WHIRLY1 acts upstream of ABA-related reprogramming of drought-induced gene expression in barley and affects stress-related histone modifications

WHIRLY1, a small plant-specific ssDNA-binding protein, dually located in chloroplasts and the nucleus, is discussed to act as a retrograde signal transmitting a stress signal from the chloroplast to the nucleus and triggering there a stress-related gene expression. In this work, we investigated the function of WHIRLY1 in the drought stress response of barley, employing two overexpression lines (oeW1-2 and oeW1-15). The overexpression of WHIRLY1 delayed the drought-stress-related onset of senescence in primary leaves. Two abscisic acid (ABA)-dependent marker genes of drought stress, HvNCED1 and HvS40, whose expression in the wild type was induced during drought treatment, were not induced in overexpression lines. In addition, a drought-related increase in ABA concentration in the leaves was suppressed in WHIRLY1 overexpression lines. To analyze the impact of the gain-of-function of WHIRLY1 on the drought-related reprogramming of nuclear gene expression, RNAseq was performed comparing the wild type and an overexpression line. Cluster analyses revealed a set of genes highly up-regulated in response to drought in the wild type but not in the WHIRLY1 overexpression lines. Among these genes were many stress- and abscisic acid (ABA)-related ones. Another cluster comprised genes up-regulated in the oeW1 lines compared to the wild type. These were related to primary metabolism, chloroplast function and growth. Our results indicate that WHIRLY1 acts as a hub, balancing trade-off between stress-related and developmental pathways. To test whether the gain-of-function of WHIRLY1 affects the epigenetic control of stress-related gene expression, we analyzed drought-related histone modifications in different regions of the promoter and at the transcriptional start sites of HvNCED1 and HvS40. Interestingly, the level of euchromatic marks (H3K4me3 and H3K9ac) was clearly decreased in both genes in a WHIRLY1 overexpression line. Our results indicate that WHIRLY1, which is discussed to act as a retrograde signal, affects the ABA-related reprogramming of nuclear gene expression during drought via differential histone modifications

In vivo inhibition of cysteine proteases provides evidence for the involvement of 'senescence-associated vacuoles' in chloroplast protein degradation during dark-induced senescence of tobacco leaves

Breakdown of leaf proteins, particularly chloroplast proteins, is a massive process in senescing leaves. In spite of its importance in internal N recycling, the mechanism(s) and the enzymes involved are largely unknown. Senescenceassociated vacuoles (SAVs) are small, acidic vacuoles with high cysteine peptidase activity. Chloroplast-targeted proteins re-localize to SAVs during senescence, suggesting that SAVs might be involved in chloroplast protein degradation. SAVs were undetectable in mature, non-senescent tobacco leaves. Their abundance, visualized either with the acidotropic marker Lysotracker Red or by green fluorescent protein (GFP) fluorescence in a line expressing the senescence-associated cysteine protease SAG12 fused to GFP, increased during senescence induction in darkness, and peaked after 2-4 d, when chloroplast dismantling was most intense. Increased abundance of SAVs correlated with higher levels of SAG12 mRNA. Activity labelling with a biotinylated derivative of the cysteine protease inhibitor E-64 was used to detect active cysteine proteases. The two apparently most abundant cysteine proteases of senescing leaves, of 40 kDa and 33 kDa were detected in isolated SAVs. Rubisco degradation in isolated SAVs was completely blocked by E-64. Treatment of leaf disks with E-64 in vivo substantially reduced degradation of Rubisco and leaf proteins. Overall, these results indicate that SAVs contain most of the cysteine protease activity of senescing cells, and that SAV cysteine proteases are at least partly responsible for the degradation of stromal proteins of the chloroplast.Instituto de Fisiología Vegeta

Drought-stress-related reprogramming of gene expression in barley involves differential histone modifications at ABA-related genes

Plants respond to drought by the major reprogramming of gene expression, enabling the plant to survive this threatening environmental condition. The phytohormone abscisic acid (ABA) serves as a crucial upstream signal, inducing this multifaceted process. This report investigated the drought response in barley plants (Hordeum vulgare, cv. Morex) at both the epigenome and transcriptome levels. After a ten-day drought period, during which the soil water content was reduced by about 35%, the relative chlorophyll content, as well as the photosystem II efficiency of the barley leaves, decreased by about 10%. Furthermore, drought-related genes such as HvS40 and HvA1 were already induced compared to the well-watered controls. Global ChIP-Seq analysis was performed to identify genes in which histones H3 were modified with euchromatic K4 trimethylation or K9 acetylation during drought. By applying stringent exclusion criteria, 129 genes loaded with H3K4me3 and 2008 genes loaded with H3K9ac in response to drought were identified, indicating that H3K9 acetylation reacts to drought more sensitively than H3K4 trimethylation. A comparison with differentially expressed genes enabled the identification of specific genes loaded with the euchromatic marks and induced in response to drought treatment. The results revealed that a major proportion of these genes are involved in ABA signaling and related pathways. Intriguingly, two members of the protein phosphatase 2C family (PP2Cs), which play a crucial role in the central regulatory machinery of ABA signaling, were also identified through this approach

Identification of genomic regions involved in tolerance to drought stress and drought stress induced leaf senescence in juvenile barley

BACKGROUND: Premature leaf senescence induced by external stress conditions, e.g. drought stress, is a main factor for yield losses in barley. Research in drought stress tolerance has become more important as due to climate change the number of drought periods will increase and tolerance to drought stress has become a goal of high interest in barley breeding. Therefore, the aim is to identify quantitative trait loci (QTL) involved in drought stress induced leaf senescence and drought stress tolerance in early developmental stages of barley (Hordeum vulgare L.) by applying genome wide association studies (GWAS) on a set of 156 winter barley genotypes. RESULTS: After a four weeks stress period (BBCH 33) leaf colour as an indicator of leaf senescence, electron transport rate at photosystem II, content of free proline, content of soluble sugars, osmolality and the aboveground biomass indicative for drought stress response were determined in the control and stress variant in greenhouse pot experiments. Significant phenotypic variation was observed for all traits analysed. Heritabilities ranged between 0.27 for osmolality and 0.61 for leaf colour in stress treatment and significant effects of genotype, treatment and genotype x treatment were estimated for most traits analysed. Based on these phenotypic data and 3,212 polymorphic single nucleotide polymorphisms (SNP) with a minor allele frequency >5 % derived from the Illumina 9 k iSelect SNP Chip, 181 QTL were detected for all traits analysed. Major QTLs for drought stress and leaf senescence were located on chromosome 5H and 2H. BlastX search for associated marker sequences revealed that respective SNPs are in some cases located in proteins related to drought stress or leaf senescence, e.g. nucleotide pyrophosphatase (AVP1) or serine/ threonin protein kinase (SAPK9). CONCLUSIONS: GWAS resulted in the identification of many QTLs involved in drought stress and leaf senescence of which two major QTLs for drought stress and leaf senescence were located on chromosome 5H and 2H. Results may be the basis to incorporate breeding for tolerance to drought stress or leaf senescence in barley breeding via marker based selection procedures. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12870-015-0524-3) contains supplementary material, which is available to authorized users

WHIRLY1 regulates aliphatic glucosinolate biosynthesis in early seedling development of Arabidopsis.

WHIRLY1 belongs to a family of plant-specific transcription factors capable of binding DNA or RNA in all three plant cell compartments that contain genetic materials. In Arabidopsis thaliana, WHIRLY1 has been studied at the later stages of plant development, including flowering and leaf senescence, as well as in biotic and abiotic stress responses. In this study, WHIRLY1 knockout mutants of A. thaliana were prepared by CRISPR/Cas9-mediated genome editing to investigate the role of WHIRLY1 during early seedling development. The loss-of-function of WHIRLY1 in 5-day-old seedlings did not cause differences in the phenotype and the photosynthetic performance of the emerging cotyledons compared with the wild type. Nevertheless, comparative RNA sequencing analysis revealed that the knockout of WHIRLY1 affected the expression of a small but specific set of genes during this critical phase of development. About 110 genes were found to be significantly deregulated in the knockout mutant, wherein several genes involved in the early steps of aliphatic glucosinolate (GSL) biosynthesis were suppressed compared with wild-type plants. The downregulation of these genes in WHIRLY1 knockout lines led to decreased GSL contents in seedlings and in seeds. Since GSL catabolism mediated by myrosinases was not altered during seed-to-seedling transition, the results suggest that AtWHIRLY1 plays a major role in modulation of aliphatic GSL biosynthesis during early seedling development. In addition, phylogenetic analysis revealed a coincidence between the evolution of methionine-derived aliphatic GSLs and the addition of a new WHIRLY in core families of the plant order Brassicales

WHIRLY1 regulates aliphatic glucosinolate biosynthesis in early seedling development of arabidopsis

WHIRLY1 belongs to a family of plant-specific transcription factors capable of binding DNA or RNA in all three plant cell compartments that contain genetic materials. In Arabidopsis thaliana, WHIRLY1 has been studied at the later stages of plant development, including flowering and leaf senescence, as well as in biotic and abiotic stress responses. In this study, WHIRLY1 knockout mutants of A. thaliana were prepared by CRISPR/Cas9-mediated genome editing to investigate the role of WHIRLY1 during early seedling development. The loss-of-function of WHIRLY1 in 5-day-old seedlings did not cause differences in the phenotype and the photosynthetic performance of the emerging cotyledons compared with the wild type. Nevertheless, comparative RNA sequencing analysis revealed that the knockout of WHIRLY1 affected the expression of a small but specific set of genes during this critical phase of development. About 110 genes were found to be significantly deregulated in the knockout mutant, wherein several genes involved in the early steps of aliphatic glucosinolate (GSL) biosynthesis were suppressed compared with wild-type plants. The downregulation of these genes in WHIRLY1 knockout lines led to decreased GSL contents in seedlings and in seeds. Since GSL catabolism mediated by myrosinases was not altered during seed-to-seedling transition, the results suggest that AtWHIRLY1 plays a major role in modulation of aliphatic GSL biosynthesis during early seedling development. In addition, phylogenetic analysis revealed a coincidence between the evolution of methionine-derived aliphatic GSLs and the addition of a new WHIRLY in core families of the plant order Brassicales