Altered starch turnover in the maternal plant has major effects on Arabidopsis fruit growth and seed composition

Abstract

Mature seeds of both the high-starch starch-excess1 (sex1) mutant and the almost starchless phosphoglucomutase1 mutant of Arabidopsis (Arabidopsis thaliana) have 30% to 40% less lipid than seeds of wild-type plants. We show that this is a maternal effect and is not attributable to the defects in starch metabolism in the embryo itself. Low lipid contents and consequent slow postgerminative growth are seen only in mutant embryos that develop on maternal plants with mutant phenotypes. Mutant embryos that develop on plants with wild-type starch metabolism have wild-type lipid contents and postgerminative growth. The maternal effect on seed lipid content is attributable to carbohydrate starvation in the mutant fruit at night. Fruits on sex1 plants grow more slowly than those on wild-type plants, particularly at night, and have low sugars and elevated expression of starvation genes at night. Transcript levels of the transcription factor WRINKLED1, implicated in lipid synthesis, are reduced at night in sex1 but not in wild-type seeds, and so are transcript levels of key enzymes of glycolysis and fatty acid synthesis. sex1 embryos develop more slowly than wild-type embryos. We conclude that the reduced capacity of mutant plants to convert starch to sugars in leaves at night results in low nighttime carbohydrate availability in the developing fruit. This in turn reduces the rate of development and expression of genes encoding enzymes of storage product accumulation in the embryo. Thus, the supply of carbohydrate from the maternal plant to the developing fruit at night can have an important influence on oilseed composition and on postgerminative growth.

Figure 1.

Effects of embryo-specific down-regulation of the activity of PGM1. A, Detection of PGM (top panel) and PGI (bottom panel) activity after native PAGE. Extracts were prepared from embryos at 12 DAF on plants grown in 16 h of light and 8 h of dark. The asterisk indicates plastidial PGM activity. Each lane contained 20 μg of protein. WT, Wild type. B, Starch accumulation in developing embryos from wild-type plants (left panel) and P_oleosin::asPGM1 lines 144 (middle panel) and 51 (right panel). Embryos at 12 DAF were stained with Lugol’s iodine solution. The asterisks indicate a zone of starch accumulation above the radicle tip of embryos from transgenic plants. Bar = 200 μm and applies to all panels. C, Fatty acid accumulation (measured as FAME) during embryo development. Black bars, the wild type; gray bars, P_oleosin::asPGM1 line 51; white bars, P_oleosin::asPGM1 line 144. Values are means ± se of measurements made on three replicates, each of 50 seeds pooled from five plants.

Figure 2.

Effect of the maternal genotype on the lipid content of Arabidopsis seeds. A, Detection of starch in leaves of Col-0 wild type (WT), sex1-3, and SEX1/sex1-3 plants at the end of the day (EOD) and the end of the night (EON). Leaves from 3-week old rosettes were stained with Lugol’s iodine solution. B, Starch content of leaves from 3-week old Col-0, sex1-3, and SEX1/sex1-3 plants at the end of the day (white bars) and the end of the night (black bars). Values are means ± se of measurements on three biological replicates. FWT, Fresh weight. C, Mature seed of sex1-3 (left) and SEX1/sex1-3 (middle), and a mature SEX1/sex1-3 embryo (right), stained with Lugol’s iodine solution. The presence of starch in a zone above the radicle tip (asterisk) was exhibited by one-quarter of the mature embryos on the same plant. Bar = 200 μm. D, Total FAME content of wild-type (+/+), SEX1/sex1-3 (+/−), and sex1-3 (−/−) mature embryos from a selfed SEX1/sex1-3 plant and of mature embryos from wild-type and sex1-3 maternal plants. Measurements were on 65 individual, genotyped embryos (segregation ratio: 13 wild-type, 31 SEX1/sex1-3, and 20 homozygous sex1 mutants). Similar results were obtained for a second, independent SEX1/sex1-3 plant. The solid line inside each box represents the average FAME content, the box represents the values between the first and third quartiles of the data set, and the whiskers show the range of values.

Figure 3.

Effect of altered starch turnover in the maternal plant on seedling growth. A, Hypocotyl length of 7-d-old dark-grown seedlings from seed of wild-type (WT), sex1-3, and pgm1-1 maternal plants. Seeds were germinated on agar in the absence (black bars) or the presence (white bars) of 2% (w/v) Suc. Values are means of measurements on 15 seedlings, and error bars represent se. Similar results were obtained with seed of independently grown plants. B, Hypocotyl length of 7-d-old dark-grown seedlings from seed of a selfed SEX1/sex1-3 plant. For comparison, seeds from wild-type and sex1-3 maternal plants grown at the same time were included. Values for SEX1/sex1-3 progeny are means of measurements on 67 individual, genotyped seedlings. Values for the wild type and sex1-3 are means of measurements on 16 and 10 seedlings, respectively, and error bars represent se.

Figure 4.

Reproductive growth of the sex1-3 mutant. A, Typical flowers from the apex of the primary flowering stem from a wild-type (WT; left) and a sex1-3 (right) plant. Images are at the same magnification. B, Time course of extension of single siliques from a wild-type (top) and a sex1-3 (bottom) plant. The picture is a montage of images of the same silique +1 and +7 h after dawn at the indicated DAF. Bar = 0.5 cm. C, Silique extension on wild-type (diamonds) and sex1-3 (squares) plants during seed development. Measurements were made on images from the start and end of each 12-h night (gray shading). Values are means ± se for eight to 18 siliques pooled from eight plants for each genotype. D, Relative growth rate of wild-type (diamonds) and sex1-3 (squares) siliques during the day and the night, calculated from the data in C.

Figure 5.

Luciferase (LUC) activity in plants expressing luc under the control of the promoter of the sugar-repressed gene At1g10070. A, LUC activity (visualized by bioluminescence, in pseudocolor) at the end of the night in 3-week-old wild-type (left) and sex1-3 (right) plants expressing pAt1g10070₁₁₀₀:LUC. The scale at the top represents bioluminescence intensity (black = low, red = high). B, LUC activity at the end of the night in the apex of the primary flowering stem of a wild-type (left) and a sex1-3 (right) plant expressing pAt1g10700₁₁₀₀:LUC. The images are at the same magnification and were taken at the same time. C, LUC activity at the end of the night in developing siliques at 3 DAF from wild-type (top) and sex1-3 (bottom) plants expressing pAt1g10070₁₁₀₀:LUC. D, Quantification of LUC activity in siliques of pAt1g10070₁₁₀₀:LUC (black bars) and sex1-3/pAt1g10070₁₁₀₀:LUC (white bars) plants at the end of the night. Values are means ± se for eight to 10 siliques pooled from five plants. WT, The wild type. E, Expression of the sugar-repressed gene At1g10070 during a 12-h night in siliques from wild-type (diamonds) and sex1-3 (squares) plants at 12 DAF. RNA was extracted from five siliques pooled from five plants. Transcript levels were measured by qRT-PCR and normalized against a UBIQUITIN10 control. Values are means of three technical replicates and are expressed relative to the wild type at the end of the day (0 h).

Figure 6.

Carbohydrate contents of siliques from wild-type (diamonds) and sex1 mutant (squares) plants during the night. Each value is for a pool of five siliques (12 DAF) from five plants, assayed in triplicate. Error bars represent se. Similar results were obtained in an independent experiment (Supplemental Fig. S8).

Figure 7.

Daily pattern of gene expression in siliques of wild-type (black bars) and sex1 (white bars) plants. RNA was extracted from five siliques pooled from five plants at 12 DAF at the end of the day (EOD) and the end of the night (EON). Transcript abundance was measured by qRT-PCR with UBIQUITIN10 as a control. Values are means of three technical replicates, and error bars represent se. Similar results were obtained in a separate experiment.

Figure 8.

Development of embryos on wild-type (black bars) and sex1 (white bars) maternal plants. Seeds were viewed under differential interference contrast optics at the indicated DAF. Results are percentages of embryos examined. The numbers of embryos examined were as follows: at 6 DAF, 153 wild type and 125 sex1; at 8 DAF, 160 wild type and 158 sex1; at 10 DAF, 159 wild type and 105 sex1; at 14 DAF, 92 wild type and 82 sex1. The developmental stages were as follows: 1, globular; 2, transition; 3, heart; 4, torpedo; 5, walking stick; 6, early cotyledon; 7, cotyledon; 8, fully grown embryo.