Polar Auxin Transport Inhibitor Research Articles

A differential distribution of auxin and flavonols in radiata pine stem seedlings exposed to inclination

Stem reorientation in response to inclination in trees has commanded the attention of the research community widely, but the molecular mechanisms of the response are still unknown. The most accepted theory is auxin redistribution in affected tissues. Flavonols have been reported as potent inhibitors of polar auxin transport; however, no reports have shown differential modulation by flavonols within the trunk. Here, transcriptional, microscopic, and chemical approaches were used to analyze the contents of flavonoids in 45° inclined radiata pine seedlings. A time course was established and samples obtained by cutting stems into apical, medial, and basal segments or by longitudinal dissection into upper and lower halves to determine polar distribution of flavonoids. The expression analysis of PrCHS, PrF3H, and PrFLS indicated induction in response to inclination; higher transcript levels were recorded at the basal zone and in the upper half of the stem. A reduction in abundance of PrARP transcripts in addition to immunospecific auxin detection at the lower half of inclined stem was found. Concomitantly, quercetin and kaempferol accumulated in the upper stem half. Our results suggest that stem reorientation is a response to a concerted and differential mechanism that modulates imbalance of auxin distribution in one side of the stem.

Development of root system architecture of Arabidopsis thaliana in response to colonization by Martelella endophytica YC6887 depends on auxin signaling

Many rhizobacteria promote plant growth by producing hormones that stimulate the development of plant root system and increase plant biomass. The aim of this study was to investigate the growth promotion activity of the bacterial strain Martelella endophytica YC6887 and elucidate the signaling pathways potentially involved in Arabidopsis interaction with M. endophytica YC6887. The growth regulation was evaluated by inoculation of strain YC6887 with wild-type Arabidopsis Col-0 seedlings and mutants defective in auxin aux1-7, axr4-2, eir1-1, ethylene ein2-1, etr1-3, jasmonic acid signaling jar1, and root hair deficient mutant rhd6. The auxin response was further determined by using transgenic line DR5::GUS and a polar auxin transport inhibitor, 1-N-naphthylphthalamic acid (NPA). M. endophytica YC6887 increased the number of lateral roots and plant biomass of Arabidopsis by producing phenylacetic acid. The growth promotion and improved lateral root development by the bacterium decreased in the auxin related mutants, whereas the ethylene and jasmonic acid mutants had a wild type response. The strain YC6887 increased root hair density in wild type Col-0 and recovered the root hair forming ability in root hair deficient mutant rhd6. Moreover, strain YC6887 treatment showed distinct response in DR5::GUS transgenic line compared to the control. Strain YC6887 lost its growth-promoting activity in the presence of NPA, an auxin transport inhibitor. This indicated that strain YC6887 activated the auxin signaling mechanism. Our results showed that M. endophytica YC6887 promoted plant growth in terms of plant biomass and root system development. Arabidopsis root system development upon M. endophytica YC6887 colonization was dependent on auxin signaling, but independent of ethylene and jasmonic acid signaling.

モモ赤肉品種における発育ステージ，光およびオーキシン輸送阻害剤が果皮および果肉の着色に及ぼす影響

モモ赤肉品種における発育ステージ，光およびオーキシン輸送阻害剤が果皮および果肉の着色に及ぼす影響

Gene expression profile of zeitlupe/lov kelch protein1 T-DNA insertion mutants in Arabidopsis thaliana: Downregulation of auxin-inducible genes in hypocotyls

Elongation of hypocotyl cells has been studied as a model for elucidating the contribution of cellular expansion to plant organ growth. ZEITLUPE (ZTL) or LOV KELCH PROTEIN1 (LKP1) is a positive regulator of warmth-induced hypocotyl elongation under white light in Arabidopsis, although the molecular mechanisms by which it promotes hypocotyl cell elongation remain unknown. Microarray analysis showed that 134 genes were upregulated and 204 genes including 15 auxin-inducible genes were downregulated in the seedlings of 2 ztl T-DNA insertion mutants grown under warm conditions with continuous white light. Application of a polar auxin transport inhibitor, an auxin antagonist or an auxin biosynthesis inhibitor inhibited hypocotyl elongation of control seedlings to the level observed with the ztl mutant. Our data suggest the involvement of auxin and auxin-inducible genes in ZTL-mediated hypocotyl elongation.

Auxin is a long-range signal that acts independently of ethylene signaling on leaf abscission in Populus.

Timing of leaf abscission is an important trait for biomass production and seasonal acclimation in deciduous trees. The signaling leading to organ separation, from the external cue (decreasing photoperiod) to ethylene-regulated hydrolysis of the middle lamellae in the abscission zone, is only poorly understood. Data from annual species indicate that the formation of an auxin gradient spanning the abscission zone regulates the timing of abscission. We established an experimental system in Populus to induce leaf shedding synchronously under controlled greenhouse conditions in order to test the function of auxin in leaf abscission. Here, we show that exogenous auxin delayed abscission of dark-induced leaves over short and long distances and that a new auxin response maximum preceded the formation of an abscission zone. Several auxin transporters were down-regulated during abscission and inhibition of polar auxin transport delayed leaf shedding. Ethylene signaling was not involved in the regulation of these auxin transporters and in the formation of an abscission zone, but was required for the expression of hydrolytic enzymes associated with cell separation. Since exogenous auxin delayed abscission in absence of ethylene signaling auxin likely acts independently of ethylene signaling on cell separation.

Effect of plant growth regulators on the accumulation of indolizidine alkaloids in Securinega suffruticosa callus cultures

In the presented work, plant growth regulators (PGRs) were tested for their influence on biomass growth and accumulation of therapeutically-relevant indolizidine alkaloids in Securinega suffruticosa (Phyllanthaceae) callus cultures. The study included 9 auxins [2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid, indole-3-acetic acid (IAA), 4-chlorophenoxyacetic acid, indole-3-butyric acid, 4-amino-3,5,6-trichloropicolinic acid, β-naphthoxyacetic acid, 1-naphthaleneacetic acid, indole-3-propionic acid, one polar auxin transport inhibitor (2,3,5-triiodobenzoic acid) and 7 cytokinins [2-isopentenyladenine, 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea, N-(2-chloro-4-pyridinyl)-N′-phenylurea, diphenylurea, zeatin, kinetin (KIN), 6-benzylaminopurine]. The respective PGRs were applied at 0.5 and 5.0 mg l−1, together with 5.0 or 0.5 mg l−1 KIN or 2,4-D (for auxins and cytokinins, respectively). The calli subjected to different phytohormone combinations (39 modifications in total) were evaluated for growth and alkaloid content, and the results tested for statistical differences. The highest concentrations of securinine (1.73 mg g−1 DW) and allosecurinine (3.11 mg g−1 DW) were recorded in the callus grown in the presence of 0.5 mg L−1 IAA and 5.0 mg L−1 KIN. To the best of the authors’ knowledge, this is the first such comprehensive report concerning the influence of PGRs on alkaloid accumulation in plant in vitro cultures.

Auxin Biosynthesis, Accumulation, Action and Transport are Involved in Stress-Induced Microspore Embryogenesis Initiation and Progression in Brassica napus.

Isolated microspores are reprogrammed in vitro by stress, becoming totipotent cells and producing embryos and plants via a process known as microspore embryogenesis. Despite the abundance of data on auxin involvement in plant development and embryogenesis, no data are available regarding the dynamics of auxin concentration, cellular localization and the expression of biosynthesis genes during microspore embryogenesis. This work involved the analysis of auxin concentration and cellular accumulation; expression of TAA1 and NIT2 encoding enzymes of two auxin biosynthetic pathways; expression of the PIN1-like efflux carrier; and the effects of inhibition of auxin transport and action by N-1-naphthylphthalamic acid (NPA) and α-(p-chlorophenoxy) isobutyric acid (PCIB) during Brassica napus microspore embryogenesis. The results indicated de novo auxin synthesis after stress-induced microspore reprogramming and embryogenesis initiation, accompanying the first cell divisions. The progressive increase of auxin concentration during progression of embryogenesis correlated with the expression patterns of TAA1 and NIT2 genes of auxin biosynthetic pathways. Auxin was evenly distributed in early embryos, whereas in heart/torpedo embryos auxin was accumulated in apical and basal embryo regions. Auxin efflux carrier PIN1-like gene expression was induced in early multicellular embryos and increased at the globular/torpedo embryo stages. Inhibition of polar auxin transport (PAT) and action, by NPA and PCIB, impaired embryo development, indicating that PAT and auxin action are required for microspore embryo progression. NPA also modified auxin embryo accumulation patterns. These findings indicate that endogenous auxin biosynthesis, action and polar transport are required in stress-induced microspore reprogramming, embryogenesis initiation and progression.

A strigolactone signal is required for adventitious root formation in rice.

Strigolactones (SLs) and their derivatives are plant hormones that have recently been identified as regulating root development. This study examines whether SLs play a role in mediating production of adventious roots (ARs) in rice (Oryza sativa), and also investigates possible interactions between SLs and auxin. Wild-type (WT), SL-deficient (d10) and SL-insensitive (d3) rice mutants were used to investigate AR development in an auxin-distribution experiment that considered DR5::GUS activity, [(3)H] indole-3-acetic acid (IAA) transport, and associated expression of auxin transporter genes. The effects of exogenous application of GR24 (a synthetic SL analogue), NAA (α-naphthylacetic acid, exogenous auxin) and NPA (N-1-naphthylphalamic acid, a polar auxin transport inhibitor) on rice AR development in seedlings were investigated. The rice d mutants with impaired SL biosynthesis and signalling exhibited reduced AR production compared with the WT. Application of GR24 increased the number of ARs and average AR number per tiller in d10, but not in d3. These results indicate that rice AR production is positively regulated by SLs. Higher endogenous IAA concentration, stronger expression of DR5::GUS and higher [(3)H] IAA activity were found in the d mutants. Exogenous GR24 application decreased the expression of DR5::GUS, probably indicating that SLs modulate AR formation by inhibiting polar auxin transport. The WT and the d10 and d3 mutants had similar expression of DR5::GUS regardless of exogenous application of NAA or NPA; however, AR number was greater in the WT than in the d mutants. The results suggest that AR formation is positively regulated by SLs via the D3 response pathway. The positive effect of NAA application and the opposite effect of NPA application on AR number of WT plants also suggests the importance of auxin for AR formation, but the interaction between auxin and SLs is complex.

Negative phototropism is seen in Arabidopsis inflorescences when auxin signaling is reduced to a minimal level by an Aux/IAA dominant mutation, axr2

Inflorescences of a dominant mutant of Arabidopsis Aux/IAA7, axr2, showed negative phototropism with a similar fluence response curve to the positive phototropism of wild-type stems. Application of a synthetic auxin, NAA, and an inhibitor of polar auxin transport, NPA, increased and decreased respectively the magnitude of the phototropic response in the wild type, while in axr2 application of NAA reduced the negative phototropic response and NPA had no effect. Decapitation of the apex induced a small negative phototropism in wild-type stems, and had no effect in axr2 plants. Inflorescences of the double mutants of auxin transporters, pgp1 pgp19, showed no phototropic response, while decapitation resulted in a negative phototropic response. These results suggest that negative phototropism can occur when the level of auxin or of auxin signaling is reduced to a minimal level, and that in plant axial organs the default phototropic response to unilateral blue light may be negative. Expression of axr2 protein by an endodermis-specific promoter resulted in agravitropism of inflorescences in a similar way to that of axr2, but phototropism was normal, confirming that the endodermis does not play a critical role in phototropism.

Auxin and physical constraint exerted by the perianth promote androgynophore bending in Passiflora mucronata L. (Passifloraceae).

The androgynophore column, a distinctive floral feature in passion flowers, is strongly crooked or bent in many Passiflora species pollinated by bats. This is a floral feature that facilitates the adaptation to bat pollination. Crooking or bending of plant organs are generally caused by environmental stimulus (e.g. mechanical barriers) and might involve the differential distribution of auxin. Our aim was to study the role of the perianth organs and the effect of auxin in bending of the androgynophore of the bat-pollinated species Passiflora mucronata. Morpho-anatomical characterisation of the androgynophore, including measurements of curvature angles and cell sizes both at the dorsal (convex) and ventral (concave) sides of the androgynophore, was performed on control flowers, flowers from which perianth organs were partially removed and flowers treated either with auxin (2,4-dichlorophenoxyacetic acid; 2,4-D) or with an inhibitor of auxin polar transport (naphthylphthalamic acid; NPA). Asymmetric growth of the androgynophore column, leading to bending, occurs at a late stage of flower development. Removing the physical constraint exerted by perianth organs or treatment with NPA significantly reduced androgynophore bending. Additionally, the androgynophores of plants treated with 2,4-D were more curved when compared to controls. There was a larger cellular expansion at the dorsal side of the androgynophores of plants treated with 2,4-D and in both sides of the androgynophores of plants treated with NPA. This study suggests that the physical constraint exerted by perianth and auxin redistribution promotes androgynophore bending in P.mucronata and might be related to the evolution of chiropterophily in the genus Passiflora.

P026 Naturally-occurring inhibitors of auxin polar transport from Saussurea costus : Relevance to the isolation and identification, and the mode of action

P026 Naturally-occurring inhibitors of auxin polar transport from Saussurea costus : Relevance to the isolation and identification, and the mode of action

Exploring the role of auxin in the androgynophore movement in Passiflora.

The flowers of the species belonging to the genus Passiflorashow a range of features that are thought to have arisen as adaptations to different pollinators. Some Passiflora species belonging to the subgenus Decaloba sect. Xerogona, show touch-sensitive motile androgynophores. We tested the role of auxin polar transport in the modulation of the androgynophore movement by applying auxin (IAA) or an inhibitor of auxin polar transport (NPA) in the flowers. We recorded the movement of the androgynophore during mechano-stimulation and analyzed the duration, speed, and the angle formed by the androgynophore before and after the movement, and found that both IAA and NPA increase the amplitude of the movement in P. sanguinolenta. We hypothesize that auxin might have a role in modulating the fitness of these Decaloba species to different pollination syndromes and demonstrate that an interspecific hybrid between insect- and hummingbird-pollinated Xerogona species present a heterosis effect on the speed of the androgynophore movement.

ABA plays essential roles in regulating root growth by interacting with auxin and MAPK signaling pathways and cell-cycle machinery in rice seedlings

In this study, we examined the relationship between abscisic acid (ABA), auxin, and mitogen-activated protein kinase (MAPK) signaling and the cell cycle in rice (Oryza sativa L. cv. Zhonghua No. 11) roots. Root system growth was significantly promoted by 0.1 nM ABA but markedly inhibited by 0.3 mM Tungstate (TS, an ABA biosynthesis inhibitor). Detection of plants treated with DRB (an RNA synthesis inhibitor, 5,6-dichlorobenzimidazole 1-β-d-ribofuranoside), TIBA (2,3,5-triiodobenzoic acid, an inhibitor of polar auxin transport), or BFA (brefeldin, a protein transport inhibitor) using DR5-GUS staining revealed that ABA regulates the distribution of auxin via transcription and transport pathways. The expression of some auxin- and cell cycle-related genes, as well as several ABA or MAPK genes, was differentially regulated in roots by ABA and TS at 7 or 11 days, indicating that a certain level of ABA action may primarily target specific genes involved in MAPK and auxin signaling and the cell cycle at a given developmental stage. The results suggest that an appropriate level of ABA determines auxin homeostasis through controlling gene expression and the distribution of auxin, and that ABA regulates the auxin and MAPK signaling pathways and influences cell-cycle progression. Overall, ABA plays positive roles in regulating root system growth by modulating MAPK and auxin signaling and the cell cycle. Our findings help elucidate the integrative effects of ABA, auxin, and MAPK signaling and the cell cycle on root growth. We propose a working model for the role of ABA in root system growth.

Differential behavior within a grapevine cluster: decreased ethylene-related gene expression dependent on auxin transport is correlated with low abscission of first developed berries.

In grapevine, fruit abscission is known to occur within the first two to three weeks after flowering, but the reason why some berries in a cluster persist and others abscise is not yet understood. Ethylene sensitivity modulates abscission in several fruit species, based on a mechanism where continuous polar auxin transport across the pedicel results in a decrease in ethylene perception, which prevents abscission. In grapevine, flowering takes about four to seven days in a single cluster, thus while some flowers are developing into berries, others are just starting to open. So, in this work it was assessed whether uneven flowering accounted for differences in berry abscission dependent on polar auxin transport and ethylene-related gene expression. For this, flowers that opened in a cluster were tagged daily, which allowed to separately analyze berries, regarding their ability to persist. It was found that berries derived from flowers that opened the day that flowering started – named as “first berries” – had lower abscission rate than berries derived from flowers that opened during the following days – named as “late berries”. Use of radiolabeled auxin showed that “first berries” had higher polar auxin transport, correlated with lower ethylene content and lower ethylene-related transcript abundance than “late berries”. When “first berries” were treated with a polar auxin transport inhibitor they showed higher ethylene-related transcript abundance and were more prone to abscise than control berries. This study provides new insights on fruit abscission control. Our results indicate that polar auxin transport sustains the ability of “first berries” to persist in the cluster during grapevine abscission and also suggest that this could be associated with changes in ethylene-related gene expression.

Timing Is Everything: Highly Specific and Transient Expression of a MAP Kinase Determines Auxin-Induced Leaf Venation Patterns in Arabidopsis

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules present in all eukaryotes. In plants, MAPK cascades were shown to regulate cell division, developmental processes, stress responses, and hormone pathways. The subgroup A of Arabidopsis MAPKs consists of AtMPK3, AtMPK6, and AtMPK10. AtMPK3 and AtMPK6 are activated by their upstream MAP kinase kinases (MKKs) AtMKK4 and AtMKK5 in response to biotic and abiotic stress. In addition, they were identified as key regulators of stomatal development and patterning. AtMPK10 has long been considered as a pseudo-gene, derived from a gene duplication of AtMPK6. Here we show that AtMPK10 is expressed highly but very transiently in seedlings and at sites of local auxin maxima leaves. MPK10 encodes a functional kinase and interacts with the upstream MAP kinase kinase (MAPKK) AtMKK2. mpk10 mutants are delayed in flowering in long-day conditions and in continuous light. Moreover, cotyledons of mpk10 and mkk2 mutants have reduced vein complexity, which can be reversed by inhibiting polar auxin transport (PAT). Auxin does not affect AtMPK10 expression while treatment with the PAT inhibitor HFCA extends the expression in leaves and reverses the mpk10 mutant phenotype. These results suggest that the AtMKK2-AtMPK10 MAPK module regulates venation complexity by altering PAT efficiency.

Block of ATP-binding cassette B19 ion channel activity by 5-nitro-2-(3-phenylpropylamino)-benzoic acid impairs polar auxin transport and root gravitropism.

Polar transport of the hormone auxin through tissues and organs depends on membrane proteins, including some B-subgroup members of the ATP-binding cassette (ABC) transporter family. The messenger RNA level of at least one B-subgroup ABCB gene in Arabidopsis (Arabidopsis thaliana), ABCB19, increases upon treatment with the anion channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), possibly to compensate for an inhibitory effect of the drug on ABCB19 activity. Consistent with this hypothesis, NPPB blocked ion channel activity associated with ABCB19 expressed in human embryonic kidney cells as measured by patch-clamp electrophysiology. NPPB inhibited polar auxin transport through Arabidopsis seedling roots similarly to abcb19 mutations. NPPB also inhibited shootward auxin transport, which depends on the related ABCB4 protein. NPPB substantially decreased ABCB4 and ABCB19 protein levels when cycloheximide concomitantly inhibited new protein synthesis, indicating that blockage by NPPB enhances the degradation of ABCB transporters. Impairing the principal auxin transport streams in roots with NPPB caused aberrant patterns of auxin signaling reporters in root apices. Formation of the auxin-signaling gradient across the tips of gravity-stimulated roots, and its developmental consequence (gravitropism), were inhibited by micromolar concentrations of NPPB that did not affect growth rate. These results identify ion channel activity of ABCB19 that is blocked by NPPB, a compound that can now be considered an inhibitor of polar auxin transport with a defined molecular target.

The role of auxin transporters in monocots development.

Auxin is a key regulator of plant growth and development, orchestrating cell division, elongation and differentiation, embryonic development, root and stem tropisms, apical dominance, and transition to flowering. Auxin levels are higher in undifferentiated cell populations and decrease following organ initiation and tissue differentiation. This differential auxin distribution is achieved by polar auxin transport (PAT) mediated by auxin transport proteins. There are four major families of auxin transporters in plants: PIN-FORMED (PIN), ATP-binding cassette family B (ABCB), AUXIN1/LIKE-AUX1s, and PIN-LIKES. These families include proteins located at the plasma membrane or at the endoplasmic reticulum (ER), which participate in auxin influx, efflux or both, from the apoplast into the cell or from the cytosol into the ER compartment. Auxin transporters have been largely studied in the dicotyledon model species Arabidopsis, but there is increasing evidence of their role in auxin regulated development in monocotyledon species. In monocots, families of auxin transporters are enlarged and often include duplicated genes and proteins with high sequence similarity. Some of these proteins underwent sub- and neo-functionalization with substantial modification to their structure and expression in organs such as adventitious roots, panicles, tassels, and ears. Most of the present information on monocot auxin transporters function derives from studies conducted in rice, maize, sorghum, and Brachypodium, using pharmacological applications (PAT inhibitors) or down-/up-regulation (over-expression and RNA interference) of candidate genes. Gene expression studies and comparison of predicted protein structures have also increased our knowledge of the role of PAT in monocots. However, knockout mutants and functional characterization of single genes are still scarce and the future availability of such resources will prove crucial to elucidate the role of auxin transporters in monocots development.

Auxin represses stomatal development in dark-grown seedlings via Aux/IAA proteins.

Stomatal development is tightly regulated through internal and external factors that are integrated by a complex signalling network. Light represents an external factor that strongly promotes stomata formation. Here, we show that auxin-resistant aux/iaa mutants, e.g. axr3-1, exhibit a de-repression of stomata differentiation in dark-grown seedlings. The higher stomatal index in dark-grown axr3-1 mutants when compared with the wild type is due to increased cell division in the stomatal lineage. Excessive stomata in dark-grown seedlings were also observed in mutants defective in auxin biosynthesis or auxin perception and in seedlings treated with the polar auxin transport inhibitor NPA. Consistent with these findings, exogenous auxin repressed stomata formation in light-grown seedlings. Taken together, these results indicate that auxin is a negative regulator of stomatal development in dark-grown seedlings. Epistasis analysis revealed that axr3-1 acts genetically upstream of the bHLH transcription factors SPCH, MUTE and FAMA, as well as the YDA MAP kinase cascade, but in parallel with the repressor of photomorphogenesis COP1 and the receptor-like protein TMM. The effect of exogenous auxin required the ER family of leucine-rich repeat receptor-like kinases, suggesting that auxin acts at least in part through the ER family. Expression of axr3-1 in the stomatal lineage was insufficient to alter the stomatal index, implying that cell-cell communication is necessary to mediate the effect of auxin. In summary, our results show that auxin signalling contributes to the suppression of stomatal differentiation observed in dark-grown seedlings.

The polar auxin transport inhibitor TIBA inhibits endoreduplication in dark grown spinach hypocotyls

We addressed the question of whether an additional round of endoreduplication in dark-grown hypocotyls is a common feature in dicotyledonous plants having endopolyploid tissues. Ploidy distributions of hypocotyl tissues derived from in vitro-grown spinach (Spinacia oleracea L. cv. Atlas) seedlings grown under different light conditions were analyzed by flow cytometry. An additional round of endoreduplication (represented by 32C cells) was found in the dark-grown hypocotyl tissues. This response was inhibited by light, the intensity of which is a crucial factor for the inhibition of endoreduplication. The higher ploidy cells in cortical tissues of the dark-grown hypocotyls had larger cell sizes, suggesting that the additional round of endoreduplication contributes to hypocotyl elongation. More importantly, a polar auxin transport inhibitor, 2,3,5-triiodobenzoic acid (TIBA), strongly inhibits endoreduplication, not only in spinach but also in Arabidopsis. Because other polar auxin transport inhibitors or an auxin antagonist show no or mild effects, TIBA may have a specific feature that inhibits endoreduplication.

Crosstalk between ABA, auxin, MAPK signaling, and the cell cycle in cadmium-stressed rice seedlings

We studied the link between abscisic acid (ABA), auxin, MAPK (mitogen-activated protein kinase) signaling, and the cell cycle in cadmium (Cd)-stressed rice (Oryza sativa L. cv. Zhonghua No. 11) roots. ABA can partially compensate for root growth inhibition and counteract over-accumulation of auxin caused by Cd. GUS staining of rice plants harboring DR5-GUS showed that ABA affects auxin distribution in Cd-stressed roots. Detection using DRB (5,6-dichlorobenzimidazole 1-β-d-ribofuranoside, an RNA synthesis inhibitor), MG132 (a protein degradation inhibitor), BFA (brefeldin A, a protein transport inhibitor), and TIBA (2,3,5-triiodobenzoic acid, a polar auxin transport inhibitor) revealed that ABA regulates the distribution of auxin via transcription, protein degradation, and transport pathways under Cd stress. Several genes related to ABA and MAPK, key components of the auxin signaling pathway, and the cell cycle were differentially regulated by Cd plus ABA vs. Cd plus tungstate (TS) (a ABA biosynthesis inhibitor) at 7 days or 11 days of treatment in roots, indicating that ABA levels affect the transcription of these genes, and that these genes are differentially regulated by ABA in rice seedlings at different developmental stages. Furthermore, the expression of some of these genes differed between Cd + ABA/TS-treated plants and plants treated with TS or ABA alone, suggesting that ABA signaling serves specific functions in the regulation of gene expression under Cd stress. Overall, these results suggest that ABA coordinates auxin and MAPK signaling and the cell cycle in response to Cd stress. The ABA signal transduction pathways in Cd-stressed rice plants are discussed.