Conserved, divergent and heterochronic gene expression during Brachypodium and Arabidopsis embryo development.

KEY MESSAGE: Developmental and transcriptomic analysis of Brachypodium embryogenesis and comparison with Arabidopsis identifies conserved and divergent phases of embryogenesis and reveals widespread heterochrony of developmental gene expression. Embryogenesis, transforming the zygote into the mature embryo, represents a fundamental process for all flowering plants. Current knowledge of cell specification and differentiation during plant embryogenesis is largely based on studies of the dicot model plant Arabidopsis thaliana. However, the major crops are monocots and the transcriptional programs associated with the differentiation processes during embryogenesis in this clade were largely unknown. Here, we combined analysis of cell division patterns with development of a temporal transcriptomic resource during embryogenesis of the monocot model plant Brachypodium distachyon. We found that early divisions of the Brachypodium embryo were highly regular, while later stages were marked by less stereotypic patterns. Comparative transcriptomic analysis between Brachypodium and Arabidopsis revealed that early and late embryogenesis shared a common transcriptional program, whereas mid-embryogenesis was divergent between species. Analysis of orthology groups revealed widespread heterochronic expression of potential developmental regulators between the species. Interestingly, Brachypodium genes tend to be expressed at earlier stages than Arabidopsis counterparts, which suggests that embryo patterning may occur early during Brachypodium embryogenesis. Detailed investigation of auxin-related genes shows that the capacity to synthesize, transport and respond to auxin is established early in the embryo. However, while early PIN1 polarity could be confirmed, it is unclear if an active response is mounted. This study presents a resource for studying Brachypodium and grass embryogenesis and shows that divergent angiosperms share a conserved genetic program that is marked by heterochronic gene expression.

A WOX/Auxin Biosynthesis Module Controls Growth to Shape Leaf Form.

A key challenge in biology is to understand how the regional control of cell growth gives rise to final organ forms. Plant leaves must coordinate growth along both the proximodistal and mediolateral axes to produce their final shape. However, the cell-level mechanisms controlling this coordination remain largely unclear. Here, we show that, in A. thaliana, WOX5, one of the WUSCHEL-RELATED HOMEOBOX (WOX) family of homeobox genes, acts redundantly with WOX1 and WOX3 (PRESSED FLOWER [PRS]) to control leaf shape. Through genetics and hormone measurements, we find that these WOXs act in part through the regional control of YUCCA (YUC) auxin biosynthetic gene expression along the leaf margin. The requirement for WOX-mediated YUC expression in patterning of leaf shape cannot be bypassed by the epidermal expression of YUC, indicating that the precise domain of auxin biosynthesis is important for leaf form. Using time-lapse growth analysis, we demonstrate that WOX-mediated auxin biosynthesis organizes a proximodistal growth gradient that promotes lateral growth and consequently the characteristic ellipsoid A. thaliana leaf shape. We also provide evidence that WOX proteins shape the proximodistal gradient of differentiation by inhibiting differentiation proximally in the leaf blade and promoting it distally. This regulation allows sustained growth of the blade and enables a leaf to attain its final form. In conclusion, we show that the WOX/auxin regulatory module shapes leaf form by coordinating growth along the proximodistal and mediolateral leaf axes.

The Transcriptional Landscape of Polyploid Wheats and Their Diploid Ancestors during Embryogenesis and Grain Development.

Modern wheat production comes from two polyploid species, Triticum aestivum and Triticum turgidum (var durum), which putatively arose from diploid ancestors Triticum urartu, Aegilops speltoides, and Aegilops tauschii How gene expression during embryogenesis and grain development in wheats has been shaped by the differing contributions of diploid genomes through hybridization, polyploidization, and breeding selection is not well understood. This study describes the global landscape of gene activities during wheat embryogenesis and grain development. Using comprehensive transcriptomic analyses of two wheat cultivars and three diploid grasses, we investigated gene expression at seven stages of embryo development, two endosperm stages, and one pericarp stage. We identified transcriptional signatures and developmental similarities and differences among the five species, revealing the evolutionary divergence of gene expression programs and the contributions of A, B, and D subgenomes to grain development in polyploid wheats. The characterization of embryonic transcriptional programming in hexaploid wheat, tetraploid wheat, and diploid grass species provides insight into the landscape of gene expression in modern wheat and its ancestral species. This study presents a framework for understanding the evolution of domesticated wheat and the selective pressures placed on grain production, with important implications for future performance and yield improvements.plantcell;31/12/2888/FX1F1fx1.

Evolution, Initiation, and Diversity in Early Plant Embryogenesis.

During embryogenesis in plants, cell identities are specified de novo, starting from a single cell. By combining imaging, genomic profiling, and genetics, principles of early plant development have been unraveled in the dicotyledonous plant Arabidopsis. A central emerging question, however, is how well zygotic embryogenesis in Arabidopsis reflects homologous processes in other plant species, including early diverging, non-flowering, and non-seed plants. Here, we consider plant embryogenesis with an emphasis on its evolutionary history, the diverse modes of its initiation, and the concepts in pattern formation among morphologically distinct plant groups. Furthermore, we explore challenges and future directions in plant embryogenesis research.

A Growth-Based Framework for Leaf Shape Development and Diversity.

How do genes modify cellular growth to create morphological diversity? We study this problem in two related plants with differently shaped leaves: Arabidopsis thaliana (simple leaf shape) and Cardamine hirsuta (complex shape with leaflets). We use live imaging, modeling, and genetics to deconstruct these organ-level differences into their cell-level constituents: growth amount, direction, and differentiation. We show that leaf shape depends on the interplay of two growth modes: a conserved organ-wide growth mode that reflects differentiation; and a local, directional mode that involves the patterning of growth foci along the leaf edge. Shape diversity results from the distinct effects of two homeobox genes on these growth modes: SHOOTMERISTEMLESS broadens organ-wide growth relative to edge-patterning, enabling leaflet emergence, while REDUCED COMPLEXITY inhibits growth locally around emerging leaflets, accentuating shape differences created by patterning. We demonstrate the predictivity of our findings by reconstructing key features of C. hirsuta leaf morphology in A. thaliana. VIDEO ABSTRACT.

LMI1 homeodomain protein regulates organ proportions by spatial modulation of endoreduplication.

How the interplay between cell- and tissue-level processes produces correctly proportioned organs is a key problem in biology. In plants, the relative size of leaves compared with their lateral appendages, called stipules, varies tremendously throughout development and evolution, yet relevant mechanisms remain unknown. Here we use genetics, live imaging, and modeling to show that in Arabidopsis leaves, the LATE MERISTEM IDENTITY1 (LMI1) homeodomain protein regulates stipule proportions via an endoreduplication-dependent trade-off that limits tissue size despite increasing cell growth. LM1 acts through directly activating the conserved mitosis blocker WEE1, which is sufficient to bypass the LMI1 requirement for leaf proportionality.

A Molecular Framework for the Embryonic Initiation of Shoot Meristem Stem Cells.

The establishment of pluripotent stem cells is a key event during plant and animal embryogenesis, but the underlying mechanisms remain enigmatic. We show that in the flowering plant Arabidopsis thaliana, expression of the shoot meristem stem cell marker CLV3 becomes detectable in transition stage embryos. Surprisingly, the key regulator of stem cell homeostasis WUSCHEL (WUS) is expressed but dispensable for stem cell initiation. Rather, the WUS paralog WOX2, a regulator of embryo patterning initiated in the zygote, functions in this process by shielding stem cell progenitors from differentiation. WOX2 upregulates HD-ZIP III transcription factors required for shoot identity and balances cytokinin versus auxin hormone pathways, revealing that classical plantlet regeneration procedures recapitulate the natural induction mechanism. Our findings link transcriptional regulation of early embryo patterning to hormonal control of stem cell initiation and suggest that similar strategies have evolved in plant and animal stem cell formation.

Transcriptional activation of Arabidopsis axis patterning genes WOX8/9 links zygote polarity to embryo development.

In most flowering plants, the apical-basal body axis is established by an asymmetric division of the polarized zygote. In Arabidopsis, early embryo patterning is regulated by WOX homeobox genes, which are coexpressed in the zygote but become restricted to apical (WOX2) and basal (WOX8/9) cells. How the asymmetry of zygote division is regulated and connected to the daughter cell fates is largely unknown. Here, we show that expression of WOX8 is independent of the axis patterning signal auxin, but, together with the redundant gene WOX9, is activated in the zygote, its basal daughter cell, and the hypophysis by the zinc-finger transcription factor WRKY2. In wrky2 mutants, egg cells polarize normally but zygotes fail to reestablish polar organelle positioning from a transient symmetric state, resulting in equal cell division and distorted embryo development. Both defects are rescued by overexpressing WOX8, indicating that WRKY2-dependent WOX8 transcription links zygote polarization with embryo patterning.

The asymmetric division of the Arabidopsis zygote: from cell polarity to an embryo axis.

During plant embryogenesis, a simple body plan consisting of shoot and root meristem that are connected by the embryo axis is set up by the first few rounds of cell divisions after fertilization. Postembryonically, the elaborate architecture of plants is created from stem cell populations of both meristems. Here, we address how the main axis (apical-basal) of the plant embryo is established from the single-celled zygote and the role that the asymmetric division of the zygote plays in this process. We will mainly draw on examples from the model plant Arabidopsis, for which several key regulators have been identified during the last years.

New insights into the organization, recombination, expression and functional mechanism of low molecular weight glutenin subunit genes in bread wheat.

The bread-making quality of wheat is strongly influenced by multiple low molecular weight glutenin subunit (LMW-GS) proteins expressed in the seeds. However, the organization, recombination and expression of LMW-GS genes and their functional mechanism in bread-making are not well understood. Here we report a systematic molecular analysis of LMW-GS genes located at the orthologous Glu-3 loci (Glu-A3, B3 and D3) of bread wheat using complementary approaches (genome wide characterization of gene members, expression profiling, proteomic analysis). Fourteen unique LMW-GS genes were identified for Xiaoyan 54 (with superior bread-making quality). Molecular mapping and recombination analyses revealed that the three Glu-3 loci of Xiaoyan 54 harbored dissimilar numbers of LMW-GS genes and covered different genetic distances. The number of expressed LMW-GS in the seeds was higher in Xiaoyan 54 than in Jing 411 (with relatively poor bread-making quality). This correlated with the finding of higher numbers of active LMW-GS genes at the A3 and D3 loci in Xiaoyan 54. Association analysis using recombinant inbred lines suggested that positive interactions, conferred by genetic combinations of the Glu-3 locus alleles with more numerous active LMW-GS genes, were generally important for the recombinant progenies to attain high Zeleny sedimentation value (ZSV), an important indicator of bread-making quality. A higher number of active LMW-GS genes tended to lead to a more elevated ZSV, although this tendency was influenced by genetic background. This work provides substantial new insights into the genomic organization and expression of LMW-GS genes, and molecular genetic evidence suggesting that these genes contribute quantitatively to bread-making quality in hexaploid wheat. Our analysis also indicates that selection for high numbers of active LMW-GS genes can be used for improvement of bread-making quality in wheat breeding.

Physical mapping and identification of a candidate for the leaf rust resistance gene Lr1 of wheat.

Lr1 is a dominant leaf rust resistance gene located on chromosome 5DL of bread wheat and the wild species Aegilops tauschii. In this study, three polymorphic markers (WR001, WR002, and WR003) were developed from resistance gene analogs (RGAs) clustering around the Lr1 locus. Using these and other markers, Lr1 was mapped to a genetic interval of 0.79 cM in Ae. tauschii and 0.075 cM in wheat. The CAPS marker WR003, derived from LR1RGA1, co-segregated with Lr1 in both mapping populations of wheat and Ae. tauschii. For isolation of Lr1, two genomic BAC libraries (from Ae. tauschii and hexaploid wheat) were screened using the tightly flanking marker PSR567F and a set of nested primers derived from the conserved region of the RGA sequences. Approximately 400 kb BAC contig spanning the Lr1 locus was constructed. The LR1RGA1 encoding a CC-NBS-leucine-rich repeat (LRR) type of protein was the only one of the four RGAs at the Lr1 locus, which co-segregated with leaf rust resistance. Therefore, it represents a very good candidate for Lr1. The allelic sequences of LR1RGA1 from resistant and susceptible lines revealed a divergent DNA sequence block of approximately 605 bp encoding the LRR repeats 9-15, whereas the rest of the sequences were mostly identical. Within this sequence block, the 48 non-synonymous changes resulted in 44 amino acid differences. This indicates that LR1RGA1 likely evolved through one or more recombination or gene conversion events with unknown genes.

Extensive de Novo genomic variation in rice induced by introgression from wild rice (Zizania latifolia Griseb.).

To study the possible impact of alien introgression on a recipient plant genome, we examined >6000 unbiased genomic loci of three stable rice recombinant inbred lines (RILs) derived from intergeneric hybridization between rice (cv. Matsumae) and a wild relative (Zizania latifolia Griseb.) followed by successive selfing. Results from amplified fragment length polymorphism (AFLP) analysis showed that, whereas the introgressed Zizania DNA comprised <0.1% of the genome content in the RILs, extensive and genome-wide de novo variations occurred in up to 30% of the analyzed loci for all three lines studied. The AFLP-detected changes were validated by DNA gel-blot hybridization and/or sequence analysis of genomic loci corresponding to a subset of the differentiating AFLP fragments. A BLAST analysis revealed that the genomic variations occurred in diverse sequences, including protein-coding genes, transposable elements, and sequences of unknown functions. Pairwise sequence comparison of selected loci between a RIL and its rice parent showed that the variations represented either base substitutions or small insertion/deletions. Genome variations were detected in all 12 rice chromosomes, although their distribution was uneven both among and within chromosomes. Taken together, our results imply that even cryptic alien introgression can be highly mutagenic to a recipient plant genome.