Jose M. Barrero

The Short-Chain Alcohol Dehydrogenase ABA2 Catalyzes the Conversion of Xanthoxin to Abscisic Aldehyde

Mutants able to germinate and perform early growth in medium containing a high NaCl concentration were identified during the course of two independent screenings and named salt resistant (sre) and salobreño (sañ). The sre and sañ mutants also were able to germinate in high-osmoticum medium, indicating that they are osmotolerant in a germination assay. Complementation analyses revealed that sre1-1, sre1-2, sañ3-1, and sañ3-2 were alleles of the abscisic acid (ABA) biosynthesis ABA2 gene. A map-based cloning strategy allowed the identification of the ABA2 gene and molecular characterization of four new aba2 alleles. The ABA2 gene product belongs to the family of short-chain dehydrogenases/reductases, which are known to be NAD- or NADP-dependent oxidoreductases. Recombinant ABA2 protein produced in Escherichia coli exhibits a Km value for xanthoxin of 19 μM and catalyzes in a NAD-dependent manner the conversion of xanthoxin to abscisic aldehyde, as determined by HPLC–mass spectrometry. The ABA2 mRNA is expressed constitutively in all plant organs examined and is not upregulated in response to osmotic stress. The results of this work are discussed in the context of previous genetic and biochemical evidence regarding ABA biosynthesis, confirming the xanthoxin→abscisic aldehyde→ABA transition as the last steps of the major ABA biosynthetic pathway.

INCURVATA2 Encodes the Catalytic Subunit of DNA Polymerase α and Interacts with Genes Involved in Chromatin-Mediated Cellular Memory in Arabidopsis thaliana

Cell type–specific gene expression patterns are maintained by the stable inheritance of transcriptional states through mitosis, requiring the action of multiprotein complexes that remodel chromatin structure. Genetic and molecular interactions between chromatin remodeling factors and components of the DNA replication machinery have been identified in Schizosaccharomyces pombe, indicating that some epigenetic marks are replicated simultaneously to DNA with the participation of the DNA replication complexes. This model of epigenetic inheritance might be extended to the plant kingdom, as we report here with the positional cloning and characterization of INCURVATA2 (ICU2), which encodes the putative catalytic subunit of the DNA polymerase α of Arabidopsis thaliana. The strong icu2-2 and icu2-3 insertional alleles caused fully penetrant zygotic lethality when homozygous and incompletely penetrant gametophytic lethality, probably because of loss of DNA polymerase activity. The weak icu2-1 allele carried a point mutation and caused early flowering, leaf incurvature, and homeotic transformations of sepals into carpels and of petals into stamens. Further genetic analyses indicated that ICU2 interacts with TERMINAL FLOWER2, the ortholog of HETEROCHROMATIN PROTEIN1 of animals and yeasts, and with the Polycomb group (PcG) gene CURLY LEAF. Another PcG gene, EMBRYONIC FLOWER2, was found to be epistatic to ICU2. Quantitative RT-PCR analyses indicated that a number of regulatory genes were derepressed in the icu2-1 mutant, including genes associated with flowering time, floral meristem, and floral organ identity.

Anatomical and Transcriptomic Studies of the Coleorhiza Reveal the Importance of This Tissue in Regulating Dormancy in Barley

The decay of seed dormancy during after-ripening is not well understood, but elucidation of the mechanisms involved may be important for developing strategies for modifying dormancy in crop species and, for example, addressing the problem of preharvest sprouting in cereals. We have studied the germination characteristics of barley (Hordeum vulgare ‘Betzes’) embryos, including a description of anatomical changes in the coleorhiza and the enclosed seminal roots. The changes that occur correlate with abscisic acid (ABA) contents of embryo tissues. To understand the molecular mechanisms involved in dormancy loss, we compared the transcriptome of dormant and after-ripened barley embryos using a tissue-specific microarray approach. Our results indicate that in the coleorhiza, ABA catabolism is promoted and ABA sensitivity is reduced and that this is associated with differential regulation by after-ripening of ABA 8′-hydroxylase and of the LIPID PHOSPHATE PHOSPHATASE gene family and ABI3-INTERACTING PROTEIN2, respectively. We also identified other processes, including jasmonate responses, cell wall modification, nitrate and nitrite reduction, mRNA stability, and blue light sensitivity, that were affected by after-ripening in the coleorhiza that may be downstream of ABA signaling. Based on these results, we propose that the coleorhiza plays a major role in causing dormancy by acting as a barrier to root emergence and that after-ripening potentiates molecular changes related to ABA metabolism and sensitivity that ultimately lead to degradation of the coleorhiza, root emergence, and germination.

Gene expression profiling identifies two regulatory genes controlling dormancy and ABA sensitivity in Arabidopsis seeds

Seed dormancy is a very important trait that maximizes the survival of seed in nature, the control of which can have important repercussions on the yield of many crop species. We have used gene expression profiling to identify genes that are involved in dormancy regulation in Arabidopsis thaliana. RNA was isolated from imbibed dormant (D) and after-ripened (AR) ecotype C24 seeds, and then screened by quantitative RT-PCR (qRT-PCR) for differentially expressed transcription factors (TFs) and other regulatory genes. Out of 2207 genes screened, we have identified 39 that were differentially expressed during the first few hours of imbibition. After analyzing T-DNA insertion mutants for 22 of these genes, two displayed altered dormancy compared with the wild type. These mutants are affected in genes that encode a RING finger and an HDZip protein. The first, named DESPIERTO, is involved in ABA sensitivity during seed development, regulates the expression of ABI3, and produces a complete loss of dormancy when mutated. The second, the HDZip (ATHB20), is expressed during seed germination in the micropylar endosperm and in the root cap, and increases ABA sensitivity and seed dormancy when mutated.

Grain dormancy and light quality effects on germination in the model grass Brachypodium distachyon

• Lack of grain dormancy in cereal crops such as barley and wheat is a common problem affecting farming areas around the world, causing losses in yield and quality because of preharvest sprouting. Control of seed or grain dormancy has been investigated extensively using various approaches in different species, including Arabidopsis and cereals. However, the use of a monocot model plant such as Brachypodium distachyon presents opportunities for the discovery of new genes related to grain dormancy that are not present in modern commercial crops. • In this work we present an anatomical description of the Brachypodium caryopsis, and we describe the dormancy behaviour of six common diploid Brachypodium inbred genotypes. We also study the effect of light quality (blue, red and far-red) on germination, and analyse changes in abscisic acid levels and gene expression between a dormant and a non-dormant Brachypodium genotype. • Our results indicate that different genotypes display high natural variability in grain dormancy and that the characteristics of dormancy and germination are similar to those found in other cereals. • We propose that Brachypodium is an ideal model for studies of grain dormancy in grasses and can be used to identify new strategies for increasing grain dormancy in crop species.

Transcriptomic analysis of wheat near-isogenic lines identifies PM19-A1 and A2 as candidates for a major dormancy QTL

BackgroundNext-generation sequencing technologies provide new opportunities to identify the genetic components responsible for trait variation. However, in species with large polyploid genomes, such as bread wheat, the ability to rapidly identify genes underlying quantitative trait loci (QTL) remains non-trivial. To overcome this, we introduce a novel pipeline that analyses, by RNA-sequencing, multiple near-isogenic lines segregating for a targeted QTL.ResultsWe use this approach to characterize a major and widely utilized seed dormancy QTL located on chromosome 4AL. It exploits the power and mapping resolution afforded by large multi-parent mapping populations, whilst reducing complexity by using multi-allelic contrasts at the targeted QTL region. Our approach identifies two adjacent candidate genes within the QTL region belonging to the ABA-induced Wheat Plasma Membrane 19 family. One of them, PM19-A1, is highly expressed during grain maturation in dormant genotypes. The second, PM19-A2, shows changes in sequence causing several amino acid alterations between dormant and non-dormant genotypes. We confirm that PM19 genes are positive regulators of seed dormancy.ConclusionsThe efficient identification of these strong candidates demonstrates the utility of our transcriptomic pipeline for rapid QTL to gene mapping. By using this approach we are able to provide a comprehensive genetic analysis of the major source of grain dormancy in wheat. Further analysis across a diverse panel of bread and durum wheats indicates that this important dormancy QTL predates hexaploid wheat. The use of these genes by wheat breeders could assist in the elimination of pre-harvest sprouting in wheat.

Roles for blue light, jasmonate and nitric oxide in the regulation of dormancy and germination in wheat grain (Triticum aestivum L.)

Abscisic acid (ABA) plays a central role in seed dormancy and transcriptional regulation of genes coding for ABA biosynthetic and degradation enzymes is responsible for control of ABA content. However, little is known about signalling both before and after ABA regulation, in particular, how environmental signals are perceived and transduced. We are interested in these processes in cereal grains, particularly in relation to the development of strategies for controlling pre-harvest sprouting in barley and wheat. Our previous studies have indicated possible components of dormancy control and here we present evidence that blue light, nitric oxide (NO) and jasmonate are major controlling elements in wheat grain. Using microarray and pharmacological studies, we have found that blue light inhibits germination in dormant grain and that methyl jasmonate (MJ) and NO counteract this effect by reducing dormancy. We also present evidence that NO and jasmonate play roles in dormancy control in vivo. ABA was reduced by MJ and this was accompanied by reduced levels of expression of TaNCED1 and increased expression of TaABA8′OH-1 compared with dormant grain. Similar changes were caused by after-ripening. Analysis of global gene expression showed that although jasmonate and after-ripening caused important changes in gene expression, the changes were very different. While breaking dormancy, MJ had only a small number of target genes including gene(s) encoding beta-glucosidase. Our evidence indicates that NO and MJ act interdependently in controlling reduction of ABA and thus the demise of dormancy.

A Role for Barley CRYPTOCHROME1 in Light Regulation of Grain Dormancy and Germination

This work reveals that the photoreceptor CRYPTOCHROME1 (CRY1), but not CRY2, is involved in the perception of blue light in dormant barley grains, which inhibits germination by impeding ABA decline in the embryo. It is well known that abscisic acid (ABA) plays a central role in the regulation of seed dormancy and that transcriptional regulation of genes encoding ABA biosynthetic and degradation enzymes is responsible for determining ABA content. However, little is known about the upstream signaling pathways impinging on transcription to ultimately regulate ABA content or how environmental signals (e.g., light and cold) might direct such expression in grains. Our previous studies indicated that light is a key environmental signal inhibiting germination in dormant grains of barley (Hordeum vulgare), wheat (Triticum aestivum), and Brachypodium distachyon and that this effect attenuates as after-ripening progresses further. We found that the blue component of the light spectrum inhibits completion of germination in barley by inducing the expression of the ABA biosynthetic gene 9-cis-epoxycarotenoid dioxygenase and dampening expression of ABA 8’-hydroxylase, thus increasing ABA content in the grain. We have now created barley transgenic lines downregulating the genes encoding the blue light receptors CRYTOCHROME (CRY1) and CRY2. Our results demonstrate that CRY1 is the key receptor perceiving and transducing the blue light signal in dormant grains.

Dormancy in cereals (not too much, not so little): about the mechanisms behind this trait

As in other cultivated species, dormancy can be seen as a problem in cereal production, either due to its short duration or to its long persistence. Indeed, cereal crops lacking enough dormancy at harvest can be exposed to pre-harvest sprouting damage, while a long-lasting dormancy can interfere with processes that rely on rapid germination, such as malting or the emergence of a uniform crop. Because the ancestors of cereal species evolved under very diverse environments worldwide, different mechanisms have arisen as a way of sensing an appropriate germination environment (a crucial factor for winter or summer annuals such as cereals). In addition, different species (and even different varieties within the same species) display diverse grain morphology, allowing some structures to impose dormancy in some cereals but not in others. As in seeds from many other species, the antagonism between the plant hormones abscisic acid and gibberellins is instrumental in cereal grains for the inception, expression, release and re-induction of dormancy. However, the way in which this antagonism operates is different for the various species and involves different molecular steps as regulatory sites. Environmental signals (i.e. temperature, light quality and quantity, oxygen levels) can modulate this hormonal control of dormancy differently, depending on the species. The practical implications of knowledge accumulated in this field are discussed.

The microRNA pathway genes AGO1, HEN1 and HYL1 participate in leaf proximal-distal, venation and stomatal patterning in Arabidopsis.

We isolated Arabidopsis thaliana mutants with incurved vegetative leaves. Positional cloning of incurvata8 (icu8), icu9 and icu15 has identified them as new loss-of-function alleles of the HYPONASTIC LEAVES1 (HYL1), ARGONAUTE1 (AGO1) and HUA ENHANCER1 (HEN1) genes, respectively, which encode known components of the microRNA pathway. The morphological and histological characterization of these mutants and of dicer-like1-9 indicates that small RNAs participate in the proximal-distal and adaxial-abaxial patterning of leaves, as well as in stomatal number establishment. The abnormal vasculature of ago1 and hyl1 leaves also suggests a role for AGO1 and HYL1 in venation patterning. Our mutants expand the allelic series of AGO1, HYL1 and HEN1, and might help to understand the developmental and cellular significance of miRNA-mediated posttranscriptional regulation.