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.     

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.     

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.     

Contrasting Rootstock-Mediated Growth and Yield Responses in Salinized Pepper Plants (Capsicum annuum L.) Are Associated with Changes in the Hormonal Balance

Salinity provokes an imbalance of vegetative to generative growth, thus impairing crop productivity. Unlike breeding strategies, grafting is a direct and quick alternative to improve salinity tolerance in horticultural crops, through rebalancing plant development. Providing that hormones play a key role in plant growth and development and stress responses, we hypothesized that rootstock-mediated reallocation of vegetative growth and yield under salinity was associated with changes in the hormonal balance. To test this hypothesis, the hybrid pepper variety (Capsicum annuum L. “Gacela F1”) was either non-grafted or grafted onto three commercial rootstocks (Creonte, Atlante, and Terrano) and plants were grown in a greenhouse under control (0 mM NaCl) and moderate salinity (35 mM NaCl) conditions. Differential vegetative growth versus fruit yield responses were induced by rootstock and salinity. Atlante strongly increased shoot and root fresh weight with respect to the non-grafted Gacela plants associated with improved photosynthetic rate and K⁺ homeostasis under salinity. The invigorating effect of Atlante can be explained by an efficient balance between cytokinins (CKs) and abscisic acid (ABA). Creonte improved fruit yield and maintained the reproductive to vegetative ratio under salinity as a consequence of its capacity to induce biomass reallocation and to avoid Na⁺ accumulation in the shoot. The physiological responses associated with yield stability in Creonte were mediated by the inverse regulation of CKs and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. Finally, Terrano limited the accumulation of gibberellins in the shoot thus reducing plant height. Despite scion compactness induced by Terrano, both vegetative and reproductive biomass were maintained under salinity through ABA-mediated control of water relations and K⁺ homeostasis. Our data demonstrate that the contrasting developmental and physiological responses induced by the rootstock genotype in salinized pepper plants were critically mediated by hormones. This will be particularly important for rootstock breeding programs to improve salinity tolerance by focusing on hormonal traits.     

The Effect of Sodium Butyrate on Adventitious Shoot Formation Varies among the Plant Species and the Explant Types

Histone acetylation plays an important role in plant growth and development. Here, we investigated the effect of sodium butyrate (NaB), a histone deacetylase inhibitor, on adventitious shoot formation from protoplast-derived calli and cotyledon explants of tobacco (Nicotiana benthamiana) and tomato (Solanum lycopersicum). The frequency of adventitious shoot formation from protoplast-derived calli was higher in shoot induction medium (SIM) containing NaB than in the control. However, the frequency of adventitious shoot formation from cotyledon explants of tobacco under the 0.1 mM NaB treatment was similar to that in the control, but it decreased with increasing NaB concentration. Unlike in tobacco, NaB decreased adventitious shoot formation in tomato explants in a concentration-dependent manner, but it did not have any effect on adventitious shoot formation in calli. NaB inhibited or delayed the expression of D-type cyclin (CYCD3-1) and shoot-regeneration regulatory gene WUSCHEL (WUS) in cotyledon explants of tobacco and tomato. However, compared to that in control SIM, the expression of WUS was promoted more rapidly in tobacco calli cultured in NaB-containing SIM, but the expression of CYCD3-1 was inhibited. In conclusion, the effect of NaB on adventitious shoot formation and expression of CYCD3-1 and WUS genes depended on the plant species and whether the effects were tested on explants or protoplast-derived calli.     

A Novel Thylakoid Ascorbate Peroxidase from Jatrophacurcas Enhances Salt Tolerance in Transgenic Tobacco

Ascorbate peroxidase (APX) plays an important role in the metabolism of hydrogen peroxide in higher plants. In the present study, a novel APX gene (JctAPX) was cloned from Jatropha curcas L. The deduced amino acid sequence was similar to that of APX of some other plant species. JctAPX has a chloroplast transit peptide and was localized to the chloroplasts by analysis with a JctAPX-green fluorescent protein (GFP) fusion protein. Quantitative polymerase chain reaction (qPCR) analysis showed that JctAPX was constitutively expressed in different tissues from J. curcas and was upregulated by NaCl stress. To characterize its function in salt tolerance, the construct p35S: JctAPX was created and successfully introduced into tobacco by Agrobacterium-mediated transformation. Compared with wild type (WT), the transgenic plants exhibited no morphological abnormalities in the no-stress condition. However, under 200 mM NaCl treatment, JctAPX over-expressing plants showed increased tolerance to salt during seedling establishment and growth. In addition, the transgenic lines showed higher chlorophyll content and APX activity, which resulted in lower H₂O₂ content than WT when subjected to 400 mM NaCl stress. These results suggest that the increased APX activity in the chloroplasts from transformed plants increased salt tolerance by enhancing reactive oxygen species (ROS)-scavenging capacity under short-term NaCl stress conditions.     

Interplay between Ca2+/Calmodulin-Mediated Signaling and AtSR1/CAMTA3 during Increased Temperature Resulting in Compromised Immune Response in Plants

Changing temperatures are known to affect plant–microbe interactions; however, the molecular mechanism involved in plant disease resistance is not well understood. Here, we report the effects of a moderate change in temperature on plant immune response through Ca²⁺/calmodulin-mediated signaling. At 30 °C, Pst DC3000 triggered significantly weak and relatively slow Ca²⁺ influx in plant cells, as compared to that at 18 °C. Increased temperature contributed to an enhanced disease susceptibility in plants; the enhanced disease susceptibility is the result of the compromised stomatal closure induced by pathogens at high temperature. A Ca²⁺ receptor, AtSR1, contributes to the decreased plant immunity at high temperatures and the calmodulin-binding domain (CaMBD) is required for its function. Furthermore, both salicylic acid biosynthesis (ICS) and salicylic acid receptor (NPR1) are involved in this process. In addition to stomatal control, AtSR1 is involved in high temperature-compromised apoplastic immune response through the salicylic acid signaling pathway. The qRT-PCR data revealed that AtSR1 contributed to increased temperatures-mediated susceptible immune response by regulating SA-related genes in atsr1, such as PR1, ICS1, NPR1, as well as EDS1. Our results indicate that Ca²⁺ signaling has broad effects on the molecular interplay between changing temperatures as well as plant defense during plant–pathogen interactions.