Mutations in the Arabidopsis homolog of LST8/GβL, a partner of the target of Rapamycin kinase, impair plant growth, flowering, and metabolic adaptation to long days

Abstract

The conserved Target of Rapamycin (TOR) kinase forms high molecular mass complexes and is a major regulator of cellular adaptations to environmental cues. The Lethal with Sec Thirteen 8/G protein β subunit-like (LST8/GβL) protein is a member of the TOR complexes, and two putative LST8 genes are present in Arabidopsis thaliana, of which only one (LST8-1) is significantly expressed. The Arabidopsis LST8-1 protein is able to complement yeast lst8 mutations and interacts with the TOR kinase. Mutations in the LST8-1 gene resulted in reduced vegetative growth and apical dominance with abnormal development of flowers. Mutant plants were also highly sensitive to long days and accumulated, like TOR RNA interference lines, higher amounts of starch and amino acids, including proline and glutamine, while showing reduced concentrations of inositol and raffinose. Accordingly, transcriptomic and enzymatic analyses revealed a higher expression of genes involved in nitrate assimilation when lst8-1 mutants were shifted to long days. The transcriptome of lst8-1 mutants in long days was found to share similarities with that of a myo-inositol 1 phosphate synthase mutant that is also sensitive to the extension of the light period. It thus appears that the LST8-1 protein has an important role in regulating amino acid accumulation and the synthesis of myo-inositol and raffinose during plant adaptation to long days.

Figure 1.

Distances between LST8 Protein Sequences. Tree showing the average distances based on sequence identities between plant and algae LST8 protein sequences using the COBALT multiple alignment tool (Papadopoulos and Agarwala, 2007). The alignment is available as Supplemental Data Set 1 online.

Figure 2.

Localization of a LST8-GFP Fusion Protein after Transient Transformation of Cotyledons from a RabC1-RFP–Expressing Arabidopsis Line. Each row is derived from a time-lapse series (5-s intervals). The arrows indicate mobile dots. Scale bar = 10 μm. (A) RFP-specific fluorescence from the RabC1-RFP construct that labels the endosomes. (B) GFP-specific fluorescence from the 35S:LST8-GFP construct. (C) RFP and GFP signals were merged.

Figure 3.

GUS Staining of Transformed Arabidopsis Plants Carrying a pLst8:GUS Construct Containing 1 kb of LST8-1 Promoter. Plantlet (A), primary root tip (B), emerging secondary root (C), aerial part (D), close-up on a leaf showing staining of stomatal guard cells (E), emerging leaves and stipules ([F], indicated by arrows), and flowers ([G] and [H]).

Figure 4.

Complementation of a Yeast lst8 Mutant with the Arabidopsis LST8-1 cDNA. A yeast lst8 mutant strain expressing the Saccharomyces cerevisiae LST8 cDNA under the control of an inducible Gal promoter was used for complementation studies. On a permissive, Gal-containing medium, the yeast lst8 mutant strain is able to grow (A), but on selective, Glc-containing medium, the yeast lst8 mutant strain containing an empty transformation vector fails to grow ([B], bottom part). The expression of the Arabidopsis LST8-1 cDNA in the yeast lst8 mutant strain fully restores the ability to grow on Glc medium ([B], top part). [See online article for color version of this figure.]

Figure 5.

Arabidopsis LST8-1 Interacts with the C-Terminal FRB-Kinase Domain of TOR. (A) Yeast two-hybrid assay with the TOR FRB-kinase domain as bait and the LST8-1 protein as prey. C1-, pADH::GAL4BD pADH::GAL4AD-LST8; C2-, pADH::GAL4BD-FRBK pADH::GAL4AD; C+, pADH::GAL4BD-HSD1pADH::GAL4AD-GAPC2; 1, 2, 3, independent double yeast transformants with pADH:GAL4BD-TOR/FRB and pADH:GAL4AD-LST8-1. (B) Split-luciferase assay in Arabidopsis cotyledons after transient expression. Relative light emission of the different split-luciferase protein pairs. Luciferase (LUC) activity was monitored with at least two independent infiltration experiments per tested interactions. The mean of the experiments is shown together with the corresponding sd values.

Figure 6.

Impact of T-DNA Insertions on Transcription of the LST8 Genes. (A) Analysis of LST8-1 and LST8-2 expression levels in lst8-1-1 and lst8-1-2 mutants by RT-PCR. The reference constitutive gene is EF1a (Elongation factor 1a). See Methods for details. WT, wild type. (B) Analysis of LST8-1 expression level in lst8-1-1 and lst8-1-2 mutants by quantitative real-time RT-PCR. Arbitrary units are calculated relative to the EF1a expression level. Values are the mean of at least three independent repetitions ± sd.

Figure 7.

Phenotype of the lst8-1 Insertion Mutants. (A) to (D) The control wild-type plants are on the left, and the lst8-1-1 mutant plants are on the right. (A) Plants cultivated in growth chambers for 4 weeks under SD conditions. (B) Plants grown in vitro for 7 d under LD conditions. (C) Plants cultivated in the greenhouse for 6 weeks under LD conditions (winter). (D) Plants cultivated for 4 weeks under SD conditions as in (A) followed by 1 week under LD conditions. (E) and (F) lst8-1-1 mutant grown in the greenhouse under LD conditions. lst8-1 mutants develop multiple meristems ([E], indicated by red arrows), become bushy, and produce several stems (F).

Figure 8.

Development of Multiple Meristems in lst8-1-1 Mutant Plants. Sections of the apical meristem zone were performed and observed after resin embedding and NBB staining (A) or paraffin embedding and Schiff reagent staining (B). Arrows indicate multiple meristems. [See online article for color version of this figure.]

Figure 9.

Influence of Long-Day Conditions on Nitrogen Assimilation in lst8 Mutants. Nitrate content (A), NiR (B), total NR (C), and Gln synthetase (GS; [D]) activities after transfer to LD conditions of wild-type and lst8-1-2 mutant plants. Plants were grown under controlled conditions. Values are the mean of at least three independent repetitions ± sd. The values for nitrate concentrations (A) have been fitted to a regression line, and the corresponding slope is indicated. Statistically different values between the wild type and lst8-1-2 are indicated by a star (Student’s t test). FW, fresh weight; LD+n, number of days under LD conditions.

Figure 10.

Diurnal Variations in Soluble Sugar and Starch Content during the Transition from SDs to LDs in the Wild Type and the lst8-1-2 Mutant. Plants were first grown under controlled SD conditions and harvested at the beginning (morning [m]) and end (night [n]) of the day preceding the shift to LD. Plants were again harvested at day 2 after the start of LD conditions. Results are mean of at least three different samples ± sd. Statistically different values between the wild type (WT) and lst8-1-2 are indicated by a star (Student’s t test). FW, fresh weight.

Figure 11.

Kinetic Analysis of Phloem Labeling Using CF Diacetate. CF was applied on cotyledons of plants grown in vitro under LDs. Fluorescence was recorded every 3 s. Magnification shows the labeling of conductive tissues inside the root. WT, wild type.

Figure 12.

Leaf Metabolite Contents after Transfer to LD Conditions of Wild-Type and lst8-1-2 Mutant Plants. Values are derived from normalized areas of specific peaks after GC-MS experiments (see Methods for details). Plants were grown in controlled growth chambers. Values are the means of three independent repetitions ± sd. Darker bars correspond to the mutant plants. LDn, number of days under LD conditions (16 h light); SD, 8 h light.

Figure 13.

Differentially Expressed Genes in the Transcriptomic Analysis of lst8-1 Mutants Using CATMA Arrays. For each condition, gene expression in the mutant samples was compared with that in wild-type samples grown under the same light regimes as references. 1, lst8-1-2 to wild type in SD; 2, lst8-1-2 to wild type in LD for 2 d; 3, wild type in LD to wild type in SD (reference); 4, lst8-1-2 in LD to lst8-1-2 in SD; 5 to 8, same as 1 to 4, except with the lst8-1-1 mutant. (A) Differentially expressed genes were ordered from the lowest to the highest ratio with the wild type LDs (LD after 2 d) to SDs comparison as reference (see Methods for the definition of differentially expressed genes). (B) Differentially expressed genes in the comparison between lst8-1-2 and wild-type grown under LD conditions, which were also differentially expressed in a mips1 mutant compared with wild-type plants grown in LD and in TOR ethanol-inducible RNAi lines induced by ethanol for 24 h (see Methods for details). Only genes that are found in common between at least two comparisons were retained for this analysis. Data were obtained from the CatDB database and from Meng et al. (2009).

Figure 14.

Differentially Expressed Genes in lst8-1 Mutants. Transcriptome comparisons were performed between leaves of lst8-1 mutants and wild-type plants grown either in SDs (8 h) or transferred to LDs (16 h) between wild-type plants grown in SDs and transferred to LDs and between mips1 mutants and the corresponding wild type in LDs, and between the TOR ethanol-inducible RNAi lines and the corresponding control line (mean of 6-3 and 5-2 RNAi lines induced for 24 h with ethanol). Genes showing opposite variations when compared with the wild type in SDs or LDs, or of special interest, were selected among differentially expressed transcripts in the lst8-1 mutants. The results are the mean of the intensity ratios for lst8-1-1 and lst8-1-2 mutants and are presented as log2 ratios. Experiments were run in duplicate. A color code was used to visualize the data.