The Key Role of Glutamate Dehydrogenase 2 (GDH2) in the Control of Kernel Production in Maize (Zea mays L.).

The agronomic potential of glutamate dehydrogenase 2 (GDH2) in maize kernel production was investigated by examining the impact of a mutation on the corresponding gene. Mu-insertion homozygous and heterozygous mutant lines lacking GDH2 activity were isolated and characterized at the biochemical, physiological and agronomic levels. In comparison to the wild type and to the homozygous ghd2 mutants, the heterozygous gdh2 mutant plants were characterized by a decrease in the root amino acid content, whereas in the leaves an increase of a number of phenolic compounds was observed. On average, a 30 to 40% increase in kernel yield was obtained only in the heterozygous gdh2 mutant lines when plants were grown in the field over two years. The importance of GDH2 in the control of plant productivity is discussed in relation to the physiological impact of the mutation on amino acid content, with primary carbon metabolism mostly occurring in the roots and secondary metabolism occurring in the leaves.

CROWN ROOTLESS1 binds DNA with a relaxed specificity and activates OsROP and OsbHLH044 genes involved in crown root formation in rice.

In cereals, the root system is mainly composed of post-embryonic shoot-borne roots, named crown roots. The CROWN ROOTLESS1 (CRL1) transcription factor, belonging to the ASYMMETRIC LEAVES2-LIKE/LATERAL ORGAN BOUNDARIES DOMAIN (ASL/LBD) family, is a key regulator of crown root initiation in rice (Oryza sativa). Here, we show that CRL1 can bind, both in vitro and in vivo, not only the LBD-box, a DNA sequence recognized by several ASL/LBD transcription factors, but also another not previously identified DNA motif that was named CRL1-box. Using rice protoplast transient transactivation assays and a set of previously identified CRL1-regulated genes, we confirm that CRL1 transactivates these genes if they possess at least a CRL1-box or an LBD-box in their promoters. In planta, ChIP-qPCR experiments targeting two of these genes that include both a CRL1- and an LBD-box in their promoter show that CRL1 binds preferentially to the LBD-box in these promoter contexts. CRISPR/Cas9-targeted mutation of these two CRL1-regulated genes, which encode a plant Rho GTPase (OsROP) and a basic helix-loop-helix transcription factor (OsbHLH044), show that both promote crown root development. Finally, we show that OsbHLH044 represses a regulatory module, uncovering how CRL1 regulates specific processes during crown root formation.

Impacts of environmental conditions, and allelic variation of cytosolic glutamine synthetase on maize hybrid kernel production.

Cytosolic glutamine synthetase (GS1) is the enzyme mainly responsible of ammonium assimilation and reassimilation in maize leaves. The agronomic potential of GS1 in maize kernel production was investigated by examining the impact of an overexpression of the enzyme in the leaf cells. Transgenic hybrids exhibiting a three-fold increase in leaf GS activity were produced and characterized using plants grown in the field. Several independent hybrids overexpressing Gln1-3, a gene encoding cytosolic (GS1), in the leaf and bundle sheath mesophyll cells were grown over five years in different locations. On average, a 3.8% increase in kernel yield was obtained in the transgenic hybrids compared to controls. However, we observed that such an increase was simultaneously dependent upon both the environmental conditions and the transgenic event for a given field trial. Although variable from one environment to another, significant associations were also found between two GS1 genes (Gln1-3 and Gln1-4) polymorphic regions and kernel yield in different locations. We propose that the GS1 enzyme is a potential lead for producing high yielding maize hybrids using either genetic engineering or marker-assisted selection. However, for these hybrids, yield increases will be largely dependent upon the environmental conditions used to grow the plants.

A transposon surveillance mechanism that safeguards plant male fertility during stress.

Although plants are able to withstand a range of environmental conditions, spikes in ambient temperature can impact plant fertility causing reductions in seed yield and notable economic losses1,2. Therefore, understanding the precise molecular mechanisms that underpin plant fertility under environmental constraints is critical to safeguarding future food production3. Here, we identified two Argonaute-like proteins whose activities are required to sustain male fertility in maize plants under high temperatures. We found that MALE-ASSOCIATED ARGONAUTE-1 and -2 associate with temperature-induced phased secondary small RNAs in pre-meiotic anthers and are essential to controlling the activity of retrotransposons in male meiocyte initials. Biochemical and structural analyses revealed how male-associated Argonaute activity and its interaction with retrotransposon RNA targets is modulated through the dynamic phosphorylation of a set of highly conserved, surface-located serine residues. Our results demonstrate that an Argonaute-dependent, RNA-guided surveillance mechanism is critical in plants to sustain male fertility under environmentally constrained conditions, by controlling the mutagenic activity of transposons in male germ cells.

Transcriptome profiling of laser-captured crown root primordia reveals new pathways activated during early stages of crown root formation in rice.

Crown roots constitute the main part of the rice root system. Several key genes involved in crown root initiation and development have been identified by functional genomics approaches. Nevertheless, these approaches are impaired by functional redundancy and mutant lethality. To overcome these limitations, organ targeted transcriptome analysis can help to identify genes involved in crown root formation and early development. In this study, we generated an atlas of genes expressed in developing crown root primordia in comparison with adjacent stem cortical tissue at three different developmental stages before emergence, using laser capture microdissection. We identified 3975 genes differentially expressed in crown root primordia. About 30% of them were expressed at the three developmental stages, whereas 10.5%, 19.5% and 12.8% were specifically expressed at the early, intermediate and late stages, respectively. Sorting them by functional ontology highlighted an active transcriptional switch during the process of crown root primordia formation. Cross-analysis with other rice root development-related datasets revealed genes encoding transcription factors, chromatin remodeling factors, peptide growth factors, and cell wall remodeling enzymes that are likely to play a key role during crown root primordia formation. This atlas constitutes an open primary data resource for further studies on the regulation of crown root initiation and development.

NADH-GOGAT Overexpression Does Not Improve Maize (Zea mays L.) Performance Even When Pyramiding with NAD-IDH, GDH and GS.

Maize plants overexpressing NADH-GOGAT were produced in order to determine if boosting 2-Oxoglurate production used as a carbon skeleton for the biosynthesis of amino acids will improve plant biomass and kernel production. The NADH-GOGAT enzyme recycles glutamate and incorporates carbon skeletons into the ammonium assimilation pathway using the organic acid 2-Oxoglutarate as a substrate. Gene pyramiding was then conducted with NAD-IDH and NADH-GDH, two enzymes also involved in the synthesis of 2-Oxoglurate. NADH-GOGAT overexpression was detrimental for shoot biomass production but did not markedly affect kernel yield. Additional NAD-IDH and NADH-GDH activity did not improve plant performance. A decrease in kernel production was observed when NADH-GDH was pyramided to NADH-GOGAT and NAD-IDH. This decrease could not be restored even when additional cytosolic GS activity was present in the plants overexpressing the three enzymes producing 2-Oxoglutarate. Detailed leaf metabolic profiling of the different transgenic plants revealed that the NADH-GOGAT over-expressors were characterized by an accumulation of amino acids derived from glutamate and a decrease in the amount of carbohydrates further used to provide carbon skeletons for its synthesis. The study suggests that 2-Oxoglutarate synthesis is a key element acting at the interface of carbohydrate and amino acid metabolism and that its accumulation induces an imbalance of primary carbon and nitrogen metabolism that is detrimental for maize productivity.

Inference of the gene regulatory network acting downstream of CROWN ROOTLESS 1 in rice reveals a regulatory cascade linking genes involved in auxin signaling, crown root initiation, and root meristem specification and maintenance.

Crown roots (CRs) are essential components of the rice root system. Several genes involved in CR initiation or development have been identified but our knowledge about how they organize to form a gene regulatory network (GRN) is still limited. To characterize the regulatory cascades acting during CR formation, we used a systems biology approach to infer the GRN controlling CR formation downstream of CROWN ROOTLESS 1 (CRL1), coding for an ASL (asymmetric leaves-2-like)/LBD (LOB domain) transcription factor necessary for CR initiation. A time-series transcriptomic dataset was generated after synchronized induction of CR formation by dexamethasone-mediated expression of CRL1 expression in a crl1 mutant background. This time series revealed three different genome expression phases during the early steps of CR formation and was further exploited to infer a GRN using a dedicated algorithm. The predicted GRN was confronted with experimental data and 72% of the inferred links were validated. Interestingly, this network revealed a regulatory cascade linking CRL1 to other genes involved in CR initiation, root meristem specification and maintenance, such as QUIESCENT-CENTER-SPECIFIC HOMEOBOX, and in auxin signalling. This predicted regulatory cascade was validated in vivo using transient activation assays. Thus, the CRL1-dependant GRN reflects major gene regulation events at play during CR formation and constitutes a valuable source of discovery to better understand this developmental process.

Control of Maize Vegetative and Reproductive Development, Fertility, and rRNAs Silencing by HISTONE DEACETYLASE 108.

Histone deacetylases (HDACs) catalyze the removal of acetyl groups from acetylated histone tails that consequently interact more closely with DNA, leading to chromatin state refractory to transcription. Zea mays HDA108 belongs to the Rpd3/HDA1 HDAC family and is ubiquitously expressed during development. The newly isolated hda108/hda108 insertional mutant exhibited many developmental defects: significant reduction in plant height, alterations of shoot and leaf development, and alterations of inflorescence patterning and fertility. Western blot analyses and immunolocalization experiments revealed an evident increase in histone acetylation, accompanied by a marked reduction in H3K9 dimethylation, in mutant nuclei. The DNA methylation status, in the CHG sequence context, and the transcript level of ribosomal sequences were also affected in hda108 mutants, while enrichment in H3 and H4 acetylation characterizes both repetitive and nonrepetitive transcriptional up-regulated loci. RNA-Seq of both young leaf and anthers indicated that transcription factor expression is highly affected and that the pollen developmental program is disrupted in hda108 mutants. Crosses between hda108/hda108 and epiregulator mutants did not produce any double mutant progeny indicating possible genetic interactions of HDA108 with distinct epigenetic pathways. Our findings indicate that HDA108 is directly involved in regulation of maize development, fertility, and epigenetic regulation of genome activity.

Maternal control of nutrient allocation in plant seeds by genomic imprinting.

Imprinted genes are commonly expressed in mammalian placentas and in plant seed endosperms, where they exhibit preferential uniparental allelic expression. In mammals, imprinted genes directly regulate placental function and nutrient distribution from mother to fetus; however, none of the >60 imprinted genes thus far reported in plants have been demonstrated to play an equivalent role in regulating the flow of resources to the embryo. Here we show that imprinted Maternally expressed gene1 (Meg1) in maize is both necessary and sufficient for the establishment and differentiation of the endosperm nutrient transfer cells located at the mother:seed interface. Consistent with these findings, Meg1 also regulates maternal nutrient uptake, sucrose partitioning, and seed biomass yield. In addition, we generated an imprinted and nonimprinted synthetic Meg1 ((syn)Meg1) dosage series whereby increased dosage and absence of imprinting both resulted in an unequal investment of maternal resources into the endosperm. These findings highlight dosage regulation by genomic imprinting as being critical for maintaining a balanced distribution of maternal nutrients to filial tissues in plants, as in mammals. However, unlike in mammals, Meg1 is a maternally expressed imprinted gene that surprisingly acts to promote rather than restrict nutrient allocation to the offspring.

Duplicate maize Wrinkled1 transcription factors activate target genes involved in seed oil biosynthesis.

WRINKLED1 (WRI1), a key regulator of seed oil biosynthesis in Arabidopsis (Arabidopsis thaliana), was duplicated during the genome amplification of the cereal ancestor genome 90 million years ago. Both maize (Zea mays) coorthologs ZmWri1a and ZmWri1b show a strong transcriptional induction during the early filling stage of the embryo and complement the reduced fatty acid content of Arabidopsis wri1-4 seeds, suggesting conservation of molecular function. Overexpression of ZmWri1a not only increases the fatty acid content of the mature maize grain but also the content of certain amino acids, of several compounds involved in amino acid biosynthesis, and of two intermediates of the tricarboxylic acid cycle. Transcriptomic experiments identified 18 putative target genes of this transcription factor, 12 of which contain in their upstream regions an AW box, the cis-element bound by AtWRI1. In addition to functions related to late glycolysis and fatty acid biosynthesis in plastids, the target genes also have functions related to coenzyme A biosynthesis in mitochondria and the production of glycerol backbones for triacylglycerol biosynthesis in the cytoplasm. Interestingly, the higher seed oil content in ZmWri1a overexpression lines is not accompanied by a reduction in starch, thus opening possibilities for the use of the transgenic maize lines in breeding programs.