Live-cell RESOLFT nanoscopy of transgenic Arabidopsis thaliana.

Subdiffraction super-resolution fluorescence microscopy, or nanoscopy, has seen remarkable developments in the last two decades. Yet, for the visualization of plant cells, nanoscopy is still rarely used. In this study, we established RESOLFT nanoscopy on living green plant tissue. Live-cell RESOLFT nanoscopy requires and utilizes comparatively low light doses and intensities to overcome the diffraction barrier. We generated a transgenic Arabidopsis thaliana plant line expressing the reversibly switchable fluorescent protein rsEGFP2 fused to the mammalian microtubule-associated protein 4 (MAP4) in order to ubiquitously label the microtubule cytoskeleton. We demonstrate the use of RESOLFT nanoscopy for extended time-lapse imaging of cortical microtubules in Arabidopsis leaf discs. By combining our approach with fluorescence lifetime gating, we were able to acquire live-cell RESOLFT images even close to chloroplasts, which exhibit very strong autofluorescence. The data demonstrate the feasibility of subdiffraction resolution imaging in transgenic plant material with minimal requirements for sample preparation.

CRISPR/Cas9-mediated knockout of Populus BRANCHED1 and BRANCHED2 orthologs reveals a major function in bud outgrowth control.

The TCP-type transcription factors BRANCHED1 and BRANCHED2 shape plant architecture by suppressing bud outgrowth, with BRANCHED2 only playing a minor role in Arabidopsis. Here, we investigated the function of orthologs of these genes in the model tree Populus. We used CRISPR/Cas9 to generate loss-of-function mutants of previously identified Populus BRANCHED1-1 and BRANCHED2-1 candidate genes. BRANCHED1-1 mutants exhibited strongly enhanced bud outgrowth. BRANCHED2-1 mutants had an extreme bud outgrowth phenotype and possessed two ectopic leaves at each node. While BRANCHED1 function is conserved in poplar, BRANCHED2, in contrast to its Arabidopsis counterpart, plays an even more critical role in bud outgrowth regulation. In addition, we identified a new, not yet reported association of this gene to leaf development.

Knockdown of strigolactone biosynthesis genes in Populus affects BRANCHED1 expression and shoot architecture.

Plant architecture is modified by a regulatory system that controls axillary bud outgrowth. Key components in this system are strigolactones (SLs) and BRANCHED1, which inhibit bud outgrowth. Their role has been described in herbaceous model systems, including Arabidopsis, rice and pea. However, a role in woody perennial species, including the model tree poplar, has not been unequivocally proven. In this study, we tested a role for SLs in Populus × canescens by treatment with the synthetic SL GR24. We generated MORE AXILLARY BRANCHING4 (MAX4) knockdown lines to study the architectural phenotype of poplar SL biosynthesis mutants and the expression of SL-regulated genes. We show that GR24 is perceived by the model tree poplar. MAX4 knockdown lines exhibit typical SL deficiency symptoms. The observed changes in branching pattern, internode length and plant height can be rescued by grafting. We identified putative poplar BRANCHED1 and BRANCHED2 genes and provide evidence for a regulation of BRANCHED1 by SLs. Our results suggest a conservation of major regulatory mechanisms in bud outgrowth control in the model tree poplar. This may facilitate further research, pinpointing the role of SLs and BRANCHED1 in the complex regulation of bud outgrowth in trees.

Tissue- and Cell-Specific Cytokinin Activity in Populus × canescens Monitored by ARR5::GUS Reporter Lines in Summer and Winter.

Cytokinins play an important role in vascular development. But knowledge on the cellular localization of this growth hormone in the stem and other organs of woody plants is lacking. The main focus of this study was to investigate the occurrence and cellular localization of active cytokinins in leaves, roots, and along the stem of Populus × canescens and to find out how the pattern is changed between summer and winter. An ARR5::GUS reporter construct was used to monitor distribution of active cytokinins in different tissues of transgenic poplar lines. Three transgenic lines tested under outdoor conditions showed no influence of ARR5::GUS reporter construct on the growth performance compared with the wild-type, but one line lost the reporter activity. ARR5::GUS activity indicated changes in the tissue- and cell type-specific pattern of cytokinin activity during dormancy compared with the growth phase. ARR5::GUS activity, which was present in the root tips in the growing season, disappeared in winter. In the stem apex ground tissue, ARR5::GUS activity was higher in winter than in summer. Immature leaves from tissue-culture grown plants showed inducible ARR5::GUS activity. Leaf primordia in summer showed ARR5::GUS activity, but not the expanded leaves of outdoor plants or leaf primordia in winter. In stem cross sections, the most prominent ARR5::GUS activity was detected in the cortex region and in the rays of bark in summer and in winter. In the cambial zone the ARR5::GUS activity was more pronounced in the dormant than in growth phase. The pith and the ray cells adjacent to the vessels also displayed ARR5::GUS activity. In silico analyses of the tissue-specific expression patterns of the whole PtRR type-A family of poplar showed that PtRR10, the closest ortholog to the Arabidopsis ARR5 gene, was usually the most highly expressed gene in all tissues. In conclusion, gene expression and tissue-localization indicate high activity of cytokinins not only in summer, but also in winter. The presence of the signal in meristematic tissues supports their role in meristem maintenance. The reporter lines will be useful to study the involvement of cytokinins in acclimation of poplar growth to stress.

SUPPRESSOR OF APICAL DOMINANCE1 of Sporisorium reilianum Modulates Inflorescence Branching Architecture in Maize and Arabidopsis.

The biotrophic fungus Sporisorium reilianum causes head smut of maize (Zea mays) after systemic plant colonization. Symptoms include the formation of multiple female inflorescences at subapical nodes of the stalk because of loss of apical dominance. By deletion analysis of cluster 19-1, the largest genomic divergence cluster in S. reilianum, we identified a secreted fungal effector responsible for S. reilianum-induced loss of apical dominance, which we named SUPPRESSOR OF APICAL DOMINANCE1 (SAD1). SAD1 transcript levels were highly up-regulated during biotrophic fungal growth in all infected plant tissues. SAD1-green fluorescent protein fusion proteins expressed by recombinant S. reilianum localized to the extracellular hyphal space. Transgenic Arabidopsis (Arabidopsis thaliana)-expressing green fluorescent protein-SAD1 displayed an increased number of secondary rosette-leaf branches. This suggests that SAD1 manipulates inflorescence branching architecture in maize and Arabidopsis through a conserved pathway. Using a yeast (Saccharomyces cerevisiae) two-hybrid library of S. reilianum-infected maize tissues, we identified potential plant interaction partners that had a predicted function in ubiquitination, signaling, and nuclear processes. Presence of SAD1 led to an increase of the transcript levels of the auxin transporter PIN-FORMED1 in the root and a reduction of the branching regulator TEOSINTE BRANCHED1 in the stalk. This indicates a role of SAD1 in regulation of apical dominance by modulation of branching through increasing transcript levels of the auxin transporter PIN1 and derepression of bud outgrowth.

Shaping plant architecture.

Plants exhibit phenotypical plasticity. Their general body plan is genetically determined, but plant architecture and branching patterns are variable and can be adjusted to the prevailing environmental conditions. The modular design of the plant facilitates such morphological adaptations. The prerequisite for the formation of a branch is the initiation of an axillary meristem. Here, we review the current knowledge about this process. After its establishment, the meristem can develop into a bud which can either become dormant or grow out and form a branch. Many endogenous factors, such as photoassimilate availability, and exogenous factors like nutrient availability or shading, have to be integrated in the decision whether a branch is formed. The underlying regulatory network is complex and involves phytohormones and transcription factors. The hormone auxin is derived from the shoot apex and inhibits bud outgrowth indirectly in a process termed apical dominance. Strigolactones appear to modulate apical dominance by modification of auxin fluxes. Furthermore, the transcription factor BRANCHED1 plays a central role. The exact interplay of all these factors still remains obscure and there are alternative models. We discuss recent findings in the field along with the major models. Plant architecture is economically significant because it affects important traits of crop and ornamental plants, as well as trees cultivated in forestry or on short rotation coppices. As a consequence, plant architecture has been modified during plant domestication. Research revealed that only few key genes have been the target of selection during plant domestication and in breeding programs. Here, we discuss such findings on the basis of various examples. Architectural ideotypes that provide advantages for crop plant management and yield are described. We also outline the potential of breeding and biotechnological approaches to further modify and improve plant architecture for economic needs.

Phosphatidylinositol 4,5-bisphosphate influences PIN polarization by controlling clathrin-mediated membrane trafficking in Arabidopsis.

The functions of the minor phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] during vegetative plant growth remain obscure. Here, we targeted two related phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) PIP5K1 and PIP5K2, which are expressed ubiquitously in Arabidopsis thaliana. A pip5k1 pip5k2 double mutant with reduced PtdIns(4,5)P2 levels showed dwarf stature and phenotypes suggesting defects in auxin distribution. The roots of the pip5k1 pip5k2 double mutant had normal auxin levels but reduced auxin transport and altered distribution. Fluorescence-tagged auxin efflux carriers PIN-FORMED (PIN1)-green fluorescent protein (GFP) and PIN2-GFP displayed abnormal, partially apolar distribution. Furthermore, fewer brefeldin A-induced endosomal bodies decorated by PIN1-GFP or PIN2-GFP formed in pip5k1 pip5k2 mutants. Inducible overexpressor lines for PIP5K1 or PIP5K2 also exhibited phenotypes indicating misregulation of auxin-dependent processes, and immunolocalization showed reduced membrane association of PIN1 and PIN2. PIN cycling and polarization require clathrin-mediated endocytosis and labeled clathrin light chain also displayed altered localization patterns in the pip5k1 pip5k2 double mutant, consistent with a role for PtdIns(4,5)P2 in the regulation of clathrin-mediated endocytosis. Further biochemical tests on subcellular fractions enriched for clathrin-coated vesicles (CCVs) indicated that pip5k1 and pip5k2 mutants have reduced CCV-associated PI4P 5-kinase activity. Together, the data indicate an important role for PtdIns(4,5)P2 in the control of clathrin dynamics and in auxin distribution in Arabidopsis.

Stabilization of cytokinin levels enhances Arabidopsis resistance against Verticillium longisporum.

Verticillium longisporum is a vascular pathogen that infects the Brassicaceae host plants Arabidopsis thaliana and Brassica napus. The soilborne fungus enters the plant via the roots and colonizes the xylem of roots, stems, and leaves. During late stages of infections, Verticillium spp. spread into senescing tissue and switch from biotrophic to a necrotrophic life style. Typical symptoms of V. longisporum-induced disease are stunted growth and leaf chlorosis. Expression analyses of the senescence marker genes SENESCENCE-ASSOCIATED GENE12, SENESCENCE-ASSOCIATED GENE13, and WRKY53 revealed that the observed chlorosis is a consequence of premature senescence triggered by Verticillium infection. Our analyses show that, concomitant with the development of chlorosis, levels of trans-zeatin decrease in infected plants. Potentially, induction of cytokinin oxidase/dehydrogenase expression by Verticillium infection contributes to the observed decreases in cytokinin levels. Stabilization of Arabidopsis cytokinin levels by both pharmacological and genetic approaches inhibits Verticillium proliferation and coincides with reduced disease symptom development. In summary, our results indicate that V. longisporum triggers premature plant senescence for efficient host plant colonization.

Asymmetric gibberellin signaling regulates vacuolar trafficking of PIN auxin transporters during root gravitropism.

Gravitropic bending of plant organs is mediated by an asymmetric signaling of the plant hormone auxin between the upper and lower side of the respective organ. Here, we show that also another plant hormone, gibberellic acid (GA), shows asymmetric action during gravitropic responses. Immunodetection using an antibody against GA and monitoring GA signaling output by downstream degradation of DELLA proteins revealed an asymmetric GA distribution and response with the maximum at the lower side of gravistimulated roots. Genetic or pharmacological manipulation of GA levels or response affects gravity-mediated auxin redistribution and root bending response. The higher GA levels at the lower side of the root correlate with increased amounts of PIN-FORMED2 (PIN2) auxin transporter at the plasma membrane. The observed increase in PIN2 stability is caused by a specific GA effect on trafficking of PIN proteins to lytic vacuoles that presumably occurs downstream of brefeldin A-sensitive endosomes. Our results suggest that asymmetric auxin distribution instructive for gravity-induced differential growth is consolidated by the asymmetric action of GA that stabilizes the PIN-dependent auxin stream along the lower side of gravistimulated roots.

Verticillium infection triggers VASCULAR-RELATED NAC DOMAIN7-dependent de novo xylem formation and enhances drought tolerance in Arabidopsis.

The soilborne fungal plant pathogen Verticillium longisporum invades the roots of its Brassicaceae hosts and proliferates in the plant vascular system. Typical aboveground symptoms of Verticillium infection on Brassica napus and Arabidopsis thaliana are stunted growth, vein clearing, and leaf chloroses. Here, we provide evidence that vein clearing is caused by pathogen-induced transdifferentiation of chloroplast-containing bundle sheath cells to functional xylem elements. In addition, our findings suggest that reinitiation of cambial activity and transdifferentiation of xylem parenchyma cells results in xylem hyperplasia within the vasculature of Arabidopsis leaves, hypocotyls, and roots. The observed de novo xylem formation correlates with Verticillium-induced expression of the VASCULAR-RELATED NAC DOMAIN (VND) transcription factor gene VND7. Transgenic Arabidopsis plants expressing the chimeric repressor VND7-SRDX under control of a Verticillium infection-responsive promoter exhibit reduced de novo xylem formation. Interestingly, infected Arabidopsis wild-type plants show higher drought stress tolerance compared with noninfected plants, whereas this effect is attenuated by suppression of VND7 activity. Together, our results suggest that V. longisporum triggers a tissue-specific developmental plant program that compensates for compromised water transport and enhances the water storage capacity of infected Brassicaceae host plants. In conclusion, we provide evidence that this natural plant-fungus pathosystem has conditionally mutualistic features.

Transcription factor WRKY23 assists auxin distribution patterns during Arabidopsis root development through local control on flavonol biosynthesis.

Gradients of the plant hormone auxin, which depend on its active intercellular transport, are crucial for the maintenance of root meristematic activity. This directional transport is largely orchestrated by a complex interaction of specific influx and efflux carriers that mediate the auxin flow into and out of cells, respectively. Besides these transport proteins, plant-specific polyphenolic compounds known as flavonols have been shown to act as endogenous regulators of auxin transport. However, only limited information is available on how flavonol synthesis is developmentally regulated. Using reduction-of-function and overexpression approaches in parallel, we demonstrate that the WRKY23 transcription factor is needed for proper root growth and development by stimulating the local biosynthesis of flavonols. The expression of WRKY23 itself is controlled by auxin through the Auxin Response Factor 7 (ARF7) and ARF19 transcriptional response pathway. Our results suggest a model in which WRKY23 is part of a transcriptional feedback loop of auxin on its own transport through local regulation of flavonol biosynthesis.

Inositol trisphosphate-induced Ca2+ signaling modulates auxin transport and PIN polarity.

The phytohormone auxin is an important determinant of plant development. Directional auxin flow within tissues depends on polar localization of PIN auxin transporters. To explore regulation of PIN-mediated auxin transport, we screened for suppressors of PIN1 overexpression (supo) and identified an inositol polyphosphate 1-phosphatase mutant (supo1), with elevated inositol trisphosphate (InsP(3)) and cytosolic Ca(2+) levels. Pharmacological and genetic increases in InsP(3) or Ca(2+) levels also suppressed the PIN1 gain-of-function phenotypes and caused defects in basal PIN localization, auxin transport and auxin-mediated development. In contrast, the reductions in InsP(3) levels and Ca(2+) signaling antagonized the effects of the supo1 mutation and disrupted preferentially apical PIN localization. InsP(3) and Ca(2+) are evolutionarily conserved second messengers involved in various cellular functions, particularly stress responses. Our findings implicate them as modifiers of cell polarity and polar auxin transport, and highlight a potential integration point through which Ca(2+) signaling-related stimuli could influence auxin-mediated development.

Clathrin mediates endocytosis and polar distribution of PIN auxin transporters in Arabidopsis.

Endocytosis is a crucial mechanism by which eukaryotic cells internalize extracellular and plasma membrane material, and it is required for a multitude of cellular and developmental processes in unicellular and multicellular organisms. In animals and yeast, the best characterized pathway for endocytosis depends on the function of the vesicle coat protein clathrin. Clathrin-mediated endocytosis has recently been demonstrated also in plant cells, but its physiological and developmental roles remain unclear. Here, we assessed the roles of the clathrin-mediated mechanism of endocytosis in plants by genetic means. We interfered with clathrin heavy chain (CHC) function through mutants and dominant-negative approaches in Arabidopsis thaliana and established tools to manipulate clathrin function in a cell type-specific manner. The chc2 single mutants and dominant-negative CHC1 (HUB) transgenic lines were defective in bulk endocytosis as well as in internalization of prominent plasma membrane proteins. Interference with clathrin-mediated endocytosis led to defects in constitutive endocytic recycling of PIN auxin transporters and their polar distribution in embryos and roots. Consistent with this, these lines had altered auxin distribution patterns and associated auxin transport-related phenotypes, such as aberrant embryo patterning, imperfect cotyledon specification, agravitropic growth, and impaired lateral root organogenesis. Together, these data demonstrate a fundamental role for clathrin function in cell polarity, growth, patterning, and organogenesis in plants.

Silencing of Vlaro2 for chorismate synthase revealed that the phytopathogen Verticillium longisporum induces the cross-pathway control in the xylem.

The first leaky auxotrophic mutant for aromatic amino acids of the near-diploid fungal plant pathogen Verticillium longisporum (VL) has been generated. VL enters its host Brassica napus through the roots and colonizes the xylem vessels. The xylem contains little nutrients including low concentrations of amino acids. We isolated the gene Vlaro2 encoding chorismate synthase by complementation of the corresponding yeast mutant strain. Chorismate synthase produces the first branch point intermediate of aromatic amino acid biosynthesis. A novel RNA-mediated gene silencing method reduced gene expression of both isogenes by 80% and resulted in a bradytrophic mutant, which is a leaky auxotroph due to impaired expression of chorismate synthase. In contrast to the wild type, silencing resulted in increased expression of the cross-pathway regulatory gene VlcpcA (similar to cpcA/GCN4) during saprotrophic life. The mutant fungus is still able to infect the host plant B. napus and the model Arabidopsis thaliana with reduced efficiency. VlcpcA expression is increased in planta in the mutant and the wild-type fungus. We assume that xylem colonization requires induction of the cross-pathway control, presumably because the fungus has to overcome imbalanced amino acid supply in the xylem.

Salt stress affects xylem differentiation of grey poplar (Populus x canescens).

In this study the impact of salt stress on the physiology and wood structure of the salt-sensitive Populus x canescens was investigated. Two weeks of salt stress altered wood anatomy significantly. The xylem differentiation zone was reduced and the resulting vessels exhibited reduced lumina. To understand this phenomenon, ion composition, levels of corresponding transcripts and of the stress hormone ABA were analysed. With increasing sodium and chloride concentrations, a general reduction of potassium was found in roots and shoots, but not in leaves. Consequently, the corresponding K+ channel transcripts in roots favoured K+ release. The overall osmolarity in leaves was up to fourfold higher than in roots or shoots. Therefore, adjustment of the K+/Na+ balance seemed not to be required in leaves. Sodium increased gradually from roots to shoots and then to leaves indicating that sodium storage took place first in roots, then in shoots, and finally in leaves to protect photosynthesis from salt effects as long as possible. Since leaf abscisic acid levels markedly increased, stomatal closure seemed to limit CO2 uptake. As a consequence, diminished nutrient supply to the cambium in combination with lowered shoot K+ content led to decreased vessel lumina, and a reduction of the radial cambium was observed. Thus, xylem differentiation was curtailed and the development of full size vessels was impaired.

GH3::GUS reflects cell-specific developmental patterns and stress-induced changes in wood anatomy in the poplar stem.

GH3 genes related to the auxin-inducible Glycine max (L.) Merr. GmGH3 gene encode enzymes that conjugate amino acids to auxin. To investigate the role of GH3 enzymes in stress responses and normal wood development, Populus x canescens (Ait.) was transformed with the promoter-reporter construct GH3::GUS containing a GH3 promoter and the 5' UTR from soybean. beta-Glucuronidase (GUS) activity was present in the vascular tissues of leaves and in developing lateral roots and was inducible in silent tissues by external auxin application. A decrease in GUS activity from the stem apex to the bottom corresponded to decreases in auxin concentrations in these tissues. High auxin concentration and high GH3::GUS activity were present in the pith tissue, which may provide storage for auxin compounds. GH3 reporter was active in ray cells, paratracheal parenchyma cells, maturing vessels and in cells surrounding maturing phloem fibers but not in the cambium and immature phloem, despite high auxin concentrations in the latter tissues. However, the GH3 promoter in these tissues became active when the plants were exposed to abiotic stresses, like bending or salinity, causing changes in wood anatomy. We suggest that adjustment of the internal auxin balance in wood in response to environmental cues involves GH3 auxin conjugate synthases.

Gradual soil water depletion results in reversible changes of gene expression, protein profiles, ecophysiology, and growth performance in Populus euphratica, a poplar growing in arid regions.

The responses of Populus euphratica Oliv. plants to soil water deficit were assessed by analyzing gene expression, protein profiles, and several plant performance criteria to understand the acclimation of plants to soil water deficit. Young, vegetatively propagated plants originating from an arid, saline field site were submitted to a gradually increasing water deficit for 4 weeks in a greenhouse and were allowed to recover for 10 d after full reirrigation. Time-dependent changes and intensity of the perturbations induced in shoot and root growth, xylem anatomy, gas exchange, and water status were recorded. The expression profiles of approximately 6,340 genes and of proteins and metabolites (pigments, soluble carbohydrates, and oxidative compounds) were also recorded in mature leaves and in roots (gene expression only) at four stress levels and after recovery. Drought successively induced shoot growth cessation, stomatal closure, moderate increases in oxidative stress-related compounds, loss of CO2 assimilation, and root growth reduction. These effects were almost fully reversible, indicating that acclimation was dominant over injury. The physiological responses were paralleled by fully reversible transcriptional changes, including only 1.5% of the genes on the array. Protein profiles displayed greater changes than transcript levels. Among the identified proteins for which expressed sequence tags were present on the array, no correlation was found between transcript and protein abundance. Acclimation to water deficit involves the regulation of different networks of genes in roots and shoots. Such diverse requirements for protecting and maintaining the function of different plant organs may render plant engineering or breeding toward improved drought tolerance more complex than previously anticipated.

Gene expression and metabolite profiling of Populus euphratica growing in the Negev desert.

BACKGROUND: Plants growing in their natural habitat represent a valuable resource for elucidating mechanisms of acclimation to environmental constraints. Populus euphratica is a salt-tolerant tree species growing in saline semi-arid areas. To identify genes involved in abiotic stress responses under natural conditions we constructed several normalized and subtracted cDNA libraries from control, stress-exposed and desert-grown P. euphratica trees. In addition, we identified several metabolites in desert-grown P. euphratica trees. RESULTS: About 14,000 expressed sequence tag (EST) sequences were obtained with a good representation of genes putatively involved in resistance and tolerance to salt and other abiotic stresses. A P. euphratica DNA microarray with a uni-gene set of ESTs representing approximately 6,340 different genes was constructed. The microarray was used to study gene expression in adult P. euphratica trees growing in the desert canyon of Ein Avdat in Israel. In parallel, 22 selected metabolites were profiled in the same trees. CONCLUSION: Of the obtained ESTs, 98% were found in the sequenced P. trichocarpa genome and 74% in other Populus EST collections. This implies that the P. euphratica genome does not contain different genes per se, but that regulation of gene expression might be different and that P. euphratica expresses a different set of genes that contribute to adaptation to saline growth conditions. Also, all of the five measured amino acids show increased levels in trees growing in the more saline soil.

Populus euphratica displays apoplastic sodium accumulation, osmotic adjustment by decreases in calcium and soluble carbohydrates, and develops leaf succulence under salt stress.

Populus euphratica Olivier is known to exist in saline and arid environments. In this study we investigated the physiological mechanisms enabling this species to cope with stress caused by salinity. Acclimation to increasing Na+ concentrations required adjustments of the osmotic pressure of leaves, which were achieved by accumulation of Na+ and compensatory decreases in calcium and soluble carbohydrates. The counterbalance of Na+/Ca2+ was also observed in mature leaves from field-grown P. euphratica trees exposed to an environmental gradient of increasing salinity. X-ray microanalysis showed that a primary strategy to protect the cytosol against sodium toxicity was apoplastic but not vacuolar salt accumulation. The ability to cope with salinity also included maintenance of cytosolic potassium concentrations and development of leaf succulence due to an increase in cell number and cell volume leading to sodium dilution. Decreases in apoplastic and vacuolar Ca2+ combined with suppression of calcineurin B-like protein transcripts suggest that Na+ adaptation required suppression of calcium-related signaling pathways. Significant increases in galactinol synthase and alternative oxidase after salt shock and salt adaptation point to shifts in carbohydrate metabolism and suppression of reactive oxygen species in mitochondria under salt stress.