Effect of ABA deficiency on the expansion of hypocotyl cells.

<p>The influence of ABA on the elongation of epidermal cells of etiolated hypocotyls in <i>sit</i> and WT (cv. Rheinlands Ruhm) was investigated using SEM imaging analysis. Triangles represent all measured values (n = 471) for each genotype. The bars within the boxes indicate the median values in each case, while the boxes’ upper and lower boundaries indicate the boundaries of the first and third quartiles. The Mann-Whitney test was used to prove statistical significance (p < 0.01).</p

The effects of ABA on the growth of etiolated hypocotyls.

<p>A) The effect of ABA-deficiency and exogenous ABA on hypocotyl growth in the <i>sit</i> mutant. Germinated seeds of the WT (cv. Rheinlands Ruhm) mutant were transferred to untreated media (control) and media supplemented with 100 nM ABA and grown in the dark for 4 days. The results shown in the figure represent the medians of normalized length of hypocotyls from three independent experiments; the error bars represent the boundaries of the first and third quartiles. The “WT control” was set as 100% hypocotyl length and all other values (medians, quartiles) are expressed as percentages of this value. To prove statistical significance the Mann-Whitney test was performed independently (<i>sit</i> versus WT) for control and ABA treated samples. Asterisks denote values that differ significantly (Mann Whitney test; p < 0.01, n = 136). B) The hypocotyl elongation of the <i>sit</i> mutant treated with various concentrations of ABA. Germinated seeds were transferred to untreated media (control) and media supplemented with ABA and grown in the dark for 4 days. The results shown in the figure represent the medians of normalized length of hypocotyls from two independent experiments; the error bars represent the boundaries of the first and third quartiles. The sample “control” was set as 100% hypocotyl length and all other values (medians, quartiles) are expressed as percentages of this value. To prove statistical significance the Kruskal-Wallis ANOVA with multiple post-hoc comparison was performed. Asterisks denote values that differ significantly from “control” sample (p < 0.01; n = 232). C) The hypocotyl elongation of the WT (cv. Rheinlands Ruhm) treated with various concentrations of ABA. Germinated seeds were transferred to untreated media (control) and media supplemented with ABA and grown in the dark for 4 days. The results shown in the figure represent the medians of normalized length of hypocotyls from two independent experiments; the error bars represent the boundaries of the first and third quartiles. The sample “control” was set as 100% hypocotyl length and all other values (medians, quartiles) are expressed as percentages of this value. To prove statistical significance the Kruskal-Wallis ANOVA was performed. Asterisks denote values that differ significantly from “control” sample (p < 0.01, n = 200). D) The effect of ABA-deficiency and exogenous ABA on hypocotyl growth in the <i>not</i> mutant. Germinated seeds of the WT (cv. Lukullus) mutant were transferred to untreated media (control) and media supplemented with 50 nM ABA and grown in the dark for 4 days. The results shown in the figure represent the medians of normalized length of hypocotyls from one independent experiment. The “WT control” was set as 100% hypocotyl length and all other values (medians, quartiles) are expressed as percentages of this value. To prove statistical significance the Mann-Whitney test was performed independently (<i>not</i> versus WT) for control and ABA treated samples. Asterisks denote values that differ significantly (Mann Whitney test; p < 0.03, n = 79).</p

Simplified scheme of ABA biosynthesis and catabolism.

<p>Selected enzymatic steps in ABA biosynthesis are shown. The names of the genes encoding the enzymes that catalyze each step in tomato and <i>Arabidopsis</i> are indicated; the names of genes examined in this work are underlined. The conversion of phytoene to ß-carotene is mediated by phytoene desaturase (PED); this step is blocked by fluridone. Zeaxanthin epoxidase (ZEP) catalyzes the synthesis of violaxanthin, which is then converted to neoxanthin. The subsequent synthesis of xanthoxin is catalyzed by 9-<i>cis</i>-epoxycarotenoid dioxygenase (NCED), which is encoded in the gene <i>LeNCED1</i> in tomato and disrupted in <i>notabilis</i> mutant. Whereas the previous steps occur in plastids, xanthoxin is transported to the cytosol where it is converted to the abscisic aldehyde by short-chain dehydrogenase/reductase (SDR). The final step of ABA biosynthesis is the oxidation of abscisic aldehyde to ABA by an abscisic aldehyde oxidase (AAO), which is encoded in genes that are disrupted in the <i>sitiens</i> and <i>flacca</i> tomato mutants. ABA degradation (shown in the red frame) is mediated by ABA 8´-hydroxylase (A8H, cytochrome P450 monooxygenase), whose product spontaneously isomerizes to phaseic acid. The genes encoding ABA 8´-hydroxylase in tomato are <i>SlCYP707A1–SlCYP707A4</i>. Dashed arrows represent missing steps in the pathway. These schemes are modified according to Kitahata and co-authors [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117793#pone.0117793.ref032" target="_blank">32</a>] and Nambara and Marion-Poll [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117793#pone.0117793.ref070" target="_blank">70</a>].</p

Regulation of ABA metabolism during seed germination and post-germination growth.

<p>Free ABA levels are reported as relative means [a.u.] ± SE based on three independent experiments. All values are expressed relative to those for the dark-grown sample at the 120 hours time point, which was assigned a value of 100 a.u. (A). The fold changes in the expression of the <i>LeNCED1</i> (B) and <i>SlCYP707A3</i> (C) genes are reported as the means of three independent experiments ± SE. <i>Tip41like</i> and <i>PP2ACs</i> were used as housekeeping genes. Relative quantification was performed using the expression levels for the dark-grown sample at 120 hours as a reference. The red arrow marks the point of seed germination. Figure D) shows the different stages of tomato seedling development considered in this work. The first is seed imbibition (6h–48h), which is followed by seed germination (72h). Both of these steps occurred in darkness in all experiments. The seedlings were then separated (96h–120h) into two groups: one was kept in darkness (d) to induce skotomorphogenesis while the other was grown under continuous BL (bl) to induce de-etiolation. White bars indicate a distance of 1 mm.</p

The effects of ABA on endoreduplication.

<p>The influence of ABA on nuclear ploidy was investigated using hypocotyl segments of <i>sit</i> and WT seedlings grown in darkness on untreated media and media containing 100 nM ABA (A). Triangles represent all measured values (n = 40) for each genotype and treatment. The bars within the boxes indicate the median values in each case, while the boxes’ upper and lower boundaries indicate the boundaries of the first and third quartiles. Three independent experiments were performed. The Kruskal-Wallis ANOVA test with multiple comparisons revealed that only the <i>sit</i> control sample differed significantly from the WT control (p < 0.01). No other significant differences were found. (B) Analysis of the expression of the <i>SlKRP1</i> and <i>SlKRP3</i> genes based on the mean of three independent experiments ± SE. <i>Tip41like</i> and <i>PP2ACs</i> were used as housekeeping genes. All expression values are quoted relative to those for the “WT control” sample.</p

Endogenous Abscisic Acid Promotes Hypocotyl Growth and Affects Endoreduplication during Dark-Induced Growth in Tomato (<i>Solanum lycopersicum</i> L.)

<div><p>Dark-induced growth (skotomorphogenesis) is primarily characterized by rapid elongation of the hypocotyl. We have studied the role of abscisic acid (ABA) during the development of young tomato (<i>Solanum lycopersicum</i> L.) seedlings. We observed that ABA deficiency caused a reduction in hypocotyl growth at the level of cell elongation and that the growth in ABA-deficient plants could be improved by treatment with exogenous ABA, through which the plants show a concentration dependent response. In addition, ABA accumulated in dark-grown tomato seedlings that grew rapidly, whereas seedlings grown under blue light exhibited low growth rates and accumulated less ABA. We demonstrated that ABA promotes DNA endoreduplication by enhancing the expression of the genes encoding inhibitors of cyclin-dependent kinases <i>SlKRP1</i> and <i>SlKRP3</i> and by reducing cytokinin levels. These data were supported by the expression analysis of the genes which encode enzymes involved in ABA and CK metabolism. Our results show that ABA is essential for the process of hypocotyl elongation and that appropriate control of the endogenous level of ABA is required in order to drive the growth of etiolated seedlings.</p></div

The effects of ABA deficiency on biosynthesis and overall CK levels in etiolated tomato seedlings.

<p>Overall CK levels are quoted as the mean of the relative values [a.u.] ± SE for three independent experiments. All measurements are expressed in relation to those for the “WT control” sample, which was assigned a value of 100a.u. (A). The expression of the <i>SlLOG2</i> gene is presented as the mean of three independent experiments ± SE. <i>Tip41like</i> and <i>PP2ACs</i> were used as housekeeping genes. Expression was quantified in relation to that of the specified reference genes in the “WT control” sample (B).</p

Effect of <i>sit</i> and <i>not</i> mutations on the endogenous ABA content in etiolated hypocotyls.

<p>The dark-grown hypocotyls of <i>sit</i> and <i>not</i> mutants and corresponding WTs were harvested five days after germination and ABA content was measured. Free ABA levels are reported as relative means [a.u.] ± SE based on two biological repeats. The mutant values are expressed relative to those for corresponding WT, which were assigned a value of 100 a.u.</p

Sequences of primer combinations used in this study.

<p>Sequences of primer combinations used in this study.</p

Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells, Growth, Photosynthetic Capacity, and Biosynthesis of Plastidic Cytokinins in Arabidopsis

<div><p>Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated <i>pgi1-3</i>, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, <i>PGI1</i>. Starch content in <i>pgi1-3</i> source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of <i>pgi1-2</i>, a T-DNA insertion pPGI null mutant. Starch deficiency of <i>pgi1</i> leaves could be reverted by the introduction of a <i>sex1</i> null mutation impeding β-amylolytic starch breakdown. Although previous studies showed that starch granules of <i>pgi1-2</i> leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of <i>pgi1-2</i> and <i>pgi1-3</i> mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic β-amylase encoding genes in <i>pgi1</i> leaves, which was accompanied by increased β-amylase activity. Both <i>pgi1-2</i> and <i>pgi1-3</i> mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P)H/NAD(P) ratios in <i>pgi1</i> leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in <i>pgi1</i> leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of <i>pgi1</i> leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs.</p></div