Giuseppina Falasca

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.

Functional Characterization of OsMADS18, a Member of the AP1/SQUA Subfamily of MADS Box Genes

MADS box transcription factors controlling flower development have been isolated and studied in a wide variety of organisms. These studies have shown that homologous MADS box genes from different species often have similar functions. OsMADS18 from rice (Oryza sativa) belongs to the phylogenetically defined AP1/SQUA group. The MADS box genes of this group have functions in plant development, like controlling the transition from vegetative to reproductive growth, determination of floral organ identity, and regulation of fruit maturation. In this paper we report the functional analysis of OsMADS18. This rice MADS box gene is widely expressed in rice with its transcripts accumulated to higher levels in meristems. Overexpression of OsMADS18 in rice induced early flowering, and detailed histological analysis revealed that the formation of axillary shoot meristems was accelerated. Silencing of OsMADS18 using an RNA interference approach did not result in any visible phenotypic alteration, indicating that OsMADS18 is probably redundant with other MADS box transcription factors. Surprisingly, overexpression of OsMADS18 in Arabidopsis caused a phenotype closely resembling the ap1 mutant. We show that the ap1 phenotype is not caused by down-regulation of AP1 expression. Yeast two-hybrid experiments showed that some of the natural partners of AP1 interact with OsMADS18, suggesting that the OsMADS18 overexpression phenotype in Arabidopsis is likely to be due to the subtraction of AP1 partners from active transcription complexes. Thus, when compared to AP1, OsMADS18 during evolution seems to have conserved the mechanistic properties of protein-protein interactions, although it cannot complement the AP1 function.

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 and cytokinin control formation of the quiescent centre in the adventitious root apex of arabidopsis

Background and Aims Adventitious roots (ARs) are part of the root system in numerous plants, and are required for successful micropropagation. In the Arabidopsis thaliana primary root (PR) and lateral roots (LRs), the quiescent centre (QC) in the stem cell niche of the meristem controls apical growth with the involvement of auxin and cytokinin. In arabidopsis, ARs emerge in planta from the hypocotyl pericycle, and from different tissues in in vitro cultured explants, e.g. from the stem endodermis in thin cell layer (TCL) explants. The aim of this study was to investigate the establishment and maintenance of the QC in arabidopsis ARs, in planta and in TCL explants, because information about this process is still lacking, and it has potential use for biotechnological applications. Methods Expression of PR/LR QC markers and auxin influx (LAX3)/efflux (PIN1) genes was investigated in the presence/absence of exogenous auxin and cytokinin. Auxin was monitored by the DR5::GUS system and cytokinin by immunolocalization. The expression of the auxin-biosynthetic YUCCA6 gene was also investigated by in situ hybridization in planta and in AR-forming TCLs from the indole acetic acid (IAA)-overproducing superroot2-1 mutant and its wild type. Key Results The accumulation of auxin and the expression of the QC marker WOX5 characterized the early derivatives of the AR founder cells, in planta and in in vitro cultured TCLs. By determination of PIN1 auxin efflux carrier and LAX3 auxin influx carrier activities, an auxin maximum was determined to occur at the AR tip, to which WOX5 expression was restricted, establishing the positioning of the QC. Cytokinin caused a restriction of LAX3 and PIN1 expression domains, and concomitantly the auxin biosynthesis YUCCA6 gene was expressed in the apex. Conclusions In ARs formed in planta and TCLs, the QC is established in a similar way, and auxin transport and biosynthesis are involved through cytokinin tuning.

The proline biosynthetic genes P5CS1 and P5CS2 play overlapping roles in Arabidopsis flower transition but not in embryo development

Overexpression of the proline biosynthetic gene P5CS1 results in early flowering in Arabidopsis. However, the p5cs1 loss-of-function mutant exhibits a modest delay in flowering, suggesting that P5CS2, a duplicated P5CS1 gene present in the Arabidopsis, may also play a role in flower transition. In situ mRNA hybridizations and quantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed that P5CS1 and P5CS2 are expressed at similar levels and with the same pattern of expression in vegetative and floral shoot apical meristems as well as in axillary meristems. Arabidopsis lines homozygous for the p5cs1 mutant and simultaneously heterozygous for the p5cs2 mutation showed a stronger late-flowering phenotype than p5cs1 single mutants, confirming that also P5CS2 plays a role in flower transition and supporting the notion of overlapping functions of the two P5CS genes in this developmental process. P5CS1 and P5CS2 have identical messenger RNA (mRNA) distributions also in embryos, but only p5cs2 mutant embryos exhibit alterations of the cellular division planes and consequently stop developing. This suggests a specific role of P5CS2 in embryogenesis and an involvement of proline in cell division. Accordingly, exogenous proline accelerated organ growth and meristem formation, and stimulated expression of the cell cycle-related protein CYCB1;1.

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

Highlight AtABCC3 detoxifies cadmium by transporting phytochelatin–cadmium complexes into the vacuoles, and it can functionally complement abcc1 abcc2 mutants.

Root histogenesis from tobacco thin cell layers

SummaryInternode stem expiants ofNicotiana tabacum cv. Samsun, consisting of eight cell layers: epidermis, subepidermal chlorenchyma, collenchyma and cortical parenchyma (i.e., thin cell layers), were cultured under conditions inducing rhizogenesis. The aim was to investigate the histological sequence of adventitious root formation in this system. The earliest cytological events in culture (12 h) were nucleolar extrusions and amitotic nuclear divisions. Though not restricted to a specific cell layer, the two phenomena were more frequent in the subepidermal chlorenchyma, and characterized the first phases (12-96 h) of cell proliferation mainly occurring in this layer. Amitoses were followed by the formation of thin walls within the original cells, resulting in the formation of intracellular clusters. These subepidermal clusters were separated by enlarged cells of the parent tissue, whose nuclei showed nucleolar extrusion. At day 3 the first mitoses were observed in cells having abundant starch inclusions. Amitotic divisions also continued, but less frequently. The increasing frequency of mitoses in the subepidermal chlorenchyma (day 4), as well as in the two underlying collenchymatous layers, contributed to the growth of the superficial clusters, in which small clumps of meristematic cells were formed; these, later (day 9), gave rise to root domes. The 5th cell layer remained undivided for a relatively long time (two weeks). The 6th and 7th layers proliferated mitotically later (from day 8 onwards) than the superficial layers and formed root domes following the same histological sequence. Wound callus, generated by the innermost layer, increased markedly in the last two weeks of culture and concomitantly formed vascular clumps surrounded by meristematic layers; these produced root primordia which were frequently anomalous (day 26–27). Regardless of its origin (i.e., superficial or deep layers of the expiant, or wound callus cells), root tip formation was always preceded by the differentiation of a sheath of starch-containing cells, from which the root cap developed.

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.

Histological analysis of adventitious rooting in Arabidopsis thaliana (L.) Heynh seedlings

ABSTRACT Adventititous rooting is essential for the post-embryonic growth of the root apparatus in various species. In Arabidopsis thaliana, adventitious rooting has been reported in some mutants, and auxin seems to be the inducer of the process. The objective of the study was to identify the tissues involved in adventitious rooting in the most commonly used ecotypes for molecular and genetic studies (i.e. Columbia, Wassilewskija and Landsberg erecta) both in the presence and absence of exogenous auxin. Seedlings of the three ecotypes were grown under various conditions. When grown under 16 hours light/day for 11 days, all seedlings showed adventitious roots, both with and without auxin, however, both adventitious and lateral rooting were enhanced by exogenous auxin (2 µM naphthaleneacetic acid). Independently of the presence of auxin and of the ecotype, the hypocotyl pericycle produced adventitious roots directly (i.e., according to the same pattern of lateral root formation by the pericycle cells in the primary root). However, in the presence of auxin, roots of indirect origin also, and mainly, formed and their formation was preceded by the exfoliation of the tissues external to the stele. Exfoliation was caused by cell hypertrophy, separation, and disintegration, which mainly involved the endodermis. At the exfoliation site, the pericycle, with a minor contribution of a few endodermal cells, produced the callus from which indirect roots arose. The finding that adventitious rooting occurs in the absence of auxin (all ecotypes) indicates that this process is part of the normal root apparatus in Arabidopsis, with the hypocotyl pericycle as the target tissue of the process. Exogenous auxin alters adventitious rhizogenesis mainly affecting the endodermis response.

Two SERK genes are markers of pluripotency in Cyclamen persicum Mill

The genetic basis of stem cell specification in somatic embryogenesis and organogenesis is still obscure. SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) genes are involved in embryogenesis and organogenesis in numerous species. In vitro culture of Cyclamen persicum immature ovules provides a system for investigating stem cell formation and maintenance, because lines forming either organs or embryos or callus without organs/embryos are available for the same cultivar and plant growth regulator conditions. The present aim was to exploit this property of cyclamen cultures to understand the role of SERK(s) in stem cell formation and maintenance in somatic embryogenesis and organogenesis in vitro, in comparison with expression in planta. CpSERK1 and CpSERK2 were isolated from embryogenic callus. CpSERK1 and CpSERK2 levels by RT-PCR showed that expression is high in embryogenic, moderate in organogenic, and null in recalcitrant calli. in situ hybridizations showed that the expression of both genes started in clumps of pluripotent stem cells, from which both pre-embryogenic aggregates and organ meristemoids derived, and continued in their trans-amplifying, meristem-like, derivatives. Expression declined in organ meristemoids, in parallel with a partial loss of meristematization. In mature somatic embryos, and in shoot and root primordia, CpSERK1 and CpSERK2 were expressed in meristems, and similar patterns occurred in zygotic embryo and primary meristems in planta. The results point to SERK1 and SERK2 as markers of pluripotency in cyclamen. It is proposed that the high expression of these genes in the trans-amplifying derivatives of the stem cells maintains a pluripotent condition leading to totipotency and, consequently, somatic embryogenesis.