Christine Rochat

An integrated overview of seed development in Arabidopsis thaliana ecotype WS

This work is part of a research program aiming at identifying and studying genes involved in Arabidopsis thaliana seed maturation. We focused here on the Wassilewskija ecotype seed development and linked physiological and biochemical data, including protein, oil, soluble sugars, starch and free amino acid measurements, to embryo development, to obtain a complete and thorough reference data set. A. thaliana seed development can be divided into three stages. During early embryogenesis (i.e. morphogenesis), seed weight and lipid content were low whereas important amounts of starch were transiently accumulated. In the second stage, or maturation phase, a rapid increase in seed dry weight was observed and storage oils and proteins were accumulated in large quantities, accounting for approximately 40% of dry matter each at the end of this stage. During the third and last stage (late maturation including acquisition of desiccation tolerance), seed dry weight remained constant while an acute loss of water took place in the seed. Storage compound synthesis ended concomitantly with sucrose, stachyose and raffinose accumulation. This study revealed the occurrence of metabolic activities such as protein synthesis, in the final phase of embryo desiccation. A striking correlation between peaks in hexose to sucrose ratio and transition phases during embryogenesis was observed.

Storage reserve accumulation in Arabidopsis: metabolic and developmental control of seed filling.

Abstract In the life cycle of higher plants, seed development is a key process connecting two distinct sporophytic generations. Seed development can be divided into embryo morphogenesis and seed maturation. An essential metabolic function of maturing seeds is the deposition of storage compounds that are mobilised to fuel post-germinative seedling growth. Given the importance of seeds for food and animal feed and considering the tremendous interest in using seed storage products as sustainable industrial feedstocks to replace diminishing fossil reserves, understanding the metabolic and developmental control of seed filling constitutes a major focus of plant research. Arabidopsis thaliana is an oilseed species closely related to the agronomically important Brassica oilseed crops. The main storage compounds accumulated in seeds of A. thaliana consist of oil stored as triacylglycerols (TAGs) and seed storage proteins (SSPs). Extensive tools developed for the molecular dissection of A. thaliana development and metabolism together with analytical and cytological procedures adapted for very small seeds have led to a good description of the biochemical pathways producing storage compounds. In recent years, studies using these tools have shed new light on the intricate regulatory network controlling the seed maturation process. This network involves sugar and hormone signalling together with a set of developmentally regulated transcription factors. Although much remains to be elucidated, the framework of the regulatory system controlling seed filling is coming into focus.

Very-Long-Chain Fatty Acids Are Involved in Polar Auxin Transport and Developmental Patterning in Arabidopsis

This work identifies the immunophilin PASTICCINO1 as a member of the complex necessary for very-long-chain fatty acid synthesis and demonstrates that fatty acids are directly involved in auxin carrier distribution during organogenesis. Very-long-chain fatty acids (VLCFAs) are essential for many aspects of plant development and necessary for the synthesis of seed storage triacylglycerols, epicuticular waxes, and sphingolipids. Identification of the acetyl-CoA carboxylase PASTICCINO3 and the 3-hydroxy acyl-CoA dehydratase PASTICCINO2 revealed that VLCFAs are important for cell proliferation and tissue patterning. Here, we show that the immunophilin PASTICCINO1 (PAS1) is also required for VLCFA synthesis. Impairment of PAS1 function results in reduction of VLCFA levels that particularly affects the composition of sphingolipids, known to be important for cell polarity in animals. Moreover, PAS1 associates with several enzymes of the VLCFA elongase complex in the endoplasmic reticulum. The pas1 mutants are deficient in lateral root formation and are characterized by an abnormal patterning of the embryo apex, which leads to defective cotyledon organogenesis. Our data indicate that in both tissues, defective organogenesis is associated with the mistargeting of the auxin efflux carrier PIN FORMED1 in specific cells, resulting in local alteration of polar auxin distribution. Furthermore, we show that exogenous VLCFAs rescue lateral root organogenesis and polar auxin distribution, indicating their direct involvement in these processes. Based on these data, we propose that PAS1 acts as a molecular scaffold for the fatty acid elongase complex in the endoplasmic reticulum and that the resulting VLCFAs are required for polar auxin transport and tissue patterning during plant development.

gurke and pasticcino3 mutants affected in embryo development are impaired in acetyl-CoA carboxylase

Normal embryo development is required for correct seedling formation. The Arabidopsis gurke and pasticcino3 mutants were isolated from different developmental screens and the corresponding embryos exhibit severe defects in their apical region, affecting bilateral symmetry. We have recently identified lethal acc1 mutants affected in acetyl‐CoA carboxylase 1 (ACCase 1) that display a similar embryo phenotype. A series of crosses showed that gk and pas3 are allelic to acc1 mutants, and direct sequencing of the ACC1 gene revealed point mutations in these new alleles. The isolation of leaky acc1 alleles demonstrated that ACCase 1 is essential for correct plant development and that mutations in ACCase affect cellular division in plants, as is the case in yeast. Interestingly, significant metabolic complementation of the mutant phenotype was obtained by exogenous supply of malonate, suggesting that the lack of cytosolic malonyl‐CoA is likely to be the initial factor leading to abnormal development in the acc1 mutants.

Purification, characterization and physiological role of sucrose synthase in the pea seed coat (Pisum sativum L.)

The seed coat is a maternal organ which surrounds the embryo and is involved in the control of its nutrition. This study with pea (Pisum sativum L.) was conducted to understand more fully the sucrose/starch interconversions occurring in the seed coat. The concentrations of soluble sugars, the starch content, and the activities of the sucrose-metabolizing enzymes, sucrose synthase (Sus; EC 2.4.1.13), alkaline and soluble acid invertase (EC 3.2.1.26) and sucrose-phosphate synthase (SPS; EC 2.4.1.14) were compared at four developmental stages during seed filling. Among the four enzymes, only Sus activity was very high and strongly correlated with the starch concentration in the seed coat. Sucrose synthase catalyses the cleavage of sucrose in the presence of UDP into UDP-glucose and fructose. Sucrose synthase was purified from pea seed coats in a three-step protocol, consisting of diethylaminoethyl-Sephacel chromatography, gel filtration and affinity chromatography. The enzyme was characterized at the biochemical and molecular levels. Sucrose synthase exhibits biochemical properties which allow it to function in the direction of both sucrose cleavage and synthesis. The mass-action ratio of its four substrates was close to the theoretical equilibrium constant at the four developmental stages we studied. A labelling experiment on seed coats has shown that Sus activity is reversible in vivo and can produce 37% of neosynthesized sucrose in the seed coat cells (minimum value). It is concluded that Sus could play a central role in the control of sucrose concentration in the seed coat cells in response to the demand for sucrose in the embryo during the development of the seed.

Characterization of a tissue-specific and developmentally regulated β-1,3-glucanase gene in pea (Pisum sativum)

As part of a search for seed coat-specific expressed genes in Pisum sativum cv. Finale by PCR-based methods, we identified and isolated a cDNA encoding a β-1,3-glucanase, designated PsGNS2. The deduced peptide sequence of PsGNS2 is similar to a subfamily of β-1,3-glucanases, which is characterized by the presence of a long amino acid extension at the C-terminal end compared to the other β-1,3-glucanases. PsGNS2 is expressed in young flowers and in the seed coat and is weakly expressed in vegetative tissues (roots and stems) during seedling development. It is not inducible by environmental stress or in response to fungal infection. In developing pea flowers the transcript is detectable in all four whirls. In the seed coat the expression is temporally and spatially regulated. High abundance of the transcript became visible in the seed coat when the embryo reached the late heart stage and remained until the mid seed-filling stage. In situ hybridization data demonstrated that the expression of PsGNS2 is restricted to a strip of the inner parenchyma tissue of the seed coat, which is involved in temporary starch accumulation and embryo nutrition. This tissue showed also less callose deposits than the other ones. The 5′ genomic region of PsGNS2 was isolated and promoter activity studies in transgenic Medicago truncatula showed a seed-specific expression. Highest activity of the promoter was found in the seed coat and in the endosperm part of the seed.

Amino Acid Transport

Depending on the species, nitrogen may be reduced either by symbiosis with bacteria (legumes, see Chap. 3.1), directly after root uptake from the soil (see Chap. 2), or after xylem transport of the root nitrate to the leaf (most herbaceous species, e.g. Gossypium, Xanthium). Some plants also reduce nitrate partly in the roots and partly in the leaves (Picea, barley, maize). Although the different patterns of reduction result in different patterns of nitrogen transport, one feature common to all plants is that the two main conducting systems (xylem and phloem) are involved, and that xylem/phloem exchanges may occur along the path (Pate 1980). This ensures a constant and efficient recycling of nitrogen among the different organs. As will be detailed below, amino acids play a major role in nitrogen transport. In some cases, especially in the remobilisation of nitrogen from the endosperm to the growing embryo, the transport of small peptides (up to four to five amino acids) also has physiological significance. Although the spectra of amino acids found in the xylem and in the phloem are similar, the xylem usually contains low amino acid concentrations (3–20 mM in Urtica,Rosnick-Shimmel 1985), whereas much higher concentrations are found in the phloem, e.g. 100 mM in rice (Hayashi and Chino 1990) and 60 to 140 mM in different sugar beet varieties (Lohaus et al. 1994).