Hypersensitivity of an Arabidopsis sugar signaling mutant toward exogenous proline application

Abstract

In transgenic Arabidopsis a patatin class I promoter from potato is regulated by sugars and proline (Pro), thus integrating signals derived from carbon and nitrogen metabolism. In both cases a signaling cascade involving protein phosphatases is involved in induction. Other endogenous genes are also regulated by both Pro and carbohydrates. Chalcone synthase (CHS) gene expression is induced by both, whereas the Pro biosynthetic Delta(1)-pyrroline-5-carboxylate synthetase (P5CS) is induced by high Suc concentrations but repressed by Pro, and Pro dehydrogenase (ProDH) is inversely regulated. The mutant rsr1-1, impaired in sugar dependent induction of the patatin promoter, is hypersensitive to low levels of external Pro and develops autofluorescence and necroses. Toxicity of Pro can be ameliorated by salt stress and exogenously supplied metabolizable carbohydrates. The rsr1-1 mutant shows a reduced response regarding sugar induction of CHS and P5CS expression. ProDH expression is de-repressed in the mutant but still down-regulated by sugar. Pro toxicity seems to be mediated by the degradation intermediate Delta(1)-pyrroline-5-carboxylate. Induction of the patatin promoter by carbohydrates and Pro, together with the Pro hypersensitivity of the mutant rsr1-1, demonstrate a new link between carbon/nitrogen and stress responses.

Figure 1

Regulation of the class I patatin promoter by amino acids. Pat(B33)-Gus plants were cultured on MS medium containing 30 mm Glc and 15 mm Gln, citrulline, or Pro. After 30 d, samples were harvested to quantify GUS activity in roots and leaves. In this and all subsequent diagrams each column represents the mean of five independent measurements. Error bars indicate sd. Shaded bars, Leaf; white bars, root.

Figure 2

Quantification of GUS-activity in roots of Pat(B33)-Gus (shaded bars) and rsr1-1 (white bars) plants, cultured on MS medium containing 30 mm Glc. Samples were taken after 30 h of incubation time on 200 mm Pro in the presence or absence of 0.4 μm okadaic acid. Plants were pretreated for 2 h with the inhibitor before Pro was added.

Figure 3

Toxicity of exogenously supplied Pro. The mutant rsr1-1 is hypersensitive to Pro: After 9 d on MS medium containing 15 mm Pro and 30 mm Glc, extensive lesions were visible and root growth was almost completely inhibited (A). After 12 d the seedlings died (B). Under the same conditions, Pat(B33)-Gus showed almost no lesions after 9 d (C). On higher Pro concentrations (40 mm/30 mm Pro/Glc), Pat(B33)-Gus also showed lesions in various organs (D after 9 d; E after 15 d). Excised leaves of the Arabidopsis C24 wild type showed similar lesions when incubated for 48 h on agar plates containing 200 mm Pro (F). Prior to the appearance of necrotic tissue (G, I, and L), autofluorescent compounds accumulated (H, K, and M; excitation 470 nm). L and M, Left, Root of Pro-untreated plant; right, root of Pro-treated plant.

Figure 4

Amelioration of Pro toxicity by salt stress. Growth of Pat(B33)-Gus (upper halves of the plates) and rsr1-1 (lower halves) on MS medium containing 20 mm Glc supplemented with different salt concentrations (A and B, no salt; C and D, 50 mm NaCl; E and F, 100 mm NaCl) in the presence (B, D, and F) or absence of Pro (A, C, and E).

Figure 5

Pro-specific induction of the patatin promoter was blocked by the presence of salt. GUS activity was quantified in roots of 30-d-old plants incubated in liquid MS medium for 30 h in the presence of 200 mm Pro, 100 mm NaCl, or a combination of both.

Figure 6

Amelioration of Pro toxicity by Glc but not by non-metabolizable 3Omeg. Four-week-old Arabidopsis seedlings grown in the presence of 30 mm Glc without (A) or with 15 mm Pro (B). Pat(B33)-Gus (upper half of the plates) bleached in the presence of 15 mm Pro, the mutant rsr1-1 (lower half of the plates) was already dead. Increase of the Glc concentration to 120 mm enabled rsr1-1 to survive and Pat(B33)-Gus to grow normally (C), whereas a combination of 30 mm Glc and 90 mm 3Omeg was ineffective (D). E, Internal Pro content positively correlated with increasing external Glc concentration. Pro increase was more pronounced in Pat(B33)-Gus (shaded bars) than in rsr1-1 (white bars).

Figure 7

P5C induces Pro-like damages in Arabidopsis. A, Reduction of chlorophyll fluorescence of P5C-treated Pat(B33)-Gus plant (1 mm for 9 h) and B, P5C-untreated Pat(B33)-Gus plant. Plants treated for 3 d with 1 mm P5C showed brown dead tissue (C), whereas after treatment with 100 mm Pro only minor damage appeared (D). The yellow color in C derives from 2,4-dinitrophenylhydrazine hydrochloride double salt of P5C.

Figure 8

RNA gel-blot analysis of the expression of the ProDH, P5CS, and CHS genes in 3-week-old non-bolting Pat(B33)-Gus and rsr1-1 plants after various treatments. Plants were pre-cultured on 2MS medium for 30 d, and transferred to liquid MS medium containing 200 mm Pro, Suc, or sorbitol, respectively. Root and leaf material was harvested after 8 and 24 h of incubation. Fifteen micrograms of total RNA was loaded in each lane.

Figure 9

Genetic analysis of pro^HS2-1, a second Pro-hypersensitive mutant. Homozygous pro^HS2-1 plants (center) were crossed to Pat(B33)-Gus, wild-type Arabidopsis ecotype Col0 and rsr1-1. F₁ progeny and parental lines were cultured on MS medium supplemented with 30 mm Glc and 20 mm Pro. Pro hypersensitivity of pro^HS2-1 was complemented in all crosses.

Figure 10

Working model for the position of AtRSR1 in the regulatory network connecting Pro and carbohydrate signaling with the patatin promoter. AtRSR1 is induced by sugars and Pro degradation, but in the latter case the actual effector remains unclear. Activation of AtRSR1 leads to repression of ProDH expression and induces the patatin promoter. Endogenous target genes other than ProDH remain to be identified. Toxicity of Pro derives from the overproduction of P5C in the absence of stress. The mutation in AtRSR1 abolishes feedback inhibition of Pro degradation and thereby produces hypersensitivity toward Pro. Amelioration of toxicity can be achieved by hyperosmolar conditions that reduce ProDH transcript levels or by supply of metabolizable carbohydrates that potentially interfere on a different level.