Kamil Frankowski

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.

Ethylene, auxin, and abscisic acid interactions in the control of photoperiodic flower induction in Pharbitis nil

Interactions between indole-3-acetic acid (IAA), abscisic acid (ABA), and ethylene (ET) in the photoperiodic flower induction of a short-day (SD) plant Pharbitis nil were investigated. It was shown that both IAA and ABA applied just before and during the first half of the 16-h-long inductive night inhibited flower induction in P. nil. Ethylene is also thought to be a strong flowering inhibitor of SD plants but only when it is applied in the second half of the inductive night. The application of IAA just before the inductive night decreased the content of endogenous ABA in cotyledons only during the first half of the inductive night. Additionally, the application of 2-aminoethoxyvinylglycine (AVG) — an ethylene biosynthesis inhibitor — partially reversed the inhibitory effect of IAA and ABA on flowering. The results suggest that the mechanisms of P. nil flower inhibition by IAA and ABA might be independent. However, both the hormones influenced ethylene production which directly inhibited flowering. We also show that ABA applied on the cotyledons of P. nil seedlings just before the inductive night caused a clear increase in the expression of PnACS1 and PnACS2 genes (encoding enzymes involved in ethylene biosynthesis) from the first hours after its application. The transcripts of PnACO1 and PnACO3 genes were also increased but their maximal values were shifted in relation to the PnACS1 and PnACS2. The data presented here strongly support the idea that both IAA and ABA inhibit P. nil flowering through the modulation of ethylene biosynthesis.

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.

Molecular cloning of the BLADE-ON-PETIOLE gene and expression analyses during nodule development in Lupinus luteus

The BLADE-ON-PETIOLE (BOP) genes have been recently shown to play an essential role in many physiological processes, including embryogenesis, meristem determinacy, leaf patterning and nodule development. In our research we used Lupinus luteus, a plant with great agronomic potential due to its high protein content and nitrogen fixation ability. In this work, LlBOP in L. luteus was identified for the first time and its expression during nodule development was analyzed. The high expression levels of LlBOP and LlLbI (LEGHEMOGLOBIN), essential to nitrogen-fixing symbiosis, were noted in the developing root nodules and were correlated with the occurrence of leghemoglobin. All of these data indicate that LlBOP is an important regulator of root nodule formation and functioning in L. luteus.

Profiling the BLADE-ON-PETIOLE gene expression in the abscission zone of generative organs in Lupinus luteus

The great agronomic potential of Lupinusluteus, species widely cultivated in many European countries as well as Australia, is strongly affected by premature and excessive generative organ abscission, mainly flowers. The unwanted process takes place in a specialized group of cells, called abscission zone (AZ). During their development they become competent to respond to external and internal factors, including phytohormones. Recently it was shown that the formation of AZ cells in Arabidopsisthaliana is coordinated by transcription factors, BLADE-ON-PETIOLE (BOPs). There is no such data, excluding tobacco plants, about BOP-dependent regulation of organ abscission in crop plants. In this work, we examined LlBOP mRNA content during generative organs AZ development and functioning. The high accumulation of LlBOP transcript was accompanied by the differentiation of morphologically distinct cells at the base of the mature flower pedicel. Moreover, enhanced LlBOP expression was observed in the active AZ, and was regulated by factors, which can strongly affect generative organ abscission. All these data indicate that LlBOP is involved in the abscission zone formation and functioning in L. luteus.

The influence of abscisic acid on the ethylene biosynthesis pathway in the functioning of the flower abscission zone in Lupinus luteus.

Flower abscission is a highly regulated developmental process activated in response to exogenous (e.g. changing environmental conditions) and endogenous stimuli (e.g. phytohormones). Ethylene (ET) and abscisic acid (ABA) are very effective stimulators of flower abortion in Lupinus luteus, which is a widely cultivated species in Poland, Australia and Mediterranean countries. In this paper, we show that artificial activation of abscission by flower removal caused an accumulation of ABA in the abscission zone (AZ). Moreover, the blocking of that phytohormones biosynthesis by NDGA (nordihydroguaiaretic acid) decreased the number of abscised flowers. However, the application of NBD - an inhibitor of ET action - reversed the stimulatory effect of ABA on flower abscission, indicating that ABA itself is not sufficient to turn on the organ separation. Our analysis revealed that exogenous ABA significantly accelerated the transcriptional activity of the ET biosynthesis genes ACC synthase (LlACS) and oxidase (LlACO), and moreover, strongly increased the level of 1-aminocyclopropane-1-carboxylic acid (ACC) - ET precursor, which was specifically localized within AZ cells. We cannot exclude the possibility that ABA mediates flower abscission processes by enhancing the ET biosynthesis rate. The findings of our study will contribute to the overall basic knowledge on the phytohormone-regulated generative organs abscission in L. luteus.

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.

The Generative Development of Traditional and Self-Completing (Restricted Branching) Cultivars of White Lupin (Lupinus Albus L.), Yellow Lupin (L.Luteus L.) and Narrow-Lafed Lupin (L. Angustifolius L.) Grown under Different Phytotron Conditions

ABSTRACT Increasing the number of flowers and pods set, as well as reducing the intensity of their abortion, is of crucial importance for the yielding of leguminous plants. This study examined the effects of the type of soil used and mineral fertilization applied on the generative development of the traditional and self-completing (restricted branching) cultivars of white lupin (Lupinus albus L.), yellow lupin (L. luteus L.) and narrow-lafed lupin (L. angustifolius L.) cultivated under controlled phytotron conditions. Experiments carried out under such conditions allow for the elimination of variable environmental factors affecting the course of plant ontogenesis in field cultivation, and enable unambiguous interpretation of the biochemical and molecular influence of a selected factor on the physiological process studied. For the first time, the influence of different cultivation factors on generative development of traditional and selfcompleting (restricted branching) cultivars of lupins under phytotrone was examined. The research results presented here indicate that each of the selected lupin cultivars has its own characteristic cultivation conditions that are optimal for its generative development. The largest number of flowers were formed by the traditional cultivars of L. luteus and L. angustifolius, as well as the self-completing (restricted branching) cultivars of L. luteus and L. albus grown in class IIIa soil material. The lowest flower abortion rate was observed in L. albus grown in class V soil material, in L. luteus grown in class IIIa soil material, and in L. angustifolius grown in class IVa soil material. Regardless of the cultivation conditions applied, in all of the lupin cultivars examined the first pods to be set were characterized by the lowest abortion rate. The results obtained allowed for the development of lupin phytotron cultivation models for the purposes of research on generative development control.

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.