As characterized by direct sequence comparison described in Chapter 4, the newly isolated cDNA fragments are most probably homologues of floral identity genes, LFY, AP 1, PI, and AG for Sophora and Clianthus. The most interesting questions to be asked for this study were: In which part of the plant are these genes expressed? And if they express in floral tissues, do they express only in the specific floral organs defined by their role of floral identity? To answer these two questions, quantitative expression of four floral identity genes in a variety of vegetative and reproductive tissues, and then in different floral organs of Sophora and Clianthus were analysed using real-time peR assay.
5.3.3.1 Expression of floral identity genes in different vegetative and reproductive tissues of Sophora
Vegetative tissues including adult leaves and shoot tips, and reproductive tissues including inflorescences 2-3 mm in length, early stage flower buds 2-3 mm in length,
mid-stage flower buds 1 8-20 mm in length, and seed pods 2-3 weeks after flowering were used in this experiment. Inflorescences were sampled in November 2002. Early stage flower buds, mid-stage flower buds, and seed pods were harvested in July, September and October 2003, respectively. Adult leaves and shoot tips were harvested in January 2003,
at least one month after the end of the inflorescence initiation period to avoid the
contamination of inflorescence primordia in the shoot tips. Result showed that there was a distinct expression profile for each gene, represented by relative mRNA concentration among different tissue samples (Fig. 5. 17).
STLFY activity was detected in all tested tissues. The expression level was very low in adult leaves and young seed pods, representing 0.08% and 0.07% of its peak level. Low level of expression (5. 1 %) was also detected in vegetative shoot tips. Compared to its highest level of expression in early stage flower buds, STLFY expressed at 16.2% in inflorescences, and at 0. 1 7% in mid-stage flower buds.
In contrast to STLFY, no STAP 1 activity was detected in adult leaves, vegetative shoot tips, and seed pods. Similar to STLFY, STAPl expressed at 1 6.2% in inflorescences
compared to the highest expression level in early stage flower buds. Only low level (6.2%) of STAP 1 activity was detected in mid-stage flower buds.
The highest STPI expression was detected in mid-stage flower buds, while the expression levels in early stage flower buds and inflorescence were 38% and 0.6% of the highest expression, respectively. Similar to STAP 1, no STPI activity was detected in leaves, vegetative shoot tips, and seed pods.
Similar to STPI, the highest STAG expression was detected in mid-stage flower buds. However, high STAG expression level (74%) was also detected in early stage flower buds while low expression ( 1 .2%) was detected in inflorescences. At for STAPl and STPI, no STAG activity was detected in leaves and vegetative shoot tips. However, STAG also expressed in seed pods at 2. 1 % of its highest level, being 30-fold higher than that of STLFY, the only other gene expressed in seed pods.
In summary, only STLFY activity was detected in leaves and vegetative shoot tips, and both STLFY and STAG expressed in seed pods, all at low levels. The highest expression was detected in early stage flower buds for STLFY and STAP 1, and in mid-stage flower buds for STPI and STAG. In expressed tissues types, expression levels of each gene varied
100 STLFY STAP1 - 1 0 � 0 - c: 0 ·en U) 1 � c. >< Cl) Cl) > 0.1 :;:::: ca Q; a: 0.01 0 A 8 C 0 E F A 8 C 0 E F 100 STPI STAG 10 - eft. - c: 0 ·en 1 U) � c. >< Cl) Cl) 0.1 > :;:::: ca Q; a: 0.01 o A 8 c o E F A 8 c o E F Tissue types
Figure 5.17 Relative expression (Iogarithmized scale) of four Sophora floral identity genes in vegetative and reproductive tissues
A: Leaves; B: Vegetative shoot tips; C: Inflorescences 2-3 mm in length; D: Early stage flower buds of 2-3 mm in length; E: Mid-stage flower buds 18-20 mm in length; F: Young legumes 2-3 weeks after flowering.
5.3.3.2 Expression of floral identity genes in different vegetative and reproductive tissues of Clianthus
Tissue types comparable to those of Sophora were tested for the four Clianthus floral identity genes using real-time peR assay. Those used were shoot tips, adult leaves, and inflorescences harvested in May 2003, and early stage flower buds, mid-stage flower buds, and seed pods sampled in July, September and October 2003 , respectively. As for their
Sophora homologues, a distinct expression profile was obtained for each gene (Fig. 5. 1 8).
All tested tissues had CMLFY expression at relative mRNA levels from 1 .7% in seed pods, to 100% in early stage flower buds. Low CMLFY activity was also detected in leaves and vegetative shoot tips, representing 2-3% of its mRNA level in floral primordia. CMLFY also expressed at high level (45%) in inflorescences, although at a low level (7%) in mid-stage flower buds.
No CMAP 1 activity was detected in adult leaves, vegetative shoot tips, and seed pods. Apart from its highest expression level in early stage flower buds, CMAP 1 also expressed at a high level (82%) in mid-stage flower buds. A CMAP 1 mRN A level of 9% of its highest expression was detected in inflorescences.
CMP! expression was not detected in leaves, vegetative shoot tips, and seed pods. In comparison with mid-stage flower buds in which CMP! expressed at its highest level, only 19% and 1 .5 % mRNA were detected in early stage flower buds and inflorescences, respectively.
The highest CMAG expression was detected in mid-stage flower buds, whereas 35% and 2.4% relative expression were detected in early stage flower buds and inflorescences, respectively. No CMAG expression was detected in adult leaves and vegetative shoot tips. However, a CMAG expression as high as 35% was detected in seed pods.
In summary, only CMLFY expression was detected in leaves and vegetative shoot tips. While all the four genes expressed at different level in inflorescence and floral buds, the highest expression was detected in early stage flower buds for CMLFY and CMAP 1, and in mid-stage flower buds for CMP! and CMAG Both CMLFY and CMAG activity were detected in seed pods, at much higher level than those for their Sophora homologues.
1 00 CMFLY CMAP1 1 0 - � 0 - c: .2 1 en en � Q. )( Cl) 0.1 Cl) > ; ca Cii er: 0.01 o A B c o E F A B c o E F 1 00 CMPI CMAG 1 0 - � 0 - , c: 0 '0 en Cl) ... Q. )( Cl) 0.1 Cl) > ; � Cl) er: 0.01 o A B c o E F A B c o E F Tissue types
Figure 5.18 Relative expression (Iogarithmized scale) of four Clianthus floral identity genes in vegetative and reproductive tissues
A: Leaves; B : Vegetative shoot tips; C: Inflorescences 2-3 mm in length; D: Early stage flower buds of 2-3 mm in length; E: Mid-stage flower buds 1 0- 1 2 mm in length; F: Young seed pods 2-3 weeks after flowering.
5.3.3.3 Expression of floral identity genes in different floral organs of Sophora
The objective was to test the function of Sophora floral identity gene homologues in specifying and identifying different flower organs, and to elucidate the applicability of the ABC model in woody leguminous species. Therefore, the expression profiles of STAP 1, STPI, and STAG, the representative Sophora homologues of each of the ABC class genes, together with their upstream controlling gene homologue STLFY, and an internal control gene, ST18S, were determined in four whorls of flower organ tissue using real-time PCR assay. Flower buds 1 8-20 mm in length showing petal tip sampled in September 2003 were used in this experiment. Sepals, petals, stamens, and carpel s were excised from the same flower bud. To avoid cross contamination among floral organs, only the upper half of each floral organ was used for RNA isolation. Before comparing the expression levels between tissue samples, melting curves of all PCR products were checked to confirm the nature of the amplified products.
The results showed that expression of the internal control gene ST18S was relatively stable across the four organ tissues, with a variation of expression level of only 40% detected between organ types (Fig. 5 . 1 9 and Fig. 5 .20).
STLFY expressed in all of the four flower organs, although a near 2-fold difference was detected between different organ samples. Higher mean STLFY expression was detected in sepals and stamens than in carpels and petals, although this did not achieve statistical significance.
Highest STAP 1 expression was detected in sepals. Petals also had high STAP 1 mRNA concentration, representing 77% of that of sepals. No STAP 1 activity was detected in stamens, and a very low level of STAP 1 activity was detected in c arpels, representing less than 111000 of its highest level (Fig. 5.20). The real-time quantification curves (Fig. 5 . 2 1 A) showed that PCR products accumulated normally only i n sepal and petal templates. When stamen and carpel templates were used, the PCR reactions generated much delayed and abnormal accumulating curves, with shorter logarithmic phases and low plateau level. The melting curve analysis indicated that the PCR reaction from stamens generated a product with similar melting temperature to that for the no template control. However, the PCR reaction using cDNA template from carpels did generate the same PCR products as templates from sepals and petals, possessing the same melting temperature (Fig. 5.21 B).
While similar STPI expression level was present in stamens ( 1 00%) and petals (96%), no STPI activity was detected in sepals and carpels (Fig. 5. 19, 5 .20).
Highest STA G expression was detected in stamens, followed by that in carpels (76%). No STAG activity was detected in sepals, and a trace expression of STAG, representing less than 1/37000 of its highest level, was detected in petals (Fig. 5 . 1 9, 5 .20).
In summary and excluding trace expressions (mRNA concentration below 0. 1 % of their corresponding highest expression levels), STAP 1 expressed only in sepals and petals, STPI expressed only in petals and stamens, and STAG expressed only in stamens and carpels.
Represented by the relative mRNA concentrations calculated using average values of the expression in each tissue, gene activities varied enormously among different genes at this developmental stage. ST18S expression was over l oo,OOO-fold higher than that of STLFY.
A difference of 40000-fold was detected among the four target genes. Similar amounts of gene product were detected for STAP 1 and STAG which were 7-to 8-fold lower than that of STPI, and over SOOO-fold higher than that of STLFY (Fig. 5 .20).