Knockdown of CELL DIVISION CYCLE16 reveals an inverse relationship between lateral root and nodule numbers and a link to auxin in Medicago truncatula

Abstract

The postembryonic development of lateral roots and nodules is a highly regulated process. Recent studies suggest the existence of cross talk and interdependency in the growth of these two organs. Although plant hormones, including auxin and cytokinin, appear to be key players in coordinating this cross talk, very few genes that cross-regulate root and nodule development have been uncovered so far. This study reports that a homolog of CELL DIVISION CYCLE16 (CDC16), a core component of the Anaphase Promoting Complex, is one of the key mediators in controlling the overall number of lateral roots and nodules. A partial suppression of this gene in Medicago truncatula leads to a decrease in number of lateral roots and a 4-fold increase in number of nodules. The roots showing lowered expression of MtCDC16 also show reduced sensitivity to phytohormone auxin, thus providing a potential function of CDC16 in auxin signaling.

Figure 1.

Structural analysis of the M. truncatula CDC16 gene. A, Gene structure of the MtCDC16 gene. Translated regions of exons are shown as gray boxes. The boldface line before the translational start site represents the 5′-untranslated and the putative promoter regions. The 500-bp fragment indicated below, which includes portions of exon 1 and the 5′-untranslated region, marks the area that was used for constructing the RNAi vector. The location of the two primer sets (P1-P1' and P2-P2') used for RT-PCR analysis of MtCDC16 transcripts is indicated. B, Alignment of the predicted M. truncatula CDC16 protein with the Arabidopsis CDC16 protein. Blocks of amino acid sequences marked in orange indicate the TPR motifs. C, Graphical representation of the distribution of TPR motifs within M. truncatula CDC16 and Arabidopsis CDC16 proteins. Gray boxes indicate the TPR motifs. D, Southern-blot analysis to estimate the copy number of MtCDC16 in M. truncatula genome.

Figure 2.

MtCDC16-suppressed (Mtcdc16i) roots show decreased lateral root number and root length. A. rhizogenes carrying the pRNAi-GUS or pRNAi-CDC16 construct was used to transform L416 seedlings (see “Materials and Methods”). A, Control root (gusi), showing normal primary and lateral root growth. B, MtCDC16-suppressed root with a moderate phenotype [Mtcdc16i (moderate)], showing reduced lateral root development. C, MtCDC16-suppressed root with a severe phenotype [Mtcdc16i (severe)], showing extreme reduction in root length and lateral roots. D, Root length and lateral root numbers from three independent sets of control (gusi) and Mtcdc16i roots. Error bars represent sd from the mean. E, Quantitative RT-PCR analysis of MtCDC16 expression in Mtcdc16i roots. P1 indicates the primers designed within region used for the RNAi construct, and P2 primers were designed downstream of the region used for the RNAi construct.

Figure 3.

Severe suppression of MtCDC16 results in aberrant cell morphology in the root meristem. A and B, Median longitudinal, toluidine blue-stained sections of the root tips of a control root (A) and an Mtcdc16i (severe) root (B). The double-sided bracket marks the root apical meristem of the control root and the corresponding region of the Mtcdc16i root. C and D, Higher-magnification image of the bracketed regions in A and B. C shows regularity in cell morphology at the root apical meristem in the control roots. D shows irregularity in cell morphology in comparable zone of the Mtcdc16i (severe) root. Bars = 50 μm.

Figure 4.

Partial suppression of MtCDC16 results in hypernodulation phenotype. A and B, Control roots (A) and Mtcdc16i roots (B) inoculated with S. meliloti and grown in an aeroponic chamber for 4 weeks. C, Acetylene reduction assay. Nitrogenase activity of intact nodules on control roots and Mtcdc16i roots was measured on a whole-root basis. The error bars indicate sd from the mean from three independent experiments. D, Quantitative RT-PCR analysis shows significant suppression of MtCDC16 in Mtcdc16i roots and nodules. Each point represents the mean of three replicates with error bars representing the sd.

Figure 5.

Longitudinal sections of root nodules from Mtcdc16i and control roots. Longitudinal section of a control nodule (A) and Mtcdc16i (moderate) nodule (B). The length of the infection zones is designated by brackets. Nodule zones are labeled according to Vasse et al. (1990): meristem (I), infection zone (II), interzone (asterisk), nitrogen-fixing zone (III). Bar = 0.5 mm.

Figure 6.

Promoter-GUS fusion analysis of MtCDC16 promoter activity in transgenic hairy roots of M. truncatula. The expression of GUS is indicated by blue staining. A to D, GUS activity in the uninoculated plant roots. GUS expression observed at the root tip (arrowhead) of primary root (PR; A), lateral root primordium (LRP; B), emerging lateral root (ELR; C), and at the tip (arrowhead) of fully emerged lateral root (LR; D). E to G, Induction of GUS during nodule development. Blue staining observed in the nodule primordium (NP; E), emerging nodule (EN; F), and at the meristematic zone (arrowhead) of a mature nodule (N; G). Bars = 50 μm.

Figure 7.

Auxin response phenotype of MtCDC16. A, Quantitative RT-PCR analysis to estimate the transcript abundance of MtCDC16 in wild-type roots in the presence and absence of 2,4-D. Each point represents the mean of three replicates with error bars representing the sd. B and C, Histochemical GUS staining patterns of CDC16:GUS in transgenic hairy roots in the absence (B) and presence (C) of 0.5 μm 2,4-D. Bars = 50 μm.

Figure 8.

Auxin resistance phenotype of MtCDC16. A, Root growth assay for auxin resistance. Inhibition of root growth in the presence of 0.5 μm 2,4-D was assayed in control (gusi) and Mtcdc16i roots. B, Quantitative RT-PCR analysis to estimate the transcript abundance of TC106907 and TC101963 in gusi and Mtcdc16i (moderate) roots in the presence and absence of 5 μm 2,4-D. In A and B, each point represents the mean of three replicates with error bars representing the sd.