Physiological and transcriptomic analyses reveal novel insights into the cultivar-specific response to alkaline stress in alfalfa (Medicago sativa L.).

Soil alkalization severely limits plant growth and development, however, the mechanisms of alkaline response in plants remain largely unknown. In this study, we performed physiological and transcriptomic analyses using two alfalfa cultivars (Medicago sativa L.) with different sensitivities to alkaline conditions. The chlorophyll content and shoot fresh mass drastically declined in the alkaline-sensitive cultivar Algonquin (AG) following alkaline treatment (0-25 mM Na2CO3 solution), while the alkaline-tolerant cultivar Gongnong NO.1 (GN) maintained relatively stable growth and chlorophyll content. Compared with AG, GN had higher contents of Ca2+ and Mg2+; the ratios of Ca2+ and Mg2+ to Na+, proline and soluble sugar, as well as higher enzyme activities of peroxidase (POD) and catalase (CAT) under the alkaline conditions. Furthermore, transcriptomic analysis identified three categories of alkaline-responsive differentially expressed genes (DEGs) between the two cultivars: 48 genes commonly induced in both the cultivars (CAR), 574 genes from the tolerant cultivar (TAR), and 493 genes from the sensitive cultivar (SAR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that CAR genes were mostly involved in phenylpropanoid biosynthesis, lipid metabolism, and DNA replication and repair; TAR genes were significantly enriched in metabolic pathways, such as biosynthesis of amino acids and secondary metabolites including flavonoids, and the MAPK signaling pathway; SAR genes were specifically enriched in vitamin B6 metabolism. Taken together, the results identified candidate pathways associated with genetic variation in response to alkaline stress, providing novel insights into the mechanisms underlying alkaline tolerance in alfalfa.

Silicon Priming Created an Enhanced Tolerance in Alfalfa (Medicago sativa L.) Seedlings in Response to High Alkaline Stress.

Alkaline stress as a result of higher pH usually triggers more severe physiological damage to plants than that of saline stress with a neutral pH. In the present study, we demonstrated that silicon (Si) priming of alfalfa (Medicago sativa L.) seedlings increased their tolerance to high alkaline stress situations. Gongnong No. 1 seedlings were subjected to alkaline stress simulated by 25 mM Na2CO3 (pH 11.2). Alkaline stress greatly decreased the biomass and caused severe lodging or wilting of alfalfa seedlings. In contrast, the application of Si to alfalfa seedlings 36 h prior to the alkaline treatment significantly alleviated the damage symptoms and greatly increased the biomass and chlorophyll content. Because of being concomitant with increasing photosynthesis and water use efficiency, decreasing membrane injury and malondialdehyde content, and increasing peroxidase and catalase ascorbate activities in alfalfa leaves, thereby alleviating the triggered oxidative damage by alkaline stress to the plant. Furthermore, Si priming significantly decreased the accumulation of protein and proline content in alfalfa, thus reducing photosynthetic feedback repression. Si priming significantly accumulated more Na in the roots, but led to a decrease of Na accumulation and an increase of K accumulation in the leaves under alkaline stress. Meanwhile, Si priming decreased the accumulation of metal ions such as Mg, Fe, Mn, and Zn in the roots of alfalfa seedlings under alkaline stress. Collectively, these results suggested that Si is involved in the metabolic or physiological changes and has a potent priming effect on the alkaline tolerance of alfalfa seedlings. The present study indicated that Si priming is a new approach to improve the alkaline tolerance in alfalfa and provides increasing information for further exploration of the alkaline stress response at the molecular level in alfalfa.

A Multi-year Beneficial Effect of Seed Priming with Gibberellic Acid-3 (GA3) on Plant Growth and Production in a Perennial Grass, Leymus chinensis.

Seed priming is a widely used technique in crops to obtain uniform germination and high-quality seedlings. In this study, we found a long-term effect of seed priming with gibberellic acid-3 (GA3) on plant growth and production in Leymus chinensis. Seeds were germinated on agar plates containing 0-200 µM GA3, and the germinated seedlings were transplanted to clay planting pots and grown for about one year. The clonal tillers grown from the mother plants were transplanted to field conditions in the second year. Results showed that GA3 treatment significantly increased seed germination rate by 14-27%. GA3 treatment also promoted subsequent plant growth and biomass production, as shown by a significant increase in plant height, tiller number, and fresh and dry weight in both pot (2016) and field (2017) conditions. It is particularly noteworthy that the growth-promoting effect of a single seed treatment with GA3 lasted for at least two years. In particular, GA3 treatment at 50 µM increased aboveground fresh and dry weight by 168.2% and 108.9% in pot-grown conditions, and 64.5% and 126.2% in field-grown conditions, respectively. These results imply a transgenerational transmission mechanism for the GA-priming effect on clonal offspring growth and biomass production in L. chinensis.

Stunted Growth Caused by Blast Disease in Rice Seedlings Is Associated with Changes in Phytohormone Signaling Pathways.

In response to pathogen attack, plants prioritize defense reactions generally at the expense of plant growth. In this work, we report that changes in phytohormone signaling pathways are associated with the stunted plant growth caused by blast disease in rice seedlings. Infection of rice seedlings with blast fungus Magnaporthe oryzae (race 007.0) at the four-leaf stage (three true leaves) resulted in considerable inhibition of the growth of the upper uninfected distal leaves; the length of leaf blade and leaf sheath of the sixth and seventh leaf was reduced by 27 and 82%, and 88 and 72%, respectively, compared to that in the uninoculated plant control. Interestingly, cutting off the blast-infected fourth leaf blade within 2 days post inoculation (dpi) significantly rescued the inhibition of leaf growth, implying that an inhibitory substance(s) and/or signal was generated in the blast-infected leaves (fourth leaf) and transmitted to the upper distal leaves (sixth and seventh) during the 2-dpi period that induced growth inhibition. Expression analysis of marker genes for phytohormone pathways revealed acute activation of the jasmonate (JA) and abscisic acid (ABA) signaling pathways, and repression of auxin, gibberellic acid (GA) and salicylic acid (SA) signaling pathways, in the sixth leaf. The genes related to cell wall expansion were also significantly downregulated. In the blast-infected fourth leaf, JA pathway was activated within 2 dpi, followed by activation of ABA pathway 3 dpi. Further, leaf inhibition caused by blast infection was partially rescued in the rice mutant line coleoptile photomorphogenesis 2 (cpm2), which is defective in the gene encoding allene oxide cyclase (OsAOC). These results indicate that the JA signaling pathway is at least partly involved in the growth inhibition processes. Collectively, our data suggest that, upon pathogen attack, rice seedlings prioritize defense reactions against the infecting pathogen by temporarily ceasing plant growth through the systemic control of phytohormone pathways.

Root Damage under Alkaline Stress Is Associated with Reactive Oxygen Species Accumulation in Rice (Oryza sativa L.).

Alkaline stress (high pH) severely damages root cells, and consequently, inhibits rice (Oryza sativa L.) seedling growth. In this study, we demonstrate the accumulation of reactive oxygen species (ROS) in root cells under alkaline stress. Seedlings of two rice cultivars with different alkaline tolerances, 'Dongdao-4' (moderately alkaline-tolerant) and 'Jiudao-51' (alkaline-sensitive), were subjected to alkaline stress simulated by 15 mM sodium carbonate (Na2CO3). Alkaline stress greatly reduced seedling survival rate, shoot and root growth, and root vigor. Moreover, severe root cell damage was observed under alkaline stress, as shown by increased membrane injury, malondialdehyde accumulation, and Evan's Blue staining. The expression of the cell death-related genes OsKOD1, OsHsr203j, OsCP1, and OsNAC4 was consistently upregulated, while that of a cell death-suppressor gene, OsBI1, was downregulated. Analysis of the ROS contents revealed that alkaline stress induced a marked accumulation of superoxide anions ([Formula: see text]) and hydrogen peroxide (H2O2) in rice roots. The application of procyanidins (a potent antioxidant) to rice seedlings 24 h prior to alkaline treatment significantly alleviated alkalinity-induced root damage and promoted seedling growth inhibition, which were concomitant with reduced ROS accumulation. These results suggest that root cell damage, and consequently growth inhibition, of rice seedlings under alkaline stress is closely associated with ROS accumulation. The antioxidant activity of superoxide dismutase, catalase, peroxidase, and ascorbate peroxidase increased under alkaline stress in the roots, probably in response to the cellular damage induced by oxidative stress. However, this response mechanism may be overwhelmed by the excess ROS accumulation observed under stress, resulting in oxidative damage to root cells. Our findings provide physiological insights into the molecular mechanisms of alkalinity-induced damage to root cells, and will contribute to the improvement of alkaline stress tolerance in rice plants.

Priming effect of abscisic acid on alkaline stress tolerance in rice (Oryza sativa L.) seedlings.

Saline-alkaline stress is characterized by high salinity and high alkalinity (high pH); alkaline stress has been shown to be the primary factor inhibiting rice seedling growth. In this study, we investigated the potential priming effect of abscisic acid (ABA) on tolerance of rice seedlings to alkaline stress simulated by Na2CO3. Seedlings were pretreated with ABA at concentrations of 0 (control), 10, and 50 µM by root-drench for 24 h and then transferred to a Na2CO3 solution that did not contain ABA. Compared to control treatment, pretreatment with ABA substantially improved the survival rate of rice seedlings and increased biomass accumulation after 7 days under the alkaline condition. ABA application at 10 µM also alleviated the inhibitory effects of alkaline stress on the total root length and root surface area. Physiologically, ABA increased relative water content (RWC) and decreased cell membrane injury degree (MI) and Na(+)/K(+) ratios. In contrast, fluridone (an ABA biosynthesis inhibitor) decreased the RWC and increased MI in shoots under the alkaline conditions. These data suggest that ABA has a potent priming effect on the adaptive response to alkaline stress in rice and may be useful for improving rice growth in saline-alkaline paddy fields.

[Effects of storage conditions and sowing methods on seed germination of Leymus chinensis].

This paper studied the effects of four storage conditions (dry storage at 4 degrees C, dry storage at room temperature, storage in moist non-saline-alkali soil at 4 degrees C, and storage in moist saline-alkali soil at 4 degrees C), three germination substrates (filter paper, non-saline-alkali soil, and saline-alkali soil), and two sowing methods (sowing on and in the substrates) on the seed germination of Leymus chinensis. The results showed that compared with the dry storage at room temperature, dry storage at 4 degrees C improved the germination velocity significantly, but had less effect on germination rate. When stored in moist saline-alkali soil, the germination rate was only 28.8%, in contrast to 73.7% in non-saline-alkali soil. With different sowing methods, the seed germination rates of L. chinensis was in the order of sowing on the surface of non-saline-alkali soil (89.3%) > between papers (68.0%) > on papers (64.0%) > in non-saline-alkali soil (61.3%) > on the surface of saline-alkali soil (25.6%) > in saline-alkali soil (6.0%).