The WHIRLY (WHY) DNA/RNA binding proteins fulfil multiple but poorly characterised functions in leaf development. WHY1 transcript levels were highest in the bases of 7-day old barley leaves. Immunogold labelling revealed that the WHY1 protein was more abundant in the nuclei than the proplastids of the leaf bases. Transcript and metabolite profiling analysis of barley lines (W1-1 and W1-7) lacking WHY1, which show delayed greening compared to the wild type. While the transcript profile of leaf development was largely unchanged in W1-1 and W1-7 leaves, there were differences in levels of several transcripts encoding transcription factors associated with chloroplast development. These include a barley homologue of the Arabidopsis GATA transcription factor that regulates stomatal development, greening and chloroplast development, NAC1, two transcripts with similarity to Arabidopsis GLK1 and two transcripts encoding ARF transcriptions factors with functions in leaf morphogenesis and development. Chloroplast proteins were less abundant in the W1-1 and W1-7 leaves than the wildtype. The levels of TCA cycle metabolites and GABA were significantly lower in WHY1 knockdown leaves than the wild type. We conclude that WHY1 functions in the nuclei of the cells in the leaf bases contributes to the regulation of chloroplast development.

Photoperiod and circadian controls play crucial roles in the regulation of chloroplast biogenesis. To understand more about the regulation of this process, we compared the greening of the first leaves of wild type barley and two WHIRLY1 (WHY1)-deficient lines. Seedlings were grown in darkness for 4 days prior and then exposed to light at the beginning of the photoperiod on the 5th day or under standard photoperiod conditions. The accumulation of chlorophyll, as well plastid-encoded photosynthetic transcripts and proteins was delayed in the WHY1-deficient lines under standard photoperiod conditions because of defects in plastid gene expression, ribosomal processing and photosynthetic protein accumulation. The acquisition of full photosynthetic capacity was delayed by about 11 days in the first leaves and the newly forming leaves of the WHY1-deficient lines compared to the wild type. However, the light-dependent accumulation of pigments, transcripts and photosynthetic proteins was similar in all lines when etiolated seedlings were exposed to light. These results demonstrate that WHY1 is required for the integration of photoperiod-dependent signalling and chloroplast development in barley leaves.