Identification of two chickpea multidrug and toxic compound extrusion transporter genes transcriptionally upregulated upon aluminum treatment in root tips.

Aluminum (Al) toxicity poses a significant challenge for the yield improvement of chickpea, which is an economically important legume crop with high nutritional value in human diets. The genetic basis of Al-tolerance in chickpea remains unclear. Here, we assessed the Al-tolerance of 8 wild Cicer and one cultivated chickpea (PBA Pistol) accessions by measuring the root elongation in solution culture under control (0 µM Al3+) and Al treatments (15, 30 µM Al3+). Compared to PBA Pistol, the wild Cicer accessions displayed both tolerant and sensitive phenotypes, supporting wild Cicer as a potential genetic pool for Al-tolerance improvement. To identify potential genes related to Al-tolerance in chickpea, genome-wide screening of multidrug and toxic compound extrusion (MATE) encoding genes was performed. Fifty-six MATE genes were identified in total, which can be divided into 4 major phylogenetic groups. Four chickpea MATE genes (CaMATE1-4) were clustered with the previously characterized citrate transporters MtMATE66 and MtMATE69 in Medicago truncatula. Transcriptome data showed that CaMATE1-4 have diverse expression profiles, with CaMATE2 being root-specific. qRT-PCR analyses confirmed that CaMATE2 and CaMATE4 were highly expressed in root tips and were up-regulated upon Al treatment in all chickpea lines. Further measurement of carboxylic acids showed that malonic acid, instead of malate or citrate, is the major extruded acid by Cicer spp. root. Protein structural modeling analyses revealed that CaMATE2 has a divergent substrate-binding cavity from Arabidopsis AtFRD3, which may explain the different acid-secretion profile for chickpea. Pangenome survey showed that CaMATE1-4 have much higher genetic diversity in wild Cicer than that in cultivated chickpea. This first identification of CaMATE2 and CaMATE4 responsive to Al3+ treatment in Cicer paves the way for future functional characterization of MATE genes in Cicer spp., and to facilitate future design of gene-specific markers for Al-tolerant line selection in chickpea breeding programs.

Novel Sources of Tolerance to Aluminium Toxicity in Wild Cicer (Cicer reticulatum and Cicer echinospermum) Collections.

In acid soils, the toxic form of aluminium, Al3+, significantly inhibits root growth and elongation, leading to less water and nutrient uptake. Previous research had shown differential Al toxicity tolerance among cultivated Cicer arietinum L. (chickpea); however, the potential for developing tolerant cultivars is limited by the narrow genetic diversity of cultivated chickpeas. Recent collections from Turkey of wild Cicer species, Cicer reticulatum, and Cicer echinospermum, have increased the available gene pool significantly, but there has been no large-scale screening of wild Cicer for acid tolerance or Al3+ toxicity tolerance. This study evaluated 167 wild Cicer and 17 Australian chickpea cultivars in a series of screenings under controlled growth conditions. The pH of 4.2 and Al concentrations of 15 and 60 µM Al were selected for large-scale screening based on dose response experiments in a low ionic strength nutrient solution. The change in root length showed better discrimination between tolerant and sensitive lines when compared with shoot and root dry weights and was used as a selection criterion. In a large-scale screening, 13 wild Cicer reticulatum accessions had a higher root tolerance index (≥50%), and eight had higher relative change in root length (≥40%) compared with PBA Monarch, which showed greater tolerance among the Australian domestic cultivars screened. In general, C. reticulatum species were found to be more tolerant than C. echinospermum, while genetic population groups Ret_5, Ret_6, and Ret_7 from Diyarbakir and Mardin Province were more tolerant than other groups. Among C. echinospermum, Ech_6 from the Siv-Diyar collection site of the Urfa Province showed better tolerance than other groups. In this first detailed screening of aluminium toxicity tolerance in the new wild Cicer collections, we identified accessions that were more tolerant than current domestic cultivars, providing promising germplasm for breeding programs to expand chickpea adaptation to acid soils.

Variation of Cicer Germplasm to Manganese Toxicity Tolerance.

After aluminum, manganese toxicity is the most limiting factor for crops grown in acidic soils worldwide. But overall, research on Mn toxicity is still limited. The poor acid tolerance of chickpea may be related to Mn toxicity, but there has been no previous screening of chickpea germplasm (nor in its wild Cicer relatives, Cicer reticulatum and Cicer echinospermum) for tolerance to Mn toxicity. A screening technique was developed for tolerance to Mn toxicity using three released cultivars of chickpea (Cicer arietinum L), Ambar, PBA HatTrick, and PBA Striker; one accession each of C. reticulatum and C. echinospermum; and lupin (Lupinus angustifolius) as a Mn-tolerant check, with eight Mn concentrations of 2, 25, 50, 100, 150, 200, 250, and 500 µM Mn as MnSO4 in a low-ionic-strength nutrient solution. The plants were harvested at 14 and 28 days after Mn treatments. The nutrient uptake in shoots (young, old leaves, and the rest of the shoot) and roots was investigated. The best discrimination between tolerant and intolerant Cicer genotypes based on relative shoot dry weight, root dry weight, total root length, and scoring of toxicity symptoms was achieved at 150 µM Mn after 14 days of growth in Mn solution. Among the chickpea cultivars, the greater relative plant growth (both shoot and root) of Ambar and PBA Striker at 100-200 µM Mn contrasted with that of PBA HatTrick, while the C. echinospermum accession was more tolerant to Mn toxicity than C. reticulatum. Manganese tolerance in both domestic cultivars and wild accessions was associated with internal tolerance to excess Mn following greater uptake of Mn and translocation of Mn from roots to shoots.

Genotypic variation among chickpea and wild Cicer spp. in nutrient uptake with increasing concentration of solution Al at low pH.

In many acidic soils, high concentrations of toxic Al3+ hamper plant growth by restricting root growth which in turn restricts water and nutrient absorption. Previous research showed variation among chickpea (Cicer arietinum L.) and wild Cicer species in root elongation at 15 µM Al or more, but effects on nutrient absorption have not been examined. The variation in nutrient uptake of two chickpea varieties (PBA HatTrick and PBA Striker) and two wild Cicer species (C. echinospermum (C. echi) and C. reticulatum (C. reti)) was determined in low pH (4.2) nutrient solution with increasing Al concentrations (0, 7.5, 15, 30 µM Al). While C. echi, PBA HatTrick and PBA Striker had thicker roots and more lateral roots compared to C. reti, C. reti had greater aluminium tolerance index (AlTI) at 15 and 30 µM Al. The C. echi had higher uptake of root and shoot Al (7.5, 15 and 30 µM Al), P and S (15 and 30 µM Al) while its uptake was marginally lower for Mg, Ca (all Al treatments) and K (15 and 30 µM Al). By contrast, C. reti contained higher shoot Ca concentration at 15 and 30 µM Al and it had lower root Al uptake. Manganese uptake by C. reti roots and shoots were high enough to induce moderate Mn toxicity at 0 and 7.5 µM Al. Therefore, in response to Al toxicity, C. reti maintained greater AlTI and restricted Al uptake while increasing Ca uptake.

The Park Grass Experiment and next-generation approaches: local adaptation of sweet vernal grass revisited.

Long-term ecological experiments provide unique opportunities to observe the effects of natural selection. The Park Grass Experiment at Rothamsted Experiment Station in Hertfordshire, UK, is the longest running ecological experiment that incorporates fertilization treatments and has been ongoing since 1856. In the 1970s, local adaptation was observed in the grass Anthoxanthum odoratum to the elevated soil aluminium levels of the fertilized plots. Gould et al. (2014) have utilized this system to reevaluate the extent of local adaptation, first documented nearly 45 years ago (Snaydon), and to use emerging molecular approaches to identify candidate genes for the adaptation. From their work, they identify several plausible candidate loci for aluminium tolerance. This work shows the power of long-term field-based trials in a scientific age concentrated on rapidly emerging molecular techniques often utilized in short, narrowly focused laboratory or controlled environment experiments. The current study clearly illustrates the benefits gained by combining these molecular approaches within long-term monitoring experiments that can be regularly revisited in a changing world and used to address questions on evolutionary scales.