Academic literature on the topic 'Auxines de synthèse'

Abstract Auxins as phytohormons, responsible for coordination of growing processes, also contribute to the formation of tension wood (TW) in trees, but the mechanism of this process is still unclear. In this study, it has been tried to induce TW formation in erect hybrid poplar trees (in opposite to inclined or bended trees) by treatment with exogenous indole-3-acetic acid (IAA, as one of the auxins) or N-1-naphthylphthalamic acid (NPA) as an auxin transport inhibitor. The treatment with IAA resulted in TW formation and, as expected, NPA treatment did not. The gelatinous fiber formation and higher cellulose content in the cell wall were unambiguous indicators for TW formation. Real-time polymerase chain reaction (PCR) analysis revealed that genes of PIN1, ABCB1, and AUX2 involved in polar auxin transport were highly expressed in trees treated with exogenous IAA. Moreover, expressions of cellulose biosynthesis related genes of UGP1, UGP2 and CesA13 were strongly up-regulated. These observations indicate that the accelerated intercellular polar auxin transport caused by exogenous IAA is accounted for TW formation, i.e. the signal transduction of auxin is affected, which then facilitates cellulose biosynthesis. In contrast, the transcript abundances of PIN1 and all selected cellulose synthases (CesAs) were decreased after NPA treatment via inhibiting the cellular auxin efflux with negative effects on plant’s primary growth. These results are interpreted that TW formation is closely associated with the acceleration of intercellular polar auxin transport.

Substituted quinolinecarboxylic acids, including quinclorac (BAS 514H), are a new class of highly selective auxin herbicides, which are chemically similar to naturally occurring compounds isolated from plants and soils. Quinclorac is used in rice to control important dicot and monocot weeds, particularly barnyardgrass. The herbicide has also been developed for application in turfgrass areas, spring wheat, and chemical fallow. Quinclorac is readily absorbed by germinating seeds, roots, and leaves and is translocated in the plant both acropetally and basipetally. By mimicking an auxin overdose, quinclorac affects the phytohormonal system in sensitive plants. The compound stimulates the induction of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase activity and thus promotes ethylene biosynthesis. In susceptible dicots, increased levels of ethylene trigger an accumulation of abscisic acid (ABA), which, as part of the intrinsic auxin activity of quinclorac, plays a major role in growth inhibition and the induction of epinasty and senescence. In sensitive grasses, such as barnyardgrass species, large crabgrass, broadleaf signalgrass, and green foxtail, quinclorac leads particularly to an accumulation of tissue cyanide, formed as a co-product during increased ACC and ethylene synthesis. This causes phytotoxicity characterized by the inhibition of root and particularly shoot growth with tissue chlorosis and subsequent necrosis. These effects were not observed in tolerant rice and a resistant biotype of barnyardgrass. No significant differences in uptake, translocation, or metabolism of quinclorac between resistant and sensitive grasses were found. Hence, a target-site-based mechanism of selectivity is suggested. The induction process of the ACC synthase activity plays the primary role in the selective herbicide action of quinclorac. This is a common effect of auxin herbicides and auxins, which lead to the accumulation of cyanide and/or ABA depending on the plant species and tissues, the compound concentration in the tissue, and their biological activity.

Indole synthase (INS), a homologous cytosolic enzyme of the plastidal tryptophan synthase A (TSA), has been reported as the first enzyme in the tryptophan-independent pathway of auxin synthesis. This suggestion was challenged as INS or its free indole product may interact with tryptophan synthase B (TSB) and, therefore, with the tryptophan-dependent pathway. Thus, the main aim of this research was to find out whether INS is involved in the tryptophan-dependent or independent pathway. The gene coexpression approach is widely recognized as an efficient tool to uncover functionally related genes. Coexpression data presented here were supported by both RNAseq and microarray platforms and, hence, considered reliable. Coexpression meta-analyses of Arabidopsis genome was implemented to compare between the coexpression of TSA and INS with all genes involved in the production of tryptophan via the chorismate pathway. Tryptophan synthase A was found to be coexpressed strongly with TSB1/2, anthranilate synthase A1/B1, phosphoribosyl anthranilate transferase1, as well as indole-3-glycerol phosphate synthase1. However, INS was not found to be coexpressed with any target genes suggesting that it may exclusively and independently be involved in the tryptophan-independent pathway. Additionally, annotation of examined genes as ubiquitous or differentially expressed were described and subunits-encoded genes available for the assembly of tryptophan and anthranilate synthase complex were suggested. The most probable TSB subunits expected to interact with TSA is TSB1 then TSB2. Whereas TSB3 is only used under limited hormone conditions to assemble tryptophan synthase complex, putative TSB4 is not expected to be involved in the plastidial synthesis of tryptophan in Arabidopsis.

Mature pollen from Petunia hybrida contains significant levels of 1-aminocyclopropane-1-carboxylic acid (ACC), and this ACC is thought to play a role in pollination-induced ethylene by the pistil. We investigated the developmental accumulation of ACC in anthers and pollen. The level of ACC in anthers was very low until the day before anthesis, at which time it increased 100-fold. A 1.1-kb partial ACC synthase cDNA clone (pPHACS2) was amplified from total RNA isolated from mature anthers by reverse transcriptase, followed by polymerase chain reaction using oligonucleotide primers synthesized to conserved amino acid sequences in ACC synthases. The expression of pPHACS2 mRNA during anther development was correlated with the accumulation of ACC and was localized to the pollen grain. The pPHACS2 cDNA was used to identify the PH-ACS2 gene from a library of genomic DNA fragments from Petunia hybrida. PH-ACS2 encoded an ACC synthase transcript of four exons interrupted by three introns. The ACC synthase protein encoded by the PH-ACS2 gene shared >80% homology with ACC synthases from tomato (LE-ACS3) and potato (ST-ACS1a). A chimeric PH-ACS2 promoter-β-glucuronidase (GUS) gene was used to transform petunia and transgenic plants were analyzed for GUS activity. GUS staining was localized to mature pollen grains and was not detected in other tissues. Despite similarities to LE-ACS3, we did not detect GUS activity under conditions of anaerobic stress or in response to auxin. A series of 5-prime-flanking DNA deletions revealed that sequences within the PH-ACS2 promoter were responsible for pollen-specific expression.

A field experiment was conducted in 2009–2010 at Pennsylvania and Maryland locations, and repeated it in 2010–2011 to test the effectiveness of POST-applied herbicides at fall and spring timings on seeded hairy vetch in winter wheat. A total of 16 herbicide treatment combinations was tested that included synthetic auxins, acetolactate synthase (ALS) inhibitors, and a protoporphyrinogen oxidase inhibitor. Spring applications tended to be more effective than fall applications. Among synthetic auxins, clopyralid (105 g ae ha−1) and treatments containing dicamba (140 g ae ha−1) were effective at both timings, resulting in greater than 90% hairy vetch control at wheat harvest. Pyroxsulam and prosulfuron applied at 18 g ai ha−1 provided the most effective hairy vetch control (> 90%) at both application timings among ALS inhibitors. Spring applications of several herbicides provided moderate (> 80%) to high (> 90%) levels of hairy vetch control, including: 2,4-D amine (140 g ae ha−1), mesosulfuron-methyl (15 g ai ha−1), tribenuron-methyl (13 g ai ha−1), and thifensulfuron/tribenuron-methyl treatments (16 and 32 g ai ha−1). Winter wheat injury was evaluated, but symptoms were negligible for most treatments. Winter wheat yields declined with increasing hairy vetch biomass. Fall herbicides may be prioritized to reduce hairy vetch competition during the fall and early spring growing season. Our research has established that several synthetic auxin and ALS-inhibiting herbicides, applied POST in fall or spring, can be safely used in winter wheat to control hairy vetch in an integrated weed management program.

Herbicide-resistant weeds have been identified and recorded on every continent where croplands are available. Despite the diversity of weed communities, it is of interest how selection has led to the same consequences in distant regions. Brassica rapa is a widespread naturalized weed that is found throughout temperate North and South America, and it is a frequent weed among winter cereal crops in Argentina and in Mexico. Broadleaf weed control is based on glyphosate that is used prior to sowing and sulfonylureas or mimic auxin herbicides that are used once the weeds have already emerged. This study was aimed at determining whether a convergent phenotypic adaptation to multiple herbicides had occurred in B. rapa populations from Mexico and Argentina by comparing the herbicide sensitivity to inhibitors of the acetolactate synthase (ALS), 5-enolpyruvylshikimate-3-phosphate (EPSPS), and auxin mimics. Five B. rapa populations were analyzed from seeds collected in wheat fields in Argentina (Ar1 and Ar2) and barley fields in Mexico (Mx1, Mx2 and MxS). Mx1, Mx2, and Ar1 populations presented multiple resistance to ALS- and EPSPS-inhibitors and to auxin mimics (2,4-D, MCPA, and fluroxypyr), while the Ar2 population showed resistance only to ALS-inhibitors and glyphosate. Resistance factors ranged from 947 to 4069 for tribenuron-methyl, from 1.5 to 9.4 for 2,4-D, and from 2.7 to 42 for glyphosate. These were consistent with ALS activity, ethylene production, and shikimate accumulation analyses in response to tribenuron-methyl, 2,4-D, and glyphosate, respectively. These results fully support the evolution of the multiple- and cross-herbicide resistance to glyphosate, ALS-inhibitors, and auxinic herbicides in B. rapa populations from Mexico and Argentina.

Corn poppy (Papaver rhoeas L.) and cornflower (Centaurea cyanus L.) are two overwintering weed species found in crop fields in Europe. They are characterised by a similar life cycle, similar competitive efforts, and a spectrum of herbicides recommended for their control. This review summarises the biology and herbicide resistance phenomena of corn poppy and cornflower in Europe. Corn poppy is one of the most dangerous dicotyledonous weeds, having developed herbicide resistance to acetolactate synthase inhibitors and growth regulators, especially in Mediterranean countries and Great Britain. Target site resistance to acetolactate synthase inhibitors dominates among herbicide-resistant poppy biotypes. The importance of non-target site resistance to acetolactate synthase inhibitors in this species may be underestimated because non-target site resistance is very often associated with target site resistance. Cornflower, meanwhile, is increasingly rare in European agricultural landscapes, with acetolactate synthase inhibitors-resistant biotypes only listed in Poland. However, the mechanisms of cornflower herbicide resistance are not well recognised. Currently, herbicides mainly from acetolactate synthase and photosystem II inhibitors as well as from synthetic auxins groups are recommended for the control of both weeds. Integrated methods of management of both weeds, especially herbicide-resistant biotypes, continue to be underrepresented.

Ethylene is the plant hormone that controls several features of plant growth and development. The rate-limiting step in its synthesis is the formation of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) from S-adenosylmethionine (AdoMet), catalyzed by ACC synthase. We have isolated a complementary DNA sequence encoding ACC synthase from zucchini (Cucurbita) fruits. The biological activity of the clone was confirmed by the ability of the cloned sequence to direct ACC synthase activity in Escherichia coli and yeast. In vivo studies using the ACC cDNA as probe showed that the ACC synthase gene is induced by a diverse group of inducers, including wounding, Li+ ions, and the plant hormone auxin.

A population of oriental mustard from Port Broughton in South Australia was reported as not being controlled by 2,4-D. Dose response experiments determined this population was resistant to both 2,4-D and MCPA, requiring greater than 20 times more herbicide for equivalent control compared to a known susceptible population (from Roseworthy, South Australia) and a population resistant only to the acetohydroxyacid synthase (AHAS)-inhibiting herbicides (from Tumby Bay, South Australia). The Port Broughton population was also found to be resistant to three chemical groups that inhibit AHAS; however, the level of resistance was lower than the known acetolactate synthase–resistant population from Tumby Bay. Herbicides from other modes of action were able to control the Port Broughton population. Assays of isolated AHAS from the Port Broughton population showed high levels of resistance to the sulfonylurea and sulfonamide herbicide groups, but not to the imidazolinone herbicides. A single nucleotide change in the AHAS gene that predicted a Pro to Ser substitution at position 197 in the protein was identified in the Port Broughton population. This population of oriental mustard has evolved multiple resistance to AHAS-inhibiting herbicides (AHAS inhibitors) and auxinic herbicides, through a mutation in AHAS and a second nontarget-site mechanism. Whether the same mechanism provides resistance to both AHAS inhibitors and auxinic herbicides remains to be determined. Multiple resistance to auxinic herbicides and AHAS inhibitors in the Port Broughton population will make control of this population more difficult.

The phytohormone auxin regulates nearly all aspects of plant growth and development. Tremendous achievements have been made in elucidating the tryptophan (Trp)-dependent auxin biosynthetic pathway; however, the genetic evidence, key components, and functions of the Trp-independent pathway remain elusive. Here we report that the Arabidopsis indole synthase mutant is defective in the long-anticipated Trp-independent auxin biosynthetic pathway and that auxin synthesized through this spatially and temporally regulated pathway contributes significantly to the establishment of the apical–basal axis, which profoundly affects the early embryogenesis in Arabidopsis. These discoveries pave an avenue for elucidating the Trp-independent auxin biosynthetic pathway and its functions in regulating plant growth and development.

Le coquelicot (Papaver rhoeas) est une adventice cosmopolite très répandue dans les cultures de céréales d’hiver en Europe qui présente un haut potentiel d’invasion et de propagation dans les cultures. Il est principalement contrôlé par les herbicides inhibiteurs de l’ALS et les herbicides auxiniques. L’utilisation intensive de ces deux modes d’action à conduit à l’évolution de la résistance dans de nombreuses populations de coquelicot à travers l’Europe. La résistance aux herbicides implique deux catégories de mécanismes : la résistance liée à la cible (RLC) et la résistance non liée à la cible (RNLC). Chez le coquelicot, seuls des mécanismes de RNLC ont été identifiés, mais les gènes spécifiques restent inconnus. Ce travail de thèse a donc plusieurs objectifs : (i) identifier et potentiellement valider les déterminants génétiques de la résistance aux herbicides auxiniques chez le coquelicot et (ii) évaluer la présence de résistance aux herbicides auxiniques dans des populations françaises de Coquelicot. Dans une première partie, nous avons caractérisé phénotypiquement le matériel végétal disponible via des tests biologiques de sensibilités aux herbicides (Chapitre 1) pour évaluer la situation de la résistance des coquelicots aux herbicides auxiniques en France. Nous avons montré que la résistance au 2,4-D en France était répandue, voire très bien installée dans certaines zones. Nous avons également identifié deux parcelles en Italie et en Grèce où des plantes résistantes à l’halauxifène-méthyl ont été détecté, suggérant un début d’évolution de la résistance à ce nouvel herbicide de synthèse. Les populations avec un ratio équilibré d’individus résistants et sensibles ont été utilisées pour la production de matériel végétal pour les approches de biologie moléculaire de la deuxième partie.Dans une deuxième partie, nous avons étudié la résistance constitutive au 2,4-D et à l’halauxifène-méthyl parmi 14 populations via le séquençage de l’ARN (RNAseq) (Chapitre 2). Nous avons montré que les profils d’expression des plantes sensibles et résistantes étaient propres à chaque population. Parmi les gènes différentiellement exprimés chez les plantes résistantes, certaines familles de gènes potentiellement impliqués dans la métabolisation des herbicides (CYP450, GST, transporteurs ABCs etc.) ou des cascades de régulation (facteurs de transcription, protéines kinases) ont été identifiées. Sur la base de ces résultats, le niveau d’expression de ces gènes à été validé via une approche de RT-qPCR à partir d’un échantillon plus large de plantes. L’ensemble des résultats indiquent qu’il existe potentiellement une grande variété de mécanismes de résistance inter- et intra-population. Le deuxième RNAseq (Chapitre 3) visait à étudier la réponse transcriptomique des plantes résistantes et sensibles entre 4h et 48h après l’application du 2,4-D dans deux populations. Nous avons identifié une grande diversité de gènes et de familles de gènes spécifiquement induits chez les plantes résistantes des deux populations, mais leur rôle dans la résistance n’a pas pu être vérifié. Comme dans la résistance constitutive, il peut potentiellement s’agir d’enzymes de détoxication, de transporteurs, voire de potentiels gènes cibles de l’auxine ou de gènes associés à la réponse générale au stress. De plus, le 2,4-D induit une réponse rapide qui est détectable dans les 4h suivant le traitement quels que soient le phénotype et la population. Enfin, la comparaison des gènes différentiellement exprimés de façon constitutive entre les deux approches de RNAseq démontre que l’absence de gènes communs est potentiellement due à une diversité élevée de mécanismes de résistance intra- et -inter populations, ou au fait que les mécanismes qui contribuent le plus à la résistance sont dû à des mutations de structure
Corn poppy (Papaver rhoeas) is a very widespread cosmopolitan weed in winter crops cereal in Europe which has a high potential for invasion and spread in crops. It is mainly controlled by ALS inhibitor herbicides and auxin herbicides. The intensive use of these two modes of action has led to the evolution of resistance in many poppy populations across Europe. Herbicide resistance involves two categories of mechanisms: target-site-based resistance (TSR) and non-target-site-based resistance (NTSR). In poppy, only NTSR mechanisms have been identified, but the specific genes remain unknown. This work therefore has several goals: (i) identify and potentially validate the genetic determinants of resistance to auxin herbicides in corn poppy and (ii) evaluate resistance status to auxin herbicides in French poppy populations.In a first part, we phenotypically characterized the plant material available using herbicides sensitivity bioassays (Chapter 1) to assess the resistance status of poppies to auxin herbicides in France. We have shown that resistance to 2,4-D in France was widespread, even very well established in certain areas. We also identified two areas in Italy and Greece where resistant plants to halauxifen-methyl were detected, suggesting the beginning of the evolution of resistance to this new synthetic herbicide. Populations with a balanced ratio of resistant and sensitive individuals were used for plant material production for the molecular biology approaches of the second part.In a second part, we studied constitutive resistance to 2,4-D and halauxifen-methyl among 14 populations via RNA sequencing (RNAseq) (Chapter 2). We showed that the expression profiles of sensitive and resistant plants were specific to each population. Among the genes differentially expressed in resistant plants, some gene families potentially involved in the metabolism of herbicides (CYP450, GST, ABC transporters, etc.) or regulatory cascades (transcription factors, protein kinases) have been identified. Based on these results, the expression level of these genes was validated via an RT-qPCR approach using a larger sample of plants. All the results indicate that there is potentially a wide variety of inter- and intra-population resistance mechanisms.The second RNAseq (Chapter 3) aimed to study the transcriptomic response of resistant and sensitive plants between 4h and 48h after the application of 2,4-D in two populations. We identified a large diversity of genes and gene families specifically induced in resistant plants from both populations, but their role in resistance could not be verified. As in constitutive resistance, these can potentially be detoxification enzymes, transporters, or even potential auxin target genes or genes associated with the general stress response. In addition, 2,4-D induces a rapid response which is detectable within 4 hours following treatment regardless of the phenotype and population.Finally, the comparison of constitutively differentially expressed genes between the two RNAseq approaches demonstrates that the absence of common genes is potentially due to a high diversity of intra- and -inter population resistance mechanisms, or to the fact that the mechanisms that contribute the most to resistance are due to structural mutations

Les isoprénoïdes constituent une vaste famille de constituants cellulaires synthétisés chez la plupart des organismes vivants. Chez les plantes, la biosynthèse des isoprénoïdes est réalisée dans trois compartiments : le plaste, le cytoplasme-réticulum endoplasmique et la mitochondrie. Nous avons orienté nos travaux vers l'étude moléculaire et fonctionnelle du rôle de 2 types d'isoprénoïdes cytosoliques (dolichols et stérols) au cours du développement chez les plantes. Pour mener à bien notre étude, nous avons créé des lignées mutantes « knockdown » via la technique de !'ARN interférence (RNAi) et caractérisé des mutant~d'insertion T-DNAs « knockout » pour les gènes d'intérêt chez Arabidopsis.Dans le premier chapitre, nous montrons que les isoprénoïdes sont impliqués de façon indirecte dans la Nglycosylation de protéines via le Dolichol-P-Mannose (Dol-P-Man) dont la synthèse est catalysée par la dolichol phosphate mannose synthase (DPMS). Nous démontrons que chez les plantes, la DPMS est organisée en un complexe hétéromérique localisé dans le réticulum endoplasmique (RE) qui comprend 3 sous unités DPMS1, DPMS2 et DPMS3 codées par 3 gènes. Seule DPMS1 possède une activité catalytique. Les lignées DPMS 1-RNAi et dpms 1 présentent une hypo N-glycosylation des protéines, une forte chlorose et une inhibition de la croissance racinaire. Ces traits sont associés à une hypersensibilité à l'ammonium et à une induction de la« unfolded protein response »au niveau du RE. L'ensemble de ces données montrent que les gènes DPMS jouent un rôle important dans la N-glycosylation des protéines et le développement des plantes.Dans le deuxième chapitre, nous avons porté notre attention sur le rôle des intermédiaires de biosynthèse des stérols («SBls») dans la régulation du développement des plantes en choisissant comme cible ERG28,une protéine impliquée dans le complexe enzymatique de déméthylation en C-4 des stérols « SC4DM ». Nous montrons que ERG28 est localisée dans le RE et assemble 3 enzymes du complexe« SC4DM », la«sterol 4a-methyl oxidase ». la « 4a-carboxysterol-C3-dehydrogenase/C4-decarboxylase » et la « sterone ketoreductase ». Nous démontrons que la perte de fonction de ERG28 dans les lignées ERG28-RNAi eterg28 se traduit par des phénotypes caractéristiques d'une inhibition du transport polaire de l'auxine« PAT»(différenciation d'inflorescence de type «PIN», perte de dominance apicale, fusion des feuilles et inhibition du développement racinaire ... ). Ces phénotypes sont corrélés à l'accumulation de méthylène-cycloartanol-4-carboxy-4-méthyl (MCCM), un« SBI »qui inhibe de façon spécifique le« PAT». Ces données mettent en évidence un nouveau type d'interaction entre l'auxine et les stérols
Isoprenoids represent important cell constituents synthesized in many living organisms. ln plants, isoprenoid biogenesis occurs in three compartments : plastids, the endoplasmic reticulum-cytosol and mitochondrie.We focused on the molecular and functional studies of the role of Iwo cytosolic isoprenoids ( dolichol andsterol) in the development of plants. The key Io our strategy is the targeted silencing of specific Arabidopsis genes using the RNAi technology (knockdown) and the identification of T-DNA insertion mutants (knockout). ln the first chapter, we show that isoprenoids are involved indirectly in protein N-glycosylation via Dolichol P-Mannose derived from dolichol phosphate mannose synthase (DPMS). We demonstrate that plant DPMSis organized as a heteromeric enzyme complex localized in the endoplasmic reticulum (ER) and consists of DPMS1 acting as the catalytic core and two interacting subunits DPMS2 and DPMS3. The DPMS1-RNAiand dpms1 lines display an altered N-glycosylation pattern and exhibit extensive chlorosis, strong inhibition of root growth and hypersensitivity to ammonium. These phenotypic defects are associated with an «unfolded protein response» in the ER. These data demonstrate that the DPMS genes are essential for the protein N-glycome and plant development. ln the second chapter, we focused on the potentiel roles of sterol biosynthetic intermediates (SBls) in plant development using ERG28 protein, a component of the sterol C-4 demethylation (SC4DM) complex, as a target. We demonstrate that ERG28 is localized in ER and tethers 3 enzymes, sterol 4alpha-methyl oxidase, 4alpha carboxysterol-C3-dehydrogenase/C4- decarboxylase and sterone ketoreductase. We show that the Arabidopsis ERG28-RNAi and erg28 lines develop the hallmarks of altered polar auxin transport (PAT) including the differentiation of pin-like inflorescences, the loss of apical dominance, leaf fusion and inhibition root growth. The observed phenotypes correlate with the accumulation of methylene-cycloartanol-4-carboxy-4-methyl, a cryptic SBI. Our data provide a new level of interaction between sterols and auxin

The project investigated the molecular genetic and biochemical basis of pollination-induced senescence of Phalaenopsis flowers. This experimental system offered unique advantages in that senescence is strictly regulated by pollination, providing the basis to experimentally initiate and synchronize senescence in populations of flowers. The postpollination syndrome in the Phalaenopsis orchid system was dissected by investigating the temporal and spatial regulation of ACC synthase gene expression. In the stigma, pollen-borne auxin induces the expression of the auxin-regulated ACC synthase (PS-ACS2) gene, resulting in ACC synthesis within 1 h following pollination. Newly formed ACC is oxidized by basal constitutive ACC oxidase to ethylene, which then induces the expression of the ethylene-regulated ACC synthase(PS-ACS1) and oxidase (ACO1) genes for further autocatalytic production of ethylene. It is speculated that during the 6-h period following pollination, emasculation leads to the production or release of a sensitivity factor that sensitizes the cells of the stigma to ethylene. ACC and ethylene molecules are translocated from the stigma to the labellum and perianth where ethylene induces the expression of PS-ACS1 and ACO1 resulting in an increased production of ACC and ethylene. Organ-localized ethylene is responsible for inrolling and senescence of the labellum and perianth. The regulation of ethylene sensitivity and signal transduction events in pollinated flowers was also investigated. The increase in ethylene sensitivity appeared in both the flower column and the perianth, and was detected as early as 4 h after pollination. The increase in ethylene sensitivity following pollination was not dependent on endogenous ethylene production. Application of linoleic and linoleic acids to Phalaenopsis and Dendrobium flowers enhanced their senescence and promoted ethylene production. Several major lipoxygenase pathway products including JA-ME, traumatic acid, trans-2-hexenal and cis-3-hexenol, also enhanced flower senescence. However, lipoxygenase appears to not be directly involved in the endogenous regulation of pollination-induced Phalaenopsis and Dendrobium flower senescence. The data suggest that short-chain saturated fatty acids may be the ethylene "sensitivity factors" produced following pollination, and that their mode of action involves a decrease in the order of specific regions i the membrane lipid bilayer, consequently altering ethylene action. Examination of potential signal transduction intermediates indicate a direct involvement of GTP-binding proteins, calcium ions and protein phosphorylation in the cellular signal transduction response to ethylene following pollination. Modulations of cytosolic calcium levels allowed us to modify the flowers responsiveness to ethylene.

Introduction Previous research had suggested that benzoxazinoids, a class of defensive metabolites found in maize, wheat, rye, and wild barley, are not only direct insect deterrents, but also influence other areas of plant metabolism. In particular, the benzoxazinoid 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxa- zin-3(4H)- one (DIMBOA) was implicated in: (i) altering plant growth by interfering with auxin signaling, and (ii) leading to the induction of gene expression changes and secondary plant defense responses. The overall goal of this proposal was to identify mechanisms by which benzoxazinoids influence other aspects of plant growth and defense. Specifically, the following hypotheses were proposed to be tested as part of an approved BARD proposal: Benzoxazinoid breakdown products directly interfere with auxin perception Global changes in maize and barley gene expression are induced by benzoxazinoid activation. There is natural variation in the maize photomorphogenic response to benzoxazinoids. Although the initial proposal included experiments with both maize and barley, there were some technical difficulties with the proposed transgenic barley experiments and most of the experimental results were generated with maize. Summary of major findings Previous research by other labs, involving both maize and other plant species, had suggested that DIMBOA alters plant growth by interfering with auxin signaling. However, experiments conducted in both the Chamovitz and the Jander labs using Arabidopsis and maize, respectively, were unable to confirm previously published reports of exogenously added DIMBOA effects on auxin signaling. Nevertheless, analysis of bx1 and bx2 maize mutant lines, which have almost no detectable benzoxazinoids, showed altered responses to blue light signaling. Transcriptomic analysis of maize mutant lines, variation in inbred lines, and responses to exogenously added DIMBOA showed alteration in the transcription of a blue light receptor, which is required for plant growth responses. This finding provides a novel mechanistic explanation of the trade-off between growth and defense that is often observed in plants. Experiments by the Jander lab and others had demonstrated that DIMBOA not only has direct toxicity against insect pests and microbial pathogens, but also induces the formation of callose in both maize and wheat. In the current project, non-targeted metabolomic assays of wildtype maize and mutants with defects in benzoxazinoid biosynthesis were used to identify unrelated metabolites that are regulated in a benzoxazinoid-dependent manner. Further investigation identified a subset of these DIMBOA-responsive compounds as catechol, as well as its glycosylated and acetylated derivatives. Analysis of co-expression data identified indole-3-glycerol phosphate synthase (IGPS) as a possible regulator of benzoxazinoid biosynthesis in maize. In the current project, enzymatic activity of three predicted maize IGPS genes was confirmed by heterologous expression. Transposon knockout mutations confirmed the function of the maize genes in benzoxazinoid biosynthesis. Sub-cellular localization studies showed that the three maize IGPS proteins are co-localized in the plastids, together with BX1 and BX2, two previously known enzymes of the benzoxazinoid biosynthesis pathway. Implications Benzoxazinoids are among the most abundant and effective defensive metabolites in maize, wheat, and rye. Although there is considerable with-in species variation in benzoxazinoid content, very little is known about the regulation of this variation and the specific effects on plant growth and defense. The results of this research provide further insight into the complex functions of maize benzoxazinoids, which are not only toxic to pests and pathogens, but also regulate plant growth and other defense responses. Knowledge gained through the current project will make it possible to engineer benzoxazinoid biosynthesis in a more targeted manner to produce pest-tolerant crops without negative effects on growth and yield.