Cold Induction of EARLI1, a Putative Arabidopsis Lipid Transfer Protein, Is Light and Calcium Dependent

As sessile organisms, plants must adapt to their environment. One approach toward understanding this adaptation is to investigate environmental regulation of gene expression. Our focus is on the environmental regulation of EARLI1, which is activated by cold and long-day photoperiods. Cold activation of EARLI1 in short-day photoperiods is slow, requiring several hours at 4ºC to detect an increase in mRNA abundance. EARLI1 is not efficiently cold-activated in etiolated seedlings, suggesting that photomorphogenesis is necessary for its cold activation. Cold activation of EARLI1 is inhibited in the presence of the calcium channel blocker lanthanum chloride or the calcium chelator EGTA. Addition of the calcium ionophore Bay K8644 results in cold-independent activation of EARLI1. These data suggest that EARLI1 is not an immediate target of the cold response, and that calcium flux affects its expression. EARLI1 is a putative secreted protein and has motifs found in lipid transfer proteins. Over-expression of EARLI1 in transgenic plants results in reduced electrolyte leakage during freezing damage, suggesting that EARLI1 may affect membrane or cell wall stability in response to low temperature stress

Jasmonate promotes auxin-induced adventitious rooting in dark-grown Arabidopsis thaliana seedlings and stem thin cell layers by a cross-talk with ethylene signalling and a modulation of xylogenesis

Background: Adventitious roots (ARs) are often necessary for plant survival, and essential for successful micropropagation. In Arabidopsis thaliana dark-grown seedlings AR-formation occurs from the hypocotyl and is enhanced by application of indole-3-butyric acid (IBA) combined with kinetin (Kin). The same IBA + Kin-treatment induces AR-formation in thin cell layers (TCLs). Auxin is the main inducer of AR-formation and xylogenesis in numerous species and experimental systems. Xylogenesis is competitive to AR-formation in Arabidopsis hypocotyls and TCLs. Jasmonates (JAs) negatively affect AR-formation in de-etiolated Arabidopsis seedlings, but positively affect both AR-formation and xylogenesis in tobacco dark-grown IBA + Kin TCLs. In Arabidopsis the interplay between JAs and auxin in AR-formation vs xylogenesis needs investigation. In de-etiolated Arabidopsis seedlings, the Auxin Response Factors ARF6 and ARF8 positively regulate AR-formation and ARF17 negatively affects the process, but their role in xylogenesis is unknown. The cross-talk between auxin and ethylene (ET) is also important for AR-formation and xylogenesis, occurring through EIN3/EIL1 signalling pathway. EIN3/EIL1 is the direct link for JA and ET-signalling. The research investigated JA role on AR-formation and xylogenesis in Arabidopsis dark-grown seedlings and TCLs, and the relationship with ET and auxin. The JA-donor methyl-jasmonate (MeJA), and/or the ET precursor 1-aminocyclopropane-1-carboxylic acid were applied, and the response of mutants in JA-synthesis and -signalling, and ET-signalling investigated. Endogenous levels of auxin, JA and JA-related compounds, and ARF6, ARF8 and ARF17 expression were monitored. Results: MeJA, at 0.01 μM, enhances AR-formation, when combined with IBA + Kin, and the response of the early-JA-biosynthesis mutant dde2–2 and the JA-signalling mutant coi1–16 confirmed this result. JA levels early change during TCL-culture, and JA/JA-Ile is immunolocalized in AR-tips and xylogenic cells. The high AR-response of the late JA-biosynthesis mutant opr3 suggests a positive action also of 12-oxophytodienoic acid on AR-formation. The crosstalk between JA and ET-signalling by EIN3/EIL1 is critical for AR-formation, and involves a competitive modulation of xylogenesis. Xylogenesis is enhanced by a MeJA concentration repressing AR-formation, and is positively related to ARF17 expression. Conclusions: The JA concentration-dependent role on AR-formation and xylogenesis, and the interaction with ET opens the way to applications in the micropropagation of recalcitrant species

Histology and symplasmic tracer distribution during development of barley androgenic embryos

The present study concerns three aspects of barley androgenesis: (1) the morphology and histology of the embryos during their development, (2) the time course of fluorescent symplasmic tracers’ distribution, and (3) the correlation between symplasmic communication and cell differentiation. The results indicate that barley embryos, which are developing via an androgenic pathway, resemble their zygotic counterparts with respect to their developmental stages, morphology and histology. Analysis of the distribution of the symplasmic tracers, HPTS, and uncaged fluorescein indicates the symplasmic isolation of (1) the protodermis from the underlying cells of the late globular stage onwards, and (2) the embryonic organs at the mature stage of development

Reductions in mesophyll and guard cell photosynthesis impact on the control of stomatal responses to light and CO2

Transgenic antisense tobacco plants with a range of reductions in sedoheptulose-1,7-bisphosphatase (SBPase) activity were used to investigate the role of photosynthesis in stomatal opening responses. High resolution chlorophyll a fluorescence imaging showed that the quantum efficiency of photosystem II electron transport (Fq′/Fm′) was decreased similarly in both guard and mesophyll cells of the SBPase antisense plants compared to the wild-type plants. This demonstrated for the first time that photosynthetic operating efficiency in the guard cells responds to changes in the regeneration capacity of the Calvin cycle. The rate of stomatal opening in response to a 30 min, 10-fold step increase in red photon flux density in the leaves from the SBPase antisense plants was significantly greater than wild-type plants. Final stomatal conductance under red and mixed blue/red irradiance was greater in the antisense plants than in the wild-type control plants despite lower CO2 assimilation rates and higher internal CO2 concentrations. Increasing CO2 concentration resulted in a similar stomatal closing response in wild-type and antisense plants when measured in red light. However, in the antisense plants with small reductions in SBPase activity greater stomatal conductances were observed at all Ci levels. Together, these data suggest that the primary light-induced opening or CO2-dependent closing response of stomata is not dependent upon guard or mesophyll cell photosynthetic capacity, but that photosynthetic electron transport, or its end-products, regulate the control of stomatal responses to light and CO2. © 2008 The Author(s)

Construction of plant transformation vectors carrying beet necrotic yellow vein virus coat protein gene (ii)- plant transformation

Fragments containing the coat protein gene of beet necrotic yellow vein virus were cloned in two plant transformation vectors: pCAMBIA3301M with the bar gene as selectable marker, and pCAMBIA1304M, with resistance to hygromycin. Three constructs were made of each vector: CPL, containing coat protein gene with leader sequence; CPS with coat protein gene, and CPSas with coat protein gene in antisense orientation. Vectors pC3301MCPL, pC3301MCPS. and pC3301MCPSas were used in Agrobacterium—mediated transformation of Nicotiana tabacum (tobacco), Nicotiana excelsior and Nicotiana benthamiana. Regenerants that developed roots on selective media were tested for the presence of CP fragments and the bar gene, but most regenerants were nontransformed (50-83% escapes). After all rooted plants had been selfed, and T1 seed germinated on selective media, only plants descending from one N. excelsior regenerant transformed with pC3301MCPS were positive for presence of bar gene and CPS fragment. Tobacco and Nicotiana benthamiana were transformed with constructs pC1304MCPS and pC1304MCPSas. Transformation efficiency was much higher and approximately 50% of regenerants that rooted on media with 20 mg l−1 hygromycin were positive for the presence of CP fragments. All T1 plants were positive for presence of CP fragments

Quince (Cydonia oblonga) in vitro plant root formation through an automated temporary inmersion system, and its acclimation

Artículo científicoQuince (Cydonia oblonga) is a non-traditional fruit tree found in Costa Rica that has therapeutic and nutritional properties; however its slow growth and root formation prevents the production of a homogeneous population when using conventional farming techniques. Hence, the aim of this research project was to generate uniform plant material in a reduced time span using a temporary immersion bioreactor system (RITAS ®). A semisolid rooting MS culture medium supplemented with 0.1 mg L-1 NAA; 0.3 mg L-1 IBA and 3% sucrose (pH 6.5), developed in the Centro de Investigación en Biotecnología (CIB), Instituto Tecnológico de Costa Rica (ITCR), in Cartago, was used as a reference medium. Four different variations in the sucrose concentration (1%, 2%, 3%, and 4%) were performed in liquid medium. Each trial was evaluated with in vitro plants which had been previously exposed to the culture medium of the corresponding treatments, in a stationary mode and for a 15 day long period, and with in vitro plants without any previous treatment (a total of eight treatments). The comparison of the root formation percentages evidenced the clear effect of sucrose concentration used, with the best results obtained when using the 2% sucrose trial with no pre-treatment (73.3%). The in vitro plants were acclimated in cylinders made out of peat, have previously been disinfected with fungicide, and placed in a humidity chamber at a 20.5°C average temperature and a 75,5% relative humidity for the establishment of weekly fertilizing cycles. The acclimation process generated an 80% survival rate, since several seedlings experienced stem strangulation caused by a fungal attack. The conidiophores identified through optical and scanning electron microscopy evidenced the presence of Cladosporium spp., which was controlled with carbendazim and iprodione fungicides

Transcriptional Analysis of Arabidopsis thaliana Response to Lima Bean Volatiles

Exposure of plants to herbivore-induced plant volatiles (HIPVs) alters their resistance to herbivores. However, the whole-genome transcriptional responses of treated plants remain unknown, and the signal pathways that produce HIPVs are also unclear.Time course patterns of the gene expression of Arabidopsis thaliana exposed to Lima bean volatiles were examined using Affymetrix ATH1 genome arrays. Results showed that A. thaliana received and responded to leafminer-induced volatiles from Lima beans through up-regulation of genes related to the ethylene (ET) and jasmonic acid pathways. Time course analysis revealed strong and partly qualitative differences in the responses between exposure at 24 and that at 48 h. Further experiments using either A. thaliana ET mutant ein2-1 or A. thaliana jasmonic acid mutant coi1-2 indicated that both pathways are involved in the volatile response process but that the ET pathway is indispensable for detecting volatiles. Moreover, transcriptional comparisons showed that plant responses to larval feeding do not merely magnify the volatile response process. Finally, (Z)-3-hexen-ol, ocimene, (3E)-4,8-dimethyl-1,3,7-nonatriene, and (3E,7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene triggered responses in A. thaliana similar to those induced by the entire suite of Lima bean volatiles after 24 and 48 h.This study shows that the transcriptional responses of plants to HIPVs become stronger as treatment time increases and that ET signals are critical during this process