HKT1;1 and HKT1;2 Na+ Transporters from Solanum galapagense Play Different Roles in the Plant Na+ Distribution under Salinity

Salt tolerance is a target trait in plant science and tomato breeding programs. Wild tomato accessions have been often explored for this purpose. Since shoot Na⁺/K⁺ is a key component of salt tolerance, RNAi-mediated knockdown isogenic lines obtained for Solanum galapagense alleles encoding both class I Na⁺ transporters HKT1;1 and HKT1;2 were used to investigate the silencing effects on the Na and K contents of the xylem sap, and source and sink organs of the scion, and their contribution to salt tolerance in all 16 rootstock/scion combinations of non-silenced and silenced lines, under two salinity treatments. The results show that SgHKT1;1 is operating differently from SgHKT1;2 regarding Na circulation in the tomato vascular system under salinity. A model was built to show that using silenced SgHKT1;1 line as rootstock would improve salt tolerance and fruit quality of varieties carrying the wild type SgHKT1;2 allele. Moreover, this increasing effect on both yield and fruit soluble solids content of silencing SgHKT1;1 could explain that a low expressing HKT1;1 variant was fixed in S. lycopersicum during domestication, and the paradox of increasing agronomic salt tolerance through silencing the HKT1;1 allele from S. galapagense, a salt adapted species.     

Root-to-Shoot Long-Distance Mobile miRNAs Identified from Nicotiana Rootstocks

Root-derived mobile signals play critical roles in coordinating a shoot’s response to underground conditions. However, the identification of root-to-shoot long-distance mobile signals has been scant. In this study, we aimed to characterize root-to-shoot endogenous mobile miRNAs by using an Arabidopsis/Nicotiana interfamilial heterograft in which these two taxonomically distant species with clear genetic backgrounds had sufficient diversity in differentiating miRNA sources. Small RNA deep sequencing analysis revealed that 82 miRNAs from the Arabidopsis scion could travel through the graft union to reach the rootstock, whereas only a very small subset of miRNA (6 miRNAs) preferred the root-to-shoot movement. We demonstrated in an ex vivo RNA imaging experiment that the root-to-shoot mobile Nb-miR164, Nb-miR395 and Nb-miR397 were targeted to plasmodesmata using the bacteriophage coat protein MS2 system. Furthermore, the Nb-miR164 was shown to move from the roots to the shoots to induce phenotypic changes when its overexpressing line was used as rootstock, strongly supporting that root-derived Nb-miR164 was able to modify the scion trait via its long-distance movement.     

Overexpression of a Weed (Solanum americanum) Proteinase Inhibitor in Transgenic Tobacco Results in Increased Glandular Trichome Density and Enhanced Resistance to Helicoverpa armigera and Spodoptera litura

In this study we produced transgenic tobacco plants by overexpressing a serine proteinase inhibitor gene, SaPIN2a, from the American black nightshade Solanum americanum under the control of the CaMV 35S promoter using Agrobacterium tumefaciens-mediated transformation. SaPIN2a was properly transcribed and translated as indicated by Northern blot and Western blot analyses. Functional integrity of SaPIN2a in transgenic plants was confirmed by proteinase inhibitory activity assay. Bioassays for insect resistance showed that SaPIN2a-overexpressing transgenic tobacco plants were more resistant to cotton bollworm (Helicoverpa armigera) and tobacco cutworm (Spodoptera litura) larvae, two devastating pests of important crop plants, than the control plants. Interestingly, overexpression of SaPIN2a in transgenic tobacco plants resulted in a significant increase in glandular trichome density and a promotion of trichome branching, which could also provide an additional resistance mechanism in transgenic plants against insect pests. Therefore, SaPIN2a could be used as an alternative proteinase inhibitor for the production of insect-resistant transgenic plants.     

Ectopic Expression of CsSUN in Tomato Results in Elongated Fruit Shape via Regulation of Longitudinal Cell Division

Fruit shape, an important agronomic trait of cucumber (Cucumis sativus L.), is tightly controlled by a series of genes such as CsSUN, a homologue of SlSUN that is responsible for the tomato (Solanum lycopersicum) fruit shape via the modulation of cell division. However, the direct genetic evidence about the CsSUN-mediated regulation of fruit shape is still scarce, limiting our mechanistic understanding of the biological functions of CsSUN. Here, we introduced CsSUN into the round-fruited tomato inbred line ‘SN1′ (wild type, WT) via the Agrobacterium tumefaciens-mediated method. The high and constitutive expression of CsSUN was revealed by real-time PCR in all the tested tissues of the transgenic plants, especially in the fruits and ovaries. Phenotypic analyses showed that the ectopic expression of CsSUN increased fruit length while it decreased fruit diameter, thus leading to the enhanced fruit shape index in the transgenic tomato lines relative to the WT. Additionally, the reduction in the seed size and seed-setting rate and the stimulation of seed germination were observed in the CsSUN-expressed tomato. A histological survey demonstrated that the elongated fruits were mainly derived from the significant increasing of the longitudinal cell number, which compensated for the negative effects of decreased cell area in the central columellae. These observations are different from action mode of SlSUN, thus shedding new insights into the SUN-mediated regulation of fruit shape.     

Effects of Elevated Peroxidase Levels and Corn Earworm Feeding on Gene Expression in Tomato

Microarray analysis was used to measure the impact of herbivory by Helicoverpa zea, (corn earworm caterpillar) on wild-type and transgenic tomato, Solanum lycopersicum, plants that over-express peroxidase. Caterpillar herbivory had by far the greatest affect on gene expression, but the peroxidase transgene also altered the expression of a substantial number of tomato genes. Particularly high peroxidase activity resulted in the up-regulation of genes encoding proteinase inhibitors, pathogenesis-related (PR) proteins, as well as proteins associated with iron and calcium transport, and flowering. In a separate experiment conducted under similar conditions, real-time quantitative polymerase chain reaction (qPCR) analysis confirmed our microarray results for many genes. There was some indication that multiple regulatory interactions occurred due to the interaction of the different treatments. While herbivory had the greatest impact on tomato gene expression, our results suggest that levels of expression of a multifunctional gene, such as peroxidase and its products, can influence other gene expression systems distinct from conventional signaling pathways, further indicating the complexity of plant defensive responses to insects.     

Lack of NPR1 Increases Vascular Endothelial Adhesion through Induction of Integrin Beta 4

Natriuretic peptide receptor 1 (NPR1) serves as a modulator of vascular endothelial homeostasis. Interactions between monocytes and endothelial cells may initiate endothelium dysfunction, which is known as an early hallmark of atherosclerosis. In this study, we performed RNA-sequencing analysis for the aorta of Npr1 knockout (Npr1^+/−) mice and found that differentially expressed genes were significantly related to cell adhesion. This result was supported by an increased expression of intercellular adhesion molecule 1 (ICAM-1) in the aortic endothelium of Npr1^+/− mice. Moreover, we observed that the knockdown of NPR1 increased ICAM-1 expression and promoted THP-1 monocyte adhesion to human umbilical vein endothelial cells (HUVECs). NPR1 overexpression decreased ICAM-1 expression and inhibited the adhesion of monocytes to HUVECs treated by TNF-α (a cell adhesion inducer). Further analysis showed that adhesion-related genes were enriched in the focal adhesion signaling pathway, in which integrin beta 4 (Itgb4) was determined as a key gene. Notably, ITGB4 expression increased in vascular endothelium of Npr1^+/− mice and in NPR1-knockdown HUVECs. The deficiency of ITGB4 decreased ICAM-1 expression and attenuated monocyte adhesion to NPR1-knockdown endothelial cells. Additionally, a reduced NPR1 and an increased ITGB4 expression level were found in an atherosclerosis mouse model. In conclusion, our findings demonstrate that NPR1 deficiency increases vascular endothelial cell adhesion by stimulating ITGB4 expression, which may contribute to the development of atherosclerosis.     

Effects of the Chloroplast Fructose-1,6-Bisphosphate Aldolase Gene on Growth and Low-Temperature Tolerance of Tomato

Tomato (Solanum lycopersicum) is one of the most important greenhouse vegetables, with a large cultivated area across the world. However, in northern China, tomato plants often suffer from low-temperature stress in solar greenhouse cultivation, which affects plant growth and development and results in economic losses. We previously found that a chloroplast aldolase gene in tomato, SlFBA4, plays an important role in the Calvin-Benson cycle (CBC), and its expression level and activity can be significantly altered when subjected to low-temperature stress. To further study the function of SlFBA4 in the photosynthesis and chilling tolerance of tomato, we obtained transgenic tomato plants by the over-expression and RNA interference (RNAi) of SlFBA4. The over-expression of SlFBA4 led to higher fructose-1,6-bisphosphate aldolase activity, net photosynthetic rate (Pn) and activity of other enzymes in the CBC than wild type. Opposite results were observed in the RNAi lines. Moreover, an increase in thousand-seed weight, plant height, stem diameter and germination rate in optimal and sub-optimal temperatures was observed in the over-expression lines, while opposite effects were observed in the RNAi lines. Furthermore, over-expression of SlFBA4 increased Pn and enzyme activity and decreased malonaldehyde (MDA) content under chilling conditions. On the other hand, Pn and MDA content were more severely influenced by chilling stress in the RNAi lines. These results indicate that SlFBA4 plays an important role in tomato growth and tolerance to chilling stress.     

Genome-Wide Characterization and Expression Analysis of Pathogenesis-Related 1 (PR-1) Gene Family in Tea Plant (Camellia sinensis (L.) O. Kuntze) in Response to Blister-Blight Disease Stress

Pathogenesis-related 1 (PR-1) proteins, which are defense proteins in plant–pathogen interactions, play an important role in the resistance and defense of plants against diseases. Blister blight disease is caused by Exobasidium vexans Massee and a major leaf disease of tea plants (Camellia sinensis (L.) O. Kuntze). However, the systematic characterization and analysis of the PR-1 gene family in tea plants is still lacking, and the defense mechanism of this family remains unknown. In this study, 17 CsPR-1 genes were identified from the tea plant genome and classified into five groups based on their signal peptide, isoelectric point, and C-terminus extension. Most of the CsPR-1 proteins contained an N-terminal signal peptide and a conserved PR-1 like domain. CsPR-1 genes comprised multiple cis-acting elements and were closely related to the signal-transduction pathways involving TCA, NPR1, EDS16, BGL2, PR4, and HCHIB. These characteristics imply an important role of the genes in the defense of the tea plant. In addition, the RNA-seq data and real-time PCR analysis demonstrated that the CsPR-1-2, -4, -6, -7, -8, -9, -10, -14, -15, and -17 genes were significantly upregulated under tea blister-blight stress. This study could help to increase understanding of CsPR-1 genes and their defense mechanism in response to tea blister blight.