Among plant species analyzed, P. vulgaris was the only one that showed a binding-site penalty score lower than 5, corresponding to a score recommended to consider a small RNA-target binding as probably functional. For other species, the high penalty scores, ranging from 7.5 to 9, indicate a very low probability for the existence of a functional miR1511/ALS3 regulatory node .A previous study about the response of different Andean and Mesoamerican common-bean cultivars to AlT showed that Andean genotypes are more tolerant to this abiotic stress, as compared to Mesoamerican genotypes . Our phylogenetic analysis revealed that all the Andean genotypes present a deleted version of the MIR1511 that would result in the absence of functional mature miR1511 . Previous work from our group showed that common-bean miR1511 expression responds to AlT stress . Here we analyzed the regulation of miR1511 and ALS3, as well as the early effects of AlT in roots of common-bean plants from the Mesoamerican BAT93 genotype vs. Andean G19833 genotype, with a deleted MIR1511 . Common-bean plantlets from BAT93 and G19833 genotypes were grown in hydroponic conditions either in control or AlT treatments, for up to 48 hrs. First, we performed the expression analysis of miR1511 and ALS3 target gene, using qRT-PCR . In AlT-stressed BAT93 plants, the transcript accumulation level of mature miR1511 gradually decreased, reaching more than half at 24 hours post-treatment ,vertical grow while at 48 hpt it returned to values close to those of time 0 . As expected, G19833 plants did not express mature miR1511 . The transcript level of ALS3 target gene increased in AlT treatment. The ALS3 transcript accumulation was significantly higher in G19833 roots, which lack miR1511, compared to BAT93 roots .
At 6 hpt, ALS3 expression in G19833 roots almost doubled and remained unchanged up to 48 hpt, when transcript accumulation in BAT93 and G1988 roots reached similar levels . To further study the role of miR1511/ALS3 in the physiological reaction of common-bean plants to high Al levels, we aimed to over express the miR1511 precursor in transgenic roots. As long as stable transformation of Phaseolus vulgaris plants is, to date, nearly impossible, we chose to use BAT93 and G19833 composite plants -with untransformed aerial organs and transgenic roots . As long as common bean is recalcitrant to stable transformation, this method is an alternative to demonstrate miRNA functionality . The miR1511-overexpressing composite plants as well as control plants, transformed with empty vector , were grown in AlT and control treatments. The expression level of miR1511 and ALS3 were determined by qRT-PCR in roots from composite plants harvested at 48 hpt . A two-fold accumulation of miR1511 transcript was observed in BAT93 OE1511 roots from plants grown in either treatment, compared to EV . In G19833 EV roots, the absence of miR1511 was confirmed, however a significant accumulation of miR1511 mature transcript was observed in OE1511 roots, albeit at lower levels than the level from BAT93 OEmiR1511 roots . In control treatment, both genotypes showed lower expression level of ALS3 in OEmiR1511 vs. EV roots. In addition, increased ALS3 transcript levels were observed in AlT stressed roots from both genotypes, as compared to control treatment . The primary and earliest symptoms of plants subjected to AlT stress is an inhibition of lateral root formation and root growth due to the alteration of root cell expansion and elongation, inhibiting cell division . On this basis, we analyzed the root architecture phenotype of BAT93 and G19833 OEmiR1511 and EV plants, grown under AlT or control treatments for 48 h . The growth rate of root length, width and area as well as the number of lateral roots, was calculated from the difference of each value at 48 hpt vs. time 0. The BAT93 EV plants under AlT showed decreased rates of each root parameter , thus indicating the drastic effect of high Al on root development. In contrast, G19833 EV plants showed higher tolerance to AlT evidenced by similar rate of the root length, area, width and lateral root number in stress vs control treatments . These results are in agreement with those previously reported indicating a higher tolerance to AlT of Andean common-bean genotypes compared to Mesoamerican genotypes . Surprisingly, in G19833 plants genetically engineered for the expression of mature miR1511, the effect of root phenotype was evident.
The rate of root length, area, width and lateral root number of G19833 OEmiR1511 AlT-stressed plants significantly decreased as compared to EV plants, showing reduced levels similar to those from BAT93 stressed plants . In A. thaliana, primary root growth inhibition under phosphate limitation or AlT is mediated by ALS3 and LPR1, a ferroxidase . LPR1 acts downstream of ALS3 and its expression is epistatic to ALS3 expression . To determine if LPR1 could be involved in the different response to AlT of BAT93 vs. G19833 plants, we measured the accumulation of LPR1 transcripts in similar AlT conditions as those from Figure 4. The transcript level of LPR1 gene decreased in AlT treatment. In AlT BAT93 roots, the transcript level of LPR1 gradually decreased reaching half of the initial expression at 48 hpt. In AlT G19833 roots, the LPR1 expression was significantly lower compared to BAT93 roots from 6 hpt to 24 hpt . At 48 hpt, LPR1 transcript reached similar levels in roots from both genotypes . The LPR1 expression pattern was opposite to the ALS3 expression profile in AlT-stressed roots , indicating an epistatic relation between these two genes and the possible participation of LPR1 together with ALS3 in the control of common-bean root growth under AlT. In order to determine if miR1511 indirectly controls LPR1 expression via the regulation of ALS3 transcript, we evaluated the LPR1 transcripts accumulation in transgenic roots from OEmiR1511 and EV composite plants, growing in Alt vs control conditions. In both BAT93 and G19833 roots, a significant increase of LPR1 transcript accumulation was observed in OEmiR1511 roots from plants grown in either treatment, compared to EV roots . In AlT treatment, roots from both genotypes showed significant lower LPR1 transcript level compared to roots from control condition. Again, LPR1 expression pattern was the opposite compared to that of ALS3 in the same transgenic root samples , thus indicating the probable epistatic relationship between these two genes and the indirect regulation of miR1511 on LPR1 expression.In plants, microRNA genes have a higher birth and death rates than protein-coding genes . For various authors, the miRNAs’ evolution rate generates a reservoir of adaptability for gene regulation . Due to this high evolutionary turnover rate, new miRNA families and members emerge, while others lose their regulatory role and disappear from genomes of phenotypically close species or genotypes. In soybean, MIR1511 is subjected to this mechanism. Htwe et al.,reported two altered versions of MIR1511 alleles in some annual wild soybean genotypes, whereas no deletion was found in G. max and perennial wild soybean MIR1511. Here, we report a similar phenomenon for P. vulgaris MIR1511 genotypic variations. Only part of the MW1 subgroup of P. vulgaris Mesoamerican genotypes and all the Andean genotypes analyzed displayed a 58 bp-deletion in the miR1511 precursor gene compared to the corresponding sequence of P. dumosus, P. coccineus, the PhI gene pool and the rest of P. vulgaris Mesoamerican genotypes .
As MIR1511 is present in non-legume species, the most parsimonious hypothesis to explain the evolution process associated with this event is to consider a deletion of part of miR1511 precursor sequence. In contrast to soybean, where probably two deletion events were required for the generation of two alternative MIR1511 alleles, our results suggest a single deletion event in the common ancestor of a part of MW1 Mesoamerican subgroup and the Andean genotypes for the generation of a different allele of miR1511 precursor gene. This single MIR1511 deletion event hypothesis supports the Mesoamerican model proposed by Ariani and colleagues where the Mesoamerican gene pool is the ancestral population from which the other gene pools have derived. The fact that the PhI gene pool contains an untruncated version, as do the other closely-related Phaseolus species included in this analysis, further confirms the sister-species status of the PhI gene pool, now known as P. debouckii . P. debouckii also contains ancestral, i.e., non-derived, sequences for phaseolin seed protein and chloroplast DNA . Based on the MIR1511 phylogenetic history presented here , we propose an addendum to this model where AW gene pool genotypes derived from one, or more,indoor growers member of the MW1 Mesoamerican subgroup. A clear distinct geographical distribution pattern was observed among the P. vulgaris genotypes featuring the MIR1511 deletion and the ones with an unaltered allele . MIR1511 deletion occurred in genotypes originating from the northern and southern extreme limits of the common-bean distribution in Latin American area. Such distribution pattern correlates with the annual precipitation pattern reported for the American continent , indicating that genotypes with MIR1511 deletion originated from areas with significantly less precipitation as compared to areas where genotypes with unaltered MIR1511 originated . Drought makes soil not suitable for agriculture; it tends to increase soil concentration of different compounds that would result in plant toxicity, including aluminum toxicity, which is an important plant growth-limiting factor . The harsh soil conditions of areas where P. vulgaris genotypes lacking of MIR1511 originated probably forced these common-bean populations to adapt and favored selection of genotypes with higher AlT tolerance. In this work, we showed the experimental validation of a target gene for P. vulgaris miR1511. We validated the miR1511-induce cleavage of ALS3 transcript, an ABC transporter participating in Al detoxification in plants . However, additional action of miR1511 by translation repression of ALS3 cannot be excluded. Other proposed target genes for P. vulgaris miR1511 are not related to plants AlT response and show high binding-site penalty score, thus improbable to be considered as functional in the AlT response.We interpret that the MIR1511 deletion resulting in lack of mature miR1511 allowed common-bean adaptation to high Al in the soils by eliminating the negative regulation of ALS3 transcript and the accumulation of LPR1, in the first 48 hpt, thus increasing its tolerance to AlT and favoring plant growth. Interestingly, similar characteristics hold for the soybean MIR1511-deletion case where the origin of soybean genotypes featuring a MIR1511-altered allele is geographically correlated with areas susceptible to high Al concentration in soil due to presence of drought in these regions .
High aluminum levels in soil mainly affect plant roots; aluminum can be allocated to different sub-cellular structures thus altering the growth of the principal root and the number of lateral roots . In this sense, it has been observed that AlT-stressed plants favor the transport of chelated Al to vacuoles and from roots, through the vasculature, to aerial tissues that are less sensitive to Al accumulation . In Arabidopsis and other plants, ALS1 and ALS3, from the ATP-binding cassette transporter family, are involved in Al detoxification and enhance tolerance to AlT. ALS3 is located in the tonoplast, the plasma membrane of root cortex epidermal cells, and in phloem cells throughout the plant . It has been shown that Arabidopsis als3 mutants are more sensitive to AlT exhibiting extreme root growth inhibition, compared to wild type plants . Recent studies on the role of Arabidopsis ALS3 in root growth inhibition revealed its regulation via the inhibition of the STOP1-ALMT1 and LPR1 pathways, which indirectly control ROS accumulation in roots via the modulation of Fe accumulation . Furthermore, Arabidopsis ALS3 expression is induced by excess Al , a phenomenon we observed in common-bean plants as well . Common-bean ALS3 expression doubled after 6 hours under AlT in roots from G19833 plants, while in stressed roots from BAT93 plants a similar level was reached only after 48 h of treatment . The opposite expression profile was found for the ALS3-epistatic gene LPR1, in the same samples . Our data on the different ALS3 and LPR1 expression level from both genotypes indicate that the absence of the negative regulator miR1511 in G19833 plants allows a faster response to AlT. Although the level of mature miR1511 decreased in BAT93 roots up to 24 h of after exposure to high Al, this level seems sufficient to induce degradation of ALS3 transcript, which showed reduced levels compared to G19833 roots, and an increase of LPR1 expression . Our analysis of root architecture in common-bean plants showed that G19833 Andean genotype plants are more tolerant to AlT as compared to Mesoamerican BAT93 plants .