Arabidopsis AtGSTF2 is regulated by ethylene and auxin, and encodes a glutathione S-transferase that interacts with flavonoids

Expression of the Arabidopsis glutathione S-transferase (GST) gene AtGSTF2 is induced by several stimuli, but the function of this GST remains unknown. We demonstrate that AtGSTF2 expression is also induced by glutathione, paraquat, copper, and naphthalene acetic acid (NAA) via a mechanism independent of ethylene perception, as determined by analysis of the ethylene-insensitive etr1 mutant. Deletion analyses identified two promoter regions important for regulation of AtGSTF2 expression in response to several of these inducers. Previous studies have suggested that AtGSTF2 interacts with indole-3-acetic acid (IAA) and the auxin transport inhibitor 1-N-naphthylphthalamic acid (NPA). We show that recombinant AtGSTF2 directly binds IAA, NPA, and the artificial auxin NAA. As NPA may act as an endogenous flavonoid regulator of auxin transport, competition between NPA and flavonoids for binding to AtGSTF2 was examined. Both quercetin and kaempferol competed with NPA for AtGSTF2 binding, indicating that all three compounds bind AtGSTF2 at the same site. In transgenic Arabidopsis seedlings, AtGSTF2::GUS expression occurred at the root-shoot transition zone and was induced in this region, as well as at the root distal elongation zone, after treatment with IAA. In wild-type seedlings, AtGSTF2 is localized near the plasma membrane of cells in the root-shoot transition zone. However, both AtGSTF2::GUS expression and localization of AtGSTF2 protein were disrupted in flavonoid-deficient tt4 seedlings. Our results indicate that AtGSTF2 is involved not only in stress responses but also in development under normal growth conditions.     

Organ-specific expression of glutathione S-transferases and the efficacy of herbicide safeners in Arabidopsis

The functions of plant glutathione S-transferases (GSTs) under normal growth conditions are poorly understood, but their activity as detoxification enzymes has been harnessed in agriculture for selective weed control. Herbicide safeners protect monocot crops from herbicide injury but have little effect on weedy monocot or dicot species. Protection by safeners is associated with expression of herbicide-metabolizing enzymes including GSTs, but the basis for selective action of safeners between monocots and dicots is not known. To address this question we have studied the response of Arabidopsis (Arabidopsis thaliana) to various safeners. Benoxacor, fenclorim, and fluxofenim did not protect Arabidopsis from herbicide injury but did induce RNA expression of the glutathione-conjugate transporters encoded by AtMRP1, AtMRP2, AtMRP3, and AtMRP4. These safeners also induced the organ-specific expression of AtGSTU19 and AtGSTF2, two previously characterized Arabidopsis GSTs from different classes of this enzyme family. RNA hybridization, immunoblot, and reporter gene analyses indicated expression of AtGSTU19 induced by safeners predominated in roots. To test the hypothesis that increased expression of AtGSTU19 would be sufficient to provide tolerance to chloroacetamide herbicides, a chimeric gene was produced containing the open reading frame for this GST driven by a constitutive promoter. Plants containing this transgene had a modest increase in AtGSTU19 protein, predominantly in roots, but this had no effect on tolerance to chloroacetamide herbicides. The localized induction of GSTs by safeners in roots of Arabidopsis may explain why these compounds are unable to provide herbicide tolerance to dicot plant species.     

Interactions between jasmonates and ethylene in the regulation of root hair development in Arabidopsis

Root hair formation is an important model with which to study cell patterning and differentiation in higher plants. Ethylene and auxin are critical regulators of root hair development. The role of jasmonates (JAs) was examined in Arabidopsis root hair development as well as their interactions with ethylene in this process. The results have shown that both methyl jasmonate (MeJA) and jasmonic acid (JA) have a pronounced effect on promoting root hair formation. However, the effect of MeJA and JA on root hair formation was blocked by ethylene inhibitors Ag+ or aminoethoxyvinylglycine (AVG). The stimulatory effects of MeJA and JA were also diminished in ethylene-insensitive mutants etr1-1 and etr1-3. Furthermore, the JA biosynthesis inhibitors ibuprofen and salicylhydroxamic acid (SHAM) suppressed 1-aminocyclopropane-1-carboxylic acid (ACC)-induced root hair formation, and decreased the root hairs in seedlings of the ethylene over-producing mutant eto1-1. These results suggested that JAs promote root hair formation, through an interaction with ethylene.     

Expression of AtPRP3, a proline-rich structural cell wall protein from Arabidopsis, is regulated by cell-type-specific developmental pathways involved in root hair formation

The tightly regulated expression patterns of structural cell wall proteins in several plant species indicate that they play a crucial role in determining the extracellular matrix structure for specific cell types. We demonstrate that AtPRP3, a proline-rich cell wall protein in Arabidopsis, is expressed in root-hair-bearing epidermal cells at the root/shoot junction and within the root differentiation zone of light-grown seedlings. Several lines of evidence support a direct relationship between AtPRP3 expression and root hair development. AtPRP3/beta-glucuronidase (GUS) expression increased in roots of transgenic seedlings treated with either 1-aminocyclopropane-1-carboxylic acid (ACC) or alpha-naphthaleneacetic acid (alpha-NAA), compounds known to promote root hair formation. In the presence of 1-alpha-(2-aminoethoxyvinyl)glycine (AVG), an inhibitor of ethylene biosynthesis, AtPRP3/GUS expression was strongly reduced, but could be rescued by co-addition of ACC or alpha-NAA to the growth medium. In addition, AtPRP3/GUS activity was enhanced in ttg and gl2 mutant backgrounds that exhibit ectopic root hairs, but was reduced in rhd6 and 35S-R root-hair-less mutant seedlings. These results indicate that AtPRP3 is regulated by developmental pathways involved in root hair formation, and are consistent with AtPRP3's contributing to cell wall structure in Arabidopsis root hairs.     

Auxin and ethylene response interactions during Arabidopsis root hair development dissected by auxin influx modulators

The plant hormones auxin and ethylene have been shown to play important roles during root hair development. However, cross talk between auxin and ethylene makes it difficult to understand the independent role of either hormone. To dissect their respective roles, we examined the effects of two compounds, chromosaponin I (CSI) and 1-naphthoxyacetic acid (1-NOA), on the root hair developmental process in wild-type Arabidopsis, ethylene-insensitive mutant ein2-1, and auxin influx mutants aux1-7, aux1-22, and double mutant aux1-7 ein2. Beta-glucuronidase (GUS) expression analysis in the BA-GUS transgenic line, consisting of auxin-responsive domains of PS-IAA4/5 promoter and GUS reporter, revealed that 1-NOA and CSI act as auxin uptake inhibitors in Arabidopsis roots. The frequency of root hairs in ein2-1 roots was greatly reduced in the presence of CSI or 1-NOA, suggesting that endogenous auxin plays a critical role for the root hair initiation in the absence of an ethylene response. All of these mutants showed a reduction in root hair length, however, the root hair length could be restored with a variable concentration of 1-naphthaleneacetic acid (NAA). NAA (10 nM) restored the root hair length of aux1 mutants to wild-type level, whereas 100 nM NAA was needed for ein2-1 and aux1-7 ein2 mutants. Our results suggest that insensitivity in ethylene response affects the auxin-driven root hair elongation. CSI exhibited a similar effect to 1-NOA, reducing root hair growth and the number of root hair-bearing cells in wild-type and ein2-1 roots, while stimulating these traits in aux1-7and aux1-7ein2 roots, confirming that CSI is a unique modulator of AUX1.     

Induction of glutathione S-transferases in Arabidopsis by herbicide safeners

Herbicide safeners increase herbicide tolerance in cereals but not in dicotyledenous crops. The reason(s) for this difference in safening is unknown. However, safener-induced protection in cereals is associated with increased expression of herbicide detoxifying enzymes, including glutathione S-transferases (GSTs). Treatment of Arabidopsis seedlings growing in liquid medium with various safeners similarly resulted in enhanced GST activities toward a range of xenobiotics with benoxacor, fenclorim, and fluxofenim being the most effective. Safeners also increased the tripeptide glutathione content of Arabidopsis seedlings. However, treatment of Arabidopsis plants with safeners had no effect on the tolerance of seedlings to chloroacetanilide herbicides. Each safener produced a distinct profile of enhanced GST activity toward different substrates suggesting a differential induction of distinct isoenzymes. This was confirmed by analysis of affinity-purified GST subunits by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. AtGSTU19, a tau class GST, was identified as a dominant polypeptide in all samples. When AtGSTU19 was expressed in Escherichia coli, the recombinant enzyme was highly active toward 1-chloro-2,4-dinitrobenzene, as well as chloroacetanilide herbicides. Immunoblot analysis confirmed that AtGSTU19 was induced in response to several safeners. Differential induction of tau GSTs, as well as members of the phi and theta classes by safeners, was demonstrated by RNA-blot analysis. These results indicate that, although Arabidopsis may not be protected from herbicide injury by safeners, at least one component of their detoxification systems is responsive to these compounds.     

Auxin and ethylene are involved in the responses of root system architecture to low boron supply in Arabidopsis seedlings

Changes in root architecture are one of the adaptive strategies used by plants to compensate for nutrient deficiencies in soils. In this work, the temporal responses of Arabidopsis (Arabidopsis thaliana) root system architecture to low boron (B) supply were investigated. Arabidopsis Col-0 seedlings were grown in 10 μM B for 5 days and then transferred to a low B medium (0.4 μM) or control medium (10 μM) for a 4-day period. Low B supply caused an inhibition of primary root (PR) growth without altering either the growth or number of lateral roots (LRs). In addition, low B supply induced root hair formation and elongation in positions close to the PR meristem not observed under control conditions. The possible role of auxin and ethylene in the alteration of root system architecture elicited by low B supply was also studied by using two Arabidopsis reporter lines (DR5:GUS and EBS:GUS) and two Arabidopsis mutants with impaired auxin and ethylene signaling (aux1-22 and ein2-1). Low B supply increased auxin reporter DR5:GUS activity in PR tip, suggesting that low B alters the pattern of auxin distribution in PR tip. Moreover, PR elongation in aux1-22 mutant was less sensitive to low B treatment than in wild-type plants, which suggests that auxin resistant 1 (AUX1) participates in the inhibition of PR elongation under low B supply. From all these results, a hypothetical model to explain the effect of low B treatment on PR growth is proposed. We also show that ethylene, via ethylene-insensitive 2 (EIN2) protein, is involved in the induction of root hair formation and elongation under low B treatment.     

Auxin and ethylene promote root hair elongation in Arabidopsis

Genetic and physiological studies implicate the phytohormones auxin and ethylene in root hair development. To learn more about the role of these compounds, we have examined the root hair phenotype of a number of auxin- and ethylene-related mutants. In a previous study, Masucci and Schiefelbein (1996) showed that neither the auxin response mutations aux1 and axr1 nor the ethylene response mutations etr1 and ein2 have a significant effect on root hair initiation. In this study, we found that mutants deficient in either auxin or ethylene response have a pronounced effect on root hair length. Treatment of wild-type, axr1 and etr1 seedlings with the synthetic auxin, 2,4-D, or the ethylene precursor ACC, led to the development of longer root hairs than untreated seedlings. Furthermore, axr1 seedlings grown in the presence of ACC produce ectopic root hairs and an unusual pattern of long root hairs followed by regions that completely lack root hairs. These studies indicate that both auxin and ethylene are required for normal root hair elongation.     

Germinating pigeonpea (Cajanus cajan) seeds secrete factor(s) having antiethylene-like effects

Germinating pigeonpea seeds were found to release factor(s), which dramatically altered the growth pattern of rice seedlings. This included an increased sensitivity of roots to gravity, an increase in seminal root length, and increased density of branch roots on the seminal root and suppression of root hair formation. Furthermore, the known characteristic responses of rice to ethylene (namely, coleoptile growth promotion and induction of senescence) were inhibited by the presence of these factor(s). This altered growth pattern of rice seedlings was similar to that brought about by the ethylene-antagonist, silver nitrate. Further, the change in growth pattern in rice was specifically reversed by ethrel. In addition, in wild-type Arabidopsis thaliana these factors suppressed formation of root hair, the development of which is known to be promoted by ethylene. In contrast, in a mutant A. thaliana which shows constitutive ethylene response ( ctr1-1 ), the factors failed to inhibit root hair formation. These observations indicated that germinating pigeonpea seeds secrete factor(s), which have antiethylene-like effects.     

Hormones act downstream of TTG and GL2 to promote root hair outgrowth during epidermis development in the Arabidopsis root.

The Arabidopsis root produces a position-dependent pattern of hair-bearing and hairless cell types during epidermis development. Five loci (TRANSPARENT TESTA GLABRA [TTG], GLABRA2 [GL2], ROOT HAIR DEFECTIVE6 [RHD6], CONSTITUTIVE TRIPLE RESPONSE1 [CTR1], and AUXIN RESISTANT2 [AXR2]) and the plant hormones ethylene and auxin have been reported to affect the production of root hair and hairless cells in the Arabidopsis root. In this study, genetic, molecular, and physiological tests were employed to define the roles of these loci and hormones. Epistasis tests and reporter gene studies indicated that the hairless cell-promoting genes TTG and GL2 are likely to act early to negatively regulate the ethylene and auxin pathways. Studies of the developmental timing of the hormone effects indicated that ethylene and auxin pathways promote root hair outgrowth after cell-type differentiation has been initiated. The genetic analysis of ethylene-and auxin-related mutations showed that root hair formation is influenced by a network of hormone pathways, including a partially redundant ethylene signaling pathway. A model is proposed in which the patterning of root epidermal cells in Arabidopsis is regulated by the cell position-dependent action of the TTG/GL2 pathway, and the ethylene and auxin hormone pathways act to promote root hair outgrowth at a relatively late stage of differentiation.     

乙烯在植物形态发育中的作用(综述)

乙烯对植物生长发育的许多方面,如根的形成、花的诱导、器官衰老脱落均起重要的调节作用。拟南芥根表皮中的乙烯是根毛发育的一个正调控因子。兰花授粉诱导花被萎蔫过程的早期反应是提高了对内源乙烯水平的敏感性。     

Profilin plays a role in cell elongation, cell shape maintenance, and flowering in Arabidopsis

Profilin (PFN) is an ubiquitous, low-M(r), actin-binding protein involved in the organization of the cytoskeleton of eukaryotes including higher plants. PFNs are encoded by a multigene family in Arabidopsis. We have analyzed in vivo functions of Arabidopsis PFN by generating transgenic plants carrying a 35S-PFN-1 or 35S-antisense PFN-1 transgene. Etiolated seedlings underexpressing PFN (PFN-U) displayed an overall dwarf phenotype with short hypocotyls whose lengths were 20% to 25% that of wild type (WT) at low temperatures. Light-grown PFN-U plants were smaller in stature and flowered early. Compared with equivalent cells in WT, most cells in PFN-U hypocotyls and roots were shorter, but more isodiametric, and microscopic observations of etiolated PFN-U hypocotyls revealed a rough epidermal surface. In contrast, light-grown seedlings overexpressing PFN had longer roots and root hair although etiolated seedlings overexpressing PFN were either the same size or slightly longer than WT seedlings. Transgenic seedlings harboring a PFN-1-GUS transgene directed expression in root and root hair and in a ring of cells at the elongating zone of the root tip. As the seedlings matured PFN-1-GUS was mainly expressed in the vascular bundles of cotyledons and leaves. Our results show that Arabidopsis PFNs play a role in cell elongation, cell shape maintenance, polarized growth of root hair, and unexpectedly, in determination of flowering time.     

The expression patterns of arabinogalactan-protein AtAGP30 and GLABRA2 reveal a role for abscisic acid in the early stages of root epidermal patterning

In the Arabidopsis root, patterning of the epidermal cell types is position-dependent. The epidermal cell pattern arises early during root development, and can be visualized using reporter genes driven by the GLABRA (GL)2 promoter as markers. The GL2 gene is preferentially expressed in the differentiating hairless cells (atrichoblasts) during a period in which epidermal cell identity is believed to be established. We show that AtAGP30 is also expressed in atrichoblasts. This gene encodes an arabinogalactan-protein (AGP) that is known to play a role in root regeneration and increases abscisic acid (ABA)-response rates. Although the expression level of this gene is regulated by the plant growth factors ABA and ethylene, only ABA was found to affect the tissue-specific pattern of expression. ABA also disrupts the expression pattern of the GL2::GUS (beta-glucuronidase) reporter gene. Our results indicate that ABA regulates epidermal cell-type-specific gene expression in the meristematic zone of the Arabidopsis root, while ethylene is known to act at later stages of epidermal differentiation. Despite its effects on the early stages of root epidermal patterning, ABA does not affect root hair formation on mature wild-type epidermal cells, suggesting that other developmental cues, like positional information, can progressively over-ride the ABA-mediated disruption of early epidermal patterning.     

生长素和乙烯互作调控硝酸铵诱导的根毛分叉

以野生型拟南芥(WT)及其生长素和乙烯不敏感型突变体(aux1-7、axr1-3、etr1-1和etr1-3)为实验材料,采用固体培养法研究了高浓度硝酸铵对根毛发育的影响,以揭示其调控根毛发育的机制。结果表明:(1)随着外源硝酸铵浓度的逐渐增加,拟南芥根毛伸长受阻,产生大量的分叉根毛。(2)高浓度硝酸铵条件下,外源活性氧或活性氧产生抑制剂二苯基氯化碘(DPI)的添加能抑制高浓度硝酸铵诱导的分叉根毛产生。(3)高浓度硝酸铵条件下,外源生长素或乙烯合成前体物质1-氨基-环丙烷-1-羧酸(ACC)处理能恢复根毛的正常生长,解除高浓度硝酸铵诱导根毛分叉现象。(4)高浓度硝酸铵条件下,外源生长素处理乙烯不敏感型突变体或ACC处理生长素不敏感型突变体均能抑制突变体分叉根毛的形成。研究表明,活性氧、生长素和乙烯都参与了高浓度硝酸铵对根毛发育的过程调控;在硝酸铵诱导的根毛分叉中生长素和乙烯存在相互作用,在缺乏生长素信号通路时,乙烯能够发挥补充作用抑制分叉根毛的产生;在缺乏乙烯信号通路时,生长素也可以弥补缺失乙烯的作用抑制根毛的分叉,但是需要更高浓度的生长素才能充分抑制分叉根毛的产生。