Wyatt Paul

Premature dissolution of the microsporocyte callose wall causes male sterility in transgenic tobacco.

Male sterility in a petunia cytoplasmic male sterile line has been attributed to the early appearance of active callase, a beta-1,3-glucanase, in the anther locule. This leads to premature dissolution of the callose walls surrounding the microsporogenous cells. We have mimicked this aspect of the petunia line in transgenic tobacco by engineering the secretion of a modified pathogenesis-related vacuolar beta-1,3-glucanase from the tapetum prior to the appearance of callase activity in the locule. Plants expressing the modified glucanase from tapetum-specific promoters exhibited reduced male fertility, ranging from complete to partial male sterility. Callose appearance and distribution are normal in the male sterile transgenic plants up to prophase I, whereupon callose is prematurely degraded. Meiosis and cell division occur normally. The resultant microspores have an abnormally thin cell wall that lacks sculpturing. The tapetum shows hypertrophy. Male sterility is probably caused by bursting of the aberrant microspores at a time corresponding to microspore release. These results demonstrate that premature callose degradation is sufficient to cause male sterility and suggest that callose is essential for the formation of a normal microspore cell wall.

The isolation and characterisation of the tapetum-specific Arabidopsis thaliana A9 gene

The Brassica napus cDNA clone A9 and the corresponding Arabidopsis thaliana gene have been sequenced. The B. napus cDNA and the A. thaliana gene encode proteins that are 73% identical and are predicted to be 10.3 kDa and 11.6 kDa in size respectively. Fusions of an RNase gene and the reporter gene β-glucuronidase to the A. thaliana A9 promoter demonstrated that in tobacco the A9 promoter is active solely in tapetal cells. Promoter activity is first detectable in anthers prior to sporogenous cell meiosis and ceases during microspore premitotic interphase.The deduced A9 protein sequence has a pattern of cysteine residues that is present in a superfamily of seed plant proteins which contains seed storage proteins and several protease and α-amylase inhibitors.

The molecular biology of anther differentiation

Abstract This review attempts to summarise recent progress in our understanding of androgenesis in plants. There are two major divisions within this work. The first is a cytomorphological description of anther development and gametogenesis in model hermaphrodite plants. This is intended as a background against which to view the molecular biological information; this aspect constitutes the second main division in this chapter. The approach taken in describing these recent advances is intended to reflect, more or less, how these were made in practice. Gene cloning via cDNA library construction from whole anther organs or microspores represents the main technique, and therefore the merits of this approach, as applied to the developing anther, are discussed. In particular, the influence of cDNA library construction and screening methods on the type or class of genes identified to date are highlighted. The information generated from gene cloning is divided roughly into three sections. The first of these describes how the synthesis of specific mRNAs has provided valuable insights into the physiology and ontogeny of anther development. The physical and functional properties of cloned cDNAs cognate to mRNAs expressed within the anther are the subject of the second section. The third of these sections deals with anther-expressed genes, including analysis in transgenic plants. Finally, the commercial spin-off from this part of plant science, artificial male sterility, is discussed.

Patterns of gene expression in developing anthers of Brassica napus

The relationship between bud length, anther length and stage of anther development has been investigated in Brassica napus using a series of cytological markers that define steps in the process of male gametogenesis. It was determined that bud length is directly related to anther length and that anther or bud length is tightly linked to the stage of male gametogenesis within the anther. This simple correlation has enabled the construction of cDNA libraries representing transcripts expressed in defined stages of anther development, and the detailed examination of the developmental pattern of expression of anther RNAs.Two anther cDNA libraries were constructed, one from anthers of 1.2–1.8 mm long buds (sporogenesis library) and one from anthers of 1.8–4.0 mm long buds (microspore development library). A total of 19 independent cDNAs have been isolated by differential screening whose temporal expression patterns overlap and which together cover the stages of anther development from pre-meiotic microsporocytes to tri-nucleate pollen grains. The pattern of expression of each of these clones is unique and indicates that stages of anther development which cannot be easily distinguished by light microscopy can be recognised by virtue of the absence or presence of certain RNAs. Three cDNAs isolated from the sporogenesis library have been shown by in situ hybridisation to be tapetum-specific. In contrast, five clones isolated from the microspore development library are microspore-specific. These clones exhibit a pattern of expression different to those previously described in that their transcripts are absent in mature pollen grains. Thus these RNAs are probably required in microspore development rather than for the growth of the germinating pollen grain.

Duplicate Maize Wrinkled1 Transcription Factors Activate Target Genes Involved in Seed Oil Biosynthesis

WRINKLED1 (WRI1), a key regulator of seed oil biosynthesis in Arabidopsis (Arabidopsis thaliana), was duplicated during the genome amplification of the cereal ancestor genome 90 million years ago. Both maize (Zea mays) coorthologs ZmWri1a and ZmWri1b show a strong transcriptional induction during the early filling stage of the embryo and complement the reduced fatty acid content of Arabidopsis wri1-4 seeds, suggesting conservation of molecular function. Overexpression of ZmWri1a not only increases the fatty acid content of the mature maize grain but also the content of certain amino acids, of several compounds involved in amino acid biosynthesis, and of two intermediates of the tricarboxylic acid cycle. Transcriptomic experiments identified 18 putative target genes of this transcription factor, 12 of which contain in their upstream regions an AW box, the cis-element bound by AtWRI1. In addition to functions related to late glycolysis and fatty acid biosynthesis in plastids, the target genes also have functions related to coenzyme A biosynthesis in mitochondria and the production of glycerol backbones for triacylglycerol biosynthesis in the cytoplasm. Interestingly, the higher seed oil content in ZmWri1a overexpression lines is not accompanied by a reduction in starch, thus opening possibilities for the use of the transgenic maize lines in breeding programs.

Maternal Control of Nutrient Allocation in Plant Seeds by Genomic Imprinting

Imprinted genes are commonly expressed in mammalian placentas and in plant seed endosperms, where they exhibit preferential uniparental allelic expression. In mammals, imprinted genes directly regulate placental function and nutrient distribution from mother to fetus; however, none of the >60 imprinted genes thus far reported in plants have been demonstrated to play an equivalent role in regulating the flow of resources to the embryo. Here we show that imprinted Maternally expressed gene1 (Meg1) in maize is both necessary and sufficient for the establishment and differentiation of the endosperm nutrient transfer cells located at the mother:seed interface. Consistent with these findings, Meg1 also regulates maternal nutrient uptake, sucrose partitioning, and seed biomass yield. In addition, we generated an imprinted and nonimprinted synthetic Meg1 ((syn)Meg1) dosage series whereby increased dosage and absence of imprinting both resulted in an unequal investment of maternal resources into the endosperm. These findings highlight dosage regulation by genomic imprinting as being critical for maintaining a balanced distribution of maternal nutrients to filial tissues in plants, as in mammals. However, unlike in mammals, Meg1 is a maternally expressed imprinted gene that surprisingly acts to promote rather than restrict nutrient allocation to the offspring.

empty pericarp4 Encodes a Mitochondrion-Targeted Pentatricopeptide Repeat Protein Necessary for Seed Development and Plant Growth in Maize

The pentatricopeptide repeat (PPR) family represents one of the largest gene families in plants, with >440 members annotated in Arabidopsis thaliana. PPR proteins are thought to have a major role in the regulation of posttranscriptional processes in organelles. Recent studies have shown that Arabidopsis PPR proteins play an essential, nonredundant role during embryogenesis. Here, we demonstrate that mutations in empty pericarp4 (emp4), a maize (Zea mays) PPR-encoding gene, confer a seed-lethal phenotype. Mutant endosperms are severely impaired, with highly irregular differentiation of transfer cells in the nutrient-importing basal endosperm. Analysis of homozygous mutant plants generated from embryo-rescue experiments indicated that emp4 also affects general plant growth. The emp4-1 mutation was identified in an active Mutator (Mu) population, and cosegregation analysis revealed that it arose from a Mu3 element insertion. Evidence of emp4 molecular cloning was provided by the isolation of four additional emp4 alleles obtained by a reverse genetics approach. emp4 encodes a novel type of PPR protein of 614 amino acids. EMP4 contains nine 35–amino acid PPR motifs and an N-terminal mitochondrion-targeted sequence peptide, which was confirmed by a translational EMP4–green fluorescent protein fusion that localized to mitochondria. Molecular analyses further suggest that EMP4 is necessary to regulate the correct expression of a small subset of mitochondrial transcripts in the endosperm.

The Maize Transcription Factor Myb-Related Protein-1 Is a Key Regulator of the Differentiation of Transfer Cells

Transfer cells are highly modified plant cells specialized in the transport of solutes. They differentiate at many plant exchange surfaces, including phloem loading and unloading zones such as those present in the sink organs and seeds. In maize (Zea mays) seeds, transfer cells are located at the base of the endosperm. It is currently unknown how apical-basal polarity is established or why the peripheral cells at the base of the endosperm differentiate into transfer instead of aleurone cells. Here, we show that in epidermal cells committed to develop into aleurone cells, the ectopic expression of the transfer cell-specific transcriptional activator Myb-Related Protein-1 (MRP-1) is sufficient to temporarily transform them into transfer cells. These transformed cells acquire distinct transfer cell features, such as cell wall ingrowths and an elongated shape. In addition, they express a number of MRP-1 target genes presumably involved in defense. We also show that the expression of MRP-1 is needed to maintain the transfer cell phenotype. Later in development, an observed reduction in the ectopic expression of MRP-1 was followed by the reversion of the transformed cells, which then acquire aleurone cell features.

The promoter of ZmMRP-1, a maize transfer cell-specific transcriptional activator, is induced at solute exchange surfaces and responds to transport demands.

Transfer cells have specializations that facilitate the transport of solutes across plant exchange surfaces. ZmMRP-1 is a maize (Zea mays) endosperm transfer cell-specific transcriptional activator that plays a central role in the regulatory pathways controlling transfer cell differentiation and function. The present work investigates the signals controlling the expression of ZmMRP-1 through the production of transgenic lines of maize, Arabidopsis, tobacco and barley containing ZmMRP-1promoter:GUS reporter constructs. The GUS signal predominantly appeared in regions of active transport between source and sink tissues, including nematode-induced feeding structures and at sites of vascular connection between developing organs and the main plant vasculature. In those cases, promoter induction was associated with the initial developmental stages of transport structures. Significantly, transfer cells also differentiated in these regions suggesting that, independent of species, location or morphological features, transfer cells might differentiate in a similar way under the influence of conserved induction signals. In planta and yeast experiments showed that the promoter activity is modulated by carbohydrates, glucose being the most effective inducer.

Correct processing of the kiwifruit protease actinidin in transgenic tobacco requires the presence of the C-terminal propeptide.

A 35S cauliflower mosaic virus promoter and a tapetum-specific promoter were used to direct the synthesis in tobacco of preproactinidin and a derivative that lacked a C-terminal extension. Preproactinidin was processed into a form that migrated identically on protein gels with mature actinidin extracted from kiwifruit. This protein was proteolytically active in vitro, and high-level accumulation of this protein appeared to be detrimental to plant growth. Plants expressing an actinidin cDNA construct that lacked the sequence encoding the C-terminal propeptide were phenotypically normal but accumulated N-proactinidin, which was proteolytically active in vitro but did not self-cleave to mature actinidin. In transgenic tobacco, the C-terminal extension of actinidin is therefore required for correct processing.