Agata Kućko

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.

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.

Photoperiod and ethylene-dependent expression of gibberellin biosynthesis gene InEKO1 during flower induction of Ipomoea nil

Ent-kaurene oxidase (EKO) catalyze three sequential oxidations in the early steps of gibberellin biosynthesis pathway. In this research, a cDNA sequence of InEKO1 gene in the model short-day plant Ipomoea nil was identified. Our studies revealed that inductive conditions for flowering caused an increase in the transcriptional activity of the examined gene in the cotyledons–the main organs for the perception of the photoperiodic stimulus. In contrast, in the second half of the 16 h long inductive night and after that, a decreased amount of InEKO1 mRNA in the apexes was detected. What is more, ethylene, the key inhibitor of flower induction in I. nil, elevated the InEKO1 expression exclusively in the cotyledons between 10 and 14 h of the inductive night.

Methyl jasmonate-dependent senescence of cotyledons in Ipomoea nil

Jasmonic acid methyl ester (JAMe) has been recently shown to play a crucial role in many physiological processes. In this paper, we focused on cotyledon senescence in Ipomoea nil and revealed that JAMe and darkness are the main factors stimulating the process examined. What is more, we showed that mefenamic acid (a jasmonate biosynthesis inhibitor) reverses the stimulatory effect of darkness on senescence. In plants growing under dark conditions, stimulation of JASMONIC ACID CARBOXYL METHYLTRANSFERASE (InJMT) expression and, consequently, an increase in JAMe content, have been observed. In turn, the level of jasmonic acid (JA) gradually decreased. Moreover, dark-grown seedlings demonstrated a lower PSII functional activity and a reduced chlorophyll content and autofluorescence. All of these data suggest that JAMe is a signal molecule controlling the senescence of cotyledons in I. nil.

INFLORESCENCE DEFICIENT IN ABSCISSION-like is an abscission-associated and phytohormone-regulated gene in flower separation of Lupinus luteus

Abscission is a natural process that occurs to facilitate shedding of no longer needed organs, but on the other hand, can be triggered by certain environmental conditions, e.g. biotic or abiotic stresses. Regardless of the stimuli, organ shedding takes place specifically at the abscission zone (AZ). A signaling pathway that controls this process in Arabidopsis thaliana from ligand to receptors has been proposed. However, knowledge concerning the influence of plant hormones on these molecular elements still remains enigmatic. Excessive and premature flower abscission in the crop species Lupinus luteus L. is a process of substantial interest to the agricultural industry, as it can affect yield. Our strategy combined molecular studies, comprehensive ultrastructural and histological analysis, as well as exogenous hormone treatment to describe the contribution of the Lupinus IDA-like gene in flower abscission. In the AZ of the naturally abscised flowers, the differentiation of morphologically distinct cells characterized by progressive degradation processes was accompanied by LlIDL mRNA accumulation. A similar effect was observed following early steps of AZ activation and after abscisic acid or ethylene treatments. These phytohormones, previously pointed out as key stimulators of flower separation, altered the temporal expression pattern of LlIDL. Exogenous EPIP peptide synthesized on the basis of LlIDL sequence, significantly increased flower abortion rate, which indicates that this motif governs protein activity. In conclusion, our data provide new evidence for LlIDA involvement in both the early and late events of flower abscission supported by detailed spatiotemporal characterization of AZ cell structure and ultrastructure.

Photoperiodic flower induction in Ipomoea nil is accompanied by decreasing content of gibberellins

The involvement of gibberellins (GAs) in the control of flower induction in the short-day plant Ipomoea nil has been investigated. To clarify the molecular basis of this process, we identified the full-length cDNAs of the InGA20ox3 and InGA2ox1 genes, which encode enzymes responsible for GA biosynthesis and catabolism, respectively. We studied the expression patterns of both genes and determined the tissue and cellular immunolocalisation of gibberellic acid (GA3) in the cotyledons of 5-day-old seedlings growing under inductive and non-inductive photoperiodic conditions. In the second half of the inductive night, which is crucial for flower induction in I. nil, InGA20ox3 expression decreased, whereas InGA2ox1 mRNA accumulated, which indicates that photoperiod regulates the activity of both genes. Furthermore, these changes are correlated with GA3 level. Thus, our results support the thesis that the proper balance between the expression of the InGA20ox3 and InGA2ox1 genes and low GA3 content correlate with photoperiodic flower induction in I. nil.

Gibberellic acid affects the functioning of the flower abscission zone in Lupinus luteus via cooperation with the ethylene precursor independently of abscisic acid

The abscission of plant organs is a phytohormone-controlled process. Our study provides new insight into the involvement of gibberellic acid (GA3) in the functioning of the flower abscission zone (AZ) in yellow lupine (Lupinus luteus L.). Physiological studies demonstrated that GA3 stimulated flower abortion. Additionally, this phytohormone was abundantly presented in the AZ cells of naturally abscised flowers, especially in vascular bundles. Interesting interactions among GA3 and other modulators of flower separation were also investigated. GA3 accumulated after treatment with the ethylene (ET) precursor 1-aminocyclopropane-1-carboxylic acid (ACC). Abscisic acid (ABA) treatment did not cause such an effect. Furthermore, the expression of the newly identified LlGA20ox1 and LlGA2ox1 genes encoding 2-oxoglutarate-dependent dioxygenases fluctuated after ACC or ABA treatment which confirmed the existence of regulatory crosstalk. GA3 appears to cooperate with the ET precursor in the regulation of AZ function in L. luteus flowers; however, the presented mechanism is ABA-independent.