Expansin gene loss is a common occurrence during adaptation to an aquatic environment

Expansins comprise a superfamily of plant cell wall loosening proteins that can be divided into four individual families (EXPA, EXPB, EXLA and EXLB). Aside from inferred roles in a variety of plant growth and developmental traits, little is known regarding the function of specific expansin clades, for which there are at least 16 in flowering plants (angiosperms); however, there is evidence to suggest that some expansins have cell‐specific functions, in root hair and pollen tube development, for example. Recently, two duckweed genomes have been sequenced (Spirodela polyrhiza strains 7498 and 9509), revealing significantly reduced superfamily sizes. We hypothesized that there would be a correlation between expansin loss and morphological reductions seen among highly adapted aquatic species. In order to provide an answer to this question, we characterized the expansin superfamilies of the greater duckweed Spirodela, the marine eelgrass Zostera marina and the bladderwort Utricularia gibba. We discovered rampant expansin gene and clade loss among the three, including a complete absence of the EXLB family and EXPA‐VII. The most convincing correlation between morphological reduction and expansin loss was seen for Utricularia and Spirodela, which both lack root hairs and the root hair expansin clade EXPA‐X. Contrary to the pattern observed in other species, four Utricularia expansins failed to branch within any clade, suggesting that they may be the result of neofunctionalization. Last, an expansin clade previously discovered only in eudicots was identified in Spirodela, allowing us to conclude that the last common ancestor of monocots and eudicots contained a minimum of 17 expansins.     

Root hair-specific expansins modulate root hair elongation in rice

Root hair growth requires intensive cell‐wall modification. This study demonstrates that root hair‐specific expansin As, a sub‐clade of the cell wall‐loosening expansin proteins, are required for root hair elongation in rice (Oryza sativa L.). We identified a gene encoding EXPA17 (OsEXPA17) from a rice mutant with short root hairs. Promoter::reporter transgenic lines exhibited exclusive OsEXPA17 expression in root hair cells. The OsEXPA17 mutant protein (OsexpA17) contained a point mutation, causing a change in the amino acid sequence (Gly104→Arg). This amino acid alteration is predicted to disrupt a highly conserved disulfide bond in the mutant. Suppression of OsEXPA17 by RNA interference further confirmed requirement for the gene in root hair elongation. Complementation of the OsEXPA17 mutant with other root hair EXPAs (OsEXPA30 and Arabidopsis EXPA7) can restore root hair elongation, indicating functional conservation of these root hair EXPAs in monocots and dicots. These results demonstrate that members of the root hair EXPA sub‐clade play a crucial role in root hair cell elongation in Graminaceae.     

Boron toxicity is alleviated by hydrogen sulfide in cucumber (Cucumis sativus L.) seedlings

Boron (B) is an essential micronutrient for plants, which when occurs in excess in the growth medium, becomes toxic to plants. Rapid inhibition of root elongation is one of the most distinct symptoms of B toxicity. Hydrogen sulfide (H₂S) is emerging as a potential messenger molecule involved in modulation of physiological processes in plants. In the present study, we investigated the role of H₂S in B toxicity in cucumber (Cucumis sativus) seedlings. Root elongation was significantly inhibited by exposure of cucumber seedlings to solutions containing 5 mM B. The inhibitory effect of B on root elongation was substantially alleviated by treatment with H₂S donor sodium hydrosulfide (NaHS). There was an increase in the activity of pectin methylesterase (PME) and up-regulated expression of genes encoding PME (CsPME) and expansin (CsExp) on exposure to high B concentration. The increase in PME activity and up-regulation of expression of CsPME and CsExp induced by high B concentration were markedly reduced in the presence of H₂S donor. There was a rapid increase in soluble B concentrations in roots on exposure to high concentration B solutions. Treatment with H₂S donor led to a transient reduction in soluble B concentration in roots such that no differences in soluble B concentrations in roots in the absence and presence of NaHS were found after 8 h exposure to the high concentration B solutions. These findings suggest that increases in activities of PME and expansin may underlie the inhibition of root elongation by toxic B, and that H₂S plays an ameliorative role in protection of plants from B toxicity by counteracting B-induced up-regulation of cell wall-associated proteins of PME and expansins.     

Expression of a plant expansin is involved in the establishment of root knot nematode parasitism in tomato

A group of plant proteins, expansins, have been identified as wall-loosening factors and as facilitators of cell expansion in vivo. The root knot nematode Meloidogyne javanica establishes a permanent feeding site composed of giant cells surrounded by gall tissue. We used quantitative PCR and in situ localization to demonstrate the induction of a tomato (Lycopersicon esculentum cv. VF36) expansin (LeEXPA5) expression in gall cells adjacent to the nematode feeding cells. To further characterize the biological role of LeEXPA5 we have generated LeEXPA5-antisense transgenic roots. The ability of the nematode to establish a feeding site and complete its life cycle, the average root cell size and the rate of root elongation were determined for the transgenic roots, as well as the level of LeEXPA5 expression in non-infected and nematode-infected roots. Our results demonstrated that a decrease of LeEXPA5 expression reduces the ability of the nematode to complete its life cycle in transgenic roots. We suggest that a plant-originated expansin is necessary for a successful parasitic nematode–plant interaction.     

Plant‐like bacterial expansins play contrasting roles in two tomato vascular pathogens

Expansin proteins, which loosen plant cell walls, play critical roles in normal plant growth and development. The horizontal acquisition of functional plant‐like expansin genes in numerous xylem‐colonizing phytopathogenic bacteria suggests that bacterial expansins may also contribute to virulence. To investigate the role of bacterial expansins in plant diseases, we mutated the non‐chimeric expansin genes (CmEXLX2 and RsEXLX) of two xylem‐inhabiting bacterial pathogens, the Actinobacterium Clavibacter michiganensis ssp. michiganensis (Cmm) and the β‐proteobacterium Ralstonia solanacearum (Rs), respectively. The Cmm Δ CmEXLX2 mutant caused increased symptom development on tomato, which was characterized by more rapid wilting, greater vascular necrosis and abundant atypical lesions on distant petioles. This increased disease severity correlated with larger in planta populations of the Δ CmEXLX2 mutant, even though the strains grew as well as the wild‐type in vitro. Similarly, when inoculated onto tomato fruit, Δ CmEXLX2 caused significantly larger lesions with larger necrotic centres. In contrast, the Rs Δ RsEXLX mutant showed reduced virulence on tomato following root inoculation, but not following direct petiole inoculation, suggesting that the RsEXLX expansin contributes to early virulence at the root infection stage. Consistent with this finding, Δ RsEXLX attached to tomato seedling roots better than the wild‐type Rs, which may prevent mutants from invading the plant's vasculature. These contrasting results demonstrate the diverse roles of non‐chimeric bacterial expansins and highlight their importance in plant–bacterial interactions.     

Effects of transgenic soybean on growth and phosphorus acquisition in mixed culture system

Transgenic soybean plants overexpressing the Arabidopsis purple acid phosphatase gene AtPAP15 (OXp) or the soybean expansin gene GmEXPB2 (OXe) can improve phosphorous (P) efficiency in pure culture by increasing Apase secretion or changing root morphology. In this study, soybean‐soybean mixed cultures were employed to illuminate P acquisition among plants in mixed stands of transgenic and wild‐type soybean. Our results showed that transgenic soybean plants were much more competitive, and had greater growth and P uptake than wild‐type soybean in mixed culture in both low P calcareous and acid soils. Furthermore, OXe plants had an advantage in calcareous soils when mixed with OXp, whereas the latter performed much better in acid soils. In soybean‐maize mixed culture, transgenic soybean had no impact on maize growth compared to controls in both acid and calcareous soils with different P conditions. As for soybean in mixed culture, OXp plants had no significant advantages regardless of P availability or soil type, while P efficiency improved in OXe in calcareous soils compared to controls. These results imply that physiological traits could be easily affected by the mixed maize. Transgenic soybean plants with enhanced root traits had more competitive advantages than those with improved root physiology in mixed culture.     

Overexpression of β‐expansin gene GmEXPB2 improves phosphorus efficiency in soybean

Soybean (Glycine max) is an important oil crop in agricultural production, but low phosphorus (P) availability limits soybean growth and production. Expansin is a family of plant cell wall proteins and involved in a variety of physiological processes, including cell division and enlargement, root growth and leaf development. To test the potential effects of expansins on crop production, we have developed soybean transgenic plants overexpressing a soybean β‐expansin gene GmEXPB2, which was significantly induced by phosphate (Pi) starvation. The results indicated that constitutive overexpression of GmEXPB2 promoted leaf expansion, sequentially stimulated root growth and consequently resulted in improved P efficiency in the transgenic plants under P‐limited conditions in hydroponics. In particular, when tested in calcareous (CS) and acid soils (AS), the two GmEXPB2 transgenic soybean lines showed above 25 and 40% increases in plant dry weight and P content, respectively to wild‐type plants in low‐P CS, but not in AS. To our knowledge, this is the first report in which improvement of P efficiency could be achieved through constitutive overexpression of an endogenous EXPB gene in soybean. These findings suggest that genetic modification of root and leaf traits might be a suitable strategy for improving crop production in low‐P soils.     

Characteristics of the binding of a bacterial expansin (BsEXLX1) to microcrystalline cellulose

Plant expansin proteins induce plant cell wall extension and have the ability to extend and disrupt cellulose. In addition, these proteins show synergistic activity with cellulases during cellulose hydrolysis. BsEXLX1 originating from Bacillus subtilis is a structural homolog of a β‐expansin produced by Zea mays (ZmEXPB1). The Langmuir isotherm for binding of BsEXLX1 to microcrystalline cellulose (i.e., Avicel) revealed that the equilibrium binding constant of BsEXLX1 to Avicel was similar to those of other Type A surface‐binding carbohydrate‐binding modules (CBMs) to microcrystalline cellulose, and the maximum number of binding sites on Avicel for BsEXLX1 was also comparable to those on microcrystalline cellulose for other Type A CBMs. BsEXLX1 did not bind to cellooligosaccharides, which is consistent with the typical binding behavior of Type A CBMs. The preferential binding pattern of a plant expansin, ZmEXPB1, to xylan, compared to cellulose was not exhibited by BsEXLX1. In addition, the binding capacities of cellulose and xylan for BsEXLX1 were much lower than those for CtCBD3. Biotechnol. Bioeng. 2013; 110: 401–407. © 2012 Wiley Periodicals, Inc.     

Stable transformation of Lithospermum erythrorhizon by Agrobacterium rhizogenes and shikonin production of the transformants

Seedling hypocotyls of Lithospermum erythrorhizon were infected with Agrobacterium rhizogenes (strain 15834) harboring a binary vector with an intron-bearing the β-glucuronidase (GUS) gene driven by cauliflower mosaic virus (CaMV) 35S promoter as well as the hygromycin phosphotransferase (HPT) gene as the selection marker. About 20% of the hairy roots isolated were hygromycin resistant and had co-integrated GUS and HPT genes in their Lithospermum genomic DNA. Because GUS activity was detected in almost all the hygromycin-resistant root tissues, the CaMV 35S promoter seems to be ubiquitously active in L. erythrorhizon hairy roots. In pigment production medium M9, the hairy root cultures had shikonin productivity similar to that of cell suspension cultures of Lithospermum. They also showed light-dependent inhibition of shikonin biosynthesis similar to that of Lithospermum cell cultures. These findings suggest that this hairy root system transformable with A. rhizogenes is a suitable model system for molecular characterization of shikonin biosynthesis via reverse genetics. Received: 2 March 1998 / Revision received: 25 May 1998 / Accepted: 8 July 1998     

Alkaloid patterns and biosynthetic capacity of root cultures from some pyrrolizidine alkaloid producing Senecio species

Root cultures of Senecio vulgaris, S. vernalis, S. erucifolius and S. squalidus were established. The patterns of pyrrolizidine alkaloids found in these root cultures were analyzed by high-resolution GC and GC-MS and compared with the alkaloids present in the respective plants. In vitro cultured roots produce alkaloid patterns and accumulate quantities which are comparable to those found in soil grown plants. With the exception of the otonecine derivative senkirkine all pyrrolizidines accumulate as N-oxides. Only senkirkine is partially released into the medium. The cultures incorporate biosynthetic precursors, e.g. ¹⁴C-labelled putrescine or spermidine with high efficiency into the alkaloids. Senecionine N-oxide was found to be the main product of biosynthesis. Evidence is presented that senecionine N-oxide is directly transformed into senkirkine, the main alkaloid of S. vernalis root cultures.Abbreviations GC Gas chromatography - MS Mass spectroscopy - PND Phosphorous-Nitrogen-Detector - FID Flame Ionization Detector - fr.wt Fresh weight     

The isolation of wall-bound proteins regulating yield threshold tension in glycerinated hollow cylinders of cowpea hypocotyl

To isolate and purify the factor regulating the yield threshold tension (y) through acidification of the cell wall, proteins were extracted from hypocotyls of Vigna unguiculata L. Their effects on the pH‐dependencies of the wall extensibility (φ) and y were examined with reconstitution experiments by incorporating them into the heat‐denatured glycerinated hollow cylinders (GHCs). The wall mechanical properties of the reconstituted GHCs were determined using stress–strain experiments. Only the proteins extracted with 1 kmol m^–3 of NaCl from the wall of elongation region restored the pH‐dependencies of φ and y once extinguished with heat‐denaturation, but proteins extracted from the other cell constituents or from the mature region of hypocotyl affect neither properties. Fractionation of the wall‐bound proteins by a hydrophobic column chromatography showed that the two different fractions affected φ or y independently. The sodium dodecyl sulphate‐polyacrylamide gel electrophoresis showed that the active fraction which restored the pH‐dependency of y still consists of two proteins of 30 and 32 kDa after purification by the sequential fractionation with cation‐exchange and gel filtration. These two proteins were named as ‘yieldin 30’ and ‘32’. Western blotting analysis using the rabbit‐antiserum against the cucumber expansin indicated that the yieldins are independent of cucumber expansin.     

Tanshinone biosynthesis in Salvia miltiorrhiza and production in plant tissue cultures

Salvia miltiorrhiza Bunge (Lamiaceae) root, generally called Danshen, is an important herb in Chinese medicine widely used for treatment of cardiovascular diseases. Diterpenoid tanshinons are major bioactive constituents of Danshen with notable pharmacological activities and the potential as new drug candidates against some important human diseases. The importance of Danshen for traditional and modern medicines has motivated the research interest over two decades in the biosynthesis and biotechnological production of tanshinones. Although diterpenes in plants are presumably derived from the non-mevalonate (MVA) pathway, tanshinone biosynthesis in S. miltiorrhiza may also depend on the MVA pathway based on some key enzymes and genes detected in the early steps of these pathways. Plant tissue cultures are the major biotechnological processes for rapid production of tanshinones and other bioactive compounds in the herb. Various in vitro cultures of S. miltiorrhiza have been established, including cell suspension, adventitious root, and hairy root cultures, which can accumulate the major tanshinones as in the plant roots. Tanshinone production in cell and hairy root cultures has been dramatically enhanced with various strategies, including medium optimization, elicitor stimulation, and nutrient feeding operations. This review will summarize the above developments and also provide our views on future trends.     

Improved metabolic action of a bacterial lysine decarboxylase gene in tobacco hairy root cultures by its fusion to arbcS transit peptide coding sequence

The gene of a bacterial lysine decarboxylase (ldc) fused to arbcS transit peptide coding sequence (tp), and under the control of the CaMV 35S promoter, was expressed in hairy root cultures ofNicotiana tabacum. The fusion of theldc to the targeting signal sequence improved the performance of the bacterial gene in the plant cells in many respects. Nearly all transgenic hairy root cultures harbouring the35S-tp-ldc gene contained distinctly higher lysine decarboxylase activity (from 1.5 to 30 pkat LDC per mg protein) than those which had been transformed with constructs in which the gene had been directly cloned behind the CaMV 35S promoter. The higher enzyme activity led to the accumulation of up to 0.7% cadaverine on a dry mass basis. In addition, part of the cadaverine pool was used for increased biosynthesis of anabasine, an alkaloid which was hardly detectable in control cultures. The best line contained anabasine levels of 0.5% dry mass, which could further be enhanced by feeding of lysine.