Trehalose 6-phosphate is required for the onset of leaf senescence associated with high carbon availability

Abstract

Trehalose 6-phosphate (T6P) is an important regulator of plant metabolism and development. T6P content increases when carbon availability is high, and in young growing tissue, T6P inhibits the activity of Snf1-related protein kinase (SnRK1). Here, strong accumulation of T6P was found in senescing leaves of Arabidopsis (Arabidopsis thaliana), in parallel with a rise in sugar contents. To determine the role of T6P in senescence, T6P content was altered by expressing the bacterial T6P synthase gene, otsA (to increase T6P), or the T6P phosphatase gene, otsB (to decrease T6P). In otsB-expressing plants, T6P accumulated less strongly during senescence than in wild-type plants, while otsA-expressing plants contained more T6P throughout. Mature otsB-expressing plants showed a similar phenotype as described for plants overexpressing the SnRK1 gene, KIN10, including reduced anthocyanin accumulation and delayed senescence. This was confirmed by quantitative reverse transcription-polymerase chain reaction analysis of senescence-associated genes and genes involved in anthocyanin synthesis. To analyze if the senescence phenotype was due to decreased sugar sensitivity, the response to sugars was determined. In combination with low nitrogen supply, metabolizable sugars (glucose, fructose, or sucrose) induced senescence in wild-type and otsA-expressing plants but to a smaller extent in otsB-expressing plants. The sugar analog 3-O-methyl glucose, on the other hand, did not induce senescence in any of the lines. Transfer of plants to and from glucose-containing medium suggested that glucose determines senescence during late development but that the effects of T6P on senescence are established by the sugar response of young plants.

Figure 1.

Senescence-dependent changes in T6P content in leaves 9 and 10 of wild-type Col-0 (black circles) and transgenic plants expressing the TPS gene, otsA (white circles), or the TPP gene, otsB (black triangles). Data are means ± se of three plants. Asterisks indicate significant differences (ANOVA) between the three genotypes for each time point: * P ≤ 0.05, ** P ≤ 0.01. Different letters indicate significant differences between the genotypes (P ≤ 0.05; Tukey’s pairwise comparison). FW, Fresh weight.

Figure 2.

Phenotypes of wild-type Col-0 and transgenic plants expressing the TPS gene, otsA, or the TPP gene, otsB. A, Rosettes of plants grown for 55 d. B, Individual leaves of plants grown for 52 d.

Figure 3.

Senescence-dependent changes in leaves 9 and 10 of wild-type Col-0 (black circles) and transgenic plants expressing the TPS gene, otsA (white circles), or the TPP gene, otsB (black triangles). A, Chlorophyll content. B, F_v/F_m. Data are means of four plants ± se. Asterisks indicate significant differences (ANOVA) between the three genotypes for each time point: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Different letters indicate significant differences between the genotypes (P ≤ 0.05; Tukey’s pairwise comparison).

Figure 4.

Senescence-dependent changes in gene expression determined by quantitative RT-PCR in leaves 9 and 10 of wild-type Col-0 (black circles) and transgenic plants expressing the TPS gene, otsA (white circles), or the TPP gene, otsB (black triangles). A, Cytosolic glutamine synthetase 1;4 (GLN1;4; At5g16570). B, High-affinity nitrate transporter 2.5 (NTR2.5; At1g12940). C, Leucoanthocyanidin dioxygenase (LDOX; At4g22880). D, Anthocyanin pigment 2 protein (PAP2 = MYB90; At1g66390). E, Senescence-associated gene 12 (SAG12; At5g45890). F, Chlorophyll a/b-binding protein (LHCB1;4; At2g34430). G, Fructose-bisphosphate aldolase (FBP aldolase; At4g26530). Expression ratios are presented relative to Col-0 values on day 32. Data are means of three plants ± se. Asterisks indicate significant differences (ANOVA) between the three genotypes for each time point: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Different letters indicate significant differences between the genotypes (P ≤ 0.05; Tukey’s pairwise comparison).

Figure 5.

Sugar contents during senescence in wild-type Col-0 (black circles) and transgenic plants expressing the TPS gene, otsA (white circles), or the TPP gene, otsB (black triangles). A, Glc. B, Fru. C, Suc. Data are means ± se of three plants. Asterisks indicate significant differences (ANOVA) between the three genotypes for each time point: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Different letters indicate significant differences between the genotypes (P ≤ 0.05; Tukey’s pairwise comparison). FW, Fresh weight.

Figure 6.

Response of wild-type Col-0 and transgenic plants expressing the TPS gene, otsA, or the TPP gene, otsB, to growth for 49 d on low-nitrogen (4.7 mm; Low N) or high-nitrogen (30 mm; High N) medium without or with the addition of 111 mm Glc.

Figure 7.

Whole-rosette F_v/F_m values in wild-type Col-0 (black circles) and transgenic plants expressing the TPS gene, otsA (white circles), or the TPP gene, otsB (black triangles), during growth on low-nitrogen (4.7 mm; Low N; C and D) or high-nitrogen (30 mm; High N; A and B) medium without (A and C) or with (B and D) the addition of 111 mm Glc. Data are means of at least 16 plants ± sd.

Figure 8.

Whole-rosette F_v/F_m values during growth on low-nitrogen medium without the addition of sugar (black circles) or with the addition of 111 mm 3-OMG (white circles), Glc (black triangles), Fru (white triangles), or 55.5 mm Suc (black squares). A, Wild-type plants (Col-0). B, Transgenic plants expressing the TPS gene, otsA. C, Transgenic plants expressing the TPP gene, otsB. Data are means of at least 12 plants ± sd.

Figure 9.

Effect of transfer to and from Glc-containing medium on whole-rosette F_v/F_m values. A, Wild-type plants (Col-0). B, Transgenic plants expressing the TPS gene, otsA. C, Transgenic plants expressing the TPP gene, otsB. Plants were transferred from low-nitrogen medium containing 111 mm Glc to medium containing 111 mm sorbitol (Glc-Stl; white symbols) or vice versa (Stl-Glc; black symbols) after 1 week (1w; circles) or 3 weeks (3w; triangles). Data are means of plants from five plates ± se.