Differential effects of NAA and 2,4-D in reducing floret abscission in cestrum (Cestrum elegans) cut flowers are associated with their differential activation of Aux/IAA homologous genes

Abstract

Background and aims: A previous study showed that the relative effectiveness of 2,4-dichlorophenoxyacetic acid (2,4-D) compared with that of 1-naphthaleneacetic acid (NAA) in reducing floret bud abscission in cestrum (Cestrum elegans) cut flowers was due to its acropetal transport. The aim of the present study was to examine if the differential effect of these auxins on floret abscission is reflected in the expression of Aux/IAA genes in the floret abscission zone (AZ).

Methods: cDNAs were isolated by PCR-based cloning from the floret AZ of auxin-treated cut flowers. The expression patterns of the cDNAs in various tissues and the effect of indole-3-acetic acid (IAA), applied with or without cycloheximide, on their expression in the floret AZ were examined by northern blot analysis. The regulation of transcript accumulation in the floret AZ in response to NAA or 2,4-D was measured by real-time PCR during auxin pulsing of cut flowers and vase life, concomitantly with floret abscission.

Key results: Six isolated cDNAs were identified to represent Aux/IAA homologous genes, designated as Cestrum elegans (Ce)-IAA1 to Ce-IAA6. Four Ce-IAA genes were characterized as early auxin-responsive genes (ARGs), and two (Ce-IAA1 and Ce-IAA5) as late ARGs. Only Ce-IAA5 was AZ-specific in floret buds. A temporal regulation of Ce-IAA transcript levels in the floret AZ was found, with 2,4-D inducing higher expression levels than NAA in floret buds. These Ce-IAA expression levels were negatively correlated with floret abscission.

Conclusions: The differential transport characteristics of NAA and 2,4-D in cestrum cut flowers were reflected in differential activation of the Ce-IAA genes identified in the floret AZ. Therefore, Aux/IAA genes can be used as molecular markers to measure auxin activity, which reflects free auxin level in the AZ. Two of the identified genes, Ce-IAA1 and Ce-IAA5, may also have a regulatory role in abscission.

Fig. 1.

Sequence alignment of the primary protein sequence encoded by six cestrum Aux/IAA genes (Ce-IAA1–Ce-IAA6) and representative Aux/IAA protein sequences from Arabidopsis (At-IAA6, Q38824; At-IAA7, CAB46059; At-IAA8, AAC49047), tobacco (Nt-iaa2·3, AAD32142; Nt-iaa4·3, AAD32144; Nt-iaa4·5, AAD32145; Nt-iaa28, AAD32146) and hybrid aspen (Ptt-IAA8, CAC84712). Alignment was limited to the partial available sequence of Ce-IAA proteins and full-length sequences of the representative proteins. The sequences were aligned using ClustalW software (Thompson et al., 1994). The black and grey shaded areas represent at least 50% amino acid identity or similarity, respectively. The conserved basic residues that comprise the NLS sequence in domains II and IV, and the conserved amino acids of the protein destabilization element in domain II (box), which are the basic features of Aux/IAA proteins, are indicated.

Fig. 2.

Effect of IAA and/or CHX on induction of five Ce-IAA genes in cestrum floret AZ. Explants (3–4 mm long) were excised from the floret AZ and incubated overnight on 1 % agar plates for depletion of the endogenous auxin. The samples were then incubated for 1 h in either 1/2 MS, 1/2 MS containing 10 µm IAA or 50 µm CHX, or were pre-incubated for 30 min in 1/2 MS with 50 µm CHX and then transferred for an additional 1 h to 1/2 MS containing 10 µm IAA and 50 µm CHX. In all the treatments the 1/2 MS medium contained 0·5 % agarose. Untreated control samples were incubated for 1 h on moist Whatman No. 1 paper. Total RNA (20 mg) was denatured, loaded on each lane, blotted onto Hybond N+ membranes and hybridized with gene-specific Ce-IAA probes amplified from the 3′UTR of the clone.

Fig. 3.

Expression patterns of four Ce-IAA genes in various organs or tissues excised from untreated (A) or 2,4-D-treated (B) cestrum cut flowers. The shoots (30 cm long) were pulsed for 24 h with 0·2 mm 2,4-D at 20 °C in the observation room. Samples were collected from the different organs or tissues of control and 2,4-D-treated shoots. Total RNA (20 mg) was denatured, loaded per each lane, blotted onto Hybond N+ membranes and hybridized with gene-specific Ce-IAA probes amplified from the 3′UTR of the clone.

Fig. 4.

Effect of NAA or 2,4-D pulsing of cestrum cut flowers on expression of six Ce-IAA genes in the AZ of floret buds during pulsing and subsequent vase life. The cut flowers were pulsed with 0·2 mm of the different auxin solutions in 0·02 % 8-HQC for 24 h and then transferred to a TOG-6 solution during vase life. Total RNA was extracted from the AZ of floret buds during pulsing and subsequent vase life. The level of gene expression was determined by RT-PCR. The transcript level was comparatively quantified vs. ribosomal 18S cDNA, which served as an internal reference (housekeeping gene) to normalize gene expression. The primer pairs used for each gene and for the 18S gene are presented in Table 1. The relative amount of each specific transcript is in reference to its own level in untreated controls (0 time = 1·0). The results represent average values of duplicate RT-PCR reactions for the same cDNA in one representative experiment.

Fig. 5.

Effect of NAA or 2,4-D pulsing of cestrum cut flowers on expression of six CeIAA genes in the AZ of open florets during pulsing and subsequent vase life. The cut flowers were pulsed with the auxins, and total RNA was extracted from the AZ of open florets and assayed as detailed in Fig. 4. The results represent average values of duplicate RT-PCR reactions for the same cDNA in one representative experiment.

Fig. 6.

Effect of NAA or 2,4-D pulsing of cestrum cut flowers on abscission of floret buds (A) and open florets (B) during vase life. The cut flowers (30 cm long) bearing mainly floret buds (A) or open florets (B) were pulsed with 0·2 mm of the different auxin solutions in 0·02 % 8-HQC for 24 h and then transferred to a TOG-6 solution during vase life. At the indicated time intervals, the inflorescence heads were placed into polyethylene bags and tapped gently. Abscised floret buds or open florets were then counted and their accumulated number is presented as percentage of total floret buds or open florets per inflorescence. Data represent the mean values of six shoots ± s.e.