Spatial co-transcriptomics reveals discrete stages of the arbuscular mycorrhizal symbiosis.

The symbiotic interaction of plants with arbuscular mycorrhizal (AM) fungi is ancient and widespread. Plants provide AM fungi with carbon in exchange for nutrients and water, making this interaction a prime target for crop improvement. However, plant-fungal interactions are restricted to a small subset of root cells, precluding the application of most conventional functional genomic techniques to study the molecular bases of these interactions. Here we used single-nucleus and spatial RNA sequencing to explore both Medicago truncatula and Rhizophagus irregularis transcriptomes in AM symbiosis at cellular and spatial resolution. Integrated, spatially registered single-cell maps revealed infected and uninfected plant root cell types. We observed that cortex cells exhibit distinct transcriptome profiles during different stages of colonization by AM fungi, indicating dynamic interplay between both organisms during establishment of the cellular interface enabling successful symbiosis. Our study provides insight into a symbiotic relationship of major agricultural and environmental importance and demonstrates a paradigm combining single-cell and spatial transcriptomics for the analysis of complex organismal interactions.

Genetic and Phenotypic Analysis of Shoot Apical and Floral Meristem Development.

The shoot apical and floral meristems (SAM and FM, respectively) of Arabidopsis thaliana contain reservoirs of self-renewing stem cells that function as sources of progenitor cells for organ formation during development. The primary SAM produces all the aerial structures of the adult plant, while the FMs generate the four types of floral organs. Consequently, aberrant SAM and FM activity can profoundly affect vegetative and reproductive plant morphology. The embedded location and small size of Arabidopsis meristems make accessing these structures difficult, so specialized techniques have been developed to facilitate their analysis. Microscopic, histological, and molecular techniques provide both qualitative and quantitative data on meristem organization and function, which are crucial for the normal growth and development of the entire plant.

Interactive CLV3, CLE16 and CLE17 signaling mediates stem cell homeostasis in the Arabidopsis shoot apical meristem.

The ability of plants to grow and form organs throughout their lifetime is dependent on their sustained stem cell activity. These stem cell populations are maintained by intricate networks of intercellular signaling pathways. In Arabidopsis thaliana, the small secreted peptide CLAVATA3 (CLV3) controls shoot apical meristem (SAM) maintenance by activating a signal transduction pathway that modulates the expression of the homeodomain transcription factor WUSCHEL (WUS). Here, we demonstrate that two CLV3-related peptides, CLE16 and CLE17, restrict stem cell accumulation in the absence of CLV3. CLE16 and CLE17 contribute independently to SAM maintenance and organ production in clv3 plants at all stages of development. We show that CLE16 and CLE17 signal through a subset of CLV3 receptors, the BARELY ANY MERISTEM (BAM) receptor kinases, and act upstream of WUS. Our study reveals that CLE16 and CLE17 function in a mechanism that partially compensates for CLV3 to maintain stem cell homeostasis and plant resiliency, and expands the potential targets for enhancing yield traits in crop species.

A reevaluation of the role of the ASIL trihelix transcription factors as repressors of the seed maturation program.

Developmental transitions are typically tightly controlled at the transcriptional level. Two of these transitions involve the induction of the embryo maturation program midway through seed development and its repression during the vegetative phase of plant growth. Very little is known about the factors responsible for this regulation during early embryogenesis, and only a couple of transcription factors have been characterized as repressors during the postgerminative phase. Arabidopsis 6b-INTERACTING PROTEIN-LIKE1 (ASIL1), a trihelix transcription factor, has been proposed to repress maturation both embryonically and postembryonically. Preliminary data also suggested that its closest paralog, ASIL2, might play a role as well. We used a transcriptomic approach, coupled with phenotypical observations, to test the hypothesis that ASIL1 and ASIL2 redundantly turn off maturation during both phases of growth. Our results indicate that, contrary to what was previously published, neither of the ASIL genes plays a role in the regulation of maturation, at any point during plant development. Analyses of gene ontology (GO)-enriched terms and published transcriptomic datasets suggest that these genes might be involved in responses during the vegetative phase to certain biotic and abiotic stresses.

The signaling peptide-encoding genes CLE16, CLE17 and CLE27 are dispensable for Arabidopsis shoot apical meristem activity.

The shoot apical meristem produces all of the leaves, stems and flowers of a flowering plant from a reservoir of stem cells at its growing tip. In Arabidopsis, the small polypeptide signaling molecule CLAVATA3 (CLV3), a member of the CLV3/EMBRYO SURROUNDING REGION-RELATED (CLE) gene family, is a key component of a negative feedback loop that maintains stem cell activity in shoot and floral meristems throughout development. Because in some plant species multiple CLE genes are involved in regulating shoot apical meristem activity, we tested the hypothesis that CLE genes other than CLV3 might function in stem cell homeostasis in Arabidopsis. We identified three Arabidopsis CLE genes expressed in the post-embryonic shoot apical meristem, generated loss-of-function alleles using genome editing, and analyzed the meristem phenotypes of the resulting mutant plants. We found that null mutations in CLE16, CLE17 or CLE27 affected neither vegetative nor reproductive shoot meristem activity under normal growth conditions, although CLE27 appears to slightly prolong vegetative growth. Our results indicate that the CLE16, CLE17 and CLE27 genes have largely redundant roles in the Arabidopsis shoot apical meristem and/or regulate meristem activity only under specific environmental conditions.

Peptide signaling molecules CLE5 and CLE6 affect Arabidopsis leaf shape downstream of leaf patterning transcription factors and auxin.

Intercellular signaling mediated by small peptides is critical to coordinate organ formation in animals, but whether extracellular polypeptides play similar roles in plants is unknown. Here we describe a role in Arabidopsis leaf development for two members of the CLAVATA3/ESR-RELATED peptide family, CLE5 and CLE6, which lie adjacent to each other on chromosome 2. Uniquely among the CLE genes, CLE5 and CLE6 are expressed specifically at the base of developing leaves and floral organs, adjacent to the boundary with the shoot apical meristem. During vegetative development CLE5 and CLE6 transcription is regulated by the leaf patterning transcription factors BLADE-ON-PETIOLE1 (BOP1) and ASYMMETRIC LEAVES2 (AS2), as well as by the WUSCHEL-RELATED HOMEOBOX (WOX) transcription factors WOX1 and PRESSED FLOWER (PRS). Moreover, CLE5 and CLE6 transcript levels are differentially regulated in various genetic backgrounds by the phytohormone auxin. Analysis of loss-of-function mutations generated by genome engineering reveals that CLE5 and CLE6 independently and together have subtle effects on rosette leaf shape. Our study indicates that the CLE5 and CLE6 peptides function downstream of leaf patterning factors and phytohormones to modulate the final leaf morphology.

Characterization of inulin hydrolyzing enzyme(s) in commercial glucoamylases and its application in lactic acid production from Jerusalem artichoke tubers (Jat).

A high inulinase activity was found in three commercially available glucoamylase enzymes. Its origin was investigated and two proteins in the commercial glucoamylases were identified as the potential enzymes showing inulinase activity. One of the commercial glucoamylases, GA-L New from Genencor, was used for Jerusalem artichoke tubers (Jat) hydrolysis and a high hydrolysis yield of fructose was obtained. The simultaneous saccharification and lactic acid fermentation (SSF) of Jat was carried out using GA-L New as the inulinase and Pediococcus acidilactici DQ2 as the fermenting strain. A high lactic acid titer, yield, and productivity of 111.5 g/L, 0.46 g/g DM, and 1.55 g/L/h, respectively, were obtained within 72 h. The enzyme cost using the commercial glucoamylase as inulinase was compared to that using the typical inulinase and a large profit margin was identified. The results provided a practical way of Jat application for lactic acid production using cheap commercial glucoamylase enzyme.

Simultaneous saccharification and high titer lactic acid fermentation of corn stover using a newly isolated lactic acid bacterium Pediococcus acidilactici DQ2.

A lactic acid bacterium with high tolerance of temperature and lignocellulose derived inhibitor was isolated and characterized as Pediococcus acidilactici DQ2. The strain used in the simultaneous saccharification and fermentation (SSF) for high titer lactic acid production at the high solids loading of corn stover. Corn stover was pretreated using the dry sulphuric acid pretreatment, followed by a biological detoxification to remove the inhibitors produced in the pretreatment. The bioreactor with a novel helical impeller was used to the SSF operation of the pretreated and biodetoxified corn stover. The results show that a typical SSF operation at 48 °C, pH 5.5, and near 30% (w/w) solids loading in both 5 and 50 L bioreactors was demonstrated. The lactic acid titer, yield, and productivity reached 101.9 g/L, 77.2%, and 1.06 g/L/h, respectively. The result provided a practical process option for cellulosic lactic acid production using virgin agriculture lignocellulose residues.