Effect of plant growth promoting bacterium; Pseudomonas putida UW4 inoculation on phytoremediation efficacy of monoculture and mixed culture of selected plant species for PAH and lead spiked soils

Plant growth promoting bacteria (PGPB) enhanced phytoremediation (PEP) is an attractive remedial strategy for the remediation of polycyclic aromatic hydrocarbon (PAH) and heavy metal (HM) contaminated sites. The effect of PGPB; Pseudomonas putida UW4 inoculation on the phytoremediation efficiency of Medicago sativa, Festuca arundinacea, Lolium perenne, and mixed plants (L. perenne and F. arundinacea) was assessed. This involved two contaminant treatments; “PAH” (phenanthrene; 300?mg·kg^?1, fluoranthene; 200?mg·kg^?1, and benzo[a]pyrene; 5?mg·kg^?1) and “PAH?+?HM” (‘PAH’ treatments +100?mg of Pb/kg). PGPB inoculation significantly enhanced root biomass yield of F. arundinacea in PAH treatment, and the mixed plant shoot biomass and L. perenne root biomass yields of the PAH?+?HM treatment. PGPB significantly enhanced dissipation of phenanthrene and fluoranthene for M. sativa-PAH?+?PGPB treatment and fluoranthene for F. arundinacea-PAH?+?HM?+?PGPB treatment. In others, PGPB inoculation either had no impact or inhibited PAH dissipation. PAH dissipation for the single and mixed plant treatments with PGPB inoculation were not different. The efficiency of PEP is dependent on different factors such as PGPB inoculum biomass, plant species, plant–microbe specificity and type of contaminants. Exploiting PEP technology would require proper understanding of plant tolerance and growth promoting mechanisms, and rhizosphere activities.     

Effects of GA3 on Plant Physiological Properties,Extraction, Subcellular Distribution and Chemical Forms of Pb in Lolium perenne

The effects of growth-promoting hormone gibberellic acid 3 (GA₃) on physiology, Pb phytoextraction, and metal detoxification mechanisms in Lolium perenne were studied. Results showed that addition of GA₃ alone at lower doses (1 or 10 μM) facilitated antioxidant defense of L. perenne under Pb stress, decreased the toxicity of Pb in plant shoot by increasing the proportion of Pb in cell wall, hence significantly enhanced photosynthesis and plant growth, as well as Pb uptake and accumulation in L. perenne (P < 0.05). However, these indicators showed the opposite changes when treated with GA₃ at a higher dose (100 μM). Of the total Pb in plant shoot, 36–51% was associated with cell wall, and 31–40% was soluble fraction, while 41.4–49.7% was NaCl extractable, 24.6–35.4% HAc extractable followed by other fractions. These findings suggest that Pb fixation by pectates and proteins in cell wall and sequestration in vacuole are responsible for Pb detoxification in plant, and the GA₃ at 1 μM appears to be optimal for enhancing Pb phytoextraction by L. perenne from Pb polluted soils.     

柠檬酸和EDTA-Na2对黑麦草吸收Pb及营养元素特性的影响

为探究柠檬酸或EDTA-Na_2对Pb污染下黑麦草(Lolium perenne L.)吸收Pb和营养元素特性的影响,对水培黑麦草进行不同处理,研究黑麦草一些生理生化指标的变化。结果表明,与对照相比,Pb处理降低黑麦草干重,增加质膜透性和根系脱氢酶活性,且在叶和根中积累Pb,而叶和根中6种营养元素含量的变化不尽相同。与Pb处理同时加入低浓度的柠檬酸或EDTA-Na_2对其生长影响较小,且叶片的Pb积累量较低;而同时加入高浓度的柠檬酸或EDTA-Na_2,虽然强化Pb在叶片中的积累,但是加重了生长的抑制作用和营养元素的稳态失衡;1 mmol L~(–1)的柠檬酸强化叶片积累Pb的效应强于同浓度的EDTA-Na_2,而5和10 mmol L~(–1)柠檬酸的强化作用则弱于同浓度的EDTA-Na_2。因此,适当浓度的柠檬酸或EDTA-Na_2在治理Pb污染环境中具有一定作用。     

Effect of Medicago sativa L. and compost on organic and inorganic pollutant removal from a mixed contaminated soil and risk assessment using ecotoxicological tests

Several Gentle Remediation Options (GRO), e.g., plant-based options (phytoremediation), singly and combined with soil amendments, can be simultaneously efficient for degrading organic pollutants and either stabilizing or extracting trace elements (TEs). Here, a 5-month greenhouse trial was performed to test the efficiency of Medicago sativa L., singly and combined with a compost addition (30% w/w), to treat soils contaminated by petroleum hydrocarbons (PHC), Co and Pb collected at an auto scrap yard. After 5 months, total soil Pb significantly decreased in the compost-amended soil planted with M. sativa, but not total soil Co. Compost incorporation into the soil promoted PHC degradation, M. sativa growth and survival, and shoot Pb concentrations [3.8 mg kg^?1 dry weight (DW)]. Residual risk assessment after the phytoremediation trial showed a positive effect of compost amendment on plant growth and earthworm development. The O₂ uptake by soil microorganisms was lower in the compost-amended soil, suggesting a decrease in microbial activity. This study underlined the benefits of the phytoremediation option based on M. sativa cultivation and compost amendment for remediating PHC- and Pb-contaminated soils.     

Alfalfa plant-derived biostimulant stimulate short-term growth of salt stressed Zea mays L. plants

Background & Aims

The effects of an alfalfa plant (Medicago sativa L.) hydrolysate-based biostimulant (EM) containing triacontanol (TRIA) and indole-3-acetic acid (IAA) were tested in salt-stressed maize plants.

Methods

Plants were grown for 2 weeks in the absence of NaCl or in the presence (25, 75 and 150 mM). On the 12th day, plants were supplied for 48 h with 1.0 mg L^?1 EM or 11.2 μM TRIA.

Results

EM and TRIA stimulated the growth and nitrogen assimilation of control plants to a similar degree, while NaCl reduced plant growth, SPAD index and protein content. EM or TRIA increased plant biomass under salinity conditions. Furthermore, EM induced the activity of enzymes functioning in nitrogen metabolism. The activity of antioxidant enzymes and the synthesis of phenolics were induced by salinity, but decreased after EM treatment. The enhancement of phenylalanine ammonia-lyase (PAL) activity and gene expression by EM was consistent with the increase of flavonoids.

Conclusion

The present study proves that the EM increases plant biomass even when plants are grown under salinity conditions. This was likely because EM stimulated plant nitrogen metabolism and antioxidant systems. Therefore, EM may be proposed as bioactive product in agriculture to help plants overcome stress situations.     

Efficacy of Biosolids in Assisted Phytostabilization of Metalliferous Acidic Sandy Soils with Five Grass Species

The role of sewage sludge as an immobilising agent in the phytostabilization of metal-contaminated soil was evaluated using five grass species viz., Dactylis glomerata L., Festuca arundinacea Schreb., F. rubra L., Lolium perenne L., L. westerwoldicum L. The function of metal immobilization was investigated by monitoring pH, Eh and Cd, Pb, and Zn levels in column experiment over a period of 5-months. Grasses grown on sewage sludge-amendments produced high biomass in comparison to controls. A significant reduction in metal uptake by plants was also observed as a result of sewage sludge application, which was attributed to decreased bioavailability through soil stabilisation. We have observed that the sludge amendment decreased metal bioavailability and concentrations in soil at a depth of 25 cm, in contrast to untreated columns, where metal concentrations in the soil solution were very high.     

Variation in seedling growth of 11 perennial legumes in response to phosphorus supply

Phosphorus (P) deficiency is a major problem for Australian agriculture. Development of new perennial pasture legumes that acquire or use P more efficiently than the current major perennial pasture legume, lucerne (Medicago sativa L.), is urgent. A glasshouse experiment compared the response of ten perennial herbaceous legume species to a series of P supplies ranging from 0 to 384 µg g^?1 soil, with lucerne as the control. Under low-P conditions, several legumes produced more biomass than lucerne. Four species (Lotononis bainesii Baker, Kennedia prorepens F.Muell, K. prostrata R.Br, Bituminaria bituminosa (L.) C.H.Stirt) achieved maximum growth at 12 µg P g^?1 soil, while other species required 24 µg P g^?1. In most tested legumes, biomass production was reduced when P supply was ≥192 µg g^?1, due to P toxicity, while L. bainesii and K. prorepens showed reduced biomass when P was ≥24 µg g^?1 and K. prostrata at ≥48 µg P g^?1 soil. B. bituminosa and Glycine canescens F.J.Herm required less soil P to achieve 0.5 g dry mass than the other species did. Lucerne performed poorly with low P supply and our results suggest that some novel perennial legumes may perform better on low-P soils.     

Phytoremediation of Oil-Sludge–Contaminated Soil

The aim of this research was to select plant species that could be effective in the phytoremediation of a former oil-sludge pit. Seven crop plants (Triticum aestivum L., Secale cereale L., Avena sativa L., Hordeum vulgare, Sorghum bicolor L. Moench, Panicum miliaceum L., and Zea mays L.), five wild grasses (Lolium perenne L., Bromopsis inermis, Agropyron cristatum L., Agropyrum tenerum L., and Festuca pratensis Huds.), and three legumes (Medicago sativa L., Trifolium pratense L., and Onobrychis antasiatica Khin.) were screened for phytotoxicity, including the assessment of germination, shoot biomass, and root biomass, in a pot experiment. The estimation of oil-sludge degradation in the root zone of the tested plants showed that rye accelerated cleanup most effectively, degrading all of the main contaminant fractions in the oil sludge by a total of 52%. Although alfalfa had a lower phytoremediation potential than did rye, it maintained large numbers of soil microorganisms, including polycyclic aromatic hydrocarbon degraders, in its rhizosphere. Rye and alfalfa were chosen for a large-scale study to remediate an oil-sludge pit on the grounds of a petroleum refinery. Remediation monitoring confirmed the effectiveness of rye: the oil-sludge content decreased consistently for 3 years and remained low in comparison with the results from other plant species.     

Competition between Lolium perenne and Digitaria sanguinalis: Ecological consequences for harbouring an endosymbiotic fungus

Abstract. This study examined the influence of the fungal endophyte Neotyphodium lolii on the competitive interactions between its perennial, cool season host, Lolium perenne (perennial ryegrass), and a warm season, annual grass, Digitaria sanguinalis (large crabgrass), in densely planted stands (>1000 plant.m^?2) in the glasshouse. Endophyte infection had little or no effect on L. perenne tiller production, above‐ or below‐ground biomass or root: shoot ratio in monoculture. However, endophyte infection significantly reduced L. perenne tiller production and above‐ground biomass in mixtures with D. sanguinalis. Conversely, D. sanguinalis had significantly higher above‐ground biomass and yielded more seed (g) when competing with endophyte infected L. perenne. An apparent trade‐off between allocation of resources to reproductive vs root tissues was observed in D. sanguinalis– root: shoot ratio was significantly lower when competing with endophyte infected L. perenne. Results indicate negative ecological consequences for harbouring the fungal endophyte when competing with the fast growing annual grass in newly established stands. These findings underscore the existence of a physiological cost of harbouring the fungal endophyte which is often overlooked.     

Enhanced Pb Absorption by Hordeum vulgare L. and Helianthus annuus L. Plants Inoculated with an Arbuscular Mycorrhizal Fungi Consortium

The effect of an arbuscular mycorrhizal fungi (AMF) consortium conformed by (Glomus intraradices, Glomus albidum, Glomus diaphanum, and Glomus claroideum) on plant growth and absorption of Pb, Fe, Na, Ca, and ³²P in barley (Hordeum vulgare L.) and sunflower (Helianthus annuus L.) plants was evaluated. AMF-plants and controls were grown in a substrate amended with powdered Pb slag at proportions of 0, 10, 20, and 30% v/v equivalent to total Pb contents of 117; 5,337; 13,659, and 19,913 mg Pb kg^?1 substrate, respectively. Mycorrhizal root colonization values were 70, 94, 98, and 90%, for barley and 91, 97, 95, and 97%, for sunflower. AMF inoculum had positive repercussions on plant development of both crops. Mycorrhizal barley absorbed more Pb (40.4 mg Pb kg^?1) shoot dry weight than non-colonized controls (26.5 mg Pb kg^?1) when treated with a high Pb slag dosage. This increase was higher in roots than shoots (650.0 and 511.5 mg Pb kg^?1 root dry weight, respectively). A similar pattern was found in sunflower. Plants with AMF absorbed equal or lower amounts of Fe, Na and Ca than controls. H. vulgare absorbed more total P (1.0%) than H. annuus (0.9%). The arbuscular mycorrizal consortium enhanced Pb extraction by plants.     

Development and mapping of a public reference set of SSR markers in Lolium perenne L.

We report on the characterization and mapping of 76 simple sequence repeat (SSR) markers for Lolium perenne. These markers are publicly available or obtained either from genomic libraries enriched for SSR motifs or L. perenne expressed sequence tag (EST) clones. Four L. perenne mapping populations were used to map the SSR markers. A consensus linkage map of the four mapping populations containing 65 of the SSR markers is presented, together with primer information and a quality score indicating the usefulness of the SSR marker in different populations. The SSR markers identified all seven L. perenne linkage groups.     

LEGUME-GRASS INTERCROPPING PHYTOREMEDIATION OF PHTHALIC ACID ESTERS IN SOIL NEAR AN ELECTRONIC WASTE RECYCLING SITE: A FIELD STUDY

A field experiment was conducted to study the phytoremediation of phthalic acid esters (PAEs) by legume (alfalfa, Medicago sativa L.)-grass (perennial ryegrass, Lolium perenne L. and tall fescue, Festuca arundinacea) intercropping in contaminated agricultural soil at one of the largest e-waste recycling sites in China. Two compounds, DEHP and DnBP, were present in the soil and in the shoots of the test plants at much higher concentrations than the other target PAEs studied. Over 80% of ‘total’ (i.e., all six) PAEs were removed from the soil across all treatments by the end of the experiment. Alfalfa in monoculture removed over 90% of PAEs and alfalfa in the intercrop of the three plant species contained the highest shoot concentration of total PAEs of about 4.7 mg kg^?1 DW (dry weight). Calculation of phytoextraction efficiency indicated that the most effective plant combinations in eliminating soil PAEs were the three-species intercrop (1.78%) and the alfalfa monocrop (1.41%). Phytoremediation with alfalfa was effective in both monoculture and intercropping. High bioconcentration factors (BCFs) indicated the occurrence of significant extraction of PAEs by plants from soil, suggesting that phytoremediation may have potential for the removal of PAEs from contaminated soils.     

C and N allocation in soil under ryegrass and alfalfa estimated by 13C and 15N labelling

Background and Aims

Below-ground translocated carbon (C) released as rhizodeposits is an important driver for microbial mobilization of nitrogen (N) for plants. We investigated how a limited substrate supply due to reduced photoassimilation alters the allocation of recently assimilated C in plant and soil pools under legume and non-legume species.

Methods

A non-legume (Lolium perenne) and a legume (Medicago sativa) were labelled with ¹⁵N before the plants were clipped or shaded, and labelled twice with ¹³CO₂ thereafter. Ten days after clipping and shading, the ¹⁵N and ¹³C in shoots, roots, soil, dissolved organic nitrogen (DON) and carbon (DOC) and in microbial biomass, as well as the ¹³C in soil CO₂ were analyzed.

Results

After clipping, about 50 % more ¹³C was allocated to regrowing shoots, resulting in a lower translocation to roots compared to the unclipped control. Clipping also reduced the total soil CO₂ efflux under both species and the ¹³C recovery of soil CO₂ under L. perenne. The ¹⁵N recovery increased in the shoots of M. sativa after clipping, because storage compounds were remobilized from the roots and/or the N uptake from the soil increased. After shading, the assimilated ¹³C was preferentially retained in the shoots of both species. This caused a decreased ¹³C recovery in the roots of M. sativa. Similarly, the total soil CO₂ efflux under M. sativa decreased more than 50 % after shading. The ¹⁵N recovery in plant and soil pools showed that shading has no effect on the N uptake and N remobilization for L. perenne, but, the ¹⁵N recovery increased in the shoot of M. sativa.

Conclusions

The experiment showed that the dominating effect on C and N allocation after clipping is the need of C and N for shoot regrowth, whereas the dominating effect after shading is the reduced substrate supply for growth and respiration. Only slight differences could be observed between L. perenne and M. sativa in the C and N distribution after clipping or shading.     

A Modified Self-thinning Equation to Describe Size/Density Relationships for Defoliated Swards

Use of the self-thinning rule to describe size/density compensation(SDC) in defoliated swards is examined. It is shown that defoliationrelated variation in leaf area and associated morphogeneticchanges in plant structure necessitate slope corrections, designatedC_a and C_r , respectively. The theory predicts that reduced leafarea in more heavily defoliated swards will result in SDC atslopes more negative than -3/2 (variable leaf area SDC), andthat there will be a transition to -3/2 (constant leaf area)SDC at higher herbage mass. Empirical data from previous experiments with Lolium perenneL. and Medicago sativa L. are examined, and appear to confirmthe theoretical predictions, including the slope change at thepoint of transition from variable to constant leaf area SDC.This transition point, designated d_i , is subject to interspecificvariation and is related to the mature shoot size of a particularspecies. Some applications of this theory are discussed, andin particular a sward productivity index is proposed.Copyright1995, 1999 Academic Press Variable leaf area self-thinning, size/density compensation, Lolium perenne, Medicago sativa, sward productivity index     

Efficiency of Opuntia ficus in the phytoremediation of a soil contaminated with used motor oil and lead,compared to that of Lolium perenne and Aloe barbadensis

Industrial pollutants such as heavy metals and hydrocarbons in soils represent a serious concern due to their persistence and negative effects on the environment, affecting cellular processes in living organisms and even causing mutations and cancer. The main objectives of this work were to evaluate the efficiency of Opuntia ficus in the phytoremediation of a soil polluted with used motor oil. Two other species, one with different and one with similar characteristics, relatively, were used for comparison purposes: Lolium perenne and Aloe barbadensis. The effect of the plants on lead solubility and bioaccumulation, the biomass production of each specie and the microbial counts and bacterial identification for each experiment was studied. Total petroleum hydrocarbons (TPH) were measured every 5 weeks throughout the 20-week phytoremediation experiment. At the end of the experiment soluble Pb, Pb extracted by the plant species, microbiological counts, total biomass and bacterial species in soil were analyzed. Even though Lolium perenne showed the highest TPH removal (47%), Opuntia ficus produced the highest biomass and similar removal (46%). Since Opuntia ficus requires low amounts of water and grows fast, it would be a suitable option in the remediation of soils polluted with hydrocarbons and/or heavy metals.     

基于容器分区处理探究黑麦草生长对喀斯特不同土壤生境和水分的响应

为了探究不同水分条件下喀斯特地区分布不均、厚薄不一土壤小生境对禾本科草本植物生长的影响,用3种不同深度的容器(对照深度CK,深土D和浅土S)两两组合为6种复合容器(CK-CK、CK-S、CK-D、D-D、S-D和S-S)以实现容器分区,研究了黑麦草的根系生长、生物量积累及其分配特征。结果表明:1)在水分充足(W_0)条件下,组合了浅土容器和深土容器的处理中,黑麦草的根系生长(根长、根直径、根表面积和根生物量)均低于对照容器(CK-CK),且有浅土容器的组合处理(S-S,S-D,CK-S)受抑制程度大于有深土容器的组合处理(CK-D,D-D);当水分含量降低后,即中水(W_1)和低水(W_2)条件下,有深土容器的组合[D-D和(或)CK-D]根系生长与对照相比显著增加,而有浅土容器的组合[S-S和(或)CK-S]根系生长与对照相比显著降低。2)对比同一处理不同容器分区中黑麦草生长指标发现,在水分充足情况下,深土容器和浅土容器均会抑制植物生长,而当水分减少,S区根系生长被严重抑制,但D区根系增长优势明显。3)水分充足条件下,根冠比未受到显著影响;当水分降低时,组合了深土容器的处理根冠比均有升高的趋势,组合了浅土容器的处理根冠比有降低趋势。由此可见,不同土壤生境带来的物理空间限制会影响植物根系生长和生物量积累与分配,但水分的减少会改变根系生长及生物量积累对不同土壤生境的响应:在水分充足时,土壤物理空间是影响根系生长和生物量积累与分配的主要因子,黑麦草主要发展浅层根系。而当水分减少时,黑麦草根系在浅层土壤中无法获取供给生长代谢活动的足量水分,更倾向于将有限的有机物分配给根,通过根系伸长、表面积和体积增大、直径增粗等策略加强水分吸收,从而增强对干旱的抗逆性,提高对土壤和水分异质性的适应。     

Growth Substances Isolated from Tubers of Cyperus esculentus var. aureus

A methanolic extract of tubers of Cyperus esculentus Ten. var. aureus Richt. contains compounds which inhibit growth of oat coleoptile sections and germination of Beta vulgaris L., Lotus corniculatus L., Lolium perenne L., Pisum sativum L., Trifolium repens L., Lactuca sativa L. and Lycopersicum esculentum Ludwig. p-Hydroxybenzoic acid, vanillic acid, syringic acid, ferulic acid and p-coumaric acid were identified. Four other active compounds which could not be identified were also isolated.     

Response of a root hemiparasite to elevated CO2 depends on host type and soil nutrients

Although elevated CO₂ may affect various forms of ecological interactions, the effect of elevated CO₂ on interactions between parasitic plants and their hosts has received little attention. We examined the effect of elevated CO₂ (590 μl l⁻¹) at two nutrient (NPK) levels on the interactions of the facultative root hemiparasite Rhinanthus alectorolophus with two of its hosts, the grass Lolium perenne and the legume Medicago sativa. To study possible effects on parasite mediation of competition between hosts, the parasite was grown with each host separately and with both hosts simultaneously. In addition, all combinations of hosts were grown without the parasite. Both the parasite and the host plants responded to elevated CO₂ with increased growth, but only at high nutrient levels. The CO₂ response of the hemiparasite was stronger than that of the hosts, but depended on the host species available. With L. perenne and M. sativa simultaneously available as hosts, the biomass of the parasite grown at elevated CO₂ was 5.7 times that of parasites grown at ambient CO₂. Nitrogen concentration in the parasites was not influenced by the treatments and was not related to parasite biomass. The presence of the parasite strongly reduced both the biomass of the hosts and total productivity of the system. This effect was much stronger at low than at high nutrient levels, but was not influenced by CO₂ level. Elevated CO₂ did not influence the competitive balance between the two different hosts grown in mixture. The results of this study support the hypothesis that hemiparasites may influence community structure and suggest that these effects are robust to changes in CO₂ concentration. Received: 17 August 1998 / Accepted: 3 March 1999     

Variation in fine root biomass of three European tree species: Beech (Fagus sylvatica L.), Norway spruce (Picea abies L. Karst.), and Scots pine (Pinus sylvestris L.)

Abstract

Fine roots (<2 mm) are very dynamic and play a key role in forest ecosystem carbon and nutrient cycling and accumulation. We reviewed root biomass data of three main European tree species European beech, (Fagus sylvatica L.), Norway spruce (Picea abies L. Karst.) and Scots pine (Pinus sylvestris L.), in order to identify the differences between species, and within and between vegetation zones, and to show the relationships between root biomass and the climatic, site and stand factors. The collected literature consisted of data from 36 beech, 71 spruce and 43 pine stands. The mean fine root biomass of beech was 389 g m^?2, and that of spruce and pine 297 g m^?2 and 277 g m^?2, respectively. Data from pine stands supported the hypothesis that root biomass is higher in the temperate than in the boreal zone. The results indicated that the root biomass of deciduous trees is higher than that of conifers. The correlations between root biomass and site fertility characteristics seemed to be species specific. There was no correlation between soil acidity and root biomass. Beech fine root biomass decreased with stand age whereas pine root biomass increased with stand age. Fine root biomass at tree level correlated better than stand level root biomass with stand characteristics. The results showed that there exists a strong relationship between the fine root biomass and the above-ground biomass.     

Direct shoot formation in spontaneously occurring root pseudonodules of alfalfa (Medicago sativa L.)

Summary A simple and rapid procedure for direct organogenesis from root nodulelike structures of alfalfa (Medicago sativa L.) line SGg, spontaneously induced on growth regulator-free Gamborg (B₅) medium, was developed. Prolific adventitious shoot initiation was obtained using a combination of 1.0 mg/liter TIBA and 0.5 mg/liter 2iP. Transfer of shoots to a medium containing 0.5 mg/liter ABA and reduced concentration of TIBA (0.5 mg/liter) before rooting markedly stimulated shoot development. Regenerated shoots rooted easily and revealed the early appearance of nodulelike structures on basal medium (B₅) lacking growth regulators. Analysis of endogenous growth regulator levels of SGg roots maintained on growth regulators free media, showed that spontaneous shoot appearances was correlated with high cytokinin-to-auxin ratios.