Differential effects of salinity and osmotic stress on the plant growth-promoting bacterium Gluconacetobacter diazotrophicus PAL5.

Plant growth-promoting bacteria (PGPB) represent a promising alternative to the massive use of industrial fertilizers in agriculture. Gluconacetobacter diazotrophicus is a PGPB that colonizes several plant species. Although this bacterium is able to grow at high sucrose concentrations, its response to environmental stresses is poorly understood. The present study evaluated G. diazotrophicus PAL5 response to stresses caused by sucrose, PEG 400, NaCl, KCl, Na2SO4 and K2SO4. Morphological, ultrastructural and cell growth analysis revealed that G. diazotrophicus PAL5 is more sensitive to salt than osmotic stress. Growth inhibition and strong morphological changes were caused by salinity, in consequence of Cl ion-specific toxic effect. Interestingly, low osmotic stress levels were beneficial for bacterial multiplication, which was able to tolerate high sucrose concentrations, Na2SO4 and K2SO4. Our data show that G. diazotrophicus PAL5 has differential response to osmotic and salinity stress, which may influence its use as inoculant in saline environments.

Passion fruit flowers: Kunitz trypsin inhibitors and cystatin differentially accumulate in developing buds and floral tissues.

In order to better understand the physiological functions of protease inhibitors (PIs) the PI activity in buds and flower organs of passion fruit (Passiflora edulis Sims) was investigated. Trypsin and papain inhibitory activities were analyzed in soluble protein extracts from buds at different developmental stages and floral tissues in anthesis. These analyses identified high levels of inhibitory activity against both types of enzymes at all bud stages. Intriguingly, the inhibitory activity against both proteases differed remarkably in some floral tissues. While all organs tested were very effective against trypsin, only sepal and petal tissues exhibited strong inhibitory activity against papain. The sexual reproductive tissues (ovary, stigma-style and stamen) showed either significantly lower activity against papain or practically none. Gelatin-SDS-PAGE assay established that various trypsin inhibitors (TIs) homogenously accumulated in developing buds, although some were differentially present in floral organs. The N-terminal sequence analysis of purified inhibitors from stamen demonstrated they had homology to the Kunitz family of serine PIs. Western-blot analysis established presence of a ∼60 kDa cystatin, whose levels progressively increased during bud development. A positive correlation between this protein and strong papain inhibitory activity was observed in buds and floral tissues, except for the stigma-style. Differences in temporal and spatial accumulation of both types of PIs in passion fruit flowers are thus discussed in light of their potential roles in defense and development.

Wound response in passion fruit (Passiflora f. edulis flavicarpa) plants: gene characterization of a novel chloroplast-targeted allene oxide synthase up-regulated by mechanical injury and methyl jasmonate.

The induction of a chloroplast-localized 13-lipoxygenase (13-LOX) in passion fruit leaves in response to methyl jasmonate (MeJa) was previously reported. Since allene oxide synthase (AOS) is a key cytochrome P450 enzyme in the oxylipin pathway leading to AOS-derived jasmonates, the results above led in turn to an investigation of AOS in our model plant. Spectrophotometric assays showed that 24 h exposure of MeJa caused a high increase in 13-hydroperoxy linolenic acid (13-HPOT) metabolizing activity in leaf tissue. Western analysis using polyclonal antibodies against tomato AOS strongly indicate that, at least a part of the 13-HPOT metabolizing capacity can be attributed to AOS activity. We cloned the cDNA from a novel AOS encoding gene from passion fruit, named PfAOS. The 1,512 bp open reading frame of the AOS-cDNA codes a putative protein of 504 amino acid residues containing a chloroplast target sequence. Database comparisons of the deduced amino acid sequence showed highest similarity with dicot AOSs. Immunocytochemistry analysis showed the compartmentalization of AOS in chloroplasts of MeJa treated leaves, corroborating the predicted subcellular localization. Northern analysis showed that AOS gene expression is induced in leaf tissue in response to mechanical wounding and exposure to MeJa. In addition, such treatments caused an increase in papain inhibitor(s) in leaf tissue. Taken together, these results indicate that PfAOS may play an important role in systemic wound response against chewing insect attack. Furthermore, it can be useful as a tool for understanding the regulation of jasmonates biosynthesis in passion fruit.