NnSUS1 encodes a sucrose synthase involved in sugar accumulation in lotus seed cotyledons.

Fresh lotus seeds are gaining favor with consumers for their crunchy texture and natural sweetness. However, the intricacies of sugar accumulation in lotus seeds remain elusive, which greatly hinders the quality improvement of fresh lotus seeds. This study endeavors to elucidate this mechanism by identifying and characterizing the sucrose synthase (SUS) gene family in lotus. Comprising five distinct members, namely NnSUS1 to NnSUS5, each gene within this family features a C-terminal glycosyl transferase1 (GT1) domain. Among them, NnSUS1 is the predominately expressed gene, showing high transcript abundance in the floral organs and cotyledons. NnSUS1 was continuously up-regulated from 6 to 18 days after pollination (DAP) in lotus cotyledons. Furthermore, NnSUS1 demonstrates co-expression relationships with numerous genes involved in starch and sucrose metabolism. To investigate the function of NnSUS1, a transient overexpression system was established in lotus cotyledons, which confirmed the gene's contribution to sugar accumulation. Specifically, transient overexpression of NnSUS1 in seed cotyledons leads to a significant increase in the levels of total soluble sugar, including sucrose and fructose. These findings provide valuable theoretical insights for improving sugar content in lotus seeds through molecular breeding methods.

The Arabidopsis RLCK VI_A2 Kinase Controls Seedling and Plant Growth in Parallel with Gibberellin.

The plant-specific receptor-like cytoplasmic kinases (RLCKs) form a large, poorly characterized family. Members of the RLCK VI_A class of dicots have a unique characteristic: their activity is regulated by Rho-of-plants (ROP) GTPases. The biological function of one of these kinases was investigated using a T-DNA insertion mutant and RNA interference. Loss of RLCK VI_A2 function resulted in restricted cell expansion and seedling growth. Although these phenotypes could be rescued by exogenous gibberellin, the mutant did not exhibit lower levels of active gibberellins nor decreased gibberellin sensitivity. Transcriptome analysis confirmed that gibberellin is not the direct target of the kinase; its absence rather affected the metabolism and signalling of other hormones such as auxin. It is hypothesized that gibberellins and the RLCK VI_A2 kinase act in parallel to regulate cell expansion and plant growth. Gene expression studies also indicated that the kinase might have an overlapping role with the transcription factor circuit (PIF4-BZR1-ARF6) controlling skotomorphogenesis-related hypocotyl/cotyledon elongation. Furthermore, the transcriptomic changes revealed that the loss of RLCK VI_A2 function alters cellular processes that are associated with cell membranes, take place at the cell periphery or in the apoplast, and are related to cellular transport and/or cell wall reorganisation.

Enzymes contributing to the hydrogen peroxide signal dynamics that regulate wall labyrinth formation in transfer cells.

Transfer cells are characterized by an amplified plasma membrane area supported on a wall labyrinth composed of a uniform wall layer (UWL) from which wall ingrowth (WI) papillae arise. Adaxial epidermal cells of developing Vicia faba cotyledons, when placed in culture, undergo a rapid (hours) trans-differentiation to a functional epidermal transfer cell (ETC) phenotype. The trans-differentiation event is controlled by a signalling cascade comprising auxin, ethylene, apoplasmic reactive oxygen species (apoROS), and cytosolic Ca2+. Apoplasmic hydrogen peroxide (apoH2O2) was confirmed as the apoROS regulating UWL and WI papillae formation. Informed by an ETC-specific transcriptome, a pharmacological approach identified a temporally changing cohort of H2O2 biosynthetic enzymes. The cohort contained a respiratory burst oxidase homologue, polyamine oxidase, copper amine oxidase, and a suite of class III peroxidases. Collectively these generated two consecutive bursts in apoH2O2 production. Spatial organization of biosynthetic/catabolic enzymes was deduced from responses to pharmacologically blocking their activities on the cellular and subcellular distribution of apoH2O2. The findings were consistent with catalase activity constraining the apoH2O2 signal to the outer periclinal wall of the ETCs. Strategic positioning of class III peroxidases in this outer domain shaped subcellular apoH2O2 signatures that differed during assembly of the UWL and WI papillae.

A Förster resonance energy transfer sensor for live-cell imaging of mitogen-activated protein kinase activity in Arabidopsis.

The catalytic activity of mitogen-activated protein kinases (MAPKs) is dynamically modified in plants. Since MAPKs have been shown to play important roles in a wide range of signaling pathways, the ability to monitor MAPK activity in living plant cells would be valuable. Here, we report the development of a genetically encoded MAPK activity sensor for use in Arabidopsis thaliana. The sensor is composed of yellow and blue fluorescent proteins, a phosphopeptide binding domain, a MAPK substrate domain and a flexible linker. Using in vitro testing, we demonstrated that phosphorylation causes an increase in the Förster resonance energy transfer (FRET) efficiency of the sensor. The FRET efficiency can therefore serve as a readout of kinase activity. We also produced transgenic Arabidopsis lines expressing this sensor of MAPK activity (SOMA) and performed live-cell imaging experiments using detached cotyledons. Treatment with NaCl, the synthetic flagellin peptide flg22 and chitin all led to rapid gains in FRET efficiency. Control lines expressing a version of SOMA in which the phosphosite was mutated to an alanine did not show any substantial changes in FRET. We also expressed the sensor in a conditional loss-of-function double-mutant line for the Arabidopsis MAPK genes MPK3 and MPK6. These experiments demonstrated that MPK3/6 are necessary for the NaCl-induced FRET gain of the sensor, while other MAPKs are probably contributing to the chitin and flg22-induced increases in FRET. Taken together, our results suggest that SOMA is able to dynamically report MAPK activity in living plant cells.

Expression and properties of the mitochondrial and cytosolic forms of fumarase in sunflower cotyledons.

Fumarase (EC 4.2.1.2) is encoded in sunflower (Helianthus annuus L.) by two genes (FUM1 and FUM2) expressing correspondingly the mitochondrial and the cytosolic form. Both forms have been purified from sunflower cotyledons and characterized. Three quarters of fumarase activity is located in the mitochondrial and one quarter in the cytosolic fraction. The cytosolic form has lower pH optimum than the mitochondrial form, it possesses higher affinity to malate, activated by Mn2+ and less efficiently by Mg2+ while the mitochondrial form is activated only by Mg2+. It is proposed that the mitochondrial form is involved in the respiratory processes linked to the tricarboxylic acid cycle and the cytosolic form participates in the utilization of succinate produced in the glyoxylate cycle providing the flux to gluconeogenesis in germinating sunflower seeds.

Mitogen-activated protein kinases concentrate in the vicinity of chromosomes and may regulate directly cellular patterning in Vicia faba embryos.

MAIN CONCLUSION: Mitogen-activated protein kinases seem to mark genes which are set up to be activated in daughter cells and thus they may play a direct role in cellular patterning during embryogenesis. Embryonic patterning starts very early and after the first division of zygote different genes are expressed in apical and basal cells. However, there is an ongoing debate about the way these different transcription patterns are established during embryogenesis. The presented data indicate that mitogen-activated protein kinases (MAPKs) concentrate in the vicinity of chromosomes and form visible foci there. Cells in the apical and basal regions differ in number of foci observed during the metaphase which suggests that cellular patterning may be determined by activation of diverse MAPK-dependent genes. Different number of foci in each group of separating chromatids and the specified direction of these mitoses in apical-basal axis indicate that the unilateral auxin accumulation in a single cell may regulate the number of foci in each group of chromatids. Thus, we put forward a hypothesis that MAPKs localized in the vicinity of chromosomes during mitosis mark those genes which are set up to be activated in daughter cells after division. It implies that the chromosomal localization of MAPKs may be one of the mechanisms involved in establishment of cellular patterns in some plant species.

C4 photosynthesis and transition of Kranz anatomy in cotyledons and leaves of Tetraena simplex.

PREMISE OF THE STUDY: Tetraena simplex is an independently evolved C4 species in the Zygophylloideae (Zygophyllaceae) and a characteristic forb of saline flats in hot and sandy desert habitats. During early ontogeny, the species had a morphological shift from planar cotyledons (dorsiventral symmetry) to terete, succulent leaves (radial symmetry). We tested whether this shift had a corresponding change in internal Kranz anatomy and tissue patterning. METHODS: For a comprehensive characterization of C4 photosynthesis across early ontogeny in T. simplex, structural and ultrastructural anatomical properties and localization patterns, activities, and immunoblotting of key C4 photosynthetic enzymes were compared in mesophyll and bundle sheath tissues in cotyledons and leaves. KEY RESULTS: Cotyledons and leaves possessed different types of Kranz anatomy (atriplicoid type and a "Tetraena" variant of the kochioid type, respectively), reflecting the change in leaf morphology. In bundle sheath cells, key differences in ultrastructural features included increased organelle numbers and chloroplast thylakoid stacking. C4 enzymes had strict tissue-specific localization patterns within bundle sheath and mesophyll cells in both cotyledons and leaves. The decarboxylase NAD-ME maintained the highest activity, increasing from cotyledons to leaves. This classified T. simplex as fully C4 across ontogeny and a strictly NAD-ME biochemical subtype. CONCLUSIONS: Tetraena simplex cotyledons and leaves showed differences in Kranz type, with associated progression in ultrastructural features, and differing activities/expression levels of C4 enzymes. Furthermore, leaves characterized a new "Tetraena" variation of the kochioid Kranz anatomy.

Flavonol rhamnosylation indirectly modifies the cell wall defects of RHAMNOSE BIOSYNTHESIS1 mutants by altering rhamnose flux.

Rhamnose is required in Arabidopsis thaliana for synthesizing pectic polysaccharides and glycosylating flavonols. RHAMNOSE BIOSYNTHESIS1 (RHM1) encodes a UDP-l-rhamnose synthase, and rhm1 mutants exhibit many developmental defects, including short root hairs, hyponastic cotyledons, and left-handed helically twisted petals and roots. It has been proposed that the hyponastic cotyledons observed in rhm1 mutants are a consequence of abnormal flavonol glycosylation, while the root hair defect is flavonol-independent. We have recently shown that the helical twisting of rhm1 petals results from decreased levels of rhamnose-containing cell wall polymers. In this study, we found that flavonols indirectly modify the rhm1 helical petal phenotype by altering rhamnose flux to the cell wall. Given this finding, we further investigated the relationship between flavonols and the cell wall in rhm1 cotyledons. We show that decreased flavonol rhamnosylation is not responsible for the cotyledon phenotype of rhm1 mutants. Instead, blocking flavonol synthesis or rhamnosylation can suppress rhm1 defects by diverting UDP-l-rhamnose to the synthesis of cell wall polysaccharides. Therefore, rhamnose is required in the cell wall for normal expansion of cotyledon epidermal cells. Our findings suggest a broad role for rhamnose-containing cell wall polysaccharides in the morphogenesis of epidermal cells.

Identification and expression analysis of starch branching enzymes involved in starch synthesis during the development of chestnut (Castanea mollissima Blume) cotyledons.

Chinese chestnut (Castanea mollissima Blume) is native to China and distributes widely in arid and semi-arid mountain area with barren soil. As a perennial crop, chestnut is an alternative food source and acts as an important commercial nut tree in China. Starch is the major metabolite in nuts, accounting for 46 ~ 64% of the chestnut dry weight. The accumulation of total starch and amylopectin showed a similar increasing trend during the development of nut. Amylopectin contributed up to 76% of the total starch content at 80 days after pollination (DAP). The increase of total starch mainly results from amylopectin synthesis. Among genes associated with starch biosynthesis, CmSBEs (starch branching enzyme) showed significant increase during nut development. Two starch branching enzyme isoforms, CmSBE I and CmSBE II, were identified from chestnut cotyledon using zymogram analysis. CmSBE I and CmSBE II showed similar patterns of expression during nut development. The accumulations of CmSBE transcripts and proteins in developing cotyledons were characterized. The expressions of two CmSBE genes increased from 64 DAP and reached the highest levels at 77 DAP, and SBE activity reached its peak at 74 DAP. These results suggested that the CmSBE enzymes mainly contributed to amylopectin synthesis and influenced the amylopectin content in the developing cotyledon, which would be beneficial to chestnut germplasm selection and breeding.

Two Acyltransferases Contribute Differently to Linolenic Acid Levels in Seed Oil.

Acyltransferases are key contributors to triacylglycerol (TAG) synthesis and, thus, are of great importance for seed oil quality. The effects of increased or decreased expression of ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1) or PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE (PDAT) on seed lipid composition were assessed in several Camelina sativa lines. Furthermore, in vitro assays of acyltransferases in microsomal fractions prepared from developing seeds of some of these lines were performed. Decreased expression of DGAT1 led to an increased percentage of 18:3n-3 without any change in total lipid content of the seed. The tri-18:3 TAG increase occurred predominantly in the cotyledon, as determined with matrix-assisted laser desorption/ionization-mass spectrometry, whereas species with two 18:3n-3 acyl groups were elevated in both cotyledon and embryonal axis. PDAT overexpression led to a relative increase of 18:2n-6 at the expense of 18:3n-3, also without affecting the total lipid content. Differential distributions of TAG species also were observed in different parts of the seed. The microsomal assays revealed that C.sativa seeds have very high activity of diacylglycerol-phosphatidylcholine interconversion. The combination of analytical and biochemical data suggests that the higher 18:2n-6 content in the seed oil of the PDAT overexpressors is due to the channeling of fatty acids from phosphatidylcholine into TAG before being desaturated to 18:3n-3, caused by the high activity of PDAT in general and by PDAT specificity for 18:2n-6. The higher levels of 18:3n-3 in DGAT1-silencing lines are likely due to the compensatory activity of a TAG-synthesizing enzyme with specificity for this acyl group and more desaturation of acyl groups occurring on phosphatidylcholine.

Isolation and characterization of genes encoding leucoanthocyanidin reductase (FeLAR) and anthocyanidin reductase (FeANR) in buckwheat (Fagopyrum esculentum).

Proanthocyanidins (PAs) are a major group of flavonoids synthesized via the phenylpropanoid biosynthesis pathway, however the pathway has not been fully characterized in buckwheat. Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) are involved in the last steps of PA biosynthesis. To isolate the genes for these enzymes from buckwheat we performed PCR using degenerate primers and obtained cDNAs of ANR and LAR, which we designated FeANR and FeLAR1. A search for homologs in a buckwheat genome database with both sequences returned two more LAR sequences, designated FeLAR2 and FeLAR3. Linkage analysis with an F2 segregating population indicated that the three LAR loci were not genetically linked. We detected high levels of PAs in roots and cotyledons of buckwheat seedlings and in buds and flowers of mature plants. FeANR and FeLAR1-3 were expressed in most organs but had different expression patterns. Our findings would be useful for breeding and further analysis of PA synthesis and its regulation in buckwheat.

Plant growth regulators induced urease activity in Cucurbita pepo L. cotyledons.

This study is aimed to investigate the activity of urease (EC 3.5.1.5, urea amidohydrolase) that catalyzes the hydrolysis of urea in 5-day-old Cucurbita pepo cotyledons subjected to various concentrations of different growth regulators. The treatment of C. pepo cotyledons with different concentrations (100-600 µmol) of different auxins [indole-3-acetic acid (IAA), indole butyric acid (IBA), indole propionic acid (IPA) and naphthalene acetic acid (NAA)]; or with different concentrations (100-300 µmol) of different cytokinins [kinetin, zeatin and benzyladenine (6-BA)] resulted in a significant increase of urease activity, compared to control. The optimal effects were recorded for each of 500 µmol of IAA and 300 µmol of zeatin treatments. A gradual increase in urease activity was detected in cotyledons treated with various concentrations (0.2-1.0 mM) of 28-homobrassinolide (HBL), in relative to control. A substantial increase in urease activity was observed in cotyledons subjected to different concentrations of triazole (10-60 mg L(-1)), containing either triadimefon (TDM) or hexaconazole (HEX), compared to control. The combination of 300 µmol zeatin with any of protein inhibitors, namely 5-fluorouridine (FUrd), cordycepin and α-amanitin, resulted in the alleviation of their inhibitory effect on the urease activity.

A mutation of casein kinase 2 α4 subunit affects multiple developmental processes in Arabidopsis.

KEY MESSAGE: Arabidopsis CK2 α4 subunit regulates the primary root and hypocotyl elongation, lateral root formation, cotyledon expansion, rosette leaf initiation and growth, flowering, and anthocyanin biosynthesis. Casein kinase 2 (CK2) is a conserved tetrameric kinase composed of two α and two ß subunits. The inhibition of CK2 activity usually results in severe developmental deficiency. Four genes (CKA1-CKA4) encode CK2 α subunit in Arabidopsis. Single mutations of CKA1, CKA2, and CKA3 do not affect the normal growth of Arabidopsis, while the cka1 cka2 cka3 triple mutants are defective in cotyledon and hypocotyl growth, lateral root development, and flowering. The inhibition of CKA4 expression in cka1 cka2 cka3 background further reduces the number of lateral roots and delays the flowering time. Here, we report the characterization of a novel knockout mutant of CKA4, which exhibits various developmental defects including reduced primary root and hypocotyl elongation, increased lateral root density, delayed cotyledon expansion, retarded rosette leaf initiation and growth, and late flowering. The examination of the cellular basis for abnormal root development of this mutant revealed reduced root meristem cells with enhanced RETINOBLASTOMA-RELATED (RBR) expression that promotes cell differentiation in root meristem. Moreover, this cka4-2 mutant accumulates higher anthocyanin in the aerial part and shows an increased expression of anthocyanin biosynthetic genes, suggesting a novel role of CK2 in modulating anthocyanin biosynthesis. In addition, the complementation test using primary root elongation assay as a sample confirms that the changed phenotypes of this cka4-2 mutant are due to the lack of CKA4. Taken together, this study reveals an essential role of CK2 α4 subunit in multiple developmental processes in Arabidopsis.

Characterization of the legumains encoded by the genome of Theobroma cacao L.

Legumains are cysteine proteases related to plant development, protein degradation, programmed cell death, and defense against pathogens. In this study, we have identified and characterized three legumains encoded by Theobroma cacao genome through in silico analyses, three-dimensional modeling, genetic expression pattern in different tissues and as a response to the inoculation of Moniliophthora perniciosa fungus. The three proteins were named TcLEG3, TcLEG6, and TcLEG9. Histidine and cysteine residue which are part of the catalytic site were conserved among the proteins, and they remained parallel in the loop region in the 3D modeling. Three-dimensional modeling showed that the propeptide, which is located in the terminal C region of legumains blocks the catalytic cleft. Comparing dendrogram data with the relative expression analysis, indicated that TcLEG3 is related to the seed legumain group, TcLEG6 is related with the group of embryogenesis activities, and protein TcLEG9, with processes regarding the vegetative group. Furthermore, the expression analyses proposes a significant role for the three legumains during the development of Theobroma cacao and in its interaction with M. perniciosa.

Endoplasmic reticulum localization and activity of maize auxin biosynthetic enzymes.

Auxin is a major growth hormone in plants and the first plant hormone to be discovered and studied. Active research over >60 years has shed light on many of the molecular mechanisms of its action including transport, perception, signal transduction, and a variety of biosynthetic pathways in various species, tissues, and developmental stages. The complexity and redundancy of the auxin biosynthetic network and enzymes involved raises the question of how such a system, producing such a potent agent as auxin, can be appropriately controlled at all. Here it is shown that maize auxin biosynthesis takes place in microsomal as well as cytosolic cellular fractions from maize seedlings. Most interestingly, a set of enzymes shown to be involved in auxin biosynthesis via their activity and/or mutant phenotypes and catalysing adjacent steps in YUCCA-dependent biosynthesis are localized to the endoplasmic reticulum (ER). Positioning of auxin biosynthetic enzymes at the ER could be necessary to bring auxin biosynthesis in closer proximity to ER-localized factors for transport, conjugation, and signalling, and allow for an additional level of regulation by subcellular compartmentation of auxin action. Furthermore, it might provide a link to ethylene action and be a factor in hormonal cross-talk as all five ethylene receptors are ER localized.

Identification of a Highly Specific Isoflavone 7-O-glucosyltransferase in the soybean (Glycine max (L.) Merr.).

Isoflavone conjugates [7-O-ß-D-glucosides and 7-O-(6″-malonyl-ß-D-glucosides) of daidzein and genistein] accumulate in soybean roots and serve as the stored precursors of isoflavones (aglycons), which play very important roles in the rhizobia-mediated nodulation of this plant. Thus far, the isoflavone 7-O-glucosyltransferase (GmIF7GT or GmUGT1) has been biochemically characterized and is believed to be involved in isoflavone conjugate biosynthesis. The soybean genome encodes many other glycosyltransferase homologs (GmUGTs) that are related to GmUGT1; however, their catalytic properties, substrate specificities, and role(s) in isoflavone conjugation are unknown. In this study, nine different GmUGT1-related GmUGT cDNAs were isolated; six of these cDNAs belonged to two distinct phylogenetic subgroups (A and B), and these six were functionally characterized. The results showed that GmUGT4, a representative of subgroup A, encoded a UGT that was highly specific for isoflavones showing kcat and kcat/Km values for daidzein of 5.89 ± 0.65 s(-1) and 2.91 × 10(5) s(-1)M(-1), respectively. Moreover, GmUGT4 was expressed in the roots (mainly in lateral roots) of the 7-day-old seedlings and seeds, both of which contained abundant amounts of isoflavone conjugates. By contrast, GmUGT1 and GmUGT7, which were subgroup B members, encoded enzymes with broad glucosyl-acceptor specificities and were mainly expressed in the aerial portions (cotyledons and hypocotyls) of the seedlings. In the present study, we proposed that the role of isoflavone glucosylation in a soybean plant is assigned to different GmUGT members in an organ/tissue-dependent manner. We also established the functional importance of GmUGT4 in the biosynthesis of isoflavone conjugates in lateral roots that make a major contribution to overall N2 fixation.

Ribonuclease J is required for chloroplast and embryo development in Arabidopsis.

Chloroplasts perform many essential metabolic functions and their proper development is critically important in embryogenesis. However, little is known about how chloroplasts function in embryogenesis and more relevant components need to be characterized. In this study, we show that Arabidopsis Ribonuclease J (RNase J) is required for chloroplast and embryo development. Mutation of AtRNJ led to albino ovules containing aborted embryos; the morphological development of rnj embryos was disturbed after the globular stage. Observation of ultrastructures indicated that these aborted embryos may result from impaired chloroplast development. Furthermore, by analyzing the molecular markers of cell fate decisions (STM, FIL, ML1, SCR, and WOX5) in rnj embryos, we found that this impairment of chloroplast development may lead to aberrant embryo patterning along the apical-basal axis, indicating that AtRNJ is important in initiating and maintaining the organization of shoot apical meristems (SAMs), cotyledons, and hypocotyls. Moreover, the transport and response of auxin in rnj embryos was found to be disrupted, suggesting that AtRNJ may be involved in auxin-mediated pathways during embryogenesis. Therefore, we speculate that RNJ plays a vital role in embryo morphogenesis and apical-basal pattern formation by regulating chloroplast development.

Efficient use of energy in anoxia-tolerant plants with focus on germinating rice seedlings.

Anoxia tolerance in plants is distinguished by direction of the sparse supply of energy to processes crucial to cell maintenance and sometimes to growth, as in rice seedlings. In anoxic rice coleoptiles energy is used to synthesise proteins, take up K(+) , synthesise cell walls and lipids, and in cell maintenance. Maintenance of electrochemical H(+) gradients across the tonoplast and plasma membrane is crucial for solute compartmentation and thus survival. These gradients sustain some H(+) -solute cotransport and regulate cytoplasmic pH. Pyrophosphate (PPi ), the alternative energy donor to ATP, allows direction of energy to the vacuolar H(+) -PPi ase, sustaining H(+) gradients across the tonoplast. When energy production is critically low, operation of a biochemical pHstat allows H(+) -solute cotransport across plasma membranes to continue for at least for 18 h. In active (e.g. growing) cells, PPi produced during substantial polymer synthesis allows conversion of PPi to ATP by PPi -phosphofructokinase (PFK). In quiescent cells with little polymer synthesis and associated PPi formation, the PPi required by the vacuolar H(+) -PPi ase and UDPG pyrophosphorylase involved in sucrose mobilisation via sucrose synthase might be produced by conversion of ATP to PPi through reversible glycolytic enzymes, presumably pyruvate orthophosphate dikinase. These hypotheses need testing with species characterised by contrasting anoxia tolerance.

Endogenous factors regulating poor-nutrition stress-induced flowering in pharbitis: The involvement of metabolic pathways regulated by aminooxyacetic acid.

The short-day plant pharbitis (also called Japanese morning glory), Ipomoea nil (formerly Pharbitis nil), was induced to flower by poor-nutrition stress. This stress-induced flowering was inhibited by aminooxyacetic acid (AOA), which is a known inhibitor of phenylalanine ammonia-lyase (PAL) and the synthesis of indole-3-acetic acid (IAA) and 1-aminocycropropane-1-carboxylic acid (ACC) and thus regulates endogenous levels of salicylic acid (SA), IAA and polyamine (PA). Stress treatment increased PAL activity in cotyledons, and AOA suppressed this increase. The observed PAL activity and flowering response correlate positively, indicating that AOA functions as a PAL inhibitor. The inhibition of stress-induced flowering by AOA was also overcome by IAA. An antiauxin, 4-chlorophenoxy isobutyric acid, inhibited stress-induced flowering. Both SA and IAA promoted flowering induced by stress. PA also promoted flowering, and the effective PA was found to be putrescine (Put). These results suggest that all of the pathways leading to the synthesis of SA, IAA and Put are responsive to the flowering inhibition by AOA and that these endogenous factors may be involved in the regulation of stress-induced flowering. However, as none of them induced flowering under non-stress conditions, they may function cooperatively to promote flowering.