Quantitative Analysis of Plant Cytosolic Calcium Signals in Response to Water Activated by Low-Power Non-Thermal Plasma

Non-thermal plasma technology is increasingly being applied in the plant biology field. Despite the variety of beneficial effects of plasma-activated water (PAW) on plants, information about the mechanisms of PAW sensing by plants is still limited. In this study, in order to link PAW perception to the positive downstream responses of plants, transgenic Arabidopsis thaliana seedlings expressing the Ca²⁺-sensitive photoprotein aequorin in the cytosol were challenged with water activated by low-power non-thermal plasma generated by a dielectric barrier discharge (DBD) source. PAW sensing by plants resulted in the occurrence of cytosolic Ca²⁺ signals, whose kinetic parameters were found to strictly depend on the operational conditions of the plasma device and thus on the corresponding mixture of chemical species contained in the PAW. In particular, we highlighted the effect on the intracellular Ca²⁺ signals of low doses of DBD-PAW chemicals and also presented the effects of consecutive plant treatments. The results were discussed in terms of the possibility of using PAW-triggered Ca²⁺ signatures as benchmarks to accurately modulate the chemical composition of PAW in order to induce environmental stress resilience in plants, thus paving the way for further applications in agriculture.     

FvNST1b NAC Protein Induces Secondary Cell Wall Formation in Strawberry

Secondary cell wall thickening plays a crucial role in plant growth and development. Diploid woodland strawberry (Fragaria vesca) is an excellent model for studying fruit development, but its molecular control of secondary wall thickening is largely unknown. Previous studies have shown that Arabidopsis NAC secondary wall thickening promoting factor1 (AtNST1) and related proteins are master regulators of xylem fiber cell differentiation in multiple plant species. In this study, a NST1-like gene, FvNST1b, was isolated and characterized from strawberry. Sequence alignment and phylogenetic analysis showed that the FvNST1b protein contains a highly conserved NAC domain, and it belongs to the same family as AtNST1. Overexpression of FvNST1b in wild-type Arabidopsis caused extreme dwarfism, induced ectopic thickening of secondary walls in various tissues, and upregulated the expression of genes related to secondary cell wall synthesis. In addition, transient overexpression of FvNST1b in wild-type Fragaria vesca fruit produced cells resembling tracheary elements. These results suggest that FvNST1b positively regulates secondary cell wall formation as orthologous genes from other species.     

Single Amino Acid Exchange in ACTIN2 Confers Increased Tolerance to Oxidative Stress in Arabidopsis der1–3 Mutant

Single-point mutation in the ACTIN2 gene of the der1–3 mutant revealed that ACTIN2 is an essential actin isovariant required for root hair tip growth, and leads to shorter, thinner and more randomly oriented actin filaments in comparison to the wild-type C24 genotype. The actin cytoskeleton has been linked to plant defense against oxidative stress, but it is not clear how altered structural organization and dynamics of actin filaments may help plants to cope with oxidative stress. In this study, we characterized root growth, plant biomass, actin organization and antioxidant activity of the der1–3 mutant under oxidative stress induced by paraquat and H₂O₂. Under these conditions, plant growth was better in the der1–3 mutant, while the actin cytoskeleton in the der1–3 carrying pro35S::GFP:FABD2 construct showed a lower bundling rate and higher dynamicity. Biochemical analyses documented a lower degree of lipid peroxidation, and an elevated capacity to decompose superoxide and hydrogen peroxide. These results support the view that the der1–3 mutant is more resistant to oxidative stress. We propose that alterations in the actin cytoskeleton, increased sensitivity of ACTIN to reducing agent dithiothreitol (DTT), along with the increased capacity to decompose reactive oxygen species encourage the enhanced tolerance of this mutant against oxidative stress.     

Dual Reproductive Cell-Specific Promoter-Mediated Split-Cre/LoxP System Suitable for Exogenous Gene Deletion in Hybrid Progeny of Transgenic Arabidopsis

Genetically modified (GM) crops possess some superior characteristics, such as high yield and insect resistance, but their biosafety has aroused broad public concern. Some genetic engineering technologies have recently been proposed to remove exogenous genes from GM crops. Few approaches have been applied to maintain advantageous traits, but excising exogenous genes in seeds or fruits from these hybrid crops has led to the generation of harvested food without exogenous genes. In a previous study, split-Cre mediated by split intein could recombine its structure and restore recombination activity in hybrid plants. In the current study, the recombination efficiency of split-Cre under the control of ovule-specific or pollen-specific promoters was validated by hybridization of transgenic Arabidopsis containing the improved expression vectors. In these vectors, all exogenous genes were flanked by two loxP sites, including promoters, resistance genes, reporter genes, and split-Cre genes linked to the reporter genes via LP4/2A. A gene deletion system was designed in which NCre was driven by proDD45, and CCre was driven by proACA9 and proDLL. Transgenic lines containing NCre were used as paternal lines to hybridize with transgenic lines containing CCre. Because this hybridization method results in no co-expression of the NCre and CCre genes controlled by reproduction-specific promoters in the F1 progeny, the desirable characteristics could be retained. After self-crossing in F1 progeny, the expression level and protein activity of reporter genes were detected, and confirmed that recombination of split-Cre had occurred and the exogenous genes were partially deleted. The gene deletion efficiency represented by the quantitative measurements of GUS enzyme activity was over 59%, with the highest efficiency of 73% among variable hybrid combinations. Thus, in the present study a novel dual reproductive cell-specific promoter-mediated gene deletion system was developed that has the potential to take advantage of the merits of GM crops while alleviating biosafety concerns.     

人工microRNAs干扰At3A06基因增强了拟南芥对菌核病的敏感性

基于本研究室自主开发的油菜防御cDNA芯片分析结果,油菜基因Bn3A06受菌核病诱导后特异上调表达。序列比对发现,该基因与拟南芥基因At3A06高度同源,后者编码289个氨基酸,功能未知。本研究用花序浸染法将At3A06基因的人工微RNA（amiRNA）干扰载体（该载体包含一个除草剂草胺磷抗性基因）转化拟南芥。经草胺磷筛选获得3株T1代转基因植株,PCR检测均为阳性;荧光定量PCR检测表明,与野生型对照相比,3个转基因植株的At3A06基因的表达量均显著降低;对其T2代进行菌核病抗性鉴定,结果显示转基因株系对菌核病的敏感性均显著增强（P〈0.05）。推测At3A06基因可能参与了植物的防御反应,与植株对核盘菌抗性相关。     

Glucosinolate and Trichome Defenses in a Natural Arabidopsis lyrata Population

Glucosinolates (GS) and trichomes contribute to plant resistance against insect herbivores in the model Arabidopsis thaliana. The functional and genetic characteristics of herbivore defense, however, can differ even between closely related species. In a quantitative genetic experiment with the out-crossing perennial Arabidopsis lyrata spp. petraea, we measured constitutive GS composition, trichome density, leaf thickness, and plant resistance in four different herbivore interactions. In a single population of A. lyrata, we found heritable variation for trichome density as well as GS amount and carbon side-chain elongation ratios associated with activity in methylthioalkylmalate synthase (MAM). Unexpectedly, heritabilities for indole GS in A. lyrata were high and less affected by differences in plant age and environment than aliphatic GS. We found significant heritability in plant resistance to the specialist Plutella xylostella and generalist Trichoplusia ni, but not to the specialists Pieris brassicae and Phyllotreta cruciferae. Analyses of phenotypic and genetic correlations between candidate defense traits and insect resistance suggested that A. lyrata resistance was conferred by a combination of indole GS amount and trichome density, and, to a lesser extent, aliphatic GS ratios and leaf thickness. Variation in the most abundant compound, the aliphatic 3-hydroxypropyl GS, had little impact on A. lyrata herbivore resistance. The contribution of defense traits to resistance depended on the experimental herbivory context, and resistances were weakly correlated. A diversified defense strategy is likely to be important for long-lived individuals of A. lyrata that are subject to attack by many different herbivores in nature.     

拟南芥谷氨酸-tRNA合成酶(AtGluRS)不能取代酵母GluRS

将拟南芥细胞质谷氨酸-tRNA合成酶（AtGluRS）引入酵母细胞，研究其与酵母GluRS进行功能互补的可能性．无功能的酵母GluRS会导致酵母细胞死亡．将携带AtGluRS的pACT2-AtGluRS或pBridge（dNLS）-AtGluRS表达载体转化到含有能表达酵母谷氨酸．tRNA合成酶的pRS316-yGluRS载体的gluRS缺陷型酵母菌株中，若拟南芥GluRS能取代酵母中的该同源蛋白，酵母细胞中的载体pRS316-yGluRS在非选择性培养条件下将会很快丢失．然而，在经过3d的非选择性培养后，发现酵母细胞没有因拥有AtGluRS而失去提供GluRS功能的载体pRS316-yGluRS．可见，不同物种的GluRS之间，即使其具有很高的同源性，它们在演化过程中可能已形成了各自不同的特性，导致了拟南芥AtGluRS不能对酵母GluRS起功能互补作用．提示一些同源蛋白在异源生物体中不能发挥其正常功能．     

Rates of exocytosis and endocytosis in Arabidopsis root hairs and pollen tubes

Exocytosis and endocytosis are pivotal in many biological processes, but remain difficult to quantify. Here we combine a new algorithm for estimating vesicle size with a detailed morphological analysis of tip-growing cells, in which exocytosis is highly localized and therefore more readily quantified. Cell preservation was rendered as life-like as possible by rapid freezing. This allowed us to produce the first estimates of exocytosis rates in the root hairs and pollen tubes of the model plant Arabidopsis. To quantify exocytosis and endocytosis rates during cell growth, we measured the diameter of vesicles located in the tips of Arabidopsis root hairs and pollen tubes and the widths of cell walls and the cell lumen in longitudinal thin transmission electron microscopic sections. In addition, we measured growth velocities of Arabidopsis root hairs and pollen tubes, using video microscopy. The number of exocytotic vesicles required for cell wall expansion, and the amount of excess membrane inserted into the plasma membrane to be internalized, were estimated from the values that were obtained. The amount of excess membrane that is inserted into the plasma membrane during cell growth was estimated as 86.7% in root hairs and 79% in pollen tubes. This membrane has to be recycled by endocytosis. From counting of the total number of vesicles that is present in thin EM sections through the pollen tube tip, we estimated the average number of vesicles that is present in the tip of pollen tubes. By calculating the total amount of membrane and cell wall material that is required for continued cell growth, assuming that all vesicles are exocytotic, we estimated that pollen tubes continue to grow for 33 s when delivery of vesicles to the tip is inhibited. We arrested vesicle delivery to the tip by application of cytochalasin D. After cytochalasin D application, pollen tubes continued to grow for 30-40 s, which is in the same range as the estimated value of 33 s and shows that in this time frame, the availability of exocytotic vesicles is not a limiting factor.     

Genome-Wide Analysis of the Soybean Calmodulin-Binding Protein 60 Family and Identification of GmCBP60A-1 Responses to Drought and Salt Stresses

Calmodulin-binding protein 60 (CBP60) members constitute a plant-specific protein family that plays an important role in plant growth and development. In the soybean genome, nineteen CBP60 members were identified and analyzed for their corresponding sequences and structures to explore their functions. Among GmCBP60A-1, which primarily locates in the cytomembrane, was significantly induced by drought and salt stresses. The overexpression of GmCBP60A-1 enhanced drought and salt tolerance in Arabidopsis, which showed better state in the germination of seeds and the root growth of seedlings. In the soybean hairy roots experiment, the overexpression of GmCBP60A-1 increased proline content, lowered water loss rate and malondialdehyde (MDA) content, all of which likely enhanced the drought and salt tolerance of soybean seedlings. Under stress conditions, drought and salt response-related genes showed significant differences in expression in hairy root soybean plants of GmCBP60A-1-overexpressing and hairy root soybean plants of RNAi. The present study identified GmCBP60A-1 as an important gene in response to salt and drought stresses based on the functional analysis of this gene and its potential underlying mechanisms in soybean stress-tolerance.