The apple Ca2+/H+ exchanger MdCAX2L-2 functions positively in modulation of Ba2+ tolerance.

Calcium is an essential element for plant growth and development, and it plays an important role in the responses of plants to abiotic stress. High concentrations of heavy metal ions in soil significantly affect the yield and quality of crops and pose human health threats when these ions accumulate in edible organs. The Ca2+/H+ exchanger (CAX) family is a class of transporters that mediate the transmembrane transport of both Ca2+ and metal ions, and they are widely involved in regulating plant growth and development and stress responses. Here, we cloned an AtCAX2 ortholog, MdCAX2L-2, from apple. It is constitutively expressed in various apple tissues and significantly induced by Ca2+ and Ba2+ treatments. The MdCAX2L-2 protein is located in the vacuolar membrane in both plant and yeast cells. Overexpression of MdCAX2L-2 enhanced the tolerance of the yeast mutant K667 to high concentrations of Ca2+ and Ba2+. In addition, the role of MdCAX2L-2 in modulating Ba2+ tolerance was identified using MdCAX2L-2-overexpressing transgenic Arabidopsis plants and apple calli. Comparison of growth phenotypes and stress-related physiological indexes under BaCl2 treatment indicated that MdCAX2L-2 could enhance the Ba2+ tolerance of plants by promoting Ba2+ compartmentalization into the vacuoles and eliminating excess ROS. Our results provide insights that will aid future studies examining the function of CAX proteins in regulating stress tolerance in fruit crops, as well as their underlying mechanisms.

MdNAC104 positively regulates apple cold tolerance via CBF-dependent and CBF-independent pathways.

Low temperature is the main environmental factor affecting the yield, quality and geographical distribution of crops, which significantly restricts development of the fruit industry. The NAC (NAM, ATAF1/2 and CUC2) transcription factor (TF) family is involved in regulating plant cold tolerance, but the mechanisms underlying these regulatory processes remain unclear. Here, the NAC TF MdNAC104 played a positive role in modulating apple cold tolerance. Under cold stress, MdNAC104-overexpressing transgenic plants exhibited less ion leakage and lower ROS (reactive oxygen species) accumulation, but higher contents of osmoregulatory substances and activities of antioxidant enzymes. Transcriptional regulation analysis showed that MdNAC104 directly bound to the MdCBF1 and MdCBF3 promoters to promote expression. In addition, based on combined transcriptomic and metabolomic analyses, as well as promoter binding and transcriptional regulation analyses, we found that MdNAC104 stimulated the accumulation of anthocyanin under cold conditions by upregulating the expression of anthocyanin synthesis-related genes, including MdCHS-b, MdCHI-a, MdF3H-a and MdANS-b, and increased the activities of the antioxidant enzymes by promoting the expression of the antioxidant enzyme-encoding genes MdFSD2 and MdPRXR1.1. In conclusion, this study revealed the MdNAC104 regulatory mechanism of cold tolerance in apple via CBF-dependent and CBF-independent pathways.

The cold-stress responsive gene DREB1A involved in low-temperature tolerance in Xinjiang wild walnut.

Background: Low-temperatures have the potential to be a serious problem for plants and can negatively affect the normal growth and development of walnuts. DREB1/CBF (Dehydration Responsive Element Binding Protein 1/C-repeat Binding Factor), one of the most direct transcription factors in response to low-temperature stress, may improve the resistance of plants to low-temperatures by regulating their functional genes. However, few studies have been conducted in walnut. The Xinjiang wild walnut is a rare wild plant found in China, with a large number of excellent trait genes, and is hardier than cultivated walnuts in Xinjiang. Methods: In this work, we identified all of the DREB1 members from the walnut genome and analyzed their expression levels in different tissues and during low-temperature stress on the Xinjiang wild walnut. The JfDREB1A gene of the Xinjiang wild walnut was cloned and transformed into Arabidopsis thaliana for functional verification. Results: There were five DREB1 transcription factors in the walnut genome. Among them, the relative expression level of the DREB1A gene was significantly higher than other members in the different tissues (root, stem, leaf) and was immediately un-regulated under low-temperature stress. The overexpression of the JfDREB1A gene increased the survival rates of transgenic Arabidopsis lines, mainly through maintaining the stability of cell membrane, decreasing the electrical conductivity and increasing the activities of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Additionally, the expression levels of cold-inducible genes like AtKIN1, AtERD10, AtRD29A, AtCOR15A and AtCOR47, were significantly increased. These results showed that the JfDREB1A gene may play an important role in the response to cold stress of the Xinjiang wild walnut. This study contributes to our understanding of the molecular mechanism of the Xinjiang wild walnut's response to low-temperature stress and will be beneficial for developing walnut cultivars with improved cold resistance.

Transcriptome profiling based on Illumina- and SMRT-based RNA-seq reveals circadian regulation of key pathways in flower bud development in walnut.

Flower bud development is a defining feature of walnut, which contributes to the kernel yield, yield stability, fruit quality and commodity value. However, little is known about the mechanism of the flower bud development in walnut. Here, the stages of walnut female flower bud development were divided into five period (P01-05) by using histological observation. They were further studied through PacBio Iso-Seq and RNA-seq analysis. Accordingly, we obtained 52,875 full-length transcripts, where 4,579 were new transcripts, 3,065 were novel genes, 1,437 were consensus lncRNAs and 20,813 were alternatively spliced isoforms. These transcripts greatly improved the current genome annotation and enhanced our understanding of the walnut transcriptome. Next, RNA sequencing of female flower buds at five periods revealed that circadian rhythm-plant was commonly enriched along with the flower bud developmental gradient. A total of 14 differentially expressed genes (DEGs) were identified, and six of them were confirmed by real-time quantitative analysis. Additionally, six and two differentially expressed clock genes were detected to be regulated by AS events and lncRNAs, respectively. All these detected plant circadian genes form a complex interconnected network to regulate the flower bud development. Thus, investigation of key genes associated with the circadian clock could clarify the process of flower bud development in walnut.

Integrated transcriptomic and proteomic analysis reveals the complex molecular mechanisms underlying stone cell formation in Korla pear.

Korla pear (Pyrus sinkiangensis Yü) is a landrace selected from a hybrid pear species in the Xinjiang Autonomous Region in China. In recent years, pericarp roughening has been one of the major factors that adversely affects fruit quality. Compared with regular fruits, rough-skin fruits have a greater stone cell content. Stone cells compose sclerenchyma tissue that is formed by secondary thickening of parenchyma cell walls. In this work, we determined the main components of stone cells by isolating them from the pulp of rough-skin fruits at the ripening stage. Stone cell staining and apoptosis detection were then performed on fruit samples that were collected at three different developmental stages (20, 50 and 80 days after flowering (DAF)) representing the prime, late and stationary stages of stone cell differentiation, respectively. The same batches of samples were used for parallel transcriptomic and proteomic analysis to identify candidate genes and proteins that are related to SCW biogenesis in Korla pear fruits. The results showed that stone cells are mainly composed of cellulose (52%), hemicellulose (23%), lignin (20%) and a small amount of polysaccharides (3%). The periods of stone cell differentiation and cell apoptosis were synchronous and primarily occurred from 0 to 50 DAF. The stone cell components increased abundantly at 20 DAF but then decreased gradually. A total of 24,268 differentially expressed genes (DEGs) and 1011 differentially accumulated proteins (DAPs) were identified from the transcriptomic and proteomic data, respectively. We screened the DEGs and DAPs that were enriched in SCW-related pathways, including those associated with lignin biosynthesis (94 DEGs and 31 DAPs), cellulose and xylan biosynthesis (46 DEGs and 18 DAPs), S-adenosylmethionine (SAM) metabolic processes (10 DEGs and 3 DAPs), apoplastic ROS production (16 DEGs and 2 DAPs), and cell death (14 DEGs and 6 DAPs). Among the identified DEGs and DAPs, 63 significantly changed at both the transcript and protein levels during the experimental periods. In addition, the majority of these identified genes and proteins were expressed the most at the prime stage of stone cell differentiation, but their levels gradually decreased at the later stages.

Full-length transcriptome and targeted metabolome analyses provide insights into defense mechanisms of Malus sieversii against Agrilus mali.

Malus sieversii is the wild progenitor for many cultivars of domesticated apple and an important germplasm resource for breeding. However, this valuable species faces a significant threat in the areas north of the Tianshan Mountains in China, by the invasion of Agrilus mali, a destructive pest of apple trees belonging to the family Buprestidae. Our preliminary study has has shown that there may be resistance to this insect in M. sieversii plants in the field, but the corresponding molecular mechanisms remain unclear. In this study, we compared the response of insect-resistant and insect-susceptible plants of M. sieversii to insect feeding using full-length transcriptome and targeted metabolome. 112,103 non-chimeric full-length reads (FLNC) totaling 10.52 Gb of data were generating with Pacific Biosciences SingleMolecule, Real-Time (PacBio SMRT) sequencing. A total of 130.06 Gb data of long reads were acquired with an Illumina HiSeq. Function annotation indicated that the different expressed genes (DEGs) were mainly involved in signal transduction pathway of plant hormones and in the synthesis of compounds such as terpenes, quinones, flavonoids, and jasmonic acid. Through targeted metabolome analysis resistant strains showed higher levels of trans-cinnamic acid, caffeine and ferulic acid after pest infestation. This study helps to decipher the transcriptional changes and related signaling paths in M. sieversii after an insect feeding, which lays a foundation for further research on molecular mechanisms of insect resistance in apples.