Cytological and Transcriptomic Analyses Reveal Important Roles of CLE19 in Pollen Exine Formation

Abstract

The CLAVATA3/ESR-RELATED (CLE) peptide signals are required for cell-cell communication in several plant growth and developmental processes. However, little is known regarding the possible functions of the CLEs in the anther. Here, we show that a T-DNA insertional mutant, and dominant-negative (DN) and overexpression (OX) transgenic plants of the CLE19 gene, exhibited significantly reduced anther size and pollen grain number and abnormal pollen wall formation in Arabidopsis (Arabidopsis thaliana). Interestingly, the DN-CLE19 pollen grains showed a more extensively covered surface, but CLE19-OX pollen exine exhibited clearly missing connections in the network and lacked separation between areas that normally form the lacunae. With a combination of cell biological, genetic, and transcriptomic analyses on cle19, DN-CLE19, and CLE19-OX plants, we demonstrated that CLE19-OX plants produced highly vacuolated and swollen aborted microspores (ams)-like tapetal cells, lacked lipidic tapetosomes and elaioplasts, and had abnormal pollen primexine without obvious accumulation of sporopollenin precursors. Moreover, CLE19 is important for the normal expression of more than 1,000 genes, including the transcription factor gene AMS, 280 AMS-downstream genes, and other genes involved in pollen coat and pollen exine formation, lipid metabolism, pollen germination, and hormone metabolism. In addition, the DN-CLE19(+/+) ams(2/2) plants exhibited the ams anther phenotype and ams(+/2) partially suppressed the DN-CLE19 transgeneinduced pollen exine defects. These findings demonstrate that the proper amount of CLE19 signal is essential for the normal expression of AMS and its downstream gene networks in the regulation of anther development and pollen exine formation.Pollen grains are generated in the male reproductive organ anther of flowering plants and are essential for plant fertility. In the model plant Arabidopsis (Arabidopsis thaliana), pollen grains are ellipsoidal, and the pollen surface is covered by a reticulate exine. In contrast, rice (Oryza sativa) pollen grains are globular and have a smooth surface without the reticulate structure. A well-organized pollen wall structure is essential for the physical and chemical stability of the mature pollen by providing protection for pollen grains from environmental stresses, such as desiccation and microbial attacks (Scott et al., 2004). The pollen wall is composed mainly of three layers: the intine layer, the exine layer, and the pollen coat (Zinkl et al., 1999;Edlund et al., 2004;Blackmore et al., 2007). The intine is the innermost layer of the pollen wall immediately adjacent to the plasma membrane of the pollen vegetative cell and is composed mainly of pectin, cellulose, hemicellulose, hydrolytic enzymes, and hydrophobic proteins (Scott et al., 2004). Both the exine layer and the pollen coat layer are basically of a lipidic nature. The exine is largely formed from acyl lipid and phenylpropanoid precursors, which together form the mixed stable biopolymer known as sporopollenin (Gui...

Shifting the limits in wheat research and breeding using a fully annotated reference genome

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Identification and Functional Analysis of the CLAVATA3/EMBRYO SURROUNDING REGION (CLE) Gene Family in Wheat

“…It has a role in enabling the CLE peptide to rotate or bend [6]. Previous research has also shown that a glycine-to-threonine substitution at residue 6 gave the strongest antagonistic effect in the wild type [30]. In addition, the CLE domain also showed high conservation at residues 1, 4, 5, 9 and 11 in wheat, rice and Arabidopsis , and the residues are critical for the function of endogenous CLV3 in Arabidopsis .…”

“…AtCLE45 regulates both root protophloem and pollen development [24,29]. AtCLE19, apart from its role in controlling the root meristem, regulates cotyledon establishment, endosperm development and pollen-wall formation in Arabidopsis [30,31]. Tracheary Element Differentiation Inhibitory Factor (TDIF), a CLE-like peptide, was originally isolated as a suppressor of tracheary-element differentiation from the Zinnia elegans mesophyll cell-culture medium.…”

Arabidopsis Novel Microgametophyte Defective Mutant 1 Is Required for Pollen Viability via Influencing Intine Development in Arabidopsis

“…For callose examination, tetrads were dissected from the anthers and stained with 0.01% (w/v) of aniline blue as described previously ( Lu et al, 2014 ). Semi-thin sections of anthers from the flowers of mutants, RNAi lines, and the wild type were fixed overnight in FAA (50% of ethanol, 5.0% of glacial acid, and 3.7% of formaldehyde), dehydrated in a graded ethanol series (2 × 50, 70, 85, 95, 2 × 100%), and then embedded in Technovit 7100 resin (Heraeus Kulzer) as previously described ( Zhang et al, 2007 ; Wang et al, 2017 ). The embedded tissues were sectioned (1μm thick) using a motorized RM2265 rotary microtome (Leica Microsystems).…”