Article Figures & Data
Figures
- Fig. 1.
Alignment of the representative sequences of theChlMe1 and ChlMe2 3′-UTRs obtained by TAIL-PCR. A, 3′-UTR sequences of ChlMe1. The sequence from F. trinervia was presented for theC4(3-4) group, F. pringlei ChlMe1-2 for the C3(3-4) group, and F. brownii ChlMe1-1 for theC3-C4(5-6)group. B, 3′-UTR sequences of ChlMe2. The sequence fromF. trinervia was presented for theC4(3-4) group, F. pringlei ChlMe2-2 for the C3(3-4) group, and F. brownii ChlMe2-3 for theC3-C4(5-6)group. The sequences included were immediately after the stop codon and upstream of the polyadenylation site, as defined by the F. trinervia ChlMe1 and the F. pringlei ChlMe2 cDNAs (Börsch and Westhoff, 1990; Lipka et al., 1994). F. ang(3-4) was the single species examined of theC3-C4(3-4)group. Dashes at the beginning of F. ang(3-4) ChlMe1sequence represent unknown sequence; the remaining dashes represent deletions. Black bars indicate regions with high variability. Stars in A indicate the C4-specific nucleotides. See text for more details.
- Fig. 2.
Southern analysis of genomic DNAs fromFlaveria spp. using probes specific for ChlMe1(A) and ChlMe2 (B). The different species are grouped together according to types of photosynthesis. Within each of the C3-C4 groups, the species are placed in order of increasing C4 capabilities (Ku et al., 1991). Note that F. brownii and F. vaginata have been characterized as C4-like. RI, EcoRI; H3, HindIII; Xb, XbaI. Numbers at left indicate sizes in kilobase pairs.
- Fig. 3.
Changes in the levels of ChlMe1 andChlMe2 mRNAs during leaf development. A, F. pringlei leaves. B, F. trinervia leaves. Relative quantitative RT-PCR was used to analyze ChlMe1 andChlMe2 mRNA levels in the same samples obtained from leaves of various lengths. 18S rRNA was used as the internal control in each sample. The results shown were the average of at least two experiments. Error bars represent se.
- Fig. 4.
Translational fusion constructs using 5′ and 3′ regions of F. trinervia ChlMe1 and F. pringlei ChlMe2 for F. bidentis transformation. ThegusA gene encoding GUS was used in all constructs as a reporter. The lengths of the 5′ regions are specified with the start codon as +1. The black box labeled I represents the first intron. The length of each region is shown in relative proportion. See “Materials and Methods” for more details. Ftr, F. trinervia; Fpr,F. pringlei.
- Fig. 5.
Histochemical localization of GUS activity in leaves and cotyledons of F. bidentis seedlings transformed with the construct ChlMe1-nos. A, A 4-week-old seedling showing strong staining in both cotyledons and true leaves. B, High-magnification, paradermal view of a cleared seedling true leaf showing intense blue staining around veins. C, One-week-old cotyledons grown under different light conditions. Illumination enhanced staining somewhat in BS cells and suppressed staining in M cells. D, Relative GUS activity in consecutive leaves taken from a single branch of a greenhouse-grown plant transformed with ChlMe1-nos. Shown are data for two independent transformants. Gray bars represent the length of each leaf measured from the tip to the end of the petiole. Black points show GUS activity obtained from each leaf, plotted relative to the maximal point.
- Fig. 6.
Histochemical localization of GUS activity in leaves and cotyledons of F. bidentis seedlings and mature plants transformed with the construct ChlMe2-nos. A, A 3-week-old seedling showing basipetal down-regulation of GUS expression in leaves. B, Dark-field view of a longitudinal section of a primary leaf (approximately 1.5 mm in length). Under dark-field illumination, the crystalline GUS product appears as bright pink spots. Staining was uniform in the basal, non-differentiated region of the leaf and increased in region proximal to developing veins. C, High magnification of a region in (B) with strong staining. Staining was restricted to cells with high chloroplast number (i.e. BS cells and M cells surrounding BS cells). D, Consecutive leaves of a mature plant (youngest leaf at left). Note the absence of basipetal down-regulation of GUS expression. Staining was strongest in young leaves and down-regulated in older leaves. E, One-week-old cotyledons grown under different light conditions. Illumination was required for GUS expression in cotyledons but not in the first true leaves. F, Relative GUS activity in consecutive leaves taken from a single branch of a greenhouse-grown plant transformed with ChlMe2-nos. Shown are data for two independent transformants. Gray bars represent the length of each leaf measured from the tip to the end of the petiole. Black points show GUS activity obtained from each leaf, plotted relative to the maximal point.
- Fig. 7.
Histochemical localization of GUS activity in non-photosynthetic tissues of F. bidentis seedlings transformed with the constructs ChlMe1-nos (A–D) andChlMe2-nos (E–H). A, Dark-field view of a longitudinal section through the SAM, where staining was absent. Under dark-field illumination, the crystalline GUS product appears as bright pink spots. B, A root tip showing staining in the vascular bundle and columnellar cells, but not in the root meristem. C, An upper portion of a root showing GUS staining in the phloem. D, A cross-section of a stem showing strong staining in the phloem. E, Dark-field view of a longitudinal section through SAM showing similar staining as the surrounding tissues. F, An apical portion of a dark-grown seedling showing strong staining in the arrested first leaves and SAM. G, A root tip of soil-grown seedling showing strong staining throughout the whole tip, including the root meristem. H, A cross-section of a stem showing staining in the vascular bundles, particularly in the xylem.
