DOH1, a class 1 knox gene, is required for maintenance of the basic plant architecture and floral transition in orchid

Abstract

We report here the isolation and identification of an orchid homeobox gene, DOH1, from Dendrobium Madame Thong-In. Analyses of its sequence and genomic organization suggest that DOH1 may be the only class 1 knox gene in the genome. DOH1 mRNA accumulates in meristem-rich tissues, and its expression is greatly downregulated during floral transition. In situ hybridization analysis demonstrates that DOH1 is also expressed in the incipient leaf primordia and is later detected in the same region of the inflorescence apex, as in DOMADS1. Overexpression of DOH1 in orchid plants completely suppresses shoot organization and development. Transgenic orchid plants expressing antisense mRNA for DOH1 show multiple shoot apical meristem (SAM) formations and early flowering. In addition, both the sense and antisense transformants exhibit defects in leaf development. These findings suggest that DOH1 plays a key role in maintaining the basic plant architecture of orchid through control of the formation and development of the SAM and shoot structure. Investigations of DOMADS1 expression in the SAM during floral transition reveal that the precocious flowering phenotype exhibited by DOH1 antisense transformants is coupled with the early onset of DOMADS1 expression. This fact, together with the reciprocal expression of DOH1 and DOMADS1 during floral transition, indicates that downregulation of DOH1 in the SAM is required for floral transition in orchid and that DOH1 is a possible upstream regulator of DOMADS1.

Figure 1.

Sequence Comparison of Class 1 knox Proteins. (A) Alignment of the deduced amino acid sequences of DOH1 and its homologs from maize (KN1), Arabidopsis (STM), and rice (OSH1). Black boxes indicate identical amino acids, and dashes indicate gaps introduced to maximize the alignment. The positions of the first amino acid residues are shown on the left. (B) Phylogenetic analysis of class 1 knox proteins. Orchid class 1 knox protein is indicated by the asterisk. The horizontal branch length is proportional to the estimated number of base substitutions. Bootstrap values (>50%) in 100 replicates are indicated next to the nodes. Genus and species are given in parentheses behind the corresponding protein.

Figure 2.

Genomic DNA Gel Blot Analysis of the DOH1 Gene in Wild-Type Plants. A DNA gel blot containing 20 μg of orchid genomic DNA digested with various restriction enzymes was hybridized at low stringency with the digoxigenin-labeled DOH1 DNA probe. The sizes of the DNA markers are given at right in kilobases. A, ApaI; E, EcoRV; P, PstI; Sa, SmaI; Sp, SspI; Su, StuI; X, XbaI.

Figure 3.

RNA Gel Blot Analysis of the DOH1 Gene in Wild-Type Plants. (A) RNA gel blot analysis of the DOH1 gene in different orchid tissues. An RNA gel blot containing 10 μg of total RNA in each lane was hybridized with the digoxigenin-labeled full-length DOH1 DNA probe. RNA was isolated from roots (R; length, 1 cm), vegetative stems (VS; length, 0.4 cm), transitional stems (TS; length, 0.4 cm), young leaves (YL; length, 0.5 cm), old leaves (OL; length, 2 cm), VSAMs (V; 6 weeks old; length, 1.5 mm), TSAMs (T; 12 weeks old; length, 2 mm), and flowers (F; length, 1.5 cm). (B) Time course of DOH1 expression in orchid development. Lanes are labeled according to the age (in weeks) of tissues. From left to right, total RNA (25 μg per lane) was successively extracted from thin sections of protocorms (P; 0 weeks old; length, 1 mm), PLBs (PLB; 2 weeks old; length, 4 to 5 mm), VSAMs (VSAM; 4, 6, and 8 weeks old; ength, 1.5 mm), TSAMs (TSAM; 9 and 12 weeks old; length, 2 mm), and inflorescence meristems (IM; 14 weeks old; length, 3 to 5 mm). Hybridization was performed as in (A). The rRNAs, stained by methylene blue, indicate the amount of total RNA loaded in each lane.

Figure 4.

In Situ Localization of DOH1 Transcripts in Shoot Apices and Developing Floral Buds. (A) A 6-week-old vegetative shoot apex. (B) A 9-week-old transitional shoot apex. (C) and (D) A 15-week-old inflorescence apex. (E) and (F) A 16-week-old young floral bud. (G) and (H) An 18-week-old developed floral bud. (I) and (J) Part of a 20-week-old mature flower. Sections in (A), (B), (D), (F), (H), and (J) were hybridized with the DOH1 antisense RNA probe; those in (C), (E), (G), and (I) were hybridized with the DOH1 sense RNA probe. In (A) through (F), bright-field illumination was used for visualization of hybridization signals; in (G) through (J), dark-field illumination was used. am, apical meristem; b, bract; c, column; fm, floral meristem; fp1, first floral primordium; fp2, second floral primordium; fp3, third floral primordium; im, inflorescence meristem; lp, leaf primordium; p, petal; s, sepal. .

Figure 5.

Phenotypes of Transgenic Orchid Plants Overexpressing the DOH1 Gene. (A) Shoot development of a typical 35S::DOH1se orchid transformant (top) and a wild-type orchid plant (bottom). Plantlets that had been in culture for 4, 5, 6, and 8 weeks are laid out from left to right. Arrows point to the SLTs. (B) Leaf from a wild-type plant (left) and the CLLs from 35S::DOH1se orchid transformants (middle and right). The split tips of transgenic CLLs are marked with arrows. (C) Scanning electron micrograph of a typical SAM of the 35S::DOH1se transformant. The first and only CLL is initiated surrounding the SAM (arrow). (D) Higher magnification image of (C), showing the mound-shaped SAM and the trichomes (arrows) on the adaxial surface of the CLL. (E) Median longitudinal section through the young shoot apex of a typical transformant, showing the only surrounding CLL and the emerging SLT (arrow). (F) Median longitudinal section through the developing shoot apex of a typical transformant. Arrow points to the broadening base of the SLT. (G) Median longitudinal section through the young shoot apex of a wild-type plant, showing the surrounding CLL and the SAM (arrow) with the emerging leaf primordia (LP). (H) and (I) Scanning electron micrograph of the adaxial surface of the mature CLL from a typical transformant showing trichomes (H) and the mature CLL from a wild-type plant showing no trichomes (I). .

Figure 6.

Phenotypes of DOH1 Antisense Transgenic Orchid Plants. (A) Shoot development of a typical 35S::DOH1as transformant (top) and a wild-type plant (bottom). Peripheral leaves of the transformant were removed to facilitate viewing of the multiple-shoot phenotype. Plantlets that had been in culture for 3, 4, 5, 6, and 8 weeks are laid out from left to right. (B) Leaf phenotypes of a wild-type plant (far left) and several 35S::DOH1as transformants. (C) Top view of a 35S::DOH1as transformant. Arrows point to both of the shoots that originated simultaneously from the same meristem region. Leaves initiated surrounding both of the shoots are marked with asterisks. (D) Side view of a 35S::DOH1as transformant, showing the simultaneous growth of three separate young shoots from the same meristem region. Peripheral leaves encircling the shoots were dissected for viewing the multiple-shoot phenotype. Arrows indicate the initiated roots. (E) Scanning electron micrograph of formation of the SAM of a wild-type plant. The new emerging SAM sheathed by the CLL is indicated by an arrow. (F) Scanning electron micrograph of the shoot tip of a young wild-type plant. (G) Scanning electron micrograph of the SAM formation of a typical 35S::DOH1as transformant. Split SAMs sheathed by the CLL are marked by arrows. (H) Scanning electron micrograph of the young plantlet of a typical 35S::DOH1as transformant. Arrows indicate the young shoots simultaneously developing from the same meristem region. (I) Median longitudinal section through the young shoot apex of a typical transformant. Two new SAMs flanked by developing leaf primordia (LP) are marked with arrows. (J) Median longitudinal section through the developing shoot apex of a typical transformant. Positions at which new leaf primordia (LP) will initiate from the respective SAMs (S) are marked with asterisks. .

Figure 7.

DNA Gel Blot Analysis of the Orchid DOH1 Sense and Antisense Transgenic Plants. A gel blot containing genomic DNA from independent transgenic lines (10 μg per lane) digested with SmaI was hybridized with the digoxigenin-labeled full-length DOH1 DNA probe. DNA was isolated successively from 35S::DOH1se transformants (lines se1, se7, and se17), 35S::DOH1as transformants (lines as4 and as10), and wild-type plants (wt). The endogenous DOH1 gene band present in all of the plants is indicated with an arrow. The sizes of the DNA markers are given at right in kilobases.

Figure 8.

RNA Gel Blot Analysis of the Orchid DOH1 Sense and Antisense Transgenic Plants. (A) Expression of DOH1 in orchid 35S::DOH1se transformants. Total RNA was isolated from mature CLLs from wild-type plants (wt) and the independent 35S::DOH1se transgenic lines (lines se1, se7, se17, se28, and se33). The gel blot, containing 10 μg of total RNA in each lane, was hybridized with the digoxigenin-labeled full-length DOH1 DNA probe. (B) Expression of the DOH1 sense and antisense genes in 35S::DOH1as transformants. Total RNA was isolated from 6-week-old VSAMs (length, 1.5 mm) from wild-type plants (wt) and independent 35S::DOH1as transformants (lines as4, as10, as15, as23, as27, as31, and as38). The gel blot, containing 20 μg of total RNA in each lane, was hybridized with the digoxigenin-labeled full-length DOH1 RNA sense probe (SP) or antisense probe (AP). The rRNAs, stained by methylene blue, indicate the amount of total RNA in each lane.

Figure 9.

RNA Gel Blot Analysis of the DOMADS1 Gene in Orchid 35S::DOH1as Transformants. (A) Expression of DOMADS1 in TSAMs from wild-type and 35S::DOH1as transgenic plants. RNA was isolated from 10-week-old TSAMs (l) from wild-type plants (wt) and independent transformants (lines as4, as10, as15, as23, as27, as31, and as38). The gel blot, containing 20 μg of total RNA in each lane, was hybridized with the digoxigenin-labeled RNA probe derived from the 3′-specific region of the DOMADS1 gene. (B) Time course of DOMADS1 expression in a plant of the 35S::DOH1as transgenic line as31. The temporal schemes of main events during the development of wild-type and DOH1 antisense transgenic plants are illustrated above the RNA gel blot results. The horizontal bars in the graph indicate the different developmental phases of orchids. The average time of each phase was defined by calculating these values in 100 wild-type plants and in all 25 independent 35S::DOH1as transformants that could produce flowers. The time of onset of expression of DOMADS1 in wild-type and transgenic line as31 plants is labeled with an asterisk and an ×, respectively. Total RNA (30 μg per lane) was extracted from thin sections of protocorms (0 weeks old; l), VSAMs (6 weeks old; ), TSAMs (8 and 10 weeks old; l), and inflorescence meristems (12, 14, and 16 weeks old; ) from a transgenic line as31 plant. Hybridization was performed as in (A). The amount of total RNA loaded in each lane was determined by comparing the staining of the rRNAs with methylene blue.

Figure 10.

Histochemical Analysis of GUS Activity in Longitudinal Sections of the Apical Meristem from Orchid Plants Harboring DOMADS1::GUS Constructs. (A) A 9-week-old transitional shoot apex from a transgenic line as31 plant carrying DOMADS1::GUS constructs. (B) A 9-week-old transitional shoot apex from a wild-type plant carrying DOMADS1::GUS constructs. (C) An 11-week-old inflorescence apex from the same plant as in (A). (D) A 13-week-old floral bud from the same plant as in (A). Blue staining in (A), (C), and (D) indicates GUS activity. am, apical meristem; b, bract; c, column; fm, floral meristem; fp, floral primordium; im, inflorescence meristem; lp, leaf primordium; p, petal; s, sepal. .

Figure 11.

Scheme of the Orchid Plant Architecture of Wild-Type Plants and 35S::DOH1se and 35S::DOH1as Transformants. Compared with wild-type plants, 35S::DOH1se transformants grow without initiation of lateral organs from the SAM and do not enter the reproductive stage, whereas 35S::DOH1as transformants exhibit the simultaneous growth of multiple shoots with their own lateral organs and inflorescences. Leaves in the graph are not labeled. C, CLL; I, inflorescence; S, SAM.