Classification and quantification of leaf curvature

Abstract

Various mutants of Arabidopsis thaliana deficient in polarity, cell division, and auxin response are characterized by certain types of leaf curvature. However, comparison of curvature for clarification of gene function can be difficult without a quantitative measurement of curvature. Here, a novel method for classification and quantification of leaf curvature is reported. Twenty-two mutant alleles from Arabidopsis mutants and transgenic lines deficient in leaf flatness were selected. The mutants were classified according to the direction, axis, position, and extent of leaf curvature. Based on a global measure of whole leaves and a local measure of four regions in the leaves, the curvature index (CI) was proposed to quantify the leaf curvature. The CI values accounted for the direction, axis, position, and extent of leaf curvature in all of the Arabidopsis mutants grown in growth chambers. Comparison of CI values between mutants reveals the spatial and temporal variations of leaf curvature, indicating the strength of the mutant alleles and the activities of the corresponding genes. Using the curvature indices, the extent of curvature in a complicated genetic background becomes quantitative and comparable, thus providing a useful tool for defining the genetic components of leaf development and to breed new varieties with leaf curvature desirable for the efficient capture of sunlight for photosynthesis and high yields.

Fig. 1.

Diagrams of direction, axis, position, and extent of leaf curvature. Global curvature is designated for a leaf as a whole, while local curvature describes a part of a leaf. (A) Upward direction (UD) of leaf curvature. (B) Transverse axis (TA) and longitudinal axis (LA) of a curved leaf. (C) Positions of curvature in a leaf with apical, lateral, central, or basal curvature. (D) The distance between lateral margins of a leaf before flattening (ab) and after flattening (a′b′). (E) A schematic diagram of a roughly spoon-shaped leaf to show how ab and a′b′ were measured for global curvature. (F) A schematic diagram indicating how ab and a′b′ were measured for local wavy curvature. (G) Classification of curvature according to direction, axis, position, and extent of leaf curvature.

Fig. 2.

The rosette phenotypes of the mutant and transgenic lines, showing the leaf curvature caused by the mutant alleles. The mutant and transgenic lines were described in the Material and methods. Columbia (Col), Landsberg (Ler), and Nossen (Nos) plants were used as wild-type controls. Scale bars=1.00 cm.

Fig. 3.

Curvature indices (CIs) of the mutant leaves along the transverse (A) and longitudinal (B) axes. The seventh leaves of plants at the bolting stage (stage 5.1) were globally measured (Boyes et al., 2001). WT-N, Nossen; WT-L, Landsberg; WT-C, Columbia; 2mCNA, CNA::2mCNA plant; 2mREV, REV::2mREV plants; 7mTCP4, TCP4::7mTCP4; 5mARF16, ARF16::5mARF16 plants. Asterisks indicate significant differences between the mutant and the ild type (*P <0.05) by Student's t-tests. Error bars represent the standard deviation (SD). The number of leaves measured for each genotype was >20. Two asterisks indicate significant differences (t-test, **P <0.01).

Fig. 4.

Curvature indices (CIs) of local regions from leaves on different nodes and at different stages. (A) Local CI of jba-1D and jaw-1D leaves along the transverse axis. (B) Local CI of jba-1D and jaw-1D leaves along the longitudinal axis. (C) Global CI of hyl1 and jaw-1D leaves along the transverse axis. (D) Global CI of hyl1 and jaw-1D leaves along the longitudinal axis. The number of leaves measured for each genotype was >20. Error bars indicate the SD.

Fig. 5.

The phenotypes of the plants and leaves at different stages, showing the changes in the types of curvature. (A) Phenotypes of seedling (S), rosette, and flowering plant (F) of hyl1 mutants. (B) Phenotypes of leaves, showing curvature of cotyledons (C), and first, seventh, and 11th leaves of hyl1 mutants. (C) Phenotypes of seedling (S), rosette, and flowering plant (F) of jaw-1D mutants. (D) Phenotypes of leaves, showing curvature of cotyledons (C), and first, seventh, and 11th leaves of jaw-1D mutants.

Fig. 6.

The blade length-to-width (BLW) ratios (A) and petiole-to-blade length (PBL) ratios (B) of the mutant and transgenic lines. Leaf width and length and petiole length were measured on the plants at the bolting stage. WT-N, Nossen; WT-L, Landsberg; WT-C, Columbia; 2mCNA, CNA::2mCNA plants; 7mTCP4, TCP4::7mTCP4; 5mARF16, ARF16::5mARF16 plant. Error bars indicate the SD.

Fig. 7.

Accumulation of miRNAs and expression of target genes. (A) Accumulation of miRNAs in activation-tagged mutants of miR166g, miR319a, and miR160c genes. (B) RT-PCR analysis showing the expression levels of REV, TCP4, and ARF16 in mismatch mutants and gain-of-function miRNA mutant plants. (C) Transverse curvature of HYL1-R1, HYL1-R2, and HYL1-R3 lines. (D) RT-PCR analysis showing the expression levels of HYL1 and REV in three lines of 35S::HYL1 plants.