Ectopic maltase alleviates dwarf phenotype and improves plant frost tolerance of maltose transporter mutants

Abstract

Maltose, the major product of starch breakdown in Arabidopsis (Arabidopsis thaliana) leaves, exits the chloroplast via the maltose exporter1 MEX1. Consequently, mex1 loss-of-function plants exhibit substantial maltose accumulation, a starch-excess phenotype and a specific chlorotic phenotype during leaf development. Here, we investigated whether the introduction of an alternative metabolic route could suppress the marked developmental defects typical for mex1 loss-of-function mutants. To this end, we ectopically expressed in mex1 chloroplasts a functional maltase (MAL) from baker's yeast (Saccharomyces cerevisiae, chloroplastidial MAL [cpMAL] mutants). Remarkably, the stromal MAL activity substantially alleviates most phenotypic peculiarities typical for mex1 plants. However, the cpMAL lines contained only slightly less maltose than parental mex1 plants and their starch levels were, surprisingly, even higher. These findings point to a threshold level of maltose responsible for the marked developmental defects in mex1. While growth and flowering time were only slightly retarded, cpMAL lines exhibited a substantially improved frost tolerance, when compared to wild-types. In summary, these results demonstrate the possibility to bypass the MEX1 transporter, allow us to differentiate between possible starch-excess and maltose-excess responses, and demonstrate that stromal maltose accumulation prevents frost defects. The latter insight may be instrumental for the development of crop plants with improved frost tolerance.

Figure 1

The NTT1 N-terminus targets yeast MAL to the chloroplast. Transient expression of the hybrid construct with C-terminally fused GFP in Arabidopsis protoplasts. The GFP fluorescence accumulates in close proximity to the chlorophyll autofluorescence, which is indicative for a stromal localization. (A) GFP fluorescence. (B) chlorophyll autofluorescence. (C) merge of A and B. (D) greyscale. Scale bars represent 10 µm.

Figure 2

Identification of cpMAL expressing lines and determination of MAL activity. (A) cpMAL mRNA accumulation in mex1 (control) and 24 individual cpMAL expressing lines. (B) MAL activity in extracts prepared from mex1 and the cpMAL lines 9 and 16, respectively (given in units, U = 1 µmole/min). Three technical replicates were used for RT-qPCR; expression levels are given as ct-values normalized to the two chosen reference genes. Five biological replicates were used for the enzyme activity test. Standard errors are displayed and significance of differences between MAL activity in mex1 and in the two selected cpMAL lines were analyzed by student t test: ***P-value <0.001.

Figure 3

Plastidic MAL activity alleviates the dwarf phenotype of 4 weeks old mex1 mutants. (A) Rosette phenotype of representative WT (Col-0), mex1 and _cpMAL overexpressor plants (cpMAL9 and cpMAL16). Mean rosette diameter (B), leaf number (C) and fresh weight (D) of 20 plants per line. Standard errors are given. Rosettes have been digitally extracted for comparison, scale bar represents 1 cm. Significance of differences between the growth of mex1 and the remaining plant lines was analyzed by Student t test: ***P-value <0.001.

Figure 4

Plastidic MAL activity alleviates photosynthetic defects of the mex1 mutant. (A) representative images of minimum fluorescence (F₀) and maximum quantum yield (F_v/Fm) of rosette leaves from Arabidopsis wild-type (Col-0), mex1, and cpMAL lines 9 and 16. (B) values of the photosynthetic parameter F_v/F_m. (C) values of the photosynthetic parameter non-photosynthetic quenching (NPQ). Plants were grown for 4 weeks under standard light intensity (125 µmol photons m⁻²s⁻¹). Data represent the mean values of ten leaves from at least six biological replicates. Standard errors are given. Scale bars represent 1 cm. Statistical analysis of differences between wild-type and mutant levels was performed with one-tailed t test: *P ≤ 0.05; **P ≤ 0.01 and ***P ≤ 0.001.

Figure 5

Diurnal levels of selected carbohydrates in wild-type, mex1, and cpMAL plants. (A) maltose levels; (B) starch levels; (C) glucose levels; (D) fructose levels; (E) sucrose levels. Plants were grown for 4 weeks under standard light intensity (125 µmol photons m⁻²s⁻¹). Each data point represents mean values of ten leaves taken from at least four biological replicated plants. Standard errors are given.

Figure 6

Analysis of inflorescence development of wild-type and mutant plants. Determination of differences in the onset of bolting. Plants were cultivated for 4 weeks under standard conditions (120 µE, 10 h light/14 h dark) and transferred to LD conditions (250 µE, 16 h light/8 h dark) for flowering induction. When its shoot length reached 1 cm, the corresponding plant was defined as bolted. Horizontal arrows indicate when plants reached 100% bolting, vertical arrows indicate day of harvest. Data represent the mean ± SEM of three replicates with ≥8 plants each.

Figure 7

Analysis of the freezing tolerance of wild-type and mutant plants. (A) Representative picture of a set of wild-type and mutant plants after 3 weeks’ recovery from −10°C freezing. (B) quantification of the survival rate. Scale bar represents 1 cm. Statistics: n = 12 sets (of at least eight plants per line), data represent the mean ± SEM: *P ≤ 0.05; **P ≤ 0.01 and ***P ≤ 0.001, estimated by student’s t test.