Maria Grazia Annunziata

Salinity Stress and Salt Tolerance

Salinity is one of the most serious factors limiting the productivity of agricultural crops, with adverse effects on germination, plant vigour and crop yield (R Munns & Tester, 2008). Salinization affects many irrigated areas mainly due to the use of brackish water. Worldwide, more than 45 million hectares of irrigated land have been damaged by salt, and 1.5 million hectares are taken out of production each year as a result of high salinity levels in the soil (R Munns & Tester, 2008). High salinity affects plants in several ways: water stress, ion toxicity, nutritional disorders, oxidative stress, alteration of metabolic processes, membrane disorganization, reduction of cell division and expansion, genotoxicity (Hasegawa, Bressan, Zhu, & Bohnert, 2000; R. Munns, 2002; Zhu, 2007). Together, these effects reduce plant growth, development and survival. During the onset and development of salt stress within a plant, all the major processes such as photosynthesis, protein synthesis and energy and lipid metabolism are affected (Parida & Das, 2005). During initial exposure to salinity, plants experience water stress, which in turn reduces leaf expansion. The osmotic effects of salinity stress can be observed immediately after salt application and are believed to continue for the duration of exposure, resulting in inhibited cell expansion and cell division, as well as stomatal closure (T. J. Flowers, 2004; R. Munns, 2002). During long-term exposure to salinity, plants experience ionic stress, which can lead to premature senescence of adult leaves, and thus a reduction in the photosynthetic area available to support continued growth (Cramer & Nowak, 1992). In fact, excess sodium and more importantly chloride has the potential to affect plant enzymes and cause cell swelling, resulting in reduced energy production and other physiological changes (Larcher 1980). Ionic stress results in premature senescence of older leaves and in toxicity symptoms (chlorosis, necrosis) in mature leaves due to high Na+ which affects plants by disrupting protein synthesis and interfering with enzyme activity (Hasegawa, Bressan, Zhu, & Bohnert, 2000; R. Munns, 2002; R Munns & Termaat, 1986). Many plants have evolved several mechanisms either to exclude salt from their cells or to tolerate its presence within the cells. In this chapter, we mainly discuss about soil salinity, its effects on plants and tolerance mechanisms which permit the plants to withstand stress, with particular emphasis on ion homeostasis, Na+ exclusion and tissue tolerance. Moreover we give a synthetic overview of the two major approaches that have been used to improve stress tolerance: exploitation of natural genetic variations and generation of transgenic plants with novel genes or altered expression levels of the existing genes. A fundamental biological understanding and knowledge of the effects of salt stress on plants is necessary to provide additional

Loss of Starch Granule Initiation Has a Deleterious Effect on the Growth of Arabidopsis Plants Due to an Accumulation of ADP-Glucose

ADP-Glc in the starch-deficient mutant ss3/ss4 sequesters adenine nucleotides, which limits photophosphorylation, leads to photooxidative stress, and causes the chlorotic and stunted phenotypes. STARCH SYNTHASE4 (SS4) is required for proper starch granule initiation in Arabidopsis (Arabidopsis thaliana), although SS3 can partially replace its function. Unlike other starch-deficient mutants, ss4 and ss3/ss4 mutants grow poorly even under long-day conditions. They have less chlorophyll and carotenoids than the wild type and lower maximal rates of photosynthesis. There is evidence of photooxidative damage of the photosynthetic apparatus in the mutants from chlorophyll a fluorescence parameters and their high levels of malondialdehyde. Metabolite profiling revealed that ss3/ss4 accumulates over 170 times more ADP-glucose (Glc) than wild-type plants. Restricting ADP-Glc synthesis, by introducing mutations in the plastidial phosphoglucomutase (pgm1) or the small subunit of ADP-Glc pyrophosphorylase (aps1), largely restored photosynthetic capacity and growth in pgm1/ss3/ss4 and aps1/ss3/ss4 triple mutants. It is proposed that the accumulation of ADP-Glc in the ss3/ss4 mutant sequesters a large part of the plastidial pools of adenine nucleotides, which limits photophosphorylation, leading to photooxidative stress, causing the chlorotic and stunted growth phenotypes of the plants.

Durum wheat seedling responses to simultaneous high light and salinity involve a fine reconfiguration of amino acids and carbohydrate metabolism

Durum wheat plants are extremely sensitive to drought and salinity during seedling and early development stages. Their responses to stresses have been extensively studied to provide new metabolic targets and improving the tolerance to adverse environments. Most of these studies have been performed in growth chambers under low light [300-350 µmol m-2 s-1 photosynthetically active radiation (PAR), LL]. However, in nature plants have to face frequent fluctuations of light intensities that often exceed their photosynthetic capacity (900-2000 µmol m-2 s-1 ). In this study we investigated the physiological and metabolic changes potentially involved in osmotic adjustment and antioxidant defense in durum wheat seedlings under high light (HL) and salinity. The combined application of the two stresses decreased the water potential and stomatal conductance without reducing the photosynthetic efficiency of the plants. Glycine betaine (GB) synthesis was inhibited, proline and glutamate content decreased, while γ-aminobutyric acid (GABA), amides and minor amino acids increased. The expression level and enzymatic activities of Δ1-pyrroline-5-carboxylate synthetase, asparagine synthetase and glutamate decarboxylase, as well as other enzymatic activities of nitrogen and carbon metabolism, were analyzed. Antioxidant enzymes and metabolites were also considered. The results showed that the complex interplay seen in durum wheat plants under salinity at LL was simplified: GB and antioxidants did not play a main role. On the contrary, the fine tuning of few specific primary metabolites (GABA, amides, minor amino acids and hexoses) remodeled metabolism and defense processes, playing a key role in the response to simultaneous stresses.

Durum Wheat Roots Adapt to Salinity Remodeling the Cellular Content of Nitrogen Metabolites and Sucrose

Plants are currently experiencing increasing salinity problems due to irrigation with brackish water. Moreover, in fields, roots can grow in soils which show spatial variation in water content and salt concentration, also because of the type of irrigation. Salinity impairs crop growth and productivity by inhibiting many physiological and metabolic processes, in particular nitrate uptake, translocation, and assimilation. Salinity determines an increase of sap osmolality from about 305 mOsmol kg−1 in control roots to about 530 mOsmol kg−1 in roots under salinity. Root cells adapt to salinity by sequestering sodium in the vacuole, as a cheap osmoticum, and showing a rearrangement of few nitrogen-containing metabolites and sucrose in the cytosol, both for osmotic adjustment and oxidative stress protection, thus providing plant viability even at low nitrate levels. Mainly glycine betaine and sucrose at low nitrate concentration, and glycine betaine, asparagine and proline at high nitrate levels can be assumed responsible for the osmotic adjustment of the cytosol, the assimilation of the excess of ammonium and the scavenging of ROS under salinity. High nitrate plants with half of the root system under salinity accumulate proline and glutamine in both control and salt stressed split roots, revealing that osmotic adjustment is not a regional effect in plants. The expression level and enzymatic activities of asparagine synthetase and Δ1-pyrroline-5-carboxylate synthetase, as well as other enzymatic activities of nitrogen and carbon metabolism, are analyzed.

Reproductive failure in Arabidopsis thaliana under transient carbohydrate limitation: flowers and very young siliques are jettisoned and the meristem is maintained to allow successful resumption of reproductive growth

The impact of transient carbon depletion on reproductive growth in Arabidopsis was investigated by transferring long-photoperiod-grown plants to continuous darkness and returning them to a light-dark cycle. After 2 days of darkness, carbon reserves were depleted in reproductive sinks, and RNA in situ hybridization of marker transcripts showed that carbon starvation responses had been initiated in the meristem, anthers and ovules. Dark treatments of 2 or more days resulted in a bare-segment phenotype on the floral stem, with 23-27 aborted siliques. These resulted from impaired growth of immature siliques and abortion of mature and immature flowers. Depolarization of PIN1 protein and increased DII-VENUS expression pointed to rapid collapse of auxin gradients in the meristem and inhibition of primordia initiation. After transfer back to a light-dark cycle, flowers appeared and formed viable siliques and seeds. A similar phenotype was seen after transfer to sub-compensation point irradiance or CO2 . It also appeared in a milder form after a moderate decrease in irradiance and developed spontaneously in short photoperiods. We conclude that Arabidopsis inhibits primordia initiation and aborts flowers and very young siliques in C-limited conditions. This curtails demand, safeguarding meristem function and allowing renewal of reproductive growth when carbon becomes available again.

Transcription Factors and Genes in Abiotic Stress

Plants are constantly exposed to changes in environmental conditions. When these changes are rapid and extreme, plants generally perceive them as stresses. Abiotic stresses are the most serious factors limiting the productivity of agricultural crops, with adverse effects on germination, plant vigour and crop yield. Responses to abiotic stresses are not linear pathways, but are complicated integrated circuits involving the interaction of additional cofactors and/or signalling molecules to coordinate a specified response to a given stimulus. The regulation of these responses requires proteins operating in signal transduction pathways, such as transcriptional factors, which modulate gene expression by binding to specific DNA sequences in the promoters of respective target genes. This type of transcriptional regulatory system is called regulon. At least four different regulons that are active in response to abiotic stresses have been identified. Dehydration-responsive element binding protein 1 (DREB1)/C-repeat binding factor (CBF) and DREB2 regulons function in ABA-independent gene expression, whereas the ABA-responsive element (ABRE) binding protein (AREB)/ABRE binding factor (ABF) regulon functions in ABA-dependent gene expression. In addition to these major pathways, other regulons, including the NAC and MYB/MYC regulons are involved in abiotic stress-responsive gene expression. Transcription factors (TFs) are powerful targets for genetic engineering in abiotic stress resistance in crop plants and many studies have been done in the last two decades on this topic. The aim of this book chapter is to give a comprehensive and up-to-date literature review in this field.

Getting back to nature: a reality check for experiments in controlled environments

The carbon and nitrogen metabolism of Arabidopsis plants grown in sunlight differs from plants grown with artificial light, even when the spectral quality and sinusoidal profile of sunlight are approximated experimentally.

Circadian, Carbon, and Light Control of Expansion Growth and Leaf Movement

A 3D imaging platform was used to analyze the contribution of clock and light signaling to the regulation of diurnal changes in rosette expansion rate and leaf angles in Arabidopsis. We used Phytotyping4D to investigate the contribution of clock and light signaling to the diurnal regulation of rosette expansion growth and leaf movement in Arabidopsis (Arabidopsis thaliana). Wild-type plants and clock mutants with a short (lhycca1) and long (prr7prr9) period were analyzed in a T24 cycle and in T-cycles that were closer to the mutants’ period. Wild types also were analyzed in various photoperiods and after transfer to free-running light or darkness. Rosette expansion and leaf movement exhibited a circadian oscillation, with superimposed transients after dawn and dusk. Diurnal responses were modified in clock mutants. lhycca1 exhibited an inhibition of growth at the end of night and growth rose earlier after dawn, whereas prr7prr9 showed decreased growth for the first part of the light period. Some features were partly rescued by a matching T-cycle, like the inhibition in lhycca1 at the end of the night, indicating that it is due to premature exhaustion of starch. Other features were not rescued, revealing that the clock also regulates expansion growth more directly. Expansion growth was faster at night than in the daytime, whereas published work has shown that the synthesis of cellular components is faster in the day than at nighttime. This temporal uncoupling became larger in short photoperiods and may reflect the differing dependence of expansion and biosynthesis on energy, carbon, and water. While it has been proposed that leaf expansion and movement are causally linked, we did not observe a consistent temporal relationship between expansion and leaf movement.

Genome-wide Association Mapping Reveals that Specific and Pleiotropic Regulatory Mechanisms Fine-tune Central Metabolism and Growth in Arabidopsis

Many enzyme activity and metabolite quantitative trait loci (QTL) colocalize, including a pleiotropic QTL at ACCELERATED CELL DEATH6 that points to a trade-off between defense and central metabolism. Central metabolism is a coordinated network that is regulated at multiple levels by resource availability and by environmental and developmental cues. Its genetic architecture has been investigated by mapping metabolite quantitative trait loci (QTL). A more direct approach is to identify enzyme activity QTL, which distinguishes between cis-QTL in structural genes encoding enzymes and regulatory trans-QTL. Using genome-wide association studies, we mapped QTL for 24 enzyme activities, nine metabolites, three structural components, and biomass in Arabidopsis thaliana. We detected strong cis-QTL for five enzyme activities. A cis-QTL for UDP-glucose pyrophosphorylase activity in the UGP1 promoter is maintained through balancing selection. Variation in acid invertase activity reflects multiple evolutionary events in the promoter and coding region of VAC-INV. cis-QTL were also detected for ADP-glucose pyrophosphorylase, fumarase, and phosphoglucose isomerase activity. We detected many trans-QTL, including transcription factors, E3 ligases, protein targeting components, and protein kinases, and validated some by knockout analysis. trans-QTL are more frequent but tend to have smaller individual effects than cis-QTL. We detected many colocalized QTL, including a multitrait QTL on chromosome 4 that affects six enzyme activities, three metabolites, protein, and biomass. These traits are coordinately modified by different ACCELERATED CELL DEATH6 alleles, revealing a trade-off between metabolism and defense against biotic stress.

Ty1-copia group retrotransposons and the evolution of retroelements in several angiosperm plants: evidence of horizontal transmission

The phylogenetic relationships among thirty-seven new Ty1-copia group retrotransposons in seven angiosperm plants were examined by reverse transcriptase and ribonuclease H sequence analysis. Distribution pattern of the retrotransposons of closely related plant species generally reflects a close phylogenetic relationship. In contrast, we found that several retrotransposon sequences from the same genome exhibited a high degree of divergence and had a relatively high degree of identity versus retrotransposon sequences from widely divergent species, including an ancestral phytopathogen fungus. This finding supports the hypothesis that the horizontal transmission from phytopatogen organism to the host flowering plants could have played a role in the evolutionary dynamics of Ty1-copia group retrotransposons.