Abstract
Hordeum vulgare (barley) hordoindolines (HINs), HINa, HINb1, and HINb2, are orthologous proteins of wheat puroindolines (PINs) that are small, basic, cysteine-rich seed-specific proteins and responsible for grain hardness. Grain hardness is, next to its protein content, a major quality trait. In barley, HINb is most highly expressed in the mid-stage developed endosperm and is associated with both major endosperm texture and grain hardness. However, data required to understand the spatio-temporal dynamics of HIN transcripts and HIN protein regulation during grain filling processes are missing. Using reverse transcription quantitative PCR (RT-qPCR) and proteomics, we analyzed HIN transcript and HIN protein abundance from whole seeds (WSs) at four [6 days after pollination (dap), 10, 12, and ≥20 dap] as well as from aleurone, subaleurone, and starchy endosperm at two (12 and ≥20 dap) developmental stages. At the WS level, results from RT-qPCR, proteomics, and western blot showed a continuous increase of HIN transcript and HIN protein abundance across these four developmental stages. Miroscopic studies revealed HIN localization mainly at the vacuolar membrane in the aleurone, at protein bodies (PBs) in subaleurone and at the periphery of starch granules in the starchy endosperm. Laser microdissetion (LMD) proteomic analyses identified HINb2 as the most prominent HIN protein in starchy endosperm at ≥20 dap. Additionally, our quantification data revealed a poor correlation between transcript and protein levels of HINs in subaleurone during development. Here, we correlated data achieved by RT-qPCR, proteomics, and microscopy that reveal different expression and localization pattern of HINs in each layer during barley endosperm development. This indicates a contribution of each tissue to the regulation of HINs during grain filling. The effect of the high protein abundance of HINs in the starchy endosperm and their localization at the periphery of starch granules at late development stages at the cereal-based end-product quality is discussed. Understanding the spatio-temporal regulated HINs is essential to improve barley quality traits for high end-product quality, as hard texture of the barley grain is regulated by the ratio between HINb/HINa.
Highlights
In barley, hordoindolines (HINs) are described as PIN ortholog proteins
In order to explore the dynamics of HIN transcripts during endosperm development, we analyzed first the levels of HINa and both HINb transcripts in developing whole seed (WS). cDNA alignment analyses of HINa, HINb1, and HINb2 showed that the DNA sequence HINa is different to HINb1 and HINb2, but the cDNA sequences of HINb1 and HINb2 are very similar (Figure 1A)
No significant changes of the abundance of HIN transcripts could be observed in WSs during development, even though both HIN transcripts showed a trend to increase during development, especially the HINa transcripts (Figure 1B)
Summary
Hordoindolines (HINs) are described as PIN ortholog proteins. Genetic studies have associated HIN proteins to both major endosperm texture and grain hardness (Darlington et al, 2001; Takahashi et al, 2010). HIN transcripts are endosperm specific; their mRNA level increased from 14 to 20 days after pollination (dap) in the endosperm and in the aleurone, and declined at 30 dap. Both HINa and HINb transcripts are more abundant in the endosperm than in the aleurone between 14 and 20 dap. No evidence was found that HINa plays a role in grain texture in barley as it was described for PINa (Darlington et al, 2001)
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