Capping the hydroxyl groups (-OH) of α-cellulose to reduce Hy-groscopicity for accurate 18O/16O measurement by EA/Py/IRMS.

Obtaining an accurate measurement of 18O/16O at natural abundance level for land plants-derived α-cellulose with the currently popular EA/Py/IRMS (elemental analysis/pyrolysis/isotope ratio mass spectrometry) method is a challenge due to the hygroscopic nature of the exposed hydroxyl groups, as the 18O/16O of adsorbed moisture is usually different from that of the α-cellulose and the relative amount of adsorbed moisture is sample- and relative humidity-dependent. To minimize the hygroscopicity-related measurement error, we capped the hydroxyl groups of α-cellulose by benzylation to various degrees and found that the 18O/16O ratio of α-cellulose increased with the degree of benzyl substitution (DS), consistent with the theoretical prediction that a reduced presence of exposed hydroxyl groups should lead to a more accurate (and therefore more reliable) α-cellulose 18O/16O measurement. We propose the establishment of a moisture adsorption-degree of substitution or percentage of oxygen-18O/16O ratio equation, based on the measurement of C%, O% and δ18O of variably capped α-cellulose, so that a robust correction can be made in a plant species- and laboratory conditions-specific manner. Failure to do so will lead to an average underestimate of α-cellulose δ18O by 3.5 mUr under "average" laboratory conditions.

On the Chemical Purity and Oxygen Isotopic Composition of α-Cellulose Extractable from Higher Plants and the Implications for Climate, Metabolic, and Physiological Studies.

The 18O/16O ratio of α-cellulose in land plants has proved of interest for climate, environmental, physiological, and metabolic studies. Reliable application of such a ratio may be compromised by the presence of hemicellulose impurities in the α-cellulose product obtainable with current extraction methods, as the impurities are known to be isotopically different from that of the α-cellulose. We first compared the quality of hydrolysates of "α-cellulose products" obtained with four representative extraction methods (Jayme and Wise; Brendel; Zhou; Loader) and quantified the hemicellulose-derived non-glucose sugars in the α-cellulose products from 40 land grass species using gas chromatography-mass spectrometry (GC/MS). Second, we performed compound-specific isotope analysis of the hydrolysates using GC/Pyrolysis/IRMS. These results were then compared with the bulk isotope analysis using EA/Pyrolysis/IRMS of the α-cellulose products. We found that overall, the Zhou method afforded the highest purity α-cellulose as judged by the minimal presence of lignin and the second-lowest presence of non-glucose sugars. Isotopic analysis then showed that the O-2-O-6 of the α-cellulose glucosyl units were all depleted in 18O by 0.0-4.3 mUr (average, 1.9 mUr) in a species-dependent manner relative to the α-cellulose products. The positive isotopic bias of using the α-cellulose product instead of the glucosyl units stems mainly from the fact that the pentoses that dominate hemicellulose contamination in the α-cellulose product are relatively enriched in 18O (compared to hexoses) as they inherit only the relatively 18O-enriched O-2-O-5 moiety of sucrose, the common precursor of pentoses and hexoses in cellulose, and are further enriched in 18O by the (incomplete) hydrolysis.

Combining Heat Stress with Pre-Existing Drought Exacerbated the Effects on Chlorophyll Fluorescence Rise Kinetics in Four Contrasting Plant Species.

Although drought and high temperature are two main factors affecting crop productivity and forest vegetation dynamics in many areas worldwide, little work has been done to describe the effects of heat combined with pre-existing drought on photochemical function in diverse plant species. This study investigated the biophysical status of photosystem II (PSII) and its dynamic responses under 2-day heat stress during a 2-week drought by measuring the polyphasic chlorophyll fluorescence rise (OJIP) kinetics. This study examined four contrasting species: a C3 crop/grass (wheat), a C4 crop/grass (sorghum), a temperate tree species (Fraxinus chinensis) and a tropical tree species (Radermachera sinica). Principal component analysis showed that the combination of heat and drought deviated from the effect of heat or drought alone. For all four species, a linear mixed-effects model analysis of variance of the OJIP parameters showed that the deviation arose from decreased quantum yield and increased heat dissipation of PSII. The results confirmed, in four contrasting plant species, that heat stress, when combined with pre-existing drought, exacerbated the effects on PSII photochemistry. These findings provide direction to future research and applications of chlorophyll fluorescence rise OJIP kinetics in agriculture and forestry, for facing increasingly more severe intensity and duration of both heat and drought events under climate change.

One-Pot Extractive Transesterification of Fatty Acids Followed by DMOX Derivatization for Location of Double Bonds Using GC-EI-MS.

Fatty acids are an essential structural and energy storage component of cells and hence there is much interest in their metabolism, requiring identification and quantification with readily available instrumentation, such as GC-MS. Fatty acid methyl esters (FAMEs) can be generated and extracted directly from biological tissue, in a one-pot process, and following high resolution GC, their respective chain length, degrees of unsaturation, and other functionalities can be readily identified using EI-MS. Defining the positions of the double bonds in the alkyl chain requires conversion of the FAMEs into their respective dimethyloxazoline (DMOX) derivatives. Following EI, this derivative allows charge retention on the heterocycle, and concomitant charge remote fragmentation of the alkyl chain to yield key double bond position identifying ions. The protocols described herein have been applied to the identification and quantification of fatty acids harvested from microalgae grown to produce biofuels and to the screening of salt tolerant Arabidopsis mutants.

The effect of processing medium on the 2 H/1 H of carbon-bound hydrogen in α-cellulose extracted from higher plants.

RATIONALE: Although the 2 H/1 H ratio of the carbon-bound hydrogens (C-Hs) in α-cellulose extracted from higher plants has long been used successfully for climate, environmental and metabolic studies, the assumption that bleaching with acidified NaClO2 to remove lignin before pure α-cellulose can be obtained does not alter the 2 H/1 H ratio of α-cellulose C-Hs has nonetheless not been tested. METHODS: For reliable application of the 2 H/1 H ratio of α-cellulose C-H, we processed plant materials representing different phytochemistries and photosynthetic carbon assimilation modes in isotopically contrasting bleaching media (with an isotopic difference of 273 mUr). All the isotope ratios were measured by elemental analyzer/isotope ratio mass spectrometry (EA/IRMS). RESULTS: Our results show that H from the bleaching medium does appear in the final pure α-cellulose product, although the isotopic alteration to the C-H in α-cellulose due to the incorporation of processing H from the medium is small if isotopically "natural" water is used to prepare the processing medium. However, under prolonged bleaching such an isotope effect can be significant, implying that standardizing the bleaching process is necessary for reliable 2 H/1 H measurement. CONCLUSIONS: The currently adopted method for removing lignin for α-cellulose extraction from higher plant materials with acidified NaClO2 bleaching is considered acceptable in terms of preserving the isotopic fidelity if isotopically "natural" water is used to prepare the bleaching solution.

Headspace solid-phase microextraction coupled to gas chromatography-mass spectrometry with selected ion monitoring for the determination of four food flavoring compounds and its application in identifying artificially scented rice.

The adulteration of rice using synthetic aromatic flavorings to fraudulently imitate commercially valuable fragrant rice varieties has attracted extensive attention from regulatory authorities around the world. In order to get convincing evidence of adulteration, appropriate scientific analytical methods need to be developed. In this study, a simple and efficient headspace solid-phase microextraction (HS SPME) technique coupled with gas chromatography mass spectrometry using selected ion monitoring (GC-MS-SIM) for the determination of four food flavoring compounds which are possibly used as adulterants is proposed. The HS SPME operating under optimized conditions increased the selectivity and sensitivity of the analysis by eliminating matrix interferences. The method presented adequate precision and linearity with limits of detection ranging from 0.5 to 10 ng/mL. This HS SPME/GC-MS-SIM method is directly applicable to the analysis of volatiles in rice and has the advantages of minimal pretreatment. It was applied successfully to the analysis of six rice flavoring essences, ten fragrant rice and four artificially scented rice samples.

Calcium homeostasis during hibernation and in mechanical environments disrupting calcium homeostasis.

To maintain calcium homeostasis during physical inactivity, precise coordination is necessary between different organs of the body. There are a number of factors which alter an organism's calcium balance, such as growth, aging, physical inactivity and acquired or inherited disorders which ultimately lead to bone loss. In non-hibernating mammals, physical inactivity causes bone loss which may not be completely recoverable during the lifespan of an individual despite a resumption of activity. Extreme physical inactivity and nutritional deprivation are two other important factors that lead to bone loss in non-hibernating mammals. The mechanism of bone loss is still poorly understood, however, there is some evidence which shows that during hibernation, smaller mammals (ground squirrels, bats, and hamsters) undergo bone loss. While on the other hand, hibernating bears do not show any sign of bone loss and retain their bone structure and strength. This may be due to differences in their hibernation patterns, as smaller mammals may excrete calcium throughout the hibernation period, which ultimately leads to bone loss, whereas bears seem to have a more developed and advanced mechanism to prevent calcium loss and maintain their bone structure. In this review, we summarize calcium homeostasis and its adaptive mechanisms with reference to bone loss in hibernating as compared to non-hibernating mammals. We also review the effect of microgravity and simulated microgravity on bone physiology and subsequent adaptation.

ERECTA receptor-kinases play a key role in the appropriate timing of seed germination under changing salinity.

Appropriate timing of seed germination is crucial for the survival and propagation of plants, and for crop yield, especially in environments prone to salinity or drought. However, the exact mechanisms by which seeds perceive changes in soil conditions and integrate them to trigger germination remain elusive, especially once the seeds are non-dormant. In this study, we determined that the Arabidopsis ERECTA (ER), ERECTA-LIKE1 (ERL1), and ERECTA-LIKE2 (ERL2) leucine-rich-repeat receptor-like kinases regulate seed germination and its sensitivity to changes in salt and osmotic stress levels. Loss of ER alone, or in combination with ERL1 and/or ERL2, slows down the initiation of germination and its progression to completion, or arrests it altogether under saline conditions, until better conditions return. This function is maternally controlled via the tissues surrounding the embryo, with a primary role being played by the properties of the seed coat and its mucilage. These relate to both seed-coat expansion and subsequent differentiation and to salinity-dependent interactions between the mucilage, subtending seed coat layers and seed interior in the germinating seed. Salt-hypersensitive er105, er105 erl1.2, er105 erl2.1 and triple-mutant seeds also exhibit increased sensitivity to exogenous ABA during germination, and under salinity show an enhanced up-regulation of the germination repressors and inducers of dormancy ABA-insensitive-3, ABA-insensitive-5, DELLA-encoding RGL2, and Delay-Of-Germination-1. These findings reveal a novel role of the ERECTA receptor-kinases in the sensing of conditions at the seed surface and the integration of developmental, dormancy and stress signalling pathways in seeds. They also open novel avenues for the genetic improvement of plant adaptation to changing drought and salinity patterns.

Occurrence and distribution of unsubstituted B-ring flavanones in Eucalyptus foliage.

A group of plant specialised metabolites (PSMs) collectively known as unsubstituted B-ring flavanones (UBFs) have previously been found in the foliage of some species from the genus Eucalyptus L'Hér. (Myrtaceae), specifically from the subgenus Eucalyptus (monocalypts). Captive feeding studies using artificial diets suggest that these compounds may potentially influence the feeding preferences of marsupial folivores, such as koalas. Understanding natural variation in the composition and concentration of UBFs in eucalypt foliage is a first step to deciding whether, through their effects on herbivory, they might have broader effects on ecosystem dynamics. We used ESI-LCMS/MS and HPLC to characterise and quantify UBFs in 351 individual trees from 25 monocalypt species. We found large variation in the total UBF concentration both between and within species. For example, the mean concentration of UBFs in Eucalyptus muelleriana was 0.2 mg g-1 dry wt, whereas it was 105.7 mg g-1 dry wt, with a range of 78.2-141.3 mg g-1 dry wt, in Eucalyptus mediocris. Different eucalypt species contained different subsets of ten UBFs, and three species showed potential chemotypic variation between individuals within species. Our results suggest that UBFs naturally vary between monocalypt species and individuals at concentrations that could realistically be expected to affect the feeding dynamics of marsupial eucalypt folivores. UBFs could be measured relatively rapidly and cheaply in future studies using near-infrared reflectance (NIR) spectroscopy, as we were able to successfully predict the total UBF concentration of samples from their NIR spectra, with an r2 value of 0.98 and a standard error of prediction (SEP) of 6.07. This work further solidifies NIR spectroscopy as a powerful tool enabling ecologists to analyse the chemical composition of large numbers of samples.

The Synthesis of ³H-Labelled 8-Azido-N6-Benzyladenine and Related Compounds for Photoaffinity Labelling of Cytokinin-Binding Proteins.

The biology of the group of plant hormones termed cytokinins is reviewed to reveal areas where further studies of cytokinin-binding proteins could be significant. Such areas include: inhibition of human tumour cell growth by cytokinin ribosides, the role of cytokinins in the development of diverse micro-organisms including the cyanobacteria and Mycobacterium tuberculosis, the very rapid responses of plant cells to exogenous cytokinins, and other aspects of cytokinin plant biology. Photoaffinity labelling (PAL) coupled to the recent advances in HPLC of proteins and mass spectral analysis and sequencing of proteins, may have relevance to these areas. To facilitate PAL, we present experimental details for two methods for synthesis of 8-azido-N6-benzyladenine, which has the azido affinity group in the preferred position of the purine ring. Synthesis from [2-³H]adenosine yielded the above-mentioned PAL reagent with ³H in the purine ring and also gave labelled 9-riboside and 8-azido-N6,9-dibenzyladenine. 8-Azido-N6-benzyladenine was also prepared from 6,8-dichloropurine by a facile synthesis, which would allow a label to be sited in the benzyl group where substituents can also be introduced to vary cytokinin activity. The use of inactive cytokinin analogues in assessing the significance of PAL is discussed.

Novel Position-Specific 18O/16O Measurement of Carbohydrates. I. O-3 of Glucose and Confirmation of 18O/16O Heterogeneity at Natural Abundance Levels in Glucose from Starch in a C4 Plant.

The 18O/16O ratio at both molecular and positional levels in the carbohydrates of higher plants is a reliable proxy for the plant growth environment, and a potential indicator of the plant photosynthetic carbon assimilation mode, and its physiological, biochemical and metabolic status. The lack of exploitable nuclear resonance in 18O and 16O and the extremely low 17O abundance make the NMR-based PSIA (position-specific isotopic analysis) a significant challenge. In this Article, an alternative three-step wet chemistry based method for accessing the 18O/16O of glucose O-3 is presented. The O atoms (OH groups) at positions 1, 2, 5, and 6 were first protected by acetonation (converting glucose to 1,2;5,6-di- O-isopropylidene-glucofuranose). The protected glucose was then esterified at O-3 by thionoformylation. Subsequent Barton-McCombie deoxygenation quantitatively removed the O-3 from the protected sugar. Mass balance was then applied to calculate the 18O/16O of O-3 using the isotopic values of the protected sugar before and after the deoxygenation step. The method is innovative in that (i) isolation and purification of individual compounds for 18O by EA/Pyrolysis/IRMS analysis is unnecessary as the reaction mixture can be analyzed on a GC/Pyrolysis/IRMS; (ii) sample quantity is dramatically reduced; and (iii) the approach to access the O-3 isotopic signal can be easily expanded to other positions within glucose and other sugars. It was shown that O-3 is enriched by 12 mUr relative to the molecular average (O-2-O-6) for a glucose of C4 photosynthetic origin. We highlighted the potential applications of the intramolecular O isotopic heterogeneity of glucose this method revealed.

Promotion of mammalian angiogenesis by neolignans derived from soybean extracellular fluids.

Excessive or insufficient angiogenesis is associated with major classes of chronic disease. Although less studied, small molecules which can promote angiogenesis are being sought as potential therapeutics for cardiovascular and peripheral arterial disease and stroke. Here we describe a bioassay-directed discovery approach utilising size exclusion and liquid chromatography to purify components of soybean xylem sap that have pro-angiogenic activity. Using high resolution accurate mass spectrometry and nuclear magnetic resonance spectroscopy, the structure of two pro-angiogenic molecules (FK1 and FK2) were identified as erythro-guaiacylglycerol-8-O-4'-(coniferyl alcohol) ether (eGGCE), and threo-guaiacylglycerol-8-O-4'-(coniferyl alcohol) ether (tGGCE). These two molecules, which are coniferyl neolignan stereoisomers, promoted in vitro angiogenesis in the µM to nM range. Independently sourced samples of eGGCE and tGGCE exhibited comparable pro-angiogenic activity to the soybean derived molecules. The cellular mode of action of these molecules was investigated by studying their effect on endothelial cell proliferation, migration, tube formation and adhesion to the extracellular matrix (ECM) components, fibronectin and vitronectin. They were found to enhance endothelial cell proliferation and endothelial cell tube formation on Matrigel, but did not affect endothelial cell migration or adhesion to fibronectin and vitronectin. Thus, this study has identified two coniferyl neolignan stereoisomers, eGGCE and tGGCE, as pro-angiogenic molecules, with eGGCE being less active than tGGCE.

Plasticity of photosynthetic heat tolerance in plants adapted to thermally contrasting biomes.

In many biomes, plants are subject to heatwaves, potentially causing irreversible damage to the photosynthetic apparatus. Field surveys have documented global, temperature-dependent patterns in photosynthetic heat tolerance (PHT ); however, it remains unclear if these patterns reflect acclimation in PHT or inherent differences among species adapted to contrasting habitats. To address these unknowns, we quantified seasonal variations in Tcrit (high temperature where minimal chlorophyll-a fluorescence rises rapidly, reflecting disruption to photosystem II) in 62 species native to 6 sites from 5 thermally contrasting biomes across Australia. Tcrit and leaf fatty acid (FA) composition (important for membrane stability) were quantified in three temperature-controlled glasshouses in 20 of those species. Tcrit was greatest at hot field sites and acclimated seasonally (summer > winter, increasing on average 0.34 °C per °C increase in growth temperature). The glasshouse study showed that Tcrit was inherently higher in species from warmer habitats (increasing 0.16 °C per °C increase in origin annual mean maximum temperature) and acclimated to increasing growth temperature (0.24 °C °C-1 ). Variations in Tcrit were positively correlated with the relative abundance of saturated FAs, with FAs accounting for 40% of Tcrit variation. These results highlight the importance of both plastic adjustments and inherent differences determining contemporary continent-wide patterns in PHT .

On the contributions of photorespiration and compartmentation to the contrasting intramolecular 2H profiles of C3 and C4 plant sugars.

Compartmentation of C4 photosynthetic biochemistry into bundle sheath (BS) and mesophyll (M) cells, and photorespiration in C3 plants is predicted to have hydrogen isotopic consequences for metabolites at both molecular and site-specific levels. Molecular-level evidence was recently reported (Zhou et al., 2016), but evidence at the site-specific level is still lacking. We propose that such evidence exists in the contrasting 2H distribution profiles of glucose samples from naturally grown C3, C4 and CAM plants: photorespiration contributes to the relative 2H enrichment in H5 and relative 2H depletion in H1 & H6 (the average of the two pro-chiral Hs and in particular H6,pro-R) in C3 glucose, while 2H-enriched C3 mesophyll cellular (chloroplastic) water most likely contributes to the enrichment at H4; export of (transferable hydrogen atoms of) NADPH from C4 mesophyll cells to bundle sheath cells (via the malate shuttle) and incorporation of 2H-relatively unenriched BS cellular water contribute to the relative depletion of H4 & H5 respectively; shuttling of triose-phosphates (PGA: phosphoglycerate dand DHAP: dihydroacetone phosphate) between C4 bundle sheath and mesophyll cells contributes to the relative enrichment in H1 & H6 (in particular H6,pro-R) in C4 glucose.

Hydrogen isotopic differences between C3 and C4 land plant lipids: consequences of compartmentation in C4 photosynthetic chemistry and C3 photorespiration.

The 2 H/1 H ratio of carbon-bound H in biolipids holds potential for probing plant lipid biosynthesis and metabolism. The biochemical mechanism underlying the isotopic differences between lipids from C3 and C4 plants is still poorly understood. GC-pyrolysis-IRMS (gas chromatography-pyrolysis-isotope ratio mass spectrometry) measurement of the 2 H/1 H ratio of leaf lipids from controlled and field grown plants indicates that the biochemical isotopic fractionation (ε2 Hlipid_biochem ) differed between C3 and C4 plants in a pathway-dependent manner: ε2 HC4 > ε2 HC3 for the acetogenic pathway, ε2 HC4 < ε2 HC3 for the mevalonic acid pathway and the 1-deoxy-D-xylulose 5-phosphate pathway across all species examined. It is proposed that compartmentation of photosynthetic CO2 fixation into C4 mesophyll (M) and bundle sheath (BS) cells and suppression of photorespiration in C4 M and BS cells both result in C4 M chloroplastic pyruvate - the precursor for acetogenic pathway - being more depleted in 2 H relative to pyruvate in C3 cells. In addition, compartmentation in C4 plants also results in (i) the transferable H of NADPH being enriched in 2 H in C4 M chloroplasts compared with that in C3 chloroplasts for the 1-deoxy-D-xylulose 5-phosphate pathway pathway and (ii) pyruvate relatively 2 H-enriched being used for the mevalonic acid pathway in the cytosol of BS cells in comparison with that in C3 cells.

A role for jasmonates in the release of dormancy by cold stratification in wheat.

Hydration at low temperatures, commonly referred to as cold stratification, is widely used for releasing dormancy and triggering germination in a wide range of species including wheat. However, the molecular mechanism that underlies its effect on germination has largely remained unknown. Our previous studies showed that methyl-jasmonate, a derivative of jasmonic acid (JA), promotes dormancy release in wheat. In this study, we found that cold-stimulated germination of dormant grains correlated with a transient increase in JA content and expression of JA biosynthesis genes in the dormant embryos after transfer to 20 (o)C. The induction of JA production was dependent on the extent of cold imbibition and precedes germination. Blocking JA biosynthesis with acetylsalicylic acid (ASA) inhibited the cold-stimulated germination in a dose-dependent manner. In addition, we have explored the relationship between JA and abscisic acid (ABA), a well-known dormancy promoter, in cold regulation of dormancy. We found an inverse relationship between JA and ABA content in dormant wheat embryos following stratification. ABA content decreased rapidly in response to stratification, and the decrease was reversed by addition of ASA. Our results indicate that the action of JA on cold-stratified grains is mediated by suppression of two key ABA biosynthesis genes, TaNCED1 and TaNCED2.

Factors Altering Pyruvate Excretion in a Glycogen Storage Mutant of the Cyanobacterium, Synechococcus PCC7942.

Interest in the production of carbon commodities from photosynthetically fixed CO2 has focused attention on cyanobacteria as a target for metabolic engineering and pathway investigation. We investigated the redirection of carbon flux in the model cyanobacterial species, Synechococcus elongatus PCC 7942, under nitrogen deprivation, for optimized production of the industrially desirable compound, pyruvate. Under nitrogen limited conditions, excess carbon is naturally stored as the multi-branched polysaccharide, glycogen, but a block in glycogen synthesis, via knockout mutation in the gene encoding ADP-glucose pyrophosphorylase (glgC), results in the accumulation of the organic acids, pyruvate and 2-oxoglutarate, as overflow excretions into the extracellular media. The ΔglgC strain, under 48 h of N-deprivation was shown to excrete pyruvate for the first time in this strain. Additionally, by increasing culture pH, to pH 10, it was possible to substantially elevate excretion of pyruvate, suggesting the involvement of an unknown substrate/proton symporter for export. The ΔglgC mutant was also engineered to express foreign transporters for glucose and sucrose, and then grown photomixotrophically with exogenous organic carbon supply, as added 5 mM glucose or sucrose during N- deprivation. Under these conditions we observed a fourfold increase in extracellular pyruvate excretion when glucose was added, and a smaller increase with added sucrose. Although the magnitude of pyruvate excretion did not correlate with the capacity of the ΔglgC strain for bicarbonate-dependent photosynthetic O2 evolution, or with light intensity, there was, however, a positive correlation observed between the density of the starter culture prior to N-deprivation and the final extracellular pyruvate concentration. The factors that contribute to enhancement of pyruvate excretion are discussed, as well as consideration of whether the source of carbon for pyruvate excretion might be derived from photosynthetic CO2 fixation or from remobilisation of existing carbon stores.

Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula Cytokinin Perception Mutant cre1.

Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation.

Jasmonic acid is associated with resistance to twospotted spider mites in diploid cotton (Gossypium arboreum).

The twospotted spider mite (Tetranychus urticae Koch) is capable of dramatically reducing the yield of cotton crops and is often difficult and expensive to control. This study investigated and compared two important plant hormones, jasmonic acid (JA) and salicylic acid (SA), as constitutive and/or induced defence response components in a mite susceptible commercial cotton cultivar, Sicot 71 (Gossypium hirsutum L.) and a resistant diploid cotton BM13H (Gossypium arboreum L.). Foliar application of JA and methyl jasmonate (MeJA) reduced the mite population and leaf damage but application of other potential elicitors, SA and methyl salicylate (MeSA) did not. The concentrations of JA and SA in leaf tissues of induced and non-induced Sicot 71 and BM13H were quantified by liquid chromatography coupled to electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS). The JA content was constitutively higher in BM13H than Sicot 71 and also highly induced by mite infestation in BM13H but not in Sicot 71. However, SA was not significantly induced in either BM13H or Sicot 71. The expression levels of JA related genes, LOX, AOS and OPR were measured by quantitative PCR and elevated expression levels of JA related genes were detected in mite-infested BM13H. Therefore, JA and MeJA were implicated as key biochemical components in both the constitutive and induced defence responses of BM13H to spider mites.

Alteration of flavonoid accumulation patterns in transparent testa mutants disturbs auxin transport, gravity responses, and imparts long-term effects on root and shoot architecture.

Flavonoids have broad cross-kingdom biological activity. In Arabidopsis, flavonoid accumulation in specific tissues, notably the root elongation zone and root/shoot junction modulate auxin transport, affect root gravitropism, and influence overall plant architecture. The relative contribution made by aglycones and their glycosides remains undetermined, and the longer-term phenotypic effects of altered flavonoid accumulation are not fully assessed. We tested Arabidopsis thaliana mutants that accumulate different flavonoids to determine which flavonoids were causing these affects. Tandem mass spectrometry and in situ fluorescence localisation were used to determine the in vivo levels of aglycones in specific tissues of 11 transparent testa mutants. We measured rootward and shootward auxin transport, gravitropic responses, and identified the long-term changes to root and shoot architecture. Unexpected aglycone species accumulated in vivo in several flavonoid-pathway mutants, and lower aglycone levels occurred in transcription factor mutants. Mutants accumulating more quercetin and quercetin-glycosides changed the greatest in auxin transport, gravitropism, and aerial tissue growth. Early flavonoid-pathway mutants showed aberrant lateral root initiation patterns including clustered lateral root initiations at a single site. Transcription factor mutants had multiple phenotypes including shallow root systems. These results confirm that aglycones are present at very low levels, show that lateral root initiation is perturbed in early flavonoid-pathway mutants, and indicate that altered flavonoid accumulation affects multiple aspects of plant architecture.