Balanced cell proliferation and expansion is essential for flowering stem growth control

Abstract

The postembryonic development of aboveground plant organs relies on a continuous supply of cells from the shoot apical meristem. Previous studies of developmental regulation in leaves and flowers have revealed the crucial role of coordinated cell proliferation and differentiation during organogenesis. However, the importance of this coordination has not been examined in flowering stems. Very recently, we attempted to identify regulatory factors that maintain flowering stem integrity. We found that the increased cell number in clavata (clv) mutants and the decreased cell size in de-etiolated (det)3-1 resulted in flowering stems that were thicker and thinner, respectively, than in wild-type (WT) plants. Interestingly, in the cell proliferation- and cell expansion-defective double mutant clv det3-1, the flowering stems often exhibited severe cracking, resulting in exposure of their inner tissues. In this study, further quantification of the cellular phenotypes in the cotyledons and leaves revealed no differences between det3-1 and clv3 det3-1. Together, the above findings suggest that the clv3 mutation in a det3-1 background primarily affects flowering stems, while its effect on other organs is likely negligible. We propose that the coordination between cell proliferation and differentiation is not only important during leaf development, but also plays a role in the growth control of Arabidopsis flowering stems.

Plant organs such as stems, leaves, and flowers are derived throughout a plant's life from the shoot apical meristem (SAM).¹ During organogenesis, the number of stem cells in the SAM is regulated by the CLV-WUS feedback circuit, whereby the peptide CLV3 is perceived by 3 receptors: CLV1, CLV2-SOL2/CRN, and RPK2.^2,3 Dynamic features of proliferative cell growth and differentiation have been investigated during leaf development in previous reports.^4-8 For example, the failure of plants to produce appropriate numbers and/or sizes of cells often results in abnormal leaves with an altered size, shape, or morphology.^9-12 Developmental biology has advanced greatly since the completion of the Arabidopsis genome sequence and the generation of publicly available large-scale mutant collections. It is believed that coordination (involving cell- and non-autonomous pathways) between cell proliferation and differentiation is essential to produce leaves with appropriate shapes and sizes.^6-15 Nonetheless, despite advances in our understanding of leaf development, flowering stems have not been well-characterized.

Isolation and characterization of clv3-101 det3-1 mutants

To address the above issue and identify novel regulatory factors that contribute to the maintenance of flowering stem integrity, we mutagenized det3-1 mutant seeds. Given the severe dwarf stem phenotype of the det3-1 mutant, we attempted to identify enhancers of det3-1 with novel morphological abnormalities in their flowering stems. We isolated A#26-2; det3-1, a mutant line that exhibited cracks and swelling in its flowering stems (Fig. 1A).¹⁶ DNA sequencing revealed that A#26-2; det3-1 contains a mutation in CLV3 (a novel mutant allele of clv3) and that all phenotypic aberrations, including stem cracking, are mimicked by the clv3-8, clv1-4, and clv2-1 alleles when paired with det3-1.¹⁶ To the best of our knowledge, the A#26-2 single mutant (A#26-2 sm) represents the first clv3 allele in a Columbia-0 (Col-0) background. Therefore, for convenience and with analogy to the names of other clv alleles in a Col-0 background (e.g., clv1-101 and clv2-101), A#26-2 sm will hereafter be referred to as clv3-101. Whereas the clv3 mutant, which had an enlarged SAM, formed thickened flowering stems and flowers with increased organ numbers, the det3-1 mutant exhibited thin flowering stems that contained few and small cells (Fig. 1B).¹⁶ Interestingly, despite severe damage due to stem cracking, all of the double mutants (clv3-101 det3-1; clv1-4 det3-1; clv2-1 det3-1; and clv3-8 det3-1) grew normally and were fertile (Fig. 1C).

Figure 1.

Stem cracking does not affect plant viability and fertility. (A) Stem development in clv3-101 det3-1 mutant plants. Image of a swollen (left panel) and cracked stem (right panel) produced by scanning electron microscopy. The images show stem portions dissected from the first internode at the base of the flowering stem. Scale bar, 1 mm. (B) Histological cross-sections revealed an increase in inner stem volume. WT, det3-1, clv3-8, and clv3-8 det3-1 plants were grown for 35 days after sowing (DAS), and their stems were collected and fixed for histological cross-sectioning. The images show a juxtaposed one-quarter of representative stem cross-sections of each genotype that were double-stained with safranin and toluidine blue (some of the images are modified from Maeda et al. [2014], Figure S6). Scale bar, 250 μm. (C) The gross phenotype of several double mutants. The mutants det3-1, clv3-101 det3-1, clv1-4 det3-1, clv2-1 det3-1, and clv3-8 det3-1 were grown for 65 DAS on rockwool. All of the double mutants were healthy and their growth was not affected by stem cracking. Scale bar, 2 cm.

To increase our understanding of the effects of the above mutations on other organs, the length of the petioles, the cell number, and the sizes of the cotyledons and first leaves were determined. Our results indicate that the lengths of the petioles and flowering stems were comparable between det3-1 and clv3-101 det3-1 (Fig. 2). While the flowering stem thickness was clearly enhanced in clv3-101 det3-1 compared to det3-1 (Fig. 1B), flowering stem elongation was similar in both genotypes (Fig. 2B).¹⁶ These results are suggestive of a differential effect of SAM cell mass increases that preferentially promote stem thickening rather than stem elongation in a det3-1 background. Moreover, although the cell number was increased slightly in the cotyledons and first leaves of clv3-101 det3-1 compared to det3-1, the overall organ and cell sizes were not significantly affected (Fig. 3). Therefore, stem cracking, which is known to primarily affect flowering stems, may be the result of stem indeterminate growth and/or cambium activity, which are not observed in leaves with determinate growth. Taken together, while the clv3 mutation in the det3-1 background resulted in noticeable flowering stem thickening, its effect on other organs (e.g., leaves) is likely negligible.

Figure 2.

Gross morphological phenotypes of clv3-101 det3-1. (A) Photos of WT, det3-1, and clv3-101 det3-1 mutant seedlings grown for 14 DAS on rockwool. Scale bar, 1 cm. (B) The petiole lengths for both cotyledons and for the first leaves were determined at 25 DAS. The flowering stem lengths were determined at 49 DAS. All data are given as means and the standard deviation (n = 8). Asterisk, 2-tailed Student's t-test at P < 0.05 between det3-1 and clv3-101 det3-1. F.L., first leaf.

Figure 3.

Cellular phenotypes of clv3-101 det3-1. WT, det3-1, and clv3-101 det3-1 mutant plants were grown for 25 DAS on rockwool. The cotyledons and first leaves from 16 individuals per genotype were dissected, fixed, and cleared for histological analysis. The palisade mesophyll tissue cell numbers and sizes were determined in mature cotyledons (A–C) and first leaves (D–F). The data represent the means and standard deviation (n = 16). Asterisk, 2-tailed Student's t-test at P < 0.005 between det3-1 and clv3-101 det3-1. F.L., first leaf.

Scanning electron microscopic analyses of flowering stem epidermal cells revealed clear differences between the WT and clv mutant plants. The cells were narrower and longer in clv3-101, clv3-8, and clv1-4 (but not in clv2-1) compared to those in wild type.¹⁶ Based on the restricted expression of CLAVATA genes and their role in limiting excessive cell proliferation at the SAM, it is possible that the different cell shape in these mutants represents a specific response to the thickened flowering stems. This agrees with the observation that the epidermal cells in the flowering stems of clv2-1 (exhibiting the weakest phenotype among the clv mutants examined so far) were indistinguishable from those of wild type.¹⁶

Flowering stems: An emerging model for coordinated growth studies

In plants, classical mutagenesis and screening strategies have been used to identify single-gene lesions that cause visible morphological phenotypes. The cloning of causal genes and characterization of their molecular function can be used to examine the observed phenotypes. However, the apparent organ size does not necessarily reflect the number and size of cells due to compensatory events between these 2 parameters.^7,9-12,17,18 Cell number and size variations theoretically fall into 8 different classes if we assume that each parameter remains unchanged, increases, or decreases.⁵ While some mutant classes were easily isolated (e.g. those showing a decreased cell number and increased cell size), other mutant classes (e.g., those showing an increased cell number and cell size) were nearly absent.^5,7,9 In most cases, double or triple mutants are constructed due to genetic redundancy and gene duplication. These approaches rendered our interpretations unilateral, and could not be used to examine genetically distant pathways. Based on these results, using det3-1 (which shows a decrease in cell size and a clear flowering stem phenotype) simplified the identification of stem-specific phenotypes.

Coordinated growth (between cell proliferation and differentiation) at the tissue, organ, and organismal levels plays an important role in developmental biology; however, the consequences of lost coordination have been addressed in typical plant model organs, including leaves and roots. Plant leaves and most floral organs (which can be considered modified leaves) display flattened structures. It has previously been observed that coordination between cell proliferation and expansion is common during leaf development, but also occurs in flowering stems.¹⁶ Additional studies are required to examine the mechanism behind stem cracking at the molecular level.

Final remarks and future prospects

Mechanical perturbation is known to inhibit inflorescence stem elongation in both herbaceous (including Arabidopsis thaliana) and woody plants, and has implications for how plants adjust their size and form to survive in mechanically perturbed environments.¹⁹ In fact, the effects of mechanical perturbations on the biomechanics, primary growth, and secondary tissue development of inflorescence stems of Arabidopsis have been addressed.¹⁹ Changes in the mechanical properties of mechanically perturbed plants are associated with significant changes in tissue geometry, size, and position of the pith, lignified interfascicular tissue, and cortex, as well as a reduction in the density of lignified cells.¹⁹ Therefore, to maintain upright axes under conditions of severe mechanical perturbation, Arabidopsis developed “short and flexible” rather than “short and rigid” flowering stems. ¹⁹ Importantly, cross-sections of stems showing interfascicular regions revealed that clv3-8 det3-1 fibers are underdeveloped with thin walls compared to wild type, det3-1, and clv3-8 (Fig. 1B).¹⁶ In this case, interfascicular regions may represent mechanically weak points that can crack when challenged by an outward mechanical stress exerted by the expanding inner pith cells. This is possible since cracks on the surface often spread in parallel to the primary flowering stem.¹⁶ The hypothesis could be confirmed by constructing double mutants between clv3 and fragile fiber8 and determining whether they exhibit stem cracking.²⁰ In addition, serial cross-sectioning of clv3-8 det3-1 flowering stems at regions spanning the cracks should increase our understanding of whether cracks form preferentially in the interfascicular regions between 2 neighboring vascular bundles. However, why fibers of the interfascicular regions in clv3-8 det3-1 are underdeveloped remains unclear. Overall, the molecular basis of stem cracking requires further characterization.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We would like to thank the Ministry of Education, Culture, Sports, Science and Technology of Japan [Grant-in-Aid for Young Scientists (B) (to A.F.); Scientific Research on Priority Areas (to H.T.); Creative Scientific Research (to H.T.); Grant-in-Aid for Scientific Research on Innovative Areas (to A.F., G.H., S.S. and H.T.)] for the funding that made this work possible.