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Background and Aims The C4Urochloa species (syn. Brachiaria) and Megathyrsus maximus (syn. Panicum maximum) are used as pasture for cattle across vast areas in tropical agriculture systems in Africa and South America. A key target for variety improvement is forage quality: enhanced digestibility could decrease the amount of land required per unit production, and enhanced lipid content could decrease methane emissions from cattle. For these traits, loss-of-function (LOF) alleles in known gene targets are predicted to improve them, making a reverse genetics approach of allele mining feasible. We therefore set out to look for such alleles in diverse accessions of Urochloa species and Megathyrsus maximus from the genebank collection held at the CIAT. Methods We studied allelic diversity of 20 target genes (11 for digestibility, nine for lipid content) in 104 accessions selected to represent genetic diversity and ploidy levels of U. brizantha, U. decumbens, U. humidicola, U. ruziziensis and M. maximum. We used RNA sequencing and then bait capture DNA sequencing to improve gene models in a U. ruziziensis reference genome to assign polymorphisms with high confidence. Key Results We found 953 non-synonymous polymorphisms across all genes and accessions; within these, we identified seven putative LOF alleles with high confidence, including those in the non-redundant SDP1 and BAHD01 genes present in diploid and tetraploid accessions. These LOF alleles could respectively confer increased lipid content and digestibility if incorporated into a breeding programme. Conclusions We demonstrated a novel, effective approach to allele discovery in diverse accessions using a draft reference genome from a single species. We used this to find gene variants in a collection of tropical grasses that could help reduce the environmental impact of cattle production.

Background and AimsUrochloa (syn. Brachiaria, and including some Panicum and Megathyrus) is a genus of tropical and subtropical grasses widely sown as forage to feed ruminants in the tropics. A better understanding of the diversity among Urochloa spp. allow us to leverage its varying ploidy levels and genome composition to accelerate its improvement, following the example from other crop genera.MethodsWe explored the genetic make-up and population structure in 111 accessions, which comprise the five Urochloa species used for the development of commercial cultivars. These accessions are conserved from wild materials from collection sites at their centre of origin in Africa. We used RNA-seq, averaging 40M reads per accession, to generate 1,167,542 stringently selected SNP markers that tentatively encompassed the complete Urochloa gene pool used in breeding.Key ResultsWe identified ten subpopulations, which had no relation with geographical origin and represented ten independent gene pools, and two groups of admixed accessions. Our results support a division in U. decumbens by ploidy, with a diploid subpopulation closely related to U. ruziziensis, and a tetraploid subpopulation closely related to U. brizantha. We observed highly differentiated gene pools in U. brizantha, which were not related with origin or ploidy. Particularly, one U. brizantha subpopulation clustered distant from the other U. brizantha and U. decumbens subpopulations, so likely containing unexplored alleles. We also identified a well-supported subpopulation containing both polyploid U. decumbens and U. brizantha accessions; this was the only group containing more than one species and tentatively constitutes an independent “mixed” gene pool for both species. We observed two gene pools in U. humidicola. One subpopulation, “humidicola-2”, was much less common but likely includes the only known sexual accession in the species.ConclusionsOur results offered a definitive picture of the available diversity in Urochloa to inform breeding and resolve questions raised by previous studies. It also allowed us identifying prospective founders to enrich the breeding gene pool and to develop genotyping and genotype-phenotype association mapping experiments.HIGHLIGHTWe clarified the genetic make-up and population structure of 111 Urochloa spp. forage grasses to inform cultivar development.

Urochloa (including Brachiaria, Megathyrus and some Panicum) tropical grasses are native to Africa and are now, after selection and breeding, planted worldwide, particularly in South America, as important forages with huge potential for further sustainable improvement and conservation of grasslands. We aimed to develop an optimized approach to determine ploidy of germplasm collection of this tropical forage grass group using dried leaf material, including approaches to collect, dry and preserve plant samples for flow cytometry analysis. Our methods enable robust identification of ploidy levels (coefficient of variation of G0/G1 peaks, CV, typically &lt, 5%). Ploidy of some 348 forage grass accessions (ploidy range from 2x to 9x), from international genetic resource collections, showing variation in basic chromosome numbers and reproduction modes (apomixis and sexual), were determined using our defined standard protocol. Two major Urochloa agamic complexes are used in the current breeding programs at CIAT and EMBRAPA: the ’brizantha’ and ’humidicola’ agamic complexes are variable, with multiple ploidy levels. Some U. brizantha accessions have odd level of ploidy (5x), and the relative differences in fluorescence values of the peak positions between adjacent cytotypes is reduced, thus more precise examination of this species is required. Ploidy measurement of U. humidicola revealed aneuploidy.

Feruloylation of arabinoxylan (AX) in grass cell walls is a key determinant of recalcitrance to enzyme attack, making it a target for improvement of grass crops, and of interest in grass evolution. Definitive evidence on the genes responsible is lacking so we studied a candidate gene that we identified within the BAHD acyl-CoA transferase family. We used RNA interference (RNAi) silencing of orthologs in the model grasses Setaria viridis (SvBAHD01) and Brachypodium distachyon (BdBAHD01) and determined effects on AX feruloylation. Silencing of SvBAHD01 in Setaria resulted in a c. 60% decrease in AX feruloylation in stems consistently across four generations. Silencing of BdBAHD01 in Brachypodium stems decreased feruloylation much less, possibly due to higher expression of functionally redundant genes. Setaria SvBAHD01 RNAi plants showed: no decrease in total lignin, approximately doubled arabinose acylated by p-coumarate, changes in two-dimensional NMR spectra of unfractionated cell walls consistent with biochemical estimates, no effect on total biomass production and an increase in biomass saccharification efficiency of 40-60%. We provide the first strong evidence for a key role of the BAHD01 gene in AX feruloylation and demonstrate that it is a promising target for improvement of grass crops for biofuel, biorefining and animal nutrition applications.

Photosynthesis and associated signalling are influenced by the dorso-ventral properties of leaves. The degree of adaxial/abaxial symmetry in stomatal numbers, photosyn- thetic regulation with respect to light orientation and the total section areas of the bundle sheath (BS) cells and the surrounding mesophyll (M) cells on the adaxial and abaxial sides of the vascular bundles were compared in two C4 (Zea mays (maize) and Paspalum dilatatum) and one C3 (Triticum turgidum (Durum wheat)) monocotyledonous species. The C3 leaves had a higher degree of dorso-ventral symmetry than the C4 leaves.Photosynthetic regulation was the same on each side of the wheat leaves, as were stomatal numbers and the section area of the BS relative to that of the M cells (BS/M section area ratio). In contrast, photo- synthetic regulation in maize and P. dilatatum leaves showed a marked surface-specific response to light orienta- tion. Compared to the adaxial sides of the C4 monocotyle- donous leaves, the abaxial surfaces had more stomata and the BS/M section area ratio was significantly higher. Differ- ences in dorso-ventral structure, particularly in Kranz anatomy, serve not only to maximize photosynthetic capac- ity with respect light orientation in C4 monocotyledonous leaves but also allow adaxial and abaxial-specific signalling from the respective M cells.

Mitochondrial electron transport pathways exert effects on carbon-nitrogen (C/N) relationships. To examine whether mitochondria-N interactions also influence plant growth and development, we explored the responses of roots and shoots to external N supply in wild-type (WT) Nicotiana sylvestris and the cytoplasmic male sterile II (CMSII) mutant, which has a N-rich phenotype. Root architecture in N. sylvestris seedlings showed classic responses to nitrate and sucrose availability. In contrast, CMSII showed an altered 'nitrate-sensing' phenotype with decreased sensitivity to C and N metabolites. The WT growth phenotype was restored in CMSII seedling roots by high nitrate plus sugars and in shoots by gibberellic acid (GA). Genome-wide cDNA-amplified fragment length polymorphism (AFLP) analysis of leaves from mature plants revealed that only a small subset of transcripts was altered in CMSII. Tissue abscisic acid content was similar in CMSII and WT roots and shoots, and growth responses to zeatin were comparable. However, the abundance of key transcripts associated with GA synthesis was modified both by the availability of N and by the CMSII mutation. The CMSII mutant maintained a much higher shoot/root ratio at low N than WT, whereas no difference was observed at high N. Shoot/root ratios were strikingly correlated with root amines/nitrate ratios, values of < 1 being characteristic of high N status. We propose a model in which the amine/nitrate ratio interacts with GA signalling and respiratory pathways to regulate the partitioning of biomass between shoots and roots.

Nodule numbers are regulated through systemic auto-regulatory signals produced by shoots and roots. The relative effects of shoot and root genotype on nodule numbers together with relationships to organ biomass, carbon (C) and nitrogen (N) status, and related parameters were measured in pea (Pisum sativum) exploiting natural genetic variation in maturity and apparent nodulation intensity. Reciprocal grafting experiments between the early (Athos), intermediate (Phonix) and late (S00182) maturity phenotypes were performed and Pearson's correlation coefficients for the parameters were calculated. No significant correlations were found between shoot C/N ratios and plant morphology parameters, but the root C/N ratio showed a strong correlation with root fresh and dry weights as well as with shoot fresh weight with less significant interactions with leaf number. Hence, the root C/N ratio rather than shoot C/N had a predominant influence on plant morphology when pea plants are grown under conditions of symbiotic nitrogen supply. The only phenotypic characteristic that showed a statistically significant correlation with nodulation intensity was shoot length, which accounted for 68.5% of the variation. A strong linear relationship was demonstrated between shoot length and nodule numbers. Hence, pea nodule numbers are controlled by factors related to shoot extension, but not by shoot or root biomass accumulation, total C or total N. The relationship between shoot length and nodule numbers persisted under field conditions. These results suggest that stem height could be used as a breeding marker for the selection of pea cultivars with high nodule numbers and high seed N contents.

Targeting transgene expression to specific cells and tissues is desirable for a number of reasons (Gittins et al., 2000) not least of which is the requirement to reduce perceived risks with GM technology. In fruit crops, where invariably the product is consumed fresh, this assumes unparalleled importance. To this end, the ability of several heterologous and homologous gene promoters to drive expression of the βglucuronidase (gusA) marker gene in the vegetative tissues of transgenic apple (Malus pumila, Mill.) have and are being tested. These promoters originally drove expression in leaves (Rubisco, small subunit (SSU), RBCS3C from tomato, SRS1 from soybean), in vascular tissue (rolC, and CoYMV) and in root tissue (extA from Brassica). Homologous promoters from apple were cloned for expression in fruit. Transgenic lines were produced by Agrobacterium-mediated transformation and the levels of gusA activity in the vegetative tissues of young plants were compared with those using the CaMV 35S promoter to drive expression of the same gene. Quantitative GUS data were related to the copy number of transgene loci assessed by Southern blotting. The precise location of GUS activity in each tissue was identified by staining of whole leaves and tissue sections with the chromogenic substrate X-Gluc. Light-regulation and patterns of expression are recorded in various vegetative tissues of apple.

The ability of the heterologous promoters, rolCP and CoYMVP, to drive expression of the gusA reporter gene in the vegetative tissues of apple (Malus pumila Mill.) has been studied using transgenic plants produced by Agrobacterium-mediated transformation. Replicate plants of each transgenic clone were propagated in soil to a uniform size and samples of leaf, petiole, stem, and root were taken for the measurement of β-glucuronidase (GUS) activity by fluorometric assay. The levels of expression were compared with those in tissues of a representative clone containing the CaMV 35S promoter. These quantitative GUS data were related to the copy number of transgene loci assessed by Southern blotting. The CoYMV promoter was slightly more active than the rolC promoter, although both expressed gusA at a lower level than the CaMV 35S promoter. In clones containing the rolC promoter with multiple transgene loci, expression values were generally among the highest or lowest in the range. The precise location of GUS activity in each tissue was identified by staining of whole leaves and tissue sections with a chromogenic substrate. This analysis demonstrated that with both the rolC and CoYMV promoters the reporter gene activity was primarily localised to vascular tissues, particularly the phloem. Our results indicate that both promoters would be suitable to drive the expression of transgenes to combat pests and diseases of apple that are dependent on interaction with the phloem.

Seeds were obtained from seven natural populations of Acacia dealbata, three natural populations of A. mangium and a seed orchard of A. mangium, representing the natural range of the two species. Polyploids were discovered in two of the seven populations of A. dealbata. The 2C DNA amount for diploid A. dealbata (2n = 2x = 26) was 1·74 pg, and for diploid A. mangium (2n = 2x = 26) was 1·30 pg. A naturally occurring tetraploid of A. dealbata (2n = 4x = 52) had a 2C DNA amount of 3·41 pg and a naturally occurring triploid genotype had a 2C DNA amount of 2·53 pg. The use of colchicine and oryzalin was investigated as a means of producing higher frequencies of tetraploids of both A. mangium and A. dealbata for incorporation into breeding programmes. Colchicine treatment gave tetraploid frequencies up to 29 % for A. dealbata seedlings, and up to 18 % for A. mangium seedlings. In contrast, no tetraploid A. mangium was detected following oryzalin treatment, and the low frequencies of tetraploids observed in A. dealbata could be attributed to their natural occurrence.

It is desirable that the expression of transgenes in genetically improved crops is restricted to the tissue requiring the encoded activity. To this end, the ability of several heterologous and homologous gene promoters to drive expression of the ?-glucuronidase (gusA) marker gene in the vegetative tissues of transgenic apple (Malus pumila Mill.) and commercial strawberry (Fragaria x ananassa) was tested. These promoters originally drove expression in leaves (Rubisco) small subunit (SSU), RBCS3CP from tomato, SRS1P from soybean, vascular tissue, rolCP, and CoYMVP and tissue root, extAP (Brassica) and PsMTAP (Pisum). Homologous promoters from apple were cloned for expression in fruit and from strawberry for expression in stamens, petals and roots. Transgenic lines were produced by Agrobacterium-mediated transformation and the level of gusA expression in the vegetative tissues of young plants was compared with that using the CaMV 35S promoter. Quantitative GUS data were related to the copy number of transgene loci assessed by Southern blotting. The precise location of GUS activity in each tissue was identified by staining of whole leaves and tissue sections with the chromogenic substrate X-Gluc. Light-regulation and patterns of expression are recorderd in various vegetative tissues of apple.

The Brassica napus extensin A gene is highly expressed in root tissue of oilseed rape. In an attempt to identify an effective root-specific promoter for biotechnological applications, we have examined the ability of the −940 extA promoter to drive expression of the gusA reporter gene in the vegetative tissues of apple (Malus pumila Mill cv. Greensleeves). Transgenic apple lines were produced by Agrobacterium tumefaciens-mediated transformation and GUS activity was analysed both quantitatively and qualitatively. The extA promoter was active in all tissues of young plants in all 15 clones examined. However Southern blot data suggested that only a proportion of the population contained the entire promoter and that others had suffered deletions of unknown length. This may have contributed to the variation seen in the quantitative and qualitative expression of GUS. Specific GUS activity was highest in the stems where it approached, and in some clones, exceeded that using the constitutive CaMV 35S promoter. Histochemical analysis confirmed that GUS was localised to tissues involved in structural support of the stem. Staining was particularly intense at nodal junctions where high tensile stress is exerted on the tissues. Maturing phloem tissues showed localisation of expression to the phloem parenchyma cells and phloem fibres. Transverse sections of the root revealed staining of primary procambial tissues including the young endodermis but no staining was seen in the cortex. Although the −940 extA promoter is clearly not root-specific in apple, it is likely to have useful biotechnological applications in tree species.

The cell walls of grasses such as wheat, maize, rice, and sugar cane, contain large amounts of ferulate that is ester-linked to the cell wall polysaccharide glucuronoarabinoxylan (GAX). This ferulate is considered to limit the digestibility of polysaccharide in grass biomass as it forms covalent linkages between polysaccharide and lignin components. Candidate genes within a grass-specific clade of the BAHD acyl-coA transferase superfamily have been identified as being responsible for the ester linkage of ferulate to GAX. Manipulation of these BAHD genes may therefore be a biotechnological target for increasing efficiency of conversion of grass biomass into biofuel. Here, we describe the expression of these candidate genes and amounts of bound ferulate from various tissues and developmental stages of the model grass Brachypodium distachyon. BAHD candidate transcripts and significant amounts of bound ferulate were present in every tissue and developmental stage. We hypothesize that BAHD candidate genes similar to the recently described Oryza sativa p-coumarate monolignol transferase (OsPMT) gene (PMT sub-clade) are principally responsible for the bound para-coumaric acid (pCA), and that other BAHD candidates (non-PMT sub-clade) are responsible for bound ferulic acid (FA). There were some similarities with between the ratio of expression non-PMT/PMT genes and the ratio of bound FA/pCA between tissue types, compatible with this hypothesis. However, much further work to modify BAHD genes in grasses and to characterize the heterologously expressed proteins is required to demonstrate their function.

The transcriptome of the developing starchy endosperm of hexaploid wheat (Triticum aestivum) was determined using RNA-Seq isolated at five stages during grain fill. This resource represents an excellent way to identify candidate genes responsible for the starchy endosperm cell wall, which is dominated by arabinoxylan (AX), accounting for 70% of the cell wall polysaccharides, with 20% (1,3;1,4)-β-d-glucan, 7% glucomannan, and 4% cellulose. A complete inventory of transcripts of 124 glycosyltransferase (GT) and 72 glycosylhydrolase (GH) genes associated with cell walls is presented. The most highly expressed GT transcript (excluding those known to be involved in starch synthesis) was a GT47 family transcript similar to Arabidopsis (Arabidopsis thaliana) IRX10 involved in xylan extension, and the second most abundant was a GT61. Profiles for GT43 IRX9 and IRX14 putative orthologs were consistent with roles in AX synthesis. Low abundances were found for transcripts from genes in the acyl-coA transferase BAHD family, for which a role in AX feruloylation has been postulated. The relative expression of these was much greater in whole grain compared with starchy endosperm, correlating with the levels of bound ferulate. Transcripts associated with callose (GSL), cellulose (CESA), pectin (GAUT), and glucomannan (CSLA) synthesis were also abundant in starchy endosperm, while the corresponding cell wall polysaccharides were confirmed as low abundance (glucomannan and callose) or undetectable (pectin) in these samples. Abundant transcripts from GH families associated with the hydrolysis of these polysaccharides were also present, suggesting that they may be rapidly turned over. Abundant transcripts in the GT31 family may be responsible for the addition of Gal residues to arabinogalactan peptide.

Cellular redox homeostasis is a hub for signal integration. Interactions between redox metabolism and the ABSCISIC ACID-INSENSITIVE-4 (ABI4) transcription factor were characterized in the Arabidopsis thaliana vitamin c defective1 (vtc1) and vtc2 mutants, which are defective in ascorbic acid synthesis and show a slow growth phenotype together with enhanced abscisic acid (ABA) levels relative to the wild type (Columbia-0). The 75% decrease in the leaf ascorbate pool in the vtc2 mutants was not sufficient to adversely affect GA metabolism. The transcriptome signatures of the abi4, vtc1, and vtc2 mutants showed significant overlap, with a large number of transcription factors or signaling components similarly repressed or induced. Moreover, lincomycin-dependent changes in LIGHT HARVESTING CHLOROPHYLL A/B BINDING PROTEIN 1.1 expression were comparable in these mutants, suggesting overlapping participation in chloroplast to nucleus signaling. The slow growth phenotype of vtc2 was absent in the abi4 vtc2 double mutant, as was the sugar-insensitive phenotype of the abi4 mutant. Octadecanoid derivative-responsive AP2/ERF-domain transcription factor 47 (ORA47) and AP3 (an ABI5 binding factor) transcripts were enhanced in vtc2 but repressed in abi4 vtc2, suggesting that ABI4 and ascorbate modulate growth and defense gene expression through jasmonate signaling. We conclude that low ascorbate triggers ABA- and jasmonate-dependent signaling pathways that together regulate growth through ABI4. Moreover, cellular redox homeostasis exerts a strong influence on sugar-dependent growth regulation.

Environmental controls on leaf NAD status remain poorly understood. Here, we analyzed the effects of two key environmental variables, CO(2) and nitrogen, on leaf metabolite profiles, NAD status and the abundance of key transcripts involved in de novo NAD synthesis in wild-type (WT) Nicotiana sylvestris and the CMSII mutant that lacks respiratory complex I. High CO(2) and increased N supply both significantly enhanced NAD(+) and NADH pools in WT leaves. In nitrogen-sufficient conditions, CMSII leaves were enriched in NAD(+) and NADH compared to the WT, but the differences in NADH were smaller at high CO(2) than in air because high CO(2) increased WT NADH/NAD(+). The CMSII-linked increases in NAD(+) and NADH status were abolished by growth with limited nitrogen, which also depleted the nicotine and nicotinic acid pools in the CMSII leaves. Few statistically significant genotype and N-dependent differences were detected in NAD synthesis transcripts, with effects only on aspartate oxidase and NAD synthetase mRNAs. Non-targeted metabolite profiling as well as quantitative amine analysis showed that NAD(+) and NADH contents correlated tightly with leaf amino acid contents across all samples. The results reveal considerable genotype- and condition-dependent plasticity in leaf NAD(+) and NADH contents that is not linked to modified expression of NAD synthesis genes at the transcript level and show that NAD(+) and NADH contents are tightly integrated with nitrogen metabolism. A regulatory two-way feedback circuit between nitrogen and NAD in the regulation of N assimilation is proposed that potentially links the nutritional status to NAD-dependent signaling pathways.

Photosynthesis is an important driver of ecosystem sustainability in the face of climate change. Monocotyledonous crop species with C4 photosynthesis such as maize (Zea mays L; corn) and sugar cane are crucial for future food security and biofuel crop requirements, while C4 pasture grasses such as Paspalum are central to natural ecosystems. The global demand for corn will exceed that for wheat and rice by 2020, making it the world's most important crop. Light-driven photosynthesis supports plant biomass production, but plants have also evolved safety valve mechanisms that attenuate the absorption of potentially lethal levels of excess light. The array of survival responses that enables leaves to evade photoinhibition is complex and involves chloroplast and leaf movement as well as the molecular rearrangements that facilitate thermal energy dissipation. Here we report a novel morphological mechanism that allows C4 monocotyledonous leaves to regulate photosynthesis independently on each surface with respect to incident light allowing better adaptation to water deficits and light stress. We show that under abaxial illumination as occurs when monocotyledonous leaves curl in response to water stress the stomata close and photosynthetic metabolism shuts down on the adaxial surface of C4 leaves but these parameters increase in function to the abaxial surface. We discuss how this regulation confers a survival advantage to the C4 relative to C3 leaves which are unable to regulate their dorso-ventral functions in relation to light.

Improvement in photosynthesis per unit leaf area has been difficult to alter by breeding or genetic modification. We report large changes in photosynthesis in Nicotiana tabacum transformed with E. coli genes for the trehalose pathway. Significantly, photosynthetic capacity (CO2 assimilation at varying light and CO2, and quantum yield of PSII electron transport) per unit leaf area and per leaf dry weight were increased in lines of N. tabacum transformed with the E. coli gene otsA, which encodes trehalose phosphate synthase. In contrast, transformation with otsB, which encodes trehalose phosphate phosphatase or Trec, encoding trehalose phosphate hydrolase, produced the opposite effect. Changes in CO2 assimilation per unit leaf area were closely related to the amount and activity of Rubisco, but not to the maximum activities of other Calvin cycle enzymes. Alterations in photosynthesis were associated with trehalose 6-phosphate content rather than trehalose. When growth parameters were determined, a greater photosynthetic capacity did not translate into greater relative growth rate or biomass. This was because photosynthetic capacity was negatively related to leaf area and leaf area ratio. In contrast, relative growth rate and biomass were positively related to leaf area. These results demonstrate a novel means of modifying Rubisco content and photosynthesis, and the complexities of regulation of photosynthesis at the whole plant level, with potential benefits to biomass production through improved leaf area.

Ascorbic acid (AA) fulfils many essential functions in plants. It is a key antioxidant and an important reducing substrate for a number of enzymes. The effects of low AA on plant architecture and leaf ultrastructure were studied in Arabidopsis thaliana mutants, which have constitutively moderately low (vtc1) or very low (vtc2) leaf AA contents compared with the wild type. Shoot development was comparable in all accessions over the first 14 d of growth. The production of primary roots was slightly different in vtc1, vtc2, and wild-type plants. However, the most notable difference was that a high proportion of the primary roots of the vtc2 plants grown on soil had lost the wild-type responses to gravity. The vtc mutants showed the antagonistic interaction between nitrate and sugar in the regulation of lateral root (LR) development that was observed in the wild type. However, the vtc2 mutants produced greater numbers of longer LRs than wild-type or vtc1 plants at all levels of nitrate. At later stages of development, the vtc rosettes were smaller than those of the wild type and the leaves showed intracellular structural changes that are consistent with programmed cell death (PCD). PCD symptoms such as nuclear chromatin condensation, the presence of multivesicular bodies, and extensive degradation and disorganization of the grana stacks were observed in 8-week-old vtc2 leaves and in 10-week-old vtc1 leaves. The data presented here illustrate the importance of tissue AA contents in regulating whole plant morphology, cell structure, and development.

The Swi1 and Swi3 proteins are required for mat1 imprinting and mating-type switching in Schizosaccharomyces pombe, where they mediate a pause of leading-strand replication in response to a lagging-strand signal. In addition, Swi1 has been demonstrated to be involved in the checkpoint response to stalled replication forks, as was described for the Saccharomyces cerevisiae homologue Tof1. This study addresses the roles of Swi1 and Swi3 during a replication process perturbed by the presence of template bases alkylated by methyl methanesulfonate (MMS). Both the swi1 and swi3 mutations have additive effects on MMS sensitivity and on the MMS-induced damage checkpoint response when combined with chk1 and cds1, but they are nonadditive with hsk1. Cells with swi1, swi3, or hsk1 mutations are also defective in slowing progression through S phase in response to MMS damage. Moreover, swi1 and swi3 strains show increased levels of genomic instability even in the absence of exogenously induced DNA damage. Chromosome fragmentation, increased levels of single-stranded DNA, increased recombination, and instability of replication forks stalled in the presence of hydroxyurea are observed, consistent with the possibility that the replication process is affected in these mutants. In conclusion, Swi1, Swi3, and Hsk1 act in a novel S-phase checkpoint pathway that contributes to replication fork maintenance and to survival of alkylation damage.

Genes for trehalose metabolism are widespread in higher plants. Insight into the physiological role of the trehalose pathway outside of resurrection plant species is lacking. To address this lack of insight, we express Escherichia coli genes for trehalose metabolism in Arabidopsis thaliana , which manipulates trehalose 6-phosphate (T6P) contents in the transgenic plants. Plants expressing otsA [encoding trehalose phosphate synthase (TPS)] accumulate T6P whereas those expressing either otsB [encoding trehalose phosphate phosphatase (TPP)] or treC [encoding trehalose phosphate hydrolase (TPH)] contain low levels of T6P. Expression of treF (encoding trehalase) yields plants with unaltered T6P content and a phenotype not distinguishable from wild type when grown on soil. The marked phenotype obtained of plants accumulating T6P is opposite to that of plants with low T6P levels obtained by expressing either TPP or TPH and consistent with a critical role for T6P in growth and development. Supplied sugar strongly inhibits growth of plants with reduced T6P content and leads to accumulation of respiratory intermediates. Remarkably, sugar improves growth of TPS expressors over wild type, a feat not previously accomplished by manipulation of metabolism. The data indicate that the T6P intermediate of the trehalose pathway controls carbohydrate utilization and thence growth via control of glycolysis in a manner analogous to that in yeast. Furthermore, embryolethal A. thaliana tps1 mutants are rescued by expression of E. coli TPS, but not by supply of trehalose, suggesting that T6P control over primary metabolism is indispensable for development.

It is desirable that the expression of transgenes in genetically modified crops is restricted to the tissues requiring the encoded activity. To this end, we have studied the ability of the heterologous ribulose-1,5- bisphosphate carboxylase/oxygenase (Rubisco) small- subunit (SSU) gene promoters, RBCS3CP (0.8 kbp) from tomato (hycopersion esculentum Mill.) and SRS1P (1.5 kbp) from soybean (Glycine max (h.) Mers.), to drive expression of the b-glucuronidase (gusA) marker gene in apple (Malus pumila Mill.). Transgenic lines of cultivar Greensleeves were produced by Agrobacterium-mediated transformation and the level of gusA expression in the vegetative tissues of young plants was compared with that produced using the cauliflower mosaic virus (CaMV) 35S promoter. These quantitative GUS data were assessed for their relationship to the copy number of transgene loci. The precise location of GUS activity in leaves was identified histochemically. The heterologous SSU promoters were active primarily in the green vegetative tissues of apple, although activity in the roots was noticeably higher with the RBCS3C promoter than with the SRS1 promoter. The mean GUS activity in leaf tissue of the SSU promoter transgenics was approxi- mately half that of plants containing the CaMV 35S promoter. Histochemical analysis demonstrated that GUS activity was localised to the mesophyll and palisade cells of the leaf. The influence of light on expression was also determined. The activity of the SRS1 promoter was strictly dependent on light, whereas that of the RBCS3C promoter appeared not to be. Both SSU promoters would be suitable for the expression of transgenes in green photosynthetic tissues of apple.

Main conclusion Methyl-jasmonate induces large increases in p-coumarate linked to arabinoxylan in Brachypodium and in abundance of GT61 and BAHD family transcripts consistent with a role in synthesis of this linkage. Jasmonic acid (JA) signalling is required for many stress responses in plants, inducing large changes in the transcriptome, including up-regulation of transcripts associated with lignification. However, less is known about the response to JA of grass cell walls and the monocot-specific features of arabinoxylan (AX) synthesis and acylation by ferulic acid (FA) and para-coumaric acid (pCA). Here, we show that methyl-jasmonate (MeJA) induces moderate increases in FA monomer, > 50% increases in FA dimers, and five–sixfold increases in pCA ester-linked to cell walls in Brachypodium callus. Direct measurement of arabinose acylated by pCA (Araf-pCA) indicated that most or all the increase in cell-wall pCA was due to pCA ester-linked to AX. Analysis of the RNA-seq transcriptome of the callus response showed that these cell-wall changes were accompanied by up-regulation of members of the GT61 and BAHD gene families implicated in AX decoration and acylation; two BAHD paralogues were among the most up-regulated cell-wall genes (seven and fivefold) after 24 h exposure to MeJA. Similar responses to JA of orthologous BAHD and GT61 transcripts are present in the RiceXPro public expression data set for rice seedlings, showing that they are not specific to Brachypodium or to callus. The large response of AX-pCA to MeJA may, therefore, indicate an important role for this linkage in response of primary cell walls of grasses to JA signalling. Electronic supplementary material The online version of this article (10.1007/s00425-018-2968-9) contains supplementary material, which is available to authorized users.