Intron-mediated alternative splicing of Arabidopsis P5CS1 and its association with natural variation in proline and climate adaptation

Abstract

Drought-induced proline accumulation is widely observed in plants but its regulation and adaptive value are not as well understood. Proline accumulation of the Arabidopsis accession Shakdara (Sha) was threefold less than that of Landsberg erecta (Ler) and quantitative trait loci mapping identified a reduced function allele of the proline synthesis enzyme Δ(1)-pyrroline-5-carboxylate synthetase1 (P5CS1) as a basis for the lower proline of Sha. Sha P5CS1 had additional TA repeats in intron 2 and a G-to-T transversion in intron 3 that were sufficient to promote alternative splicing and production of a nonfunctional transcript lacking exon 3 (exon 3-skip P5CS1). In Sha, and additional accessions with the same intron polymorphisms, the nonfunctional exon 3-skip P5CS1 splice variant constituted as much as half of the total P5CS1 transcript. In a larger panel of Arabidopsis accessions, low water potential-induced proline accumulation varied by 10-fold and variable production of exon 3-skip P5CS1 among accessions was an important, but not the sole, factor underlying variation in proline accumulation. Population genetic analyses suggest that P5CS1 may have evolved under positive selection, and more extensive correlation of exon 3-skip P5CS1 production than proline abundance with climate conditions of natural accessions also suggest a role of P5CS1 in local adaptation to the environment. These data identify a unique source of alternative splicing in plants, demonstrate a role of exon 3-skip P5CS1 in natural variation of proline metabolism, and suggest an association of P5CS1 and its alternative splicing with environmental adaptation.

Fig. 1.

A chromosome 2 QTL causes reduced proline accumulation in Sha. (A) Proline accumulation across a range of ψ_w for Ler and Sha seedlings as well as NILs generated by twice backcrossing RILs 56 and 68 (Fig. S2) to Ler. Data are means ± SE, n = 6–10. (B) Plot of LOD scores for ψ_w-induced proline of the Ler × Sha RIL population. The gray line is for proline at −0.7 MPa and the darker line is for −1.2 MPa. The dashed line indicates the significance threshold (LOD = 2.5).

Fig. 2.

Differences in P5CS1 intron sequence are the basis of the Pro-W2 QTL and are sufficient to promote production of nonfunctional exon 3-skip P5CS1 transcript. (A) Diagram of the 5′ portion of the two alternative P5CS1 transcripts detected by 5′-RACE analysis. Boxes indicate exons with dark portions indicating the coding region. Gray boxes indicate possible coding region of the exon 3-skip transcript starting from an alternative downstream ATG. The number of clones of each variant found is indicated along with sequence of the 3′ ends of P5CS1 introns 2 and 3; see Dataset S2 for complete intron 2 and 3 sequence. (B) Semiquantitative RT-PCR using primers indicated in A to detect P5CS1 splice variants before low ψ_w treatment (0 h) or after 10 and 96 h at −1.2 MPa. Actin2 was used as a control gene. P5CS1 protein in samples from the same treatments was detected by Western blot (50 μg protein loaded). Tubulin antibody was used as a loading control. (C) Proline content of Ler × p5cs1-4 or Sha x p5cs1-4 F₁ seedlings after 96 h at −1.2 MPa. For both Ler and Sha, reciprocal crosses gave identical results and combined data are shown. Data are means ± SE (n = 6–9) with P value of the comparison of the two sets of F₁ seedlings indicated. Western blot detection of P5CS1 and tubulin (Lower) are also shown. (D) Proline and P5CS1 level of plants transformed with P5CS1 genomic fragments (gP5CS1). Sha was transformed with the Ler genomic P5CS1 clone (Sha/Ler-gP5CS1) and p5cs1-4 transformed with either Ler or Sha gP5CS1(p5cs1-4/gP5CS1). Note that p5cs1-4 is in the Col-0 genetic background. Proline data are combined from three-to-five independent T₃ homozygous lines and are means ± SE (n = 6–12); Western blot is for one representative transgenic line. (E) Exon 3-skip P5CS1 after 96 h at -1.2 MPa in F₁ seedlings of Ler and Sha crossed to p5cs1-1 (1–1) or p5cs1-4 (1–4). RT-PCR was performed for 24 cycles when both P5CS1 transcripts were in the linear range of amplification. Quantitation of exon 3-skip percentage is indicated by numbers along bottom of the gel. Reciprocal crosses gave similar results and representative data are shown. (F) Percentage of exon 3-skip P5CS1 transcript produced by Ler or Sha gP5CS1 in p5cs1-4. For both gP5CS1 constructs, data shown are means ± SE of three to four independent T₃ homozygous lines. (G) Proline content and P5CS1 level for p5cs1-4 transformed with either the exon 3-skip or full-length (F.L.) P5CS1 cDNA under control of the 35S promoter (p5cs1-4/35S:P5CS1). Proline data are means ± SE (n = 6–12) of three-to-four independent T₃ homozygous lines and Western blot is from one representative transgenic line. Western blot of additional transgenic lines is shown in Fig. S3.

Fig. 3.

Alternative splicing of P5CS1 is a major factor underlying variation in low ψ_w-induced proline accumulation between Arabidopsis accessions. (A) Proline contents of 180 Arabidopsis accessions after 96-h low ψ_w (−1.2 MPa) treatment. Box encompasses the 25–75 percentiles with the solid line indicating the median and dashed line the mean proline content of all accessions. Whiskers show the 10–90 percentiles and dots indicate outliers. Proline data for each accession is listed in Dataset S1. (B) Percentage of exon 3-skip P5CS1 mRNA as related to size of P5CS1 intron 2 insertion. Percentage of exon 3-skip P5CS1 was determined for seedlings exposed to −1.2 MPa for 96 h. The intron 2 insertion size was estimated by PCR and gel electrophoresis. Presentation of data are as described in A. n = 129 for the −2 to +4 insertion size and n = 9 for +6 to +8. Exon 3-skip percentage and intron 2 insertion size for each accession are listed in Dataset S1. (C) P5CS1 intron 2 and 3 sequences of selected accessions having varying intron 2 insertion size and exon 3-skip P5CS1 percentage. Complete intron 2 and 3 sequence alignments can be seen in Dataset S2. (D) Exon 3-skip P5CS1 and P5CS1 protein level of selected accessions. (Upper) RT-PCR using primers a and c (Fig. 2A). (Lower) Western blot detection of P5CS1 and tubulin (loading control). (E) Relationship of Pro accumulation at −1.2 MPa to percentage of exon 3-skip P5CS1 mRNA. Triangles indicate accessions where the exon 3-skip P5CS1 mRNA could not be detected (N.D., not detected).

Fig. 4.

Association of exon 3-skip P5CS1 percentage and proline content with climatic factors. (A) Negative association of exon 3-skip P5CS1 percentage with the PC2 of climate (SI Materials and Methods and Dataset S3). Accessions where exon 3-skip P5CS1 could not be detected are shown as squares. Only accessions with SNP data and reliable collection locations (SI Materials and Methods) are included (n = 76). (B) Positive association of proline content at low ψ_w with climate PC2. (C) Increased frequency of exon 3-skip P5CS1 in accessions from more variable temperature environments. Mean diurnal temperature range was the climate variable most strongly associated with exon 3-skip P5CS1 percentage (Dataset S4). (D) Proline accumulation had an opposite, but weaker, correlation to mean diurnal temperature range and an overall weaker association with climate variables (Dataset S5).