Stefania Grillo

SOS2 Promotes Salt Tolerance in Part by Interacting with the Vacuolar H+-ATPase and Upregulating Its Transport Activity

ABSTRACT The salt overly sensitive (SOS) pathway is critical for plant salt stress tolerance and has a key role in regulating ion transport under salt stress. To further investigate salt tolerance factors regulated by the SOS pathway, we expressed an N-terminal fusion of the improved tandem affinity purification tag to SOS2 (NTAP-SOS2) in sos2-2 mutant plants. Expression of NTAP-SOS2 rescued the salt tolerance defect of sos2-2 plants, indicating that the fusion protein was functional in vivo. Tandem affinity purification of NTAP-SOS2-containing protein complexes and subsequent liquid chromatography-tandem mass spectrometry analysis indicated that subunits A, B, C, E, and G of the peripheral cytoplasmic domain of the vacuolar H+-ATPase (V-ATPase) were present in a SOS2-containing protein complex. Parallel purification of samples from control and salt-stressed NTAP-SOS2/sos2-2 plants demonstrated that each of these V-ATPase subunits was more abundant in NTAP-SOS2 complexes isolated from salt-stressed plants, suggesting that the interaction may be enhanced by salt stress. Yeast two-hybrid analysis showed that SOS2 interacted directly with V-ATPase regulatory subunits B1 and B2. The importance of the SOS2 interaction with the V-ATPase was shown at the cellular level by reduced H+ transport activity of tonoplast vesicles isolated from sos2-2 cells relative to vesicles from wild-type cells. In addition, seedlings of the det3 mutant, which has reduced V-ATPase activity, were found to be severely salt sensitive. Our results suggest that regulation of V-ATPase activity is an additional key function of SOS2 in coordinating changes in ion transport during salt stress and in promoting salt tolerance.

Interaction of SOS2 with Nucleoside Diphosphate Kinase 2 and Catalases Reveals a Point of Connection between Salt Stress and H2O2 Signaling in Arabidopsis thaliana

ABSTRACT SOS2, a class 3 sucrose-nonfermenting 1-related kinase, has emerged as an important mediator of salt stress response and stress signaling through its interactions with proteins involved in membrane transport and in regulation of stress responses. We have identified additional SOS2-interacting proteins that suggest a connection between SOS2 and reactive oxygen signaling. SOS2 was found to interact with the H2O2 signaling protein nucleoside diphosphate kinase 2 (NDPK2) and to inhibit its autophosphorylation activity. A sos2-2 ndpk2 double mutant was more salt sensitive than a sos2-2 single mutant, suggesting that NDPK2 and H2O2 are involved in salt resistance. However, the double mutant did not hyperaccumulate H2O2 in response to salt stress, suggesting that it is altered signaling rather than H2O2 toxicity alone that is responsible for the increased salt sensitivity of the sos2-2 ndpk2 double mutant. SOS2 was also found to interact with catalase 2 (CAT2) and CAT3, further connecting SOS2 to H2O2 metabolism and signaling. The interaction of SOS2 with both NDPK2 and CATs reveals a point of cross talk between salt stress response and other signaling factors including H2O2.

Acclimation to low water potential in potato cell suspension cultures leads to changes in putrescine metabolism

Changes in levels and biosynthesis of di- and polyamines are associated with stress responses in plant cells. The involvement of these molecules was investigated here in cultured potato (Solanum tuberosum L.) cells grown in medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin, and acclimated or not to low water potential. The diamine (putrescine) and polyamine (spermidine and spermine) status in cells gradually acclimated to increasing concentrations (up to 20 %, w/v) of polyethylene glycol (PEG) Mr 8000, was compared with that of unacclimated cells abruptly exposed (shocked) or not (controls) to 20 % (w/v) PEG. After a 72-h subculture, the free and perchloric acid (PCA)-soluble conjugated di- and polyamine pattern in acclimated cells was not dramatically different from that of controls, but PCA-insoluble conjugated putrescine was 14-fold higher than in controls. In shocked cells, a strong reduction in free putrescine and spermidine/spermine titres occurred. Arginine (ADC, EC 4.1.1.19) and ornithine (ODC, EC 4.1.1.17) decarboxylase activities were not substantially altered in shocked cells compared with controls, while in PEG-acclimated cell populations they increased about 3-fold, both in the soluble and particulate fractions. S-Adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21) and diamine oxidase (DAO, EC 1.4.3.6) activities followed a similar pattern to each other in that their activities were enhanced 2- and 3-fold, respectively, in acclimated cells over unacclimated controls. Ethylene production was also enhanced in acclimated cells. These results indicate that, with respect to di- and polyamines, acquired tolerance to low water potential in potato cells leads principally to changes in putrescine biosynthesis and conjugation which may be involved in ensuring cell survival.

Physical Stresses in Plants

The influence of high temperature stress (heat shock or HS) and other environmental stress agents on gene expression of soybean seedlings has been extensively studied. The sequence analysis of HS genes has revealed a high degree of conservation among individual members of several heat shock protein (HSP) families and different classes within a family, but some interesting differences have been noted. These studies have also revealed complex patterns of regulation of expression of the HS genes and accumulation of the HSPs. Based primarily upon the deduced amino acid sequence of the HSPs, immunological cross-reactivity, and intracellular localization, the complex group of low molecular weight (LMW) HSP genes have been organized into multiple classes. In soybean several eDNA and genomic clones encoding 20 to 24 kD LMW HSPs have been isolated which represent new classes of the LMW HSP gene super family based on nucleotide/amino acid sequence and cell fractionation analyses. The mRNAs transcribed from these genes are of lower abundance than those for the 15 to 18 kD Class I and II proteins, and these genes occur as small multigene (i.e. three to four) classes or subfamilies. The mRNAs of three of these classes of LMW HSP genes are translated on ERbound ribosomes and possess hydrophobic leader sequences. The presence of a consensus ER retention sequence on two of these proteins indicates that they probably reside within the ER. The third protein lacks the consensus ER retention signal and presumably is translocated to an as yet unidentified location. The mRNA representing a fourth LMW gene class is translated on unbound cytoplasmic ribosomes, and the predicted protein has aN-terminal sequence with properties similar to that of some proteins which are translocated into mitochondria. Early studies with soybean seedlings indicated that some 22 to 24 kD HSPs are localized in mitochondria. Differential induction by amino analog treatment indicates that genes assigned to the same class based on amino acid similarity and localization can be regulated differently. The possible role of the multiple classes on LMW 15 to 24 kD HSPs in protein protection

Asg1 is a stress-inducible gene which increases stomatal resistance in salt stressed potato

The identification of critical components in plant salt stress adaptation has greatly benefitted, in the last two decades, from fundamental discoveries in Arabidopsis and close model systems. Nevertheless, this approach has also highlighted a non-complete overlap between stress tolerance mechanisms in Arabidopsis and agricultural crops. Within a long-running research program aimed at identifying salt stress genetic determinants in potato by functional screening in Escherichia coli, we isolated Asg1, a stress-related gene with an unknown function. Asg1 is induced by salt stress in both potato and Arabidopsis and by abscisic acid in Arabidopsis. Asg1 is actively transcribed in all plant tissues. Furthermore, Asg1 promoter analysis confirmed its ubiquitous expression, which was remarkable in pollen, a plant tissue that undergoes drastic dehydration/hydration processes. Fusion of Asg1 with green fluorescent protein showed that the encoded protein is localized close to the plasma membrane with a non-continuous pattern of distribution. In addition, Arabidopsis knockout asg1 mutants were insensitive to both NaCl and sugar hyperosmotic environments during seed germination. Transgenic potato plants over-expressing the Asg1 gene revealed a stomatal hypersensitivity to NaCl stress which, however, did not result in a significantly improved tuber yield in stress conditions. Altogether, these data suggest that Asg1 might interfere with components of the stress signaling pathway by promoting stomatal closure and participating in stress adaptation.

Adaptation of Potato Cells to Low Water Potential and Changes in Membrane Fatty Acid Composition and Fluidity

Disruption of cellular membranes has been observed in plant tissues in response to intense water stress, with loss of membrane functionality. A stepwise adaptation allows biochemical and structural changes of cellular membranes and maintenance of membrane fluidity, which is a prerequisite of acquired tolerance (1). While an extensive literature on changes in membrane fatty acid composition induced by varying external temperatures is available (2), much less is known about the changes induced in plants under drought conditions. It has been reported that, besides variation of the major classes of lipids, the level of unsaturation of fatty acids (FA) decreases (2). The data reported deal with the changes observed in potato cells which were gradually adapted to increasing low water potentials induced by PEG 8000, which reduces free water concentration extracellularly (3). Adapted cells were able to grow actively in the presence of 20% PEG and maintained a normal ultra-structure of the main cellular and sub-cellular constituents. FA of the main extra-chloroplast phospholipids were found to be more saturated in PEG-adapted cells, compared to those in unadapted cells. These changes were associated to an increase of microviscosity of isolated protoplasts from adapted cells, as determined by steady-state fluorescence anisotropy by using diphenil-hexatriene (DPH) as a membrane probe (4).

Regulation of gene expression in response to drought and osmotic shock

We have studied the response of potato cell suspension culture to PEGmediated low water potential. Gradual adaptation to high concentrations of PEG 8000 (20%) did not affect growth and endogenous ABA content of potato cells. On the contrary, direct exposure to 20% PEG (osmotic shock) drastically reduced growth in unadapted cells and also induced a six-fold increase in ABA level within 5 days from the imposition of the stress. 2D-electrophoretic pattern of in vivo labeled proteins of adapted cells was very similar to’ that of control cells. Only few polypeptides were down-regulated and the synthesis of at least 31 individual polypeptides was increased upon adaptation. Osmotic shock as well as ABA treatment of unadapted cells determined an overall reduction of protein synthesis. Most of the induced polypeptides in PEG-shocked cells were also found to be induced in unadapted cells upon treatment with exogenous ABA. However, it was possible to identify some polypeptides specifically induced in osmotically shocked cells, a group of which was also detected in adapted cells. The comparison of protein electrophoretic patterns demonstrated t h a t most changes observed in PEG adapted cells were not mediated by ABA. Long-term adaptation of potato cells to PEG enhanced both the transcript and protein level of osmotin, a 26 kD protein reported to accumulate in salt-adapted tobacco cells. In osmotic shocked cells a dramatic increase of osmotin transcripts was also detected, which however did not correlate with the level of t h e protein.

Genes for tolerance to water and osmotic stress in glycophyte plants

Abstract Most of the crops are extremely susceptible to environmental stresses, because they have been selected for a high yield performance under optimal growth conditions. Not tolerant plants (glycophytes) respond to changes in the environment with complex mechanisms, involving a network of genes, which are either up- or down-regulated. Some of the genes induced are common between glycophytes and typically tolerant plants, e.g. xerophytes and halophytes, supporting the contention that stress tolerance mechanisms are ubiquitous. In this paper the changes in gene expression observed in potato cells upon abrupt imposition of water stress compared to the changes induced during a gradual acclimation to the same conditions are reported. Data on the involvement or not of the phytohormone abscisic acid in controlling such changes are also reported. The regulation of specific genes (osmotin, Em) during the stress have been studied in glycophyte plants, such as tomato and wheat.