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

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

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 Physiological Changes and Histone Modifications in Barley Primary Leaves at HSP17 Gene

Stress-inducible genes undergo epigenetic modifications under stress conditions. To investigate if HSP17, of which transcripts accumulate in plant cells under stress, is regulated through epigenetic mechanisms under drought stress, 5-day-old barley (Hordeum vulgare cv. Carina) plants were subjected to progressive drought through water withholding for 22 days. Changes in physiological status and expression of HSP17 gene were monitored in primary leaves of control and drought-treated plants every two days. Twelve days after drought started, control and drought-treated plants were analyzed by chromatin-immunoprecipitation using antibodies against three histone modifications (H3K4me3, H3K9ac, and H3K9me2) and H3 itself. Already after four days of drought treatment, stomatal conductance was severely decreased. Thereafter, maximum and quantum yield of photosystem II (PSII), regulated and non-regulated energy dissipation in PSII, and later also chlorophyll content, were affected by drought, indicating the stress-induced onset of senescence. At the 12th day of drought, before leaf water content declined, expression of HSP17 gene was increased two-fold in drought-treated plants compared to the controls. Twelve days of drought caused an increase in H3 and a loss in H3K9me2 not only at HSP17, but also at constitutively transcribed reference genes ACTIN, PROTEIN PHOSPHATASE 2A (pp2A), and at silent regions BM9, CEREBA. In contrast, H3K4me3 showed a specific increase at HSP17 gene at the beginning and the middle part of the coding region, indicating that this mark is critical for the drought-responsive transcription status of a gene

Drought Stress-Related Physiological Changes and Histone Modifications in Barley Primary Leaves at HSP17 Gene

Stress-inducible genes undergo epigenetic modifications under stress conditions. To investigate if HSP17, of which transcripts accumulate in plant cells under stress, is regulated through epigenetic mechanisms under drought stress, 5-day-old barley (Hordeum vulgare cv. Carina) plants were subjected to progressive drought through water withholding for 22 days. Changes in physiological status and expression of HSP17 gene were monitored in primary leaves of control and drought-treated plants every two days. Twelve days after drought started, control and drought-treated plants were analyzed by chromatin-immunoprecipitation using antibodies against three histone modifications (H3K4me3, H3K9ac, and H3K9me2) and H3 itself. Already after four days of drought treatment, stomatal conductance was severely decreased. Thereafter, maximum and quantum yield of photosystem II (PSII), regulated and non-regulated energy dissipation in PSII, and later also chlorophyll content, were affected by drought, indicating the stress-induced onset of senescence. At the 12th day of drought, before leaf water content declined, expression of HSP17 gene was increased two-fold in drought-treated plants compared to the controls. Twelve days of drought caused an increase in H3 and a loss in H3K9me2 not only at HSP17, but also at constitutively transcribed reference genes ACTIN, PROTEIN PHOSPHATASE 2A (pp2A), and at silent regions BM9, CEREBA. In contrast, H3K4me3 showed a specific increase at HSP17 gene at the beginning and the middle part of the coding region, indicating that this mark is critical for the drought-responsive transcription status of a gene

The Barley Heavy Metal Associated Isoprenylated Plant Protein HvFP1 Is Involved in a Crosstalk between the Leaf Development and Abscisic Acid-Related Drought Stress Responses

The heavy metal associated isoprenylated plant proteins (HIPPs) are characterized by at least one heavy metal associated (HMA) domain and a C-terminal isoprenylation motif. Hordeum vulgare farnesylated protein 1 (HvFP1), a barley HIPP, is upregulated during drought stress, in response to abscisic acid (ABA) and during leaf senescence. To investigate the role of HvFP1, two independent gain-of-function lines were generated. In a physiological level, the overexpression of HvFP1 results in the delay of normal leaf senescence, but not in the delay of rapid, drought-induced leaf senescence. In addition, the overexpression of HvFP1 suppresses the induction of the ABA-related genes during drought and senescence, e.g., HvNCED, HvS40, HvDhn1. Even though HvFP1 is induced during drought, senescence and the ABA treatment, its overexpression suppresses the ABA regulated genes. This indicates that HvFP1 is acting in a negative feedback loop connected to the ABA signaling. The genome-wide transcriptomic analysis via RNA sequencing revealed that the gain-of-function of HvFP1 positively alters the expression of the genes related to leaf development, photomorphogenesis, photosynthesis and chlorophyll biosynthesis. Interestingly, many of those genes encode proteins with zinc binding domains, implying that HvFP1 may act as zinc supplier via its HMA domain. The results show that HvFP1 is involved in a crosstalk between stress responses and growth control pathways

Knockdown of WHIRLY1 Affects Drought Stress-Induced Leaf Senescence and Histone Modifications of the Senescence-Associated Gene HvS40

The plastid-nucleus located protein WHIRLY1 has been described as an upstream regulator of leaf senescence, binding to the promoter of senescence-associated genes like HvS40. To investigate the impact of WHIRLY1 on drought stress-induced, premature senescence, transgenic barley plants with an RNAi-mediated knockdown of the HvWHIRLY1 gene were grown under normal and drought stress conditions. The course of leaf senescence in these lines was monitored by physiological parameters and studies on the expression of senescence- and drought stress-related genes. Drought treatment accelerated leaf senescence in WT plants, whereas WHIRLY 1 knockdown lines (RNAi-W1) showed a stay-green phenotype. Expression of both senescence-associated and drought stress-responsive genes, was delayed in the transgenic plants. Notably, expression of transcription factors of the WRKY and NAC families, which are known to function in senescence- and stress-related signaling pathways, was affected in plants with impaired accumulation of WHIRLY1, indicating that WHIRLY1 acts as an upstream regulator of drought stress-induced senescence. To reveal the epigenetic indexing of HvS40 at the onset of drought-induced senescence in WT and RNAi-W1 lines, stress-responsive loading with histone modifications of promoter and coding sequences of HvS40 was analyzed by chromatin immunoprecipitation and quantified by qRT-PCR. In the wildtype, the euchromatic mark H3K9ac of the HvS40 gene was low under control conditions and was established in response to drought treatment, indicating the action of epigenetic mechanisms in response to drought stress. However, drought stress caused no significant increase in H3K9ac in plants impaired in accumulation of WHIRLY1. The results show that WHIRLY1 knockdown sets in motion a delay in senescence that involves all aspects of gene expression, including changes in chromatin structure