The 3-ketoacyl-CoA synthase WFL is involved in lateral organ development and cuticular wax synthesis in Medicago truncatula

Plant Molecular Biology - A 3-ketoacyl-CoA synthase involved in biosynthesis of very long chain fatty acids and cuticular wax plays a vital role in aerial organ development in M. truncatula....     

The effect of phenyl acetic acid on shoot bud induction, elongation and rooting of chickpea

A highly efficient protocol for plant regeneration from cotyledonary node of two chickpea (Cicer arietinum L.) cultivars ICCV-10 and Annigeri used phenylacetic acid (PAA). The Murashige and Skoog (MS) medium supplemented with 2.0 mg dm⁻³ 6-benzylaminopurine (BAP) and 1.0 mg dm⁻³ PAA was used for induction of bud formation. Buds were elongated on MS medium supplemented either with only 0.75 mg dm⁻³ gibberellic acid (GA₃) or 0.2 mg dm⁻³ GA₃ + 0.6 mg dm⁻³ PAA. The elongated shoots were then transferred onto rooting medium containing 1 mg dm⁻³ PAA. The frequency of multiple shoot induction and rooting was higher in Annigeri as compared to ICCV-10. The complete plantlets with well-developed roots were transferred to pots containing sterilized soil and sand in the ratio 3:1 where they survived (74 %) and set normal seeds.     

Differential Expression of Proteoglycans in Tissue Remodeling and Lymphangiogenesis after Experimental Renal Transplantation in Rats

Background

Chronic transplant dysfunction explains the majority of late renal allograft loss and is accompanied by extensive tissue remodeling leading to transplant vasculopathy, glomerulosclerosis and interstitial fibrosis. Matrix proteoglycans mediate cell-cell and cell-matrix interactions and play key roles in tissue remodeling. The aim of this study was to characterize differential heparan sulfate proteoglycan and chondroitin sulfate proteoglycan expression in transplant vasculopathy, glomerulosclerosis and interstitial fibrosis in renal allografts with chronic transplant dysfunction.

Methods

Renal allografts were transplanted in the Dark Agouti-to-Wistar Furth rat strain combination. Dark Agouti-to-Dark Agouti isografts and non-transplanted Dark Agouti kidneys served as controls. Allograft and isograft recipients were sacrificed 66 and 81 days (mean) after transplantation, respectively. Heparan sulfate proteoglycan (collXVIII, perlecan and agrin) and chondroitin sulfate proteoglycan (versican) expression, as well as CD31 and LYVE-1 (vascular and lymphatic endothelium, respectively) expression were (semi-) quantitatively analyzed using immunofluorescence.

Findings

Arteries with transplant vasculopathy and sclerotic glomeruli in allografts displayed pronounced neo-expression of collXVIII and perlecan. In contrast, in interstitial fibrosis expression of the chondroitin sulfate proteoglycan versican dominated. In the cortical tubular basement membranes in both iso- and allografts, induction of collXVIII was detected. Allografts presented extensive lymphangiogenesis (p<0.01 compared to isografts and non-transplanted controls), which was associated with induced perlecan expression underneath the lymphatic endothelium (p<0.05 and p<0.01 compared to isografts and non-transplanted controls, respectively). Both the magnitude of lymphangiogenesis and perlecan expression correlated with severity of interstitial fibrosis and impaired graft function.

Interpretation

Our results reveal that changes in the extent of expression and the type of proteoglycans being expressed are tightly associated with tissue remodeling after renal transplantation. Therefore, proteoglycans might be potential targets for clinical intervention in renal chronic transplant dysfunction.     

A Virus-Induced Gene Silencing Screen Identifies a Role for Thylakoid Formation1 in Pseudomonas syringae pv tomato Symptom Development in Tomato and Arabidopsis

Pseudomonas syringae pv tomato DC3000 (Pst DC3000), which causes disease in tomato (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana), produces coronatine (COR), a non-host-specific phytotoxin. COR, which functions as a jasmonate mimic, is required for full virulence of Pst DC3000 and for the induction of chlorosis in host plants. Previous genetic screens based on insensitivity to COR and/or methyl jasmonate identified several potential targets for COR and methyl jasmonate. In this study, we utilized Nicotiana benthamiana and virus-induced gene silencing to individually reduce the expression of over 4,000 genes. The silenced lines of N. benthamiana were then screened for altered responses to purified COR. Using this forward genetics approach, several genes were identified with altered responses to COR. These were designated as ALC (for altered COR response) genes. When silenced, one of the identified genes, ALC1, produced a hypersensitive/necrosis-like phenotype upon COR application in a Coronatine-Insensitive1 (COI1)-dependent manner. To understand the involvement of ALC1 during the Pst DC3000-host interaction, we used the nucleotide sequence of ALC1 and identified its ortholog in Arabidopsis (Thylakoid Formation1 [THF1]) and tomato (SlALC1). In pathogenicity assays performed on Arabidopsis thf1 mutant and SlALC1-silenced tomato plants, Pst DC3000 induced accelerated coalescing necrotic lesions. Furthermore, we showed that COR affects ALC1 localization in chloroplasts in a COI1-dependent manner. In conclusion, our results show that the virus-induced gene silencing-based forward genetic screen has the potential to identify new players in COR signaling and disease-associated necrotic cell death.In nature, plants come in contact with numerous microbes that are potential pathogens. Active plant defense mechanisms, in general, involve a complex network of three genetically distinct signaling pathways, known as the salicylic acid (SA), jasmonic acid (JA), and ethylene pathways (Kunkel and Brooks, 2002; Glazebrook, 2005). Pathogens, in turn, have coevolved by developing mechanisms that suppress plant defense pathways by secreting virulence factors. Several pathovars of Pseudomonas syringae produce phytotoxins. In plants, these phytotoxins generally induce chlorosis (e.g. coronatine [COR], phaseolotoxin, and tabtoxin; Mitchell, 1976; Gnanamanickam et al., 1982; Levi and Durbin, 1986) or necrosis (e.g. syringomycin and syringopeptin; Paynter and Alconero, 1979; Iacobellis et al., 1992). Bacterial toxins act as virulence factors and contribute to increased disease severity by facilitating bacterial movement in planta (Patil et al., 1974), lesion size (Bender et al., 1987; Xu and Gross, 1988), pathogen multiplication (Bender et al., 1987; Feys et al., 1994; Mittal and Davis, 1995), and suppression of plant defense (Uppalapati et al., 2007, 2008).COR, a phytotoxin produced by P. syringae pv tomato DC3000 (Pst DC3000), is induced in the presence of the plant host metabolites such as malic, citric, shikimic, and quinic acids, which are present in leaf extracts and apoplastic fluids of tomato (Solanum lycopersicum; Li et al., 1998). COR contributes to the virulence of Pst DC3000 in Arabidopsis (Arabidopsis thaliana), tomato, collard (Brassica oleracea var viridis), and turnip (Brassica rapa var utilis; Brooks et al., 2004; Elizabeth and Bender, 2007; Uppalapati et al., 2007). It has been shown that COR has structural and functional resemblance to 12-oxo-phytodienoic acid, methyl jasmonate (MeJA), and related derivatives known as the jasmonates (JAs; Feys et al., 1994; Weiler et al., 1994; Uppalapati et al., 2005). MeJA is a plant growth hormone that plays a key role in plant defense response to biotic and abiotic stress (Howe et al., 1996; McConn et al., 1997; Vijayan et al., 1998; Truman et al., 2007).During a compatible interaction with a host, Pst DC3000 infection results in the activation of the JA signaling pathway (Zhao et al., 2003; Laurie-Berry et al., 2006; Uppalapati et al., 2007). This causes the suppression of the SA pathway owing to its antagonistic relation with the JA pathway (Kloek et al., 2001; Kunkel and Brooks, 2002; Zhao et al., 2003; Uppalapati et al., 2007). The suppression of the SA pathway during the Pst DC3000-host interaction is thought to be caused by COR, which functions as a molecular mimic of JAs (Feys et al., 1994; Bender et al., 1999; Staswick and Tiryaki, 2004).Pst DC3000 causes disease on several plant species including tomato and Arabidopsis. A typical symptom on tomato leaves is bacterial speck, which includes necrosis surrounded by a chlorotic halo (Mittal and Davis, 1995; Zhao et al., 2003). In Arabidopsis, the infected area exhibits water-soaked lesions accompanied by diffused chlorosis (Mittal and Davis, 1995; Brooks et al., 2004). Pst DC3000 infection also causes chlorosis in other plants belonging to the Brassicaceae family, such as collard and turnip (Elizabeth and Bender, 2007). In addition to chlorosis, Pst DC3000-infected collard plants exhibit water-soaked lesions and anthocyanin, suggesting that Pst DC3000 elicits unique responses in different plants. Studies have shown that tomato plants inoculated with a COR-defective mutant of Pst DC3000 did not develop typical chlorotic symptoms; furthermore, COR contributed to pathogen fitness and disease development in a SA-independent manner (Uppalapati et al., 2007). Tomato leaf tissues treated with purified COR show chlorosis (Gnanamanickam et al., 1982; Uppalapati et al., 2005, 2007). Unlike tomato, purified COR does not elicit chlorosis on Arabidopsis leaves (Bent et al., 1992; Mach et al., 2001). However, in Arabidopsis, COR is required for full disease symptom development and pathogen fitness in a SA-dependent manner (Kloek et al., 2001; Brooks et al., 2004). These results suggest that COR functions as an important virulence factor in tomato and Arabidopsis, although it functions differently in these hosts.More recently, we have demonstrated a role for COR-induced effects on photosynthetic machinery and reactive oxygen species (ROS) in modulating necrotic cell death during bacterial speck disease of tomato (Ishiga et al., 2009a). Despite our present understanding of COR function, it is not clear how chlorosis impacts or benefits pathogen virulence. Furthermore, the identity of host molecular targets for COR and the downstream signaling cascades that ensue are not well understood. Based on similarities between COR and JAs in terms of structure and function (Feys et al., 1994; Uppalapati et al., 2005), it seems likely that COR and JA interact with at least one common host receptor (Katsir et al., 2008). Thus, in addition to furthering our understanding of disease development, studies aimed at understanding the molecular mechanism of COR may provide information on JA-mediated plant defense.In an effort to identify plant proteins that are the molecular targets of COR, we used a tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) as a fast-forward genetics tool (Liu et al., 2001a, 2001b; Anand et al., 2007) to screen a Nicotiana benthamiana cDNA library for genes that are involved in the response to COR. We identified a N. benthamiana gene, ALC1 (for altered COR response), that when silenced displayed an unexpected hypersensitive/necrosis-like phenotype rather than a typical chlorotic phenotype in response to COR application. ALC1 has homology to an Arabidopsis gene, Thylakoid Formation1 (THF1; Wang et al., 2004). The pathogenicity assays performed in this study indicate that loss of ALC1/THF1 leads to accelerated cell death in response to Pst DC3000 infection in both tomato and Arabidopsis.     

The Trans-Acting Short Interfering RNA3 Pathway and NO APICAL MERISTEM Antagonistically Regulate Leaf Margin Development and Lateral Organ Separation,as Revealed by Analysis of an argonaute7/lobed leaflet1 Mutant in Medicago truncatula

Leaf shape elaboration and organ separation are critical for plant morphogenesis. We characterized the developmental roles of LOBED LEAFLET1 by analyzing a recessive mutant in the model legume Medicago truncatula. An ortholog of Arabidopsis thaliana ARGONAUTE7 (AGO7), Mt-AGO7/LOBED LEAFLET1, is required for the biogenesis of a trans-acting short interfering RNA (ta-siRNA) to negatively regulate the expression of AUXIN RESPONSE FACTORs in M. truncatula. Loss of function in AGO7 results in pleiotropic phenotypes in different organs. The prominent phenotype of the ago7 mutant is lobed leaf margins and more widely spaced lateral organs, suggesting that the trans-acting siRNA3 (TAS3) pathway negatively regulates the formation of boundaries and the separation of lateral organs in M. truncatula. Genetic interaction analysis with the smooth leaf margin1 (slm1) mutant revealed that leaf margin formation is cooperatively regulated by the auxin/SLM1 (ortholog of Arabidopsis PIN-FORMED1) module, which influences the initiation of leaf margin teeth, and the TAS3 ta-siRNA pathway, which determines the degree of margin indentation. Further investigations showed that the TAS3 ta-siRNA pathway and NO APICAL MERISTEM (ortholog of Arabidopsis CUP-SHAPED COTYLEDON) antagonistically regulate both leaf margin development and lateral organ separation, and the regulation is partially dependent on the auxin/SLM1 module.