These data agree with those reported by Blair et al. . 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, 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.Phaseolus vulgaris G19833 and BAT93 MIR1511 sequences were obtained from the Phaseolus vulgaris release v2.1, from Phytozome 12 database, and from NCBI Whole Genome Shotgun database ,danish trolley respectively. Sequences for the phylogenetic tree display in Figure 1 were obtained from the SNPhylo analysis performed by Ariani et al. , based on the collection of representative SNPs located in sequences at least 5 kb from an annotated feature .
Only sequences from non-admixed genotypes were selected for the analysis , constituting a total of 87 sequences from three Phaseolus species , including three populations for the Mesoamerican , one for the Andean , and one for the Northern Peru–Ecuador P. vulgaris gene pool. Sequence alignment was performed thanks to MUSCLE algorithm .Accession PI430191 was used as out group for rooting the phylogenetic tree. Evolutionary analyses were conducted in MEGA X . GBS sequence reads from the 87 selected genotypes were pre-processed as previously described and mapped to the BAT93 MIR1511 sequence using the BWA-MEM algorithm as described by Ariani et al. . MIR1511 was considered complete if at least one read was mapped to the miR1511 mature sequence and at least another read mapped to another part of the miR1511 precursor sequence. When at least one read was mapped to another part of the miR1511 precursor sequence and none on the miR1511 mature sequence, MIR1511 was considered deleted. For further supporting this analysis we performed an additional validation for the samples with a complete BAT93 MIR1511 sequence. In brief, the reads aligning to the BAT93 MIR1511 sequence were re-mapped to the G19833 reference genome. Alignment results showed a partial alignment to these reads in the homologous region containing the truncated MIR1511 in the G19833 reference, up to the deletion visible in Figure 1a . 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. One-third of the fields on earth contain calcareous soil. Plants grown in calcareous soils that are low in iron availability demonstrate decreased growth and yield. Under conditions of low Fe availability, rice plants induce transcriptional responses that promote the uptake of Fe from the soil as ferric Fe–mugineic acid phytosiderophore chelates and ferrous Fe ions.
Thus, an understanding of the mechanisms by which plants such as rice respond to Fe deficiency is required to maintain plant yields and prevent food shortages. In our previous studies, Kobayashi et al. identified two Fe-deficiency-responsive cisacting elements , which confer Fe deficiency-induced expression in rice roots and leaves . We also found two rice transcription factors, IDE-binding factors 1 and 2 , that bind to IDE1 and IDE2, respectively . IDEF1 and IDEF2 belong to uncharacterized branches of the plant-specific transcription factor families ABI3/VP1 and NAC, and they exhibit novel sequence recognition properties. IDEF1 and IDEF2 transcripts are constitutively expressed in both roots and leaves. Transgenic rice plants that express IDEF1 under the control of the IDS2 promoter were found to be tolerant to early Fe deficiency in hydroponic culture and calcareous soil. IDEF1 regulates the ferrous ion transporter gene OsIRT1, the Fe-deficiency-induced transcription-factor gene OsIRO2, and other genes related to Fe deficiency. IDEF2 regulates the metal nicotianamine transporter gene OsYSL2 as well as other Fe-deficiency-related genes. Nevertheless, the specific mechanisms and tangential pathways affected by these key transcription factors have not been elucidated fully. Therefore, delineation of the expression patterns and characteristics of IDEF1 and IDEF2 could help elucidate the response of rice plants to Fe deficiency. To understand the mechanisms by which plants respond to Fe deficiency, we examined the expression patterns of IDEF1 and IDEF2 by promoter–GUS analysis under Fe-sufficient and Fe-deficient conditions. This information could be critical for the creation of rice varieties that grow in problem soils. We analyzed the spatial expression patterns of IDEF1 and IDEF2 during the germination, vegetative, and seed-maturation stages by histochemical localization of GUS staining as described by Inoue et al. and Nozoye et al. . Two kb of the 5’ region upstream of the translation start site was used as the promoter sequence for IDEF1, whereas 2 kb of the 5’ region upstream of the transcription start site was used as the promoter sequence for IDEF2. Rice was transformed with the IDEF1 promoter–GUS or the IDEF2 promoter– GUS by an Agrobacterium-mediated method, and T1 or T2 seeds were obtained for use in the analysis. To induce Fe deficiency, 28-day-old plants were cultivated hydroponically without Fe– EDTA 1-12 days before harvest. For analysis during the flowering and maturing periods, IDEF1 and IDEF2 seeds were cultured in Fe-sufficient artificial soil with fertilizer, and developing seeds were progressively sampled for GUS expression analysis. IDEF1 and IDEF2 expression was observed in both the endosperm and embryo during the early seed germination period. IDEF2 expression was induced in the leaf primordium during germination. IDEF1 and IDEF2 regulate genes related to Fe deficiency as well as other unknown genes. The expression patterns of some Fe-deficiency-induced genes have been investigated during the early germination period . The expression patterns of IDEF1, IDEF2, and OsNAS1 were similar; all were expressed in the embryo and endosperm. Conversely, other Fedeficiency-induced genes such as OsNAS2, OsNAS3, OsNAAT1, OsDMAS1, OsYSL2, and OsIRT1 were not expressed in endosperm tissues. It is speculated that genes involved in phytosiderophore biosynthesis and Fe transport are differentially regulated in the germination and vegetative stages under low Fe conditions. IDEF1 or IDEF2 promoter–GUS plants were grown in hydroponic culture under Fedeficient or Fe-sufficient conditions. The expression patterns of IDEF1 and IDEF2 were found to be similar despite Fe availability. In leaf blades of IDEF1 lines, strong expression was observed in mesophyll cells and in small vascular bundles. Interestingly,vertical aeroponic tower garden the main vascular bundle was not stained in either Fe-sufficient or Fe-deficient leaf samples. It is assumed that the principle function of the main vascular bundle is to transport water and nutrients and that Fe is needed in mesophyll cells and small vascular bundles for photosynthesis. In contrast to IDEF1, IDEF2 was highly expressed in vascular bundles but not in mesophyll cells. In the inner layers of the stem/leaf sheath of IDEF1 lines, mesophyll and small vascular cells demonstrated dense staining, indicating high IDEF1 expression. In root sections, IDEF1 and IDEF2 lines showed strong GUS staining in the secondary roots, which emerge under conditions of Fe deficiency.
This finding suggests that IDEF1 and IDEF2 induce Fe-deficiency-responsive genes such as OsIRT1 and OsIRO2 in secondary roots and that this may play an important role in the uptake and utilization of Fe in low Fe conditions. GUS staining was also found inside the vascular bundles of root sections in IDEF1 and IDEF2 lines. IDEF1 and IDEF2 lines were cultured in soil to investigate spatial expression patterns during the flowering and maturation periods. Prior to anthesis, IDEF1 line pollen showed high expression. After fertilization, the ovary was found to be heavily stained. Expression was also observed in the vascular bundles of the husk during the flowering and early seed development stages. There was strong staining of the embryo and the aleurone layer in the late progress maturation stages . In embryos, the scutellum and leaf primordium were densely stained. Similar to IDEF1, IDEF2 was expressed in most pollen in the flowering stage. IDEF2 was also expressed in immature seeds just after flowering and in the dorsal vascular sections in the late maturation stage. In flowering plants, a primary role for boron is to form a diester cross-link between two monomers of rhamnogalacturonan-II , a pectic polysaccharide present in the cell walls of all vascular plants . Rhamnogalacturonan-II is a structurally complex domain of pectin , which comprises 12 different monosaccharides that are linked together by at least 20 different glycosidic linkages . Nevertheless, its structure is largely conserved in vascular plants . The majority of RG-II exists in the wall as a dimer that is generated by forming a borate diester between the D-apiose of side chain A of two RG-II molecules. The inability of RG-II to properly assemble into a dimer results in the formation of cell walls with abnormal biochemical and bio-mechanical properties and has a severe impact on plant productivity.Nevertheless, the mechanisms that drive the interactions between borate and RG-II are poorly understood . There is increasing evidence that alteration of RG-II structure and cross-linking have severe impacts on plant growth, development and viability. To date, the only characterized RG-II biosynthetic enzymes are the rhamnogalacturonan xylosyl transferases , which catalyze the transfer of xylose from UDP-xylose to fucose to form ɑ-xylose–fucose in vitro . Inactivation of RGXT1 and -2 has no discernible effect on plant growth or RG-II structure , implying redundancy of function, whereas mutations affecting RGXT4 lead to defects of root and pollen tube growth that are lethal . Mutations that prevent the synthesis of UDP-Api and CMP-Kdo are also lethal and provide further evidence for the essential role of RG-II in plant growth . In the dwarf Arabidopsis mutant murus 1 L-galactose replaces L-fucose in several cell wall polysaccharides, including RGII, because the plant is unable to produce GDP-fucose in its shoots as it lacks GDP-D-mannose-4,6-dehydratase GMD1 . This has been shown to result in the incomplete formation of the A side-chain of RG-II, which in turn reduces the stability of the borate cross-linked dimer . Thus, the structural integrity of RG-II is probably important for its biological functions. Pectic and hemicellulosic polysaccharides are synthesized in the Golgi apparatus using activated donor substrates, typically in the form of nucleotide diphosphate-linked sugars . However, most NDP sugars are synthesized in the cytosol . Thus, NDP-sugar transporters are required to provide substrates for glycan synthesis . The Golgi-localized NST sub-family, which forms part of clade IIIa of the NST/triose phosphate transporter super family , comprises four members related to GONST1 , the first nucleotide sugar transporter described in Arabidopsis . The members of this family are the only Arabidopsis NSTs that contain a predicted GDP-binding motif .