Using excised leaves to screen lucerne for salt tolerance

Abstract

Salinity affects many physiological processes at all levels of plant structural organization. Being a physiologically and genetically complex trait, salinity tolerance implies a coordinated contribution of multiple mechanisms, making plant screening for salt tolerance extremely difficult. In this work, we show how the use of excised leaves can fulfill that task. We argue that, by adding NaCl directly to the transpiration stream, the protective effects of several mechanisms regulating Na⁺ delivery to the shoot are eliminated, enhancing PSII exposure to salinity treatment and resulting in a significant decline in leaf photochemistry (Fv/Fm characteristics). We suggest that measuring Fv/Fm characteristics on excised salt-treated leaves provides an opportunity to evaluate the efficiency of vacuolar Na⁺ compartmentation, arguably the most important feature for salt tolerance. We also explain the observed decline in Fv/Fm values as salt-induced structural damage to chloroplasts caused by oxidative stress.

The bottleneck of every breeding program is the availability of efficient screening techniques. Combining a highly predictive method with simplicity, low cost and ability to evaluate hundreds if not thousands of samples per day is a highly challenging task. The problem is further exacerbated manifold when multiple physiological traits contribute to plant tolerance. This is exactly the case for salinity tolerance.^1,2 It is estimated that salinity affects the level of transcription of ∼8% of all genes;³ of these, fewer then 25% are salt stress specific.⁴ Numerous physiological traits have been suggested to be used as physiological “markers” for plant screening for salt tolerance including seed germination characteristics, growth rates, plant biomass reduction, rate of CO₂ assimilation, stomatal conductance characteristics, changes in chlorophyll content, chlorophyll fluorescence characteristics, leaf elemental content, ultrastructural changes, and various electrophysiological characteristics.^5–12 Among these, the chlorophyll fluorescence method has often been seen as the most attractive tool for plant breeders, because measurements of the maximal photochemical efficiency of PSII in dark-adapted samples (so-called Fv/Fm value¹³) take only a few seconds using fully automated pulse amplitude-modulated fluorometers. There have been numerous attempts to use chlorophyll fluorescence characteristics (specifically, the Fv/Fm value) to screen plants for salt tolerance (reviewed in ref. 14). However, these attempts only had limited success, with results being often very controversial.^14–17 It is becoming increasingly evident that, in intact plants, PSII is well protected against salinity stress, and is the last “line of defense” that fails only when nothing else can be done to protect it from the detrimental effects of salinity. Multiple mechanisms such as restriction of the unidirectional Na⁺ uptake by roots, active Na⁺ extrusion from the cytosol to the external media, efficient cell- and tissue-specific Na⁺ compartmentation, prevention of Na⁺ transport to the shoot, and recirculation of Na⁺ back to the roots through the phloem^3,18,19 may all contribute to protection of PSII against salt stress.

The contribution of some of the above protective mechanisms may be reduced, or completely eliminated, when salt stress is imposed directly onto leaves (e.g., by immersing petioles of excised leaves into NaCl solution). Indeed, under these conditions, when plants have no ability to exclude Na⁺ from the transpirational stream, a significant amount of Na⁺ is expected to rapidly accumulate in leaves. Unless some genotypes have efficient mechanisms to sequester it in the epidermis and/or mesophyll vacuoles, elevated cytosolic Na⁺ levels are expected to affect leaf photochemistry and thus be reflected in the changes of Fv/Fm characteristics. This paper provides supportive evidence for this hypothesis and investigates some of the mechanisms underlying the detrimental effects of salinity on leaf photochemistry at the ultrastructural level in lucerne (Medicago sativa L.) following our recent study on multiple mechanisms contributing to salt tolerance in this species.¹⁷

Adding of 150 mM NaCl into the transpiration stream of lucerne cultivars for 48 h resulted in a significant decline in Fv/Fm characteristics (Fig. 1A). This is in contrast to our previous report that none of these genotypes showed a significant reduction in Fv/Fm ratio after 5 weeks of NaCl stress in hydroponically grown plants.¹⁷ The decline in Fv/Fm values in excised leaves correlated strongly (significant at p < 0.05) with the decline in shoot dry weight (DW) in hydroponically-grown plants (Fig. 1B) suggesting that the use of excised leaves may be a rather efficient way of predicting plant performance under saline conditions (and, hence, be used for screening genotypes for salt tolerance).

Figure 1.

(A) chlorophyll fluorescence characteristics of excised leaves of five lucerne genotypes after 48 h exposure to NaCl treatment. Plants were grown for 50 days as described elsewhere.¹⁷ The fourth most recently developed leaf was excised, cut to the same petiole length, and immersed into plastic vials containing 0 (control) or 150 mM NaCl (treatment) solutions and kept for 48 h under low light (60 µmol m⁻² s⁻¹ at the leaf level; cold fluorescent tubes). Mean ± SE (n = 10). (B) correlation between Fv/Fm data from excised leaves (as above) and the reduction in plant biomass (shoot dry weight; expressed as % of control) from hydroponically grown plants after 5 weeks of 80 mM NaCl treatment (reviewed in ref. 17).

To understand the possible reasons for the observed decline in Fv/Fm characteristics, ultrastructural differences between control and NaCl-treated leaf mesophyll cells were studied. Substantial morphological changes to various cellular structures were observed in response to saline treatment (Fig. 2).

Figure 2.

Effect of salinity on leaf ultrastructure in the WL516 lucerne cultivar. (A) the continuous “end-to-end” distribution of thin chloroplasts in control tissue. Bar = 5 µm. (B) discrete “bulbous” chloroplasts of NaCl-treated tissue. Bar = 5 µm. (C) a single thin chloroplast within control tissue. The granal stacks (G) are interspersed with dark, electron dense spherical plastoglobuli (P). Starch grains (SG) are few in number and formed between granal stacks, displacing them. The plant cell wall (PCW) is discernible from the stroma (S). Bar = 1 µm. (D) a single “bulbous” chloroplast within NaCl-treated tissue. Starch grains (SG) interspersed among grana (G) are more numerous. The plant cell wall (PCW) appears to be much thicker. Bar = 1 µm. (E) in control tissue, the thylakoids with grana (G) are densely packed and intergranal thylakoids (IT) are distinct and numerous. Bar = 200 nm. (F) a chloroplast within NaCl-treated tissue. Both the thylakoids comprising grana (G) and intergranal thylakoids (IT) are very loosely arranged compared to those of the control tissue. Note the abundant plastoglobuli (P) and large starch granule within (SG). Bar = 200 nm.

A continuous ‘end-to-end’ distribution of chloroplasts around the cell's periphery was observed in control plants (Fig. 2A). In contrast, the chloroplasts of NaCl-treated cells appeared ‘bulbous’ and discrete (Fig. 2B). It has been argued that the rounding of chloroplasts may be due to swelling and that this causes the stroma of chloroplasts to be less dense and the granal thylakoids appearing expanded.²⁰ NaCl-treated cells also displayed extensive but thin peripheral cytoplasmic regions devoid of chloroplasts. Overall, the chloroplasts of control tissue contained fewer starch granules (Fig. 2C) compared to the ‘bulbous’ chloroplasts of NaCl-treated plants (Fig. 2D), similar to previous reports on some other species.²¹

Grana, composed of layered thylakoids, appeared to be much more regular and densely stacked and intergranal thylakoids were distinct and well-developed in chloroplasts of control tissue (Fig. 2E). In NaCl-treated cells, grana were disrupted and the thylakoids within them were loosely and unevenly stacked. Intergranal thylakoids were difficult to distinguish from the background stroma (Fig. 2F). Starch granules accumulated in greater numbers in NaCl-treated mesophyll cells (compare Fig. 2C and D). These changes in thylakoid structure are usually typical for oxidative stress damage^22,23 and point to salinity-induced ROS production as a possible reason for the observed decline in Fv/Fm characteristics shown in Figure 1.

Although chloroplasts are potentially the most powerful source of oxidants, they are exposed to oxidative stress more so than any other organelle because of their internal O₂ concentrations, especially in the thylakoid membranes. Chloroplasts are therefore particularly prone to generating activated oxygen species.^24,25 With limited availability of CO₂ to chloroplasts due to stomatal closure, ROS build up and in-turn can cause peroxidation of chloroplast lipids, damage to thylakoid membranes and pigment breakdown which leads to the reduction or loss of carbon-fixing ability of chloroplasts.²⁵ All these changes will be reflected in Fv/Fm decline, as observed in this study (Fig. 1).

Overall, our results reported here suggest that eliminating the contribution of several mechanisms regulating Na⁺ delivery to the shoot, by using excised leaves, may enhance PSII exposure to salinity treatment and allow using Fv/Fm measurements as an efficient screening tool for salt tolerance. Given the fact that none of the species can fully prevent Na⁺ build-up in the shoot, leaf ability to compartmentise excessive Na⁺ in the vacuole is arguably the most important feature for salt tolerance.¹⁸ Measuring Fv/Fm characteristics from salt-treated excised leaves gives, therefore, a nice opportunity to evaluate the efficiency of such compartmentation.

Addendum to: Smethurst CF, Rix K, Garnett T, Auricht G, Bayart A, Lane P, Wilson SJ, Shabala S. Multiple traits associated with salt tolerance in lucerne: revealing the underlying cellular mechanisms. Funct Plant Biol. 2008;35:640–650. doi: 10.1071/FP08030.