Aluminum-dependent root-growth inhibition in Arabidopsis results from AtATR-regulated cell-cycle arrest

Aluminum (Al) toxicity is a global problem severely limiting agricultural productivity in acid-soil regions comprising upwards of 50% of the world's arable land [1, 2]. Although Al-exclusion mechanisms have been intensively studied [3-9], little is known about tolerance to internalized Al, which is predicted to be mechanistically complex because of the plethora of predicted cellular targets for Al(3+)[2, 10]. An Arabidopsis mutant with Al hypersensitivity, als3-1, was found to represent a lesion in a phloem and root-tip-localized factor similar to the bacterial ABC transporter ybbm, with ALS3 likely responsible for Al transfer from roots to less-sensitive tissues [10-12]. To identify mutations that enhance mechanisms of Al resistance or tolerance, a suppressor screen for mutants that mask the Al hypersensitivity of als3-1 was performed [13]. Two allelic suppressors conferring increased Al tolerance were found to represent dominant-negative mutations in a factor required for monitoring DNA integrity, AtATR[14-17]. From this work, Al-dependent root-growth inhibition primarily arises from DNA damage coupled with AtATR-controlled blockage of cell-cycle progression and terminal differentiation because of loss of the root-quiescent center, with mutations that prevent response to this damage resulting in quiescent-center maintenance and sustained vigorous growth in an Al-toxic environment.     

<Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic"> ALS1</Emphasis> encodes a root tip and stele localized half type ABC transporter required for root growth in an aluminum toxic environment

Aluminum toxicity in acid soils severely limits crop productivity through inhibition of root growth and, consequently, shoot development. Several Arabidopsis mutants were previously identified as having roots with Al hypersensitivity, suggesting that these represent deleterious mutations affecting genes required for either Al tolerance or resistance mechanisms. For this report, the als1-1 mutant was chosen for further characterization. The phenotype of als1-1 is most obviously presented in Al challenged roots, as evidenced by exaggerated root growth inhibition in conjunction with increased expression of Al-responsive genes compared to wt. Using a map-based cloning approach, the als1-1 mutation was isolated and found to represent a deleterious amino acid substitution in a previously uncharacterized half type ABC transporter, At5g39040, which is expressed in a non-Al dependent manner in all organs tested. GUS-dependent analyses revealed that ALS1 expression is primarily localized to the root tip and the vasculature throughout the plant. Concomitant with this, an ALS1: GFP fusion accumulates at the vacuolar membrane of root cells, indicating that ALS1 may be important for intracellular movement of some substrate, possibly chelated Al, as part of a mechanism of Al sequestration.     

ALS3 encodes a phloem-localized ABC transporter-like protein that is required for aluminum tolerance in Arabidopsis

Aluminum (Al) toxicity in acid soils is a worldwide agricultural problem that severely limits crop productivity through inhibition of root growth. Previously, Arabidopsis mutants with increased Al sensitivity were isolated in order to identify genes important for Al tolerance in plants. One mutant, als3, exhibited extreme root growth inhibition in the presence of Al, suggesting that this mutation negatively impacts a gene required for Al tolerance. Map-based cloning of the als3-1 mutation resulted in the isolation of a novel gene that encodes a previously undescribed ABC transporter-like protein, which is highly homologous to a putative bacterial metal resistance protein, ybbM. Northern analysis for ALS3 expression revealed that it is found in all organs examined, which is consistent with the global nature of Al sensitivity displayed by als3, and that expression increases in roots following Al treatment. Based on GUS fusion and in situ hybridization analyses, ALS3 is primarily expressed in leaf hydathodes and the phloem throughout the plant, along with the root cortex following Al treatment. Immunolocalization indicates that ALS3 predominantly accumulates in the plasma membrane of cells that express ALS3. From our results, it appears that ALS3 encodes an ABC transporter-like protein that is required for Al resistance/tolerance and may function to redistribute accumulated Al away from sensitive tissues in order to protect the growing root from the toxic effects of Al.     

Arabidopsis mutants with increased sensitivity to aluminum.

Al-sensitive (als) mutants of Arabidopsis were isolated and characterized with the aim of defining mechanisms of Al toxicity and resistance. Most als mutants selected on the basis of root growth sensitivity to Al were recessive, and together the mutants constituted eight complementation groups. Also, in most als mutants, Al sensitivity appeared to be specific for Al relative to La (another trivalent cation), except als2, which was more sensitive to La than wild type. The tendency of roots on mutant seedlings to accumulate Al was examined by staining with morin and hematoxylin, dyes used to indicate the presence of Al. A significant increase in morin staining was observed in als5, consistent with its increased sensitivity to Al. Unexpectedly, als7 and als4 showed less morin staining, suggesting that the roots on these mutants accumulate less Al than wild type seedlings after exposure to Al-containing solutions. Roots of wild-type seedlings produce callose in response to AlCl3 concentrations that inhibit root growth. Only als5 accumulated more callose than wild type in response to low levels (25 mu M) of AICI3 However, als4 and als7 did not accumulate callose at this AlCl3 concentration even though root growth was significantly inhibited. The lack of callose accumulation in als4 and als7 suggests that there is not an obligatory relationship between callose deposition and Al-induced inhibition of root growth.     

Overexpression of an Arabidopsis magnesium transport gene, AtMGT1, in Nicotiana benthamiana confers Al tolerance

Aluminium (Al) toxicity is the most important limiting factor for crop production in acid soil environments worldwide. In some plant species, application of magnesium (Mg(2+)) can alleviate Al toxicity. However, it remains unknown whether overexpression of magnesium transport proteins can improve Al tolerance. Here, the role of AtMGT1, a member of the Arabidopsis magnesium transport family involved in Mg(2+) transport, played in Al tolerance in higher plants was investigated. Expression of 35S::AtMGT1 led to various phenotypic alterations in Nicotiana benthamiana plants. Transgenic plants harbouring 35S::AtMGT1 exhibited tolerance to Mg(2+) deficiency. Element assay showed that the contents of Mg, Mn, and Fe in 35S::AtMGT1 plants increased compared with wild-type plants. Root growth experiment revealed that 100 microM AlCl(3) caused a reduction in root elongation by 47% in transgenic lines, whereas root growth in wild-type plants was inhibited completely. Upon Al treatment, representative transgenic lines also showed a much lower callose deposition, an indicator of increased Al tolerance, than wild-type plants. Taken together, the results have demonstrated that overexpression of ATMGT1 encoding a magnesium transport protein can improve tolerance to Al in higher plants.     

The Arabidopsis cell cycle checkpoint regulators TANMEI/ALT2 and ATR mediate the active process of aluminum-dependent root growth inhibition

Aluminum (Al) toxicity is a global issue that severely limits root growth in acidic soils. Isolation of suppressors of the Arabidopsis thaliana Al-hypersensitive mutant, als3-1, resulted in identification of a cell cycle checkpoint factor, ALUMINUM TOLERANT2 (ALT2), which monitors and responds to DNA damage. ALT2 is required for active stoppage of root growth after Al exposure, because alt2 loss-of-function mutants fail to halt root growth after Al exposure, do not accumulate CyclinB1;1 in the root tip, and fail to force differentiation of the quiescent center. Thus, alt2-1 mutants are highly tolerant of Al levels that are severely inhibitory to the wild type. The alt2-1 allele is a loss-of-function mutation in a protein containing a putative DDB1-binding WD40 motif, previously identified as TANMEI, which is required for assessment of DNA integrity, including monitoring of DNA crosslinks. alt2-1 and atr loss-of-function mutants, the latter of which affects the cell cycle checkpoint ATAXIA TELANGIECTASIA-MUTATED AND RAD3-RELATED, are severely sensitive to DNA crosslinking agents and have increased Al tolerance. These results suggest that Al likely acts as a DNA-damaging agent in vivo and that Al-dependent root growth inhibition, in part, arises from detection of and response to this damage by TANMEI/ALT2 and ATR, both of which actively halt cell cycle progression and force differentiation of the quiescent center.     

Al Inhibits Both Shoot Development and Root Growth in als3, an Al-Sensitive Arabidopsis Mutant

In als3, an Al-sensitive Arabidopsis mutant, shoot development and root growth are sensitive to Al. Mutant als3 seedlings grown in an Al-containing medium exhibit severely inhibited leaf expansion and root growth. In the presence of Al, unexpanded leaves accumulate callose, an indicator of Al damage in roots. The possibility that the inhibition of shoot development in als3 is due to the hyperaccumulation of Al in this tissue was examined. However, it was found that the levels of Al that accumulated in shoots of als3 are not different from the wild type. The inhibition of shoot development in als3 is not a consequence of nonspecific damage to roots, because other metals (e.g. LaCl3 or CuSO4) that strongly inhibit root growth did not block shoot development in als3 seedlings. Al did not block leaf development in excised als3 shoots grown in an Al-containing medium, demonstrating that the Al-induced damage in als3 shoots was dependent on the presence of roots. This suggests that Al inhibition of als3 shoot development may be a delocalized response to Al-induced stresses in roots following Al exposure.     

Isolation and characterization of a rice mutant hypersensitive to Al

Rice (Oryza sativa L.) is a highly Al-resistant species among small grain crops, but the mechanism responsible for the high Al resistance has not been elucidated. In this study, rice mutants sensitive to Al were isolated from M(3) lines derived from an Al-resistant cultivar, Koshihikari, irradiated with gamma-rays. Relative root elongation was used as a parameter for evaluating Al resistance. After initial screening plus two rounds of confirmatory testing, a mutant (als1) was isolated from a total of 560 lines. This mutant showed a phenotype similar to the wild-type plant in the absence of Al. However, in the presence of 10 microM Al, root elongation was inhibited 70% in the mutant, but only 8% in the wild-type plant. The mutant also showed poorer root growth in acid soil. The Al content of root apices (0-1 cm) was much lower in the wild-type plant. The sensitivity to other metals including Cd and La did not differ between the mutant and the wild-type plants. A small amount of citrate was secreted from the roots of the mutant in response to Al stress, but there was no difference from that secreted by the wild-type plant. Genetic analysis of F(2) populations between als1 and wild-type plants showed that the Al-resistant seedlings and Al-sensitive seedlings segregated at a 3 : 1 ratio, indicating that the high sensitivity to Al in als1 is controlled by a single recessive gene. The gene was mapped to the long arm of chromosome 6, flanked by InDel markers MaOs0619 and MaOs0615.     

Adequate phenylalanine synthesis mediated by G protein is critical for protection from UV radiation damage in young etiolated Arabidopsis thaliana seedlings

Etiolated Arabidopsis thaliana seedlings, lacking a functional prephenate dehydratase1 gene (PD1), also lack the ability to synthesize phenylalanine (Phe) and, as a consequence, phenylpropanoid pigments. We find that low doses of ultraviolet (UV)-C (254 nm) are lethal and low doses of UV-B cause severe damage to etiolated pd1 mutants, but not to wild-type (wt) seedlings. Furthermore, exposure to UV-C is lethal to etiolated gcr1 (encoding a putative G protein-coupled receptor in Arabidopsis) mutants and gpa1 (encoding the sole G protein alpha subunit in Arabidopsis) mutants. Addition of Phe to growth media restores wt levels of UV resistance to pd1 mutants. The data indicate that the Arabidopsis G protein-signalling pathway is critical to providing protection from UV, and does so via the activation of PD1, resulting in the synthesis of Phe. Cotyledons of etiolated pd1 mutants have proplastids (compared with etioplasts in wt), less cuticular wax and fewer long-chain fatty acids. Phe-derived pigments do not collect in the epidermal cells of pd1 mutants when seedlings are treated with UV, particularly at the cotyledon tip. Addition of Phe to the growth media restores a wt phenotype to pd1 mutants.     

A multidrug and toxic compound extrusion (MATE) transporter modulates auxin levels in root to regulate root development and promotes aluminium tolerance

MATE (multidrug and toxic compound extrusion) transporters play multiple roles in plants including detoxification, secondary metabolite transport, aluminium (Al) tolerance, and disease resistance. Here we identify and characterize the role of the Arabidopsis MATE transporter DETOXIFICATION30. AtDTX30 regulates auxin homeostasis in Arabidopsis roots to modulate root development and Al-tolerance. DTX30 is primarily expressed in roots and localizes to the plasma membrane of root epidermal cells including root hairs. dtx30 mutants exhibit reduced elongation of the primary root, root hairs, and lateral roots. The mutant seedlings accumulate more auxin in their root tips indicating role of DTX30 in maintaining auxin homeostasis in the root. Al induces DTX30 expression and promotes its localization to the distal transition zone. dtx30 seedlings accumulate more Al in their roots but are hyposensitive to Al-mediated rhizotoxicity perhaps due to saturation in root growth inhibition. Increase in expression of ethylene and auxin biosynthesis genes in presence of Al is absent in dtx30. The mutants exude less citrate under Al conditions, which might be due to misregulation of AtSTOP1 and the citrate transporter AtMATE. In conclusion, DTX30 modulates auxin levels in root to regulate root development and in the presence of Al indirectly modulates citrate exudation to promote Al tolerance.     

Interaction of aluminium and drought stress on root growth and crop yield on acid soils

Background

Aluminium (Al) toxicity and drought stress are two major constraints for crop production in the world, particularly in the tropics. The variation in rainfall distribution and longer dry spells in much of the tropics during the main growing period of crops are becoming increasingly important yield-limiting factors with the global climate change. As a result, crop genotypes that are tolerant of both drought and Al toxicity need to be developed.

Scope

The present review mainly focuses on the interaction of Al and drought on root development, crop growth and yield on acid soils. It summarizes evidence from our own studies and other published/related work, and provides novel insights into the breeding for the adaptation to these combined abiotic stresses. The primary symptom of Al phytotoxicity is the inhibition of root growth. The impeded root system will restrict the roots for exploring the acid subsoil to absorb water and nutrients which is particularly important under condition of low soil moisture in the surface soil under drought. Whereas drought primarily affects shoot growth, effects of phytotoxic Al on shoot growth are mostly secondary effects that are induced by Al affecting root growth and function, while under drought stress root growth may even be promoted. Much progress has recently been made in the understanding of the physiology and molecular biology of the interaction between Al toxicity and drought stress in common bean (Phaseolus vulgaris L.) in hydroponics and in an Al-toxic soil.

Conclusions

Crops growing on acid soils yield less than their potential because of the poorly developed root system that limits nutrient and water uptake. Breeding for drought resistance must be combined with Al resistance, to assure that drought resistance is expressed adequately in crops grown on soils with acid Al-toxic subsoils.     

Two Arabidopsis srfr (suppressor of rps4-RLD) mutants exhibit avrRps4-specific disease resistance independent of RPS4

RPS4 specifies the Arabidopsis disease resistance response to Pseudomonas syringae pv. tomato expressing avrRps4 and was cloned based on the identification of RLD as a naturally occurring susceptible accession. To dissect the molecular and genetic basis of disease resistance, we used a genetic approach to identify suppressor mutations that reactivate the avrRps4-triggered defense response in RLD. In this report, we describe two non-allelic srfr (suppressor of rps4-RLD) mutants, srfr1 and srfr3, that were susceptible to virulent P. syringae pv. tomato strain DC3000, but resistant to DC3000 expressing avrRps4. In quantitative bacterial growth assays, growth of DC3000 was similar in wild-type control and both mutant lines, indicating that basal resistance was not enhanced in srfr1 and srfr3. Growth of DC3000 (avrRps4) was approximately 30-fold lower in srfr1 and srfr3 than in RLD, but intermediate compared with fully resistant Col-0 and transgenic RLD containing RPS4-Col. The srfr1 and srfr3 mutants did not develop spontaneous lesions prior to inoculation or constitutively express the pathogenesis-related gene PR-1. Therefore, srfr1 and srfr3 constitute novel avr-specific mutants that differ from previously described Arabidopsis mutants with elevated disease resistance. The srfr1 and srfr3 mutations were recessive, and both mapped to the bottom of chromosome IV. Genetic analysis indicated that resistance in srfr1 and srfr3 was independent of the rps4-RLD allele, but dependent on a second gene in RLD. We propose that SRFR1 and SRFR3 are negative regulators of avrRps4-triggered gene-for-gene disease resistance.     

Protein geranylgeranyltransferase I is involved in specific aspects of abscisic acid and auxin signaling in Arabidopsis

Arabidopsis (Arabidopsis thaliana) mutants lacking a functional ERA1 gene, which encodes the beta-subunit of protein farnesyltransferase (PFT), exhibit pleiotropic effects that establish roles for protein prenylation in abscisic acid (ABA) signaling and meristem development. Here, we report the effects of T-DNA insertion mutations in the Arabidopsis GGB gene, which encodes the beta-subunit of protein geranylgeranyltransferase type I (PGGT I). Stomatal apertures of ggb plants were smaller than those of wild-type plants at all concentrations of ABA tested, suggesting that PGGT I negatively regulates ABA signaling in guard cells. However, germination of ggb seeds in response to ABA was similar to the wild type. Lateral root formation in response to exogenous auxin was increased in ggb seedlings compared to the wild type, but no change in auxin inhibition of primary root growth was observed, suggesting that PGGT I is specifically involved in negative regulation of auxin-induced lateral root initiation. Unlike era1 mutants, ggb mutants exhibited no obvious developmental phenotypes. However, era1 ggb double mutants exhibited more severe developmental phenotypes than era1 mutants and were indistinguishable from plp mutants lacking the shared alpha-subunit of PFT and PGGT I. Furthermore, overexpression of GGB in transgenic era1 plants partially suppressed the era1 phenotype, suggesting that the relatively weak phenotype of era1 plants is due to partial redundancy between PFT and PGGT I. These results are discussed in the context of Arabidopsis proteins that are putative substrates of PGGT I.     

Aluminum induces low phosphate adaptive responses and modulates primary and lateral root growth by differentially affecting auxin signaling in Arabidopsis seedlings

Aims

The aims of this work were to investigate the aluminum (Al) and phosphate (P) interactions in the regulation of root system architecture of Arabidopsis thaliana seedlings and the contribution of auxin signaling in primary and lateral root growth in response to Al toxicity.

Methods

Detailed analyses of root system architecture and cell division were performed in Arabidopsis WT seedlings and in low phosphorus insensitive mutants lpi1-3 and lpr1-1 lpr2-1 in response to Al. Expression studies of P-deficiency regulated phosphate transporter AtPT2 were also conducted. The role of auxin as a mediator of root morphogenetic changes by Al was evaluated by using the auxin-signaling mutants tir1, tir1 afb2 afb3, and arf7 arf19.

Results

Al inhibited primary root growth by affecting cell cycle progression and causing differentiation of cells in the root meristem. These effects were reduced in low phosphorus insensitive lpi1-3 and low phosphate resistant lpr1-1 lpr2-1 Arabidopsis mutants. Al also activated the expression of the low phosphate-induced P transporter AtPT2 in roots. Lateral root formation by Al decreased in tir1 afb2 afb3 while arf7 arf19 mutants were highly resistant to Al in both primary root inhibition and lateral root induction.

Conclusions

Our results suggest that lateral root formation in response to Al toxicity and P deficiency may involve common signaling mechanisms, while a pathway involving ARF7 and ARF19 is important for primary root growth inhibition by Al.     

Two dominant photomorphogenic mutations of Arabidopsis thaliana identified as suppressor mutations of hy2

By screening suppressor mutants of the hy2 mutation of Arabidopsis thaliana , two dominant photomorphogenic mutants, shy1-1D and shy2-1D , for two genetic loci designated as SHY1 and SHY2 ( s uppressor of hy 2 mutation) have been isolated. Both of these non-allelic, extragenic suppressor mutations of hy2 are located on chromosome 1 of the Arabidopsis genome. Both mutations suppress the elongated hypocotyl phenotype of hy2 by light-independent inhibition of hypocotyl growth as well as by increasing the effectiveness of light inhibition of hypocotyl elongation. The shy1-1D mutation is partially photomorphogenic in darkness with apical hook opening and reduced hypocotyl elongation. The shy2-1D mutant displays highly photomorphogenic characteristics in darkness such as true leaf development, cotyledon expansion, and extremely reduced hypocotyl growth. In regard to hypocotyl elongation, however, the shy2-1D mutation is still light sensitive. Examination of red/far-red light responses shows that the shy1-1D mutation suppresses the hypocotyl elongation of the hy2 mutation effectively in red light but not effectively in far-red light. The shy2-1D suppresses hypocotyl elongation of the hy2 mutation effectively in both red and far-red light. Both mutations can also suppress the early-flowering phenotype of hy2 and have a distinct pleiotropic effect on leaf development such as upward leaf rolling. The data obtained suggest that SHY1 and SHY2 represent a novel class of components involved in the photomorphogenic pathways of Arabidopsis . This is the first report on the identification of dominant mutations in the light signal transduction pathway of plants.     

Interactions between abscisic acid and ethylene signaling cascades

We screened for mutations that either enhanced or suppressed the abscisic acid (ABA)-resistant seed germination phenotype of the Arabidopsis abi1-1 mutant. Alleles of the constitutive ethylene response mutant ctr1 and ethylene-insensitive mutant ein2 were recovered as enhancer and suppressor mutations, respectively. Using these and other ethylene response mutants, we showed that the ethylene signaling cascade defined by the ETR1, CTR1, and EIN2 genes inhibits ABA signaling in seeds. Furthermore, epistasis analysis between ethylene- and ABA-insensitive mutations indicated that endogenous ethylene promotes seed germination by decreasing sensitivity to endogenous ABA. In marked contrast to the situation in seeds, ein2 and etr1-1 roots were resistant to both ABA and ethylene. Our data indicate that ABA inhibition of root growth requires a functional ethylene signaling cascade, although this inhibition is apparently not mediated by an increase in ethylene biosynthesis. These results are discussed in the context of the other hormonal regulations controlling seed germination and root growth.     

Ethylene stimulates nutations that are dependent on the ETR1 receptor

Ethylene influences a number of processes in Arabidopsis (Arabidopsis thaliana) through the action of five receptors. In this study, we used high-resolution, time-lapse imaging to examine the long-term effects of ethylene on growing, etiolated Arabidopsis seedlings. These measurements revealed that ethylene stimulates nutations of the hypocotyls with an average delay in onset of over 6 h. The nutation response was constitutive in ctr1-2 mutants maintained in air, whereas ein2-1 mutants failed to nutate when treated with ethylene. Ethylene-stimulated nutations were also eliminated in etr1-7 loss-of-function mutants. Transformation of the etr1-7 mutant with a wild-type genomic ETR1 transgene rescued the nutation phenotype, further supporting a requirement for ETR1. Loss-of-function mutations in the other receptor isoforms had no effect on ethylene-stimulated nutations. However, the double ers1-2 ers2-3 and triple etr2-3 ers2-3 ein4-4 loss-of-function mutants constitutively nutated in air. These results support a model where all the receptors are involved in ethylene-stimulated nutations, but the ETR1 receptor is required and has a contrasting role from the other receptor isoforms in this nutation phenotype. Naphthylphthalamic acid eliminated ethylene-stimulated nutations but had no effect on growth inhibition caused by ethylene, pointing to a role for auxin transport in the nutation phenotype.     

Overexpression of an auxilin-like gene (F9E10.5) can suppress Al uptake in roots of Arabidopsis

Plants resistant to aluminium (Al) stress were isolated from Arabidopsis thaliana enhancer-tagged mutant lines. Compared with the parental Col-7 control line, one of the resistant candidates, #355-2, showed a higher expression of the F9E10.5 gene (At1g75100) on chromosome 1, a lower Al content in whole roots, and a shorter root hair length (approximately 30%). Both Al influx and associated oxidative stress occurred in root hairs, as well as in root tips of Col-7; however, they were seen only in root tips of #355-2. Transgenic plants overexpressing the F9E10.5 gene showed a slightly higher Al resistance than their parental control line (Ler). The F9E10.5 gene encodes an auxilin-like protein related to the clathrin-uncoating process in endocytosis. Microscopic observation indicated that both Al ion influx and endocytosis activity were lower in root hair cells of the #355-2 line than in those of Col-7. These results suggested that overexpression of this auxilin-like protein inhibits endocytosis in root hair cells by a disturbance of the transport system as in animal cells shown previously. It was also suggested that a part of the Al influx occurred via endocytosis in root hair cells in Arabidopsis. The Al resistance in the #355-2 line may therefore be due to a lower Al uptake via endocytosis in the root hair region.