Regulation of growth in rice seedlings

Etiolated rice seedlings (Oryza sativa L.) exhibited marked morphological differences when grown in sealed containers or in containers through which air was passed continuously. Enhancement of coleoptile and mesocotyl growth and inhibition of leaf and root growth in the sealed containers (enclosure syndrome) were accompanied by accumulation of CO₂ and C₂H₄ in and depletion of O₂ from the atmosphere. Ethylene (1 l 1^–1), high levels of CO₂, and reduced levels of O₂ contributed equally to the increase in coleoptile and mesocotyl growth. The effect of enclosure could be mimicked by passing a gas mixture of 3% O₂, 82% N₂, 15% CO₂ (all v/v), and 1 l l^–1) C₂H₄ through the vials containing the etiolated seedlings. The effects of high CO₂ and low O₂ concentrations were not mediated through increased C₂H₄ production. The enclosure syndrome was also observed in rice seedlings grown under water either in darkness or in light. The length of the rice coleoptile was positively correlated with the depth of planting in water-saturated vermiculite. The length of coleoptiles of wheat, barley, and oats was not affected by the depth of planting. In rice, the length of coleoptile was determined by the levels of O₂, CO₂, and ethylene, rather than by light. This regulatory mechanism allows rice seedlings to grow out of shallow water in which the concentration of O₂ is limiting.     

Gaseous factors involved in the enhanced elongation of rice coleoptiles under water

Abstract. Elongation responses of intact coleoptiles of rice (Oryza sativa L. ev. Sasanishiki) explants to various gases were examined under submerged conditions in continuously flowing gas-saturated incubation media. Reduced O₂ tension (hypoxia). CO₂ and especially C₂H₄ significantly stimulated coleoptile elongation; the optimal concentrations of O₂, CO₂ and C₂H₄ when applied singly were 0.07 m³ m^-3, 0.10 m³ m^-3, and 3 cm³, respectively. However, in addition to these gases other as yet unknown factors were involved in the enhanced elongation of rice coleoptiles under water. The actions of CO₂ and C₂H₄, unlike that of hypoxia, were accompanied by increases in dry weight of the coleoptiles. The effect of C₂H₄ occurred independently of O₂ concentrations, whereas that of CO₂ occurred above 0.08 m³ m^-3O₂. Maximum elongation of rice coleoptiles under submerged conditions was obtained when the flowing medium was saturated with a gas mixture containing 0.10 m³ m^-3 O₂, 0.10 m³ m^-3 CO₂ and 10 cm³ m^-3 C₂H₄, greatly surpassing elongation in static media. However, elongation in static media was greater than that in a closed atmosphere. The intercellular C₂H₄ concentration in explants growing in static media was higher than that in a closed atmosphere. These results showed that the coleoptile elongation of rice seedlings under water may be regulated by the accumulation of CO₂ and C₂H₄ in and around the seedlings under hypoxic conditions.     

Ethylene generation and reversal of ethylene effects during development in vitro of rapid-cycling Brassica campestris L

A rapid-cycling line of Brassica compestris L was grown from seed to maturity in vitro. Ethylene accumulated in culture containers throughout the growth period when these were tightly sealed (i.e. closed with aluminum foil, tight plastic caps, parafilm or rubber stoppers), whereas no C₂H₄ was detected in vented containers (i.e. closed with foam or cotton plugs, or loosely fitted plastic caps). Under both closure conditions, mean levels of CO₂, O₂ and N₂ were similar to atmospheric levels. The accumulation of C₂H₄ in sealed containers was associated with inhibition of plant development. Plants grown in a sealed condition had swollen hypocotyls, fewer and smaller leaves and either abortive flowers or no floral buds whereas plants in vented containers developed normally to maturity. Significant reversion to normal development was observed when plants growing in sealed containers were treated with 2,5-norbornadie e (NBD), an anti-C₂H₄ compound. Treatment with NBD offers a promising way of regulating C₂H₄ effects in vitro.     

Ethylene and the growth of rice seedlings

Etiolated whole rice seedlings enclosed in sealed vials produced ethylene at a rate of 0.9 picomole per hour per seedling. When 2-centimeter-long shoots were subdivided into 5-millimeter-long sections, the sections containing the tip of the shoot evolved 37% of the total ethylene with the remaining 63% being produced along a gradient decreasing to the base of the shoot. The tip of the coleoptile also had the highest level of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid and of the ethylene-forming enzyme activity. Ethylene is one of the factors controlling coleoptile elongation. Decapitation of the seedling reduced ethylene evolution to one-third its original level and inhibited coleoptile growth. In short-term experiments, the growth rate of decapitated seedlings was restored to almost that of intact seedlings by application of ethylene at a concentration of 10 microliters per liter. Apart from ethylene, O₂ also participates in the control of coleoptile growth. When rice seedlings were grown in a gas mixture of N₂ and O₂, the length of the coleoptiles reached a maximum at a concentration of 2.5% O₂. Lower and higher concentrations of O₂ reduced coleoptile growth. The effect of exogenous ethylene on coleoptile growth was also O₂ dependent.     

Light or ethylene treatments induce transverse cell enlargement in etiolated maize mesocotyls

Dark-grown maize seedlings (hybrid WF-9 × 38-11) exposed for 1 or more hours to white light and then returned to darkness developed mesocotyls with enlarged apical diameters. This swelling response was an all-or-none response, and the fraction of the seedling population that showed the response depended on seedling age at irradiation. Irradiation of the coleoptile alone was nearly as effective in causing this response as was irradiation of the nodal region of the epicotyl, but irradiation of the mesocotyl base was ineffective. Removal of the coleoptile prior to irradiation did not prevent the formation of the light-induced swelling. Exogenously applied C₂H₄ (10 microliters per liter) for 24 hours in dark also induced swelling of the mesocotyl. The swelling induced in the intact seedlings was localized in the apical mesocotyl tissues with either light or C₂H₄ treatment, and maximal response to both treatments occurred with 3- to 4-day-old seedlings. Swelling of the mesocotyl was the result of transverse cell enlargement, not increase of cell numbers. The evidence suggests that light and C₂H₄ induce mesocotyl swelling in intact maize shoots by a common mechanism.     

Further Observations on the Effect of Carbon Dioxide on the Growth of Etiolated Avena Seedlings

Oat seedlings when grown in 5 per cent. CO² show an increasedgrowth of the mesocotyl and a reduced growth of the coleoptile.To elucidate this effect the following data, for both treatedand control plants, have been obtained: (1) fresh and dry weightincreases in the whole plants and in the separate organs, (2)water contents, and (3) progressive depletion of the endosperm. The enhanced growth of the mesocotyl in CO² was closely, correlatedwith dry-matter content but not with water content. The depletionof the endosperm was reduced by treatment with CO², and of thesmaller amount of carbohydrate entering the embryo a higherproportion remained in the mesocotyl; coleoptile growth wasconsequently diminished. The distribution of dry matter betweenthe coleoptile and mesocotyl must be an important factor intheir reciprocal growth behaviour.     

The Analysis of Correlative Growth in the Etiolated Oat Seedling in Relation to Carbon Dioxide and Nutrient Supply

To overcome the reduced extension growth of the coleoptile whichoccurs when oats are grown in air enriched with 5 per cent.CO¹, plants have been provided with nutrients via the roots.2 per cent, sucrose, glucose or mannitol so applied furtherpromoted the mesocotyl and further depressed the coleoptile.Root growth was also depressed. To induce promotion of coleoptile growth by externally appliedsucrose, seedlings were heated in darkness at 40° C. for3 hours so restricting selectively the growth of the mesocotyl.Promotion of the coleoptile, however, was not observed. Application of mixed Na and K nitrates occasioned an immediategrowth promotion of doleoptile and leaves in both the presenceand absence of CO², and also a.much less pronounced promotionof the mesocotyl in CO²; there was no effect in air. This enhancedgrowth of the coleoptile and leaves was coupled with a correspondinglygreater dry weight and also with an increased outflow of reservesfrom the endosperm into the plumule. Thus, while externally applied sugars seemed not to reach thecoleoptile, those made available from the endosperm as a resultof improved nitrogen supply were rapidly translocated to it.Simultaneous provision to the roots of nitrate and sucrose didnot improve the absorption and translocation of sugar. An analysis of covariance has been computed using the mesocotyland coleoptile length data together with the outflow from theendosperm and the conclusions so derived are discussed in relationto the problem of growth integration in etiolated oat seedlings.     

Effects of 2,5-norbornadiene on cocklebur seed germination and rice coleoptile elongation in response to CO2 and C2H4

To examine in more detail the mechanisms of cocklebur (Xanthium pennsylvanicum Wallr.) seed germination and rice (Oryza sativa L. cv. Sasanishiki) coleoptile elongation that were responsive to both C₂H₄ and CO₂, the effects of NBD (2,5-norbornadiene), a cyclic olefin known as a competitive inhibitor of C₂H₄, on those phenomena were tested under various conditions. NBD strongly inhibited germination of cocklebur seeds and their axial and cotyledonary growth. The NBD effects were significantly negated by endogenously evolved and exogenously applied CO₂ regardless of incubation temperature. Similarly, the inhibitory NBD effect was negated by C₂H₄ at 23°C, but at 33°C a low concentration (3 1/L) of C₂H₄ rather enhanced the inhibitory NBD effect. This phenomenon reflected the growth responses of the tip zone of axial tissues in cocklebur seeds to NBD and C₂H₄, in which both gases were antagonistic in regulating the axial growth at 23°C but additive in inhibiting it at 33°C. Maximal negation of these inhibitory NBD effects was brought about by simultaneous application of CO₂ and C₂H₄. Similarly, elongation of rice coleoptiles was suppressed by NBD, and when they were immature, its inhibitory action was counteracted by both C₂H₄ and CO₂, especially during simultaneous application. However, the inhibitory NBD effect was completely negated by C₂H₄ applied alone at concentrations above 500 1/L regardless of the physiological age of coleoptiles. These inhibitory NBD effects are additional evidence suggesting that C₂H₄ acts as a growth regulator in both cocklebur seed germination and rice coleoptile elongation. That NBD was capable of counteracting CO₂ action in some cases but was incapable of negating inhibitory C₂H₄ action, such as that observed in cocklebur seeds, suggests that NBD acts with some side effects besides being a competitive inhibitor of C₂H₄ actions.     

Inhibitory action of red light on the growth of the maize mesocotyl: evaluation of the auxin hypothesis

Brief irradiation of 3-d-old maize (Zea mays L.) seedlings with red light (R; 180 J m^-2) inhibits elongation of the mesocotyl (70–80% inhibition in 8 h) and reduces its indole-3-acetic acid (IAA) content. The reduction in IAA content, apparent within a few hours, is the result of a reduction in the supply of IAA from the coleoptile unit (which includes the shoot apex and primary leaves). The fluence-response relationship for the inhibition of mesocotyl growth by R and far-red light closely resemble those for the reduction of the IAA supply from the coleoptile. The relationship between the concentration of IAA (1–10 M) supplied to the cut surface of the mesocotyl of seedlings with their coleoptile removed and the growth increment of the mesocotyl, measured after 4 h, is linear. The hypothesis that R inhibits mesocotyl growth mainly by reducing the IAA supply from the coleoptile is supported. However, mesocotyl growth in seedlings from which the coleoptiles have been removed is also inhibited by R (about 25% inhibition in 8 h). This inhibition is not related to changes in the IAA level, and not relieved by applied IAA. In intact seedlings, this effect may also participate in the inhibition of mesocotyl growth by R. Inhibition of cell division by R, whose mechanism is not known, will also result in reduced mesocotyl elongation especially in the long term (e.g. 24 h).Abbreviations FR far-red light - IAA indole-3-acetic acid - P_fr phytochrome in the far-red-absorbing form - P_r phytochrome in the red-absorbing form - R red light     

Committee for the study of the Scottish Flora

Summary

Three different oxygen-containing germination environments demonstrate the profound influence exerted by environmental oxygen (0₂) on growth and plastogenesis in coleoptiles of light-germinated rice seedlings. Coleoptile greening is extensive in low numbers of seedlings germinated in a sealed, initially air-saturated, static water environment and in large numbers of seedlings germinated under unagitated water underambient gaseous exchange conditions. In seedlings germinated in air (?21% 0₂), coleoptile greening is sparse and extension growth is much reduced compared with coleoptile extension growth of the submerged seedlings. Coleoptile greening and shoot and root growth are completely inhibited under hypoxia resulting from large numbers of germinating seedlings competing for the limited 0₂ supply in the sealed, initially air-saturated, static water environment. Coleoptile extension growth is highest under hypoxia and lowest under ?21% 0₂. The observations presented here demonstrate that 0₂ stress and non- stress conditions serve as environmental signals which influence growth behaviour and plastogenesis in coleoptiles of light-germinated rice seedlings.     

Toxicity in leaves of rice exposed to cadmium is due to hydrogen peroxide accumulation

The production of H₂O₂ in detached rice leaves of Taichung Native 1 (TN1) caused by CdCl₂ was investigated. CdCl₂ treatment resulted in H₂O₂ production in detached rice leaves. Diphenyleneiodonium chloride (DPI) and imidazole (IMD), inhibitors of NADPH oxidase (NOX), prevented CdCl₂-induced H₂O₂ production, suggesting that NOX is a H₂O₂-genearating enzyme in CdCl₂-treated detached rice leaves. Phosphatidylinositol 3-kinase inhibitors wortmanin (WM) or LY294002 (LY) inhibited CdCl₂-inducted H₂O₂ production in detached rice leaves. Exogenous H₂O₂ reversed the inhibitory effect of WM or LY, suggesting that phosphatidylinositol 3-phosphate is required for Cd-induced H₂O₂ production in detached rice leaves. Nitric oxide donor sodium nitroprusside (SNP) was also effective in reducing CdCl₂-inducing accumulation of H₂O₂ in detached rice leaves. Cd toxicity was judged by the decrease in chlorophyll content. The results indicated that DPI, IMD, WM, LY, and SNP were able to reduce Cd-induced toxicity of detached rice leaves. Twelve-day-old TN1 and Tainung 67 (TNG67) rice seedlings were treated with or without CdCl₂. In terms of Cd toxicity (leaf chlorosis), it was observed that rice seedlings of cultivar TN1 are Cd-sensitive and those of cultivar TNG67 are Cd-tolerant. On treatment with CdCl₂, H₂O₂ accumulated in the leaves of TN1 seedlings but not in the leaves of TNG67. Prior exposure of TN1 seedlings to 45^oC for 3 h resulted in a reduction of H₂O₂ accumulation, as well as Cd tolerance of TN1 seedlings treated with CdCl₂. The results strongly suggest that Cd toxicity of detached leaves and leaves attached to rice seedlings are due to H₂O₂ accumulation.     

Regulation of growth in stem sections of deep-water rice

Submergence in water greatly stimulates internodal elongation in excised stem sections of deep-water rice (Oryza sativa L. cv. Habiganj Aman II) and inhibits growth of leaf blades and leaf sheaths. The highest rates of internodal growth have been observed in continuous light. Very little growth occurs in submerged sections kept in darkness or incubated under N₂ in the light. The effect of submergence on the growth of deep-water rice is, at least in part, mediated by C₂H₄, which accumulates in the air spaces of submerged sections. This accumulation results from increased C₂H₄ synthesis in the internodes of submerged sections and reduced diffusion of C₂H₄ from the tissue into the water. Increased C₂H₄ levels accelerate internodal elongation and inhibit the growth of leaves. Compounds capable of changing the rate of C₂H₄ synthesis, namely aminoethoxyvinylglycine, an inhibitor of C₂H₄ synthesis, and 1-aminocyclopropane-1-carboxylic acid, the immediate, precursor of C₂H₄, have opposite effects on growth of internodes and leaves. The enhancement of internodal elongation by C₂H₄ is particularly pronounced in an atmosphere of high CO₂ and low O₂. The increase in C₂H₄ synthesis in internodes of submerged sections is primarily triggered by reduced atmospheric concentrations of O₂. The rate of C₂H₄ evolution by internodes isolated from stem sections and incubated in an atmosphere of low O₂ is up to four times greater than that of isolated internodes incubated in air. In contrast, C₂H₄ evolution from the leaves is reduced under hypoxic conditions. The effect of submergence on growth of stem sections of deep-water rice can be mimicked by exposing non-submerged sections to a gas mixture which is similar to the gaseous atmosphere in the internodal lacunae of submerged sections, namely 3% O₂, 6% CO₂, 91% N₂ (by vol.) and 1 l l^-1 C₂H₄. Our results indicate that growth responses obtained with isolated rice stem sections are similar to those of intact deep-water rice plants.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine     

C(2)H(4): Its Incorporation and Metabolism by Pea Seedlings under Aseptic Conditions

The effects of various treatments on the recently reported system in pea (Pisum sativum cv. Alaska), which results in (a) the incorporation of ¹⁴C₂H₄ into the tissue and (b) the conversion of ¹⁴C₂H₄ to ¹⁴CO₂, was investigated using 2-day-old etiolated seedlings which exhibit a maximum response. Heat treatment (80 C, 1 min) completely inhibited both a and b, whereas homogenization completely inhibited b but only partially inhibited a. Detaching the cotyledons from the root-shoot axis immediately before exposing the detached cotyledons together with the root-shoot axis to ¹⁴C₂H₄ markedly reduced both a and b. Increasing the ¹⁴C₂H₄ concentration from 0.14 to over 100 μl/l progressively increased the rate of a and b with tissue incorporation being greater than ¹⁴C₂H₄ to ¹⁴CO₂ conversion only below 0.3 μl/l ¹⁴C₂H₄. Reduction of the O₂ concentration reduced both a and b, with over 99% inhibition occurring under anaerobic conditions. The addition of CO₂ (5%) severely inhibited ¹⁴C₂H₄ to ¹⁴CO₂ conversion without significantly affecting tissue incorporation. Exposure of etiolated seedlings to fluorescent light during ¹⁴C₂H₄ treatment was without effect. Similarly, indoleacetic acid, gibberellic acid, benzyladenine, abscisic acid, and dibutyryl cyclic adenosine monophosphate had no significant effect on either a or b.     

Enhancement of Ethylene Release from Leaf Tissue during Glycolate Decarboxylation : A Possible Role for Photorespiration

When leaf discs of Xanthium strumarium L. and Salvia splendens L. are incubated in sealed flasks in the light, more C₂H₄ gas is released in the presence of added CO₂ (30-200 millimolar NaHCO₃) than without CO₂. In Salvia, the maximum rate of C₂H₄ release occurs when sufficient CO₂ (above 125 millimolar NaHCO₃) is added to saturate photosynthesis confirming previous studies. The maximum rate of C₂H₄ release from illuminated discs is similar to the rate in the dark with or without CO₂ in both species. Glycolate enhances a CO₂-dependent C₂H₄ evolution from illuminated leaf discs. However, the maximum rate of C₂H₄ release with glycolate is the same as that observed with saturating CO₂. When photosynthesis is inhibited by darkness or by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, glycolate has no effect.

Studies with [2,3-¹⁴C]-1-aminocyclopropane-1-carboxylic acid (ACC) show that the pattern of C₂H₄ release and the specific activity of the ¹⁴C₂H₄ in the presence and absence of glycolate is similar to that described above, indicating that glycolate does not alter uptake of the exogenously supplied precursor (ACC) or stimulate C₂H₄ release from an endogenous source at appreciable rates. Glycolate oxidase in vitro generates H₂O₂ which stimulates a slow breakdown of ACC to C₂H₄, but since exogenous glycolate is oxidized to CO₂ in both the light and the dark it is argued that the glycolate-dependent increase in C₂H₄ release from illuminated leaf discs is not mediated directly by the action of enzymes of glycolate catabolism. The effects of glycolate and CO₂ are not easily explained by changes in stomatal resistance. The data support the view that glycolate decarboxylation at subsaturating levels of CO₂ in the light stimulates C₂H₄ release by raising the CO₂ level in the tissue.

     

d- rice seedling development in periodically stirred water in sealed environments

Summary

Large numbers of germinating seedlings of two rice cultivars exhibited d^- type development consisting solely of completely white coleoptile growth after 15 days in the light in both static and periodically stirred water environments in completely sealed jars. Thus, whether germinated in static or agitated water in the sealed environments, coleoptile greening and subsequent normal seedling development were inhibited by hypoxic stress caused by the large numbers of germinating rice seedlings competing for the limited environmental oxygen supply. Consequently, the evidence presented points away from the formation of unfavourable oxygen diffusion gradients in static water environments in the sealed jars as having been responsible for d type seedling development observed in previous investigations. Continuous aeration of the 15-day-old static and periodically stirred aquatic environments, respectively, resulted in complete reversal of all d seedlings into normal green plants.     

Effects of the interaction between ozone and carbon dioxide on gas exchange,ascorbic acid content,and visible leaf symptoms in rice leaves

Tropospheric ozone (O₃) decreases photosynthesis, growth, and yield of crop plants, while elevated carbon dioxide (CO₂) has the opposite effect. The net photosynthetic rate (P _N), dark respiration rate (R _D), and ascorbic acid content of rice leaves were examined under combinations of O₃ (0, 0.1, or 0.3 cm³ m⁻³, expressed as O⁰, O^0.1, O^0.3, respectively) and CO₂ (400 or 800 cm³ m⁻³, expressed as C⁴⁰⁰ or C⁸⁰⁰, respectively). The P _N declined immediately after O₃ fumigation, and was larger under O^0.3 than under O^0.1. When C⁸⁰⁰ was combined with the O₃, P _N was unaffected by O^0.1 and there was an approximately 20 % decrease when the rice leaves were exposed to O^0.3 for 3 h. The depression of stomatal conductance (g _s) observed under O^0.1 was accelerated by C⁸⁰⁰, and that under O^0.3 did not change because the decline under O^0.3 was too large. Excluding the stomatal effect, the mesophyll P _N was suppressed only by O^0.3, but was substantially ameliorated when C⁸⁰⁰ was combined. Ozone fumigation boosted the R _D value, whereas C⁸⁰⁰ suppressed it. An appreciable reduction of ascorbic acid occurred when the leaves were fumigated with O^0.3, but the reduction was partially ameliorated by C⁸⁰⁰. The degree of visible leaf symptoms coincided with the effect of the interaction between O₃ and CO₂ on P _N. The amelioration of O₃ injury by elevated CO₂ was largely attributed to the restriction of O₃ intake by the leaves with stomatal closure, and partly to the maintenance of the scavenge system for reactive oxygen species that entered the leaf mesophyll, as well as the promotion of the P _N.     

Glucose metabolism in anaerobic rice seedlings

More than 80% of the radioactivity from [U-¹⁴C]glucose metabolised by anaerobic rice seedlings or by excised roots or coleoptiles was recovered as ethanol plus CO₂; less than 5% was recovered as water-soluble acidic components. Rates of ¹⁴CO₂ formation from [U-¹⁴C]glucose were similar in roots and coleoptiles in both N₂ and air atmospheres. More ¹⁴CO₂ was formed from [U-¹⁴C]glucose than could be accounted for by ethanolic fermentation, and the specific yields of ¹⁴CO₂ from [6-¹⁴C]glucose and [1-¹⁴C]glucose gave unusually high C-6/C-1 ratios (1.7) in the anaerobic coleoptile. The results may indicate that appreciable pentan synthesis occurs in the anaerobic coleoptile.     

Involvement of glutathione in heat shock– and hydrogen peroxide–induced cadmium tolerance of rice (Oryza sativa L.) seedlings

The role of reduced glutathione (GSH) in heat shock (HS)- and H₂O₂-induced protection of rice (Oryza sativa L., cv. Taichung 1) seedlings from Cd stress was investigated. HS- and H₂O₂-pretreatment resulted in an increase in GSH content in leaves of rice seedlings. Addition of exogenous GSH under non-HS conditions, which resulted in an increase in GSH in leaves, enhanced subsequent Cd tolerance of rice seedlings. Pretreatment with buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis, which effectively inhibited GSH content induced by HS and H₂O₂, reduced subsequent Cd tolerance. Furthermore, the effect of BSO on HS- and H₂O₂-induced GSH accumulation and toxicity by subsequent Cd stress can be reversed by the addition of GSH. The time-course analyses of HS in rice seedlings demonstrated that the accumulation of H₂O₂ preceded the increase in GSH. Based on the data obtained in this study, it could be concluded that the early accumulation of H₂O₂ during HS signals the increase in GSH content, which in turn protects rice seedlings from oxidative damage caused by Cd.     

MAPKs as a cross point in H2O2 and auxin signaling under combined cadmium and zinc stress in rice roots

Previously, we have reported the role of MAPKs (mitogen-activated protein kinases) under cadmium stress. This work continue to explore the relationship between MAPKs, H₂O₂, auxin signaling, and OsHMA and OsZIP gene expression in rice (Oryza sativa L.) roots under combined cadmium (Cd) and zinc (Zn) stress. Compared with Cd, Cd+Zn reduced Cd levels but increased Zn accumulation in the roots. Three OsMAPK genes were negatively regulated, while two OsHMA and two OsZIP genes were positively regulated by MAPK pathways under Cd+Zn stress. Transgenic rice expressing DR5-GUS exhibited enhanced GUS activity in H₂O₂-, PD (MAPKK inhibitor PD98059)-, or (Cd+Zn)-treated roots, which also exhibited increased H₂O₂ concentrations, whereas GUS staining decreased in roots in response to Cd+Zn+PD, DMTU (N,N′-dimethylthiourea, a H₂O₂ scavenger), or Cd+Zn+DMTU treatment, with reduced H₂O₂ levels. GUS levels were consistent with H₂O₂ levels, suggesting that MAPK pathway-mediated auxin redistribution occurs via H₂O₂, and H₂O₂ functions downstream of MAPK but upstream of auxin signaling pathways. Furthermore, MAPK pathways serve specific functions in regulating the expression of some key genes of auxin signaling (OsYUCCA, OsPIN, OsARF, and OsIAA) under Cd+Zn stress. Overall, MAPK cascades function in the integration of metal transport, H₂O₂ generation, and auxin signaling in rice seedlings grown under Cd+Zn stress.