Effects of hypergravity conditions on elongation growth and lignin formation in the inflorescence stem of Arabidopsis thaliana

The effects of hypergravity on elongation growth and lignin deposition in secondary cell walls of the Arabidopsis thaliana (L.) Heynh. inflorescence stem were examined in plants grown for 3 days after exposure to hypergravity in the direction from shoot to root at 300 g for 24 h. The content of acetylbromide-extractable lignins in a secondary cell wall fraction prepared by enzyme digestion of inflorescence stem segments removing primary cell wall components was significantly increased by the hypergravity stimulus. Xylem vessels, particularly in a region closer to the base of the inflorescence stem, increased in number. Gadolinium chloride at 0.1 mM, a blocker of mechanoreceptors, partially suppressed the effect of hypergravity on lignin deposition in the secondary cell wall fraction. These results suggest that mechanoreceptors are responsible for hypergravity-induced lignin deposition in secondary cell walls in A. thaliana inflorescence stems.     

Flavonoid-related regulation of auxin accumulation in<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis>-induced plant tumors

Agrobacterium tumefaciens-induced plant tumors accumulate considerable concentrations of free auxin. To determine possible mechanisms by which high auxin concentrations are maintained, we examined the pattern of auxin and flavonoid distribution in plant tumors. Tumors were induced in transformants of Trifolium repens (L.), containing the beta-glucuronidase ( GUS)-fused auxin-responsive promoter ( GH3) or chalcone synthase ( CHS2) genes, and in transformants of Arabidopsis thaliana (L.) Heynh., containing the GUS-fused synthetic auxin response element DR5. Expression of GH3::GUS and DR5::GUS was strong in proliferating metabolically active tumors, thus suggesting high free-auxin concentrations. Immunolocalization of total auxin with indole-3-acetic acid antibodies was consistent with GH3::GUS expression indicating the highest auxin concentration in the tumor periphery. By in situ staining with diphenylboric acid 2-aminoethyl ester, by thin-layer chromatography, reverse-phase high-performance liquid chromatography, and two-photon laser-scanning microscopy spectrometry, tumor-specific flavones, isoflavones and pterocarpans were detected, namely 7,4'-dihydroxyflavone (DHF), formononetin, and medicarpin. DHF was the dominant flavone in high free-auxin-accumulating stipules of Arabidopsis leaf primordia. Flavonoids were localized at the sites of strongest auxin-inducible CHS2::GUS expression in the tumor that was differentially modulated by auxin in the vascular tissue. CHS mRNA expression changes corresponded to the previously analyzed auxin concentration profile in tumors and roots of tumorized Ricinus plants. Application of DHF to stems, apically pretreated with alpha-naphthaleneacetic acid, inhibited GH3::GUS expression in a fashion similar to 1-N-naphthyl-phthalamic acid. Tumor, root and shoot growth was poor in inoculated tt4(85) flavonoid-deficient CHS mutants of Arabidopsis. It is concluded that CHS-dependent flavonoid aglycones are possibly endogenous regulators of the basipetal auxin flux, thereby leading to free-auxin accumulation in A. tumefaciens-induced tumors. This, in turn, triggers vigorous proliferation and vascularization of the tumor tissues and suppresses their further differentiation.     

Hormone interactions and regulation of PsPK2::GUS compared with DR5::GUS and PID::GUS in Arabidopsis thaliana

The putative pea PINOID homolog, PsPK2, is expressed in all growing plant parts and is positively regulated by auxin, gibberellin, and cytokinin. Here, we studied hormonal regulation of PsPK2::GUS expression compared with DR5::GUS and PID::GUS in Arabidopsis. PsPK2::GUS, DR5::GUS, and PID::GUS expression in Arabidopsis shoots is mainly localized in the stipules, hydathodes, veins, developing leaves, and cotyledons. Unlike DR5::GUS, PsPK2::GUS, and PID::GUS are weakly expressed in root tips. Both DR5::GUS and PsPK2::GUS are induced by different auxins and are more sensitive to methyl indole acetic acid, 4-chloro-indole acetic acid, and α-naphthalene acetic acid than others. GA(3) has no significant effect on GUS activity in DR5::GUS-transformed seedlings compared to the control, but induction by auxin and gibberellin in combination is synergistic. Cytokinin increases auxin transport in Arabidopsis seedlings. Auxin, gibberellin, and cytokinin all increase GUS activity in shoots of PsPK2::GUS transformed plants compared to the control. However, only auxin and gibberellin increase GUS activity in PID::GUS shoots. In conclusion, auxin, gibberellin, and cytokinin positively regulate PsPK2 expression in shoots, but not in roots. Auxin and gibberellin also upregulate AtPIN1 and LEAFY expression, which is similar to PsPIN1 and Uni in pea. With minor exceptions, the orthologous genes from both species are regulated similarly.     

Auxin redistribution and shifts in PIN gene expression during Arabidopsis grafting

Auxin is important in the development of plant vascular tissues. Reconnection of vascular bundles between scion and stock is a primary aim of grafting, and polar auxin transport greatly affects the formation of a continuous vascular model. The role of auxin in the process of graft-union development was studied by grafting the seedlings of Arabidopsis thaliana (L.) Heynh. DR5:GUS marker plants, which exert the auxinspecific responses. Auxin induced the DR5:GUS expression in the vascular bundles around graft surface and stimulated the formation of multiple vascular bundle reconnections on the third day after grafting (DAG). DR5:GUS expression was delayed for one day in both scion and stock and dramatically declined by the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). Vascular bundle reconnection was observed only on the 4th DAG. These results suggest that auxin stimulates the reconnection of the vascular bundles, whereas NPA inhibits it. We studied the role of PIN proteins in graft development by grafting seedlings of PIN:GUS plants. PIN had different expression patterns in the graft process. Expression levels of PIN genes were analyzed by real-time PCR. All PIN genes had the higher expression level at the third DAG. We conclude that auxin stimulates the development of graft unions, and the patterns of expressions of PIN family genes can affect the development of graft-union by controlling the auxin flow.     

Arabidopsis PLC2 is involved in auxin‐modulated reproductive development

Phospholipase C (PLC) is an enzyme that plays crucial roles in various signal transduction pathways in mammalian cells. However, the role of PLC in plant development is poorly understood. Here we report involvement of PLC2 in auxin‐mediated reproductive development in Arabidopsis. Disruption of PLC2 led to sterility, indicating a significant role for PLC2 in reproductive development. Development of both male and female gametophytes was severely perturbed in plc2 mutants. Moreover, elevated auxin levels were observed in plc2 floral tissues, suggesting that the infertility of plc2 plants may be associated with increased auxin concentrations in the reproductive organs. We show that expression levels of the auxin reporters DR5:GUS and DR5:GFP were elevated in plc2 anthers and ovules. In addition, we found that expression of the auxin biosynthetic YUCCA genes was increased in plc2 plants. We conclude that PLC2 is involved in auxin biosynthesis and signaling, thus modulating development of both male and female gametophytes in Arabidopsis.     

Cell type identity in<Emphasis Type="Italic">Arabidopsis</Emphasis> roots is altered by both ascorbic acid-induced changes in the redox environment and the resultant endogenous auxin response

Redox plays a critical role in controlling many cellular processes of plant growth and development. To understand the effect of changes in redox on cell-type determination in the root meristem, we examined the influence of a strong reducing agent -ascorbic acid (AA) - on both the expression patterns of several cell type-specific promoters and the endogenous auxin sensitivity of auxin-responsive DR5::GUS transgenic plants. AA treatment altered the regular expression of columella-specific markers. Moreover, when the same treatment was applied to the DR5::GUS lines, normal expression of the GUS reporter was completely abolished in the auxin maximum, while exogenous auxin restored AA-driven depletion of that maximum. Interestingly, the level of DHA (dehydroascorbate, an oxidized form of AA) in the AA-treated roots was greatly increased. This indicates that changes in cell-type specificity and the sensitivity to endogenous auxin may result from an increase in the cellular DHA that is metabolized from exogenously supplied AA. Therefore, we propose that redox changes in the root meristem alter auxin homeostasis, perhaps causing a change in cell types within the root meristem.     

Brassinosteroids interact with auxin to promote lateral root development in Arabidopsis

Plant hormone brassinosteroids (BRs) and auxin exert some similar physiological effects likely through their functional interaction, but the mechanism for this interaction is unknown. In this study, we show that BRs are required for lateral root development in Arabidopsis and that BRs act synergistically with auxin to promte lateral root formation. BR perception is required for the transgenic expression of the beta-glucuronidase gene fused to a synthetic auxin-inducible promoter (DR5::GUS) in root tips, while exogenous BR promotes DR5::GUS expression in the root tips and the stele region proximal to the root tip. BR induction of both lateral root formation and DR5::GUS expression is suppressed by the auxin transport inhibitor N-(1-naphthyl) phthalamic acid. Importantly, BRs promote acropetal auxin transport (from the base to the tip) in the root. Our observations indicate that BRs regulate auxin transport, providing a novel mechanism for hormonal interactions in plants and supporting the hypothesis that BRs promote lateral root development by increasing acropetal auxin transport.     

Hypergravity stimulus enhances primary xylem development and decreases mechanical properties of secondary cell walls in inflorescence stems of Arabidopsis thaliana

BACKGROUND AND AIMS: The xylem plays an important role in strengthening plant bodies. Past studies on xylem formation in tension woods in poplar and also in clinorotated Prunus tree stems lead to the suggestion that changes in the gravitational conditions affect morphology and mechanical properties of xylem vessels. The aim of this study was to examine effects of hypergravity stimulus on morphology and development of primary xylem vessels and on mechanical properties of isolated secondary wall preparations in inflorescence stems of arabidopsis. METHODS: Morphology of primary xylem was examined under a light microscope on cross-sections of inflorescence stems of arabidopsis plants, which had been grown for 3-5 d after exposure to hypergravity at 300 g for 24 h. Extensibility of secondary cell wall preparation, isolated from inflorescence stems by enzyme digestion of primary cell wall components (mainly composed of metaxylem elements), was examined. Plants were treated with gadolinium chloride, a blocker of mechanoreceptors, to test the involvement of mechanoreceptors in the responses to hypergravity. KEY RESULTS: Number of metaxylem elements per xylem, apparent thickness of the secondary thickenings, and cross-section area of metaxylem elements in inflorescence stems increased in response to hypergravity. Gadolinium chloride suppressed the effect of hypergravity on the increase both in the thickness of secondary thickenings and in the cross-section area of metaxylem elements, while it did not suppress the effect of hypergravity on the increase in the number of metaxylem elements. Extensibility of secondary cell wall preparation decreased in response to hypergravity. Gadolinium chloride suppressed the effect of hypergravity on cell wall extensibility. CONCLUSIONS: Hypergravity stimulus promotes metaxylem development and decreases extensibility of secondary cell walls, and mechanoreceptors were suggested to be involved in these processes.     

Arabidopsis SMALL AUXIN UP RNA63 promotes hypocotyl and stamen filament elongation

Auxin regulates plant growth and development in part by activating gene expression. Arabidopsis thaliana SMALL AUXIN UP RNAs (SAURs) are a family of early auxin-responsive genes with unknown functionality. Here, we show that transgenic plant lines expressing artificial microRNA constructs (aMIR-SAUR-A or -B) that target a SAUR subfamily (SAUR61-SAUR68 and SAUR75) had slightly reduced hypocotyl and stamen filament elongation. In contrast, transgenic plants expressing SAUR63:GFP or SAUR63:GUS fusions had long hypocotyls, petals and stamen filaments, suggesting that these protein fusions caused a gain of function. SAUR63:GFP and SAUR63:GUS seedlings also accumulated a higher level of basipetally transported auxin in the hypocotyl than did wild-type seedlings, and had wavy hypocotyls and twisted inflorescence stems. Mutations in auxin efflux carriers could partially suppress some SAUR63:GUS phenotypes. In contrast, SAUR63:HA plants had wild-type elongation and auxin transport. SAUR63:GFP protein had a longer half-life than SAUR63:HA. Fluorescence imaging and microsomal fractionation studies revealed that SAUR63:GFP was localized mainly in the plasma membrane, whereas SAUR63:HA was present in both soluble and membrane fractions. Low light conditions increased SAUR63:HA protein turnover rate. These results indicate that membrane-associated Arabidopsis SAUR63 promotes auxin-stimulated organ elongation.     

DR5 as a reporter system to study auxin response in Populus

Key message

Auxin responsive promoter DR5 reporter system is functional in Populus to monitor auxin response in tissues including leaves, roots, and stems.

Abstract

We described the behavior of the DR5::GUS reporter system in stably transformed Populus plants. We found several similarities with Arabidopsis, including sensitivity to native and synthetic auxins, rapid induction after treatment in a variety of tissues, and maximal responses in root tissues. There were also several important differences from Arabidopsis, including slower time to maximum response and lower induction amplitude. Young leaves and stem sections below the apex showed much higher DR5 activity than did older leaves and stems undergoing secondary growth. DR5 activity was highest in cortex, suggesting high levels of auxin concentration and/or sensitivity in this tissue. Our study shows that the DR5 reporter system is a sensitive and facile system for monitoring auxin responses and distribution at cellular resolution in poplar.     

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.     

Evidence for altered polar and lateral auxin transport in the gravity persistent signal (gps) mutants of Arabidopsis

Plant shoots do not respond when they are reoriented relative to gravity at 4 degrees C. However, when returned to vertical at room temperature, these organs bend in response to the previous cold gravistimulation. The inflorescence stem of the Arabidopsis thaliana gravity persistent signal (gps) mutants respond abnormally after the cold gravistimulation: gps1 does not bend when returned to room temperature, gps2 bends the wrong way and gps3 over-responds, curving past the predicted angle. In wild type and the mutants, basipetal auxin transport in the inflorescence stem was abolished at 4 degrees C but restored when plants were returned to room temperature. In gps1, auxin transport was increased; in both gps2 and gps3, no significant difference was found when compared to wild type. Expression of the auxin-inducible P(IAA2)::GUS reporter gene, indicated that auxin-induced gene expression was redistributed to the lower side of the inflorescence stem in wild type after gravistimulation at 4 degrees C. In gps1, no asymmetries in P(IAA2)::GUS expression were seen. In gps2, P(IAA2)::GUS expression was localized to the upper side of the stem and in gps3, asymmetric P(IAA2):GUS expression was extended throughout the elongation zone of the inflorescence stem. These results are consistent with altered lateral Indole-3-acetic-acid (IAA) gradients being responsible for the phenotype of each mutant.     

The Arabidopsis dual-affinity nitrate transporter gene AtNRT1.1 (CHL1) is regulated by auxin in both shoots and roots

The AtNRT1.1 (CHL1) gene of Arabidopsis encodes a dual-affinity nitrate transporter and contributes to both low and high affinity nitrate uptake. Localization studies have shown that CHL1 expression is preferentially targeted to nascent organs and growing regions of roots and shoots in Arabidopsis. In roots, CHL1 expression is concentrated in the tips of primary and lateral roots and is activated during lateral root initiation. In shoots, strong CHL1 expression is found in young leaves and developing flower buds. These findings suggest that CHL1 expression might be regulated by a growth signal such as the phytohormone auxin. To test this, auxin regulation of CHL1 was examined. Using transgenic Arabidopsis plants containing CHL1::GUS/GFP DNA constructs, it was found that treatment with exogenous auxin or introduction of the auxin overproducing mutations (yucca and rooty) resulted in a strong increase in CHL1::GUS/GFP signals in roots and leaves. When mature roots were treated with auxin to induce lateral root formation, CHL1::GFP signals were dramatically enhanced in dividing pericycle cells and throughout primordia development. RNA blot analysis showed that CHL1 mRNA levels in whole seedlings increase within 30 min of auxin treatment. The distribution of CHL1 expression in Arabidopsis roots and shoots was found to be similar to that of DR5::GUS, a synthetic, auxin-responsive gene. These results indicate that auxin acts as an important signal regulating CHL1 expression and contributes to the targeting of CHL1 expression to nascent organs and root tips in Arabidopsis.     

Potassium carrier TRH1 is required for auxin transport in Arabidopsis roots

Disruption of the TRH1 potassium transporter impairs root hair development in Arabidopsis, and also affects root gravitropic behaviour. Rescue of these morphological defects by exogenous auxin indicates a link between TRH1 activity and auxin transport. In agreement with this hypothesis, the rate of auxin translocation from shoots to roots and efflux of [3H]IAA in isolated root segments were reduced in the trh1 mutant, but efflux of radiolabelled auxin was accelerated in yeast cells transformed with the TRH1 gene. In roots, Pro(TRH1):GUS expression was localized to the root cap cells which are known to be the sites of gravity perception and are central for the redistribution of auxin fluxes. Consistent with these findings, auxin-dependent DR5:GUS promoter-reporter construct was misexpressed in the trh1 mutant indicating that partial block of auxin transport through the root cap is associated with upstream accumulation of the phytohormone in protoxylem cells. When [K+] in the medium was reduced from 20 to 0.1 mm, wild type roots showed mild agravitropic phenotype and DR5:GUS misexpression in stelar cells. This pattern of response to low external [K+] was also affected by trh1 mutation. We conclude that the TRH1 carrier is an important part of auxin transport system in Arabidopsis roots.     

Alteration of auxin polar transport in the Arabidopsis ifl1 mutants

The INTERFASCICULAR FIBERLESS/REVOLUTA (IFL1/REV) gene is essential for the normal differentiation of interfascicular fibers and secondary xylem in the inflorescence stems of Arabidopsis. It has been proposed that IFL1/REV influences auxin polar flow or the transduction of auxin signal, which is required for fiber and vascular differentiation. Assay of auxin polar transport showed that the ifl1 mutations dramatically reduced auxin polar flow along the inflorescence stems and in the hypocotyls. The null mutant allele ifl1-2 was accompanied by a significant decrease in the expression level of two putative auxin efflux carriers. The ifl1 mutants remained sensitive to auxin and an auxin transport inhibitor. The ifl1-2 mutant exhibited visible phenotypes associated with defects in auxin polar transport such as pin-like inflorescence, reduced numbers of cauline branches, reduced numbers of secondary rosette inflorescence, and dark green leaves with delayed senescence. The visible phenotypes displayed by the ifl1 mutants could be mimicked by treatment of wild-type plants with an auxin polar transport inhibitor. In addition, the auxin polar transport inhibitor altered the normal differentiation of interfascicular fibers in the inflorescence stems of wild-type Arabidopsis. Taken together, these results suggest a correlation between the reduced auxin polar transport and the alteration of cell differentiation and morphology in the ifl1 mutants.     

Arabidopsis thickvein mutation affects vein thickness and organ vascularization, and resides in a provascular cell-specific spermine synthase involved in vein definition and in polar auxin transport

Polar auxin transport has been implicated in the induction of vascular tissue and in the definition of vein positions. Leaves treated with chemical inhibitors of polar auxin transport exhibited vascular phenotypes that include increased vein thickness and vascularization. We describe a recessive mutant, thickvein (tkv), which develops thicker veins in leaves and in inflorescence stems. The increased vein thickness is attributable to an increased number of vascular cells. Mutant plants have smaller leaves and shorter inflorescence stems, and this reduction in organ size and height is accompanied by an increase in organ vascularization, which appears to be attributable to an increase in the recruitment of cells into veins. Furthermore, although floral development is normal, auxin transport in the inflorescence stem is significantly reduced in the mutant, suggesting that the defect in auxin transport is responsible for the vascular phenotypes. In the primary root, the veins appear morphologically normal, but root growth in the tkv mutant is hypersensitive to exogenous cytokinin. The tkv mutation was found to reside in the ACL5 gene, which encodes a spermine synthase and whose expression is specific to provascular cells. We propose that ACL5/TKV is involved in vein definition (defining the boundaries between veins and nonvein regions) and in polar auxin transport, and that polyamines are involved in this process.     

Use of an inducible reporter gene system for the analysis of auxin distribution in the moss<Emphasis Type="Italic"> Physcomitrella patens</Emphasis>

The plant hormone auxin plays a major role in a variety of growth and developmental responses, even in the more ancient plants-for example, cell differentiation in mosses. Nevertheless, almost nothing is known about the distribution of auxin during moss development. To address this question, we characterised auxin distribution in the moss Physcomitrella patens using auxin-inducible reporter gene systems. Stable transgenic Physcomitrella plants were produced expressing the beta-glucuronidase (GUS) gene driven by the auxin-inducible promoters GH3 and DR5, respectively. Both fusions showed remarkable differences with respect to auxin-induced promoter strength and expression kinetics. A detailed characterisation of the GUS expression pattern in different developmental stages revealed that the highest auxin concentrations were in dividing and ontogenetic young cells.