Abscisic acid- and ethylene-induced abscission of yellow lupine flowers is mediated by jasmonates.

The appropriate timing of organ abscission determines plant growth, development, reproductive success, and yield in relation to crop species. Among these, yellow lupine is an example of a crop species that loses many fully developed flowers, which limits the formation of pods with high-protein seeds and affects its economic value. Lupine flower abscission, similarly to the separation of other organs, depends on a complex regulatory network functioning in the cells of the abscission zone (AZ). In the present study, genetic, biochemical, and cellular methods were used to highlight the complexity of the interactions among strong hormonal stimulators of abscission, including abscisic acid (ABA), ethylene, and jasmonates (JAs) precisely in the AZ cells, with all results supporting that the JA-related pathway has an important role in the phytohormonal cross-talk leading to flower abscission in yellow lupine. Based on obtained results, we conclude that ABA and ET have positive influence on JAs biosynthesis and signaling pathway in time-dependent manner. Both phytohormones changes lipoxygenase (LOX) gene expression, affects LOX protein abundance, and JA accumulation in AZ cells. We have also shown that the signaling pathway of JA is highly sensitive to ABA and ET, given the accumulation of COI1 receptor and MYC2 transcription factor in response to these phytohormones. The results presented provide novel information about the JAs-dependent separation of organs and provide insight and details about the phytohormone-related mechanisms of lupine flower abscission.

Glomus sp. and Bacillus sp. strains mitigate the adverse effects of drought on maize (Zea mays L.).

Maize (Zea mays L.) is an economically important source of food and feed. This species is highly sensitive to drought, which is the most limiting factor for the biomass yield of a crop. Thus, maize cultivation methods should be improved, especially by environment-friendly agricultural practices, such as microorganisms. Here, we provide evidence that Glomus sp. and Bacillus sp. modulate maize response to drought. Inoculation of maize seeds by these microorganisms restored the proper photosynthetic activity of the plant under drought and stabilized the osmoprotectant content of the leaf. The beneficial effect of Glomus sp. and Bacillus sp. was also related to the stabilization of cell redox status reflected by hydrogen peroxide content, antioxidant enzymes, and malondialdehyde level in leaves. As we revealed by several methods, shaping maize response to drought is mediated by both microorganism-mediated modifications of cell wall composition and structure of leaves, such as downregulating pectin, affecting their methylation degree, and increasing hemicellulose content. Overall, we provide new information about the mechanisms by which Glomus sp. and Bacillus sp. induce drought tolerance in maize, which is a promising approach for mitigating abiotic stresses.

EPIP as an abscission promoting agent in the phytohormonal pathway.

Understanding the mechanisms underlying the activation of the abscission zone (AZ) responsible for organ separation from plant body in crop species will help improve their yielding and economic importance. Special attention has been given recently to the role of the INFLORESCENCE DEFICIENT IN ABSCISSION protein, particularly its functional fragment, EPIP peptide. Its stimulatory effect on abscission in different crops has been demonstrated. Recently we described the role of EPIP in the redox, lipid, and pectin-related events taking place in AZ of Lupinus luteus flowers, which undergo massive abscission in natural conditions. To further examine EPIP contribution in AZ functioning, here, we analyze its impact on the ultrastructural changes, synthesis of two hormonal abscission stimulators - abscisic acid (ABA) and ethylene (ET), and the appearance of phosphoproteins. As our results show, the response of flower AZ to exogenous EPIP involves the induction of distinct modifications related to the one hand with upregulation of cell activity but on the other hand degradation processes and possible autophagy. Furthermore, the EPIP stimulated biosynthesis pathways of ABA and ET precisely in AZ cells. In addition, progressive phosphorylation of proteins has been observed under EPIP influence. The highly accumulated ones were identified as those, related to primary metabolism and reactive oxygen species homeostasis, and their role in abscission has been discussed. To summarizing, the presented detailed description of EPIP action in AZ cells in combination with our previous data offers new insights into its regulatory function and provides opportunities to counteract excessive flower abscission in lupine.

Jasmonate-Dependent Response of the Flower Abscission Zone Cells to Drought in Yellow Lupine.

Lipid membranes, as primary places of the perception of environmental stimuli, are a source of various oxygenated polyunsaturated fatty acids-oxylipins-functioning as modulators of many signal transduction pathways, e.g., phytohormonal. Among exogenous factors acting on plant cells, special attention is given to drought, especially in highly sensitive crop species, such as yellow lupine. Here, we used this species to analyze the contribution of lipid-related enzymes and lipid-derived plant hormones in drought-evoked events taking place in a specialized group of cells-the flower abscission zone (AZ)-which is responsible for organ detachment from the plant body. We revealed that water deficits in the soil causes lipid peroxidation in these cells and the upregulation of phospholipase D, lipoxygenase, and, concomitantly, jasmonic acid (JA) strongly accumulates in AZ tissue. Furthermore, we followed key steps in JA conjugation and signaling under stressful conditions by monitoring the level and tissue localization of enzyme providing JA derivatives (JASMONATE RESISTANT1) and the JA receptor (CORONATINE INSENSITIVE1). Collectively, drought-triggered AZ activation during the process of flower abscission is closely associated with the lipid modifications, leading to the formation of JA, its conjugation, and induction of signaling pathways.

The acceleration of yellow lupine flower abscission by jasmonates is accompanied by lipid-related events in abscission zone cells.

Yellow lupine is an economically important crop. This species has been used as a great model for abscission processes for several years due to extreme flower abortion, which takes place in the abscission zone (AZ). AZ activation involves modifications of cell walls, membranes, and cellular structure. In this paper, we applied physiological, molecular, biochemical, and instrumental methods to explore lipid-associated changes and the possible involvement of lipid-derived phytohormones - jasmonates (JAs) - in flower AZ activation. Our comprehensive analyses revealed that natural abscission is accompanied by the upregulation of peroxidase, which reflects a disruption of redox balance and/or lipids peroxidation in AZ cell membranes. Redox imbalance was confirmed by appearance of malondialdehyde. Lipid-related processes involved the specific localization and increased level and activity of lipase and LOX, enzymes associated with cell membrane rupture, and JA biosynthesis. Lipid-hydrolyzing phospholipase D, implicated previously in abscission, is also found in naturally active AZs. Observed changes are accompanied by the accumulation of jasmonates, both free jasmonic acid and its methyl ester. The JA derivative exhibited higher biological activity than the nonconjugated form. Overall, our study shed new light on the lipid and phytohormonal regulation of AZ functioning supporting a role of JAs during abscission-associated events.

Remodeling of Cell Wall Components in Root Nodules and Flower Abscission Zone under Drought in Yellow Lupine.

We recently showed that yellow lupine is highly sensitive to soil water deficits since this stressor disrupts nodule structure and functioning, and at the same time triggers flower separation through abscission zone (AZ) activation in the upper part of the plant. Both processes require specific transformations including cell wall remodeling. However, knowledge about the involvement of particular cell wall elements in nodulation and abscission in agronomically important, nitrogen-fixing crops, especially under stressful conditions, is still scarce. Here, we used immuno-fluorescence techniques to visualize dynamic changes in cell wall compounds taking place in the root nodules and flower AZ of Lupinus luteus following drought. The reaction of nodules and the flower AZ to drought includes the upregulation of extensins, galactans, arabinans, xylogalacturonan, and xyloglucans. Additionally, modifications in the localization of high- and low-methylated homogalacturonans and arabinogalactan proteins were detected in nodules. Collectively, we determined for the first time the drought-associated modification of cell wall components responsible for their remodeling in root nodules and the flower AZ of L. luteus. The involvement of these particular molecules and their possible interaction in response to stress is also deeply discussed herein.

EPIP-Evoked Modifications of Redox, Lipid, and Pectin Homeostasis in the Abscission Zone of Lupine Flowers.

Yellow lupine is a great model for abscission-related research given that excessive flower abortion reduces its yield. It has been previously shown that the EPIP peptide, a fragment of LlIDL (INFLORESCENCE DEFICIENT IN ABSCISSION) amino-acid sequence, is a sufficient molecule to induce flower abortion, however, the question remains: What are the exact changes evoked by this peptide locally in abscission zone (AZ) cells? Therefore, we used EPIP peptide to monitor specific modifications accompanied by early steps of flower abscission directly in the AZ. EPIP stimulates the downstream elements of the pathway-HAESA and MITOGEN-ACTIVATED PROTEIN KINASE6 and induces cellular symptoms indicating AZ activation. The EPIP treatment disrupts redox homeostasis, involving the accumulation of H2O2 and upregulation of the enzymatic antioxidant system including superoxide dismutase, catalase, and ascorbate peroxidase. A weakening of the cell wall structure in response to EPIP is reflected by pectin demethylation, while a changing pattern of fatty acids and acyl lipids composition suggests a modification of lipid metabolism. Notably, the formation of a signaling molecule-phosphatidic acid is induced locally in EPIP-treated AZ. Collectively, all these changes indicate the switching of several metabolic and signaling pathways directly in the AZ in response to EPIP, which inevitably leads to flower abscission.

Drought Disrupts Auxin Localization in Abscission Zone and Modifies Cell Wall Structure Leading to Flower Separation in Yellow Lupine.

Drought causes the excessive abscission of flowers in yellow lupine, leading to yield loss and serious economic consequences in agriculture. The structure that determines the time of flower shedding is the abscission zone (AZ). Its functioning depends on the undisturbed auxin movement from the flower to the stem. However, little is known about the mechanism guiding cell-cell adhesion directly in an AZ under water deficit. Therefore, here, we seek a fuller understanding of drought-dependent reactions and check the hypothesis that water limitation in soil disturbs the natural auxin balance within the AZ and, in this way, modifies the cell wall structure, leading to flower separation. Our strategy combined microscopic, biochemical, and chromatography approaches. We show that drought affects indole-3-acetic acid (IAA) distribution and evokes cellular changes, indicating AZ activation and flower abortion. Drought action was manifested by the accumulation of proline in the AZ. Moreover, cell wall-related modifications in response to drought are associated with reorganization of methylated homogalacturonans (HG) in the AZ, and upregulation of pectin methylesterase (PME) and polygalacturonase (PG)-enzymes responsible for pectin remodeling. Another symptom of stress action is the accumulation of hemicelluloses. Our data provide new insights into cell wall remodeling events during drought-induced flower abscission, which is relevant to control plant production.

Disruption of the Auxin Gradient in the Abscission Zone Area Evokes Asymmetrical Changes Leading to Flower Separation in Yellow Lupine.

How auxin transport regulates organ abscission is a long-standing and intriguing question. Polar auxin transport across the abscission zone (AZ) plays a more important role in the regulation of abscission than a local concentration of this hormone. We recently reported the existence of a spatiotemporal sequential pattern of the indole-3-acetic acid (IAA) localization in the area of the yellow lupine AZ, which is a place of flower detachment. In this study, we performed analyses of AZ following treatment with an inhibitor of polar auxin transport (2,3,5-triiodobenzoic acid (TIBA)). Once we applied TIBA directly onto the AZ, we observed a strong response as demonstrated by enhanced flower abscission. To elucidate the molecular events caused by the inhibition of auxin movement, we divided the AZ into the distal and proximal part. TIBA triggered the formation of the IAA gradient between these two parts. The AZ-marker genes, which encode the downstream molecular components of the inflorescence deficient in abscission (IDA)-signaling system executing the abscission, were expressed in the distal part. The accumulation of IAA in the proximal area accelerated the biosynthesis of abscisic acid and ethylene (stimulators of flower separation), which was also reflected at the transcriptional level. Accumulated IAA up-regulated reactive oxygen species (ROS) detoxification mechanisms. Collectively, we provide new information regarding auxin-regulated processes operating in specific areas of the AZ.

Molecular and Hormonal Aspects of Drought-Triggered Flower Shedding in Yellow Lupine.

The drought is a crucial environmental factor that determines yielding of many crop species, e.g., Fabaceae, which are a source of valuable proteins for food and feed. Herein, we focused on the events accompanying drought-induced activation of flower abscission zone (AZ)-the structure responsible for flower detachment and, consequently, determining seed production in Lupinus luteus. Therefore, detection of molecular markers regulating this process is an excellent tool in the development of improved drought-resistant cultivars to minimize yield loss. We applied physiological, molecular, biochemical, immunocytochemical, and chromatography methods for a comprehensive examination of changes evoked by drought in the AZ cells. This factor led to significant cellular changes and activated AZ, which consequently increased the flower abortion rate. Simultaneously, drought caused an accumulation of mRNA of genes inflorescence deficient in abscission-like (LlIDL), receptor-like protein kinase HSL (LlHSL), and mitogen-activated protein kinase6 (LlMPK6), encoding succeeding elements of AZ activation pathway. The content of hydrogen peroxide (H2O2), catalase activity, and localization significantly changed which confirmed the appearance of stressful conditions and indicated modifications in the redox balance. Loss of water enhanced transcriptional activity of the abscisic acid (ABA) and ethylene (ET) biosynthesis pathways, which was manifested by elevated expression of zeaxanthin epoxidase (LlZEP), aminocyclopropane-1-carboxylic acid synthase (LlACS), and aminocyclopropane-1-carboxylic acid oxidase (LlACO) genes. Accordingly, both ABA and ET precursors were highly abundant in AZ cells. Our study provides information about several new potential markers of early response on water loss, which can help to elucidate the mechanisms that control plant response to drought, and gives a useful basis for breeders and agronomists to enhance tolerance of crops against the stress.

Spatio-temporal localization of LlBOP following early events of floral abscission in yellow lupine.

The phenomenon of excessive flower abscission in yellow lupine is a process of substantial interest to the agricultural industries, because it substantially affects the yield. The aim of this work was to provide an analysis of the changes taking place precisely in the abscission zone (AZ) during early stages of flower separation. We put particular emphasis on mRNA accumulation of BOP (BLADE ON PETIOLE) gene encoding a transcriptional factor so far considered to be essential for AZ formation. Our results show that the AZ displays a particular transcriptional network active in the specific stages of its function, as reflected by the expression profile of LlBOP. Noteworthy, spatio-temporal LlBOP transcript accumulation in the elements of pedicel vascular tissue reveals divergent regulatory mechanism of its activity. We have also found that AZ cells accumulate reactive oxidative species following abscission and what is more, become active due to the increasing amount of uridine-rich small nuclear RNA, accompanied by poly(A) mRNA intensive synthesis. Our paper is a novel report for BOP involvement in the AZ functioning in relation to the whole transcriptional activity of AZ and overall discussed regarding BOP role as a potential mobile key regulator of abscission.

Spatio-temporal IAA gradient is determined by interactions with ET and governs flower abscission.

The abscission zone (AZ) is a specialized tissue that usually develops at the base of an organ and is highly sensitive to phytohormones, e.g., abscisic acid (ABA), ethylene (ET), and gibberellins (GAs). A current model of organ abscission assumes that the formation of an auxin gradient around the AZ area determines the time of shedding; however, that thesis is supported by studies that are primarily concerned with auxin transporters. To better understand the events underlying the progression of abscission, we focused for the first time on indole-3-acetic acid (IAA) distribution following AZ activation. We performed a series of immunolocalization studies in proximal and distal regions of floral AZ cells in yellow lupine, which is an agriculturally important legume. The examined phytohormone was abundant in natural active AZ cells, as well as above and below parts of this structure. A similar gradient of IAA was observed during the early steps of abscission, which was induced artificially by flower removal. Surprisingly, IAA was not detected in inactive AZ cells. This paper is also a consequence of our comprehensive studies concerning the phytohormonal regulation of flower abscission in yellow lupine. We present new data on interactions between IAA and ET, previously pointed out as a strong modulator of flower separation. The detailed analysis shows that disruption of the natural auxin gradient around the AZ area through the application of synthetic IAA had a positive effect on ET biosynthesis genes. We proved that these changes are accompanied by an accumulation of the ET precursor. On the other hand, exposure to ET significantly affected IAA localization in the whole AZ area in a time-dependent manner. Our results provide insight into the existence of a spatio-temporal sequential pattern of the IAA gradient related to the abscission process; this pattern is maintained by interactions with ET. We present new valuable evidence for the existence of conservative mechanisms that regulate generative organ separation and can help to improve the yield of agronomically significant species in the future.

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.

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 phytohormone's 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.

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.

[Abscisic acid metabolism]. / Metabolizm kwasu abscysynowego.

Abscisic acid is one of the plant hormones that determines normal growth and development, i.e. seeds ripening and germination, stomata opening and closure, flowering and stress responses. An appropriate level of endogenous ABA plays a key role in the regulation of most of these processes. Its content in a particular tissue is a balance between the rate of its biosynthesis, oxidative degradation and formation of inactive derivatives (mainly ester). The progress on ABA metabolism was relatively slow in the past. Application of modern molecular biology methods let the most of genes encoding enzymes involved in the regulation of ABA metabolism be identified and contributed to the understanding of its action.

[Jasmonate biosynthesis--the latest discoveries]. / Biosynteza jasmonianów u roslin--najnowsze odkrycia.

Jasmonates are plant hormones involved in many growth and development processes. They also participate in plant defense responses. Current progress in the study on biosynthesis and signaling of jasmonates has contributed to the understanding of the mechanisms regulating concentration of these hormones in the cell. Sustaining a proper level of jasmonates allow the plant to respond appropriately to changing conditions. It is possible due to the large number of enzymes and genes involved in biosynthesis of these hormones as well as multilevel control of their expression.