The Arabidopsis AtRaptor genes are essential for post-embryonic plant growth

Abstract

Background: Flowering plant development is wholly reliant on growth from meristems, which contain totipotent cells that give rise to all post-embryonic organs in the plant. Plants are uniquely able to alter their development throughout their lifespan through the generation of new organs in response to external signals. To identify genes that regulate meristem-based growth, we considered homologues of Raptor proteins, which regulate cell growth in response to nutrients in yeast and metazoans as part of a signaling complex with the target of rapamycin (TOR) kinase.

Results: We identified AtRaptor1A and AtRaptor1B, two loci predicted to encode Raptor proteins in Arabidopsis. Disruption of AtRaptor1B yields plants with a wide range of developmental defects: roots are thick and grow slowly, leaf initiation and bolting are delayed and the shoot inflorescence shows reduced apical dominance. AtRaptor1A AtRaptor1B double mutants show normal embryonic development but are unable to maintain post-embryonic meristem-driven growth. AtRaptor transcripts accumulate in dividing and expanding cells and tissues.

Conclusion: The data implicate the TOR signaling pathway, a major regulator of cell growth in yeast and metazoans, in the maintenance of growth from the shoot apical meristem in plants. These results provide insights into the ways in which TOR/Raptor signaling has been adapted to regulate plant growth and development, and indicate that in plants, as in other eukaryotes, there is some Raptor-independent TOR activity.

Figure 1

Raptor proteins in eukaryotes are highly conserved. (A) Similarity plot of Raptor homologues from the vascular plants Arabidopsis, Medicago truncatula and Oryza sativa, the fungus S. pombe (Mip1p), and mammals. The X-axis represents residue number; the Y-axis represents percent identity at that residue from 0% (0) to 100% (1). (B) Schematic diagram showing the position of the Raptor N-terminal Conserved / putative Caspase domain (RNC/C) region, HEAT repeats (H), and WD-40 repeats (WDx7) common to all Raptor proteins. (C) Phylogeny of plant, animal and fungal Raptor proteins. Bootstrap values, calculated using both parsimony (left) and maximum likelihood (right) are shown to the left of the clades they describe. The two Arabidopsis Raptor proteins, AtRaptor1A and AtRaptor1B, resolve as a single clade with 100% confidence. The alignment was generated using Megalign (DNAStar), the similarity plot was generated from this alignment using VectorNTI, and bootstrap values were calculated using PAUP*4.0b.

Figure 2

AtRaptor loci and insertion allele characterization. (A) AtRaptor1A and AtRaptor1B loci. Genomic sequence is depicted as a thin central line. Thick blocks indicate exons. Coding exons span the central line; exons encoding untranslated regions are fully below the central line. The positions of the T-DNA insertions are depicted with inverted triangles. (B) Reverse-Transcribed RNA-template Polymerase Chain Reactions (RT-PCR) on plants homozygous for both wild-type AtRaptor alleles (Col), the AtRaptor1A insertion allele (A-) or the AtRaptor1B insertion allele (B-), using primers spanning the AtRaptor1A insertion site, the AtRaptor1B insertion site, or control primers. Both AtRaptor insertion alleles abolish accumulation of the wild-type transcript from their locus.

Figure 3

Seedling root phenotype of AtRaptor1B-/- (B-) mutants. (A), (B). Col and B- seedlings on growth medium, four days after germination. The B- root has not penetrated the medium and is thick, hairy and coiled. (C) Col, A- and B- seedlings at 8 days after germination in light. Scale bar is in mm. (D) Same genotypes and age as C, grown in the dark. (E) Measurements of populations grown as in C, D. Root length is indicated in tan; shoot length is dark green. (F) B- and Col seedlings grown on vertical plates for 12 days, and then returned to horizontal growth for three days. B- roots are thin, straight and hairless on vertical plates (compare to 3B), and revert to coiled growth only in tissue generated after being placed horizontally. (G) Quantification of results in (F). B- seedlings grown on vertical plates are intermediate in length between flat-grown B- seedlings and Col seedlings. (H), (I) Col and B- root tips, viewed under bright field microscopy. Scale bar = 100 μm. B- root tips contain all the cell types seen in Col root tips, but the overall morphology is blunt and rounded compared to Col.

Figure 4

AtRaptor1B-/- plants grow slowly. (A), (B) Col and B- plants at 15 days after germination on soil. (C) B- plants bolt later than Col or A-. Shown are shoots from plants 1 month after germination. (D) Growth curve of Col, A- and B- plants. The X-axis represents time after production of the first leaf. The Y-axis represents the number of rosette leaves up to 11; presence of a floral bud is 12; number of cauline leaves plus 12 is 13–16, and values above 16 are the number of shoot apices harboring flowers plus 15. B- plants show slower leaf initiation, later bolting (though at a similar rosette leaf number as Col and A-) and later flowering.

Figure 5

AtRaptor1B-/- plants show altered shoot architecture. (A) B- plant at flowering. The primary shoot apex, center, has ceased growth and is surpassed by axillary branches. Compare to Col, A- in 3C. (B) Col, A- and B- primary shoot length. (C) Mature B- plant, showing a bushy phenotype due to decreased primary shoot growth and increased branching. (D) Shoot apex of the plant in C, magnified 4.5×. Axillary branches outgrow the primary shoot, which arrests in a whorl of sterile flowers. (E) Col, A- and B- cauline and rosette branch number. (F) Col, A- and B- cauline and rosette branch length. B- primary shoots are smaller than Col or A-, and secondary shoots initiate more frequently than Col or A- but are not significantly longer than Col or A-.

Figure 6

AtRaptor accumulation pattern. (A) AtRaptor transcripts accumulate throughout the floral shoot apex, stem and differentiating floral buds. Accumulation is not confined to dividing or meristematic cells, but fades in intensity away from the apex. (B) Adjacent tissue slice, probed with actin. AtRaptor and actin transcript accumulation patterns differ. (C) In silico analysis of AtRaptor1A (left) and AtRaptor1B (right) accumulation from 1434 developmental gene chip experiments. Results are given by developmental stage (X-axis) and in terms of gene chip-normalized expression levels (Y-axis). Expression levels are shown to scale. Developmental stages are as follows: 1, 1.0–5.9 days; 2, 6.0–13.9 days; 3, 14.0–17.9 days; 4, 18.0–20.9 days; 5, 21.0–24.9 days; 6, 25.0–28.9 days; 7, 29.0–35.9 days; 8, 36.0–44.9 days; 9, 45.0–50.0 days. Analyses performed via the genevestigator website .

Figure 7

AtRaptor1A-/- 1B-/- double mutants. (A) Col, A-, B- and A-B- seedlings at seven days on growth medium with no sucrose. (B) 1A-/- 1B+/- progeny germinated on growth medium supplemented with 0%, 1% or 6% sucrose. Shown for each treatment is an A-B- seedling and an A-/- B+ sibling. A-B- seedlings on 1% sucrose show significant root growth and minimal leaf buds. Scale bar for A, B = 5 mm. (C) Quantification of results in (B). (D) A-B- root tips grown on 1% sucrose lack an epidermal cell layer. (E) A-B- roots form root hairs on 1% sucrose. Scale bar = 100 μm. Compare to 3H, I. (F, G, H, I) Scanning electron microscopy on Col, A-B- shoot apices from growth medium plates. As in (B), A-B- seedlings show minimal (SAM) activity. Primordia for leaves 1 and 2 form but do not expand significantly. Scale bar = 50 μm (F, G) or 15 μm (H, I).

Figure 8

TOR functions in two complexes in eukaryotes. (A) TOR participates in two complexes in yeast and mammals. The first of these, TORC1, regulates cell growth in response to nutrient and hormonal signals. Raptor is integral for TORC1 activity. The second of these, TORC2, regulates cytoskeletal organization. Its activity is nutrient-independent, and Raptor is not a component of TORC2. (B) Model of TOR function in plants. Embryonic development is indicated by the single horizontal arrow from zygote to seedling; meristem-driven post-embryonic development is indicated by the arrows emanating from the seedling root and shoot apices. TOR, acting independent of Raptor in a putative complex homologous to yeast and mammalian TORC2, is essential for embryonic development. TOR via TORC2 may play a role in post-embryonic development as well; the embryonic lethal AtTOR knockout phenotype precludes a definitive answer on this point. Raptor activity, in a putative plant homologue of TORC1, is dispensable for embryonic development but is essential for meristem-driven post-embryonic growth.