Combinatorial interactions between LBD10 and LBD27 are essential for male gametophyte development in Arabidopsis

Abstract

The LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) genes encodes a unique class of transcription factors that play roles in diverse aspects of lateral organ development in plants. The Arabidopsis LBD gene family comprises 42 members and biological functions of most of the LBD genes are unknown. Our molecular genetic analysis and a variety of functional assays including expression analysis, genetic transmission and pollen viability assays, and pollen development analysis demonstrated that LBD10 co-acts with SIDECAR POLLEN(SCP)/LBD27 to control an early stage of microspore development but also plays a distinct role at later bicellular and tricellular pollen stages and that these 2 LBD genes are essential for Arabidopsis pollen development. We also showed that LBD10 and LBD27 interact with each other to be localized into the nucleus. Our subcellular localization analysis of LBD10 in comparison with LBD27 during pollen development indicated that regulated protein degradation may be involved in determining spatially and temporally distinct and overlapping expression patterns of these LBD transcription factors, contributing to distinct and combinatorial roles of LBD10 and LBD27 in Arabidopsis pollen development.

Abbreviations

ASL

asymmetric leaves 2-like

LBD

lateral organ boundaries domain

SCP

sidecar pollen

A pollen grain is a male gametophyte formed in anthers, the male parts of flowers. During pollen development in Arabidopsis, the diploid pollen mother cell undergoes meiosis to produce tetrad encapsulating tapetum cell. The unicellular haploid microspores released are polarized by microtubule-dependent migration of the nucleus, which undergoes an asymmetric cell division called Pollen Mitosis I (PMI) to produce bicellular pollen, consisting of a larger vegetative nucleus and a smaller generative cell engulfed within the vegetative cell cytoplasm. The generative cell undergoes a symmetric cell division called Pollen Mitosis II (PMII) to produce tricellular pollen, which has a vegetative cell and 2 sperm cells.^1,2 The vegetative cell nucleus and the 2 sperm cells migrate into the pollen tube to the embryo sac for double fertilization.³ Recent studies have revealed several components and the transcription factors which play important roles during male gametophyte development in Arabidopsis.^1,2 GEMINI POLLEN1, a member of microtubule-associated protein family, is involved in nuclear positioning and asymmetric division,⁴ and TWO-IN-ONE, a homolog of FUSED kinase, plays a role in cytokinesis of the male gametophyte.⁵ The R2R3 MYB transcription factor DUO1 and a nuclear protein DUO3 control proliferation of male germ line.^6,7

Analysis of lbd27 mutant indicated that LBD27 plays a role in the correct timing and orientation of the asymmetric microspore division.⁸ Several LBD gene family members have been shown to play important roles in diverse aspects of lateral organ development in Arabidopsis, rice and maize.⁹ In Arabidopsis, LBD6 functions in the establishment of adaxial-abaxial polarity,¹⁰ LBD30 is involved in embryogenesis,¹¹ and LBD16, LBD18, LBD29 and LBD33 control lateral root development.^12-14 As the Arabidopsis eFP browser showed that LBD10 expression was specifically and strongly increased during pollen development, we conducted functional analysis of LBD10 gene as well as lbd10 lbd27 double mutant analysis for siliques, pollen morphology, thin-sections of anther, and DAPI-staining of mature pollen, showing that LBD10 synergistically interacts with LBD27 to control pollen development and that these 2 LBD genes are essential for pollen function.¹⁵ Analysis of mature pollen grains and developmental analysis indicated that LBD10 is involved in microspore polarization prior to the first asymmetric division and also in germ cell mitosis. These results indicated that LBD10 plays an overlapping role with LBD27 during PMI, but also plays a distinct role during PMII. Expression analysis of LBD10:GFP and LBD27:RFP as well as H2B:GFP fusion proteins under the control of its own promoter during pollen development in transgenic Arabidopsis showed that LBD10 and LBD27 are expressed in both the vegetative cell and germ cell nuclei throughout the pollen development but display distinct subcellular accumulation. The results showed that LBD27:RFP was detected in the central nucleus of microspores but no longer detected by early stage of bicellular pollen, whereas LBD10:GFP was detected in the central nucleus of early microspore and the nucleus of mid-late bicellular pollen, but at a later stage of development, the LBD10 GFP signal was detected only in the vegetative cell nucleus. These results indicate that protein degradation mechanism might operate to control spatially and temporarily differential accumulation of LBD10 and LBD27 for their distinct and overlapping roles during pollen development. Degradation of cell cycle inhibitors by SCF^FBL17 E3 ubiquitin ligase has been shown to control twin sperm-cell production.¹⁶ Recent studies have demonstrated that degradation of repressors by SCFs through the ubiquitin-proteasome pathway plays a key role in plant hormone auxin, jasmonate, gibberellin, ethylene and abscisic acid signaling.^17,18 Circadian clock and photomorphogenesis are also regulated by proteasome-mediated protein degradation.^18,19 It will be interesting to investigate if SCFs are involved in determining differential accumulation and subcellular localization of LBD10 and LBD27 for their biological functions.

A variety of studies demonstrated that nuclear localization of some transcription factors is mediated by dimerization with their binding partners for displaying different cellular responses.^20-24 Our protein-protein interaction studies using bimolecular fluorescence complementation assays have shown that LBD10 and LBD27 are localized in the nucleus as a heterodimer in Arabidopsis protoplasts. Confocal microscopy and fluorescence microscopy showed that both LBD10 and LBD27 exist in the nucleus of the early microspores and polarized microspores. These results indicate that a combinatorial regulation of LBD10 and LBD27 at the early stage of pollen development is exerted in part through the heterodimerization-mediated nuclear translocation. The mRNA accumulation from 20 different LBD genes had been detected in floral organs.²⁵ Arabidopsis pollen RNA-seq data sets have also shown that in addition to LBD10 and LBD27, the mRNAs from other LBDs including LBD22, LBD25, and LBD36, were significantly detected in pollen.²⁶ Heterodimerization between 2 different transcription factors facilitates nuclear localization and contributes to transcriptional regulation by modulating DNA-binding specificity and affinity and/or the recruitment of different binding partners.²⁷ Thus the protein-protein interactions among these LBD transcription factors may provide redundancy as well as diversity to their biological functions during pollen developmental process.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This study was supported by grants from the Next-Generation BioGreen 21 Program (PJ01104701), the Rural Development Administration, and the Basic Science Research Program (grant no. 2013R1A1A2062335) through the National Research Foundation of Korea, funded by the Ministry of Education, Science, and Technology of Korea, Republic of Korea to J. Kim.