Development of endosperm transfer cells in barley

Abstract

Endosperm transfer cells (ETCs) are positioned at the intersection of maternal and filial tissues in seeds of cereals and represent a bottleneck for apoplasmic transport of assimilates into the endosperm. Endosperm cellularization starts at the maternal-filial boundary and generates the highly specialized ETCs. During differentiation barley ETCs develop characteristic flange-like wall ingrowths to facilitate effective nutrient transfer. A comprehensive morphological analysis depicted distinct developmental time points in establishment of transfer cell (TC) morphology and revealed intracellular changes possibly associated with cell wall metabolism. Embedded inside the grain, ETCs are barely accessible by manual preparation. To get tissue-specific information about ETC specification and differentiation, laser microdissection (LM)-based methods were used for transcript and metabolite profiling. Transcriptome analysis of ETCs at different developmental stages by microarrays indicated activated gene expression programs related to control of cell proliferation and cell shape, cell wall and carbohydrate metabolism reflecting the morphological changes during early ETC development. Transporter genes reveal distinct expression patterns suggesting a switch from active to passive modes of nutrient uptake with the onset of grain filling. Tissue-specific RNA-seq of the differentiating ETC region from the syncytial stage until functionality in nutrient transfer identified a high number of novel transcripts putatively involved in ETC differentiation. An essential role for two-component signaling (TCS) pathways in ETC development of barley emerged from this analysis. Correlative data provide evidence for abscisic acid and ethylene influences on ETC differentiation and hint at a crosstalk between hormone signal transduction and TCS phosphorelays. Collectively, the data expose a comprehensive view on ETC development, associated pathways and identified candidate genes for ETC specification.

Keywords: ABA; TCS; barley seed; cell wall ingrowths; endosperm transfer cells; ethylene; nutrient transport; tissue-specific analysis.

FIGURE 1

Morphological and ultrastructural analysis of barley ETC development. Light microscopy (E,G,I,M,Q,U), DIC fluorescence microscopy (F,J,N,R,V), scanning electron (K,O,S,W) and transmission electron microscopy (H,L,P,T,X) images show ETC differentiation from cellularization (3/4 DAF) until the establishment of transfer cell structure (12 DAF). (A) Median cross section of a barley grain at 7 DAF depicts grain tissues and (B) magnification shows the transfer region of barley grains. (C) Volume of ETCs from 5 to 14 DAF, (D) scanning electron picture of an endosperm cell at 10 DAF. At 3/4 DAF, cellularization in the region of the syncytium facing the nucellar projection has just started (E,G). Cells contain a dense cytoplasm, rich in organelles of the endomembrane secretory system, and segments of cell walls are apparent but do not border complete cells (arrow, H). At 5 DAF, cellularization of the ETC region is completed; up to three rows of cells facing the NP are apparently different from the other cells of the endosperm (I). Cell walls appear subtle and thin; branching and deposition of cell wall ingrowths are not visible (K). Cells show a strong compartmentation and multiple mitochondria predominantly adjacent to the cell wall (arrows, L). At 7 DAF, cell walls are clearly thickened (M) and first branches of ribbed cell wall depositions covering the inner surface of walls can be detected (O). At 10 DAF, area of cell walls further increase and cells are completely lined by massive walls (Q,R). Cell walls show parallel rib-shaped depositions characteristic for the flange-type ingrowths (S). ER appeared to be constantly striped and nestled to the thickened walls (T). At 12 DAF, cell wall and ingrowths emerge asymmetrically and cover the main part of the cell (U–X). The cytoplasm is reduced compared to earlier stages (X). CW, cell wall; CWI, CW ingrowths; ER, endoplasmic reticulum; Mi, mitochondria; ETC, endosperm transfer cells; NP, nucellar projection; SE, starchy endosperm; SY, syncytium; V, vacuole (images modified after Thiel et al., 2012a).

FIGURE 2

Expression profiles of transporter genes in developing ETCs as determined by microarray analysis. Transcript levels are median-centered and log₂-transformed. Transcript levels are indicated by color code: yellow, high; blue, low [data were selected from transcriptome analysis presented by Thiel et al. (2012a)].

FIGURE 3

Phylogenetic relationship of TCS elements from barley, Arabidopsis and rice and expression profiles of selected genes in barley ETCs. (A) Barley sequences are highlighted by blue boxes. (a) Histidine kinases, colors indicate different subgroups. Arabidopsis PDK was used as outgroup. (b) HPt elements, protein sequences of Z. mays and T. aestivum were additionally included in the alignment. Yeast HPt protein YPD1 was used as outgroup. (c–e) Type-A, -B, -C response regulators, amino acid sequences of selected maize response regulators were included in the alignment. (B) Expression profiles of barley TCS elements in ETCs as determined by qRT-PCR analyses. Expression profiles of candidate genes were grouped by clusters. [Images are reproduced from Thiel et al. (2012b) with permission of PLoS org.].

FIGURE 4

Transcript analyses of HvHK1 and putatively interacting transcription factors. (A) In situ hybridization specifies the expression of HvHK1 in 3 DAF grains exclusively in the part of the cellularizing endosperm facing the nucellar projection. (B) Transcriptional response of HvHK1 to hormones was analyzed by in vitro experiments. Grains were incubated in MS medium supplemented with the indicated concentrations of hormones for 16h. The ΔΔCt value was calculated from qRT-PCR analysis and represents the log₂-ratio compared to control conditions. (C) Expression profiles of putative interacting transcription factors and ABA signaling elements in ETCs. Transcript levels were determined by qRT-PCR analyses and relative expression is given in the log₁₀ scale. Transcripts numbers indicate the contig identifier in the 454 transcriptome assembly [Image in C is reproduced from Thiel et al. (2012b)].

FIGURE 5

Hypothetical model of cellular processes and signals determining ETC differentiation from cell specification to the establishment of transfer cell morphology. The figure summarizes results from different kinds of tissue-specific analyses. Further explanations are given in the text.