Lotus tenuis tolerates combined salinity and waterlogging: maintaining O2 transport to roots and expression of an NHX1-like gene contribute to regulation of Na+ transport

Salinity and waterlogging interact to reduce growth for most crop and pasture species. The combination of these stresses often cause a large increase in the rate of Na+ and Cl− transport to shoots; however, the mechanisms responsible for this are largely unknown. To identify mechanisms contributing to the adverse interaction between salinity and waterlogging, we compared two Lotus species with contrasting tolerances when grown under saline (200 mM NaCl) and O2-deficient (stagnant) treatments. Measurements of radial O2 loss (ROL) under stagnant conditions indicated that more O2 reaches root tips of Lotus tenuis, compared with Lotus corniculatus. Better internal aeration would contribute to maintaining Na+ and Cl− transport processes in roots of L. tenuis exposed to stagnant-plus-NaCl treatments. L. tenuis root Na+ concentrations after stagnant-plus-NaCl treatment (200 mM) were 17% higher than L. corniculatus, with 55% of the total plant Na+ being accumulated in roots, compared with only 39% for L. corniculatus. L. tenuis accumulated more Na+ in roots, presumably in vacuoles, thereby reducing transport to the shoot (25% lower than L. corniculatus). A candidate gene for vacuole Na+ accumulation, an NHX1-like gene, was cloned from L. tenuis and identity established via sequencing and yeast complementation. Transcript levels of NHX1 in L. tenuis roots under stagnant-plus-NaCl treatment were the same as for aerated NaCl, whereas L. corniculatus roots had reduced transcript levels. Enhanced O2 transport to roots enables regulation of Na+ transport processes in L. tenuis roots, contributing to tolerance to combined salinity and waterlogging stresses

Aerenchymatous phellem in hypocotyl and roots enables O2 transport in Melilotus siculus

• Aerenchymatous phellem (secondary aerenchyma) has rarely been studied in roots. Its formation and role in internal aeration were evaluated for Melilotus siculus, an annual legume of wet saline land. • Plants were grown for 21 d in aerated or stagnant (deoxygenated) agar solutions. Root porosity and maximum diameters were measured after 0, 7, 14 and 21 d of treatment. Phellem anatomy was studied and oxygen (O(2)) transport properties examined using methylene blue dye and root-sleeving O(2) electrodes. • Interconnecting aerenchymatous phellem developed in hypocotyl, tap root and older laterals (but not in aerial shoots), with radial intercellular connections to steles. Porosity of main roots containing phellem was c. 25%; cross-sectional areas of this phellem were threefold greater for stagnant than for aerated treatments. Root radial O(2) loss was significantly reduced by complete hypocotyl submergence; values approached zero after disruption of hypocotyl phellem below the waterline or, after shoot excision, by covering hypocotyl phellem in nontoxic cream. • Aerenchymatous phellem enables hypocotyl-to-root O(2) transport in M. siculus. Phellem increases radially under stagnant conditions, and will contribute to waterlogging tolerance by enhancing root aeration. It seems likely that with hypocotyl submerged, O(2) will diffuse via surface gas-films and internally from the shoot system

Growth responses of Melilotus siculus accessions to combined salinity and root-zone hypoxia are correlated with differences in tissue ion concentrations and not differences in root aeration

Soil salinity and root-zone hypoxia often occur together in saline landscapes. For many plants, this combination of stresses causes greater increases in Na+ and Cl− in shoots, and decreases in K+, than from salinity alone. These changes in ion concentrations from combined salinity and hypoxia can have more adverse consequences for growth than from salinity alone. The herbaceous forage legume Melilotus siculus naturally occurs in saline soils prone to waterlogging; however, accessions differ in their tolerances, although all form high levels of aerenchyma. We hypothesised that tolerance to combined salinity and hypoxia would be associated with either greater aerenchyma formation in roots or the innate ability of the accessions to regulate tissue ion concentrations. Fifteen accessions of M. siculus were grown in nutrient solution with two salinities (0 or 200 mM NaCl) and two aeration treatments (aerated or hypoxic) for 21 days. Dry mass (shoot and root), root porosity and ion concentrations (Cl−, Na+, K+) in shoots and roots were assessed. In the M. siculus accessions variation in the shoot dry mass under saline–hypoxic conditions was negatively correlated with shoot Cl− and Na+, and positively correlated with the shoot K+. Shoot ion concentrations under saline–hypoxic conditions were related to concentrations under saline–aerated conditions, but not to the porosity of the main root, which was relatively high (∼18 to 25%). Differences in the tolerance of M. siculus accessions to combined salinity and root-zone hypoxia were mediated by variation in the plants’ ability to regulate ions, and were not related to variation in root porosity, which was relatively high in all accessions. The interaction between salinity and hypoxia was not detrimental to M. siculus, a waterlogging tolerant species

Comparisons of annual pasture legumes in growth, ion regulation and root porosity demonstrate that Melilotus siculus has exceptional tolerance to combinations of salinity and waterlogging

Annual pasture legumes with high tolerance of combined salinity and waterlogging are needed for saline areas in many rain-fed agricultural regions. Melilotus siculus is an annual legume from saline marshy areas of the Mediterranean with potential use in saline pastures and, based on descriptions of its native habitat, was hypothesised to tolerate combined salinity and waterlogging. Experiments compared M. siculus to Trifolium michelianum and Medicago polymorpha, other annual pasture legumes with reported salt or waterlogging tolerances, with 0–450 mM NaCl in hydroponics or in sand culture. Tolerance to combined salinity and waterlogging was also evaluated at 150, 400 and 550 mM NaCl in a stagnant deoxygenated nutrient solution. M. siculus was substantially more salt tolerant than the other two species. At 450 mM NaCl, shoot DM of M. siculus was 30% of control, compared with only 15% for the other species. M. siculus was also the most tolerant species to combined salinity and stagnant treatment, and produced new leaves even after 14 d in stagnant nutrient solution with 550 mM NaCl (∼ sea water salinity). In comparison, T. michelianum and M. polymorpha only survived up to 5 d in stagnant solution with 400 mM NaCl. Even at only 150 mM NaCl in stagnant solution, shoot DM was just 30% of control for T. michelianum and M. polymorpha, compared to 60% for M. siculus. Tolerance to combined salinity and waterlogging was associated with higher root porosity and regulation of shoot Na+ and Cl−, with the more tolerant M. siculus having similar shoot Na+ and Cl− concentrations for both aerated-saline and stagnant-saline treatments. In summary, M. siculus is a pasture legume that can grow at high salinity (up to 550 mM) even when in combination with waterlogging, as root porosity and associated O2 transport presumably enables continued regulation by the roots of Na+ and Cl− entry under these dual stress conditions

Variation in salinity tolerance, early shoot mass and shoot ion concentrations within Lotus tenuis: Towards a perennial pasture legume for saline land

Perennial legumes are needed for productive pastures in saline areas. We evaluated 40 lines of Lotus tenuis for tolerance to salinity at both germination and vegetative growth stages. Salt tolerance during the early vegetative stage was assessed in a sand-tank experiment with NaCl concentrations of 0–450 mm NaCl for 5 weeks. Most L. tenuis lines were more salt tolerant and had at least 50% lower shoot Na+ plus Cl– (% dry mass (DM)) compared with some other common pasture legumes, Medicago sativa, M. polymorpha and Trifolium subterraneum. Within L. tenuis significant variation in salt tolerance was found, with C50 values (concentrations of NaCl that decreased shoot dry matter to 50% of control) ranging from ~100 to 320 mm. Shoot concentrations of Cl–, Na+ and K+ did not always correlate with salt tolerance; some tolerant lines had low shoot Na+ and Cl– (and thus better nutritive value), while others tolerated high shoot Na+ and Cl–. We also found variation within L. tenuis for salt tolerance of seeds, with lines ranging from 0 to 70% germination after recovery from a prior exposure to 800 mm NaCl for 15 days. There was no relationship between salinity tolerance of scarified seeds and subsequent growth of seedlings; therefore, testing of seeds alone would not be an appropriate screening method for salt tolerance in L. tenuis. This study of 40 L. tenuis lines has shown significant genetic variation for salt tolerance within this species, and we have identified key lines with potential to be productive in saltland pasture systems

EST-derived SSR markers from defined regions of the wheat genome to identifyLophopyrum elongatumspecific loci

Lophopyrum elongatum, a close relative of wheat, provides a source of novel genes for wheat improvement. Molecular markers were developed to monitor the introgression of L. elongatum chromosome segments into hexaploid wheat. Existing simple sequence repeats (SSRs) derived from genomic libraries were initially screened for detecting L. elongatum loci in wheat, but only 6 of the 163 markers tested were successful. To increase detection of L. elongatum specific loci, 165 SSRs were identified from wheat expressed sequence tags (ESTs), where their chromosomal positions in wheat were known from deletion bin mapping. Detailed sequence analysis identified 41 SSRs within this group as potentially superior in their ability to detect L. elongatum loci. BLASTN alignments were used to position primers within regions of the ESTs that have sequence conservation with at least 1 similar EST from another cereal species. The targeting of primers in this manner enabled 14 L. elongatum markers from 41 wheat ESTs to be identified, whereas only 2 from 124 primers designed in random positions flanking SSRs detected L. elongatum loci. Addition and ditelosomic lines were used to assign all 22 markers to specific chromosome locations in L. elongatum. Nine of these SSR markers were assigned to homoeologous chromosome locations based on their similar position in hexaploid wheat. The remaining markers mapped to other L. elongatum chromosomes indicating a degree of chromosome rearrangements, paralogous sequences and (or) sequence variation between the 2 species. The EST-SSR markers were also used to screen other wheatgrass species indicating further chromosome rearrangements and (or) sequence variation between wheatgrass genomes. This study details methodologies for the generation of SSRs for detecting L. elongatum loci