Mechanisms of Cl-transport in plants are poorly understood, despite the importance of minimizing Cl -toxicity for salt tolerance. This review summarizes Cl -transport processes in plants that contribute to genotypic differences in salt tolerance, identifying key traits from the cellular to whole-plant level. Key aspects of Cl -transport that contribute to salt tolerance in some species include reduced net xylem loading, intracellular compartmentation and greater efflux of Cl -from roots. We also provide an update on the biophysics of anion transport in plant cells and address issues of charge balance, selectivity and energy expenditure relevant to Cl -transport mechanisms. Examples are given of anion transport systems where electrophysiology has revealed possible interactions with salinity. Finally, candidate genes for anion transporters are identified that may be contributing to Cl -movement within plants during salinity. This review integrates current knowledge of Cl -transport mechanisms to identify future pathways for improving salt tolerance.

The control of Na(+) and Cl(-) uptake from soils, and the partitioning of these ions within plants, is an essential component of salinity tolerance. Genetic variation in the ability of roots to exclude Na(+) and Cl(-) from the transpiration stream flowing to the shoot has been associated with salinity tolerance in many species. The maintenance of a high uptake of K(+) is also essential, so measurements of Na(+), K(+) or Cl(-) are frequently used to screen for genetic variation in salinity tolerance. As these ions are not bound covalently to compounds in cells, they can be readily extracted with dilute acid. Na(+) and K(+) can be measured in a dilute nitric acid extract using a flame photometer, by atomic absorption spectrometry or by inductively coupled plasma (ICP)-atomic emission spectrometry. Cl(-) can be measured in the same acid extract with a chloridometer or colorimetrically using a spectrophotometer.

Salinity and waterlogging interact to reduce growth of poorly adapted species by, amongst other processes, increasing the rate of Na(+) and Cl(-) transport to shoots. Xylem concentrations of these ions were measured in sap collected using xylem-feeding spittlebugs (Philaenus spumarius) from Lotus tenuis and Lotus corniculatus in saline (NaCl) and anoxic (stagnant) treatments. In aerated NaCl solution (200 mM), L. corniculatus had 50% higher Cl(-) concentrations in the xylem and shoot compared with L. tenuis, whereas concentrations of Na(+) and K(+) did not differ between the species. In stagnant-plus-NaCl solution, xylem Cl(-) and Na(+) concentrations of L. corniculatus increased to twice those of L. tenuis. These differences in xylem ion concentrations, which were not caused by variation in transpiration between the two species, contributed to lower net accumulation of Na(+) and Cl(-) in shoots of L. tenuis, indicating that ion transport mechanisms in roots of L. tenuis were contributing to better 'exclusion' of Cl(-) and Na(+) from shoots, compared with L. corniculatus. Root porosity was also higher in L. tenuis, due to constitutive aerenchyma, than in L. corniculatus, suggesting that enhanced root aeration contributed to the maintenance of Na(+) and Cl(-) 'exclusion' in L. tenuis exposed to stagnant-plus-NaCl treatment. Lotus tenuis also had greater dry mass than L. corniculatus after 56 d in NaCl or stagnant-plus-NaCl treatment. Thus, Cl(-) 'exclusion' is a key trait contributing to salt tolerance of L. tenuis, and 'exclusion' of both Cl(-) and Na(+) from the xylem enables L. tenuis to tolerate, better than L. corniculatus, the interactive stresses of salinity and waterlogging.

Lotus tenuis (Wadst. & Kit.) is a perennial legume widely grown for pasture in the flood-prone and salt affected Pampa region of Argentina. The physiology of salt and waterlogging tolerance in L. tenuis (four cultivars) was evaluated, and compared with Lotus corniculatus (three cultivars); the most widely cultivated Lotus species. Overall, L. tenuis cultivars accumulated less Na + and Cl -, and more K + in shoots than L. corniculatus cultivars, when exposed to 200 mM NaCl for 28 days in aerated or in stagnant solutions. Root porosity was higher in L. tenuis cultivars due to greater aerenchyma formation. In a NaCl dose-response experiment (0-400 mM NaCl in aerated solution), L. tenuis (cv. Chaja) accumulated half as much Cl -in its shoots than L. corniculatus (cv. San Gabriel) at all external NaCl concentrations, and about 30% less shoot Na + in treatments above 250 mM NaCl. Ion distributions in shoots were determined for plants at 200 mM NaCl. L. tenuis (cv. Chaja) again accumulated about half as much Cl -in old leaves, young leaves and stems, compared with concentrations in L. corniculatus (cv. San Gabriel). There were not, however, significant differences between the two species for Na + concentrations in the various shoot tissues. The higher root porosity, and maintenance of lower shoot Cl -and Na + concentrations in L. tenuis, compared with L. corniculatus, contributes to the greater tolerance to combined salt and waterlogging stress in L. tenuis. Moreover, significant variation for tolerance to combined salinity and waterlogging stress was identified within both L. tenuis and L. corniculatus.

Summary• Internal root aeration enables waterlogging-tolerant species to grow in anoxic soil. Secondary aerenchyma, in the form of aerenchymatous phellem, is of importance to root aeration in some dicotyledonous species. Little is known about this type of aerenchyma in comparison with primary aerenchyma.• Micro-computed tomography was employed to visualize, in three dimensions, the microstructure of the aerenchymatous phellem in roots of Melilotus siculus. Tissue porosity and respiration were also measured for phellem and stelar tissues. A multiscale, three-dimensional, diffusion-respiration model compared the predicted O 2 profiles in roots with those measured using O 2 microelectrodes.• Micro-computed tomography confirmed the measured high porosity of aerenchymatous phellem (44-54%) and the low porosity of stele (2-5%) A network of connected gas spaces existed in the phellem, but not within the stele. O 2 partial pressures were high in the phellem, but fell below the detection limit in the thicker upper part of the stele, consistent with the poorly connected low porosity and high respiratory demand.• The presented model integrates and validates micro-computed tomography with measured radial O 2 profiles for roots with aerenchymatous phellem, confirming the existence of nearanoxic conditions at the centre of the stele in the basal parts of the root, coupled with only hypoxic conditions towards the apex.

Tolerance to waterlogging is an important breeding objective for barley (Hordeum vulgare L.); however, it is a complex quantitative trait. It is difficult to screen large numbers of lines in the field due to environmental variability, and it is also challenging to screen large numbers in controlled conditions if yield data are to be collected. The direct measurement of traits that contribute to waterlogging tolerance, such as aerenchyma development in roots, may offer advantages especially if molecular markers can be developed to screen breeding populations. A doubled haploid population from a cross between Franklin and YuYaoXiangTian Erleng was screened for adventitious root porosity (gas-filled volume per unit root volume) as an indicator of aerenchyma formation. A single QTL for root porosity was identified on chromosome 4H which explained 35.7 and 39.0 % of phenotypic variation in aerated and oxygen-deficient conditions, respectively. The nearest marker was EBmac0701. This QTL is located in the same chromosomal region that contributed to tolerance when the same population was screened in an earlier independent soil waterlogging experiment. Comparative mapping revealed that this QTL is syntenic with the Qaer1.02-3 QTL in maize and the Sub1A-1 gene in rice, which are associated with aerenchyma formation (maize) and submergence tolerance (rice), respectively. This is the first report of a QTL for root porosity in barley which elucidates a major mechanism of waterlogging tolerance

Summary• 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.

BackgroundBituminaria bituminosa is a perennial legume species from the Canary Islands and Mediterranean region that has potential as a drought-tolerant pasture species and as a source of pharmaceutical compounds. Three botanical varieties have previously been identified in this species: albomarginata, bituminosa and crassiuscula. B. bituminosa can be considered a genomic 'orphan' species with very few genomic resources available. New DNA sequencing technologies provide an opportunity to develop high quality molecular markers for such orphan species.Results432,306 mRNA molecules were sampled from a leaf transcriptome of a single B. bituminosa plant using Roche 454 pyrosequencing, resulting in an average read length of 345 bp (149.1 Mbp in total). Sequences were assembled into 3,838 isotigs/contigs representing putatively unique gene transcripts. Gene ontology descriptors were identified for 3,419 sequences. Raw sequence reads containing simple sequence repeat (SSR) motifs were identified, and 240 primer pairs flanking these motifs were designed. Of 87 primer pairs developed this way, 75 (86.2%) successfully amplified primarily single fragments by PCR. Fragment analysis using 20 primer pairs in 79 accessions of B. bituminosa detected 130 alleles at 21 SSR loci. Genetic diversity analyses confirmed that variation at these SSR loci accurately reflected known taxonomic relationships in original collections of B. bituminosa and provided additional evidence that a division of the botanical variety bituminosa into two according to geographical origin (Mediterranean region and Canary Islands) may be appropriate. Evidence of cross-pollination was also found between botanical varieties within a B. bituminosa breeding programme.ConclusionsB. bituminosa can no longer be considered a genomic orphan species, having now a large (albeit incomplete) repertoire of expressed gene sequences that can serve as a resource for future genetic studies. This experimental approach was effective in developing codominant and polymorphic SSR markers for application in diverse genetic studies. These markers have already given new insight into genetic variation in B. bituminosa, providing evidence that a division of the botanical variety bituminosa may be appropriate. This approach is commended to those seeking to develop useful markers for genomic orphan species.