Analysis of synthesis mutants demonstrates an ascorbate requirement for growth under low light and for high light-dependent anthocyanin accumulation, but no consistent effects on photoinhibition or zeaxanthin accumulation were found.

The late embryogenesis abundant (LEA)5 protein is predominantly expressed in Arabidopsis leaves in the dark, the levels of LEA5 transcripts decreasing rapidly upon illumination. LEA5 is important in plant responses to environmental stresses but the mechanisms involved have not been elucidated. We therefore explored LEA5 functions in Arabidopsis mutants (lea5) and transgenic Arabidopsis plants constitutively expressing LEA5 (OEX 2-5), as well as in transgenic barley lines expressing the Arabidopsis LEA5 gene. The OEX 2-5 plants grew better than controls and lea5 mutants in the presence of the prooxidants methyl viologen and menadione. Confocal microscopy of Arabidopsis mesophyll protoplasts expressing a LEA5-YFP fusion protein demonstrated that LEA5 could be localized to chloroplasts as well as mitochondria in Arabidopsis protoplasts. Tandem affinity purification (TAP) analysis revealed LEA5 interacts with the chloroplast DEAD-box ATP-dependent RNA helicase 22 (RH22) in Arabidopsis cells. Split YFP analysis confirmed the interaction between RH22 and LEA5 in chloroplasts. The abundance of translated protein products in chloroplasts was decreased in transgenic Arabidopsis plants and increased in lea5 knockout mutants. Conversely, the abundance of translated mitochondrial protein products was increased in OEX 2-5 plants and decreased in lea5 mutants. Mitochondrial electron transport rates were higher in the OEX 2-5 plants than the wild type. The transformed barley lines expressing the Arabidopsis LEA5 had increased seed yields, but they showed a greater drought-induced inhibition of photosynthesis than controls. Taken together, these data demonstrate that LEA5 regulates organellar translation, in order to enhance respiration relative to photosynthesis in response to stress.

The WHIRLY (WHY) DNA/RNA binding proteins fulfil multiple but poorly characterised functions in leaf development. Here we show that 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. To identify transcripts associated with leaf development we conducted hierarchical clustering of differentially abundant transcripts along the developmental gradient of wild-type leaves. Similarly, metabolite profiling was employed to identify metabolites exhibiting a developmental gradient. A comparative analysis of transcripts and metabolites in barley lines (W1-1 and W1-7) lacking WHY1, which show delayed greening compared to the wild type revealed that the transcript profile of leaf development was largely unchanged in W1-1 and W1-7 leaves. However, 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 tricarboxylic acid cycle metabolites and GABA were significantly lower in WHY1 knockdown leaves than the wild type. This study provides evidence that WHY1 is localized in the nuclei of leaf bases, contributing the regulation of nuclear-encoded transcripts that regulate 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.