Two poplar calcineurin B-like proteins confer enhanced tolerance to abiotic stresses in transgenic Arabidopsis thaliana

Calcium is a critical component in a number of plant signal transduction pathways and the calcineurin B-like protein (CBL) family is a unique group of calcium sensors regulating a family of CBL-interacting protein kinases (CIPKs). In this study, two poplar CBL genes, PeCBL6 (GenBank acc. No. DQ907710) and PeCBL10 (GenBank acc. No. DQ899956), were characterized in transgenic Arabidopsis thaliana, particularly with regard to its role in abiotic stress resistance. Expression of the two CBL genes in poplar was induced by cold, drought, or high salinity, but not by abscisic acid (ABA) treatment. In Arabidopsis thaliana, PeCBL6 was found in the nucleus and PeCBL10 in the tonoplast. Transgenic Arabidopsis plants overexpressing PeCBL6 or PeCBL10 showed enhanced tolerance to high salinity, drought and low temperature. These results suggested that PeCBL6 and PeCBL10 may function as positive regulators of salt, drought and temperature responses.     

Jasmonic acid does not mediate root growth responses to wounding in Arabidopsis thaliana

Jasmonic acid (JA) is a crucial plant defence signalling substance that has recently been shown to mediate herbivory-induced root growth reduction in the ecological model species Nicotiana attenuata . To clarify whether JA-induced reduction of root growth might be a general response increasing plant fitness under biotic stress, a suite of experiments was performed with the model plant Arabidopsis thaliana . JA bursts were elicited in leaves of A. thaliana in different ways. Root growth reduction was neither induced by foliar application of herbivore oral secretions nor by direct application of methyl jasmonate to leaves. Root growth reduction was observed when leaves were infected with the pathogen Pseudomonas syringae pv. tomato, which persistently induces the JA signalling pathway. Yet, high resolution growth analyses of this effect in wild type and JA biosynthesis knock-out mutants showed that it was elicited by the bacterial toxin coronatine that suggests ethylene- but not JA-induced root growth reduction in A. thaliana . Overall, the results demonstrate that the reaction of root growth to herbivore-induced JA signalling differs among species, which is discussed in the context of different ecological defence strategies among species.     

Effect of reduced arginine decarboxylase activity on salt tolerance and on polyamine formation during salt stress in Arabidopsis thaliana

Polyamines have been suggested to play an important role in stress protection. However, attempts to determine the function of polyamines have been complicated by the fact that, dependent on the conditions, polyamine contents increase or decrease during stress. To determine the importance of polyamine formation during salt stress, we analysed polyamine contents and salt tolerance in two Arabidopsis thaliana mutants, spe1-1 and spe2-1 (Watson et al. Plant J 13: 231–239, 1998), with reduced activity of arginine decarboxylase (EC 4.1.1.19), an important enzyme in polyamine synthesis. Polyamines accumulated in wild-type plants (Col-0 and Ler-0) that were pre-treated with 100 m M NaCl before transfer to 125 m M NaCl, but not in plants that were directly transferred to 125 m M NaCl without prior treatment with 100 m M NaCl. This shows that polyamine accumulation depends on acclimation to salinity. The salt treatment that induced polyamine accumulation in wild-type plants did not lead to polyamine accumulation in the spe1-1 and spe2-1 mutants. Decreased fresh weight, chlorophyll content and photosynthetic efficiency indicated that the spe1-1 mutant was more severely affected by salt stress than its wild type, Col-0. In the spe2-1 mutant decreased salt tolerance compared to its wild type, Ler-0, became apparent as bleaching under severe salt stress. The present results demonstrate that decreased polyamine formation due to lower arginine decarboxylase activity leads to reduced salt tolerance.     

Diversity and salt tolerance of native Acacia rhizobia isolated from saline and non‐saline soils

Re‐establishing native vegetation in stressed soils is of considerable importance in many parts of the world, leading to significant interest in using plant–soil symbiont interactions to increase the cost‐effectiveness of large‐scale restoration. However, effective use of soil microbes in revegetation requires knowledge of how microbe communities vary along environmental stress gradients, as well as how such variation relates to symbiont effectiveness. In Australia, shrubby legumes dominate many ecosystems where dryland salinity is a major issue, and improving plant establishment in saline soils is a priority of regional management agencies. In this study, strains of rhizobial bacteria were isolated from a range of Acacia spp. growing in saline and non‐saline soils. Replicates of each strain were grown under several salinity levels in liquid culture and characterized for growth and salt tolerance. Genetic characterization of rhizobia showed considerable variation among strains, with salt tolerance and growth generally higher in rhizobial populations derived from more saline soils. These strains showed markedly different genetic profiles and generic affiliations to those from more temperate soils, suggesting community differentiation in relation to salt stress. The identification of novel genomic species from saline soils suggests that the diversity of rhizobia associated with Australian Acacia spp. is significantly greater than previously described. Overall, the ability of some symbiotically effective strains to tolerate high salinity is promising with regard to improving host plant re‐establishment in these soils.     

Arabidopsis thaliana AtHAL3: a flavoprotein related to salt and osmotic tolerance and plant growth

The 26S proteasome is a multi-subunit ATP-dependent protease responsible for degrading most short-lived intracellular proteins targeted for breakdown by ubiquitin conjugation. The complex is composed of two relatively stable subparticles, the 20S proteasome, a hollow cylindrical structure which contains the proteolytic active sites in its lumen, and the 19S regulatory particle (RP) which binds to either end of the cylinder and provides the ATP-dependence and the specificity for ubiquitinated proteins. Among the approximately 18 subunits of the RP from yeast and animals are a set of six proteins, designated RPT1-6 for regulatory particle triple-A ATPase, that form a distinct family within the AAA superfamily. Presumably, these subunits use ATP hydrolysis to help assemble the 26S holocomplex, recognize and unfold appropriate substrates, and/or translocate the substrates to the 20S complex for degradation. Here, we describe the RPT gene family from Arabidopsis thaliana. From a collection of cDNAs and genomic sequences, a family of genes encoding all six of the RPT subunits was identified with significant amino acid sequence similarity to their yeast and animal counterparts. Five of the six RPT sub- units are encoded by two genes; the exception being RPT3 which is encoded by a single gene. mRNA for each of the six proteins is present in all tissue types examined. Five of the subunits (RPT1 and 3-6) complemented yeast mutants missing their respective orthologs, indicating that the yeast and Arabidopsis proteins are functionally equivalent. Taken together, these results demonstrate that the RP, like the 20S proteasome, is functionally and structurally conserved among eukaryotes and indicate that the plant RPT subunits, like their yeast counterparts, have non-redundant functions.     

Hyperactive spruce budworm antifreeze protein expression in transgenic Drosophila does not confer cold shock tolerance

Drosophila melanogaster, a freeze intolerant and cold shock sensitive insect, was transformed with the hyperactive insect antifreeze protein gene (AFP) from the spruce budworm, Choristoneura fumiferana. Transformation P-element constructs (pCasper) were made with CfAFP 337 isoform DNA using a strong constitutive promoter, Actin 5c. This is the first report of insect AFP used to transform another insect. Properly folded active insect AFP was only detected when signal sequences were used to target proteins to the endoplasmic reticulum for secretion into the hemolymph. The 18 residue Drosophila binding protein signal sequence (BiP) constructs resulted in transformed fly lines with significantly higher AFP expression in hemolymph than when the native C. fumiferana AFP signal sequence was used. The resultant transgene fly lines have the highest levels of thermal hysteresis, 0.8 degrees C, seen for any engineered Drosophila. Despite the high level of expression, even higher than some overwintering fish with natural levels of endogenous AFP, the transformants did not display any cold shock resistance compared to controls or low AFP expressing lines. These results indicate that insect AFP alone cannot protect Drosophila from cold shock and may not be useful for Drosophila cryopreservation.