Yuanyuan Bu

Conserved V-ATPase c subunit plays a role in plant growth by influencing V-ATPase-dependent endosomal trafficking.

In plant cells, the vacuolar-type H(+)-ATPases (V-ATPase) are localized in the tonoplast, Golgi, trans-Golgi network and endosome. However, little is known about how V-ATPase influences plant growth, particularly with regard to the V-ATPase c subunit (VHA-c). Here, we characterized the function of a VHA-c gene from Puccinellia tenuiflora (PutVHA-c) in plant growth. Compared to the wild-type, transgenic plants overexpressing PutVHA-c in Arabidopsis thaliana exhibit better growth phenotypes in root length, fresh weight, plant height and silique number under the normal and salt stress conditions due to noticeably higher V-ATPase activity. Consistently, the Arabidopsis atvha-c5 mutant shows reduced V-ATPase activity and retarded plant growth. Furthermore, confocal and immunogold electron microscopy assays demonstrate that PutVHA-c is mainly localized to endosomal compartments. The treatment of concanamycin A (ConcA), a specific inhibitor of V-ATPases, leads to obvious aggregation of the endosomal compartments labelled with PutVHA-c-GFP. Moreover, ConcA treatment results in the abnormal localization of two plasma membrane (PM) marker proteins Pinformed 1 (AtPIN1) and regulator of G protein signalling-1 (AtRGS1). These findings suggest that the decrease in V-ATPase activity blocks endosomal trafficking. Taken together, our results strongly suggest that the PutVHA-c plays an important role in plant growth by influencing V-ATPase-dependent endosomal trafficking.

Identification and Characterization of a PutAMT1;1 Gene from Puccinellia tenuiflora

Nitrogen is one of the most important limiting factors for plant growth. However, as ammonium is readily converted into ammonia (NH3) when soil pH rises above 8.0, this activity depletes the availability of ammonium (NH4 +) in alkaline soils, consequently preventing the growth of most plant species. The perennial wild grass Puccinellia tenuiflora is one of a few plants able to grow in soils with extremely high salt and alkaline pH (>9.0) levels. Here, we assessed how this species responds to ammonium under such conditions by isolating and analyzing the functions of a putative ammonium transporter (PutAMT1;1). PutAMT1;1 is the first member of the AMT1 (ammonium transporter) family that has been identified in P. tenuiflora. This gene (1) functionally complemented a yeast mutant deficient in ammonium uptake (2), is preferentially expressed in the anther of P. tenuiflora, and (3) is significantly upregulated by ammonium ions in both the shoot and roots. The PutAMT1;1 protein is localized in the plasma membrane and around the nuclear periphery in yeast cells and P. tenuiflora suspension cells. Immunoelectron microscopy analysis also indicated that PutAMT1;1 is localized in the endomembrane. The overexpression of PutAMT1;1 in A. thaliana enhanced plant growth, and increased plant susceptibility to toxic methylammonium (MeA). Here, we confirmed that PutAMT1;1 is an ammonium-inducible ammonium transporter in P. tenuiflora. On the basis of the results of PutAMT1;1 overexpression in A. thaliana, this gene might be useful for improving the root to shoot mobilization of MeA (or NH4 +).

Arabidopsis cysteine proteinase inhibitor AtCYSb interacts with a Ca2+-dependent nuclease, AtCaN2

AtCYSb physically interacts with AtCaN2 by two hybrid (View interaction)

Adverse effect of urease on salt stress during seed germination in Arabidopsis thaliana.

Seed germination is a critical stage in the development of crops that grow in saline soils. We noticed that seeds of an Arabidopsis urease mutant have significantly increased salt stress tolerance. To understand why, we treated the wild type (WT) with a urease inhibitor and found that its salt stress tolerance was also improved. We hypothesized that urease acting on urea generates NH4 +, which probably exacerbates salt stress. As expected, the urease inhibitor significantly decreased the NH4 + level in WT seeds. These findings suggest that blocking urease activity improves salt tolerance during seed germination by lowering the concentration of NH4 +.

An efficient method for stable protein targeting in grasses (Poaceae): a case study in Puccinellia tenuiflora.

BackgroundAn efficient transformation method is lacking for most non-model plant species to test gene function. Therefore, subcellular localization of proteins of interest from non-model plants is mainly carried out through transient transformation in homologous cells or in heterologous cells from model species such as Arabidopsis. Although analysis of expression patterns in model organisms like yeast and Arabidopsis can provide important clues about protein localization, these heterologous systems may not always faithfully reflect the native subcellular distribution in other species. On the other hand, transient expression in protoplasts from species of interest has limited ability for detailed sub-cellular localization analysis (e.g., those involving subcellular fractionation or sectioning and immunodetection), as it results in heterogeneous populations comprised of both transformed and untransformed cells.ResultsWe have developed a simple and reliable method for stable transformation of plant cell suspensions that are suitable for protein subcellular localization analyses in the non-model monocotyledonous plant Puccinellia tenuiflora. Optimization of protocols for obtaining suspension-cultured cells followed by Agrobacterium-mediated genetic transformation allowed us to establish stably transformed cell lines, which could be maintained indefinitely in axenic culture supplied with the proper antibiotic. As a case study, protoplasts of transgenic cell lines stably transformed with an ammonium transporter-green fluorescent protein (PutAMT1;1-GFP) fusion were successfully used for subcellular localization analyses in P. tenuiflora.ConclusionsWe present a reliable method for the generation of stably transformed P. tenuiflora cell lines, which, being available in virtually unlimited amounts, can be conveniently used for any type of protein subcellular localization analysis required. Given its simplicity, the method can be used as reference for other non-model plant species lacking efficient regeneration protocols.

Overexpression of AtOxR gene improves abiotic stresses tolerance and vitamin C content in Arabidopsis thaliana

BackgroundAbiotic stresses are serious threats to plant growth, productivity and result in crop loss worldwide, reducing average yields of most major crops. Although abiotic stresses might elicit different plant responses, most induce the accumulation of reactive oxygen species (ROS) in plant cells leads to oxidative damage. L-ascorbic acid (AsA, vitamin C) is known as an antioxidant and H2O2-scavenger that defends plants against abiotic stresses. In addition, vitamin C is also an important component of human nutrition that has to be obtained from different foods. Therefore, increasing the vitamin C content is important for improving abiotic stresses tolerance and nutrition quality in crops production.ResultsHere, we show that the expression of AtOxR gene is response to multiple abiotic stresses (salt, osmotic, metal ion, and H2O2 treatment) in both the leaves and roots of Arabidopsis. AtOxR protein was localized to the Endoplasmic Reticulum (ER) in yeast and Arabidopsis cells by co-localization analysis with ER specific dye. AtOxR-overexpressing transgenic Arabidopsis plants enhance the tolerance to abiotic stresses. Overexpression of AtOxR gene resulted in AsA accumulation and decreased H2O2 content in transgenic plants.ConclusionsIn this study, our results show that AtOxR responds to multiple abiotic stresses. Overexpressing AtOxR improves tolerance to abiotic stresses and increase vitamin C content in Arabidopsis thaliana. AtOxR will be useful for the improvement of important crop plants through moleculer breeding.