Programmed Cell Death in Developing Brachypodium distachyon Grain

The normal developmental sequence in a grass grain entails the death of several maternal and filial tissues in a genetically regulated process termed programmed cell death (PCD). The progression and molecular aspects of PCD in developing grains have been reported for domesticated species such as barley, rice, maize and wheat. Here, we report a detailed investigation of PCD in the developing grain of the wild model species Brachypodium distachyon. We detected PCD in developing Brachypodium grains using molecular and histological approaches. We also identified in Brachypodium the orthologs of protease genes known to contribute to grain PCD and surveyed their expression. We found that, similar to cereals, PCD in the Brachypodium nucellus occurs in a centrifugal pattern following anthesis. However, compared to cereals, the rate of post-mortem clearance in the Brachypodium nucellus is slower. However, compared to wheat and barley, mesocarp PCD in Brachypodium proceeds more rapidly in lateral cells. Remarkably, Brachypodium mesocarp PCD is not coordinated with endosperm development. Phylogenetic analysis suggests that barley and wheat possess more vacuolar processing enzymes that drive nucellar PCD compared to Brachypodium and rice. Our expression analysis highlighted putative grain-specific PCD proteases in Brachypodium. Combined with existing knowledge on grain PCD, our study suggests that the rate of nucellar PCD moderates grain size and that the pattern of mesocarp PCD influences grain shape.

Small RNA and Degradome Analyses Uncovering the Extensive Effects of miRNAs and Targets in Early Developing Grains of Common Wheat

Background: The development of grains is important for wheat production, and wheat (Triticum aestivum) is one of the staple food crops worldwide. MicroRNAs (miRNAs), as a kind of small regulatory RNAs, play important roles during plant growth and development. Although the development of plant grain/seed is widely researched, there is limited knowledge on miRNAs’s regulation of early developing wheat grains. Results: In the present study, miRNAs and their targets were explored in early developing grains of wheat cultivar “Bainong 4199” at 7DAP and 14 DAP using high-throughput small RNA and degradome sequencing. A total of 105 known and 79 novel miRNAs were identified, including 46 known and 32 novel miRNAs from 7 DAP library and 87 known and 78 novel miRNAs from 14 DAP library, respectively. Expression analysis of miRNAs revealed that 39 miRNAs including 19 known and 20 novel miRNAs were differentially expressed between 7 DAP and 14 DAP. In total, 266 targets for 40 known wheat miRNAs, 152 targets for 13 other known plant miRNAs and 258 targets for 25 novel miRNAs were predicted across small RNA and degradome analyses. For differentially expressed miRNAs, 23 targets were predicted to be cleaved by 7 miRNAs, including 3 known and 4 novel miRNAs. Majority of the miRNAs potentially regulated multiple targets, whereas some miRNAs only acted on a single target gene. Functional analyses showed that miRNAs and their targets widely participated in the regulations of early wheat grain development and metabolism. The expression patterns of the randomly selected miRNAs and targets were validated using quantitative real-time polymerase chain reaction, and showed consistent and reliable results. Conclusion: This study suggests that quite a few known and novel miRNAs and their targets play extensive roles during the early grain development of common wheat. Understanding of miRNA-mediated regulatory network involved in wheat grain development will help us to elucidate the molecular mechanisms underlying wheat grain development and carry out ingenious molecular improvements in wheat breeding.

Reinvestigation of grain weight genes TaTGW6 and OsTGW6 casts doubt on their role in auxin regulation in developing grains

The THOUSAND-GRAIN WEIGHT 6 genes (TaTGW6 and OsTGW6) are reported to result in larger grains of wheat and rice by reducing production of indole-3-acetic acid (IAA) in developing grains. However, a critical comparison of data on TaTGW6 and OsTGW6 with other reports on IAA synthesis in cereal grains requires that this hypothesis be reinvestigated. Here, we show that TaTGW6 and OsTGW6 are members of a large gene family that has undergone major, lineage-specific gene expansion. Wheat has nine genes, and rice three genes encoding proteins with more than 80% amino acid identity with TGW6 making it difficult to envisage how a single inactive allele could have a major effect on IAA levels. TGW6 is proposed to affect auxin levels by catalysing the hydrolysis of IAA-glucose (IAA-Glc). However, we show that developing wheat grains contain undetectable levels of ester IAA in comparison to free IAA and do not express an IAA-glucose synthase. Previous work on TGW6, reported maximal expression at 20 days after anthesis (DAA) in wheat and 2 DAA in rice. However, we show that neither gene is expressed in developing grains. Instead, TaTGW6, OsTGW6 and their close homologues are exclusively expressed in pre-emergence inflorescences; TaTGW6 is expressed particularly in microspores prior to mitosis. This combined with evidence for high levels of IAA production from tryptophan in developing grains demonstrates TaTGW6 and OsTGW6 cannot regulate grain size via the hydrolysis of IAA-Glc. Instead, their similarity to rice strictosidine synthase-like (OsSTRL2) suggests they play a key role in pollen development.

Expression of TaTAR2.3-1B, TaYUC9-1 and TaYUC10 correlates with auxin and starch content of developing wheat grains

The role of auxin in developing grains of wheat (Triticum aestivum) is contentious with contradictory reports indicating either positive or negative effects of IAA (indole-3-acetic acid) on grain size. In addition, the contributions to the IAA pool from de novo synthesis via tryptophan, and from hydrolysis of IAA-glucose are unclear. Here we describe the first comprehensive study of tryptophan aminotransferase and indole-3-pyruvate mono-oxygenase expression during wheat grain development from 5 to 20 days after anthesis. A comparison of expression data with measurements of endogenous IAA via combined liquid chromatography-tandem mass spectrometry with heavy isotope labelled internal standards indicates that TaTAR2.3-1B, TaYUC9-A1, TaYUC9-B, TaYUC9-D1, TaYUC10-A and TaYUC10-D are primarily responsible for IAA production in developing grains. Furthermore, we show that IAA synthesis is controlled by genes expressed specifically in developing wheat grains as has already been reported in rice (Oryza sativa) and maize (Zea mays). Our results cast doubt on the proposed role of THOUSAND-GRAIN WEIGHT gene, TaTGW6, in promoting larger grain size via negative effects on grain IAA content. The work on TaTGW6 has overlooked the contribution of the dominant IAA biosynthesis pathway. Although IAA synthesis occurs primarily in the endosperm of wheat grains, we show that the TaYUC9-1 group is also strongly expressed in the embryo. Within the endosperm, TaYUC9-1 expression is highest in aleurone and transfer cells, supporting data from other cereals suggesting that IAA has a key role in differentiation of these tissues.

Thaumatin-Like Protein (TLP) Gene Family in Barley: Genome-Wide Exploration and Expression Analysis during Germination

Thaumatin-like Proteins (TLPs) are known to play a vital role in plant defense, developmental processes and seed germination. We identified 19 TLP genes from the reference genome of barley and 37, 28 and 35 TLP genes from rice, Brachypodium and sorghum genomes, respectively. Comparative phylogenetic analysis classified the TLP family into nine groups. Localized gene duplications with diverse exon/intron structures contributed to the expansion of the TLP gene family in cereals. Most of the barley TLPs were localized on chromosome 5H. The spatiotemporal expression pattern of HvTLP genes indicated their predominant expression in the embryo, developing grains, root and shoot tissues. Differential expression of HvTLP14, HvTLP17 and HvTLP18 in the malting variety (Morex) over 16–96 h of grain germination revealed their possible role in malting. This study provides a description of the TLP gene family in barley and their possible involvement in seed germination and the malting process.

Bioinformatic and experimental evidence for suicidal and catalytic plant THI4s

Like fungi and some prokaryotes, plants use a thiazole synthase (THI4) to make the thiazole precursor of thiamin. Fungal THI4s are suicide enzymes that destroy an essential active-site Cys residue to obtain the sulfur atom needed for thiazole formation. In contrast, certain prokaryotic THI4s have no active-site Cys, use sulfide as sulfur donor, and are truly catalytic. The presence of a conserved active-site Cys in plant THI4s and other indirect evidence implies that they are suicidal. To confirm this, we complemented the Arabidopsistz-1 mutant, which lacks THI4 activity, with a His-tagged Arabidopsis THI4 construct. LC–MS analysis of tryptic peptides of the THI4 extracted from leaves showed that the active-site Cys was predominantly in desulfurated form, consistent with THI4 having a suicide mechanism in planta. Unexpectedly, transcriptome data mining and deep proteome profiling showed that barley, wheat, and oat have both a widely expressed canonical THI4 with an active-site Cys, and a THI4-like paralog (non-Cys THI4) that has no active-site Cys and is the major type of THI4 in developing grains. Transcriptomic evidence also indicated that barley, wheat, and oat grains synthesize thiamin de novo, implying that their non-Cys THI4s synthesize thiazole. Structure modeling supported this inference, as did demonstration that non-Cys THI4s have significant capacity to complement thiazole auxotrophy in Escherichia coli. There is thus a prima facie case that non-Cys cereal THI4s, like their prokaryotic counterparts, are catalytic thiazole synthases. Bioenergetic calculations show that, relative to suicide THI4s, such enzymes could save substantial energy during the grain-filling period.