Characterization of a Null Allelic Mutant of the Rice <i>NAL1</i> Gene Reveals Its Role in Regulating Cell Division

<div><p>Leaf morphology is closely associated with cell division. In rice, mutations in <i>Narrow leaf 1 (NAL1)</i> show narrow leaf phenotypes. Previous studies have shown that <i>NAL1</i> plays a role in regulating vein patterning and increasing grain yield in <i>indica</i> cultivars, but its role in leaf growth and development remains unknown. In this report, we characterized two allelic mutants of <i>NARROW LEAF1 (NAL1)</i>, <i>nal1-2</i> and <i>nal1-3</i>, both of which showed a 50% reduction in leaf width and length, as well as a dwarf culm. Longitudinal and transverse histological analyses of leaves and internodes revealed that cell division was suppressed in the anticlinal orientation but enhanced in the periclinal orientation in the mutants, while cell size remained unaltered. In addition to defects in cell proliferation, the mutants showed abnormal midrib in leaves. Map-based cloning revealed that <i>nal1-2</i> is a null allelic mutant of <i>NAL1</i> since both the whole promoter and a 404-bp fragment in the first exon of <i>NAL1</i> were deleted, and that a 6-bp fragment was deleted in the mutant <i>nal1-3</i>. We demonstrated that <i>NAL1</i> functions in the regulation of cell division as early as during leaf primordia initiation. The altered transcript level of G1- and S-phase-specific genes suggested that <i>NAL1</i> affects cell cycle regulation. Heterogenous expression of <i>NAL1</i> in fission yeast (<i>Schizosaccharomyces pombe</i>) further supported that <i>NAL1</i> affects cell division. These results suggest that <i>NAL1</i> controls leaf width and plant height through its effects on cell division.</p></div

Phenotypes of <i>nal1-2</i>.

<p>(A) Morphology of wild-type (WT; Nipponbare, left) and <i>nal1-2</i> (right) plants at the heading stage (bar = 5 cm). (B) Comparison of leaf width between the WT (left) and <i>nal1-2</i> (right) (bar = 5 mm). (C) Comparison of leaf length between the WT (left) and <i>nal1-2</i> (right) (bar = 5 cm). (D) Comparison of seed phenotype between the WT (left) and <i>nal1-2</i> (right) (bar = 0.5 mm). (E, F) Transverse sections through the middle part of the mature upper second leaves of WT (E) and <i>nal1-2</i> plants (F) (bars = 1 mm). Asterisk and circle in E and F indicate large vein and small vein, respectively.</p

Histological analyses of leaves.

<p>(A, B) The abaxial epidermis of the upper second leaf blade of wild-type (WT) (A) and <i>nal1-2</i> (B) plants (bars = 50 μm). (C–F) Cross sections through the middle part of the upper second leaf blade of WT (C, E) and <i>nal1-2</i> (D, F) plants. Red double-headed arrows indicate the thickness of the leaf mesophyll adjacent the midrib (C, D) and small veins (E, F). Red curves in C and D outline the parenchyma cell layers (bars = 50 μm). (G) Magnified images of the red-boxed regions in A (left) and B (right). Black arrows show the position of stomatal guard cells (bar = 50 μm). (H–L) Comparison of the leaf blade width (H), epidermal cell number (I), epidermal cell size (J), leaf mesophyll thickness (K), and the number of mesophyll cell layers (L) between the WT and <i>nal1-2</i>. Epidermal cell number was calculated by dividing each leaf blade width by the corresponding epidermal cell width. Data are shown as means ± standard error (SE) (H, I, K, and L, n ≥ 10; J, n ≥ 500). Student’s t-test was used to analyze significant differences between the WT and <i>nal1-2</i>. *, 0.01 ≤ P < 0.01; **, P < 0.01.</p

Morphometric analysis of the <b><i>nal1-2</i></b> mutant.

<p>Phenotypes of <i>nal1-2</i> were measured at filling stage. Seconde leaf is the upper second leaf. Values are the mean ± standard error (SE) (n≥15). Asterisks reveal the significance of differences between wild-type and <i>nal1-2</i> plants, which is obtained by Student’s <i>t</i>-test: *, 0.01 ≤ <i>P</i> < 0.01; **, <i>P</i> < 0.01.</p><p>Morphometric analysis of the <b><i>nal1-2</i></b> mutant.</p

Proliferative status of cell divisions in the internode of <i>nal1-2</i>.

<p>(A) The lengths of the panicle and internodes are compared between the wild type (WT; left) and <i>nal1-2</i> (right; bar = 5 cm). (B, C) Cross sections through the middle part of the internode III in the WT (B) and <i>nal1-2</i> (C). Red double-headed arrows indicate the thickness of the internode III. Bars = 0.5 mm. (D) Comparison of longitudinal sections of internode III between the WT (left) and <i>nal1-2</i> (right). Red and green boxes outline parenchyma cells (bar = 50 μm). (E, F) Images of transverse sections of internode are zoomed-in. Cell layers of the WT (E) and <i>nal1-2</i> (F) are contoured by red curves (bars = 100 μm). (G–L) Comparison of internodes length (G), internodes thickness (H), parenchyma cell size along the transverse axis (I), the number of internode III cell layers (J), and parenchyma cell length along the longitudinal axis (K) in internode III between the WT and <i>nal1-2</i>. I–IV represents the upper four internodes, respectively. Results are shown as means ± standard error (SE) (G–J, n = 10; I, n ≥ 230; K, n ≥ 200). Asterisks represent significant differences between WT and <i>nal1-2</i> plants based on Student’s t-test: *, 0.01 ≤ P < 0.01; **, P < 0.01. (L) The number of cells along the longitudinal axis in internode III. Data were estimated by dividing the average internode length by the average parenchyma cell length.</p

Heterologous expression of <i>NAL</i>1 in yeast (<i>S</i>. <i>Pombe</i>).

<p>(A, B) Growth of <i>S</i>. <i>pombe</i> transformed with <i>pREP1-NAL1</i>, <i>pREP1-nal1-3</i>, and the empty vector <i>pREP</i>1 in the presence (+thiamine, the expression of gene is repressed) and absence of thiamine (-thiamine, the expression of gene is induced). (A) Spreading yeast cells on the plate. (B) Spotting yeast cells on the plate after a serial of dilution. (C) Phenotype of yeast cells under bright and UV light microscopy in the absence of thiamine. LM, light microscopy; UV, observation of 4’,6-diamidino-2-phenylindole (DAPI) stained cells under UV light (bars = 10 μm).</p

Map-based cloning of <i>nal1-2</i>.

<p>(A) The gene responsible for the <i>nal1-2</i> phenotype was located in an approximately 67-kb region on chromosome 4 (chr. 4). Vertical lines indicate the positions of molecular markers and the number of recombinants. (B) Seven putatively functional open reading frames (ORFs) and a retrotransposon within the fine mapping region. Red broken lines represent the position of the deleted fragment. The deleted region is modified by fragmentation. Green arrows represent putatively functional genes and the black arrow points to the retrotransposon. (C) Both the whole promoter and part of the first exon of LOC_Os04g52479 (<i>NAL1</i>) were deleted in the <i>nal1-2</i> mutant. A 6-bp deletion located in the first exon was also detected in the <i>nal1-3</i> mutant. The letters ATG and TAG represent the start and stop codon, respectively. The red arrow shows the edge of the deleted fragment. Yellow and blue boxes represent the regulatory region and ORF, respectively. Gray lines indicate the positions of primers designed for semiquantitative RT-PCR analyses. (D, E) Genetic complementation of <i>nal1-2</i>. From left to right are WT, <i>nal1-2</i>, and two representative lines of transgenic plant. (D) Gross morphology at the early heading stage showing complementation of plant height. (E) Central part of flag leaves showing complementation of leaf width. (F) The transcript level of <i>NAL1</i> was detected by semi-quantitative RT-PCR analyses. Total RNAs were isolated from the first two leaves of WT and <i>nal1-2</i> plants. Ubiquitin was used as an amplification control.</p