Functional analysis of the group 4 late embryogenesis abundant proteins reveals their relevance in the adaptive response during water deficit in Arabidopsis

Abstract

Late-Embryogenesis Abundant (LEA) proteins accumulate to high levels during the last stages of seed development, when desiccation tolerance is acquired, and in vegetative and reproductive tissues under water deficit, leading to the hypothesis that these proteins play a role in the adaptation of plants to this stress condition. In this work, we obtained the accumulation patterns of the Arabidopsis (Arabidopsis thaliana) group 4 LEA proteins during different developmental stages and plant organs in response to water deficit. We demonstrate that overexpression of a representative member of this group of proteins confers tolerance to severe drought in Arabidopsis plants. Moreover, we show that deficiency of LEA proteins in this group leads to susceptible phenotypes upon water limitation, during germination, or in mature plants after recovery from severe dehydration. Upon recovery from this stress condition, mutant plants showed a reduced number of floral and axillary buds when compared with wild-type plants. The lack of these proteins also correlates with a reduced seed production under optimal irrigation, supporting a role in fruit and/or seed development. A bioinformatic analysis of group 4 LEA proteins from many plant genera showed that there are two subgroups, originated through ancient gene duplication and a subsequent functional specialization. This study represents, to our knowledge, the first genetic evidence showing that one of the LEA protein groups is directly involved in the adaptive response of higher plants to water deficit, and it provides data indicating that the function of these proteins is not redundant to that of the other LEA proteins.

Figure 1.

Transcript and protein accumulation patterns of the AtLEA4 gene family during embryogenesis, germination, and seedling establishment. A, Semiquantitative RT-PCR analysis of AtLEA4 transcripts using total RNA (2 μg) from flowers and buds, developing siliques, seeds, and seedlings. ACTIN2 (ACT2) transcript was used as a loading control. B, Western-blot analysis using specific antibodies against each of the AtLEA4 proteins and protein extracts (15 μg) from the same stages of development described in A. Molecular mass markers are indicated (MW). Arrowheads indicate higher molecular mass bands, which were recognized specifically by the corresponding antibodies. Reversible stain with Ponceau red after transfer is shown as a loading reference. These results are representative of five independent experiments.

Figure 2.

Transcript and protein expression patterns of the AtLEA4 gene family from seedlings after 12-h treatments with ABA or abiotic stress. A, Northern-blot analysis using specific probes for each AtLEA4 transcript and total RNA (20 μg) from 2-week-old plants grown in vitro. Hybridization with 28S rRNA was used as a loading control. B, Western-blot analysis using specific antibodies for each AtLEA4 protein and total extracts (15 μg) from the same treatments as in A. Molecular mass markers are indicated (MW), and arrowheads show higher molecular mass bands, which were specifically recognized by the corresponding antibodies. Reversible stain with Ponceau red was used as a loading reference. These results are representative of three independent experiments.

Figure 3.

Phenotypic analysis of 35S::AtLEA4-5::NOS adult plants under dehydration and after recovery from drought. A, Western-blot analysis using antibodies against AtLEA4-5 and total protein extracts (15 μg) from the wild type (WT) and homozygous independent transgenic lines (lines 2–4, 6, and 7) under optimum irrigation. B, Western blot as in A using drought-treated plants in the adult stage. Molecular mass markers (MW) are indicated. Reversible stain after transfer with Ponceau red is shown as a loading reference. C, RWC of adult plants under irrigation or subjected to drought. One-way ANOVA showed significant differences between groups for drought-treated plants (P = 0.0065). Bars indicate means ± se (n = 3). D, Whole plant biomass under irrigation or after 10 d of recovery from severe drought. Significant differences were found between groups in the plant biomass recovered after stress (P = 0.0007). Bars indicate means ± se (n = 3). E, Number of floral and axillary buds per plant under irrigation (n = 3) and after recovery from drought (n = 9). Bars indicate means ± se. Significant differences between groups were found under irrigation (P < 0.0001) and after recovery from stress (P = 0.0033). Different letters above each bar indicate statistically significant differences using Dunnett’s posttests (P < 0.05).

Figure 4.

Reduction in the expression levels of AtLEA4-5 transcript and its corresponding protein in the transposon insertion mutant (dSpm). A, Northern-blot analysis using a specific probe for AtLEA4-5 and total RNA (10 μg) from dry seeds of wild-type (Wt) and dSpm mutant plants. Reversible stain with methylene blue after transfer was used as a loading reference. B, Western-blot analysis using specific antibodies against AtLEA4-5 and total protein extracts (10 μg) from dry seeds of wild-type and dSpm mutant plants. C, Western-blot analysis using protein extracts from roots under dehydration (5 μg) of wild-type and dSpm mutant plants. D, Western-blot analysis using total protein extracts (10 μg) from adult plants under dehydration, showing wild-type, homozygous transgenic lines with constitutive expression of AtLEA4-5 protein in the wild-type background (OE), transposon insertion in the AtLEA4-5 gene (dSpm), and its complementation with a 35S::AtLEA4-5::NOS construct (Comp). For B to D, reversible staining with Ponceau red after transfer was used as a loading reference.

Figure 5.

Accumulated germination percentage of the wild type (WT) and AtLEA4 transgenic lines under optimal growth conditions or under stress. Germination was quantified by radicle emergence using seeds of homozygous lines plated on standard MS medium (control [A]), MS medium with 0.35 m mannitol (osmotic stress [B]), or MS medium with 0.25 m NaCl (ionic + osmotic stress [C]). Transgenic lines used were the 35S::AtLEA4-5::NOS construct in the wild-type background (OE 4-5) and the 35S::AtLEA4-5::NOS construct in the dSpm mutant background (dSpm Compl). Also, insertion mutant in the AtLEA4-5 gene (dSpm 4-5) and double mutant in AtLEA4-1 and AtLEA4-2 genes silenced with an a-miR construct (miR 4-1/2) were analyzed. Seeds were stratified for 3 d and incubated in a growth chamber at 25°C for the indicated times. Error bars indicate se of three replicates (n = 300), which were fit to a sigmoidal dose-response curve. Significant differences between genotypes were found in three parameters of the curve fit (steepness of the curve, Y value at the top plateau, and X value when the response is halfway between bottom and top) at P > 0.0011 (A), P < 0.0001 (B), and P < 0.0001 (C). [See online article for color version of this figure.]

Figure 6.

Phenotypic analysis of adult plants with altered accumulation levels of the AtLEA4 protein family. Seedlings grown in vitro for 2 weeks were transplanted to a low-water-retention substrate and kept under optimum irrigation with nutrient solution until flowering under greenhouse conditions. Wild type (WT) and homozygous lines were grown in the same pot. Dehydration was followed by loss of water from the substrate, and pots were rotated in the tray every 2 d to maintain uniform water loss during drought treatment. A, Biomass of whole plants under control conditions (well-irrigated plants) or after 6 d of recovery from stress. One-way ANOVA was applied for each treatment to compare the performance of the different lines. This analysis showed significant differences between lines after recovery from drought (P < 0.0001). Bars indicate means ± se (n = 8). B, Number of axillary and floral buds per plant under optimum irrigation (n = 4) or after 6 d of recovery from stress (n = 8). Significant differences between groups were found using one-way ANOVA under control conditions (well-irrigated plants; P = 0.0095) and after recovery from stress (P < 0.0001). Bars indicate means ± se. Different letters show significant differences between bars (P < 0.05) as indicated by Tukey’s posttests. Homozygous lines were used in all experiments: transposon insertion in the AtLEA4-5 gene (dSpm 4-5), posttranscriptional gene silencing single mutant with RNAi-directed silencing of the AtLEA4-5 gene (RNAi 4-5), posttranscriptional gene silencing double mutants in AtLEA4-1 and AtLEA4-2 genes using an a-miR (a-miR 4-1/2), and the resulting F2 crosses from mutants of RNAi with a-miR (triple mutant). This figure also shows data from lines ectopically overexpressing AtLEA4-5 protein (35S::AtLEA4-5::NOS) in the wild type (OE 4-5) or in the AtLEA4-5::dSpm mutant background (Compl dSpm). The numbers in parentheses indicate the homozygous line used for the phenotypic analysis as shown in Figures 4 and 7.

Figure 7.

Posttranscriptional gene silencing of AtLEA4 genes using an a-miR (a-miR 4-1/2) to silence AtLEA4-1 and AtLEA4-2 or RNAi to silence AtLEA4-5 transcripts (RNAi 4-5). A, Northern blot using antisense probe of a-miR 4-1/2 and RNA (20 μg) from homozygous transgenic seedlings grown under optimal conditions to confirm the constitutive expression of mature a-miR 4-1/2. Wild type (Wt) and RNAi 4-5 lines (lanes 3–5) were used as controls. Reversible staining with methylene blue after transfer was used as a loading reference. B, Western blot using specific antibodies for AtLEA4-1 and AtLEA4-2 proteins and total protein extracts (10 μg) from adult plants grown under drought to show their accumulation levels in the wild type and in homozygous silenced plants (lanes 2–4). F, Flower; L, leaf; R, root. The arrow shows the proteins migrating with the expected molecular mass (MW) for the corresponding monomer size, and the arrowhead shows the higher molecular mass band specifically detected with AtLEA4-2 antibodies. The selected lines for further phenotypic analysis were lines 3 and 4. C, Western blot using antibodies against AtLEA4-5 protein and total protein extracts (10 μg) from homozygous RNAi 4-5 seedlings (lanes 1–5) grown in vitro for 2 weeks and immersed in liquid MS medium, where they were treated for 8 h without (C, for control) or with 25% PEG solution (S, for stress), showing different silencing levels. The selected lines for further phenotypic analyses were those showing the lower AtLEA4-5 protein accumulation (lanes 3–5). Reversible staining with Ponceau red after transfer was used as a loading reference in B and C.

Figure 8.

Total seed production of AtLEA4 single (RNAi 4-5, dSpm 4-5), double (a-miR 4-1/2), and triple mutants grown under optimal irrigation. Plants were germinated in vitro and transplanted to develop and set seeds under optimum irrigation conditions. Seeds from the wild type (WT) and two independent homozygous lines from each construct used in this study were harvested throughout the productive cycle until senescence. The transgenic lines overexpressing the AtLEA4-5 gene in the dSpm mutant (dSpm 4-5) and wild-type backgrounds (OE 4-5) are shown as controls. Bars indicate means ± se (n = 5). The numbers in parentheses show the lines selected from each construct (as indicated in Figs. 4 and 7). Significant differences among genotypes were determined by one-way ANOVA (P < 0.0001) statistical analysis. Different letters show significant differences between groups as indicated by Dunnett’s posttests (P < 0.05).

Figure 9.

Consensus phylogenetic tree of LEA4 protein sequences from plants. The phylogenetic tree was obtained from analyses with several programs from the Phylip suite of phylogenetic programs. Bootstrapped data sets were obtained with Seqboot using 100 repetitions. The reconstruction from 74 proteins and translated EST sequences showed two conserved subgroups that diverged before the appearance of vascular plants. AtLEA4-1 and AtLEA4-2 (black arrowheads) belong to subgroup 4A, whereas AtLEA4-5 (white arrowhead) belongs to subgroup 4B. The numbers (1–0) next to each taxon indicate the presence of motifs, but their arrangement within the protein sequence is shown in Supplemental Table S3, where motif 10 corresponds to “0” in this figure.