The D-type cyclin CYCD4;1 modulates lateral root density in Arabidopsis by affecting the basal meristem region

Abstract

Root cell division occurs primarily in the apical meristem, from which cells are displaced into the basal meristem, where division decreases and cell length increases before the final differentiation zone. The organization of the root in concentric files implies coordinated division and differentiation of cell types, including the xylem pole pericycle cells, which uniquely can resume division to initiate lateral roots (LR). Here, we show that D-type cyclin CYCD4;1 is expressed in meristematic pericycle protoxylem poles and is required for normal LR density. Cycd4;1 mutants also show a displacement of the apical/basal meristem boundary in the pericycle and longer pericycle basal meristem cells, whereas other cell layers and overall meristem size and root growth are unaffected. Auxin is proposed to separately prepattern and stimulate LR initiation. Stimulation is unimpaired in cycd4;1, suggesting CYCD4;1 requirement for normal spacing but not initiation. Both pericycle cell length and LR density phenotypes of cycd4;1 are rescued by low concentrations of applied auxin, suggesting that the basal meristem has a role in determining LR density. We further show CYCD4;1 is rate-limiting for sucrose-dependent LR formation, since CYCD4;1 expression is sucrose-dependent and wild-type roots fully phenocopy cycd4;1 in sucrose absence. We conclude that CYCD4;1 links meristem pericycle cell behavior to LR density consistent with a basal meristem prepatterning model and that D-type cyclins can confer division potential of defined cell types through cell-specific expression patterns.

Fig. 1.

Expression and gene structure of CYCD4;1. (A) The CYCD4;1 promoter is active in the pericycle cells of the root meristem as shown by GFP signal. (B) CYCD4;1 is expressed in the xylem poles of the pericycle in the apical meristem (CYCD4;1 promoter driving GUS reporter). (C) Gene structure of CYCD4;1. Boxes indicate exons, and the position of the T-DNA insertions in the CYCD4;1 mutants are shown. PCR amplicons from homozygote mutant plants were purified and sequenced using insert specific primers, showing that both mutants had tandem head-to-head insertion events within the CYCD4;1 locus. The cycd4;1-1 has a T-DNA insertion at position −25 bp from the initiation codon and cycd4;1-20 in the fourth exon.

Fig. 2.

The phenotype of the cycd4;1 null-mutants. (A) cycd4;1 show a decreased number of LR. (B) Cell lengths in different zones of the root meristem. Average WT and cycd4;1-1 cell lengths in epidermis (ep), cortex (co), and endodermis (en) for cells 1–10 and 11–20 counted from the QC are shown in upper left table. Main figure: Average pericycle cell lengths are shown for cells 1->40 from QC for WT and cycd4;1 mutants grown on 0 nM and 10 nM exogenous auxin. Note (Inset) that cells of the basal meristem in cycd4;1-1 are enlarged only in the absence of auxin and that the start of net elongation takes place closer to the QC (arrowhead) than in WT. Significant (t test P < 0.05) changes were observed between cycd4;1-1 without NAA and the other condition for cell number 17 to 25. Lines below the x-axis indicate basal meristem regions of WT and cycd4;1-1. The apical meristem and rapid elongation zones lie to the left and right of the basal meristem region, respectively. (C) Cell length and position from the QC of the pericycle cells (pc) in the cycd4;1-1 and WT and epidermis cells in the WT (ed) as comparison. (D) LR density at 10 DAG in WT and cycd4;1-1 upon treatment of increasing levels of auxin. Asterisks indicate differences are significant (t test P < 0.05).

Fig. 3.

Effect of auxin and sucrose on the cycd4;1-1 phenotype. (A) Pericycle cell lengths in the aux1 and slr mutants compared to WT and cycd4;1-1. Aux1-7 plants were treated were grown without and with 10 nM NAA. Cell lengths are significantly different (t test P < 0.05) compared to WT for cells 14–26 (both aux1), 17–26 (cycd4;1-1), and 25–30 (slr). (B–E) CYCD4;1 promoter driving GUS-GFP reporter in 10-day-old roots on different sucrose and light conditions. (B) Roots grown for 9 days in the dark (−suc) transferred to the dark (−suc). (C) Roots grown for 9 days in the dark (−suc) transferred to the light (−suc). (D) Roots grown for 9 days in the dark (−suc) transferred to the dark (+suc). (E) Roots grown in the light (+suc) transferred to the light (+suc). (F) LR density at 10 DAG in WT and cycd4;1-1 grown on media containing different sucrose concentrations. (G) Average pericycle cell lengths in WT and cycd4;1-1 grown on media containing different sucrose concentrations. Inset shows enlargement of the basal meristem region of the curves. No significant differences were found between WT on 0.75% and 1.5% sucrose, except for cell numbers 29–31 (t test P < 0.05). No significant changes (t test P < 0.05) were found between WT on 0% sucrose and the cycd4;1-1 conditions. Cell lengths are significantly different (t test P < 0.05) between WT on sucrose and WT without sucrose or the cycd4;1-1 conditions between cells 21–27.