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Agronomic traits controlling the formation, architecture and physiology of source and sink organs are main determinants of rice productivity. Semi-dwarf rice varieties with low tiller formation but high seed production per panicle and dark green and thick leaves with prolonged source activity are among the desirable traits to further increase the yield potential of rice. Here, we report the functional characterization of a zinc finger transcription factor, OsGATA12, whose overexpression causes increased leaf greenness, reduction of leaf and tiller number, and affects yield parameters. Reduced tillering allowed testing the transgenic plants under high density which resulted in significantly increased yield per area and higher harvest index compared to wild-type. We show that delayed senescence of transgenic plants and the corresponding longer stay-green phenotype is mainly due to increased chlorophyll and chloroplast number. Further, our work postulates that the increased greenness observed in the transgenic plants is due to more chlorophyll synthesis but most significantly to decreased chlorophyll degradation, which is supported by the reduced expression of genes involved in the chlorophyll degradation pathway. In particular we show evidence for the down-regulation of the STAY GREEN RICE gene and in vivo repression of its promoter by OsGATA12, which suggests a transcriptional repression function for a GATA transcription factor for prolonging the onset of senescence in cereals.

Nitrogen is an essential mineral nutrient and is required in great abundance for plant growth and development. Insufficient nitrogen triggers extensive physiological and biochemical changes in plants which constitute a set of adaptive responses to nitrogen limitation. In this study, to determine the genome-wide transcriptome response to nitrogen limitation, Arabidopsis plants were grown with limiting (3 mM) and sufficient (10 mM) nitrate, respectively, and their gene expression profiles were analyzed using Affymetrix GeneChip arrays. In addition to inducing the adaptive responses in Arabidopsis, nitrogen limitation altered the expression levels of 629 genes with 340 up-regulated and 289 down-regulated. The up-regulated group included the genes involved in protein degradation and the biosynthesis of anthocyanin and phenylpropanoids. The down-regulated group contained the genes functioning in photosynthesis and in the synthesis of nitrogenous macromolecules such as chlorophyll, proteins, amino acids and nucleotides. Numerous nitrogen limitation responsive genes encode transcription factors, signal transduction components, and proteins required for hormone synthesis and response. The Arabidopsis nitrogen limitation adaptation mutant (nla) is defective in developing the nitrogen limitation adaptive responses. The microarray analysis revealed that the absence of the functional NLA in the nla mutant extensively altered its responsive transcriptome to nitrogen limitation. In this mutant 1122 genes were up-regulated and 622 repressed. It was also found that the nla mutant phenotype was associated with the early induction of senescence-associated genes. This study presents a genome-wide view of Arabidopsis transcriptome response to nitrogen limitation and its regulation by NLA, and provides information to probe the molecular mechanism controlling plant adaptability to nitrogen limitation.

Background High density stress, also known as intraspecies competition, causes significant yield losses in a wide variety of crop plants. At the same time, increases in density tolerance through selective breeding and the concomitant ability to plant crops at a higher population density has been one of the most important factors in the development of high yielding modern cultivars. Results Physiological changes underlying high density stress were examined in Oryza sativa plants over the course of a life cycle by assessing differences in gene expression and metabolism. Moreover, the nitrogen limitation was examined in parallel with high density stress to gain a better understanding of physiological responses specific to high density stress. While both nitrogen limitation and high density resulted in decreased shoot fresh weight, tiller number, plant height and chlorophyll content, high density stress alone had a greater impact on physiological factors. Decreases in aspartate and glutamate concentration were found in plants grown under both stress conditions; however, high density stress had a more significant effect on the concentration of these amino acids. Global transcriptome analysis revealed a large proportion of genes with altered expression in response to both stresses. The presence of ethylene-associated genes in a majority of density responsive genes was investigated further. Expression of ethylene biosynthesis genes ACC synthase 1, ACC synthase 2 and ACC oxidase 7 were found to be upregulated in plants under high density stress. Plants at high density were also found to up regulate ethylene-associated genes and senescence genes, while cytokinin response and biosynthesis genes were down regulated, consistent with higher ethylene production. Conclusions High density stress has similar but greater impact on plant growth and development compared to nitrogen limitation. Global transcriptome changes implicate ethylene as a volatile signal used to communicate proximity in under dense population growth condition and suggest a role for phytohormones in high density stress response in rice plants. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-681) contains supplementary material, which is available to authorized users.

Self-incompatibility (SI) in Brassica is controlled by the S locus. The specificity of the SI response is controlled on the stigma side by the S receptor kinase (SRK) and on the pollen side by the SCR (S locus cysteine-rich) protein, but other proteins might be involved in the process of self-pollen rejection. In this study, we show that the AtPP gene linked to the S locus of Brassica napus is expressed in the stigmas of SI lines. AtPP has a developmental pattern of expression similar to the SRK gene. The AtPP protein has similarity with members of an Arabidopsis protein family and with an S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase, which is a plant defense-related protein of Clarkia breweri representing a new class of methyltransferases. A member of the AtPP gene family is present in the homeolog region of the S locus in Arabidopsis. Therefore, this gene might have co-evolved with S genes from an ancestral S locus of Brassicaceae. Possible functions of the AtPP protein in the self-recognition process are discussed.

Self-incompatibility (SI) promotes outbreeding in flowering plants, and in Brassica SI is genetically controlled by the S locus. Self-incompatible Brassica and self-fertile Arabidopsis belong to the same crucifer family. In addition, a comparative analysis reveals a high degree of microsynteny between the B. campestris S locus and its homologous region in Arabidopsis--with the notable exception that the Brassica SI genes, SLG and SRK, are missing. Brassica ARC1 encodes a component of the SRK signal transduction pathway leading to self-pollen rejection, and no closely related ARC1 homolog has been identified in Arabidopsis. The purpose of the research reported here was to introduce Brassica SI components into Arabidopsis in an attempt to compensate for the missing genes and to investigate whether the SI phenotype can be transferred. Inserts of approximately 40 kb from the fosmid clones F20 and F22, which span the B. napus W1 SLG-SRK region, were cloned into the plant transformation vector pBIBAC2. Transgenic plants were generated that expressed the Brassica SI genes in the flower buds. In addition, the endogenous, SLG-like, gene AtS1 was not co-suppressed by the Brassica SLG transgene. No SI phenotype was observed among the T1 BIBAC2-F20 and BIBAC2-F22 transgenic plants. When the ARC1 gene was transformed into BIBAC2-F20 or BIBAC2-F22 plants, the resulting BIBAC2-F20-ARC1 and BIBAC2-F22-ARC1 plants still set seeds normally, and no rejection response was observed when self-incompatible B. napus W1 pollen was placed on BIBAC2-F20-ARC1 or BIBAC2-F22-ARC1 Arabidopsis stigmas. Taken together, our results suggest that complementing Arabidopsis genome with Brassica SLG, SRK and ARC1 genes is unlikely to be sufficient to transfer the SI phenotype.

The expression of nitrite reductase (NiR; EC 1.7.7.1), the second enzyme in the nitrate assimilatory pathway, is regulated by nitrate as well as by end-products of nitrate assimilation, namely, glutamine (Gln) and asparagine (Asn). Nitrate induces expression of the NiR gene. Previously, using deletion analysis of the spinach (Spinacia oleracea L.) NiR gene promoter in transgenic tobacco (Nicotiana tabacum L.) and in-vivo dimethyl sulfate footprinting, we had identified the region between −230 bp and −180 bp as being critical for nitrate inducibility of this gene. In the present study, we show that the region from +1 to +67, which forms part of its untranslated leader, is important for minimal induction in the presence of nitrate. Electrophoretic mobility shift assays reveal concentration-dependent and competitive binding of a factor in tobacco nuclear extracts to this region. In the presence of Gln or Asn, the expression of spinach NiR is repressed. This repression is observed with the full-length NiR promoter (−3100 bp) as well as with the shortest promoter (−230 bp) that gives nitrate induction, which includes the +67 bp leader sequence. The repressed expression of the gene is not the result of reduced nitrate accumulation in the presence of the nitrogen metabolites.

As part of the Brassicaceae self-incompatibility response, callose is deposited in the stigma papillar cells. To determine if callose plays an important role in the rejection of incompatible pollen by the stigma, transgenic Brassica napus. L. plants were produced which express the tobacco β-1,3-glucanase cDNA (the enzyme which degrades callose) in the stigma papillae. Using aniline blue fluorescence, little or no callose was detected in the papillar cells of transgenic stigmas. However, the self-incompatibility system appeared to be unaffected based on the lack of pollen tube growth and the subsequent lack of seed set. The transgene had no effect on compatible pollinations. Thus, while callose deposition is associated with the B. napus self-incompatibility response, it is not required for the rejection of incompatible pollen.

Nitrite reductase (NiR) is the second enzyme in the nitrate assimilatory pathway reducing nitrite to ammonium. The expression of the NiR gene is induced upon the addition of nitrate. In an earlier study, a 130 bp upstream region of the spinach NiR gene promoter, located between −330 to −200, was shown to be necessary for nitrate induction of β-glucuronidase (GUS) expression in tissue-specific manner in transgenic tobacco plant [28]. To further delineate the cis-acting elements involved in nitrate regulation of NiR gene expression, transgenic tobacco plants were generated with 5′ deletions in the−330 to −200 region of the spinach NiR gene promoter fused to the GUS gene. Plants with the NiR promoter deleted to −230 showed a considerable increase in GUS activity in the presence of nitrate, indicating that the 30 bp region between −230 to −200 is crucial for nitrate-regulated expression of NiR. In vivo DMS footprinting of the −300 to −130 region of the NiR promoter in leaf tissues from two independent transgenic lines revealed several nitrate-inducible footprints. Footprinting within the −230 to −181 region revealed factor binding to two adjacent GATA elements separated by 24 bp. This arrangement of GATA elements is analogous to cis-regulatory sequences found in the promoters of nitrate-inducible genes of Neurospora crassa, regulated by the NIT2 Zn-finger protein. The −240 to −110 fragment of the NiR promoter, which contains two NIT2 consensus core elements, bound in vitro to a fusion protein comprising the zinc finger domain of the N. crassa NIT2 protein. The data presented here show that nitrate-inducible expression of the NiR gene is mediated by nitrate-specific binding of trans-acting factors to sequences preserved between fungi and higher plants.

An S-receptor kinase (SRK) gene associated with self-incompatibility in a Brassica napus subsp. oleifera line has been characterized. The SRK-A14 cDNA shows the highest levels of homology in the 5' end to the SLG-A14 cDNA present at the same locus. RNA blot analysis shows that the SRK-A14 gene is expressed predominantly in the pistil, and at lower levels in the anthers. The predicted amino acid sequences from the extracellular domain of the SRK-A14 gene and three other SRK genes were compared. The different SRK extracellular domains were for the most part very similar, with the exception of two variable regions containing a high level of amino acid alterations. These extracellular domains also contain a region of similarity to the immunoglobulin domains present in members of the immunoglobulin superfamily. These findings may define regions of the SRK protein that are necessary for interactions between SRK and other proteins.

A self-incompatible canola-quality Brassica napus ssp. oleifera line (W1) was generated by introgressing the S-locus from a self-incompatible B. campestris plant into the Westar cultivar. Using the polymerase chain reaction (PCR) with primers derived from conserved regions in S-locus glycoprotein (SLG) alleles, the central region of the active SLG gene (910) was obtained. The remaining portions of the cDNA for this 910 gene were subsequently cloned using the PCR-rapid amplification of cDNA ends (RACE) procedure. Sequence analysis revealed that the 910 cDNA show a high degree of sequence similarity to SLG alleles associated with Class I self-incompatible lines. The 910 gene was found to be absent in the original self-compatible cv. Westar (B. napus) and segregated with self-incompatibility in a mixed population generated from a cross between self-incompatible W1 and self-compatible Westar. RNA blot analysis indicated that high levels of 910 mRNAs were present in the stigma as buds approached anthesis. Thus, the SLG allele of W1 transferred from B. campestris via backcrosses to a line of cv. Westar has been identified.

Nitrite reductase is the second enzyme in the nitrate assimilatory pathway. The transcription of this gene is regulated by nitrate as well as a variety of other environmental and developmental factors. Genomic clones containing the entire nitrite reductase gene have been isolated from a spinach genomic library and sequenced. The sequence is identical in the transcribed region to a previously isolated spinach NiR cDNA clone (Back et al., 1988) except for the presence of three introns. The analysis of the genomic clones and DNA blot hybridization demonstrates that there is a single NiR gene per haploid genome in spinach. This is in contrast to what has been found for other plant species. The transcription initiation site has been determined by S1 mapping and the 5' upstream region has been used to regulate the GUS reporter gene in transgenic tobacco plants. This gene was found to be regulated by the addition of nitrate in the transgenic plants.

The main nitrogen source for most higher plants is soil nitrate. Prior to its incorporation into amino acids, plants reduce nitrate to ammonia in two enzymatic steps. Nitrate is reduced by nitrate reductase to nitrite, which is further reduced to ammonia by nitrite reductase. In this paper, the complete primary sequence of the precursor protein for spinach nitrite reductase has been deduced from cloned cDNAs. The cDNA clones were isolated from a nitrate-induced cDNA library in two ways: through the use of oligonucleotide probes based on partial amino acid sequences of nitrite reductase and through the use of antibodies raised against purified nitrite reductase. The precursor protein for nitrite reductase is 594 amino acids long and has a 32 amino acid extension at the N-terminal end of the mature protein. These 32 amino acids most likely serve as a transit peptide involved in directing this nuclear-encoded protein into the chloroplast. The cDNA hybridizes to a 2.3 kb RNA whose steady-state level is markedly increased upon induction with nitrate.

The translocation of secretory proteins across the endoplasmic reticulum involves the recognition and cleavage of an amino-terminal extension called the signal sequence1. The structure of signal peptides appears to be ubiquitous in having a very hydrophobic central core2, so that the signal sequence in secretory proteins from one organism could possibly be recognized by the processing and transport apparatus of another. We therefore wished to investigate whether a protein, α-amylase, one of several hydrolytic enzymes secreted from the aleurone of wheat into the endosperm during germination, could be processed and secreted in an active form from the yeast Saccharomyces cerevisiae, secretion being dependent upon the plant signal sequence. Here, synthesis of α-amylase was by inserting a cDNA clone coding for the entire α-amylase structural gene3 into a yeast expression vector4. The α-amylase protein coded for by this gene fusion has the signal sequence located internally, not at the N-terminal end of the polypeptide. Nevertheless, it is processed and the processed form is secreted into the medium in an active form. There are potential industrial applications for yeast that secrete a functional α-amylase.