The Arabidopsis thaliana homolog of yeast BRE1 has a function in cell cycle regulation during early leaf and root growth

Abstract

Chromatin modification and transcriptional activation are novel roles for E3 ubiquitin ligase proteins that have been mainly associated with ubiquitin-dependent proteolysis. We identified HISTONE MONOUBIQUITINATION1 (HUB1) (and its homolog HUB2) in Arabidopsis thaliana as RING E3 ligase proteins with a function in organ growth. We show that HUB1 is a functional homolog of the human and yeast BRE1 proteins because it monoubiquitinated histone H2B in an in vitro assay. Hub knockdown mutants had pale leaf coloration, modified leaf shape, reduced rosette biomass, and inhibited primary root growth. One of the alleles had been designated previously as ang4-1. Kinematic analysis of leaf and root growth together with flow cytometry revealed defects in cell cycle activities. The hub1-1 (ang4-1) mutation increased cell cycle duration in young leaves and caused an early entry into the endocycles. Transcript profiling of shoot apical tissues of hub1-1 (ang4-1) indicated that key regulators of the G2-to-M transition were misexpressed. Based on the mutant characterization, we postulate that HUB1 mediates gene activation and cell cycle regulation probably through chromatin modifications.

Figure 1.

Plant Phenotypes of the hub1 and hub2 Mutants and the Corresponding Wild Types, Ler and Col, Grown in Soil in a Growth Chamber.

Figure 2.

Leaf and Root Phenotypes of hub1 and hub2. (A) Juvenile and adult fully expanded leaves of Ler (top panel) and hub1-1 (bottom panel). (B) Rosette weight of hub1-1, hub1-4, hub1-3, hub2-1, Ler, and Col plants grown in soil (n = 48 plants) and harvested at inflorescence emergence stage. FW, fresh weight; DW, dry weight. Bars indicate mean ± sd. (C) Absolute palisade cell number across half of the first and second expanded leaves (n = 3). Bars indicate mean ± sd. ***, Statistical difference at P < 0.001 from the t test. (D) Transverse section through the central part of the first leaf of plants (26 DAS) grown in vitro. mv, midvein. (E) Primary root growth kinetics of mutants hub1-1 and hub2-1 and wild types Ler and Col grown under in vitro conditions (n = 8). (F) Root cortex cell production per hour in Ler and hub1-1 seedlings grown in vitro. Bars indicate mean ± sd (n = 1200).

Figure 3.

Kinematic Analysis of Leaf and Root Growth of the First Leaf Pair of the Wild Type Ler and the hub1-1 Mutant. (A) Root growth rate. (B) Leaf lamina area. (C) Epidermal cell number on the abaxial side of the leaf. (D) Relative leaf expansion rate. (E) Average CDRs of the epidermal cells on the abaxial side of the leaf. (F) Stomatal index on the abaxial side of the leaf. Error bars indicate sd (n = 5).

Figure 4.

Effect of hub1-1 on Nuclear DNA Content during the Development of the First Leaf Pair by Flow Cytometry Analysis. (A) Ler. (B) hub1-1 mutant.

Figure 5.

GO Analysis of 934 Downregulated Genes in Shoot Apices of hub1-1 in the ATH1 Microarray Experiment. The yellow to orange color of the circles correspond to the level of significance of the overrepresented GO category from 0.05 and below according to a multiple t test with false discovery rate–corrected P value. The size of the circle is proportional to the number of genes in the category.

Figure 6.

Identification of the HUB1 Gene. (A) Map-based cloning strategy. Eight amplified fragment length polymorphism markers (SM) analyzed on 20 F2 phenotypically mutant plants identified the mutation in a 293-kb interval on chromosome 2. Recombinants used for fine-mapping delimited the locus to regions of 97 and 27 kb flanked by SNP markers (CER). The last interval of the 27-kb region contained four genes that were sequenced and allowed identification of the hub1-1 mutation at the end of exon 16 of At2g44950, which was designated HUB1. Black boxes and triangles represent exons and T-DNA insertions, respectively. The RING domain position is indicated with a black line. (B) Allelism test. Phenotype of the hub1-1/hub1-4 and hub1-1/hub1-3 heterozygotes grown in vitro, their respective parents, and the corresponding wild type (Ler for hub1-1 and Col for hub1-4 and hub1-3). (C) Complementation test of the hub1-1 mutant transformed with the p35S-HUB1 construct. (D) H2B ubiquitination assay by HUB1. Protein gel blot hybridization with HA antibody against HA-ubiquitin. Lane 1, no H2B substrate; lane 2, all components present; lane 3, no E1 enzyme; lane 4, no E2 (Rad6) enzyme; lane 5, no E3 HUB1; and lane 6, no ubiquitin.