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Plants have evolved to grow under prominently fluctuating environmental conditions. In experiments under controlled conditions, temperature is often set to artificial, binary regimes with constant values at day and at night. This study investigated how such a diel (24 hr) temperature regime affects leaf growth, carbohydrate metabolism and gene expression, compared to a temperature regime with a field‐like gradual increase and decline throughout 24 hr. Soybean (Glycine max) was grown under two contrasting diel temperature treatments. Leaf growth was measured in high temporal resolution. Periodical measurements were performed of carbohydrate concentrations, carbon isotopes as well as the transcriptome by RNA sequencing. Leaf growth activity peaked at different times under the two treatments, which cannot be explained intuitively. Under field‐like temperature conditions, leaf growth followed temperature and peaked in the afternoon, whereas in the binary temperature regime, growth increased at night and decreased during daytime. Differential gene expression data suggest that a synchronization of cell division activity seems to be evoked in the binary temperature regime. Overall, the results show that the coordination of a wide range of metabolic processes is markedly affected by the diel variation of temperature, which emphasizes the importance of realistic environmental settings in controlled condition experiments., Dynamically changing temperature affects soybean leaf growth and regulatory mechanisms in non‐intuitive ways. The results may help improving knowledge transfer between experiments performed under controlled conditions and experiments performed in the field.

The carbon isotopic composition (δ13C) of foliage is often used as proxy for plant performance. However, the effect of NO3– vs. NH4+ supply on δ13C of leaf metabolites and respired CO2 is largely u...

The carbon isotopic composition (δ

Mutation breeding has a long-standing history and in some major crop species, many of the most important cultivars have their origin in germplasm generated by mutation induction. For almost two decades, methods for TILLING (Targeting Induced Local Lesions IN Genomes) have been established in model plant species such as Arabidopsis (Arabidopsis thaliana L.), enabling the functional analysis of genes. Recent advances in mutation detection by second generation sequencing technology have brought its utility to major crop species. However, it has remained difficult to apply similar approaches in forage and turf grasses, mainly due to their outbreeding nature maintained by an efficient self-incompatibility system. Starting with a description of the extent to which traditional mutagenesis methods have contributed to crop yield increase in the past, this review focuses on technological approaches to implement TILLING-based strategies for the improvement of forage grass breeding through forward and reverse genetics. We present first results from TILLING in allogamous forage grasses for traits such as stress tolerance and evaluate prospects for rapid implementation of beneficial alleles to forage grass breeding. In conclusion, large-scale induced mutation resources, used for forward genetic screens, constitute a valuable tool to increase the genetic diversity for breeding and can be generated with relatively small investments in forage grasses. Furthermore, large libraries of sequenced mutations can be readily established, providing enhanced opportunities to discover mutations in genes controlling traits of agricultural importance and to study gene functions by reverse genetics.

White clover (Trifolium repens L.) is one of the most important legumes for fodder production in temperate climates, particularly in intensive pasture systems. Like many other forage legumes, it lacks the energy content to maximize productivity of modern ruminant livestock breeds. White clover produces water-soluble carbohydrates and starch in its leaves as a diurnal product of photosynthesis. However, little is known about the genetically encoded variability of diel changes in carbohydrate content. We assessed the amount of glucose, fructose, sucrose, and starch in the leaves of 185 plants of a genetically diverse white clover population. Water-soluble carbohydrates only provided on average 10.6% of dry weight (DW) of the total analyzed non-structural carbohydrate (NSC) content at the end of the day (ED), while starch supplied 89.4% of the NSC content. The top 5% of individuals accumulated over 25% of their DW as starch at ED. The leaf starch content at ED showed up to a threefold difference between genotypes, with a repeatability value of 0.95. Our experiments illustrate both the physical potential of white clover to serve as a competitive energy source to meet the demand of modern ruminant livestock production and the genetic potential to improve this trait by breeding.

l-Ascorbic acid (AsA) is an essential nutrient in the human diet. It is required for growth and repair of tissues, and severe deficiency can lead to debilitating diseases such as scurvy. Although relatively cheap to synthetically produce, ascorbate deficiencies are common throughout developed and developing countries. In plants, AsA is a key component of multiple antioxidant systems, which defend plant cells from biotic and abiotic stresses, and is therefore an attractive target for biofortification in crops. AsA metabolism is complex due to the existence of multiple biosynthetic pathways, which can contribute differing amounts of AsA depending on the plant species, organ, tissue type, and stress conditions. In addition, several pathways of oxidation and recycling of AsA exist, which together allow subcellular compartments to tailor AsA accumulation and turnover. Biotechnological approaches in model and crop species have been successful in increasing AsA concentrations, but these gains have not reached the potential some species of plants have to produce AsA and that could benefit industrial and public stakeholders. Given recent improvements in our understanding of AsA biosynthesis as well as the advancement in novel breeding technology, there is renewed potential to overcome limitations in AsA biofortification. Here we attempt to connect the current biological knowledgebase with novel technologies and crop resources to provide a strategy to improve plant-synthesized AsA in the world food system.

Eukaryotic cells require mechanisms to establish the proportion of cellular volume devoted to particular organelles. These mechanisms are poorly understood. From a screen for plastid-to-nucleus signaling mutants in Arabidopsis thaliana, we cloned a mutant allele of a gene that encodes a protein of unknown function that is homologous to two other Arabidopsis genes of unknown function and to FRIENDLY, which was previously shown to promote the normal distribution of mitochondria in Arabidopsis. In contrast to FRIENDLY, these three homologs of FRIENDLY are found only in photosynthetic organisms. Based on these data, we proposed that FRIENDLY expanded into a small gene family to help regulate the energy metabolism of cells that contain both mitochondria and chloroplasts. Indeed, we found that knocking out these genes caused a number of chloroplast phenotypes, including a reduction in the proportion of cellular volume devoted to chloroplasts to 50% of wild type. Thus, we refer to these genes as REDUCED CHLOROPLAST COVERAGE (REC). The size of the chloroplast compartment was reduced most in rec1 mutants. The REC1 protein accumulated in the cytosol and the nucleus. REC1 was excluded from the nucleus when plants were treated with amitrole, which inhibits cell expansion and chloroplast function. We conclude that REC1 is an extraplastidic protein that helps to establish the size of the chloroplast compartment, and that signals derived from cell expansion or chloroplasts may regulate REC1.

The Arabidopsis arc1 (accumulation and replication of chloroplasts 1) mutant has pale seedlings and smaller, more numerous chloroplasts than the wild type. Previous work has suggested that arc1 affects the timing of chloroplast division but does not function directly in the division process. We isolated ARC1 by map-based cloning and discovered it encodes FtsHi1 (At4g23940), one of several FtsHi proteins in Arabidopsis. These poorly studied proteins resemble FtsH metalloproteases important for organelle biogenesis and protein quality control but are presumed to be proteolytically inactive. FtsHi1 bears a predicted chloroplast transit peptide and localizes to the chloroplast envelope membrane. Phenotypic studies showed that arc1 (hereafter ftsHi1-1), which bears a missense mutation, is a weak allele of FtsHi1 that disrupts thylakoid development and reduces de-etiolation efficiency in seedlings, suggesting that FtsHi1 is important for chloroplast biogenesis. Consistent with this finding, transgenic plants suppressed for accumulation of an FtsHi1 fusion protein were often variegated. A strong T-DNA insertion allele, ftsHi1-2, caused embryo-lethality, indicating that FtsHi1 is an essential gene product. A wild-type FtsHi1 transgene rescued both the chloroplast division and pale phenotypes of ftsHi1-1 and the embryo-lethal phenotype of ftsHi1-2. FtsHi1 overexpression produced a subtle increase in chloroplast size and decrease in chloroplast number in wild-type plants while suppression led to increased numbers of small chloroplasts, providing new evidence that FtsHi1 negatively influences chloroplast division. Taken together, our analyses reveal that FtsHi1 functions in an essential, envelope-associated process that may couple plastid development with division.

We previously provided evidence that plastid signaling regulates the downstream components of a light signaling network and that this signal integration coordinates chloroplast biogenesis with both the light environment and development by regulating gene expression. We tested these ideas by analyzing light- and plastid-regulated transcriptomes in Arabidopsis (Arabidopsis thaliana). We found that the enrichment of Gene Ontology terms in these transcriptomes is consistent with the integration of light and plastid signaling (1) down-regulating photosynthesis and inducing both repair and stress tolerance in dysfunctional chloroplasts and (2) helping coordinate processes such as growth, the circadian rhythm, and stress responses with the degree of chloroplast function. We then tested whether factors that contribute to this signal integration are also regulated by light and plastid signals by characterizing T-DNA insertion alleles of genes that are regulated by light and plastid signaling and that encode proteins that are annotated as contributing to signaling, transcription, or no known function. We found that a high proportion of these mutant alleles induce chloroplast biogenesis during deetiolation. We quantified the expression of four photosynthesis-related genes in seven of these enhanced deetiolation (end) mutants and found that photosynthesis-related gene expression is attenuated. This attenuation is particularly striking for Photosystem II subunit S expression. We conclude that the integration of light and plastid signaling regulates a number of END genes that help optimize chloroplast function and that at least some END genes affect photosynthesis-related gene expression.

Light and plastid signals promote chloroplast biogenesis and are among the most potent inducers and repressors of photosynthesis-related gene expression, respectively. These signals can be likened to a 'gas and brake system' that promotes efficient chloroplast biogenesis and function. Recent findings indicate that a particular plastid signal can 'rewire' a light signaling network, converting it from an inducer into a repressor of particular photosynthesis-related genes. Therefore, a plastid signal appears to be an endogenous regulator of light signaling rather than a signal acting independently from light. This integration of light and plastid signals may allow plants to proactively manage chloroplast dysfunction when performing chloroplast biogenesis and maintenance in adverse light conditions.

Here we describe the methodology of using virus-induced gene silencing (VIGS) as a powerful and scalable tool to screen the function of genes that participate in adaptation to drought. Silencing of endogenous gene expression in Nicotiana benthamiana is achieved by systemic infection of the aerial parts of the plant with a virus engineered to contain homologous fragments of the target gene(s) of interest. Silenced plant material can be consistently produced with little optimization in less than 1 month without specialized equipment, using only simple cloning and transformation techniques. Although maximal silencing is localized to only a few leaves, when whole plants are subjected to water stress, the tissue from these silenced leaves can be characterized for physiological, biochemical, and transcriptional responses to determine the role of the candidate genes in drought tolerance.

Here we describe the methodology of using virus-induced gene silencing (VIGS) as a powerful and scalable tool to screen the function of genes that participate in adaptation to drought. Silencing of endogenous gene expression in Nicotiana benthamiana is achieved by systemic infection of the aerial parts of the plant with a virus engineered to contain homologous fragments of the target gene(s) of interest. Silenced plant material can be consistently produced with little optimization in less than 1 month without specialized equipment, using only simple cloning and transformation techniques. Although maximal silencing is localized to only a few leaves, when whole plants are subjected to water stress, the tissue from these silenced leaves can be characterized for physiological, biochemical, and transcriptional responses to determine the role of the candidate genes in drought tolerance.

A new method has been developed to rapidly determine the total inorganic carbon concentration (gaseous [CO2] + aqueous [CO(2)] + [HCO3-] + [CO3(2)-]) in developing seeds. Seeds are rapidly dissected and homogenized in 1 N HCl in gas-tight vials. The headspace gas is then analyzed by infrared gas analysis. Developing rapeseed (Brassica napus L.) and soybean [Glycine max (L.) Merr.] seeds were analyzed and found to have up to 40 and 12 mM total inorganic carbon, respectively. These concentrations are ca. 600-2000-fold higher than in ambient air or values reported for leaves. Carbon dioxide concentrations in rapeseed peaked during the stage of maximum oil synthesis and declined as seeds matured. The consequences for seed metabolism, physiology and carbon economy are discussed.

The biosynthesis of thylakoid lipids in eukaryotic photosynthetic organisms often involves enzymes in the endoplasmic reticulum (ER) and the chloroplast envelopes. Two pathways of thylakoid lipid biosynthesis, the ER and the plastid pathways, are present in parallel in many species, including Arabidopsis, but in other plants, e.g. grasses, only the ER pathway is active. The unicellular alga Chlamydomonas reinhardtii diverges from plants like Arabidopsis in a different way because its membranes do not contain phosphatidylcholine, and most thylakoid lipids are derived from the plastid pathway. Here, we describe an acylated derivative of sulfolipid, 2′-O-acyl-sulfoquinovosyldiacylglycerol (ASQD), which is present in C. reinhardtii. Although the fatty acids of sulfoquinovosyldiacylglycerol (SQDG) were mostly saturated, ASQD molecular species carried predominantly unsaturated fatty acids. Moreover, directly attached to the head group of ASQD was preferentially an 18-carbon fatty acid with four double bonds. High-throughput robotic screening led to the isolation of a plasmid disruption mutant of C. reinhardtii, designated Δsqd1, which lacks ASQD as well as SQDG. In this mutant, the SQD1 ortholog was completely deleted and replaced by plasmid sequences. It is proposed that ASQD arises from the sugar nucleotide pathway of sulfolipid biosynthesis by acylation of the 2′-hydroxyl of the sulfoquinovosyl head group. At the physiological level, the mutant showed increased sensitivity to a diuron herbicide and reduced growth under phosphate limitation, suggesting a role for SQDG and/or ASQD in photosynthesis as conducted by C. reinhardtii, particularly under phosphate-limited conditions.

The Arabidopsis arc1 (accumulation and replication of chloroplasts 1) mutant has pale seedlings and smaller, more numerous chloroplasts than the wild type. Previous work has suggested that arc1 affects the timing of chloroplast division but does not function directly in the division process. We isolated ARC1 by map-based cloning and discovered it encodes FtsHi1 (At4g23940), one of several FtsHi proteins in Arabidopsis. These poorly studied proteins resemble FtsH metalloproteases important for organelle biogenesis and protein quality control but are presumed to be proteolytically inactive. FtsHi1 bears a predicted chloroplast transit peptide and localizes to the chloroplast envelope membrane. Phenotypic studies showed that arc1 (hereafter ftsHi1-1), which bears a missense mutation, is a weak allele of FtsHi1 that disrupts thylakoid development and reduces de-etiolation efficiency in seedlings, suggesting that FtsHi1 is important for chloroplast biogenesis. Consistent with this finding, transgenic plants suppressed for accumulation of an FtsHi1 fusion protein were often variegated. A strong T-DNA insertion allele, ftsHi1-2, caused embryo-lethality, indicating that FtsHi1 is an essential gene product. A wild-type FtsHi1 transgene rescued both the chloroplast division and pale phenotypes of ftsHi1-1 and the embryo-lethal phenotype of ftsHi1-2. FtsHi1 overexpression produced a subtle increase in chloroplast size and decrease in chloroplast number in wild-type plants while suppression led to increased numbers of small chloroplasts, providing new evidence that FtsHi1 negatively influences chloroplast division. Taken together, our analyses reveal that FtsHi1 functions in an essential, envelope-associated process that may couple plastid development with division.

Summary • When plastids experience dysfunction they emit signals that help coordinate nuclear gene expression with their functional state. One of these signals can remodel a light-signaling network that regulates the expression of nuclear genes that encode particular antenna proteins of photosystem II. These findings led us to test whether plastid signals might impact other light-regulated processes. • Photomorphogenesis was monitored in genomes uncoupled 1 (gun1), cryptochrome 1 (cry1), and long hypocotyl 5 (hy5), which have defects in light and plastid signaling, by growing Arabidopsis thaliana seedlings under various light conditions and either treating or not treating them with antibiotics that induce chloroplast dysfunction and trigger plastid signaling. • It was found that plastid signals that depend on GUN1 can affect cotyledon opening and expansion, anthocyanin biosynthesis, and hypocotyl elongation. We also found that plastid signals that depend on CRY1 can regulate cotyledon expansion and development. • Our findings suggest that plastid signals triggered by plastid dysfunction can broadly affect photomorphogenesis and that plastid and light signaling can promote or antagonize each other, depending on the responses studied. These data suggest that GUN1 and cry 1 help to integrate chloroplast function with photomorphogenesis.

Plastid signals are among the most potent regulators of genes that encode proteins active in photosynthesis. Plastid signals help coordinate the expression of the nuclear and chloroplast genomes and the expression of genes with the functional state of the chloroplast. Here, we report the isolation of new cryptochrome1 (cry1) alleles from a screen for Arabidopsis thaliana genomes uncoupled mutants, which have defects in plastid-to-nucleus signaling. We also report genetic experiments showing that a previously unidentified plastid signal converts multiple light signaling pathways that perceive distinct qualities of light from positive to negative regulators of some but not all photosynthesis-associated nuclear genes (PhANGs) and change the fluence rate response of PhANGs. At least part of this remodeling of light signaling networks involves converting HY5, a positive regulator of PhANGs, into a negative regulator of PhANGs. We also observed that mutants with defects in both plastid-to-nucleus and cry1 signaling exhibited severe chlorophyll deficiencies. These data show that the remodeling of light signaling networks by plastid signals is a mechanism that plants use to integrate signals describing the functional and developmental state of plastids with signals describing particular light environments when regulating PhANG expression and performing chloroplast biogenesis.