Differential gene expression patterns and physiological responses improve adaptation to high salinity concentration in pepper accessions.

High salinity decreases the productivity of crops worldwide. Pepper is particularly sensitive to high salt concentrations. Herein, we subjected three tolerant pepper accessions (C12, B14 and A25) to high sodium chloride concentration (70 mM NaCl). The aerial and root biomass, leaf and root osmotic potential (Ψπ ), Na+ , Cl- , K+ and proline concentrations and the relative expression of the putative genes CaSOS1, CaHKT1, three CaNHXs and CaP5CS were measured. Different salinity tolerance strategies depending on the pepper accession were identified. In C12, tolerance was attributed to the accumulation of Na+ in vacuoles and endosomes by the activation of vacuolar CaNHXs genes and the reduction in Ψπ ; additionally, the activation of CaHKT1 and CaSOS1 in leaves and roots moved and accumulated Na+ ions in the xylem and xylem parenchyma cells (XPC) as well as expulsed it out of the root cells. A25 accession, on the contrary, was specialized in compartmentalizing Na+ ions in root and leaf vacuoles and root XPC by the up-regulation of CaNHXs and CaHKT1, respectively, avoiding a toxic accumulation in leaves. Finally, B14 accession moved and accumulated Na+ in xylem and XPC, reducing its concentration in roots by the activation of CaSOS1 and CaHKT1. This study shade light on different tolerance mechanisms of pepper plants to overcome salt stress.

New insights into short-term water stress tolerance through transcriptomic and metabolomic analyses on pepper roots.

In the current climate change scenario, water stress is a serious threat to limit crop growth and yields. It is necessary to develop tolerant plants that cope with water stress and, for this purpose, tolerance mechanisms should be studied. NIBER® is a proven water stress- and salt-tolerant pepper hybrid rootstock (Gisbert-Mullor et al., 2020; López-Serrano et al., 2020), but tolerance mechanisms remain unclear. In this experiment, NIBER® and A10 (a sensitive pepper accession (Penella et al., 2014)) response to short-term water stress at 5 h and 24 h was studied in terms of gene expression and metabolites content in roots. GO terms and gene expression analyses evidenced constitutive differences in the transcriptomic profile of NIBER® and A10, associated with detoxification systems of reactive oxygen species (ROS). Upon water stress, transcription factors like DREBs and MYC are upregulated and the levels of auxins, abscisic acid and jasmonic acid are increased in NIBER®. NIBER® tolerance mechanisms involve an increase in osmoprotectant sugars (i.e., trehalose, raffinose) and in antioxidants (spermidine), but lower contents of oxidized glutathione compared to A10, which indicates less oxidative damage. Moreover, the gene expression for aquaporins and chaperones is enhanced. These results show the main NIBER® strategies to overcome water stress.

Short-term water stress responses of grafted pepper plants are associated with changes in the hormonal balance.

Phytohormones play an important role in regulating the plant behavior to drought. In previous studies, NIBER® pepper rootstock showed tolerance to drought in terms of production and fruit quality compared to ungrafted plants. In this study, our hypothesis was that short-term exposure to water stress in young, grafted pepper plants would shed light on tolerance to drought in terms of modulation of the hormonal balance. To validate this hypothesis, fresh weight, water use efficiency (WUE) and the main hormone classes were analyzed in self-grafted pepper plants (variety onto variety, V/V) and variety grafted onto NIBER® (V/N) at 4, 24, and 48h after severe water stress was induced by PEG addition. After 48h, WUE in V/N was higher than in V/V, due to major stomata closure to maintain water retention in the leaves. This can be explained by the higher abscisic acid (ABA) levels observed in the leaves of V/N plants. Despite the interaction between ABA and the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), in relation to stomata closure is controversial, we observed an important increase of ACC at the end of the experiment in V/N plants coinciding with an important rise of the WUE and ABA. The maximum concentration of jasmonic acid and salicylic acid after 48h was found in the leaves of V/N, associated with their role in abiotic stress signaling and tolerance. Respect to auxins and cytokinins, the highest concentrations were linked to water stress and NIBER®, but this effect did not occur for gibberellins. These results show that hormone balance was affected by water stress and rootstock genotype, where NIBER® rootstock displayed a better ability to overcome short-term water stress.

Grafting Enhances Pepper Water Stress Tolerance by Improving Photosynthesis and Antioxidant Defense Systems.

Currently, limited water supply is a major problem in many parts of the world. Grafting peppers onto adequate rootstocks is a sustainable technique used to cope with water scarcity in plants. For 1 month, this work compared grafted peppers by employing two rootstocks (H92 and H90), with different sensitivities to water stress, and ungrafted plants in biomass, photosynthesis, and antioxidant response terms to identify physiological-antioxidant pathways of water stress tolerance. Water stress significantly stunted growth in all the plant types, although tolerant grafted plants (variety grafted onto H92, Var/H92) had higher leaf area and fresh weight values. Var/H92 showed photosynthesis and stomata conductance maintenance, compared to sensitive grafted plants (Var/H90) and ungrafted plants under water stress, linked with greater instantaneous water use efficiency. The antioxidant system was effective in removing reactive oxygen species (ROS) that could damage photosynthesis; a significant positive and negative linear correlation was observed between the rate of CO2 uptake and ascorbic acid (AsA)/total AsA (AsAt) and proline, respectively. Moreover, in Var/H92 under water stress, both higher proline and ascorbate concentration were observed. Consequently, less membrane lipid peroxidation was quantified in Var/H92.

Uncovering salt tolerance mechanisms in pepper plants: a physiological and transcriptomic approach.

BACKGROUND: Pepper is one of the most cultivated crops worldwide, but is sensitive to salinity. This sensitivity is dependent on varieties and our knowledge about how they can face such stress is limited, mainly according to a molecular point of view. This is the main reason why we decided to develop this transcriptomic analysis. Tolerant and sensitive accessions, respectively called A25 and A6, were grown for 14 days under control conditions and irrigated with 70 mM of NaCl. Biomass, different physiological parameters and differentially expressed genes were analysed to give response to differential salinity mechanisms between both accessions. RESULTS: The genetic changes found between the accessions under both control and stress conditions could explain the physiological behaviour in A25 by the decrease of osmotic potential that could be due mainly to an increase in potassium and proline accumulation, improved growth (e.g. expansins), more efficient starch accumulation (e.g. BAM1), ion homeostasis (e.g. CBL9, HAI3, BASS1), photosynthetic protection (e.g. FIB1A, TIL, JAR1) and antioxidant activity (e.g. PSDS3, SnRK2.10). In addition, misregulation of ABA signalling (e.g. HAB1, ERD4, HAI3) and other stress signalling genes (e.g. JAR1) would appear crucial to explain the different sensitivity to NaCl in both accessions. CONCLUSIONS: After analysing the physiological behaviour and transcriptomic results, we have concluded that A25 accession utilizes different strategies to cope better salt stress, being ABA-signalling a pivotal point of regulation. However, other strategies, such as the decrease in osmotic potential to preserve water status in leaves seem to be important to explain the defence response to salinity in pepper A25 plants.

Effect of Grafting on the Production, Physico-Chemical Characteristics and Nutritional Quality of Fruit from Pepper Landraces.

Grafting is a widely utilized agronomical technique to improve yield, disease resistance, and quality of fruit and vegetables. This work aims to assess the effect of grafting and fruit ripening on the production, physico-chemical characteristics, and nutritional quality of fruit from Spanish local pepper landraces. Landraces "Cuerno," "Sueca," and "Valencia" were used as scions, and "NIBER®" as the rootstock. Two ripening stages of the fruits were sampled: green and red. Grafting improved the yield and marketable quality and did not negatively influence the physico-chemical and nutritional characteristics of the fruit. It was noteworthy that the bioactive compound contents and antioxidant capacity were more related to maturity stage and genotype, and red fruit had a higher antioxidant capacity than green fruit. However, in all the scions, grafting significantly enhanced lycopene content in both red and green fruit. Another important effect of grafting was the volatile compound composition evidenced by discriminant analyses, which was characterized for the first time in the fruit of these landraces. The rootstock and scion combination could be a way to improve not only the production, but also the fruit quality of peppers.

Physiological characterization of a pepper hybrid rootstock designed to cope with salinity stress.

In pepper crops, rootstocks that tolerate salt stress are not used because available commercial rootstocks offer limited profits. In this context, we obtained the hybrid NIBER®, a new salinity-tolerant rootstock that has been tested under real salinity field conditions for 3 years with 32%-80% higher yields than ungrafted pepper plants. This study aimed to set up the initial mechanisms involved in the salinity tolerance of grafted pepper plants using NIBER® as a rootstock to study root-shoot behavior, a basic requirement to develop efficient rootstocks. Gas exchange, Na+/K+, antioxidant capacity, nitrate reductase activity, ABA, proline, H2O2, phenols, MDA concentration and biomass were measured in ungrafted plants of cultivar Adige (A), self-grafted (A/A), grafted onto NIBER® (A/N) and reciprocal grafted plants (N/A), all exposed to 0 mM and 70 mM NaCl over a 10-day period. Salinity significantly and quickly decreased photosynthesis, stomatal conductance and nitrate reductase activity, but to lower extent in A/N plants compared to A, A/A and N/A. A/N plants showed decreases in the Na+/K+ ratio, ABA content and lipid peroxidation activity. This oxidative damage alleviation in A/N was probably due to an enhanced H2O2 level that activates antioxidant capacity to cope salinity stress, and acts as a signal molecule rather than a damaging one by contributing a major increase in phenols and, to a lesser extent, in proline concentration. These traits led to a minor impact on biomass in A/N plants under salinity conditions. Only the plants with the NIBER® rootstock controlled the scion by modulating responses to salinity.

Pepper Rootstock and Scion Physiological Responses Under Drought Stress.

In vegetables, tolerance to drought can be improved by grafting commercial varieties onto drought tolerant rootstocks. Grafting has emerged as a tool that copes with drought stress. In previous results, the A25 pepper rootstock accession showed good tolerance to drought in fruit production terms compared with non-grafted plants and other rootstocks. The aim of this work was to study if short-term exposure to drought in grafted plants using A25 as a rootstock would show tolerance to drought now. To fulfill this objective, some physiological processes involved in roots (rootstock) and leaves (scion) of grafted pepper plants were analyzed. Pepper plants not grafted (A), self-grafted (A/A), and grafted onto a tolerant pepper rootstock A25 (A/A25) were grown under severe water stress induced by PEG addition (-0.55 MPa) or under control conditions for 7 days in hydroponic pure solution. According to our results, water stress severity was alleviated by using the A25 rootstock in grafted plants (A/A25), which indicated that mechanisms stimulated by roots are essential to withstand stress. A/A25 had a bigger root biomass compared with plants A and A/A that resulted in better water absorption, water retention capacity and a sustained CO2 assimilation rate. Consequently, plants A/A25 had a better carbon balance, supported by greater nitrate reductase activity located mainly in leaves. In the non-grafted and self-grafted plants, the photosynthesis rate lowered due to stomatal closure, which limited transpiration. Consequently, part of NO3 - uptake was reduced in roots. This condition limited water uptake and CO2 fixation in plants A and A/A under drought stress, and accelerated oxidative damage by producing reactive oxygen species (ROS) and H2O2, which were highest in their leaves, indicating great sensitivity to drought stress and induced membrane lipid peroxidation. However, drought deleterious effects were slightly marked in plants A compared to A/A. To conclude, the A25 rootstock protects the scion against oxidative stress, which is provoked by drought, and shows better C and N balances that enabled the biomass to be maintained under water stress for short-term exposure, with higher yields in the field.

Salt-tolerant rootstock increases yield of pepper under salinity through maintenance of photosynthetic performance and sinks strength.

The performance of a salt-tolerant pepper (Capsicum annuum L.) accession (A25) utilized as a rootstock was assessed in two experiments. In a first field experiment under natural salinity conditions, we observed a larger amount of marketable fruit (+75%) and lower Blossom-end Root incidence (-31%) in commercial pepper cultivar Adige (A) grafted onto A25 (A/A25) when compared with ungrafted plants. In order to understand this behavior a second greenhouse experiment was conducted to determine growth, mineral partitioning, gas exchange and chlorophyll a fluorescence parameters, antioxidant systems and proline content in A and A/A25 plants under salinity conditions (80 mM NaCl for 14 days). Salt stress induced significantly stunted growth of A plants (-40.6% of leaf dry weight) compared to the control conditions, while no alterations were observed in A/A25 at the end of the experiment. Accumulation of Na(+) and Cl(-) in leaves and roots was similar in either grafted or ungrafted plants. Despite the activation of protective mechanisms (increment of superoxide dismutase, catalase, ascorbate peroxidase activity and non-photochemical quenching), A plants showed severely reduced photosynthetic CO2 assimilation (-45.6% of AN390) and substantial buildup of malondialdehyde (MDA) by-product, suggesting the inability to counteract salt-triggered damage. In contrast, A/A25 plants, which had a constitutive enhanced root apparatus, were able to maintain the shoot and root growth under salinity conditions by supporting the maintained photosynthetic performance. No increases in catalase and ascorbate peroxidase activities were observed in response to salinity, and MDA levels increased only slightly; indicating that alleviation of oxidative stress did not occur in A/A25 plants. In these plants the increased proline levels could protect enzymatic stability from salt-triggered damage, preserving the photosynthetic performance. The results could indicate that salt stress was vanished by the lack of negative effects on photosynthesis that support the maintained plant growth and increased marketable yield of the grafted plants.

Some rootstocks improve pepper tolerance to mild salinity through ionic regulation.

Grafting has been proposed as an interesting strategy that improves the responses of crops under salinity. In pepper, we reported increased fruit yield of the commercial 'Adige' cultivar under salinity when grafted onto accessions Capsicum chinense Jacq. 'ECU-973' (12) and Capsicum baccatum L. var. pendulum 'BOL-58' (14), whereas no effect was observed when grafted onto accession Capsicum annuum L var. 'Serrano' (5). We also analysed the physiological and biochemical mechanisms related to the tolerance conferred by these rootstocks. Responses to salinity (40 mM NaCl) were studied in the different plant combinations for 30 days by determining water relations, mineral content, proline accumulation, photosynthetic parameters, nitrate reductase activity and antioxidant capacity. Higher salt tolerance was achieved when the 'Adige' cultivar was grafted onto the 12 genotype, which allowed not only lower Na(+) and Cl(-) accumulation in the scion, but also ion selectivity maintenance, particularly Na(+)/K(+) discrimination. These traits led to a minor negative impact on photosynthesis, nitrate reductase activity and lipid peroxidation in grafted scion leaves. This work suggests that using tolerant pepper rootstocks that maintain the scion's ion homeostasis is a promising strategy to provide salinity tolerance and can consequently improve crop yield.

Rootstock alleviates PEG-induced water stress in grafted pepper seedlings: physiological responses.

Recent studies have shown that tolerance to abiotic stress, including water stress, is improved by grafting. In a previous work, we took advantage of the natural variability of Capsicum spp. and selected accessions tolerant and sensitive to water stress as rootstocks. The behavior of commercial cultivar 'Verset' seedlings grafted onto the selected rootstocks at two levels of water stress provoked by adding 3.5 and 7% PEG (polyethylene glycol) was examined over 14 days. The objective was to identify the physiological traits responsible for the tolerance provided by the rootstock in order to determine if the tolerance is based on the maintenance of the water relations under water stress or through the activation of protective mechanisms. To achieve this goal, various physiological parameters were measured, including: water relations; proline accumulation; gas exchange; chlorophyll fluorescence; nitrate reductase activity; and antioxidant capacity. Our results indicate that the effect of water stress on the measured parameters depends on the duration and intensity of the stress level, as well as the rootstock used. Under control conditions (0% PEG) all plant combinations showed similar values for all measured parameters. In general terms, PEG provoked a strong decrease in the gas exchange parameters in the cultivar grafted onto the sensitive accessions, as also observed in the ungrafted plants. This effect was related to lower relative water content in the plants, provoked by an inefficient osmotic adjustment that was dependent on reduced proline accumulation. At the end of the experiment, chronic photoinhibition was observed in these plants. However, the plants grafted onto the tolerant rootstocks, despite the reduction in photosynthetic rate, maintained the protective capacity of the photosynthetic machinery mediated by osmotic adjustment (based on higher proline content). In addition, water stress limited uptake and further NO3(-) transfer to the leaves. Increased nitrate reductase activity in the roots was observed, mainly in plants grafted onto the sensitive rootstocks, as well as the ungrafted plants, and this was associated with the lessened flux to the leaves. This study suggests that PEG-induced water stress can be partially alleviated by using tolerant accessions as rootstocks.