Waldemar Wojciechowski

Light- and IAA-regulated ACC synthase gene (PnACS) from Pharbitis nil and its possible role in IAA-mediated flower inhibition.

The light- and indole-3-acetic acid (IAA)-regulated 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene (PnACS) from Pharbitis nil was isolated. Here, it was shown that the gene was expressed in cotyledons, petioles, hypocotyls, root and shoot apexes both in light- and dark-grown seedlings. The highest expression level of PnACS was found in the roots. IAA applied to the cotyledons of P. nil seedlings caused a clear increase of PnACS messenger accumulation in all the organs examined. In this case, the most IAA-responsive were the hypocotyls. Our studies revealed that the PnACS transcript level in the cotyledons exhibited diurnal oscillations under both long-day (LD) and short-day (SD) conditions. IAA applied at the beginning of inductive darkness caused a dramatic increase in the expression of PnACS, suggesting that the inhibitory effect of IAA on P. nil flowering may result from its stimulatory effect on ethylene production.

The involvement of InMIR167 in the regulation of expression of its target gene InARF8, and their participation in the vegetative and generative development of Ipomoea nil plants

The plant hormone auxin plays a critical role in regulating plant growth and development. Recent advances have been made that having improved our understanding of auxin response pathways, primarily by characterizing the genes encoding auxin response factors (ARFs) in Arabidopsis. In addition, the expression of some ARFs is regulated by microRNAs (miRNAs). In Arabidopsis thaliana, ARF6 and ARF8 are targeted by miR167, whereas ARF10, ARF16 and ARF17 are targeted by miR160. Nevertheless, little is known about any possible interactions between miRNAs and the auxin signaling pathway during plant development. In this study, we isolated the miR167 target gene InARF8 cDNA from the cotyledons of the short day plant (SDP) Ipomoea nil (named also Pharbitis nil). Additionally, the In-miR167 precursor was identified from the I. nil EST database and analyses of InARF8 mRNA, In-pre-miR167 and mature miR167 accumulation in the plants vegetative and generative organs were performed. The identified cDNA of InARF8 contains a miR167 complementary sequence and shows significant similarity to ARF8 cDNAs of other plant species. The predicted amino acid sequence of InARF8 includes all of the characteristic domains for ARF family transcription factors (B3 DNA-binding domain, AUX/IAA-CTD and a glutamine-rich region). Quantitative RT-PCR reactions and in situ hybridization indicated that InARF8 was expressed primarily in the shoot apices, leaf primordia and hypocotyls of I. nil seedlings, as well as in flower pistils and petals. The InARF8 transcript level increased consistently during the entire period of pistil development, whereas in the stamens, the greatest transcriptional activity occurred only during the intensive elongation phase. Additionally, an expression analysis of both the precursor In-pre-miR167 molecules identified and mature miRNA was performed. We observed that, in most of the organs examined, the InARF8 expression pattern was opposite to that of MIR167, indicating that the genes activity was regulated by mRNA cleavage. Our findings suggested that InARF8 and InMIR167 participated in the development of young tissues, especially the shoot apices and flower elements. The main function of MIR167 appears to be to regulate InARF8 organ localization.

Cross talk between phytohormones in the regulation of flower induction in Pharbitis nil

Application of gibberellic acid (GA3) on the cotyledons of 5-d-old Pharbitis nil reversed the inhibitory effect of both abscisic acid (ABA) and ethylene on flowering. Application of GA3 slightly decreased ethylene production and did not affect the endogenous ABA content in the cotyledons during the night. However, it reversed the stimulating effect of ABA on ethylene production.

The role of PnACO1 in light- and IAA-regulated flower inhibition in Pharbitis nil

In this study, the first ACC oxidase (PnACO1) cDNA from model short-day plant Pharbitis nil was isolated. The expression pattern of PnACO1 was studied under different conditions (photoperiod and auxin), an adequate balance of which determines P. nil flowering. It was shown that the gene was transcribed in all the examined organs of the 5-day-old seedling and was strongly activated by auxin. Our results also revealed that PnACO1 transcript accumulation in the cotyledons showed diurnal oscillations under both LD and SD conditions. On the basis of presented and previously obtained data, we suggest that flowering inhibition evoked by IAA in P. nil results from its stimulatory effect on both ACC synthase and oxidase gene expression and, consequently, enhances ethylene production.

Impact of InMIR319 and light on the expression of InTCP4 gene involved in the development of Ipomoea nil plants

Abstract MicroRNAs regulate gene expression by guiding the cleavage or attenuating the translation of target mRNAs. In Arabidopsis thaliana, the subset of class II TCP genes (plant-specific group of transcription factors) contains an miR319-binding site. One of them, AtTCP4, regulates negatively leaf growth and positively leaf senescence. In addition, miR319 targeting of TCP4 is critical for petal and stamen development and affects flowering time. The aim of this work was to identify the cDNA of InTCP4 gene and In-miR319 precursor in Ipomoea nil (Pharbitis nil). The cDNA sequence of InTCP4 shows a significant similarity to the cDNA members of the TCP family of other plant species and contains nucleotides complementary to miR319. The identified sequence In-pre-miR319 creates a long hairpin structure and mature miRNA sequence is located in a similar place as in precursors found in other plant species. Accumulation of InTCP4 mRNA and In-pre-miR319 was examined in various organs of I. nil plants. We found that the InTCP4 is strongly expressed in cotyledons of I. nil seedlings while the In-pre-miR319 accumulates mainly in the hypocotyls of seedlings. Moreover, we investigate the role of InTCP4 in the flowering induction, flower development and cotyledon senescence in I. nil. We indicate that the InTCP4 expression is controlled by both light/clock and miR319. Both InTCP4 and InMIR319 probably participate in the regulation of such processes as do their homologues in other plant species, the development of cotyledons, leaves and flower elements. The main function of InMIR319 seems to be the regulation of InTCP4 organ localization.

De novo Transcriptome Profiling of Flowers, Flower Pedicels and Pods of Lupinus luteus (Yellow Lupine) Reveals Complex Expression Changes during Organ Abscission.

Yellow lupine (Lupinus luteus L., Taper c.), a member of the legume family (Fabaceae L.), has an enormous practical importance. Its excessive flower and pod abscission represents an economic drawback, as proper flower and seed formation and development is crucial for the plants productivity. Generative organ detachment takes place at the basis of the pedicels, within a specialized group of cells collectively known as the abscission zone (AZ). During plant growth these cells become competent to respond to specific signals that trigger separation and lead to the abolition of cell wall adhesion. Little is known about the molecular network controlling the yellow lupine organ abscission. The aim of our study was to establish the divergences and similarities in transcriptional networks in the pods, flowers and flower pedicels abscised or maintained on the plant, and to identify genes playing key roles in generative organ abscission in yellow lupine. Based on de novo transcriptome assembly, we identified 166,473 unigenes representing 219,514 assembled unique transcripts from flowers, flower pedicels and pods undergoing abscission and from control organs. Comparison of the cDNA libraries from dropped and control organs helped in identifying 1,343, 2,933 and 1,491 differentially expressed genes (DEGs) in the flowers, flower pedicels and pods, respectively. In DEG analyses, we focused on genes involved in phytohormonal regulation, cell wall functioning and metabolic pathways. Our results indicate that auxin, ethylene and gibberellins are some of the main factors engaged in generative organ abscission. Identified 28 DEGs common for all library comparisons are involved in cell wall functioning, protein metabolism, water homeostasis and stress response. Interestingly, among the common DEGs we also found an miR169 precursor, which is the first evidence of micro RNA engaged in abscission. A KEGG pathway enrichment analysis revealed that the identified DEGs were predominantly involved in carbohydrate and amino acid metabolism, but some other pathways were also targeted. This study represents the first comprehensive transcriptome-based characterization of organ abscission in L. luteus and provides a valuable data source not only for understanding the abscission signaling pathway in yellow lupine, but also for further research aimed at improving crop yields.

Molecular and cytological characterization of ZTL in Ipomoea nil

The ZEITLUPE (ZTL) protein is involved in the control of circadian period, hypocotyl elongation and flowering time in Arabidopsis thaliana. The aim of the present work was the identification of the InZTL gene and localization of its mRNA in the model short-day plant Ipomoea nil. The deduced InZTL protein of 622 amino acid residues contained a LOV domain at the N-terminal part, followed by an F-box domain and six carboxy terminal kelch repeats. Amino acid sequence of InZTL showed 84 % homology with Mesembryanthemum crystallinum ZTL (McZTL) and 83 % with Arabidopsis thaliana ZTL (AtZTL). Fluorescence in situ hybridization (FISH) to InZTL mRNA showed its high accumulation in the vascular bundles as well in the guard cells of the cotyledon. Immunolocalization of ZTL protein indicated a similar distribution pattern of ZTL protein as InZTL mRNAs.