Nitrogen recycling from the xylem in rice leaves: dependence upon metabolism and associated changes in xylem hydraulics

Abstract

Measurements of amino acids in the guttation fluid and in the xylem exudates of cut leaves from intact plants provide evidence of the remarkable efficiency with which these nitrogenous compounds are reabsorbed from the xylem sap. This could be achieved by mechanisms involving intercellular transport and/or metabolism. Developmental changes in transcripts and protein showed that transcripts for phosphoenolpyruvate carboxykinase (PEPCK) increased from the base to the leaf tip, and were markedly increased by supplying asparagine. Supplying amino acids also increased the amounts of protein of PEPCK and, to a lesser extent, of pyruvate, Pi dikinase. PEPCK is present in the hydathodes, stomata and vascular parenchyma of rice leaves. Evidence for the role of PEPCK was obtained by using 3-mercaptopicolinic acid (MPA), a specific inhibitor of PEPCK, and by using an activation-tagged rice line that had an increase in PEPCK activity, to show that activation of PEPCK resulted in a decrease in N in the guttation fluid and that treatment by MPA resulted in an increase in amino acids in the guttation fluid and xylem sap towards the leaf tip. Furthermore, increasing PEPCK activity decreased the amount of guttation fluid, whereas decreasing PEPCK activity increased the amount of xylem sap or guttation fluid towards the leaf tip. The findings suggest the following hypotheses: (i) both metabolism and transport are involved in xylem recycling and (ii) excess N is the signal involved in modulating xylem hydraulics, perhaps via nutrient regulation of water-transporting aquaporins. Water relations and vascular metabolism and transport are thus intimately linked.

Keywords: Amides; amino acids; asparagine; hydathode; phosphoenolpyruvate carboxykinase; rice (Oryza sativa); xylem..

Fig. 1.

Amino acid concentrations of xylem exudate and guttation sap in 7-d-old first leaves of O. sativa. The xylem exudate and guttation fluid measurements are from the combined volume of 24 plants. Values are the mean ±SE from three independent experiments. All measurements of amino acid contents in the xylem exudate are significantly different at the 5% level when compared to the respective guttation sap.

Fig. 2.

Amino acid and amide contents in guttation fluid at the tip (entire amount collected immediately after the 12h dark period) or xylem sap both at and various distances from the base (collected at 1h intervals in the light period) in 7-d-old primary leaves of O. sativa fed water (control), 10mM L-alanine, 10mM L-glutamine, or 10mM L-asparagine. The xylem exudate and guttation fluid measurements are from the combined volume of 24 plants. Values are the mean ±SE from three independent experiments. All xylem exudate and guttation sap amino acid contents are significantly different at the 5% level when compared to the control. Note break in Y-axis. Mean guttation and xylem exudate volumes (μl) were as follows: water, base to tip, 60, 11.5, 0.2, 41; alanine, 101, 58, 53, 135; glutamine, 112, 62, 45, 70; asparagine, 140, 111, 62, 103.

Fig. 3.

Relative expression of various genes compared to snU3 RNA in 7-d-old control (●), asparagine-fed (∆) and glutamine-fed (□) first leaves of O. sativa. Results are expressed as means ±SE of three biological replicates.

Fig. 4.

Immunoblots showing the effect of nitrogenous compounds or malate on the abundance of the PEPCK 3, PEPC, NADP-ME, NAD-ME and PPDK proteins in 0.5cm leaf sections taken from base to tip of the first leaf of 7-d-old leaves of O. sativa. Loadings on gels contained the soluble protein content of 0.33mg of FW of tissue. Numbers on the left hand Y-axis refer to molecular masses (kDa). Note that the image is constructed from different immunoblots.

Fig. 5.

PEPCK is present in the stomata, s, hydathode, h, and vascular tissue (xylem parenchyma, xp; phloem parenchyma, pp). Transverse sections of rice after immunodiazostaining with pre-immune (A and C) and PEPCK serum (B and D). Serum used in panel B was a peptide antibody to the chromosome 3 isoform in rice. Serum used in panel D was a protein antibody to PEPCK in cucumber that was more sensitive to PEPCK located in the vascular tissue but less sensitive to PEPCK located in the stomata and hydathode of rice. Bars: 20 μm for A, B; 30 μm for C, D.

Fig. 6.

Schematic presentation of the roles of transport and the possible metabolic role of PEPCK in nitrogen recycling from the xylem. Transport involves unloading of amino acids and amides (N) from the xylem, passage through the vascular parenchyma between the xylem and phloem, and loading into the phloem. Metabolism involves similar transport processes in conjunction with metabolism in the vascular parenchyma. Specifically, the role of PEPCK would involve the metabolism of OAA (oxaloacetate) deriving from Asp and, ultimately, Asn. N released by Asn and Asp metabolism could then be also transported into the phloem.