Long-distance phloem transport of glucosinolates in Arabidopsis

Abstract

Glucosinolates are a large group of plant secondary metabolites found mainly in the order Capparales, which includes a large number of economically important Brassica crops and the model plant Arabidopsis. In the present study, several lines of evidence are provided for phloem transport of glucosinolates in Arabidopsis. When radiolabeled p-hydroxybenzylglucosinolate (p-OHBG) and sucrose were co-applied to the tip of detached leaves, both tracers were collected in the phloem exudates at the petioles. Long-distance transport of [(14)C]p-OHBG was investigated in wild-type and transgenic 35S::CYP79A1 plants, synthesizing high amounts of p-OHBG, which is not a natural constituent of wild-type Arabidopsis. In both wild-type and 35S::CYP79A1 plants, radiolabeled p-OHBG was rapidly transported from the application site into the whole plant and intact p-OHBG was recovered from different tissues. The pattern of distribution of the radioactivity corresponded to that expected for transport of photoassimilates such as sucrose, and was consistent with translocation in phloem following the source-sink relationship. Radiolabeled p-OHBG was shown to accumulate in the seeds of wild-type and 35S::CYP79A1 plants, where p-OHBG had been either exogenously applied or endogenously synthesized from Tyr in the leaves. p-OHBG was found in phloem exudates collected from cut petioles of leaves from both wild-type and 35S::CYP79A1 plants. Phloem exudates were shown to contain intact glucosinolates, and not desulphoglucosinolates, as the transport form. It is concluded that intact glucosinolates are readily loaded into and transported by the phloem.

Figure 1

Loading and export of radiolabeled p-OHBG. A, [³H]p-OHBG and [¹⁴C]Suc were applied simultaneously to the tips of single detached leaves and the phloem exudates were subsequently collected by incubating the leaf petioles in 15 mm EDTA for 6 h. Values (±5%) represent the radioactivity of ³H or ¹⁴C in the phloem exudates as percentage of the total radioactivity applied. B, Time course of export of radioactivity after application of [³H]p-OHBG to a single either young or mature leaf of flowering plants. At given time points, the donor leaves were excised from four to six plants and the radioactivity in the remaining parts of the plant was measured.

Figure 2

Long-distance transport and distribution of radioactivity in wild-type Arabidopsis 24 h after application of [¹⁴C]p-OHBG to mature rosette leaves of flowering plant. A, Photograph (left) and autoradiography (right) of Arabidopsis incubated for 24 h after application of [¹⁴C]p-OHBG to leaves (arrows). B, Measurement of radioactivity in methanol extracts of different tissues. Four plants were sampled and dissected. C, Qualitative TLC analysis of radiolabeled p-OHBG in methanol extracts of different tissues from a single plant. The position of intact p-OHBG is indicated (arrow). D, Photograph (left) and autoradiogram (right) of labeled Arabidopsis intact and crushed siliques. E, Non-labeled controls for D.

Figure 3

The content of [¹⁴C]p-OHBG in seeds and vegetative tissues of wild-type and 35S::CYP79A1 plants. The plants were fed with either [¹⁴C]Tyr or [¹⁴C]p-OHBG at the vegetative stage and harvested at withering stage. A, Quantitative measurement of total radioactivity in methanol extracts of vegetative tissues (V) and seeds (S) of wild-type and 35S::CYP79A1 plants. Insert: 1, [¹⁴C]p-OHBG standard; 2, methanol extract of wild-type rosette leaves 3 weeks after application of [¹⁴C]Tyr; 3, methanol extract of 35S::CYP79A1 rosette leaves 3 weeks after application of [¹⁴C]Tyr; 4, [¹⁴C]Tyr standard. B, TLC analysis of radiolabeled desulpho p-OHBG in vegetative tissues (V) and seeds (S) of wild-type and 35S::CYP79A1 plants. Methanol extracts of vegetative tissues and seeds were applied to DEAE column and sulfatase treated (see “Materials and Methods”). Fifteen-microliter aliquots of 250 μL DEAE eluate were analyzed by TLC. The position of desulpho p-OHBG is indicated (arrow). C, LC-mass spectrometry (MS) UV trace (229 nm) of desulphoglucosinolates in seeds (upper trace) and vegetative tissues (lower trace) of wild-type plants fed with [¹⁴C]p-OHBG. The samples are identical to those in lane 4 and 5 in B. Arrow indicates the position of desulpho p-OHBG (D). Mass spectrum of peak 6 in C confirming the expected [M+Na]⁺ ion at m/z 368 corresponding to desulpho p-OHBG. The numbers correspond to the desulphoglucosinolates of the following glucosinolates: 1, 3-Hydroxypropylglucosinolate; 2, 3-methylsulphinylpropylglucosinolate (3MSOP); 3, 4-hydroxybutylglucosinolate; 4, 4-methylsulphinylbutylglucosinolate (4MSOB); 5, 5-methylsulphinylpentylglucosinolate; 6, p-OHBG; 7, 6-methylsulphinylhexylglucosinolate; 8, 7-methylsulphinylheptylglucosinolate; 9, 4-methylthiobutylglucosinolate; 10, indol-3-ylmethylglucosinolate; 11, 8-methylsulphinyloctylglucosinolate; 12, 4-methoxyindol-3-ylmethylglucosinolate; 13, 3-benzoyloxypropylglucosinolate; 14, 6-methylthiohexylglycosinolate; 15, 4-benzoyloxybutylglucosinolate; 16, 7-methylthioheptylglucosinolate; 17, 8-methylthiooctylglucosinolate.

Figure 4

HPLC and LC-MS analysis of major glucosinolates in phloem exudates and methanol extracts of rosette leaves of wild-type and 35S::CYP79A1 plants. A through F, HPLC UV trace (229 nm). G and H, LC-MS UV-trace (229 nm). A, Glucosinolates in phloem exudates of wild-type leaves collected in distilled water. B, Glucosinolates in phloem exudates of 35S::CYP79A1 leaves collected in distilled water. C, Glucosinolates in phloem exudates of wild-type leaves collected in 15 mm EDTA. D, Glucosinolates in phloem exudates of 35S::CYP79A1 leaves collected in 15 mm EDTA. E, HPLC of glucosinolates in rosette leaves of wild-type plants. F, HPLC of glucosinolates in rosette leaves of 35S::CYP79A1 plants. G, LC-MS of phloem exudates without sulfatase treatment of both wild-type (lower trace) and 35S::CYP79A1 leaves (upper trace). H, LC-MS of sulfatase-treated phloem exudates of 35S::CYP79A1 leaves. The numbers correspond to the desulphoglucosinolates of the following glucosinolates: 1, 3MSOP; 2, 4MSOB; 3, 5-methylsulphinylpentylglycosinolate; 4, p-OHBG; 5, benzylglucosinolate, internal standard; 6, 7-methylsulphinylheptylglucosinolate; 7, indol-3-ylmethylglycosinolate; 8, 5-methylthiopentylglucosinolate; 9, 8-methylsulphinyloctylglucosinolate; 10, 4-methoxyindol-3-ylmethylglucosinolate; 11, 1-methoxyindol-3-ylmethylglucosinolate; 12, 8-methylthiooctylglucosinolate.