A GDSL-motif Esterase/Lipase Affects Wax and Cutin Deposition and Controls Hull-Caryopsis Attachment in Barley.

The cuticle covering aerial organs of land plants is well known to protect against desiccation. Cuticles also play diverse and specialized functions, including organ separation, depending on plant and tissue. Barley shows a distinctive cuticular wax bloom enriched in ß-diketones on leaf sheaths, stem nodes and internodes and inflorescences. Barley also develops a sticky surface on the outer pericarp layer of its grain fruit leading to strongly adhered hulls, 'covered grain', important for embryo protection and seed dispersal. While the transcription factor-encoding gene HvNUDUM (HvNUD) appears essential for adherent hulls, little is understood about how the pericarp cuticle changes during adhesion or whether changes in pericarp cuticles contribute to another phenotype where hulls partially shed, called 'skinning'. To that end, we screened barley lines for hull adhesion defects, focussing on the Eceriferum (= waxless, cer) mutants. Here, we show that the cer-xd allele causes defective wax blooms and compromised hull adhesion, and results from a mutation removing the last 10 amino acids of the GDS(L) [Gly, Asp, Ser, (Leu)]-motif esterase/lipase HvGDSL1. We used severe and moderate HvGDSL1 alleles to show that complete HvGDSL1 function is essential for leaf blade cuticular integrity, wax bloom deposition over inflorescences and leaf sheaths and pericarp cuticular ridge formation. Expression data suggest that HvGDSL1 may regulate hull adhesion independently of HvNUD. We found high conservation of HvGDSL1 among barley germplasm, so variation in HvGDSL1 unlikely leads to grain skinning in cultivated barley. Taken together, we reveal a single locus which controls adaptive cuticular properties across different organs in barley.

Conserved signalling components coordinate epidermal patterning and cuticle deposition in barley.

Faced with terrestrial threats, land plants seal their aerial surfaces with a lipid-rich cuticle. To breathe, plants interrupt their cuticles with adjustable epidermal pores, called stomata, that regulate gas exchange, and develop other specialised epidermal cells such as defensive hairs. Mechanisms coordinating epidermal features remain poorly understood. Addressing this, we studied two loci whose allelic variation causes both cuticular wax-deficiency and misarranged stomata in barley, identifying the underlying genes, Cer-g/ HvYDA1, encoding a YODA-like (YDA) MAPKKK, and Cer-s/ HvBRX-Solo, encoding a single BREVIS-RADIX (BRX) domain protein. Both genes control cuticular integrity, the spacing and identity of epidermal cells, and barley's distinctive epicuticular wax blooms, as well as stomatal patterning in elevated CO2 conditions. Genetic analyses revealed epistatic and modifying relationships between HvYDA1 and HvBRX-Solo, intimating that their products participate in interacting pathway(s) linking epidermal patterning with cuticular properties in barley. This may represent a mechanism for coordinating multiple adaptive features of the land plant epidermis in a cultivated cereal.

The relationship between the expression pattern of starch biosynthesis enzymes and molecular structure of high amylose maize starch.

Two high amylose (HAM) inbred lines with apparent amylose contents of 55 % and 62 %, respectively, were selected to explore the relationship between molecular structure and gene expression of starch-synthase involved enzymes. GPC analysis of debranched starches showed that the HAM starches (HAMSs) had shorter amylose chains and longer amylopectin chains than normal maize starch (NMS). FACE analysis showed that these HAMSs had a higher content of amylopectin chains of DP > 21. Quantitative Real-Time PCR analysis showed that the HAM lines had specifically low expression of the starch branching enzyme IIb (SBEIIb), and the starch synthase IIIa (SSIIIa) homologue, and high expression of the isoamylase 2 (ISA2), potentially suppressing the generation of amylopectin molecules through deficient branching and excessive debranching process, thereby increasing the relative amylose content. A high expression of GBSS1 was potentially associated with increased short amylose chain lengths in HAMSs.

The distribution pattern of endopolyploidy in maize.

KEY MESSAGE: We discovered that endopolyploidization is common in various organs and tissues of maize at different development stages. Endopolyploidy is not specific in maize germplasm populations. Endopolyploidy is caused by DNA endoreplication, a special type of mitosis with normal DNA synthesis and a lack of cell division; it is a common phenomenon and plays an important role in plant development. To systematically study the distribution pattern of endopolyploidy in maize, flow cytometry was used to determine the ploidy by measuring the cycle (C) value in various organs at different developmental stages, in embryos and endosperm during grain development, in roots under stress conditions, and in the roots of 119 inbred lines from two heterotic groups, Shaan A and Shaan B. Endopolyploidy was observed in most organs at various developmental stages except in expanded leaves and filaments. The endosperm showed the highest C value among all organs. During tissue development, the ploidy increased in all organs except the leaves. In addition, the endopolyploidization of the roots was significantly affected by drought stress. Multiple comparisons of the C values of seven subgroups revealed that the distribution of endopolyploidization was not correlated with the population structure. A correlation analysis at the seedling stage showed a positive relationship between the C value and both the length of the whole plant and the length of main root. A genome-wide association study (GWAS) identified a total of 9 significant SNPs associated with endopolyploidy (C value) in maize, and 8 candidate genes that participate in cell cycle regulation and DNA replication were uncovered in 119 maize inbred lines.

Evolutionary, structural and expression analysis of core genes involved in starch synthesis.

Starch is the main storage carbohydrate in plants and an important natural resource for food, feed and industrial raw materials. However, the details regarding the pathway for starch biosynthesis and the diversity of biosynthetic enzymes involved in this process are poorly understood. This study uses a comprehensive phylogenetic analysis of 74 sequenced plant genomes to revisit the evolutionary history of the genes encoding ADP-glucose pyrophosphorylase (AGPase), starch synthase (SS), starch branching enzyme (SBE) and starch de-branching enzyme (DBE). Additionally, the protein structures and expression patterns of these four core genes in starch biosynthesis were studied to determine their functional differences. The results showed that AGPase, SS, SBE and DBE have undergone complicated evolutionary processes in plants and that gene/genome duplications are responsible for the observed differences in isoform numbers. A structure analysis of these proteins suggested that the deletion/mutation of amino acids in some active sites resulted in not only structural variation but also sub-functionalization or neo-functionalization. Expression profiling indicated that AGPase-, SS-, SBE- and DBE-encoding genes exhibit spatio-temporally divergent expression patterns related to the composition of functional complexes in starch biosynthesis. This study provides a comprehensive atlas of the starch biosynthetic pathway, and these data should support future studies aimed at increasing understanding of starch biosynthesis and the functional evolutionary divergence of AGPase, SS, SBE, and DBE in plants.

Bivariate flow cytometric analysis and sorting of different types of maize starch grains.

Particle-size distribution, granular structure, and composition significantly affect the physicochemical properties, rheological properties, and nutritional function of starch. Flow cytometry and flow sorting are widely considered convenient and efficient ways of classifying and separating natural biological particles or other substances into subpopulations, respectively, based on the differential response of each component to stimulation by a light beam; the results allow for the correlation analysis of parameters. In this study, different types of starches isolated from waxy maize, sweet maize, high-amylose maize, pop maize, and normal maize were initially classified into various subgroups by flow cytometer and then collected through flow sorting to observe their morphology and particle-size distribution. The results showed that a 0.25% Gelzan solution served as an optimal reagent for keeping individual starch particles homogeneously dispersed in suspension for a relatively long time. The bivariate flow cytometric population distributions indicated that the starches of normal maize, sweet maize, and pop maize were divided into two subgroups, whereas high-amylose maize starch had only one subgroup. Waxy maize starch, conversely, showed three subpopulations. The subgroups sorted by flow cytometer were determined and verified in terms of morphology and granule size by scanning electron microscopy and laser particle distribution analyzer. Results showed that flow cytometry can be regarded as a novel method for classifying and sorting starch granules. © 2017 International Society for Advancement of Cytometry.