SUMMARYIt 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 auxinperception 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.

HighlightAtABCC3 detoxifies cadmium by transporting phytochelatin–cadmium complexes into the vacuoles, and it can functionally complement abcc1 abcc2 mutants.

In addition to the full-length transcript , a splice variant () of the auxin response factor gene has been reported. Here, we identified an intron-retaining variant of, , whose translated product is imported into the nucleus and has tissue-specific localization in By inducibly expressing each variant in flowers, we show that fully complements the short-stamen phenotype of the mutant and restores the expression of , encoding a key regulator of stamen elongation. By contrast, the expression of and had minor or no effects on stamen elongation and expression. Coexpression of and in both the wild type and caused premature anther dehiscence: We show that is responsible for increased expression of the jasmonic acid biosynthetic gene and that is responsible for premature endothecium lignification due to precocious expression of transcription factor gene Finally, we show that ARF8.4 binds to specific auxin-related sequences in both the and 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.

SummaryThe aims of this work were to obtain a human antibody against the tumour-associated antigen tenascin-C (TNC) and to compare the yield and quality of plant-produced antibody in either stable transgenics or using a transient expression system. To this end, the characterization of a full-sized human immunoglobulin G (IgG) [monoclonal antibody H10 (mAb H10)], derived from a selected single-chain variable fragment (scFv) and produced in plants, is presented. The human mAb gene was engineered for plant expression, and Nicotiana tabacum transgenic lines expressing both heavy (HC) and light (LC) chain were obtained and evaluated for antibody expression levels, in vivo assembly and functionality.Affinity-purified H10 from transgenics (yield, 0.6-1.1 mg/kg fresh weight) revealed that more than 90% of HC was specifically degraded, leading to the formation of functional antigen-binding fragments (Fab). Consequently, H10 was transiently expressed in Nicotiana benthamiana plants through an Agrobacterium -mediated gene-transfer system. Moreover, the use of the p19 silencing suppressor gene from artichoke mottled crinkle virus raised antibody expression levels by an order of magnitude (yields of purified H10, 50-100 mg/kg fresh weight). Approximately 75% of purified protein consisted of full-sized antibody functionally binding to TNC ( K D = 14 n M ), and immunohistochemical analysis on tumour tissues revealed specific accumulation around tumour blood vessels. The data indicate that the purification yields of mAb H10, using a transient expression system boosted by the p19 silencing suppressor, are exceptionally high when compared with the results reported previously, providing a technique for the over-expression of anticancer mAbs by a rapid, cost-effective, molecular farming approach.

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.

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 nonviable 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.Keywords: Anther development; Arabidopsis; auxin transport; endothecium lignification; tapetum differentiation Citation: Cecchetti V, Brunetti P, Napoli N, Fattorini L, Altamura MM, Costantino P, Cardarelli M (2015) ABCB1 and ABCB19 auxin transporters have synergistic effects on early and late Arabidopsis anther development.

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.

Main conclusionThe heterologous expression ofAtPCS1in 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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-015-2428-8) contains supplementary material, which is available to authorized users.