Fluorimetric-Based Method to Detect and Quantify Total S-Nitrosothiols (SNOs) in Plant Samples.

Accumulating experimental evidence indicates that S-nitrosylation (technically S-nitrosation) events have a central role in plant biology, presumably accounting for much of the widespread influence of nitric oxide (NO) on developmental, metabolic, and stress-related plant responses. Therefore, the accurate detection and quantification of S-nitrosylated proteins and peptides can be particularly useful to determine the relevance of this class of compounds in the ever-increasing number of NO-dependent signaling events described in plant systems. Up to now, the quantification of S-nitrosothiols (SNOs) in plant samples has mostly relied on the Saville reaction and the ozone-based chemiluminescence method, which lacks sensitivity and are very time-consuming, respectively. Taking advantage of the photolytic properties of S-nitrosylated proteins and peptides, the method described in this chapter allows simple, fast, and high-throughput detection of SNOs in plant samples.

Transcriptional foliar profile of the C3-CAM bromeliad Guzmania monostachia.

Guzmania monostachia is an epiphytic tank bromeliad that displays the inducible CAM photosynthesis under stressful conditions and had the highest stomata density in the leaf apex, while the base portion has the highest density of trichomes, which are specialized structures used to acquire water and nutrients from the tank solution. In order to correlate the genetic factors behind these morpho-physiological characteristics along the leaf blade of G. monostachia, a comparative transcriptome analysis was performed to identify the functional enriched pathways and unigenes that could play a role in the apical, middle and basal leaf portions. A total of 653 million reads were used for de novo transcriptome assembly, resulting in 48,051 annotated unigenes. Analysis of differentially expressed genes (DEGs) among distinct leaf regions revealed that 806 DEGs were upregulated in the apex compared to the middle portion, while 9685 DEGs were upregulated in the apex and 9784 DEGs were upregulated in the middle portions compared to the base. Our outcomes correlated some DEGs and identified unigenes with their physiological functions, mainly suggesting that the leaf apex was related to the regulation of stomatal movement, production of chlorophyll, cellular response to stress, and H2O2 catabolic process. In contrast, the middle portion showed DEGs associated with the transport of amino acids. Furthermore, DEGs from the leaf base were mainly correlated with responses to nutrients and nitrogen compounds, regulation of potassium ion import, response to water deprivation, and trichome branching, indicating that, at least in part, this leaf portion can replace some of the root functions of terrestrial plants. Therefore, possibly candidate unigenes and enriched pathways presented here could be prospected in future experimental work, opening new possibilities to bioengineer non-inducible CAM plants and/or improve the fertilization use efficiency by increasing leaf nutrient acquisition of crop plants.

Involvement of aquaporins on nitrogen-acquisition strategies of juvenile and adult plants of an epiphytic tank-forming bromeliad.

MAIN CONCLUSION: Depending on the N source and plant ontogenetic state, the epiphytic tank-forming bromeliad Vriesea gigantea can modulate aquaporin expression to maximize the absorption of the most available nitrogen source. Epiphytic bromeliads frequently present a structure formed by the overlapping of leaf bases where water and nutrients can be accumulated and absorbed, called tank. However, this structure is not present during the juvenile ontogenetic phase, leading to differences in nutrient acquisition strategies. Recent studies have shown a high capacity of the bromeliad Vriesea gigantea, an epiphytic tank-forming bromeliad, to absorb urea by their leaves. Since plant aquaporins can facilitate the diffusion of urea through the membranes, we cloned three foliar aquaporin genes, VgPIP1;1, VgPIP1;2 and VgTIP2;1 from V. gigantea plants. Through functional studies, we observed that besides water, VgTIP2;1 was capable of transporting urea while VgPIP1;2 may facilitate ammonium/ammonia diffusion. Moreover, aiming at identifying urea and ammonium-induced changes in aquaporin expression in leaves of juvenile and adult-tank plants, we showed that VgPIP1;1 and VgPIP1;2 transcripts were up-regulated in response to either urea or ammonium only in juvenile plants, while VgTIP2;1 was up-regulated in response to urea only in adult-tank plants. Thereby, an ontogenetic shift from juvenile to adult-tank-forming-plant appears to occur with metabolic changes regarding nitrogen metabolism regulation. Investigating urea metabolism in wild species that naturally cope with organic N sources, such as V. gigantea, may provide the knowledge to modify nitrogen use efficiency of crop plants.

The contribution of weak CAM to the photosynthetic metabolic activities of a bromeliad species under water deficit.

The Crassulacean acid metabolism (CAM) can be a transitory strategy for saving water during unfavourable conditions, like a dry season. In some cases, CAM can also contribute to the maintenance of photosynthetic integrity, even if carbon gain and growth are impaired. CAM occurs in different intensities, being stronger or weaker depending on the degree of nocturnal malic acid accumulation. For example, Guzmania monostachia is an epiphytic tank bromeliad that shows an increase in its nocturnal organic acid accumulation and a variable CAM behaviour when exposed to water deficit. In this context, this study aimed at investigating whether the weak CAM displayed by this species may mitigate the harmful effects of water limitation on its photosynthetic activity. To this, bromeliads were submitted to well-watered and water deficit conditions. Guzmania monostachia plants under water deficiency conditions showed a reduction on atmospheric carbon assimilation without exhibiting changes in PSII integrity and carbohydrate production while showed an increase in nocturnal malic acid accumulation. Additionally, spots with high PSII efficiency in the leaf portion with a greater nocturnal malic acid accumulation were observed in plants exposed to water shortage conditions. These high-efficiency spots might be associated with a greater malate decarboxylation capacity. Also, the malic acid contributed to approximately 50% of the total carbon assimilated under water deficit. These results suggest that weak CAM may participate in photo-protection and it appears to meaningfully contribute to the overall carbon balance, being an important metabolic strategy to maintain plant fitness during water deficit periods.

Spatial patterns of photosynthesis in thin- and thick-leaved epiphytic orchids: unravelling C3-CAM plasticity in an organ-compartmented way.

BACKGROUND AND AIMS: A positive correlation between tissue thickness and crassulacean acid metabolism (CAM) expression has been frequently suggested. Therefore, this study addressed the question of whether water availability modulates photosynthetic plasticity in different organs of two epiphytic orchids with distinct leaf thickness. METHODS: Tissue morphology and photosynthetic mode (C3 and/or CAM) were examined in leaves, pseudobulbs and roots of a thick-leaved (Cattleya walkeriana) and a thin-leaved (Oncidium 'Aloha') epiphytic orchid. Morphological features were studied comparing the drought-induced physiological responses observed in each organ after 30 d of either drought or well-watered treatments. KEY RESULTS: Cattleya walkeriana, which is considered a constitutive CAM orchid, displayed a clear drought-induced up-regulation of CAM in its thick leaves but not in its non-leaf organs (pseudobulbs and roots). The set of morphological traits of Cattleya leaves suggested the drought-inducible CAM up-regulation as a possible mechanism of increasing water-use efficiency and carbon economy. Conversely, although belonging to an orchid genus classically considered as performing C3 photosynthesis, Oncidium 'Aloha' under drought seemed to express facultative CAM in its roots and pseudobulbs but not in its leaves, indicating that such photosynthetic responses might compensate for the lack of capacity to perform CAM in its thin leaves. Morphological features of Oncidium leaves also indicated lower efficiency in preventing water and CO2 losses, while aerenchyma ducts connecting pseudobulbs and leaves suggested a compartmentalized mechanism of nighttime carboxylation via phosphoenolpyruvate carboxylase (PEPC) (pseudobulbs) and daytime carboxylation via Rubisco (leaves) in drought-exposed Oncidium plants. CONCLUSIONS: Water availability modulated CAM expression in an organ-compartmented manner in both orchids studied. As distinct regions of the same orchid could perform different photosynthetic pathways and variable degrees of CAM expression depending on the water availability, more attention should be addressed to this in future studies concerning the abundance of CAM plants.