The full-length Auxin Response Factor 8 isoform ARF8.1 controls pollen cell wall formation and directly regulates TDF1, AMS and MS188 expression.

Auxin Response Factor 8 plays a key role in late stamen development: its splice variants ARF8.4 and ARF8.2 control stamen elongation and anther dehiscence. Here, we characterized the role of ARF8 isoforms in pollen fertility. By phenotypic and ultrastructural analysis of arf8-7 mutant stamens, we found defects in pollen germination and viability caused by alterations in exine structure and pollen coat deposition. Furthermore, tapetum degeneration, a prerequisite for proper pollen wall formation, is delayed in arf8-7 anthers. In agreement, the genes encoding the transcription factors TDF1, AMS, MS188 and MS1, required for exine and pollen coat formation, and tapetum development, are downregulated in arf8-7 stamens. Consistently, the sporopollenin content is decreased, and the expression of sporopollenin synthesis/transport and pollen coat protein biosynthetic genes, regulated by AMS and MS188, is reduced. Inducible expression of the full-length isoform ARF8.1 in arf8-7 inflorescences complements the pollen (and tapetum) phenotype and restores the expression of the above transcription factors. Chromatin immunoprecipitation-quantitative polymerase chain reaction assay revealed that ARF8.1 directly targets the promoters of TDF1, AMS and MS188. In conclusion, the ARF8.1 isoform controls pollen and tapetum development acting directly on the expression of TDF1, AMS and MS188, which belong to the pollen/tapetum genetic pathway.

Arsenic accumulation in Pteris vittata: Time course, distribution, and arsenic-related gene expression in fronds and whole plantlets.

In this work, arsenic (As) accumulation and distribution over time in Pteris vittata young fronds from adult plants and in whole plantlets, grown on a highly contaminated As-soil, was determined by µ-XRF. A linear increase in As content up to 60 days was found in young fronds at different times, and a progressive distribution from the apex to the base of the fronds was observed. In whole plantlets, As signal was detectable from 9 to 20 days in the apex of a few fronds and fiddleheads. Later, up to 60 days, As was localized in all fronds, in the rhizome and in basal part of the roots. The dynamics of expression of As-related genes revealed a good correlation between As content and the level of the As (III)-antiporter PvACR3 transcript in plantlets roots and fronds and in young fronds. Moreover, the transcription of As (V)-related gametophytic genes PvGAPC1, PvOCT4 increases over time during As accumulation while PvGSTF1 is expressed only in roots. Here, we demonstrate the suitability of the µ-XRF technique to monitor As accumulation, which allowed us to propose that As is initially directly transported to fiddleheads and apex of fronds, is later distributed to the whole fronds and simultaneously accumulated in the rhizome and roots. We also provide indications on the expression of candidate genes possibly involved in As (hyper)accumulation.

Arabidopsis TWISTED DWARF1 regulates stamen elongation by differential activation of ABCB1,19-mediated auxin transport.

Despite clear evidence that a local accumulation of auxin is likewise critical for male fertility, much less is known about the components that regulate auxin-controlled stamen development. In this study, we analyzed physiological and morphological parameters in mutants of key players of ABCB-mediated auxin transport, and spatially and temporally dissected their expression on the protein level as well as auxin fluxes in the Arabidopsis stamens. Our analyses revealed that the FKBP42, TWISTED DWARF1 (TWD1), promotes stamen elongation and, to a lesser extent, anther dehiscence, as well as pollen maturation, and thus is required for seed development. Most of the described developmental defects in twd1 are shared with the abcb1 abcb19 mutant, which can be attributed to the fact that TWD1-as a described ABCB chaperone-is a positive regulator of ABCB1- and ABCB19-mediated auxin transport. However, reduced stamen number was dependent on TWD1 but not on investigated ABCBs, suggesting additional players downstream of TWD1. We predict an overall housekeeping function for ABCB1 during earlier stages, while ABCB19 seems to be responsible for the key event of rapid elongation at later stages of stamen development. Our data indicate that TWD1 controls stamen development by differential activation of ABCB1,19-mediated auxin transport in the stamen.

Phyto-Beneficial Traits of Rhizosphere Bacteria: In Vitro Exploration of Plant Growth Promoting and Phytopathogen Biocontrol Ability of Selected Strains Isolated from Harsh Environments.

Beneficial interactions between plants and some bacterial species have been long recognized, as they proved to exert various growth-promoting and health-protective activities on economically relevant crops. In this study, the growth promoting and antifungal activity of six bacterial strains, Paenarthrobacter ureafaciens, Beijerinckia fluminensis, Pseudomonas protegens, Arthrobacter sp., Arthrobacter defluii, and Arthrobacter nicotinovorans, were investigated. The tested strains resulted positive for some plant growth promoting (PGP) traits, such as indole-3-acetic acid (IAA), 1-aminocyclopropane-1-carboxylate-deaminase (ACC-deaminase), siderophore production, and solubilization of phosphates. The effect of the selected bacteria on Arabidopsis thaliana seedlings growth was assessed using different morphological parameters. Bacterial activity against the phytopathogenic fungal species Aspergillus flavus, Fusarium proliferatum, and Fusarium verticillioides was also assessed, since these cause major yield losses in cereal crops and are well-known mycotoxin producers. Strains Pvr_9 (B. fluminensis) and PHA_1 (P. protegens) showed an important growth-promoting effect on A. thaliana coupled with a high antifungal activity on all the three fungal species. The analysis of bacterial broths through ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) confirmed the presence of potential PGP-compounds, among these are desferrioxamine B, aminochelin, asperchrome B, quinolobactin siderophores, and salicylic acid.

Light controls stamen elongation via cryptochromes, phytochromes and COP1 through HY5 and HYH.

In Arabidopsis, stamen elongation, which ensures male fertility, is controlled by the auxin response factor ARF8, which regulates the expression of the auxin repressor IAA19. Here, we uncover a role for light in controlling stamen elongation. By an extensive genetic and molecular analysis we show that the repressor of light signaling COP1, through its targets HY5 and HYH, controls stamen elongation, and that HY5 - oppositely to ARF8 - directly represses the expression of IAA19 in stamens. In addition, we show that in closed flower buds, when light is shielded by sepals and petals, the blue light receptors CRY1/CRY2 repress stamen elongation. Coherently, at flower disclosure and in subsequent stages, stamen elongation is repressed by the red and far-red light receptors PHYA/PHYB. In conclusion, different light qualities - sequentially perceived by specific photoreceptors - and the downstream COP1-HY5/HYH module finely tune auxin-induced stamen elongation and thus male fertility.

A Newly Identified Flower-Specific Splice Variant of AUXIN RESPONSE FACTOR8 Regulates Stamen Elongation and Endothecium Lignification in Arabidopsis.

In addition to the full-length transcript ARF8.1, a splice variant (ARF8.2) of the auxin response factor gene ARF8 has been reported. Here, we identified an intron-retaining variant of ARF8.2, ARF8.4, whose translated product is imported into the nucleus and has tissue-specific localization in Arabidopsis thaliana By inducibly expressing each variant in arf8-7 flowers, we show that ARF8.4 fully complements the short-stamen phenotype of the mutant and restores the expression of AUX/IAA19, encoding a key regulator of stamen elongation. By contrast, the expression of ARF8.2 and ARF8.1 had minor or no effects on arf8-7 stamen elongation and AUX/IAA19 expression. Coexpression of ARF8.2 and ARF8.4 in both the wild type and arf8-7 caused premature anther dehiscence: We show that ARF8.2 is responsible for increased expression of the jasmonic acid biosynthetic gene DAD1 and that ARF8.4 is responsible for premature endothecium lignification due to precocious expression of transcription factor gene MYB26 Finally, we show that ARF8.4 binds to specific auxin-related sequences in both the AUX/IAA19 and MYB26 promoters and activates their transcription more efficiently than ARF8.2. Our data suggest that ARF8.4 is a tissue-specific functional splice variant that controls filament elongation and endothecium lignification by directly regulating key genes involved in these processes.

An auxin maximum in the middle layer controls stamen development and pollen maturation in Arabidopsis.

Here, we investigated the role of auxin distribution in controlling Arabidopsis thaliana late stamen development. We analysed auxin distribution in anthers by monitoring DR5 activity: at different flower developmental stages; inhibiting auxin transport; in the rpk2-3 and ems1 mutants devoid of middle layer (ML) or tapetum, respectively; and in the auxin biosynthesis yuc6 and perception afb1-3 mutants. We ran a phenotypic, DR5::GUS and gene expression analysis of yuc6rpk2 and afb1rpk2 double mutants, and of 1-N-naphthylphthalamic acid (NPA)-treated flower buds. We show that an auxin maximum, caused by transport from the tapetum, is established in the ML at the inception of late stamen development. rpk2-3 mutant stamens lacking the ML have an altered auxin distribution with excessive accumulation in adjacent tissues, causing non-functional pollen grains, indehiscent anthers and reduced filament length; the expression of genes controlling stamen development is also altered in rpk2-3 as well as in NPA-treated flower buds. By decreasing auxin biosynthesis or perception in the rpk2-3 background, we eliminated these developmental and gene expression anomalies. We propose that the auxin maximum in the ML plays a key role in late stamen development, as it ensures correct and coordinated pollen maturation, anther dehiscence and filament elongation.

Overexpression of AtPCS1 in tobacco increases arsenic and arsenic plus cadmium accumulation and detoxification.

MAIN CONCLUSION: The heterologous expression of AtPCS1 in tobacco plants exposed to arsenic plus cadmium enhances phytochelatin levels, root As/Cd accumulation and pollutants detoxification, but does not prevent root cyto-histological damages. High phytochelatin (PC) levels may be involved in accumulation and detoxification of both cadmium (Cd) and arsenic (As) in numerous plants. Although polluted environments are frequently characterized by As and Cd coexistence, how increased PC levels affect the adaptation of the entire plant and the response of its cells/tissues to a combined contamination by As and Cd needs investigation. Consequently, we analyzed tobacco seedlings overexpressing Arabidopsis phytochelatin synthase1 gene (AtPCS1) exposed to As and/or Cd, to evaluate the levels of PCs and As/Cd, the cyto-histological modifications of the roots and the Cd/As leaf extrusion ability. When exposed to As and/or Cd the plants overexpressing AtPCS1 showed higher PC levels, As plus Cd root accumulation, and detoxification ability than the non-overexpressing plants, but a blocked Cd-extrusion from the leaf trichomes. In all genotypes, As, and Cd in particular, damaged lateral root apices, enhancing cell-vacuolization, causing thinning and stretching of endodermis initial cells. Alterations also occurred in the primary structure region of the lateral roots, i.e., cell wall lignification in the external cortex, cell hypertrophy in the inner cortex, crushing of endodermis and stele, and nuclear hypertrophy. Altogether, As and/or Cd caused damage to the lateral roots (and not to the primary one), with such damage not counteracted by AtPCS1 overexpression. The latter, however, positively affected accumulation and detoxification to both pollutants, highlighting that Cd/As accumulation and detoxification due to PCS1 activity do not reduce the cyto-histological damage.

Cadmium-inducible expression of the ABC-type transporter AtABCC3 increases phytochelatin-mediated cadmium tolerance in Arabidopsis.

The heavy metal cadmium (Cd) is a widespread environmental contaminant with harmful effects on living cells. In plants, phytochelatin (PC)-dependent Cd detoxification requires that PC-Cd complexes are transported into vacuoles. Here, it is shown that Arabidopsis thaliana seedlings defective in the ABCC transporter AtABCC3 (abcc3) have an increased sensitivity to different Cd concentrations, and that seedlings overexpressing AtABCC3 (AtABCC3ox) have an increased Cd tolerance. The cellular distribution of Cd was analysed in protoplasts from abcc3 mutants and AtABCC3 overexpressors grown in the presence of Cd, by means of the Cd-specific fluorochromes 5-nitrobenzothiazole coumarin (BTC-5N) and Leadmium™ Green AM dye. This analysis revealed that Cd is mostly localized in the cytosol of abcc3 mutant protoplasts whereas there is an increase in vacuolar Cd in protoplasts from AtABCC3ox plants. Overexpression of AtABCC3 in cad1-3 mutant seedlings defective in PC production and in plants treated with l-buthionine sulphoximine (BSO), an inhibitor of PC biosynthesis, had no effect on Cd tolerance, suggesting that AtABCC3 acts via PCs. In addition, overexpression of AtABCC3 in atabcc1 atabcc2 mutant seedlings defective in the Cd transporters AtABCC1 and AtABCC2 complements the Cd sensitivity of double mutants, but not in the presence of BSO. Accordingly, the level of AtABCC3 transcript in wild type seedlings was lower than that of AtABCC1 and AtABCC2 in the absence of Cd but higher after Cd exposure, and even higher in atabcc1 atabcc2 mutants. The results point to AtABCC3 as a transporter of PC-Cd complexes, and suggest that its activity is regulated by Cd and is co-ordinated with the activity of AtABCC1/AtABCC2.

ABCB1 and ABCB19 auxin transporters have synergistic effects on early and late Arabidopsis anther development.

Arabidopsis abcb1 abcb19 double mutants defective in the auxin transporters ABCB1/PGP1 and ABCB19/PGP19 are altered in stamen elongation, anther dehiscence and pollen maturation. To assess the contribution of these transporters to stamen development we performed phenotypic, histological analyses, and in situ hybridizations on abcb1 and abcb19 single mutant flowers. We found that pollen maturation and anther dehiscence are precocious in the abcb1 but not in the abcb19 mutant. Accordingly, endothecium lignification is altered only in abcb1 anthers. Both abcb1 and abcb1 abcb19 stamens also show altered early development, with asynchronous anther locules and a multilayer tapetum. DAPI staining showed that the timing of meiosis is asynchronous in abcb1 abcb19 anther locules, while only a small percentage of pollen grains are non-viable according to Alexander's staining. In agreement, TAM (TARDY ASYNCHRONOUS MEIOSIS), as well as BAM2 (BARELY ANY MERISTEM)-involved in tapetal cell development-are overexpressed in abcb1 abcb19 young flower buds. Correspondingly, ABCB1 and ABCB19 mRNA localization supports the observed phenotypes of abcb1 and abcb1 abcb19 mutant anthers. In conclusion, we provide evidence that auxin transport plays a significant role both in early and late stamen development: ABCB1 plays a major role during anther development, while ABCB19 has a synergistic role.

Auxin polar transport in stamen formation and development: how many actors?

In flowering plants, proper development of stamens, the male reproductive organs, is required for successful sexual reproduction. In Arabidopsis thaliana normally six stamen primordia arise in the third whorl of floral organs and subsequently differentiate into stamen filaments and anthers, where male meiosis occurs, thus ending the early developmental phase. This early phase is followed by a late developmental phase, which consists of a rapid elongation of stamen filaments coordinated with anther dehiscence and pollen maturation, and terminates with mature pollen grain release at anthesis. Increasing evidence suggests that auxin transport is necessary for both early and late phases of stamen development. It has been shown that different members of PIN (PIN-FORMED) family are involved in the early phase, whereas members of both PIN and P-glycoproteins of the ABCB (PGP) transporter families are required during the late developmental phase. In this review we provide an overview of the increasing knowledge on auxin transporters involved in Arabidopsis stamen formation and development and we discuss their role and functional conservation across plant species.

Auxin controls Arabidopsis anther dehiscence by regulating endothecium lignification and jasmonic acid biosynthesis.

It has been suggested that, in Arabidopsis, auxin controls the timing of anther dehiscence, possibly by preventing premature endothecium lignification. We show here that auxin content in anthers peaks before the beginning of dehiscence and decreases when endothecium lignification occurs. We show that, in the auxin-perception mutants afb1-3 and tir1 afb2 afb3, endothecium lignification and anther dehiscence occur earlier than wild-type, and the gene encoding the transcription factor MYB26, which is required for endothecium lignification, is over-expressed specifically at early stages; in agreement, MYB26 expression is reduced in naphthalene acetic acid-treated anthers, and afb1 myb26 double mutants show no endothecial lignification, suggesting that auxin acts through MYB26. As jasmonic acid (JA) controls anther dehiscence, we analysed how auxin and JA interact. In the JA-defective opr3 mutant, indehiscent anthers show normal timing of endothecium lignification, suggesting that JA does not control this event. We show that expression of the OPR3 and DAD1 JA biosynthetic genes is enhanced in afb1-3 and tir1 afb2 afb3 flower buds, but is reduced in naphthalene acetic acid-treated flower buds, suggesting that auxin negatively regulates JA biosynthesis. The double mutant afb1 opr3 shows premature endothecium lignification, as in afb1-3, and indehiscent anthers due to lack of JA, which is required for stomium opening. By treating afb1 opr3 and opr3 inflorescences with JA, we show that a high JA content and precocious endothecium lignification both contribute to induction of early anther dehiscence. We propose that auxin controls anther dehiscence timing by negatively regulating two key events: endothecium lignification via MYB26, and stomium opening via the control of JA biosynthesis.

Cadmium tolerance and phytochelatin content of Arabidopsis seedlings over-expressing the phytochelatin synthase gene AtPCS1.

Previous studies demonstrated that expression of the Arabidopsis phytochelatin (PC) biosynthetic gene AtPCS1 in Nicotiana tabacum plants increases the Cd tolerance in the presence of exogenous glutathione (GSH). In this paper, the Cd tolerance of Arabidopsis plants over-expressing AtPCS1 (AtPCSox lines) has been analysed and the differences between Arabidopsis and tobacco are shown. Based on the analysis of seedling fresh weight, primary root length, and alterations in root anatomy, evidence is provided that, at relatively low Cd concentrations, the Cd tolerance of AtPCSox lines is lower than the wild type, while AtPCS1 over-expressing tobacco is more tolerant to Cd than the wild type. At higher Cd concentrations, Arabidopsis AtPCSox seedlings are more tolerant to Cd than the wild type, while tobacco AtPCS1 seedlings are as sensitive as the wild type. Exogenous GSH, in contrast to what was observed in tobacco, did not increase the Cd tolerance of AtPCSox lines. The PC content in wild-type Arabidopsis at low Cd concentrations is more than three times higher than in tobacco and substantial differences were also found in the PC chain lengths. These data indicate that the differences in Cd tolerance and in its dependence on exogenous GSH between Arabidopsis and tobacco are due to species-specific differences in the endogenous content of PCs and GSH and may be in the relative abundance of PCs of different length.

Auxin regulates Arabidopsis anther dehiscence, pollen maturation, and filament elongation.

We provide evidence on the localization, synthesis, transport, and effects of auxin on the processes occurring late in Arabidopsis thaliana stamen development: anther dehiscence, pollen maturation, and preanthesis filament elongation. Expression of auxin-sensitive reporter constructs suggests that auxin effects begin in anthers between the end of meiosis and the bilocular stage in the somatic tissues involved in the first step of dehiscence as well as in the microspores and in the junction region between anther and filament. In situ hybridizations of the auxin biosynthetic genes YUC2 and YUC6 suggest that auxin is synthesized in anthers. In agreement with the timing of auxin effects, the TIR1, AFB1, AFB2, and AFB3 auxin receptor-encoding genes are transcribed in anthers only during late stages of development starting at the end of meiosis. We found that in tir1 afb triple and quadruple mutants, anther dehiscence and pollen maturation occur earlier than in the wild type, causing the release of mature pollen grains before the completion of filament elongation. We also assessed the contribution of auxin transport to late stamen developmental processes. Our results suggest that auxin synthesized in anthers plays a major role in coordinating anther dehiscence and pollen maturation, while auxin transport contributes to the independent regulation of preanthesis filament elongation.

ROX1, a gene induced by rolB, is involved in procambial cell proliferation and xylem differentiation in tobacco stamen.

The Agrobacterium rhizogenes oncogene rolB mimics the effects of auxin in that it increases the sensitivity of transformed cells to this hormone. Here we isolated a tobacco gene, ROX1, acting downstream of rolB. We show that plants with reduced levels of ROX1 mRNA, due to the expression of a 35S-driven ROX1-antisense construct, have flowers with stamens and pistils longer than normal because of an increased number of cells. Localized expression of rolB in anthers results in overexpression of ROX1 and reduced growth of stamens, due to a reduced number of cells. In addition, the longer stamens of antisense plants show a delayed xylem differentiation in the lateral bundles, primarily of the junction region between anther and filament, while the shorter stamens of ROX1-overexpressing plants show a precocious differentiation of xylem cells in the same tissues. Expression of ROX1 in stamens peaks at early stages of stamen growth, and ROX1 mRNA is localized mostly in anther procambial cells. The sequence of ROX1 shares a conserved element with a number of plant genes, including TED3, which is involved in xylem differentiation. These results point to a role of ROX1 in the balance between proliferation of procambial cells and xylem differentiation during stamen development.

Overexpression of Arabidopsis phytochelatin synthase in tobacco plants enhances Cd(2+) tolerance and accumulation but not translocation to the shoot.

Phytochelatins (PCs) are metal binding peptides involved in heavy metal detoxification. To assess whether enhanced phytochelatin synthesis would increase heavy metal tolerance and accumulation in plants, we overexpressed the Arabidopsis phytochelatin synthase gene (AtPCS1) in the non-accumulator plant Nicotiana tabacum. Wild-type plants and plants harbouring the Agrobacterium rhizogenes rolB oncogene were transformed with a 35S AtPCS1 construct. Root cultures from rolB plants could be easily established and we demonstrated here that they represent a reliable system to study heavy metal tolerance. Cd(2+) tolerance in cultured rolB roots was increased as a result of overexpression of AtPCS1, and further enhanced when reduced glutathione (GSH, the substrate of PCS1) was added to the culture medium. Accordingly, HPLC analysis showed that total PC production in PCS1-overexpressing rolB roots was higher than in rolB roots in the presence of GSH. Overexpression of AtPCS1 in whole seedlings led to a twofold increase in Cd(2+) accumulation in the roots and shoots of both rolB and wild-type seedlings. Similarly, a significant increase in Cd(2+) accumulation linked to a higher production of PCs in both roots and shoots was observed in adult plants. However, the percentage of Cd(2+) translocated to the shoots of seedlings and adult overexpressing plants was unaffected. We conclude that the increase in Cd(2+) tolerance and accumulation of PCS1 overexpressing plants is directly related to the availability of GSH, while overexpression of phytochelatin synthase does not enhance long distance root-to-shoot Cd(2+) transport.

Expression of rolB in tobacco flowers affects the coordinated processes of anther dehiscence and style elongation.

The effect of auxin on stamen and pistil development in tobacco flowers was investigated by means of the localized expression of rolB (root loci B), an Agrobacterium oncogene that increases auxin sensitivity in a cell-autonomous fashion. When rolB is driven by the promoter of the meiosis-specific Arabidopsis gene DMC1 (disrupted meiotic cDNA 1), expression occurs earlier in male than in female developing organs, resulting in a delay in anther dehiscence with respect to normal timing of pistil development. As a consequence of this developmental uncoupling, self-pollination is prevented in pDMC1:rolB plants. Histological analysis of pDMC1:GFP plants indicates that in tobacco, this promoter is active not only in meiocytes but also in somatic tissues of the anther. In contrast, simultaneous expression of rolB in anther and pistil somatic tissues, achieved by expressing a construct containing rolB under the control of the promoter of the petunia gene FBP7 (floral binding protein 7), results in a concomitant delay of both anther dehiscence and pistil development without affecting self-pollination of the plants. Analysis of plants harboring the pFBP7:GUS construct shows that in tobacco, this promoter is active not only in the ovules, as described for petunia, but also in pistil and anther somatic tissues involved in the dehiscence program. The delay in anther dehiscence and pistil development could be phenocopied by exogenous application of auxin. Jasmonic acid (JA) could not rescue the delay in anther dehiscence. These results suggest that auxin plays a key role in the timing of anther dehiscence, the dehiscence program is controlled by the somatic tissues of the anther, and auxin also regulates pistil development.