The nucellus: between cell elimination and sugar transport.

The architecture of the seed is shaped by the processes of tissue partitioning, which determines the volume ratio of maternal and zygotic tissues, and nutrient partitioning, which regulates nutrient distribution among tissues. In angiosperms, early seed development is characterized by antagonistic development of the nucellus maternal tissue and the endosperm fertilization product to become the main sugar sink. This process marked the evolution of angiosperms and outlines the most ancient seed architectures. In Arabidopsis, the endosperm partially eliminates the nucellus and imports sugars from the seed coat. Here, we show that the nucellus is symplasmically connected to the chalaza, the seed nutrient unloading zone, and works as both a sugar sink and source alongside the seed coat. After fertilization, the transient nucellus accumulates starch early on and releases it in the apoplasmic space during its elimination. By contrast, the persistent nucellus exports sugars toward the endosperm through the SWEET4 hexose facilitator. Finally, we analyzed sugar metabolism and transport in the transparent testa 16 mutant, which fails to undergo nucellus cell elimination, which shed light on the coordination between tissue and nutrient partitioning. Overall, this study identifies a path of sugar transport in the Arabidopsis seed and describes a link between sugar redistribution and the nucellus cell-elimination program.

Post-flowering biotic and abiotic stresses impact nitrogen use efficiency and seed filling in Arabidopsis thaliana.

Nitrogen (N) is an essential nutrient that plants require for the synthesis of amino acids, proteins, and many other important metabolites. Plant metabolism and growth are consequently dependent on the amount of N that is assimilated and distributed from source leaves to developing sinks, such as fruits and seeds. The environmental stresses enhanced by climate change deeply influence seed yield and seed composition, and may disturb N use efficiency (NUE) in pants. We aimed to investigate plant responses to extreme climates with regard to NUE, N remobilization efficiency, and seed composition. By studying a collection of Arabidopsis genotypes showing a range of C:N ratios in seeds, we investigated the impact of different post-flowering growth conditions (control, heat, drought, low nitrate availability, induced senescence, and induced plant defense) on seed yield, N allocation in organs, NUE, and N remobilization efficiency. We analysed how post-flowering stresses could change seed filling and showed that post-flowering stresses change both the range of N and C concentrations and the C:N stoichiometry in seeds. Using a new trait, called delta seed composition, we measured the deviation in C:N stoichiometry of each genotype and revealed the genetic determinism of the C:N stoichiometry. Altogether, the results indicate that extreme climate impacts NUE dramatically in plants and generates different bottlenecks in N fluxes during seed filling.

ACCELERATED CELL DEATH 6 Acts on Natural Leaf Senescence and Nitrogen Fluxes in Arabidopsis.

As the last step of leaf development, senescence is a molecular process involving cell death mechanism. Leaf senescence is trigged by both internal age-dependent factors and environmental stresses. It must be tightly regulated for the plant to adopt a proper response to environmental variation and to allow the plant to recycle nutrients stored in senescing organs. However, little is known about factors that regulate both nutrients fluxes and plant senescence. Taking advantage of variation for natural leaf senescence between Arabidopsis thaliana accessions, Col-0 and Ct-1, we did a fine mapping of a quantitative trait loci for leaf senescence and identified ACCELERATED CELL DEATH 6 (ACD6) as the causal gene. Using two near-isogeneic lines, differing solely around the ACD6 locus, we showed that ACD6 regulates rosette growth, leaf chlorophyll content, as well as leaf nitrogen and carbon percentages. To unravel the role of ACD6 in N remobilization, the two isogenic lines and acd6 mutant were grown and labeled with 15N at the vegetative stage in order to determine 15N partitioning between plant organs at harvest. Results showed that N remobilization efficiency was significantly lower in all the genotypes with lower ACD6 activity irrespective of plant growth and productivity. Measurement of N uptake at vegetative and reproductive stages revealed that ACD6 did not modify N uptake efficiency but enhanced nitrogen translocation from root to silique. In this study, we have evidenced a new role of ACD6 in regulating both sequential and monocarpic senescences and disrupting the balance between N remobilization and N uptake that is required for a good seed filling.

Arabidopsis Seed Content QTL Mapping Using High-Throughput Phenotyping: The Assets of Near Infrared Spectroscopy.

Seed storage compounds are of crucial importance for human diet, feed and industrial uses. In oleo-proteaginous species like rapeseed, seed oil and protein are the qualitative determinants that conferred economic value to the harvested seed. To date, although the biosynthesis pathways of oil and storage protein are rather well-known, the factors that determine how these types of reserves are partitioned in seeds have to be identified. With the aim of implementing a quantitative genetics approach, requiring phenotyping of 100s of plants, our first objective was to establish near-infrared reflectance spectroscopic (NIRS) predictive equations in order to estimate oil, protein, carbon, and nitrogen content in Arabidopsis seed with high-throughput level. Our results demonstrated that NIRS is a powerful non-destructive, high-throughput method to assess the content of these four major components studied in Arabidopsis seed. With this tool in hand, we analyzed Arabidopsis natural variation for these four components and illustrated that they all displayed a wide range of variation. Finally, NIRS was used in order to map QTL for these four traits using seeds from the Arabidopsis thaliana Ct-1 × Col-0 recombinant inbred line population. Some QTL co-localized with QTL previously identified, but others mapped to chromosomal regions never identified so far for such traits. This paper illustrates the usefulness of NIRS predictive equations to perform accurate high-throughput phenotyping of Arabidopsis seed content, opening new perspectives in gene identification following QTL mapping and genome wide association studies.

QTL meta-analysis in Arabidopsis reveals an interaction between leaf senescence and resource allocation to seeds.

Sequential and monocarpic senescence are observed at vegetative and reproductive stages, respectively. Both facilitate nitrogen (N) remobilization and control the duration of carbon (C) fixation. Genetic and environmental factors control N and C resource allocation to seeds. Studies of natural variation in Arabidopsis thaliana revealed differences between accessions for leaf senescence phenotypes, seed N and C contents, and N remobilization efficiency-related traits. Here, a quantitative genetics approach was used to gain a better understanding of seed filling regulation in relation to leaf senescence and resource allocation. For that purpose, three Arabidopsis recombinant inbred line populations (Ct-1×Col-0, Cvi-0×Col-0, Bur-0×Col-0) were used to map QTL (quantitative trait loci) for ten traits related to senescence, resource allocation, and seed filling. The use of common markers across the three different maps allowed direct comparisons of the positions of the detected QTL in a single consensus map. QTL meta-analysis was then used to identify interesting regions (metaQTL) where QTL for several traits co-localized. MetaQTL were compared with positions of candidate genes known to be involved in senescence processes and flowering time. Finally, investigation of the correlation between yield and seed N concentration in the three populations suggests that leaf senescence disrupts the negative correlation generally observed between these two traits.

WRINKLED transcription factors orchestrate tissue-specific regulation of fatty acid biosynthesis in Arabidopsis.

Acyl lipids are essential constituents of all cells, but acyl chain requirements vary greatly and depend on the cell type considered. This implies a tight regulation of fatty acid production so that supply fits demand. Isolation of the Arabidopsis thaliana WRINKLED1 (WRI1) transcription factor established the importance of transcriptional regulation for modulating the rate of acyl chain production. Here, we report the isolation of two additional regulators of the fatty acid biosynthetic pathway, WRI3 and WRI4, which are closely related to WRI1 and belong to the APETALA2-ethylene-responsive element binding protein family of transcription factors. These three WRIs define a family of regulators capable of triggering sustained rates of acyl chain synthesis. However, expression patterns of the three WRIs differ markedly. Whereas only WRI1 activates fatty acid biosynthesis in seeds for triacylglycerol production, the three WRIs are required in floral tissues to provide acyl chains for cutin biosynthesis and prevent adherence of these developing organs and subsequent semisterility. The targets of these WRIs encode enzymes providing precursors (acyl chain and glycerol backbones) for various lipid biosynthetic pathways, but not the subsequent lipid-assembling enzymes. These results provide insights into the developmental regulation of fatty acid production in plants.

Natural variation in seed very long chain fatty acid content is controlled by a new isoform of KCS18 in Arabidopsis thaliana.

Oil from oleaginous seeds is mainly composed of triacylglycerols. Very long chain fatty acids (VLCFAs) are major constituents of triacylglycerols in many seed oils and represent valuable feedstock for industrial purposes. To identify genetic factors governing natural variability in VLCFA biosynthesis, a quantitative trait loci (QTL) analysis using a recombinant inbred line population derived from a cross between accessions Bay-0 and Shahdara was performed in Arabidopsis thaliana. Two fatty acid chain length ratio (CLR) QTL were identified, with one major locus, CLR.2, accounting for 77% of the observed phenotypic variation. A fine mapping and candidate gene approach showed that a key enzyme of the fatty acid elongation pathway, the ß-ketoacyl-CoA synthase 18 (KCS18), was responsible for the CLR.2 QTL detected between Bay-0 and Shahdara. Association genetics and heterologous expression in yeast cells identified a single point mutation associated with an alteration of KCS18 activity, uncovering the molecular bases for the modulation of VLCFA content in these two natural populations of Arabidopsis. Identification of this kcs18 mutant with altered activity opens new perspectives for the modulation of oil composition in crop plants.

Insights from ANA-grade angiosperms into the early evolution of CUP-SHAPED COTYLEDON genes.

BACKGROUND AND AIMS: The closely related NAC family genes NO APICAL MERISTEM (NAM) and CUP-SHAPED COTYLEDON3 (CUC3) regulate the formation of boundaries within and between plant organs. NAM is post-transcriptionally regulated by miR164, whereas CUC3 is not. To gain insight into the evolution of NAM and CUC3 in the angiosperms, we analysed orthologous genes in early-diverging ANA-grade angiosperms and gymnosperms. METHODS: We obtained NAM- and CUC3-like sequences from diverse angiosperms and gymnosperms by a combination of reverse transcriptase PCR, cDNA library screening and database searching, and then investigated their phylogenetic relationships by performing maximum-likelihood reconstructions. We also studied the spatial expression patterns of NAM, CUC3 and MIR164 orthologues in female reproductive tissues of Amborella trichopoda, the probable sister to all other flowering plants. KEY RESULTS: Separate NAM and CUC3 orthologues were found in early-diverging angiosperms, but not in gymnosperms, which contained putative orthologues of the entire NAM + CUC3 clade that possessed sites of regulation by miR164. Multiple paralogues of NAM or CUC3 genes were noted in certain taxa, including Brassicaceae. Expression of NAM, CUC3 and MIR164 orthologues from Am. trichopoda was found to co-localize in ovules at the developmental boundary between the chalaza and nucellus. CONCLUSIONS: The NAM and CUC3 lineages were generated by duplication, and CUC3 was subsequently lost regulation by miR164, prior to the last common ancestor of the extant angiosperms. However, the paralogous NAM clade genes CUC1 and CUC2 were generated by a more recent duplication, near the base of Brassicaceae. The function of NAM and CUC3 in defining a developmental boundary in the ovule appears to have been conserved since the last common ancestor of the flowering plants, as does the post-transcriptional regulation in ovule tissues of NAM by miR164.

The evolutionary-developmental analysis of plant microRNAs.

MicroRNAs (miRNAs) control many important aspects of plant development, suggesting these molecules may also have played key roles in the evolution of developmental processes in plants. However, evolutionary-developmental (evo-devo) studies of miRNAs have been held back by technical difficulties in gene identification. To help solve this problem, we have developed a two-step procedure for the efficient identification of miRNA genes in any plant species. As a test case, we have studied the evolution of the MIR164 family in the angiosperms. We have identified novel MIR164 genes in three species occupying key phylogenetic positions and used these, together with published sequence data, to partially reconstruct the evolution of the MIR164 family since the last common ancestor of the extant flowering plants. We use our evolutionary reconstruction to discuss potential roles for MIR164 genes in the evolution of leaf shape and carpel closure in the angiosperms. The techniques we describe may be applied to any miRNA family and should thus enable plant evo-devo to begin to investigate the contributions miRNAs have made to the evolution of plant development.

PROCERA encodes a DELLA protein that mediates control of dissected leaf form in tomato.

Leaves of seed plants can be described as simple, where the leaf blade is entire, or dissected, where the blade is divided into distinct leaflets. Mechanisms that define leaflet number and position are poorly understood and their elucidation presents an attractive opportunity to understand mechanisms controlling organ shape in plants. In tomato (Solanum lycopersicum), a plant with dissected leaves, KNOTTED1-like homeodomain proteins (KNOX) are positive regulators of leaflet formation. Conversely, the hormone gibberellin (GA) can antagonise the effects of KNOX overexpression and reduce leaflet number, suggesting that GA may be a negative regulator of leaflet formation. However, when and how GA acts on leaf development is unknown. The reduced leaflet number phenotype of the tomato mutant procera (pro) mimics that of plants to which GA has been applied during leaf development, suggesting that PRO may define a GA signalling component required to promote leaflet formation. Here we show that PRO encodes a DELLA-type growth repressor that probably mediates GA-reversible growth restraint. We demonstrate that PRO is required to promote leaflet initiation during early stages of growth of leaf primordia and conversely that reduced GA biosynthesis increases the capability of the tomato leaf to produce leaflets in response to elevated KNOX activity. We propose that, in tomato, DELLA activity regulates leaflet number by defining the correct timing for leaflet initiation.

Negative regulation of KNOX expression in tomato leaves.

Leaves of seed plants can be described as simple, where the leaf blade is entire, or dissected, where the blade is divided into distinct leaflets. Both simple and dissected leaves are initiated at the flanks of a pluripotent structure termed the shoot apical meristem (SAM). In simple-leafed species, expression of class I KNOTTED1-like homeobox (KNOX) proteins is confined to the meristem while in many dissected leaf plants, including tomato, KNOX expression persists in leaf primordia. Elevation of KNOX expression in tomato leaves can result in increased leaflet number, indicating that tight regulation of KNOX expression may help define the degree of leaf dissection in this species. To test this hypothesis and understand the mechanisms controlling leaf dissection in tomato, we studied the clausa (clau) and tripinnate (tp) mutants both of which condition increased leaflet number phenotypes. We show that TRIPINNATE and CLAUSA act together, to restrict the expression level and domain of the KNOX genes Tkn1 and LeT6/Tkn2 during tomato leaf development. Because loss of CLAU or TP activity results in increased KNOX expression predominantly on the adaxial (upper) leaf domain, our observations indicate that CLAU and TP may participate in a domain-specific KNOX repressive system that delimits the ability of the tomato leaf to generate leaflets.

KNOX action in Arabidopsis is mediated by coordinate regulation of cytokinin and gibberellin activities.

The shoot apical meristem (SAM) is a pluripotent group of cells that gives rise to the aerial parts of higher plants. Class-I KNOTTED1-like homeobox (KNOX) transcription factors promote meristem function partly through repression of biosynthesis of the growth regulator gibberellin (GA). However, regulation of GA activity cannot fully account for KNOX action. Here, we show that KNOX function is also mediated by cytokinin (CK), a growth regulator that promotes cell division and meristem function. We demonstrate that KNOX activity is sufficient to rapidly activate both CK biosynthetic gene expression and a SAM-localized CK-response regulator. We also show that CK signaling is necessary for SAM function in a weak hypomorphic allele of the KNOX gene SHOOTMERISTEMLESS (STM). Additionally, we provide evidence that a combination of constitutive GA signaling and reduced CK levels is detrimental to SAM function. Our results indicate that CK activity is both necessary and sufficient for stimulating GA catabolic gene expression, thus reinforcing the low-GA regime established by KNOX proteins in the SAM. We propose that KNOX proteins may act as general orchestrators of growth-regulator homeostasis at the shoot apex of Arabidopsis by simultaneously activating CK and repressing GA biosynthesis, thus promoting meristem activity.

Evolution of leaf developmental mechanisms.

Leaves are determinate organs produced by the shoot apical meristem. Land plants demonstrate a large range of variation in leaf form. Here we discuss evolution of leaf form in the context of our current understanding of leaf development, as this has emerged from molecular genetic studies in model organisms. We also discuss specific examples where parallel studies of development in different species have helped understanding how diversification of leaf form may occur in nature.

Comparative molecular and functional analyses of the tobacco cyclin-dependent kinase inhibitor NtKIS1a and its spliced variant NtKIS1b.

In all eukaryotes, cell cycle progression is controlled by cyclin-dependent kinases (CDKs) whose activity is regulated at several levels including inhibition by CDK inhibitors. Here, we report a comparative molecular and functional analysis of the tobacco (Nicotiana tomentosiformis) CDK inhibitor, NtKIS1a, and its spliced variant, NtKIS1b. The C-terminal end of NtKIS1a shares strong sequence similarity with mammalian CIP/KIP inhibitors, which is not the case for NtKIS1b. Consistent with this, NtKIS1a but not NtKIS1b inhibits in vitro the kinase activity of CDK/cyclin complexes, and tobacco (Nicotiana tabacum) D-type cyclins and an A-type CDK are NtKIS1a, but not NtKIS1b, interacting partners. Although both NtKIS1a and NtKIS1b transcripts are mainly found in flowers and more precisely in stamens, NtKIS1b transcript levels are cell cycle regulated, whereas those of NtKIS1a remain constant during the cell cycle. NtKIS1a and NtKIS1b fused to fluorescent proteins are localized in the nucleus when transiently expressed in onion epidermal cells. Furthermore, there is no competition for their nuclear localization when they are simultaneously overexpressed. In vitro competition toward CDK kinase activity suggests that NtKIS1b is a strong competitor of NtKIS1a. Arabidopsis plants overexpressing NtKIS1a-green fluorescent protein (GFP) or NtKIS1b-GFP fusion proteins were obtained. In these plants, the fusion proteins are still localized in the nucleus. Interestingly, NtKIS1a-GFP-overexpressing plants display strong morphological modifications and a reduced CDK kinase activity, whereas NtKIS1b-GFP-overexpressing plants display a wild-type phenotype including a wild-type CDK kinase activity. Our results strongly suggest that the inhibition of the kinase activity is responsible for the phenotypic modifications.

The CDK inhibitor NtKIS1a is involved in plant development, endoreduplication and restores normal development of cyclin D3; 1-overexpressing plants.

Plant development requires stringent controls between cell proliferation and cell differentiation. Proliferation is positively regulated by cyclin dependent kinases (CDKs). Acting in opposition to CDKs are CDK inhibitors (CKIs). The first tobacco CKI (NtKIS1a) identified was shown to inhibit in vitro the kinase activity of CDK/cyclin complexes and to interact with CDK and D-cyclins. However, these features, which are common to other plant and animal CKIs already characterised, did not provide information about the function of NtKIS1a in plants. Thus, to gain insight into the role of NtKIS1a and especially its involvement in cell proliferation during plant development, we generated transgenic Arabidopsis thaliana plants that overexpress NtKIS1a. These plants showed reduced growth with smaller organs that contained larger cells. Moreover, these plants displayed modifications in plant morphology. These results demonstrated that plant organ size and shape, as well as organ cell number and cell size, might be controlled by modulation of the single NtKIS1a gene activity. Since in mammals, D-cyclins control cell cycle progression in a CDK-dependent manner but also play a CDK independent role by sequestering the CKIs p27(Kip1) and p21(Cip1), we tested the significance of cyclin D-CKI interaction within a living plant. With this aim, NtKIS1a and AtCycD3;1 were overexpressed simultaneously in plants by two different methods. Our results demonstrated that overexpression of the CKI NtKIS1a restores essentially normal development in plants overexpressing AtCycD3;1, providing the first evidence of cyclin D-CKI co-operation within the context of a living plant.