Genetic ablation of root cap cells in Arabidopsis

Abstract

The root cap is increasingly appreciated as a complex and dynamic plant organ. Root caps sense and transmit environmental signals, synthesize and secrete small molecules and macromolecules, and in some species shed metabolically active cells. However, it is not known whether root caps are essential for normal shoot and root development. We report the identification of a root cap-specific promoter and describe its use to genetically ablate root caps by directing root cap-specific expression of a diphtheria toxin A-chain gene. Transgenic toxin-expressing plants are viable and have normal aerial parts but agravitropic roots, implying loss of root cap function. Several cell layers are missing from the transgenic root caps, and the remaining cells are abnormal. Although the radial organization of the roots is normal in toxin-expressing plants, the root tips have fewer cytoplasmically dense cells than do wild-type root tips, suggesting that root meristematic activity is lower in transgenic than in wild-type plants. The roots of transgenic plants have more lateral roots and these are, in turn, more highly branched than those of wild-type plants. Thus, root cap ablation alters root architecture both by inhibiting root meristematic activity and by stimulating lateral root initiation. These observations imply that the root caps contain essential components of the signaling system that determines root architecture.

Figure 1

(A–D) GUS-stained primary root tips of line 102-1 at 0 hour (imbibed) (A), 10 hours (B), 1 day (C), and 4 days (D) after transfer to 22°C. (E) Longitudinal section of a stained primary root tip of a transgenic plant expressing the GUS gene from the root cap-specific promoter. (F) The columella and lateral root cap cells expressing the GUS gene in E are indicated by large and small red circles, respectively. (Bar = 20 μm.)

Figure 2

The Ds insertion site and a diagram of the constructs used for Arabidopsis transformation. (A) Sequence at the Ds insertion site. The putative initiator ATG of the RCP1 gene is shown in boldface. The 8-bp Ds target site duplication is underlined by arrows. The underlined TAA is a stop codon in-frame with the initiator ATG. The 5′ end of the RCP1 expressed sequence tag cDNA is indicated by a bent arrow at the stop codon. One of the ScaI restriction sites used to isolate the root cap promoter is shown. (B) The 1.4-kb ScaI promoter fragment of the RCP1 gene was fused to a GUS gene containing nuclear localization signal (nlsGUS) and the DT-A^tsM gene, each of which is followed by a transcriptional terminator (NOS) taken from a gene for nopaline synthase. ScaI* indicates the location of the restriction site shown in A.

Figure 3

Longitudinal sections of primary root tips. One-day-old root tips of wild-type (A) and transgenic (D) plants; 2-day-old root tips of wild-type (B) and transgenic (E) plants; 4-day-old root tips of wild-type (C) and transgenic (F) plants. Division of quiescent cells was occasionally observed in transgenic roots, as seen in D (arrow). (Bar = 20 μm.)

Figure 4

Differentiation of root cells in transgenic and wild-type plants. Wild-type (A and B) and DT-A^tsM (C and D) seedlings were grown on MS agar medium for 4 days. (Bar = 50μm.)

Figure 5

Root growth rates. Seedlings were grown on vertical MS agar plates. The mean values of roots of wild-type (No-0) and DT-A^tsM plants were plotted (n = 32 and 32 roots, respectively).

Figure 6

Direction of primary root growth. Wild-type and DT-A^tsM (SSD) seedlings were grown on the surface of a vertical MS agar plate for 7 days. The angle of the primary root from the vertical was determined and plotted as a point on the circular graph. The numbers inside the graph are the numbers of plants whose root angles from the vertical are within each 30° range.

Figure 7

Distribution of the number of lateral roots. Wild-type and DT-A^tsM seedlings were grown on MS agar medium for 14 days. The number of lateral roots on 69 wild-type plants (No-0) and on 61 (line 2-1) and 55 (line 6-5) transgenic plants was counted under a dissecting microscope. Each histogram shows the distribution of the number of primary lateral roots, secondary lateral roots, and tertiary lateral roots. The sum of the number of primary, secondary, and tertiary lateral roots was for each wild-type and DT-A^tsM transgenic plant (total).