A group of CLE peptides regulates de novo shoot regeneration in Arabidopsis thaliana.

Known for their regulatory roles in stem cell homeostasis, CLAVATA3/ESR-RELATED (CLE) peptides also function as mediators of external stimuli such as hormones. De novo shoot regeneration, representing the remarkable plant cellular plasticity, involves reconstitution of stem cells under control of stem-cell regulators. Yet whether and how stem cell-regulating CLE peptides are implicated in plant regeneration remains unknown. By CRISPR/Cas9-induced loss-of-function studies, peptide application, precursor overexpression, and expression analyses, the role of CLE1-CLE7 peptides and their receptors in de novo shoot regeneration was studied in Arabidopsis thaliana. CLE1-CLE7 are induced by callus-induction medium and dynamically expressed in pluripotent callus. Exogenously-applied CLE1-CLE7 peptides or precursor overexpression effectively leads to shoot regeneration suppression, whereas their simultaneous mutation results in enhanced regenerative capacity, demonstrating that CLE1-CLE7 peptides redundantly function as negative regulators of de novo shoot regeneration. CLE1-CLE7-mediated shoot regeneration suppression is impaired in loss-of-function mutants of callus-expressed CLAVATA1 (CLV1) and BARELY ANY MERISTEM1 (BAM1) genes, indicating that CLV1/BAM1 are required for CLE1-CLE7-mediated shoot regeneration signaling. CLE1-CLE7 signaling resulted in transcriptional repression of WUSCHEL (WUS), a stem cell-promoting transcription factor known as a principal regulator of plant regeneration. Our results indicate that functionally-redundant CLE1-CLE7 peptides genetically act through CLV1/BAM1 receptors and repress WUS expression to modulate shoot-regeneration capacity, establishing the mechanistic basis for CLE1-CLE7-mediated shoot regeneration and a novel role for CLE peptides in hormone-dependent developmental plasticity.

The Toughest Material in the Plant Kingdom: An Update on Sporopollenin.

The extreme chemical and physical recalcitrance of sporopollenin deems this biopolymer among the most resilient organic materials on Earth. As the primary material fortifying spore and pollen cell walls, sporopollenin is touted as a critical innovation in the progression of plant life to a terrestrial setting. Although crucial for its protective role in plant reproduction, the inert nature of sporopollenin has challenged efforts to determine its composition for decades. Revised structural, chemical, and genetic experimentation efforts have produced dramatic advances in elucidating the molecular structure of this biopolymer and the mechanisms of its synthesis. Bypassing many of the challenges with material fragmentation and solubilization, insights from functional characterizations of sporopollenin biogenesis in planta, and in vitro, through a gene-targeted approach suggest a backbone of polyhydroxylated polyketide-based subunits and remarkable conservation of biochemical pathways for sporopollenin biosynthesis across the plant kingdom. Recent optimization of solid-state NMR and targeted degradation methods for sporopollenin analysis confirms polyhydroxylated α-pyrone subunits, as well as hydroxylated aliphatic units, and unique cross-linkage heterogeneity. We examine the cross-disciplinary efforts to solve the sporopollenin composition puzzle and illustrate a working model of sporopollenin's molecular structure and biosynthesis. Emerging controversies and remaining knowledge gaps are discussed, including the degree of aromaticity, cross-linkage profiles, and extent of chemical conservation of sporopollenin among land plants. The recent developments in sporopollenin research present diverse opportunities for harnessing the extraordinary properties of this abundant and stable biomaterial for sustainable microcapsule applications and synthetic material designs.

CYP77B1 a fatty acid epoxygenase specific to flowering plants.

Contrary to animals, little is known in plants about enzymes able to produce fatty acid epoxides. In our attempt to find and characterize a new fatty acid epoxygenase in Arabidopsis thaliana, data mining brought our attention on CYP77B1. Modification of the N-terminus was necessary to get enzymatic activity after heterologous expression in yeast. The common plant fatty acid C18:2 was converted into the diol 12,13-dihydroxy-octadec-cis-9-enoic acid when incubated with microsomes of yeast expressing modified CYP77B1 and AtEH1, a soluble epoxide hydrolase. This diol originated from the hydrolysis by AtEH1 of the epoxide 12,13-epoxy-octadec-cis-9-enoic acid produced by CYP77B1. A spatio-temporal study of CYP77B1 expression performed with RT-qPCR revealed the highest level of transcripts in flower bud while, in open flower, the enzyme was mainly present in pistil. CYP77B1 promoter-driven GUS expression confirmed reporter activities in pistil and also in stamens and petals. In silico co-regulation data led us to hypothesize that CYP77B1 could be involved in cutin synthesis but when flower cutin of loss-of-function mutants cyp77b1 was analyzed, no difference was found compared to cutin of wild type plants. Phylogenetic analysis showed that CYP77B1 is strictly conserved in flowering plants, suggesting a specific function in this lineage.

Function of the HYDROXYCINNAMOYL-CoA:SHIKIMATE HYDROXYCINNAMOYL TRANSFERASE is evolutionarily conserved in embryophytes.

The plant phenylpropanoid pathway generates a major class of specialized metabolites and precursors of essential extracellular polymers that initially appeared upon plant terrestrialization. Despite its evolutionary significance, little is known about the complexity and function of this major metabolic pathway in extant bryophytes, which represent the non-vascular stage of embryophyte evolution. Here, we report that the HYDROXYCINNAMOYL-CoA:SHIKIMATE HYDROXYCINNAMOYL TRANSFERASE (HCT) gene, which plays a critical function in the phenylpropanoid pathway during seed plant development, is functionally conserved in Physcomitrium patens (Physcomitrella), in the moss lineage of bryophytes. Phylogenetic analysis indicates that bona fide HCT function emerged in the progenitor of embryophytes. In vitro enzyme assays, moss phenolic pathway reconstitution in yeast and in planta gene inactivation coupled to targeted metabolic profiling, collectively indicate that P. patens HCT (PpHCT), similar to tracheophyte HCT orthologs, uses shikimate as a native acyl acceptor to produce a p-coumaroyl-5-O-shikimate intermediate. Phenotypic and metabolic analyses of loss-of-function mutants show that PpHCT is necessary for the production of caffeate derivatives, including previously reported caffeoyl-threonate esters, and for the formation of an intact cuticle. Deep conservation of HCT function in embryophytes is further suggested by the ability of HCT genes from P. patens and the liverwort Marchantia polymorpha to complement an Arabidopsis thaliana CRISPR/Cas9 hct mutant, and by the presence of phenolic esters of shikimate in representative species of the three bryophyte lineages.

The Class II KNOX genes KNAT3 and KNAT7 work cooperatively to influence deposition of secondary cell walls that provide mechanical support to Arabidopsis stems.

The transcription factor KNOTTED ARABIDOPSIS THALIANA7 (KNAT7) is a Class II KNOTTED1-like homeobox (KNOX2) gene that, in interfascicular fibres, acts as a negative regulator of secondary cell wall biosynthesis. In addition, knat7 loss-of-function mutants display an irregular xylem (irx) phenotype, suggesting a potential positive regulatory role in xylem vessel secondary cell wall deposition. Although our understanding of the role of KNAT7 is evolving, the function(s) of the closely related KNOX2 genes, KNAT3, KNAT4, and KNAT5, in secondary wall formation still remain unclear. We found that all four Arabidopsis KNOX2 genes were expressed in the inflorescence stems. However, only the knat3 knat7 double mutants showed a phenotype, displaying an enhanced irx phenotypes relative to the single mutants, as well as decreased interfascicular fibre cell wall thickness. Moreover, knat3 knat7 double mutants had reduced stem tensile and flexural strength compared with wild-type and single mutants. In contrast, KNAT3 overexpression resulted in thicker interfascicular fibre secondary cell walls in inflorescence stems, suggesting a potential positive regulation in interfascicular fibre secondary wall development. This work identifies KNAT3 as a potential transcriptional activator working together with KNAT7 to promote secondary cell wall biosynthesis in xylem vessels, while concurrently acting antagonistically with KNAT7 to influence secondary wall formation in interfascicular fibres.

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.

Polypeptide signaling molecules in plant development.

Intercellular communication mediated by small signaling molecules is a key mechanism for coordinating plant growth and development. In the past few years, polypeptide signals have been shown to play prominent roles in processes as diverse as shoot and root meristem maintenance, vascular differentiation, lateral root emergence, and seed formation. Signaling components such as CLV1 and the IDA-HAE/HSL2 signaling module have been discovered to regulate distinct developmental processes in different tissues. Recent studies have also uncovered novel polypeptide-receptor interactions, intracellular components and downstream target genes, adding complexity to our picture of polypeptide signaling networks. Finally, new families of plant polypeptides, such as the GLV/RGF/CLEL and ESF factors, are being identified, the functions of which we are only beginning to understand.

The biosynthesis, composition and assembly of the outer pollen wall: A tough case to crack.

The formation of the durable outer pollen wall, largely composed of sporopollenin, is essential for the protection of the male gametophyte and plant reproduction. Despite its apparent strict conservation amongst land plants, the composition of sporopollenin and the biosynthetic pathway(s) yielding this recalcitrant biopolymer remain elusive. Recent molecular genetic studies in Arabidopsis thaliana (Arabidopsis) and rice have, however, identified key genes involved in sporopollenin formation, allowing a better understanding of the biochemistry and cell biology underlying sporopollenin biosynthesis and pollen wall development. Herein, current knowledge of the biochemical composition of the outer pollen wall is reviewed, with an emphasis on enzymes with characterized biochemical activities in sporopollenin and pollen coat biosynthesis. The tapetum, which forms the innermost sporophytic cell layer of the anther and envelops developing pollen, plays an essential role in sporopollenin and pollen coat formation. Recent studies show that several tapetum-expressed genes encode enzymes that metabolize fatty acid derived compounds to form putative sporopollenin precursors, including tetraketides derived from fatty acyl-CoA starter molecules, but analysis of mutants defective in pollen wall development indicate that other components are also incorporated into sporopollenin. Also highlighted are the many uncertainties remaining in the development of a sporopollenin-fortified pollen wall, particularly in relation to the mechanisms of sporopollenin precursor transport and assembly into the patterned form of the pollen wall. A working model for sporopollenin biosynthesis is proposed based on the data obtained largely from studies of Arabidopsis, and future challenges to complete our understanding of pollen wall biology are outlined.

Arabidopsis VASCULAR-RELATED UNKNOWN PROTEIN1 regulates xylem development and growth by a conserved mechanism that modulates hormone signaling.

Despite a strict conservation of the vascular tissues in vascular plants (tracheophytes), our understanding of the genetic basis underlying the differentiation of secondary cell wall-containing cells in the xylem of tracheophytes is still far from complete. Using coexpression analysis and phylogenetic conservation across sequenced tracheophyte genomes, we identified a number of Arabidopsis (Arabidopsis thaliana) genes of unknown function whose expression is correlated with secondary cell wall deposition. Among these, the Arabidopsis VASCULAR-RELATED UNKNOWN PROTEIN1 (VUP1) gene encodes a predicted protein of 24 kD with no annotated functional domains but containing domains that are highly conserved in tracheophytes. Here, we show that the VUP1 expression pattern, determined by promoter-ß-glucuronidase reporter gene expression, is associated with vascular tissues, while vup1 loss-of-function mutants exhibit collapsed morphology of xylem vessel cells. Constitutive overexpression of VUP1 caused dramatic and pleiotropic developmental defects, including severe dwarfism, dark green leaves, reduced apical dominance, and altered photomorphogenesis, resembling brassinosteroid-deficient mutants. Constitutive overexpression of VUP homologs from multiple tracheophyte species induced similar defects. Whole-genome transcriptome analysis revealed that overexpression of VUP1 represses the expression of many brassinosteroid- and auxin-responsive genes. Additionally, deletion constructs and site-directed mutagenesis were used to identify critical domains and amino acids required for VUP1 function. Altogether, our data suggest a conserved role for VUP1 in regulating secondary wall formation during vascular development by tissue- or cell-specific modulation of hormone signaling pathways.

The interplay of lipid acyl hydrolases in inducible plant defense.

Lipid acyl hydrolases (LAH) have received recently increased attention in the context of plant defense. Multiple structurally unrelated gene families have been annotated in Arabidopsis as encoding potential lipid deacylating enzymes with numerous members being transcriptionally activated upon biotic stress. Confirming in silico predictions, experimental data have illustrated the wide subcellular distribution of LAHs indicating they likely interact with distinct membrane systems to initiate specific cellular responses. While recombinant LAHs are active in vitro on a small set of polar lipids, precise knowledge of in vivo substrates and hydrolysis products is generally lacking. Functional analysis of a few LAHs has revealed their roles in initiating oxylipin biosynthesis, cell death execution, signalling or direct antimicrobial activity. The picture emerging is that pathogenic challenge triggers a complex network of lipid hydrolysis events across the cellular compartments resulting in changes in membrane structures and release of signal precursors involved in the building-up of an adequate immune response.

LAP6/POLYKETIDE SYNTHASE A and LAP5/POLYKETIDE SYNTHASE B encode hydroxyalkyl α-pyrone synthases required for pollen development and sporopollenin biosynthesis in Arabidopsis thaliana.

Plant type III polyketide synthases (PKSs) catalyze the condensation of malonyl-CoA units with various CoA ester starter molecules to generate a diverse array of natural products. The fatty acyl-CoA esters synthesized by Arabidopsis thaliana ACYL-COA SYNTHETASE5 (ACOS5) are key intermediates in the biosynthesis of sporopollenin, the major constituent of exine in the outer pollen wall. By coexpression analysis, we identified two Arabidopsis PKS genes, POLYKETIDE SYNTHASE A (PKSA) and PKSB (also known as LAP6 and LAP5, respectively) that are tightly coexpressed with ACOS5. Recombinant PKSA and PKSB proteins generated tri-and tetraketide α-pyrone compounds in vitro from a broad range of potential ACOS5-generated fatty acyl-CoA starter substrates by condensation with malonyl-CoA. Furthermore, substrate preference profile and kinetic analyses strongly suggested that in planta substrates for both enzymes are midchain- and ω-hydroxylated fatty acyl-CoAs (e.g., 12-hydroxyoctadecanoyl-CoA and 16-hydroxyhexadecanoyl-CoA), which are the products of sequential actions of anther-specific fatty acid hydroxylases and acyl-CoA synthetase. PKSA and PKSB are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the PKS genes displayed pollen exine layer defects, and a double pksa pksb mutant was completely male sterile, with no apparent exine. These results show that hydroxylated α-pyrone polyketide compounds generated by the sequential action of ACOS5 and PKSA/B are potential and previously unknown sporopollenin precursors.

Analysis of TETRAKETIDE α-PYRONE REDUCTASE function in Arabidopsis thaliana reveals a previously unknown, but conserved, biochemical pathway in sporopollenin monomer biosynthesis.

The precise structure of the sporopollenin polymer that is the major constituent of exine, the outer pollen wall, remains poorly understood. Recently, characterization of Arabidopsis thaliana genes and corresponding enzymes involved in exine formation has demonstrated the role of fatty acid derivatives as precursors of sporopollenin building units. Fatty acyl-CoA esters synthesized by ACYL-COA SYNTHETASE5 (ACOS5) are condensed with malonyl-CoA by POLYKETIDE SYNTHASE A (PKSA) and PKSB to yield α-pyrone polyketides required for exine formation. Here, we show that two closely related genes encoding oxidoreductases are specifically and transiently expressed in tapetal cells during microspore development in Arabidopsis anthers. Mutants compromised in expression of the reductases displayed a range of pollen exine layer defects, depending on the mutant allele. Phylogenetic studies indicated that the two reductases belong to a large reductase/dehydrogenase gene family and cluster in two distinct clades with putative orthologs from several angiosperm lineages and the moss Physcomitrella patens. Recombinant proteins produced in bacteria reduced the carbonyl function of tetraketide α-pyrone compounds synthesized by PKSA/B, and the proteins were therefore named TETRAKETIDE α-PYRONE REDUCTASE1 (TKPR1) and TKPR2 (previously called DRL1 and CCRL6, respectively). TKPR activities, together with those of ACOS5 and PKSA/B, identify a conserved biosynthetic pathway leading to hydroxylated α-pyrone compounds that were previously unknown to be sporopollenin precursors.

A BAHD acyltransferase is expressed in the tapetum of Arabidopsis anthers and is involved in the synthesis of hydroxycinnamoyl spermidines.

BAHD acyltransferases catalyze the acylation of many plant secondary metabolites. We characterized the function of At2g19070, a member of the BAHD gene family of Arabidopsis thaliana. The acyltransferase gene was shown to be specifically expressed in anther tapetum cells in the early stages of flower development. The impact of gene repression was studied in RNAi plants and in a knockout (KO) mutant line. Immunoblotting with a specific antiserum raised against the recombinant protein was used to evaluate the accumulation of At2g19070 gene product in flowers of various Arabidopsis genotypes including the KO and RNAi lines, the male sterile mutant ms1 and transformants overexpressing the acyltransferase gene. Metabolic profiling of flower bud tissues from these genetic backgrounds demonstrated a positive correlation between the accumulation of acyltransferase protein and the quantities of metabolites that were putatively identified by tandem mass spectrometry as N(1),N(5),N(10)-trihydroxyferuloyl spermidine and N(1),N(5)-dihydroxyferuloyl-N(10)-sinapoyl spermidine. These products, deposited in pollen coat, can be readily extracted by pollen wash and were shown to be responsible for pollen autofluorescence. The activity of the recombinant enzyme produced in bacteria was assayed with various hydroxycinnamoyl-CoA esters and polyamines as donor and acceptor substrates, respectively. Feruloyl-CoA and spermidine proved the best substrates, and the enzyme has therefore been named spermidine hydroxycinnamoyl transferase (SHT). A methyltransferase gene (At1g67990) which co-regulated with SHT during flower development, was shown to be involved in the O-methylation of spermidine conjugates by analyzing the consequences of its repression in RNAi plants and by characterizing the methylation activity of the recombinant enzyme.