Screening for the salt-tolerant wheat lines with ethylene insensitivity was efficient and effective
Wheat (Triticum aestivum L.) is a staple crop in the world, but is only moderately salt tolerant . However, salt stress was reported to affect one-fifth of irrigated agricultural land in the world , it is of great importance to cultivate salt-tolerant varieties in order to improve the global wheat production. In this research, new wheat germplasm was generated by EMS mutagenesis based on a popular wheat variety ‘Luyuan 502’. The wheat variety is a semi-winter mid-maturing variety with cuboid spikes, long awns, white shells, white grains, strong growth, and excellent resistance to cold stress, rust and powdery mildew diseases, and is wildly planted in the northern part of Chinese Huanghuai winter wheat region, including Shandong Province, central and southern Hebei Province, and central and southern Shanxi Province of China. In this study, a large mutant pool was generated and screened for salt resistant lines.
Ethylene is one of plant hormones and plays an important role in plant salt-tolerance [29, 33, 34]. In dicotyledonous plants, the etiolated seedlings treated with ethylene precursor ACC showed inhibition of root and hypocotyl elongation, swelling of the hypocotyl and exaggeration of the apical hook, which is known as ‘triple response’ [35, 36]. However, in monocotyledonous plants, the responses is more complex. For example, in rice, ethylene only inhibits root growth, but promotes coleoptile growth of etiolated rice seedlings, which is collectedly known as ‘double response’ [33, 37–39]. Unfortunately, less has been known about ethylene related responses in wheat. In the present study, we tested the mutagenized wheat lines and found their root length was sensitive to the ethylene treatment. As a result, we used the root length of etiolated wheat seedlings to screen the ethylene insensitive mutants (Fig. 2G - I). Based on the relationship between ethylene insensitivity and salt tolerance in wheat, we screened the EMS-mutagenized pool and obtained 11 salt-tolerant wheat lines with high yield (Table 1). Our results proved the efficiency and effectiveness of the screening for salt tolerance and ethylene insensitivity in the wheat.
Transcriptomic analysis for alteration of the responsive gene expressions in salt-tolerant pathway
It was reported that salt stress induces ion imbalance and hyperosmotic effects to increase ROS concentration, damage chloroplast construct, exaggerate enzyme inefficiency, decrease photosynthesis, and accelerate photorespiration [9, 11, 40]. Plants also evolve in several responses to alleviate the negative effects of salt stress, which contribute to survival of plants in the salt stress. The salt-tolerant mechanism of new wheat mutant lines was excavated by transcriptome analysis in this research.
The transcriptomes of 2-week-old seedlings of the ethylene-insensitive salt-tolerant wheat lines 2–7 and 1–29 treated with 150 mM NaCl were compared with those of the wild type samples. The multiple comparisons of WT-vs-WT-S, 2-7-vs-2-7-S, and 1-29-vs-1-29-S showed the expression of CABs, PERs/PODs, BGLUs, CYP707s, ZEPs, and THICs in the antenna proteins in photosynthesis, biosynthesis of secondary metabolites, cyanoamino acid metabolism, carotenoid biosynthesis, thiamine metabolism, and cutin, suberine and wax biosynthesis pathways were significantly changed (Fig. 4; Supplementary material 2–4).
CABs bind chlorophyll a and b to make up light harvesting antenna complex, which absorbs light and transfer excitation energy to photosystems I and II in chloroplasts . In high plants, CABs are encoded by multiple genes in high plants. Such as, Arabidopsis contains 10 CABs, and tomato has 16 CABs in the genome [41, 42]. Recently, it was reported that the expression of CABs is induced by salt, indicating a significant role of CABs in salt resistance . In this research, 40 CABs including TRIAE_CS42_1AL_TGACv1_000383_AA0010660, TRIAE_CS42_1AL_TGACv1_000708_AA0017440, TRIAE_CS42_1BL_TGACv1_030603_AA0095440, TRIAE_CS42_1BL_TGACv1_032695_AA0133030, TRIAE_CS42_1DL_TGACv1_061419_AA0194670, and TRIAE_CS42_1DL_TGACv1_061593_AA0199400 were showed in the salt-tolerant pathway of wheat (Supplementary material 2–4). Due to the limited information about CABs in salt response, more studies definitely need to be done in the future.
PERs/PODs are enzymes that catalyze substrate oxidation with hydrogen peroxide as an electron receiver. In this research, 32 PERs/PODs including TRIAE_CS42_5DL_TGACv1_434349_AA1434390, TRIAE_CS42_5DL_TGACv1_435881_AA1455860, TRIAE_CS42_5DL_TGACv1_436164_AA1458860, TRIAE_CS42_6AL_TGACv1_472947_AA1527400, and TRIAE_CS42_7AL_TGACv1_556904_AA1773120, were detected in the salt-tolerant pathway (Supplementary material 2–4).
The BGLUs belong to subfamily I of glycoside hydrolases, which hydrolyze beta-glycosidic bonds to release terminal glucosyl residues from glycosides, oligosaccharides, and disaccharides . The BGLUs have been proved playing roles in plant growth, development, and biotic and abiotic resistance by releasing glucose from oligosaccharides in cell wall polysaccharides to change cell wall structures, activating defense compounds from inactive glycosides to defense against herbivores and fungi, releasing plant hormones from its inactive glyconjugates to satisfy the plants requirement, or releasing scent compounds from involatile precursor [45–47]. The specific function of a certain BGLU enzyme in plant growth, development, biotic and abiotic resistance depends on its expression pattern, substrate specificity, and different localization . The BGLUs also have been reported induced by salt treatment. For example, the CsBGLU12 in Crocus sativus was significantly induced by salt, and its transient overexpression leaves of tobacco accumulated antioxidant flavonols, which confer tolerance to salt stresses by alleviation ROS accumulation . In this research, 41 BGLUs including TRIAE_CS42_2BL_TGACv1_130794_AA0418050, TRIAE_CS42_3B_TGACv1_222633_AA0768330, TRIAE_CS42_3B_TGACv1_223344_AA0780610, TRIAE_CS42_3DL_TGACv1_250039_AA0861010, TRIAE_CS42_4AS_TGACv1_307764_AA1023360, and TRIAE_CS42_5AL_TGACv1_374195_AA1193240, were detected (Supplementary material 2–4). Considering the diverse functions of BGLUs in plants, the molecular mechanism of these BGLUs in salt-tolerance pathway definitely need to be explored.
WRKY transcription factors, which regulate genes by binding to a DNA cis-acting element called W-box, have important roles in response to salt stress . Overexpression of the wheat TaWRKY2 and TaWRKY19 in Arabidopsis led to stronger salt responses . It was also reported that salt induced expression of TaWRKY10, and overexpression of TaWRKY10 in tobacco led to enhance salt resistance including increased germination rate, root length, survival rate, relative water content, proline production, and soluble sugar contents, as well as decreased ROS and MDA contents . In addition, TdWRKY1, 3, 4, and 5 isolated from durum wheat were also induced by high-salt treatment, suggesting their possible roles in salt response . In this research, 12 WRKYs coding genes including TRIAE_CS42_7DS_TGACv1_623759_AA2055930, XLOC_076596, TRIAE_CS42_1AL_TGACv1_001348_AA0029060, TRIAE_CS42_1AL_TGACv1_002809_AA0045280, and TRIAE_CS42_1DL_TGACv1_062218_AA0210650, were also detected in the salt-tolerance pathway (Supplementary material 2–4), suggesting that they may play important roles in salt tolerance in wheat plants.
The wheat ERFs in response to ethylene and salt stress
ERF transcription factors regulate diverse biological processes in plant growth, development, abiotic and biotic stress responses by activating genes with GCC-box in the promoter [53, 54]. To date, 117 ERFs have been identified in bread wheat and only few ERFs including TaERF1, TaERF3, TaPIE1, TaPIEP1, TaERF4, and TaERF8-2B have been characterized. In details, TaERF8-2B, playing roles in plant growth and development, regulated plant architecture and yield related traits, and associates with plant height, heading date and 1000 kernel weight (TKW) . Overexpression of TaPIE1 in wheat accumulated high soluble sugars and proline contents, and improved freezing and necrotrophic pathogen Rhizoctonia cerealis tolerance . Overexpression of ERF transcription factor TaPIEP1 in wheat enhanced resistance to fungal pathogen Bipolaris sorokiniana .
The wheat ERF transcription factors were also reported playing roles in abiotic resistance. Overexpression of TaERF3 in bread wheat promoted tolerance to salt and drought stresses . TaERF1 contributes to increase drought, cold, and salt tolerance in transgenic Arabidopsis plants . While another ERF transcription factor TaERF4 might have opposite roles with TaERF1 and TaERF3 in plant abiotic tolerance, it was reported that overexpression of TaERF4 in Arabidopsis enhanced salt sensitivity, indicating the complex roles of ERF transcription factors in plant abiotic resistance .
Based on transcriptome sequencing, 9 novel wheat ERFs in response to ethylene and salt stress were identified in this research. Heatmap analysis showed that the expression levels of 2 ERFs TRIAE_CS42_5DL_TGACv1_432926_AA1394650 and TRIAE_CS42_6AS_TGACv1_486327_AA1559760 decreased significantly after salt treatment, suggesting they may paly negative regulation roles in ethylene regulated salt tolerance in wheat (Fig. 7C). At the same time, the expression levels of the other 7 ERFs including TRIAE_CS42_3DL_TGACv1_250510_AA0869450, TRIAE_CS42_6DL_TGACv1_526341_AA1680010, TRIAE_CS42_7AL_TGACv1_556681_AA1768690, TRIAE_CS42_7AS_TGACv1_569387_AA1814810, TRIAE_CS42_7DL_TGACv1_606411_AA2010000, TRIAE_CS42_7DS_TGACv1_621751_AA2025220, and TRIAE_CS42_7DS_TGACv1_623962_AA2057840, increased significantly after salt treatment, indicating their positive roles in ethylene regulated salt tolerance in wheat (Fig. 7C). Comparing with the previous results, it is suggested that the wheat ERFs may function differently in regulation of stress responses. Considering most of ERF transcription factors improve abiotic tolerance in crops without causing undesirable growth phenotypes , it is possible to manipulate the ERFs in response to ethylene and salt stress for improvement of wheat salt-tolerance breeding in the future.