Cold stress is an important factor that limits apple production.  In this study, we examined the tissue-cultured plantlets of apple rootstocks ‘M9T337’ and ‘60-160’, which are resistant and sensitive to cold stress, respectively.  The enriched pathways of differentially expressed genes (DEGs) and physiological changes in ‘M9T337’ and ‘60-160’ plantlets were clearly different after cold stress (1°C) treatment for 48 h, suggesting that they have differential responses to cold stress.  The differential expression of WRKY transcription factors in the two plantlets showed that MdWRKY40is and MdWRKY48 are potential regulators of cold tolerance.  When we overexpressed MdWRKY40is and MdWRKY48 in apple calli, the overexpression of MdWRKY48 had no significant effect on the callus, while MdWRKY40is overexpression promoted anthocyanin accumulation, increased callus cold tolerance, and promoted the expression of anthocyanin structural gene MdDFR and cold-signaling core gene MdCBF2.  Yeast one-hybrid screening and electrophoretic mobility shift assays showed that MdWRKY40is could only bind to the MdDFR promoter.  Yeast two-hybrid screening and bimolecular fluorescence complementation showed that MdWRKY40is interacts with the CBF2 inhibitor MdMYB15L through the leucine zipper (LZ).  When the LZ of MdWRMY40is was knocked out, MdWRKY40is overexpression in the callus did not affect MdCBF2 expression or callus cold tolerance, indicating that MdWRKY40is acts in the cold signaling pathway by interacting with MdMYB15L.  In summary, MdWRKY40is can directly bind to the MdDFR promoter in order to promote anthocyanin accumulation, and it can also interact with MdMYB15L to interfere with its inhibitory effect on MdCBF2, indirectly promoting MdCBF2 expression, and thereby improving cold tolerance.  These results provide a new perspective for the cold-resistance mechanism of apple rootstocks and a molecular basis for the screening of cold-resistant rootstocks.

Rye (Secale cereale L., 2n=2x=14, RR) is a significant genetic resource for improving common wheat because of its resistance to multiple diseases and abiotic-stress tolerant traits.  The 1RS chromosome from the German cultivated rye variety Petkus is critical in wheat breeding.  However, its weakened disease resistance highlights the need to identify new resources.  In the present study, a novel derived line called D27 was developed from common wheat and Mexico Rye.  Cytological observations characterized the karyotype of D27 as 2n=42=21 II.  Genomic in situ hybridization indicated that a pair of whole-arm translocated Mexico Rye chromosomes were inherited typically in the mitotic and meiosis stages of D27.  Experiments using fluorescence in situ hybridization (FISH) and gliadin electrophoresis showed that D27 lacked wheat 1DS chromosomes.  They were replaced by 1RS chromosomes of Mexico Rye, supported by wheat simple-sequence repeat markers, rye sequence characterized amplified region markers, and wheat 40K SNP array analysis.  The wheat 1DS chromosomes could not be detected by molecular markers and wheat SNP array, but the presence of rye 1RS chromosomes was confirmed.  Agronomic trait assessments indicated that D27 had a higher tiller number and enhanced stripe rust and powdery mildew resistance.  In addition, dough properties analysis showed that replacing 1DS led to higher viscosity and lower dough elasticity in D27, which was beneficial for cake making.  In conclusion, the novel cytogenetically stable common wheat–Mexico Rye T1DL·1RS translocation line D27 offers excellent potential as outstanding germplasm in wheat breeding programs focusing on disease resistance and yield improvement.  Additionally, it can be valuable for researching the rye 1RS chromosome’s genetic diversity. 

Drought and salt stresses, the major environmental abiotic stresses in agriculture worldwide, affect plant growth, crop productivity, and quality.  Therefore, developing crops with higher drought and salt tolerance is highly desirable.  This study reported the isolation, biological function, and molecular characterization of a novel maspardin gene, OsMas1, from rice.  The OsMas1 protein was localized to the cytoplasm.  The expression levels of OsMas1 were up-regulated under mannitol, PEG6000, NaCl, and abscisic acid (ABA) treatments in rice.  The OsMas1 gene was introduced into the rice cultivar Zhonghua 11 (wild type, WT).  OsMas1-overexpression (OsMas1-OE) plants exhibited significantly enhanced salt and drought tolerance; in contrast, OsMas1-interference (OsMas1-RNAi) plants exhibited decreased tolerance to salt and drought stresses, compared with WT.  OsMas1-OE plants exhibited enhanced hypersensitivity, while OsMas1-RNAi plants showed less sensitivity to exogenous ABA treatment at both germination and post-germination stages.  ABA, proline and K⁺ contents and superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and photosynthesis activities were significantly increased.  In contrast, malonaldehyde (MDA), hydrogen peroxide (H₂O₂), superoxide anion radical (O₂^-·), and Na⁺ contents were significantly decreased in OsMas1-OE plants compared with OsMas1-RNAi and WT plants.  Overexpression of OsMas1 up-regulated the genes involved in ABA signaling, proline biosynthesis, reactive oxygen species (ROS)-scavenging system, photosynthesis, and ion transport under salt and drought stresses.  Our results indicate that the OsMas1 gene improves salt and drought tolerance in rice, which may serve as a candidate gene for enhancing crop resistance to abiotic stresses.

Psathyrostachys huashanica Keng (2n=2x=14, NsNs) is regarded as a valuable wild relative species for common wheat cultivar improvement because of its abundant beneficial agronomic traits.  However, although the development of many wheat–P. huashanica-derived lines provides a germplasm base for the transfer of excellent traits, the lag in the identification of P. huashanica chromosomes in the wheat background has limited the study of these lines.  In this study, three novel nondenaturing fluorescence in situ hybridization (ND-FISH)-positive oligo probes were developed.  Among them, HS-TZ3 and HS-TZ4 could specifically hybridize with P. huashanica chromosomes, mainly in the telomere area, and HS-CHTZ5 could hybridize with the chromosomal centromere area.  We sequentially constructed a P. huashanica FISH karyotype and idiogram that helped identify the homologous groups of introduced P. huashanica chromosomes.  In detail, 1Ns and 2Ns had opposite signals on the short and long arms, 3Ns, 4Ns, and 7Ns had superposed two-color signals, 5Ns and 6Ns had fluorescent signals only on their short arms, and 7Ns had signals on the intercalary of the long arm.  In addition, we evaluated different ways to identify alien introgression lines by using low-density single nucleotide polymorphism (SNP) arrays and recommended the SNP homozygosity rate in each chromosome as a statistical pattern.  The 15K SNP array is widely applicable for addition, substitution, and translocation lines, and the 40K SNP array is the most accurate for recognizing transposed intervals between wheat and alien chromosomes.  Our research provided convenient methods to distinguish the homologous group of P. huashanica chromosomes in a common wheat background based on ND-FISH and SNP arrays, which is of great significance for efficiently identifying wheat–P. huashanica-derived lines and the further application of Ns chromosomes