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High-molecular-weight glutenin subunits (HMW-GS) are major components of seed storage proteins (SSPs) and largely determine the processing properties of wheat (Triticum aestivum) flour. HMW-GS are encoded by the GLU-1 loci and regulated at the transcriptional level by interaction between cis-elements and transcription factors (TFs). We recently validated the function of conserved cis-regulatory modules (CCRMs) in GLU-1 promoters, but their interacting TFs remained uncharacterized. Here we identified a CCRM-binding NAM-ATAF-CUC (NAC) protein, TaNAC100, through yeast one-hybrid (Y1H) library screening. Transactivation assays demonstrated that TaNAC100 could bind to the GLU-1 promoters and repress their transcription activity in tobacco (Nicotiana benthamiana). Overexpression of TaNAC100 in wheat significantly reduced the contents of HMW-GS and other SSPs as well as total seed protein. This was confirmed by transcriptome analyses. Conversely, enhanced expression of TaNAC100 increased seed starch contents and expression of key starch synthesis-related genes, such as TaGBSS1 and TaSUS2. Y1H assays also indicated TaNAC100 binding with the promoters of TaGBSS1 and TaSUS2. These results suggest that TaNAC100 functions as a hub controlling seed protein and starch synthesis. Phenotypic analyses showed that TaNAC100 overexpression repressed plant height, increased heading date, and promoted seed size and thousand kernel weight. We also investigated sequence variations in a panel of cultivars, but did not identify significant association of TaNAC100 haplotypes with agronomic traits. The findings not only uncover a useful gene for wheat breeding but also provide an entry point to reveal the mechanism underlying metabolic balance of seed storage products., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

As the frequency of extreme environmental events is expected to increase with climate change, identifying candidate genes for stabilizing the protein composition of legume seeds or optimizing this in a given environment is increasingly important. To elucidate the genetic determinants of seed protein plasticity, major seed proteins from 200 ecotypes of Medicago truncatula grown in four contrasting environments were quantified after one-dimensional electrophoresis. The plasticity index of these proteins was recorded for each genotype as the slope of Finlay and Wilkinson's regression and then used for genome-wide association studies (GWASs), enabling the identification of candidate genes for determining this plasticity. This list was enriched in genes related to transcription, DNA repair and signal transduction, with many of them being stress responsive. Other over-represented genes were related to sulfur and aspartate family pathways leading to the synthesis of the nutritionally essential amino acids methionine and lysine. By placing these genes in metabolic pathways, and using a M. truncatula mutant impaired in regenerating methionine from S-methylmethionine, we discovered that methionine recycling pathways are major contributors to globulin composition establishment and plasticity. These data provide a unique resource of genes that can be targeted to mitigate negative impacts of environmental stresses on seed protein composition., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Plant asparaginyl endopeptidases (AEPs) are expressed as inactive zymogens that perform maturation of seed storage protein upon cleavage-dependent autoactivation in the low-pH environment of storage vacuoles. The AEPs have attracted attention for their macrocyclization reactions, and have been classified as cleavage or ligation specialists. However, we have recently shown that the ability of AEPs to produce either cyclic or acyclic products can be altered by mutations to the active site region, and that several AEPs are capable of macrocyclization given favorable pH conditions. One AEP extracted from Clitoria ternatea seeds (butelase 1) is classified as a ligase rather than a protease, presenting an opportunity to test for loss of cleavage activity. Here, making recombinant butelase 1 and rescuing an Arabidopsis thaliana mutant lacking AEP, we show that butelase 1 retains cleavage functions in vitro and in vivo. The in vivo rescue was incomplete, consistent with some trade-off for butelase 1 specialization toward macrocyclization. Its crystal structure showed an active site with only subtle differences from cleaving AEPs, suggesting the many differences in its peptide-binding region are the source of its efficient macrocyclization. All considered, it seems that either butelase 1 has not fully specialized or a requirement for autocatalytic cleavage is an evolutionary constraint upon macrocyclizing AEPs., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

¹³C metabolic flux analysis (MFA) has become the experimental method of choice to investigate the cellular metabolism of microbes, cell cultures and plant seeds. Conventional steady-state MFA utilizes isotopic labeling measurements of amino acids obtained from protein hydrolysates. To retain spatial information in conventional steady-state MFA, tissues or subcellular fractions must be dissected or biochemically purified. In contrast, peptides retain their identity in complex protein extracts, and may therefore be associated with a specific time of expression, tissue type and subcellular compartment. To enable 'single-sample' spatially and temporally resolved steady-state flux analysis, we investigated the suitability of peptide mass distributions (PMDs) as an alternative to amino acid label measurements. PMDs are the discrete convolution of the mass distributions of the constituent amino acids of a peptide. We investigated the requirements for the unique deconvolution of PMDs into amino acid mass distributions (AAMDs), the influence of peptide sequence length on parameter sensitivity, and how AAMD and flux estimates that are determined through deconvolution compare to estimates from a conventional GC-MS measurement-based approach. Deconvolution of PMDs of the storage protein β-conglycinin of soybean (Glycine max) resulted in good AAMD and flux estimates if fluxes were directly fitted to PMDs. Unconstrained deconvolution resulted in inferior AAMD and flux estimates. PMD measurements do not include amino acid backbone fragments, which increase the information content in GC-MS-derived analyses. Nonetheless, the resulting flux maps were of comparable quality due to the precision of Orbitrap quantification and the larger number of peptide measurements., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

In Arabidopsis thaliana, MAIGO 2 (MAG2) is involved in protein transport between the endoplasmic reticulum (ER) and the Golgi apparatus via its association with the ER-localized t-SNARE components SYP81/AtUfe1 and SEC20. To characterize the molecular machinery of MAG2-mediated protein transport, we explored MAG2-interacting proteins using transgenic A. thaliana plants expressing TAP-tagged MAG2. We identified three proteins, which were designated as MAG2-INTERACTING PROTEIN 1-3 [MIP1 (At2g32900), MIP2 (At5g24350) and MIP3 (At2g42700)]. Both MIP1 and MAG2 localized to the ER membrane. All of the mag2, mip1, mip2 and mip3 mutants exhibited a defect in storage protein maturation, and developed abnormal storage protein body (MAG body) structures in the ER of seed cells. These observations suggest that MIPs are closely associated with MAG2 and function in protein transport between the ER and Golgi apparatus. MIP1 and MIP2 contain a Zeste-White 10 (ZW10) domain and a Sec39 domain, respectively, but have low sequence identities (21% and 23%) with respective human orthologs. These results suggest that the plant MAG2-MIP1-MIP2 complex is a counterpart of the triple-subunit tethering complexes in yeast (Tip20p-Dsl1p-Sec39p) and humans (RINT1-ZW10-NAG). Surprisingly, the plant complex also contained a fourth member (MIP3) with a Sec1 domain. There have been no previous reports showing that a Sec1-containing protein is a subunit of ER-localized tethering complexes. Our results suggest that MAG2 and the three MIP proteins form a unique complex on the ER that is responsible for efficient transport of seed storage proteins., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

The barley Risø16 mutation leads to inactivation of cytosolic ADP-Glc pyrophosphorylase, and results in decreased ADP-Glc and endospermal starch levels. Here we show that this mutation is accompanied by a decrease in storage protein accumulation and seed size, which indicates that alteration of a single enzymatic step can change the network of storage metabolism as a whole. We used comprehensive transcript, metabolite and hormonal profiling to compare grain metabolism and development of Risø16 and wild-type endosperm. Despite increased sugar availability in mutant endosperm, glycolytic intermediates downstream of hexose phosphates remained unchanged or decreased, while several glycolytic enzymes were downregulated at the transcriptional level. Metabolite and transcript profiling also indicated an inhibition of the tricarboxylic acid cycle at the level of mitochondrial nicotinamide adenine dinucleotide (NAD)-isocitrate dehydrogenase and an attendant decrease in alpha-ketoglutarate and amino acids levels in Risø16, compared with wild type. Decreased levels of cytokinins in Risø16 endosperm suggested co-regulation between starch synthesis, abscisic acid (ABA) deficiency and cytokinin biosynthesis. Comparative cis-element analysis in promoters of jointly downregulated genes in Risø16 revealed an overlap between metabolic and hormonal regulation, which leds to a coordinated downregulation of endosperm-specific and ABA-inducible gene expression (storage proteins) together with repression by sugars (isocitrate dehydrogenase, amylases). Such co-regulation ensured that decreased carbon fluxes into starch lead to a coordinated inhibition of glycolysis, amino acid and storage proteins biosynthesis, which is useful in the prevention of osmotic imbalances and oxidative stress due to increased accumulation of sugars., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Chitinases are a class of ubiquitous proteins that are widely distributed in plants. Defense is the major natural role for chitinases, primarily against fungal pathogens. Little is known regarding their non-defensive roles in seeds. In this study, a new class III chitinase from pomegranate seeds (pomegranate seed chitinase, PSC) was isolated and purified to homogeneity. The native state of PSC is a monomer with a molecular weight of approximately 30 kDa. This chitinase naturally binds calcium ions with high capacity and low affinity, suggesting that PSC is a calcium storage protein. Consistent with this idea, its amino acid sequence (inferred from cDNA) is rich in acidic amino acid residues, especially Asp, similar to reported calcium storage proteins. The presence of calcium considerably improves the stability of the protein but has little effect on its enzymatic activity. Transmission electron microscopy analyses indicate that, similar to phytoferritin, this enzyme is widely distributed in the stroma of amyloplasts of the embryonic cells, suggesting that amyloplasts in seeds could serve as an alternative plastid for calcium storage. Indeed, the transmission electron microscopy results showed that, within the embryonic cells, calcium ions are mainly distributed in the stroma of the amyloplasts, consistent with a role for PSC in calcium storage. Thus, the plant appears to have evolved a new plastid for calcium storage in seeds. During seed germination, the content of this enzyme decreases with time, suggesting that it is involved in the germination process., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Understanding the quantitative control of fatty acid desaturation during the biosynthesis of seed storage oil has become a priority area for research, as a consequence of its importance for both human health and the substitution of mineral oil for industrial applications. We have analysed the genome structure of two mutants in Arabidopsis thaliana that show substantially elevated content of the omega-3 polyunsaturated fatty acid linolenic acid in their seed oil. In one, rfc4, sequences totalling approximately 2 Mb from chromosome 2 have been duplicated and inserted into chromosome 3. In the other mutant, ife, chromosome 2 sequences totalling approximately 1.4 Mb have been duplicated and inserted into a linked position. In both cases, the duplications encompass the FAD3 locus, which encodes the linoleate desaturase responsible for the biosynthesis of linolenic acid for accumulation in seed storage oil. The results show that mutagens such as fast neutrons (used for the induction of rfc4) and T-DNA (used for the induction of ife, which is not linked to the T-DNA present in the line) can result in the duplication of very large genome segments. They also show that increasing the dosage of the FAD3-containing genomic region results in an increase in the linolenic acid content of seed oil. Consequently, screening methods for duplication of FAD3 orthologues in oil crops may be an appropriate approach for the identification of germplasm for breeding varieties with increased proportions of linolenic acid in the oil that they produce., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Rice glutelins are synthesized at the endoplasmic reticulum (ER) as precursors (pro-glutelins), and are transported to protein storage vacuoles, where they are processed into mature proteins. The molecular basis of this process is largely unknown. Here, we report the isolation of a rice mutant, gpa1, that accumulates 57 kDa pro-glutelins in seeds and whose endosperm has a floury appearance. Transmission electron microscopy analysis showed that the gpa1 endosperm cells have an enlarged ER lumen and a smaller protein body II (PBII), and accumulated three types of newly generated subcellular structures. Moreover, a proportion of glutelins in the gpa1 endosperm cells were not delivered to PBII, and instead were mis-targeted to two of the newly generated structures or secreted. The gene corresponding to the gpa1 mutation was found to be OsRab5a, which encodes a small GTPase. In Arabidopsis protoplasts, OsRab5a protein was found to co-localize predominantly with AtVSR2, a molecular marker for the pre-vacuolar compartments (PVC). We conclude that OsRab5a plays an essential role in trafficking of storage protein to PBII, possibly as part of its function in organizing the endomembrane system in developing endosperm cells of rice., (© 2010 The Authors. The Plant Journal © 2010 Blackwell Publishing Ltd.)

Two different gene families have been proposed to act as sorting receptors for vacuolar storage cargo in plants: the vacuolar sorting receptors (VSRs) and the receptor homology-transmembrane-RING H2 domain proteins (RMRs). However, functional data on these genes is scarce and the identity of the sorting receptor for storage proteins remains controversial. Through a genetic screen we have identified the mtv2 mutant, which is defective in vacuolar transport of the storage cargo VAC2 in shoot apices. Map-based cloning revealed that mtv2 is a loss of function allele of the VSR4 gene. We show that VSR1, VSR3 and VSR4, but not the remaining VSRs or RMRs, participate in vacuolar sorting of VAC2 in vegetative tissues, and 12S globulins and 2S albumins in seeds, an activity that is essential for seedling germination vigor. Finally, we demonstrate that the functional diversification in the VSR family results from divergent expression patterns and also from distinct sorting activities of the family members., (© 2010 The Authors. The Plant Journal © 2010 Blackwell Publishing Ltd.)

Seeds play a fundamental role in colonization of the environment by spermatophytes, and seeds harvested from crops are the main food source for human beings. Knowledge of seed biology is therefore important for both fundamental and applied issues. This review on seed biology illustrates the important progress made in the field of Arabidopsis seed research over the last decade. Access to 'omics' tools, including the inventory of genes deduced from sequencing of the Arabidopsis genome, has speeded up the analysis of biological functions operating in seeds. This review covers the following processes: seed and seed coat development, seed reserve accumulation, seed dormancy and seed germination. We present new insights in these various fields and describe ongoing biotechnology approaches to improve seed characteristics in crops.

The rice (Oryza sativa L.) basic leucine Zipper factor RISBZ1 and rice prolamin box binding factor (RPBF) are transcriptional activators of rice seed storage protein (SSP) genes in vivo. To ascertain the functions of these trans-activators in seed development, knock-down (KD) transgenic rice plants were generated in which the accumulation of RISBZ1 and RPBF was reduced in an endosperm-specific manner by co-suppression (KD-RISBZ1 and KD-RPBF). The accumulation of most SSPs changed little between individual KD mutants and wild-type plants, whereas a double KD mutant (KD-RISBZ1/KD-RPBF) resulted in a significant reduction of most SSP gene expression and accumulation. The reduction of both trans-activators also caused a greater reduction in seed starch accumulation than individual KD mutants. Storage lipids were accumulated at reduced levels in KD-RISBZ1 and KD-RISBZ1/KD-RPBF seeds. KD-RPBF and KD-RISBZ1/KD-RPBF seeds exhibited multi-layered aleurone cells. Gene expression of DEFECTIVE KERNEL1 (OsDEK1), CRINKLY4 (OsCR4) and SUPERNUMERARY ALEURONE LAYER 1 (OsSAL1) rice homologues was decreased in the KD mutants, suggesting that these genes are regulated by RISBZ1 and RPBF. These phenotypes suggest that combinatorial interactions between RISBZ1 and RPBF play an essential role during grain filling. The functional redundancy and compensation between RISBZ1 and RPBF possibly account for weak effects on the SSP levels in single KD mutants, and help maintain various processes during seed development in rice. Physical interaction between RISBZ1 and RPBF may ensure that these processes are carried out properly.