Cosuppression of eukaryotic release factor 1-1 in Arabidopsis affects cell elongation and radial cell division

Abstract

The role of the eukaryotic release factor 1 (eRF1) in translation termination has previously been established in yeast; however, only limited characterization has been performed on any plant homologs. Here, we demonstrate that cosuppression of eRF1-1 in Arabidopsis (Arabidopsis thaliana) has a profound effect on plant morphology, resulting in what we term the broomhead phenotype. These plants primarily exhibit a reduction in internode elongation causing the formation of a broomhead-like cluster of malformed siliques at the top of the inflorescence stem. Histological analysis of broomhead stems revealed that cells are reduced in height and display ectopic lignification of the phloem cap cells, some phloem sieve cells, and regions of the fascicular cambium, as well as enhanced lignification of the interfascicular fibers. We also show that cell division in the fascicular cambial regions is altered, with the majority of vascular bundles containing cambial cells that are disorganized and possess enlarged nuclei. This is the first attempt at functional characterization of a release factor in vivo in plants and demonstrates the importance of eRF1-1 function in Arabidopsis.

Figure 1.

Wild-type and broomhead phenotypes. A, A mature wild-type plant (left) and a broomhead plant exhibiting a severe phenotype (right). B, Wild-type inflorescence showing regular spacing of flowers along the internode. C, Reduced internode elongation in the broomhead phenotype. Wild-type (D) and broomhead (E) plants are identical during juvenile rosette development. However, at the late adult vegetative stage, broomhead transgenics (G) are distinguishable from wild-type plants (F) by the production of anthocyanin in the veins of the rosette leaves.

Figure 2.

Variation in the broomhead phenotype at the whole plant level and in the broomhead-like structures. A to D, Broomhead plants showing growth retardation of the primary bolt at the rosette level and the production of numerous auxiliary bolts (A), prominent broomhead-like structures on the inflorescence apices (B), an intermediate severity phenotype and the presence of multiple auxiliary bolts (C), and a weak phenotype with broomhead-like structures appearing later in development (D). E to H, Inflorescence stems showing a severe reduction in internode elongation (E) and a reversion of the phenotype with newly formed inflorescences exhibiting an increase in internode elongation (F–H).

Figure 3.

Northern analysis of wild-type, broomhead, and nonphenotype tissues. A, Northern blot of total RNA prepared from different tissues (se, seedlings; rl, rosette leaves; cl, cauline leaves; st, stem; b, buds; r, roots) of wild-type (WT) and broomhead (B) plants. The membrane was hybridized with a radiolabeled eRF1-1 probe and subsequently stripped and hybridized with an AtDRG2 probe. B, Total RNA was isolated from bud tissue of nonphenotype transgenics (N1–N6) in six independent eRF1-1 overexpressor lines. Wild-type and broomhead RNA bud samples were included as controls. A ribosomal probe for both blots was used to verify equal sample loading.

Figure 4.

Southern-blot analysis of four independent broomhead lines. Genomic DNA was extracted from broomhead lines and digested with EcoRI, SalI, or HindIII, transferred to a membrane, and probed with a radiolabeled eRF1-1 probe.

Figure 5.

GUS staining pattern and northern analysis of eRF1-1:GUS transgenic Arabidopsis plants. A, An eRF1-1:GUS transgenic plant exhibiting a severe broomhead phenotype and failing to show any GUS activity. B, A broomhead cluster from a less severe transgenic line revealing GUS activity in developing seeds. C and D, Nonphenotype transgenics showing GUS activity in all plant tissues (C) and minimal GUS activity in rosette leaves (D). E, Close-up of GUS staining pattern in rosette leaves. F, A mostly GUS negative nonphenotype plant exhibiting GUS activity in developing seeds. G, Northern analysis of total RNA isolated from bud tissue of wild-type plants (lane 1), CaMV 35S-GUS (transgenic control, lane 2), and eRF1-1:GUS transgenics (lanes 3, 4, and 5). eRF1-1:GUS transgenics were grouped according to their GUS expression pattern and phenotype: GUS positive nonphenotype plants (lane 3), GUS negative nonphenotype plants (lane 4), and GUS negative broomhead plants (lane 5). The cauline leaves of nonphenotype transgenics were stained at the time of bud collection to ensure the separation into GUS positive and negative samples. The membrane was hybridized with a probe corresponding to the full-length eRF1-1 transcript to allow detection of both the eRF1-1:GUS transgene and the endogenous eRF1-1 gene. The membrane was then stripped and rehybridized to the 3′ untranslated region (UTR) of the eRF1-1 transcript to only enable detection of the endogenous gene. Hybridization with a ribosomal probe was used to show equal loading in each lane. An arrowhead indicates the eRF1-1:GUS transcript and the endogenous eRF1-1 is indicated by an asterisk.

Figure 6.

Relative expression levels of eRF1-1, eRF1-2, and eRF1-3 family members in nonphenotype and broomhead transgenic plants. eRF1-1, eRF1-2, and eRF1-3 transcript levels were quantified in wild-type, broomhead, and nonphenotype plants using quantitative real-time PCR. The graphs show the relative values, with respect to wild-type plants, of eRF1-2 and eRF1-3 (A) and eRF1-1 (B) in broomhead plants (black bars) and nonphenotype plants (white bars). Values shown are means ± se.

Figure 7.

Anatomy of inflorescence stems and apical meristems of wild-type and broomhead plants. A and B, Longitudinal sections of the mid-region of wild-type (A) and broomhead (B) inflorescence stems. C, Enlargement of the fascicular cambial region in B showing disorganization of the cambium. Enlarged nuclei are marked by arrows. D and E, Longitudinal sections of wild-type (D) and broomhead (E) apical meristems. F and G, Transverse sections of the mid-region of wild-type (F) and broomhead (G) stems. The arrows indicate lignification of the disorganized fascicular cambial region. H and I, Enlargement of the vascular bundles from F and G. H, Wild type; I and J, broomhead vascular bundles. The fascicular cambial region displays active, controlled cell division in I and disorganized cell growth with enlarged nuclei in J. The arrow in J indicates lignification of the disorganized fascicular cambial region. K and L, Phloroglucinol-HCl staining of stem sections through the mid-region of wild-type (K) and broomhead (L) plants. Phloroglucinol-HCl stains lignified tissue red/pink. Ectopic lignification in the cambial region of broomhead plants is indicated by arrows. pi, pith; co, cortex; fc, fascicular cambium; if, interfascicular fibers; pc, phloem cap cells; ps, phloem sieve cells; ifp, interfascicular fiber precursors. Bars = 100 μm in A, B, D to G, K, and L, 50 μm in H to J, and 10 μm in C.

Figure 8.

eRF1-1 expression pattern as revealed by staining of eRF1-1 promoter-GUS transgenic lines and northern analysis. eRF1-1 expression was detected in germinating seeds (A), the vascular tissues of young seedlings (B), rosette leaf veins (C), cauline leaf veins (D), the inflorescence stem (E), roots (F), pollen grains (J), trichomes (K), and guard cells (L). The shoot apical meristem (B) and the root meristem (F) also show GUS activity. G, Transverse section through the mid-region of an inflorescence stem revealing GUS expression is mainly restricted to the vascular bundles and the interfascicular region. H, Enlargement of a vascular bundle from G, displaying GUS expression in phloem (p), fascicular cambium (fc), xylem parenchyma (xp), and interfascicular parenchyma (ip) cells. I, Transverse section through the elongating region of an inflorescence stem showing a more uniform pattern of expression across all cell types. M, Northern blot of total RNA isolated from different tissues (se, seedlings; yrl, young rosette leaves; orl, old rosette leaves; ycl, young cauline leaves; ocl, old cauline leaves; st, stem; b, buds; ofl, open flowers; si, siliques; r, roots) of wild-type plants. The membrane was hybridized with a radiolabeled eRF1-1 probe and then subsequently stripped and rehybridized with a ribosomal probe to show equal sample loading.