Lecture 5b: Abiotic stress: salt stress Flashcards

Question

Stress-tolerance traits have to be in the breeding population

Answer 1

Breeding has limited genetic diversity and the gene pool breeders have available in elite varieties. What happens if the traits/genes for a trait are not present in a population? Stress tolerant genes missing from modern cultivars are often present in wild population Lopes, M.S., Araus, J.L., van Heerden, P.D.R., and Foyer, C.H. (2011). Enhancing drought tolerance in C4 crops. J. Exp. Bot. 62: 3135 – 3153 by permission of Society of Experimental Biology

Answer 2

Salt storage in the vacuole: *NHX1 is a vacuolar Na+/H+ antiporter that moves sodium to the vacuole *Against a gradient - so needs energy *Coupled to pumps like AVP1 (vacuole proton pyrophosphatase) that built up a proton gradient Note: The SOS3/SCaBP8-SOS2 module may also positively regulate the activities of NHX The overall scheme in this slide is taken from a Nature Genetics review (http://www.nature.com/nrg/journal/v9/n6/full/nrg2342.html) Genetic approaches to crop improvement: responding to environmental and population changes Proton pumps like AVP1 generate protons to fuel the NHX1 antiporter. (The protons can also be generated by a vacuolar ATPase). So you can either express more antiporter or you can increase the proton drive. Both have been done. See following slides. Cellular responses to Na+ toxicity. It is thought that Na+ is passively transported into the cytosol by non-selective cation channels (NSCCs). To decrease cytosolic Na+ concentration, two mechanisms operate. One is to exclude Na+ across the plasma membrane. SOS1, a Na+–H+ exchanger located at the plasma membrane, is the only transporter identified in plants that carries out this function. Another mechanism is to compartmentalize Na+ into vacuoles, where Na+ is less toxic. This is performed by the vacuolar Na+&–H+ antiporter, NHX1. Both transporter activities require an H+ gradient across the membranes, which is generated either by a plasma membrane H+-ATPase or by a vacuolar H+-ATPase (yellow ovals), and by vacuolar H+-pyrophosphatase (AVP1). Accumulation of osmoprotectants (for example, betaine, proline and sugar alcohols) and heat shock proteins (HSPs) are also induced by salt stress. Overexpression of SOS1, NHX1, and AVP1, as well as HSPs or proteins for the biosynthesis of osmoprotectants, has been reported to enhance salt tolerance in several plant species.

Answer 3

AtNHX1 is the first studied plant vacuolar protein that can mediate Na+ transport into vacuoles (Apse MP, Aharon GS, Snedden WA, Blumwald E (1999) Science 285: 1256-1258) Fig. 3. Salt treatment of wild-type plants and plants overexpressing AtNHX1. Transgenic T3 plants from the lines overexpressing AtNHX1 were used in assessing salt tolerance. Twenty wild-type plants and 20 of each of the three lines of transformed plants overexpressing AtNHX1 were grown on a short-day cycle (8 hours light, 16 hours dark). Each set of 20 plants was divided into five groups (labeled A through E) of four plants each. We applied 25 ml of a diluted nutrient solution (1/8 MS salts) every other day over the 16-day watering treatment. The control group received no NaCl supplementation. The remaining groups were watered with nutrient solution supplemented with NaCl. The concentrations of NaCl supplementation were increased stepwise by 50 mM every 4 days for each group, to the indicated maximum: (A) control, (B) 50 mM NaCl, (C) 100 mM NaCl, (D) 150 mM NaCl, and (E) 200 mM NaCl. The salt-tolerance phenotype was observed in three independent transgenic lines tested. The transgenic line shown (2') is representative of the three tested lines as are the plants from each treatment group. (A) (upper panel) Wild-type plants. (B) (lower panel) Plants overexpressing AtNHX1. (Inset) Northern blot of RNA isolated from leaves of wild-type (wt) and three independent lines (2'; 3'; 4') of transgenic plants grown in the absence of NaCl. RNA was probed with AtNHX1 cDNA. Equal amounts of total RNA were present in each sample (30 µg). In all plants, the endogenous 2.1 kilobase (kb) transcript was detected. An additional transcript of ~1.7 kb that corresponds to the predicted open reading frame of AtNHX1 can be seen only in the transgenic plants. Apparent molecular size (kb) of the transcripts is indicated to the left.

Answer 4

*Elevated vacuolar solute content -> greater water retention drought tolerance *At high Na+ concentrations, enhances the Na+ sequestration into the vacuole – salt survival sodium sequestration in vacuole also improves water retention Data from two separate papers. The first shows it works in Arabidopsis. The second shows tomato. As well as conferring salt tolerance as might be expected, the increased proton gradient across the vacuolar membrane appears to improve solute uptake (this is hypothesised in the paper but a clear argument is not given as to why) and the authors think this is why water is better retained in AVP1 overexpressers (next slide) and the plants are more drought tolerant. The plants also have improved root growth (a likely indirect consequence of the H translocating activity affecting auxin movement) and this helps too. Figure on the left is from : Gaxiola R A et al. PNAS 2001;98:11444-11449 (http://www.pnas.org/content/98/20/11444.short) Wild-type and AVP1 transgenic plants deprived of water. Eight wild-type plants and eight of each of the two AVP1-overexpressing transgenic lines (AVP1-1 and AVP1-2) were grown on soil in a 16-h light/8-h dark cycle at 21°C. Plants were watered with a diluted nutrient solution (1/8 MS Murashige & Skoog salt mixture) every 4 days for 4 weeks. At week 5, the plants were watered and then transferred to a 24°C growth chamber with no further addition of water. The photograph corresponds to plants at day 10 of water-deficit stress (A) and the same plants 24 h after rewatering (B). WT, wild type. Figure on the right is from a paper 4 years later in which the AVP1 from Arabidopsis was expressed in tomato. These XAVP1-D plants (see below and next slide) are not just over-expressers but they have a gain of function mutation that increases the effectiveness of the AVP1 protein. (It’s a mutation at its active site). From: Park et al., PNAS 2005 vol 102, https://www.pnas.org/content/102/52/18830 (From the paper: “Of note, the AVP1D gene is the E229D gain-of-function mutant of the AVP1 gene that has a coordinated increase of both PPi hydrolytic activity and PPi-dependent H+-translocation”) Figure legend: Control and transgenic tomato plants respond differently to an imposed soil water deficit. Ten-week-old, well watered plants were subjected to imposed water stress by withholding irrigation. In each panel, images of six control plants are shown on the left and three plants each of lines XAVP1D-1 (white arrows) and XAVP1D-2 (green arrows) are shown on the right. (A) Photographs were taken after 13 days of stress. Pots were irrigated to field capacity on day 13, and photographs were taken 1 day (B) and 4 days (C) after rewatering.

Answer 5

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1323196/ From the same tomato paper as on previous slide. Transgenic plants maintain greater leaf water potentials and take up greater amounts of water during imposed soil water deficits. Sets of control and transgenic plants were subjected to water deficit stress as described in the legend of Fig. 3. In all cases, pots were irrigated to field capacity on day one. (A) Leaf water potentials of four control plants and a total of eight transgenic plants (representing lines XAVP1D-1, XAVP1D-2, and XAVP1D-5 from Fig. 1) were monitored during the imposed stress. Results are shown as means ± SE of leaf water potential measured in control and (pooled) transgenic plants. (B) Photograph of representative control and XAVP1D plants taken on day 5 of the imposed stress. (C) Water uptake of control and transgenic plants during an imposed period of soil water deficit. The experiment shown in A was repeated with a second set of control and transgenic plants (representing lines XAVP1D-1 and XAVP1D-2); in this case, four transgenic and four control plants were tested. Water uptake measurements are shown for periods of the stress during which transgenic plants displayed significantly greater leaf water potentials (i.e., on days 5, 6, and 8, as was the case during the stress regime shown in A). The leaf water potentials with corresponding standard error values (in MPa) of control and transgenic plants measured on the day that water uptake was measured (i.e., on day 5, day 6, and day 8) are shown above the bars representing water uptake values.

Answer 6

extreme salt environment AVP1 variant has high tol. Article in Plant Biotechnology journal 2014 by Schilling et al. Expression of the Arabidopsis vacuolar H+-pyrophosphatase gene (AVP1) improves the shoot biomass of transgenic barley and increases grain yield in a saline field http://onlinelibrary.wiley.com/doi/10.1111/pbi.12145/full Legend for this figure: Shoot biomass of wild-type and transgenic barley expressing AVP1 in a saline field trial (a) Shoot biomass (g DW/plant) of wild-type (cv. Golden Promise) and four transgenic barley lines expressing AVP1 (35S-AVP1-1a, 35S-AVP1-1b, 35S-AVP1-2 & 35S-AVP1-3) after 12 weeks growth in the low- (white bars) and high-salinity (grey bars) field. (b) Image of shoot growth of a representative wild-type (cv. Golden Promise) and transgenic barley expressing AVP1 (35S-AVP1-1b) at high salinity. Values are the mean ± s.e.m (n = 12) with asterisks (*) indicating a significant difference (one-way ANOVA, LSD, P < 0.05).

Answer 7

Nguyen, N.T., Vu, H.T., Nguyen, T.T., Nguyen, L.-A.T., Nguyen, M.-C.D., Hoang, K.L., Nguyen, K.T. and Quach, T.N. (2019) ‘Co-expression of Arabidopsis AtAVP1 and AtNHX1 to improve salt tolerance in soybean’, Crop Science, [online] Available at: https://doi.org/10.2135/cropsci2018.10.0640 Nguyen et al Crop science, Vol. 59, 2019; 1133 The build up of stored sodium resulted in toxicity in adult plants – suggesting sequestering is less effective than depositing Both transgenes were expressed using a strong constitutive promoter – CamV35S to drive expression All transgenic lines have increased Na+ and K+ accumulation in leaves and root Greater accumulation of Na+ in leaves and roots, may limit long-term tolerance to salinity -> no benefit on final yield Fig. 2. Responses of transgenic soybean expressing AtAVP1 and AtNHX1 to NaCl stress. (A) Growth of wild-type (DT26) and transgenic plants under 0 and 100 mM NaCl. Pictures were taken after 28 d under 100 mM NaCl stress. (B) Plant height, shoot dry weight (DW) and root DW of WT and transgenic plants overexpressing AtAVP1 (A) and AtNHX1 (N) and both genes (AN). Salt treatments started at V2, and measurements were taken after 35 (for the 100 mM NaCl treatment) and 14 d (for the 200 mM NaCl treatment), when the leaf scorch score of the WT reached 5.0. A common lowercase letter indicates no significant difference among means of soybean genotypes in the same stress treatment (mean ± SEM, Duncan’s test, p < 0.05). Fig. 5. Transgenic and wild-type soybean at maturity. Top: Plants growing under control (2.3 dS m−1) and salt stress (7.5 dS m−1) in greenhouse conditions in summer 2016. Bottom: Shoot dry weight (DW) and seed yield were measured at harvest. A common lowercase letter indicates no significant difference among means of soybean genotypes in the same stress treatment (mean ± SEM, Duncan’s test, p < 0.05).

Answer 8

Gaxiola, R.A., Fink, G.R. and Alper, S.L. (2011) ‘Transgenic plants overexpressing a plant vacuolar pyrophosphatase’, US Patent 8003852 B2, issued 23 August 2011.

Answer 9

Drought and salinity key points *In response to both stressors, plants often exhibit stomatal closure as a protective mechanism to conserve water, limiting gas exchange. *Both drought and salt stress result in water scarcity for plants, impacting essential physiological processes. *Both drought and salt stress can cause cellular damage due to the accumulation of reactive oxygen species, impacting plant cell structures. *Both drought and salt stress trigger molecular signalling pathways involved in stress response and adaptation (ROS, phytohormones (ABA, BR and others), compatible solutes) *Both stress conditions commonly result in stunted growth, as plants redirect resources to cope with the stress *Improving tolerance to one, may also lead to the improvement of other stresses that involve osmotic stress/water deficits – more on that next week