Characterization of harpin<Subscript>Xoo</Subscript> induced hypersensitive responses in non host plant,tobacco

Harpin proteins encoded by hrp genes are bacterial protein elicitors that can stimulate hypersensitive response (HR) in non-host plants. HR-related pathogen resistance involves a complex form of programmed cell death (PCD). It is increasingly viewed as a key component of the hypersensitive disease response of plants. Currently, the evidence of harpin proteins-induced PCD is deficient though it exhibits phenotypic parallels with HR, and the mechanism of harpin proteins-induced PCD is not well understood. In this study, we demonstrate that harpin_Xoo protein from Xanthomonas oryzae pv. oryzae of rice bacterial blight expressed and isolated from bacterial cells acted as an agent to induce PCD in infiltrated tobacco plants. Treatment of tobacco leaves with harpin_Xoo induced typical PCD-related morphological and biochemical changes including cell shrinkage and nuclear DNA degradation. We further analyzed the expression of several genes in signal transduction pathway of PCD in tobacco plants by real-time qRT-PCR analysis using EF-1α as an endogenous control. Our results showed that the expression of NtDAD1 was down-regulated and the expression of BI-1, tpa1 and aox1 was up-regulated following the infiltration of harpin_Xoo into tobacco leaves. Our data suggest that harpin_Xoo can induce PCD with the coordination of PCD-related genes in infiltrated tobacco leaves, providing evidence to further investigate the signal transduction pathways of HR and PCD.     

Establishment of somaclonal variants of Robusta coffee with reduced levels of cafestol and kahweol

Cafestol (caf) and kahweol (kah) are two diterpenes uniquely associated with the unsaponified lipid fraction of coffee brew and are reported to be responsible for an increase in serum cholesterol and triglyceride levels. The plant growth regulators (PGRs) indole-3-acetic acid (IAA), N ⁶-benzyladenine (BA), and thidiazuron (TDZ); the plant growth-promoting agents silver nitrate, triacontanol (TRIA), and coconut water; and some PGR antagonists such as lovastatin, paclobutrazol, and 2,3,5-triiodobenzoic acid (TIBA) were used to determine the variation of caf and kah in somatic embryos of Robusta coffee (Coffea canephora, CxR variety). Embryogenic (EG) medium was comprised of half-strength Murashige and Skoog basal components (½MS) supplemented with 2.85 μM IAA and 1.11 μM BA. After an 8-wk culture, somatic embryos were subjected to diterpene extraction and HPLC analysis of caf and kah profiles. TRIA-supplemented (5 μg L^?1) EG medium devoid of IAA reduced the levels of caf and kah by 18–24 and 48–55%, respectively, in coffee somatic embryos. Similarly, the combination of 2.85 μM IAA, 2.27 μM TDZ, and 5–10% coconut water in ½MS basal medium drastically reduced the caf and kah levels in somatic embryos. There was 60–75% reduction in both caf and kah in the presence of 5 μM TIBA, followed by 56–62% reduction in the presence of 10 μM silver nitrate. In contrast, there was 25–32% elevation of caf and kah in EG medium supplemented with 5 μM paclobutrazol. In this study, for the first time, somaclonal variants of C. canephora with reduced levels of diterpenes caf and kah were established. Furthermore, these lines exhibited consistency in their metabolite profiles when cultivated under greenhouse conditions. In-depth investigations at physiological level are warranted in order to elucidate the actual mechanism of these PGR inhibitors on alterations in endogenous pools of diterpenes in coffee somatic embryos.     

TIBA affects the induction of direct somatic embryogenesis from leaf explants of Oncidium

To study the effect of auxin on direct somatic embryogenesis from leaf cultures ofOncidium `Gower Ramsey', 1-cm-long explants have been cultured in vitro testing IAA, 2,4-, quercetin, TIBA and PCIB. On a modified MS medium devoid of plant growth regulators, leaf cells of three regions (leaf tips, adaxial sides and cut ends) formed somatic embryos. After 8 weeks in culture, the frequencies of embryo-forming explants were 55, 52.5 and 30 % on leaf tips, adaxial sides and cut ends, respectively, and the numbers of embryos per dish was 89.3. Except for TIBA, other growth regulators (IAA, 2,4-, quercetin, PCIB) and their combinations tested, all retarded direct embryo formation. In the presence of 0.1 and 0.5 μM TIBA, leaf tip, adaxial sides and cuts end of explants gave almost the same embryogenic response as the control. However, 10 and 27.5 % of explants were induced to form embryos from abaxial sides, and these explants did not form embryos on cut ends. In addition, after 8?weeks in culture, TIBA at 0.5?μM highly promoted the mean numbers of embryos per dish to 134.2.     

Identification of dehydrocostus lactone and 4-hydroxy-β-thujone as auxin polar transport inhibitors

The survey of naturally occurring of auxin polar transport regulators in Asteraceae was investigated using the radish (Raphanus sativus L.) hypocotyl bioassay established in this study. Significant auxin polar transport was observed when radiolabeled indole-3-acetic acid (IAA) was applied at the apical side of radish hypocotyl segments, but not when it was applied at the basal side of the segments. Almost no auxin polar transport was observed in radish hypocotyl segments treated with synthetic auxin polar transport inhibitors of N-(1-naphthyl)phthalamic acid (NPA) and 9-hydroxyfluorene-9-carboxylic acid (HFCA) at 0.5 μg/plant. 2,3,5-Triiodobenzoic acid (TIBA) at 0.5 μg/plant was less effective than NPA and HFCA, and p-chlorophenoxyisobutyric acid (PCIB) at 0.5 μg/plant had almost no effect on auxin polar transport in the radish hypocotyl bioassay. These results strongly suggest that the radish hypocotyl bioassay is suitable for the detection of bioassay-derived auxin polar transport regulators. Using the radish hypocotyl bioassay and physicochemical analyses, dehydrocostus lactone (decahydro-3,6,9-tris-methylene-azulenol(4,5-b)furan-2(3H)-one) and 4-hydroxy-β-thujone (4-hydroxy-4-methyl-1-(1-methylethyl)-bicyclo[3.1.0]hexan-3-one) were successfully identified as auxin polar transport inhibitors from Saussurea costus and Arctium lappa, and Artemisia absinthium, respectively. About 50 and 40 % inhibitions of auxin polar transport in radish hypocotyl segments were observed at 2.5 μg/plant pre-treatment (see “Materials and methods”) of dehydrocostus lactone and 4-hydroxy-β-thujone, respectively. Although the mode of action of these compounds in inhibiting auxin polar transport has not been clear yet, their possible mechanisms are discussed.     

Osmotic stress stimulates shoot organogenesis in callus of rice (Oryza sativa L.) via auxin signaling and carbohydrate metabolism regulation

This study aimed to clarify the possible mechanism of endogenous phytohormone signaling and carbohydrate metabolism during shoot organogenesis induced by osmotic stress in rice (Oryza sativa L. cv. Tainung 71) callus. Non-regenerable calli derived from Tainung 71 immature embryos were inoculated on Murashige and Skoog medium containing 10 μM 2,4-D. They turned to highly regenerable calli (HRC) (regeneration frequency more than 75 %) with lower calli fresh weight and water content when 0.6 M sorbitol was supplemented into the medium. The regeneration frequency was prominently decreased to 25 % while an auxin transport inhibitor, 2,3,5-triiodobenzoic acid (TIBA), was added into the sorbitol-treated medium. It suggested that endogenous auxin signal may be involved in the induction of HRC under osmotic stress treatment. As well, HRC showed high levels of glucose, sucrose, and starch and high expression of cell wall-bound invertase 1, sucrose transporter 1 (OsSUT1), OsSUT2, PIN-formed 1, and late embryogenesis abundant 1 (OsLEA1) genes. Their expressions are all dramatic inhibited except OsLEA1 under TIBA treatment. It suggests a key role of auxin may be linked to the effect of shoot regeneration under osmotic stress treatment. Therefore, we present a putative hypothesis for regenerable calli induction by osmotic stress treatment in rice. Osmotic stress may regulate endogenous levels of auxin interacting with abscisic acid, then affect carbohydrate metabolism to trigger callus initiation and further shoot regeneration in rice.     

Overexpressing CYP71Z2 Enhances Resistance to Bacterial Blight by Suppressing Auxin Biosynthesis in Rice

Background

The hormone auxin plays an important role not only in the growth and development of rice, but also in its defense responses. We’ve previously shown that the P450 gene CYP71Z2 enhances disease resistance to pathogens through regulation of phytoalexin biosynthesis in rice, though it remains unclear if auxin is involved in this process or not.

Methodology and Principal Findings

The expression of CYP71Z2 was induced by Xanthomonas oryzae pv. oryzae (Xoo) inoculation was analyzed by qRT-PCR, with GUS histochemical staining showing that CYP71Z2 expression was limited to roots, blades and nodes. Overexpression of CYP71Z2 in rice durably and stably increased resistance to Xoo, though no significant difference in disease resistance was detected between CYP71Z2-RNA interference (RNAi) rice and wild-type. Moreover, IAA concentration was determined using the HPLC/electrospray ionization/tandem mass spectrometry system. The accumulation of IAA was significantly reduced in CYP71Z2-overexpressing rice regardless of whether plants were inoculated or not, whereas it was unaffected in CYP71Z2-RNAi rice. Furthermore, the expression of genes related to IAA, expansin and SA/JA signaling pathways was suppressed in CYP71Z2-overexpressing rice with or without inoculation.

Conclusions and Significance

These results suggest that CYP71Z2-mediated resistance to Xoo may be via suppression of IAA signaling in rice. Our studies also provide comprehensive insight into molecular mechanism of resistance to Xoo mediated by IAA in rice. Moreover, an available approach for understanding the P450 gene functions in interaction between rice and pathogens has been provided.     

Bud emergence and shoot growth from mature citrus nodal stem segments

Bud emergence and shoot growth from adult phase citrus nodal cultures were studied using Citrus mitis (calamondin), Citrus paradisi (grapefruit), and Citrus sinensis (sweet orange). The effects of 6-benzyladenine (BA), indole 3-acetic acid (IAA), and citrus type on shoot quality and growth of mature bud explants from greenhouse grown trees were determined using a 2-component mixture-amount × citrus type experiment. BA increased shoot number and IAA improved shoot growth. The best shoot quality (fewer shoots but large shoots) was obtained with 1 μM IAA for calamondin, 15.5 μM IAA for sweet orange, and 30 μM IAA for grapefruit. Grapefruit exhibited substantial leaf abscission compared to calamondin and sweet orange. Four factors (AgNO₃, silver thiosulphate (STS), CaNO₃, or gelling) were screened individually for their efficacy in reducing leaf abscission. Five factors (AgNO₃, gelling, MS ion concentration, plant growth regulator and venting) were investigated to identify potential combinations for reducing leaf abscission and maximizing shoot growth and bud emergence. The factor combination identified as most effective in minimizing leaf drop, promoting shoot growth, and maximizing bud emergence for grapefruit was 2 mg l^?1 AgNO_3, Gelrite, 1 × MS ion concentration, 30 μM IAA, and vented.     

In vitro shoot growth, direct organogenesis and somatic embryogenesis promoted by silver nitrate in Coffea dewevrei

Direct differentiation of shoot buds in Coffea dewevrei was evident from the seedling shoots with collar region and also from collar region end of hypocotyl segments in presence of 40 μM AgNO₃, 8.88 μM of BA and 2.85 μM of IAA. Apart from this, shoot end of hypocotyl explants mainly supported yellow friable callus or somatic embryos. Subsequent transfer to the same medium induced secondary somatic embryogenesis. The collar region of the hypocotyl explants not only showed direct organogenesis by producing 1–3 shoots per explant and also able to produce globular somatic embryos and embryogenic yellow friable callus. Similarly direct somatic embryogenesis along with yellow friable embryogenic callus formation on 1/2 strength MS medium comprising 1.47 μM IAA, 2.22 μM BA and 40 μM AgNO₃ was noticed from cut portion of in vitro leaf and stalk of regenerated plants. The microshoots rooted well upon subculturing onto the same medium in 6 weeks and showed 60 % survival in green house and resumed growth upon hardening.     

Hormone-Induced Gene Expression During Gravicurvature of <Emphasis Type="Italic">Brassica</Emphasis> Roots

To investigate the spatial and temporal dependence of hormonal regulation during gravitropism, we compared the effects of root cap application of indole-3-acetic acid (IAA) and abscisic acid (ABA) with gene expression changes occurring naturally during gravitropic reaction of Brassica rapa roots. The expression of auxin, ABA, and metabolism-related genes in the tip, elongation zone, and maturation zone varied with time, location, and hormone concentration and characterized polar auxin transport. IAA was transported readily shootward and inhibited growth more than ABA. Expression of PIN3 and IAA5 in the elongation zone showed downregulation on the convex but upregulation on the concave side. Both PIN7 and IAA5 responded near maximally to 10^?8 M IAA within 30 min, suggesting that auxin activates its own transport system. Ubiquitin 1 (UBQ1) responded after a lag time of more than 1 h to IAA. The metabolic control gene Phosphoenolpyruvate carboxylase 1 (PEPC1) was more sensitive to ABA but upregulated by high concentrations of either hormone. The time course and duration of gene activation suggests that ABA is not involved in gravitropic curvature, differential elongation is not simply explained by IAA-induced upregulation, and that reference genes are sensitive to auxin.     

Influence of 2,3,5-triiodobenzoic acid on the transport and metabolism of IAA in lupin hypocotyls

The influence of 2,3,5-triiodobenzoic acid (TIBA) on the transport and metabolism of indolyl-3-acetic acid (IAA) was studied in etiolated lupin (Lupinus albus L) hypocotyls. Double isotope-labeled IAA [(5-³H)-IAA plus (1-¹⁴C)-IAA] was applied to the cut surface of decapitated seedlings. This confirmed that the species mobilized was unaltered IAA and permitted us to measure the in vivo decarboxylation of applied IAA. A pretreatment with TIBA applied to the cut surface produced a partial or drastic inhibition in the basipetal IAA movement at 0.5 or 100 M, respectively. Since TIBA inhibits auxin polar transport by interfering with the efflux carrier, the above results suggest that 100 M TIBA is sufficient to saturate the binding sites in the transporting cells. Compared to the control plants, in vivo decarboxylation of IAA was enhanced in 0.5 M TIBA-treated plants, while no decarboxylation was detected after treatment with 100 M TIBA. The in vitro decarboxylation of (1-¹⁴C)-IAA catalyzed by purified peroxidase was moderately activated by 100 M and unaffected by 0.5 M TIBA. The paradoxical effect of TIBA in vivo vs in vitro assays suggests that the in vivo effect of TIBA on IAA oxidation might be the consequence of the action of TIBA on the auxin transport system. Thus, transport reduction by 0.5 M TIBA caused a temporary accumulation of IAA in that apical region of the hypocotyl which has the highest capacity to decarboxylate IAA. In the presence of 100 M TIBA, a concentration which presumably saturates the efflux carriers, most of the added IAA can be expected to be located in the transporting cells where, according to the present data, IAA decarboxylation cannot take place.     

Optimization of <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of shoot tip explants of green gram (<Emphasis Type="Italic">Vigna radiata</Emphasis> (L.) Wilczek)

Shoot tip explants prepared from seedlings of ML-267 genotype of green gram were inoculated on MSB5 medium supplemented with BAP (0–20 μM) individually or in combination with minimal concentration of auxins (NAA/IAA/IBA) for adventitious shoots formation. BAP alone without auxins was observed to be efficient in multiple shoot induction and optimum shoot proliferation was achieved on MSB5 medium containing 10 μM BAP with 100?% shoot induction frequency. 3-day-old explants gave best shoot multiplication response and the mean shoot number decreased significantly in 4-day and 5-day-old explants. The induced shoots rooted profusely on ½ MSB5?+?2.46 µM IBA and about 90?% of the plantlets survived after acclimatization and set seed normally. Shoot tip explants infected with A.tumefaciens (LBA4404) harboring pCAMBIA 2301?+?AnnBj1 recombinant vector. Various factors which influence the competence of transformation were optimized based on the frequency of transient GUS expression in shoot tip explants. Optimum levels of transient GUS expression were recorded at pre-culture of explants for 2 days, infection for 10 min with Agro-culture of 0.8 OD and co-cultivation for 3 days on co-cultivation medium containing 100 µM acetosyringone in dark at 23?°C. Putative transformed shoots were produced on selection medium (shoot inductionmedium with100 mg/l kanamycin and 250 mg/l cefotaxim). PCR analysis confirmed the presence of AnnBj1, nptII, and uidA genes in T₀ plants. Stable GUS activity was detected in flowers of T₀ plants and leaves of T₁ plants. PCR analysis of T₁ progeny revealed AnnBj1 gene segregated following a Mendelian segregation pattern.     

Effect of auxin on mesocotyl elongation of dark-grown maize under different seeding depths

In order to elucidate the physiological mechanism of maize mesocotyl elongation induced by auxin under different seeding depths, seeds of five maize inbred lines, including 3681-4 line tolerant to deep seeding, were treated with IAA and triiodobenzoic acid (TIBA) under seeding depths of 20 or 2 cm. Under deep seeding conditions, maize mesocotyls grew by 1.5–2.0 times faster than under shallow seeding conditions. IAA (10⁻⁶ to 10⁻⁴ M) applied to roots stimulated mesocotyl elongation only of 3681-4 line and only under deep seeding conditions. TIBA (10⁻⁵ and 10⁻⁴ M) applied to roots inhibited mesocotyl elongation in all lines, but only 3681-4 was sensitive to 10⁻⁶ M TIBA. IAA promoted only cell elongation, and TIBA inhibited both cell elongation and cell division. After IAA and TIBA treatments, endogenous IAA content changed in parallel with the mesocotyl growth rate under different seeding depths. Furthermore, ABP1 gene expression changed in parallel with the mesocotyl growth rate under deep seeding conditions. Therefore, deep seeding tolerance of 3681-4 line was achieved due to auxin-regulated rapid mesocotyl elongation.     

Effects of indole-3-acetic acid and auxin transport inhibitors on the style curvature of three Alpinia species (Zingiberaceae)

The effects of indole-3-acetic acid (IAA) and the auxin transport inhibitors 2, 3, 5-triiodobenzoic acid (TIBA) and 1-N-naphthylphthalamic acid (NPA) on the style curvature of Alpinia platychilus, A. blepharocalyx, and A. mutica were studied. Exogenous IAA stimulated the style curvature movement of the anaflexistylous morph (ana-morph) and cataflexistylous morph (cata-morph) of three Alpinia species in light, but had no effect in the dark. Treatment with auxin efflux inhibitors (NPA and TIBA) before flower opening did not affect the first curvature of the two morphs in darkness; however, the subsequent second movement of the ana-morph was enhanced by NPA or TIBA, while the second movement of the cata-morph was completely inhibited. After the first curvature, NPA and TIBA treatments at 06:00?hours (before significant illumination) and 11:00?hours (after the styles were illuminated for 4?h) increased the second curvature of the ana-morph, but significantly decreased that of the cata-morph. The effect at 06:00?hours was more significant than the effect at 11:00?hours. These results suggested that auxin and auxin transport affected the style curvature in a different way in the two morphs, and two morphs had distinct mechanisms for style movement at different times.     

Location of transported auxin in etiolated maize shoots using 5-azidoindole-3-acetic Acid

A study was undertaken using the photoaffinity labeling agent, tritiated 5-azidoindole-3-acetic acid ([³H],5-N₃IAA), to identify cells in the etiolated maize (Zea mays L.) shoot which transport auxin. Transport of [³H],5-N₃IAA was shown to be polar, inhibited by 2,3,5-triiodobenzoic acid (TIBA) and essentially freely mobile. There was no detectable radiodecomposition of [³H],5-N₃IAA within tissue kept in darkness for 4 hours. Shoot tissue which had taken up [³H],5-N₃IAA was irradiated with ultraviolet light to covalently fix the photoaffinity labeling agent within cells that contained it at the time of photolysis. Subsequent microautoradiography showed that all cells contained radioactivity; however, the amount of radioactivity varied among different cell types. Epidermal cells contained the most radioactivity per area, approximately twofold more than other cells. Parenchyma cells in the mature stelar region contained the next largest amount and cortical cells, sieve tube cells, tracheary cells, and all cells in the leaf base contained the least amount of the radioactive label. Two observations suggest that the auxin within the epidermal cells is transported in a polar manner: (a) the amount of auxin in the epidermal cells is greatly reduced in the presence of TIBA, and (b) auxin accumulates on the apical side of a wound in the epidermis and is absent on the basal side. While these results indicate that auxin in the epidermis is polarly transported, this tissue cannot be the only pathway since the epidermis is only a small fraction of the shoot volume. The greater than twofold difference between the concentration of auxin in the epidermal and subtending cells demonstrates that physiological differences in the concentration of auxin can occur between adjacent cells.     

Effect of Cyclanilide on Auxin Activity

Cyclanilide is a plant growth regulator that is registered for use in cotton at different stages of growth, to either suppress vegetative growth (in combination with mepiquat chloride) or accelerate senescence (enhance defoliation and boll opening, used in combination with ethephon). This research was conducted to study the mechanism of action of cyclanilide: its potential interaction with auxin (IAA) transport and signaling in plants. The activity of cyclanilide was compared with the activity of the auxin transport inhibitors NPA and TIBA. Movement of [³H]IAA was inhibited in etiolated corn coleoptiles by 10 μM cyclanilide, NPA, and TIBA, which demonstrated that cyclanilide affected polar auxin transport. Although NPA inhibited [³H]IAA efflux from cells in etiolated zucchini hypocotyls, cyclanilide had no effect. NPA did not inhibit the influx of IAA into cells in etiolated zucchini hypocotyls, whereas cyclanilide inhibited uptake 25 and 31% at 10 and 100 μM, respectively. Also, NPA inhibited the gravitropic response in tomato roots (85% at 1 μM) more than cyclanilide (30% at 1 μM). Although NPA inhibited tomato root growth (30% at 1 μM), cyclanilide stimulated root growth (165% of control at 5 μM). To further characterize cyclanilide action, plasma membrane fractions from etiolated zucchini hypocotyls were obtained and the binding of NPA, IAA, and cyclanilide studied. Cyclanilide inhibited the binding of [³H]NPA and [³H]IAA with an IC₅₀ of 50 μM for both. NPA did not affect the binding of IAA, nor did IAA affect the binding of NPA. Kinetic analysis indicated that cyclanilide is a noncompetitive inhibitor of both NPA and IAA binding, with inhibition constants (K _i) of 40 and 2.3 μM, respectively. These data demonstrated that cyclanilide interacts with auxin-regulated processes via a mechanism that is distinct from other auxin transport inhibitors. This research identifies a possible mechanism of action for cyclanilide when used as a plant growth regulator.