Differential regulation of RNA levels of gibberellin dioxygenases by photoperiod in spinach

Abstract

Previous work with spinach (Spinacia oleracea) has shown that the level of gibberellin (GA) 20-oxidase is strongly up-regulated by long days (LD). In the present work, the effect of photoperiod on expression of other GA dioxygenases was investigated and compared with that of GA 20-oxidase. Two GA 2-oxidases and one GA 3-oxidase were isolated from spinach by reverse transcription-polymerase chain reaction with degenerate primers and by 5'- and 3'-rapid amplification of cDNA ends. As determined by high-performance liquid chromatography with on-line radioactivity detection, the SoGA3ox1 gene product catalyzed 3beta-hydroxylation of GA(9) to GA(4) and GA(20) to GA(1). The SoGA2ox1 and the SoGA2ox2 gene products catalyzed 2beta-hydroxylation of GA(9) to GA(51) and GA(20) to GA(29). The product of GA(20) metabolism by SoGA3ox1 was identified as GA(1) by gas chromatography-mass spectrometry, whereas the products of GA(1) and GA(20) metabolism by SoGA2ox1 and SoGA2ox2 were identified as GA(8) and GA(29), respectively. SoGA2ox1 also metabolized GA(53) to GA(97). The levels of SoGA20ox1 transcripts were greatly increased in all organs tested in LD conditions, but the levels of SoGA3ox1 transcripts were only slightly increased in blades and petioles. A decrease in the levels of the SoGA2ox1 transcripts in young leaves and tips in LD conditions is opposite to the expression pattern of the SoGA20ox1. Expression of SoGA20ox1 in petioles and young leaves was strongly up-regulated by a supplementary 16 h of light, but the levels of SoGA3ox1 and SoGA2ox1 transcripts did not change. It is concluded that regulation and maintenance of GA(1) concentration in spinach are primarily attributable to changes in expression of SoGA20ox1.

Figure 1

The early-13-hydroxylation pathway from GA₁₂ to GA₈ of GA biosynthesis and deactivation in spinach. GA₅₃ is converted to GA₂₀ by GA 20-oxidase via GA₄₄ and GA₁₉. GA₂₀ can be converted to GA₁ by GA 3-oxidase. GA₁₂, GA₅₃, GA₂₀, and GA₁ can be deactivated by 2β-hydroxylation to GA₁₁₀, GA₉₇, GA₂₉, and GA_8, respectively.

Figure 2

Alignment of the deduced amino acid sequences for the SoGA3ox1 gene from spinach with other GA 3-oxidases. Conserved regions are boxed in black. Asterisks indicate the putative Fe²⁺-binding motif at the active site of 2-oxoglutarate-dependent dioxygenases (Thomas et al., 1999). SoGA3ox1, GA 3-oxidase from spinach (GenBank accession no. AF506280); NtGA3ox, GA 3-oxidase from tobacco (Nicotiana tabacum; accession no. BAA89316); StGA3ox, GA 3-oxidase from potato (Solanum tuberosum; accession no. AAK91507); PsGA3ox, GA 3-oxidase from garden pea (GenBank accession no. AAC96015); AtGA3ox, GA 3-oxidase from Arabidopsis (accession no. T51691); CmGA3ox and CmGA23ox, GA 3-oxidases from pumpkin (Cucurbita pepo; accession nos. AAB64347 and CAB92914); and OsGA3ox, GA 3-oxidase from rice (Oryza sativa; accession no. BAB62072).

Figure 3

Alignment of the deduced amino acid sequences for the SoGA2ox1 and SoGA2ox2 genes from spinach with other GA 2-oxidases. Conserved regions are boxed in black. Asterisks indicate the putative Fe²⁺-binding motif at the active site of 2-oxoglutarate-dependent dioxygenases (Thomas et al., 1999). SoGA2ox1 and SoGA2ox2, GA 2-oxidases from spinach (accession nos. AF506281 and AF506282); PcGA2ox1, GA 2-oxidase from runner bean (accession no. AT132438); PsGA2ox1 and PsGA2ox2, GA 2-oxidases from garden pea (accession nos. AF100954 and AF100955); AtGA2ox1, AtGA2ox2, and AtGA2ox3, GA 2-oxidases from Arabidopsis (accession nos. AJ132435, AJ132436, and AJ132437); OsGA2ox1, GA 2-oxidase from rice (accession no. BAB40934).

Figure 4

Expression patterns of the SoGA20ox1, SoGA3ox1, SoGA2ox1, and SoGA2ox2 genes in various organs of spinach plants grown in SD and after 8 LD. Northern blots were prepared by separating 30 μg of total RNA of each organ through 1.2% (w/v) agarose gels containing formaldehyde, followed by transfer to Hybond-N⁺ membranes. The blots were separately hybridized to ³²P-labeled cDNAs of SoGA20ox1, SoGA3ox1, SoGA2ox2, or SoActin. After RT-PCR of SoGA2ox1 and SoActin, 10 μL of the PCR reaction products were separated through 2.0% (w/v) agarose gel by electrophoresis and hybridized to a ³²P-labeled cDNA of SoGA2ox1 or SoActin. The numbers under the blots indicate the relative amount of each transcript after standardization using SoActin as a loading control. The value for each gene transcript in blades of spinach grown in SD conditions was arbitrarily set at 1.0.

Figure 5

Time course of SoGA20ox1, SoGA3ox1, and SoGA2ox1 expression after spinach plants were transferred from SD to LD conditions. GA20ox1 transcripts increased with increasing duration of LD treatment in petioles (A), young leaves (B), and tips (C). GA3ox1 transcripts increased with increasing duration of LD treatment in petioles (A), but they did not change in young leaves (B), and tips (C) after exposure to increasing numbers of LD. GA2ox1 transcripts increased with increasing duration of LD treatment in petioles (A), but they decreased in young leaves (B), and tips (C). The numbers under the blots indicate the relative amount of transcript after standardization using SoActin as a loading control. The value of each transcript level in petioles in SD was arbitrarily set at 1.0.

Figure 6

Effects of light and darkness on the transcript level of SoGA20ox1, SoGA3ox1, and SoGA2ox1 in petioles (A), young leaves (B), and tips (C). Spinach plants were harvested at the end of the 8-h high-intensity light period (0 h) and after transfer from SD or LD conditions to 16 h of darkness (16 D) or to 16 h of weak incandescent light (16 L). The numbers under the blots indicate the relative amount of each transcript after standardization using SoActin as a loading control. The value of each transcript level in petioles at 0 h in SD was arbitrarily set at 1.0.

Figure 7

Expression of SoGA20ox1, SoGA3ox1, and SoGA2ox1 in male flowers (♂), female flowers (♀), stems (St), and shoot tips (ShT). Spinach plants were harvested after more than 3 weeks in LD conditions. In this case, the shoot tips included the upper 1 cm of the shoots; stems were the next 1 cm of the elongated stems. The numbers under the blots indicate the relative amount of each transcript after standardization using SoActin as a loading control. The value of the transcript level of each gene in male flowers was arbitrarily set at 1.0.