Yuzhen Han

Fertilization in maize indeterminate gametophyte1 mutant

Summary.Mature embryo sacs of the maize mutant indeterminate gametophyte1 displayed different cellular patterns compared to those of the wild type. About 40% of the ig1 embryo sacs contained three or more synergids and two or more egg cells at the micropylar end. During fertilization in embryo sacs with two synergids, both of them frequently degenerated and were penetrated by two pollen tubes. 75% of the embryo sacs containing three or more synergid cells were penetrated by two or more pollen tubes, although most of them had only one degenerated synergid. Multiple fusions between the sperm cells and eggs frequently occurred in the same embryo sac, which subsequently generated multiple embryos. There were two or more central cells in about 33% of ig1 embryo sacs. The largest central cell was usually adjacent to the egg apparatus and contained two unfused polar nuclei, while those extra central cells located at the chalazal end usually had a single nucleus. Fertilization occurred only between the male gamete and the largest binucleate central cell. The extra central cells eventually degenerated after fertilization.

RRP41L, a Putative Core Subunit of the Exosome, Plays an Important Role in Seed Germination and Early Seedling Growth in Arabidopsis

In prokaryotic and eukaryotic cells, the 3′-5′-exonucleolytic decay and processing of RNAs are essential for RNA metabolism. However, the understanding of the mechanism of 3′-5′-exonucleolytic decay in plants is very limited. Here, we report the characterization of an Arabidopsis (Arabidopsis thaliana) transfer DNA insertional mutant that shows severe growth defects in early seedling growth, including delayed germination and cotyledon expansion, thinner yellow/pale-green leaves, and a slower growth rate. High-efficiency thermal asymmetric interlaced polymerase chain reaction analysis showed that the insertional locus was in the sixth exon of AT4G27490, encoding a predicted 3′-5′-exonuclease, that contained a conserved RNase phosphorolytic domain with high similarity to RRP41, designated RRP41L. Interestingly, we detected highly accumulated messenger RNAs (mRNAs) that encode seed storage protein and abscisic acid (ABA) biosynthesis and signaling pathway-related protein during the early growth stage in rrp41l mutants. The mRNA decay kinetics analysis for seed storage proteins, 9-cis-epoxycarotenoid dioxygenases, and ABA INSENSITIVEs revealed that RRP41L catalyzed the decay of these mRNAs in the cytoplasm. Consistent with these results, the rrp41l mutant was more sensitive to ABA in germination and root growth than wild-type plants, whereas overexpression lines of RRP41L were more resistant to ABA in germination and root growth than wild-type plants. RRP41L was localized to both the cytoplasm and nucleus, and RRP41L was preferentially expressed in seedlings. Altogether, our results showed that RRP41L plays an important role in seed germination and early seedling growth by mediating specific cytoplasmic mRNA decay in Arabidopsis.

Paternal cytoplasmic transmission in Chinese pine ( Pinus tabulaeformis )

Summary.In this paper, the stages of normal sexual reproduction between pollen tube penetration of the archegonium and early embryo formation in Pinus tabulaeformis are described, emphasizing the transmission of parental cytoplasm, especially the DNA-containing organelles – plastids and mitochondria. The pollen tube growing in the nucellus contained an irregular tube nucleus followed by a pair of sperm cells. The tube cytoplasm contained abundant organelles, including starch-containing plastids and mitochondria. The two sperm cells differed in their volume of cytoplasm. The leading sperm, with more cytoplasm, contained abundant plastids and mitochondria, while the trailing one, with a thin layer of cytoplasm, had very few organelles. The mature egg cell contained a great number of mitochondria, whereas it lacked normal plastids. At fertilization, the pollen tube penetrated into the egg cell at the micropylar end and released all of its contents, including the two sperms. One of the sperm nuclei fused with the egg nucleus, whereas the other one was retained by the receptive vacuole. Very few plastids and mitochondria of male origin were observed around the fusing sperm and egg nuclei, while the retained sperm nucleus was surrounded by a large amount of male cytoplasm. The discharged tube cytoplasm occupied a large micropylar area in the egg cell. In the free nuclear proembryo, organelles of maternal and paternal origins intermingled in the neocytoplasm around the free nuclei. Most of the mitochondria had the same features as those of the egg cell, but some appeared to be from sperm cells and tube cytoplasm. Plastids were obviously of male origin, with an appearance similar to those of the sperm or tube cells. After cellularization of the proembryo, maternal mitochondria became more abundant than the paternal ones and the plastids enlarged and began to accumulate starch. The results reveal the cytological mechanism for paternal inheritance of plastids and biparental inheritance of mitochondria in Chinese pine.

The AtTudor2, a protein with SN-Tudor domains, is involved in control of seed germination in Arabidopsis

The 4SN-Tudor domain protein is an almost ubiquitous eukaryotic protein with four Staphylococcal nuclease domains at the N terminus and a Tudor domain towards the C terminus. It has been found that Tudor-SN protein has multiple roles in governing gene expression during cell growth and development in animals. In plant, although Tudor-SN orthologs have been found in rice, pea and Arabidopsis, and are associated with cytoskeleton, their roles in growth and development are poorly understood. In this study, we investigated the function of Arabidopsis Tudor-SN protein, AtTudor. Our results indicated that the expression of AtTudor2 in seeds was evidently higher than in other tissues. Furthermore, we found that the expression of a key enzyme for GA biosynthesis, AtGA20ox3, was downregulated obviously in AtTudor2 T-DNA insertion mutant and AtTudor1/AtTudor2 RNAi transgenic lines. Together, our results suggest that AtTudor2 is involved in GA biosynthesis and seed germination of Arabidopsis.

Two SUMO Proteases SUMO PROTEASE RELATED TO FERTILITY1 and 2 Are Required for Fertility in Arabidopsis

SUMO proteases regulate plant fertility through multiple pathways. In plants, the posttranslational modification small ubiquitin-like modifier (SUMO) is involved in regulating several important developmental and cellular processes, including flowering time control and responses to biotic and abiotic stresses. Here, we report two proteases, SUMO PROTEASE RELATED TO FERTILITY1 (SPF1) and SPF2, that regulate male and female gamete and embryo development and remove SUMO from proteins in vitro and in vivo. spf1 mutants exhibit abnormal floral structures and embryo development, while spf2 mutants exhibit largely a wild-type phenotype. However, spf1 spf2 double mutants exhibit severe abnormalities in microgametogenesis, megagametogenesis, and embryo development, suggesting that the two genes are functionally redundant. Mutation of SPF1 and SPF2 genes also results in misexpression of generative- and embryo-specific genes. In vitro, SPF1 and SPF2 process SUMO1 precursors into a mature form, and as expected in vivo, spf1 and spf2 mutants accumulate SUMO conjugates. Using a yeast two-hybrid screen, we identified EMBRYO SAC DEVELOPMENT ARREST9 (EDA9) as an SPF1-interacting protein. In vivo, we demonstrate that EDA9 is sumolyated and that, in spf1 mutants, EDA9-SUMO conjugates increase in abundance, demonstrating that EDA9 is a substrate of SPF1. Together, our results demonstrate that SPF1 and SPF2 are two SUMO proteases important for plant development in Arabidopsis (Arabidopsis thaliana).

Polysomes are associated with microtubules in fertilized eggs of Chinese pine (Pinus tabulaeformis)

Summary.Transmission electron microscopy of immunogold-labeled Chinese pine egg cells before and after fertilization revealed that polysomes are associated with microtubules (MTs) from fertilization to the 2-nucleate embryo stage. Ribosome aggregates of various size and shape were randomly distributed in the cytoplasm of the eggs before fertilization. Single MTs or clusters were observed to be free of polysomes at this stage. Upon fertilization, all polysomes were attached to MTs, and this association persisted until the formation of the polarized embryo. Thereafter, the polysomes spread into the cytoplasm and no polysome-MT association was observed in the embryo. Some of the polysomes were attached to one end of the MTs, while others appeared to form contacts along their entire length. No polysome-microfilament association was observed at any stage of the development. The polysome-MT association may provide a mechanism for MT-dependent mRNA localization in early embryo development of this plant.

Overexpression of a LAM Domain Containing RNA-Binding Protein LARP1c Induces Precocious Leaf Senescence in Arabidopsis

Leaf senescence is the final stage of leaf life history, and it can be regulated by multiple internal and external cues. La-related proteins (LARPs), which contain a well-conserved La motif (LAM) domain and normally a canonical RNA recognition motif (RRM) or noncanonical RRM-like motif, are widely present in eukaryotes. Six LARP genes (LARP1a-1c and LARP6a-6c) are present in Arabidopsis, but their biological functions have not been studied previously. In this study, we investigated the biological roles of LARP1c from the LARP1 family. Constitutive or inducible overexpression of LARP1c caused premature leaf senescence. Expression levels of several senescence-associated genes and defense-related genes were elevated upon overexpression of LARP1c. The LARP1c null mutant 1c-1 impaired ABA-, SA-, and MeJA-induced leaf senescence in detached leaves. Gene expression profiles of LARP1c showed age-dependent expression in rosette leaves. Taken together, our results suggest LARP1c is involved in regulation of leaf senescence.

Orientation of microtubules suggests a role in mRNA transportation in fertilized eggs of Chinese pine (Pinus tabulaeformis)

Summary.Polysomes become associated with microtubules (MTs) in egg cells of Chinese pine upon fertilization, providing direct evidence for MT-based intracellular mRNA and polysome localization. We have investigated by immunoelectron microscopy the orientation and spatial distribution of MTs and their association with polysomes in the fertilized egg cells. There is a perinuclear accumulation of MTs and polysomes in the zygote soon after fertilization. At this time, some of the MTs are perpendicular to the nuclear envelope and directly connected to the outer membrane or nuclear-pore complexes (NPC) at one end, and the other ends reach to the outer tier or cortical MTs that are parallel to the long axis of the zygote. The polysomes in the perinuclear region show the same spatial and temporal pattern as the MTs. Immunolocalization of the mRNA-binding protein hnRNP indicates that the mRNAs are loaded onto the nucleus-associated MTs immediately after their export from the nuclear-pore complexes. The polysomes and mRNAs are then transported from these MTs to the outer tier and/or cortical MTs, where they further localize to the polar region of the cell.