The miR1511 over expression in transgenic BAT93 roots increased the root growth sensitivity to Al and, moreover, an increased sensitivity to AlT was observed in G19833 composite plants engineered for miR1511 expression . These data support the hypothesis that miR1511 induces degradation of ALS3 transcripts thus delaying the adequate root response to AlT stress. Therefore, absence of miR1511 resulting in diminished ALS3 transcript degradation appears to be an evolutionary advantage to Al contamination in soils, leading to an inhibition of the LPR1 pathways, a faster relocation of chelated Al to vacuole and Al-tolerant aerial tissues and a lesser effect on root growth, a phenomenon that partially explains why P. vulgaris Andean genotypes are more resistant to AlT than Mesoamerican ones . Overall, the current results about AlT in arid environments, combined with previous results by other authors, illustrates the complexity of adaptation to drought conditions. Tolerance of these conditions encompass mechanisms of growth and development like root depth and reshaping of the root profile, persistent growth despite drought conditions , continued translocation of photosynthesis from pod walls into seeds resulting in a high pod harvest index , and Al detoxification under unfavorable edaphic conditions . In turn,hydroponic channel this knowledge helps designing and interpreting experiments in the introgression of genetic diversity for drought tolerance from wild to domesticated common-bean and breeding drought tolerant common bean, in general .
In conclusion, our study reports an original case of the gene evolution of a single MIRNA the MIR1511, within Phaseolus vulgaris and close relatives, allowing adaptation to the aluminum toxicity abiotic stress. Seeds from the common bean Mesoamerican cv BAT93 and Andean accession G19833 were surface sterilized in 10% commercial sodium hypochlorite for 5-10 min and finally rinsed 5–6 times in sterile distilled water. Subsequently seeds were germinated on moist sterile paper towels at 30°C for 2-3 days in darkness. Plants were grown in hydroponic system under controlled environmental conditions as previously described . For control treatment, the hydroponic trays contained 8 L of full-nutrient Franco & Munns solution . For the AlT stress treatment, plantlets were allowed to adapt to the hydroponic culture for 4 days, then the nutrient solution was supplemented with 70 μM AlCl3. For both treatments the pH of the solution was adjusted to 4.5 and was controlled throughout the experiment. Common-bean composite plants with transgenic roots were generated as described below and grown in similar control or AlT conditions as those described for un-transformed plants. miR1511 overexpression in common-bean transgenic roots was performed using the pTDTO plasmid . The expression cassette is driven by the 35S cauliflower mosaic virus promoter. A red fluorescent protein was used as transformation efficiency reporter gene. Precursor of miR1511 was obtained using common bean root cDNA as template and the specific primers, which are listed in Table S2. After purification, PCR products were digested by XhoI and BamHI and inserted into the pTDTO vector.
The resulting OEmiR1511 plasmid, and the corresponding empty vector , were introduced into Agrobacterium rhizogenes K599 by electroporation. Then, these transformed bacteriawas used as vector of plant transformation, as described previously . Total RNA was isolated from 100 mg 48 hpt root tissue using mirVana™ miRNA Isolation Kit , following the manufacturer’s recommendations. Three biological replicates, from plants grown under the same conditions, were analyzed. For mature miRNA quantification , cDNAs were prepared from total RNA that was polyadenylated and reversely transcribed using the NCode miRNA First-Strand Synthesis Kit according to the manufacturer’s instructions. For mRNA detection, cDNA was first obtained with Superscript III Reverse transcriptase and oligo‐dT for priming cDNA preparation. Ten-fold dilutions of resulting cDNAs were then used for qRT-PCR experiments, using SYBR Green qPCR Master Mix. The reactions were analyzed using Applied Bio-system 7300 real-time thermocycler . qPCR cycles were set as follows: 94°C for 1 min, then40 cycles of 94°C for 20 s and 60°C for 60s. Two technical replicates were performed for each reaction. The “comparative Ct method” and normalization by geometrical mean of three housekeeping genes and U6 sRNA, were used for relative accumulation level of mRNA transcripts and mature miRNAs, respectively. The sequences of primers used for PCR amplification are listed in Table S2.. To assess the significance of differential expression of the mean values from three biological replicates for each condition, Mann Whitney statistical tests were performed . The architecture of transgenic roots from composite plants under control or AlT treatments was analyzed by determining the growth rate of root length, width, and area, as well as the number of lateral roots formed, using the ImageJ software. For both treatments root measurements were done at the beginning of the experiment and after 48 hrs of growth. As mentioned before, AlT treatment plants were first adapted to hydroponic culture in control treatment .
They were then taken out from this culture to be quickly photographed -for subsequent root architecture analysis- and were introduced into a hydroponic culture under AlT treatment for 48 hpt to be harvested and photographed again. Data of growth rate of each parameter represent the difference of the values at 48 and 0 hpt. Each root architecture parameter was determined in transgenic roots from 10 to 15 composite plants for each treatment. Statistical analyses were performed using the Mann-Whitney null hypothesis statistical test. The dictionary definition of the prefix ‘nano’ indicates that the object or dimension it describes is in the range of 10-9 of the dimensions it is described by, and it is as such that nanobubbles have become popular, under a misnomer. Many prefer the name ultrafine bubbles, since the size range of nanobubbles begins at one micron, and usually goes down much further to only as few as 10 nanometers in diameter. Since their discovery as the remains of collapsing micro-bubbles and of their persistence after formation, attempts have been consistently to understand the mechanics of their dissolution and stability, to enable the design of systems that use them to our advantage. The first field to experience benefits due to micro- and nanobubbles was agriculture, and the use of nanobubble water was well-documented by several studies since the year 2000, showing increased growth and quality of root vegetables grown in hydroponic systems, as well as the cultivation of tomatoes in soil. Further benefits were also demonstrated with pisciculture, showing increased sizes of the fish cultured in nanobubble water, due to an increase in the dissolved oxygen content. Similar benefits were also demonstrated in the case of shrimp breeding, due to the same phenomenon. However, all of these systems were simply a case of using the equipment for generating micro-bubbles without much control, and to permit them to dissolve into the water without regard for optimization. Indeed,hydroponic dutch buckets without any parameters to measure the rate of dissolution and the generation and stability of the generated bubbles, it is not possible to optimize such a system. Thus, ongoing research has focused on the generation, stability and control of these bubbles for diverse application in the fields of drug delivery, water treatment, energy storage, and various others. The second technological application of micro-bubbles was for the treatment of water based on the release of hydroxide ions from collapsing micro-bubbles, which shed light on one particular area, which was a promising candidate for explaining the stability of the nanobubble. The focus of stability was discovered by measurement of the zeta-potential of the first micro-bubbles of about 2 microns in diameter, which was found to be about -35 mV, and is still thought to be the cloud of ions that exists around a nanobubble. This suggested a role of the surrounding cloud of ions in their stability, in particular their ability to inhibit diffusion of the gas into the fluid, which has given rise to several theories regarding the mechanism of the ions’ stabilizing influence. Several approaches have also been made for specific cases such as surface nanobubbles and electrochemically generated bubbles, which involve several scenarios of diffusion and shrinkage. The work that is outlined here will summarize and look for theoretical evidence and alternatives to the presented theories, as well as present a new argument for the mechanism of stability of bulk nanobubbles, which seeks to incorporate and explain as many of the observed behaviors of nanobubble systems as possible.Several theoretical approaches have been proposed, many of which are highly specific to the circumstances for which the study was conducted, and none thus far have proposed an overarching theory as to the formation and evolution of bulk nanobubbles. As far back as 1997 Ljunggren and co-workers proposed theoretical explanations for colloid-sized gas bubbles based on diffusion of the gas into the liquid, which could now be considered nanobubbles.
Seddon et. al. also contributed to the emerging idea around the same time, but there have been few contributions to understanding their stable presence since then. Explanations for specific cases of phenomena such as surface nanobubbles, nanobubbles generated electrochemically, and so forth have been offered so far. Early on, the YoungLaplace equation was used to describe nanobubble stability, but the internal pressures required are far higher than would be possible at ambient temperature for the amount of gas that is contained within the bubble. Attard and co-workers analysed the thermodynamic stability of bulk nanobubbles, but it was found that the radius of nanobubbles could not be accurately predicted from thermodynamic considerations, nor was an expression offered for the rate of decrease in nanobubble size. Brenner and Lohse presented a model for predicting the radius of surface bubbles based on the dynamic equilibrium between diffusion into and out of nanobubbles situated at a surface. Further work in specific cases by Weijs and Lohse suggested the use of increased length scales to counter the problem of high internal pressures due to the relatively high surface tension of a bubble in that size range. Sverdrupand colleagues offered explanations as to the rates of decrease in size based on diffusion in all directions possible through the gas-water interface at the nanobubble surface. In their models they consider the possibility of diffusion both into and out of the nanobubble, with a sufficiently high mass transfer coefficient. Their models consist of a combination of Henry’s Law and Taylor series expansion. The equations are plotted, taking time as a function of radius and show coherence with previous models given by Ljunggren. However, no comparisons with experimental data are provided. The size of the nanobubble depends on the balance of the surface forces which are holding it together. The Young-Laplace equation seems inadequate to completely describe the phenomenon as it requires extremely high internal pressures of the gas to balance the surface tension that causes the nanobubble to shrink, as summarized by Attard and coworkers . However, the interface through which the diffusion occurs has thus far been considered to have constant properties of being composed only of water molecules and gas molecules. Yasui and colleagues also detail several theories that attempt to explain bulk nanobubble stability, based on the armoured bubble model, a particle crevice theory, a skin theory, the dynamic equilibrium model and electrostatic repulsion. Among these theories, it appears that electrostatic repulsion has the most experimental support. Studies of interfaces between water and practically all surfaces such as glass are negatively charged, assumed to be due to the accumulation of hydroxide ions physisorbed to the monolayer as reported by Zangi and Engberts. Thus, it is reasonable to suppose that the water-gas interface is also negatively charged due to similar congregation of hydroxide ions at the bubble surface. Furthermore, studies conducted by Takahashi and others have shown that nanobubbles are indeed negatively charged, with oxygen nanobubbles having a zeta potential about -35 mV. Thus, it is evident that hydroxide ions physisorbed onto the surface of the nanobubble play a role in the interactions between the molecules present there. Jin et. al. proposed a model for bulk nanobubble stability involving the electrostatic repulsion, terming the pressure due to the electrostatic force as Maxwell pressure. One rationale involving the surface charge density of a bulk nanobubble has been proposed by Ahmed and colleagues that involves electrostatic repulsion balancing the surface tension. In the following, we consider a theory of electrostatic repulsion and what it requires of the conditions imposed for nanobubbles to have the long-term stability that has been observed experimentally.