Xiaomu Niu

Ion Homeostasis in NaCl Stress Environments

Homeostasis can be defined as the tendency of a cell or an organism to maintain internal steady state, even in response to any environmental perturbation or stimulus tending to disturb normality, because of the coordinate responses of its constituent components. Typically, ions constantly flux in and out of cells in a controlled fashion with net flux adjusted to accommodate cellular requirements, thus creating an ionic homeostasis. When plant cells are exposed to salinity, mediated by high NaCl concentrations, kinetic steady states of ion transport for Na+ and Cland other ions, such as K+ and Ca2+, are disturbed (Binzel et al., 1988). High apoplastic levels of Na+ and Clalter aqueous and ionic thermodynamic equilibria, resulting in hyperosmotic stress, ionic imbalance, and toxicity. Thus, it is vital for the plant to re-establish cellular ion homeostasis for metabolic functioning and growth, that is, to adapt to the saline environment. Comparisons of what have been interpreted to be adaptive responses among various species lead to the conclusion that some salt-tolerant plants have evolved specialized complex mechanisms that allow adaptation to saline stress conditions. In fact, these unique mechanisms, such as salt glands, exist in few plant species and cannot be presumed to be ubiquitously functional for salt adaptation of all plants. However, intrinsically cellular-based mechanisms appear to be common to all genotypes and are a requisite for salt tolerance. Of paramount importance are those mechanisms that function to regulate ion homeostasis while mediating osmotic adjustment through the accumulation and intracellular compartmentation of ions that are predominant in the external environment. In this update we will focus principally on Na+ homeostasis in sodic environments; however, we also include discussions of H+, K+, Ca2+, and Clbecause of the interrelationship of these ions with Na+ homeostasis. Ion transport processes across the plasma membrane and the tonoplast will be emphasized because these are presumed to be most essential for the control of intracellular Na+ uptake and vacuolar compartmentation.

Antifungal activity of tobacco osmotin has specificity and involves plasma membrane permeabilization

Osmotin protein is able to inhibit in vitro the growth of a number of unrelated pathogens. A survey of 31 isolates representing 18 fungal genera indicated that sensitivity may be determined at the genus level. Hyphal growth of Aspergillus flavus, Aspergillus parasitica, Rhizoctonia solani and Macrophomina phaseolina was highly resistant to osmotin whereas the growth of Bipolaris, Fusarium and Phytophthora species was very sensitive. Of all fungi tested Trichoderma longibrachiatum hyphal growth was most inhibited by osmotin treatment. Osmotin either induced spore lysis, inhibited spore germination or reduced germling viability in seven fungal species that exhibited some degree of sensitivity in hyphal growth inhibition tests. The species-specific growth inhibition was correlated with the ability of osmotin to dissipate the fungal membrane pH gradient. Both growth inhibition and pH gradient dissipation by osmotin were sensitive to NaCl and other inorganic cations. Cells of T. longibrachiatum were insensitive to osmotin after plasmolysis, suggesting that the cell wall may be a component of the mechanism by which osmotin permeabilizes the plasma membrane and kills fungal cells.

NaCl Regulation of Plasma Membrane H+-ATPase Gene Expression in a Glycophyte and a Halophyte

NaCl regulation of plasma membrane H+-ATPase gene expression in the glycophyte tobacco (Nicotiana tabacum L. var Wisconsin 38) and the halophyte Atriplex nummularia L. was evaluated by comparison of organ-specific mRNA abundance using homologous cDNA probes encoding the ATPases of the respective plants. Accumulation of mRNA was induced by NaCl in fully expanded leaves and in roots but not in expanding leaves or stems. The NaCl responsiveness of the halophyte to accumulate plasma membrane H+-ATPase mRNA in roots was substantially greater than that of the glycophyte. Salt-induced transcript accumulation in A. nummularia roots was localized by in situ hybridization predominantly to the elongation zone, but mRNA levels also increased in the zone of differentiation. Increased message accumulation in A. nummularia roots could be detected within 8 h after NaCl (400 mM) treatment, and maximal levels were severalfold greater than in roots of untreated control plants. NaCl-induced plasma membrane H+-ATPase gene expression in expanded leaves and roots presumably indicates that these organs require increased H+-electrochemical potential gradients for the maintenance of plant ion homeostasis for salt adaptation. The greater capacity of the halophyte to induce plasma membrane H+-ATPase gene expression in response to NaCl may be a salt-tolerance determinant.

Two Wound-Inducible Soybean Cysteine Proteinase Inhibitors Have Greater Insect Digestive Proteinase Inhibitory Activities than a Constitutive Homolog

Diverse functions for three soybean (Glycine max L. Merr.) cysteine proteinase inhibitors (CysPIs) are inferred from unique characteristics of differential regulation of gene expression and inhibitory activities against specific Cys proteinases. Based on northern blot analyses, we found that the expression in leaves of one soybean CysPI gene (L1) was constitutive and the other two (N2 and R1) were induced by wounding or methyl jasmonate treatment. Induction of N2 and R1 transcript levels in leaves occurred coincidentally with increased papain inhibitory activity. Analyses of kinetic data from bacterial recombinant CysPI proteins indicated that soybean CysPIs are noncompetitive inhibitors of papain. The inhibition constants against papain of the CysPIs encoded by the wound and methyl jasmonate-inducible genes (57 and 21 nM for N2 and R1, respectively) were 500 to 1000 times lower than the inhibition constant of L1 (19,000 nM). N2 and R1 had substantially greater inhibitory activities than L1 against gut cysteine proteinases of the third-instar larvae of western corn rootworm and Colorado potato beetle. Cysteine proteinases were the predominant digestive proteolytic enzymes in the guts of these insects at this developmental stage. N2 and R1 were more inhibitory than the epoxide trans-epoxysuccinyl-L-leucylamide-(4-guanidino)butane (E-64) against western corn rootworm gut proteinases (50% inhibition concentration = 50, 200, and 7000 nM for N2, R1, and E-64, respectively). However, N2 and R1 were less effective than E-64 against the gut proteinases of Colorado potato beetle. These results indicate that the wound-inducible soybean CysPIs, N2 and R1, function in host plant defense against insect predation, and that substantial variation in CysPI activity against insect digestive proteinases exists among plant CysPI proteins.

Plasma-membrane H+-ATPase gene expression is regulated by NaCl in cells of the halophyte Atriplex nummularia L.

An Atriplex nummularia L. cDNA probe encoding the partial sequence of an isoform of the plasma-membrane H+ -ATPase was isolated, and used to characterize the NaCl regulation of mRNA accumulation in cultured cells of this halophyte. The peptide (447 amino acids) translated from the open reading frame has the highest sequence homology to the Nicotiana plumbaginifolia plasma-membrane H+-ATPase isoform pma4 (greater than 80% identity) and detected a transcript of approximately 3.7 kb on Northern blots of both total and poly(A)+ RNA. The mRNA levels were comparable in unadapted cells, adapted cells (cells adapted to and growing in 342 mM NaCl) and deadapted cells (cells previously adapted to 342 mM NaCl that are now growing without salt). Increased mRNA abundance was detected in deadapted cells within 24 h after exposure to NaCl but not in unadapted cells with similar salt treatments. The NaCl up-regulation of message abundance in deadapted cells was subject to developmental control. Analogous to those reported for glycophytes, the plasma-membrane H+-ATPase are encoded by a multigene family in the halophyte.

Transgenic peppermint (Mentha×piperita L.) plants obtained by cocultivation with Agrobacterium tumefaciens

Abstract The first transgenic peppermint (Mentha×piperita L. cultivar Black Mitcham) plants have been obtained by Agrobacterium-mediated transformation by cocultivation with morphogenically responsive leaf explants. Basal leaf explants with petioles, from leaves closest to the apex of in-vitro-culture-maintained shoots (5 cm), exhibited optimal shoot organogenetic responsiveness on medium supplemented with thidiazuron (8.4 µm). Shoot formation occurred at sites of excision on the leaf blade and petiole either directly from cells of the explant or via a primary callus. Analyses of transient GUS activity data indicated that DNA delivery by microprojectile bombardment was more effective than Agrobacterium infection. However, no transgenic plants were obtained from over 22,000 leaf explants after particle bombardment. Cocultivation of leaf explants with Agrobacterium strain EHA 105 and kanamycin selection produced transgenic plants. Greater transient and stable -glucuronidase (GUS) activities were detected in explants or propagules transformed with the construct where gusA was driven by the pBISN1 promoter rather than a CaMV 35S promoter. Eight plants were subsequently regenerated and verified as transgenic based on detection of the nptII transgene by PCR and Southern blot analyses. The Southern analyses indicated that the plants were derived from eight unique transformation events. All transgenic plants appeared morphologically normal. Analyses of GUS activities in leaves sampled from different portions of these transgenic plants, 10 months after transfer to the greenhouse, indicated that six out of the eight original regenerants were uniformly transformed, i.e., did not exhibit chimeric sectors.

NaCl-Induced Alterations in Both Cell Structure and Tissue-Specific Plasma Membrane H+ -ATPase Gene Expression

NaCl-induced plasma membrane H+-ATPase gene expression, which occurs in roots and fully expanded leaves of the halophyte Atriplex nummularia L. (X. Niu, M.L. Narasimhan, R.A. Salzman, R.A. Bressan, P.M. Hasegawa [1993] Plant Physiol 103: 713–718), has been differentially localized to specific tissues using in situ RNA hybridization techniques. Twenty-four-hour exposure of plants to 400 mM NaCl resulted in substantial accumulation of H+ pump message in the epidermis of the root tip and the endodermis of the root elongation/differentiation zone. In expanded leaves, NaCl induction of plasma membrane H+-ATPase message accumulation was localized to bundle-sheath cells. Ultrastructural analyses indicated that significant cytological adaptations in root cells included plasmolysis that is accompanied by plasma membrane invaginations, formation of Hechtian strands and vesiculation, and vacuolation. These results identify specific tissues that are involved in the regulation of Na+ and Cl- uptake into different organs of the halophyte A. nummularia and provide evidence of the intercellular and interorgan coordination that occurs in the mediation of NaCl adaptation.

Bioengineering mint crop improvement

Essential oils, synthesized and stored in leaf glandular trichomes, of the Mentha species are valuated commercially as additives for food products, cosmetics and pharmaceuticals. Mint production and oil yield is attenuated by both biotic and abiotic stresses. Consequently, there is need for development of cultivars with pest resistance and stable oil quality. Most mint cultivars are natural hybrids vegetatively propagated. Their sterility impairs the success of conventional breeding and to date, the application of irradiation mutation techniques have not resulted in the release of new commercially acceptable cultivars for widespread use. The paper summarizes the state of mint biotechnology by discussing advancements related to in vitro culture and genetic transformation, generation of herbicide resistant plants, and strategies for enhancing disease resistance and essential oil biosynthesis.

Bar-expressing peppermint (Mentha × Piperita L. var. Black Mitcham) plants are highly resistant to the glufosinate herbicide Liberty

Weed control is a substantial economic input for production of mint oils, the most commercially important of which are obtained from peppermint. The objective of this research is to obtain peppermint plants resistant to the broad-spectrum herbicide glufosinate, which can be used for development of economically efficacious weed control strategies and, perhaps, serve as a paradigm in perennial crops. The bar gene, which encodes phosphinothricin acetyltransferase (PAT) which inactivates glufosinate-ammonium or phosphinothricin (PPT), was constructed into Agrobacterium tumefaciens binary vectors under the nopaline synthase (NOS) or a chimeric promoter containing a trimer of the OCS-upstream-activating sequence (UAS) to a MAS promoter/activator region[(OCS)3MAS]. A total of 142 independent transgenic peppermint (cv. Black Mitcham) plants were obtained (107 and 35 were obtained with pGPTV (and pCAS1) and pATC940 vectors, respectively) and evaluated for herbicide resistance in the greenhouse after foliar application of glufosinate herbicide Liberty as the commercial product. All transgenic plants exhibited substantially less herbicide symptom development than non-transgenic Black Mitcham or untransformed tissue cultured-derived plants, albeit variation for herbicide resistance occurred amongst the transformed lines. Plants from 35 of the 142 lines were selected at random and all were PCR-positive for the presence of bar. Five lines, that were least affected, exhibited no injury symptoms to Liberty concentrations that are 4 times the standard level for control of weeds in peppermint fields. The most resistant transgenic plants had the greatest steady-state PAT mRNA levels and PAT activities. No experimental difference in herbicide resistance was evident between plant populations obtained with pGPTV (pCAS1)-bar or pATC940-bar vector. However, 4 of 35 lines transformed with (ocs)3MAS-bar exhibited maximal resistance while only 1 of 107 NOS-bar lines has comparable resistance. These herbicide resistant peppermint plants will facilitate development of post-emergent herbicide control strategies that use newer generation herbicides, like glufosinate, which have reduced environmental and product residual because of metabolism by microbes and the transgenic plants.

Salt-Sensitive Mutants of Chlamydomonas reinhardtii Isolated after Insertional Tagging.

We describe the isolation of salt-sensitive Chlamydomonas reinhardtii mutants by insertional mutagenesis using the nitrate reductase (Nit1) gene. The plasmid pMN24, containing Nit1, was used for transformation of 305CW15 (nit1 cw15 mt+), and transformants were selected for complementation of the nit- phenotype. From 6875 nit+ colonies, four transformants (S4, S18, S46, and S66) were isolated that exhibited both Na+ and Li+ sensitivity (sod-), and another transformant (S33) was selected that exhibited sensitivity to Li+ but not Na+ (lit-) based on relative growth comparisons with the wild-type strain. S33, S46, and S66 were no more growth inhibited by sorbitol than was 305CW15. In comparison, S4 and S18 exhibited substantial growth inhibition in medium supplemented with sorbitol. Genetic analyses indicated that the salt-sensitive mutants were each defective in a single recessive gene. The mutant genes in S4 (sod1), S33 (lit1), and S66 (sod3) are linked to a functional copy of Nit1 and are presumably tagged with a pMN24 insertion.