The Arabidopsis L-Type Amino Acid Transporter 5 (LAT5/PUT5) Is Expressed in the Phloem and Alters Seed Nitrogen Content When Knocked Out

Abstract

The Arabidopsis L-type Amino Acid Transporter-5 (LAT5; At3g19553) was recently studied for its role in developmental responses such as flowering and senescence, under an assumption that it is a polyamine uptake transporter (PUT5). The LATs in Arabidopsis have a wide range of substrates, including amino acids and polyamines. This report extensively studied the organ and tissue-specific expression of the LAT5/PUT5 and investigated its role in mediating amino acid transport. Organ-specific quantitative RT-PCR detected LAT5/PUT5 transcripts in all organs with a relatively higher abundance in the leaves. Tissue-specific expression analysis identified GUS activity in the phloem under the LAT5/PUT5 promoter. In silico analysis identified both amino acid transporter and antiporter domains conserved in the LAT5/PUT5 protein. The physiological role of the LAT5/PUT5 was studied through analyzing a mutant line, lat5-1, under various growth conditions. The mutant lat5-1 seedlings showed increased sensitivity to exogenous leucine in Murashige and Skoog growth medium. In soil, the lat5-1 showed reduced leaf growth and altered nitrogen content in the seeds. In planta radio-labelled leucine uptake studies showed increased accumulation of leucine in the lat5-1 plants compared to the wild type when treated in the dark prior to the isotopic feeding. These studies suggest that LAT5/PUT5 plays a role in mediating amino acid transport.

Keywords: Arabidopsis; L-type amino acid transporter; LAT; LAT5; PUT5; amino acid translocation; amino acids; nitrogen; polyamine uptake transporter; seed nitrogen.

Figure 1

In silico characterization of the Arabidopsis LAT5/PUT5 protein. (a) A cartoon representation showing the 12 transmembrane domains in the LAT5/PUT5 protein with both c- and n- termini in the cytoplasmic side. (b) Domains conserved in the LAT5/PUT5 protein. The black bar shows the length of the amino acid sequence. The blue and red bars show the position and extent of the domains in LUT5/PUT5 protein. The blue bars show the conserved domains identified in the Pfam database and the red bars show the conserved domains identified in the NCBI conserved domain database. (c) Putative physical and functional associations of the LAT5/PUT5 with other proteins. The thickness of the gray line indicates the confidence level of the association between the two proteins. Thicker lines indicate higher confidence.

Figure 2

Organ- and tissue-specific expression analyses of the LAT5/PUT5. (a) Organ-specific quantitative RT–PCR detects the LAT5/PUT5 transcripts in all organs. The relative quantitation (RQ) was calculated using an average expression of the LAT5/PUT5 in whole plants as a calibrator and Actin2 as an endogenous control. The error bar represents the average difference between the highest and lowest RQ value of three biological replications each with three technical replications. (b–j) Tissue-specific expression of the LAT5/PUT5 based on GUS activity under the LAT5/PUT5 promoter. (b) GUS activity is present in the rosette leaf at the seeding stage; (c) Mature rosette leaf shows GUS stain; (d) A cross-section of the rosette leaf shows GUS stain in the mesophyll cells, concentrating more in the minor veins; (e) In mature stem, GUS stain is not visible on the stem surface; (f) A transverse section of a mature stem shows GUS stain in the phloem; (g) A longitudinal section of the stem shows GUS stain present in the phloem and absent in the xylem; (h) In the flower, GUS stain is present in the sepal and stamen filament; (i) In siliques, GUS stain is present along the vascular pattern in the fruit carpel; (j) Circles in the cross-section of a developing silique show the positions of the vascular tissues where GUS activity is present. GUS stain is present in the replum and the secondary vasculature in the carpel; (k) In mature plants, a GUS stain is present in the mature primary and lateral roots; (l) A GUS stain is present in the seedling roots and root tips. c, Cortex; cl, Carpel; e, Epidermis; mn, Minor Vein; mj, major vein r, Replum; p, Phloem; x, Xylem.

Figure 2

Organ- and tissue-specific expression analyses of the LAT5/PUT5. (a) Organ-specific quantitative RT–PCR detects the LAT5/PUT5 transcripts in all organs. The relative quantitation (RQ) was calculated using an average expression of the LAT5/PUT5 in whole plants as a calibrator and Actin2 as an endogenous control. The error bar represents the average difference between the highest and lowest RQ value of three biological replications each with three technical replications. (b–j) Tissue-specific expression of the LAT5/PUT5 based on GUS activity under the LAT5/PUT5 promoter. (b) GUS activity is present in the rosette leaf at the seeding stage; (c) Mature rosette leaf shows GUS stain; (d) A cross-section of the rosette leaf shows GUS stain in the mesophyll cells, concentrating more in the minor veins; (e) In mature stem, GUS stain is not visible on the stem surface; (f) A transverse section of a mature stem shows GUS stain in the phloem; (g) A longitudinal section of the stem shows GUS stain present in the phloem and absent in the xylem; (h) In the flower, GUS stain is present in the sepal and stamen filament; (i) In siliques, GUS stain is present along the vascular pattern in the fruit carpel; (j) Circles in the cross-section of a developing silique show the positions of the vascular tissues where GUS activity is present. GUS stain is present in the replum and the secondary vasculature in the carpel; (k) In mature plants, a GUS stain is present in the mature primary and lateral roots; (l) A GUS stain is present in the seedling roots and root tips. c, Cortex; cl, Carpel; e, Epidermis; mn, Minor Vein; mj, major vein r, Replum; p, Phloem; x, Xylem.

Figure 3

Phenotypic analysis of the lat5-1 on soil and plate culture. (a) Visual presentation of the lat5-1 showing reduced leaf growth compared to the WT; (b) the homozygous lat5-1 mutant plants show no difference in the total number of rosette leaves; (c) but show reduced leaf size, measured by the leaf width; (d) and reduced biomass, compared to the WT under normal growth condition on fertilized soil. Photos were taken when the plants were five weeks old. For leaf biomass, leaves were cut off and dried at 70 °C for two hours before being weighed. The error bar represents the standard deviation of five replicates. (e) Leaf mesophyll cell protoplasts in the lat5-1 are smaller compared to the WT. The error bar represents the standard deviation of the diameter of 100 cells from three individual plant protoplast cultures. (f) The mutant lat5-1 seedlings are more sensitive to exogenous leucine compared to the WT. Variable concentrations of L-leucine were added to a nitrogen-free growth medium with 1 mmol·L⁻¹ nitrate as the source of nitrogen. (g) A quantitative representation shows significantly increased sensitivity of the lat5-1 seedlings to exogenous leucine compared to the WT, measured by the reduction in fresh weight of biomass. The relative percentage of growth was calculated assuming the biomass obtained by the corresponding WT as 100%. The error bar represents the standard deviation of three to five biological replications with two seedlings per replication. Photos were taken when the seedlings were two-weeks old. Each growth condition had three or more plate replications. Each growth experiment was repeated at least twice with similar results. * p < 0.05; ** p < 0.01; *** p < 0.001. DW, Dry weight; FW, Fresh weight; M, mol·L⁻¹.

Figure 4

Radio-labelled leucine uptake study shows significantly increased accumulation of leucine in lat5-1 compared to the WT when seedlings were treated in dark prior to the isotopic feeding and allowed to export. (a) Seedlings grown under normal light/dark regime show decreased accumulation of ¹⁴C- and ³H- in the lat5-1 compared to the WT. (b) Seedlings, kept in dark for 27 h prior to the isotopic feeding, show increased accumulation of both ¹⁴C- and ³H- in the lat5-1 compared to the WT. (c) Seedlings, kept in dark for 27 h prior to the isotopic feeding, show significantly increased accumulation of both ¹⁴C- and ³H- in the lat5-1 compared to the WT when uptake was followed by export. Error bar represents standard deviation of three biological replications. * p < 0.05.

Figure 5

The lat5-1 shows increased free amino acid content in the siliques and increased nitrogen content in the seeds, compared to the WT. (a) The total free amino acid concentration is increased in both stem and silique and decreased in the leaves in the lat5-1 compared to the WT. The total free amino acids detected in the WT was arbitrarily taken as 100% to calculate the relative amount in the lat5-1. Error bar represents the standard deviation of three biological replications. (b) The seed weight is significantly increased in the lat5-1, compared to the WT. (c) The percent of nitrogen content is significantly increased in the lat5-1, compared to the WT. Error bar represents the standard deviation of nine replications from three individual plants with 100 seeds per replication. * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001.