EXORDIUM-LIKE1 promotes growth during low carbon availability in Arabidopsis

Abstract

Little is known about genes that control growth and development under low carbon (C) availability. The Arabidopsis (Arabidopsis thaliana) EXORDIUM-LIKE1 (EXL1) gene (At1g35140) was identified as a brassinosteroid-regulated gene in a previous study. We show here that the EXL1 protein is required for adaptation to C- and energy-limiting growth conditions. In-depth analysis of EXL1 transcript levels under various environmental conditions indicated that EXL1 expression is controlled by the C and energy status. Sugar starvation, extended night, and anoxia stress induced EXL1 gene expression. The C status also determined EXL1 protein levels. These results suggested that EXL1 is involved in the C-starvation response. Phenotypic changes of an exl1 loss-of-function mutant became evident only under corresponding experimental conditions. The mutant showed diminished biomass production in a short-day/low-light growth regime, impaired survival during extended night, and impaired survival of anoxia stress. Basic metabolic processes and signaling pathways are presumed to be barely impaired in exl1, because the mutant showed wild-type levels of major sugars, and transcript levels of only a few genes such as QUA-QUINE STARCH were altered. Our data suggest that EXL1 is part of a regulatory pathway that controls growth and development when C and energy supply is poor.

Figure 1.

EXL1 expression in response to C availability. A, Expression profiles from publicly available microarray data (Bläsing et al., 2005; Gibon et al., 2006; Osuna et al., 2007; Usadel et al., 2008). The data were normalized using RMA and log₂ transformed. The means of two or three replicates are shown. The se is shown if three replicates were available. Expression of EXO is shown for comparison. FN, Full nutrition; C-starv, C starvation. B, Quantitative RT-PCR analysis of EXL1 transcript levels in wild-type (Col-0) shoots. Plants were grown in soil under the low-total-irradiance regime (4 h of light [60 μmol m⁻² s⁻¹]/20 h of dark). RNA was extracted from shoots of 28-d-old plants. eIF1α cycle threshold values were subtracted from respective cycle threshold values of the gene of interest. Subsequently, differences were subtracted from an arbitrary value (i.e. 40). Higher numbers indicate higher transcript levels. A difference of 1 unit indicates a 2-fold change. Error bars indicate se of the gene of interest in three technical replicates. The data shown are from one experiment representative of three independent biological replicates. eN, End of night; eD, end of day. C, Quantitative RT-PCR analysis of EXL1 transcript levels in wild-type (Col-0) shoots. Plants were grown in soil under short-day conditions (8 h of light [140 μmol m⁻² s⁻¹]/16 h of dark). RNA was extracted from 35-d-old plants. Technical details are as given for B. [See online article for color version of this figure.]

Figure 2.

EXL1:HA expression in response to different Suc levels and light regimes. A, Relative EXL1:HA transcript levels in shoots. Plants carrying the pEXL1::EXL:HA and 35S::EXL1:HA constructs were grown in synthetic medium supplemented with 0.2%, 0.5%, 1%, 3%, and 5% (w/v) Suc. RNA was extracted from 14-d-old plants. Technical details are as given for Figure 1B. The data shown are from one experiment representative of three independent biological replicates. B, Relative EXL1:HA transcript levels in roots. RNA was extracted from the same plants as in A. C, Relative EXL1:HA transcript levels in shoots. Plants were grown in soil under light-limited conditions (4 h of light [60 μmol m⁻² s⁻¹]/20 h of dark) and subjected to extended night. RNA was extracted from 35-d-old plants. eD, End of day; eN, end of night. D, Relative EXL1:HA transcript levels in shoots. Plants were grown in soil under long-day conditions (16 h of light/8 h of dark) and subjected to extended night. RNA was extracted from 28-d-old plants.

Figure 3.

Western-blot analysis of EXL1:HA protein levels. Plants carrying the pEXL1::EXL:HA construct were grown under different conditions. The EXL1:HA fusion protein was detected using a monoclonal anti-HA antibody and an enhanced chemiluminescence detection system. Similar results were obtained for independent transgenic lines. A, Plants were grown in synthetic medium supplemented with 0.2%, 0.5%, 1%, 3%, and 5% (w/v) Suc. Protein was extracted from the same shoot material as used for transcript analysis (Fig. 2A). B, Plants were grown in long-day conditions as described in the legend of Figure 2D. eD, End of day; eN, end of night. C, Plants were grown under light-limited conditions as described in the legend of Figure 2C.

Figure 4.

Biomass production in response to Suc and BR. Plants were grown in synthetic medium supplemented with 0.2% or 1% (w/v) Suc and brassinosteroids (50 nm castasterone [CS] or 50 nm brassinolide [BL]). Fresh weight was determined after 16 d. Results are means ± se (n = 5 plates per genotype and treatment containing 10 plants each). Biomass of exl1 plants was significantly reduced in the control treatment with 0.2% Suc (denoted with an asterisk; t test, P < 0.01). Percentage differences in BR treatments with 0.2% Suc supply were significantly larger in exl1 in comparison with the wild type (t test, P < 0.05).

Figure 5.

Leaf growth in response to Suc and BR. Plants were grown in synthetic medium supplemented with 0.2% (w/v) Suc and brassinosteroids (50 nm brassinolide [BL] or 50 nm 24-epibrassinolide [EBL]). Length and width of primary leaves were determined after 16 d. Results are means ± se (n = 5 plates per genotype and treatment containing 10 plants each). Mutant values denoted with an asterisk are significantly different from those of their wild type (t test, P < 0.05). Percentage differences of leaf length in BR treatments were significantly larger in exl1 in comparison with the wild type (t test, P < 0.05).

Figure 6.

Dark development phenotypes. A, Seedlings were grown in half-concentrated MS medium supplemented with 0.05% or 0.15% (w/v) Suc for 3 weeks in the dark. Only the largest seedlings of a representative experiment are shown. B, Seedlings were grown in half-concentrated MS medium supplemented with 0.15% (w/v) Glc for 3 weeks in the dark. Development of plants was grouped into three categories: elongated hypocotyls with closed cotyledons (black bars), opened cotyledons (gray bars), and formation of true leaves (dark gray bars). The experiment was carried out independently three times. The observed responses from one representative experiment are shown.

Figure 7.

Phenotype of the exl1 mutant under growth-limiting light conditions. A, Wild-type, exl1, exl1-D, and complemented exl1 plants were raised for 2 weeks in standard conditions and then grown for 2 weeks under light-limited conditions (4 h of light [60 μmol s⁻¹ m⁻²]/20 h of dark). B, Fresh weight (FW) and dry weight (DW) of plants shown in A. Results are means ± se (n = 6 individuals per genotype). Mutant values denoted with an asterisk are significantly different from those of their wild type (t test, P < 0.01). [See online article for color version of this figure.]

Figure 8.

QQS transcript levels in wild-type and exl1 plants. A, Wild-type and exl1 plants were grown in soil under the low-total-irradiance regime (4 h of light [60 μmol m⁻² s⁻¹]/20 h of dark). For the wild type, the same cDNAs were used as for EXL1 analysis (Fig. 1B). eN, End of night; eD, end of day. B, QQS expression in a diurnal cycle (end of night to 16 h of dark) and in extended night in wild-type and exl1 plants. eN corresponds to 16 h of dark. The same wild-type cDNAs were used as for EXL1 analysis (Fig. 1C).

Figure 9.

Survival rates after extended night and anoxic stress. A, Plants were raised under short-day conditions (8 h of light/16 h of dark) and subjected to different periods of continuous darkness. After retransfer to a greenhouse with long-day conditions, plants were grown for 1 week prior to determining survival rates. Five independent experiments were analyzed. Results are mean survival rates ± se (n = 15 plants per genotype, time, and experiment). Mutant values denoted with an asterisk are significantly different from those of their wild type (t test, P < 0.01). B, Seven-day-old plants were subjected to anoxia for 8 and 10 h. After the treatment, plants were transferred to ambient air, and survival rates were determined after a 10-d recovery period. Results are mean survival rates ± se (n = 4 plates containing at least 50 seedlings each). Mutant values denoted with an asterisk are significantly different from those of their wild type (t test, P < 0.01).