs) in plants are well established, little is known about GA action on carbon metabolism and the available reports seem contradictory. We studied the effects of GA deficiency in mutants of tomato (Solanum lycopersicum L.) on rates of carbon acquisition and the allocation of acquired carbon to growth and respiration of leaves, stems and roots. Carbon budgets were calculated from 24 h measurements of photosynthesis and respiration. The partitioning of nitrogen compounds to leaves, stems and roots, which strongly influences carbon budgets, was also studied. The GA‐deficient mutants acquired less carbon per unit plant mass per day than did the wild type and used a larger fraction of it for root growth and root respiration. To find out to what extent these changes were just consequences of restriction of growth, the experiment was repeated at a low exponential nitrate addition rate, which forced all genotypes to grow at the same rate. Under these conditions, the low‐GA mutants still photosynthesized and respired faster and partitioned more carbon to root growth than the wild type did. The reasons for the observed differences in carbon economies between the wild type and the low‐GA mutants are discussed.     

Plant growth, nutrient acquisition and mycorrhizal symbioses of a waterlogging tolerant legume (Lotus glaber Mill.) in a saline-sodic soil

Seedlings of Lotus glaberMill., were grown in a native saline-sodic soil in a greenhouse for 50 days and then subjected to waterlogging for an additional period of 40 days. The effect of soil waterlogging was evaluated by measuring plant growth allocation, mineral nutrition and soil chemical properties. Rhizobiumnodules and mycorrhizal colonisation in L. glaberroots were measured before and after waterlogging. Compared to control plants, waterlogged plants had decreased root/shoot ratio, lower number of stems per plant, lower specific root length and less allocation of P and N to roots. Waterlogged plants showed increased N and P concentrations in plant tissues, larger root crown diameter and longer internodes. Available N and P and organic P, pH and amorphous iron increased in waterlogged soil, but total N, EC and exchangeable sodium were not changed. Soil waterlogging decreased root length colonised by arbuscular mycorrhizal (AM) fungi, arbuscular colonisation and number of entry points per unit of root length colonised. Waterlogging also increased vesicle colonisation and Rhizobium nodules on roots. AM fungal spore density was lower at the end of the experiment in non-waterlogged soil but was not reduced under waterlogging. The results indicate that L. glaber can grow, become nodulated by Rhizobium and colonised by mycorrhizas under waterlogged condition. The responses of L. glaber may be related its ability to form aerenchyma.