Plasma membrane H+ -ATPases-mediated cytosolic proton gradient regulates pollen tube growth.

The polar growth of pollen tubes is essential for the delivery of sperm cells during fertilization in angiosperms. How this polar growth is regulated has been a long-standing question. An in vitro pharmacological assay previously implicated proton flux in pollen tube growth, although genetic and cellular supporting evidence was lacking. Here, we report that protons form a gradient from the pollen tube tip to the shank region and this gradient is generated by three members of Arabidopsis H+ -ATPases (AHAs). Genetic analysis suggested that these AHAs are essential for pollen tube growth, thus providing new insight into the regulation of polar growth.

Why are ATP-driven microtubule minus-end directed motors critical to plants? An overview of plant multifunctional kinesins.

In plants, microtubule and actin cytoskeletons are involved in key processes including cell division, cell expansion, growth and development, biotic and abiotic stress, tropisms, hormonal signalling as well as cytoplasmic streaming in growing pollen tubes. Kinesin enzymes have a highly conserved motor domain for binding microtubule cytoskeleton assisting these motors to organise their own tracks, the microtubules by using chemical energy of ATP hydrolysis. In addition to this conserved binding site, kinesins possess non-conserved variable domains mediating structural and functional interaction of microtubules with other cell structures to perform various cellular jobs such as chromosome segregation, spindle formation and elongation, transport of organelles as well as microtubules-actins cross linking and microtubules sliding. Therefore, how the non-motor variable regions specify the kinesin function is of fundamental importance for all eukaryotic cells. Kinesins are classified into ~17 known families and some ungrouped orphans, of which ~13 families have been recognised in plants. Kinesin-14 family consisted of plant specific microtubules minus end-directed motors, are much diverse and unique to plants in the sense that they substitute the functions of animal dynein. In this review, we explore the functions of plant kinesins, especially from non-motor domains viewpoint, focussing mainly on recent work on the origin and functional diversity of motors that drive microtubule minus-end trafficking events.

The Arabidopsis TRM61/TRM6 complex is a bona fide tRNA N1-methyladenosine methyltransferase.

tRNA molecules, which contain the most abundant post-transcriptional modifications, are crucial for proper gene expression and protein biosynthesis. Methylation at N1 of adenosine 58 (A58) is critical for maintaining the stability of initiator methionyl-tRNA (tRNAiMet) in bacterial, archaeal, and eukaryotic tRNAs. However, although research has been conducted in yeast and mammals, it remains unclear how A58 in plant tRNAs is modified and involved in development. In this study, we identify the nucleus-localized complex AtTRM61/AtTRM6 in Arabidopsis as tRNA m1A58 methyltransferase. Deficiency or a lack of either AtTRM61 or AtTRM6 leads to embryo arrest and seed abortion. The tRNA m1A level decreases in conditionally complemented Attrm61/LEC1pro::AtTRM61 plants and this is accompanied by reduced levels of tRNAiMet, indicating the importance of the tRNA m1A modification for tRNAiMet stability. Taken together, our results demonstrate that tRNA m1A58 modification is necessary for tRNAiMet stability and is required for embryo development in Arabidopsis.

TICKET attracts pollen tubes and mediates reproductive isolation between relative species in Brassicaceae.

In flowering plants, pollen tubes are attracted to the ovule by secreted peptides to release the sperm cells for double fertilization. This process is species-specific and acts as an important stage of reproductive isolation between species. Here we identified a cysteine-rich peptide TICKET2 in Arabidopsis thaliana and its orthologs in Arabidopsis lyrata and Capsella rebella that can attract the conspecific pollen tubes, but not the pollen tubes of relative species in Brassicaceae. Genetic knockout of the AtTICKET subclade compromised the pollen tube attraction efficiency. This study identified a new pollen tube attracting signal and shed light on the molecular basis of reproductive isolation.

Maternal control of suspensor programmed cell death via gibberellin signaling.

Plant embryos are generated and develop in a stable and well-protected microenvironment surrounded by maternal tissue, which is vital for embryogenesis. However, the signaling mechanisms responsible for maternal tissue-to-proembryo communication are not well understood. Here, we report a pathway for maternal tissue-to-proembryo communication. We identify a DELLA protein, NtCRF1 (NtCYS regulative factor 1), which regulates suspensor programmed cell death (PCD). NtCRF1 can bind to the promoter of NtCYS and regulate the suspensor PCD-switch module NtCYS-NtCP14 in response to gibberellin (GA). We confirm that GA4, as a primary signal triggering suspensor PCD, is generated in the micropylar endothelium by the transient activation of NtGA3oxs in the maternal tissue. Thus, we propose that GA is a maternal-to-proembryo communication signal that is decoded in the proembryo by a GID1-CRF1-CYS-CP14 signaling cascade. Using this mode of communication, maternal tissue precisely controls the embryonic suspensor PCD and is able to nurse the proembryo in a stage-dependent manner.

New Insights into 5hmC DNA Modification: Generation, Distribution and Function.

Dynamic DNA modifications, such as methylation/demethylation on cytosine, are major epigenetic mechanisms to modulate gene expression in both eukaryotes and prokaryotes. In addition to the common methylation on the 5th position of the pyrimidine ring of cytosine (5mC), other types of modifications at the same position, such as 5-hydroxymethyl (5hmC), 5-formyl (5fC), and 5-carboxyl (5caC), are also important. Recently, 5hmC, a product of 5mC demethylation by the Ten-Eleven Translocation family proteins, was shown to regulate many cellular and developmental processes, including the pluripotency of embryonic stem cells, neuron development, and tumorigenesis in mammals. Here, we review recent advances on the generation, distribution, and function of 5hmC modification in mammals and discuss its potential roles in plants.

Gametophytic Pollen Tube Guidance: Attractant Peptides, Gametic Controls, and Receptors.

Pollen tube guidance in flowering plants is a unique and critical process for successful sexual reproduction. The pollen tube that grows from pollen, which is the male gametophyte, precisely navigates to the embryo sac, which is the female gametophyte, within the pistil. Recent advances have clarified the molecular framework of gametophytic pollen tube guidance. Multiple species-specific attractant peptides are secreted from synergid cells, the proper development and function of which are regulated by female gametes. Multiple receptor-like kinases on the pollen tube tip are involved in sensing species-specific attractant peptides. In this Update article, recent progress in our understanding of the mechanism of gametophytic pollen tube guidance is reviewed, including attraction by synergid cells, control of pollen tube guidance by female gametes, and directional growth of the pollen tube by directional cue sensing. Future directions in the study of pollen tube guidance also are discussed.

Arabidopsis CBP1 Is a Novel Regulator of Transcription Initiation in Central Cell-Mediated Pollen Tube Guidance.

In flowering plants, sperm cells are delivered to the embryo sac by a pollen tube guided by female signals. Both the gametic and synergid cells contribute to pollen tube attraction. Synergids secrete peptide signals that lure the tube, while the role of the gametic cells is unknown. Previously, we showed that CENTRAL CELL GUIDANCE (CCG) is essential for pollen tube attraction in Arabidopsis thaliana, but the molecular mechanism is unclear. Here, we identified CCG BINDING PROTEIN1 (CBP1) and demonstrated that it interacts with CCG, Mediator subunits, RNA polymerase II (Pol II), and central cell-specific AGAMOUS-like transcription factors. In addition, CCG interacts with TATA-box Binding Protein 1 and Pol II as a TFIIB-like transcription factor. CBP1-knockdown ovules are defective in pollen tube attraction. Expression profiling revealed that cysteine-rich peptide (CRP) transcripts were downregulated in ccg ovules. CCG and CBP1 coregulate a subset of CRPs in the central cell and the synergids, including the attractant LURE1. CBP1 is extensively expressed in multiple vegetative tissues and specifically in the central cell in reproductive growth. We propose that CBP1, via interaction with CCG and the Mediator complex, connects transcription factors and the Pol II machinery to regulate pollen tube attraction.

SPOROCYTELESS is a novel embryophyte-specific transcription repressor that interacts with TPL and TCP proteins in Arabidopsis.

Germlines in plants are formed de novo during post-embryonic development, while little is known about the mechanism that controls this process. In Arabidopsis, the earliest gene controlling this process is SPOROCYTELESS (SPL). A decade ago, we showed that loss of SPL function abolished sporogenesis in both male and female organs of Arabidopsis. However, its function is unclear up to now. In this study, we showed that SPL belongs to a novel transcription repressor family specific in embryophyte, which consists of 173 members in the land plants so far. All of them contain a conserved SPL-motif in their N-terminal and an ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif in the C-terminal, therefore designated as SPL-like, EAR-containing proteins (SPEARs). Consistently, SPL acts as a transcriptional repressor in yeast and tobacco cells, and SPEAR proteins are able to form homodimer and/or heterodimer with each other in vitro. Furthermore, SPEARs interact with the TOPLESS (TPL) co-repressors via the EAR motif and TCP family transcription factors in yeast cells. Together, we propose that SPL and SPEARs most likely belong to a novel transcription repressor family in land plants which may play a variety of developmental roles in plants.

Small RNA profiling in two Brassica napus cultivars identifies microRNAs with oil production- and development-correlated expression and new small RNA classes.

MicroRNAs (miRNAs) and small interfering RNAs are important regulators of plant development and seed formation, yet their population and abundance in the oil crop Brassica napus are still not well understood, especially at different developmental stages and among cultivars with varied seed oil contents. Here, we systematically analyzed the small RNA expression profiles of Brassica napus seeds at early embryonic developmental stages in high-oil-content and low-oil-content B. napus cultivars, both cultured in two environments. A total of 50 conserved miRNAs and 9 new miRNAs were identified, together with some new miRNA targets. Expression analysis revealed some miRNAs with varied expression levels in different seed oil content cultivars or at different embryonic developmental stages. A large number of 23-nucleotide small RNAs with specific nucleotide composition preferences were also identified, which may present new classes of functional small RNAs.

Ovule development in Arabidopsis: progress and challenge.

Female gametophyte, the central core of the ovule, is a simple seven-celled reproductive structure. Its stereotyped ontogeny provides a traceable model system to study mechanisms controlling cell growth, cell division, cell fate, pattern formation, and perhaps the function of essential genes in plants. An auxin concentration gradient was demonstrated for the first time in the embryo sac to control gametic cell fate. Mutant analysis also indicates a role of RNA processing in the mitotic progression of the gametophytic generation and cell fate determination in the embryo sac. Combined studies of genetics and transcriptome analysis revealed recently that epigenetic pathways play a critical role in female gametophyte development. In addition, the discovery that a large number of small secreted cysteine-rich proteins are enriched in embryo sac is of special interest. Except these insights and progresses, challenge ahead is to reveal the signaling pathways and their interactions that lead to the patterning of the female gametophyte.

SLOW WALKER3, encoding a putative DEAD-box RNA helicase, is essential for female gametogenesis in Arabidopsis.

RNA helicases are adenosine tri-phosphatases that unwind the secondary structures of RNAs and are required in almost any aspect of RNA metabolism. They are highly conserved from prokaryotic to eukaryotic organisms. However, their precise roles in plant physiology and development remain to be clarified. Here we report that the mutation in the gene SLOW WALKER3 (SWA3) results in the slow and retarded progression of mitosis during megagametogenesis in Arabidopsis. SWA3 is a putative RNA helicase of the DEAD-box subfamily. Mutant megagametophyte development is arrested at four- or eight-nucleate stages, furthermore, one of the synergids in about half of the mutant embryo sacs displays abnormal polarity, with its nucleus locating at the chalazal end, instead of the micropylar end in the wild-type. Transmission of the mutation through female gametophytes is severely reduced in swa3. However, a small portion of mutant embryo sacs are able to develop into mature and functional female gametophytes when pollination was postponed. The SWA3 in Arabidopsis is a homolog of Dbp8 in yeast. Dbp8 interacts with Efs2 and is essential for biogenesis of 18S rRNA in yeast. Our data suggest that SWA3 may form a complex with AtEfs2 and take roles in ribosomal biogenesis as RNA helicase during megagametogenesis in Arabidopsis.

Transgenic expression of DwMYB2 impairs iron transport from root to shoot in Arabidopsis thaliana.

In plant, iron uptake and homeostasis are tightly regulated to ensure its absorption from soil and to avoid excess iron in the cell. Many genes involved in this process have been identified during past several years, but there are many problems remain unsolved in the genetic regulation of whole plant iron trafficking and allocation. MYB transcription factors contain tandem repeats of a approximately 50 amino acid DNA-binding motif (R) and are involved in the regulation of many aspects of plant development, hormone signaling and metabolism. Here, we report that the ectopic expression of orchid R2R3-MYB gene DwMYB2 in Arabidopsis thaliana confers the transgenic plants hypersensitivity to iron deficiency. In DwMYB2 transgenic plants, the iron content in root is two-fold higher compared to that in wild-type root, while the reverse is true in shoot. This imbalance of iron content in root and shoot suggested that the translocation of iron from root to shoot was affected by the expression of DwMYB2 in the transgenic plants. Consistently, gene chip and reverse transcription-polymerase chain reaction analysis revealed that the ferric-chelate reductase gene, AtFRO2, and the iron transporter gene, AtIRT1 and AtIRT2, are up-regulated by DwMYB2 expression, while other potential iron transporters such as AtIREG1, AtFRD3 and NRAMP1 are down-regulated. In addition, the expression of several putative peptide transporters and transcription factors are also altered in the 35S::DwMYB2 transgenic lines. These data provide us insight into the whole plant translocation of iron and identify candidate genes for iron homeostasis in plants despite the fact that a heterologous gene was expressed.

Arabidopsis GLUTAMINE-RICH PROTEIN23 is essential for early embryogenesis and encodes a novel nuclear PPR motif protein that interacts with RNA polymerase II subunit III.

Precise control of gene expression is critical for embryo development in both animals and plants. We report that Arabidopsis thaliana GLUTAMINE-RICH PROTEIN23 (GRP23) is a pentatricopeptide repeat (PPR) protein that functions as a potential regulator of gene expression during early embryogenesis in Arabidopsis. Loss-of-function mutations of GRP23 caused the arrest of early embryo development. The vast majority of the mutant embryos arrested before the 16-cell dermatogen stage, and none of the grp23 embryos reached the heart stage. In addition, 19% of the mutant embryos displayed aberrant cell division patterns. GRP23 encodes a polypeptide with a Leu zipper domain, nine PPRs at the N terminus, and a Gln-rich C-terminal domain with an unusual WQQ repeat. GRP23 is a nuclear protein that physically interacts with RNA polymerase II subunit III in both yeast and plant cells. GRP23 is expressed in developing embryos up to the heart stage, as revealed by beta-glucuronidase reporter gene expression and RNA in situ hybridization. Together, our data suggest that GRP23, by interaction with RNA polymerase II, likely functions as a transcriptional regulator essential for early embryogenesis in Arabidopsis.

A novel class of MYB factors controls sperm-cell formation in plants.

In contrast to animals, the plant male germline is established after meiosis in distinctive haploid structures, termed pollen grains. The germline arises by a distinct asymmetric division of the meiotic products . The fates of the resulting vegetative and generative cells are distinct. In contrast to the larger vegetative cell, arrested in the G1 phase of the cell cycle, the smaller generative cell divides once to produce the two male gametes or sperm cells. Sperm cells are delivered to the female gametes by the pollen tube, which develops from the vegetative cell. In spite of recent efforts to understand pollen development , the molecular pathway controlling sperm-cell ontogenesis is unknown. Here, we present the isolation of DUO1, a novel R2R3 MYB gene of Arabidopsis, as the first gene shown to control male gamete formation in plants. DUO1 is specifically expressed in the male germline, and DUO1 protein accumulates in sperm-cell nuclei. Mutations in DUO1 produce a single larger diploid sperm cell unable to perform fertilization. DUO1 appears to be evolutionarily conserved in several plant species and defines a new subfamily of pollen-specific MYB genes.

Characterization, expression and phylogenetic study of R2R3-MYB genes in orchid.

cDNA fragments representing 21 R2R3-MYB genes were isolated by RT-PCR from the Dendrobium orchid hybrid Woo Leng. Six full-length cDNA clones were obtained from a flower cDNA library, four of which, DwMYB1, DwMYB2, DwMYB8 and DwMYB10, represent typical plant R2R3-MYB genes. The conceptual DwMYB4 protein is truncated at the C-terminal region and contains the R2 repeat and the N-terminal half of the R3 repeat (R2R3'). DwMYB4 expression is restricted to flowers. DwMYB9 contains an 8 amino acid N-terminal deletion in the R2 repeat (R2'R3) and is expressed at high levels in mature flower and inflorescence, but at very low levels in young flower buds. DwMYB8 and DwMYB10 show similar expression patterns and share very high sequence similarity in the N-terminal part of the MYB domain. Analysis of amino acid substitution indicated that the pattern and type of substitution between Arabidopsis and maize are quite different. Maize may have more conserved substitution in the MYB(BRH) domain than Arabidopsis.