Differential effects of sucrose and auxin on localized phosphate deficiency-induced modulation of different traits of root system architecture in Arabidopsis

Abstract

Phosphorus, one of the essential elements for plants, is often a limiting nutrient in soils. Low phosphate (Pi) availability induces sugar-dependent systemic expression of genes and modulates the root system architecture (RSA). Here, we present the differential effects of sucrose (Suc) and auxin on the Pi deficiency responses of the primary and lateral roots of Arabidopsis (Arabidopsis thaliana). Inhibition of primary root growth and loss of meristematic activity were evident in seedlings grown under Pi deficiency with or without Suc. Although auxin supplementation also inhibited primary root growth, loss of meristematic activity was observed specifically under Pi deficiency with or without Suc. The results suggested that Suc and auxin do not influence the mechanism involved in localized Pi sensing that regulates growth of the primary root and therefore delineates it from sugar-dependent systemic Pi starvation responses. However, the interaction between Pi and Suc was evident on the development of the lateral roots and root hairs in the seedlings grown under varying levels of Pi and Suc. Although the Pi+ Suc- condition suppressed lateral root development, induction of few laterals under the Pi- Suc- condition point to increased sensitivity of the roots to auxin during Pi deprivation. This was supported by expression analyses of DR5uidA, root basipetal transport assay of auxin, and RSA of the pgp19 mutant exhibiting reduced auxin transport. A significant increase in the number of lateral roots under the Pi- Suc- condition in the chalcone synthase mutant (tt4-2) indicated a potential role for flavonoids in auxin-mediated Pi deficiency-induced modulation of RSA. The study thus demonstrated differential roles of Suc and auxin in the developmental responses of ontogenetically distinct root traits during Pi deprivation. In addition, lack of cross talk between local and systemic Pi sensing as revealed by the seedlings grown under either the Pi- Suc- condition or in the heterogeneous Pi environment highlighted the coexistence of Suc-independent and Suc-dependent regulatory mechanisms that constitute Pi starvation responses.

Figure 1.

Effect of Pi and Suc availability on morphophysiological traits. Wild-type seedlings were grown for 7 d on vertically oriented agar petri plates containing P+S+, P+S−, P−S+, or P−S− nutrient. Data are presented for fresh weight; n = 6 replicates of 20 seedlings each (A); shoot-to-root fresh weight biomass ratio, n = 6 replicates of 20 seedlings each (B); soluble Pi content, n = 6 replicates of 100 to 150 seedlings each (C); leaf area, n = 4, ▪ = 1 mm² (D); and anthocyanin content, n = 6 replicates of 100 to 150 seedlings each (E). Values are means ± se. Different letters indicate that the means differ significantly (P < 0.05).

Figure 2.

Role of Suc in Pi deficiency-induced determinate primary root growth. A, Transgenic CycB1;1∷uidA Arabidopsis seedlings, grown on different nutrient media, were scanned sequentially at indicated time intervals and after replenishment with P+S+ nutrient for temporal analysis of primary root growth. B, RSAs are representative of 20 seedlings each of P+S+ (a), P+S− (b), P−S+ (c), and P−S− (d). Lateral roots were spread under a stereomicroscope to present their architectural details. At the bottom (e–h), → indicates primary root length at the time of transfer to P+S+ for replenishment. Scale bar = 1 cm. C, Histochemical GUS staining of primary root tips of CycB1;1∷uidA seedlings grown for 7 d on P+S+ (a), P+S− (b), P−S+ (c), and P−S− (d) and after their replenishment (e–h) with P+S+ for 7 d. GUS-stained seedlings were observed under compound microscopy (20× objective). Photographs are representative of at least 10 to 12 seedlings. Scale bar = 50 μm. D, Real-time PCR analysis of the relative expression levels of Pht1;4 and PLDZ2 in seedlings grown on vertically oriented agar petri plates under different nutrient conditions for 7 d. 18S ribosomal RNA was used as an internal control. Data presented are the means of three technical replicates ± se. [See online article for color version of this figure.]

Figure 3.

Effect of auxin availability on Pi deficiency-induced determinate growth of the primary root. A, Transgenic DR5∷uidA Arabidopsis seedlings were grown on different nutrient media for 7 d. GUS-stained hypocotyl-root junction and tips of primary and lateral roots were observed under compound microscopy (20× objective). Photographs are representative of eight to 10 seedlings for each of the treatments. Scale bar = 0.1 mm. B, Free IAA content in upper and lower roots (4 mm) of seedlings grown in different nutrient media for 7 d. Values are mean ± sd from 500 seedlings per replicate, n = 3. *, Means differ significantly (P < 0.05). C, Polar transport of [³H] IAA assay 2, 4, and 6 mm from the root tips of the seedlings grown under different nutrient conditions for 3 d. Values are means ± sd from 10 seedlings per replicate (n = 3). D, Representative RSAs of 20 seedlings each of transgenic CycB1;1∷uidA Arabidopsis seedlings grown for 7 d in 0.1 μm IAA-supplemented nutrient medium. •, Primary root length (average = 2.4 cm; n = 80) at the time of transfer to auxin-supplemented nutrient medium. Scale bar = 1 cm. E, Ten of the transgenic CycB1;1∷uidA seedlings for each of the treatments shown in D were used for histochemical GUS staining of the primary root tip. F, The rest of them were transferred to P+S+ for 7 d for replenishment. →, Primary root length achieved in auxin-supplemented different nutrient media at the time of replenishment. Some of the lateral roots growing near the root tip were removed to expose the primary root. Scale bars = 100 μm and 50 μm in A and E, respectively; 1 cm in D and F.

Figure 4.

Effect of Pi and/or Suc availability on lateral root development. Wild-type seedlings grown under different nutrient conditions were documented for the number of primary and higher order laterals after 7 d (A), 14 d (B), and replenishment with P+S+ for 7 d (C). Values are means ± se; n = 20. Different letters indicate that the means differ significantly (P < 0.05).

Figure 5.

Effect of Pi and/or Suc availability on the expression of auxin-responsive reporter DR5∷uidA in the root system. Transgenic DR5∷uidA Arabidopsis seedlings were grown on different nutrient media for 7 d followed by histochemical GUS staining. Schematic illustration of the seedling depicting different regions (a, b, c, and d) on the root system that were scored for the number of areas showing GUS expression. a, Region between the primary root tip and the youngest lateral root. b, Region between the hypocotyl-root junction and the youngest lateral root. c, Primary lateral roots. d, Second-order lateral roots. Data correspond to the average number of areas in the designated regions showing GUS expression in 20 seedlings each for different treatments. Values are means ± se.

Figure 6.

Auxin transport mutant alters Pi and Suc response on lateral root development. Wild-type and auxin transport mutants pgp19 (A–C) and tt4-2 (D–F) were grown on different nutrient media for 7 d. A and D, Lateral roots were spread to reveal the architectural details. RSAs are representative of 20 seedlings each for different treatments. •, Root length (average = 1.98 cm; n = 80) at the time of transfer to different nutrient media. Scale bar = 1 cm. Data are presented for the number of lateral roots (B and E) and total lateral root length (C and F). Values are mean ± se; n = 20. Different letters indicate that the means differ significantly (P < 0.05).

Figure 7.

Effect of Pi and Suc availability on root hair development. Development of root hairs in a 5-mm section from the primary root tip of the wild-type seedlings grown on different nutrient media for 2 d. Data are presented for the number of root hairs (B) and root hair length (C). Scale bar = 0.5 mm. Values are means ± se; n = 20. Different letters indicate that the means differ significantly (P < 0.05).

Figure 8.

Effects of homogenous and heterogenous Pi environment on local and systemic Pi starvation responses. A and B, Transgenic Pht1;4∷uidA Arabidopsis seedlings were grown on different nutrient media for 7 d. RSAs are representative of 20 seedlings each for different treatments. •, Root length (average = 1.8 cm; n = 80) at the time of transfer to different nutrient media. Scale bar = 1 cm. Insets in A show the primary and lateral root tips of the seedlings analyzed for GUS expression of Pht1;4∷uidA. Scale bar in the inset = 100 μm. B, Seedlings stained with BCIP for evaluation of in vivo APase activity. C, Root growth on divided agar petri plates containing P+S− medium in the upper compartment and P+S+, P−S+, or P−S− in the lower compartment. •, Root length (average = 3.7 cm; n = 80) at the time of transfer to divided plates. Scale bar = 1 cm. Inset shows the primary root tips of the seedlings analyzed for GUS expression of CycB1;1∷uidA (a) and Pht1;4∷uidA (b). Scale bar in the inset = 100 μm. D, Increase in primary root length of the seedlings subsequent to their transfer to divided plates. Values are means ± se; n = 20. Different letters indicate that the means differ significantly (P < 0.05).