Hao Hu

Summary: Abscission is predetermined in specialized cell layers called the abscission zone (AZ) and activated by developmental or environmental signals. In the grass family, most identified AZ genes regulate AZ anatomy, which differs among lineages. A YABBY transcription factor, SHATTERING1 (SH1), is a domestication gene regulating abscission in multiple cereals, including rice and Setaria. In rice, SH1 inhibits lignification specifically in the AZ. However, the AZ of Setaria is nonlignified throughout, raising the question of how SH1 functions in species without lignification. Crispr‐Cas9 knockout mutants of SH1 were generated in Setaria viridis and characterized with histology, cell wall and auxin immunofluorescence, transmission electron microscopy, hormonal treatment and RNA‐Seq analysis. The sh1 mutant lacks shattering, as expected. No differences in cell anatomy or cell wall components including lignin were observed between sh1 and the wild‐type (WT) until abscission occurs. Chloroplasts degenerated in the AZ of WT before abscission, but degeneration was suppressed by auxin treatment. Auxin distribution and expression of auxin‐related genes differed between WT and sh1, with the signal of an antibody to auxin detected in the sh1 chloroplast. SH1 in Setaria is required for activation of abscission through auxin signaling, which is not reported in other grass species.

Abscission, known as shattering in crop species, is a highly regulated process by which plants shed parts. Although shattering has been studied extensively in cereals and a number of regulatory genes have been identified, much diversity in the process remains to be discovered. Teff (Eragrostis tef) is a crop native to Ethiopia that is potentially highly valuable worldwide for its nutritious grain and drought tolerance. Previous work has suggested that grain shattering in Eragrostis might have little in common with other cereals. In this study, we characterize the anatomy, cellular structure, and gene regulatory control of the abscission zone (AZ) in E. tef. We show that the AZ of E. tef is a narrow stalk below the caryopsis, which is common in Eragrostis species. X-ray microscopy, scanning electron microscopy, transmission electron microscopy, and immunolocalization of cell wall components showed that the AZ cells are thin-walled and break open along with programmed cell death at seed maturity, rather than separating between cells as in other studied species. Knockout of YABBY2/SHATTERING1, documented to control abscission in several cereals, had no effect on abscission or AZ structure in E. tef. RNA sequencing analysis showed that genes related to programmed cell death and cell wall modification are enriched in the AZ at the early seed maturity stage. These data show that E. tef drops its seeds using a unique mechanism. Our results provide the groundwork for understanding grain shattering in Eragrostis and further improvement of shattering in E. tef.

Paysonia auriculata (Brassicaceae) produces multiple hydroxy fatty acids as major components of the seed oil. We tracked the changes in seed oil composition and gene expression during development, starting 14 days after flowers had been pollinated. Seed oil changes showed initially higher levels of saturated and unsaturated fatty acids (FAs) but little accumulation of hydroxy fatty acids (HFAs). Starting 21 days after pollination (DAP) HFA content sharply increased, and reached almost 30% at 28 DAP. Total seed oil also increased from a low of approximately 2% at 14 DAP to a high of approximately 20% by 42 DAP. We identified almost all of the fatty acid synthesis and modification genes that are known from Arabidopsis, and, in addition, a strong candidate for the hydroxylase gene that mediates the hydroxylation of fatty acids to produce valuable hydroxy fatty acids (HFAs) in this species. The gene expression network revealed is very similar to that of the emerging oil crop, Physaria fendleri , in the sister genus to Paysonia . Phylogenetic analyses indicate the hydroxylase enzyme, FAH12, evolved only once in Paysonia and Physaria , and that the enzyme is closely related to FAD2 enzymes. Phylogenetic analyses of FAD2 and FAH12 in the Brassicaceae and outgroup genera suggest that the branch leading to the hydroxylase clade of Paysonia and Physaria is under relaxed selection, compared with the strong purifying selection found across the FAD2 lineages.

Directional transport of auxin is critical for inflorescence and floral development in flowering plants, but the role of auxin influx carriers (AUX1 proteins) has been largely overlooked. Taking advantage of available AUX1 mutants in green millet (Setaria viridis) and maize (Zea mays), we uncover previously unreported aspects of plant development that are affected by auxin influx, including higher order branches in the inflorescence, stigma branch number, glume (floral bract) development, and plant fertility. However, disruption of auxin flux does not affect all parts of the plant, with little obvious effect on inflorescence meristem size, time to flowering, and anther morphology. In double mutant studies in maize, disruptions of ZmAUX1 also affect vegetative development. A green fluorescent protein (GFP)-tagged construct of the Setaria AUX1 protein Sparse Panicle1 (SPP1) under its native promoter showed that SPP1 localizes to the plasma membrane of outer tissue layers in both roots and inflorescences, and accumulates specifically in inflorescence branch meristems, consistent with the mutant phenotype and expected auxin maxima. RNA-seq analysis indicated that most gene expression modules are conserved between mutant and wild-type plants, with only a few hundred genes differentially expressed in spp1 inflorescences. Using clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 technology, we disrupted SPP1 and the other four AUX1 homologs in S. viridis. SPP1 has a larger effect on inflorescence development than the others, although all contribute to plant height, tiller formation, and leaf and root development. The AUX1 importers are thus not fully redundant in S. viridis. Our detailed phenotypic characterization plus a stable GFP-tagged line offer tools for future dissection of the function of auxin influx proteins.

Directional transport of auxin is critical for inflorescence and floral development in flowering plants, but the role of auxin influx carriers (AUX1 proteins) has been largely overlooked. Taking advantage of available AUX1 mutants in Setaria viridis and maize, we uncover previously unreported aspects of plant development that are affected by auxin influx, including higher order branches in the inflorescence, stigma branch number, and glume (floral bract) development, and plant fertility. However, disruption of auxin flux does not affect all parts of the plant, with little obvious effect on inflorescence meristem size, time to flowering, and anther morphology. In double mutant studies in maize, disruptions of ZmAUX1 also affect vegetative development. A GFP-tagged construct of SvAUX1 under its native promoter showed that the AUX1 protein localizes to the plasma membrane of outer tissue layers in both roots and inflorescences, and accumulates specifically in inflorescence branch meristems, consistent with the mutant phenotype and expected auxin maxima. RNA-seq analysis finds that most gene expression modules are conserved between mutant and wildtype plants, with only a few hundred genes differentially expressed in spp1 inflorescences. Using CRISPR-Cas9 technology, we disrupted SPP1 and the other four AUX1 homologs in S. viridis . SvAUX1/SPP1 has a larger effect on inflorescence development than the others, although all contribute to plant height, tiller formation, leaf, and root development. The AUX1 importers are thus not fully redundant in S. viridis . Our detailed phenotypic characterization plus a stable GFP-tagged line offer tools for future dissection of the function of auxin influx proteins. One sentence summary: Mutations in a single auxin importer gene Spp1/SvAUX1 uncover broad and unexpected effects in nearly all aspects of the development of shoots, inflorescences, and flowers.

Knocking out a lignin-associated gene in poplar reveals a compensatory pathway for lignification with links to thiol redox balance and sulfur assimilation.

The lignin biosynthetic pathway is highly conserved in angiosperms, yet pathway manipulations give rise to a variety of taxon-specific outcomes. Knockout of lignin-associated4-coumarate:CoA ligases(4CLs) in herbaceous species mainly reduces guaiacyl (G) lignin and enhances cell wall saccharification. Here we show that CRISPR-knockout of4CL1inPopulus tremula×albapreferentially reduced syringyl (S) lignin, with negligible effects on biomass recalcitrance. Concordant with reduced S-lignin was downregulation offerulate 5-hydroxylases(F5Hs). Lignification was largely sustained by 4CL5, a low-affinity paralog of 4CL1 typically with only minor xylem expression or activity. Levels of caffeate, the preferred substrate of 4CL5, increased in line with significant upregulation ofcaffeoyl shikimate esterase1. Upregulation ofcaffeoyl-CoA O-methyltransferase1and downregulation ofF5Hsare consistent with preferential funneling of 4CL5 products toward G-lignin biosynthesis at the expense of S-lignin. Thus, transcriptional and metabolic adaptations to4CL1-knockout appear to have enabled 4CL5 catalysis at a level sufficient to sustain lignification. Finally, genes involved in sulfur assimilation, the glutathione-ascorbate cycle and various antioxidant systems were upregulated in the mutants, suggesting cascading responses to perturbed thioesterification in lignin biosynthesis.One sentence summary: Knockout of lignin-associated4CL1inPopulusreveals a 4CL5-dependent, caffeate-modulated compensatory pathway for lignification with links to thiol redox balance and sulfur assimilation.

Summary: Abscission is a process in which plants shed their parts, and is mediated by a particular set of cells, the abscission zone (AZ). In grasses (Poaceae), the position of the AZ differs among species, raising the question of whether its anatomical structure and genetic control are conserved. The ancestral position of the AZ was reconstructed. A combination of light microscopy, transmission electron microscopy, RNA‐Seq analyses and RNA in situ hybridisation were used to compare three species, two (weedy rice and Brachypodium distachyon) with the AZ in the ancestral position and one (Setaria viridis) with the AZ in a derived position below a cluster of flowers (spikelet). Rice and Brachypodium are more similar anatomically than Setaria. However, the cell wall properties and the transcriptome of rice and Brachypodium are no more similar to each other than either is to Setaria. The set of genes expressed in the studied tissues is generally conserved across species, but the precise developmental and positional patterns of expression and gene networks are almost entirely different. Transcriptional regulation of AZ development appears to be extensively rewired among the three species, leading to distinct anatomical and morphological outcomes.

Setaria viridis (green foxtail) and its domesticated relative S. italica (foxtail millet) are diploid C4 panicoid grasses that are being developed as model systems for studying grass genomics, genetics, development, and evolution. According to archeological evidence, foxtail millet was domesticated from green foxtail approximately 9,000 to 6,000 YBP in China. Under long-term human selection, domesticated foxtail millet developed many traits adapted to human cultivation and agricultural production. In comparison with its wild ancestor, foxtail millet has fewer vegetative branches, reduced grain shattering, delayed flowering time and less photoperiod sensitivity. Foxtail millet is the only present-day crop in the genus Setaria, although archeological records suggest that other species were domesticated and later abandoned in the last 10,000 years. We present an overview of domestication in foxtail millet, by reviewing recent studies on the genetic regulation of several domesticated traits in foxtail millet and discuss how the foxtail millet and green foxtail system could be further developed to both better understand its domestication history, and to provide more tools for future breeding efforts.

Plant cell walls exhibit architectural and compositional changes throughout their development and in response to external cues. While tubulins are involved in cell wall biogenesis, much remains unknown about the scope of their involvement during the orchestration of this resource-demanding process. A transgenic approach coupled with cell wall compositional analysis, RNA-seq and mining of publicly available diurnal gene expression data was used to assess the involvement of tubulins in poplar leaf cell wall biogenesis. Leaf cell walls of transgenic poplar lines with constitutive overexpression of α-tubulin (TUA) exhibited an increased abundance of homogalacturonan, along with a reduction in xylose. These changes were traced to altered expression of UDP-glucuronic acid decarboxylase (GADC) in the transgenic leaves. A model is postulated by which altered diurnal control of TUA through its constitutive overexpression led to a metabolic tradeoff affecting cellular utilization of GADC substrate UDP-glucuronic acid. While there were no effects on cellulose, hemicellulose or lignin abundance, subtle effects on hemicellulose composition and associated gene expression were noted. In addition, expression and enzymatic activity of pectin methylesterase (PME) decreased in the transgenic leaves. The change is discussed in a context of increased levels of PME substrate homogalacturonan, slow stomatal kinetics and the fate of PME product methanol. Since stomatal opening and closing depend on fundamentally contrasting microtubule dynamics, the slowing of both processes in the transgenic lines as previously reported appears to be directly related to underlying cell wall compositional changes that were caused by tubulin manipulation.