The Mode of Sucrose Degradation in Potato Tubers Determines the Fate of Assimilate Utilization

Cytosolic (U-IN-2) or apoplasmic (U-IN-1) targeting of yeast invertase in potato tubers leads to a reduction in sucrose and an increase in glucose content, but specific phenotypical changes are dependent on the subcellular targeting of the enzyme. Cytosolic expression leads to a more severe phenotype with the most striking aspects being reduced starch content and increased respiration. Despite extensive research, the regulatory mechanisms leading to these changes remain obscure. Recent technological advancements regarding potato transcriptional and genomic research presented us with the opportunity to revisit these lines and perform detailed gene expression analysis, in combination with extensive metabolic profiling, to identify regulatory networks underlying the observed changes. Our results indicate that in both genotypes reduced UDP-glucose production is associated with a reduced expression of cell wall biosynthetic genes. In addition, U-IN-1 tubers are characterized by elevated expression of senescence-associated genes, coupled to reduced expression of genes related to photosynthesis and the cytoskeleton. We provide evidence that increased respiration, observed specifically in U-IN-2 tubers, might be due to sugar signaling via released trehalose-6-phosphate inhibition of the SnRK1 complex. In both genotypes, expression of the plastidic glucose-6-phosphate transporter (GPT) is significantly down-regulated. This leads to a shift in the cytosolic to plastidic glucose-6-phosphate ratio and hence might limit starch synthesis but also the oxidative pentose phosphate pathway. This might explain the observed changes in several additional plastid localized pathways, most notably reduced expression of fatty acid biosynthetic genes and an accumulation of shikimate. Interestingly, a strict negative correlation between invertase and GPT expression could be observed in a wide range of potato tubers. This reciprocal regulation may be part of a more general switch controlling energy versus storage metabolism, suggesting that the fate of assimilate utilization is coordinated at the level of sucrose degradation

Comparative transcriptome analysis coupled to X-ray CT reveals sucrose supply and growth velocity as major determinants of potato tuber starch biosynthesis

<p>Abstract</p> <p>Background</p> <p>Even though the process of potato tuber starch biosynthesis is well understood, mechanisms regulating biosynthesis are still unclear. Transcriptome analysis provides valuable information as to how genes are regulated. Therefore, this work aimed at investigating transcriptional regulation of starch biosynthetic genes in leaves and tubers of potato plants under various conditions. More specifically we looked at gene expression diurnally in leaves and tubers, during tuber induction and in tubers growing at different velocities. To determine velocity of potato tuber growth a new method based on X-ray Computed Tomography (X-ray CT) was established.</p> <p>Results</p> <p>Comparative transcriptome analysis between leaves and tubers revealed striking similarities with the same genes being differentially expressed in both tissues. In tubers, oscillation of granule bound starch synthase (GBSS) expression) was observed which could be linked to sucrose supply from source leaves. X-ray CT was used to determine time-dependent changes in tuber volume and the growth velocity was calculated. Although there is not a linear correlation between growth velocity and expression of starch biosynthetic genes, there are significant differences between growing and non-growing tubers. Co-expression analysis was used to identify transcription factors positively correlating with starch biosynthetic genes possibly regulating starch biosynthesis.</p> <p>Conclusion</p> <p>Most starch biosynthetic enzymes are encoded by gene families. Co-expression analysis revealed that the same members of these gene families are co-regulated in leaves and tubers. This suggests that regulation of transitory and storage starch biosynthesis in leaves and tubers, respectively, is surprisingly similar. X-ray CT can be used to monitor growth and development of belowground organs and allows to link tuber growth to changes in gene expression. Comparative transcriptome analysis provides a useful tool to identify transcription factors possibly involved in the regulation of starch biosynthesis.</p

Simultaneous silencing of isoamylases ISA1, ISA2 and ISA3 by multi-target RNAi in potato tubers leads to decreased starch content and an early sprouting phenotype

<div><p>Isoamylases hydrolyse (1–6)-alpha-D-glucosidic linkages in starch and are involved in both starch granule formation and starch degradation. In plants, three isoamylase isoforms with distinct functions in starch synthesis (ISA1 and ISA2) and degradation (ISA3) have been described. Here, we created transgenic potato plants with simultaneously decreased expression of all three isoamylases using a chimeric RNAi construct targeting all three isoforms. Constitutive expression of the hairpin RNA using the 35S CaMV promoter resulted in efficient silencing of all three isoforms in leaves, growing tubers, and sprouting tubers. Neither plant growth nor tuber yield was effected in isoamylase-deficient potato lines. Interestingly, starch metabolism was found to be impaired in a tissue-specific manner. While leaf starch content was unaffected, tuber starch was significantly reduced. The reduction in tuber starch content in the transgenic plants was accompanied by a decrease in starch granules size, an increased sucrose content and decreased hexose levels. Despite the effects on granule size, only little changes in chain length composition of soluble and insoluble glucose polymers were detected. The transgenic tubers displayed an early sprouting phenotype that was accompanied by an increased level of sucrose in parenchyma cells below the outgrowing bud. Since high sucrose levels promote sprouting, we propose that the increased number of small starch granules may cause an accelerated turnover of glucan chains and hence a more rapid synthesis of sucrose. This observation links alterations in starch structure/degradation with developmental processes like meristem activation and sprout outgrowth in potato tubers.</p></div

Impact of <i>ISA</i> silencing on tuber sprouting.

<p>Tubers of 5 plants each from wild type (WT) and transgenic lines 7, 16 and 39 were stored after harvest at room temperature in darkness. A) Sprouting kinetics. To monitor the impact on dormancy length, 2–5 similar sized tubers from each plant were picked (n = 13–20) and their sprouting behaviour was regularly scored over a 15-week period until 100% sprouting had been reached in wild-type tubers. A tuber was considered to sprout when sprouts of 2 mm length became visible. B) Photographs of transgenic (lines 7, 16, 39) and control tubers taken after 13 weeks of storage showing that the transgenic lines sprout earlier than the wild-type controls (WT). C) Number of sprouts per tuber. Number of sprouts formed per tuber were counted from 13–20 individual tubers. Values represent the mean +/- SE. Significant differences to wild type were calculated using two-tailed t-test assuming equal variances and are indicated by asterisks (**p<0.01, *p<0.05).</p

Schematic representation of the chimeric RNAi hairpin construct.

<p>Schematic representation of the chimeric RNAi hairpin construct.</p

Starch structure determined as relative chain length distribution.

<p>Starch from sprouting tubers was either isolated from parenchyma associated with the sprout (A) or (B) from parenchyma not associated with the sprout. Line 7 (blue), line 16 (red), line 39 (grey) and the wild-type control (WT; black). Values represent mean +/- SE of 3–4 biological replicates.</p

Impact of reduced expression of <i>ISA</i> isogenes on soluble sugars and starch content in source leaves.

<p>Metabolite contents in wild type and <i>ISA</i>- silenced plants were determined at the end of the light period (after 16h light) (light grey) and at the end of the dark period (after 8h darkness) (dark grey). A) starch B) sucrose, C) hexoses. Values represent the mean +/- SE of 5 biological and 2 technical replicates each.</p

Starch granule size distribution.

<p>Size distribution was determined as pixels’ size from ca. 150 starch granules of each replicate using ImageJ software as schematically shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181444#pone.0181444.g005" target="_blank">Fig 5</a>. A) Granules were compiled into groups and the number of granules within a specific range (e.g. >25, >50, >75, etc.) were counted, calculated as relative percentage of total number and values cumulatively plotted. The inset shows the median granule size of each genotype. B) Bar charts of selected groups of relative starch granule sizes subjected to t-test test analysis. Values represent means +/- SE from 3 individual tubers (n = 3). Significant differences to wild type are indicated by asterisks (*p<0.05). Line 7 (light grey), line 16 (dark grey), line 39 (white) wild-type control (WT; black).</p

Determination of the starch granule size.

<p>Starch granules from freshly harvested tubers were extracted, stained with iodine and inspected under the microscope. A) representative micrographs of iodine-stained starch granules derived from tubers of wild-type and <i>ISA</i>-silenced lines 7, 16 and 39. B) Representative image illustrating the segmentation of images by means of the ImageJ software tool to determine the relative size of each 2D starch granule. The software allows for the manual separation of granules connected to each other in the image.</p