Natasha L. Teakle

Mechanisms of Cl- transport contributing to salt tolerance

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.

Measuring Soluble Ion Concentrations (Na + , K + , Cl − ) in Salt-Treated Plants

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.

Root aeration via aerenchymatous phellem: three-dimensional micro-imaging and radial O2 profiles in Melilotus siculus.

• 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 near-anoxic conditions at the centre of the stele in the basal parts of the root, coupled with only hypoxic conditions towards the apex.

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.

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 O(2)-deficient (stagnant) treatments. Measurements of radial O(2) loss (ROL) under stagnant conditions indicated that more O(2) 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 O(2) transport to roots enables regulation of Na(+) transport processes in L. tenuis roots, contributing to tolerance to combined salinity and waterlogging stresses.

Leaf gas films delay salt entry and enhance underwater photosynthesis and internal aeration of Melilotus siculus submerged in saline water

A combination of flooding and salinity is detrimental to most plants. We studied tolerance of complete submergence in saline water for Melilotus siculus, an annual legume with superhydrophobic leaf surfaces that retain gas films when under water. M. siculus survived complete submergence of 1 week at low salinity (up to 50 mol m(-3) NaCl), but did not recover following de-submergence from 100 mol m(-3) NaCl. The leaf gas films protected against direct salt ingress into the leaves when submerged in saline water, enabling underwater photosynthesis even after 3 d of complete submergence. By contrast, leaves with the gas films experimentally removed suffered from substantial Na(+) and Cl(-) intrusion and lost the capacity for underwater photosynthesis. Similarly, plants in saline water and without gas films lost more K(+) than those with intact gas films. This study has demonstrated that leaf gas films reduce Na(+) and Cl(-) ingress into leaves when submerged by saline water - the thin gas layer physically separates the floodwater from the leaf surface. This feature aids survival of plants exposed to short-term saline submergence, as well as the previously recognized beneficial effects of gas exchange under water.

Improvement of salt and waterlogging tolerance in wheat: comparative physiology of Hordeum marinum-Triticum aestivum amphiploids with their H. marinum and wheat parents

Hordeum marinum Huds. is a waterlogging-tolerant halophyte that has been hybridised with bread wheat (Triticum aestivum L.) to produce an amphiploid containing both genomes. This study tested the hypothesis that traits associated with waterlogging and salinity tolerances would be expressed in H. marinum-wheat amphiploids. Four H. marinum accessions were used as parents to produce amphiploids with Chinese Spring wheat, and their responses to hypoxic and 200mM NaCl were evaluated. Relative growth rate (RGR) in the hypoxic-saline treatment was better maintained in the amphiploids (58-71% of controls) than in wheat (56% of control), but the amphiploids were more affected than H. marinum (68-97% of controls). In hypoxic-saline conditions, leaf Na+ concentrations in the amphiploids were lower than in wheat (30-41% lower) but were 39-47% higher than in the H. marinum parents. A strong barrier to radial oxygen loss formed in basal root zones under hypoxic conditions in two H. marinum accessions; this barrier was moderate in the amphiploids, absent in wheat, and was weaker for the hypoxic-saline treatment. Porosity of adventitious roots increased with the hypoxic treatments; values were 24-38% in H. marinum, 16-27% in the amphiploids and 16% in wheat. Overall, the amphiploids showed greater salt and waterlogging tolerances than wheat, demonstrating the expression of relevant traits from H. marinum in the amphiploids.

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.

Evaluation and breeding of tedera for Mediterranean climates in southern Australia

Abstract. Tedera (Bituminaria bituminosa C.H. Stirton var. albomarginata and var. crassiuscula) has been identified as one of the most productive and drought-tolerant species of herbaceous perennial legumes based on 6 years of field evaluation in Western Australia in areas with Mediterranean climate and annual rainfall ranging from 200 to 600 mm. Importantly, tedera demonstrated broad adaptation to diverse soils, and some accessions have shown moderate levels of tolerance to waterlogging and salinity. Tedera exhibits minimal leaf shedding during summer and autumn. Economic modelling strongly suggests that giving livestock access to green tedera in summer and autumn will dramatically increase farm profit by reducing supplementary feeding. The breeding program (2006–12) evaluated the available genetic diversity of tedera for its field performance in seven nurseries with 6498 spaced plants in total covering a wide variation in rainfall, soils and seasons. Best overall plants were selected using a multivariate selection index generated with best linear unbiased predictors (BLUPs) of dry matter cuts and leaf retention traits. The breeding program also evaluated tedera for grazing tolerance, grazing preference by livestock, waterlogging tolerance, seed production, cold tolerance, disease susceptibility and presence of secondary compounds. Tedera is a diploid, self-pollinated species. Therefore, 28 elite parents were hand-crossed in several combinations to combine outstanding attributes of parents; F1 hybrids were confirmed with the aid of highly polymorphic, simple sequence repeat markers. The F1s were progressed to F4s by single-seed descent breeding. Elite parent plants were selfed for two generations to be progressed in the breeding program without hybridisation. Over time, selections from the crossing and selfing program will deliver cultivars of three ideotypes: (i) drought-tolerant, (ii) cold- and drought-tolerant, (iii) waterlogging- and drought-tolerant.