Functional analysis of the Arabidopsis PAL gene family in plant growth, development, and response to environmental stress

Abstract

Phenylalanine ammonia-lyase (PAL) catalyzes the first step of the phenylpropanoid pathway, which produces precursors to a variety of important secondary metabolites. Arabidopsis (Arabidopsis thaliana) contains four PAL genes (PAL1-PAL4), but there has been no genetic analysis to assess the biological functions of the entire gene family. Here, we report the generation and analysis of combined mutations for the four Arabidopsis PAL genes. Contrary to a previous report, we found that three independent pal1 pal2 double mutants were fertile and generated yellow seeds due to the lack of condensed tannin pigments in the seed coat. The pal1 pal2 double mutants were also deficient in anthocyanin pigments in various plant tissues, which accumulate in wild-type plants under stress conditions. Thus, PAL1 and PAL2 have a redundant role in flavonoid biosynthesis. Furthermore, the pal1 pal2 double mutants were more sensitive to ultraviolet-B light but more tolerant to drought than wild-type plants. We have also generated two independent pal1 pal2 pal3 pal4 quadruple knockout mutants, which are stunted and sterile. The quadruple knockout mutants still contained about 10% of the wild-type PAL activity, which might result from one or more leaky pal mutant genes or from other unknown PAL genes. The quadruple mutants also accumulated substantially reduced levels of salicylic acid and displayed increased susceptibility to a virulent strain of the bacterial pathogen Pseudomonas syringae. These results provide further evidence for both distinct and overlapping roles of the Arabidopsis PAL genes in plant growth, development, and responses to environmental stresses.

Figure 1.

Structures and mutants for the PAL genes. A, Exon and intron structures of the four PAL genes. The exons are indicated with rectangles and the introns with lines. The locations of T-DNA or transposon insertions in the pal mutants are indicated. B, Expression of PAL genes in the wild type and pal mutants. Semiquantitative RT-PCR was performed with total RNA isolated from 10-week-old inflorescence stems of the wild type and pal single and quadruple mutants. The experiment was repeated twice with similar results.

Figure 2.

PAL activity in the wild type and pal mutants. Crude extracts were prepared from 10-week-old inflorescence stems of the wild type and indicated pal mutants, and PAL activity was determined with [¹⁴C]Phe and is expressed in pmol cinnamic acid (CA) s⁻¹ mg⁻¹ protein. Means and se were calculated from average PAL activities determined from three experiments with five plants per experiment for each genotype. According to Duncan's multiple range test (P = 0.05), means of PAL activities do not differ significantly if they are indicated with the same letter.

Figure 3.

Phenotypes of the pal1 pal2 double mutants. A, Lack of anthocyanin pigments in the pal1 pal2 double mutants. Wild-type and pal1 pal2 double mutant plants were grown in soil without fertilization at 19°C under a 12-h-light/12-h-dark photoperiod. The photograph was taken about 8 weeks after germination. B, Inflorescences and siliques of wild-type and pal1 pal2 double mutant plants. C, Seeds of wild-type and pal1 pal2 double mutant plants. D, Vanillin staining of immature seeds for detection of catechins and proanthocyanidins.

Figure 4.

Enhanced sensitivity of the pal1 pal2 double mutants to short-term UV-B light treatment. Four-week-old wild-type and pal1 pal2 double mutant plants were irradiated with 1.24 μmol m⁻² s⁻¹ UV-B light for 4 h. The photograph was taken 1 d after UV-B light treatment. The experiment was repeated twice with similar results.

Figure 5.

Enhanced sensitivity of the effects of UV-B light on the efficiency of PSII photochemistry and photoprotection between wild-type and double mutant plants. A, Images of F_v/F_m, Φ_PSII, q_P, and NPQ of plants before and after exposure to UV-B light at 1.24 μmol m⁻² s⁻¹ for 4 h. Chlorophyll fluorescence images were taken immediately after UV-B light treatment. The false color code depicted at the bottom of the images ranged from 0 (black) to 1.0 (purple). B, Average values for the respective chlorophyll fluorescence images. Data are means of five replicates (±sd). According to Duncan's multiple range test (P = 0.05), means of the photosynthetic parameters do not differ significantly before UV-B light exposure if they are indicated with the same lowercase letter and do not differ significantly after UV-B light exposure if they are indicated with the same uppercase letter.

Figure 6.

Enhanced drought tolerance of the pal1 pal2 mutants. Seven-week-old Arabidopsis plants (eight plants for each genotype) were moved into a walk-in growth chamber with approximately 50% humidity. The photograph of representative plants was taken 2 weeks after withholding watering. The experiment was repeated twice with similar results.

Figure 7.

Phenotypes of the pal1 pal2 pal3 pal4 quadruple mutants. A, Six-week-old wild-type and pal1 pal2 pal3 pal4 quadruple mutant plants. B, Flowering plants of the wild type and pal1 pal2 pal3 pal4 quadruple mutants. C, Lignin staining with phloroglucinol/HCl in the basal part of 3-month-old inflorescence stems of wild-type and pal1 pal2 pal3 pal4 quadruple mutant plants. D, Lignin content of 3-month-old inflorescence stems of wild-type and pal1 pal2 pal3 pal4 quadruple mutant plants. Lignin content was determined as acetyl bromide-soluble lignin. Means and se were calculated from three average lignin contents determined from three experiments with five plants per experiment for each genotype. According to Duncan's multiple range test (P = 0.05), means of lignin contents do not differ significantly if they are indicated with the same letter.

Figure 8.

Basal and pathogen-induced accumulation of total SA. A, Basal SA contents in wild-type (Col-0) and mutant plants. B, Pathogen-induced SA contents in wild-type (Col-0) and mutant plants. Plants were infiltrated with avirulent Pst DC3000 avrRpt2 (OD₆₀₀ = 0.02 in 10 mm MgCl₂). Inoculated leaves were harvested 24 h later for total SA determination. Means and se were calculated from average SA contents determined from three experiments with four to six plants per experiment for each genotype. According to Duncan's multiple range test (P = 0.05), means of SA contents do not differ significantly if they are indicated with the same letter.

Figure 9.

Responses of Arabidopsis pal mutants to P. syringae. A, Disease symptom development. Plants were infiltrated with a suspension of Pst DC3000 (OD₆₀₀ = 0.0002 in 10 mm MgCl₂). Photographs of representative inoculated leaves were taken at 3 d post inoculation (dpi). B, Altered bacterial growth. Pathogen inoculation was performed as in A. Samples were taken at 0 dpi (white bars) or 3 dpi (black bars) to determine the growth of the bacterial pathogen. Means and se were calculated from 10 plants for each treatment. According to Duncan's multiple range test (P = 0.05), means of colony-forming units (cfu) at 0 dpi do not differ significantly if they are indicated with the same lowercase letter and means of colony-forming units at 3 dpi do not differ significantly if they are indicated with the same uppercase letter.