An RNA-Seq transcriptome analysis of orthophosphate-deficient white lupin reveals novel insights into phosphorus acclimation in plants

Abstract

Phosphorus, in its orthophosphate form (P(i)), is one of the most limiting macronutrients in soils for plant growth and development. However, the whole-genome molecular mechanisms contributing to plant acclimation to P(i) deficiency remain largely unknown. White lupin (Lupinus albus) has evolved unique adaptations for growth in P(i)-deficient soils, including the development of cluster roots to increase root surface area. In this study, we utilized RNA-Seq technology to assess global gene expression in white lupin cluster roots, normal roots, and leaves in response to P(i) supply. We de novo assembled 277,224,180 Illumina reads from 12 complementary DNA libraries to build what is to our knowledge the first white lupin gene index (LAGI 1.0). This index contains 125,821 unique sequences with an average length of 1,155 bp. Of these sequences, 50,734 were transcriptionally active (reads per kilobase per million reads ≥ 3), representing approximately 7.8% of the white lupin genome, using the predicted genome size of Lupinus angustifolius as a reference. We identified a total of 2,128 sequences differentially expressed in response to P(i) deficiency with a 2-fold or greater change and P ≤ 0.05. Twelve sequences were consistently differentially expressed due to P(i) deficiency stress in three species, Arabidopsis (Arabidopsis thaliana), potato (Solanum tuberosum), and white lupin, making them ideal candidates to monitor the P(i) status of plants. Additionally, classic physiological experiments were coupled with RNA-Seq data to examine the role of cytokinin and gibberellic acid in P(i) deficiency-induced cluster root development. This global gene expression analysis provides new insights into the biochemical and molecular mechanisms involved in the acclimation to P(i) deficiency.

Figure 1.

Transcripts differentially expressed due to P_i. A total of 2,128 transcripts were identified as differentially expressed between P_i-sufficient and P_i-deficient tissues. To be considered differentially expressed, the transcript must have RPKM ≥ 3 in at least one tissue, 2-fold or greater change between tissues, and P ≤ 0.05. A, A total of 1,342 transcripts differentially expressed due to P_i in leaves. B, A total of 904 transcripts differentially expressed due to P_i in roots. C, Heat map of Z scores (number of sd) illustrating expression profiles of 2,128 transcripts differentially expressed due to P_i. Red indicates high expression, yellow indicates intermediate expression, and white indicates low expression. See also Supplemental Tables S1 and S2.

Figure 2.

Adjustments in root metabolism promote acclimation to P_i deficiency. Shown are modifications in white lupin cluster root metabolism that facilitate acclimation to P_i deficiency as evidenced by transcript expression. Increased expression in PdCR was confirmed by qPCR as indicated by fold change in parentheses. Red arrows represent genes known to be up-regulated due to P_i deficiency. Increased Suc metabolism via glycolysis and organic acid production provide carbon for malate and citrate exudation into the rhizosphere. Organic acids lost through exudation are replenished through anaplerotic pathways involving phosphoenolpyruvate carboxylase (PEPC) and a glyoxylate-like cycle malate synthase (MS). One-carbon metabolism is enhanced through a formate and THF pathway. The THF pathway contributes to Met and ethylene production. Increased expression of transcripts involved in phospholipid degradation releases P_i for recycling and carbon for acetyl-CoA synthesis. Acetyl-CoA and glyoxylate provide carbon for malate synthesis through malate synthase. Formate may be carboxylated to glyoxylate by a putative glyoxylate synthase (Glox-S). Additional abbreviations are as follows: Suc synthase (SS); phosphoenolpyruvate carboxylase kinase (PEPCK); phosphoenolpyruvate carboxylase carboxykinase (PEPCRK); malate dehydrogenase (MDH); citrate synthase (CS); formamidase (FORM); formate dehydrogenase (FDH); Ala glyoxylate transaminase (AGTA); THF deformylase (THFD); methylene-THF reductase (MTHFR); S-adenosyl-Met synthase (SAM Synth); aminocyclopropane synthase (ACC Synth); aminocyclopropane oxidase (ACC Oxidase); phospholipase A (PLA1); glycerophosphodiester-phosphodiesterase (GPX-PDE); acid phosphatase (AP); monogalactosyldiacylglycerol synthase (MGDG synthase); sulfoquinovosyltransferase (SQD).

Figure 3.

Heat map of expression profiles of transcripts involved in acclimation to P_i deficiency. Expression, represented by Z scores, is shown for selected transcripts differentially expressed due to P_i deficiency. To be considered differentially expressed, the transcript must have RPKM ≥ 3 in at least one tissue, 2-fold or greater change between tissues, and P ≤ 0.05. Red indicates high expression, yellow indicates intermediate expression, and white indicates low expression. Transcripts have been grouped by function to clarify the role of each group in the P_i deficiency response. For more details, see Supplemental Table S8.

Figure 4.

CK application impairs P_i stress-induced cluster root formation. All root samples were collected at 14 DAE of shoots from quartz/sand growth medium. CK-treated plants were given 10⁻⁶ or 10⁻⁷ m BA at 3, 6, and 8 DAE. A, P_i-deficient control plant. Bar = 10 cm. B, P_i-deficient plant treated with 10⁻⁶ m BA. Note the reduction in cluster roots on the BA-treated plant. C, BA application reduces the percentage of root weight of cluster roots from 20% in P_i-deficient control plants (b) to approximately 10% (±se), comparable to those of P_i-sufficient plants (a). Columns with the same lowercase letter are not significantly different (P = 0.05).

Figure 5.

Effects of GA₃ and Paclo on white lupin cluster root development. All root samples were collected at 14 DAE of shoots from quartz/sand growth medium. GA-treated plants were treated with 10⁻⁶ m GA at 3, 6, 9, and 12 DAE. Paclo-treated plants were given 1 mg of Paclo (per each 6-L pot) at 5 DAE. A and B, Roots of P_i-sufficient and P_i-deficient control plants. C and D, Roots of P_i-sufficient and P_i-deficient plants treated with 10⁻⁶ m GA. E and F, Roots of P_i-sufficient and P_i-deficient plants treated with Paclo. Bars = 5 cm. G, Mean number of cluster roots per plant (±se). Note that Paclo-treated P_i-sufficient plants have increased cluster root formation mimicking that of P_i-deficient plants. GA had no effect on mean cluster root number but does appear to decrease rootlet density (Supplemental Fig. S7). Columns with the same lowercase letter are not significantly different (P = 0.05).

Figure 6.

Candidate genes for use as P_i deficiency indicators. The heat map illustrates the expression pattern, represented by Z scores, in our lupin RNA-Seq data of sequences identified as differentially expressed in multiple microarray experiments and lupin RNA-Seq data due to P_i deficiency. Red indicates high expression, yellow indicates intermediate expression, and white indicates low expression. All transcripts, except the ferritin sequences, are up-regulated in PdCR. Transcripts 47701, 31197, 79787, 35670, and multiple ferritin sequences are up-regulated in PdL.