Restriction of cytosolic sucrose hydrolysis profoundly alters development, metabolism, and gene expression in Arabidopsis roots

Abstract

Plant roots depend on sucrose imported from leaves as the substrate for metabolism and growth. Sucrose and hexoses derived from it are also signalling molecules that modulate growth and development, but the importance for signalling of endogenous changes in sugar levels is poorly understood. We report that reduced activity of cytosolic invertase, which converts sucrose to hexoses, leads to pronounced metabolic, growth, and developmental defects in roots of Arabidopsis (Arabidopsis thaliana) seedlings. In addition to altered sugar and downstream metabolite levels, roots of cinv1 cinv2 mutants have reduced elongation rates, cell and meristem size, abnormal meristematic cell division patterns, and altered expression of thousands of genes of diverse functions. Provision of exogenous glucose to mutant roots repairs relatively few of the defects. The extensive transcriptional differences between mutant and wild-type roots have hallmarks of both high sucrose and low hexose signalling. We conclude that the mutant phenotype reflects both low carbon availability for metabolism and growth and complex sugar signals derived from elevated sucrose and depressed hexose levels in the cytosol of mutant roots. Such reciprocal changes in endogenous sucrose and hexose levels potentially provide rich information about sugar status that translates into flexible adjustments of growth and development.

Keywords: Arabidopsis; hexose; neutral invertase; root; root transcriptome; sucrose; sugar signalling.

Fig. 1.

Reduced root length and apical meristem size in the cinv1 cinv2 mutant. (A) Phenotype of 7-day-old seedlings grown vertically on medium without glucose. (B) Root length measurements of wild-type (black symbols), cinv1 (light grey symbols), cinv2 (dark grey symbols), and cinv1 cinv2 (white symbols) seedlings on medium without glucose. Values are means of 25–30 measurements ±SD. (C) Confocal images of 4-day-old roots stained with propidium iodide. Wild-type (left) and cinv1 cinv2 (right). Arrows indicate the boundary between the division zone (the meristem) and the elongation zone of the root (the point at which a given epidermal cell is twice the length of the cell immediately below). Scale bar=100 µm. (D) Lengths of mature epidermal cells in roots of seedlings grown with or without glucose. Arrows indicate walls between adjacent epidermal cells. Scale bar=10 µm. Cropped and reorientated images are shown for ease of comparison. The original, unedited micrographs used to make this composite are provided in Supplementary Fig. S2. (E) Meristem cell numbers in 4-day-old roots grown in medium without glucose (blue bars) or with 55 mM glucose (orange bars). Values are means of 50–60 measurements ±SD. For both genotypes, values with and without glucose were statistically significantly different, and for both treatments values for the two genotypes were statistically significantly different (Student’s t-test, P<0.0002). (F) Root lengths of wild-type (left graph) and cinv1 cinv2 (right graph) seedlings grown for 7 d on different concentrations of glucose (red symbols), fructose (blue symbols), mannitol (grey symbols), and 3-O-methylglucose (white symbols). Values are means ±SD of 25–30 measurements. (G) Stimulation of root growth of 7-day-old seedlings by different concentrations of glucose. For each sugar concentration, the mean values for root length for 7-day-old wild-type (black symbols) and cinv1 cinv2 (white symbols) seedlings grown on mannitol were subtracted from the equivalent value for roots grown on glucose [data from (F)] to give glucose-specific root extension.

Fig. 2.

Developmental abnormalities in roots of the cinv1 cinv2 mutant. (A) Overview of cell positions and cell fate in the root meristem. Arrows indicate cell types as follows: green, stele initials; blue, quiescent centre; orange, columella stem cells in D1 position; yellow, columella differentiated cells in D2 position. Two examples each of wild-type (upper panel) and cinv1 cinv2 (lower panel) roots from 4-day-old plants are shown. (B) Differentiation status of columella cells in 4-day-old seedlings. Roots were stained with propidium iodide. Arrows indicate columella stem cells.

Fig. 3.

Expression of root meristem markers in cinv1 cinv2 roots. Confocal images of 4-day-old roots stained with propidium iodide. Expression of PIN1–GFP is lower in cinv1 cinv2 than in wild-type roots in the absence of glucose, but similar when 55 mM glucose is exogenously supplied. Expression of DR5:GFP, WOX5:GFP, PLT1:PLT1–YFP, and PLT2:PLT2–YFP is similar in cinv1 cinv2 and wild-type roots.

Fig. 4.

Differences in transcript levels between 4-day-old wild-type and cinv1 cinv2 roots. (A) Left: numbers of differentially expressed genes (DEGs) in the absence of glucose in the growth medium (Total–Glc) and in the presence of glucose in the growth medium (Total+Glc), and estimates from these data of numbers of genes differentially expressed whether or not glucose was added (Glc independent), number differentially expressed only in the absence of glucose (Glc suppressed), and number differentially expressed only in the presence of glucose (Glc only). Orange bars: numbers of genes expressed at a higher level in mutant than in wild-type roots. Blue bars: numbers of genes expressed at a lower level in mutant than in wild-type roots. (B) Degree of overlap of individual DEGs in the –Glc and +Glc datasets; (C) Numbers of genes differentially expressed in the presence versus the absence of glucose (glucose-DEGs). Left, glucose-DEGs in wild-type and mutant roots; right, numbers of individual glucose-DEGs exclusive to wild-type or mutant roots (Wt only and mutant only, respectively); colours are as for (A). (D) Degree of overlap of individual glucose-DEGs in the wild-type and mutant datasets. (E) Degree of overlap of the wild-type and mutant glucose-DEGs with each other and with a set of previously identified glucose-responsive genes (+Glc datasets). In overlaps, bold type indicates the number of genes that changed in the same direction between two datasets, and italics indicates the number of genes that changed in opposite directions.